It is argued that the current state of affairs on terminology is unsatisfactory. It is suggested that the way ahead needs to be determined by consideration of the purposes of classification and the key concepts that should shape diagnosis.
Both papers in this special issue (Reilly
First, both papers slip into statements about diagnosing ‘children’. In my opinion, that is unacceptable. Diagnoses, surely, must refer to the disorders or patterns shown by a child. It is demeaning to imply that the diagnosis incorporates all that matters about that individual. That is particularly the case when research findings have shown that language impairment at age 4 years often proves to be transient rather than persistent (
The next need is to consider the purpose of classification. In the American Psychiatric Association’s (
Before turning to that issue, it is necessary to ask whether it might be best to leave things as they are. Bishop argues that would be unsatisfactory because so many different terms are being used, with no clear indication whether they mean the same problem or a range of rather different problems. I agree. Reilly
Reilly
Are there other descriptors that should be added? I think not. ‘Specific’, as commonly used now (as with SLI) implies a ‘pure’ language impairment, and that is not supported by any of the available evidence. To exclude cases with a specific pathogenic genetic cause sounds reasonable at first sight because the concept is of a multifactorial causation. Nevertheless, in my opinion, that is best dealt with in an accompanying descriptive text rather than through some exclusionary criterion.
The same applies to other possible exclusionary terms such as social disadvantage or profound deafness or profound intellectual impairment, at least so far as clinical usage is concerned. For research purposes, however, it will usually be necessary to have a set of rules to deal with the matter.
Reilly
The testing is also less straightforward than it might seem at first sight. To begin with, there is a far from perfect agreement among tests of language. Which do you advise to be used and why? Secondly, the findings will be hugely influenced by the particular populations studied. Should it include, for example, cases of autism? Once more, the choice of population needs to be guided by the concepts, and not by some preference for ‘normal’ populations.
I congratulate Reilly
Michael Rutter has no relevant interests but in the past he was a member of both ICD-11 and DSM-5 committees; e-mail:
Many researchers in the area of SLI are already using a broader definition of SLI, and do not use this term as a declaration that their study participants have a pure profile. The effort to acknowledge this broader outlook through the alternative term ‘language impairment’ would create more problems of boundary confusion than it would solve.
I agree with Reilly and colleagues that the label ‘specific language impairment’ is often applied to children who show subtle weaknesses in areas that go beyond language, and that these children cannot be empirically distinguished from those children who would match the original ‘pure’ profile of SLI. However, I disagree that an interim solution to this issue is to remove the word ‘specific’ and use instead the term ‘language impairment’. My position is shaped by two observations. First, researchers in the area of SLI are already using a broader definition of SLI in practice, and adopt SLI to distinguish their participants from those who fall into traditional clinical categories, not as a declaration that these participants have a pure profile. Second, the effort to acknowledge this broader outlook through the alternative term ‘language impairment’ would create more problems of boundary confusion than it would solve.
Since at least the 1970s, SLI researchers had been documenting weaknesses in non-linguistic areas such as symbolic play and mental imagery, which already cast doubt on the meaning of a nonverbal IQ of 85. The problems with 85 became more obvious with the work of Tomblin and colleagues, who, in the process of trying to determine the prevalence of SLI (as traditionally defined), found little reason to distinguish children with language impairments according to IQ level. Other characteristics were likewise found to be non-categorical. Based on this information and related data from other labs,
In making their case, Reilly
Reilly
I should also point out that the use of a significant (e.g., 1 SD) discrepancy between language and nonverbal IQ scores predates the appearance of the term ‘specific language impairment’ in the literature. Furthermore, this large-discrepancy criterion is no longer common in SLI research, and, from my experience in the United States, the clinical agencies that continue this misinformed practice have never employed the term ‘specific language impairment’.
I believe that the subtle problems in non-linguistic areas are now considered part of the SLI phenotype. Put differently, we have accepted the point that the children whose profiles do not fit the ‘pure’ profile of SLI of the earlier literature are not demonstrably different and can be included in studies of children with SLI. Paradoxically, where a line is drawn despite a fuzzy boundary is in the use of ‘impairment’—as in the authors’ retention of this word in ‘language impairment’. Inspection of the authors’ figures 2 and 3 reveals that there is no clear psychometric basis for separating the children into impairment and non-impairment groups.
We do so for practical reasons—to single out children that we believe should be given extra assistance. The use of SLI also has a practical basis—to identify children with language deficits who do not fall into categories such as intellectual disability, traumatic brain injury or autism spectrum disorder.
So why ‘SLI’ rather than some plausible alternative label? One alternative seen in the literature is ‘primary language impairment’. This label avoids creating an image of a pure profile, though it implies that at least one secondary impairment exists. Yet I believe that the weaknesses often seen in conjunction with the language deficit are not of sufficient magnitude to justify terms such as ‘motor impairment’ or ‘cognitive impairment’. And if a weakness in another ability area (e.g., nonverbal cognition) did warrant an ‘impairment’ classification, the scientific basis for regarding the language impairment as ‘primary’ would not be clear.
Despite its flaws, the use of ‘SLI’ holds an important advantage. As Bishop notes, this term is employed in far more instances in the published literature than alternative terms. This was also apparent to me (
Our field once went through a phase during which, in an attempt to avoid the ‘medical model’, rather imprecise terms were applied to children with language deficits. One had to dig deeply to discover whether the participants approximated children with SLI or were instead exhibiting intellectual disability or autism. Although we have moved beyond that phase, adopting a general term such as ‘language impairment’ will introduce new problems. Consider two questions of current relevance that might be asked: Do children with attention-deficit/hyperactivity disorder (ADHD) and a language impairment have the same language profile or the same source of language difficulty as children with SLI? Do children with autism spectrum disorder (ASD) and a language impairment have the same language profile or the same source of language difficulty as children with SLI? If we remove ‘specific’ we have: Do children with ADHD/ASD and a language impairment have the same language profile or the same source of language difficulty as children with language impairment? To make sense, we must say instead: ‘as children with language impairment who do not have ADHD/ASD’ (note that the alternative ‘as children with language impairment only’ might be confused with traditional SLI). Is this an improvement? I fear that our ability to communicate with the public and other disciplines will be made even more difficult if we adopt a generic label such as ‘language impairment’.
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Difficulties with the definition of SLI are shared with other neurodevelopmental disorders. Although a functional definition of language impairment (LI) could meet service needs, it remains important to identify its primary or ‘core’ features. The definition should recognize that LI can be observed in pure form (‘selective’) or co-occur with other risk factors in order to clarify targets for intervention.
Reilly and colleagues propose that the term ‘specific language impairment’ (SLI) should be abandoned because there is no evidence for a circumscribed category of language disorder in children whose cognitive skills are within normal limits. Exclusionary criteria, they argue, should be relaxed and the term replaced by ‘plain’ language impairment (LI) with
A key issue for the definition of LI is that it is heterogeneous and often co-occurs with other disorders. In this respect, it is no different from other neurodevelopmental disorders which are typically heritable, show early onset and persist through the life span (
DSM-5 has solved the problem of the lack of clear boundaries between the disorders of dyslexia, dysgraphia and dyscalculia by banding them together under a category of ‘Specific Learning Disorder’. The strength of this strategy is that it recognizes frequent co-morbidities but also accommodates the fact that core components of individual disorders differ (e.g., phonological deficits in dyslexia and non-verbal number deficits in dyscalculia). The obvious weakness is that these learning disorders are separated in the classification system from Language Disorder, which is listed among Communication Disorders, and yet a language disorder is frequently the root cause of a learning disorder (
Thus, while the need for consensus surrounding the definition of ‘SLI’ is accepted, this should go beyond identifying the cut-off at which it appears to be associated with the risk of poor outcome, to defining core features. There is clearly no one SLI. However, if it is assumed that multiple risk factors accumulate toward a threshold for diagnosis, there is every reason to continue to search for core cognitive and linguistic profiles. As Bishop (
Finally, Reilly
So should we abandon the term SLI? On balance, we should not. As Bishop’s companion paper states, SLI is the term most often used by researchers and surely research should guide practice? More generally, oral language is a critical foundation for learning and to ensure engagement with classroom instruction. Children with language impairments are at high risk of educational failure and hence poor career prospects; the cost to the nation is significant not least in terms of unemployment and adult well-being. Globally, professionals need to speak to policymakers with a single voice to make a case for the identification of children whose language skills are not at the level required for them to engage with education regardless of terminology. The analogy with dyslexia is useful: researchers understand that this disorder primarily affects decoding and is associated with phonological deficits, but lay understanding is different. Arguably, what matters is that governments are aware of ‘dyslexia’ and that legislation demands arrangements are made for affected individuals.
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This commentary reflects on some issues which arise from the short- and long-term recommendations made by Reilly
There is no doubt that the diagnosis of specific language impairment has raised lots of challenges for the practising speech and language therapists (SLT) in the UK and beyond for many years and for families in accessing speech and language therapy services (
I would suggest, however that the removal of the term ‘specific’ does not solve the challenges for the clinician in the identification of this group of children and in the subsequent decision-making process in relation to the type and level of intervention these cases require. The effect is a broadening of the diagnostic category thereby adding more clinical confusion. Although causation is not easily identified for this group, the clinical reality is that the profile of the child who is currently diagnosed with SLI does present differently to the child where the aetiology
Reilly
In terms of proposed new criteria, Reilly
There is consensus around the need to review the current criteria of SLI and that these need to be considered in the longer-term by setting out an action plan to systematically test out the boundaries of the diagnostic category. I also wholly support the idea that this can only be achieved successfully through global collaboration. In terms of our diagnostic label, however, what of the argument for the
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Reilly and colleagues have made a compelling case for the field to move away from the diagnostic category of specific language impairment. An important step is for researchers to acknowledge the heterogeneity of language impairment rather than rail against it. I argue that a failure of the research community to embrace a new paradigm will reflect a significant failure of leadership.
Reilly
The paper was an invigorating read in that it articulates the anxieties felt by many over the ‘SLI’ label, clearly outlines the evidence for why it must be abandoned, and provides recommendations for short- and long-term action. I wholeheartedly agree that an immediate switch to the term ‘language impairment’ (LI) and the adoption of well-defined inclusionary rather than exclusionary criteria, are important first steps in addressing the current state of discord. The longer-term goal of shifting health and educational services from a diagnostic-based funding paradigm to a model based on the level of functional impairment is clearly optimal for a broad phenotype such as LI.
Reilly
The case of autism spectrum disorder (ASD) is illustrative here. From the earliest description of autism in 1943 to the present day, there has been a widely held view that the behaviours associated with the disorder occur more often together than would be expected by chance, and therefore there will be a single cause that explains the non-random co-occurrence of these symptoms. However, like SLI, the behavioural and biological evidence never seemed to accord with this fervently held view. The behavioural heterogeneity in ASD spans the entire range of verbal and nonverbal abilities, with considerable variability also present in the level of repetitive mannerisms, motor impairments and social behaviours. Similarly, while twin studies identified that ASD is a highly heritable condition, no single causal pathway has been identified that accounts for more than 1% of affected individuals.
Yet, despite the overwhelming evidence for the behavioural and aetiological heterogeneity of ASD, for many years the research literature continued to be dominated by ‘between groups’ methodologies, in which people with ASD were recruited and then compared as a group against control participants on a given predictor or outcome variable. The focus was on identifying points of difference/similarity
The field of LI can learn from the ASD experience. While ‘SLI’ provides a neat set of criteria that researchers can use to group children, it is a diagnostic label that hides considerable behavioural and aetiological heterogeneity. Reilly
Expanding the diagnostic criteria from SLI to LI—in particular, relaxing the exclusions on nonverbal impairment and social disadvantage—would likely increase the phenotypic heterogeneity among participant samples even further. Researchers need to embrace this variability rather than rail against it. This may mean shifting the research emphasis, at least in the short-term, from ‘between groups’ designs (LI versus typically developing controls) to methodologies that seek to understand the quantitative and qualitative characteristics of individuals across the LI spectrum. Of course, such a paradigm shift within the research field will not be without resistance, particularly given the current scientific climate that craves straightforward findings that can be distilled easily to the general public. Exploring heterogeneity within the LI diagnosis will yield considerably more complex than simple findings, and researchers, journal editors and grant reviewers must be brave enough to accept this.
The duty of a scientist is to observe evidence and make further predictions based on these data. Reilly and colleagues have provided compelling evidence that SLI is neither a clinically or biologically valid diagnosis, and it is a scientist’s responsibility to heed these data. A failure of the research community to embrace a new paradigm will reflect a significant failure of leadership and present a major impediment to achieving the positives outcomes that we all desire for people with LI.
Andrew Whitehouse is supported by a Career Development Fellowship from the National Health and Medical Research Council (1004065); e-mail:
The identification of children’s language difficulties has implications for the nature and amount of additional support provided in schools. Terminology should allow for communication between professionals across health and education and with researchers. Classification should reflect the nature, degree and persistence of the language impairment and allow for changing developmental needs.
Reilly
In this commentary we focus on what might be considered an educational perspective, a perspective notably absent from both Reilly
Many of the points raised by Reilly
Flexible systems reflect the reality of children’s development but raise challenges for commissioning of services and supporting learning in schools. To do this accurately it is important to consider identification, intervention, and the impact of language learning needs. While many students with language learning difficulties experience a range of continuing difficulties many do not. Importantly and missing from both commentaries is the role of moderating and mediating variables in influencing achievement and well-being.
So how do we identify language-learning needs? Interestingly the reviews present different approaches. Reilly
From an educational perspective a clear advantage of accurate identification is that appropriate support and curriculum differentiation can be put in place. The identification of language learning difficulties provides a framework for differentiation. In addition these difficulties need also to be identified within the context of effective pedagogy (
Identification of language impairment is influenced by within-child factors including gender and age as well as absolute and relative levels of impairment; contextual factors including social disadvantage, teachers’ knowledge and skills, and local policies (
Identifying language-learning difficulties for teaching and learning is crucial. Establishing inclusionary criteria will support policy and practice. Teachers offer a unique perspective on the struggles the children have in accessing the curriculum (
Recognition that the ways in which problems with language learning impact on children and young people will vary across development is also important (
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Repeatedly debated and discussed, a powerful label is needed to reflect the needs and impact of language difficulties—not least for children and their families. Language impairment, while a relevant alternative to SLI, may present issues in determining exactly which children would be included. A dynamic approach to assessment, supported by a skilled workforce, would help the decision-making process.
In a 21st-century world saturated with communication, it is a curious paradox that we tie ourselves up in knots when we try to explain children’s communication difficulties. We need a clear and compelling descriptor that children and young people, their families, practitioners and the wider world can understand and use.
Children and young people we work with rarely, if ever, describe themselves as having SLI, SLCN or any of the labels we use to describe language difficulties. Families have similar issues; in the words of a frustrated parent: ‘they say he has a speech/language “need” … it’s more than just a “need” it’s a real difficulty and disability for him’.
As the leading children’s communication charity in the UK, I CAN’s mission is to raise awareness of the scale and impact of language difficulties. Our commentary reflects this and we welcome the chance to be part of the discussion around ‘the spaghetti junction’ of terminology.
Our view is that the spaghetti is not helping.
Language difficulties in children are the most frequently reported special educational need (SEN) in primary aged children in England (Department for Education 2013); more than autism and dyslexia. Yet there are fewer research studies, less public awareness, and these children often receive less support despite similar impacts on educational and social outcomes (Dockrell
Despite being generic, speech, language and communication needs (SLCN) as a label has served us well over the last decade. In England it has taken us through the Bercow Review of Services for Children and Young People with Speech, Language and Communication Needs (
SLI as a label has
We know that with the right support children and young people with language difficulties make good progress (
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We feel there will still be confusion about the group of children we identify with language impairment. Along with others, we have favoured a Russian doll approach which sees language difficulties as subgroups within groups allowing us a sense of size. If ‘language impairment’ becomes the term describing all children with language difficulties, we will need qualifiers to distinguish between ‘language impairment as part of another condition’ and ‘language impairment as the primary area of need’. We note the term ‘primary language impairment’ emerging in the literature (e.g.,
Assessing over two time points may allow for the developmental nature of language difficulties, but we challenge how practical this is even in the early years. A dynamic approach to assessment, which looks at
At the risk of adding more questions than answers, we remain optimistic about finding the Holy Grail—a single term that will both give us a powerful label and ensure that all stakeholders have an understanding of the changing nature and the impact of childhood language difficulties. We agree wholeheartedly with the need for further refinement of definitions and criteria, that these should be based within frameworks that acknowledge the changing nature of children’s needs, but also based firmly on what works for children and their families.
Crucially, however, given how difficult it can be to identify often ‘hidden’ language difficulties, and the fact that currently the first point of contact for children will not be a specialist in language difficulties, we cannot risk over-complicating the system. The success of any system, therefore, relies on a well trained early years and school workforce coupled with the clinical expertise of speech and language therapists to tease out differences, identify key features, judge responsiveness and plan appropriate intervention.
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Reilly
The target articles by Reilly
Another problem with relying on standardized tests for diagnosis or determining service eligibility is that most scores are normally distributed, making decisions about where to draw the line between typical and impaired language fairly arbitrary. Both target articles highlight the importance of ‘functional’ impairment in interpreting standardized test scores.
The criterion advocated by Reilly
One might argue that this is not a problem—supporting children at possible risk of language impairment doesn’t hurt anyone, right? I would argue that when resources are scarce, it is crucial to target provision on those who are unlikely to resolve spontaneously. One mechanism for doing so is to combine standardized assessment with parent/teacher report of language in everyday settings (
An important avenue for future research and professional debate is how and when speech–language therapy services should intervene. Reilly
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Reilly
We welcome this discussion and in particular Reilly
Theoretically it is hard to argue against much of what Reilly
From a clinician’s perspective it is obvious that the verbal/non-verbal discrepancy criteria for SLI has only been accepted within our profession and by no one else. Teachers and parents in particular are more interested in functional outcomes.
The emphasis on the development of inclusion criteria which is based more on the child or young person’s needs is also a positive step, and fits much more with government and societal expectations.
We support the need for a clearly defined diagnosis. Whilst labelling does have potentially negative connotations, for young people with long-term conditions, understanding their condition is an important step on the way to self management. Bishop (
Reilly
Using non-verbal IQ as a criteria and the differential diagnosis of delay versus disorder have been central components of many services’ prioritization systems. Removing these sends shockwaves through the profession and there is a risk that without robust diagnostic systems in place there will be significant confusion amongst practitioners. Who will we prioritize? What will be the point of differential diagnosis? Careful but urgent action is required if professionals are not to lose direction and consequent credibility with families.
Reilly
Other conditions such as ASD/ADHD have formal routes to diagnosis as outlined by the National Institute for Health and Care Excellence (NICE). This gives them a prominence and clarity. Whilst for LI we do not have that, we do need greater guidance. A more robust diagnostic process agreed by consensus will increase quality for children and families as well as benefitting researchers. Whilst Reilly
It is apparent that the term ‘specific language impairment’ needs to change. Even if the criteria were altered the ‘specific’ element naturally skews the casual listener to the current definition. There are however problems with many of the alternatives. ‘Language impairment’ is deficient as the term also relates to adult onset conditions and as Bishop makes the point search terms are important and a three word term is stronger. ‘Primary language impairment’ emphasizes language as the main area of need, but in the UK may be confused with primary schooling as well as sharing an acronym with pragmatic language impairment. If primary language impairment was adopted in diagnostic terms there could be PLI and ‘secondary language impairment’ or alternately PLI versus ‘language impairment associated with other conditions’. This would help as a first step towards subcategorization of language impairment.
At this watershed time it is potentially an opportune time to generate a new term. Autism was first used in 1943. A new term would take some time to be understood but once established would be easily searchable and not carry any misleading connotations.
Although taking place in an academic journal the discussion of terminology is in effect a rebranding exercise. Thus it may be appropriate to apply marketing-style approaches to the development of a new term, including consultation with parents and young people.
Whatever the outcome the LI field needs broad consensus agreement across all groups. We suggest that the short- and long-term aims be developed into a robust international plan. It needs a planned approach to ensure that all in the field are aware of the direction of travel. Whilst well argued, the changes proposed are significant. Without a coordinated approach towards a clear term, definition and diagnostic pathway there is a risk that the language impairment field may suffer an extended period of confusion which will be of detriment to all, including the children, young people and families we aim to support.
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Understanding the many poorly studied families of
The book is no 8 in the CBS Biodiversity Series and is well laid out and well illustrated. A great deal of effort has gone into researching and compiling this book and the authors are warmly congratulated.
One important aspect is whether the family is clearly outlined and the authors start with a historical review. Certainly in this aspect the family is well documented and the difficulty in understanding the family is clearly stated. A precise diagnosis for the family is also provided although several other genera not mentioned in the book might well fit into this description. The family is also compared with similar families, i.e.,
Because almost all attempts to isolate taxa of
The main context of this book discusses each genus and this is most thorough and will be very useful for mycologists. The following inclusions are provided for each genus; history, differences between species, ecology, comparison with other genera, diagnostic features, detailed description, notes, and illustrations. In all the book deals with 34 accepted genera, two excluded genera and several doubtful genera. Descriptions of genera are very thorough and are based on the type species and all are very well illustrated (it would be nice if they had been in colour).
There are some nice Tables in the book, including Table 1.5 which lists host families on which the
This is an excellent book and starts to deal with the immense complexity of the
The book should be available in all universities and colleges where mycology is taught and where work on mycology is carried out, especially where there is a need to work with and identify plant pathogens.
In Persoonia 21, Reflections unfortunately some typing errors ocurred in the following titles, for which we apologise. The correct entries are given below:
Douanla-Meli C. 2007.
Frisch A, Lange U, Staiger B. 2007.
Kärnefelt I, Thell A. 2007.
Previous proposals to change the name of the
To address these issues, new proposals were recently made by D.L. Hawksworth and colleagues and published in both Taxon and Mycotaxon (
A whole day is planned to be set aside for the Nomenclature Session in Edinburgh, and all full registrants at the Congress will be able to vote on all formal proposals made to that date, copies of which it is envisaged will be made available at the Registration Desk. Unlike International Botanical Congresses, no system of weighted ‘institutional votes’ will be allowed. Hawksworth (in litt.) suggests that the IMA Executive Committee to nominate a Chair, Vice-Chair, and Rapporteur for the Session for ratification or change by those present at the start of the Session.
In any event, it has been agreed that Mycotaxon will now publish all proposals relating to the nomenclature which are included in Taxon as simultaneously as possible, starting with the April–June 2009 volume.
It has been shown that pantethine, the disulphide of pantetheine, rescues a Drosophila model of pantothenate kinase–associated neurodegeneration (PKAN). However, the pharmacology of pantethine is known to be incompatible with its use as a possible replacement therapeutic for this neurodegenerative disease: the compound is known to be unstable in serum and is unlikely to permeate the blood–brain barrier. In an attempt to overcome these pharmacological difficulties, we successfully synthesized 4–thiobutyltriphenylphosphonium pantetheine (TBTP–pantetheine), a pantetheine derivative, and compared its serum stability and pharmacological properties to those of pantethine.
It is shown that TBTP–pantetheine is stable under aqueous conditions; however, the compound is enzymatically degraded in fetal calf serum at a higher rate than its parent compound pantethine. In a PAMPA system (Parallel Artificial Membrane Permeability Assay, an in vitro setup to determine passive membrane transport), TBTP–pantetheine shows increased lipophilicity by a significantly higher tendency to associate with the membrane; however, permeability coefficients are not significantly different. We conclude that TBTP–pantetheine is unable to overcome the pharmacological hurdles emerging from a pantethine–based therapy for PKAN. Nevertheless, this research shows that pharmacological properties can be altered by derivatization of the parent molecule pantethine thus strengthening the rationale to synthesize and test novel derivatives of pantetheine and related molecules within TIRCON, in order to facilitate a possible replacement strategy in the future.
Background: Pantothenate kinase associated neurodegeneration (PKAN) is the most common defect causing NBIA in children. Despite advances in the research of novel therapies for PKAN, validated international clinical rating scales to be used in clinical trials are lacking, and significant gaps concerning phenotype–genotype correlations and disease progression persist.
Main Objective: We aimed to identify and genetically characterize the Spanish population with NBIA and to design and validate a quantitative method for clinical assessment of PKAN patients.
Methodology: We performed a cross–sectional multi–centre study, with NBIA patient recruitment through professional associations. Design of a Disease Rating Scale for PKAN (PKAN–DRS) including four sub–scales: cognitive, behavioral, physical, and functional assessment. Items were scored from 0 (normal) to 3 or 4 (maximum severity), (total scores 0 (no disease) to 140 (maximum severity)). For validity and reliability assessment, four independent examiners rated PKAN patients using recorded videotapes. Sanger sequencing of NBIA genes was performed in undiagnosed NBIA families.
Results: To date, 37 NBIA patients (mean age 20 years, range 3–52; 13 males) have been identified at 15 centres in Spain: PKAN (N=22); PLAN (N=8); undiagnosed NBIA (N=7). The PKAN–DRS was applied to 10 PKAN patients (mean age 29, 5 years, range 14–53), total scores ranging from 35 to 67. Physical scores positively correlated with functional scores, and age at disease onset inversely correlated with total scores. The c.1583C>T mutation in the PANK2 gene was found in homozygosis in 4 gypsy patients from two consanguineous families from Northern Spain, suggesting a common origin for this nucleotide change.
Perturbation of iron homeostasis and accumulation of iron in specific regions of the brain play an important role in neurodegenerative processes in all forms of NBIA, Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS) and others. Iron deposition, whether a primary or secondary pathological event, may underlie or contribute to progression of neurodegeneration by promoting generation of reactive oxygen species (ROS), leading to oxidative cell damage and brain inflammation.
ApoPharma's marketed iron chelator, deferiprone, is a brain–permeable 3,4–hydroxypyridinone (HP) with favourable activity in multiple animal models of neurodegenerative disease. Deferiprone has recently been shown to be effective in clinical trials in PD, and is being studied in pantothenate kinase–associated neurodegeneration (PKAN) and ALS patients. We have now developed a series of new HP iron chelators with improved properties. Model HPs penetrate the blood–brain barrier, cells and subcellular organelles, and selectively chelate Fe(III) with an affinity that facilitates transfer of iron to endogenous acceptors for reuse, avoiding significant systemic iron depletion.
Using cellular and animal models of neurological cell protection, PD and AD, and an ataxic ceruloplasmin–hephestin double knockout (DKO) mouse, we have demonstrated the efficacy of selected HPs in alleviating brain iron deposition, neuroinflammation, and ROS–related cellular damage. Significant effects on disease–related endpoints included improvement of cognitive function in mouse AD and motor function in rat PD models, and prevention of ataxia in the DKO mouse. These findings may be generalized to other neurodegenerative disorders in which brain iron accumulation leads or contributes to disease pathology.
Pantothenate kinase 2 catalyzes the key, rate–limiting step in coenzyme A biosynthesis, therefore mutations in the gene encoding this enzyme have the potential to disrupt a number of metabolic processes by disrupting cellular CoA pools. Elevations in lactate suggest that mutations in this mitochondrial enzyme could cause general mitochondrial dysfunction.
Particularly relevant for neurodegenerative processes is the relationship between mitochondria and iron. The mitochondrion upholds the synthesis of iron–sulfur clusters and heme, therefore a fraction of incoming iron must go through this organelle before reaching its final destination. In turn, the mitochondrial respiratory chain is the source of reactive oxygen species (ROS) derived from leaks in the electron transport chain. The co–existence of both iron and ROS in the secluded space of the mitochondrion makes this organelle particularly prone to hydroxyl radical–mediated damage. Additionally, mitochondrial dysfunction and ROS production promotes the selective degradation of damaged mitochondria by autophagy which must be associated to increased mitochondrial biogenesis for compensating the loss of mitochondrial mass.
Our project aims to understand the molecular mechanism and modulation of mitophagy and mitochondrial biogenesis in cellular models of NBIA.
Characterization of iron metabolism alterations, mitochondrial dysfunction and the molecular mechanisms involved in mitophagy activation in fibroblasts harbouring
Molecular analysis of pathways involved in mitochondrial biogenesis as a compensatory mechanism in response to mitophagy activation in fibroblasts harbouring
Screening of modulators of autophagy and mitochondrial biogenesis from commercial libraries capable of restoring the physiopathological alterations in
PKAN is the most common form of Neurodegeneration with Brain Iron Accumulation (NBIA); it is associated with mutations in the
Neurodegeneration with brain iron accumulation (NBIA) constitutes a group of neurodegenerative diseases characterized by a prominent extrapyramidal movement disorder, intellectual deterioration and characteristic iron deposition in the basal ganglia. Ten genes have been identified so far, but limited information exists regarding expression and function of these genes within the human brain.
To address possible relationships between known NBIA genes, predict their functions, and identify overlapping pathways, we used a systems–biology approach based on whole transcriptome gene expression analysis. As part of the UK Human Brain Expression Consortium (UKBEC), we analysed the expression profiles of 101 neuropathologically normal individuals (10 brain regions each). Weighted gene co–expression network analysis (WGCNA) was used to cluster genes into co–expression modules. The overrepresentation of NBIA transcripts in basal ganglia modules (substantia nigra and putamen studied) was assessed.
Six modules containing NBIA genes were found for the substantia nigra, but there was no evidence of significant overrepresentation. Two putamen modules were significantly enriched for NBIA transcripts, namely the brown (PANK2, ATP13A2, C19orf12, COASY; P= 0.003) and green (FTL, DCAF17, FA2H; P= 0.021) modules. Enrichment analysis of these two putamen modules revealed an overrepresentation of gene ontology terms and KEGG pathways, including: brown – synaptic vesicle endocytosis and axon cargo transport (biological processes), synaptic membrane (cellular component), and synaptic vesicle cycle KEGG pathway; green – ensheathment of neurons, neuronal action potential and oligodendrocyte development (biological processes), myelin sheath (cellular component), and cadherin binding (molecular function). Our data suggests shared processes and pathways in NBIA gene networks.
The NBIA Alliance is the informal international umbrella organization for NBIA lay advocacy groups. Launched in 2012 under the patronage of the European Union Seventh Framework Programme (FP7 EU) project TIRCON (Treat Iron–Related Childhood–Onset Neurodegeneration), the NBIA Alliance’s main objectives are to increase awareness of NBIA and to support cooperative research into NBIA Disorders. As an interface and contact point for all people in public and health policy with a special interest in NBIA, the NBIA Alliance and its members are bringing together patients, clinicians, researchers, and further stakeholders. The NBIA Alliance comprises 6 legal entities in France, Germany, Italy, the Netherlands, Spain, and the United States, and one patient group still under development in the United Kingdom. New NBIA advocacy groups are just emerging in Argentina and Canada.
The long–standing groups, Hoffnungsbaum e.V. in Germany and the NBIA Disorders Association in the U.S., are partners in TIRCON and have been fostering the establishment of further national NBIA lay advocacy groups and of the international NBIA Alliance. Herein these advocacy groups that are lesser–known to the international NBIA community introduce themselves and describe their impact on NBIA research.
AISNAF (Associazione Italiana Sindromi Neurodegenerative da Accumulo di Ferro) was established in 2006 as the third NBIA patient organization. It is a contact point for NBIA families in Italy, has raised awareness on NBIA and co–funded scientific symposia as well as NBIA research projects. It aims to help harmonize and spread clinical best practices in Italy.
The French AIDNAI (Association Internationale de Dystrophie Neuro Axonale Infantile), launched in 2000 as a patient organization for NBIA subtype PLAN, supported research in PLAN, and extended its scope to all NBIA Disorders when becoming member of the NBIA Alliance in 2012.
Inspired by an NBIA Alliance meeting at the 2nd joint NA/NBIA symposium in Ede (the Netherlands) in October 2012, the Dutch Stichting IJzersterk was established in 2013. Using social media as an additional platform resulted in contact with 10 patient families within a year and fostered successful fundraisers for NBIA research.
The establishment of the Spanish ENACH Asociación (Asociación de Enfermedades Neurodegenerativas por Acumulación Cerebral de Hierro) in 2013, following the set–up of the NBIA Alliance, is focused on gaining visibility and extensive networking among NBIA families, researchers and physicians. A Scientific and Medical Advisory Board is already in place. Identifying a large patient cohort in Spain, the involvement of 15 hospitals, setting up a research infrastructure and 4 ongoing research projects have been achieved within less than 2 years.
Additionally, NBIA UK, NBIA Canada and an Argentinian patient representative have taken their first steps to setup organizations or to join the NBIA community.
Neurodegeneration with Brain Iron Accumulation (NBIA) is a group of ultra–rare neurological movement disorders characterized by high iron levels in the basal ganglia and a progressive degeneration of the nervous system. Currently, there is no cure or proven therapy for any of the NBIA forms.
Due to the lack of treatment options, the international European Union Seventh Framework Programme (FP7 EU) consortium TIRCON (Treat–Iron–Related–Childhood–Onset–Neurodegeneration) is dedicated to the investigation of NBIA. 13 partners from eight countries are conducting a clinical therapy study and set up a patient registry/ biobank in order to improve the infrastructure for NBIA research and care.
The lay advocacy groups, NBIA Disorders Association (NBIADA, USA) and Hoffnungsbaum e.V. (HoBa, Germany), are full partners in TIRCON. Based upon their experience in such collaborative projects, NBIADA and HoBa have been promoting the creation of further patient groups worldwide. The NBIA Alliance, launched during the TIRCON kickoff meeting in 2012, is the first informal international umbrella association for NBIA lay advocacy groups. Currently, seven NBIA patient organizations are involved in the Alliance. Its main objectives are to increase awareness for NBIA among the health care community by translating scientific knowledge into patient–friendly information and to support cooperative research. The NBIA Alliance is an information platform and a contact point for patients and clinicians where dedicated structures are not yet in place. The poster presents the launch, development, goals, activities, constraints and future options of the NBIA Alliance. It will underline the mutual empowerment of the NBIA patients' movement and the NBIA research and care community in TIRCON.
Neurodegeneration with brain iron accumulation (NBIA) comprises a heterogeneous group of disorders with radiologically discernible brain iron. Phospholipase–associated neurodegeneration (PLAN) is the second most common subtype, accounting overall for 20% patients with NBIA. Three clinical subtypes are recognised, including classical infantile neuroaxonal dystrophy (INAD), atypical neuroaxonal dystrophy (NAD) and
It is well recognized that PLAN is a devastating condition with an aggressive and relentless disease course. Children with INAD have progressive cognitive and motor difficulties, culminating in significant morbidity and reduced life expectancy. Currently, there are no disease–modifying agents to halt the progression of this disorder, let alone a cure, and therefore all available treatments for PLAN currently focus on symptom palliation to maximize quality of life.
At Great Ormond Street Hospital, we provide a tertiary service for many children with PLAN. I will discuss our current rationale for treatment strategies in PLAN, namely the management of spasticity, dystonia and secondary complications. I will also present data regarding the first reported use of deep brain stimulation in atypical NAD.
The paucity of disease–ameliorating treatments in PLAN has driven us to seek novel therapeutic strategies, and to that aim, we are currently undertaking a proof–of–concept gene therapy study in the INAD mouse model. This murine model recapitulates many of the clinical and histopathological features of the human phenotype, thereby rendering the model ideal for our study project. We will perform systemic intravenous delivery of an AAV9 viral vector to deliver therapeutic
Background: Neurodegeneration with brain iron accumulation (NBIA) comprises a clinically and genetically heterogeneous group of progressive disorders characterised by high brain iron. Different genetic forms of NBIA encountered in childhood and early adolescence have been reported including pantothenate kinase–associated neurodegeneration (PKAN),
Methods: We undertook (i) clinical assessment and/or detailed analysis of the clinical notes and (ii) mutational analysis for relevant candidate genes.
Results: We studied 37 childhood–onset cases (5 familial and 26 sporadic) and confirmed a genetic diagnosis of NBIA in 19 (51.4%) patients (3 PKAN, 6 PLAN, 1 MPAN, 6 BPAN, 3 patients with a novel unpublished gene defect). Beside the classical progressive motor symptoms and cognitive regression, many patients had additional extrapyramidal features, ocular findings and neuropsychiatric features. We also identified unusual/rare clinical features in classical disease (e.g. absent eye–of–the–tiger sign in PKAN) and a number of atypical disease presentations (e.g. atypical PLAN, early recognition of BPAN in infancy).
Conclusion: This review highlights that NBIA is both clinically and genetically heterogeneous, and for many cases in the UK the genetic aetiology is yet to be discovered. Further gene identification will significantly improve the understanding of disease mechanisms as well as identifying novel therapeutic targets for these neurodegenerative disorders.
We have studied brain iron loading and related changes in a mouse model with mutations in the iron–regulatory genes Hfe and transferrin receptor 2 (Tfr2) and without known mutations in NBIA–related genes.
Brain iron and ferritin levels were increased (>1.7–fold, p<0.025, n≥5 mice/group) and iron distribution altered in male Hfe-/-xTfr2mut AKR mice at 12 weeks of age after 3 weeks on a high iron diet (2% carbonyl iron) compared to age– and gender–matched controls. Methods used included inductively coupled–atomic emission spectroscopy, non–heme iron assay, ferritin immunoblotting, micro X–ray fluorescence mapping of metal distribution and micro X–ray absorption near edge spectroscopy analysis of iron species.
Brain RNA arrays revealed transcript decreases (p<0.05) for five NBIA–related genes: phospholipase A2, group VI (Pla2g6, 1.7–fold), fatty acid 2–hydroxylase (Fa2h, 1.4–fold), chromosome 19 open reading frame 12 (C19orf12, 1.3–fold), ATPase13A2 (1.2–fold) and ceruloplasmin (Cp, 1.2–fold). Four other NBIA–related transcripts for pantothenate kinase 2 (PANK2) and CoA synthase (COASY), both involved in CoA metabolism, WD–repeat domain 45 (implicated in autophagy) and the nuclear transmembrane protein DCAF17 were unchanged. Aside from the ferroxidase Cp, transcripts showing changes are distinguished by having myelin–related roles. At least 6 other myelin–related transcripts also decreased (p<0.05; ≥1.2–fold).
These findings provide evidence that excess iron loading can cause decreases in the expression of a subset of the genes implicated in NBIA, notably those genes which may also have roles pertaining to myelin. Iron excess may therefore cause changes which resemble, although to a lesser degree, the effects of pathogenic, loss–of–function, NBIA–causing mutations in these genes.
Neurodegeneration with brain iron accumulation (NBIA) includes a clinically and genetically heterogeneous group of disorders with progressive extrapyramidal signs and neurological deterioration, characterized by iron accumulation in the basal ganglia. In our biobank, which is part of the Telethon Biobank Network, a considerable number of clinically associated NBIA cases without a genetic diagnosis are stored. In order to characterize this group we are studying our cohort of patients by the TruSeq Custom Amplicon panel (MiSeq Illumina platform) formed by 16 NBIA–associated genes. We screened, using Sanger sequencing, one of three affected sisters for the most commonly NBIA–associated genes, identifying a heterozygous mutation in
Reviewing the sisters’ clinical history we observed differences between one sister and the other two with respect to age of onset and type of symptoms, differences that segregated appropriately with the different genetic findings in
The sister carrying only the p. Gly521Arg is likely to be affected by a different disease and show a different phenotype due to the combined effect of the mutation and unknown susceptibility factors. The sequencing of the NBIA panel for this sister could be a powerful approach to complete the molecular diagnosis.
Abnormal accumulation of brain iron has been detected in various neurodegenerative diseases, but the contribution of iron overload to pathology remains unclear. In a group of distinctive brain iron overload diseases known as Neurodegeneration with Brain Iron Accumulation (NBIA), nine disease genes have been identified. In the NBIAs, brain iron accumulation was observed with MRI and at autopsy in the globus pallidus and other brain regions in patients and was associated with severe dystonia and gait abnormalites. Only two of these diseases, aceruloplasminemia and neuroferritinopathy, are directly caused by abnormalities in iron metabolism, which affect mainly astrocytes in the former and neurons in the latter. Understanding the early molecular pathophysiology of these diseases should provide insights into the role of iron and to the design of specific therapeutic approaches.
Pantothenate kinase–associated neurodegeneration (PKAN) is a rare inherited disease characterized by progressive movement impairment and iron accumulation in specific areas of the brain. Currently there are no treatment options for this devastating autosomal recessive disorder. Affected individuals carry mutations in pantothenate kinase, the first enzyme required to synthesize Coenzyme A, suggesting that impaired metabolism of Coenzyme A is one of the causes of the disease. This is further underscored by the recent finding that patients with mutations in Coenzyme A synthase, the last enzyme of the Coenzyme A biosynthesis pathway suffer from comparable symptoms as PKAN patients. Coenzyme A is an essential metabolic cofactor required for over 100 metabolic reactions. Why mutations in genes coding for Coenzyme A biosynthesis enzymes induce neurodegeneration is currently not clear. We and others have investigated
Frontotemporal lobar degeneration (FTLD) represents a group of neurodegenerative dementias mainly affecting people younger than 65 years of age. Up to 50% of patients have a family history of dementia and in most cases familial segregation is compatible with autosomal dominant inheritance. Although mutations in seven genes have been identified, the genetic cause remains unknown in about 60% of familial FTLD. We aimed to find novel FTLD genes in a whole genome sequencing approach in 13 unrelated familial patients and three relatives affected with FTLD, selected from a Belgian cohort of 590 FTLD patients. The genome sequences were annotated using GenomeComb and variants were filtered and prioritized using multiple genetic and functional criteria. Analysis of selected variants in all FTLD patients and 1314 matched controls revealed the presence of two novel coding missense variants (p.W395C and p.A444P) in the vacuolar protein sorting 13 homolog C gene (
From their start in 2002, and until very recently, the international neuroacanthocytosis (NA) symposia reported little tangible progress with respect to understanding the specific disease mechanisms that we anticipate will eventually form the basis of causal treatment. Our association with colleagues from the neurodegeneration with brain iron accumulation (NBIA) field was not only a strategic alliance within the rare disease field but also a stimulus to speed the pace of discovery, 20 and 13 years, respectively, after the discovery of the genes for McLeod syndrome (MLS:
The small, close NA community, organized around the Advocacy for Neuroacanthocytosis Patients (www.naadvocacy.org), had been successful in teaching our clinical colleagues about the NA syndromes, offering the free Western blot testing service to facilitate diagnosis of ChAc (instructions: www.euro–hd.net/html/na/diseases/chac), and providing information to fellow scientists. However, only with the European Multidisciplinary Initiative on Neuroacanthocytosis (EMINA) which terminated in early 2014, and its currently active successor, EMINA–2, have new basic science discoveries come at a quicker pace. The most recent development is the very welcome interest by the European collaboration program in science and technology (COST) and its “proteostasis action” (proteostasis–2014.cipf.es).
The current NA symposium presented a number of potential ChAc models, in yeast, slime mould, drosophila, and human induced pluripotent stem cells. New insights were presented into the roles of
There has been great progress in the NBIA syndromes, especially in pantothenate kinase–associated neurodegeneration (PKAN), with the identification of new syndromes caused by novel genes, with significant progress in disease models, and with treatment being offered in the TIRCON project. Given the new genetic data and the well–organized patient cohorts with NBIA syndromes, the relationship of PKAN and the other syndromes to acanthocytosis may be further elucidated once blood smears and acanthocyte counts are systematically performed in these subjects. These findings will also inform us about the future utility of NA and NBIA cooperation.
Cohen syndrome is an autosomal recessive disorder caused by mutations in the gene
The third joint symposium was held in Stresa, Italy, October 30 to November 1, 2014.
Erythrocyte structure and function depend upon interactions between proteins in and associated with the membrane, centered around band 3. This protein harbours physiological removal signals and, as a main component of the links between the cytoskeleton and the lipid bilayer, plays a central role in deformability. Also, as a binding partner of hemoglobin and key glycolysis enzymes, band 3 is the link between the oxygen–dependent trade–off between ATP production and NADPH–mediated protection against oxidative damage. Phosphorylation of band 3 by Lyn and Syk kinases is involved in the regulation of these processes.
The presence of acanthocytes in the blood of patients with chorea–acanthocytosis (ChAc) and other NA syndromes provides the rationale for attempts to identify the molecular causes and consequences of acanthocytosis. The results of this approach indicate that a disturbance of the interaction between band 3 and other membrane–cytoskeleton proteins is at the center of the causes of the acanthocyte shape. The pathway leading to this disturbance includes abnormal membrane recruitment of and band 3 phosphorylation by Lyn kinase. In most patients, the presence of acanthocytes does not have clinical consequences, but various in vitro analyses show functional alterations. These alterations are not restricted to the acanthocytes in ChAc patients, but are also present in patients with other NA and NBIA syndromes. A morphological, functional and molecular comparison of the acanthocytes and non–acanthocytes of the same patient, and of erythrocytes of various NA and NBIA patients, provides additional possibilities to identify the neuropathological mechanisms of these disorders.
The yeast Vps13 protein (Vps13p) is the prototype of a family of molecules that includes human VPS13A, for which loss of function results in chorea–acanthocytosis. The VPS13 gene was first identified for its requirement in retrograde transport from the late endosome to the Golgi in yeast. Further genetic, cell biological and biochemical studies identified Vps13p as a 357 kDa cytosolic protein implicated in regulating forward transport from the trans Golgi network (TGN) to the late endosome and required for a late stage in sporulation (Brickner, J.H & Fuller, R.S. 1997. J. Cell Biol. 139:23–36). Using cell–free fusion and transport assays (Brickner et al. 2001. J. Cell Biol. 155:969–978; Blanchette, J.M., et al., 2004. J. Biol. Chem. 279:48767–73), we find that Vps13p is directly required for TGN homotypic fusion and TGN to late endosome vesicular transport. Extracts from cells with deletions or temperature–sensitive mutations in VPS13 are defective in these fusion and transport reactions but are complemented by Vps13p that has been purified to near homogeneity from yeast cells. Soluble, purified Vps13p is monomeric and is in complex with the small calmodulin–like protein, Cdc31p (yeast centrin). Cdc31p is required for both cell–free reactions. Under reaction conditions, purified Vps13p binds to yeast membranes in an ATP–stimulated fashion. The binding of Vps13p to phosphorylated phospholipids and a low resolution electron microscopic structure for the protein derived from negative stained (uranyl formate) samples will be discussed.
This work has been supported in part by the Protein Folding Disease Fast Forward Initiative, the Advocacy for Neuroacanthocytosis Patients, the Endowment for the Basic Sciences at the University of Michigan Medical School and NIH RO1 grant GM50915 (RSF).
Chorea–acanthocytosis (ChAc) is an autosomal recessive inherited disease caused by loss–of–function mutations in the VPS13A gene, which encodes for chorein protein. The yeast homolog of VPS13A functions in vesicle and mitochondrial trafficking. These processes are aberrantly regulated in Huntington disease (HD), which is phenotypically very similar to ChAc, and suggests that they play an important role in ChAc pathogenesis. Therefore, this study focuses on vesicle and mitochondria trafficking. Live imaging of neurons differentiated from induced pluripotent stem cells (iPSCs) revealed a decreased lysosome count in neurites and reduced motility of mitochondria. Mitochondria were abnormally shortened, reminiscent of vesicles. Characterization of patient mitochondria by respirometry showed diminished function. It is possible that the disease progression of HD and ChAc share a common underlying mechanism. We are currently investigating genes identified as hallmarks of trafficking impairment in HD using qPCR and cytoskeleton dynamics via immunocytochemistry and Western blot.
Introduction: The European Multidisciplinary Initiative on Neuroacanthocytosis (EMINA) has provided detailed clinical characterization of the different NA syndromes and collected valuable brain and muscle samples of chorea–acanthocytosis (ChAc) patients. ChAc is caused by loss–of–function mutations within the gene
Overall goal: The overall goal of the EMINA–2 consortium is to dissect the molecular pathophysiology of ChAc caused by mutations in the
Experimental approach: To use patient material, patient–specific induced pluripotent stem (iPS) cell models and animal models (VPS13A-/- mice, Drosophila models) of ChAc for studying the effects of VPS13A mutations on intracellular signalling pathways and cytoskeleton formation. The EMINA–2 consortium now combines the clinical excellence of EMINA including the characterized patient material (blood, brain and muscle samples) with basic cell and molecular biology as well as animal model research to characterize the molecular events leading to neurodegeneration in ChAc.
Significance: The results will contribute to an understanding of the molecular pathophysiology of ChAc as a model not only for NA syndromes but also for inclusion body–independent neurodegenerative diseases of the basal ganglia. This knowledge will help to generate novel causal treatment approaches in ChAc and eventually other neurodegenerative diseases of the basal ganglia.
Chorea–acanthocytosis (ChAc) is a progressive multisystem neurodegenerative disorder which manifests with several devastating symptoms. The complex nature of motor and psychological disorders on one hand, and the rarity of the disease on the other, makes physicians and researchers unable to reach a definite solution for patients' sufferings. Thus, most of our knowledge in management of this disease is based on small case series and personal experiences. Among the wide range of complications in our ChAc patients, oral problems have been very challenging to tackle. Dysarthria and dysphagia are two major problems that affect most patients. The first one disturbs social relationships, and the second one puts patients at high risk of aspiration pneumonia. In addition, dysphagia reduces food intake and the ensuing malnutrition leaves patients vulnerable to infectious and metabolic disorders. Bruxism, not uncommon in ChAc, causes early tooth flattening and decay. Lip– and tongue–biting, habitual or dystonic in nature, is not only painful and deteriorate pre–existing dysphagia, but may result in serious oral cavity infection. Finally, drooling is both embarrassing and negatively affects speech. Control of these complications with speech therapy, mouth guards, anti–cholinergic drugs, and botulinum toxin injection into salivary glands and tongue muscles are at best modestly effective. Overtreatment worsens speech and swallowing and can be dangerous. Frequent visits, gradual dose adjustments, and trials of different treatment methods are necessary factors to achieve acceptable treatment results. In this presentation, I review and share our experiences in controlling the above symptoms in Iranian patients.
Chorea–acanthocytosis (ChAc) is a hereditary neurodegenerative disorder, one of the neuroacanthocytosis syndromes (NA). One of the hallmarks of NA is the presence of circulating acanthocytes, generation of which is still under investigation. Recently, we reported increased Tyr–phosphorylation state of the red blood cell (RBC) membrane proteins from ChAc patients, related to abnormal activation of Lyn, an Src family kinase (SFKs) (Blood 118; 5652; 2011). In the context of international collaboration, we further characterized Lyn signaling pathway in RBC from ChAc patients. In ChAc RBCs, we found a weakness of ankyrin–based multiprotein complex bridging the membrane to the cytoskeleton, contributing to the generation of acanthocytes. We then evaluated the state of Lyn (active–inactive) in the cytoplasmic fraction from RBC of ChAc patients. In ChAc RBCs we found higher levels of phospho–Lyn–396, corresponding to active Lyn, compared to controls. We then evaluated whether classical Lyn inhibitors such as PP2 or Dasatinib, a pharmacological Lyn inhibitor, might block Lyn in ChAc RBCs. We found that both PP2 (0.1μM) or Dasatinib (0.1 μM) were able to efficiently inhibit Lyn in both ChAc and healthy RBCs. These data suggest that in ChAc (i) the abnormal activation of Lyn affects RBC membrane mechanical stability weakening both multiprotein complexes bridging the membrane to the cytoskeleton; (ii) Lyn activity is inhibited by either PP2 or Dasatinib, suggesting Lyn as possible new therapeutic target in ChAc.
This work was supported by NA advocacy and Telethon grant GP13005 and Erare–JTC 2012 ("EMINA2").
* These two authors contributed equally.
Chorea–acanthocythosis is caused by loss–of–function mutations in the VPS13A gene. Functional annotation of VPS13A as a protein involved in vesicle transport and lysosomal degradation largely relies on data collected in yeast, and the mammalian VPS13A protein remains largely uncharacterized. We chose an ecotropic lentiviral knock–down approach to study VPS13A function in human cell lines and primary cells. Knock–down efficacies of different shRNA constructs were analysed by Q–PCR and Western blotting, and a reduction below 20% of control VPS13A protein levels could be obtained. The most efficient constructs were used for knock–down of VPS13A in HeLa cells and iPS cells. In HeLa cells, a distinct change in cell morphology was observed in response to VPS13A shRNA treatment. Preliminary analysis points towards problems with cellular adhesion or migration, which are observed in response to the VPS13A knock–down. An in–depth analysis of the phenotype, which fits well with previous reports on cytoskeletal abnormalities in VPS13A–deficient cells, is currently on–going.
In yeast, VPS13 plays roles in at least three distinct processes. First, VPS13 is required for the trafficking of certain proteins to the vacuole, the yeast equivalent of the lysosome. Second, during the developmental process of sporulation, the Vps13 protein translocates to the prospore membrane where it is required for proper morphogenesis of this compartment. Third, though usually dispensable for growth, VPS13 becomes essential for cell viability in genetic backgrounds that lack endoplasmic reticulum/mitochondrial contact sites, suggesting a role for VPS13 in mitochondrial homeostasis.
These three phenotypes may reflect distinct functions of VPS13 or they may indicate a requirement for the same molecular function in different processes at different locations within the cell. In either case, these activities are separable genetically. For example, we have identified alleles of VPS13 that are defective only in the mitochondrial function as well as yeast strains in which the VPS13 mutant displays the mitochondrial and sporulation phenotypes, but no trafficking defect.
To better understand the molecular basis for the different roles of VPS13, we have begun a structure–function analysis of the protein to generate mutants defective in only one aspect of VPS13 function and to correlate these phenotypes with changes in protein–protein interactions as determined by co–immunoprecipitation and mass spectrometry. In addition, to seek insight into what function(s) of VPS13 is relevant to chorea–acanthocytosis, cognate mutations corresponding missense alleles found in ChAc patients have been introduced into the yeast gene. Examination of the effect of these mutations on different VPS13 functions will be described.
Chorea–acanthocytosis (ChAc) is a rare neurodegenerative disease characterized by progressive movement disorder, cognitive and behaviour changes, seizures and myopathy. The typical clinical features are limb chorea and oral dystonia. Here we report the clinical features of two brothers affected with ChAc. Their parents were healthy; however, non–specific movement disorders were reported in relatives. The 47 year–old man was affected by a mild mental retardation and a drug–resistant temporal lobe epilepsy diagnosed since he was 27 years–old. At age 45 years–old he started to develop choreic movements and dystonia affecting upper limbs and oral region. Dysarthria, dysphagia, asymmetric plastic rigidity and areflexia at upper and lower limbs were also present. Brain MRI showed mild enlargement of ventricular cavities associated with focal malacia of the right temporal lobe. A pacemaker was implanted after a cardiac arrest when he was 45 years–old. In contrast his 53 year–old sister started to complain progressive impaired gait with frequent falls and slurred speech in her 50s. In few years she developed dysphagia and progressive cognitive impairment. A neurological examination disclosed the presence of extrapyramidal signs characterised by bradykinesia and mask–like expressionless face, without involuntary movements. Brain MRI showed bilateral hyperintensities of striatum on T2–weighted images. Both patients presented acanthocytes in peripheral blood and Western blot analysis showed reduced chorein expression. This report highlights the wide intra–familial phenotypic variability of ChAc and suggests the consideration of ChAc in the differential diagnosis of juvenile parkinsonism and temporal epilepsy with mental retardation.
Chorea–acanthocytosis (ChAc) is an autosomal recessive neurodegenerative disorder due to mutation of the VPS13A (CHAC) gene on chromosome 9q21 encoding chorein. Clinical features include mixed movement disorders (chorea, dystonia with prominent orofacial involvement and self–mutilation, tics, parkinsonism, head thrusts), eye movement abnormalities suggestive of brain stem involvement, seizures, subcortical dementia and psychiatric features with impairment of frontal lobe function. Myopathy and neuropathy are often present. Neuroimaging strongly resembles that in Huntington disease. Blood tests reveal the presence of acanthocytosis in the blood smear but this may not always be detectable. Elevated creatine kinase is also typical and may be detected before the appearance of neurological symptoms or signs. Protein assays or genetic testing can confirm the diagnosis.
ChAc belongs to the group of neuroacanthocytosis syndromes alongside with X–linked McLeod syndrome, pantothenate kinase–associated neurodegeneration and Huntington disease–like 2. Furthermore, the differential diagnosis includes other forms of inherited chorea (Huntington disease, other forms of the Huntington–like disorders, other syndromes of neurodegeneration with brain iron accumulation [NBIAs], Wilson disease and others), but acquired causes should also be considered.
Yeast Vps13 was first discovered as a protein involved in the delivery of proteins from Golgi apparatus to the vacuole. Null mutant vps13D displayed defects in localization of several proteins, such as vacuolar carboxypeptidase Y and Golgi apparatus proteins Kex2, Vps10 and Ste13. Vps13 is also required for sporulation. However, the molecular function of Vps13 is unknown. In humans there are four homologs of yeast Vps13 and mutations in VPS13A and VPS13B cause the hereditary disorders chorea–acanthocytosis and Cohen syndrome, respectively.
We showed that Vps13D mutant has impairment of endocytosis. The null mutant shows delay in internalization of FM4–64 lipophylic dye from the plasma membrane to vacuole, and slower growth on canavanine–containing medium, indicating defective endocytosis of Can1 permease. This mutant also has defects in actin cytoskeleton organization: actin patches are not well polarized and abnormal actin clumps are present in cells. Dynamic actin cytoskeleton is important for endocytosis but also in selective autophagy pathways. Selective cytoplasm–to–vacuole (Cvt) pathway was only weakly delayed, as documented by Western blot analysis of preApe1 maturation. Nonselective autophagy, measured by GFP–Atg8 degradation, was not defective when compared with control strain. A Vps13D strain also showed defects in vacuole staining by CMAC, a pH–sensitive dye. This mutant was impaired in vacuolar localization of Vph1 protein, the subunit of vacuolar ATP–ase, and showed slower growth on rich medium at low pH. This may indicate disturbances in pH homeostasis of cells which in turn may affect various trafficking steps.
McLeod Syndrome (MLS) is one of the neuroacanthocytosis syndromes, characterized by hematological, immunohematological, neuro–psychiatric and other cardinal symptoms. The genetic defect is located on the short arm of X chromosome at position Xp21.1 (XK). Currently 27 aberrations such as single nucleotide polymorphism, stop codons, insertion/deletion mutation and splice site mutations are recognized. There is no well–defined genotype–phenotype correlation of XK mutation. Clinical manifestations are heterogeneous and variable, from asymptomatic carrier status to debilitating neuro–psychiatric and cardiodegenerative sequelae. Lead diagnostic findings are the pathognomonic aberrations of the Kell and Kx blood group antigen systems as well as red cell acanthocytosis. In addition, X–chromosomal inheritance with carrier status of the mothers of involved males is pivotal for MLS diagnosis. Immunohematological phenotyping of Kell antigens and flow–cytometric assessment of expression of Kell glycoprotein, as well as structured molecular characterization of the X–chromosomal defect, constitute the key findings for the diagnosis of MLS. When there are extended deletions of Xp21.1, involving centromeric or teleomeric genes such as the CYBB, RPGR, OTC or DMD loci, the prototypic hematological findings of MLS may be dominated by the phenotypic manifestations of affected neighbouring genes of XK(contiguous gene syndrome).
Cellular and subcellular manifestation of XK mutations in hematological as well as extra–erythropoietic tissues will be reviewed. KEL/XK –KO mouse models, as well as assessment of transmembrane cation transport regulation of MLS red blood cells, may give insights into the pathomechanism of MLS manifestation in extra–hematological organs. Clinical aspects of MLS patients, in addition to patients with X–linked CGD syndrome as examples of contiguous gene syndrome, will be presented.
The X–linked McLeod neuroacanthocytosis syndrome (MLS) is characterized by the association of erythrocyte acanthocytosis and progressive striatal neurodegeneration. As the autosomal recessive chorea–acanthocytosis (ChAc), MLS has a Huntington disease–like phenotype consisting of a hyperkinetic, mostly choreatic, movement disorder, psychiatric manifestations and cognitive decline, with a relentlessly progressive course over several decades. In addition, MLS patients may have a multi–system involvement including motor–dominant axonal neuropathy, myopathy and cardiomyopathy. McLeod syndrome is exceptionally rare with an estimated prevalence of less than 1 to 5 per 1,000,000 inhabitants. In the last years, it has been recognized that MLS has a worldwide distribution. MLS is caused by mutations in the XK gene. Although the association of the acanthocytic membrane abnormality with selective striatal degeneration suggests a common pathogenetic pathway, the possible mechanisms by which these mutations cause striatal neurodegeneration are still a matter of debate and research. The diagnosis of MLS involves (often negative) blood smears to detect acanthocytosis, but also the determination of serum creatine kinase (CK), which is elevated in virtually all MLS patients. Cerebral MRI may demonstrate striatal atrophy. Kell and Kx blood group antigens are reduced or absent, thus delivering an accurate diagnosis of MLS. Identification of a distinct mutation in the XK gene is confirmatory. The course of McLeod syndrome is relentlessly progressive, and there is no curative therapy yet known. However, regular cardiologic studies and avoidance of transfusion complications are mandatory. The hyperkinetic movement disorder may be treated as in Huntington disease. Other symptoms including psychiatric manifestations should be managed in a symptom–oriented manner.
We have recently reported a novel alteration in erythrocyte Mg2+ and K+ homeostasis in cells from Xk knockout mice, a model of McLeod Syndrome (MLS). We now show that in an untreated, asymptomatic MLS subject alterations in cellular ion content were associated with changes in the activity of 4 major cation transporters in erythrocytes. There was an 18% increase in erythrocyte K+ (245.3–298.5 mmol/Kg Hb), Mg2+ (5.6–7.5 mmol/Kg Hb) and total Ca2+ levels (0.29–0.52 mmol/Kg Hb) when compared to control cells, suggesting altered ion homeostasis. Consistent with this hypothesis, Na+–independent Mg2+ permeability (2.0 to 0.6 mmol/1013 cell x h) was significantly reduced, implying an abnormality in Mg handling. We also observed that Na/Mg exchanger (NME) activity was significantly decreased. It has been shown that Kell/XK complex are phosphorylated by Casein kinase II (CKII). We observed that TBB (4,5,6,7–tetrabromobenzotriazole), a specific CKII blocker, did not block Big ET–3–stimulated NME activity in Xk–mutated erythrocytes, unlike in control cells. These results suggest that alterations in CKII signaling mediate disordered Mg levels in MLS. K+ transport mediated by the Gardos channel was increased when compared to controls (18 to 26.8 mmol/1013 cell x h). In addition, the volume–stimulated and Cl independent K/Cl co–transporter was almost two–fold lower when compared to controls. These results are in agreement with a higher K+ content in these cells. In contrast, no differences in Na transporter activities were observed. Thus, our results provide novel insights into potential mechanisms by which alterations in Kx protein lead to abnormalities in erythrocytes transporters, which may play critical roles in acanthocytosis development.
Introduction: Pantothenate kinase–associated neurodegeneration (PKAN) is the major genetic subtype of Neurodegeneration with Brain Iron Accumulation (NBIA), accounting for 35–50% of cases. The progressive nature and intractable dystonia in this ultra–rare disorder make it a challenge to manage. To date, there is no comprehensive guideline for the diagnosis and management of these patients.
Aims and Methods: We aimed to develop a best practice guideline for PKAN, leveraging the wisdom of a small pool of experts, to facilitate holistic management. Our team comprised multidiscipinary professionals (neurologists, geneticists, surgeons, genetic counsellors, therapists and others) and family members with practical PKAN expertise. These experts drew on their direct experiences and trials and errors with PKAN to address key issues. A detailed PubMed search was also utilised to identify and review all relevant published data. The team developed, drafted, and prepared a consensus guideline for publication.
Results: Management of PKAN patients is complex and needs to be tailored from diagnosis to end–stage disease. Our guideline addressed these relevant issues:
Diagnostic evaluation
Initial management (medical, genetic implications, psychosocial)
Pharmacologic and surgical management
Monitoring for disease complications
Emergency management
Educational management
Nutrition
Psychosocial support
Conclusion: A comprehensive document outlining the care and management of patients with PKAN benefits the entire NBIA community. This guideline not only serves to support new families as they are diagnosed, but also provides clear guidance to physicians and allied healthcare professionals. The experience we gain will inform the development of similar guidelines for other forms of NBIA.
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In this retrospective consecutive cohort study, we attempted to determine the point prevalence of urine bilirubin and urobilinogen in blunt abdominal trauma patients and its utility as a predictor of intraabdominal injury.
A total of 986 consecutive trauma patients were screened, of which 698 (71%) were adult blunt trauma patients. 516 (52%) of these patients underwent a urinalysis and CT scan of the abdomen and pelvis or operative exploration of the abdomen and were eligible for inclusion. Urinalysis results were examined for the presence of hemoglobin, urobilinogen and urine bilirubin. CT scan results and operative reports were examined for evidence of liver and spleen laceration, as well as bowel or mesenteric injury. Patient medical records were reviewed for history of preexisting liver or biliary pathology. spleen lacerations, or bowel injuries. Preexisting liver or biliary conditions, such as hepatitis, gallstones, or previous cholecystectomy were not statistically associated with elevation of urine bilirubin (p=0.53) and urobilinogen (p=0.06).
Urinalysis was positive for urobilinogen in 28 (5.43%) patients and urine bilirubin in 15 (2.91%). 19 (4%) patients were found to have liver lacerations, 28 (5%) to have spleen lacerations, and 15 (3%) to have bowel or mesenteric injury. Eight (29%) patients with urobilinogen (p=0.002) and five (33%) patients with urine bilirubin (p=0.005) were found to have liver lacerations,
Our results indicate that urobilinogen (OR=3.58; 95% conf interval=1.29, 9.02), urine bilirubin (OR=4.32; 95% conf interval=1.11, 14.45), and hemoglobin (OR=3.41; 95% conf interval=1.65, 7.75) on initial urinalysis after blunt trauma are independent predictors of intraabdominal injury. Initial urinalysis results may be useful in guiding imaging studies and further evaluation of blunt trauma patients. Future research is necessary to clarify the clinical significance of this connection.
With vaccination advances and herd immunity, the vast majority of initially positive Emergency Department (ED) cerebrospinal fluid (CSF) cultures are subsequently determined to be nonpathogenic contaminants. Clinical follow-up in these patients is costly, time-consuming, and unnecessary if their cultures do not demonstrate a pathogen with potential to cause acute bacterial meningitis. We hypothesized that, in immunocompetent patients with normal gram stain, protein, glucose, and white blood cell (WBC) count, all positive cultures are contaminants and need no further care.
Using standard data forms, we retrospectively reviewed charts of 169 immunocompetent ED patients of any age who had positive CSF cultures from January 1, 2000 to December 31, 2004. Of these, 135 had CSF WBC ≤7, normal chemistries, and negative initial gram stains. Our primary outcome measure was the proportion of final culture results that represent true infection (Streptococcus pneumoniae, S. agalactiae, Neisseria meningitidis, Listeria monocytogenes, Escherichia coli, Salmonella species, and Haemophilus influenzae) versus contamination (Staphylococcus epidermidis, Streptococcus bovis, Propionibacterium acnes, viridans streptococci, Enterobacter, Flavobacterium, Enterococcus, Corynebacterium, and non-anthrax Bacillus). In addition, we report the rate of subsequent phone follow-up, repeat ED visits, repeat lumbar punctures, and non-therapeutic antibiotics. Results: Of the 135 positive cultures studied, 134 were contaminants. One remaining culture revealed S. agalactiae (Group B Strep) in an adult patient. Repeat lumbar puncture in this patient did not grow any organism. The initial “positive” culture was therefore deemed to be a contaminant. Thus, all 135 positive cultures were contaminants. The most common organisms isolated were coagulase-negative staphylococcus (n=52), P. acnes (n=33), viridans streptococci (n=14) and Corynebacterium (n=8). Unnecessary clinical follow-up activities were as follows: phone calls (49%), repeat ED visits (12%), repeat LPs (10%), and initiation of non-therapeutic antibiotics (3%).
In our study, all positive CSF samples in patients with negative lumbar punctures were determined to be contaminants. If validated with a larger sample, follow-up may be unnecessary in patients with positive CSF cultures if their initial lumbar puncture was normal.
Previous research has shown a correlation between elevated intracranial pressure (EICP) and an increase in optic nerve sheath diameter (ONSD). Few studies have analyzed the use of bedside ultrasound in measuring the ONSD of patients with suspected EICP. This study seeks to assess the ease of measurement of ONSD in Emergency Department patients, show the distribution of ONSD as measured by beside ultrasound, and compare the accuracy of this ONSD to non-contrast head CT for the diagnosis of EICP.
Patients undergoing head computed tomography (CT) in an academic emergency department were prospectively identified. Emergency department physicians were trained on measuring ONSD with a 10-minute slide presentation. Informed consent was obtained and the patient’s ONSD was measured in both eyes by a physician blinded to the CT results. Measurements were taken on the closed eyelids of supine patients using a 10-MHz linear probe. The ONSD was measured 3mm posterior to the globe. CT scans consistent with EICP were defined as one or more of the following: a midline shift of greater then 3mm, sulci effacement, abnormal cisterns or a collapsed third ventricle. The mean value, standard deviation and range of the ONSD were identified.
From April 2005 to January 2006, 210 patients undergoing head CT in the emergency department were identified and enrolled in the study. Mean ONSD of left eyes was 4.74 mm (SD 1.28) with a range from 1.7 to 8.3 mm. Mean ONSD of the right eyes was 4.72 mm (SD 1.17) with a range from 1.8 mm to 8.2 mm. 9 patients (4%) were identified as having changes on head CT consistent with EICP. Mean ONSD of their left and right eyes respectively was 5.42 mm (SD 1.3, range 4.0 to 7.4 mm) and 5.12mm (SD 0.75, range 4.0 to 6.4).
Previous studies indicate that the mean ONSD in normal adult patients is 5.0mm. Our prospective study demonstrates a mean of 4.75 mm in patients without EICP. Further enrollment of patients with signs of EICP on CT scan is required for analysis of ONSD in these patients.
To evaluate how the ultrasound findings in first trimester vaginal bleeding predict the pregnancy outcome in patients who present to a University Emergency Department (ED).
Prospective Cohort Study
University Emergency Department
All women over thirteen years of age who presented to the ED complaining of first trimester vaginal bleeding between April 2004 and January 2005 were eligible for the study. Patients who were diagnosed with ectopic pregnancy, molar pregnancy, or were not pregnant were excluded. Also excluded were patients who elected to terminate the pregnancy, did not wish to participate in the study, or were unable to be contacted for follow-up.
All women underwent a standard workup for first trimester vaginal bleeding. This included a serum BHCG and an ultrasound (transabdominal or transvaginal) performed by emergency physicians. The ultrasound results were recorded as no definitive intrauterine pregnancy (NDIUP) where no gestational sac, yolk sac or fetus was visualized either in the uterus or elsewhere, intrauterine pregnancy (IUP) where there was visualization of an intrauterine gestational sac greater than 5 mm with either a fetal pole or yolk sac, live intrauterine pregnancy (LIUP) where an intrauterine pregnancy was identified with fetal heart motion or abnormal intrauterine pregnancy where an intrauterine gestational sac 10 mm or greater with no gestational sac or 16 mm or greater with no fetal pole was identified. The women were then followed up by either telephone contact or medical records to determine the outcome of the pregnancy.
Live birth
A total of 124 women were enrolled. Of the 70 that met inclusion criteria, 23 (32%) carried to term and delivered live babies. None of the babies carried to term were stillborn. Of the 23 live births, 18 (78%) were noted to have a documented live intrauterine pregnancy at the time of the ED visit. The other findings included intrauterine pregnancy (13%) and no definitive intrauterine pregnancy (8%). None of the abnormal intrauterine pregnancies resulted in a live birth.
In women with first trimester vaginal bleeding the likelihood of carrying the pregnancy to term does appear to be related to the findings on ultrasound. Women with a documented live intrauterine pregnancy have a much greater chance of delivery than women with an abnormal intrauterine pregnancy.
Acute appendicitis is the most common abdominal surgical emergency. The aim of this project is to evaluate the accuracy of emergency physicians using bedside ultrasound for appendicitis (BUSA).
All physicians attended a 1-hour didactic lecture on BUSA. Patients undergoing testing for appendicitis were approached for inclusion; this consisted of a 5- minute BUSA. In addition to the BUSA, all patients received at least one: Radiology performed ultrasound (RUS), CT scan, appendectomy, or telephone follow-up. BUSA results were kept confidential from surgeons and radiologists.
A total of 126 patients were analyzed. In 44 cases BUSA was positive. Of these, 37 agreed with the surgical pathology report (true positives), while 7 were found to be negative (false positives). In 82 cases BUSA was negative. Of these, 62 were determined not to have appendicitis (true negatives), and 20 received appendectomies showing appendicitis by pathology (false negatives). Sensitivity for BUSA was 65% [95% CI 52–76], specificity was 90% [95% CI 81–95], positive predictive value was 84% [95% CI 71–92], and negative predictive value was 76% [95% CI 65–84]. The likelihood ratio of a positive BUSA was 6.4 (95% CI 3.1–13.2).
One hour of focused training is not sufficient for the emergency physician to use BUSA as a solitary screening modality.
Pediatric cases comprise approximately 22% of rattlesnake envenomations in the U.S. The recent introduction of Crotaline Fab antivenom and withdrawal from the market of the traditional antivenom preparation has changed the way rattlesnake envenomation is treated. Although in some hospitals Crotaline Fab antivenom may be the only antivenom currently available, there is little data regarding its use in children. Our objective is to provide the first data regarding safety and effectiveness of this new drug in the pediatric population.
Data was collected prospectively and retrospectively for all pediatric rattlesnake envenomations treated at two urban hospitals during the year 2001. Cases were included if there were signs of envenomation at presentation, patient age 13 years or less, and administration of Crotaline Fab antivenom. Cases were excluded if Antivenin Crotalidae Polyvalent was given. Primary outcome variables were snakebite severity scores throughout the course of therapy, number of vials of Crotaline Fab antivenom given, occurrence of allergic reactions, need for surgical therapy, and the presence of permanent sequelae or serum sickness identified at telephone follow-up.
In the 12 study cases, age ranged from 14 months to 13 years. (mean=6.9, sd=4.2) Presentation snakebite severity scores ranged from 2 to 9. (mean=5.3, sd=2.3) Total Crotaline Fab antivenom doses ranged from 4 to 22 vials. (mean=12.7, sd=5.4) Initial control of symptoms was achieved with 4–16 vials (mean=7.7, sd=3.7) and severity scores stabilized or improved within 24 hours in all patients. Recurrence of local swelling occurred in one case despite scheduled doses of antivenom. No cases required surgical intervention and no permanent sequelae were identified. No immediate or delayed hypersensitivity reactions occurred.
In this group of pediatric patients treated for rattlesnake envenomation, Crotaline Fab antivenom was safe and appeared to be effective.
Spinal cord injury without radiographic abnormality (SCIWORA) has been predominantly reported in case series of pediatric patients. Our objective was to better define the incidence and characteristics of patients with SCIWORA, using the
We conducted a prospective, observational study of all Emergency Department patients selected by physicians for plain cervical radiography after blunt trauma at 21 medical centers in the United States. Data available for analysis included the patient demographics, presence or absence of the NEXUS criteria, as well as the reports of all cervical spine-imaging studies performed. SCIWORA was strictly defined as the presence of spinal cord injury (SCI) as shown by magnetic resonance imaging (MRI), when a complete and technically adequate plain radiographic series revealed no injury.
Of the 34,069 patients entered into the NEXUS database, there were 818 (2.4%) with SCI, including 27 (0.08%) patients with SCIWORA. Although the participating sites enrolled over 3000 pediatric patients (<18 years old), including 30 with SCI, there were no cases of SCIWORA in children. The most common findings on MRI, among patients with SCIWORA, were central disc herniation, spinal stenosis, and cord edema or contusion. The central cord syndrome was specifically described in 10 (37%) of the 27 cases.
Using strict, objective criteria, we found that SCIWORA was an uncommon disorder, and occurred only in adult patients in the large NEXUS cohort. This is likely due to a much higher overall incidence of SCI in adults compared to children.
To determine which EM program format (PGY 1–3, 2–4 or 1–4) favors fellowship training or academic career.
Mailed survey of 122 program directors (PDs) of US EM residencies regarding number of graduates (1995–2000) who pursued fellowships, community practice (teaching/non-teaching), academics, advanced degree, and further residency, both immediately and after 3–5 years.
54.9% of programs responded regarding 2518 graduates (49.8% of all graduates). More 4-year format graduates pursued academics initially (2–4 vs.1–3, OR=1.45,95% CI: 1.15–1.82; 1–4 vs.1–3, OR=2.20, CI: 1.71–2.82). PGY 1–4 favored academics vs. 2–4 (OR=1.52, CI: 1.14–2.02). 19.4% of 1–3 graduates chose academics, vs. 25.8% for 2–4, and 34.6% for 1–4. PGY 1–4 favored fellowship vs. both 2–4 and 1–3 (OR= 2.23, CI: 1.29–3.88, and OR=2.53, CI: 1.62–3.94, respectively). There was no difference in fellowship pursuit between PGY 1–3 and 2–4 programs. In all, 4.3% of 1–3 residents pursued fellowships, vs. 4.8% of 2–4, and 10.1% of 1–4. For 1995–97 graduates, more PGY 1–4 format graduates were still in academics vs.1–3 and 2–4 (OR=1.88, CI: 1.28–2.83 and OR=1.68, CI: 1.04–2.72 respectively); there was no difference between PGY 1–3 and 2–4 formats. 219 of the 232 graduates (94.3%) who started out in academics remained there 3–5 years later. In aggregate, 5.2% of graduates pursued fellowships, while 23.1% pursued academics initially. 46/219 (21.0%) academic physicians from the 1995–97 classes were fellowship trained.
4-year formats, especially 1–4, favored fellowships and academics more than the 1–3 format. Fellowship pursuit was uncommon (4–10% of graduates), while 25–35% chose academics. Most new academic EPs, are not fellowship trained. Response rate and recall bias are limitations. Further study should elucidate reasons for these associations.
Etomidate has become a preferred medication for adult rapid sequence intubation (RSI) due to its minimal effect on blood pressure. The manufacturer does not yet recommend its use in children under 10 due to a lackof data. Potential risks of etomidate include an association with myoclonus, seizures, emesis, and adrenal insufficiency. This study further elucidates the risk-benefit profile of etomidate for RSI in young children.
Trained abstractors reviewed the medical records for all children under 10 who received etomidate for RSI between July 1996 and October 1999. The study took place at a level one pediatric trauma center.
105 children, with an average age of 3, received a median dose of 0.32 mg/kg of etomidate. Indications for RSI included: trauma (64), pulmonary disease (23), ALOC (15), and “other” (11). The average blood pressure and pulse increased 4 mmHg (systolic), 7 mmHg (diastolic), and 10 bpm within 10 minutes of receiving etomidate. Complications included 3 patients who vomited within 10 minutes of etomidate administration (95% C.I. = 0.6% to 8.1%) −1 prior to, and 2 after intubation. There were no cases of myoclonus or status epilepticus. 43 patients received steroids during the hospital course, none for suspected adrenal insufficiency. Three patients died, all from severe brain injury. 1 in 5 patients had multiple attempts at intubation, but there were no failures.
In children less than 10, etomidate maintains the appeal of hemodynamic stability, and appears to have a low risk of adrenal insufficiency, myoclonus, and status epilepticus. The association between etomidate and emesis remains unclear. Thus, for clinical situations in which minimal blood pressure changes during RSI are critical, etomidate appears to have a favorable risk-benefit profile for these patients.
The American College of Surgeons has advocated digital rectal examination (DRE) in the evaluationof all adult trauma patients. Its clinical utility however is unclear. This study sought to determine if physical exam findings could allow omission of the DRE in an identifiable subset of adult trauma patients.
Trained abstractors reviewed the ED records of all adult trauma activations at a level I trauma center during 2000. Variables included the result of the rectal exam, physical examination, and discharge diagnoses.
There were 733 adult trauma activations in 2000. 51 were excluded (6 records could not be located, 44 were intubated prior to arrival in the ED, 1 had pre-existing T12 paraplegia, 1 had spilled blood contaminating the rectum). 10 patients had DRE “deferred”, 2 patients refused DRE, 6 patients died prior to a DRE, and 56 had no documentation of a DRE. There were 41 abnormal DREs (6.7%) in the remaining 607 patients. Abnormal findings included: 39 with decreased or absent tone, 1 with perianal lacerations, and 1 with a high-riding prostate. 7 of the 41 patients with abnormal DRE had no significant injury on discharge (false positives). 16 had spinal abnormalities, 16 had severe head injury, 1 had a urethral disruption, and 1 had peri-rectal lacerations (true positives). All patients with a true positive DRE had at least one of the following: abnormal neurological exam as evidenced by either extremity weakness or chemical paralysis for intubation (19), GCS < 15 (11), or extremity paresthesias (1), blood at the urethral meatus (1), complaints of rectal pain (1), or age over 75 (1).
Few trauma patients have abnormal DREs. Of those that do, all true positive abnormal DREs in this sample could be predicted by: an abnormal neurological exam, urethral blood, rectal pain, or age over 75. These results must be verified by a validation set.
The goal of this study was to determine if the addition of parenteral opiate medications in combination with methohexital for moderate/procedural sedation in the ED increases the risk of respiratory or cardiovascular complications.
We conducted a review of an existing single ED database of all patients who underwent moderate and procedural sedation in which methohexital was administered over a 2-year period. This database included data on patient demographics, procedure, sedation medications, pre-, intra-, and post-procedure vitals signs and monitoring changes, procedural success, complications and management. Patients were stratified into 2 groups: those who were pre-medicated with parenteral opiates and those who were not. Significant respiratory and cardiovascular abnormalities and complications were defined aprior to data collection and analysis. Power analysis determined that 108 cases were needed to detect a 25% increase in complication rates. Statistical analysis was performed using Fisher’s exact with p<0.05 considered significant (STATA 6.0).
During the study period, there were 114 patients who received methohexital, of whom 65 received parenteral opiate pre-medication (primarily morphine and fentanyl) and 49 did not. Overall rate of respiratory or cardiovascular complications was 15.9% with no significant difference between those who received opiate pre-medication and those who did not (18.7% vs. 11.0% respectively, p=.20). All complications were transient and managed without any long-term sequelae. Overall procedural success was 81% with no difference between the 2 groups (p=.50).
In this study, the addition of parenteral opiate pre-medication with methohexital for moderate procedural sedation in the ED did not result in any increase in respiratory or cardiovascular complications nor decrease in procedural success. These findings need further validation with a larger, randomized study.
To determine if pressure-immobilization (PI) delays mortality and/or elevates intracompartmental pressure after artificial, intramuscular
We prospectively studied 20 pigs using a randomized, controlled design. After the pigs were anesthetized,
The dose of venom resulted in 100% mortality. The mean survival times (minutes ± SD) were 189 ± 33 with PI and 155 ± 23 without. The effect size (the difference between the 2 groups) was 34 minutes (95% CI = 6 to 62, P = 0.021). The mean intracompartmental pressures (mmHg ± SD) were 67 ± 13 with PI and 24 ± 5 without (effect size: 43 mmHg, 95% CI = 32 to 53, P < 0.0005). The mean circumferences (cm ± SD) were 14.3 ± 0.8 with PI and 19.1 ± 1.0 without (effect size: −4.8 cm, 95% CI = −5.7 to −3.9, P < 0.0005).
PI resulted in significantly longer survival, less swelling, and higher intracompartmental pressures.
Overcrowding is a reflection of the status of the safety net, and diversion is a reflection of overcrowding. As core safety net providers it is our job to try and ensure the integrity of the safety net. In order to define the problem of overcrowding we must have objective data. The objective of this study was to analyze yearly diversion hours in a large metropolitan city and to determine if the diversion times in Los Angeles (LA) County are increasing.
This was a retrospective observational review of diversion data obtained from Redinet. Redinet is the central computer link in the LA county area used to negotiate ambulance traffic, and all diversions must be documented in Redinet. Six years of diversion data (collected on a monthly basis) were reviewed. The data collected included hours on diversion for each month, county versus private hospital and total hours on diversion/year.
Diversion totals for each year (in hours) were 577 in ‘95, 651 in ‘96, 740 in ‘97, 883 in ‘98, 1,130 in ‘99, 1,624 in ‘00 out of 8,760 total hours available. The average rate of increase was 17%. There was a 281% increase in the amount of diversion time over a 6-year period. Private hospitals show an increase each year in diversion of 14%. County hospitals show an increase in diversion per year of 19%. Currently, County hospitals in LA are on Diversion 35% of the time. In the year 2000, hospitals across the board in LA County were on diversion all day 1 out every 5.4 days.
In LA County the “Safety Net” is progressively Unraveling with diversion times trending up each year. County Hospitals in LA are especially being affected by emergency department overcrowding with dramatically increasing diversion times. This may have substantial consequences unless measures to halt and reverse this trend are initiated.
To determine predictors of overall patient satisfaction (PtSat) at an urban County Emergency Department (ER) with emphasis on three specific components of medical care (Timing, Thoroughness, and Compassion/Respect).
Cross-sectional prospective study of patients (Pts) who completed treatment over six consecutive days in August 2000. Overall PtSat (a 5 question composite variable accounting for acquiescence bias) was assessed using a multilingual (English, Spanish, or Chinese) questionnaire.373 Pts completed the survey: − 55% female, mean age 42, avg. medical acuity 1.6 (3-pt scale), avg. health status 2.9 (5-pt scale), mean LOS 9.4 (+/−7.3) hours, 64% uninsured.
Our findings demonstrate overall very favorable PtSat: 88% indicated that they “would recommend this ED.” On a 5-pt scale, mean Overall PtSat, was 3.5 (between “good” and “very good”). Questions on “length of time” (wait, treatment, and time spent with provider) had a mean of 3.2. Questions on thoroughness/quality of care had a mean of 3.7. Finally, patients were especially pleased on avg. with their interactions with staff & providers, at a mean score of 3.9. Multivariate regression on all three components of care demonstrated significant positive correlation with Overall PtSat and a reasonably good fit, explaining just over half the variance (Adj. R2 =.51). A larger modeladding demographic, health, and economic factors improved fit (Adj. R2 =.64) and satisfaction with the three medical care components remained the strongest significant predictors (Time, B=.25, Respect, B=.20, Thoroughness, B=.17). Other positive and significant predictors included Health Status, and Age. Female gender and the Number of Previous ER Visits reduced PtSat.
Patients were very satisfied with patient/provider interactions in this urban emergency department and patient satisfaction did not suffer from prejudicial disparities. Satisfaction with Time is the most important predictor of ER PtSat. PtSat was only minimally affected by demographic & socioeconomic factors.
Most patients admitted to the hospital from the emergency department with syncope do not have myocardial infarction yet routine current practice is to draw serial cardiac enzymes.
To assess the value of serial cardiac enzymes in elderly patients who present to the ED with syncope.
A retrospective chart review was performed on consecutive patients age 65 and over presenting with syncope to a teaching hospital ED between 7/1/98 and 6/30/99. Charts were screened for presenting history, cardiac risk factors and outcomes including acute coronary syndromes, myocardial infarction and death. Patients returning to the ED or admitted as an inpatient within 72 hours of discharge were recorded as well.
Of 497 visits, 327 patients met the study criteria, with 99% of charts available for review. 212 patients (65%) had CPK drawn and 12% had Troponin I (TnI) as well. Two patients, 0.94%, (95% confidence interval: 0.01%–3.36%) had positive cardiac enzymes during their hospitalization. CPK was positive in both and TnI, drawn in one patient, was also positive. One of these patients had chest discomfort in addition to a syncopal event. The other patient had dementia and could not recall the details surrounding her syncopal event. In addition, her baseline EKG demonstrated a LBBB, limiting the interpretation of the EKG.
Cardiac enzymes may be of little additional value if drawn routinely on elderly patients who are admitted to the hospital from the emergency department with syncope, unless they have other signs or symptoms suggestive of myocardial ischemia by history such as chest pain or dyspnea, or by EKG, i.e. new STTW abnormalities, ST elevation, or an EKG that is uninterpretable for ischemia.
Our institution has limited telemetry, stepdown and ICU bed capacity. Patients are commonly monitored in the ED for prolonged periods until a bed of proper acuity is available. There are approximately 45,000 visits per year in the main ED and another 35,000 in the Urgent Care Clinic.
Assess gurney occupancy time relative to the length of stay (LOS)in the ED.
LOS was calculated from our patient tracking system for 21,688 patients that were seen in the main ED during the first 6 months of 2001. We subsequently divided the patients into 5 groups: Group 1 had an ED visit of 6 hours (hrs) or less. Group 2 had a stay of 6–12 hrs, Group 3 stayed 12–24 hrs, Group 4 stayed between 24–48 hrs, and Group 5 stayed > 48 hrs. The percentage of patients in each of the groups was calculated for each month separately, and subsequently averaged over the 6 months. The midpoint of length of stay for each category was multiplied by the percentage of patients in each category for the first 6 months of 2001. The product was termed “Standardized Gurney Hours”. This number was normalized to obtain “Standardized Gurney Occupancy” (SGO).
52% of patient visits were placed into Group 1, and accounted for 17% of the SGO. Group 2 represented 28% of the patients, resulting in 28% of the SGO. Group 3 corresponded to 14% of patients and 29% of the SGO. Group 4 was 5% of the patients and represented 19% of the SGO, and Group 5 was 1% of the patients and represented 7% of the SGO. When Groups 4 and 5 were combined (i.e., patient stay > 24 hrs), they represented 6% of the patients with 26% of the Standardized Gurney Occupancy.
As the ED visit becomes prolonged, the SGO increases exponentially. Those patients that leave the department in less than 6 hrs comprised more than half of the ED visits, and utilized only 17% of the SGO. By comparison, patients who stayed more than 24 hr were 6% of the patients and utilized over 25% of the SGO. This underscores the need of a hospital to have facilities in place to expediently transfer patients out of the ED to the floor or ICU.
To compare success and complication rates between ETC and ETTs by paramedics. Placement with successful ventilation was the primary outcome, with complication rates, survival to admission and discharge, and aspiration pneumonia as secondary measures.
Retrospective review of three years of EMS runsheets for all patients where an ECT was attempted. Abstracters were hypothesis-blinded, trained and monitored (Kappa =.7–1.0). This EMS system uses the ETC primarily as an alternative airway to failed tracheal intubation. Pharmacologically assisted intubation is not used. ETT patients were selected from the EMS QA database for the same period. We reviewed the charts of 19 ECT patients.
ECT insertion was attempted on 200 patients: 140 (70%) successful, 55 (27.5%) failed (some for multiple reasons), and 5 not recorded. An ETT was attempted for 169 patients: 152 (90%) successful, 17 (10%) failed. ECT location was noted in 104: 83 (80%) esophageal, 21 (20%) tracheal. Inability to determine placement of the ETC was due to emesis from both ports in 28 cases and inability to pass the ECT occurred in 29. The ECT caused one patient dental trauma, and one ETC placement was temporally related to the onset of subcutaneous emphysema. Blood in the ETC from pre-existing active upper GI bleed occurred in 10 patients (5.3%) and 6 tubes (3.2%) dislodged. Ninety-one runsheets noted disposition; 18 (19.8%) survived to hospital admission. Of 19 ECT hospital charts reviewed, 5 survived to admission, none to discharge. Average admission ABGs were pH 7.02/p02 288/pC02 57/HC03 15. Length of stay was 1–21 days, with aspiration pneumonia present in 2/5.
Similar to previous reports, ETT success was greater than ECT. In an earlier study, EMT-Ds had a higher ECT success rate (155/195 or 79%) than para-medics here (70%) (p=.04, OR 1.67 (95% CI 1.02–2.70). Successful ECT use may depend on local experience and level of specific training, rather than comprehensive para-medic training.
To elicit the practice patterns of board-certified emergency medicine physicians when a parent demands to sign out their child against medical advice.
E-mail requests were submitted to board certified, emergency physicians requesting their participation in an anonymous, web-based electronic survey. Physicians who agreed to participate were presented with a fictitious case scenario describing a mother who brings her febrile, lethargic 4-month-old infant into an emergency department. The infant’s vital signs were reported to be temperature 104.5 F, heart rate 150, respiratory rate 30 and blood pressure 84/60. The mother, who does not appear to be intoxicated or confused, becomes very upset that the nurse is unable to establish an intravenous line after two attempts. Angrily, she proceeds to pick-up the infant and leave the Emergency Department prior to a complete medical evaluation by the physician. After being presented this fictitious case scenario, subjects were then asked how they would manage the situation if the infant and mother had presented to them in their usual practice setting.
Of the 674 board certified emergency medicine physicians who correctly completed the survey, 214 (32%) physicians stated that they would allow mother to leave with infant and take no further actions; 214 (32%) physicians stated that they would request the mother to signrelease from medical liability forms prior to leaving; 131 (19%) stated that they would allow mother to leave with infant but report the case to Child Protective Services afterwards; 115 (17%) physicians stated that they would call security immediately to prevent mother and infant from leaving. The data was analyzed using the Chi-squared test and found to be statistically significant (p< .05).
This study suggests that a single ‘standard of care’ is not being practiced in emergency medicine with regards to the management of parents signing out children AMA and that structured guidelines are needed.
An area of the skull exposed by burn injury has been covered by various methods including local flap, skin graft, or free flap surgery. Each method has disadvantages, such as postoperative alopecia or donor site morbidities. Due to the risk of osteomyelitis in the injured skull during the expansion period, tissue expansion was excluded from primary reconstruction. However, successful primary reconstruction was possible in burned skull by tissue expansion.
From January 2000 to 2011, tissue expansion surgery was performed on 10 patients who had sustained electrical burn injuries. In the 3 initial cases, removal of the injured part of the skull and a bone graft was performed. In the latter 7 cases, the injured skull tissue was preserved and covered with a scalp flap directly to obtain natural bone healing and bone remodeling.
The mean age of patients was 49.9±12.2 years, with 8 male and 2 female. The size of the burn wound was an average of 119.6±36.7 cm2. The mean expansion duration was 65.5±5.6 days, and the inflation volume was an average of 615±197.6 mL. Mean defect size was 122.2±34.9 cm2. The complications including infection, hematoma, and the exposure of the expander were observed in 4 cases. Nonetheless, only 1 case required revision.
Successful coverage was performed by tissue expansion surgery in burned skull primarily and no secondary reconstruction was needed. Although the risks of osteomyelitis during the expansion period were present, constant coverage of the injured skull and active wound treatment helped successful primary reconstruction of burned skull by tissue expansion.
Burn wounds require different treatments depending on their location and severity. The scalp is the only area in the body with dense hair patterns and reconstructive options are therefore limited. Scalp wounds with calvarial exposure present a complex clinical challenge and require a systematic approach for reconstruction. Local flaps are ideal for small wounds but are rarely adequate for larger defects. Skin grafts can be a useful option, but require removal of the outer table, thus increasing the complexity and risks of the reconstruction. Skin grafts on trabecular bone also break down easily. A free flap is an option, but requires microvascular expertise and a complex, multi-staged reconstructive effort with the possibility of donor-site morbidity. Scalp-tissue expansion is a validated, reliable, and safe technique for the treatment of alopecia and has been commonly used in secondary reconstruction in skull injury [
From January 2000 to 2011, 10 patients presented to the Hangang Sacred Heart Hospital with a burned scalp and an exposed skull after sustaining an electrical burn injury.
Rectangular type expanders (Nagosil, Nagor Ltd., Glasgow, UK) were used. The injection port of the expander was replaced with a mini dome valve prior to use. The air within the expander was removed completely, and the four corners of the expander were folded gently to prevent perforation of the flaps by the expander. The horizontal and vertical sides of the space where the expander would be inserted were larger than the entire area by approximately 2 to 3 cm when compressed without sharply folding the four corners of the tissue expander. These sides were drawn in order to prevent the folding of the tissue expander during its insertion. Upon the insertion of the tissue expander, an incision was made at the boundary of the skull's injured area. The length was approximately half of the compressed tissue expander. Through this incision, a small pouch was made by subgaleal dissection; a silicone tube was placed by preparing a tunnel that penetrated the skin area away from the tissue expander. Hemostasis was performed on bleeding areas by electrocautery. The absence of bleeding was verified by washing with saline; a tissue expander was inserted into the prepared pouch. Prior to the insertion, leakage tests were performed using saline, and the air within the tissue expander was removed. After the insertion, the expander was inflated with 20 to 30 mL saline up to the volumein which the tissue expander unfolded, a drain was installed, and a two-layer suture was performed. After surgery, a Hemovac (Zimmer Inc., Warsaw, IN, USA) was used to prevent hematoma, and it was maintained from 5 to 7 days after surgery. Starting 2 weeks after the insertion, 5 to 10 mL saline was injected all at once, 4 to 5 times a week at our outpatient clinic for approximately 9 to 11 weeks while assessing the development of side effects (e.g., flares in the flap, paleness, necrosis). During the expansion period, constant skull coverage was maintained with foam dressing materials such as Mepilex (Mölnycke Health Care AB, Goteborg, Sweden) and an antibiotic ointment such as Bactroban (GlaxoSmithKline, Middlesex, United Kingdom) or Teramycin (PT Pfizer Inc., New York, NY, USA) to prevent skull desiccation or the development of osteomyelitis. In addition, frequent and active wound treatment was performed by wound dressing and irrigation with normal saline 3 to 4 times a week. Preventive antibiotics were prescribed during the expansion period. When the expansion reached the desired level, the tissue expander was removed. Granulation tissue and necrotic tissue were removed and the injured skull was resected or burred by a diamond burr (Asculap Inc., Tuttlingen, Germany). The affected area was covered with a displacement, advancement, or rotation scalp flap. In the initial period, after the removal of injured skull tissue, costal cartilage or adjacent bone tissue was divided into two and grafted to the skull defect. In 7 cases which were done recently, the injured skull was preserved and covered by a scalp flap directly. By preserving the injured skull and covering it with expanded vascular-rich scalp tissue, the burned skull was successfully primarily reconstructed in all 7 cases.
The sex of the patients, their age, injured sites, the type and number of tissue expanders used, the duration of the expansion period, expansion volume, surgery outcomes, complications, and the length of the follow-up period are shown in
Out of 10 patients, 8 were male. The mean age of the patients was 49.9±12.2 years (
A 47-year-old male patient was admitted due to a scalp injury (10×15 cm) caused by an electric burn. Two tissue expanders (400 mL and 500 mL) were inserted. For 63 days, saline (up to 950 mL) was injected, while necrotic soft tissue and necrotic bone tissue were removed. A bone graft was performed by using a bihalved rib, followed by covering the area with an expanded flap by advancing and rotating. In the 3D CT performed 1 year after surgery, well-engrafted bones were detected. In the follow-up observation 10 years after surgery, satisfactory results were observed in the volume and direction of hair growth (
A 49-year-old male patient sustained a scalp injury (13×12 cm) caused by an electric burn. An incision was made along the parietal area of the scalp injury where 1 tissue expander (500 mL) was inserted. After surgery, 615 mL of saline was injected for 70 days. After the removal of necrotic tissues, injured bone was covered by advancing a scalp flap. One year after surgery the scalp showed natural bone healing and remodeling. After surgery, osteomyelitis was not observed during a 5-year follow-up period (
Numerous methods have been introduced for the reconstruction of the burned skull. For small lesions (<3 cm), primary closure can be effected [
Most third or fourth degree burns that could induce skull injury are primarily electric burns caused by a current higher than 22,000 volts. Even in an electric burn injury, the extremities such as the hand and foot are the areas most commonly injured; cases with skull injury are rare. Hence, during the 10-year follow-up observation period, only 10 cases could be examined. Thus, it is necessary to obtain more clinical research results by securing more cases.
Reconstruction was successfully performed on 10 patients with burned skulls by using a tissue expander in the acute stage. Even in cases of an exposed skull and when the occurrence of osteomyelitis was suspected, by combining aggressive treatment and the prevention of osteomyelitis with tissue expansion surgery, the burned skull could be reconstructed successfully. During that time, efforts should be made to rapidly expand tissue and treat the wound. Through such efforts, the burned skull could be successfully reconstructed by a one-stage tissue expansion surgery with acceptable complications and a good long-term outcome.
No potential conflict of interest relevant to this article was reported.
Tissue expansion with bone graft
(A) Full expanded state of two expanders. (B) Harvested and bihalved rib bone. (C) Debridement including dead cortical bone. (D) Rib graft to skull defect. (E) Immediately after expander removal and local flap operation. (F) 3D CT image postoperative 1 year. The grafted bone is visibly viable. (G) After 10 years of follow-up. Good hair volume and direction.
Tissue expansion without bone graft
(A) Before operation, 13×12 cm skull defect. (B) After one tissue expander insertion. (C) After full expansion. (D) Immediately after expander removal and local flap. (E) Bone scan postoperative 1 year. No evidence of osteomyelitis. (F) After 5 years follow-up. Good hair volume and direction.
Case summary
RCT, rectangular.
In the extremities of premature infants, the skin and subcutaneous tissue are very pliable due to immaturity and have a greater degree of skin laxity and mobility. Thus, we can expect wounds to heal rapidly by wound contraction. This study investigates wound healing of full-thickness defects in premature infant extremities.
The study consisted of 13 premature infants who had a total of 14 cases of full-thickness skin defects of the extremities due to extravasation after total parenteral nutrition. The wound was managed with intensive moist dressings with antibiotic and anti-inflammatory agents. After wound closure, moisturization and mild compression were performed.
Most of the full-thickness defects in the premature infants were closed by wound contraction without granulation tissue formation on the wound bed. The defects resulted in 3 pinpoint scars, 9 linear scars, and 2 round hypertrophic scars. The wounds with less granulation tissue were healed by contraction and resulted in linear scars parallel to the relaxed skin tension line. The wounds with more granulation tissue resulted in round scars. There was mild contracture without functional abnormality in 3 cases with a defect over two thirds of the longitudinal length of the dorsum of the hand or foot. The patients' parents were satisfied with the outcomes in 12 of 14 cases.
Full-thickness skin defects in premature infants typically heal by wound contraction with minimal granulation tissue and scar formation probably due to excellent skin mobility.
Premature infants are live-born babies delivered before 37 complete weeks of gestation [
The current study consisted of 13 premature infants (14 cases) with full-thickness skin defects due to extravasation of total parenteral nutrition (
Local and systemic treatments were performed after the infants were referred from the Department of Pediatrics. For local treatment, conservative management was performed and the defects healed by secondary intention. After cleansing the wound with normal saline, a sufficient amount of an antibiotic and anti-inflammatory ointment mixture was topically applied to the whole region of the wound site, followed by a saline-moistened gauze dressing. This treatment was performed twice a day during the acute phase and once a day during the convalescent phase. The necrotic tissue was removed when it was clearly demarcated. For systemic treatment, we administered prophylactic antibiotics during the acute stage and ascorbic acid mixed with fluids at a daily dose of 50 to 120 mg as the antioxidants. After wound closure, topical oil moisturization and mild compression were applied until the scars were stabilized.
We reviewed the size of the defect, duration of closure, healing pattern, and shape of the scar from the medical records and photographs. At a final follow-up visit, we evaluated the degree of the parents' satisfaction with the treatment outcomes by showing photographs of the initial defects and the final scar. The degree of satisfaction was graded at one of four levels: excellent, good, fair, and poor.
Among all of the 14 cases of full-thickness defects in the 13 premature infants, the defects were present on the hand or the wrist in 5 cases, on the foot or the ankle in 8 cases, and on the elbow in 1 case. The size of the defects ranged from 0.5 cm to three-fourths of the longitudinal length of the dorsum of the hand or foot. The defects had completely closed 14 to 55 days after injury. The follow-up periods ranged from 3 to 41 months (
The vast majority of the defects closed by wound contraction. The direction of wound contraction was usually parallel to the longitudinal axis of the extremity, that is, vertical to the relaxed skin tension line (RSTL). The geometric shape of the defect gradually changed to elliptical. The shapes of the scars were pinpoint in 3 cases, linear in 9 cases, and round in 2 cases.
The defects smaller than 1 cm2 healed by wound contraction without granulation tissue formation and resulted in pinpoint scars (
Two cases resulted in a round scar (
Mild contracture due to wound contraction was observed in 2 cases with linear scars on the dorsum of the hand and foot (
The degree of the parents' satisfaction was excellent in 9 cases of pinpoint scars and linear scars, good in 3 cases of depressed or mild contracted linear scars, fair in 2 cases of round hypertrophic scars, and poor in 0 cases.
Cutaneous closed wounds heal without scar formation in the early gestational fetus [
Premature infants, delivered before 37 completed weeks of gestation, have premature skin (red, thin, smooth, shiny, and wrinkled with lanugo), less subcutaneous fat, and lower muscle tone than a full-term infant [
In the current study, wound healing of the full-thickness skin defects due to extravasation injury in the premature infant extremities usually seemed to have several characteristics of less inflammatory response, less granulation tissue formation, maximum wound contraction, minimal epithelialization, and consequently minimal scar formation.
Wound contraction is a process in which the surrounding skin is pulled circumferentially toward an open wound [
In premature infants, however, the skin is still thin, reddish, and wrinkled with a small amount of subcutaneous fat due to immaturity [
Inflammation and granulation tissue formation play key roles in adult wound healing, but not in that of an early fetus [
In the current study, most of the full-thickness skin defects in the premature infants, who received both initial and continuous wound management, had no or minimal inflammation or granulation tissue formation, which resulted in pinpoint or linear scars. The minimally formed granulation tissue did not show a beefy-red appearance but was slightly protruded with a shiny surface despite of no bacterial growth in the wound culture. It was also not adhered to the marginal skin until wound closure. In contrast, there was an inflammatory response around the wound in the case of delayed initial treatment or discontinued wound management. Consequently, granulation tissue increased, gradually turned to a beefy-red appearance, and adhered to the wound margin during wound contraction. After epithelialization, this led to the appearance of a hypertrophic scar. Thus, we guessed that more granulation tissue, especially in a beefy-red appearance, probably inhibits wound contraction due to marginal adhesion and may result in an unsatisfactory round scar formation. In other words, reduction of the inflammatory response might play an important role in inhibiting granulation tissue and scar formation similar to fetal wound healing.
Local wound management in premature infants optimizes healing by maintaining a physiologic local wound environment, which is characterized by adequate moisture, normal body temperature, bacterial balance, and neutral to mildly acidic pH [
The total number of scars was inversely related to the gestational age and directly related to the duration of intensive care due to the significant lesions caused by the multiple invasive procedures for their survival [
This study had limitations, including the lack of an animal model research basisand a quantitative biochemical study. The quantitative extent of wound contraction in premature infants compared with those of adults remains somewhat unclear. Therefore, more studies are needed to reveal more precisely the wound healing process of premature infants.
This article was presented at the 1st Research and Reconstructive Forum on May 12-13, 2011 in Deajeon, Korea.
No potential conflict of interest relevant to this article was reported.
A case of a pinpoint scar
(A) Full-thickness skin defect 34 days after extravasation injury. (B) Eighteen months after treatment.
The cases of linear scars
(A) Extravasation on the left dorsum of the foot. A full-thickness skin defect was noted 18 days later. (B) Three months after treatment, there was a linear scar without contracture. (C) A full-thickness skin defect of three-fourths of the hand dorsum in longitudinal length. (D) Five months after treatment, a linear scar with mild contracture was observed, but no functional deformity remained.
A case of a round hypertrophic scar with mild contracture
(A) A full-thickness defect of two-thirds of the right hand dorsum in longitudinal length 18 days after extravasation injury. (B) Ten weeks after treatment.
Summary of full-thickness skin defects due to extravasation injury in 13 preterm infants
GP, gestational period; Rt, right; RH, round hypertrophic; contr, mild contracture; Lt, left; PP, pinpoint; L, linear.
a)The length between the wrist to the metacarpophalangeal joint; b)The length between the ankle to the metatarsophalangeal joint.
Many topical hemostatics are widely applied for bleeding control. They can be classified into two categories according to their mechanism of action on the clotting cascade in a biologically active or passive manner. Passive hemostatics include cellulose and gelatin. We performed an experimental study to compare the effect of passive hemostatics in wound healing by applying them to a rectus abdominis muscle defect of white mice.
Surgicel is a sterile absorbable knitted fabric prepared by the controlled oxidation of regenerated cellulose. Spongostan is an absorbable hemostatic gelatin sponge. In 30 mice, a 1×1 cm defect was created on the rectus abdominis muscle and the materials were applied in three ways: control group, cellulose (Surgicel) group, gelatin (Spongostan) group. For the histologic analysis, biopsies were performed at 3 and 28 days.
After 3 days, the cellulose group showed limited granulation formation with acute inflammatory reactions similar to the control group. At the 28th day, moderate amounts of granulation tissue formation was observed with milder inflammatory reactions than the control group. In the gelatin group, after 3 days, gelatin remnants were observed surrounded by severe inflammatory changes. After 28 days, the same quantity of gelatin remnants could be still observed.
This study suggests that cellulose is associated with minimal morbidity in wound healing, while the use of gelatin shows severe adverse tissue reactions with delayed wound healing. Consequently, cellulose is better than gelatin when considering wound healing.
Nowadays, many kinds of hemostatics are used in various surgical fields. Generally, in the choice of hemostatics, how quickly and how effectively it works are important factors. For this reason, many hemostatic studies have been performed to compare hemostatic effects
Thirty 8-week-old, 280 to 300 g, white male mice from the ICR (CrljBgi: CD1) strain living in the same conditions were used in the study. After a 1 week period of adjustment in the laboratory, the study was begun.
1) Cellulose (Surgicel, absorbable oxidized regenerated cellulose, Johnson & Johnson, Arlington, TX, USA). 2) Gelatin (Spongostan, absorbable hemostatic gelatin sponge, Johnson & Johnson).
40 mg/kg of pentobarbital sodium (Hanlim Pharmaceutical, Seoul, Korea) and 20 mg/kg of ketamine hydrochloride (Ketar) (Yuhan, Seoul, Korea) were injected into the intraperitoneal cavity of white mice for anesthesia. We removed the abdominal hair and used a 10% povidone-iodine and 70% alcohol solution for disinfection and then made an incision with a No.15 scalpel in the abdomen and made a 1×1 cm full layer muscle defect in the left rectus abdominis muscle by separating the subcutaneous fat (
30 white mice were randomly classified to 3 groups, each composed of 10 mice. Control group, no care was provided for the defects; cellulose group, the muscle defect site was treated with cellulose (Surgicel); gelatin group, the muscle defect site was treated with gelatin (Spongostan). The cellulose or gelatin was applied in almost the same volume to each animal. The abdominal wound was sutured with 6-0 nylon and dressing was done with 10% povidone-iodine and 70% alcohol solution. The mice were caged, one mouse in one cage, and observed for 28 days. During the experiment, feed (5L79, PMI Inc., St. Louis, MO, USA) and water was provided ad libitum.
On postoperative days 3 and 28, we selected 5 mice from each group and took tissue, including wound site. The tissue taken from each mouse was fixed with 10% formalin solution for more than 6 hours, and stained with hematoxylin and eosin stain. We observed any proliferation of granulation tissue, the degree of inflammation, and the presence or absence of foreign bodies, and we measured the degree of wound healing in each group by comparing it with the control group.
We compared the wound healing effect of each of the two hemostatics by observing any proliferation of granulation tissue, the degree of inflammation, and the presence or absence of giant cells and foreign bodies. Each groups showed similar histologic findings.
Three days after inducing the wounds, similarly to the control group, proliferation of granulation tissue was rarely found and acute inflammatory cells had infiltrated the tissue (
Twenty-eight days after inducing the wounds, we could not find cellulose debris. Compared to the control group, the proliferation of granulation tissue was more severe while the signs of inflammation were lower (
Three days after inducing wound, proliferation of granulation tissue is rarely found and focal inflammatory cell is infiltrated to tissue (
Twenty-eight days after inducing the wounds, the debris of gelatin was found, and giant cells and modified macrophages around the debris are found. Compared to the control group, there was less proliferation of granulation tissue and there was much more inflammation and a greater foreign body reaction (
Wound healing generally goes through an inflammation phase, epithelial phase, proliferation phase, and maturation phase [
When methods such as compression, suture, and electrocautery are not effective or cannot be performed for hemostasis in primary wound care, hemostatics can be helpful. Hemostatics can be classified into two types; one type including cellulose, gelatin, and collagen acts as a physical structure within which platelets can aggregate and stops bleeding indirectly; another type, such as thrombin, acts directly on the last step of the coagulation pathway [
Surgicel, a knitted fabric prepared by the controlled oxidation of regenerated cellulose, is a light/pale yellow color sterile gauze-type absorbable hemostatic. It is clinically effective as an adjuvant for capillary and venous hemorrhage in various fields of operation, and is absorbed without leaving any foreign body after being used for wound hemostasis in peeling, tissue biopsy, nail avulsion, and trauma [
Spongostan, composed of a gelatin ingredient, is a hemostatic in the form of an absorbant sponge. It has been reported that Spongostan is effective in hemostasis [
The results from our study showed that cellulose, compared to gelatin, does not hinder the wound healing process as much. However, to understand the clinical significance of this finding, factors such as the degree of granulation tissue proliferation and the degree of necrosis and inflammation should have been quantified to compare the wound healing effect.
This study compared the wound healing effect of cellulose and gelatin, agents known to be effective in bleeding control, with the control group by applying each of them to mice with muscle defects. The results showed that gelatin, compared to cellulose, greatly delayed the wound healing. Therefore, when hemostatics are needed, cellulose, instead of gelatin, is recommended, considering its effects on wound healing.
This article was supported by Wonkwang Institute of Clinical Medicine in 2011.
This article was presented at the 1st Research and Reconstructive Forum on May 12-13, 2011 in Deajeon, Korea.
No potential conflict of interest relevant to this article was reported.
Anatomical drawing of a wound-induced rat
A No.15 scalpel was used for incision at the abdomen and a 1×1 cm full layer muscle defect was made in the left rectus abdominis muscle. Cellulose or gelatin was then applied to each defect site. Each abdominal wound was sutured with 6-0 nylon and daily wound dressing was performed with a 10% povidone-iodine and 70% alcohol solution.
Three days after inducing the wound (H&E, ×200)
(A) Control group. (B) Cellulose group, acute inflammatory cells infiltrated the tissue (white arrow) similarly to the control group. (C) Gelatin group, focal inflammatory cells infiltrated (white arrow) the tissue.
Twenty-eight days after inducing the wound (H&E, ×200)
(A) Control group, exuberant granulation tissue formation can be recognized with infiltration of chronic inflammatory cells (white arrow). (B) Cellulose group, the wounded muscular bundles are well repaired with no granulation tissue formation (white arrow). (C) Gelatin group, the image shows gelatin remnants (vertical white arrow) and there is an extensive foreign-body reaction to the amorphous basophilic material (horizontal white arrow).
The evaluation of a breast after breast reconstruction depends on a surgeon's subjective criteria. We used computed tomography (CT) scans to obtain an objective evaluation of the postoperative results by measuring the breast volume of patients who had undergone breast reconstruction using pedicled transverse rectus abdominis myocutaneous (TRAM) flaps. This research will help in the objective postoperative evaluation of reconstructed breasts, and also in the preoperative flap size designs.
A total of 27 patients underwent breast reconstruction using pedicled TRAM flaps after mastectomy from September 2007 to July 2010. Of these, 10 patients who were followed up and underwent CT scans 2 or more times during the follow-up period were included in this study. We evaluated the change in breast volume over time using CT scans, and the interval breast volume change between CT scans.
All of the 10 patients' reconstructed breasts showed a volume decrease over time. The breast volume changes in the intervals between CT scans were as follows: 5.65% decrease between the first CT and second CT scan, 2.3% decrease between the second CT and third CT scan, (statistically significant) and 1.89% decrease between the third CT and forth CT scan. (not statistically significant).
This research shows the possibility of objectively evaluating the postoperative breast volume changes. The findings will be helpful in designing the size of TRAM flaps to use on defects after mastectomy. Based on these results, we should also closely observe the reconstructed breast volume for at least 2 years.
Breast reconstruction with flap surgery has fewer complications compared with breast reconstruction with artificial implants, such as implant displacement, infection, and capsular contracture [
Therefore, in the current study, postoperative breast volume changes were measured after breast reconstruction using transverse rectus abdominis myocutaneous (TRAM) flap. The results of these measurements should be helpful for deciding the flap size based on the defect after mastectomy and more objectively evaluating the postoperative results.
A total of 27 patients underwent immediate breast reconstructions using TRAM flaps after mastectomy from September 2007 to July 2010. The patients that were examined using positron emission tomography/computed tomography (PET/CT) or magnetic resonance imaging (MRI) were excluded, and the patients followed up using CT were included in the study. The patients who had received radiation therapy after mastectomy were also excluded.
CT scans were routinely performed for surveillance in the general surgery department to detect metastasis and recurrence of cancer. Therefore, CT scans did not need to be performed for our breast measurement study.
Consecutive CT scans were performed at 1 mm intervals, 1 mm thickness at 120 kV, and 170 mAs with a v64 channel multidetector computed tomography (MDCT) device (LightSpeed, GE Healthcare, Milwaukee, WI, USA) while the patients were lying supine, holding their breaths, and raising both hands over their heads. The breast volumes were examined using the Infinitt program (Infinitt Health Care, Seoul, Korea). We checked each section of the breast such as the section visible in a CT axial view, and changed each section into a three-dimensional image and measured the volume. The same surgeon checked the volume 3 times for each patient, and the median value was set as the breast volume.
When we measured the breast volume, we set the range of the breast from the posterior margin, anterior of the pectoralis major to the anterior margin, skin, and nipple-areola complex. On the axial view, we set the range of the breast from the starting part of the breast crest to the ending part of the breast crest. The medial side of the breast was the lateral border of the sternum, and the lateral side of the breast was the mid-axillary line (
We compared the changes in breast volume over time. In all 10 patients, the first CT scan was performed at about 9 months after breast reconstruction (average, 9.2 months), and the interval period between CT scans was about 1 year (average, 11.7 months).
We evaluated the breast volume change between each CT scan. We defined the interval volume change (VC) between the first CT and second CT scan as "VC1" (n=10), the volume change between the second CT and third CT as "VC2" (n=7), and the volume change between third CT and fourth CT as "VC3" (n=4) (
We evaluated the mean change of VC and VC in proportion to the breast volume in the earlier CT. We used the Wilcoxon signed-rank test for statistical verification.
All 10 patients underwent modified radical mastectomy in the general surgery department of our hospital. All of the patients underwent pedicled TRAM flap using the contralateral superior epigastric artery. All 10 patients were diagnosed with invasive ductal carcinoma. The patients were categorized according to stage as follows: stage I, 2 patients; stage IIa, 7 patients; and stage IIIa, 1 patient. All 10 patients were treated with the same regimen of chemotherapy and hormone therapy (
Of the 10 patients, the volume of the breast that had the lesion decreased on average over time (
The goal of breast reconstruction is for the breast that had the lesion to resemble the preoperative breast. For this, symmetry of both breasts needs to be satisfied. During the postoperative follow-up period, the evaluation criteria of the changes in the breast and symmetry of both breasts depends almost entirely on the surgeon's subjective criteria.
The measurement of breast volume is clearly necessary when performing breast reconstruction and mammoplasty. Using this measurement, we determined the size of the breast implant and the increase of the patient's postoperative satisfaction. Many methods are available to measure breast volume, but no standard method has yet been established; therefore, at our hospital, we depend on the surgeon's subjective decision when making preoperative plans or postoperative predictions.
In a prior study, Tegrmeier [
The method for measuring breast volume with an imaging device involves less discomfort for patients, and we can ignore the changes caused by the patient's position. CT is less expensive and more cost-effective than MRI. CT has been long used for follow-up evaluations on the recurrence of breast cancer after breast resection and metastasis of localized lymph node.
The measurement of volume using a CT scan was introduced previously in the diagnosis of a blow-out fracture. Lee et al. [
To satisfy the symmetry of both breasts following breast reconstruction, Stevenson and Goldstein [
Amir et al. [
Based on these results, surgeons and patients were more satisfied with the results obtained by applying an over-correction of the flap size when the design of the flap was made.
In this study, inter-observer bias was minimized because only one examiner measured all of the breast volumes. Intra-observer bias was also minimized by using a mean value of three separate measurements.
Measuring breast volume using CT scans also depends on the examiner's subjective judgment, and the inaccuracy of measurements can cause apparent differences between breast volumes. This method considered only the volume of breasts in evaluating symmetry and did not consider the shapes of the breasts.
This study has some limitations. Because only a few cases were examined, the results cannot be broadly generalized. Moreover, the follow-up periods were short and the radiation effect on the flap was ignored; there was also the risk of unnecessary radiation exposure. Therefore, this study's findings are limited to generalization to the following types of patients: patients who want immediate breast reconstruction after mastectomy, patients who have a high risk potential for local or distant metastasis, which should be observed using CT scanning during the follow-up period, and patients who do not need adjuvant radiation therapy after breast reconstruction.
However, measuring the volume of the breast using a CT scan is cost-effective and does not cause patient discomfort. Therefore, this method is an objective and effective method for measuring breast volume and can be very helpful in designing the size of TRAM flaps on the defect after mastectomy.
No potential conflict of interest relevant to this article was reported.
Author's method for measuring the breast volume
(A) The boundary of the breast is selected in each axial view section of computed tomography. (B) The anterior view of a 3-dimensional (3D) reconstructed image. (C) The basal view of a 3D reconstructed image. (D) The breast volume is measured.
This figure shows what we evaluated and what is VC
We defined "volume change (VC)" as interval volume change between computed tomography (CT) scans. In this study, VC was breast volume change proportional to breast volume in the former CT scan. TRAM, transverse rectus abdominis myocutaneous.
Volume change of the reconstructed breast using TRAM flap in patient 7
(A) Measurement value of the breast on the first CT. (B) Measurement value of the breast in the fourth CT, 3 years and 4 months later. This figure shows the volumetric decrease of the breast over time. TRAM, transverse rectus abdominis myocutaneous; CT, computed tomography.
This figure shows that breast volume changed in the interval between CT scans
The first (5.65%) and second (2.3%) breast volume changes are statistically significant. However, the third breast volume change (1.89%) is not statistically significant. These results mean that the reconstructed breast volume change after 3 years can be ignored; therefore, the volume of a reconstructed breast should be closely observed for at least 2 years. TRAM, transverse rectus abdominis myocutaneous; CT, computed tomography.
Clinical information of the study group
IDC, invasive ductal carcinoma; CTx, chemotherapy; HTx, hormone therapy; RTx, radiotherapy.
Volumetric changes of the reconstructed breasts using TRAM flap
Values are presented as mean±standard deviation.
TRAM, transverse rectus abdominis myocutaneous; CT, computed tomography; OP, operation.
Adult acute lymphoblastic leukemia (ALL) is one of the intractable diseases in hematological malignancies, showing about 30% long-term survival by intensive chemotherapies with high incidence of relapse. Allogeneic hematopoietic stem cell transplantation (allogeneic HSCT) has been therefore applied to many such patients, showing better responses than chemotherapy and autologous HSCT, although there were some controversial results regarding a timing of allogeneic HSCT, especially in standard-risk ALL patients. In general, a better outcome (about 40% to 60% long-term survival) is obtained when adult high-risk patients in first complete remission (CR1) or second complete remission (CR2) received allogeneic HSCT using conventional myeloablative conditioning (MAC) regimens.Long-termsurvival decreased depending on the remission status; namely, about 20-40% in CR2, 10-20% in ≧CR3, and about 10%in non-CR. The most common MAC regimens for allogeneic HSCT in ALL patients are 120 mg/kg cyclophosphamide (CY) plus 12 to 13.2 gray (Gy) fractionated total body irradiation (TBI). By these conditioning regimens, the 3-year overall survival (OS) rate of about 50% was obtained when it was performed in CR1.In fact, when we analyzedthedata for 515 patients aged 15 to 59 years who received allogeneic HSCT in CR1 or CR2 with CY/TBI regimen between 1993 and 2007 from the Japan Society for Hematopoietic Cell Transplantation data base and the Japan Marrow Donor Program data base, the 5-year OS rate was 55.2%. In contrast, we have used medium-dose VP-16 in addition to conventional CY/TBI regimen to eradicate minimal residual disease. This regimen showed an excellent outcome in adult ALL patients transplanted in CR1, resulting in the 3-year OS rate of 89.2% without increasing the 3-year relapse rate (8.1%) and transplant-related mortality (TRM) rate (5.4%). Reduced-intensity conditioning (RIC) regimens are also applied for adult ALL patients with favorable outcomes (the 2- to 3- year OS rate: 30% to 60%); however, the 2- to 3-year relapse rate (20% to 40%) and TRM rate (20% to 30%) are still high. Therefore, a suitable conditioning regimen in allogeneic HSCT for adult ALL patients aged younger 50 years old in CR1 appears to be medium-dose VP/CY/TBI, while RIC regimens are preferable for patients aged over 50 years old or younger patients with some complications.
Haploidenticalfamily donor is an alternative for whom indicated for allogeneic hematopoietic stem cell transplantation (allo-HSCT). Beneficially that is readily available in comparison to matched unrelated donor (MUD) searching. We describe our experience with allo-HSCT in 21 adult patients (age: 16-61 y/o median 26) with high-risk hematological diseases. Among them, 18 patients (86%) with acute leukemia, 1 lymphoma and 2 severe aplastic anemia (SAA).Nineteen (90%) were parental donors and 2 filial donors. Fourteen (67%) were HLA-DR identical. Hematopoietic stem cells were mobilized by G-CSF and collected peripherally, which was not manipulated in vitro. Among 19 patients with hematological malignancies, 15 (79%) received myeloablative conditioning and 4 reduced intensity conditioning, but they all received ATG containingimmunoablation therapy before allo-HSCT. Post-transplantationalGvHD prophylaxis consisted of cyclosporine and MTX/MMF. All patients had successful trilineage engraftment. G-CSF was routinely given after transplantation. However, delayed graft failure developed in 2 SAA patients and they were successfully rescued by second allo-HSCT with the same donor. Three (14%) patients had Grade III/IV acute GvHD,while3 in 11 (27%) evaluable patients suffered from chronic GvHD. All patients hadcytomegalovirus (CMV) hyperantigenemia and 2 developed CMV peumonitis. Epstein-Barr virus (EBV) viremia was found in 10 patients, with onset time from Day 18 to Day 272. Among them, 4 patients developed post transplantation lymphoproliferative disorder (PTLD). Fourteen (67%) patients died post allo-HSCT. The cause of death included 6 (43%) relapse, 4 (29%) infection, 2 (14%) PTLD, 1 (7%) GvHD and 1 (7%) poor graft function. The overall survival (OS) for 19 patients with hematological malignancies at 1 and 2 yr was 49% and 42% respectively. That is similar in comparison with 62 high risk leukemia patients who received MUD allo-HSCT during the same period at the same BMT unit, their OS at 1 and 2 yr was 46% and 44% respectively.In conclusion, our results suggest that haploidenticalfamily donors are feasible alternatives for allo-HSCT in patients withhigh-risk hematological diseases under an intensive immunoablation conditioning regimen.
Hematopoietic stem cell transplantation (HSCT) has been applied in China for about 30 years, the number of HSCT has been increasing, donor and stem cell sources were expanded, indication of diseases and patients for HSCT extended.Up to now, 102 medical units nationwide have earned the certificate to perform HSCT. The annual increase rates were 8.8% to 10.8%. Matched sibling donor is 41%, mismatched related/haploidentical donor is 24%, unrelated volunteer donor is 16%, and umbilical cord blood is 2%. The indications of major disease entities are acute myeloid leukemia (AML, 35%), acute lymphobastic leukemia (ALL, 25%), chronic myeloid leukemia (CML, 21%), and myelodysplastic syndrome (MDS, 8%). The most encouraging outcome of related haploidentical HSCT in the world has been proved in the novel system without ex vivo T-depletion due to the contributions that Chinese doctors made.In contrast to CD34+ selected related haplotype transplants, Peking University developed an unmanipulatedhaploidentical transplantation with granulocyte colony-stimulating factor (G-CSF) primed marrow grafts and G-CSF mobilized peripheral blood grafts (G-PB) as stem cell source. Comparable incidence of graft-versus-host disease (GVHD), transplantation- related mortality (TRM) and the probability of survival were found among patients who received haplotype transplants and those from HLA identical sibling or unrelated HSCT. Consequently, the total number of related haploidentical HSCT is increasing during the past four years, and now accounts for about 30% of that of whole allogeneic HSCT. The Chinese Stem Cell Donor Database Management Center was established in 2001. As of August 31, 2010, there are 1 149 189 volunteers in the Chinese Marrow Donor Program (CMDP), with 1807 blood stem cell cases.
The present case report represents a successful attempt to induce transplantation tolerance to organ allograft by combined administration of donor hematopoietic cells and kidney based on in vivo deletion of alloreactive host-vs-graft and graft-vs-host alloreactive T cells following nonmyeloablative conditioning. We were able to induce mixed and eventually full donor chimerism and tolerance of kidney allograft in a 15-year-old male with ESRD after cisplatin treatment and autologous HSCT for mediastinal germ cell tumor. Our approach to induce tolerance was based on preferential depletion of alloreactive T cells induced by exposure to donor’s alloantigens and administration of cyclophosphamide at day 2 & day 3 after stem cells infusion. Additional non-specific immunosuppression as part of the conditioning included exposure to 2 fractions of TLI, treatment with alemtuzumab (monoclonal anti-CD52) and short-term conventional IS treatment to avoid early graft loss, due to request of IRB. Using this approach, with rapid tapering of all conventional IS treatment the patient maintains good renal functions without evidence of both acute and chronic rejection for 25 months off all medications.
Though the first Indian HSCT was done at Tata Memorial Hospital way back in 1983, even today HSCT programme in India is in its infancy. In the first 28 years only 4015 transplants have been done. In USA (population 307 million), 16,974 HSCTs were done in 2008. In comparison, in India with a population of 1.2 billion, 438 transplants were done in 2008. Presently, there are approximately 25 centers undertaking HSCT in India.
As opposed to majority of transplants done worldwide being autologous, 60% of transplants done in India are allogeneic. The main indications for allogeneic transplants were Thallasemia Major 26% & acute leukemias 33%; and for autologous transplants were multiple myeloma 50% & lymphomas 31%. Most of the allogeneic HSCTs in India are now done with PBSC as source of stem cells. However, as compared to developed countries, still most donors in India are HLA matched siblings. Very few haplo, MUD or cord blood transplants have been done. Major transplant centers like CMC, Vellore & TMH, Mumbai have started venturing in this field. However, the extremely high cost of such transplant is still a major source of hindrance.
Though we are improving by leaps and bounds, one of the major reasons for slow progress in the field of HSCT in India is the limited number of Tertiary Cancer Centers and shortage of trained personnel, both medical and paramedical, in this field. Cost is also a major issue. Though the cost of transplant in India is around USD $20,000 which is a fraction of the cost for such a transplant in USA, this is still beyond the reach of the majority of patientsin India, which has a per capita GDP of $3608 (as opposed $ 48,665 of USA & 34,646 of Japan). With no medical insurance system, most patients have to pay from their pockets.
Most of the SCTs done in India are done as part of routine “service”. Except at CMC Vellore, hardly any “research” is being undertaken. This also reflects in the fact that a pub med search for “HSCT & India” yielded only 111 publications. Also most published work is in the preliminary stage and it may take some time before the translational research reaches to the bedside. HSCT is by no means an ideal mode of therapy. What we need in the future is better way to decrease GVHD without affecting the GVL effect. More use of simpler cellular therapies like dendritic cells, mesenchymal stem cells, NK cells, immunomodulators etc. to achieve this and improve outcome of malignant conditions, and hopefully limit the use of HSCT only for non-malignant conditions!
Worldwide Network for Blood and Marrow Transplantation (WBMT) is planning the first global level workshop for the promotion of hematopoietic stem cell transplantation (HSCT) in developing countries under the collaboration with World Health Organization (WHO) in Hanoi, Vietnam, on Nov. 10-12, 2011. The preparatory meetings for this Vietnam Workshop 2011 were held in Phuket, Orland, Hawaii and Paris under the attendance of the representatives from Vietnam, WBMT (APBMT/EBMT/CIBMTR/ WMDA etc) and WHO. The goals of the workshop were discussed and were set as followings; 1) Create awareness among government policymakers about the value of hematopoietic stem cell (HSC) transplantation in developing healthcare systems, 2) Encourage the integration of HSC transplantation within the Healthcare Policy of developing countries, 3) Establish the basic ethical, medical and infrastructure requirements for providing HSC transplantation within a developing healthcare system, 4) Create a model for achieving goals 1) – 3) that can be replicated throughout the WHO regions of the world, 5) Optimize existing transplant programs. Through almost bi-weekly telephone conference, the actual constructions of the workshop have been finalized and those are the followings; 1) Establishing a Transplant Program, 2) Indication for Transplant and Patient Selection, 3) Donor Selection, 4) Graft Processing, 5) Developing an Outcomes Database, 6) Dissemination of Information, 7) Inspection and accreditation( AHCTA). At this moment, 60 attendees from foreign countries (guests from developing countries and speakers from experienced countries) and 100 domestic (Vietnam) attendees are expected. This trial is experimental but we expect that it would be a fruitful one not only for developing countries but also for advanced countries in the field of HSCT.
Some of patients with autoimmune disease (AD), havingprogressive skin sclerosis or interstitial pneumonia (IP) etc., are severely damaged or fatal from the treatment-resistant disease progression. To induce remission in such severe AD by eradicationof autoreactive lymphocytes andby immune reconstitution, we underwent a phase I-II trial to elucidate feasibility and efficacy of autologous hematopoietic stem cell transplantation (auto-HSCT) for severe AD. Nineteen patients with systemic sclerosis (SSc), 3 patients with dermatomyositis and a patient with Wegener’s granulomatosis (WG) were enrolled. Peripheral blood stem cells (PBSCs) were mobilized with cyclophosphamide (CY, 4 g/m2) and G-CSF. After collecting PBSCs more than 2x10^6CD34+cells/kg by apheresis, they were cryopreserved until autographting. CD34+ cells were immunologically selected in 11 SSc patients, 2 dermatomyositis patients and a WG patient just after apheresis. All of the patients were treated with high-dose CY (200 mg/kg) and received auto-HSCT. There was no treatment-related mortality. As toxicity, there were a variety of post-transplant infections such as adenoviral hemorrhagic cystitis, herpes zoster and cytomegaloviral antigenemia. In patients with SSc, skin sclerosis was markedly improved in all of the patients within 6 months and the improvement was sustained for60 months after auto-HSCT. Vital capacity was significantly increasedat 48 months after HSCT and KL-6, a marker for IP, was significantly decreased during 12-60 months after HSCT. A titer of anti-Scl-70 was significantly decreased during 1-60 months after HSCT. Progression-free and overall 5-year survivals were 66% and 93%, respectively. In patients with dermatomyositis, progressive IP and skin ulcers were dramatically improved after auto-HSCT. In a patient with WG, the size of the left orbital granuloma decreased. To study the mechanism for durable effects, we analyzed the immune reconstitution after HSCT in 11 SSc patients with CD34+ selection. The number of CD8+ T cells recovered as fast as a month, in contrast, that of CD4+ T cells was severely suppressedfor 60 months after HSCT. The number of CD4+CD45RO+ memory T cells recovered earlier than those of CD4+CD45RA+ naïve T cells. The number of B cells recovered to the baseline at 12 months after HSCT. Of note, CD4+ Th1 cells became predominant, and Th1/Th2 ratio was significantly increased during 1-60 months after HSCT. CD34-selected auto-HSCT was more effective on skin sclerosis and was more strongly associated with viral infection than unmanipulated auto-HSCT. In conclusion, auto-HSCT is feasible and effective in the treatment of severe AD.
Mesenchymal stem cells (MSCs) are multipotent progenitor cells that have emerged as a promising therapeutic modality for tissue regeneration and repair. The interest in MSC therapy has been further raised by the observation that MSCs are able to modulate immune responses in vitro and in vivo. These properties may be used in clinical therapy in the context of allogeneic stem cell transplantation or treatment of auto-immune disorders. MSCs are known to secrete a number of cytokines and regulatory molecules implicated in regulation of hematopoiesis. These characteristics have generated clinical interest to use MSCs to enhance hematopoietic stem cell engraftment. MSCs suppress the proliferation of T cells induced by alloantigens or mitogens and MSCs have been reported to induce T cell division arrest, to inhibit the differentiation and maturation of dendritic cells, and to decrease the production of inflammatory cytokines. Allogeneic MSCs may prolong skin allograft survival in immunocompetent baboonsand may prevent the rejection of allogeneic tumor cells in immunocompetent mice. The mechanisms underlying these effects of MSCs have not been clearly identified.
MSC therapy is associated with several potential risks, including immunogenicity, malignant transformation and ectopic tissue formation. Although initial publication suggested the possibility of human MSCs in culture to transform into malignant cells, recent evidence indicates that such transformation in-vitro is an extremely rare event. Prolonged expansion of MSCs in-vitro does not result in emergence of independent clones and cytogenetic analysis or comparative genomic hybridisation studies do not reveal the structural abnormalities. Altogether, the experimental and clinical data do not support the possible malignant transformation as a result of prolonged expansion in culture.
In pilot studies, encouraging results have been reported for patients with graft rejection after haplo-identical stem cell transplantation and steroid resistant graft-versus-host disease (GVHD), treated with bone marrow-derived and ex-vivo expanded MSCs. A majority of the patients (70%) showed a clinical response on MSC treatment and this response was associated with improved survival (Le Blanc et al. 2008). A recent follow-up study performed in children transplanted in Pavia (Italy) and Leiden (the Netherlands) suggest that the interval between onset of GHVD and treatment with MSCs may be an important variable determining response to MSC treatment. Early MSC treatment after the diagnosis or steroid refractory acute GVHD was associated with a higher probability of obtaining complete remission. These preliminary data may indicate that timing of MSC treatment is a critical determinant of efficacy and outcome.
Bone marrow transplantation is accepted as an effective therapeutic modality for genetic and acquired diseases of the hematopoietic system. This cellular therapy is founded on the principle that bone marrow contains hematopoietic stem cells that can repopulate the blood. Over the past two decades, we have recognized that bone marrow also contains mesenchymal stem/progenitor cells. In principle, then, marrow transplantation as a means of transplanting mesenchymal cells could be used to treat disorders of mesenchymal tissues, in an analogous fashion to hematopoietic cell therapy. Using osteogenesis imperfecta (OI), a genetic disorder of bone, as a model system, we demonstrated that transplantation of unmanipulated bone marrow could functionally engraft in recipient bone and provide measurable clinical benefits. Based on the notion that mesenchymal stromal cells (MSCs) generated the donor-derived osteopoiesis, we subsequently intravenously infused allogeneic, gene-marked MSCs obtained frommarrow donors. Measured engraftment of the genemarked MSCs in bone wasexceedingly low; nonetheless, it was associated with an acceleration of growth in the children. Animal models of osteopoietic engraftment and differentiation suggest that nonadherent marrow cells (NAMBCs) are superior to MSCs forosteopoietic engraftment and differentiation to osteoblasts as systemic cell therapy of bone. Moreover, the mechanism of osteopoietic engraftment may be an important factor governing the durability of the osteopoietic graft. This animal data led to our third clinical trial which unambiguously demonstrated that marrow mononuclear cell “boosts” in our OI children can lead to osteopoietic engraftmentand striking clinical benefits in a subset of patients suggesting potent mesenchymal progenitor activity. Moreover, these data suggest that the therapeutic benefit of marrow mononuclear cell therapymay be collagen-mutation dependent. In an animal model of OI, we demonstrate that the NABMCs engraft and differentiate to osteoblasts contributing normal collagen to the OI bone matrix, consistent with our clinical data suggesting a collagen-mutationdependent mechanism. Additional murine data suggests that the cytokine therapy may be able to induce osteopoietic differentiation of donor marrow cells after BMT. Our clinical trials and murine models collectively suggest that bone marrow cell therapy is an effective strategy for the treatment of OI and possible other disorders of bone. The development of broadly applicable marrow cell therapy for bone will depend, in part, on better knowledge of the transplantation biology of marrow cells and osteopoietic differentiation, which will be most effectively uncovered by both innovative laboratory research as well as scientifically-based clinical trials.
Human gamma-delta T cells (γ δ T cells), which are subgroups of peripheral blood T cells comprising less than 10% of peripheral blood mononuclear cells (PBM), are known to defend the body against infection. Seventy percent of peripheral blood γ δ T cells express Vγ2 and Vδ2 from among the variable elements of T cell receptors (Vγ2Vδ2 T cells) and these Vγ2Vδ2 T cells are activated in a TCR-dependent manner by several small phosphorylatedor aminated alkyl molecules. We have synthesized more than 60 kinds of monoethyl-pyrophosphate derivatives and 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP) which exhibit potent stimulation of γ δ T. cells more than 100-fold that of isopentenyl pyrophosphate (IPP) which is one of the natural ligands of Vγ2Vδ2 T cells.We also generated in vitro culture systems of Vγ2Vδ2 T cells, and these cultured Vγ2Vδ2 T cells showed a strong anti-tumor effect.
Many tumor cells up-regulate the mevalonate-pathway. IPP which is one of the intermediate metabolisms of the pathway accumulates in tumor cells and Vγ2Vδ2 T cells and recognizes IPP of the TCR and exhibitscytotoxity. Nitrogen containing bisphosphonates, such as zoledronic acid (Zol), inhibit Farnesyl-pyrophosphate (FPP) synthetase which is one of the important enzymes in the mevalonate pathway. Inhibition of FPP synthetase resulted in accumulation of IPP in the tumor cells which Vγ2Vδ2 T. cells recognize and then kill them.
We conducted a phase I/IIaclinical trial of adoptive transfer of V□2V□2□ T cells to patients with advanced renal cell carcinoma. Eleven patients who had undergone nephrectomy and had lung metastasis were enrolled. Peripheral blood Vγ2Vδ2 T cells obtained from the patients were stimulated
Adoptive immunotherapyadministrating autologous or allogeneic
Among lots of effector cells for cancer treatment, cytokine-induced killer (CIK)cells are shown to be a heterogeneous population, and themajor population expresses both the T cell marker CD3 andthe NK cell marker CD56. Also, CIK cells have potent cytolytic activity and exhibits non major histocompatibility complex(MHC) restricted lysis of target tumor cells.
Our products, Immuncell-LCoR which is derived from peripheral blood mononuclear cells and cultured under well defined culture conditions for 2-3 week, has characteristics bearing cytotoxic T cells(CTL) and CIK cells. We have recently reported that Immuncell-LC could suppress the growth ofa variety of solid tumors including HCC tumor in animals or
Today, I will present introduction of Immuncell-LCoR for adoptive immunotherapy including our preclinical results and clinical design and our developments to improve immunotherapy efficacy for cancer.
A novel method of expansion and activation of natural killer (NK) cells for clinical application under good manufacturing practice (GMP) compliance was established. Purified NK cells from 100 mL of peripheral blood were stimulated with interleukin-2 (IL-2) and anti-CD3 antibody (OKT-3) in a closed culture bag system for 14 days. More than 1x109 of highly pure population of CD16+/CD56+ NK cells were obtained while contaminants such as T cells, B cells, or monocytes were analyzed by less than 0.5 percent.
Expanded NK cells showed potent efficacy on killing tumor cells. In response to target cells, effector cytokines such as IFN-□ and TNF-□ were efficiently secreted 10-fold more compared to resting NK cells. Adhesion molecules, DNAM-1; activating receptors, NKG2D; and natural cytotoxicity receptors (NCRs), NKp30, NKp44, and NKp46 were up-regulated by
For non-clinical testing for toxicity and kinetics, human NK cells were adoptively transferred into SCID mice via tail vein. The administrated NK cells were mainly found in the lung, liver, and spleen for 48 hours and a few were circulated in the blood stream. One week after transfer, human NK cells were hardly detected by immunohistochemistry, flow cytometry or PCR. There were no reports concerning any significant adverse effects of NK cell transfusion.
In conclusion, an innovative process of large-scale expansion of NK cells with up-regulated expression of cytotoxic effector molecules was developed. These
“This study was supported by a grant of the Korea Healthcare technology R&D Project, Ministry of Health and Welfare, Republic of Korea. (A062260).”
Metastatic colorectal cancer remains lethal in the majority of patients despite effective chemotherapy and monoclonal antibodies, underscoring the need for additional therapies. Infiltration of tumor by T cells has been associated with longer survival of colorectal cancer patients in numerous studies. This finding leads to the hypothesis that adaptive immunity is capable ofcontrolling colorectal cancers and that activation of T cell responses against colorectal cancers (using active specific immunotherapy or “therapeutic cancer vaccines”) results in clinical benefit. Indeed, various vaccination strategies have been reported to increase T cell infiltration into murine and human tumors including colorectal cancer. We have performed preclinical studies and NCI-sponsored clinical trials demonstrating that autologous DC modified with viral vectors encoding tumor antigens induce more potent T cell responses than DC loaded with peptides or conventional mRNA. We developed a broadly applicable and clinically feasible strategy to modify autologous DC using a new generation of recombinant pox vectors encoding a tumor antigen and TRICOM (triad of co-stimulatory molecules B7.1, ICAM-1, LFA-3). Early clinical studies demonstrated safety and immunogenicity. We have completed an NCI-funded, multicenter, randomized phase II clinical trial of heterologous prime-boost vaccination with recombinant pox vectors encoding CEA and MUC-1 plus injection site doses of GM-CSF versus heterologous prime-boost vaccination with autologous DC loaded ex vivo with pox vectors for patients with resected hepatic and pulmonary metastases of CRC (n=74). The purpose of the study was to “pick a winner” between either the DC loaded with the poxvectors or the poxvectors given with GM-CSF to choose the strategy to be used in a phase III trial. A preplanned endpoint was to determine overall survival for the vaccine strategies and also, to compare this survival with that of a contemporary control group of similar patients who were not vaccinated. We demonstrated improved overall survival in patients with metastatic CRC vaccinated against carcinoembronic antigen (CEA) and MUC1 compared to non-vaccinated controls. Notably, this strategy was effective in the setting of minimal tumor burden following complete metastasectomy. Unfortunately, the majority of patients are unsuitable for complete metastasectomy and will not only have detectable tumor, but also suffer from significant immunologic dysfunction, partly characterized by significant numbers of regulatory T cells (Tregs). Consequently, we hypothesize that patients will benefit from cancer immunotherapy in the setting of minimal tumor burden, or in combination with immunomodulatory strategies that circumvent the immunologic dysfunction.
Sipuleucel-T is an autologous active cellular immunotherapy designed to stimulate an immune response to prostate cancer. It is FDA approved for the treatment of men with asymptomatic or minimally symptomatic castrate resistant prostate cancer, based on prolongation of overall survival. Sipuleucel-T is manufactured by culturing a patient’s peripheral blood mononuclear cells, including antigen presenting cells, with a recombinant protein comprising a tumor-associated antigen (prostatic acid phosphatase) and granulocyte-macrophage colony stimulating factor. Treatment consists of 3 infusions at approximately 2-week intervals, resulting in a prime-boost pattern of immune activation, with a robust antigen-specific cellular and humoral immune response. Specifically, antigen presenting cells are activated after culture with the recombinant protein, as evidenced by an increase in CD54 expression in the first dose at Week 0, that is further upregulated in subsequent doses at Week 2 and Week 4. Activated APC-associated cytokines and chemokines are produced in the media during manufacture of sipuleucel-T, and activated T-cell-associated cytokines are detected in the media of the second and third products. Antigenspecific T-cell proliferative and IFN gamma ELISPOT memory responses are evident after administration of the first sipuleucel-T dose. There are correlations between product parameters as well as peripheral immune responses and overall survival. .Adverse events are generally mild to moderate and resolve within 2 days. As the first autologous cellular immunotherapy to demonstrate a survival benefit, sipuleucel-T is an important new treatment for men with asymptomatic or minimally symptomatic castrate resistant prostate cancer.
In recent publications, human proof of concept for Chimeric Antigen Receptor (CAR) engineered T cells was demonstrated in a pilot study with CLL patients.[1,2] Three patients were treated with expanded autologous T cells virally-transfected with a CAR molecule that is specific for recognition of the CD19, a molecule expressed solely on B-cells and on leukemia / lymphoma cells. Twelve months subsequent to treatment, two of the patients exhibit Complete Response and one patient exhibits Partial Response with >90% reduction in tumor burden.
Recognizing the safety and toxicity of virus-mediated delivery of CAR, MaxCyte has developed an alternative approach to introducing the CAR as an messenger RNA (mRNA) molecule and using MaxCyte cell loading technology platform to enable a robust, cGMP compliant, method of manufacturing for clinical / commercial delivery of CAR-engineered T- and NK-cells.
In collaborations with investigators at the University of Pennsylvania (Philadelphia, PA), using CD19 targeted CAR mRNA loading into ex vivo expanded T cells, it has been demonstrated that the in vitro and in vivo antitumor activity of α-CD19 CAR mRNA loaded T cells is equivalent to that of T cells transfected with α-CD19 CAR encoded by lentiviral vectors.[3] Similar pre-clinical studies using α-Mesothelin CAR mRNA loaded T cells demonstrate safety and anti-tumor efficacy directed against tumors that specifically over-express Mesothelin;[4] a tumorassociated-antigen preferentially expressed on multiple solid tumors including mesothelioma, ovarian cancer, pancreatic cancer and GI cancers. The safety and therapeutic activity of α-. Mesothelin CAR mRNA loaded T cells is under investigation in human clinical studies at the University of Pennsylvania
In parallel collaborations with investigators at the St Jude Childrens Research Hospital (Memphis, TN), using CD19 targeted CAR mRNA loading into ex vivo expanded NK cells, it has been demonstrated that the in vitro and in vivo anti-tumor activity of α-CD19 CAR mRNA loaded NK cells is equivalent to that of T cells transfected with α-CD19 CAR encoded by retroviral vectors.[5] These pre-clinical studies form the basis for filing of an IND application with the US FDA, for initiation of human clinical trials to evaluate the safety and therapeutic activity of α-CD19 CAR mRNA loaded NK cells for treatment of AML in pediatric population.
The presentation will provide a summary of translational experiences leading to the IND filings for Chimeric Antigen Receptor (CAR) messenger RNA (mRNA) loaded T- and NK-cell immunotherapy
Mesenchymal stromal cells (MSCs), frequently termed mesenchymal stem cells, are one of the most commonly investigated cells in cell therapy. We reported the first clinical trial of allogeneic MSC infusions in a study of MSC therapy for children with osteogenesis imperfecta (OI), a genetic disorder of collagen type I. The children showed an acute acceleration of growth following the cell therapy. Curiously, despite the striking clinical outcome, very low levels of donor MSCs were detected in the patients’ bone suggesting that the mechanism underlying the clinical effect of MSC therapy is unrelated to osteopoietic differentiation. We reasoned that since bone growth is initiated by chondrocytes in the growth plate (GP), MSCs may be directly acting at GP. However, in our murine model, MSCs do not engraft and differentiate to chondrocytes in the GP. Moreover, freshly isolated primary chondrocytes cocultured with MSCs do not proliferate greater than controls suggesting that MSCs do not secrete a soluble mediator that directly stimulates GP chondrocyte activity. We then considered that the growth may be the result of a complex metabolic pathway initiated by a MSC secreted soluble mediator. To test this hypothesis, we infused mice with MSCs and obtained serum at day 7. Chondrocytes assayed in media supplemented with serum taken from MSC-infused mice stimulated greater chondrocytes proliferation than controls (P<0.01). Analysis of proliferating cell nuclear antigen (PCNA) in the GP of mice infused with MSCs revealed significantly more proliferating cells compared to controls (P=0.006). To determine whether our findings were applicable to OI, we transplanted MSCs into OI mice. Four weeks after the transplantation, we observed a significantly increased lumbar vertebral length (P=0.0008) as well as total body weight (P=0.0025). Based on these findings, we hypothesized that MSC therapy may protect articular cartilage (AC) from developing osteoarthritis (OA) by stimulating the proliferation of chondrocytes in the AC. To examine this idea, we transplanted MSCs into a murine model of OA once per week for four weeks, beginning at the time of OA induction. The joints were then evaluated with the Osteoarthritis Research Society International (OARSI) scoring system. The joints treated with MSCs resulted in significantly a lower score than controls (P<0.0001) with proliferating chondrocytes evident in the AC. Our data indicate that the mechanism of both MSCstimulated linear growth and delayed development of OA is the secretion of a soluble factor that initiates a metabolic pathway resulting in chondrocyte proliferation in GP and AC.
Hepatocellular carcinoma (HCC) represents one of the major global health concerns affecting both developing and developed nations. Though currently hepatectomy and orthotopic liver transplantation could provide a potentially curative treatment for HCC, only a small proportion of patients will be eligible for these treatments since HCC is often detected at an advanced stage. Additionally, the observed cure rates for these treatments are elusive due to the high incidence of recurrence and salvage therapies for recurrent disease are not curative. Hence, the development of innovative therapeutic platforms for clinical applications is urgently needed. An emerging cancer therapeutic tool is the mesenchymal stem cell (MSC)-based therapy. MSC is a potentially good candidate due to its ability to target tumor cells and integrating into the stroma. Their easiness of expansion in culture is an additional advantage. Recently oncolytic measles viruses (MV) have been demonstrated to have potent oncolytic activity against several types of human cancers and their toxicity has also been reported to be minimal. Oncolytic MV targets CD46+ cells. MV uses the hemagglutinin envelope glycoprotein to infect cancer cells via the cellular CD46 receptor and the fusion envelope glycoprotein to trigger fusion of the viral cell membranes for virus entry. Expression of these fusogenic hemagglutinin and fusion proteins on surfaces of virus-infected cells results in massive intercellular fusion with uninfected neighbouring CD46+ to generate the characteristic MV-induced cytopathic effects of syncytia formation. We have demonstrated that most of the human HCC tested are CD46+. We are therefore exploiting the ability of the Edmonston vaccine lineage of MV as a CD46+-selective oncolytic agent for HCC cancer therapy. In this presentation, the homing ability of MSC was investigated in an orthotopic human HCC xenograft model. Results showed that systemic delivery of oncolytic MV-loaded human bone marrow (BM)- derived MSC preferentially home at tumor sites and the overexpression of CD46 on cell surfaces of human HCC cells resulted in the preferential killing of the tumor cells. Pre-existing antiviral antibodies in cancer patients can potentially neutralize the MV and decrease its antitumor potency. Hence, the ability of the BM-derived MSC to protect the “naked” MV viruses from antibody neutralization is also being investigated.
In Japan, cord blood is the first choice as an alternative donor of hematopoietic stem cell transplantation (HSCT) for high-risk leukemia patients, when appropriate donors (HLA-full match or 1 locus mismatch donors) are not available. However, there are several problems in cord blood transplantation (CBT), especially in early phase of transplantation, such as higher infection and relapse rate and late recovery of platelet, etc. Haplo-SCT is another alternative method and has been developed in many countries. The problem of naked haplo-SCT is severe graft– versus-host disease (GvHD) caused by uncontrolled allo-reactivity of donor T cells and that of T-cell depleted haplo- SCT is higher infection and higher relapse rate caused by delay of immune reconstitution (IR).
We are challenging T-cell depleted Haplo-SCT combined with HSV-TK gene modified T cell add-back therapy and we had experiences of the best-case scenario and the worst-case scenario in the first two cases.
The first case is a 47 y.o. female ALL patient and IR was achieved after three times T-cell “add-back”. GvHD appeared on the same day of IR and administration of GCV completely cured GvHD. This patient is alive without relapse or viral/fungal infection for 12 months. The second case is a 59 y.o. male AML patient and he died from viral infection before achievement of IR. These two cases suggested that add-back therapy with HSV-TK gene modified T cells is essential for successful Haplo-SCT, and HSV-TK/GCV system is useful to control GvHD caused by addbacked T cells.
I will show the detail of the cases and discuss the benefits of T-cell depleted Haplo-SCT combined with HSV-TK gene modified T cell add-back therapy.
Recently there are several big findings in the field of engineered T cell immunotherapy for cancers and viral infections using T cell receptor (TCR) and others. The Gene Medicine Unit of Takara Bio Inc. is focusing on the development of such engineered T cell immunotherapy for cancer and HIV infection. Our proprietary technologies are being used for these developments, including gene transduction method using RetroNectin, a recombinant human fibronectin fragment, siRNA technology for high expression of TCR genes in T cells, and our genes such as MazF, E. coli RNase. In the TCR gene therapy, we are now focusing on the use of MAGE-A4 or WT1 tumor antigen-specific, HLA-A24 restricted TCR. In Japan, we are doing and planning several clinical studies to treat cancer patients, in which TCR gene-modified autologous T cells are used for esophageal cancer or hematological malignancies, and HSV-TK (Herpes Simplex Virus thymidine kinase) gene-modified donor T cells are used for hematological malignancies. Also, we will perform Takara-sponsored clinical trials to use the gene-modified T cells as pharmaceuticals after testing for the quality and storing at a stable condition. In the future, we will go forward to perform such clinical trials to determine a better method to give better efficacy in the treatment of cancer patients in Asia including China and Korea.
T cells may be genetically modified to target tumor associated antigens through the retroviral introduction of genes encoding artificial T cells receptors, termed chimeric antigen receptors (CARs), specific to these targeted antigens. We have previously demonstrated in xenotransplant models of systemic human CD19+ B cell tumors in immunecompromised SCID-Beige mice, that human T cells engineered to express the 19-28z CAR specific to the B cell antigen CD19 can efficiently eradicate disease. Based on these findings we have initiated clinical trials treating patients with relapsed or refractory chronic lymphocytic leukemia (CLL) of B cell acute lymphoblastic leukemia (B-ALL). Meanwhile, we furthered out pre-clinical studies by generating a transgenic C57BL6(mCD19-/- hCD19+/- ) immune competent modified syngeneic T cell murine mouse model. Studies using this tumor model demonstrate requisite need for prior cyclophosphamide conditioning chemotherapy to allow for eradication of systemic EL4(hCD19) tumors and predicted normal B cell aplasias. Based on these findings we modified our clinical trials to include prior cyclophosphamide conditioning. Promising clinical trial results from our institution will be presented and compared to recently published clinical trial results from other institutions (the National Cancer Institute, and the University of Pennsylvania) similarly utilizing autologous T cells modified with CD19-targeted CARs. Finally, preclinical studies will be presented demonstrating enhanced efficacy of tumor targeted T cells additionally modified to express the IL-12 cytokine gene. These studies demonstrate potential enhanced anti-tumor efficacy in the absence of prior conditioning chemotherapy and may hold promise for a more robust anti-tumor effect in future clinical trials.
Several surgical approaches to repair cartilage defects have been reported such as reattachment of a detached osteochondral fragment to the lesion, microfracture, mosaicplasty and ACI. We treated eight cartilage defects with meniscal transplantation from 1990 to 1995. Then we started to perform transplantation of tissue-engineered cartilage made ex vivo for the treatment of osteochondral defects of the joints (110 cases) as a second generation of chondrocyte transplantation from 1996. Sixty knees who had received transplantation of tissue-engineered cartilage for cartilage defects were followed up for at least 5 years. Although the clinical results were satisfactory, we need the surgical approaches to treat large cartilage defects with less invasive technique.
One of the less invasive surgical procedures to treat large cartilage defects is microfarcture or drilling. However, these techniques under arthroscopy are not sufficient to repair cartilage defects with hyaline cartilage. I think that there are two weak points such as insufficient number of mesenchymal stem cells and early overloading on the treated area. I would like to show our novel approaches using an external magnetic field to deliver precisely injected cells with magnetic beads to an articular defect and articulated distraction device for reducing the load.
Corneal diseases are one of the major causes of total or partial blindness in developing countries. The number of patients registered for cadaver corneal transplantation in India alone is close to 100000 per year. According to the registries, 90% of the patients are unable to get a transplantation done, due to lack of donor corneas. When the data on the disease pattern of such patients was analyzed, nearly one-third had stand-alone endothelial disease, one-third epithelial disease and the remaining one-third only had a total damage of the cornea requiring a pan keratoplasty per se. Therefore we have come with solutions for addressing the corneal endothelial and epithelial diseases using novel nanomaterial based in vitro expanded corneal limbal stem cells and corneal endothelial precursors respectively.
Stem cells have two important properties that distinguish themselves from other types of cells; they can both proliferate without changing their phenotypes indefinitely, and they also can differentiate into one or more new kind of cells depending on the culture conditions. Thus, stem cell therapy could be most effective to treat the diseases that are marked by the loss of cells. In last decade, various types of stem cells have been identified from the preimplantation stage embryos, fetus, placenta, and adult tissues. Moreover, it is now almost a common practice to make induced pluripotent stem cells (iPS) from various adult somatic cells using only a few defined factors.
Recent advances in the stem cell biology, including the development of optimized cell type specific culture systems, and the broader understandings of biochemical and molecular signals involved in cell self-renewal and differentiation have made the cell based therapy closer to the practical uses. As of now, at least 180 adult stem cell therapies are being used or tested in real life in the forms of bone marrow stem cell, mesenchymal stem cell, cord blood stem cell, fetal brain stem cell and fat stem cell transplantations.
CHA Health Systems, Korea is developing feasible and applicable stem cell therapies for various clinical studies with leading research institutes in ACT Inc, USA. We are now extensively studying human embryonic stem cells (hES), neural stem cells, and various adult stem cells for medical applications. In addition, CHA Health Systems recently developed CHA-StationTM, an integrated cell separation system and established comprehensive human stem cell banks such as germ cell bank, fetus stem cell bank, embryo bank, and adult stem cell bank.
Human cord blood-derived Mesenchymal Stem Cells (hUCB-MSCs) are a robust source for not only giving rise to a new cell type in need but also for secreting various trophic factors which will signal the activation of host’s own stem cells for producing required cell types and drive the regenerative process.
MEDIPOST has made R&D efforts for over 10 years for developing adult stem cell drugs to meet unmet medical needs in areas such as osteoarthritis, stroke, Alzheimer’s disease, bronchopulmonary dysplasia (BPD) and improving the efficacy of hematopoietic stem cell transplantation (HSCT).
Through rigorous pre-clinical studies and clinical trials, we have proven the safety and efficacy of hUCB-MSCs asadult stem cell drug materials. The drug products under development include CARTISTEM® for cartilage regeneration which has completed Phase III clinical trial in Korea and is awaiting Biologies License Application (BLA) approval from the KFDA. Once approved, CARTISTEM® will become available as the world’s first hUCBMSC derived adult stem cell drug for treating osteoarthritis and will be marketed in Korea in 2012. CARTISTEM® has also received an Investigational New Drug (IND) clearance from the US-FDA in February 2011 and the Phase l/lla clinical trial will soon commence in the US. Other hUCB-MSC products named PNEUMOSTEM® for treating Bronchopulmonary Dysplasia (BPD) and NEUROSTEM® for treating Alzheimer’s Disease (AD) are both undergoing Phase I clinical trials in Korea.
Since the discovery of ABO blood type by Karl Landsteiner, blood transfusion has been used extensively in clinical practice as one of standard therapeutic interventions. Over the century, blood transplantation has been dependent on blood donation, and the risks associated with it such as immune reactions or infections have not been completely resolved. In addition to these problems, changes in social structure leading to low birthrate and aging population in a society like ours have made it difficult to maintain sufficient amount of donated blood constantly. We wondered whether we would still be doing blood transfusion using donated blood 50 years from now and as an alternative to it, we started to search for a possibility of using pluripotent stem cells as a source of blood cells. Our initial target is platelets because they are difficult to preserve and always in demand.
Recently, we established an in vitro culture system whereby hESCs or hiPSCs can be differentiated into hematopoietic progenitors within “sac”-like structures, which we termed ES-sacs or iPS-sacs. The cells inside these sacs were capable of differentiating into mature blood cells including megakaryocytes and functional platelets (Takayama et al., Blood, 2008). Furthermore, we found that these human iPSC-derived platelets can form thrombus together with mouse platelets in vivo (Takayama et al. J. Exp. Med. 2010). These results collectively indicated that hESCs and hiPSCs can be potential sources for platelet production and that this system will contribute to understanding the mechanism of various genetic platelet disorders and to development of novel cell/gene therapy.
Cellular therapeutics has become increasingly complex, moving from simple collection, volume reduction, and cryopreservation of hematopoietic cells to manufacturing of a variety of specialized cellular products for cancer immunotherapy, regenerative medicine, and cell-based gene therapies. From January 2011, the University of Alabama at Birmingham Cell Therapy Laboratory has been transitioning from a small hematopoietic processing laboratory into a fully cGMP-compliant multipurpose cell manufacturing facility. This transition has been driven by the increasing demand for advanced cell manufacturing services both within and outside of the bone marrow transplantation program and by the institution’s need to offer sophisticated academic and industry based multicenter cell therapy trials to our patients. In addition to the construction of an new cell manufacturing facility, additional challenges were upgrading all core operational standard operating procedures; opening a compliance, regulatory, and quality service within the cell therapy laboratory; and addressing the changes in both the culture of our laboratory and the services that are impacted by these changes. Implementation of these changes in practice are taking place while continuing to support collection and processing for a pediatric and adult bone marrow transplant program that serves approximately 120 patients per year. These challenges are being addressed through a close partnership with our investigators, the UAB facilities planning service, external compliance/regulatory consultants, and our principal diagnostic and manufacturing equipment suppliers. Through this collaborative effort we have incorporated new industry and NIH-funded multicenter trials as well as initiated two new cGMP manufacturing protocols for trials unique to our institution. The first institution-specific trial will evaluate ex vivo expanded/activated haploidentical gamma/delta T cells in the treatment of glioblastoma multiforme. The scientific rationale for this trial has been extensively addressed in both in vitro and animal studies and in preliminary discussions with FDA and represents a simple manufacturing process ideal for the initial work of a new laboratory. The second trial will evaluate gene correction of induced pluripotent cells (iPSc) from patients with nonmalignant genetic hematopoietic diseases followed by re-differentiation of the corrected iPSc into hematopoietic progenitors for autologous transplantation. This manufacturing process is complex and has been greatly enhanced by the availability of a manufacturing system with total bio-containment suitable for extended cell manipulation and culture protocols. In summary, the challenges that exist to transitioning an existing hematopoietic processing laboratory to a cGMP manufacturing facility can be successfully addressed through partnerships with employees, academic and hospital based stakeholders, consultants, and high-value suppliers.
The Biomedical Division of SANYO Electric Co., Ltd. has been engaged in the business of CPC (Cell Processing Centre = GMP grade clean room) since 2001 and we have successfully installed more than 100 facilities of these across Japan.
SANYO’S objective in this area is to provide an aseptic environment to users who manipulate human cells that are not usually feasible to sterilise at the final stage. In the CPC, it is required to design and construct a grade B room that is enclosed by grade D or C rooms because safety cabinets, CO2 incubators, centrifuges and cell monitoring microscope and such must be placed in a grade B room in the GMP facilities.
On the contrary, the space requirement for the traditional CPC and its running costs (electricity for air conditioning, consumables such as 2nd non-shedding gown, etc) are always issues to operate the entire CPC facilities. Due to these issues, a compactly designed yet the high performance device which offers the same level of or better aseptic environment as the CPC was gradually and increasingly hoped for by many customers.
SANYO Electric Co., Ltd has started the marketing of CPWS systems overseas at the beginning of 2011 and we successfully installed a first unit in the US this year.
In order to address these issues, SANYO introduced the unique CPWS (Cell Processing Work Station) systems in the field in 2004 and has been supplying them in Japan. The work area of CPWS system is totally closed therefore it can offer a high level of aseptic condition. Also, a CO2 incubator and a centrifuge can be directly connected to the CPWS system and this design makes it possible to achieve an aseptic environment in the CO2 incubator and the centrifuge along with the main work area. The whole area can be decontaminated by a validatable hydrogen peroxide decontamination cycle, and on top of the closed design, the CPWS system realises the high level aseptic condition.
The CPWS system which utilises the closed system conducts the air exchange only through the integrated HEPA filters therefore it eliminates the requirement of a grade B room. This contributes the smaller installation space compared to the traditional CPC facilities as well as less electricity since the air conditioning system can be smaller and also 2nd gowns (consumables) are not required to wear. These benefits can support the low cost operation of facilities and this unique CPWS concept has been well accepted by many users in Japan.
It is important for the industrialization of the regenerative medicine to develop automated cell processing technologies. Especially, it is necessary not only to culture high-quality cells, but also to avoid the contamination of cells. In the research field, the skilled human technician has operated the cell processing in the clean environment. However, the human operation costs a great deal. The automated system will reduce the cost and provide high quality cells stably without contamination. We have developed the Robotized- Cell Processing eXpert system: R-CPX using our clean robot system. In R-CPX, the operation area can be cleaned by V-PHP (Vapor-Phase Hydrogen Peroxide), and cells from plural donors can be cultivated in parallel. Using the cleaning process after the donor’s cell process, another donor’s cell can be operated without contamination. R-CPX operates some devices automatically according to the same procedure that the skilled technician do. We have developed the automated protocol for several kinds of cell processes. And, we realized the automated process for mesenchymal stem cell and the corneal epithelial cell sheet. The culture of mesenchymal stem cell by the automatic process shows the same result of the manual operation. The corneal regeneration process needs such a delicate operation that a small amount of medium liquid is supplied to the small cell dish slowly. This research has been supported by New Energy and Industrial Technology Development Organization (NEDO).
50 The 2nd Meeting of Senior Advisor Clinical Development Services Quintiles Transnational Japan K.K. Co-Sponsor : Quintiles Transnational Japan K.K. Hidemi Aida Clinical development in Asia, from CRO point of view Asia is rapidly becoming a new frontier for drug development as Western companies seek to develop and register their products in Asia, while emerging Asian companies seek new capabilities to globalize products. As Asia continues to grow, the need for improved healthcare will drive the need for new medicines. To fully capitalize on the R&D opportunities presenting themselves in the growing markets of Asia, the biopharmaceutical industry will need to relinquish its faltering “go-at-it-alone” linear model of drug discovery and development in which one firm controls all the pieces, and shift towards a “wheel-and-spoke” model of multiple strategic partnerships with other biopharma and service providers companies with complementary strengths and capabilities.
Asian biopharmaceutical companies face challenges that are a blend of those faced by the multinational pharmaceuticals and small biotechs. They too face rising R&D costs, regulatory hurdles and diminishing returns, but their key challenge has been the inability to globalize their clinical development programs. An historic example is the Japanese pharmaceutical industry that has excelled in discovery, product development and commercialization within the confines of Japan, but internationally Japanese companies have few products and virtually no market share.
The past decade witnessed a sharp increase in the number of clinical trials being run in Asia. However, this increase has primarily been the “off-shoring” of patient enrollment from the West to Asia because of the cheaper and easier access to patients, while the study concept and drug development strategies remained in the US and in Western Europe. However, akin to trends in other industries, this initial phase of off-shoring of low cost, low skilled operations will soon be followed by the demand for more highly technical and knowledge-based drug development capabilities in Asia.
As part of this change it will be important to fracture the linear, monolithic “go-at-it-alone” model of biopharma -from drug discovery through development to commercialization - in favour of a model replete with multiple partnerships and strategic alliances . In the latter, companies will focus on their core strengths, and partner or outsource all other activities to other firms better at performing those tasks. This model will lend itself to the principle of “how can we work to win together?”
As part of this paradigm it will not only be important for CROs to work together with biopharma abut it will also be imperative that investigators are an integral part of the team that will deliver new and better medicines to patients faster
Our pilot clinical trial was performed in 6 chronic stroke patients (stroke duration more than 6 months) by implanting autologous hematopoietic stem cells into the infarct brain. The results show that the damaged corticospinal tract might be regenerated within 3 to 6 months after the transplantation. However, the corticospinal tract regeneration and motor improvement were much better in young stroke patients (age less than 60 years old) than the elder (age more than 61 years old). We hypothesized that the age dependent regeneration of the corticospinal tract may be related to the molecular systems of the chemoattractants and stress proteins.
The most common and strongest chemoattractant protein is the stromal-cell-derived factor-1 alpha (SDF-1 alpha) and its receptor CXCR4. These two proteins together with cellular prion protein (PrPsc) are upregulated in the acute injured brain and stem cells. Stress induced protein such as secretoneurin is also upregulated during acute brain injury. In animal studies, overexpression of these proteins not only protects the injured brain from neural apoptosis, but also attracts bone marrow as well as neural stem cell to the injured site. Stem cells secrete various nurotrophic factors, angiogenic factors, stress proteins, and chemoattractants to enhance neuroprotection, tractogenesis and neuroplasticity. In addition, stem cells may also rejuvenate the surrounding cells.
We are currently conducting a randomized, double blind control trial for chronic stroke patients by implanting CD34 stem cells into the infarct brain. Our preliminary results show that there were a significant increment of corticospinal tract fiber density ratio (CTFDR) assessed by diffusion tensor image (DTI) in treatment group than control. The neurological improvements were better in the treatment group as compared to control patients in regarding to NIHSS, ESS, EMS, and Barthel Index. Details will be presented in the meeting.
This randomized control study demonstrates that a therapeutic strategy using G-CSF combined with autologous implantation of PBSCs mobilized by G-CSF transplantation for old stroke patients is safe, feasible, and shows preliminary evidence of improved neurological outcomes.
I will give a brief overview of the claims, economics, and geographical scope of the unregulated global industry (or black market) centered on the commercialization of stem cell injections for which there is no rigorous evidence of safety and/or efficacy. In particular, I will focus on recent regulatory developments and shifts in marketing tactics and regions targeted.
Natural killer (NK) cells are a promising tool for cancer immunotherapy because of their potent killing response to a broad range of tumor cells through diverse receptor-ligand interactions. We present NK cell therapy for patients with cancer using a novel method for ex vivo NK cell expansion we developed at our clinic founded in 2004.
Our method induces vigorous expansion of CD3-CD56+ NK cells without using accessory cells or fetal bovine serum. Peripheral blood mononuclear lymphocytes were stimulated with GMP-regulated medical drugs in the presence of autologous plasma, and expanded in NKGM medium (Kohjin Bio) containing interleukin-2. After 14 days of culture in C02-permeable bags (1L × 2), the median NK cell yield was 5.9 × 109 cells (range; 1.6-11.0 × 109 cells, n = 25) from 20 ml peripheral blood of healthy donors, and its median expansion was 859-fold (range; 296- to 2700-fold) of the initial input NK cells. In vitro cytotoxicity against K562 was 60.3% (median, n = 5) at an E:T ratio of 1:1, and in vivo cytotoxicity against human pancreatic cancer cell lines was also observed in a murine model.
Although medical practice without national approval is able to be performed by medical doctor’s discretion in Japan, weestablished the ethics committee of New City Osaki Clinic as a third party to estimate the clinical protocol for NK cell therapy. In addition to approval by the committee, patients gave written informed consent before NK cell therapy. To ensure safety and traceability of cell products, we saved all records of culture process and introduced a bar-code system to avoid cross-contamination during a GMP-based aseptic culture procedure.
We have treated 448 cancer patients until May 2011. The only adverse event was transient fever (up to 38°C) in around 20% of the patients, occurring 1-2 h after the infusion. The survival benefit was evaluated in 33 patients with advanced pancreatic cancer who received at least 4 infusions of NK cells combined with/without chemotherapy (gemcitabine and/or S-1). The median overall survival rate was 16.0 months (range: 4-52). The 1-year survival rate was 64%. Immunologic monitoring revealed a significant increase in both NK activity and the number of NK cells and NKG2D+ cells in the patients showing clinical benefit.
The described method is easy and safe, and would provide practical NK cell therapy exerting ADCC activity in antibody therapy, or cytolysis in an allogeneic setting.
The role of donor lymphocyte infusion (DLI) in the prophylaxis of relapse has not been defined. We retrospectively analyzed the data of 88 patients with advanced stage acute leukemia after HLA-mismatched/haploidentical hematopoietic stem cell transplantation (HSCT) whose treatment did (n=61) or did not (n=27) include prophylactic DLI. The two groups were compared with respect to relapse and overall survival. Further, a detailed analysis of risk factors was performed.
The 2-year cumulative incidence of relapse in patients receiving prophylactic DLI and not receiving prophylactic DLI were 36% and 55% (P = 0.017). Estimated survival at 3 years was 31% for patients receiving prophylactic DLI and 11% for patients not receiving prophylactic DLI (P=0.001). The three-year probability of LFS was also higher in patients receiving prophylactic DLI (22%) than in patients not receiving prophylactic DLI (11%) (P = .003). Multivariate analysis for relapse showed that use of prophylactic DLI after transplantation was an independent prognostic factor (p=0.025). Higher OS was associated with use of prophylactic DLI (P = 0.002), acute myeloid leukemia (P = 0.027) and female sex (P = 0.023). Our results suggest that the prophylactic modified DLI may increase survival after HLA-mismatched/haploidentical HSCT for advanced-stage acute leukemia.
Objects: To investigate the profile of donor lymphocyte infusion (DLI)-associated acute graft-versus-host disease (GVHD) in haploidentical T-cellreplete hematopoietic stem cell transplantation (HSCT).
Methods: Consecutive 124 patients receiving modified DLI after haploidentical T-cell-replete HSCT were enrolled.
Results: The cumulative incidence of DLI-associated acute GVHD was 53.2% for grade II to IV and 28.4% for grade III to IV. Multivariate analysis demonstrated that the duration of GVHD prophylaxis after DLI was related to the development of DLI-associated grade III to IV acute GVHD (P<0.05). The cumulative incidence of grade III to IV acute GVHD in patients with prophylaxis more than 6 weeks, 4 - 6 weeks, 2 – 4 weeks and less than 2 weeks were 9.3%, 14.4%, 31.6% and 49.5%, respectively (P=0.018). Besides, DLI-associated grade III to IV acute GVHD was the only risk factor for overall survival (P=0.038, OR=2.869) and transplant-related mortality (P=0.018, OR=3.296), but not risk factor for relapse after DLI (P=0.840).
Conclusion: This was the first study to investigate the incidence and risk factors of DLI-associated acute GVHD in haploidentical T-cell-replete HSCT, and for the first time confirmed that the duration of GVHD prophylaxis after DLI was the only risk factor for the development of grade III to IV acute GVHD. DLI with GVHD prophylaxis more than 4 weeks was associated with a lower incidence of grade ? to ? acute GVHD. Besides, this study also showed that DLI-associated grade III to IV acute GVHD was the only risk factor for inferior OS and higher TRM.
Furthermore, almost all expanded cells displayed functional cell surface molecules such as NKG2D and CD16 implicated in cytotoxicity and antigen dependent cell cytotoxicity (ADCC). Indeed, the expanded NK cells showed strong cytotoxicity against various tumor cell lines such as leukemia, lung, breast, melanoma, and rectal, and also ADCC in combination with tumor antigen specific antibody in vitro. In largescale culture system, we could obtain 109 ~ 1010 cells containing a high proportion (>90%) of CD3-CD56+-NK cells from 50mL of peripheral blood. Next, we tested for the ability of this system to expand NK cells from advanced cancer patients’ blood. As a result, the mean of total cell expansion rate after 21-22 days culture was 669-fold (range 191 ~ 1710 : n=5) and more than 94% of CD3-CD56+-NK cells were contained in all cases.
Background: Cytokine-induced killer (CIK) cells are highly efficient cytotoxic effector cells generated by culturing PBMCs in the presence of anti- CD3 mAb, IL-2 and IFN-?, and they characteristically contain the “NK-like T cell” population expressing both T cell marker CD3 and NK cell marker CD56 (CD3+CD56+). Among CIK cells, NK-like T cells (CD3+CD56+) are the main effector cells and demonstrate the most potent cytolytic activity to various types of tumor cells. Therefore, it is suggested that the technology for increasing the NK-like T cells proportion in CIK cells is a key to success for enhancing the clinical effects. We have previously reported the efficient T-cell expansion method using immobilized anti-CD3mAb and RetroNectin® (RN). In this method, NK-like T cells are not so much in expanded T cells even though a large amount of naive T cells can be obtained. Here, we show a novel expansion method for producing CIK cells by two step stimulations, with initial anti-CD3mAb/RN and second “supplement X”.
Results: By combining anti-CD3mAb/RN (1st) and supplement X (2nd) stimulations, PBMCs proliferated 250-700 fold on day14-15, and the expanded cells contained higher proportion (40-60%) of CD3+CD56+ than ones proliferated without second stimulation by “supplement X” (10- 20%). Most of the expanded cells expressed both CD8 and NKG2D, and showed strong cytotoxicity and IFN-? release. Next, we evaluated which population of CD3+CD56+ or CD3+CD56- cells showed strong cytotoxicity. As a result, CD3+CD56+ was more potent than CD3+CD56- population. Further, we established large-scale culture system using gas-permeable culture bags, CultiLife®215 and CultiLife®Eva. With this system, we could obtain a large number of CIK cells (>1010) containing high proportion of NK-like T cells (CD3+CD56+) ( ? 50%) from 50mL of blood.
Conclusion: We expect that this newly devised method for CIK expansion would be an effective tool for CIK-based immunotherapy.
PURPOSE: Natural killer (NK) cells play a crucial role of the innate immune system, and clinical studies to treat advanced malignancies using NK cells are emerging worldwide; however, overall purity and numbers of NK cells and their antitumor potencies are unsatisfactory as to obtain clinical outcome. We therefore attempted to establish an efficient method to purify and expand the highly activated NK cells ex vivo.
METHODS: Under approval of the institutional ethical committee, peripheral blood mononuclear cells (PBMC) were collected from healthy volunteers and patients with advanced malignancies. CD3+-cells were depleted by magnetic beads, and CD3-depleted PBMC (CD3-PBMC) could be expanded in the presence of high concentration hIL-2 and 5% human autoserum for 14days to expand NK cells. The characteristics of these CD3-/CD56+ NK cells were subjected to the expression of surface markers, CD107a mobilization and cell-mediated cytotoxicity against K562 cells in vitro compared with those of primary NK cells.
RESULTS: CD3-PBMC could be expanded in the presence of high concentration hIL-2 and the majority of the expanded cells were CD3-/ CD56+ (>90%) after 14days culture. The expanded NK cells expressed various killer-cell immunoglobulin-like receptors (KIRs), natural cytotoxicity receptors (NCRs), CD69, NKG2D, and CD16. CD107a mobilization assay and cytotoxicity assay revealed that expanded NK cells could kill K562 target cells more efficiently than that seen by primary NK cells.
CONCLUSION: We have established new methods to deal with the current issues underlying NK-based immunotherapy. Overall purity of expanded NK cells was sufficiently expressing various receptors, including KIRs and NCRs. Moreover, these cells showed strong cytotoxic activity against K562; and importantly, we could obtain these NK cells from PBMCs from patients with advanced malignancies using this method. Therefore, this novel method may contribute to provide a new tool for clinical cancer immunotherapy.
Herceptin is a widely used antibody-drug for current breast cancer treatment. However, a number of patients respond poorly to Herceptin, making the development of a new therapy that enhances the effect of Herceptin necessary. The antitumor effect of Herceptin arises from the antibodydependent cellular cytotoxity (ADCC) of Natural Killer (NK) cell. Hence a therapy that combines NK cell activity with Herceptin is a promising approach, and it is this possibility that we have examined in this study. First, a method for expansion of NK cells ex vivo was established, and then the expanded cells were evaluated for Herceptinmediated ADCC against the breast cancer cell lines. In our experiment, the NK cells were cultured from CD3-negative cells obtained from peripheral blood mononuclear cells by magnetic bead selection using a newly-developed culture medium and closed bag system. The fresh cells on day0 and the expanded cells were evaluated for surface markers, intracellular cytokine production and Herceptinmediated ADCC against the breast cancer cell lines MCF-7 and MDA-MB-231. After expansion for 14-23 days, the percentage of CD56+ cells was higher than 99% and the CD3+ cells stayed below 0.1%, and the pure population of NK (CD3-/CD56+) cells increased to 31.9 ± 12.0 fold. Expression of NK activation markers NKG2D, CD107a, Perforin, and GranzymeB were extremely high before and after expansion. The cultured NK cells showed a high level of cytotoxity against the breast cancer cell lines and an even higher level in the presence of Herceptin. In conclusion, a novel NK cell expansion method was established and the expanded NK cells showed a high level of Herceptin-mediated ADCC. These findings suggest that NK cell therapy combined with Herceptin would be a novel clinical immunotherapy for breast cancer patients with a poor response to Herceptin..
Aims: ADCC is an important mechanism of therapeutic monoclonal antibody drugs (mAbs) for anti-cancer. Natural killer (NK) cells and γδT cells are major effectors of ADCC through Fc receptor (CD16). We aim to develop a new method with simultaneous expansion of NK cells and γδT cells (NK/ γδT) for the purpose of combination immunotherapy with mAbs.
Methods: NK/ γδT were simultaneously cultivated from PBMCs in the presence of OK432, zoledronate and IL-2 with immobilized anti-CD16 antibody without any feeder layers.
Results: Median of expansion rate of NK/ γδT cultured from healthy donors (n=3) for 3 weeks were 3425 times and 2256 times, respectively. A large number of CD16+ cells were obtained. CD16 expressed in most of the NK cells and a part of γδT cells. In particular, the percentage of CD16+ γδT cells from the new method is higher than that from a conventional expansion method using zoledronate. NK/ γδT had high cytotoxicity against CD20+ cell lines and induced the ADCC by Rituxmab.
Conclusion: We simultaneously expanded NK/ γδT possessing high ADCC effects in vitro. These cells may be useful immune cells for a combination immunotherapy with mAbs
We have conducted clinical trials of adoptive γδT cell therapy for the treatment of cancer. Peripheral blood mononuclear cells (PBMC) were harvested by apheresis and γδT cells had been expanded ex vivo for 14 days by culture with zoledronate (5 μM) and IL-2 (1000 IU/ml). Patients received >1x109 γδT cells every 2 weeks. Infused γδT cells gradually accumulated in patients’ peripheral blood and accounted for 10% of PBMC as late as 3 months after the final injection, even without IL-2 administration in vivo. These cells maintained the ability to release cytotoxic granules (detected by CD107 assay) and produce IFN-γ in response to re-stimulation. To determine the factors contributing to γδ T cell survival in vivo, we investigated their expression of receptors for common γ chain (γc) family cytokines at the time of infusion. IL-2Rβ and γc, but not IL-7R α, IL-15Rα or IL-21R, were present on these cells, and although the IL-2Rα was upregulated at the initiation of culture, it was only very weakly expressed at the time of transfer. When γδ T cells were maintained for longer periods in culture, IL-15 but not low dose IL-2 and IL-7 supported their survival. Because IL-15 can be transpresented with IL-15Ra, γδT cell expressing IL-2RIβ and γc can respond more efficiently to IL-15 than IL-2. These results suggest that tissue-specific γc-dependent signals in response to IL-15 might affect the survival and function of infused γδ T cells, and hence materially influence the success of immunotherapy.
Currently, the development of FVIII antibodies (FVIII inhibitors) remains the most significant treatment complication for hemophilia A patients. These inhibitory antibodies usually target functional epitopes on FVIII, thus rendering FVIII replacement therapy ineffective. We have used the hemophilia A mouse model to evaluate a novel strategy for the induction of reduced immunological responsiveness to FVIII. With a view to establishing immune tolerance to the FVIII, autologous blood outgrowth endothelial cells (BOECs) transduced with a lentiviral vector encoding canine FVIII (cFVIII) were mixed with Matrigel and injected subcutaneously (2 x106 cells/injection) into hemophilia A mice that had previously been infused repeatedly with cFVIII in order to develop a robust anti-cFVIII inhibitors (25-45 BUs). The levels of inhibitors began to decline within 6 weeks of BOEC implantation in all mice and after 2 or 3 implantations of transduced BOECs the inhibitor levels in all mice had declined to < 5BUs. When these partially tolerized mice were re-challenged with the intravenous injection of cFVIII, they showed a rapid increase of FVIII inhibitor titers. In contrast, partially tolerized mice that were re-challenged with subcutaneous cFVIII showed no increase in inhibitor titers. Increased numbers of CD4+CD25+Foxp3+ regulatory T cells were detected in the spleen (4%) and subcutaneous draining lymph nodes (7%) of the partially tolerized mice and adoptive transfer of CD4+ splenocytes and draining node lymphocytes from these animals to naïve hemophilic mice transferred tolerance to cFVIII. These results indicate that genetically-modified BOECs implanted subcutaneously in a Matrigel scaffold may provide a novel immunemodulating therapy for elimination of FVIII inhibitors in hemophilia A patients.
Background: Transplantation of bone marrow Hematopoietic Stem cells are indispensable to replace the blood forming system in hematological malignancies. The engraftment after transplantation depends on total CD34+ count. We report a comparative analysis of the of CD34+ cells from bone marrow of people of different age groups from within India.
Materials and Methods: Patients enrolled for autologous bone marrow mononuclear cell injection for spinal cord injury with normal vital parameters were included in this study. The Bone Marrow (BM) tapped from the posterior iliac crest preserved under 10 deg Celsius was transported to a central lab within 12 hours of harvesting. The Mononuclear cells (MNC) were isolated using density fractionation method and the CD34+ cells were analyzed using flow cytometry. The isolation of MNCs and flow cytometry for all the samples were done in a single laboratory set-up following all GMP and GLP protocols.
Results: The total number of samples evaluated was 332 and the CD34+ Cell percentage in the various age groups were 1.0569%,in those belonging to the age group of 0-20 years it was 1.04% in the 21-40 year group, 0.96% in the 41-60 year group and 0.93% in the 61-80 year group. The difference in CD34+ cells percentage across these five groups showed a decreasing trend but didn’t reach a statistical significance.
Conclusion: Our results imply that the percentage of CD34+ cells decrease with increasing age. This trend needs further analysis in a larger population. With the recent literatures suggesting Colony Forming Units (CFU) as a strong independent predictor of engraftment in cord blood or bone marrow transplantation, we are extending this study to assess the CFU in bone marrow sam ples of various age groups which may reveal further relevance of their engraftment capability.
Introduction: Sipuleucel-T is an autologous cellular immunotherapy designed to stimulate an immune response to prostate cancer and is FDAapproved for the treatment of asymptomatic or minimally symptomatic metastatic castrate resistant (hormone refractory) prostate cancer (mCRPC). Sipuleucel-T is manufactured from peripheral blood mononuclear cells (PBMCs) isolated from fresh leukapheresis at weeks (wks) 0, 2, and 4. PBMCs are cultured ex vivo with a recombinant fusion protein, PA2024 (composed of prostatic acid phosphatase [PAP] and granulocyte-macrophage colony-stimulating factor [GM-CSF]). Control patients (pts) received a product made without PA2024 culture. In the Phase III IMPACT trial, pts who received sipuleucel-T had a 4.1 months improvement in median survival (HR=0.78; 95% confidence interval: 0.61, 0.98; P=0.03) compared to pts who received control product. Here, we assessed the immune response profiles generated by sipuleucel-T and correlate product parameters and posttreatment immune responses with overall survival (OS).
Methods: Prior to infusion in pts, all lots of sipuleucel-T and control product were evaluated for antigen presenting cell (APC) activation, as assessed by the upregulation of CD54. In addition, large CD54+ cell content and the total nucleated cell (TNC) count were also determined. Culture supernatants from sipuleucel-T or the control product were assayed for cytokine production (IL-2, TNF α, and IFN γ ), while cellular immune responses (T cell proliferation and IFN γ ELISPOT) were assessed against the antigens (PA2024 and PAP) in pre-culture cells, in a subset of pts, at wks 2 and 4. Antibody responses were measured by ELISA to the antigens. Post-treatment cellular and humoral immune responses were assayed at wks 6, 14, and 26.
Results: APC activation was evident in sipuleucel-T, but not in control pts. The magnitude of APC activation in sipuleucel-T pts was greater at wk 2 (P<0.001) and wk 4 (P<0.001) compared to wk 0. Cellular immune responses to PA2024 and PAP were not evident in the sipuleucel-T pts at wk 0, but were detected at wk 2 and increased at wk 4, whereas control pts had no responses against either antigen. Cytokines were not present in control cultures, but were present in sipuleucel-T cultures at wks 2 and 4 only. Post-treatment proliferative responses to both antigens increased from pretreatment in sipuleucel-T pts only and were maintained through wk 26 (P<0.001). Post-treatment IFN γ ELISPOT responses to both antigens were also present in sipuleucel-T pts only and were maintained through wk 26 (P<0.001 compared to pre-treatment), demonstrating a memory T cell response. Antibody responses were only evident in sipuleucel-T pts after treatment and were maintained up to wk 26 (P<0.001). Both antibody responses seroconverted to IgG, indicating a memory B cell phenotype. Each lot release parameter of sipuleucel-T correlated with OS: CD54 upregulation (P=0.123), cumulative CD54+ cell count (P=0.016), and TNC count (P=0.008). In addition, there was evidence of correlations between OS and cellular responses (proliferation at week 14 [P=0.057] and IFN? ELISPOT at week 26 [P=0.049]), and humoral response at week 6 (P=0.079).
Conclusions: Sipuleucel-T is a product that is designed to elicit an antigen-specific memory T cell response and a long-lasting humoral response. These responses imply the presence of functional antigen-specific T cells generated as consequence of priming by the first infusion, and boosting by the second and third infusions. There is evidence of correlations between product parameters and OS as well as immune responses and OS.
[PURPOSE]: Dendritic cells (DCs) play a crucial role in maintaining the immune system. Though DC-based cancer immunotherapy has been suggested to hold potential to treat various malignancies, clinical efficacies are still insufficient in many human trials. We proved that this antitumor effect depends on the number of DCs (Kato T., et al. Neoplasia 2010), and it is necessary to prepare an enough number of DCs for effective treatments of tumors. In this study, therefore, we attempted to expand functional human DCs ex vivo with new technologies.
[Materials and Methods]: Peripheral blood mononuclear cells (PBMCs) were obtained from healthy volunteers and cancer patients. CD3-depleted PBMCs were expanded and differentiated into DCs in the presence of cytokine cocktails for several weeks by floating cultivation. Expanded DC properties were analyzed, and compared with those of conventional DC.
[RESULTS]: Total cells increased approximately 10 – 100 fold after 5 weeks culture and >80% of expanded cells expressed CD11c. Thus, by this method, 10 – 100 times more CD11c+ cells could be obtained than conventional procedures could. As are seen in conventional DCs, expanded DCs showed dendrites after maturation, and endocytotic activities. Expanded DCs also expressed HLADR, adhesion molecules, and co-stimulatory molecules and produced inflammatory cytokines as well as conventional DCs did. Functionally, MLR assay revealed that expanded DCs could stimulate allogenic T-cell proliferation to the same extent as conventional DCs.
[CONCLUSIONS]: We established a new culture method to expand human DCs. Expanded DC had properties that were required to obtain therapeutic gain. We expect that this technology will be able to contribute largely to both basic and clinical research of human cancer immunotherapy. DC expansion technology will improve therapeutic gain of cancer and alleviate patients’ burden of apheresis.
For successful dendritic cell (DC) therapy of cancer, it is important to choose the optimal method to load DCs with antigens to induce strong antitumor immunity, particularly tumor-specific CTL responses. In this study, we performed side-by-side comparisons of two antigen loading methods, coincubation (Co) and electroporation (Ep), using murine bone marrow DCs. First we examined the uptake of FITC-dextran, and showed more efficient uptake of FITC-dextran by DCs loaded by Ep than those by Co. Next, to compare the efficiency to prime CD4 and CD8 T cells, DCs were loaded with OVA by Co or Ep, and then cocultured with OVA-specific TCR-transgenic OT-I CD8 T cells or OT-II CD4 T cells. OVA-Co-DCs induced moderate expansion and cytokine secretion of OT-II T cells, but had little effect on OT-I T cells. In contrast, OVA-Ep-DCs potently stimulated both OT-I and OT-II T cells to expand and secrete cytokines, such as IL-2 and IFN-?. Using monoclonal antibody specific for MHC classl/OVA peptide (257-264) complex, we confirmed efficient presentation of the OVA peptide on MHC class I by OVA-Ep-DCs. These results suggest that DCs loaded with tumor antigens by Ep would have greater abilities to induce anti-tumor immunity including CTL responses than those by Co. Now we are comparing in vivo anti-tumor effects of DCs loaded with tumor antigens by these two methods.
[Background]Mesenchymal stem cells (MSCs) have been highlighted in the field of regenerative medicine for their availability and multipotency. However, it has been reported that MSC may associate with cell-infusion-related events including thromboembolism after being administered intravenously. This study investigated the procoagulant property of MSCs.
[Methods]As a preliminary study, we administered 1.5 x 105 cells of murine adipose-derived MSC (ASCs) to wild-type mice via tail vein, and found that 11 out of 13 mice died within 24 h after injection. This fatal event was found to be associated with thromboembolism in the lung, heart, or liver. In order to investigate precise mechanism of this ASC-related event, we assessed ASCs-derived procoagulant properties with (1) citrated whole-blood using ROTEM and (2) citrated plasma using a coagulometer. We also performed the immunostaining and gene expression analysis of ASCs for mouse tissue factor (TF), and a clotting assay using normal plasma (NP) and factor VII-deficient plasma (F7DP).
[Results]ROTEM analysis demonstrated significant shortenings of CT and CFT, and increased a-angle in ASCs samples. Plasma clotting assay also showed the procoagulant property of ASCs in a cell-number dependent manner. We also found that ASCs expressed high TF mRNA, and TF was strongly expressed around the cell surface. ASCs also shortened the clotting time of NP, while the time of F7DP was unable to be shortened as clearly as that of NP. Further investigations were conducted using human ASCs and found that human ASCs also possess strong procoagulant activity triggered by TF.
[Conclusions]ASCs express TF around the cells, and TF may trigger the activation of extrinsic coagulation pathway, leading unexpected thromboembolism. An optimal inhibition for TF by some anti-coagulant agents is recommended for securing the clinical safety of systemic ASCs administration.
Placenta derived mesenchymal stem cells (PDMCs) had been demonstrated by using different protocols. Recently, we newly developed a novel type of MSCs from the chorionic membrane of the human term placenta, named pcMSCs.
In comparison of pcMSC and PDMCs, pcMSCs are distinguished with its unique resources of chorionic layers, rather than the whole, unseparted placenta of PDMCs. RTPCR results indicate pcMSCs express various isoforms of HLA-G, including membrane forms (HLA-G1, -G2, -G3, G4) and soluble forms (HLA-G5, and –G6). Moreover, immunohistochemistry (IHC) and immunofluorescence (IFC) showed that HLA-Gs was strongly expressed only on the chorionic membrane. It was feasible to speculate that pcMSCs may secret more HLA-Gs than PDMCs.
In our preliminary studies, pcMSCs were used as therapeutic cells to treat paraquat induced acute lung injury in animal models. After the therapy, the results showed that at day-6, the survival rates of mice increased from 8% to 35% and decreased the infiltration of neutrophil into lung tissues and alveolar spaces; however, other therapies, including treatment with dexamethasone and other cell types did not increase the survival rates. These results indicated that pcMSC cell therapy significantly restored lung function.
Since HLA-G had been documented as potential immunoregulary molecules, we hypothesized that pcMSCs may decrease neutrophils infiltration in lung tissues through the action of HLA-G.s. This interaction between HLA-G and HL60 cells were examined using a co-culture migration system. It showed pcMSCs down-regulated the migration of differentiated neutrophil-like HL-60 cells, while administration of HLA-G neutralizing antibody blocked the down-regulation. Hence, it was speculated that HLA-G was the main molecule to inhibit the migration of neutrophil.
The results suggest that HLA-G molecules were important in alleviation of acute lung injury by pcMSC therapy in mice model.
In driving hES cell technology towards widespread applications considerable effort has been focused on the improvement of culture conditions and on enabling efficient differentiation. We have established two technologies which will better enable researchers to achieve these aims.
The use of feeder based protocols for the creation, expansion and banking of hES cell lines are well established. The advances in technology for feeder free culture have predominantly relied on the cultivation of pluripotent cells in colony based systems, commonly used in conjunction with ill-defined matrices. We have developed a process to allow enzymatic single cell passaging in a medium in which Wnt3a and bFGF are interchangeable by a method utilising small molecule control of beta-catenin interaction with its binding partners in concert with additional growth factor supplementation. This medium can be partnered with chemically defined or synthetic matrix components to provide exemplary consistency of phenotype within and between passages. Following extended passage in the Pluripro® P300 system cells retain the undifferentiated phenotype, as evidenced by expression of markers of the undifferentiated state, and capacity for in-vitro differentiation into the three germ lineages. In addition we have formulated a range of growth factors (including FGF2) with enhanced efficacy at a level of multiple orders of magnitude, when comparing equivalent molar concentrations, through multivalent conjugation to carrier substrates (STAR technology).
The development of improved culture systems for pluripotent cells and the increased efficacy of growth factors in inducing and establishing differentiated progeny are essential requirements if stem cell technology is to fulfil its potential and overcome technical and economic barriers. Taken together, these two advancements have the potential to transform stem cell research and the development of cellular therapeutics.
Patient specific human induced pluripotent stem (hiPS) cells not only provide a promising tool for cellular disease models in general, but also open up the opportunity to establish cell-type specific systems for personalized medicine. One of the crucial prerequisites for these strategies, however, is a fast and efficient reprogramming strategy from easy accessible somatic cell populations. Keratinocytes from plucked human hair had been introduced as a superior cell source for reprogramming purposes compared to the widely used skin fibroblasts. The starting cell population is however limited and thereby further optimization in terms of time, efficiency and quality is inevitable. Here we show that rat embryonic fibroblasts (REFs) should replace mouse embryonic fibroblasts as feeder cells in the reprogramming process. REFs enable a significantly more efficient reprogramming procedure as shown by colony number and total amount of SSEA4- positive cells. We successfully produced k-hiPS cells from various donors. The arising k-hiPS cells display the hallmarks of pluripotency such as expression of stem cell markers and differentiation into all three germ layers. The increased reprogramming efficiency using REFs as a feeder layer occurred independent of the proliferation rate in the parental keratinocytes and acts, at least in part, in a non-cell autonomous way by secreting factors known to facilitate pluripotency such as Tgfb1 Inhba and Grem1. Hence, we provide an easy to use and highly efficient reprogramming system which could be very useful for a broad application to generate human iPS cells.
The generation of induced pluripotent cells (iPS) from adult somatic cells provides tremendous advantages for the study of disease specific models and in the future may become a source of cells for regenerative therapies. Many methods of developing iPSs are currently in use and each possesses inherent difficulties resulting in low frequencies of fully reprogrammed cells. We will present a brief review of epigenetic processes involved in reprogramming followed by a description of easy and efficient lentiviral methods which incorporate a single polycystronic vector and use a Cre-mediated excision process to eliminate the exogenous transgenes. We will further discuss culture protocols we have developed that optimize generation and characterization of iPS cells.
Tendon-derived stem cells are commonly considered to be of mesenchymal origin, having the capacity to differentiate into adipocytes, chondrocytes, osteoblasts and tendon cells. Earlier on we have shown that human tendon perivascular cells express markers associated with neural stem cells such as Nestin and Musashi1.
Here we describe a so far unrecognized type of human tendon perivascular stem cells (hTPSC) expressing markers commonly associated with embryonic stem cells (ESC). Tissue samples from intact human biceps-, supraspinatus and semitendinosus tendons were obtained with patients´ informed consents, tissue donors were aged from 18-73 (n=10) years. By immunohistochemistry and single cell PCR we demonstrate that hTPSCs express Oct4, Nanog, Klf4, Cmyc and Sox2 in vivo. In cell culture, these cells give rise to clonal spheroid cell aggregates harbouring cells expressing the stem cell markers mentioned and markers associated with all three embryonic germ layers, such as Insulin and Glucagon (endoderm), Collagens type 1 and 3 (mesoderm) and GFAP and Galactosyl ceramidase (ectoderm). In differentiation experiments, hTPSC can give rise to adipocytes, osteoblasts, oligodendrocytes, astrocytes and insulin producing cells.osteoblasts, oligodendrocytes, astrocytes and insulin producing cells.experiments, hTPSC can give rise to adipocytes, osteoblasts, oligodendrocytes, astrocytes and insulin producing cells. Collagens type 1 and 3 (mesoderm) and GFAP and Galactosyl ceramidase (ectoderm). In differentiation experiments, hTPSC can give rise to adipocytes, osteoblasts, oligodendrocytes, astrocytes and insulin producing cells. Despite their ESC-like marker expression and their mulitpotency, these cells do not form tumors upon injection into immunodeficient mice.
Interestingly, these cells seem to persist up to an old age. They could even be detected in tendon tissue from a 73 year old donor.
These findings suggest that TPSC represent a more undifferentiated cell type than mesenchymal stem cells.
An important issue that will have to be addressed in the future is to learn more about the nature of the niche that keeps these cells undifferentiated throughout life. Also the role of these cells in tendon degeneration and healing needs to be examined.
Moreover, hTPSC may be a valuable source for future applications in tissue engineering and cell therapy.
Current paradigms in cancer therapy attempt to eliminate all malignant cells of a tumor lesion. The cancer stem cell (CSC) paradigm, however, predicts that tumors are initiated by a minor subset of cancer initiating cells and are maintained by a few, so far less identified cancer maintaining cells. In this contribution, however, we demonstrate that specific elimination of a less than a 2% subset of melanoma maintaining cells eradicates established melanoma lesions without targeting the tumor cell mass. The tumor stem cell subset is selectively eliminated from tumor lesions by adoptive transfer of cytotoxic T cells redirected by an engineered chimeric antigen receptor. Targeted elimination of the minority of tumor cells which co-express HMW-MAA (MCSP) and CD20 eradicated established melanoma lesions in longterm despite the bulk of tumor cells. Targeting of any random cancer cell subset was not effective. HMW-MAA+ CD20+ melanoma subset cells were found in about 4 out of 5 melanoma biopsies of different histology and clinical grade. Our data provide first evidence that progressing melanoma is maintained by a minority of cells, the targeted elimination of which results in tumor eradication
Mammalian epidermis consists of the interfollicular epidermis (IFE) with associated hair follicles (HF) and sebaceous glands (SG) (Blanpain and Fuchs, 2009).
Functional SGs are important for barrier acquisition and protection against pathogens. Upon maturation, cells of the SG (sebocytes) disintegrate and release sebum (Niemann, 2009). This requires constant replenishment of differentiated cells and implies stem or progenitor cells to be involved in SG-renewal.
Previously, our lab has demonstrated that individual bulge stem cells and their descendants constantly regenerate the SG under homeostatic conditions in adult skin (Petersson et al, 2011).
In contrast, little is known about the cellular origins and molecular mechanisms crucial for the development of this organ and the entire pilosebaceous unit. We have investigated the cellular processes during SG formation in more detail. Analysis of the spatio-temporal organisation of stem and progenitor compartments was performed during morphogenesis of the pilosebaceous unit. Our results suggest a dynamic expression of distinct HF stem cell markers during the formation of the HF and the SG. For instance, progenitor markers Lrig1 and Sox9 are first coexpressed by epidermal progenitor cells and become confined to distinct compartments as soon as the SG starts to form. We provide evidence that proliferation of Lrig1 positive progenitor cells drives SG development at the upper part of the HF. In contrast, the MTS24/Plet1 progenitor cell pool is not likely to contribute to the initial stages of SG formation. Our data clearly demonstrate that different stem and progenitor compartments within the HF are established at different time-points during development.
Furthermore, by studying the process of SG morphogenesis in more detail, we found that in HFs of mouse tail skin, two prominent SG arise from one single cluster of precursor cells.
Finally, to identify the cellular origin of the SG, we have performed lineage tracing experiments during epidermal morphogenesis. First results indicate that SGs originate from IFE.
Further analysis will help to unravel the cellular and molecular signals governing establishment of distinct stem cell compartments and SG formation.
Notch signaling is a crucial cell-cell communication pathway affecting cell-lineage decisions, proliferation, apoptosis, self-renewal and differentiation processes. In the canonical pathway, Notch is thought to mediate its function via the transcription factor RBP-J mainly by increasing the expression of the target proteins of the Hes/Hey family. Recently, we described novel cell context dependent Notch1 target genes that comprise a high percentage of transcription factors. Among these we found Sox9 to be regulated under various differentiation conditions.
Here, we describe that during mesodermal differentiation as embryoid bodies this Notch1 induced upregulation of Sox9 had its maximum at day 4 and diminished at day 10. Furthermore, by inhibition of protein synthesis as well as luciferase experiments with RBP-J binding site mutants of Sox9 promoter reporter constructs, Sox9 was shown to be a direct target gene. To further investigate the role of Sox9 for Notch1 mediated effects, we employed a siRNA strategy to specifically quench the Sox9 peak induced by Notch1 induction without affecting induction of other Notch1 target genes. For chondrogenic differentiation we found that a temporary activation of Notch1 during the early stages of EB formation resulted in a strong increase in chondrogenic differentiation during later stages. This increase in cartilage development could be entirely reversed by the application of Sox9 siRNA, whereas the known blockage of cardiac differentiation by Notch1 was unaffected by the reduced Sox9 levels. In another set of experiments we used the same Sox9 siRNA strategy to investigate the role of Sox9 in Notch-mediated cell lineage decisions during neuroectodermal development. Notch is known to play a role in the decision between glial and neuronal cells as well as for the induction of neural crest differentiation, though the molecular basis if these effects are not understood. Our experiments revealed that in neuroectodermal differentiation conditions Sox9 is also a direct target gene of Notch1. Notch1 induction led to a strong increase in glial cell formation while inhibiting the generation of neurons. Quenching of the Sox9 peak induced by Notch1 signaling using Sox9 siRNA led to a significant decrease of glial cells, demonstrating the pivotal role of Sox9 in mediating Notch1 signals in this context.
In summary, our data indicate that the effects of Notch signaling are not only mediated through the well-described Hes/Hey family. The novel direct Notch1 target gene Sox9 described here plays an essential role, both, in chondrocytic development as well as in neuroectodermal differentiation, thus emphasizing the importance of alternative mechanisms in Notch signal transduction.
This study analyzes the effects of sitagliptin +/- G-CSF treatment on survival and myocardial regeneration after myocardial infarction in a mouse model. The SDF-1- CXCR4 axis is a key mechanism of cardiac homing of stem cells. G-CSF is known to mobilize bone-marrow-derived stem cells into peripheral blood, whereas sitagliptin is a DPP-IV (dipeptidylpeptidase IV)-inhibitor- Sitagliptin therefore prevents the essential stem cell homing factor SDF-1 from being cleaved.
Acute myocardial infarction was induced by surgical occlusion of the left descending artery in 10-11 weeks old male C57BL/6 mice. Sitagliptin was administered per os in a titrated dose regimen with blood levels measured by LCM/ M. DPP-IV activity was analyzed by enzyme activity assays. Saline and G-CSF were injected intraperitoneally for 6 days following myocardial infarction. The effects of the dual therapy as well as the effects of sole sitagliptin treatment on cardiac stem cell mobilization and homing was measured by flow cytometry. The impact on neovascularization and cell proliferation was analyzed by immunohistochemistry and the treatment benefits on infarct size was assessed by histology. The treatment impact on cardiac function and survival was analyzed by millar tip catheterization and the Kaplan-Maier-method.
Enzyme activity assays revealed a significant decrease in DPP-IV enzyme activity after sitagliptin application.
Sitagliptin+G-CSF as well as sole sitagliptin therapy Enzyme activity assays revealed a significant decrease in DPP-IV enzyme activity after sitagliptin application. Sitagliptin+G-CSF as well as sole sitagliptin therapy enhanced both mobilisation and cardiac homing of BMCs. Cell proliferation (Ki67+) and neovascularization were increased in both treatment groups, resident cardiac stem cells were stimulated and cardiac remodelling was significantly decreased. Dual therapy consisting of sitagliptin and G-CSF as well as sole sitagliptin application significantly reduced infarct size, had a positive impact on myocardial function and improved survival compared to sole G-CSF or saline application. The beneficial effects seen were most remarkable for the dual therapy group, but also significant for sole sitagliptin administration. Additional application of the CXCR4-antagonist AMD3100 reversed the beneficial treatment effects of both treatment regimens back to baseline. This suggests specificity of the treatment effects to the CXCR4-axis.
This is the first study showing that combined application of G-CSF and Sitagliptin and yet sole sitagliptin administration increases cardiac homing of stem cells, induces neovascularization, reduces cardiac remodelling, enhances cell proliferation, has a positive impact on cardiac function and improves survival after acute myocardial infarction. Combined administration of sitagliptin and G-CSF and even sole sitagliptin application has beneficial effects on cardiac regeneration beyond its known anti-diabetic potential and may be a new therapeutic regimen after myocardial infarction.
The endosteal hematopoietic stem cell (HSC) niche consists of cellular components including osteoblasts, osteoclasts and mesenchymal stromal cells as well as extracellular matrix components (ECM) and chemokines. HSCs reside in this niche in close contact to their surroundings. In the process of stem cell mobilization, a proteolytic microenvironment contributes to the release of hematopoietic stem and progenitor cells (HSPCs) from their niches, the precise molecular mechanism, however, is still not known. In the present study we characterized the expression and secretion of a member of the matrix metalloproteinase (MMP) family, the collagenase MMP-8, in the context of the endosteal stem cell niche identifying a new component of the proteolytical environment during mobilization.
We analyzed blood serum samples of HSPC donors treated with G-CSF for several days. The elevated number of cycling CD34+ HSPCs correlated very strongly with an increased serum concentration of MMP-8. Circulating CD34+ HSPCs can also be found physiologically in umbilical cord blood. An ELISA analysis of umbilical cord blood serum showed also elevated MMP-8 serum concentration. Next, we investigated the influence of MMP- 8 on the adhesive interaction of HSPCs with osteoblasts. After binding of fluorescently labeled CD34+ HSPCs to primary osteoblasts, activated MMP-8 was added. The assay revealed a strong reduction of HSPC attachment to osteoblasts after incubation with activated MMP-8 compared to non-activated MMP-8. We hypothesized that this reduction in cell attachment was due to the degradation of cell adhesion and/or extracellular matrix (ECM) molecules. A strongly adhesive ECM component synthesized by human osteoblasts is laminin 511 (LM-511). MMP-8 is able to proteolytically digest recombinant LM-511 as shown by silver staining after SDS-PAGE, but this degradation had no impact on cell adhesion. A detailed analysis of the cleavage products using peptide mass fingerprint analysis showed that the important integrin-binding sites are still intact. CXCL12α, a chemokine with an important role in HSPC migration, was also tested as a potential substrate for MMP-8. MALDI-TOF analysis revealed that MMP-8 can process the N-terminal end of CXCL12α. In cell migration assays using non-degraded and MMP-8 processed CXCL12α as a stimulus, a strong inhibitory effect on HSPC migration through the degradation by MMP-8 was exhibited.
In summary, we provide strong evidence that the matrix metalloproteinase MMP-8, which can be released from granulocytes, can drastically reduce adhesion of HSPCs to osteoblasts. Furthermore, we suggest that the proteolytic degradation of cell-cell interactions and the important chemokine CXCL12α by MMP-8 can support mobilization of HSPC out of their niche.
Cardioprotective actions of estrogen are well recognized for many years. Recent studies indicate a novel role of estrogen receptors (ER) in stem/precursor cell-involved cardiac repair. Taking into account that cardiac c-kit+ precursor cells are mainly recruited from bone marrow (BM) c-kit+ cell populations, we aimed here to elucidate the functional importance of ERα in BM c-kit+ precursor cells after ischemic heart injury. The c-kit+ cells were isolated from femurs and tibias of male wistar rats 7 days after myocardial infarction (MI) by magnetic activated cell sorting in combination with fluorescent activated cell sorting (FACS). After MI, the percentage of BM c-kit+ cells increased by 2.11 fold. BM c-kit+ cells, which expressed both intracellular and membrane (m)ERα, were shown to inhibit apoptosis of co-cultured cardiomyocytes in a paracrine manner. In addition, ERα stimulation could improve paracrine cardioprotection by BM c-kit+ cells. According to the expression of mERα, BM c-kit+ cells were further sorted using FACS into c-kit+mERα+ and c-kit+mERα- cell populations. Notably, BM c-kit+mERα+ cells were more potent in supporting paracrine cardioprotection than ckit+ mERα- cells both in vitro and in vivo. Futher analysis revealed that BM c-kit+mERα+ cells were characterized by increased production of cardioprotection cytokines including IL-6 and IL-10. Importantly, blocking IL-6, but not IL-10, by neutralizing antibody abolished the protective effect of BM ckit+ mERα+ cell in supporting cardiomyocytes, indicating IL-6 was responsible for BM c-kit+mERα+ cell-mediated paracrine cardioprotection. Finally, the c-kit+mERα+ cell population was verified in peripheral blood of patients with heart failure. Thus, this work explains a novel cardioprotective mechanism mediated by BM c-kit+mERα+ cells via paracrine IL-6.
Desmin, a type three intermediate filament protein is expressed in all types of muscle cells and contributes to homeostasis in the adult heart. Knock out of the desmin gene lacks a clear embryonic phenotype; however, upregulation of desmin expression resulted in an increased number of developing cardiomyocytes and a temporal restricted upregulation of the cardiogenic transcription factor Nkx2.5 in cardiac progenitor cells during a small window in time. Thus we hypothesize that desmin may directly promote cardiogenic commitment and myocardial differentiation. To test whether desmin influences transcriptional regulation of the Nkx2.5 gene during cardiomyogenesis we choose embryoid bodies as a model system to study the short-lived function of desmin in nascent cardiogenic cells. Desmin indeed interacts with regulatory DNA elements of the Nkx2.5 gene, is present in nuclei of cardiac progenitor cells, activates the Nkx2.5 gene via the cardiac specific enhancer element in fibroblasts, myoblasts, and cardiomyocytes, and rescues Nkx2.5 related haploinsufficiency during cardiomyogenesis. These results attribute a new dimension to the role of desmin in commitment and differentiation of progenitor cells to cardiomyocytes, by demonstration of its participation in the transcriptional regulation of the Nkx2.5 gene.
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited cardiac disorder characterized by emotional and physical stress-induced ventricular tachyarrhythmia, syncope and sudden cardiac death in children and young adults. In many cases, CPVT has been linked to mutations in the cardiac ryanodine receptor type 2 gene (RYR2) encoding a Ca2+ channel in the membrane of the sarcoplasmic reticulum (SR) leading to excessive Ca2+ release from the SR during diastole upon catecholaminergic stimulation. Here we report the generation of induced pluripotent stem cell (iPSC) lines from a CPVT patient carrying the novel heterozygous autosomal dominant mutation p.F2483I in the RYR2 gene. Single cardiomyocytes derived from CPVT iPS cells revealed arrhythmias and delayed after depolarizations (DADs) after isoproterenol stimulation. Arrhythmic response to adrenergic stimulation was also observed in multicellular beating clusters. Patient iPS derived cardiomyocytes exhibit higher amplitudes and longer durations of spontaneous Ca2+ release events at basal state when compared to control cardiomyocytes. In addition, the Ca2+-induced Ca2+ release events continued after repolarization and were abolished by increasing the cytosolic cAMP levels with forskolin. This study demonstrates the suitability of iPSCs in modeling RYR2- related inherited cardiac disorders in vitro and opens new opportunities for drug development, to optimize patient treatment, and pinpoint the role of different RYR2 mutations in disease pathogenesis.
Stem cell therapy is a promising approach treating endstage heart failure. Mesenchymal stem-cells (MSCs) injected intra- myocardially differenciate into capillaries while glandular stem cells, derived from pancreas, parotis or submandibularis, transform into cardio- myocytes. Major questions of applying stem cell therapy in a failing myocardium are the intra-myocardial homing and the development of gap junctions. The following study will deal with these 2 questions.
Glandular stem cells were characterized by red PKH26 and MSCs by green PKH67 makers. A mix of one million of each cell type was injected into three locations of the goat's myocardium of the left ventricle. Intra-myocardial homing of glandular stem cells and MSCs (CD133+) were evaluated in 6 female goats after 1 and 3 hours after intra-myocardial injection. Additionally from 6 female goats myocardium was harvested with injected stem cells after 6 weeks.
Furthermore glandular stem cells of goats were co-cultured with goat´s myocardium for 48 h and kept in culture for 3 weeks. An immune-histological staining of connexin 43 (gap junctions) was performed on these cultured glandular stem cells.
Having used a mix of intra-myocardial injection of GSCs and MSCs, MSCs showed a significant cell migration into the surrounding myocardium, more expressed after 3 hours than after one hour. After 6 weeks, within the frozen myocardial slices 76,4% of the marked stem cells were identified as GSCs (red) but only 23,6% as green MSCs (P≤0,05). Additionally in cell cultures glandular stem-cells being in contact with myocardium developed connexin 43 mainly in that part of the cell membrane being in cell to cell contact. These primarily results of course need more research work concerning connexin 43 expression after an intramyocardial injection in a big animal model.
Due to a significant better intra-myocardial residence of GSCs in comparison to MSCs combined with the ability expressing connexin 43 (gap junctions), glandular stem cells might become a very promising treatment option for the repair of irreversible damaged myocardium.
The proliferative potential of pluripotent stem cell derived cardiomyocytes is limited and reasonable yields for novel therapeutic options have yet to be achieved. In a first attempt of such “cardiovascular forward programming” using pluripotent stem cells, we have previously shown that MesP1 represents a master regulator sufficient to induce cardiovasculogenesis (David et al., Nat Cell Biol, 2008). In ES cells MesP1 overexpression resulted in significantly increased numbers of beating cardiomyocytes and of endothelial cells. Our experiments revealed a prominent function of MesP1 within a gene regulatory cascade causing Dkk-1 mediated blockage of canonical wntsignalling. Our findings suggest a mechanism for cardiovascular specification highly conserved in vertebrates initiated via MesP genes with prominent factors such as Nkx acting further downstream. Detailed patch clamping analyses showed electrophysiological characteristics corresponding to all subtypes of cardiac ES cell differentiation in Nkx2.5 as well as MesP1 programmed embryoid bodies (EBs) but fractions of cardiomyocytes had distinct characteristics: MesP1 forced the appearance of early/intermediate type cardiomyocytes (~60%) in comparison to control cells whereas Nkx2.5 led to preferentially differentiated ventricular cells (~80%) (David et al., Cardiov Res, 2009). In order to unravel the regulation of MesP1 expression we have now analysed Eomes and Brachyury(T) as its potential inducers. We demonstrate that the MesP1 positive cell population is derived from the Brachyury(T) positive fraction in the embryo as well as in ES cells. Likewise, loss of Brachyury(T) causes a dramatic decrease of MesP1 expression accompanied by reduced cardiac markers. Using EMSA, ChIP and reporter assays we found a 3.4 kb proximal MesP1 promoter fragment, directly bound and activated by Brachyury(T) via a T responsive element (David et al., Cardiov Res, 2011). To characterize the cellular progeny eliminating an overexpression situation we used this promoter fragment for isolating MesP1 positive cells from differentiating pluripotent stem cells via magnetic cell sorting based on a deleted CD4 surface marker. This yielded a highly pure common cardiovascular progenitor population with the potential to form all three cardiovascular lineages: cardiomyocytes, endothelial cells and smooth muscle cells. Electrophysiological and pharmacological parameters of the derived cardiomyocytes affirm the pivotal role of MesP1 during the earliest cardiovasculogenic events: by far most of the cardiomyocytes (~94%) corresponded to the desirable multipotent early/intermediate type highly exceeding the numbers achieved by the above described forward programming via MesP1 (~60%) (David et al., in revision).
Finally, I will address ongoing work transferring our forward programming approach described above to the enrichment of pacemaker cells, which may become useful for biological treatment of the “sick sinus syndrome”.
The ability of stem cells to differentiate into various somatic cell phenotypes makes them an attractive source for tissue engineering and regenerative medicine. Focusing on a potential therapeutic application of these cells, a non ‐ invasive and rapid method for the characterization of the cell's differentiation state is needed. The aim of this study was to determine, if Raman‐spectroscopy is a suitable, tool to identify different cell types, cellular activity or even to detect the differentiation states of stem cells in a noninvasive and label-free manner.
To determine differences in the biochemical component patterns, Raman‐spectra of the cell groups were obtained and analyzed with principal component analysis (PCA) and the support vector machine (SVM) using the Opus and Unscrambler software. To verify the actual cells and cell states biological tests were used, like immunofluorescence labeling and flow cytometry. For cell viability analysis we combined a temperature induction of apoptosis and necrosis in the cell lines SAOS-2 and SW-1353 with flow cytometry and fluorescence labeling for apoptosis and necrosis. For cell identification of MSC and fibroblasts flow cytometry and cell culture for cellular differentiation was used to reference to the Raman spectroscopic results. For the analysis of stem cell differentiation we used undifferentiated murine ESCs (feeder ‐free cell line CCE), differentiated embryoid body (EB) ‐ derived cells and ESC ‐ derived cardiovascular cells. Murine embryonic fibroblasts (MEFs) served as controls. The differentiation states of the cells were confirmed by immunofluorescence staining employing antibodies against pluripotency markers (Oct4 and SSEA) and CD31, a marker that labels cells, which are committed to the cardiovascular lineage.
The results show that the Raman spectroscopy can be used to determine small differences in the cells, which can be correlated to cell viability, differentiation of cell types, for example MSC and fibroblasts and to analyze stem cell differentiation.
Staphylococcus aureus (S. aureus), Streptococcus pyogenes (S. pyogenes) and enterobacteria such as Escherichia coli (E. coli) belong to the most frequent initiators of wound infection. It is known that wound healing involves specific differentiation of resident mesenchymal progenitor cells. Since it is unknown which effects bacteria and bacterial compounds exert on differentiation of these stem/progenitor cells, we investigated the influence of the above-listed bacteria on the differentiation state of mesenchymal stem cells from adipose tissue (adipose tissue-derived stem cells/ASC) in vitro.
S. aureus, S. pyogenes, and E. coli (isolated from infected wounds) were exposed to ASC (isolated from liposuctionderived adipose tissue) in passage 4 under standard cell culture conditions. Cytotoxicity was tested by live/dead staining, whereas adherence and internalization of bacteria on or in ASC were tested in an antibiotic protection assay. Furthermore, effects of vital and heat-inactivated bacteria and bacterial cell wall compounds were monitored by assays for metabolic activity (MTS), proliferation and osteogenic and adipogenic differentiation. Statistics were performed by Mann-Whitney-U-Test.
Co-incubation of ASC with bacteria adherence and internalization of the bacteria. Gram-positive species (S. aureus, S. pyogenes) were much more effective in adherence and internalization than the Gram-negative E. coli. In the short run (up to 24 h), none of the bacterial species tested executed cytotoxic effects. In long term experiments (14 d), adipogenically or osteogenically stimulated ASC were exposed to heat-inactivated bacteria, bacterial lysates, and bacterially conditioned cell culture media. Exposure of inactivated E. coli or their membrane compound lipopolysaccharide (LPS) to ASC induced an increase in proliferation, reduction of metabolic activity, increased osteogenic and decreased adipogenic differentiation. In contrast, inactivated Gram-positive bacteria and their cell wall component LTA did not induce clear effects.
The effects observed may be mediated by Toll-like receptors belonging to the innate immune system and serving for the recognition of pathogenic structures, among them bacterial compounds. ASC express toll like receptors TLR1 - TLR6 and TLR9. The recognition of LPS is by TLR4, recognition of LTA is by TLR2. Thus, specific stimulation of ASC by E. coli or LPS is presumably mediated by activation of TLR4. Since Gram-positive bacteria and LTA did not induce clear effects, although the receptor is expressed, further studies are necessary.
This work is financially supported by the EU, the Federal State of Mecklenburg-Vorpommern and the research funding FORUN of the Medical Faculty, University of Rostock.
Transplantation of mesenchymal stem cells (MSCs) derived from adult bone marrow has been proposed as a potential therapeutic approach for postinfarction left ventricular (LV) dysfunction. However, age-related functional decline of stem cells has restricted their clinical benefits after transplantation into the infarcted myocardium. The limitations imposed on patient cells could be addressed by genetic modification of stem cells. This study was designed to improve our understanding of genetic modification of human bone marrow derived mesenchymal stem cells (hMSCs) by polyethylenimine (PEI, branched with Mw 25 kDa), one of non-viral vectors that show promise in stem cell genetic modification, in the context of cardiac regeneration for patients. We optimized the PEI-mediated reporter gene transfection into hMSCs, evaluated whether transfection efficiency is associated with gender or age of the cell donors, analyzed the influence of cell cycle on transfection, and investigated the transfer of therapeutic vascular endothelial growth factor gene (VEGF) hMSCs were isolated from patients with cardiovascular disease aged from 41 to 85 years. Optimization of gene delivery to hMSCs was carried out based on the particle size of the PEI/DNA complexes, N/P ratio of complexes, DNA dosage and cell viability. The highest efficiency with the cell viability near 60% was achieved at N/P ratio 2 and 6.0Ag DNA/cm2. The average transfection efficiency for all tested samples, middle-age group (< 65y), old-age group (>65y), female group and male group was 4.32%, 3.85%, 4.52%, 4.14% and 4.38%, respectively. The transfection efficiency didn't show any correlation either with the age or the gender of the donors. Statistically, there were two subpopulations in the donors; and transfection efficiency in each subpopulation was linearly related to the cell percentage in S-phase. No significant phenotypic differences were observed between the two subpopulations. Furthermore, PEI-mediated therapeutic gene VEGF transfer could significantly enhance the expression level.
Recent findings indicate that Endothelial Progenitor Cells (EPCs) differentiate towards an endothelial like cell type and accelerate re-endothelialization of blood vessels. Healthy veins and arteries are usually lined by a single layer of endothelial cells. This cellular coating, called endothelium, has to be intact for hemocompatibility and prevention of thrombi formation. In order to create intact endothelium within autologous tissue engineered vessels patient's own healthy veins have to be sacrificed to harvest endothelial cells (ECs). Hence EPCs could become an important substitute for ECs in tissue engineering of grafts. Anyhow there is still an ongoing discussion about endothelial progenitor cells and their phenotypes. On the other hand it has already been shown that late outgrowths EPCs are very similar to ECs. Thus we exposed late outgrowth EPCs from human peripheral blood to defined shear stress within our bioreactor and examined them.
We developed a novel bioreactor system that provides defined levels of shear stress. Basically the device consists of two coaxial cylinders with different sizes. The inner one rotates and impels medium in the gap between both cylinders. This results in shear stress for the cells that are seeded on the inner wall of the outer cylinder. EPCs were exposed to laminar shear stress of 0.45 Pa for 24h. Then expression of endothelial specific proteins PECAM-1 and VEGF-R2 was estimated by immunocytochemistry and quantitative real-time PCR. Also we monitored the shape of the cells with the systems own bright field microscope.
EPCs were influenced by the shear stress treatment. To our surprise bright field microscopy images revealed that late outgrowth EPCs being exposed to shear stress did not orientate in direction of flow within 24h. Interestingly, they rather flattened themselves. PECAM-1 and VEGF-R2 expression of EPCs was lower after shear stress. In addition these results were underscored by comparative real-time PCR data.
Our new bioreactor system is a suitable device for investigation of cells under defined shear stress conditions. Observation of EPCs revealed that they flatten under the influence of shear stress instead of aligning in direction of flow. However taking the drop of PECAM-1 and VEGF-R2 expression into consideration that was noticeable at both translation and transcription levels, doubts arouse about the endothelial character of used EPCs. Therefore undeniably more investigation will be necessary in order to judge the future role of human peripheral blood EPCs in tissue engineering. Without doubt our system can match all needs of future experiments as shear stress can be adjusted to any desired level. Moreover the device permits long time cultivation as medium exchange can be done via incorporated ports very easily. To be brief we proved the feasibility of our bioreactor and demonstrated an odd behavior of heatedly discussed EPCs under shear stress.
Fibrin gel has proven a valuable scaffold for Tissue Engineering. Complex geometries can be produced by injection moulding, it offers effective cell seeding and can be produced autologously. In order to evaluate its suitability for respiratory Tissue Engineering, we examined proliferation and differentiation of respiratory epithelial cells on fibrin gel in comparison to culture on collagen-coated, microporous membranes.
Respiratory epithelial cells were isolated from ovine tracheae using published protocols and expanded to passage two. Consequently, cells were seeded onto fibrin gels and collagen coated polyethylene membranes, Proliferation was assessed every 24 hours using a Casy Cell Counter for 6 days. Differentiation was evaluated by measurement of transepithelial electrical resistance, livemicroscopy for ciliary motion analysis and histology. Cells were kept in culture for 4 weeks on the respective surfaces.
Respiratory epithelial cells formed a confluent layer by day 4 on both surfaces as revealed by live microscopy. Proliferation showed no significant difference with respect to surface. The transepithelial electrical resistance increased during the first 6 days of culture, falling to a steady level by day 10. No significant differences could be shown with respect to resistance between the two groups. Live-microscopy revealed the development of ciliae by day 18 with ciliary motion being observed by day 24 in both groups. Histology showed a differentiated respiratory epithelium.
We hypothesized that respiratory epithelium might proliferate and differentiate as well on fibrin gel as it does on collagencoated, microporous membranes. In our study, we could not demonstrate any difference in cell proliferation or differentiation when grown on either surface. Thus, we concluded that fibrin gel might prove a suitable scaffold for respiratory Tissue Engineering and merits further investigation.
We investigated the effect of implantation of engineered heart tissue (EHT) in heart failure in vivo..
EHT was created from neonatal rat cardiomyocytes, collagen, matrigel and media. After cultivation time (14 days) electrically stimulated EHT started to contract spontaneously and developed force (0.44 ±0.13mN). Histological analysis revealed the presence of troponin I and connexin 43 positive, cross striated cardiomyocytes, besides pre-formed vessels and connective tissue.
We induced DCM by application of doxorubicin in rats for 6 weeks. Cardiac function was controlled by echocardiography during the experiment. On 80 days animals were separated into 3 groups: 1. Day 80-group with animals to examine the status of DCM on day 80 (n=6), 2. EHT-group (n=13), 3. Sham-group (n=12).
In EHT-group we implanted EHT around the beating heart and in Sham-group animals underwent the same surgery without EHT-implantation.
One month after operation hemodynamic measurements were performed (Millar catheter). We examined the LV (left ventricular) +dp/dt max as measurement for contractility, under control conditions and under dobutamine (0.2mg/kg) for stimulation of β-adrenergic receptors (β-AR). Subsequently the hearts were prepared for epicardial electrical mapping analysis and finally for histological analysis.
Echocardiography revealed a significant impairment of heart function on day 80 (33.1±0.7%) measured as fractional shortening (FS) as compared to healthy animals (41.9±0.9%, p< 0.05). After surgery in EHT-group FS increased by +4.6±1.3% . In comparison, Sham-group exhibited further decrease in FS (-7.5±3.7%, p< 0.05). Hemodynamic measurements indicated a decrease in LV dp/dt max in Day 80-group (4698±370mmHg) and in Sham-group (5336±723mmHg) compared to healthy animals (6470±246mmHg, p< 0.05), but not in EHT-group (7840±672mmHg). Contractility analysis revealed that the dobutamine-induced increase in contractility was abolished in DCM (healthy: 12350±1619mmHg/s vs. Day 80: 7050±1045mmHg/s, p< 0.05), but was restored in EHT-group (12579±2892mmHg/s), while it further declined in Shamgroup (5824±543mmHg/s). The restored dobutamine response indicated that the long-term hemodynamic situation in DCM was improved by EHT-implantation. Additionally, mapping analysis exhibited electrical coupling of EHT with the recipient heart. EHT was tightly ingrown into the native myocardium and was connected to the coronary system. In vivo EHT showed organised collagen structure, elastic fibres, and troponin I and connexin 43 positive cardiomyocytes.
The restored dobutamine response indicated that DCMinduced increase in sympathic activity with consequent β-AR down-regulation can be reversed by EHT-implantation, which demonstrated an improvement in hemodynamic regulation and cardiac function. (BMBF: 0313909)
Goal was to engineer biological, arterial grafts with antithrombotic, autologous endothelial luminal surface combined with extraluminal smooth vascular muscle layer (VSMC) and to test invivo.
(1) Different decellularisation methods described in literature were compared to identify the most suitable one with focus on the preservation of extracellular fibre matrix. (2) Endothelial precursor cells (EPC), isolated from bone marrow and VSMC from small venous segments of donor animals were cultivated. Cells were seeded sandwich-like on homologous decellularized venous scaffolds and conditioned under pulsatile circulation in a bioreactor. (3) The semiautologous grafts were implanted in carotidal position on both sides in five Beagle dogs (n=10; group 2) as interposition. A group of five animals, receiving only acellular grafts in both carotid positions (n=10; group 1) served as controls.
Comparison of four in literature described decellularisation methods showed different preservation of elastic and collagen fibres compared with native veins, whereas decellularity was similar in all methods. This forced us to choose a decellularization protocol with the best preservation of the extracellular matrix. The invivo experiments showed in group 1 (control) already after one week a complete thrombotic occlusion of the decellularized implants, whereas in group 2 9/10 semiautologous grafts were patent after 98±4 days in ultrasound, angiography and histology (p=0,0001)..
A complete incorporation of semiautologous grafts in the surrounding tissue could be shown. The seeding with two different cell types preserved an aneurysmatic degeneration under arterial conditions with patency without anticoagulation.
The percutaneous stent angioplasty of peripheral vessels is well established as clinical routine. Unfortunately the patency rates of small-calibre grafts (Δ 6mm) are still unsatisfying, especially in the lower limb reg ion. The aim of the BioStent concept is to overcome restenosis. The basic approach is total separation of atherosclerotic plaques from the blood stream. The second and essential approach is to form an intact, functional and active endothelial cell layer. The proposed concept bases on the combination of a selfexpanding nitinol stent with the principles of vascular Tissue Engineering: The moulding process of vascular grafts, based on a fibrin gel scaffold, allows complete integration of a self-expanding stent structure within the tissue-engineered vessel. With this completely new principle the major causes of restenosis (1.) the foreign body reaction, (2.) the cell proliferation with ingrowths in the lumen and (3.) acute thrombosis by hemo-incompatibility will be prevented. The reason is total exclusion of the atherosclerotic surface from the blood stream and the coating of the neolumen with a functional endothelial cell layer, including antithrombotic function of the endothelial cells..
Small-calibre (6mm) BioStents were made by combining a self-expanding nitinol stent with a thin fibrin-based tissueengineered blood vessel.
Remodelling of the fibrin scaffold with mature autologous proteins was tested by histological analyses. A confluent endothelial cell monolayer lining the luminal surface of the Biostent was shown by scanning electron microscopy.
A thin coverage of about 200Cm completely wrapping the stent structure was achieved. Scanning electron microscopy revealed that the total surface was covered by a confluent layer of endothelial cells.
The present feasibility study shows successful combination of a self-expanding nitinol-stent with a fibrin-based tissueengineered blood vessel. The next step in this project will be animal studies in a sheep model.
Thus first stents have already been implanted and we are looking forward to the results. Based on the results of this study the system will be improved. Our current stent construct has a diameter of 6 mm and is supposed for peripheral blood vessels.
Aim of further studies will be to reduce the diameter of the stent for coronary stent application. Finally, our tissue engineered stent platform is well suited to be used in other applications.
Recently, we already started projects based on the BioStent concept that aim to improve endobronchial and urogenital stenting.
Soft tissue regeneration by mesenchymal stem cells and biomaterial scaffolds has been an emerging technology in regenerative medicine today. In the present study, we have evaluated the hepatomimetic potential of a novel biodegradable, biocompatible, polymeric nanofiber scaffold made up of PLLA (Poly-L-Lactide)-collagen and fibrin clot extracted from blood. Such a novel scaffold fabricated by electrospinning technique was evaluated for fiber morphology, composition, mechanical property, biomagnetic property, biocompatibility and hepatic differentiation potential in vitro.
The nanofibers were fabricated by electrospinning and their morphology was observed under a scanning electron microscope. The tensile properties of nanoscaffold were evaluated with a mechanical testing system. Elemental composition of the nanofiber scaffold (fibrinogen, iron content, albumin) was assessed biochemically. Biomagnetic behaviour of nanoscaffold was studied using a vibrating sample magnetometer.
The proliferation of MSCs seeded on electrospun nanofiber was assessed quantitatively by MTT assay. Hepatic differentiation was carried out with a novel hepatogenic conditioning media, derived from goat liver extract, with EGF and FGF-4 pre-induction. Differentiation of MSCs to hepatocyte like cells on nanoscaffold was confirmed by immunofluorescence and electron microscopy.
Morphological analysis reveals the blended nanofibers have a fiber diameter of 381± 0.171 nm. The amount of iron and fibrinogen present in nanofiber scaffold was significant compared to original electrospinning solution. SDS PAGE analysis of nanofiber components reveals two bands of 57 kDa and 48 kDa, which represents the fibrinogen. The magnetic behavior of the electrospun nanofiber reveals a significant magnetic hysteresis loop to a varying magnetic field (-2000 to 3000 G) at room temperature. This is the first report describing biomagnetic property of a nanoscaffold derived from blood components. Morphological characterization of the MSCs seeded on nanoscaffolds by scanning electron microscopy shows better cell entrapment and infiltration onto nanofibers and their enhanced proliferation. The morphology of cells on nanofibrous scaffold is more of hepatocyte like and it was confirmed by immunofluorescence analysis for the albumin expression. Cells on nanoscaffold showed higher expression compared to that on TCP.
All the above results support enhanced morphological and functional hepatic differentiation of MSCs on a novel biomagnetic and biocompatible nanoscaffold, making its future therapeutic application widespread. This study indicates that in hepatic failure, autologous blood clot derived similar nanoscaffold impregnated with autologous bone marrow derived mesenchymal cells may be a therapeutic option for augmenting liver regeneration, if implanted at orthotopic site.
Cardiomyocytes are an important target in drug Discovery and Safety Pharmacology. Many drugs can interfere with human cardiac ion channels and trigger fatal arrhythmia. However, as animal cells do have a different set of cardiac ion channels than human cells the aim for a long time has been to establish a human cell based assay. Here we compare the properties of iPS cell derived cardiomyocytes, ES cell derived cardiomyocytes with a non mammalian primary cardiomyocyte based assay and heterologous expression systems.
We compare results from cardiac tissue recordings and Langendorff hearts with our datasets obtained from a variety of stem cell derived cardiomyocytes for a panel of reference compounds.
Currently the International Organization for Standardization (ISO) is developing a new standard document (ISO 13022) which will address the requirements for risk management for medical products based on viable human cells.
By definition of the relevant European Directives and Regulations products including human tissues and cells are currently considered as medicinal products in Europe, and standards are not commonly used for medicinal products in the European Community. However, there is an obvious benefit in a close coordination of the work on regulatory guidelines in Europe and internationally harmonized standards for all manufacturers of these products who are focusing on the international market.
The standard ISO 13022 specifies a procedure to identify the hazards and hazardous situations associated with such products, to estimate and evaluate the resulting risks, to control these risks, and to monitor the effectiveness of that control. Furthermore, it outlines the decision process for the residual risk acceptability, taking into account the balance of residual risk, and expected medical benefit as compared to available alternatives. It will cover viable human materials of autologous origin as well as allogeneic human material.
The document is intended to provide requirements and guidance on risk management related to the hazards typical of medical products manufactured utilizing viable human materials such as:
contamination by bacteria, moulds or yeasts and parasites, contamination by viruses, contamination by agents causing Transmissible Spongiform Encephalopathies (TSE), contaminating material responsible for undesired pyrogenic, immunological or toxicological reactions, decomposition of the product and degradation products caused by inadequate handling including procurement, packaging, storage, transport and application, and complications resulting from the mix up of human raw materials.
These considerations apply to all stages from donor selection to application of the product.
Information on the status of the standard document and the potential for participation in the developing process will be further topics of the presentation.
Differentiation of embryonic stem cells (ESCs) is a comprehensive biological process controlled by many regulators. A better understanding of differentiation process is an essential base for development research, tissue engineering and pharmaceutical oriented ESC research. Recent studies demonstrated that microRNAs (miRNA) play a central role in the regulation of ESC differentiation. In the present study, a transgenic murine ESC line was applied as a model for miRNA regulation study in cardiomyocyte specific differentiation. miRNA expression was profiled using high throughput microarray platforms for samples obtained at undifferentiated stage (day0) and different time points during cardiomyocyte specific differentiation and maturation (day12, day19 and day26). 50 miRNAs were identified as plausibly different expressed during the process from ESCs to mature cardiomyocytes and therefore recognized as candidate regulator miRNAs involved in cardiomyocyte specific differentiation procedure. Parallel to miRNA profiling, a genome wide transcriptome analysis was performed for the same samples. This analysis discovered regulation effects between undifferentiated ESCs and cardiomyocytes on transcriptional level. Functional analysis of regulated genes and miRNA target genes reveals possible regulatory pathway of candidate miRNAs. Although it is widely believed that miRNA regulators act on both transcriptional and translational levels, this study concentrated on finding correlation between miRNA regulator and target gene transcript. Regulated miRNA target genes showed enrichment in important pathways involved in cardiomyocyte specific differentiation process.
Further studies can surely complete the scenario of cardiomyocyte specific ESC differentiation.
Embryonic stem (ESC) and induced pluripotent stem (iPSC) cells are attractive sources for ex vivo generation of cardiomyocytes. These systems are well-suited for developmental studies, high throughput drug screenings and cell replacement approaches. Activation of calciumactivated potassium channels (SKCas) via the small molecule 1-EBIO (1-Ethyl-2-benzimidazolinone ) leads to an induction of mesodermal differentiation and an enrichment of cardiac pacemaker cells. To date, these findings were restricted to mouse embryonic stem cells, thus limiting a broad applicability.
It is obvious that this strategy could be a powerful approach for clinical and research applications.
Therefore, we have validated our previous findings using human induced pluripotent stem cells derived from plucked human hair.
We assessed SKCa-function via the small molecule 1-EBIO in terms of cardiac differentiation in human pluripotent cells and provide the following results: (i) the generation of virusfree human iPS cells as a potential source for patient-specific iPS cells and (ii) the successful transfer of the SKCa activation assay to the human system in terms of rapid remodeling of the actin cytoskeleton, inhibition of proliferation and induced commitment to the cardiac lineage upon SKCa activation.
Our data clearly demonstrate the generation of SKCa-induced cardiac pacemaker cells from human iPSCs and overcomes specific limitations for potential applications. With our system using human iPSCs we further identify new strategies for the generation of autologous cells for cell replacement applications, thereby, providing a key milestone on the path to making cardiac subtypes in men.
The implementation of the sinuses of Valsalva for both aortic and pulmonary position is a crucial step towards the development of functional tissue engineered heart valves with optimal hemodynamic performance and reduced risk of thrombi formation. However, the implementation of these features is not standard in tissue engineered heart valves. In our laboratory we aim at the realization of autologous heart valves starting from materials isolated from the patient (fibrinogen and cells) and shaped into 3D geometries by moulding techniques. We present a new fabrication method that results in the realization of a heart valve scaffold reproducing the complete complex geometry of a semilunar valve. Our concept consists of a mould in two parts: a ventricular part and a vascular part which contains three removable bulbs representing the sinuses of Valsalva (
Mould with three removable sinuses of Valsalva
The moulds were designed with the 3D CAD software Pro/Engineer (PTC, Needham, MA, USA) and manufactured by rapid prototyping. The cell embedded fibrin gel valves were produced by polymerizing a fibrinogen solution in TBS (10 mg/ml) with CaCl2, thrombin and ovine umbilical cord derived fibroblasts (10x106 /ml) suspended in TBS. Afterwards, the tissue engineered heart valve was placed in a static bioreactor to be cultured on the mould for 14 days in order to avoid cell-mediated tissue contraction before transferring it to a bioreactor for dynamic cultivation of a duration of 30 days.
After the polymerization of the fibrin gel had occurred, first the vascular part and subsequently the three removable sinuses of Valsalva were removed. The construct was successfully released without any tearing despite the poor mechanical properties of the hydrogel. We obtained a conduit presenting the complex geometry of a semilunar valve with a three leaflet valve and the sinuses of Valsalva cast. The scaffold consisted of a single piece without the need for suturing any of the parts together. After static and dynamic cultivation the tissue was well developed and the conduit demonstrated a good compliance.
The presence of the sinuses of Valsalva in the aortic and the pulmonary root is fundamental for the correct functioning of semilunar heart valves. The implementation of the sinuses in tissue engineered valves will lead to an improvement of the valve function and an increased durability. Ongoing research focuses on the culturing, mechanical conditioning in bioreactors and evaluation of the scaffolds in vitro. Further evaluation will include immunohistology and determination of hydroxyproline and DNA content.
Tissue engineering is a multidisciplinary science that merges different fields of sciences like cell biology, physics, chemistry, material sciences, genomics, proteomics, engineering and medicine together to create biological substitutes of native tissues for doing research in lab or to use for medical application.
Tissue engineering is a promising approach to overcome the problems of shortage of organs and tissues for replacing or repairing damaged tissues or organs. Although the history of tissue engineering goes back into the last century but it is nearly 3 decades since accelerating into research in this field especially with the experimental and clinical goals.
The main progress in this field arise from 1990s that since that time tissue engineering turns from just a laboratory research field into a rapidly growing industry. With industry application of tissue engineering more research funds invests into the research labs and therefore this field of science due to earlier mentioned reasons and especially due to regenerative medicine and clinical application needs has an extremely promising future.
In the past tissue engineering considered as a part of biomedical sciences but it is now more considered as a multidisciplinary field of science. Tissue engineering is now used in many fields of biological sciences and medical sciences and has many experimental and clinical applications ( e.g. to replace or repair organs or tissues like bone and cartilage,…).
In this presentation the past of tissue engineering and its application in regenerative medicine will briefly discussed and then I spend more time on the myocardial tissue engineering (MTE) as the main scope of this presentation.
Cardiovascular diseases (CVDs) and especially myocardial infarction (MI) are the number one cause of mortality and morbidity in the developing countries. It is also one of the main cause of mortality and morbidity in the developing and rest of the world countries.
There are many routine therapy methods following MI like oxygen therapy, beta -blockers medication therapy or injection of Heparin or to administrate anti platelet medications like Aspirin or Clopidogrel to inhibit blood clots in the coronary artery to restore blood flow in the blocked coronary.
Myocardial scar formation and myocardial infarct size are large and the damages are irreversible. To help the damaged myocardial tissue back to normal function also seems need new therapy approaches in addition to previous routine therapies that earlier mentioned.
Myocardial tissue engineering needs some constructs that they should have those like biocompatibility, Mechanical integrity, biodegradability, being cell friendly, biomimetic and also have fabrication capability.
In this presentation after this introduction I discuss advantages and disadvantages and success and failures of MTE in details. The goal of this presentation is to give a better idea on the past, present and the future of the MTE.
Ectopic expression of certain transcription factors can reprogram somatic cells to a pluripotent state. It has been shown that hematopoietic stem cells can be reprogrammed with a higher efficiency than differentiated blood cells. Similar findings have not been demonstrated in other primary organ systems. Moreover, molecular characteristics in the cellular hierarchy of tissues that influence reprogramming capacities need to be delineated. Here, we analyzed the influence of the differentiation stage of freshly isolated, murine liver cells on the reprogramming efficiency.
Liver progenitor cell (LPC)-enriched cell fractions from fetal and adult liver showed a significantly increased reprogramming efficiency compared to unsorted fetal liver cells, when 3 or 4 reprogramming factors were transduced for the generation of iPS cells.
The reprogramming efficiency of fetal LPCs reached up to 30% in the 4-factor experiments. LPC-derived iPS cells showed all hallmark features of pluripotency. Increased reprogramming efficiencies of LPCs correlated with the expression of reprogramming factors (Klf4 and c-Myc in fetal LPCs and c-Myc in adult LPCs) as well as with an elevated expression of BAF-complex members (Baf155, Brg1) in adult and fetal LPCs. The increased reprogramming efficiency of LPCs compared to differentiated liver cells occurred independent of the proliferation rate in the freshly isolated cell populations.
The current study provides the first experimental evidence that the differentiation stage impairs reprogramming efficiency in freshly isolated cells from mouse liver. The data indicate that LPCs carry intrinsic, cell proliferation-independent characteristics allowing highly efficient reprogramming. LPCs could serve as a novel cell source for fast and efficient iPS formation.
Urea Cycle Disorders (UCD) are inherited errors of metabolism with deficiencies of one of the six enzymes involved in the urea cycle. Patients suffering from UCDs cannot detoxify nitrogen and have a poor prognosis, especially if the onset of the disease occurs in the neonatal period. Mortality reaches about 85% after 10 years in these children, most of the survivors showing severe neurological impairment. The current therapeutic concepts include dietary restrictions and treatment with ammonia scavenger drugs, which are, however, often not sufficient to prevent UCD patients from recurrent hyperammonemic crisis. Early orthotopic liver transplantation (OLT) is a definite cure of the metabolic disease but a risky procedure in small children. Liver cell transplantation aims to provide an additional therapeutic option especially for the group of very young patients.
Liver cell transplantation is applied to children with UCD in a clinical trial in Germany, after previous experience gained from individual therapeutic attempts. The children treated so far received 1-6 intraportal infusion of liver cell suspension after surgical catheter placement, with concomitant immunosuppression. The liver cells were isolated from donated organs (donor age, 5 days to 55 years) in a GMP compliant manufacturing procedure.
Intraportal liver cell infusion was well tolerated in all nine study patients and four cases of therapeutic attempts treated so far. The procedure was feasible even in neonates (n = 4) and no complications were noted during the infusions, which were carried out with ongoing control of peripheral oxygen saturation and portal blood circulation. In most patients the clinical situation stabilised after treatment over a period of up to 21 months or until OLT. Further efficacy evaluation using laboratory, biochemical, and immunohistological methods is currently still ongoing in order to quantify the engraftment of donor hepatocytes in the recipient’s liver.
Intraportal liver cell transplantation is feasible and safe in children with UCD. In parallel to a broader clinical use of LCT based on clinical studies, research on suitable liver stem cells should be promoted to overcome the limited availability of adult hepatocytes, with concomitant improvement of repopulation.
Hematopoietic BMSCs are involved in hepatic regeneration after liver resection. We previously demonstrated peripheral mobilisation of CD133+/CD45+ hematopoietic BMSCs after extended forms of clinical hepatectomy. In this study we correlated peripheral CD133+/CD45+cell mobilisation with the extent of resected liver volume and its regain, with paracrine factors participating in hepatic regeneration like hepatic growth factor (HGF), CXCL12 (SDF-1) and alpha feto protein (AFP). Additionally the potential mobilising capacity of HGF and SDF-1 for these stem cells was analyzed.
Peripheral progenitor mobilisation was investigated by FACS analyses in 30 hepatectomy patients. Exact extend of liver volume loss and regain by day 21 after hepatic resection was determined by CT scan volumetry. 20 patients with resection volume of less than 20% (group A; n=20) were compared to 10 patients with a resection extend of 30-67% (group B; n=10). HGF, SDF-1 and AFP levels in patient's serum were determined by ELISA technology. Mobilising capacity of HGF and SDF-1 for CD133+/CD45+ BMSCs was investigated in
In group B we observed increased serum levels of HGF, SDF-1 in the first 6h and of AFP beyond 24h if compared to group A. Beyond an augmented peripheral mobilisation of CD133+/CD45+ cells from day 2 on in the large resection group, levels of early CD133+/CD45+ stem cell mobilisation correlated directly with the level of hepatic volume regain by day 21. The mobilisation of CD133+/CD45+ cells on day 4 after extended liver resection in group B was directly correlated with the levels of AFP serum levels on day 6 after hepatectomy. In
These data suggest HGF and SDF-1 to play a role for mobilisation of CD133+ stem cells from BM in the liver regenerative scenario subsequent to hepatic resection. The increase of AFP serum levels supports the activation of stem cells after extended liver resections since AFP is considered to be a marker of “stemness” in hepatic regeneration. Whether peripheral mobilisation of CD133+ BMSCs that correlates with levels of liver regeneration after hepatectomy results in integration of those cells in intra-hepatic stem cell compartments, as possibly suggested here by AFP expression data, needs to be further explored.
Caspase-activation plays a fundamental role in the maintenance of tissue homeostasis by clearing injured cells to maintain tissue homeostasis through receptor mediated apoptosis. An important role of caspase8 during immunemediated liver injury and regeneration was previously demonstrated by our laboratory. We aim to examine its role during hepatic stem cell (Oval cells) activation, a process closely related to liver regeneration..
For this approach, hepatocyte specific conditional caspase8 knockout (casp8Δhepa) animals and mice with an ubiquitous deletion of caspase8 (casp8ΔMx) were compared in the DDC (3,5-diethocarbonyl-1,4- dihydrocollidine) model of oval cell activation and secondary sclerosing cholangitis. Oval cells were isolated and characterized by flow cytometry (FACS) and real time PCR. Liver tissue was further analysed by IHC.
Higher transaminases and bilirubin levels were observed in Casp8ΔMx levels compared to wild type (WT) and control, while casp8Δhepa animals were protected after 4-weeks of DDC-feeding. Additionally, histological analysis revealed reduced liver injury and immune-cell infiltration in Casp8Δhepa mice
Thereby caspase3 and -8 activities were reduced in both knockout strains as demonstrated by caspase3 and -8 assay, suggesting other mechanisms being responsible for the phenotype. Correlating to the stronger liver injury, casp8ΔMx mice displayed more proliferation in periportal areas where LPC emerge and reside as indicated by 5-fold higher BrdU incorporation rate and significantly higher CD133, Cyclin A, D and E mRNA expression. The following analysis of hepatic progenitor cells by flow cytometry (sca-1, OC-1, -2 and -3) as well as by immunohistochemistry (CK-19) unrevealed a significant stronger (5-fold) oval cell activation in casp8ΔMx mice, while casp8Δhepa had significantly less then WT mice after 4-weeks of DDC-feedinig. Deletion of caspase8 itself was not evident in isolated progenitor cells. Preliminary data now point to an additionally enhanced infiltration of immunecells (CD45, CD11b, CD4) in casp8ΔMx mice. This finally resulted in a stronger fibrosis progression of the underlying sclerosing cholangitis induced by DDC in casp8ΔMx mice, as evidenced by an enhanced expression of collagen and α- SMA.
Our data suggest a differential role of death receptor mediated liver injury through caspase8 activation in individual liver cell types. While hepatocyte specific knockout provided protection from liver damage an ubiquitous deletion of caspase8 triggered more injury and inflammation. This was finally related to a significantly stronger activation of the liver progenitor cell compartment and more tissue remodelling.
Free radical production and oxidative stress are known to increase in liver during aging, and may contribute to the oxidative damage, which plays an important role in the aging process. These changes increase during menopausal condition in females when the level of estradiol is decreased. The objective of this study was to observe the changes in activities of membrane linked ATPases (Na+K+ ATPase, Ca2+ATPase), antioxidant enzymes (superoxide dismutase, glutathione-Stransferase), lipid peroxidation levels, lipofuscin content and membrane fluidity occurring in livers of female rats of 3, 12 and 24 months age groups, and to see whether these changes are restored to 3 months control levels rats after exogenous administration of17-β-estradiol (E2).
The aged rats (12 and 24 months) were given subcutaneous injection of E2 (0.1Ag/g body weight) daily for one month. After 30 days of hormone treatment, experimental animals of all the groups were sacrificed and livers were isolated for further study. The results obtained in the present work revealed that normal aging was associated with significant decrease in the activities of membrane linked ATPases, antioxidant enzymes, membrane fluidity and an increase in lipid peroxidation and lipofuscin content in livers of aging female rats.
The present study showed that E2 treatment reversed the changes to normal levels. E2 treatment may be beneficial in preventing some of the age related changes in the liver by increasing antioxidant defenses.
More than 40 years have gone since Friedenstein described the so called colony forming unit fibroblasts. At least in the late 1990ties, a boom of stem cell research aiming for clinical application was trigger mainly but not only by publications of the groups of Pittenger, Caplan, Bruder and Prokop et al.. These publications include information of the last 20 years about the multipotency of mesenchymal progenitor cells and emphasize the significance of the human bone marrow as a cellular source for tissue regeneration. At those days “mesenchymal stem cells” (MSCs) seems to be a promising candidate to regenerate almost every local musculo-skeleteal defect. Getting more precise and detail data about the stem cell biology including differentiation potential, intracellular signaling, homing behavior and immunosuppression stem cell research was strongly promoted by industrial grants but also by public funding. There was great euphoria and hope to treat even systemic diseases such as osteoporosis, osteogenesis imperfecta and others diseases sufficiently by cell therapeutics.
In contrast to the rapid increase in knowledge and a spirit of optimism in MSC research, the regulatory authorities observe the clinical application of autologous progenitor cells more critically and sometimes with suspect. This skepticism seems to be based on the indistinct use of the word “stem cell” in many publications but also on a lack of clinical reliable data justifying a broad application besides healing attempts. In addition, the use of and research on embryonic stem cells was controversly discussed in public leading to new regulations and laws on the national and European level. Third, due to a lack of money in the health care systems insurance companies did not promote or reimburse new treatment concepts including tissue regeneration by MSCs.
Especially during the last few years controversy has arisen regarding the role and relevance of MSC in orthopaedic surgery. In contrast to the limited results in cartilage regeneration, bone regeneration was successful as shown in several clinical case series and is therefore one major focus for potential clinical application. Some investigators as the authors of this abstract suggested that the application of bone marrow aspiration concentrate (BMAC) is a valuable tool to stimulate local bone formation.
This hypothesis is based on recent data which demonstrate that a) MSCs are not the exclusive source of osteoblast, b) hematopoietic cells promote osteoblastic differentiation and c) both cell lineages are located next to each other in their physiological niches.
However, it is questionable if the relatively small number of living spongious MSC is sufficient to induce relevant bone regeneration in vivo.
Therefore osteoconductive or osteoinductive bone substitutes are essential mimicking as a scaffold and osteopromoting microenvironment. In a review of the literature and the presentation of own in vitro but also in vivo data it is demonstrated that especially progenitors from the adult bone marrow are still promising for the treatment of defined musculoskeletal disorders. Compared to tissues with a complex three dimensional histoarchitecture such as cartilage or muscle, bone regeneration seems to be most effective. However, controlled and prospective clinical trials are strongly required to establish orthopaedic cell therapy as a standard procedure in the near future.
The pourpose of this study was to evaluate the efficacy and the survival of local injection of allogenic MSC marked with Red Fluorescent Protein (RFP) (Lentigen-Italy) in collagenase induced tendonitis in the ovine Achille's tendon. The study was performed after the approval by the National Animal Care and Use Committee
Four sheep (2 years old, female, 45 bwt) have been enrolled in the study. After some days for the acclimatation, the sheep have been investigated to exclude any previous Achilles' tendon lesion. Three weeks before starting of the study one sheep was randomly selected for Bone Marrow harvesting for MSCs cultivation. The MSCs obtained has been trasfected with a lentivirus for integration of a gene for expression of Red Fluorescent Protein (RFP). After a week the other 3 sheep was injected in both Achilles' tendon with 400 U.I. of Collagenase IA of Cl. hystoliticum (Sigma-Aldrich-Italy). After two weeks the left Achilles' tendon of each sheep was injected with a solution of 6x106 RFPMSCs ( MSCRFP ) in 1 ml of fibrine glue (TISSUCOL , Baxter). The remaining tendons were used as negative control and received the same volume of saline solution as placebo. At 3-4-6 weeks from the treatment the tendons were harvested and evaluated for morphology, collagen I and III expression, and visualized at fluorescence microscope to assess RFP expression of the grafted MSCRFP. The results of these investigations evidenced the presence of MSCRFP in the treated tendons respect to the control ones at 3, 4 and 6 weeks after the treatment. Moreover, the RFP positive tissue showed high expression of collagen I and low collagen III with good morfphology in comparison to the lesions treated with placebo. The presence of high expression of collagen I and low collagen III with good morfopholgy, in term of restored tendon architecture, can be related to the MSCRFP injected into tendon lesions, as a large number of cells can survive in the site of injection.
These results showed that intralocal administration of MSCs into the tendon lesion can lead to a good effect on injured tendon . The local infusion delivery entails injecting MSCs directly into the tissue of interest and guarantees a higher number of engrafted cells and optimal therapuetic effect.
Besides the survival of high numbers of positive RFP -MSCs in treated samples have been demonstrated at 3, 4 (Fig.1) and 6 weeks from the treatment. We have evaluated also that quality of tendon healing in MSCRFP treated tendons has been based on a better architecture of collagen fibers and high expression of collagen I respect to collagen III (Fig.2), related to the control tendons.
The data obtained in this study confirm that MSCs allograft have a positive effect on tendon healing, its lack of significant immunogenicity permitting allogenic transplantation without immunosuppressive drugs and that the local injection in the tendon allows the survival of MSCs with good engraftment effincency.
Understanding pathways that control normal bone development is imminent for deducing novel therapeutic targets, which could be aimed at disease intervention in the clinic. The Wnt signaling pathway for instance plays a central role in osteogenesis and human disease including bone cancer, with β-catenin (CatnB) being a key mediator of the canonical signaling cascade. In contrast to the classic canonical Wnt ligand Wnt3a, non-canonical Wnt5a may trigger inactivation of CatnB transcriptional activity in a cell context specific manner. While older evidence places canonical Wnt signaling at the regulation of bone formation, newer studies suggest an involvement of Wnt5a in the regulation of mesenchymal commitment. Here, we use murine embryonic stem cells (ESCs), an extremely versatile stem cell that undergoes all developmental stages from an unspecified cell to a mature matrix calcifying osteoblast when differentiated in vitro, as a model to investigate how canonical and non-canonical Wnt ligands affect CatnB levels and regulate proper osteogenic progression.
Using stable T-Brachyury::GFP and LEF/TCF::GFP ESC lines and recombinant Wnt ligands, we were able to show that CatnB transcriptional activity was up-regulated during early mesendoderm formation concomitantly with T-Brachyury expression (differentiation day 3-5) carried by Wnt3a. In contrast, addition of Wnt5a between differentiation days 5 to 7 led to enhanced matrix calcification and expression of bone specific genes. Western blot analysis of fractionated protein lysates revealed diminished nuclear CatnB levels and decreased LEF/TCF transcriptional activity upon Wnt5a supplementation.
A stable ESC line encoding for a GFP reporter combined with a Wnt5a promoter was then used for FACS sorting to confirm the role of Wnt5a in osteogenic commitment. We were able to show that Wnt5a::GFP+ progenitors sorted on day 7 after differentiation initiation were characterized by an enhanced mineralization capacity when compared to GFP- cells. In addition, a second Wnt3a responsive phase was detected that correlated with subsiding Wnt5a expression and enhanced expression of genes specific for mesenchymal cells.
In summary, different members of the Wnt family seem to function at different time points during differentiation to enhance osteogenesis.
For tissue engineering applications it is essential that the used stem cells elicit immunomodulatory functions. It is well known that bone marrow mesenchymal stem cells avoid immune responses by the release of different immunosuppresive factors.
Since jaw periosteal cells (JPCs) represent a suitable stem cell source for bone regeneration in the oral and maxillofacial surgery we analysed the expression of MHC molecules and immunosuppressive factors in untreated, osteogenic and adipogenic differentiated JPCs.
By FACS analysis undifferentiated JPCs were shown to be MHC class I positive, MHC class II negative and they expressed intracellularly intermediate levels of HLA-G being a MHC class I-like protein. After induction of osteogenesis JPCs maintained basically their MHC expression whereasinduction of adipogenesis led to decreased levels of MHC class I and HLA-G expression. By microarray analysis we detected elevated expression levels of immunosuppressive factors such as Galectin-1 (Gal-1) and -3, prostaglandin E2 receptor and synthase, semaphorin 3A and hepatocyte growth factor (HGF) during JPC osteogenesis. These expression patterns were validated by quantitative RT-PCR, proteome arrays and/or western blot.
Our preleminary data show that JPCs might elicit immunosuppressive functions by the release of Gal-1, -3, HGF and HLA-G. Moreover, they maintain their MHC expression during in vitro expansion and osteogenesis but not during adipogenesis.
Ligaments link bone to bone, whereas tendons connect muscles to bone. Both tissues transmit the forces developed by muscle contractions across joints, stabilize these or produce motion. Tendons originate in muscle (musculo-tendinous junction) and insert into bone forming an osteotendinous junction (OTJ, enthesis). Ligament-tobone and tendon-to-bone interfaces (entheses, OTJs) serve to dissipate stress between soft tissue and bone. Surgical reconstruction of these interfaces is an issue of considerable importance as they are prone to injury and the integration of bone and tendon/ligament is in general not satisfactory. Soft tissues like cartilage, tendons and ligaments are poorly vascularized and heal slowly. Tendon-to-bone healing is a rather slow process and involves complex biological activities between these nonhomogenous soft and hard tissues.
As a novel mean, to regenerate tendon-bone attachment sites we report here the stem cell-dependent spontaneous formation of fibrocartilaginous and fibrous entheses in heterotopic locations of the mouse if progenitors possess a tenogenic and osteo-/chondrogenic capacity. The tenogenic and osteo-/chondrogenic competence was mediated by viral expression of the biologically active Smad8 signaling mediator (Smad8ca) and of Bone Morphogenetic Protein 2 (BMP2) in murine and human mesenchymal stem cells (MSCs). Modified MSCs were implanted in subcutaneous or intramuscular sites of the mouse.
The stem cell-dependent enthesis formation was characterized histologically by immunohistological approaches and by in situ hybridization. We have already described the Smad8-dependent formation of tendon-like structures before (Hoffmann et al., J. Clin. Invest., 2006). Using this system, we were able to initiate the MSC-dependent generation of fibrocartilaginous tendon-bone junctions of murine MSCs after heterotopic implantations of modified stem cells. Viral modification of human MSCs (hMSCs) exhibited a similar result: hMSCs expressing both the biologically-active Smad8ca and BMP2 generate ectopic tendon-bone insertions, however, without fibrocartilaginous elements. Moreover, it could be demonstrated that Smad8ca expression alone was sufficient for the formation of tendon/ligament-like structures without bony elements.
We conclude that the Smad8/BMP2-dependent modification of MSCs may contribute to regenerative therapies for the stem cell-dependent establishment of tendon-bone attachment sites destroyed in accidents or by chronic inflammatory diseases
As in vitro creation of large pieces of bone still remains a challenge, use of hybrid-implants offers a bridge between tissue engineering and complete metal or ceramic implants. Regarding flexibility and stability Nitinol is a promising material for bone implants. Adipose derived stem cells (ASCs) carry the potential for osteogenic differentiation and offer an alternative to mesenchymal stem cells as they are easy available from every patient. Pre settlement of an implant with autologous ASCs might prevent inflammatory reactions on the implant and might shorten the time to osseointegration.
The implant structure should allow ingrowth of ASCs and vessels, but still has to fulfill its stabilization duty. So the implant needs a high-resolved inner structure. Selective laser melting (SLM) offers a high-resolution manufacturing process for individual implant design.
Meshes and cage-like endostructured implants were processed by SLM with web width down to 509m. Mesh width varied between 250 and 500μm. The implants were colonized with ASCs and cultured for 24h up to 6 weeks. Osteogenic differentiation was induced chemically (by culture media) or mechanically (by application of pressure on the cages in customized clamps). Cells on the Nitinol structures were analyzed by immunfluorescence, histology and scanning electron microscopy.
ASCs adhere, proliferate and differentiate osteogenically on Nitinol structures. Osteogenic differentiation can be induced chemically or mechanically, which was demonstrated by expression of osteogenic factors as BMP-6. Furthermore, calcium inclusions can be detected. Visual appearance and measured vitality of cells are normal.
Whether used as full or foamy implant the general problems of Nitinol are the same as with other implant materials:
Long time of osseointegration or insufficient integration, inflammatory reactions, cell death in sponge forms because of insufficient cell supply (lack of blood vessels). These problems could be solved by the combination of a cellular bioactivation of the surface with an alternative implant structure.
As the work at hand shows, osteogenic differentiation of ASCs can be induced by mechanical stress. No predifferentiation in vitro or the use of growth factors in vivo is needed. This might shorten the time to osseointegration in vivo.
Cells show high vitality as the open structure allows optimal nutrition and oxygen supply.
Autologous ASCs and Nitinol are a promising combination for processing customized osteoimplants with a cage-like structure.
In the bone marrow, hematopoietic stem cells (HSC) reside in endosteal and vascular niches. Their interactions with the niches are essential for the maintenance of HSC number and properties. The molecular composition and the matrix elasticity of the microenvironment play important roles in the differentiation of adult and embryonic stem cells. However, the mechanisms responsible for how elasticity plays a role in the HSC niche are still unclear. Osteoblasts, the major cellular component of the endosteal HSC niche, flatten during HSC mobilization when stimulated by the nervous system. We hypothesized (I) that a flat osteoblast during mobilization has different elastic properties from a “normal” osteoblast under steady-state conditions and (II) that the HSC in contact with the osteoblasts can react after sensing this change in elasticity during mobilization.
KG-1a cells were obtained form DSMZ and human hematopoietic stem and progenitor cells were isolated from umbilical cord blood after informed patients consent. Confocal and atomic force microscopy were applied in a simplified in vitro model of the endosteal stem cell niche in order to investigate the cell height and elasticity of osteoblasts.
Polyethylene glycol diacrylate hydrogels with tunable elasticity were developed and applied for cell adhesion and migration studies.
We could show, that the osteoblast flattening during mobilization is accompanied by osteoblast stiffening, demonstrating that during mobilization not only biochemical but also mechanical properties of the niche are modulated. HSC are able to sense differences in the elasticity of their underlying substrate and therefore respond with altered adhesion and migration, which could facilitate the exit of HSC from the niche.
Concluding from our data, we propose the following model for how the nervous system uses the elasticity of the osteoblasts in the endosteal HSC niche to modulate HSC adhesion and migration: During mobilization, signals from the nervous system lead to osteoblast flattening and stiffening. The HSC in the niche respond to the change in elasticity of their cellular support with increased adhesion and migration, which facilitates the egress of HSC from their niche. Thus, matrix elasticity is an important factor in regulating the retention of HSC in the endosteal niche and should be considered in attempts to propagate HSC in vitro for clinical applications.
During normal aging, brain experiences structural, molecular, and functional alterations. Aging in females and males is considered as the end of natural protection against age related diseases like osteoporosis, coronary heart disease, diabetes, Alzheimer's disease and Parkinson's disease. Protection from age-related disorders is provided by several factors, including estrogens. These changes increase during menopausal condition in females when the level of estradiol is decreased.
The objective of this study was to observe the changes in activities of superoxide dismutase (SOD), glutathione S-transferase (GST), Ca2+ATPase, intracellular calcium levels, DNA degradation and glucose transporter 4 (GLUT4) occurring in brains of female albino Wistar rats of 3 months (young), 12 months (adult) and 24 months (old) age groups, and to see whether these changes are restored to normal levels after exogenous administration of estradiol.
The aged rats (12 and 24 months old) (n= 8 for each group) were given subcutaneous injection of 17-β-estradiol (0.1μg/g body weight) daily for one month. Controls animals received The aged rats (12 and 24 months old) (n= 8 for each group) were given subcutaneous injection of 17-β- estradiol (0.1Cg/g body weight) daily for one month. Controls animals received an equal volume of vehicle. After 30 days of hormone treatment experimental animals of all the groups were sacrificed and brains were isolated for further study.
The results obtained in the present work revealed that normal aging was associated with significant decrease in the activities of Na+K+ ATPase, SOD, GST, Ca2+ATPase and GLUT4 levels in the brains of aging female rats, and an increase in DNA degradation and intracellular calcium levels. Administration of E2 brought these changes to near normalcy.
It can therefore be concluded that E2's beneficial effects seemed to arise from its antioxidant and antilipidperoxidative effects, implying an overall neuroprotective and anti-aging action. The results of this study will be useful for pharmacological modification of the aging process and applying new strategies for control of age related disorders.
Seasonal spawning and ambisexual (sex changing) teleost fish species show postembryonic stem cell based differentiation processes which are thought to be strictly related to foetal (intraovum and intrauterine) development of homeothermic vertebrates. Seasonal gonadal recrudescens and the initial steps of heterologous gonadal tissue development in the course of sex change share common features. Both processes are based on motile stem cells. These cells can be isolated from fish brain and seem to be of neuroectodermal origin.
Histological examination of fish during the gonadal recrudescent or sex inverting period and primary cell cultures derived from fish of the corresponding status show that migrating stem cells are connected to their origin via tunnelling nanotubes / filodia in the 10 mm range. In appropriate cell culture systems an intra syncytial transport of granula and a extra cellular transport of cytoplasm components originating from the primary cells along the tunnelling nanotubes is recognizable. Although these cells are derived from somatic adult fish transcription of genes which are thought to be strictly related to embryonic development of oviparous vertebrates could be observed in stem cell bearing tissues and corresponding tissue cultures. Parallel investigation of the Koi Herpes Virus (Cy HV- 3) disease of carp suggests that stem cells under investigation are the primary target and virus depot in latent infected fish.
We conclude that postembryonic proliferation of stem (germ) cells is under the control of a long distance signalling pathway. The results implicate that spatial and temporal separation of gene amplification -transcription and - translation could play a role in control of adult stem cell and stem cell transmitted viral infections. The robustness and reproducibility of our tissue culture systems represent a promising and innovative tool to investigate the physiological integration of stem cells with embryonic characteristics on the organisational level of a somatic adult organism.
We have found that phenotypically-defined, plastic-adherent human dermal mesenchymal stem cells (MSCs) have immunomodulatory capacities. To unravel, whether regulatory T cells are involved, total adherent dermal cells, adherent dermal MSC subsets and, as a control, bone marrow-derived (BM-) MSCs, which all lack the expression of CD80/86, were subjected to functional in vitro assays with CD25-depleted naive helper T (Th) cells and stimulation with αCD3 or αCD3/CD28 mAbs. Using flow cytometry, T cell proliferation was visualized via carboxyfluorescein succinimidyl ester dilution and the differentiation of dividing cells into regulatory T cells was evaluated by counterstaining of the transcription factor FoxP3. Stimulation of naive Th cells with αCD3/CD28 and coculture with dermal cells or BM-MSCs led to a marginal increase of FoxP3-expressing T cells, while stimulation with αCD3alone significantly increased the FoxP3 expression from a value of 4.09%±1.26 in stimulated T cells alone, to 15.36%±1.81 after coculture with dermal cells (P< 0.003, n=7) and 10.98%±2.0 with BM-MCSs (P< 0.02, n=6). Interestingly, CD90+ dermal cells induce more FoxP3 expression in Th cells than CD90- cells (19.14%±4.02 vs. 8.11%±1.70, n=3). In line with this observation, a higher percentage of FoxP3-expressing Th cells were detected upon coculture with CD271- dermal cells (> 90% coexpressed CD90) compared with CD271+ cells (50-70% coexpressed CD90; 14.56%±2.54 vs. 9.62%±2.56, n=3).
Our data suggest that skin-resident MSCs may coordinate the interactions that are necessary to initiate the induction and proliferation of regulatory T cells to maintain the immune homeostasis in human skin and to prevent excessive immunopathology.
Very small embryonic-like (VSEL) cells have been described as putatively pluripotent stem cells present in murine bone marrow and human umbilical cord blood (hUCB) and as such are of high potential interest for regenerative medicine. However, there remain some questions concerning the precise identity and properties of VSEL cells, particularly those derived from hUCB. For this reason, we have carried out an extensive characterisation of purified populations of VSEL cells from a large number of UCB samples. Consistent with a previous report, we find that VSEL cells are CXCR4+, have a high density, are indeed significantly smaller than HSC and have an extremely high nuclear/cytoplasmic ratio. Their nucleoplasm is unstructured and stains strongly with Hoechst 33342.
A comprehensive FACS screen for surface markers characteristic of embryonic, mesenchymal, neuronal or hematopoietic stem cells revealed negligible expression on VSEL cells. These cells failed to expand in vitro under a wide range of culture conditions known to support embryonic or adult stem cell types and a microarray analysis revealed the transcriptional profile of VSEL cells to be clearly distinct both from well-defined populations of pluripotent and adult stem cells and from the mature hematopoietic lineages. Finally, we detected an aneuploid karyotype in the majority of purified VSEL cells by fluorescence in situ hybridisation. These data support neither an embryonic nor an adult stem cell like phenotype, suggesting rather that hUCB VSEL cells are an aberrant and inactive population that is not comparable to murine VSEL cells.
Cell culture protocols have specific requirements in regenerative medicine. Human platelet lysate (HPL) has proven as effective substitute for fetal calf serum (FCS) without the risk of xenogeneic immune reactions or transmission of bovine pathogens. Heparin needs to be added as anticoagulant before addition of HPL to culture medium - otherwise HPLmedium forms a gel within few hours. Here, we demonstrated that such HPL-gels provide a suitable 3D-matrix for cell culture which - apart from heparin - consists of the same components as the overlayered culture medium.
Mesenchymal stromal cells (MSC) grew in several layers at the interface between HPL-gel and HPL-medium without contact to any artificial biomaterials. Notably, proliferation of MSC was much higher on HPL-gel compared to tissue culture plastic (TCP).
Furthermore, the frequency of initial fibroblastoid colony forming units (CFU-f) was increased on HPL-gel. The viscous consistency of HPL-gel enabled passaging with a convenient harvesting and re-seeding procedure by pipetting of cells together with their HPL-matrix - this method does not require washing steps and can easily be automated. Alternatively, plasmin could be added to dissolve HPL-gels. The immunophenotype and in vitro differentiation potential of MSC were not affected by culture-isolation on HPL-gel. Taken together, HPL-gel has many advantages over conventional plastic surfaces: it facilitates enhanced CFU-f outgrowth, increased proliferation rates, higher cell densities and nonenzymatic passaging procedures for culture expansion of MSC.
Over the last decade, cord blood (CB) has become a viable option for haematopoietic stem cell (HSC) transplantation. The use of CB is hampered by the insufficient number of SC per graft required for adult patients. Therefore, several strategies have been followed in order to ex vivo expand the number of HSC.
Here we use biomaterials for CB-HSC expansion. First, seventeen degradable biopolymers and synthetic polymers from our biomaterial bank (Neuss et al., Biomaterials 2008) were assessed for basic compatibility. Viability, cytotoxicity and apoptosis were analyzed using a standardized method (Ferreira MV et al., Journal of Biomolecular Screening 2011).
A group of six compatible materials was selected further. PVDF, Texin, Resomer LR704, Resomer RG503, PCL and Fibrin were used to culture HSC during 7 days. Long-term viability, cytotoxicity and apoptosis were measured. CFSE staining was used to evaluate cell proliferation on the material surfaces. Together with CFSE, the immunophenotype of the cultured cells was analyzed by using the progenitor markers CD133, CD38 and CD34. Colony-forming unit assays and morphology (SEM) were also evaluated. Hematopoietic reconstitution of NOD/SCID mice is currently under assessment (Ferreira MV, Biomaterials 2011, submitted).
To evaluate the contribute of each biomaterial to the potential of expansion, proliferation curves (CellTiter-Blue® Cell Viability Assay) were created for HSC cultured over a period of 17 days in each of the materials, either using a standard culture cocktail or a previously optimized cocktail (Ferreira MV, Journal of Tissue engineering and Regenerative Medicine 2011, in revision). So far, HSC cultured in fibrin proliferate better and exhibit a more primitive phenotype in comparison to the control (TCPS). In the future, 3D scaffolds will be tested for HSC expansion.
Various transmittable diseases from donor blood led to explore in-vitro manufacturing, cost effective, clinically applicable RBCs in large scale with several defined protocols worldwide. Commercially available alternative blood substitutes and medicines to increase the blood constituents are unstable with less oxygen carrying capacity and have not been successful in replacing donor derived RBCs. Large scale production of RBCs in-vivo depends on potential cell sources, bio-physiological parameters, erythroid inducers and sterile, non reactive, non -tumorogenic expansion media, to produce stable and functionally efficient RBCs. We formulated an efficient bone marrow milieu considering the physiological factors, favouring the differentiation of haematopoietic progenitors to clinically applicable and pathogen free mature RBCs.
The mononuclear cells were isolated from cord blood and bone marrow of living human donor with a pre-informed consent. The cells were nourished with media derived from human plasma of same blood group, bone marrow milieu and erythropoietic differentiation inducers (following laboratory proprietary protocol). The Temperature, Oxygen concentration, pH, Osmolarity of the cultures were maintained optimally. Finally, the cultured RBCs were subjected to various assays for cell proliferation, differentiation, haemoglobin content, and structural and functional properties of cultured RBCs such as oxygen affinity and dissociation ability etc.
The day 4 onward burst colony forming units were observed proving the erythroid lineage , which was monitored and documented on daily basis till the transformation happened from nucleated to enucleated adult RBC formation on 17th day. The scalability of the functional RBCs appeared to be 15 times of the originally seeded mononuclear cell number (2x106/ml of culture media) in the culture flask and it was reproducible. The cells were in majority mature RBCs (95% by FACS) with adult haemoglobin, with trivial presence of other lineages. The adult haemoglobin had been proved by haemoglobin electrophoresis. Cultured RBCs were morphologically and functionally efficient. The scalability of production was assured in bioreactor culture expansion with indigenous novel biocompatible nano-fiber scaffold. Oxygen affinity and dissociation were efficient with five sets of experiments.
This method of producing RBCs in large scale with a simple in-vitro reproducible protocol for making scalable and functional RBCs which is devoid of WBCs would be advantageous for frequently transfused patients, who are suffering from blood dyscrasias and exsanguination due to severe injury.
Further in-vivo stability evaluation with various animal experiments and clinical trials would make the possibility of clinically applicable, off the shelf packed RBCs which do not require screening for viral diseases as the cell source and media are pre-tested.
Mesenchymal stem cells (MSC) control hematopoietic stem cell (HSC) differentiation and proliferation by production of growth factors/cytokines, extracellular matrix (ECM) as well as cell-cell-interactions. It was shown that the co-culture of bone marrow-derived MSC (BM-MSC) with umbilical cord HSC (UC-HSC) can be utilized for cord blood expansion purposes as they preserve the necessary hematopoietic microenvironment. For clinical transplantation, it may be preferred to obtain HSC and MSC from the same donor, thereby eliminating complications resulting from a HSC and MSC genetic mismatch. In a previous study, we established a three-dimensional bone microenvironment using MSC and a collagen matrix (Schneider et al., Biomaterials 2010). In the current study we analyzed the potential of UC-MSC to act as a stromal support for UC-HSC compared to BM-MSC in our established three-dimensional bone marrow niche.
As previously described, we generated 3D-collagen gels with and without embedded UC- and BM-MSC. Enrichment for CD34+ cells from umbilical cord bloodwas performed by using immunomagnetic beads. We analyzed cell divisions of HSC by using cell division tracking with CFSE. Further, we analyzed HSC migration and immunophenotypic differentiation as well as remodelling of the extracellular matrix by MSC. We demonstrated a positive influence of the cell-free, three-dimensional collagen gel on HSC proliferation in comparison to monolayer culture.
The proliferation rate was even enhanced when HSC were co-cultured on collagen gels containing UC-MSC or BM-MSC. Nevertheless, the co-culture of HSC and UC-MSC also accelerated the differentiation of HSC. CD34 expression decreased after a cultivation period of up to 14 days, whereas CD38 expression was up-regulated as well as the expression of the differentiation markers CD13, CD45 and CD56. In contrast, in co-culture with BM-MSC the more primitive CD34(+) CD38(-) fraction of HSC was maintained in longterm-culture. Histological analysis demonstrated that UC-MSC as well as BM-MSC might have a chemotactic influence on HSC as migration into the collagen gel was accelerated by MSC. Migrated HSC showed the typical cobblestone-like pattern of proliferating hematopoietic progenitor cells in the collagenous matrix. Besides, HSC maintained their differentiation potential in the collagenous matrix and showed signs of maturation. Interestingly, in particular UC-MSC caused intensive ECM remodelling (immunohistochemistry) and produced a dense matrix of fibronectin, osteopontin and collagen I, typical for the bone marrow niche.
Concluding, in our three-dimensional culture system BM-MSC support in particular the maintenance of the primitive CD34+ HSC subpopulation in long-term culture while UC-MSC accelerate proliferation, differentiation and matrix remodelling. Our culture system is an optimal system for studying HSC differentiation, proliferation and matrix remodelling in physiological and pathophysiological processes.
Poor MSCs survival after transplantation is one of the major challenges in their therapeutic application. Therefore, it is necessary to augment MSCs in order to improve the efficacy of cell therapy. In this study, we manipulated MSCs with a cytoprotective factor i.e Nrf2 and evaluate its efficiency flowing MSCs transplantation to cisplatin-induced acute kidney injury in a rat model.
Mesenchymal stem cells were isolated from bone marrow. Nrf2 was isolated and TOPO cloned into the pENTR vector. The recombinant vector was transfered into pAD/CMV/V5- DEST vector by gateway technology. Recombinant adenovirus was produced in AD293 cells, followed by infecting into MSCs. The Nrf2 engineered MSCs were exposed to hypoxic, serum deprived and oxidative stress conditions followed by evaluation of the cells viability and apoptosis. Finally, Nrf2-MSCs were transplanted to cisplatin-induced acute kidney injury in rats and their therapeutic efficacy was assayed by blood urea nitrogen, creatinine determination and morphologic analysis.
Nrf2 was successfully expressed in MSCs. Nrf2-MSCs retained their multi-differentiation capacity. Comparing to the controls, following exposure of the Nrf2 transduced cells to hypoxia, serum deprivation and oxidative stress conditions, cell viability was found to be higher, while their apoptosis rate was lower. Nrf2 alleviates cisplatin-induced acute kidney injury.
Our findings are good demonstration of how an understanding of the cellular stress response can be leveraged for practical application. Enhanced anti-apoptotic and anti oxidative capabilities of MSCs following Nrf2 infection via adenoviral vectors, could be a graft cell death prevention strategy in transplantation and may emerge an alternative plan for stem cell therapy.
Transplantation of human islets can cure type 1 diabetes, but amongst other factors, the shortage of donor islets limits its wide-scale application. Differentiation of beta cells from pluripotent sources is an attractive alternative source,but the efficiency of in vitro differentiation protocols is limited. We established a robust in vitro process to direct mouse-derived pluripotent embryonic stem cell (mESC) differentiation into pancreatic islet bodies. Two factors proved effective in increasing differentiation efficiency. First, the addition of a high concentration of Activin A during embryoid body formation increased differentiation into definitive endoderm (DE). Using a transgenic mouse cell line carrying a Sox17-Cherry fusion (SCF) knock-in, we were able to analyze the amount of DE cells by fluorescence- activated cell sorting.
Activin A increased the number of SOX17-positive cells approximately 2-fold compared to the control group without Activin A treatment. A second factor that markedly increased islet body differentiation was the use of 3D culture.
Embryoid bodies enriched for definitive endoderm were transferred into 3D culture using Matrigel. By day 28 of differentiation, these three-dimensional islet bodies expressed pancreatic lineage markers PDX1, NGN3 and CPeptide,and a yield of seven islet bodies per EB were obtained. Whilst the generation of complex three-dimensional organ tissues in vitro remains a major challenge for translational studies, it is hoped that similar modifications may also improve differentiation of beta cells from human ES and iPS cells and eventually allow for transplantation of pluripotent derived islet bodies.
Diabetes and obesity are risk factors for tendinopathy, tendon injury and impaired tendon healing. So far, the mechanisms of tendon impairment in diabetic patients remain a matter of debate. Altered metabolic parameters as well as changes in the vasculature may play a role.
Along our search for stem cells suitable for tendon repair, we discovered a tendon-derived cell type readily differentiating towards insulin producing cells in vitro. Here we describe a population of tendon cells expressing the pancreatic ß-cell associated proteins Insulin and Glucagon in vivo.
Tissue samples from intact human biceps-, supraspinatus, and semitendinosus tendons were obtained with patients´ informed consents, tissue donors were aged from 23-63 (n=5) years.
By immunocytochemistry, Laser Capture Microdissection and quantitative PCR we show that these cells are mainly located in the perivascular area, but also in the dense, collagenous parts of tendon tissue. Single cell PCR analysis of isolated tendon perivascular cells reveals a coexpression of Insulin with the stem cell-associated markers Oct4 and Nanog. Both Insulin and Insulin-related mRNA are significantly reduced in Achilles tendons of rats treated with 60μg/kg of the ß-cell specific cytostatic agent Streptozotocin 5 days after the treatment. Biomechanical testing revealed that this treatment also results in a significant (p=0,018) reduction in tendon tensile strength of 39,9% (n=12) after 5 days.
In our hands, enzymatically released tendon cells neither show glucose-dependent insulin secretion nor staining with Dithizone, a zinc-chelating agent commonly used for the detection of pancreatic ß-cells.
These findings indicate that, despite several similarities with pancreatic ß-cells, these tendon- derived cells have different properties and functions.
Whether these cells are affected by changing metabolic parameters or diabetes, or whether these cells may be a potential source for tissue engineering or cell therapy in diabetes treatment will have to be elucidated by further experiments.
The mammalian embryo is said to have a high amount of plasticity, as observed by the ability of the embryo to develop in a wide variety of conditions in vitro. Following fertilization, many changes occur to an early stage embryo in regards to metabolism and embryonic nutrition. The requirement for glucose (Glc) during development is thought to be for the biosynthesis of macromolecules such as phospholipids and nucleic acids, however studies have shown that diabetic Glc levels can be toxic to the developing embryo. For instance, newborns of diabetic mothers may exhibit skeletal malformations.
Embryonic stem cells (ESCs) are pluripotent cells that are capable of modeling osteogenesis and thus often serve researchers to study embryonic skeletal development. Against this background, we aimed to gain insight into the molecular mechanism whereby Glc may influence the process of osteogenesis.
Murine ESCs were differentiated in media containing either physiological (1.0 g/l) or diabetic (4.5 g/l) concentrations of D-glucose while simultaneously being triggered into osteogenesis using 1alpha,25(OH)2 vitamin D3. The osteogenic differentiation ability of the cells was evaluated by histochemical stainings, quantitative bone marker gene expression and flow cytometric analyses at distinct time points of maturity.
In addition, the calcification of the secreted extracellular matrix was quantified and signaling pathway activation studied.
mRNA expression and van Gieson staining suggested that more collagen was produced and secreted by cells differentiated in diabetic conditions, while matrix calcification was severely impaired, suggesting that cells were prevented from maturation. Instead, cells differentiated in physiological Glc conditions showed normal osteogenic progression,including stage specific activity of alkaline phosphatase. Follow-up studies then revealed that the Glc-mediated osteogenic impairment was already detectable at the level of an osteoprogenitor state. Western blotting then allowed us to correlate persisting Col I mRNA expression to elevated protein levels of AKT and activated AKT (phosphor-S473),which has been previously reported to control Col I mRNA expression in other cell types. Stage-specific blockage of AKT activation using the AKT inhibitor 124005 restored normal Col I mRNA levels and led to a calcification rescue in diabetic conditions.
In conclusion, we were able to show that diabetic Glc inhibits the formation of matrix calcifying osteoblasts in embryonic stem cell cultures possibly through AKT mediated persistent expression of Col I mRNA.
Vascular calcification in chronic kidney disease (CKD) patients has emerged as a tightly regulated, coordinated and osteoblastic process resembling bone morphogenesis The current study is based on the hypothesis that mesenchymal stem cells (MSC) constitute critical cells for pro-calcific ECM remodeling in CKD patients. Human MSC were cultured in media supplemented with pooled sera from either healthy or uremic patients (20%). Exposure to uremic serum enhanced the proliferation of MSC (cell counting, BrdU incorporation) whereas apoptosis and necrosis were not affected (annexin V and 7-AAD staining). Uremic serum exposed MSC recapitulated osteogenesis by matrix calcification and expression of bone-related genes (BMP2-receptor, ALP, osteopontin, Runx2) within 35 days. The uremic serum-induced osteogenesis was shown to be BMP2/4 dependent and was completely blocked by a BMP2/4 neutralizing antibody or its natural antagonist.
NOGGIN. Calcification and matrix remodelling were further analysed in a collagen-embedded osteogenesis model recapitulating the vascular collagen I/III environment.
The uremic serum induced calcification was shown to occur along collagen fibres as shown by SEM, energy-dispersive X-ray spectroscopy and von Kossa staining and was accompanied by extensive matrix remodelling.
MSC acquired a myofibroblastic phenotype, contracted the collagenous matrix and extensively remodelled the collagenous matrix by producing components of the ECM.
These changes in the artificial vascular wall were comparable to remodelling process observed in arteries of CKD patients (n=12) compared to vessels of young children (n=10).
Concluding, uremic serum induced in a BMP2/4-dependent manner an osteoblast-like phenotype in MSC accompanied by matrix remodelling and calcification comparable to the remodelling and calcification in CKD patients' arteries.
Gene targeting by homologous recombination via customized zinc-finger nucleases (ZFN) is a powerful method to manipulate the genome and correct genetic defects. Although efficiency of ZFN based homologous recombination has been shown to be significantly higher than by means of conventional gene targeting, the selection of suitable clones still requires cells that proliferate in culture. Clinically applicable ZFN-based gene correction in patient-specific cells was hardly possible so far, due to the inability to sufficiently expand most adult (stem and progenitor) cells
Regenerative medicine is in need of a solid animal model as a link between rodents and human to evaluate functionality, immunogenicity and clinical safety of embryonic stem cells (ESCs) or reprogrammed cell types. The common marmoset monkey (
For reprogramming bone-marrow derived mesenchymal cells of the common marmoset were used. In the presence of TAV, SB431542, PD0325901 and ascorbic acid, the cells were reprogrammed via a lentiviral SFFV driven quadcistronic vector system in mTESR medium and transferred onto mouse embryonic feeder cells at day 21.
The cells obtained showed typical ESC-like morphology and were positive for alkaline phosphatase and the endogenous pluripotency markers Oct3/4, Nanog, Sox2, KLF4 and MYC while exogenous genes were downregulated. From passage 19 on cells were also differentiated successfully to embryoid bodies. Marker genes and morphology of early neuronal progenitors, adipocytes, chondrocytes, osteogenic cells and megakaryocytes could be observed so far.
These cells appear to be fully reprogrammed iPS cells of the common marmoset which display pluripotency markers and can be differentiated into various cell types. They are a promising tool for animal models close to human for preclinical studies in the field of regenerative medicine.
Therapeutic application of pluripotent stem cells and their derivatives requires large quantities of cells generated in defined conditions which cannot be produced via adherent culture. Standardized fully controlled stirred tank reactors are widely used in biopharmaceutical industry for culture volumes of up to 10.000 L. However, standard cell lines used for the production or recombinant factors are usually homogenous and robust and therefore relative easy to expand. In contrast, cultures of hPSC (including hiPSC/hESC) are much more demanding, particularly regarding maintenance of pluripotency and translation to suspension. A direct transfer of these complex cultures to established stirred bioreactor systems is therefore challenging. In recent publications we have identified a defined culture medium that resulted in up to six-fold increase in cell numbers within 4-7 days 1-3 The culture system is based on initial single cell dissociation which is critical for standardized process inoculation of suspension cultures. Cells maintained a stable karyotype, expression of pluripotency markers and their potential to differentiate into derivatives of all three germ layers1-3. Here we will present unpublished results on successful process translation to stirred bioreactors. These data provide key steps to establish scalable mass expansion of hiPS/hES aiming at the translation of stem cell research to clinical practice. Experiments to optimize culture conditions regarding control of cell aggregation, feeding strategies and other parameters will also be presented.
The present experiment was carried out to study the role of different culture conditions on development of embryonic stem cell colony from early stage IVF derived embryonic embryos in buffalo. The 8-16 cell stage IVF embryos were made zona free and the clumped blastomere were cultured on matrigel and fibronectin coated tissue culture dish in culture condition viz. Control (C-I), M-I:C-I+ stem cell factor, SCF),M-II: C-I+SCF+bFGF and M-III:CI+SCF+bFGF+IGF1) and were cultured at 37oC, 5% CO2 and 90% relative humidity in CO2 incubator. Once the clumped blastomere cells multiplied and made stem cell colony, they were passaged mechanically. A total of 16 number of zona free embryos were cultured in each medium on matrigel or fibronectin coated dish. It was also observed that the ES cell colony formation was better on matrigel than on fibronectin.
However on both matrigel and fibronectin, the efficiency of embryonic blastomemere attachment, stem cell colony formation and propagation were significantly higher when ES cell medium was supplemented with only stem cell factor SCF(M-I) or supplemented with SCF+bFGF+IGF1 (M-III) followed by supplementation of SCF+bFGF (M-II) as compared to control media. Media-I and III were found to be significantly (P< 0.01) better as compared to Media-II, however, there was no significant difference between Media-II and control. The better ES cell colony formation on matrigel as well as on Medai-1 and 3 than fibronectin and Medium 2 and control may be due to the fact that the pluripotent gene expression was better in former conditions. It can be concluded that buffalo ES cell colonies can be established in ES cell medium supplemented with SCF alone or SCF+ bFGF4+IGF1 on matrigel and fibronectin synthetic matrices.
Pluripotent stem cells are capable of differentiating into all three germ layers namely, endoderm, mesoderm, and ectoderm. Since the reprogramming of somatic cells into pluripotent state has been very well established, induced pluripotent stem (iPS) cells hold the great promise in custom-tailored regenerative medicine therapies. Molecular and functional characterization of iPS cells revealed a strong correlation between aberrant expression of genes from Dlk1-Dio3 imprinting cluster and the germ-line transmission ability. Moreover, defects in the imprinting status of Dlk1-Dio3 gene cluster was also observed in iPS cells generated by various independent research groups, making it as a common defect occurring during reprogramming. The activation of Gtl2 expression (Gtl2on), one of the genes from Dlk1-Dio3 cluster, is currently being used as a bona fide marker of authentic pluripotent cells competent of germ-line transmission.
Recently, we have shown that during reprogramming germcell (GC) marker genes expression precede the expression of pluripotency-related genes. Hence, in the present study we aimed to analyze the role of GC marker genes in reprogramming and also in the activation of Gtl2 gene. Screening of several GC marker genes revealed that Stella, a maternal factor with a role in imprinting maintenance, as a potential candidate. We found that the expression of Stella along with the expression of known reprogramming factors (Oct4, Sox2, Klf4 and c-Myc) reduces the time needed for reprogramming and also increases the efficiency of iPS generation. Interestingly, addition of Stella to the reprogramming factor cock-tail leads to the generation of 1all Gtl2on iPS cells. Currently, we are investigating these cells for their pluripotent cell characteristics and germ-line transmission competency.
Induced pluripotent stem cells (iPSCs) may represent an ideal cell source for future regenerative therapies. Clearly, the development of iPSC-based therapeutic concepts requires suitable animal models. Nonhuman primates show the highest similarities to humans concerning the physiological, cellular and molecular level and macaques are frequently used in preclinical research and pharmacology. Considering the establishment of allogeneic / autologous cell transplantation models, we have generated iPSCs from cynomolgus monkeys (M. fascicularis, cyiPSC). Instead of the commonly human immunodeficiency virus (HIV) based reprogramming vectors that show poor transduction of simian cells, cynomolgus skin fibroblasts were reprogrammed using simian immunodeficiency virus (SIV) based vectors encoding OCT4, SOX2, KLF4, and c-myc. Resulting cyiPSCs show all typical characteristics of primate embryonic stem cells (ESCs). Culture characteristics and cell / colony morphology is almost identical to cynomolgus ESCs and cyiPSCs stain positive for typical pluripotency markers such as OCT4, NANOG, and TRA-1-60. CyiPSCs differentiate in vivo and in vitro into derivatives of all three germlayers. Furthermore, differentiation into functional cardiomyocytes could be demonstrated. Our data indicate that the generated cyiPSCs are highly similar to human iPSCs and should therefore represent an excellent cell source for allogeneic / autologous preclinical cell transplantation models.
This study was induced to investigate the possibility of reprogramming mesenchymal bone marrow stem cells (BMSCs) into endothelial cells by application of growth factors into culture media.
Isolation of BMSCs were performed by bone marrow aspiration of the mini-pig femur. Bone marrow was filtered with a 100μm cell strainer to eliminate bone remains and tissue fragments. After centrifugation pellet was dissolved in PBS-EDTA solution and the mononuclear cells were isolated by density gradient centrifugation with histopaque. Additionally, cells were washed with media and centrifuged further for 5min. The pellet was solved in medium containing endothelial cell growth factor supplement (10%). Medium was changed every second day. Confluent cells were analysed for the expression of eNOS by immunfluorescence staining and Western blot. Furthermore, the functionality of cells to release NO was examined by spectrophotometric investigation.
Isolated BMSCs cultured with endothelial cell growth factor exhibited a typical cobblestone-like endothelial cell phenotype. Immunfluorescence staining and Western blot analysis with eNOS antibody showed eNOS expression in the same manner than venous endothelial cells. Investigations of the NO release exhibited also the functionality of endothelial-like BMSCs to liberate NO, which is an essential feature for the functionality of endothelial cells.
The reprogramming of isolated pig BMSCs into endotheliallike cells with endothelial characteristics was possible by adding of endothelial cell growth factor. In future studies BMSCs could be a source for production functional endothelial cells.
This study aimed to explore relevant factors to optimise the composition of a serum-free defined culture medium intended to support the expansion of human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC) under feeder-free conditions
Mesenchymal stem cells (MSC) are multipotent adult stem cells. Bone-marrow derived MSCs are able to differentiate at least into osteocytes, adipocytes and chondrocytes
Preliminary experiments suggest that oxidative stress could be involved in this quiescent phase after isolation. By characterizing different isolates from C57BL/6 mice,general characteristics concerning proliferation and differentiation were analyzed. The cell populations displayed a heterogeneous marker profile, though all isolates did not show the expression of hematopoietic markers (CD34, CD45). Surprisingly, cell isolates displayed an increased proliferation in later passages. The cell populations consisted of unipotent, bipotent and tripotent cells with the tripotent isolates being considered as mMSC according to Pittenger. The osteogenic differentiation of the cells was analyzed in more detail by measuring the level of mineralization at different time points. Taken together, the isolation of mesenchymal stromal cells from C57BL/6 mice results in a heterogeneous cell population consisting of various precursor cells.
The better understanding of early human embryonic development has been hampered with the availability of samples and eventually addressed with in vivo embryonic development (ED). As an initial step we have recapitulated the early human ED with multilineage differentiation of human embryonic stem cells (hESC) integrated with sensitive genomics approach in our prior publication. To further explore the human ED and its subsequent application in toxicogenomics we have assigned the time kinetic hESC ED assays. The global transcriptional profiling and the subsequent gene ontology analysis reveal that from day 3 the developmental biological processes were identified. Also, the gene signatures identified during the embryonic development were used for the assessment of early developmental toxicity. The known developmental toxicants and the negative compound were treated at sublethal concentration during the Embryoid body development for 14 days. To demonstrate the toxicological application positive compounds methyl mercury, valproicacid and negative compounds mannitol were treated at IC10 and four times lower than IC10 concentrations. The principal component analysis showed, mannitol clustering close to control and the positive compounds showed significant number of differentially expressed transcripts. The further in depth analysis of gene expression data provided the proof of concept that this platform can demonstrate the developmental toxicity.
Many characteristics of human embryonic stem cells (hESCs) including pluripotency are useful tools for studying the harmful effects of reference compounds during early development. The early embryogenesis is recapitulated by pluripotent embryonic stem cells when differentiated in vitro, which makes hESC an effective tool for developmental toxicology and embryotoxicology. All-
In rat and mice, All-
The aim of this study was to investigate whether continuous electrical stimulation affects electrophysiological properties and orientation of fetal cardiomyocytes (FCMs) in culture.
FCMs at day 14.5 p.c. were harvested from murine hearts and electrically stimulated for six days in culture using a custom made stimulation chamber. A stimulation frequency of 10 Hz was applied continuously during cultivation. Subsequently, action potentials of FCMs were recorded with glass microelectrodes. Additionally, alpha-actinin immuno-stainings of the cultivated FCMs were performed and the angles between the electrical field lines and the longitudinal axis of the cells were measured. All data are presented as mean ± SEM.
Action potential duration to 50% repolarisation (APD50) of stimulated FCMs (37.34±1.43 ms, n=29) was significantly decreased as compared to controls (52.81±1.65 ms, n=21, p< 0.001). APD90 of stimulated cells (102.10±4.32 ms, n=29) was also significantly lower as compared to controls (148.30±11.21 ms, n=21, p< 0.001). The ratio of APD50/APD90 of stimulated FCMs (38.63±2.17 %, n=29) did not differ from controls (38.06±3.49 %, n=21, p=0.78). Spontaneous beating frequency of stimulated (1.86±0.17 Hz, n=29) and non-stimulated cells (2.17±0.17 Hz, n=21, p=0.22) was comparable. AP amplitude of electrically stimulated cells (67.42±1.47 mV, n=29) was equal to that of non-stimulated cells (71.28±1.35 mV, n=21, p=0.07). Maximal diastolic potential of stimulated cells (-56.87±0.96 mV, n=29) was not different from that of non stimulated cells (-58.55±1.02 mV, n=21, p=0.25). Maximal upstroke velocity (Vmax) of stimulated cells (25.78±3.97 V/s, n=29) and of non-stimulated cells (21.13±4.16 V/s, n=21) was comparable (p=0.43). The angles between the electrical field lines and the longitudinal axis of the stimulated cells (8.44±1.13°, n=31) were significantly lower as compared to the non-stimulated cells (49.43±3.33°, n=44, p< 0.001).
The decrease of APD50 and APD90 observed in stimulated fetal cardiomyocytes in vitro corresponds to the electrophysiological maturation of fetal cardiomyocytes in vivo. To our knowledge, continuous electrical stimulation is the first condition leading to an electrophysiological maturation and parallel alignment of cultured murine cardiomyocytes.
This implies that the electrophysiological maturation of cardiomyocytes in vivo may at least partially depend on electrical activity.
Malignant brain tumors, in particular the glioblastoma mutliforme (GBM), are amongst the most lethal solid human tumors. They grow invasively, develop resistance to radiation and chemotherapy, and frequently recur. Recently, GBM tissue was classified into four distinct molecular entities, the classical, mesenchymal, neural and proneural subtype based on a combined genome, transcriptome and proteome analysis. It remains unclear whether stem-cell like tumor cells (SCLC), which represent a small subfraction of the GBM, could be grouped into similar categories and whether GBMs harbor different types of SCLC.
We screened 80 human brain tumor biopsies for the presence of SCLC by immunological techniques and isolated SCLC from different glioma entities, such as glioblastoma mutlifirme (GBM) and gliosarcoma (GSarc). SCLC were expanded in serum-free medium and transplanted into SCID mice. Expression of stemness factors and neural markers was investigated by immunocytochemistry, FACS, Western blot analysis and real time PCR. Single cells from SCLC cultures were seeded in 96 well plates, clonally expanded and subsequently subjected to phenotype analysis.
We identified stem cell-like cells (SCLC) in all brain tumor biopsies analyzed so far. SCLCs that gave rise to proliferating cultures were transplanted into the brain of SCID mice to prove their tumorigenic potential. All SCLC expressed the intermediary filaments Nestin and the transcription factor Sox2. Co-expression of the intermediary filament GFAP or the marker CD133 (prominin-1), however, varied largely. The histostructural organization of Nestin+/GFAP- and Nestin+/GFAP+ cells differed between the various glioma tissues. This indicated that malignant gliomas show a high heterogeneity with respect to the presence and localization of SCLC. This also translated to SCLC cultures derived from GBM and GSarc biopsies. Several SCLC cultures exclusively contained Nestin+/GFAP+ cells, whereas others encompassed only Nestin+/GFAP- cells. Mixed forms were also observed. In these cultures the ratio of Nestin+/GFAP+ to Nestin+/GFAP- was remarkably stable over many passages. The same phenomenon was observed for the presence of CD133+ and CD133- cells. In addition, we found co-expression of Nestin and CD44 in almost all SCLC subtypes, which is in contrast to data published in the novel classification of molecular GBM entities. Clonal expansion of single SCLC cells from selected cultures revealed different degrees of heterogeneity, supporting the view that SCLC subtypes may co-exist in the same culture and presumably the same tumor.
First and second author = Contributed equally
Malignant brain tumors are amongst the most lethal solid tumors. The standard treatment consists of surgical resection followed by adjuvant radio- and chemotherapy. It has been proposed that glioma cells with stem cell features, designated SCLC hereafter, contribute to initiation and progression of primary and recurrent glioma. Regulation of SCLC and non-SCLC growth and the crosstalk amongst themremains largely elusive.
Up to now, we screened 80 human brain tumors for the presence and features of SCLC. Weexpanded brain tumor cells in vitro and analyzed the expression of receptor tyrosine kinases and peptide growth factorsin SCLC and non-SCLC by means of immunological staining as well as Western blot analysis and real time PCR. In addition, we investigated mitogen dependence (growth curves, proliferation and cell survival assays) and differentiation (immunocytochemistry) in the absence of exogenously provided growth factors.
We identified stem cell-like cells (SCLC) in all brain tumor biopsies analyzed so far. These cellsexhibited a substantial heterogeneity with respect to morphology, proliferation rate, growth modus and the expression of neural markers and stemness factors. Therefore, we refer to these cells as SCLC subtypes. Both, SCLC and non-SCLCfrom the same tumor co-expressed several receptor tyrosine kinases (RTK), includingEGFR/HER1 and the SCF (stem cell factor) receptor cKIT. Co-expression of RTKs and their corresponding ligandswas observed in several cultures, indicating that SCLC and non-SCLC might stimulate themselves by autocrine mechanisms. Besides the fulllength receptors, a truncated EGFR/HER1 and cKIT was observed in several cultures, indicating that these brain tumor cells may be activated in the absence of HER1 ligands and SCF, respectively. Secretion of the growth factors EGF and SCF was observed using cytokine ELISA techniques. In keeping with this, thedependence ongrowth factorssupplied with the medium was low and varied amongst the various SCLC and non-SCLC cultures. In addition, the RTK expression level of SCLC and non-SCLC from the same tumor differed. Together our data suggest that the human brain tumors cellsexertintracrine and autocrine growth control and cross-talkvia paracrine mechanisms.
Adipose tissue is easily accessible and abundant source of stem cells and can be harvested during liposuction. Aspiration of liposuction alone does not significantly alter the viability of Adipose Derived Stem Cells (ADSCs). However, liposuction is usually performed under general anaesthetic with infiltration of local anaesthetic fluid. That is why the aim of this study was to assess the influence of anaesthetic fluid on ADSCs viability.
Adipose tissue was collected during liposuction. Briefly, ADSCs were isolated by collagenase digestion (0.075%, 30 min in 37°C) and followed centrifugal separation. Cells were cultured in DMEM/HAM'S F-12 medium supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic. After 14 days, cells were seeded on 24-wells plate (1x104 cells/well) and cultured in complete medium for 24 h. Then cells were exposed on anaesthetic fluid (NaCl 0.9%, Adrenaline 0.1%, Articaine hydrochloride 4%,. Natrium bicarbonicum 8.4%) for 1 hour. Cells in control were cultured in complete medium. The viability of cells was assessed by MTT assay
ADSCs in control had a regular shape and size. Cells were elongated and spindle-shaped, with numerous cytoplasmatic lamellipodia. After incubation with anaesthetic fluid decrease in cell number, compared to control was observed. Cells began to detach from the well surface. The viability of examined cells was 57,6% and was much lower comparing to control.
Incubation of ADSCs with anaesthetic fluid significantly decreased their viability. It can be a limitation of adipose tissue as a source of stem cells. There is a necessary to optimize the method of adipose tissue collection for tissue engineering and regenerative medicine purposes.
Recent literature and experimental data, have demonstrated the significance of cancer stem cell-like cells (CSC-like cells), as an entity of circulating tumor cells (CTCs). Nanog, Okt 3/4, SOX2, Nestin and CD34 constitute specific markers of CSC-like cells and pilot studies showed a possible relationship between these transcription factors and clinical assessment of patients. The present study aims to determine the change of the above markers in correlation with the stage of the disease.
In the first panel of this study, CTCs from more than forty (40) patients with breast cancer in different stages, according to TNM classification system, have been isolated. The quantification of CSC-like cells in CTCs cultures followed and in the second panel, real-time qPCR has been used for molecular analysis. Gene-specific primers for each marker and for endogenous gene (18S rRNA) have been designed and evaluated in reactions with positive control samples. The analysis has been performed by using relative quantification, normalized to the reference gene.
A first evaluation of the data suggests that there is no linear relationship between the gene expression of transcription factors and the stage of the disease. In most markers, the higher value is noted in stages II-III. Nanog's expression varies, while Okt3/4 seems to be overexpressed in stage II. The lower value of Nestin has been observed in stage IV, whereas for CD34 transcription factor has been observed in stage I.
CSC-like cells may generate tumors through the stem cell processes of self-renewal and differentiation into multiple cell types. There are also resistant to chemotherapy, thus making difficult the treatment. This study demonstrates the correlation between gene expression of transcriptional markers, which is expressed particular in those cells, and the stage of disease in breast cancer. Concerning the above data, the question of whether these factors might be the target of new drugs arises. Further studies to confirm the above in a larger scale of samples, need to be performed.
Zinc finger proteins are among the most abundant proteins in eukaryotic genomes. Their functions are extraordinarily diverse and include DNA recognition, RNA packaging, transcriptional regulation, apoptosis, protein folding and assembly, and lipid binding. Zfp819 was identified in our comparative transcriptome analysis of undifferentiated and differentiated ESCs as a novel gene which is expressed highly in pluripotent cells but not in their differentiated counterparts. The protein encoded by Zfp819 belongs to C2H2-zinc finger (C2H2-Znf) family of proteins and bears a functional KRAB (Krueppel-associated box) domain on N terminal region of the protein, yet the function is not known. Recently, Zfp819 was shown to be being highly expressed in partially and fully reprogrammed induced pluripotent cells (iPSCs), but not in parental somatic cells. Therefore, these results suggest the possible crucial role for Zfp819 in establishments and maintenance of pluripotency.
To study the role of Zfp819 in pluripotency network, we altered the expression of Zfp819 by either overexpression or down-regulation and found no overt changes in the expression of key pluripotency marker genes. Unexpectedly, the expression of retroviral control elements such as Line1 and IAP gag were significantly up regulated upon Zfp819 down regulation in mouse ES cells. Further, to identify the interaction partners of Zfp819 in pluripotent cells, we performed a yeast two-hybrid screen on ESCs cDNA library with the N-terminal part of Zfp819 as bait. The identification of several putative interaction proteins suggest that Zfp819 might function as a transcriptional and cell cycle/apoptosis regulator. The characterization of interaction between Zfp819 and its putative interaction proteins is currently in progress. Further studies aimed at identifying the DNA targets bound by Zfp819 are in progress to unveil the role of Zfp819 in transcriptional regulation.
The biological phenomenon of cell fusion plays a fundamental role in several physiological and pathophysiological processes. In cancer cell fusion has been linked to the origin of more malignant tumor hybrid cells exhibiting an increased metastatogenic behavior as well as an enhanced drug resistance. Thereby, tumor hybrid cells could originate from the fusion between two tumor cells or between a tumor cell and a normal cell, such as a macrophage or an adult stem cell.
In the present work the migratory behavior and induction of signal transduction pathways of two hybrid cell lines (M13HS-3 and M13HS-8) in comparison to the parental cells (M13SV1-EGF-Neo breast stem cells and HS578THyg breast cancer cells) towards EGF and CCL21 was investigated.
All cell lines were positive for EGFR and HER2 expression. However, detectable levels of HER3 were solely found on M13SV1-EGFP-Neo breast stem cells and hybrid clone 8. By contrast, M13SV1-EGFP-Neo were negative for CCR7, whereas M13HS-2, M13HS-8 and HS578T-Hyg cells do express the CCL21 receptor. Cell migration is prerequisite for metastasis formation and both factors have been demonstrated to be involved in the metastatic spreading of disseminated breast cancer cells. Particular for CCL21 it is assumed that this chemokine plays a crucial in mediating the specific metastatic spreading of breast cancer cells into regional lymph nodes. All investigated cell lines responded to EGF stimulation with an increased locomotory activity, whereas solely the hybrid cells responded to CCL21 treatment with an increased migratory activity.
Co-treatment of cells with EGF and CCL21 revealed an additive effect on the migration of M13HS-2 and M13HS-8 hybrid cells. Analysis of PLC activity by flow cytometrybased calcium measurements showed that both parental cell lines and the M13HS-8 hybrid cell line exhibited increased cytosolic calcium levels upon EGF stimulation. By contrast, CCL21 did not lead to elevated intracellular calcium levels in any of the analyzed cell lines indicating that PLC was not activated. Western Blot analysis of AKT and MAPKp42/44 and their phosphorylated counterparts showed increased pAKT and pMAKPp42/44 levels in EGF and CCL21 treated HS578T-Hyg breast cancer cells and M13HS-8 hybrid cells, but not in M13HS-2 hybrids. By contrast, solely EGF treatment resulted in increased pAKT and pMAKPp42/44 levels in M13SV1-EGFP-Neo cells, whereas CCL21 had no effect.
We conclude from our data that they clearly depict the random nature of cell fusion resulting in unique hybrid cells exhibiting unique properties. The finding that hybrid cells, but not the parental tumor cells, responded to CCL21, which has been associated with lymph node metastasis of breast cancer suggests that cell fusion is a mechanism how tumor (hybrid) cells could become metastatic.
Notch proteins are transmembrane receptors that influence cell fate decisions, differentiation, proliferation and apoptosis in many developmental systems. After ligand binding and activation, the Notch intracellular domain (NIC) is cleaved from the cytoplasmic membrane and translocates into the nucleus to act as a transcription factor. NIC binds to DNA via the adapter protein RBP-J (also termed CBF-1) and converts the transcriptional repressor RBP-J into a transcriptional activator. Recently we have shown that Notch signaling regulates the expression of genes playing key roles in cell differentiation, cell cycle control and apoptosis in a highly context dependent manner. Epigenetic events like histone modification, DNA methylation and chromatin remodeling are tightly involved in the control of gene expression.
To investigate the role of chromatin modifications in cell-context dependent Notch1 target gene activation, we determined the chromatin modifications present at the regulatory regions of genes that we identified to be regulated in embryonic stem cells (ESC) by activated Notch1 using public databases. We found that the promoter regions of Notch1 target genes are marked by an enrichment of H3K4me3, an activating modification, and bivalent domains, containing an activating (H3K4me3) and repressing (H3K27me3) modification. We further screened the regulatory regions of the Notch1 target genes Hes5, Sox9 and Pax6 for potential RBP-J binding sites and analyzed the chromatin marks and their changes upon Notch1 activation in ESC using ChIP. Upon activation of Notch1 signaling for 15 min we observed changes in H3K4me3 and H3K27me3 at the RBP-J binding sites of the Notch1 target genes Hes5, Sox9 and Pax6. These results suggest that Notch1 signaling may alter the epigenetic marks, in particular the bivalent domains at the regulatory regions of its target genes. To further determine which of the RBP-J binding sites are used for Notch1 signaling at certain stages of development, we performed luciferase experiments with Sox9 RBP-J binding site mutant promoter constructs. When the two RBP-J binding sites located +40bp and -325bp relative to the transcription start site of Sox9 were mutated, the expression upon Notch1 activation was reduced compared to the wildtype Sox9 promoter.
Our data indicate that epigenetic regulation at Notch1 target genes involves changes in histone lysine methylation (H3K4me3, H3K27me3) upon Notch1 activation that may promote gene expression. Results from luciferase reporter constructs revealed an important role for the two RBP-J binding sites near the transcription start site for Notch1 mediated Sox9 upregulation with the possibility that they may act in a synergistic manner.
Chronic wounds represent a major problem in medicine today as their incidence is continuously increasing due to an aging population. Physiological wound healing is a complex biological process proceeding from the stage of inflammation through proliferation to maturation. It requires a well-orchestrated interaction of mediators, resident and infiltrating cells. In this context, mesenchymal stem cells play a crucial role as they are attracted to the wound site and influence wound healing processes via direct cell-cell interaction as well as paracrine secretion and transdifferentiation. In chronic wounds these finely tuned mechanisms are disturbed by mediators of the wound environment.
We analysed the effect of acute and chronic wound fluid on adipose-derived stem cells (ADSCs). Therefore we successfully developed protocols to harvest wound fluid from acute and chronic wounds. We investigated the proliferation and migration capacity of ADSCs under the influence of acute and chronic wound fluid (AWF; CWF) using MTT test and transwell migration assay.
AWF and CWF positively influence ADSC migration. However, AWF has a significantly stronger chemotactic impact on ADSCs than CWF. Proliferation of ADSCs is inversely influenced by AWF and CWF, respectively. Whereas proliferation is stimulated by AWF, CWF has a negative effect on ADSC proliferation over time.
These results give an insight into impaired ADSC function in chronic wounds. The less stimulating effect of CWF on ADSC proliferation and migration compared to AWF might be one reason for an insufficient wound healing process in chronic ulcers.
Bone marrow-derived stem cells have been shown to play a role in cardiac tissue regeneration. Cardiac ischemia causes HIF-1-dependent overexpression of stem cell homing factor SDF-1, and thus, stem cells are recruited to the area of tissue injury. Direct intramyocardiac transplantation may support this physiological process. In animal models as well as phase I / II clinical trials, transplantation of human bone marrow stem cells selected for the surface marker CD 133 led to improved heart function.
Besides SDF-1, however, various other factors such as chemokines, cytokines and nonpeptide molecules are released during cardiac injury. Elevated levels of extracellular adenosine-triphosphat (ATP) have been detected in ischemic vs. normal myocard. In the present study, we investigated the influence of extracellular ATP on CD 133+ cell migration.
Sternal bone marrow samples were obtained from cardiac surgery patients who had given their informed consent. CD133+ cells were isolated by density centrifugation and MACS isolation. Cell purity was evaluated by flow cytometry. In vitro migration of untreated or preconditioned CD133+ cells towards SDF-1 and ATP was evaluated in a Boyden chamber followed by microscopic analyses. The influence of ATP on CD133+ cell viability was examined by Annexin/7-AAD staining detected via flow cytometry. Cellular proliferation after preconditioning was verified by CFU assay.
ATP as well as SDF-1 induced significant CD 133+ cell migration. The effect of ATP was dose-dependent, 100μM ATP enhanced cellular migration 6,3 fold compared to control (medium). CD133+ cells contained small numbers of apoptotic (13,6 % ± 4,78) and necrotic (4,13 % ± 1,38) cells. Preconditioning with 100 μM ATP for 16h lowered apoptosis rate slightly, yet insignificantly (8 % ± 1,63 apoptosis, 4,2 % ± 2,61 necrosis). We detected no influence of 100 μM ATP on viability or proliferation as evaluated by CFU count. Moreover, we investigated a possible “priming” effect of ATP preconditioning on migration towards SDF-1. Incubation of CD133+ cells with 100 μM ATP for 16h resulted in slightly enhanced migration towards SDF-1 (5,73 % ± 1,11 ATP vs. 3,89 ± 0,44 control). The difference, however, was not significant.
We demonstrated that besides the well-known stem cell homing factor, SDF 1, ATP induces the migration of CD 133+ cells in vitro. Extracellular ATP may contribute to CD133+ cell migration towards ischemic tissue in vivo. Our data do not provide any evidence for a negative impact of ATP on CD133+ cells. Further studies are necessary to clarify the mechanism of ATP-induced migration.
PTH administration after myocardial infarction (MI) is known to attenuate myocardial function, survival and cardiac remodeling. These effects mainly resulted from an increase of mobilization and homing of CD34+/CD45+ stem cells into the ischemic myocardium. PTH related stem cell mobilization was shown to be related to an endogenous GCSF release. The aim of our study is to determine the role of G-CSF on the cardioprotective effects of PTH.
G-CSF +/+ (C57BL/6) and G-CSF -/- mice were treated with PTH (80 μg/kg/d) for 6 days after inducing a MI by LAD ligation. The myocardial homing factor SDF-1 was analyzed on day 2 with ELISA. Stem cell populations in peripheral blood and heart were examined on day 6 by FACS. Cardiac function and immuhistochemistry were investigated on day 6 and day 30.
PTH treatment resulted in a significant increase of CD45+/CD34+ cells in peripheral blood in G-CSF +/+, but not in G-CSF -/- mice. However, a significant increase of SDF-1 and enhanced migration of CD45+/CD34+ cells into the ischemic myocardium was revealed after PTH administration in both, G- G-CSF +/+ and G-CSF -/- mice. Enhanced stem cell homing was associated with an ameliorated cardiac function and post-MI survival after PTH treatment. Furthermore, infarct size, wall thickness and neovascularisation showed a significant improvement in both groups 30 days after MI.
The cardioprotective effects of PTH could be shown to be independent from endogenous G-CSF release and therefore from stem cell mobilization. This puts more emphasis on the role of stem cell homing into the ischemic myocardium
Small cell lung carcinomas (SCLC) are highly aggressive, invasive and early metastasizing tumors. The process of metastasis is going along with phenotypical changes. Cells have to detach from the primary tumor, invade the surrounding stroma, enter the circulation and escape the detection of the immune system until reaching metastatic sites. Epithelial-mesenchymal transition (EMT) processes, initially known from embryonic development, have recently been described to play an important role for these processes. EMT is going along with phenotypical changes and disbanding cell-cell connections that result in a transition from a more epithelial phenotype to a more mesenchymal-like appearance. It is described that the reverse process mesenchymal-epithelial transition (MET) happens at the site of metastasis, and it is postulated that these processes are linked to radio- and chemoresistance. There is very little knowledge on EMT processes and its impact on metastasis in SCLC.
SCLC cell lines NCI-H69, NCI-H82, NCI-N592 are usually forming floating cell clusters when cultured in RPMI 10%FCS, only very few cells are growing adherent to tissue culture flask (3-7%). FACS analysis shows different subpopulations of size and density within a cell line. We started to analyze the phenotypical morphology of the cell lines NCI-H69, NCI-H82, NCI-N592 and NCI-H446. The addition of low concentrations of BrdU to the culture is inducing a phenotypical change to mainly adherent growing cells. These changes are accompanied by changes of typical EMT markers on gene and protein levels.
For the induction of phenotypical changes 10μM BrdU was added to the culture medium for 14-21 days. Medium was changed every second day by centrifuging und resuspension. Once the majority of cells appear adherent BrdU was not added anymore. The cells remain adherent and show normal growth patterns. We show that the phenotypical changes go along with changes in membrane structures like Tight Junctions, Desmosome, Gap Junctions, Vimentin and Cadherins. Mesenchymal markers are upregulated in adherent cells, whereas epithelial markers are downregulated. We are postulating that under the influence of BrdU SCLC cell lines undergoing a mesenchymal shift. Our in vitro model might be interesting to study in vivo incidences of EMT.
Mesenchymal stem cells (MSC) can only be culture expanded for a limited time until they reach a senescent state, the so called “Hayflick-limit”, which is accompanied by growth arrest, cell enlargement and reduced differentiation capacity. Therefore, culture associated changes in MSC may hamper their therapeutic potential. In this study, we have analyzed genetic and epigenetic changes upon long-term culture of MSC from human adipose tissue. The fibroblastoid colony-forming unit (CFUf) frequency and the differentiation potential were already significantly impaired within the initial passages. Relevant chromosomal aberrations were not detected by karyotyping and SNP-microarrays and this supports the notion that human MSC possess relatively little genomic instability. Subsequently, we have compared DNA-methylation profiles with the Infinium HumanMethylation27 Bead Array and the profiles differed markedly in MSC derived from adipose tissue and bone marrow, indicating that the epigenetic makeup of MSC is highly dependent on the tissue of origin. Highly consistent senescence-associated modifications at specific CpG sites, especially in developmental genes, occurred already within the early expansion phase (between the passages 5 and 10). Remarkably, these DNA-methylation changes are highly enriched in regions with repressive histone modifications such as trimethylation of H3K9, H3K27 and EZH2 targets. These results indicate that cellular aging is not just a random accumulation of cellular defects, but that it is precisely regulated by epigenetic means in the course of culture expansion.
Embryoid bodies (EBs) are an accepted model of
We suppressed the PI3K regulatory subunit p85α using a small interfering RNA (Pik3r1 siRNA) and examined the effects on EB development in hanging drop culture. We observed a 150% increase in the volume of the treated EBs within 24 h, compared to the negative controls. FACS assays showed that this increase in volume is not due to increased cellular proliferation. Instead, the increase in volume appears to be due to reduced cellular aggregation and adherence. This is further shown by our observation that 40% of treated EBs form twin instead of single EBs,and that they have a significantly reduced ability to adhere to culture dishes when plated. A time course over the first 96 h reveals that the impaired adherence is transient and explained by an initial 12-hour delay in EB development. Quantitative PCR expression analysis suggests that the adhesion molecule integrin-β1 (ITGB1) is transiently downregulated by the p85α suppression.
Up to now, the function of the regulatory subunit p85α was rather poorly understood. Here, we provide first evidence that its gene Pik3r1 is involved in early embryoid development. We found that suppressing p85α leads to a delay in forming compact EBs, accompanied by a transient inability of the EBs to undergo normal cell-cell and cell-substrate adhesion.
Establishment of a hepatocyte-like in vitro system is a topical issue because of a need for a reproducible and accessible tool for early drug discovery and toxicological screenings. Also, such a system could prospectively serve as a source of hepatocytes feasible for clinicaltransplantations. In contrast to primary hepatocytes, which rapidly lose their functionality, embryonic stem (ES) cell-derived hepatocyte-like cells could represent a convenient material for both clinical trials and drug- and toxicology testing. Attempts to differentiate hepatocytes from ES cells are mostly based on application of defined-composed media containing growth factors and cytokines. However,the main associated problem is still an efficient differentiation and, in particular, selection of differentiated cells reliably imitating hepatic features. In this study we aimed to establish a system for generation of hepatocyte precursors capable of development to hepatocyte-like cells.
We used stable transgenic clones of murine ES cellspossessing a bicistronic expression vector which contains the eGFP reporter gene and a puromycin resistance cassette under control of a common α-fetoprotein (AFP)gene promoter. Thus, in our experimental model,differentiating endoderm-derived hepatic progenitor- or hepatocyte-like cells with the activated AFP promoter coordinately developed eGFP fluorescence and puromycin resistance, allowing for their “live” monitoring and antibiotic selection. For high-yield generation of target cells, spinner flask cultivation mode was used.
Cell cultures were mainly investigated by real time qRTPCR analysis, fluorescent immunostaining, and some functional assays.
We established an up-scaled embryoid body (EB) culture,in which eGFP-expressing cells developing in the outer rim of EBs could be preliminary selected by exposure to puromycin and then separated as aggregates by collagenase treatment. In the course of further cultivation and selection in spinner flasks using specifically optimized rotation parameters, they formed growing spheroids that comprised actively proliferating cells. RNA analysis of the spheroid culture revealed expression of hepatic precursor and hepatocyte marker genes, comparable to that in fetal liver. Once plated onto collagen I substrate, the spheroids outgrew as colonies expressing hepatic proteins albumin and α-1-antitrypsin, the main hepatic cytokeratin CK-18,cell membrane protein of periportal hepatocytes Ecadherin,and, at a lower level, liver specific organic anion transporter Lst-1. Glycogen-storing and indocyanine green up-taking cells were detected as well.
The data obtained suggest differentiation of hepatocyte precursor cells and likely also of a fraction of more advanced differentiated cells in the established spheroid culture. This confirms a feasibility to produce and to select cells, which are able to develop to hepatocytes, using high-yield dynamic suspension cultures facilitating early hepatic differentiation.
The ability to ex-vivo produce respiratory epithelial cell types would offer new therapeutic options to treat pulmonary injuries and diseases, including genetic surfactant deficiencies and cystic fibrosis. Pluripotent stem cells (PSCs) may represent a suitable cell source but the efficiency of existing differentiation protocols towards lung epithelial cells is still low. Therefore, we aimed at identifying key factors which drive the differentiation of murine embryonic (mESCs) as well as murine induced pluripotent stem cells (miPSCs) into airway epithelial Clara cells and alveolar epithelial type II (ATII) cells. We made use of miPSC clones established from two different strains of transgenic CCSP-rtTA2s/GFP-tetO7-lacZ mice which either confer transgene expression predominantly in Clara cells or ATII cells.
Keratinocyte growth factor (KGF, FGF-7), a member of the fibroblast growth factor family, is known to be a strong growth factor for primary airway and alveolar epithelial cells. Similar to KGF glucocorticoids also have a stimulating effect on the maturation of primary lung epithelial cells and this effect is even potentiated by the simultaneous addition of a cAMP analog and isobutylmethylxanthine. We thus hypothesized that the addition of KGF either alone or together with the three-factor combination dexamethasone, 8-bromoadenosine-cAMP and isobutylmethylxanthine (DCI) might support the pulmonary differentiation of mESCs and miPSCs. Indeed, we identified a synergistic effect of KGF and DCI on the differentiation into lung epithelial cell types with a surprising need for an early KGF supplementation already starting at day 0 or 5 of differentiation; suggesting a so far unknown effect of KGF on the generation of lung epithelial (progenitor) cells from PSCs per se during the early phase of differentiation when lung epithelial cells are not yet originated. This synergistic effect resulted in increased mRNA expression of the lung epithelial specific markers surfactant protein (SP-)B, SP-C and Clara cell secretory protein (CCSP). Affimetrix microarray analyses further confirmed the positive effect of KGF and DCI on pulmonary cell generation from PSCs. Finally, electron microscopy revealed the presence of ATII-like cells which contained composite body-like and more mature lamellar body-like structures, typical organelles of ATII cells. Those lamellar body-like structures had striking similarities with lamellar bodies of ATII cells of the adult rodent lung. In addition, cells were identified with ultrastructural features typical for Clara cells.
In conclusion, we were able to demonstrate a synergistic effect of KGF and DCI on the differentiation of PSCs resulting in increased expression of respiratory marker genes and cells with ultrastructural features typical for ATII and Clara cells. The successful generation of lung epithelial cells from iPSCs paves the way for an autologous cell therapy of lung diseases.
Current research in the field of tissue engineering is focused on the production of large-scale amounts of pluripotent stem cell-derivatives (e.g. cardiomyocytes, hepatocytes, insulin-producing cells). To realize this, stem cell aggregation in microwells is a powerful approach yielding highly reproducible differentiation units in large quantities. We have developed a rapid and inexpensive soft lithography method to fabricate silicone masters, which can be used for agarose/DMEM patterning within a petri dish. Resulting non-adhesive “AgarOwell” plates containing800 microwells on one 10 cm dish were used for the aggregation of 1000 murine iPS cells / well into embryoid bodies (EBs). Comparison to the “gold standard” hanging drop method, led to similar results concerning harvesting efficiency (almost 100%), size distribution of EBs, cardiac differentiation efficiency (98-99% beating EBs on day 8) and marker expression. Thus, the presented method offers an inexpensive, fast and reproducible approach for large quantity/high quality embryoid body formation.
The differentiation of adipose tissue-derived mesenchymal stem cells (AT-MSC) into neuron-like cells on an engineered biomaterial has been used in an effort to construct a cell-seeded scaffold that could be used as a permissive bridge that promotes axonal regeneration and the re-establishment of damaged connections after spinal cord injury. Modifications of poly(2-hydroxyethyl methacrylate) (PHEMA) with laminin derived peptide sequence IKVAV (Ile-Lys-Val-Ala-Val) have been developed to create highly superporous hydrogel scaffolds that promote cell-surface interactions.
AT-MSC were isolated from Wistar rats and seeded on IKVAV-PHEMA hydrogels as well as on control laminincoated glass cover slips. The neuronal differentiation was induced by using Neurobasal media containing B27 and growth factors - EGF, bFGF and NGF during 11 days. The results were evaluated by qPCR and immunocytochemistry.
During neural differentiation, the increased expression of the neuronal markers ß-III-tubulin, MAP2 and nestin, GFAP, and the trophic factors BDNF, GDNF, EGF and FGF was observed in cells seeded on IKVAV-PHEMA hydrogels.
Immunocytochemisty revealed that during differentiation, the cells on IKVAV-PHEMA hydrogels became positive for MAP2, while they remained positive for nestin, ß-III-tubulin, NCAM, NG2 and p75. Neuronal differentiation on laminin-coated coverslips resulted in decreased immunostaining for nestin and ß-III-tubulin, while the cells remained positive for NCAM, NG2 and p75.
The results suggest that the IKVAV sequence has a supportive effect on neuronal differentiation and that IKVAV-modified hydrogels can be used as cell-seeded 3D scaffolds for neural tissue regeneration.
Supported by: GAČR: P304/11/0731, P304/11/0653, 108/10/1560, 309/08/H079; 1M0538 and IAA500390902.
Adipose-derived stromal / stem cells (ASCs) possess a multilineage differentiation potential, can be used from an autologous origin and are, therefore, attractive candidates for clinical applications to repair or regenerate damaged tissues and organs. Adipose tissue as a stem cell source is ubiquitously available and has several advantages compared to other sources. It is easily accessible in large quantities with a minimal invasive harvesting procedure and the isolation of ASCs yields a high amount of stem cells, which is essential for stem cell-based therapies and tissue engineering. Differentiation of ASCs into cell types of mesodermal origin has been shown in a variety of studies. The plasticity of ASCs towards cells of the mesodermal lineage has been shown by their differentiation into chondrocytes, osteoblasts, adipocytes, and myocytes. Their potential to differentiate into lineages with nonmesodermal origin is even more exciting: ASCs are also able to differentiate into cells of ecto- and endodermal origin. Various
Adipose derived mesenchymal cell (Ad-MSCs) is a promising source for autologous cell therapy because of their safety and lower risk of tumorogenesis. But Ad-MSCs from old donors are less pluripotent and do not proliferate as good as the ones from young donors. We previously found this age-related loss of capacity had a link to the epigenetic and pluripotency status, and consequently lead to their lower hepatic and osteogenic differentiation capacity. The aim of the present study was to find out whether old Ad-MSCs could be modified epigenetically by 5-Azacytidine (AZA) or BIX 01294 (BIX) to improve their biological function after differentiation into hepatocyte or osteoblasts like cells.
Ad-MSCs were isolated from abdominal adipose tissue of young (≤45yrs) and old (>45yrs) donors according to the ethical guidelines of TU Munich. Each group was pretreated with AZA (5, 20 μM) and BIX (0.1, 0.2 μM) over 24 and 48 h and then analysed immunocytochemically for genomic distribution of 5-methylcytosine and 5-hydroxymethylcytosine and expression of pluripotency genes ((Oct4, Nanog, Sox2, c-Myc) and TET oncogenes (1, 2, 3) responsible for hydroxymethylation. To verify differentiation potential into hepatic and osteogenic lineages, specific enzymatic and metabolic parameters were tested post in vitro differentiation. This included assessment of CYP activity, urea production and alkaline phosphatase activity for hepatic function and Alizarin Red and von Kossa staining for osteoblast-like structure.
Both AZA and BIX treatment significantly decreased the global DNA methylation of Ad-MSCs, most prominent in cells derived from old donors (AZA caused a decrease by 30%, BIX by over 60%). At 48h treatment of AZA and BIX led to an increase of TET1 expression and a higher 5-hmC level in AdMSCs, which was associated with changes in the expression of pluripotency related genes: BIX treatment gave rise to a significant (p< 0.05 vs. untreated) elevated Nanog, c-Myc and Oct4 expression in 'old` Ad-MSCs. Furthermore, pretreatment of 'old' Ad-MSCs with AZA or BIX and a subsequent differentiation into hepatocyte or osteoblast-like cells resulted in a significant improvement of function and activity of liver and bone-related markers: pretreated hepatocyte-like-cells demonstrated an elevated urea and glucose metabolic capacity as well as Phase I/II enzyme activity. In some cases AZA/BIX-treated cells even reached similar CYP450 activities and urea levels as seen in primary human hepatocytes. Similarly, AP activity, Alizarin Red and von Kossa staining in osteoblast-like-cells from old donors were found improved in comparison to non-AZA/BIX-treated cells.
AZA and BIX treatment seems to be a promising approach to modify human Ad-MSCs from old donors up to the level of cells from young donors. This approach would be safer because it avoids viral transfection as used in iPS technology thus reducing the risk of tumorogenesis
A dedicated goal in the interdisciplinary area of biomedical engineering and stem cell research is the establishment of complex tissues that resemble their in vivo counterparts.
During embryonic development, the skin forms as a result of reciprocal interactions between mesoderm and ectoderm in the presence of bone morphogenetic protein-4 (BMP-4). Embryonic stem cells (ESC) are able to recapitulate developmental steps in vitro. Recently, germline-derived pluripotent stem cells (gPSC) were generated from explanted murine testis without introducing exogenous transcription factors with tumorigenic potential. Hence, gPSC represent a promising and novel cell type for tissue engineering.
In the current study, we combined mesenchymal-epithelial interactions by cultivating gPSC and ESC air-exposed and on three-dimensional organotypic skin equivalents. We analyzed the differentiation capacity as well as the epidermal cell fate of gPSC and ESC in the presence and absence of BMP-4. Interestingly, gPSC displayed a stronger and accelerated propensity to differentiate compared to ESC by qt-RT-PCR. Under organotypic culture conditions of skin, gPSC generated complex tubulocystic structures, lined by a multilayered, stratified (CK5/6+, CK8/18-) squamous epithelium in ≥50%. In contrast, ESC underwent apoptosis (as shown by staining against caspase 3) under the same epidermal differentiation pressure and only formed small tubulocystic structures lined by simple, CK8/18+ epithelia. Furthermore, gPSC produced a dense eosinophilic basal membrane surrounding the epithelial structures and spontaneously differentiated into mesodermal and endodermal lineages. The addition of BMP-4 neither enhanced the epithelial nor epidermal differentiation of the stem cells. Given that monolayer cultures induced an epithelial but not an epidermal differentiation of both pluripotent cell types, the air-liquid interphase appears to be a strong and essential stimulus for epidermal differentiation of gPSC.
Concluding, we successfully differentiated gPSC into a multilayered, squamous epithelium on organotypic skin equivalents and demonstrated their extensive differentiation potential resembling to early embryogenesis. Challenges for future studies will be the derivation of pure populations of keratinocytes, as well as the elimination of undifferentiated, pluripotent cells to reduce the risk of tumor/teratoma formation.
Adult newts (Urodela) are able of eye regeneration even after complete removal of the original neural retina (NR). Because of this, visual system of the newt is a unique
This work was supported by Russian Foundation for Fundamental Research (Grant № 11-04-00728; 11-04-00125).
Satellite cells are myogenic stem cells responsible for growth, repair, and maintenance of skeletal muscles at all stages of ontogenesis. Isolated satellite cells
During the reprogramming of differentiated cells into induced pluripotent stem cell (iPS) by defined transcription factors, generation of other specific cell types was recently reported (Efe
Using viral transduction of mouse fibroblast derived from different parts of the body (i.e. tail tips, ears, abdominal pars) as well as from the whole body, we delivered transcription factors: Oct4, Sox2, Klf4, and c-Myc, the classical factors frequently used for the generation of iPS cells. We further tested other factors and conditions (BMP-4, Activin, DCA, FGF-2, Wnt inhibitors, VEGF, serum concentration, including different timing of treatment) that could potentially enhance cardiac differentiation. In order to confirm cardiogenesis, we performed immunostaining for specific cardiac markers. Functional characterization of derived cardiomyocytes is being done in order to control cardiac-specific functionality. Thus, development of action potentials, responses to the beta-adrenergic agonists as well as release of calcium from the sarcoplasmic reticulum is being investigated.
Our preliminary results indicate that within ~10 days from transgene induction and subsequent periodical stimulation by other factors, mouse embryonic fibroblasts (MEFs) are reprogrammed to spontaneously contracting clusters of cardiomyocytes. Process of newly emerging clusters in the fibroblast culture continues over period of several days. Before the detection of the first cardiomyocytes no intermediate iPS stage is observed. Our immuno-cytological experiments indicate positive signals of differentiated cardiomyocytes for the sarcomeric protein troponin I (TnI) as well as for the cardiac sarcoplasmic protein ryanodine receptor (RyR2) and others.
Our first results indicate the possibility to activate early cardiac reprogramming from MEFs. In addition, our results could offer optimal conditions to enhance cardiogenesis and platform for derivation of specific and functional mouse cardiomyocytes. Transdifferentiation of MEF has the potential for being a reproducible method for generating cardiomyocytes in a quick and reliable way.
Despite improved heart failure therapy, there remains a lack of clinically relevant concepts for structural regeneration of the diseased heart muscle. We have previously reported that Pressure controlled Intermittent Sinus Coronary Occlusion (PICSO) - a coronary sinus intervention, reduces infarct size clinically and induces washout clearing the ischemic/reperfused microcirculation. There is experimental and clinical evidence that besides the hemodynamic “mechanistic” effect another molecular pathway may exist influencing myocardial regeneration thus improving clinical outcome. We hypothesize that PICSO is a clinically feasible form activating coronary venous endothelium and its underlying mode of action is similar to the common understanding of stem cell research.
We analyzed the regenerative potential of hitherto unknown molecules released in the Coronary venous blood of heart failure patients after 20 minutes of PICSO to confirm our hypothesis of reactivating developmental processes (“embryonic recall”) by inducing proliferation in cell cultures as surrogate for regenerative pathways.
Serum from healthy volunteers and Cardiomyopathy patients enrolled during the surgical intervention of resynchronization therapy, was collected pre and post 20 minutes of PICSO and compared to controls (n>10). Serum levels of
Levels of NT-proBNP and IL-6 were seen in serum samples from cardiomyopathy patients increased after PICSO therapy. Serum IL-6 levels were associated with NT-ProBNP levels. Interestingly, 3 of 5 test serum samples showed also a significant increase in the capability of proliferation of human fibroblast cells compared to both control groups. In addition, cell scratch test showed increase migration capability (P>0.05) for test serum samples.
Levels of established cardiovascular biomarkers (IL-6 and NT-ProBNP) could indicate an induction of the SAFE-pathway, leading to mitochondrial integrity and explaining reduction in infarct size. The
Human amniotic membrane has already been applied in a lot of clinical trials due to its beneficial properties and is still of great interest for a multitude of areas of research. Tissue engineering strategies usually require cell isolation and combination with a suitable carrier substrate. Alternatively, the cell sheet technology enables transplantation of expanded cells without the use of carrier materials. In contrast, our approach is to differentiate stem cells within intact amniotic membrane (AM), which constitutes a pre-formed sheet of stem cells, without prior cell isolation. We have previously demonstrated osteogenic differentiation of stem cells within intact human AM in vitro. Beside bone, regeneration of cartilage is an important scope in orthopedic trauma. For this reason, also the chondrogenic differentiation potential of human AM was investigated. In vitro chondrogenic differentiation of human AM biopsies was induced by culture in Mesenchymal Stem Cell Chondrocyte Differentiation Medium (C, Lonza) supplemented with transforming growth factor beta 3 (Lonza), optionally supplemented with fibroblast growth factor 2 (FGF-2, R&D Systems; CF), and the medium described by Tallheden (T) for 16 weeks. Membrane viability was quantified by EZ4U-Nonradioactive Cell Proliferation and Cytotoxicity Assay (Biomedica). To determine chondrogenesis, cartilage-specific collagen II and glycosaminoglycans (GAG) were demonstrated by alcian blue staining of paraffin embedded histological sections. Furthermore, the amount of GAG was quantified using BlyscanTM Sulfated Glycosaminoglycan Assay (Biocolor). The highest hAM-viability was sustained in the medium described by Tallheden [d56: control medium (Co): 15.12%±1.47%; C: 24.56±4.26%; CF: 21.71%±13.31%; T: 56.18%±19.14% compared to d0]. Regardless of the medium applied, Alcian blue stainings of all AM-samples revealed accumulation of GAGs in the membranes. Nevertheless, stainings of samples cultured in control medium were less intense, whereas CF appeared to show the most intense staining. Quantitative evaluation showed that in the control medium (Co) the amount of GAG was decreasing during culture (52.0%±14.5% on d56 compared to d0). When culturing in C, the GAG-amount was increased compared to Co at any timepoint and remained stable throughout the culture period. Noticeable, 3-fold increased GAG production compared to Co was obtained when adding FGF-2 to the medium. Chondrogenic differentiation of intact human amniotic membrane could be induced by chondrogenic medium (C) and further increased with supplemented FGF-2. Thus, these results are another promising step towards using intact human amnion with its residing stem cells for tissue engineering.
The auditory midbrain implant (AMI) is a novel prosthesis directly activating the auditory neurons within the inferior colliculus for hearing restoration in patients with bilateral neural deafness. As a response to insertion trauma, the formation of a reactive fibrillary sheath around the electrode is observed. From cochlear implant-related research it is known that fibrosis or gliosis can impair electrode-tissue-interaction. Structuring of the surface of the electrode, though, can hinder unspecific growth of fibrotic tissue within the cochlea. In order to test the influence of surface structuring on retrocochlear neuronal tissue, however, an adequate in vitro model is missing. Thus, aim of the study was the development of a dissociated inferior colliculus culture to study interactions between neurons and structured surfaces. Colliculi inferiores were isolated from neonatal rats and were seeded after dissociation in laminin-coated 96-well-plates. After a cultivation period of 2-5 days, cells were fixed and stained immunocytochemically. Using glial- and neuron-specific antibodies such as against S100, MAP2, Tuj1 and GAP43 the established cultures were characterized morphologically. We could demonstrate adhesion and survival of dissociated neurons and glial cells isolated from the inferior colliculus. Using this in vitro model, interactions between cells of the inferior colliculus and surface modifications of electrodes to be implanted could be investigated in order to minimize reactive gliosis formation around the implantation site and potentially for a more effective hearing restoration.
Regeneration is a promising and also complex phenomena in biology and medicine. All species have some kind of capacity for regenerating but the level for this capacity depends on species and tissues are varied.
Regenerative biology is the process of replacing or regenerating animal cells, tissues or organs to restore or establish normal function. Methods for that are different but it could be by stimulating the body to repair or by replacing damaged tissues or organs.
The main strategies for regenerative biology in general and especially in cardiac regenerative biology are based on the two main following approaches: 1- Cell therapy (e.g. stem cell therapy) and 2- Tissue therapy (e.g. tissue engineering).
Stem cells unlike other cell types in the body have two main important properties: 1- They are self- renewal and unspecialized cells. 2- They have unlimited potency.
There are two main types of stem cells: 1- Embryonic stem cells (ESCs) and 2- Adult stem cells (ASCs). In this presentation a brief history and the recent findings of administration of ASCs on the treatment of the heart will be reviewed.
In some vertebrates such as Zebra fish cardiac regeneration was reported following severe injury of heart. The mechanism for this cardiac regeneration has not fully understood but it has suggested that it could be based on divisions of cardiomyocytes.
In animals the idea of regenerating damaged heart tissue after myocardium infarction (MI) using stem cell transplantation is very promising.
Adult stem cells are multipotent and can substitute to repair damaged tissues. Due to lack of ethical concerns they widely are used in the therapy of patients across the world in clinical trials for treatment of different diseases like cardiovascular disease (CVDs), however, adult stem cell therapy is not still a routine approach for treatment of diseases.
As before any clinical studies to undertake experimental studies are necessary, therefore to review the results of such experiments are very important too.
In this oral presentation I explain recent findings on regenerative biology strategies with emphasize on the treatment of heart diseases (especially MI) with stem cells in experimental models.
Cell cultures for cell therapy and regenerative medicine products are mainly performed in the presence of fetal calf serum (FCS). Due to its animal origin, the use of FCS bears the risk of prion disease transmission or immunogenicity of xenogeneic proteins. Several studies are already published that replaced FCS by human serum or use of defined media where recombinant growth factors were added. Due to its high concentrations in growth factors (GF), human platelet lysate (hPL) also turned out to be a potential substitute for FCS.
Our aim was to develop a production process according to GMP requirements for hPL to be used in cell culture. At the beginning, hPL was made out of outdated thrombocyte pools. The concentrates were stored at -80°C and thawed in a water bath at +37°C to cause platelet lysis. hPL was compared to FCS in several proliferation studies including adipose tissue derived stem cells (ASC), amniotic mesenchymal stroma cells, fibroblasts and chondrocytes. Furthermore, differentiation of ASC in presence of hPL as well as redifferentiation of chondrocytes cultured with hPL was achieved. In all these studies lower amounts of hPL were needed to obtain equal or superior results compared to FCS.
However, there is only a low amount of thrombocyte pools which need to be discarded and heparin needs to be added to the medium to avoid gel formation. Therefore, process adaptation was performed. Buffy coats with too low plasma levels that would be discarded are used. The plasma is replaced by 0.9% sodium chloride solution and volume reduction as well as increase in platelet concentration was achieved by centrifugation. Finally, several freeze thaw cycles were performed to achieve the highest yield in GF.
Preliminary cell culture results with fibroblasts and ASC showed that the modified hPL is also better than FCS but the ideal final concentration in the media needs to be determined. For process control, sterility testing is implemented. Measurement of pH-value, platelet concentration, and total protein content are defined as quality control parameters. Additionally, ELISA for platelet derived GF BB, transforming GF β1, insulin-like GF 1, epidermal GF and basic fibroblast GF were performed. We were able to define a GMP compliant production process for hPL that obtains better results in cell culture than FCS. Thus, hPL can be used in cell culture to avoid the use of non-human products in cell therapy and regenerative medicine.
The development of functional myocardial patches requires several different tools, the most complex currently being the bioreactor. An ideal bioreactor for this task imitates the conditions inside the beating human heart so that the conditioned cells begin to beat, mimicking cardiomyocytes.
The bioreactor engineered in our laboratory addresses two important variables in tissuegenesis of cardiac tissue. Firstly, the cell-seeded patches are stimulated using an electrical circuit. The electrical current imitates the ECG of physiological heart tissue in-vivo. Secondly the flow, created by a piston inside the bioreactor, is programmed to mimic the flow in the living heart. To achieve this, an ideal biomimetic flow curve is produced by programming the piston movement. The bioreactor consists of a Plexiglas shell with a custom built piston in the medium chamber. At the top of this chamber there is a ring for patch fixation.
The architecture of the system allowed easy visualization of the medium flow as well as inspection of the ring for patch fixation. Seven day function testing showed that the bioreactor permits effective and sterile conditioning. Microbiological analysis of the used medium revealed no bacterial contamination. During this trial run various amplitudes and cardiac outflow rates were successfully tested.
Unlike any other current bioreactor in myocardial tissue engineering our bioreactor achieves simultaneous electrical and mechanical stimulation. Both these stimuli as well as chemical signaling have been shown to be vital to cardiac tissue development. Combining chemical differentiation and electromechanical conditioning of stem cells in this bioreactor we hope to create a beating myocardial patch.
Collagen abnormalities have been shown to be a cause of the cutaneous changes taking place during physiological aging with reduction of thickness and alterations in its biomechanical properties. Concomitantly, increased elevated MMP expression with a related reduction in collagen synthesis are common associated mechanisms. Thus, although the exact mechanism of cutaneous aging is unclear, it is known that intrinsic biological skin aging results in a loss of collagen and an increase in MMP-1 expression. Moreover, very recently it has been found that a significant decrease in mitochondrial membrane potential in samples from aged donors, accompanied by a significant increase in ROS levels.his study tested the activity of LD-1227 (containing caviar-derived homogenate added with.
CoQ10-selenium component, CaviarLieri®, Lab-Dom, Switzerland) in aged human skin and its role on skin mitochondria function Human dermal fibroblasts were obtained from healthy donors over 70y old and treated with LD-1227 for 72 h. As compared to baseline, LD-1227 caused a robust (>67%) collagen type I synthesis (p< 0.001) and decreased fibronectin (p< 0.05) with significant fibronectin mRNA downregulation (p< 0.05, r=0.78). A significant collagen mRNA overexpression occurred with LD-1227 treatment (p< 0.05). Mitochondria cytosolic ATP level decreased in aged skin samples (p< 0.05 vs young control) but this phenomenon was reverted by LD-1227 (< p 0.01). These data show that LD-1227 modify the extracellular-matrix milieu in aged skin and beneficially affect mitochondrial function.
Today glutaraldehyd fixed pulmonary valves are implanted in clinical trails, however limited by absence of regeneration, remodelling and growth potential. This feasibility study was performed to evaluate deliver-related tissue distortion of tissue engineered (TE) heart valves during implantation.
The use injectable TE heart valves was mounted on a self-expanding nitinol stent (n=7) and delivered into the pulmonary position of seven pigs (26-31 kg), performing a stenotomy or limited lateral thoracotomy. Prior to implantation, the injectable TE heart valve was crimped by using an applicator. The positioning of the implant was guided by fluoroscopy. Hemodynamic measurements were performed by epicardial echocardiography, angiography and invasive pressure measurements. Finally, the animals were sacrificed and the injectable TE heart valves were inspected by gross examination and histology.
Orthotropic delivery of the implanted TE heart valves (diameter 19mm) were successfully performed in all, expect in one due to valve migrated become of size discrepancy. Angiographically all other valves (n=6) showed normal valve function, supported by epicardial echochardiography in which no increase flow velocity was measured, neither valve regurgitation. Invasive pressure measurements showed a mean pressure gradient of 5mmHg. Histological evaluation demonstrated no integrity changes of extracellular matrix and absence of collagen and elastin distortion.
Transcatheter implantation of an injectable TE heart valve seems to be possible without tissue distortion due to the delivery system.
To find an alternative treatment option for heart failure we produced engineered tissue from mesenchymal stem cells (MSC) to enhance the immunological compatibility after implantation in rat heart.
We used a rat bone marrow derived mesenchymal stem cell line (MSC, Gibco) to produce engineered tissues (MSC-ET). Low-passage (P6) MSCs were analyzed for phenotype characteristics by flow cytometry and adipogenic differentiation.
Cultured MSCs were mixed with matrigel, collagen and serum containing media and were casted into a circular structure for creating MSC-ETs. After 5 days of consolidation time the MSC-ETs were electrically stimulated (1Hz, 1mV and 0,1mA) for further 6 days. Subsequently MSC-ETs (n=3) were implanted around the beating heart of immunosupressed adult rats. After 1 month the rat hearts were surgically excised.
In vitro and in vivo MSC-ETs were histologically examined for collagen and elastic-fibers. By immunohistochemical investigation we measured expression of cardiac connexins (Cx40, Cx43 and Cx45), vessel associated von Willebrandt factor and mesenchymal stem cell-marker CD90.
At day 14 in vitro MSC-ETs (n=4) did not exhibit any contractions. Histological analysis showed rings containing 64.57±2.04% collagen tissue, but no elastic fibres. Furthermore we exhibited 19.7±8.1 micro vessels / 1mm².
In vivo 2 of 3 MSC-ETs were clearly distinguishable from the native heart. Our investigation showed that MSC-ETs in vivo consisted of less collagen (27.43±9.56%) than in vitro, but developed elastic fibers (0.93±0.18%). The number of micro vessels showed an increase up to 39.3± vessels /1mm². We also found CD90 and Cx43 expressing cells in vivo and in vitro.
Surprisingly, in 1 of 3 MSC-ET-transplanted rats the heart was completely surrounded by a huge, undifferentiated, pleomorphic sarcoma. Histological investigations of the growth and location of the tumour indicated that the tumour was part of the MSC-ET. Analysing the grade of the sarcoma by using the FNCLCC-grading system revealed, that the neoplasm was a high grade malignant sarcoma (total score= 7/8).
Dedifferentiated sarcoma cells lost the CD90 expression utterly, which certainly might indicate the malignant transformation.
Additionally, the majority of sarcoma cells were positive for the cardiac connexins (Cx40, Cx43, Cx45), but at the tumour-heart border their expression was left. Therefore, we hypothesize that the tumour was not able to communicate with the heart, which gives the sarcoma cells the opportunity to infiltrate healthy tissue.
Low passage MSCs of a MSC-ET can dedifferentiate into a high grade malignant sarcoma in vivo. However, it remains unclear whether the transformation might be inborn to these cells or whether it might be caused by the treatment of these cells during the process of ET formation.
Carbohydrate hydrogels are attractive scaffold materials for myocardial tissue engineering since they provide high biocompatibility and chemical versatility adjustable to specific tissue demands.
Human amniotic fluid stem cells (hAFSCs) have become an attractive stem cells source for medical therapy. The aims of this study were the isolation and characterization of human amniotic fluid stem cells.
The human amniotic fluid was obtained from 23 patients in age from 19 to 45 years, during planned amniopunctions. Patients were divided into the following age groups: 1th - patients in age from 19 to 29, 2nd - from 30 to 39 and 3rd - from 40 to 45. The average of age in the following groups was respectively: 26 years (SD - Standard Deviation = 3,7), 35 years (SD = 2,7) and 43 (SD = 2,2). The samples were centrifuged at 350g for 10 min. After washing with PBS, cells were gently suspended and plated. When reached confluence the number of isolated cells and their phenotype were assessed. The phenotype was evaluated using: proliferation rate, differentiation potential and clonogenicity.
Human amniotic fluid-derived stem cells were allowed to adhere to plastic culture dishes and the proliferation rate ranged between 5x103 to 15x103 cells during 7 days of culture. Cells cultured in medium supplemented with differentiation factors toward osteogenic lineage showed change of morphology, from fibroblast-like to polygonal, after 5 days. hAFSCs formed an average of 8,2 ± 1,7 colonies. The clonogenicity that correlates with number of stem cells was 0,16%. The average of cells number was respectively: in the 1st group - 20x104 (SD = 11x104), 2nd - 46x103 (SD = 15x103) and in the 3rd - 97x103 (SD = 38x103). The average of amniotic fluid amount was respectively: in the 1st group - 1ml (SD = 0,6), 2nd - 1,2ml (SD = 0,5) and 3rd - 2ml (SD = 0). The following results give the 30% of success rate.
In conclusion, even if further investigations are required, the results obtained in this study support the finding that amniotic fluid contains rapidly growing cells with high differentiation potential. On the other hand, from such an amount of amniotic fluid the number of isolated cells is small, so the effectiveness of culture establishment is to little and limits their application in
Arsenic and lead are ubiquitously distributed in environment. Human exposure to arsenic and lead is increased due to rapid industrialization and their use in formulation of many products. Presence of arsenic in drinking water is serious health problem in many countries. Arsenic and lead are known to have genotoxic and mutagenic effects and cause different cancers. Liposuction material is rich source of stem cells. In the present study cytotoxic and genotoxic effects of arsenic and lead were tested on Adipose derived mesenchymal stem cells (AMSCs). Cells were exposed to 1-10 μg/ml and 10-100 μg/ml concentration of arsenic and lead respectively for 6, 12, 24 and 48 h and cytotoxic effects were measured by neutral red uptake assay. Genotoxic effects of arsenic and lead were tested by comet assay. There was gradual decrease in growth of cells with increase in time and concentration of arsenic. There was also change in morphology of cells and cell became round at higher arsenic concentrations (10 μg /ml). Decrease in growth of AMSCs was observed on lead exposure but lead proved to be less cytotoxic when cells were exposed for longer duration. No change in morphology of lead exposed cells was observed. DNA damage was observed in metal treated cells. Different parameters of comet assay were investigated for control and treated cells which indicated more DNA damage in arsenic treated cells as compared to lead. Intact nucleus was observed in control cells. Present study clearly demonstrate that both arsenic and lead have cytotoxic and genotoxic effects on AMSCs while arsenic have more deleterious effects on AMSCs as compared to lead.
Testing of compounds for potential neurotoxic properties is required for all drugs and chemicals in Europe. Due to the complexity of neurotoxic effects especially systemic repeated dose toxicology testing is frequently performed in animals. Here we present a high throughput and high content screening platform based on stem cell derived neuronal networks cultured on microelectrode arrays. this assays allow parallell recording of nine neuronal networks with 28 electrodes each for extended periods of time. thus e can demonstrate repeated dose chronic toxicity as well as acute toxic effects with one integrated assay.
Cardiovascular diseases (CVDs) are the leading cause of death in the developed countries. CVDs have different types and among them myocardium infarction (MI) is the most frequent form. MI occurs when the blood flow in coronary artery blocked. It might happen due to different reasons but some parameters like age, gender, smoking, lack of physical activity can be involve on the frequency of that.
The main strategies for the treatment of MI are: Oxygen therapy and beta- blockers medication treatment. The main goal for those therapy methods is to restore blood flow in the blocked coronary as fast as possible. The other strategies are like injection of Heparin and to administration of some medication with anti platelet like Aspirin or Clopidogrel and finally in acute condition to undertake the heart surgery is necessary.
To undertake new approaches as supplementary approaches in addition to routine therapy methods seems promising. Among them stem cell therapy is a less invasive, less expensive seems promising approach to accelerate treatment of the patients following MI.
There are two main types of stem cells: 1- Embryonic stem cells (ESCs) and 2- Adult Stem Cells (ASCs).
To discuss ESCs is out of scope of this review but ASCs there are different types of that but the most important type of ASCs is bone marrow stem cells (BMSc). BMSc are delivered to two main sources of 1- hematopoietic stem cells (HSCs) and 2-Mesenchymal stem cells (MSCs).
In patients after MI heart function will reduce like left ventricular ejection fraction (LVEF) and some the other important parameters also affect too, therefore to undertake a new approach to help routine treatments following MI is an important strategy. The first time Strauer et al in 2002 reported successfully clinical administration of BMSc and since then many researches across the world have done more promising experimental and clinical on this topic.
The goal for this review poster is to determine the effects of administration of MSCs on the therapy of patients following MI according to recent published studies on this topic.
Hear Failure (HF) is one of the leading causes of death worldwide and especially in developed countries. When heart muscle does not capable to supply blood flow then HF occurs. Changes in human life style (e.g. lack of physical exercise), health problems (e.g. diabetes, hypertension,), smoking, age, and gender are most important risk factors of HF. HF mostly occur in men but the overall prevalence in both sexes are the same because women after HF more survive.
As HF is a chronic cardiovascular disease (CVD) therefore the treatment of that needs a life time management. In other words a patient suffer from HF needs to be under treatment for long time. As HF has different symptoms depends on them different therapy strategies will undertake by clinicians. A combination of some medications is most common but in severe HF cases to undertake heart surgery is a must.
The main medications are used for HF therapy are: 1- Angiotensin- Converting enzyme (ACE) inhibitors 2- Angiotensin II receptor blockers (ARBs) 3- Beta- blockers 4- Digoxin.
If the HF be more chronic then heart surgery like coronary bypass surgery or in the most severe case heart transplant is the final option save the life of patient. In addition to routine therapies for HF patients it seems that new approaches can be useful. Among them stem cell therapy is a promising method. Stem cells are a group of cells that have the renewal capabilities and they can repair damaged cells and tissues.
There are two main types of stem cells. 1- Embryonic stem cells (ESCs) and 2- Adult stem cells (ASCs). In this review poster we only discuss the effects of the administration of mesenchymal stem cells (MSCs) that are main type of ASCs on HF patients.
In animal models the administration of MSCs led to improvement on heart function in animals with HF condition. In clinical studies also the administration of MSCs on HF patients is promising.
The goal of this review poster is to present an up-to-date on the effects of administration of MSCs on the therapy of patients with HF.
In the subventricular zone (SVZ) neurogenesis is regulated by the coordinated proliferation and differentiation of different precursor types: slowly proliferating type B cells, rapidly dividing transit-amplifying type C cells and type A neuroblasts. Previous evidence suggests that g-aminobutyric acid (GABA) released by neuroblasts provides a feedback signal inhibiting the proliferation of type A and B cells by activation of GABAA receptors, while the effects of GABA on type C cells are unknown. Here we used levels of EGFR expression and lineage specific markers to purify stem cells and neuroblasts from the postnatal SVZ. Clonal assays and analysis of mice lacking expression of orphan receptor Tlx antigen showed that cells expressing high levels of EGFR (EGFRhigh) directly correlated with stem cell activity
Mesenchymal stem cells (MSC) are multipotent adult stem cells. They can be isolated from bone marrow (BM), fat tissue, cord blood (CB) and other tissues. Although these cells gain importance as potential sources for stem cell-based therapies, the developmental potential of MSCs from different origins and from different culture conditions are not well characterised. Therefore the BMBF-funded START-MSC (
Fat-MSC and CB-MSC were isolated by plastic adherence, BM-derived MSCs were FACS-sorted for the marker CD271. CD271 is considered a potential marker for BM-MSCs. To analyse the developmental potential of human MSCs (hMSC) from different origins (BM CD271+, CB, fat), we injected 5-10 hMSC of each type into murine embryonic day (E) 3.5 blastocysts and analysed the progeny of injected hMSCs in different tissues on embryonic day E16.5 by human-specific real-time PCR.
We were able to detect progeny of injected hMSCs in several embryonic tissues of E16.5 embryos. Donor positive tissues were detected after injection of all three hMSC types. Positive tissues showed low-level donor cell engraftment (between 2-20 human cells per 10.000 murine cells). Further we observe that CD271+ BM cells generated less frequent engraftment compared to hMSC derived from CB or fat tissue. The quantification of donor signals by real time PCR indicated that the injected cells underwent proliferation.
All three MSC types that were analysed showed engraftment in developing murine embryos. So far we could not detect enhanced engraftment of distinct tissues. The injection of pre-selected CD271+ BM cells shows the least frequent donor positive tissues. To further address the question whether donor cells are functionally integrated into recipient tissues we are planning combined immunohistochemistry and
Recent literature reported the isolation of spermatogonial stem cells from adult mouse testis and their response to differentiating culture conditions in a pluripotent manner similar to embryonic stem cells. Aim of the present study was to show the possibility of isolation and differentiation of adult spermtogonial stem cells from human testicular parenchyma into the different tissues of all of the three human germ layers.
Regular testicular parenchymas from orchiectomy specimens, determined by a pathologist, were collected. Cells were cultured in knockout culture medium with GDNF. After 7-10 days the medium was changed to basic medium with LIF. Clusters formed which contain the spermatogonial stem cells and for higher purification a MACS seperation with CD49f followed. The isolated cells were transposed into specific established culture mediums and protocols for neuronal, pancreatic, osteogenic and myogenic differentiation of embryonic stem cells. The precursor cells were transplanted in utero and adult nude mice for testing the potency of the cells.
Adult spermatogonial stem cells could spontaneously differentiate into derivatives of all three primary germ layers: differentiation towards insulin producing cells, osteogenic cells, smooth muscle cells, and neuronal live cells was confirmed by PCR analysis, ELISA and immunohistochemistry. Transplanted cells showed characteristics of neural and pancreatic tissues.
Adult spermatogonial stem cells seem to keep their pluripotency and plasticity throughout life. Human stem cells from testicular tissue may allow individual cell based therapy without the ethical problems associated with human embryonic stem cells and immunological problems of nowadays transplantation of allografts.
The existence of specialised regulatory microenvironments or niches that sustain stable stem cell populations is well documented in many tissues. However, the specific mechanisms by which niche support cells govern stem cell maintenance remain largely unknown. We have show that the Jak/Stat signalling pathway acts in somatic, support cells of the Drosophila ovary to maintain the adjacent germline stem cells (GSCs) in an undifferentiated state. In addition, we have demonstrated that removal of this pathway in support cells leads to stem cell loss by differentiation. Further, the ectopic activation of Jak/Stat signalling in support cells augments dpp mRNA levels and increases the range of Dpp signalling, a BMP2 homologue known to act as a niche extrinsic factor required for GSC survival and division in Drosophila. As a consequence, the GSC niche is expanded and tumours of stem cells are produced. Our results provide strong evidence for a model in which Jak/Stat signalling in support cells regulates dpp transcription and thus niche size.
In the bone marrow stem cell niche, osteoblasts lining the endosteum are of major importance in supporting hematopoietic stem cell maintenance and controlling self-renewal. Here, we report on the expression of the fibulins, highly conserved calcium-binding glycoproteins that are components of the extracellular matrix of human osteoblasts. We also provide insights into their functional relevance in the stem cell niche. All six members of the fibulin family were detected by RT-PCR. The expression of fibulin-1 and -2 was verified by Western blot analysis. Two-color immunofluorescence staining revealed a co-localization of both fibulins with fibronectin in an extracellular meshwork. Fibulin-2, but not fibulin-1, appeared to be instantly degraded by active metalloproteases present in osteoblast cultures. Fibulin-2 was shown to be a substrate for MMP-2, which was constitutively secreted by primary osteoblasts, as assessed by gelatin zymography. Whereas fibulin-2 exhibited cell binding properties towards the hematopoietic progenitor cell line KG1a, fibulin-1 was capable of modulating adhesion to fibronectin, resulting in the disturbance of fibronectin-induced cell signaling pathways, as analyzed with an anti-phosphotyrosine antibody. Thus, fibulins seem to be important components of the extracellular matrix of osteoblasts, which are likely to influence the adhesive properties of the stem cell niche towards hematopoietic stem cells.
Mouse or human embryonic stem (mES/hES) cell lines are usually derived and propagated on inactivated feeder cell layers. Human feeder cell layers such as foetal human fibroblasts or post-natal human skin fibroblasts have lately been used in order to eliminate the risk of foreign protein contamination during hES propagation. However, it is impossible to conclusively evaluate the beneficial or detrimental effects of feeder cell layers on propagation and subsequent differentiation of stem cells without the possibility to dissociate feeder and stem cells. Furthermore, feeder cells themselves might have a detrimental effect in clinical application, since feeder cells might be carried along during
Stem cells (D3) were grown on neomycin resistant
The sensitivity of the nested RT-PCR approach was demonstrated by amplifying
We have therefore developed a method that for the first time allows the complete and reproducible separation from stem and feeder cells at an early time point and the analysis of distinct stem cell colonies. Complete separation from stem and feeder cells is essential in regenerative cell replacement therapies. Separation of stem and feeder cells is also an important prerequisite in the unambiguous analysis of differentiation mechanism.
Many regenerative medicine research efforts focus on the treatment of severe medical conditions with cells derived from the patient’s own bone marrow (BM). The effectiveness of these autologous BM cell therapies is often limited by the number of stem and progenitor cells in the final product that contain tissue regenerating potential. One strategy to address the issue of stem and progenitor cell number is to culture the patient’s BM cells, prior to implantation, in a standardized and reproducible cell manufacturing process. Here we describe a closed, automated Good Manufacturing Practice (GMP)-compliant process that begins with a small bone marrow aspirate from the patient and through a series of process steps generates a patient-specific dose of Tissue Repair Cells (TRCs) that contains a higher number and higher percentage of stem and progenitor cells than the original BM.
Bone marrow mononuclear cells (MNCs) from a small sample of the patient’s BM (30-50 ml) are isolated by Ficoll gradient centrifugation using an automated device to deplete the erythrocyte and granulocyte components of the BM. The resulting MNCs are then inoculated into a cell culture cassette and cultivated for 12 days in a completely closed automated cell manufacturing system that provides a completely computer-controlled culture environment through the regulation of temperature and gas and media exchange. This cell environmental control together with the single-pass perfusion technology, enables the replication of early-stage stem and progenitor cells while preventing their differentiation into mature cells. The resulting cell product shows significantly increased populations of stem and progenitor cells and decreased populations of mature cells. CD90+ mesenchymal stem cells, represent a key cell population that undergoes significant expansion under this perfusion culture and has been observed to increase up to 48-fold in cell number and up to 200-fold in CFU-F forming capability during the manufacturing process. The stem cell content of the product has been proved through the ability of specific sub-populations to differentiate into adipocytes, osteoblasts or cells with endothelial characteristics.
In conclusion the combination of closed and automated technologies can be used to support the production of a validated and GMP-compliant cellular product for therapeutic use.
Diabetic foot syndrome (DFS) appears as a concomitant illness of diabetes mellitus. Diabetes is one of the main causes of non-traumatic amputation in Germany due to severe peripheral arterial occlusive disease (PAOD) with chronic critical limb ischemia (CLI) being the most abundant problem. Ulceration occurs as failure of the microcirculation and progressive occlusion of the larger blood vessels exacerbates preexisting microvascular abnormalities.
For the first time megadoses of pluripotent stem and progenitor cells - Tissue Repair Cells (TRCs) - are used in the treatment of diabetic ulcers to induce revascularisation of the affected limb and to enhance local perfusion. As a concomitant result wound healing, which is highly dependent on perfusion, should occur and lead to preservation of the limb. DFS patients with chronic limb ischemia and without the option of surgical or interventional revascularisation are being recruited and randomized to either the transplant group or the control group within this clinical trial. Approximately 40 ml of bone marrow is harvested from the iliac crest, followed by preparation and culture of mononuclear cells. Application sites of the enriched mesenchymal bone marrow cells (BMCs) or Tissue Repair Cells (TRCs) are intramuscular (i.m.) into the gastrocnemius of the affected limb or intraarterial (i.a.) into the femoral artery. Of the 26 patients have been enrolled in the study. 10 patients have completed the 12 month follow-up, 2 patients were immediately dropped from the study due to low bone marrow quality, 1 patient from the control group and 1 patient from the BMC i.a. left the study to receive major amputations, 1 patient withdrew his consent and the other 11 patients are still in the follow-up period. To measure the therapeutic effects of the various treatment arms patients were evaluated for ABI, TcpO2, and reactive hyperemia (Laser Doppler and BOLD). Patients also underwent imaging with angiographic methods..All patients treated with BMCs or TRCs, independent of application method, showed tendential improvement of microcirculation (reactive hyperemia) in the affected foot as well as complete primary wound healing. No serious adverse events related to the treatment were noted. In addition, no calcification of soft tissue has been observed.The transplantation of expanded autologous bone marrow stem cells as well as mesenchymal bone marrow cells in diabetic patients with critical limb ischemia induced chronic tissue ulcers proved to be safe and shows tendential improvement of microcirculation and complete wound healing.
Osteonecrosis of the femoral head involves the death of cells in trabecular bone and marrow, fracture of subchondral bone, and often leads to total hip replacement. Retrospective clinical studies have shown that osteonecrosis in 80-90% of affected patients progresses to destroy the femoral head, usually within 2-3 years after diagnosis. There are no effective treatment options for terminating or reversing the disease process.
Reports using autologous bone marrow tissue injected percutaneously into the necrotic area have shown a high rate of success in early stage osteonecrosis. As a more standardized alternative to fresh bone marrow, Aastrom Inc. has developed a proprietary automated process to expand autologous bone marrow cells
A new procedure has been developed to deliver BRC/matrix compositions into the femoral head to treat osteonecrosis. This abstract outlines the clinical formulations, the surgical procedure, safety profiles and clinical outcome of the product in 3 compassionate use patients with necrosis of the femoral head.
Transplantation of allogeneic stem cells of bone marrow or peripheral blood origin has proven to be the only curative therapy for a variety of malignant as well as non-malignant diseases. The authors describe recent advances in the field of stem cell selection techniques that have enabled the use of selected cell components for differential clinical applications under GMP conditions. The reconstitution of a functional immune system over time has been carefully monitored in several pediatric cohorts following transplantation of positively selected stem cells by V beta spectratyping of the emerging T cell repertoire as well as by TREC analysis.
We here report mayor scientific contributions that have rapidly revolutionized today’s haematopoietic stem cell transplantation strategies: Bone marrow, stimulated apheresis product or umbilical cord blood stem cells- which graft works best in the adult transplant setting? Does HLA disparity really limit transplantation success? CD34 selection, CD3/19 depletion and T cell therapies - how to puzzle the perfect cell graft. The stem cell recipient of increasing age: What is the appropriate conditioning chemotherapy with little toxicity?
Pathophysiological models developed from animal studies form the basis of our understanding of the development of stroke.
Based on fully matured neuroblastoma cells (SH-SY5Y) we established a unique
We employed the post hypoxic cell cultures for direct co-culturing with various fractions of stem cell containing mononuclear cells (MNC) from human umbilical cord blood (HUCB). Over the course of 3 days, all applicated MNC-fractions provided significant protection from neuronal apoptosis and also triggered the retaining of neuronal characteristics such as forming networks.
Clear neuroprotection was also detectable when MNC were affixed in impermeable cell-culture-inserts. In direct as well as in indirect co-cultures MNC induced an alteration in cytokine and chemokine concentrations. Our data suggest that the neuroprotective effects of MNC might result from direct cell-cell contacts and/or the adjustment of specific soluble mediators.
Acute graft-versus-host disease (aGVHD) is the major limitation for a broader application of allogeneic hematopoietic stem cell transplantation (allo-HSCT). In aGVHD alloreactive donor T-cells attack the recipient’s gastrointestinal tract, liver and skin. To address this unusual tissue tropism we developed luciferase transgenic (
A.B. and St. S. contributed equally to this study.
CD34 glycoprotein in human hematopoiesis is expressed on a subset of progenitor cells capable of self-renewal, multilineage differentiation and reconstitution. Nucleolin is an abundant factor of growing and cancerous cells, involved in regulation of gene transcription and RNA metabolism, whose transcripts are enriched in murine hematopoietic stem cells, as opposed to differentiated tissue. Here we investigated the involvement of nucleolin in gene regulation in human CD34-positive hematopoietic cells.
Interactions of nucleolin with CD34 promoter region were detected by EMSA and chromatin immunoprecipitation, and activation of the CD34 promoter region - by co-transfections of a nucleolin cDNA expression vector together with CD34 promoter reporter constructs. Levels of gene expression were monitored by immunoblotting, real-time RT-PCR and FACS.
Human bone marrow-derived CD34-positive cells KG1, stably transfected with a nucleolin cDNA expression vector, were employed. CD34-positive cells from apheresis collections were enriched using CD34 MicroBeads.
It is shown that, in human CD34-positive hematopoietic cells, nucleolin increases the abundance of the CD34 and Bcl-2 transcripts and proteins, and cell surface CD34 protein expression is enhanced by nucleolin. Nucleolin-mediated activation of the CD34 gene transcription results from sequence-specific interactions with the CD34 promoter region.
Our study reveals a novel role of nucleolin for gene regulation in CD34-positive hematopoietic cells and likely for the maintenance of these cells. We report that nucleolin is a crucial activator of the CD34 gene transcription. We suggest that nucleolin may be involved in controlling basic functions of CD34-positive hematopoietic cells, and its deregulation may contribute to the development of acute myelogenous leukemias.
Mesenchymal stem cells (MSC) are suitable candidates for the cell-based reconstruction of different tissues including cartilage and have been isolated from different sources such as synovium, adipose tissue and bone marrow. These cells were assumed to be similar with regard to self-renewal, multidifferentiation potential and surface epitopes. For the clinical application of MSCs for articular cartilage repair, functional suitability and phenotypic stability are crucial factors. In this study, we compared the
MSC were isolated from adipose tissue (ATSC), bone marrow (BMSC) and synovium (SMSC) and were cultured under chondrogenic, osteogenic and adipogenic conditions. Differentiation was evaluated based on histochemical staining, immunohistology and gene expression analysis. After 5 weeks of chondrogenesis, MSC were transplanted subcutaneously into SCID mice. Four weeks later, explants were analysed by histology.
ATSC and SMSC showed reduced chondrogenic differentiation potential compared to BMSC and required combined application of TGF-beta 3 plus BMP-6 for successful chondrogenic differentiation. Chondrogenesis was always associated with up-regulation of hypertrophic markers like Col X.
Our findings indicate that adipose tissue, bone marrow and synovium provide heterogenous MSC populations. SMSC, like ATSC, required more stringent conditions for chondrogenesis when compared with BMSC. None of the
Bone-marrow derived human mesenchymal stem cells (hMSCs) will probably be a valuable tool for regenerative medicine in the future. In-vitro cultured hMSCs show restricted proliferation and differentiation capability. In this study we demonstrate, that age of donors is a main limiting factor for in-vitro expansion and differentiation of hMSCs, but extracellular matrix (ECM) proteins (ECM-gel, Laminin-1, -5, -S, collagen IV, fibronectin), hypoxic conditions (1% and 5% O2), the use of human serum instead of fetal calf serum (FCS), and CD 271+ selection have positive effects on growth rates and differentiation capacity.
Cells were analyzed via histochemical and immunohistochemical staining procedures, telomerase activity detection, different flow cytometry protocols (incl. STRO-1, Oct-3/4 and Alkaline Phosphatase expression), quantitative photometrical and fluorometrical assays, and RT-PCR.
HMSCs, selected from young donors (age 2138), had significant higher proliferation and differentiation potential, compared to a group of elder (44-89) donators. Basement-membrane ECM proteins lead to a 103 fold higher in-vitro expansion and each protein had defined ascending effects on the process of differentiation; these effects were inhibited by antibodies against receptors of the particular protein. Hypoxic conditions, human serum, and CD 271 positive selection had also direct, significant effects on proliferation and the maintenance of differentiation capacity, and the combination of mentioned factors yielded again in a significant higher cumulation for the contemplated attributes. Cultivated samples were investigated for proliferation behaviour. The subunit of so-called RS-cells (flow-cytometry: light forward and side scatter population; small cells: 7-13 µm diameter) was exclusively responsible for growth of hMSCs cultures and showed better differentiation features. Each detected subpopulation originated from RS cell subunit. At least this in-vitro model was used for the expansion of peripherial blood extracted circulating cells after G-CSF stimulation, and CD 133 positive selection and/or Interleukin-6 (IL-6) treatment. Selected cells showed flow cytometric characteristics of hMSCs and could successfully cultivated, and differentiated into osteoblasts, chondrocytes and adipocytes. Without positive selection or IL-6 usage no cells with hMSCs properties were detected.
ECM-components, hypoxia, human serum, and magnetic bead associated cell selection are valuable constituents of an model for effective cultivation of mesenchymal stem cells extracted from different sources.
Cell trafficking between fetus and mother during pregnancy resulting in a situation of maternal microchimerism is a widespread phenomenon in mammalians including humans. We have investigated the trafficking of pregnancy
In the brain, PAPCs coexpress mature neuronal specific markers indicating neuronal differentiation potential. In contrast glial cell markers are not coexpressed. Some PAPCs were found to express stem/progenitor or immature neuronal markers such as nestin, doublecortin, and PS-NCAM. Further evidence for neuronal maturation comes from an increasing axonal/dendritic complexity over time. PAPC integration into the hippocampus is organotypical with neurons developing axonal/dendritic polarity which is indistinguishable from endogenous hippocampal neurons. In summary, PAPCs migrate to the maternal brain where they integrate and differentiate predominantly towards the neuronal linage. PAPCs undergo a process of maturation and express stem/progenitor or immature neuronal cell specific markers when they are phenotypically immature. Mature neuronal markers are coexpressed when cells are phenotypically more mature. This might indicate that integration and maturation of PAPCs into the brain follows mechanisms similar to that observed during natural adult neurogenesis.
Furthermore, we find PAPC in maternal organs such as pancreas, small intestine as well as kidney where, like in the brain, cells integrate organotypically. In the pancreas we found evidence for the coexpression of exocrine lineage markers which suggests pancreatic differentiation of PACs into acinar cells. We conclude that PAPCs as a population demonstrate multipotent lineage differentiation potential, a feature typical for stem cells. The fetal cell transfer model mimics an autologous transplantation paradigm and as such is an invaluable tool to study the integration, survival, differentiation, maturation and functionality of stem cells and their derivatives in different host tissues.
Allogeneic stem cell transplantation for patients with hematologic malignancies has proofed to be one of the most powerful forms of immunotherapy. Studies comparing T-cell depleted versus undepleted grafts convincingly demonstrated the role of T-cells in the graft-versus-leukaemia effect at the cost of increased graft-versus-host-disease. It has been an elusive goal of research to prevent GVHD without dramatically increasing the chance of relapse or major transplant complications. New insights in the regulatory mechanisms and dynamics of various T-cell subsets now open avenues for dissecting the T-cell response adapted to the patient's requirements: 1) supportive antigen-specific T-cell therapy provides protection against viral and fungal antigens; 2) regulatory T-cells may be used to modulate GVHD; 3) T-cells against minor histocompatibility antigens and leukaemia-associated antigens can provide targeted immunotherapy with a reduced risk of increased GVHD. We have developed methods to prime and expand antigen-specific cells from the naive T-cell repertoire and to specifically select for these T-cells on the basis of their CD137 expression upon stimulation. In consequence various epitopes of the leukaemia-associated transcription factor Wilms Tumor antigen 1 have been described that will be of further use for leukaemia-specific T-cell therapy. Furthermore the use of novel cytokine combinations in vitro results in a T-cell phenotype with a favourable CD28 expression, which increases the chances that adoptively transferred T-cells persist in vivo.
Human embryonic stem cells (hESC) hold great promise for regenerative medicine. Presumably, however, they would be rejected by the immune system of the recipient, as hESC would be transplanted into an allogeneic setting. Surprisingly, previous reports had shown reduced immune reactivity against hESC, with reduced proliferation and reduced cytotoxicity. Underlying cellular immune mechanisms are however not well defined. Interestingly, the inhibitory effect was shown to be soluble factor-independent, as fixation of hESC did not abrogate this inhibitory effect.
We used well established
We found that the presence of hESC could reduce specifically T-cell proliferation against allogeneic and xenogeneic antigens, as well as against the much stronger antibody-mediated TCR activation. Reduced T-cell proliferation in presence of hESC was neither due to an increase in apoptosis, nor to an increase in FoxP3+ regulatory T cells. Rather, we showed that T cell stimulation in the presence of hESC resulted in an increase in the proportion of surface CTLA-4 expressing T cell. CTLA-4 is a major inhibitor of T cell proliferation. Concomitantly, we also found an increase in IL-10 secreting T cells. IL-10, a major regulator of inflammation, inhibits T-cell proliferation and plays a role in tolerance induction after hematopoietic stem cell transplantation. Moreover, IL-10 has also been shown to be an effector of CTLA-4-mediated immune regulation.
Our results thus strongly suggest that the reducing effect of hESC on T-cell proliferation is mediated through upregulation of these two potent immune-regulatory molecules. Moreover, this effect was mediated by membrane-associated molecules on the hESC, as only fixed cells were used. It will be now interesting to characterize such molecules and to specifically address whether their presence would be maintained throughout terminal differentiation of hESC, as for transplantation no undifferentiated hESC would be transplanted.
Stem cells are commonly defined as being undifferentiated and capable of self-renewal. Although indefinite self-renewal is often regarded as an intrinsic and unique stem cell trait, accumulating evidence, points to the possibility that stem cells also arise through processes of de-differentiation. Therefore, the classical presentation of cell populations as being organized in a hierarchical manner, toped by a stem cell that gradually differentiates in an irreversible mode, should be re-examined. If progenitor cells that have already begun to differentiate, may turn back and become stem cell, then stemness is a state rather than a cellular entity. Our recent studies on multipotent stromal cells (MSC), show that such cell populations do not differentiate in a hierarchical manner, and that their differentiation mode is compatible with a phase space model. One example is that the capacity of MSC to support hemopoiesis, which is regarded as one direction of MSC differentiation, may be either a property of the undifferentiated MSC itself, or alternatively a property of fully differentiated osteogenic products of the MSC. An additional issue which is a focus of intense controversy, is the finding that bone marrow-derived stem cells may give rise to cells of other organs and tissues. The same is true for muscle-derived stem cells, neural stem cells and skin stem cells. I recently suggested that pluripotency that entails plasticity, rather than any other trait, is a hallmark of the stem state. Plasticity is thus a state in which the cells harbor a potential to give rise to a multitude of lineages and choose among the options available within the three germ layers. Several studies demonstrated the plastic nature of some adult tissue stem cells, such as the mesenchymal stem cells from the bone marrow, or the skin progenitor cell. These studies strongly suggest that the commonly accepted notion, that adult stem cells should be classified as entities distinct from pluripotent stem cells, is questionable. It is suggested that the underlying properties of cells in the stem state are: being undifferentiated, dependent on their niche for asymmetrical divisions and self-renewal and capable of differentiation into a wide range of cellular entities. These properties are not shared by cells within tumors that are capable of giving rise to new tumors upon transplantation, the tumor initiating cells (TIC). Indeed, unlike stem cells that depend on their niche and are lost following serial transplantation, TIC proliferate indefinitely. Most importantly, TIC have a very limited differentiation potential and are certainly not pluripotent. They therefore represent a biological entity that is biologically different from stem cells.
Hypertrophic differentiation of mesenchymal stem cells (MSCs) presents a major problem in TGF-b-driven
Chondrogenic induction of hBMSC spheroid cultures was modified by addition of factors suspected to stimulate or inhibit chondrogenic hypertrophy. Hypertrophic differentiation was assessed by immunohistochemical analysis (collagen type I, -II, -X, alcian blue), RT-PCR (Col1A1, Col2A1, Col10A1, MMP-13) and quantification of ALP activity up to 6 weeks of differentiation. After 6 weeks of culture under chondrogenic conditionsmicromasses were transplanted subcutaneously in SCID mice for 4 weeks and analyzed histologically (alizarin red) thereafter.
Chondrogenic differentiation as confirmed by positive staining of type II collagen and alcian blue was achieved after supplementing chondrogenic medium with TGF-b3. None of the other growth factors (BMP-2, -4, -6, -7, aFGF, IGF-I) led to chondrogenesis, alone, whereas combination with TGF-b results in chondrogenesis without suppressing collagen type X expression. Combinations of TGF-b with PTHrP or bFGF suppressed collagen type X staining and ALP induction. However, they also prevented the differentiation to chondrocyte-like cells when added from day 0. Delayed addition of PTHrP or bFGF rescued chondrogenesis and suppressed ALP activity along with expression of other hypertrophic markers.
PTHrP and bFGF are attractive anti- hypertrophic factors able to modulate the chondrogenic effect of TGF-b on MSC
Pluripotency describes the ability of stem cells to self-renew and to give rise to all cell types of the developing embryo. Pluripotent embryonic stem cells (ESCs) are established from the inner cell mass of blastocysts and can be maintained
CCE and BL6 ESCs were routinely grown on murine embryonic fibroblasts (MEFs) and E14 ESCs without MEFs on gelatine-coated dishes in the presence of LIF and 15% FCS. Upon FCS reduction (10%) and MEF- as well as LIF-removal differentiation was induced. Loss of pluripotency was measured by CFC assay, for which appropriate numbers of differentiated ESCs were plated onto MEFs and colonies were counted. Gene expression of pluripotency- and differentiation-associated genes was determined via
Upon differentiation induction ESCs quickly lose their pluripotent character and their potential to form colonies on MEFs. The expression of pluripotency-associated genes
Together, these data indicate that the end of pluripotency is characterized by an unexpectedly low level of heterogeneity within the pool of differentiating ES cells regarding histone H4 acetylation, proliferation and gene expression. Further studies shall address the question whether the end of pluripotency is a defined point of no return or whether there is a time window of ambiguity.
HMGA proteins are abundant architectural chromatin proteins. They bind to AT-rich DNA and localize preferentially in heterochromatin. Their acidic C-terminal domain is considered to be relevant in interactions between HMGA and other factors. In addition, HMGA proteins are expressed particularly in early embryos and undifferentiated cells. In differentiated cells they are downregulated and high HMGA expression levels in adult cells correlate with tumor progression and malignancy.
We used C2C12 mouse myoblast cells as a model system to investigate the influence of HMGA1 on differentiation and chromatin plasticity. Therefore, we created C2C12 cells stably expressing HMGA1a-EGFP and performed knock down of HMGA1 by siRNA. Using bimolecular fluorescence complementation (BiFC), we examined protein-protein interaction between HMGA and HP1 in living cells.
We showed for the first time that HMGA1 expression is gradually downregulated after induction of myogenesis. However, persistent HMGA expression inhibited myogenesis. This inhibition can be explained by an HMGA- dependent misexpression of several genes that are required for proper differentiation. RNAi experiments demonstrated that downregulation of HMGA proteins is necessary to restore proper gene expression and to release the myogenic program.
Furthermore, BiFC analyses revealed that the acidic C-terminus of HMGA interacts with the chromodomain of HP1. Photobleaching experiments indicated that the HP1 residence in heterochromatin dependents on the presences of functional HMGA proteins. Moreover, HP1 binding properties during early myogenic differentiation variegate dependent on the expression level of HMGA1.
Together, our data indicate that the differential expression of HMGA proteins and their capacity to interact with HP1 proteins participates in the regulation of heterochromatin maintenance and plasticity during differentiation. We propose that downregulation of HMGA proteins is required to allow chromatin plasticity and remodeling and to enable cells to enter the myogenic differentiation program.
Ageing of the organism besides functional impairment of mature somatic cells may be a matter of impaired regeneration as a consequence of altered self renewal of stem cells. The latter is caused by endogenous (cellular) and systemic (humoral) factors. These factors can involve cell damage and repair mechanisms, altered differentiation and induction of transdifferentiation towards alternative stem cell differentiation pathways, and epigenetic changes causing silencing of important key signalling genes.
Both stem cells and somatic cells are subjected to genomic and proteomic damage which accumulates and leads to the induction of cellular fail safe mechanisms, e.g. apoptosis and replicative senescence. Thus the suppression of tumour development by such fail safe programs occurs at the cost of proliferative and regenerative capacity. Monogenetic models of progeria suggest that altered DNA repair can cause ageing and progeria syndromes. Both loss of function mutations of DNA repair enzymes as well as enhanced DNA damage due to an increased load of reactive oxygen species (ROS) initiate cellular fail safe programs, but could also promote tumorigenesis, if proliferative stimuli overcome cell cycle control check points. There is recent evidence that quiescent stem cells can also accumulate damage because the quiescent state results in an attenuation of checkpoint control and DNA damage responses for repair or apoptosis. Thus in spite of sufficient numbers of stem cells their regeneration capacity may be severely impaired. The cellular load of ROS is controlled by environmental and nutritional factors but also by ROS metabolizing enzymes, which also can be targets of polymorphisms and mutations. Thus active control mechanisms in cells are essential to maintain their genomic and proteomic integrity.
Age-related switching into preferred stem cell differentiation pathways can also augment the ageing process of an organism, due to the lack of specific functions of the resulting tissue. Augmented Wnt signalling was reported to drive muscle stem cells towards fibrosis rather than muscle regeneration. Preferred adipogenic differentiation of mesenchymal stem cells and adipogenic transdifferentiation of osteogenic precursors represent another similar scenario. Epigenetic dysregulation may be another hallmark of ageing where the expression of growth and stemness factors can be impaired by DNA methylation processes and may also contribute to alterations of differentiation processes.
Adult and embryonic stem cells are both discussed as tools for cell based therapeutic strategies. Cell damage and ageing processes may occur during the ex vivo / in vitro handling procedures. Thus research on the maintenance of stem cell integrity appears to be extremely important. Strategies of differentially addressing healthy versus presenescent stem could be established to avoid forced proliferation of damaged cell populations. Exciting new data about a protective function of enhanced activity of the Arf/p53 (Cdkn2a locus (Arf) and p53) tumor suppressor pathway against ageing may promise that anti ageing protection can be even engineered in target stem cells to foster the security and minimize the risk of inducing malignancies in regenerative therapeutic strategies.
Aldosterone is a well known risk factor associated with hypertension and cardiovascular injury. Bone marrow derived endothelial progenitor cells (EPC) play an important role in neovascularization and endothelial repair implicating a possible role as target for the prevention/therapy of vascular diseases.
We examined the effects of aldosterone as well as the mineralocorticoid receptor (MR) antagonist eplerenone on EPC number and function
Human EPCs expressed the MR both at gene and protein level. Aldosterone treatment of human EPC in vitro impaired cellular function, which could be rescued by MR antagonism. EPC from patients with primary hyperaldosteronism had a significant impairment in migratory potential compared with age-matched healthy controls. Likewise, incubation of peripheral blood mononuclear cells with aldosterone (1-100 nmol/l) in vitro reduced EPC formation and development of colony forming units (CFU) in a concentration dependent manner. Co-treatment with the selective MR antagonist eplerenone alleviated this effect. Aldosterone reduced EPC migratory capacity and increased the intracellular production of reactive oxygen species (ROS), which was attenuated by MR blockade. While the protein kinase (PK) C inhibitor chelerythrine had no effect, co-treatment with the PKA inhibitor H-89 completely alleviated aldosterone effects on EPC migratory capacity and ROS production. Finally, eplerenone treatment improved number and function of circulating EPC in rats with secondary hyperadosteronism due to heart failure.
We have shown impaired EPC number and function in both primary and secondary hyperaldosteronism. In vitro, aldosterone impaired EPC formation and function in a MR- and PKA-dependent manner involving ROS formation. In vivo and clinical data additionally show a rescue of EPC function in hyperaldosteronism by specific targeting of the MR receptor. Beneficial effects of MR antagonists in cardiovascular disease prevention and therapy may be mediated in part by improved EPC biology.
Impairment of the proliferation potential of mesenchymal stem cells (MSCs) may account for age-related deficiencies in bone regeneration contributing to bone-related diseases like osteoporosis. As a model for cellular aging, extended
MSCs were isolated from bone marrow and incubated in standard propagation medium. Additional MSC cultures were supplemented with 2 or 6 ng/ml IGF2 to check for a possible rescue of replicative senescence. For each incubation, monolayers were passaged as soon as they reached confluence and RNA was isolated in parallel with each passaging. Gene expression levels of IGF signaling components and putatively IGF2-responsive genes were evaluated by RT-PCR.
MSC populations of eight different donors were subjected to
Reproducible down-regulation of IGF2 expression and some IGF2-responsive genes during
Fat-derived mesenchymal stem cells are a promising source of stem cells for cell transplantation. In the present study it is shown that undifferentiated human fat-derived stem cells display robust oscillations of intracellular calcium [Ca2+]i which may be associated with differentiation processes. [Ca2+], oscillations were dependent on extracellular calcium and calcium release from intracellular stores since they were abolished in calcium-free medium and in the presence of the store-depleting agent thapsigargin. [Ca2+]i oscillations were apparently regulated by the inositol 1,4,5-trisphosphate (InsP3) pathway and dependent on gap junctional coupling since they were abolished in the presence of the phospholipase C antagonist U73,122, in the presence of the InsP3 receptor antagonist 2-aminoethoxydiphenyl borate (2-APB) as well as in the presence of the gap junction uncouplers 1-heptanol and cabenoxolone. Preincubation with the nitric oxide (NO) synthase inhibitors NG-monomethyl-L-arginine (L-NMMA), N(G)-nitro-L-arginine methyl ester (L-NAME), and diphenylen iodonium (DPI), synchronized [Ca2+]i oscillations between individual cells within the area of inspection, whereas the NO-donor S-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside (SNP) were without effects. The synchronization of [Ca2+]i oscillations was due to an improvement of intracellular coupling since fluorescence recovery after photobleaching (FRAP) experiments revealed increased reflow of the fluorescence indicator calcein into the bleached area in the presence of the NOS inhibitor DPI. In summary our data demonstrate that intracellular NO levels regulate synchronization of [Ca2+]i oscillations in undifferentiated fat-derived stem cells by controlling gap junctional coupling.
The original notion that the bone marrow stroma would include a population of multipotent progenitors of skeletal tissues has evolved into the notion of a “mesenchymal stem cells”, which would be found not only in the bone marrow but also in other mesoderm derivatives in the adult organism. Until recently, self-renewal, one of the key defining properties of stem cells, remained undemonstrated, leaving an aura on uncertainty around the very notion of mesenchymal stem cell. Using the Melanoma Cell associated Adhesion Molecule (MCAM, CD146) as a marker suited to follow the fate of explanted clonogenic progenitors through ex vivo expansion and in vivo transplantation, we were able to show that MCAM expressing clonogenic progenitors in the bone marrow stroma do self-renew in vivo, generate multiple skeletal tissues, and regenerate a compartment of subendothelial stromal cells identical to the ones originally explanted as CFU-Fs. A unique feature of bone marrow stromal clonogenic progenitors is their ability to establish the hematopoietic microenvironment (HME) at heterotopic sites, which makes skeletal progenitors in the bone marrow stroma a key component of the hematopoietic stem cell “niche”. MCAM-expressing clonogenic progenitors (CFU-Fs) are found in, and can be purified from, a variety of other mesodermal derivatives, including skeletal muscle. Here, MCAM-expressing CFU-Fs represent a non-canonical, self-renewing myogenic progenitor, distinct from satellite cells, and endowed with an amazing capacity for spontaneous myogenic differentiation. Both in the bone marrow stroma and in skeletal muscle, cells that express MCAM in situ are located to the abluminal side of microvessels (sinusoids in the bone marrow). From all these data, a novel picture emerges in which a system of clonogenic progenitors endowed with tissue specific differentiation potential and self- renewal capacity exist in different tissues, with the same anatomical location and a closely similar surface phenotype. Their nature as microvascular subendothelial cells provides a novel clue as to the developmental mechanisms that lead to the establishment of progenitor/stem cells in post-natal tissues.
Pluripotent mesenchymal stem cells (MSC) are the source for the regeneration of bone and cartilage. In osteoporosis anabolic and antiresorptive drugs are applied which respectively stimulate the differentiation of MSC to osteoblasts (e.g. PTH and Teriparatide) or inhibit osteoclasts (e.g. bisphosphonates (BP)). The amino-bisphosphonate zoledronic acid (ZA) inhibits the farnesylsynthase of osteoclasts which leads to inhibition of posttranslational prenylation of proteins like Ras and Rho resulting in osteoclast apoptosis. It was shown that a once-yearly infusion of ZA 5 mg during a 3-year period significantly reduced the risk of osteoporotic fractures. Osteoblasts and precursor cells are also targets of bisphosphonates but little is known about short-time and chronic effects of ZA on MSC cultures and their osteogenic offspring.
To clarify the influence of ZA on MSC, cells were treated with 5, 20 and 50 µM ZA for 6 and 16 h and the gene expression pattern was determined by RT-PCR. Additionally, MSC were treated with 5 ? 50 µM ZA for 3 h and the osteogenic differentiation potential of MSC was analyzed after 4 weeks in osteogenic medium. Cells were harvested and osteogenic marker genes were amplified by RT-PCR. Mineralization of the cell monolayer was analyzed by alizarin red staining. For chronic stimulation with BP MSC were treated ) with 5, 20 and 50 µM ZA for 24, 48 and 72 h and apoptosis and proliferation capacity of the cells were determined.
Stimulation of MSC with 50 µM ZA for 6 to 16 h increased the expression of Dkk1 and runx3 and decreased the expression of L1CAM. Short-time treatment of MSC with 20 and 50 µM ZA for 3 h stimulated osteogenic differentiation capacity after 4 weeks compared to untreated controls which was shown by alizarin red staining. Osteogenic markers as runx3 and osteopontin were upregulated dose-dependently while osteocalcin was downregulated. In contrast, long-time treatment of MSC with 20 and 50 µM for 48 and 72 h led to increased apoptosis and decreased proliferation capacity. After 24 h no effect was detected.
We show here that a 3 h exposure to µM concentrations of ZA is already sufficient to enhance osteogenic differentiation of MSC in vitro while long-time exposure to ZA impairs MSC proliferation and induces MSC apoptosis. In osteoporosis treatment this might be of clinical relevance to better determine the dosing and the upper threshold of BP accumulation in the bone microenvironment when constantly high local BP concentrations start to impair osteogenic effects in bone.
Supported by Novartis.
Crohn’s disease (CD) is associated with a higher prevalence of osteoporosis, a complication that is increasingly recognized as a significant source of morbidity also in these patients (pts.). Predicting those individuals at high risk remains controversial, yet disease activity is thought to be one of the main contributing factors. We used primary human mesenchymal progenitor cells directed to the osteoblastic phenotype to investigate the effect of serum of seven pts. during the active phase in CD on expression of osteoblast related genes. Four of the investigated pts. had osteoporosis or osteopenia (CD-opo), three showed no obvious bone disease (CD-con).
Mesenchymal progenitor cells obtained from a healthy patient were grown up to confluence in DMEM supplied with 10% FCS, glutamine, penicillin/streptomycin. Cells were seeded at 105/ml in 6 well plates. Supplementation was switched to 1% human serum (healthy proband). When reaching confluence, cells were stimulated with 10 mM? P-glycerolphosphate and 10 µM ascorbic acid and human serum was changed to eight different CD sera (four CD-opo, three CD-con, one healthy control). After 3 and 14 days cell were harvested and mRNA was isolated using TRIZOL. Marker expression was determined with real-time PCR and beta-actin was used for normalization.
A clear difference in gene expression profile between sere from CD pts. and from the healthy proband was detectable. Sera from CD-opo pts. induced RANKL gene expression 100 fold compared to healthy proband and 50 fold compared to CD-con. Osterix and RUNX 2 expression increased 2 to 10 fold, respectively, by serum from all CD pts., whereas no difference was seen for alkaline phosphatase and interleukin 6. Collagen and osteocalcin gene expression seemed to be influenced without reaching significant effects.
Serum from pts. with CD induced RANKL gene expression reflecting activation of bone resorption. This induction was largest in CD-opo pts. suggesting RANKL as one of the main contributing factors in this condition. Osteoblastic transcription factors osterix and RUNX2 were also induced by inflammatory activity but did not differentiate between CD- opo and CD-con. No effect on late osteoblast differentiation markers was detectable. Our results suggest that factors in the serum of pts. with active CD lead to bone disease mediated by mesenchymal progenitor cells.
Neurogenesis persists throughout adulthood in two restricted regions of the mammalian central nervous system: the subventricular zone of the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus. The generation of new neurons from neural stem cells is controlled by signals derived from the local microenvironment. We previously showed that Wnt-proteins are key regulators of adult hippocampal neurogenesis. Specifically, we found that Wnt-signaling stimulates neuronal fate determination and proliferation of neuronally committed precursor cells. We have now identified several transcription factors, which are prominently expressed in immature newborn neurons of the adult dentate gyrus. In this presentation, we will discuss the potential function of these transcription factors as well as their potential interaction with Wnt-signaling in adult hippocampal neurogenesis.
Neural stem cells (NSCs) are found in the developing as well as in the adult brain. They are self-renewing cells that maintain the capacity to differentiate into all major brain-specific cell types, such as glial cells and neurons. However, it is still unclear whether these cells are capable of gaining full functionality, which is one of the major rerequisites for NSC-based cell replacement strategies of neurological diseases. The ability to establish and maintain polarized excitatory synaptic contacts would be one of the basic requirements for intercellular communication and functional integration into existing neuronal networks. In primary cultures of hippocampal neurons it has already been shown that synaptogenesis is characterized by a well ordered, time-dependent targeting and recruitment of pre- and postsynaptic proteins. In this study, we investigated the expression and localization of important pre- and postsynaptic proteins including Bassoon and synaptophysin, as well as proteins of the ProSAP/Shank family, in differentiating rat fetal mesencephalic NSCs.
Moreover, we analyzed the ultrastructural features of neuronal cell-cell contacts during synaptogenesis. We show that NSCs express and localize cytoskeletal and scaffolding molecules of the pre- and postsynaptic specializations, in a well defined temporal order.
Current pharmacotherapy of Parkinson’s disease does improve symptoms and extends the patients life expectancy, but does not provide a cure for this detrimental disease. Alternative approaches include cell replacement therapy. Human bone marrow stromal cells (MSC) are not capable of neuroectodermal differentiation per se. Though, in recent years several reports have claimed to achieve a neural transdifferentiation of these easily accessible stem cells. Here we report our protocol to
Upon trypsination MSC are cultured in suspension in serum-free P48F medium with 5 μ heparin, FGF2, and EGF (20 ng/ml each), under 3% oxygen (instead of 21%).
Initially, we identified changes of gene expression upon conversion of MSC by mRNA gene chip analysis. In respect to their genome-wide transcriptome, both MSC and MSC-NSC were similar to each other, but differed significantly from adult human NSC. On closer examination we could identify several “neural” genes (e.g. EAAT1, GFAP, MAOA, MAP2, MBP, NES, NOT1, NSE, PAX6, PTCH, SNCB) and dopaminergic genes (e.g. Nurr1, TH) being up-regulated in MSC-NSC and confirmed these data by real-time RT-PCR. Further investigations revealed that these changes were due to the specific culture conditions (suspension, serum-withdrawal, and hypoxia). Additionally, several neurotrophic factors (e.g. HGF, VEGFa) are up-regulated on both mRNA and protein level, reaching concentrations significantly higher than in human adult cortex. Though lacking therapeutic effects in a mouse model of amyotrophic lateral sclerosis due to limited survival of intrathecally transplanted cells, we observed a directed migration of MSC-NSC
Based on their neural gene expression profile MSC-NSC share noticeable similarities with adult human NSC arguing for a partial transdifferentiation. The converted cells release neurotrophic factors at high concentrations and further investigations will reveal whether the cells synthesize and release dopamine. Presenting a relatively simple epigenetic protocol to induce a cell phenotype with potentially improved neurotherapeutic properties, MSC-NSC might be a valuable resource for future treatment of neurodegenerative diseases by autologous transplantation.
The enteric nervous system (ENS) is part of the PNS and regulates the blood flow and the peristaltic and secretory activity of the gut. There is evidence that the ENS is also involved in the function of the mucosal immune system and influences the epithelial stem cell compartment of intestinal mucosa.
Several groups have demonstrated that multipotent, self-renewing ENS progenitor cells persist in the fetal and postnatal gut of humans and rodents. These neural progenitors can be expanded and differentiated in neurons and glia cells under appropriate cell culture conditions.
An important role for the regulation of stem cells and progenitor cells has been attributed to the canonical Wnt pathway. The Wnt signaling cascade is initiated upon binding of the secreted Wnt ligand to a member of the family of Frizzled (Fzd) transmembrane receptors and a specific co-receptor of the family of low-density lipoprotein receptors.
We have recently identified Fzd-4 expressing cells in the ENS of human small and large intestine. We further characterized, positive cells from human perinatal und adult gut samples using RT-PCR, immunohistochemistry and FACS. Additionally, Fzd-4 positive cells were separated by MACS-Sorting to investigate the proliferation and differentiation capacity of selected cells
Understanding molecular mechanisms that control differentiation of multipotent mesenchymal stromal cells or mesenchymal stem cells (MSCs) is central to the development of novel therapies. Many studies have successfully identified critical factors involved in initiating and maintaining lineage-specific transcriptional changes although the role of post-transcriptional gene silencing during differentiation remains unclear.
In the last decade, it has emerged that RNA may play a major role in the regulation of gene and protein expression in all eukaryotic cells. The transcriptome describes the repertoire of RNAs transcribed from the genome of a particular organism. Gene expression profiling, using defined cell populations, can determine which mRNA species are transcribed, the proteins they encode (though not necessarily translated) and the potential functional consequences. However mature mRNAs represent only about 2% of the total transcriptional output of complex genomes and there is growing evidence that non-coding RNAs profoundly influence gene expression profiles and therefore cellular function.
MicroRNAs (miRNAs) are a superfamily of evolutionary conserved, small non-coding RNAs that bind to the 3’ UTR of mRNAs to inhibit protein translation. Consequently, miRNAs can influence a broad range of biological activities, which are instrumental in conferring tissue identity and function. We have determined the expression function of miRNAs during osteogenic differentiation of MSCs and osteoprogenitors in vitro. Expression was determined by microarray analysis of 984 known and predicted miRNAs using RNA isolated from undifferentiated cells or following differentiation in the presence of osteogenic supplements (dexamethasone, ascorbic acid and beta-gycerolphosphate) over 21 days. Differentiation was confirmed by induction of alkaline phosphatase, von-Kossa positive mineralisation and qRT-PCR, revealing temporally relevant changes in expression of osteogenic markers (cbfa-1, alkaline phosphatase, type-I collagen, osteopontin and osteocalcin). The analyses revealed constitutive and differentiation-dependent expression of different miRNAs. The functional role of miRNAs was determined using anti-mir microRNA inhibitors transfected into progenitors before osteogenic stimulation and bioinformatic analyses using predictive algorithms were used to identify potential miRNA targets. Our evidence suggests an important functional role of miRNAs during osteogenesis which may have pervasive implications for our understanding of MSC biology.
Mechanical boundary conditions as well as the function of mesenchymal stem cells (MSCs) play a pivotal role during bone regeneration. However, the molecular mechanisms underlying the dependency of the regeneration process on mechanical loading remain unclear. This study has therefore investigated how matrix metalloproteases (MMP) activity is influenced by mechanical stimulation and whether MMP activity affects MSC behaviour.
MSCs were isolated from bone marrow aspirates and subsequently characterised functionally and by flow cytometry. The influence of mechanical stimulation was analysed in a bioreactor system that aims to resemble the early phase of bone healing (compression of 10 kPa, 1Hz, 3 days) using a fibrin/cancellous bone construct. A furin inhibitor was added to investigate the mechanism of MMP regulation. RNA and protein expression were determined by means of gene arrays and ELISAs. Gelatinolytic activity of MMPs was detected by zymography. Functional assays investigating migration, proliferation and differentiation were supplemented with specific inhibitors for MMP-2, MMP-3 and MMP-13. Three independent experiments using different MSC donors with at least duplicates in each were conducted.
Mechanical stimulation of MSCs led to an upregulation of their extracellular gelatinolytic activity, which was consistent with the increased protein levels seen for MMP-2, -3, -13 and TIMP-2. However, mRNA expression levels of MMPs/TIMPs showed no changes in response to mechanical stimulation. The furin protease was proved to be involved in the regulation of MMP-2. Specific inhibition of MMP-2, -3 and -13 showed MMP-13 to be associated with osteogenic differentiation.
The observed independency of mRNA and protein expression levels indicates the involvement of post transcriptional processes for MMP regulation, such as the alteration in MMP activity demonstrated for MMP-2. To summarise, the results of this study suggest that MSC function is controlled by MMP activity, which in turn is regulated by mechanical stimulation of these cells. Thus MMP/TIMP balance seems to play an essential role in transferring mechanical signals into MSC function. In the future, details of this cascade are to be unravelled before such understanding could be translated into stimulation of bone healing even in unloaded clinical conditions.
Osteoarthritis (OA) is such a widespread complication of age that it is expected to become the fourth leading cause of disability by the year 2020. The prevalence and the incidence rates are significantly higher in women. The healthy, homogeneous population of chondrocytes in articular cartilage changes in the pathogenesis of OA to a heterogeneous mixture of OA chondrocytes, newly emerging elongated chondrocytes and progenitor cells, mainly found adjacent to the OA defect.
With the help of fluorescence associated flow cytometry (FACS), micro-array and real time RT-PCR we characterized the different cell types from different patients to evaluate their chondrogenic and differentiation potential according to the gender.
In vivo, elongated chondrocytes from women and men exhibit a more fibroblast-like expression pattern than the OA chondrocytes. Micro-array analysis (Affymetrix Human Genome U133A 2.0) of healthy cartilage tissue and of the cell populations of OA tissues revealed gender specific differences: e.g., SOX-9, collagen type II and COMP turned out to be regulated; whereas fibronectin, integrin alpha 5 and collagen type I expression was unregulated between women and men. FACS analyses demonstrated progenitor cells from late stages of human OA positive for “so called” stem cell markers. Tested for their differentiation potential in vitro, the OA chondrocytes were not able to differentiate even after dedifferentiation on plastic. In contrast, approximately 20% of the elongated chondrocyte population and 75% of the progenitor cell population can be differentiated into chondrocytes, osteocytes and adipocytes.
We present evidence that the elongated chondrocytes from late stages of OA originate from the progenitor cell population. These progenitor cells have a strong chondrogenic potential which can be enhanced by suppressing their osteogenic differentiation. Their differentiation potential and their matrix production capacity differ between women and men, which is in line with the differences in the prevalence and the incidence rates of OA. The regulatory influences leading to these differences need further investigations to allow a better understanding of the pathogenesis of OA and hopefully to reveal new aspects for therapeutic interventions. The results support our opinion that the progenitor cells seem to be a promising starting point for new concepts of a cell biological treatment of OA.
Mesenchymal stem cells (MSCs) are multipotent adult stem cells capable to differentiate into osteoblasts. Therefore, they represent attractive cell sources for tissue engineering applications, especially for bone replacement. Proteoglycans (PGs) exhibit a crucial role for matrix assembly and remodeling. Nevertheless, since bone development is a highly dynamic and complex process, the regulation of the extracellular matrix (ECM) formation remains elusive. Consequently, the aim of this study was to investigate the mRNA expression levels of genes involved in PG assembly. Hence, we analyzed mRNA and protein expressions of crucial PG core proteins and key enzymes involved in glycosaminoglycan (GAG) biosynthesis in different stages of osteogenesis.
Human MSCs were induced to differentiate into osteoblasts in appropriate medium. The progress of osteogenesis was confirmed using histological stainings. The mRNA levels of osteogenic marker genes as well as the investigated target genes were analyzed by real-time RT-PCR. The xylosyltransferase activity was monitored in the culture supernatant.
For one of the rate-limiting enzymes in GAG biosynthesis xylosyltransferase I (XT-I), maximal mRNA expression levels (3.89 ± 0.83-fold increase) and elevated enzyme activities (285 ± 17 dpm/μg DNA) were observed 10 days after osteogenic induction, simultaneously to the beginning mineralization of the ECM, whereas the highly homologous protein XT-II showed no specific alterations. The differential expression of chondroitin sulfate, dermatan sulfate and heparan sulfate chains were determined by analyzing the mRNA expression of EXTL2 (α-1,4-
The tight synchronized expression profiles demonstrate the coordinated biosynthesis of the PGs and underline their importance for the formation of bone and for the osteogenic stem cell differentiation. Furthermore our results indicate that prominent assembly and remodeling processes occur especially in later stages of osteogenesis and in parallel to the mineralization of the ECM.
The isolation and culture of embryonic stem (ES) cells has opened the possibility of generating unlimited numbers of any cell type. This is of particular importance in neurobiology as homogeneous cell populations are not available in sufficient quantities. In addition, ES cells can be genetically manipulated or isolated carrying relevant mutations. Thus, wild-type and mutant neurons may be compared and mechanisms causing the loss of specific cell types in neurodegenerative diseases identified.
We have devised a neuronal differentiation procedure of mouse ES cells
We use this system to analyze signaling pathways in neurodegeneration. In contrast to neuronal cell lines we can address synaptic function and neurite de- and regeneration. For example, using ES cells that we isolated from
Our studies show the highly predictive value of the differentiation system for the
Zygotes with two genomes from the same sex generated by the exchange of paternal (androgenetic; AG) or maternal (gynogenetic; GG) pronuclei are not competent to develop into viable offspring. Despite the restricted developmental potential of uniparental cells probably reflecting the different roles of maternal and paternal genomes during development, uniparental zygotes develop into blastocyst from which embryonic stem cells (ESC) can be generated. The neural differentiation potential of GG ESCs is well characterised while the developmental potential of AG ES cells is less clear.
Our study investigates the potential of murine AG ESCs in comparison to GG and biparental (normal fertilised; N) ESCs to differentiate into neural progenitor/stem cells utilizing an
We observe that AG ESCs similar to GG and N ESCs differentiate
Our data show that uni- and bi-parental ESCs do not differ in their
Neural stem cells (NSCs) are self-renewing multipotent precursors capable of giving rise to both neurons and macroglia. NSCs persist postnatally in the subventricular zone (SVZ) the germinal epithelium lining the lateral ventricle. In the anterior SVZ (SVZa), three main types of precursors drive the process of neurogenesis (type B, C and A) leading to the generation of olfactory inhibitory interneurons throughout adulthood. Type B cells, the primary stem cell type divides rarely giving rise to type C cells or transit amplifying cells that by rapid divisions generate committed migratory neuroblasts also known as type A cells. Although both type B and C cells are clonogenic
NPCs were proliferated in NSA medium containing EGF (20 ng/ml), FGF-2 (10ng/ml) and B27 (2%). For differentiation, NPCs were cultured on poly-L-lysine (PLL)-coated chamber slides with NSA medium containing 1% FCS and 2 ng/ml FGF-2. Lentivirus was produced in 293FT cell line by cotransfection of lentiviral construct and packaging plasmids for Dlx2 gene delivery to NPCs. EGFR+ cells or infected cells were sorted by FACS. Gene expression level was analyzed by qPCR.
EGFR expressing cells were isolated from both the anterior and the hippocampal SVZ of postnatal and embryonic mice at day 18 of embryonic development (E18). Despite they were both capable of forming clones, cells derived from the hippocampal SVZ expressed lower levels of EGFR and Dlx-2 mRNA and were less neurogenic compared to their SVZa counterpart. Forced expression of Dlx2 promoted clone forming capacity, proliferation, and neurogenesis in precursors derived from the SVZa but not in precursors obtained from the hippocampal SVZ. Importantly the effect of Dlx-2 overexpession in SVZa precursors was mediated by activation of EGFR.
Taken together, these data suggest that SVZa and hippocampal SVZ precursors are intrinsically different. Furthermore Dlx2 promotes transit-amplifying properties only in precursors derived from the SVZa.
78 patients aged 3-18 were treated for DMD. Diagnosis was confirmed by the genetic analysis.
Patients were administered mesenchymal, ectodermal, and endodermal stem cells isolated from germ layers of 4-8 weeks old cadaverous embryos’ systems and organs; amounts administered - 0,5-3 ml, cell count - 0,1-100x105/ml.
In the course of the first two months after the treatment, strength of different muscle groups increased by 100-700% and was maintained for 8-15 months. Besides, reported was also ROM increase, within the limits of the patient’s stage: stage II patients reported improved gait quality, ability to step on the heel and walk the stairs, raise from the floor with more confidence, and endure bigger loads during the day. Observed were also decreased pseudohypertrophy and strain of forearms and calves, contractures of knee and ankle joints, subsidence of myocardiopathy manifestations and respiratory insufficiency, especially in stage IVa-IVb patients.
DMD patients require continuous treatment (at least, once every 6-8 months) aimed at aversion of muscular atrophy progression. Termination of the treatment results in further progression of the disease and limitation of physical activity. Transplantation of embryonic stem cells terminates DMD progression, notwithstanding the stage of the process, and results in marked improvements.
Nanoscale materials are the fundamental building blocks and functional subunits of cells, including subcellular organelles and extracellular matrix components. Currently, there is growing recognition of the importance of understanding and incorporating nanobiology into biomedical applications. This issue is of particular importance in the emerging field of regenerative medicine, the goal of which is to develop methods to repair, replace, and regenerate diseased, injured, or non-functional tissues. Towards this goal, stem or progenitor cells have been considered a highly desirable candidate cell type, because of their expandability and potential to be induced toward specific cell differentiation lineages. A key requirement in tissue engineering and regenerative medicine is that ultimately the “regenerate tissue” needs to be a three-dimensional structure. In weight-bearing musculoskeletal tissues, this requirement is particularly critical. Musculoskeletal disorders affect one out of seven Americans. This severe disease burden underscores the need to develop novel and effective treatment protocols. This seminar will present the excitement as well as the challenges in the field of skeletal tissue engineering and regeneration, specifically the application of adult stem cells and nanomateriral scaffolds. The biology of adult stem cells, particularly the mechanisms regulating their proliferation
Duchene muscular dystrophy (DMD) is a most severe form of muscular dystrophy, which is inherited as a sex-linked recessive trait and affects 1/3500 of newborn males. Molecular genetic studies indicate that DMD is the result of mutations in the huge gene that encodes dystrophin. In order to confirm the results obtained from mouse model, which did not provide clinical sings of the disease, it has been proposed that muscular dystrophy in the golden retriever dog may be homologous to human to clinical trials. To compare two types of adult stem cells, Umbilical Cord CD 34+ and Dental Pulp Stem Cells (IDPSC), as potential multipotent stem cells for cell therapy use, by the evaluation of their skeletal myogenic potential, migration ability and capacity to restore dystrophin function in skeletal muscle cells of dystrophic young dogs.
Each cell type was analyzed according to their morphology, ultrastructure (confocal, immunohistochemistry and TE microscopy), and cell culture ability.
By TEM, canine umbilical cord progenitor cells had an immature cell structure, differing from all primitive blood components. Cell cultures showed poor proliferation. Also, cells, obtained by density solution and magnetic separation, were used for injections into the biceps femuralis or the femural artery. After 60 days, tissue biopies failed to demonstrate the presence of dystrophin either by imunnohistochemistry or by protein blotting. Conversely, the analysis of tissue biopsies of animals injected with IDPSC showed denser cell engraftment, as indicated by both the presence of DiI-stained cells and anti-IDPSC antibody positive labeling. Clinical aspects were considered relevant, with the demonstration of significant differences depending on the route of injection and cell type.
The efficacy of arterial injection of pulp dental cells to treat muscular dystrophy demonstrated that canine multipotent stem cells have great potential for cell therapy, promising to become a new trend for therapeuthical approaches aiming muscular dystrophy.
With increased participation in sports, the frequency of anterior cruciate ligament (ACL) ruptures is rapidly increasing. Currently over 100,000 patients rupture their ACL each year in the USA. The ACL fails to heal after rupture, and loss of ACL function leads to knee instability, loss of proprioceptive function, and osteoarthritis in over 60% of patients. Ligament reconstruction with biologic grafts such as autologous patellar tendon or hamstring tendon is the gold standard but does not restore the complex architecture and biomechanics of the ACL. A number of growth factors such as transforming growth factor (TGF-??, insulin-like growth factor-1 (IGF-1), fibroblast growth factor-2 (FGF-2), and respectively, bone morphogenetic proteins-12 and -13 (BMP-12 and -13) have been evaluated for their ability to stimulate different aspects of ligament repair, including outgrowth, cell division, and collagen synthesis. In this study the ligament differentiation of human mesenchymal stem cells (hMSCs) transduced with BMP-12 and embedded in collagen hydrogel was investigated.
Human MSCs from bone marrow were transduced at confluency with 10 vp/cell of adenovirus encoding BMP-12. Afterwards, genetically modified hMSCs were placed at 3 x 105 cells per 200 μl collagen type I hydrogel (Arthro Kinetics plc, Esslingen, Germany). After 21 days in culture histochemical, immunohistochemical, and RT-PCR analyses of the hydrogel constructs were performed.
Histochemical (H&E, Azan, Masson Goldner, van Gieson) and immunohistochemical analyses revealed elongated fibroblast-like cells embedded in a ligament-like matrix. The genetical modification of MSCs with BMP-12 resulted in a weak staining for collagen type III and V and elastin. In contrast, a strong staining for tenascin and vimentin was detected. Furthermore, specific ligament marker like biglycan, collagen type III and V, decorin, elastin, tenascin, tenomodulin, and vimentin were expressed as shown in the RT-PCR analyses.
Gene transfer with BMP-12 in combination with a collagen type I hydrogel lead to the development of a ligament-specific extracellular matrix. However, the use of genetically modified hMSCs could be an effective strategy for the formation of genetically optimized hydrogel constructs for the ligament repair.
Matrix-based autologous stem cell transplantation marks a promising approach for the treatment of chondral lesions, although none of the clinically used biomaterials reflects both biochemical composition and ultrastructural aspects of articular cartilage. Recent findings have shown that electrospun three-dimensional nanofibrous structures provide a biomimetic environment for seeded cells. In this study, we investigated the fabrication of a collagen type II-based nanofibrous scaffold via electrospinning for the potential use in stem cell based cartilage repair strategies.
Electrospun collagen type II (bovine) scaffolds were fabricated using different solvents, hexafluoropropanol (HFP) or acetic acid, with varying concentrations of collagen type II. Morphological characteristics of the different scaffolds were assessed by scanning electron microscopy. Optimized nanofiber scaffolds were subject to chemical cross-linking using hexamethylene-diisocyanate to prevent rapid biodegradation. Cross-linked scaffolds were seeded with 40,000 MSCs and cultured for 21 days in basal medium. Cell proliferation and cell mediated contraction was assayed over time.
In general, nanofibers produced using HFP showed a larger diameter (400-3000 nm) when compared to fibers spun from acetic acid (150-650nm), which also appeared more homogeneous. In addition, fiber size increased with higher collagen II concentration for each solvent. Cross-linking of the scaffolds was necessary to stabilize and prevent the structure from rapid biodegradation. An increase in cell number during the culture period was seen for both types of scaffolds, a 2.1-fold increase when using acetic acid, and a 1.41-fold increase when using HFP as solvent. After 21 days, cell-mediated contraction in the acetic acid derived scaffold was 23%, and 12% when using HFP as the solvent.
We have successfully electrospun collagen type II using a weak acid solvent to produce collagen type II-based nanofibrous scaffolds. Our results show that fiber sizes are dependent on fabrication parameters. By using cross-linking reagents, the collagen type II nanofibers are structurally stable during the culture period, and support continuous cell proliferation. Taken together, these findings suggest that promising electrospun collagen type II nanofibers have biologically favorable structure and properties and represent tissue engineered scaffolds applicable for cartilage repair.
Teratomas are benign tumours derived from pluripotent embryonic stem cells and contain a variety of organ structures. Sacrococcygeal teratomas (SCTs) are the most frequent solid tumours of the newborn with an incidence of approximately 1:35.000. To find a novel source for human embryonic stem cells, SCTs were examined.
1. A histological survey was performed on 20 SCTs. 2. Tissue from a SCT of a newborn girl was obtained intra-operatively. After generation of a cell suspension, cells were expanded in selective media to facilitate the cultivation of stem cells contained therein. After a few cell passages, a homogeneous population of stem cells was isolated.
1. Histological analysis of the 20 SCTs revealed that in particular, caudal tissues (Fallopian tube, vaginal epithelium) and the outgrowth of pancreatic tissue with islets from colonic crypts were encountered in 50% of cases. Stem cells were successfully demonstrated in paraffin sections by using nanog, Oct4, SSEA-4, and nestin antibodies.
2. Immunofluorescence and RT-PCR analyses revealed that the isolated cells were positive for nanog, Oct4, SSEA-4, and Stella, and thus showed traits of embryonic stem cells. The isolated stem cells spontaneously grew as spheroids. First differentiation assays showed that
Sacrococcygeal teratomas represent a novel source for human embryonic stem cells.
During cell division type III intermediate filament proteins such as desmin and vimentin are disassembled by phosphorylation of their amino-terminal domains. Embryonic stem cells (ESCs) have a high mitotic index, a very short cell cycle and do not synthesize type III IF proteins with the exception of vimentin. We hypothesize that type III intermediate filament proteins, different from vimentin, might not only be dispensable but also detrimental for proliferation or phenotypic maintenance of ESCs, and these effects might be mediated by their amino-terminal domains.
Mouse ESCs were stable transfected with a constitutively expressed desminect and a mutant desmin1-48 transgene, respectively. Proliferation of ESCs was analysed and localisation of desmin and vimentin in mouse ESC, in differentiating ESCs in embryoid bodies and in mouse cardioblast like stem cells isolated from embryonic and neonatal heart was studied by confocal scanning immunofluorescence microscopy.
Constitutive expression of desmin led to the immediate death of all but one ESC clone. Low level synthesis of desmin in one surviving ESC line caused severe growth retardation. Constitutive synthesis of desminΔ 1-48, lacking the amino-terminal domain, allowed establishing ESC lines which were not hindered in their proliferation. Synthesis of desmin or desminΔ1-48 did not alter the stem cell phenotype and was not sufficient to induce differentiation of ESCs. As vimentin, desmin was diffusely distributed in the cytoplasm and most surprisingly, in the nucleus of a significant fraction of ESCs. DesminΔ1-48 never entered the nucleus and formed aggregates in the cytoplasm of ESCs. Desmin but not desminΔ1-48 was also found to localise in the cell nucleus of a small fraction of putative precursor cells of cardiomyocytes in embryoid bodies.
Desmin influences the cell cycle but not the maintenance of the pluripotent phenotype of ESCs. The amino-terminus of desmin is required for its nuclear localization and for the negative effect of desmin on proliferation and survival of ESCs. Localisation of desmin in the nucleus of cells differentiating to cardiomyocytes provides some physiological significance and supports our previous findings that desmin influences expression of cardiomyocyte specific transcription factor genes and corroborates PETER TRAUB’s numerous data demonstrating a specific interaction of type III intermediate filament proteins with DNA.
Embryonic stem cells (ESCs) of murine and human origin have been shown to be able to differentiate into derivatives of all three germ layers and also into extraembryonic trophoectoderm. ESC-derived trophoblasts may represent an extremely useful in vitro model to investigate placental morphogenesis and implantation events during embryonic development. Therefore, the aim of our study was to investigate, whether monkey ES cells are able to form trophoectoderm in vitro similar to mouse and human ESCs.
Rhesus monkey ESCs (RESCs) were maintained and passaged using standard protocols. To initiate differentiation, non-differentiated RESC colonies were detached and transferred into agarose-coated culture plates to form embryoid bodies (EBs). After 2d of suspension culture in 80% αMEM, 20% FCS, 1 mM Glutamine, 0.1 mM β-Mercaptoethanol and 1% non-essential amino acids, the EBs were plated onto tissue culture plates coated with 0.1% Gelatin in 80% IMDM, 20% FCS, 1 mM Glutamine, 0.1 mM β-Mercaptoethanol and 1% non-essential amino acids. In the course of 2-3 weeks the expression of trophoblast markers was assessed by means of semi-quantitative RT-PCR, immunofluorescence and electron microscopy.
Based on the experimental conditions used to differentiate human ES-cells into cardiomyocytes, we developed a protocol, aiming at the differentiation of RESCs into trophoblasts. Using an EB-based approach, effective differentiation into trophoblasts was achieved. To characterize the trophoblast-like cells, semi-quantitative RT-PCR analyses were performed. The expression of several trophoblast marker genes including human chorionic gonadotrophin α (αHCG), caudal type homeobox transcription factor 2 (cdx2), eomesodermin (eomes) fibroblast growth factor receptor 2 (FGFR-2) and HAND-1 could be detected. Immunofluorescence staining showed expression of cdx2 and cytokeratin Endo-A (Troma-1), whereas the endodermal marker α-fetoprotein was not expressed. The ultra structural analysis of cystic structures, most likely formed due to the expression of fluid-pumping channels as typical for early trophoblasts, showed that the differentiated cells resembled cells of the trophoectoderm. More detailed analyses of the gene expression and of the ultra structures are ongoing.
This is the first study demonstrating the successful differentiation of RESCs towards cells of the trophoblast lineage. RESC-derived trophoblasts represent a valuable tool to investigate differentiation and function of early human trophoblast.
Non-human primate embryonic stem cells (ESC) are an invaluable tool for preclinical cell replacement therapy approaches. Moreover, primate ESC can be used to study specific problems of early primate development. Since ES cell lines show slight yet significant differences in their differentiation potential and molecular characteristics, it is important to have several different ES cell lines from one species at the same time allowing comparative studies. Thus, the best cell line for each intended application can be chosen.
We have characterized a novel ES cell line (CJES001) from the common marmoset
Blastocyst collection and immunosurgery were performed as described before by Sasaki et al. (2005). CJES001 cells expressed alkaline phosphatase (AP), stage specific embryonic antigens (SSEA)-1, -3, and -4, tumor rejection antigen (TRA) 1-60, and TRA 1-81, as well as the transcription factors SOX-2 and OCT4, indicating pluripotency. Teratoma formation in immunodeficient mice as well as embryoid body (EB) formation revealed the potential to differentiate into derivatives of all three embryonic germ layers. Karyotyping analysis showed presence of a complete set of female chromosomes at passages >70, and a roughly 5-fold increase of telomerase activity was detected by real time PCR when compared to cultured primary cells of the marmoset.
Interestingly, also strong expression of germ cell specific genes was detected in CJES001 cells at the mRNA level by RT-PCR and at the protein level by immunofluorescence indicating that these cells also develop germ line cells in addition to several types of somatic cells. Due to the fact that different ESC lines exhibit different preferences with regard to germ layer differentiation, we now may have a model cell line available for germ cell formation.
In conclusion, we report the establishment of a novel ES cell line from the common marmoset, which can be used to evaluate the potential of cell replacement therapy in a preclinical nonhuman primate model. Moreover, basic cell and developmental studies can be facilitated by these cells.
Osteoarthritis (OA) is such a widespread complication of age that it is expected to become the fourth leading cause of disability by the year 2020. The healthy, homogeneous population of chondrocytes in articular cartilage changes in the pathogenesis of OA to a heterogeneous mixture of OA chondrocytes, newly emerging elongated chondrocytes and progenitor cells, mainly found adjacent to the OA defect. In vivo, elongated chondrocytes show a more fibroblast-like expression pattern than the OA chondrocytes. Micro-array analysis (Affymetrix Human Genome U133A 2.0) of healthy cartilage tissue and of the cell populations of OA tissues revealed differences in the expression profiles of these three cell types. FACS analyses demonstrated progenitor cells from late stages of human OA positive for stem cell markers. Tested for their differentiation potential in vitro, the OA chondrocytes turned out to be unable to differentiate. In contrast, approximately 20% of the elongated chondrocyte population and 75% of the progenitor cell population can be differentiated into chondrocytes, osteocytes and adipocytes. There is evidence that the percentages of differentiable cells in populations sorted by FACS for stem cell markers, e.g. CD29, CD44, CD90, CD105, are highly different. The expression profile of “so called” markers for asymmetric cell division might have an influence on these results. RNA silencing experiments show, that the suppression of genes associated with osteocyte differentiation enhances the chondrogenic potential of the progenitor cells. When seeded on the surface of OA defects the progenitor cells attach and integrate in short time. We present evidence that the elongated chondrocytes from late stages of OA originate from the progenitor cell population. These progenitor cells have a strong chondrogenic potential enhanced by suppressing their osteogenic differentiation. The regulatory influences leading to these differences need further investigations to allow for a better understanding of the pathogenesis of OA and hopefully to unravel new cell biological therapeutic interventions.
Local or systemic recruitment of adult stem cells is a central process for tissue regeneration and repair. The respective knowledge concerning non-hematopoetic stem cells, however, is just beginning to be elucidated. Multipotent mesenchymal stromal cells (MSCs) for example have been identified in postnatal bone marrow and various tissues or organs throughout the body and it has been suggested that their presence within the perivascular niche may contribute to tissue distribution. In blood MSCs circulate in very low numbers under physiologic conditions, however, an increase has been observed after trauma. On the other hand systemic application does not only lead to “homing” into the bone marrow but also to sites of tissue lesions.
To address the basic question whether human bone marrow-derived MSCs are able to respond to soluble chemoattractive ligands we analyzed their migratory response to various growth factors released during bone remodeling using a modified Boyden chamber assay. These studies indicated that BMP-2/4, different PDGF-isoforms (most potently PDGF-BB) and IGF-I/II were chemoattractive for human MSCs. Interestingly IGFBP5 had a stimulatory effect itself and enhanced the influence of IGF-I in contrast to IGFBP3. Osteogenic differentiation partly decreased (e.g. PDGF-BB) and partly increased (e.g. IGF-I) the respective chemotactic responses. Since bone formation
Endothelial progenitor cells (EPCs) are a promising tool for applications in tissue engineering, treatment of cardiovascular diseases and cancer therapy. A major limitation until today is the small number of EPCs which can be obtained from one patient. The cysteine-rich protein 61 (CYR61/CCN1) is a matricellular signalling protein which belongs to the CCN-family. Here we describe the improved ex vivo propagation of EPCs obtained from peripheral blood in the presence of CYR61 [0.5 ug/μl].
EPCs were isolated from peripheral blood of 25 healthy volunteers via ficoll gradient centrifugation. Treatment of the cells with CYR61 occurred in different concentrations (0.05 μg/ml to 1.5 μg/ml) and at different time points. CYR61 treated and untreated control cells were characterised using FACS analysis, immunohistochemistry, RT-PCR, uptake of acLDL and concurrent staining for ulex lectin. Additionally, cell treatment with the CYR61 mutants with defect binding sides for the integrins αvβ3 (B3) and α6β1 (T1) was performed.
EPCs respond to CYR61 treatment in a dose dependent manner. Treatment with 0.5 μg/ml CYR61 resulted in a 7-fold increased cell number within one week, compared to unstimulated control cells. Both CYR61 treated and untreated cells show the same EPC marker expression of CD34, CD133 and KDR in FACS analysis, immunohistochemistry and RT-PCR, respectively. Uptake of acLDL and concurrent staining for ulex lectin proved the EPC phenotype of the cultivated cells. Treatment of EPCs with the CYR61 mutants B3 and T1 showed less but not significant results in cell number compared to CYR61 treatment.
The marker expression of CD34, CD133 and KDR as well as the uptake of acLDL and concurrent staining with ulex lectin in both the CYR61 treated and untreated cells clearly demonstrates that CYR61 treatment does not affect EPCs phenotype. CYR61 treatment at different time points indicated that the observed effect of improved cell number is a result of increased cell adhesion rather than enhanced cell proliferation. The results of the EPCs treatment with the CYR61 mutants B3 and T1 indicate that the integrins αvβ3 and α6β1 are not involved in the CYR61 effect on EPCs.
We conclude that the angiogenic inducer CYR61 is a promising factor for
Cell-based therapies are a promising approach to face multiple medical questions. Therefore, non-invasive monitoring of those cells is an important issue. Today, even single cells can be monitored non-invasively with MRI using superparamagnetic iron oxide (SPIO) particles as MR markers. In MR images SPIOs cause hypointensities which, unfortunately, can also be induced by other causes, e.g. blood clots. Thus, monitoring of SPIO labeled cells is ambiguous. Recently, perfluorocarbon (PFC) particles have shown their potential as non-ambiguous MR markers for detection of cells
Cells from the human mesenchymal stem cell line hMSC-TERT were double-labeled. First, cells were labeled with perfluoro-15-crown-5-ether (PF15C) emulsions using electroporation. Subsequently, the PFC labeled cells were co-labeled with SPIO particles (VSOPs) via incubation. 1H/19F-MRI was preformed on a 7 T Bruker Biospec with a homebuilt, double-resonant birdcage-coil. For high resolution 1H-MR images Gradient-Echo sequences were used. 19F Turbo-Spin-Echo experiments were performed with lower resolution to identify/confirm the location of the double-labeled cells. 19F-MRS was used to quantify the intracellular PF15C content. Furthermore, the intracellular iron quantity was measured with mass-spectroscopy. Cell apoptosis and proliferation rates were determined using luminescence tests. Histological analyses of the double-labeled TERT cells were performed with haematoxylin-eosin (H&E) and Prussian blue staining.
The double-labeled cells can be detected easily
1H-MR images and 19F-MR images allow the visualization of the labeled cells and could be acquired in experiments lasting only a few minutes. The labeled cells appear as dark spots or regions in the 1H images and can be distinctly identified using 19F-MRI. Double-labeling thus allows to monitor labeled cells non-ambiguously with high spatial resolution and may lead to quantitative cell imaging in the future using 19F-MRS.
Stem/progenitor cells with the potential to differentiate into insulin producing cells in vitro and/or in vivo were described in pancreatic islets, pancreatic ducts, among the population of pancreatic acinar cells and within adult or fetal pancreas without further specification. In some instances progenitor cells in pancreatic ducts were thought to expand and differentiate into insulin producing cells in response to specific stimuli. Whereas in the case of acinar cells a de-differentiation appears to be the first step followed by re-differentiation into β-cells. The origin of the bona fide stem cell however remains somewhat elusive and a recent report questioned the entire concept of β-cell stem/progenitor cells with studies using genetic lineage tracing experiments. With this approach it has been shown that pre-existing β-cells rather than adult stem/progenitor cells retained a proliferative capacity and may thus represent the major source of new β--cells in adult life, at least in mice. In this study the authors almost excluded the possibility of stem or progenitor cells to play a role in β-cell replacement in adult life. This extreme position however may not be justified by the data, given the fact that the study was not designed to identify precursor cells per se but rather to provide evidence for or against their participation in β-cell regeneration. And, this study is in conflict with numerous recent in vivo and in vitro studies suggesting the existence of pancreatic stem/precursor cells. Today, it is not evident which of the concepts will pass the test of time.
Cell-based therapy of type 1 diabetes mellitus resides on beta cell replacement and islet regeneration strategies. Various cell types of embryonic, foetal and adult origin have been investigated. So far, the most adequate alternative cell source is still to be defined. Human islet pancreatic derived cells (hIPC) have been demonstrated to differentiate
Gene expression pattern was analysed in hIPCs and hMSC by means of cDNA microarray hybridization (Affymetrix U133 Plus 2.0 chip) and stem cell marker genes were confirmed by RT-PCR. hIPCs and hMSC were expanded and cultivated in specific pancreatic endocrine promoting conditions (high glucose serum free medium, Activin-A, s-cellulin, hHGF, exendin-4 and nicotinamid). After 5 days in differentiation medium, islet gene markers were investigated by means of RT-PCR and immunocytochemistry in both cell populations.
Both cell types expressed key genes of a cluster of stemness-associated genes
Our results provide evidence for similar stemness character of the two stromal populations. Both, hIPCs and hMSCs can be induced
Strategies for cell based-therapy of type 1 diabetes mellitus are based on pancreatic islet replacement and islet regeneration. Human islet-derived precursor cells (hIPC) expressing nestin and c-met have been investigated as an additional source of beta-cells. These cells have been demonstrated to differentiate in vitro into insulin producing cells and are assumed to be of endodermal origin. In continuation of previous work we provide further evidence that hIPCs share a common phenotype with human bone marrow derived mesenchymal stem cells (hMSC) and, in addition, bear the potential to differentiate along mesenchymal maturation pathways.
hIPC and hMSC phenotyping was perfomed by FACS and immunocytochemistry. Gene expression was examined by cDNA array analysis (Affymetrix U133 Plus Chip). hIPCs were expanded and subjected to osteogenic, chondrogenic and adipogenic differentiation media. Differentiation markers were analysed by RT-PCR and immunocytochemistry.
Both cell types express nestin and c-met. hIPCs display a mesenchymal immunophenotype (SH3+, SH2+, CD29+, CD44+, CD54+, CD90+) and a gene expression pattern similar to hMSC. Moreover, hIPCs could be induced to differentiate in vitro towards the osteogenic (osteocalcin, alkaline phosphatase, day 28), adipogenic (LPL, PPARgamma, day 14) and chondrogenic (collagen IX, X, day 21) lineages.
Our results demonstrate that hIPCs and hMSCs share common phenotypes and similar mesenchymal differentiation capacities supporting the occurrence of endodermal to mesodermal transition in epithelial precursor cells. Understanding the molecular mechanisms that enable these cells to cross the traditional germ layer boundaries will help to develop effective strategies for in vitro generation and differentiation of specific phenotypes for the use in cell therapeutic approaches.Moreover, if hIPCs represent a population of pancreatic cells with stem cell/regeneration potential, these cells could be induced
Over the past decade methods have been developed to engineer spontaneously contracting, force-generating 3-dimensional cardiac muscle tissues, both as a target validation model in drug development and as material for cardiac replacement therapy. The present techniques depend on primary cardiac cells from newborn rat or mouse or embryonic chick that have no significant capacity for cell division. Consequently, cardiac tissue engineering at present is essentially a mean to reassemble a heart tissue
Characterization of gene expression signatures for cardiomyocytes derived from embryonic stem cells will help to define their early biologic processes.
A transgenic alpha-myosin heavy chain (MHC) embryonic stem cell lineage was generated, expressing puromycin resistance and enhanced green fluorescent protein (EGFP) under the control of the alpha-MHC promoter. A puromycin-resistant, EGFP-positive, alpha-MHC-positive cardiomyocyte population was isolated with over 92% purity. RNA was isolated after electrophysiological characterization of the cardiomyocytes.
Comprehensive transcriptome analysis of alpha-MHC-positive cardiomyocytes in comparison with undifferentiated alpha-MHC embryonic stem cells and the control population from 15-day-old embryoid bodies led to identification of 884 upregulated probe sets and 951 downregulated probe sets in alpha-MHC-positive cardiomyocytes. A subset of upregulated genes encodes cytoskeletal and voltage-dependent channel proteins, and proteins that participate in aerobic energy metabolism. Interestingly, mitosis, apoptosis, and Wnt signaling-associated genes were downregulated in the cardiomyocytes. In contrast, annotations for genes upregulated in the alpha-MHC-positive cardiomyocytes are enriched for the following Gene Ontology (GO) categories: enzyme-linked receptor protein signaling pathway (GO:0007167), protein kinase activity (GO:0004672), negative regulation of Wnt receptor signaling pathway (GO:0030178), and regulation of cell size (0:0008361). They were also enriched for the Biocarta p38 mitogen-activated protein kinase signaling pathway and Kyoto Encyclopedia of Genes and Genomes (KEGG) calcium signaling pathway.
The specific pattern of gene expression in the cardiomyocytes derived from embryonic stem cells reflects the biologic, physiologic, and functional processes that take place in mature cardiomyocytes. Identification of cardiomyocyte-specific gene expression patterns and signaling pathways will contribute toward elucidating their roles in intact cardiac function.
Prolonged myocardial ischemia results in myocardial dysfunction, even after successful revascularisation. We have reported that retrograde application of embryonic EPCs, derived from murine d 7.5 embryos (Tie-2+, c- Kit+, Sca-1+, flk-1 low, MHC-1-), provide rapid cardioprotection via soluble factors. Now, we investigated the role of Thymosin beta 4 (TB4) as paracrine factor mediating the cardioprotective potential of eEPCs.
In vitro, neonatal rat cardiomyocytes (NRCMs) were subjected to hypoxia (4h)/reoxygenation (1h) in the absence or presence of eEPCs with or without TB4 shRNA transfection, which decreased of TB4 mRNA levels to 20% of control cells. In vivo, pigs (n=6 per group) underwent percutaneous LAD occlusion for 60 min. After 55 min of ischemia either saline solution or eEPCs (5 * 10 6 DiI labeled cells) ± TB4 shRNA, or TB4 protein (15mg/animal) were applied via selective pressure-regulated retroinfusion into the anterior interventricular vein. Infarct size and functional reserve (sonomicrometry at 150/min atrial pacing) were determined after 24h reperfusion. Myeloperoxidase (MPO) levels were obtained to analyze inflammatory cell invasion.
In vitro, survival of NRCMs was increased from 32 ± 4 to 90 ± 2%, when eEPC were present.TB4 shRNA, but not control shRNA, abolished this effect (45 ± 7%), whereas TB4 protein restored it (85±3%). In vivo, eEPCs significantly decreased infarct size compared to control group (36±3% vs. 58±5% of AAR, p<0.05), but not in the presence of TB4-shRNA (48 ± 7% of AAR). Moreover, TB4 protein retroinfusion alone was as effective as eEPC application (36±4%). Functional reserve of the infarcted area was enhanced after eEPC (33±4 SES, % of RCx) or TB4 protein application (39±5% SES) compared to control (10±3%), except eEPCs transfected with TB4 shRNA (19±5% SES). Retroinfusion of eEPCs or TB4 protein significantly reduced MPO levels in the ischemic tissue (1996±546, 1455±197 U/g tissue MPO) compared to control (3323±388 U/g tissue MPO) or TB4 shRNA treated eEPCs (5449±829 U/g tissue MPO).
Our findings reveal that early cardioprotection after ischemia may be achieved by embryonic EPC retroinfusion, unless TB4 levels are largely reduced. Consistently, TB4 protein suffices to provide significant cardioprotection after ischemia and reperfusion.
Stem cell therapy holds great promise for the replacement of damaged or dysfunctional myocardium. Peroxisome proliferators-activated receptors (PPARα, -β and ??)) are nuclear receptors which have been shown to participate in cell differentiation. The aim of this study was to investigate the role of PPARα in cardiomyogenesis during the differentiation of mouse embryonic stem (ES) cell-derived embryoid bodies (EBs). When EBs were treated with PPARα agonists (WY14643, GW7647 and Ciprofibrate) a significant increase in cardiomyogenesis was observed. In contrast, the PPARα antagonist MK886 decreased the number and the size of beating foci. The effect of the PPARα agonists was abolished when EBs were pre-incubated with the free radical scavengers Vitamin E (trolox) and N-(2-mercapto-propionyl)-glycine, indicating the involvement of reactive oxygen species (ROS). Furthermore, we observed an increase in ROS when EBs were treated with PPARα agonists, and consequently a decrease in intracellular ROS when EBs were treated with MK886. The effect of PPAR? agonists on intracellular ROS was attenuated by the NADPH-oxidase inhibitor DPI and Apocynin, indicating the involvement of NADPH oxidase. Also, we observed a significant increase in mRNA expression of fetal cardiac genes and membrane potential of mitochondria after treatment with PPARα agonists. In summary our data indicate, that PPAR? stimulation induces cardiomyogenesis in ES cells using a pathway that involves ROS and NADPH oxidase.
Beta-cell replacement represents the ultimate cure for type 1 diabetes, however it is limited by availability of organ donors. Adult human islets are difficult to propagate in culture, and efforts to expand them result in loss of beta-cell phenotype. We have recently shown that cells from adult human islets of multiple donors can be significantly expanded in tissue culture in a simple medium for at least 16 population doublings, without a change in replication rate or noticeable cell mortality, representing an expansion of over 65,000-fold. Microarray studies revealed extensive changes in gene expression in the expanded cells, compared with normal islets. Indirect evidence suggested that a significant fraction of the replicating cells were derived from dedifferentiated β cells. However, genetic lineage-tracing studies in mouse models failed to document expansion of cultured beta cells. We are currently developing lineage-tracing tools for studying cultured human beta cells. In a screening of compounds capable of inducing redifferentiation of the expanded cells we have shown that betacellulin restores β-cell gene expression and insulin content in cells from part of the donors. These methods may allow transplantation of functional islet cells from single donors into multiple recipients.
The shortage of human islets as well as allograft rejection hampers islet transplantation for treatment of type 1 diabetes. Recent findings suggest that bone marrow derived mesenchymal stem cells (MSC) have the capacity to differentiate into a variety of cell types including endocrine cells of the pancreas, which could provide an abundant source of autologous cells for this procedure. Our aim was to investigate if MSC isolated from different patients with type 1 diabetes harbour the potential to adopt a pancreatic endocrine phenotype in order to accelerate a bench-to-bedside approach regarding the future use of bMSC for transplantation purposes.
MSC were isolated from three patients with diabetes mellitus type 1 and expanded in DMEM with 10% FBS and FGF (5ng/ml). For induction of differentiation, cells were incubated for three days in serum free differentiation medium (DMEM/F12) supplemented with factors known to enhance β-cell differentiation. Total RNA was extracted on days 0, 1, 2 and 3 and subjected to quantitative real time RT-PCR. Immunocytochemistry (ICC) was performed with undifferentiated and differentiated cells from day 3 using an anti human c-peptide antisera.
Upon induction of differentiation, the formation of islet-like clusters was observed and quantitative RT-PCR analysis revealed thatvarious key transcription factors needed for the development of pancreatic endocrine cells were either constitutively expressed (Isl-1, Pax6) or up-regulated (Ngn-3, Ipf-1) during the 3 days differentiation period. Additionally, an induction of the islet protein genes (insulin, somatostatin, glucagon and glut-2) was observed. Furthermore, ICC indicated the presence of C-peptide positive cells in differentiated cells.
bMSC isolated from patients with diabetes mellitus type 1 are able to differentiate into a pancreatic endocrine phenotype in vitro by sequential expression of key developmental genes. The yield of islet hormones however was very low, indicating that the current strategies for isolation and differentiation need substantial improvements.
In the course of 1-5 years, observed were18 patients (6 women and 12 men) with diabetes mellitus (DM) complicated by hyperinsulinism and dyslipidemia, mean age being 43.5±5.8 years, duration of the disease ? 1.2±0.5 years. 40% of these patients managed their condition by diet alone, others were undergoing monotherapy with minimal dosages of sulphonylureas. All patients presented with lasting morning hyperglycemia, absence of glycosuria, above normal C-Peptide (5.4±1.3 ng/ml), HbA1C concentration ? 8.1±1.5%. All patients showed changed blood lipid profiles with elevated cholesterol level, in the mean by 35%, increased LDL and VLDL concentration by mean 30%. The symptoms of the disease were minimal and manifested in the form of functional disturbances and psychophysiological reactions. Patients were treated by transplantations of hematopoietic and non-hematopoietic mesenchymal and endodermal embryonic stem cells isolated from growth zones of 4-8 weeks old cadaverous embryos’ internal organs. Suspensions were administered intravenously, in the amount of 0.5-3.0 ml, cell count0.1-100x 105/ml.
In the course of the first post-transplant week, 44% of patients reported elevation of glycemia by 15-25%, then decrease, without additional glycemic therapy. Within a month, 84% of patients reported decreased weakness and better mood. Decrease of morning hyperglycemia began after 2-3 months, and was accompanied by lowered C-Peptide. Normalization of the above indices, as well as lowering of HbA1C concentration, was reported in 5-7 months after the initial treatment; at the same time period observed was positive tendency in normalization of lipid metabolism. Lasting normalization of carbohydrate and lipid metabolism in 78% of patients was reported after 10-12 months, and was accompanied by discontinuance of hyperglycemic medications. Further 2-year observation of 60% of the above patients confirmed sustainability of the effects.
Ca2+ is believed to serve as an important mediator of differentiation. We therefore investigated the role of L-type Ca2+ channels during directional differentiation of murine embryonic stem (ES) cells into cardiomyocytes
The murine ES cell line D3, engineered to express EGFP under control of the a-myosin heavy chain promoter, was cultivated according to standard protocols. Embryoid bodies (EBs) were differentiated into spontaneously beating cardiomyocytes either under control conditions or under presence of 10 μM nifedipine, a known L-type Ca2+ channel blocker. Differentiation was monitored using fluorescence microscopy, immunocytochemistry and flow cytometry. The expression of cardiac genes was studied applying the reverse transcription-polymerase chain reaction (RT-PCR). The classical whole cell configuration of the patch clamp technique was used for action potential (AP) and L-Type Ca2+ current measurements.
EBs cultured in the presence of nifedipine exhibited a markedly decreased number of EGFP-positive cardiomyocytes from 3.16±0.3% in control, n=5 and 0.36% in nifedipine-treated conditions, n=5 (flow cytometry data). This inhibitory effect was related to reduced gene expression of a- cardiac MHC, ANF and MLC-2V. With patch clamp experiments we found after extensive nifedipine washout a prolongation of the APD90 (66.8±0.8 ms in nifedipine-treated EBs (n=10) as compared to 43.1±0.4 ms in control conditions, n=29). We have also observed a significantly decreased of L-type Ca2+ current amplitude (~ 75%), a right shift of the current-voltage (
Blockage of L-type Ca2+ channels inhibits differentiation of ES cells into cardiomyocytes.
Acute graft-versus-host disease (aGVHD) is the major limitation for a broader application of allogeneic hematopoietic stem cell transplantation (allo-HSCT). In aGVHD alloreactive donor T-cells attack the recipient’s gastrointestinal tract, liver and skin. To address this unusual tissue tropism we developed luciferase transgenic (
A.B. and St. S. contributed equally to this study.
Bone marrow (BM) derived stem cells improve cardiac function after myocardial infarction (MI). Recently, parathyroid hormone (PTH) was shown to regulate the stem cell niche in the bone marrow. Therefore, in a murine model of MI we analyzed the influence of PTH treatment on survival, functional parameters as well as stem cell homing to the heart.
12-24 h after MI, PTH was injected daily for two weeks. 6 and 30 days after the surgical procedure, pressure volume relationships were investigated in vivo. Heart tissues were investigated by immunhistochemistry and RT-PCR. Cardiac Homing was studied by flow cytometry.
PTH treatment resulted in a significant improvement of post MI survival and myocardial function associated with less reduction of LV wall thickness and smaller infarct size. These effects were mediated by an enhanced homing of CXCR4 enriched CD45+/CD34+ BM derived stem cells into the ischemic heart facilitated by a PTH mediated upregulation of SDF-1a. However, infiltrated inflammatory CD45+/CD34-cells revealed a strongly reduced expression of CXCR4. In contrast to the BM niche, myocardial ischemia alone or after PTH treatment did not promote proliferation of resident cardiac CD45-/CD34-/c-kit+ and even decreased the number of CD45-/CD34-/Sca-1+ stem cells in the heart. Immunohistologically, PTH treated hearts revealed an increased neovascularization of CD31+ capillaries in the borderzone, which could be explained by an upregulation of VEGF-A and VEGF-receptor1 mRNA. Furthermore, PTH treatment enhanced expression of IGF-1 receptor protein primarily localized on cardiomyocytes in the borderzone, which was related to a reduced number of apoptotic cells.
PTH treatment after MI exerts beneficial effects on survival and myocardial function associated with an augmented homing of CXCR4 enriched CD45+/CD34+ stem cells towards an increased SDF-1a gradient in the myocardium. Paracrine effects of these cell populations correlated with an improved neovascularization and cell survival via VEGF and IGF-1 dependent pathways. Since PTH is already used in patients with osteoporosis our findings may have direct impact on the initiation of clinical studies in patients with ischemic heart disease.
Over the past few years matrix-coupled autologous chondrocyte transplantation (MACT) has been developed and applied in clinical practice. Here, proliferated chondrocytes are seeded into biomechanical stable 3D-matrices which act as the scaffold and provide a temporary structure for the cellular organization. Clinical application of MACT is one of the main surgical procedures for repairing cartilage defects; however, limitation of chondrocyte availability and their decreased proliferative potential have been an issue. Over the last few years the research on bone marrow derived mesenchymal stem cells (bmMSC) has become a subject of extensive research because of their ability to differentiate into multiple lineages of tissues in vivo and in vitro. A major area of interest is the application of bmMSC in cartilage tissue engineering. Few studies have applied stem cell derived cartilage in small animal models such as the mouse or the rabbit. However, these animal models are unsuitable for the preclinical study of a focal cartilage defect because of their thin cartilage layer. The objective of the in vitro experiments is to investigate the behaviour of ovine bmMSC based collagen (CaReSR, Arthro Kinetics plc, Esslingen) hydrogels under TGF-s3 addition compared to fully autologous cultivated primary chondrocyte scaffolds with respect to the composite material properties, the cell viability and the deposition of extracellular matrix components.
During 4 weeks of autologous culture, aggregate properties were quantitatively verified, cell viability and the expression of cartilage markers were assayed. Different microscopic techniques (CLSM, SEM) indicated a subdivision of bmMSC based scaffolds into a central construct region with uniformly distributed stem cells with low levels of apoptosis, and peripheral layers of proliferative cells, which undergo chondrogenic differentiation. Immunohistochemical staining and quantitative measurements of sulfated glycosaminoglycans (s-GAG) of bmMSC hydrogels showed a significant increase in matrix deposition, mainly in outer areas. Furthermore, semi-quantitative RT-PCR of bmMSC specimens reflected a constant collagen type I activity with enhanced collagen type II, aggrecan and Sox9 expression which would suggest hyaline-like cartilage formation. We propose the application of bmMSC seeded collagen hydrogels for the repair of a focal articular cartilage defect with a diameter of 5 mm in the medial femoral condyles of adult Merino-sheep.
Growth hormone (GH) treatment of GH-deficient patients improves endothelial function and reduces overall cardiovascular risk. Mechanistically, the GH-dependent insulin-like growth factor-1 (IGF-1) stimulates activity of the endothelial NO synthase, which regulates in part vasoprotective endothelial progenitor cells (EPC). Low IGF-1 and EPC concentrations in patients have recently been correlated with impaired vascular function and development of coronary artery disease.
Here we tested our hypothesis, that treatment with recombinant human (rh) GH increases NO bioavailability and subsequently augments circulating EPC levels in healthy male volunteers (mean age 57.4 ± 1.4 years; n=16). We measured markers of NO bioavailability, such as cyclic guanosine monophosphate (cGMP), vascular endothelial growth factor (VEGF), nitrate, nitrite and asymmetric dimethylarginine (ADMA) as well as EPC numbers and blood pressure before and after a ten day treatment of 0.4 mg rhGH/per day.
rhGH treatment increased levels of IGF-1 by 2-fold (p<0.0001) and of the IGF-1 binding protein 3 by 19%±2.7% (p<0.001). Concentrations of the sensitive NO mediator cGMP (12.69±1.04 vs 14.79±1.00 μM cGMP/mM creatinine; p=0.03) and of VEGF (19.0±1.0 vs 21.4±1.4 pg/ml; p<0.05) were increased after rhGH treatment. In contrast, plasma concentrations of the endogenous NOS inhibitor ADMA were reduced (p<0.05), whereas plasma nitrate levels tended to be increased. The rise in NO bioavailability was closely correlated with a 2-fold increase in circulating CD133+/VEGFR2+ EPC. These findings were also observed in mice treated with GH for 7 days. Importantly, blocking of the IGF-1 receptor in vivo abolished the GH-mediated effects on markers of increased NO bioavailability.
In healthy middle-aged subjects rhGH treatment enhances overall nitric oxide bioavailability and subsequently EPC numbers. Animal data demonstrate increased NO availability to be mediated via an increase in IGF-1 plasma levels. Our proof-of-principle study warrants further clinical trials to test a potential therapeutic effect of recombinant human GH in patients with various cardiovascular diseases, impaired NO bioavailability and EPC.
Chondrogenic defects in articular joints, such as the knee, are common and a wide spread problem with little regenerative potential. Although various attempts have been made to regenerate articular cartilage through mesenchymal stem cells (MSC), our understanding of the mechanisms involved in chondrogenesis is still limited. However, for successful tissue engineering a better understanding is required. Therefore, the aim of this study was to examine in vitro the possible interaction and communication that occurs between MSC and primary chondrocytes (PCH) in order to gain more insight into the complicated mechanism of chondrogenesis.
Bone marrow MSC were cultured in a ratio of 1:1 with autologous PCH. To be able to evaluate the cell interaction, MSC were membrane labelled with a red fluorescent dye and PCH with a green fluorescent dye and monolayer co-cultures monitored in vitro over a period of seven days. High density co-cultures were evaluated with light microscopy, electronmicroscopy and immunoelectronmicroscopy
In high density co-culture an even distribution of MSC/PCH is observed. After 7 days cartilage-nodules form. Already after 1 day in monolayer co-culture, both cell types extend long directed pseudopodia, intensively searching for the other cell type. A few MSC/PCH engage already in close cell-cell contacts. It is noticeable that PCH proliferate strongly in the vicinity of MSC. At day 3, PCH proliferate even more actively around the MSC. Many cell processes can be observed and the intensity of cell contacts increases further. A few PCH now contain small red vesicles and some cells are bicoloured suggesting cell-fusion. At day 4 the number of PCH strongly increases. Furthermore, PCH without contact to MSC now contain red vesicles suggesting the uptake of soluble factors produced by the MSC.
We could show that there are active interactions between MSC and PCH leading to adequate communication. This intensive interaction through the co-culture might be an important stimulus for the chondrogenic differentiation of MSC and opens up new exciting possibilities for cartilage tissue engineering.
Adult stem/progenitor cells have been isolated from different tissues such as bone marrow, adipose tissue, muscle and peripheral blood. Adipose tissue-derived stem/progenitor cells have the potential to differentiate in vitro into osteogenic, chondrogenic and adipogenic cell types. They may therefore prove to be an easily accessible and abundant source of stem/progenitor cells.
We isolated and characterized adult stem/progenitor cells from human cervical subdermal fatty tissue, which was obtained during tracheostomy, and analysed their adipogenic, osteogenic and chondrogenic differentiation in vitro. The isolated stem/progenitor cells were characterized using (a) fluorescence activated cell sorting, (b) histochemical- and immunhistochemical staining as well as RT-PCR analysis and, (c) determination of their proliferation rate during passaging. Asking the question whether close cell-cell interactions favor human stem/progenitor cell differentiation prior to lineage specific induction, osteogenic, adipogenic and chondrogenic properties of the isolated stem/progenitor cells were analyzed using the standardized three-dimensional mesenchymal microsphere (MMS) -model of stem cell cultivation.
Using the standardized MMS-model of stem cell differentiation as well as classical monolayer protocols, we were able to demonstrate that subdermal adipose tissue derived stem/progenitor cells differentiate along the osteogenic, adipogenic and chondrogenic lineages. These results were confirmed by the expression of tissue-specific genes like osteopontin, osteocalcin, PPARγ, aP2, C/EBP, collagen type II, and aggrecan. Immunhistochemical staining revealed cells to be positive for osteopontin, bone sialoprotein as well as collagen type II and collagen type X. Comparison of MMS-cultivation to monolayer and micro mass body protocols demonstrated MMS cultivated stem/progenitor cells to exhibit tissue-related attributes favoring multilineage differentiation prior to lineage specific induction. Standardized MMS differentiation might therefore offer a model of three-dimensional adult stem/progenitor cell differentiation, considering cell-cell interactions as well as multilineage differentiation.
One important parameter for bone marrow mesenchymal stem cell (bmMSC) fate including stemness and differentiation is the oxygen environment. The oxygen tension inside the bone marrow in vivo is 5% on average. We investigated the influence of low oxygen tension during expansion of bmMSC on following chondrogenic differentiation in standard pellet culture and in clinical applied collagen gel (Arthro Kinetics plc). The differentiation was performed at commonly used 21% ambient oxygen conditions.
Ovine bmMSCs were expanded at ambient 5% pO2 and 21% pO2 as control. The proliferation potential was analysed by colony forming unit ? fibroblast (CFU-F) assay. The second passage cells were used for chondrogenic differentiation in pellet cultures and collagen gels. Differentiation was accomplished at normoxic conditions with supplementation of 10ng/ml TGF-β3 for up to 21 days. Chondrogenic differentiation was assessed by immunhistochemical staining (aggrecan, type II collagen), measurement of sulfated glycosaminoglycans (sGAG) and quantitative real-time RT-PCR (type I collagen, type II collagen, type X collagen, aggrecan).
Ovine bmMSC expanded at low oxygen showed an approximately two times higher CFU-F number compared to normoxic conditions.
After expansion at 5% pO2, pellets showed an increased chondrogenic differentiation compared to 21% pO2 as shown by immunhistochemistry, production of sGAG, and gene expression. The amount of sGAG in pellets was 35% ± 6% higher after hypoxic proliferation. The cells in pellet culture had a more rounded shape and produced distinct more collagen type II compared to cells expanded at 21% pO2.
Our results suggest that maintaining ovine bmMSCs in vitro at low oxygen environment is more physiological and increases the following chondrogenic differentiation. These findings contribute to the knowledge of stem cell cultivation, and they might be a potent approach to improve chondrogenic differentiation of cartilage tissue engineering from bmMSCs.
Bone marrow is the most commonly used source for the isolation of human mesenchymal stem cells (hMSC). Similar cells can also be isolated from various other tissues including adipose tissue, dental pulp and umbilical cord. Umbilical cord-derived stem cells are believed to have a higher proliferation and differentiation capacity due to their primitive developmental stage. We isolated hMSC from bone marrow and the Wharton’s Jelly of umbilical cords and compared the cells regarding their surface epitopes, proliferation and differentiation.
HMSC were isolated from bone marrow according to standard protocols (Pittenger et al., Science, 1999) and from the Wharton’s Jelly of umbilical cords. To this end, 2 cm pieces of umbilical cords were opened lengthwise with a scalpel. The matrix was scraped off and incubated with 2 mg/ml collagenase for 16 h, trypsinized for 30 min, washed and seeded on T75 culture flasks. Proliferation was analyzed using XTT tests. The differentiation into adipocytes, osteoblasts and chondrocytes was performed according to standard protocols (Pittenger et al., 1999). Surface epitopes were analyzed by flow cytometry. Spontaneous 3D aggregation and differentiation within cellular aggregates was investigated by histological staining and immunohistochemistry.
The flow cytometric analysis of the surface epitopes CD51, CD54, CD73, CD90 and CD105 showed that the isolated cells from bone marrow and umbilical cord displayed an hMSC phenotype. Cells could be readily differentiated into adipocytes, osteoblasts and chondrocytes. Interestingly, the umbilical cord-derived cells spontaneously formed 3D aggregates when cultured under post-confluent conditions. All cells of these aggregates were viable and spontaneously differentiated into several specialized cell types akin to the well-known differentiation of embryoid bodies.
The umbilical cord-derived hMSC resemble bone marrow-derived hMSC and additionally show a spontaneous 3D aggregation and differentiation in vitro. These results indicate that umbilical cord-derived hMSC are a valuable tool for cell-based therapy, but due to their spontaneous differentiation capacity, teratoma formation might be possible.
Adult human haematopoietic stem cells possess high plasticity that was demonstrated with respect to their differentiation into organ specific cell types. Using differential experimental design, studies have indicated that adult stem cells can differentiate into hepatocytes-like cells which differ in hepatic marker expression profiles and functional properties. However, methods are still being developed for induction of stem cell differentiation and the contribution of direct cell-cell-contact or soluble factors is under investigation. For hepatic differentiation of CD34+ cord blood cells we investigated models with respect to contributions of direct stem cell - hepatocyte ? interactions and secreted factors from hepatocyte culture.
CD34+ stem cells were cocultured with different cell types as murine hepatocytes line AML-12, human hepatocarcinoma HepG2 and murine hepatocytes. Flow cytometric analysis using dye transfer technique showed that stem cells get in contact with hepatocytes. Video imaging revealed a high CD34+ motility. FACS and Western Blot techniques demonstrated that CD34+ cells express Connexin43 and the hepatocytes Connexin32. Through these gap junctions cells may communicate and perhaps support differentiation into hepatic-like cells. Futhermore, conditioned cell culture media from HepG2 cells induced early endodermal differentiation (SOX17, FOXA2, AFP) of CD34+ stem cells, suggesting the involvement of secreted soluble factors. Conditioned hepatocyte media and coculture of CD34+ cells with hepatic cells represents a valuable model for studying hepatic differentiation processes.
Embryonic stem cells are characterized by pluripotency, the ability to form every cell type of the body. This feature gives them a great potential in regeneration therapy. However, the mechanisms that regulate and control the process of differentiation into a defined cell lineage are still poorly understood. The application of embryonic stem cells for research or clinical therapy is linked to several ethical and immunological problems enhancing the need for other approaches. One alternative is the use of spermatogonial stem cells that have recently been shown in mice to be pluripotent. We have established a system for induced directed differentiation using a spermatogonial cell line from medakafish.
The spermatogonial cell line SG3 was obtained from testis of adult medakafish and is stable in culture in the presence of a complex mix of growth factors. Directed differentiation was induced by chemical treatment, modulation of culture conditions or transfection with expression vectors for presumed or known lineage specific masterregulators of differentiation. Status of differentiation was investigated by morphological analysis and by examination of gene expression patterns.
Our experiments demonstrate that SG3 cells are capable of spontaneous differentiation and of formation of other cell types than sperm. Attempts to induce directed differentiation resulted in the formation of cells showing morphological features of melanocytes, neurons, osteoblasts, and adipocytes. We also obtained cells capable of phagocytosis that therefore seem to be macrophages.
The use of stem cells for regeneration therapy requires at first methods to generate specific cell types from stem cells. Our results give indications for directed differentiation by chemical treatment, modulation of culture conditions, and transfection. Moreover, this study demonstrates that medaka spermatogonia are pluripotent and therefore may be ascribed an equal status to embryonic stem cells. This finding, together with compatible data published for mouse spermatogonia, strongly suggests the possibility for human pluripotent spermatogonia.
The proper ratio of organic and inorganic components within the bone matrix is an extremely important factor that both influences development and functionality of bone tissue. Focusing on the influence of extracellular calcium concentrations on the bone matrix formation and mineralization we were looking for suitable methods to better control the process of human bone tissue generation in vitro.
For the experiments primary human osteoprogenitor cells were used which were derived either from a heterotopic ossification or from trabecular bone. Cells were grown in ZKT-I medium (in-house formulation*) containing 0.89 mmol/l Ca2+ or in ZKT-I modifications with twice and four times increased Ca2+ concentrations. All media were additionally supplemented with human serum, dexamethasone, ascorbate-2-phosphate, and β-glycerophosphate. Cell growth and vitality, type I collagen synthesis, alkaline phosphatase (AP) activity, Ca2+ consumption and Ca2+ accumulation in the extracellular matrix (ECM) were monitored over a period of 31 days.
Increased Ca2+ concentrations in the media resulted in an accelerated collagen matrix mineralization and a higher mineralization level. Scanning electron microscopy carried out in the middle of cultivation showed different stages of mineral crystal formation dependent on the Ca2+ concentration in the extracellular environment. Moreover, the increase of Ca2+ content in culture media caused reduction of collagen I synthesis and lowering of AP activity. Therefore, bone-forming cells produced more ECM with a lower degree of mineralization using medium with a low Ca2+ content. Less ECM with a higher degree of mineralization was generated in medium with a Ca2+ content of about 1.8 mmol/l. An even higher extracellular Ca2+ concentration of 3,3 mmol/l was associated with non-physiological mineralization or calcification as well as a decline of cell growth and intensification of apoptosis.
Ca2+ concentrations in culture media have a substantial impact on the ECM synthesis and mineralization. We believe that the reduction of collagen matrix formation and decrease of AP activity are two of numerous regulatory reactions which counteract the non-physiological mineralization. In the context of bone tissue engineering media with lower or slightly increased Ca2+ content may be used to influence and to improve the ratio of organic and inorganic components of artificial bone tissue.
The alveolar epithelium of the lung is composed of two cell types, alveolar type-1 epithelial cells (AT1 cells) and alveolar type-2 epithelial cells (AT2 cells). AT2 cells are known for their ability to produce pulmonary surfactant and to serve as progenitor cells for AT1 cells. Transplantation of murine embryonic stem cell (mESC)-derived AT2 cells would provide a new therapeutic option to treat pulmonary injuries and disorders which are associated with loss of AT2 cells and impaired surfactant production, respectively.
Based on our recently established completely serum-free differentiation protocol we are searching for factors which efficiently enhance the generation of mESC-derived AT2-like cells. In this context the keratinocyte growth factor (KGF) is a promising candidate. KGF is a member of the fibroblast growth factor family and has been shown to be a strong growth factor for AT2 cells in vitro and in vivo. We hypothesize that KGF either supports the differentiation of AT2-like cells from mESCs and/or induces the proliferation of mESC-derived AT2-like cells. Studies concerning the appropriate concentration and the right point in time for the application of KGF during the differentiation process are ongoing. Furthermore, proteomic analyses of culture supernatants will help in the identification of further AT2 cell inducing factors.
Recent studies demonstrate that Muller glial cells (MGCs) are capable of dedifferentiation into retinal progenitor cells (RPCs). Therefore we isolated MGCs and RPCs and characterized the activation state of selected pathways by quantitative real-time reverse transcription (qRT) PCR.
RPCs and MGCs were isolated from retinas of C57/BL6 mice between postnatal days P0 to P10 (for RPCs) and P8 to P12 (for MGCs). Isolated cells were cultured and used for analysis after passage four. We performed a reverse transcription (RT)-PCR for Nestin, Musashi 1, Sox 2 as RPC specific markers and for the gene expression of GFAP and Vimentin as MGC specific markers. To analyze mRNA profiles, a qRT-PCR array was established for signal transduction pathways Wnt, FGF, Hedgehog, Notch and TGF-β.
We proved successful isolation of RPCs and MGCs. Gene expression was determined for specific markers for the Wnt, FGF, Hedgehog, Notch and TGF β signal transduction pathways. However gene expression levels for WNT5, Gli3, Shh, Dll1, Hes5, BMP2 and BMP7 were upregulated in RPCs compared to MGCs, but no differentially gene expression was observed for MGCs.
We demonstrate successful isolation of RPCs and MGCs from the postnatal murine retina. All investigated signal transduction pathways seem to be activated in RPCs and MGCs. Interestingly enough, gene expression of selected signal transduction pathways were increased in RPCs in comparison to MGCs. These data may suggest that MGCs have more restricted capacities for cell differentiations than RPCs.
Multiple tissue niches in the human body are now recognized to harbour adult stem cells. Here, we have examined a panel of putative stem cell/progenitor markers on progenitor cells derived from mechanically and enzymatically processed adult human scalp skin, which may serve as a convenient and abundant source of adult human progenitor cell populations.
After removal of the epidermis, adherent growing cells of a purposely heterogenous mixture of intradermal cells (including sweat glands, fibroblasts, and hair follicle-derived cells), could be propagated for more than 20 passages in serum-supplemented medium. Cells stained positive for Vimentin, and weak expression of nestin, GFAP or neuronal proteins could be occasionally observed in a very small subfraction of the culture.
When grown to confluency a small subfraction of the cells (less than 1%) within the culture began to express low levels of SMA. These Sma+ cells did not undergo significant morphological changes.
The cells could be also maintained in media containing FCS and the mitogens bFGF, EGF or PDGF AB. Interestingly, human skin-derived stem/progenitor cells obtained in this manner exhibited a strong commitment to the mesodermal lineage, since a large subfraction of them expressed high levels of a-SMA (about 50%) and underwent dramatic morphological changes.
Replacement of serum by the supplement BIT9500 affected cell growth in two ways: Firstly, growth as monolayer cultures was changed towards a growth pattern that resembled formation of foci. Secondly, proliferation was impaired and eventually stopped after 2 to 3 weeks. Adding back FCS during the first two weeks restored normal growth pattern and proliferation, indicating that the culture had not undergone terminal growth arrest or differentiation. Growth supports such as fibronectin and laminin or otherwise coated flasks and glass coverslips did not affect growth or marker expression.The diverse cell populations generated in this manner were then tested for spontaneous differentiation markers by immunocytochemistry and RT-PCR. This intriguingly differentiation potential of progenitor cells contained in normal, aging human scalp skin (mean age: 50+/- 5 years) warrants systemic follow-up, since it raises the possibility that adult human skin-derived progenitor cells can serve multiple cell-based therapy purposes for regenerative medicine.
The dental follicle is an ectomesenchymal tissue surrounding the developing tooth germ. Previously our group reported the isolation of precursor cells (DFPCs) derived from the dental follicle of human third molar teeth, which are fibroblast-like, colony forming and plastic adherent cells (
The mechanisms of nuclear extrusion, the unique ability of Erythrocytes, are poorly researched. For a better understanding of erythroid differentiation and enucleation process, we performed an oligonucleotide microoarray analysis of human CD34+ cells during cytokine-induced in vitro erythropoiesis. Several genes were found to be differentially up- or down-regulated during erythropoiesis belonging to different functional categories.
Human CD34+ cells were isolated and cultured over 21 days in a three-phase liquid assay (d1-7: SCF, TPO, FLT3-L; d8-14: SCF, EPO, IGF-1; d15-21: EPO). Cell growth and differentiation was evaluated by flow cytometry and cytospin preparations. Cells were harvested on days 7, 10, 14 and 17 and RNA was isolated. Gene expression was analyzed by Miltenyi Biotech using topic-defined PIQR Stem Cell Microarray. The measurements were compared to day 0 as a control. A pathway analysis was performed to identify genes which are involved in erythroid differentiation, such as genes involved in pro-and anti apoptotic and enucleation processes.
A total of 4 experiments were performed for the gene arrays. At the end of the culture more than 95% of the cells were glycophorin A positive and therefore clearly of erythroid nature. According to cytospins, cells showed morphological characteristics of normoblasts (43%) and reticulocytes (52%). Several genes and gene families were identified as being differentially regulated during erythropoiesis. Genes belonging to ribosome cluster were significantly down-regulated during erythropoiesis with 18 genes including DDX21. Other annotations which were enriched were mitosis and stress with 14 genes (including BUB1B) and 42 genes (including NFκ-family), respectively.
Associated with an appropriate in vitro erythropoiesis assay, we were able to identify a large amount of genes (about 73) which were significantly up- or down-regulated. RT-PCRs of mRNA were performed to reproduce our observation. Further experiments should confirm our preliminary observations during RBC development.
Embryonic stem cells (ESCs) have gained significant attention over the last few years. Due to their unlimited proliferation potential and pluripotency they represent a cell population that could be potentially used in cellular therapies for the treatment of otherwise incurable diseases, such as degenerative bone diseases. The classical non-canonical wnt molecule wnt5a has been associated with bone development, but three sub-pathways exist that vary depending on the cell context. Here, we have characterized the downstream targets of wnt5a in ESCs during the course of osteoblast differentiation.
We have used murine ESCs as a model to investigate how wnt5a affects normal osteogenic development thereby identifying molecular processes that could be used to drive differentiation more efficiently in vitro. Previously, we have successfully shown that recombinant wnt5a protein can support osteogenesis when administered during the time of osteoprogenitor commitment during ESC culture. In the current study, we used a reporter wnt5a::GFP ESC line as readout to characterize prospective downstream targets of wnt5a. Specifically, inhibitors to PKC and CamKII were administered to ESC cultures.
Proliferation, cAMP levels and gene expression was measured in early osteoblasts and could be directly correlated to the degree of mineralization in mature osteoblasts. Specifically, proliferation of osteoprogenitors seems to be regulated by PKC, whereas osteoblast-specific gene expression is controlled through CAMKII.
Eventually, our results suggest that the non- canonical wnt signaling pathway regulates osteoprogenitor maturation through PKC and CamKII.
In order to gain insights in the mechanisms of “sternness”, an important aspect in stem cell research is, how the maintenance of the potential for self-renewal and the prevention of spontaneous differentiation in stem cells is achieved. In murine embryonic stem (mES) cells, one member of the family of signal transducers and activators of transcription proteins, STAT3, is needed to keep mES cells in the ?stemness”-status. The latent transcription factor STAT3 is activated by the leukaemia inhibitory factor (LIF) and consequently imported into the nucleus. In contrast to mES-cells, activated nuclear STAT3 is neither necessary nor sufficient to keep human embryonic stem (hES) cells in the undifferentiated status. In this work, we analyzed the localization and thereby the activity of STAT3 in embryonic stem cells of medaka fish (MES) and during early embryonic development of medaka.
The embryonic stem cell line MESI was obtained from blastulae of medaka fish (Oryzias latipes) and remains undifferentiated in cell culture in presence of a complex mix of growth factors. STAT3-activity in MESI-cells and early embryos of medaka fish was investigated via immunofluorescence.
Ectopic expression of an eGFP-tagged STAT3 resulted in dominantly cytoplasmic and thus inactive localization in MESI. Analyses in medaka embryos of blastula-stage demonstrated that STAT3 is inactive in all cells.
Investigations on embryos before 1024-cell-stage revealed that STAT3 has a wave-like import into the nucleus between 64-cell-stage and 512-cell-stage with a peak at 64-cell-stage. No increase of nuclear STAT3-levels was detected before 64-cell-stage or after 512-cell-stage.
The assays on MESI cells and blastula-staged medaka embryos demonstrated that STAT3 is not activated in medaka stem cells. Consequently, these data indicate that activated STAT3 is not necessary for stem-cell status in medaka. Hence, the mouse-system is the only vertebrate stem cell system depending on active STAT3. Furthermore, the import-wave of STAT3 spanning 64-cell-stage to 512-cell-stage medaka embryos argues for a specific process in medaka development which is depending on nuclear presence of activated STAT3.
Cell Sheet Engineering is emerged as a novel technique which allows creating of replacement three dimensional tissue structures without the use of biodegradable scaffolds. It was already showed, that cell culture surfaces based on temperature responsive polymer films allow for the none enzymatic harvesting of viable Adult Cell Sheets, which can be used for tissue reconstruction and transplantation surgery.
The experimental method is based on growing confluent sheets from Human Placenta Mesenchymal Stem Cells [HPMSC] on conducting substrates, for example [ITO] coated with polyelectrolyte multilayer films consisting of Poly-L-Lysine [PLL] and Hyaluronic Acid [HA]. After reaching confluence intact dense stem cell sheets will be either directly differentiated into adult cell types and then detached from the surfaces of substrates or alternatively firstly detached from the surfaces of substrates by applying of potential and then differentiated as intact three dimensional structures.
Dense viable stem cell sheets were created on the experimental conducting substrates coated with polyelectrolyte multilayer films of different compositions. Experimental substrates were stratificated by analysis of viability, vitality, grade of confluence, amount, and morphology of stem cells. The ability of confluent viable stem cell sheets to be differentiated into adult cell types, for example Chondrocytes, Osteocytes or Cardiomyocytes will be analyzed.
Cell Sheet Engineering is a new promising and universal methodology that allows obtaining intact assemblies of stem cells which can be harvested from the substrates and differentiated into three dimensional adult tissue structures destined for transplantation. This method can also be applied for construction of cell sheets from the adult cell types to obtain three dimensional tissue structures for assembling of complex tissue models, such as an in vitro model of adult angiogenesis, or for drug screening applications.
The wound coverage of extensive burn injuries and other deep skin defects represents a major problem in dermatology. The main problem is set by low availability of autologous material provided by the patient for covering the site of injury. Therefore the development of skin equivalents for transplantation consisting of dermal and epidermal components to support the regeneration of skin is of high interest. The aim of our work is to support the engraftment of skin equivalents by implanting endothelial vessels in the dermal layer of skin transplants.
ADSC were immobilised in a bioartificial collagen-elastin matrix and cultured under dynamic flow conditions in the presence or absence of VEGF (
Our results show that the flow rate and the VEGF concentration have a high impact on the development of vessels formed by ADSC in the matrix. The vessels formed by ADSC under flow rates of 1.2ml/24h and VEGF concentration of 50ng/ml were about 100μm to 130μm deep and stable for several days. Most important, ADSC differentiated into endothelial cells and formed vessel like structures under these conditions. This very successful in vitro vascularisation of dermal skin equivalents with endothelial differentiated ADSC may offer the possibility to support the engraftment of skin transplants by advancing the neovascularisation in vivo.
Erythropoiesis proceeds in so called hematopoietic microenvironments or niches, in which stroma cells produce extra cellular matrix molecules. To investigate the influence of these molecules on proliferation and differentiation we used an established in vitro Erythropoiesis system in co-culture with different matrix proteins.
Human CD34+ cells were isolated and cultured over 16 days in a two-phase liquid assay (d1-d8 SCF, EPO, IGF-1, steroids; d9-d16 EPO, insulin) in 6 well plates coated with Laminin, Collagen, Fibronectin or Extra Cellular Matrix (ECM). An uncoated plate was used as a negative control. Cell viability was determined by trypan blue staining and microscopic examination. To measure cell proliferation, vital cells were enumerated using a Neubauer counting chamber. Cell growth and differentiation was evaluated by flow cytometry and cytospin preparations on each day of analysis.
A total of 5 experiments were performed. At the end of the culture more than 95% of the cells of the control culture were glycophorin A positive and therefore clearly of erythroid nature. According to cytospins, cells showed morphological characteristics of normoblasts (50%) and reticulocytes (45%). The cells cultured with ECM showed the best vitality (91%) compared with the other cultures. The proliferation rate assessed in different culture conditions showed no significant difference. On the last day of culturing the control culture showed the highest amount of erythroid cells compared to cells cultured on proteins.
The hematopoietic niche or microenvironment provides in vivo the optimum condition for proliferation and differentiation of erythroid precursers. Our aim was to optimize the in vitro Erythropoiesis by co-cultivating the erythroid precursor with extra cellular matrix proteins. However our experiments showed that the in vitro Erythropoiesis could not be optimized by adding matrix proteins to the culture.
Multiple tissue niches in the human body are now recognized to harbour adult stem cells. Here, we have examined a panel of putative stem cell/progenitor markers on progenitor cells derived from mechanically and enzymatically processed adult human scalp skin, which may serve as a convenient and abundant source of adult human progenitor cell populations.
After removal of the epidermis, adherent growing cells of a purposely heterogenous mixture of intradermal cells (including sweat glands, fibroblasts, and hair follicle-derived cells), could be propagated for more than 20 passages in serum-supplemented medium. The diverse cell populations generated in this manner were then tested for spontaneous differentiation markers by immunocytochemistry and RT-PCR.
When grown to confluency a small subfraction of the cells (less than 1%) within the culture began to express low levels of SMA. These Sma+ cells did not undergo significant morphological changes.
The cells could be also maintained in media containing FCS and the mitogens bFGF, EGF or PDGF AB. Interestingly, human skin-derived stem/progenitor cells obtained in this manner exhibited a strong commitment to the mesodermal lineage, since a large subfraction of them expressed high levels of a-SMA (about 50%) and underwent dramatic morphological changes.
Replacement of serum by the supplement BIT9500 affected cell growth in two ways: Firstly, growth as monolayer cultures was changed towards a growth pattern that resembled formation of foci. Secondly, proliferation was impaired and eventually stopped after 2 to 3 weeks. Adding back FCS during the first two weeks restored normal growth pattern and proliferation, indicating that the culture had not undergone terminal growth arrest or differentiation.
Growth supports such as fibronectin and laminin or otherwise coated flasks and glass coverslips did not affect growth or marker expression.
This intriguingly differentiation potential of progenitor cells contained in normal, aging human scalp skin (mean age: 50+/- 5 years) warrants systemic follow-up, since it raises the possibility that adult human skin-derived progenitor cells can serve multiple cell-based therapy purposes for regenerative medicine.
How about cells that are ontogenetically completely immature and far from being “neuronally” differentiated, do they have a cholinergic system, and if so, what does this mean functionally? It is already established that cholinesterases (ChEs) appear in every embryonic blastema at a very early stage of development, independent from innervation. Here, we analysed the expression and possible functions of the cholinergic system in the embryonic stem cells (ESCs).
In order to better define the onset of cholinergic markers expression during development, we examined their expression in two murine embryonic stem cell lines (CGR8 and D3) and in differentiating embryoid bodies (EBs) by HPLC, RT-PCR, histochemistry and enzyme activity measurements; their functions were analysed by acetylcholine (ACh) supplementation, by inhibition of muscarinic and nicotinic ACh receptors and of ChEs and by overexpression of AChE.
ESCs expressed all cholinergic components, including ACh and its receptors. Amounts of pmol ACh could be detected in both stem cells lines and increased during EB differentiation. Acetyl- (AChE) and Butyrylcholinesterase (BChE) mRNA and activity were present already in undifferentiated ESCs; BChE was always higher than AChE. Along with differentiation into EBs, AChE mRNA and activity increased. A significant increase in cell viability and proliferation was observed when ACh was added to the medium, the effect being concentration-dependent; specific inhibition of cholinesterases led to the same results.
This suggests that ACh functions as a signal molecule in ESCs, controlling basic cellular processes like cell proliferation and cholinesterases act as regulators of its concentration. With these results we could show that mouse embryonic stem cells, a non-neuronal tissue, express a cholinergic system; its functions are not related to synaptic transmission, but to developmental processes.
The Embryonic Stem Cell Test (EST) represents a validated in vitro system for the classification of chemical compounds according to their embryotoxic potential. It is based on the evaluation of beating cardiomyocytes in embryoid body (EB) outgrowths in comparison to cytotoxic effects on D3 murine embryonic stem cells and 3T3 fibroblasts. To improve the predictive potential of the EST and to avoid false-negative classifications, further cell type-specific endpoints of differentiation have to be established. Recent attempts employ RT-PCR methods or FACS analyses based on antibody staining of neuronal markers, but these techniques are cost-intensive and need a lot of hands-on time. Both aspects are disadvantageous in future routine applications. Hence we tested the feasibility of detecting neuronal differentiation by using a simple fluorescent staining of Nissl substance, which is specific for neuronal cells and can therefore be used to distinguish them from non-neuronal cells.
EBs were produced from D3 suspension cultures within 4 days. Neuronal differentiation was induced by incubating EBs for 4 days with retinoic acid (RA). Then EBs were transferred to TC-dishes and further cultured for at least 9 days. To detect neuronal cells in EB outgrowths, NeuroTrace? fluorescent Nissl stain was used together with DAPI. To confirm the specificity of staining, a coculture of embryonic neuronal cells from chicken retina with a human Nissl-negative keratinocyte cell line was used. Additionally, RT-PCR was performed to monitor the expression of other neuronal markers such as tyrosine hydroxylase.
RA treatment of EBs led to a pronounced spot-like staining of the Nissl substance similar to that usually found in fully differentiated neuronal cells. A few neuronal cells spontaneously developed also in RA-untreated EBs and could clearly be detected by Nissl staining. In monolayer cocultures, a weak homogeneous background staining of nuclei and cytosol could also be observed in HaCaT keratinocytes, but chicken embryo retina cells displayed a significantly stronger but still homogeneous staining of cells.
A reliable identification of neuronal cells by fluorescent Nissl staining is possible in combination with morphological criteria. For future applications in routine testing, image analysis employing threshold-based algorithms or FACS analysis should be used to standardize the evaluation procedure.
To obtain information on the embryotoxicity of compounds, testing is performed in vivo using pregnant rats or in vitro using cultured embryos or cells. Taking advantage of the potential of embryonic stem cells to differentiate in culture, the embryonic stem cell test (EST) with a permanent murine embryonic stem cell line (D3) has been developed. This test is based on the determination of several indicators of embryotoxicity as the inhibition of differentiation is combined with the study of differences in the cytotoxic response of embryonic and adult cells.
The differentiation assay with D3 cells was performed after adding several concentrations of test compounds according to the validated EST protocol, published by ECVAM, 2002. After 10 days of exposure, the number of EBs with beating myocards was assessed microscopically. In parallel, cytotoxicity assays were performed with embryonic D3 cells and 3T3 fibroblasts, representing adult tissue.
4 model compounds were examined: penicillin G (non-embryotoxic in vivo), LiCl (weakly embryotoxic), retinoic acid and 5-fluorouracil (both strongly embryotoxic). The results obtained with these agents were in line with previously published data. However, the concurrent cytotoxicity tests for discriminating between cytotoxic and specifically embryotoxic effects turned out to be a confounding factor. The addition of the test compounds shortly after passage of the cell cultures (as described in the ECVAM protocol) strongly increased their apparent cytotoxicity. This was particularly the case with t-chlorobutanol, another compound tested. It is widely used as a drug preservative but its embryotoxicity is still under investigation [Smoak, 1993].
The observed increase in cytotoxicity presumably is either the result of an interference of the agent with cell adhesion and spreading or of a much higher sensitivity of freshly passaged cells due to sublethal plasma membrane damage. Either way, this effect resulted in an overestimation of cytotoxicity for some test compounds thereby giving rise to an erroneous underestimation of embryotoxicity. Thus, in routine testing this confounder would increase the risk of generating false-negative results regarding the embryotoxicity of test compounds. We propose that this particular aspect of the present EST protocol should be subjected to further investigations.
PTH is an effective anabolic agent of bone in patients with osteopenia. Recently, the presence of PTH1-Rc was demonstrated during early stages of antler bone regeneration. We also found that PTH1-Rc is present in mesenchymal cells derived from fresh marrow transplanted into demineralized bone matrix cylinder (DBM) in a rat ectopic bone regeneration model, during early stage of endochondral bone formation. Elevated expression of PTH1-Rc was detected at days 3 and 7 where mesenchymal cells from bone marrow are adhering on the DBM surface, proliferate and produce osteo and chondro progenitors. At later stages, the level of PTH1-Rc expression is relatively reduced. We further studied the temporal expression of PTH1-Rc in marrow derived MSC in culture. Bone marrow was obtained from 2 month old DA rat femurs and plated at 9x105 cell/cm2. After 4 days in culture, 10-8M dexamethasone was added to half of the dishes. Immunostaining of PTH1-Rc was performed after 7, 14 and 21 days in culture. We observed that at day 7 most of the cultured cells expressed PTH1-Rc. At 14 and 21 days, PTH1-Rc was expressed mostly in small, round cells while in the more differentiated fibroblast-like cells the receptor was not detected. In dexamethasone treated cultures the expression of PTH1-Rc declined. Taken together, we propose that PTH and PTHrP increase the pool of osteo-progenitors by activating the PTH1-Rc on MSCs.
Cell sheet engineering has emerged as a novel technique for creation of artificial, three-dimensional model tissues without the need for biodegradable scaffolds. Others could show that cell culture on temperature-responsive polymer films allows non-enzymatic harvesting of intact mesenchymal stem cell sheets, which can be further surgically transplanted for tissue reconstruction (Miyahara et al. Nat Med 2006; 12:459-65). We have begun to explore new methodology that builds on the use of electric charge to accomplish non-enzymatic harvest of cell sheets.
We used charge-sensitive arrangements of polyelectrolyte multilayer films (PEMs) that were obtained by piling up to 8 alternating layers of poly-L-lysine (PLL) and hyaluronic acid (HA). Some PEM layers were additional formed with a top layer of PLL-PEG-RGD or adsorbed fibronectin. Tests were performed with placenta-derived mesenchymal stem cells (HPMSC). The cells were cultured for 48hrs, then examined for morphology (phase microscopy), viability (life/death stain) and vitality (WST-assay).
Stratification of nine different surfaces revealed following principal requirements (1) HPMSC adhered and grew out on HA but not on PLL surfaces (2) Fibronectin precoating, or alternatively, PLL-PEG-RGD layers, are necessary to mediate efficient adhesion and outgrowth.
These are first steps into development of new technology for cell sheet engineering. We showed that the charge-sensitive arrangements of PEM cell do indeed function as substrates for human mesenchymal stem cells. Next steps are whether stem cell sheets grown on PEMs can be differentiated prior to transplantation which is under current investigation.
Purification of endothelial cells and identification of genes involved in endothelial differentiation are of great interest as it could be beneficial for therapeutic applications. Mouse embryonic stem (ES) cells serve as a potential source of endothelial cells (EC) for transcriptomic analysis. ECs were isolated by immuno-magnetic separation using platelet endothelial cell-adhesion molecule-1 (PECAM1, also known as CD31). When plated on matrigel, CD31+ cells were able to form capillary-like structures. Immunostaining also revealed the presence of endothelial-specific markers like CD31, VE-cadherin and the characteristic ability of the endothelial cells to uptake DiI Ac-LDL. Quantitative and semi-quantitative PCR analysis further confirms the presence of endothelial markers along with hematopoietic markers. In order to explore the known and novel transcripts involved in vasculogenesis and angiogenesis, large-scale microarray analysis were performed to determine the expression profiles of CD31+ cells which have been compared with murine endothelial cell line (1G11 cells). Several differentially expressed genes were identified which includes some well-known genes important for endothelial differentiation, angiogenesis and hematopoiesis. Moreover, our gene ontology (GO) analysis from the complete transcriptome have identified several genes involved in the signalling transduction pathways and the biological processes that are enriched in CD31+ endothelial cells giving an insight into the cellular and molecular mechanisms underlying the vascular development in mouse ES cells. Further investigation in this field can be extended by gain-of-function and loss-of-function analysis that may allow examining the role of genes in vascular development.
Fibroblasts represent a cell source having the potential of differentiating into other cell types apart from the fibroblast line. Fibroblasts are among those adult cells, which are easily to acquire and a variety of cell culture and tissue engineering methods are already available. Our research activities focus on a distinct differentiation step -from the fibroblast to the myofibroblast (MF)- due to the high clinical relevance. Because the MF is a key player during the development of tissue fibrosis (i.e. liver fibrosis, arteriosclerosis, excessive scarring after wounding) on the one hand, and is believed to support tissue regeneration (organ- and wound healing) on the other hand, it is a promising approach to develop molecular and biochemical methods, enabling the characterization and handling of MF. The detailed knowledge of the process of MF differentiation (MFD) is a prerequisite for subsequent strategies aiming at the development of tissue engineered soft tissue substitutes.
Differentiation of fibroblasts to MFs can easily be assessed after cell culture by the detection of α-smooth-muscle-actin among fibroblasts either by FACS or fluorescence immunocytochemistry. Human wound explants were cultured and the presence of MF among other cell types was characterized and correlated to the healing status (n=10). In addition the effect of human dermal microvascular endothelial cells (HDMEC) on MFD was investigated in 2D- (n=10) and 3D- (matrigel; n=3) co-cultures. Our experiments aiming to actively control MF differentiation included the application of transforming growth factor β1 (TGFβ1) in a soluble (n=8) as well as in a covalently immobilized form. Cell culture substrates were two kinds of aldehyde- (n=5, n=6) and epoxy-functionalized (n=6) culture slides.
Enhanced MFD was observed in cultures from human wound explants compared to normal human dermal fibroblasts.
Areas, where fibroblasts were in contact with HDMEC in co-culture showed an enhanced differentiation potential, which was likely influenced by the detected secretion of endothelin-1 by HDMEC. The 3D-growth of HDMEC in matrigel -manifesting in elongated bundles- seemed to be supported by accompanying MF.
Soluble TGFβ1 was most effective in triggering MFD at a concentration of 1 ng/ml. Covalently immobilized TGFβ1 also remained biologically active leading to enhanced MFD.
The data show that the characterization of the MFD rate of human wounds is a promising diagnostic tool in the future. Furthermore it is possible to control MFD actively by soluble and covalently immobilized TGFβ1. Investigation of the MFD process also reveals basic differentiation phenomena and strategies transferable to differentiation strategies of stem cells from other origin.
Mesenchymal stem cells (MSCs) are a widely used cell source in tissue engineering and regenerative medicine due to their ability to be expanded and differentiated easily in vitro as well as in vivo. MSCs can be obtained from a variety of tissues including adipose tissue. This is easy to obtain and it is capable of yielding cell numbers substantial enough to obviate extensive expansion in culture. Static cell culture with the aim of tissue engineering applications has been proven to be disadvantageous since constructs grown in static cell culture lack the mechanical stability native tissue exhibits. Mechanical stimulation has therefore become a substantial tool in tissue engineering to accustom cells to their future physically active environment. Moreover, differentiation of stem and progenitor cells has been proven to be supported by mechanical strain.
Adipose tissue derived mesenchymal stem cells (adMSCs) were subjected to a cyclic mechanical strain with frequency of 1 Hz and an elongation of 5%. Time schemes of strain were varied and included 15 min, 60 min, 2 h, 4 h and 8 h and a repetitive strain of thrice 15 min, 60 min, 2 h, 4 h and 8 h with each having an intermission of double the strain time (i.e., 30 min, 60 min, and 4 h, 8 h and 16 h respectively). Osteogenic marker expression (collagen I, cbfa1/runx2, bone sialoprotein 2, Osteopontin, bone morphogenetic proteins 2 and 4, osteocalcin, alkaline phosphatase) after strain was monitored via reverse transcriptase polymerase chain reaction. Collagen III expression was monitored as indication for repair mechanisms induced by the cells after cell damage.
Osteogenic marker expression was unique after each applied time scheme. Considerable differences of osteogenic marker expression compared to control cells were determined after thrice 15 min and once 60 min of strain. Repetition of strain resulted in lower collagen III expression compared to singular strain indicating cellular accustomization.
Mechanical strain was shown to have distinct effects on the osteogenic differentiation status of adipose tissue derived mesenchymal stem cells. Different time schemes were applied reaching from singular short time strain to a repeated long time strain. It was shown that strain duration has to be considered carefully for each experimental setup keeping in mind that mechanical load effects cellular processes including differentiation.
Adipose tissue-derived stem cells (ASC) have multilineage differentiation capacity. Therefore, ASC may have the potential to be used for various clinical applications, including bone, cartilage, and cardiac tissue regeneration. Recent studies revealed a cell population within freshly isolated ASC showing a distinct expression of CD34, a well-known marker of hematopoietic stem cells. These cells are able to perform osteogenic and adipogenic differentiation after specific stimulation.
An important step during regeneration of tissues is the vascular supply. It is induced by pro-angiogenic factors (e.g. vascular endothelial growth factor/VEGF) released by tissues requiring blood vessels.
Since ASC are an interesting cell type for tissue engineering applications and since the characteristics of ASC-subpopulations are rather unidentified, we have compared CD34-negative and CD34-positive human ASC regarding their capacity of adipogenic and osteogenic differentiation. Furthermore, the release of VEGF was analyzed in both adipogenic and osteogenic differentiation. In principle, both ASC-subpopulations performed adipogenic and osteogenic differentiation (quantified by oil red O and alkaline phosphatase staining). Significant variations in the differentia-tion degrees were not detectable. Interestingly, adipogenic stimulation did not lead to significant deviations in VEGF release (assayed by ELISA) whereas osteogenic stimulation induced a sig-nificant downregulation of VEGF release in both ASC-subpopulations by ca. 50%.
Since blood vessel formation is crucial in tissue development and repair, the role of VEGF downregulation during osteogenic differentiation in vitro remains unclear. One explanation could be an intricate regulatory control of VEGF release during the different phases of osteogenic differentiation. Thus, knowledge about the regulation of angiogenesis-inducing factors in ASC-subpopula-tions and osteogenic differentiation of stem cells in general remains fragmentary and needs further investigation.
This work is financially supported by the European Union and the Federal State Mecklenburg-Vorpommern.
With aging, the gain of fatty tissue in the bone marrow and the concomitant loss of bone mass are inversely related. Transdifferentiation of (pre-)osteoblasts is likely to contribute to the fatty degeneration. Elucidation of the hitherto unknown molecular mechanisms initiating this process could help to address novel therapeutic targets that inhibit adipogenesis and enhance osteogenesis. After establishment of a cell culture system of human mesenchymal stem cells (MSCs) that allows for transdifferentiation of (pre-)osteoblasts into adipocytes and vice versa, we aimed here to identify gene products that initiate transdifferentiation.
Isolation of MSCs, adipogenic and osteogenic (trans)differentiation, cytochemical staining and mRNA expression analyses were performed using standard methods. RNA was isolated 3 h and 24 h after initiation of adipogenic transdifferentiation of pre-osteoblasts as well as of osteogenic transdifferentiation of adipocytes and used for Affymetrix microarray analyses. Duplicate microarray analyses were evaluated by signal log ratios and change p-values and re-evaluated by RT-PCR.
Reproducible and often reciprocal regulation patterns for adipogenic and osteogenic transdifferentiation were observed for 414 and 922 genes, respectively. A combined evaluation of all 16 GeneChips was enabled by the development of a bioinformatic scoring scheme that ranked regulated genes according to their potential relevance for initiation of transdifferentiation taking reproducibility, high degree of regulation and distinct reciprocity between both transdifferentiation directions into account. Members of Wnt, IGF and FGF signaling were detected amongst the highly ranked gene products. Functional examination of FGF1 as one of the highly ranked potential key factors confirmed its inhibitory effect on adipogenic transdifferentiation as expected by microarray results.
The identification of potential key molecules associated with transdifferentiation of osteoblasts and adipocytes was enabled by the combination of Affymetrix software and further bioinformatic analysis. The principle usability of our ranking was proven by the positive functional testing of FGF1. Further functional examination of individual, highly-ranked genes with reciprocal regulation in the different transdifferentiation directions could uncover novel signaling pathways and factors, which initiate these processes.
At cultivation of stem cells, defined in-vitro conditions are important. Differentiation largely depends on the biological environment and growth conditions. Therefore, monitoring of essential parameters like oxygen and pH is of vital importance and enables systemic optimization of culture conditions for complex differentiation processes.
Differentiation of haematopoietic and neuronal stem cells needs exact hypoxic conditions to guarantee reproducible results. The SDR SensorDishR Reader enables for the first time non-invasive online monitoring of dissolved oxygen (DO) and pH in 24-well multidishes. Optical sensors for DO or pH are located at the bottom of each well and read out through the bottom of the multidish by a small and robust 24-channel reader. This reader can be placed in an incubator or on a shaker for continuous monitoring during the whole period of cultivation.
The application of the SensorDish Reader is demonstrated with examples for cultivation of human embryonic stem cells and haematopoietic stem/progenitor cells (HSPC). The cells were cultivated directly in the OxoDishR for online oxygen monitoring. Different oxygen levels of the gas phase were applied and the dissolved oxygen in the medium was measured for the whole time of the cultivation. The influence of opening the ncubator, lifting the lid of the multidish, and of media change was also investigated as well as different cell concentrations.
With the SDR SensorDishR Reader, negative effects can be detected and resolved in time. Effects which had not been considered before can now be obtained and regarded, e.g. too high oxygen content in the medium can be avoided by media change under hypoxic incubator atmosphere. The non-invasive method of the system guarantees continuous monitoring during the whole cultivation and reduces the risk of contamination.
The identification of appropriate cell types is necessary to establish cell-based therapies for muscular dystrophy. From the applicative point of view, the ideal cell population should be: easy to obtain, expandable in vitro to the large number of cells presumably required for systemic treatment (1 x 109 or more), able to reach skeletal muscle through a systemic route, and should be able to differentiate into skeletal muscle cells in vivo with high efficiency. In this context, the resemblance of umbilical cord stromal cells (UCSC) with pericytes, a cell type recently identified as myogenic precursor, prompted us to determine the surface antigen expression and to investigate the differentiation potential of UCSC.
Perivascular and stromal cells were isolated from 15 human umbilical cords and expanded in culture until they reached senescence. Cells were characterized by flow cytometry and their differentiation potential was investigated under suitable conditions.
The isolated cell populations consisted of adherent cells with fibroblastoid morphology which could be cultured for >20 passages equivalent to >50 population doublings. Flow cytometric analysis revealed that the cells express mesenchymal stem cells markers (CD73, CD90, CD105) at high levels but do not express hematopoietic/leucocyte antigens (CD34, CD45, CD117, HLA-DR). When properly stimulated, UCSC differentiated into the osteogenic, chondrogenic and adipogenic lineage. Furthermore the cells express markers also expressed by pericyte-like cells (desmin, smooth muscle actin, alkaline phosphatase, CD146). Preliminary results indicate that UCSC can be induced to express myogenin. In addition, they express a number of integrins and adhesion molecules (CD29, CD49a-e, CD51, CD61, CD44).
UCSC show an extensive in vitro proliferation which indicates that cells from a single umbilical cord may generate enough cells to treat a paediatric patient with muscular dystrophy. The cells express some of the proteins that enable leucocytes to adhere and cross the endothelium which is a prerequisite for diffusion into the interstitium of skeletal muscles. In addition, UCSC could not only be differentiated into the osteogenic, chondrogenic and adipogenic lineage but seemed to contain precursors with myogenic potential. Because of these features, UCSC might be considered as a valuable source for the isolation of potent cells for future cell therapies of muscular dystrophy.
In patients suffering from therapy-resistant wounds, transplantation with split-thickness skin grafts is hampered because of the limited availability of donor skin as well as mechanical long-term stability of the grafts. Alternative tissue engineered concepts (EpiDexO) use the hair follicle as origin for the transplantation of follicular keratinocytes which are presumed to correspond to stem cells of the outer root sheath, however; had not yet been characterized upon their stem cell properties.
Biopsies from human scalp were characterized for the expression of stem cell markers, previously identified in the murine hair follicle. Keratinocytes were isolated and expanded from human plucked hair follicles, according to the EpiDexO-protocol. The expression of keratinocyte precursor cells as well as differentiation markers were analyzed immunohistochemically in multilayered sheets as well as in primary and serial cultures of follicular keratinocytes. By clonality assays, clonogenic potential of serial cultures was investigated morphologically as well as quantitatively.
We could identify cytokeratin 15 (CK15), follistatin, and CD200 as markers for bulge-stem cells of the scalp hair follicle (however, not CD34, nestin, and cytokeratin 19), transferrin and p63 as markers for transient amplifying cells. In EpiDexO-sheets expression of CK15, follistatin, s1-integrin, transferrin, and p63 could be identified according to normal human epidermis. However, during serial culture, number of holoclones diminished and CK15 was almost completely lost within the secondary passage.
Follicular keratinocytes seem to be an appropriate origin to transplant keratinocyte precursor cells. However, mechanisms should be identified to enhance stem cell pool of such transplantation concepts.
A Rotary Cell Culture System (RCCS) allows the creation of a microgravity environment of low shear force, high-mass transfer and 3-dimensional cell culture of various cell types. Aim of this study was to evaluate the growth pattern and the secretory function of rabbit lacrimal gland acinar cells in a microgravity environment using a RCCS.
Lacrimal gland acinar cells from New Zealand White rabbits of both sexes were isolated and cultured in a RCCS up to 28 days. Cells were analysed by light and electron microscopy at day 7, 14, 21 and 28. Secretory function was tested by measuring the s-hexosaminidase activity.
After seeding to the RCCS, the lacrimal gland cells formed spheroidal aggregates. The acinar cells inside the spheroids retained their histotypic features, but in the center of the spheroids groups of necrotic cells became more abundant during the culture period. The evaluation of the secretory function showed a response to stimulation with carbachol until day 7.
Acinar lacrimal gland cells can be successfully cultured in a RCCS up to 28 days, with a secretory response to carbachol up to 7 days. A simulated microgravity environment allows to maintain long-term cultures of lacrimal gland acinar cells and promises opportunities for further applications in basic and applied cell research on lacrimal gland cells.
The secretion of the lacrimal gland provides 95% of the aqueous tears, which are essential for lubrication, nutrition and protection of the ocular surface. Long term studies of acinar lacrimal gland cells in-vitro are complicated by low proliferation rate and fast loss of cell function on plastic. Aim of this study was to evaluate the growth pattern and the secretory function of lacrimal gland acinar cells on amniotic membrane in a rabbit model.
Lacrimal gland acinar cells from Chinchilla Bastard- and New Zealand White rabbits of both sexes were isolated and cultured on denuded amniotic membrane. Cells were analysed by light and electron microscopy. Secretory function was tested by measuring the s-hexosaminidase activity.
Three days after seeding to the amniotic membrane, the acinar cells had attached to each other and formed small cluster. Cell clusters consisted of 2-5 cell layers and the cells showed fine granulation in their cytoplasm, typical for secreting cells. Between day 7 and 14 cell clusters increased in size and acini-like structures with a central lumen were found. Cells showed polarity, with a basal nucleus and apical secretory granules. Between day 21 and 28 still acini-like structures were found inside the cell clusters. Accumulation of secretory material in the central lumen and desmosome formation connecting the apical cell structures was ) frequently evident. However, the number of cytoplasmatic granules decreased and on parts of the AM cell morphology changed to flat, spindle shaped cells with a small nucleus. Stimulation with carbachol showed a strong s-hexosaminidase release until day 7, with a decreasing secretory function detectable until day 21.
Acinar lacrimal gland cells can be successfully cultured on amniotic membrane up to 28 days, with a secretory response to carbachol up to 21 days. This model may be used for further experimental work, to elucidate cellular mechanisms in normal and diseased lacrimal tissue.
Hematopoietic stem cells (HSCs) from umbilical cord blood (UCB) can easily be collected without harming the patient. Former studies showed that these cells can be turned into cells which share characteristics with embryonic stem cells. We are aiming at optimizing this method using a population of naive cells from UCB for further expansion, differentiation and transplantation in the field of cell based therapies.
Isolated HSCs from UCB were cultured in-vitro by using a mixture of the cytokines LIF, FGF and TGF. By comparing different culture strategies such as using different media, different amount of additives (Glutamine, FCS) and different mixtures of the mentioned cytokines we study efficient ways to propagate and expand cells with embryonic phenotype. Up to now, cells were analyzed by flow cytometry, immunohistochemistry and RT-PCR using hematopoietic markers and embryonic stem cells markers.
Critical factors in this method are the basic media and the cell concentration. IMDM supplemented with 2 mM Glutamine, 10-20% FCS, and a cell concentration of 5x105/ml showed optimal results. For cell expansion the isolated HSCs were treated with LIF for several days. To promote the expression of embryonic cell markers additional cytokines were introduced. To generate suitable cells for transplantation we also work on strategies to avoid any animal substances in our culture system.
It is desirable to set up an easy method to generate a homogeneous population of embryonic-like cells derived from HSCs of UCB in order to use them in future clinical cell based therapies. It seems that our cells express stem cells markers such as SSEA-4 and are expandable. However, a lot more work needs to be done to assure that these cells (and/or their differentiated offspring’s) will not cause more harm than good after clinical use (e.g. transplantation).
Fabry disease is an X-linked lysosomal storage disorder caused by a deficiency of the enzyme alpha-galactosidase resulting in progressive intracellular accumulation of glycosphingolipids. Deposition of glycosphingolipids in the vascular system may lead to alterations in vascular function and endothelial progenitor cells (EPC).
In patients with Fabry disease (n=23; 38.9±12.4 years) and in healthy age-matched controls (n=21; 40.8±8.9 years), vascular function was assessed by peripheral arterial tonometry (PAT) based on finger plethysmography. Vascular function was defined as the ratio of pulse wave amplitude (PWA) during reactive hyperemia relative to baseline. In addition, the duration of increased PWA until return to baseline levels was determined as an additional marker for vasodilatory capacity. The augmentation index was calculated by an automatized determination of the reflected pulse wave as a marker for arterial stiffness. Left ventricular wall thickness was assessed by echocardiography. Vasoregenerative capacity was estimated by investigating number and function of circulating EPC.
Patients with Fabry disease had an increased PAT hyperemia ratio (2.44±0.13 vs 1.96±0.1; p=0.008), whereas the duration of increased PWA during hyperemia was significantly reduced compared with healthy controls (222.4s±54.3s vs 300.5s±52.2s; p<0.01). Fabry patients had a significant increase in arterial stiffness with increasing age, whereas this was not observed in healthy controls. Duration of increased PWA was inversely correlated with increased left ventricular end-diastolic posterior wall thickness (standardized coefficient -0.58, p=0.029). In contrast, PAT hyperemia ratio, basic blood parameters, glomerular filtration rate, gender and age were not correlated with left ventricular hypertrophy. Finally, migratory potential of circulating EPC was impaired by 46±12% (p<0.05) in Fabry patients compared with healthy age-matched controls. Underlying molecular mechanisms and therapeutic options are currently investigated.
The present data point to an early pathologic involvement of the vascular system including impairment of the vasoregenerative reserve in Fabry disease. This was may aid our understanding of the progressive development of cardiovascular disease in patients with Fabry disease.
Mesenchymal stem cells (MSCs) have the capacity to proliferate and differentiate into chondrocytes, osteoblasts and adipocytes. For this reason, MSCs are considered to be of great clinical potential in cell based regenerative strategies for the skeletal system. Questions arise to what extent MSCs are subject to age related changes and whether MSCs based strategies should be limited to patients of selected age groups. The aim of this study was to elucidate the effect of age on MSC-frequency, cell proliferation and differentiation capacity in vitro.
Fifteen donors from 7-85 years were involved in this study. The age range was covered by three groups of 5 donors each. Group 1 ranged from 7-12 years, group 2 from 20-55 years and group 3 included 5 donors from 60-85 years. Mononuclear cells from bone marrow were isolated and CFU-F number was determined. Subcultures were prepared to assess single cell cloning efficiency and proliferation rate. Chondrogenic differentiation was assessed by glycosaminoglycan (GAG) quantification and histologic/immunohistologic evaluation of Alcian blue and Collagen Type II. For osteogenesis, cells were evaluated for membrane bound alkaline phosphatase (ALP) and Alizarin Red S. For adipogenesis, lipid accumulation was quantified by Oil Red O staining.
No correlation was found between CFU-F-frequency or proliferation rate of MSCs and donor age. A negative correlation of single cell cloning efficiency with age was, however, observed (r2= -0,604; p<0,01). While no correlation was obvious between adipogenic differentiation capacity of MSCs and age, activity of bone related ALP (r2=0,413; p<0,05) and calcium deposition (r2=0,721; p<0,001) showed a positive correlation with age. Furthermore we observed significant correlations between ALP activity and calcium deposition (r2=0,6; p<0,01) and between proliferation rate and lipid accumulation (r2=-0,422; p<0,05).
MSC proliferative potential and differentiation capacity to adipogenic lineage did not change with age. Osteogenic capacity correlated positively with increasing donor age, which sheads a promising light on the potential application of cell based bone remodelling strategies also in patients of older age. It seems promissing to further study MSC populations from donors of older age to gain a better understanding about the molecular mechanisms regulating age-related changes in connective tissue biology and their possible consequences for regenerative medicine.
In different variants of cell therapy with stem cells (SC) and their derivatives, both allogeneic (e.g., obtained from cord blood) and autologous materials are used. And though the last one is “own”, after its multiplication ex vivo for reaching sufficient amount for therapeutic effect, the cells obtained differ from the native cells and therefore are taken by the organism in somewhat other manner. The reaction of an organism on the introduction of allogeneic and natively autologous (but multiplied ex vivo) cells is realized both by direct cell and humoral interactions. Such reaction significantly determines success or failure of the treatment. On the other hand, many factors are involved into the organism response, and it is different in various individuals. To evaluate such response, its personalization is necessary.
Due to restricted access to the biological material of an individual, we chose influence of blood plasma on cell growth as the most informative from the possible accessible variants. Model objects were rabbits. As the most difficult patients for cell therapy are aged groups, model objects were chosen from the population of healthy rabbits more than 4 years old. Blood serum was testes in 3 variants: native, heated at 56oC during 40 min and filtered through nitrocellulose membrane with pore diameter 0,22 mkm. The last variant actually represented adsorption: plasma volume for filtration was not more than 1 ml and nitrocellulose in non-specific absorber. The prepared plasma was added to DMEM to the final concentration 0,3 ? 5% with twofold dilutions. Controls were the same concentrations of fetal calf serum (FCS) and medium without any serum. Test culture for control and experimental samples was cell line RK-13 (rabbit kidney). Culturing was performed in standard conditions during 72-96 h. Evaluation was based on growth rate and character as well as on cell morphology.
These showings varied significantly both among different individuals and for the same animal in various time intervals. The diversities varied from complete cell death to proliferation, morphology changing, growth character and attachment to substrate. Growth with FCS was always standard in all experiments.
As plasma concentration in human organism is at least 20 fold higher in comparison with the experiments presented, even moderate differences in SC and their derivatives from the native cells can meet any reaction of the host during their introducing. Even personalization made not directly before introducing, is not always adequate.
Bone is a unique and highly regenerative tissue in vertebrates. The process of bone healing and regeneration is thought to be influenced by the action of mesenchymal stem cells (MSCs). It is known from clinical and animal studies that the regeneration potential of bone decreases with age. Therefore, the aim of this study was to analyse the relationship between donor age and the functional behaviour of MSCs. In addition, the underlying molecular mechanisms were investigated by proteome analysis.
MSCs were obtained from 3 weeks (yMSCs) and 12 month (oMSCs) old male Spraque Dawley rats. Cells were isolated from bone marrow of the femoral and tibial bones, and selected by plastic adherence. MSCs were characterised by flow cytometry (CD44+; CD73+, CD90+ and CD45-). The numbers of colony forming units (CFUs) were determined four days after isolation. The migration capacity was measured in a modified Boyden chamber assay. Senescent cells were identified by their s-galactosidase activity. The osteogenic differential potential was analysed by measurement of alkaline phosphatase (AP) and the matrix mineralization (Alizarin Red) after incubation in osteogenic medium. The proteomes of yMSCs and oMSCs after cultivation in expansion media and after an osteogenic stimulus were investigated by high resolution 2D-PAGE followed by mass spectrometric protein identification as described by Klose and colleagues.
No difference in the MSCs typical surface marker profile was observed between yMSCs and oMSCs. In contrast, total number as well as number of AP-positive CFUs was significantly decreased in old animals. The migration capacity of oMSCs was lower than that of yMSCs. Proliferation and differentiation capacity of MSCs were independent of donor age. Higher passage cultures of oMSCs contained higher numbers of senescent cells. The proteome analysis of low passage oMSCs and yMSCs demonstrated differential expression of 34 proteins (expansion medium) and 64 proteins (osteogenic medium). According to functional annotation clustering, age related molecular function were associated with cytoskeleton organization and antioxidant defence. Notably, potential target proteins were found which are known to be associated to bone physiology.
In summery, we could show that MSC number, function as well as protein expression changes with the donor age. Results might be relevant for the development of innovative therapies.
We generated, on titanium surfaces, self-assembled layers of vertically orientated TiO2 nanotubes with defined diameters between 15 and 100 nm and show that adhesion, spreading, growth and differentiation of mesenchymal stem cells are critically dependent on the tube diameter. A spacing less than 30 nm with a maximum at 15 nm provided an effective length scale for accelerated integrin clustering / focal contact formation and strongly enhances cellular activities compared to smooth TiO2 surfaces. Cell adhesion and spreading were severely impaired on nanotube layers with a tube diameter larger than 50 nm, resulting in dramatically reduced cellular activity and a high extent of programmed cell death. Thus, mesenchymal stem cells seems to be very sensitive to nano-scale microenvironment such as a lateral spacing geometry representing 30-50 nm of nanotube diameter as a critical borderline for cell fate.
Participation of mesenchymal stromal progenitor cells (MSC) in tissue reparation may occur under reduced oxygen tension, the degree and duration of which may determine MSC functional characteristics. We showed that 96h hypoxia (5% O2) had a stimulating effect on rat bone marrow MSC which manifested itself in reduced heterogeneity within MSC cultures, an increase in cell proliferation, a decrease in percentage of damaged cells, maintenance of cells immunophenotype and initial stages of differentiation. Moreover MSC stood long-term hypoxia with no increase in the amount of damaged cells and capability of further differentiation. This may be accounted for the fact that 5% oxygen level simulates in vivo oxygen tension and can be regarded as an approximation of physiological conditions rather than hypoxic impact. The aim of this study was to evaluate the effects of anoxia on rat bone marrow-derived MSC.
Cells used in the experiments were at 1-4 passages and cultivated either in normoxia (95% air + 5% CO2), hypoxia (95% gas mixture of 95%N2 + 5%CO2 and 5% O2) or anoxia (95% N2 + 5% CO2). MSC morphology and proliferation were evaluated using videomicroscopy. MSC viability and surface markers expression were assessed by flow cytometry. Osteogenic and adipogenic differentiation MSC was determined as to alkaline phosphatase (AP) activity or lipid droplets formation within cells respectively in response to differentiation stimuli. 96h anoxia didn’t lead to changes in MSC morphology, proliferation rate and immunophenotype; didn’t increase significantly the percentage of damaged cells despite some activation of apoptosis; didn’t inhibit the initial stages of stimulated adipogenic and osteogenic differentiation despite the suppression of AP activity showing that rat bone marrow MSC is a population resistant to a significant decrease in oxygen tension. Further cultivation of MSC in anoxic environment led to their progressive damage mainly by necrosis in contrast to apoptosis as the main damaging mechanism under 96h anoxia and antiapoptotic effect of hypoxia. It may come in line with the data that proapoptotic effect of low oxygen tension depends on HIF stabilization occurring mostly at less than 5% oxygen tension while antiapoptotic hypoxia effect is provided regardless of HIF.
Tooth development is driven by a complex crosstalk between epithelium and neural crest-derived mesenchyme via signalling molecules, transcription factors and influences of the extracellular matrix. Despite knowledge of tooth development in rodents, the knowledge on gene expression important for a comprehensive tooth development in humans remains fragmentary. The aim of this study was to gain more insight into the molecular mechanisms that control differentiation of human tooth, and to clarify whether different compartments (niches) show inherent differences in their expression levels of stemness markers due to different cell populations.
Human impacted third molars were divided into the operculum, periodontal ligament, developing pulp and ? as novel ? the pad like tissue beneath the pulp. We characterized the expression level of all compartments by real-time PCR of 16 different genes. In addition we performed whole genome expression arrays to clarify whether the coding of stem cells derived from different compartments is maintained in vitro.
The expression of Msx2 and HNK1 in all compartments confirms their ectomesenchymal origin. With regard to markers for ectomesenchyme and tooth development every single compartment held its own signature of gene expression. The expression of stemness markers such as nanog or Oct4 pointed to multipotent / pluripotent features. The differences in relative gene expression turned out to be dynamic along the progress in tooth development. In vitro, cell cultures derived from dental pulp and pad like tissue showed substantial differences in their respective gene expression profiles on a whole genome scale.
Concerning a set of 16 genes leads to the impression of dynamic opposed by rather quiescent compartments. Differences observed in cell cultures derived from pulp and pad like tissue may account to the fact that both compartments share in general the same cell populations but vary with respect to their relative abundance. On the other hand, it is conceivable that cells of both compartments are committed due to instructive signalling of their respective niches. Is has to be explored in further experiments whether this is reflected by differences in e.g. differentiation capacities to other tooth derived stem cells.
Stem cell status is maintained and ended by mixtures of signals received and interpreted by the cells. The often referred to stem cell niche is such a mixture of signals and varies strongly for most stem cell types. Our approach to identifying the signaling mechanisms underlying these different effects include inter- as well as intraspecies comparison of signal-cascade statuses in stem cells.
Using an array of different stem cell types from the lower vertebrate medaka (Oryzias latipes) and the mouse, we have compared the signalling activity as well as the signal-complex make-up and its consequences on upkeep of stemness versus differentiation. We used approaches such as co-immunoprecipitation, HPLC fractionation, immunofluorescence and real-time PCR as well as live-imaging of fluorescently tagged signal-mediators such as STAT3.
We were able to demonstrate that the pluripotency of medaka stem cells as well as medaka blastula embroys are independent of STAT3 activity. Furthermore, biochemical analyses of the signalling complexes downstream of BMP2 revealed the presence of STAT3. Next, we found STAT3 modified and re-located to the cytoplasm as a result of ligand-engagement of BR1a in embryonic stem cells. Alteration of the composition of the complex also changed differentiation behaviour of neural stem cells.
While our comparison between pathways necessary for pluripotency in both mouse and medaka revealed strong differences both in vivo and in vitro, all the systems analysed thus far share a common, huge signal-integrator complex. Amongst other proteins, this complex includes the BMP-receptor 1a and STAT3. Treatment of stem cells with different BMP ligands results in re-localisation and secondary modification of STAT3. The presented data a hence first evidence for a functional crosstalk between these pathways.
Addressing the hypothesis that the murine embryonic microenvironment can induce differentiation in human tumour cells, we developed an in vitro coculture system for established human AML cell lines in combination with a variety of stromal cell lines derived from different murine embryonic hematopoietic sites. Coculture-induced effects contain decreased proliferation and colony formation capabilities, further AML cells show differentiated morphologies and the up-regulation of myelo-monocytic lineage markers. Our aim now is to uncover mechanistical details of molecular and cellular interactions between leukemic cells and their stroma, which may lead to the identification of new paradigms for active control and reprogramming of leukemic cells.
Irradiated murine stroma cells were plated to confluence. On the next day, human leukemic cells were seeded onto stromal cells. After 3 days AML cells were harvested from the feeder layer and analysed for the expression of myelo-monocytic lineage markers by FACS. In Flow Chamber experiments as well as by adding neutralising antibodies directed against VLA-4 and VCAM-1 to the coculture system, the influence of these cell adhesion molecules on differentiation status was analysed. Also, cytokine profiles of differentiation-supporting and non-supporting stroma cell lines were prepared and differentially expressed cytokines (e.g. TGF-b) were analysed for their involvement in differentiation induction by supplementation to human AML cell cultures.
Flow Chamber experiments show strongly reduced adhesion of AML cells to the stromal layer when VCAM-1 is blocked on stroma cells. On the other hand, blocking of VLA-4 on AML cells seems not to be effective. Regarding the effect of soluble factors, first results indicate, that TGF-b, though per se not sufficient to induce differentiation in human AML cells, is able to increase coculture-induced effects on differentiation. For other cytokines like TNF-a, RANTES, IL-6 or LIF we could not show any effects so far.
Our preliminary results suggest that VCAM-1 but not VLA-4 is involved in adhesion of human AML cells to murine embryonic stromal cells. That poses the question whether VCAM-1 is necessary for the coculture-induced differentiation. The cytokine studies have to be repeated with a broader panel of cytokine concentrations. In addition analyses of participating signal transduction pathways are planned to give more information about differences in signaling in human AML cells upon coculture.
We generated two tumorigenic cell lines from neurosphere cultures of rodent SVZ stem cells. These cell lines, termed R2303 and R2902, express stem cell markers (such as Nestin, CD133, Musashi-1 and Sox2) and can be differentiated into neuronal and glial phenotypes. However, expression of stem cell markers and proliferation continues when these cells are differentiated. Transplantation of R2303 cells into syngenic, immunocompetent rats demonstrated the potential to form malignant tumors. These tumors showed streak necrosis, invasion into surrounding brain parenchyma and expression of undifferentiated (Nestin) as well as neuronal (Map2) markers. Karyotyping revealed a high grade mosaicism and massive structural and numeric chromosomal aberrations. We identified the platelet-derived growth factor receptor alpha as potential candidate for the continued proliferation of R2303 and R2902 cells, as this receptor is not downregulated during differentiation and is also prominently expressed within tumors. Moreover, RNAi knockdown of the PDGF receptor alpha resulted in growth reduction of R2303 cells. These cell lines may provide a link between brain and cancer stem cells. Furthermore, rapid in vitro expansion of adult stem cells (e.g. for stem cell therapies) needs to be closely monitored for the occurrence of aberrant cell types.
Under observation (9 years) was a group of patients with 1.5 ?20-year history of confirmed rheumatoid arthritis (RA) presenting drug intolerance or inefficiency of routine methods, and marked limitation of functional capacity. For treatment, used were cryopreserved embryonic stem cell (ESC) suspensions containing hematopoietic and non-hematopoietic mesenchymal stem cells. ESC suspensions were prepared from organs of 4-8 weeks old embryonic cadavers obtained from legal abortions. Total nucleated cell count ? 10-400x106/ml, mononuclear cell count ? 10-100x104/ml. After the treatment, all patients reported the Syndrome of Early Post-Transplantation Improvements manifested by decreased general weakness, subsidence of pain, improved and more optimistic mood, improved appetite and normalized sleep formula. This method of treatment allowed for the decrease of the degree of the inflammatory activity, from high (14 points) to minimal (3 points), and achievement of clinical remission in 85% of cases.
Within 1 year, patients under observation reported decrease of pain, joint, and inflammatory indices - main clinical criteria of RA, functional capacity increase manifested by the ability of performing everyday activities impossible prior to this treatment, improved gait, ability to use public transport and continue full-time work. Subsequent transplantations were performed in 62% of cases: 80% of them were aimed at functional capacity and life quality improvement, and only 20% were performed for the decrease of the disease activity.
Immunocorrecting effect of ESC was observed in all the patients. Reported was increase of T-suppressor count (CD8+); helper/suppressor ratio CD4+/CD8+ decreases over a period of 360 days. Prior to the treatment, rheumatoid factor titer amounted to 3.2 U, and within 360 days, it decreased to 1.02 U. Bioethical principles on application of embryonic cell suspensions were strictly adhered to at all the stages of the research.
The variety of commercially available biomaterials for tissue engineering is continually expanding. For bone regeneration constructs using human jaw periosteal cells (JPC) the extent of osteoinductive ability of different three-dimensional scaffolds is not yet established. We compared collagen (Coll), open-cell polylactic acid (OPLA) and calcium phosphate (CaP) 3D-scaffolds for their suitability and osteoinductivity of human JPC.
Human JPC were seeded onto Coll, OPLA and CaP scaffolds (BD Biosciences). JPC proliferation was analyzed by a colorimetric assay. Cell adhesion and spreading was visualized by scanning electron microscopy (SEM). Live-measurements of oxygen consumption within the cell-seeded scaffolds showed maximal time period of in vitro culturing. For further osteogenic differentiation experiments only OPLA scaffolds were used. Gene expression of JPC during osteogenesis was examined by quantitative PCR and element analysis of formed nodules by EDX spectrometry.
Maximal proliferation rates were detected within OPLA scaffolds. 12 days after in vitro culturing we detected the highest oxygen consumption through proliferating JPC within the scaffolds. 15 days after in vitro culturing oxygen concentrations were increasing again, probably due to cell death by apoptosis. SEM clearly showed a greater and multilayered JPC growth and the formation of specific nodules under osteogenic conditions. EDX spectrometry revealed that nodules containted of calcium with or without phosphorus.
Polylactid acid seemed to be the most beneficial biomaterial for JPC growth among the analyzed scaffolds. The time period for in vitro culturing should not exceed 14-15 days because of possible transplantation of apoptotic cells. We demonstrated that JPC growing within OPLA scaffolds were able to mineralize in vitro. We concluded that analyzed OPLA scaffolds provide a promising environment for bone substitutes using human JPC.
Bone marrow stromal cells (BMSCs) are the major cellular component of the marrow stroma and include the mesenchymal progenitors. Their in vitro adherence ability allows isolation and expansion of multipotent colonies (CFU-F). BMSCs display osteogenic commitment, which can be modulated by culture conditions. The aim of this study was to evaluate the expression of osteogenic phenotypic traits in BMSCs to assess their potential use in tissue-engineering strategies.
BMSCs were isolated from canine marrow aspirates after plating at a density of 6-9 x 107 cells per 150-cm2 flasks. After 24 hours medium was replaced and changed three times per week until cultures became ~90% confluent. The number of progenitors was quantified on day 14 by counting CFU-Fs with ? 50 cells. The master gene of osteogenic commitment CBFA1, type I collagen, alkaline phosphatase (ALP) and gene products characterizing the osteoblastic phenotype, such as osteonectin were quantified by RT-PCR. The expression of fibronectin, type I collagen, decorin, biglican, osteonectin, osteopontin and bone sialoprotein (BSP) was investigated on days 7, 14 and 21 by immunocytochemistry and ALP by cytochemistry. BMSCs were also incubated for 21 days under osteogenic conditions to induce Ca deposition that was further analyzed by nuclear fast red staining, scanning electron microscopy (SEM) and by energy dispersive X-ray analysis (EDXA).
BMSCs obtained from CFU-F expansion displayed spindle shape morphology with ~30% of the colonies positive for ALP on day 14. On passaging, the levels of mRNA expression of osteoblast-related markers decreased while CBFA-1 levels remained constant. The expression of osteopontin, BSP, type I collagen and fibronectin did not change substantially over time in culture. Decorin and biglycan were variably expressed. Calcium deposits, detected by nuclear fast red staining as amorphous structures, were confirmed by EDXA analysis and were identified on SEM as 10 μm spheroid structures encased by fibers and extracellular matrix.
A number of in vitro methods are employed to determine the differentiation ability and commitment of BMSCs with the osteogenic phenotype. Our data demonstrate that canine BMSCs, after ex-vivo expansion express CBFA-1 and other osteoblast-related gene products that can be taken as evidence of the osteogenic commitment. This biological property strongly supports the use of BMSCs for the reconstruction of bone defects as well as in general veterinary regenerative medicine.
Our clinical aim is to develop a one-step procedure for autologous chondrocyte transplantation, i.e. harvesting, isolation and reimplantation of CCs performed in one surgical procedure. Consequently there wouldn’t be an increase in cell number. The use PRP, a mitogenic agent, might compensate for this. The aim of this study was to test the influence of PRP on proliferation and differentiation of freshly isolated CCs. In parallel, we submitted MSC to the same experimental set-up.
Cartilage from adult sheep was collected and CCs isolated via digestion. After removal of undigested cartilage, cells were directly submitted to the experiment. MSCs were obtained using a standardized protocol, expanded and dealt with analogous to CCs. PRP extracts were produced according to published protocols. We had six different treatment groups: (1) Cells cultured as a micromass (MM); (2) same as (1) but with the addition of activated PRP; (3) cells suspended in a fibrin sealant; (4) cells suspended in a PRP clot; (5) cells in monolayer (ML); (6) same as (5) but with addition of activated PRP. Chondrogenicity was assessed via quantification of collagen type II mRNA (Col_II_mRNA) and immunohistochemical analysis.
MM culture best conserved the chondrogenic phenotype of CCs. Addition of PRP diminished significantly the amount of expressed Col_II_mRNA. Culturing freshly isolated CCs in ML led to a significant reduction of Col_II_mRNA compared to MM; here, addition of PRP had only a weak influence on chondrogenicity. For MSCs, MM culture showed the highest level of Col_II_mRNA. Addition of PRP reduced expression rate. Still, a 3D-growth together with PRP led to a higher expression level of Col_II_mRNA compared to ML.
For both cell types a proliferative effect of PRP was observed
Freshly isolated CCs combined with PRP lose their chondrogenic phenotype during in vitro culture. Loss of chondrogenicity seems to be connected with an increase in proliferation. According to our results it might be possible that MSCs combined with PRP could improve the healing of cartilage defects in vivo.
Murine mesenchymal progenitors (C3H10T?) stably expressing BMP2 and the constitutively active SMAD8ca, a member of the family of mediators transferring signals of TGF-beta/BMP growth factors undergo differentiation into cells with tendon and ligament morphology
Literature:
Hoffmann, A. et al. (2006). Neotendon Formation Induced by Manipulation of the Smad8 Signaling Pathway in Mesenchymal Stem Cells.
The success of matrix-based autologous cell implantation for the treatment of articular cartilage defects is dependent on the lateral integration and the deposition of cartilage specific extracellular matrix components. Due to the limited availability of healthy chondrocytes and dedifferentiation during expansion culture, the need for an alternative cell is obvious. In this study, using a bovine in vitro integration model, agarose constructs laden with MSCs, differentiated and dedifferentiated chondrocytes were compared concerning their applicability in matrix-based cartilage repair strategies.
Circular native cartilage rings with a centred 4mm hole were fabricated from knee cartilage of calves. In the first group cell-laden agarose constructs (10x106cells/ml) seeded with either de-/differentiated chondrocytes (0, 2, 5 or 8 population doublings), or MSCs were implanted into the cartilage rings. These hybrid constructs were cultured for 28 days in a chemically defined, serum free medium (CM-). In a second group, constructs were initially kept as free-swelling culture in CM-supplemented with TGF-s3 (CM--+) for 21 days and then implanted. Four weeks after implantation lateral integration, biomechanical properties and accumulated glycosaminoglycans/bulk collagen were assessed within the constructs.
Untreated scaffolds seeded with dedifferentiated chondrocytes or MSCs revealed significant lower integration compared to differentiated chondrocytes. In comparison, in the pre-treated group MSC-seeded cell-carriers showed stronger integration into the surrounding native cartilage than those seeded with dedifferentiated chondrocytes. Remarkably, pre-treated MSC-seeded constructs showed distinct matrix deposition, mechanical properties and integration that were significantly higher compared to constructs seeded with dedifferentiated chondrocytes.
These results show that chondrocytes lose their ability to deposit cartilage specific extracellular matrix components within the implant with increasing number of population doublings, resulting in inferior mechanical properties and integration. In addition, dedifferentiated chondrocytes exhibit inferior responsiveness to anabolic factors, such as TGF-s3. MSCs are able to undergo chondrogenic differentiation during the pre-treatment with CM+ and show superior accumulation of cartilage specific extracellular matrix after implantation, supporting their applicability in matrix-based cartilage repair strategies.
(Support: NIH NIAMS Intramural Research Program ? Z01 AR41131)
Therapeutical concepts of local gene therapy for bony defects need to be verified in vivo. Since permanent genetic alterations, i.e. transgenic animals are realised most easily in mice, an in vivo model for bone healing should be a mouse model, too. Existing mouse fracture models are either externally fixed osteotomies with very narrow bony gaps or internally fixed non reproducible fractures that will always heal spontaneously. This pilot study aimed to establish a mouse model for the investigation of bone healing in a critical size bony defect of the femur.
25 BL/6 mice underwent femoral osteotomy creating externally fixed bony gaps of 3.5mm. Bone healing was studied radiographically and histologically for up to 6 weeks. Fixateur design was optimised to allow for highest reproducibility, widest extension of the bony defect, sufficient mechanical stability, and for stereotactical injection of cell containing material into the defect.
For the established model critical size of the bony defect could be demonstrated for a period of a least 6 weeks. The constructed external fixateur proved to be sufficiently mechanically rigid. The surgical procedure appeared to be safe (loss of one out of 25 mice perioperatively). Injection of bone marrow stromal cells into the defect led to partial ossification within 3 weeks.
Standardised critical size bone defects are necessary to evaluate bone healing using gene therapy based therapeutical strategies. With the proposed external fixation bone healing in a highly reproducible critical size bone defect of the murine femur may be analysed for a least 6 weeks. Stereotactical injection into the bone defect of e.g. genetically manipulated cells with increased osteogenic competence is easily possible and qualifies the models for a great variety of experimental settings.
In this work, we specifically wished to characterize the differentiation potential of the cells that migrate out of the anterior cruciate ligament (ACL) compared to marrow derived mesenchymal stem cells (MSCs) in order to gain insights how to stimulate a more effective healing response of the ACL cells after injury.
Cell isolation: ACL fibroblasts cells were isolated by explant cultures and MSCs were recovered by adhaerent culture of bone marrow aspirate which (informed consent and IRB approval). The recovered cells were placed in monolayer cultures in complete DMEM media. 2nd passage cells were used for all experiments. Cell surface antigens: FACS analyses have been performed using monoclonal antibodies for: CD14, CD29, CD31, CD34, CD44, CD45, CD90, CD105, CD106, CD133. Multilineage differentiation cultures: Osteogenesis was induced by monolayer cultures in complete DMEM supplemented with dexamethasone, s-glycerophosphate, ascorbate, and recombinant BMP-2 (25 ng/mL). The adipogenic phenotype was induced by supplementation with dexamethasone, insulin, indomethacine and IBMX in monolayer culture. Chondrogenesis was induced by aggregate cultures maintained in serum-free medium containing dexamethasone, ascorbate, proline, sodium pyruvate, and recombinant TGF-s1 (10 ng/mL). Negative control cultures were also maintained in the respective media without supplements, and all cultures were maintained for three weeks. Phenotype characterization: Lineage specific differentiation was analyzed by RT-PCR (COL I, II, IX, X, SOX-9, ALP, OC, Cbfa1, LPL, PPARg2) and histology and immunohistochemistry (H&E, Alcian Blue, ALP, Oil Red O, COL I, II, X).
FACS analyses revealed positive staining for the markers CD 29, 44, 90, 105, 106 for both cell types, but to a lesser extend in the ACL cells. All other markers were negative for both cell types. Following three weeks of differentiation culture, the ACL cells revealed a strong chondrogenic, adipogenic and osteogenic differentiation potential, such as the MSCs, as shown by the respective histological, immunohistochemical and RT-PCR analyses. In contrast, the respective negative control cultures for the ACL cells and MSCs, which were maintained without any media supplements, were negative for the tissue specific markers.
The study was designed to characterize the cells migrating out of the bulk ACL tissue. Our study revealed an almost similar surface antigen expression profile compared to marrow derived-MSCs and an equivalent multilineage mesenchymal differentiation potential of these cells. This might be used in order to augment a favourable healing response toward healing of ACL defects by using e.g. growth factors. Global gene expression analyses (Affymetrix HG-U133_Plus) are underway to further characterize the stemness of the ACL cells on a molecular level.
The subacromial bursa plays key roles in the gliding mechanism of the shoulder and can be efficiently used to augment repair procedures after rotator cuff tears. In this work, we specifically wished to characterize the cells that can be isolated from subacromial bursa tissue and to evaluate their potential to differentiate into the various mesenchymal lineages compared adult marrow-derived mesenchymal stem cells (MSCs).
Bursa cells were isolated from 5 bursae subacromiales by collagenase digestion and marrow-derived MSCs were recovered by adhaerent culture of bone marrow aspirate from 5 different donors. Tissues and aspirates were retrieved from patients undergoing rotator cuff repair surgery and total hip arthroplasty after informed consent and local IRB approval. Monolayer cultures were analyzed by flow cytometry (CD34, CD53, CD73, CD90, CD105, CD106, CD133, CD144, CD166, and Stro-1). Osteogenesis was induced by supplementation with dexamethasone, s-glycerophosphate, ascorbate, and recombinant BMP-2 (25 ng/mL), and adipogenesis was induced by supplementation with dexamethasone, insulin, indomethacine and IBMX in monolayer culture. Chondrogenesis was induced by pellet cultures in serum-free medium containing dexamethasone, ascorbate, proline, sodium pyruvate, and recombinant TGF-s1 (10 ng/mL). Negative control cultures were also maintained in the respective media without supplements, and all cultures were maintained for three weeks. The lineage differentiation was analyzed by RT-PCR (COL I, II, IX, X, SOX-9, ALP, OC, Cbfa1, LPL, PPARg2), histology and immunohistochemistry (H&E, Alcian Blue, Alizarin red, ALP, Oil Red O, COL I, II and X).
Light microscopy and flow cytometry revealed a mainly fibroblastic appearance and similar positive stainings for the markers CD 73, 90, 105, 106, 133, 144, 166 for both cell types. Following three weeks of differentiation culture, the bursa cells and MSCs revealed a strong chondrogenic, adipogenic and osteogenic differentiation potential, as shown by the respective histological, immunohistochemical and RT-PCR analyses. In contrast, the respective negative control cultures for the bursa cells and MSCs, which were maintained without any media supplements, were negative for the tissue specific markers.
Cells retrieved from subacromial bursa tissue reveal an almost similar surface antigen expression profile compared to marrow derived-MSCs and an almost equivalent multilineage mesenchymal differentiation potential for all lineages observed. Therefore bursa cells have to be considered adult multilineage progenitor cells. This knowledge might be used in order use these cells to augment of rotator cuff repairs. Global gene expression analyses (Affymetrix HG-U133_Plus) are underway to further characterize the stemness of the bursa-derived cells.
The success of surgical reconstruction of rotator cuff tears is limited due to the lack of ability to heal spontaneously, the degree of muscular atrophy, and the fatty degeneration. Injecting autologous myogenic progenitor cells or multipotent mesenchymal stem cells (MSCs) into the area of fatty infiltration could be a crucial factor in improving the so far unsatisfactory outcome of surgical reconstruction.
Myogenic progenitor cells and MSCs were isolated using protocols described by Blau & Webster (1981) and Noth et al. (2002), respectively. Both cell types were labelled with very small superparamagnetic iron oxide nanoparticles (VSOPs). The stability of labelling during cell expansion was determined with iron specific histochemical staining (Prussian blue). VSOP labelled, as well as non-labelled control cells were injected into a muscle specimen from a rabbit’s rotator cuff, and magnetic resonance imaging (MRI) was performed using a 7 T high-field MR spectrometer. In the MRI experiments 2D FLASH-sequences with echo times of TE = 8 ms and repetition times of TR = 689 ms were employed. A nominal spatial resolution of 137 x 137 μm2 was achieved with a slice thickness of 1 mm.
Intracellular uptake of iron oxide particles after incubation with VSOPs was shown with Prussian blue staining. Neither the myogenic progenitor cells nor the MSCs showed any loss of labelling within the first 6 cell divisions. Also, the proliferation capacity of both cell types was not influenced by the labelling. After the injection of VSOP labelled cells into a muscle specimen, the cells could be detected successfully with MR imaging. In a long term study, the VSOP labelled MSCs embedded in a tissue equivalent (collagen type I hydrogel) could be detected for more than 20 weeks using MRI.
Both, myogenic progenitor cells and MSCs were successfully labelled with iron oxide particles and used for MR cell tracking. Running experiments with the rotator-cuff defect model will show if the labelled cells will integrate themselves into the area of fatty degeneration and contribute to the regeneration of the muscle.
The non-invasive monitoring of cell behavior in vivo is still one major issue in cell-based therapies in orthopedic surgery. In particular, detailed information about the localization, proliferation, and differentiation of the transplanted cells in the target tissue is of utmost importance. In this study, magnetically labeled human mesenchymal stem cells (hMSCs) were embedded in collagen type I hydrogels and visualized in vitro using high-resolution magnetic resonance imaging (MRI).
Very small superparamagnetic iron oxide nanoparticles (VSOPs) were used to label hMSCs. Particle incorporation was demonstrated using fluorescent dye-labeled VSOPs. Stability of labeling during expansion of cells was determined with iron specific histological staining (Prussian blue). To assess whether the iron oxide particles are incorporated into the cells or only attached to the cellular surface, transmission electron microscopy (TEM) was performed. The influence of VSOP-labeling on the differentiation ability of hMSCs was determined histologically and on the mRNA level. For in vitro MR imaging, different concentrations of both magnetically labeled and unlabeled hMSCs were embedded in collagen type I hydrogels (Arthro Kinetics AG, Esslingen). MRI was performed at different time points using a Bruker 11.7 T MR spectrometer.
After incubation of hMSCs with VSOPs, fluorescent dye-labeled particles could be detected within the cells. This was confirmed with iron specific Prussian blue staining and TEM. During expansion in monolayer culture, a distinct reduction of VSOP-labeling appeared after 5 to 9 cell divisions, based on the dilution of label upon cell division. Compared to unlabeled cells, no inhibition of adipogenic, osteogenic, and chondrogenic differentiation of magnetically labeled hMSCs could be detected. Both, VSOP-labeled and unlabeled cells showed a specific matrix production and similar expression levels of according genes. After embedding of magnetically labeled hMSCs in collagen type I hydrogels, where no proliferation takes place, the cells could be detected with a MR spectrometer over at least 20 weeks. Controls with unlabeled cells were also visible in the MR images, but didn’t exhibit the typical signal intensity decrease conditioned by VSOP.
The results of our study show that the incorporation of VSOPs in hMSCs leads to an efficient cellular label with no adverse effect on their stem cell function. VSOP-labeled hMSCs can be successfully detected in a three-dimensional construct in vitro using high-field MRI. Further investigations have to reveal how long labeled cells can be visualized and monitored reliably in vivo.
To repair cartilage lesions, modern cell-based treatments focus on the employment of various cell types like Bone Marrow Stromal Cells (BMSCs) and chondrocytes. To be successful on the long term, stability of the cell phenotype in the generated cartilage is required. Recent studies show that besides BMSCs, chondrocytes are also multipotent. In this study we analyze the stability of the chondrogenic phenotype of BMSCs, and primary and dedifferentiated chondrocytes. To induce a chondrogenic phenotype the 3 cell types are pre-cultured in chondrogenic medium. To test the phenotypic stability of these cells an assay was used where chondrogenic medium was switched to adipogenic or control medium. Finally, we evaluate and compare the phenotype of the different cell types by RT-PCR and histology.
Human BMSCs were expanded for 3 passages. Primary chondrocytes from human knee joints were used immediately, or dedifferentiated by expansion for 2 passages in monolayer. The 3 cell types were pre-cultured for 10 days on chondrogenic medium, both in 2-d monolayer and 3-d alginate beads. For the following ten days cells remained on chondrogenic medium or switched to adipogenic or control medium (no chondrogenic factors). At day 20, histology and RT-PCR analysis were used to evaluate the effects of the medium switch of the cells.
Collagen 2 (COL2) expression confirmed that chondrogenesis was induced on chondrogenic medium in all cell types after 10 days and even more obviously after 20 days. When chondrogenic medium was switched to adipogenic, a tempering of chondrogenic phenotype (COL2) in all cell types was observed. Furthermore, primary and dedifferentiated chondrocytes showed decreased expression of the hypertrophic marker collagen 10 in the adipogenic medium, while BMSCs did not. Furthermore the switch to adipogenic medium gave elevated aP2 expression in all 3 cell types. This occurred most clearly in the BMSCs, which was confirmed by histology, as BMSCs were able to create lipid vacuoles when switched to adipogenic medium, while primary and dedifferentiated chondrocytes were not.
These results indicate that the chondrogenic phenotype is not stable after 10 days of chondrogenic culture. BMSCs seem less stable than primary and dedifferentiated chondrocytes. Future plans include creating a more stable cartilage phenotype in BMSCs, via switching experiments with a longer chondrogenic pre-culture time.
61 patients, mean age 48,4±16,3, were treated for ALS. Duration of the disease: up to 1 year ? 10 patients, 2 years ? 18, 3 years ? 29, over 3 years ? 4. Course of treatment envisaged administration of 4 cell suspensions containing mesenchymal, ectodermal, and endodermal stem cells obtained from growth zones of 4-8 weeks old cadaverous embryos’ systems and organs; amounts administered varied from 0,5 to 4 ml, cell count - from 0,1 to 100x105/ml. During the first course of treatment, 67% of patients reported decreased weakness, improved appetite, decreased fasciculations, decreased spasticity, and higher spirits. In the course of 2 months, 34% of patients observed increased range of motions in the extremities, decreased muscular rigidity, lower reflexes, decreased number of fasciculating muscle zones, better endurance of daily loads, less expressed dysphagia and dysarthria. In 3-6 months, 58% of patients reported aggravation of condition and reversion of relevant symptoms. II treatment was performed in 24 cases, III course ? in 8 cases, IV course ? in 3 cases.
Embryonic stem cells do not terminate degenerative process, but provide for palliation of ALS course.
The best results were achieved in the patients with up to 1 year ALS history.
Treated and observed (2-8 years) were patients (14 women, 10 men) with 2-3-year history of relapsing-remitting multiple sclerosis, mean age being 31.2±3.8, duration of remissions ?3.4±1.2 months. Most often, observed were pyramidal and sensory disturbances, and ophtalmological symptoms. Indications for stem cell treatment were as follows: ineffective medicinal therapy, Interferon intolerance, development of the new foci; aggravation of neurological symptoms. For treatment, used were embryonic stem cell suspensions (ESCS) containing stem cells of mesenchymal and ectodermal origin obtained from active growth zones of 4-8 weeks old embryonic cadavers’ organs. Suspensions were administered in the amount of 1-3 ml, cell count being 0.1-100x105/ml. In the course of treatment, applied were 2-4 different suspensions, mode of administration being intracavitary, intravenous, and subcutaneous. After treatment, syndrome of early post-transplant improvement was observed in 70% of patients, its main manifestations being decreased weakness, improved appetite and mood, decreased depression. In the course of first post-treatment months, positive dynamics was observed in the following aspects: nystagmus, convergence disturbances, spasticity, and coordination. In such symptoms as dysarthria, dysphagia, and ataxia, positive changes occurred at much slower rate. In general, the treatment resulted in improved range and quality of motions in the extremities, normalized muscle tone, decreased fatigue and general weakness, and improved quality of life. Forth, 87% of patients reported no exacerbations, no aggravation of neurological symptoms, and no further progression of disability. MRI performed in 1-2 years after the initial treatment, showed considerable subsidence of focal lesions, mean by 31%, subsidence of gadolinium enhanced lesions by 48%; T2-weighted images showed marked decrease of the foci’s relative density.
The subgranular zone of the hippocampal dentate gyrus and the subventricular zone adult of the adult mammalian brain harbour neural stem cells (adult NSCs), which provide a lifelong source for neurons and glial cells. To elucidate their potential usage in neuroregenerative medicine, several technical problems need to be solved, including the development of optimized protocols for cell expansion and differentiation.
rNSC-1b, a subclone of an NSC cell line established from the brain of an adult rat, was grown in defined media containing a serum supplement and growth factor cocktails. Cocktails of the growth factors EGF, bFGF, PDGF and LIF and neurotrophic factors, as well as several growth matrices were analyzed for their effects on cell expansion and differentiation. Immunocytochemistry, RT-PCR and Western blot analysis were performed to monitor gene expression during expansion and differentiation of rNSC-1.
Underlining their neural stem cell character, rNSC-1 cells co-express Nestin, GFAP and S100, as well as Oct4 and Sox2. Moreover, rNSC-1 cells express the pro-neuronal factors HES5 and prox-1 reflecting their commitment to the neural lineage. In addition, they express REST, an inhibitor of the neuronal phenotype which parallels the fact that neuronally differentiating cells are absent in rNSC-1 cultures at this stage. During expansion rNSC-1b cultures exhibit a high proliferation rate and preferentially grow as neurospheres. Adherent growth can be triggered by polyornithin, matrix proteins such as fibronectin, laminin or Matrigel, but also by the application of cyclic AMP or FCS. Presence of increasing concentrations of FCS resulted in increasing numbers of cells that differentiated into astroglia. Adherent cell growth per se, however, did not induce neural differentiation. Presence of bFGF, but not of the other growth factors was absolutely required for rNSC-1 expansion. Neural differentiation could be efficiently induced by addition of a mix of cAMP and retinoic acid and led to the generation of cells expressing glial or neuronal markers.
Stable in vitro expansion of adult rat stem cells committed to the neural lineage required the presence of bFGF but not of EGF, PDGF or LIF. Neural differentiation was inducible by exposure to cAMP/retinoic acid and resulted in the production of neuronal and glial derivatives
Dental follicle precursor cells (DFPCs) are plastic adherent cells, which are capable of differentiation toward dental cells, like cementoblasts and periodontal ligament cells. These ectomesenchymal cells express neural stem cell markers Nestin and Notch-1 that specify DFPCs also as potential neural progenitor cells. We investigated the gene expression pattern of typical neural and glial cell markers in DFPCs and investigated their neural differentiation potential.
DFPCs were isolated from dental follicles and cultured until passage five. For different stimulations of neural differentiation a serum replacement medium (SRM) was used in combination with various mixtures of commercially available supplements B27, N2 and G5 and growth factors fibroblast growth factor (FGF)-2 and epidermal growth factor (EGF). Total RNA was isolated from DFPCs before and after neural differentiations. Gene expression was determined by quantitative reverse transcription (qRT)-PCR for neural and glial cell markers beta Tubulin III, Neurofilament, Nestin, MAP2 and GFAP.
Investigated neural and glial cell markers were found to be expressed in undifferentiated DFPCs. After induction of neural cell differentiation DFPCs became either long drawn out shaped cells or formed neurosphere-like structures after cell detachment from the cell culture surface. Gene expression increased for neural cell markers tested.
Previous studies have suggested that DFPCs may also be able to differentiate into neural prescusor cells. Here, we demonstrate that DFPCs express typical neural cell markers and differentiate toward neural like cells after treatment with SRM based cell culture media. This supports the earlier suggestions and shows that DFPCs indeed are capable of differentiation into neuroectodermal cells.
Observed were 12 patients, 7 men and 5 women (mean age ? 58±6.3), with clinical manifestations of metabolic syndrome. All patients presented stage I-II hypertension, carbohydrate metabolism disturbances: impaired glucose tolerance (IGT) ? 8 and mild type II diabetes mellitus (DM) (morning fasting hyperglycemia, aglycosuria) ? 3, with hyperinsulinemia and elevated C-Peptide (4.7±1.6 ng/ml). Lipid metabolism disturbances were reported in all the patients: elevated cholesterol, triglycerides, increased concentration of low density lipoproteids (LDL), very low density lipoproteids (VLDL), decreased concentration of high density lipoproteids (HDL), and abdominal obesity (BMI?30.8±1.6 kg/m2), waist ? relatively W-94.8±2.3, M-105.7±2.9 cm). Transplanted were embryonic hematopoietic and non-hematopoietic mesenchymal and endodermal stem cells obtained from germ layers of 4-8 weeks old cadaverous embryos’ internal organs and sorted thereafter. 1-3 ml of cell suspensions were administered intravenously, cell concentration ? 0.1-100x105/ml.
Patients were observed for 1-5 years. In 1-2 months, 83% of patients reported gradual BP decrease with subsequent stabilization in the course of the next 2-3 months ? 135-140/80-90 mm Hg, with parallel 1.5-2-fold decrease of hypertension medications. In 7-9 months, glucose tolerance test (glycemia level 2 hours after glucose load was 7.2±0.3 mmol/l) revealed the improvement of carbohydrate metabolism in IGT patients. Decrease of morning hyperglycemia in type II DM patients usually started in 2-3 month, normalization was observed in 5-7 months. C-Peptide concentration gradually decreased, beginning from after 2-3 months, and normalized within 6-9 months. Normalization of blood lipids was reported in 75% of patient after 9-12 months, with parallel decrease of triglycerides, cholesterol, LDL, VLDL, and HDL increase.
Medical imaging ideally serves a 2 fold purpose in the application of cell therapies. It facilitates the delivery of the cells and allows monitoring of the therapy over time. Practically many problems need to be still overcome. For example direct cell labeling affects the proliferation of the cells. Immunoresponse limits usually the survival of cells and thereby the efficiency of the therapy. Protective Microcapsules avoid direct cell labeling and provide immunoprotection. In our study we examined the imaging properties of Fe or Flourine labeled Microcapsules.
Imaging was performed on a standard MAGNETOM Avanto with 1.5T and a MAGNETOM Trio with 3T field strength. Microcapsules of ~300μm size were synthesized and loaded with 80.9 ± 4.9ng Fe per capsule. Alternatively a second sample of Microcapsules where labeled with Fluorine. The Fe loaded capsules were embedded in a grid shaped Gel phantom starting with different concentrations. The Fluorine labeled capsules were embedded in Gel tubes. Imaging was done with standard balanced Gradient Echo sequences (TrueFISP) and off Resonance Imaging approaches. For Fluorine imaging simple Tx/Rx loop coils with 4cm and 8cm diameter for 1.5 and respectively 3T have been used.
Both types of capsules could be imaged without significant problems. Iron labeling allowed the detection of a single capsule. The fluorine label permitted detection of a cluster of 6 capsules. S/N with Fe labels was significantly higher. Encapsulation of pancreatic islet sustained viability of the cells equally to pure cells. The porosity of the capsules allowed the cells to take up their normal function of insulin production.
This work demonstrates the feasibility of new concepts for cellular therapies based on image guidance and therapy monitoring. The approach has the potential to overcome major limitations of cell therapies.
Adult stem cells are investigated as alternative source for beta-cell replacement therapy. So far, no consistent differentiation capacity for insulin producing cells has been shown in human bone marrow-derived mesenchymal stem cells (hMSC). Here we investigated the ability of hMSC-TERT cell line to differentiate into insulin-producing cells under regulation of hNgn3 and hPdx-1.
hMSC-Tert endocrine progenitor potential was analysed. Subsequently, stably overexpressing hNgn3 and/or hPDX-1 cell lines were generated (hMSC-TN, hMSC-TP and hMSC-TN/P). Islet gene regulation and protein synthesis were analysed by RT-PCR, Western Blotting, reporter gene assays and immunocytochemistry. Insulin content and secretion were evaluated by ELISA.
hMSC-Tert expressed progenitor cell markers, nestin and c-met and displayed pancreatic endocrine gene expression under specific culture conditions. Generated cell lines highly overexpressed the ectopic genes along with regulation of multiple islet genes, including insulin. In hMSC-Tert, Ngn3 induced expression of endogenous Pdx-1. hMSC-TP revealed direct activation of insulin gene. Coexpression of Ngn3 and Pdx-1 did not show synergistic effect on insulin expression efficiency. Insulin was expressed, produced and stored under regulation of hNgn3 and/or Pdx-1. However, no glucose dependent insulin secretion was observed in these cells.
In a human system of MSCs: introduction of key endocrine transcription factors is able to induce differentiation towards insulin-producing phenotypes; hNgn3 is able to trigger pancreatic endocrine differentiation cascade, lying upstream of Pdx-1; higher endocrine maturation must be achieved, in order to obtain functional hMSC that are suitable to the cell-based therapy of type1 diabetes.
Emerging evidence suggests that Erythropoietin (EPO) protects the myocardium from ischemic injury and promotes beneficial remodelling. However, the role of EPO and its receptor (EPO-R) in mediating cardiac regeneration remains unclear. We hypothese that stem cell homing and proliferation modulated by EPO could contribute to its cardio-protective effects. After permanent left ventricular myocardial infarction (MI), Epoetin-α (3000 U/kg) was injected along the infarction border. At six weeks after MI, cardiac functionality was measured by pressure-volume loops in left and right ventricles. Infarction size, angiogenesis and pathologic effects were evaluated. Gene expressions of EPO-R, SDF-1, CXCR-4, c-kit, eNOS, TNF-α, IL-8, Integrin-β and CdK4 were analyzed by RT-PCR at different time points of the first week (24h, 48h, 96h and 7 days). Our findings indicated improved left ventricular function both at baseline levels and under Dobutamine stress (dp/dt maximum and minimum, tau, cardiac output, stroke work, ejection fraction n=11-14, p<0.05) and decreased right ventricular wall stress (maximum and endsystolic pressure n=5-8, p<0.05). Infarction size was reduced from 27.8±3.4% to 20.1±2.8% (n=6-8, p<0.01). Capillary density was enhanced from 257.7±24.5 to 338.5±35.9 (vessels per square mm, n=6-8, p<0.05). Mortality was decreased from 29.0% to 22.2% (n=53-69). No thrombosis was observed in the intramural myocardium. EPO-R was down regulated in infarcted, peri-infarcted and non infarcted areas at all time points (n=7, p<0.05). Cardiac SDF-1?, CXCR-4 and eNOS expressions were increased at 24 hours. C-kit was up regulated significantly at 48 hours compared to 24 hours in the EPO treated hearts. In untreated hearts, c-kit expression remained constant. Proinflammatory cytokines (TNF-α, IL-8 and Integrin-β)? were down regulated. Cell cycle re-entry marker (CdK4) was increased at 24 hours in non infarcted zones. In conclusion, we demonstrate intramyocardium Epoetin-α injection induces an earlier up regulation of stem cell chemoattractants, reduces inflammation, enhances angiogenesis and restores heart function after MI.
Clinical studies suggest that transplantation of total bone marrow (BM) after myocardial infarction (MI) is feasible and potentially effective. However, focusing on a defined BM-derived stem cell type may enable a more specific and optimized treatment. Multilineage differentiation potential makes BM-derived multipotent adult progenitor cells (MAPCs) a promising stem cell pool for regenerative purposes. We analyzed the cardioregenerative potential of human MAPCs in a murine model of myocardial infarction.
Human MAPCs were selected by negative depletion of CD45+/Glycophorin+ BM cells and plated on fibronectin coated dishes. In vitro, stem cells were analyzed by RT-PCR. In vivo, we transplanted hMAPCs (5x105) by intramyocardial injection after MI in immunodeficient SCID beige mice. 6 and 30 days after the surgical procedure, pressure volume relationships were investigated in vivo. Heart tissues were analyzed immunhistochemically.
RT-PCR experiments on early hMAPCs passages evidenced an expression of Oct 4, a stem cell marker indicating pluripotency. In later passages, cardiac markers (Nkx2.5, GATA4, MLC-2v, MLC-2a, ANP, TnT, TnI, Cx 43) and smooth muscle cell markers (SMA, SM22α) were expressed. Transplantation of hMAPC into the ischemic border zone after MI resulted in an improved cardiac function at day 6 (EF 26 vs. 20%) and day 30 (EF 30 vs. 23%). Co-localisation of hMAPC marker vimentin and SMA in immunhistochemistry demonstrated that hMAPCs integrate into vessels in the peri-infarct region and show smooth-muscle-like characteristics. Furthermore, engrafted hMAPCs formed cardiomyocyte-like cells expressing cardiac proteins Nkx 2.5 and MHC. The proliferation marker Ki67 was absent in immunohistochemistry, and teratoma formation was not found indicating no tumorous potential of transplanted hMAPCs in the tumor-sensitive immunodeficient SCID model.
Transplantation of human MAPCs after MI ameliorates myocardial function, which may be explained by hMAPCs` potential to integrate into vessels and myocardial structures in the borderzone and form smooth-muscle-cell-like as well as cardiomyocyte-like cells. Lack of evidence of tumorous potential in the tumor-sensitive SCID model indicates, that hMAPC may deliver not only an effective, but also safe stem cell pool for the treatment of MI.
Heart failure is a leading cause of morbidity and mortality worldwide. Coronary artery disease (CAD) is currently one of the most common identifiable cause of heart failure. Mortality after myocardial infarction depends on the time when reperfusion of the infarct-related artery is performed. If this is not done rapidly, necrosis, scar formation and left ventricular remodelling occur. Today, cell-based therapy either by direct application or mobilisation has gained a lot of interest and is believed to improve left ventricular function after myocardial infarction.
We screen various available patent data bases to give an overview on different patent applications of cell-based therapies in cardiovascular medicine.
The first patents presented are about application of stem cells for heart failure or for inducing neoangiogenesis. Patents have been filed that differ between stem cell therapy alone or stem cell therapy in combination with other factors such as growth hormones. Stem cell therapy alone mostly consists of various steps: 1) isolation of cells from a patient. This is done by muscle or bone marrow biopsy, lipoaspirate or taking circulating blood from the patient. 2) Cultivation and/or expansion of the favoured cell type. These include lineage negative (Lin-) cells (Freyman, US7097833), adipose tissue-derived stem cells (Strem, MX5009044A), bone marrow-derived stem cells (Furcht et al., US7015037) or others. 3) Transplantation of the cell type. This is solved either by direct injection into the specific organ or intravenous administration. Stem cell therapy in combination with other factors requires an additional step. In this setup, accessory factors, like growth hormones (PDGF, FGF, PDGF) or other factors like adrenomedulin are given either to the isolated cells or are directly given to the patient. Other authors are using the administration of growth hormones or other factors like nicotine or nicotine-receptor agonists alone to mobilize stem cells from the bone marrow (Cooke et al., US6720340). We also present an overview about current patent applications of cardiomyocytes generation from embryonic or adult stem cells. Various cell types can be isolated and used to generate myocytes, e.g. cells from the endometrial membrane of the uterus or mesenchymal stem cells obtained from menstrual blood (Umezawa et al., WO06078034A1), TVEM-expanded CD34+/CD38- cells from the peripheral blood (Rudd, WO 06093860A3) or myogenic stem cells from adipose tissue (Geng et al., WO06017320A3). In addition, mesenchymal or myogenic stem cells used for myocyte generation need to be taken into culture before administration to the patient. Culture conditions for cardiac cells differ from those for other cell types. Isolated cells are cultured, e.g. in rotating bioreactor vessels (Rudd, WO 06093860A3) or engrafted on a histoengineered scaffold material of thermoplastic resin having a porous three-dimensional network and application of a pulsatile flow (Nakayama et al., JP2006246770A2). After a certain period of cultivation, myoblasts are then re-transplanted into the patient. We finally report patents in the field of cell culture methods for human stem cells that are used for generation of different types of cardiovascular cells.
A systematic overview on the current patent situation about use of stem cells in cardiovascular medicine enables optimized future decision making in the development of novel strategies for improved regenerative medicine.
Tissue engineering of cardiac tissue requires the concerted action of appropriate scaffolding biomaterials and functional cardiomyocytes together with a well-developed vascular network to ensure adequate oxygenation. In this study we describe the origin and characterization of a progenitor like cell that is present in cardiomyocyte cultures after isolation of the cardiomyocyte population of neonatal murine hearts.
Cardiomyocytes (CM) were cultured from enzymatically digested neonatal murine hearts after depletion of the majority of fibroblasts by adherence to plastic. The cardiomyocyte enriched cell population was characterized by immunohistochemistry, FACS analysis and transmission electron microscopy (TEM). Cell proliferation was measured by quantitating BrdU incorporation. CD31+ cells were isolated/depleted by magnetic cell sorting (MACS).
Freshly isolated CM enriched cells contained 90% cells positive for the cardiac markers troponin and α-actinin, and 10% cells positive for CD31, which is commonly expressed on endothelial cells. During culture of the CM-enriched cells, cobblestone areas were observed. Due to high proliferative activity of the cobblestone forming cells, these cobblestone areas increased rapidly in size and number after 7 days of culture. Further characterization of these areas showed that they were positive for CD31, and partly positive for uptake of DiI acetylated LDL. This indicated an endothelial phenotype of the cobblestone forming cells. Remarkably, TEM of the cobblestone areas also showed the presence of progenitor like cells and immature cardiomyocytes. Depletion of CD31+ cells resulted in a strong decrease in the number of cobblestone areas after culture, which implies that the cobblestone areas originate from CD31+ cells or a CD31+ subpopulation.
Overall, these data show that the neonatal heart contains a CD31+ cobblestone forming cell that may have the capacity to differentiate into both endothelial cells and cardiomyocytes. Therefore, these cells may have clinical potential in cardiac regeneration.
Loss of cardiomyocytes during myocardial infarction leads to decreased heart function. Replacing the injured myocardium with a muscle tissue created in vitro has gained increasing attention. Yet, the optimal cell source to engineer a functional and contractile cardiac tissue has to be defined. In this regards, mesenchymal bone marrow stem cells (MSCs) present numerous advantages: easy accessibility, large expansion capacity, high plasticity and aptitude to differentiate into cardiomyocytes. In the present study we investigated different strategies to induce the differentiation of rat MSCs into cardiac-like cells.
Isolated from tibial and femoral rat bone marrow, MSCs were separated from hematopoietic stem cells by their capacity to adhere to plastic. MSCs were cultured for 14 days with 20% FBS and FACS analysis were performed. To induce their differentiation into cardiomyocytes, different strategies were evaluated: (1) chemical treatments with 5-azacytidin, TSA or ascorbic acid; (2) culture with cardiomyocytes (CM)-conditioned medium. Phenotype characterization was evaluated by immunostaining for specific cardiac proteins: cTnT, cMHC, alpha-actinin sarcomeric and titin, and by RT-PCR for specific transcription factors (GATA4, Nkx2.5) and structural proteins (cTnT, beta-MHC).
After 14 days, at least 98% of the cell population was positive for CD90, CD29, and CD44, negative for CD109, CD31, and about 40% were positive for CD45. Cells rapidly spread in culture and punctuated localization of alpha-actinin was detected by immunofluorescence in pseudopods and at the periphery of spreading cells. We detected an increased gene expression of cTnT, beta-MHC, desmin and Nkx2.5 after 4 weeks compared to initially seeded cells. There was no difference between non-treated cells, chemical and conditioned medium treatments. This expression decreased after 12 weeks. The immunostaining revealed only a weak presence of related proteins in a small number of cells.
Our results showed that cardiac differentiation of MSCs was initiated independently of the treatments and concerned only a sub-population of the heterogeneous MSCs population. Further investigation will be undertaken to define this sub-population with cardiac progenitor characteristics. Differentiation of cells toward cardiocytes was mainly observed at the gene levels. Additional stimuli such as mechanical stretch may be necessary to promote further cell differentiation and maturation.
Ischemic heart disease as the most frequent cardiovascular disease still implies a major health burden to western countries. In a certain amount of patients, conventional revascularization therapy is unsuccessful leaving these patients with disabling angina pectoris or congestive heart failure. Theoretically combining TMLR for signalling induction and pain relief and stem cell injection for regeneration support offers a promising treatment strategy for there otherwise intractable patient suffering from ischemic heart disease.
Five patients with TML and stem cell therapy were evaluated. The pelvic bone was punctured and 300 ml of crude bone marrow were aspirated. Patients were operated “off pump”. Between 11 and 25 laser channels were created. In between the channels the stem cells were applied with 6 to 12 injections. Cell selection was carried out using magnetic cell separation. The patient follow up was 14.2±6.9 (mean+SEM).
NYAH classification improved significantly between preoperatively (mean 3.4) and three (mean 1.,8) and six month (mean 1.8) postoperatively. When patients were asked to rank their complains on a scale from 1 to 10, all patients described an immediate improvement postoperatively (7.4 preoperatively, 3.6 at three month and 3.2 at six month). An improvement in left ventricular ejection fraction was as well observed (48.8±13 preoperatively, 59.6±13 at three month (p=0.028) and 61.6±17 at six month (p=0.038 to preoperatively). The effect on LVEDV varied between the patients and did not reach statistic significance.
In summery, the presented investigation demonstrated an increase in LVEF and better clinical performance by intramyocardial CD 133+ stem cells injection and TML therapy. The reason remains uncertain and may be explained by paracrine mechanism. It is still uncertain how long the observed effect will last, or if the better cardiac performance will lead to better life expectancy.
Bone marrow derived stem cells either transplanted or mobilised by cytokines improve cardiac function after myocardial infarction (MI). Besides of its classical function in erythropoiesis recent reports have shown additional effects of EPO like antiapoptotic effects and stem cell mobilisation. These effects are known to improve myocardial regeneration after MI. Therefore, we analysed in a murine model of surgically induced MI the influence of EPO treatment on survival, functional parameters as well as stem cell mobilisation and homing. Methods: Human EPO (3000 IE/kg) was injected intraperitoneally immediately after ligation of the left anterior descendens (LAD) as well as on the two consecutive days (1000 IE/kg) ? a dose which did not significantly affect erythropoiesis. 6 and 30 days after the surgical procedure, pressure volume relationships were investigated in vivo. Cardiac tissues were further analysed by histology. To show the impact on stem cell mobilisation and homing as well as serum cytokine levels FACS and ELISA was performed.
EPO treated animals showed a significant improvement of survival post MI (62% vs. 36%). FACS data demonstrated mobilisation of CD31, c-kit and Sca-1 positive stem cells and homing of Sca-1 and CXCR4 positive stem cells was enhanced after EPO treatment. Serum levels of G-CSF were significantly increased after EPO administration, whereas SDF-1 levels were decreased and VEGF remained unchanged. Histology of EPO treated hearts showed less reduction of LV wall thickness and a smaller size of infarction at day 30 (22% vs. 42%). In addition, myocardial function of PTH treated mice was improved (EF: 23% vs. 15%) and elasticity demonstrated a less degree of infarct remodelling (Elasticity: 21.0 mmHg/μl vs. 8.1 mmHg/μl).
We have shown that EPO application after MI ameliorates myocardial function. This may be explained - beside direct effects via the EPO receptor - by mobilisation and homing of bone marrow-derived stem cells and a change in the serum cytokine pattern, which may lead to improved neovascularization and cell survival. Therefore, EPO treatment presents a promising non-invasive approach to ameliorate heart failure post MI.
Aortic aneurysm is a common disease among elderly people and is usually found in patients with bicuspid aortic valve (BAV) a decade earlier compared with those having normal aortic valve. So far, only the surgical therapy of aortic aneurysm is available and the need for preventative measures is evident. This study investigated the differentially expressed markers in vascular smooth muscle cells (VSMCs) isolated from diseased aorta.
Diseased aorta removed directly after surgery treated over night with 0.5% collagenase solution and resuspended in smooth muscle cell growth medium. Dissolved cells grown at 37°C in an atmosphere of 5% CO2 in air. 25cm2 cell culture flasks with 8x106cells/ml were harvested and were spreaded on chamber slides for 24h. Immunohistochemical techniques were used to detect alpha- smooth muscle actin, beta-tubulin, tie-2, VEGF R1, and glial fibrillary acidic protein (GFAP).
There was 2 to 3-fold decrease in the GFAP in aneurysmatic tissues compared to nonaneurysmatic (p = 0.002). However, the expression of alpha- smooth muscle actin was increased in aneurysmatic tissues (4.8±0.5 vs 6.9±1.2, p = 0.002)
Stem cell markers were expressed in diseased aorta, the potential strategies to promote the regression of thoracic aneurysm will be here discussed.
PR domain containing 4 (PRDM4) is the human and mouse homolog of rat Schwann cell factor 1 (SC1) and belongs to the PRDM gene family of transcriptional repressors. The PR domain defines a family of transcription factors involved in cell differentiation and tumorgenesis. SC1/PRDM4 is a cytoplasmic interaction partner of the p75NTR receptor, and it is activated by ligand bindung to this receptor. It shows a differential cytoplasmic and nuclear distribution, and its presence in the nucleus correlates strongly with inhibition of mitosis. It has been shown that SC1/PRDM4 acts as a transcriptional repressor which requires the presence of its zinc finger domains. The PR domain and zinc finger domains are also necessary to direct SC1’s nuclear localization. Last, SC1/PRDM4 represses the promoter of a promitotic gene, cyclin E, suggesting a mechanism for how growth arrest is regulated by SC1/PRDM4.
NSCs obtained from mouse forebrain embryos at E12 were grown as free-floating neurospheres, dissociated by mechanical trituration and subsequently plated on rV polyornithin-laminin-coated cover slides. Chimeric lentiviral vector plasmid pGJ3p containing SC1/PRDM4 cDNA and the expression plasmid VSV-G were transfected into 293T cells for virus production. The supernatant containing different lentiviruses was applied to NSCs. For PRDM4/SC1 siRNA, short RNA hairpins were cloned into the self-inactivating lentiviral vector pSIH1-H1-copGFP shRNA.
Overexpression of SC1/PRDM4 in mouse embryonic neural stem cells inhibits mitosis and induces apoptosis. Absence of p75NTR did not protect NSCs from apoptosis caused by SC1/PRDM4 overexpression. Apoptosis cannot be prevented by neurotrophic factor signalling, but it is absent in Bax-/- neural stem cells. Knockdown of SC1/PRDM4 expression by siRNA lentiviruses in NSCs significantly increases the number of pH3 positive cells, thus providing further evidence for the role of SC1/PRDM4 in the regulation of cell cycle. PRDM4/SC1-siRNA treatment also modulated the number of arrested cells with apoptotic nuclei in comparison to control snapshot treatment.
Our data suggest that SC1/PRDM4 acts as a key regulator of neural stem cells differentiation and mediates neurotrophin effects via p75NTR and possibly also other signals that determine cell cycle fate during neural differentiation In addition, these data provide evidence for the molecular basis of a link between cell cycle exit and cell death in neural differentiation. Next steps will be to investigate SC1/PRDM4 function in mouse models for further exploration of its role in neural stem cell renewal and neurogenesis in the peripheral and central nervous system.
Closure of burn wound is the primary requirement in order to reduce morbidity and mortality that are otherwise very high due to non-availability of permanent wound covering materials. Sheets of cultured epidermis grown from autologous epidermal keratinocyte stem cells are accepted world over as one of the best wound covering materials. In a largely populated country like ours where burn casualties occur more frequently due to inadequate safety practices, there is a need for indigenous research inputs to develop such methodologies. The technique to culturing epidermal sheets in vitro involves the basic Reheinwald-Green method with our own beneficial inputs. The technique employs attenuated 3T3 cells as feeders for propagating keratinocyte stem cells that are isolated from the epidermis of an initial skin biopsy of about 5 cm2 from the patient. The cultures are then maintained in Dulbecco’s modified Eagle’s medium strengthened with Ham’s F12 formula, bovine fetal serum and various specific growth-promoting agents and factors in culture flasks under standard culture conditions. The primary cultures thus established would be serially passaged to achieve the required expansion. Our major inputs are into the establishment of (1) an efficient differential trypsinization protocol to isolate large number epidermal keratinocytes from the skin biopsy, (2) a highly specific, unique and foolproof attenuation protocol for 3T3 cells and (3) a specialized and significant decontamination protocol. The fully formed epidermal sheet as verified by immuno-histochemical and light & electron microscopic studies, is lifted on to paraffin gauze by incubating in a neutral protease. The graft is then ready to be transported to the operating theatre for autologous application. We have a capability of growing cultured epidermal sheets sufficient enough to cover 40 per cent burn wound in 28 days. The preliminary small area clinical applications undertaken so far revealed quicker healing proving the importance and usefulness of the method.
With this new approach a large number of moderate to severely burned patients could be saved in several burn centers across our country with reduced hospitalization period. However, the cell based therapeutic option in burn-wound healing by the application of
With this aim, we are presently attempting to create such a scaffold using Mebiol gel, which could support the cultured epidermis for better transfer to the wound bed. Additionally, the usefulness of Mebiol gel in growing epidermal sheets without the necessity of FBS and/or animal origin feeder cells but using human feeder cells will also be tested.
Oral submucous fibrosis is a progressive oral disease first described by Pindborg and Sirsat 3 decades ago. It is a premalignant condition. The signs and symptoms depend on the involvement of the different sites in the oral cavity. The patient feels burning sensation for normal diet and trismus which may be so severe. If not properly treated the risk of malignant change in advanced cases of OSMF is relatively high. Wide ranges of treatment such as medical management, surgical therapy and physiotherapy have been attempted in the past, but none of them has proved to be a cure for this chronic fibrotic disease.
Histopathologically as the disease progresses, (i) change in the morphology of collagen, (ii) increased accumulation of amorphous collagen, and (iii) decreased collagen degradation results in decrease in number of blood vessels are observed in the affected area compared to the normal area. With an aim of bringing more blood supply to the affected area which is expected to bring more nutrients and help in collagen degradation, earlier application of vasodilators and studies with curcumin have been done, but still with - no significant outcome.
As an alternative approach to improve the blood circulation, we have tried Autologous bone marrow stem cells which have been earlier applied in several diseases such as ischemic peripheral vascular diseases, ischemic heart diseases etc with proven improvement in angiogenesis.
A 38 year old patient with oral submucosal fibrosis, proven by histopathology, and endothelial markers was injected 175 million BMMNCs into the area affected. The paramaters such as blanching, fibrous band have significantly improved, 4 weeks after the injection. We could observe positive changes clinically to prove the improvement. The mouth opening has improved to 35 mm from the previous 30.0 mm. Further histopathology and SEM studies with larger samples are done for establishing stem cell therapy’s safety and efficacy.
Articular cartilage, the load-bearing tissue in diarthrodial joints, when damaged due to trauma could lead to osteoarthritis. At present Autologous Cartilage Implantation is an established method in which patients own chondrocytes are isolated and then implanted after in vitro expansion over the affected area with bovine or porcine collagen matrix. This procedure results in more of Collagen Type I during in vitro expansion, which eventually becomes fibrocartilage. Also it requires growth factors. We have in this study tried growing human Chondrocytes without growth factors using synthetic scaffolds to grow more Collagen Type II
Human cartilage specimens were harvested through arthroscopy from the non-weight bearing area of the knee joint from 13 patients who underwent surgical procedures of the knee joint after getting their informed consent. The tissues were transported in saline taking 1 hour to laboratory and subjected to digestion with Collagenase type II for 16~18 Hrs. The chondrocyte cells obtained after dissociation were divided into two groups for culture. Gr. I were embedded in a Thermogelation polymer (TGP) and Gr. II in basal culture media (DMEM + Ascorbic Acid) without using any growth factors. The Group II cells were viable only for 4 weeks and then started degenerating. The TGP-Chondrocytes scaffolds were grown for 16 weeks and the specimens were harvested at 4, 8, 12 and 16-week intervals and their morphology and molecular characteristics were studied by H&E staining, S-100 protein analysis and RT-PCR.
Human chondrocytes could be cultured in both TGP (group I) and Basal culture media (group II). The Gr. I cells were viable upto the 16th week while the Group II chondrocytes started degenerating after the 4 week. Both the groups were proven positive for S-100 protein, a Chondrocyte specific marker protein; Gr. II specimens after 4 weeks, and Gr. I specimens after 4, 8, 12 and 16 weeks. RT-PCR study of the cells of group I were positive for TGF beta 3 (Proliferation, differentiation, and other functions), GR beta, GR alpha (Development, metabolism and immune Response) (glucocorticoid receptor alpha), AGGF (Apoptosis), VDR (Vitamin D3 Receptor), Col II (Type II Collagen).
We have established a methodology by which Human chondrocytes could be cultured in vitro without any growth factors for a period of 16 weeks in a polymer-hydrogel scaffold. Upon further confirmation of their characteristics, the TGP grown chondrocytes can be used for autologous implantation to repair damaged cartilage area as the Collagen Type II which grows better without growth factors in the scaffold, eventually will become Hyaline cartilage is expected to give a longer disease free duration than the present method of ACI.
We studied 100 patients with Spinal Cord injury (SCI) after Autologous Stem cell Injection in the Spinal fluid with a Follow up of 6 months post Stem cell injection. There were 69 males and 31 females; age ranging from 8 years to 55 years. Time after Spinal Injury ranged from 11 years - 3 months (Average: 4.5 years). The Level of Injury ranged from Upper Thoracic (T1-T7) - 34 pts, Lower thoracic (T7-T12) -45 pts, Lumbar -12, Cervical-9 pts. All patients had an MRI Scan, urodynamic study and SSEP (somatosensory Evoked Potential) tests before and 3 months after Stem cell Injection.
80% of patients had Grade 0 power in the Lower limbs and rest had grade 1-2 power before stem cell injections. 70% of cases had complete lack of Bladder control and 95% had reduced detrusor function.
We Extracted CD34 and CD 133 marked Stem cells from 100 ml of Bone marrow Aspirate using Ficoll Gradient method with Cell counting done using flowcytometry.15 ml of the Stem cell concentrate was injected into the Lumbar spinal fluid in aseptic conditions. The CD 34/CD45 counts ranged from 120-400 million cells in the total volume.
6 months after Injection, 8 patients had more than 2 grades of Motor power improvement, 3 are able to walk with support. 1 patient with T12/L1 injury was able to walk without support. 12 had sensory tactile and Pain perception improvement and 8 had objective improvement in bladder control and Bladder Muscle contractility. A total of 18 patients had reported or observed improvement in Neurological status. 85% of patients who had motor Improvement had Lesions below T8. MRI, SSEP and Urodynamic Study data are gathered at regular intervals.
This study shows that Quantitative and qualitative Improvement in the Neurological status of paralyzed patients after Spinal cord injury is possible after autologous bone marrow Stem cell Injections in select patients. There was no report of Allodynia indicating the safety of the procedure. Further studies to (i) quantify the neurological and vascular damage and to standardize the dosage, (ii) Identify Mechanism of action of each group of Stem cells
Liver Cirrhosis is the end stage of chronic liver disease which may happen due to alcoholism, viral infections due to Hepatitis B, Hepatitis C viruses and is difficult to treat. Liver transplantation is the only available definitive treatment which is marred by lack of donors, post operative complications such as rejection and high cost. Autologous bone marrow stem cells have shown a lot of promise in earlier reported animal studies and clinical trials. We have in this study administered in 22 patients with chronic liver disease, autologous bone marrow stem cell whose results are presented herewith.
In 22 patients with chronic liver diseases of Child B-C category, Autologous Stem Cell has been transfused, upon getting approval from the ethics committee and informed consent. Under short GA, 200-300ml of bone marrow was tapped. The bone marrow stem cells were isolated using density gradient fractionation method and processed, suspended as per protocols earlier published (Terai et al., doi:10.1634/stemcells.2005-0542).The processing was done in cGMP facility under stringent aseptic precautions. Endotoxin test clearance was obtained (<0.25EU) and CD34+ analysis was performed using FACS. A cell count of 240 to 1068 X 106 was administered intravenously through the median cubital vein. Liver function tests, ultrasound and CT were performed before the administration, thereafter at 4 and 8 weeks of infusion. The protocols used were the same as used by Terai et al., Yamaguchi University, Japan.? Acites, albumin, bilirubin, radio lucency of liver and overall quality of life were studied in all these patients. Liver biopsies were not done due to lack of patient compliance. Standard work up of chronic liver disease by viral marker, copper, Alfa Feto Protein etc., were performed.
The administration of the transfusion did not have any adverse reactions in the patients. 67% of patients showed an increase of serum albumin that was significant, 73% showed significant reduction of ascites, 32% showed drop in bilirubin. The overall quality of life of index (QULI) was significantly improved in 82% of the patients.
Autologous bone marrow stem cells administered in chronic liver disease patients has yielded significantly good results and has been safe. Although a pilot clinical trial, the study shows promises for newer exciting cell based therapies for chronic liver disease.
Patients with end stage heart failure have very few treatment options. The long waiting times for transplant and the complications associated with immunosuppression has led to the search for alternatives. Subsequent to the isolation and characterization of stem cells, tremendous advances have been made and the safety and feasibility of autologous bone marrow derived stem cells has been proven in preclinical studies. Clinical studies have also shown mobilized cells repair the infracted heart, improving function and survival. We have started a clinical study to evaluate the efficacy of bone marrow derived stem cells. Bone-marrow was aspirated from the right iliac crest and the stem cells were isolated by density gradient method and suspended according to the mode of delivery.
From Jan 2007 till date 10 patients (8 adults, 2 children, age) with end stage cardiovascular disorder of varied etiology (Ischemic left ventricular dysfunction - 6 patients, Primary pulmonary hypertension - 2 patients, Dilated cardiomyopathy -1 patient, Biventricular non-compaction -1 patient) underwent stem cell therapy. All patients were evaluated and cardiac function was measured by using echocardiography and thallium scintigraphy. There were no procedure related complications. These patients are being regularly followed-up and one patient who has completed 6-month follow-up has shown improvement in perfusion as well as increase in ejection fraction of 10%. Stem cell therapy in patients with end-stage cardiovascular disorder might be a promising tool by means of angiogenesis and other paracrine mechanisms.
Nuclear study images of one of the patients included in the study. The upper one before and lower one six months after the injection.
The Ejection fraction was 16% (Pre-treatment image on top) which increased to 22% after Autologous Bone Marrow Stem Cell Therapy (Post-treatment-bottom image) according to the author.
Nanotechnology is the manipulation or self-assembly of individual atoms, molecules, or molecular clusters into structures to create materials and devices with new or vastly different properties. Nanotechnology can work from the top down (which means reducing the size to the nanoscale e.g. marble block to a statue or the bottom up (which involves manipulating individual atoms and molecules into nanostructures ie. we add matter till we end with the final or the desired product. Nanotechnology has been described as the new industrial revolution and both developed and developing countries are investing heavily in this technology to secure a market share.
The economy of the future will be a “knowledge-based economy” and maximizing knowledge through education, research and innovation will be important goals. Nanotechnology has a vast potential to revolutionize the critical sectors of the economy viz. health and agriculture: the subjects chosen for this talk. New tools for the treatment of diseases, disease detection, smart sensors and smart delivery systems for both mankind and agriculture are being developed and it will help to combat the viruses and other pathogens that effect both humans and the agriculture sector. It is expected that In the near future it will become possible through nanotechnology to increase the efficiency of drugs and pesticides and yet using lower doses. In the recent years applications of nanoscale biotechnology to food systems have been identified and are being pursued. Some of the studies are directed towards developing nano-sensing technology for detecting trace components in food, preserving food nutrients for an extended term and developing technology for preserving functional components without negatively impacting the palatability of food.
The talk will cover what is nanobiotechnology, why we study nanobiotechnology and how to create the nanoengineered materials and what we can expect to get out of nanobiotechnology. The work in some of these areas being carried out at the Indian Institute of Chemcical Technology will also be covered.
In recent years regenerative biology has reached to greater heights due to its therapeutic potential in treating degenerative diseases; as they are not curable by modern medicine. With the advent of research in stem cells and developmental biology the regenerative potential of adult resident stem cells is becoming clearer. The long term objective of regenerative medicine or cell therapy is to treat patients with their own stem cells. These stem cells could be derived from the diseased organs such as skin, liver, pancreas etc. or from reservoirs of multipotent stem cells such as bone marrow or cord blood.
Manipulating the ability of tissue resident stem cells as well as from multipotent reservoirs such as bone marrow, umbilical cord and cord blood to give rise to endocrine cells may open new avenues in the treatment of diabetes. A better understanding of stem cell biology would almost certainly allow for the establishment of efficient and reliable cell transplantation experimental programs in the clinic. We show here that multipotent mesenchymal stem cells can be isolated from various sources such as the bone marrow, placenta, umbilical cord. Upon stimulation with specific growth factors they differentiate into islet like clusters (ILCs). When ILCs obtained from the above mentioned sources were transplanted in experimental diabetic mice, restoration of normoglycemia was observed within three weeks of transplantation with concomitant increase in the body weight. These euglycemic mice exhibited normal glucose tolerance test indicating normal utilization of glucose.
Allthough the MSCs isolated from all the sources had the same characteristics; they showed significant differences in their islet differentiation potential. ILCs isolated for the human bone marrow did not show any pancreatic hormones in vitro, but upon transplantation they matured into insulin and somatostatin producing hormones. Placental MSCs as well as ILCs showed insulin trascripts indicating their readiness towards islet lineage.
These studies point towards futuristic therapeutic approach of auto-transplantation of bone marrow for diabetes. Our studies demonstrate that human bone marrow, umbilical cord and placenta have the potential to differentiate into islets. These alternative sources of stem cells for islet neogenesis will form the basis for generating large number of islets required for transplantation for diabetes reversal.
Since 2007, we had started clinical trial using mesenchymal stem cell (MSCs) for the treatment of aseptic bone necrosis as a first clinical trial permitted by Japanese Health, Labour and Welfare Ministry.
Aseptic bone necrosis of the femoral head commonly occurs in patients with two to four decades, causing severe musculoskeletal disability. Although its diagnosis is easy with X-ray and MRI, there has been no gold standard invented for treatment of this disease. MSCs represent a stem cell population in adult tissues that can be isolated and expanded in culture, and differentiate into cells with different nature. Combination with β-tri-calcium phosphate and vascularized bone graft, we succeeded to treat bone necrosis of the femoral head.
Regenerative medicine using stem cells is hopeful and shed a light on intractable disease. To become widespread, Basic, Translational, Application, and Developmental study is needed.? From an experience of cell therapy using MSCs, we started to research induced pluripotent stem cell (iPS) for clinical application.
Autologous Immune Enhancement therapy (AIET) is one of the lesser used modality of therapy in our country for advanced cancers. AIET uses patient’s own immune cells such as NK cells and T-Lymphocytes to fight against cancer.
To study the effectiveness of AIET in Advanced Carcinoma of pancreas.
A 40 years old male was diagnosed with inoperable locally advanced Carcinoma of the head of pancreas when he was investigated for obstructive jaundice. Palliative bypass surgery was done, since his tumor was inoperable. Following his surgery he received 6 cycles of adjuvant chemotherapy and 3 cycles of AIET. After completion of chemotherapy and 2 cycles of AIET therapy his tumor marker decreased significantly with decrease in tumor size. So re-laparotomy was attempted for removal of tumor, which was unsuccessful. After a long gap he received his 3rd cycle of AIET. He was irregular in receiving his AIET treatment. He survived for 18 months after the diagnosis.
The median life span of patients following diagnosis of advanced Carcinoma of pancreas is usually not more than 3-6 months. Longer survival of our patient could be due to AIET, but we need further studies to prove it. There were no adverse effects encountered due to AIET.
Stem Cell Therapy (SCT) although in vogue since the early seventies for Hematological malignancies, is slowly evolving and extending into other fields. In Spinal Cord Injury its indications and uses are still getting refined.
In our centre, after extensive review of literature drew up our own inclusion/exclusion criteria which was approved by our ethics committee. We selected six patients with Dorsal Spinal Cord Injury for this study. Epidemiological and clinical data was collected and Videography of the patient’s initial presentation was done. Clinical assessment was carried out by a team of specialist including the Neurosurgeon, Neurologist, Urologist and Physiotherapist.100 ml of Bone marrow harvested from the posterior iliac crest was sent in cold preservation to NCRM, Chennai. Bone Marrow Mono Nuclear cells were isolated and CD34+ cells quantified using FACS. The isolated cells were injected through lumbar puncture. One of the six patients received two sittings of SCT, while all others have undergone only one sitting. The patients were followed on a monthly basis during which regular neurological reassessment was done and domiciliary physiotherapy was also given.
One patient who was given two sittings of SCT made very good improvement and is now ambulant with the aid of an Orthosis. Two other patients who received one sitting of SCT had objective sensory and mild motor improvement; Three other patients had no improvement. There were no adverse reactions in any of them.
SCT is an evolving option for Spinal Cord Injury patients and although it is too early to comment, results as of date show a positive influence. The numbers of variables are many, but the results are promising and definitely warrant further clinical trials.
The adult Bone Marrow Stem Cells (BMSCs) have distinct advantages over the other types of stem cells. They are multipotent, can be stored for upto 10 years and considered to be one of the best sources of hematopoietic and mesenchymal stem cells in an adult body. Genetically inherited diseases such as Retinitis Pigmentosa and Degenerative diseases such as Age Related Macular Degeneration remain unsolved as no definitive treatment is available to repair the damages caused to the RPE and Photoreceptors as of now. In this scenario, the technique of Bone Marrow aspiration & isolation of Mono Nucleated Cells (MNCs) & intra-vitreal injection of a very small volume of MNCs in human retinal disorders has been standardized and is safe and feasible for human studies (Mohanty
Ciliary Pigment Epithelium was harvested from donor eyes from Aditya Jyot Eye Hospital, Mumbai and was taken to and grown at NCRM lab. The cells were grown in the earlier reported methodology of Brenda
The CPE derived Retinal stem cells grew well in the lab. However, the practical difficulties of harvesting the same in patients limited our further steps in this study.
Cadaver eye RPE cells were harvested and grown using polymer scaffolds after transporting them over 6 to 12 Hrs. The RPEs were grown on conventional methods and in polymer scaffolds and were subjected to RT-PCR.
Human RPEs were able to grow without amniotic membrane and the same was proven by RT-PCR. This would make it possible for the peripheral RPEs taken from patients to be stored and later expanded and used for replacing the diseased cells of the central portion of the retina in future, without having to harvest the RPEs again.
Bone marrow mono nuclear cells were isolated and transported in cold containers (4-8oC) over a period of 6-12 Hrs and viability was evaluated.
The bone marrow mononuclear cells were viable up to 12 Hrs in our methodology with a viability of more than 95% making it possible for cells isolated from Chennai centre to be taken to Mumbai or any other destination within a reach of 12 Hrs for application as reported in earlier studies.
The in-vitro expansion of RPEs without Human Amniotic Membrane is expected to open up a new possibility for treating the Retinal Degenerative Diseases. However an animal study is needed before clinical application. Intra vitreal application of Bone Marrow Mono Nuclear cells to treat RP and AMD as reported earlier are considered safe. We plan to undertake treatment and long term follow-up of more numbers of patients with RP and AMD.
Ocular surface disorders due to limbal stem cell deficiency are an important cause of ocular morbidity and visual loss. Although autologous limbal stem cell transplants have helped in the management of unilateral disease, allografts in those with bilateral disease often fail due to immunological reasons. The use of autologous buccal epithelium cultivated on amniotic membrane has been described as a useful approach in the management of this condition. It is the purpose of this study to explore the feasibility of using a novel thermo-gelatin polymer (TGP) as a substrate to culture these cells, and to characterize them using RNA extraction and RT-PCR.
Oral cheek mucosal biopsies were obtained from 5 adult patients undergoing Modified Osteo-Odonto Keratoprosthesis surgery. The specimens were transported to the laboratory in transport medium. The cells were released using enzymatic digestion and seeded in both convention culture medium and TGP. The resulting cellular growth was characterized using RNA extraction and RT-PCR.
Cells could be cultured from 4 of the 5 specimens. In one specimen, contamination occurred and this was discarded. In the other specimens, the cheek epithelial cells could be cultured in both the conventional culture medium and TGP, with equal ease. RT-PCR revealed the presence of K3, a marker for epithelial cells, and GAPDH indicating the presence of some adipose tissue as well.
Buccal mucosal epithelial cells in culture on the 28th day in novel TGP synthtic scaffold
Histopathological examination and H&E staining of the cells grown in the method as shown above in Picture A
It is possible to culture autologous cheek mucosal epithelial cells using TGP, a synthetic scaffold, without the need for other biological substrates. Since the specimens are obtained from the oral cavity, stringent asepsis is required. Further studies are required for histopathological characterization of the cultured cells and to create a model for delivery onto the ocular surface of eyes with bilateral surface disease due to limbal stem cell deficiency.
Bullous Keratopathy (BK) affects thousands of people in India every year. Though in early stages it is manageable medically, advanced disease warrants either total corneal transplantation or partial thickness transplantation for which a donor-cadaver cornea is necessary. Amano et al have reported the successful treatment of BK in animal models using in-vitro expanded human corneal endothelial precursors; though the rabbits had to be kept facing eye down to allow gravity assisted settling of the cells to the summit of the cornea where the damage had been created. For successful treatment using the above method, a human being has to lie prone with eyes immobilized for 24-36 Hrs. This is extremely discomforting and hence not practical. Corneal endothelium removed from the button and transported at varying temperature conditions for 48Hrs was successfully cultured in NCRM and this was reported earlier. We are working on a suitable scaffold to retain the cells in situ until their attachment to the damaged portion of the corneal endothelium enabling it to heal without the patient having to lie prone. With such capability, we envisage to make a corneal endothelial precursor/stem cell (CES) bank named as CESBANK to make in-vitro expanded CES available for patients with corneal diseases, most commonly Bullous Keratopathy (BK).
The CESBANK will have a (i) Total ophthalmology diagnosis clinic, (ii) Operation theater and an out-patient clinic equipped for handling corneal procedures, (iii) An in-patient ward for post-CES transplantation patients, (iv) A cGMP cell screening, serology, immunology & molecular characterization lab, (v) Cell processing, expansion and cryopreservation lab, (vi) An eye bank, (vii) Documentation & networking facility (viii) Teaching accessories with lecture rooms, web & telecon capability (ix) A world class faculty for clinical & research projects. Earlier proven methodologies of corneal endothelial harvesting will be undertaken in CESBANK along with the collaborating hospitals. Studies comparing presently available scaffolds with novel nanomaterials for the application of CES into the affected portion of the eye will be undertaken. Long term cryopreservation and transportation to and fro from longer distances will be standardized to enlarge the network.
Ninety thousand patients are in the backlog every year, waiting for corneal transplantation in India, of which 30,000 may benefit from CESBANK. The CESBANK, as per the present plan, would be able to provide expanded CES for at least 14000 eyes to be treated every year when fully functional, provided it gets adequate number of donor eyes.
The present project when implemented could make one donor eye be usable to more than 5 to 14 recipient eyes. By increasing the awareness for eye donation, more patients with corneal endothelial diseases awaiting donor cornea could be treated.
The prevalence of acquired hearing loss is very high. About 10% of the total population and more than one third of the population over 65 years suffer from debilitating hearing loss. The most common type of hearing loss in adults is idiopathic sudden sensorineural hearing loss (ISSHL). In the majority of cases, ISSHL is permanent and typically associated with loss of sensory hair cells in the organ of Corti. Following the loss of sensory hair cells, the auditory neurons undergo secondary degeneration. Sensory hair cells and auditory neurons do not regenerate throughout life, and loss of these cells is irreversible and cumulative.
However, recent advances in stem cell biology have gained hope that stem cell therapy comes closer to regenerating sensory hair cells in humans. A major advance in the prospects for the use of stem cells to restore normal hearing comes with the recent discovery that hair cells can be generated ex vivo from embryonic stem (ES) cells, adult inner ear stem cells and neural stem cells. Furthermore, there is increasing evidence that stem cells can promote damaged cell repair in part by secreting diffusible molecules such as growth factors. These results suggest that stem-cell-based treatment regimens can be applicable to the damaged inner ear as future clinical applications.
Previously we have established an animal model of cochlear ischemia in gerbils and showed progressive hair cell loss up to 4 days after ischemia. Auditory brain stem response (ABR) recordings have demonstrated that this gerbil model displays severe deafness just after cochlear ischemia and gradually recovers thereafter. These pathological findings and clinical manifestations are reminiscent of ISSHL in humans. In this study, we have shown the effectiveness of stem cell therapy by using this animal model of ISSHL
Stem cells are undifferentiated cells that through replications have the capabilities of both self-renewal and differentiation into mature specialized cells. Broadly, there are two types of stem cells, embryonic stem cells and adult stem cells. Embryonic stem cell biology has been associated with ethical controversy and also their growth is difficult to control. Adult stem cells are located in tissues throughout the body and function as a reservoir to replace damaged or aging cells. Embryonic stem cells are by definitions, the master cells capable of differentiating into every type of cells either in-vitro or in-vivo. Several lines of evidence suggests, however, that adult stem cells and even terminally differentiated somatic cells under appropriate micro-environmental cues are able to be reprogrammed and contribute to a much wider spectrum of differentiated progeny than previously anticipated. Hematopoietic Stem Cells (HSCs), for example, from different sources have been shown to cross the tissue boundaries and give rise to the cells of the other germ layers.
In the past few years, the plasticity of adult cells in several post-natal tissues has attracted special attention in regenerative medicine. Stem cell therapies represent a new field of biomedical science which could provide in the future the cure for diseases until now considered incurable. The reconstitution of adult stem cells may be promising source for the regeneration of damaged tissues and for the resolution of organ dysfunction. However, there are two major limitations to the use of such cells:-
(i) They are rare and
(ii) Only a few types exist that can be isolated without harming the patient.
Due to the inability to efficiently and safely harvest or expand stem cells from most adult organs (e.g. liver, gastrointestinal tract, heart, brain), the majority of human stem cell trials have focused on clinical applications for HSCs, mesenchymal stem cells (MSCs), or both, which can be easily obtained in clinically sufficient numbers from peripheral blood, bone marrow, umbilical cord blood or placenta. HSCs can give rise to muscle, liver cells, astrocytes, etc, especially when co-cultured with the particular tissue progenitor cells and in presence of MSCs.
Allogeneic Stem Cell Transplantation (SCT):
Hematological Malignancies: CML, AML, ALL, NHL, HD, MDS.
Solid Tumors: Renal Cell Carcinoma, etc
Autologous Stem Cell Transplantation:
Hematological malignancies: AML, NHL, HD, MM
Solid Tumors: Neuroblastoma, EWS, RMS, etc
Non-Malignant conditions:
Hematological: Aplastic Anaemia, Fanconii Anaemia, Thalesemia, Sickle Cell Diseases, Myelofibrosis
Non Hematological: Osteopetrosis, Storage diseases (e.g. Gauchers)
Immune Disorders: SCID, HLH, etc.
Non-Malignant conditions:
Autoimmune Disorders: systemic sclerosis, multiple sclerosis, systemic lupus erythromatosis, juvenile idiopathic arthritis.
Heart Disease: HSCs expanded ex-vivo (especially after B-catenin treatment) have been found useful in the treatment of ischemic myocardial injury.
CNS: HSCs have been used in the treatment of ischemic stroke, spinal cord injury and neurological disease like Parkinson’s disease.
Diabetes Mellitus:-
Gastro-intestinal & liver diseases:-
Cirrhosis end stage liver disease
Acute Liver failure
Metabolic liver diseases
Severe Inflammatory bowel disease, Crohn’s disease.
Patients with refractory celiac disease
Kidney disease: following acute renal injury - tubular injury or glomerular injury.
Skin repair & regeneration
Generation of normal tissues: Potential therapeutic use of ex-vivo produced blood substitutes such as:
RBCs
Platelets
The outcome of HSCTs can be improved by various manipulations.
A. Before HSCT:
Expansion of HSCs using:
1. Culture with various cocktails: SCF + TPO + Flt-3l; IL-3 (promotes higher CD133 cell expansion) + IL-6 (maintains immature phenotype); G-CSF / GM-CSF + IL-3 + SCF, etc
2. cMPL agonist NR-101
3. Fetal liver stromal cells
4. MSCs: provide suitable cellular environment for in-vitro expansion of HSC & HPCs from umbilical cord blood.
5. Valproic acid; T-hoxb4-H, etc Expansion should favor cell proliferation over cell differentiation.
B. During HSCT:
Enhance engraftment by using:
1. Mesenchymal stem cell infusions
2. Valproic acid
3. Double / Triple cord blood transplants
C. After HSCT: Infusion of:
1. Dendritic Cells
2. T-Regs: for modulation autoimmune disease or for transplant tolerization; derived from CB-HPCs
3. NK-Cells
MSCs have low inherent immunogenicity. Sources: Bone marrow; Wharton’s Jelly umbilical cord.
1. Enhancement of hematopoietic engraftment provide the supportive micro-environmental niche for HSC
2. Prevention / suppression of GVHD: MSCs exert an immediate anti-inflammatory & immunomodulatory role; cause induction of transplant tolerance
3. MSCs home to damaged tissue to participate in regenerative process; rebuild diseased tissues along with HSCs
4. Foster engraftment of haploidentical HSCs
5. Cell therapy & tissue engineering
Though HSCs have been used in all the above indications under laboratory condition, in animal studies, or even in humans, there is lot that still needs to be done before HCTs become standard of care.
A pubmed search for HSCT & India yielded only 87 publications. Though the first Allogenic BMT was done at TMH long back in 1983, in the last 25 years hardly 2000 transplants have been performed in a country of over a billion population. This is compared to nearly 4000 transplants done each year world over. As of now, there are approximately 20 units doing SCT in India, the main among them being CMC, Vellore; TMH, Mumbai & AIIMS, Delhi. Apart from these centers, two other centers which have consistently published are, Stem Cell Biology Laboratory of National Institute of Immunology, Delhi & Dr. H.L. Trivedi Institute of Transplantation Sciences, Ahmedabad. However, most of published work in this field from India is in the preliminary stages and it may take some time before the translational research reaches to the bedside.
The supply of transfusable red blood cells (RBCs) is not sufficient in many countries. If erythroid cell lines able to produce transfusable RBCs in vitro were established, they would be valuable resources. However, such cell lines have not been established.
We developed a robust method to obtain differentiated cell lines following the induction of hematopoietic differentiation of mouse embryonic stem (ES) cells and established five independent hematopoietic cell lines using the method. Three of these lines exhibited characteristics of erythroid cells. Although their precise characteristics varied, each of these lines could differentiate in vitro into more mature erythroid cells, including enucleated RBCs. Following transplantation of these erythroid cells into mice suffering from acute anemia, the cells proliferated transiently, subsequently differentiated into functional RBCs, and significantly ameliorated the acute anemia.
Considering the number of human ES and induced pluripotent stem (iPS) cell lines that have been established so far, the intensive testing of a number of these lines for erythroid potential may allow the establishment of human erythroid cell lines similar to the mouse erythroid cell lines. In addition, our results strongly suggest the possibility of establishing useful cell lines committed to specific lineages other than hematopoietic progenitors from human ES and iPS cells.
The Cell Engineering Division of RIKEN BioResource Center is a not-for-profit public “Cell Bank” that accepts donation and deposit of human and animal cell materials developed by life science research community. We examine, standardize, amplify, preserve, and provide cell materials to the scientists around the world.
The stem cells such as ES cells and iPS cells are valuable in current biology and medical sciences. Thus, we are collecting such stem cell lines and aiming to contribute to the fields of developmental biology, transplantation medicine, regenerative medicine, and so on
Ever since man invented fire he has been more frequently burning himself by this creation than by the naturally occurring bushfires. It is estimated that over 1.152 million people in India suffer from burn injuries requiring treatment every year and majority of them are women aged between 16-40 years and most of them occur in the kitchen.
The treatment for burns basically involves autologous skin grafting, which originated in India more than two thousand years ago (Sushruta Samhita), is still the gold standard for the wound resurfacing, although, autografting is difficult where graftable donor sites are limited. Although, Cadaver skin, porcine or bovine xenografts are used alternatively over the past thirty years, modern approaches like the Bioengineering of skin substitutes emerged during the past 20 years as advanced wound management technologies with no social impediment. They can be broadly categorized as Acellular and Cellular biotechnological products. The acellular products like Alloderm (LifeCell Corporation), Integra (Integra Life Sciences) act like template and depend on natural regeneration, while the cellular ones are either ‘Off-the-Shelf’ products like Apligraf (Organogenesis Inc) and Orcel (Ortec International) have allogenic elements and ‘home grown’ autologous cell products like Cultured Epithelial Autograft (CEA) and epidermal-dermal composite skin use synthetic or natural non-human matrices. The CEA is based on the ex-vivo epidermal stem cell-expansion and our laboratory has been engaged in CEA technique development with innovative cost-effective approach and yielded promising preliminary clinical success.
The basic methodological approach in CEA technique which is still clinically adopted by several developed countries involves the use of growth arrested mouse dermal fibroblasts as growth supportive matrix and is thus considered a drawback as a whole. Additionally, there is no superior enough method available to augment the growth of human keratinocyte stem cells capable of producing epithelia for large-scale grafting in burns and maintain long-term functionality as a self-renewing tissue. The normal functioning of such an in vitro constructed graft under long-term artificial growth conditions is limited by the difficulties of maintaining the epidermal stem cell compartment. An apparent answer to this problem of stem cell depletion during autograft preparation would be to start with a pure population of progenitor stem cells and derive sustainable autograft from them. We have been aiming to this solution and currently attempting to isolate a pool of epidermal progenitor cells using Mebiol gel, which is a Thermo-Reversible Gelation polymer and was shown by others to support the growth of multi-potent skin-derived epithelial progenitor-1 cells. Additionally, the usefulness of Mebiol gel in maintaining epidermal stem cell compartment without FBS and/or animal origin feeder cells is being investigated by our group
A three year old male non-descriptive companion dog was presented to the Small Animal Orthopedic Unit of Madras Veterinary College Teaching Hospital (MVC) with paraplegia of fourth degree neurological deficit of hind limbs due to automobile trauma. Radiographic views were suggestive of dislocation at T8-T9 vertebral segment with fracture of L2 vertebra. Myelography confirmed the signs of abrupt stoppage of the contrast column cranial to dislocated area and was interpretive of transected spinal cord at L2 level. Construct was prepared with bone marrow mononuclear cells (BMMNC) isolated from bone marrow aspirate of femur and the cells were seeded in Thermoreversible Gelatin Polymer (TGP) at the cell processing facility of Nichi-In Centre for Regenerative Medicine (NCRM) as per GMP protocols and was engrafted after hemilaminectomy and durotomy procedures in the MVC. Postoperatively the animal was clinically stable; however the animal died on the 7th day. Autopsy revealed co-morbid conditions like cystitis, nephritis and transmissible venereal tumor. Histopathology of the engrafted area revealed sustainability of aggregated stem cells that were transplanted revealing an ideal biocompatibility of the construct prepared with bone marrow mononuclear cells and polymer hydrogel for spinal cord regeneration in dogs. Further
studies in similar cases will have to be undertaken to prove the long term efficacy.
Bone marrow derived CD34+ cells have been in clinical application in patients with haematological malignancies. One of the major problems with this treatment is the non-availability of matched donors or the necessity of multiple transfusions depending upon the pathology. Recently evidences have been accumulating to prove the safety and efficacy of autologous CD34+ cells in diseases such as myocardial dysfunction, peripheral vascular diseases and neurological certain conditions. However there are only a few reports in the literature on ex vivo expansion of the bone marrow derived CD34+ cells. We have in two different studies proven that isolated CD34+ cells from baboon bone marrow and non-isolated BMMNCs from human bone marrow could be expanded with increase in percentage of CD34+ cells using a novel scaffold.
Study I: Bone marrow was derived from healthy baboons posterior iliac crest and mononuclear cells were isolated using density gradient method. Then the BMMNCs were subjected to magnetic bead (Miltenyi Biotech) separation of CD34+ cells, which were expanded ex vivo for one week.
Study 2: A portion of the bone marrow drawn for diagnosis or bone marrow aspirated for clinical application from human patients was used after informed consent. The BMMNCs were subjected to ex vivo expansion without any further separation. Initial and post-expansion CD34+ percentage and quantity were evaluated at different intervals starting from 7 to 21 days. Both the studies used the same Thermo-Gelation Reversible Polymer Scaffold impregnated with same culture cocktails prepared in NCRM.
Study1 revealed increase in the quantity of CD34+ cells which formed colonies in the culture from the 5th day onwards. The study 2 revealed a significant increase in the total quantity of CD34+ cells percentage increased from 0.91% to 2.26% on day 7 (n=4), 1.53% to 2.93% on day14 (n=1) and 1.32% to 11.71% (n=3) on day 21on an average quantified using flow cytometry.
This study has shown that ex vivo expansion of CD34+ cells with and without isolation is feasible. This technology may become a good tool to hematooncologists who may in need of a precious matched unit to be expanded multifold or may cryopreserve the donors’ bone marrow for subsequent application to same or similar patients. Patients and health adults who haven’t had an opportunity to store their cord blood also now have an option to store their own bone marrow cells in several small aliquots which could be thus expanded for future application when needed.
Use of autologous bone marrow stem cell is a newly evolving treatment modality for end stage cardiac failure as reported in the literature. We report our experience with two patients with dilated cardiomyopathy who underwent this treatment after failure of maximal conventional therapy.
A 29 year old Male patient with history of orthopnea and PND, with a diagnosis of dilated cardiomyopathy and echocardiographic evidence of severe LV dysfunction was referred for further treatment. His echo on admission showed EF of 17% and no other abnormal findings except elevated bilirubin levels. He was in NYHA functional class IV. He received intracoronary injection of autologous bone marrow stem cells in January 2009. 254X106 cells were injected with a CD34+ of 0.20%. His clinical condition stabilized and he was discharged home. He received a second injection of 22X106 in vitro expanded stem cells with a CD34+ of 0.72% in Aug 2009. He is now in NYHA class II-III with EF 24%.
A 31year old Male patient with history of increasing shortness of breath, severe over the past 3-4 days was admitted for evaluation and treatment. His echo on admission showed EF of 20% and was in NYHA functional class IV. Coronary angiogram was normal and he was stabilized on maximal anti failure measures. He received intracoronary autologous bone marrow stem cell injection of 56X106 with a CD34+ of 0.53% in August 2009. His clinical condition stabilized over the next 10 days and he was discharged home.
In our experience of two cases of dilated cardiomyopathy, safety of intracoronary injection of autologous bone marrow stem cells both isolated and in vitro expanded has been proven in both the cases with efficacy proven in one of the cases. Long term follow-up of these two cases and inclusion of more number of similar cases where all available conventional therapies have not resulted in significant improvement for such studies are planned.
A 66yrs old Diabetic and Hypertensive female, who had Anterior Wall MI 5yrs ago and had undergone PTCA with Stent to LAD, was admitted for refractory CHF with Severe LVD 2yrs ago and the LVEF then was 25%. Coronary Angiogram was done which showed Total Occlusion of LAD and 50% Stenosis of RCA.
100ml of her bone marrow was harvested from posterior iliac crest and the BMMNCs were isolated as per cGMP protocols at NCRM, Chennai and 325X106 cells with a CD34+ count of 0.84% were injected the next day by transfemoral catheter into the coronary arteries. Post treatment she had clinical improvement. EF increased by 5%. She was in Class-II for 1 year. After 1 yr, she was admitted with severe CHF and EF had deteriorated to 20%.
This time BMMNCs isolated from the bone marrow were subjected to in vitro expansion by which the initial 0.15% CD34+ cells increased by nearly 30 fold to 4.62%. Totally 315X106 cells were injected into the coronaries. Post treatment there is clinical as well as Echo evidence of improvement and BNP level has come down by 30%.
Isolated and expanded CD34+ cells from bone marrow mononuclear cells of autologous origin, administered into the coronaries in an Ischemic Cardiomyopathy patient has been proven to be safe. The clinical and Echo cardiographic improvement that has sustained for long-term, proves the feasibility and efficacy of two consecutive autologous bone marrow stem cell applications, one isolated and the second ex vivo expanded. More case studies may be undertaken to further evaluate the results
Heart and vascular endothelial cells from human pluripotent stem cells are of interest for applications in cell therapy and cardiovascular disease. Differentiation protocols are now sufficiently refined that production of cardiomyocytes, endothelial- and smooth muscle cells is fairly efficient and reproducible. Genetically marked hESCs have been produced in which expression of eGFP is ubiquitous or under lineage specific control. We have used various tagged hESC-lines to trace cardiomyocytes following transplantation into a mouse heart after myocardial infarction and to select cardiovascular progenitors as a source of the different cardiovascular cell types. Although cardiomyocytes survive for months in the mouse heart and cause early improvements in function, these are not sustained. Cardiovascular progenitors from hESC and hiPSC however, present more immediate applications in drug discovery, toxicity and cardiovascular disease modelling. Results of these studies, in particular drug responses of hESC-derived cardiomyocytes and a hiPSC model for vascular disease, will be shown.
Our group investigates the cell biological basis underlying the proliferation
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Reprogramming of mouse and human somatic cells to induced pluripotent stem (iPS) cells has been possible with retroviral expression of the pluripotency-associated transcription factors Oct4, Sox2, Nanog and Lin28 as well as Klf4 and c-Myc. Considering that ectopic expression of some of these factors can cause tumors and expression vectors themselves are potentially mutagenic due to insertion into the host genome, reducing the number of transgenes during the generation of iPS cells is a crucial step towards their clinical applicability as an alternative pluripotent stem cell source.
The initial studies were underdone inducing pluripotency in mouse and human fibroblasts. Based on the hypothesis, that in inducing pluripotency the number of reprogramming factors can be reduced when using somatic cells that endogenously express appropriate levels of the reprogramming transcription factors, we were able to demonstrate, that mouse and human neural stem cells (NSC), that express Sox2, Klf4 and c-Myc, can be reprogrammed to iPS cells by retroviral expression of Oct4 alone. Human Oct4 iPS cells resemble human embryonic stem cells in global gene expression profiles, epigenetic status, as well as pluripotency in vitro and in vivo.
Cord blood derived cells are attractive starting populations for reprogramming, since their DNA is relatively young and they present the perspective to generate HLA-matched pluripotent stem cell banks based on existing cord blood banks. Human cord blood derived somatic stem cells (USSC) can be reprogrammed to iPS cells. These cord blood iPS cells are highly similar to human embryonic stem cells morphologically, at the molecular level by gene expression microarrays, global miRNA and epigenetic profiling, as well as in their in vitro and in vivo differentiation potential.
We will describe the current status of our strategies to induce pluripotency from mouse and human somatic cells with a minimal number of defined transcription factors and small molecules for patient-specific disease modeling and cell therapeutic applications.
Intercellular exchange of protein and RNA-containing microparticles is an increasingly important mode of cell-cell communication. Microparticles, which include exosomes, micro-vesicles, apoptotic bodies and apoptotic microparticles, are small (50 - 400 nm in diameter), membranous vesicles that can contain DNA, RNA, miRNA, intracellular proteins and express extracellular surface markers from the parental cells.
Our primary aim is to elucidate the role of microparticles in damage induced neurogenesis following traumatic brain injury in vivo.
Cerebrospinal fluid was serially centrifuged. Pelleted microparticles were analysed by FACS, electron microscopy, RT-PCR and mass spectroscopy. Quantification of miRNA was performed by Small RNA Chip (Agilent Tech.). Specific miRNAs were identified by miRNA microarrays (Affymetrix).
We verified the presence of cerebral cell derived microparticles in cerebrospinal fluid of healthy volunteers and patients of TBI by FACS analysis and electron microscopy. Microparticles contained RNA, miRNA and protein. RNA was not susceptible to RNase digestion underlining RNA to be contained in protective vesicles. Approximately 50% of the RNA content was demonstrated to be pre- and miRNA. A variety of specific miRNA species indicated in the regulation of regenerative processes were identified. Furthermore, RT-PCR analysis revealed cerebral microparticles to carry transcripts for MAP2, ß-actin and CaMKII. MAP2 mRNA was not detected in cerebral microparticles of healthy controls. The presence of ß-actin and CaMKII in microparticles might indicate neuronal RNA granules to be also packed into microparticles. Proteomic analysis indicated microparticles to carry proteins involved in motility (ankyrin-3), membrane regulation (stabilin-2, synaptotagmin), neuronal development (growth factor independence-1), as well as typical neuronal receptors (glutamate receptor). Within the first three days following traumatic brain injury 14% ± 3% of microparticles in cerebrospinal fluid was CD133+, indicating a substantial fraction of microparticles to be derived from neuronal precursor cells.
We were able to demonstrate for the first time the release of stem cell derived microparticles into cerebrospinal fluid. The detection of specific RNA transcripts and pre/miRNA underlines their predicted role for microparticles in cell-cell communication. To our knowledge there are no reports to date on the role of microparticles in the cerebral environment. We are presently studying the role of microparticle in endogenous neurogenesis following cerebral injury.
In a previous study we have identified a novel murine gene (GenBank X83587) which was found to be the mouse homologue of human Co-REST, a co-repressor to the neuronal silencer REST (repressor element-1 silencing transcription factor). Co-REST mediates the transcriptional repression of REST-responsive genes by recruiting histone deacetylases and other chromatin modifying enzymes to the repression site. In recent studies a REST-independent role of Co-REST in gene repression has been documented. A major role of the REST/Co-REST repressor complex is to restrict neuron-specific gene expression in extra-neural tissue. Accordingly, the occurrence of REST and Co-REST in neural tissue at early developmental stages has raised much interest in a potential role of this repressor complex in the timing of neural stem cell maturation.
Although expression of Co-REST has been related to long-term gene silencing mechanisms, its role in stem cell biology is still debatable. In a previous study we have shown that, in contrast to REST, Co-REST expression persists in the developing mouse CNS beyond newborn stage and - at low levels - throughout adulthood.
In this project we have been focussing on the stage-specific expression of Co-REST in the mammalian olfactory epithelium (OE), which is known to harbour a pool of highly dynamic neurogenic cells (neural stem/precursor cells, NSCs/NPCs). Using immunohistochemistry and in situ hybridization we have determined the spatio-temporal expression pattern of Co-REST in the developing and adult mouse OE. Co-expression studies have been performed using antibodies against stem cell-related transcription factors, NPC-associated cytoplasmic proteins, and neuron-specific terminal differentiation markers.
Here we have shown that the stem cell-associated proteins Sox2 and Nestin are co-localized in the young OE, showing prominent staining in basal and apical regions. Interestingly, expression of Co-REST is detectable exclusively in Sox2/Nestin-free areas. On the other hand, a considerable overlap of Co-REST and Doublecortin expression is visible in the OE from E10.5 on, which provides evidence to suggest that Co-REST may be considered a reliable marker for early neurons or neural precursor cells but does not indicate stem cell quality of OE cells.
Early mammalian heart development is characterized by transient expression of alpha-smooth muscle actin (Acta2). To date, cardiomyocytes expressing Acta2 in the early stages of
Generation of cardiomyocytes from pluripotent stem cells provides a future perspective to overcome the complications of heart transplantation including in particular the shortage of donor organs. Thereby, stem cell therapy will require the availability of various cardiac cell types. Whereas early cardiovascular precursors appear to be important for novel approaches such as reseeding decellularized hearts, direct cell transplantation may require specified terminally differentiatied cells. Our previous work demonstrated that MesP1 represents a master regulator sufficient to induce cardiovasculogenesis in pluripotent cells. Yet, a profound comprehension of the developmental processes as well as the molecular background is fundamental in order to specifically generate these cell types.
To gain further insights into MesP1 driven cardiovasculogenesis we initially compared global mRNA expression patterns of MesP1 overexpressing cells with control cells relying on the
Based on CD4 expression magnetic cell sorting (MACS) of MesP1 positive cells will be utilized for deep sequencing (RNA-Seq) and transplantation approaches.
Human adult pancreatic (parotic) stem cells were brought in a co-culture with human myocardium. The origin of the resulting toponin- I -positive cardiomyocyte-like cells was considered as unclear. Were do they derive from: adult stem cells or the added myocardium? To clarify their origin, sex-chromosomal analyses were to perform.
Male adult stem cells were harvested from pancreatic (parotic) tissue of patients undergoing operative procedures due to pancreatic (parotic) but not maligne diseases of femal patients (n=6). The cells were selected, cultured and passaged. Simultaneously with a troponin-I-staining, a Fluorescence In Situ Hybridization (FISH) is performed to evaluate the X and Y chromosome signals present in each cell. Human myocardial biopsies for co-cultures were taken from female patients.
We could show by simultaneously applied immunocytochemistry for troponin-I and FISH that human adult stem cells from pancreas and parotis with a positive immunocytochemistry for troponin-I differentiated into cardiomyocyte-like cells which were male (XY) likewise the applied glandular stem cells.
The differentiation of human adult pancreatic and parotic stem cells enhanced by a cardiomyocyte coculture is reliably proven now. These glandular stem cells might become a clinically relevant autologous source of regenerative tissue for the repair of irreversible damaged myocardium.
The purpose of this study was to determined optimal dose of BMP4 and its time period on differentiation Promordial Germ Cells (PGCs) from mouse Embryonic Stem Cells (mESCs) in vitro.
To differentiation of PGCs, EBs from mouse ES cells were cultured in concentrations 0, 5, 10 and 50 ng/ml BMP4 at the different time interval. Viability of PGCs was assayed by MTT. Also, germ cell markers Oct-4, Stella and Mvh were analyzed by flow cytometry, Immunocytochemistry, electrophoresis gel and Real Time PCR.
BMP4 at 10 ng/ml concentrations for day 4 had the best on the viability. The data of flowcytometry demonstrated most of Mvh+ cells was observed in D4B10. Immunocytochemistry of EBs in D2B5, D2B10, D4B5 and D4B10 groups identified cells that are positive to Mvh. The result of Real time PCR was illustrated that expression of gene Oct-4 in the control groups was high and it gradually decreased with adding concentrations 5 ng/ml and 10 ng/ml of BMP4 on days 2nd, 4th and 6th. The maximum expression of Stella was observed in D4B10 group. However, the expression of Stella with BMP4 concentration of 50 ng/ml on the day 2nd, 4th and 6th considerably were decreased. The expression of Mvh with BMP4 concentrations of 0 ng/ml and 50 ng/ml was none. However, the expression of Mvh were increased in D2B5, D2B10, D4B5 and D4B10 groups (not significantly).
The results propose that concentrations of 10 ng/ml BMP4 at days 4th had the optimal effects on differentiation of PGCs to mESCs.
Previously, we reported the establishment of multipotent adult germline stem cells (maGSCs) from mouse testis. Similar to mouse embryonic stem cells (ESCs), these cells are able to self-renew and differentiate into derivatives of all three germ layers. These properties make maGSCs a potential cell source for the treatment of disease such as diabetes or liver cirrhosis. In this study, we describe the efficient generation of maGSC-derived hepatic-like cells by two differentiation systems using maGSCs and ESCs. Induction into the hepatic lineage through embryoid body formation resulted in a gradually decrease of undifferentiated Oct3/4-and Nanog-positiv cells during differentiation in contrast to an increase of endoderm-and hepatocyte-specific markers. To receive a higher amount of hepatic progenitors and mature hepatocytes we established a co-culture differentiation protocol using OP9 stromal cells with activin A. By reverse transcription polymerase chain reaction and immunofluorescence we show that OP9 co-culture, and furthermore activin A treatment increased and accelerated the expression of endodermal and liver-specific genes and proteins such as SOX17, HNF4, AFP, AAT, TTR, and ALB. Flow cytometry analysis resulted in 51% AFP-, 61% DLK-1-, and 26% ALB-positive maGSCs-derived hepatic like cells at late differentiation stages. Furthermore, these maGSC-derived hepatic-like cells successfully demonstrated in vitro functions associated with mature hepatocytes including albumin-and urea-secretion, glycogen storage, uptake of acetylated low density lipoprotein (LDL), and uptake and release of indocyanine green (ICG). Taken together, these data show that maGSCs show similar endodermal differentiation capabilities as ESCs and make them to a potential autologous and alternative source for pluripotent stem cells in regenerative medicine.
Organ regeneration with stem cells requires a delicate balance between the loss of donor cell pluripotency (so that teratoma formation is avoided) and retention of donor cell proliferative capacity prior to terminal differentiation (so that a sufficient number of target cells are generate to effect a therapeutic benefit). The aim of this study is to generate proliferating cardiovascular progenitors (Flk1+ cells) from pluripotent stem cells derived from spermatogonial stem cells (SSCs) of double transgenic mice (MHC-neo/ MHC-EGFP) and to investigate their heart regeneration potential. We have generated four SSC lines with MHC-Neo-EGFP+ background from adult male mice. The SSC culture can be expanded over one year in vitro in the presence of glial cell line-derived neutrotrophic factor (GDNF) required for the maintenance of SSC self-renewal. The established SSC culture show typical SSC morphology and express SSC-specific markers like DAZL, VASA and GFR1a at both mRNA and protein levels. Furthermore, these SSCs from the MHC-Neo-EGFP+ mice were reprogrammed into pluripotent stem cells by overexpression of Oct4 alone. The reprogrammed cells showed similar characteristics as multipotent adult germline stem cells and are positive for pluripotency markers such as Oct4, Nanog, Sox2 and SSEA-1. They were able to spontaneously differentiate into cells of all embryonic germ layers in vitro by using the hanging drop method. The GFP+ beating cardiomyocytes can be selected by G418 treatment. After transplantation of these cells in SCID-beige mice, teratomas were detected 6 weeks later. In addition, we established a protocol to induce these cells to differentiate into Flk1+ cardiovascular progenitors. After coculture of these cells (n = 30000 per 10 cm dish) with OP9 cells for 6-days, about 25-35% of the cells were positive for Flk1. This efficiency is comparable to those derived from multipotent adult germline stem cells. For investigating the regenerative potential of these cells we used a myocardial infarction mouse model. We transplanted 500000 Flk1+ cells per mouse in an infarcted heart. At different time points after cell transplantation (2 days, 2 weeks, 4 weeks and 8 weeks) we took the hearts out and analysed them to identify the Flk1+ cells and their derivatives. Our first preliminary data showed that the transplanted cells survived in the infarcted heart. Investigations on the integration of the cells into the host myocardium and on the functional improvement by echocardiography are in progress.
Hematopoietic stem cells (HSCs) can either self renew or give rise to multipotent hematopoietic progenitor cells (HPCs). As the most primitive hematopoietic cells both cell types obtain the ability to reconstitute all mature blood cell types. According to the classical model of hematopoiesis, these HPCs next become either restricted to the lymphoid or to the myeloid lineage, the common lymphoid (CLPs) or common myeloid progenitor (CMPs) cells. Via more restricted HPCs, CLPs then finally give rise to T, B and natural killer (NK) cells as well as to a subset of dendritic cells (DCs), while CMPs create macrophages, granulocytes, megakaryocytes and erythrocytes as well as a second subtype of DCs.
Due to the recent characterization of HPCs containing partial myeloid and partial lymphoid developmental potentials the classical model of hematopoiesis has been challenged. A bundle of new data suggests the existence of additional or alternative developmental pathways.
Aiming to set up a functional
Since we are still interested in an
Cardiomyocytes derived from human pluripotent stem cells possess a high potential for regenerative treatment of cardiovascular diseases as well as for drug screening and drug safety tests in pharmaceutical industry. However, one of the main obstacles to overcome towards
Our aim is to systematically test and assess combinations of transgenic transcription factors for their cardiogenic potential in human pluripotent stem cells.
To this end, an optimized electroporation protocol was used to simultaneously transfect up to three different non-viral expression plasmids into hiPS or hES cells. Combining this technique with antibiotic selection allowed the establishment of transgenic cell clones. Various clones with different transgene expression levels, as tested by qPCR, were then differentiated and assessed for cardiac markers. In a second approach, hiPS cells were transiently transfected by electroporation, seeded as a monolayer and directly subjected to differentiation in a fully defined serum-free medium.
Notch signaling is a pivotal mechanism throughout development, controlling cell lineage decisions in a cell-context dependent manner. Notch ligand binding, located on a neighboring cell, results in the consecutive cleavage of the Notch receptor by an ADAM protease and γ-secretase, followed by the release of the intracellular domain of Notch (NIC), which translocates then into the nucleus and converts the RBP-J repressor into an activator of Notch target genes. Although the cell-context dependency has been proposed for a long time, little is known about which target genes are affected in which cell type and how the cell-context dependency is mediated mechanistically. Here, we employed a Tamoxifen-inducible Notch1 system in embryonic stem cells at different stages of mesodermal differentiation combined with genome-wide transcriptome analysis and qPCR verification. Furthermore, using this system we were able to screen for potential direct targets using the protein synthesis inhibitor cycloheximide. Our results revealed that a large number of the identified target genes are unique for the cell type and vary highly in dependence of other signals. Among the potential direct Notch1 targets are, in addition to the known direct Notch1 target genes of the Hes and Hey family, key regulatory transcription factors such as Sox9, Pax6, Runx1, Myf5 and Id proteins that are critically involved in lineage decisions [
Potential therapeutic applications of embryonic stem cell(ESC)-derived hepatocytes as an alternative to the transplantation of the whole liver or of primary hepatocytes is currently confirmed to be a highly topic issue. Crucial points thereby are high-yield generation and efficient selection of cells displaying hepatocyte-specific features from differentiating ESC cultures. In our study, we establish culture conditions for a large-scale production of hepatocyte-like cells from ESCs in a murine in-vitro model, using stable transgenic ESC clones which contain the live eGFP reporter gene and a puromycin resistance cassette, both driven by a common alpha-fetoprotein(AFP) gene promoter. From that clones, suspension-and adherent cultures with the activated AFP promoter were generated, which derived from the endoderm-like cell population. These cultures are supposed to express eGFP fluorescence as well as to acquire puromycin resistance, thus allowing for live monitoring of both differentiation and antibiotic selection.
We found out that a 95%-yield of eGFP-expressing embryoid bodies(EBs) can be achieved by using a spinner flask suspension culture. This culture seems to be temporally synchronised in terms of the rate of eGFP-positive cells and of their localization pattern in the outer rim of EBs. Features of cells selected from the EB culture by puromycin application were shown to be dependent on the time point of the initial drug application. Thus, the puromycin treatment of EBs on their relatively early developmental stage yielded highly eGFP-expressing cell clusters that, after their plating onto adhesive substrates, developed to a culture comprising both highly proliferative hepatocyte-precursor-like- and advanced differentiated hepatocyte-like cells. Exposure of a long-term adherent culture to the drug resulted rather in selection of a significantly more mature cell population displaying a low proliferation capacity. We also investigate the effect of different adhesive substrates on the developmental pattern of adherent cultures.
Hence, alteration of culture conditions allows for differentiation of murine ESCs toward cells of the hepatic lineage and their selection on defined developmental stages, which is of interest in regard to toxicology screening- and transplantation models.
Cytosine arabinoside (cytarabine) is an antimetabolic agent, which damages DNA when the cells cycle holds in S phase Cytarabine is mainly used in the treatment of AML and lymphomas. It has been reported to cause severe neuropsychiatric side effects in human patients under going therapy for AML. Cytarabine is also reported to be used in the study of nervous system. In the present study, we have investigated the effect of cytarabine on human embryonic stem cells (hESCs) derived randomly differentiated embryoid bodies. Cytarabine, being a developmental toxicant, exhibited cytotoxicity at high concentrations, while sub-lethal concentration of cytarabine was found to stimulate neuronal markers such as PAX6 and MAP2. In addition, low concentration of cytarabine also caused inhibition of developmental markers. To find the significant toxicity markers microarray analysis was performed with Ilumina HumanHT-12 v3 Expression BeadChip, revealing cytarabine promoted axon guidance and other pathways known to be involved in the regulation of neurogenesis in hESC embryoid bodies. In addition 124 upregulated and 416 down regulated significant genes (p ≤ 0.05) were observed in cytarabine treated EBs compared with untreated. The significantly (p ≤ 0.01) upregulated annotations for cytarabine treatment in the GO biological process relate to neuronal differentiation like neural morphogenesis, axonogenesis, nervous system development etc. In our work, it is shown that cytarabine can stimulate pathways and markers for neuronal differentiation and inhibit developmental markers.
The differentiation of human embryonic stem cells (hESC) into multiple organotypic cells has great potential in developmental biology and regenerative medicine. In recent years mouse embryonic stem cells were used for in vitro toxicity studies, in particular developmental toxicity studies. We combined hESCs with toxicogenomics for the construction of a developmental toxicity platform. hESCs were randomly differentiated into multiple lineages such as endoderm, ectoderm and mesoderm and challenged with thalidomide at sublethal doses. To identify significant toxicity markers, microarray analysis was performed with Ilumina HumanHT-12 v3 Expression BeadChip. We could identify 40 significantly upregulated and 407 significantly downregulated genes (P≤0.05) in day 14 Thalidomide treated samples compared to day 14 untreated samples. Among these transcripts, we found that germ line markers expressed on day 14 were down regulated when challenged with thalidomide. Gene ontology enrichment analysis reveals thalidomide treatment downregulated organ development, anatomical structure development, multicellular organismal development and circulatory system development in biological process. Shortlisted developmental markers were further validated with real time quantitative PCR (RT-qPCR) in independent experiments. To validate these markers a similar experiment was carried out with penicillin as a control compound where no changes were observed in the expression level. Further more, to study the interspecies differences, a similar experiment was carried out with CGR8 mouse embryonic stem cells. Interestingly we found a battery (Markers shortlisted in hESC study) of markers was significantly repressed with thalidomide at sublethal concentration in RT-qPCR studies. Thus, the multilineage differentiation of pluripotent stem cells combined with transcriptioinal profiling of developmental markers may be a strong tool for the developmental toxicity platform.
Embryonic stem cells (ES) are precursor cells with the ability of pluripotency and self-renewal and bear the capability to differentiate into tissue specialized cells found in three primary germ layers of an embryo (endoderm, mesoderm and ectoderm) in response to appropriate signals. The coax ES cells fate is controlled by both intrinsic regulator and the extracellular environment (niche), which under appropriate conditions in cell culture are apparently spontaneous, which generally are inefficient and leads to heterogeneous population of differentiated and undifferentiated cells that are not useful for cell-based therapy and moreover complicate the biological studies of particular differentiation program. A lot of work has been poured in recent years to improve the differention process leading to purified or semi purified cell lineages, most of them generally are controlled by a cocktail of growth factors, signaling molecules and/or genetic manipulation. However these protocols restrict derived cells for basic research as they utilize either xeno confined substitutes or are transgenic, limiting their therapeutic usage. Employment of small molecules gives an advantage for such application for the best reasons as they not only are cell permeable but have been wisely studied tools for modulating complex cellular pathways. In the present study, differentiation potential of murine ES cells under the pressure of chemopreventive agents was studied. Curcumin, EGCG and quercetin are phytocompounds and part of our daily intake in form of many foods or food supplements. The differentiation of ES cell was performed in sub toxic levels. It was found via elaborative gene expression and protein expression data that EGCG cause significant stimulatory effect on cardiomyogenesis and mesoderm in general. Curcumin had a stimulatory role on ectoderm in particular neuroectoderm formation seen via numerous ectoderm gene markers expression studies. Quercetin was cytotoxic and yielded to heterogeneous cell after differention. In summary we show the use of small molecules that enhances the induction of differention of cardiomyocytes and/or neuroectoderm. Our study helps to explore the effects of chemopreventive drugs in ES cell differentiation to form germ layers.
Beta-cell differentiation from embryonic stem cells involves complex signaling mechanisms. Nutritional status of the mother was shown to have a profound effect on offspring’s beta-cell numbers, islet cell size, proliferation capacity and islet-insulin content. This prompted us to investigate the role of whole-body energy sensor AMP Kinase on the expression of pancreas specific transcription factors and on beta-cell differentiation of mouse embryonic stem cells.
CGR8 mouse embryonic stem cells were used for this study. Chemical activators and inhibitors were used for modulating the AMP Kinase signaling in differentiating embryonic stem cells. RT-PCR experiments were used for monitoring the mRNA expression of several pancreas specific transcription factors (Hnf-6, Mac A, Neutrogena 3, Neuron D1, Foxa2, Pdx-1) during the course of embryonic stem cell differentiation, in the presence and absence of AMP Kinase activators and inhibitors. The relationship between TGF-beta signaling pathway and AMP Kinase signaling was studied using RT-PCR and western blot.
The results showed that modulating AMP Kinase signaling pathway of mouse embryonic stem cells has a profound influence on the expression of pancreas specific transcription factors. Inhibition of AMP Kinase during the earlier stages of embryonic stem cell differentiation increases the fold expression of pancreas specific transcription factors while activation of AMP Kinase has an opposite effect. Experiments with AMP Kinase activators show that TGF-beta mRNA expression was down-regulated by metformin, an AMP Kinase activator.
Initial experiments showed that AMP Kinase signaling pathway has a strong effect on the expression of pancreas specific transcription factors in mouse embryonic stem cells. Studying the relationship between AMP Kinase and TGF-beta pathway will help in understanding the interplay of these signaling molecules in the beta-cell differentiation of embryonic stem cells.
Present cell-based therapies of ischemic diseases are predominantly relying on autologous endothelial progenitor cells (EPCs). However, their purification from peripheral blood or bone marrow is laborious. Furthermore, patient-derived EPCs are numerically reduced and mostly functionally impaired. Consistently, the efficacy of current EPC-based therapies is low. Therefore, the evaluation of alternative sources of autologous cells suitable to promote adult vascular growth is required. Recently, we have obtained germline-derived pluripotent stem (gPS) cells from adult mouse unipotent germline stem cells. The objective of our study is to reveal whether functional endothelial-like cells (ECs) capable to support angiogenesis and arteriogenesis in ischemic diseases can be derived from this new type of pluripotent stem cells.
Briefly, PECAM-1 (CD31)-positive cells were isolated from single cell suspensions of gPS-derived embryoid bodies (EBs) by fluorescence-activated cell sorting (FACS) and subcultivated on OP9 stromal cells. Subsequently, EC-like colonies were mechanically isolated and expanded on collagen IV-coated cell culture dishes. Using FACS analysis it was demonstrated that the cells expressed the endothelial cell-specific markers PECAM-1, von Willebrand Factor, Tie2, Flk1, and vascular endothelial-cadherin. These results were confirmed by immunofluorescence staining and RT-PCR. The cells were successfully maintained in
The induction of pluripotent stem cells from differentiated, somatic cells by genetic reprogramming with the transcription factors, Oct3, Sox2, c-Myc and Klf4
Induced pluripotent stem (iPS) cells hold great promise for research and potential therapeutic applications but the question whether iPS cells actually represent an equivalent alternative to embryonic stem (ES) cells regarding its use as an
Our data shows that there are remarkable differences with respect to the chondrogenic differentiation capacity as well as the expression of pluripotency markers between undifferentiated murine and human iPS and ES cells.
Murine iPS and ES cells were cultured as cell aggregates, the so called embryoid bodies (EBs), by the hanging drop method. Both cell lines could be differentiated into chondrocytes but iPS cells were found to be significantly less efficient. Further, iPS cell aggregations were smaller and less stable compared to ES cell EBs.
The differentiation of human iPS cells is posing a challenge as established methods failed to produce EBs. EB formation by fragmentation of cell colonies was inefficient and aggregates from single cell suspension disaggregated.
The interplay between cells and their extracellular matrix (ECM) is of utmost importance in tissues like cartilage where cells are completely surrounded by ECM and cell-cell contacts thus take a minor role. A large number of these interactions are mediated by members of the integrin family. Chondrogenic development has been characterized in detail by using numerous model systems. Of these, murine embryonic stem cells (ESC) are of special importance because they are widely available without ethical concerns and because they render a large portion of animal experiments unnecessary. It has been proven that all physiologic stages of chondrogenic development are adequately mimicked by ESC in vitro. Since we found high expressions of the ECM molecule fibronectin (FN) and its major cellular receptor in cartilage, α5β1-integrin, during early chondrogenic development, namely during the formation of mesenchymal condensations that require active cell migration, we treated murine ESC either with RGD-containing blocking peptides mimicking the cell attachment domain of FN or RGE-containing control peptides to test the importance of FN-α5β1-integrin-interactions. RT-PCR analysis of α5 and FN-expression showed significant alterations demonstrating activity of the RGD-containing peptides. As shown by Alcian blue staining, the formation of chondrogenic nodules was significantly reduced although the number of PNA-positive cellular condensations and Collagen II-positive nodules remained unchanged. Confocal laser scanning microscopy did not show morphological differences between cells treated with blocking peptides and cells treated with control peptides. In addition we performed a phosphoryllation-sensitive western blot to analyze Focal Adhesion Kinase (FAK)-activity as an integrin downstream signaling target. Taken together our results suggest that interactions between FN and α5β1-integrin play an important role during early chondrogenic differentiation stages of murine ESC that is independent from the formation of mesenchymal condensations.
The Notch pathway is an evolutionary highly conserved intercellular cell communication mechanism, involved in various cell lineage determination processes during embryonic development. Here we show the influence of Notch1 signalling on chondrogenic differentiation via Sox9 as a regulatory protein. We have previously demonstrated, that stages of early chondrogenesis can be recapitulated during ES cell differentiation
Mesenchymal-epithelial interactions play a pivotal role in tubular morphogenesis and in the integrity of the kidney. During renal repair similar mechanisms may regulate cellular reorganisation and differentiation. Kidney regeneration of the renal tubular epithelium has been proven to be enhanced by treatment with mesenchymal stem cells. The mechanisms underlying this effect, however, remain unclear. Some in vivo studies suggest differentiation of MSC into tubular epithelial cells. Adipose-derived mesenchymal stem cells (ASC) can easily be isolated from lipoaspirates and possess a high plasticity towards different lineages. Previous studies of our group showed that retinoic acid (ATRA) induced initiation of epithelial differentiation of ASC.
We tested the influences of nephrogenic factors on epithelial differentiation of ASC. Therefore, ASC were cultured with media containing different concetrations of Activin A (ActA), bone morphogenic protein –7 (BMP-7), and ATRA. ASC proliferation was analyzed by a fluorimetric assay, while differentiation into epithelial lineage was assessed by morphologic changes and by the induction of characteristic markers like cytokeratin-18 (CK) and zona occludens protein 1 (ZO-1). Expression of the markers was shown by qPCR, Western blotting and immunofluorescence stainings.
Addition of a mixture of ActA, BMP-7, and ATRA induced epithelial differentiation of ASC, whereas cell proliferation was not enhanced. Expression of the epithelial markers CK-18 and ZO-1 was significantly induced after 14 days of incubation.
Our study highlighted that ASC are able to differentiate towards the epithelial lineage by cultivation with a combination of ActA, BMP-7, and ATRA. Such predifferentiated stem cells might be advantageous in regenerative medicine by improving stem cell based therapeutic options.
The ability to generate germ cells from embryonic stem cells (ESCs) provides a powerful in vitro model to study germ cell development.
The purpose of this study was to determine 10 ng/ml of BMP4 on SSEA-1 expression in process of mouse Embryonic Stem Cells (mESCs) differentiation to PGCs in vitro.
Mouse ES cells were generated as embryoid bodies (EBs) in vitro by hanging drop method .In order to differentiation of PGCs, EBs was cultured in concentration 10 ng/ml BMP4 until 4 day. Germ cell markers SSEA-1 and Mvh were analyzed by flow cytometry, Immunocytochemistry and electrophoresis gel.
By flow cytometry detection, the differentiation SSEA-1 positive cells in control and treated groups were 67% and 31% respectively. Immunohistochemical analysis of the EBs demonstrated that SSEA-1 positive cells after 4 days culture localized only in the edge of EBs. We found that addition of 10 ng/ml BMP4 increased expression of the germ cell-specific marker Mvh during differentiation of mESCs to PGCs.
We conclude that concentrations of 10ng/ml after 4 days had the effects on differentiation PGCs of mESCs.
Monitoring of stem cell homing plays a crucial role in preclinical evaluation of evolving stem cells therapies. Stem cells are wandering of desired organs and tissues very often. In case of heart is essential to visualize the right position of the delivered cells and ideal to quantify (recognize) the amount of remaining cells in the infarcted area. Most common were histological methods of recognizing remaining cells. Noninvasive methods would be more efficient and less time consuming. For tracking is very important to label the cells with particle which does not effect viability and would be specific enough to mark even a small number of cells. Bone-marrow mesenchymal cells (BMMCs) labeling methods were already described for canine, pig and rat models. Evaluation and comparison of labeling methods for rabbit BMMC is still lacking. Nuclear magnetic resonance is common cardiovascular diagnostic method with ideal recognition of heart geometry and motion. Radioactive indium can be tracked for four weeks and amount of radioactivity in the tissue is measurable. Combination of these two methods seems to be ideal.
Rabbit BMMCs were isolated and cultured for 1-3 weeks. (A) Labeling was based on incubation of cells with complex 111 Indium-tropolone for 5-15 min. Labeling efficiency was determined. Surviving of BMMCs during 1 week was monitored. Different amounts of labeled cells were placed in phantom of rabbit chest and underwent basic gamma kamera imaging. (B) Two types of iron oxide particles (Resovist or supermagnetic maghemite) were added to the BMMCs culture up to final concentration 100ug/ml. After several days particles were washed out and labeled BMMCs were placed in phantom of rabbit chest.
Results NMR imaging was evaluated. BMMCs displaying about 3 Bq/cell. Viability was not significantly decreased by this procedure. BMMCs numbers as low as 50 x 10^3 could be easily localized and imaged using gamma camera.Resovist and deadherence process resulted in surviving of 80% of cells. Cluster as small as 50 x 10^3 cells could be detected by NMR imaging.
Resovist labeling of rabbit BMMCs were proved working and optimized. These methods can be used as base for in vivo studies of tracking of cells delivered into the infarcted rabbit heart. Washout of radioactive/contrast substances does not affect our methods and viability of the cells is not impaired.
The regeneration of diseased hyaline cartilage continues to be a great challenge, mainly because degeneration—caused either by major injury or by age-related processes—can overextend the tissue’s self-renewal capacity. Adult osteoarthritic cartilage and cartilage from rheumatoid arthritis were obtained from the knee joints of patients (ages: 65 - 75 years) suffering from late-stage osteoarthritis (OA) or rheumatoid arthritis (RA) after total knee replacement. Light microscopy, ultrastructural investigations, cell isolation, cloning and immortalization, as well as multipotent differentiation experiments were performed. Furthermore, quantitative real-time RT-PCR, Western blotting, SILAC proteomics as well as RNA interference were applied. We have recently shown, that repair tissue from late stages of osteoarthritis in humans harbors a unique progenitor cell population, termed chondrogenic progenitor cells (CPCs). These exhibit stem cell characteristics such as clonogenicity, multipotency, and migratory activity. CPCs are governed by the osteogenic transcription factor runx-2 and the chondrogenic transcription factor sox-9. They show gender differences and exhibit estrogen and progesterone receptors. Treatment, especially with estrogen, at least in vitro, can enhance their chondrogenic potential. We have now isolated a similar CPC population from RA cartilage tissue, RA-CPC are regulated via IL-17 and novel TCR receptors. Our results offer new insights into the biology of progenitor cells in the context of diseased cartilage tissue and are relevant in the development of novel therapeutics for osteoarthritis and rheumatoid arthritis. DFG IMMUNOBONE Bl 1122/1-1 Koelling S, Kruegel J, Irmer M, Path J, Sadowski B, Miró X, Miosge N (2009) Migratory chondrogeniprogenitor cells from repair tissue during the late stages of human osteoarthritis Cell Stem Cell 4:324-335. Koelling S, Miosge N (2010) Sex differences of chondrogenic progenitor cells in late stages of osteoarthritis. Arthritis and Rheumatism 64:1077-1087. Koelling S, Miosge N (2009) Stem cell therapy for cartilage regeneration in osteoarthritis. Expert Opinion on Biological Therapy 9:1-7.
Conditional on apheresis techniques, stem cell products contain a considerable amount of thrombocytes. Platelets are the major source of soluble CD40 ligand (sCD40L) (1) in the blood. It has been demonstrated that CD40L is cleaved from the surface of activated platelets. sCD40L is well known to show immun-modulatory functions and high concentrations in blood products (2). Therefore we examined sCD40L concentrations in stem cell apheresis.
In four patients suffering from multiple myeloma and undergoing autologous stem cell apheresis, sCD40L concentrations were measured in peripheral blood samples before, during and after apheresis procedure and in the respective stem cell product. sCD40L concentrations were determined by a commercially available ELISA Kit (R&D Systems). In an additional approach, platelet-rich plasma (PRP) from healthy volunteers (n=6) was incubated with different pharmacological inhibitors (MMP-2/MMP-9 Inhibitor I, MMP-9 Inhibitor I, MMP-2 Inhibitor I, recombinant ADAM 10, and recombinant ADAM-17) during platelet activation.
During stem cell apheresis, a decrease in platelet count could be observed from 94,822/
During stem cell apheresis, sCD40L concentrations in peripheral blood were mainly influenced by alterations of platelet count. As known from platelet concentrates, an accumulation of sCD40L could also be observed in stem cell products pointing out the importance of sCD40L release by platelets. Additionally, these data support the hypothesis that MMP-2 might be the protease, primarily responsible for sCD40L cleavage from platelet surface.
Teratoma formation from embryonic stem (ES) cells and immune rejection of their derivatives represent a major impediment to their therapeutic use. The primary effector cells mediating immune rejection of transplanted cells are cytotoxic T cells, which recognize major histocompatibility (MHC) class I/peptide complexes on target cells. In this study we demonstrate that MHC class I molecules are expressed only at very low levels on murine ES cells and they are not induced by the interferon gamma (IFNg) despite the presence of IFNg-receptors on their cell surface. Thus, we aimed at dissecting the molecular mechanism responsible for the low level of MHC class I expression and unresponsiveness to IFNg in murine ES cells. First, we postulated that leukemia inhibitory factor (LIF), a standard component of ES cell culture media, may be a factor responsible for suppression of MHC class I expression, because of the induction of STAT3 by LIF. Indeed, removal of LIF resulted in upregulated the levels of MHC class I molecules on ES cells and its addition to differentiated cells in day 4 embryoid bodies suppressed their expression. STAT3-knockdown (STAT3-KD) in ES cells significantly increased the MHC class I expression even in the absence of IFNg and this increase was further enhanced by IFNg treatment. Using flow cytometry and STAT3-KD we demonstrated that STAT3 negatively affects IFNg-signaling by blocking STAT1 phosphorylation in ES cells. Luciferase reporter assay also indicated that GAS promoter responded strongly to IFNg in STAT3-depleted ES cells and only weakly in intact cells. Upregulation of MHC class I levels by STAT3-KD resulted in reduced lysis of ES cells by activated syngeneic natural killer (NK) cells. However, lysis of ES cells, which are normally not efficiently killed by cytotoxic T lymphocytes (CTLs), was increased by CTLs after STAT3-KD. These data indicate that STAT3 pathway plays an important role in regulating the MHC class I expression in murine ES cells and the susceptibility of these cells to lysis by NK cells and CTLs. Interfering with the inhibitory pathways that suppresses MHC class I expression may help control teratoma formation from contaminating ES cells in therapeutic cell transplants and may be used to eradicate cancer cells known to evade immune recognition by downregulating the MHC class I expression
The hormonal controls of adult epithelial stem cells
Hematopoietic stem and progenitor cells (HSPC) are known to reside in specialized niches at the endosteum in the trabecular bone. It is well established that proteases can take part in the cytokine-induced mobilization process. However, migratory processes such as the regular trafficking and induced mobilization of HSPC are not fully understood.
In the present study we showed that the osteoblast-secreted activated cathepsin X is able to reduce the direct interaction of HSPC with human bone-forming osteoblasts.
Immature cathepsin X is also bound to the cell surface of human osteoblasts. Knocking-down endogenous cathepsin X in osteoblasts with siRNA and subsequent HSPC adhesion studies led to a significant increase of HSPC binding to the adherent cells confirming its proteolytic influence on HSPC adhesion. In this context we studied the activation of cathepsin X and elucidated with different biochemical methods that cathepsin X can be activated by cathepsin L, a protease that is known to get secreted by activated osteoclasts.
Applying MALDI-TOF analysis we showed that the chemokine SDF-1, which is secreted by bone marrow stromal cells, can be readily digested with the carboxymonopeptidase cathepsin X. SDF-1 is a highly potent chemoattractant and a mediator of cell adhesion for HSPC. Migration assays with cathepsin X-digested SDF-1 showed a significant decrease in migration of HSPC compared to the non-truncated chemokine indicating that the protease is capable to inactivate SDF-1alpha. Furthermore, cathepsin X can convert the other isoform SDF-1beta to SDF-1alpha.
Current studies focus on the interaction between HSPC and their niche, especially the involvement of secreted proteases of the cathepsin family, their regulation and their extracellular substrate specificity to investigate the trafficking of HSPC in more detail.
We have previously shown that peripheral blood monocytes can be differentiated
So far it has been difficult to generate a proven stem cell line in animal which has been attributed to the lack of proper culture condition. The present experiment was carried out to study the role of different culture conditions on multiplication of early stage IVF derived embryonic cells of buffalo. The IVF embryos of 32-cell stage were made zona free. The clumped blastomere were cultured on inactivated murine embryonic fibroblast (MEF) with culture condition viz. (C-I) DMEM+ITS +FBS +LIF + SCF, (C-II) DMEM+ITS +FBS +LIF + SCF + IGF1, (C-III) DMEM+ITS +FBS +LIF + SCF +IGF1+BFGF4. The blastomere were cultured at 37oC, 5% CO2 and 90% relative humidity in CO2 incubator. Once the isolated blastomere clumped made stem cell clone, they were passaged mechanically. In the first culture condition, blastomere did not maintain for long time in culture. In culture condition-II, the multiplication was better as compared to C-I but no cell line could be derived in this culture condition also. In culture condition C-III, the cell clones could be propagated upto 3rd passage. The stem cell like clone were positive for Oct , AP and Nanog expression. The results indicated that multiplication of buffalo IVF derived embryonic cells from early stage embryos were better when they were cultured in presence of LIF, SCF, IGF1 and bFGF4 than culture condition excluding these cytokines.
So far it has been difficult to generate a proven stem cell line in animal which has been attributed to the lack of proper culture condition. The present experiment was carried out to study the role of feeder free culture conditions on multiplication of early stage IVF derived embryonic cells of goat. The blastomeres isolated from IVF embryos were cultured directly or after giving activation with 10μl of 7% ethanol/ml medium for 5 min on three types of feeder free coated plates viz. gelatin, matrigel and poly-l-lysine. Four different media were used viz. (Media-1)CR11aa supplemented with BSA, FBS, LIF, ITS (0.1%), IGF-1, bFGF, EAA (1%) and NEAA (0.5%), in Media-2; FBS supplementation was reduced to 10%; in Media-3, EAA was not incorporated, rather supplementation of NEAA was increased to 1% and in Media-4, 1mg/ml glucose was supplemented additionally. The blastomere were cultured at 37oC, 5% CO2 and 90% relative humidity in CO2 incubator. The results indicated that when blastomere were cultured on gelatin coated dish, the formation of ES cell clone were significantly higher (P<0.05) in Media-1 &3 than Media 2 & 4. However there was no significant difference between Media 1 & 3. Similarly when blastomere were cultured on matrigel, the ES cell clone formation was significantly higher (P<0.05) in Media-1 & 3 than Media 2 & 4. Again there was no significant difference was observed between Media 1 & 3 and Media 2 & 4. It was observed that there was no significant difference between gelatin and matrigel on formation of ES cell clones irrespective of media used. Further when all the media condition was considered together, there was no significant difference of ES cell clone formation when blastomere were cultured either on gelatin or matrigel. None of the embryos cultured on polylysine coated plate were found to be attached and developed to stem cell clones in any media and all of them died within few days. The result indicated that gelatin or matrigel coating could be used as a feeder free culture system for making ES cell clone of caprine.
Freshly isolated hematopoietic stem and progenitor cells (HSPCs) are small, round cells which adopt a polarized cell shape upon cultivation. Depending on the activity of the phosphoinositol-3-kinase (PI3K) they form a leading edge at the front and a uropod at the rear pole. We have recently shown that in addition to different lipid raft associated proteins, the lipid raft organizing molecules Flotillin-1 and -2 get highly concentrated at the tip of the uropod. Performing pharmaceutical inhibitor studies we dissected mechanisms controlling HSPC polarization and were able to discriminate two levels of cellular polarization. According to our observation the vast majority of freshly isolated human HSPCs, i.e. umbilical cord blood derived CD34+ cells, show a random distribution of the Flotillins and other lipid raft associated molecules like ICAM3. Upon cultivation they redistribute these molecules to form a crescent and thus become intrinsically polarized, before they adopt their characteristic morphological polarized cell shape. Using this discrimination, we obtained evidence that PI3K and atypical protein kinase C (aPKC) activities are required to organize the intrinsic polarity while the morphological polarization process also depends on protein synthesis, actin polymerization and rho-GTPases activities.
Since aPKCs form an evolutionary conserved complex with the partitioning defect proteins Par3 and Par6 as well as with the rho-GTPase Cdc42 and this complex has been found to organize cell polarity in many organisms and tissues, we decided to investigate the function of the individual components on the cell polarization process of human HSPCs next. Due to the fact that the Par/aPKC complex also coordinates asymmetric cell divisons in a number of systems and as we showed that human HSPCs can divide asymmetrically, we have started to study the impact of these proteins on the cell fate of human CD34+ cells in parallel. Our pharmaceutical studies as well as our experimental strategy within the Par/aPKC project together with some preliminary results will be presented.
Granulocyte colony-stimulating factor (G-CSF) is widely used in clinical practice for human stem cell mobilization and transplantation, and to reduce chemotherapy-induced febrile neutropenia. While the effect of G-CSF to stimulate neutrophil-progenitor cell development is well established, accumulating evidence implies additional functions of G-CSF as well as an impact on other cell-types, it is effects on stem cells are widely unknown. Our group now addressed the question about a possible effect of G-CSF on embryonic stem cells and early hematopoietic stem cells.
We used murine embryonic E14 stem cells which provide a homogeneous cell system to study cellular and molecular events that occur during early hematopoietic development. The embryonic stem cells were cultured in standard medium or medium continuously supplemented with G-CSF (50 ng/ml) in hanging drops for 3 days and the resulting embryoid bodies were kept in culture for a further period of 8 days. On days 3 (d3), 6 (d6) and 11 (d6+5), cells were harvested and separated with the Anti Sca-1- microbeads for analysis with Realtime-PCR, FACS and protein-arrays.
Our data show that G-CSF influences the early embryonic and hematopoietic cell development. Most hematopoietic genes (e.g. Gata-2, Pu1, Lmo2) are upregulated as early as day 3 in the cell pool. When the cells were separated with MACS-microbeads (sca1-antibody), we see that the stem cells show a delayed expression of hematopoietic stem cell surface-markers (e.g. CD34, CD117, CD133) and a downregulation in hematopoietic genes when stimulated with G-CSF.
Our results suggest that G-CSF is influencing hematopoietic stem cell development, and thus not only neutrophil-cell development. The protein and gene analysis of the complete cell cultures imply that G-CSF acts on these immature cells to promote hematopoietic development, and that this effect is not limited to hematopoietic cells. The downregulation of genes and stem cell markers of immature stem cells bearing the sca1-protein interestingly implies that G-CSF has a direct or indirect effect which preserves stem cell properties of the immature stem cells from the stem cell pool.
Low oxygen tension is thought to be an integral component of the human mesenchymal stem cell (MSC) native bone marrow microenvironment. MSCs (n=9) were maintained under hypoxic atmospheres (1%O2 and 5%O2) for up to ten
Mesenchymal stromal cells are (MSCs) are widespread in adult organisms and are involved in tissue repair as well as in the regulation of immunregulatory responses. Up to now it is not clear how MSCs respond to unfavorable conditions like ischemia. Toll like receptors (TLRs) play a major role in immune system, participating in the recognition of microbial pathogens and pathogen-associated components. We investigated in seven experiments the induction of TLR expression of MSCs which were exposed to low oxygen tension (1%O2 and 5%O2) without the additional substitution of classical TLR ligands like heat shock proteins or lipopolysaccharides. In flow cytometry analyses MSCs showed at 1%O2 respectively 5%O2 significantly upregulated expression of TLR2, TLR3, TLR4, TLR7, TLR8, TLR9 and TLR10. In 1%O2 treated cultures TLR3 and TLR4 expression was significantly more represented compared to 5%O2 exposed MSCs. Furthermore, the toll-like receptor associated protein MyD88 was also upregulated. Additionally, in ELISA based analyses the cytokine expression of CCL2, CXCL10, interferon-1β, interleukin-1α, interleukin-1β, interleukin-6, interleukin-8, interleukin-10 and tumor-nekrosis-factor-α was significantly enhanced, e.g. in the case of CXCL10 126.099±29.28pg/mL (1%O2) respectively 102.461±23.486pg/mL compared to normoxia (37.854±11.329pg/mL). In the supernatant of low oxygen cultures, endogeneous danger proteins heat shock protein 60 and fibronectin were found, indicating that MSCs at low oxygen tension produces these proteins. Thus, low oxygen tension may have direct effects on immunmodulatory responses of MSCs, as well as on proliferation capacity or stemness. In hypoxic cell niche of MSCs, immunmodulatory effects may suppressed by other factors.
The basement membrane extracellular matrix (ECM) is composed of proteins like collagen IV and laminin-5 and also of mobilized proteoglycans like heparansulfate proteoglycan (HP). In recent studies, the in
The failure of regeneration after spinal cord injury (SCI) has been attributed to axonal demyelination and neuronal death. Cellular replacement and white matter regeneration are both necessary for SCI repair. In this study, we evaluated the co-transplantation of olfactory ensheathing cells (OEC) and embryonic stem (ES) cell-derived motor neurons (ESMN) on contused SCI.
OEC cultured from olfactory nerve rootlets and olfactory bulbs. ESMN was generated by exposing mouse ES cells to retinoic acid and sonic hedgehog. Thirty female rats were used to prepare SCI models in five groups. Control and medium-injected groups was subjected to induce lesion without cell transplantation. OEC or ESMN or both were transplanted into the site of the lesion in other groups.
The purity of OEC culture was 95%. Motor neuron progenitor markers (Olig2, Nkx6.1 and Pax6) and motor neuron markers (Isl1, Isl2 and Hb9) were expressed. Histological analysis showed that significantly more (P<0.001) spinal tissue was spared in OEC, ESMN and OEC+ ESMN groups but the OEC+ ESMN group had a significantly greater percentage of spared tissue and myelination than other groups (P< 0.05). The numbers of ESMN in co-transplanted group were significantly higher than ESMN group (P<0.05). A significant (P<0.05) recovery of hindlimb function was observed in rats in the transplanted groups.
We found that the co-transplantation of ESMN and OEC into an injured spinal cord has a synergistic effect, promoting neural regeneration, ESMN survival and partial functional recovery.
The transcription factor Pou5f1/Oct4 controls pluripotency in mammalian ES cells, but little is known about its role in early development.
Heart failure due to loss of functional cardiomyocytes is one of the most frequent cardiovascular diseases. Understanding the genetic network that leads to functional cardiomyocytes is the first step to develop future therapies.A global transcriptome analysis yielded up-regulated genes in cardiomyocytes, which were derived from murine embryonic stem (ES) cells (Doss et al., 2007). In the present study we were on the one hand interested in a fast screen for the functional role of transcripts with unknown function (TUFs) for an intact activity of the heart. Therefore we searched for homologues in the zebrafish genome and performed a morpholino-based knockdown approach. We tested several TUFs of the zebrafish and found most of them to be expressed in the cardiovascular system. Morpholino-oligonucleotide injections caused highly specific cardiovascular defects in the majority of them such as altering of heart morphology, vascular defects or accumulation of blood cells to a different extent and penetrance. This pilot approach thus shows the potential of the zebrafish to identify TUFs in the cardiovascular system.Based on that, on the other hand we want to investigate promising candidate genes from the zebrafish approach in ES cells. ES cells are pluripotent cells which were isolated from early mouse embryos and because of their pluripotency able to differentiate into derivatives of the three primitive layers: ectoderm, endoderm and mesoderm. Using ES cells which were differentiated into three-dimensional structures, so-called embryoid bodies (EBs), we want to study early developmental stages. For this we have generated stable knockdown clones of mouse ES cells expressing a shRNA construct against the desired genes. These clones were also labeled with a green fluorescent protein (GFP) reporter to track the silencing effect. Clones, expressing GFP, were initiated to generate EBs and will be reviewed via PCR, western blotting and beating activity.
CCAAT enhancer binding protein (C/EBP) transcription factors may reprogram cells from the lymphoid lineage into the myeloid lineage. However, little is known about the molecular mechanism of this cell plasticity and its potential role in leukemogenesis.
B cell progenitors were substituted with the wild type or functional mutants of the transcription factor C/EBPβ to elucidate the mechanisms of reprogramming. After in vitro culturing in a stromal cell co-culture system that supports both, B cell and myeloid development, cells were analyzed for surface marker expression, cell morphology, protein expression, functionality and rearrangements in the immunoglobulin locus.
Our data show that 1) the long C/EBPβ isoforms LAP and LAP*, but not the short isoform LIP may reprogram B cell progenitors to mature myeloid cells; 2) distinct amino acid substitutions, and even some deletion mutants of the transactivation domain, maintain reprogramming functions; 3) the deletion mutant ΔCR3,4 is incapable of reprogramming, while the individual deletion mutants ΔCR3 and ΔCR4 may retain reprogramming functions. Reprogrammed B-to myeloid cells display common macrophage characteristics, including phagocytic activity, and exhibit rearranged IgH locus, thus confirming their B cell origin.
Our results show that the C/EBPβ regulatory domain and conserved regions (CR) 1 and 2 from the transactivation domain are dispensable for the lineage switch. CR 3 and 4 display redundant functions, as the presence of either one of them comprises reprogramming function, whereas the lack of both abrogates reprogramming. Our data suggest that the molecular mechanisms of B cell to macrophage reprogramming resides in CR3,4. It appears as important to determine the interaction partners of CR3,4 in B cells to work out the mechanism of transdifferentiation.
Notch proteins are transmembrane receptors which influence cell fate decisions, differentiation, proliferation and apoptosis in many developmental systems including neurogenesis and myogenesis. After ligand binding and activation, the Notch intracellular domain (NIC) is cleaved from the cytoplasmic membrane and translocates into the nucleus to act as a transcription factor. NIC binds to DNA via the adapter protein RBP-J (also termed CBF-1) and converts the transcriptional repressor RBP-J into a transcriptional activator. Recently we have shown that Notch signaling regulates expression of genes playing key roles in cell differentiation, cell cycle control and apoptosis in a highly context dependent manner. Epigenetic events like histone modification, DNA methylation and chromatin remodeling are tightly involved in the control of gene expression. To investigate the role of chromatin modifications for cell-context dependent activation of direct Notch target genes, we analysed the chromatin modifications present at the regulatory regions of genes that we identified to be regulated in embryonic stem cells (ESC) by activated Notch using public databases. We found that the promoter regions of Notch target genes are marked by an enrichment of H3K4me3 and bivalent domains (H3K4me3 and H3K27me3). We further screened the regulatory regions of the Notch1 target genes Hes5, Sox9 and Myf5 for potential RBP-J binding sites and analysed the modifications and their changes upon Notch activation in ESC and mesodermal cells. After activation of Notch signaling for 4 h, H3K4me3 and H3K4me2 marks at the regulatory regions of Notch tar get genes, Hes5 and Sox9 in ESC and Myf5 in mesodermal cells, were increased, suggesting that Notch signaling may lead to remodeling at the regulatory regions of its target genes. In addition, we tested the effect of LSD-1 (Lysine specific demethylase 1), a histone modifying enzyme that controls gene repression, on the expression of Notch target genes. After inhibition of LSD-1 by PCPA, Notch induced gene expression of the target genes Hes5, Hey1, Id4 and Pax6 was increased in ESC, further indicating an important role for histone modifications in the regulation of Notch target gene activation.
Somatic stem cells give rise to self-renewing and differentiating daughter cells. To maintain the regenerative capacity of given tissues over each organism’s life time, it is required that somatic stem cell pools are kept relatively constant. There is good evidence that decision processes controlling whether somatic stem cell progeny self-renew or get committed to differentiate are tightly controlled. At the example of hematopoietic stem and progenitor cells (HSPCs), the best investigated somatic stem cell system so far, it has been shown that extrinsic factors provided by the surrounding environment, the stem cell niches, are required to maintain primitive hematopoietic cell fates. In addition, at the example of human umbilical cord blood derived CD34+ cells we recently identified four different proteins that segregate asymmetrically in a proportion of dividing HSPCs, demonstrating that HSPCs can - as it has long been suggested - indeed divide asymmetrically.
Remarkably, three of the four proteins identified, the tetraspanins CD53 and CD63 as well as the transferrin receptor (CD71), are associated with the endosomal compartment. Together with recent findings in model organisms it becomes tempting to speculate that the endosomal machinery essentially participates in cell fate decision processes. To gain more insight into the underlying mechanisms, we decided to analyze the functional impact of distinct endosomal proteins on the biology of HSPCs.
Since the endosomal trafficking largely depends on the activity of Rab-GTPases, we aim to analyze the functional impact of certain Rab-GTPases on processes controlling self-renewal
Variation in gene expression at the single-cell level may be a critical parameter in understanding the differentiation of stem cells; however, single-cell samples have been difficult and expensive to analyze. BioMarkTM dynamic arrays provide a convenient and cost-effective method for performing multiple RNA expression assays on multiple single-cell samples. The BioMark 96.96 Dynamic Array enables running up to 96 real time PCR assays on up to 96 samples, i.e. up to 9,216 different reactions.
Here we demonstrate the use of gene expression assays for Stem Cell biologists and other users. These assays use the detection dye EvaGreen -a second generation dye less inhibitory than SYBR Green. Assay design involves a preamplification step (STA) followed by nested priming within the preamp product. To date, these approaches provide robust qPCR assays for a variety of eukaryotic and prokaryotic systems. Here we use that approach and EvaGreen to examine single cell gene expression.
The Notch pathway plays a pivotal role in the control of cell fate decisions during differentiation of various tissues. In early embryogenesis, activated Notch signaling favours the formation of ectoderm at the expense of mesendoderm. In the development of the nervous system, Notch promotes the maintenance of neural stem cells, inhibits differentiation into neurons and commits neural progenitors to a glial fate. Recently we have shown that Notch signaling regulates expression of genes playing key roles in cell differentiation, cell cycle control and apoptosis in a highly context dependent manner. In embryonic stem cells under neuroectodermal differentiation conditions, Notch1 activation results in the upregulation of Pax6 and Sox9 RNA and protein. Sox9 and Pax6 expression is induced by activated Notch1 also in the absence of protein synthesis, suggesting that Pax6 and Sox9 may be direct Notch1 target genes. Pax6 promotes neural stem cell maintenance and is required for the specification of a neuronal fate. Sox9 specifies glial cells, promotes glial differentiation and inhibits neuronal differentiation. To understand the molecular mechanisms by which Notch mediates the neuro-ectodermal lineage choice, we combined a tamoxifen-inducible system to activate Notch signaling in embryonic stem cells differentiating
In mammals, cell lineage specification is established at the blastocyst stage. At this stage, transcription factor Cdx2 represses pluripotency genes, thus promoting extraembryonic trophoblast fate. Recently, transcription factor Gata3 was shown to act in a parallel pathway in promoting trophoblast cell fate, suggestingthat there are more factors working in the trophoblast lineage. Here, we report that the transcription factorTcfap2c is expressed at a high level in the trophectoderm and is able to induce trophoblast fate in embryonic stem cells. Trophoblast fate induced by Tcfap2c does not require Cdx2 and vice versa, suggesting that themolecules act in alternative pathways. However, both Tcfap2c and Cdx2 are required for the upregulation of Elf5, a marker of trophoblast stem cell maintenance, suggesting that both factors are required for stable trophoblast induction. Tcfap2c-induced trophoblast-like cells are stable in long-term culture, indicating thatthey are capable of self-renewal. Tcfap2c-controlled trophoblast maintenance involves the induction of Cdx2 and the repression of the pluripotency factor Nanog. Tcfap2c-induced trophoblast-like cells differentiate to trophoblast derivatives in vitro and contribute to the trophectoderm in blastocysts in vivo. Taken together, these observations suggest that Tcfap2c and Cdx2 cooperate to override the pluripotency program and establish theextraembryonic trophoblast maintenance program in murine embryos.
A hallmark feature of embryonic stem (ES) cells is their ability for self-renewal to maintain pluripotent. ES cells can be derived and maintained independently of serum and cytokines using compounds that negatively regulate signalling pathways. Here, we report that innate Sprouty4 (Spry4) action deploys a similar strategy by inhibiting specific signalling cascades. We find that Spry4 is highly enriched in ES cells and is likewise confined to the inner cell mass of mouse blastocysts. Chromatin immunoprecipitation and overexpression assays validate
The NOD/LtSz-scid IL2Rγnull mice present an
Purified human CD34+ cells derived from frozen cord blood were transplanted into non-lethally irradiated newborn mice. Peripheral blood was collected for flow cytometric analysis using two novel eleven colour flow cytometric panel for T-/NK-cells and B-/DC-cells to analyze subtypes and differentiation status of human cells in the peripheral blood and lymphoid organs. Spleen, thymus, lymph nodes and bone marrow were analyzed by flow cytometry and plasma by ELISA measurements of human IgG, IgM, IgA.
In transplanted animals human IgM-plasma level became detectable eight weeks after transplantation and increased over time to up to 50 μ/mL. IgG and IgA-plasma level remained undetectable between week 8 and 26. At week eight after transplantation human lymphocytes became detectable, with low frequency of human T-cells and high frequency of human B-cells in peripheral blood. Between week 16 and 22 huCD45+ cells reached >40 %. Over time, the rate of the human T cells increased whereas the rate of the B-cells decreased. High frequencies of B-cells in the spleen and low frequencies in the lymph nodes were detected, whereas the T-cells showed an inversed ratio in spleen and lymph nodes. Both cell types remain stable over the time. At week 22 after transplantation 40 % of thymic cells are human T cells. This rate decreased to 25 % at week 29. Additionally, the proportion of the human CD4 and CD8 double positive T cells decreases from 50 % to 10 % at week 22 and 29, respectively. Immune histology of bone marrow, thymus, lymph nodes and spleen demonstrated presence of huCD45+, huCD4+, huCD8+, huCD11+, huCD20+ and huCD23+ cells. Especially spleen and lymph nodes showed distinctive human lymphoid structures.
NOD/LtSz-scid IL2Rγnull mice present a robust model for
To date, the generation of stable transgenic hESC lines remains inefficient and transgenic hiPS lines allowing lineage-specific enrichment of progenies have not been reported. Herein, we describe a non-viral strategy for the efficient, parallel introduction of multiple-plasmid constructs into hESC / hiPS by means of a single drug resistance. This enables, for example, the antibiotic-based purification of cardiomyocytes (CMs) that also express several reporter genes facilitating donor cell monitoring, quantification, and eventually functional assessment
_Our method allows single cell dissociation of common hESC / hiPS cultures without limiting cell vitality. Co-introduction of multiple plasmids was established by optimized electroporation. Cells were seeded in feeder-free cultures to facilitate antibiotic-based clonal selection; colonies were individually expanded and tested for the presence of multiple transgenes.
Two factors were critical for the generation of stable lines in only 12 days: high transduction efficiency (of up to 60%) and high cell vitality post transduction thereby supporting high colony recovery. On average, about 20 transgenic clones were reproducibly achieved from 1.5 million treated hESC or hiPS; about 25% of these clones carried multiple transgenes as demonstrated by PCR and functional assessment. Clones were also analysed for the expression of pluripotency markers and the capacity to differentiate into all germ-layers in vitro and in vivo.
Successful enrichment of essentially pure CMs was established following cardiomyogenic differentiation of transgenic clones. We also report the current application of these CMs for the
This is the first report of stable hiPS clones resulting in the purification of functional CMs and the parallel expression of additional marker genes. These clones were generated using a novel, fast, and highly efficient method of transgene introduction, which is of broad interest for numerous gain and loss of function applications in pluripotent stem cell research. Notably, stable multi-transgene expression was observed for at least 25 passages, the latest time point assessed. Thus, the method overcomes the common issue of vector silencing in pluripotent stem cells often associated with viral transduction methods.
Clara cells are non-mucous and non-ciliated secretory cells of the conducting airways. One of their major function is to protect the bronchiolar epithelium of mammals and the upper airways of some species such as mice by producing several products like the
Spermatongonial stem cells (SSCs) are unique cells in testis that can proliferate, differentiate and transmit genetic information to next generation. However the effect of different media and feeders on gene expression of these cells is not well known. So in this study, we compare the in vitro effect of adult Sertoli cells, fetal Sertoli cells, mouse embryonic fibroblast (MEF) and SIM mouse embryo-derived thioguanine and ouabain resistant (STO) as different feeder layers with or without GDNF, GFR- αl, bFGF on gene expression of SSCs .
For this purpose we isolated SSCs from 3-6 day mouse by enzymatic digestion, and then SSCs were enriched by magnetic activated cell sorting (MACS) against Thy-1 antibody. The purity of the isolated cells was assayed by flow cytometry with α6-integrin and β1-integrin antibodies. Adult and fetal Sertoli cells were isolated by DSA lectin. For isolation adult Sertoli cells we used 8-12 week male mouse and for fetal Sertoli cells we used 14.5 day male embryos. For MEF we used 13-16 day mouse embryos. Identities of cells were confirmed by fluorescent immunostaining against vimentin for Sertoli cells and alkaline phosphatase activity for SSCs. After enrichment, SSCs were cultured for 7 days in different feeders and media. The expression of specific genes for SSCs (α6-integrin, β1-integrin, DAZL and stra-8) was studied by RT-PCR.
The percentage of SSCs purification was 85-90%. Expression of these genes were higher significantly (p<0.05) in Sertoli cells group compared to other.
Our findings suggested that co-culture with Sertoli cells had a positive effect on in vitro culture of SSCs and had best effect on SSCs gene expression than others.
Spermatogonial stem cells have self renewal and differentiation that produce mature sperm in development and differentiation. This cells ,so uses in regenerative medicine and gene therapy. This cells is necessary because 1/250 persons have cancers in 2010 and so suggested %50 couples will have infertility until 2050.Because infertility increased gradually so maintenance SSCs are necessary .SSCs are few in testis (%0/03 ), So isolation and culture them is important,also it is more importance in some cases (cancer of testis, Obstructive asospermia)
tunica albogina removed from adult mouse tetes and cells isolated, then mechanical digestion of somniferous tubules, Enzymatic digestion did and cell suspension transferred to Percoll (Discontinious percoll centrifugation) and after cell suspantion teransfered to coated dishes with Laminin.then supernatant removed and MACS with thy-1 immunobids was done .before and after MACS isolation, cells purification percent meseared with α6 , β1 integrins. After purification, cells cultured in MEMα medium and after one week colony formation assayed by Alkalan phosphatase staining ,Immunocytochemistry ,RT-PCR and Flow cytometeric analysis. Viability of cells was done in all of steps.
purification of cells about 80% after MACS .colony assesment showed Alkalan phosphatase positive and α6, (β1 integrins in SSCs by Immunocytochemistry, Flow cytometeriy and Stra8 and DAZL by RT-PCR was detected.
MACS is the best method for isolation and purification cells . α6 , β1 integrins are the best markers for detection of Spermatogonial stem cells .
Human iPS cells are very similar to human embryonic stem (ES) cells but do not require an embryo for their derivation. They can be derived from patients with complex genetic defects to create
Tissue homeostasis and cancerous transformations are a consequence of reciprocal interactions between the epithelial tissue and lineages arising from cells with stem cell properties. This view has important consequences for the way we investigate complex multilevel systems. Motivated by experimental evidence for the dominance of individual stem cells in tissue organization, we developed a conceptual framework to analyze cross-level principles. Our analysis of cell-tissue relationships then led us to the formulation and proof of a
Hematopoietic stem cell (HSC) transplantation is a routinely used therapeutic method. Most of all currently registered stem cell-related clinical trials focus on HSCs. Selecting the most potent HSC population might improve the beneficial stem cell related therapeutic effect.
To analyze c-kit+ subpopulations for their proliferation and differentiation capacity, murine bone marrow c-kit+ cells were sorted for lin-, CD34, Sea1 and CD45. Proliferation potential was analyzed using a Colony-Forming-Unit (CFU) assay. Surface markers and gene expression were characterized by flow cytometry and real-time PCR.
Prior to CFU assay entire cell population was viable, lin- and c-kit+. Their purity exceeded 92%. The major subpopulation was CD45+CD34-Scal- (21.25 ± 4.25%), followed by CD45+CD34+Scal- (15.00 ± 4.23%). Cells positive for CD45, CD34 and Sca1 and cells negative for all three markers formed smaller populations (0.74 ± 0.40% and 1.03 ± 1.25%).
After CFU assay we found significant differences in the CFU count of analyzed subpopulations (P≤0.0026). CD45+CD34+Scal+ cells displayed the highest CFU count per utilized cell (0.470 ± 0.055; n=3), followed by CD45+CD34+Scal- cells (0.162 ± 0.019; n=5). CD45+CD34-Scal- and CD45-CD34-Scal- cells showed low CFU frequency (0.0032 ± 0.0009 and 0.0035 ± 0.0027, n=5, difference n. s.;
Interestingly, original CD45+CD34+Scal- cells after CFU displayed a larger Scal+ population than CD45+CD34+Scal+ cells (14.51 ± 5.21% versus 3.84 ± 0.72% CD34+Scal+c-kit+lin CD45+ cells). In CD45+CD34+Scal -derived cells, SPP1 and GATA2 genes, which are present in HSC niche, were upregulated whereas Wnt3a and VEGFA genes, which are mainly expressed in active and differentiating HSC, were strongly downregulated compared to cells derived from the CD45+CD34+Scal+ subpopulation.
In conclusion, we present viable lin- c-kit+ cell subpopulations, specified by differential CD45, CD34 and Seal protein surface expression, are highly heterogeneous in proliferation and differentiation capacity. CD45+CD34+Scal+ cells proliferate more readily and may differentiate more distinctively than CD45+CD34+Scal , while CD34 Seal cells display a low proliferation potential. These findings might help deepen the knowledge in c-kit+ HSC plasticity and therapeutic potential.
In the past years it has been shown that bone marrow-derived mesenchymal stem or stromal cells (BM-MSCs) have a high potential for cell-based therapies and tissue engineering applications because of their multilineage differentiation potential and their immunomodulatory properties. However, bone marrow presents several disadvantages, namely low frequency of MSCs, high donor-dependent variations in quality and the isolation procedure is painful and implies the risk of infection. In search of alternative sources of MSCs, the umbilical cord (UC) tissue gained more and more attention. Since the UC is discarded after birth, the cells are easily accessible without ethical concerns. We isolated a population of plastic-adherent mesenchymal stem cell-like cells from human UC-tissue, which exhibit a high proliferative potential and express several MSC markers, including CD44, CD73, CD90 and CD105 (negative for CD31, CD34 and CD45). Furthermore, the cells display multilineage differentiation potential.
The aim of this study was to shape the microenvironment of the cells with regard to different oxygen concentrations and cell-cell interactions with immune cells. Therefore, the oxygen consumption, as well as the metabolic activity and HIF-1α target gene expression were determined. In addition, immunomodulatory properties of MSC-like cells were analyzed by direct and indirect co-culture experiments using peripheral blood mononuclear cells (PBMC) in CFSE-based proliferation assays.
Our study revealed that UC-derived MSC-like cells consume 2-3 times less oxygen under hypoxic conditions (1.5% O2, 2.5% O2 and 5% O2) as compared to 21% O2 control. Hypoxic culture conditions caused stabilization of HIF-1a protein and subsequent regulation of its target genes, involved in glucose metabolism. Moreover, UC-derived MSC-like cells showed increased proliferation at 2.5% O2.
Furthermore, our results demonstrated that MSC-like cells do not induce proliferation of allogeneic PBMCs
Somatic stem cells are required to maintain homeostasis in different tissues. In this context stem cells give rise to differentiating cells which replace cells getting lost in the lifetime of a multi-cellular organism. To fulfil this function over a long period of time, it is essential that the pool of stem cells remains a constant size. Since both the abnormal loss as well as the uncontrolled expansion of stem cells is fatal for organisms, the decision of self-renewal
At the example of the hematopoietic system, a few transcription factors, e.g. HoxB4, AMLl/Runxl, SCL/Tall, Meisl, have been identified, taking part in the decision process self-renewal
With the aim to identify additional transcription factors required for the self-renewal process of primitive human hematopoietic cells, we have performed genome wide GeneChipTM analyses of different cell fractions, containing either primitive or more mature hematopoietic cells. We identified a number of transcription factors encoding genes which are specifically expressed in the most primitive hematopoietic cell fractions, whose function has not yet been associated with hematopoiesis. In order to characterize the early hematopoietic function of some of these candidate genes we decided to perform over expression as well as RNAi mediated knock down experiments. We are using a lentiviral strategy to genetically manipulate primary human umbilical cord blood derived CD34+ cells and analyze effects on the cell fates of transduced cells in different functional read out systems.
The discovery of so-called circulating endothelial progenitor cells (EPCs) has highly stimulated the field of vascular biology. During recent years different protocols have been applied to raise and expand such cells. However, a recent comprehensive comparison of obtained EPCs revealed that many of them are of hematopoietic origin and just mimic an endothelial cell surface phenotype. According to the published studies only the
Of note, ECFCs raised from human umbilical cord blood are phenotypically almost identical to human umbilical cord vein endothelial cells (HUVECs). Furthermore, similar to HUVECs their proliferation capacity is limited to a few passages. In this context it is often suggested that both cell types become senescent and stop to proliferate. Provided that both endothelial cell types are organized in a hierarchical manner similar to primitive hematopoietic cells, such a limited expansion would also be expected, if the culture conditions would not allow self-renewal of the more primitive cells. Favoring the latter hypothesis we aim to optimize the culture conditions for these endothelial cells.
For a number of different cell systems cell fate modulating features of biomaterials and matrices have been reported. To this end we decided to compare the impact of distinct biomaterials as well as basal membrane-derived matrices on the adherence capacity, the vitality, the cytotoxicity, the apoptosis rate and the expansion rate of primary human endothelial cells.
So far, we compared the impact of collagen V, fibrin, gelatin, heparin, hyaluron acid, laminin platelet lysate coated plastic dishes with conventional plastic dishes and a novel commercially available advanced plastic dish type from Greiner Bio-One GmbH. Additionally, we studied biological features of ECFCs expanded on different biomaterials used in tissue engineering including PVDF-gf, PTFE, PET, Texin 950, PDMS, L209S (PLLA), R203S (PDLLA), LR704, RG503, LT706, PCL, BAK1095, PEA-C and Alginate. Our current results will be presented.
Endothelial progenitor cells (EPCs) play an important role in angiogenesis, which is essential for physiological processes as well as tumor growth. The primitive hemangioblast make up only 0.1-0.5% to total CD34+ cells and have the surface phenotype CD34+, CD133+, VEGF-R2+, CD31+. Cells exhibiting these markers are predominantly found in bone marrow (BM) and in stem cell apheresates (SA). However, the exact molecular mechanisms regulating the proliferation, differentiation and migration of EPCs as well as the interaction with niche cells still remain elusive. Our study focus on the biological distinctions between CD133+ cells from the BM and SA, using to analyse expression profile for EPC surface marker and genes as well as micro RNA (miRNA).
We characterised the differences between EPCs cells from the BM and SA by using CD133+ cells separating with Anti-CD133- microbeads. These cells were cultured for 21 days and samples were obtained at day 0, 2, 4, 8, 14 and 21. Surface marker e.g. CD133, CD31, CD34 and VEGF-R2 were determined by FACS. Genexpression had been analysed by realtime PCR and analysis for stem cell miRNAs.
Results of FACS showed that CD133+ cells from BM as well as SA expressed the different surface markers in a similar level at day 0 and in all probes of the expansion culture (day 2- 21). We could show that there are no differences at day 0 in the expression of different genes which are associated with endothelial differentiation like Prominin or VEGF-R2. Only the CD133+ cells from the BM showed up regulation for e.g. Prominin and VEGF-R2 in expansion culture from day 2 to 21. Interestingly the miRNA302 is up regulated in the purify EPCs from BM at day 0 as well as at day 8 and 21 of the expansion culture.
In summary we suggest that there is no difference between the phenotype of CD133+ cells whether they from BM or SA. Also there is no difference in gene expression in the purify EPCs. Only in the expansion culture the CD133+ cells from BM showed up regulation of some genes. We suppose that CD133+ cells from BM have more stem cell potential then cells from SA. This hypothesis will be supported by the observation that only in CD133+ cells from the BM miRNA302 is up regulated. MiRNA302 is also highly expressed in embryonic cells. We postulate that CD133+ cells from BM have more stem cell potential then from SA although they have the same phenotypes.
The role of adult mesenchymal stromal cells (MSC) in tissue maintenance and regeneration has received significant attention of late. MSCs in culture undergo senescence after a certain number of cell doublings whereby the cells enlarge and finally reach terminal growth arrest. In this study we have analyzed the behavior of subpopulation of rapidly self-renewing cells (RS-) cells. 22 samples of MSCs were isolated from bone marrow of donors between 22-87 years old. In general, RS-cell fraction was significant lower after 10 days cultivation in samples obtained of donors >50 years (end passage 2: 13,74±4,45% to 22,51±6,21%; p<0.01). With the increase of passage number RS-cell fraction decreased, independent of age of donors. In samples of younger donors (<50 years), the approval of RS-cells was slighter (passage 5: 7,45±3,45% to 16,38±6,29%). The seven samples of donors under 50 years old could always been cultivated until passage 10 (100 days), while MSCs from older donors stopped proliferation before. Interestingly, after RS-cell fraction was dropped down under about 6,65%, the terminal growth arrest was reached reproducibly in all samples, indicating a essential effect of RS-cells on proliferation behavior of the whole population. In flow cytometry approaches only 0,9±0,15% of RS-cells were positive for senescence indicating staining with β-Galactosidase, while the rest of population offered passage-dependent larger numbers of positive cells. Although telomere length varied particularly in donors, an telomere-dependent correlation to the decline of RS-cell subpopulation could not be statistically verified. Thus, the study demonstrated that in
Resistance to apoptosis plays an important role in cancer therapies such as chemotherapy and radiation; whereas molecular processes for avoiding apoptosis mechanism are mostly unknown. A conventional therapy by HNSCC (
Human HNSCC cell lines were treated with Paclitaxel to induce apoptosis. Apoptosis induction was quantified by flow cytometry and western blot analysis. The processing and activation of caspase 3 was used as a read out system for Paclitaxel induced apoptosis.
Paclitaxel induced programmed cell death in each treated HNSCC cell line. Nevertheless in every experiment at least some cells avoided cell death and finally survived.
These Paclitaxel resistance subpopulation could be cancer stem cells and should be further analyzed related to stem cell characteristic and their tumor inducing potential.
Total RNA from nasal polyps was isolated and a cDNA library was synthesized. Polymerase chain reaction (PCR) was performed in order to detect stem cell markers like CD133, Nestin, ABCG2 and Sox2. Cryostat sections of nasal polyps were prepared and stained against various stem cell marker proteins, such as Oct4, using immunohistochemistry (IHC).
Using PCR we were able to detect the expression of the specific stem cell markers CD133, Nestin, ABCG2, Sox2. Furthermore, immunostaining revealed the existence Oct4-positive cells in nasal polyp tissue.
Our data strongly suggest the existence of cells with stem cell characteristics. We postulate that these cells are involved in the development and progression of
Culture medium for mesenchymal stromal cells (MSC) is frequently supplemented with fetal calf serum (FCS). FCS can induce xenogeneic immune reactions, transmit bovine pathogens and has a high lot-to-lot variability that hampers reproducibility of results. Human supplements have been used as substitutes for FCS before and several other groups demonstrated that pooled human platelet lysate (HPL) provides an attractive alternative. However, the composition of heterogeneous subpopulations might be affected by serum supplements and lot-to-lot variation of individual platelet lysates has not yet been addressed in detail.
In this study we compared the impact of serum supplements on initial fibroblastoid colony forming units (CFU-F). Subsequently, we addressed the activity of lysates from platelet units of individual donors on proliferation,
Isolation of MSC with either HPL or FCS resulted in similar CFU-F frequency, colony morphology, immunophenotype, and adipogenic differentiation potential. Osteogenic differentiation was more pronounced in HPL than in FCS. There was some variation in MSC proliferation with individual lysates but it was always higher in comparison to FCS. Proliferation of MSC correlated with the concentration of platelet-derived growth factor (PDGF) and there was a moderate association with platelet counts. All HPLs facilitated expansion for more than 20 population doublings.
Reliable long-term expansion was possible with each lysate of individual platelet units and this supports the notion that donor recipient matched or autologous HPL can be used for therapeutic MSC products. However, there was some variation in growth supportive potential and this correlated with PDGF concentration.
Stem cells (SC) are characterized by the ability to renew themselves through symmetric and asymmetric cell division and to differentiate into a diverse range of specialized cell types. This implicates their potential for therapy in regenerative medicine. However, our understanding of how SCs are maintained or replenished during life-time until senescence is poor. In this project signaling cascades involved in the self-renewal process of SCs are examined. Furthermore, since SCs display an undifferentiated phenotype, novel genetic approaches are needed to specifically address SCs. Modern genetic techniques not only enable researchers to visualize individual cells in order to identify morphological characteritics but also place them in a position to simultaneously manipulate cellular responses. In order to monitor and trace stem cells we have generated and tested novel reporters to monitor Notch as well as Oct4 positive stem cells in vitro and in vivo. In combination with conditional gene inactivation and cell ablation experiments these reporters provide new tools to characterize endogenous adult SCs and will give valuable information not only for researchers but also for clinicians in search for novel therapeutic approaches.
After an acute renal failure the kidney is able to regenerate. A role in this process is awarded to renal mesenchymal stromal cells. These cells may support the self-healing process via interaction with injured tubule cells by producing erythropoeitin (Epo) or via stem/progenitor cell attributes with direct replacement of injured cells.
The aim of this study was to isolate and characterize renal mesenchymal stromal cells
A population of mesenchymal stromal cells from adult mouse kidney was isolated by plastic adherence. The cells were characterized by different attributes: proliferation capacity (CFU-F), cell proliferation in NOX or HOX (2%O2), telomerase activity, FACS analysis, gene and protein expression, as well as differentiation capacity. To explore whether isolated renal cells (k) have similarity to mesenchymal stromal cells from other organs, they were compared to mesenchymal lung cells (l), colon cells (c), bone marrow cells (bm) and fibroblasts (fb, isolated from murine skin).
No significant difference between renal cells and cells of other mesenchymal origin were shown in CFU-F-, cell proliferation and telomerase analysis. Cell proliferation ascertainment showed a 2-fold better proliferation in NOX than HOX.
In FACS analysis, cells from kidney and colon could not be distinguished. Significant difference was shown by comparing kidney and lung (CD44 [k>l]), kidney and bone marrow (CD117 [k<bm], CD292 [k<bm], OCT ¾ [k<bm]) and fibroblasts and all other cell cultures (CD105 [f<k/l/c/bm], CD117 [f>k/l/c/bm], Sca-1[f<k/l/c/bm]).
Analysis of differences of renal cells to fibroblasts by immunoflourescence and RT-PCR showed differences in aSMA (k(+)/f-), cytokeratin (k(+)/f-), lim1 (k(+)/f+), pax8 (k+/f-), ksp (k+/f-) and erythropoeitin (k(+)/f-). The gene expression of nephrin (k+/f+) and podocin (k(+)/f-) was different to the protein expression of nephrin (k(+)/f-) and podocin (k-/f-).
Analysis of differentiation capacity showed that only single cells are able to obtain adipogenic, chondrogenic or osteogenic characteristics.
In conclusion, renal mesenchymal stromal cells can be significantly distinguished to different mesenchymal cell cultures. Only rare renal cells express Epo or are able to undergo an adipogenic, osteogenic or chondrogenic differentiation.
Hematopoietic stem cell (HSC) transplantation is a therapeutic option in the treatment of inherited diseases and leukemias. In many cases HSC transplantation is associated with complications including engraftment failure or long-term pancytopenia [
Mesenchymal stem cells have been identified as playing an important role in the support of HSC engraftment in animal models and patients due to their cytokine production [
Here, we investigated the effect of USSC on the engraftment of cotransplanted human HSC in the NOD/SCID mouse model. After 4 weeks, homing and engraftment of human cells to the bone marrow was significantly increased in mice cotransplanted with HSC and USSC (30.9%), as compared to the control group (HSC only) (5.9%, p=0.004). After 8 weeks, the median proportions of human cells detected in bone marrow were 24.2% in the cotransplanted group and 11.3% in the control group. The percentage of human cells maintaining their CD34 expression in the bone marrow was unaffected by cotransplantation of USSC. Apart from short-term entrapment in the lungs, USSC themselves could not be detected in the bone marrow or other organs. According to migration assay results and to expression of genes known to mediate HSC homing and migration, USSC showed no potential to migrate towards bone marrow. An
Cardiac cell transplantation is a promising approach for cardiac regeneration in heart failure patients. However, the ideal cell source has not been found yet. In the presented work we induced a lineage conversion of skeletal muscle derived precursors into cardiomyocytes avoiding gene manipulation, which can be easily translated into a clinical application.
Skeletal precursor cells were isolated from adult C57/BL6 mice. Following a primary expansion and purification according to a skeletal myoblast isolation protocol, the cell product was further cultured under hanging drop culture conditions. The forming cell clusters were characterized by immunohistochemistry and single cell patch-clamping.
Under hanging drop culture conditions the purified cells showed a high lineage conversion rate towards cardiomyocyte-like phenotype. Besides synchronous beating of the clusters, these cells were highly positive for cardiac troponin, connexin43, cardiac myosin heavy-chain. Electrophysiological assessment under 8 Hz stimulation showed cardiomyocyte like shape of the action-potentials.
Despite an ongoing controversial discussion about skeletal precursor cells a cell source for cardiac cell therapy, we confirmed successful lineage conversion of those cells into a cardiomyocyte-like phenotype. This provides an outstanding alternative cell source for cardiac cell therapy which can be easily translated to clinical application.
Stem cell transplantation is emerging as a promising approach for the regeneration of infarcted myocardium. However, there is little known about the fate of the transplanted cells since the means for tracking transplanted cells and measuring their therapeutic effects
Murine mesenchymal stem cells (mMSC) were isolated from bone marrow. After expansion lineage specificity was confirmed in vitro by adipo-, chondro- and osteo-differentiation and FACS-analysis for CD44 and Sca-1. mMSC were labeled with paramagnetic microspheres (Ø 1μm) and transplanted into the border zone of the infarcted myocardium subsequently after cryo-infarction.
Microspheres were phagocytosed by mMSC efficiently, without interfering with their proliferation and differentiation potential. A minimum of 50,000 transplanted mMSC could be clearly detected and co-localized by MRI up to five days after transplantation. Furthermore, compared to sham-controls, cell transplanted animals showed a significant improvement left ventricular function and reduction of the infarct scar three weeks postoperatively.
Transplantation of mesenchymal stem cells results in reduction of infarct size and improvement of left ventricular function. In addition, we could show that in vivo tracking of transplanted cells is feasible by MRI and opens new options to elucidate the mechanisms of cardiac cell therapy.
CD133(+) cells isolated from umbilical cord blood (UCB) represent an established source of transplantable hematopoietic progenitors, and subpopulations of such cells have been shown to be capable of differentiating into mesenchymal lineages. Here we investigated whether CD133(+) UCB cells may contribute to the regeneration of injured brain and skin regions. A purified population of CD133(+) cells from human UCB was expanded in a stem cell medium, labeled with a fluorescent cell tracker and administered intravenously to 9-day-old rats subjected to unilateral carotid artery ligation and subsequent hypoxia (8% O2) for 120 minutes on day 7 after birth. The capability of these cells to differentiate into the neurogenic lineage was confirmed
Gene targeting by homologous recombination via customized zinc-finger nucleases (ZFN) is a powerful method to manipulate the genome and correct genetic defects. Although efficiency of ZFN based homologous recombination has been shown to be significantly higher than by means of conventional gene targeting, the selection of suitable clones still requires cells that proliferate in culture. Clinically applicable ZFN-based gene correction in patient-specific cells was hardly possible so far, due to the inability to sufficiently expand most adult (stem and progenitor) cells
As a first step towards ZFN-based gene targeting, a non-viral gene-transfer approach with transfection rates of up to 60% and high cell vitality was established for hiPS cells. Aiming at the development of a general ZFN-based recombination approach in hiPS cells, we investigated the functionality of an eGFP specific ZFN in an eGFP transgenic hiPS cell clone. Targeting of the eGFP via non-homologous end joining resulted in up to 3% eGFPneg cells, and mRNA expression of genetically modified eGFP was shown in sorted eGFPneg hiPS cell clones.
Ultimately, the development of a generally applicable protocol for ZFN based site-specific recombination and gene correction in patient-specific hiPS cells may enable the development of cellular therapies for various genetic diseases.
Therapeutic application of stem cell derivatives requires large quantities of cells produced in defined media and cannot be produced via adherent culture. We have applied human induced pluripotent stem (hiPS) cells expressing eGFP under control of the OCT4 promoter to establish the expansion of undifferentiated human embryonic stem (hES) and hiPS cells in static suspension culture. A defined culture medium has been identified that results in up to six-fold increase in cell numbers within four days. Our culture system is based on initial single cell dissociation which is critical for standardized process inoculation. HES/hiPS cells were expanded for up to 17 passages. The cells maintained a stable karyotype, their expression of pluripotency markers and their potential to differentiate into derivatives of all three germ layers. The ability to expand hES/hiPS cells in a scalable suspension culture represents a critical step towards production in stirred bioreactors.
Standardized fully controlled stirred tank reactors are already widely used in biopharmaceutical industry for culture volumes of up to 10.000 L. However, standard cell lines used for the production or recombinant factors are usually homogenous and robust and therefore relative easy to maintain under standardized conditions.
In contrast, cultures of hiPS/hES are relatively heterogeneous, particularly when combined with differentiation. A direct transfer of these complex cultures to established bioreactor systems is therefore challenging.
Applying our experience in static suspension culture we have systematically performed translation to stirred bioreactors. This includes optimization of inoculation, control of hydrodynamics and feeding strategies to establish stepwise adaptation to scalable mass expansion of hiPS/hES cells.
In addition we are exploring direct combination of expansion and differentiation processes in stirred bioreactors aiming at the translation of stem cell research to clinical practice.
In sickle cell anemia (SA), synthesis of pathological hemoglobin S (HbS) induces sickling of red blood cells (RBC), their reduced deformation capacity and increased adherence to the endothelial wall. This results in vaso-occlusive events, responsible for much of the morbidity and mortality. Fetal hemoglobin (HbF) is known as the most potent modifier in disease severity, since it protects RBC from pathological HbS polymerization. The aim of the study was the modification of RBC pathology by the ex vivo generation of RBC, containing a high level of HbF, derived from hematopoietic stem cells (HSC) of SA patients.
Peripheral blood (PB) CD34+ HSC from SA patients were cultured over 25 days in an in-vitro erythropoiesis assay. Generated RBC were compared with native RBC from the same patient for their level of HbF (HPLC, flow cytometry), the expression of adhesion molecules CD36, Integrin a4ß1, CD239, ICAM-4, CD47, CD147 (flow cytometry), their deformation capacity (ektacytometry) and adhesion to human laminin (flow adhesion assay).
CD34+ HSC were found to be increased in PB of SA patients (~10fold) and could be isolated in a sufficient way. The applied in-vitro erythropoiesis assay was able to generate 100% enucleated RBC with an at least 16.500fold amplification. Compared to native SA-RBC, ex vivo generated RBC showed an increased HbF level (~ 5-fold). Whereas native SA-RBC showed strong adhesion to laminin, this was reduced or absent in ex vivo generated RBC. In line with this, cultured RBC showed a modified expression of adhesion molecules. In contrast to the reduced deformation of native SA-RBC, the deformation capacity of ex vivo generated RBC was also normalized.
RBC generation under ex vivo conditions allows for the modulation of the pathological phenotype in SA. Besides an increased level of HbF, ex vivo generated cells show a normalized deformation capacity and reduced adhesion to elements of the endothelial wall. Further work will be carried out to identify underlying pathways. As the most interesting clinical application, such ex vivo generated RBC might be suitable as an autologous transfusion product, able to reduce the frequency of vaso-occlusive events and to circumvent storages of compatible RBC units.
Evidence of neurogenesis in the adult enteric nervous system in the postnatal human ENS brought new perspective for cell therapy and neural regeneration. Although these cells do show a decreased plasticity in the aging gut, they are a potential source for autologeous neural stem cells. The appendix might be the appropriate location with a sufficient amount enteric nervous tissue where these cells could be easily harvested.Appendices from adults and children (age ranged from 6 month to 69 years) were obtained from two surgical centers. From each appendix tissue samles were collected and processed for immunohistochemistry. The remaining tissue was used for the isolation of myenteric and submucous plexus. Muscle and submucous layer were separated and enzymatically digested, so that pure myenteric plexus could be harvested, dissociated and seeded in 25 cm2 flasks. Every 2 days, half of the medium was replaced. After 6 day in culture free floating neurospheres as well as neurons and glia cells were easily to discriminate. The supanatant with free floating neurospheres was cultivated for another ten days. Than neurospheres were dissociated and resuspended in an extracellular matrix gel with to mimick the tissue in which the stem cells will have to be transplanted.Immunostaining for neuronal and glial markers were performed and cells analyzed. Sections from the parrafin blocks were stained for nestin, Musashi 1 and Integrin-ß1.After 6 days in cell culture first neurespheres were seen which could be cultivated up to 40 days. After dissociation of neurospheres, the neuronal spheres and isolated neuronal cells developed a intricated network with glia, neurons and interconnecting fibers, as seen in primary enteric cultures before. Immunostaining with PGP, ß-tubulin III, GFAP and S-100 showed positive stainings in different cell types. The Immunohistochemsitry of the histological preparations showed an increase of nestin postive cells in the inflamed appendices (
The IFN-γ-inducible enzyme indoleamine 2,3-dioxygenase (IDO) catalyzes the conversion of tryptophan to kynurenine. This enzyme is able to mediate antimicrobial functions which result in an inhibition of e.g.
We recently detected that IFN-γ-stimulated human MSC mediate antibacterial, antiparasitical, antiviral and immunoregulatory effects in an IDO-dependent manner. The IDO-dependency of these effects was shown by the abrogation of antimicrobial effects by the supplementation of tryptophan or by the addition of the IDO-specific inhibitor 1-methyltryptophan.
Here, it is shown that an infection with hCMV reduces the IFN-γ induced IDO-activity in human MSC. This suppressive effect of hCMV can be explained by an inhibition of the IFN-γ signalling pathway. We observed that the inhibition of IDO-activity results from a dramatic reduction of IDO transcription and translation in infected cells. Consequently, these cells were no longer able to restrict bacterial and parasitic growth and, furthermore, these hCMV-infected cells lost their IDO-mediated immunosuppressive capacity. This coherence between virus infection and inhibition of IDO-induction still gains more importance in the case of organ or haematopoetic stem cell transplantation. From clinical observations it is known that after transplantations an active hCMV infection results in an increased risk of infection and in a higher risk of transplant rejection. Both effects could be explained by the observations of the hCMV-infection’s influence on IDO-activity in our
The extracellular microenvironment plays a significant role in controlling cellular behaviour. Identification of appropriate biomaterials that support cellular attachment, proliferation and most importantly lineage-specific differentiation is critical for tissue engineering. Collagens exert important functions as cellular microenvironment and therefore make them ideally suited to use as biomaterial for tissue engineering (TE).
Here we designed collagen scaffolds from marine sources for cartilage TE. Since mesenchymal stem cells (MSCs) have multipotent capabilities including differentiation towards the chondrocytic lineage and MCSs have been used successfully in animal models to regenerate articular cartilage, we tested the MSCs concerning their cartilage-forming ability in marine collagen scaffolds.
Using adult mesenchymal stem cells from human bone marrow biopsies analyses show low cell attachment, low new extracellular matrix deposition as well as low differentiation capacity towards the chondrocytic lineage implying this as an unsuitable way for cartilage TE. However, primary adult chondrocytes seems to be a suitable cell source for engineering hyalinelike cartilage. Qualitative analyses of these bioscaffolds show chondrocyte cell adhesion to the matrix, cell proliferation as well as new matrix deposition. Based on (immuno)histological analyses this newly synthesized matrix consists of proteoglycans and collagens - the major constituents of hyaline, articular cartilage. Quantitative analyses on protein level show a collagen ratio of the chondrocyte specific marker collagen type II to the dedifferentiation marker of chondrocytes, collagen type I, of up to 50:1. From this, the
Since
Neuropathic pain is a very complex disease, involving several molecular pathways. Current available drugs have a generalized nature and act only on the temporal pain symptoms rather than being targeted towards the several mechanisms underlying the generation and propagation of pain. Nowadays, pain research is directing towards new molecular and cellular methods, such as stem cell therapy. Aim of this study was to verify whether human mesenchymal stem cell (hMSC) transplantation could be an useful cell-based therapy for neuropathic pain treatment.
We used spared nerve injury (SNI) mouse model of neuropathic pain to assess the possible use of hMSCs as anti-neuropathic tool. Bio-molecular, immunocytochemical and immunohistochemical analysis were carried out in order to verify stem cell-mediated changes in molecular mechanisms underlying pain development and maintenance.
Human MSCs were transplanted in the mouse lateral cerebral ventricle. Stem cells injection was performed 4 days after sciatic nerve surgery. Neuropathic mice were monitored 7, 10, 14, 17, and 21 days after surgery. Human MSCs were able to reduce pain like behaviours, once transplanted in cerebral ventricle. Anti-nociceptive effect was detectable from day 10 after surgery (6 days post cell injection). Transplanted MSCs reduced the mRNA levels of the pro-inflammatory interleukin IL-1ß mouse gene, astrocytic, microglial cell activation and premature senescence-associated neuronal suffering. Indeed, hMSCs were able to decrease the ß-galactosidase over-activation positive profiles in the cortex of SNI/hMSC-treated mice compared to SNI/vehicle mice.
Despite over fifty years of research there are no valid treatments over time and neuropathic pain can be classified as an incurable disease without treatment. Mesenchymal stem cell therapy represents the new promising potential treatment for neuropathic pain relief.
Stem cell therapy is actively being explored as a novel method to regenerate damaged myocardium. It has been established that the heart contains a reservoir of stem cells (c-kit+, sca-1+, isl-1+) having the capability for ex vivo and in vivo differentiation toward the vascular and cardiac lineages and showing cardiac regeneration potential. The aim of the present study is to characterize cardiac stem cells in the human heart tissue (appendix of the right atrium) harvested during coronary artery bypass grafting.
We analyzed samples of heart appendix by fluorescence-activated cell sorting and immunohistochemical method for markers of stemness (C-kit), hematopoietic cells (CD34, CD45), blood lineage markers (Lin) and tryptase. We isolated c-kit cells using explant culture and anti-c-kit antibody. Coculture with neonatal rat cardiomyocytes was used to analize cardiomyogenic potential of c-kit cells.
C-kit positive cells consist about 0,79±0,32% of total cell population of appendix of the right atrium and were largely negative for CD34 and cocktail of blood lineage markers Lin. We identified two populations of C-kit positive cells: about 60% of cells were C-kit(+)CD45(+), which might populate the heart via circulation and another one were CD45 negative (40%). Immunohistochemical research of autopsy samples of left ventricular tissue showed that majority of C-kit are distinctly positive for CD45 and tryptase, suggested that they are mast cells and only a small population of C-kit(+)CD45(-)triptase(-) cells represent human cardiac stem cells.
Using magnetic cell sorting c-kit positive cells could be successfully isolated from human heart tissue and expanded in vitro. C-kit cells undergo cardiomyogenic potential when cocultured with neonatal rat cardiomyocytes.
Thus appendix of the right atrium could be an alternative source of autologous cardiac stem cells.
Tendons are specialized tissues that connect muscle to bone and transmit the forces generated by muscle to bone, resulting in joint movements. They are characterized by a sparse vascular bed, low cell density and a decreased healing capacity. Only little is known about the molecular composition of tendon cells, their extracellular matrix and their progenitor cells. We analysed biopsies of human supraspinatus tendon (ST) from patients of various ages for the existence and the distribution of progenitor cells using immunhistochemistry. The expression of various cellular markers: for tendon cells (Scleraxis, Tenomodulin), extracellular matrix (ECM) proteins (Col1A1 and 3A1, MMP-2, -9, Lox), progenitor cells (Nestin, CD 133, CD11b, VCAM-1) and for cell differentiation (Aggrecan, Sox-9, Osterix, REST, CoREST) was studied with RT-PCR and qRT-PCR. In tendon biopsies of patients >50 years the yield of total RNA was significantly lower than in biopsies of younger patients (<50 years) and the expression frequencies of progenitor cell markers was decreasing with age.To further study tendon ageing we isolated RNA from Achilles tendons of young adult (6 weeks) and old NMRI mice (>24 months) and performed expression analysis of Nestin, Aggrecan, Scleraxis, Col1A1, Col3A1, MMP-2, MMP-9 and Lox. The expression of Scleraxis and Aggrecan was significantly down-regulated and Nestin mRNA was not detectable in tendons of old mice. The ECM proteins MMP-2, Col1A1, Col3A1 and Lox showed significant down-regulation in old mouse tendons, whereas MMP-9 was up-regulated in old mice.We also analysed biopsies of ruptured human ST for the expression of the above mentioned markers, interestingly the age dependency was not detectable anymore. Regarding to tissue degenerations, like fatty infiltrations, hypervascularization or cartilagenous modifications as well as the increase of the dense connective tissue in the ST, the expression of cellular markers revealed significant differences. Between males and females we found the most striking and highly significant differences in the expression of ECM markers. In conclusion progenitor cells are still detectable in older individuals although to a lesser extent, depending on individual susceptibility and pathological status of the patients.
Applying stem cell therapy in a failing myocardium, the dimension of an intra-myocardial cell homing is significant. Thus a comparison of the homing potential between glandular (GSCs) and mesenchymal stem cells (MSCs) was performed within the myocardium of a big animal model.
In African Bore Goats the intramyocardial homing and the engraftment of glandular stem cells and MSCs (CD133+) were evaluated. Glandular stem cells were characterized by red PKH26 respectively green PKH67 (MSCs) makers. After a left lateral thoracotomy and exposure of the left heart ventricle a mix of one million of each cell type was injected into three locations of the goat’s myocardium of the left ventricle. Myocardial samples were taken after one resp. three hours, others were harvested 6 weeks after injection (n=5). Frozen tissue slices were generated and examined for the marked cells.
Using a mix of an intra-myocardial injection of GSCs and MSCs, solely in MSCs (green) a significant cell migration into the surrounding myocardium (n=3) was observed, more expressed after 3 hours than after one hour. Additionally within 5 goats with three intra-myocardial injections in each after 6 weeks nearly all GSCs remained within the myocardium while the MSCs disappeared almost completely. Within the frozen myocardial slices 98% of the marked stem cells were identified as GSCs (red) but just 2% as green MSCs. (P≤0,05)
Due to a 98 % homing of GSCs combined with the ability developing cardiomyocyte like cells, glandular stem cells might become a very promising treatment option in the therapy of a failing myocardium.
Following acute myocardial infarction (MI), the heart suffers, beside ischemia-induced direct myocardial injury, from a subsequent indirect damage through improper in-flammatory reaction. A wealth of information indicates that the renin-angiotensin system (RAS) can interfere with acute cardiac remodelling and inflammation processes during cardiovascular injury. Given the recent observations showing that AT2 receptors (AT2R) are abundantly expressed in immunocompetent cells and involved in cell-mediated inflammatory injury, it appears likely that AT2R may exert their actions through interfering with CD4+ T cell-involved inflammatory processes in response to ischemia-induced cardiac injury.
We isolated CD4+ T cells from peripheral blood of rats with acute myocardial infarction and healthy donors using Ficoll gradient centrifugation and MACS technology. The CD4+AT2R+ and CD4+AT2R- T cell populations were further purified by FACS sorting. The purity of isolated cells and the expression of AT2R and various cytokines were confirmed using FACS analysis, cytospin staining and RT-PCR methods. To study the role of AT2R on cytokine production, the CD4+AT2R+ and CD4+AT2R- T cell were treated with AT2R agonist (compound 21), or angiotensin II in combination with AT2R antagonist PD PD123319 (PD).
Here, we defined the CD4+AT2R+ T cell subpopulation in the peripheral blood of rats and humans. These blood CD4+AT2R+ T cells were characterized by upregulated expression of transcription factor forkhead box protein FOXP3 [
Several data of the past 15 years suggest that cell fusion is not only a process of normal development and tissue homeostasis, it is also assumed to participate in cancer progression. Because of the high fusogenic capacity of cancer cells cell fusion may promote diversity in cancer cell populations. Therefore fusion of a cancer cell with another cancer cell, an immunocompetent cells or even an adult stem cell can give rise to hybrid cells with new properties.
What is the role of cell fusion in cancer progression?
Characterisation of hybrid cells derived from spontaneous fusion of murine 67NR-Hyg breast cancer with puromycin resistant bone marrow derived MSCs, from Tg(GFPU)5Nagy/J mice
Realtime PCR Arrays for analysing breast cancer and multi drug resistance associated genes revealed an increased expression of drug resistance proteins, in particular ABC-transporters. These findings correlated with high ABC-transporter mediated Rhodamine 123 efflux of hybrid cells in comparison to parental cell lines detected by FACS analysis. XTT-proliferation-assay after culturing cells over a period of 3 days among different concentrations of chemotherapeutic agents mentioned above showed an increased drug resistance of hybrid cells compared to parental cells for Doxorubicin, 17-DMAG, Etoposide and Paclitaxel. Hybrid cells exhibited an altered morphology under influence of chemotherapeutic agents, especially Doxorubicin and 17-DMAG, but even survived at concentrations of 10μM. Drug resistance of hybrid cells may be reversed by addition of 50μM Verapamil in some cases of tested chemotherapeutic agents.
We conclude that cell fusion between breast cancer cells and MSCs can give rise to hybrid cells with altered properties that direct enhanced ABC-transporter mediated drug resistance and therefore may promote cancer cell survival during chemotherapy.
Little is known about the processes by which cancer stem cells arise in the different tissues. Our analysis of aggressive squamous cell carcinomas (SCCs) of the salivary gland in human patients suggested a lspecific link to the Wnt/b-catenin and Bmp signalling systems. Using a genetically modified strain of mouse in which Wnt signalling is up-regulated and Bmp is suppressed, we found that Wnt/b-catenin promotes the transformation of normal stem cells into cancer stem cells through an epigenetic mechanism. Mouse SCCs contained high numbers of CD24+CD29+ cancer stem cells. As few as 500 of these cells sufficed to cause tumours in NOD/SCID mice. This contrasts mice in which only one of the signalling systems was altered albeit high numbers of stem cells and efficient tissue regeneration, they had no apparent tumours. We discovered that the difference of normal compared to cancer stem cells in the salivary gland is an up-regulation of specific pluripotency genes, e.g. Dppa5, as well as a global changes in trimethylated Lysine 4 and 27 of histone 3. This indicates an increase of active chromatin and a decrease in the repressive form, which suggests a mechanistic explanation for the change of cell fate. Cancer stem cells of the salivary gland grow as non-adherent spheres and retain the capacity for differentiation if b-catenin is inhibited. This depends on repressive chromatin, as shown by the fact that 5-azacytidine or HDAC-inhibitors restore stemness. Our data opens new strategies for future cancer therapies in humans.
We have shown that leukemia stem cells candidates (LSCC) can be prospectively identified by high activity of aldehyde dehydrogenase (ALDHbr) among the leukemia blasts from the marrow of patients with acute myeloid leukemia (AML). These LSCC demonstrated functional characteristics of stem cells
In this report we have studied the relationship between the frequency of LSCC at diagnosis with persistence of leukemia blasts after induction chemotherapy as well as with long-term clinical outcome. We have identified subsets among the LSCC and correlated their individual functional properties with the corresponding marker profile using single cell sorting.
The percentage of LSCC (ALDHbr) in
Thus high frequencies of LSCC at the time of diagnosis predict persistence of leukemia blasts, failure to achieve CR within the first cycle and poor overall clinical outcome and hence represent an independent poor prognostic factor.
Malignant brain tumors are amongst the most lethal solid tumors. The gold standard in therapy is concurrent radiochemotherapy, using the alkylating compound temozolomide (TMZ). It has been proposed that stem-like tumor cells mediate therapy resistance and regrowth.
We identified stem-like cells in human glioblastoma, gliosarcoma, oligodendroma and oligoastrocytoma. These cells exhibited a substantial heterogeneity with respect to proliferation rate and growth as spheres, adherent or semi-adherent cultures. Based on the expression of the intermediary filaments Nestin and GFAP, the transcription factors Sox2, Oct4, Nanog, as well as regulators and signalling molecules, such as p53, EGFR, PDGFRa, and PTEN we defined subtypes of stem-like brain tumor cells (SClC). CD133 expression varied largely between different SClC lines. We determined the responsiveness of SClC subtypes to TMZ and chloroquine and related it to the expression of the repair enzyme MGMT and key players in apoptotic and autophagic processes.
Dose curves showed that responsiveness to TMZ was significantly different. Strong responsiveness to TMZ did not only depend on the methylation status of the MGMT promoter but on additional features, some of which appeared to be related to the SClC stemness state. Co-application of chloroquine, a drug used in malaria prevention, which presumably affects autophagy, enhanced responsiveness of SClC to TMZ in a dose-dependent manner. Both, TMZ and chloroquine induced cleavage of PARP, a key player in apoptosis and a measure for caspase 3 activity. The levels of cleaved PARP, however, differed largely between the various SClC lines. Expression of Beclin and LC3B, proteins which are associated with different autophagy-associated processes were up-regulated in some but not all SClC-lines. This indicates, that different SClC subtypes respond differently to cell death inducing reagents and parallels our findings concerning the different proliferation and differentiation capacity of SClC subtypes.
The incidence of adenocarcinomas of the esophagus has increased during the last years. Many studies focused on the presence of cancer stem cells that have the capacity to regenerate tumors. Tumor initiating cells have the ability of self-renewal and proliferation, are resistant to drugs, and might express surface markers that were associated to stem cells. Moreover, recent studies demonstrated that radioresistance might be also caused by cancer stem cells (CSC). Isolation and identification of cancer stem cells in human tumors and in tumor cell lines are important steps for a further functional characterization of cancer stem cells, in order to find new ways to destroy them. The present study focused on characterization by flow-cytometry of the antigen expression of several biomarkers (CD24, CD44, CD71, CD105, CD117, CD133, CD166, CD200, EpCAM, E-cadherin, beta-catenin) associated to esophageal cells KYSE-150. Cells were cultivated in DMEM:F12 cell culture medium containing different amounts of fetal calf serum (FCS) (from 2% to 20%) or serum replacement (10% - 20%) and growth factors (bFGF, EGF). Moreover, CD44+ cells isolated after magnetic sorting using were further cultivated and antigenicity of subpopulations compared.
In addition, proliferation through cell cycle phases was also studied by using propidium iodide staining, followed by flow-citometry analysis. The results obtained demonstrated a differential expression of the biomarkers taken under study, depending on cell culture conditions used. Proliferation was also influenced since DNA analysis showed major changes in S-phase distribution. These results might add to identification of esophageal stem cells and facilitate the studies on carcinogenesis. Further studies will bring new data concerning evaluation of radio- and chemoresistance, in order to establish new protocols which might eliminate/diminish the tumours, significantly contributing to the immunotherapeutical management of esophageal tumors.
Cylindrospiradenomas are benign skin appendage tumors that can develop at multiple sites of hair follicle bearing skin as a result of Brooke-Spiegler Syndrome (BSS). The autosomal-dominant BSS is associated with mutations in the
Interestingly, BSS patients also show extensive T cell infiltrates in tumors and tumor-free regions of their scalp and strong, ectopic expression of MHC class II molecules on the ORS of their hair follicles. This suggests that inflammatory processes precede or accompany tumor formation and growth.
To test whether anti-inflammatory agents inhibit tumor growth, we established a serum-free assay normally used for hair follicle organ culture that allows the maintainence of cylindro(spiradeno)ma fragments for up to 6 days in vitro. Addition of Na-salicylate resulted in increased cell death in treated compared to untreated cylindrospiradenoma fragments. Thus, administration of anti-inflammatory agents may offer a pharmacological alternative to surgical cylindrospiradenoma management.
Glioblastoma multiforme (GBM) is one of the most lethal solid tumors. GBMs grow invasively, develop resistance to radiation and chemotherapy, and frequently recur. Recently, glioma were classified into four distinct molecular entities, the classical, mesenchymal, neural and proneural subtype based on genomic and transcriptomic data obtained with brain tumor tissue. It remains unclear whether stem-like glioma cells, which constitute a small subfraction of the brain tumor cells, could be grouped into similar categories. We analyzed the features of stem-like glioma cells from glioblastoma, gliosarcoma, astrocytoma, oligodendroma and oligoastrocytomas.
Primary cultures of glioblastoma and gliosarcoma consisted of varying amounts of different cell types. Growth in serum-depleted medium containing the growth factors EGF and bFGF resulted in progressive enrichment of stem -like cells. Co-expression of the intermediary filaments Nestin and GFAP as well as prominin-1/ CD133, factors typically found in adult neural stem cells/progenitors, was observed only in a subfraction of stem-like cells and expression of the transcriptional regulator Sox-2 varied largely. In keeping with their stemness features, coexpression of Nestin and the stemness factors Nanog or Oct4 was detected in all populations of stem-like tumor cells. Expression of neurofilaments, doublecortin, DLX, Pax6, PSA-NCAM and other neural markers was additionally observed, suggesting that the majority of the populations of stem-like cells would belong to a neural or proneural group. mRNAs of the factors Myc, Gli, PTEN, Rest, Hif1α and p53, which affect stemness and tumorigenicity, respectively, were detected in all populations of stem-like cells. The major differences, however, concerned the protein expression levels. In particular, the level of p53 protein varied largely, even though p53 RNA was present in all primary cultures, suggesting malfunctioning of the p53-pathway in a subset of glioma cells. In addition, PTEN and AKT/PKB phosphorylation differed.Together our data reveals (i) a clear heterogeneity of stem-like glioma cells from different gliomas and gliosarcomas and (ii) differences between stem-like cells and their non stem-like counterparts from the same tumors.
Malignant brain tumors are amongst the most lethal solid tumors. The standard treatment consists of surgical resection followed by adjuvant radiochemotherapy. It has been proposed that glioma cells with stem cell features contribute to both, initiation and progression of primary and recurrent glioma. It remains elusive, how their growth is regulated and how the stem-like cells, which constitute only a subfraction of the brain tumor, could contribute to tumor progression and relapse. Here we investigated the responsiveness of stem-like tumor cells to mitogens and growth factor withdrawal. Moreover, we analyzed the expression of receptor tyrosine kinases and peptide growth factors in stem-like brain tumor cells and their non-stem-like conunterparts from the same tumor.
We identified stem-like cells in all brain tumor biopsies analyzed so far. These cells exhibited a substantial heterogeneity with respect to morphology, proliferation rate, growth modus and the expression of neural markers and stemness factors. Therefore, we refer to these cells as subtypes of stem-like cells (SClC-subtypes). Both, bulk tumor cells and SClC-subtypes coexpressed several receptor tyrosine kinases (RTK), including members of the HER-family (HER: human epidermal growth factor (EGF) receptor), as well as receptors for PDGF (platelet-derived growth factor), FGF (fibroblast growth factor) and SCF (stem cell factor). Except for the SCF receptor c-kit, RTK expression appeared unrelated to the cell type, tumor type, and the WHO grade of the original tumor. SClC-subtypes were maintained in serum-free medium containing the growth factors EGF and bFGF (basic fibroblast growth factor). Withdrawal of either one or both growth factors only moderately impaired growth in a cell line-specific way, indicating that stem-like glioma cells are largely independent of the exogenous growth factor supply. This is explained by the co-expression of EGF, heparin-bound EGF, bFGF, SCF and both, PDGF A and B in a large subset of the SClC subtypes. The expression pattern showed some variation between the various SClC subtypes and amongst SClC and the corresponding bulk tumor. The responsiveness to EGF and bFGF differed, and PDGF AB mediated a cell line-specific growth reduction. Together our data suggest that the bulk tumor cells and SClC within malignant gliomas exert autocrine growth control and that the various cell populations within the tumors may cross-talk via paracrine mechanisms.
Glioblastoma multiforme (GBM) is one of the most lethal solid tumors. GBMs grow invasively, develop resistance to radiation and chemotherapy, and frequently recur. The gold standard in GBM therapy is concurrent radiochemotherapy, using the alkylating compound temozolomide (TMZ). It has been proposed that stem-like tumor cells mediate therapy resistance and regrowth. Furthermore, resistance against TMZ seemed related to an altered apoptotic and autophagic death machinery. We determined the responsiveness of subtypes of stem-like glioma cells to TMZ and chloroquine, compared it to the behaviour of the established glioma cell line U87, related it to the expression of MGMT and ACB-transporters, and analyzed apoptotic and autophagic processes.
All of the primary cultures with stem-like cells exhibited self-renewal, although growth behaviour and proliferation rate differed largely. CD133 expression varied between different cell lines. Dose curves showed that responsiveness to TMZ was significantly different between the various stem-like cell lines, as BrdU incorporation was inhibited with different efficacy. Strong responsiveness to TMZ was related to the methylation of the MGMT promoter, but not to the expression levels of the ABC-transporters analyzed or p53. Co-application of chloroquine, a drug used in malaria prevention, which presumably affects autophagy, enhanced responsiveness of stem-like cells and U87 to TMZ
Head and neck squamous cell carcinoma (HNSCC) is one of the most common solid neoplasms worldwide. Unfortunately, the mortality rates are still high due to local tumor invasion and to a high predilection for the development of relapses and metastases. The cellular and molecular mechanisms responsible for tumor aggressiveness and its response to chemo- and radiation therapies remain mostly unknown. It is becoming more and more obvious that tumor progression and metastasis are stem cell driven processes and that these ‘tumor stem cells’ have to be in the focus of innovative therapeutic and diagnostic approaches.
Our aim is to identify and characterize tumor stem cell populations in HNSCC and to analyze and visualize their migration activity and their tumor inducing potential. Therefore, cells were labelled with superparamagnetic dextran coated iron oxide nanoparticles in order to make them detectable via ‘magnetic particle imaging’ (MPI), which is a new quantitative imaging technique capable of determining the spatial distribution of superparamagnetic nanoparticles at high temporal and spatial resolution. Tumor cells’ nanoparticel uptake was corroborated using flow cytometry analysis of FITC labelled particles as well as electron microscopy. We will show the progress of our investigations.
Superparamagnetic iron oxide nanoparticles (SPIO) are of increasing interest in biomedical applications such as targeted cancer therapy, magnetic field-triggered drug release or
Cancer stem cells (CSCs) are thought to be responsible for tumor progression and therapy resistance. They have been identified in a variety of human tumors as well as in cancer cell lines. Cell lines might therefore serve as an attractive source for CSC
Altogether, 12 colorectal cancer cell lines of carcinomas and metastases were analyzed by flow cytometry using a panel of six CSC surface markers (CD326, CD133, CD44, CD166, Msi-1 and Gpr49). Expression frequency of CSC markers was divided into four categories with
All cell lines but one (HT29) showed a stable expression pattern throughout all four replicates. HT29 showed an increased expression for CD133 and CD166 over time and was thus excluded from further analyses. The majority (91%) of cell lines showed high expression for CD326. About half to one third of the cell lines expressed at high frequency CD44 and CD166 (in 45%) and CD133 (in 36%).
In contrast, most cell lines expressed Msi1 and Gpr49 at low frequency. Since CD326, Msi1, and Grp49 did not show any major expression differences in between the various cell lines, we checked for potential correlation of CD44, CD133 and CD166 expression differences with clinical parameters. However, we could not observe any significant correlation.
Colorectal cancer cell lines do harbour to a substantial amount CSCs. The frequency of such shows a distinct variability among different cell lines particularly for CD44, CD133, and CD166. This might be due to different clinical properties such as tumor progression and metastasizing as reported previously. In our study, case numbers were too small to validate such reports. Interestingly, the frequency of CSC remained considerably stable over multiple passages within the individual cell line, except for HT29. We suggest excluding HT29 from
The primary characteristics of adult stem cells are maintaining prolonged quiescence, ability to self-renew and plasticity to differentiate into multiple cell types. These properties are evolutionarily conserved from fruit fly to humans. Similar to normal tissue repair in organs, the stem cell concept is inherently impregnated in the etiology of cancer. Tumors contain a minor population of tumor-initiating cells, called “cancer stem cells” that maintain some similarities in self-renewal and differentiation recognized as features of normal adult stem cells. Therefore, various methods developed originally for the analysis and characterization of adult stem cells are being extended to evaluate cancer stem cells. Relevant methods that are used generally across normal stem cells as well as cancer stem cells are summarized
The hypothesis that cancer could be a stem cell disease actually existed for several years before definitive ‘proof-of-concept’ experiments unequivocally established their involvement in initiating the tumorigenic state. Over the last decade, the combination of fluorescence-activated cell sorting with functional read-outs e.g. in vitro colony forming units in colony initiating assays, and competitive repopulating assays in animal models has enabled the prospective purification and enrichment of cancer stem cells (CSCs) in leukemia and also in certain solid tumors. Combination of two or more of these methods for validation of cancer stem cells appears to be a promising approach for the precise isolation and analysis of cancer stem cells. However, a major limitation for research in the field of stem cells and CSCs has been the lack of research resources in the form of in vitro models for these studies
Cancer is one of the leading causes of death among the human race. No valid modalities of treatment other than surgical treatment have been established for this disease. We aimed to identify and to characterize cancer using large number of human monoclonal antibodies (mAbs) which are specific against their surface for new molecular targeted immunotherapy. In order to find proper targets for therapeutic antibodies against cancers we developed a screening strategy. We used a huge phage library of human antibodies. At the first step we comprehensively isolated many monoclonal antibodies (mAbs) that specifically bound to surface of cancer cells. Development of ICOS (Isolation of antigen/antibody complexes through organic solvent) method allowed us to succeed in isolation of a huge number of mAbs with various characteristics (Y Akahori et al. 2009). At the next step we selected clones that showed tumor-specific staining patterns in immunohistochemical (IHC) analysis by using many fresh cancer tissues reseted. Many surgeons took part in this project. Finally the antigens recognized by these clones were identified by immunoprecipitation (IP) followed by analysis with mass (MS) spectrometry (G Kurosawa et al. 2009). We have succeeded in identification of 29 tumor-associated antigens (TAAs) and in isolation of 441 human mAbs that specifically bound to one of the 29 TAAs (G Kurosawa et al. 2008). In these screenings of the library, rounds of the selection process, mixing of cells and phage particles centrifugation growth of phages, were repeated three to four times in each screening. Therefore, numbers of phages of the clones whose antigens were abundantly present on the cell surface increased during the screenings. Recently we developed a new method for isolation of clones whose antigens were less abundantly present on the cell surface. Hence, we would like to talk on these methodology and discuss regarding this “A novel antibody based approach to Cancer Treatment” in this plenary session.
There is increasing evidence that the transplanted bone marrow stromal cells (BMSC) significantly promote functional recovery after central nervous system (CNS) damage in the animal models of various kinds of CNS disorders, including cerebral infarct, brain contusion and spinal cord injury. However, there are several shortages of information when considering clinical application of BMSC transplantation for patients with neurological disorders. In this paper, therefore, we discuss what we should clarify to establish cell transplantation therapy in clinical situation and describe our recent works for this purpose.
The BMSC have the ability to alter their gene expression profile and phenotype in response to the surrounding circumstances and to protect the neurons by producing some neurotrophic factors. They also promote neurite extension and rebuild the neural circuits in the injured CNS. Using optical imaging and MRI techniques, the transplanted BMSC can non-invasively be tracked in the living animals for at least 8 weeks after transplantation. Functional imaging such as PET scan may have the potential to assess the beneficial effects of BMSC transplantation. The BMSC can be expanded using the animal protein-free culture medium, which would maintain their potential of proliferation, migration, and neural differentiation.
It is urgent issues to develop clinical imaging technique to track the transplanted cells in the CNS and evaluate the therapeutic significance of BMSC transplantation in order to establish it as a definite therapeutic strategy in clinical situation in the future
Recent studies have shown that mesenchymal stem cells isolated from post natal human dental pulp, (Dental pulp stem cells-DPSCs) which is from permanent teeth and SHED (stem cells from human exfoliated deciduous teeth), the Periodontal ligament stem cells (PDLSC) and Stem cells from root Apical papilla(SCAP)have the potential to differentiate into cells of a variety of tissues including heart, muscle, cartilage, bone, nerve, salivary glands, teeth etc(
After obtaining written informed consent, 24 teeth that were extracted for therapeutic or cosmetic reasons from 16 patients were used in this study. The specimens were transported from the clinic to NCRM lab taking 6 to 48 Hrs. For removal of the pulp tissue, the teeth were split obliquely at the Cementoenamel junction and the pulp tissue was isolated using brooches. The extracted pulp tissues were subjected to digestion using Collagenase type- I and type II at 37°C for 15- 30 minutes. The digested cells were filtered with 70μm filter and centrifuged at 1800 rpm for 10 minutes. The pellet was then suspended in Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F12 supplemented with 15% fetal bovine serum , 100 U/ml penicillin, 100 μg/ml streptomycin,2 m M L -glutamine, and 2 m M nonessential amino acids (
Viable Dental Pulp tissue-cells were obtained after transportation of up to 48 hrs and the in vitro growth of cells was initially slow but colonies were identified from the 10th day onwards. The cells were harvested at different intervals of 14-28 days for each sample based on their growth and subjected to H & E staining. The H & E staining of the cultured cells of all the samples showed positive results.
We are able to transport extracted teeth and derive viable dental pulp tissue cells after enzymatic digestion and multiply them in culture after a maximum of 48 hrs after transportation. The cells could be grown in culture with a morphology resembling dental pulp stem cells while in culture expansion and in H&E studies. Further characterization of the cells is necessary to confirm their Stemness.
The Enteric Nervous system (ENS) is a part of the Peripheral nervous system (PNS) that controls the peristaltic activity of the gut wall which is essential for propulsion of food in the digestive tract. It is composed of a large number of neurons and glial cells, distributed throughout the length of the gut. These ganglion cells develop from the neural crest in the embryo. Failure of complete colonization of the gut by these enteric neural crest cells during early development of life results in absence of ganglia or neurons in a portion of the gut, usually the colon which leads to aperistaltis and severe intestinal obstruction. This is known as Hirschsprung’s disease (HSCR) also known as congenital megacolon. HSCR affects 1 in 4500 newborns (
The postnatal gut full thickness biopsy samples of size 2-4 mm were obtained using from 13 patients undergoing gut resection surgery after informed consent. The samples were washed in Phosphate Buffer saline and using forceps, the outer smooth muscle layers along with the myenteric plexus were peeled off from the underlying tissue as strips. The strips were washed in Phosphate Buffer saline (PBS) and treated with 1mg/ml Collagenase/Dispase mixture in PBS for 30-45 min at 37°C. The digested cells were filtered with 70μm filter and the cell suspensions were centrifuged at 1800rpm for 10 mins. The pellet obtained was suspended in DMEM/F12 medium supplemented with penicillin (100U/ml), streptomycin (100 μg/ml), L-glutamine (2 mmol/L), growth factors like bFGF (20ng/ml) and EGF (20ng/ml)(2). Cell counting was done by Trypan Blue dye exclusion method and the cells were seeded in cell culture dishes coated with Fibronectin. The flasks with cells were incubated at 37°C with 5% CO2 for varying periods from 18 days-28 days. The cells were observed daily and media change was done every 2-3 days.
In all the samples, the Neurosphere like bodies (NLBs) were observed in the culture from 10th day onwards which were then subjected to histological and immunohistochemical studies. H&E staining showed positive for neural cells and Immunohistochemistry yielded positive for S-100, normally present in cells derived from the neural crest and Neuron Specific Enolase (NSE) a neuronal specific marker.
We could successfully isolate and expand Human Enteric Neuronal cells from postnatal gut biopsy samples. Further research is warranted to utilize these Enteric Neuronal Cells for Cell based therapies to treat Hirschsprung’s disease.
Recent studies have indicated that bone marrow stromal cells (BMSC) have the potential to improve neurological function when transplanted into animal model of central nervous system (CNS) disorders. However, there still exist several questions to solved prior to clinical application. In this study, therefore, we aimed to clarify the optimal delivery route of BMSC transplantation over a reasonable time window.
The rats were subjected to permanent middle cerebral artery occlusion. The BMSC were labeled with quantum dot (QD) 800. The labeled BMSC were transplanted into the infarct brain directly or intravenously at 7 days after the insult. Motor function was serially assessed. The BMSC were also tracked using near infrared (NIR) fluorescence imaging technique every week. The fate of the transplanted BMSC was examined at 5 weeks after transplantation, using Immunohistochemistry.
Direct, but not intravenous, transplantation of BMSC significantly enhanced functional recovery. NIR fluorescence imaging could visualize their migration towards cerebral infarct in directly, but not intravenously, injected animals. The findings were supported on histological analysis. Thus, the BMSC were widely engrafted in the infarct brain in the directly injected animals, but few BMSC were observed in the intravenously injected ones.
This study strongly suggests that direct transplantation of BMSC may be more beneficial in treating patients with ischemic stroke than their intravenous transplantation. Therapeutic time window must be called into account when considering the route of BMSC transplantation.
Vascular endothelial cells play a major role in wound healing and also in growth of the tumors. Angiogenesis can be a target for treating diseases that are due to either poor vascularisation or decreased blood supply as in stroke, ulcers, heart disease, etc or abnormal and increased vasculature like in tumours. Application of specific compounds that may inhibit or induce the creation of new blood vessels in the body may help in the treatment of such diseases (
Isolation and Comparison of culture characteristics of Human microvascular endothelium cultured conventionally and in novel nanomaterial scaffold and further study the morphological and molecular characteristics of microvascular endothelial cells under normal gravity against simulated micro gravity.
The human Omentum samples were obtained using surgical procedures after informed consent. The microvascular endothelial cells were isolated following the protocol described by Scott et al (
A 21% increase in number of NO positive cells was observed in the cells cultured on the novel nanopolymer, while a 1.7 fold increase in nitrite production was detected in Group I in comparison to that of Group II.
Our data suggests the presence of more NO driven nitrite in the Microvascular endothelial cells cultured in the nanopolymer. In the next phase of the study, the cells thus isolated from both the groups will be subjected to simulated microgravity of 10-3 g in a clinostat at the AUKBC Vascular Biology Lab and the morphological and molecular characteristics of Microvascular endothelial cells will be studied to identify whether the effects of microgravity on Microvascular endothelial cells is organ specific.
Patients with complete spinal cord injury at cervical level, mostly lead a life with quadriplegia. We report a case of 22 year old male, who became quadriplegic after C4-C5 injury and has improved after five injections of in-vitro expanded autologous bone marrow mononuclear cells (ABMMNC).
The patient developed complete quadriplegia following fall from height. MRI suggested C5 burst fracture with retropulsion of C5 vertebrae and cord contusion at C4-C5 and underwent decompression and fixation four days post injury. After surgical fixation, bone marrow was harvested twice and a portion of the MNCs were expanded in-vitro and injected and another portion was cryopreserved which were later expanded and injected through lumbar puncture. The first injection was given 13 days after surgery. The first specimen of BM had CD34 of 0.18% and was injected twice; once immediately after harvesting (CD34:1.57%) and later after cryopreservation for 11 months (CD34: 3.33%). The second specimen had a CD34 of 0.52% and was injected thrice; twice after harvesting with CD34 of 1.28% and 6.23% respectively and later after cryopreservation for four months (CD34: 14.03%). The expansion was done in a GMP compliant clean room using autologous serum and the specimens were subjected to aerobic culture and Endotoxin tests before each administration.
Investigations done (i)Post injury, (ii) Post decompression and (iii) one year (iv) One and half year after the injury have shown objective improvements with Light touch score (20-22-40-48), Pin prick score (20-20-32-40), Anal sensations (Nil-Nil-Present), Motor Score (11-11-22-26), Sensory level (C6-C6-C6-C6), Motor level (C5-C5-C6-C6), Neurological level (C5-C6-C6-C6), Spinal cord independence measure (9-12-45-50) and Barthel index (0-0-30-40). Repeat MRI show gliosis at C5. The ASIA score has improved from A to B with no adverse reactions.
Multiple intrathecal injections of in-vitro expanded ABMMNCs in cervical-spinal cord injury was found to be safe and gradual objective improvements have been observed over a year and a half without any adverse outcome.
We have fabricated new types of polymer hydrogels and polymer nanocomposites, i.e., nanocomposite gels (NC gels) and soft, polymer nanocomposites (M-NCs: solid), with novel organic/inorganic network structures. Both NC gels and M-NCs were synthesized by in-situ free-radical polymerization in the presence of exfoliated clay platelets in aqueous systems and were obtained in various forms such as film, sheet, tube, coating, etc. and sizes with a wide range of clay contents. Here, disk-like inorganic clay nano particles act as multi-functional cross linkers to form new types of network systems. Both NC gels and M-NCs have extraordinary optical and mechanical properties including ultra-high reversible extensibility, as well as a number of new characteristics relating to optical anisotropy, polymer/clay morphology, biocompatibility, stimuli-sensitive surfaces, micro-patterning, etc. For examples, the biological testing of medical devices, comprised of a sensitization test, an irritation test, an intracutaneous test and an in vitro cytotoxicity test, was carried out for NC gels and M-NCs. The safety of NC gels and M-NCs was confirmed in all tests. Also, the interaction of living tissue with NC gel was investigated in vivo by implantation in live goats; neither inflammation nor concrescence occurred around the NC gels. Furthermore, it was found that both N-NC gels consisting of poly (N-isopropylacrylamide)(PNIPA)/clay network and M-NCs consisting of poly(2-methoxyethyacrylate)(PMEA)/clay network show characteristic cell culture and subsequent cell detachment on their surfaces, although it was almost impossible to culture cells on conventional, chemically-cross linked PNIPA hydrogels and chemically crosss linked PMEA, regardless of their crosslinker concentration. Various kinds of cells, such ashumanhepatoma cells (HepG2), normal human dermal fibroblast (NHDF), and human umbilical vein endothelial cells (HUVEC), could be cultured to be confluent on the surfaces of N-NC gel or dried N-NC gels and M-NC film, regardless of their thickness. Also, it was found that cells cultured on the surfaces of N-NC gels and M-NCs could be detached in the forms of sheets of cells or single cells without trypsin treatment, but by just decreasing the temperature to 200C. Thus, the serious disadvantages (intractability, mechanical fragility, optical turbidity, poor processing ability, low stimulus sensitivity, etc.) associated with the conventional, chemically-cross linked polymeric materials were overcome in NC gels and M-NCs. Also, NC gels and M-NCs can be used as new types of substrata with ability of cell culture and subsequent thermo responsive cell detachment.
Although transplantation is one of the major medical achievements of the last half century, the shortage of organs and need for long term immunosuppressive therapy limits its usefulness. Advances in stem cell therapy has the potential both to overcome the shortage of organs but also to provide novel ways of reintroducing a tolerogenic state in patients who require life saving transplantation therapy.
Understanding mechanisms of rejection which involve both innate and adaptive immunity would allow for novel therapeutic approaches to eliminate or avoid the use of toxic immunosuppressive agents. The evolving era of functional genomics in organ transplantation has been supported by advances in gene profiling, sequencing, proteomics, antibody profiling and bioinformatics. Thus, heralding a new era of intelligent and personalized monitor and therapy. Molecular and cell based biomarkers and now emerging which may be useful to monitor the immune status of the patient and it is anticipated that over the next several years these will detect rejection for immune events before a transplanted organ or cell is damaged. Patterns of genomic biomarkers are also being developed which may predict patients, who achieve tolerance which may not only be useful in the setting of transplantation, but also in patients with autoimmune disease. The ultimate goal of future studies will be to identify markers with sufficient predictive value to improve graft survival, limit graft injury from under immunosuppression and reduce patient morbidity from over immunosuppression.
These approaches will also be critical for successful stem cell therapy where it is known that immunologic barriers limit its adoption.
Carlos Lima et al. who are pioneers in this field reported their clinical pilot study of olfactory mucosa transplantation for chronic spinal cord injury. They showed the safety and feasibility of it. Olfactory mucosa contains the olfactory ensheathing cells and neural stem cells. Recent studies have demonstrated the potential therapeutic role of both cells in spinal cord injury. We have already reported the effectiveness of olfactory mucosa transplantation for rat spinal cord injury. Furthermore we indicated the reconstruction of cortico-spinal tract by BDA (biotinylated dextran amine) tracer study with the olfactory mucosa transplantation. We elucidated how grafts of nasal olfactory mucosa repair the injured rat spinal cord as compared with the nasal respiratory mucosa containing no olfactory ensheathing cell and neural stem cell. The spinal cord of recipient rats (adult female Sprague-Dawlley rats; 10 rats; 160-180g) was exposed at The 8-9 level, and a contusion injury was produced using the weight drop device developed at New York University. The exposed cord was moderately contused by a 10g weight that dropped from a height of 75 mm. A couple of weeks after injury, the injury site were exposed and posterior sulcus of the spinal cord was opened. Minced olfactory mucosa or respiratory mucosa derived from GFP rats were transplanted into the sulcuses. The BBB score in each animal was observed at 1, 2, 4 and 8 weeks after the transplantation. The recovery of the hind limb movement in the olfactory mucosa transplanted rats improved significantly compared to the respiratory mucosa transplanted rats. In histological assessment, the expression of p75NGFR and GFAP was strong in the olfactory mucosa grafts at 1 and 2 weeks after the transplantation and it was decreased at 8 weeks after the transplantation. The expression of p75NGFR and GFAP was not observed in the respiratory mucosa graft. The expression of Neurofilament was observed strongly at the site in the olfactory mucosa transplanted rats. The numerous fibres strongly stained with Neurofilament were surrounding the GFP positive cells and penetrating the transplanted olfactory mucosa. There were no apparent Neurofilament stained fibres at the marginal spinal cord. As we have already reported, olfactory mucosa transplantation for spinal cord injury has a certain effectiveness for the hind limb motor recovery. In this study, we recognized the numerous axons which surround the transplanted cells and penetrate the mucosa at the transplanted site without marginal spinal white matter. Olfactory mucosa might be more suitable niche than white matter which contains inhibiting factor for axonal regeneration in spinal cord. To succeed the neuronal regenerative therapy, cells, factors and scaffold have been required. Olfactory mucosa might have all of them. We are now performing the clinical trial of olfactory mucosa transplantation for chronic complete spinal cord injuries in Japan. We could have four patients so far and recognize the voluntary EEG of their thigh.
The cartilage injuries demand novel therapeutic approaches as the success rates of the current conventional strategies for the repair of injured articular cartilages are not that encouraging. Earlier we have reported that the Thermoreversible Gelation Polymer (TGP) is an ideal scaffold for human chondrocyte expansion in vitro. In this study, we report the preliminary results of the in vitro expansion, characterization and experimental in vivo transplantation of chondrocytes in a rabbit model of cartilage injury
Nine rabbits were included in this study scheduled for two years, after approval by the ethics committee. In the first animal, Chondrocytes were isolated from the weight bearing area of patellar groove in the left hindlimb and cultured in TGP Scaffold and maintained at 37°C in 5% carbon dioxide incubator for 64 days without growth factors. Then the TGP-Chondrocyte construct was transplanted into an experimental defect created in the knee of the right forelimb of the same rabbit. After a period of 10 weeks, a biopsy was taken from the transplanted region and subjected to morphological analysis, characterization by histopathology (H&E stain) and Immunohistochemistry (S-100 staining).
The chondrocytes in the 3D TGP culture had round to oval shaped morphology without any de-differentiation which is otherwise observed in Conventional 2D cultures. A macroscopic structure which resembled cartilage was appreciated in the TGP construct in vitro after 64 days which was then transplanted to the rabbit. The H&E and Immunohistochemistry studies confirmed the presence of chondrocytes in the biopsy tissue.
Based on the results, we conclude that the TGP significantly supports the in vitro expansion of chondrocytes for a longer period and the 3D culture using TGP preserves the phenotype of the articular chondrocytes. The tissue thus grown when implanted with the TGP has engrafted well without any adverse reactions and upon confirmation of safety following completion of the entire study with adequate follow-up, human applications could be considered.
Though the transplantation of in vitro expanded human corneal endothelial precursors in animal models of endothelial damage by injecting into the anterior chamber has been reported, the practical difficulties of accomplishing such procedure in human patients have been a hurdle to clinical translation. Here we report the successful transplantation of in vitro expanded human corneal precursor cells to an animal eye using a transparent Nano-composite sheet and their engraftment.
Human Corneal endothelial cells (HCEC) were isolated from human cadaver eyes with informed consent and expanded in the lab using a sphere forming assay in a novel Thermoreversible Gelation Polymer (TGP) for 26 days. HCEC obtained by sphere forming assay were seeded in a novel Nano-composite sheet, which was made of PNIPA-NC gels by in-situ, free-radical polymerization of NIPA monomer in the presence of exfoliated clay (synthetic hectorite “Laponite XLG”) uniformly dispersed in aqueous media. After a further seven days in vitro culture of HCEC in the Nano-composite sheet, cells were harvested and transplanted on cadaver-bovine eyes (n=3). The cells were injected between the corneal endothelial layer and the Nano-composite sheet that had been placed prior to the injection in close proximity to the endothelial layer. After three hours, the transplanted Nano-composite sheets were removed from the bovine eyes and subjected to microscopic examination. The corneas were subjected to Histo-pathological studies along with controls.
HCEC formed sphere like colonies in TGP which expressed relevant markers as confirmed by RT-PCR. Microscopic studies of the Nanosheets and histopathological studies of the cornea of the Bull’s eye revealed that the HCEC got engrafted to the corneal endothelial layer of the bovine eyes with no remnant cells in the Nanosheet.
Transplantation of in vitro expanded donor human corneal endothelial cells using a transparent Nano-composite sheet was feasible in bovine eyes and the HCEC an engrafted within three hours of transplantation. Pilot human studies could be planned for utilization of this material and strategy.
Autologous Natural Killer (NK) cells and Cytotoxic T Lymphocytes (CTLs) based immune-cell therapy, otherwise called as Autologous Immune enhancement therapy (AIET), though has been in clinical practice in several developed nations since early 90s, in India it is in infancy due to lack of technological knowhow. Our institute has been providing the AIET cell expansion services since 2005 and we here in report our experience in 30 such patients of both solid tumours and hematological malignancies.
The number of AIET transfusions in each patient ranged from one to six. All the patients included had Stage III to IV malignancy. AIET was either given along with the chemotherapy or after the completion of a minimum of six cycles of chemotherapy in all the patients. 70 ml of Peripheral Blood was collected each time. The protocol followed was as per Terunuma et al (Breast Cancer 2010) which uses only the patients’ autologous plasma for expansion of the Natural Killer Cells and Cytotoxic T lymphocytes from the peripheral blood. The cells were cultured for a period of 10 to 16 days and then transfused to the patients intravenously. The cells were subjected to Flow cytometry before and after the
The percentage of NK cells and CTLs after expansion by flow cytometry ranged from 60 to 82 %. There were no adverse reactions in any of the patients following transfusion. The mean prolonged survival time was 15 months and 27% of the patients had Static non-progressive disease after the therapy. Two patients reported significant decrease in Cancer marker levels after AIET and among the terminally ill, two had more than two years survival. All the patients reported improvement in quality of life and resumption of appetite following AIET.
Optimal in vitro expansion of NK cells and CTLs of patients with stage III-IV cancer either concurrently or after chemotherapy could be accomplished using autologous serum without use of feeder layers. The
Former affiliation, when the study was done
Cardiac cell replacement therapy is a promising therapy to improve cardiac function in heart failure, a major cause of morbidity and mortality in the world. In contrast to clinically intensively tested bone-marrow cells, embryonic or induced pluripotent stem cell can be differentiated into functional cardiomyocytes (ES-CM or iPS-CM) which after transplantation can integrate into host myocardium and thereby replace lost myocardium. Since engraftment and persistence of transplanted ES-CM and iPS-CM was found to be very limited, we tried to increase it in this study by co-transplantation of non-cardiomyocytes with iPS-CM.
ES-CM and iPS-CM were derived and highly purifed from transgenic male murine embryonic or induced pluripotent stem cells using an antibiotic resistance under the control of a cardiac specific promoter. 300,000 ES-CM or iPS-CM with or without admixture of 300,000 000 syngeneic male murine mesenchymal stem cells (MSC) or syngeneic male murine embryonic fibroblasts (MEF) were intramyocardially injected into healthy hearts of syngeneic female mice. Hearts were explanted after 24h, DNA was isolated and the number of transplanted cells was determined using quantitative real-time PCR with transgene specific primers.
For every surgery day, 1 additional aliquot was mixed with an explanted heart ex-vivo and served as control and known dilutions of transgene positive in transgene negative DNA were included to derive a calibration curve.
24h after intramyocardial injection of iPS-CM, we detected 0.62±0.44% of the transplanted cell number (approx. 1900 cells), which was significantly less than in the surgery day controls (p < 0.001) and similar to the intramyocardial injection of ES-CM with 0.93±0.28% (approx. 2800 cells). After co-transplantation of MSC numbers were 2.5-fold higher with 1.54±0.23% of transplanted transgenic cells (p < 0.05 vs. iPS-CM, approx. 4600 cells). Preliminary data suggest that co-transplantation of MEF showed similar results with 1.74±0.93% or approx. 5200 cells).
Persistence of iPS-derived cardiomyocytes at 24 hours after intramyocardial injection is increased by cotransplantation of MSC, but it remains very limited. Nevertheless, this strategy could be useful to improve the efficiency of cardiac cell replacement therapy and should therefore be further investigated.
Cardiac cell replacement is a promising therapy for ischemic damaged heart tissue. A functional integration and maturation of transplanted cells prevents arrhythmias and enhances synergistic contractions of transplanted and host tissue. We compared the electrophysiological integration and properties of transplanted fetal cardiomyocytes (FCM) and induced pluripotent stem cell-derived cardiomyocytes (iPSCM), which are regarded as promising cell type for cardiac cell replacement therapy.
FCM from transgenic mice expressing eGFP under control of the α-actin promoter were isolated at day 14.5. Genetically modified murine iPSCM, expressing eGFP and a puromycin resistance under control of the alpha-MHC promoter, were purified by antibiotic selection. FCM and purified iPSCM were injected into adult mouse hearts (2 injections, ~500,000 cells per site). At different times after transplantation (2-12 days), recipients were sacrificed and viable ventricular tissue slices (thickness: 150 μm) were prepared. Slices were focally stimulated by a unipolar electrode placed in host tissue. Recordings of action potentials were performed by glass microelectrodes in transplanted cells, which could be identified by their green fluorescence, and in host cardiomyocytes within the tissue slices.
Coupling of FCM and iPSCM to host tissue could be clearly demonstrated. Some transplanted FCM and iPSCM showed no conduction blocks even at high stimulation frequencies of up to 8 Hz, while others had blocks at lower stimulation frequencies or were not electrically integrated at all. Electrical integration was better in FCM than in iPSCM. The delay between stimulation artifact and Action Potential upstroke were significantly prolonged for FCM (19,84±3,77 ms at day 6 and 11,92±2,64 ms at day 12 ) and IPSCM (35,34±8,79 ms) in comparision to host tissue (6,96±1,0 ms). Electrophysiological properties of transplanted iPSCM differed significantly from those of host cardiomyocytes (P< 0.05 for all parameters). IPSCM had a lower maximum diastolic potential (-50.1±5.6 mV vs. -70.9±5.1mV), amplitude (49.7±3.4 mV vs. 75.0±11.5 mV) and maximum upstroke velocity (11.1±1.3 V/s vs. 102.2±31.7 V/s). APD50 was longer (21.7±10.1 ms vs. 13.8±10.0 ms), APD90 was shorter (37.2±15.4 ms vs. 95.7±20.2 ms). In FCM, action potentials were fetal-like at day 6 after transplantation, but were comparable to those of adult cells at day 12.
Fetal cardiomyocytes and IPSCM are able to integrate electrically into host tissue, but conduction blocks occur and are more pronounced in iPSCM. Action potential properties of transplanted iPSCM differ considerably from those of recipient cardiomyocytes and are also more immature as compared to FCM.
Ang II interferes with cardiac remodeling via its AT1 and AT2 receptor (R). It has been shown that the AT2R is up-regulated in fetus and following cardiovascular injury, and is involved in tissue regeneration and differentiation. We have recently identified the CD117+AT2R+ cell population in rat heart and bone marrow with the potential to enhance cardiac repair/regeneration upon AT2R stimulation. In the present study, we further characterize the cardiac differentiation potential of bone marrow CD117+AT2R+ cell population in response to myocardial infarction (MI) in mice.
The CD117+AT2R+ cells were isolated from bone marrow samples of mice after acute MI, analyzed with FACS, Patch-Clamp technique, RT-PCR and immunostaining to assess their differentiation characteristics.
The isolated bone marrow CD117+AT2R+ cell population exhibited a distinct morphology in culture with a tendency to form cell aggregates. Whole-Cell-Patch-Clamp measurements detected an alteration of potassium channels in this cell population over a time span of 7 days. An increase in ion channel activation and potassium outward rectifier was also observed under defined
We demonstrate here that bone marrow CD117+AT2R+ cell population is characterized by AT2R-mediated cardiac differentiation potential, as shown by the development of ion channels and the expression of cardiac differentiation markers.
Heart failure (HF) is a serious health problem in developed countries and rest of the world. Typically in contrast with myocardium infarction (MI) that can cause suddenly, HF is a long term problem.
HF has two main types:
Systolic heart failure ( SHF) that in this condition the problem arise as the heart pump function can not work sufficiently, therefore blood flow that leaves the heart (ejection fraction) decreases. This decreases depends on the severity of HF can be vary.
Diastolic heart failure (DHF) that in this condition the heart muscle can not fill with blood as diastolic blood pressure in this situation is low. The main reason for this situation is stiffness of the heart muscle.
HF is a common health problem across the world. It has estimated that in developed countries around 2% of adults suffer from it but with increasing the age this problem becomes severe and it rise to 6-10 % in patients over the age of 65. So, it shows that age and changing in life style (e.g. lack or reducing physical exercise) and in addition the other issues like hypertension, myocardium infarction (MI) can cause this main health problem. The researches on small animals especially on rats and mouse are developing rapidly to can answer to questions arise from different aspects of HF. In addition to routine treatments for HF new approaches seems necessary to can accelerate the treatment of this heart disease.
Stem cell therapy is a new promising approach. There are two main types of stem cells. 1- Adult stem cells (ASCs) 2- Embryonic stem cells (ESCs). In human to use ESCs is a challenging issue and it can in some countries use for therapy of patients under certain regulations, however, in animal models ESCs widely use in researches. ASCs is out of scope of this review.
The goal of this review is to evaluate the recent findings of the effects of ESCs on the treatment of HF in experimental models
Myocardium infarction (MI) is the leading cause of mortality and morbidity in the developed countries. It is also one of the main cause of the mortality and morbidity in the developing countries and rest of the world. There are different risk factors for that and among them smoking, age, sex, health problems like diabetes, high blood pressure, and lack of physical exercise are more involved.
The consequence for MI is to reduce blood flow in heart tissue. There are different routine treatments for MI like oxygen therapy, injection of heparin and anti pallets medications like Aspirin or Clopidogrel to restore blood flow in the heart. There are new approaches for treatment of MI: These approaches can use as supplementary methods. Among them stem cell treatment is a promising approach. Animal models are good sources to do research for preclinical studies as with them we can mimic the clinical health problems like MI: The main advantage for stem cell administration is that it is a less expensive- less invasive promising treatment method. The supplementary treatment with stem cells in addition to the routine treatment methods is that it can improve the quality of treatment. In animals especially small animal's sudden death can arise of MI cause. There are different symptoms in animals after MI that among them myocardium scar formation, decreasing heart function, decreasing blood flow, increasing blood pressure are key parameters. MI in some animals like cats and dogs are rare but in small animals especially in rats and mouse causes after ligation when those animals are used as models to evaluate the effects of MI on them. These help researchers to have a better understanding of MI in experimental models to improve the quality of research and treatments in clinical studies. Studies shows that the administration of stem cells can improve the heart function following MI in animal models studies. There are two main types of stem cells but in this review we only review the embryonic stem cells (ESCs) and the other source (Adult stem cells) is out of scope of this review.
In this review poster we undertake a review on the recent findings on this topic in experimental studies.
Liver transplantation is the only established therapy for end-stage liver disease. However, due to the limited availability of donor organs, cell transplantation has been suggested as an alternative. Though the majority of patients waiting for liver transplantation has severe fibrosis or cirrhosis, most preclinical settings for the evaluation of cell transplantation are based on non-fibrotic liver injury. In the present study, we analyze the regenerative capacity of fetal stem/progenitor cells by transplantation into a mouse model of progressive liver fibrosis (Mdr2 -/- mice) at early and late stages of liver fibrosis.
Fetal liver stem/progenitor cells were isolated from wild-type Balb/c mice. Cells were injected intrasplenically into Mdr2 -/- mice of different ages (sixth weeks vs sixth months) after 1/3 partial hepatectomy. Mice were sacrificed at 2 and 4 months after cell transplantation and liver tissues were taken for total RNA isolation. Engraftment of transplanted fetal liver stem/progenitor cells was investigated by quantitative expression analysis of hepatocyte-specific Mdr2 using RT-PCR.
Six months old Mdr2 -/- mice reveal only faint (< 0.5%) or rather no Mdr2 expression at any time point after cell transplantation. However, Mdr2 -/- mice of a younger age display considerably higher expression levels of Mdr2 after cell transplantation, rising from maximal 1 % at 2 months after transplantation up to 4 % at 4 months after transplantation.
Repopulation of Mdr2-/- mice with fetal stem/progenitor cells demonstrated to be successful with mice of a younger age, i.e. less fibrosis. In contrast, Mdr2 -/- mice at a late stage of progressive liver fibrosis seem to be unaffected by fetal liver stem/progenitor cell transplantation. This is possibly due to a lower engraftment efficiency, as the transplanted cells fail to traverse the endothelial barrier in mice with significant matrix.
The isolation of hepatic stem cells is demanding due to the lack of specific surface markers. Previously, we identified Neighbor of Punc E 11 (Nope) as a novel oncofetal marker of stem/progenitor cells in the murine liver. In the current study we focused on the expression pattern of Nope as a potential marker for stem/progenitor cells in normal adult liver as well as after acute (partial hepatectomy) and/or chronic liver injury (Mdr2 -/- mice).
Liver tissues were obtained from adult Balb/c mice (10 weeks) and Mdr2 -/- mice of different age and stage of fibrosis. In subgroups with partial hepatectomy, livers were obtained 24 hours up to 7 days postoperatively. In selected mice, liver regeneration was modified by injection of DNA alkylating reagents 4 and 2 weeks before analysis. Expression levels of Nope were quantified using quantitative RT-PCR in homogenized liver tissue and after microdissection of bile ducts. Immunohistochemistry on cryosections was performed combining stainings for Nope with the biliary marker protein CK 19, an epithelial-specific Pancytokeratin (PanCK) and the canalicular membrane marker dipeptidylpeptidase (DPP) IV.
While normal adult liver shows only negligible expression of Nope, chronic liver injury in Mdr2 -/- mice leads to a considerably increased expression level of Nope. Co-stainings with CK19 demonstrated a bile-duct-specific expression of Nope in these mice. While acute injury in normal adult liver has no effect, an additional partial hepatectomy in the Mdr2 -/- mouse model results in sparse detection of Nope positive cell clusters if hepatocyte proliferation is blocked by DNA alkylating reagents. These Nope positive clusters are negative for CK19 but positive for PanCK and DPPIV.
Here we report the expression of the oncofetal stem/progenitor cell marker Nope in the Mdr2 -/- mouse model of progressive liver fibrosis. While Nope is restricted to bile ducts in the chronic injury model, a rare population of regenerating stem/progenitor cells arises in case of an additional acute injury if physiological regeneration is blocked. We conclude that Nope is a potential marker for stem/progenitor cells in the regenerating adult liver.
Aim of this study is to characterize the phenotypic profile of normal colon stem cells in order to compare this pattern to that found in colon cancer stem cells taking advantage of their different proliferative rate compared to non stem cells.
Normal human colon biopsies were digested, cultured in serum-free medium and stained with PKH26. Cells were stained for the stemness marker Musashi-1 (Msi-1), Cytokeratin (CK) 20, CK18, Muc-1 and Ki67 and analyzed through confocal microscopy and cytofluorimetry.
PKH26 staining allowed to identify two subsets: PKHpos e PKHneg cells. PKHpos cells showed high PKH intensity, reflecting a slow proliferation rate, whereas PKHneg lost the PKH dye, due to high proliferation rate. Most PKHpos cells lacked the expression of the differentiation marker CK20, and 5±0.9% of these cells were Msi-1+. PKHneg were >99.9% Msi-1- but Muc-1and CK20 positive. In serum-free conditions, sorted PKHneg cells died rapidly, whereas PKHpos cells persisted for up to 35 days forming spheroid-like structures, morphologically comparable to those obtained from tumor colon tissues. In the presence of serum, PKHpos cells differentiated into epithelial-like cells, and acquired CK20 and Muc-1 expression, while completely loosing Msi-1 expression. Furthermore, thanks to the different proliferative rate and markers expression, we could identify 4 different cell populations: PKHhigh/Msi+/CK20- dormant stem-like cells with low Ki67 expression, a subset of PKHpos/Msi+/CK20+ stem-like cells in active proliferation that had acquired differentiation markers and expresses high levels of Ki67, PKHlow/CK20+/Msi- actively proliferating cells that progressively lost PKH staining, eventually differentiating into terminal PKHneg/CK20+/Msi- cells.
So far, the ability to form spheroids is reported for tumor colon samples but not for normal colon tissues. Here we demonstrate that stem-like cells derived from normal colon mucosa present this ability and show some specific features of stem cells, such as long lifespan in the absence of serum, expression of stemness markers, lack of differentiation markers and ability to differentiate into different cell types. Moreover, thanks to the PKH26 assay, we identified two stem cell populations: quiescent one and active and proliferating one.
Osteoprosis is a disease characterized by low bone mass and micro-architectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture. Some studies in found vertebral strength could be better assessed with bone mineral density (BMD) while a strong correlation between failure force and BMD was observed in recent studies.
A motion segment consists of two discs and the vertebrae in between was modeled. Five distinct structural regions were simulated in the current model, namely discs annuluses, discs nucleuses, end plates, cortical and trabecular bone. The motion segment had being loaded until fracture happened. Load-displacement curve of upper disc was attained. In lower load the curve has the maximum gradient and when the stress become more than the yield stress of cortical and trabecular bone, the gradient of the curve become less.
Experimental data which were attained from this motion segment were applicable with the curve and errors were lower than 10%.
High Mobility Group Box chromosomal protein 1 (HMGB1) is a nuclear DNA-binding protein acting as a proinflammatory cytokine. It is released from necrotic cells, activated macrophages dendritic cells and platelets. HMGB1 can induce prolonged inflammation and organ failure. Due to the preparation technique haematopoietic stem cell products consist not only of CD34+ cells but also of a mixture of different cell types, e.g. after GM-GCSF stimulation of a high addition of granulocytes, showing storage induced alterations. Therefore we evaluated HMGB1 levels in apheresis- and bone marrow (BM)-derived stem cell products.
In healthy BM donors (n = 3) and peripheral stem cell (PBSC) donors (n = 3), HMGB1 levels were determined in plasma samples (anticoagulated by EDTA (1.8 mg/mL)) of peripheral blood (PB) as well as in the respective stem cell product immediately after preparation and 24h after storage. HGMB1 was measured by an commercially available ELISA-Kit.
In the peripheral blood, HMGB1 levels of BM donors were in a lower range (5.6 ng/mL ± 1 ng/mL) than the levels of PBSC donors (12.4 ng/mL ± 6.3 ng/mL). In contrast, BM-derived stem cell products showed immediately after preparation higher HMGB1 levels (212 ng/mL ± 93 ng/mL) in comparison to the respective PBSC products (74 ng/mL ± 26 ng/mL). After 24h storage an accumulation of HMGB1 could be observed in the stem cell products, independent on the preparation technique and leading to comparable HMGB1 concentrations (228 ng/mL ± 80 ng/mL (BM) vs. 247 ng/mL ± 74 ng/mL (PBSC)).
During haematopoietic stem cell preparation clear alterations of HMGB1 levels could be observed. In the peripheral blood of GM-GCSF-stimulated PBSC donors HMGB1 levels were found within a higher range in comparison to BM donors. However, after storage of the stem cell products an accumulation of HMGB1 occured independently on the preparation technique.
After hematopoietic stem cell transplantation (HSCT), regeneration of the hematopoietic system requires activation of the stem cell pool. So far, the mechanisms that recruit these cells into proliferation and self-renewal are scarcely understood. Here, we have addressed the question if activation of hematopoietic stem and progenitor cells (HPC) after autologous HSCT is mediated by systemically released cytokines and growth factors. Serum was taken from patients before chemotherapy, during neutropenia and after hematopoietic recovery. Subsequently, it was used as supplement for
The frequency of colony forming units (CFU) as well as the number of cobblestone area forming cells (CAFC) was also increased. More than 2 weeks after HSCT when hematopoietic recovery was almost completed, this stimulating effect declines to normal levels as observed with samples from before chemotherapy. Chemokine profiling revealed down-regulation of several growth factors after HSCT including platelet-derived growth factors PDGFAA, PDGF-AB and PDGF-BB, whereas expression of monocyte chemotactic protein-1 (MCP-1) increased. Metabolomic profiling was used for identification of 46 metabolites that are currently tested for their functional relevance in HPC expansion. Taken together, these results demonstrate that systemically released factors stimulate hematopoiesis after autologous HSCT. This feedback mechanism opens new perspectives for
Leukemia stem cells (LSC), similar to their normal counterparts (HSC), are well protected by adhesion to their niche in the bone marrow. Mobilization of LSC to the circulation might render them vulnerable to anti-leukemia therapy. The aim of this study was to explore mechanisms of leukemia mobilization from the BM with mobilizing agents like AMD3100 (AMD) in a pre-clinical immune deficient mouse model.
Immunodeficient mice were engrafted with the childhood pre-B-ALL leukemic cell line G2 and with primary childhood precursor-B-ALL cells from 4 patients with up to 100% of transplanted mice being engrafted. Engraftment was without prior irradiation, thereby leading to a more physiological model of human leukemias.
Treatment with AMD lead to a significant mobilization of all transplanted leukemias with a mobilization level of between 3 - 8 times above baseline. Next, we examined the role of SDF-1 release by AMD. It could already be shown, that AMD3100 releases SDF-1 in healthy mice from the bone marrow to the peripheral blood, resulting in progenitor cell mobilization (Dar et al. Leukemia 2011). In the experiments reported here, inhibition of SDF-1 action with neutralizing CXCR4 antibodies abrogated AMD-induced leukemia mobilization. Recently we also demonstrated catecholamine receptor expression on hematopoietic stem and progenitor cells and of mobilization of these cells by catecholamines (Spiegel et al. Nat. Immunol. 2007). We showed now that the G2 cell line and all 4 examined precursor-B-ALL samples express the catecholamine receptors D3, D5 and beta-2. Treatment with high doses of epinephrine alone led to leukemia mobilization
We could demonstrate the applicability of an
There is recent evidence from clinical trials that implantation of stem/progenitor cells improve limb ischemia. In the present study implantation of autologous peripheral blood mononuclear cells (PBMNCs) mobilized by granulocyte-colony stimulating factor (G-CSF) - investigated in patients with chronic limb ischemia. Twenty-four patients with chronic lower limb ischemia were enrolled and randomized (1:1) to either the implanted group or the control group. In the implanted group, the patients received subcutaneous injections of recombinant human G-CSF (300μg/day) for 5 days to mobilize stem/progenitor cells, and their PBMNCs were collected and implanted by multiple intramuscular injections into ischemic limbs while control group injected by sterile saline and receive medical treatment. All of the patients were followed up after at 12 week. At the end of the follow-up period, the main manifestations were significantly improved in the patients of the implanted group compared to the control group. Mean of rest pain decreased from 6.42±2.15 to 1.67±3.89 (P< 0.001). Mean of pain free walking distance increased from 25±29 to 409±204 (P< 0.001). Mean ankle-brachial pressure index increased from 0.45± 0.32 to 0.79± 0.38 (P = 0.005). A total of 7 of 9 limb ulcers and wounds (77.8%) of implanted patients healed after cell implantation. Two lower limb amputations occurred in the implanted patients. In contrast, eight control patients had to receive a lower limb amputation.
Cancer, the major killer disease of the century requires a multi-pronged approach and among the latest modalities of treatments, Immunotherapy occupies a promising role. Immunotherapy for cancer was first started to be practised in the NIH and cell based immunotherapy for cancer is in practice for the past three decades. [
For AIET using NK cells, the process involves separation of lymphocytes from the peripheral blood of the patient followed by selective NK cell expansion using the expansion kit (BINKIT, BIJ, JAPAN) without feeder layers and then infusion of the expanded-activated NK cells. [
We are also working on combination immunotherapy using NK cells and CTLs and also NK cells and peptide pulsed DCs. The principle behind combining NK cells and CTLs is a dual advantage approach combining the innate immune system and adaptive immune system wherein the CTLs will kill the MHC expressing cancer cells while NK cells will kill the MHC non-expressing cancer cells also. [
In our studies in NOG SCID mice, activated NK cells were shown to reduce the size of breast cancer cells (MDA-MB231) [
Cancer has to be tackled with a multipronged approach and combining NK cell and CTL cell based AIET with conventional modalities of treatments such as Surgery, Chemotherapy and Radiotherapy as well as other modalities like Hyperthermia, Proton Beam therapy and low dose chemotherapy is effective even in advanced cancers which are refractory to conventional therapeutic modalities.
In general, the preferable characteristic of the target molecules for development of cancer vaccines are high immunogenicity, very common expression in cancer cells, specific expression in cancer cells and essential molecules for cell survival (to avoid loss of expression). We previously reported that three novel HLA-A24-restricted immunodominant peptides, which were derived from three different oncoantigens, TTK, LY6K, and IMP-3,were promising targets for cancer vaccination for esophageal squamous cell carcinoma (ESCC)patients. Then, we had performed a phase I clinical trial using three HLA-A24-binding peptides and the results had been shown to be promising for ESCC. Therefore, we further performed a multicenter, non-randomized phase II clinical trial.
Sixty ESCC patients were enrolled to evaluate OS, PFS, immunological response employing ELISPOT and pentamer assays. Each of the three peptides was administered with IFA weekly. All patients received the vaccination without knowing an HLA-A type, and the HLA types were key-opened at the analysis point. Hence, the endpoints were set to evaluate differences between HLA-A*2402-positive (24(+)) and -negative (24(-)) groups.
The OS in the 24 (+) group (n=35) tended to be better than that in the 24(-) group (n=25) (MST 4.6 vs. 2.6 month, respectively, p = 0.121), although the difference was not statistically significant. However, the PFS in the 24(+) group was significantly better than that in the 24(-) group (p = 0.032). In the 24(+) group, ELISPOT assay indicated that the LY6K-, TTK-, and IMP3-specific CTL responses were observed after the vaccination in 63%, 45%, and 60% of the 24(+) group, respectively. The patients having LY6K-, TTK-, and IMP3-specific CTL responses revealed the better OS than those not having CTL induction, respectively. The patients showing the CTL induction for multiple peptides have better clinical responses.
The immune response induced by the vaccination could make the prognosis better for advanced ESCC patients.
Corneal transplantation has been in routine practice to treat corneal endothelial diseases like Bullous Keratopathy, in which either the whole cornea or the partial cornea (the endothelium alone) is transplanted from the cadaver donor to the recipient with the endothelial disease [
After HCEP transplantation, the eye balls need to be fixed 24- 36 hours facing down, to facilitate the gravity-assisted settling of the cells injected into the anterior chamber on to the endothelium which is possible in animals but difficult in humans. To overcome this hurdle, we used the nanocomposite gel sheet (D25-NC gel sheet) developed by Haraguchi [
The study was undertaken in three patients, two suffering from bullous keratopathy and one patient with congenital corneal dystrophy after proper informed consent. The right eye was affected in each patient and the transplantation of HCEP cells were done in these right eyes. 6 x 104 HCEP cells isolated from one human cadaver donor cornea were expanded using the polymer based protocol [
The eye for eyes concept has become a reality by combining the strengths of clinical expertise with cell culture and synthetic material technology in a manner which is easy to reproduce both in the laboratory and clinically.
The International Society for Disease Surveillance (ISDS) celebrates its 10th annual meeting with the arrival of the 2011 ISDS Annual Conference, ‘Building the Future of Public Health Surveillance’. This milestone in the Society's history is punctuated not only by the achievements of the disease surveillance community but also by the promise of what lies ahead. The Annual Conference brings together a community of researchers and practitioners focused on monitoring, understanding and improving population health. The abstracts appearing in this special supplement, accepted for presentation at the Annual Conference, include innovative analytical techniques, progressive public health practice and cutting edge informatics that support a timely, accurate and informed response to emerging outbreaks of disease and other health threats. The breadth of topics addressed ranges from detection of novel symptom patterns indicative of newly emerging outbreaks to development of a web platform for the text mining of clinical reports and from surveillance of the illegal wildlife trade for detecting emerging zoonoses to assessing the validity of emergency department-based influenza-like illness syndromes against confirmed laboratory results in children. In addition to the 160 contributed oral and poster presentations, the conference also features a keynote talk from Dr. Farzad Mostashari, National Coordinator for Health Information Technology, and plenary sessions on international surveillance, novel technologies and postdisaster surveillance, as well as a variety of informal round table discussions, workshops and committee meetings. The diversity of thinking represented here is great and yet the fibers of these works come together in a yarn that, woven throughout the fabric of public health surveillance, informs the best of what is and what in the future can be.
To quantify the injury burden and to identify possible risk factors using bicycle-related injury (BRI) visits at Boston emergency departments (EDs).
In May 2001, Boston released a strategic transportation plan to improve bicycle access and safety (
The Boston Public Health Commission (BPHC) syndromic surveillance system receives information from ED visits from all 10 acute care hospitals in Boston every 24 hours. Data received include visit date, demographics, ZIP code of residence, chief complaints and ICD-9 CM-coded final diagnosis. Disposition information was reported from 9 of these hospitals in 2010. BPHC collaborated with CDC's BioSense Program to specify a BRI syndromic case definition that combined chief complaint and ICD-9 CM-coded information and excluded motor cycle only related events. Subsyndromes were used to assess the type of injury and severity based upon 47 standard BioSense subsyndromes and 21 subsyndromes developed for this study.
The data sample used for this study included over 2 million visits between 2007 and 2010. Injury visits were categorized at the neighborhood level using a standard ZIP code of residence-to-neighborhood mapping. Results were stratified by age, patient neighborhood of residence, race/ethnicity, gender and disposition (2010 data only).
Over the study period, a total of 4510 ED visits were classified as BRIs (0.22%). The percentage of BRI visits increased from 0.18% in 2007 to 0.27% in 2010. The majority of injuries (69%) occurred between May and September (
Percentage of visits at Boston EDs involving bicycle injuries: 2007–2010.
Seventy-five percent of persons presenting with BRIs were male and 60% reported race/ethnicity as white. Persons aged 18–25 years represented 28% of visits and those aged 6–17 years accounted for 17%. Boston residents accounted for 52% of BRI visits; 15% were from bordering communities. One Boston neighborhood with the highest BRI rate by patient residence also has a large college student population.
Throughout the entire study period (2007–2010), nearly one quarter (1082) of BRI visits were associated with fractures and dislocations; whereas less than 10% of visits were for sprains or strain injuries. Head injuries were associated with 84 (1.9%) of BRI visits.
In 2010, 149 (11%) of the 1411 BRI visits resulted in admission, most commonly for fractures and dislocations. Twenty-two percent were among individuals aged between 50 and 59 years; 21% were among persons aged 18–24 years. Fifty-four percent of all BRI admissions were associated with fractures and dislocations. Thirty-one (2.2%) BRI visits in 2010 were associated with head injuries; of which 11 (35%) were admitted for care. For BRI visits involving falls, 8% were admitted versus 17% for BRI visits associated with a motor vehicle.
Syndromic surveillance can be used to monitor and track BRI and to inform targeted prevention activities such as education and outreach to select at-risk populations (e.g., college students). Presently, information on the environmental context of injuries, such as the precise location of the accident, is limited. As bicycle use increases, improved methods to combine syndromic surveillance, emergency medical services and public safety information are needed to identify accident ‘hot spots’ to guide implementation of preventive measures.
Injury; prevention; emergency; bicycle; syndromic surveillance
We sought to develop a method for visualizing data quality over time.
The Public Health—Seattle & King County (PHSKC) syndromic surveillance system has been collecting emergency department (ED) data since 1999. These data include hospital name, age, sex, zip code, chief complaint, diagnoses (when available), disposition and a patient and visit key. Data are collected for 19 of 20 King County EDs, for visits that occurred the previous day. Over time, various problems with data quality have been encountered, including data drop-offs, missing data elements, incorrect values of fields, duplication of data, data delays and unexpected changes in files received from hospitals. In spite of close monitoring of the data as part of our routine syndromic surveillance activities, there have occasionally been delays in identifying these problems. Since the validity of syndromic surveillance is dependent on data quality, we sought to develop a visualization to help monitor data quality over time, in order to improve the timeliness of addressing data quality problems.
SAS version 9.2 (Carey, NC) was used to create two groups of visualizations: a separate heatmap for each hospital, showing how each individual ED performs on each of 13 data quality measures and a separate heatmap for each data quality measure, showing how data quality varies by ED. The heatmaps summarize data by month and year, though other visualizations (e.g., daily or weekly) are also possible. For each row on the heatmap, a color change indicates that data quality has shifted over time. Blocks with stable color over time suggests that there has not been a change in data quality. White space on the heatmap highlights periods of time where data were not recorded by the system and can provide a visual cue for newly added EDs, hospital closures or data drop-offs. The heatmaps are generated monthly for each of 13 data quality measures. SAS code for generating the heatmaps will be provided at the session.
Two heatmaps are provided as examples of our visualization approach (see
Heatmaps showing percent of records at each ED missing patient disposition (top) and diagnosis (bottom) data over time. The heatmaps provide a visual cue for when data quality has changed (color shifts), where data are routinely unavailable (stable color over time) or missing (white space). The heatmaps are generated monthly for each of 13 data quality measures.
Syndromic surveillance systems commonly encounter problems with data quality. These problems can result in imprecise counts and can adversely affect detection of trends, outbreaks and situational awareness [2]. The heatmap visualizations have been a useful tool for PHSKC to identify problems with data quality in a timely manner. The code can be easily adapted to display other data quality measures, stratifications and data sources beyond the ED setting.
Data quality; public health practice; visualization
We sought to evaluate the quality of influenza hospitalizations data gathered by our biosurveillance system.
The Washington Comprehensive Hospital Abstract Reporting System (CHARS) has collected discharge data from billing systems for every inpatient admitted to every hospital in the state since 1987 (
We included patients with a diagnosis of influenza (ICD9 codes 487.0, 487.1, 487.8, 488.0 or a textual variant of ‘influenza’, excluding ‘
In 2008, the PHSKC system (which searches through admission and discharge diagnoses) identified 180 patients hospitalized with influenza, compared with 161 patients identified by CHARS (which is based on discharge diagnoses exclusively). Thus far, PHSKC has reviewed 46 charts from 8 hospitals to validate system accuracy; review of data from the remaining hospital is pending. We identified 3 hospitals that were transmitting incorrect data to PHSKC and requested correction and resubmission of historical data from these hospitals. Preliminary analysis revealed that 35 of the 180 influenza hospitalizations captured by the PHSKC system (19%) were missed by CHARS; however, 15 of these patients (43%) were admitted with presumptive diagnoses of influenza but were determined not to have influenza by the time of discharge. Also, 28 of 161 influenza hospitalizations (17%) captured by CHARS were missed by the PHSKC system; however, we had no means of reverse-identifying CHARS records and, therefore, could not evaluate the validity of these data by chart review.
This evaluation identified several problems with data quality, which were substantial though not universal across hospitals. We plan to continue the analysis using 2009 data, to ensure that data quality issues have been resolved. A key limitation of this analysis is that CHARS is an imperfect gold standard for identifying King County influenza admissions; we could not independently identify admissions based on laboratory data to determine which system performed better.
Data quality; influenza; hospitalizations
We gratefully acknowledge the participation of the hospitals and their assistance coordinating the chart reviews.
Our goal is to learn the underlying network structure along which a disease outbreak might spread and use the learned network to improve the timeliness and accuracy of outbreak detection.
Disease surveillance data often have an underlying network structure (e.g., for outbreaks that spread by person-to-person contact). If the underlying graph structure is known, detection methods such as GraphScan (
Our solution builds on the GraphScan (
We generated simulated disease outbreaks that spread based on the zip code adjacency graph with additional edges added to simulate travel patterns and injected these outbreaks into real-world hospital data. We evaluated detection time and spatial accuracy using the learned graphs for these simulated injects (
Comparison of detection performance of the true, learned and adjacency graphs.
We proposed a novel framework to learn graph structure from unlabeled data. This approach can accurately learn a graph structure, which can then be used by graph-based event detection methods such as GraphScan, enabling more timely and accurate detection of outbreaks, which spread based on that latent structure. Our results show that the learned graph structure is similar to the true underlying graph structure. The resulting graph often has better detection power than the true graph, enabling more timely detection of outbreaks, while achieving similar spatial accuracy to the true graph.
Event detection; biosurveillance; graph learning
This work was partially supported by National Science Foundation grants IIS-0916345, IIS-0911032 and IIS-0953330.
For an early detection and control of an infectious disease outbreak, we developed and have been operating syndromic surveillance for evacuation sites ‘evacuation site surveillance’.
In March 11, 2011, the big earthquake attacked eastern Japan followed by huge tsunami and nuclear plant accident. Consequently, a lot of people could not help living in evacuation sites. Since those evaluation sites have high density of population and were not necessarily good in sanitary condition, outbreaks of influenza, norovirus or other infectious diseases were concerned.
We developed a web-based evacuation site surveillance system with 8 syndromes including acute gastroenteric symptoms; influenza or influenza-like-illness; acute respiratory symptoms other than influenza; rash and fever; neurologic symptoms including tetanus, meningitis and encephalitis; cutaneous symptoms; wound-related infectious diseases; icterus and death. Age of the patients was classified into three categories: younger than 5 years, 5 to 64 years and 65 years old or older. Analysis by evacuation site was performed automatically, and if some aberrations were found, the system showed an alert sign on the screen of a computer. The information on patients was shared with the public health center and the local government office simultaneously.
Evacuation site surveillance started in Fukushima prefecture on March 25, 2011, and in Miyagi prefecture on May 8, 2011. About 400 sites in Miyagi prefecture were covered until the end of May. When the surveillance found an aberration, the public health center investigated the site and started taking an action for control.
This system raised awareness of infectious diseases and provided good information for risk assessment. Before the earthquake, the pharmacy surveillance and the school surveillance (only in Miyagi prefecture), which are nationwide syndromic surveillance in Japan, were operating, and these played a complementary role for evacuation site surveillance and the official surveillance. Our experience showed that it would be too late to start to develop the system from the scratch after a disaster occurred. Thus, it is essential to make a plan on activation of the system in advance in case a severe disaster occurs and to prepare and stockpile the hardware that is necessary for an early activation of evacuation site surveillance. The necessary hardware, for example, includes battery and communication tool even if electronic power, internet and (mobile) phone network are shut down. This is the next challenge.
Earthquake; evacuation site; disaster
Assess the feasibility and utility of adopting a common influenza-like illness (ILI) syndrome across participating jurisdictions in the ISDS Distribute project.
Syndromic surveillance systems were designed for early outbreak and bioterrorism event detection. As practical experience shaped development and implementation, these systems became more broadly used for general surveillance and situational awareness, notably ILI monitoring. Beginning in 2006, ISDS engaged partners from state and local health departments to build Distribute, a distributed surveillance network for sharing de-identified aggregate emergency department (ED) syndromic surveillance data through existing state and local public health systems (
Six jurisdictions provided 4 years of baseline ED data using a common ILI definition comprising 3 subsyndrome components defined by a formal code-set (Fig.
Time-series of percent of total ED visits: locally preferred ILI (top) and common ILI (bottom) syndromes for six Distribute jurisdictions.
We found less variation between jurisdictions in weekly ratios using the common ILI definition (mean 2%; range 1.5–3.1%) than locally preferred syndromes (mean 4.9%; range 1.8-8.4%), and influenza epidemic signal-to-noise ratios were comparable for most jurisdictions during the study period. The findings suggest that the common syndrome improves comparability without an overall cost in terms of epidemic signal discrimination.
The results of this common ILI assessment suggest that disparate local systems can adopt a harmonized syndrome definition allowing for meaningful comparisons and national aggregation while maintaining the ability to use local systems and definitions. The common ILI syndrome provided more directly comparable time-series, both during baseline periods and epidemics. Use of the common syndrome did not have an overall or systematic cost in terms of epidemic signal discrimination. Where the signal-to-noise ratio was not improved, differences were usually minimal. Also, the use of the common syndrome did not restrict the use of the locally defined syndromes for local detection. This collaborative pilot was useful in synthesizing local experience in the creation of a nationally harmonized ILI syndrome definition.
Influenza; surveillance; epidemiology; syndrome standard; emergency department
We describe the initial phase of the ISDS Distribute pilot for monitoring acute gastroenteritis (AGE) syndromic emergency department (ED) visits and present preliminary analysis of age-specific trends documenting a dramatic shift in AGE consistent with US rotavirus vaccine policy and use.
Epidemic AGE is a major contributor to the global burden of morbidity and mortality. Rotavirus and norovirus epidemics present a significant burden annually, with their predominant impact in temperate climates occurring during winter periods. Annually, epidemic rotavirus causes an estimated 600,000 deaths worldwide and 70,000 hospitalizations in the United States, primarily among children younger than 5 years (
The Distribute project began in 2006 as a distributed, syndromic surveillance effort networking state and local health departments to share aggregate ED based influenza-like illness (ILI) syndrome data (
All jurisdictions submitting AGE data to Distribute presented seasonal trends with predominant winter peaks. Across the pilot jurisdictions, seasonal peaks from 2003/04 to 2005/06 occurred during Mar–Apr, while 2006/07 to 2009/10 seasonal trends peaked predominantly in Dec–Feb. Overall, epidemic timing was similar across age groups; however, the shifting pattern in impact after the 2006/07 season presented a greater drop among young children.
Distribute jurisdiction AGE trends, July 2003 to May 2011, shown normalized as a measure of relative increase for 6 jurisdictions (thin lines) and as a composite mean ratio (thick black lines). The top graph shows relative increase for ED visits among all age groups. The bottom shows relative increase only for those aged younger than 2 years.
The results of the pilot suggest the Distribute model can be successfully generalized to monitoring AGE trends, specifically the age-specific timing and impact of winter-seasonal epidemic rotavirus and norovirus. The case study of 2006 rotavirus vaccine implementation and subsequent shift in timing and impact of AGE trends suggest that syndromic ED data can potentially provide a useful surveillance indicator of population-level vaccine effect.
Gastroenteritis; norovirus; rotavirus; epidemiology; emergency department
The goal of this work is to make available to the public health community an open source system that makes available in a standards-based, modular fashion the basic tools required to conduct automated indicator-based population health surveillance. These tools may be deployed in a flexible fashion on health department servers, in the Amazon EC2 cloud, or in any combination, and are coupled through well-defined standards-based interfaces.
Cost-effective, flexible and innovative tools that integrate disparate data sets and allow sharing of information between geographically dispersed collaborators are needed to improve public health surveillance practice. Gossamer Health (Good Open Standards System for Aggregating, Monitoring and Electronic Reporting of Health),
Gossamer combines work on (1) methods for automated surveillance based on summarized clinical data, such as the influenza and visit counts used in the Distribute project (1), (2) methods developed for the modularization of surveillance processes developed for the Shoki project (2), (3) methods developed for the automated processing of Health Information Exchange data (HIE) as part of the CDC HIE initiative (3) and (4) standard industry server virtualization and deployment techniques (4).
Gossamer uses code developed at UW and additional open source components. Most components are distributed under the ‘3-clause BSD license’, permitting free use, modification and redistribution. Automated modules include (1) HL7 message receipt, processing and storage, (2) compilation of line listing data from HL7 Minimum Biosurveillance Data Set (MBDS) and Meaningful use (under development) messages, (3) classification of cases into syndromes and compilation of syndrome data into indicators, (4) receipt, storage, aggregation and management of indicator data, and (5) analysis, visualization and reporting (AVR) of indicator data. Modules may be deployed locally or in the EC2 cloud and communicated using standard protocols to let deployment strategies be mixed across the system to support both sharing and shared use of components, as well as load balancing and optimization. This presentation will talk about the goals of the open source system and give underlying details of the technical implementation using virtual machines. As an example, we will discuss an application of the Gossamer system instance developed to let a state public health agency disseminate summarized laboratory test results for multiple (14) respiratory viruses (see
Gossamer Health Screenshot showing timeseries graph of positive test rates for multiple respiratory viruses.
To support existing and emergent surveillance needs, the UW has worked with local and state health jurisdictions to identify features that allow for user-defined indicators of chronic and infectious disease surveillance. An important aspect of the Gossamer Health vision is its support for public health agencies to collaborate in cross-jurisdictional surveillance efforts through both on-demand and automated sharing of standards-based data feeds.
Gossamer is a work in progress, but it is a community work. All are welcome to participate in its development.
Surveillance; open source; architecture; cloud; software
To demonstrate how rapid ad hoc sharing of surveillance data can be achieved through informatics methods developed for the Distribute project.
Cross-jurisdictional sharing of public health syndrome data is useful for many reasons, among them to provide a larger regional or national view of activity and to determine if unusual activity observed in one jurisdiction is atypical. Considerable barriers to sharing of public health data exist, including maintaining control of potentially sensitive data and having informatics systems available to take and view data.
The Distribute project (
The Gossamer system (
The Distribute and Gossamer systems have been used for ad hoc sharing in three different contexts: sharing of common ILI data for research into syndrome standardization, sharing syndromic data for specific events (2010 Olympics) and for pilot regional sharing of respiratory laboratory results. Two additional projects are underway to share specific syndromes of recent interest: alcohol-related and heat-related ED visits.
The Distribute system was initially designed to share 4 syndromes (broad and narrow ILI, and GI syndromes). To reduce barriers to entry, the Distribute project does not impose strict syndrome definitions. This lack of standardization introduces variability between jurisdictions and a pilot has been undertaken to compare sites with preferred definitions and to develop a common ILI definition. To enable the addition of a common syndrome considerable modifications to the structure of the Distribute system were required. The approach taken allowed for the use of arbitrary indicators and stratification ranges.
The Gossamer system uses a similar data storage architecture to that of the current version of Distribute, though Gossamer is more modular and better able to use external services. These features make it useful for moving beyond specific political structures or disease content areas.
The expanded data model has now been used to support the ILI standardization effort through comparison of newly contributed ‘ILI-S’ syndrome data. Distribute was also used to develop a site to allow Washington State DoH to share specific syndromic data with British Columbia during the 2010 Olympics. An instance of Gossamer demonstrated sharing laboratory results for 14 viral isolates between two states. In addition to community-driven comparisons of ILI and GI syndromes, the data model has been applied at the design level to two additional syndrome types for ad hoc data sharing: alcohol intoxication-related visits and heat exposure-related ED visits.
While built around similar data models, each system has strengths and weaknesses for ad hoc sharing of data. Advantages of the Distribute system for sharing additional data include making use of the existing trust and community that is based around the system, which reduces many barriers to sharing data and facilitates adding more community members. In addition, data feeds and administrative details are already in place.
Disadvantages of using Distribute include limitations in the common data transmission format, limitations in stratifiers and limitations in compartmentalization.
The implementation of the very similar data model in Gossamer is able to address some of these issues by various strategies including virtualization and modular architecture, while extending the flexibility which supports new applications of the data collection, quality and analysis methods developed for use with influenza syndromes in Disribute.
The 5 examples illustrate the strengths of the community of practice approach to sharing data. The Distribute and Gossamer systems illustrate how lightweight systems can be designed to easily facilitate ad hoc sharing between jurisdictions.
Informatics; surviellance; architecture; data sharing; public health practice
The goal of this session will be to briefly present two methods for comparing aggregate data quality and invite continued discussion on data quality from other surveillance practitioners and to present the range of data quality results across participating Distribute sites.
Distribute is a national emergency department syndromic surveillance project developed by the International Society for Disease Surveillance (ISDS) for influenza-like illness (ILI) that integrates data from existing state and local public health department surveillance systems. The Distribute project provides graphic comparisons of both ILI-related clinical visits across jurisdictions and a national picture of ILI.
Unlike other surveillance systems, Distribute is designed to work solely with summarized (aggregated) data, which cannot be traced back to the unaggregated 'raw' data. This and the distributed, voluntary nature of the project create some unique data quality issues, with considerable site to site variability. Together with the ISDS, the University of Washington has developed processes and tools to address these challenges, mirroring work done by others in the Distribute community.
University of Washington together with the ISDS has undertaken a comprehensive analysis of the quality of the data being received by Distribute, primarily using visual methods, examining data quality characteristics within and between sites. This process included basic exploratory analysis of data quality problems and analytical analysis of specific aspects of data quality, including the relationship between timeliness, completion and accuracy.
Considerable variability was seen between sites in terms of timeliness and completion, and completion rates did not necessarily correlate with accuracy. In our talk, we will present results comparing the quality of data between sites (sites will be unidentified), in particular comparisons between timeliness, completion and accuracy. We will also examine the types of observed relationships between timeliness, completeness and accuracy exhibited across the sites.
The purpose of this talk is to facilitate discussion between Distribute participants around data quality and the role that the ISDS can play in ensuring data quality. We will show prototypes of two features that could be hosted on the Distribute restricted site. The first feature would allow each site to compare the quality of their data (identified only to them, with site linked to the id of the user) with the remaining sites (each unidentified). The second feature would allow each site to see time series of their data together with prediction intervals for the accuracy of the ILI ratio for recent dates where the data are incomplete (see
ILI ratio timeseries calculated from incomplete data with superimposed 95% prediction interval for the complete data value for a representative site.
Our goal is to spark discussion on data quality with respect to syndromic surveillance data and, in particular, how the Distribute project can be leveraged to improve the quality of aggregate data produced by participating sites.
Data quality; surveillance; public health practice; data quality
To present exploratory tools and methods developed as part of the data quality monitoring of Distribute data and discuss these tools and their applications with other participants.
Distribute is a national emergency department syndromic surveillance project developed by the International Society for Disease Surveillance for influenza-like illness (ILI) that integrates data from existing state and local public health department surveillance systems. The Distribute project provides graphic comparisons of both ILI-related clinical visits across jurisdictions and a national picture of ILI.
Unlike other surveillance systems, Distribute is designed to work solely with summarized (aggregated) data, which cannot be traced back to the unaggregated 'raw' data. This and the distributed, voluntary nature of the project creates some unique data quality issues, with considerable site to site variability. Together with the ISDS, the University of Washington has developed processes and tools to address these challenges, mirroring work done by others in the Distribute community.
The University of Washington together with the ISDS has undertaken a comprehensive analysis of the quality of the data being received by Distribute, primarily using visual methods, examining data quality characteristics within and between sites. Several visualization tools were developed to assist in analyzing and characterizing data quality patterns for each site: upload pattern graphs (
Upload pattern plots for three sites. Each point represents an encounter date contained with in an upload file. The x-axis represents the date of the file upload and the y-axis represents the number of days prior to the upload date the encounter date was. The graph is truncated at 16 days prior to the upload date.
In our talk, we will present these visualizations and demonstrate how they can be used to discover several common and some unusual data quality patterns and issues. We will also discuss the underlying architecture that allows us to reconstruct prior views and discuss the importance of examining data quality in terms of prior data views.
Visualizations; data quality; surveillance
To describe some uses of EMR data for surveillance and situational awareness during disaster response.
During responses, an electronic medical record (EMR) allows federal emergency response staff to view and evaluate near real-time clinical encounter data. Analysis of EMR patient data can enhance situational awareness and provide decision advantage for headquarters’ staff during both domestic and international events. The EMR was utilized by field medical personnel during the response to the Haiti earthquake.
During the U.S. response to the Haiti Earthquake in January 2010, patient demographic and clinical treatment data were collected by ESF-8 responders through the EMR. Data were collected throughout the patient experience during registration, triage, treatment and discharge. Inclusion criteria for encounter records in the main analysis were entered into the EMR between January 18, 2010, and February 22, 2010, encounter occurred at one of the HHS sites in Haiti and data downloaded no later than February 23, 2010. Data were then analyzed in order to identify potential emerging conditions and operational medical needs during the entire response.
We analyzed 8925 patient encounter records entered into the EMR between January 18 and February 22, 2010. Of those records, 4612 (51.8%) were coded as female, 3995 (44.8%) as male and 303 (3.4%) were not specified. Additionally, 1444 (16.2%) of the encounters were coded as less than 6 years old, 1638 (18.3%) were coded as 6–18 years old, 4352 (48.8%) were coded as 19–49 years old, 1004 (11.2%) were coded as 50–65 years old, 283 (3.2%) were coded as more than 65 years old and 204 (2.3%) were not specified. Mean age was 27.1 (SD=19.1) years with a minimum of 1 day and a maximum of 100 years. Additionally, 6575 (75.1%) records were coded as nonurgent, 1889 (21.6%) as urgent and 295 (3.3%) as emergent. Daily surveillance of the records resulted in the identification many of suspected or confirmed symptom and disease occurrences. They included 8 cases of chicken pox/herpes zoster, 46 cases of conjunctivitis, 1 case of hemorrhagic fever, 23 guns shots wounds, 15 cases of malaria, 1 case of measles, 3 cases of meningitis, 2 cases of mumps, 53 cases of acariasis (including scabies), 1 case of typhus, 3 cases of tetanus, 3 cases of tuberculosis and 7 cases of pneumonia. We also detected 714 instances of fever and 550 instances of diarrhea.
During the 2010 earthquake response in Haiti, knowledge of the medical encounters through EMR data in the field provided indications of need for patient care. The surveillance of suspected and confirmed condition and diseases of concern allowed for timely decisions on adjustments to the response. Event burden could be quickly assessed through electronic reporting. EMR data can enhance and inform emergency response decision-making during domestic and international events and may be a useful tool for field public health and medical surveillance and situational awareness during future disaster responses.
Surveillance; disaster response; electronic medical records
To develop and evaluate a nationwide automated system for early detection of aberrations and real-time monitoring of pneumonia and influenza (P&I) mortality in Taiwan.
Influenza is a serious disease that seasonality causes substantial but varying morbidity and mortality. In Taiwan, estimates of the influenza mortality burden were based on post-hocanalyses of national mortality statistics and not available until at least six months after the corresponding epidemic. Timely monitoring and early detection of influenza-associated excess mortality can guide antiviral or vaccine interventions and help healthcare capacity planning. Beginning April 2009, Taiwan Centers for Disease Control (TCDC) has been collaborating with the Department of Health (DOH) Office of Statistics to develop an automated system for real-time P&I mortality surveillance (
Taiwan's Mortality Information Regulations require medical institutions to report any mortality to DOH through the National Death Certificate System (NDCS) within 7 days after a death certification is issued. Automated data from the NDCS were daily submitted to TCDC by secure electronic transmission and processed and analyzed using SAS Enterprise Guide 4.3 (SAS Institute Inc, Cary, NC). For each report, the underlying cause of death was determined by applying the World Health Organization classification principles (
In 2008, the number of deaths for which P&I was listed as the underlying cause in the national mortality statistics was 8,665; of these, 6,795 (78%) were reported through the NDCS. The weekly surveillance-based P&I mortality estimates had a consistently strong correlation with those obtained from mortality statistics data (correlation coefficient 0.85, p<0.0001). Eighty seven percent of the reports were received within 7 days after death (median 2 days). During the 2010–11 influenza season, an increase in mortality was observed in January 2011, with the highest weekly number of P&I deaths to be 421 (week 5 of 2011) (
Weekly numbers of pneumonia and influenza deaths, Taiwan, week 1 of 2010 through week 29 of 2011
Taiwan has established an early warning system for P&I mortality to assist with characterization of influenza severity. Integration of this mortality surveillance with data from viral surveillance, Real-Time Outbreak and Diseases Surveillance (RODS) for influenza-like illness, and surveillance for influenza with severe complications can provide policy makers with timely information during public health emergencies. Further efforts should focus on broader adoption of electronic death reporting to capture a larger percentage of deaths, educating physicians about how to complete death certificates to improve data quality, and evaluating the sensitivity and specificity of this aberration detection algorithm.
Taiwan; influenza; mortality; aberrations
To demonstrate the utility of the BioSense Program for postdisaster response surveillance.
The CDC's BioSense Program receives near real-time health care utilization data from a number of sources, including Department of Defense (DoD) healthcare facilities from around the globe and nonfederal hospital emergency departments (EDs) in the United States, to support all-hazards surveillance and situation awareness. Following the tsunami in Japan on March 11, 2011, the BioSense Program modified its surveillance protocols to monitor: (1) injuries and possible radiation-associated health effects in Japan-based DoD facilities and (2) potential adverse health effects associated with the consumption of potassium iodide (KI), a salt used to prevent injury to the thyroid gland in the event of radiation exposure, among persons attending participating EDs in the US. We present the findings from that enhanced surveillance.
The BioSense Program monitored healthcare activity in 20 DoD facilities located in Japan from March 17 through April 11, 2011. In Japan-based outpatient DoD facilities, we monitored 10 health conditions, which are associated with injuries, and possible syndromic presentations of radiation exposure, which included nausea/vomiting, diarrhea, headache, hypotension, rash, convulsion, dyspnea, dizziness and anemia. We also searched for radiation exposure-specific International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) E codes E926 and E926.9. There was a 2- to 5-day lag time between the time of patient visit and time when ICD-9-CM-coded final diagnoses were available.
To monitor healthcare utilization for potential adverse effects associated with KI exposure in the United States, we searched ED chief complaint (CC) data from all 635 nonfederal BioSense hospitals for the following keywords indicating a KI-related visit: (1) potas*, pottas*; (2) iodine, iodide; (3) KI; (4) radiation, radiation; (5) nuclea*; (6) Japan. A given ED visit was considered a match if the content of the CC met the following keyword inclusion criteria: 1 and 2, 3, 4 and 5 or 6. Perl Regular Expressions were used to take into account upper- and lower-case letters and word boundaries. CC data were updated within 0–2 days following the visit date.
To identify clusters of patient visits of interest, we used a modified version of the early aberration reporting system (EARS) C2 statistic (
From March 17 through April 11, 2011, the BioSense Team created daily reports for the CDC's Emergency Operation Center and DoD counterparts. Reports included time series graphs for each of the 10 health conditions.
Weekly diarrhea and total visits for 20 Japan DoD facilities (November 2010–April 2011).
BioSense is an adaptable electronic all-hazards public health surveillance system that can provide near real-time health situational awareness during large-scale natural disasters.
BioSense; disaster surveillance; radiation; potassium iodide; syndromic
This paper describes the use of multiple influenza surveillance data for situational awareness of influenza activity.
Much progress has been made on the development of novel systems for influenza surveillance (
A multivariate dynamic linear time series model was fitted to data on influenza-like illness (ILI) rates among networks of public and private general practitioners and school absenteeism rates, plus drop-in fever count data from designated flu clinics (DFC) that were created during the pandemic. The data streams were assumed to follow an underlying latent process with local linear trend. The estimated level and trend of the latent process reflect the magnitude and direction of influenza activity, which are then combined to infer an overall influenza activity index. Correlations between the estimated influenza level from the model and laboratory isolation rate were calculated to assess its performance before and during the 2009 pandemic.
ILI rates from public outpatient clinics and the estimated influenza level from the multivariate model had the highest correlations with laboratory isolation data before the 2009 pandemic (
Laboratory influenza detection rates from January 2004 through December 2009. The inferred influenza activity index was superimposed and color-coded from white (low) to red (high) in each panel. The vertical dashed line indicates the start of the pandemic period.
The use of a multivariate method to integrate information from multiple sources of influenza surveillance data can improve situational awareness of influenza activity, with the advantage of maintaining performance when data streams are interrupted or supplemented by additional systems during certain critical periods such as the 2009 influenza pandemic.
Sentinel surveillance; influenza; multivariate analysis; pandemic
This research was in part funded by the Research Fund for the Control of Infectious Diseases of the Food and Health Bureau of the Hong Kong SAR Government, the Area of Excellence Scheme of the University Grants Committee (grant number AoE/M–12/06) and the Harvard Center for Communicable Disease Dynamics from the National Institute of General Medical Sciences (grant number U54 GM088558).
To evaluate the sensitivity, positive predictive value (PPV), timeliness, completeness and representativeness of laboratory-confirmed influenza hospitalization data from a health information exchange with respect to traditional notifiable condition (NC) reporting.
During the 2009 H1N1 influenza pandemic, the Washington State Department of Health (DOH) temporarily made laboratory-confirmed influenza hospitalizations reportable. Reporting of influenza hospitalizations is resource intensive for hospitals and local health jurisdictions. As a result, electronic sources of influenza hospitalization data are being explored. A Regional Health Information Exchange (HIE) in Washington currently sends DOH ICD9 coded discharge diagnoses and microbiology laboratory orders and results for all patients admitted to 17 hospitals throughout Washington, including 4 of the 5 hospitals in Spokane County. The HIE hospitalization and laboratory data may be a valuable replacement for mandatory notifiable condition reporting to monitor the basic epidemiology and severity of influenza in Washington.
For the 2010–2011 influenza season, Spokane Regional Health District required hospitals to report any admissions with laboratory-confirmed influenza using traditional NC surveillance methods. Simultaneously, HIE records from four Spokane facilities were monitored for flu diagnoses (i.e., records with ICD9 487–488 listed in the working or final diagnoses) and positive flu laboratory test results (including rapid antigen, DFA, culture or PCR). Records from the NC system and the HIE were matched using facility name, age, gender, county and admission date. The medical records of cases detected by the HIE but not reported through the NC system were evaluated to determine true case status. Sensitivity and PPV were calculated for each surveillance system. Timeliness, completeness and representativeness of records received through the HIE were evaluated against NC reporting.
One hundred forty-six true laboratory-confirmed influenza cases were identified (
Case identification
HIE data are a useful resource for influenza hospitalization surveillance. They are sensitive, specific and representative of the true population of laboratory-confirmed influenza patients admitted to the hospital. It also provided data that were adequately timely and complete. Microbiology laboratory data improved the sensitivity and PPV of the Public Health Surveillance HIE feed to levels near that of NC reporting when used in combination with discharge diagnoses. Thus, for influenza, this enhanced syndromic data feed is comparable to traditional clinical surveillance.
Health information exchange; influenza; surveillance
This work would not have been possible without the support of many people. We specifically would like to thank Phill Lowe, Kimhoa Ngo, Anthony Tellez-Marfin, Bill Lober, the infection control practitioners at the participating hospitals and Inland Northwest Health Services for their contributions.
Encourage hospitals to participate in the Oregon Health Authority (OHA) emergency department (ED) syndromic surveillance system, Oregon ESSENCE.
OHA, in collaboration with the Johns Hopkins University Applied Physics Laboratory, implemented a syndromic surveillance system, Oregon ESSENCE. A critical component to developing and growing this statewide system is obtaining buy-in and voluntary participation from hospital EDs. This process involves approval at multiple levels within a hospital facility from administration to information technology (IT) staff responsible for sending electronic ED data to the Oregon ESSENCE system. Therefore, developing marketing materials that appeal to a wide range of recruitment audiences is a key step in obtaining stakeholder buy-in. OHA adopted the ISDS and CDC syndromic surveillance standards for the public health objective of the Center for Medicaid and Medicare Services (CMS) Meaningful Use Programs. However, Oregon hospitals will not receive financial incentive to participate in Oregon ESSENCE from CMS until 2014 during stage two of Meaningful Use. Consequently, this project's early years will focus on obtaining voluntary participation from hospitals.
OHA developed a recruitment packet to provide information to hospital Chief Executive Officers, Chief Information Officers, Infection Preventionists, Meaningful Use coordinators and IT staff. The packets will be distributed in a number of ways: primarily, during face-to-face meetings with hospital and public health stakeholders, and also during other forums such as meetings of the Oregon Association of Hospitals and Health Systems as well as broader Meaningful Use seminars. Recruitment folders include a brief overview of syndromic surveillance and the ESSENCE system (Welcome to Oregon ESSENCE); a description of utility (Oregon ESSENCE: Real-time Data for Public Health Action); a list of the requested variables (Oregon ESSENCE Data Fact Sheet); examples of effective uses of ESSENCE (ESSENCE success stories); a visual diagram of the data flow process (Oregon ESSENCE data flow); and a list of action steps to begin participation (Let's Roll).
We developed an informative packet of materials for a variety of audiences that is both appealing and concise. Oregon's hospitals come in all shapes and sizes, each with unique approval processes for engaging in data sharing, prioritization of voluntary public health projects, coordination of Meaningful Use efforts and IT support. Therefore, we expect that the breadth and depth of the marketing materials will be a critical component to successful recruitment of hospitals.
We developed appealing and concise information packets for a variety of audiences. While each individual may not need the full breadth of the information we are providing, depending on their role at the hospital, we anticipate that the recruitment packet provides a useful overview of Oregon ESSENCE and syndromic surveillance to a variety of hospital and public health stakeholders.
Emergency departments; data exchange; marketing
To quantify Emergency Department (ED) visits with gastrointestinal symptoms during Federal holidays in Miami-Dade.
People usually celebrate holidays by inviting family and friends to have food at home or by gathering and eating at restaurants or in other public venues. This increased exposure to food with a common source can create conditions for outbreaks of gastrointestinal illnesses. Holidays can also be targeted by bioterrorists who seek to maximize physical damage, psychological impact and publicity around dates of patriotic or religious significance. They might aim at contaminating food and water supplies, especially with CDC-defined category B agents that can cause diseases such as salmonellosis, shigellosis, cholera, crytosporidiosisand infections with
A query with the string ^vomit^,or,^diarrhea^,or,^gastroenteritis^ (VDG) was performed in the Electronic Surveillance System for Early Notification of Community-based Epidemics (ESSENCE) during a 7-day period surrounding the 10 Federal Holidays of each year of the quadrennial 2007–2010. The count of ED patients during the 7-day period was compared to the count of a 28-day background by calculating a ratio between the 2 periods for both, the 4-year average and year-specific. The analysis was broken down by age groups (0–4, 5–17, 18–64, 65 and plus and all-age). Database analysis was conducted using SAS 9.2.
President's Day and Labor Day were associated with the highest 4-year average increases (12% each). Decreases in the 4-year average only occurred around two of the holidays, Independence Day (−7%) and Memorial Day (−5%). Age groups 0–4 and 5–17 had their largest 4-year average spikes around Labor Day (+24% among 0–4 and +30% among 5–17), right after the beginning of classes as well as around President's Day (12% and 13%). The 18–age group had its largest 4-year average increases during Christmas (19%) and Thanksgivings Days (15%). As for the 65+ age group, Christmas (15%) and President's Day (14%) showed the largest increases. The span was much wider when analyzing year-specific holidays, from +40% after Martin Luther King Day in 2010 to −17% after Independence Day on the same year. Factors other than holidays could have also influenced the increases in ED visitors, such as the beginning of classes in August of each year and the H1N1 influenza epidemic in 2009. This research hinges on the comparison of the holiday period to a 28-day background. Future tracking of VDG should also be based on comparing the current holiday period to its mean of previous years to control for seasonal or day-specific effects. The availability of only 4 years of data prevented us from removing the seasonal effect in this research.
ESSENCE can help to track the incidence of gastrointestinal symptoms in the community during holiday periods. The incidence of reportable gastrointestinal diseases during holiday periods should also be ascertained in a future research.
Syndromic; surveillance; holidays; gastrointestinal; illness
To DrGuoyan Zhang and Erin O'Connell for their review and advice.
INDICATOR, an existing biosurveillance system, required an updated user interface to support more data sources and more robust reporting and data visualization.
INDICATOR provides an open source platform for biosurveillance and outbreak detection. Data sources currently include emergency department, patient advisory nurse, outpatient clinic and school absence activity. We are currently working with the University of Illinois College of Veterinary Medicine and will include veterinary data so that animal and human health data can be analyzed together.
The INDICATOR user interface must support multivariate searching, visualization and reporting. Primary users of the system are public health officials with varying degrees of technical experience. Therefore, it is critical that the INDICATOR interface be simple yet powerful. It needs to provide a clear and recognizable hierarchy of search options.
The designer met with key stakeholders, including investigators of the Champaign-Urbana public health district to determine functionality requirements. He then developed use cases and mocked up iterative designs of the user interface, refining them in response to feedback.
The usability study yielded a list of user groups, specifically county health care infectious disease (ID) specialist, county health care medical professional, Carle/McKinley ID specialist and medical professionals, school district medical professional or ID specialist, other local ID specialists, veterinary medical professional or ID specialist, ID researcher, state/federal ID specialist, high-level stakeholder and system administrator.
The study also yielded a hierarchy of searchable categories, e.g., medical diagnoses, school attendance, weather, veterinary and system-generated alerts. The user starts with one high-level category, e.g., medical diagnosis, and is then able to narrow the search by specifying data source, diagnosis, syndromic group, etc. The system provides ‘merge’ functionality to allow comparison of disparate data sources, e.g., weather and flulike illness (
User interface design for INDICATOR.
The team chose Google Web Toolkit (GWT), a well-supported open source software package for the implementation. This allowed a highly interactive user interface that required only a simple web browser for the client. For example, we are able to dynamically generate and populate widgets for searching in response to the terms previously entered by the user. To represent the hierarchical search categories, we use XML files. This enhances maintainability by allowing us to add, edit and remove terms without modifying the code.
The focus on usability yielded a design for a simple, yet powerful user interface that supports exploration of time series and spatial data.
Biosurveillance; user interface; usability; veterinary data; design
We would like to thank Carle Foundation, Christie Clinic and schools in Champaign County for sharing their data. We would also like to thank members of the One Health project at the University of Illinois College of Veterinary Medicine for their input.
Determine operational definition of emergency department (ED) visits attributable to asthma for public health surveillance.
Tracking ED asthma visits is an important part of asthma surveillance, as ED visits can be preventable and may represent asthma control failure (
We utilized all ED visit data for 2008–2009 from the state public health surveillance system (
350,341 (4.0%) of the 8.7 million ED visits had a diagnosis of asthma in 1 of the 11 Dx positions. The most common chief complaints for visits with asthma were: Dx positions 1 and 2- dyspnea and asthma, and Dx positions 3_5- injury. 69,877 (19.9%) of the asthma visits had at least 1 procedure code assigned, those with asthma or cardiac procedure code are shown in
Procedures by diagnosis position where asthma first appears.
Restricting the definition of an asthma-related ED visit to the first diagnosis position may miss a substantial proportion of the asthma-related public health burden. Further analysis is in progress to evaluate the validity of these preliminary findings.
Public health surveillance; ED data; asthma
This project is supported by a Gillings Innovation Laboratory award (UNC). State public health surveillance system data were provided by the North Carolina Public Health Data Group. The content of this paper is solely the responsibility of the authors and does not necessarily represent the official views of the NC Public Health Data Group or NC DETECT. The authors take sole responsibility for the scientific validity and accuracy of methodology, results, statistical analyses and conclusions presented.
To describe the process by which researchers request access to data sets of emergency department data from NC DETECT, the history of this process and the resulting best practices and lessons learned.
The North Carolina Division of Public Health (NC DPH) has been collecting emergency department data in collaboration with the Carolina Center for Health Informatics in the UNC Department of Emergency Medicine (CCHI) since 1999. As of August 2011, there are 113 of 115 emergency departments sending data electronically at least once daily to NC DETECT. Data elements include disposition, initial vital signs, up to 11 ICD-9-CM final diagnosis codes, up to five external causes of injury codes (E-codes), as well as the arrival date and time, patient sex and age, patient zip and county and chief complaint. As of January 2008, NC DETECT emergency department data covered 99% of the NC population and captures approximately 4.5 million ED visits each year. As a result, requests for data from researchers continue to increase. Use of the data for public health purposes is covered by the mandate requiring hospitals to submit their emergency department data to NC DPH.
Data requesters must use the ED data in NC DETECT for public health-focused studies. Data requests from commercial entities are not approved. The data request process occurs primarily via paper and e-mail, although we have implemented a centralized tracking system to store all documentation for data requests and track changes to them over time. To initiate the process, requesters view a presentation on
We currently have 31 data requests in our online tracking system. Each data request represents multiple e-mails and phone calls, iterative revisions to the data request and multiple data pulls from the NC DETECT database. Requests can be delayed when researchers request data elements that are not collected by NC DETECT, submit unclear requirements, change requirements, do not understand the challenges of processing free text data and/or add/change researchers who will be accessing the data. Requesters have used NC DETECT data to publish manuscripts on topics including the health effects of wildfires, ED visits for cancer patients, tick-borne illness and asthma, and comparison of NC ED visit data to national data, among others. Because the ED visit data are collected under a state mandate and in collaboration with the NCHA, their release and use for research is thoroughly evaluated by the DOC. The complexity of the data requests over time has resulted in changing of data use restrictions, as well as revisions of the DUAwording. We do not have enough resources to closely monitor the use of the data once they are provided to researchers. However, researchers are expected to abide by all provisions detailed in their DUA and by signing acknowledge the potential penalties for violation of the terms the agreement.
Data requests can take a considerable amount of time and iterative discussions with the requester, even with a welldefined process and clear documentation. Understanding the administrative and technical time commitments involved is important when considering making syndromic surveillance data available to external users for research.
Data sharing; data use agreements; data requests
To determine system usefulness of the ESSENCE Desktop Edition (EDE) in detecting increases in the number of dengue cases in the Philippines.
Recent events have focused on the role of emerging and reemerging diseases not only as a significant public health threat but also as a serious threat to the economy and security of nations. The lead time to detect and contain a novel emerging disease or events with public health importance has become much shorter, making developing countries particularly vulnerable to both natural and manmade threats. There is a need to develop disease surveillance systems flexible enough to adapt to the local existing infrastructure of developing countries but which will still be able to provide valid alerts and early detection of significant public health threats.
In collaboration with the Philippine National Epidemiology Center (NEC) and the Philippines-AFRIMS Virology Research Unit, Armed Forces Research Institute of Medical Sciences, the EDE program, which was developed by Johns Hopkins University-Applied Physics Lab, was introduced in the Philippines to augment the data analysis capability of the Philippine Integrated Disease Surveillance and Response (PIDSR) System. Reported significant increases in the number of suspect dengue cases/outbreaks at the municipality, provincial and regional level, which were reported to and investigated by NEC from July 1, 2011, to August 31, 2011, were used as a reference point. A defined period, 30 days prior to the date when the event was officially reported, was retrospectively analyzed. The day when an EDE alert was first triggered and the number of EDE alerts detected during this period were described. Since NEC analyzes data by morbidity week, municipalities that were detected to be above the NEC alert or epidemic threshold during a randomly selected morbidity week (week 31; reporting date of August 6, 2011) were compared with the number of EDE alerts triggered during the past 7 days before the report date of morbidity week 31.
Retrospective analysis done during the past 30 days before the event was officially reported showedthatEDEAlertswere already triggered as early as 30 days (median of 27.5 and range 14-30 days) prior to the date of the NEC report. The number of days associated with EDE 'alerts' out of the 30 days prior to the NEC official report date had a median of 9.5 days (range of 3-14 days). A total of 17 municipalities had reported dengue cases above the alert or epidemic threshold with 8/17 of these municipalities having at least 1 day in the previous week with a case count of more than 5 while 9/17 had case counts of 5 or less for all 7 days in the past week.Formunicipalities with at least 1 day with a case count of 5 or more for the previous 7 days, the median of the number of days with associated EDE alerts was 5 (range 0-7 days). For municipalities with case counts of 5 or less for all 7 prior days, no alerts were usually generated (median 0 and range 0-2 days).
A surveillance system's usefulness for outbreak detection should be correlated with its ability to increase the lead time in detecting outbreaks of public health significance, which should subsequently lead to a more timely intervention. Analysis of currently available data seem to show promising applications of EDE in early warning alert capability of impending increases in dengue cases in the Philippines though when case counts are 5 or less, alert results may not be very reliable. Validity of alerts generated by EDE for early detection of outbreaks should be further investigated using other diseases, prediagnostic/ nonclinical/nontraditional data and syndromes, taking into consideration effect of seasonality, weekly trends or holidays.
Surveillance, outbreak, dengue, predictive, validity
The author wishes to acknowledge the support of DOH-NEC Philippines, AFHSC-GEIS, USAMC-AFRIMS and JHU-APL.
We describe approaches to the evaluation of threat reduction efforts in resource-limited countries. Specifically, we present an opportunistic approach to measure the success of efforts to improve on-farm biosecurity in Uzbekistan, which should lead to a reduction of disease transmission between animals and holdings, and to humans for zoonotic conditions.
The Biological Threat Reduction Program (BTRP) of the U.S. Defense Threat Reduction Agency (DTRA) delivers interventions to enhance surveillance of especially dangerous pathogens of both humans and animals within countries of the former Soviet Union. The program targets the different stages at which threats or their impact can be reduced, for example, via (i) the reduction of exposure to threats or (ii) measures for the containment of the threat.
The program delivers training on surveillance-related subjects through regular events attended by representatives of the Ministry of Agriculture of Uzbekistan (UZ). This provides an opportunity to capture data and conduct simple interventions on specific subjects amenable to basic evaluation. Given the sensitive nature of pathogen-specific data, we focus on nondisease- specific interventions leading to the reduction of exposure to and release of any given hazard. Here, we present an opportunistic approach for capturing data, at no additional cost, to assess (i) baseline awareness of on-farm biosecurity measures among UZ veterinary officials and (ii) the impact of training on their awareness of biosecurity. We also discuss the conceptual design of a study to assess on-farm biosecurity practices in UZ.
We monitor four indicators to assess the effects of training (
Collection of data at the training events is planned to start during the summer of 2011. We will present results of the data captured at the training events from that period onward. This will provide a baseline of biosecurity awareness within the surveillance workforce and suggested check lists for on-farm biosecurity assessments.
An on-farm assessment to identify biosecurity breaches against the check list developed at the trainings can be conducted during the visits by officials to farms as part of their regular duties. This will not require significant additional resources. A record of the number of breaches is made for every farm. This constitutes the baseline biosecurity status of the farm prior to the intervention. The intervention would be in the form of advice from the visiting official and the provision of a limited amount of materials to support the implementation of biosecurity measures (e.g., poster with biosecurity messages, log book, etc.). Later visits to the farm by the official will aim to record the number of breaches and allow comparisons with the baseline. Results from this study could be taken as a proxy measure of the ROI from the training efforts.
The model of opportunistic data collection at trainings can be extended to advise other nonsensitive areas, such as some of the technical attributes of the surveillance system (timeliness, etc.). Other extensions are possible by means of expanding generic frameworks, like the one discussed here, into disease-specific ones either to assess biosecurity or the risk pathways to the introduction of a pathogen into the country, a region or a farm.
Especially dangerous pathogens; threat reduction; effect of training; biosecurity; resource-poor countries
Syndromic surveillance uses syndrome (a specific collection of clinical symptoms) data that are monitored as indicators of a potential disease outbreak. Advanced surveillance systems have been implemented globally for early detection of infectious disease outbreaks and bioterrorist attacks. However, such systems are often confronted with the challenges such as (i) incorporate situation specific characteristics such as covariate information for certain diseases; (ii) accommodate the spatial and temporal dynamics of the disease; and (iii) provide analysis and visualization tools to help detect unexpected patterns. New methods that improve the overall detection capabilities of these systems while also minimizing the number of false positives can have a broad social impact.
In this paper, we propose an inference model for determining the location of outbreaks of epidemics in a network of nodes. In our setting, the network is the NC counties where the basic model incorporates spatial geographical relationships between the counties. The model is epidemiological, by choice, to process daily flu counts from the counties in order to infer when an outbreak of flu is present in a county that is distinguishable from background counts. The methodology incorporates Gaussian Markov random field (GMRF) and spatio-temporal conditional autoregressive (CAR) modeling.
The methodology has some nice features including timely detection of outbreaks, robust inference to model misspecification, reasonable prediction performance as well as attractive analytical and visualization tool to assist public health authorities in risk assessment. Based on extensive simulation studies and synthetic data generated from a dynamic SIR model, we demonstrated that the model is capable of capturing outbreaks rapidly, while still limiting false positives.
In this paper, we have presented a new methodology that adapts the existing GMRF class of models to deal with spatio-temporal surveillance data. When the data are mainly spatial and coarsely discretized in time, simple models such as the CAR model will continue to be valuable for descriptive analysis. However, when data have a fine resolution in both the spatial and temporal dimensions, our model, which explicitly incorporates the directional nature of time by conditioning future events on past outcomes, is likely to be more insightful.
Syndromic surveillance; spatio-temporal; Markov random field; conditional autoregressive
This research was supported in part by NSF grants DMS–0914906 to the National Institute of StatisticalSciences, DMS–0914903 to Clemson University, DMS–0914603 to the University of GeorgiaResearch Foundation, DMS–0914921 to the University of South Carolina Research Foundation andDMS–0112069 to the Statistical and Applied Mathematical Sciences Institute. Any opinions, findingsand conclusions or recommendations expressed in this publication are those of the authorsand do not necessarily reflect the views of the National Science Foundation.
To demonstrate how event-based biosurveillance can be utilized to closely monitor disease emergence in an isolated rural setting, where medical information and epidemiological data are limited, for the purpose of identifying areas for public health intervention improvements.
Argus is an event-based surveillance system, which captures information from publicly available Internet media in multiple languages. The information is contextualized, and indications and warning (I&W) of disease are identified. Reports are generated by regional experts and are made available to the system's users (
Argus reports meeting the following inclusion criteria were selected retrospectively: (1) disease: plague, (2) location: Peru, (3) time period: April–October 2010. The reports were reviewed for relevant I&W of plague infection, with the goal of identifying factors that contributed to disease spread and ineffective public health response.
From the time period specified, media reported on a human plague outbreak in northern Peru where all 3 clinical forms of plague were identified (septicemic, pneumonic and bubonic); in one area, bubonic plague was registered for the first time in over a decade while pneumonic plague was reported for the first time ever in the country, according to an official (
The first human case of bubonic plague was reported in April, followed by a 2-month reporting lull from May to July. Subsequently, new media information revealed ongoing human plague cases, including nosocomial pneumonic infections which had spread from one patient to medical staff and one relative, as well as a severe lack of biosafety personal protective and laboratory equipment (
Retrospective review of Argus reports later identified 3 key factors that limited the effectiveness of disease management in the region: (
The use of an event-based methodology provided detailed insight into a localized, small-scale disease situation where limited medical and epidemiological information was available. Argus documentation of this event allowed for a retrospective review, which identified deficiencies in the current disease management system in Peru and drew attention to the potential negative impact of social and political context on public health efforts.
Surveillance; plague; emergence; intervention; isolated
Members of the Georgetown Medical Center Project Argus team, including but not limited to, Denise McAnany, Sarah Riedl and Carlos Balhana.
The purpose of this study was to develop methodology to accurately identify and track heat illness in a timely manner using syndromic surveillance data.
Heat waves have serious health impacts such as heat exhaustion, heat stroke, dehydration and death. Heat illness morbidity and mortality can be reduced with the identification of vulnerable populations and targeted public health interventions. In June and July of 2011, a heat wave occurred in Nebraska in which 28 days reached 90 F or higher. Syndromic surveillance data were used to describe heat-related illness emergency department (ED) visits during this time.
Eight hospitals currently submit syndromic surveillance ED data to Nebraska Department of Health and Human Services (NeDHHS), representing approximately 18% of all ED visits for the state. Five hospitals reported complete data for the selected study period, June 1, 2010–August 10, 2011. The three hospitals not reporting complete data for the study period were excluded. These records represent approximately 15% of all ED visits in the state for June–August. Cases of heat-related illness were identifiedusing ICD9CM diagnostic and external cause of injury codes: 992, 705.1, 708.2 and E900. Additional cases were identified from the chief complaint field using the SAS INDEX function to locate the following words within the text field: ‘HEAT’, ‘HEATED’, ‘DEHYDRATED’ and ‘HYPERTHERMIA’. Each record returned from these searches was examined to confirm the presence of heat illness. Chief complaint fields containing keywords but not involving heat-related illness, i.e., ‘applied heat to swollen ankle’, were eliminated.
A total of 21,906 and 23,222 total ED records were available from five Nebraska ED facilities during June 1, 2010–August 10, 2011. ICD9CM codes identified 182 heat-related illness records in 2010 and 227 records in 2011. Searching the chief complaint field for keywords identified 119 and 188 records in 2010 and 2011, respectively. After reviewing records to confirm presence of heat illness, 64 chief complaint-identified records were excluded in 2010, and 100 chief complaint-identified records were excluded in 2011. In 2010 and 2011, there were 220 and 293 ED records, respectively, indicating heat-realted illness from ICD9CM codes or chief complaint key words. Preliminary results suggest crude rates for heat-related illness are slightly higher in 2011 than 2010. Heat-related illness visits were found in 10.0 records per 1000 visits in 2010 and 12.6 records per 1000 visits in 2011. Combining 2010 and 2011 data, patients with heat-related ED vists were 55% male (n=273) with a median age of 34 years. Further analyses will assess correlation between heat index and heat illness in Nebraska.
The rate of heat-related illness ED visits was slightly higher in the summer of 2011 than in 2010. This system provides an effective method to identify and track heat illness. Timely identification of patients with heat illness using this system can facilitate rapid and focused public health response and reduce heat*related morbidity and mortality.
Syndromic surveillance; heat illness; heat wave
Tom Safranek, MD; Jennifer Marcum, DrPh.
We propose a new text-based spatial event detection method, the semantic scan statistic, which uses free-text data from emergency department chief complaints to detect, localize and characterize newly emerging outbreaks of disease.
Commonly used syndromic surveillance methods based on the spatial scan statistic (
We propose a new approach to detect emerging patterns of keywords in the chief complaint data. Our semantic scan statistic has three steps: automatically inferring a set of topics (probability distributions over words) from the data using Latent Dirichlet Allocation (
We compared the three semantic scan methods to the standard, prodrome-based spatial scan using synthetic disease outbreaks injected into real-world emergency department data from Allegheny County, PA. We first considered 55 different outbreak types, corresponding to all distinct ICD-9 codes with at least 10 cases, which were mapped to one of the existing prodromes. For each outbreak type, we generated spatially localized injects with chief complaints sampled from the cases with that ICD-9 code (Fig. 1). The static, dynamic and incremental methods required an average of 7.7, 7.1 and 6.9 days, respectively, to detect and were able to precisely characterize the outbreak based on the detected topic (e.g., top keywords for ICD-9 code 569.3 were ‘rectal’, ‘bleed’, and ‘bleeding’). The prodrome method achieved more timely detection (5.0 days to detect) but with much less precise characterization (e.g., ‘hemorrhagic’ for ICD-9 code 569.3). Next, we considered both randomly selected, unmapped ICD-9 codes and synthetically generated unprecedented events, such as an outbreak that makes the patient's nose turn green. The prodrome method required 10.9 days to detect these outbreaks, while the semantic scan was able to achieve much faster detection. For example, for the green nose outbreak, the static, dynamic and incremental methods detected in 6.4, 5.3 and 5.6 days, respectively. The dynamic and incremental methods correctly identified the emerging topic (keywords ‘green’, ‘nose’, ‘nasal’, etc.), while the static method did not, since the outbreak did not correspond to any of the topics learned from historical data.
The semantic scan statistic can successfully capture emerging spatial patterns in free-text chief complaint data, enabling more timely detection of novel emerging outbreaks with previously unseen patterns of symptoms. Other advantages include more accurate characterization of outbreaks (identifying a set of keywords that precisely describe the disease symptoms) and the ability to detect outbreaks without preexisting syndrome definitions. Additionally, our methods have the potential to achieve more timely detection by incorporating free-text data sources, such as Twitter and other social media tools, into the surveillance process.
Text mining; event detection; semantic scan
This work was partially supported by NSF grants IIS-0916345, IIS-0911032 and IIS-0953330.
To conduct preliminary assessment of the U.S. destination locations among newly arriving immigrants and refugees with tuberculosis (TB) classifications.
Immigrants and refugees are required to undergo TB screening before entering the United States (
We assessed destination information from the EDN system for immigrants and refugees arriving during 2009 and 2010 with TB disease (Class A TB with waiver) or a radiographic TB without positive smear or culture for TB (Class B1), or LTBI (Class B2), or contact with a TB case. The destination information was mapped with ArcGIS software to the county level and aggregated at the national level. Data were categorized by region according to the 10 Agency for Toxic Substances and Disease Registry (ATSDR) regions (
The EDN system provided 23,348 and 24,707 notifications with TB classifications during 2009 and 2010, respectively. The largest number of notifications occurred in the region comprising Arizona, California, Hawaii and Nevada (ATSDR Region 9); these increased by 8.2% from 9,058 in 2009 to 9,802 in 2010. Over 80% of these notifications are for people whose birth countries are the Philippines, Mexico and Vietnam. Notifications of persons born in Mexico decreased by 10.8% from 2009 to 2010 in ATSDR region 9 whereas those born in the Philippines and Vietnam increased by 2.8% and 72.9% respectively. Information on secondary migration was reported to the EDN system by health departments on 1307 (5.6%) and 1880 (7.6%) records for 2009 and 2010, respectively. The median number of days from arrival to updated location was 115 in 2009 and 70 in 2010.
Over one third of immigrant and refugee arrivals with TB notifications were in the region comprising Arizona, California, Hawaii and Nevada. The increase in TB notifications in this region was attributed to those born in Vietnam. Secondary migration data were available, but the relatively early change in locations may indicate corrections to initial destination data rather than true secondary migration.
Tuberculosis; immigrants; refugees; electronic disease notification; EDN
To create, implement and test a flexible methodology to generate detailed synthetic surveillance data providing realistic geospatial and temporal clustering of baseline cases.
Modern public health surveillance systems have great potential for improving public health. However, evaluating the performance of surveillance systems is challenging because examples of baseline disease distribution in the population are limited to a few years of data collection. Agent-based simulations of infectious disease transmission in highly detailed synthetic populations can provide unlimited realistic baseline data.
Dynamic social networks for the Boston area (4.1 million individuals) were constructed based on data for individuals, locations and activity patterns collected from the real world. We modeled a full season of endemic influenza-like illnesses (ILI), healthcare seeking behavior and a surveillance system for outpatient visits. The resulting in silico surveillance data contain the demographics and complete history of disease progression for all individuals in the population; those who are in a specified surveillance system create a data stream of ILI visits. Outbreaks of influenza are artificially inserted into this surveillance data. Outbreak detection using space-and-time scan statistics was used to analyze the background with and without the inserted outbreaks. The performance of the algorithm was assessed under different levels of coverage and catchment distributions. One hundred unique baseline data sets were generated. Twelve artificial outbreaks were inserted in each. Six different surveillance system designs were assessed.
Simulated ILI surveillance data for downtown Boston as captured by simulated surveillance system. Surveillance counts per day centered in each zip code location are shown as histograms within each zip code. Detection of an inserted test outbreak (red triangle) is indicated by red-bordered zip codes and a false-positive outbreak by black-bordered zip codes. A, Pseudo-ROC curves of outbreak detection. Proportion detected for each surveillance system versus proportion of all false-positives identified. B, Proportion detected versus mean days to detection across decision thresholds.
We present a robust framework for using highly detailed simulations to provide the foundation for evaluating and designing a surveillance system's ability to detect outbreaks. A small demonstration study shows that detection rates varied from 17% to 80% across the different surveillance systems. Increased coverage did not linearly improve detection probability for all surveillance systems. Surveillance systems with uniform coverage of the population did not perform better than one based on a real-world system with nonuniform coverage. Higher coverage improved the timeliness of detection but, for most cases, by only 1 or 2 days on average. Additional results can be found online (
Pseudo-ROC curves of outbreak detection. Proportion detected for each surveillance system vs. proportion of all false-positives identified.
Highly detailed simulations of infectious disease transmission can be configured to represent nearly infinite scenarios, making them a powerful tool for evaluating the performance of surveillance systems and the methods used for outbreak detection.
Computer simulation; surveillance evaluation; outbreak detection; SaTScan
The ‘wisdom of the crowd’ or the ‘crowd trial’ is a process of taking into account the collective intelligence of a large population sharing experiences regarding health issues and treatments online via social media platforms [Health 2.0], generating novel data sets comprising massive unstructured user-generated content of health reports.
Unlike regulated formal postmarketing reports, the crowd trial takes place spontaneously, continuously and on a very large scale. This crowd trial provides a snapshot of health trends and has become a proxy of postmarket clinical trials of medications and other therapies.
The purpose of this case report is to demonstrate how applying an additional data source originated from e-patient reports helps support drug surveillance and pharmacovigilance processes.
Singulair (Montelukast Sodium) is a leukotriene receptor antagonist, indicated to prevent asthma attacks in adults and children. It is also used to relieve allergies in adults and children.
Singulair was approved by the FDA in February 20, 1998. In March 2008, the FDA informed healthcare professionals of investigating the possible association between Singulair usage and behavior/mood changes, suicidality and suicide.
First Life Research (FLR) identifies, analyzes, indexes and aggregates user-generated content by collecting billions of testimonials from social networks. It utilizes cutting edge technologies for massive data aggregation and applies advanced natural language processing (NLP) techniques for continuous analyses, in order to convert this unstructured data into refined information.
With the proliferation of social networks, the web has become a warehouse of patient discussions and reports, estimated at 10 billion records and growing at a rate of 40 percent per year. These reports are spread across more than 150,000 (and growing) English-language sites, forums and blogs. FLR has searched and mapped thousands of these sites and indexed hundreds of millions of posts (currently 800M) and is engaged in refining statistical methods of signal detection that enables investigation of health trends. FLR can look at large samples and discover small changes, such as drug side effects, which may not be discovered by other means for years.
In this case, FLR detected the mentioned FDA alerts and related clinical manifestations prior to the official alert by ‘listening’ to the ‘crowd trial’, in that case, the Singulair users.
FLR signal detection in the Singulair discussions trend overtime.
This report shows that by ‘listening’ into the social web, unforeseen phenomena may be revealed. Specifically, it is evident that advanced technological solutions and signal detection algorithm were able to detect neuropsychiatric events (side effects) in the case of Singulair, more than 2 years prior to any official warning by the regulator or the manufacturer.
‘Crowd trial’ provides a dashboard of health trends and grants feedback on medications, drug safety, side effects, interactions and drug comparisons.
The insights gained and demonstrated as aforementioned can be used to support and enable better informed decision making processes, both for patients and healthcare providers.
Drug surveillance; user generated content; crowd trial; adverse drug reaction
The emerging ‘wisdom of the crowd’ analytics potentially represents a new phase and eventually new tools using data evaluations based on large scale population inputs, and it will benefit greatly all public health environment.
To cluster cities in the United States based on their levels of mortality from influenza and pneumonia.
Influenza is a major cause of mortality. In developed countries, mortality is at its highest during winter months, not only as a result of deaths from influenza and pneumonia but also as a result of deaths attributed to other diseases (e.g., cardiovascular disease). Understandably, much of the surveillance of influenza follows predefined geographic regions (e.g., census regions or state boundaries). However, the spread of influenza and its resulting mortality does not respect such boundaries.
Data on influenza and pneumonia mortality were collected from 97 cities over 11 years (1996 through 2007), as reported in the MMWR (
The average within-cluster distance for the nine census regions is 5205 units. Ignoring geography, we found that our nine largest clusters held 85 of the cities (87.6% of the total cities observed) and maintained an average within-cluster distance of 4918 units. This amounted to a 5.5% reduction in the within-cluster distance. The largest of these clusters held 33 cities from all but one census region and had a within-cluster distance of 4295 units, meaning that its within-cluster distance was 17.5% smaller than that of the mean within-cluster distance of the nine census regions, the largest of which only held 17 cities.
It is natural to think of geographic proximity as an indicator of how likely a city or region's pattern of influenza mortality mirrors that of another region. However, we hypothesize that the relatively high level of travel within the country will affect the pattern of mortality such that cities across the nation may resemble one another more closely than cities within a predefined geographic region. Our approach involved the creation of a discrepancy measure specifically designed for time series data and the application of a clustering routine that seeks to create high quality clusters (rather than high-inclusion clusters). The largest cluster held roughly a third of the observed cities and yet still had a low within-cluster distance when compared to the geographic census regions. This result suggests that many cities observe similar influenza and pneumonia mortality patterns despite varying geographical locations.
There are several limitations to this study. First, while our discrepancy works well in the presence of missing data, a preponderance of consecutive missing time points can negatively affect performance. We determined that 25 cities out of the original 122 cities reported too sporadically for analysis. Furthermore, our clustering technique depends upon the arbitrary selection of a ‘quality criterion’ that is very data driven. High quality clusters can be obtained, but this often leads to a large number of clusters. Conversely, a small number of clusters can be obtained by lowering the quality criterion. Future work will determine if we can use this time-series- clustering approach to find repeatable clusters that may or may not suggest changes to current geographic boundaries in an effort to coordinate future influenza surveillance activities.
Time series; clustering; influenza
The first results of the inventory of syndromic surveillance systems in Europe, conducted in the framework of the European project Triple-S, are presented.
Triple-S (Syndromic Surveillance Survey, Assessment towards Guidelines for Europe) was launched in 2010 for a 3-year period (
Based on the definition of the U.S. Centers for Disease Control and Prevention (
Further, an inventory of syndromic surveillance systems in Europe has been started. To identify human syndromic surveillance systems, a literature review was first performed using Pubmed and Google, identifying 40 relevant publications. A brief questionnaire was sent to Triple-S partners, the European Center for Disease Prevention and Control (ECDC) and other contact persons to identify existing, past, pilot and planned systems in the different countries and the reference person for each system. The reference persons were then asked to complete a long online questionnaire for collecting detailed information (e.g., objectives, data sources, timeliness, statistical methods for outbreak detection, reporting tools and response measures).
For the inventory of veterinary syndromic surveillance systems, a similar method was used. The brief questionnaire was sent to the European Food and Safety Agency (EFSA) focal points and chief veterinary officers of each Member State and to the members of the European College of Veterinary Public Health. Differently from the inventory of human systems, the veterinary inventory included mortality surveillance systems.
Eight site-visits to existing systems are scheduled between June 2011 and May 2012, e.g., United Kingdom, France and Denmark/Sweden. Open to 6–10 project partners and participants from all European countries, the visits offer an in-depth understanding of a variety of systems and facilitate knowledge transfer, through discussions on practical experiences with national and regional stakeholders (e.g., strengths and weaknesses of the systems, lessons learned from the operators and users and expectations of decision makers).
The first output of the project was the adoption of the definition of human and animal syndromic surveillance.
The initial results from the inventory of syndromic surveillance systems and the geographical distribution of identified systems will be presented. As a result of the literature review and the responses to the brief questionnaire for human systems, 19 active systems, 11 pilot or planned systems and 4 systems for past mass gathering events since 2000 (e.g., Olympic games) have been identified to date. For veterinary systems, only 5 systems were identified by the literature review, whereas 16 active, 7 pilot and 2 expired systems have been identified by the brief questionnaires.
The first 4-day site-visit took place in the United Kingdom (Birmingham and Glasgow) in June 2011, where there are several systems based on different data sources: emergency departments, general practitioners (Q-surveillance, Piper and SISRS), phone calls to help lines (NHS Direct, NHS24), out-of-hours primary care and pharmacy prescription data.
By December 2011, four site visits have been conducted. The synthesis of the first visited systems, including their main characteristics and the experiences and lessons learned through those visits, will be presented.
Results from the inventory and the site visits will constitute the foundation for the development of guidelines for improving syndromic surveillance in Europe.
Inventory of systems; syndromic surveillance definition; Triple-S project; site visits; Europe
The authors thank all participants to the project activities. On behalf of the Triple-S project consortium.
To present the Triple-S project, which aims to increase the European capacity for real-time or near real-time surveillance and monitoring of the health burden of expected and unexpected health-related events.
A European project to develop guidelines to strengthen public health surveillance and rapid response was launched in September 2010 for 3 years, under the name Triple-S (Syndromic Surveillance Survey, Assessment toward Guidelines for Europe). The project, co-financed by the European commission through the Executive Agency for Health and Consumers, involves 24 organizations from 13 countries (
Triple-S partners map
The Triple-S project coordinated by the French Institute for Public Health Surveillance (InVS) is divided into six work packages (WP). Three are horizontal managerial WP for coordination, dissemination and evaluation. The other three concern an inventory of existing systems (WP4), country visits (WP5) for knowledge exchange and a deeper understanding of a selected number of systems. WP4 and 5 will lead to the development of guidelines for implementing syndromic surveillance in Europe (WP6).
The inventory will identify competent organizations and reference persons for animal and human syndromic systems in the European Union. A questionnaire will allow the collection of detailed characteristics of established, pilot and planned systems.
The Triple-S consortium has adopted a proactive approach to stimulate knowledge exchange through eight country visits that include stakeholders of the systems.
The projects's final guidelines will provide scientific and technical guidance and tools for the development and im- plementation of syndromic surveillance systems for both human and animal health, according to the needs and expectations of the different EU member states taking into consideration the different data sources available and the different aims of syndromic surveillance systems. An additional outcome of the project is to build a sustainable network of organizations, which can provide support and advice on tasks related to syndromic surveillance: management, partnership with data providers and users, statistical methods, definitions of syndromes and dissemination.
For this, the triple S project will develop links with different projects in the USA (Meaningful Use and Distribute) and Canada (Ontario Risk and Behaviour Surveillance System). For organizations planning to start or reestablish a syndromic surveillance system in their own country, this network could help raise awareness of opportunities and of pros and cons of the anticipated syndromic surveillance system.
The objective of the project is not to create one single European syndromic surveillance system but to review and analyze syndromic surveillance activities across Europe to produce guidelines, while respecting the diversity of health systems and potential data sources for syndromic surveillance across Europe.
Triple-S project; syndromic surveillance systems; guidelines; inventory
To investigate the use of search volume data from Google Insight for the detection and characterization of adverse drug events.
Adverse drug events (ADEs) are a major cause of morbidity and mortality (
Drugs with a high risk of harm outweighing the therapeutic value have recently been subjected to a greater level of interest with the Food and Drug Administration's Risk Evaluation and Mitigation Strategies (REMS) (
Increasingly, Americans have been turning to the internet for health-related information, largely by the use of search engines such as Google. The volume of searches for drugs and ADEs provides a unique insight about the interest in various medications and side effects as well as longitudinal changes.
We generated a list of the 179 most commonly used drugs in the United States in 2008 based on the Agency for Healthcare Research and Quality's Medical Expenditure Panel Survey (MEPS). Using this list of drugs, we consulted MicroMedex, a drug database, for information regarding possible ADEs for each drug. Next, we then obtained search volume data from Google Insight for all possible pairs of drugs and ADEs.
Using a set of searches restricted to only the known ADEs for a given drug, we coded each ADE as either common or other as listed by MicroMedex. Based on this categorization, we conducted a Wilcoxon two-sample signed rank test. Finally, we constructed a negative binomial model to explain the number of ADEs found by Google Insights. The total number of detected ADEs was modeled using the number of common ADEs in MicroMedex, the number of other ADEs in MicroMedex and the number of prescriptions for the drug based on 2008 data from MEPS as covariates.
A second list of 149 drugs with REMS was obtained from the FDA and search volume as collected for each of the drugs. We fit a generalized linear model to the data starting 1 year before and ending 1 year after the initial REMS approval date. The model included a dummy variable indicating if the month occurred before or after the initial approval of the REMS. The interaction between this variable and the time covariate was used to determine if the REMS had any impact on interest as measured by search volume.
Both the Wilcoxon signed rank test and the negative binomial model indicate that Google Insight more readily detected common ADEs compared to the other ADEs. The Wilcoxon rank sum test indicated a shift toward more complete detection for the common ADEs compared to other ADEs (
The negative binomial had similar results. The marginal increase in the number of ADEs detected by Google at the median for both the common and other ADEs was similar at 1.27 and 1.29, respectively. However, the median values were 7 and 39, respectively.
Only 40% (59/149) of drugs with a REMS approval demonstrated a change in slope with 90% confidence. The remaining 60% (90/149) indicated no significant change in interest over the time frame.
Our data help validate the use of Google Insights and search volume as a means to estimate the relative incidence of ADEs. In addition, internet search volume can be used as a rapid means for detecting new or changing ADEs after approval. Finally, the severity and frequency of ADEs may vary within a particular drug class, and search volume may provide additional information for guiding clinicians to select a given drug within a class.
The release of the REMS failed to create a change in search volume for the majority of the drugs. This may be due to prior elevated interest as the result of previous safety alerts or may be an indication that the REMS fails to create increased awareness of the risks of the drug. Further analysis of FDA safety alerts or change point analysis may provide a greater understanding of the effect of various risk management methods.
adverse drug events, Google Insights, post-marketing surveillance
Correlation and linear regression analyses were completed to evaluate the relationship between a heat-related illness (HRI) classifier using emergency department (ED) chief complaint data and specific weather variables as predictors, in Ohio.
The ability to estimate and characterize the burden of disease on a population is important for all public health events, including extreme heat events. Preparing for such events is critical to minimize the associated morbidity and mortality (
Syndromic surveillance data from ED visits were collected and analyzed from Ohio's syndromic surveillance application, EpiCenter, for July 2011. Since the physical effects of HRI can vary greatly and affect multiple body systems, a specific classifier was created to query ED visits that were likely related to HRI and was defined as chief complaints referencing heat ‘exhaustion or exposure’, dehydration or hyperthermia. Measurements for weather variables included temperature, dew point, humidity, pressure and wind speed. The average daily values of these variables were calculated from seven geographically representative cities in Ohio and used as a surrogate for statewide data. These data were obtained from Weather Underground, which collects data from Automated Surface Observations System (ASOS) stations located at airports throughout the United States. These data were analyzed via time-series analyses and stratified by age group and gender. Correlation and linear regression analyses were performed, using SAS v 9.2 to determine which weather variables were the best predictors of HRI, as defined by ED chief complaint data.
During the third week of July 2011, Ohio experienced a heat wave with multiple heat advisories throughout its various cities. The total ED visits related to HRI peaked on July 21 (n=170, 107 males, 63 females), which was also the day with the highest maximum temperature (97.4 F). A time-series chart of these ED visits by age group is shown below. The data show that the most sensitive populations (ages 0-5 and 65 and older) were the least affected and likely were adhering to the heat advisories. The 18–39- and 40–64-year-old age groups were most affected by the heat. Pearson correlation showed a strong relationship between HRI visits and mean temperature and dew point (
These results suggest the advisories provided to the public during the heat wave in Ohio were most adhered to by the sensitive populations (very young and elderly). Middle-aged males were most susceptible to HRI during the peak of the heat wave. Temperature and dew point showed a strong relationship with HRI and were modeled as significant predictors of HRI. Additional analyses should be completed to further evaluate this relationship. Finally, obtaining patient diagnosis records from the hospital EDs would provide strength in validating the observed results.
Heat-related illness; weather; predictor; classifier; correlation
To describe steps used to build the required infrastructure to meet Public Health Meaningful Use (MU) reporting requirements for electronic syndromic surveillance, Electronic Lab Reporting (ELR), and immunization data in NH Division of Public Health Services (NH DPHS).
Under the Electronic Health Record (EHR) Incentive Program Rule, hospitals are eligible to receive incentive payments from the Centers of Medicare and Medicaid Services (CMS) provided they meet certain requirements including MU. Demonstrating MU requires meeting a core and menu set of objectives including the capability to submit electronic syndromic surveillance, ELR and immunization data in accordance with state law and practice. NH is building a NH Heath Information Exchange (HIE) to serve all NH's MU needs including those of public health. This not only represents a huge opportunity for public health to collect more data to enhance disease detection and control, improve safety, and reduce health disparities, but also presents an integration challenge.
In 2011, NH DPHS initiated a project with Orion Health to build a Rhapsody integration engine (
Orion Health contractors set up the Rhapsody server, configured data routes and built validation, filtering, and mapping logic. Mapping to HL7 2.5.1 was performed, but additional mapping to 2.3.1 was done before sending data to the syndromic surveillance application. Hospitals were directed to reroute data transmissions to the new Rhapsody VPN IP address and port, and Rhapsody was configured to pass traffic to the original surveillance application address and port. Additionally, data was sent through the normal VPN connection to compare the accuracy and performance of the new path.
This MU project generated more hospital participation than was realized prior to initiating the Rhapsody integration. Negligible syndromic surveillance processing time degradation was realized with the added Rhapsody processing. This processing allowed NH DPHS to implement it's last acute care hospital into the existing syndromic surveillance application (using Rhapsody mapping), filter existing hospital syndromic surveillance transmissions on specific patient types (preventing unwanted types), receive MU ELR and immunization data prior to expected timelines, increase hospital MU certification reimbursement without additional MU expenditure, and decrease the hospital laboratory staff reporting burden, which previously was manual.
NH DPHS was able to take advantage of opportunities and resources beyond the State of NH. The brokered Orion Health Rhapsody MU certification solution provided a lower cost certification solution to hospitals (as compared to purchasing Rhapsody or certifying their EHR). NH DPHS was able to build an expandable public health MU infrastructure easily integrated with the NH HIE. The MU REC website provided guidance, FAQ's, state rules, and allowed NH DPHS to communicate effectively with hospital partners and the NH REC, to take advantage of REC expertise, and keep all partners informed.
Informatics,; disease surveillance,; Meaningful Use
I would like to acknowledge the NH Bureau of Public Health Informatics personnel Chris Taylor, Brook Dupee, and Jerry Bardsley; and Orion Health contractors Trico Hightower, Art Ramos, Jeff Proulx, and Drew Ivan for their hard work on this project.
To illustrate development of syndromic surveillance in NH, share innovation experience with the public health community and contribute to the syndromic surveillance body of knowledge in the new public health information technology landscape.
In response to the terrorist attack of September 11, 2001, the NH Department of Health and Human Services (NH DHHS) engaged state and external partners in the design of an early warning surveillance system to support bioterrorism and emergency preparedness. Initially, NH DHHS began collecting four syndrome counts from 13 hospital emergency departments (EDs) by fax. Automation began in 2002, when an over-the-counter (OTC) syndromic surveillance pilot system was implemented by Scientific Technologies Corporation (STC). In 2003–2004 this system, the Syndromic Tracking and Encounter Management System (STEMS), was expanded to include school absentee and occupational health reports. Over time, an internal death data application was automated to query vital record deaths, and in 2005, a real-time ED surveillance pilot, the Automated Hospital ED Data System (AHEDD), was developed by STC to replace manual ED surveillance. Over the past decade, NH continued to expand the original concept with innovative approaches to identify undetected or underreported disease outbreaks.
NH's surveillance consists of assessing individual but compa- tible surveillance systems for (
The OTC pharmaceutical system was implemented with automated data processing and alerting within an enterprise architecture. Modified Shewhart charting was developed with dynamic system modeling using a knowledge base technique. Community health status was charted with a set of state syndrome variables and dynamic processes, where baselines, thresholds, trend analysis and alerts from historic data were automatically charted (
Over time, custom querying included data mining techniques adapted from the death data application, (2) to detect narrowly defined chief complaint health conditions and cluster activity. Over time, custom querying included data mining techniques adapted from the death data application, (2) to detect narrowly defined chief complaint health conditions and cluster activity. This together with a ‘Google’-like query tool allow NH surveillance staff to quickly assess any situation. Recently, a single portal infrastructure, based on AHEDD, was created to receive all external syndromic surveillance, Electronic Lab Reporting and immunization transmissions, helping hospital partners meet Meaningful Use (MU), which paves the way for integration with a statewide Health Information Exchange.
Over the past 10 years, the usefulness of NH's surveillance systems has been demonstrated repeatedly. STEMS detected influenza and school norovirus outbreaks (
Ten years of NH syndromic surveillance tool development has established a critical biosurveillance infrastructure with emergency preparedness response capability during disease outbreaks and natural disasters. These syndromic surveillance tools are now integral to the daily efforts of epidemiologists and public health professionals.
Informatics; disease surveillance; Meaningful Use
To explore the association of influenza vaccination with Influenza-like illness ( ILI) among adults aged 65 years and older
After the 2009 H1N1 influenza pandemic, CDC initiated community-based surveillance of self-reported influenza-like illness (ILI) (
Self-reported survey data from the 2010 Behavioral Risk Factor Surveillance System (BRFSS) was analyzed. Because the relationship between ILI and influenza infection is strongest during the influenza season, we limited the study sample to adults aged 65 years and older who participated between January and March 2010 (N=35,628). We adjusted for three categories of individual-level factors: sociodemographics, health behaviors, and history of chronic disease diagnoses. We used stratified, weighted multivariable logistic regression to estimate the association between receipt of the influenza vaccine in the past year and report of ILI in the past month via adjusted odds ratios (aOR) and 95% confidence intervals (95% CI).
Recent ILI was reported by 3.37% (95% CI: 3.02–3.73%) of responders. 67.7% (95% CI: 66.8–68.6%) reported receiving the influenza vaccine in the past year. After adjusting for sociodemographics, health behaviors, and chronic disease diagnoses, receipt of influenza vaccination was significantly associated with recent ILI, with vaccine recipients being more likely to report ILI (aOR=1.50, 95% CI: 1.01–2.24). Persons who are underweight (BMIB18.5, compared with normal weight) (aOR=3.21, 95% CI: 1.19–8.65), and those diagnosed with asthma (aOR=2.45, 95% CI: 1.65–3.62), coronary heart disease (aOR=1.77, 95% CI: 1.17–2.65), and stroke (aOR= 1.75, 95% CI: 1.07–2.87) were also more likely to report ILI.
Our study showed an association between influenza vaccination and influenza-like illness among persons aged 65 years and older. This is a counterintuitive finding as vaccines are known to reduce the burden of influenza. Although our study is cross-sectional and we cannot determine a causal pathway, it is possible that individuals with greater susceptibility to influenza infection (e.g., persons with chronic diseases) were more likely to get vaccinated. Indeed, these findings suggest the success of targeted public health messaging regarding the importance of vaccination among high risk individuals.
Influenza; vaccination; influenza-like illness; surveillance
To assess the impact of database development and maintenance on clinical practice and quality of care.
Cardiothoracic surgery quality improvement is a core value of healthcare provision. In order to improve quality of care, information on key indicators needs to be systematically collected and maintained. In 2006, the cardiothoracic department at AgaKhan University developed an infrastructure that would enable us to answer the more challenging research queries in cardiac surgery practice. The resulting electronic cardiothoracic database is based on the European Association of Cardiothoracic Surgeons database and the Society of Thoracic Surgeons database. While, it is currently used only at Aga Khan University, it has the potential to become a multicenter database.
We chose the following aspects of patient care to be included in the database form: presurgery patient condition and medications, anesthesia information, perfusion information, surgery information, recovery information, status of the patient at discharge and30 days and 365 days postsurgery follow-up information. Information was collected through structured questionnaire by trained data abstractor and entered into Microsoft Access software. On the basis of research hypotheses, specific data chunk was extracted and analyzed in SPSS (Statistical Package of Social Sciences) software.
From January 2006 to May 2011, there were 3418 open heart surgeries performed. Out of them, 69.63% were isolated coronary artery bypass grafting, 10.7% were isolated valve, 2.3% were valve and coronary artery bypass grafting, 15.9% were other cardiac procedures and 1.46% was combination of cardiac procedures. The overall 30-day mortality was 3.6%. Postsurgery morbidity was 21.5%, which includes 3.6% reoperation for bleeding, 0.6% neurological, 0.7% dialysis, 1.3% heart failure, 0.3% septicemia, 1.8% prolonged ventilation, 8.6% multiorgan failure, 0.9% respiratory complications, 1.7% cardiac arrest and 1.1% deep sternum wound infection. Follow-up at 30 days, patients alive were 91.6%, dead, 0.1% and lost to follow-up, 3.6% and, at 365 days, alive, 93.2%, dead, 2.1%, and lost to follow-up, 4.6%.
Before this database, there was no way to monitor mortality and morbidity. Fortunately, with the development of database, postsurgery mortality and morbidity rates could easily be generated. It helped in development of strict enforcement of protocol to reduce the mortality and morbidity rates. It also helped in controlling preventable postsurgery complications. It also helps in identification of a gap inpatient knowledge regarding the use of warfarin after heart valve surgery and deficiencies in laboratory capabilities, both causing catastrophic complications. As a result, we modified our practice in an effort to address these issues and reduce the complication rates after heart valve surgery.
Furthermore, identification of the need to quantify the midterm functional status of in-person and telephonic interview, resulting in the development of a questionnaire that has been added to our protocol 1-year postsurgery.
More meticulous record keeping, including long-term follow-up for 5 years will be collected. In addition to this, the development of a separate congenital/pediatrics cardiac surgery database will also be developed.
Updated and stringently maintained database helps to identify deficiencies in practice and provides a direction for future improvement.
Database; coronary artery bypass grafting; warfarin; mortality; quality of care; evaluation
Dr Taimur Asif Ali (resident of cardiac surgery).
To expand analytical functions of the statewide biosurveillance network for increased local situational awareness, to repurpose surveillance data analysis for strengthening clinical information decision support and to create meaningful visualizations, which are more easily understood by a broader, less technical audience.
The Ohio Department of Health (ODH) has created a biosurveillance network, which was originally based on the Real-time Outbreak and Disease Surveillance (RODSTM created by the University of Pittsburgh and is now developed as EpiCenterTM and managed by Health Monitoring Systems, Inc. (HMS) (
The need to evaluate biosurveillance data within a local, real- world context is necessary to maximize situational awareness at the community level given that: emergencies begin and end as local events, hospital emergency department demographic catchments may vary significantly within a county and some emergency department capabilities may be unique to particular hospitals. With the addition of data analysis methods, which produce intuitive summary visualizations of large data sets, it is hoped that individuals whose training is not as specialized as the EpiCenterTM users will gain a greater understanding of the biosurveillance data and its relevance to the local population.
Reusable procedures were created for MySQL™ (
Additional blocks of code were created in R™ (
The information graphics are to be assembled and annotated as an annual reference resource, Cuyahoga County Hospital Emergency Department Service Utilization Profile (HEDSUP). This output of localized biosurveillance data analysis summarizes the facility admissions, catchment demographics and symptoms using various visualizations, which aim to be understandable by an audience of nonstatistical experts.
Facility specific sections will be made available to hospital infection control practitioners and emergency department managers to evaluate patterns of service utilization and to improve data quality. Local health departments are considered to be the primary users through daily epidemiology and surveillance requiring decision support during disease outbreak investigation and response activities across all hospital emergency departments within a health jurisdiction.
The high availability of open source components cited above, the ease of integration and minimal development cost may provide low-resource environments, such as local health departments, increased functionality through a sustainable and customizable framework of modular components. The utilization of more intuitive data visualizations will shift the burden of advanced data analysis to a less technical interpretation of information by a broader user base for a greater understanding of local public health dynamics.
Visualization; open source; localized; situational awareness; biosurveillance
An increase in tuberculosis (TB) among homeless men residing in Marion County, Indiana, was noticed in the summer of 2008. The Marion County Public Health Department (MCPHD) hosted screening events at homeless shelters in hopes of finding unidentified cases. To locate men who had a presumptive positive screen, the MCPHD partnered with researchers at Regenstrief Institute (RI) to create an alert for healthcare providers who use the Gopher patient management system in one of the city's busiest emergency departments. A similar process was used at this facility to impact prescription behavior (
MCPHD and RI created a legal memorandum of understanding so that MCPHD could share the names and date of births of suspect cases to the programmers at RI. The alert went into effect in July of 2010. When a healthcare provider's search is also one of the suspected names, an alert appears on the screen informing the provider that this person should have a chest x-ray as part of a follow-up to a TB outbreak investigation. A phone number of a MCPHD nurse on call is provided. The suspect list is periodically updated to remove names of patients who have been located in other medical settings. The novel aspect of this system is that normal methods of locating these individual such as phone or address was not available. Additionally, other traditional public health methods to contact these patients had not proved successful.
Fifty-three different patients have been on the alert list since activation. Only one notification has occurred in the 13 months of activation. On December 1, 2010, the TB program reported that a provider had seen one of the suspect cases and the alert prompted the provider to order a chest x-ray and notify the MCPHD staff.
A review of a hospital patient management system revealed that 12 other patients were seen in the emergency departments while on active alert lists. Some patients were seen more than once while on the list. Some cases showed that the chest x-rays were performed as requested but the patient records did not indicate if the procedure was prompted due to the alert or because the patient presented with symptoms of TB. A review of the process is underway to better understand why these encounters did not provide a notification to the MCPHD.
Several MCPHD staff work in local homeless shelters daily looking for the suspect patients. If a staff member prompts an individual to go to the ED, that encounter is not recorded in the MCPHD Patient Management System. Therefore, the provider may ignore the TB alert on these patients since the patient was already suspected for TB.
In some instances, the patients were seen at the MCPHD TB clinic within a couple of days of being seen at the hospital ED. Again, the records do not indicate if the patient was prompted to go to the TB clinic. Interestingly, no new TB cases among this population were reported for the month of August.
Future attempts at locating patients: Since many of the identified cases have known psychiatric issues, outreach workers in the TB program are hoping to partner with the mental health community to reach some of the suspect cases. MCPHD is working with a local mental health clinic to create a similar alert in their patient management system. Also, RI may help develop another alert that includes contacts of cases, rather than suspect patients in hopes of completing the first initial screening for that segment of the population.
The most important outcome is getting the patients tested and treated if infected. Assessing the effectiveness of the alert is difficult due to a lack of encounter data. Only one-fourth of the patients (13 of the 53) visited the emergency department during the year of the alert. This is another reminder that multiple strategies must be used to reach this population for care. Health informatics can be an aid to public health in such endeavors.
Public health alert; clinical encounter; TB; automated alert
This paper characterizes a regional outbreak of tuberculosis (TB) in market swine by combining local swine producer-based information on condemned stock at slaughter with geographically broader FSIS Animal Disposition Reporting System (ADRS) data. This study aims to obtain summary information on anomalous swine TB (STB) condemns at slaughter, compare critical outbreak time frames between outbreak areas and identify the geographical spread of abnormally high STB condemns.
A case study presented at the November 2010 Iowa Annual Swine Disease Conference for Swine Practitioners detailed increases in STB lesions beginning January 2010 (
The ADRS provides weekly condemn data to VS along with information on species and total number of animals slaughtered. June 2007–May 2011 ADRS market swine data were grouped by three major swine production areas (basins) to (
Mean weekly swine TB condemn rates, which seasonally ranged from 5.7 to 21.4 per 100,000 between 2007 and 2009 (mean=10.7, SD=3.0), increased above typical levels beginning January 2010 and rose even higher beginning January 2011 (
Weekly swine TB condemn rates, four-year series.
By combining ‘on-the-ground’ practitioner-based information with geographically broader analysis of ADRS data, we identified an emerging disease situation involving TB lesions in swine. A preliminary epidemiological investigation suggests that likely principal risk sources are related to feed and ground water (
Abattoir; surveillance; syndromic
To develop and implement a web-based, county-level flu immunization record keeping system that accurately tracks nonidentifiable vaccine recipients and seamlessly uploads to the state record keeping system.
Historically, it has been the role of local health departments to administer, monitor and report flu vaccinations of its residents to the state health department. In 2009, the looming threat of an influenza outbreak (H1N1) led to the extension of the Public Readiness and Emergency Preparedness Act (PREP) (
No off the shelf software was available that met local health department system requirements and budget constraints. Thus, a collaborative team of public health professionals and database programmers convened to establish a project charter that outlined the system requirements, personnel responsible, time line and budget. A qualitative analysis of current county level systems and data helped to establish the requirements of the FITS system, as well as direct the reporting capabilities and features. The fixed budget expenditure and early deadline led to an expedited timeframe; the system was completed within 4 months.
A FITS was created in accordance with the project charter, timeline, user specifications and budget and included but was not limited to the following technologies: CentOS, Ruby and Excel. An innovative feature of the FITS system is the live lookup of national drug codes (NDC) hosted on the FDA's website, which autopopulates the FITS vaccination database with up-to-date information (
The field of public health informatics calls for the conceptualization, design, development, deployment, refinement, maintenance and evaluation of surveillance systems (
Influenza; surveillance; software; administration; recipient
The development of this system was made possible by the commitment of Stark County Health Department personnel (William Franks, Christina Gruber and Sherry Smith), Project Manager Lauren Drinkard, Programmer Michael Fleet and Project Director, R. Scott Olds.
We propose a modification to the usual inference test of the spatial scan statistic, incorporating additional information about the size of the most likely cluster found.
Spatial cluster analysis is considered an important technique for the elucidation of disease causes and epidemiological surveillance. Kulldorff's spatial scan statistic, defined as a likelihood ratio, is the usual measure of the strength of geographic clusters (
Kulldorff's spatial scan statistic for aggregated area maps searches for clusters of cases without specifying their size (number of areas) or geographic location in advance. Their statistical significance is tested while adjusting for the multiple testing inherent in such a procedure. However, as is shown in this work, this adjustment is not done in an even manner for all possible cluster sizes (
We pose a modified inference question: what is the probability that the null hypothesis is rejected for the original observed cases map with a most likely cluster of size k, taking into account only those most likely clusters of size k found under null hypothesis for comparison? This question is especially impor- tant when the p-value computed by the usual inference process is near the alpha significance level, regarding the correctness of the decision based in this inference.
Numerical experiments are made showing that the proportions of rejections of the null hypothesis differ noticeably, by employ- ing the usual critical value, compared with using the data-driven critical values. It is also shown that the computational cost of estimating the data-driven critical value may be reduced through the use of a simple interpolation.
A practical procedure is provided to make more accurate inferences about the most likely cluster found by the spatial scan statistic. The proposed method is more useful when the computed
Spatial scan statistic; inference; data-driven
We thank the agencies Fapemig, CNPq and Capes.
We describe a method to determine the partition of a map consisting of point event data, identifying all the multiple significant anomalies, which may be of high or low risk, thus monitoring the existence of possible outbreaks.
The Voronoi Based Scan (VBScan) (
In our novel approach, we use the previous VBScan recursively on the map with case-control point event data. At each recursive step, we compute two functions: (i) the likelihood ratio of the multiple components and (ii) the likelihood ratio increase since the previous step. As the first function always increases monotonically with every added component to the partition, the last function is used as a measure of the cost-benefit of adding a further region to the partition. This is done employing a multicriteria decision process, determining the nondominated partition solutions. Through Monte Carlo replications under null hypothesis, we compute the significance of the nondominated solutions and choose the best partition.
Our method was tested on several different simulated maps partitioned into different numbers of components (ranging from 2 to 4). The relative risks for each component were chosen as 3 sigma, –3 sigma and 0 sigma, corresponding respectively to high-, low- and neutral-risk spots. We evaluate the power of detection and matching (a measure of overlap between the real and detected partitions) for each set of 1000 Monte Carlo replications. The average power varies from 0.686 to 0.803, and matching varies from 0.566 to 0.850 for the several sets of simulations.
We also applied the method on a case study of dengue fever in a small Brazilian town in 2010 (
The proposed method is fast, with good partitions' accuracy determination. The dengue fever application's result shows that our method is in very good agreement with the previous analysis with VBScan, which indicates two significant high-risk clusters (the red region is the primary cluster with
Case-control; disease cluster; spatial scan statistic; space partition; dengue fever
We thank the agencies Fapemig, CNPq and Capes.
We propose a fast, exact algorithm to make detection and inference of arbitrarily shaped connected spatial clusters in aggregated area maps based on constrained dynamic programming.
The spatial scan statistic (
Many heuristics have appeared recently to compute approximate values that maximizes the logarithm of the likelihood ratio. The Fast Subset Scan (
The spatial cluster detection problem was formulated as the classic knapsack problem (
Dynamic programming relies on the principle that, in an optimal sequence of decisions or choices, each subsequence must also be optimal. During the search for a solution, it avoids full enumeration by pruning early partial decision solutions that cannot possibly lead to optimal solutions.
We propose a novel method, the Geographical Dynamic Scan (GDScan) to find optimal connected clusters. It employs an adaptation of the Nemhauser–Ullman algorithm for the 0–1 knapsack problem (
The dynamic programming algorithm is thus modified to consider (i) a geographical proximity constraint and (ii) a connectivity constraint, for each region j. Assuming that the geographical proximity of a region j contains k regions, the geographical dynamic scan guarantees the optimal solution within the collection of 2^k subsets, searching for only a small number of subsets, which depends almost linearly on k, on average.
We conducted numerical simulations showing that GDScan has good power of detection, sensitivity and positive predicted value.
An application is shown for the dataset of Chagas’ disease cases in the population at risk of puerperal women in Minas Gerais state, Brazil, in 2006 (
The nondominated sets of cluster solutions.
We show that GDScan is a fast and an efficient method to detect connected arbitrarily shaped disease clusters in aggregated area maps.
Disease cluster; dynamic programming; spatial scan statistic
The authors thank the agencies CNPq, Fapemig and Capes.
To demonstrate how event-based biosurveillance, using direct and indirect indications and warning (I&W) of disease, provides early warning and situational awareness of the emergence of infectious diseases that have the potential to cause social disruption and negatively impact public health infrastructure, trade, and the economy (
Argus is an event-based, multilingual surveillance system, which captures and analyzes information from publicly available Internet media. Argus produces reports that summarize and contextualize I&W of emerging threats and makes these reports available to the system's users (
Argus reports meeting the following inclusion criteria were reviewed: (
On May 23, a surge in EHEC infections was reported at hospitals mainly in northern Germany; the outbreak was unusual in that it caused atypically severe symptoms in adult females. By May 26, state health authorities had identified over 600 EHEC cases, including 214 severe cases with hemolytic uremic syndrome (HUS), and confirmed the causative agent as a highly virulent HUS-associated EHEC 41 strain belonging to serotype O104:H4. Faced with a rapidly growing number of cases, health authorities notified the EU of a potential public health emergency of international concern and implemented new surveillance systems (
This study highlights the challenges faced in providing a timely response to a rapidly spreading infectious disease outbreak and the role that event-based biosurveillance can play in quickly identifying areas for public health intervention. Argus reporting identified that the EHEC outbreak fundamentally challenged the public health system in Germany, by exposing deficiencies in infectious disease surveillance. More importantly, it evidenced that even a strong public health system must be able to adapt rapidly to challenges posed by the changing epidemiology of infectious diseases (
Event-based biosurveillance; infectious disease; social disruption;
Iarocci and Polakis, authors with equal contributions.Special thanks to Inna Sakhvoruk, Denise McAnany, Wai-Ling Mui and Aimee R. Reilly.
To better inform health IT standardization practices, specifically related to Meaningful Use, by describing how US public health agencies use unstructured, free-text EHR data to monitor, assess, investigate and manage issues of public health interest.
In 2010, as rules for the Centers for Medicaid and Medicare Electronic Health Record (EHR) Incentive Programs (Meaningful Use) (
On January 31, 2011, ISDS, in collaboration with the CDC BioSense Program, recommended a core set of data for public health syndromic surveillance (PHSS) to support public health's participation in Meaningful Use. This study was conducted to better support a requirement for ED CC as free-text, by investigating the relationship between the unstructured, free-text form of CC data and its usefulness in public health practice.
PHSS analysts from 40 public health agencies that contribute syndromic data to the ISDS Distribute project were asked to take an online survey.
The survey, developed in consultation with state- and local-level syndromic surveillance experts and implemented using SurveyMonkey®, consisted of 15 questions, which were crafted to obtain data in four areas: (
Participants had 2 weeks to complete the survey. ISDS staff contacted nonrespondents to encourage participation 7 and 3 days before the end of the survey period. Qualitative survey data from open-ended questions were reviewed and grouped into themes or categories.
PHSS epidemiologists or analysts from 87.5% (35 out of 40) of the Distribute-contributing health authorities completed the survey. Within the respondent group, 9 cover local jurisdictions, 25 state jurisdictions, and one was from CDC BioSense. Combined, the 35 agencies captured EHR data from 1344 ED.
Survey results revealed that 97% of participants receive ED patient CC data in free-text (
Data formats in which ED CC are received by public health authorities for syndromic surveillance. Most syndromic surveillance practitioners (97%) receive and use ED CC data in a free-text format. A significant number of survey respondents also report receiving these data as ICD-9 (34%) or as text from a drop-down menu (20%).
A majority of survey respondents (74%) reported having used free-text CC to monitor public health in over 17 different emergencies over the past 2-3 years. Most frequently, free-text CC was used to monitor the impact of H1N1, heat waves, infectious disease outbreaks, and winter storms.
Through a national survey of PHSS epidemiologists, ISDS identified that public health agencies benefit from free-text CC data, and this format needs to be maintained. ISDS also learned that as newly certified EHR systems are switching CC from free-text to a structured format, the advantages for making this transition are not fully known to public health practitioners.
Syndromic surveillance; Meaningful Use; free-text; EHR
Mr. Aaron Kite-Powell, Dr. Joseph Gibson and Ms. Julia Gunn provided valuable insight on early survey iterations.
To evaluate the utility of the Connecticut hospital emergency department syndromic surveillance system (HEDSS) to monitor gastrointestinal (GI) illness in the community.
The HEDSS system was implemented in 2004 to monitor disease activity (
Although previous studies have shown that ED syndomic surveillance is not useful for early detection of GI outbreaks (
In Connecticut,
Vomiting and diarrhea were each highly correlated with the combined GI syndrome (
There is a strong and consistent association between ED visits for GI illness and facility outbreaks, the majority of which are suspected to be caused by norovirus (
Syndromic surveillance; gastrointestinal; public health practice; evaluation
To describe the improvements in New Jersey's Emergency Department surveillance system over time.
In the summer of 2001, New Jersey (NJ) was in the process of developing surveillance activities for bioterrorism. On September 11, 2001, the United States suffered a major terrorist attack. Approximately a month later, anthrax-laced letters were processed through a NJ Postal Distribution Center (PDC). As a result of these events, the state instituted simplistic surveillance activities in emergency departments (EDs). Over time, this initial system has developed into a broader, more streamlined approach to surveillance that now includes syndromic data, e.g., Influenza-like illness (ILI), as well as the use of technology (automated surveys, real-time data connections and alert analysis), to achieve surveillance goals and provide daily information to public health partners in local health departments and DHSS response colleagues.
Daily response rates over time were analyzed to determine whether enhancements to surveillance produced any improve- ment in participation by EDs. During the timeframe used for the study, the total number of EDs varied due to facility closures and reorganizations and, therefore, daily response was measured by using the percentage of facilities responding each day versus the actual number. The study was broken into three different time periods: (1) December 2001, which is when the state's surveillance began, to August 2004, prior to updates in anticipation of the Republican National Convention in nearby New York City; (2) August 2004, when a more technologically advanced method was introduced, to April 2009; and (3) April 2009 when the Novel H1N1 Influenza A outbreak occurred and more sophisticated data collection mechanisms were implemented to present day.
With each implementation of a new form of data collection and more advanced analysis, the response rate increased (see
Facility response rates over time.
As automation in surveillance activities has increased, participation rates of facilities improved as well. Hospital staff became more engaged when there was a more defined purpose to reporting ED visits and admissions (e.g., The Republican National Convention and the H1N1 Novel Influenza A outbreak). Based on the improvements observed, the state is undertaking a project to move all NJ EDs into a real-time, syndromic surveillance system. This implementation is expected to further enhance data reporting and increase response rates beyond the current 86.4%.
Surveillance; emergency department; participation
NJ regional epidemiologists; Hospital infection control and ED staff; HMS,Inc.; DHSS OITS staff.
To describe how use of cloud computing resources can improve the timely provision of disease surveillance analyses.
Two significant barriers to greater use of syndromic surveillance techniques are computational time and software complexity. Computational time refers to the time for many methods (for example, scan statistics and A Multidirectional Optimum Ecotope-Based Algorithm [AMOEBA] statistics) to create reliable results. Software complexity refers to the difficulty of setting up and configuring suites of software to collect data, analyze it and visualize the results. Both of these barriers can be partially surmounted by the use of cloud computing resources.
We used Amazon's EC2 (Elastic Compute Cloud) services to experiment with cloud computing for syndromic surveillance. We applied two cloud service models: Infrastructure as a Service (IaaS) and Software as a Service (SaaS).
Our first goal was to apply cloud computing technologies in order to reduce computational time needed for syndromic analyses. Scan statistics, due to their reliance on Monte Carlo simulation to find confidence levels, are particularly well suited to being improved by parallel computation. We used the R package DCluster to calculate scan statistics and combined that with the SNOW (Simple Network of Workstations) package. We also experimented with using cloud computing to parallelize the AMOEBA approach to cluster detection.
Our second goal was to determine the practicality of easing the barrier of software complexity. To that end, we created software packages that include data import, analysis and visual presenta- tion of results and released them as freely available virtual machines, or images, for the public to use. The GeoViz Toolkit was one of the software packages delivered in this manner (
The GeoViz Toolkit is shown with multiple views of rates of ILI. The spatiotemporal clustering algorithms in the GeoViz Toolkit, as well as the multiple view visualizations, are examples of syndromic analytic functions that can be improved using cloud computing.
We found that both Infrastructure as a Service and Software as a Service cloud computing service models can help reduce barriers to effective use of sydromic surveillance methods. Easy provision of many computers allowed us to speed up the computational times by an order of magnitude. The creation of integrated software services to perform disease surveillance is the easiest way to deliver complex functionality.
We conclude that, in the future, cloud computing can and should play a more prominent role in disease surveillance.
Cloud computing; scan statistics; informatics
We would like to thank the NGA University Research Initiative (NURI) program and Amazon Web Services, for their support. No endorsement is implied.
To develop an algorithm to identify disease outbreaks by detecting aberrantly large proportions of patient residential zip codes outside a healthcare facility catchment area.
The Veterans Health Administration (VHA) uses the Electronic Surveillance System for the Early Notification of Community-based Epidemics (ESSENCE) to detect disease outbreaks and other health-related events earlier than other forms of surveillance (
An H1N1 influenza outbreak was identified at a Veteran Benefits Administration (VBA)-sponsored conference in Baltimore, MD, in July 2009 in which affected VBA employees (both local and from out-of-town) sought healthcare at the VA Maryland Health Care System—Baltimore Medical Center. Using ESSENCE, daily counts of ICD-9 codes related to influenza diagnoses (as defined by VA ESSENCE influenza-like illness [ILI] syndrome group) were collected from the VHA Baltimore Medical Center from March 01, 2009, to September 12, 2009. Data included case status (as defined by ICD-9 code and chart review), date and location of visit and patients’ zip code of residence. We also accessed data from the VA Planning System and Support Group to determine whether the patients’ residential ZIP code fell within the Baltimore VA Medical Center's catchment area. Using SAS, a p-chart (where the denominator was the daily number of patient ILI encounters) was run to determine days during which an aberrant proportion of patients from out-of-catchment zip codes were encountered.
An aberrant proportion of out-of-catchment zip code ILI encounters signaled an out-of-control process (or alert) on July 23, 2009, 2 days later than the beginning of the influenza outbreak at the facility (
ILI encounter dates of patients’ whose residential zip code is out of the Baltimore VA Medical Center's catchment area. The jagged solid line exceeding the dashed line (the upper confidence limit) indicates an aberrantly large proportion of out-of-catchment events over the surveillance period.
Using p-charts to detect unusual clusters of patients’ residential zip codes that fall outside of facilities catchment area is likely a method of detecting disease outbreaks previously not utilized. Future work includes running this algorithm in all VA Medical Centers to prospectively identify disease outbreaks involving increased proportions of patients residing outside of the medical center's catchment area.
Surveillance; p-chart; algorithm; signal detection; outbreak
We are grateful to Gina Oda, Eugene Elbert, and Vivian Hung for their help with this project.
To monitor community illness and detect outbreaks during the 2009 influenza A/H1N1 pandemic using a newly developed surveillance system for monitoring school absenteeism.
In April 2009, a novel strain of influenza A was detected in Mexico, which quickly spread to the United States and the rest of the world. In response to the pandemic, the New Hampshire Department of Health and Human Services (NH DHHS) developed a web-based school absenteeism reporting system to track and record overall absenteeism and influenza-like illness (ILI)-related absenteeism in New Hampshire schools.
An absenteeism reporting form was developed and placed on a NH DHHS website. Access to the reporting form website was through a secure NH Department of Education (DOE) web portal, and school nurses were asked to voluntarily report data into the system. The questionnaire asked for the number of students absent, the number of students absent for ILI, the number of staff absent and the number of staff absent for ILI in addition to the name of the reporting school (
New Hampshire Department of Health and Human Services web-based school absenteeism reporting form.
Between September 7 and December 23, 2009, statewide overall school absenteeism ranged from 0.0% to 29.3% and statewide ILI-related absenteeism ranged from 0.0% to 14.2%, both of which peaked the week of November 1–7, 2009. The observed peak of school absenteeism was consistent with data observed in other ILI surveillance systems such as over-the-counter sales of cough and cold medications and visits to emergency departments (
Indicators of influenza-like illness in New Hampshire, September 1–December 23, 2009
The newly developed school absenteeism reporting system provided important public health surveillance data to evaluate potential outbreaks or clusters of disease in communities and resulted in the detection of and response to 103 outbreaks of ILI that would not have been detected otherwise. Enhanced interaction with school nurses through the development of the surveillance system resulted in increased awareness in school populations about the influenza A/H1N1 (2009) virus. Further- more, the rapid detection and response to potential outbreaks identified using the system may have minimized the effect of ILI in the school system and the community, through heightened awareness and implementation of control measures.
Although the reporting system started as a way to monitor the impact of the 2009 influenza A/H1N1 pandemic, NH DHHS has continued to routinely monitor student absence ever since. School absenteeism surveillance has the potential to aid in early detection, and mitigation, of other communicable diseases in the school system, an important indicator of illness in the communit.
School surveillance; H1N1; influenza; New Hampshire
Google flu trends (GFT) is a novel internet-based influenza surveillance system that uses search engine query data to estimate influenza activity. This study assesses the temporal correlation of city GFT data to both confirmed cases of influenza and standard crowding indices from one inner-city emergency department (ED).
EDs supply critical infrastructure to provide medical care in the event of a disaster or disease outbreak, including seasonal and pandemic influenza (
This study was performed over 21 months (January 2009–October 2010) at an urban academic hospital with physically and administratively separate adult and pediatric EDs. We collected weekly data from GFT for the city of Baltimore, ED CDC reported standardized influenza-like illness (ILI) data, laboratory-confirmed influenza data and ED crowding indices (including patient volume, number of elopements, waiting room time and length of stay for admitted and discharged patients). Pediatric and adult data were analyzed separately using cross- correlation with GFT.
GFT correlated with both number of positive influenza tests as seen in
Temporal comparison of GFTs and hospital wide positive influenza tests.
City-level GFT shows strong correlation with local influenza cases and ED ILI visits, providing first time evidence of its utility for local ED surveillance and, potentially, response planning. Importantly, GFT correlated with several pediatric ED crowding measures as well as those for low acuity adult patients.
Influenza, Google flu trends, emergency department, crowding
Supported by the Department of Homeland Security (2010-ST-061-PA0001).
To describe a real-time reportable disease and surveillance solution focused on local public health department needs and compatible with state health departments, regardless of meaningful use certification status of health care providers.
Multiple options (
Prior to ARRA 2009/Meaningful Use, KHA, KY CHFS and ETI created an electronic disease-reporting solution for hospitals and associated local public health. The Community Surveillance project operates in 3 population centers within Kentucky and provides real-time surveillance and disease reporting for up to 18 prevalent disease conditions across more than 20 provider locations and 6 provider organizations within the Northern Kentucky, Lexington/Fayette County and Louisville Metro communities. The software solution, ETI's HealthSIS, is able to accept meaningful use certified messaging, uncertified messaging formats and custom data streams for both reportable disease and syndromic surveillance information from a single stream within each provider location. The messaging stream used for identifying reportable or syndromic conditions is a copy of the content generated by normal hospital operations; specialized or additional data input is not required from health care provider staff. HealthSIS is configured to submit only data relevant to surveillance goals of the community from source systems; this configurable filtering capability allows for reduced resource requirements, reduced data management resources, and a manageable data set for analysis.
Since mid-2008, NKIDHD receives electronic disease reports and surveillance support for what is now the 18 most prevalent disease conditions in the community. SEMC (6 facilities) has realized 75%–90% reduction in public health reporting effort as a result of the capabilities provided by HealthSIS. Since mid-2009, LFCHD receives syndromic surveillance data from ambulance runs, initially to support preparations for the 2010 Alltech-FEI World Equestrian Games; LFCHD began receiving electronic disease reports for 9 prevalent disease conditions in mid-2010 from CBH and UKMC. Since late 2009, LMPHW receives electronic disease reports for 5 prevalent disease conditions from BHE and notifications of same from JHSMH; in 2011, NHC began participating in the disease-reporting process.
Installing and maintaining electronic disease and syndromic surveillance to support public health agencies is possible without large budgets and without massive systems upgrades within health care provider organizations; benefits are clearly measurable – independent study in Washington state in 2010 confirms benefit of the concept (
Disease reporting; clinical systems; data integration
To describe the application and process developed by the Maryland Department of Health and Mental Hygiene (DHMH) Office of Preparedness and Response (OP&R) and Office of Information Technology (OIT) for monitoring and auditing the transfer of syndromic surveillance data.
The electronic surveillance system for the early notification of community-based epidemics (ESSENCE) is the web-based syndromic surveillance system utilized by DHMH. ESSENCE utilizes a secure, automated process for the transfer of data to the ESSENCE system. Data sources in the Maryland ESSENCE system include emergency department (ED) chief complaints, poison control center calls, over-the-counter (OTC) medication sales and pharmaceutical transaction data (for certain classes of antibacterial and antiviral medication). All data sources have statewide coverage and are captured daily in near real-time fashion. OIT developed a web-based application in conjunction with OP&R to allow the epidemiologists involved in the ESSENCE program to monitor and audit the transfer of this data. The application allows the user to indicate whether or not each data file has been consumed into ESSENCE for any date of the year. The user can edit these daily entries at any time to update the status of the data that have been received. The user may also query the database by data source, date and date range to generate a report. The database also contains contact information for technical and infection control staff at the hospitals that participate in the ESSENCE program. Finally, the application can also generate reports that detail which users have logged into ESSENCE, when the log-in occurred, and which pages within ESSENCE were visited.
Forty-six EDs, two major pharmacy chains, two poison control centers and the Centers for Disease Control and Prevention (CDC; through a pilot partnership), all contribute data to ESSENCE on a daily basis. Thirty-six separate SSH File Transfer Protocol (sFTP) data feeds are required to transfer and incorporate these data into ESSENCE. Beginning January 1, 2011, the web-based application developed by OIT was utilized on a daily basis to ensure that the transfer of data was monitored and recorded regularly. Each morning, an epidemiologist from OP&R logs into ESSENCE and verifies which data points have been consumed into the system. The presence or absence of each data point is then recorded in the data tracking application. An automated e-mail is generated that details which data sources are absent. This e-mail is sent to the OIT employees involved in the ESSENCE program, who then check the server to see if the data were transferred or if there was an error during the transfer and consumption process. The epidemiologists then contact each data source that failed to send the data on that particular day.
Data are presented for January 1, 2011, through July 31, 2011 (212 total days). Between the ED chief complaint data, the poison control data, the OTC medication sales data and the pharmaceutical transaction data, there are a total of 50 data points on any given day in the Maryland ESSENCE system. This amounts to a total of 10,600 possible data points that can be in the ESSENCE system during this time frame. Using the data tracking application, OP&R has managed to acquire and consume 10,527 data points or 99.31% of all possible data points for this time period. The poison control data are complete for this time frame, as is the pharmaceutical transaction data, and one of the major pharmacy chain's data. The other major pharmacy chain whose data are not complete is only missing 2 data points (99.10% complete). Twenty-five of the 46 EDs have transferred 100% of the possible data points to the ESSENCE system. All other EDs contributing data to the ESSENCE system are at least 95.75% complete.
The data tracking application developed by OIT and utilized by OP&R to monitor and audit the transfer of syndromic surveillance data has thus far been successful in ensuring data completeness. Those involved with the program have been able to monitor and document that over 99% of all possible data are incorporated into the surveillance system. The data source with the lowest completion percentage has over 95% of its data incorporated into the system. DHMH will continue to use this system moving forward to ensure that the syndromic surveillance data transfers continue to be successful.
Data; transfer; audit; ESSENCE
To evaluate the performance of several sentinel surveillance site placement algorithms for influenza-like illness (ILI) surveillance systems. We explore how these different approaches perform by capturing both the overall intensity and timing of influenza activity in the state of Iowa.
ILI data are collected via an Influenza Sentinel Provider Surveillance Network at the state level. Because participation is voluntary, locations of the sentinel providers may not reflect optimal geographic placement. This study analyzes two different geographic placement schemes––a maximal coverage model (MCM) and a K-median model, two location-allocation models commonly used in geographic information systems (GIS) (
We developed a web-based site placement calculator to aid public health officials in designing their surveillance system. We then compare the two algorithmic site placement schemes against each other by simulating the spread of influenza across the state of Iowa. Our simulations are based on an Iowa Medicaid dataset comprised two million cases classified with 30 different ILI ICD-9 codes and their corresponding geocodes from 2000 to 2008. We use the Huff Model (
Considering outbreak intensity, we show that sites chosen by our approach outperform the sites used by the IDPH. In other words, we can provide a more accurate representation of disease burden during an influenza season. However, our approach does not provide a substantial difference in the detection of the start, peak and end of the influenza season. In addition, when using the noise values generated by the EM algorithm to analyze the minimal number of sites needed to estimate the timing of the influenza season, our results were highly influenced by both the number of different ICD-9 codes considered and the number of cases considered.
We built a web-based tool to assist public health officials in designing their sentinel surveillance site network. Through simulation, we show that the sites our tool selects allow for better representation of disease burden. We also show that selecting the correct ICD-9 codes that the surveillance network should consider may be as important as selecting the locations of the sites themselves. Using our methods, we can help public health officials design surveillance systems, which are smaller and more easily managed but still detect cases that reflect reliable estimates of both disease burden and timing.
Influenza; surveillance; simulation; time series; geographic information systems
To develop a robust, sensitive and specific local, regional and national public health surveillance system utilizing an electronic clinical information system.
Although development of computerized medical record systems in the United States is a high priority, there are relatively few instances of such systems supporting disease surveillance systems. The Indian Health Service (IHS) has had an electronic record database for over 30 years; however, implementation of point of care electronic health records (EHR) and use of these data for public health surveillance have begun only over the past 4 years.
The IHS health database is distributed across the United States with most data maintained at 465 local care facilities. Among these 465 facilities, there are over 235 EHR deployments, using similar but separately maintained configurations of the IHS EHR. Data are entered into the EHR system as part of daily clinical care or transcribed from paper for results of clinical referrals or outside tests. We developed a surveillance system that identifies reportable cases, notifies providers and provides data to a dedicated national surveillance database. Cases are found using a locally deployed extension to the local data system that searches for a combination of ICD-9 codes, clinical data and laboratory data, based on Council of State and Territorial Epidemiologists (CSTE) case definitions, on a nightly basis. Reports for situational awareness and response are made locally, regionally and nationally using an a priori established priority ranking of the public health importance of a case or outbreak.
Pandemic influenza (pH1N1) was the first health condition targeted for surveillance in 2009. Through an iterative process, a combination of ICD-9 codes and measured fever at the time of visit yielded the highest sensitivity and specificity using the case definition for influenza-like illness (ILI) found in the Centers for Disease Control and Prevention's (CDC) ILInet surveillance system. Formal evaluation of ILI surveillance using review of local medical records (electronic and paper) in one region of the country found that the system had a sensitivity of 96.4% and specificity of 97.8%. IHS is expanding EHR surveillance to capture cases of chlamydia, syphilis, HIV, invasive pneumococcal disease, measles,
Development of highly sensitive and specific surveillance systems using EHR is possible when using clinical and laboratory data to supplement physician diagnosis as recorded with ICD-9 codes. Because there is minimal action required on the part of healthcare providers, EHR-based surveillance has the potential to improve the simplicity, flexibility, timeliness and reliability of most surveillance systems.
Electronic health records; surveillance; pandemic influenza
Larry Layne
Cluster finder tools like SaTScan (
Using the nonparametric intensity function (
The intrinsic variability that exists in the cases counting data for aggregated-area maps amounts to a corresponding uncertainty in the delineation of the most likely cluster found by methods based on the spatial scan statistics (
We use the intensity function to visualize the plausibility of each area of some study map to belong to a possible cluster in the map.
Minas Gerais state is located in Brazil southeastern region and composed of 853 municipalities or areas with an estimated population of 19,150,344 in 2005. The population at risk consisting of 7,033,712 adults aged 45 years or older with a total of 28,039 cases of diabetes (type I and II jointly) in the period of January 2002 to May 2011 All data were obtained from the Brazilian Ministry of Health (www.datasus.gov.br) and Brazilian Institute of Geography and Statistics (www.ibge.gov.br).
Cluster detected by circular scan for diabetes cases in Minas Gerais.
Intensity function for diabetes cases in Minas Gerais.
The intensity function map (
Given a study map with an observed number of cases distributed among its areas, the intensity function value for each area represents the importance of that particular area in delineating a possible cluster of anomalies in the map. This is shown clearly in the results obtained for diabetes cases in Minas Gerais.
Intensity function; spatial scan statistics; diabetes; delineation of spatial clusters
We acknowledge the support by Fapemig and CNPq, Brazil.
We present a new method for efficiently and accurately detecting irregularly shaped outbreaks by incorporating ‘soft’ constraints, rewarding spatial compactness and penalizing sparse regions.
The spatial scan statistic (
For a given local neighborhood with center location s c and size k, we place a bonus or penalty Δi =h(1–2di /r) on each location si , where di is that location's distance from the center, r is the neighborhood radius and h is a constant representing the strength of the compactness constraint. Each Δi can be interpreted as the prior log-odds that si will be affected, and thus the center location (di =0, Δi =h) is eh times as likely as its (k–1)th nearest neighbor (di =r, Δi =−h). We demonstrate that the penalized score function F’(S)=F(S)+Σsi ɛ S Δi can be efficiently maximized over all subsets S for each neighborhood. To do so, we show that F(S) can be written as an additive function (sum over locations) conditioned on the relative risk in region S, and therefore F’(S) is additive given the risk as well. We then jointly maximize F?(S) over all subsets S and all values of the risk.
The penalized subset scan was evaluated using emergency department (ED) data from 97 Allegheny County zip codes. We compared detection power and spatial accuracy, with and without compactness constraints, on synthetic, spatially localized outbreaks injected into the ED data. Our results show that including compactness constraints allows the penalized subset scan methods to detect outbreaks earlier and improve spatial accuracy, as compared to the unpenalized fast localized scan and circular scan, over a wide range of neighborhood sizes (
Comparison of detection performance for methods with and without soft compactness constraints.
Our results demonstrate that the incorporation of soft compactness constraints substantially improves the timeliness and accuracy of outbreak detection. Our new approach based on additive linear-time subset scanning enables efficient maximization of penalized scan statistics over subsets of the data, thus improving detection of irregular clusters.
Outbreak detection; spatial scan; penalized subset scan
This work was partially supported by NSF grants IIS-0916345, IIS-0911032, and IIS-0953330.
In this project, we present a robust methodology for evaluating the fitness of the Semi-Naïve Bayesian Network (SNBN) disease model structure used by Geographic Utilization of Artificial Intelligence in Real-Time for Disease Identification and Alert Notification (GUARDIAN)––a real-time, scalable, extensible, automated, knowledge-based infectious disease detection and diagnosis system––for determining the appropriate thresholds, which separate between different ‘goodness of fit’ categories, and for tuning the model parameters to optimize its applicability within a particular healthcare facility.
Each of the infectious diseases that GUARDIAN monitors is modeled in GUARDIAN's knowledge base as a SNBN (
General model fitness is evaluated using 10-fold cross-validation. The overall, threshold-independent, model quality is determined through analysis of the receiver operating characteristic (ROC) curve (
To tune the SNBN model parameters for a particular healthcare facility, we use a hill-climbing algorithm (
We performed the process outlined above for severe acute respiratory syndrome (SARS), because its similarity to influenza presents the largest challenge of all of the diseases in GUARDIAN's current knowledge base. Even with this challenge, the area under the ROC curve was 0.929 and was optimized to 0.962 through hill-climbing. Gaussian decomposition showed good fidelity using 5 risk categories (
Gaussian decomposition for SARS.
As we have demonstrated for SARS, GUARDIAN's infectious-disease knowledge base uses robust models, which accurately discriminate among related diseases with a high level of accuracy, confidently place each diagnosis within its appropriate relative risk category and optimize its performance for a particular healthcare facility. These results illustrate the improvement that disease-specific expert systems, such as GUARDIAN, represent over traditional trend-based anomaly detection systems. The robust process presented here is currently being used to validate GUARDIAN's existing and ever-growing knowledge base containing a number of diseases of interest for public health surveillance.
Disease model tuning; surveillance threshold creation; model fitness
To establish and maintain an active binational sentinel hospital-site surveillance system and to enhance border region epidemiology and laboratory infrastructure.
The Border Infectious Disease Surveillance (BIDS) program was established in 1999 by the Centers for Disease Control and Prevention and Mexico Secretariat of Health, following mandates from the Council of State and Territorial Epidemiologists (CSTE) and the United States–Mexico border health association to improve border surveillance. The BIDS program is a binational public health collaboration to create an active sentinel-site surveillance of infectious disease among the United States–Mexico border. It is a collaborative effort between local, state, federal and international public health agencies throughout both countries in the border region. This project is aimed at using the best aspects of both countries surveillance system.
We established a network of sentinel clinic and hospital sites along the geographical United States–Mexico border region. We utilized a shared syndromic case definition that is compatible between both countries. Standardized data collection instruments allows for exchange of surveillance data. We increased the laboratory capacity for to test for diseases of public health importance.
This effort has been successful at building a regional surveillance system. In the 2010, three pilot hospital sites were enrolled to conduct severe acute respiratory infection (SARI) surveillance. These patients were tested for viral, bacterial and important fungal infections that cause respiratory disease.
RT-PCR Test Results Among SARI Cases from All Sites by Week, October 2010 to August 2011.
A surveillance system using syndromic and CSTE case definitions allows for comparison of morbidity in the United States/Mexico border region, increased communication and bidirectional sharing of information across the border. Creating and expanding a regional surveillance system that crosses an international boundary requires coordination and collaboration from all agencies involved. These surveillance data allow for examination of the border region as one epidemiologic unit. Consistent communication with clinicians and hospital staff helps to build credibility and interest. Simplicity in surveillance procedures encourages compliance. These surveillance efforts can guide vaccine allocation planning and efficiency in evaluating illnesses that maybe vaccine preventable. Systems to share information between various states in the United States–Mexico border region are important to develop binational control strategies.
Surveillance; syndromic; border; binational; Mexico
We gratefully acknowledge the efforts of the local health departments and hospital facilities.
Imbalances in wealth, education, infrastructure, sociopolitical leadership, healthcare and demographics create opportunities and challenges when implementing public health interventions. Understanding these, while embracing ‘smart power’, one can objectively assess a country's receptivity for support. Therefore, we developed a novel conceptual framework and toolset that objectively measured opportunities and challenges to inform decision-making, specifically about future implementation of the Electronic Integrated Disease Surveillance System (EIDSS)—a computer-based system for national reporting and monitoring of reportable human and veterinary infectious diseases—in East Africa and the Middle East.
After conceptualizing and designing the toolset architecture, we gathered objective data to calculate indicators using a systematic approach from published reports; articles from peer-reviewed journals; and websites of international organizations and national Ministries in each country. We also interviewed stakeholders. Indicators were weighted to reflect the level of impact on elements and domains, and standardized baselines were established to uniformly measure outcomes. Outcomes for each element and domain were then calculated based on the weighted, indicator data.
One hundred twenty-four indicators were identified that measured 16 elements that defined 7 domains of country-specific opportunities and challenges: political will, stakeholder involvement, culture, public health functionality, healthcare, laboratory and communication infrastructure. Thirty (24%) of the 124 indicators were chosen from the reporting requirements of the 2005 International Health Regulations. In the pilot, we found various positive and negative implementation characteristics in Uganda, Kenya, Tanzania, Afghanistan, Iraq and Pakistan.
We conceived a new and useful approach to objectively analyze opportunities and challenges for public health interventions within a country. With respect to introducing EIDSS, we piloted the toolset and described a balanced view of the opportunities and challenges. Application of this novel framework should be useful for other public health interventions, and validation and further testing of the toolset should be performed.
Public health evaluation; assessment; SWOT; smart power
We aimed to assess whether web search queries are a viable data source for the early detection and monitoring of dengue epidemics.
With an estimated 500 million people infected each year, dengue ranks as one of the most significant mosquito-borne viral human diseases and one of the most rapidly emerging vectorborne diseases (
Bolivia, Brazil, India, Indonesia and Singapore were chosen for analysis based on data availability and adequate search volume. For each country, a univariate linear model was built by fitting a time series of the fraction of Google search query volume for specific dengue-related queries from that country against a ‘gold standard’ time series of dengue case counts for a time-frame within 2003–2010. The specific combination of queries used was chosen to maximize model fit. Spurious spikes in the data were also removed prior to model fitting. The final models, fit using a training subset of the data, were cross-validated against both the overall dataset and a holdout subset of the data. All search queries were fully anonymized. This methodology is similar to the approach used to develop Google flu trends (
Dengue generated over a million Google search queries every month. Some queries showed that the user was looking for more information about the disease, while others were looking for symptoms or treatments. Model-fitted ‘expected’ epidemic curves matched official case counts ‘observed’ epidemic curves quite well for all 5 countries in most most countries (
A comparison of the model-fitted and official case counts dengue epidemic curves in each country.
Web search query data were found to be capable of tracking dengue activity in Bolivia, Brazil, India, Indonesia and Singapore. Whereas traditional dengue data from official sources are often not available until after some substantial delay, web search query data are available in near real-time and could serve as a useful low-cost complement to traditional surveillance. Even if peaks are no earlier, there is value in ‘now-casting’—predicting the present where there are delays in gaining access to current official data (
Dengue; epidemics; public health
This paper addresses the problem of predicting high incidence rates of dengue fever in Peru several weeks in advance.
Dengue fever is endemic in over 100 countries and there are an estimated 50–100 million cases annually (
Predictive disease modeling attempts to exploit the complicated relationship between disease outbreaks and measurable environmental, biological, ecological and sociopolitical variables. Previous studies (
The ROC curve and positive predictive value (PPV) for the test set are shown in
ROC curve and PPV for dengue prediction.
Effective methodologies to predict outbreaks of dengue fever may facilitate public health interventions to mitigate the impact of the disease. For best results, the researchers must have access to data streams with timely, detailed and accurate values of predictor variables. High PPV is of principal importance as health officials may be unlikely to spend resources on mitigation efforts based on model predictions without evidence of accuracy on past outbreaks.
Dengue; disease prediction; logistic regression
Funding for this work is provided by the Armed Forces Health Surveillance Center (AFHSC) Division of GEIS Operations.
The project objective was to develop and test a decision support module using the multiple data sources available in the U.S. Department of Defense(DoD) version of the Electronic Surveillance System for Early Notification of Community-Based Epidemics (ESSENCE).
Block 3 of the U.S. Military ESSENCE system affords routine access to multiple sources of data. These include administrative clinical encounter records in the Comprehensive Ambulatory Patient Encounter Record (CAPER), records of filled prescription orders in the Pharmacy Data Transaction Service (PDTS), developed at the DoD Pharmacoeconomic Center, Laboratory test orders and results in HL7 format and others. CAPER records include a free-text Reason for Visit field, analogous to chief complaint text in civilian records, and entered by screening personnel rather than the treating healthcare provider. Other CAPER data fields are related to case severity. DoD ESSENCE treats the multiple, recently available data sources separately, requiring users to integrate algorithm results from the various evidence types themselves. This project used a Bayesian network (BN) approach to create an ESSENCE module for analytic integration, combining medical expertise with analysis of 4 years of data using documented outbreaks.
The strategy was to emulate a domain expert's use of ESSENCE by means of a BN whose inputs were outputs of alerting algorithms (
Major subtasks included modifying the ESSENCE chief complaint processor (
Initial findings from fusion using severity concepts in CAPER data yielded sharp alerting reduction from pure algorithmic methods, with a timeliness loss of 1 day in 2 known outbreaks, no days in 4 others. The alert reduction was dramatic in datasets from small facilities, typically reducing the alert rate from 20 per year to below 5. In larger facilities, the reduction was less dramatic but often over 50%. Time series chosen from the laboratory test and GCN groupings were tested using additional known outbreaks. Results of fusing all algorithmic output streams will be presented.
This approach avoids the need to routinely examine multiple evidence sources and encourages routine investigation, not only by reducing alerting but also by directing attention to alerts that are likely connected with serious illness, with readily accessible details on which criteria were activated.
Fusion; Bayesian network; outbreak detection; chief complaint; Department of Defense
To develop strategic objectives necessary to optimize the collection, integration and use of information across public health programs and internal and external partners for improving the overall health and safety of people and their communities.
There is national recognition of the need for cross-programmatic data as well as system coordination and integration for surveillance, prevention, response and control implementation. To accomplish this, public health must develop an informatics competency and create an achievable roadmap, supported by performance measures, for the future. Within the New York State Department of Health, Office of Public Health (OPH), a cross-organizational and cross-functional Public Health Information Management Workgroup (PHIM-WG) was formed to align public health information and technology goals, objectives, strategies and resources across OPH. In June 2011, the OPH Performance Management Initiative, funded by the Centers for Disease Control and Prevention's National Public Health Improvement Initiative, offered strategic planning workshops, funded by the Association for State and Territorial Health Officials (ASTHO), to PHIM-WG.
Senior management of the major programmatic areas within OPH including, Communicable and Chronic Disease, Family Health, HIV/AIDS, Environmental Health and Wadsworth Center Laboratory, identified representatives to participate in PHIM-WG. Informatics, information technology (IT) and information management (IM) literature was reviewed to determine a framework upon which to build the strategy (
PHIM-WG includes physician, epidemiology, program management, policy and planning, IT, quality improvement and project management representatives. IM, composed of the integration of program, processes, policy and technology, was the selected framework. An initial informatics lexicon was developed. Using CBSC, identified strategic destinations were aligned with PHAB-ES objectives, which were then adapted and aligned with the IM framework. An IM vision and strategy map, including strategic objectives and destinations, were produced. Public health IM desired state, objectives, and activities were linked to the PHAB-ES within four major community perspectives; health status, implementation, process and learning, and assets. PHIM-WG is working to produce a more-fully developed strategy and implementation plan, including engaging internal and external partners, defining associated performance metrics to measure progress to the desired state and aligning with NYSDOH strategic planning efforts.
As a cornerstone of public health, IT/IM should be and can be aligned with or directly linked to the public health essential services. The development and promotion of a common informatics lexicon and workforce engagement and training are critical to public health, especially for advancing data analysis, use, and dissemination capabilities. PHAB-ES-based IM strategic planning can be an essential first step for community collaborators to define the vision, objectives and measurable activities to advance the technology, research and practice of public health surveillance.
Informatics; information management; strategic planning; public health; cross program
NYSDOH OPH Public Health Information Management Workgroup, Nancy Schultz Consulting, ASTHO and Results That Matter (RTM) Team
To describe the complementary usage of electronic emergency department (ED) data, coroner deaths and 911 dispatch call center data in a bacterial meningitis outbreak.
Beginning on March 13, 2011, ACDC experienced an unusual increase in reported bacterial meningitis cases in Los Angeles (LA) County. Early in the investigation, there were few epidemiological links between the cases. Three cases were homeless; two resided at the same Skid Row shelter in downtown LA. ACDC assessed its syndromic surveillance databases to help gauge the scope of the outbreak and detect potentially overlooked cases.
Electronic ED chief complaints (CC) from January 1, 2011, to April 10, 2011. were queried from eight EDs within an 11-mile radius of Skid Row. Only visitors with resident zip codes that corresponded to Skid Row or that were blank to account for homelessness were included. Visits were reviewed if CC included keywords based on common meningitis symptoms and also those of confirmed cases.
Coroner deaths from the same time period were reviewed for location of death and homeless status. Real-time LA City emergency dispatch calls were also reviewed if they were made from the same homeless shelter in which the two confirmed cases resided.
Two hundred and thirty-eight ED visits met the meningitis syndrome definition; however, there was no substantial increase (
ED visits per day in customized meningitis syndrome category. Dotted vertical line indicates date of first confirmed case.
There was no overall increase in the total number of homeless coroner deaths. Two of 45 deaths took place in shelters—one death in January from ‘cardiomyopathy’ that occurred at the homeless shelter of interest, and another nonspecific shelter death in March from ‘strep pneumonia’.
Forty-one 911 ambulance calls were made from the homeless shelter associated with the confirmed meningitis cases. While there was no overall increase in call volume, one call matched a confirmed case fatality.
One limitation of ED data in this investigation is that they do not contain patient resident addresses, making restriction to the homeless or shelter residents impossible. While no additional cases were found, the absence of an increase provides validation that a large countywide outbreak had not occurred.
Both coroner and 911 call databases were more flexible than ED data, containing fields facilitating focused queries on the key epidemiological links of homelessness and shelter residence. Coroner data are limited, however, in that there is a 2-day reporting lag. While many homeless deaths were found, few had precisely reported death locations.
Many 911 calls were reported from the shelter of interest. While medical information was vague, additional details enabled ACDC to match one call to a confirmed case. Follow-up for diagnosis information is possible when ED transportation information is present. Precise caller locations make 911 calls particularly useful for investigations with a strong emphasis on location such as point source outbreaks. Querying preestablished ED visit, coroner death, and 911 call feeds can provide a relatively quick supplement to traditional outbreak investigations.
Coroner; 911 call; dispatch; emergency department; outbreak
The authors would like to thank Van Ngo for details regarding the confirmed cases in the outbreak.
This study deals with the development of statistical methodology for online surveillance of small area disease data in the form of counts. As surveillance systems are often focused on more than one disease within a predefined area, we extend the surveillance procedure to the analysis of multiple diseases. The multivariate approach allows for inclusion of correlation across diseases and, consequently, increases the outbreak detection capability of the methodology.
The ability to rapidly detect any substantial change in disease incidence is of critical importance to facilitate timely public health response and, consequently, to reduce undue morbidity and mortality. Unlike testing methods (
When small area disease data in the form of counts are available, Bayesian hierarchical Poisson models are commonly used to describe the behavior of disease (
For the joint analysis of two or more diseases, we introduce a generalization of the shared component model (
We analyze weekly emergency room discharges for acute upper respiratory infections, influenza, acute bronchitis, asthma and pneumonia in 2009. The data are available by county for the 46 counties of South Carolina. The use of a shared component model accounting for correlation across diseases provides a better overall fit. In addition, the used of the multivariate SCPO increases the statistical power for detection of outbreaks.
Public health surveillance; spatial data; Bayesian hierarchical models; joint disease mapping; conditional predictive ordinate
This work was supported by Grant Number R03CA162029 from the National Cancer Institute.
To evaluate the usefulness of utilizing real-time hospital emergency department chief complaint data to estimate counts of patients presenting at emergency departments with heat-related illness during the July 2011 heat wave in Indianapolis.
In 2004, the Marion County Public Health Department (MCPHD), which serves a county population over 890,000, began using a real-time syndromic surveillance system, ESSENCE (Electronic Surveillance System for the Early Notifica- tion of Community-based Epidemics) to assist in detecting possible disease outbreaks. Today, about 1600 emergency department visits occur daily in Marion County's 14 emergency departments. Epidemiologists from MCPHD have contributed to the city's extreme temperature plans for the last few years. While most of the previous increases in heat-related illnesses in Marion County have been attributed to prolonged heat exposure in connection with local auto races, the county had not activated the county wide emergency response plan in several years. From Tuesday, July 19 through Friday, July 22, 2011, the Marion County Extreme Temperature Plan was put into action in response to several days of a high heat index.
As the written plan indicated, a MCPHD epidemiologist checked ESSENCE every 3 hours and sent updated numbers to the Emergecny Operations Center three times a day via e-mail. The query used the following terms: ‘^heat^,or,^dehyd^,or,^hot^,andnot,^gunshot^’. This is one of several pieces of information used to guide decision making when considering opening additional cooling centers and creating press releases for the public.
The MCPHD sister's agency is the area hospital that accepts medically underserved patients. With this access to the patient system, the electronic medical records of 21 people meeting the search criteria and seeking care at this emergency department between June 1, 2011, and July 29, 2011 were reviewed. An extended time period was reviewed to see if there were obvious differences in the counts of patients or terminology used in the chief complaint once the heat wave was upon the city. Five (24%) sought care in June 2011, 16 (76%) in July. Fifty percent of those seeking care for heat-related issues were seen in a 2-day period in July. Six people (29%) developed symptoms while at work. Work-related tasks included roofing, painting, working in metal tanks and driving trucks without air conditioning. Two homeless persons (10%) sought care during the 2-day time frame when half of the cases were identified as well as four of the six who developed symptoms while at work. Alcohol and drugs may have been a contributing factor for three (14%) of those seeking care. Nine individuals (43%) were treated for medical conditions, such as chronic obstructive pulmonary disease, diabetes, urinary tract infections, gastrointestinal infections or pneumonia.
Although the current query did produce a few ‘false positives’, the MCPHD staff has decided to continue the use of the same terminology since a significant amount of the cases detected were indeed heat related. The counts from the days of the heat wave were significantly higher than in previous summer months. The use of syndromic surveillance during a heat event can provide meaningful information for decision makers in emergency preparedness.
Heat-Related Chief Complaints, Marion County, Indiana, June 1, 2011 to July 31, 2011.
Heat; syndromic; preparedness
To discuss advantages of real time surveillance system within Armed Forces, using a real outbreak case.
It is admitted that real time surveillance system permits to reduce delay of outbreak detection and preventive measures implementation (
ASTER overall architecture.
Declaration network: at the end of medical consultation, each medical staff member declares clinic signs of his patient using a numeric standardized form on computers (specific declaration software). They transmit this anonymous form to a data base located in a Military Surveillance Disease Centre in France.
Analysis network: observed data are automatically compared with historical data every 10 minutes, using current past graph method (specific analysis software), to produce alarm signals. These signals have to be analysed by epidemiologists to confirm or not the real alert about outbreak occurrence.
Data base already contained administrative data about all the soldiers present in Djibouti; and in case of illness symptoms in cause with date of onset and rapid antigenic tests results. Fifty-one cases of tonsillitis were declared during 4 days on 646 soldiers (attack rate=8%), with 18 positive streptotests on 25 performed (72%) (
Tonsillitis outbreak curve within French Armed Forces, Djibouti, June 2011.
ASTER produced an early warning signal, 7 days before the classic surveillance system, and only 1 day after the beginning of symptoms. It is based on clinic signs surveillance, which is more sensitive than disease surveillance. It permitted to perform immediately the description of the outbreak, using the real-time database without disturbing physicians and, therefore, to change the local food provider. The quality of data was good although physicians were busy because of the number of patients.
Real time surveillance; outbreak; early warning; sensitivity
To develop a statewide biosurveillance system based on emergency medical services (EMS) information, which employs both symptom-based illness categorization and spatiotemporal analysis.
The purpose of the National Collaborative for Bio-preparedness (NCB-P) is to enhance biosurveillance and situational awareness to better inform decision-making using a statewide approach. EMS represents a unique potential data source because it intersects with patients at the point of insult or injury, thus providing information on the timing and location of care. North Carolina uses a standardized EMS data collection system, the Prehospital Medical Information System (PreMIS), to collect information on EMS encounters across the state using the National EMS Information System (NEMSIS) template (
De-identified records from all EMS encounters in North Carolina from 2009 to 2010 were utilized in the project. Based upon a previous analysis of emergency department (ED) presentations, an interdisciplinary team (EMS, emergency department, epidemiology and public health) then developed an approach to assign EMS records to 1 of the 20 symptom-based illness categories (gastrointestinal illness, respiratory, etc). EMS encounter records were characterized into these illness categories using a novel text analytic program (SAS Institute, Cary, NC). Baseline patterns of EMS encounters were developed for each illness category across the state, local regions (3-digit zip code) over time. Event alerts were identified across the state and by regions in illness categories using either change detection with cumulative sum (CUSUM) analysis (3 standard deviations) or and a novel text-proportion (TAP) analysis approach (SAS Institute).
2.4 million EMS encounter records over a 2-year period were analyzed. The initial analysis focused upon gastrointestinal illness given the potential relationship of gastrointestinal distress to infectious outbreaks, food contamination and intentional poisonings (ricin). After accounting for seasonality, a significant gastrointestinal event was detected in February 2010 (see red circle on graph in
Gastrointestinal (GI) Illness Sequence of Alerts. Daily GI events in statewide EMS encounters over a two year period using change alerts of 3 standard deviations from mean (red circle), CUSUM analysis (yellow circle) and novel TAP analysis approach (Dec. 6, 2009).
Advantages of EMS data include being an early point of contact with patients and providing information on the location of insult or injury. Surveillance based on EMS information system data can detect outbreaks of illness of interest to public health. A novel text proportion technique shows promise as a useful early event detection method.
Biosurveillance; analytics; detection; emergency; preparedness
Identify the potential of Twitter as a source for monitoring and visualizing content regarding influenza-like Illness (ILI) in Spanish-speaking populations for biosurveillance purposes.
Influenza is a recurrent viral disease with potential to result in pandemics. Therefore, it is necessary to have a timely, responsive and accurate detection. The use of Twitter as a source for data mining in biosurveillance has been previously shown useful (
We implemented a system that builds upon existing technologies and services. The open source platform Ushahidi (
The prototype website operated for a pilot period of 1 month starting April 11h, 2011. A total of 473 unique occurrences worldwide were captured, of which 29% are located in Mexico (138/478) and 52% in Colombia (244/473).
We observed a higher number of incidences in Colombia relative to Mexico (
(a) Screen-capture of the visualization interface for the reports aggregated in the system. (b) Influenza virological surveillance data extracted from the Flunet site by WHO.
Our approach has promising potential for timely detection of ILI-related incidences in the areas previously underrepresented. Future work is to include different linguistic and contextual representations.
Biosurveillance; influenza; twitter; open source; spanish
ALP is supported by the International Fulbright S&T Award and CONACyT-Mexico, FSO is supported by the Fogarty International Center NIH Grant No. 1 D43 TW008443-01 and CSF is partially supported by the Gerondelis Foundation.
This project represents collaboration among the CDC BioSense Program, Tarrant County Public Health and the ESSENCE Team at the Johns Hopkins University APL. The objectives of the project are to: develop reusable meaningful use messaging software for ingestion of health information exchange data available in Tarrant County, demonstrate the use of this data for supporting surveillance, demonstrate the ability to share data for regional and national surveillance using the messaging guide model and demonstrate how this model can be proliferated among health departments that use ESSENCE by investigating the potential use of cloud technology. The presentation will outline the steps for achieving this goal.
National Health IT Initiatives are helping to advance the state of automated disease surveillance through incentives to healthcare facilities to implement electronic medical records and provide data to health departments and use collaborative systems to enhance quality of care and patient safety. While the emergence of a standard for the transfer of surveillance data is urgently needed, migrating from the current practice to a future standard can be a source of frustration.
This project will investigate tools that can be used to support ingestion and translation of public health meaningful use data in the HL7 formats. Open source tools, such as Mirth, have been identified as early candidates to support this function. After the necessary translations have been made, this project will investigate transfer methods to move the meaningful use data from a public health department to a cloud environment. With data available in the cloud, the project will then investigate methods for putting the ESSENCE system in a cloud environment as well. This will provide the collaborative team a platform to evaluate the utility of both the meaningful use data and potentially the value of having regional and national data sharing aspects available to the public health users. Finally, the team will determine the scalability and performance of a cloud environment for disseminating these tools to other jurisdictions across the country.
Early research for this project has already shown the need to redesign aspects of the ESSENCE system to support the additional meaningful use data fields. These changes involved modifications to the database design and the utilization of a more flexible configuration system. We fully expect additional modifications to be made to better support the cloud environment. These findings and the results of the public health evaluation of the system will be presented.
Public health departments will soon be flooded with mountains of new data. Having tools that can translate, transfer and utilize these new data sets effectively will be necessary. This collaborative team will research and put into practice solutions that can be used throughout the country.
Electronic medical records for public health; meaningful use; interoperability; cloud
This talk will point out the inconsistencies and misunderstandings of the word ‘standard’. Specifically, it will discuss HL7, syndrome definitions, analytical algorithms and disease surveillance systems.
Domains go through phases of existence, and the electronic disease surveillance domain is no different. This domain has gone from an experimental phase, where initial prototyping and research tried to define what was possible, to a utility phase where the focus was on determining what tools and data were solving problems for users, to an integration phase where disparate systems that solve individual problems are tied together to solve larger, more complex problems or solve existing problems more efficiently. With the integration phase comes the desire to standardize on many aspects of the problem across these tools, data sets and organizations. This desire to standardize is based on the assumption that if all parties are using similar language or technology, then it will be easier for users and developers to move them from one place to another.
Normally the challenge to the domain is deciding on a vocabulary or technology that allows seamless transitions between all involved. The disease surveillance domain has accomplished this by trying to use some existing standards, such as Health Level 7 (HL7), and trying to develop some of their own, such as chief complaint-based syndrome definitions. However, the standards that are commonly discussed in this domain are easily misunderstood. These misunderstandings are predominantly a communication and/or educational issue, but they do cause problems in the disease surveillance domain. With the increased use of these standards due to meaningful use initiatives, these problems will continue to grow and be repeated without improved understanding and better communication about standards.
After reviewing presentations and participating in many discussions at conferences and with public health officials, a number of topics were identified that many believe use or are standards. These topics included HL7, syndrome definitions, analytical algorithms definitions and the definition of what is or is not a disease surveillance system. Next, the common understandings of each were compiled and compared with actual definitions and real world experiences from users of the standards. From this, a list of misunderstandings or poorly communicated aspects of each topic was derived.
The results of this process have pointed out a number of inconsistencies with general assumed knowledge and actual truth related to many standards. The HL7 standard is just one example of a standard that is misunderstood in many aspects. Many believe that HL7 is a transport protocol, others believe that is a file format, others believe that it defines specific locations for data elements and still others believe that HL7 ‘set the language, structure and data types required for seamless integration from one system to another’ (
Standards are highly beneficial to a domain. They provide efficiency in tool development and promote interoperability between organizations. Sadly, fully understanding a standard can sometimes be difficult, and misunderstandings can allow decisions to be made on untrue assumptions about a standard. The word standard has a meaning attached to it that can easily confuse someone into believing a capability exists that actually does not. Through improved education and communication, we can benefit from these standards without getting caught in their traps.
Standards; HL7; syndrome definition; detector interfaces; system definition
To describe the Centers for Disease Control and Prevention (CDC) Countermeasure Tracking Systems (CTS) and the impact of its four closely related informatics components toward enhancing federal, state and local public health capacity to track and manage medicine and other medical or nonmedical supplies during daily operations and all hazards public health events.
Description: CDC works to save lives and protect people during major public health events. In an effort to support these processes, CDC established CTS, which is maintained within the Division of Informatics Solutions and Operations (DISO), in the Public Health Informatics and Technical Program Office (PHITPO). CTS consists of four system components, which interoperate to improve communications and event response efficiency while still functioning independently, recognizing the unique requirements and use cases for each system. Collectively, the data consolidated from these systems can show population coverage, numbers of untreated individuals, drug and equipment shortages, need for resupply and more. The web-based applications are deployed centrally at CDC and use the CDC's secure data access method for security.
The first of these components is the Inventory Management and Tracking System (IMATS), currently under development. IMATS provides state and local public health providers with a tool to track medical and nonmedical countermeasure inventory and supplies during daily operations or an event. The solution tracks quantities of inventory, monitors reorder thresholds and facilitates warehouse operations including receiving, staging and storing of inventory.
The Communications Portal is a web-based content management system in development, which consolidates important event response details into one place and will provide timely and adequate information to states and other jurisdictions. This system is complementary to the IMATS as it manages communications related to, but not limited to, Emergency Use Authorization (EUA), Investigational New Drug (IND) and recall notices.
Preparing for the future of public health surveillance also requires innovative and appropriate informatics systems that provide timely and accurate response to all-hazards events. CTS and its components are developed to assist in all types of surveillance needs.
Countermeasure; all hazards; public health; inventory; tracking
Childhood trauma is a major risk factor for the development of affective disorders later in life. We sought to determine whether this risk is linked to neurostructural changes in limbic brain regions after childhood trauma.
We recruited 49 medically healthy adult women (28.2±7.1 years of age) from the Atlanta area to include women with/without childhood trauma and with/without major depression (MDD). Childhood trauma exposure was quantified using the Childhood Trauma Questionnaire (CTQ). Lifetime and current diagnoses of MDD and posttraumatic stress disorder (PTSD) were assessed using the Structured Clinical Interview for DSM-IV (SCID). Current depressive symptoms were assessed using the Hamilton Rating Scale for Depression (HAM-D). Magnetic resonance images were acquired, preprocessed, and registered into stereotactic space. Volume analyses of the left and right amygdala were performed using the interactive software package DISPLAY developed at the Brain Imaging Center of the Montreal Neurological Institute, and a standardized segmentation protocol was applied to outline the anatomical boundaries of the amygdala. Total plasma cortisol responses to the Trier Social Stress Test (TSST) were measured.
When stratifying groups by childhood trauma exposure and MDD, women with both childhood trauma and MDD had largest right amygdala volumes compared to all other groups (interaction effect:
These findings add to the growing understanding of the neurobiological basis that may underlie the association between early adverse experience, stress vulnerability, and increased risk for affective disorders.
Previous animal experiments suggest that prenatal stress affects pregnancy outcomes and impairs cognitive functions of offspring. Our goal was to investigate how prenatal exposure to stressful life events influence pregnancy outcome and infant's physical and neurobehavioral development.
A clinical trial was performed. One thousand eight hundred and fifty-six pregnant women were willingly assessed using the Life Events Scale for Pregnant Women (LESPW) before delivery. Those whose score were more than or equal to 375 on LESPW were assigned to higher levels of psychological stress during pregnancy. One hundred and forty-two cases were selected from 1856 pregnant women controlling for variables such as gestational age, maternal age, obstetric complications, socioeconomic status, and trait anxiety. The prenatal stress (PNS) group and the control (CON) group were composed of 71 full-term infants each (1:1 pair matched). Infants’ birth weight (BW) and head circumference (HC) from both groups were assessed at birth and the neonatal neurobehavioral development was evaluated at 72 hours using the neonatal behavioral neurological assessment (NBNA).
Three hundred and twenty-seven cases from 1856 scored more than 375 on LESPW, incidence of stress was 17.62%. The proportion of undesirable pregnancy outcomes from 327 cases were 147 cases (44.95%), with threatened abortion 38 (11.62%), premature delivery 31 (9.48%), pregnancy complications 73 (22.32%), stillbirth 5 (1.53%), and low birth weight infants 120 (36.7%). The pregnancy outcomes of the non-stressed 1529 cases were undesirable in 579 cases (37.87%), with threatened abortion 123 (8.04%), premature delivery without cause 208 (13.6%), pregnancy complications 240 (15.70%), stillbirth 8 (0.52%), and low birth weight infants 159 (10.4%). BW, HC and the score of NBNA of full-term infants in the PNS were lower than those of the CON (
Pregnant women with stress were at increased risk of adverse pregnancy outcome, low birth weight infants in high levels of maternal psychosocial stress were significantly higher than that of premature birth. This shows us that PNS can induce fetal intrauterine growth retardation, and influence newborn's physical and neurobehavioral development, especially in newborn girls.
Childhood adverse experiences are known to induce persistent changes in the hypothalamic–pituitary–adrenal (HPA) axis reactivity to stress. However, the mechanisms by which these experiences shape neuroendocrine response to stress remain unclear.
We tested whether bullying victimization influenced SERT DNA methylation using a discordant monozygotic (MZ) twin design. A sub-sample of 28 MZ twin pairs discordant for bullying victimization, with data on cortisol and DNA methylation, was identified in the Environmental Risk (E-Risk) Longitudinal Twin Study, a nationally representative 1994–1995 cohort of families with twins.
Bullied twins had higher
Our study extends findings drawn from animal models, supports the hypothesis that early-life stress modifies DNA methylation at a specific CpG site in the
Stress is a complex phenomenon coordinated by several neural systems and has consequently been measured by several biomarkers. Salivary cortisol is the classical used stress biomarker representing the hypothalamic-pituitary-adrenal system. Heart rate variability (HRV), defined as the distance variability between consecutive R peaks, is increasingly used as marker of the autonomic nervous system and as a result also as a stress marker (defined as sympathetic over parasympathetic dominance). Associations between children's salivary cortisol and HRV will be examined.
In 190 children (5–10 year) of the Belgian ChiBS study salivary cortisol and HRV were sampled. Salivary cortisol samples were collected when waking up, 30 minutes and 60 minutes after wake-up and in the evening on two weekdays. HRV measurements in supine position were undertaken with Polar chest belts during 5 minutes. Apart from HRV time-domain analysis, also frequency-domain analysis was performed in the low-frequency (LF) and high-frequency (HF) bands. Multilevel growth curve modelling with adjustments for age, sex, physical activity and wake-up time was used to analyse the HRV associations with overall cortisol, cortisol awakening response (CAR) and cortisol diurnal decline.
Higher overall cortisol levels were negatively associated with mean RR, root mean square of successive differences (RMSSD), percentage of RR intervals differing more than 5 0ms (pNN50) and HF. A steeper diurnal decline was positively associated with normalised LF and the LF/HF ratio and negatively with HF. The CAR was positively associated with normalised LF and the LF/HF ratio and negatively with normalised HF.
Higher salivary cortisol levels were associated with lower parasympathetic activity. A larger CAR and steeper diurnal decline were associated with a sympathetic over parasympathetic dominance. Consequently, the two main neural stress systems (represented by cortisol and HRV) show good agreement in reflecting children's stress status, although not all parameters were significantly related. Measuring both pathways stays recommended as the pathways might be stimulated differently depending on the stressor.
Substantial evidence suggests conditions in intrauterine life may play a critical role in subsequent health and disease susceptibility related outcomes (i.e., the concept of fetal or developmental programming of health and disease). The elucidation of biological mechanisms underlying these effects is an area of active investigation. We suggest that telomere biology may represent a novel mechanism underlying the effects of a disparate set of suboptimal intrauterine exposures on various health and disease risk phenotypes. From an evolutionary-developmental perspective, energy substrate availability (i.e., nutrition) and challenges that have the potential to impact the structural or functional integrity and survival of the organism (i.e., stress) likely represent the most important environmental considerations underlying natural selection and developmental plasticity. Maternal stress and nutrition in pregnancy therefore represent attractive candidate processes in the context of fetal programming of telomere biology. Our previous work has established an important role for prenatal stress and stress-related processes in adult telomere biology
In two longitudinal birth cohorts, stress- and nutrition-related processes were assessed during pregnancy and telomere length (TL) was subsequently measured in newborns (cord blood) and infants (buccal cells).
(1) Among the nutrition-related factors, maternal lower folate levels (an essential methyl donor) and higher triglyceride concentrations in early pregnancy were significantly and independently associated with shorter newborn TL. (2) Among psychosocial stress-related measures, higher maternal pregnancy-specific stress was associated with shorter newborn TL. (3) Maternal estrogen (E3) levels during early pregnancy were associated with longer infant TL.
Taken together, our findings provide the first evidence in humans that maternal nutrition and stress-related processes during pregnancy may exert a programming effect on the newborn and infant telomere biology system.
Stress in early life is known to have a powerful direct effect on poor health in later life. This direct effect requires one or more underlying mechanisms that can maintain it across the life-course. It is therefore essential to characterize the biological mechanisms through which children may acquire such lasting vulnerability to disease, namely, the mechanisms of biological embedding. One plausible mechanism may lie in changes to DNA. New research suggests that stress exposures can accelerate the erosion of DNA segments called telomeres.
In the past 2 years, six studies provided support for an association between telomere length (TL) and childhood stress. Although these studies advance understanding of the link between childhood stress and TL, almost all studies have relied on adult measures of TL and retrospective recall of stress years after the stress was experienced raising important questions about the true nature of these findings. Interpretation of findings from cross-sectional studies of TL is ambiguous in light of recent longitudinal analyses of repeated TL measurements. These recent findings indicate that the temporal process of telomere erosion is more complex than initially assumed, and that repeated measures (not just length at one time point) are needed to measure true telomere erosion in individuals who are experiencing stress. Moreover, given the elapsed time between the putative stress exposure and the measurement of TL, it has not been clear whether telomeres began eroding during stress exposure or whether erosion occurred years later, possibly promoted by the sequelae of childhood stress or other intervening variables.
In our study, we used a longitudinal design to test the effects of violence exposure during childhood on telomere erosion in a cohort of young children. We tested the hypothesis that cumulative violence exposure would accelerate telomere erosion in children while they experienced stress.
Participants were 236 children (49% females; 42% with one or more violence exposures) recruited from the Environmental Risk (E-Risk) Longitudinal Twin Study, a nationally representative UK 1994–1995 birth cohort. Each child's mean relative telomere length was measured simultaneously in baseline and follow-up DNA buccal cells, using the quantitative PCR method for T/S ratio.
Violence was assessed as exposure to maternal domestic violence, frequent bullying victimization and physical maltreatment by an adult. We interviewed mothers (or the primary caregiver) about each exposure when the children were 5, 7, and 10 years of age and compiled a cumulative record of each child's exposure to violence. Nearly 54.2% (
To test the main hypothesis that violence exposure would accelerate telomere erosion between ages 5 and 10 years, we conducted ordinary least squares multiple regression analysis. The outcome variable was age-10 TL, controlling for baseline TL at age 5 years and sex, SES, and body mass index as covariates.
We first examined the effect of each type of violence exposure on TL separately. Children exposed to domestic violence showed slightly accelerated telomere erosion from age 5 to 10, compared with children who had not been exposed to domestic violence, but this change was not statistically significant (β= −.059, SE=.045,
Next, we tested the main hypothesis that cumulative exposure to violence will be associated with accelerated TL erosion. Children who experienced two or more kinds of violence exposure showed significant TL erosion from baseline to follow-up measurement compared with children who had one or no kinds of violence exposures (β = −0.054, SE=0.023,
This finding provides the first evidence that stress-related accelerated telomere erosion in buccal cells can be observed already at young age while children are experiencing stress. Children who experienced two or more types of violence exposure between age-5 baseline and age-10 follow-up measurements showed significantly more telomere erosion, even after adjusting for confounding factors. The results of the present study add weight to the hypothesis that exposure to stress in childhood can alter biological processes in relation to telomere erosion. Methodological strengths of this study include a longitudinal design with reliable and valid prospective assessments of multiple violence exposures during childhood and repeated measurements of TL during this same developmental period.
Major depressive disorder (MDD) and other serious mental illnesses are associated with high rates of comorbid medical illnesses. Many of these comorbid conditions are more typically seen in the aged, raising the possibility that these psychiatric illnesses are associated with accelerated aging. An emerging biomarker of cell aging and of increased risk of medical illness is leukocyte telomere length, and several studies have now characterized leukocyte telomere length in MDD and other psychiatric illnesses. Fewer psychiatric studies have characterized the activity of telomerase, an enzyme that can elongate and preserve telomeric DNA, or have investigated the biochemical mediators of accelerated telomere shortening.
Seven studies examining telomere length in MDD, three studies in schizophrenia, two studies in bipolar illness, two studies in PTSD, and one study in generalized anxiety disorder were reviewed, as were one study of telomerase activity in MDD and one study in schizophrenia. Additional studies in chronically stressed individuals and in individuals with histories of childhood adversity were also reviewed.
Shortened leukocyte telomeres have been demonstrated in MDD, bipolar illness, schizophrenia, anxiety disorders, and post-traumatic stress disorder, although in some studies, only subgroups of patients (e.g., those with longer lifetime exposure to the illness, those with poor responses to treatment, or those with preexisting histories of childhood adversity) showed shortened telomeres. Leukocyte telomere shortening is correlated with peripheral indices of increased oxidative stress and increased immune activation. Two studies (one in caregivers with high depression ratings and one in unmedicated patients with MDD) reported elevated peripheral blood mononuclear cell (PBMC) telomerase activity, perhaps representing a compensatory attempt by the body to preserve endangered telomeres. Preliminary data in MDD suggest that relatively low telomerase activity before treatment, and greater treatment-associated increases in telomerase activity, are associated with better antidepressant responses. This, plus the preliminary observation that PBMC telomerase activity is directly correlated with hippocampal volume (by MRI) in MDD, support emerging preclinical data that telomerase has intrinsic neurotrophic and antidepressant effects.
Telomere shortening in MDD and certain other psychiatric conditions may, at least partially, reflect chronic exposure to inflammation and oxidation. As such, it may be a bellwether of increased medical risk, or it may play a more direct causal role in accelerated aging. The interplay of telomere integrity and telomerase activity may be an important determinant of psychiatric and medical outcome. Overall, the data are consistent with the view that MDD and certain other psychiatric illnesses have systemic manifestations beyond the brain and call into question the dichotomy of “mental” vs. “physical” illnesses.
Chronic stressors across the life course predict accelerated pathogenesis of diseases of aging and early mortality. Telomere length, the DNA-based biomarker indicating cellular aging, is a mechanism of disease development, and shortens in a dose response fashion by duration and severity of life stressor exposures. Telomere length provides an important window in understanding a life span model of the accumulation of stress on aging. Self-reported perceived chronic stress and exposure to stressful life experiences during childhood and adulthood are related to short telomeres.
While the expectation that the accumulation of life stress leads to cellular senescence, most studies indicate cross-sectional associations between life stressors and telomere length. Findings suggesting longitudinal associations between life stressors and telomere shortening are best represented in studies associating self-reported early childhood traumatic experiences with short telomeres in adulthood. Adults reporting moderate-to-severe childhood maltreatment and stressful experiences, such as divorce and parental separation, are more likely to have significantly shorter telomeres than those reporting no childhood maltreatment. To date, only one study has prospectively demonstrated associations between traumatic experiences and telomere shortening. Five-year-old children exposed to two or more traumatic stressors, including maternal domestic violence, frequent bullying victimization and physical maltreatment by an adult, have shorter telomeres at age 10 compared with children exposed to less or no violent stressors.
No studies, however, indicate prospective effects of adulthood stressors on telomere shortening over time. Perhaps, as we suggest elsewhere, chronic stressor effects on biological pathways are rarely main effects, but rather an intricate interplay between life adversity and resiliency factors. Our work, and that of others, is increasing our understanding of how psychological stress resilience, social connections, and lifestyle may moderate relationships between life stressors and health. Here, we present evidence from two studies that support our proposed model that behavioral and psychosocial resiliency can buffer the effects of stress on telomere length, both cross-sectionally and prospectively. Cross-sectionally, we tested whether multisystem resiliency – defined as a composite of healthy emotion regulation, strong social connections, and being physically active – mitigates previously demonstrated associations between concurrent depression diagnosis and telomere length. We found support for this model, which will be presented. In a second study we examined how a lifestyle composite might buffer stress-induced telomere shortening prospectively.
Two hundred sixty-one non-smoking women between the ages of 50 and 65 were recruited for a prospective study on telomere length change over the course of the year. Leukocyte telomere length (LTL) was assayed at baseline and 12-month follow-up. Perceived stress, typical dietary practices, sleep quality, and exercise levels were self-reported at baseline, 4 months, 8 months, and 12 month follow-up. Seventeen questions about life events that may have occurred in the previous year were asked at follow-up, including events such as divorce, death of a family member, and job loss. Health events were not included as they may confound effects of stressors on telomere biology. Women with histories of cancer, who were premenopausal, or did not have complete self-report data were excluded from these analyses, leaving a final sample of 196 women.
Results indicated that perceived stress at baseline, perceived stress accumulated over the year, or accumulation of stressors over the year were unrelated to 12-month LTL, covarying baseline telomere length, age, BMI, and income level. However, findings do suggest significant interactions between markers of stress and a healthy lifestyle over the year composed of healthy dietary practices, sleep quality, and exercising. For those at one standard deviation below mean healthy lifestyle, baseline perceived stress (
In summary, healthy lifestyle factors and psychosocial resiliency may interrupt a cascade of harmful effects that accelerate cellular aging, diminishing the impact that chronic psychological or objective life stress has on health. From conception to death, we are exposed to stressors. And while stressors may shape the manifestation of resiliency factors, leading to an interrelated cluster, our work suggests that psychosocial resources and lifestyle factors can add up to multisystem resiliency, providing increasing cellular buffering from life stress. Without attending to such interactions, stress effects are often masked and missed.
Obesity is associated with increased symptoms of anxiety and depression. We hypothesised that severe obesity in pregnancy would be associated with adverse psychological health, with effects on gestational weight gain (GWG) and baby birthweight (BWT). We aimed to study mood and birth outcomes among participants in a longitudinal study of severe obesity in pregnancy.
In this study, 140 severely obese (body mass index [BMI] (mean (SD)) 44.1 (4.1) kg/m2) and 96 lean (BMI 22.6 (1.6) kg/m2) pregnant women were recruited. Ethical approval and written, informed consent were obtained. Obese women were advised about healthy eating and weight maintenance. Serial weights were recorded and GWG calculated between 16 and 36 weeks’ gestation. Women were asked to complete validated questionnaires to assess mood, including ‘satisfaction with life’, Hospital Anxiety and Depression Scale (HADS) and Spielberger State and Trait Anxiety in early (12–20 weeks’ gestation) and late pregnancy (28–32 weeks’ gestation). Term BWT (>37 weeks’ gestation) was recorded (
Obese women were significantly less satisfied with life than lean women and had higher HADS depression and anxiety scores, and state and trait anxiety scores at both time points (all
Obese women had less GWG than lean women (5.3 (6.0) vs. 10.2 (3.7) kg,
In lean, increased BWT was associated with higher BMI (
Severely obese pregnant women have more symptoms of anxiety and depression and are less satisfied with life than lean women. Increased anxiety in response to pregnancy is associated with lower BWT in all, but altered mood has differing associations with GWG in lean and obese. Understanding mood may help interventions to optimise GWG in severely obese women.
Although heritability is an important factor related to the onset of psychotic disorders, environmental factors also play a role. Early-life stress, which includes both prenatal stressful exposures and childhood maltreatment, has been suggested to have an impact on the developing brain. Cognitive alterations in schizophrenia and psychotic disorders have been described in several neuropsychological domains: attention, memory (verbal, visual and working memory), processing speed, reasoning and social cognition. Recent studies suggest that the hypothalamic–pituitary–adrenal (HPA) axis modulates cognitive functioning in patients with psychosis but that this association does not seem to be related to increased exposure to stressful events. We aimed to study whether early-life stress and the HPA axis are associated with a poorer cognitive performance in subjects with a psychotic disorder.
We studied 46 subjects with an early psychosis (aged 18–35 years), who were attending the Early Psychosis Program from Reus (HPU Institut Pere Mata, Spain). These subjects included three clinical populations: (1) first episode of psychosis (FEP,
In relation to cognition, subjects with perinatal stress showed significantly poorer cognitive performance in the attention/vigilance domain (
Early-life stress and HPA axis measures are associated with a poorer cognitive functioning in subjects with early psychosis. However, cognitive domains seem to be affected differently by early-life stress and HPA axis measures: attention/vigilance by perinatal stress; social cognition by childhood maltreatment; visual and working memory by cortisol levels.
Stress diseases such as affective disorders are often characterized by a disturbed regulation of the hypothalamus-pituitary-adrenocortical (HPA) axis. This dysregulation can be explained by an impaired function of the receptors involved in the HPA-axis regulation, for example, the glucocorticoid receptors (GR). The regulation process of the HPA axis and the GR function are influenced by several genes, for instance by NR3C1 coding for the GR and also by FKBP5, a co-chaperone in the GR-complex. Binder et al. showed that common polymorphisms in FKBP5 are associated with increased FKBP5 protein expression as well as correlation between cortisol levels and peripheral blood FKBP5 mRNA expression. Regarding the effects of FKPB5 genotypes on psychosocial stress reaction, Ising and colleagues tested healthy subjects with the Trier Social Stress Test, a standardized paradigm to induce psychosocial stress. Subjects homozygous for any of the FKBP5 variants showed an incomplete normalization of the stress induced cortisol secretion.
Recent studies demonstrated that FKBP5 and NR3C1 are involved in the endocrine stress reaction. Therefore, we expected changes of FKBP5 and NR3C1 mRNA expression in peripheral blood after exposure to a psychosocial stress situation. To address this, we performed a pilot study where we tested six healthy young men without history of psychiatric or severe somatic disorders and applied the trier social stress test (TSST). Before and after two consecutive TSSTs, we took blood samples with a venous catheter in order to measure ACTH and cortisol in plasma and mRNA expression of the candidate genes in peripheral blood. Blood cells were stabilized using PAXgene tubes, and gene expression was processed by qPCR.
Briefly after the psychosocial stress the stress hormones ACTH and cortisol increased whereas the reaction to the second TSST was lower suggesting a habituation effect. These endocrine stress responses were followed by an alteration in FKBP5 gene expression, further underlining the importance of this gene for the neuroendocrine stress reaction. NR3C1 mRNA levels did not change after the TSST.
Our preliminary data indicate an effect of psychosocial stress on the FKBP5 mRNA levels. Further research with larger samples sizes is required to replicate and extend these results.
Corticosteroids are released in response to stress and have been shown to influence affective learning in rodents and humans. Many models of pathogenesis of affective and anxiety disorders have incorporated stress and cortisol as vulnerability factors. A well-established paradigm to investigate emotional learning and memory processes is classical fear conditioning.
Here, we set out to investigate corticosteroid effects on fear acquisition and memory retention in healthy men (
We specifically targeted time-dependent effects of corticosteroids on fear conditioning in this within-subjects delay and trace conditioning paradigm by administering 10 mg hydrocortisone either 4 h or 1 h before fear acquisition. Twenty-four hours later, subjects came back for a fear memory retention test followed by extinction. Analyses show that hydrocortisone impaired trace acquisition specifically on EMG startle responses, regardless at which time point it was administered. However, we found an enhancement of EMG fear responses to the trace stimulus 24 h later only when corticosteroids were administered 4 h before acquisition.
This indicates that slow corticosteroid effects before acquisition lead to better retention of the fear memory for trace conditioning the next day.
Associations between stress levels in everyday life and controlled laboratory conditions remain a controversial topic. The aim of the present study was to compare psychological and physiological stress levels assessed in a real-life setting with laboratory stress levels.
Thirty-five healthy male students (age M = 24.4, SD = 2.6 years) took part in the study. The first part of the study consisted of a 2-day period within which subjects collected saliva and rated their stress levels on a visual analogue scale immediately after awakening, 30min later, at 9am, and then every 2 hours for a total of nine times a day while maintaining their regular daily activities. Salivary alpha-amylase (sAA) was assessed as a marker for autonomic nervous system activity at each time point. In the second part, subjects were invited to two laboratory sessions on two separate days, with randomized exposure to either a standardized stress test (cold pressor test (CPT), stress condition) or a rest condition (reading magazines). Again, sAA and subjective stress were assessed repeatedly during both conditions.
During both days, sAA levels showed a distinct diurnal rhythm, with a trough in the morning and a steady increase over the course of the day (time effect,
Stress levels in everyday life were shown to predict psychological as well as physiological stress levels in the laboratory. Furthermore, subjects with high stress levels in everyday life experienced a more pronounced psychological stress response to a laboratory stressor.
To evaluate the impact of stress on children's well-being, it is important to have valid and reliable stress assessment methods. Nevertheless, selection of an appropriate method for a particular research question may not be straightforward, as there is currently no consensus on a reference method to measure stress in children. This paper examined to what extent childhood stress can be estimated accurately by commonly applied stress measures.
Two hundred and seventy-two girls between 5 and 11 years old participated in this study as part of the ChiBS project. Child-reported estimates of stress were collected through the Coddington Life Events Scale (CLES). Serum, saliva and hair samples were collected for cortisol analyses. The intercorrelations of cortisol in the different biological samples were investigated by Spearman rank correlations and Bland-Altman plots. Next, CLES-scores, salivary and hair cortisol concentrations were compared triangularly with the true, but unknown childhood stress using the Triads method, based on pair-wise Spearman's correlation coefficients and the calculation of validity coefficients.
Serum cortisol (free and total) was positively correlated with salivary morning and AUC cortisol. Hair cortisol correlated with salivary morning and AUC cortisol, but not with serum cortisol. In relation to recent childhood stress (0–3 months ago), the highest validity coefficients were observed for salivary cortisol measurements, while for periods more distant in the past hair cortisol measurements displayed the highest validity coefficients.
This paper investigated the relationship between cortisol measurements in different biological samples, showing a lack of association and disagreement between measures of single-point, short-term cortisol versus long(er)-term cortisol. In addition, this paper examined to what extent childhood stress can be accurately estimated by stressor questionnaires and biological markers in girls. Salivary cortisol was shown to most accurately indicate true childhood stress for short periods in the past (i.e. last three months), whereas hair cortisol may be preferred above salivary measurements for periods more distant and thus for chronic stress assessment.
Stress undermines health, perhaps via activation of the hypothalamic-pituitary andrenal (HPA) axis. There is evidence that psychological factors (i.e., sense of control, familiarity, effective coping, and social support) can buffer stress effects and HPA axis activation. There is also evidence that resilience and compassionate goal orientations (striving to help others rather than promoting the self) are associated with health and well-being, perhaps via HPA-buffering effects. We utilized a laboratory model of social evaluative threat (Trier Social Stress Test, TSST) to activate the HPA axis and study the stress-buffering effects of control, familiarity/coping, and compassionate goals, testing for interactions with resilience. Cortisol results were previously presented (41st International Society of Psychoneuroendocrinology (ISPNE) annual conference). Adrenocorticotropic hormone (ACTH) responses, which have now been analyzed, have strengthened the original findings.
Healthy participants (
Overall, the type of instruction significantly interacted with resilience in modulating ACTH responses throughout the TSST (
The ACTH results mirror previously reported findings with cortisol responses to the TSST: Coping instructions increased anticipatory stress in low resilient participants, whereas compassionate goal instructions reduced stress reactivity to the TSST in high resilient participants. Further work assessing individual differences in resilience, and tailoring stress inoculation techniques accordingly, may facilitate development of more effective means of reducing the detrimental health effects of stress.
Salivary cortisol has frequently been used as a biomarker of chronic stress. The results have differed considerably between studies, which could to some extent be explained by the various definitions of chronic stress cases, ranging from patients with a clinically diagnosed condition to working individuals scoring high on burnout questionnaires. Thus, it is not possible to generalize findings in the literature to stress-related conditions encountered in the clinic and it is difficult to apply the knowledge in diagnosis and treatment. The aim of this study was to elucidate the usefulness of basal salivary cortisol as a marker of chronic stress in a clinical population with stress-related exhaustion.
We have measured salivary cortisol concentrations in two different samples of patients with a clinically diagnosed exhaustion disorder (ED). ED is defined as physical and mental exhaustion experienced for at least two weeks, caused by exposure to one or more stressors for a minimum of six months. In the first study, 162 patients (64% females) collected saliva samples at awakening and 15 minutes thereafter to assess the cortisol awakening response. This patient group was compared with 79 healthy controls (49% females). The patients repeated the saliva sampling at follow-up assessments after 3, 6, 12, and 18 months of treatment. The second study of 68 patients (79% females) included saliva samples taken at awakening, 30 minutes thereafter and at bedtime on two consecutive days to assess the diurnal profile, and follow-up assessments after 6 and 12 months. This study included 98 healthy controls (56% females). Age, sex, BMI, antidepressant use, and physical activity were considered as potential confounders.
No significant differences were found between patients and controls in salivary cortisol awakening response (first study) or diurnal profiles (second study). Furthermore, follow-up measurements in patients indicated that salivary cortisol concentrations did not change significantly during treatment.
Salivary cortisol levels, at least as measured in this study, apparently provide a rather poor reflection of the long-term stress exposure experienced by the patients in this study. Thus, basal salivary cortisol measurements are not recommended as a biomarker of stress-related exhaustion.
Recent research on stress and decision making highlights the importance of considering the reciprocal relationship of these processes. Indeed, everyday experience suggests that economic decision situations can be stressful in and by themselves, particularly if they involve psychosocially stressful elements like competition. It is, however, at present not known whether or not physiological reactions elicited by the decision situation influence the decision that is reached. According to Salvador and Costa [Salvador, A. & Costa, R. (2009). Coping with competition: Neuroendocrine responses and cognitive variables. Neurosci Biobehav Rev, 33(2), 160–170], effects of competition (i.e., positive vs. negative outcomes) critically depend on the nature of applied coping strategies that are in turn related to specific physiological changes.
Our study examined the physiological and subjective changes induced by an established economic laboratory competition paradigm in a mixed-gender sample of 104 healthy participants. A mental arithmetic task was performed first under a piece-rate payment scheme and afterwards under a tournament (i.e., winner-takes-it-all) condition (i.e., forced competition). In a third round, subjects decided how to be paid (i.e., piece rate or tournament).
Our results indicate that the laboratory paradigm indeed elicited physiological reactions that were related to the voluntary choice of competition. Participants that chose tournament were more likely to appraise the situation as challenging and showed higher sympathetic nervous system reactivity and higher testosterone increase during the game.
As these physiological changes are associated with an active coping mechanism (Salvador & Costa, 2009), we conclude that while competition is not
Chronic stress is associated with negative health outcomes and is linked with neuroendocrine changes, suppressed immunity, and central nervous system neuropathology. While human studies have illustrated the benefits of stress reduction, mechanistic understanding of how decreasing stress affects health, and disease progression remains unclear. Furthermore, prior animal studies have focused primarily on increasing stress, and few animal models of stress reduction have been fully developed. Therefore, we have developed a “calm mouse model” with caging enhancements designed to reduce murine stress.
Male
Calm, Cntl Ex, and Calm Ex animals exhibited significantly less corticosterone production than Cntl (Day 49: Calm, Mdiff 20.5 ng corticosterone metabolites/0.05 g feces (CM), CI95 11.7–29.4,
Our data indicate that both Calm and exercise caging generated reductions in physiologic stress measures in mice and that Calm animals exhibited increases in splenocyte subpopulations that may underlie changes in functional immunity. Collectively, the Calm model represents a promising approach to studying the biological effects of stress reduction in the context of health and in conjunction with disease models.
Stressful life events have been shown to have great influence on the onset or recurrence of psychotic symptoms [1]. Increased stressful life events [2], perceived stress [3], and higher cortisol levels [4,5] have been described in subjects with a first psychotic episode. The main aim of our study was to explore the relationship between stress measures and cortisol levels in subjects with early psychoses.
We included 85 subjects, aged between 18 and 35 years, who attended the Early Psychosis Program from Reus (Tarragona, Spain). All subjects were assessed at baseline using a structured clinical interview (Schedules for Clinical Assessment in Neuropsychiatry) to obtain a clinical diagnosis. We stratified the sample into three groups: Group 1, first episode of psychosis (FEP,
In all subjects, stressful life events were positively associated with perceived stress (
UHR subjects report greater levels of perceived stress when compared to those subjects with a psychotic disorder. Stressful life events and perceived stress are associated but not with plasma cortisol levels.
Preterm birth is marked by stressful environment in intra- as well as extrauterine life. Furthermore, preterms are exposed to repeated painful procedures, loud noise, and restricted contact with parents in the neonatal intensive care unit. This environment can affect hormonal and physiological systems and lead to long-term negative outcomes. Despite this, little is known about how early-life stress affects preterms later on in childhood. The goals of the current study were threefold: (1) comparing cortisol profile, including cortisol awakening response (CAR), between preterm and full-term children; (2) assessing memory, behavior, and emotion of preterms; (3) evaluating if preterms are more responsive to an acute stressor.
Basal cortisol and α-amylase (sAA) profiles, including CAR of 30 preterm children were evaluated. Salivary samples were measured on two consecutive days at four time points: awakening, 30 min post-awakening, 1600h, and 2100h. Furthermore, we assess memory functions by using the wide range assessment of memory and learning and screen behavior/emotion by using strengths and difficulties questionnaire. The results of preterms were compared to an age- and sex-matched control group (
We specifically targeted time-dependent effects of corticosteroids on fear conditioning in this withinsubjects delay and trace conditioning paradigm by administering 10 mg hydrocortisone either 4 h or 1 h before fear acquisition. Twenty-four hours later, subjects came back for a fear memory retention test followed by extinction. Analyses show that hydrocortisone impaired trace acquisition specifically on EMG startle responses, regardless at which time point it was administered. However, we found an enhancement of EMG fear responses to the trace stimulus 24 h later only when corticosteroids were administered 4 h before acquisition.
Our findings illustrate the long-lasting effects of preterm birth on the hypothalamic pituitary adrenal (HPA) axis, internalizing behavior, and memory. The findings are in line with the idea that early-life stress alters the set-point of the HPA axis thereby creating a more vulnerable phenotype.
Advanced age is associated with aberrant inflammation and innate immune cell activation that contribute to refractory responses to trauma, tissue injury, and infection. Despite clinical observations that aging impairs wound healing, few studies have examined how the innate immune response contributes to resolution of cutaneous wound infection. Using an excisional wound injury model, young (3–4 months) and aged (18–20 months), BALB/c mice received 102–3 CFU/mL per wound of
This work was supported by NIH R21 AI073987 (EJK), R01 AG018859 (EJK), T32 AG031780 (PWL), and Dr. Ralph and Marian C. Falk Medical Research Trust (EJK) and the MD/PhD Program at Loyola University, Stritch School of Medicine.
Neutrophils comprise the first line of host defense, killing invading microbes by generating reactive oxygen species (ROS) through the activable NADPH oxidase (NOX2) system. This respiratory burst oxidase is genetically absent in patients with chronic granulomatous disease, resulting in recurring severe infections with high morbidity and mortality. The phagocyte oxidase features membrane-bound catalytic components (gp91
Given the importance of H2O2 as a cellular signaling molecule and our recent finding demonstrating a positive feedback regulation of the NOX5 isoform by H2O2, we explored the possibility that the phagocytic NOX2 system is regulated by H2O2. Our data show that H2O2 induces superoxide production in human blood neutrophils and monocytes, as well as in K562 leukemia cells overexpressing NOX2 system components. We demonstrated that H2O2 stimulates NOX2-mediated superoxide generation in both neutrophils and K562/NOX2 cells via a signaling pathway involving Ca2+ influx and c-Abl tyrosine kinase acting upstream of PKCδ. Relevant to the oxidative stress associated with aging, we found recently that this pathway is dysregulated in human neutrophils from healthy older individuals. Among subjects age 20–65, there was a trend for an age-related increase in basal superoxide generation, whereas basal rates in those older than 65 were decreased relative to the younger age groups. In addition, one way ANOVA analysis across all age groups (20–87 years) demonstrated significant age-related declines in the activation levels of NOX2 by H2O2, especially for subjects older than 65.
Thus, dysregulation of phagocyte ROS production over time (i.e. failure to produce appropriate amounts of ROS in the right place at the right time) may contribute to the aging process. Identification of the signaling proteins responsible for abnormal NOX2 regulation by H2O2 will help to explore mechanisms of inflammation-associated aging phenotypes such as increased susceptibility to infection and autoimmunity, delayed phagocyte apoptosis, and chronic inflammation.
T cell function declines with age and impairs the ability to fight new infections and control persistent infection. We previously showed that regulatory T cells (Treg), an immune suppressive CD4+ T cell subset, accumulate dramatically with age in both humans and mice. Furthermore, Treg accrual in aged mice prevented control of persistent infection. Moreover, we found that decreased expression of the pro-apoptotic molecule Bim in aged Treg contributes to enhanced survival and subsequent Treg accrual. Given the role of IL-2 in Treg homeostasis in young animals, we investigated the role of IL-2 on modulating Treg survival and Bim expression in aging. We found that IL-2 is sufficient to promote accumulation of Bimlo Treg in young mice. Also, in aged mice we found that IL-2 neutralization resulted in a partial loss of Treg, but selected for CD25lo Treg. Interestingly, the remaining CD25lo Treg population had increased expression of CD122 (IL-2/IL-15Rβ). Using aged IL-15 deficient mice, we found that IL-15 partially contributed to Treg accrual. Interestingly, we found that the additional loss of Bim (IL-15-/-Bim-/-) rescued the loss of Treg in aged IL-15-deficient mice. Mechanistically, we explored the role of FOXO transcription factors in control of Bim expression in Treg. Notably, we found that deletion of FOXO1/3a/4 in
The thymus is the primary organ for thymopoiesis and the production of naïve T cells from early T cell progenitors (ETP). With age the thymus involutes, resulting in a decline in the production of naïve T cells, which contributes to a compromised immune system in the elderly leaving them more susceptible to illnesses and infection. A mechanism for thymic involution and the decline in naïve T cell production with age is not well elucidated. Previously, we showed that a decline in expression of the transcription factor Foxn1 with age correlated with reduced thymopoiesis as determined by the decline in thymocyte number. Using a transgenic mouse model in which Foxn1 is overexpressed under the human keratin 14 promoter (
This work was supported by NIH R01 AG32809 (PL), R01 AG013874 (PW), T32 AI007508 (EZ), T32 AG031780 (EZ), and an intramural pilot project grant from Loyola University, Stritch School of Medicine (PL).
Parkinson's disease (PD) is the most prevalent neurodegenerative movement disorder affecting up to 5% of the population aged 65–85 years. PD is characterized by progressive dopaminergic neurodegeneration in the nigrostriatal pathway leading to profound dopamine depletion in the substantia nigra and striatum. Evidence from different labs suggests that oxidative stress plays a role in pathogenesis of PD. Previous studies have shown that expression of ALDH1 and GPX1, which are important for clearance of aldehydes andH2O2, respectively, are reduced in the substantia nigra of PD patients. To determine the contribution of deficiency in these two genes to the pathogenesis of PD, our lab has generated mice with simultaneous deletion of Aldh1a1 and Gpx1 genes (Aldh1a1/Gpx1 KO). Young adult Aldh1a1/Gpx1 KO mice showed a significantly increased latency to fall in the automated accelerating rotarod test and increased reversal time in the pole test compared to age-matched wild type control mice. There were no significant effects on levels of dopamine and its metabolites DOPAC and HVA, dopamine transporter or vesicular monoamine transporter 2 protein in midbrain and striatum. However, levels of 4-hydroxynonenal, an end product of lipid peroxidation, were increased in the midbrain and striatum of Aldh1a1/Gpx1 KO. In summary, simultaneous deletion of Gpx1 and Aldh1a1 genes was not associated with altered measures of dopaminergic function but was associated with motor deficits in young adult mice. In addition, deficiency of Gpx1 and Aldh1a1 genes was associated with increased lipid peroxidation. Future experiments will include determining behavioral phenotypes in mice at middle and old ages, evaluating the number of dopaminergic neurons in the substantia nigra. Also, because Gpx1 is predominantly expressed in microglia and neuroinflammation has been suggested to be important for pathogenesis of PD, levels of the microglia marker Iba-1 in substantia nigra and striatum, as well as the number of Iba-1 positive cells in brain, will be determined.
Alzheimer's disease (AD), one of aging-associated diseases, is the most common dementia affecting millions of people around the world. Epidemiological studies indicate that exposure to environmental toxins, such as pesticides, is a risk factor of AD. However, little is known about how pesticide exposure may promote AD pathogenesis. In this study, we investigated the effects of paraquat exposure on cognition and amyloidogenesis in AD mouse models. Our results indicated that paraquat exposure increased the generation of H2O2 by brain mitochondria, which was directly correlated with increased mitochondrial oxidative damage and reduced mitochondrial function. Moreover, APP transgenic mice exposed to paraquat showed exacerbated cognitive impairment and elevated Aβ levels. Peroxiredoxin 3 (Prdx3) is a key enzyme for mitochondrial H2O2 removal. Our results showed that overexpression of Prdx3 prevented paraquat-induced increase in mitochondrial H2O2 generation and mitochondrial damage. Importantly, both cognitive impairment and elevation of Aβ levels induced by paraquat exposure were significantly attenuated in APP transgenic mice with Prdx3 overexpression. Therefore, our results demonstrate that increased mitochondrial H2O2 generation is a key mechanism by which pesticide exposure exacerbates AD pathogenesis.
Despite more than a century of research, the primary mechanisms involved in the pathogenesis of the most common age-related neurodegenerative disease, Alzheimer's disease (AD), remain unclear. Laboratory mice and rats do not develop this disease, and this is attributed to their short lifespan. We propose an extraordinarily long-lived rodent, the naked mole-rat as a novel, natural model for the study of AD. We assessed common risk factors for AD in this species and surprisingly found that young (2 year) naked mole-rats (NMRs) exhibited many of these risk factors: We found high levels of lipid peroxidation in NMR brains. High oxidative damage is associated with neuronal damage in human AD. We also found that levels of both soluble and insoluble Aß in the brains of NMRs were comparable or greater than levels found in a genetically manipulated common AD mouse model (3XTg). These findings prompted us to evaluate if specific brain regions commonly associated with AD (cortex, hippocampus) are better protected against these harmful affects than the relatively unaffected cerebellum. Despite their exceptional longevity, young NMRs exhibited high levels of Aß particularly in the hippocampus and this was coupled with low antioxidant defense and low proteasome activity. These factors might render the NMR hippocampus particularly susceptible to neuronal damage. In addition, we report the unique key properties of NMR Aß (sequence, aggregation propensity and toxicity) that suggest NMRs may be an important addition to the sporadic AD models studied to date.
Centrally secreted neurosteroids and peripherally secreted centrally active neurosteroids increase after stressful stimuli modulating anxiety in experimental animals and possibly in humans. Their altered secretions have been reported in panic disorders (PDs) and suggested to be a possible cause or an effect of the panic symptomatology. We have studied in two successive experiments related to PDs the secretions of neuroactive steroids. In experiment 1, we measured the neuroactive steroids in 25 women and 13 men with PD during an interictal phase to see whether their secretion is related to panic symptomatology and whether it changes after successful pharmacotherapy. In experiment 2, neuroactive steroids were examined in normal probands, 42 women and 17 men who reacted with acute panic symptomatology to inhalation of 7% CO2to see whether neurosteroid alterations precede as a cause or follow as an effect of the acute panic attacks, possibly clarifying their pathogenetic mechanism in PDs. In experiment 1, we found that compared to controls, women with PDs had significantly higher levels of progesterone in the midluteal phase of the cycle, of pregnenolone in the premenstrual phase, of 3α,5α-tetrahydroprogesterone in the follicular phase, and of 3α,5α-tetrahydrodeoxycorticosterone in the premenstrual phase, whereas in men with PDs, the plasma concentrations of progesterone and dehydroepiandrosterone were greater than in controls both in a drug-free month and during paroxetine therapy, in both cases correlating significantly with the panic scores. In experiment 2, we found a trend toward a decrease of neurosteroid concentrations during and after CO2inhalation, with no correlations between panic symptomatology and neurosteroid secretion. This would exclude that their possible alterations could be either a cause or an effect of panic attacks.
Holocaust offspring are at greater risk for the development of Posttraumatic stress disorder (PTSD) than comparison subjects, and the increased vulnerability appears to be associated with maternal PTSD. Paternal PTSD is associated with increased vulnerability to depression in Holocaust offspring. In prior studies, neuroendocrine alterations have been observed in Holocaust offspring that resemble those described in association with PTSD and PTSD risk. Holocaust offspring were more likely to show cortisol hypersuppression on the dexamethasone suppression test (DST) in association with parental PTSD even if offspring had not developed lifetime PTSD. These data imply an enhanced glucocorticoid receptor responsiveness that might be associated with PTSD risk, as has been demonstrated in other populations at risk for PTSD, such as soldiers preparing to deploy for combat. More recently, it has been of interest to consider the differential contributions of maternal vs. paternal PTSD on offspring neuroendocrinology. Such work may identify putative epigenetic changes underlying neuroendocrine alterations associated with PTSD risk.
The current study examined glucocorticoid responsiveness as reflected by the 0.50 mg dexamethasone suppression test (DST) and the lysozyme stimulation test (LST) in 81 Holocaust offspring, the majority of whom (73%) had two Holocaust exposed parents. The offspring were subdivided based on maternal and paternal PTSD. The LST is an in vitro test of glucocorticoid responsiveness carried out in live cultured lymphocytes exposed to varying doses of dexamethasone (DEX) for which a lower IC50-DEXfor lysozyme inhibition indicates increased GR responsiveness.
In this sample, there was a significant correlation between the cortisol response to DEX (expressed as cortisol decline from pre- to post-DEX), and the lysozyme IC50-DEX(
The data indicate that maternal and paternal PTSD may contribute uniquely to neuroendocrine alterations and to psychiatric and psychological outcomes in Holocaust survivor offspring.
Smaller hippocampal volume (HV) and a history of childhood trauma are important risk factors for developing post-traumatic stress disorder (PTSD) in adulthood. However, many individuals experience traumatic events throughout lifespan but do not develop a PTSD diagnosis. Few studies have assessed how these vulnerabilities might correspond with adult trauma symptoms among healthy populations. The goal of this study was to, therefore, assess how childhood trauma and HV are associated with adult trauma symptoms among young and older adults without PTSD.
Thirty-two healthy older adults and 28 young adults completed the Trauma Symptoms Checklist and the Childhood Trauma Questionnaire. Magnetic resonance imaging scans were performed and HV measurements were obtained through manual segmentation using a well-validated protocol.
Multiple regressions computed for each age group showed that in both young and older adults, childhood trauma was not associated with HV. However, in young adults, HV was significantly associated with magnitude of adult trauma symptoms such that young adults with small HV reported more adult trauma symptoms. No significant associations between HV and adult trauma symptoms were observed in older adults.
These novel results reveal that smaller HV is associated with adult trauma symptoms among healthy young adults, but not older adults. It is possible that small HV supersedes childhood trauma in explaining adulthood trauma symptoms among healthy young adults. Based on these findings, it would be interesting for future studies to investigate how hippocampal-dependent processes might contextualize concomitant trauma symptoms.
There is accumulating evidence of hypothalamic–pituitary–adrenal (HPA)-axis dysregulation in depressed children and adolescents. For example, depressed children tend to show a pattern of nonsuppression to the Dexamethasone Suppression Test, suggesting atypical feedback sensitivity of the axis. However, evidence linking HPA-axis stress reactivity during laboratory tasks and pediatric depression is more limited and contradictory, with studies showing both blunted and hyperactive cortisol responses to stress in depressed youth. These conflicting findings may been partially attributed to key group differences across studies, such as the age of the sample, depression severity, or history of maltreatment. In this talk, I discuss another key source of variability: individual differences in peak timing, as a factor that can obscure the nature of the depression-HPA-axis relation. I then compare two common modeling approaches (GLM repeated measures and Growth Curve Modeling) with a Functional Data Analysis approach in their ability to account for individual differences in peak times.
We examined depressive symptoms as predictive of HPA-axis reactivity in two different studies. The first study involved 65 children ages 6–7 participating in a large longitudinal study of the development of internalizing and externalizing symptoms. These children completed two standard laboratory stress tasks. Saliva cortisol was sampled at 5-minute intervals for 60 minutes. Internalizing symptoms were measured via the parent-completed Child Behavior Checklist. The second study involved 60 children ages 9–16 participating in a study of neuroendocrine functioning in depressed youth. These children also completed a standard laboratory stress task after which saliva cortisol was measured at 10 minute intervals. Depressive symptoms were measured via the Children's Depression Inventory. We then compared three approaches to model cortisol stress reactivity as predicted by internalizing (study 1) depressive symptoms (study 2): (1) a standard repeated measures (RM) approach, (2) a standard growth curve (GC) approach modeling change from the time of the stressor (using pre-stress time as the intercept), and (3) a functional data analysis (FDA) approach using individual peak times as a common anchor. This FDA approach involves the shifting of individual curves horizontally so that all individual peaks are anchored on a common time point. This allows for the estimation of true peaks (intercept) and acceleration towards this peak (slopes) while controlling for individual variability in peak times.
In both of our studies, the RM and the CG models failed to find a link between internalizing/depressive symptoms and atypical HPA-axis reactivity (
Our data suggest that traditional modeling approaches, such as RM and GC may not be sensitive to one type of anomaly associated with depressive symptoms. Specifically, significant individual and group variability in time-to-peak may confound the type of fixed-time comparisons obtained with RM and GC approaches. In contrast, using the FDA approach, which controls for this variability, we found that depression impacts the final post-stress peaks but not the slope towards this peak. Since peak levels are a function of initial levels (baseline), rate of activation (slope) and time to peak (duration of activation), our results suggest that internalizing/depressive symptoms are associated with anomalies in the duration of post-stress HPA-axis activation leading to elevated post-stress peaks.
Osteoarthritis (OA) is a leading cause of disability in the United States, and it afflicts more than 30 million Americans who are past middle age. Currently, there are no FDA approved drugs specific for the treatment of this debilitating age-associated degenerative joint disease. Although not a classical inflammatory arthropathy, OA is frequently associated with signs and symptoms of inflammation, including joint pain, swelling/stiffness that may lead to functional impairment. Furthermore, increased levels of inflammatory cytokines such as IL-1β, either locally in the cartilage or in the synovial fluid during OA pathogenesis, can repress the synthesis of type II collagen (COL2A1) and aggrecan (ACAN) and upregulate proteinases such as matrix metalloproteinases and the disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family of aggrecanases, thereby ultimately favoring cartilage degradation. We have previously demonstrated that mice lacking the matrilin-3 gene develop OA very early in life. Elucidating the biological mechanism by which MATN3 can delay OA is a critical first step in understanding its potential as a novel OA therapy. In our efforts to do so, we discovered that recombinant human matrilin-3 (rhMATN3) protein can inhibit several catabolic effects of IL-1β (including its downregulation of COL2A1 and ACAN and its stimulation of the major catabolic proteinase, ADAMTS-5). Furthermore, we have also recently obtained strong evidence suggesting that matrilin-3's regulation of these targets is dependent on its ability to stimulate interleukin-1 receptor antagonist (IL-1Ra): a potent IL-1β pathway inhibitor. Future studies will focus on characterizing the signal transduction pathway involved in matrilin-3 induced stimulation of IL-1Ra.
This study is supported by NIH AG 017021, AG 014399, and RR024484.
Synovial fluid (SF) contains hyaluronan (HA), an important lubricant for joint articulation at high-molecular weight (Mr) and concentration. After injury and in osteoarthritis, SF HA concentration and Mr decrease, although the biophysical mechanisms for this are unclear.
HA efflux from SF is higher after injury in a Mr-dependent manner.
The residence time of HA for various Mr was determined in
After saline injection, high Mr HA accumulated over time, whereas after HA injection, [HA] was lower (1) with decreasing Mr, (2) for ACLT compared to Non-Op knees, and (3) at day 7 compared to 28 (
The decreased HA residence time in the joint at early times after ACLT may be due to the inflammatory response in synovium following surgery and ACLT injury. This study identifies increased efflux of high-Mr HA as a biophysical mechanism of diminished HA concentration after injury and during joint inflammation, as often occurs in osteoarthritis.
NIH-NIA 1F31AG039939, NIH-NIAM
fluids: (A) normal rabbit SF, (B–G) saline injection, (H) injected HA, HA injection at days (I-N) 7 and (O-T) 28 after surgery.
Total HA by Mr (A) saline injection, (B) injected HA, and after HA injection at days (C) 7 and (D) 28. Stacked bars: (top) endogenous HA and (bottom) injected. Dashed line: normal rabbit SF.
Calculated time constants by Mr bin for Non-Op (−) and ACLT (+) data at days 7 and 28.*
Dynamic events in injured skeletal muscle lead to a robust regenerative response that is critically dependent on macrophages; however, the regulatory role of these inflammatory cells remains to be established. Given the important role of microRNA (miRNA) in macrophage biology, it seems likely that these small, non-coding RNAs are involved in macrophage-specific biological events. The present study was designed to explore this possibility by exploiting the known defect in macrophage recruitment and activation in CCR2-/- mice. Thus, the temporal expression of macrophage-related miRNA in muscle regenerative events
Research supported in part by the Veterans Administration (Merit Review Grant) and the National Institutes of Health (F30 HL110743 and R01 HL074236).
Aging-associated bone mineral density (BMD) loss is becoming a significant, worldwide social and financial health care problem. In this study, we demonstrated the beneficial role of FDA-approved prescription omega-3 fatty acids, Lovaza® against BMD loss during aging using 12-month-old C57BL/6 mice. Mice were fed with 1 and 4% of Lovaza® and placebo diets including a group with 4% regular fish oil (18/12 (EPA/DHA)) diet for 12 months. DXA scan was performed once before starting the diet at 12 month of age and again after 12 months of feeding. After analyzing the BMD of different bone regions, we found that 4% Lovaza® prevents age-associated BMD loss, while 1% Lovaza® offers mild protection. However, 1% Lovaza® showed better protection than that of 4% regular fish oil. We also measured the effect of LPS-stimulated cytokines production in bone marrow (BM) cells collected from mice fed with different experimental diets. Interestingly, 4% Lovaza® showed decreased production of pro-inflammatory cytokines, TNF-α, and IL-6 and increased production of anti-inflammatory cytokines IL-10 and IFN-γ as compared to that of other groups. We also analyzed RANKL-stimulated activation of inflammatory and pro-osteoclastogenic signaling molecules p38 MAPK and JNK in BM cells using Fast Activated Cell-based ELISA (FACE) assay system. Interestingly, both 1 and 4% Lovaza® showed reduced activation of both p38 MAPK and JNK. Furthermore, we analyzed the components of bone remodeling in serum samples for TRAP5b, PINP, RANKL, and OPG. Bone resorption marker, TRAP5b level was dramatically reduced in 4% Lovaza® treated mice, whereas bone formation marker, PINP level was unaffected. Moreover, the osteoclast stimulating factor, RANKL was significantly reduced in 4% Lovaza® treated mice, whereas the decoy receptor for RANKL, OPG level was unaffected. These data indicate that Lovaza® protects age-associated bone loss in C57BL/6 mice by modulating inflammatory signaling molecules and related bone resorption.
Age-related sarco-osteopenia involves declines in mass, quality, and strength of both muscle and bone. Because the protein deacetylase Sirtuin1 (Sirt1) has been associated with improvements in the healthspan in mice involving both muscle and bone, Sirt1 may be an excellent target for study for its potential in preventing and/or ameliorating sarco-osteopenia. We are currently exploring the effects of resveratrol (RSV), a putative activator of Sirt1, on bone. Our preliminary data demonstrate that
Preservation of mitochondrial function and reduced generation of reactive oxygen species are correlated with increased lifespan and healthspan. Recent studies in invertebrates and rodents have challenged this paradigm by demonstrating that several mitochondrial electron chain alterations are associated with increased longevity. For example, mice lacking Surfeit locus protein 1 (SURF1), a 30 kDa inner mitochondrial membrane protein essential for the assembly of electron transport chain complex IV (Cytochrome c oxidase, COX), have a 50%–80% decrease in COX activity compared to control littermates, yet increased longevity. Maintenance of mitochondrial function is essential for insulin sensitivity, which is one important correlate of longevity. In this study, we asked whether SURF1 null mice have alterations in insulin sensitivity that might contribute to the increased longevity in these mice. Compared to wild-type littermates, SURF1 null mice displayed reduced body weight that could be attributed to decreased fat mass. This reduction in adipose mass in SURF1 null mice is not due to impairments in lipid storage, rather due to increased lipolysis and fatty acid oxidation as indicated by increased phosphorylation levels of hormone sensitive lipase and acetyl CoA carboxylase. PGC1α and its downstream targets NRF1 and Tfam were elevated in the adipose tissue of SURF1 null mice, indicative of increased mitochondrial biogenesis. Increases in fatty acid oxidation and mitochondrial biogenesis are important correlates of insulin sensitivity. Consistent with this notion, a significant improvement in insulin sensitivity was observed in SURF1 null mice; however, their glucose clearance capacity remains unaffected. A strong upregulation of mitochondrial proteases Lon, ClpP, and mitochondrial Hsp60 (components of the mitochondrial unfolded protein response) was observed in the adipose tissue of SURF1 null mice suggesting the involvement of additional components in insulin sensitivity or longevity. Together, these data demonstrate a novel crosstalk between reduced complex IV activity and mitochondrial biogenesis that causes increased fatty acid oxidation and insulin sensitivity and suggest increased insulin sensitivity as a potential mechanism for increased longevity in these mice.
This work was supported by the Ellison Medical Foundation.
Recent evidence suggests that the lipopolysaccharide (LPS) receptor, toll-like receptor-4 (TLR4), and signaling pathways downstream of TLR4 play a role in skeletal muscle insulin resistance. Much data implicate saturated free fatty acids (FFA), whose circulating levels are often elevated in individuals with insulin resistance, as ligands for TLR4. Our lab recently demonstrated elevated concentrations of LPS in plasma of patients with type 2 diabetes (T2D), providing another mechanism for elevated TLR4-mediated inflammation in these individuals. TAK-242, a small-molecule antisepsis agent, binds selectively to TLR4 and inhibits its downstream signaling events. The purpose of the present study was to investigate the effect of TAK-242 on LPS and FFA-induced inflammatory pathways in skeletal muscle. L6 myotubes were preincubated with/without TAK-242 (1µM) for 1 h, prior to stimulation with 100 ng/ml of LPS for 1 h, or 400 uM stearic acid for 1 and 6 h. LPS caused an inflammatory response, as evidenced by increased phosphorylation of Jun N-terminal kinase (JNK) (2.1-fold), p38 MAPK (5.4-fold), IKBα (3.9-fold) and p65 NFkB (8.4-fold), and reduced IKBα protein levels (5.3-fold). TAK-242 completely inhibited the phosphorylation of these proteins in response to LPS. Stearic acid treatment for 1 h increased phosphorylation of JNK (1.5-fold), IKBα (74%), and p65 NFkB (94%), whereas TAK-242 partially reduced the increase in the phosphorylation of the inflammatory proteins by 22, 62, and 84%, respectively. Stearic acid treatment for 6 h increased phosphorylation of JNK (3.6-fold), p38 (6.6 fold), and p65 NFkB (1.2-fold), and TAK-242 reduced the stearic acid-mediated increases in JNK (by 38%) and p38 (by 51%), although p65 NFkB phosphorylation was not affected.
(1) TAK-242 reduces LPS- and lipid-induced inflammation in muscle cells; (2) LPS induces an inflammatory response primarily by activating TLR4; and (3) saturated FFA also works through TLR4 to induce an inflammatory response, although other mechanisms likely are involved in this process.
TAK-242 may represent a novel therapeutic approach to reduce inflammation and improve insulin action in conditions associated with insulin resistance such as obesity, aging, and T2D.
Dysfunctional mitochondria can lead to disruptions in ATP production and increase the reactive oxygen species related to aging. Previously, it was thought that dysfunctional mitochondria would lead to a decrease in lifespan; however, mutations or knockdowns resulting in a decrease in the activity of the mitochondrial electron transport chain have been shown to increase longevity in
Here, we explore a novel conserved mechanism of longevity in the mitochondrial complex IV-deficient Surf1 knockout mouse. Knockout of Surf1, which codes for a complex IV assembly factor, results in a 50%–80% decline in complex IV activity and a 20% increase in median lifespan. This dysfunction results in tissue-specific differences in superoxide and hydrogen peroxide production, membrane potential, and ATP production. Interestingly, these fibroblasts are more resistant to the superoxide generator paraquat. Here, we show that these mice have an increase in mitochondrial specific chaperones (HSP60) and proteases (Lon and CLPP) that have been linked to mtUPR providing a possible mechanism for the increased resistance to cellular stresses. Taken together, the Surf1 knockout mouse could have enhanced longevity due to a novel mechanism conserved from invertebrates to mammals.
This project was supported by a grant from the Ellison Medical Foundation.
Sleep fragmentation is a common health issue in aging populations. Most commonly studied in relation to sleep apnea, it has been demonstrated in humans that sleep fragmentation is associated with disruption of many body functions ranging from glucose metabolism to cognitive function.
In an effort to assess the effects of rapamycin on healthspan in C57BL/6 mice, we used a non-invasive assay to monitor animal activity. The Pack Laboratory developed a high-throughput model for phenotyping sleep in mice. which validates the use of activity/inactivity assessments as a measure of sleep, with sleep defined as any bout of inactivity of ≥40 seconds. Based on their results, we measured activity across a 24-h light/dark cycle to assess sleep fragmentation.
We report here that C57BL/6 mice that were started on rapamycin at 19 months of age show significant sex differences in sleep fragmentation unrelated to aging. Sex differences in sleep fragmentation are also found in aging humans; the mechanism underlying this sex difference warrants further investigation.
Inhibition of the TOR signaling pathway by rapamycin has been shown to extend measures of lifespan in genetically heterogeneous mice and C57BL/6 mice. To determine whether rapamycin treatment delays age-associated declines in healthspan, we performed a single-blinded study using C57BL/6 mice fed with encapsulated rapamycin or control diet starting at 19 months of age. We tested these mice with a series of putative healthspan assays chosen for their similarity to measures of aging and frailty in humans. Several of the assays showed significant differences between rapamycin-treated animals and controls. In some cases, rapamycin-treated animals performed significantly better than age-matched controls, whereas in other assays there were no differences. In addition to treatment effects, we also found several useful indicators of age-related decline in mice comparable to assays used to measure human frailty.
Resistance to toxins and other stressors has been shown to impact healthspan and longevity in multiple models of aging, including worms, flies, and rodents. The naked mole-rat, the longest-living rodent with a maximum lifespan of >31 years, demonstrates a profound resistance to a variety of toxins
A 30% increase in the number of older fathers during the last three decades has made the paternal age affect an increasingly important topic in aging reproductive health. Using
Evolutionary theory predicts that selection pressure declines with age. On the molecular level, one consequence of this may be that proteins needed for long life show loss-of-function point mutations in short-lived species. We used multiple regression, controlling for both body mass and shared ancestry, to find these lifespan-associated point mutations in several thousand high-quality protein alignments from 33 mammalian species. Overall, we found many more mutations in our data than would be expected by chance. We present examples of proteins that may be relevant to mammalian aging.
Measures of health span offer a more unified index of overall aging than measuring lifespan or longevity alone, as health span includes assays on a range of functional systems within the individual. The objectives of this study were to identify robust and reproducible functional measures of healthspan and to determine the interrelationship of these measures, and perhaps measures predictive of mortality. The study uses 16 different assays (e.g. body composition, metabolic rate, gait analysis, nerve conduction, kidney function) to evaluate overall health span in C57BL/6 male mice of four different ages (4, 20, 28, and 32 months of age) in a cross-sectional design. Many of these assays show significant consistent changes with age. By identifying age-related changes in these functional patterns, we can begin to develop biomarkers of aging that will help guide future directions of aging research in animal models and clinical work. For example, geriatric frailty involves the interrelationship of multiple variables – involuntary weight loss, exhaustion, lack of spontaneous activity, slow walking speed, and reduced grip strength. By measuring these variables in aging rodents, we can develop models that explore the physiology and etiology of such complex phenomena and provide critical insights into the mechanisms associated with age-related decline.
Rapamycin (rapa) has recently been found to extend lifespan in
Our previous data indicated that there were no differences in body composition, with exception in fat mass, in which only DR treatment had a significant decrease in fat mass, and both treatment showed significantly reduced phosphorylation of S6 (60%), which indicates that both treatments affected mTOR activity similarly. Also, both treatment increased levels of autophagy when measured by the ratio of LC3II/LC3I. From our microarray analysis of the liver, we observed that DR has a more dramatic effect on genes than rapa. Our principal component analysis, which provides a broad overview of changes, showed that there was no overlap between rapa and DR, but there were some shared overlap of components between AL and rapa and also between DR and DR+rapa. The gene analysis showed that DR and DR+rapa share a large overlap of genes, whereas overlap between DR and rapa is fewer. We found similar results in our pathway analysis as well. Our preliminary data indicate that although DR and rapa may have similar effects on mTOR activity and autophagy, there are still major differences at the gene and pathway level and that the differences observed among both treatments suggest that rapamycin is not a DR mimetic.
This work was supported by NIH Grant AG036613.
The San Antonio Nathan Shock Center Conferences have attracted international speakers and participants since 1995. This annual conference, held in Bandera, Texas, USA, addresses a different topic in the biology of aging each year. The venue's intimate setting, relatively remote location, and common areas are ideal for a small conference (80–100 participants), where copious informal intellectual interchange supplements that of the formal sessions. The 2011 meeting, part of an annual series sponsored by the University of Texas Health Science Center San Antonio, TX, USA, and the Nathan Shock Center of Excellence in the Biology of Aging, addressed the causes of age-associated inflammation and its effect on age-associated diseases.
It is firmly established that aging is associated with chronic low-grade inflammation. Chronic inflammation has been proposed to be a primary cause of aging, to exacerbate/accelerate age-associated diseases such as Alzheimer's disease, to alter normal immune function and thereby increase susceptibility to infection and cancer, and to be a viable pharmacological target for delayed disease progression and enhanced longevity. Age-associated inflammation is the result of a wide range of factors that include deregulation of senescent cells, environmental exposures, and age-associated illnesses. Thus, age-associated inflammation can be considered as both cause and effect during aging at the cellular and organismal level and is inseparable from almost all aspects of biological aging.
The purpose of the 2011 conference was to provide a forum for the presentation and dissemination of recent findings pertaining to age-associated inflammation. To this end, speakers were recruited who are working on exciting new studies that help to clarify the molecular mechanisms underlying age-associated inflammation, and its causal relationship to diabetes, muscle wasting, neurodegenerative diseases, cancer, and infection and immunity, as well as a target for prophylactic intervention. Importantly,
Emerging evidence has implicated the gastrointestinal flora in the pathogenesis of insulin resistance by causing chronic, low-grade endotoxemia. The goals of this study were to determine: (1) whether obese non-diabetic and obese diabetic subjects have abnormal endotoxin [lipopolysaccharide (LPS)] concentrations in plasma; (2) whether LPS promotes an inflammatory response and induces insulin resistance in human muscle, employing a primary muscle cell culture system. We measured plasma LPS concentrations and insulin-stimulated glucose metabolism (M) with the euglycemic insulin clamp (160 mU/m2.min) in 13 lean [BMI=25±1 kg/m2, age=39±2 years, fasting plasma glucose (FPG)=92±3 mg/dl, M=10.4±0.7 mg/kg.min], 9 obese non-diabetic (BMI=32±1, age=48±3, FPG=92±2, M=8.5±0.7), and 11 obese diabetic (BMI=34±1, age=48±3, FPG=151±13, M=4.2±0.5) subjects. Obese non-diabetic and diabetic subjects had elevated LPS concentrations in plasma when compared with lean healthy controls. There was an inverse correlation between LPS concentrations and insulin sensitivity (M value). Incubation of human myotubes derived from lean non-diabetic subjects with LPS caused an activation of NFκB signaling in a time-dependent manner, as evidenced by a significant increase in IL-6 and MCP1 gene expressions and phosphorylations of p38 MAPK and Jun N-terminal kinase (JNK). We, then, determined the metabolic consequence of the inflammatory response initiated by LPS in the human myotubes. We found that LPS induced a significant reduction of Akt, AS160, and GSK3 phosphorylations that were correlated with a diminished insulin-stimulated glucose uptake. Interestingly, knocking down toll-like receptor (TLR4), a receptor for LPS, reduced the LPS-induced increases in p38 and JNK phosphorylation as well as IL-6 and MCP1 mRNA expression.
(1) Obese non-diabetic and obese diabetic subjects have increased concentrations of endotoxin in plasma, and this could play a role in the pathogenesis of insulin resistance; (2) LPS directly causes an inflammatory response that leads to insulin resistance in human muscle; and (3) downregulation of TLR4 blunts the LPS-induced inflammatory response in human muscle, suggesting that pharmacologic blockade of TLR4 could be a useful strategy for the treatment of insulin resistance.
We recently showed that inhibition of mTOR by systemic, long-term rapamycin treatment blocks AD-like cognitive impairments and Aß accumulation in mice modeling AD. To determine the mechanisms by which long-term inhibition of mTOR modulates cognitive function, we examined the complete proteome of brains of control- and rapamycin-treated PDAPP mice. Members of the chaperone/heat shock response (HSR) family were overrepresented among the proteins whose abundance was increased by rapamycin treatment in transgenic brains. In agreement with the observed upregulation of heat shock proteins (HSPs), we found that HSF1 was activated, and that eIF4E-biding protein (4E-BP, which participates in shutoff of protein synthesis during heat shock) was activated, setting up conditions for the preferential translation of chaperone mRNAs in rapamycin-treated brains. Overexpression of HSF1 mimicked the effects of rapamycin, resulting in chaperone upregulation and preservation of cognitive function in PDAPP mice. Conversely, inhibition of the chaperone HSP90 strongly increased Aß levels in transgenic brain slice cultures. Taken together, our results indicate that activation of the chaperone network is sufficient to lower Aß levels and block cognitive impairments in PDAPP mice and suggest that long-term inhibition of the mTOR pathway may decrease Aß levels and preserve cognitive function by increasing the levels of chaperones in transgenic mouse brains. Maintenance of proteostasis may, therefore, be sufficient to preserve cognitive function in AD-like neurodegeneration. Our data suggest that rapamycin, already used in clinical settings, may have potential for the treatment of AD.
Decline in sensory acuity is a general hallmark of aging, which in humans decreases quality of life. We report here refinement of a sensory acuity assay in mice. Three features of the assay merit attention. First, as mice are primarily nocturnal in nature, olfaction is an important sensory modality for them. Second, our assay instead of using artificial olfactory cues employs major urinary proteins that are important in both intrasexual and intersexual communication of mice in nature. Third, the assay can be performed in the mouse's home cage, thus avoiding artifacts from distracting, novel environments. Procedurally, the assay uses serial dilutions of urine and preference for the urinary odor relative to water control to olfactory acuity. Age-related changes in olfactory acuity have not previously been reported in mice.
We have refined the assay that compares time spent sniffing a sample relative to time spent at a distilled water control, numerous times, and the most recent refinement appears to be sensitive, repeatable, and encompasses particularly informative urinary dilution ranges. Specifically, previous testing revealed that of any age, mice usually cannot distinguish urine from water at a dilution of 1:10,000 (Rendón, unpublished data). The range of experimental dilutions that have been narrowed down through successive modifications of the assay is between 1:10,000 and 1:5,000. Sampling in this range allows us to pinpoint and compare discriminatory ability and to more accurately assess the influence of factors, such as age, or drugs, on mouse olfactory acuity.
Over the last decades, periodontal diseases have been discussed as a possible risk factor for systemic diseases with an inflammatory origin.
To investigate if adult patients with periodontitis attending the dental school in Huddinge, Sweden, had more signs of systemic diseases such as cardiovascular disease, diabetes mellitus, pulmonary diseases, specifically asthma bronchiale, compared to healthy patients or those patients with gingivitis.
In this retrospective study, dental charts were examined where the periodontal diagnoses were known. A total of 325 patients with severe periodontitis and 149 patients without periodontitis born 1928–1968 were identified. The diagnosis regarding the systemic diseases was self-reported. Odd ratios for all cardiovascular diseases, diabetes mellitus and pulmonary diseases were calculated with a logistic regression model adjusted for age, gender, and smoking.
A total of 44.3% of those with severe periodontitis also had cardiovascular diseases, 19.1% had asthma bronchiale and 21.2% suffered from diabetes mellitus. Among the controls 30.9% had cardiovascular disease, 23.5% suffered from asthma and 6.7% had diabetes mellitus. Both among the controls and among those with severe periodontitis hypertension was the most frequent diagnosis. There was a significant association between periodontitis and cardiovascular disease (OR=1.79 (0.0143)), but not between asthma bronchiale and periodontitis. The risk for diabetes mellitus was greater among those with periodontitis, OR=2.95 (<0.0001).
This study shows that there is an association between periodontitis and certain systemic diseases such as cardiovascular disease and diabetes mellitus. However, the underlying mechanism is unknown and it is too early to conclude whether periodontitis is a predictor for other systemic inflammatory diseases.
In advanced forms of chronic periodontitis, changed or elevated parameters for a number of blood markers have been detected (total cholesterol, LDL, HDL, triglycerides, fibrinogen, CRP and other inflammatory mediators). As important risk markers for cardiovascular disease (CVD) these findings also indicate possible links between periodontitis and CVD. Reasonable explanations could be the direct spread of periodontal bacteria and their products into the circulation triggering the atherosclerotic process and/or the release of host derived inflammatory mediators from chronically inflamed tissues. Several inflammatory disorders (systemic lupus erythematosus, idiopathic juvenile arthritis, rheumatoid arthritis) and other chronic infections associated with inflammation, are also associated with an increased incidence of CVD. In general, exacerbations of these disorders are characterized by activation of leukocytes as well as increased or changed levels of cytokines and other inflammatory mediators which may impose injury to the arterial wall through induction of endothelial dysfunction, secondary atherogenic dyslipidemia and activation of the coagulation cascade. Peripheral blood mononuclear cells (PBMCs) (monocytes and lymphocytes) are central in the inflammation and hence in the atherosclerotic process being exposed to the same environmental factors as cells in the arterial wall. Alterations in gene expression levels of inflammatory mediators in these cells could represent an important source of biomarkers for progression of atherosclerotic disease before signs of inflammation can be seen in vivo.
This pilot study consisted of 5 patients (>43 years of age) with advanced chronic periodontitis and a control group (5; >47 years of age). All participants were otherwise healthy without medical treatment. Fasting blood samples were collected at the initial visit. Analysis of routine parameters was performed in collaboration with the Department of Medical Biochemistry, Oslo University Hospital. PBMCs were isolated, and gene expression of selected cytokines was measured using real-time PCR. Release of selected inflammatory markers was also measured from cultured patient-derived PBMCs. The study was approved by the Regional Ethics Committee.
Preliminary results from this study showed that fibrinogen levels were significantly higher in the periodontitis group as compared to those in the controls (Mann-Whitney U test). However, no significant differences between test and control group were verified for the other routine parameters investigated (CRP, total cholesterol, HDL, LDL, triglycerides, Apo A1 and Apo B). By Spearman's correlation coefficient test, a strong positive correlation was found between CRP and triglycerides, CRP and Apo B, between cholesterol and LDL, cholesterol and apoB, HDL and apo A1, LDL and apoB and between triglycerides and apoB. A negative correlation between HDL levels and fibrinogen was also detected. For the PBMCs at mRNA level an inverse significant relationship between the test and control group was detected for TNFα, TNFR2 and CXCL16.
A statistical significant difference for fibrinogen levels was detected between the periodontitis and control group. Furthermore, a strong positive correlation was found between CRP and triglycerides, CRP and Apo B, total cholesterol and HDL, LDL, apoA1 and apoB and between triglycerides and apoB. mRNA expression for the PBMCs showed an inverse significant relationship for TNFa, TNFR2 and CXCL16. Our preliminary results suggested that untreated advanced chronic periodontitis increase or change the blood levels of cytokines and other inflammatory mediators known as risk markers for CVD.
Oral mucositis (OM) is an extremely painful condition that develops in the mouths of patients undergoing cancer therapy. It is characterized by painful sores in the mouth and throat which interfere with eating, talking, and social activities. OM can be so painful that it may cause a patient to discontinue cancer therapy, thus leading to increased mortality. Yet, clinical experience shows that ulcer size and/or location might not correlate with suffering. Consequently, assessment tools such as the NCI-CTC scale or the OMAS, which are largely based on measurement and localization of oral ulcerations, do not reflect the actual ordeal of patients suffering from OM (the OM experience; OME). Therefore, it is not possible to understand how to treat or prevent OM or to improve the quality of life of those undergoing cancer therapies since OME cannot be assessed reliably. Hence, currently available methods for assessment of OM cannot be used to test the development of new therapeutics for prevention or management of OM. We suggest that a more realistic and biologically useful approach for the appraisal of OME should include clinical, patient-based as well as objective biological markers of OM to reflect the OME accurately but these have not been validated.
To develop measurements of oral albumin and polymorphonuclear neutrophil (PMN); levels; surrogate markers for epithelial ulceration, combined with the Patient Reported Oral Mucositis Scale (PROMS) to provide a more clinically-relevant estimate of the OME in contrast to methods that are based largely on subjective and clinician-based assessments of ulcer size in patients receiving cancer treatment.
Patients at Princess Margaret Hospital in Toronto, Ontario, who were diagnosed with head and neck cancer and receiving radiation therapy, with or without concomitant chemotherapy, were invited to partake in a prospective study. The protocol was approved by the University Health Network Research Ethics Board, Ref. 09-0231-CE. Using a power calculation (set at 80%, p < 0.05) it was determined that 20 patients were needed.. With patient consent, a priori inclusion and exclusion criteria were used for screening purposes. Patients were examined once, before radiation therapy and thereafter twice-weekly during a 6 or 7 week course of treatment. The final examination was performed 4 to 6 weeks after completion of treatment. OM was evaluated clinically according to two commonly used assessment tools, the NCI-CTC-criteria and the OMAS. OME was assessed at each study visit by obtaining saline mouthrinses for measurement of oral albumin and PMN levels, while the participants also completed a PROMS visual analogue scale (VAS) questionnaire focused on the OME. The clinical and biomarker measurements as well as patient-reported OME data were subjected to linear and rank correlation statistical analyses.
Fifty patients consented to participate in the study and 36 completed the whole course of sixteen clinical examinations. The change from baseline was statistically significant for all biomarkers of OM as well as for OME. All indices demonstrated similar patterns of peak OM activity at 6 and 7 weeks. A subgroup of patients still had OM 4 to 6 weeks after therapy, something not always reported with the NCI-CTC. Significant rank and/or linear relationships (p< 0.05) between clinical assessments the biomarker measurements and the OME data could be identified using the Spearman and/or Pearson product moment correlation coefficients.
Albumin and PMN measurements, coupled with PROMS VAS data correlated well. These measurements add novel and complementary psychosocial as well as biologically objective dimensions for assessment of OME that are superior to those offered by currently available unidimensional methods of assessment, which focus mainly on ulceration. Using these new methods of assessment, more realistic estimation of the OME should permit the development of more effective therapies for OM than are currently available.
Saliva may have a potential to be used in the diagnosis of oral and systemic diseases. The overall aim of the present investigation was to explore the possibility to use substances in saliva as biomarkers for systemic and oral diseases. In this preliminary report TNFα and IL-8 are related to periodontal status and smoking.
451 randomly selected adults, living in the Southernmost county of Sweden (Skåne) participated in the study. The participants were between 20 and 89 years old, 51% were women and 17% were smokers. All participants were examined as per standard examination procedures and stimulated saliva samples were collected. Digital panoramic radiographs and bite-wings were taken. The cytokines were analysed with a multiplex immune assay (Luminex Bio-Rad).
Patients with at least 1 gingival pocket ≥6 mm and ≥20% BOP had significantly higher IL-8 concentrations, 686 (±922) pg/mL vs. 459 (±643) pg/mL, p<0.01. The concentration of TNFα was lower but the difference did not reach significance, 3.5 (±8.4) pg/mL vs. 5.1 (±13.4) pg/mL. Smokers had significantly higher concentrations of TNFα, 10.0 (±23.6) vs. 3.8 (±8.6) pg/mL and lower concentrations of IL-8342.8 (±349.2) vs. 533.8 (±755.4) pg/mL, p=0.029 and p=0.001 respectively.
Periodontal inflammation was reflected in increased salivary concentrations of IL-8 while no significant association between TNFα concentrations and inflammation could be found. Smoking had a significant influence on both IL-8 and TNFα.
The epithelium of the gingiva contributes to innate immune response in several ways, for instance by producing antimicrobial peptides model.
In the present study, the secretion and localization of three epithelial peptides, human β-defensin (hBD)-2, -3, and cathelicidin (LL-37), were studied in an organotypic dento-epithelium (OD-E) model exposed to
Biofilm of
After 5 h of incubation, the biofilm of
In our OD-E epithelial antimicrobial peptide responses have different regulation and localization characteristics, similar to those known to occur in the gingival epithelium in vivo.
Inflammation associated with cancer and induced by cancer therapy is associated with clinical signs of sickness behavior that can culminate in the development of symptoms of depression. Intraperitoneal administration of lipopolysaccharide to mice induces depressive-like behavior. Lipopolysaccharide-induced depressive-like behavior is mediated by activation of indoleamine 2,3-dioxygenase (IDO) that degrades tryptophan along the kynurenine pathway and produces kynurenine metabolites such as quinolinic acid that acts as a N-Methyl-D-aspartate (NMDA) receptor agonist. The present study was carried out to determine whether the NMDA receptor antagonist ketamine alleviates lipopolysaccharide-induced depressive-like behavior.
Mice were injected intraperitoneally with ketamine or saline immediately before administration of the cytokine inducer lipopolysaccharide or saline via the same route. Their behavior and bodyweight were monitored up to 28 h post-injection. Depressive-like behavior was measured by increased immobility in the forced swim test and decreased sucrose preference for a sucrose solution. Brain, liver and plasma were collected 6 h and 28 h after treatment to measure biomarkers of inflammation.
Lipopolysaccharide induced the expression of cytokine, IDO, tryptophan 2,3-dioxygenase (TDO) and heme oxygenase-1 at the periphery and in the brain. This effect was not altered by ketamine pretreatment. Ketamine blocked the development of depressive-like behavior but had no effect on sickness behavior measured by body weight loss, reduced food intake and decreased motor activity.
These data indicate that ketamine is able to abrogate inflammation-induced depressive-like behavior by antagonising the activating effects of kynurenine metabolites on NMDA receptors.
Chronic subordinate colony housing (CSC, 19d) is an established mouse model for chronic psychosocial stress and causes glucocorticoid (GC) resistance in splenocytes and IL-4 producing peripheral lymph node cells. Here we tested the hypothesis that CSC further causes development of GC resistance at the level of the pituitary gland, which is, in turn, causally involved in the increased plasma ACTH response to prolonged heterotypic stressor exposure (4h of restraint/ shaking) following CSC exposure.
Male mice were either exposed to the CSC model or single-housed for control (SHC), in order to investigate changes at the level of the pituitary gland, measured by molecular (Western Blotting, qPCR) and in vitro techniques.
To exclude that the increased plasma ACTH secretion in response to acute heterotypic stressors is mediated by an increased responsiveness of the pituitary to hypothalamic corticotrophin releasing hormone (CRH) and/or arginine vasopressin (AVP) we first employed Western Blotting to reveal possible changes in pituitary CRH receptor 1 (CRH-R1) and AVP receptor 1b (AVP-R1b) expression. However, CRH-R1 expression was significantly lower, while AVP-R1b expression was unaffected in CSC compared with single-housed control (SHC) mice, arguing against an increased pituitary responsiveness. In order to exclude altered receptor sensitivity we are currently investigating if the
In line with the hypothesis of a reduced negative feedback inhibition, CSC compared with SHC mice showed a down-regulation of glucocorticoid receptor (GR), mRNA (qPCR) and protein (Western Blotting) expression in pituitary tissue. Although a comparable corticosterone-mediated
Scientific evidence indicating that excessive stress during human pregnancy can have long-lasting effects on mother and child is increasing. But the underlying biological mechanisms remain elusive. Recent findings suggest a key role of the hypothalamic–pituitary–adrenal axis and the placental enzyme 11β-hydroxysteroid dehydrogenase Type 2 (11β-HSD2). This enzyme inactivates cortisol (F) to cortisone (E), thereby protecting the foetus from maternal F overexposure. Studies on pregnant rats show that placental 11β-HSD2 is up-regulated following acute maternal stress but impaired after chronic stress. Whether a similar mechanism exists in humans is unclear. Therefore, we investigated the acute stress response of salivary F (SalF) in second-trimester pregnant women undergoing amniocentesis and compared this response with amniotic fluid F, E, and the E/(E+F) ratio as an index of placental 11β-HSD2 activity. Since 11β-HSD2 is also present in the adult salivary glands, we determined salivary E (SalE) and the SalE/(SalE+SalF) ratio, as a marker for salivary 11β-HSD2 activity as well and examined the association of these parameters with the amniotic fluid markers of stress.
Repeated saliva samples and an aliquot of amniotic fluid were collected from 34 healthy pregnant women (mean age=37.5, SD=3.9 years) undergoing amniocentesis for karyotyping. Changes in stress perception and state anxiety were monitored using questionnaires. Participants were re-invited for a control condition after receiving the inconspicuous test results of the amniocentesis.
Compared to the control condition, the pregnant participants showed significant increases in psychological distress during the amniocentesis. SalF and SalE increased correspondingly while SalE/(SalE+SalF) decreased. SalF correlated positively with amniotic fluid E (
The present results further our understanding of the maternal–foetal stress response considerably and suggest that during acute stress, maternal F is converted to E within the foeto-placental unit. This is most probably due to the activity of placental 11β-HSD2. Further investigation of the influence of chronic stress on the enzyme activity is essential.
Lower socioeconomic position is associated with increased risk of morbidity and premature mortality from physical and mental disorders and confers similar “transgenerational” consequences on the offspring. The effects on the offspring appear initiated prenatally as lower socioeconomic position also increases risk of prematurity and small/large body size at birth. The biological mechanisms remain, however, elusive. We hypothesized that fetoplacental stress (glucocorticoid) hormone exposure might mediate the link, as we have found in first and second generation exposures to glucocorticoids in rodent pregnancy. We therefore examined associations between socioeconomic position and placental expression of placental genes involved in glucocorticoid exposure and transfer between the mother and fetus.
Biopsies of placental tissue were obtained from 62 healthy (mothers age 32.2±[SD] years), singleton, term pregnancies (37–42 gestational weeks) a maximum of 90 min after (vaginal or caesarian) delivery, snap frozen in liquid nitrogen, and stored at −80°C. Placental mRNAs encoding glucocorticoid receptor (GR) and 11-beta hydroxysteroid dehydrogenase 1 (HSD1) and 2 (HSD2), which regenerate and inactivate glucocorticoids respectively, were determined by real-time PCR. Level of education and occupational status of the mother, indices of socioeconomic position, were obtained from hospital birth records.
Placental GR and HSD1 mRNAs increased with decreasing maternal education (unadjusted
Lower socioeconomic position is associated with higher placental GR and HSD1 gene expression. This combination will both regenerate active glucocorticoids in placenta (with potential impact locally in placental cells and by spill-over on the fetus) and increase placental sensitivity to glucocorticoids. By analogy with preclinical mechanistic studies, this may have immediate offspring and transgenerational effects on cardiometabolic and neuropsychiatric risk but adds placental glucocorticoid sensitivity and regeneration as novel processes involved.
In previous research, we found that flattened diurnal cortisol predicted early mortality with breast cancer, independent of other known risk factors, and this has since been confirmed among patients with lung cancer. In that study, loss of diurnal variation in cortisol was associated with self-reported awakenings during the night, implying an interaction with sleep disruption. This suggested that objective measures of sleep would clarify the relationship between disruption of circadian cortisol rhythms and sleep disturbance.
Here we recruited 101 women with metastatic breast cancer and 16 age- and socioeconomic status-matched controls. We measured sleep using full electroencephalographic (EEG), electro-oculographic, and electromyographic recordings in the clinical research center, where we were also able to draw blood samples throughout the night using a long IV line through a hole in the wall. Sleep measures were confirmed with two nights of home EEG recordings and 2 weeks of actigraphy.
Among 63 for whom complete cortisol and sleep data are now available, we observed a phase shift in the relationship between the peak of cortisol and wake time such that patients woke earlier than their cortisol peak. Controls woke on average 1 h 20 min before the cortisol peak, whereas patients woke 1 h 54 min before. This control–patient difference was not statistically significant. However, among all subjects, there was a significant .38 correlation between diurnal cortisol slope and time from waking to the cortisol peak, such that those who woke earlier in relation to the cortisol peak had flatter cortisol slopes. Among the patients alone, the correlation was .43. This suggests that flattening of diurnal cortisol is associated with early morning waking. In the sample of 101, we found a relationship between misalignment of preferred and actual bedtimes and disease-free interval (DFI), the time from initial breast cancer diagnosis to date of metastasis. Shorter DFI is a strong predictor of reduced survival time. Going to bed earlier or later than preferred bedtime was associated with shorter DFIs, compared with aligned bedtime (HR=3.25, 95%CI=1.17–8.98,
Thus waking ahead of the normal morning cortisol peak was related to flatter diurnal cortisol, and misalignment of preferred and actual sleep times was also associated with poor prognosis.
Sleep disturbances are among the most common symptoms of posttraumatic stress disorder (PTSD). There is growing evidence that sleep fragmentation and short sleep duration are risk factors for hyperlipidemia, diabetes, obesity, and other risk factors for vascular disease. No work has examined the association of sleep with lipid metabolism in PTSD.
A cross-sectional 2×2 design (PTSD/control × male/female) included medication-free, nonobese, medically healthy subjects. The sample was comprised of 42 individuals with current chronic PTSD (52% female;
PTSD subjects had significantly elevated total cholesterol, very-low-density lipoprotein (VLDL) cholesterol, and triglycerides relative to controls (all
The results suggest an association of sleep to cardiovascular risk factors in PTSD. Further research is needed to assess whether effective treatment of sleep in PTSD will favorably affect lipid metabolism.
The peak number of protective immune cells measured in the blood at the zenith of their diurnal rhythm is a measure of their overall capacity for immunoprotection. Rhythmic diurnal changes in blood immune cell numbers reflect a redistribution of cells from the blood to other body compartments, and back into the blood. This redistribution may be critical for leukocyte maintenance and for the surveillance and effector functions of the immune system.
We investigated diurnal changes in absolute numbers of NK cells in patients with metastatic breast cancer (MBC) (
In agreement with the literature, control subjects showed peak blood NK cell numbers at T1, with a decrease to their diurnal trough at around the sleep midpoint (T4), followed by a return to diurnal peak numbers 12 h later. Compared to controls, patients with MBC showed significantly lower peak NK cell numbers (
These results suggest that patients with MBC have reduced NK-cell-mediated immunoprotection compared to controls and that among patients, higher NK cell numbers are related to longer disease-free interval and better Karnofsky status. Patients with MBC also show decreased diurnal NK cell redistribution compared to controls, and among patients, reduced diurnal NK cell redistribution is associated with increased sleep disruption.
Joint stiffness in rheumatoid arthritis (RA) is worse in the morning and has been associated with increased secretion of the pro-inflammatory cytokine IL-6 and with decreased secretion of cortisol, suggesting that clinical symptoms may be related to hormonal and immune circadian variations. We measured 24-h blood profiles of IL-6 and cortisol in patients with RA to determine any changes in IL-6 and cortisol following a 2-week course of prednisone administered orally in a specially designed timed-release tablet (TRT).
Nine patients with active RA were clinically assessed and had 24-h blood sampling before and after a 2-week course of TRT prednisone (5 mg per day). Patients took the TRT orally at 2200 h, and the prednisone was released at 0200 h. Changes in circadian variation in cortisol and IL-6 and clinical measures were compared using random coefficient regression modelling and Wilcoxon matched-pairs signed-rank test.
IL-1ra, IL-1β, IL-4 and TNF showed no circadian variation prior to TRT prednisone. Significant alterations in circadian profiles and concentrations of IL-6 and cortisol were observed following TRT prednisone. The peak value of IL-6 fell from 42.5 to 21.3 pg/ml and occurred earlier (0134 h compared to 0827 h) (
These experiments cast new light on circadian patterns of cytokines and hormones in a chronic inflammatory disease. We propose that these changes in IL-6 and cortisol, prior to the onset of morning joint stiffness, are functionally important in mediating the improvement in joint stiffness following prednisone in patients with RA.
Using a well-established non-human primate model of anxious temperament (AT) we characterized alterations in the neural circuit that underlie the dispositional risk to develop anxiety and depression. Genetic variation encoding the
In a large cohort of young rhesus monkeys (
Regarding
These data suggest that genetic variation in
Abnormalities of hypothalamic–pituitary–adrenal (HPA) axis in depression and suicide are among the most consistent findings in biological psychiatry. However, the specific molecular mechanism associated with HPA axis abnormality in the brain of depressed or suicidal subjects is not clear. It is believed that abnormal HPA axis is caused by increased levels of corticotropin-releasing factor (CRF) and decreased levels of glucocorticoid receptor (GR) in the brain of depressed or suicide subjects. To study their role in teenage suicide, we determined the protein and gene expression of CRF, CRF receptors, and GR in the prefrontal cortex (PFC), hippocampus, and amygdala of teenage suicide victims and teenage normal control subjects.
The postmortem brain samples were obtained from the Maryland Brain Collection at the Maryland Psychiatric Research Center, Baltimore, MD, USA. Samples were obtained from 24 teenage suicide victims and 24 normal teenage control subjects. Psychological autopsy was performed and the subjects were diagnosed according to the
We observed that the protein and gene expression of the CRF was significantly increased in the PFC (Brodmann area 9) and in amygdala, but not in the hippocampus, of teenage suicide victims compared with normal control subjects. The protein and gene expression of CRF-R1 was significantly decreased in the PFC and amygdala, but not in the hippocampus, of suicide victims. We also observed a significant decrease in the protein and mRNA expression of GR in the PFC and amygdala, but not in the hippocampus, of teenage suicide victims compared with control subjects.
These results thus indicate that suicidal behavior is associated with increased CRF and decreased GR in certain specific areas of the brain of suicide victims compared with controls.
Hypothalamic-pituitary-adrenal (HPA) axis activity remains a major focus for the study of the pathophysiology of anxiety and depressive disorders. Recent developments in genetics allow for potential new avenues for assessing risk and for developing new treatments. We will address recent studies on genetics of HPA axis dysregulation in a preclinical model of anxiety/depression, the brains of suicide victims, and severely ill delusional and nondelusional depressives. Last, the development of new glucocorticoid receptor (GR) antagonists that may prove useful as therapeutics in major psychiatric disorders is reviewed.
Ned Kalin will first present data on a stress model of anxiety/depression in rhesus monkeys. Over 400 monkeys were characterized on behavior and positron emission tomography imaging in response to an intruder and were genotyped for alleles for both corticotropin-releasing hormone receptors (CRH-R1 and CRH-R2). Shyam Pandey will report on gene expression for CRH-R1, CRH-R2, GR, and the mineralocorticoid receptor (MR) in multiple brain regions of adolescents who committed suicide and in matched controls who did not. In a sample of 122 subjects, Alan Schatzberg will present data on genetic variation differences in GR and CRH-R1 between severely depressed patients (delusional and nondelusional) and healthy controls as well as on the relationship of CRH and GR alleles to mean cortisol activity collected hourly from 6 PM to 1 AM as well as from 1 AM to 9 AM. Finally, Joseph Belanoff of Corcept Therapeutics will discuss the application of medicinal stereochemistry in the development of GR antagonists with greater GR specificity and organ selectivity than those currently available.
Associations between allelic variations in HPA axis genes and behavior were observed for CRH-R1 and CRH-R2 alleles in the rhesus monkey. Decreased message expression for GR and CRH-R1 was observed in key brain regions in suicide victims. Allelic variation for CRH_R1 and GR was associated with risk for severe depression and psychosis, and GR alleles were associated with elevated cortisol levels. A number of nonsteroidal GR antagonists have been synthesized and are active in various animal models.
HPA axis remains a potential source of diagnostic tests and innovative treatment.
Major depressive disorder (MDD) and panic disorder (PD) are common and disabling medical disorders with stress and genetic components. Dysregulation of the stress response of the hypothalamic–pituitary–adrenal axis, including the corticotrophin-releasing hormone (CRH) signaling via primary receptors (CRHR1 and CRHR2), is considered to play a major role for onset and recurrence in MDD and PD.
To confirm the association of CRHR1 and CRHR2 with MDD and PD, we investigated 12 single nucleotide polymorphisms (SNPs) in MDD patients (
The SNP rs110402 and rs242924 in the CRHR1 gene and the rs3779250 in the CRHR2 gene were associated with MDD. The SNP rs242924 in the CRHR1 gene was also associated with PD. The T-A-T-G-G haplotype consisting of rs7209436 and rs173365 in CRHR1 was positively associated with MDD. The T-A haplotype consisting of rs7209436 and rs110402 in CRHR1 was positively associated with MDD. The C-C haplotype consisting of rs4722999 and rs37790 in CRHR1 was associated with PD.
These results provide support for an association of CRHR1 and CRHR2 with MDD and PD.
Mifepristone, a potent glucocorticoid receptor (GR) and progesterone receptor (PR) antagonist, has recently become the first medication approved for the treatment of Cushing's syndrome, the archetypal illness of cortisol excess. Mifepristone is also being studied for the treatment of psychotic depression in a Phase 3 study and in numerous academic studies on diseases in which GR antagonism is thought to be potentially useful. In all cases, mifepristone utility is generated by its ability to block GR and its antagonism of PR is either irrelevant or troublesome. A selective GR antagonist may confer the same benefits of mifepristone while removing an important liability.
Data are provided from animal and human studies of mifepristone and animal studies of novel, selective GR antagonists in metabolic and psychiatric diseases.
Pre-clinical studies indicate that selective GR antagonists may potentially have the same clinical utility as mifepristone in blocking cortisol while eliminating the unwanted effects of progesterone blockade.
Generalized avoidance belongs to the core symptoms of a variety of anxiety disorders such as panic disorder or posttraumatic stress disorder. However, therapy for avoidance behavior still bears many obstacles. Even though exposure-based approaches are the method of choice, they suffer from inferior patient compliance. This can be ascribed to patients’ inability to stand the high emotional load experienced during the therapeutic sessions. The situation could be much improved if (1) learning about the safety of a feared situation could be enforced, while (2) the negative effect inherent to the exposure is decreased. This would allow for the number/duration of the exposure sessions to be restricted to a minimum, and at the same time, the emotional load of the therapeutic sessions could be dampened, with direct consequences on compliance rates. So far, however, most of the treatments with anxiolytic capabilities (e.g., benzodiazepines) lead to state-dependency or amnesia, with the consequence that safety learning is attenuated, if not completely blocked.
The role of the endocannabinoid system in fear relief and safety learning was investigated in numerous animal studies employing pharmacological and genetic approaches. Behavioral experiments involved classical fear conditioning and inhibitory avoidance learning, followed by extinction training and safety learning. Mice were treated with the CB1 receptor antagonist/inverse agonist SR141716 (3 mg/kg) or the endocannabinoid uptake/degradation inhibitor AM404 (3 mg/kg). Parts of the experiments were performed with conventional and conditional mice lacking expression of CB1 receptors either in the entire brain or in distinct neuronal populations.
Our studies revealed the following key findings: (1) Endocannabinoids play an essential role in acute fear relief, once the averseness of the test situation exceeded a certain threshold. (2) These effects are mediated via CB1 on glutamatergic nerve terminals. (3) The capacity of the endocannabinoid system is limited in highly aversive situations but can be reestablished by blocking of endocannabinoid uptake/degradation. (4) At the same time, signaling via CB1 on dopamine D1 receptor positive neurons contributes not only to acute fear relief but also to safety learning in an inhibitory avoidance task. (5) The efficiency of safety learning in this task can be improved and the risk of relapse of avoidance behavior can be reduced by pharmacological enhancement of endocannabinoid signaling.
Drugs promoting endocannabinoid signaling via CB1 receptors may represent a new class of compounds that combine the advantages of “happy pills” (in terms of fear and stress relief) with those of “smart drugs” (i.e., facilitated safety learning), thus increasing compliance rates and success of exposure-based therapies in anxiety disorders.
Endocannabinoids have been shown to be important for the regulation of multiple aspects of the stress response, although the neural circuits underlying this phenomenon are not well characterized. The amygdala is rich in cannabinoid receptors and endocannabinoid content and is well seated to integrate the role of endocannabinoid signaling to the regulation of the stress response. This series of studies sought to determine the roles of the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) in the amygdala with respect to both activation and adaptation of the stress response.
For the first two experiments, male Sprague-Dawley rats were employed. For acute activation of the stress response, a 30 min exposure to restraint stress was employed, while 9 days of 30 min restraint was employed to examine adaptation of the stress response. In the third study, C57Bl/6 mice (both wild type and those deficient in the AEA degrading enzyme fatty acid amide hydrolase, FAAH) were employed.
Exposure to acute restraint stress increased the hydrolytic activity of FAAH and decreased AEA content within the amygdala. Local administration of a FAAH inhibitor (10 ng) into the basolateral amygdala (BLA) reduced stress-induced corticosterone secretion, indicating that a FAAH-mediated loss of AEA signaling in the BLA contributes to activation of the stress response. Following 9 days of repeated restraint, the corticosterone response to stress habituated, and this adaptive response was reversed by local administration of AM251 (1 µg), a CB1 receptor antagonist, into the BLA. Consistent with this, repeated restraint stress caused an increase in 2-AG content within the amygdala, indicating that a recruitment of amygdalar 2-AG signaling is required for stress adaptation. Chronic stress exposure caused an increase in FAAH activity and a reduction in AEA content within the amygdala. FAAH deficient mice did not exhibit this reduction in AEA content and were similarly protected against the ability of chronic stress to cause dendritic expansion and spine growth within the BLA, as well as heightened indices of anxiety.
These findings indicate that AEA and 2-AG signaling at the CB1 receptor within the amygdala both serve to inhibit activation of the stress response. AEA appears to serve more of a tonic, gatekeeper role, the loss of which promotes activation of the stress response. Prevention of this loss of AEA signaling, through a blockade of FAAH activity, is capable of dampening the effects of acute and chronic stress. On the other hand, 2-AG signaling is recruited by repeated restraint stress to promote habituation and adaptation of the stress response. As such, a ying-yang model exists within the amygdala with the two endocannabinoid ligands serving different roles to regulate the stress response.
There is extensive evidence that glucocorticoid hormones impair the retrieval of memory of emotionally arousing experiences. Although it is known that glucocorticoid effects on memory retrieval impairment depend on rapid interactions with arousal-induced noradrenergic activity, the neurobiological mechanism underlying this presumably nongenomically mediated glucocorticoid action remains to be elucidated. Here, we show that the hippocampal endocannabinoid system, a rapidly activated retrograde messenger system, is involved in mediating glucocorticoid effects on retrieval of contextual fear memory.
For all three experiments, male Sprague-Dawley rats were trained on a hippocampus-dependent contextual fear-conditioning (CFC) task and retention was tested 24 h later. All drugs were administered 60 min before retention testing.
Systemic injections of corticosterone (3.0 mg/kg) impaired memory retrieval of CFC training (
These findings indicate that glucocorticoid-induced memory retrieval impairment depends on functional interactions between the endocannabinoid and noradrenergic systems.
The endocannabinoid system has been shown to be an important regulator of the stress response and adaptation to stressful situations and environments in animals. Little is known, however, about the role of this system in acutely and chronically stressed humans.
We developed an HPLC-MS-MS-based method to measure plasma concentrations of the ECs anandamide (ANA), 2-arachidonoylglycerol (2-AG), the N-acyl-ethanolamides palmitoylethanolamide (PEA), oleoylethanolamide (OEA), stearoylethanolamine (SEA), and N-oleoyldopamine (OLDA), determined leucocyte cannabinoid (CB) receptor mRNA and genotyped the CB receptor genes (CB1/CB2) for known single nucleotide polymorphisms in critically ill patients.
We used these methods in a number of studies in healthy volunteers, critically ill patients, and individuals with PTSD to delineate the relationship between peripheral EC signaling and the intensity of acute and chronic traumatic stress. In a first series of experiments, we exposed healthy volunteers (
These findings point to a possible involvement of the EC system in acutely and chronically stressed humans with traumatic memories and PTSD. Additional studies of EC signaling in PTSD patients before and after therapeutic interventions could lead to novel biomarkers and to further progress in the understanding of PTSD and the multiple biological and behavioral sequelae of this complex disorder.
Over the last decade, the neuropeptide oxytocin has attracted considerable attention for its crucial role in social behavior. Motivated by animal studies showing that oxytocin attenuates stress and anxiety in rodents, numerous studies have been conducted to investigate whether oxytocin has similar effects on stress and anxiety in humans. Most of these studies have revealed that oxytocin also attenuates stress and anxiety in humans, presumably by decreasing amygdala activity during the processing of threatening stimuli. However, these studies have almost exclusively been conducted in males, leaving open whether the observed effects can be generalized to females.
To investigate how oxytocin affects amygdala-dependent threat-processing in females, we used functional magnetic resonance imaging (fMRI) to measure females’ amygdala reactivity to threatening and nonthreatening faces (study one:
There were no differences in females’ gaze behavior during stimulus processing after oxytocin as compared to placebo administration. However, after oxytocin administration females showed more amygdala activity to threatening faces (study one) and scenes (study two) than after placebo administration.
Taken together, the present findings suggest that oxytocin enhances amygdala-dependent threat-processing in females, which is in sharp contrast with previous findings showing that oxytocin attenuates amygdala-dependent threat-processing in males. Although these findings point to a possible sexual dimorphism in oxytocin-mediated threat-processing, future studies are warranted to further address this issue, preferably by directly comparing oxytocin effects on threat-processing between males and females.
Despite numerous studies on the influence of psychosocial stress on hypothalamic-pituitary-adrenal axis (HPA) system responsivity, heterogeneous results have been found with regard to depression in remission. In addition, knowledge concerning cognitive functioning in the remitted state is also narrow showing thus far inconsistent results. The present study investigated the effect of psychosocial stress on the cortisol response and cognitive performance in patients recovered from depression in comparison to healthy controls.
Eighty patients who have recovered from depression for at least 6 months (average: 31 months) and 80 healthy matched controls were investigated on the effects of psychosocial stress (TSST) on the performance in an affective go/nogo task. Cortisol responses, behavioral inhibition, reaction time performance and emotional-cognitive functioning were analyzed. We hypothesized that stress vulnerability of cognitive performance is positively correlated to HPA system responsiveness (measured by salivary cortisol) in both healthy subjects and remitted patients but larger in remitted patients compared to healthy controls.
Thus far, preliminary analyses reveal no abnormal stress-associated HPA system response in patients recovered from depression in comparison to healthy controls. However, remitted patients showed impaired attentional set shifting in the go/nogo task. This impairment was positively correlated with the duration of illness.
Our study is the first to investigate affective go/nogo task performance and effects of a stress challenge test in patients recovered from depression. Our data demonstrate that attentional set shifting deficits are not only present during acute episodes but also in remission. These deficits seem to be correlated with the duration of illness. Nonetheless, restored stress-associated HPA system function suggests recovery of the HPA system reactivity to psychosocial stress in patients remitted from depression. This in turn suggests that the observed cognitive impairment is not mediated by abnormal HPA responses.
Cognitive impairment in the area of executive functioning may be considered a specific trait marker that persists after clinical and neuroendocrinological remission.
A relationship between exposure to sexual violence and thyroid hormone alterations have been observed among women with premenstrual dysphoric disorder (PMDD), as well as women with posttraumatic stress disorder (PTSD). Women with Borderline Personality Disorder (BPD) report a high estimate of childhood trauma. The aim of the present study was to assess relationships between thyroid hormone measures and exposure to violence in childhood in females with BPD.
Ninety-two clinically euthyroid women with BPD diagnosis and at least two prior serious suicide attempts in their history were assessed with the Karolinska Interpersonal Violence Scales (KIVS). The KIVS is a new structured interview, containing four subscales with concrete examples of exposure to violence and expressed violent behavior in childhood (between 6 and 14 years of age) and during adult life (15 years or older). In addition to serum cortisol, baseline thyroid functioning was evaluated by measuring plasma thyroid stimulating hormone (TSH), free and bound Triiodothyronine (T3) and Thyroxine (T4) levels, as well as the FT3/FT4 (free T3/ free T4) ratio, by immunoassays.
The FT3/FT4 ratio showed a significant negative correlation with exposure to violence as a child.
Altered thyroid activity, especially FT3/FT4 levels, was associated with exposure to violence in childhood in suicide attempters. Severe childhood trauma-related stress may promote lasting altered thyroid levels and/or contribute to the development of psychopathology associated with BPD traits, coming to the notice of psychiatric care.
Antenatal glucocorticoid (GC) exposure has been discussed as a potent programming factor of hypothalamus–pituitary–adrenal (HPA)-axis activity producing sustained alterations in cortisol secretion throughout life. So far, the assessment of HPA-axis activity in offspring of mothers treated with synthetic GCs has been limited to a time period shortly after birth, with prematurity being an important confound in most prior studies.
The present study aimed to investigate HPA-axis reactivity of term-born children with antenatal GC exposure in a larger sample (
We observed significantly increased cortisol reactivity to acute psychosocial stress in 6–11 years old, term-born children exposed to antenatal synthetic GC treatment compared to controls (
The present study provides first evidence for long-lasting effects of antenatal synthetic GC exposure on HPA-axis reactivity in term-born children. These findings may bear important implications regarding the vulnerability for stress-related physical and psychiatric disorders, for which dysregulation of the HPA-axis has been discussed as a potential causal factor.
Problematic peer relationships in adolescence have long been linked with various psychological disorders, but there remain questions as to why adolescents with similar social experiences may suffer no psychological effects or why some respond with depression or anxiety while others become aggressive. Parenting style and level of chaos in the home environment have also been shown to have protective or detrimental effects in conjunction with social stressors. Adolescence is typified by substantial hormonal changes and maturation of both the pubertal and the stress systems. Ian Goodyer has suggested that atypical ratios of stress and pubertal hormones may be indicative of vulnerability for psychopathology. High cortisol and low DHEAS have been linked to depression, whereas the opposite has been found in those with aggression. This study is the first to examine the cortisol/DHEAS ratio as a moderator of peer stress in the development of psychopathology in adolescents. This investigation uses a biopsychosocial model to test the moderating role of parenting style, environmental chaos, and adrenal hormone ratios on the association between social stress and aggression or depression over a 1-year period.
Participants were 156 young adolescents (50% f; M age = 11 years, SD = 0.7), ethnically diverse, and predominantly middle to lower SES. Depressive symptoms, aggression, social stress, and environmental chaos were assessed via survey and interview reports from mothers and children. Parenting characteristics were assessed via mother survey. Saliva and urine samples were collected on multiple mornings to measure cortisol and DHEAS, respectively.
Cross-sectional and longitudinal analyses indicate significant main effects of parenting style, chaos, and adrenal hormone ratios in predicting depressive symptoms and aggression and significant moderating effects on the relationship between social stressors and psychopathology. High cortisol/DHEAS ratio predicted depressive symptoms and enhanced the effects of peer problems; low ratio was predictive of aggression in adolescents with high levels of peer problems.
The results of this study shed light on factors that may better explain the varying responses adolescents have to social stressors, thereby identifying adolescents at risk for psychological problems.
Alcohol Use Disorders (AUDs) are multiphasic, multifactorial, and heterogeneous disorders for which the differential risk traits have been proposed to be associated with distinct risk profiles. However, whether these profiles are distinct in terms of neuronal and hormonal mechanisms remains less understood. Behavioral evidence has demonstrated differential motivational systems mediating the response to alcohol, two of which are the psychomotor/cue for reward and the anxiety systems that are in turn exemplified by sensation seeking (SS) and anxiety sensitive (AS) individuals, respectively.
Two equally divided groups of healthy social drinker AS and SS males and females (
Salivary cortisol secretion relative to ground (AUCg) was significantly different between groups and within subjects. Gender showed a significant main effect (
These findings provide evidence for the notion that distinct risk personality profiles are associated with differential vulnerability for AUDs. They further support the self-medication theory, whereby AS individuals drink to dampen stress, rendering the former a negative reinforcer targeting and inhibiting their neural and hormonal stress circuitry.
Oxidative stress (an imbalance between free radicals and the ability to neutralize them with antioxidants) occurs in several mental illnesses, including major depression (MDD). A major antioxidant in humans is glutathione peroxidase, which reduces GSSH to GSH, increasing glutathione's ability to scavenge free radicals. The brain, and the hippocampus (HC) in particular, is particularly sensitive to oxidative stress, and HC oxidative stress (particularly in the CA1 and CA3 & dentate gyrus [CA3&DG] subfields) may contribute to major depression.
Nineteen medication-free subjects with MDD and 19 matched controls underwent 4T MRI scanning of the HC and had fasting morning venipuncture for peripheral oxidative stress assessment. Two of the MDD subjects did not have glutathione (GSH) and/or glutathione disulfide (GSSG) data. Because of the preliminary nature of the study, no corrections for multiple comparisons were applied.
Across all subjects, the antioxidant Vitamin C was directly correlated with total HC (
These exploratory data are consistent with the hypothesis that oxidative stress is related to diminished hippocampal volume, with the CA3&DG subfield perhaps being the most sensitive. The relationship of peripheraloxidative stress to local oxidative stress in the HC is unknown, but studies in humans have suggested some degree of direct correlation between blood and cerebrospinal fluid (CSF) oxidative markers, and peripheral oxidative stress measures are increased in several neurodegenerative diseases.
There is evidence that acute stress impacts decision making (DM) under risk. It has been concluded that stress prompts riskier decisions in men. However, in the DM tasks used thus far, the expected value (EV) of reward and risk of decision options are confounded and it is, therefore, unclear which component is being affected by acute stress.
We developed a new DM paradigm, in which EV of reward and risk of decision options are independent and quantifiable. Subjects (5 men, age: 31.2 ±1.92 years) completed 220 trials in which they had to repeatedly choose between a safe and a risky option associated with different EV of reward and risk. Stress was induced using the Socially Evaluated Cold Pressor Test (SECPT). Each subject received the SECPT and the corresponding control condition in random order.
Comparing the stress and control condition on a trial-by-trial basis, we found that, descriptively, gamble variance, a measure for the risk associated with decision options, was about 10% higher when subjects were stressed compared with when they received the control manipulation. EV of reward on which subjects gambled did not differ between stress and control manipulation.
Our data provide a first hint that risk but not reward processing in healthy young men might be affected by acute stress.
Obesity and the metabolic syndrome (MetS) predispose to multiple diseases and to accelerated cell aging as indexed by accelerated shortening of telomeres in peripheral blood mononuclear cells (PBMC's). Major depressive disorder (MDD) is often associated with MetS and is also associated with increased disease risk and PBMC telomere shortening. A potential role of leptin in telomere shortening has been suggested, but prior results have been inconsistent and no study has yet assessed this relationship in MDD. The goal of this study was to assess the relationship between serum leptin concentrations and PBMC telomere length in MDD and in controls and to assess whether this relationship is mediated by body-mass index (BMI) or the homeostatic model assessment of insulin resistance (HOMA-IR), two principal components of the MetS.
Eighteen medication-free MDD subjects (11 female, 7 male, mean age 37.1 + 2.7 years) and 17 healthy controls (11 female, 6 male, mean age 37.8 + 3.0 years) had blood drawn for assay of fasting morning levels of leptin, glucose, and insulin and PBMC telomere length. The groups did not differ on BMI (24.66 + 3.72 vs. 24.77 + 4.29, respectively, n.s.). Analyses were co-varied for age and sex, with and without BMI.
In the combined group, serum leptin concentrations were inversely correlated with telomere length (
Relatively high leptin concentrations, in the presence or absence of increased BMI and insulin resistance, may be a risk factor for telomere shortening. While this was demonstrated here in individuals with MDD, a similar relationship in non-depressed individuals cannot be ruled out because of the small sample size.
Affected autonomic heart regulation is implicated in the pathophysiology of cardiovascular diseases and is also associated with posttraumatic stress disorder (PTSD). However, although sympathetic hyperactivation has been repeatedly shown in PTSD, research has neglected the parasympathetic branch. The objective of this study is the long-term assessment of heart rate (HR) dynamics and its circadian changes as an index of autonomic imbalance in PTSD. Since tonic parasympathetic activity underlies long-range correlation of heartbeat interval fluctuations in healthy state, we included nonlinear (unifractal) analysis as an important and sensitive readout to assess functional alterations.
Electrocardiogram recordings over a 24-h period were conducted in 15 deployed male subjects with moderate to high levels of combat exposure (PTSD:
Subjects with PTSD showed significantly higher baseline HR, higher LF/HF ratio in frequency domain analysis, blunted differences between daytime and nighttime measures, as well as higher scaling coefficient αfast during the day, indicating diminished tonic parasympathetic activity.
This study appears to be the first combining linear and non-linear methods to assess long-period autonomic and circadian differences in HR dynamics between combatants with and without PTSD. Diminished circadian differences and blunted tonic parasympathetic activity altering HR dynamics suggest central neuro-autonomic dysregulation that could represent a possible link to increased cardiovascular mortality in PTSD.
Rumination, defined as past-centered negative thinking, has been linked to stress physiology and suggested to affect mental and physical health. Research has shown that both state and trait rumination is correlated with cortisol responses to psychosocial stress. It has not been addressed if state rumination is associated with cortisol responses to repeated stress.
Nineteen participants (aged 21–65, mean age = 53.5; nine males) were exposed to the Trier Social Stress Test (TSST) twice on consecutive days. Salivary cortisol was measured 1 min before and 1, 10, 30, 60 and 120 min post-TSST on both days. Participants provided self-reports of post-stress state rumination on both days. Participants further provided information about early adversity using the Childhood Trauma Questionnaire and self-rated depression and perceived chronic stress.
Cortisol responses were successfully induced on both days of testing (
Post-stress rumination on day 1 was correlated with hypothalamic–pituitary–adrenal (HPA) axis reactivity. Day 1 post-stress rumination was correlated with day 2 responses to the same stressor, but day 2 rumination was unrelated to the stress response on that day. This suggests that rumination has prolonged effects on stress reactivity. Other variables, such as subclinical childhood trauma, were also related to state rumination and cortisol responses. These factors are potential mediators of the relationship between state rumination and HPA axis stress reactivity.
Cortisol levels have been extensively studied in patients with posttraumatic stress disorder (PTSD), but their specific relationship to intrusive memory symptoms is unknown. Salivary alpha-amylase (sAA), an index of sympathetic activation, has never been studied in the context of PTSD. This study adopted the Trauma Film Paradigm to assess how changes in cortisol and sAA levels during memory encoding are related both to subsequent intrusive memories of the film and to individuals’ pre-existing characteristics.
Saliva samples were collected in the afternoon (considering the circadian rhythm of cortisol and sAA) from 58 healthy adult participants at baseline, during the film, and post-film. Measurements of pre-existing PTSD symptoms, dissociation and anxiety traits as well as intrusions of the traumatic film over the week following film viewing were assessed.
Results showed that cortisol levels increased, whereas sAA levels decreased in response to the film. The vividness of intrusive memories was negatively correlated with cortisol levels during and after the film. Pre-existing PTSD symptom severity was negatively correlated with cortisol levels at the post-film stage and positively correlated with sAA in both during the film and post-film stages. Moreover, dissociative traits (especially dissociative amnesia) were negatively correlated with sAA levels at baseline and during the film, while anxiety traits were positively correlated with post-film sAA levels.
This is the first study to investigate the relationship between cortisol, sAA, intrusive trauma memories and pre-existing psychological traits. The results supported the hypothesis that insufficient cortisol release in the immediate aftermath of trauma is a risk factor for the development of intrusive symptoms. The findings also shed light on how pre-existing characteristics affect physiological reactions to traumatic stimuli.
Currently, there are no effective interventions that prevent the development of posttraumatic stress disorder (PTSD) in recently traumatized individuals. The neuropeptide oxytocin is a potent regulator of two important processes disturbed in PTSD: it regulates physiological and behavioural stress and fear responses. In addition, oxytocin administration influences socio-emotional processes. Interestingly, high levels of acute distress after trauma and a lack of social support are risk factors for developing PTSD. Therefore, oxytocin administration appears to be a promising preventive treatment for PTSD, by hypothetically ameliorating dysregulated stress and fear responses as well as facilitating adaptive social functioning.
We have initiated a randomized controlled trial (RCT) to investigate the effectiveness of an intranasal oxytocin treatment regimen in preventing the development of PTSD in recently traumatized individuals at increased risk for PTSD. In addition, in the same population we are conducting an fMRI study, which will create deeper insights into the neural mechanisms through which oxytocin and social context may regulate fear responses to traumatic stress.
In this presentation, the rationale behind stimulation of the oxytocin system in recently trauma-exposed individuals at risk for PTSD will be discussed, and an outline of the RCT will be presented. In addition, preliminary pilot data of the RCT will be shown.
In a recent study, we unexpectedly found lower fasting plasma glucose concentrations in patients with stress-related exhaustion compared with healthy controls. To further elucidate the reliability of these findings we now investigated possible differences in glucose and glycated haemoglobin (HbA1c) levels between all patients with Exhaustion Disorder (ED) that entered the treatment program at the Institute of Stress Medicine, Gothenburg, Sweden between 2004 and 2010 and a healthy control population. We also investigated the development of plasma glucose during 18 months of multimodal treatment and related it to changes in symptoms of burnout, depression and anxiety.
The study included 383 patients (71% females, age 21–66 years) and 199 healthy controls (50% females, age 25–54 years). All patients fulfilled the criteria for ED, which include physical and mental exhaustion experienced for at least two weeks, caused by exposure to one or more stressors for a minimum of six months. Cardinal features are markedly reduced mental energy, impaired memory and reduced capacity to meet demands.
Blood samples were drawn in the morning after fasting since 22:00 the day before. Follow-up measurements were performed after 3, 6, 12, and 18 months of treatment in the patient group.
Fasting plasma glucose was significantly lower in the patients (4.7±0.4 mmol/L) compared with healthy controls (5.0±0.5 mmol/L), both in women and men. HbA1c did not differ between patients and controls. These results remained after controlling for age, BMI, WHR, physical activity, and antidepressant use.
In the patient group, plasma glucose levels increased significantly from inclusion to the follow-up measurements after 12 and 18 months. Changes in glucose during treatment were not related to improvement of symptoms of depression, anxiety or burnout.
We confirm our previous finding that plasma glucose levels are lower in patients with stress-related exhaustion compared with healthy controls. The increase during treatment could indicate that lower level of glucose might be a consequence of long-term stress, which is normalised during treatments. Further studies are needed to confirm if this is the case and whether this relatively small difference in glucose levels is of clinical relevance.
Deep brain stimulation (DBS) is an effective treatment for obsessive-compulsive disorder (OCD), but its mechanism of action is largely unknown. Since DBS may induce rapid symptomatic changes and the pathophysiology of OCD has been suggested to be related to the hypothalamic-pituitary-adrenal (HPA)-axis, we set out to study whether/how DBS affects the HPA-axis in OCD patients.
We studied 16 therapy-refractory OCD patients treated with DBS of the accumbal area for at least 1 year in an “on” and “off” stimulation phase, with a 1-week interval. We measured 24-h urinary excretion of cortisol, adrenalin, and noradrenalin as well as obsessive-compulsive (Y-BOCS), depressive (Ham-D), and anxiety (HAM-A) symptom scores.
Eight patients who completed the study were included in the final analysis. The comparison between DBS on and off phase revealed a change in Y-BOCS (39%), HAM-D (78%), and HAM-A (56%) scores. Median cortisol levels increased by 53% in the off phase, from 93 to 143 nmol/24 h, and correlated strongly with Y-BOCS and HAM-D changes. There was no significant change in urinary adrenaline or noradrenaline excretion.
Our findings indicate that symptom improvement in DBS for OCD patients is associated with changes in cortisol levels.
Lesbian (L), gay (G), and bisexual (B) individuals frequently report heightened distress due to discrimination, yet investigations into their physiological stress responsivity are missing from the literature. Our group recently showed that disclosing one's sexual orientation corresponds with comparatively lower psychiatric symptoms and morning cortisol levels than those who remain “in the closet.” Extending from our earlier studies, the current study investigated whether sexual minorities might manifest differential cortisol levels than heterosexuals (Hs) in response to social-evaluative threat.
Participants included 87 healthy adults (mean age 25, 54% men) identifying as L/G/B (
Results reveal that L/B women had higher cortisol levels than Hs women 40 min after stress exposure. As a group, G/B men had significantly lower cortisol levels in contrast to Hs men. The covarying effects of age, self-esteem, and disclosure status intermittingly contributed to time and group effects for both sexes.
Our findings demonstrate that relative to Hs controls (1) L/B women displayed higher cortisol levels late after TSST exposure, whereas (2) G/B men displayed lower overall cortisol levels throughout testing. We previously reported that G/B men in our sample manifested lower depressive symptoms and allostatic load based on 20 biomarkers compared to Hs men. It is possible that G/B men who are able to successfully overcome stigma may be resistant to chronic stress and stress reactivity. Yet, the opposite might be true for L/B women who displayed heightened distress during recovery that may indicate ruminative processes. These results suggest that it is important to include intrasex variations such as sexual orientation as well as unique developmental challenges such as disclosure processes in future psychoneuroendocrine studies.
A few weeks ago and for the first time in 20 years, US health officials have lowered the threshold for lead (Pb) poisoning from 10 to 5 g/dL in blood, but only in young children. However, elders are also a high-risk population when considering adverse effects of lead exposure. The association between low-level lead exposure and cognitive variability is well documented in elderly people, e.g. in the domain of attention and memory. Toxicokinetic studies have also demonstrated that the skeleton is the site of storage for around 95% of lead in the adult human body, resulting in a release of lead in blood in elderly people with bone demineralization. One potential mechanism explaining adverse health effects of lead exposure stands in its endocrine disrupting function, and a recent study has found significant associations in children between low-lead levels and cortisol reactivity to the cold pressure task. We hypothesized that this association between lead exposure and hypothalamo–pituitary–adrenal functioning could be observed in elders from the general population.
Pb levels were determined from blood samples of 78 elderly individuals (mean age = 58.37, SE = 4.01) without previous occupational Pb exposure. Diurnal cortisol was measured using salivary cortisol samples collected at home over two working days at awakening, 30 min after waking, 14: 00 h, 16: 00 h and before bedtime (∼10 pm) periods. Salivary cortisol reactivity was assessed in response to the Trier Social Stress Test (TSST).
All participants showed blood Pb levels below the threshold limit recommended by the CDC with a mean Pb of 2.6 g/dL, SE = 1.4. No association was found between lead exposure and diurnal cortisol activity (
Lead levels, even at a very low level of exposure, are associated with a blunted cortisol response to the TSST. These findings support the relationship between environmental contaminants and stress, and support the idea that regulation should be applied to the aging population. The next step will be to determine whether the association between lead exposure and cognitive variability could be explained by impairment of the stress system.
Early life stress restructures the nervous system. In rodents, the level of maternal care causes lifelong differences in central glucocorticoid (GC) sensitivity and memory. Furthermore, human adults with a history of child abuse have decreased hippocampal GC receptor gene expression and lower cortisol responses to stress. As GCs modulate memory, hypothalamic–pituitary–adrenal (HPA) axis functions altered by atypical care may influence memory.
Participants were women (
Abuse history moderated cortisol's effect on gist memory,
These findings are particularly compelling as the abuse group result contrasts with previous human memory and rodent research. However, basal vs. stress-induced cortisol may differentially affect memory. Furthermore, child abuse is likely a more profound early stressor compared to maternal neglect in rodents. In conclusion, early experience shapes how GCs affect cognitive functioning. This study is an essential step toward determining physiological and long-term effects of child abuse.
Retrospective studies show that childhood adversity is associated with systemic inflammation in adulthood. Few prospective studies have examined whether childhood adversity influences inflammation in an observable manner during childhood or adolescence and whether these effects are sustained over time.
Using longitudinal data from the Avon Longitudinal Study of Parents and Children, we examined associations between acute adverse events at seven time points prior to age 8 and inflammation at ages 10 and 15. Inflammatory markers at age 10 included interleukin-6 (IL-6;
Adverse events in middle childhood (occurring between ages 6 and 8), as well as cumulative adversity between the ages of 1.5 and 8 years, were associated with higher levels of IL-6 and CRP at age 10. Adverse events occurring in early childhood (age 1.5) or middle childhood (age 8), and cumulative adversity between the ages of 1.5 through 8 years predicted increased levels of CRP at age 15, and these associations persisted after adjustment for CRP at age 10. Some, but not all, of these associations were mediated by BMI.
This study documents that exposure to adverse events prior to age 8 is associated with elevated inflammation at age 10 and in mid-adolescence. These findings provide prospective evidence for a biological mechanism by which early experiences may shape long-term health.
In posttraumatic stress disorder (PTSD), enhanced negative feedback of the hypothalamic–pituitary–adrenal axis is a prominent finding, which has often been interpreted in the context of enhanced glucocorticoid receptor sensitivity. Neuropsychological alterations are also an important feature in PTSD. Problems particularly with learning and memory have been found, including deficits in verbal declarative memory as well as autobiographical memory. In healthy humans, most studies suggest impairing effects of glucocorticoids on memory retrieval. Up to now, studies that investigate the effects of cortisol administration on memory in patients with PTSD are rare and yielded inconclusive results.
In a placebo controlled cross-over study, we compared the effect of exogenous cortisol on memory retrieval in patients with PTSD (
Opposing effects of cortisol on memory were observed when comparing patients with controls. In controls, cortisol had impairing effects on memory retrieval, whereas in patients with PTSD cortisol had enhancing effects on memory retrieval.
The present results suggest beneficial effects of acute cortisol elevations on hippocampal mediated memory processes in PTSD. Possible neurobiological mechanisms underlying these findings are discussed.
Accumulating evidence links posttraumatic stress disorder (PTSD) with elevated inflammatory activity. However, the clinical significance of this association is unclear. Although inflammation could increase the risk of autoimmune disease, little is known about whether patients with PTSD are at increased risk of developing autoimmune disorders.
We conducted a retrospective cohort study of 673,277 Iraq and Afghanistan veterans younger than 55 years, who received VA healthcare from October 1, 2005, to March 31, 2012, with at least 1 year of follow-up. Department of Veterans Affairs administrative data were used to identify ICD-9 codes for mental health and autoimmune disorders and to obtain sociodemographic, military service, and health service utilization information. Generalized linear models were used to ascertain the association of PTSD with subsequent autoimmune diagnoses after adjusting for age, race, and number of primary care visits.
The sample was 88% male and 49% white with a mean age of 31.3 years (±8.7). PTSD was diagnosed in 206,623 (31%) veterans, and mental health disorders other than PTSD were diagnosed in an additional 132,242 (20%) veterans. Compared to veterans with no mental health diagnoses, those diagnosed with PTSD had increased risk for subsequent diagnosis with thyroiditis (adjusted relative risk [ARR]=1.74; 95% CI 1.67, 1.82), rheumatoid arthritis (ARR=1.92; 95% CI 1.67, 2.20), inflammatory bowel disease (ARR=1.32; 95% CI, 1.20, 1.46), multiple sclerosis (ARR=2.23; 95% CI, 1.88, 2.64), systemic lupus erythematous (ARR=1.81; 95% CI, 1.48, 2.23), and any of these disorders alone or in combination (ARR=1.50; 95% CI, 1.45, 1.56). Moreover, while there was an increased risk for each of these disorders in veterans with mental health disorders other than PTSD, the risk was consistently higher in those diagnosed with PTSD. Women had significantly higher risk for autoimmune disorders overall, but the pattern of results was similar in men and women.
Veterans with PTSD appear to be at increased risk for autoimmune disorders compared to those with no or other mental health diagnoses. Future prospective longitudinal cohort studies are needed to establish causality, measure inflammatory markers in conjunction with PTSD, and evaluate whether successful treatment of PTSD reduces risk of autoimmune disorders.
Neuropeptide Y (NPY) is a peptide with behaviorally relevant effects on the hippocampus and is thought to function as an endogenous anxiolytic. In prior work, we reported that veterans who had recovered from combat-related post-traumatic stress disorder (PTSD) had higher levels than those who were not combat exposed. NPY levels were significantly associated with the extent of symptom improvement, suggesting that plasma NPY levels may represent a biological correlate of resilience to, and/or recovery from, the adverse effects of trauma exposure. Cytosine methylation of the glucocorticoid gene (GR methylation) has been associated with PTSD risk and/or symptom expression. GR methylation is influenced by environmental factors that can result in enduring differences in function, including neuroendocrine regulation. As the NPY gene has glucocorticoid response elements, levels of circulating NPY represent a potential indicator of alterations in GR responsivity.
The relationship of NPY to PTSD and GR methylation was examined in two samples. In the first sample, veterans who developed PTSD following combat exposure were compared to those who did not develop PTSD. In a second sample, veterans with combat-related PTSD were assessed prior to and following a course of prolonged exposure (PE).
In the cross-sectional study, veterans with PTSD had higher NPY levels than those who never developed PTSD (
To the extent that improvement from symptomatic PTSD may involve a mobilization of endogenous mechanisms to reduce hyperarousal and other post-trauma
Nearly 34–65% of Gulf War veterans (GWV) continue to suffer from chronic multisymptom illness (CMI); novel pharmacological treatment approaches are needed to improve the health of these veterans. This study aims to determine whether mifepristone, a glucocorticoid receptor antagonist, can reverse the neuroendocrine alterations described in GWV and improve the physical health, mental health, and neurocognitive functioning of GWV with CMI.
Sixty-five GWV were enrolled into the study; 36 eligible GWV who met criteria for CMI and did not have any exclusionary medical or psychiatric conditions were randomized to receive mifepristone (200 mg/day) or matched placebo first in this crossover study. Both treatment phases lasted 6 weeks and were separated by a 4-week wash-out period. The primary clinical outcome measure was the change from treatment baseline to treatment endpoint in the physical health component score (PCS) of the veterans SF-36 health survey. Primary neurocognitive outcome measures included change in spatial working memory and verbal declarative memory as measured by the MATRICS Consensus Cognitive Battery. Additional outcome measures included change in the mental health components score (MCS) of the SF-36 and self-reported symptoms of fatigue, depression, and PTSD. Cortisol and ACTH levels and a measure of glucocorticoid sensitivity (lysozyme IC50-DEX) were also obtained to characterize the neuroendocrine response to mifepristone in GWV with CMI.
Data collection is complete; results regarding the primary and secondary clinical, neuropsychological, and neuroendocrine outcome measures will be presented.
If this study shows that mifepristone improves physical health or cognition or reduces constituent symptoms of CMI in GWV, it would suggest that mifepristone may be of therapeutic value in this population.
Evidence for distinct biological perturbations in posttraumatic stress disorder (PTSD) with severe child abuse versus no child abuse: consequences for robust biomarkers for PTSD. The identification of biomarkers for PTSD has been difficult, likely due to inter-individual differences in genetic risk factors and environmental exposures. The aim of the current study was to interrogate the influences of the environment on gene expression profiles by characterizing biological differences in PTSD after severe child abuse versus PTSD after adult trauma.
A total of 396 trauma-exposed individuals were included in this study. The PTSD symptomatic scale (PSS), clinician-administered PTSD scales (CAPS), childhood trauma questionnaire, and trauma events inventory were used to assess current clinical PTSD and childhood and adult trauma severity. Whole blood gene expression and DNA methylation was measured on Illumina Human-HT12v3 and Human Methylation 450k arrays. Analysis was performed by using R software.
Of 741 transcripts significantly associated with current PTSD severity, only 2% was associated with PTSD in both, individuals exposed to child abuse and adult trauma (
These data suggest that PTSD occurring after severe child abuse is biologically distinct from PTSD after adult trauma, therefore, accounting for different environmental variables is crucial for identification of biomarkers for PTSD.
Pain and posttraumatic stress disorder (PTSD) are highly comorbid conditions. Patients with chronic pain have higher rates of PTSD. Likewise, patients with PTSD are often diagnosed with numerous chronic pain conditions. Despite the high pain-PTSD comorbidity, the pathophysiologic mechanisms underlying this phenomenon are incompletely understood and only recently researchers have started to investigate pain-PTSD overlap using experimental pain models. The aim of the present study was to examine the activation of the pain-processing pathway in a cohort of combat PTSD compared to combat controls in response to a prolonged painful stimulus.
Novel data from the experimental pain model using intramuscular capsaicin comparing a group of 10 Operation Enduring Freedom/Operation Iraqi Freedom (OEF/OIF) veterans with combat-related PTSD and 11 matched OEF/OIF veterans without PTSD will be presented. Intramuscular capsaicin causes a prolonged, deep-aching muscle pain, resembling pain associated with chronic pain states. Following capsaicin injection into the left thigh muscle, subjects underwent functional magnetic resonance imaging scanning while whole brain perfusion was measured with arterial spin labeling (ASL). At every 10 min, samples of cerebrospinal fluid (CSF) were drawn and subjective pain ratings were measured throughout the scanning window (30 min). Following scanning, CSF draws and pain ratings continued and evidence for central sensitization was assessed by temporal summation of repeated pressure pain stimuli.
Our results show evidence for an acute form of central sensitization in the PTSD group in comparison to matched combat controls. The maximum pain response and initial pain decrease were not different between the two groups, yet significantly higher pain ratings were observed in the PTSD group 15 min postinjection of capsaicin. ASL showed significant group by time interactions within pain-processing network, whereby PTSD group maintained high levels of brain perfusion in the ventral medial frontal gyurs and other interoceptive and evaluative brain circuits throughout the second half of the scan, similar to subjective pain ratings. Furthermore, significantly higher temporal summation of pain was also noted in the PTSD compared to the control group.
We found increased sensitivity to prolonged, deep experimental pain in combat-related PTSD compared to traumatized subjects who never developed PTSD following combat. We posit that this increased pain response on behavioral, spinal, and supraspinal levels is related to a form of acute central sensitization in these individuals in response to a prolonged pain stimulus. Initial neuroimaging findings point to differential activation of frontal systems as potentially underlying pain-PTSD pathways and perhaps provide initial mechanisms for the development of testable models of perturbed pain processing in PTSD.
The gradual emergence of symptoms following exposure to traumatic events has presented a major conceptual challenge to psychiatry. The presumption that all people have significant acute symptoms is not supported by careful longitudinal research. One such study of 1,018 accident victims conducted over 2 years will be presented. The mechanism that leads to the progressive escalation of symptoms with the passage of time leading to delayed onset post-traumatic stress disorder (PTSD) involves the process of sensitisation and kindling. The acute stress response represents the neurobiological platform for the different trajectories of symptoms, and data on 49 participants will be presented, demonstrating how the acute hypothalamic–pituitary–adrenal axis reactivity and melatonin levels predict later symptoms.
The development of traumatic memories at the time of stress exposure represents a major vulnerability through repeated environmental triggering of the increasing dysregulation of an individual's neurobiology. An increasing body of evidence demonstrates how the increased allostatic load associated with PTSD. This broader perspective has important implications for developing treatments that address the underlying dysregulation of cortical arousal and neurohormonal abnormalities, following exposure to traumatic stress.
Results will be presented on a prospective longitudinal study of risk and resilience for post-traumatic stress disorder (PTSD) symptoms in 400 police academy recruits, assessed during academy training and followed during the first 7 years of police service.
Utilizing Latent Growth Mixture Modeling (LGMM) we have established three symptom trajectories, highly resilient, initially distressed with gradual improvement, and increasing distress.
We will present findings on the relations of the following predictors ascertained during academy training to the three PTSD symptom trajectories: I.Q., family histories of anxiety, depression, alcohol and drug abuse, neuroticism, personal histories of childhood or adolescent traumatic exposure, levels of awakening cortisol, fear-potentiated acoustic startle, MHPG and cortisol responses to a critical incident video challenge, sleep quality as measured by actigraphy, and candidate polymorphisms including serotonin transporter (SLC6A4), adrenergic pathway genes, ADRB1, ADRB2, ADRA2C, brain derived neurotrophic factor gene (BDNF), genes for several critical components of the hypothalamic–pituitary–adrenal (HPA) axis such as the glucocorticoid receptor (NR3C1), CRH receptor 1 (CRHR1), and FK506 binding protein 5 (FKBP5) and Catechol-
Mulivariate models of risk and resilience will be presented utilizing a multinomial logistic regression nested in the unconditional LGMM.
PTSD is associated with changes in the glucocorticoid receptor (GR) pathway. We hypothesized that altered functioning of the GR pathway is already present before development of PTSD and thus represents a biological vulnerability factor for the development of PTSD. Therefore, we investigated the predictive value of several GR pathway components for the development of high levels of PTSD symptoms.
We included a cohort of 1,032 Dutch soldiers prior to deployment to Afghanistan. GR pathway components were assessed in blood collected prior to deployment. PTSD symptoms were assessed 6 months after return.
A high GR number, high GILZ mRNA expression, and low FKBP5 mRNA expression in leukocytes prior to deployment were independently associated with development of high levels of PTSD symptoms. In addition, sensitivity of T-cells for regulation by the synthetic glucocorticoid dexamethasone was associated with development of high levels of PTSD symptoms. However, the direction of the association between dexamethasone-sensitivity and PTSD depended on the presence of co-morbid depressive symptoms.
Altered functioning of the GR pathway in leukocytes is a vulnerability factor for development of high levels of PTSD symptoms. The identification of such biological vulnerability factors for PTSD could facilitate the selection of individuals for preventive treatment within groups at risk for trauma-exposure.
There is an urgent need to develop biological and behavioral predictors of PTSD risk/resilience in individuals with high trauma exposure, such as active military duty. First, we will briefly review psychophysiological risk factors for PTSD. Second, we will describe preliminary data from a prospective study of active duty Marines examining psychophysiological responses before and after deployment to Iraq or Afghanistan. Third, we will discuss our cross-species work in animal models of PTSD risk/resilience to inform these study findings.
This study was conducted as part of a 4 h battery (clinical, psychosocial, laboratory, and psychophysiological assessments) conducted both before, and 3 and 6 months after deployment (Marine Resiliency Study) in >2,500 Marines. Here, we examined (1) effect of deployment overall on physiological reactivity measures on baseline startle, pre-pulse inhibition, and affective modulation of startle and (2) comparison of pre-deployment startle reactivity across subjects matched for combat exposure with and without PTSD symptoms, 3 months post-deployment.
We observed small but significant increases in baseline startle and pre-pulse inhibition after deployment. Startle potentiation to aversive images was also significantly increased after deployment. Importantly, baseline startle magnitude
These results support previous reports suggesting that startle reactivity may probe trait biological processes that confer risk for PTSD symptoms. To complement these findings, we (1) are conducting a similar prospective study to determine if fear conditioning and extinction performance predicts deployment-related stress disorders and (2) have developed a homologous rodent model to aid identification of potential epigenetic mechanisms underlying psychophysiological and fear-processing risk factors.
Our insight into the neurobiological dynamics underlying the processes that may over time cumulate into syndromes like burn-out and depression is rapidly developing. A recent, though important, step has been to combine the relevant parameters of multiple domains (physiological, endocrine, social/emotional) to optimize prognostic accuracy. This is of relevance as initially subtle neurobiological disturbances associated with stress may indicate the start of a negative and potentially dangerous trend, both for physical and psychological health.
On the basis of the regular monitoring of key variables of allostatic processes (like heart rate variability, corticosteroid concentrations, and psychosocial status), risks scores for internalizing development can be calculated. When repeatedly collected by means of a standardized assessment protocol, it becomes possible to conduct trend analyses, which may potentially indicate development towards aversive outcomes like burn-out (in labour environments) or for example depression.
On the basis of available data, algorithms have been developed combining diverse allostatic key variables into multi-level prognostic models (low–medium–high risk for internalizing development). On the basis of these models, a standardized assessment protocol is developed using state-of-the-art information technology to make the application as consumer friendly as possible.
Although some technical developments are necessary to optimize the potency of assessment protocols like the present one (e.g., sensor technology able to measure or estimate corticosteroid concentrations “on the spot”), the used algorithms do not only seem to provide valid prognostic information, though essential indicators for easy to apply preventive strategies as well. These could be instrumental in averting long-term negative psychological outcomes.
The self-help book industry is one of the most lucrative in North America generating profits of $10 billion annually. The main purpose of self-help books is to increase the sense of worth of the readers as well as to provide them with adequate coping strategies, so they can better negotiate their stress. Despite the popularity of this literature, no study has investigated whether it impacts on people's stress reactivity. Consequently, the goal of this study was to compare consumers and non-consumers of self-help books with regard to their physiological stress response.
Thirty-one healthy men and women aged between 18 and 65 took part in this study. Of this group, 16 reported being consumers of self-help books, whereas the other 15 participants reported not being consumers nor attracted by these books. During their afternoon visit to the laboratory, all participants were exposed to the Trier Social Stress Test, a validated psychosocial stressor. Salivary samples were taken throughout the session in order to quantify their cortisol levels. Participants also filled out different questionnaires assessing self-esteem, depressive symptomatology and personality traits.
In terms of stress reactivity, the area under the curve with respect to increase was significantly higher in consumers when compared to non-consumers. The two groups did not differ from each other in terms of depressive symptomatology and self-esteem. The consumer group scored lower on the “extraversion” personality trait compared to the non-consumer group.
Healthy consumers of self-help books are more stress reactive when facing a psychosocial stressor than non-consumers of self-help books. Although the current study design does not allow concluding about the efficacy of these books, the results nonetheless suggest that further investigation about the impact of this literature is necessary. Moreover, given the considerable amount of consumers of self-help books and their poor ability to cope with stress, there is clearly a need of increasing public awareness about effective coping strategies.
In adults, there is a robust, immediate effect of sleep on the diurnal cortisol profile. Shorter sleep duration and poorer sleep quality are associated with greater awakening response, flatter diurnal slope, and higher evening cortisol levels. Because of methodological limitations, this relation is less well-established in children and adolescents. Specifically, the use of single cortisol samples and sampling at unconventional times limit the generalizability of these findings. This study examines the influence of sleep duration, sleep quality, and daytime sleepiness on the diurnal cortisol profile in children and adolescents.
Children and adolescents aged 8–18 (
After controlling for age and day of the week, higher bedtime cortisol was associated with shorter sleep duration (
Poorer sleep quality, greater daytime sleepiness, and shorter sleep duration were related to higher bedtime cortisol. Poorer sleep quality and greater daytime sleepiness were associated with higher AUCTG and AUCI, respectively. While child-report measures of sleep were associated with cortisol, parent-report measures were not. Current findings offer insight into possible pathways linking sleep and health. Future studies should further elucidate this association by examining objective measures of sleep.
The maintenance of cardiovascular and cerebrovascular health is based on a complex relationship between the heart and the brain. While some responses to stress are vital for survival, mental stress has also been claimed to cause cardiovascular disease. The Japanese observation from the early 1990s of a reversible stress-induced cardiomyopathy, the takotsubo cardiomyopathy (TC), a peculiar type of left ventricular (LV) dysfunction triggered by an acute strong emotional or physical stressor, supports this notion. The syndrome, mostly affecting postmenopausal women, presents signs and symptoms of acute coronary syndrome without evidence of obstructive coronary artery disease. Though the definite pathophysiology of TC remains to be identified, a catecholamine overstimulation of the myocardium is thought to underlie the pathogenesis and forms the basis for treatment of this medical entity.
Direct recordings of multiunit efferent postganglionic muscle sympathetic nerve activity (MSNA) were obtained from 12 female patients, 5 in the acute (24–48 hours) and 7 in the recovery phase (1–6 months), with apical ballooning pattern and 12 healthy matched controls. MSNA was expressed as burst frequency (BF), burst incidence (BI) and relative median burst amplitude (RMBA%). All patients were investigated with ongoing medication.
MSNA was lower in patients with TC as compared to matched controls, but did not differ between the acute and recovery phase of TC. RMBA%, blood pressure and heart rate did not differ between the groups.
MSNA is shown to be lower in patients with TC compared to healthy controls, suggesting that sympathetic neuronal outflow is rapidly reduced following the initial phase of TC. A distension of the ventricular myocardium, due to excessive catecholamine release over the heart in the acute phase may increase the firing rate of unmyelinated cardiac c-fibre afferents resulting in widespread sympathetic inhibition. Such a mechanism may underlie the lower MSNA reported in our patients.
The Social Self Preservation Theory posits that situations that threaten the ‘social self’ elicit shame which, in turn, is linked to cortisol stress response. Body esteem may be one predictor of the propensity to respond with shame to stress. Hence, the present study aimed at assessing whether body esteem is associated with cortisol stress responses, and further, whether this relationship is mediated by cognitive appraisals of challenge and threat.
We exposed 44 participants (21 F, 21±2 years) to the Trier Social Stress Test (TSST). Salivary cortisol was assessed at −1, +1, +10, +30, and +50 min. Body esteem (BE) as well as subscales addressing appearance, weight, and attribution of others’ judgments were assessed with the Body Esteem Scale for Adolescents and Adults (BESAA). Appraisals of challenge and threat were assessed with the Primary and Secondary Appraisal Scale (PASA).
While the TSST successfully elicited cortisol stress responses (
Despite the strong social-evaluative component of our stress test, these findings suggest that how one feels about one's weight and overall physical appearance matters more than what one thinks others may think in this regard. Interestingly, those feelings and beliefs may be associated with gender differences in stress appraisal, such that for females, high BE may be stress protective, while for men, low BE may lead to disengagement from a stressful situation.
Hyperprolactinemia as one of the frequent adverse effects associated with the use of antipsychotics is often neglected but can interrupt the compliance of treatment (1,2). Antipsychotic-induced hyperprolactinemia in women with schizophrenia frequently results in menstrual dysfunction (3) despite its potential to block D2 receptors. However, little (or less?) is known about the effect of olanzapine on prolactin levels in women.
Ms. S, military white-collar, a 35-year-old woman with psychosis, experienced amenorrhea shortly after beginning as well as during treatment with risperidone, 6 mg/day. Previously, she had been treated with haloperidol; she recalled one other occasion when her menses had ceased for 3 months. Before treatment with risperidone, however, Ms. S had been having regular monthly menstrual periods. Medical evaluation revealed an elevated serum prolactin level (100 ng/ml), a negative pregnancy test, and normal thyroid function tests. Magnetic resonance imaging showed no evidence of pituitary adenoma. Alternative treatment with olanzapine was initiated and titrated to 20 mg/day.
After 2 months of olanzapine treatment, Ms. S's monthly menses resumed. Serum prolactin levels, although still elevated, trended downward to 86 ng/ml and 52 ng/ml after 2 and 4 months of olanzapine treatment, respectively. Although she has olanzapine-induced weight gain, her psychiatric condition remained in remission.
There are clinical trials regarding improvement of hyperprolactinemia after switching to olanzapine (4). We reviewed a case in which risperidone-induced hyperprolactinemia–amenorrhea normalized without clinical worsening after switching to olanzapine.
Amenorrhea as one of frequent adverse effects associated with the use of atypical antipyschotics is often neglected but can interrupt the compliance of treatment. There are clinical trials regarding improvement of hyperprolactinemia after switching to olanzapine while some trials regarding the opposite and the improvement with aripiprazole. Aripiprazole is an antipsychotic with partial dopamine antagonism and agonism. Its advantageous side effect profile has been described earlier. We reviewed a case in which olanzapine induced amenorrhea normalized without clinical worsening after switching to aripiprazole.
Ms. C, a 36-year-old woman with psychosis, developed menstrual dysfunction and galactorrhea soon after beginning a treatment of olanzapine, 20 mg/day. She reported having monthly menses before regimen. After 3 month of treatment, menses were absent and galactorrhea began. Ms. C was not pregnant. Her prolactin level was 157.20 ng/ml, and an MRI showed no sign of pituitary adenoma. Olanzapine medication was discontinued in the patients because of galactorrhea, and raised liver enzyme activities. Aripiprazole was initiated and titrated to 15 mg/day.
After 1 month of aripiprazole treatment, monthly menses resumed and galactorrhea resolved. The serum prolactin fell to a normal level (27.20 ng/ml). Ms. C's psychiatric condition improved and she has remission.
Aripiprazole's reduced potential to elevate prolactin may provide a treatment advantage for women with schizophrenia. Moreover, since menstrual cycles may normalize during treatment with aripiprazole, women treated with this drug may have improved fertility when compared with women receiving typical antipsychotics and olanzapine. In this case, aripiprazole treatment resulted in reduction of serum prolactin levels and resolution of galactorrhea. Further studies will be required to assess the comparative effects of aripiprazole and other antipsychotics on prolactin levels and resolution of galactorrhea.
The impact of sleep on basal Hypothalamic–Pituitary–Adrenal (HPA)-axis functioning has been well documented. Specifically, decreased sleep quality and quantity are associated with higher basal cortisol levels, one index of HPA-axis functioning. Few studies, however, have examined the impact of sleep quality and quantity on the cortisol awakening response (CAR), or the diurnal peak in cortisol that occurs shortly after awakening. Investigating this association is important given that a higher CAR is associated with an increased risk for mental and physical health problems. Therefore, the current study aims to further examine the relationship between sleep and CAR in order to gain a better understanding of sleep's impact on HPA-axis functioning.
58 undergraduate students (29 males; mean age = 18.74) were assessed over two consecutive mornings. Each morning, participants completed a daily sleep diary to assess self-reported sleep quality and total sleep time (TST) from the previous night. Saliva samples were used to obtain morning cortisol levels. Participants were asked to provide four saliva samples by spitting into salivettes. The first sample was obtained immediately after awakening. The following three samples were obtained at 30, 45, and 60 minutes after the first sample. Participants repeated this procedure on Day 2.
Multilevel growth curve modeling was used in order to examine the impact of sleep quality and quantity on CAR. Results from the current study demonstrate that TST, or the total minutes slept during the preceding night on each day was significantly associated with both the intercept and slope of the model. More specifically, lower TST was associated with lower cortisol levels at awakening (
Contrary to prior research that has reported no or a small association between sleep duration and CAR, our study shows that participants with shorter sleep duration have lower cortisol levels at awakening and a faster rate of cortisol increase following awakening. Thus, these findings suggest that the amount a person sleeps may directly impact their diurnal cortisol pattern the subsequent morning.
The analysis of steroid hormones in hair is increasingly used in psychoneuroendocrinological research as a valid and easily implementable method for the retrospective assessment of cumulative long-term hormone secretion. To determine steroid hormone concentrations in hair, most laboratories have so far relied on immunochemical assays which are fast and easy to perform, but have a reduced reliability and analytical specificity due to cross-reactivity with other substances. Furthermore, immunoassay can only measure a single steroid at one time. By contrast, liquid chromatography tandem mass spectrometry (LC-MS/MS) has better specificity, sensitivity and reproducibility, and can measure a wide spectrum of steroid hormones simultaneously. Here, we report data on the development of a new LC-MS/MS-based method for the identification of endogenous concentrations of seven steroid hormones (cortisol, cortisone, testosterone, progesterone, corticosterone, DHEA, androstendione) in human hair.
Hair samples were first washed with isopropanol. Steroid hormones were extracted from 10 mg whole hair by 1.8 ml methanol incubation at room temperature. One milliliter methanol was transferred to a new tube and evaporated to dryness. Then the extraction was resuspended with 0.25 ml water, 0.20 ml of which was injected into the machine for analysis.
The limits of detection were 0.1 pg/mg (cortisol), 0.1 pg/mg (cortisone), 0.4 pg/mg (testosterone), 0.9 pg/mg (progesterone), 0.4 pg/mg (corticosterone), 9.0 pg/mg (DHEA), 0.1 pg/mg (androstendione). Linear ranges were 0.5–100 pg/mg (cortisol), 0.5–100 pg/mg (cortisone), 2–100 pg/mg (testosterone), 4–100 pg/mg (progesterone), 2–100 pg/mg (corticosterone), 40–1000 pg/mg (DHEA) and 0.5–100 pg/mg (androstendione).
This LC-MS/MS method provides a highly specific analytical strategy for the detection of seven endogenous hormones in human hair and is thus likely to further enhance the accuracy of future research in this field.
The validity of experimentally induced panic attacks as a model to study the pathophysiology of panic disorder has been questioned. Unspecific, unpleasant and aversive effects as well as specific patterns of psycho vegetative symptoms pointing to different subtypes of panic disorder have been observed. These findings raise the question of challenge paradigms as a valuable tool to identify different vulnerabilities in patients with panic disorder.
We compared the two most widely studied panicogenic drugs sodium lactate and cholecystokinin tetrapeptide (CCK-4) with placebo in 25 patients with panic disorder and age- and gender-matched healthy control subjects. To measure psychophysiological changes, we repeatedly administered the Acute Panic Inventory (API) and visual analogue scales for anxiety and arousal. Cardiovascular (heart rate and blood pressure) and neuroendocrine (ACTH, Cortisol and prolactin) data were recorded simultaneously.
In patients with panic disorder, 18 out of 26 experienced a sodium lactate- or a CCK-4 induced panic attack. Lactate or CCK-4-induced symptoms and induced panic attacks were only correlated in healthy controls, but not in patients with panic disorder. (Analysis of sodium lactate- and CCK-4-induced changes of cardiovascular and neuroendocrine parameters is in progress at the moment and results will be presented).
The mechanisms of lactate and CCK-4 induced panic attacks are distinct in panic disorder patients but not in healthy controls. Different neurobiological vulnerabilities may be uncovered by different challenges and may indicate differential response to specific therapeutic interventions as well.
A major control demand in successful dual-task performance is the task-specific separation of task-goal representations and of the related stimulus-response translation processes. Although these cognitive control processes of task shielding and the physiological effects of acute stress share substantial neural commonalities such as their relation to the prefrontal cortex (PFC), direct empirical evidence of how specific PFC-related cognitive control processes involved in dual-tasking are influenced by acute stress is still missing. Therefore, the present study investigated the impact of acute psychosocial stress on task shielding in dual-task performance.
Fifty-six healthy subjects were exposed to either an acute psychosocial stressor (the Trier Social Stress Test) or a standardised control situation prior to a dual task. The individual physiological stress response was monitored by analysing salivary α-amylase (sAA) and cortisol as markers of sympathetic nervous-system and hypothalamus–pituitary–adrenal (HPA-)axis activity, respectively. Task shielding was assessed by the amount of interference of Task 2 processing on prioritised Task 1 performance (between-task interference).
Following successful stress induction, as indicated by increases in sAA and cortisol, stressed individuals displayed increased between-task interference relative to controls. This result was further substantiated by a correlation between treatment-related increase in cortisol, but not sAA, and between-task interference.
Acute psychosocial stress reduces task shielding, and thus allows for more between-task interference in dual-task performance. We interpret this finding as a shift in cognitive control states from a more serial resource-demanding to a more parallel resource-efficient task-processing mode. The results further suggest a potential role of the HPA-stress response for the development of the observed control adjustment.
Leukocyte telomere length (LTL), a marker of cellular aging, has been proposed as a pathogenic mechanism by which depression may confer increased risk of adverse cardiovascular events. Prior studies have suggested that depression and depressive symptoms are associated with shorter LTL, but these studies are limited by small sample sizes, selective enrollment of participants (e.g. psychiatric outpatients), and lack of adjustment for cardiovascular risk factors and other covariates. The present study examines the association of LTL with depression and depressive symptoms in a large, populations-based cohort.
Participants included 2225 apparently healthy individuals from the 1995 Nova Scotia Health Survey (NSHS95) population-based study. Depressive symptoms were assessed by the Center for Epidemiological Studies-Depression (CES-D) scale. LTL was assessed by a real-time polymerase chain reaction method. Linear regression analyses were used to examine the association between LTL and depressive symptoms, probable depressive disorder (CES-D ≥ 10 or CES-D ≥ 16), and specific depressive symptom clusters (depressed affect, somatic concerns, positive affect, and interpersonal problems). These analyses were adjusted for clinical and demographic factors thought to potentially confound the association between depression and LTL, including: age, sex, body mass index, Framingham risk score, and previous ischemic heart disease.
In an unadjusted model, each 1-point increase on the CES-D was significantly associated with a 3.49 base pair
Concurrent depressive symptoms were not independently associated with LTL in a large population-based study. These results suggest that the excess risk of cardiovascular disease risk associated with concurrent depression may not be due to accelerated cellular aging.
Cortisol is a stress-related hormone that interacts with peripheral and neural systems. Although cortisol is important for short-term stress responses, chronically high cortisol is hypothesised to underlie the long-term effects of chronic stress, including decreased dendritic arborisation in rodent prefrontal cortex (PFC). Here we examined the relationship between variation in stress-induced cortisol levels and regional brain volume in 592 young rhesus monkeys.
Cortisol was quantified from blood samples taken from 592 rhesus monkeys (µ-age: 1.88 years; sex: 265 F) immediately after a human intruder presented their profile to the monkey for 30 min. T1-weighted structural MRI scans, taken within 2 weeks of testing, were transformed to an atlas-based study specific template using ANTS (
Those subjects with higher cortisol had significant decreases in cortical volume. More specifically, there was a significant negative relationship (FDR,
These results provide novel evidence that higher cortisol is associated with less PFC volume in young primates, and builds on previous work examining the relationship between stress and cortical thickness in older adult humans. These data, collected in very young animals, demonstrate that PFC volume and cortisol are negatively associated early in life. These findings are particularly interesting because the PFC regions identified here undergo substantial development throughout late childhood and early adulthood. Moreover, because changes in PFC are thought to underlie the emergence of adult-like cognitive and emotional functioning, our findings may have great relevance to the later development of affective psychopathology.
Cortisol is critical for survival and reflects a primary mechanism by which emotions can influence immune responses throughout the periphery. Although cortisol release is adaptive in response to stress, chronically increased cortisol is known to have negative effects on both body and brain. Here we use a large sample of rhesus monkeys to examine individual differences in stress-related cortisol, in relation to white matter (WM) structure within a distributed brain network. We correlated individual differences in stress-induced cortisol with diffusion tensor imaging (DTI) measures of WM microstructure in 330 young rhesus monkeys.
330 young rhesus macaques were scanned. Diffusion-weighted imaging was performed using a GE SIGNA 3T scanner. Scanning parameters were
Individual differences in cortisol were correlated with DTI-measured FA in the internal capsule (
Our data suggest that naturally occurring increased levels of cortisol are associated with structural differences in key WM regions that coordinate long-range connectivity. Because these connections are important for adaptive and maladaptive stress responses, these findings are highly relevant to understanding the development of stress-related psychopathology.
Chronic stress has been related to lower telomerase, an enzyme that helps preserve the integrity of DNA and slow immunological aging. However, it is unknown whether daily psychological processes reflecting healthy emotion regulation protect against stress-related immune-aging.
We examined basal telomerase activity in a sample of 72 healthy premenopausal women across a range of stress levels, including 35 mothers caring for a child with autism and 37 low-stress control mothers of healthy children. Participants completed a nightly diary over the course of a week, reporting their exposure to positive and negative events. Then they rated the extent to which they employed various emotion-regulation strategies in response to these events. Within-subject weekly means for all measures were calculated. In addition, composite scores for positive affect in response to positive daily events and negative affect in response to daily stressors were calculated, and weekly means obtained. Depressive symptoms were assessed using the Inventory of Depressive Symptoms. On day 4 of the study week, a fasting blood draw was performed to measure peripheral blood mononuclear cells (PBMC) telomerase activity.
Higher telomerase activity was significantly associated with the use of more resilient emotion regulation strategies, including more positive emotional responses to positive daily events (
These are the first findings to link daily emotion-regulation processes to telomerase activity. Daily emotion regulation strategies characterized by greater engagement with the positive and lower emotional suppression are associated with increases in telomerase, which may contribute to resilient immune cell aging. Emotion regulation, particularly in relation to the use of strategies that maintains a positive outlook in the face of stressful life exposures, may protect against cell aging.
To test how genomic loci identified in genome-wide association studies (GWAS) influence the developmental progression of smoking behavior.
A 38-year prospective longitudinal study of a representative birth-cohort.
The Dunedin Multidisciplinary Health and Development Study was conducted in Dunedin, New Zealand.
A total of 1037 male and female study members participated in this study.
We assessed genetic risk with a multi-locus genetic risk score (GRS). The GRS was composed of single-nucleotide polymorphisms identified in three meta-analyses of GWAS of smoking quantity phenotypes.
Smoking initiation, conversion to daily smoking, progression to heavy smoking, nicotine dependence (Fagerstrom Test of Nicotine Dependence), and cessation difficulties were evaluated at eight assessments spanning ages 11–38 years.
GRS was unrelated to smoking initiation. However, individuals at higher genetic risk were more likely to convert to daily smoking as teenagers, progressed more rapidly from smoking initiation to heavy smoking, persisted longer in smoking heavily, developed nicotine dependence more frequently, were more reliant on smoking to cope with stress, and were more likely to fail in their cessation attempts. Further analysis revealed that two adolescent developmental phenotypes – early conversion to daily smoking and rapid progression to heavy smoking – mediated associations between the GRS and mature phenotypes of persistent heavy smoking, nicotine dependence, and cessation failure. The GRS predicted smoking risk over and above family history.
Initiatives that disrupt the developmental progression of smoking behavior among adolescents may mitigate genetic risks for developing adult smoking problems. Future genetic research may maximize discovery potential by focusing on smoking behavior soon after smoking initiation and by studying young smokers.
The cellular enzyme elomerase replenishes telomeric DNA, which can be lost during repeated mitoses or during exposure to inflammation and oxidation. However, telomerase may have other, non-canonical functions, including (in animal models) antidepressant and neurogenesis-enhancing effects. In this study, we determined the relationship between telomerase activity [(measured in peripheral blood mononuclear cells (PBMCs)] and hippocampal (HC) volume in depressed individuals (MDDs) and matched controls.
Nineteen medication-free subjects with MDD and 17 matched healthy controls underwent 4T MRI scanning and fasting morning venipuncture for assessment of unstimulated PBMC telomerase activity. Due to the exploratory nature of the study, corrections for multiple comparisons were not applied.
Hippocampal volume was smaller, but not significantly so, in the MDDs than the controls. As reported previously, MDD subjects had significantly higher PBMC telomerase activity than the controls (
The relationship between telomerase activity in PBMCs and telomerase activity in the HC is unknown. Nonetheless, these results are consistent with emerging preclinical data that telomerase may have neurotrophic and antidepressant effects, may facilitate the neurotrophic effects of brain derived neruotrophic factor (BDNF) and may reverse certain signs of aging, and with clinical data that telomerase may be associated with favorable antidepressant responses. Our finding of significant telomerase/HC correlations only in the MDD subjects raises the possibility that telomerase may play a compensatory or reparative role in this disease.
We investigated whether the habitual physical activity (PA) level had an impact on the acute effects of a short bout of 12 minutes of intensive exercise on cognitive performance and testosterone (T) concentration in primary school children. We further looked for associations between the T concentration and cognitive performance.
42 students of a fourth grade (9–10 years of age) were randomly assigned to an experimental group (EG,
After the intervention participants of the experimental group showed better performances in the d2-test of concentration compared to control. We further observed a significant group (EG, CG), test (pre, post), activity level (high, low) interaction indicating a different pre- to post-test development in T concentration for high- and low-active participants in the EG and CG. Post hoc pairwise comparisons revealed that after acute exercise the T concentration decreased only in habitually low-active children.
The results indicate that the intensive exercise only interacted with the hypothalamic-pituatary-gonadal (HPG) axis in habitually low-active preadolescents, but had a beneficial effect on cognitive performance for all participants independent of their activity level.
Recent meta-analyses have stimulated an active debate on whether the serotonin transporter gene-linked polymorphic region (5-HTTLPR) is associated with an elevated vulnerability to psychiatric diseases on exposure to environmental adversity. As a potential mechanism explaining genotype-depended differences in stress sensitivity, altered stress-induced activation of the hypothalamus-pituitary-adrenal (HPA) axis has been investigated in several experimental studies, with most of the studies comprising small samples.
We evaluated the association of 5-HTTLPR genotype and cortisol reactivity to acute psychosocial stress by applying a meta-analytical technique based on 11 relevant data sets (total
The present meta-analysis indicates a small (
Our meta-analytical results are consistent with a wide variety of experimental studies indicating a significant association between 5-HTTLPR genotype and intermediate phenotypes related to stress sensitivity. Future studies are needed to clarify the consistency of this effect and to further explore whether altered HPA-axis stress reactivity reflects a potential biological mechanism conveying an elevated risk for the development of stress-related disorders in S allele carriers.
Neonatal maternal separation (MS) leads to a long-term dysfunction of the hypothalamic–pituitary–adrenal (HPA) axis in the offspring. Consumption of highly palatable food (HPF) strongly activates the brain reward center and modulates the HPA response to stress. In this study, we have examined the effects of HPF free access during adolescence and youth on the adverse psycho-emotional behaviors by MS experience in rats.
Male and female SD rat pups were separated from dam for 3 h daily during PND 2–14 (MS) or left undisturbed (NH). Half of NH and MS pups had free choices of cookie (HPF) and chow from PND 28, the rest half received chow only. All rats (n = 8–10 in each group) were subjected to behavioral tests during young adulthood (PND 54–59), and the feeding conditions continued until the end of behavioral sessions.
Maternal separation (MS) experience suppressed ambulatory activity both in male and female rats, and HPF access restored it only in males. Caudal grooming was reduced and rostral grooming increased by MS, and HPF access restored them both in males and females. HPF access did not alter MS-induced anxiety-like behaviors during elevated plus maze test both in male and females. Immobility duration during Porsolt swim test was increased by MS experience both in males and females, and HPF access restored it only in females, not in males.
Results demonstrate that free access to HPF during adolescence and youth may partly improve MS-induced anxiety-like behaviors both in male and female offspring, and depression-like behaviors only in females.
The impact of psychosocial stress on a variety of negative health outcomes is well documented, with much of the current research efforts directed at possible mechanisms. For example, psychosocial stress in humans has recently been associated with DNA damage that plays a role in the etiology of negative health outcomes, and also with changes in DNA transcription to messenger Ribonucleic Acid (mRNA). We have become interested in one putative regulatory element in mRNA translation to proteins: microRNA (miRNA). In this study, we aimed to investigate the relationship between psychosocial stress and changes in gene expression changes on the miRNA level, and to further investigate whether stressful life events and personality traits moderate these relationships.
Using a pre-post design, 36 adults were exposed to standardized psychosocial stress in the laboratory (Trier Social Stress Test [TSST]) and completed measures on perceived and chronic stress. In addition, cortisol levels were determined from saliva samples obtained prior to stressor and at eight time points during recovery. Before and after the TSST, subjects underwent a total of three blood draws from which peripheral blood mononuclear cells (PBMCs) were extracted in order to determine miRNA gene expression levels, using the Affymetrix Genechip 2.0 microRNA array. RNA was extracted from each sample and gene expression was measured by hybridization to the miRNA microarray. In an effort to identify a miRNA expression profile for the acute stress response, we compared miRNA expression changes at baseline (before onset of the stressor) with miRNA expression at the two time points following the stressor.
The acute psychosocial stressor produced a higher cortisol response in a subset of the study participants (high responders). We expect these individuals to exhibit significant changes in miRNA expression from baseline to post-stress. We further hypothesize that these changes will be most significant for miRNAs that regulate expression of genes associated with the cortisol stress response.
Our study aims to identify a miRNA signature of social stress and to correlate differences in miRNA expression with psychological variables such as early life stress and resilience, which may function to mitigate the stress response.
Exposure to chronic social stress is a strong predictor of postpartum depression and anxiety. Recent studies have described a chronic social stress (CSS) rodent model for postpartum depression where the repeated exposure of lactating dams to novel male intruders attenuates both the display of maternal care and growth during lactation and increases self-grooming, a measure of anxiety. Investigation of the adult female offspring of these affected dams reveals an attenuated nursing efficiency that is associated with decreases in central oxytocin, prolactin, and vasopressin gene expression.
The current study continued the characterization of the (CSS) model by expanding the analyses to include milk intake, saccharin intake (a measure of anhedonia), and gene expression of the stressed dams.
CSS decreased maternal care and saccharin intake, attenuated pup milk intake by 40%, and altered gene expression in lactating dams.
It is concluded that CSS is an ethologically and translationally relevant model for postpartum depression and anxiety, as well as associated impairments in nursing.
The low-dose dexamethasone-suppression test (DST) has originally been introduced by Yehuda et al..
We here report data on the salivary cortisol responses to awakening (CAR) to the DST in healthy subjects (
We observed stepwise highly significant differences among these three populations with respect to both supersuppression (< 2 nmol/l) and escape (> 6 nmol/l) of cortisol levels. Amazingly, a supersuppression was most frequently observed in healthy subjects, while an escape was most prevalent in inpatients, less common in outpatients, and rare in healthy subjects. While none of the healthy subjects got a PHQ diagnosis, inpatients and outpatients showed an average of 1.8 and 1.9 diagnoses, respectively, but did not differ with respect to the type and degree of stress pathology. Thus, the DST may rather be considered an unspecific test of dysregulations of the pituitary–adrenal axis.
Many research studies observed a supersuppression of cortisol levels in hypocortisolemic subjects with stress related disorders, such as post traumatic stress disorder (PTSD), fibromyalgia, chronic pelvic pain. These subjects commonly express symptoms of fatigue, pain, and an enhanced stress sensitivity, but seem to be protected against deleterious effects of cortisol on organ functions. Such a protective effect may possibly explain our observation that hypocortisolemia and supersuppression are less common in inpatients and outpatients. However, the increasing number of escapes from healthy subjects to outpatients and inpatients was not unexpected. We discuss these findings by applying an additional analysis of endophenotypes.
Rodent studies indicate that the orbital and medial prefrontal cortex have an inhibitory control on the hypothalamic–pituitary–adrenal (HPA) axis, by restraining the acute stress response and facilitating negative feedback inhibition, which can also affect its basal tone of activity. Similarly in humans, extent of damage to the medial prefrontal cortex correlates negatively with cortisol levels. However, lesions of the orbital frontal cortex (OFC) in adult monkeys resulted in no effects on HPA activity. In the present study, we assessed the effects of neonatal OFC lesions on emotional and HPA reactivity to an acute stressor.
Subjects received bilateral aspiration lesions of orbital frontal areas 11 and 13 (Neo-Oasp,
In the presence of the HI, Neo-Oasp animals exhibited less species typical defensive freezing responses as compared to controls (Group:
Results indicate that OFC damage in infancy alters emotional behaviors as well as basal but not stress reactive HPA axis function.
Hormonal contraception has been the subject of numerous research studies. Despite the fact pharmaceutical companies advertise the physical side effects of the medication both positive (e.g. improved acne, reduced ovarian cancer risk) and negative (e.g. increased risk of stroke, weight gain), the knowledge of the potential psychological effects are often based on Internet searches or less than credible resources. The range of empirical support for the effects of the medications on mood has been beneficial including improved mood to negative such as mental health distress. Aside from psychological research, sexual side effects including reduced libido and reduced sexual responsiveness have also been reported. The majority of the research on hormonal contraceptives has been conducted in a clinical setting. It is unclear if the preceding findings would be found with females self-selecting to use the medication as opposed to paid study participants in a clinical setting.
In order to gain a greater understanding of the relationship between hormonal contraceptive usage and affect, an Internet survey with females of childbearing age (age range: 17–48,
Contrary to previous findings, results from this correlational study suggest no effects of hormonal contraceptive use on psychological distress or mood. Females using hormonal contraception did, however, report higher scores for sexual satisfaction and increased sexual activity.
While this investigation was not experimental and, therefore, causation cannot be determined, females using hormonal contraception may be relieved that this research suggests that these drugs do not lead to psychologically harmful side effects and sexuality may be improved with usage. More research is needed to confirm these findings.
Multiple sclerosis (MS) as an inflammatory demyelinating disease of the brain and the spinal cord is associated with a high prevalence rate of major depressive disorder (MDD). Psychological stress has been linked to MS pathogenesis as well as relapse risk in established disease. Moreover, MS diagnosis itself may be a potential trigger for the development of posttraumatic stress disorder (PTSD). Both MDD as well as PTSD have been linked to altered hypothalamic-pituitary-adrenal (HPA) axis regulation and consecutively to elevated or lowered cortisol secretion. This study explores associations of PTSD and MDD with HPA activity in patients with MS.
In a cross-sectional sample of female MS patients, psychological comorbidities were diagnosed using the structured clinical interview (SCID). Circadian salivary cortisol profiles (AUC) and the cortisol awakening response (CAR) as markers of HPA axis activity were assessed over the course of 2 days. On the third day, low dose oral dexamethasone suppression was examined (post-Dex CAR/AUC).
Forty-nine patients with relapsing-remitting MS were included. Eleven patients fulfilled diagnostic criteria for current MDD. A total of 14 patients were diagnosed with PTSD, 7 of whom developed PTSD related to MS-diagnosis. Patients with PTSD were not currently depressed. Importantly, patients with comorbid psychological disorders showed significantly lower coping resources such as self-efficacy, sense of coherence, and social support. While no significant differences were found in most measures of cortisol secretion between the three groups, we observed a trend for higher CAR after dexamethasone suppression in MS patients with PTSD.
The present study indicates a high frequency of MDD and PTSD in MS and associations to reduced salutogenetic resources. These comorbidities might be linked to different aspects of HPA axis dysregulation and could be associated to different biological pathways.
The second generation antipsychotic drug olanzapine is an effective pharmacological treatment for psychosis. However, there is an increasing awareness that the use of the drug is commonly associated with serious metabolic side-effects in patients, including hyperglycemia, glucose intolerance and insulin resistance, and places patients at risk for developing cardiometabolic disorders, such as Type 2 diabetes. These side effects have been accurately modelled in rodent paradigms. We and other groups have demonstrated previously that olanzapine causes significant glucose intolerance and insulin resistance in rats.
In the present study, we directly compared three distinct classes of antidiabetic drugs, which included metformin (100 and 500 mg/kg, PO), rosiglitazone (6 and 30 mg/kg, PO) and glyburide (2 and 20 mg/kg, PO), on olanzapine-induced glucose dysregulation and insulin resistance. Adult female rats (
Both doses of olanzapine caused pronounced glucose dysregulation and insulin resistance that were significantly reduced by treatment with metformin and rosiglitazone; however, glucose tolerance did not fully return to control levels. In contrast, glyburide failed to reverse olanzapine-induced glucose intolerance, despite significantly increasing insulin levels.
These findings indicate that oral hypoglycemic drugs which influence hepatic glucose metabolism, such as metformin and rosiglitazone, are more effective in regulating olanzapine-induced glucose dysregulation than those affecting primarily insulin secretion, such as glyburide. The current model may also be used to better understand the biological mechanism of glucose dysregulation caused by olanzapine and how it can be reversed.
Recent research suggests that atypical cortisol awakening response (CAR) is an outcome of negative factors, such as post traumatic stress disorder (PTSD) (Johnson et al., 2008) and insomnia (Backhaus et al., 2004), in adults. While a positive CAR is present in the vast majority of adults, less is known about the normal development of this response in children. Without a clear sense of typical emergence in early childhood, it is uncertain if risk factors can be standardly associated with the magnitude of CAR during development. Our study aims to expand current research on CAR to children under the age of 8 years. We aim to understand how CAR manifests in young children and how methodological, familial, and child-specific factors contribute to positive CAR (responders).
Fifty-two children (54% female) and mothers participated, ranging in age from 1 to 8 years (M: 4.88, SD: 1.72). Mothers were asked to complete several questionnaires and were sent a “cortisol packet” with instructions to obtain child saliva samples when they awoke (T1) and 45 minutes later (T2) across 2 days. “Responders” were identified as children whose cortisol levels increased from T1 to T2.
No difference in responder group by child age was found in this sample. It may suggest a step-like model, such that emergence of positive CAR would begin in infancy (38%: Saridjan, 2010) and that rates may start to increase only around age 10 (60%: Freitag, 2009) until they stabilize in adulthood (75%: Wust, 2000). In contrast to previous literature, methodological variables, such as daily routine and time between samples (Griefahn & Robens, 2011), were not significantly associated with responder status. However, results were consistent with Saridjan and colleagues (2010), demonstrating that lower family income was associated with greater likelihood of being a responder. Maternal psychopathology had no effect on child CAR status. Interestingly, for child-specific factors, internalizing and externalizing scores had the opposite effect such that, for every increase in externalizing score the risk of being in the responder group increased by 13.5%; however, for every increase in internalizing score, the risk of being a responder decreased by 10%.
Future research should aim to understand the effects of pure internalizing and externalizing versus comorbidity on cortisol in larger samples.
Acute and chronic stress is commonly reported by HIV-seropositive (HIV + ) individuals and may contribute to cognitive dysfunction that interferes with treatment adherence and daily functioning. Here we present data from two studies aimed at characterizing the effects of stress and stress hormones on cognition in HIV+ women.
Six hundred and forty-five HIV+ and 345 at risk HIV-, predominantly African-American participants of the Women's Interagency HIV Study (WIHS), completed the 10 item Perceived Stress Scale (PSS-10) and a comprehensive neurocognitive test battery including measures of verbal learning and memory, verbal fluency, psychomotor speed, executive function, fine motor skills, working memory, attention, and concentration. High stress was defined by scores in the top quartile. Salivary cortisol levels were assessed concurrently with the neurocognitive battery and PSS-10 in 25 HIV+ women as a pilot at the Chicago WIHS.
After adjusting for relevant demographic and behavioral characteristics, HIV+ women performed worse than HIV- women on measures of verbal learning, memory and attention (
Our findings indicate that HIV is associated with verbal memory difficulties among women and that high perceived stress may exacerbate the effect of HIV infection on poor memory performance. Longitudinal assessments are underway to determine the robustness of these associations.
High blood glucose levels increase individuals’ susceptibility to age-related diseases and mortality. However, the molecular mechanisms by which hyperglycemia impairs cellular function is unclear. Emerging research suggests that mitochondrial dysfunction may be a potential allostatic mechanism that mediates the deleterious effects of hyperglycemia via dynamic pathways. Mitochondria are ubiquitous organelles that are the primary producer of cellular energy and, therefore, central to health and disease. In addition, mitochondria contain their own genetic material – the mitochondrial DNA (mtDNA). Importantly, mutations in mtDNA cause human neurological diseases, and hyperglycemia can impair mitochondrial DNA and function. Here, we hypothesized that in patients with inherited mtDNA mutations, those with poor glucose homeostasis or with diabetes would present with more severe neurological symptoms than those with normal glucose balance.
A literature review and retrospective study of 86 patients with the mtDNA 3243A > G mutation was conducted. We assessed glucose homeostasis and neurological symptoms using the Newcastle Mitochondrial Disease Assessment Scale (NMDAS), and Chi-squared statistics were used to compare the incidence of neurological symptoms in patients with or without glucose intolerance.
In patients with pre-existing mitochondrial disease, the incidence of neurological symptoms, including cerebellar ataxia (OR: 9.52; 95% CI: 2.03-44.52) and peripheral neuropathy (OR: 3.91; 95% CI: 1.43-10.76) were greater in those with glucose intolerance than in those with no diagnosed glucose intolerance. In addition, a dose–response relationship linked the severity of glucose intolerance and incidence of cerebellar ataxia.
These preliminary results suggest that mtDNA mutations may render brain tissue more susceptible to glucose toxicity. Given that the accumulation of mtDNA damage occurs with senescence, our findings may have implications for resilience in the elderly. In addition to metabolic stress (i.e., hyperglycemia), mitochondrial functions are modulated by mediators of stress (i.e., cortisol), suggesting that integration of psychoneuroendocrine mediators can occur within mitochondria, thus contributing to the “wear and tear” of allostatic load. In this presentation, we introduce
Emotions have been associated with production of pro-inflammatory cytokines such as TNF-alpha and IL-6; more precisely, negative emotions with greater cytokines level whereas positive emotions with lower level of pro-inflammatory cytokines. This may have significant repercussions on individuals’ health specifically for those who are subjected to chronic inflammation as in the case of HIV positive persons. In fact, HIV virus itself produces greater TNF-alpha production, which in its turns might promote greater IL-6 levels. Thus, this work was conducted in order to verify whether along with HIV association with pro-inflammatory cytokines production, negative and positive emotions, as well as their “balance”, might be associated with cytokines level.
Participants to this cross-sectional study were 90 individuals with HIV diagnosis. Emotions were assessed through the Italian version of Derogatis Affects Balance Scale edited by the first two authors of this work. The biomarkers included were viral load, TNF-alpha, and IL-6. Individuals also self-reported whether they were under antiretroviral therapy.
A Structural Equation Model was performed in order to test if negative and positive emotions and their balance (that consisted in the ratio of positive/negative emotions) along with viral load were associated with cytokines level. Results indicated that viral load (β=0.538,
Taken together, these results may indicate that together with virus effect in producing greater inflammation, emotions may also contribute to it. Negative emotions could then promote greater inflammation whereas, when individuals experience more positive emotions than negative, inflammation might be reduced.
Pregnant women with stress are at increased risk of adverse pregnancy outcomes. And prenatal stress (PNS) can influence newborn's mental development. Earthquake exposure is a special stressor for pregnant women. Ningqing County is one of the worst-hit areas of the Wenchuan Earthquake (8.0 magnitude) happened in Sichuan Province, China on 12 May 2008. To explore the related influencing factors of children's mental development whose mother exposed to the earthquake, we investigated the mental development of children born after the Wenchuan Earthquake in Ningqiang County.
A total of 86 children aged 0–3 years were recruited in this study. They were randomly selected and were screened by “the mental developmental screening test (DST)” for children aged 0–6 years compiled by Children's Hospital of Fudan University. For ease of interpretation, children were classified in four different groups based on ages: 0-year-old; 1-years-old; 2-years-old; and 3-years-old.
Among the 86 children, there were 54 boys and 32 girls. The mean Development Quotient (DQ) score was 98.59±2.08. Twenty two (including 18 boys and 4 girls) children's DQ score was <85, and they shared the percentage of 25.6. The mean Mental Development Index (MI) score was 97.35±1.64. Seventeen children's MI score was less than 85, and they shared the percentage of 19.8. The incidence of DQ < 85 was significantly higher than the national urban average (14.9%) (
Earthquake possibly contributes to the retardation of children born after Wenchuan Earthquake in Ningqiang County of China.
An eight-magnitude earthquake struck Wenchuan, Sichuan Province of China on 12 May 2008. Ningqing County is one of the worst-hit areas. Earthquake exposure was a special stressor for pregnant women. But little is known about what the earthquake influence on pregnant women and the degree of the influence. Only if we understand people's mentation after a natural disaster that can we help them to get over it. To assess the impact of the earthquake on the mental health, we investigated the impact of the Wenchuan Earthquake on pregnant women who lived in Ningqiang County.
Women who had babies within 3 years after earthquake in Ningqiang County were randomly recruited in this study. Seventy five women were screened by employing Life Events Scale for Pregnant Women (LESPW) compiled by Yan Gao et al. in 2005 and 87 women were screened by employing Post-traumatic Stress Disorder Self-rating Scale (PTSD-SS) compiled by Xianchen Liu et al. in 1998.
Among 75 women, the mean score of LESPW was 297.08±21.95, and 30.67% women's score was equal or greater than 375. This result was significantly higher than that of Xiaomei Li's report of general pregnant women in Weifang (223.18±129.30)(
The Wenchuan earthquake brings lasting adverse influences to pregnant women in Ningqiang county of China within three years.
Previous work in our and other laboratories has demonstrated dysregulated immune function and cellular oxidative stress responses in subjects with Major Depressive Disorder (MDD). In the current study, we determined whether there were differences in the transcriptional regulation of these pathways in leukocytes from subjects with MDD, and healthy controls.
We used genome-wide transcriptional profiling (Affymetrix U133 Plus 2 oligonucleotide arrays) and promoter-based bioinformatic strategies (TELiS) to assess transcription factor (TF) activity in leukocytes from 15 unmedicated MDD patients versus 19 age-, gender-, and ethnicity-matched healthy controls, prior to initiation of antidepressant therapy, and after 8 weeks of sertraline treatment.
Bioinformatic analysis of 39,000 differentially expressed genes indicated increased transcriptional activity of cAMP Response Element-Binding (CREB) factor, Interferon Response Factors, and the oxidative stress-responsive Nuclear Factor (erythroid-derived 2)-like 2 (NRF2). Eight weeks of antidepressant therapy was associated with significant reductions in Hamilton Depression Rating Scale scores and reduced activity of NRF2, but not CREB or interferon response factor activities. Several other transcriptionally regulated pathways previously associated with depression, including the glucocorticoid receptor (GR), nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB), and early growth response proteins 1-4 (EGR1-4) pathways, showed either no significant differences as a function of disease or treatment, or activities that were opposite to those previously hypothesized in previous research. Quantitative RT-PCR analysis confirmed the expression profiling data.
These results are consistent with the hypothesis that oxidative stress and innate antiviral responses may be involved in MDD by activating immune cell transcriptional pathways, and that successful antidepressant therapy may result in reduced oxidative stress responses at the level of gene transcription.
Bariatric procedures such as laparoscopic adjustable gastric banding (LAGB) can markedly decrease body adiposity in severely obese adolescents, but relatively little is known about the short-term effects of such procedures on meal-related hormonal response.
Participants completed a fixed size breakfast meal and fasting concentrations of appetitive hormones (leptin, insulin/glucose, ghrelin, PYY) were measured. PYY, ghrelin, and visual analog scale (VAS) ratings of fullness, hunger, nausea, and desire to eat were assessed immediately before the meal and 15, 30, 60, and 90 minutes afterwards.
A total of 10 normal-weight controls (age: 15.4±2.0 years, BMI: 21.3±1.7 kg/m2;
Despite short-term decreases in body mass index post-LAGB, few changes were observed in appetitive hormones prior to or following a standardized breakfast. In addition, subjective ratings of fullness, hunger, nausea, and desire to eat did not differ between surgical candidates and normal-weight controls, but following LAGB, adolescents reported significantly more nausea. Future studies should examine longer-term effects of LAGB on appetitive hormones.
Posttraumatic Stress Disorder (PTSD) is a complex mental disorder with functional and structural changes in the brain that may result from mitochondria-centered responses to harmful stresses. PTSD is an ongoing issue in the military. However, at present, there is no biological tool for PTSD diagnosis. Diagnosis for PTSD is established on the basis of clinical history and mental status examination, using a clinically structured interview based on a symptom checklist or patient self-report. It is often under-diagnosis. The clinical assessment would benefit substantially from a more objective means to identify PTSD patients. Here, we present evidence that there are significant differences of expression profiles of mitochondria-focused gene in the blood between PTSD and non-PTSD control US military service members.
Using a mitochondria-focused gene cDNA array, we examined the expression profiles of 1170 mitochondria-focused genes across samples from subjects with (
Significance tests demonstrated different expression levels in 26 genes between PTSD and non-PTSD controls. A relationship analysis found that among the 26 genes, the expression levels of five genes were significantly correlated with the total PCL score in the PTSD subjects.
The expression of mitochondria-focused gene fingerprints and dysregulated genes in the blood of PTSD patients warrants a large size study to determine their clinical utility in military population.
Deployed military personnel are at risk for (mental) health problems, including post traumatic stress disorder (PTSD), major depressive disorder (MDD), and fatigue. We hypothesized that development of these conditions is associated with biological vulnerability factors. Therefore, we assessed whether the development of PTSD, depressive and/or fatigue symptoms in response to military deployment could be predicted by glucocorticoid (GC) signalling in leukocytes and the capacity of peripheral blood cells to produce cytokines.
We included 1,032 Dutch military personnel prior to deployment to Afghanistan. Symptom severity was assessed 6 months after return. In blood collected prior to deployment, we assessed GC signalling in leukocytes (glucocorticoid receptor [GR] number, target gene mRNA expression, and GC-sensitivity) and cytokine production upon stimulation with LPS, PHA, or IL-1β.
We identified different vulnerability factors for development of a high level of PTSD, depressive, and fatigue symptoms. PTSD symptom development was predicted by high GC signalling in leukocytes. In contrast, depressive symptom development was associated with low GC signalling in T-cells and high T-cell cytokine production capacity. Finally, development of fatigue was associated with low GC signalling in monocytes prior to deployment and high reactivity of monocytes to IL-1β after deployment.
The identified vulnerability factors for the development of high levels of PTSD, depressive, and fatigue symptoms were condition specific. This indicates PTSD, depression, and fatigue have different underlying biological mechanisms. Moreover, the results suggest that the biological profile prior to stress/trauma exposure may not only determine
Stress provokes physiological alterations, which are thought to mediate the development, maintenance, and progression of several disorders. Social support is thereby thought to possess a buffer function, decreasing the physiologic effects of stress. In our previous work, we were able to show a buffering effect of perceived social support (PSS) on the stress response of salivary α-amylase (sAA), an index of sympathetic nervous system activity. The aim of the present longitudinal study was to examine the effects of alterations in PSS on baseline and stress levels of sAA.
Swiss male recruits (
The TSST-G induced a significant increase in sAA activity on both occasions, while military service resulted in an overall decrease in PSS. Changes in PSS were associated with alterations in baseline and stress responses of sAA: recruits with a decrease in PSS over the 10-week period revealed an increase in baseline activity (
Our findings show that military service is associated with a decrease in PSS. This decrease is related to alterations of sympathetic nervous system activity, characterized by an increased tone and decreased responsiveness. This longitudinal study emphasizes the need to boost psychosocial resources during military service.
Although tricyclic antidepressants (TCAs) are not recommended as first line therapy for depression in patients with coronary heart disease (CHD), they are still occasionally prescribed. Rationales may include resistance to other classes of antidepressants, previous response to TCAs, or treatment continuation after onset of a CHD. Despite their antidepressive effectiveness, TCAs may worsen cardiovascular prognosis because of autonomic side effects. Here, we examined potential adverse effects of TCAs on autonomic function as marked by heart rate variability (HRV) and norepinephrine (NE) levels.
A total of 956 outpatients with stable CHD, 44 used TCAs. All patients were prospectively followed for 7.2±2.6 years. Standard deviation of all normal RR intervals (SDNN) as a measure of HRV was calculated from 24 h-electrocardiographic recordings. NE levels were measured in plasma and 24 h-urinary samples. We also calculated hazard ratios for all-cause mortality.
Users of TCAs had an increased risk of mortality compared to non-users (
Use of TCAs was associated with increased mortality in patients with CHD. Unfavourable changes in autonomic function as marked by low HRV and high NE levels might be a potential mechanism.
Cognitive deficits and alterations in cortisol secretion are characteristic features of major depression disorder (MDD). The cortisol awakening response (CAR) is altered in depression and crucially depends on hippocampus function, a brain area closely related to cognitive function.
We examined 21 MDD patients without medication, 20 MDD patients treated with antidepressants, and 41 healthy control subjects (HC), matched for age, gender, and years of education. We applied several neuropsychological tests. Salivary cortisol levels were measured on two consecutive days at awakening, and 30 min and 60 min after awaking.
Both patient groups did not differ in severity of depression (
The magnitude of the CAR is strongly associated with impaired memory function in unmedicated depressed patients even though CAR was not significantly increased in these patients. In contrast, medicated patients showed a blunted CAR and unimpaired cognitive function compared to controls. These results suggest that antidepressant treatment may reduce CAR and partially restore memory function even if depressive psychopathology is still present.
Telomeres cap and protect the ends of chromosomes from fusion. Excessively shortened telomeres are associated with telomere dysfunction and chromosomal instability (CIN), DNA damage and an increased risk of degenerative diseases of ageing. Psychological stress has been strongly associated with accelerated telomere shortening, consistent with a wealth of evidence that chronic stress impacts negatively on health, possibly contributing to initiation of cancers, cardiovascular disease and neurodegenerative disorders such as Alzheimer's disease. Risk for these disorders is increased by deficiency in micronutrients, such as folate, an essential co-factor required for accurate replication of DNA and maintenance of methylation (epigenome) patterns, providing protection against CIN.
The aim of this preliminary study was to test the hypothesis that chronic exposure to the stress hormone cortisol impacts deleteriously on telomere length (TL) and that this effect would be further aggravated by folate (Vitamin B9) deficiency. Human lymphocytes from 3 males and 3 females (aged 53±3 years) were cultured
Cells cultured in FA-replete medium and chronically exposed to 550 or 1300 nM cortisol displayed longer TL at day 12 than cortisol-free controls (
The results of this study do not support the hypothesis that cortisol, folate deficiency or their interaction can explain telomere shortening associated with psychological stress. Further analyses are being performed to determine if cortisol causes changes in CIN or epigenome status and the extent to which these effects correlate with TL.
There is increasing evidence that oxytocin promotes empathy in humans. However, research on oxytocin and emotion recognition, a fundamental component of empathy, has yielded inconsistent results. Part of the problem is that studies have focused on limited, and varying, categories of emotional stimuli. Therefore, we investigated the effect of intranasal oxytocin on the identification of seven basic emotions (happiness, sadness, fear, excitement, surprise, disgust, and anger) using social and non-social stimuli, and we explored the effect of oxytocin on conceptual understanding of emotion.
Eighty-two participants were administered a 24IU dose of intranasal oxytocin or placebo in a double-blind experiment. Participants completed the perceiving (faces, designs) and understanding (blends, changes) emotion components of the Mayer-Salovey-Caruso Emotional Intelligence Test (MSCEIT) 120 minutes after drug administration.
Contrary to our prediction, standardized scores for accurately detecting emotions during the faces task of the MSCEIT were lower following oxytocin administration than placebo (
Oxytocin appears to influence the recognition of facial expressions of emotion by increasing the perceived intensity of the emotion, while having no effect on more complex processing (i.e., understanding emotion). The present findings further support the view that oxytocin influences social information processing by increasing the salience of emotional stimuli, which may have positive or negative effects depending on context.
Considerable research has focused on the relationship of anxiety with alterations in the hypothalamic-pituitary-adrenal (HPA) acute stress response. Findings, however, differ among studies on adults and children, and among different types of anxiety. This study investigates the relationship of anxiety symptom severity with HPA reactivity to the cold pressor task (CPT) in preadolescent children. We hypothesize that children with increased symptoms of anxiety will have increased cortisol (HPA) reactivity to the CPT.
A social-evaluative adaptation of the CPT was used to elicit HPA acute stress reactivity among 42 children (26 female, 16 male) aged 8–12 years (mean age, 10 years) recruited from a child anxiety disorders clinic (
Maximum cortisol TR and AUCi did not differ between children recruited from the anxiety disorders clinic and the community. Among all subjects, maximum TR was significantly greater for those with high anxiety symptoms on the STAI-T (
Results suggest that increased anxiety symptom severity is associated with greater cortisol reactivity to acute stress in preadolescent children. Moreover, findings were similar among youth recruited from the clinic and the community, thus providing additional evidence of the high prevalence of anxiety in children and the potential associated risk of alterations in physiological stress reactivity among those with more severe symptoms.
Prior theory and research suggests that psychosocial stress during development may contribute to vulnerability to problems in adulthood in domains of both mental and physical health through alterations in both automatic attentional and psychobiological stress reactivity processes. We hypothesized that childhood exposure to family conflict would be related to variations in hypothalamic–pituitary–adrenal (HPA)-axis activity patterns and would moderate the relation between attentional bias to threat and biological stress reactivity to acute laboratory stress exposure.
A sample of young adult female participants (
Exposure to family conflict during development was significantly positively correlated with baseline cortisol in the sample as a whole (
Results suggest that exposure to chronic stress early in development shapes later perception and interpretation of environmental cues as stressful and has an enduring impact on biological reactivity to acute stress. These findings expand on earlier work by Luecken and colleagues presenting a combined cognitive-affective model to link characteristics of the family environment during development to alterations in psychological and physiological stress reactivity processes in adulthood, which may ultimately underlie illness vulnerability.
Early life stress (ELS) poses a risk for mental disorders and aging-related physical diseases. Accelerated biological aging reflected in shorter leukocyte telomere length (LTL) may underlie these risks. Yet, studies examining associations between ELS and LTL have been scanty, elusive, and retrospective. We examined if objectively documented ELS in childhood, retrospectively, reported traumatic experiences across the lifespan and whether their combination is associated with LTL in later adulthood.
ELS, traumatic experiences, and LTL were present in 1,486 participants of the Helsinki Birth Cohort Study, born between 1934 and–44 in Helsinki, Finland. Of them 215 were recorded as separated temporarily from their parents in childhood. The separations took place during World War II when Finnish children were voluntarily evacuated unaccompanied by their parents to temporary foster care abroad (mean age at and length of separation 4.6 and 1.7 years, respectively). Traumatic experiences across the lifespan were self-reported at age 63.2 years (SD = 2.8) using the Traumatic Experiences Checklist, and LTL was measured at age 61.5 years (SD = 2.9) using real-time quantitative polymerase chain reaction (PCR) method.
LTL did not differ significantly by separation status or by having experienced traumas (
ELS and traumatic experiences may, in combination, contribute to accelerated cellular aging and shed light into the underlying mechanisms linking ELS and early traumatic experiences with mental disorders and aging-related diseases.
Recently, the hypothalamic-pituitary-adrenal axis activated by psychological stress has been reported to be involved in irritable bowel syndrome (IBS) symptoms. In the present study, we investigated the effect of prolonged stress (for two weeks) on salivary adrenal hormones in individuals with IBS.
The participants were 23 female college students (mean age = 18.8 years), including 10 individuals with IBS met Rome II criteria and 13 individuals without IBS (control group), and they were scheduled for a two-week teaching practice at kindergarten. Participants were asked to collect their saliva immediately (T1), 30 min after awakening (T2), and before sleep (T3) at date of a month before (D1), 3 days after (D2), 7 days after beginning of teaching practice (D3), and several days after the end of it (D4). They also completed perceived stress scale (PSS) (D1–D4). Comparisons between groups and between dates in PSS score were done using 2-way analysis of variance. Linear mixed model was applied to analyze the effects of the presence of IBS, time, and date on salivary adrenal hormones (cortisol, dehydroepiandrosterone [DHEA], DHEA-sulfate [DHEA-S]).
There were significant effects of day in PSS (
Cortisol:DHEA ratio in participants of the present study responded to prolonged stress, and individuals with IBS showed higher cortisol:DHEA ratio at 30 min after awakening than individuals without IBS during experiment period. These results of this study using prolonged stressor differs from that of our previous study using acute stressor that individuals with IBS showed lower DHEA-S level and DHEA-S:DHEA ratio throughout the experiment.
Altered long-term secretion of glucocorticoid hormones is believed to play a pivotal role in linking chronic stress to cardiometabolic risk. Despite experimental data supporting this link, previous epidemiological field studies have often yielded inconsistent results. Amongst other things, this is likely to be related to methodological limitations in the assessment of glucocorticoid secretion over prolonged periods of time. The measurement of glucocorticoids in hair may constitute a major advancement here, enabling the assessment of cumulative hormone levels over periods of up to six months. Here we will present first data from a large industry-funded cohort study investigating links between work-related stress, long-term glucocorticoid secretion and cardiometabolic risk factors.
Hair samples were obtained from 1315 employees of the airline manufacturing industry and assayed for cortisol (F) and cortisone (E) concentrations using liquid chromatography with tandem mass spectrometry detection (LC-MS/MS). In addition, relevant anthropometric, psychosocial and physiological biomarkers of cardiometabolic risk were assessed.
Results reveal positive associations of hair F and E concentrations with measures of central obesity (body mass index, waist-to-hip ratio), resting systolic and diastolic blood pressure as well as fasting morning blood levels of glucose, glycated haemoglobin (HbA1c), C-reactive protein and high-density lipoprotein (negative). Significant positive associations with low-density lipoprotein and triglyceride levels were only seen for hair E but not for hair F. These findings are in line with current conceptions suggesting an important role of aberrant glucocorticoid secretion in the development of cardiometabolic risk. Implications of these data for hair analysis as an important future tool in epidemiological field research will be discussed.
Intranasal oxytocin attenuates cortisol levels during social stress inductions. However, no research to date has documented the dose–response relationship between intranasal oxytocin administration and cortisol, and researchers examining intranasal oxytocin have not examined the cortisol response to physical stress. We, therefore, examined the effects of 24 and 48 IU of intranasal oxytocin on the cortisol response to vigorous exercise.
Seventeen males participated in a randomized, placebo-controlled, double-blind, and within-subject experiment. Participants engaged in vigorous exercise for 60 minutes following the administration of placebo or intranasal oxytocin on three occasions. Saliva samples and mood ratings were collected at 8 intervals across each session.
Salivary cortisol concentrations changed over time, peaking after 60 minutes of exercise [Quadratic:
This is the first study to demonstrate that the effect of intranasal oxytocin on salivary cortisol is dose-dependent, and that intranasal oxytocin attenuates cortisol levels in response to physical stress. Future research using exogenous oxytocin will need to consider the possibility of dose–response relations.
Accumulating research indicates that oxytocin (OT) plays a key role in human social cognition and behavior. Inspection of the data, however, suggests that the social effects of OT often depend on contextual factors, including person characteristics. For example, some studies show that OT is helpful for avoidantly attached individuals, who are less socially engaged, whereas other studies show that OT exacerbates chronic interpersonal insecurities in anxiously attached individuals, who are preoccupied with closeness. Such variability raises questions about the mechanism by which OT influences human social behavior. Drawing upon animal research on OT and the other-directed (e.g., maternal) behavior, we propose that OT induces a similar shift in focus away from self and toward others in humans. This theory would explain some of the person-specific effects of OT since becoming more other- and less self-oriented should be helpful for avoidant individuals who are excessively focused on the self to the exclusion of others, but could be hurtful for anxious individuals who are already overly other focused and have no sense of self.
Thirty-one males received 24 IU intranasal OT/placebo in a randomized, double-blind, crossover trial and then completed tasks assessing the implicit cognitive accessibility and explicit self perceptions of agency (self orientation) and communion (other orientation). Individual differences in attachment were assessed at baseline.
OT significantly decreased the cognitive accessibility of agency (self) and increased the cognitive accessibility of communion (other). Similarly, OT significantly decreased the endorsement of agency traits (arrogant) and increased the endorsement of communal traits (kind, warm, caring). Critically, this OT-induced shift from self to other differentially affected avoidant and anxiously attached participants, with avoidant individuals, who are generally low in communion, showing the largest increase in communion following OT, but anxious individuals, who are generally low in agency, showing even further reduction in agency/sense of self.
These data shed light on the variability in extant research on the social effects of oxytocin in humans and help explain both the beneficial and potentially harmful effects of OT.
The neuropeptide oxytocin (OT) is well known for its positive effects on dampening stress responses and increasing prosocial behavior. This view of OT has led to increased interest in its application for the treatment of a wide range of disorders that include autism, anxiety, trauma, and schizophrenia. However, paradoxical findings in recent work have revealed that OT may play a more nuanced role in regulating physiology and behavior. This work is beginning to shed new light on contextual factors that may influence the direction of OT's effects.
In the present study, we used female prairie voles (
We found that OT pretreatment prior to a stressor was associated with changes in behavior, plasma hormone concentrations, and patterns of functional coupling between brain areas known to be critically involved in stress responses and social cognition.
This talk will discuss recent work, from our lab and others, that examines the role of OT at the interface of stress and social behavior.
Childhood maltreatment negatively impacts brain development, often producing transgenerational continuity of abusive parenting and increased risk for a range of psychiatric disorders. The biological basis for these far-reaching effects is not currently understood, but evidence suggests traumatic events could affect behavioral trajectories through changes in gene expression that are mediated by DNA methylation.
To explore this, we exposed male and female infant rats to nurturing or adverse caregiving environments. We measured changes in DNA methylation and gene expression in developing and adult animals. Candidate genes were selected according to their role in brain plasticity, responsiveness to stress, and association with several psychiatric disorders.
Exposure to adverse caregiving environments induced long-lasting changes in cortical DNA methylation and expression of the brain-derived neurotrophic factor (BDNF) gene. In addition, females exposed to adverse caregiving environments later mistreated their own offspring, and their offspring likewise displayed altered DNA methylation. We are currently investigating the impact of nurturing vs. adverse caregiving environments on epigenetic gene regulation within a larger behaviorally relevant brain network (the medial prefrontal cortex, central/basolateral amygdala, dorsal vs. ventral hippocampus). Preliminary biochemistry data indicate caregiving experiences trigger epigenetic changes that differ between brain regions, sexes, and gene locus.
These findings demonstrate the remarkable ability of early-life caregiving environments to produce distinct epigenetic modifications across behaviorally relevant brain regions. Our work as well as that of others suggests that DNA methylation serves as a biological pathway linking early-life adversity to long-term (and perhaps multigenerational) changes in neurobiology and behavior.
Childhood maltreatment is linked to multiple metabolic and immunological abnormalities. Experimental research in animal models showed that stressful experiences in early life may also be associated with impaired leptin response to physiological stimuli, such as adiposity and inflammation. Therefore, we tested if maltreated children showed leptin deficiency.
We assessed leptin and C-reactive protein in dried blood spots and anthropometric measures from 170 twelve-year-old participants of the Environmental Risk (E-Risk) Study. Childhood maltreatment was prospectively assessed through repeated interviews with mothers in the first decade of study participants’ life.
Maltreated children showed a trend towards lower leptin levels than non-maltreated children. Furthermore, maltreated Children showed reduced leptin response to increasing inflammation and adiposity levels. These findings could not be explained by key potential confounders or pre-existing abnormalities in energy homeostasis.
Childhood maltreatment is associated with leptin deficiency, which could contribute to previously reported metabolic and immune abnormalities. Exposure to childhood trauma among pregnant women is associated with increased placental CRH production over gestation.
There is an insufficient understanding of the neurobiology of post-traumatic stress disorder (PTSD). Therefore, the development of an animal model of PTSD that takes into account clinical features of the disorder is of value toward enhancing our understanding of the mechanisms, and in the development of novel treatments, of emotional trauma.
Adult male rats were administered chronic psychosocial stress composed of two 1-hour periods of inescapable exposure to a cat, in conjunction with daily unstable pair housing, over a 31 day period. The rats were then given a battery of tests, including measures of behavior (anxiety testing, startle response), cognition (predator-based fear memory and new memory testing), hormone levels (basal and evoked glucocorticoids), responses to pharmacological agents (dexamethasone and yohimbine) and cardiovascular activity (blood pressure/heart rate). In addition, we measured epigenetic alterations (methylation) of the brain-derived neurotrophic factor (BDNF) gene.
Psychosocially stressed rats exhibited a PTSD-like phenotype. The stressed rats exhibited a strong fear-conditioned memory of the two cat exposures, an increase in behavioral signs of anxiety, an exaggerated startle response, increased blood pressure, greater sensitivity to yohimbine and a hippocampus-dependent memory impairment, relative to controls. In addition, stressed rats exhibited reduced basal glucocorticoid levels, greater sensitivity to dexamethasone and hypermethylation of the BDNF gene in the hippocampus.
These findings demonstrate that intense psychosocial stress produced dramatic changes in physiology and behavior in rats which are comparable to those observed in people diagnosed with PTSD. This rat model, therefore, may enhance our understanding of the mechanisms underlying human trauma and in the development of more effective pharmacotherapy for people with PTSD.
Context processing imbues appropriate salience to the stimuli that is encountered. This ability enables us to hide from a predator in the wild, but to enjoy a visit to the zoo, although the lion may look the same in both contexts. Failures in contextual processing can lead to inappropriate fear responses rooted in failures to use safety cues, consider internal states, anticipate events, or appraise them properly. Posttraumatic stress disorder (PTSD) is associated with exaggerated fear, unwanted recollection, and inappropriate emotional and social responses. We proposed that PTSD pathophysiology involves deficits in context processing and examined this hypothesis using PTSD animal model and fMRI studies in patients with PTSD.
Using validated animal model of PTSD, we examined fear conditioning, fear extinction and context-dependent extinction recall, and fear renewal in single prolonged stress (SPS)-exposed animals. We further examined the relationships between glucocorticoid receptors (GRs) upregulation in SPS, and fear renewal deficits were observed. Using 3T fMRI paradigm, we examined fear conditioning, fear extinction, extinction recall, and fear reinstatement in PTSD patients and trauma-exposed control subjects.
In humans, we found that fear-conditioning procedures activated fear-associated brain regions, but PTSD patients had similar fMRI activation maps to trauma-exposed controls during fear conditioning and extinction. However, they exhibited decreased responses to contextual signals of safety and danger. In animal work, we found that the SPS-exposed animals exhibited normal levels of conditioning and extinction, but specific deficits in context-dependent extinction recall and fear renewal. In “dismantling” studies, only animals exposed to full SPS and that demonstrated largest upregulation of GR in hippocampus and prefrontal cortex exhibited fear renewal deficits.
These results demonstrate contextualization deficits in PTSD subjects. PTSD animal model findings mirror those observed in PTSD patients and further implicate specific molecular targets in defined brain regions in contextualization deficits. Together, this set of studies demonstrates the combined power of translational research into trauma psychopathology.
Animal research has challenged the permanency of memory by suggesting that reactivation (retrieval) of a specific memory may return it to a labile state from which it must be “re-consolidated” if it is to persist. Pharmacologically blocking reconsolidation offers the therapeutic possibility of weakening traumatic memories in post-traumatic stress disorder (PTSD).
We have been testing the above hypothesis using systemic drugs approved for human use. In rats we employ classical conditioning consisting of pairing a tone CS with a shock US on Day 1 (acquisition), presenting the tone without shock on Day 2 (reactivation) followed by drug, and then re-presenting the CS alone on Days 3 and 10 (tests). We have also used slice electrophysiology to measure the increase in cortico- and thalamo-lateral amygdala synaptic efficacy as a result of the tone-shock association, and then decrement in this efficacy following reconsolidation blockade. In PTSD subjects, we have administered oral drug along with verbal or written narration of the traumatic event (reactivation) and subsequently measured the strength of the traumatic memory via psychophysiological recording during script-driven imagery, and/or symptom report.
In animal work, we have found that the antiglucocorticoid receptor mifepristone, the protein synthesis inhibitor rapamycin, and the alpha-2-adrenergic autoreceptor agonist clonidine all block partially reconsolidation of conditioned fear. Clonidine does so in a dose-dependent manner. Additionally, rapamycin reverses the synaptic enhancement described above, providing an underlying physiological basis for reconsolidation blockade. In humans, we have found that traumatic memory reactivation plus double-blind, placebo-controlled propranolol reduce physiologic responding during script-driven imagery, and that six weekly open-label propranolol plus memory reactivation sessions reduce PTSD symptoms to a similar degree as current cognitive behavioral treatments. A double-blind clinical trial is underway.
The above results show progress toward a clinical application of reconsolidation blockade, but much more needs to be done before efficacy is demonstrated.
The identification of molecular post-traumatic stress disorder (PTSD) susceptibility pathways associated with different patterns of behavioral response to trauma is essential to an understanding of the neurobiology of PTSD and can pave the design for new treatments. Although several genes have been reported to be differentially expressed in PTSD, methodological constraints have limited the interpretation, for example, variation in the type or magnitude of trauma exposure, inter-individual genetic variation, and tissue specificity of response. Animal models are useful in delineating some of these issues. In this study, we used a unique animal model of PTSD with ecological and population validity. Adult rats were exposed briefly to predator scent stress, which mimics a threatening situation. Rats respond heterogeneously to this type of traumatic stress behaviorally and physiologically, similar to human response variability. In this model, two behavioral extremes can be studied – vulnerable and resistant ‘subtypes’.
Sprague-Dawley rats were exposed to the scent of cat urine. The outcome measures included behavior in an elevated plus-maze and the acoustic startle response 7 days after exposure. Cut-off behavioral criteria classified exposed rats according to their behavioral response as those with ‘extreme behavioral response’ and ‘minimal behavioral response’ (MBR), with unexposed rats as controls. From the tissue obtained 24 h after the behavioral tests, basal gene expression using Illumina BeadArrays was evaluated for whole blood and three brain areas: amygdala, anterior cortex and hippocampus. For data quality control and differential expression analysis, we used R and LIMMA (as included in MeV software), respectively. Pathway analysis was performed with ingenuity.
There was only minimal overlap in gene expression across brain regions and gender demonstrating the existence of distinct tissue-specific susceptibility pathways in male and female rats. Among the differentially expressed genes, the ones regulated by the glucocorticoid receptor (e.g., FKBP5, Per-1, and NPY) were particularly over-represented (especially in blood and hippocampus), indicating that glucocorticoid regulation is involved in vulnerability and resistance to trauma. The observed gene expression profiles may also indicate the over-representation of discrete functional biological clusters and pathways (e.g., MAPK signaling and circadian rhythm).
Glucocorticoid-related gene expression is underlying the different response pattern following trauma, with distinct regional/structural differences between male and female rats.
Post-traumatic stress disorder (PTSD) arises from the interaction of genetic and environmental factors. Thus, a better understanding of the molecular etiology of PTSD would be greatly facilitated by the development of animal models that explore gene×environment interaction in the context of traumatic stress. To this end, we have identified a new mouse genetic model for stress vulnerability that may provide novel insight into the neurobiology of PTSD. Our studies focus on mice that are deficient for TIA-1, a prion-related RNA binding protein that regulates the expression of multiple target genes in the mammalian brain.
TIA-1 KO mice and wild-type littermates are generated from TIA-1 heterozygous crosses. All behavioral (fear conditioning, open field, elevated-plus maze, forced-swim test) and electrophysiological (hippocampal field recordings) experiments are conducted in accordance with standard protocols.
Under baseline conditions, TIA-1 KO mice are indistinguishable from wild-type controls in all behavioral and neuroendocrinological measures evaluated thus far. However, several weeks after exposure to contextual fear conditioning, TIA-1 KO mice demonstrate increased anxiety and despair-like behavior, as well as abnormal glucocorticoid production. Moreover, these phenotypes are observed predominantly in female animals. Electrophysiological studies reveal aberrant synaptic plasticity in the ventral hippocampus of knockout animals in response to corticosterone treatment, consistent with a critical role for TIA-1 in normal emotional memory formation in the hippocampus during stress. Finally, molecular data suggest that TIA-1 may regulate alternative splicing of the glucocorticoid receptor, which is known to be important for both hypothalamic–pituitary–adrenal (HPA) axis function and hippocampal synaptic plasticity during stress.
TIA-1 KO mice recapitulate several key features of chronic PTSD observed in humans. Thus, our studies demonstrate that TIA-1-deficient mice represent a useful model in the study of gene×environment interaction during traumatic stress, and may contribute to our knowledge of the molecular basis of PTSD. Finally, because individuals with PTSD are also susceptible to substance abuse, we, therefore, discuss the utility of TIA-1 knockout mice in the study of PTSD and comorbid substance use disorders.
Relatively little is known about how lasting qualities of the individual (e.g., traits and/or one's past history) moderate the effects of cortisol on emotional learning. We hypothesized that cortisol would have more pronounced effects on memory formation in individuals who show greater levels of trait negative affect (Trait NA) or who experienced early life separation (ELS).
In Study 1, involving 42 healthy adults (22 women), we examined how Trait NA moderated the effects of cortisol administration (IV-administered 0.1 mg/kg/30 min hydrocortisone; CORT) vs. placebo on memory formation for unpleasant and neutral photographs. In a preliminary study (Study 2), in 18 depressed adults (10 women), we examined how ELS (because of parental divorce or permanent separation) moderated the effects of CORT (15 mg orally administered hydrocortisone) vs. placebo on memory formation for positive and negative words.
In Study 1, we found that in women with higher Trait NA, CORT facilitated memory formation. In women with lower levels of Trait NA, CORT had no effects of memory formation. Study 2 revealed that in depressed women with ELS, CORT facilitated memory formation for negative words. Specifically, CORT (vs. placebo) biased memory in a negative direction by an average of 4.2 (SD=0.73) words in women with ELS. In depressed women without ELS, CORT had no effect on memory formation. In both studies, 1 & 2, effects were less robust in men or trended in the opposite direction, which may represent true sex differences or may be because of confounding factors, such as differences in cortisol elevations for men vs. women.
In summary, our data suggest that lasting qualities of individuals, such as Trait NA or history of early separation, moderate cortisol's effects on emotional memory. Further investigation into how variation in personal traits and past experiences moderate cortisol's effects on emotional cognition is one important step in elucidating why some individuals are more sensitive than others to the detrimental (e.g., negatively biasing) effects of stress on emotional cognition and memory. These data may also inform research regarding the use of corticosteroid receptor ligands in treatment for psychopathology. Individual differences in affective style or past experiences may predict therapeutic response to corticosteroid receptor ligands.
Enhanced memory for emotional events is a well-known phenomenon. From an evolutionary perspective, it is an adaptive mechanism, as it helps to remember threatening as well as pleasurable experiences. Stress hormones are important players in the regulation of emotional memory. Specifically, in animals and in humans, glucocorticoids enhance memory consolidation of emotionally arousing experiences but impair memory retrieval. Glucocorticoid actions are partly mediated by glucocorticoid receptors in the hippocampus, amygdala and prefrontal cortex, key brain regions for emotional memory. Here, we investigated whether the BclI polymorphism of the glucocorticoid receptor gene is associated with emotional memory in healthy young subjects. This polymorphism has been previously related to traumatic memories and posttraumatic stress disorder (PTSD) symptoms in patients who underwent heart surgery (Hauer et al., 2011).
To assess memory performance, we used a picture-learning task consisting of learning and recalling emotional and neutral photographs on two consecutive days in 841 healthy young subjects. Genotyping of the BclI polymorphism was done with Pyrosequencing on a PyroMark ID System.
The BclI variant was associated with short-delay recall of emotional pictures: GG-carriers showed increased emotional memory performance as compared to CG- and CC-carriers. We did not find a genotype-dependent difference in recall performance for neutral pictures.
These findings indicate that the BclI polymorphism contributes to inter-individual differences in emotional memory in healthy young subjects and suggest a genetic link between emotional memory in healthy humans and traumatic memory in patients who underwent cardiac surgery.
The inability to store fearful memories into their original encoding context may be an important vulnerability factor for developing anxiety disorders like posttraumatic stress disorder (PTSD). Such altered memory
In a double blind, placebo-controlled design, 60 men were randomly assigned to one out of three possible groups. 1) In the rapid cortisol group, participants received 10mg hydrocortisone 30 min before completing the “associative imagination task” (AIT), 2) while the slow cortisol group received the drug 240 min before the task. 3) A third group received placebo at both times. During the AIT task, participants were instructed to vividly imagine 30 neutral and 30 negative words in unique background pictures. Approximately, 24 h later, participants completed two surprise memory tests: cued retrieval and recognition. Crucially, to assess memory contextualization, half of the negative and neutral words were tested in intact contexts, whereas the other half of the words were tested in rearranged context combinations.
Recognition data showed that negative memories were generally less context-bound than neutral memories. Moreover, cortisol exerted time-dependent effects on contextual dependency of
This study shows distinct time-dependent effects of cortisol on the contextualization of negative memories. These results suggest that non-genomic effects of cortisol may underlie impaired memory contextualization observed in PTSD, whereas genomic effects of cortisol may open avenues for cortisol as a protective agent against (traumatic) fear memory generalization.
Stress and stress hormones (e.g., cortisol) modulate memory processes in both facilitative and deleterious ways, but mechanisms of memory enhancement are not yet fully understood. Specific effects of cortisol may be of particular interest, given its importance in a range of stress-related illness that is associated with cognitive changes.
To elucidate potential mechanisms of memory facilitation via pharmacological manipulation; healthy participants underwent functional magnetic resonance imaging after oral-administration of 100 mg of hydrocortisone (
Cortisol at encoding enhanced subsequent memory only for face-building combinations but not for faces or buildings alone. Cortisol increased anterior hippocampal activation and a mediation analysis suggests that cortisol enhancement of conjunctive memory was at least partially mediated through this enhanced hippocampal activity. Cortisol memory enhancement was also associated with increased hippocampal interconnectivity.
These results support the hypothesis that the hippocampus may be an integral participant in cortisol's memory facilitation effects, with potential implications for psychopathologies like posttraumatic stress disorder, that involve stress-axis, hippocampal, and memory abnormalities.
The stress hormone cortisol is known to modulate different memory processes. In general, high levels of cortisol increase memory consolidation, especially for emotional material. For a long time, memories were thought to be stable and resistant to changes after their consolidation was completed. However, recent evidence suggests that consolidated memories are subject to modulation upon their recall (reactivation). This suggests that memory reactivation opens a window of opportunity allowing the memory trace to be modulated in a lasting manner. Given that cortisol is an important modulator of memory, we were interested in investigating how it could modulate reactivated memories.
In the first study, 32 healthy men and women encoded a slideshow containing neutral and negative segments. Two days later, all participants recalled the slideshow (reactivation) and were randomly assigned to a stressor, the Trier Social Stress Test (TSST) or a control condition. Memory was reassessed immediately after that, as well as 5 days later. For the second study, 33 healthy men were exposed to the slideshow (as described above). Three days later, they were either administered with a placebo or metyrapone (a cortisol synthesis inhibitor). All participants had to recall the slideshow when the medication was active, and 4 days later. The third study assessed the impact of real negative news from the media. Fifty-six healthy men and women who were media consumers were either exposed to real negative news or to real neutral ones. Subsequently, they were all exposed to the TSST and their memory for the news was assessed 24h later.
Study 1 demonstrated that emotional memory was enhanced following stress and this effect was still observed 5 days later. Study 2 results showed that memory reactivation of the emotional material was lower in the metyrapone group and this deficit was still present 4 days later. Study 3 demonstrated that women who were exposed to real negative news remembered this news more and were more stress reactive to a subsequent stressor.
Memory is an active process that can be updated upon its reactivation and cortisol can act as a modulator of this process. Results of our studies will be discussed with regards to their relevance to the clinical domain, more particularly for posttraumatic stress disorder.
Memory functions involve three stages: encoding, consolidation, and retrieval. Modulating effects of glucocorticoids (GCs) have been consistently observed for encoding and retrieval. However, little is known on how GCs affect consolidation.
In Study I, after encoding emotional and neutral texts, cortisol or placebo was intravenously infused while participants were either awake (
In Study I, retention of temporal order within the texts was enhanced when cortisol was infused during the wake phase but impaired when it was infused during sleep. In Study II, blockade of MRs impaired free recall of both texts and pictures, especially for emotional material. In contrast, blockade of GRs resulted in better memory retrieval.
Study I points toward fundamentally different mechanisms of cortisol on hippocampal memory consolidation, depending on the brain state. Study II and III indicate opposing roles of MRs and GRs in memory retrieval.
Stressful experiences during early life can remodel brain circuitry underlying behavioral adaptation with consequences for resilience and vulnerability to emotional and cognitive disorders. At least in the rodent this apparent permanent outcome of early adversity can be modulated by maternal influences and depends on the later life environmental context with the stress hormones of the hypothalamus-pituitary-adrenal axis in the driver's seat. A frequently investigated model is the animal that has experienced as pup reduced maternal care. Such a period of early neglect enhances the pup's responsivity to adverse emotional experiences, an effect that can even be detected within families and was found to advance prematurely the development of emotional and fear circuitry. Alternatively, enhanced care is capable of overridingthe lasting impact of neurotoxicity in early life. For instance, the frequently reported adverse effect of early life treatment with dexamethasone (as life-saving treatment of prematurely born infants) is strikingly attenuated by enhanced maternal care induced by daily handling.
Dr. Nederhof will review recent animal and human studies supporting the cumulative stress and mismatch hypotheses. Dr. Parker will present behavioral and neuroendocrine data from monkey studies supporting the idea of stress inoculation following early exposure to moderate stressors. Dr. Bagot will address the importance of later-life context when investigating the effects of early-life experience using rats exposed as pups to varying levels of maternal care and ex-vivo electrophysiology. Dr. Vinkers will demonstrate in healthy volunteers a modulation by genotype and gender of the accumulating effects of stress on psychiatric outcome
The cumulative stress hypothesis states that aversive experiences early in life predispose individuals to be more vulnerable to aversive challenges later in life. Indeed, adversity has consistently been associated with psychopathology; however, it is neither a determinative nor a sufficient explanation. The mismatch hypothesis provides an alternative explanation; adverse experiences early in life trigger adaptive processes, thereby rendering an individual to be better adapted to adversity later in life.
A review of both the animal and human literature on the interaction between early and later adversity and its relation with psychopathology.
In the animal literature, support for both the cumulative stress hypothesis and the mismatch hypothesis was found. The human literature is characterized by a general paucity of interaction studies. Convincing evidence for individual differences in sensitivity to early programming suggests that both hypotheses might be true but applicable to different individuals.
The cumulative stress hypothesis is proposed to apply to individuals who were not or only to a small extent programmed by their early environment, the mismatch hypothesis to individuals who experienced strong programming effects.
Early exposure to severe stress in childhood increases the incidence of mood and anxiety disorders in adulthood. Far less researched, but of equal importance, is the theory that moderate early stress exposure instead of increasing vulnerability results in subsequent resilience. Variously described as inoculating, immunizing, steeling, or toughening, the notion that moderate postnatal stress exposure strengthens resistance to subsequent stressors has far-reaching implications for understanding the pathogenesis and prevention of stress-related affective disorders. Although the psychobiology of stress-inoculation-induced resilience in humans is largely unknown, new insights have emerged from seven studies of monkeys previously exposed to moderate postnatal stress compared to no-stress control-rearing conditions. Evidence from these studies indicates that early exposure to moderate stressors that temporarily stimulate anxiety and activate the hypothalamic-pituitary-adrenal (HPA) axis leads subsequently to diminished negative arousal, prosocial tendencies, enhanced cognitive control, increased curiosity, larger prefrontal cortical volumes, and attenuated HPA axis activation. In contrast to rodents, rearing differences in the development of neuroendocrine stress resistance in monkeys are more closely related to differences in prior stress exposure than to differences in maternal care. In addition, unlike in rodents, no rearing differences in glucocorticoid feedback sensitivity were observed in monkeys, suggesting that the neural basis of stress resistance in primates may differ from that in rodents. Finally, results from a pharmacological study, which further support the key role of acute early anxiety exposure in promoting the development of subsequent behavioral indications of resilience, will be presented. Findings from these non-human primate studies support the intriguing hypothesis that moderate early stress exposure may likewise provide a pathway to resilience in humans.
Variations in maternal care in the rat associate with robust differences in hippocampal synaptic plasticity and learning in the offspring. In addition, differences in stress reactivity associate with variations in maternal care. However, the potential influence of stress on hippocampal function is often overlooked in studies of effects of early life experience. Previously, we found differential modulation of hippocampal function and plasticity by stress in adult offspring exposed to varying levels of maternal care. N-methyl-D-aspartate receptors (NMDAR) regulate synaptic plasticity, and NMDAR function is modulated by stress and CORT. We hypothesised that altered NMDAR function underlies the interaction of maternal and stress effects on hippocampal synaptic plasticity.
We used electrophysiology to examine NMDAR-dependent LTP and NMDAR synaptic function in adult offspring of mothers that varied in the frequency of pup licking/grooming (LG), i.e., High or Low LG.
Under basal conditions, long-term potentiation (LTP) was impaired in the hippocampal dentate gyrus of Low LG offspring relative to High LG offspring. Synaptic NMDAR function was enhanced in Low LG offspring with no change in α-amino-3-hydroxy-methyl-4-isoxazole propionic acid receptor function (AMPAR). NMDAR antagonism by low concentration APV rescued the basal LTP deficit in Low LG offspring and inhibited LTP in High LG offspring. Stress-level CORT (100nM) rapidly enhanced LTP in offspring of Low LG rats and impaired LTP in offspring of High LG rats. CORT robustly increased NMDAR function in High LG offspring, eliminating the maternal effect. CORT did not affect NMDAR function in Low LG offspring. Thus, Low LG offspring exhibit basally elevated NMDAR function coupled with insensitivity to CORT modulation indicative of a chronic alteration of NMDAR.
These results suggest that low maternal care exerts a lasting effect on hippocampal plasticity through enhanced function of NMDAR in synapses. The blunted effect of CORT on synaptic NMDAR in Low LG rats could be adaptive in promoting cognitive functioning in challenging conditions, such as the improved contextual fear conditioning previously observed in these rats.
Stress exposure increases the risk for the development of depression. The leading hypothesis is that stress exposure only increases the risk in individuals combining a vulnerable genetic background with (repeated) stress exposure. However, it is currently unknown which (combination) of stressful life events are the most etiologically relevant to predict depressive symptoms. Preliminary evidence suggests that repeated cumulative stress may have the highest impact.
We aimed to determine the effects of repeated stress exposure on depressive symptoms (Beck Depression Inventory) in a cross-sectional sample of healthy subjects (
Childhood trauma, later life stressors, and current daily hassles independently and cumulatively contributed to depressive symptoms (continuous and dichotomized). High cumulative stress exposure was associated with depressive symptoms (
Cumulative exposure to three independent stressors contributed to the development of depressive symptoms in a healthy sample. Thus, our data implicate the existence of a multiple hit model in which independent but cumulative stress exposure leads to increased depressive symptoms. Multiple assessments across various stress domains may increase the validity and reliability of stress exposure compared to a single assessment. Importantly, the MR haplotype moderated the cumulative effects of stress on depressive symptoms, confirming and extending earlier studies, which showed that the MR haplotype 2 is associated with reduced optimism and a blunted stress response.
Animal research has indicated that oxytocin is involved in social bonding, stress regulation, and positive physiologic adaptations that may be linked with greater longevity and successful aging. Because of its potential role in promoting positive human social behavior, recent research has focused on whether oxytocin may lead to improved social and emotional functioning for various mental disorders. Moreover, given its apparent anti-stress effects, some investigators have posited that oxytocin may provide the basis for the observed beneficial effects of positive social relationships on health. While knowledge of the effects of oxytocin in healthy humans remains limited, the emerging research has raised some doubt as to whether effects are uniformly prosocial or stress-reducing. Moreover, gender differences in these stress-related effects have been speculated but not tested in humans. In this study, we examine whether oxytocin enhances salutary responses to social stress and compare effects between men and women.
Hypotheses were tested with a placebo-controlled, double-blind experiment, using a between subjects 2 (male vs. female)×2 (oxytocin vs. placebo) design. Participants (
Analyses were conducted using two-way analysis of variance models (or ANCOVA if covariates such as age or estradiol were included). Participants given oxytocin, relative to placebo, responded to social stress with a
Findings indicated that oxytocin stimulates an approach-oriented cardiovascular profile during social stress, suggesting mechanisms by which oxytocin might influence health. However, before considering oxytocin as therapeutic or uniformly beneficial, greater understanding of possible gender dimorphic effects is needed.
Oxytocin is associated with both social cognition/perception and affiliation and with stress response regulation. Both stress response regulation and social factors influence response to trauma. While some social/interpersonal factors may mitigate response to trauma (e.g., social support), others can contribute to trauma-related symptoms (e.g., interpersonal avoidance). While most research on the effects of oxytocin shows a positive influence on social cognition/affiliation, other research suggests that this influence varies with context (e.g., oxytocin is associated with decreased trust/affiliation in the context of social threat). Furthermore, some studies of endogenous oxytocin levels show positive relationship between with symptoms of trauma related disorders (e.g., depression and posttraumatic stress disorder (PTSD)).
We present data from a sample (
We found that, in a highly traumatized sample, peripheral oxytocin was positively associated with both perceived social support and with levels of depression and PTSD. One way of understanding this data is the idea that oxytocin is associated with salience of social cues. In some circumstances (e.g., relationships with trusted family members), oxytocin may positively influence social perceptions/behaviors. In other circumstance (e.g., threatening interpersonal contexts), it may be a marker for trauma-related symptoms such a depression and PTSD. Many of our study participants live in unsafe neighborhoods and have been exposed to repeated interpersonal trauma. Interpersonal trauma, particularly in childhood, may increase the likelihood social cues are perceived as threatening. Thus, these data point to the need for complex models in understanding relationship of trauma response and oxytocin and in efforts to use oxytocin in the treatment of trauma exposed individuals.
Women are twice as likely as men to develop Posttraumatic stress disorder (PTSD). Abnormal acquisition of conditioned fear has been suggested as a mechanism for the development of PTSD. While some studies of healthy humans suggest that women are either no different or express less conditioned fear responses during conditioning relative to men, differences in the acquisition of conditioned fear between men and women diagnosed with PTSD has not been examined.
Thirty-one participants (18 men, 13 women) with full or subsyndromal PTSD completed a fear-conditioning task. Participants were shown computer-generated colored circles that were paired (CS + ) or unpaired (CS − ) with an aversive electrical stimulus, and skin conductance levels were assessed throughout the task.
Repeated measures ANOVA analyses indicated a significant sex by stimulus interaction during acquisition,
In contrast to studies of healthy individuals, we found enhanced acquisition of conditioned fear in women with PTSD. Greater fear conditioning in women may either be a pre-existing vulnerability trait or an acquired phenomenon that emerges in a sex-dependent manner after the development of PTSD. Characterization the underlying mechanisms of these differences is needed to clarify sex-related differences in the pathophysiology of PTSD.
In healthy individuals, the magnitude of the startle-eyeblink reflex to a brief, acoustic probe is modulated by the valence (goodness/badness) of a photographic picture and this modulation shows a linear, stepwise pattern. Startle amplitude is smallest during pleasant, largest during unpleasant, and intermediate during neutral pictures. Animal work shows that startle modulation is mediated by the brain's fear/defensive circuit which is centered on the amygdala—a region important in valence and emotion regulation. We previously reported (Hazlett et al., 2007) exaggerated affective startle modulation during processing of unpleasant (e.g., “suicidal”) but not neutral words in individuals with borderline personality—a disorder characterized by emotion dysregulation and suicidal behavior.
In light of this work, we present preliminary data from an ongoing Department of Defense DoD-sponsored study examining whether suicidal behavior confers defensive hyperreactivity measured as increased affective startle modulation during emotional pictures, irrespective of diagnosis. We examined 40 age- and gender-matched veterans with varying levels of suicidal behavior: ideators (I):
Compared with the I and SA groups, the MA group showed startle hyperreactivity during unpleasant pictures (MA > I:
These findings indicate a spectrum of hyperreactivity during unpleasant picture viewing that parallels the severity of suicidal behavior: Startle amplitude during unpleasant pictures progressively increased from Is at the minimum, to SAs, and finally MAs at the extreme. Affective startle may provide a useful non-verbal, psychophysiological biomarker for suicidal behavior. Correlations between affective startle, symptom severity, and emotion regulation will also be presented.
Post-traumatic stress disorder (PTSD) is associated with exaggerated autonomic responses to sudden, loud acoustic stimuli, particularly under conditions characterized by ambiguous threat. However, it is not clear if such exaggerated responses are a stable vulnerability factor for developing PTSD or a feature of current PTSD that resolves with symptom resolution. We investigated this issue by comparing autonomic startle responses to acoustic stimuli under low, medium, and high threat conditions in Gulf War Veterans with and without current and past PTSD.
Our sample included three groups: no PTSD (
Individuals with current PTSD had significantly higher heart rate responses compared with the no PTSD group in the low,
These results suggest that exaggerated autonomic startle responses under conditions of low and medium threat vary as a function of current PTSD severity and are not a marker of PTSD vulnerability. Moreover, the data suggest that this startle paradigm with varying threat levels may be a useful index of hyperarousal of fear-related neurocircuitry in PTSD.
Because posttraumatic stress disorder (PTSD) and blast-mild traumatic brain injury (mTBI) commonly are comorbid in veterans of the wars in Iraq and Afghanistan, attributing persistent behavioral or other symptoms to one or the other entity remains controversial. Here, we asked if multimodal neuroimaging would reveal persistent functional or structural abnormalities in veterans who had experienced blast-mTBIs in Iraq and/or Afghanistan and, if so, could any such abnormalities be attributed to comorbid PTSD.
Thirty-four blast-mTBI veterans (26 with PTSD and 8 without PTSD) and 16 Iraq and/or Afghanistan-deployed veterans without blast or blunt impact mTBI or PTSD were studied. Each veteran underwent magnetic resonance diffusion tensor (DTI) and magnetization transfer/cross-relaxation imaging (MT-CRI), as well as fluorodeoxyglucose positron emission tomography (FDG-PET); structured clinical assessments of blast and combat exposure, psychiatric diagnoses, and posttraumatic stress disorder symptoms; neurologic evaluations; and self-report scales of postconcussive symptoms (PCS), combat exposure, depression, sleep quality, and alcohol use.
Blast-mTBI veterans exhibited reduced fractional anisotropy in the genu and splenium of the corpus callosum on DTI; reduced macromolecular-bound proton fraction (a brain putative measure of myelin integrity) in white and gray matter and multiple regions of interest on MT-CRI; and parietal, somatosensory, and visual cortex hypometabolism on FDG-PET. The presence of PTSD in mTBI veterans had no effect on DTI or MT-CRI structural brain biomarkers. The only effect of PTSD on FDG-PET functional biomarkers was lower glucose metabolism in visual cortices bilaterally.
Iraq and Afghanistan combat veterans with blast-mTBI exhibit abnormalities of brain white matter structural integrity and macromolecular organization and regional cortical glucose metabolism years after blast exposure. Although comorbid PTSD was associated with lower visual cortex metabolism, it had no effect on structural biomarkers. These findings are consistent with recent neuropathologic evidence of cortical tauopathy and neuronal degeneration in a small sample of Veterans with blast-mTBI.
We previously reported that hippocampal volume was associated with current, but not lifetime post traumatic stress disorder (PTSD) symptom severity. In this study, we further investigated the role of current versus remitted PTSD on the volumes of other brain regions previously implicated in PTSD.
Magnetic resonance imaging data from a 4 Tesla scanner of 191 veterans (75 trauma unexposed, 43 trauma exposed without PTSD, 39 trauma exposed with PTSD, 34 trauma exposed recovered from PTSD) were analyzed with FreeSurfer software program (version 4.5).
Veterans with current PTSD had smaller hippocampal, caudal anterior cingulate, insula, and corpus callosum volumes than trauma unexposed veterans (
The finding that current but not lifetime PTSD accounted for the volumes of multiple brain regions suggests either that smaller brain volume is a vulnerability factor that either impedes recovery from PTSD; or less likely, that recovery is accompanied by a wide-spread restoration of brain tissue.
Increased GR sensitivity has been associated with PTSD severity in several studies, and in a recent report, was found to predict the extent of improvement in severity of PTSD symptoms from pre- to post-treatment. These findings provided the rational for an investigation of two GR genotype polymorphisms know to affect GR sensitivity in relation to PTSD severity and treatment response. The BclI (rs41423247) single nucleotide polymorphism (SNP) is an intronic restriction fragment length polymorphism located 646 bp downstream from GR exon 2. SNP 9β (rs6198) is located on exon 9β of the GR. It is believed to increase the stability of splice variant GRβ, an inhibitor of the wild-type GRα. Carriers of the BclI minor allele have been associated with an increased sensitivity to glucocorticoids (GCs), whereas 9β carriers have been linked to relatively diminished sensitivity. We studied these polymorphisms in the context of a treatment study in which participants were evaluated prior to and following treatment.
For the treatment study, subjects were randomized into two treatment conditions, weekly prolonged exposure therapy and a minimal attention, in which participants received a weekly phone call to evaluate symptom severity and monitor for safety. Clinical outcome was assessed using pre- and post-treatment Clinician Administered PTSD Scale (CAPS) total scores. Pre-treatment genotype data were available for 27 of 36 receiving prolonged exposure and for 9 or 13 who received the minimal attention condition. DNA was isolated from lymphocytes using Ficoll-Paque Plus (Amersham Pharmacia Biotech) and extracted (using FlexGene DNA Kit, Qiagen); genotyping of SNPs BclI and 9β was performed using the allelic discrimination technique with custom designed probes and primers according to the published genomic sequences, with results that did not differ from Hardy–Weinberg equilibrium.
The presence of the G allele in the BclI SNP was inversely associated with lifetime, but not current, total CAPS score for treatment completers (
The BclI polymorphism is related to a less severe lifetime course of PTSD, and is a substantial predictor of positive outcome in response to short-term psychotherapy. The relation of this genotype to recovery from PTSD was even stronger than pre-treatment clinical severity or type of treatment. The result suggests that BclI genotype may be a useful biomarker in the selection of potential treatment candidates. This work should be repeated in additional, and larger samples for verification.
Hypothalamus–pituitary–adrenal (HPA) axis reactivity, which has been considered a potential endophenotype for psychiatric disorders, is commonly investigated by repeated-measures designs utilizing frequent sampling of salivary cortisol in temporal proximity to psychosocial stressors. To remove sources of cortisol variance, which are not related to HPA axis reactivity, researchers often utilize classification criteria to identify individuals who show no cortisol response (non-responders), for example, baseline-to-peak distances of 2.5 nmol/l. However, such classification criteria have not been systematically evaluated with regard to their classification performance.
As a first step, we fitted an autoregressive latent trajectory model to cortisol data, which was obtained from longitudinally sampled saliva of 504 participants, of which 309 were exposed to the Trier Social Stress Test. Different sources of time-series variance were accounted for by modeling of initial cortisol levels, amplitude of the subsequently occurring secretory episodes, and continuous cortisol elimination. Assuming zero-amplitudes for individuals who show no stress response, a mixture distribution was implemented for secretory episodes, resulting in appropriate classifications of cortisol responders, or non-responders. Then, as a second step, we evaluated the classification performance of various proposed classifiers by constructing receiving operator characteristics.
Results reveal (a) that covariance and mean structure of cortisol time-series can be sufficiently accounted for by the proposed model, allowing to infer on endocrine parameters that can barely be extracted by conventional analyses and (b) that the 2.5 nmol/l criterion is suboptimal in terms of simultaneously minimizing false-positive and false-negative classifications and inferior as opposed to other classifiers.
To maintain the low number of false positives, but to increase true-positive classifications, we suggest to lower the conventional baseline-to-peak classification threshold to 1.5 nmol/l. Furthermore, classification performance can be increased by adjusting baseline-to-peak differences for initial cortisol levels.
Common gene variants predisposing for altered dopamine (DA) neurotransmission are candidates for schizophrenia-susceptibility genes, although genome-wide studies so far showed a weak association of these variants with schizophrenia. It has actually become apparent that the expression of psychotic symptoms in schizophrenia is associated with the exposure of the genetically predisposed individuals to environmental risk factors during development such as early life adversity and upbringing in an unfavorable social environment. Furthermore, it has been postulated that genetic predisposition can promote not only vulnerability in response to negative environmental input, but also resilience in response to positive environmental stimulation.
We decided to test this hypothesis in the apomorphine-susceptible (APO-SUS) rat line, which was selected from Wistar rats on the basis of an extremely enhanced stereotypic gnawing response to administration of the dopamine agonist, apomorphine (APO-gnawing). The parental strain was used for comparison. Adult rats exposed as pups to poor maternal care and to post-weaning social-isolation rearing were examined for pre-pulse inhibition of acoustic startle (PPI), T-maze spontaneous alternation, contextual fear-conditioning and stress hormonal responses to a conditioned emotional stressor.
Adult APO-SUS rats that had experienced poor maternal care as judged from low maternal licking and grooming (LG) scores showed dramatically enhanced stress-induced ACTH levels in the face of modest increases in circulating corticosterone (CORT) and prolactin levels. These low LG offspring also developed a basal PPI-deficit, reduced acoustic startle and impaired contextual fear-conditioning, but showed enhanced short-term memory. Additional isolation rearing abolished entirely basal PPI and impaired short-term memory in these individuals. High LG offspring, on the contrary, displayed enhanced PPI in both rearing conditions that was reduced only after CORT-challenge, while the low LG was resistant to CORT. Maternal LG history alone in Wistar rats had limited effects on the behaviour or stress response of offspring. When low maternal LG history was combined with post-weaning social isolation, basal APO-gnawing was decreased and PPI increased. High LG offspring reared in isolation displayed, however, the highest APO-gnawing and the lowest PPI levels among rats reared in social isolation. An injection of high dose CORT in the adult low LG offspring reduced PPI, whereas the high LG group was resistant to the acute effects of CORT.
If exposure to negative social environment accumulates, a schizophrenia-like phenotype, characterized by a severe deficit in sensorimotor gating and brain glucocorticoid-resistance, precipitates in the genetically predisposed individuals while the non-predisposed individuals are resilient. However, the same genetically predisposed individuals are sensitive to positive environment as well, where they improve their phenotype and outperform the controls, which do not change. This is the first animal model to find strong evidence for a differential susceptibility to stress in development depending on genetic predisposition.
Telomere length (TL) is an important marker of cellular aging that can be examined from birth to death and provide information about health status and disease risk. TL shortens in early childhood with the majority of the shortening occurring by age 4. TL is associated with stress and obesity in adults. It is possible that exposure to early life stressors and excess adiposity from birth and the first year of life may impact the rate of telomere shortening. Few studies have examined TL in the first years of life, and none of them have examined stress and obesity in infants.
We examined TL by qPCR using genomic DNA from dried blood spots in a sample of 109 four-year-old, low-income Latino children and their mothers. TL is expressed as T/S (the ratio of telomeric product vs. single copy gene product). This group of children and their mothers were recruited prenatally in San Francisco at which time socio-demographic and health history was assessed. In addition, child weight and length and maternal body mass index (BMI) have been assessed annually from birth with the child's weight and length measured also at birth and 6 months of age. Maternal depressive symptoms were assessed prenatally, at 4–6 weeks postpartum and annually throughout the follow-up period. Child behavior was evaluated using the child behavior checklist (CBCL) at 3 and 4 years of age for internalizing and externalizing behaviors. Student's
In bivariate analysis, being obese at 6 months of age (weight/length ≥ 95th percentile) and being obese at both 6 and 12 months of age were associated with shorter TL at age 4 (1.62±0.36 versus 1.84±0.34,
In this population of low-income Latino children, obesity in the first year of life was associated with shorter telomere length at age 4, independent of sex mother's TL and mother's depression. Thus, obesity early in life may shape TL, whereas obesity in the toddler and preschool years may be less associated with obesity at age 4.
Life-threatening experiences, including the observations of severe trauma and/or violence, coupled with feelings of extreme fear and helplessness can result in posttraumatic stress disorder (PTSD). Unpredictability, uncontrollability, and novelty are considered key factors in eliciting and influencing the intensities of the stress responses. Personal coping strategies may affect resilience and susceptibility to stressors and, in PTSD patients, may also affect responses to stressors such as cortisol secretion. Recent interest in PTSD models focuses on the drivers of susceptibility versus resilience factors and the identification of potential targets for prevention and/or treatment of PTSD. Following a traumatic event, most individuals experience at least some symptoms of PTSD. However, many trauma survivors who develop PTSD recover over the course of months.
Systems and integrative biology approaches were applied to characterize the development of PTSD using an animal model of repeated social trauma/stress. Behavioral, physiological, and histopathological consequences of repeated social stress were evaluated using a modified "6 hour box-in-box resident-intruder" model. At the end of the stress episodes, mouse blood samples and organs were collected and brains were dissected into 17 different regions. Transciptomic, metabolomic, proteomic, and epigenomic changes were analyzed using microarrays.
Pan-omic analyses of this mouse model that simulate aspects of PTSD revealed that genes involved in axonal guidance signaling, apoptosis, inflammation, corticotropin releasing hormone signaling, synaptic long-term depression, dendritic branching, and cardiac hypertrophy were upregulated in stressed mice compared to the control. Suppressed transcripts were involved in synaptic long-term potentiation, lymphocyte activation, gap junction signaling, and glucocorticoid receptor pathway.
We characterized the regulation patterns of genes, metabolites, protein, DNA methylation, and their associated networks in a mouse model of PTSD. These patterns can be used as part of a diagnostic panel for the development of PTSD and for the validation of therapeutic interventions.
Individuals with disordered eating have the highest mortality rate of any psychiatric disorder and females make up the majority of the eating disordered population. Tobacco smokers have lower BMIs than do their non-smoking counterparts, and adolescent and adult females report using tobacco to lose or maintain body weight. Multiple biobehavioural factors contribute to this nicotine-body weight relationship, rodent studies suggest that reduced food intake following nicotine exposure may be a primary factor.
To examine the effects of nicotine on body weight changes in response to different food types.
We used an oral nicotine administration paradigm to investigate body weight changes in the presence of standard chow, high sweet and high fat foods in adolescent (
Adult mice weighed more but ate less food than did adolescents (
These findings suggest, in females, the appetite suppression qualities of nicotine differ based on age, with nicotine exposure actually increasing food consumption in adolescents. Nicotine's effects on food intake do not result in body weight changes in either age group.
One in five UK women is obese at antenatal booking. Maternal obesity increases risk of offspring obesity, behavioural and metabolic disorders. Animal studies suggest male offspring are more vulnerable to these effects than females. We hypothesised that this is mediated by altered action of maternal glucocorticoids, key regulators of development and stimulators of appetite and weight gain.
Serum cortisol levels were measured at 16, 28 and 36 weeks gestation in
Cortisol levels were significantly lower throughout pregnancy in obese than lean (
Lower circulating cortisol levels in obese pregnancy, together with the more effective placental barrier to maternal glucocorticoids may be a mechanism contributing to higher birthweight in offspring of obese women. In lean women, dietary composition may regulate cortisol levels during pregnancy.
A recent study showed that a mineralocorticoid receptor (MR) gene variant, MR haplotype 2, was associated with higher levels of dispositional optimism, less thoughts of hopelessness, and lower risk of depression in women but not in men. Mice lacking the MR in the forebrain, MRCaMKCre mice, were generated to further investigate behavioral sex differences with and without the MR. Here, the hypothesis that sex differences would disappear after deletion of the MR was tested.
We used male (
At the end of the conditioning, all mice acquired the fear-motivated response. During the first minutes of the memory test, both male and female MRCaMKCre mice remembered and feared the context more than the control mice. Furthermore, female MRCaMKCre mice were not able to extinguish this memory even on the second day of memory testing. The female mutants could also not discriminate between cue (more freezing) and context periods (less freezing). In contrast, male MRCaMKCre and control mice showed extinction and were capable to discriminate, although extinction of the MRCaMKCre mice started slower.
The loss of forebrain MR does not eliminate sex differences but rather results in large differences in emotional and cognitive behaviors between female and male mice. This finding suggests a role of this receptor in the female prevalence of stress- and anxiety-regulated disorders.
Adverse exposures that influence growth in prenatal and early postnatal periods are thought to influence vulnerability to chronic diseases via their effects on the neuroendocrine system. In humans, assessment of the underlying mechanisms has been restricted. The aim of the present study was to investigate the effects of adverse early life exposures, specifically maternal mood, on hypothalamic–pituitary–adrenal (HPA) axis, sympathetic nervous system (SNS) and parasympathetic nervous system (PNS) responses to an acute physiological stressor. In addition, we conducted a preliminary examination into whether effects varied by time of exposure and sex.
A total of 139 individuals (mean age 15.12 years) were recruited from the Avon Longitudinal Study of Parents and Children (ALSPAC) birth cohort. Participants underwent the CO2 stress test, and indices of the PNS, SNS and HPA axis were measured. Pre-existing data on mothers’ demographic and psychosocial factors during pregnancy (18 and 32 weeks) and postnatally (8 weeks and 8 months) were extracted, as were participants’ clinical and demographic data at birth.
Increases in both prenatal and postnatal anxiety and depression were associated with greater SNS reactivity to the stressor and slower recovery, as well as blunted HPA axis responses. Programming effects on the SNS appeared restricted to male offspring only. No consistent relationships were evident for any of the measures of pre-stress function.
We have found preliminary evidence that both pre- and postnatal maternal anxiety and depression have sustained programming effects on the SNS and HPA axis. Effects on the SNS were restricted to male offspring.
Long-acting injectable (LAI) antipsychotics have been developed to increase compliance in schizophrenia. Risperidone-LAI was the first LAI atypical antipsychotic, as a biweekly injection. Paliperidone Palmitate (PP) is a recently developed LAI atypical antipsychotic that is administered monthly. PP is hydrolized to paliperidone (9-hydroxyrisperidone), the primary active metabolite of risperidone. Although both risperidone and paliperidone are associated with increases in prolactin levels, there is limited information regarding whether there are differences in sexual functioning between both compounds. We aimed to study whether there are changes in prolactin levels and/or sexual function after switching from LAI-risperidone to LAI-paliperidone.
We have studied 12 psychopathologically stable subjects with a psychotic disorder (
There was a significant reduction in prolactin levels from baseline to the 3-month assessment. Those subjects with higher prolactin levels seemed to show a greater reduction. In relation to sexual dysfunction, although some cases improved notably in ASEX scores, the reduction was not significant in all the samples. None of the subjects reported worsening in psychotic symptoms.
In our sample, the switch from LAI-risperidone to paliperidone reduced prolactin levels during a 3-month period. However, changes in prolactin levels were not associated with a significant improvement in sexual functioning.
Subjects with psychosis are at risk for metabolic syndrome, mainly secondary to antipsychotic treatment although overeating also plays a role. The hypothalamic–pituitary–adrenal axis, which is involved in visceral obesity, may also affect energy balance through affecting energy intake and the risk for stress-induced hyperphagia. The main aim of the study was to study the association between salivary cortisol, overeating, and obesity in a sample of subjects with early psychosis.
We evaluated 53 participants (mean age: 23.5 years, 42% females) who were attending to the Early Psychosis Program from Reus (HPU Institut Pere Mata, Tarragona, Spain). All participants met criteria for a psychotic disorder or were considered at risk mental states after administration of the Schedules of Clinical Assessment in Neuropsychiatry (SCAN) or the Comprehensive Assessment of At Risk Mental States (CAARMS). Dietary habits were assessed by a dietician who administered the Eating Disorders module of the SCAN to explore two types of overeating: (1) Grazing, defined as repeatedly eating small amounts of food between mean meals (2) Binging, defined as consuming large quantities of food in a very short period of time. As grazing and binging can coexist in the same patient, we recoded overeating in four categories: (1) no overeating, (2) only grazing, (3) only binging and (4) grazing and binging. BMI and waist circumference were registered. A fasting morning saliva sample (9 h) was obtained. Salivary cortisol levels were determined by ELISA. Statistical analyses were performed with SPSS v.17.0. Kruskal–Wallis test was used to test differences between groups in continuous variables. Chi-squared tests were used to test differences between groups in categorical data.
Of 53 participants, 37 (69.8%) reported grazing and/or binging. Salivary cortisol was not associated with BMI or waist circumference. Of all participants, the group reporting both grazing and binging showed increased salivary cortisol levels (
In young participants with early psychosis, overeating (coexistence of grazing and binging episodes) is associated with greater BMI and increased morning salivary cortisol levels.
Research has attempted to link atypical hormone patterns to behavior problems in adolescents with varying success. Exploring the interactive effects of hypothalamic–pituitary–adrenal (HPA) and hypothalamic–pituitary–gonadal (HPG) hormones may help clarify findings. Given the lasting influence of early life stress on hormones and mental health, considering the effect maternal depression (MD) may lend additional clarification.
Longitudinal data from 346 youth (171 males) were used to examine these associations. MD was measured during infancy and preschool with the Center for Epidemiologic Studies Depression scale. Morning levels of cortisol, dehydroepiandrosterone (DHEA), and testosterone were assessed at age 15 years. Internalizing and externalizing at 15, 16, 17, and 18 years were assessed with multi-informant report on the MacArthur Health and Behaviors Questionnaire using variables measuring number of symptoms (severity) and preponderance of one symptom type over the other (directionality). A two-level hierarchical linear model examined how neuroendocrine measures, early MD, and sex independently and jointly influenced mental health trajectories.
For severity, a two-way interaction (
Results support the benefits of examining multiple hormones in the prediction of mental health problems and suggest additional hormone risk patterns are present in individuals exposed to early life MD.
In the central nervous system, the serotonin (5HT) neurotransmitter system plays a key role in the regulation of mood and emotion. Alterations in the 5HT system are thought to contribute to psychopathologies. In addition, drugs targeting the 5HT system are effective in the treatment of depression and anxiety disorders. Children with anxious temperament (AT) are characterized by excessive shyness, worrying, and avoidant behavior. This temperament, when stable across development, increases the risk of later developing depression and anxiety disorders. Using a well-established, nonhuman primate model of AT, we tested whether variations in the 5HT system predict individual differences in AT. We focused on the central nucleus region of the amygdala (CeA) because we have established that metabolic activity in this region is predictive of AT.
Using Affymetrix GeneChip® rhesus macaque genome arrays, we assessed gene expression from CeA tissue in 24 young male rhesus monkeys phenotyped for AT. Robust regression analysis was performed with correction for multiple comparisons across all annotated transcripts that are part of the neuroactive ligand pathway (KO04080) in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.
As hypothesized, variation in gene expression predicted individual differences in AT. Specifically, of the thirteen 5HT receptors assessed, only the 5HT2C receptor (5HT2C;
Previous work by others has shown robust levels of 5HT2C receptor mRNA and radioligand binding in the monkey CeA. In addition, rodent models have indicated a role for the 5HT2C receptor in anxiety-like responding. Our findings suggest that higher levels of 5HT2C receptor gene expression are associated with lower levels of AT. Increased expression of the gene encoding for this receptor may facilitate 5HT signaling in the amygdala thereby promoting adaptive responses and resilience to potentially anxiety provoking situations.
Although pessimistic individuals are at increased risk for developing cardiovascular disease, the biological mechanisms underlying this effect, and the social-environmental factors that modify these effects, remain unclear. To address this issue, we examined how pessimism, defined as the generalized tendency to expect negative outcomes, interacts with life stress exposure to predict metabolic health.
Seventy-one pre-menopausal mothers – 35 had a child with autism and 36 did not – participated in the study. They ranged from 28 to 51 years-old (
As predicted, pessimism and cumulative life stress exposure were each independently associated with greater metabolic risk, independent of age, income and caregiver status (Pessimism:
These results suggest that pessimistic individuals living under high levels of stress may have the greatest risk for cardiovascular disease and highlight pessimistic beliefs as a possible treatment target for reducing stress-related disease burden.
A Casabé, CG Roehrborn, LF Da Pozzo, S Zepeda, RJ Henderson, S Sorsaburu, C Henneges, DG Wong, L Viktrup. J Urol 2014;191:727–733
Since the launch of the 5-alpha reductase inhibitor finasteride in 1992, medical management of lower urinary tract symptoms (LUTSs) secondary to benign prostatic hyperplasia (BPH) has continued to evolve. Today, in addition to 5-alpha reductase inhibitors, both alpha-blockers and phosphodiesterase type-5 inhibitors are approved for the treatment of men with BPH/LUTS. Recently, various guidelines have recommended combination of a 5-alpha reductase inhibitor with an alpha-blocker to achieve earlier and greater LUTS improvement when compared with monotherapy of either group of drug alone. However, there is an increased risk of sexual dysfunction related to this combination of therapies that constructs the major concern related to this treatment option.
Tadalafil is a long-acting phosphodiesterase type-5 inhibitor that has recently been approved for the treatment BPH/LUTS. Clinical studies have demonstrated that tadalafil 5 mg once daily results in clinically significant improvements in BPH/LUTS with the additional benefit of restoring erectile function in patients with concomitant erectile dysfunction. In order to assess whether the combination of a 5-alpha reductase inhibitor with tadalafil may result in early LUTS improvement with fewer sexual side effects, Casabé et al. conducted a randomized, double-blind, placebo-controlled, multicenter study. The authors randomized 695 men to receive either tadalafil 5 mg or placebo once daily co-administered with finasteride 5 mg once daily over the course of 26 weeks. The results of this study demonstrated that tadalafil 5 mg coadministered with finasteride resulted in more rapid and more significant LUTS improvement when compared with finasteride coadministered with placebo. Moreover, the finasteride/tadalafil combination significantly improved erectile function in men who had erectile dysfunction prior to initiation of treatment. These results support that once daily coadministration of tadalafil with finasteride can be the treatment of choice for men with LUTS and erectile dysfunction symptoms.
TM Pisansky, SL Pugh, RE Greenberg, N Pervez, DR Reed, SA Rosenthal, RB Mowat, A Raben, MK Buyyounouski, LA Kachnic, DW Bruner. JAMA 2014;311:1300–1307
All currently available prostate cancer treatment modalities include the risk of erectile dysfunction (ED), and a significant amount of prostate cancer patients experiences ED after radiotherapy. In this multicenter, stratified, placebo-controlled, double-blind, parallel-group study, Pisansky et al. aimed to determine whether tadalafil 5 mg once daily maintains spontaneous erectile function in men treated with radiotherapy for prostate cancer. The authors randomized 242 men whose prostate cancer was treated with radiation to receive either daily tadalafil 5 mg treatment or placebo for 24 weeks. The International Index of Erectile Function (IIEF) was administered before radiotherapy, at week 2, week 4, between weeks 20 and 24, between weeks 28 and 30, and 1 year thereafter. IIEF did not demonstrate any statistically significant difference between the two groups. The authors concluded that daily use of tadalafil 5 mg compared with placebo did not result in improved erectile function, and they did not recommend this treatment modality to prevent ED in these patients.
JP Mulhall, N Verma, S Deveci, R Tal, K Kobylarz, A Müller. BJU Int 2014;113:656–661
Surgical or chemical castration is used as a standard therapy for metastatic cancer, and it is associated with impaired erectile function due to the imbalance between smooth muscle and collagen in the corpora cavernosa. Taking the beneficial effect of sildenafil citrate on penile tissue after cavernous nerve injury into consideration, Mulhall et al. assessed whether this molecule would result in preservation of erectile function and tissue structure in the rat penis after castration. They used 60 male Sprague-Dawley rats divided into three groups (sham, bilateral orchidectomy, and bilateral orchidectomy + sildenafil). The authors measured maximum intracavernosal pressure–mean arterial blood pressure (ICP/MAP) ratio, evaluated smooth muscle–collagen (SM–C) ratio, and defined apoptotic indices (AIs) on 7th and 28th postoperative days. The authors demonstrated that the ICP/MAP ratio was highest in the sham group, whereas it was lowest in the orchidectomy group. Rats treated with sildenafil after orchidectomy showed statistically significant improvements in their ICP/MAP ratio over the corresponding control groups; however, their ICP/MAP ratio was lower than that of rats in the sham group. On the other hand, the authors could not detect any significant differences in either SM–C ratio or AIs between the orcidectomy and orchidectomy + sildenafil groups. Considering the increased ICP/MAP ratios in the treatment group, the authors concluded that daily treatment with sildenafil may prevent or reduce the damage to penile tissue after castration.
EA Salem, EH Elkady, A Sakr, AM Maarouf, L Bendary, S Khalil, A Shahin, H Kamel. Urology 2014;84:1374–1377
The optimal graft material to be used in the penile incision and grafting surgery for the treatment of Peyronie Disease (PD) continues to be a matter of debate. Although various materials have been used for grafting procedures (i.e., dermis, vein, cadaveric and bovine pericardium, dura mater, synthetic materials, and porcine small intestine submucosal extracellular matrix), there is no consensus about the ideal graft in patients with PD. Synthetic grafts are associated with inflammatory and infectious complications, whereas tissue-engineered grafts are costly. In spite of their favorable outcomes low cost, the high incidence of harvest site side effects of autologous grafts limit their wide spread use.
In this study, Salem et al. evaluated the use of lingual mucosa graft (LMG) in 17 patients with PD. The angle of deformity ranged from 45 to 70 degrees with a mean angle of 60 degrees. All patients were assessed with the International Index of Erectile Function-5 (IIEF-5) preoperatively. Postoperative evaluation included IIEF-5 score and the assessment of penile deformity after 1 month, and then every 3 months until 18 months. Donor site complications were also reported. The authors found only mild and transient donor site complications such as pain and numbness. Fifteen of the 17 patients (88.2%) had straightening of the penis at follow-up. Two patients had mild residual curvature of the penis (20 degrees) which was treated conservatively. Erectile function was maintained in 16 patients; 94% of patients reported satisfaction with the procedure. This report is the first study to analyze the use of LMG for PD, and excellent results were obtained. The advantages of this technique include easy accessibility of the tongue mucosa compared with the deeply seated buccal mucosa, lower costs compared with “off the shelf” graft materials, and lower donor site morbidity. These results must be analyzed with caution because of the short follow-up period, small sample size, and single institution nature of the study. As all patients in this series had curvature less than 70 degrees, the applicability of this technique to more severe curvature is unclear. Further studies on lingual grafts for PD are warranted.
J Klap, M Schmid, KR Loughlin. J Urol 2015;193:403–414
For many years, testosterone was believed to play a major role in the development of prostate cancer (PCa). In the last decades, serious controversies have emerged about the true relationship between testosterone and PCa. In this review article, the authors looked for the answers to the two main questions: (i) Is there any link between serum testosterone and incidence, grade, and aggressiveness of PCa? (ii) What is the true risk of testosterone replacement therapy (TRT) in patients with primary hypogonadism that already treated PCa or are in active surveillance program?
The authors reviewed the literature from 1994 to 2014 and analyzed the results of 45 articles were selected and analyzed, of which 18 and 17 showed a relationship between PCa and low and high total testosterone, respectively. On the other hand, 10 studies showed no relationship between testosterone and PCa.
The discrepancy in these results may be attributable to biological and/or methodological factors. The first biological explanation is the Saturation Model, which states PCa cells are sensitive to fluctuations from very low doses of testosterone. As levels increase, the androgen receptors become saturated, and additional testosterone does not influence androgen-driven changes in prostate tissue growth. Additional biological factors that must be considered include the functional activity of androgen receptors in prostate tissue and the theoretical role of intraprostatic testosterone and dihidrotestosterone. Finally, the effects of estrogens on carcinogenesis of PCa are incompletely elucidated; some of the biological effects of testosterone on the prostate may be mediated by aromatization of testosterone to estradiol.
The main methodological failure of this analysis is that only three of the 45 studies collected blood for testosterone assay in the morning and only one study collected two or more samples. Thus, testosterone identified as low in some studies may have been normal on another day or at another hour during the day. No definitive conclusions can be made about the relationship about testosterone and PCa from these trials. Additional trials must be done to overcome the methodological obstacles and clarify biological hypothesis.
There are relatively few papers on TRT in patients previously treated for PCa. It is advisable that patients considering TRT after treatment for PCa be extensively counseled on the controversies surrounding TRT in this situation; patients who wish to proceed should sign informed consent forms and be followed very closely with serial digital rectal examination and PSAs. There are only two studies of TRT in patients in active surveillance for PCa with contradictory results. TRT may be considered in such cases, but it must be made clear that the long-term risks are unknown, and there exists the possibility of PCa progression from this treatment.
FH Silva, C Lanaro, LO Leiria, RL Rodrigues, AP Davel, MA Claudino, HA Toque, E Antunes. Am J Physiol Heart Circ Physiol 2014;307:H1393–1400
The role of sympathetic neurotransmission in erectile dysfunction (ED) is increasingly becoming a hot topic in sexual medicine, particularly in the field of penile rehabilitation program after radical prostatectomy. The leading theory dictates that nitric oxide (NO) is a negative modulator of sympathetic neurotransmission. Vasoconstriction and increased corporal smooth muscle tone produced by sympathetic signals are enhanced by impaired NO bioavailability. This enhancement of hypercontractility in cavernosal tissues and their supplying arteries promotes the development of ED.
In the present article, the authors investigated the role of oxidative stress in the pathogenesis of ED focusing on its link with the development of an aberrant sympathetic neurotransmission in the erectile tissue. The authors report that ED in middle-aged rats (10 months old) is associated with up-regulation of mRNA coding for tyrosine hydroxylase (a specific marker for sympathetic nerve), and increased phenylephrine-induced contractions in organ bath studies, along with alpha1-adrenoceptor-mediated cavernosal vasoconstriction. Furthermore, decreased expression of soluble guanylyl cyclic in cavernosal smooth muscle was also reported.
NADPH oxidase is localized in sympathetic nerve fiber endings, which indicates a superoxide-mediated mechanism in neurovascular control. Interestingly, the present study reported that the treatment with the NADPH oxidase inhibitor “apocynin” normalized the reactive oxygen species (ROS) levels and restored the tyrosine hydroxylase mRNA expression and hence the sympathetic cavernosal contractions. These results demonstrate a role for the elevated oxidative stress contributing to ED in middle-aged rats. Excess of superoxide could promote a higher tyrosine hydroxylase expression, whereas in the latter, it reduces NO bioavailability and sGC expression, which in turn causes ED by mechanisms involving impairment of relaxations and facilitation of contraction. Based on these data, the authors concluded that antioxidant therapies might represent an interesting approach to manage ED in aging population.
While the results presented provide a clear message on the role of oxidative stress in sympathetic hyperactivity in aging induced ED, this study is not devoid of limitations. The major study limitations include the lack of an in vivo evaluation of erectile function and the “age” of the rat selected for mimicking the middle age human condition. Without an in vivo evaluation, it not possible to evaluate the degree of erectile function impairment in the rats. Moreover, a 10-month-old rat is equivalent to a 28-year-old man, and older animals (20–22 months) should have been selected to study the age-dependent effects on erectile function.
GN Yin, MJ Choi, WJ Kim, MH Kwon, KM Song, JM Park, ND Das, KD Kwon, D Batbold, GT Oh, GY Koh, KW Kim, JK Ryu, JK Suh. Proc Natl Acad Sci U S A 2014;111:E2731–2740
Much progress over the past two decades has improved our understanding of the molecular pathophysiology of erectile dysfunction (ED). However, few medical therapies have been developed since the introduction of phosphodiesterase inhibitors in 1998. Nonetheless, our improved understanding of erectile function and dysfunction has yielded new drug targets over the past several years, including extracellular-related kinase, guanylate cyclase, the RhoA/Rho-kinase pathway, and the endothelin and angiotensin receptors. In 1996, nerve injury-induced protein 1 (Ninjurin 1, Ninj1) was identified as an induced factor after axotomy in neurons and Schwann cells that promote neurite extension [
Rather than merely focusing on surgical nerve injury, the same group more recently evaluated the role of Ninj1 in mice with diabetes-induced ED, finding that Ninj1 inhibition facilitates erectile function in this setting as well [
When evaluating the effects of Ninj1 inhibition on nerve content, the authors found not only more neurofilaments in mouse penis undergoing Ninj1 inhibition but higher levels of neuronal nitric oxide synthase (nNOS) in Ninj1-inhibited penis. Preservation of nNOS is significant given that nNOS release from nerve terminals initiates penile erection. These effects on nerve preservation were mediated by secretion of nerve growth factors (NGFs) including brain-derived neurotrophic factor, NGF, and neurotrophin 3, which were markedly upregulated during Ninj1 inhibition.
The authors then expanded their investigations to Ninj1 knockout mice, which had preserved erectile function even in the setting of STZ treatment, further confirming a role for Ninj1 in ED pathogenesis. Finally, while the presence of Ninj1 and Ang1-Tie2 are important in regulation of diabetes-induced ED, the entire pathway has not yet been described. To identify Ninj1 target genes, the authors used gene expression microarray technologies in MCECs exposed to high and normal glucose conditions, and with and without knockdown of Ninj1 using siRNA, finding numerous genes that may play a role in the pathway.
This study is significant in that it describes a novel pathway regulating both angiogenesis as well as neurogenesis in the penis and suggests a novel therapeutic target in the setting of ED. The detailed mechanisms by which Ninj1 is induced, the components of the Ninj1 pathway, and the mechanisms of angiogenesis and neurogenesis mediated by this pathway remain to be defined. In addition, the role of the Ninj1 pathway in humans is currently unknown. It will indeed be interesting to see what future investigations yield in determining whether Ninj1 is a bona fide regulator of human erectile function, and therefore a target for future therapies.
KJ Derefinko, JR Peters, TA Eisenlohr-Moul, EC Walsh, ZW Adams, DR Lynam. Arch Sex Behav 2014;43:1149–1158
The outcomes of risky sexual behavior have a long-term negative impact on individuals' private and public lives. Young adults are at particular risk for these outcomes. A sample of 135 undergraduate men (Mage = 19.5) participated in an experimental study aimed at assessing the unique predictive value of impulsivity-related traits, behavioral markers of risk taking, and physiological reactivity in three distinct indicators of risky sex: number of sexual partners, having sex with strangers, and irregular condom use. For the first time, three approaches, usually considered individually in the study of sexual risk taking, were incorporated in a single design: (i) trait markers of risky sex (evaluated by self-report), (ii) behavioral markers of risky decisions (measured by experimental means, usually involving some level of awareness), and (iii) physiological reactivity to emotional stimuli (measured experimentally, involving no awareness by the participant). In this study, participants answered to a set of self-report measures assessing impulsivity (negative urgency, positive urgency, and sensation seeking), participated in a behavioral risk taking task (the Ballon Analogue Risk Task [BART]) and in a reward-seeking Go/No-Go task, and underwent a psychophysiological evaluation (skin conductance reactivity) to pleasant vs. unpleasant stimulation. Scores on these markers were used as predictors of the mentioned indicators of risky sex. Sensation seeking (β = 0.19) and risk taking on the BART (β = 0.18) significantly predicted number of sexual partners (
L Liao, S Lunn, M Baker. Sex Relation Ther 2015;30:167–180
How do men appraise menopause? A lot has been written about women's perceptions of menopause and so little about the male perspectives. In this qualitative study, eight heterosexual male partners (Mage = 51.5) described their perceptions about midlife menopause. Male partners' views of menopause were analyzed through thematic analysis (TA); this method is particularly suitable when no structured empirical data exist. Participants were interviewed by a white female psychologist in her early thirties, either at the participant's home or the interviewer's workplace. The following general topics emerged:
Menopause was seen as a process, something to be entered into and completed. During this process, women were described as emotionally charged. Simultaneously, some men appraised menopause as a chance of personal growth and a period in which women can expand into a more liberating life.
Men recognized that menopause is a shared process implying a transition to a new dyadic stage. Yet men felt they were pulled unprepared into this process. They felt helplessness. They were expected to support their partners but were left unsupported by the system. Also, men alluded to some positive and negative outcomes in sex. Some expressed preoccupation about the partner's loss of libido. But, on the other hand, menopause was also regarded as positive (e.g., liberation from the contraceptive constraints).
Menopause was referred as a taboo topic possibly because of its overlap with sexual phenomena. Men do not talk about it because they feel embarrassed. Menopause was regarded as a sign of diminished female sexuality. Men felt they had to respect the partners' privacy rather than discuss this topic. Finally, most interviewees agreed that there is a lack of general knowledge on this subject; menopause was defined as a nebulous area and not many options exist to clarify men's doubts. Women seem to be the only target of information and support.
In all, men lack knowledge on menopause because of a social etiquette that defines who is expected to receive such information. Yet men want to know more about menopause as they too have to go through this process. Although this was a first attempt to understand men's perspectives about menopause, findings support the role of professionals in assisting male partners of menopausal women.
Barret C, Whyte C, Comfort J, Lyons A, Crameri, P. Sex Relation Ther 2015;30:131–142
We very much enjoyed reading a letter by Drs Joulaei and Motazedian published in an earlier issue of your journal (
Although the PHC in Iran has a high level of political and medical commitment in the control and prevention of several diseases, its limitations cannot be ignored (
MARPS do not tend to be seen in public and are less likely to refer to public clinics which may reveal their HIV status to the general population. We believe the stigma surrounding HIV/AIDS is really profound and most people in the family-oriented context of Iran care deeply about the issues of “face” at both individual and family levels. Most People Living with HIV (PLHIV) worry that the disclosure of their HIV status to the community can negatively affect themselves and their families in the terms of employment and the level of social support they would receive. As a result, assuming that PLHIV would approach a PHC clinic is a little bit far-fetched (
On the other hand, the majority of MARPS live in cities; even those who may live in rural areas tend to migrate to cities where they can choose an isolated life and avoid the stigmatized look of those around them. In other words, the effectiveness of PHC in rural areas in reaching MARPS can be questioned. Therefore, we assume we should be more realistic when considering PHC for controlling HIV/AIDS. We doubt that we can put the burden of HIV/AIDS on the shoulders of PHC.
We think a partial vertical approach should still be taken into account. Despite the disadvantages of a vertical health program, we still believe their defined goals and objectives with measureable outcomes as well as high accountability and the rate of success in limited timeframe make them a better option in the context of Iran. We do not recommend an entire isolated vertical care and treatment system. In our model, we suggest a vertical system in providing special care and treatment to those who are at a higher risk of infection, as well as PLHIV along with their families. This system should be compatible with the rules and regulations of a comprehensive PHC with appropriate links to formal components of the national health system in order to access its data and resources, benefit from its settings, and seek support from the personnel of the system.
All in all, considering that HIV is not only an infection, but a complex and multifaceted issue, reaching the MARPS through only the PHC system in Iran is not that achievable and despite its effectiveness, PHC cannot have a key role in this regard. We recommend the authors address such limitations to PHC in Iran, and suggest a more comprehensive scheme to approach this complicated issue.
Swine flu is the present problem of the world. In the tropical countries, swine flu is still pandemic.
The author hereby reports and discusses on a pediatric case of swine flu. The case is a 7 year-old boy (body weight 28 kg) presenting to the primary care center in Bangkok, Thailand with the complaint of high fever, non productive cough, nausea, vomiting and malaise. His body temperature was 39.2 °C. His throat was red and lung clear. The attending physician performed tourniquet test and got positive result. The boy was referred to the hospital for further proper management. At the hospital, complete blood count was done and no thrombocytopenia could be detected. In this case, the finalized diagnosis as swine flu was derived. The Real time PCR test was done to confirm new H1N1 influenza virus infection (confirmation was performed at Thai Department of Medical Science). During hospitalization, investigations done to rule out co-existing dengue fever or other infections included hemoculture, dengue serological study (paired serum test) and Chikungunya serological study (results of all tests were negative). Chest X ray was also done in this case and there was no lung involvement. This case was treated by antiviral drug (Oseltamivir 60 mg twice daily) and got full recovery within 10 days. Of interest, high fever and flu like symptoms are non significant and several tropical diseases can have this presentation. In Thailand, dengue infection is common and tourniquet test is helpful in screening and diagnosis[
Erythromycin has been the classic drug of choice for Streptococcal pharyngitis in case of allergy to penicillin.
To determine the resistance rate of Group A beta hemolytic Streptococci (GABHS) to erythromycin in Mashhad (population 2,500,000), we performed a biphasic research, composed of two retrospective (April 1998- March 1999 and April 2003-March 2005) and a prospective studies. In the first part, we collected GABHS positive cultures and their (disk diffusion) antibiograms from three medical diagnosis laboratories in Mashhad. In the second phase, in April 2005, throat cultures were taken from 204 elementary, high school and college students; antibiograms were done by both disk diffusion and E-test.
In the retrospective study, during 1998-1999, thirty-seven of 62 (59.67%) GABHS isolated samples and during 2003-2005, thirty-six of 44 (81.87%) GABHS isolates were resistant to erythromycin. The mean age of the 2003-2005 group was 25.5 years. The resistance rate in children less than 10 years old was 71.42% and in the older ones 88.23% (
In the prospective study, 76 of 204 (37.3%) throat samples were positive for GABHS and both the E-test and disk diffusion method showed that 73.7% (56 of 76) of the isolates were resistant to erythromycin. The mean age for this group was 14.63 years. Although the rate of erythromycin resistance was higher in the high school students, the difference was not statistically significant (
The rate of Erythromycin resistance among GABHS in Mashhad between 1998 and 2005
The highest rate of erythromycin resistance for GABHS (98.8%) has been reported from China[
In our study there was 100% concordance in the erythromycin resistance of GABHS by disc diffusion and E-test methods. Other studies have also shown such a close identity between the two methods[
What is the cause of this high grade of erythromycin resistance? The rate of macrolide resistance is closely related to the extent at which these agents are used; at the time of our study (2005) macrolides were not among the most common antibiotics that were being used in Iran. According to the report of the food and drug organization of the Ministry of Health of Iran (
Age is an important factor in the rate of antibiotic resistance. Significant negative correlation has been found between the age of patients and the erythromycin-resistance[
We conclude that GABHS has a high rate of resistance to erythromycin in Mashhad, and although we couldn't find a reasonable explanation for this high degree of resistance, erythromycin can no longer be used for empirical treatment of streptococcal pharyngitis in this city. This project was funded by the Research Vice Chancellor of Mashhad University of Medical Sciences.
I read with great interest the article by Heydarian et al [
The authors described that bottles of drug and placebo were similar in size and shape. However, it is well known that, the most important factor in zinc trials have been problem in blinding due to bad taste of zinc salts. There is also no description of the adverse events in the study (even in the zinc group), which is really surprising[
The authors also described that, as studies using zinc have found conflicting results in pneumonia, they conducted this trial to find if zinc benefits another acute lower respiratory tract infection (ALRTI), namely acute bronchiolitis. But they also did not find any beneficial effect. This can be explained as follows. It is known that, benefit from zinc administration seems to increase after 100 hours of illness, and might be inherent in the mechanism of the zinc effect [
Though the authors do not have any explanation why respiratory distress symptoms were improved significantly 24 hours after therapy in the control group, the same has not been shown in the Table 2 (I could not find any result to be significant between the two groups in the table, even after 48 or 72 hrs).
Lastly, the authors describe that, zinc administration does not improve clinical manifestations and hospitalization, in subjects with normal serum zinc level before treatment. However, this is not true if we consider the following points. First, plasma zinc levels are not the most reliable indicators of zinc status in the body[
To conclude, more research is needed in this area before any firm conclusion can be made. For the result to be meaningful, above factors must be taken care of.
None
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There are three myths about the role that the public can play in determining their health, said Professor Marcus Longley, Director, Welsh Institute for Health and Social Care, and Professor of Applied Health Policy, University of Glamorgan: they cannot weigh risks and benefits; scientific uncertainty is dangerous for lay people; and most people would simply rather not know.
As with all myths there is an element of truth in these statements but modern thinking about public health is more nuanced. An independent report based on the latest evidence from behavioural economics and psychology concluded that ‘People do not smoke or drink too much because they are ignorant, stupid or perverse – rather, it is the combination of the enjoyment that they get from these things and wider social or other environmental factors that mean they find it hard to adopt healthier behaviours [
The goal of public health policy has long been to increase life expectancy but this has been pursued at the cost of quality of life in later years [
What do we understand by ‘risky’? The risk of fatality for a 50 year-old man taking aspirin to prevent a coronary event is 10 per 100,000 person-years of use. This is about the same risk as being a passenger in a car (11 per 100,000 person-yrs) and far lower than for a motor-cyclist (450 per 100,000 person-years [
Five years ago, before the association between aspirin and cancer risk reduction was publicised, Professor Longley and Professor Elwood convened a Citizens’ Jury to consider what information should be provided to the public and how it should be delivered [
In this trial, the 16-member jury considered the case of cardioprevention with low dose aspirin. Jury members were a diverse group with contrasting views pre-trial views about aspirin but they collaborated effectively to develop 10 recommendations (
Considering the three myths, Professor Longley said that the Citizen’s jury showed that the public can weigh scientific evidence, though they need a process and support to do so and this is resource-intensive. They can deal with uncertainty – jurors wanted to be informed even about issues that were controversial among scientists. It was less clear whether the jury refuted the assertion that people would rather not know.; Professor Longley suspected that health was not an important issue for some people.
Questions remain. How can the views of vulnerable people be represented? What are the resource implications for clinicians? Can this approach work for issues that are less clear than the aspirin debate? Do health professionals and the public share a common understanding about what constitutes ‘health’, and is it more important to some people than others?
Citizen’s jury recommendations on the use of low-dose aspirin for cardioprevention [
There is good evidence to support the use of aspirin as secondary prevention of cardiovascular events but not for primary prevention in healthy people at low risk. The case for primary prevention in people at high risk (eg the elderly, those with diabetes or multiple risk factors) is unclear. Unsurprisingly, the public is confused by positive and negative headlines about aspirin, acknowledged Professor Jane Armitage, Professor of Clinical Trials and Epidemiology, Clinical Trials Surveillance Unit, Oxford.
Summarising the evidence for secondary prevention, she said the Antithrombotic Trialists’ Collaboration had shown, using patient-level data, that antiplatelet treatment reduced major coronary events and ischaemic each by one-third and vascular mortality by one-sixth [
There are two possible strategies for avoiding a first vascular event in individuals at low-risk: start aspirin immediately and continue for life, or delay aspirin until definite signs or symptoms of vascular disease emerge. Immediate treatment may avoid first heart attacks and strokes but exposes users to an increased risk of cerebral haemorrhage and gastrointestinal bleeding, for many years.
The assessment of the potential benefit of aspirin must take into account the changing background incidence of vascular events and alternative interventions. Vascular mortality has declined substantially in many countries among younger adults and both vascular and coronary heart disease among 70–79 year-olds (
There have been six large studies of primary prevention with aspirin, with approximately 660,000 person-years of follow-up in a population of 95,000 largely healthy people, of whom about one-third were over 65 years old [
Current management guidelines recommend life-long aspirin to all those whose 10-year risk of coronary heart disease is predicted to be 10–20 per cent. However, this recommendation assumes that bleeding risk remains approximately constant (about 0.1%/year) for everyone or is determined only by age. However, the risk of bleeding is also increased by comorbidity (such as diabetes), smoking, raised blood pressure and being a man. There are, therefore, several factors that can increase the risk of bleeding events. It is also evident that the absolute benefits of primary prevention with aspirin are greatly reduced when given with other drugs which halve the risk coronary events in individuals at moderate or high risk (
Professor Armitage summarised the arguments by saying that primary prevention with statins and anti-hypertensive drugs can halve the risk of vascular events. The benefits of adding aspirin to this strategy are small and are associated with small risks of bleeding. It is therefore not appropriate to recommend aspirin for a large proportion of the general population. On the other hand, there are sufficient grounds for individuals to make a rational choice to take aspirin if they consider the benefits to be worth the risks for themselves. Studies now underway will provide evidence in important subgroups such as people with diabetes and older people in coming years.
Mortality trends for vascular disease (left) and CHD (right) aged 70–79.
RCTs of aspirin for primary prevention.
Gastro-intestinal bleed (or other major extracranial bleed) in primary prevention trials.
The effect of concurrent treatment on the efficacy of primary prevention with aspirin in individuals at moderate and high risk (A=aspirin, C=controls [
Dr Hugh Turner, GP in County Durham and a Fellow of the Royal Society for Public Health, said that he had come to the meeting hoping to find a clear message about using aspirin that he could take home to his patients. Tongue in cheek, he summarised the conclusions of the meeting in the form of advice to one of his patients in this way. “There is good evidence that aspirin, when taken (sitting upright) every day as an oblong tablet of 75 mg–300 mg, reduces cancer risk after 5 years. Taking it at night may increase the benefit. Before starting, have a test for
Dr Turner’s point was that there is clearly room to improve the clarity of the information that patients receive about aspirin, and he added that there is also a need to educate health professionals.
In discussion, participants expressed reluctance to see aspirin replace the five healthy behaviours but it was acknowledged that most people will not make the necessary changes to their lifestyle. Public health initiatives, though they have the potential to achieve a greater risk reduction than aspirin, are not popular with the public.
This report was funded by an unrestricted educational grant from Novartis Pharma AG.
This includes expenditure on R&D funded by grants or in-licensed to other companies or institutions, and proportional expenses for joint ventures. R&D refers to personnel-related costs, such as salaries, consumables and a suitable share of expenditure to account for administration, depreciation, rent, but capital R&D expenditure is excluded.
This includes complete products and bulk sales as well as royalties from licensed out medicinal products.
PhRMA reports on new medicines in development for cancer.
Reversion of cells to an immature or a less differentiated form, as occurs in most malignant tumours.
Regression of a specialised cell or tissue to a simpler, more embryonic, unspecialised form. Dedifferentiation may occur before the regeneration of appendages in plants and certain animals and in the development of some cancers.
The war on cancer. In:
Such as arsenic trioxide.
Such as Sugen’s SU5416 and SU6668.
AACR-NCI-EORTC Molecular Targets and Cancer Therapeutics Nov 17–23 2003. Abstract Numbers: A87 & 102.
Defined as the adjustment of the immune response to a desired level, as in immunopotentiation, immunosuppression or induction of immunological tolerance.
Are a category of signalling molecules that are used extensively in cellular communication. They are proteins, peptides or glycoproteins.
AACR-NCI-EORTC Molecular Targets and Cancer Therapeutics Nov 17–23 2003. Abstract Number: A18
Adjuvants are pharmacological or immunological agents that modify the effect of other agents (e.g. drugs, vaccines) whilst having few if any direct effects when given by themselves.
Glioblastoma multiforme (GBM) is the most common and most aggressive type of primary brain tumour in humans.
Population and GDP figures from UN. 2007e. World Population Prospects 1950–2050: The 2006 Revision. Database. Department of Economic and Social Affairs, Population Division. New York. Accessed July 2009.
The major network of Experimental Cancer Medicine Centres (ECMCs) was established in 2006 across the United Kingdom to bring together laboratory and clinical patient-based research to speed up the development of new therapies and biomarkers by evaluating new drugs and individualising patient treatment. It is a joint initiative between Cancer Research UK and the Departments of Health in England, Scotland, Wales and Northern Ireland. See:
See
See:
EROCAN+Plus has the objective to support the harmonisation of European cancer research and EUSTIR to integrate research and to develop a common European strategy on breast cancer. See:
See:
L’ Institut National du Cancer. See:
See:
For four of the drugs, this was not possible, and only four ‘quintiles’ could be used.
The * denotes all terms linked to the primary word/term.
A paper with two UK and one French addresses would count unity for each on an integer count basis and 0.67 and 0.33, respectively, on a fractional count basis.
When production of clinical trial supplies are contracted out to a third party, there is an obligation by the trial sponsor to audit the facility ensuring operation to appropriate standards and for QP to confirm clinical trial materials are made to EU GMP standards. This is a major issue for products being made outside the EU (e.g. USA), as an EU QP needs to release the product and confirm it conforms to EU GMP standards regardless of its acceptability elsewhere (e.g. US FDA).
See:
The two cultures is the title of an influential 1959 Rede lecture by the scientist and novelist CP Snow Its thesis was that the breakdown of communication between the ‘two cultures’ of modern society – the sciences and the humanities – was a major hindrance to solving the world’s problems.
Pearson Commission on International Development.
For example NCI/NIH Developmental Therapeutics Programme (
This chapter builds on the methods outlined in: Kanavos P, Li G, Vandoros S. (2008). The Value of Pharmaceutical Innovation: Perspectives and Outlook, Paper submitted to LIF LSE Health, mimeo, December. The chapter extends and updates the evidence in that paper to 2009.
Variation in uptake and diffusion can signify the sub-optimal use of technology. Excess use is signified when the costs outweigh benefits for any additional level of technology diffusion or use. Under-use can occur when the foregone benefits outweigh the costs of additional diffusion or use. Both scenarios are sub-optimal, potentially resulting in economic costs and/or reduced health outcomes.
Estimates of minimum €500 million annually (2008).
And which could be equal to the highest rate of generic penetration at the molecule level in a given country.
Association of British Pharmaceutical Industry (UK)
Actual citation impact
American Cancer Society (USA)
Agency for Healthcare Research and Quality (USA)
American Institute for Cancer Research (USA)
Amelioration du Service Medical Rendu (France)
Biotechnology and Biological Sciences Research Council (UK)
Bacillus Calmette-Guerin
Children’s Cancer and Leukaemia Group
Canadian Cancer Research Alliance (Canada)
Centers for Disease Control and Prevention (USA)
Chronic myeloid leukaemia
Centre for Medicines Research
Cooperative research and development agreements
Cancer Research UK (UK)
Cancer stem cells
Common Scientific Outcome
Disability adjusted life years
Dendritic cell
Magnetic resonance imaging
Department of Health and Human Services Agencies (USA)
Deoxyribonucleic acid
Department of Defense (USA)
Department of Energy (USA)
Department of Health (UK)
European Commission
Experimental Cancer Medicine Centres
European and Developing Countries Clinical Trials Partnership
European Federation of Pharmaceutical Industries and Associations
Epidermal growth factor receptor
European Free Trade Association
European Medicines Agency
Environmental Protection Agency (USA)
Economic and Social Research Council (UK)
European Union
US Food and Drug Administration (USA)
Federation of European Cancer Societies
Framework Programme
Global Alliance for Vaccines and Immunisations
Gross Domestic Product
Global Fund to Fight AIDS, Tuberculosis and Malaria
Good manufacturing process
Hepatitis B
Health Care Financing Administration (USA)
Human epidermal growth factor receptor 2
Human immunodeficiency virus
Human papilloma virus
Initial Cancer Research Partners
International Cancer Research Portfolio
Interferon, alpha/gamma
Interleukin-2
International Medicines Initiative
Institut National du Cancer (France)
Institute of Medicine
Institute of Medicine (USA)
Intellectual property rights
Intellectual property rights
Innovative Therapies for Children with Cancer
Japan Pharmaceutical Manufacturers Association
Market Authorisation
Monoclonal antibodies
Multi-drug resistance
US Medical Expenditure Panel (USA)
Medical Research Council (UK)
National Agency for Space Exploration (USA)
New chemical entity
National Cancer Institute (USA)
National Cancer Research Institute (UK)
New drug applications
Non governmental organisations
National Health Service (UK)
National Institute of Health (NIH)
Natural killer
New molecular entity
Non-small cell lung cancer
National Science Foundation (USA)
% of papers on clinical guidelines
% of papers on government policy
% of papers in mass media
% of papers in text-books
Potential citation impact
Positron emission tomography
Pharmaceutical Research and Manufacturers of America
Paediatric investigation plan
% of reviews
Quality assurance
Quality control
Research and Development
RAND Corporation’s Research and Development in the US database
Research funding organisations
Research level journals
Research level titles
Ribonucleic acid
Return on investment
Rest of world
Southern Europe New Drug Organisation
French Society of Paediatric Oncology
International Society of Paediatric Oncology
Summary of Product Characteristics
Supplementary protection certificate
Serotonin re-uptake inhibitors
Strengths, Weaknesses, Opportunities and Threats
Tricyclic anti-depressants
Tumour infiltrating lymphocytes
Tumour necrosis factor, alpha
Target related affinity profiling
Utility of journals
United States of America
US Department of Agriculture (USA)
US Patent and Trademark Office (USA)
Department for Veterns’ Affairs (USA)
Vascular endothelial growth factor
World Health Organisation
Web of Science
‘Mediterranean diet’ has been associated with reduced risk not only of cardiovascular disease but also of cancer at various sites. Selected aspects of the Mediterranean diet have been related to cancer risk; a series of case-control studies conducted in Italy since the early 1980s on over 15,000 cases of 20 cancer sites, and a similar number controls. For most epithelial cancers, and particularly for digestive tract cancers, the risk decreased with increasing vegetable and fruit consumption, with relative risks between 0.3 and 0.7 for the highest versus the lowest tertile. A number of vitamins and other micronutrients or food components (including carotenoids, lycopene and flavonoids) showed an inverse relation with cancer risk. With reference to flavonoids, our data provided support for a protective role of flavanones on upper aerodigestive tract, proanthocyanidins on gastric cancer, flavonols and proanthocyanidins on colorectal, flavonols and flavones on breast, and isoflavones on ovarian cancers. However, the components responsible for the favourable effect of a diet rich in vegetables and fruit remain largely undefined. Fish, and consequently, a diet rich in n-3 fatty acids also tended to be favourable diet indicators. In contrast, subjects reporting frequent red meat intake showed elevated risks for several common neoplasms. Whole grain food (and hence fibre) intake was related to reduced risk of several cancers, particularly of the upper digestive tract, but refined grain intake and, consequently, glycaemic load and glycaemic index were associated with increased risk of different types of cancers. Olive oil—the major common characteristic of diet in various parts of the Mediterranean—is a major source of monounsaturated fats in Mediterranean countries but also an important source of several micronutrients and food components. It appears to be a favourable indicator of the risk of various common cancers, particularly of the (upper) digestive tract but also of the colorectum, breast and ovary.
In this report, I will aim to define the characteristics of the nursing appraisal that facilitates a nutritional assessment of cancer patients.
I will begin with some important quotes, the first being one from Florence Nightingale (this year marking the one hundredth anniversary of her death) in 1860 in her publication ‘Notes on Nursing’, a founding text for modern nursing care (so much so that the introduction reads ‘What it is, and What it is not’), devotes an entire chapter to ‘Taking Food’.
Virginia Henderson in the declination of what constitutes the thinking behind the production of theory (of the most notable in International circles), identifies 14 nursing care needs that are common to every human being, which include eating and drinking.
We can, therefore, say that the concept of nutritional needs is historically recognised as part of specific nursing interventions, which are to be activated where required or where potentially required. Today, pre-registration nursing courses require the study of nutrition, and the documentation systems require detailed records of the characteristics of that need.
Time passes quickly and looking at the present day and the context here in Lombardy, we cannot fail to note, always more stringently, that health care organisations, and their employees are required to demonstrate (through compliance with standards) the processes to which we subject our patients (or to put it as Virginia Henderson—the beneficiaries of our intervention). The definition of specific standards for nutrition and nutritional therapy (e.g. AOP 1.6, COP 5, PFE 4, etc.) are an example.
I have deliberately quoted the context of Lombardy for the implementation of the standards of Joint Commission, but I must mention the urban context as the Local Health Agency of Milan has adopted the Guidelines in the Prevention of Malnutrition.
With all of these requirements, comes the task of analysing patient’s notes regarding aspects that make reference to diet, supplemented by research to develop a thesis of a student dietician with the same goal.
In agreement with the Dietetic service of the EIO, we decided to deal with this issue by allowing a multidisciplinary team, including nurses and dieticians, to move from the concept of identification of nutritional needs, towards an assessment of the nutritional status of the oncology patient. This requires a multidisciplinary approach in which there is clarity regarding completeness and accuracy of data to be collected, definition of responsibility and definition of timeframes (in this case, there is the necessity to consider continuity of care). With regards to completeness and accuracy of data collection, we have included a focus of interest on the evaluation of nutritional status in the process that has led to the development of new nursing documentation (today paper, tomorrow computerized and integrated with other health care professionals notes). This project of revision anticipates the passage towards a documentation oriented to the identification of a Nursing Minimum Data Set (NMDS) and to the identification of existing specific references to nutritional status (e.g. weight loss in the last three–six months, glucose intolerance, etc.). The working group, drawing on the scientific production by the Oncology Nursing Association has led to the development of an NMDS of 48 outcomes, grouped into 13 categories, one of which is that of NUTRITIONAL STATUS.
In relation to the accuracy of data collection, we have focused the discussion on what are the necessary data to investigate and in which phases of the nursing assessment. We have identified the need to investigate normal weight, weight changes in the last three months, calculation of the body mass index, capacity/limitations in the assumption of food, reporting of food intake in order to cover calorific needs and the definition of ‘items’ in order to activate a dietetic consultation/referral. For the definition of this last aspect, we made a choice that was in line with the policy and characteristics of the EIO and have used the terminology of the Common Terminology Criteria for Adverse Events (CTCAE version n°4) from the National Cancer Institute (USA).
The definition of levels of responsibility is a direct consequence of the adoption of valid nursing documentation (Palese
Finally, we have considered the theme of time frames, or ‘What time is appropriate to enable the assessment of nutritional requirements’; in EIO, this is already active during pre-operative outpatient visit with the participation of the Nursing Case Manager in the detection of weight loss (or also weight gain in some cases), reporting restrictions on food intake and consequent activation of a dietician referral and counselling to allow corrective interventions.
During hospitalization, supervision is provided as outlined previously, while during discharge, multidisciplinary collaboration is fundamental to initiate an effective health education intervention relating to the clinical condition of the patient.
During the outpatient phase, patients continue to undergo assessment in the detection of outcomes relating to the dietetic consultation and multidisciplinary collaboration (important collaboration with the enterostomy). We have provided a telephone follow-up service for hospitalized patients, but, above all, we aim for more, in terms of completeness of the service, activation of a 24-h outpatient service—this service was created with the goal of allowing direct access to the EIO, in case of urgent problems, without having to pass via a general medical doctor. This provides access for patients previously treated at the Institute, which include also those patients with problems relating to nutrition and to the use of safeguards to promote food intake.
Palese A, Cabarcas GR, Dotti R and Riboli O. Documentazione scritta (consegne e piani di assistenza). Assistenza infermieristica e ricerca 2006; 25: 109–114.
The prevalence of malnutrition in patients with gastrointestinal malignancy has been reported to range from 26% to 57%; weight loss is often present since the beginning of the disease and, frequently, the nutritional status further deteriorates throughout the treatments. Malnutrition is known to be a risk factor in the overall treatment of patients with gastrointestinal cancer, and it has a negative impact on the final prognosis [
Enteral nutrition (EN), a cost effective tool, is demonstrated to reduce post-operative infectious complications in malnourished patients undergoing major gastrointestinal surgery [
The aim of this study is to evaluate the efficacy of home enteral nutrition (HEN) on the nutritional status, the quality of life and tolerance to chemotherapy in malnourished patients who undergo major gastrointestinal surgery for malignancy. The primary objective is the maintenance of nutritional status after hospital discharge, and secondary objectives are the improvement in quality of life and tolerance to chemotherapy.
Inclusion Criteria: both genders over 18 years of age, documented cancer of the gastrointestinal tract (oesophagus, stomach, pancreas, biliary tract), candidate for major elective surgery, pre-operative nutritional risk score ≥ 3 and written informed consent.
Exclusion Criteria: Age < 18 years, Karnofsky index < 60, renal failure (ongoing haemodialysis or plasma creatinine > 3 mg/dl), respiratory failure (arterial blood PaO2 < 70 mmHg), ASA score = 4 or 5, Child-Pugh class C, short bowel syndrome, pregnancy, emergency operation and foreign residence or residence in Italian region with no regulation designed for home artificial nutrition.
General design issues: Phase III, mono-centre, controlled, open-label, two-parallel groups, randomized, clinical trial and the type of comparison is observation. The study is promoted by the National Cancer Institute and the European Institute of Oncology, Milan, Italy.
All patients fulfilling the selection criteria will be included and randomized in the study; the planned enrolment is 140 patients (70 in each group) over a period of two years. All patients will continue observation for six months after discharge. In all patients included in the study, a fine needle catheter jejunostomy will be implanted at the end of the scheduled surgery; NE will start on post-operative day 1 and progressively increase, with the objective of supplying 25 kcal/kg b.w. and 0.2–0.25 g nitrogen/kg b.w. daily. Before discharge patients will be randomized to receive either home enteral nutrition (treatment group) or nutritional counselling (control group). In the HEN group, the planned nutrition will cover the basal energy requirement calculated with the Harris Benedict formula, it will be administrated preferentially through an integration of oral diet. Enteral Nutrition will include any standard polymeric formula providing 1–1.5 kcal/ml with carbohydrates 50%–60%, lipids 25%–35% and protein 12%–20%. HEN can be withdrawn in the treated group, after two months from discharge, whenever a weight gain ≥ 5% is reported, and oral alimentation is regular and adequate. In the control group, specific nutritional indications including total energy and protein requirements will be provided to patients and high-calorie oral supplements will be prescribed whenever necessary.
The HEN can be started in patients of the control group if a further weight loss ≥5% is reported. Anthropometric, biochemical and functional measures will be used for assessment and monitoring of the nutritional status, before discharge, and one, two, four and six months after discharge. Quality of life is assessed at hospital admission and two months after discharge, using the self-administrated FAACT questionnaire; the tolerance to chemotherapy is evaluated by the ratio CT planned/CT administered; numbers and degrees of side effects were scored in accordance to WHO classification for toxicity.
Calcitriol (1,25(OH)D), the biologically active form of vitamin D, has been shown to be an anti-proliferative, pro-differentiation, pro-apoptotic agent and an inhibitor of cell migration. The vitamin D receptor (VDR) is a crucial mediator for the cellular effects of vitamin D and additionally interacts with other cell-signalling pathways that influence cancer development. Alterations in vitamin D receptor expression and in the synthesis and catabolism of vitamin D metabolites are involved in the growth regulation of tumours, thus, compromising calcitriol sensitivity and signalling.
Cultured melanoma cells can synthesise 1,25(OH)D from 25-hydroxyvitamin D (25(OH)D), express the VDR, and the anti-proliferative and pro-differentiation effect of 1,25(OH)D have been shown in human malignant melanoma (MM) cells. The 1,25(OH)D molecule has been shown to induce apoptosis in a human MM cell line
Epidemiological data indicate that vitamin D deficiency is relatively common in Europe. In an Italian study, we found that 85% of the participants had insufficient level of circulating 25(OH)D. We show that vitamin D supplementation was associated with a significant reduction (7%) in total mortality in healthy subjects. When we looked at cancer risk, we showed consistent inverse relationship between serum 25-hydroxyvitamin D levels and colorectal cancer incidence. For breast cancer, results were heterogeneous by study design; in fact, serum 25(OH)D level are measured after the diagnosis of cancer and a reverse causation problem can occur.
We also showed, through a meta-analysis of the association between the two most studied VDR polymorphisms (
A cohort study investigated the hypothesis that higher vitamin D levels reduce the risk of relapse from melanoma. The survival analysis showed that higher 25(OH)D levels, at diagnosis, were associated with lower Breslow thickness and were independently protective of relapse and death. There was also evidence of interaction between
In early supplementation trials, the lack of effect on cancer incidence has been attributed to insufficient vitamin D supplementation, stressing the need to better study vitamin D bioavailability.
In order to assess whether vitamin D supplementation could improve prognosis of Cutaneous Malignant Melanoma (CMM), we planned an Italian multi-centre trial in stage II resected melanoma patients, evaluating the effect of vitamin D supplementation and monitoring changes in 25(OH)D.
We intend also to study in detail the biology of VDR on vitamin D metabolism and in relation to CMM prognosis, looking at the association between 25(OH)D and VDR polymorphisms with Breslow thickness, the most important prognostic factor of CMM. In fact, tumour thickness could be the result of an effect on cell proliferation, and this study could give more information on functional effects of VDR and on the mechanisms that potentially make the vitamin D important in cancer development. We will study gene–gene interactions and changes in vitamin D metabolites, investigating how metabolism and biology of vitamin D interacts with environmental, anthropometrical and nutritional factors and contributes to cancer progression.
Some of the findings from this study will be of large interest also for a wide spectrum of cancers.
In a contest of multidisciplinary approach, an adequate nutritional support should be considered of fundamental importance as supportive therapy in patients treated with radiotherapy for malignant tumours. Radiation therapy, in fact, produces an acute mucosae inflammation and, after the end of treatment, a chronic soft tissues fibrosis, which could produce a transient or a definitive alteration of food intake and/or food absorption. For these reasons, patients taking radiation therapy should be evaluated prior to treatment and offered specialized nutritional counselling in order to optimize the treatment and prevent radiation-related side effects. In head and neck and thoracic regions, acute radiotherapy-related dysphagia is due to the inflammatory process of irradiated tissue (oral cavity, pharyngeal and oesophagus mucosa) with the development of mucositis, oedema and pain. After the end of treatment, late dysphagia might be due to soft tissues fibrosis, chronic lymphedema and damage to neural tissue. Any hindrance to oral food intake should be therefore prevented and adequately supported. During the first phases of pharyngeal mucositis, oral supplementation is used when oral intake is still permitted. Subsequently, when patients cannot adequately swallow, feeding by tube is usually given. Tube feeding nutrition can be performed by a nasogastric tube (generally not preferred and used only for short period because of its negative cosmetic impact and because of the high risk of mucosal injury, sinusitis and aspiration) or by gastrostomy (percutaneous endoscopic gastrostomy – PEG).
Apart from the patient’s quality of life, the clinically relevant negative impact of dysphagia and the subsequent weight loss in response to anti-cancer therapies is well known [
Radiation therapy to the abdominal or pelvic region has different consequences. These patients may experience radiation-induced enteritis resulting in intestinal malabsorption. Also, for gastrointestinal tumours, a recent study, although performed with a limited number of patients, showed that the worsening of nutritional status resulted to be associated with worse results of anti-cancer therapies [
In conclusion, controlled clinical trials and Institutional guidelines are strongly recommended in order to optimize the nutritional assessment and intervention for oncologic patients undergoing radiation therapy.
Over the years, enteral nutrition (EN) has gained considerable popularity due to its favourable effects on the digestive tract, its lower cost and rate of complications compared to parenteral nutrition.
However, clinicians are often faced with contradictory data and difficult decision making when having to determine the optimal modalities of EN administration. An example of such a difficulty is the precise definition of the expected morbidity rate of needle catheter jejunostomy (NCJ). Some findings suggest that NCJ is associated with a significant rate of early and late complications; in some instances, they may be life threatening and require surgical intervention. In this scenario, the role of NCJ in providing nutritional therapy to depleted patients undergoing major gastrointestinal procedures should be re-examined.
We prospectively evaluated the rate and type of early and late complications associated with NCJ in patients undergoing surgical treatment for upper gastrointestinal malignancies.
Three hundred and eighty consecutive patients underwent NCJ implant according to Delany’s method at the end of their scheduled surgical procedure (total/subtotal gastrectomy = 242; duodenopancreatectomy = 45; esophagectomy = 78; other procedures = 15). The EN program was started on post-operative day (pod) 1 in the surgical ICU with 15 ml/h and progressed to the optimal goal of 80 ml/h by post-operative day 5, using a nutritionally complete liquid polymeric diet providing 1 kcal/ml.
Clinical examination and adverse gastrointestinal symptoms were recorded on a daily basis.
Short-term complications directly attributable to the NCJ were defined as tube leakage with intra-abdominal or intraparietal spillage, intra-abdominal abscess, small bowel obstruction at the catheter site, tube blockage or dislodgement or soft tissue infection. The NCJ was always removed in the outpatient clinic after hospital discharge. Regular follow-up visits were conducted in all cases until the patient died or the study was closed, in order to detect long-term complications, including operations to correct adhesions or small bowel obstruction.
All the Patients received the EN program for a mean of 14 pod (range: 8–46); a single short-term complication of the NCJ has been observed in this series (tube blockage; third pod). An easy resolution has been obtained with percutaneous replacement under x-ray control. No late complications have been detected after a mean follow-up of 24.01 months (range: 2–42).
Extensive effort has been made to improve outcome of patients undergoing major surgical procedures of the upper gastrointestinal tract, and nutrition support is now recognized an essential component of the clinical management of these patients. The ability to deliver nutrition enterally after an operation has been a significant advance and the NCJ gained popularity, leading some investigators to recommend prophylactic jejunostomy at the time of any high-risk abdominal operation, emphasizing the substantial safety and the relatively low cost of this additional procedure. However, a few studies investigated prospectively the rate of complications arising from the NCJ, and limited data are provided from the medical literature coming from randomized trials. Zapas
A small prospective randomized study has investigated the safety and efficacy of enteral feeding by jejunostomy in patients undergoing elective esophagogastrectomy [
As the benefits of early enteral feeding over total parenteral nutrition in terms of reduction of septic morbidity and lower costs are indisputable and demonstrated by a number of randomized trials, a possible solution to overcome the NCJ-related morbidity could be the reconsideration of the NCJ as the sole way to provide effective enteral nutrition in the post-operative period, in favour of an intrajejunal feeding initiated via a nasally passed tube. No data coming from well-designed randomized trials are presently available about this specific issue; nevertheless, the compliance of the patients towards this approach should be considered as a possible, major limiting factor, as at least 27% of nasally passed tubes for enteral nutrition are expected to be voluntarily or accidentally removed within a few days after placement [
Finally, a large series (500 consecutive cases) recently published by a monocentric experience [
Cancer of the lung is the most common type of cancer in the world [
As part of the COSMOS study (Continuous Observation of Smoking Subjects) designed to implement a comprehensive screening programme for early diagnosis of lung cancer in high-risk volunteers in North West Italy [
The COSMOS study is a single-centre non-randomized lung cancer screening trial, conducted in Northern Italy. Details of participants, screening protocol and diagnostic work up have been published elsewhere [
At baseline, usual diet before the enrolment was measured by a semi-quantitative food frequency questionnaire originally developed for the EPIC-Italy study [
The average daily quantities of foods, energy and nutrients consumed by participants have been calculated using a computer program (Pale et al, 2003), non-including supplement intake. Nutrient intake has been calculated according to a specifically developed Italian food composition database for epidemiological study [
Cox proportional Hazards regression was used to analyse the association between nutrients intake and risk of lung cancer. We divided the consumption of nutrients (macro- and micronutrients) according to quartiles of consumptions. Multivariate models were adjusted for age, sex, smoking status, number of pack-years, past history of emphysema or chronic obstructive pulmonary disease (COPD) and total energy intake.
During 19,616 person-years of observation, 134 participants of the COSMOS study were diagnosed with lung cancer. Initially, 5203 subjects were enrolled, but dietary information was obtained for 4363 participants (84%). After exclusion of patients with abnormal dietary values (outliers), the final study population included 4337 subjects (
Mean energy intake reported by the study population was 2254 kcal (9428 kJ) per day (
In our study, no significant association was found between either macro- or micronutrients and lung cancer risk, when we compared the highest versus lowest quartiles of consumption (
The null association reported may be explained in part by the strong association between lung cancer and smoking, influencing the effect of the single nutrients. In addition, smokers tend to have a less healthy diet and to be leaner than non-smokers, but our population results to be health conscious. We found a high percentage of supplement users, and overweight and obese (together 56%). These factors may have influenced our results. Further analysis needs to investigate the associations between dietary nutrients and the risk of lung cancer risk in subgroups (sex, smoking status, histological subtype o) in our population. To complete the analysis, we will also include nutrients intake (vitamins and minerals) coming from supplements.
Demographic characteristic, BMI and smoking habits in COSMOS participants
Median energy and nutrients intake from food in all study population (4337) compared to the national recommendations [
Hazard ratios (HRs) (95% CI)ç for lung cancer, according to quartiles of consumption of specific nutrients for all participants (4337)
Breast cancer (BC) is the most common malignancy in women and is their leading cause of death from cancer. In high-income countries, the incidence of BC has increased steadily over the past decades, but BC mortality is declining, suggesting a benefit from early detection and more effective treatment. BC survivors are constantly increasing, and research investment for the identification of modifiable factors associated with BC recurrences is increasing too. Western lifestyle, characterized by low levels of physical activity and a diet rich in refined carbohydrates, animal fats and protein is associated with high prevalence of metabolic syndrome, insulin resistance and high serum levels of sex hormones and growth factors. All these factors are strongly related with BC risk and BC prognosis but are potentially modified through diet and lifestyle.
The present work summarizes the metabolic, hormonal and dietary correlates of increased risk of BC and BC recurrences.
Obesity is associated with an increased risk of BC after menopause, while no association or slightly reduced BC risk has been found before menopause. Preventing weight gain in adulthood, however, would decrease the overall burden of BC. Obesity has been shown to adversely affect prognosis in both pre- and post-menopausal BC, after controlling for clinical and pathological prognostic factors. High serum levels of steroid sex hormones and of bio-available insulin-like growth factor I (IGF-I) are associated with an increased risk of BC. Furthermore, insulin and markers of insulin resistance, such as abdominal obesity, high blood glucose, high serum level of testosterone, and metabolic syndrome, may affect both BC incidence and prognosis. Sedentary lifestyle is associated with increased BC risk, both before and after menopause. Women who practice regularly at least some physical activity decrease their BC risk by 30% or more, and there is increasing evidence that physical activity may protect also against BC recurrences. The association between dietary fat intake and BC is an highly controversial topic in epidemiology. Results of the Women Initiative on Nutrition Study (WINS), a randomized dietary prevention trial, suggested a strong benefit of dietary fat reduction on relapse-free survival with the strongest protection for hormone receptor-negative BC. The Women’s Healthy Eating and Living (WHEL) Study tested whether a dietary pattern high in vegetables, fruit and fiber and low in fat might reduce BC relapses in women with early-stage BC. The primary analyses of the WHEL study did not demonstrate an event-free survival advantage in patients randomized in the dietary arm. This trial, however, was isocaloric and the intervention group did not loose weight.
The briefly reviewed factors are strongly related to diet and lifestyle and may be potentially modified. Our DIANA (Diet and Androgens) intervention trials demonstrated that a sustainable dietary modification aimed at lowering insulin levels, based on Mediterranean and macrobiotic dietary principles, can reduce body weight, metabolic syndrome and the bioavailability of sex-hormones and growth factors. Together with other studies showing that Mediterranean diet can revert metabolic syndrome, these results suggest that dietary changes should be recommended for both BC prevention and treatment.
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This is an Open Access article distributed under the terms of the Creative Commons Attribution License (
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (
This is an Open Access article distributed under the terms of the Creative Commons Attribution License
This is an Open Access article distributed under the terms of the Creative Commons Attribution License
This is an Open Access article distributed under the terms of the Creative Commons Attribution License
This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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As eye care workers, we don't treat eyes, we treat people. Similarly, eye care does not take place in a vacuum; it is part of the wider health care system.
It is possible to forget this ‘bigger picture’ when our work demands such a lot of us. However, as we think about it, we will be surprised how much the work we do depends on the wider health system and, in turn, how much our eye care system can contribute to the wider health system.
The World Health Organization emphasises the need to have the health systems perspective or framework in mind when we plan and deliver eye care services.
This is not as complicated as it may sound! It means spending a little time thinking about each of the different but related elements that make up the building blocks of the health system.
We can think about each of these within our eye programme. But more importantly, the health systems perspective encourages us to think about how our eye programme interacts - both positively and negatively - with nearby, parallel health services and the wider health system such as the local hospital or national health system.
We need accurate information to plan and monitor eye services. SOUTH AFRICA
Health information is one of the six components of a health care system. We need information about each of the components in order to plan and monitor - with the ultimate aim of providing a better service and eliminating avoidable blindness.
A health management information system (HMIS), if well designed, can help us to manage all the information needed by (and generated by) the eye care programme - whether at local, district, or national level. Whether we are planning a new HMIS or evaluating an existing HMIS, the health systems framework can be a useful thinking tool.
It can help us to determine what questions the HMIS should be able to answer (which in turn determines how we design it) and plan the implementation of our HMIS.
Central to both of these is an awareness of how our planned HMIS will integrate with any others in existence; or, where there are no others, how ours can be extended to support other health services.
In order to know how well we are doing, we need information on how many people we are reaching (the quantity). Simple counts (or tallies) of the number of patients seen, screened, referred, and treated over the last day, month, or year will give us some idea of how we are doing. Information about the quality of our services is equally important. Table
It is vital that this information is shared with those who are responsible for the work behind the numbers. This is both to acknowledge work well done and to plan ways of improving our work where necessary. The information collected should also be used by us and not just be passed on to others. The authors recommend holding periodic review meetings to analyse the information, identify problems or constraints, and decide on the steps we must take to do better.
At the level of the individual eye unit, we recommend that the person who collects the information should also analyse and present it. For example, the person who completes the daily register could be trained to analyse the data and present it at the monthly eye care team meeting. This will help them collect the information more carefully. They could look at how common different conditions are or the areas most patients come from. This will bring life to what could otherwise be a boring duty.
Your HMIS should try to capture the following:
Who is employed where, and to do what? In collecting information on human resources, you should look at all workers who contribute to the work, not just health workers. For example, you should consider records clerks, equipment technicians, and administrators in different districts.
How many patients are seen at different eye units and by different staff members?
Where are there long waiting times? You may improve the flow of patients by moving staff within a particular hospital, or by assigning staff to a clinic or hospital where there are more patients and longer waiting times and waiting lists.
The HMIS may be able to help you keep track of stock levels and stock used (this will be covered in detail in Issue 76, December 2011), as well as what equipment is available and functioning (see Issue 73, September 2010).
Your HMIS should capture information to produce financial reports which can be used to manage income generated, to manage budgets, and for reports to donors.
A summary of the above information will be invaluable to make decisions about all aspects of the eye programme and help you when reporting to your manager, the hospital leadership, the ministry of health, or a donor. It is good to spend time thinking about your reporting requirements when you design the HMIS.
Examples of information for monitoring the quantity and quality of eye care services provided
| Eye care services | Information for monitoring quantity of work | Information for monitoring quality of work |
|---|---|---|
| Number of patients seen requiring medical treatment | Proportion of people needing services who are coming for treatment (are we meeting the need?) | |
| Number of trichiasis operations done | Number of repeat trichiasis operations done | |
| Number of refractions done | Proportion of patients with 6/6 distance corrected vision | |
| Number of glasses prescribed or dispensed | Proportion of patients with J2 near corrected vision | |
| Number of cataract operations performed | Proportion of eyes achieving vision 6/18 or better at last postoperative visit | |
| Number of cataract operations performed on blind patients | Proportion of eyes failing to see 6/60 at last postoperative visit |
Do not destroy existing systems, but build on their strengths.
(Adapted from
Make an inventory of the forms, log books and other tools used to record and summarise data at different levels.
Assess the quality of the data being collected using the existing forms/ formats at different levels.
Determine the problems encountered with the current system of data collection, including problems with timing and flow of information.
Define the different roles/functions of each level, for each of the major programmes; these will determine what information they need. For example, at the village level, information may be needed for case finding and service delivery; at the district level, information may be needed for monitoring and supervision. At the provincial level, information may be needed for programme planning and evaluation, and at the national level, for policy formulation.
Develop a first draft of the form, ensuring that it will produce all the data you need.
Get feedback from staff who will be using the form and make improvements where necessary.
Assess the advantages and disadvantages of manually processing the data compared to using computers. Consider the cost, the availability of personnel (and their training, particularly at the lowest level), as well as the availability of technical support.
Pre-test any software and develop a training programme for staff.
Feed back to staff involved in the HMIS not only its outputs, but also who is using the information, what they are using and how.
Do not destroy existing systems; rather build on the strengths and learn from the weaknesses of what already exists.
Consult with staff when you design the forms and/or computer system so that you are sure they understand them and know how to use them.
Collect only the data which you are sure you will use.
Ensure that the people collecting the data understand what it will be used for. Report the results back to them on a regular basis - this will help to keep staff motivated.
The most effective data collection and reporting tools are simple and short.
Data which is incomplete or incorrectly collected is worse than useless - it can give you false information.
Not all data should be generated through the routine system of data collection. Data that are not frequently needed or are required only for certain subsets of the population can be generated through special studies and sample surveys.
The development of the HMIS is always a work in progress. It is a dynamic endeavour where managers and workers strive for constant improvement.
In 1997, the World Health Organization formed the Global Alliance to Eliminate Blinding Trachoma by 2020 (GET 2020), a coalition of governmental, non-governmental, research, and pharmaceutical partners. In 1998, the World Health Assembly urged member states to map blinding trachoma in endemic areas, implement the SAFE strategy (which stands for surgery for trichiasis, antibiotics, facial-cleanliness and environmental change, such as clean water and latrines) and collaborate with the global alliance in its work to eliminate blinding trachoma.
Over these past 13 years, much progress has been made. Pfizer Inc has committed to donating the Zithromax necessary for eliminating blinding trachoma by 2020, non-governmental organisations have scaled up their support to national programmes to implement the SAFE strategy, and some trachoma-endemic countries are now close to reaching their intervention goals. Since the Pfizer donation began in 1999 through the International Trachoma Initiative (ITI), more than 155 million Zithromax treatments have been distributed.
But trachoma remains a blinding scourge. It is still believed to be endemic in 57 countries (Figure
Trachoma-endemic countries
We only have ten years left to reach the goal of eliminating blinding trachoma. In order to achieve this, all endemic countries must have the full scale SAFE strategy in place by 2015 to allow enough time for implementation to have an impact. Enormous challenges lie ahead of us. Some of the remaining endemic countries are in conflict or have just come out of conflict, and lack infrastructure and resources to fully address the disease. Even countries free of conflict lack financial resources for the epidemiological surveys to determine which districts need intervention, or to support intervention in endemic districts. Implementing the SAFE strategy can be a strain on resources as well, since providing access to clean water and latrines is not inexpensive.
However, we believe that, together, we can overcome these challenges and reach our goal.
In this issue of the
The first article in the series is about national trachoma task forces. We hope that this new series will provide tools to assist those who are implementing programmes to eliminate blinding trachoma.
Mass distribution of antibiotics is one of the four components of the SAFE strategy. ETHIOPIA
Many years ago, in Central Asia, the
Through the role of Editor, I have had the opportunity and privilege of meeting and working with many colleagues and friends, truly an international community of health professionals who have sufficient vision and selfless purpose to work together, seeking to eliminate avoidable blindness worldwide.
During the past twenty years, the
It has been the ethos and purpose of the
Two eye nurses browse a copy of the
A reader survey conducted recently showed that, for 73% of our readers, this journal is their only source of up-to-date information
Perhaps appropriately, the last twenty years have seen a growth in the development of eye care services – policies for disease control, human resource development, and infrastructure and appropriate technology provision – all central to VISION 2020: The Right to Sight. It is an encouraging thought that the
My first encounter with the
The seven Exchange articles that follow are based on the dissertations of students at the International Centre for Eye Health, London School of Hygiene and Tropical Medicine, who graduated in 2008.
The Jabel Awliya camp for internally displaced persons (IDPs). SUDAN
Following decades of civil war, approximately two million internally displaced persons (IDPs) are living in and around Khartoum, the capital city of Sudan; 400,000 have settled in the four official camps, and the rest live in 30 ‘squatter areas’ scattered around the city. These communities are poor, vulnerable, and at greater risk of avoidable or preventable blinding eye disease.
Our study aimed to evaluate the distribution, availability, and accessibility of eye care services in the camps for displaced persons. This descriptive situation analysis of human resources and infrastructure is a necessary first step towards providing adequate and sustainable services.
All four official IDP camps surrounding Khartoum were included in our situational analysis. Quantitative data on human resources and infrastructure was collected, using a checklist, from the ministry of health and from the available services in the visited camps. Qualitative data aimed to explore the behaviour of IDPs in seeking eye care; they included focus-group discussions with mothers of school-aged children, semistructured interviews with functionally blind IDPs, and semi-structured interviews with health care staff working in the camps.
Virtually no eye care services were found in the camps. The only permanent service found in all the visited camps was a single eye clinic in a camp housing around 150,000 people, which was integrated within a primary health care unit. When assessing the IDPs' barriers to accessing medical eye care, we found that the main ones were: poverty, the absence of services, the lack of an accompanying individual, the fear of surgery, and customs and beliefs. Given the absence of services and appropriate health care cadres, as well as the inability of IDPs to afford even subsidised basic eye drops, existing health care staff felt inadequate because they could provide very little help.
In conclusion, future service planning in the area should be directed towards affordable eye care services for the IDPs. Health service planners also need to look into the reasons behind the absence of service provision: lack of commitment, funding, or personnel.
An interview in an IDP camp. SUDAN
The visual prognosis of suppurative keratitis (SK) in the developing world is extremely poor, making this disease a significant cause of monocular blindness. Current standard practice in managing SK is empiric therapy with topical antibiotics, followed by a modification of this therapy based on clinical response and on microbiological results of corneal scrapings. Empiric therapy needs to be based on a knowledge of likely aetiological organisms, as these vary significantly both geographically and with time.
The aim of this study was to describe the epidemiology, clinical presentation, and microbiology of SK at Groote Schuur Hospital, in Cape Town, as recent data of this nature is not available in South Africa or Southern Africa. We conducted a retrospective review of clinical records of all patients admitted for inpatient therapy (186 patients, 210 admissions) over a three-year period (2005–2007).
The most commonly identified risk factors for SK were: neurotrophic cornea (28% of risk factors), ocular surface disease (23%), and trauma (21%). Trauma (p=0.004) and retroviral disease (p=0.001) were more commonly identified in the under-60 age group, and ocular surface disease (p=0.0001) in people over 60. Retroviral disease was more common in women (p=0.009) and trauma more common in men (p=0.0001).
Visual acuity in the affected eye was <6/60 for 78% of patients on admission and for 44% on final follow-up. Binocular acuity was <6/60 for 13% of patients on admission and for 5% on follow-up. Thirty per cent of patients underwent acute surgery on one or more admissions.
Cultures were positive for 75% of specimens. Bacteria were isolated in 89% of these, fungi in 6%, and both bacteria and fungi in 5%. Gram-positive organisms constituted 69% of all bacteria isolated. The sensitivity of Gram staining (proportion of actual positives which are correctly identified as such) for cases of bacterial keratitis in this setting was low (27%) and for fungal microscopy even lower (19%).
In conclusion, SK is a blinding condition in Cape Town, with patients presenting with advanced disease and frequently requiring acute surgical intervention. The spectrum of organisms isolated was very similar to that in other temperate regions. In this study, microscopy (bacterial and fungal) had a very low sensitivity, which is concerning.
Corneal blindness due to vitamin A deficiency (VAD) is a leading cause of preventable blindness in children in developing countries. This study aimed to evaluate whether VAD is a public health problem in children aged 6–72 months in the rural and underprivileged Thatta District, in Sindh Province, Pakistan. Another aim was to identify risk factors for VAD, and to determine the coverage of distribution of high-dose vitamin A.
Purposive sampling was used to identify high-risk areas. The caregivers of children were interviewed to collect information about demographics and risk factors of VAD. Both eyes of children were examined using torch and magnifying loupe. Height, weight, and mid upper-arm circumference were measured. A blood sample was obtained from every tenth child and clinical case in order to measure serum retinol levels, using high performance liquid chromatography.
Ocular examination in preschool children. PAKISTAN
Measuring height to assess nutritional status. PAKISTAN
Out of the 619 children examined, 18 (2.9%, 95% CI 1.58–4.22) were xerophthalmic. Mean serum retinol was 27.56
Five hundred and ninety-eight (96.6%) children had received vitamin A supplementation and 504 (81.4%) were immunised against measles.
Two hundred and twenty-seven (36.7%) mothers had night blindness (a symptom of VAD) during a recent or last pregnancy. In many locations, women thought this was a normal phenomenon of pregnancy.
Univariate analysis for individual risk factors was performed, but only age group was statistically significant (x2=11.97, p=0.0001), as xerophthalmia rates increased with age.
Despite high vitamin A coverage, VAD is a public health problem in preschool children and pregnant women in selected underprivileged rural areas of Pakistan. Malnutrition is widespread and levels of illiteracy among mothers were extremely high.
More studies are required with adequate sample size to identify associated risk factors. Health education and promotion activities should be run in rural areas of Pakistan to increase awareness regarding night blindness.
There are very few data on childhood blindness in Iran, a lower-middle-income country with a population of around 70.5 million people. There is currently no national programme for the prevention of childhood blindness.
Our research team undertook to pilot the key informant method in three out of the nine counties in the province of Lorestan. The aims of our study were, firstly, to establish the feasibility of a key informant survey in Iran and, secondly, to provide estimates of the prevalence and causes of childhood blindness in the area.
Around 120 community health workers were trained by the author to act as key informants, to identify and refer blind children from their own communities. Two ophthalmologists then examined the children to verify that they were blind and to diagnose the cause of blindness.
Our study confirmed the feasibility of a key informant survey in Iran: the method was time- and cost-efficient, it was well received by local health authorities, and it produced credible estimates of blindness.
Training session on the key informant method. IRAN
Our findings were consistent with previous research conducted in Iran and in the Middle-Eastern Crescent. Out of around 110 children referred for examination, a total of 27 blind children were recruited, yielding an estimated prevalence of 0.06%, so that around 10,800 children were estimated to be blind in Iran.
The leading cause of blindness was retinal disorders (40.7%), followed by disorders of the whole globe (33.3%). The majority of cases had a hereditary aetiology (70.4%), which showed a significant association with family history of blindness (p=0.002). The vast majority of blind children had a history of parental consanguinity (92.6%). Importantly, almost 50% of the cases were potentially avoidable, and 45.8% of blind school-aged children were not attending school.
Given the encouraging outcomes of this pilot study, the next rational step is to conduct a full-scale, district-level key informant survey in Lorestan. This will contribute to the planning of a national programme for the prevention of childhood blindness in Iran. Our recommendations also included the support of ongoing work in genetic counselling and of family planning and maternal and child health services.
Incidence figures, i.e. the number of new cases arising during a specific period of time, are important for the long-term planning of eye care programmes.
We conducted a follow-up after three years of all the subjects aged 40 years and above seen during the 2005 national blindness and low vision baseline survey in Bauchi State, Nigeria. Our aim was to assess the three-year incidence of blindness, low vision (visual acuity (VA)<6/18–3/60), and cataract, as well as the three-year progression of cataract in this State.
Original clusters and subjects were traced and identified using enumeration records. Presenting VA was measured using the reduced LogMar chart and, for those with VA<6/12, VA was then measured using a pinhole. For all subjects, lens opacity was graded using the Mehra Minassian classification; for those with VA<6/12, pupillary dilation was performed and lens opacity assessed using the simplified WHO grading system as in the baseline survey.
We found that the three-year cumulative incidence of blindness in persons was 5.50% (95% CI 2.34–8.66), while that of low vision and blindness together was 19% (95% CI 13.56–24.44). Most of low vision was due to uncorrected refractive error. Of those who were normal at baseline, two subjects (1.19%) became blind, and 27 (16.16%) developed new low vision. Among those with low vision at baseline, nine (27.27%) new cases of blindness developed. The three-year cumulative incidence of bilateral cataract blindness was 5.33% (95% CI 1.94–11.96). The three-year progression of lens opacities was similar for nuclear and cortical opacities, at 2.39% and 2.99% respectively. The three-year mortality in the cohort was 10.3% and there was no gender difference or relationship with visual status at baseline.
Response rate was best in clusters where there had been some service provision or contact since the baseline survey.
This study aimed to describe the extent of the use of traditional eye medicines (TEM) in the Bukavu ophthalmic district, in the Democratic Republic of Congo, with a view to collaborating with traditional healers in eye care promotion at community level.
A descriptive cross-sectional study of 470 consecutive patients who attended eye centres was carried out. This was combined with a qualitative study. Non-probabilistic convenience sampling was used to collect quantitative data, through a questionnaire and a clinical examination of patients. Qualitative information was gathered through in-depth interviews with six traditional healers.
Interviewing a traditional healer. DEMOCRATIC REPUBLIC OF CONGO
Eighty-four (17.90%) patients reported using TEM for the current disease episode before attending eye care services. There was no significant association for age, gender, or place of residence. However, the level of education (primary school and below) was significant for association with usage of TEM.
The provider of TEM was in 72.6% of cases a relative, friend, or acquaintance (‘non-professional healer’) and in 27.4% of cases a professional traditional healer (affiliated with the Congolese Association of Healers).
Among the 84 subjects who reported using TEM, 46.4% stated preference as the reason for this choice, 33.3% reported proximity as the reason, and 15.50% reported cost. No patient declared a lack of awareness of the existence of eye care services in the district.
Amongst users, TEM was used for the following conditions: 34.5% used it for acute conditions (conjunctivitis, corneal ulcers), 22.7% for chronic loss of vision (cataract and glaucoma), and 42.8% for trauma and posterior segment disease. People who chose to use TEM presented late at the hospital. 19% of those who had used TEM were blind (visual acuity <3/60) compared to 8.8% of non-users.
In conclusion, the health education of the population and the integration of traditional healers into primary eye care programmes are critical for reducing the harmful effects of traditional eye medicine. The healers interviewed expressed the willingness to collaborate with the existing eye care programme. However, much of the use of traditional eye medicine is not due to healers, but to the home practices in the region. If we are to solve this problem, the cost of services remains a key barrier to address.
It is indeed with great honour and humility that I have accepted the invitation to write the Foreword to this supplement of the Global Health Action, exclusively dedicated to a collection of articles generated from the research project of the INDEPTH Network, Climate and Mortality (CLIMO). I hope that future data analyses will also generate insights on migration as an effect of weather and climate change.
In 2009/10, the Social and Human Sciences Sector of UNESCO, through its office in Ghana, and in the context of its regional activities in Africa, provided modest financial support to INDEPTH Network, which initiated a research activity designed to analyse data on temperature, rainfall, and mortality with a view to a scientific understanding of a potential nexus between and among them, and consequently, use the findings to inform policy making in member states in Africa.
The activity brought together researchers from several HDSS centres in Africa, affiliated with INDEPTH Network, along with counterparts from India and Bangladesh, as well as partners in the North, to use existing data-sets to improve our understanding of climate and mortality. This supplement provides a first step in this direction as it analyses the effect that temperature and rainfall have on the subsequent risk of dying.
The United National Framework Convention on Climate change mandated the Intergovernmental Panel on Climate Change (IPCC) to regularly assess the state of the entire evidence available on the scientific basis of climate change, its impacts on life on the planet including human health and the most effective policy responses. However, this assessment report is only as good as the state of research, which—in the health field—is particularly patchy, as far as low-income countries are concerned.
UNESCO's mission is to contribute to the building of peace, the eradication of poverty, sustainable development and intercultural dialogue through education, science, culture, communication, and information. I am particularly proud that the work to which we modestly contributed to was not only one of knowledge generation but also included a strong component of research capacity strengthening. Young African and Asian scientists were trained in state-of-the art statistical tools and guided in the data analysis process as well as in the skills of writing up the results in scientific papers.
It is my hope that the scientific evidence presented in this supplement will assist policy making in the countries represented in the studies, to tackle the challenges of climate change. I would therefore like to congratulate INDEPTH Network and all the researchers involved in the studies, and hope that future opportunities will continue to make it possible to strengthen our collaboration.
This supplement of
Severe and sometimes devastating consequences are considered to be associated with future climate change, with the largest potential impacts occurring in areas with the least means to adapt. Impacts on health range from those related to malnutrition, fresh water scarcity, and changes in the range and transmission of many infectious diseases to those directly related to weather or climate extremes, such as those caused by tropical storms, floods or heat waves (
Understanding future hazards and their health impacts is important for adaptation and mitigation policies. The former seeks to build climate change resilience into health systems; the latter brings the information on adverse health impacts into the climate policy debate as a further motivation to reduce net emissions (
The current understanding of health impacts from climate and weather is much better in high-income countries, while there is a strong belief that the impacts will be much more severe in low- and middle-income countries (LMICs). Although this belief is likely to be true, it still relies on limited empirical evidence. At present there is a lack of studies on the current health impacts from climate and weather on the population health in LMICs, particularly in rural Africa and Asia (
The INDEPTH network has collected high-quality standardized data on demographics and mortality for geographically defined populations over many years, particularly in regions of Africa and Asia (
In high-income areas, many studies have focused on the relationship between variability in weather, mortality and health, identifying stressors such as extreme heat and humidity (
On these premises the INDEPTH Network, together with a number of south–south and north–south collaborating institutions, initiated a research and capacity-strengthening workshop in Nouna, Burkina Faso, in February 2011. Fifteen out of the 42 INDEPTH network member Health and Demographic Surveillance Systems (HDSSs) were represented by their analysts during a one-week workshop. The workshop was funded by UNESCO, Ghana, and UNESCO was represented at the workshop by Dr. Abdul Lamin. The facilitators of the workshop were from the INDEPTH Network secretariat, Accra, Ghana; Umeå University, Sweden; Heidelberg University, Germany; Columbia University, USA; and the Nouna Health Research Centre, Burkina Faso. The aim of the group was to work towards a special issue in the journal
Subsequently, a second follow-up workshop, funded by Doris Duke Charitable Foundation, was organised in Accra in May 2012. In between the two workshops, the analysts continued the work on their data with mentorship from the facilitators and commenced paper writing. The objective of the second workshop was to help the analysts develop their writing and scientific presentation skills in order to meet the standards of the upcoming scientific supplement in
The facilitators laid out the objectives of the analyses and corresponding papers to be invited for submission to the supplement in
The HDSS-associated analysts and researchers who contribute to the supplement have a wide geographical spread ranging from the west of sub-Saharan Africa to Bangladesh: Nouna HDSS (
The past, present and potentially future climatological contexts of the study areas are described in one of the papers (
In their study, Egondi and co-workers describe the direct and delayed associations between cold and warm temperatures and the amount of rainfall on daily mortality in the informal settlements of Nairobi, Kenya, over the period 2003–2008 (
Azongo and co-workers have studied the relationship between daily mortality and temperature and rainfall including delays up to a few weeks between exposure and events (
In the study by Diboulo's group, the authors describe how the population mortality from northern Burkina Faso (bordering to the Saharan desert) is affected by different weather conditions on a daily scale over the period 1999–2009 (
In a study from Rufiji, Tanzania, Mrema and co-workers the authors describe how rainfall and temperature on a monthly timescale are related to mortality (
Ingole's group studied daily population level mortality in relation to weather in the rural area of Vadu, India, 2003–2010 (
The longest daily time series studied as part of the supplement is based on data from the Matlab and the Abhoynagar areas in Bangladesh, 1983–2009. Using the Matlab registers, Lindeboom and co-workers describe the associations between mortality, temperature, rainfall and cyclones (
Overall, the results from the site-specific analyses are in line with many studies from more developed countries. They show that meteorological conditions are related to mortality days and/or weeks later. On the other hand, many of the studies also show that children under 5 years of age appear sensitive to mortality during extreme weather conditions. Some of the studies indicate a surprising absence of mortality impacts related to hot temperature. This may be related to the low prevalence of non-communicable diseases in the early stage of the epidemiological transitions (
Relating the supplemental findings to the current body of evidence (mainly from developed regions), it is important to consider the state of demographic and epidemiological transitions in the study population. Most certainly, this is particularly important when considering future impacts of weather and climate change on non-communicable diseases, as such diseases are likely to become much more prevalent in the future (
Further studies are needed to study these relationships in more detail and to compare the results between the HDSS areas. For example, using cause-specific data and identifying factors related to increased vulnerability and susceptibility to get a better understanding of the causal pathways of these effects, and also on how to enhance the local resilience to the current environmental stressors, are important next steps. A mixed methods approach using qualitative studies of people's perceptions, current knowledge of weather and climate change-related health risks, and describing peoples means to adapt, together with quantitative studies describing and enumerating the health impacts would benefit both local and national policies and decision making by jointly describing a width and depth, and possibilities for adaptation in the communities. Studies should also be designed to better understand the global and local health impacts following from climate change. For example, using projections of disease and deaths based on epidemiological transitions and climate change scenarios can help the development of longer-term adaptation programmes and global policies.
The INDEPTH network provides a possibility to expand the observation period onwards. As where longer time series are and become readily available, it is important to study the health effects related to climate variability and change directly, for example in a detection and attribution to past to present climate change framework.
Further research should develop the use of climate and weather information in increasing public health preparedness (e.g. early warning systems) to, for example, extreme climate and weather events and infectious outbreaks. The INDEPTH network offers an opportunity for development and evaluation of such interventions based on the retrospective registers and prospective data collection.
The joint work of the INDEPTH network members, analysts and facilitators described in this supplement is therefore a first step in building capacity for enhancing the long-term resilience for the population in resource-poor settings of Africa and Asia to climate and climate change. Our collaborative effort using INDEPTH data to study the effects of weather on mortality is an important first step in the challenging and hitherto under-explored road of what the health impacts of climate change in low-income countries are and how populations in theses resource-poor countries can be protected from them.
A second branch of data analysis, currently underway, refers to the link between weather and migration patterns: hence the acronym for the full study CLIMIMO (Climate Migration and Mortality).
Dual infection with HIV-1 and HIV-2, which is not uncommon in West Africa, has important implications for transmission, progression, and antiretroviral therapy. Few studies have examined HIV viral dynamics in this setting.
We compared HIV-1 and HIV-2 viral loads from 65 dually infected, antiretroviral therapy-naïve Senegalese subjects. Participants provided demographic information and blood, oral fluid, and cervicovaginal lavage (CVL) or semen samples for virologic and immunologic testing. Associations between HIV-1 and HIV-2 levels in plasma, PBMC, oral and genital samples were assessed using linear regression models with generalized estimating equations to account for subjects with multiple samples over time.
In analyses adjusting for CD4 count, age, sex, and commercial sex work, HIV-1 RNA levels were significantly higher than HIV-2 levels in semen (β=2.05 log10 copies/ml, 95% CI 0.44 to 3.66), CVL (β=1.37, 95% CI 0.83 to 1.91), and oral fluids (β=1.93, 95% CI 1.56 to 2.30). HIV-1 and HIV-2 PBMC viral DNA loads were similar in those with normal immune function (CD4 counts above 500 cells/µl) (β=0.17 log10 copies/µg of PBMC DNA, 95% CI–0.58 to 0.24), but compared to those with high CD4 counts, subjects with CD4 counts below 500 cells/µl had higher HIV-1 and lower HIV-2 levels. In plasma, subjects with CD4 counts above 500 cells/µl had mean HIV-1 plasma RNA viral loads 0.87 log10 copies/ml higher (95% CI 0.35 to 1.38) than HIV-2, while among subjects with CD4 counts between 200 and 500 cells/µl or below 200 cells/µl, this difference increased to 4.28 and 4.35 log10 copies/ml (95% CIs 2.51 to 6.04 and 2.67 to 6.04), respectively.
Our data are consistent with the hypothesis that with decreasing CD4 counts and HIV disease progression, HIV-1 may out-compete HIV-2 in dually-infected individuals. This finding may help explain the differences in epidemiology between HIV-1 and HIV-2.
Many HIV databases and applications focus on a limited domain of HIV knowledge. Since even a “simple” organism like HIV represents a very complex system with many interacting elements, the fractured structure of existing databases and applications likely limits our ability to investigate and understand HIV. To facilitate research, therefore, we have built HIVToolbox, which integrates much of the knowledge about HIV proteins and presents the data in an interactive web application. HIVToolbox allows quick and convenient hypotheses generation, experiment interpretation, and potential new drug structure creation.
HIVToolbox was built as a standard three-tier J2EE web application, consisting of
HIVToolbox was used to create several new hypotheses about HIV-1 integrase, including predicting the location of a CK2 phosphorylation site, which was later confirmed by experiment. A new version of HIVToolbox support display of the 3D locations of drug resistant mutations on surface plots of HIV proteins and the drug binding sites for structures of complexes of HIV proteins with drugs.
HIVToolbox is an open-access web application that allows virologists and structural biologists to access detailed information about HIV-1 proteins, such as sequence, structure, functional sites and relationships, homology, drug binding sites, and drug resistant mutations, and to immediately see the relationships between any or all of them. Weblink: [
Protease with drug Amprenavir shown [Interactive HIV protein page].
Analysis of human monoclonal antibodies (mAbs) developed from HIV-1 infected donors have enormously contributed to the identification of neutralization sensitive epitopes on the HIV-1 envelope glycoprotein. The third variable region (V3) is a crucial target on gp120, primarily due to its involvement in co-receptor (CXCR4 or CCR5) binding and presence of epitopes recognized by broadly neutralizing antibodies.
Thirty-three HIV-1 seropositive drug naive patients (18 males and 15 females) within the age range of 20-57 years (median=33 years) were recruited in this study for mAb production. The mAbs were selected from EBV transformed cultures with conformationally constrained Cholera-toxin-B containing V3C (V3C-CTB) fusion protein. We tested the mAbs for their binding with HIV-1 derived proteins and peptides by ELISA and for neutralization against HIV-1 viruses by TZM-bl assays.
We isolated three anti-V3 mAbs, 277, 903 and 904 from the cells of different individuals. The ELISA binding revealed a subtype-C and subtype-A specific binding of antibody 277 and 903 while 904 exhibited cross reactivity also with subtype-B V3. Epitope mapping of mAbs with overlapping V3 peptides showed exclusive binding to V3 crown. The antibodies displayed high and low neutralizing activity against 2/5 tier 1 and 1/6 tier 2 viruses respectively. Overall, we observed a resistance of the tier 2 viruses to neutralization by the anti-V3 mAbs, despite exposure of the epitopes recognized by these antibodies on the native viruses, as determined by intact virion binding assay with two representative subtype-C and B viruses (Du156.12 and JRFL).
Our study suggests that the anti-V3 antibodies derived from subtype-C infected Indian patients display neutralization potential against tier 1 viruses. Defining the epitope specificities of these mAbs and further experimental manipulations will be helpful in identification of epitopes, unique to clade C or shared with non-clade C viruses, for immunogen design.
One major obstacle to induce bNAbs resides in the high variability of the viral envelope and structural mechanisms hiding crucial epitopes. Besides, maturation of bNAbs against HIV represents a difficult process that can be impaired by the immunodeficiency associated with HIV infection. We have explored the hypothesis that preserved B cell function in LTNPs could result in the production of bNAbs at higher frequency and increased affinity in comparison with HIV progressors.
Samples (142) from the cohort of LTNPs (median RNA copies/ml: 87, median CD4+: 802 cells/µl) were kindly provided by the HIV BioBank integrated in the Spanish AIDS Research Network (RIS). A control population of 191 untreated patients (median RNA copies/ml: 10,241, median CD4+: 567 cells/µl) from Hospital Clinic, Barcelona, was analyzed. Sera at 1/200 and 1/2000 dilutions were preincubated with Env recombinant viruses harboring a luciferase gene and then added to U87.CD4.CXCR4/CCR5. bNAbs specificities were studied by ELISA using mutated gp120 that abrogates antibody binding, competition ELISA with biotinylated antibodies, neutralization assays with mutated viruses and peptide competition neutralization assays.
The percentage of elite neutralizers was higher in the LTNPs (9.3%) than in the control population (3.7%). Broadly neutralizing sera were screened for the presence of epitope-specific antibodies. CD4 binding site antibodies were detected in several sera. To determine whether these antibodies were responsible for broad neutralization, competition neutralization assays using RSC3 (antigenically resurfaced glycoprotein containing the CD4bs) were performed. RSC3 addition inhibited neutralization mediated by 16.7% of sera in LTNPs and 12.5% sera of the control population. Anti-MPER antibodies were detected in 50% individuals of both populations, including several sera with 4E10-like antibodies. Glycan-dependent HIV-1 NAbs were more abundant in LTNPs (66%) than in control population (37%).
Broad humoral immune responses against HIV-1 were more common among LTNP than a control population of untreated HIV-1-infected donors.
HIV/SIV primarily infect activated CD4+ T cells, but can infect macrophages. Because of the relatively small percentage of infected macrophages, the interaction between antigen-specific CD8+ T cells and infected macrophages in HIV/SIV infection has been poorly studied. We, therefore, sought to determine whether SIV-specific CD8+ T cells could control viral replication in infected macrophages.
We wanted to ascertain whether
Surprisingly, both
It is possible, therefore, that while AIDS virus-infected macrophages only constitute a small percentage of all virus-infected cells, they may be relatively resistant to CD8+ T cell-mediated lysis and continue to produce virus over long periods of time. Thus, macrophages could actually be contributing significantly to viral production. Induction of HIV/SIV-specific CD8+ T cells capable of killing infected macrophages or preventing establishment of the macrophage reservoir HIV might be critical for controlling viral replication.
Toll-like receptors (TLRs) are transmembrane receptors that activate cells of the innate immune systems upon recognition of pathogen-associated molecular patterns. The TLR4 is an essential component of the innate immune response to various microorganisms. We investigated the impact of TLR4 polymorphism on development of opportunistic diseases in HIV-infected patients.
The presence of TLR4 Asp299Gly single nucleotide polymorphisms (SNPs) was determined in a cohort of 180 antiretroviral treatment-naive HIV-1 infected patients and evaluated in relation to the occurrence of opportunistic infections. TLR4 genotyping was performed by real-time PCR.
One hundred sixty-five patients were homozygous for the wild-type genotype (AA); 15 patients (8,3%) were heterozygous for the Asp299Gly SNP (AG).
TLR4 polymorphism was associated with more frequent development of the opportunistic infections, such as active tuberculosis (OR=3.27; 95% CI [1.21–10.29]), herpes zoster (OR=4.15; 95% CI [1.24–7.29]) and toxoplasmosis (OR=6.23; 95% CI [1.19–18.67]) compared with genotype AA.
In addition, TLR4 SNP was associated with development of opportunistic diseases among individuals with CD4 cell count of>100 cells/mm3, compared with homozygous HIV-infected patients (OR, 5.25; 95%, CI [2.28–10.47]).
This study suggests a greater risk of developing of active tuberculosis and other opportunistic infections in patients with the Asp299Gly TLR4 polymorphism.
Diagnosis of acute HIV infection (AHI) is uncommon in resource limited settings. This abstract describes acute HIV infection in women in three East African countries.
Women at high risk of infection were recruited from ‘hot spots’ in Kericho (rural Kenya), Kampala (urban Uganda) and Mbeya (rural Tanzania). HIV negative eligible women were prospectively screened twice a week using HIV nucleic acid testing. A positive test led to entry into an intensive one-month diagnostic verification phase to definitively establish HIV infection status. Clinical and laboratory assessments were performed semiweekly. Supportive care and symptomatic treatment was provided.
Overall, 1197 high-risk volunteers have enrolled to date with 37 cases of AHI identified (31 prior to detectable antibodies). Mean age at HIV acquisition was 24.4 years (range 18–34). Only six reported unprotected sex with a known HIV positive partner. Crude incidence was 2.77/100 PY (95% CI:±0.87). Of the 37 AHI cases; 14 presented with malaria-like symptoms (all smear negative), 7 flu-like symptoms while 16 had 1–2 mild complaints (8) or no symptoms (8). Overall, AHI cases were evaluated at 302 visits and at least one symptom was reported in only 75 visits (24.8%). Pregnancy did not increase the frequency of symptoms but dehydration due to vomiting resulted in 2 of the 3 hospitalizations observed.
Identification of AHI is feasible in East Africa. Young, rural, females are most vulnerable. Individuals with clinical syndromes suggestive of malaria, but excluded by microscopy, should raise index of suspicion for AHI. The majority of cases had few or no symptoms or brief non-specific symptoms not requiring medical intervention. Screening protocols based on malaria syndromic presentation would not identify the majority of AHI cases.
Due to Lesotho's high adult HIV prevalence (23%), considerable resources have been allocated to the HIV/AIDS response, while resources for non-communicable diseases have lagged. Since November 2011, the Elizabeth Glaser Pediatric AIDS Foundation (EGPAF) has supported Lesotho Ministry of Health to roll out Family Health Days (FHDs), an innovative strategy to increase community access to integrated health services, with a focus on hard-to-reach areas where immunization coverage, HIV service uptake, and screening and treatment for chronic diseases are low.
Services were provided at mobile service delivery points from 17th October to 25th November 2011. Delivery points located in rural setting were staffed by multi-disciplinary teams of doctors, nurses, community workers, nutritionists, AIDS officers, and pharmacists (30-40 health professionals present).
During this campaign, 8,396 adults were tested for HIV (67.3% female; 32.6% male). In all, 588 (7%) tested HIV-positive (6.7% female; 7.1% male). Among those testing HIV-positive, 68.5% (403) received CD4 testing and 36.6% were enrolled into HIV care at their nearest clinics. A total of 324 ART defaulters were identified and linked back to care. Follow-up with referral facilities showed 100% of patients (defaulters and newly enrolled) linked to care were enrolled at a facility. Standard immunizations were administered to 990 children. 4,454 adults (24.7% male; 75.3% female) were screened for hypertension, and of those screened, 24.2% had elevated blood pressure and were linked to care centers. Addtitionally, 3,045 adults had blood sugar tests (27.0% males; 73.0% females); 3.1% had elevated blood sugar and were linked to care facilities.
Offering integrated services within hard-to-reach communities can increase access to a variety of critical health services, including those for non-communicable diseases, and can link ART clients lost to follow-up back to facilities. This approach will be scaled up throughout Lesotho as a strategy to reach all populations in the country.
The high burden of undiagnosed HIV in sub-Saharan Africa limits treatment and prevention efforts. Community-based HIV testing campaigns can address this challenge and provide an untapped opportunity to identify non-communicable diseases (NCDs). We tested the feasibility and diagnostic yield of integrating NCD and communicable diseases into a rapid HIV testing and referral campaign for all residents of a rural Ugandan parish.
A five-day, multi-disease campaign, offering diagnostic, preventive, treatment and referral services, was performed in May 2011. Services included point-of-care screening for HIV, malaria, TB, hypertension and diabetes. Finger-prick diagnostics eliminated the need for phlebotomy. HIV-infected adults met clinic staff and peer counselors on-site; those with CD4≤100/µL underwent intensive counseling and rapid referral for antiretroviral therapy (ART). Community participation, case-finding yield, and linkage to care three months post-campaign were analyzed.
Of 6,300 residents, 2,323/3,150 (74%) adults and 2,020/3,150 (69%) children participated. An estimated 95% and 52% of adult female and male residents participated respectively. Adult HIV prevalence was 7.8%, with 46% of HIV-infected adults newly diagnosed. Thirty-nine percent of new HIV diagnoses linked to care. In a pilot subgroup with CD4≤100, 83% linked and started ART within 10 days. Malaria was identified in 10% of children, and hypertension and diabetes in 28% and 3.5% of adults screened, respectively. Sixty-five percent of hypertensives and 23% of diabetics were new diagnoses, of which 43% and 61% linked to care, respectively. Screening identified suspected TB in 87% of HIV-infected and 19% of HIV-uninfected adults; 52% percent of HIV-uninfected TB suspects linked to care.
In an integrated campaign engaging 74% of adult residents, we identified a high burden of undiagnosed HIV, hypertension and diabetes. Improving male attendance and optimizing linkage to care require new approaches. The campaign demonstrates the feasibility of integrating hypertension, diabetes and communicable diseases into HIV initiatives.
Planned Parenthood Federation of Nigeria (PPFN) is implementing the Global Fund Round 9 project as Principal Recipient. In 2011, PPFN was challenged with lack of HCT RTK, FP and STI commodities resulting in poor performance.
In creating change to optimise PPFN performance, the Rapid Emergency Scale Up Plan was developed to meet the backlog of unmet targets in three weeks; the
This abstract therefore seeks to document lesson learnt through the plan surpassing PPFN HCT services.
PPFN designed a plan showing targets linked to each clinic. A cumulative target for all clinics within a cluster was assigned to PPFN staff in each of the 35 states. This was replicated by region. To determine if each staff would meet its targets, Kotter's 8 step models was applied.
PPFN had the following targets for 2011: provide service in 486 health facilities and ensure that and 225,800 HCT clients were provided with FP and STI services.
By using FP/STIs services as entry point, PPFN met its target by 110% for HCT services, 213% for FP/STIs services with 88% of facilities providing services
The paper indicates integration of services as a way to meet client's needs in a challenging environment and therefore increases performance while optimizing resources.
While policy and implementation support for SRHR/HIV integration is increasing, significant questions and uncertainties remain about what such programming means in practice. This is particularly the case in concentrated HIV epidemics, where little is still known about what integration should look like for key populations, including sex workers, men who have sex with men (MSM), transgenders, injection drug users (IDUs) and people living with HIV (PLHIV). While integration is a desirable goal in the long-run particularly for clinical services, joining programs and systems that are not ready could compromise quality and access for these groups that already face difficulty in obtaining appropriate services for both HIV and SRH needs.
India HIV/AIDS Alliance undertook a global review of over 160 resources available on the websites of selected national and international organisations, including NGOs, technical support agencies and UN agencies. The resources included case studies, mappings, toolkits, policy briefings and program reports.
The review identified the most common challenges in designing and implementing SRH/HIV integrated programs for key populations. These included stigma and discrimination, low levels of demand, lack of rights-based approaches, low attention to gender inequality, low understanding of key populations' specific SRH needs, lack of capacity and sensitivity among service providers, lack of strong referral systems and inadequate resources for additional interventions. The review highlighted key steps that organisations can take to successfully integrate SRHR and HIV in their responses for key populations.
While SRH/HIV integrated programs present an important opportunity to respond to the unmet needs of key populations, integration that is premature, overly rapid or too large-scale risks compromising rather than enhancing key populations' access to high quality HIV and SRH services. Good practice principles, including gender equality, human rights-based approaches, meaningful involvement of communities, for work with key populations are particularly critical in effective HIV/SRHR integration.
The San Antonio Nathan Shock Center Conferences have attracted international speakers and participants since 1995. This annual conference, held in Bandera, Texas, addresses a different topic in the biology of aging each year. The venue's intimate setting, relatively remote location and common areas are ideal for a small conference (80–100 participants) where informal intellectual interchange supplements that of the formal sessions. The 2012 meeting, part of an annual series sponsored by the Nathan Shock Center of Excellence in the Biology of Aging and the Barshop Institute for Longevity and Aging Studies at the University of Texas Health Science Center San Antonio, addressed the concept that healthy aging and assessment of physiological performance are important parameters, in addition to longevity, to measure quality of life with increasing age.
The purpose of the 2012 conference was to provide a forum for the presentation and discussion of various assays of measuring physiological performance and function and determining what assays of function could be used to asses healthspan of a mouse. Longevity is a precise endpoint (binary, the individual is either alive or it is dead), but the true goal of aging research is to increase the health of the elderly, not their longevity. That is, the goal is to enhance and extend healthspan, defined as the portion of our lives spent free of serious illnesses and disabilities. The assumption is that the only way an organism can increase its lifespan is by increasing its healthspan. This is a plausible assumption, but it still needs to be proven each time a manipulation is assessed for its potential for translation into humans. While the invertebrate models are particularly useful in genetic studies, they are generally not very good models for mammalian health, physiology, disease susceptibility, etc. Mice age with a constellation of diseases and functional losses that in some aspects resemble those observed in humans. Therefore, the conference focused on healthspan measures in mice. To this end, speakers were recruited who are working on assays (both simple and complex) to evaluate the functional status of various organ and physiological systems that are important in the health/physiological performance in mice and/or humans. In addition, attention was given to clarification of the molecular mechanisms underlying physiological decline, and its causal relationship to metabolic changes, muscle wasting, neurodegenerative diseases, cardiovascular disease, cancer, and inflammation and immunity, as well as targets for prophylactic intervention. Thus, the conference gave investigators a panel of assays that would allow them to determine the effect of genetic or pharmacological/nutritional manipulation on healthspan.
Abstracts from posters presented at the meeting are presented in this special abstract issue to provide an overview of the breadth and depth of the program.
Vascular pathology is a major feature of Alzheimer's disease (AD) and other dementias. We recently showed that chronic administration of the target-of-rapamycin (TOR) inhibitor rapamycin, a drug that extends lifespan and delays aging, prevented the development of AD-like disease in mice modeling AD. Here we show that rapamycin administrated after the onset of AD-like deficits reversed brain vascular breakdown through endothelial nitric oxide (NO) synthase activation and NO-dependent vasodilation, decreased cerebral amyloid angiopathy and brain microhemorrhages, and improved memory in AD mice. These data suggest a mechanism by which chronic rapamycin ameliorates established AD-like deficits through the preservation of brain vascular integrity and function. Rapamycin, an FDA-approved drug already used in the clinic, may have promise as a therapy for AD and possibly for vascular dementias.
A widely accepted cause of the functional losses that accompany aging is decreased brain metabolism (i.e., glucose oxidative capacity in mitochondria). It is generally believed that preserving bioenergetics is critical for optimizing lifespan and healthspan. Interventions have been introduced to preserve metabolism in aging process. Caloric restriction (CR) perhaps is the most well-studied one for various model organisms of extended longevity. In addition, in the neuronal system of rats (F344BNF1), CR also enhances cognitive function. However, the underlying physiology in the brain remains unclear. In the study, we used carbon-13 magnetic resonance spectroscopy (C-13 MRS) to investigate CR effect on brain metabolism in aged rats (24 months of age). CR-treated and control F344BNF1 rats (N = 6 for each group) were purchased from NIA. C-13 labeled glucose was continuously infused through the femoral vein of the rat for two hours and MRS was acquired simultaneously. The results show that CR rats had significantly increased oxidative metabolism rate (Voxi) in neurons (p < 0.01) and neurotransmission rate (glutamate-glutamine recycling rate; Vcyc) (p < 0.01) compared to the controls. The aged CR rats’ Voxi (4.5 µmol/g/min) and Vcyc (2.2 µmol/g/min) were comparable to those of young control rats reported in literature. However, CR and control rats did not have significant difference of glucose uptake and lactate production in the brain. The results suggest that alternative fuel subtract (e.g., ketone bodies) may be used to meet the brain energy demand. Our data provide a possible explanation of CR-induced increased lifespan and healthspan in rats.
Chronic administration of rapamycin by transgenic (Tg) PDAPP mice allows them to perform better in hippocampal-associated learning and memory tasks compared with controls. We found, using conventional brain slice methods, that rapamycin had no significant effect on excitatory synaptic transmission, neuronal excitability, or the induction of long-term potentiation (LTP) in the CA1 region of the hippocampus. Surprisingly, we observed no significant effect on LTP in the control Tg group compare with wild type (Wt). We were concerned that some factor, such as stress due to transportation, might have enhanced the likelihood for LTP. To test for this possibility, we examined the relationship between stimulus strength and the magnitude of LTP induction. It is well known that LTP is a function of stimulus strength before induction due to the properties of NMDA receptors; with greater depolarization there is more calcium influx and, in turn, larger LTP. However, we found no correlation for either of our non-rapa control groups (Wt or Tg). In contrast, there was a correlation when animals were administered rapamycin, and the correlation was greater for Wt over Tg animals. Our working hypothesis is that stress, possibly due to transport, depressed inhibitory circuits lowering the threshold for LTP induction. Monte Carlo simulations comparing the amount of LTP produced by variations in the ratio of excitation to inhibition (E/I) support this hypothesis. Chronic rapamycin may protect the hippocampal network from dis-inhibition, maintaining E/I to sustain normal cognitive function.
Chronic treatment with the mTOR inhibitor rapamycin (“Rapa”) extends lifespan in mice. Whether Rapa slows specific aging processes to increase “health span” is unknown. During aging, visual performance declines, and retinal neurons decrease in number. Here, we find and quantify a specific age-related decline in vision in mice. We also show that Rapa does not prevent this decline in visual function or affect neuron number. Instead, Rapa was detrimental to vision. Vision was tested using optokinetic tracking (“OKT”) to measure spatial frequency threshold at maximum contrast (“SPFT”) of the head-tracking behavior to horizontally drifting sinusoidal gratings. Male B6 mice were tested at 5, 21, 29, and 33 months of age (“mos”). Two other lines of mice were fed chow ad libitum containing micro-encapsulated rapamycin from 3 until 18 mos. In another group of B6 mice treated with rapamycin from 4 to 25 mos, retinas were immunostained with markers to count neuron subtypes. During normal aging, OKT SPFT significantly declined by 31%. Rapa did not protect against this age-related OKT decline in either treated strain but significantly decreased OKT performance for male, but not female, mice at 18 mos. Rapa male mice had decreased IPL thickness in the retinal periphery, but numbers of dopaminergic and cholinergic amacrine neurons and retinal ganglion cells were unchanged. Thus, Rapa does not prevent age-related declines in OKT visual function or in retinal neuron number. It instead causes an OKT SPFT deficit in male mice. These findings suggest Rapa does not increase vision health span during aging.
Decline in sensory acuity is a general hallmark of aging, which in humans decreases quality of life. We report here creation and successful utilization of a novel sensory acuity assay in mice. Three features of the assay merit attention. First, as mice are primarily nocturnal in nature, olfaction is an important sensory modality for them. Second, our assay instead of using artificial olfactory cues employs major urinary proteins, which are important in both intrasexual and intersexual communication of mice in nature. Third, the assay can be performed in the mouse's home cage, thus avoiding artifacts from distracting, novel environments. Procedurally, the assay uses serial dilutions of urine and preference for the urinary odor relative to a water control to measure olfactory acuity. Age-related changes in olfactory acuity have not previously been reported in mice. We created this assay, which compares time spent sniffing a sample relative to time spent at a distilled water control. It has been used numerous times and proves to be sensitive, repeatable and encompass particularly informative urinary dilution ranges. Specifically, previous testing revealed that of any age, mice usually cannot distinguish urine from water at a dilution of 1:10,000 (Rendón, unpublished data). The range of experimental dilutions between 1:10,000 to 1:5,000 has been narrowed down through successive modifications. Sampling in this range, we have detected a clearly defined age-related decline in mouse olfactory acuity. Therefore, this assay will serve useful in assessing changes in health span of mice and can be combined with therapeutic agents to assist in evaluation of their effect on health span.
The accumulation of oxidative damage is a proposed mechanism regulating the aging process and the development of disease. Proteins are sensitive to such oxidative stress, which can cause them to accumulate, altering conformational structure, and thus the function, of cellular proteins. Methionine sulfoxide reductase A (MsrA) plays an important role in the antioxidant defense, but is unique in that it repairs protein oxidative damage. MsrA reduces methionine sulfoxide residues to non-oxidized methionine, thus participating in the antioxidant defense system of cells specifically by protecting proteins from oxidative stress. We have found that mice that lack MsrA (MsrA−/−) and mice that over express MsrA (MsrATg) are phenotypically similar to wildtype (WT) mice under normal conditions, but that MsrA levels can regulate susceptibility to oxidative stress. Because these mice are grossly normal, this suggests that excess methionine oxidation may not occur under these physiological conditions. In vivo, increasing adiposity has been associated with increases in oxidative stress, altered redox signaling and increased oxidative damage to cellular macromolecules in several disease models, including obesity-induced metabolic diseases. When placed on a high fat (HF) diet, MsrA−/− mice become more insulin resistant than WT mice whereas MsrATg mice are protected from development of insulin resistance. The increase in insulin resistance in MsrA−/− mice fed HF diets correlated with reduced insulin-stimulated signaling in the insulin signaling pathway. We found that HF fed MsrA−/− mice had reduced phosphorylation of both insulin receptor and Akt with administration of insulin under high fat fed conditions. Also, increased insulin sensitivity seen in the HF fed MsrATg mice correlated with an increase in insulin-stimulated signaling in the insulin signaling pathway. These results suggest that oxidative damage, specifically to proteins, may play an important role in obesity-induced insulin resistance. To address how protein oxidation may cause insulin resistance, we have utilized in vitro studies in primary myoblasts to test the effect of MsrA on oxidative stress-induced insulin resistance. By utilizing these models, this study will test the hypothesis that MsrA can regulate the development of insulin resistance by repairing oxidative damage in proteins involved in the insulin signaling pathway in vitro. Insulin resistance can be induced in vitro by H2O2. In this study, skeletal muscle precursor cells isolated from MsrA−/−, MsrATG, and WT mice, and then differentiated into myotubes, were tested for resistance to oxidative stress. Insulin signaling protein phosphorylation correlates with in vivo signaling observations, determined by western blot after insulin stimulation. Our hypothesis is that the level of protein oxidation can be correlated with the degree of insulin resistance in this system. Protein oxidation can be globally measured in the cell using a carbonyl assay. Once labeled, individual proteins can also be measured for total carbonyl content via immune precipitation. Because oxidation of proteins can lead to a decline in their function, these studies will focus on both function of the insulin signaling proteins isolated from these models as well as oxidation status of these proteins.
Loss of skeletal muscle function is severely debilitating and sarcopenia profoundly affects the quality of life in the aged population. Impaired mitochondrial energetics in skeletal muscle is associated with loss of function and increased mitochondrial oxidative stress. To explore age-related mitochondrial energy deficits we use chronic (transgenic) and acute (pharmacological) targeting of mitochondrial oxidative stress. Previous work demonstrated that mitochondrial targeted catalase (mCAT) delays the onset of age-related pathology and extends lifespan in mice. However, little is known about how the relationship of mitochondrial energetics and cellular redox status changes with age. We demonstrate that there is a decline in mitochondrial quality in aged permeabilized skeletal muscle, particularly in the fast-twitch extensor digitorum longus, that was prevented in mice expressing mCAT. We also demonstrate that acute treatment (~1hr) of aged mice with the mitochondria-targeted small peptide SS-31 results in immediate improvement of skeletal muscle energy metabolism and performance. These results provide further evidence that decreased mitochondrial function with age may be due to an altered oxidative status of mitochondria and we propose that there are two facets of mitochondrial deterioration with age: a structural component that is attenuated with long-term expression of MCAT, and a regulatory component dependent upon the oxidative status of the cell that is rapidly reversible with acute treatment of SS-31. These results suggest that the oxidative state of skeletal muscle is a practical therapeutic target, and raises questions about how oxidative status and mitochondrial content affect the adaptive and pathological response of mitochondrial metabolism to age.
Recently, our laboratory made the surprising observation that overexpressing Cu/ZnSOD [Tg(SOD1-SD)+/0] in Sprague-Dawley (SD) rats resulted in a significant increase in lifespan and a reduction in age-related pathologies. The purpose of this study was to determine why overexpressing Cu/ZnSOD increases lifespan in SD rats. The Tg(SOD1-SD)+/0 rats showed lower levels of oxidative damage to DNA and lipids in vivo and higher resistance to oxidative stress in vitro. Both Tg(SOD1-SD)+/0 and wild-type rats showed an age-related increase in body fat and Cu/ZnSOD overexpression did not attenuate adiposity. Interestingly, Tg(SOD1-SD)+/0 rats showed a significant increase in insulin sensitivity at a young age and lower plasma glucose levels at an old age. To further investigate the role of Cu/ZnSOD overexpression on aging, we generated transgenic rats with F344 overexpressing Cu/ZnSOD [Tg(SOD1-F344)+/0]. Tg(SOD1-F344)+/0 rats showed similar levels of Cu/ZnSOD overexpression to Tg(SOD1-SD)+/0. The Tg(SOD1-F344)+/0 rats showed lower levels of oxidative damage to lipids in vivo, however, neither Tg(SOD1-F344)+/0 nor wild-type rats showed any age-related changes in body fat, insulin insensitivity, and plasma glucose levels. Furthermore, Tg(SOD1-F344)+/0 rats showed little increase in lifespan compared to wild-type rats. Our results are very exciting because these data indicate that overexpression of Cu/ZnSOD could be more protective against oxidative stress and could attenuate aging and age-related diseases under obese conditions in mammals. (Supported by grants from the VA Merit Review, the American Federation for Aging Research, and the Glenn Foundation)
A reduced ability to effectively regulate glucose metabolism is one of the most common markers of declining healthspan in aging mammals. Advancing age is an independent risk factor in the development of glucose intolerance, insulin resistance, and diabetes mellitus. Understanding the mechanisms responsible for this could significantly contribute to developing effective therapeutics or preventatives for those most at risk. Our data support a hypothesis that oxidation of proteins involved in insulin signaling may play a significant role in this process. Using a cell culture model, we show that oxidative stress inhibits the cellular response to insulin. The binding of insulin to insulin receptor normally promotes auto-phosphorylation of the β-subunit which regulates downstream insulin signaling through its kinase activity. Our data show that oxidative stress inhibits insulin signaling partly by causing oxidative damage that inhibits this process. Oxidative stress promotes formation of protein carbonyl adducts within insulin receptor; these adducts lead to diminished auto-phosphorylation function. We then addressed whether insulin receptor oxidation occurs in vivo with metabolic dysfunction. Insulin receptor isolated from high fat-fed C57BL/6 mice also show significantly elevated insulin receptor oxidative damage and reduced auto-phosphorylation function. Our preliminary studies suggest a similar process of oxidative damage is associated with reduced glucose metabolism in aging mice. These data support the idea that accumulating oxidative damage is a common molecular mechanism by which several primary risk factors (i.e., obesity, aging) promote insulin resistance. Targeting therapeutics that reduce/remove/repair oxidative damage might then develop as a valuable treatment option among the geriatric population.
Longevity and aging are influenced by common intracellular signals of the insulin and IGF-1 pathway. Enhanced availability of IGF-1 is promoted by cleavage of IGF binding proteins (IGFBPs) by proteases, including the pregnancy-associated plasma protein-A (PAPP-A). PAPP-A (-/-) mice live 30% longer than their normal littermates and have decreased bioactive IGF-1 on normal diets. Our objective was to elucidate the effects of a high-fat (58 % kcal)/ high-sucrose (25.5 % kcal) diet that promotes obesity and increase pro-inflammatory cytokines in normal and PAPP-A(-/-) female littermates. The results indicate that PAPP-A (-/-) mice fed a high energy diet are more glucose tolerant than normal littermates fed a low energy diet (P ≤ 0.05) while insulin tolerance did not change. The high energy diet increased IGF-1 levels in PAPP-A (-/-) mice compared to littermates (-/-) fed a low energy diet (P ≤ 0.002). PAPP-A (-/-) mice fed with a high energy diet had lower levels of pro-inflammatory cytokines (IL-2, IL-6 and TNF-a) compared to normal littermates fed a high energy diet (P < 0.05). In contrast, anti-inflammatory cytokine levels (IL-4 and adiponectin) were higher in PAPP-A (-/-) mice fed a high energy diet compared to normal littermates on high energy diet (P < 0.05). We conclude that PAPP-A (-/-) mice when compared to normal littermates are resistant to the effects of diet-induced metabolic dysfunction. Furthermore, high energy fed PAPP-A (-/-) mice have higher levels of anti-inflammatory cytokines and lower levels of inflammatory cytokines, possibly rescuing them from the detrimental effects of a high energy diet.
Obesity is a major risk factor for the development of age-related metabolic diseases. The mammalian target of the rapamycin (mTOR) pathway plays critical roles in eukaryotic cell growth, survival, and translation and hyperactivation of mTOR pathway due to excess nutrients causes insulin resistance, a major risk factor for type 2 diabetes. Rapamycin is a potent inhibitor of mTOR pathway suggesting its beneficial effects on metabolism. Paradoxically, rapamycin treatment causes glucose intolerance in mice. While most of the studies focus on the effect of rapamycin on metabolism in normal mice, no study has addressed the metabolic effects of rapamycin in diabetic mouse models. Here, we are studying the effects of rapamycin in db/db mice, a model of diabetic dyslipidemia. Administration of rapamycin for 9 months, starting at 2 months of age, significantly reduced body weight (43%) in female db/db mice compared to db/db mice fed the control diet (eudragit), due to a reduction in fat mass. This reduction in fat mass is not due to alterations in fat synthesis (PPARξ and SREBP1) or fatty acid transport (CD36 and FATP1) or lipolysis (P-HSL/HSL ratio), rather due to increased levels fatty acid oxidation as indicated by increased levels of carnitine palmitoyltransferase I (CPT1, 5-folds), large-chain acyl-coenzyme A dehydrogenase (LCAD, 2.5-folds) and medium-chain acyl-coenzyme A dehydrogenase (MCAD, 1.5-folds) in rapamycin-fed db/db mice compared to eudragit-fed db/db mice. Consistent with this observation, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1-α) are significantly up-regulated both at the transcriptional and translational levels. In addition, markers of mitochondrial biogenesis CREB-regulated transcription coactivator 3 (CRTC3), nuclear respiratory factor 1 (NRF1) and estrogen-related receptor alpha (ERRα) were significantly elevated in the adipose tissue of rapamycin-fed db/db mice. While rapamycin did not decrease the levels of circulating triglycerides and glucose in db/db mice, levels of circulating free fatty acid was significantly reduced and adiponectin levels were significantly increased by rapamycin treatment, suggesting improved insulin sensitivity. Finally, insulin sensitivity assessed by insulin tolerance test showed significant improvement in insulin sensitivity in rapamycin-fed db/db mice. In summary, our study demonstrates for the first time that rapamycin exhibits anti-obesity effect in db/db mice and improves insulin sensitivity due to the up-regulation of the mitochondrial biogenesis and increased fatty acid oxidation in the white adipose tissue.
Cytochrome c oxidase (COX) is an essential transmembrane protein complex in the mitochondrial respiratory electron chain. Mutations in genes responsible for the assembly of COX are associated with Leigh syndrome, cardiomyopathy, spinal muscular atrophy and other fatal metabolic disorders in humans. Paradoxically, mice lacking the COX assembly protein SURF1 show increased longevity associated with upregulation of mitochondrial biogenesis and stress response pathways despite significant reductions in COX activity. Here we asked whether a mouse model of cytochrome c oxidase deficiency due to a mutation in the sco2 gene, a copper chaperone that is required for the activity of COX would have similar molecular and physiologic changes. A complete knockout of the sco2 gene in mice is embryonic lethal, however mice harboring a mutated sco2 knock-in (KI) allele that is commonly found in human patients with sco2 mutations is viable, and despite the 30–60% reduction in COX activity, no significant phenotypic abnormalities are readily apparent. Interestingly, these mice have a decrease in lean mass and increase in fat mass. Preliminary evidence suggests that these mice are insulin resistant and glucose intolerant as compared to wild-type mice. The sco2 KI/KI mice also have decreased running endurance on the treadmill suggesting that these mice have muscle weakness. Interestingly, the COX-deficient mice do not have changes in the blood lactate levels suggesting that these mice do not upregulate glycolysis to compensate for decreased rates of respiration. This is counter to studies done in another COX deficient Surf1-/- mice, illuminating the complex nature of mitochondrial dysfunction on physiology. Results from this study will further our understanding of the role of complex IV in physiological outcomes due to mitochondrial dysfunction.
Vitamin D insufficiency, sarcopenia of aging, and obesity exert profound impacts on physical performance and overall healthspan. Although human clinical studies have demonstrated significant relationships between vitamin D and physical performance, they contain confounding factors such as age, obesity, diet, and lifestyle that make understanding the specific pathophysiology difficult. Therefore, we are developing a novel mouse model capable of isolating individual and combinatorial impacts of vitamin D insufficiency, aging and obesity on physical performance. We provided 6 month-old male mice with either 1000IU or 125IU vitamin D3/kg chow over 4 months. Longitudinal serum 25-OH vitamin D measurements show levels change rapidly (both depletion and repletion) and consistently to the degree of supplementation, allowing for comparisons between sufficient and insufficient mice. Furthermore, our data indicate body weight and fat percentage are higher in vitamin D insufficient mice after 4 months. Additionally, our data suggest that vitamin D insufficient mice have higher levels of IL-6 and TNF- in their epididymal fat tissue. Rotarod treadmill performance and grip strength were similar regardless of vitamin D status. However, we found that elderly mice (24 months) exhibit functional decline compared to young mice despite both groups being sufficient (25-OHD ≥ 30 ng/ml). These data lay the foundation for our continuing investigation on vitamin D insufficiency, aging, obesity and physical performance and will further our understanding of the underlying mechanisms driving health span decline.
Synucleinopathies are age-related neurodegenerative disorders characterized by expression of pathological α-synuclein inclusions. Synucleinopathies include Parkinson's disease (PD), multiple system atrophy and dementia with Lewy bodies (DLB), which affect millions of patients worldwide. Parkinsonian motor symptoms like rigidity and slow movement are common in synucleinopathies. A53T mutation is the first α-synuclein mutation linked to PD, and it is linked to both sporadic and familial PD. Autophagy is reduced in PD brain. Levels of mTOR are increased and ATG7 levels are reduced in DLB brain. Rapamycin, an mTOR inhibitor and autophagy enhancer, is protective in mouse models of neurodegenerative diseases like Alzheimer's disease and PD. Rapamycin reduces a-synuclein accumulation and neurodegenerative phenotype in neuronal cells. Feeding rapamycin diet extends mouse lifespan and the mechanisms are hypothesized to be mediated via delaying age-related diseases including neurodegenerative diseases. The aim of the study is to determine whether long-term feeding rapamycin diet at the dose that extends mouse lifespan attenuates motor deficits in neuronal A53T α-synuclein transgenic mice, which express human A53T α-synuclein richly in brain and spinal cord and develop motor deficits. Mouse diet containing microencapsulated rapamycin (14 ppm in diet; 2.25 mg/kg body weight/day) or the microencapsulation material was fed to age-matched wild type and A53T mice from 13 weeks of age. After 24 weeks of treatment, rapamycin improved performance on forepaw stepping adjustment test, accelerating rotarod test and pole test in both genders of A53T mice. Rapamycin also increased front stride length in male A53T mice. In conclusion, rapamycin attenuated motor deficits in the A53T mice. Further experiments will determine whether the effects of rapamycin are through reducing human α-synuclein expression in brain regions that control and regulate motor function including motor cortex, spinal cord, midbrain, striatum and cerebellum. In addition, it is reported that rapamycin improves myelination in explant cultures from neuropathic mice. Thus, effect of rapamycin on demyelination in A53T mice will also be determined in the brain regions mentioned above.
Parkinson's disease (PD) is the second most prevalent neurodegenerative disease. Degeneration of dopamine neurons within the substantia nigra, leads to a substantial decrease in dopamine release in the substantia nigra and the striatum, as well as impaired motor function. Motor symptoms associated with PD include resting tremor, rigidity, and bradykinesia. Although the cause of this disorder remains unclear, several lines of evidence implicate mitochondrial dysfunction and oxidative stress. Major cytosolic enzymes ALDH1 (aldehyde dehydrogenase 1) and GPX1 (glutathione peroxidase 1), are involved in the metabolism of biogenic aldehydes and the reduction of hydrogen peroxide, respectively. ALDH1 is selectively expressed in the midbrain and found to be co-localized with tyrosine hydroxylase within the substantia nigra and ventral tegmental area. Gene profiling studies have been reported showing a decreased expression of ALDH1 in surviving dopaminergic neurons of PD patients. GPX1 gene expression in the substantia nigra of PD patients is also markedly reduced. Therefore, we hypothesize that deletion of both Aldh1a1 and Gpx1 will lead to the accumulation of reactive oxygen species and highly reactive biogenic aldehydes leading to motor deficits. To test this hypothesis, our lab crossed two mouse lines deficient in Aldh1a1 and Gpx1 genes. Here we report impaired locomotor function in Aldh1a1 x Gpx1 knockout mice. These data are consistent with the idea that elevated levels of reactive oxygen species and/or biogenic aldehydes may lead to motor deficits similar to those found in Parkinson's disease.
Traumatic Brain Injury (TBI) is a known risk factor for ALS. The goal of this study is to elucidate the mechanism linking TBI to motor neuron disease, by testing the hypothesis that TBI will accelerate disease progression in animal models of ALS. We used the well-characterized mouse models of familial ALS (G93A SOD1) and sporadic ALS (TDP43, TDP25) to study the effect of TBI on ALS progression. Mice were subjected to a closed head traumatic brain injury and magnetic resonance imaging (MRI) was used 3 days after injury to characterize structural central nervous system pathology and the severity of brain injury. Histological techniques showed neuronal loss (NeuN), astrocyte infiltration (GFAP) and edema (Nissl) following mild TBI in wildtype (WT) and transgenic mice (TG). Our preliminary results indicate that TBI leads to a reduction in grip strength, decreased rotarod performance and muscle denervation via electromyography abnormalities. Also, we have characterized an acceleration of disease related weight loss and overall disease score following TBI in G93A mice. Our results are the first to show that TBI, in an animal model of ALS, results in significantly increased muscle denervation and potentiates disease onset and progression. This work is supported by an individual fellowship grant, 1F31NS080508-01, as well as the Barshop Institute for Longevity and Aging.
Mechanical inactivity or disuse causes muscle loss and bone loss in both men and women; however, it is not known whether food restriction (FR) has any effect on mechanical inactivity-associated muscle and bone loss. Disuse-associated musculoskeletal atrophy could be associated with nerve injury. The present study aimed to investigate the effect of 40% FR on sciatic nerve injury associated muscle and bone loss and also to analyze if there is any time dependent effect of FR after sciatic nerve injury. Two-month-old male C57BL/6 mice were randomly allocated into two groups: (1) ad libitum (AL) (2) 40% FR fed lab chow for 8 months. The left hind limb of each mouse was then subjected to sciatic nerve crush to induce mechanical inactivity of the particular leg. After different time points (2 days, 7 days, 14 days, 21 days, 28 days and 42 days) of mechanical inactivity, mice were sacrificed and analyzed for muscle mass (wet weight) and bone mass (dual energy x-ray absorptiometry (DXA)). AL fed mice showed significant loss of gastrocnemius and tibia due to mechanical inactivity whereas, FR mice showed protection of both gastrocnemius and tibia from inactivity associated loss. Interestingly, this gastrocnemius and tibial loss protection was stable up to 42 days of mechanical inactivity, we have tested. This data suggests that FR may be beneficial in case of disuse situation commonly happened during aging. Further studies are necessary to determine the musculoskeletal quality and the molecular mechanisms involved in FR mediated protection of musculoskeletal loss due to disuse.
Oxidative stress is implicated in loss of muscle mass with age in the CuZnSOD deficient mice (Sod1-/-). However, the mechanisms of oxidative stress-dependent loss in muscle mass are currently unknown. Since oxidative stress is considered to be an important contributor to muscle atrophy and muscle activity is dependent upon nerve stimulation, this study proposes that oxidative stress damages protein integrity which leads to impaired nerve conduction velocity and myelination. To test our hypothesis, we chose the Sod1-/- mouse model and control c57bl/6 mouse to determine declines in nerve conduction velocity and myelination. Gastrocnemius muscle isolated from the Sod1-/- mice have significant atrophy at 6 and 18 months of age. Sciatic nerve conduction velocity was significantly impaired at both 6 and 18 months of age in the Sod1-/- mice. 6 month old Sod1-/- mice had reduced axon and fiber diameter with what appeared to be changes in myelin morphology which by 18 months of age resulted in reduced myelin thickness and increased g-ratio (axon/fiber diameter). Also, the sciatic nerves from the Sod1-/- mice exhibited significant global increase in protein carbonyls and alteration in exposure of surface hydrophobic domain in proteins. Taken together, these data suggest that loss in nerve conduction velocity and myelin might play a significant functional outcome in gastrocnemius atrophy.
Half of all Americans over the age of 50 either already have or will develop osteoporosis and osteoporotic fracture is associated with increased mortality rates. Fracture can be considered a chronic condition as complications from fracture can extend well past healing of the initial fracture, thus preventing fracture is required for prolonging healthspan. Bone mineral density (BMD) is highly correlated to fracture risk and environmental factors, such as diet impact BMD. As diet can be modulated, identification of what types of and how dietary constituents decrease BMD will increase our general knowledge about the etiology of osteoporosis and could illuminate opportunities to intercede to prevent fracture. Preliminary studies have suggested that a high fat diet negatively impacts bone mass, but it remains unknown which type of fat mediates these negative effects. In this study, we specifically examined the consequence of increased cholesterol intake on bone mass and osteoblast maturation. We determined that dietary cholesterol, independent of other types of dietary fat negatively impacts on BMD in C57BL/6J female mice. We then established that dietary cholesterol appears to decrease the marrow osteoblast progenitor pools in the femur. In the vertebrae, a high cholesterol diet was associated with a decrease in trabecular bone thickness and with an increase in osteoclastic activity in the vertebrae. Together, this shows that dietary cholesterol, independent of other types of dietary fat, negatively impacts bone mass. In the femur, cholesterol affects the osteoblast linage where as in the vertebrae its effects are mediated via osteoclastic bone resorption.
Maintenance of skeletal muscle regenerative capacity is crucial for preservation of muscle mass and function with age. Skeletal muscle precursor (SMP) cells are myogenic stem cells that play a predominant role in muscle regeneration. These cells are located beneath the basal lamina of the myofiber and respond to tissue injury with activation, differentiation and fusion into an existing myofiber. Previous studies have identified a panel of cell surface markers to isolate pure populations of SMP cells from mice with minimal contamination by flow cytometry. Using this technique, the current study assessed the impact of age on SMP content and function. By analyzing male C57Bl/6 mice aged 18–100 weeks on a standard ad libitum diet, it was found that the SMP population decreases by 20–60% with age, depending on the muscle depot. The greatest decline was found in the triceps bracii, which is composed predominantly of Type IIb fibers (fast glycolytic). Furthermore, the regenerative capacity of isolated cells was impaired in older mice, as measured by proliferation and differentiation of SMP cells into myofibers. This study highlights the negative effect of aging on skeletal muscle stem cells. Future work will explore interventions to prevent the loss of regenerative capacity with age.
Osteoporosis is a silent disease characterized by excess bone resorption by osteoclasts compared to bone formation by osteoblasts. Our lab has shown that old male mice deficient in caspase-2 (Casp2-/-), a cysteine protease involved in apoptosis, exhibit severe age-related osteoporosis. Interestingly, these mice also have higher numbers of osteoclasts compared to age-matched wild-type (WT) mice. This could mean that more osteoclasts are being created or less osteoclasts are dying in Casp2-/- animals compared to WT. However, the role of caspase-2 in osteoclasts remains to be elucidated. We hypothesize that caspase-2 plays a dual role in both osteoclast apoptosis and differentiation. With regards to apoptosis, caspase-2 as an upstream component of the apoptotic pathway has been well described in a variety of cell types. Furthermore, cells lacking caspase-2 have been shown to be more resistant to oxidative stressors. Here, we show that osteoclasts derived from Casp2-/- mice are more resistant to 6 hour treatment with various doses of the general stressor H2O2 and the mitochondrial stressor rotenone compared to osteoclasts from WT mice. Osteoclasts are formed through macrophage fusion that is spurred by the osteoblast-derived cytokines CSF-1 and RANKl. We show that macrophages from Casp2-/- animals form increased numbers of osteoclasts compared to WT. In addition, we have seen that caspase-2 levels decrease in macrophages after RANKl stimulation, suggesting that low caspase-2 expression may be important during osteoclast differentiation. Delineating the role of caspase-2 in the osteoclast may provide new information that will aid in the development of novel osteoporosis treatments.
Neuromuscular junction (NMJ) degeneration and muscle atrophy occur with age and in various neuromuscular diseases. Previously we have demonstrated that mice deficient in Cu/Zn superoxide dismutase (CuZnSOD or SOD1) exhibit age-dependent NMJ degeneration, muscle weakness and functional motor deficits. The purpose of this study was 1) to determine whether these changes are associated with alterations in NMJ neurotransmission; 2) to determine whether the NMJ phenotype is a cell-autonomous trait of CuZnSOD deficiency in muscles or neurons. Electrophysiological studies of CuZnSOD knockout mice (KO) demonstrate pathological decrement in compound muscle action potential (CMAP) amplitude with repetitive nerve stimulation (RNS), which is indicative of faulty neurotransmission. To test the second aim, we utilized tissue specific knockout and transgenic mice of SOD1. Neuron-specific SOD1 knockout mice (NKO) developed a moderate reduction in muscle mass, while muscle-specific SOD1 knockout mice (MKO) showed no muscle atrophy. Neither NKO nor MKO mice showed alterations in RNS, suggesting the NMJ deficits in KO mice may be a synergistic effect from both cell types. However MKO mice exhibit multiple characteristics of myopathy including denervation potentials, central nuclei and increased muscle damage upon exercise. It suggests that CuZnSOD plays an essential role in maintaining skeletal muscle integrity. Meanwhile, neuronal SOD1 overexpression rescued muscle atrophy and aberrant CMAP parameters in the KO mice. In conclusion, the complete NMJ phenotype in KO mice is likely caused by deficiency of CuZnSOD in both muscle and neurons. Our data indicate that muscle atrophy in KO mice may be secondary to the neuronal defect.
Aging is associated with chronic low-grade inflammation, due in part to the pro-inflammatory secretory profile of replicative senescent cells (i.e. senescence associated secretory phenotype [SASP]). Paradoxically, macrophages from aged animals fail to produce the pro-inflammatory cytokines necessary to recruit and activate other immune cells and have poor bacteria killing ability (i.e. age-dependent macrophage dysfunction [ADMD]). A recent publication examining LPS-induced macrophage anergy [Park SH, et al., Nat. Immunol. 2011, 22:12(7): 607–15] triggered us to test the hypothesis that pro-inflammatory cytokines produced by senescent cells may be responsible for ADMD. Bone-marrow derived and J774A.1 macrophages exposed to senescent type II epithelial lung cells (A549 cell line) overnight demonstrated a decreased ability to kill Streptococcus pneumoniae, a gram-positive bacteria and the leading cause of community-acquired pneumonia, versus those exposed to normal cells in vitro. J774A.1 macrophages, but not bone-marrow derived macrophages, exposed to filtered conditioned media from senescent cells also showed an attenuated ability to kill bacteria versus controls. Likewise, they demonstrated an inability to produce de novo Interleukin-6 following stimulation with ethanol-killed pneumococci. Ongoing studies are focused on determining the component produced by senescent cells that is responsible for macrophage dysfunction.
The ubiquitin proteasome system (UPS) is responsible for the controlled cleavage of damaged and misfolded proteins and antigen-producing peptides. Commonly reported declines in efficiency of the UPS with age may play a critical role in age-associated dysfunction of protein homeostasis and immune function. The longest-lived rodents, naked mole-rats (Heterocephalus glaber), maintain robust, cancer-free good health for 75% of their 32 year lifespan suggesting that decline in protein homeostasis, observed in other animals, is attenuated or delayed. We compared age-related changes in proteasome activity in whole cell and sub-fractionated lysates from spleen tissues of naked mole-rats and physiologically age-matched mice. Naked mole-rat lysates, as well as cytosolic and nuclear fractions had significantly higher proteasome chymotrypsin-like (ChTL) and trypsin-like (TL) activity than those of age-matched mouse samples. The age-related decline in naked mole-rat ChTL and TL proteasome activity in spleen lysates was negligible; in contrast mice showed a significant age-related decline. By 70% of maximum lifespan proteasome activity of naked mole-rat was unchanged (p > 0.05) whereas mouse declined by 48% (p < 0.02). Similar age-related species differences were observed in all three fractions. Attenuation of age-related UPS decline in naked mole-rats was further supported by sustained maintenance of the 26S proteasome with age, and higher levels of constitutive and immunoproteasome-related proteasome subunits in the naked mole-rat compared to mice. Given the importance of the spleen in immune function, high and sustained UPS in splenic tissue may contribute significantly to prolonged good health in this extraordinary long-lived rodent.
Natural aging processes cause gradual degradation or senescence of the immune system at the humoral and cellular levels. A diminished immune capacity due to aging correlates clinically with decreased vaccine efficacy and increased susceptibility to infection and cancer. Due to this loss in immunity the protective capacity of new vaccines should be determined in older individuals. Animal models for vaccine development should embody the immunosenescent effects observed in aging humans. A baboon model was tested by immunizing young (5–6 years of age) and old (17–22 years) animals with the LcrV and F1 candidate vaccine antigens from Yersinia pestis. Contrary to the expected loss of immunity, older baboons demonstrated high antibody titers and exhibited strong T cell proliferation, particularly in response to LcrV. These findings suggest that aging has less effect on the baboon immune system. The cellular and cytokine responses to antigen stimulation were measured to better characterize the effects of aging on T cell fine specificity. T cell proliferation and IFN-γ ELISpots were used to map which of 32 overlapping synthetic F1 peptides stimulated T cells from the immunized baboons. Spectratype analysis of T cell receptor (TCR) expression indicates no age associated loss in T cell activation of the overall repertoire diversity. Currently, F1-specific T cell lines are being generated using herpesvirus papio transformed B cell lines as antigen presenting cells. Future efforts will focus on characterizing the TCR repertoire of an F1-speific response.
The anti-tumor action of calorie restriction (CR) and the possible underlying mechanisms on tumor growth were investigated using ethylnitrosourea (ENU)-induced glioma in rat. ENU was given transplacentally at gestational day 15. The brain from 4, 6, and 8-month-old rats fed either ad libitum (AL) or calorie restricted diets (40% restriction of total calories compared to AL rats) were studied. Tumor burden was assessed by comparing the size and number of gliomas present in the brain. Immunohistochemical analysis was used to detect the lipid peroxidation products [4-hydroxy-2-nonenal (HNE), malondialdehyde (MDA), and acrolein] and nitrotyrosine to document oxidative stress, levels of glycated end products, cell proliferation activity (PCNA), and cell death (ssDNA) associated with the development of gliomas. The results showed that the number of gliomas did not change with age in the AL groups; however, the average size of the gliomas was significantly larger in the 8-month-old group compared to that of the younger groups. Immunopositive areas for HNE, MDA, acrolein, nitrotyrosine, and glycated end products increased with the growth of gliomas. The CR group showed both reduced number and size of gliomas, less accumulation of oxidative damage, and less glycated end products compared to the AL group. Furthermore, the CR group showed less PCNA positive and more ssDNA positive cells. Interestingly, we also discovered that the anti-tumor effects of CR were associated with less accumulation of hypoxia inducible factor-1α (HIF1α) levels and a reduction in the mammalian target of rapamycin (mTOR) signaling. Our results are very exciting because they could not only demonstrate the anti-tumor effects of CR on oligodendroglioma, but also indicate the possible underlying mechanisms, i.e., anti-tumor effects of CR could be mediated by the changes in redox-sensitive and/or nutrient sensing signaling pathways. (Supported by grants from the VA Merit Review, the American Federation for Aging Research, the Glenn Foundation, and San Antonio Nathan Shock Center)
Our laboratory has conducted the first detailed study on the effect of overexpressing or down-regulating thioredoxin 1 (Trx1: cytosol) or thioredoxin 2 (Trx2: mitochondria) on aging. Interestingly, we found that the Trx2Tg mice showed an extension of median lifespan compared to wild-type mice, although we observed little increase in survival of the Trx1Tg mice. The extension of lifespan of Trx2Tg mice was correlated to less reactive oxygen species (ROS) production from mitochondria and less oxidative stress. These data show that overexpressing Trx in the mitochondria may be more important than in the cytosol on aging because mitochondria are a major source of ROS. When we tested the effects of reduced levels of Trx in cytosol or mitochondria on aging, we surprisingly observed the reversed effects, i.e., an increase in survival of the Trx1KO mice compared to wild-type mice, while the Trx2KO mice showed little effects on lifespan. The extension of lifespan of Trx1 KO mice was associated with less cancer compared to wild-type mice at 22–24 months of age. These results indicate that reduced cancer in the Trx1KO mice could be one of the contributing factors of extended lifespan. Our data are exciting in that we show 1) overexpressing Trx in the mitochondria increases lifespan, but overexpressing Trx in the cytosol has little effect on lifespan, which is similar to the results of mCAT mice; and 2) down-regulating Trx in the cytosol increases lifespan and reduces cancer, but down-regulating Trx in mitochondria has no effect on lifespan or cancer. These paradoxical, but intriguing results could indicate that the Trx2Tg and Trx1KO mice attenuate aging through different mechanisms, e.g., protection of mitochondria against oxidative stress and reduced age-related pathology, e.g., cancer. (Supported by grants from the VA Merit Review, the American Federation for Aging Research, and the Glenn Foundation)
Long-lived animal models across multiple phyla have a marked resistance to toxins and other xenobiotics. The longest-lived rodent, the naked mole-rat, has a maximum lifespan of 32 years and is the size of mouse yet lives almost 8 times longer. During their very long lifespan, naked mole-rats show minimal declines in many physiological and molecular age-related characteristics, and most interestingly, an incidence of spontaneously occurring cancer has never been reported. Naked mole-rats are also very resistant to an extensive array of toxins in vitro. We hypothesize that cytoprotective mechanisms in this species are contributing to their protection. We focus on pathways regulated by nuclear factor-erythroid 2-related factor-2 [Nrf2] as the key cytoprotective signaling pathway facilitating this broad resistance to cytotoxins and stressors. This ubiquitously expressed and highly conserved transcription factor has been heavily researched with regards to toxin resistance and cancer, and has been shown to interact with p21 and tumor suppressor p53, implying a role for Nrf2 in cell cycle regulation and cancer progression. Naked mole-rats show an in vitro and in vivo constitutive upregulation of Nrf2-cytoprotective signaling as well as resistance to toxins in both fibroblasts and whole animals. These long-lived rodents also show pronounced resistance to carcinogenesis in vivo and our data reveal that oncogenic and apoptotic activation may be more sensitive in naked mole-rats. By utilizing the naked mole-rat as a model of impeccable healthspan and lengthened lifespan, we may not only identify novel mechanisms that contribute to toxin resistance and cancer prevention, but also longevity.
The molecular mechanisms behind aging are complex, and one emerging theory asserts that aging occurs as a result of changes in the epigenetic landscape. Here we test the hypothesis that dietary restriction (DR) mediates its anti-aging effects through epigenetic modifiers and modifications. To test the hypothesis that DR mediates its protective effects through epigenetic modifiers, we used surgical nerve crush to model the denervation that occurs in aging skeletal muscle. We demonstrate that DR, even when initiated after surgery, protects against denervation-induced muscle atrophy as measured by gastrocnemius wet weight. DR inhibited the induction of histone deacetylase 4 (HDAC4), a known mediator of atrophic signaling in skeletal muscle. Using the general HDAC inhibitor sodium butyrate (NaBu), we demonstrate that pharmacologically inhibiting HDACs protects against the muscle loss induced by nerve crush, thus mimicking the effects of dietary restriction. To investigate the effects of aging and DR on histone modifications, we analyzed liver histones from young and old animals fed ad libitum or dietary restricted for acetylation at specific residues. We found an age-related decrease in histone H3K9 acetylation, and importantly this decrease was prevented by dietary restriction. To simulate the increase in histone acetylation seen with dietary restriction, we fed old animals the HDAC inhibitor NaBu which resulted in reduced fat mass and increased glucose tolerance over time, consistent with known effects of dietary restriction. Overall, our data support the epigenetics theory of aging and indicates that dietary restriction uses epigenetic mechanisms to protect against age-related pathologies. (This work was funded by the UTHSC at San Antonio Biology of Aging Training Grant to Steve N. Austad (MEW T32AG021890-10).
Protein homeostasis has been implicated in the aging process in a variety of model organisms. We are utilizing a range of marine bivalve mollusk species, with lifespans ranging from under a decade to over five hundred years, in a comparative study to investigate the hypothesis that long life requires superior proteome stability. These ages can be individually determined by counting growth rings in the shell. This experimental system provides a unique opportunity to study closely related organisms with vastly disparate longevities, including the longest lived animal, and their relative proteome stabilities. Specifically, we are testing their ability to maintain structure and function under various stressors, as well as prevent protein damage and aggregation. Furthermore, the influence of each species’ isolated metabolite fraction is being investigated on each of these proteostasis aspects. Protein damage and unfolding were quantified by incorporation of two fluorescent probes, specific for carbonyls and exposed hydrophobic surfaces. Preservation of function was measured by representative enzyme activity, such as GAPDH, when stressed in-vitro. Stress induced aggregation of both endogenous proteins and exogenous, aggregation prone bait proteins were also. The bait proteins used include amyloid beta, the aggregation prone peptide associated with Alzheimer's disease. The macromolecules facilitating enhanced proteostasis in the longest lived animal species could have dramatic importance to various age-related protein diseases.
Rapamycin (Rapa) and dietary restriction (DR) are two manipulations consistently shown to increase the lifespan of mice. To investigate whether Rapa and DR affect similar pathways in mice, we compared the effects of feeding mice ad libitum (AL), Rapa, DR, or a combination of Rapa and DR (Rapa + DR) on the transcriptome and metabolome of the liver. The principle component analysis of the transcriptome shows that Rapa and DR are distinct groups. Of the 2724 genes that significantly change with either Rapa or DR compared to mice fed AL, 79% are unique to DR or Rapa; only 21% of the genes are common to DR and Rapa. A similar observation was made when genes were grouped into pathways by Ingenuity Pathway analysis; 76% of the pathways are uniquely changed by DR or Rapa. The metabolome shows an even greater difference between Rapa and DR; only 6% of the metabolites that change significantly from AL mice are common to Rapa and DR. Interestingly, the number of genes significantly changed in Rapa + DR mice compared to AL mice was twice as large as the number of genes significantly altered by either DR or Rapa. In summary, while both Rapa and DR increase lifespan, their global effect on liver is quite different and a combination of Rapa and DR results in alterations in a large number of genes that are not significantly changed by either manipulation alone.
A key component of my research is to develop new generations of techniques to understand how oxidative stress-mediated protein oxidation and perturbation of functional structure contribute to aging and age-related diseases. Over the past nine years, I have been actively involved in developing techniques related to measurement of protein oxidation and conformational changes that occur during aging and in disease conditions (Chaudhuri et al. 2001, 2006; Pierce et al. 2006, 2008; Perez et al. 2009; Salmon et al. 2009; Perez et al. 2010; Bhattacharya et al. 2011; Wei et al. 2012). One of the common and unique aspects of all these technologies is the use of fluorescent molecules as probes to detect changes in protein oxidation and conformation. As fluorescent probes in general give high quantum yield, it helps to identify and quantify the potential target proteins that are present in low level and have subtle changes in conformation in any patho-physiological condition. Development of these techniques is an important part of biological research considering the fact that the oxidative stress plays an important role in aging and various diseases including Alzheimer's, Parkinson's, ALS, cancer, heart disease, arthritis, etc. As a result, many investigators are interested in determining the underlying mechanism of the role of imbalanced protein thiol homeostasis; protein oxidation and alteration of conformation contribute to aging and diseases. Most importantly, researchers want to determine if the imbalanced protein homeostasis can be modulated by experimental manipulations, such as calorie restriction and pharmacological intervention. These new sets of techniques will give investigators the necessary tools to delve into the molecular mechanisms involved in aging and age-related diseases.
Caspase-2 has been shown to play a role in aging, neurodegeneration and cancer. The contributions of capase-2 have been attributed to its regulatory role in apoptotic and non-apoptotic processes including cell-cycle, DNA repair, lipid biosynthesis, and regulation of oxidant levels in cells. Recently, our lab demonstrated that caspase-2 modulates autophagy during oxidative stress. Here we report the novel finding that caspase-2 is an endogenous repressor of autophagy. Knockout (KO) or knockdown of caspase-2 resulted in upregulation of autophagy in variety of cell types and tissues. Reinsertion of caspase-2 in caspase-2-knockout mouse embryonic fibroblast (MEF's), suppressed autophagy suggesting its role as a negative regulator of autophagy. Loss of caspase-2-mediated autophagy involved down regulation of mTOR and upregulation of AMPK activation; knocking-down of AMPK1/2 inhibited autophagy. Interestingly, siRNA-mediated knockdown of ATG5 and ATG7 failed to inhibit autophagy induced by the loss of caspase-2 suggesting involvement of the non-canonical pathway of autophagy. Our results also indicate involvement of enhanced intracellular reactive oxygen species levels, down regulation of p38 and upregulation of ERK/MEK activation in autophagy-induction due to loss of caspase-2. In response to a variety of apoptotic stimuli that induce caspase-2-mediated apoptosis, caspase-2-KO cells demonstrated further upregulation of autophagy compared to WT MEFs. Enhanced autophagy improved the survival of caspase-2-deficient cells, which maintained high ATP levels. In conclusion, we document a novel role for caspase-2 as a negative regulator of autophagy, which may provide important insight into the role of caspase-2 in aging, neurodegeneration and cancer. The current findings are the first to provide evidence for regulation of caspase activity by autophagy and thus broaden the molecular basis for the observed polarization between autophagy and apoptosis.
C57BL6 mice were studied in youth (4–6 mo), middle-age (18 mo) and old-age (26–32 mo). Albuminuria increased, and, rise in serum cystatin C indicated that renal clearance function fell with aging; there was marked heterogeneity. Kidney hypertrophy and expansion of glomerular and tubulo-interstitial matrix were progressive. Increased mRNA correlated with increase in type III collagen in middle-aged and old mice, suggesting transcriptional regulation. In old-age, increase in mRNA correlated with type I collagen protein; however, in middle age, type I collagen was increased despite unchanged mRNA. Data from ChIP analysis of binding of transcription inhibitors ZEB1/ZEB2 to the type Iα2 promoter, and, polysome assay for mRNA translation did not explain type I collagen increase in middle-age. Thus, decreased degradation could lead to type I collagen increment in middle-age. Matrix changes coincided with TGFβ/SMAD3 activation. SMAD3 binding to collagen type Iα2 promoter was increased. Since microRNAs (miRs) control protein synthesis, we studied TGFβ-regulated miRs. The renal cortical content of miR-21 and miR-200c was increased but that of miR-192, miR-200a or miR-200b was unchanged suggesting selectivity. Increase in miR-21 and miR-200c was associated with reduced expression of their targets, Sprouty-1 and ZEB2, respectively; another miR-21 target, PTEN, was unchanged. Sprouty and ZEB2 inhibit growth factor signaling and expression of miR-21, respectively. Conclusion: Distinct transcriptional and post-transcriptional mechanisms contribute to kidney matrix protein increment in middle and old age. Kidney integrity is essential for maintenance of health span. Understanding mechanisms contributing to renal senescence could identify targets for intervention to improve health span.
The development of animal models targeting different components of the TOR signaling pathway has accelerated our understanding of the role of mTOR in animal development, metabolism, diseases, and aging. In addition, the discovery of mTOR inhibitors has further enhanced our ability to define the role of mTOR signaling in various patho-physiological conditions and to develop therapeutic strategies for the treatment of different diseases. Here, we report the development and characterization of a new mouse model, which overexpresses TSC1 (named Tsc1tg mice), part of the mTOR inhibitor complex TSC1/2 (tuberous sclerosis complex 1/2). Overexpression of TSC1 stabilized TSC2 and inhibited mTOR signaling in most tissues including the heart, liver, kidney, skeletal muscle and spleen. The levels of several important cell signaling pathways were found altered in Tsc1tg mice. The body weight of Tsc1tg mice exhibits slight gender difference, with significant increase in male mice at both young and advanced ages while only slight increase in female mice at both ages, when compared to age-matched wild type littermates. Body composition of Tsc1tg mice exhibits an age-associated change; with significantly higher fat mass but lower lean mass at advanced ages. At 4–8 months of age, Tsc1tg mice have normal cardiac function as measured by echocardiography. But, when challenged with isoproterenol, Tsc1tg mice developed less cardiac hypertrophy than age-matched wild type littermates. Importantly, Tsc1tg mice performed significantly better with treadmill test. Finally, the immune response of Tsc1tg mice exhibit subtle changes over wild type control mice. In conclusion, this model will be very useful to study the role of mTOR in such diseases that are associated with a deregulation of mTOR signaling, including cancer, cardiovascular diseases, and metabolic disorders. It will also be an interesting model to study the role of mTOR in mammalian aging, complementary to the rapamycin-feeding approach.
Sex differences in life and health span are ubiquitous in humans. Women in the developed world live longer than men even if heart disease, the number one cause of death in men, were completely eliminated. Analogously, female mice respond better to a number of senescence-retarding genetic or pharmacological interventions. Particularly notable in this respect, inhibition of TOR signaling via deletion of S6K1 improves both life- and health span in female mice but has no discernible effect in males. Here we show that aging male and female C57BL/6 mice respond to rapamycin in an age and sex-specific manner. There is a larger and more robust effect on longevity in females compared with males and measures of health span have multiple age and sex-specific effects. Age, sex and age · sex-specific differences in body composition, rotarod performance, gait, measures of activity, sleep and metabolism were observed in animals treated with enteric rapamycin (=e-rapa) relative to controls. There has been very little research on the basic biological mechanisms involved in sex differences in aging, in part because past research suggested that laboratory mice and rats do not show clear consistent trends in sex-specific longevity or health span. Our results suggest that sex differences in some measures of mouse health span may only become apparent late in life and that there are sex-specific responses to senescence-retarding treatments that merit further exploration.
Loss of mitochondrial function with age has been implicated as an influencing factor in the aging process. However, studies from model organisms ranging from yeast to mammals have shown that moderate disruption of the electron transport chain can enhance longevity. In the Surf1 knockout mouse, there is a 50–75% decline in cytochrome c oxidase (complex IV of the electron transport chain) activity and a 20% extended median lifespan. Previous studies of fibroblasts from long-lived rodents have shown a correlation between increased resistance to cellular stresses and longevity. Here we investigate whether fibroblasts from Surf1 knockout animals are more resistant to stress than wild type controls. Interestingly, these results are dependent upon the passage of the cells. Early passage (<2) cells show little difference between Surf1 KO and control cells. However, in later passaged fibroblasts, KO cells are significantly more resistant to paraquat, a superoxide radical generator, than control fibroblasts. Additionally, in vivo administration of diquat (50mg/kg), a superoxide radical generator, resulted in a significantly attenuated increase in markers of oxidative stress. Animals sacrificed six hours post diquat treatment resulted in a 24% increase in liver F2-isoprostanes in wild type animals. However, in the Surf1 KO livers the increase in F2-isoprotanes was 15%. This suggests that Surf1 KO mice are more resistant to exogenous oxidative stress than wild type control animals. (This work is supported by a grant from the Ellison Medical Foundation.)
Metformin, a drug commonly prescribed to treat type-2 diabetes, has been found to extend healthspan, delay cancer incidence and progression and to increase lifespan in laboratory animals. We show here that treatment with metformin (0.1% w/w in diet) starting at one year of age extends healthspan and lifespan in male mice, while a higher dose (1% w/w) was toxic. Treatment with metformin mimicked some of the benefits of calorie restriction, such as improved physical performance, increased insulin sensitivity, and reduced LDL and cholesterol levels without a decrease in caloric intake. At a molecular level, metformin increased AMP-activated protein kinase activity and increased antioxidant protection, resulting in reductions in both oxidative damage accumulation and chronic inflammation. Our results indicate that these actions may contribute to the beneficial effects of metformin administration on health span and lifespan. These findings are in agreement with current epidemiological data and raise the possibility of metformin-based interventions to promote healthy aging.
We tested the effects of two Class I histone deacetylase inhibitors (HDAcI) on the longevity of normal-lived (Ra) and long-lived (La) strains of Drosophila melanogaster. Only deleterious effects are noted when the first HDAcI tested (sodium butyrate, NaBu) is fed to the La strain at any developmental or adult stage. When fed to the Ra strain, this drug also has negative effects when administered over the entire larval and/or adult life span, or when administered over the adult health span only. Importantly, however, it significantly decreases mortality rates and increases longevity when administered only in the adult transition or senescent spans. A different HDAcI (suberoylanilide hydroxamic acid, SAHA) administered to the same strain also showed significant late-life extending effects, suggesting that this is not an isolated effect of one drug. These results suggest that the stage-specific gene regulatory mechanisms affected by NaBu or SAHA are those intimately involved in inducing gene expression patterns characteristic of a healthy senescence. Epigenetically active molecules, if given at the appropriate stage, allow the fly to shift from a senescent span characterized by a high age-specific mortality rate to one with a lower age-specific mortality rate. These studies may provide an experimental basis with which to shed light on the fraility syndrome affecting some aging organisms.
Feeding larvae of a normal-lived strain, but not a long-lived strain, with curcumin induces an extended adult health span with significantly increased median and maximum longevities. This phenotype shows no additive effect on longevity when combined with an adult dietary restriction (DR) diet, suggesting that curcumin and DR operate via the same or overlapping pathways for this trait. This treatment significantly slows the age-specific mortality rate so that it is comparable with that of genetically selected long lived animals. The larval treatment also enhances the adults’ geotactic activity in an additive manner with DR, suggesting that curcumin and DR may use different pathways for different traits. Feeding the drug to adults during only the health span also results in a significantly extended health span with increased median and maximum life span. This extended longevity phenotype is induced only during these stage-specific periods. Feeding the drug to adults over their whole life results in a weakly negative effect on median longevity with no increase in maximum life span. There are no negative effects on reproduction, although larval curcumin feeding increases development time; but it apparently accelerates the normal late-life neuromuscular degeneration seen in the legs. Gene expression data from curcumin-fed larvae shows that the TOR pathway is inhibited in the larvae and the young to midlife adults, although several other genes involved in longevity extension are also affected. These data support the hypothesis that curcumin acts as a stage-specific DR mimetic neutriceutical; and suggest that the search for DR mimetics may be enhanced by the use of stage-specific screening of candidate molecules.
Mitochondrial mutations in Caenorhabditis elegans can lead to either a shortening of lifespan or, unexpectedly, lifespan extension. Long-lived mitochondrial mutants (Mit mutants) live twice as long as wild-type animals, have delayed development, and reduced adult sizes. We have used a GC-MS-based metabolic footprinting approach to show that Mit mutants employ a common metabolism, distinct from wild-type animals and from short-lived mitochondrial mutants. The hallmark feature of the Mit metabolism is overproduction of pyruvate and various branched-chain ketoacids. We postulate that these compounds may act as mitokines, signaling molecules emanating from the mitochondria, to result in organismal lifespan extension. We have shown that at least four compounds found in the exometabolome of the Mit mutants can delay development when administered to wild type animals. At least one of these compounds, pyruvate, has also previously been reported to increase lifespan when fed to worms. Lifespan studies on the remaining compounds are underway. We have recently begun additional studies to determine whether the Mit mutants also produce a characteristic profile of ascarosides. Ascarosides are small signaling molecules based on the dideoxysugar ascarylose, and compose the pheromone which signals dauer development in C. elegans. This alternate larval state is resistant to stress and is considered non-aging, since upon leaving the dauer state animals live out their normal lifespan. Interestingly, we found a complete absence of one ascaroside in short-lived mitochondrial mutants. Experiments are underway to determine whether this molecule is capable of recovering lifespan in these mutants.
Emerging evidence suggests that both diet composition and genetic make-up have a key role in the beneficial effects of calorie restriction (CR). CR-mediated improvements in health and/or longevity may not be universal, even within species. Furthermore the responsiveness to CR may depend on subtleties of the treatment protocol, diet composition or the “intensity of CR”. In this study we determined the differential response to CR levels of DBA/2J mice. We are testing two main hypotheses: (i) that a milder CR intervention will provide beneficial effects on lifespan and healthspan in DBA/2 mice and (ii) regardless of the lack of effect on longevity, there are healthspan benefits even at the higher CR level. Male and female DBA/2J and C57BL/6 mice on one of three diets: ad libitum (AL), 20% CR, 40% CR starting at 6 months of age. Preliminary data indicates that in female mice there is no difference in median lifespan extension between 20% and 40% CR. In male mice it appears that 20% CR is more beneficial in extending median lifespan. Insulin levels are significantly lower in all DBA mice compared to their C57BL/6 counterparts. CR lowers insulin levels in all groups. We observed a stepwise decrease in insulin resistance with increasing CR, but only in males. In female mice, there was no difference in insulin levels between 20% or 40% CR groups. These results indicate that DBA/2J do respond to CR and supports the idea that there is an “ideal” CR dose for a particular strain.
The Health Span Study data are an unprecedented cross-sectional window into the biology of rodent aging, and our newly-developed Health Span Database makes it possible to organize, curate, share, and analyze this information in ways that would have otherwise not been practical. Here we present a range of measurements (e.g. body composition, grip strength, and gait analysis) that significantly change with chronological age of the animal. We go on to identify measurements that are positively and negatively correlated with each other, which can be used to construct a performance score for the corresponding organ systems with a minimum of redundant variables, irrelevant variables, and untested assumptions about the data. This in turn sets the stage for choosing variables from which the chronological age of an animal can be estimated. An animal whose actual age is greater than its estimated age can be interpreted as being healthier for its age an animal whose actual age is lower than its estimated age. We present several such sets of candidate variables. The software portion of the Healthspan database is freely available from the first author under the GNU Public License v2. Keywords: aging, healthspan, functional assessment, animal studies of aging, longevity, bioinformatics, variable selection, physiology of aging.
Sleep fragmentation is associated with aging in human populations. As part of a larger study designed to find robust, reproducible assays of health span, we used a sleep phenotyping method developed by Pack et al. (2006) to assess age-related changes in sleep patterns. Using EEG and video monitoring, Pack et al. (2006) developed and validated a simple an operational definition of sleep as a bout of /inactivity lasting ≥40s. Using their method, we developed a sleep fragmentation index by measuring the number of bouts of sleep per hour of sleep (=sleep fragmentation index) during the light and dark phases over a 24-hour period. We then used this technique to measure sleep fragmentation in 4, 20, 28, and 32-month-old male and female C57BL/6 mice to explore sex, age-related changes in sleep and sleep disruption. In combination with other assays, age-related changes in sleep patterns may offer a relatively simple, non-invasive tool for assessing healthspan in aging mice.
Reduction of target of rapamycin (TOR) signaling has been shown to extend lifespan in invertebrates as well as in adult mice. In other genetic models of longevity in invertebrates and mice, specific manipulations in the nervous system are sufficient to extend lifespan. To determine whether the reduction of mammalian TOR (mTOR) signaling in mature neurons of adult mice is sufficient to extend lifespan and improve health span we inducibly knocked out The mTOR complex 1 specific protein, Raptor, in adult mouse neurons after brain development was complete (2.5 months). Cre-mediated recombination of genomic DNA was detected in brain, but not in liver, and Raptor protein levels were significantly reduced after induction of Cre expression. To determine whether decreasing Raptor in neurons affected health span, we measured body mass composition, metabolism, motor coordination, muscle strength, and brain metabolite concentrations. While no significant differences in motor coordination, strength or body weight were observed among experimental groups, genetic reduction of Raptor in neurons of adult mice induced significant changes in body composition, with neuronal Raptor knock-out males becoming significantly leaner than non-transgenic controls. Adult neuronal Raptor, conditional knock-outs also showed increased levels of neuronal N-acetylaspartate, a marker of neuronal health and function. Future experiments will determine if decreased mTOR complex I signaling in adult mouse neurons is sufficient to extend lifespan and improve health span. Included in the evaluation of health span will be measures of neurological function as determined by electrophysiological and behavioral experiments.
While frailty has long been recognized by physicians in the clinical setting, only recently has effort been made to standardize and quantify definitions of frailty. Fried et al. 2001 and others have used multiple measures in the hopes of developing an easily used index to evaluate age-related risks of morbidity and mortality. Among the most commonly used measures are activity, walking speed, involuntary weight loss and strength (grip strength). In order to assess age-related frailty, as opposed to ill-health more generally, two conditions should be met: firstly, the traits measured should change with age; secondly, the traits should have predictive value for increased risk of morbidity and mortality. Here we assess a combination of several potential measures of frailty in mice, including motor function (e.g. walking speed), activity (e.g. spontaneous activity), strength (e.g. grip strength), body composition and caloric expenditure (e.g. resting metabolic rate) to determine whether age-related morbidity and mortality in C57BL/6 mice can be predicted using a multivariate analysis to produce a relatively non-invasive measure of health similar to the frailty index used with humans.
Adipose tissue has dynamic endocrine and secretory functions that play significant roles in the regulation of metabolism and health span. There is increasing evidence that different adipose depots may have different, and often contradictory, effects on this regulation. For example, visceral white adipose is largely thought to be detrimental to metabolism whereas subcutaneous white adipose has been shown to have several beneficial effects. Furthermore, aging is associated with a loss of subcutaneous adipose tissue, accumulation of fat in intra-abdominal visceral adipose tissue and ectopic accumulation of fat in other tissues such as muscle and liver. Adipocyte precursor cells (pre-adipocytes) play a significant role in the homeostatic regulation of adipose tissue. In this study, we tested whether differences in mitochondrial function among the pre-adipocyte pools of each adipose depot might be a determining factor in depot-specific functional differences. Pre-adipocytes from visceral and subcutaneous adipose depots were isolated and sub-cultured from young C57BL/6J mice. Using the Seahorse bioanalyzer, we found that mitochondrial respiration rate is high and reserve capacity is low in visceral depot-derived pre-adipocytes relative to those from subcutaneous depots. In addition, we found that pre-adipocytes from visceral depots are significantly more sensitive to oxidative stress
Every year, millions of people are diagnosed with traumatic brain injury (TBI). Of those, many have repetitive TBI (rTBI). Athletes and soldiers are at particular risk for rTBI, and those with rTBI commonly exhibit psychological symptoms including depression, anxiety, poor impulse control, and suicidal ideation. Some also exhibit signs of neuro-degeneration. Currently, treatments to stem the long-term consequences of TBI have been unsuccessful. Astrocytes play a central role in neuronal support and are excellent targets for therapy after TBI. We previously showed that the purinergic agonist 2-methylthioadenosinediphosphate (2MeSADP) effectively reduces astrocyte edema following TBI in mice. We hypothesize that 2MeSADP can similarly reduce the potential long-term effects of repetitive TBI. Here, we report our preliminary findings of a novel rTBI mouse model. Three-month-old mice were subjected to five closed-skull cortical impacts over 5 days. One year after rTBI, behavior was analyzed using a variety of tests. We found that control rTBI mice exhibited more anxiety-like phenotypes compared to 2MeSADP and sham-treated mice in the open field test and the three chambered social anxiety test. Fewer control TBI mice were able to complete the vertical pole test, suggesting an impairment of motor coordination. Continuing experiments will involve histological analysis of brain tissue, and further elucidation of the behavioral phenotype in a second cohort of mice. To our knowledge, this is the first report of a long-term analysis of rTBI in mice.
This work was supported by a UTHSCSA Center for Biomedical Neuroscience pilot grant.
Although co-infection with both hepatitis B and hepatitis C in HIV is a common phenomenon, it should be remembered that the livers with those living with HIV are under attack from various other areas as well. These include the HIV virus itself which may infect stellate cells leading to the generation of fibrosis, antiretroviral therapy, other clinical medications, recreational drug use, other infections as well as traditional risk factors, principally alcohol. Approximately four million individuals are estimated to be co-infected with HIV and hepatitis B. Fortunately, there are agents which are active against both HIV and hepatitis B infection. The majority of individuals will have suppressed hepatitis B replication through receiving HAART containing tenofovir with either lamivudine or entricitabine. Alternative therapeutic options for individuals who are co-infected include pegylated interferon, entecavir, telbivudine and adefovir. Entecavir and telbivudine also have potential anti-HIV activity and therefore should only be administered with highly active antiviral therapy against HIV. Adefovir may be prescribed as monotherapy for hepatitis B alone, although it is less potent than other but at the dose utilised there is no activity against HIV. It is essential that all individuals with hepatitis B are monitored for the development of hepatoma due to an increased frequency of development and progression of this tumour type in those co-infected with hepatitis B. HIV and hepatitis C co-infection is common due to shared routes of transmission and similar geographical distributions. Although hepatitis C does not impact on the prognosis of HIV disease, HIV is associated with more rapid progression of fibrosis and hence hepatic complications associated with hepatitis C infection. Globally there is an epidemic of acute hepatitis C in HIV-infected homosexual men, which is fuelling the numbers who are co-infected. Hepatitis C infection is associated with complications associated with HIV disease including a higher risk of cardiovascular disease, diabetes, renal dysfunction, and osteoporosis with bone fractures. The development of the protease inhibitors telaprevir and boceprevir for the treatment of hepatitis C has translated into improvements in the outcome of therapy in the hepatitis C mono-infected population; although only small studies are available, the results in the co-infected population appear to be similar. In addition, second generation protease inhibitors have been associated with early response rates, even higher than with telaprevir and boceprevir in the co-infected populations. The generation of interferon-sparing regimens is also of great promise, and clinical studies have recently commenced in the HIV-infected population. It is essential that individuals with HIV are tested for hepatitis B and for hepatitis C and vice-a-versa, and that these are repeated frequently, so that individuals who are or do become co-infected can benefit from the positive outcomes associated with these new treatments.
Recently approved HCV protease inhibitors (PIs) have drug interaction liabilities with key HIV antiretrovirals (ARVs), specifically, ritonavir-boosted PIs. Sofosbuvir (SOF, formerly GS-7977), a NS5B nucleotide inhibitor, is in Phase 3 clinical development for the treatment of chronic HCV infection. We conducted a study to evaluate potential interactions between SOF and common HIV ARVs of varying 3rd agent classes (NNRTI, PI/r and integrase inhibitor-based).
In a Phase 1, fixed sequence, 4-cohort study, healthy volunteers received a single dose of SOF 400 mg before and after 14 days of Atripla (ATR, EFV/FTC/TDF 600/200/300 mg QD; n = 17, fasted), or 10 days of rilpivirine (RPV, 25 mg QD, n = 17, fed), darunavir/ritonavir (DRV/r, 800/100 mg QD, n = 19, fed), or raltegravir (RAL, 400 mg BID, n = 19, fasted). Geometric means ratio% (GMR%, combination vs alone) of PK parameters AUCinf and Cmax for SOF and GS-331007 (circulating nucleoside metabolite of SOF), or AUCtau, Cmax, and Ctau for TFV, FTC, EFV, RPV, DRV/r, or RAL were evaluated against a predetermined 70–143% equivalence boundary. Safety assessments were conducted throughout the study.
A total of 16 and 17 subjects completed the ATR and RPV cohorts, respectively, and 18 subjects completed the DRV/r and RAL cohorts. The most frequent adverse events were dizziness, headache, diarrhea, nausea, and constipation, predominantly during ATR dosing. All but one event were mild and most were consistent with known EFV adverse events. Overall, SOF was well tolerated with very few treatment-emergent events during the administration of SOF with ARVs. Co-administration of HIV ARVs and SOF did not result in clinically significant changes in SOF and/or GS-331007 exposure. ATR slightly decreased SOF and GS-331007 Cmax (~20–23%), RPV, DRV/r, and RAL modestly increased SOF exposure by 21 to 45% with no effect on GS-331007. SOF slightly increased TFV Cmax (~25%), modestly decreased RAL AUCtau (~27%) and Cmax (~43%), but did not affect RAL Ctau, and had no other effect on PK parameters of FTC, TFV, EFV, RPV, or DRV/r.
No clinically significant DDIs were observed between SOF and EFV, RPV, DRV/r, RAL or the standard-of-care backbone of FTC/TDF. These data support potential use of SOF with a variety of ARV regimens in the HIV/HCV co-infected population.
Life expectancy (LE) is an important indicator of health used widely by government and healthcare agencies to monitor trends over time and to determine resource allocation, as well as by insurance companies and pension providers. LE of the HIV-positive population has increased dramatically since the introduction of combination antiretroviral therapy (cART); indeed, it is now believed that LE may be similar to that of the general population in some subgroups. There are, however, specific subgroups in which LE remains substantially impaired. The impact of HIV and of cART on mortality may be expressed in several ways. LE itself provides an estimate of the average additional number of years that an individual would be expected to live beyond a particular age. However, the detrimental impact of HIV may also be described in terms of the number of years of life lost or the gains in LE if HIV were to be eliminated as a cause of morbidity in the population. My presentation will start with a description of the different methods that researchers have used to describe the mortality outcomes of those with HIV, and the impact of cART on these. I will then consider how LE in the HIV-positive population has changed over time, and will describe the impact of demographic factors (e.g., gender, age, ethnic group) on LE. To investigate the circumstances under which LE may return to normal levels, I will also consider the potential impact of timely diagnosis and linkage into care, continued engagement with care, optimal initiation of cART, and maintenance of viral suppression on LE. Finally, I will discuss some of the limitations of the approaches used to estimate LE, with particular emphasis on the confounding effects of lifestyle and behavioural factors when making any comparison with LE in the general population.
Human immunodeficiency virus (HIV) infection has become a chronic long-term manageable condition. Subsequently with ageing of this population, comorbidities such as renal impairment are becoming more prevalent. Kidney diseases are emerging as significant causes of morbidity and mortality with older age, black ethnicity, hypertension, diabetes, low CD4+ cell count, HCV co-infection and high viral load remaining important risk factors for kidney disease in the HIV-infected population. HIV-associated nephropathy (HIVAN) is an uncommon but important cause of renal disease whose outcome can be improved by HAART. Having started treatment, HIV-infected individuals require lifelong antiretroviral medication but long-term data on the renal toxicity of HAART are limited. HIV affects the kidney directly or indirectly by drug-related effects that are directly nephrotoxic or lead to changes in renal function by inducing metabolic vaculopathy and renal damage. Historically, indinavir, mainly through renal crystal and stone formation, affected renal function but more recently cohort data have implied that atazanavir and lopinavir are also linked to renal dysfunction through a similar mechanism. Randomized clinical trials and post-marketing data supported the renal safety of tenofovir in relatively healthy HIV-positive individuals. However, overall tenofovir use is associated with 0.5–2.5% risk of acute kidney injury (AKI). The most frequent mechanism of tenofovir-induced kidney failure is thought to be tubular necrosis due to mitochondrial toxicity. Other mechanisms by which antiretroviral drugs cause renal impairment are complex and not completely understood. Effects on drug transporters leading to unfavourable drug interactions have been proposed. The increase in drug levels of tenofovir when given with ritonavir may be part of the reason why regimens of boosted PIs and tenofovir are associated with abnormalities in eGFR. Calculations estimating clearance of creatinine are widely accepted as the best measure of kidney function when using a 24-hour collection of urine. As urine collections are rarely performed, formulae to calculate eGFR using serum creatinine, demographic, and other factors have been developed. Interpretation of these formulae is complicated, and distinguishing normal from abnormal function in the individual patient is inherently difficult. The most used formulae are the Cockcroft–Gault formula, the Modification of Diet in Renal Disease” (MDRD) formula, or The Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula. All use serum creatinine as the basis of their calculation and hence any tubular contribution to plasma creatinine levels are included in their estimation of GFR. None of these equations have been systematically validated in HIV-infected patients. The evaluation of glomerular filtration using these formulae which include plasma creatinine to estimate GFR do not take into account that 10–20% of creatine is secreted through the tubules and the proportion rises with worsening glomerular function. The actual or real glomerular filtration rate (aGFR) is difficult to measure requiring isotopic or intravenous infusions such as iohexol or detailed imaging. Another reason for the eGFR to become abnormal when the aGFR remains unaffected is the inhibition of tubular enzymes. Blocking enzymes responsible for the tubular secretion of creatinine will lead to rises in serum creatinine and a fall in eGFR. This can lead to physicians believing that drugs have caused renal impairment but actually there is no change in aGFR. Dolutegravir by inhibiting OCT 2 and ritonavir and cobicistat by inhibiting MATE-1 renal transporters can all show this phenomenon with median falls in eGFR after starting these compounds of around 10-15 ml/min. These effects usually reach a steady state by two to four weeks. Physicians need to be aware of this and be able to differentiate between the expected changes in eGFR due to any enzyme inhibition and any changes due to true renal disease. Measuring tubular dysfunction is problematic and rely on increases in urine creatinine protein ratios, normoglycemic glycosuria, the presence of increased amounts of low-molecular-weight proteins such as β2-microglobulin or light chains, aminoaciduria, and inappropriate amounts of uric acid or phosphorus, coupled with a reduced phosphate re-absorption rate. It has been suggested that urine dipstick for glucose and protein and serum phosphate be used as monitoring tools to trigger further investigation of tubular dysfunction. With the ageing of the HIV population and the use of co-medications that could alter renal function, the issue of monitoring and interpreting and understanding laboratory data to safely prescribe antiretrovirals will come even more into focus.
The introduction of highly active antiretroviral therapy resulted in the dramatic reduction of HIV/AIDS-associated mortality and the redefinition of HIV infection as a chronic condition. Nowadays, life expectancy of new diagnosed HIV individuals is approaching that of the uninfected population. Therefore, HIV management is now faced by challenges associated with age-related comorbidities, in particular cardiovascular disease (CVD). It was suggested that HIV individuals are at higher risk of developing CVD compared with the general population, which can be explained by several factors: a) HIV individuals have a higher prevalence of smoking and other unhealthy lifestyle behaviours, b) some antiretroviral drugs have been linked to direct cardiovascular risk due to their effect on lipids or other not well-understood effects, and c) HIV might additionally contribute both directly or indirectly to a higher cardiovascular risk. Uncontrolled HIV viraemia is usually associated with chronic inflammation, chronic immune activation and immunosenescence, which some authors have defined as an “accelerated ageing” status. As the HIV-positive population continues to age, and HIV infection among older people is not an uncommon phenomenon, the risk of CVD will continue to increase. However, opportunities for preventive care and screening for and treatment of CVD are frequently missed. Case management and integrated care can help them confront the physical and psychosocial aspects of the disease and reduce marginalization and the dual stigma of both ageing and HIV.
Within the last decade, the HIV epidemic amongst women has been steadily growing in the Americas, representing in some areas almost half of HIV-infected individuals. Although biological factors determine a higher vulnerability to HIV infection in women other elements seem to play as major determinants in this increasing dynamic of the HIV women's epidemic. There are epidemiological factors that are shared among HIV-infected women in different countries: belonging to minority groups, being less educated, exposed to domestic violence, high rates of poverty and difficult access to health care. These same factors hinder diagnosis and HIV care, even in countries where access to treatment is universal. Opportunities for early HIV-diagnosis are also limited; only during pregnancies in countries with MTCT prevention programmes; for most women HIV serology is performed when a partner or a child has been diagnosed, or when they arrive severely ill to a hospital. Care of HIV-infected women needs to address gender-related health problems, particularly HPV co-infection. As patients survive longer with HAART, other health problems of ageing women need also to be surveyed and screened: osteoporosis, other gynecologic malignancies and metabolic disturbances among them. MTCT programmes have achieved enormous success in high-income countries. Although enormous efforts have been made to extend this success to low- and middle-income countries, there is still a wide lag in many countries, and children born HIV-positive are still counted in their hundreds in Latin-America. This field needs further advocacy to achieve universal MTCT prevention.
While an outright cure or a preventive vaccine for HIV/AIDS remains elusive, remarkable advances in HIV treatment have been achieved over the past two decades. Most significant among these advances is the development of highly active antiretroviral therapy (HAART). Two international clinical trials presented at the 1996 International AIDS Conference in Vancouver, served as the cornerstone for the emergence of triple therapy regimens based on the use of two nucleosides plus either a protease inhibitor or a non-nucleoside reverse transcriptase inhibitor (aka highly active antiretroviral therapy or HAART) as the new standard of care [1–3]. In each case the novel triple therapy regimen used among treatment-naïve individuals fully suppressed HIV replication and, therefore, rendered the number of viral copies present in a patient's blood undetectable. As a result, the CD4 cell counts recovered and the disease was placed into a long-term remission. In the province of British Columbia (BC), within three years of the implementation of HAART, the BC Centre for Excellence in HIV/AIDS (BC-CfE) documented an 85% reduction in HIV/AIDS mortality among patients engaged in treatment. Similar results have been observed in other resource-rich environments, and more recently in resource-limited settings, where HAART has become available.
More recently, evidence has accumulated that the viral load suppression achieved by HAART has a marked impact on decreasing HIV transmission. Already in August 2006, we proposed in a viewpoint article in
The International AIDS conference held in Vancouver in 1996, where modern HAART was born, was held under the motto “One world, one hope”. Unfortunately, the world is falling short of this goal. Albeit it is encouraging that eight million people living with HIV in low- and middle-income countries have started antiretroviral treatment since 2001 – people that would probably have died otherwise – millions of people living with HIV still lack access to treatment and care. Many thousands of women, men and children continue to die as a consequence of our failure as a global community. Therefore, we need to plan aiming to honour the commitments and keep the promises made several times in different high level meetings, conferences and in an incredible number of declarations, papers and documents. As of today, ARV guidelines in so-called rich countries are not matched by WHO guidelines and several local countries recommendations. Of note these differences are not limited to the “When to start” question, but also include the list of preferred drugs, the recommended monitoring tools and the management of co-morbidities. In the era of treatment as prevention, the benefits of HAART expansion are no longer limited to the individual and has become a strategic public health tool in the struggle against HIV/AIDS. So, the gap between rich and poor countries’ guidelines becomes even more unfair and painful. The world needs to move ahead and make the goal of “One world, one hope” a reality.
Infectious agents that are endemic to specific geographic regions provide the clinical syndrome of AIDS with unique clinical and epidemiologic characteristics often not well recognized in different publications. In Latin America and the Caribbean, the infectious agents that play significant roles in the HIV epidemic in certain geographic areas are
No abstract available.
Transgender women are disproportionately affected by HIV, with prevalence and incidence rates consistently found to exceed those of men who have sex with men (MSM). Of particular concern are prevalence rates of over 50% found among young transgender women of colour. Despite this, the medical and psychosocial needs of transgender persons remain largely unmet, and few culturally appropriate and targeted HIV-prevention interventions exist for this population. This presentation will review the structural factors, including stigma, discrimination, and criminalization, as well as individual level factors, that lead to elevated HIV risk. These factors also negatively impact on access to medical care and affect all points of the HIV treatment cascade. Innovative evidence-based strategies that have been successfully implemented among transgender women, resulting in greater uptake of HIV testing, engagement, and retention in care will be described.
Social determinants of health are factors which can be changed and controlled by policy and are largely responsible for the differences in the health outcomes in diverse populations and groups. This framework provides to explain health inequities and the impact of social injustice in health. In this presentation, the social determinants of health framework will be used to describe the impact of the socio-economic and political contexts on HIV transmission risks in different populations. Particular attention will be placed to the impact of these social determinants in framing HIV-related inequities and vulnerability. Examples from prisoners and other populations in Caribbean Region will be used to describe the impact of social determinants of health on selected HIV-related outcomes including access and retention in care. Strategies and interventions to reduce inequities and address vulnerabilities associated with HIV/AIDS will be discussed.
Animal models of extended longevity have a shared characteristic of enhanced cytotoxin resistance. The longest lived rodent, the naked mole rat (∼31 years), is extremely resistant to cancer and a wide array of xenobiotics
In persons with advanced HIV infection, the initiation of antiretroviral therapy occasionally provokes an inflammatory illness called immune response inflammatory syndrome (IRIS). In some instances, this is manifested as deterioration of an already recognized opportunistic illness (paradoxical IRIS) and in others, the inflammatory response allows recognition of an opportunistic illness that was sub-clinical before ART initiation (unmasking IRIS). In either setting, the occurrence of IRIS underscores the two edged nature of the immune response – at the same time both responsible for defence against microbial pathogens and also responsible for the signs and symptoms of infection. While IRIS is clearly an inflammatory disorder, the precise mechanisms responsible for its appearance as viral replication are attenuated and systemic immunity restored remain incompletely defined.
The 20th Conference on Retroviruses and Opportunistic Infections was held in Atlanta, Georgia, in March 2013. Important new data on the pathogenesis, prevention, and treatment of HIV were presented and are reviewed here. In the realm of pathogenesis and HIV eradication, a study of patients with acute HIV infection showed that those treated with combination antiretroviral therapy (cART) during Fiebig Stage I had undetectable proviral DNA and negative viral outgrowth assays when tested after a year on therapy. Immediate treatment of an infant with evidence for maternal transmission of HIV led to apparent cure of the infant. A randomized study confirmed previous observations that intensification of cART with raltegravir leads to a transient increase in 2-LTR circles, strongly suggesting ongoing viral replication in at least some patients. Studies in HIV prevention included the disappointing results of the VOICE trial, which failed to show evidence of protection against HIV infection in women given oral or topical pre-exposure prophylaxis; poor adherence appears to explain this result. By contrast, studies in the macaque model demonstrated high rates of protection against vaginal SHIV challenge using a rilpivirine-containing vaginal ring, and against rectal challenge using a long-acting (monthly) injectable formulation of a novel integrase inhibitor. A study of the investigational integrase inhibitor dolutegravir in cART-experienced, integrase inhibitor-naïve patients showed that dolutegravir was superior to raltegravir when combined with ritonavir-boosted darunavir. The Second Line study of lopinavir/ritonavir (LPV/r) plus two nucleoside reverse transcriptase inhibitors (NRTI) versus LPV/r plus raltegravir as second-line cART showed the two regimens to be similar in overall efficacy. Lastly, the OPTIONS study showed that NRTI do not add significantly to the activity of salvage cART regimens that include more than two active agents from other classes.
Substance use is frequently encountered in HIV-infected individuals. The introduction of effective cART has led to a significant decrease in AIDS-related mortality and morbidity. However, these benefits may be less pronounced in drug users who may have reduced access to, or be less adherent with, cART. The objective of this study was to evaluate the frequency of self-reported substance use and its relationship to cART adherence using a questionnaire administered to outpatients attending an HIV clinic in Buenos Aires.
A rapid clinical screening tool was adapted from a previously validated questionnaire [1] and translated into Spanish for use in CCASAnet (Caribbean, Central and South America network for HIV epidemiology). The questionnaire was administered to all patients presenting for laboratory testing at Hospital Juan A. Fernandez, the CCASAnet site in Buenos Aires, between 15 Feb 2013 and 15 Mar 2013. Univariate comparisons of demographics and substance use were made between those patients on cART with and without missed doses. Chi-square tests were used for categorical variables.
The questionnaire was administered to 228 HIV-infected patients in care during a one-month period: 159 (69.7%) were male and median age was 41.4 years (IQR: 33–48.8). Among 173 patients receiving cART, 33 (14.5%) reported missed ART doses in the previous seven days (median missed doses 2 [IQR: 2–11]). Use of marijuana, cocaine, alcohol, and cigarettes within the previous seven days was self-reported by 11.4%, 6.6%, 36.8%, and 39.9% of patients, respectively. Compared to non-use, cocaine and alcohol use were associated with missed ART doses (cocaine use in those who missed/did not miss doses: 18.2% vs. 2.8%, P:0.04; alcohol use 48.5% vs. 29.6% p:0.043). Marijuana use was not associated with missed doses.
This pilot study confirms previous reports showing the linkage between substance abuse and lack of adherence. The point of care questionnaire was easily implemented and widely accepted and may be useful for identifying patients with increased risk for suboptimal adherence. Specific and culturally appropriate interventions are required to address this important barrier. Further analyses will assess the association of substance use and virologic suppression. This study was partially supported by CCASAnet/IeDEA, Grant awarded number UO1AI069923.
Several studies conclude that patients with HIV on antiretroviral treatment have higher risk of cardiovascular disease. The objective of this study was to assess cardiovascular risk in patients from Clinica Condesa, an HIV clinic in Mexico.
This retrospective study included HIV-infected patients from Clinica Condesa with initial visit between 2009 and 2011 and had a complete lipid profile. The smoking status and systolic blood pressure were obtained from the patient's clinical file. General demographic information, total cholesterol, HDL cholesterol, and antiretroviral treatment were obtained from an integrated clinical and laboratorial database. The Framingham group assessment charts were used to evaluate cardiovascular risk, which was then categorized in high (>20), medium (10 to 20) and low (<10). Hypertriglyceridemia was defined as triglycerides >150 mg/dL, total hypercholesterolemia as >200 mg/dL, and low HDL cholesterol as <45 mg/dL. For statistical analysis, the 20.0 SPSS software was used.
Out of 399 patients included, 19 (5%) were women and 380 (95%) men. The total mean age was 37.7; 41.1 for women and 37.5 for men. Current smokers were 5 (1.25%) women and 200 (50%) men. The average systolic blood pressure was 110 SD±8, HDL cholesterol 37.7 SD±10.7, and total cholesterol 164 SD±91. Only 4 (1%) men had high risk, 1 (0.3%) woman and 20 (5%) men were in medium, while 18 women (5%) and 354 men (89%) showed low risk. Comparisons of low group vs. medium and high group and found no differences in gender (95 vs. 96%), smokers (57 vs. 68%), low HDL cholesterol (79 vs. 88%), or antiretroviral therapy (85 vs. 60% on EFV/FTC/TDF). Differences were found in age, low-risk group had on average 36.7 while high and medium group had 54, prevalence of hypertriglyceridemia 55% (ICI 50% ICS 60%) vs. 96%(ICI 82% ICS 99%) and total hypercholesterolemia 12% (ICI 9% ICS15%) vs. 64% (ICI 44% vs. 81%).
Although a low prevalence of high cardiovascular risk was found in Clinica Condesa, the prevalent metabolic alterations related to medium and high risk should be specially regarded. As the patients are predominantly young, growing older increases the risk factors for cardiovascular disease.
Motor neurons form a specialized synapse with skeletal muscle known as the neuromuscular junction (NMJ), and degeneration of the NMJ has been implicated in disease and aging. Histone deacetylases mediate NMJ-regulated gene transcription and are involved in neurogenic muscle atrophy, although their role in age-related muscle atrophy is not known. HDAC4 and HDAC5 knockout mice are protected against surgical denervation, and pharmacological inhibition of histone deacetylases is protective in multiple models of neuromuscular disease. In this study, we examined the effect of butyrate, a histone deacetylase inhibitor, on muscle atrophy during sciatic nerve crush and age-related muscle atrophy. We demonstrate that butyrate increases histone acetylation
This work is funded by the UTHSC at San Antonio Biology of Aging Training Grant (MEW T32AG021890-10).
Synucleinopathies, including Parkinson's disease (PD), multiple system atrophy and dementia with Lewy bodies, are age-related neurodegenerative disorders characterized by pathological a-synuclein inclusions. Synucleinopathies have common motor deficits and affect millions of patients worldwide. The A53T human a-synuclein mutation is linked to both sporadic and familial PD. Rapamycin, an mTOR inhibitor, reduces human a-synuclein accumulation and neurodegenerative phenotype
The accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates a signal transduction cascade called the unfolded protein response (UPR). The most immediate response is the attenuation of protein synthesis, initiated by autophosphorylation of PKR-like ER kinase (PERK). Recently, our group reported that calcineurin (CN) strengthened the UPR by binding to PERK and enhancing its autophosphorylation (Bollo et al. PLoSOne 5 (8): e11925). Here, we report that CN-A beta protects astrocytes from ER stress, likely by enhancing autophosphorylation of PERK. First, we found that levels of phosphorylated-PERK and CN-A beta were significantly increased in astrocytes within 1 hour of oxygen and glucose deprivation (OGD). Second, overexpression of CN-A beta significantly increased the viability of wild-type astrocytes during OGD (1 hr), but not that of PERK−/ − astrocytes. Third, co-immunoprecipitation showed that CN-A beta preferentially interacted with PERK in ER-stressed astrocytes. Fourth, experiments with recombinant proteins demonstrated that PERK autophosphorylation and oligomerization were increased in the presence of CN-A beta. Finally, rapamycin-induced dimerization of CFP-FRB-cytochrome5 (ER anchor) and YFP-FKBP-CN-A beta inside human astrocytes increased PERK phosphorylation. Taken together, we suggest a novel physiological function of the classic phosphatase CN-A beta is to bind PERK and enhance the early UPR.
This work is funded by NIH R01s AG29461-06 and AG007218-25.
The subventricular zone (SVZ) is the largest reservoir of neural stem cells (NSCs) in the adult brain. Neurogenesis is reduced during aging and this decline in NSC function is thought to contribute to reduced brain function. Here, we propose immune activation and cellular senescence as contributors to age-related declines in NSC function. We observed a sharp decrease in neurogenesis in mice between the ages of 6 and 12 months. Neuroblast number was also reduced with age. We observed the appearance of β-galactosidase-positive cells, a marker of cellular senescence concomitant with reductions in neurogenesis. Senescent cells secrete factors into the extracellular environment, a phenomenon known as the senescence associated secretory phenotype (SASP). The SASP has been associated with upregulated inflammation and immune activation due to proinflammatory factors. Microglia are the prominent immune cells in the brain. Microglial activation results in the release of proinflammatory cytokines. There was no change in the number or percent of microglia cells relative to other cells in the SVZ during aging. However, both morphological changes and increased CD68 expression were observed during aging indicative of microglia becoming more activated. Interestingly, a short-term diet of encapsulated rapamycin, an inhibitor of the mTOR pathway, resulted in reduced cellular senescence in the aged brain compared to the young and an overall increase in NSC proliferation. Our results suggest that during aging there is increased inflammation in the SVZ, which may be an important contributor to declines in neural stem cell function.
Increased mitochondrial reactive oxygen species (ROS) is believed to play a key role in cognitive decline associated with aging and Alzheimer's disease, but the mechanism remains unclear. Inflammasomes are multiprotein oligomers that recognize damage-associated molecular patterns (DAMPs) to activate caspase-1, thereby allowing for cleavage and subsequent release of proinflammatory cytokines IL-1β and IL-18. Among the inflammasomes, the NLRP3 inflammasome plays a key role in sensing oxidative stress such as increased mitochondrial ROS. We are interested in studying the role of mitochondrial ROS in cognitive decline associated with aging and Alzheimer's disease using mouse models exposed to paraquat, a mitochondrial ROS inducer. Our results indicate that exposure to paraquat results in exacerbated cognitive decline in both WT mice and APP/PS1 mice. To determine whether paraquat may regulate expression of the NLRP3 inflammasome to impair cognition, we measured the relative mRNA levels of ASC and Nlrp3, two major components of the NLRP3 inflammasome, in brains from paraquat-exposed animals by qPCR. We found that paraquat-exposed WT mice had higher levels of both ASC and Nlrp3 mRNA than control WT mice. Paraquat-exposed APP/PS1 mice also had increased levels of ASC and Nlrp3 mRNA compared with control APP/PS1 mice. Our results further showed that paraquat treatment increased ASC and Nlrp3 mRNA levels in primary astrocytes. Thus, our data suggest that increased expression of NLRP3 inflammasome may play an important role in mediating cognitive decline induced by mitochondrial ROS.
The mechanistic target of rapamycin (mTOR) is a major regulator of cell growth and metabolism. mTOR assembles with Raptor or Rictor to form mTOR complex 1 (mTORC1) and 2 (mTORC2), respectively. Reduced activity of the TOR pathway extends invertebrate lifespan, and, in mice, pharmacologic reduction of mTOR signaling during adulthood, as well as germline reduction of mTOR or S6K1 extending lifespan. In other models of extended longevity, selective reduction of function in the nervous system is sufficient to extend lifespan. The role of mTORC1 signaling from the nervous system in the control of lifespan and metabolism in mammals, however, has not yet been determined. Because neuronal-specific Raptor null mice die perinatally, to explore this question we generated mice expressing a tamoxifen-inducible Cre recombinase in neurons in combination with individual homozygous floxed alleles of genes in the mTORC1 pathway (mTORfl/fl, Raptorfl/fl, and S6K1fl/fl). Cre recombinase was partially active without tamoxifen stimulation, resulting in the genetic ablation of 20–40% of the target floxed alleles during development. A greater than 20% neuronal knockdown of any of the target genes in the mTORC1 pathway reduced fat content in both sexes. Knockdown of mTOR or Raptor in neurons also significantly reduced body size as well as lower fasting and resting blood glucose levels. These data agree with previously reported data for the germline knockout of S6K1, which also exhibited reduced fat mass, but unlike the germline S6K1−/ − animals, body weight in neuronal-specific S6K1 knockdown mice was unchanged. Only neuronal knockdown of mTOR increased oxygen consumption. Lower glucose and increased lactate in blood were observed in WT and neuronal S6K1 knockdown mice, but not in neuronal mTOR and Raptor knockdown mice challenged with treadmill exercise, indicating altered muscle metabolism as a consequence of neuronal reduction of mTOR or Raptor. Remarkably, S6K1 knockout mice ran farther on the treadmill than WT mice before they reached exhaustion. In contrast, neuronal knockdown of mTOR and Raptor resulted in decreased exercise capacity. Survival to weaning age was reduced but adult mortality was unchanged for Raptor or S6K1 neuronal knockdown mice. Conversely, neuronal mTOR knockdown mice were weaned at the expected Mendelian rates but were short lived, with less than 10% surviving beyond 300 days.
Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by the degeneration of nigral dopaminergic neurons leading to motor dysfunction. Several lines of evidence implicate mitochondrial dysfunction, oxidative stress, and protein aggregation in the pathology of PD. Recent evidence, including evidence from our laboratory, suggests that shifts in dopamine and/or aldehyde metabolism lead to aldehyde accumulation, which may contribute to parkinsonism. Aldehyde cytotoxicity may be a result of their long half-life and their ability to cross cell membranes both locally and distant. Aldehyde dehydrogenase (Aldh) is the primary pathway for the detoxification of aldehydes. Mice with mutations for two midbrain isoforms of Aldh, cytosolic Aldh1a1 and mitochondrial Aldh2, exhibit age-dependent neurochemical and behavioral deficits, and respond to levodopa treatment. Aldehyde scavengers such as
Alzheimer's disease (AD) is the most prevalent neurodegenerative disease. The majority of AD cases develop sporadically with unknown etiology. Over the past decade, a strong link between type 2 diabetes (T2D) and AD has been established; however, the mechanisms responsible for this relationship remain unknown. The mammalian target of rapamycin (mTOR) plays a key role in maintaining protein homeostasis, regulating energy metabolism, and diabetic insulin resistance. We hypothesized that mTOR may serve as a mechanistic link between AD and T2D. To explore this potential association, we supplemented the drinking water of 3xTg-AD mice, a transgenic mouse model of AD, with 20% sucrose. A second cohort of mice was given sucrose in conjunction with an mTOR inhibitor, rapamycin. All were compared to age and genotype-matched mice on control diet. We found that the sucrose treatment significantly increased fat mass, impaired glucose tolerance, and altered urine concentration all of which are consistent with physiological changes that occur in T2D. Additionally, sucrose exacerbated AD-like cognitive impairment and Aß and tau brain pathology. Impressively, rapamycin mitigated the sucrose-induced increase in pathology and cognitive impairment. Our results provide compelling
To investigate the effects of pigment epithelium-derived factor (PEDF) peptides on muscle regeneration, a rat myonecrosis model of a single injection of bupivacaine into the soleus muscle was employed. PEDF peptides were delivered by bolus injection of alginate-based sustained release regiment. The muscle fiber proliferation was assayed by the incorporation of BrdU in the proliferating nuclei. The soleus muscle specimens were also stained for satellite cell marker, Pax7, so as to investigate the muscle regeneration activity. Results suggested that the administration enhances the proliferative activities of muscle fibers and/or satellite cells, which in turn may promote the muscle to regenerate. There were higher percentages of muscle fibers containing centrally located nuclei in animals treated with the PEDF peptides than with vehicle. Moreover, on average, diameters of muscle fibers from animals treated with PEDF peptides were larger than those from animals in the blank or bolus control group. These indicated that PEDF peptides stimulate both the proliferation and maturation of muscle stem cells. Gene expression profiling and inhibitor assay on C2C12 cells revealed that the stimulation on muscle progenitor cells by PEDF is mediated by PI3KAKT and mitogen-activated protein kinases (MAPK) p38.
Nuclear receptor interaction protein (NRIP) is a calcium-dependent calmodulin (CaM) binding protein (Ca+2/CaM). To investigate the functional role of NRIP in skeletal muscle, we first generated conventional NRIP knock out mice. Here, we demonstrate that NRIP-deficient mice lose muscle strength from the assays of
As life expectancy rises in developed countries, sarcopenia and dynapenia pose significant threats for the aged, both physically and economically. Current research methods used to study age-related muscle wasting may include limitations, including model organisms that exhibit muscle growth characterized by a pre-defined growth plateau. The lack of model organisms for sarcopenia which exhibit continual growth potential with age has possibly limited advances in understanding this condition for two reasons: (1) the presence of a growth asymptote prevents the discovery of novel genes and/or regulatory pathways involved in the continuation of muscle fiber recruitment throughout adulthood; and (2) the inherent growth plateau may lead to sarcopenia itself, as sustained muscle growth throughout adulthood predicts the presence of myogenic precursor cells resistant to aging. Therefore, we propose a unique muscle growth model for the study of sarcopenia that takes advantage of natural continued muscle fiber recruitment. Evidence from a comparative approach utilizing the subfamily
Sarcopenia, the severe loss of skeletal muscle mass and function, is a serious health problem affecting aged individuals. Sarcopenic individuals are weak and frail, which predisposes them to disability and death. The mechanisms responsible for muscle wasting during aging are poorly understood. Muscle maintenance depends primarily on muscle stem cells (satellite cells) that exit quiescence during regeneration and replicate to generate daughter cells that either differentiates to maintain muscle or return to quiescence for self-renewal. Controversy surrounds whether the satellite cell pool is heterogeneous in regards to self-renewal, replication or differentiation. Furthermore, during aging it is unclear whether there is a decline in satellite cell number, function or whether cell fate is altered for a subpopulation of cells. To examine satellite cell heterogeneity
Growth of proliferative cells is dependent on regulation of protein synthesis and subsequent cell division. In post-mitotic tissues such as cardiac and skeletal muscle, growth is accomplished by increased protein synthesis and the donation of nuclear DNA by stem cells. Regardless of proliferative capacity, growth and DNA synthesis are associated with increased protein synthesis. Our central hypothesis is that somatic maintenance, which we define as increased protein synthesis in the absence of growth, is associated with slowed aging. To pursue this central hypothesis, we simultaneously measure both protein synthesis and DNA synthesis using deuterium oxide. Using this approach, we have reproducibly measured DNA synthesis in laboratory rodent cardiac tissue. When comparing several long-lived rodent models, we have shown that the rate of new DNA synthesis in the heart is altered and, in some cases, this is an age-dependent or sex-dependent finding. The observed changes in DNA synthesis in cardiac tissue, which is predominantly populated by terminally differentiated, post-mitotic myocytes, suggests that cardiac muscle has either greater proliferative potential than is perhaps appreciated, or that the new DNA synthesized can be accounted for by donation from cardiac progenitor cells. We will present a comparative analysis of DNA synthesis in cardiac tissue from several long-lived models and the current results of ongoing experiments to identify the origin of the new DNA.
Traditional aging models are short lived, a useful trait for tracking changes over the course of their lifespan. However, it is within the exceptional long-lived models that we can expect to find the mechanisms necessary to resist aging, as evolution has provided them. To this end, we are utilizing a range of marine bivalve mollusk species, with lifespans ranging from under a decade to over 500 years, in a comparative study to investigate the hypothesis that a long life requires superior proteome stability. These ages can be individually determined by counting growth rings in the shell. This experimental system provides a unique opportunity to study closely related organisms with vastly disparate longevities, including the longest lived animal.
We are testing the long-lived bivalve's ability to maintain protein structure and function under various stressors, and identifying the macromolecules responsible for this protection. As protein homeostasis has been implicated in the aging process and age-related diseases, these macromolecules could have dramatic medical value. Preservation of protein function is measured by representative enzyme activity, such as GAPDH, when stressed
Caspase-2 is a protein that is well known for its involvement in cellular apoptosis. However, its function in osteoclasts, cells that resorb bone, has not yet been described. In this work, the role of caspase-2 in osteoclast differentiation and apoptosis is evaluated. During the differentiation process from macrophage precursors, caspase-2 levels decrease so that protein expression is low in the mature osteoclast. Interestingly, when caspase-2 is knocked down in the RAW 264.7 macrophage cell line, osteoclast formation is increased as shown by higher levels of the osteoclast marker Cathepsin K. Furthermore, in cells derived from global
Genetic manipulations allow controlled interventions into aging progression by changing the genetic composition of model organisms. Such manipulations can include the addition of the ‘anti-aging’ genes, the inactivation of the ‘pro-aging’ genes, or the replacement of the ‘pro-aging’ allelic variants of certain genes with their ‘anti-aging’ counterparts. The genome engineering approaches that can be used to add or replace genes are based on site-specific nucleases and DNA recombinases. Our results indicate that the latter approaches, which currently utilize primarily Flp/
Induced pluripotent stem (iPS) cells have the potential to improve health with age. IPS cells may be useful for biological teeth engineering. The critical cells in tooth development are neural crest-derived dental mesenchyme. Here, we utilize pharmacological molecules to assess iPS cells derived from the common marmoset (
Approach: To assess neural differentiation, we utilized an embryoid body generation assay in combination with a cocktail treatment of active small molecules: DMH1 (BMP inhibitor), SB431542 (activin/nodal/TGF-beta inhibitor), BIO (GSK-3 beta inhibitor), Y-27632 (ROCK/Rho inhibitor) and all-
Results: With 6 days of treatment with bFGF withdrawal and GSK-3 beta inhibitor, we found significant increases in the mRNA levels of neural markers
Conclusions: Results indicate that FGF inhibits neural differentiation, while GSK-3 beta inhibition promotes neuralization as indicated by
The naked mole rat (
Over the last few decades, there was a 30% increase in the number of fathers over the age of 35 years. At this age, a father has the potential to transmit twice as many mutations to his child as a 20-year-old father. Paternal mutations continue to double approximately every 16 years, a phenomenon known as the paternal age effect. The driving force of the paternal age effect is mutagenesis. Base excision repair activity is essential for maintaining a low mutant frequency in the male germline. However, spermatogenic cells isolated from old mice display a 50% decrease in base excision repair activity, with a concomitant increase in mutant frequency, as compared to cells prepared from young mice. Reduced base excision repair activity appears to be mediated by reduced AP endonuclease 1 (APE1), a key base excision repair protein. Mice heterozygous for Ape1 show an increased germline mutant frequency as young adults, while APE1 transgenic mice are protected from age-dependent increases in spontaneous mutagenesis. Our objective is to delineate the mechanism/s mediating reduced APE1 abundance in spermatogenic cells with increasing age. In pachytene spermatocytes and round spermatids obtained from old mice, there is a significant 35 and 25% reduction, respectively, in APE1 abundance as compared to young mice. The age-related decrease in APE1 abundance is not accompanied by a reduction in Ape1 transcript abundance, thereby suggesting that post-transcriptional or post-translational regulation is involved. In somatic cells, p53 plays a role in regulating Ape1 abundance. There is a significant increase in p53 activation in spermatogenic cell populations obtained from old mice. APE1 expression is reduced by 40% in spermatogenic populations obtained from p53 null mice, relative to wild-type mice. Combined, these results indicate a strong relationship between APE1 abundance, germline mutagenesis, and p53 activation contributing to the paternal age effect.
Nearly two decades have passed since the initial discovery that disruption of the mitochondrial electron transport chain can unexpectedly increase lifespan. Much research has been done to find the mechanisms behind this seeming paradox, but while many factors have been shown to be required, none has proven sufficient. The mitochondrial unfolded protein response (UPRmt) under the control of ATFS-1 has received much attention for its role in Mit mutant longevity, but it is not the only retrograde response induced by mitochondrial dysfunction. We show that the ATFS-1 pathway itself divaricates into separable pathways, one being the UPRmt and the other being the activation of SKN-1. Moreover, we have found a novel retrograde pathway independent of ATFS-1. This MAPK signaling cascade acts in a parallel and compensatory manner with the ATFS-1 network, such that if one pathway is deactivated, the other is turned up to compensate.
Parkinson's disease is a neurodegenerative condition affecting every sixth person older than 70 years in the United States. Current therapeutic approaches provide succour from symptoms, but not a cure. Stem cells, with their propensity to regenerate the dopaminergic neurons (which are actually depleted in this condition) are offering new hope. Steady improvement in the ‘Off’/‘On’ unified Parkinson's disease rating scale (UPDRS), Hoehn & Yahr scores, Schwab and England scores, absence of tumorigenic growth on MRI as well as the absence of any AE's/SAE's following stereotactic transplantation of bone marrow derived mesenchymal stem cells (BM-MSC's) in the sub-lateral ventricular zone (in our earlier study) have infused tremendous optimism and galvanized us to undertake the present, open-labeled, non-randomized, single-center study to evaluate further on the positive outcomes on a larger scale. The current study plans to track the administered BM-MSCs, employing
Participation in physical activity can effectively prevent muscle loss and functional decline that occurs with age. Thus, understanding the mechanistic basis for preservation of skeletal muscle health with exercise may provide clues for developing therapeutic tools to combat age-related disabilities. We have demonstrated that mesenchymal stem cells (MSCs) accumulate in the muscle of young mice in response to a single bout of eccentric exercise and indirectly facilitate muscle repair and vessel growth via paracrine factor release. The observation that mechanical strain can potently influence growth factor release from freshly isolated, muscle-derived MSCs (mMSCs) provided the impetus to utilize physiological preconditioning as a method to improve mMSC viability and function in the aged muscle microenvironment. In this study, mMSCs isolated from young mouse muscle were preconditioned (10% multiaxial strain, 5 hr) and immediately transplanted into the gastrocnemius/soleus complex of 24-month-old mice. Separate groups of mice receiving saline or non-strained mMSCs served as controls. Although satellite cell number and fiber size were unchanged in all groups, vessel size and number of NMJs were greater in mice injected with preconditioned mMSCs versus controls one week post-injection (
The results of the HPTN 052 study, which showed antiretroviral treatment (ART) is highly effective in reducing HIV transmission, have been hailed as a “game-changer” in the fight against HIV, leading to calls for significant scaling up of treatment-as-prevention (TasP). But it is unclear how TasP could be financed, given flat-lining support for global HIV programs. We assess if TasP is indeed a game-changer against HIV, or if comparable benefits can be obtained at a lower cost by scaling up existing interventions such medical male circumcision (MMC). We also assess the impact of TasP in combination with MMC. Since MMC is currently being scaled up in many countries in sub-Saharan Africa, the effectiveness of TasP in conjunction with MMC is a highly policy-relevant question.
We formulate a new mathematical model to overcome challenges in predicting the effectiveness of untried mass interventions (lack of a historical epidemic trajectory) and in predicting the combined effectiveness of different prevention interventions. Our model uses simple behavioral assumptions to estimate new HIV infections instead of estimating parameters by fitting a curve to a disease history.
For South Africa, a combination of high ART coverage at CD4< 350/ml and circumcision coverage provides approximately the same HIV incidence reduction as TasP (defined as universal ART for all HIV-infected persons) at a cost ~ $5 billion less over 2009–2020. Circumcision outperforms high ART coverage at CD4<350/ml (and TasP) significantly in cost per infection averted—$1096 compared to $6790 per infection averted. Further, circumcision increases in cost-effectiveness over time and becomes cost saving after 2040.
The preventive benefits of ART are largely reaped with high ART coverage at CD4<350/ml. Expanding circumcision coverage first is most cost effective, and then scaling up ART under current guidelines is more cost-effective for preventing HIV infections than scaling up TasP.
The Total Control of Epidemic (TCE) Program of Humana People to People aims to reduce spread of HIV and its impact by systematically engaging individuals and communities to take control of their own risk factors, while increasing access to prevention, treatment and support services. Implemented in close partnership with respective Ministries of Health and National AIDS Councils across Sub-Saharan Africa and Asia, TCE has made major impact in HIV control.
The TCE model works through two primary strategies:
a)
b)
Since the first TCE pilot in Zimbabwe in 2000, 11 million people were covered in 11 countries. 28 million individual HIV counselling sessions were delivered. As a result, 2 million people were tested for HIV and received their results. Over 500,000 women attended PMTCT services. More than 600,000 community activists were trained and engaged in community mobilization activities. In Blantyre District of Malawi, 4 times increase of PMTCT utilization within 3 years and in Ehlanzeni District of South Africa, 6 times increase of PMTCT utilization within 4 years after TCE implementation were observed.
With an average cost of U$ 2/person/year, the TCE model represents a cost effective community-based intervention for HIV control and care with proven results, that should be replicated across the most HIV affected countries.
Traditional HIV Prevention Programs have not adequately addressed the issues of women in substance abuse recovery. In response, Greenhope Services for Women, Inc developed “Phenomenal Woman”, an HIV prevention program for women recently released from prison and/or mandated to receive residential substance abuse treatment.
The target population for “Phenomenal Woman” is African-American and Latina women demonstrating relatively high HIV risk behaviors receiving residential substance abuse treatment at Greenhope. The development of “Phenomenal Women” involved incorporating key components from “SISTA”, other HIV prevention programs, the “Helping Women Recover” curriculum, and newly created components. Formative research including: three focus groups, one key informant interview and two pilot cohorts were conducted to identify and pretest curriculum components and participant retention strategies.
“Phenomenal Woman” is comprised of 5 group sessions and a booster session held 45 days after program completion. Its primary aims include: enhancing women's sense of self, increasing HIV/STI knowledge, as well as attitudes, self-efficacy, and behaviors related to sexuality, safe and sober sex practices, and spirituality. Utilizing the formative feedback received (n=42 participants), the program creatively uses tangible objects (music, affirmation sheets, and meditation rocks) to reinforce the key messages of strength, resilience, and making healthy decisions regarding safe and sober sex behaviors. The program was implemented with 76 participants with retention rate of 100% across the five initial sessions and 72% at the booster session.
Greenhope's “Phenomenal Woman” is an example of effectively developing and delivering curricula through the adaptation of evidence-based curricula and the creation of new components to address a target population's needs for HIV prevention and substance abuse relapse prevention. Through high levels of administrative support, participant retention rates were maximized. Accordingly, “Phenomenal Woman” holds considerable promise for dissemination to other agencies with similar target populations.
Mortality among people living with HIV remains high despite a decline in AIDS deaths since effective therapy. We investigate recent trends and predictors of non-AIDS related causes of deaths in a national cohort of people accessing HIV care.
Analysis of national HIV surveillance data of adults (aged 15+) diagnosed with HIV between 1997–2010 in England & Wales. Death reports were linked to the Office for National Statistics and underlying causes of death were categorised into AIDS, non-AIDS-related cancers, non-AIDS-related infections, liver disease, cardiovascular disease and stroke (CVD), accident/suicide, and other causes. Cox regression models were fitted to identify significant (p<0.05) predictors of non-AIDS deaths and adjusted hazard ratios (HR).
Of 70,906 persons diagnosed from 1997 to 2010, 3,815 (5.4%) died during 318,620 person-years of follow up. 1,865 (51%) died due to non-AIDS-related causes; increasing as a proportion from 48% of all deaths in 2000 to 59% in 2010. The most frequently reported causes of death werenon-AIDS-related infections (448,24%) (286 pneumonia, 115 septicaemia, 47 other respiratory infections), CVD (309,17%), non-AIDS-related cancers (283,15%), accident or suicide (267,14%), liver diseases (144,8%), and other causes (414, 22%). predictors of non-AIDS related death were injecting drug use (HR 4.2[3.5–5.0], late diagnosis (CD4<350cells/ul) (HR 1.2 [1.1–1.3]),<3 years exposure to HAART (HR 3.1[2.8–3.5]) and previous AIDS diagnosis (HR 2.1[1.9–2.4]). In 2010, non-AIDS related mortality rate was 3.6 per 1,000 HIV diagnosed persons age 15–59, compared to 1.6 per 1,000 in the general population age 15–59 of England and Wales.
Non-AIDS deaths account for half of deaths among HIV-infected adults and rates are double those of the general population. Late diagnosis and a history of AIDS were strong predictors of non-AIDS related deaththis requires further investigation. Prompt HIV care could reduce all-cause mortality of people living with HIV, not only AIDS-related deaths.
HIV epidemic in Russia remains concentrated mostly among injection drug users (IDUs). Little is known about the extent to which sexual partnerships bridge between IDUs and the general population and create the potential for epidemic generalization.
IDUs in two Russian cities, Novosibirsk and Ivanovo (N=593) were recruited via respondent-driven sampling. A modified one-step snowball strategy was used to recruit IDU's sex-partners who do not themselves use drugs (PIDU, N=82). Sexual behaviors of all participants were assessed using an interviewer-administered questionnaire. All participants provided blood specimens for HIV and HCV testing.
In Ivanovo HIV prevalence among IDUs was 34% and among PIDUs - 6.8% (p≤0.001). In Novosibirsk the corresponding prevalences were lower- 3.8% and 8.7% (n.s.). In both cities large proportions of IDUs reported sexual partnerships with non-IDUs - 49.7% in Ivanovo vs. 62.7% in Novosibirsk (p≤0.001) and fewer than 1 in 4 IDUs reported constant condom use in such partnerships. PIDUs in Novosibirsk also tended to have other sexual relationships with non-IDU (52.2%) while in Ivanovo this behavior was less common (6.8%; p≤0.001). Based on the proportion of IDUs and their sex-partners reporting sexual connections with non-IDUs and mean number of such partners, we estimate that for every 100 IDUs in Ivanovo there are about 70 linked PIDUs and about 14 PIDUs downstream sex-partners from general population. For Novosibirsk the corresponding estimates are much greater 118 and 95 respectively.
This pilot study results shows that two IDU populations with significantly different HIV prevalence both form sexual partnerships with non-IDUs and practice unsafe sexual behavior within such type of partnerships. However the proportion of PIDUs who form partnerships with other non-IDUs that therefore could lead to epidemic generalization is very different between two cities and this difference needs to be considered when estimating the spread of HIV into the general population.
Initiatives aimed at behaviour change of key populations such as the female sex workers (FSWs) are pivotal in reducing the transmission of HIV. A 5-year implementation research to establish the predictor factors of behaviour change among FSWs in Kenya was initiated by the African Medical Research Foundation (AMREF) with Sida and DfID support, whose follow up results are presented in this paper.
This cross-sectional survey interviewed 156 female sex workers (FSWs) identified through snowball sampling. The measurement of behaviour change was based on: the consistent use of condoms with both regular and non regular clients, reduced number of clients, routine checks for STIs, and involvement in alternative income generating activities. The adjusted odds ratios at 95% confidence interval computed during binary logistic regression analysis was used to determine the behaviour change predictor factors.
Most FSWs (84%) had participated in AMREF's integrated intervention programme for at least one year, with 4.4 years as the average duration. The results indicated that 59.1% had gone through behaviour change during the project's life cycle. The adjusted odds ratio showed that the FSWs with secondary education or more were 2.23 times likely to change behaviour, protestants were 4.61 times, being in sex work for>4 years were 2.36 times, FSWs with good HIV prevention knowledge were 4.37 times, and those engaged in alternative income generating activities were 2.30 times more likely to change their behaviour compared to respective counterparts.
Behaviour change among FSWs was possible and is associated with the level of education, religious affiliation, number of years in sex work and one's level of HIV prevention knowledge. A re-orientation on the peer education programme to focus on HIV preventive measures beyond use of condoms is emphasized.
Condom use as a means to prevent HIV infection has significantly increased over the past decade among all age groups in South Africa. However, little is known about what motivates the behaviour in South Africa. This study investigated the prevalence and demographic, psychosocial, and behavioural determinants of condom use among people aged 15 years and older in South Africa who were sexually active over the 12 months prior to the survey.
Data from the 2008 national HIV population-based survey was used. This was a cross-sectional survey which was conducted using a multi-stage stratified sampling approach. Univariate analysis and multiple logistic regression were used to identify factors associated with condom use at last sexual intercourse. A total of 5072 respondents, 46.0% males and 54.0% females, who indicated having had sex in the last 12 months were involved in the study.
Overall, there was no gender difference found in condom use: males (64.6%) vs females (60.4%). The multiple logistic regression analyses indicated that youth aged 15–24 years and students’ learners were more likely to use condoms at last sex. (AOR=2.2528, AOR=2.4358, p< 0.05). Whites, Coloureds and Indians were less likely than Africans to use condoms (AOR=0.2125, AOR=0.3000, AOR=0.4511, p< 0.05). Likewise, married people and those whose current relationships exceeded a year were less likely to use condoms (AOR=0.2782, AOR=0.3239, p=0.00). Finally, having only one regular sexual partner significantly reduces the odds of condom use (AOR=0.2855, p=0.021).
It is important to understand more about the nuances of condom use so that programmes can target those at greatest risk of infection such as African adults and people in stable relationships, especially those that might also be involved in other risky behaviours such as multiple concurrent sexual relationships.
HIV-discordant couples represent possible targets for HIV transmission prevention strategies, such as pre-exposure prophylaxis and “treatment as prevention.” However, the population-level impact of such strategies on HIV incidence will depend on the relative contribution of discordant couples to HIV spread in a given setting.
We used a deterministic, compartmental model to estimate the proportion of incident HIV cases arising within stable discordant partnerships in Lilongwe, Malawi. The model, which was based on detailed behavioral and viral load data from a sexually transmitted infections clinic in Lilongwe, included sexual contact within and outside of steady partnerships, as well as changes in HIV transmissibility across infection stages. We used a Bayesian melding approach to fit the model to empirical HIV prevalence estimates, to account for input uncertainty, and to calculate 95% credible intervals around model outputs. The best-fitting model included a “lower-risk” and a “higher-risk” group, with average partnership lengths of 2.5 years and 1.3 months, respectively. We considered partnerships in the lower-risk group to represent stable partnerships. Among those stable partnerships, we estimated the proportions that are currently HIV-discordant, HIV-concordant-negative, and HIV-concordant-positive. We also calculated the proportion of all incident cases arising within the stable, HIV-discordant couples.
Based on the best-fitting (i.e., the mode) model simulation, 48.5% (95% credible interval 48.4%–75.2%) of the adult population of Lilongwe, Malawi belongs to a stable partnership. Among the stable partnerships, an estimated 5.0% (3.2%–11.7%) are HIV-discordant, 94.4% (85.5%–96.3%) are concordant-negative, and 0.6% (0.5%–3.0%) are concordant-positive. Only 4.5% (3.3%–39.5%) of all incident infections are estimated to occur within stable, HIV-discordant partnerships annually.
The small proportion of transmission events occurring within stable, HIV-discordant couples in our model suggests that interventions targeted specifically to this group should be complemented by strategies reaching other targets.
HIV prevalence among pregnant women in Southern Africa is extremely high. Some studies suggest that sexual risk of HIV acquisition during pregnancy is doubled and that infected women may be more likely to transmit HIV to male partners. Also, the association between high maternal viral load implies that HIV infections acquired during pregnancy carry higher risk of mother-to-child transmission (MTCT). We analyze the potential benefits from extending HIV prevention to pregnant women in addition to non-pregnant women using wide-scale microbicide interventions as an example.
A transmission dynamic model is designed to assess the impact of microbicide usage in high HIV prevalence settings and estimate prevented cumulative fractions of new HIV infections (CPF), fractions of infections acquired during pregnancy, and fractions of MTCT over 10 years.
Consistent use of 70% efficacious microbicide by 60% of the non-pregnant women may prevent 36–39% and 11% new infections in women and men over 10 years, respectively, assuming no increase in sexual HIV risk to either partner during pregnancy (RRsex/preg=1). It may also prevent 5–10% MTCT depending on the increase in MTCT risk when HIV is acquired during pregnancy compared to before pregnancy (RRMTCT/preg). The usage of microbicides by pregnant women may increase the absolute CPF by 5% (RRsex/preg=1) to 10% (RRsex/preg=2) and reduce the number of HIV infections during pregnancy by 40% to 70% in different scenarios. It may add between 5% (RRsex/preg=1, RRMTCT/preg=1) and 22% (RRsex/preg=2, RRMTCT/preg=4) reduction in MTCT.
Providing HIV prevention tools to pregnant women in the context of wide-scale interventions would be desirable as it would help to increase the effectiveness of the intervention, especially if sexual risk during pregnancy is elevated. It would significantly reduce the number of HIV infections acquired during pregnancy and help prevent MTCTs but should not be a substitute for PMTCT.
Botswana's HIV prevalence is 17.6% among the general population and the incidence rate is 2.9%. In 2009, Botswana's Tertiary Education Council, recognizing that HIV interventions for College/University (tertiary) students were few/fragmented, conducted a study to understand student behaviours, needs and gaps in services. Born in the 1990s, this generation of youth is the first of its kind-a generation who has grown up with HIV-infected, heavily affected and message-fatigued.
Between 2009–2010, TEC conducted a study of 10% of tertiary students using self-administered surveys (N=4312). Classes were randomly selected from 32 institutions and surveys were augmented by 28 post-survey FGDs. Participation was voluntary, anonymous and counselling was offered. Survey questions were qualitative and quantitative.
57.0% of participants were female and 63.0% were aged 20–24. HIV knowledge was high (over 90% responded correctly to 9/11 knowledge questions) but satisfaction with current HIV interventions was low (44.9%) and 38.5% said condoms are never available on campus. 82.5% were sexually active and 45.0% had already engaged in unprotected sex. 53.9% knew their HIV status and 49.4% knew their partner′s status. 33.7% reported that they were engaging in MCP. Key findings from the FGDs include: a) campuses are sexualized spaces b) students are involved in transactional relationships c) students do not perceive themselves to be at risk for HIV and d) campus interventions are few and irrelevant.
Irrespective of increased knowledge and impact from AIDS deaths within their families while they were young children, the sexual practices that gave rise to the current HIV epidemic in Botswana persist among tertiary youth. The study raises serious reservations about the assumption that youth are making behaviour changes. It also exposes gaps in service provision and questions the strength and relevance of current interventions to youth in a country with a staggering incidence rate.
It is increasingly recognized that HIV and hepatitis C (HCV) risk, such as syringe sharing, occurs in the context of relationships between (at least) two people.
In San Francisco from 2006 to 2011, we enrolled 53 injecting partnerships that were HCV-discordant at baseline based on RNA testing. Partnerships were defined as having injected together 5 or more times in the prior month. Each partnership completed monthly interviews for one year. Generalized linear models were used to examine correlates of two outcomes (1) receptive syringe sharing (RSS) and (2) receptive cooker-sharing (RCS) reported by the HCV-negative partner.
Participants had a median age of 26 (IQR:23–28) and median years injecting of 8 (IQR:3–10). Within partnerships, 62% were female-male, 36% were male-male, 2% were female-female; the median number of times per day partners injected together was 2 (IQR:1–3), 25% were sexual and injecting partnerships and 48% lived together. RSS and RCS was more likely to occur in partnerships that lived together (OR=2.27, 95% CI: 1.42–3.63; OR=2.79, 95% CI: 1.70–4.58) and in sex and injection partnerships (OR=2.76, 95% CI: 1.75–4.36; OR=2.50, 95% CI: 1.55–4.02); partnerships who lived together and had sex were at even greater odds of risk (OR=4.15, 95% CI: 2.57–6.73; OR=3.52, 95% CI: 2.20–5.65). Among partnerships who lived together, living within unstable housing (e.g., on the street, park, or shelter) increased the risk for RSS (OR=2.22, 95% CI: 1.28–2.57) and RCS (OR=1.66, 95% CI: 1.70–2.36) compared to partnerships who lived in an apartment, hotel, or group home. Pooling money to purchase drugs also was positively associated with both outcomes (OR=1.82, 95% CI: 1.38–3.58; OR=2.11, 95% CI 1.41–3.16). Duration of the relationship, gender composition of the partnership, HIV-status, or HCV-status was not associated with either of the outcomes.
Few studies have examined HIV-risk behaviors within drug-using partnerships. Our results suggest that relationship factors may influence risk within injecting partnerships and are an important consideration in the design of relationship-based interventions.
After more than two-decades of war and displacement in Northern Uganda, over one million Internally Displaced Peoples (IDPs) are returning to their home villages. However, thousands of IDPs are only halfway home, living in transit camps near their villages. This population in transition provided a unique opportunity to assess the influence of conflict on HIV infection among young people surviving displacement and abduction in post-conflict Northern Uganda.
In 2010, a cross-sectional demographic and behavioral survey was conducted with 384 young people aged 15–29, residing in transit camps in one of two sub-counties in Gulu District. Biological specimens for HIV were collected for Rapid testing in the field and Confirmatory testing in a lab. Multivariable logistic regression identified factors significantly associated with HIV infection.
Of the 384 participants sampled, 192 (50%) were female and 107 (27.9%) were former child soldiers. Overall HIV prevalence was alarmingly high at 12.8%. HIV prevalence among females was 15.6%, 9.9% among males, and 12.1% among former child soldiers. In multivariable logistic regression, HIV positivity was significantly associated withnon-consensual 1st sex (Adjusted Odds Ratio [AOR]: 9.9, 95% Confidence Interval [CI]: 1.7, 18.06); sub-county A (AOR: 2.9, 95%: CI1.3, 6.7); STI symptoms past 12 months (AOR: 2.4, 95%: CI1.4, 6.2); practicing dry sex (AOR: 2.3, 95%: CI1.0, 5.1); age (AOR: 1.2, 95%: CI1.1, 1.3); thinking you could protect yourself from HIV (AOR: .29, 95%: CI.12, .69); and number of HIV tests in lifetime (AOR: .86, 95%: CI.81, .91).
In post-conflict Northern Uganda it has been observed that NGOs focused on relief who previously supported HIV/AIDS prevention and treatment have shuttered operations, leaving gaps in care. This, coupled with other strains of post-conflict resettlement, has produced a generation of young people ‘lost in transition’, leaving them at heightened risk of contracting HIV/AIDS. Applicable post-conflict HIV/AIDS programming is urgently required.
Hormonal contraceptives (HC) have been associated with risk of HIV-1 seroconversion in women and their partners. We examined the association between injectable HC (DMPA), combined oral contraceptives (COC), and no hormonal contraceptive use (NH) with genital tract mucosal immunity biomarkers in women with STI/RTI who did or did not become HIV infected.
Biomarkers were quantified in cervical swabs from 832 HIV-uninfected reproductive-aged Ugandan and Zimbabwean women with documented HC use, HIV/STI behavioral risk factors, STI/RTI signs and symptoms, and were correlated with HIV-1 seroconversion at next visit.
Women who had both signs and symptoms of STI/RTI had higher beta-defensin (BD)2 and lower SLPI levels in cervical secretions compared to STI-free women. Both of these changes were associated with HIV seroconversion (occurring among 24% of women at the next visit). Among women with BV, odds of top quartile BD2 levels were higher in the OC and DMPA users than in the NH group. When compared to NH, DMPA use was associated with lower levels of the anti-inflammatory regulator IL-1RA overall and, in women with intermediary vaginal microflora, HSV-2 and NG, with significantly lower odds of top quartile IL-1RA concentrations.
OC and DMPA differentially modulate levels of cervical protective immune mediators, altering responses to STI/RTIs, and providing insight into possible biological mechanisms for higher risk of HIV acquisition.
We recently reported that women in HIV-1 serodiscordant partnerships using injectable contraceptives had a 2-fold increase in HIV-1 acquisition risk (aHR=2.05, p=0.04; Heffron et al.,
We used Cox proportional hazards regression, adjusting for age, male partner's plasma viral load, and time-dependent unprotected sex and pregnancy, to compare HIV-1 incidence in women using injectable contraception to women reporting no hormonal method. Sensitivity analyses included1) additional or alternative covariates to adjust for sexual behavior, 2) restricting to follow-up periods when unprotected sex was reported, 3) censoring visits after a woman switched her contraceptive method (to minimize the effect of changing methods on risk), and 4) restricting to consistent injectable users outside of South Africa (with access to only injectable depot medroxyprogesterone acetate [DMPA]).
In all models, the approximately 2-fold increase in HIV-1 acquisition risk persisted. Neither additional adjustment for coital frequency (aHR=2.06, p=0.04) nor adjustment for the male partner's report of unprotected sex (where accuracy is less likely influenced by the woman's contraceptive choice, aHR=2.03, p=0.07) influenced the results. When limited to periods when unprotected sex was reported or including only visits prior to the first contraceptive switch, the aHR were 2.29 (p=0.17) and 2.62 (p=0.07), respectively. Finally, when only consistent, presumed DMPA users were retained (i.e. South Africans excluded), injectable contraception was associated with a nearly 4-fold increased HIV-1 risk (aHR=3.93, p=0.01).
Injectable contraception continued to be associated with increased HIV-1 risk despite numerous methods to control for potential imprecision in contraceptive exposure measurement and/or key behavioral confounders. Some analyses had p-values >0.05 due to reduced statistical power but the magnitude of association continued to be as strong as that seen in our primary analytic model.
There are data suggesting that use of hormonal contraception (HC) might affect the risk of HIV acquisition in HIV-negative women.
We conducted a systematic review of the epidemiological literature on the association between HC and HIV acquisition. We systematically searched for relevant articles in any language published or in press by December 15, 2011, evaluated study quality, assessed the association of study findings with various methodological features, and synthesized the evidence.
We identified twenty relevant studies, eight of which met minimum quality criteria. Of these, only one reported a statistically significant association between use of oral contraceptive pills (OCPs) and HIV acquisition. No studies reported statistically significant associations between use of norethisterone enantate (Net-En) and HIV acquisition, but data were limited. Estimates for depot medroxyprogesterone acetate (DMPA) or non-specified contraceptive injectables and HIV acquisition were heterogeneous, and we consider factors including analysis of condom-use information, length of inter-survey interval, and analysis of serodiscordant couples as possible reasons for heterogeneity.
Overall, current evidence does not suggest an association between OCP use and HIV acquisition. No currently available evidence suggests an association between Net-En and HIV acquisition, though data are limited. Evidence assessing DMPA or non-specified injectable contraception and risk of HIV acquisition is inconsistent; it does not establish a clear causal association with HIV acquisition, nor does it definitively rule out the possibility of an effect. Concerns remain about the potential for residual confounding, even within otherwise high-quality studies. Many women at risk of HIV have a critical need for safe and effective means of pregnancy and infection prevention, and it is imperative that clients and providers are informed that HC does not protect against HIV or other STIs.
Prevention of unintended pregnancy remains a key concern for women living with HIV, both as a core strategy to prevent mother-to-child transmission of HIV and to decrease maternal and neonatal morbidity and mortality through lower birth rates, improved birth spacing, and lower rates of unsafe abortion. However, there are theoretical concerns about the effect of various contraceptive methods on HIV disease progression.
We conducted a systematic review to determine whether HIV-infected women who use hormonal contraception are at increased risk of HIV disease progression compared with those who do not use hormonal contraception. We searched PUBMED and EMBASE for articles published in peer-reviewed journals through December 15, 2011 for evidence relevant to all hormonal contraceptive methods and HIV disease progression.
Twelve reports of eleven studies met inclusion criteria. One randomized controlled trial (RCT) found increased risk of a composite outcome of declining CD4 count or death among hormonal contraceptive users when compared with copper IUD users. Ten observational studies reported no increased risk of HIV disease progression, as measured by mortality, time to CD4 below 200, time to initiation of antiretroviral therapy, increased HIV-RNA viral load, or decreased CD4 count with hormonal contraceptive use compared with non-use.
One RCT found that hormonal contraceptive use was associated with increased risk of HIV disease progression when compared with IUD use, but this study had important methodological shortcomings. Cohort studies consistently found no association between hormonal contraceptive use and HIV disease progression compared with non-use of hormonal contraceptives. Thus, the preponderance of evidence indicates that HIV-positive women can use hormonal contraceptive methods without concerns related to HIV disease progression. Prevention of unintended pregnancy through safe and effective contraceptive use among women with HIV remains a public health priority to safeguard maternal health and prevent mother-to-child transmission of HIV.
There is a general consensus that people with severe mental illness(SMI) are more likely to have a history of childhood sexual abuse and are at increased risk for HIV. For this study, we hypothesized that our homeless, mentally ill participants reporting CSA would be more likely to report current engagement in HIV-risky behaviors, history of STI's, and associated psychopathology than their non-CSA counterparts. Furthermore, we hypothesized that those who had experienced CSA in early to middle childhood (EMCSA) would be more likely to report these challenges than those who had experienced CSA in adolescence (ACSA).
As part of a NIH-funded RCT(1R01MH084696-01A2 PI Brady) primary and secondary prevention trial for adults with SMI at-risk for HIV transmission, ninety participants were administered an assessment battery which included the Structured Clinical Interview for DSM-IV, Demographic Inventory, and Timeline Followback. Chi-square analyses were used to test our hypotheses and analyze the relation between our variables of interest.
There were no significant differences in current HIV-risky behaviors between CSA/non-CSA participants nor for EMCSA/ACSA participants. However, CSA participants were more likely to present with PTSD{
CSA was associated with an increased likelihood of presenting with associated psychological sequelae and STI's, namely Chlamydia. Further, EMCSA participants were more likely to present with HIV disease than AMCA participants. Our research contributes to literature outlining the pernicious impact CSA has on physical and mental health in the severely mentally ill homeless population. Future studies should identify potential moderators of the CSA-health risk relationship (e.g., gender).
The numbers of street children vary from 30,000 to 100,000 in Ukraine. Their vulnerability to health-related risks, including HIV/AIDS, substance and drug abuse, was a subject of the baseline study in 2008. Based on its findings, UNICEF implemented a comprehensive approach to HIV-service delivery in four pilot cities and repeated the survey to assess the effectiveness of interventions in 2011.
Behavioral survey among street-based adolescents (N=805, age 10–19, 565 boys, 240 girls) was conducted using location-based network and convenience sampling. Data were disaggregated by age and gender. Comparative data analysis was applied to learn the behavioral and knowledge changes. The client satisfaction questionnaire was used to define the service access barriers.
Street adolescents are highly vulnerable to HIV-infection: 22% injected drugs, 65% of girls provided commercial sex services; 7% of boys had sex with men; only 13% always used condom with casual sexual partners. Social vulnerability factors hinder access to medical and social services: two-thirds of respondents didn't have a permanent place of residence and were not covered by medical services. 46% didn't have an ID, 54% didn't have an education certificate. The piloted interventions caused the positive behavioral change and knowledge increase: a share of those, who correctly identified the ways of HIV transmission, has increased for 10%; a share of those, who were tested for HIV during the last year and received the result, has almost doubled. The biggest increase in HIV-testing is among girls: every sixth tested in 2008, every third in 2011.
Study confirmed effectiveness and sustainability of implemented interventions and suggested a roll-out-strategy to the country. This is of critical importance as a significant number of street children remains uncovered by services and has a low level of knowledge about HIV/AIDS, HIV-service organizations and places, where support is provided and testing is available.
This study investigated how gender, multicultural and multi-religious factors influenced the teaching and learning of HIV/AIDS education.
The qualitative case study utilised 6 primary schools from Kakuma Refugee Camp and its host community. The sample had 617 respondents from 9 nationalities, including 356 male and 160 female pupils. Interviews, observation, FGDs, documentary analysis and drawings generated data. The research proposal and tools underwent ethical review.
Cultural and religious tendencies of same gender clustering denied Muslim Somali pupils an opportunity to work together as partners in addressing pertinent and effective strategies in HIV/AIDS education. Unlike the Christian Turkana and Ugandan girls who seemed open and outgoing in HIV/AIDS education activities, Somali and Ethiopian Muslim girls remained quiet, reserved and shy as a way of showing respect to the male, a behaviour that jeopardised HIV/AIDS education. Christian Sudanese and Turkana boys and girls interacted more freely, hence learnt better. Gender influenced perceptions of pupils on HIV/AIDS education content and pedagogy. While boys seemed vocal, uncontrolled and eager to discuss sex and condoms, girls preferred discussing love and care of people living with HIV/AIDS. Refugee boys produced culturally and linguistically diverse resource materials that were easily understood across the cultural groups while portraying males as innocent victims and females as potentially responsible for the spread of HIV. Notably, pupils received different and conflicting messages on similar topics depending on the teacher's religious background. While older teachers were perceived as ‘parents’, young male teachers were seen as having a hidden ‘sex agenda’.
In conclusion, gender, culture and religion, influence the learning of HIV/AIDS education in refugee schools in a complex manner, which if not understood and controlled could have negative implications. The study recommends pre-service multicultural teacher education and training on how to make HIV/AIDS education gender-responsive.
From 2005, the Québec Ministry of Education cut what was five (5) hours of sex education (STIs, HIV/AIDS, gender, sexual diversity, etc.) per year from the secondary school curriculum. Consequently, in the context of the education reform, teachers holding specializations in English and Art, Science & Technology and Moral and Religious Education were persuaded to integrate sexuality in their course.
Being highly sexualized sites, high schools act as a channel for sexual initiation and exploration. Thus, teachers can be catalysts to providing valuable and life altering information around HIV/AIDS to their students. Through a qualitative case study, teacher narratives were collected to identify their classroom structure; strategies; awareness of HIV/AIDS; and the challenges encountered when discussing the subject in their classroom. Overall, implicating communication processes were an essential factor in uncovering the subtle, yet, uncomfortable silences found in this study.
The surface-level understanding around HIV/AIDS and a lack of consistent training and access to accurate resources identified how teachers understood and valued HIV/AIDS information. Ultimately, such familiarity corresponded to how their students comprehended the virus and viewed the marginalized communities most affected.
Theoretical frameworks connected to Paulo Freire's
School-based, teacher led Sexuality Education (SE) is effective in promoting and protecting young people's sexual health. In sub-Saharan Africa, much of SE is provided through school-based, teacher-led programmes. The aim of this study was to find out what topics in Comprehensive Sexuality Education (CSE) were acceptable and not acceptable to the teachers.
This study was part of an assessment of the status of Sexual and Reproductive Health and Rights in three regions (Tanga, Singida and Iringa) in Tanzania. Respondents were purposively obtained from 6 purposively selected primary and secondary schools in three purposively selected wards. A total of 45 teachers participated in Focus Group Discussions and Group Interviews. Data were analyzed using Nvivo software.
Six topics in CSE were consistently rejected by teachers. These are homosexuality, masturbation as an alternative to sexual intercourse, condom use, sexual pleasure and enjoyment, sexual behavours other than intercourse and appropriate and inappropriate touching. Three major reasons were given to why they rejected these topics. First, they explained that if students are taught about these topics, they may practice them and that would fuel sexual activity among them. Second, since they have to teach practically, teachers explained that demonstrating these topics would be an embarrassment to them and to the students. Third, they reported that these topics are against sexual norms of the communities where they (teachers) and students come from.
With these findings, it is important to rethink the position of teachers in the delivery of CSE. Being “teachers” does not exclude individuals from abiding by sexual norms of their community. The rejection of these topics indicates that teachers still adhere to sexual norms of their communities. In order to strengthen CSE programmes, programmes need to work on sexual norms that may hinder the delivery and success of CSE in schools.
HIV-1 subtype C is the most common subtype worldwide and the dominant subtype in southern Africa. It has been hypothesized that increased transmissibility could explain the explosive spread of subtype C in southern Africa. We assessed the risk of heterosexual HIV-1 transmission in subtype C compared with other subtypes among serodiscordant couples.
We performed a nested case-control study within a multinational prospective cohort of stable HIV-1 serodiscordant couples from 7 countries in eastern and southern Africa (Partners in Prevention HSV/HIV Transmission Study). Cases were defined as couples in which phylogenetically-linked HIV-1 transmission occurred within the partnership; four controls for each case were selected proportionally from non-transmitting couples by the study site and gender distribution of the full study cohort. HIV-1 subtype in the HIV-1 infected partner was determined through consensus partial
HIV-1 subtype distribution was comparable between cases (N=123) vs. controls (N=459): subtype A (45% vs. 44%), subtype C (38% vs. 39%), subtype D (15% vs. 14%), subtype G (1% vs. 1%), and circulating recombinant forms (CRF, 2% vs. 3%). Subtype C was not associated with an increased likelihood of HIV-1 transmission (
In this multinational study of HIV-1 transmission among stable heterosexual African HIV-1 serodiscordant couples, we found no evidence of increased transmission risk for subtype C in adjusted analyses.
In sub-Saharan Africa, where BCG vaccine is routinely given around birth to protect from disseminated TB, almost a third of infants are HIV-exposed. HIV preferentially infects activated CCR5+CD4+ cells. Highly HIV-exposed, seronegative individuals have relatively decreased peripheral CD4 T cell activation. We hypothesized that routine BCG vaccination of HIV-exposed neonates causes non-specific CD4+T cell activation.
HIV-exposed, uninfected South African infants were randomized to receive either BCG at birth or at 8 weeks of age, and blood was collected at birth, 2 and 6 weeks. Unstimulated PBMCs were stained with viability dye, anti-CD3, -CD4, -CD8, -CCR5, the activation markers CD38 and HLA-DR, and the proliferation marker Ki67. PBMC and plasma cytokine mRNA and protein levels were measured using RTPCR and Luminex respectively.
At 6 weeks of age, there was a significantly higher HLA-DR expression and CCR5, HLA-DR and CD38 co-expression on CD4+ T cells (p=.02 and p=.01 respectively; n=94) in the infants who had received BCG at birth compared to unvaccinated infants (delayed arm). In contrast, there was no difference in CD8 T cell activation between BCG-vaccinated and unvaccinated infants. The CCR5 agonist, MIP1β, was significantly higher at 6 weeks in plasma of unvaccinated infants (p=.02). There were no differences in plasma IFN-a, IFN-g, MCP-1, TNF-a, IL-8, GMCSF or IP-10 levels, nor differences in PBMC IFN-a, IFN-g, RANTES, TNF-a, IL-8, IL-10, TGF- β, OAS or IP-10 mRNA levels, between vaccinated and unvaccinated infants.
BCG vaccination induces non-specific CD4+ T cell activation and down-regulation of MIP-1β expression. This elevated T cell activation may increase HIV infections in breastfed infants and may contribute to rapid disease progression. Further research regarding the risks and benefits of BCG vaccination in HIV-exposed infants is needed to inform policy and practice.
The dilemma of infant feeding in HIV context of poor resource setting remains unresolved and the practice of replacement feeding may happen to be a curse by lowering child survival. Appropriate infant feeding counseling can reverse such risk as well as limiting spill-over (WHO, 2010.)
to describe infant feeding intents of HIV positive women and determine the appropriateness of choice of those opting for formula-feeding after the counseling process.
Routine infant feeding counseling of HIV positive mother offered by short-course trained counselors during the pregnancy or in the early-post partum. Intents were assessed using a generic acceptable, feasible, affordable sustainable, secure (AFASS) score composed of 7 variables grading from 0 to 2:type of energy, source of water, kind of latrines, disclosure to the partner, monthly income, ability to prepare bottle feeding and to give a reason for non breastfeeding. An AFASS score above 10/14 was considered as appropriate for formula feeding.
950 women were included for cohort characteristic. Among 924 women counseled, 63% intended to formula feed their babies while 37% planned to breastfeed. The AFASS criteria>10 was met by 87% who intent to practice formula-feeding compared to 57% of those who intent to breastfeed. Women counseled during post-partum were more likely than others to opt for artificial feeding (p< 0.001). Formula-feeding choice was more appropriate in women counseled during pregnancy vs after delivery (p=0.02). The determinants of choosing replacement feeding were tertiary education (p< 0.001), no previous exclusive-breastfeeding, HIV-status disclosure (p< 0.001) and AFASS score>10 (OR: 5; 95% CI: 3–6).
in Djoungolo, after infant feeding counseling, replacement feeding intent is mostly appropriate fitting mother's environment and livelihood. In addition, the desire to breastfeed remains real as more than 1/2 women who choose to breastfeed met the conditions to practice formula feeding.
Although evidence reveals that most heterosexual HIV transmission in sub-Saharan Africa takes place within marriage or cohabitation, approaches for people living with HIV (PLHIV) focus primarily on individuals. The Faithful House (TFH) is a couples-based, skills-building curriculum used with 45,000 couples in twelve countries, recently modified to address PLHIV issues. Research examined the effect of TFH on attitudes and behaviors to provide evidence for a couples-based approach for more holistic PLHIV programming.
Participants, using convenience sampling, from HIV programs in four regions of Ethiopia were randomly distributed between intervention and control groups. The intervention group participated in TFH workshop for PLHIV. Both groups completed surveys at baseline and three months post-intervention which was analyzed using STATA.
The study surveyed 378 individuals with a mean age of 35.2. Most couples (88%) were either married or cohabitating. All participants had been tested for HIV with 90% testing positive. Intervention participants (193) reported significant changes (p< 0.01) in the quality of their relationship, including improved communication and joint decision-making about child care, finances and sexual negotiation. Intervention participants had statistically significant improvements in medication adherence (18% non-adherent at baseline versus 10% at three-months) and percentage diagnosed with sexually transmitted infections in the past three months (7.3% decreased to 4.7%). Of males with pregnant partners, 94% in the intervention group attended antenatal care visits compared with 36% in the control. Intervention participants also reported statistically significant decreases (p<0.05) in violent behaviors including insulting, shoving, and forcing sex.
The modified TFH curriculum had a positive impact on attitudes and behaviors affecting the physical and relationship health of PLHIV couples. These preliminary results indicate potential for couples-based approaches for more holistic programming for PLHIV. Continued evaluations are critical in determining sustained impact on health status outcomes, attitudes and actual behavior change.
As part of a government-sponsored pilot initiative of ‘treatment as prevention’, recent efforts have been made to improve access to HIV prevention and care, including HIV testing, to vulnerable sub-populations in Vancouver, Canada. This study assessed the association between geographic factors measuring access to HIV testing sites and having a recent HIV test among hidden street- and off-street sex workers(SWs) in Vancouver.
Baseline data were used, including an interviewer-administered questionnaire, HIV/STI testing and geographic location data, from an open prospective cohort of SWs recruited in 2010 in Metropolitan Vancouver (“An Evaluation of Sex Workers’ Health Access”[AESHA]). Access was measured by density of testing sites within a catchment surrounding SWs’ place of solicitation (radius=distance travelled in 15 minutes of combined bus/walking) and time to travel to nearest testing site. Bivariate and multivariable logistic regression was used to identify if density and time were independently associated with recent HIV testing (in the last year). Adjusted odds ratios and 95% confidence intervals were reported (AOR:[95% CIs]).
In total, 291 seronegative SWs from Vancouver City were included, with 69.4% (202) reporting a recent HIV test. In bivariate analysis, having a recent HIV test was significantly associated with a higher density of testing sites (p< 0.001) and time to nearest testing site (p=0.05). After adjusting for key confounders (recent injection drug use, age and sexual identity), having a recent HIV test was significantly associated with increased density of HIV testing sites: the probability of having a recent HIV test increased by 2% for each increase in one testing site (1.02[1.01–1.04]).
Our results highlight the importance of physical availability of HIV testing sites within sex work environments to facilitate use of HIV prevention and care among SWs. Increased mobile and safer-environment interventions that facilitate access to voluntary and confidential HIV testing at outdoor and indoor sex work venues remain a critical priority.
HIV/AIDS is a leading cause of death for African-American women in the United States between the ages of 25 and 34 years. Studies have suggested that HIV-related stigma impacts morbidity and mortality rates because it contributes to poor treatment utilization for various groups of people with HIV. Despite these findings, there are no intervention studies investigating stigma reduction strategies for African-American women living with HIV.
We implemented an adapted version of the International Center for Research on Women's HIV Stigma Toolkit for African-American women living with HIV, with intervention modules led by an African-American woman living with HIV. Twenty-four participants attended workshop sessions split across 2 weekday afternoons, discussed issues “triggered” by videos that were produced specifically for the intervention, learned stigma reducing mechanisms from each other, and practiced using these mechanisms in role plays. Participants completed a measure of internalized stigma before, immediately after, and 1-week after workshop participation.
The intervention demonstrated feasibility and the women enthusiastically accepted the intervention. The women reported decreased stigma from the start of the workshop to immediately after (
Findings suggest that the Unity workshop holds promise for reducing internalized stigma for African-American women living with HIV.
This study examined the effectiveness of psycho-education in Family-to-Family Project on family relationship and emotional quotient (EQ) of adolescents in HIV families in two representative provinces in Thailand, Chiangrai and Nakorn Ratchasima. The intervention included core elements identified by Thai Ministry of Public Health and University of California, Los Angles, USA for improving physical and mental health, family relationship, and social outcomes for HIV-affected families.
The sample consisted of 194 adolescents (aged 12–17 years) in 402 HIV-affected families. A randomized controlled trial with pre-test and post-test was performed during December 2006 - January 2009. Adolescents were randomly assigned into 2 groups: (a) adolescents whose parent and caregiver(s) attended psycho-education and (b) adolescents whose parent and caregiver(s) did not attend psycho-education. The instrument was a set of questionnaire including: family relationship and EQ. Data were collected prior to the beginning of the program, and at 24-month follow-up. They were analyzed using t-test and logistic regression statistics.
The study findings suggested, when controlled sex, age and education, family relationship and EQ of adolescents before and after the intervention in each group did not vary significantly. Family relationship had significantly positive relationship with EQ on the “happy” subscale [adjusted Odds Ratio (OR)=12.03, 95% Confidence Interval(CI)=3.21–45.02] and total EQ [adjusted OR=9.57, 95% CI=2.28–40.17]. However, it did not relate to EQ on the “good” subscale [adjusted OR=3.13, 95% CI=0.57–17.12] or the “competence” subscale [adjusted OR=1.41, 95% CI=0.158–12.57].
Family relationship is a protective asset in happiness and total EQ of adolescents in HIV-affected families. However, larger and robust trials are needed to further determine the effectiveness of psycho-education for Thai HIV families. This study was funded by the National Institute of Nursing Research (grant NINR R01-NR009922).
Serodiscordant couples are an important source of new HIV infections. This analysis explores the prevalence of HIV serodiscordance among couples, HIV-testing behavior, and factors associated with serodiscordance in Mozambique.
A nationally representative HIV seroprevalence survey was conducted in Mozambique in 2009. Couples who were married or cohabiting were identified, their HIV status compared, and factors associated with concordant positivity versus serodiscordance were measured. Data were analyzed using cross-tabulation and logistic regression.
15% of 2,468 couples in Mozambique had one or both members HIV infected. Serodiscordant couples outnumbered concordant positive couples two to one (10.2% vs. 4.9%). Among discordant couples, only 15% reported that both members of the couple had been tested for HIV and received the results. Overall, 6% of discordant couples used a condom the last time they had sex with each other (compared with 3% of all couples). Condom use was positively associated with exposure to HIV testing and with HIV-seropositivity in the female partner. The only consistent risk factor for couples being concordant positive vs. discordant in the multivariate analysis was a history of self-reported symptoms of sexually transmitted infections (STIs). For example, in the binomial model, if either member of the couple had an STI, the couple was less likely to be discordant vs. concordant positive (adjusted odds ratio 0.40; 95% confidence interval 0.18, 0.89).
One in every ten couples in Mozambique is HIV-serodiscordant. The HIV-negative partners in this group are at high risk for becoming infected; however, few discordant couples know that they are discordant, and are therefore unlikely to take protective measures.
Understanding concurrent drug consumption and unprotected anal intercourse (UAI) behavior among MSM, would contribute to develop tailored interventions for HIV prevention.
A HIV Sentinel Surveillance was conducted in five cities of Peru in June–October 2011. In this survey, MSM with high-risk behavior who were unaware of their HIV serostatus, participated for the assessment of HIV and syphilis status, and demographics and sexual behavior patterns using computer-assisted self-interview. We report socio demographics, sexual risk behaviors, and other characteristics for not using a condom during anal sex associated drug consumption. A multinomial adjusted logistic regression analysis was conducted to assess potential associations between concurrent drug consumption and UAI behavior with other factors.
Among 5,575 participants, 800 (14.3%) reported concurrent drug consumption and UAI behavior in the last three months. Of them, 43.3% were < 25 year-old and 86.6% had high school or a higher education level. This behavior was independently associated with high school or higher education (OR2.27; 95% CI1.34–3.86); self-identification as sex worker (OR2.29; 95% CI1.39–3.77); and self-identification as bisexual or heterosexual (OR2.77; 95% CI1.34–5.75).
Tailored strategies to reduce risky sex behavior associated with drug consumption are urgently needed among MSM in Peru and must target subpopulations at the highest risks.
China initiated Methadone Maintenance Treatment (MMT) program in 2004, serving more than 343,000 clients by 2011. High dropout rate was one of the special challenges that needed to be addressed urgently. The aim of this study is to assess MMT clients’ dropout rate and to identify factors associated with dropout over the six-year period.
A prospective cohort study was conducted in eight MMT clinics of China from March 2004 to June 2010, involving 1511 drug users who were enrolled in MMT program in 2004. Dropout and its predictors were examined using Chi-square tests and Cox Proportional Hazards regression models.
Over the six-year follow-up period, 972 (64.3%) clients dropped out of treatment and clients were more likely to leave treatment within 1.5 years after enrollment. The leading cause of dropout was being arrested (48.7%), followed by emigration/being out of town for work (10.5%) and self-withdrawn (10.3%). Adjusting Hazard Ratio (HR) indicated that clients with high daily dose (>60mg) (P<0.0001, HR=0.38, 95% Confidence Interval (CI): 0.29–0.51), having relatives receiving MMT (P=0.027, HR=0.72, 95% CI:0.54–0.96), and higher urine morphine positive result (P<0.001, HR=0.63, 95% CI:0.52–0.76) were less likely to drop out, whereas clients with needles sharing behaviors, and frequent contact with current drug users had higher risk of dropout over the six-year period (P< 0.05).
Specific interventions to decrease dropout are needed to focus on clients with lower daily dose, sharing needles with others and more frequent contact with current drug users. The preventive role of family support on dropout should be emphasized.
A controversial aspect of HIV/AIDS epidemiology has been whether it is relative wealth or relative poverty that is a key driver of the epidemic in sub-Saharan Africa. We hypothesised that the social epidemiology of HIV in Africa is changing from a situation where more new infections are acquired by those of relatively high socioeconomic position (SEP), to one where those of relatively low SEP are at greater risk. We suggested this pattern is compatible with the inverse equity hypothesis from child heath that suggests those of higher socioeconomic position benefit first from new health interventions.
We analysed Demographic and Health Survey (DHS) data from sub-Saharan African countries with two surveys measuring HIV prevalence or with a second expected in the next two years. We inspected the pattern of HIV prevalence by SEP, indicated by education status. In the countries with two surveys we calculated the percentage risk difference for HIV prevalence stratified by education and sex.
Data were available for eleven countries: four with two surveys and seven expecting a second survey within two years. In the first/only survey, higher SEP is broadly associated with higher HIV prevalence. In countries with two surveys, HIV prevalence has risen in the no education group in all cases except among women from Lesotho, and fallen among those with secondary education in all cases except women from Malawi.
Available evidence strongly suggests that in the early phase of the epidemic HIV infections were concentrated among those of higher SEP. Our analysis supports the inverse equity hypothesis that new infections will increasingly concentrate in people of lower SEP because of lower access to public health interventions. Data that will be available within the next two years will further test this hypothesis. The inverse equity hypothesis has important implications for policy and resource allocation.
Swaziland has the highest HIV prevalence in the world. Many Swazis are chronically food insecure. Globally and within southern Africa, food insecurity has been linked to high-risk sexual behaviors, difficulty with antiretroviral (ARV) adherence, higher rates of mother-to-child transmission, and more rapid HIV progression.
In-depth interviews were conducted with 20 HIV+ female sex workers (SWs) in Swaziland. Interviews took place in four different regions of the country, and were designed to learn about context, experiences, and health service needs amongst Swazi sex workers. Interviews were coded in Atlas.ti.
Hunger was a consistent, major theme in our informants' lives. Women cited their own hunger or that of their children as the impetus to begin sex work, and as a primary force in continuing to sell sex. Nearly all informants requested food-related services (parcels, grants, or education) when asked about desired programming. Good nutrition and the ability to eat “healthy” or ”balanced“ foods was seen as an important means of controlling HIV disease progression. Informants discussed difficulty in adhering to ARVs when faced with taking pills on an empty stomach. Across interviews, discussions of CD4 counts and ARV adherence intertwined with discussions of poverty, hunger and healthy foods. Food security and food sharing were also seen as important expressions of social networks, which many SWs felt they had trouble accessing as a result of both their HIV status and profession.
Informants described a risk cycle of hunger driving sex work driving HIV infection. The two latter in turn drive an increased need for ‘healthy foods' and an alienation from social networks which offer material and emotional support against hunger. Poverty and food security are concrete, vital issues in the lives of SWs living with HIV in Swaziland, issues that cannot be ignored when conceptualizing risk or designing services.
Despite recent efforts to increase HIV testing in sub-Saharan Africa, poor testing availability and limited uptake in refugee populations persists. Refugees require additional focused efforts because many have suffered human rights violations putting them at increased HIV- risk. Our objective was to qualitatively study refugees' utilization of services in a refugee settlement where HIV/AIDS services are available.
Open-ended interviews were conducted with HIV-infected refugees living in Nakivale Refugee Settlement in southwest Uganda. Interviews focused on: (1) accessibility of HIV/AIDS-related testing and care; (2) experiences of ART adherence; and (3) perspectives on how to improve access to testing and care, adherence, and retention. Data were collected at the Nakivale HIV/AIDS Clinic from March to July of 2011 and included patient (N=73) and staff (N=4) interviews, and observations of clinical activities. For this analysis, category construction methods were used to analyze the data relating to HIV testing.
Refugees, because of competing daily hardships, do not prioritize HIV testing. Refugees living with HIV/AIDS often present to clinic for testing and initial evaluation with very advanced disease. Reported barriers to HIV testing for Nakivale refugees reflected in the data include: difficulty physically accessing testing facilities; fear of stigma associated with HIV+ diagnosis; low self-perceived risk of disease, and lack of knowledge regarding the potential benefits of medical therapy. Given the competing priorities for survival, HIV tests are not obtained until special circumstances lead to temporary priority shifting.
Understanding how HIV testing fits among the survival priorities of refugees will help in designing effective interventions. To increase HIV testing for refugees, efforts should aim to intervene in the survival cycle to encourage priority-shifting. Intervention approaches may include improving accessibility, providing incentives and increasing HIV-related education.
There is paucity of published data on ways in which HIV in children receiving Highly Active Antiretroviral Therapy (HAART) impacts other children living within the same households. We investigated the psychosocial impact of HIV on the siblings of HIV infected children.
Data were collected using pre-tested interviewer administered questionnaires and focus group discussions. Twelve 12 HIV treatment sites which account for over 90% of children receiving HAART in Botswana participated. HIV affected children were defined as those aged 6-18 years who were living in the same household as documented HIV-infected children. Ethical approval was obtained from the Botswana Ministry of Health and Baylor College of Medicine.
Of the 258 HIV affected children, 251 (97.3%) were attending school; 206 (79.8%) and 52 (20.2%) had been fully or partially disclosed to respectively. 153 (59.3%) were siblings of the HIV-infected children, 79 (30.6%) were cousins and 26 (10%) were related in other ways. 223 (86.4%) had lived together with the HIV-infected children for longer than 5 years. 11 (4.3%) said that living with an HIV-infected child made them feel different because of stigma, playing caregiver roles, fear of contracting HIV, and feeling sad. 65 (25.4%) faced various problems, including: worrying about the HIV infected child; receiving less attention from caregivers; and experiencing stress due to adherence-related issues, stigma, and family disharmony. They coped by crying, talking to an adult relative, talking to the HIV-infected child or isolating themselves from others. 230 (89%) felt sad or scared/anxious whenever the HIV-infected child was sick. 254 (98.4%) reported playing caregiver roles, such as reminding or giving medications to the infected children.
Although HIV affected children are not the prime targets of paediatric HIV interventions, they face many psychosocial challenges. Programs and policies aimed at ameliorating the impact of HIV should take these findings into account.
The last century's economic and political upheavals are widely believed to have reduced African and particularly South African families' cohesion and ability to function collectively. AIDS has compounded this threat to the resilience of households and wider family networks. We explore the resilience of families to AIDS and demonstrate that theories of social capital, family obligation and reciprocity can help to explain access to familial support in rural KwaZulu-Natal, South Africa.
Data were collected over a 7-month period from a small sample of households dealing with AIDS illness or death using in-depth interviews and participant observation. Retrospective and prospective data about households' experiences were analysed using framework analysis and the development of household case studies for comparisons.
Affected households and individuals drew on family relationships for financial and material support and physical care. Close family members, often sharing a common sense of home and family, were the most important source of care and support. Their greatest motivation was a strong moral obligation to family, associated with norms of familial assistance. Support from other family varied depending on whether levels of mutual trust, investment in social capital and physical proximity, enabled negotiated reciprocal exchange. Families suffering from conflicting obligations, conflict, severe poverty or extreme illness were more likely to be excluded from these networks and suffered because of their inability to secure familial support.
Despite examples of exclusion, we demonstrate that social capital, reciprocity and a sense of family obligation persist in families responding to the impacts of AIDS, contributing to them maintaining cohesion, collective functioning and ultimately ensuring their resilience. Interventions to support the treatment, care and well-being of sick individuals need the flexibility to be able to both support families in their efforts to provide these services and address the needs of people without access to supportive family networks.
Despite substantial data globally suggesting expansion of sex work markets in settings with recent political, economic and social transitions, there exists almost no data on HIV prevalence among SWs in post-conflict settings, particularly in sub-Saharan Africa. Of further concern, given increasing efforts to criminalize HIV and sex work in much of sub-Saharan Africa, there remains an important need to understand the HIV prevention and treatment needs of sex workers.
In 2011, 400 young women SWs (>14 years of age) completed baseline interview-administered questionnaires and confidential HIV testing. SWs were recruited through extensive peer outreach (current/former SWs), ethnographic mapping and time-location sampling to street and indoor sex venues. Bivariate and multivariate analyses were conducted to examine associations with HIV seropositivity.
Of a total of 400 young women SWs, the median age was 21 years (IQR15–26). The HIV seroprevalence was 34% (136), of whom 59% (80) were new HIV seroinfections, and 32% ( 44) were on ART. The majority (96%) solicited clients in bars, 41% (165) in lodges, and 35% (183) along truck stop. A high number (56%, 222) of SWs reported difficulty accessing condoms in the last 6months. HIV seropostivity was associated in bivariate analysis with supporting dependent children (OR=3.30, 1.57–6.89). In multivariate analyses, HIV seropositivity was associated with older age (AOR=1.18, 95% CI1.10–1.26), lower sex work income (AOR=0.99, 0.99–0.99) and reduced likelihood of having a ‘sugar daddy’ (AOR=0.49, 0.25–0.99).
Young women SWs are experiencing an alarming rate of HIV infection in Northern Uganda, with more than half unaware of their HIV seropositivity status. These data suggest critical gaps in HIV prevention, treatment and care services and an urgent need for social and structural HIV interventions that support and reach isolated young women SWs, including reducing stigma, policy reform, and peer-led outreach initiatives.
Black MSM (BMSM) constitute <1% of the U.S. population, but >25% of incident HIV infections. Enhanced understanding of factors associated with new interventions can provide data to inform programs needed to address this disproportionate epidemic.
Between 7/09 and 10/10, BMSM were enrolled in 6 U.S. cities to evaluate feasibility of a multi-component prevention intervention. This analysis focuses on the correlates of being newly diagnosed with HIV, including multivariable logistic regression. HIV testing was performed at study sites; central confirmation of results is underway.
HPTN 061 enrolled 1553 BMSM, whose median age was 39; 43% self-identified as gay/homosexual, 41% bisexual, 3% transgender, 10% straight/heterosexual. Of 96% who agreed to be tested, 10% indicated they were previously HIV-infected (PHIV), while 12% were newly diagnosed with HIV (NHIV). Compared to PHIV, NHIV were younger less likely to use marijuana poppers stimulants or inject drugs reported less internalized homophobia and lower levels of religious affiliation NHIV were more likely to be diagnosed with syphilis and anogenital gonorrhea/chlamydia than PHIV, and more likely to be diagnosed with syphilis or anogenital chlamydia than HIV-uninfected BMSM. Compared to HIV-uninfected BMSM, NHIV BMSM were more likely to be older unemployed engage in unprotected anal intercourse have multiple bacterial STDs, and were more likely to come from cities other than San Francisco.
Structural, behavioral, and biological factors (e.g. unemployment, unprotected anal sex, and STDs, but not increased substance use) are associated with new infections among American BMSM, who differ behaviorally from men who have previously been diagnosed. Given the high rates of HIV infection among BMSM, culturally-tailored programs that encourage repeated HIV/STD testing, engagement in care, and innovative prevention strategies addressing current risks are urgently needed to decrease further spread.
Black/ African American MSM (BMSM) in the United States are affected by HIV at dramatically disproportionate rates compared to MSM of other race/ethnicities. Current HIV incidence estimates in this group are needed to appropriately target prevention efforts.
In 2009–10, HPTN 061 recruited BMSM in Atlanta, Boston, Los Angeles, New York City, San Francisco and Washington D.C. for a feasibility study of a multi-component intervention to reduce HIV infection. Participants reporting ≥1 episode of unprotected anal intercourse with a man in the past six months were evaluated at baseline, 6 and 12 months. HIV status at enrollment was based on real-time testing performed at study sites and confirmatory testing at the HPTN Network Laboratory. HIV incidence based on HIV seroconversion was calculated as number of events/person-years. Confidence intervals were calculated using exact methods.
Of 1553 BMSM enrolled, 174 reported a prior HIV diagnosis and 46 refused HIV testing or a specimen was not available at baseline. Of those without a prior HIV diagnosis (n=1333), 1168 were HIV-uninfected, and 165 (12.4%) were newly diagnosed at baseline (including 3 with acute HIV infection). Among the 1168 HIV uninfected men at baseline, 26 acquired HIV infection during follow up for a 2.8% annual HIV incidence rate (95% CI: 1.8–4.1%). HIV incidence was higher among men <30 yrs (5.9%; 95% CI: 3.6–9.1%) compared men >30 yrs (1.0%; 95% CI: 0.4–2.2%), men identifying as exclusively gay/homosexual (5.0%; 95% CI: 2.6–8.8%) compared to bisexual (1.5%; 95% CI: 0.4–3.7%) and men reporting unprotected receptive anal sex (4.9%; 95% CI: 3.0–7.4%) compared to those not (1.0%; 95% CI: 0.3–2.4%).
In the largest cohort of prospectively-followed BMSM in the US, HIV incidence was high, particularly among young and gay-identified BMSM. Targeted and tailored culturally appropriate combination HIV prevention strategies incorporating behavioral, social and biomedical based interventions are urgently needed to lower these rates.
In Zambia, HIV prevalence in women (15–24) is twice that of same aged men. A gender perspective is critical for designing interventions that recognize gendered-risks to HIV. The Zambia-led Prevention Initiative (ZPI) is initiating community-level interventions through targeted approaches that address gender disparities and power imbalances. The baseline survey, part of the ZPI evaluation plan, identifies key areas for targeting.
A total of 1,060 men (aged 15–59) and 1,700 women (aged 15–49) participated in a survey conducted in 4 provinces. Provinces and districts were purposively selected; households and individuals were randomly selected. The survey included questions related to gender-based violence, rape myths that blame women for rape (i.e.,
68% of females ever experienced either or both physical (35%) or sexual abuse (39%); only 15% sought help. 62% of all respondents endorsed at least 1 of 4 rape myths and 37% supported inequitable gender norms. Those with 2+ partners (46%) in the last 12 months were more likely to support inequitable norms than those with 1 partner (39%) or no partners (26%) (p< 0.001). Nearly 20% of men think that contraception is women's business; 32% think women who use contraception are promiscuous. When women were asked about their last birth, 50% indicated they wanted to wait or did not want the pregnancy at all. Unintended pregnancies were highest among unmarried younger females (77–85%) compared to married women aged 25+ (50%).
Inequitable gender norms are pervasive and may affect women's vulnerability to HIV and gender-based violence. ZPI is addressing power imbalances between men and women that contribute to HIV risk and focusing on male norms and behaviors that contribute to gender-based violence.
Gender based violence (GBV) is a human rights violation and public health problem impacting women's health globally and is interconnected with the HIV epidemic. GBV is associated with reduced adherence, poor treatment outcomes, and mortality in women with HIV.
Using marginal structural survival models (MSMs) we evaluated the effect of sexual, physical, or emotional abuse in the past 12 months on mortality among 2,222 (1,642 HIV-infected and 580 seronegative) participants in the Women's Interagency HIV Study (WIHS), an ongoing cohort study. Mortality data were confirmed by National Death Index Plus registry match. MSMs were used to estimate the mortality hazard ratio and survival curves from baseline (1994/95 or 2001/02) through 2007, controlling for sociodemographic, behavioral, and clinical factors.
Overall, 437 (19.7%) women died between 1994 and 2007 and had abuse data available in the year prior to death. Compared to survivors, women who died were more likely to be older, HIV-infected and not treated with highly active antiretroviral therapy; have lower nadir and current CD4; to have engaged in transactional sex and used drugs and tobacco; be depressed, report lower cognitive function, and have a history of pre-study abuse including childhood sexual abuse. Accounting for these fixed and time-varying confounders, recent abuse was independently associated with all cause mortality (HR 1.54; CI 1.18, 2.02); findings remained significant in analyses stratified by pre-baseline abuse history, and by HIV serostatus. This effect was greater in uninfected (HR 4.39; CI 1.78, 10.82) than HIV infected women (HR 1.42; CI 1.07, 1.89).
Mortality risk is significantly elevated for women exposed to GBV and appreciable even in the context of a high death rate due to HIV infection. Interventions to address GBV should remain a public health priority. Further research is needed to identify possible biologic pathways underlying abuse related sequelae.
As northern Uganda emerges from decades of war and displacement, a growing number of young women engage in sex work (SW) for survival. With the escalating rates of HIV, the normalization of violence in northern Uganda creates a high occupational risk environment for young SWs. We aim to investigate the prevalence of client violence and associations with HIV risks among a cohort of SWs in Gulu, northern Uganda.
We conducted an analysis of baseline data (questionnaire and HIV screening) of SWs enrolled in a prospective cohort. Young women (≥14 years) who exchanged sex for resources in the last 30 days were recruited through ethnographic mapping, time-location sampling and peer outreach (current SWs) to SW venues. Bivariate and multivariate logistic regression modeled associations with physical and/or sexual violence by clients among SWs.
Of 400 SWs, the median age was 21 (IQR: 19–25). The majority were Acholi (92.3%) with 66.5% (266) having formerly lived in displacement camps, 34.0% (136) living with HIV. In the last six months, 83.7% (335) had experienced violence by clients: 69.0% (276) forced unsafe sex, 28.8% (115) stabbed, and 18.8% (75) raped. In multivariate logistic regression, client violence was independently associated with rushing negotiations with a client due to police presence (a-OR: 3.58, 95% CI: 1.68–7.64), inconsistent condom use by regular and one time clients (a-OR: 3.53, 95% CI: 1.89–6.86), and older age (a-OR:1.08, 95% CI: 1.01–1.17).
The SW risk environment in northern Uganda is characterized by extreme occupational violence directly associated with a 3.5-fold increase risk of non-condom use. The criminalization of sex work in Uganda contributes to 3.5-fold increased odds of client violence from rushing negotiation due to police presence. Structural interventions (decriminalization and enforcement-based approaches) must be integrated into the HIV response, both as a human rights and public health imperative.
Malawi has a generalized HIV epidemic with approximately 11.0% of adults living with HIV, though preliminary data highlight significant HIV-related vulnerabilities among key populations, such as MSM. There is limited understanding of vulnerabilities among MSM; this study aimed to fill this gap and provide population-based estimates of HIV prevalence and associations of infection among MSM in Malawi.
339 men reporting anal sex with another man in the previous year were accrued into a respondent-driven-sampling study from August 2011-March 2012 in Blantyre. Study activities included a structured survey instrument and biological assessment of HIV and syphilis.
Participants were a mean age of 25.1yrs. (range: 18–49), 46.6% were unemployed, over half were gay-identified (61.9%), and 10.3% (35/339) were currently married to a woman. Participants reported a mean of 3 male sex partners in the last 12mo. (range: 1–50). Concurrent relationships were common 30.4% (99/326) reported recent partnerships with two or more men and 14.7% (48/306) reported concurrent partnerships that included at least one female. HIV prevalence was 14.8% (49/330); among those with HIV infection, 91% (45/49) were unaware of their HIV status and 39.9% (19/39) had never tested for HIV. Nearly 60% (176/304) reported that vaginal sex was the highest risk form of sex, indicating low knowledge of transmission risk. Multivariate analysis demonstrated that age ≥26yrs (aOR4.26, CI: 2.17–9.47), history of imprisonment (aOR: 1.72; CI0.82–3.58), and having≥1 child (aOR: 2.25; 95% CI: 1.50–7.01) were associated with HIV infection, while rural residency (aOR0.32; 95% CI: 0.11–0.93) and secondary education or higher (aOR: 0.81; 95% CI: 0.41–1.63) were inversely associated.
As of May, 2012, the changing government in Malawi publicly announced intention to decriminalize homosexuality. The data here reinforce the need to take advantage of this opportunity to provide services to MSM, given the limited HIV-related knowledge and high-risk practices. This study demonstrates that MSM are an important population in Malawi's HIV epidemic and deserve targeted HIV prevention services.
We examined knowledge, attitudes, and practices toward use of daily PrEP among MSM and factors associated with their willingness to take PrEP if available and offered for free or covered by health insurance.
Between August–December 2011, MSM in two U.S. metropolitan areas heavily impacted by HIV (Miami, Florida and Washington, D.C.) were recruited and interviewed through venue based sampling for the CDC National HIV Behavioral Surveillance study. Multivariable logistic regression analysis assessed demographic, socioeconomic, drug use and sexual risk correlates of being very willing to take PrEP for each city.
The samples included 321 in Miami (median age=29;18% black, 10% white, 71% Hispanic, 1% Other) and 323 in Washington D.C. (median age=32, 28% black, 49% white, 13% Hispanic, 10% Other). Fifteen percent of men in Miami and 30% in Washington D.C. had heard information about PrEP, few knew anyone who had taken PrEP (3% in both cities), and none reported having taken it themselves. Almost half (49%) of MSM in Miami and almost two-thirds (61%) in Washington D.C. reported they would be willing to take PrEP. In Miami, only non-injection drug use in the past year was associated with decreased willingness to use PrEP (OR=.59, 95% CI (.36, .96). In Washington, D.C.,>33 years of age (OR=.45, 95% CI (.28, .74) and having fewer sexual partners (OR=.57, 95% CI (.33, .98) were associated with decreased willingness to use PrEP; non-injection drug use (OR=1.67, 95% CI (1.02, 2.73) was associated with increased willingness to use PrEP.
Awareness and use of PrEP in these two US HIV epicenters is low; innovative strategies are needed to inform and educate MSM about this new prevention strategy. Willingness to use PrEP may be impacted by drug use and sexual risk behaviors. Future studies are needed to understand how non-injection drug use may impact PrEP use.
HIV interventions targeting most-at-risk populations (MARPs) in Egypt have focused primarily on behavior change through awareness campaigns. The lack of information about the MSM population continues to be a challenge for HIV prevention and care efforts.
FHI 360 with (funding from the Drosos and Ford Foundations) established three “comprehensive care centers” (CCCs) to provide harm-reduction services for MARPs. Data were collected and analyzed for all the MSM who visited the CCCs.
From July 2008 to December 2011, 1,303 MSM visited the CCCs. Of these, 92.7% were referred by peer-educators through community outreach. The HIV prevalence among the MSM was 4.1%. About 46% were 16 to 24 years old and more than half of them had received some university education. Ever-being married to a female was reported by 13.0% of the MSM, whereas 9.5% were divorced and 22.7% had “a steady partner, but were not living together.” About 42.6% reported “ever-injecting drugs,” 69.0% shared needles and 83.7% shared paraphernalia. Alcohol was consumed by 43.7%. In the past year, about half of the MSM had commercial sex partners and 93.6% had non-commercial sex partners. Moreover, 17.5% reported exchanging sex for drugs. In the past six months, less than one quarter of the MSM used a condom during sexual intercourse with their regular partners and 31.9% used a condom with non-regular partners. About 70.7% were willing to use condoms, however only 14.0% reported condom use during their last sexual encounter.
Connections between MSM and the general population have the potential to spread the HIV epidemic beyond the MARPs. Unsafe injection of drugs and risky sexual behaviors among MSM should be addressed in future programs. There is a need to expand the CCC model for MARPs in Egypt.
Migrant populations from countries with generalised HIV epidemics represent a considerable proportion of the heterosexually acquired cases of HIV/AIDS in EU/EEA (European Union/European Economic Area) countries. A better understanding of the epidemiology of HIV among migrants is essential to tailor HIV prevention and treatment programmes.
Results from European HIV/AIDS surveillance data from 2010 and qualitative surveys on national HIV programmes and policies were used to assess the situation of HIV in migrant populations.
Overall, one-third of the heterosexually acquired HIV cases were diagnosed in individuals originating from countries with generalised epidemics and this proportion varies by country, but is >60% in Belgium, Sweden, Malta, and the UK. Studies in France, Spain and the UK describe higher prevalence of late HIV diagnosis (CD4 count <350 cells/mL3 at HIV diagnosis) in migrants compared to non-migrants and in ethnic minorities compared to the non-minority population. Half of EU/EEA countries report that they have legal, regulatory and policy barriers for migrants to access HIV treatment, care and support. Seventy-five percent of countries indicated that migrants are an important subpopulation in the national response to HIV. However, only 40% collect information on the uptake of HIV testing and only 50% on access to ART among migrants.
Evidence suggests that, in some European countries, migrants from countries with generalised HIV epidemics are disproportionally affected by HIV and do not access testing or treatment services as readily as other populations. There is a need for concerted action at a European level to gather better evidence for decision-making and to develop better measures of HIV transmission among migrants after arrival to the EU/EEA in order to improve HIV prevention resource allocation. There is also a need for strong political leadership in order to further develop and expand programmes for migrants from countries with generalised HIV epidemics.
Disclosure of seropositivity within a partnership is a pre-requisite for couples counseling and the tailoring of prevention interventions, such as the use of treatment as prevention. In this study, we used a mixed methods approach to describe the rates and determinants of disclosure within partnerships.
The Multi-Country African Testing and Counseling for HIV study was a cross-sectional study designed to compare clients’ experiences of HIV testing services in Burkina Faso, Kenya, Malawi, and Uganda. Face-to-face questionnaires were administered and included multiple-choice and open-ended questions. Our analysis focuses on a sub-set of married/cohabiting participants reporting awareness of their seropositive status. We explore the influence of stigma through a multilevel logistic regression model.
Of the 477 participants, 85.3% [95%CI 82.1–88.5] reported ever disclosing their serostatus. However, only 52.6% [95%CI 48.1–57.1] reported disclosing to their partners. We observed a significant variation between countries-participants in Kenya and Burkina Faso disclose at similar levels, while lower levels of disclosure were reported in Malawi and Uganda. Analysis of the open-ended responses among HIV positives who did divorce, revealed that disclosure can create serious rifts with partners. Perceived community stigma was approximately reported at same levels across the four countries, but levels of self-stigma differed. Patients were more likely to report self-stigma in Burkina Faso, while they were the least likely to report self-stigma in Malawi. However, none of our stigma indicators was associated with disclosure. At individual level, those with a lower education and member of a support group disclose less than the rest.
Seropositive patients tend to disclose to people in their support network. However, disclosure within the partnership is less common. We found no relationship between stigma and disclosure to partners. A tailored disclosure support and advice will be essential for programs looking at implementing treatment as prevention in serodiscordant couples.
Internal stigma or self-stigmatization is a critical problem among PLHIV as it usually leads to low self-esteem, a sense of worthlessness and depression, etc. The objective of this study is to assess the level of internal stigma among HIV-positive adults in Ethiopia
This study utilized a nationally representative two-stage cluster sampling method to collect data from 3360 PLHIV (68% of them women) sample cases.
More than half of the PLHIV blamed themselves and reported to have low self-esteem. More than 40 percent of the PLHIV feel ashamed and guilty because of their HIV status. PLHIV residing in rural areas have higher likelihood of feeling guilty than PLHIV residing in urban areas. One out of five of the PLHIV felt suicidal, in connection with their HIV positive status. Abandoning aspirations/life goals, in connection to their HIV-positive status was reported by substantial proportion of PLHIV. For instance, the proportion of PLHIV who took the decision not to have (any more) children was 59 percent,), not to have sex was 40 percent and not to get married 37 percent. Significantly higher proportion of female PLHIV were noted to have taken all these decisions compared to males (P=0.000). Almost one-quarter of PLHIV revealed that they have isolated themselves from their family and/or friends as a result of their being HIV-positive. Nearly equal proportion of PLHIV have decided to withdraw from their education/training and stopped working (12 percent) because of their HIV-status.
Internal stigma and its negative consequences are very common among PLHIV in Ethiopia. This may deter PLHIV from active participation in socio-economic activities of the community out of a fear of having their status revealed or being discriminated against. In order to address the problem, peer-to-peer support groups, skills building, network building, counselling, training, should be given attention.
Previous research has revealed a negative impact of orphanhood and HIV-related stigma on the psychological well-being of children affected by HIV/AIDS. Little is known about psychological protective factors that can mitigate the effect of orphanhood and HIV-related stigma on psychological well-being. This research examines the relationships among several risk and protective factors for depression symptoms using structural equation modeling.
Cross-sectional data were collected from 755 AIDS orphans and 466 children of HIV-positive parents aged 6–18 years in 2006–2007 in rural central China. Participants reported their experiences of traumatic events, perceived HIV-related stigma, perceived social support, future orientation, trusting relationships with current caregivers, and depression symptoms.
We found that the experience of traumatic events and HIV-related stigma had a direct contributory effect on depression among children affected by HIV/AIDS. Trusting relationships together with future orientation and perceived social support mediated the effects of traumatic events and HIV-related stigma on depression. The final model demonstrated a dynamic interplay among future orientation, perceived social support and trusting relationships. Trusting relationships was the most proximate protective factor for depression. Perceived social support and future orientation were positively related to trusting relationships.
We conclude that perceived social support, trusting relationships, and future orientation offer multiple levels of protection that can mitigate the effect of traumatic events and HIV-related stigma on depression. Trusting relationships with caregivers provides the most immediate source of psychological support. Future prevention interventions seeking to improve psychological well-being among children affected by HIV/AIDS should attend to these factors.
The deleterious impacts of racism, sexism and HIV-related stigma on well-being are widely documented, yet most research has examined these forms of stigma separately. Rising HIV infection rates among Black African Caribbean women in Canada underscore the importance of understanding factors associated with quality of life (QOL). We used a feminist intersectional approach to examine the influence of racism, sexism and HIV-related stigma on QOL among HIV-positive Black African Caribbean women in Ontario, Canada.
We conducted a community-based multi-method study triangulating qualitative and quantitative methods. Building on qualitative findings regarding stigma from 15 focus groups with HIV-positive women (n=104) in Ontario, we implemented a cross-sectional survey with HIV-positive Black African Caribbean women in three Ontario cities. Multiple linear regression (MLR) analyses were conducted to measure associations between independent (block 1: racism, sexism, HIV-related stigma; block 2: resilient coping, social support) and dependent (total QOL; QOL domains: physical; psychological; level of independence; social relationships; environment; personal beliefs) variables.
Survey participants (n=163; mean age=40.7 years, SD=8.8) reported frequent/everyday experiences of racism (29.4%) and sexism (22.6%) and high HIV-related stigma (disclosure: 84.4%; personalized: 54.7%; public attitudes 40.4%; negative self-image: 27.6%). In MLR analyses, racism, sexism and HIV-related stigma were associated with lower QOL scores (total; psychological; level of independence; social relationships; environment; personal beliefs). Resilient coping and social support accounted for a significant variance of higher QOL scores (total; psychological; social relationships; environment) after controlling for the effects of racism, sexism and HIV-related stigma.
HIV-positive Black African Caribbean women experience pervasive racism, sexism and HIV-related stigma associated with reduced QOL; social support and resilient coping were associated with higher QOL. Interventions tailored for HIV-positive Black African Caribbean women should aim to strengthen protective factors, such as resilient coping and social support, and challenge stigma and discrimination associated with HIV, race/ethnicity and gender.
Female bar/spa workers in the Philippines face continued risk of sexually transmitted infections (STIs), yet Philippine Congress recently rejected a Reproductive Health Bill. Little is known about differences in condom attitudes among workers in different venues and the effects of social support on their condom attitudes. This study assesses socio-structural and individual factors associated with condom attitudes among female bar/spa workers in the Greater Metro Manila Area, Philippines.
Female bar/spa workers (N=498) from 54 venues underwent interview-led surveys as part of a larger intervention study. Multiple hierarchical linear regression analyses, adjusted for individuals nested within venues, were conducted to assess socio-behavioral (age, education, length of time employed as an entertainer, alcohol, and substance use) and socio-structural (venue type, manager support, peer support, establishment condom rule, condom availability at establishment, and social support) factors associated with condom attitudes.
Participants were aged 18–60 years. Over 90% indicated using condoms every time while having vaginal and anal sex could lower their chances of contracting HIV/AIDS. However, nearly 70% considered condoms too expensive to use regularly. Over 60% said condom usage depended on males in their culture. Over half thought condoms caused pain or discomfort. A like proportion indicated that condom usage never or only occasionally went against their religion. In multivariate analyses, positive attitudes toward condoms were associated with co-worker peer support (0.35, p<.01) and working in spa/saunas vs. night club/bars (1.34, p<.01). Total social support increased the effect of manager support on condom attitudes (0.02, p<.02). Poorer condom attitudes were associated with substance use (−9.66, p<.001).
Socio-structural workplace factors (peer support, social support, and venue type) over individual factors (excepting substance use) influenced condom attitudes. Attention to socio-structural interventions may be necessary to improve condom attitudes among female bar and spa workers, especially those involved in sex work.
In the third decade of the HIV epidemic in the United States, the population of persons living with HIV/AIDS (PLHIV) has aged. While 50 and older was initially created as the “older age” category, that cut-point is no longer informative. Understanding the health status of different adult age-groups of PLHIV will assist in providing the types of healthcare services needed by an aging population.
A convenience sample of 2,182 PLHIV was enrolled from HIV clinics and service organizations in the United States, Canada, Puerto Rico, Namibia, China, and Thailand from February 2010 to July, 2011. This subanalysis of U.S. participants (N=1293) assessed differences in PLHIV in three adult age groups: 40–49 (n=687, 53%); 50–59 (514, 40%); and 60 and older (n=92, 7%).
Participants' mean ages were 44.7, 53.4, & 63.9 years; 71%, 73% & 79% male, no differences in race/ethnicity, 64%, 71% & 87% reported other medical conditions, 81%, 82% & 91% were taking HIV medications, and 52%, 46% & 40% were diagnosed with AIDS. No differences were found in social capital (measured by the Social Capital scale, Onyx & Bullen) or physical functioning (measured by SF-12); there were significant differences in mental functioning (measured by SF-12) in that mental health improved with age (means=43.6, 45.4, & 50.8), depressive symptoms (measured on CES-D) declined with age (means=22.2, 20.5, & 14.8) and treatment self-efficacy (measured on the HIV-ASES) improved with age (means =91.3, 96.8, & 100.5).
The oldest group of PLHIV has better mental health although they are living with multiple comorbidities, perhaps because they view themselves as survivors. Although fewer older persons have AIDS, 91% are taking HIV medications and their treatment self-efficacy is significantly higher than the younger age group (F=9.091, p=.000). It might be time to reexamine the possibility of creating peer groups between middle and older aged PLHIV.
In 2011, the World Health Organization (WHO) recommended that at-risk MSM (reporting unprotected anal intercourse in the last 6 months, partner with STI or multiple partners) should be presumptively treated for asymptomatic anorectal
We assessed the presence genitounirary and rectal symptoms, and determined the prevalence and 3-month incidence of urethral and rectal NG- and CT- infections by NAAT-screening of urine and rectal swab samples collected from at-risk MSM followed in a cohort study in Coastal Kenya. Men with syndromic or NAAT-confirmed received cefixime (400 mg stat) and doxycycline (100 mg, bd, 7 days), risk reduction counseling, and advice on partner treatment.
Of 277 at-risk MSM (97% of total meeting WHO criteria), 38 (13.7%, 95% confidence interval (CI) 9–18) had asymptomatic infections, including 28 (10.1%, 95% CI7–14) who had asymptomatic anorectal NG- or CT. Only 4 (1.4%, 95% CI 0–4) men had symptomatic infections, including 3 that were NAAT-confirmed (2 NG- and 1 NG-CT co-infection). Of 214 at-risk MSM re-screened at a median 93 days (Inter quartile range 84–103), 22 (10.3%) had an asymptomatic NG- or CT-infection, including 11 men who were treated at baseline. The 3-month incidence of any NG or CT infection was 37.0 (95% CI 24.8–55.3); any NG-infection, 12.3 (95% CI 6.2–24.7) and any CT-infection 27.8 (95% CI 17.5–44.1) per 100 person-years.
For every 10 at-risk MSM meeting criteria for presumptive treatment, 1 asymptomatic anorectal infection would be treated in this population. Upon re-screening at 3 months, 1 out of 10 at-risk MSM had asymptomatic NG- and CT- infections. Periodic presumptive treatment every 3 months should be considered for at-risk MSM in the absence of NAAT screening.
Persons living with HIV/AIDS (PLWHA) who acquire new STDs pose a risk for enhanced transmission of both HIV and STDs. Some state and local STD and HIV programs prohibit data sharing that would identify these individuals due to concerns with data security and confidentiality. Significant resources are dedicated to partner services (PS) for syphilis cases however, due to resource limitations, few if any gonorrhea (GC) cases in high morbidity areas receive this intervention.
To describe the frequency of HIV co-infection among gonorrhea cases in 4 cities/regions in the US with varied GC and HIV epidemiology.
A probabilistic method was used to match the HIV and STD surveillance databases at the New York City Department of Health (NYC), Department of Health, Washington DC (DC), Miami/Dade County Health Department (MDC), and Arizona Department of Health Services (AZ). Person and diagnosis events from the matched HIV-STD datasets included 2000–2008.
During 2000–2008, 4.6% (9,471/205,689) of reported GC cases occurred among persons with previously diagnosed (eg. preexisting) HIVNYC (5.5%, 5,930/107,786), DC (7.3%, 1,312/17,910), MDC (4%, 1,504/40,214), and AZ (2%, 725/39,779). Overall, white male GC cases had the highest HIV co-infection in each jurisdictionNYC (22%, 592/2,680), DC (11% 1,000/9,540), MDC (11%, 339/3,080), and AZ (7%, 397/5,501). The overall HIV-GC co-infection rates increased over the study period from 3% (367/12,314) to 7% (752/10,553) in NYC, 6.4% (142/2,211) to 6.7% (155/2,302) in DC, 2% (91/3,917) to 4% (165/4,265) in MDC, and 0.7% (31/4,400) to 3% (91/3,486) in AZ.
Retrospective data integration identified many co-infected HIV/GC cases, and indicated HIV co-infection rates are increasing. Real time access to integrated HIV and STD surveillance would allow better targeting of public health interventions to subgroups of the population posing highest risk for transmitting HIV in their jurisdictions. Local staffing patterns and effectiveness would need to be evaluated to determine the feasibility of interventions such as integrated PS.
A rapid HCV test was recently approved and CLIA-waived by FDA and is now being deployed to identify HCV infection in populations at risk for both HIV and HCV. The presence of HIV co-infection has been reported previously to reduce sensitivity of some HCV antibody tests. We report here on the results of a study of the efficacy of a rapid HCV test in identifying HCV infection in HIV positive and HIV negative cohorts.
A total of 1660 subjects at risk for HCV infection, or with signs and/or symptoms of hepatitis, were prospectively tested by the OraQuick® HCV Rapid Antibody Test using fingerstick blood. Of these, 444 (26.7%) self-reported as HIV positive upon enrollment into the study. Performance of the rapid test in HIV positive and negative cohorts was assessed by comparison with HCV status determined by laboratory testing from a contemporaneous venous blood draw, using an algorithm of EIA, recombinant immunoblot assay (RIBA®) and PCR.
Of 444 HIV positive subjects, 211 (47.5%) were also HCV positive, compared to an HCV seroprevalence of 41.8% (508/1216) in HIV negative subjects. Among HIV positive subjects, intravenous drug use (49.1%) and high risk sexual behavior (39.6%) were the most prevalent risk factors. Agreement between the rapid test and laboratory tests in identifying HCV infected subjects was indistinguishable between HIV positive (98.1%) and HIV negative (98.6%) populations (p=0.629).
The rapid test performed comparably to laboratory tests for prospective identification of HCV infection in at-risk subjects. Sensitivity of the OraQuick® rapid test for HCV antibodies was not compromised in HIV infected individuals. The deployment of a rapid HCV test may expand testing in populations at risk for both HIV and HCV infection and may be an important tool in raising public health awareness of HCV prevalence.
Data on HIV and hepatitis B virus (HBV) and hepatitis C virus (HCV) co-infection among children in Africa are scarce. We evaluated the seroprevalence of HBV and HCV among healthy HIV-uninfected children and HIV-infected children in the Kilimanjaro Region of northern Tanzania.
HBV and HCV markers were assessed on banked serum and plasma samples from HIV-negative children ages 1 month to 18 years and HIV-infected children on highly active antiretroviral therapy (HAART) a minimum of six months from 1 to 16 years of age. HBV markers included hepatitis B surface antigen (HBsAg), hepatitis B surface antibody, and hepatitis B core antibody (HBcAb). Infection was defined as a single positive HBsAg or HBcAb result. HCV infection was assessed by anti-HCV ELISA. Validation studies were performed on all assays prior to use and all were FDA-approved.
Samples from 560 children were available for testing. Of 394 HIV-negative children, 36 (9.1%) were HBV-infected, and of 161 HIV-infected children, 33 (20.5%) were HBV-infected. Children with HIV were 2.6 times more likely to be HBV positive (95% CI 1.53, 4.29) than children without HIV (p=0.0002). None of the 560 samples was positive for anti-HCV antibody.
HBV seroprevalence is high among children in the Kilimanjaro Region, with a significantly higher prevalence among HIV-infected children. Routine screening for HBV should be performed among HIV-infected children. Patients with co-infection require closer monitoring of liver transaminases due to hepatic toxicities associated with antiretroviral therapy, and must be provided with appropriate HAART which will target both viruses. Catch-up immunization with HBV vaccine should be considered for older HIV-infected children.
ince 2000 there is growing evidence that HCV has emerged as an STI among HIV positive MSM. Here we present a 15 year overview of the HCV epidemic among MSM visiting a large STI clinic in the Netherlands.
During waves of the bi-annual cross-sectional anonymous survey (1995–2010), participants were interviewed and tested for HIV and HCV antibodies. Additional HCV RNA tests were performed in all HIV-positive MSM. Determinants of HCV infection were analysed using logistic regression. HCV incidence was estimated using the window period from HCV RNA detection until HCV-antibody development. Phylogenetic analysis of obtained HCV NS5B sequences was performed to determine HCV genotype and to characterise HCV transmission networks among HIV-positive MSM.
Anti-HCV prevalence among HIV-positive MSM gradually increased from 2.8% in 1995 to 3.8% in 2003 and reached its peak in 2008 (17.3%). The HCV incidence was highest in 2006 (14.0/100 PY; 95% CI5.02–37.69) and decreased thereafter, although not significantly. Fisting in 2007/2008 was more strongly associated (aOR 2.62, 95% CI1.20–5.71) with HCV infection than fisting in 2009/2010 (aOR 0.98, 95% CI 0.47–2.03). In addition, Chlamydia, IDU, UAI and age were independently associated with HCV. Phylogenetic analysis revealed a high degree of MSM-specific clustering from 2000 onwards. HCV prevalence among HIV-negative MSM remained stable (around 0.5%, 2007–2010).
HCV prevalence among HIV-positive MSM significantly increased untill 2007, but appears to be levelling off in recent years. This levelling off might partly be explained by increased testing and HCV treatment uptake. The effect of fisting became less strong over time, but both risk factor analysis and phylogenetic analysis continue to support ongoing sexual transmission of HCV among HIV-positive MSM. Monitoring of HCV in both HIV-positive and HIV-negative MSM remains needed to guide prevention in order to halt this epidemic.
Less is known about the correlates of HCV clearance in Chinese injected drug users (IDUs) who are co-infected with HIV. And it remains unknown whether HCV genotypes affect the spontaneous clearance of HCV. This study was designed to determine which factors could significantly affect the clearance of HCV and whether the genotypes could exert different influences on the clearance.
The cross-sectional survey was carried out on 528 HIV-positive IDUs patients in Yunnan Province. Their information on demographic, HIV infection route, HAART, TB and HBV coinfections, CD4+T cell counts, HIV and HCV viral loads, HCV genotypes, alanine aminotransferase (ALT) levels was collected from participants. Logistic regression was performed to identify the correlates for HCV clearance defined as HCV seropositive and RNA negative.
456 out of 528 HIV-infected subjects (86.4%) were identified as HCV seropositive, including 357 (78.3%) HCV RNA positive and 99 (21.7%) RNA negative. The HCV clearance was significantly associated with the presence of chronic HBV infection (p<0.0001), higher CD4+T-cell counts (p<0.05) and was greatly reduced with higher ALT levels (p<0.05). Interestingly, the clearance of HCV genotype 1 was enhanced in higher CD4+T-cell counts (p=0.065), whereas the clearance of HCV genotype 6 were dramatically facilitated by chronic HBV infection (p<0.005), no significant association was identified with the clearance of genotype 3.
Our results suggested that the reserved host immune function and HBV competition could improve the clearance of HCV in HIV-infected subjects whereas the damage level in liver suggested the non-clearance of HCV; For the first time, we demonstrated that the clearances of different HCV genotypes were facilitated by different factors. These data have important implications for the management of HCV/HIV coinfected subjects.
HIV/AIDS among adults 50 and older in Uganda is on the increase partly due to newly diagnosed infections in this age group as well as improved survival owing to antiretroviral therapy, for many HIV infected persons. Although national programmes are in place to ensure access to medical services, older people living with HIV have unique unmet healthcare needs. The objectives of this study were to identify the healthcare needs of older Ugandans living with HIV and to gather recommendations for improvements by health service providers.
Data regarding healthcare needs were collected from HIV positive adults aged 50 years and older attending two large non-governmental outpatient clinics in Kampala and Masaka districts, Uganda between March 2011 and June 2011. Individual in-depth open-ended qualitative interviews focus group discussions were conducted. Observations of clinic interactions were also recorded. Interview transcripts were analyzed using thematic content analysis.
The mean age of the respondents was 65 years, 50% were female, (n=40). Respondents expressed multiple age-related healthcare needs that may differ from their younger counterparts. Needs increased with higher age. Both men and women attributed double stigma from HIV and old age as a major factor affecting disclosure and seeking healthcare for HIV. 60% of the respondents expressed anxiety about their future access to healthcare, the lack of social services and end of life care. Lack of transport and food access issues compromised respondents’ adherence to antiretroviral therapy.
Older people living with HIV have unique healthcare needs which health promotion programmes should consider meeting through appropriate and innovative approaches such as preventing and managing age-related chronic illnesses, palliative care, developing age-friendly services and settings. Research to further explore the impact of these healthcare needs on the quality of life of older PLWHA is required.
The health of prisoners is an issue of global concern. Increased socio-demographic and behavioural risk factors prior to incarceration and poor prison living conditions contribute to increased prevalence of transmissible infections. Little is known about the health of prisoners in Mexico City. This study sought to establish prevalence data and risk factors to identify those currently needing healthcare and to inform future policy.
This cross-sectional study was carried out in 4 Mexico City prisons, June to December 2010. Ethical approval was granted prior to starting; participation was voluntary, confidential and based on informed consent.
Participants were offered HIV, Hepatitis B, C and Syphilis testing. A representative sample completed a questionnaire on socio-demographic characteristics and risk behaviours. Positive results were delivered with counselling and treatment or referral with consent. Data was analysed using Stata.
76.8% (15,517/20,196) men and 92.9% (1,779/1914) women participated. Complete data sets were available for 98.8%. Prevalence of HIV (0.7%), syphilis (Anti-TP Ab 4.1%; VDRL 2.0%), Hepatitis B (HBcAb 3.0%; HBsAg 0.2%) and Hepatitis C (3.2%) was higher in the study population compared with national data. The relative increase was greater for HIV and syphilis amongst women, Hepatitis C in men, and all infections in younger participants. Questionnaire data (1934 men, 520 women) demonstrated lower educational levels, increased smoking and substance use compared to national data. High levels of unsterile tattoing, physical abuse and a history of sexual violence were found.
The study identified that health screening is acceptable to Mexico City prisoners and feasible on a large-scale. It demonstrated increased prevalence of HIV and other infections compared to national data, though low rates compared to international data. Individual participants benefited from earlier diagnosis, treatment and support. The data collected will also enable the formation of improved policy for this vulnerable group of individuals.
After two decades of living with HIV a generation of around 7,000 children from Romania nosocomially infected in communist era, turned into young adults. Hyper protection from the family, combined with discrimination in education services, developing social services, and a focus on medical aspects represented the environment these children grew. Between 2004 and 2010 the Global Fund financed social programs for their professional integration, increasing the percentage of those availing of cash benefits from 30% to 66%. In 2010 people living with HIV (PLHIV) and NGOs protested over ARV treatment interruptions and budgetary cuts due to economic crisis, although WHO estimated the treatment coverage at 83% in 2009.
A nationally representative clinic-based research among PLHIV was carried out in 2011 (March–June) to determine their access to treatment, care and support (N=618, ±4%). Measures of treatment interruptions, adherence and access to services were included, as well as demographics variables. Sample was weighted according to subjects’ surveillance center registration.
The vast majority of the sample (71.7%) is young (18–24 years), with more than 6 years of known seropositivity (82.1%), receiving ARV for more than 6 years (78.7%), with more than 2 changes in their treatment plan (80.1%), and with their main source of income as cash benefits (80.5%). Unintended treatment interruptions were spread (65.2%, average interruption lasting 38 days), resulting in more visits to regional center (49.2% more than once a month), higher expenses and the need for new combinations of ARV. Discontinuous adherence was reported by 42.2%. Access to social services was high (over 80%).
While Romania is a low prevalence country, and ensures high coverage of ARV treatment, authorities must pay attention at continuous treatment access for PLHIV, and support of treatment adherence to prevent deterioration of the health status and ensure universal access for PLHIV.
HIV controllers, maintaining low plasma HIV RNA levels (<2000 copies/ml) in the absence of antiretroviral therapy, are also more likely to spontaneously clear HCV infection. HLAB57, the major histocompatibility class I gene, is highly enriched in HIV controllers and is associated with HCV spontaneous clearance. Whether HLAB57 explains the increased prevalence of spontaneous HCV clearance observed in HIV controllers remains unclear.
Patients in the Study of the Consequences of Protease Inhibitor Era (SCOPE) were tested for anti-HCV using enzyme immunoassay (EIA3) and HCV RNA using discriminatory HCV transcription-mediated amplification assay (Norvatis®). We compared the proportion of HIV controllers and HIV non-controllers with serological evidence of HCV clearance (anti-HCV+/RNA−) by chi-square tests and assessed whether HLAB57 status explains the increased prevalence of HCV clearance in HIV controllers using adjusted prevalence ratio (APR) with Poisson regression models.
Of 279 HIV/HCV seropositive subjects, 48 were HIV controllers. HIV controllers were significantly more likely to have evidence of spontaneous HCV clearance than HIV non-controllers (58% vs. 38%, p=0.01). While HIV controllers were more likely to have HLAB57 than HIV non-controllers, (33% vs. 10%, p<0.01), HLAB57 was not significantly associated with HCV clearance among all participants (39% vs. 55% p=0.06), and there was no evidence of increased prevalence of HCV clearance among HLAB57+ vs. HLAB57- in HIV controllers (p=0.83). In multivariate analyses adjusted for HLAB57, age, gender, and race/ethnicity, HCV clearance was significantly more likely in HIV controllers than HIV non-controllers (APR 1.44; 95% CI 1.04–2.0; p=0.026).
HIV controllers are more likely to spontaneously clear HCV than HIV-non-controllers even when controlling for HLAB57 status. Immunologic factors other than HLAB57 must contribute to the control of HIV and HCV in HIV controllers. Identifying these factors may support the development of novel treatments for and effective vaccines against both viruses.
Evidence for HIV transmission channels, such as between men who have sex with men (MSM), or between MSM and heterosexual females, has traditionally been obtained from epidemiological data. Newer techniques, such as phylogenetic analysis of HIV sequence data, can provide biological evidence for the transmission channels suggested by self-reports and case studies. The objective of this study was to use phylogenetic analyses to characterize HIV transmission channels in Los Angeles County (LAC).
The study population consisted of 1407 LAC cases newly diagnosed with HIV-1 in 2009 and 2010 for whom genetic sequencing data (
Sixteen clusters, representing 49 cases, were identified. Each of these clusters comprised 3–4 cases. All were subtype B. Of the cases within these clusters, 86% were male, 49% were older than 30 years, 47% were Hispanic, 60% were US-born and 67% were MSM. We identified 4 distinct clusters, namely “MSM Only” (56%), “MSM/IDU” (6%), “MSM/ Female or Male Heterosexual” (6%) and finally “IDU/HET female” (6%).
The results of our phylogenetic analysis provide biological evidence for the major HIV transmission channels that have previously been established by traditional epidemiological data. While this method is still relatively new and standardized criteria for cluster identification remain largely undefined, our findings demonstrate that phylogenetic analysis has potential to serve as an additional source of information to validate the descriptions of local HIV epidemics inferred from self-reported behavioral data and case studies.
The high number of recombinants detected in the HIV-1 pandemic can be an indication that infection with multiple subtypes is common. Estimations about the frequency of multiple infections is essential, especially in places where multiple subtypes co-circulate together with a population that may have a high percentage of individuals carrying virus resistant to HAART, which may interfere with treatment to HIV infections and could lead to progression of the disease.
The present study investigated the presence of multiple infections in a population composed of 47 patients undergoing HAART enrolled in the National STD/ AIDS, the Ministry of Health, Brazil. The detection of multiple infections was carried out using a RFLP assay for the protease gene of the virus, which is capable of distinguishing between an infection caused by a single or more than one subtype of HIV-1 according to different patterns of enzyme digestion. Samples exhibiting multiple infections were cloned, sequenced and submitted to phylogenetic analysis for the protease gene of HIV-1.
According to the protease gene analysis we were able to indentify 17 HIV-1 multiple infections out of 47 samples. Multiple infections were analyzed and the majority was composed by recombinant viruses (94%), and only one was composed of pure viruses belonging to subtypes B and F (6%).
This is the first study that reports the prevalence of multiple infections and intersubtypes recombinants in a population undergoing HAART, enrolled in the STD Program sponsored by Brazilian Ministry of Health and, therefore, with free access to antiretroviral drugs. Cases of multiple infections speed HIV-1 genetic diversity rates through recombination, and may help to generate viruses showing a combination of resistance mutation profiles. Considering the patients carrying multiple infections, it may lead to an increased acquisition of drug-resistant isolates.
Sex workers are been arrested by police due to soliciting and selling sex under the vagrancy ordinance in Sri Lanka. There are some reports that sex workers are arrested by police while they are keeping condoms. It has been observed that there were some misunderstandings about condoms and that they are not considered as a medical device. Special training was conducted for training instructors in the police to educate this matter.
Three days participatory-based special training programme were conducted for 206 training instructors to achieve the objective in four instances. The training module was developed and included with various sessions using different education methods including role plays, group work and brain storming sessions. The pre and post test questionnaire was used to assess the difference of knowledge, attitude and willingness to support the national HIV/AIDS plan while handling sex workers
Total knowledge score on HIV transmission, prevention and misconception of the police officers showed significant increase from 69.5(SD+16.1) at pre intervention stage to post intervention stage 86.6(SD +13.08). Total attitude score on handling sex workers with the intention of HIV prevention showed significant increase from pre intervention to post intervention (P= 0.001). Willingness to support the national HIV/AIDS plan while handling sex workers indicated significant increase from pre intervention to post intervention (P=0.0001). All officers have positive attitude towards condoms and knowledge about vagrancy ordinance were improved after the intervention.
The special training was effective to improve the knowledge, attitudes about HIV/AIDS and improve the wiliness to support national HIV/AIDS plan while correct handling sex workers. It is recommended to provide necessary communication materials for training instructors in island wide to carry out continuous education for police officers.
Women's low or unpaid role in providing care for people living with HIV in the form of home-based care while central to the response to HIV been has increasingly been recognised as entrenching unequal gender relationships. Frameworks to map out the pathways have suggested that this process occurs in three domains: financial costs, opportunity costs and physical and emotional costs. Yet such a framework has a tendency to ignore the broader conceptualisation of gender inequalities that include violence against women.
We undertook 5 focus groups with 45 home-based carers in South Africa as part of a larger study focused on how to transform home-based care organisations from spaces of reproducing inequalities to transforming gender relationships. We conducted thematic analysis on the data exploring factors that could support or hinder gender equality.
As with other studies, our data showed home-based carers suffered economic costs related to their work, often spending their own money to provide for their patients. In addition, few received stipends or other financial support either from donors, government or NGOs. Many carers also related the extreme physical and emotional costs of the work they did; again reflecting what is already known about providing care. However, home-based carers also spoke about the central role of gender-based and sexual violence that they feared, risked and experienced both while working and sometimes within the organisations they worked in. Such relationships of violence undermined their ability to envision their work as spaces for women's empowerment and transformation.
To move women's participation in home-based care from one of reinforcing gender inequalities to empowering women requires those supporting home-based carers to envision a wider conceptualisation of the gender inequalities home-based carers face, that includes the centrality of gender- and sexual-violence, and actively work to programme and tackle these.
Psychiatric disorders are prevalent among HIV-infected individuals and may have deleterious effects on HIV care and outcomes. We sought to examine the impact of depression on retention in care and viral suppression among racial/ethnical diverse HIV-infected patients in a large managed care organization.
We evaluated a cohort of HIV- infected adults who received care and had at least 8 months' membership in health care plan in 2010 in Kaiser Permanente Southern California. History of depression prior to 2010 was identified by International Classification of Diseases, Ninth Revision Clinical Modification (ICD-9 CM) codes for depressive symptoms. Retention in care was defined as ≥ 2 CD4 and/or HIV-1 RNA tests at least 90 days apart in 2010. Viral suppression was defined as undetectable viral load or HIV-1 RNA<50 copies/mL at the last test in 2010 while on antiretroviral therapy (ART). In multivariable analyses, Poisson regression with robust error variances was used to estimate rate ratios (RR) and 95% confidence intervals (CI) for retention and logistic regression was used to estimate odds ratios (OR) for failure of viral suppression, adjusted for age, gender, race/ethnicity, mode of HIV infection, ever AIDS diagnosis, and medical center of care.
Among 6,455 patients (90% men, 49% white), 51% had a history of diagnosed depression, and 77% were retained in care. Among 4403 patients on ART with ≥ 1 viral load measurement, 86.3% were virally suppressed. In multivariable analysis, patients with depression were less likely to be retained in care (RR=0.89, 95% CI=0.86–0.91). In stratified analysis by gender, the adverse impact of depression on retention appeared to be larger among women (RR=0.82, 95% CI=0.75–0.89) than among men (RR=0.89, 95% CI=0.87–0.92). Depression was significantly associated with increased risk of failure of viral suppression (OR= 1.25, 95% CI=1.04–1.49). In stratified analysis, depression appeared to be associated with increased risk of failure of viral suppression among women (OR= 2.65, 95% CI=1.30–5.39) than among men (OR=1.15, 95% CI=0.96–1.38), and the adverse impact appeared to be larger among patients who were not retained in care (retained: OR=1.20, 95% CI=0.99–1.45; not retained: OR=1.37, 95% CI=0.83–2.27).
Depression was prevalent in this cohort and worsened retention and viral control. Retention in care may modify the adverse impact of depression on viral suppression. Interventions are needed to alleviate depression and thus enhance retention and improve HIV outcomes.
Previous research (amongst others our own published in AIDS and Social Science & Medicine) has demonstrated gender differences in various antiretroviral treatment (ART) outcomes including, adherence, quality-of-life, disclosure and retention. However, little longitudinal, in-depth research has been done to investigate the why and how of these differences.
The current study firstly aims to explore the impact of masculinity and femininity on ART experiences, in both a longitudinal and in-depth manner. In addition, the study investigates the complex interrelationships between gender constructs whereby males and females potentially influence-through intricate household dynamics- the ART experiences of the other gender.
Based on a previous explorative quantitative study, we conducted a longitudinal qualitative study whereby 12 male and 12 female ART patients were repeatedly interviewed >over the course of 1 year in the Free State province of South Africa.
Despite several cases of lipodystrophy and other side-effects among women due to ART, they seem to adhere and lead healthy lifestyles, often in the absence of a male partner. Male patients reported a clear notion of masculinity that required males to be in control and act strong, highly sexual and economically productive, thus conflicting with the role of ‘good patient’ that is expected to accept being HIV-positive, take instructions, and engage in health-enabling behaviors. The majority of men interviewed overcame these barriers and internalized their treatment, albeit with fewer side-effects and more support in terms of reminding and accompanying them to the clinic (mostly by females).
The study findings have both theoretical and practical relevance. Theoretically, the longitudinal impact of gender and household dynamics on the ART career draws attention to the sociological processes influencing ART outcomes. Practically, the study demonstrates that policy interventions aimed at improving long-term ART outcomes should incorporate the (interactions between) gender-specific illness and treatment experiences.
Asian gay men make up the highest group in MSM/gay HIV notification after Anglo background, constituting a priority group for HIV health promotion in Australia (MHAHS, 2011). Multiple challenges exist as issues of stigma, migration, isolation, in addition to sexual stereotyping and homophobia within the gay and ethnic communities respectively, complicates this work.The immediacy of these issues renders HIV health promotion a second order issue. Underrepresentation in HIV health promotion work, research and general media also contributes to their invisibility.
Multicultural HIV & Hepatitis C Service (MHAHS).
The “A-Men” publication was developed with an aim to engage lived experiences and visibility of Asian gay identities as an entry point to promoting sexual health and wellbeing. The approach was two-pronged: a resource production engaging broader issues facing Asian gay men - the person-centric approach; and the developmental process that involves and builds community ownership - ‘for the community, by the community'. With $5000 community arts funding from Sydney city council, the resource was developed and distributed in-print and online.
A total of 70 volunteers from 9 different Asian ethnicities were involved in the production.1000 hard-copies were launched in March and distributed through community and service providers. 1600 users accessed the online version to date. Volunteer feedback (n=58) showed increased social-connectedness, self-esteem, pride, HIV awareness and knowledge of services. 2 community focus groups (n=62)reported similar trends: greater community engagement and dialogue around wellbeing and self-esteem.
A broader exploration of lived realities was effective in enlivening HIV messaging with Asian gay men. Through engaging intersections of identity, art and community building, a ‘biggest bang for your buck' can be achieved in a resource limited setting. Through cultural change, "A-Men" addressed the barriers to HIV awareness and wellbeing, in the process building a more resilient and empowered community.
Evidence suggests that men who have sex with men (MSM) account for most new HIV infections in Lebanon and act as a bridge to transmission in the general population. In a region where same-sex sexual activity is highly stigmatized, a better understanding of the psychosocial factors that influence sexual risk behavior and HIV testing in MSM is essential for HIV prevention initiatives to be effective.
An exploratory, qualitative study was conducted with a sample of 31 MSM living in Beirut. Semi-structured interviews examined relationships with family and friends, comfort with and disclosure of sexual orientation, sexual behavior, and HIV testing. All interviews were recorded, transcribed, and coded in Atlas-ti to identify themes and extract counts.
All men self-identified as homosexual (77.4%) or bisexual (22.6%), although 32.3% also have sex with women, and a majority (64.5%) reported some discomfort with their sexual orientation. The men described a strong heterosexual social norm, causing some to feel guilty and lead a “double life” where they conceal their sexual orientation by publically feigning attraction or dating women, or dissociating from effeminate men. Comfort with and disclosure about sexual orientation appeared to be linked with sexual risk behaviors and HIV testing. As compared to the remaining sample (n=18), the 13 participants who were both uncomfortable with their sexual orientation and had not disclosed to either parent reported higher rates of unsafe sex (69.2% versus 33.3%), more annual sexual partners (mean=36.7 versus 17.0), and lower rates of being HIV-tested (53.8% versus 88.9%) and discussing HIV risk with their sex partners (38.5% versus 88.9%).
These findings reveal the influence of sexual identity acceptance and disclosure on condom use and HIV testing in the context of high societal stigma, and suggest the need for HIV prevention interventions to facilitate progression through these psychosocial processes of sexual development.
Evidence indicates that MSM experience higher rates of intimate partner violence (IPV) than non-MSM; however, no study has examined what defines IPV among MSM, or the associations between MSM-specific IPV and sexual risk-taking
Seven focus group discussions were held with 84 MSM, generating 30 types of IPV, including HIV-specific IPV. These were tested using a cross-sectional survey of 1,074 MSM. Factor analysis generated five domains of MSM-specific IPV, including a domain of HIV-related IPV. Six logistic regression models for unprotected anal intercourse (UAI) were created (n=626). Models controlled for age, race, education, employment, HIV status, and gay identity, with the key covariate being receipt or perpetration of three different IPV measurements: MSM-specific, Conflict Tactics Scale (CTS), and World Health Organization (WHO).
The MSM-specific IPV definition included HIV-specific typologies of violence not currently included in any definition of IPV, e.g., not disclosing HIV-positivity to a partner, and captured significantly higher reporting of IPV compared to WHO (46.2% v. 13.5% receipt of IPV; 31.9% v. 8.5% perpetration of IPV). Reporting receipt of neither WHO-defined IPV nor CTS-defined IPV was significantly associated with UAI, but reporting receipt of MSM-specific IPV significantly increased odds of UAI (OR: 1.80, 95% CI: 1.27, 2.54). Two models demonstrated a significantly increased risk of UAI among men reporting IPV perpetration (WHO OR: 1.74, 95% CI: 0.91, 3.31; CTS OR: 1.67, 95% CI: 1.06, 2.63; MSM-specific OR: 1.82, 95% CI: 1.26, 2.64).
This study provides for the first time a MSM-specific definition of IPV that captures HIV-related violence not represented in standard definitions. MSM who reported MSM-specific IPV had increased odds of UAI at last sex. These findings suggest that MSM experiencing IPV are at higher risk for HIV seroconversion, although this association may not be recognized by using non-MSM-specific definitions of IPV that do not include HIV-related violence.
Sex workers have endured a high burden of HIV infection across concentrated and generalized HIV epidemics, with a range of interventions implemented to varying degrees of success. A comprehensive, community empowerment-based HIV prevention intervention emphasizes sex worker mobilization to address structural factors related to sex worker rights and HIV risk, and typically includes peer education, condom social marketing, and STI/HIV screening and treatment. Meta-analysis demonstrated a 51% reduction in inconsistent condom use among female sex workers (FSWs) associated with such interventions. We used a deterministic model (Goals) to model the impact on HIV among FSWs and the adult population, when the community empowerment interventions were scaled up among FSWs in Kenya, Thailand, Brazil, and Ukraine.
Models were parameterized with published data or provided by country experts and calibrated against UNAIDS estimations. The intervention was increased from baseline coverage over a 5-year period (5–65% coverage in Kenya and Ukraine; 10–70% in Thailand and Brazil), while other HIV interventions were held constant. Impacts are observed from 2012–2016 and compared to status quo, when all interventions are held constant.
Increasing intervention coverage among FSWs in Brazil averted 1,830 infections among FSWs and 4,740 among adults between 2012–16, compared to status quo. Increased coverage averted a cumulative 10,800 FSW and 20,680 adult infections in Kenya. In Thailand, 220 FSW and 730 adult infections were cumulatively averted. A cumulative 2,220 infections among FSWs and 6,920 infections among adults are averted with increased coverage in Ukraine. Impacts vary and were influenced by HIV prevalence in different risk groups, risk behaviors, and population size.
The community empowerment intervention for FSWs demonstrated impacts among sex workers and the adult population epidemics across these distinct settings. Findings confirm the centrality empowerment in prevention strategies, as the intervention is rights affirming and may require fewer resources, compared to other interventions.
Clients of female sex workers (FSW) is key group in India's HIV prevention programme due to their bridging role in HIV transmission to women in the general population. With limited evidence on intervention effects, we evaluate the impact of Avahan's behaviour change communication strategy on consistent condom use among clients of FSW.
We analyzed data from 2009 Integrated Biological and Behavioural Assessment survey among clients of FSWs sampled at hotspots using time-location-cluster-sampling in Andhra Pradesh, Maharashtra and Tamil Nadu (n=5045). We used propensity score matching to estimate the impact of media messages on clients’ consistent condom use with all partners (except their spouse); having heard/seen messages on condoms and on STIs were considered as the exposures. We stratified the analyses by usual pick-up place (street or brothel).
Clients of street-based FSWs were older (31 vs 29.5 yrs, p<0.05) and better educated (77% vs 61% secondary and above, p<0.001) than clients at brothels. Exposure to messages on condoms (95%) was too high to detect an association with condom use. Exposure to STI messages was 77% among street-soliciting clients compared to 51% among those frequenting brothels. Only among the brothel-soliciting clients we could demonstrate an effect44% of exposed clients used condoms consistently vs 30% among the matched controls (p<0.001). The observed differential is most likely due to the local environmentmore intense and varied exposure to safer sex messages at brothels including stronger interpersonal communication with FSWs more effectively negotiating condom use.
Media messages on STIs are not sufficient to change clients’ behaviour. With no effect among the more educated clients of street based sex workers, the results show that without an enabling environment, clients will not act on information received. More needs to be done to reach clients of sex workers, especially those of street-based FSWs.
Although combination prevention strategies are receiving growing attention, there is little evidence to inform their implementation, particularly for injection drug-using (IDU) populations. We constructed a complex systems dynamic model to assess various strategies for reducing HIV transmission among IDU.
We modeled HIV transmission in a dynamic network of IDU and non-IDU over a thirty-year time period (1992–2021). In the model, “agents” engage in risk behavior and interact with simulated prevention interventions (i.e., needle and syringe exchange programs [NSP], HIV testing, antiretroviral treatment [ART], substance abuse treatment). The model was constructed to represent the adult New York metropolitan statistical area (MSA) population, and calibrated by comparing HIV prevalence and incidence against historical validated MSA-level data. We obtained annualized incidence estimates from Monte Carlo simulations to examine the consequences of different intervention scenarios on a hypothetical population of 150,000.
The model closely approximated published 1992–2011 data for HIV prevalence and incidence among IDU. Under current scenarios, HIV incidence among IDU residing in the New York MSA is estimated to be 3.7 per 1000 (95% CI1.0–6.3 per 1000) in 2021. Scenarios in which coverage of only one intervention was increased resulted in decreased HIV incidence at 2021, with expanded NSPs showing the lowest incidence rate (2.4 per 1000), followed by increased substance abuse treatment availability (2.8 per 1000), earlier initiation of HAART and improved adherence (2.9 per 1000), and increased access to HIV testing (3.5 per 1000). Combining all scenarios resulted in the largest absolute reduction in HIV incidence (1.7 per 1000, 95% CI0.0–3.9 per 1000) by 2021.
Our results demonstrate that combination interventions have the greatest potential to reduce HIV transmission among IDU. Although further research is required to determine cost-effectiveness, combining and bringing to scale existing evidence-based interventions may well be a highly effective strategy to reduce new infections.
The recent HIV Prevention Trial Network (HPTN) study052 strengthens the evidence base for HIV treatment as a prevention strategy. We developed an operations research model of HIV prevention, with conservative effectiveness assumptions, to assist decision-making by public health authorities. We evaluated whether focused deployment of the “test and treat” strategy (i.e., TLC-Plus) among various populations would be cost-effective.
Using an epidemic compartmental model of HIV transmission, we simulated implementation of TLC-Plus strategies, including immediate anti-retroviral therapy (ART). We evaluated the impact of deployment among different populations. Outcomes included the number of infections averted over 20 years compared to the base case (no intervention) and the cost per infection averted. Base case assumptions regarding HIV testing and the proportion infected but undiagnosed were varied as a sensitivity analysis. We assume that a baseline proportion (89%) of HIV-infected persons are diagnosed; we make realistic assumptions about linkage to care, adherence, and retention in care.
Generalized implementation of a TLC-Plus strategy averted 19% of new infections (from 59,109 to 47,690) over 20 years with a cost-per-infection averted of US$1.56 million. Focused deployment of the prevention package to specific populations reduced the absolute number of infections averted but often resulted in a program with favorable value, defined as a cost-per-infection averted<US$360,000. A TLC-PLUS intervention with a budget of approximately $22 million per year could prevent 5% of infections over 20 years (compared to base case), whereas a budget of ~US$1 billion per year would be required to attain the maximum prevention impact.
Focusing TLC-Plus strategies on specific high prevalence populations or neighborhoods may be more favorable than generalized implementation given the lower cost per infection averted and current budget limitations. The model supports ongoing efforts to focus scale-up of TLC-Plus and early ART initiation for all persons newly-diagnosed with HIV.
Preexposure prophylaxis (PrEP) is a promising biomedical strategy to reduce HIV transmission. However, many caveats such as the potential risk of sexual disinhibition and non-compliance need to be considered. The objective of this survey was to explore the sociodemographic and behavioral factors associated with the adoption of PrEP among both MSM and heterosexual seronegative partners.
Pre-piloted self-administered survey was conducted among seronegative partners in a Ryan White Clinic in South Carolina from 2010–2011. Bivariate and multivariable analyses were used to explore the data.
A total of 89 seronegative partners completed the survey. The median age was 42 years (IQR32–50) and a majority were males (56%), blacks (70%) and heterosexual (74%). A majority of respondents were willing to use PrEP, if available (94%); however, 26% suggested that they would be more likely to have unprotected sex with HIV-positive partner while using PrEP and 27% suggested that it will be difficult to take daily dose of PrEP and consistently use condoms as well. Multivariable results suggested that ‘
There is high acceptability among seronegative partners for PrEP. However, there is a substantial risk of sexual disinhibition and non-compliance while using PrEP that may be reduced by ongoing education.
HIV transmission among injecting and non-injecting drug users (IDU, NIDU) is a significant public health problem. Continuing propagation in endemic settings and emerging regional outbreaks have indicated the need for comprehensive and coordinated HIV prevention. We describe the development of a conceptual framework and calibration of an agent-based model (ABM) to examine how combinations of interventions may reduce HIV transmission among drug-using populations.
A multidisciplinary team of researchers from epidemiology, sociology, geography, and mathematics developed a conceptual framework based on prior ethnographic and epidemiologic research. An ABM was constructed and calibrated through an iterative design and verification process. In the model, “agents” represent IDU, NIDU, and non-drug users who interact with each other within risk networks, engaging in sexual and, for IDUs, injection-related risk behavior over time. Agents also interact with simulated HIV prevention interventions (e.g., syringe exchange programs, substance abuse treatment) and initiate antiretroviral treatment (ART) in a stochastic manner. The model was constructed to represent the New York metropolitan statistical area (MSA) population, and calibrated by comparing output trajectories for various outcomes (e.g., IDU/NIDU prevalence, HIV prevalence and incidence) against previously validated MSA-level data.
The model closely approximated HIV trajectories in IDU and NIDU observed in New York City between 1992 and 2002, including a steady decrease in HIV prevalence among IDUs. Exploratory results are consistent with empirical studies demonstrating that the effectiveness of a combination of interventions, including syringe exchange expansion and ART provision, dramatically reduced HIV prevalence among IDUs during this time period.
Complex systems models of adaptive HIV transmission dynamics can be used to explore the collective benefits of hypothetical combination prevention interventions. Future work will seek to inform novel strategies that may lead to more effective and equitable HIV prevention strategies for drug-using populations.
Monitoring and evaluation of prevention programs in urban centers often disregard population transience and demographic shift. These factors are often confounding variables, and need to be properly addressed to form a clear picture on intervention efficacy over long periods of time. In DC, we investigate the efficacy of prevention programs as a factor of HIV/AIDS rates in the city, to determine the proportion of rate change in HIV attributed to gentrification alone.
Ordinary Least Squares spatial regression was used to determine the relationship between HIV/AIDS rates in each neighborhood by gentrification index. Other explanatory variables such as mode of transmission and ethnicity were excluded from the analysis due to high levels of spatial autocorrelation. The resulting regression was a bivariate comparison between gentrification and HIV/AIDS rates. Mapping and data analysis were conducted in ArcGIS© and R™ respectively.
676 cases (representing 64% of all cases in 2010) were mapped across 39 neighborhoods. The bivariate model created by Ordinary Least Squares spatial regression reported a R2 value of 32%. The correlation coefficient of r=0.51 shows a significant positive association between gentrification and HIV rates (p<0.05).
Approximately 31% of the variation in neighborhood rates of HIV/AIDS in DC can be explained by gentrification and demographic shift alone. Thus, gentrification is a significant factor in explaining how HIV/AIDS rates vary by neighborhood. The findings underscore the need to consider gentrification when using M&E to advise programmatic priorities to accurately how public health interventions affect new diagnoses. Next steps will involve including suburban data to track HIV/AIDS outcomes due to gentrification in the metropolitan area.
Globally, YMSM are uniquely vulnerable to HIV. Many are dependent on family that may not understand or accept their sexuality, forcing them to hide their sexual behavior or risk losing housing and financial support. YMSM also may have limited access to information regarding sexual health and legal rights. Despite this vulnerability, YMSM's distinct needs are often overlooked by efforts to address HIV among general youth and general MSM.
In 2010, the Global Forum on MSM & HIV (MSMGF) conducted a global online survey of MSM and their service providers. Among 5,066 survey participants, 1,488 were YMSM (age 30/younger). YMSM respondents were from: Asia (65%), Latin America (10%), Australasia (9%), North America (6%), Europe (4%), and Africa (3%). Chi-square and Wilcoxon rank-sum were used to evaluate differences between YMSM and other MSM. Multivariable regression was used to identify predictors of access to HIV prevention services.
HIV-prevalence among YMSM was 14%. Most YMSM (67%) had two or more sexual partners in the past year; 33% were homeless/unstably housed. Compared to other MSM, YMSM reported lower “easy access” to evidence-based HIV prevention strategies: free HIV testing (53% vs. 37%[p<0.001]), condoms (47% vs. 36%[p<0.001]), lubricants (33% vs. 21%[p<0.001]), HIV behavioral interventions (38% vs. 21%[p<0.001]). Compared to other MSM with HIV, YMSM with HIV reported lower “easy access” to antiretroviral medications (59% vs. 33%[p<0.001]). YMSM also had significantly higher levels of perceived homophobia (p< 0.001) and internalized homophobia (p<0.001) than other MSM. Perceived homophobia was the strongest predictor (0.38; 95% CI=0.3–0.4) of compromised access to HIV prevention services among YMSM.
YMSM are at increased risk for HIV due to a number of factors. Limited access to HIV services and high levels of homophobia may exacerbate vulnerability. Programs must be developed to address the unique needs of YMSM.
In India, many MSM either are married or expect to marry. The present study aimed to identify strategies for the national HIV programme to promote the health of women partners of MSM both in terms of preventing HIV transmission and in promoting early HIV diagnosis and treatment.
Qualitative field research used a collective case study design to collect data in 2010/11 from 11 sites in seven states among 401 MSM (HIV prevention outreach workers and beneficiaries) through 57 focus groups (n=364; 46% married) and 37 key informant interviews. Women partners of MSM could not be recruited for the study. Potential interventions proposed are based on the inferences drawn by synthesising both the literature review and qualitative data (thematic analysis).
Both literature review and qualitative data showed that women partners of MSM can include wives, casual, regular, paid or paying partners, and they may be at higher risk of HIV due to their male partners' high-risk sexual behaviours, which include having a large number of male and/or female partners combined with inconsistent condom use. Field data revealed that self-identified MSM may enter into heterosexual marriage with willingness, under compulsion (e.g., family pressure) or with indifference. Importantly, qualitative data offer new insights into whether disclosure of sexuality and/or HIV status among MSM is significant in preventing HIV transmission to female partners.
HIV prevention messages aimed at MSM ought to explicitly promote awareness of the risks/vulnerabilities faced by female partners. Such strategies ought to particularly include counselling and HIV testing approaches that address the complexities of safer sex practices with both male and female sexual partners. Developing interventions for single MSM that raises awareness of the HIV risks faced by their women partners and implementing interventions that explicitly target married MSM and their wives are key to effective, future HIV prevention programming.
Few studies have documented female sex workers' (FSWs) efforts to engage in safe behaviors in the context of their multiple identities with sexual partners in India. Recognizing the intricacies of women's perceptions of risks and benefits within diverse relationships and moving beyond condom promotion are crucial for HIV prevention.
An exploratory qualitative study was conducted with female sex workers in India. Sixty FSWs participated in 5 focus groups and 25 semi-structured in-depth interviews in addition to completing a short questionnaire that captured basic socio-demographic information.
The majority (60%) of FSWs was based in rural areas and was Hindu (90.3%). Respondents' mean age was 30.2 years, with an average of 6.4 years of education. Only 28% reported being legally married or living with a partner and 82% had at least one child. Further, for 58%, sex work was the main income source. Findings implied a complex interplay of factors that determined FSWs' abilities and willingness to avoid unsafe sexual behaviors with paying clients, regular/trusted clients, and non-paying intimate partners. Often, this was dictated largely by economic hardships, emotional attachments, social norms, perceived lack of power as well as the threat of violence and to a less extent by the knowledge of HIV risk. Further, discrimination and exploitation by community members including police combined with the need to hide their sex worker identities often put FSWs in situations that compromised their efforts to avoid risky behaviors.
A deeper understanding of the multifaceted exchanges between the various partner types that heighten FSWs' vulnerabilities is critical when planning HIV prevention interventions in addition to adopting a human-rights framework and recognizing women's agency in safeguarding health. Challenging popular notions of sex workers as undeserving of substantive support and individual rights would go a long way in ensuring the effectiveness of HIV prevention efforts.
Male partners of female sex workers (FSW) have traditionally been viewed as holding power and decision-making authority regarding condom use due to the transactional nature of their relationships. The aim of this study is to explore relationship and gender dynamics between FSW and their regular partners and consider how these dynamics influence HIV vulnerability.
We conducted qualitative interviews with FSW (n=20) and regular partners (n=20). FSW were recruited from an ongoing cohort study and regular partners were referred by non-participating FSW in the cohort. Regular partners were defined as trusted and/or intimate partners with whom the referring FSW reported sex with at least 4 times in the last 3 months. We used narrative and thematic analysis to describe the relationship history and identify emergent themes.
Both FSW and regular partners described emotional, sexual, and economic attachments to each other. Regular partners, however, described greater emotional and sexual attachment. While most men provided some economic support to their FSW partner, they were aware that due to their limited economic resources, they were not able to provide for all of their partner's needs. In contrast, FSW displayed more pragmatic attitudes, emphasizing the importance of both the material and emotional support provided by regular partners. Some FSW clearly stated that while they had the strongest emotional attachment to their regular partner, they had to maintain other partnerships in order to survive.
Findings challenge hegemonic norms of masculinity between FSW and their regular partners and highlight the intersection between socio-economic position and relationship power. HIV prevention efforts targeting FSW and regular partners must address both emotional and socio-economic dimensions of relationships and their influence on HIV protective behaviors.
For some women who engage in female sex work (FSW) life remains bound up in the work as they age. We explored the reasons for entering and staying in FSW through life histories of older women (35 years and above) at high risk of HIV in Kampala, Uganda.
100 women were purposively selected from a cohort of 1027 high-risk women. Life history interviews and in-depth interviews on their life and work, including sexual practices, were conducted with each woman. Data were analyzed using thematic content analysis. Informed consent and ethical clearance were obtained.
Sixteenwomen were aged over 35, and had joined sex work as young adults usually because of financial need. They continued to take an active part in sex work. These women perceived sex work to be addictive, because of the quick access it could give to cash. Some women confided that they found their continued sex work humiliating and they really wanted to give up because they were too old and they feared that their children might find out what they did. However, faced with recurrent economic demands, paying children's school fees and buying food, these women had few alternatives. Most wished for a ‘husband’ who could help them. In the absence of such support or alternative livelihoods, giving up FSW was not an option. As they aged, they commanded lower rates from clients. Some augmented their living by selling alcohol and providing labour in the community, including child care to younger sex workers. All these women were at risk of HIV, STIs and sexual violence.
Sex work remains a key structural driver of HIV among some vulnerable older women. Sexual violence and high risk behaviour characterized sexual relations. There is an urgent need for interventions to support such middle-aged and older women.
Findings from recent clinical trials evaluating the efficacy of antiretrovirals for pre-exposure prophylaxis (PrEP) against HIV among women are mixed, and none has included U.S. women. In addition, there is a dearth of research on knowledge, attitudes, and likelihood of use of PrEP among women in the U.S., who comprise 27% of new infections in the country. Thus PrEP′s potential as an HIV prevention strategy for American women is unknown.
We conducted a focus group study with 92 women in 4 U.S. cities between March and April 2012. Participants were recruited by local, woman-serving CBOs that also hosted the groups, which were led by trained facilitators. Participants completed a questionnaire with demographic and behavioral information. Focus group questions elicited basic understanding of PrEP, attitudes about its administration and uptake, barriers to use, and targeting and marketing. All sessions were digitally recorded and transcribed, and transcripts were analyzed to identify predominant themes and any demographic or site differences.
Across sites, almost no women had heard of PrEP prior to the study; but once informed about it, nearly all found it an important option. Key considerations in deciding to take PrEP were cost (including who would pay), efficacy (most wanted 85–99%), and side effects (including interaction with contraceptives). PrEP was seen as "additional," not substitute, protection to condoms. Young women were the most commonly named target population, and schools the best venue for informing them. Mistrust of men was a main motivator for PrEP among women in Oakland and Washington; and churches were seen as a key barrier to PrEP in Memphis.
Women at risk for HIV in the U.S. are likely to use PrEP if it is known to be highly effective, doesn′t cost too much, doesn′t have significant side effects, and is promoted by peers and CBOs.
HIV prevalence among fishermen in Malaysia is ten times that of the general population. Combining Rhodes' “risk environment” framework to assess contextual features influencing HIV risk and Berkman et al.'s comprehensive model of the mediating effects of social networks on health, this paper assesses the social relationships among drug-using fishermen in Malaysia as a key axis along which HIV risk decision-making occurs.
Data were collected in Kuantan, one of the busiest fishing ports on the East coast of Malaysia and from two nearby fishing villages. Twenty-eight fishermen reporting drug use during their last fishing trip or upon returning to shore participated in in-depth interviews. Fishermen were selected based on the type of fishing vessel they worked on (traditional vs. commercial) and gear used (hook/line vs. trawlers/purse seiners) to draw upon different social networks and spheres of influence. Data were coded thematically and analyzed to inform key research questions around drug use and aspects of social networks.
Economics, occupational culture, punitive policies and stigma shaped the social networks of drug-using fishermen. Social networks, in turn, shaped HIV risk perceptions and behavior through the following pathways: 1) social influence - through the introduction to drugs, male homosociality, and peer pressure; 2) social support - drug users relied on other drug users for information and emotional needs; 3) social engagement - other drug users provided a sense of community and shared identity; and 4) access to resources and goods, like drugs.
These results suggest that aspects of social networks both constrain and enable HIV risk behavior among Malaysian fishermen by mediating the relationships between social structures and individual behaviors. Understanding the role of network influence in drug use and HIV risk among fishermen is essential to developing HIV prevention interventions that are appropriate for the unique needs of this highly interconnected population.
Drug users often use multiple substances and are involved in sexual risk behaviors, and thus are at high risk of sexually acquiring or transmitting HIV. Studies on typologies of drug use are limited, especially those that examine the relationship between typologies of polydrug use and unsafe sex practices.
Using respondent driven sampling, 248 rural drug users were recruited in rural areas of the Midwest. They participated in a longitudinal, natural history study of illicit drug use and health service utilization. Latent Class Analysis was used to identify a typology of poly-drug use and assess differences in sexual risk for HIV transmission between these classes.
All stimulant users consumed multiple drugs in the previous 30 days and many (63.7%) reported unsafe sex (condomless sex). Three latent classes were identified: Class 1 (N=107): all used powder cocaine, 90% used marijuana, 42.2% used crack-cocaine, 23.0% used heroin, and few used other stimulants and methamphetamine; Class 2 (N=67): all used crack-cocaine, 72.3% used marijuana, 20.8% used powder cocaine, 90.0% used heroin, few used other stimulants, and no methamphetamine; Class 3 (N=74): all used methamphetamine, 92.6% used marijuana, 74.2% used crack-cocaine, 88.1% used power-cocaine, 17.7% used heroin, and 49.1% used other stimulants. Although the patterns of drug use practice among the classes vary, the prevalence (64.5%, 67.2% and 59.5%, for Classes 1, 2, and 3, respectively) of unsafe sex practices was not significantly different across the classes (p=0.6213).
Rural stimulant users can be classified into three latent classes: primary cocaine users, primary crack users, and heavy multiple stimulant users. High prevalence of unsafe sex practices was reported across all classes. While much attention has been given to HIV risk reduction efforts among urban stimulant users, HIV prevention and intervention efforts for rural stimulant user are still greatly needed.
Accurate methods for estimating HIV incidence are needed to track the epidemic, identify high-risk groups, and evaluate HIV prevention interventions. We evaluated a multi-assay algorithm (MAA) for identifying recent HIV infections and estimating population-level incidence.
We analyzed 1,782 samples from 709 adults in the United States with known duration of HIV infection (0–8 years post-seroconversion; HIVNET 001, MACS, and ALIVE cohorts). The MAA included the BED capture immunoassay (BED-CEIA), an antibody avidity assay, HIV viral load, and CD4 cell count. Logistic regression with cubic splines was used to model the probability of being classified as recently infected by the MAA. We calculated the window period of the MAA (mean duration of time that individuals were classified as recently infected). We compared the accuracy of the BED-CEIA and the MAA for identifying recent infections. In the HIVNET 001 Vaccine Preparedness Cohort, we directly compared the MAA incidence estimate (cross-sectional analysis of samples from the 18-month visit), to the observed incidence based on analysis of HIV seroconversion during follow-up.
The MAA window period was 141 days (95% CI 94–150 days). Among individuals infected >1 year, 17.2% and 0.4% of 1,474 samples were misclassified as recent by the BED-CEIA and MAA, respectively. Among individuals infected >5 years, 13.6% and 0% of 345 samples were misclassified as recent by the BED-CEIA and MAA, respectively. In HIVNET 001, the annual incidence estimate based on the MAA was 0.97% (95% CI0.51%–1.71%), which is essentially identical to the incidence observed in longitudinal follow-up of the cohort (1.04%, 95% CI0.70%–1.55%).
The MAA is sensitive for detecting recent HIV infection, has a low rate of false-recent misclassification, and accurately estimated HIV incidence in a cohort study. The MAA is potentially a powerful tool for determining HIV incidence in clade B epidemics.
Swaziland has the highest estimated national HIV prevalence rates in the world. The Swaziland HIV Incidence Measurement Survey (SHIMS) provides the first national-level HIV incidence estimates based on prospectively observed seroconversions among participants in a population-based longitudinal cohort.
A nationally representative sample of men and women, age 18–49, underwent household-based, rapid HIV testing from December 2010–June 2011, including counseling to reduce HIV infection risk. Socio-demographic and behavioral characteristics were also assessed through a questionnaire survey. HIV-uninfected individuals were invited to enroll in a cohort and retested for HIV and surveyed again approximately six months later. Longitudinal incidence was calculated as events/person-years [PY]×100%.
A total of 18,154 men and women, representing approximately 7% of the adult population, were surveyed, and 11,944 tested HIV-negative and enrolled in the cohort. Among these, 10,949 (91.7%) were retested for HIV after a mean follow-up of 6.5 months, resulting in 6,054 PY of observation. There were 146 seroconversions, resulting in a weighted, national HIV incidence estimate of 2.4% (95% CI, 2.1–2.7%). Incidence was higher in women (3.1%) than in men (1.7%), overall, and was highest among women 20–24 yrs (4.2%) and men 30–34 yrs (3.1%).
The highest HIV incidence rate (4.2%) in Swaziland is among women 20–24 yrs. Among men, the peak HIV incidence rate (3.1%) occurs among those 10 years older. Study-related risk reduction counseling may have extraneously altered participants′ risk behaviors and affected the observed incidence rates. These findings reinforce the need for effective HIV prevention interventions, such as voluntary medical male circumcision, which provides a direct protection against HIV acquisition for men and an indirect protection for women. As HIV prevalence may remain high independent of HIV incidence rates, efforts to diagnose and treat persons already HIV-infected with antiretroviral therapy, are also paramount to curb Swaziland′s severe HIV epidemic.
HIV in the United Kingdom is concentrated in distinct population sub-groups. Monitoring changes in transmission patterns among these groups informs prevention and testing efforts. We present HIV new diagnoses and alongside novel incidence surveillance data in England and Northern Ireland.
National surveillance of new diagnoses in adults in England and Northern Ireland between January 2009 and June 2011 linked an avidity test. The Recent Infection Testing Algorithm (RITA) utilises the avidity result adjusted for baseline CD4 count and AIDS diagnosis to identify infections that probably acquired in the preceding 6 months.
14,682 adults were diagnosed with HIV over the period45% were men who have sex with men (MSM), 51% were heterosexual, and 2% were IDUs. One in ten was aged 15–29, 33% aged 25–34, 42% aged 35–49, and 15% aged 50+. Over the 30 months, new diagnoses in MSM increased as heterosexuals declined. A RITA result was available for 33% (4,877) of new diagnoses (these persons had similar demographic characteristics compared to those not tested). Overall, one in six (15%) had recently acquired their infection. Recent infection was more common in younger adults (25% persons aged 15–24 and 20% persons aged 25–34). MSM had the highest proportion of recent infections (23%), followed by heterosexuals (10%) and people who inject drugs (4%). One in three (31%) MSM aged under 35 acquired their infection recently, compared to one in seven (13%) aged over 50. Among heterosexuals, 20% of women aged 15–24 and 15% of men aged 25–34 had recently acquired their infection.
Surveillance data on new HIV diagnoses coupled with results from RITA indicate high rates of ongoing transmission among MSM, particularly in young MSM. Transmission patterns among young heterosexuals must also be observed closely and in the context of testing patterns.
US CDC guidelines recommend at least annual HIV testing for those at high risk. Nonadherence to testing guidelines and late diagnosis of infection may contribute to HIV transmission.
HPTN 061 is a feasibility study of a multi-component HIV prevention intervention for at-risk black MSM in 6 US cities. At enrollment, participants were offered HIV testing. Participants reporting past HIV-uninfected or unknown status at enrollment and no HIV testing within the prior 12 months were considered nonadherent to HIV testing guidelines. Participants with newly diagnosed HIV and CD4 <200 at time of diagnosis were considered to have late diagnosis. Predictors of nonadherence to testing guidelines and late diagnosis were analyzed using logistic regression models and Fisher′s exact tests. HIV test data are from baseline testing performed at study sites; confirmatory testing is underway.
HPTN 061 enrolled 1553 black MSM. At enrollment, 1384 reported no prior HIV diagnosis, and 1335 (96%) of those participants agreed to testing; 98% were male, 3% transgender, 100% black, 8% Latino, with a median age of 39 years; 38% reported incomes below US poverty level, and 67% were unemployed. Among those tested, 309 (23%) reported no HIV test in prior 12 months; testing nonadherence was highest in Atlanta (30%), lowest in DC (18%) and Boston (17%); 185 (14%) reported never testing prior to enrollment. Nonadherence to HIV testing guidelines was independently associated with age ≥35, (AOR 1.96 [95% CI 1.40–2.75]), unemployment (AOR 1.47 [1.05–2.05]), and living in Atlanta (AOR 1.85 [1.07, 3.21]). 167 (13%) participants had a new HIV diagnosis at enrollment; median CD4 was 445 at diagnosis. Of 157 (94%) with CD4 data, 28 (18%) had a late HIV diagnosis.
Nonadherence to HIV testing guidelines, undiagnosed infection and late diagnosis were common, underscoring the need for additional HIV testing and prevention efforts for this population.
Bangladesh is thus far regarded as a low HIV prevalence country with increased vulnerability due to several risk factors including high demand for commercial sex. Male clients of female sex workers (FSWs) are considered to be a potential bridge population to transmit HIV and other sexually transmitted infections (STIs) to the general population. The objectives of this paper are to describe the correlation between illicit substances (ISs), unsafe sexual behavior and self-reported symptoms suggesting STIs among male clients of FSWs in Bangladesh.
A cross-sectional study was conducted among 1565 male clients of FSWs during November 2005 to July 2006 in three sex trade settings (streets, hotels, and brothels) in Bangladesh. No personal identification of the clients was recorded and only verbal consent was taken in the structured questionnaire administered by male interviewers. Bivariate and multivariate data analysis was performed.
Among the male clients of FSWs, 36.2% aged ≤24 years, nearly 30% never attended school, and above one-half (54.6%) of them were never-married. Nearly one-third (30%) used ISs. Cannabis (60.6%), alcohol (51.9%), heroin (6.8%), sleeping pills (5.7%), and injecting drugs (1.5%) were used by these clients. Consistent condom use with non-marital sexual partners in the last one month from the interview date was reported by only 13.2% of those who used ISs. Almost one-half(45%) of the IS-users experienced the symptom of STIs in the last 12 months prior to the interview date. The IS-users were 1.7 times more likely to have symptom of STIs than the non-users (adj. OR=1.70, 95% CI: 1.29–2.20).
Use of ISs was a significant risk factor for having symptom of STIs among the male clients of FSWs and they are less likely to use condoms consistently during non-marital sexual relationship. There is an urgent need to implement targeted intervention to promote safer sex behavior for male clients of FSWs in Bangladesh, especially those who use ISs.
Injecting drug use has emerged as an important route for HIV transmission in India. The Government of India currently estimates that there are 200,000 PWID (or IDUs) in India (NACO, 2010), though other studies indicate the numbers could be much higher. Surveillance studies estimate HIV prevalence among PWID at 9.2% (NACO, 2010). Understanding the patterns of drug use and other behaviors by PWID is critical to successful intervention design. India HIV/AIDS Alliance conducted a baseline with PWID in three states (Delhi, Manipur and Haryana) on drug use and behavior patterns, as part of the Hridaya project, the India component of the five-country, Dutch government-funded Community Action on Harm Reduction program.
A cross sectional survey was conducted with PWID in three states. 181 respondents were selected through systematic random sampling using client information made available by partner NGOs at selected sites. Data were analyzed using SPSS software.
60% of PWID interviewed have injected drugs for more than 5 years; 32% inject daily and 33% at least once a week in the last 30 days. 22% of PWID have injected using needles or syringes previously used by another person. In the last 12 months, 55% of respondents had sexual intercourse with regular partners; 22.9% with casual partners; and 26.6% with commercial sex partners. During last sexual contact, 70% used condoms with regular partners; 64% with casual partners; and 51.7% with commercial sex partners respectively. Of the 100 respondents who disclosed their HIV status, 21% reported being HIV-positive.
Unsafe injection practices along with low condom usage are putting PWID at dual HIV risk. These findings support the development of focused prevention strategies that address both injection drug use and sexual behavior as part of Hridaya's harm reduction approach.
Thailand continues to experience dual epidemics of injection drug use and HIV/AIDS. In response, Thailand has relied on heavy-handed enforcement-based approaches. Although policing is known to contribute to the production of drug-related harm among people who inject drugs (IDU), the impact of police violence on HIV risks among IDU has not been fully characterized.
Using multivariate logistic regression, we identified the prevalence and correlates of experiencing police violence among IDU who participated in the Mitsampan Community Research Project (Bangkok) in 2009 and 2011.
Among the 637 unique IDU who enrolled in the study between June 2009 and October 2011, 153 (24.0%) were female, and the median age was 37 years. In total, 239 (37.5%) participants reported having ever been beaten by police. In multivariate analyses, exposure to police beatings was independently associated with male gender (Adjusted Odds Ratio [AOR]=9.25), younger age (AOR=1.65), study enrollment in 2011 (AOR=1.95), obtaining income from illegal sources (AOR=2.12), a history of imprisonment (AOR=4.52), syringe sharing (AOR=1.91) and overdose (AOR=1.73) (all
A high proportion of community-recruited IDU participating in this study reported being beaten by police, and this form of police violence appears to have increased in recent years. Further, experiencing police violence was independently associated with indicators of drug-related harm, including syringe sharing and overdose. These findings suggest that the over-reliance on enforcement-based approaches is contributing to ongoing human rights violations at the hands of police and the perpetuation of the HIV epidemic among Thai IDU. These findings indicate the need for greater police oversight and a shift toward the implementation of evidence-based programs focused on the HIV- and addiction-related needs of IDU.
Injecting drug use has been documented in 158 countries and is a major contributor to HIV epidemics. People who inject drugs have poor and inequitable access to HIV services. The Global Fund to Fight AIDS, Tuberculosis and Malaria is the leading multilateral donor for HIV programmes and a major funder of harm reduction interventions. This presentation will discuss findings from the first detailed analysis of Global Fund investments in harm reduction (which is being published in the
The full list of over 1,000 Global Fund grants was analysed to identify HIV grants that contain activities for people who inject drugs. Data were collected from the detailed budgets agreed between the Global Fund and grant recipients. Relevant budget lines were recorded and analysed in terms of the resources allocated to different interventions.
120 grants from 55 countries and territories contained activities for people who inject drugs worth a total of US$ 361 million, increasing to US$ 430 million after projections were made for grants that had yet to enter their final phase of funding. Two-thirds of the budgeted US$ 361 million was allocated to core harm reduction activities as defined by the United Nations. Thirty-nine of the 55 countries were in Eastern Europe and Asia. Only three countries with generalised HIV epidemics had grants that included harm reduction activities.
This study represents the most comprehensive assessment of Global Fund investments in harm reduction, data which are important for policy-makers, advocates and researchers alike. This funding, while substantial, falls short of the estimated needs. Investments in harm reduction must increase if HIV transmission among people who inject drugs is to be halved by 2015.
According to the latest update on the global state of harm reduction published in 2010, OST is available in 70 countries and territories around the world. The global coverage is estimated at the level of between 6 and 12 OST clients per 100 PWID. However, in most countries of Central and Eastern Europe and Central Asia coverage is limited due to long-term pilots and lack of systematic scale-up. The purpose of this study is to provide an up-to-date overview of the state of OST service provision in Eurasia, with a particular focus on access and quality issues.
Data were collected in 28 countries between August and November 2011 in order to capture some of the most recent developments related to OST service provision in Eurasia. We conducted an on-line survey using a semi-structured questionnaire in Russian and English, reviewed available literature and interviewed key stakeholders.
Essential OST-related data including year of OST introduction, OST medications, number of clients, number of sites in prison and community settings, geographic coverage, and availability of take-home doses were analysed for all 28 countries. Based on this analysis, countries were divided into three groups: i) countries with sustained availability of OST programmes; ii) countries with limited availability and sustainability of OST programmes; and iii) countries with no OST programmes. Within each of the three groups, we identified challenges and opportunities for scaling up OST services, improving their quality and/or strategies for overcoming existing opposition.
While some progress has been made and more countries introduced OST over the past three years, numerous challenges still exist. The opportunities for increasing access to and improving quality of OST programmes are related to six areas, ranging from national ownership of OST service provision to protection from police harassment and violation of human rights of OST clients and service providers.
Since 2004, China has rapidly scaled up Methadone Maintenance Treatment (MMT) from 8 to 716 clinics with 332,996 cumulatively enrolled drug users by September 2011. The average methadone dose is lower than the minimum recommended dose (60mg) in Chinese national guidelines. Lower methadone dosing may result in higher drop-out and continued HIV risk behavior. On average, 7.8% of MMT clients are HIV-infected.
A qualitative study was conducted in 2011 to determine reasons behind current dosing practices. Key informant interviews were conducted in 10 MMT clinics in 3 provinces (Guangdong, Guangxi, and Guizhou) among current and former MMT clients, their family members, and service providers. All interviews were conducted using a field guide and each interview lasted around 45 minutes. Interviews were recorded and transcripts were analyzed using a coding scheme and thematic sorting.
Interviews with clients and their family members revealed that they did not view drug users as patients with a chronic disease in need of long-term treatment; they expected MMT to end drug use in a short time. They also thought that methadone was more addictive than heroin and that methadone withdrawal was more severe and long-lasting. This contributed to their preference for lower and tapering methadone dosing. Interviews with MMT providers revealed that many doctors did not have previous experience in drug use treatment. Lack of experience and insufficient training led to client-dominated, lower methadone dosing and the common misperception “the lower dose, the safer and better for patients.”
Factors contributing to lower methadone dosing include clients and their families’ misperceptions as well as providers’ limited experience and training in MMT. To improve the performance of MMT in China, it is recommended that capacity building for providers be improved and education and counseling of clients and their family members be strengthened.
Rates of HIV are increasing among youth throughout Kenya. Research regarding how these youth have coped with their diagnosis and remained resilient is critical to the development of interventions that promote their health and well-being. The purpose of this study was to explore resilience among urban/slum youth newly diagnosed with HIV in Kenya.
Adolescents (ages 18–25) who were diagnosed with HIV within the past two years were recruited from ten villages in the Kibera informal settlement in Nairobi, Kenya. Six gender-specific focus groups (N=53; 26 female, 27 male) were conducted. Questions elicited specific and contextual data regarding participants' experiences, beliefs, and behaviors as youth newly diagnosed with HIV.
Qualitative data were analyzed first separately, and then collaboratively, by two teams of analysts in Kenya and U.S.A. using a phenomenological framework. Seven primary thematic areas of active coping strategies were reported: 1)
These data demonstrate that Kenyan youth who are newly diagnosed with HIV demonstrate resilience across multiple dimensions. In addition to
Few studies have examined the preventive effect of expanding antiretroviral therapy (ART) in Asian epidemics. Viet Nam has a concentrated epidemic with the highest HIV prevalence observed in people who inject drugs (PWID). Viet Nam aims to maximize the survival and preventive benefits of ART and plans an operational provincial pilot. Optimal targets and strategy need to be identified to achieve the goal.
We constructed a mathematical model (deterministic transmission model) to explore the effects of expansion of ART, early ART for serodiscordant couples irrespective of CD4 count and harm reduction interventions on the concentrated epidemic in Viet Nam. HIV prevalence trends and population size of PWID, female sex workers (FSWs) and their male clients, men having sex with men (MSM) and low risk women in Can Tho province, Viet Nam, were used in the model.
Compared to the current ART scenario (50% coverage, CD4 at ART initiation at 100 cells/mm3), achieving 80% ART coverage at CD4 threshold at 350 cells/mm3 will lead to approximately 20% reduction in new HIV infection annually, and biannual testing and immediate ART with 80% coverage will lead to 38% reduction. Early ART for serodiscordant couples potentially reduces annual HIV incidence in low risk women over 40% if high coverage is achieved. Opioid substitution therapy (OST) and needle syringe program (NSP) for PWID will reduce new infection in all the subpopulations.
Our modelling suggests that ART, combined with NSP and OST for PWID, could have a major impact on Viet Nam's HIV epidemic. National programs should consider further expansion of ART and earlier ART initiation to enhance ART's preventive impact. It is essential to address access barriers for key affected populations (e.g. discrimination, punitive policies) and to employ approaches that respect people's rights during this expansion.
HPTN 052 demonstrated a 96% decrease in HIV transmission when infected persons in serodiscordant relationships initiated HAART at study entry compared to those randomized to delayed treatment. However, this benefit could be attenuated if HIV-infected participants subsequently increased unprotected sex without virological control.
Between 06/2007 and 05/2010, 1763 HIV serodiscordant couples were enrolled in 9 countries in Africa, Asia and the Americas, and followed for a median of 2 years. The current analyses compared the sexual behavior of HIV-infected participants before and after they initiated HAART, and examined trends to evaluate whether risk taking changed over time by GEE models.
At enrollment, 4.0% of HIV-infected participants in the early treatment group (E) and 5.7% in the delayed arm (D) self-reported unprotected vaginal intercourse (UVI) with their primary partner within the past week. At 3 months, 2.9% of E participants did, compared to 3.0% of D participants (p=0.9). Over 2 years, UVI decreased among all participants (=−0.015, p=0.04), and the time trend was similar in both arms. Participants engaging in UVI were more likely to be female (AOR=1.6, 95%CI 1.1–2.4), from South America vs. Asia, AOR=16.0, 95%CI 8.2–31.3), from Africa vs. Asia(AOR=8.8, 95%CI 5.0–15.6), use substances (AOR=2.2,95% CI 1.3–3.9), and have a lower viral load at enrollment (AOR=0.7, 95% CI 0.6–0.9). After 2 years, 91% of E participants were virologically suppressed, compared with 22% of D participants. Self-reported unprotected anal intercourse was uncommon (<0.3% at baseline, and no change over time). Only 21% of participants on HAART who engaged in UVI or UAI had detectable plasma viremia.
Participants randomized to early HAART and those who subsequently initiated HAART did not increase risk taking over several years. The decrease in sexual risk taking, coupled with effective virologic suppression, suggest that earlier initiation of HAART could have sustained effects in decreasing HIV transmission.
An attractive approach for restoring CTL activity to HIV-1 infected individuals is the adoptive transfer of autologous CD8 T cells that have been transduced with an HIV-1 specific TCR. Previously, we described an HLA-A2-SL9 specific TCR that when introduced into CD8 T cells could control HIV-1 replication in an
By surface plasmon resonance, we defined the Kd of this wild type TCR to be 6.7µM and t1/2 to be 2.7s. Using phage display, a panel of affinity enhanced A2-YV9 TCRs were obtained with Kd values ranging from 5.1 to 0.3µM.
When introduced into CD8 T cells by lentiviral vectors, the A2 YV9 specific TCRs were highly specific for the wild type epitope. In contrast to what we previously determined for CD8 T cells transduced with the wild type A2-SL9 specific TCR, we observed that the A2-YV9 specific T cells could respond in a polyfunctional manner by simultaneously producing TNF-α, IFN-γ, IL-2, and MIP1-β when presented with a wide range of peptide concentrations. Moreover, affinity enhanced A2-YV9 specific CD8 T cells were able to recognize and respond to several variants of the wild type sequence, including those responsible for resistance to NNRTI and NRTI such as Nevirapine, Didanosine and Efavirenz.
Together, our data suggest that adoptive transfer of these A2-YV9 specific CD8 T cells presents great potential for augmenting available treatments and imparting additional control to HIV-1 infected individuals experiencing drug resistance.
HIV Controllers are rare individuals who spontaneously control HIV replication in the absence of antiretroviral therapy. To identify parameters of the CD4 response that may contribute to viral control, rather than merely reflect a persistently low viremia, we compared the T helper profile in two groups of patients with more than 10 years of viral suppression: HIV Controllers from the ANRS CO18 cohort (n=26) and efficiently treated patients (n=16).
Cells specific for immunodominant Gag and CMV peptides were evaluated for the production of 10 cytokines and cytotoxicity markers, and were also directly quantified
HIV Controller CD4+ T cells were characterized by a higher frequency of IFN-γ production, perforin+/CD107a+ expression, and polyfunctionality in response to Gag peptides. While IL-4, IL-17, and IL-21 production did not differ between groups, treated patients cells produced more IL-10 in response to Gag and CMV peptides, pointing to persistent negative immunoregulation after long-term antiretroviral therapy. Gag293 tetramer-positive cells were detected at high frequency (0.15%) and correlated positively with IFN-γ producing CD4+ T cells in the Controller group (R=0.84; P=0.01). Tetramer-positive cells were fewer in the HAART group (0.04%) and did not correlate with IFN-γ production, supporting the notion of a persistent immune dysfunction in HIV-specific CD4+ T cells of treated patients.
HIV Controllers maintained a population of highly efficient Th1 effectors directed against Gag in spite of a persistently low antigenemia, while patients treated in the long-term showed a loss of CD4 effector functions.
We previously reported that HIV Controllers harbored a unique population of CD4+ T cells expressing high avidity TCRs directed against Gag293. We propose that high avidity drives continuous Th1 effector differentiation in response to low antigen concentrations and explains the persistence of an activated antiviral response in HIV Controllers.
Natural control of HIV infection is associated with CD8+ T cell responses to HIV core antigens, encoded by
Plasma from 32 HIV controllers (HIV RNA<2000 copies/mL, including 14 elite controllers) and 21 ART-naive non-controllers (CD4+ T cell count<100/uL) were assayed for IgG1 and IgG2 antibodies to non-recombinant HIV proteins by western blot (WB) assay and to recombinant (r) p55 (Gag) and gp140 (Env) by ELISA. A positive antibody response was defined as a WB band score of 4 or an ELISA optical density >2SD of non-HIV sera for rp55 antibodies. Antibodies to rgp140 were titred. Controllers were HLA typed by sequenced-based typing using genomic DNA.
Controllers had a positive IgG1 or IgG2 antibody response to one or more core protein (p17, p24) on WB more often than non-controllers (75% vs 28.6%, p=0.0016 and 22% vs 0, p=0.034, respectively). Also, 12.5% of controllers but no non-controllers had a positive IgG2 antibody to rp55 (p=0.14). When results of WB assays and ELISA were combined, 34% of controllers but no non-controllers had positive IgG2 antibodies to core antigens (p=0.04). Positive IgG2 antibodies to core antigens were more common in patients without ‘protective’ HLA-B alleles (57%) than patients with these alleles (16.5%) (p=0.026). Positive IgG1 antibodies to
An isotype switched IgG antibody response to HIV core antigens is associated with control of HIV infection in patients without ‘protective’ HLA-B alleles.
A recent genome-wide association study (GWAS) of host determinants for HIV-1 disease revealed that single nucleotide polymorphisms (SNPs) near genes
We investigated the effects of 13 SNPs in the
A haplotype in the
This study provided novel evidence supporting a direct role of ZRND1 in modulating HIV and identified a
Understanding the early events during heterosexual HIV transmission at the genital mucosa is necessary to develop a safe and efficacious HIV microbicide or vaccine. A recent workshop highlighted the benefits of studying Highly Exposed Seronegative (HESN) individuals in order to identify and describe correlates of HIV protection. In an HESN cohort of commercial sex workers in Nairobi, Kenya, we have described a state of reduced systemic T cell immune activatio termed Immune Quiescence. However, the extent of Immune Quiescence at the genital mucosal is not known. This study characterized the female genital mucosal profile of cells, cytokines and chemokines involved in immune activation and lymphocyte recruitment among HESN.
CVL and plasma from commercial sex workers from the Majengo clinic in Nairobi, Kenya (57 HIV- followed for<3 years; 68 HIV infected and 55 HESN followed for >7 years) were analysed for the presence of 22 cytokines/chemokines and five antiproteases previously associated with resistance to HIV infection. Activation of cervico vaginal cells was analysed by multiparametric flow cytometry.
HESN women have a unique pattern of mucosal chemokine/cytokine expression. HESN subjects showed lower expression of MIG, IP-10 and IL-1a as well as higher levels of antiproteases. Among the HESN women there was a distinct chemokine gradient between the blood and genital mucosa relative to control women.
MIG and IP-10 are important regulators of T cell trafficking to the genital mucosa while IL-1a is an indicator of immune activation. The reduced levels of these cytokines/chemokines together with the unique correlations observed with antiprotease expression among HESN women suggest that the Immune Quiescent phenotype extends to the female genital tract. Reducing the number of activated CD4+ T cells in the FGT could limit cellular targets for HIV infection and may be an important component to resisting HIV infection.
For 26 years a group of HESN women from Nairobi, Kenya who can be epidemiologically described as relatively resistant to HIV infection have provided clues towards the identification of natural correlates of protection against HIV-1 infection. Studies of these HIV-1-resistant women suggest they posses a unique mucosal environment which includes the overexpression of specific antiproteases and a unique proinflamatory cytokine expression patern. Here we describe how these factors contribute to protection against HIV infection during mucosal transmission.
Cervical lavage fluid (CVL) from 277 women were collected from 76 HIV-1-resistant, 120 HIV-1 uninfected, and 97 HIV-1 infected women. CVL protein was analyzed both independently by SELDI-TOF MS and as pooled groups by 2D-LC-FTICR MS. Of the more than 350 unique proteins identified 29 proteins were differentially expressed (> 2-fold cutoff) between HIV-1-resistant women and controls. These findings were confirmed by traditional ELISA and quantitative Western Blot (WB) analysis.
The majority of overexpressed proteins were serpins, their breakdown products (p=2.2 x 10−8), and other antiproteases, as well as innate factors with known anti-HIV-1 activity. The overexpression of specific serpins and an epithelial-derived antiproteases was confirmed by ELISA and WB (p=0.004, p=0.05, and p=0.02). Underexpressed proteins in HIV-resistant women included inflammatory proteases and immune response factors. Cytokine/chemokine analysis revealed that antiprotease expression correlated with pro-inflammatory cytokines (p<0.0001). However, this was independent of the elevated antiprotease expression observed in HIV-resistant women who in fact expressed reduced levels of certain inflammatory chemokines (p=0.018).
HIV-1-resistant women have elevated acute phase response antiproteases that may regulate inflammation in the female genital tract. Coupled with elevated expression of anti-viral proteins, this may provide a mucosal environment less susceptible to HIV-1. These antiproteases might contribute to a natural protective environment against HIV-1-infection. Understanding this mechanism could aid in microbicide or therapeutic development.
Herpes simplex virus type 2 (HSV-2), the most frequent cause of genital ulcer disease (GUD), has been shown to play a more important role than any other sexually transmitted infections (STIs) in driving HIV prevalence in Africa. In turn, HIV-1 infection leads to more frequent HSV-2 reactivations and shedding. The exact immune mechanisms involved in this virological negative immuno-synergy are unknown. In the present study we sought to assess whether HIV co-infection would affects HSV-specific T cell immunity.
Nineteen HSV peptides, derived from HSV-2 glycoproteins gB and gD, were used to analyze the frequency and the magnitude of HSV-2-specific IFN-γ-producing CD4+ and CD8+ T cell responses in 30 HSV-2 seropositive patients and 17 HSV-2 seronegative individuals in a cohort of heterosexual Senegalese HIV-discordant couples, using ELISpot assay. HIV RNA viral load has been run for HIV infected subjects and CD4 count ran for all subjects using a flow cytometry method.
The magnitude and frequency HSV-2-specific T cell responses was compared between 21 HSV-2 co-infected with HIV-1 and 9 HSV-2 mono-infected individuals. A significantly higher magnitude of IFN-γ-producing T cell responses were observed in HSV-2 infected patients compared to seronegative individuals (median, 61 vs. 0 spots/106 PBMC,
Our finding suggest that co-infection with HIV-1 in HSV-2-infected patients might be associated with reduced HSV-2 cellular immune responses. However, the interaction between HIV and HSV-2 appears complex, and precise longitudinal studies will be required to dissect their exact temporal relationship.
HIV-infection leads to GALT CD4+ T-cell depletion that persists despite prolonged antiretroviral therapy (ART). SBI is a medical food that neutralizes bacterial antigens and reduces gut inflammation in animal models.
Subjects on ART with diarrhea and a thorough negative GI-workup received SBI (EnteraHealth, Ankeny,IA,USA) 2.5 grams BID for 8 weeks. 4-hour urine disaccharide gut permeability and absorption test and duodenal biopsies were obtained before and at 8-weeks. Immunohistochemistry for CD3/CD4 was performed on biopsies and flow cytometry was performed on duodenal single-cell suspensions and PBMCs for lymphocyte subsets. Markers of bacterial translocation and cytokine levels were measured in plasma. Stool was collected for 16S rDNA quantification and sequencing. Median values (interquartile ranges) and nonparametric analysis are reported.
All 8 subjects experienced resolution of GI-related symptoms. D-xylose absorption increased in 7/8 subjects and in those with improvement, the absorption levels increased from 31.4 mgs (28.5, 38.8) to 41.5 mgs (33.7, 45.2)(
SBI improved GI absorption and increased GALT CD4+ T-lymphocyte density. Further research is needed to demonstrate whether alterations in gut microbiota or inflammatory milieu impact mucosal immunology. Longer studies are needed to examine the mechanisms of SBI in GALT immune reconstitution and improvement in HIV enteropathy.
Interleukin (IL)-21 regulates three immunological functions - Th17 cell homeostasis, differentiation of memory B cells and antibody-secreting plasma cells, and long-term maintenance of functional CD8+T-cells - that are compromised in pathogenic HIV and SIV infections. Since IL-21 availability is reduced during infection, we hypothesized that
We infected 12 rhesus macaques with SIVmac239 (i.v.), and then treated 6 of them with rhesus rIL-21-IgFc (50mg/kg, s.c., once weekly for 5 weeks) during the early infection (from day 14 to 42 post-infection). Effects of IL-21 on viral load, immune activation, homeostasis of T-cells and their main subsets as well as T-cell cytotoxic potential have been evaluated in blood and mucosa by q-PCR, IHC, and flow cytometry up to 6 months post-infection. Mann-Whitney test and Spearman correlation were used for statistical analyzes.
IL-21 treatment was safe and did not increase plasma viral load or systemic immune activation. Compared to untreated animals, IL-21 treatment resulted in (i) increased expression of Perforin and GrB in total and virus specific CD8+T-cells of various anatomical sites (P<0.05); and (ii) improved mucosal immunity, with higher levels of Th17 CD4+T-cells (P<0.01) during the treatment period. Interestingly, improved Th17 homeostasis was associated with limited proliferation of intestinal CD4+and CD8+T-cells (P<0.05) and reduced plasmatic level of LPS (P<0.01) at 6 months post-infection.
IL-21 treatment during acute SIV infection beneficially impacts on the cytotoxic potential of T-cells and intestinal immunity - including increased homeostasis of Th17 cells, reduced levels of T-cell activation and limited microbial translocation - without undesirable effects on viral load. IL-21 should be further explored as a potential immunomodulator to be used in HIV infection in the context of ART or as part of new HIV vaccine strategies.
The majority of new HIV infections are acquired via heterosexual transmission. Certain individuals remain seronegative despite repeated high-risk exposure to HIV. The correlates of protection in exposed seronegative(ESN) individuals remain unclear. The purpose of this study was to determine the breadth and persistence of HIV-specific CTL responses in blood and cervical mucosa of ESN women.
The ESN cohort included 30 female partners of antiretroviral naïve HIV+ men. ESN women were followed longitudinally for 3-6 months. Controls included sero-concordant couples (n=17) and low-risk seronegative women (n=30). PBMC and mucosal mononuclear cells (MMC) from cervical cytobrush were stimulated with HIV Gag and Env (Clade C) peptide pools; IFN-g production was measured by intracellular cytokine staining. IFN-g production by>0.1% of cells after background subtraction was considered positive. Samples with <400 total events and/or<10 positive events were considered equivocal.
At baseline, PBMC from 2 of 30(7%) ESN and MMC from 3 of 17 (18%) ESN responded to stimulation with HIV antigens. During follow-up, 17 PBMC and 7 MMC samples were analyzed. Of these, PBMC from 2 ESN(12%) and MMC from 1 ESN(14%) responded to Gag and/or Env. In HIV+ women of concordant couples, responses were detected in 50% of PBMC and 40% of MMC. No responses were detected in low-risk controls. Median CD4 and plasma viral loads of male partners of ESN women were 477 cells/µL (range 238-1,090) and 26,300 copies/mL (range 400-750,000); values for male partners in sero-concordant couples were 520(range 132-1,321) and 31,850 (range 400-492,000).
These findings confirm that HIV-specific T-cell responses can be detected in PBMC and MMC from ESN women. These responses could play a protective role; however, they may simply be indicative of antigen exposure. A small percentage of ESN women continued to show positive responses upon longitudinal follow-up, arguing against a protective role for these responses.
A characteristic feature of the early phase of mammalian cells to metabolic stress is an increase in the rate of glucose uptake and metabolism. Glucose transporter 1 (Glut1) is the major glucose transporter in T cells and its expression is increased on CD4+T cells during chronic HIV-1 infection in vivo (Palmer et al., Abstract 1, IAS, 2012). We therefore seek to determine the impact of increased Glut1 expression on glucose metabolism in CD4+T cells from HIV-1-infected subjects.
The cell surface expression of Glut1 and glucose uptake (2-NBDG) was monitored in CD4+T cells from HIV-1 infected treatment naïve or HIV- controls subjects by flow cytometry. Hexokinase and glycolytic activity was measured by the intracellular concentrations of Glucose-6-phosphate (G-6-P) and L-lactate, respectively. Intracellular PTEN, pAkt (T308) and pAkt (S473) levels determined PI3K-mTOR activity. In vitro HIV-1 infection was performed on PBMCs activated with anti-CD3/CD28 microbeads and IL-7 and incubated with the CXCR4-using NL4.3-GFP virus.
Basal glucose uptake, G-6-P, L-lactate, intracellular p-Akt (T308) and p-Akt (S473) were significantly higher in CD4+Glut1+ vs CD4+Glut1- cells. This corresponded with an overall increased glucose uptake and glycolysis and lower levels of PTEN expression in CD4+T cells from HIV-1+subject vs seronegative individuals. Anti-CD3/CD28-induced Glut1 expression on CD4+ T cells was sensitive to specific inhibition of the Class1B PI3K-y and mTORC1 pathways which also blocked HIV-1 infection of CD4+T cells in vitro.
CD4+T cells from HIV-1 infected patients have increased glucose uptake and glycolytic activity mediated at least in part by the PI3k-y-mTORC1 pathway. Increased Glut1 cell surface expression and glycolysis in CD4+T cells may increase their susceptibility to HIV-1 infection and foster their depletion due to hypermetabolism. Approaches to normalize Glut1 expression or glycolysis in CD4+T cells may offer a platform for interventions to slow HIV-1 disease progression.
Progressive depletion of CD4 T cells is a hallmark of AIDS yet the underlying mechanism remains poorly understood. In human lymphoid cultures, most of the dying cells correspond to abortively infected resting CD4 T cells (
Human lymphoid aggregated cultures (HLACs) prepared with human tonsil and spleen were used to recapitulate many of the conditions encountered by HIV
>95% human lymphoid CD4 T cells that die in HLAC are abortively, not productively, infected. These nonpermissive resting cells accumulate incomplete cytosolic viral transcripts that trigger an innate immune response resulting in interferon-beta production and activation of caspase-3 and caspase-1. Most cells die as a consequence of caspase-1-mediated pyroptosis, an intensely inflammatory form of programmed cell death where cytoplasmic contents and pro-inflammatory cytokines (IL-1b) are released. Unexpectedly, HIV-induced CD4 T-cell death and release of inflammatory mediators can be blocked by addition of certain oral sulfonylureas like glimepiride that inhibit P2X7 ion channels, and are FDA-approved for the treatment of type II diabetes.
1. Abortively infected lymphoid CD4 T cells do not die due to the action of an HIV virulence factor(s), but rather because of host innate immune response launched against viral DNA produced in these cells.
2. This response likely evolved to protect the host from spread of infection, but the involvement of pyroptosis appears to trigger additional rounds of cell recruitment, infection, cell death, and inflammation.
3. CD4 T-cell depletion is blocked by P2X7 inhibitors that may interfere with pyroptosis. Such agents might be clinically useful in combination with classical antiretrovirals, particularly in HIV-infected subjects displaying rapid progression of disease or broad drug resistance.
Increased Tryptophan (Trp) catabolism into kynurenine (Kyn) and/or 3-hydroxykynurenine (3OH-kyn) by indoleamine 2,3 dioxygenase (IDO), contributes significantly to the persistent immune activation during HIV infection and plays a detrimental role in T cell responses in advanced AIDS. We herein studied Trp catabolism in elite controllers comparing to other well established groups of HIV patients with different disease outcomes.
Plasma samples from elite controllers (EC) (n=21), ART-naive (n=96), ART-successfully treated (ST) (n=18), and healthy controls (n=51) were collected. All these groups were standardized for nutritional status (albumin levels and body mass index). Levels of Trp, Kyn and 3OH-kyn were measured using isotope dilution tandem mass spectrometry and the markers of Trp catabolism (Kyn/Trp and 3OH-kyn/Trp ratios) were correlated to clinical data.
In ART-naive patients, viral load was positively associated with Kyn, 3OH-kyn levels and the markers of Trp catabolism (p<0.0001). In addition, these patients presented significantly lower Trp levels compared to controls (p=0.0002) and to ST (p=0.042). Accordingly, both Trp metabolites and Kyn/Trp and 3OH-kyn/Trp ratios were more enhanced in ART-naive
Elite controllers show a unique regulation of immunosuppressive Tryptophan catabolism. These findings may be relevant for HIV viral control and eradication.
In view of the role interleukin (IL)-7 plays in T-cell survival, homeostasis and function it is no surprise expression of the IL-7 receptor alpha-chain (CD127) is tightly regulated. We previously showed that expression of CD127 is suppressed on CD8 T-cells in HIV+ patients and that this suppression is mediated by both IL-7 and the HIV Tat protein. IL-7 down-regulates CD127 transcripts and surface protein through two distinct mechanisms. In this study we examine the mechanism by which IL-7 down-regulates the CD127 gene at the level of transcription.
CD8 T-cells from HIV-negative volunteers were treated with IL-7 (0.1−10 ng/ml) in the presence or absence of various inhibitors. CD127 transcripts were quantified by qPCR. STAT-5 phosphorylation was measured by flow cytometry. Nuclear run-on assays were utilized to measure the rate of CD127 gene transcription. Candidate CD127 repressors were identified using PCR arrays, qPCR, Western and siRNA-mediated gene knock-down assays.
IL-7 attenuates levels of CD127 transcripts in CD8 T-cells in a time- and dose-dependent manner. Both the full-length transcript and the splice-variant encoding the secreted isoform of CD127 are suppressed by IL-7. We show by nuclear run-on assay that IL-7 suppresses the rate of transcription of the CD127 gene and found no evidence that IL-7 affects the stability of CD127 mRNA. Further, the suppression of CD127 transcripts is dependent on JAK kinase activity and phosphorylation of STAT-5 but not STAT-3. Notably, cycloheximide blocked IL-7's ability to down-regulate CD127 transcripts suggesting IL-7 stimulates the
Upon binding to its receptor, IL-7 activates the JAK/STAT-5 signaling and induces the expression of a transcriptional repressor which suppresses CD127 gene transcription.
Interleukin (IL)-7 plays essential roles in T-cell development, homeostasis and activation. Disruption of this cytokine pathway likely contributes to HIV-induced immune deficiency. We previously showed that IL-7 and the HIV Tat protein reduce the half-life of the IL-7 receptor alpha-chain (CD127) in human CD8 T-cells but the mechanism directing CD127 to the proteasome is not yet understood. In this study we examined roles of SOCS proteins in regulating CD127 expression.
CD8 T-cells isolated from healthy HIV-negative volunteers were treated with IL-7 (0.1–10 ng/ml) in the presence or absence of various inhibitors. SOCS1-7 and CIS transcripts were examined by qPCR and protein expression was measured by Western. The interaction of SOCS proteins with CD127 was examined by Co-IP. Surface CD127 protein expression was measured by flow cytometry. Intracellular localization of SOCS and CD127 protein was examined by confocal microscopy.
IL-7 induces the expression of SOCS1-3 and CIS transcripts in CD8 T-cells via the JAK/STAT-5 signaling pathway in a time- and dose-dependent manner with SOCS2 transcripts increasing 300-fold within 3 hours. While induction of SOCS2 and SOCS3 mRNA was transient, SOCS1 and CIS transcripts remained elevated over baseline for at least 48 hours. Western blot analysis confirmed increased protein expression of the induced SOCS genes. Preliminary data on CD8 T-cells isolated from HIV+patients indicate that the IL-7-mediated up-regulation of SOCS transcripts is significantly decreased compared to healthy controls. IL-7 induces rapid phosphorylation and internalization of CD127 followed by proteasomal degradation. By Co-IP we show SOCS proteins induced by IL-7 physically interact with CD127 and study their cellular localization by confocal microscopy. We hypothesize this interaction directs the receptor to the proteasome.
IL-7 induces the expression of SOCS1-3 and CIS genes through the JAK/STAT-5 pathway. Through physical interaction with CD127, SOCS proteins may direct CD127 to the proteasome for degradation.
Nonhuman primate natural hosts for simian immunodeficiency viruses (SIV) develop a non-resolving chronic SIV infection, but do not develop AIDS. While several hypotheses, such as down-modulation of immune activation and differential surface expression of SIV receptors, have been suggested as putative mechanisms to explain the nonprogressive nature of SIV infection in natural hosts, mechanisms underlying high and maintained levels of plasma viremia without apparent loss of target cells in natural hosts remain unclear.
Here, we have used flow cytometric sorting, quantitative real-time PCR, immunohistochemistry, and in situ hybridization to study viral infectivity and production within subsets of peripheral blood and lymph node-resident CD4+ T cells in cohorts of chronically SIVsmm-infected sooty mangabeys and SIVsmE53-infected rhesus macaques.
We find: (1) infection frequencies among PB and LN-resident CD4+ T cells in chronically SIVsmE543-infected RM are significantly higher than those in SIVsmm-infected SM; (2) differential virus targeting is observed among anatomical LN niches and among individual CD4+ T cell subsets in SIV-infected RM and SM; (3) lymph node resident TFH cells are preferentially SIV-infected in RM, but these cells are not preferentially infected in SM and; (4) infectivity of central and effector memory CD4+ T cells is associated with plasma viremia in RM while infectivity of only effector memory CD4+ T cell infectivity is associated with plasma viremia in SM.
These data provide mechanistic insights into the maintenance of immunological function among chronically SIV-infected natural hosts for SIV, provide an explanation as to how natural hosts are able to maintain high levels of plasma viremia without apparent loss of target cells, and may lead to novel gene therapy interventions to recapitulate the natural host phenotype to animals that are susceptible to SIV-induced disease.
Transcription of the HIV-1 genome yields an initial pre-mRNA which undergoes complex alternative splicing to produce over multiple spliced mRNAs. Analysis of host cell factors important for HIV replication by genome-wide siRNA screens has emphasized that HIV replication is extremely sensitive to the complement of available splicing factors, suggesting that HIV splicing may be an attractive target for therapeutic intervention. Here, we have used single molecule amplification and third generation sequencing to characterize splice patterns for the patient isolate HIV89.6 under different conditions.
We infected HOS cells or primary T-cells and carried out single molecule PCR in emulsionsto minimize competition among amplicons. We then used Pacific Biosciences single molecule sequencing to analyze message populations.
Primary sequence read lengths averaged 1.6 Kb, and 5% of reads were over 3.8 Kb. Over 100 different messages was documented, more than doubling the previous number. The HIV sequence reads confirmed all of the known major splice junctions and identified new splice junctions, which create new ORFs in the 89.6 isolate. The presence of these new transcripts was confirmed using RT-PCR. The ORFs encode a novel form of Tat with an altered carboxy terminus and a Rev-Nef fusion (dubbed “Ref”) containing the amino terminal helix of Rev and the carboxy terminal portion of Nef. Using this assay, we found that HIV splicing differed between different cell types, differed between different human donors, and changes over time after infection.
These data illustrate how the diversity and promiscuity of splicing in HIV allows the virus to respond to different cellular environments and provides a continuous supply of evolutionary novelty that can potentially serve as a substrate for natural selection.
HIV-1 infected cells have evolved strategies to delay apoptosis but the exact mechanism is unknown. One explanation could be the enhancement of Bcl-2 levels. MicroRNAs (miRNAs) are small non-coding RNAs that participate in the innate immune response. Several cellular miRNAs target viral mRNAs, leading to a decreased viral replication, but viruses may also counteract this effect. In the case of HIV-1, Tat has been described as an RNAi suppressor, although this statement remains controversial. To get better insight into this issue and into the effect of Tat in protection to apoptosis, we have analyzed the miRNA expression pattern in Jurkat cells expressing different forms of Tat.
The miRNA expression profile of Jurkat cells with stable expression of HIV-1 full-length Tat101 (two-exon protein) or Tat72 (exon 1 isoform) was analyzed with a two color-based array of miRNAs (Exiqon). Differences in miRNA expression were then confirmed by qRT-PCR.
Global down-regulation of cellular miRNAs due to the expression of Tat was not observed. Instead, several specific miRNAs were dis-regulated due to the expression of Tat101 or Tat72, although the expression of the second exon granted a higher modification. We confirmed that cellular miR-21 and miR-222 were up-regulated in Jurkat Tat101 cells. miR-21 plays an important role in apoptosis. Since a higher resistance to FasL-mediated apoptosis was observed in Jurkat Tat101, we established a correlation between this protection and the increased levels of Bcl-2 in Jurkat Tat101. Regarding miR-222, it regulates cell cycle progression. In agreement with this, Jurkat Tat101 cells showed less proliferation capacity than control cells.
HIV-1 Tat does not show a general RNAi suppressor activity but it increases specifically several human miRNAs, conferring cells protection to apoptosis. This phenomenon was dependent on the expression of full-length Tat.
Throughout the years many labs have discovered important factors that contribute to the transcription of HIV-1. Most of these factors have been well characterized and their significance has been validated. Some of the critical factors involved in Tat activated transcription include RNA Pol II (associated with LTR), chromatin remodeling complexes (i.e. SWI/SNF), acetyltransferases (i.e. CBP, p300, pCAF), NFκB and components of pTEF-b. Surprisingly, almost none of the published literature has focused on finding these factors as complexes from genuine HIV-1 infected cells. Here we show presence of undiscovered complexes unique to HIV-1 infected cells which may serve as targets of inhibition.
We utilized a combination of HIV infected cell lines and primary latent cells (both T-cells and monocyte/macrophages) to define changes of protein complexes in infected cells. We utilized large quantities of infected cell lysates for size-exclusion chromatography to find novel kinases (i.e. cdk9/T complexes), and chromatin remodeling complexes, among others in presence of high salt (500 mM).
We found that there are novel cdk9/T complexes ranging from 2 MDa to ~300 KDa present only in T-cells that produce virus. Other components of the pTEF-b are also examined and found to be mostly unchanged in infected vs uninfected cells. Other novel complexes present only in infected cells included kinases for the NFkB pathway and SWI/SNF proteins. Many of these proteins are extremely stable and are targets of drug inhibition. Using a small panel of drugs, we find that many of the kinases utilized for transcription of HIV-1 have varying IC50s depending on the size of the complex and their protein partners.
HIV-1 infected cells contain many novel protein complexes that are yet to be discovered. Here we use a simple method of size exclusion to discover novel complexes that could potentially be used as targets of inhibition in therapeutics.
Latently-infected resting CD4+ T-cells are a major barrier to the eradication of HIV infection. These cells are enriched in lymphoid tissue compared to blood. We hypothesized that interactions between dendritic cells (DC) and resting CD4+ T-cells are critical for the establishment and maintenance of HIV latency.
Resting CD4+ T-cells labelled with eFluor670 were cultured alone or with syngeneic DC for 24h prior to infection with a CCR5-tropic, EGFP-reporter virus. Non-proliferating (eFluor670hi), non-productively-infected (EGFP-) CD4+ T-cells were sorted on day 5 post-infection. Latent infection was re-activated and amplified by co-culturing sorted cells with mitogen stimulated PBMC.
Infection of resting CD4+ T-cells in the presence of myeloid (m)DC significantly increased latent infection of non-proliferating CD4+ T-cells compared to infection of T-cells cultured alone (p=0.0005, n=11). Latent infection was not increased in resting CD4+ T-cells co-cultured with plasmacytoid DC (n=11) or monocyte-derived-dendritic-cells (n= 2). Co-culture of mDC with memory (CD45RO+) CD4+ T-cells but not naïve (CD45RO-) CD4+ T-cells resulted in latency (n=6). eFluor670hiEGFP- CD4+ T-cells that had been co-cultured with mDC showed a significant increase in the expression of CD69 (p=0.01, n=8) and PD-1 (p=0.007, n=10), but did not express HLA-DR or Ki67. Treatment of the mDC-T-cell co-cultures with blocking antibodies to the chemokines CCL19 and CXCL10 (shown to induce latent infection in resting CD4+ T-cells); the chemokine receptor CXCR3; or the adhesion molecule LFA-1 (binds to ICAM) led to no changes in the frequency of latently-infected CD4+ T-cells (n=5). When mDC-T-cell contact was prevented using transwells the number of latently-infected CD4+ T-cells was reduced (n=3).
mDC play a key role in the establishment and/or maintenance of HIV latency in resting memory CD4+ T-cells. Our results suggest this is likely to be mediated through DC-T-cell contact via alternative pathways to ICAM-LFA-1 binding.
HIV-1 penetrates the central nervous system (CNS) during early infection, establishing a viral reservoir. While macrophages and microglia represent the sites of productive HIV-1 infection, astrocytes undergo a restricted/latent infection. We recently demonstrated that astrocytes are extensively infected and may therefore constitute a significant potential reservoir of HIV-1 within the CNS. Here, we analyzed HIV-1 promoters (LTR) from matched CNS and non-CNS compartments to determine their role in virus restriction within CNS-derived cells. Determining the regulatory mechanisms of CNS-derived LTRs is essential to understanding the CNS as a HIV-1 viral reservoir, and in developing strategies aimed at HIV-1 eradication.
HIV-1 LTR sequences from a cohort of HIV-1 autopsy subjects consisting of matched CNS- and non-CNS-derived isolates were examined and their activity was determined in T-cells and SVG astrocyte cells. Electrophoretic mobility shift assays (EMSA) were used to analyze transcription factor binding activity within the core and basal promoter regions of the LTR.
CNS-derived LTRs demonstrated restricted basal transcriptional activity in both T-cells and SVG cells, and non-CNS-derived LTRs showed decreased activity in SVG cells. Restricted basal activity mapped to the three Sp binding motifs, previously shown to be essential for both Tat-independent and Tat-dependent activation of the LTR in T-cells. Further repression in astrocytes was observed due to elevated levels of the repressor Sp3 in SVG cells.
The reduced transcriptional activity observed for CNS-derived HIV-1 promoters was found to correlate with a reduction in Sp1 binding, which mapped to mutations within the core Sp binding motif. Transcriptional activity in SVG cells was further regulated by Sp3, which outcompeted Sp1 for Sp-motif binding. These data suggest that CNS-derived viruses have a reduced capacity to initiate viral transcription in astrocytes and highlights that unique transcriptional mechanisms exist within the CNS, ultimately affecting the fate of viral infection and the development of latency.
Latent reservoirs of HIV-1 consist of cells harboring dormant, stably integrated viral genomes that can be reactivated after cell stimulation. Latent HIV-1 evades immune responses and resists anti-retroviral therapy. CD4+ T cells are the major reservoir of latent HIV-1. These cells are very rare and lack distinctive markers, which has hindered their characterization.
We developed an in vitro model suitable to investigate HIV-1 latency in CD4+ T cells. In our system CD4+ T cells are activated with dendritic cells and antigen, infected in vitro with HIV-1, and then brought back to quiescence through a resting phase in the presence of interleukin-7. During the resting phase, the latently infected cells generated with our system lack expression of activation markers; do not undergo cellular proliferation and do not sustain viral replication. We sorted latently infected and uninfected cells from the same cell culture at the end of the resting phase, and profiled their entire transcriptome.
The results of this microarray analysis revealed profound differences between latently infected and uninfected cells derived from the same culture. First, a number of genes involved in all major cellular metabolic pathways are down-modulated in latently infected cells. Second, several messengers involved in gene expression (including chromatin organization, transcription, translation, post-translational modification, transport and assembly) are also down-regulated in latently infected cells. Third, genes involved in signal transduction, cell activation, cell proliferation and cell cycle progression are down-modulated in latently infected cells. Finally, several genes encoding cell surface molecules are differently expressed in latently infected vs. uninfected cells.
The establishment of HIV-1 latency does not simply entail suppression of HIV-1 expression, and the return to cell quiescence. Rather, HIV-1 appears to exploit and/or promote suppression of all cellular functions, leading to cell quiescence and viral latency. These results may have therapeutic implication for viral eradication.
HIV-1 cure remains elusive despite HAART due to the reservoirs of proviral DNA integrated into the human genome. Efforts to cure HIV-1 therefore need to aim at eliminating proviral DNA from cellular reservoirs. The first epigenetic signal identified in virus infected and uninfected cells has been promoter methylation. Compelling evidence confirms that specific promoter methylation can lead to gene silencing. Previous studies have examined HIV-1 epigenetics mostly in vitro.
We determined methylation patterns in HIV-1 proviral genomes from PBMCs obtained from 21 individuals with a spectrum of disease progression. The CpGs in the long terminal repeats (LTRs) of proviral DNA were investigated by bisulfite sequencing in up to 85 genomic variants per individual. This approach facilitates the study of the full range of CpG methylation and sequence variability of HIV-1 proviruses under conditions of natural selection in human populations.
In patients with advanced disease, the HIV-1 proviruses remained essentially unmethylated in their LTRs. In one long-term nonprogressor, the percentage of methylated proviruses varied from 0–77% at different times after infection. More important and unexpected was the detection of three specific LTR-located CpG dinucleotides that had been selectively mutated to TpAs in>20 out of the 32 samples analyzed. Comparison to 11 HIV-1 LTR sequences in the Los Alamos HIV database demonstrated that mutations in the sites identified by our study occurred more frequently than at other locations, although the mutations were different from TpAs.
These specific CpGs, possibly including their abutting sequences, might indicate weak spots in the proviral genomes whose sacrifice by mutation to TpAs could enhance the HIV-1 potential for long-term proviral survival. These data suggest that the sites of the mutated CpGs occurring at conserved sites may serve as potential targets for therapeutic interventions to eliminate integrated proviruses.
Grants: DFG-DO165/28-1; NIH-UO1-AI35004
We have demonstrated that HIV latency can be established in resting CD4+ T-cells infected after incubation with the CCR7 ligand, CCL19. The aim of this study was to identify the sites of viral integration in this model and to determine the relationship of integration sites to transcription factor binding sites and cellular gene expression.
Resting CD4+ T-cells were incubated with either IL2/PHA, CCL19, or in media alone (unactivated). After 2 days, cells were infected with NL4.3 and the presence of integrated DNA was confirmed at day 4. Total cellular DNA was purified and provirus-host junctions cloned and sequenced and mapped on the human genome using the UCSC Genome Browser. Gene expression in each cell type was determined using Illumina microarrays. Comparisons were made between these in vitro conditions and CD4 T-cells from HIV-infected patients on antiretroviral therapy (ART). Gene ontology was analysed using ClueGo.
We identified unique integration sites in infected CCL19-treated (n=247), IL2/PHA (n=432) and unactivated (n=133) T-cells. Integration occurred in transcriptionally active genes and most were involved in cellular housekeeping and cell signalling pathways. Integration sites were a similar distance from CpG islands, CTCF, pol II and histone methylation and acetylation sites. Compared to IL2/PHA-activated and unactivated cells, integration in CCL19-treated cells was further away from regions with high transcriptional activity (including transcriptional start sites (TSS); (p< 0.0001)) and closer to Long Interspersed Nuclear Elements (p< 0.0001), H4K20me3 (p< 0.0001) (a methylation site mapped to heterochromatin) and H4R3me2 (p< 0.0001; involved in priming gene expression). CCL19 treated cells and patient derived cells were similar in some but not all integration site patterns.
HIV integration occurred in transcriptionally active genes in all culture conditions although integration patterns in CCL19-treated latently infected cells were distinct. The significance of unique integration site selection in the setting of latency warrant further investigation.
SIVagm infection of rhesus macaques (RMs) provides a model of functional cure: initial high level viremia (108 copies/ml) and massive mucosal CD4+ T cell depletion are followed by durable control of SIVagm replication, complete recovery of CD4+ T cells, normalization of T-cell activation and seroreversion. The advantage of this model is that the functional cure occurs in all SIVagm-infected RMs. Immune control of SIVagm replication can be temporary reversed by experimental CD8-cell depletion.
Our objectives were to further characterize the RM/SIVagm model of functional cure by: (i) assessing the level of persistent chronic SIVagm viremia using qPCR with single copy sensitivity (SCA); (ii) determining whether rebounding virus after CD8-cell depletion is replication-competent by inoculation of uninfected RMs; and (iii) characterizing the diversity of rebounding virus using single genome sequencing (SGS).
SCA revealed low level viremia, averaging 20 copies/ml (range 10–30), in RMs 9 month after viremia became undetectable by conventional qPCR. Inoculation of new RMs with plasma collected during virus rebound after CD8 cell depletion resulted in peak viremia of 108–109 SIVagm RNA copies/ml, followed by control of viremia with kinetics similar to that following infection with high titer SIVagm stock virus. SGS of rebound plasma virus after CD8 cell depletion revealed sequence homogeneity consistent with clonality. Rebound virus was genetically similar to unpassaged SIVagm used to infect RMs, suggesting that the viral reservoirs that were the source of the rebounding virus were seeded early after infection.
These findings further validate SIVagm-infected RMs as a model of functional cure of replication-competent retrovirus infection. Deciphering the mechanisms of control may identify new strategies to achieve functional cure. This model is well suited to assess new therapeutic strategies to deplete viral reservoirs without the complexity of multidrug antiretroviral therapy.
Integration into host cell chromosomal DNA is considered an essential step in the replication of retroviruses, yet HIV-1 replication in vivo or in vitro generates one to two orders of magnitude more copies of unintegrated viral DNA (uDNA) than successfully integrated proviruses (iDNA). These extrachromosomal species are reported to possess limited capacity for gene expression and to be a replicative dead end. Resting CD4+ cells are the major targets of early infection following mucosal transmission, and resting memory CD4+ T cells constitute the major reservoir of latent infection. The cytokine environment in mucosal and lymphoid compartments facilitates HIV-1 infection of CD4+ T cells in the absence of TCR mediated activation.
We employed a combination of HIV-1 reporter viruses, flow cytometry and quantitative PCR to analyze HIV-1 early and late gene expression and virus production in purified peripheral blood CD4+ T cells.
We find that resting CD4+ T cells rendered permissive to HIV-1 replication by cytokines IL-2, IL-4, IL-7 or IL-15 provide a reservoir for the persistence of unintegrated HIV-1 DNA. Nonproliferating cells containing uDNA could generate de novo HIV-1 and transmit virus efficiently to uninfected cells, resulting in recombination between viruses. uDNA generated an order of magnitude less virus than integrated proviruses, but cells generating virus from uDNA survived and produced virus longer. Vpr packaged in virions was necessary for initial gene expression from uDNA. Subsequent T cell receptor/coreceptor-mediated activation substantially increased early viral gene expression from uDNA, but an increase in virus production was observed only when activation-induced cell proliferation was inhibited by de novo Vpr generated from the uDNA template. Activation through the T cell receptor or HDAC inhibitors in combination with Prostratin efficiently activated latent uDNA several weeks after infection of resting T cells.
We propose unintegrated HIV-1 as a potential reservoir of inducible virus.
A thorough understanding of the molecular mechanisms governing HIV-1 latency is essential in the development of rational therapeutics for the eradication of the virus. Evidence is accumulating that histone methylation regulates HIV latency. The multi-domain protein UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1), a key epigenetic regulator for maintaining DNA methylation patterns, has also been reported to interact with histone 3 lysine 9 methylated histones. This study investigates the role of UHRF1 in the transcriptional silencing of latent HIV-1 provirus.
293T cells were co-transfected with wild type HIV-1 long terminal repeat (LTR) and either with constructs encoding wild type or mutant forms of human UHRF1, treated with tumor necrosis factor alpha (TNF-α) and the promoter activity was determined by the dual luciferase assay. The presence of UHRF1 at the LTR was assessed by chromatin immunoprecipitation assay using sheared chromatin lysates from latently infected cells, ACH-2 and J-Lat 6.3. Small interfering RNA (siRNA) experiments were conducted using TZM-bl cells, which contain a chromatin-integrated HIV-1 LTR, to confirm the influence of UHRF1 on the HIV-1 LTR.
We observed that UHRF1 inhibited both basal and the induced HIV-1 gene expression by TNF-α. Chromatin immunoprecipitation assay revealed the presence of UHRF1 at the vicinity of the HIV-1 LTR and UHRF1 occupancy was reduced upon activation. Meanwhile, knockdown of UHRF1 expression modestly increased basal LTR activity.
Results suggest that UHRF1 contributes to the transcriptional silencing of latent HIV-1 provirus and further elucidate the underlying molecular mechanisms that maintain latency.
Replication-competent HIV persists in patients who are treated with highly active antiretroviral therapy (HAART). One significant reservoir of this persistent virus is within rare latently infected CD4+ T cells. However, the infrequent nature of these cells makes them challenging to study directly in infected patients, and clinical attempts to completely eliminate this viral reservoir have not been successful. Therefore improved models for HIV latency and eradication strategies are needed. The humanized bone marrow-liver-thymus (BLT) mouse provides robust multi-lineage immune reconstitution with human cells. When infected with HIV, these mice can also serve as an
BLT mice were infected with HIV and assessed for the presence of latently-infected cells. Cells were stimulated ex vivo with a variety of canonical and novel latency activators. Infected mice were also treated with HAART and then assessed for the presence of activation-inducible virus.
Up to 3% percent of human cells in spleen, peripheral blood, and thymus/liver implants in HIV-infected BLT mice harbored latent HIV. This virus was integrated, activation-inducible, and replication competent. The latently-infected cells were also responsive to stimulation with protein kinase C activators and latency-activating nanoparticles. Furthermore, activation-inducible virus was detectable in HAART-treated mice, although at lower frequencies than in untreated mice.
The humanized BLT mouse provides a versatile system for
Evidence from India suggests that a comprehensive approach to SRHR has been lacking and that current responses have not sufficiently decreased vulnerability and sexual and reproductive ill-health among PLHIV. Previous studies with PLHIV have shown high rates of unmet contraceptive needs, untreated STIs and lack of knowledge and skills on safer sex and broader positive prevention.
803 people living with HIV aged 15–49 (352 men; 401 women; 50 transgender/hijra) were interviewed in five districts in four states of India (Andhra Pradesh, Gujarat, Maharashtra and Tamilnadu). Lists of PLHIV covered by local CBOs were used as the sampling frame for the selection of respondents. The required number of respondent households was arrived at using systematic random sampling.
Approximately one quarter of all respondents reported STI-related symptoms in the previous three months which is a cause of concern. Also, a relatively small proportion of mothers reported seeking maternal health advice during their last pregnancy (12% in Andhra Pradesh; 21% in Gujarat; 44% in Maharashtra; 27% in Tamilnadu). 10% of respondents reported having been advised against having children because of their HIV status indicating a lack of accurate or comprehensive information available at health facilities. Awareness of contraceptive methods ranged between 55% and 98%; similarly, STI-related knowledge levels ranged between 56% and 91%. Reported condom use with non-regular partners ranged between 56% and 91%.
The study highlighted unmet SRHR needs of PLHIV in the four states. While most subjects reported frequent condom use, STI-related symptoms indicate unsafe sexual behavior. SRH and HIV-related knowledge levels are low, and respondents also reported SRH-related rights violations. This study confirms the need for interventions for positive prevention along with specific SRH programming for PLHIV as part of India's HIV response.
There is a dearth of literature on the challenges and coping mechanisms of school-age HIV infected children. The purpose of this study was to investigate challenges faced by HIV infected children in Botswana and to assess the coping mechanisms they employ to cope and survive.
The study was conducted among HIV-infected children and adolescents aged 6–18 years, using a cross-sectional design, in 12 antiretroviral therapy sites accounting for over 90% of all HIV-infected children receiving Highly-Active Antiretroviral Therapy in Botswana. Data were gathered using a structured pretested interviewer administered questionnaire and focus group discussions. Focus group discussions were held until saturation was achieved. Quantitative data were analyzed using descriptive statistics and cross-tabulations. Qualitative data were grouped into mutually exclusive categories according to themes emerging from the data. Ethical approval was obtained from the Botswana Ministry of Education & Skills Development, the Botswana Ministry of Health's Health and Research Development Committee and Baylor College of Medicine's Institutional Review Board.
984 HIV infected children participated between August 2010 and February 2011. The children were confronted with various challenges, including: poor school grades; poor school attendance due to medical appointments; poor nutrition; unanswered questions of how they became infected; and stigma or altered relationships with other children and teachers. In response to these challenges HIV infected children developed or adopted various coping mechanisms including: non-disclosure of HIV status, self-isolation; non-participation in school trips; attempting to treat HIV like any other disease; talking to close relatives or friends; and adoption of positive attitudes toward life.
HIV infected children faced many serious psychological and social challenges in living with HIV. These challenges elicit both positive and negative coping and survival strategies. Support systems for the children should aim to build on the positive coping strategies while ameliorating the negative ones.
Approximately 70000 people are living with HIV (PLH) in Peru. Over half live in Lima-Callao and one half are gay/bisexual men and transgender people. While they are the focus of care and prevention services, not much is known about their sexuality, sexual and reproductive health (SRH) needs and access. A community-based collaborative study involving the Peru Positive Network took place to assess sexuality and SRH status and access of PLH in Peru.
Trained PLH volunteers in Lima-Callao and 6 inner cities conducted 814 structured interviews in 2011 (48.7% in Lima-Callao, 72% biological males). This analysis focuses on 273 males reporting being gay and bisexual (40.8%) and compares Lima-Callao (n=134) with other cities (n=139).
Participants in Lima-Callao were slightly older; 40% in Lima-Callao and 60% elsewhere lacked health insurance. Overall 32% had regular partners-in Lima-Callao 95% of partners were men, while elsewhere 21% of those were women and 17% trans; 42% of regular partners were seropositive, but serostatus of another 20% was reported as unknown outside Lima. Participants had disclosed serostatus to 82% in Lima-Callao but 60% elsewhere. 88% reported penetrative sex in last 6 months-up to 40% unprotected outside Lima; condom use was unrelated to partner serostatus. Among those with only casual partners, 28% reported unprotected sex in prior month; disclosure was negligible. Participants recognized a shared responsibility in prevention, including through treatment adherence. 21% and 8% had heard about barebacking and serosorting, respectively, but few had practiced them. 15% had been diagnosed STIs in prior year, primarily herpes and syphilis. Sexual health advice received by participants often encouraged abstinence (30%).
HIV+ gay/bisexual men in Peru remain sexually active; serostatus disclosure is limited, particularly with casual partners; unprotected sex and STIs are common. Often sexual health services inappropriately advise abstinence and insufficiently address prevention and disclosure.
An increasing number of children living with HIV are growing into adolescence where they face new sets of challenges. In resource constrained settings, few psychosocial and sexual and reproductive health (SRH) services provide age-appropriate support for these adolescents.
A large multi-country cross-sectional observational study was conducted in Malawi, Mozambique, Zambia and Zimbabwe, looking at the experience of adolescents aged 10-19 [n=1600 (boys=790, girls=810)]. Gender specific experiences from rural and urban settings were explored in each country. Data was collected using in-depth interviews with adolescents (n=150) and guardian/parents (n=46), focus group discussions (n=68), participant observation (3 months) and a knowledge, attitude and practice survey (n=1498). Data was analysed through a triangulation approach.
Preliminary analyses reveal that key challenges for adolescents living with HIV include accessing information on SRH; disclosing their status to peers; talking about sex and HIV in the context of a relationship, within families and with health service providers; planning to have children; and emotional well-being. HIV represents a complex challenge for adolescents who lack safe and non-judgemental environments to talk about safer-sex options and pregnancy, whilst it is assumed by families and health providers that sexual activities should not start before adulthood. Few health services provide support tailored to adolescents; health providers give out inappropriate information and frequently breach confidentiality, resulting in further marginalising adolescents living with HIV.
Despite variations between the four countries and in rural vs. urban settings, the attitude of health care providers and the lack of family-centred approach result in inadequate support for adolescents when they first experience sexual and love relationships. The study provides evidence that new policies and funding for HIV in low income settings must include safer-sex advice and SRH interventions, training for health-workers and families in order to provide adolescents with skills to make choices about relationships, safer-sex and HIV disclosure.
Early diagnosis of HIV and prompt access to antiretroviral therapy (ART) are critical in ensuring optimal care for HIV patients. We produce key indicators aimed at monitoring access, and the quality of care delivered, to adults living with HIV in the UK.
Analyses of national surveillance data included linking reports of newly diagnosed adults to annual surveys of adults accessing HIV care and laboratory CD4 data. Deaths reports were linked from the Office for National Statistics. Proportions are presented among adults with relevant information available. Data are rounded to the nearest 100.
Late diagnosis: 6,600 were newly diagnosed in 2010, of whom half were diagnosed late (CD4<350 per mm3) in 2010 (heterosexual men 65%, women 58% and men who have sex with men (MSM) 39%) and 28% had CD4<200. Late presenters had 10-fold increased risk of dying within a year compared to those diagnosed promptly (4% vs 0.4%).
Prompt integration into care: Using the first CD4 count as a proxy, overall 98% of 6,600 adults newly diagnosed in 2010 were integrated into HIV care within 3 months, with little difference across exposure groups.
Immunological response: Among 53,400 adults receiving care for more than a year by 2010, 81% had a CD4>350 regardless of ART (heterosexual men 74%, women 82% and MSM 85%)
Viral suppression. Among 6,000 adults who started ART in 2009, 85% remained on treatment and had undetectable viral load by 2010 (heterosexual men 84%, women 85% and MSM 89%).
We have developed robust measures of patient care from routine surveillance data, enabling comparisons overtime and between population groups. The NHS provides excellent access to high quality HIV care regardless of patient characteristics, contrasting with other high-income countries. Expanding testing to reduce late diagnosis remains vital in improving the life expectancy of HIV patients.
Treatment advances and HIV transmission feed a growing population of older adults with HIV. Little attention has been given to differences within this group.
One in twenty UK-based over 50s with HIV (n=410) surveyed. Sample was accurate for demographics of wider group. Data from 106 heterosexual over 50s, comprised of 51 HW and 55 HBME.
Health: HW 3x as likely be recently diagnosed. HBME diagnosed in last 6-9 years 5x HW (51.0/10.0%). HBME twice as likely to have low CD4 count when first diagnosed. HW have higher count generally. HW♂ /HBME♀ most likely to have low CD4 count now, or not know count. HW♂ more than twice as likely not to be attending a treatment centre, despite reporting significantly higher occurrence of extreme pain/discomfort.
HW less likely to be taking medication for HIV (84.3/98.1%), have higher pill burden, find it harder to remember to take HIV treatment (15.5/5.7%), have side-effects from medication (20.8/13.3%) and more problems with walking, (56.0/42.6%), taking care of themselves (27.5/18.8%) and performing everyday activities (56.0/39.2%).
HBME♀ have more other long-term health conditions (HBME♂ have the least), but only 50% are taking medication for these (70% HW♂ are).
HBME higher levels of depression (28.8/21.6%), particularly HBME♀ (34.2%), but HW three times as likely to rate emotional/psychological health as poor.
Financial/Social Exclusion: HBME all of those with no income and 3x not enough income for basic needs (45.3/15.7%).
HW more likely to own their own home (47.1/18.2%). HBME more likely to be homeless, living in asylum support housing or renting council flat/house.
Socially, HW more likely to disclose to Partner (16.4/4.5%), family members (16.4/12.7%) or friends (16.4/12.7%). HBME more likely to seek support from non-family.
There are major health, financial and social differences between HW and HBME. These factors need to be considered when targeting support for these groups.
In communities severely affected by HIV many children are growing-up without their parents in skipped-generation households. Often these are headed by their grandparents. This research aims to critically examine what it means for children to grow-up in these households and which roles and responsibilities they assume.
The data for this qualitative and quantitative study were collected in Misangwa, a small community in the rural part of the Copperbelt Province in Zambia. Data collection was conducted by means of household surveys, in-depth interviews, Focus Group Discussions, and observational techniques. Respondents were members of the younger and the older generation found in 65 skipped-generation households. Their lives were followed over the course of thirteen months.
Between 2001 and 2009 the number of skipped-generation households in Misangwa has increased. The proportion of children living in these households also increased significantly. The experiences and roles these children have are complex. It was found that especially older children actively impact the situation in their homes and are often wholly responsible for many aspects of daily life in the households. They work on the fields and are engaged in income-generation. Given the social isolation of many skipped-generation households a focus on households, rather than families, can be justified.
Literature often characterizes fostering as one-directional. This research examined skipped-generation households using ecological theories of child development. It found that that it is often children, and not guardians, who care for others in the households. Depending on age, personal characteristics and granted freedoms the older children provide care and support to the members of their households. In a sense these people assumes the roles and are associated with the missing middle generation. The research has brought to the surface a complexity that calls a different focus on kinship and caring with attention for diversity, historicity and experience.
The District of Columbia Department of Health (DCDOH) has supported HIV test and treat activities by increasing the number of tests performed and emphasizing earlier linkage to care. Data on the impact of these efforts on viral suppression (VS) and continuity of care are limited. This analysis sought to identify factors associated with VS among newly diagnosed HIV-infected persons.
HIV-infected persons diagnosed from 2006–2007 were identified in the DCDOH HIV/AIDS surveillance database. Cases with an initial detectable viral load (VL) followed by at least one additional VL test prior to 12/31/10 were included. VS was defined as VL<400 copies/mL. Bivariate analyses and multivariate logistic regression were performed to detect differences between those who were VS and those who were not. Among VS cases, Cox proportional hazards ratios and Kaplan-Meier survival analysis were conducted to identify predictors of VS.
Of 988 newly diagnosed cases, 66% achieved VS prior to 12/31/2010. VS cases were significantly more likely to be ≥50 years of age at diagnosis (19% vs. 11%, p=0.008) and in continuous care, defined as 2 visits 3 months apart within 12 months (32% vs. 22%, p=0.002). Cases concurrently diagnosed with AIDS were also more likely to achieve VS (73% vs. 62%, p<.0001). Multivariate logistic regression revealed that MSM (aOR 1.62, 95% CI 1.1,2.4) were significantly more likely to achieve VS than heterosexuals. Among those achieving VS, survival analysis found that those=50 years of age at diagnosis (aHR 1.44 95% CI 1.2, 1.8), those linked to care within 3 months (aHR 1.35 95% CI 1.1, 1.6), and those in continuous care (aHR 1.73 95% CI 1.5,2.1) were significantly more likely to achieve VS.
These findings demonstrate the importance of earlier linkage and continuity of care in achieving viral suppression and highlight the need for more navigation programs targeted to HIV-infected persons.
The evidence linking concurrent sexual partnerships (CP) to HIV transmission prompted the Government of Botswana to launch a National Campaign in 2009 to reduce CP. The campaign aimed to increase risk perceptions associated with concurrent partnerships and to reduce CP.
This study tested whether exposure to campaign messages among targeted adults aged 18–35 (a) reduced concurrent partnerships, (b) increased risk perceptions associated with CPs, and (c) reduced behavioral factors known to be associated with CP. A national cross-sectional survey was conducted in 2011 using a two-stage cluster sampling. Individuals were randomly sampled from households within enumeration areas (n=1237 adults). Coarsened exact matching was used to create statistically equivalent groups of exposed and non-exposed individuals, based on radio/TV ownership and place of residence. Logistic regression was used on the matched sub-sample (n=1138) to ascertain the effect of intervention exposure, accounting for gender, education, occupation, and age.
Half of the respondents were female; 90% were single; 66% had secondary education; 26% had tertiary education; 11% reported having CP in the past 6 months; and 69% were exposed to at least one intervention message. Exposure to campaign messages was associated with increased risk perceptions associated with CP (OR=1.49;95%CI=1.11–2.01). There was no impact of the campaign message on reducing CP. However, exposure was associated with increased self-efficacy for condom use (OR=1.38;95%CI=1.02–1.88) and HIV testing (OR=1.6; 95%CI=1.06–2.42). In additional analysis, those who reported having CP were more likely to use condoms consistently (OR=2.3; 95%CI=1.7–3.1).
We found positive impacts of the intervention on increased risk perception and condom self-efficacy. CP is rooted in deeply entrenched social norms and thus may require a longer intervention period to see a reduction. The finding that CP was associated with condom use suggests that combined intervention messages would be more appropriate than a stand-alone CP message.
Notifying partners about HIV exposure is an important component of HIV prevention. CDC recommends programs should use surveillance data and disease reporting systems to identify newly diagnosed persons. Texas law mandates that local health departments elicit partners, notify them of possible exposure to HIV, and offer HIV counseling and testing. In Houston, the HIV/STD Surveillance Program coordinates with HIV/STD Prevention Program to identify persons with HIV infection and provide Partner Services (PS). These activities can prevent the spread of disease and improve individual health as well as the health of the community.
Using HIV surveillance data from the HIV registry, the Houston HIV/AIDS Surveillance Program identified individuals newly diagnosed with HIV. These patients were referred to HIV Prevention Program to conduct Partner Services. Partners identified were contacted by Disease Intervention Specialists and confidentially notified of possible exposure.
Using prevention data from the STD registry, patient and partner outcome information were generated. Numbers of patient interviews and number of partners notified and tested were tabulated.
Using the HIV surveillance data, the number of HIV positive patients referred for PS increased from 78% in 2005 to 96% in 2008. The percentage of providers who “opt-out” of health department-based PS has decreased from 22% to 4%.
From Prevention data, the number of patients interviewed increased from 521 in 2005 to 1,299 in 2010. More significantly, the percentage of partners notified has increased from 57% to 79% and the number of partners tested increased from 74% to 92%.
Using HIV Surveillance data for initiating Partner Services has proven to be an effective tool for HIV prevention activities. However, using surveillance data to initiate PHFU should be guarded with strict protocol to avoid inadvertent breaches in confidentiality.
HIV-related stigma and discrimination is widespread in health care settings and poses a barrier for HIV prevention and control. The objective of the intervention was to reduce service providers' stigmatizing attitudes and behaviors toward people living with HIV (PLH).
The study was conducted in 2008-2011 in 40 county-level hospitals in two provinces of China. The hospitals were matched into pairs, with one hospital in each pair randomly assigned to the intervention condition after the baseline assessment. For intervention hospitals, we identified and trained about 20% of the popular opinion leasers (POL) among service providers to disseminate stigma reduction messages within their hospital. At the same time, universal precaution supplies were provided to all participating hospitals. A total of 1,760 participants, 44 from each hospital, were assessed at baseline, 6, and 12 months. Main outcomes of the intervention included general prejudicial attitude towards PLH, avoidance intent to treat PLH, and perceived institutional support in the hospitals.
Significant improvements for the intervention group were observed in reducing prejudicial attitude (Estimate 2.401, SE=0.220; P<.0001), reducing avoidance intent (Estimate=1.098, SE=0.174; P<.0001), and increasing institutional support (Estimate=0.390, SE=0.131; P=.0029) at 6 months after controlling for age, gender, occupation, prior contacts with PLH, and province. The intervention effects were sustained and strengthened at 12 months.
Reduction in stigmatizing attitudes and behaviors among service providers can be achieved by an intervention implemented in health care settings. The intervention has the potential to be integrated into the health care systems in China and other countries.
AIDS stigma inflicts hardship and suffering on People-Living-with-HIV (PLHIV), reducing the likelihood of HIV testing, treatment and disclosure. Stigma can be particularly harmful in health care settings. This study was designed to examine and reduce AIDS stigma among nurses and nursing students in India.
We interviewed 369 nurses in Mumbai and Bangalore, assessing stigma, endorsement of coercive measures and discrimination toward PLHIV. Based on these results, we developed, implemented and evaluated a 2-session stigma reduction intervention, co-facilitated by PLHIV, for 50 intervention and 50 control nursing students.
Casual transmission misconceptions were common, with 28% of nurses believing HIV could be transmitted by sharing a glass and 26% HIV by sharing toilets with PLHIV. 70% agreed that people who were infected via sex/drugs deserved their infections. Almost all (96–99%) endorsed mandatory testing for sex workers and surgery patients and most stated that HIV-infected men (77%) and women (73%) should not be allowed to get married and that HIV-infected women should not be allowed to have children (76%). 88% said they would treat PLHIV differently from other patients, taking unnecessary precautions when drawing blood. significantly more participants worried about acquiring HIV at work (41%) than in their personal lives (14%, p<0.001). Multiple regression analyses showed that worries about occupational transmission, negative feelings toward PLHA and transmission misconceptions were significantly associated with AIDS stigma.
Nursing students showed similar misconceptions and stigma levels as the nurses. Following the intervention, students showed significant pre-post decrease in all stigma scores, including blame, endorsement of coercive measures, and intent to discriminate (all p<0.01). In contrast, pre-post scores among control students remained the same.
These findings demonstrate high levels of AIDS stigma in these health-care settings, that may be reduced by a brief intervention using a human rights framework, focusing on underlying variables and involving PLHIV.
Stigma is a hindrance to care for people living with HIV (PLHA) in Egypt. To understand how gender affects stigma among healthcare workers (HCWs), a study was conducted to identify the differences in HIV related misconceptions and attitudes of male (M) and female (F) doctors.
A cross-sectional study was conducted in two tertiary care hospitals in Cairo from October–December 2010. All doctors working in surgical departments were invited to participate; three hundred thirty two accepted (210 males, 122 females). Data was collected on socio-demographic information and HIV related misconceptions and attitudes. A chi square test was used to examine the differences between male and female doctors.
Both male and female doctors had misconceptions about HIV modes of transmission. Significant differences (p<.005) were observed regarding transmission through mosquito bites (F: 44.3%, M: 31.9%), sharing cups and spoons with a PLHA (F: 28.7%, M: 19.0%), sharing toilets with a PLHA (F: 27.9%, M 16.7%) and serving food to a PLHA (F: 14.8%, M: 6.7%).
Differences were also significant in terms of blame and refusal to deliver care: 40% of females would not treat a PLHA (vs. 20% males), 50% would refuse to treat an injecting drug user with HIV (vs 36.8% males), and 57.4% would not treat a sex worker (vs. 40.7% males).
Female doctors had more misconceptions about HIV modes of transmission and judgmental attitudes compared with males. Further studies should explore factors influencing stigma among female HCWs in order to address it accordingly.
Evidence is mixed on the benefit of medical male circumcision (MMC) for women. Questions remain whether there is direct benefit in reducing HIV acquisition among women; whether men put women at risk due to early resumption of sex; whether mass rollout of MMC leads to risk compensation in ways that compromise women's ability to negotiate condom use; and whether MMC increases patriarchal behaviors such as gender-based violence. We present data from various studies to address these questions and make the case for women's shareholding position in MMC.
We reviewed over 30 published and ongoing studies on MMC for evidence on early resumption of sex, risk compensation, benefits of MMC to women, and women's views of and reactions to MMC rollout in different African countries.
About 10 studies have returned the verdict of no risk compensation among circumcised men. In addition, there are few but scientifically solid evidence emerging showing up to 47% direct benefit of MMC in reducing HIV transmission to women, particularly when sex is not resumed early. However, four studies show consistent evidence of early resumption of sex before wound healing, ranging from 24-42% overall, 63-76% among married/cohabiting men, and 12-48% among HIV-positive men. About 15 studies indicate that women support MMC though misconceptions are still common, with up to 77% unaware of the need for sexual abstinence. Some believe that scale-up of MMC translates into reduced risk hence less need for safe sex.
With the current scale up of MMC for HIV prevention in 14 African countries, there is no evidence of risk compensation; however, early resumption of sex and women's low risk perception because their partners are circumcised may undermine the benefits of MMC to women. MMC programs should target women with correct and complete information and advocate for their involvement in partners' decisions on circumcision.
Namibia's antiretroviral treatment (ART) programme has expanded rapidly, enrolling 68,000 adult patients on treatment by December 2009, with 35% taking stavudine-based regimens. In 2010 the Ministry of Health & Social Services (MoHSS) adapted WHO recommendations and began implementing new ART guidelines that changed ART eligibility criteria and preferred first line regimens. Thus, MoHSS enlisted the support of the Supply Chain Management System (SCMS) to analyse the supply chain implications of the changes and provide support for a smooth transition.
Using MS Excel(r) spreadsheets, SCMS modelled implications of increasing the CD4 threshold for ART initiation and changing to tenofovir-based first line regimens. Backed by Namibian Government funding commitment, CMS accelerated internal procurement processes to avail tenofovir-based formulations, reviewed stock control parameters to manage reduced demand for stavudine and expanded ARV storage capacity to cope with the increased volumes of ARVs. Monitoring systems were intensified including quarterly tracking of trends in consumption and treatment regimens.
Within 8 months, CMS had managed to accumulate adequate quantities of tenofovir formulations, clearing the way for the national roll-out of the new ART guidelines starting September 2010. As a result, over 34% of the approximately 90,000 adults on ART were taking tenofovir-based regimens one year later. In addition, a potential wastage of about US$ 950,000 worth of stavudine formulations was prevented and stock outs of tenofovir formulations avoided.
Interventions to optimise existing storage space at CMS created an additional 328 pallet locations, a 74% increase in racked storage capacity in five rooms for ARVs and general pharmaceuticals.
A rapid response was achievable due to the Namibian government's commitment and the agility of the supply chain. Timely availability of information through robust monitoring systems established to capture changing consumption patterns informed procurement planning and minimised the risks associated with change in ART guidelines.
Crisis followed the October 2010 Presidential elections in Cote d'Ivoire. International sanctions were applied, the banking system collapsed, security deteriorated and internal displacement prevented access to healthcare. SCMS, a PEPFAR program administered by USAID, adapted the national HIV/AIDS supply chain strategies to respond to changing conditions and to avoid treatment interruption
From the outset, SCMS established a crisis-committee whose daily calls between the field and Washington focused on information sharing with the Ministry of Health (MOH), Mission, USG team, Implementing Partners (IPs) and international donors.
SCMS jointly reviewed supply plans and stock-status reports with international donors and IPs and adjusted planned orders to meet urgent national demands. Under threat of air, land and sea border closings, SCMS re-routed all deliveries though its' Regional Distribution Center (RDC) in Accra, Ghana. The RDCs, in turn, pulsed commodities into the country as demand required and security permitted.
Meanwhile, in country, the MOH provided patients with two months treatment instead of the usual one. The distribution mechanisms, however, were disrupted and SCMS together with IPs created an interim distribution plan in which IPs picked up their products and distributed them to sites.
Deliveries continued throughout the crisis; no health facility experienced a stockout of critical products and treatment interruption was averted. Donor collaboration ensured planned deliveries met patient needs. The RDC in neighboring Ghana provided flexibility to maximize in-country stock levels, arrange last-minute flights and make urgent deliveries. On-the-ground collaboration with the MOH and IPs ensured medicines were not only available at service delivery points, but in the patients' hand.
By creatively leveraging in-country knowledge, regional resources and procurement volumes of supplies, ARVs and other critical products reached patients throughout the crisis.
Currently, HIV/AIDS care support from donors is shifting from direct service delivery to systems strengthening of country-owned programs. However, frequent stock-outs of HIV supplies occur, as, in many cases, the systems for supply of HIV- and non-HIV-related commodities remain separate, and health facilities do not order for HIV commodities when required. The Strengthening Uganda's Systems for Treating AIDS Nationally (SUSTAIN) Project worked closely with and supported 17 public hospitals in Uganda to harmonize ordering for commodities from the governments' National Medical Stores to improve availability and service delivery.
SUSTAIN utilized four low-cost interventions to strengthen supply chain management systems: identification of focal persons responsible for submission of commodity orders to the national stores; training of the order focal persons in ART logistics management followed by onsite coaching and mentoring; telephone text message reminders to the focal persons one week prior to order deadlines; and development of an order checklist to harmonize submission of HIV- and non-HIV-related commodities.
From January to October 2011, average ordering rates for all commodities improved from 46% to 71%. Improvements were noted in order submission rates for HIV-related commodities: from 29% to 81% for ARVs, 24% to 69% for PMTCT commodities, and 22% to 63% for HIV test kits. 54% of facilities harmonized submission of orders for all commodities, an improvement from 12% in January 2011. Improved submission of orders resulted in increased availability of HIV-related commodities and quality of care.
Improving availability of commodities and supplies available through the national system can be achieved by supporting hospitals to utilize low cost, simple innovations to improve ordering rates.
The countries of the Caribbean have collectively committed to the goal of eliminating mother-to-child transmission of HIV and syphilis as public health problems by 2015, by improving the quality of care for pregnant women and babies, including wider and timelier treatment for HIV and syphilis.
Sub regional analysis of the health systems HIV response and progress to the eliminiation initiative (EI) was conducted for selected countries in the English Caribbean. Data on the monitoring events ( 7 output, 5 outcome & 3 impact indicators) for the EI were collected. 2009–2010 data on the HIV health sector progress towards universal access were analysed.
At the end of 2010 Jamaica, Bahamas and Trinidad & Tobago have reported reducing congenital syphilis cases below the target of<0.5 per 1,000 births. None of the countries reported reduced rates of MTCT of HIV to<2%, and pediatric HIV cases <0.3 per 1,000 live births. Rates of HIV screening for pregnant women range from 93% (Belize) to 95% (Jamaica), however only 24.7%–45% of pregnant women accessed ANC at clinics in their first trimester. 76%–96% of women receiving prenatal care in the four countries studied were screened for syphilis in 2010; coverage with ARV medication increased from 40% of HIV-positive pregnant women in 2005 to 92% in 2010, (P<0.001). The percentage of pregnant women diagnosed with syphilis who receive appropriate treatment range from 50% to 100%. Pregnant women having at least one visit with a skilled ANC attendant raised from 60% to 95%, ( P<0.001).
With increase quality service coverage and strengthened commitment, it now seems feasible by 2015 to eliminate new HIV infections and CS among children in some countries of the Caribbean. Challenges include the continued access of ARVs, other essential supplies and human resources.
Providing quality sexually transmitted infection (STI) services acceptable and accessible to high-risk individuals like female sex workers (FSWs) and men who have sex with men (MSM) continues to be a challenge in India. Franchising services through preferred private providers has significantly improved coverage; however, data on quality of care is limited. This paper presents results from a quality assessment of community-preferred private providers contracted in a targeted Pathfinder International-led HIV intervention program with more than 25,000 FSWs and 11,000 MSM in ten districts of Maharashtra.
Pathfinder used data from a clinical quality assessment tool implemented as part of quality assurance and supportive supervision, completed once each quarter for every preferred provider (March 2007 to April 2011). A composite weighted scale was developed with a maximum score of 70 and indicators assigned to six broad areas of assessmentaccess, clinic environment, structural, service delivery, technical, and community involvement. A mean aggregate quality score was computed in each area and also by type of provider, intervention, and setting to assess levels and trends of quality of STI care provision.
Analysis of 290 clinic assessment records for a total of 227 preferred private providers showed progressively increasing trends in all quality areas of assessment irrespective of provider, setting, and intervention type. The average quality score increased from 39.5 in 2007 to 62 by 2011. Non-allopathic and private providers in rural settings showed consistently lower quality scores than allopathic and urban providers, respectively. However, the difference was not statistically significant. An overall 1.5-fold improvement in the quality score was observed, with scores consistently above 78% in the last three years.
Through continuous supportive supervision it is possible to ensure quality of STI care provided by private providers in HIV interventions with FSWs and MSM.
The number of people who inject drugs (PWID) has grown substantially in Tanzania, with increasing significance for HIV control. We determined HIV and hepatitis C (HCV) prevalence, service access and risk behaviours among PWID in Temeke District, Tanzania.
Researchers administered a quantitative survey alongside rapid HIV/HCV antibody testing to PWID in Temeke. HIV, HCV and coinfection prevalence with 95% CIs was calculated with descriptive analyses of service access and risk behaviours.
267 PWID (87% males) reported a median injecting duration of five years (IQR=3–9). HIV prevalence was 34.8% (95%CI=29.1–40.9); 29.9% (95%CI=24.0–36.2) among males and 66.7% (95%CI=49.0–81.4) among females. HCV prevalence was 27.7% (95%CI=22.4–33.5) and similar for males and females. HIV/HCV coinfection prevalence was 16.9% (95%CI=12.6–21.9); 15.2% (95%CI=10.8–20.4) among males and 27.8% (95%CI=14.2–45.2) among females.
Most (73%) positive HIV tests were previously undiagnosed. Over half PWID (53%) had no HIV testing history and 76% had not tested in the past two years. One-third of PWID (34%) reported not knowing where to access HIV testing. Two-thirds (66%) had not heard of HCV and 98% had never tested for HCV.
Common sharing behaviours included injecting with a used syringe (42%) and sharing other equipment (17%) or mixing containers (14%). Nearly one-third (30%) of HIV positive PWID reported recent unprotected sex. About half PWID reported having not received condoms (52%) or clean injecting equipment (54%) in the past 12 months.
HIV prevalence among PWID in Temeke was substantially higher than population estimates (7% males, 10% females). Low HCV awareness and high HIV/HCV coinfection prevalence raise concerns for accelerated HCV disease progression and future liver disease burden in this population. Injecting and sexual risk behaviours, undiagnosed HIV and HCV, and limited access to testing, condoms and clean injecting equipment demand urgent scaling up of prevention and treatment services targeting PWID in Tanzania.
HIV prevalence among Tanzanian adults is 6%; with mother-to-child HIV transmission (MTCT) accounting for one in ten infections. Men play a decisive role in women's utilization of health services, but male involvement with their partners in the prevention of MTCT (PMTCT) program is low. To address this issue, the ACQUIRE Project Tanzania (ATP) has supported the Ministry of Health and Social Welfare since 2008 to involve men in antenatal care (ANC) services in Iringa, the region with the country's highest HIV prevalence (16%). Interventions include partner invitation letters, posters encouraging male attendance, prioritizing clients accompanied by partners, improving couple counseling rooms, training providers on PMTCT couple counseling, and engaging villages to develop local strategies. This evaluation assesses uptake of PMTCT services by pregnant women and their partners before and after interventions began.
A repeated cross sectional study was conducted in 354 facilities in Iringa, Tanzania between 2008 and 2011. Facility-level data on PMTCT service uptake were collected to assess partner HIV testing before and after male involvement interventions. Data were analyzed using descriptive statistics.
The number of ANC partners tested annually for HIV increased from 1,746 in 2007/08 to 10,559 in 2008/09, and 20,758 in 2009/10. Prior to interventions 7% of ANC partners were tested; after interventions 30% of partners tested in 2008/09, 40% in 2009/10 and 50% in 2010/11. While the number of partners tested increased the proportion testing positive decreased below the regional rate from 16% in 2007/08 to 10% in 2010/11. Male participation was higher in facilities with strong community support; for example up to 80% of ANC clients at Mtwango Dispensary were accompanied by partners consenting to test.
Locally initiated interventions on male involvement are crucial for the reduction of MTCT and men can be engaged to utilize and support PMTCT services.
In the United States (US), HIV screening is recommended for all pregnant women. Repeat screening is recommended in jurisdictions with elevated rates of HIV and for women with known HIV risk. This study examined the numbers of women seroconverting prior to pregnancy (PTP) and during pregnancy (DP) and the associated mother-to-child HIV transmission (MCT).
Data from HIV-infected women who delivered live infants from 2005–2010 in 15 US areas that conduct the Centers for Disease Control and Prevention's Enhanced Perinatal Surveillance (EPS) were used. EPS data were linked with National HIV Surveillance data reported through June 2011 to replace missing test dates. We determined the number of PTP- and DP-seroconverters as well as those who could not be classified due to missing testing data. DP-seroconverters had both a documented negative HIV test followed by a positive test during pregnancy or labor/delivery, whereas PTP-seroconverters were diagnosed HIV positive before pregnancy. We calculated the number of MCT among both seroconverter groups. Estimated annual percent change was used to examine trends in the percentages of DP-seroconverters as well as MCT in both seroconverter groups.
Among 10,308 HIV-infected women with live births, 124 (1.2%) were DP-seroconverters, 7,235 (70.2%) were PTP-seroconverters and 2,949 (28.6%) were unclassifiable. A statistically significant 25.0% estimated annual increase in the percent of DP-seroconverters was observed from 2005–2010 (95% CI12.3%–39.1%). MCT occurred among 2.0% of all deliveries; of these, MCT among DP-seroconverters (12.9%) was eight times that among PTP-seroconverters (1.6%)(Z=9.3, p<0.0001). Non-significant decreases in the percent of MCT were observed in both DP- (p=0.8) and PTP-seroconverters (p=0.1) from 2005–2010.
From 2005–2010, there was a significant increase in the percent of women seroconverting during pregnancy. Efforts for consistent early prenatal HIV testing and repeat third-trimester testing should be enhanced and monitored to assure universal timely provision of MCT prophylaxis.
The 2010 WHO PMTCT guidelines highlight the importance of CD4 testing for all pregnant HIV+ women, and strongly recommend lifelong antiretroviral therapy (ART) for everyone with CD4<350 cells/mm3. We examined 2009 and 2010 national data in India, where an estimated 43,000 HIV-positive pregnant women require PMTCT services annually, to assess CD4 testing practices and inform programme policy.
Antenatal HIV testing data were routinely collected from HIV Integrated Counselling and Testing Centres (ICTCs). ICTC and ART centre records in each state were abstracted to evaluate the number and proportion of HIV-infected pregnant women who received CD4 testing, proportion with CD4 count<350 cells/mm3, and number initiated on ART.
Of 5,807,778 pregnant women tested across India in 2009, 18,692 (0.32%) were detected HIV-positive. Among HIV+ pregnant women, 10,192 (54.5%) received CD4 testing, of whom 3,082 (30.2%) had a CD4 count<350 cells/mm3. In 2010, 16,204 (0.24%) of 6,877,617 tested pregnant women were detected HIV-positive. Among these, 9,917 (61.2%) received CD4 assessment, and 3,934 (39.7%) had a CD4 count<350 cells/mm3. In 2010, 2,292 pregnant women started lifelong ART, representing 58% of those with CD4<350 cells/mm3.
Among those tested, approximately 40% of pregnant HIV+ women in India require ART for their own health. However, almost 40% of detected women did not receive timely CD4 assessment or linkage to treatment. In light of these data, the Indian national programme has opted to provide a single maternal triple antiretroviral prophylaxis regimen to all HIV+ pregnant women, irrespective of CD4 count. This “test and treat” approach can be initiated before CD4 results are known, potentially reducing losses to follow-up and delays in ART initiation. Expansion of CD4 assessment remains a high priority in India, as CD4 results will still guide maternal ART duration (lifelong vs. until cessation of breastfeeding).
Prevention of mother-to-child transmission (PMTCT) programs are expanding throughout Africa. It is important to define modifiable barriers in order to improve PMTCT programs. There are few community-based studies that sample a broad population of mothers, including those who never access clinics.
Community-based surveys were conducted in February-June 2011 among women who delivered within the prior year under the Kenya KEMRI-CDC Health and Demographic Surveillance System. Rates of uptake of antenatal care (ANC), HIV testing and maternal/infant antiretrovirals at most recent pregnancy were determined, and barriers were identified in multivariate regression.
Overall, 616/652 (95%) of women accessed ANC. Eighty-four percent of women with negative/unknown HIV status were tested for HIV, 89% of these at first ANC visit. Among 216 HIV-seropositive women, 82% took antiretrovirals for PMTCT (72% antenatally, 62% at labor, 72% postpartum, but only 55% at all three times); 79% of infants received PMTCT antiretrovirals. Receipt of antiretrovirals during labor was significantly associated with facility delivery (69% facility-based vs. 55% non-facility, p<0.03). In multivariate analysis, primary education (5.83; 1.64–20.67) and fewer previous pregnancies (0.65; 0.50–0.85) were associated with accessing ANC. Knowing partner was HIV-tested (4.37; 1.62–11.73), believing MTCT can be prevented (3.31; 1.21–9.06) and having iron roof or better (4.35; 1.17–16.20) were associated with uptake of HIV testing. Among HIV-positive women, knowing partner was HIV-tested (2.39; 1.02–5.61) and ANC attendance (7.81; 1.22–50.02) were associated with uptake of maternal antiretrovirals.
In this community-based survey, uptake of HIV testing and antiretrovirals was relatively high. Although most HIV-infected women received antiretrovirals, fewer received a complete course. Education, PMTCT knowledge, and partner testing correlated with ANC/PMTCT uptake. Our findings suggest that partner testing initiatives may have benefit for PMTCT. Planned initiation of universal antiretroviral therapy for pregnant women in Kenya may improve antiretroviral coverage during labor.
The content and views in this abstract are solely the responsibility of the authors and do not necessarily represent the official views of the affiliated organizations, United States Government, or Government of Kenya.
People living with HIV (PLHIV) face various problems dealing with their relatives. Most often PLHIV in Armenia do not disclose their status to anyone. The disclosure of a childs HIV status or the parents to their children is a particularly sensative issue often leading to pshycological and interpersonal tensions, which leave influence on the health and child-parent relationships. To prevent the above “Positive People Armenian Network” NGO (PPAN) has carried psychological work with PLHIV and their children.
10 trainings with HIV affected children were conducted. It was aimed at extension of the emotional and volitional spheres. 10 children 8–13 years old were selected (8 parents were HIV+, 2 HIV-). The age group was selected as the middle childhood years are significant in reviewing children-parents relationship. Children in middle childhood years are emotionally less active than teenagers. Design of meetings was planned (fables-discussions, games, art-therapeutic exercises). They were aimed at decreasing emotional tension, empathy development, value system identification, emotional harmony. Parenting works were also carried out.
During 10 group meetings it was possible to establish psychological intimacy. Group thinking was strengthened through art-therapeutic techniques “the group tree”. Subsequent works have focused on skill development. Through discussion of Fables and art-therapy childrens vision was enlarged and turned versatile, developing the moral values.
Children learned to understand their emotions properly, see themselves on the underside for understanding others’ reactions through. Parents noted the positive change in children-parents relations with and in school environment.
The values and skills obtained during the meetings will help the children for further disclosure and acceptance of parents’ status, to manage their emotions and feelings not allowing the parents illness to cause pshycological problems in themselves. Currently individual work with parents and children is still ongoing. More similar and parent-children mixed group meetings are planned for the future.
HIV care and treatment services in Ethiopia are mostly facility based and, services are not extended to or followed up at community level. To address this challenge, AIDSRelief introduced the Community-Based Treatment Support Services (CBTS) based on the principle of continuous patient and family engagement in care and treatment to assist HIV patients achieve optimal anti-retroviral therapy (ART) adherence and maintain low rates of loss to follow-up (LTFU) at St. Luke hospital in Oromia.
The CBTS model is composed of adherence counselor, case managers, community nurses and community volunteers (CVs) with distinct responsibilities. This team, established at the facility level, was trained to assist establishment of community level support for HIV patients.
At the facility, the team leads patients through structured treatment preparation prior to ART initiation; conduct home visits; sets up appointment system and same day follow-up for patients who miss clinic appointments; and provides supportive counseling to assist status disclosure and stigma reduction. CVs focus on providing home visits, LTFU tracing, awareness raising, early identification and counseling for OIs, and referring pregnant mothers for prevention-of-mother-to-child-transmission (PMTCT) programs.
Following CBTS start-up in St. Luke hospital, the LTFU rate declined rapidly from 53.1 LTFU/1,000 patient months of follow-up (PMFU) in December 2010 to 47.3 LTFU/ 1,000 PMFU in March 2011. The retention rate has also shown statistically significant increase from 74.8% in March 2009 to 79.6% in March 2011.In addition, the overall patient crude retention rate has increased from 34.4% to 47.5%. Moreover, cross-directional linkages to other community-based programs supporting PLHIV have improved and community-wide disclosure among pregnant women has been promoted.
This experience proves that higher adherence rates can be attained through the CBTS model structured care programs. Such innovative community programs could to be scaled up and replicated for substantial country wide results.
Community support systems are effective structures to provide care and support to most vulnerable children and PLWHA. However, sustainability of such initiatives can be weak. The purpose of this study was to get insight into sustainability issues of World Vision's community based care and support programming in Eastern and Southern Africa.
A qualitative review has recently been conducted, including 13 key informant interviews and 54 focus group discussions with community initiative members, non-governmental organization staff and government representatives in Ethiopia, Kenya, Mozambique, Uganda and Zambia.
The study results indicate that the sustainability of community based organizations depends on several issues, one of the key factors being vertical linking to government structures. These links can take different shapes, ranging from loose cooperation between government officers and community organizations to government representatives becoming members of community organizations’ executive or coordinating boards. Such cooperation can even lead to a full adoption and institutionalization of community care and support initiatives into regional or national government structures, including further roll-out of such systems. The findings further indicate that any form of government structure cooperation with community based care and support systems can improve the community organizations’ sustainability and increase the service delivery of both sides.
As it seems, the stronger the link to government structures, the higher the community groups’ sustainability. However, sustainability also depends on the quality and availability of resources within such cooperation, as well as on the individual commitment of the partners. And finally there is also need to consider the possible pitfalls in government structure co-option, like the loss of access to external NGO funding and a politicization or restructuring of community groups, which can change their initial focus or even undermine and end volunteers’ efforts in providing care and support to vulnerable children and PLWHA.
TB/HIV twin epidemics should be addressed jointly at community level. PLAN Foundation engaged community volunteers (CVs) for improving access to TB/HIV prevention, treatment and care services in 5 Local Government Areas (LGAs) of Ibadan, Nigeria.
24 communities were selected from high TB-prone areas, high density areas and hardly-reached communities. A consensus meeting involving various community stakeholders was held during which 50 CVs were selected to facilitate active referral of TB suspects to DOTS centres and linkage with HIV testing services in ART clinics and other healthcare facilities in the areas.
The CVs were trained on the basics of TB, suspects’ identification, defaulter tracing, adherence support and TB/HIV relationships. They were involved in providing support and follow-up services for smear-positive cases, linkage with HIV testing and treatment for dually-infected persons; and organizing community mobilization.
Monthly CVs review meetings were held while PLAN Foundation staff provided supportive supervision to the CVs in communities.
There were steady and significant increases in number of TB suspects referred by CVs between project onset in June 2011 and October 2011. TB clients referred to DOTS centres for screening were: June-47; July-56; August-118; September-274; October-238 totaling 733 for the period. Of these, 78 of the suspects were smear positive cases while 25 were dual infected with TB and HIV.
Engaging CVs is effective in identifying, referring, supporting and following up TB suspects and persons infected with TB and HIV. There is the need for linkage of TB and HIV services as a strategy for detecting HIV infection among people with TB.
Community mobilization through CVs helps to increase awareness about TB and HIV in communities.
There should be improved support and motivation for CVs as vital linkages to communities TB/HIV interventions.
Effectively reaching PLHIV in rural, hard-to-reach areas is considered critical to improving access to health services and enrolment in care and treatment, which are needed to improve PLHIV's quality of life. Health facilities are located at least 20 kilometers from a farming population in Lamu District, Kenya. Infrastructure is poor and transport is unavailable; therefore, access to information and health services is an uphill battle. PLHIV there have had no option but to default treatment or share drugs.
Through Ministry of Health (MOH) structures, community sensitization was undertaken and trained clinical staff made available. Technical and financial support to conduct monthly mobile clinics was provided through AIDS, Population, and Health Integrated Assistance (APHIA
From June 2010 to December 2011, 304 people were tested for HIV and 6% were found positive. Cumulatively 52 (48 active) patients have received HIV care (18 new) and 37 of them are actively on HAART (20 new). Patient retention is 92% and 87% have disclosed their serostatus to their sexual partners. All patients have had a CD4 test and 6-month monitoring is regularly conducted. The patients have formed a vibrant support group for psychosocial support and economic empowerment.
With structured links to health systems and community involvement, sustainable models of outreach clinics for remote and hard-to-reach areas can be developed to achieve universal access to HIV prevention, care, and treatment. The approach has the potential to promote ART adherence, patient retention on care and treatment, and stigma reduction in the community.
There is a sense of urgency to identify instruments that can promote the health and development of children in poor resource and high HIV prevalence areas of sub-Saharan Africa. This has led to a surge in technical programmes, such as cash transfers, whose new ideas and resources may not necessarily resonate with local support structures. This paper describes the effects of a community-led cash transfer programme in Manicaland Province, Zimbabwe, and explores how community members appropriated ‘techno-fixes’ in ways that benefits them and achieve buy-in.
We report on data from 35 individual interviews and three focus group discussions, involving 24 key informants, 24 cash transfer beneficiaries, of which four are children, and 14 non-beneficiaries.
A thematic network analysis found the cash transfer programme to have had a substantial effect on children's schooling and education, mainly through improved access, concentration and ultimately performance, but also on their health and well-being, primarily through improved food intake, access to healthcare and reduced levels of stress and worry at a household-level. The programme was also said to provide the beneficiaries with an important platform to reduce levels of poverty, primarily through investment opportunities arising from having greater disposable income. Encouraged by local participation in the programme, conditionalities and witnessing its positive effects, both benefiting and non-benefiting community members spoke enthusiastically about the programme and highlighted their preference for conditional cash transfers-indicating community-wide buy-in and involvement.
We conclude that despite their invasiveness, cash transfer programmes can, if implemented through local community structures, be appropriated at a community-level in ways that facilitate health-enabling and supportive social environments for vulnerable children and youth in sub-Saharan Africa.
The advent of UNAIDS “Treatment as Prevention” policy of global antiretroviral (ARV) scale-up is not only life-saving for HIV-positive health care users, but may also hold significant potential to improve health care worker clinical practices, professionalism, and workplace morale. This research investigates the impact of ARV service delivery programming on health care workers′ clinical practices and personal and workplace morale in a rural western Ugandan setting.
As part of a larger community study (N=142, 48% female), individual qualitative interviews were conducted with health care workers (n=15, 47% female) from Rwimi clinic in Kabarole District, during a four-month period. Clinic practices were also assessed through 40 hours of participant observation. Findings were coded and analyzed using Attride-Stirling′s Thematic Network Analysis, and further mapped for discussion using the Social World Triad, a conceptual model developed for this study.
ARVs are seen as being a positive, helpful force which allows health care workers to re-frame their clinical practice and professional identity to one of mastery over the illness. Their representations of HIV/AIDS are correspondingly beginning to change, to that of an illness which is manageable and controllable with drugs, thereby providing health care workers with renewed hope and a sense of clinical competence not felt since before the HIV/AIDS era. Professional practice is improving through more positive clinical interactions between health care workers and clients, which could lead to improved health service programming uptake by the community.
The clinical use of ARVs has also improved the personal coping strategies, workplace morale, and professionalism of health care workers at this clinic. Such findings encourage increased advocacy and community outreach efforts regarding ARV service provsion, and suggest that ARVs could be utilized in concert with broader social and biomedical interventions and programming, as a social change agent to reduce stigma and improve health service uptake.
As the efforts to tackle the HIV/AIDS pandemic have become increasingly more refined, the dialogue about the ways in which the local understandings of gender and sexualities and dynamics of stigma shape the success or failure of treatment and prevention efforts has come to the fore. This presentation examines one such example in which the complex social, political, and health entanglements affect—in differing and often unexpected ways—the efforts to provide HIV-related prevention and treatment services. Specifically, it provides a qualitative socio-political analysis of the debates around the emergence of an HIV-focused clinic in Mexico City, where after the approval of a local law more comprehensive health care services for trans individuals were also to be delivered.
It is based on fourteen months of ethnographic research in Mexico City conducted in two phases spanning 2009–2011. It draws on qualitative interviews, observations and analysis of relevant textual documents, and provides a qualitative “bottom-up” analytical approach to the gathered data.
The results show that the emergence of a transgender health program delivered in what is known as the HIV clinic sparked a heated controversy between supporters and detractors. Due to the intersections between structural vulnerabilities and existing dynamics of stigma around HIV, many trans women—and particularly sex workers—prevented themselves from accessing HIV-related and other health care services available to them in this hospital. Many of them did not want to be seen as potentially HIV positive individuals, as this may have brought both negative social and labour consequences into their lives.
The predicament between accessing or not health services in a clinic tainted with stigma had direct implications for the successful prevention and treatment of HIV and other illnesses. This study supports the view that local sociocultural and political dimensions need to be fully considered when envisioning HIV-specific programs.
Relatively few systematic intervention studies have tested the efficacy of female condom (FC) promotion. We compared the efficacy of an enhanced two-session cognitive-behavioral intervention versus a single-session 'basic' intervention to promote the FC among female students at a South African tertiary institution.
A total of 296 women students were randomized to either the two-session (N=147) or one-session (N=149) intervention. Both arms received HIV/STI/pregnancy prevention information in group settings, including information on FC use and modeling of FC insertion. The two-session additionally covered partner negotiation, FC insertion practice, and personal goal-setting to achieve HIV/pregnancy prevention. Both groups had access to male and female condoms. Intervention effects were assessed at 2.5 and 5 months post-intervention. Data were analyzed using GLM, with GEE to adjust for within-subject correlation.
Both groups reported significant reductions in the number of vaginal intercourse occasions unprotected by either male or female condoms from baseline to the 2.5 and 5-month follow-ups. There were no differences between groups at either time point. Similarly, the number of FCs used increased significantly in both groups at first and second follow-ups; differences between groups again were non-significant at both time points.
These findings suggest that both interventions decreased unprotected sex and increased the number of FCs used. The shorter, single-session group-based intervention, which could easily be delivered in a clinic waiting room, holds promise in resource-constrained settings. The relatively short follow-up period does not allow us to rule out the possibility of differential longer-term effects between groups.
As medical male circumcision services are scaled up in sub-Saharan Africa, understanding circumcision's impact on sexual satisfaction, attitudes and behavior of female partners of recently circumcised men is crucial to women's well-being and program success.
We conducted a longitudinal study of behavioral risk compensation following circumcision among 18–35 year-old men in Western Kenya. Men circumcised during the study were asked to refer their female partners to be interviewed for the study.
We recruited 101 women (median age 21; IQR 19–24) who were in a relationship with the referring man before and after his circumcision. All female participants reported being satisfied with their partner's decision to become circumcised and his sexual performance after circumcision. Ninety-six percent were satisfied with the appearance of partner's penis and 91% reported enjoying sex more after circumcision. Most women (84%) reported having no or small chance of getting HIV; 38% attributed this low risk to their partner's new circumcision status. Eighty-eight percent felt more protected from sexual diseases after their partners′ circumcision. Overall, women and men held similar beliefs about circumcision. However, attitudes that could potentially lead to risk compensation were reported more frequently by women than mennow that circumcision is available, condom use is less necessary (7% men, 35% women, OR=7.00; 95%CI 2.93–16.73); I am less worried about HIV (16% men, 36% women, OR=2.88; 95%CI 1.47–5.65); I am more likely to have more than one partner (6% men, 18% women, OR=4.42; 95%CI 1.42–13.75); I am more likely to have sex without a condom (6% men, 18% women, OR=3.33; 95%CI 1.26–8.78).
Women have favorable attitudes toward male circumcision. While men are counseled about the partial protection of circumcision against HIV during the procedure, they do not appear to share this information with their partners. There is need to target women with education on male circumcision.
In 3 randomized trials, medical male circumcision (MMC) reduced HIV acquisition in heterosexual men in sub-Saharan Africa by ~60%. At the end of the trials men initially randomized to control (delayed circumcision) were free to become circumcised. Men may choose circumcision differentially, which may confound observed efficacy. We estimated the 66-month efficacy of MMC against HIV among men retained int he Kisumu randomiezd trial, accounting for time-varying confounding and loss to follow-up.
From 2002–2005, 2,784 men aged 18–24 were enroled and randomized 1:1 to immediate circumcision or control. At trial end in December 2006, control men were offered free circumcision. Follow-up continued through September 2010. HIV status, STIs, and behavior were assessed at baseline and 6-month intervals thereafter. Cox proportional hazards regression incorporating time-dependent inverse probability of treatment weights (IPTW) generated through marginal structural modeling estimated the efficacy of circumcision on HIV risk.
The cumulative 66-month HIV incidence was 7.21% [95% CI 5.90–8.75%] 4.34% among circumcised men, 10.9% among uncircumcised men. The crude hazard ratio (HR) of HIV seroconversion for circumcised vs. uncircumcised men was 0.38 [95% CI0.25–0.55]. In conventional Cox regression adjusted for variables significant at the p<0.05 level (age, condom use, incident HSV-2, GUD, penile injuries, incident gonorrhea or chlamydia), the HR was 0.46 [95% CI0.31–0.69]. In IPTW-adjusted multivariable Cox regression, the HR was 0.43 [95% CI0.26–0.71].
The efficacy of MMC was sustained at 57% at 66 months, similar to overall findings of the 3 trials under conditions of randomization (58% at 24 months). The effectiveness of circumcision against HIV acquisition in program implementation may vary due to differential selection of circumcision. These findings provide the best evidence to date of the long-term efficacy of circumcision against HIV acquisition.
The effect of the roll-out of voluntary medical male circumcision (VMMC) in Southern and Eastern Africa on HIV prevalence is unknown. Three years after the beginning of the Orange Farm (South Africa) VMMC roll-out (ANRS-12126), the project′s impact on HIV prevalence over time was assessed.
Two cross-sectional surveys were conducted, one in 2007–2008 (n=1971), before the beginning of the roll-out, and one in 2010–2011 (n=3268). The response rates exceeded 80%. Male residents aged 15 to 49 were randomly sampled, interviewed, assessed for circumcision status, counselled and tested for HIV. Blood samples were tested for HIV and for recent HIV infection using the BED assay. Prevalence ratios (PR) were calculated using multivariate (aPR) and propensity score weighted (wPR) Poisson regression models to assess the association of HIV prevalence and recent HIV infections with time and circumcision status. Covariates comprised age group, ethnic group, religion, having children, alcohol consumption, education level, age at first sexual intercourse, marital status and occupation.
In three years, male circumcision prevalence standardized on age increased from 17.0% to 53.9%. In the 2007–2008 and 2010–2011 surveys, the propensity weighted effect of circumcision status on HIV prevalence was wPR=0.37 (95% CI0.19–0.58) and wPR=0.48 (95%CI0.35-0.65), respectively. HIV prevalence standardized on age decreased from 12.5% to 9.3%, aPR= 0.74 (95% CI0.60–0.91), among participants aged 15 to 49, and from 6.2% to 4.2%, aPR=0.64 (95% CI0.49–0.85), among participants aged 15 to 29. Propensity analyses showed that the intervention avoided 536 (95% CI 135–1318) HIV infections in 2011 among the 52,000 adult men living in Orange Farm.
This study shows for the first time that the roll-out of VMMC in Africa can, if successfully promoted, lead to a decrease of HIV prevalence over time, detectable three years after its onset.
Male circumcision (MC) reduces men's risk of HIV infection, but conventional techniques require a high degree of surgical skill. The Shang Ring provides a minimally-invasive, simpler and faster approach that requires neither hemostasis nor suturing and potentially reduces training time. Trial objectives were to compare pain, acceptability, safety and ease of use of the Shang Ring versus conventional techniques.
The Shang Ring consists of inner and outer rings, with eversion of the foreskin over the inner ring before application of the outer ring and excision of the foreskin from the underside. Ring removal is done one week later. We conducted a randomized controlled trial of the Shang Ring versus conventional MC techniquesforceps guided technique in Homa Bay, Kenya, and dorsal slit technique in Lusaka, Zambia. We used a visual analog scale to evaluate pain at five time points in the first 48 hours after surgery. Clinicians graded adverse events as mild, moderate and severe based on PSI/WHO criteria, and took photographs to document adverse events and healing.
We circumcised 200 men at each site. The duration of Shang Ring procedures was significantly shorter at both sites (p<0.0001), with medians of 7 versus 20 minutes. At the two-day visit, pain was similar between groups at both sites. Adverse event rates were 3% among both study groups. At the 60-day visit, more men in the Shang Ring group were “very satisfied” with cosmetic appearance, 96% vs 85% (p<0.02) in Kenya, and 97% vs 70% (p<0.01) in Zambia. Regarding ease-of-use, five of six clinicians had a "strong preference" for the Shang Ring and considered it "much easier" to use, and none preferred the conventional technique.
Scale-up of MC in Africa should be easier when providers begin routine use of devices such as the Shang Ring.
Systematic efforts to develop a national GIPA policy can help change the HIV landscape towards improving the quality of life for PLHIV in Malawi. The GIPA Report Card, a measurement tool, which assesses the application of the GIPA principle in the national response to HIV, was conducted in Malawi to identify specific barriers and opportunities to involving PLHIV in the development of policies and delivery of programmes.
The Malawian Network of People Living with HIV conducted a nationwide assessment. The study included knowledge about the principle, PLHIV involvement in policy development at district level, inclusion of GIPA in the National Action Framework, universal access targets, and the involvement of PLHIV in research. The data collectors, all PLHIV, also visited institutions that have HIV workplace programmes.
The respondents represented varied institutions: 33% PLHIV; 27% Civil Society Organisations, 14% government departments or other public institutions; 5% UN agencies; 9% International NGOs, 5% parastatal organisations; and 5% private sector. Respondents stated there were fewer efforts in ensuring that the principle is practiced at local and community levels. Inclusion of GIPA in the Malawi HIV Extended National Action Framework signals the increase in efforts by the government to embrace the principle. However, there is no proactive communication and outreach campaign regarding the principle. Stigma and discrimination remain the greatest barrier to the GIPA.
Malawi must increase awareness and funding to support activities highlighting the meaningful involvement of PLHIV and to address the barriers to the effective application of the GIPA. A multi-sectoral communication's strategy and systematic efforts to develop national GIPA guidelines would help to promote the involvement of PLHIV and contribute to the quality of Malawi's HIV response.
Despite the growing number of women infected with HIV/AIDS in China, insufficient attention to women living with HIV/AIDS in the Global Fund's strategy and structure continue to increase the vulnerability and participation of women. No expenses in the budget are designated for projects focused on women, and all 11 NGO representatives in the Country Coordinating Mechanism (CCM) are men.
The Henan Women's Network works to mainstream gender-based approaches into China's national AIDS strategy in order to help women gain improved access to HIV/AIDS prevention, treatment and care services. The Network used three major strategies to advocate within the national Global Fund mechanism as entry point for broader national advocacy: working with female PLWHA to conduct needs assessment, and strengthen collaboration between women's health organizations; submit recommendations and mobilize international organizations including UNWOMEN and UNAIDS to incorporate gender concepts into the revision of CCM regulations; and cooperate with the All China Women's Federation on gender rights awareness trainings for health departments, the Federation and female PLWHA.
After incorporation of a gender strategy into the CCM regulations and a targeted call for applications from women, 40% of 2011 CCM NGO consultants are women. Their increased participation in decision-making process will further the development of gender-based approaches in budget and strategy.
Actual needs of women living with HIV/AIDS have been successfully passed on through a local grassroots network. Integrated support from international organizations, the government, scholars, experts and community organizations was instrumental in achieving consensus in a gender-based strategy. Moving forward, capacity-building for women living with HIV/AIDS need to enable greater participation, and needs assessments will need to clarify suitable targets for women. Budgets promulgated by the CCM need to define specific expenses to implement gender-based strategies.
Rwandan women and girls are disproportionately impacted by HIV and comprise the largest percentage of those infected. Progress has been made to promote gender equality and women's participation in decision-making. However, women living with HIV (WLHIV) remain underrepresented in the national HIV response. Experience shows that HIV policies and programs often fail when women are excluded from shaping their content and direction.
The authors examined three case studies to determine best practices for ensuring meaningful involvement of WLHIV (MIWA): (1) National Accelerated Plan for Women, Girls, Gender Equality and HIV (2010–2014); (2) EC-UN Women “Supporting Gender Equality in the Context of HIV/AIDS” program; and (3) National Strategic Plan for the Elimination of Mother-to-Child Transmission of HIV (2011–2015).
Participatory processes (workshops, focus groups, and in-depth interviews) involving women, including WLHIV, were used to identify key priorities, activities, and HIV strategies for the National Accelerated Plan (2010) and eMTCT Strategy (2012). The EC-UN Women project fostered HIV-positive women's leadership through training, technical assistance, advocacy planning sessions, and the development of formal mechanisms within national AIDS coordinating bodies that include women.
These case studies demonstrate that WLHIV's active participation in national HIV policies and strategies development increases their visibility and ensures that their needs and rights are prioritized (e.g. requirement of future participation of women and girls in national HIV decision-making). Such practices enable WLHIV to be directly involved in implementation, monitoring, and evaluation of strategies they helped to develop (e.g. by aligning their own programs and budgets to the National Accelerated Plan).
These Rwandan cases highlight MIWA's positive impact and the need for concentrated commitment to actively involve WLHIV in decision-making. Deliberate programming is needed to ensure that women and girls are empowered and equipped to transform national agendas to protect and promote their needs and rights in the HIV context in all countries.
The UK is currently reforming its benefits system to encourage people with health conditions to return to work. However, research indicates that unscrupulous employers were using pre-employment health questionnaires to discriminate against people with health conditions in the recruitment process. Research from 2006 found that when a non-disabled person and a disabled person who disclosed their disability applied for the same job, non-disabled applicants were invited to twice as many interviews. Research also shows that fewer than 50% of people diagnosed with HIV in the UK are in employment.
NAT brought together a coalition of organisations including mental health and HIV charities, to campaign to prohibit the use of health questionnaires before a job offer. The Equality Bill was identified as an appropriate piece of legislation to outlaw the use of these questionnaires. Media coverage of the impact of discrimination on people living with HIV and mental health conditions raised the profile of the campaign. The coalition submitted evidence to Parliamentary Select Committees including international examples and secured cross party support for the campaign in the House of Commons. After initially rejecting the idea, the Government met with the coalition and representatives living with HIV to listen to our concerns.
The Government accepted the evidence presented by the coalition and tabled an amendment to the Equality Bill prohibiting the use of pre-employment health questionnaires. The Equality Act came into force in October 2010 and it is now unlawful for employers to ask health related questionnaires before a job offer. This is a significant step forward in ending discrimination in recruitment for people living with HIV.
There are significant gains to be made in building coalitions with organisations supporting people with other health conditions that face similar stigma and discrimination to campaign to reform legislation.
The crises of preventable maternal death and HIV are interrelated. In Uganda, a country with HIV treatment coverage rates of 47%, 16 women die unnecessarily in childbirth daily, one in five new HIV infections is through vertical transmission, and untreated HIV contributes to 25% of preventable maternal deaths. In addition, stigma as well as lack of basic health equipment such as gloves for midwives contributes to reluctance of health professionals to attend to HIV positive mothers, particularly in remote areas.
Civil society networks in Uganda formed a diverse coalition to implement a legal and advocacy strategy to challenge the epidemic of preventable maternal death in Uganda. Combining a Constitutional Court challenge with a challenge in the court of public opinion, the coalition used grassroots mobilization, media work, budget monitoring and advocacy in Parliament to create a common platform for health justice advocacy, prioritizing issues ranging from HIV treatment access to the shortage of nurses. The multiple factors that contribute to preventable maternal death, including HIV, created multiple opportunities for high impact civil society advocacy.
This new civil society coalition has been able to break down walls between disease- and condition-specific advocacy and implement bold campaigns to scale up access to essential health services in Uganda. In one year, the coalition has supported an historic legal challenge on the right to health and preventable maternal death; has successfully advocated for increased investments in increased professional health worker recruitment, remuneration and equitable deployment; and has generated national and international press attention to the human right to health in Uganda.
We have developed an approach to advocacy in Uganda that joins civil society focusing on a wide range of diseases and conditions in synergy. Uniting around key policy opportunities has resulted in important progress in the struggle for health for all.
The purpose of the study was to identify barriers to and opportunities for evidence-based policy on illicit drugs in four sub-Saharan African countries-Tanzania, South Africa, Nigeria and Ghana. Small-scale studies indicate that HIV transmission is linked to injection and non-injection drug use in all of these countries.
A policy case study for each country was developed through in-person semi-structured interviews with key informants, including policy-makers, academic experts, NGO representatives and representatives of international organizations.
The lack of population-based data and trend data on the extent of drug use and drug injection is an impediment to policy-making, leading to denial at policy levels. In all four countries, the law allows the police wide latitude in search, seizure and arrest of people accused or suspected of committing minor drug crimes, who can receive long prison sentences. Except in Tanzania, there are very limited affordable options for evidence-based drug treatment, especially of opioid dependence. Pressure from the US and other external actors to demonstrate interdiction successes may contribute to the domination of the policing-based drug policy. South Africa's drug policy-making has been walled off from HIV policy-making bodies. Ghana is seeking to generate population-based data on the extent of drug injection as health authorities bemoan the weakness of mental health and drug treatment services.
Criminalization and policing dominate drug policy in most of these countries, impeding evidence-based responses to HIV. There is an urgent need to build capacity among policy-makers and civil society to rethink entrenched policing-based approaches to drug use and to raise awareness of affordable evidence-based services for people who use drugs. The advances made in Tanzania's methadone program, including new awareness by the police of the importance of public health approaches, should be an occasion for learning across the continent.
Cameroon is among the 22 countries with the highest burden of PMTCT unmet needs. In response to the global commitment to eliminate new infections among children and keep their mothers alive, Cameroon decided to develop a bold PMTCT scale-up plan aligned with the global MTCT elimination targets. To develop an evidence based plan, the MoH with support from its partners conducted an analysis of programme bottlenecks, disparities using an innovative tool based on marginal bottleneck budgeting (MBB).
The tool was used to analyze bottlenecks and disparities on the demand and supply of MNCH and PMTC services. Key steps included: advocacy and consensus building on the methodology, key tracer interventions; consensus on bottlenecks and disparities, causal analysis and identification of corrective actions.
Key bottlenecks are presented in
The bottleneck analysis has proven to be a powerful method for identifying and analyzing disparities and factors impeding the performance of MNCH and PMTCT programmes. To generate accurate information and draw relevant conclusions quality data are a prerequisite. This was one of the major challenges which were addressed by actively collecting data by field actors. Choices and decisions on key bottlenecks should be based on their potential impact on programme performance, relevance and feasibility of corrective strategies, and availability of resources for action. Participation of all level-stakeholders from the onset of the process increases ownership, and recognition of findings. Results from the analysis were precious inputs for the development of the national mother to child transmission elimination Plan. The plan provides a framework for strategic vision and coordination for all actors and is therefore a structured road map to achieving the set targets for PMTCT by 2015.
In 2006, the estimated prevalence of circumcised men in Swaziland was 8%. Between 2007 and 2011, approximately 35,000 men underwent voluntary medical male circumcision, including 11,000 men circumcised in 2011 during a national HIV prevention campaign, “Soka Uncobe”. The Swaziland HIV Incidence Measurement Survey (SHIMS), a nationally representative, household-based survey of men and women in Swaziland, conducted independently from the circumcision campaign, assessed health behaviors and HIV prevalence.
Between December 2010 and June 2011, SHIMS participants were surveyed about demographic, clinical and behavioral factors, including self-reported circumcision status by men. All participants underwent HIV testing.
Of 18, 212 adults surveyed in SHIMS, 7075 (39%) were men and 16% (1105/7075) were circumcised. Median age at circumcision was 20 y (interquartile range (IQR) 8, 17), median time since circumcision was 1.1 y (IQR 0.6-8.0), and 172 men (16%) reported circumcision in the prior 6 months. Prior HIV testing was more commonly reported by circumcised compared to uncircumcised men (77% vs. 49%, p<0.001). Circumcised men were slightly more likely than uncircumcised men to report always using a condom in the prior 6 months (39% vs. 33%, p=0.003) and the number of reported sex partners in the prior 6 months did not differ (1.4 vs. 1.5). Circumcised men had a significantly lower HIV prevalence compared to uncircumcised men (14% vs. 24%, PR=0.61, 95% CI=0.52–0.70).
While the prevalence of male circumcision in Swaziland remains low, it has doubled in the past 5 years from 8% to 16%. Circumcised men in Swaziland do not report riskier sexual behavior and are more likely to have been tested for HIV, compared to uncircumcised men. HIV prevalence was significantly lower in circumcised men, reinforcing the evidence for a protective effect of male circumcision provided as a population-level (non-research) intervention.
Similar to many developed countries, syphilis has re-emerged in Australia. Dramatic increases in syphilis have overwhelmingly affected men who have sex with men (MSM) and appear likely to have contributed to increased HIV transmission. In response, high MSM caseload clinics in Melbourne, Australia, have maintained strategies to increase routine syphilis serology for MSM since 2005. We examined trends in syphilis testing and incidence among MSM attending high caseload clinics in Melbourne to investigate the effect of this intervention.
Syphilis testing data among MSM attending high caseload clinics in Melbourne (January 2007 to December 2010) were analysed. Testing rates and infectious syphilis incidence were determined over time and disaggregated by HIV status and MSM reporting ‘high risk’ behaviours. Poisson regression was used to test the significance of trends.
Over four years 16,806 syphilis tests among 6,441 HIV positive MSM and 24,142 syphilis tests among 17,440 HIV negative MSM were conducted. Average annual increases in testing among HIV positive and negative MSM were 7% (95%CI=6%–8%) and 12% (95%CI=11%–13%), respectively. Between 2007 and 2010 infectious syphilis incidence declined from 6.07 to 2.58 per 100PY among HIV positive MSM (21% average annual decline), and from 3.27 to 0.96 per 100PY among HIV negative MSM (29% average annual decline). Incidence declined even more substantially in ‘high risk’ HIV negative MSM – 4.10 to 0.76 per 100PY among those reporting >10 partners in the previous 6 months and 4.29 to 0.82 per 100PY among those reporting inconsistent condom use. All testing and incidence trends were statistically significant (p<.01).
Enhanced routine syphilis testing appears to have contributed to increased syphilis detection and subsequent reductions in syphilis incidence among MSM. These findings support recommendations that syphilis can be controlled among MSM by sustaining high frequency testing, with important implications for HIV control among MSM.
In 2010, Hispanic migrants accounted for 37% of the total Hispanic population in the U.S. Relatively little is known about the high-risk sexual behaviors that may place Hispanic migrant men who have sex with men (HM-MSM) at risk for infection with sexually transmitted diseases (STDs).
During 2005–2007, 2576 Hispanic migrants were surveyed at three community-based organizations offering services to migrant communities in five states with small Hispanic populations. Participants reported demographic characteristics, sexual risk behaviors, migration patterns, and STD diagnoses (syphilis, Chlamydia and gonorrhea) in the past year. Factors associated p<0.05 with an STD diagnosis in the past 12 months among HM-MSM in bivariate analyses were included in a multivariate logistic regression model.
Of 1550 Hispanic migrant men surveyed who reported having sex in the past 12 months, 353 (23%) reported sex with a man. Of these 353 men, 26% reported being married, 46% self-identified as being heterosexual, and 21% as bisexual. In the past 12 months, 58% reported anonymous sex, 57% reported having sex under the influence of alcohol or drugs, 40% reported unprotected receptive anal sex, and 8% reported receiving money or goods for sex. Twenty-nine (8.2%) men received an STD diagnosis. Only marital status and receiving money or goods for sex were significantly associated with the outcome variable in bivariate analyses. In the multivariate logistic regression model, men who reported receiving money or goods for sex had increased odds of an STD diagnosis (adjusted odds ratio=3.1, 95% confidence interval=1.1–8.7); marital status was not significantly associated with STD diagnosis.
The prevalence of sexual risk behaviors and STD diagnoses was high in this population. STD prevention interventions tailored to non-gay identifying MSM are needed for HM-MSM. Work is needed to understand factors contributing to participation in exchange sex among HM-MSM.
Antiretroviral pre-exposure prophylaxis (PrEP) for HIV-1 prevention was highly effective in reducing HIV-1 acquisition by HIV-1 uninfected partners in African heterosexual HIV-1 serodiscordant couples in the Partners PrEP Study (efficacy67% for oral TDF PrEP and 75% for oral FTC/TDF PrEP). In that study, the placebo arm incidence was 2%/year. However, PrEP was not effective in two studies among high-risk African women (incidence 5-6%/year) leading some to hypothesize that PrEP would not have efficacy in high-incidence subpopulations.
The Partners PrEP Study is a randomized, three-arm trial of daily oral TDF and FTC/TDF PrEP provided to HIV-1 uninfected members of serodiscordant couples from Kenya and Uganda. At enrollment, HIV-1 infected partners were not eligible for antiretroviral therapy under national guidelines. We assessed the efficacy of PrEP among the subset of couples who had higher baseline risk characteristics. Higher-risk couples were defined using a previously-reported risk score composed of baseline factors associated with HIV-1 transmission (Kahle CROI 2012), including age of the HIV-1 uninfected partner, number of children together, circumcision status of the male HIV-1 uninfected partner, married and/or cohabiting, self-reported unprotected sex, and plasma HIV-1 RNA concentrations in the HIV-1 infected partner.
Among 4747 HIV-1 serodiscordant couples in the Partners PrEP Study, 1085 (22.9%) were classified as higher-risk. HIV-1 incidence was 5.0/100 person-years (95% CI 3.3–7.2, 28 transmissions) among those assigned placebo, 1.3/100 person-years (95%CI 0.5-2.8, 7 transmissions) among those assigned TDF PrEP, and 1.1/100 person-years (95%CI 0.4–2.4, 6 transmissions) among those assigned FTC/TDF PrEP, resulting in an estimated PrEP HIV-1 protection efficacy of 72% for TDF PrEP (95%CI 35-88%, p=0.02) and 78% for FTC/TDF PrEP (95%CI 46–91%, p=0.006).
PrEP provided substantial protection against HIV-1 acquisition for higher-risk HIV-1 serodiscordant couples. Higher-risk HIV-1 serodiscordant couples could be a priority population for PrEP implementation.
The iPrEx study demonstrated that pre-exposure prophylaxis (PrEP) can reduce HIV incidence among at-risk men who have sex with men (MSM). However, risk compensation (RC) could negate the benefits of PrEP.
After the release of iPrEx results, North American members (n=5035) of an Internet social network for MSM completed an online survey regarding PrEP. Demographics, sexual risk behaviors, PrEP interest, and anticipated RC with PrEP use were assessed through self-report. Multivariable logistic regression procedures adjusted for age, race/ethnicity, and education examined the association between sociodemographic variables, sexual risk behaviors and anticipated RC.
The mean age was 39 (SD=12.8), 90% were from the US, 84% were homosexual/gay and 84% were white. Ninety-three percent completed some college, 68% earned ≥$30,000/year, 25% had a history of depression, and 5% had received substance abuse treatment. Sixty-one percent indicated unprotected anal intercourse with ≥1 male partner in the prior 3 months (UAI-3), and 24% reported UAI after ≥5 drinks. On a scale of 1 (no risk) to 10 (high risk), the average self-perceived risk of HIV acquisition was 3.3 (SD=2.3). Seventy-five percent reported interest in using PrEP. While using PrEP, 20% anticipated they would decrease condom use for insertive anal sex, whereas 14% indicated they would for receptive anal sex. Factors associated with increased odds of anticipated RC for insertive anal sex were UAI-3 (aOR=1.58; 95% CI1.22–2.04; p=0.0005) and prior substance abuse treatment (aOR=2.04; 95% CI1.32–3.16; p=0.002). Factors associated with increased odds of anticipated RC for receptive anal sex were UAI-3 (aOR=1.57; 95% CI1.16–2.13; p=0.004), UAI after ≥5 drinks (aOR=1.43; 95% CI1.09-1.88; p=0.01) and increased self-perceived risk of HIV acquisition (aOR=1.10; 95% CI1.05-1.17; p=0.0003).
A substantial minority of MSM using an Internet social network anticipate increased unprotected anal sex with PrEP use. Interventions to minimize risk compensation are warranted.
Northern Thailand has a high burden HIV epidemic among MSM and TG. Oral PrEP with Truvada has demonstrated efficacy in preventing HIV among MSM in Chiang Mai, Thailand - an iPrEX trial site. Determinants of PrEP acceptability are needed to gauge the potential uptake of this prevention strategy.
From January to February 2012, 238 MSM and TG participants, who self-reported as HIV-uninfected or of unknown status, completed a self-administered survey on hand-held computers. Participants were recruited by venue-day-time sampling and asked to rate their likelihood of using PrEP at 50% efficacy. PrEP acceptability was defined as "very likely" to use PrEP. Odds ratios and 95% CIs were calculated to identify determinants of acceptability.
131 MSM and 107 TG responded, with mean ages of 23.7 and 21.8, respectively. 29% of MSM and 34% of TG had unprotected anal sex with a male/TG partner in the past 6 months. Prior awareness of PrEP was high, at 66% among both MSM and TG. 41% of MSM and 37% of TG were "very likely" to use PrEP; including respondents who were "somewhat likely" to use PrEP, the rates increased to 75% and 77% of MSM and TG, respectively. Among MSM, factors associated with PrEP acceptability included a prior history of STIs (AOR 3.9; 95%CIs 1.6-9.8) and strong confidence in taking daily medications for 1 year (AOR 2.7; 95%CIs 1.2-6.2). Among TG, factors associated with acceptability included prior awareness of PrEP (AOR 3.3; 95%CIs 1.2-9.0) and having private insurance (AOR 5.0; 95%CIs 1.3-19.0).
PrEP acceptability was higher among MSM and TG who were aware of their HIV/STI risk and of PrEP, among those who had strong medication management skills and private insurance. These findings suggest that knowledge of PrEP and perceived HIV risk, as well as financial support for PrEP, are important determinants of uptake.
The U.S. Food & Drug Administration prohibits men who have sex with other men (MSM) from donating blood. The policy does not consider the potential donor′s HIV status, sexual activity, or relationship status. The current policy allows other populations at elevated risk of HIV to less restrictive deferrals, or no deferral at all. This type of policy reinforces incorrect information about the spread of HIV. Most countries have permanent deferrals of MSM blood donors despite chronic blood shortages. Reform of U.S. blood donation guidelines is necessary to maximize blood donations and improve blood safety protocols.
Advocates, including public health and blood bank communities, blood product recipients, and NGOs, have pushed for a more equitable policy. In May 2010, GMHC joined with leading hemophilia advocates, who also represent a population disproportionately impacted by HIV, to raise awareness of blood donation guidelines in agreement that: the current deferral of all MSM results in a large number of potentially eligible blood donations; and that there are some high-risk activities permissible from heterosexual donors under the current policy. Both perspectives were presented to the Advisory Committee on Blood Safety and Availability (ACBSA) in June 2010.
The ACBSA recommended that sufficient data were not present at that time to change the policy banning MSM, that current blood donation guidelines are “suboptimal”, and that a robust research agenda be pursued in order to carefully examine alternative policies. Government agencies are now implementing the ACBSA's recommendations to identify a subset of low-risk MSM blood donors. In tandem with U.S. momentum for reform, a number of other countries, including the United Kingdom, have made reforms to their policies to allow MSM to become blood donors.
International momentum to revise blood donation guidelines for MSM advances efforts to maximize blood safety, increases blood donation, and decreases stigma of MSM.
Political leadership is known to affect HIV prevention and treatment. In this presentation, we aim to show the dimensions of political governance that are associated with ART coverage using country-level data.
Percentage of ART coverage from 2004 to 2009 as estimated by UNAIDS is analysed as the outcome measure. High-income countries as defined by the World Bank in 2009 are excluded from the analyses. Measures of political governance and GDP per capita (log-transformed) from the World Bank, adult literacy rate (UNESCO, reflected and log-transformed), gender inequality index from World Development Report 2011, skilled birth attendance at delivery compiled by World Bank (reflected and log-transformed) and disability adjusted life years (DALY) due to non-HIV causes and international (HIV and non-HIV) health funding per capita from the Institute of Health Metrics and Evaluation were fitted separately as predictors in multi-level linear models, in which year of reporting or estimation and HIV prevalence from UNAIDS are included. Missing data were imputed for fitting a final model that includes year, HIV prevalence and variables that remained statistically significant.
Higher levels of ART coverage are significantly associated with higher GDP per capita (p< 0.001), lower gender inequality (p=0.003), higher literacy rate (p=0.011), higher percentage of births attended by skilled attendants (p=0.002) and less DALY due to non-HIV causes (p=0.033). It is also highly significantly associated with better political governance in all six dimensions( political stability, voice and accountability, control of corruption, rule of law, government effectiveness and regulatory quality) (p< 0.001). In the final model, only political stability and voice and accountability remained as statistically significantly associated with higher levels of ART coverage.
The analyses indicate that better political governance are crucial determinants of ART coverage in a country.
Gender inequalities are a key driver of the epidemic, yet despite this being recognised for many years, global policies to change these often have had little effect. Using the UNAIDS’
We conducted semi-structured interviews with 16 southern African and global gender and HIV/AIDS policy makers, researchers and activists who had been involved in the development or implementation of the
Barriers to the development and implementation of the
Our study suggests that despite the
We reviewed the laws of the 50 states and compiled those vocational and professional licensing laws (e.g. for licensure to be an acupuncturist, barber, massage therapist, midwife, cosmetologist, nurse practitioner, doctor, dentist, etc.) that discriminate against people living with HIV/AIDS and/or those with a communicable, contagious, or infectious disease.
The statutes, regulations, and cases were identified in Westlaw using the following search terms: “HIV,” “Human immunodeficiency virus,” “communicable,” “contagious,” “infectious,” and “disease.” The volume includes laws that explicitly discriminate against people living with HIV/AIDS (PLWHA) as well as laws that do so implicitly through restrictions concerning communicable, contagious, or infectious diseases. While we did attempt to include all relevant state law, this does not promise to be an exhaustive list.
The results of this survey reveal a deeply rooted HIV stigma that has been embraced by policy makers in a majority of the states. Thirty-five of the fifty states have licensing requirements that explicitly discriminate against people living with HIV/AIDS for one or more of the fifteen vocations considered here, and forty-four states have requirements that either discriminate against PLWHA or discriminate against people living with infectious, communicable, or contagious diseases. These laws lack any basis in the science of HIV transmission, and maintaining them only validates the ignorance and bias that spawned them.
Licensing criteria that discriminate against people living with HIV/AIDS violate federal law. States that allow these discriminatory laws to remain on the books are ignoring scientific consensus and promoting HIV stigma. If PLWHA in the United States are to enjoy the same employment opportunities that other individuals enjoy, then states must examine their licensing procedures and alter or remove any discriminatory requirements until all licensing practices are in compliance with the Americans with Disabilities Act and guidance from the Department of Justice.
The southern United States has the highest numbers of adults and adolescents living with HIV and the highest death rates from AIDS. Numerous southern cities are in the top ten for HIV infection rates. Yet state laws and policies fuel the epidemic and exacerbate pervasive stigma and discrimination.
This long-term, multi-method investigation is based on hundreds of interviews with people living with HIV and AIDS, their advocates, public health officials, legislators, judges and medical providers, as well as extensive legal and policy analysis.
Multiple state laws and policies create an ‘environment of risk’ that has made the South the nation′s leading region for both HIV infection and deaths from AIDS. In the South, where poverty levels are the highest in the nation and fewer people have access to health insurance state policies are contributing to the disproportionate impact of HIV on minority communities. The failure to invest in public health, Medicaid and HIV-specific programs, particularly housing and transportation for a rural population; insistence on failed abstinence-based education; criminalization of HIV exposure; homophobic laws that drive MSM and transgender people underground and away from essential health services and the highest incarceration rates in the nation are some of the human rights violations that undermine public health efforts and fuel the epidemic in southern states.
Laws and policies in the southern U.S. are undermining human rights and conflict with the goals and objectives of the National HIV/AIDS Strategy. This interactive session will identify strategies for challenging these policies at the federal, state and local level.
Recent trial results confirm potential for antiretrovirals to prevent HIV-infection. As programme implementers initiate pre-exposure prophylaxis (PrEP) demonstration projects and consider national plans, research regarding the acceptability of PrEP in at-risk communities is needed to evaluate its likely uptake and potential impact in key populations. Intermittent dosing (iPrEP) deserves particular consideration due to its requirement for sexual planning.
Seven focus groups with 6-8 participants per group (n=46) were conducted. Five were conducted with adults from a high-incidence community and two informant groups with counselors experienced in working with high-risk populations. Attitudes towards oral PrEP, iPrEP, microbicide gel and sexual planning were explored. Focus group data were transcribed, coded and analyzed utilizing framework analysis. Participants self-completed a sexual planning survey.
52% were female and 48% male, with a mean age of 28 years. Barriers to PrEP acceptability included perception of antiretrovirals as treatment, side effects, stigma, risk compensation, safety and efficacy of a new product and dislike of medication. The primary facilitator of PrEP uptake was its potential for non-consensual use. iPrEP was favoured over daily PrEP for perceived ease of use with fewer dosing days. Oral PrEP was largely preferred over microbicide gel since it was perceived as not requiring disclosure and not coitally dependent, although others noted microbicides could enhance sex. On surveys, 64% of days on which sex occurred were reportedly 'planned'. Men were significantly more likely to be able to predict sex (75% vs. 32% of women). The median number of sex days per week was two and highest activity days were Friday and Saturday.
PrEP appeared acceptable to target users although barriers need addressing. Oral iPrEP was preferred over other regimens. Sexual planning was fair overall, although more than a third of sex days were unplanned and women fared worse. iPREP seems feasible in interval-specific form.
Non-occupational post exposure prophylaxis (nPEP) to HIV has been available for over a decade. Few institutions have a coordinated system for providing this service. Since 2009, St. Luke's Roosevelt Hospital (SLR) has provided nPEP in its 2 emergency departments and 3 affiliated outpatient clinics (Centers for Comprehensive Care, CCC). As of 2010, SLR adopted an nPEP protocol developed by the NYC Department of Health through a funded grant.
An extensive, retrospective chart review was conducted to describe the characteristics of adult patients who received nPEP in the ED and affiliated outpatient clinics from 12/2009 to 2/2012. Characteristics of patients who undertook nPEP and completed follow-up were compared using Fisher Exact and Wilcoxon tests. Factors associated with completing the nPEP course were analyzed using multivariate logistic regression.
216 unique encounters occurred for nPEP (median age=29; range=18-62). 34 encounters were from 13 patients with repeat nPEP visits (mean=2; range 2-3). 138 patients initially presented in ED (64%). Majority were male (n=180; 83%), white (n=99; 46%), and insured (n=134; 63%).133 of male patients (65%) reported sex with other men as their HIV risk exposure. Median time from exposure to presentation was 24 hours (range1-74 hours). 116/138 (84%) patients referred to the CCC completed 4-week follow-up. 109/116 (94%) patients completed the 28-day nPEP course. For patients who completed nPEP, there were no significant differences seen by race (
nPEP is a chemo-prophylaxis intervention that can prevent HIV acquisition and transmission and provides additional opportunities for synergistic, behavioral interventions. A multi-disciplinary approach is necessary to coordinate care, follow-up, and supportive counseling.
This study examines how HIV/AIDS funds, including vertical Global Public Goods for Health (GPGH), might be leveraged to contribute to production of horizontal GPGH through health system strengthening. Specifically, it addresses the following questions: 1)How can vertical (disease-specific funds) be used to decrease the impact of funding asymmetries between vertical and horizontal GPGH and support the production of horizontal GPGH? 2)How would complementary activities be evaluated with regard to their contribution to health system strengthening?
Global Fund grant information was used to examine the extent to which HIV/AIDS grants are currently contributing to system strengthening and capacity building at the national level beyond their disease-specific areas and if the inclusion of health system strengthening components influences grant performance. In addition, a framework is developed to evaluate the contribution of HIV/AIDS programs to the production of GPGH through the horizontal strengthening of national health systems.
In the bivariate analysis, the number of health system strengthening components was significantly related to performance. However, as more variables were added the significant disappears. The grant characteristics significant across the models were grant duration and region. While the results regarding the positive association between health system strengthening components and grant performance are inconclusive, the inclusion of health system strengthening components doesn′t appear to interfere with a grants ability to meet its disease specific performance targets.
The results from this analysis indicate the need for additional research to examine the relationship between health systems, health system strengthening, and HIV/AIDS program performance. The critical step would be to examine how the national subsystems are contributing to the international level through governance, financing or provision of goods and services related to GPGH.
Diagram#1.
Diagram#2.
Rapid scale-up of antiretroviral therapy (ART) in Southern Africa has put enormous strain on health systems. Information about acquired drug resistance in treated individuals is important to monitor quality of programmes and to ensure that ART policies remain appropriate. The majority of resistance data so far have come from urban, hospital-based programmes; limited data have been reported from rural treatment programmes.
Adult (≥16 years) HIV-infected individuals with virological failure (2×VL>1000 copies/ml) on first-line NNRTI-based ART were enrolled from all 17 primary health care clinics of the Hlabisa ART Programme. Genotypic resistance testing was performed using the in-house SATuRN/Life Technologies system. Sequences were analysed and genotypic susceptibility scores (GSS) were calculated using RegaDB and Stanford HIVDB 6.0.5 algorithms.
187 adults enrolled between Dec 2010 and Dec 2011; median age 37 years (IQR 31–45); 70% female. Median time on ART 41 months (IQR 31–53); median time on failing regimen 30 months (IQR 20–42). 120 (64%) had never achieved full virological suppression (VL≤50 copies/ml). 160 (86%) individuals had ≥1 drug resistance mutation; 149 (80%) and 153 (82%) respectively had NRTI and NNRTI mutations. 72 (38%) had at least one thymidine analogue mutation (TAM) and 32 (17%) had ≥3 TAMs. 14 (7%) had other NRTI mutations that might impact on second-line therapy (K65R-12 (6%); Q151M-3 (2%)). The standard second-line regimen was substantially compromised (defined as GSS≤1.5) in 33 (18%) individuals.
There are high levels of acquired drug resistance associated with prolonged virological failure in this rural primary health care programme. Standard second-line regimens would be significantly compromised in almost one in five adults. This suggests a role for genotypic resistance testing in routine care but, more importantly, it highlights the need for increased attention to quality of care and adherence to virological monitoring guidelines.
Since 2004, the HIV Vaccines and Microbicides Resource Tracking Working Group has employed a comprehensive methodology to track trends in R&D investments and expenditures for biomedical HIV prevention options, including HIV vaccines, microbicides, PrEP, treatment as prevention and adult voluntary medical male circumcision.
R&D data were collected on annual disbursements by public, private and philanthropic funders for product development, clinical trials and trial preparation, community education and policy advocacy efforts in order to estimate annual investment in HIV prevention R&D. Investment trends were assessed and compared by year, prevention technology type, funder category and geographic location.
The period from 2000 through 2010 saw significant growth in funding support for HIV prevention research and development. However, in 2011 HIV prevention research began to see increased funding pressures as governments worldwide decreased or flat-lined budgets in global health and as philanthropic donors worked to revise their investment strategies. Funders also had to deal with greater competition among funding priorities within the field of HIV prevention and with other major global disease needs. Despite various pressures, HIV prevention research progressed significantly in 2011, with new findings and promising new trials.
Monitoring funding trends for HIV prevention research is particularly important in this time of economic uncertainty, especially as the scientific community has articulated a clear path to the end of the HIV epidemic. Such monitoring permits identification of investment needs, prioritization of research areas and assessment of the impact of public policies that increase or decrease investment. It also provides the fact base for advocacy around spending levels and allocations that will sustain investments in the research required to build on the success of recent trials; bring novel HIV prevention candidates into the pipeline; and, support follow-on clinical trials needed to assure the safety, immunogenicity, efficacy and acceptability of new HIV prevention products.
Map 3.
MVC is a potent CCR5 co-receptor antagonist that is being considered for pre-exposure prophylaxis for HIV prevention. We evaluated in macaques the pharmacokinetic (PK) profile of MVC and assessed efficacy of MVC in preventing rectal SHIV transmission.
The PK profile of oral MVC (44 mg/kg) was evaluated at first dose in plasma and rectal secretions in 12 macaques. Half-life of CCR5-bound MVC was measured using a MIP1-β internalization assay. Relationship between MVC concentration, CCR5 occupancy, and protection from infection was evaluated in vitro. Efficacy of MVC in preventing rectal virus transmission was investigated using a macaque model consisting of weekly SHIV162p3 exposures. Six macaques received a dose of MVC 24h before each virus exposure and a second dose 2h thereafter. Four untreated macaques were controls. Infection was monitored by serology and PCR.
MVC PK profile in macaques was similar to humans. MVC concentrations peaked at 2h in plasma (median=451 ng/ml) and at 5–48h in rectal secretions (median=2,329 ng/ml). MVC AUC0–24h in rectal secretions was 7.5 times as high as in plasma (median=12,720 and 1,685 ngxhr/ml, respectively). At day 4, MVC concentration in rectal secretions (median=44 ng/ml) was 21.8 times as high as the IC50 value, and was sufficient to fully occupy CCR5 in PBMCs. The half-life of CCR5-bound MVC in PBMCs was 2.6 days. Despite this favorable PK profile, 5/6 animals receiving MVC were infected during 5 rectal SHIV exposures as did 3/4 controls.
We identified a dose of MVC that results in local and systemic drug exposures comparable to humans receiving 300 mg. Despite high and durable MVC concentrations in rectal secretions, MVC did not prevent SHIV infection in macaques. This model suggests that higher MVC concentrations are needed for rectal protection and highlights the need to better understand the mechanism of low efficacy and identify protective MVC regimens.
Clinical trials have demonstrated antiretroviral therapy (ART) is highly efficacious in preventing HIV transmission among sero-discordant couples. We examined the effectiveness of ART as prevention in a rural program in Uganda where viral load (VL) testing is not available.
We conducted a cohort study of HIV sero-discordant couples aged ≥18 years, where the positive partner was a client of The AIDS Support Organization in Jinja, Uganda. In one group of couples the positive partner was eligible for ART because of CD4 cell count ≤250 cells/ µL or a WHO Stage III or IV disease. In the second group, the infected partner was not yet ART-eligible. Both groups received regular HIV risk-reduction counseling and condoms. The uninfected partner was HIV tested every three months. We conducted VL testing and genotyping of transmitted viruses.
A total of 586 couples were enrolled, of which 352 (60%) of the positive participants received ART during the study. The median duration of ART-use at enrollment was 2.5 years and the median duration of follow-up was 1.3 years. ART couples were older than non-ART couples (median 42 vs. 40 years for men; 36 vs. 33 years for women; p<0.001, for both). ART couples were more likely to report condom-use at last sex (74% vs. 66%; p=0.038) and had longer duration in relationships than non-ART couples (median 12 vs. 9 years; p=0.003). There were no differences between the two groups in terms of male circumcision status, polygymy status, pregnancy intentions or injectable contraception-use. We found 9 new infections among partners of ART participants and 8 new infections in partners of non-ART participants, for an incidence rate ratio of 1.16 (p=0.564).
ART-use was not associated with reduced risk of HIV transmission in sero-discordant couples in a rural program in Uganda without VL testing.
The enabling legal environment has long been identified as critical to rights-based responses to HIV. In the 2011 Political Declaration on HIV/AIDS, UN Member States committed to supporting legal services as well as monitoring the impact of the legal environment on HIV prevention, treatment, care and support. Research shows that legal services in developing countries remain under-resourced, of variable quality, ad hoc and reactive, often with poor case documentation and weak links with the Health and Justice Ministries.
A study on HIV and legal empowerment was commissioned to place the program in a broader access to justice paradigm. In each country, a comprehensive needs analysis or an environmental scan was undertaken to determine the needs, available services and gaps in HIV-related legal services. A local organization already providing legal services was given technical and financial support to improve quality and scale up, including documentation of their work. Regional and international consultations were held to promote sharing on management challenges, and substantive issues such as criminal law, needs of women and girls, and access to treatments for HIV. An independent end of program evaluation was undertaken in early 2012.
A review of results in program countries and other contexts revealed success factors such as: engagement of outreach workers to create demand for services; women friendly and child friendly services can increase uptake; opportunities exist to mainstream HIV-related legal services into existing government services; service (such as child care) can increase access and assist sustainability, particularly for female clients; technical assistance can improve case documentation and hence advocacy for science-based policy and law reform.
Legal empowerment and access to justice approaches are essential elements of the enabling legal and policy environment for the response to HIV. They are needed to help take prevention, care, treatment and support to scale.
HPTN 052 provided clear evidence that early antiretroviral therapy (ART) in HIV-infected partners in serodiscordant couples decreases the risk of sexual transmission (RR=0.04). Before HPTN 052, ecological studies and models suggested that sexual transmission was lower in discordant couples in which the infected partner was on ART. To estimate ART effectiveness from the observational literature, we conducted a systematic review.
We used standard Cochrane methods to search electronic databases and conference proceedings without language limits and identified cohort and case-control studies of ART use in HIV-discordant couples. Two authors independently examined all identified studies and abstracted data using a standardized form.
We retrieved 1814 references from which 24 were potentially eligible. Seven observational studies met our inclusion criteria. These studies identified 436 episodes of HIV transmission, 71 among treated couples and 365 among untreated couples. The summary rate ratio was 0.34 [95% CI0.13, 0.92], with substantial heterogeneity (I2=73%). After excluding two studies with inadequate person-time data, we estimated a summary rate ratio of 0.16 [95% CI 0.07, 0.35] with no noted heterogeneity (I2=0%). We performed subgroup analyses to see if the effect of ART on prevention of HIV transmission differed by the index partner′s CD4 cell count. Among couples in which the infected partner had ≥350 CD4 cells/µL, we estimated a rate ratio of 0.02 [95% CI 0.00, 2.87]. In this subgroup, all 61 transmissions were in untreated couples. Tests for interaction between CD4 subgroups and infection risk were not significant.
HPTN 052 has proven that ART is a potent intervention for prevention of HIV in discordant couples in which the index partner has between 350 and 550 CD4 cells/µL. The earlier observational literature is supportive of the findings of HPTN 052, although the estimated effectiveness is eight-fold less than that found in the trial.
In many countries police practices are dramatically out of sync with declared government policies to prevent HIV by promoting access to condoms. Police confiscation and destruction of condoms held by sex workers and outreach workers impair sex workers’ access to condoms. Police treatment of condoms as evidence of criminal activity and as tools for extortion threatens sex workers’ ability to safely carry condoms and negotiate their use with clients.
In a joint research effort, community groups in Kenya, Namibia, Russia, South Africa, the United States, and Zimbabwe administered detailed questionnaires to 139 sex workers and 40 outreach workers.
Research revealed that police routinely stop and search sex workers and confiscate their condoms. In some locations, police destroy sex workers’ condoms by burning them, cutting them up, driving on them, or throwing them in the garbage. Police confiscation and destruction of sex workers’ condoms increases their vulnerability to HIV. Sex workers reported having unprotected sex with clients after police took their condoms. In some cases, police use condom possession as justification to detain or arrest a person on charges of engaging in sex work, or as leverage to extort bribes or sexual favors. Sex workers sometimes do not carry condoms or refuse condoms from health care providers because they fear condoms will get them in trouble with police. Police further impede sex workers’ access to condoms by surveilling and harassing outreach workers and taking or destroying condoms meant for distribution.
Policing practices are undermining messages of condom accessibility and acceptability crucial for disease prevention and the promotion of individual and public health. Policing tactics and guidelines must be brought into alignment with international best practices to prevent HIV. As a first step, governments should eliminate laws that criminalize sex work and other structural obstacles to police reform.
Due to the current legal frame work of sex work in Bangladesh sustainable HIV prevention interventions among Female Sex workers (FSWs) is a major challenge. It is clear from experience gained so far that social marginalization & disempowerment that characterize FSWs community are the key vulnerabilities that need to be addressed before any interventions related to HIV can be successfully adopted by them with ownership.
The first ever national congress of FSWs in Bangladesh organized by Save the Children was participated in by 1000 FSW around the country from 29 FSWs networks. They discussed legal and policy issues related to sex work and its consequences. This allowed an opportunity for shared experience from leading national, international organizations working with FSWs for last 20 years to have an analysis of the legal framework of sex work among Bangladesh.
Hotel or Street, in any settings FSWs are not in a legal position to say no and or insist client for negotiating safe sex. According to anti- prostitution law two or more FSWs cannot work together which interferes with the ability to form collective resistance to violence, abuse & unsafe sex. On the other hand Police and Municipality law creates fear of Arrest hinders community outreach and FSW is hidden: this means it is difficult to identify, contact & build trust and avoiding arrest prevails over safer sex. Also Police powers harasses peer educators, carrying condoms seen as ‘promoting', ‘soliciting' & ‘carrying on' sex work. Due to criminalization of Brothels prevention program cannot ensure 100% condom use, frequent raids disrupt HIV services.
The legal and policy framework requires radical restructuring to reduce legal sanctions and social marginalization and allow capacity building will work for sex workers community to build a sustainable HIV AIDS program in partnership with other stakeholders including government in Bangladesh.
In the past several years FTA negotiations have proliferated across the developing world. Why have PLHIV been protesting these FTAs and what provisions do they contain that could really impact access to medicines? Is this all conjecture or is there evidence to show the impact?
FTAs negotiated by the EU, US and Japan were examined. Chapters on intellectual property have been analysed. Evidence from FTAs already in existence and their impact on access to medicines was collected. FTA negotiations with India, Malaysia, Vietnam, Thailand, Brazil and South Africa were covered.
Although there are some variations in the intellectual property provisions demanded in FTAs by the EU and US their primary aim is to decrease the space for generic production of medicines including ARVs. Provisions like data exclusivity (a new monopoly on off-patent medicines), extention of patent terms beyond 20 years, enforcement measures that will have a chilling effect on generic production, interfere with judicial independence and put shipments of medicines at risk of being seized feature in these FTAs. In addition the investment chapter puts government actions to ensure access to medicines or provision of healthcare at risk of MNC's legal cases filed in private, international arbitration.
In 2001 the price of ARVs crashed from $10,000 per person per year to $350. In 2001, governments around the world signed the Doha Declaration stating that international trade rules on intellectual property should not come in the way of providing access to medicines for all. FTAs go well beyond these international trade rules that are already resulting in higher prices of newer ARVs and medicines for Hepatitis C. Understanding the nitty gritty of the demands made in these FTAs is crucial to effective advocacy on the FTAs and for the proper involvement and consultation of PLHIV and health groups by their governments.
There has been widespread concern about the impact of patents on access to low-cost generic antiretroviral (ARV) drugs in low- and middle-income countries (LMIC). Patents can hinder competition, which has provided sustainable price reductions for the older generation, less-widely-patented ARVs. However, until recently, limited data on patent status in developing countries hampered a full understanding of the implications of pharmaceutical patenting in these countries.
We compared patenting trends for 12 molecules invented pre-1995 and 12 invented post-1995, the year the WTO TRIPS Agreement came into force. We used data from the Medicines Patent Pool ARV Patent Database, the most comprehensive and regularly updated source of such information, providing information on 69 countries accounting for 84% of PLHIV in LMICs. We analyzed trends regarding: the number of molecules for which patents were applied for in a given territory, the number of LMICs with ARV patents, the expected date of patent expiration, and the implications for the development of and access to fixed-dose combinations (FDCs).
Since 1995, pharmaceutical companies are filing for patents in a greater number of LMICs and for a greater number of molecules, such that newer ARVs are more widely-patented than older drugs. A patent on just one drug in a 2- or 3-drug FDC can pose barriers for accessing the entire FDC, and such patents can pose problems in up to 80% of territories for which data was available.
Since 1995, pharmaceutical companies are filing for patents in a greater number of LMICs and for a greater number of molecules, such that newer ARVs are more widely-patented than older drugs. A patent on just one drug in a 2- or 3-drug FDC can pose barriers for accessing the entire FDC, and such patents can pose problems in up to 80% of territories for which data was available.
The french speaking countries of African ‘have been struck the hard by the pandemic. In recent years, however, the governments have made concerted efforts in combating the disease. With the support of international organisations programs on treatment, care and education have increased. However, a recurrent problem has been the difficulties of making HIV/AIDS medicines accessible. Stringent intellectual property laws act as a stumbling block to access to HIV/AIDS medicines.
Presentation of a panoramic view of the problems created by stringent intellectual property laws and how they affect access to HIV/AIDS medicines. Analysis of various approaches to facilitate access to HIV/AIDS medicines. Proffering recommendations for reforms.
Demonstrating that the patent regime of the Bangui Agreement to which most Francophone African countries have signed is hampering access to HIV/IADS medicines and that there is an urgent need for reform.
Though the coming into force of the WTO and the TRIPS Agreement ushered in a new dynamic on when and how countries had to use certain measures to provide, promote and protect access to HIV/AIDS medicines, french speaking African countries still have certain limited policy spaces for which to manoeuver and improve access to medicines. Most of them are signatories to international and regional treaties which creates rights to health obligations. Subsequent developments after coming into force of TRIPS have tended to tilt towards supporting access to medicines. The sad problem though is regional policy incoherence and countries’ entering into treaties with TRIPS plus provisions. Fortunately, international law provides enough justification for enacting access to medicines pieces of legislation. Making maximum use of TRIPS flexibilities, incorporating human rights perspectives in intellectual property treaties, forging a strong alliance with civil society are just some of the measures needed by french African countries in increasing access to HIV/AIDS medicines.
Over the past decade, the issue of counterfeit medicines has been seen as a burgeoning public health concern, requiring immediate action. Some decisionmakers have advocated for addressing the counterfeit issue through legislative measures targeting intellectual property enforcement. During this process, the term “counterfeit medicine” has been defined in ambiguous and confusing ways that have numerous legal interpretations, and thus intended and unintended consequences. One particular issue is the conflating of counterfeit medicine with generic drugs, which has and will continue to impede access to generic ARVs in developing countries.
My study consists of a political and legal analysis of three anti-counterfeiting efforts and their impacts on access to generic ARVs in developing countries, with a particular focus on East Africa.
My analysis begins with a brief discussion and overview of some definitions of counterfeit medicine used by WHO and under the TRIPS agreement. The analysis will then explore anti-counterfeiting efforts in Kenya, in the East African Community, and in Anti-counterfeiting Trade Agreement (ACTA). I then explore an example in each context of how these measures have or will impede access to generic ARVs in developing countries.
Based on this analysis, the anti-counterfeiting measures examined pose a real and dangerous threat to accessing generic ARVs in developing countries. The counterfeit definitions used conflate generics with counterfeit medicines. Thus, a clearer and more concise definition of counterfeit is needed to avoid negative implications for generic ARVs. Finally, the real public health concern at issue here is addressing the safety quality and efficacy of medicines, and anti-counterfeiting measures do not adequately address these concerns. Therefore, rather than trying to deal with substandard medicines through intellectual property rights enforcement, other policies need to be developed that not only address the real issue, but also do not pose a threat to accessing generic ARVs.
Since 2006, Brazil has attempted to negotiate lower prices with the patentee of Efavirenz in two grounds: a) EFZ was being sold at more affordable prices in countries with the same level of development and with less people in need of treatment than Brazil; b) generic versions were much cheaper. Considering the Brazilian policy of universal access of ART for PLWH, the offer of reducing 30% on the drug cost was still unsatisfactory (US$ 1,14 per pill).
Comparison of prices and case study aproach. The reference price of comparison was the last proposed by Merck for Brazil in 2007 of US$ 1,14 per pill. The total savings presented exclude the 1,5% of royalties that Brazil paid due to the Compulsory Licence.
The government strategies to maintain a sustainable coverage of treament for those in need allowed firstly to issue a compulsory licence to import generic versions of EFZ, and thereafter to estimulate local production by the government industries. The savings with EFZ 200mg, purchased of international generic supliers, were of US$ 1.042.568,99; with EFZ 600mg, imported of international generic suplies, of US$ 63.992.537,16 and with EFZ 600mg locally manufactured of US$ 29.763.749,72. Amounting to a total saving of US$ 94.798.855,87 from 2007–2011.
The economic savings that were enabled with the compulsory licence were essential for the increased access to Efavirenz in Brazil. It is important to emphasize that the adoption of mechanism also boosts the strenght of the government to negotiate prices of other medicines and to stimulate the capacity of national pharmaceutical production and the transferring of technology.
Morocco is facing a rising HIV epidemic among people using drugs (PUD). HIV prevalence among PUD ranks from 7% to 38% according to recent studies as HIV prevalence in general population is estimated to be 0.1%. This major change in epidemic pattern led Moroccan stakeholders to pay more attention to PUD. A first national harm reduction (HR) strategic plan (2008–2011) has allowed some needles and syringes programs as well as methadone for 80 people out of other HR services.
But PUD are facing legal and social barriers acting as potential barriers for access to HIV prevention, care and treatment. Country is in need to assess those barriers in order to design a more comprehensive approach to mitigate and reverse HIV among PUD.
National HIV/AIDS community based organization ALCS has managed a cross sectional study in 3 North cities. 300 PUD recruited through outreach programs were asked to answer a questionnaire including 9 questions designed to assess human right violation from police officers, health professional and closed environment. Human right violation was defined as a break in national endorsed civil rights or break in national health good practices.
99.8% of PUD have been victims of at least one of those 9 violations with following sub scores: 87% from police officers (eg. non legal custody) and 49% from health professional (eg. non access to emergency hospital units). 60% has faced situation closed to human exploitation with drug smugglers or other PUD (including sexual intercourse against drug). Famility is also a major cause of human right violations.
Human Right should be considered as a central component of HR interventions in Morocco. At a time the country is designing HR scaling up it could improve PUD access to health services and alleviate family burden.
In Macedonia, sex workers are among the most discriminated communities. Due to self stigma and no trust in the system, our experience shows that SWs almost never report perpetrators or demand protection via litigation. This made us aware about the complexity of raising awareness to litigate cases of human rights breaches.
In November 2008 a systematic human rights violation by state happened when 23 SWs were unlawfully arrested and forcibly tested for HIV/HepC. Seven SWs were found HCV positive and criminally charged. Encouraging sex workers to demand justice, HOPS developed a complex model of legal, psycho-social and media support.
After the incident, HOPS created intervention team of lawyer, outreach worker, social worker and psychologist for free information and support for emotional trauma and possible lawsuits. For the first time 13 SWs raised 2 court cases against institutions and media, and defended themselves in the criminal case. 3 years after, 2 cases finished, 1 is ongoing, but the SWs successfully resisted fears and remained motivated to follow through. Consequently, 7 criminally charged SWs were sentenced on parole, and the civil suit against state institutions was won.
SW in Macedonia are at risk for rights violation. Strategic litigation is a powerful protection tool, and while free legal aid is crucial, effective support should address emotional and other aspects of marginalization and resisting the state without fearing victimization. Trusted outreach and social workers, skilled psychologists and peer support must be part of the supportive model, involving the media and allies to create supportive environment.
Promoting the success of this model nationally and regionally, in order for organizations working with marginalized groups to adopt it in motivating them to report violence and use disposable mechanisms, including litigation. These positive outcomes will empower SWs in our country to seek justice.
HIV criminalization laws are championed as protecting women. But HIV-positive women are increasingly prosecuted under these laws, resulting in unique violations of their sexual, reproductive and parental rights. 38 U.S. states and territories have criminal HIV exposure and transmission laws that increase HIV stigma and hinder HIV testing. Yet few tools exist to support anti-HIV-criminalization advocates, especially women.
In 2011, the Positive Justice Project (PJP) - a consortium working to end abuses of the criminal law against HIV-positive people - conducted community forums focused on HIV-positive women in four states with active HIV-specific criminal law prosecutions: Louisiana, Kansas, Missouri, and Illinois. HIV-positive advocates, public health officials, and attorneys attended. Local advocates, especially HIV-positive women, engaged to reframe the HIV criminalization conversation. Panels of local and national experts opened each forum and provided legal advocacy tools - fact sheets, model legislation, and “know your rights” cards - to eliminate local HIV criminal laws. Participants then developed advocacy strategies.
Since October 2011, 125 people have been reached through PJP forums; thousands more through PJP and partner websites. PJP forums increased community knowledge of the impact of HIV criminalization laws on women, and provided general and women-specific advocacy tools to build an infrastructure at the state level to monitor and challenge existing laws. With technical assistance from PJP, HIV-positive women founded local advocacy groups in the four forum states to eliminate state criminalization laws; began monitoring criminal prosecutions and discriminatory practices; and put a woman's face to the consequences of HIV criminal laws.
HIV-positive women's engagement in turning the tide of the epidemic by personally advocating for elimination of criminal HIV exposure and transmission laws has increased awareness of the breadth of the laws' harms. Further policy research and political will is needed to support advocates to fight discriminatory criminal laws.
The established critique of criminalizing HIV exposure/transmission focuses on issues of public health impact and human rights. Although important, this critique offers little guidance on how to practically oppose criminalization in local settings. Successful advocacy requires developing a literature on the impacts of strategies undertaken by advocates in various jurisdictions. This paper contributes to that literature through an analysis of the social organization of criminal law reform in Ontario, the centre of Canadian HIV-related prosecutions.
This paper is based on the experiences of members of the Ontario Working Group on Criminal Law and HIV Exposure (CLHE), a key participant in advocacy against criminalization in the province. Drawing on a studies in the social organization of knowledge perspective (Smith, 1990), it systematically identifies the key factors shaping CLHE's activities and reflects on the outcomes of CLHE's principal strategies.
CLHE has emphasized the dissemination of research and political lobbying to create a stronger presence, in the criminal justice system, of scientific evidence on factors that reduce HIV transmission risk (a key element of the legal test in cases of HIV non-disclosure). Four initiatives have been key: (1) The mobilization of criminal defence lawyers and the identification of qualified expert witnesses have resulted in favourable judicial decisions; (2) a campaign to establish prosecutorial guidelines has been an effective community mobilization tool, but has stalled at implementation due to lack of provincial buy-in; (3) a proactive media campaign has, for the first time in Canada, generated widespread favourable mainstream media coverage of opposition to criminalization; (4) political lobbying has helped prevent harmful interventions in 2 cases recently heard by the Supreme Court of Canada.
Science-based criminal law reform led by a community organization can enhance legal defense, mobilize allies and impact media coverage, but faces challenges of engagement with provincial criminal justice authorities.
The HIV epidemic has raised new and complex legal challenges, leading to a wide range of judgements on HIV-related matters. Given the diversity of issues and the quickly evolving science, judges need more opportunities to take stock of developments relevant to the cases before them. If properly informed and supported, the judiciary can help create the legal and social environment necessary to roll back the HIV epidemic.
An advisory committee of sitting and retired judges, judicial trainers, and legal experts from around the world was constituted to inform the scope, format and content of the handbook. Issue experts prepared chapter outlines and draft texts which were shared with the advisory committee for detailed feedback. The final text includes case summaries, key principles, plain language explanations of the science and treatment of HIV, statistics, and factors to consider when adjudicating cases.
The result is a comprehensive and user-friendly handbook for the judiciary, suitable for use as a reference manual and in judicial training sessions throughout the world. Issues addressed include: discrimination, criminalization of HIV exposure, sexual assault and domestic violence, drug policy and the rights of people who use drugs, women's rights in family and property law, HIV-related treatment and healthcare, key populations at higher risk of HIV exposure, and judging during the HIV epidemic.
The handbook for judges is a unique resource with significant potential to provide judges with a contextualized understanding of key HIV-related issues, and thus improve rights protections for people living with and affected by HIV. This potential will be maximized through judicial training sessions and commitment by governments to include the judicial sector in strategies to attain universal access. The tools and training needed by judges are distinct from existing HIV-related resources therefore targeted, sensitive approaches are needed.
It is well-documented that discrimination in women's access to property and inheritance hinders an effective response to HIV. However, in southern Africa, where HIV prevalence still remains high, numerous laws on property and inheritance rights explicitly discriminate against women. Law reform efforts are slow and in many cases have been ineffective resulting in discriminatory laws remaining in place. Approaching courts through litigation as a means of striking down such laws is underutilized in southern Africa.
In an attempt to accelerate the revision and/or striking down of laws discriminating against women in property and inheritance rights, we are using litigation before domestic courts to effectively strike down laws that discriminate against women with respect to property and inheritance in southern Africa.
A number of cases have been filed. A case challenging a customary law rule denying women the opportunity to inherit from their father's estate is in the Botswana High Court; the Lesotho Constitutional Court will hear a challenge to a law denying women the right to succeed to chieftainship; and the Malawi Constitutional Court is expected to issue a decision on whether courts are permitted to only take monetary contribution to property into account when determining property distribution at the time of divorce. In each of these cases, the relief sought is a change in the discriminatory law. In all cases, the countries have constitutional provisions protecting women from discrimination. Decisions in the cases are expected by June 2012.
Law reform measures are critical to revise laws that facially discriminate against women with respect to property and inheritance rights. However, for relatively fast and effective reform, more advocates should approach courts asking them to strike down discriminatory laws.
In Australia, debate continues about how to make best use of antiretroviral-based prevention or ‘treatment as prevention’. We assessed the attitudes towards antiretroviral-based prevention, including PrEP, of Australia's primary affected populationgay men. We analysed whether HIV-positive and HIV-negative men held different attitudes to PrEP and HIV treatments.
A national, online cross-sectional survey was conducted in April–May 2011. The survey included 29 attitudinal items about PrEP, medicines, condoms and HIV treatments. Differences between the mean scores for each attitudinal item were assessed with ANOVA. Multiple linear regression (MLR) was used to identify independent differences in attitudes between HIV-positive and HIV-negative men.
1041 men were included in the analysis (88.3% HIV-negative and 11.7% HIV-positive). The mean age was 33.3 years (SD=10.8; range 18–69). Most of the sample (94.8%) identified as gay and a minority (4.3%) as bisexual. HIV-positive and HIV-negative men agreed on 13 items, such asPrEP is effective in preventing HIV, that it would make people less responsible, that it should be provided free of charge and is less effective than condoms. MLR analysis indicated nine independent differences between the two groups. HIV-negative men more strongly disagreed that HIV drugs should be restricted to HIV-positive people or people who have difficulty using condoms. HIV-positive men agreed and HIV-negative men disagreed that taking HIV treatments was straightforward and HIV-negative men were more sceptical about whether HIV treatment or an undetectable viral load prevented HIV transmission.
Australian HIV-positive and HIV-negative gay men have mixed attitudes towards PrEP and HIV treatments. Despite doubts, HIV-negative men in particular are keen to see PrEP made available. There are intriguing differences related to ‘treatment as prevention’ while HIV-positive men are enthusiastic about the benefits of HIV treatment, HIV-negative men remain sceptical about the effectiveness of HIV treatment in preventing transmission.
It has been documented that being HIV positive put the social safety net under strain preventing people from accessing HIV care. Yet, to date, the scope of epidemiological research on this topic remains limited. We tested the hypothesis that family and community cohesion were associated with uptake of HIV testing and treatment.
This research uses data from a randomized cross-sectional study of 2443 individuals sampled in communities and ART clinics in rural and urban Zambia. Associations were examined using a two step multivariable logistic regression approach. Initially independent thematic models, adjusted for sociodemographic variables, were created to examine the effect of social support, stigma, poverty and beliefs. Variables with p<0.2 in the thematic models were included in a comprehensive multivariable model for each outcome.
978 participants (53% women and 44% men) in the community reported to be tested for HIV. A total of 691, including those attending the clinics, reported to be on ART (64% women and 36% men). In the final comprehensive models, adjusted for all covariates, the risk of not being tested was associated with poor community engagement (OR=1.5 95% CI=1.12–1.91), low attendance of religious services (OR=1.3 95% CI=1.08–1.47), problems with the neighbors (OR=1.2 95% CI=1.06–1.32) and living in urban areas (OR=2.8 95% CI=2.06–3.61). Protective factors were being women and married. The strongest predictors for non-uptake of ARVs were domestic violence (OR=1.3 95% CI=1.04–1.65) and trust in traditional medicines (OR=1.8 95% CI=1.33–.2.47). Being widowed or divorced showed a statistically significant protective effect. Anticipated social stigma (OR=1.2 95% CI=1.08–1.36) was a significant risk factor for testing in the thematic model but the effect did not hold when adjusted to all covariates. Stigma was not associated with uptake of ARVs.
Community engagement and community cohesion were important predictors for uptake of HIV testing while family cohesion and trust in traditional medicines played a bigger role for treatment initiation. Targeted community programs to improve social cohesion on community and family levels can play a critical role in increasing access to HIV/AIDS services.
HIV testing is a core component of global efforts to control the HIV epidemic, although in some settings testing rates remain unacceptably low. In response, several countries have adopted peer-delivered HIV testing as a means of increasing access to testing
Using data derived from the Mitsampan Community Research Project in Bangkok, Thailand between July and October 2011, three multivariate logistic regression models were constructed to identify factors associated with willingness to receive peer-delivered pre-test counseling, rapid HIV testing, and post-test counseling.
Among our sample of 350 IDU, 44%, 38%, and 37% were willing to receive peer-delivered pre-test counseling, rapid HIV testing, and post-test counseling, respectively. In multivariate analyses, factors associated with peer-delivered pre-test counseling includedmale gender (adjusted odds ratio [AOR]=0.52), binge use (AOR=2.39), and experiencing barriers accessing health services (AOR=4.86) (all p< 0.05). Factors associated with rapid HIV testing includedbinge use (AOR=2.23), incarceration (AOR=2.35), avoiding HIV tests (AOR=0.32), experiencing barriers accessing health services (AOR=4.86), and having been to the Mitsampan Harm Reduction Center (AOR=1.76) (all p<0.05). Lastly, binge use (AOR=2.49), incarceration (AOR=1.98), and avoiding HIV tests (AOR=0.26) (all p<0.05) were significantly associated with peer-delivered post-test counseling.
We found that a substantial proportion of Thai IDU were willing to receive peer-delivered HIV testing and counseling. Individuals engaged in high intensity drug use, with a history of incarceration, and those experiencing barriers to health care were most willing to access peer-delivered HIV testing services. These findings highlight the potential of peer-delivered testing to compliment existing HIV testing programs that serve IDU.
With 14.5% HIV prevalence, the southern region of Malawi is in urgent need of theoretically informed campaigns to promote behavior change. Since 2010, the BRIDGE II Project has run a mass media campaign with a potential national listenership of 70% in conjunction with community-based and interpersonal communication interventions that facilitate behavioral choices around HIV prevention in over 340 BRIDGE II communities in 11 districts in southern Malawi. We present midterm evaluation results on two key outcomes promoted by the campaigncondom use and HIV testing.
A first-of-its-kind household-based longitudinal study was conducted in December, 2011 among 685 adults (56% female, average age=30.2 years,
Those who remained in the sample were less educated (
HIV testing and condom use significantly improved at midterm, in comparison to baseline, and exposure to the BRIDGE II programs was significantly associated with these outcomes. Multiple sexual partnerships, another intervention-targeted outcome, were too few to analyze in this sample. Further analyses will explore the role of interpersonal discussion and community mobilization activities in propagating intervention messages. Overall, mass media messages, coupled with community activities, appear to show promise in the fight against AIDS.
Between 2003 and 2006, a large randomized trial was conducted with 328 schools in Western Kenya to compare the effectiveness of two programs conducted either in isolation or combined: 1) training three teachers per primary school on the national HIV/AIDS curriculum; and 2) providing free school uniforms to students in grades 6 and above to reduce dropout rates. We assess the long-term impact of these two programs on transmission of Herpes Simplex Virus type 2 (HSV-2).
The sample includes 19,310 youths enrolled in grade 6 in 2003 in one of the 328 primary schools. A cross-sectional survey to measure HIV and HSV-2 prevalence and behavioral outcomes was administered between February 2009 and March 2011, six to eight years after the interventions. During a first wave of surveying, 54% of the youths could be successfully surveyed. Of the remainder, 29% were randomly selected for “intensive tracking”, and 81% of those were successfully surveyed. We use sampling weights to account for this sampling strategy.
The HIV prevalence was 0.17% among males (average age: 20.33) and 1.56% among females (average age: 19.93). The HSV-2 prevalence was 7.14% among males and 11.79% among females. Students in schools where both programs were implemented were less likely to be infected with HSV-2 than those in control schools (OR=0.837, p-value: 0.056, 95% CI=[0.697–1.00]). This effect was more pronounced for females (OR=0.812, p-value: 0.10, 95% CI=[0.63–1.04]) than for males (OR=0.888, p-value: 0.37, 95% CI=[0.68–1.15]). No significant differences in HSV-2 prevalence were detected between youth in the control group and those in schools receiving only one of the programs.
The national HIV/AIDS curriculum for primary school does not seem sufficient, by itself, to reduce risky sexual behaviors among youths. Ensuring that youths can stay in school appears a necessary complement.
Timely disclosure of HIV status amongst HIV discordant couples is important to prevent new infections. In Pakistan, a combination of patriarchal norms and individual level financial constraints, limit VCT for wives of positive men. We explored the response to conditional cash transfer on disclosure, condom use and VCT compared to baseline
Using clinic records we identified sexually active married men receiving care>6 months who had 1) not disclosed their status to their spouses, 2) their spouses had received no VCT. Baseline condom use among the presumed serodiscordant couples was recorded from initial visit/counseling notes. CCT covered travel/accommodation (US $ 14) to bring the spouse for VCT and encouraged the men to voluntarily disclose status beforehand. Study participants completed a questionnaire on demographics, counseling history, duration of HIV, and reasons for absent disclosure/spouse VCT.
138 men (60%) had spouses with unknown/never tested status. In this group baseline disclosure to wife was 29%, condom use 8%, median duration of clinic visits 14.3 months. 38% men reported travel costs as the main reason for lack of spouse VCT. From the138 men; 94(68%) men received CCT, which was <20% of their monthly income. 53 (56%) brought their spouses for VCT; 19 (20%) reported testing elsewhere, and 22 (24%) did not comply. CCT improved disclosure of HIV status 62% (p<0.05), and condom use 13% (p< 0.08). Factors associated with positive response to CCT were men <50 years, good ART compliance, and prior self-disclosure of status to one family member (p<0.05).
A targeted, low cost CCT ($14) can potentially help avert HIV infections in wives of positive men through promoting VCT and disclosure. Further studies are needed to review effectiveness, spillover, ethical aspects, and sustainable means to overcome financial and social constraints among this most vulnerable group of women.
To evaluate the impact and cost-effectiveness of needle-syringe exchange programs (NSEPs) in Eastern Europe and Central Asia (EECA) in preventing HIV and hepatitis C virus (HCV) infections among injecting drug users (IDUs).
A data triangulation process was conducted across eight countries in EECA. This informed a health economic analysis incorporating a mathematical model of HIV and HCV transmission and disease progression among IDUs. We compared the epidemiological outcomes and costs of NSEP coverage with scenarios of no NSEPs, with counterfactual receptive sharing determined based on an empirical relationship of associations with syringe availability in each country. Outcomes included numbers of HIV and HCV infections averted, lifetime healthcare costs from a health sector perspective, and cost per QALY gained. Discounting was applied at 0% and 3%.
There were substantially increased financial investments in NSEPs over 2005–2010. The average number of needle-syringes distributed, and proportion of IDUs reached, across all eight countries increased by more than 300%. For all eight countries, the reported level of receptive sharing decreased with increases in the per capita distribution of needle-syringes. NSEPs were estimated to avert 10–40% of HIV infections across the eight countries; a lower percentage of HCV infections were averted (~5–25% for six countries and slightly higher in two countries). NSEPs were found to already be cost-saving or cost-effective, with respect to HIV alone in the short-term, in four of eight countries, borderline cost-effective in two countries, but not yet cost-effective in two countries. When considering the additional health benefits of averted HCV infections, or the lifetime benefits of HIV infections averted, NSEPs were very cost-effective to cost-saving in all countries.
There is strong evidence that NSEPs have been effective in reducing risk, leading to reduced HIV and HCV infections averted, and are a very cost-effective public health strategy in EECA.
Globally, 30% of new global HIV infections involve injecting drug users (IDUs) and in many countries, including Vietnam, the HIV epidemic is concentrated among IDUs. We conducted a randomized controlled trial to evaluate whether a community-based network-oriented behavioral peer intervention could reduce injection and sexual HIV risk behaviors among IDUs and their network members.
419 HIV-negative index IDU aged 18–49 years and 516 of their injecting and sexual network members were enrolled in this randomized controlled trial in Thai Nguyen, Vietnam. Each index participant was randomly assigned to receive a series of six small group peer educator-training sessions and two 6-month booster sessions in addition to HIV testing and counseling (HTC) (intervention; n=210) or HTC only (control; n=209). Follow-up was conducted at 3, 6, 9 and 12 months post-intervention.
The proportion of unprotected sex dropped significantly from 49% to 27% between baseline and 3-month visit among index-nonindex pairs. However, at 12 months, post-intervention, intervention participants had a 14% greater decline in unprotected sex relative to the control (Wald test=10.8, df=4, p=.03). This intervention effect was fully accounted for by trial participants who had baseline and 12-month visits but missed some in-between visits, and for whom the control subjects were significantly more likely to report unprotected sex at 12 months compared to intervention subjects. The proportion of observed needle-sharing dropped significantly between baseline and visit 1 (14% vs 3%) and persisted until 12 months but there was no difference across trial arms (Wald test=3.74, df= 3, p=0.44).
Decreased sexual and injecting risk behaviors noted in all arms between baseline and visit 1 may be associated with the HTC received by all participants. Missing some intermittent visits may account for higher reported unprotected sex among control participants at 12 months.
The FDA is considering licensing OraQuick, a rapid, oral fluid, HIV antibody test that provides results in 20 minutes, for over-the-counter sale (“home test” or HT). We studied whether HIV-uninfected, non-monogamous gay and bisexual men living in New York City who never or rarely use condoms would test their partners prior to receptive anal intercourse (RAI) as a harm-reduction approach.
After baseline assessment and self-testing in our offices, participants received 16 HT kits to take home as an option to use with sex partners for three months, after which they were interviewed.
Of the ethnically diverse 32 men enrolled, 28 completed all study procedures and 27 used HT kits before intercourse with approximately 100 partners. Kits were used at participants’ and partners’ homes and occasionally in public places. Nine sexual partners were found to be infected; five of them were unaware of their status. Participants showed empathy for partners found to be infected; no sexual intercourse took place after someone's infection was detected. A majority of participants said that having HT kits and using them shifted their own perceptions of risk and led to changes in their risk practices. Very few problems occurred related to HT use. Most participants expressed a strong desire to continue using the test and frustration that they could not buy it freely. Testing had high acceptability among ethnic minority participants and ethnic minority sex partners.
MSM at high risk can use HT to screen sexual partners, and many partners will agree to take the test. Use of HT results in detection of previously unknown infections and avoidance of HIV exposure. Making HT available within networks where high-risk sexual practices are common may be a cost-efficient and effective way to identify previously undetected cases. HT may become an important harm reduction technology.
Willing to understand what drives the HIV epidemic among MSM in Switzerland, the Swiss Federal Office of Public Health (SFOPH) commissioned the development of a mathematical model (MM). On the basis of its results, the SFOPH developed an Urgent Action Plan in order to break the chains of new HIV infections among MSM.
The MSM epidemic was remodeled from 1980 to 2010 on the basis of data coming from the Swiss Gay Survey, the Swiss HIV Cohort and the SFOPH surveillance system. Furthermore, the MM developed scenarios on how the HIV epidemic could evolve depending on the changes that were made in the HIV prevention work among MSM.
According to the MM, in 2010, “only” 13% of the infected MSM were unaware of their HIV infection yet were the origin of 80% of the new infections. Moreover, the MM indicates that the average time to diagnose is only 2.2 years. Finally, if nothing changes, the number of MSM needing antiretroviral treatment would double in the following ten years.
The small proportion of MSM that seem to be the origin of most new transmissions tend to indicate that the spread of HIV is mainly driven by Primary HIV Infection (PHI) among MSM. Based on this result, the SFOPH developed an Urgent Action Plan organised in three action fields. The first action field aims to reduce the MSM
Recent studies indicate high rates of HIV infection among transgender persons in the United States. However, the scarce availability of national surveillance data on transgenders limits our understanding of HIV's impact on this population. This study describes the patterns of HIV testing events among transgenders served in CDC-funded HIV prevention programs nationally.
In 2008, CDC launched an expanded set of National HIV Monitoring and Evaluation (NHM&E) testing data requirements that included a gender variable, with an option for a transgender category. Using 2008
Of the 5,522,689 CDC-funded HIV testing events reported in 2008
Among transgender testing events that included HIV risk information, the most frequently reported risk behaviors included sex without using a condom (56%), sex while intoxicated and/or high on drugs (22%), and exchange of sex for drug, money, or other materials (14%).
NHM&E data indicate that transgender testing events represent a small percentage of the overall testing events but have higher levels of HIV positivity than male and female testing events. HIV testing and positivity varied by race, ethnicity, and age. These findings underscore a great need for expanding targeted HIV prevention services that are responsive to the needs and socio-demographic characteristics of transgender persons.
Transgender persons (TG) face considerable marginalization and are at significant risk of HIV. The Avahan program included TG as a key focus population for the prevention interventions implemented since 2004 in India. The evaluation of this intervention included two rounds of bio-behavioral surveys for tracking outcomes among TG in Tamil Nadu.
Cross sectional bio-behavioral surveys termed Integrated Behavioral and Biological Assessments (IBBA) were conducted in 2006 (R1) and 2009 (R2). Those who self-identified as TG, 18 years and older, were sampled using probability based sampling methods across five districts in Tamil Nadu. After voluntary consent, face-to face interview was used to collect behavioral risk information and blood and urine samples were collected to test for HIV and STIs.
807 Transgender were interviewed during both rounds of IBBA. The decrease of HIV prevalence among TG between R1 and R2 was not significant12% and 9.8% (p=0.49), respectively. However, prevalence of lifetime syphilis (any RPR and TPHA positive) decreased significantly from 16.6% in R1 to 4.2% in R2 (p< 0.001). Proportion of respondent who reported having been contacted by program peer educators increased between R1 and R275% and 83% (p=0.02), respectively, whereas reported visits to program STI clinics decreased from 75% in R1 to 45% in R2 (p=0.00). No significant change was observed in reported consistent condom use (CCU) with regular male partners (34% in R1 and 47% in R2; p=0.06), but the proportion of TG who reported last time condom use with paying partners decreased from 93% in R1 to 80% in R2 (p< 0.001). About 61% of TG reported CCU with paying partners in R2.
Increased peer contacts did not result in improved condom use. Innovative behavior change communication strategies and structural interventions are required to increase access to services among TG for reducing vulnerabilities to HIV and STIs.
HIV testing is a major part of HIV prevention efforts across the globe. In US and World Health Organization (WHO) HIV testing guidelines, opt-out testing has replaced voluntary confidential testing as a means of combating low rates of HIV testing. The success of opt-out testing is not assured, particularly due to financial, legal, operational, and cultural barriers, such as limited reimbursements for testing, obstructive laws that require written consent, limited access to healthcare, and poor compliance by care providers in testing patients perceived to be low-risk for HIV infection. Alternative methods of promoting HIV testing warrant analysis in comparison to opt-out testing.
HIV testing policies were hypothesized empirically and identified in the scientific and popular press. Each method was analyzed for consistency with WHO policy on HIV testing, which emphasizes the '3 Cs' of proper human rights-centered HIV testing. These conditions require that HIV testing must be: confidential, complemented by counseling, and informed, voluntary consent.
We identified incentivized testing, anonymous mandatory testing, and names-based mandatory testing as alternatives to voluntary confidential and opt-out testing. Incentivized testing, in which a person's “willingness” to be tested is rewarded, e.g., with money, is voluntary but potentially coercive to those of lower socioeconomic status. Incentivized testing is in limited use but large-scale implementation would need to be names-based in order to avoid significant fraud. Anonymous mandatory testing using de-identified codes that allow verification that a person has undergone HIV testing preserves confidentiality but is involuntary. Names-based mandatory testing violates confidentiality and volition.
Though each HIV testing policy provides counseling, opt-out testing remains the only ethical approach for the promotion of HIV testing as it avoids coercion and loss of privacy. Financial, legal, operational, and cultural barriers to greater uptake of opt-out testing must be removed so that ethically unacceptable HIV testing policies are avoided.
Study participants are reimbursed for transport costs incurred when travelling for clinic visits. Reimbursement also compensates for time spent in the clinic and is important for participant motivation and retention. In impoverished communities, determining adequate reimbursement is challenging. Under-compensation leads to low recruitment/retention rates, high mortality and missed trial endpoints. Ethical Committees cap reimbursements as overcompensation leads to undue influence. We sought to examine reimbursement practices, their impacts on costs and study participation in an ongoing tuberculosis vaccine trial.
Mothers in rural Western Kenya were approached to recruit their infants. Once enrolled, they come for seven scheduled visits and an average of two unscheduled/sick visits in a span of two months. Reimbursement was set at a rate of $4 per visit regardless of distance and cost incurred. Distances to clinic were measured by study staff. Furthest distance covered is 30km costing $6. Where the reimbursement rate was unacceptable to the mother given cost or critical study visits would be missed, mothers were ferried to clinic by designated vehicles at a cost of $18 per subject.
Of 144 participants enrolled after 7 months, 57(40%) were under-compensated when compared to the costs incurred in attending study visits and expressed dissatisfaction with amounts reimbursed. Of these, retention rate was 99%.118 of sick participants attended clinic and 30(20%) sought care elsewhere due to cost. Participant satisfaction was not measured.
High retention rates were achieved despite under-compensation but at a cost three times higher compared to cost of directly reimbursing the mothers. Given the challenges of conducting trials in areas with limited infrastructure, there's need for continuing dialogue between investigators and Ethical Committees regarding amounts given to participants which may be based on a gradient of reimbursement according to distances travelled. There's room for educating mothers/participants on research ethics and non monetary research participation benefits.
Ensuring informed consent is a complicated component of research, particularly with HIV prevention research conducted in poor settings. An inherent challenge characteristic of the informed consent process for HIV prevention studies is making sure that subjects understand that participation does not increase exposure to HIV and does not necessarily protect them from HIV. It is important to continuously monitor the informed consent process.
In June- September, 2011, gender-specific in-depth interviews (n=20) were held with interviewees who had been purposively selected from participants who had exited a vaginal HIV prevention study in Harare, Zimbabwe. An interview guide was used to elicit views around the informed consent process. Discussions were conducted in mother tongue and audio-recorded. Audio-recorded data were transcribed, translated verbatim into English, coded using NVivo 8 and analysed using grounded theory principles.
Key information about study was given, as subjects articulated study aims well. However, it appeared that the informed consent process had been rushed and some participants had not had enough time to decide. Moreover, some participants reported that due to both excitement and anxiety, they had felt pressured to sign consent forms before comprehending some aspects of the study. Some mentioned that they had found it difficult to ask questions about the study. Both the study procedure and duration had not been fully explained. There were mixed feelings on importance of male partner involvement in decision-making around study participation, with some feeling that spouses should have been involved and others stating that partner consultation did not matter.
This study elicited some of the issues that characterise the informed consent process for clinical trials conducted in poor settings. It also highlighted the need for researchers' ingenuity in order to come up with strategies that tailor the informed process to suit the specific needs and circumstances of individual participants.
Conducting community-based research with people living with HIV requires maintaining both individual and collective ethical standards. Research teams need to be cognisant of individual and community conceptions of informed consent, risks and benefits, and confidentiality. Although much has been written about challenges to obtaining ethics approval for HIV CBR, there have been fewer opportunities to describe ethical issues which emerge over the 'life' of these projects. This presentation addresses this gap by describing the ethical tensions which emerged during an HIV CBR study conducted in a hospital setting.
Using a case study analysis, this presentation draws on field notes, minutes from team meetings, and reflexive memos to offer a critical chronology of decisions made to improve ethical practice in one HIV CBR study. Various disciplinary (nursing, social work, public health) and stakeholder (clinician, coordinator, researcher, participant) perspectives are highlighted and contrasted to show how 'ethics' was understood and negotiated.
We discuss the range of issues that emerged at the intersections of HIV, drug use, negotiating clinical space, and piloting innovative arts-based methods. Issues of cognition and HIV required the use of a multi-step informed consent process. Research team members balanced the need to support participants with reminders while being conscious of not creating undue pressure to participate. Maintaining confidentiality proved challenging because of the way space and time are managed in a clinical setting. The sensitive nature of the study and proposed group data collection necessitated changing the study design from group to individual data collection techniques to protect confidentiality. Interestingly, this happened despite having undertaken preliminary community consultations to determine the best approach.
Our findings highlight the importance of understanding and integrating both individualistic and communitarian ethical concerns, remaining flexible about design issues, and maintaining clear and open lines of communication between all project stakeholders.
The implementation of this model comes from the minimum interventions of the Peruvian health system towards HIV/AIDS positive children and adolescents. The objective is to establish a program that provides children and adolescents with a caring, quality and confidential service. The model would contribute to reduce stigma and discrimination towards this specific HIV/AIDS positive population in South Lima.
The implementation of the model required training sessions to HIV positive children and adolescents of poor families, family members of low educational level, some quechua speakers and teachers from local schools. The topics of the trainings were the following: 1) human rights, stigma and discrimination, 2) self-care for children and adolescents, 3) diagnosis management, 4) psychotherapeutic issues with family members. Teachers and community leaders participate on a specific training on sensitization and advocacy.
31 HIV positive children and adolescents were trained on self-care and human rights. 28 families of HIV positive children received psychosocial support and their capacities and knowledge have been strengthened. 45 community leaders are more knowledgeable and sensitive on topics related to HIV/AIDS stigma and discrimination and can reproduce the training. 73 teachers are trained on topics related to HIV and children, stigma and discrimination and reproduced training to students, family members, in order to reduce rejection of HIV positive children and adolescents within their communities.
HIV/AIDS positive children and adolescents are more assertive and self-confident towards stigma and discrimination. Families and fathers of HIV positive children and adolescents are prepared to assume responsibilities, self-care and care of their children. An informed community and school would strengthen the process of reduce stigma and discrimination and reinforce adherence to treatment.
In the 2000s, France's policy on illegal drug users has become increasingly stringent. The National AIDS Council wanted to assess the impact of this punitive policy on risk reduction policy targeting drug users, notably injection drug users.
Around thirty hearings were held with representatives of the relevant authorities (Ministries of Home Affairs, Justice, and Health), HIV/AIDS control associations, associations specialized in addiction and related issues, and drug user self-help groups. A review of the literature was also undertaken.
Policies on illegal drugs have focused on strengthening sanctions against drug users. From 2001 to 2007 the number of arrests for drug offences increased by 106%. Accusations relating to drug use represented 85% of all offences during the period in question. Consequently, the cost of this punitive policy, estimated at 590 million Euros in 1995
The punitive policy on drug users has been made a top policy and budget priority, with risk reduction policy relegated to second place. This policy has hindered the development of pragmatic risk reduction measures, such as medically supervised injection centres, or prison-based needle exchange programs, adapted across the country to support the most vulnerable populations.
In 2003, France adopted a law against procuring and soliciting in order to discourage the practice of prostitution. The National AIDS Council wanted to assess its impact on sex workers in terms of prevention.
Around thirty hearings were held with representatives of the relevant authorities (Ministries of Home Affairs, Social Affairs, and Health), specialist police units and associations involved in the subject: associations campaigning and working on health and community health, social reintegration, abolition of prostitution, and sex workers' unions. A field investigation was also carried out within a community health association. Finally, all gray literature was analyzed.
The 2003 law has undermined sex workers, the majority of whom are foreigners, without directly protecting the victims of procuring. Accusations against sex workers multiplied twenty-fold in the first half of the 2000s, before dropping slightly. There has been no increase in accusations for procuring. The activity has shifted to more isolated locations, under clandestine conditions with greater exposure to violence. The prevention work carried out by community health associations has become increasingly difficult, partly due to uncertainties regarding the availability of subsidies. The police and health authorities have not managed to cooperate effectively.
The introduction of a punitive law has complicated prevention work targeting sex workers. In a hostile legislative context, it seems important to support and reinforce community health actions by means of a comprehensive approach combining prevention, access to health care, social and human rights.
HIV infection is higher among sex workers than in the general population, yet sex workers often lack access to HIV prevention services. A police practice that interferes with HIV prevention is using condoms as evidence of prostitution.
A multi-method investigation of the practice of using condoms as evidence of prostitution was conducted in New York City, Washington, D.C., Los Angeles and San Francisco between October 2011 and April 2012. In-depth interviews with hundreds of sex workers, their advocates, outreach workers, police, prosecutors, judges, and public health officials were conducted, combined with legal and policy analysis.
Police engage in the routine and widespread practice of using the presence of condoms on individuals to profile them as prostitutes, to threaten arrest and as a basis for arrest on charges related to prostitution. In San Francisco, police and city regulators use condoms as a basis for arrest in clubs frequented by transgendered persons, in massage parlors and other businesses. In New York City, prosecutors introduce condoms into evidence to support prostitution charges. In Washington D.C. police confiscate condoms found on persons they identify as sex workers. In Los Angeles, sex workers follow what they believe to be the “three-condom rule”, fearing arrest for exceeding a routinely enforced limit. In each city these practices take place in a wider context of police harassment and abuse of sex workers and those perceived to be engaging in sex work, including verbal and physical degradation and other misconduct. Transgendered women and LGBT youth are particularly targeted.
Human rights abuses make some sex workers less likely to carry condoms, particularly those most vulnerable to arrest, such as undocumented immigrants. The criminalization of condoms interferes with the right to health and undermines public health objectives of universal access to disease prevention.
Many jurisdictions continue to inappropriately prosecute people living with HIV (PLHIV) for non-disclosure of HIV-positive status, alleged exposure and non-intentional transmission. Although most HIV-related criminal cases are framed by prosecutors and the media as being cases of ′deliberate′ HIV transmission, the vast majority have involved neither malicious intent nor has transmission actually occurred or the route of transmission been adequately proven.
This global overview of HIV-related criminal laws and prosecutions is based on latest data from GNP+ Global Criminalisation Scan and media reports collated on criminalhivtransmission.blogspot.com. Final ranking will be based on the total number of prosecutions by July 1 2012 per 1000 PLHIV.
At least 63 countries have HIV-specific criminal laws and at least 48 countries have used HIV-specific (n=19) or general laws (n=31) to prosecute HIV non-disclosure, exposure or transmission. Despite growing national and international advocacy, prosecutions have not diminished, particularly in high-income countries, with the greatest numbers in North America. Since 2010, prosecutions have taken place in Belgium and Republic of Congo for the first time. In 2011, although HIV-specific laws were suspended in Denmark and rejected in Guyana, Romania passed a new HIV-specific criminal statute. In Africa, the continent with the most HIV-specific criminal laws but with few known prosecutions, Guinea, Togo and Senegal have revised their existing HIV-related legislation or adopted new legislation in line with UNAIDS guidance.
Given the lack or inadequacy of systems to track HIV-related prosecutions in most places, it is not possible to determine the actual number of prosecutions for every country in the world. These data should be considered illustrative of a more widespread, but generally undocumented, use of criminal law against people with HIV. Improved monitoring of laws, law enforcement, and access to justice is still required to fully understand impact on HIV response and PLHIV.
In Canada, the exposure and/or transmission of HIV is punishable by criminal law. The objective of this study was to investigate the impact of HIV/AIDS criminalization on awareness, prevention, and stigma as outlined in the literature and from the perspectives and experiences of stakeholders in the HIV sector in Ontario, Canada.
For the scoping review, we searched 11 databases, and reviewed (in duplicate) the results for any articles addressing topics related to HIV criminalization. We then identified stakeholders (policy/content experts, executive directors and frontline workers from community-based HIV/AIDS organizations) and invited a purposive sample to participate in one-on-one, semi-structured interviews.
The search yielded 1301 results; 147 articles were included, most of which were case reports, editorials, commentaries and essays (n=136), with only 11 primary studies. Interviews explored perspectives on criminalization and its impact on prevention efforts, community awareness of prosecutions, and stigmatization of people living with HIV/AIDS. Findings highlight confusion regarding behaviours constituting “significant risk,” resulting in difficulties in the application of legal precedent, as well as uncertainty regarding HIV knowledge in the general public. Findings also highlighted uncertainty regarding the behavioural changes attributable to criminalization, but suggest that it contributes to disincentives for testing and disclosure, fears of secondary disclosure and false accusation, strained therapeutic relationships, HIV related stigma, and barriers for promoting shared responsibility for safer sex. Participants recommended guideline development to ensure optimal use of criminal law in case related to HIV/AIDS non-disclosure.
This study provides further insight into the impact of HIV/AIDS criminalization in the community. Further research is necessary to characterize the impact from the perspective of people living with HIV/AIDS and to inform ongoing policy discussions.
Sociologists have recently become interested in the rise in prosecutions across the country under criminal HIV disclosure statutes. These law vary in their specifics, but generally make it a crime for HIV-positive individuals to have sex without first disclosing their HIV-positive status. Yet, despite a number of sociological explorations of how HIV-positive individuals relate to these laws, we know very little about how these laws are actually applied on the ground. This paper addresses this gap by exploring how legal actors in Michigan trial courts struggle to frame disclosure cases in terms of medicine and/or the law.
Using a database of prosecutions provided by the Michigan State Police, this study analyzes court transcripts from forty prosecutions for HIV non-disclosure. The police dataset included the sentencing dates, counties, and the court′s disposition for cases prosecuted between 1992 (the year the law first was prosecuted) and 2010. In order to request their corresponding court transcripts, these data were cross-referenced with local news reports in order to identify the defendants. This research design was reviewed (and deemed exempt from further review) by the University of Michigan′s Institutional Review Board.
I argue that these cases can ultimately be understood as a contest to frame the disease as disease or crime. Prosecutors frame defendants as what Thomas Shevory has called “HIV monsters” using legal discourses of negligence, harm, and morality. Their accusers, on the other hand, are framed as undeserving and helpless victims. Defense attorneys attempt to diffuse these arguments by relying on medical discourses that frame disease as victimless and deserving of treatment, not punishment.
This analysis is the first empirical study to show how HIV disclosure cases are legally argued on the ground. Moreover, this study demonstrates empirically how medical problems become transformed into crime, a poorly understood social process.
Laws criminalizing certain behaviors, and HIV non-disclosure, exposure and transmission are resurgent in developed and developing countries around the world. This study set out to understand how laws impact health seeking behavior and what coping mechanisms result amongst different key populations and to recommend policy changes.
A qualitative four-part questionnaire was developed and used in 27 focus groups carried out by community members during August and September 2011 in all regions of the world. More than 240 participants (51% PLHIV) from 32 countries, selected by their affiliation with a key population, took part in the study. Participants were asked non-identifying information such as HIV status, associated population group, gender, and housing situation. The hundreds of pages of feedback were analyzed for common themes across geographic region and population grouping. Common trends were then verified to develop key findings.
Results show that individuals are being deterred from seeking health services when their behavior is criminalized. Evidence revealed that laws criminalizing HIV and certain behaviors promote the persecution of people living with HIV. HIV-specific criminal laws are reinforcing or increasing the stigma and discrimination that inhibit access to health services and undermine treatment. Evidence also demonstrated that legal protections for people living with HIV and key affected populations are insufficient or unenforced.
The current goals of accessing HIV prevention, treatment and care services are being undermined where the legal environment is unable to enforce laws protective of individuals at high risk of or living with HIV. Measures to be taken include: supporting anti-stigma and HIV education campaigns, specifically within the judicial system and amongst law enforcement; opposing and repealing HIV criminal laws; and promoting access to legal aid. Quantitative prevention research on HIV criminal laws and rates of HIV testing would be useful in statistically proving what was discussed in this study.
In 2010, Action for Health Initiatives (ACHIEVE), Inc. conducted a research wherein evidence was found that the broad language of national anti-prostitution and anti-trafficking laws hinder effective HIV prevention initiatives among sex workers in the city. Considered as “victims”, sex workers are “rescued” while establishments are raided by police authorities using condoms as evidence of prostitution or trafficking. These hamper condom distribution and outreach activities of HIV prevention workers.
To address this issue, ACHIEVE, in partnership with the Quezon City Health Department (QCHD), held a series of consultative workshops, individual meetings and interfacing meetings with the Philippine National Police (PNP) officers, Quezon City Police Department (QCPD) officials and the local entertainment establishment organization in January 2011. The consultations sought to build partnership by forming memorandum of agreement (MOA) between QCPD and QCHD to end the conflicting law enforcement practice of using condom as evidence of prostitution.
Bureaucracy, strict hierarchy within the Police Department and the very fast turnover of officials were identified as constraints in partnership-building with the police. However, the support given by the Quezon City Health Department and the sex work establishment owners association and the community help in convincing police officers and sensitizing them during the process despite their strong moral positions against sex work. After seven months, a MOA was drafted subject for adoption by PNP Chief of Police.
While the MOA was refused to be signed by the PNP Chief of Police, the process opened many doors for partnerships and for alternative next steps. These include increased support from the Quezon City STI/AIDS Council and garnered support from police officials of various QCPD and PNP offices who were not previously partnered with. The Quezon City Committee on Health also committed to provide support in implementing rights-based and issue-based HIV education among frontline police officers.
With USAID and Global Fund support, Deloitte Consulting LLP assessed existence and effectiveness of internal controls for government and non-governmental pharmaceutical supply chains in selected African countries. Within these countries, Deloitte traced inventory movements and volumes of selected ARVs, RTKs, and other commodities from the point of entry along the supply chain to end-user consumption.
The Deloitte team assessed a variety of sites, including central, provincial, and district warehouses; rural health posts; government, mission, and private hospitals; points of entry; and transportation companies. Site sampling methodology weighted provinces and districts by expected risk areas for diversion, population size, number of sites, and volume of medicine dispensed.
ARVs, RTKs and other commodities (e.g., anti-malarials, essential medicines, ITNs) were traced. Assessments incorporated a structured questionnaire, documentation sampling and review, and end-to-end reconciliation of inventory movements across each level of the supply chain. The evaluation focused on eight areas to determine control strength: Control Environment, Monitoring and Evaluation, Port Arrival and Customs Clearing, Transportation, Receiving, Storage, Distribution, and People. Scores were rolled up to province and country level in these eight areas to yield a final risk score for each country's supply chain.
Assessed supply chains were generally found to be in the second quartile of maturity (with a score between 25 and 50 of 100 points), indicating overall weakness in internal controls. Amongst assessed countries, trends were inconsistent at the central, provincial or district levels. Common to all countries, sub-district facilities had very weak controls, leading to greater losses compared to upstream facilities.
Country governments and donors must improve documentation practices, particularly at the lowest level of the supply chain for ARV and RTK consumption. Strengthening sub-district facility controls will decrease stock-outs, diversion, and loss due to expiry, increasing available resources for health programming and improving patient outcomes.
There is a clear and growing body of evidence for increased prevalence of cardiovascular disease (CVD) in HIV patients as a result of viral effects or of antiretroviral medications (ART). This pilot was implemented to assess the feasibility of integrating routine screening of cardiovascular risk factors in an HIV clinic setting in order to inform programmatic approaches.
HIV-positive clients enrolled in the facility between May 2010 and August 2011 (12,177 clients) were targeted for clinical CVD screening. Those found with specified CVD risk factors were referred for laboratory evaluation of their fasting blood sugar and lipid profile. In addition, behavioral and biomedical interventions were provided, including referrals to an on-site cardiac clinic for clients with a ten-year CVD risk of developing a fatal or non-fatal cardiovascular event of ≥20% (WHO/ISH AFR-D CVD risk assessment charts). Follow-up annual and biannual CVD risk assessments were also scheduled for clients with <20% and ≥20% ten-year CVD risks respectively.
From May 2010 to August 2011 a total of 1,033 HIV positive clients aged between 17 and 70 years were randomly selected for screening; 82.7% were receiving ART. Of those screened, 205 (19.8%) were identified with one or more CVD risk factors and sent for laboratory evaluation; 42% of whom had a CVD risk of <20%, and 2.4% a CVD risk of ≥20%. The most common risk factors identified were age (25.7%), male sex (25.9%), high BMI (21.8%), and hypertension (15.2%). Although all risk factors were more prevalent in those receiving ART, there was no statistically significant difference with the ART-naive. Mean serum total cholesterol levels were found to increase as duration on ART increased.
We conclude that integration of screening for cardiovascular diseases in ART clinic settings is feasible and essential in order to improve the life expectancy of HIV-positive individuals.
Zambia has the world's second highest annual cervical cancer incidence and mortality rates[1] coupled with one of the world's highest HIV prevalence rates at 14.3%.[2] Research has shown an association between HIV infection and a high incidence of invasive cervical cancer with rapid progression. In the Zambia military population where the seroprevalence rate is estimated to be two times higher than the national rate, women are at greater risk of cervical cancer and HIV co-infection.
PCI and the Zambia Defence Force (ZDF) adopted the “screen-and-treat” model of the Cervical Cancer Prevention Program in Zambia (CCPPZ) and integrated it into an existing ZDF mobile HIV counseling and testing (CT) unit. The pilot was implemented in 14 ZDF units using existing health facilities and equipment. ZDF nurses were trained on the CCPPZ protocols and used an opt-out approach to enroll women accessing mobile CT services. Nurses screened consenting women using visual inspection with acetic acid (VIA) and provided same-visit treatment using cryotherapy (“screen-and-treat”).
A total of 839 women accessed mobile CT services; 560 women (67%), median age 35, consented to being screened. Of the 560 screened, 16% (88/560) were found with abnormal cervical lesions of which 11% (62/560) were eligible for on-site cryotherapy and were treated immediately while 5% (26/560) were referred to the Gynecologic Cancer Prevention Unit at University Teaching Hospital and of these 92% (24/26) completed the referral. The prevalence of HIV among women screened was 20% (113/560).
The high proportion of women (67%) that accepted screening for cervical cancer and the high proportion of women completing the referral, show that existing HIV prevention interventions provide a springboard for rapidly reaching women with timely, life-saving and easily adaptable cervical cancer prevention and treatment programs such as “screen and treat”.
[1] GLOBOCAN 2008
[2] Zambia Demographic and Health Survey (2007)
Zimbabwe is one of the countries with the highest HIV prevalence (13.75%) in the world. It has a strong policy and strategic framework and a National AIDS Trust Fund (NATF) created through an Act of Parliament. All employed individuals and companies in Zimbabwe pay 3% of their taxable income on monthly and quarterly for former and later respectively. The money support the national response to HIV and AIDS and the fund has emerged as an innovative and potentially sustainable internal response resourcing approach.
The study was conducted in Zimbabwe and used a combination of secondary literature, key informant interviews (Ministry of Health, NAC, Private Sector and Public Service authorities), focus group discussions (beneficiaries) and observations (Health facilities).
The NATF is an innovative approach for a self financed/sustained HIV and AIDS response in Zimbabwe. Currently 25% of all people on ART are supported by the fund apart from financing some interventions under prevention, advocacy and monitoring and evaluation. Assisted the country achieve Global Fund grant targets though funding the acceleration of implementation. Strengthened and improved effectiveness of NAC's coordinating capacity. Fund's full potential yet to be realised as the informal sector (with estimated 80% of the employable population) is not contributing and the economy operating 40% of its capacity. NATF is a regional best practice and four countries Tanzania, Botswana, Kenya and Zambia have visited Zimbabwe to learn about the levy.
The NATF demonstrates potential countries have to generate internal resources to sustain their responses. In the context of diminishing the external global financial support, countries need innovative approaches to avoid reversing response gains. Countries with large informal sectors need to ensure contribution by these as they also benefit from services and commodities. The success of this approach rests on genuine political commitment and private sector collaboration.
For a small NGO supporting a community health worker program in rural villages, ensuring accurate timely data collection is difficult. Paper based systems are time-consuming to collect and often provide incomplete data. A simple, cost-effective monitoring system using mobile phones can speed up data collection and provide better data. This project aimed to use electronic reporting to better capture the quality of support (social, family, personal) provided by community health workers.
The project adapted paper-based monthly reporting tools already in use for implementation on the mobile phone platform using open access Frontline software. 38 CHWs (21% of the total number) were selected from a diverse set of locations. Staff built buy in from participating CHWs for the new technology and trained them on the system. Data was collected through SMS to a central database. CHWs received an initial sum of phone credit and were reimbursed based on the number of messages received, regardless of accuracy. To assess the comparative benefit of the system, SIC measured the collection and accuracy of CHWs using paper surveys.
One month after initiation, participating CHWs had sent monthly patient information by text for 37.2% of their clients with 9.8% of messages having data errors. For paper, at one month, the percentage of monthly patient reports collected was 6%. At three months, a higher percentage of paper reports (78.7%) had been collected than electronic (45.0%). Paper surveys were two and a half times as likely to have missing information.
The mobile phone system has a clear advantage in timeliness of data. Over time, however, consistency with mobile phone reporting declined because of difficulties with reimbursements and challenges with system maintenance Next steps are adjusting the reimbursement system and evaluating whether the system is better suited for use in ongoing reporting, annual evaluation or not at all.
Shortages of health professionals in low-income countries make task shifting to community health workers (CHWs) crucial to AIDS care and treatment. Multiple studies of CHW utilization reveal poor linkages to the formal health sector, lack of clinical decision making support in the field and weak integration of information gathered at the community level with the national health information system.
A pilot study was performed at a public HIV clinic in Nairobi that provided CHWs with a mobile telephone system (ClinipakMobile) loaded with surveys that support appropriate clinical decision making and link to an electronic patient information database. Each CHW underwent a one-day training on use of ClinipakMobile and used ClinipakMobile at home visits with clients living with HIV. Variables assessed were: ability of the CHW to use ClinipakMobile based on standardized role-play assessments, CHW satisfaction surveys, ability of the server to store clinical data, and patient-based outcomes assessed by an end-of-study chart review of CHW referrals.
Seventeen CHWs participated. The CHWs quickly learned and retained skills in using ClinipakMobile across three time points (week 0, 3 and 6) and expressed great satisfaction. The database maintained function and integrity with only one down-time period of 24 hours. CHW patient interviews using ClinipakMobile recorded multiple “red flag” answers for antiretroviral non-adherence (21%), side effects (30%) and opportunistic infection symptoms (33%) resulting in 30% of patients receiving ClinipakMobile-recommended referrals to the health facility. The chart review revealed these problems were infrequently noted by clinicians at follow-up visits.
ClinipakMobile is an easy-to-use mhealth tool to improve CHW functioning to care for PLWHA. Busy clinicians may not note important clinical “red flag” that CHWs record at home visits. In the future, patient information gathered on ClinipakMobile should be downloaded from the server daily and given directly to clinicians to improve care during clinic visits.
Studies of HIV-infected people show the great efficacy of anti-retroviral therapy (ART) at preventing HIV transmission to uninfected sexual partners. High ART uptake may produce an additional secondary fall in population transmission through reducing ‘community viral load’. In the UK, HIV care is free and 95% of the diagnosed HIV-infected are established and retained in care. Using comprehensive nationwide information, we examined whether high ART uptake was associated with a fall in HIV transmission among MSM.
HIV surveillance data, and sequential Bayesian multi-parameter evidence synthesis (s-MPES), which combines direct and indirect information from multiple sources, was used to measure the recent trend in the number and proportion of diagnosed HIV-infected MSM who were infective (viral load >1500 copies/ml) and estimate the number and proportion of undiagnosed HIV-infected MSM who were infective. These combined trends were related to indicators of HIV incidence and the trend in HIV incidence estimated through an s-MPES extension. Data relate to 2006-10 in the United Kingdom.
In 2010, an estimated 40,100 MSM were HIV-infected; 74% were diagnosed and in these, ART uptake was 80%. Between 2006 (when ART uptake was 71%) and 2010, the overall proportion of infective MSM decreased from 47% to 35%, while the number infective remained around 14,000. Estimated annual MSM HIV incidence rose from 0.5% in 2002 to 0.9% in 2008. In 2010, 62% of the estimated infective were undiagnosed, 33% were diagnosed but untreated, and 5% were on ART.
Optimal ART uptake in recent years, consistent with clinical guidelines, has led to a fall in the proportion infective without any apparent effect on numbers infective or on the estimated HIV transmission rate. High ART uptake may moderate but not control HIV transmission in MSM, and behavioural interventions and increased testing remain a prevention priority.
Stopping transmission of HIV to stable partners is a main focus of positive prevention initiatives. In January 2008, the Swiss Commission on AIDS issued a statement indicating that positive individuals with effective antiretroviral treatment and without STD's are not considered sexually infectious. At the same time the importance of the negative partner's decision about precautions such as condom use was emphasized. The aim of this qualitative study was to explore how negative partners, living in stable sero-different partnerships, make everyday decisions about dealing with the HIV transmission risk.
The Study was conducted at one center of the Swiss HIV Cohort Study (SHCS). At medical consultations, patients were asked to provide written study information to their stable partners. Partners willing to participate were recruited consecutively. Open ended interviews were conducted with each participant and transcribed verbatim. Transcripts were analyzed using thematic analysis according to Brown and Clark (2006).
Ten Caucasian women and seven men, born in different European countries, gave insight views on how they deal with their perceived HIV transmission risk. The mean age in this group was 43.3 years (range29-66y). The duration of their relationship ranged from 2-24 years. Lifestyles and sexual preferences were diverse.
Participants experienced significant changes between phases of relative security and insecurity associated with the possible risk of HIV transmission. To deal with feelings of insecurity, different types of decision making were describeda) condom commitment; b) role governed commitment; c) flexible adaption and d) incongruent adaption.
To our knowledge this is one of the first studies focusing perceptions of negative partners in sero-different couples after the Swiss statement. Participants narratives provided insights into different decision making strategies, which provide guidance for individualized counseling and systematic prevention interventions.
In India, females constitute 39% of People Living with HIV/AIDs (PLHAs) (0.9 million). Andhra Pradesh state (AP) with 73% rural population, has the highest number of PLHAs (2007) (0.5 million), and high HIV prevalence among pregnant women (>1%) and female sex workers (9.74%) (HSS 2007).
The HIV/AIDs prevention component was integrated with the public sector's largest community based Health and Nutrition Program (HNP) involving women Self Help Groups (SHGs), aimed at empowering rural women on HIV/AIDs prevention and reducing stigma. Three innovative modules developed in story form with flipcharts, addressed social norms, gender, personal hygiene, reproductive health, foetal sex determination, fertilization, HIV and stigma and discrimination. Master trainers trained state and district level HNP staff, who trained sub district level staff, who in turn trained SHG women at village level.
3,244 HNP state and district level staff were trained, reaching 438,622 (86%) of SHG women in rural areas. More than 40,000 SHG women who perceived themselves at risk for HIV got tested. HIV/AIDs health resource directories were developed for all 23 districts in AP. Case studies (150) were shared at state/district advocacy meetings. Indira Kranti Patham-Society for Elimination of Rural Poverty (IKP-SERP) has planned a phased scale up of this project across AP in 2012.
With reductions in funding globally for HIV/AIDs programs, integration of HIV prevention within existing community based programs will support sustainability, community ownership, gender issues and in achievement of millennium development goals.
Cash transfer programmes with (CCT) and without (CT) conditions may improve the well-being and development of orphans and other children made vulnerable by HIV (OVC) in sub-Saharan African populations but evidence of effectiveness in Africa remains limited.
Cluster-randomised controlled trial in eastern Zimbabwe, with 30 clusters divided into ten socio-economically matched triplets. One cluster from each triplet was randomly assigned to each of three study arms-CT, CCT or control. Vulnerable households in the CT and CCT arms received bimonthly payments of $18 plus $4 per child for up to a maximum of 3 children. In the CCT arm, a 10% penalty was applied where households failed to comply with conditions regarding birth registration, immunisation and school attendance. We investigated the health, education and social effects of cash transfers.
9,811 children aged 0–17 years living in vulnerable households were recruited. After adjustment for cluster and baseline covariates, the CT programme reduced the proportion of children aged 0–4 years without birth certificates (−4.8%; 95%CI −13.3% to 3.8%) and with incomplete vaccination records (−2.1%; −10.7% to 6.5%) and the proportions of children aged 6–12 years (−7.4%, −15.4% to 0.6%) and 13–17 years (−8.5%; −15.0% to −2.0%) attending school less than 80% of days in the last month. The CCT programme produced larger, and more frequently statistically significant, reductions in the primary outcomesit reduced the proportions of children aged 0–4 years without a birth certificate (−16.8%; −24.9% to −8.7%) and with incomplete vaccination records (−4.1%; −12.3% to 4.1%), and the proportions of children aged 6–12 years (−8.1%; −17.0% to −0.7%) and 13–17 years (−10.0%; −16.6% to −3.5%) attending school less than 80% of days in the last month.
Cash transfers, particularly conditional transfers, increased birth registration and school attendance in a low income, high HIV prevalence setting.
With advances in the treatment of HIV, vertically infected young women are surviving into adulthood. Like other teens, many are sexually active at an early age, have multiple sexual partners, and use condoms inconsistently or infrequently. These young women face possible pregnancy and the risk of second generation mother-to-child transmission.
This was a retrospective review of the medical records of perinatally infected young women, followed longitudinally in a comprehensive university based Pediatric AIDS Program. Data related to reproductive health decisions, demographic and health variables were examined.
89 perinatally infected young women between the ages of 12–25 were seen between July 1, 2006 and December 31, 2010. Among this cohort there were a total of 44 pregnancies resulting in 26 live births, 10 terminations and 9 miscarriages. All exposed infants were HIV negative. Eight women were in committed relationships at the time of their pregnancy; three of the pregnancies were planned. Among the 42 pregnancies, only 6 partners (13.6%) were aware of the mother's HIV status at the time of pregnancy. Sixteen percent of the mothers of girls who became pregnant were alive as compared with 36% of the mothers of young women who did not become pregnant during the interval.
In this group of young women with perinatally transmitted HIV infection none of their infants were infected with HIV, indicating good adherence with HAART during pregnancy. The low rate of disclosure to partners is a serious issue with public health implications. Challenges of negotiating relationships for young women will be discussed. Demographic variables including as age, education, mother's vital status, will be examined as predictors of pregnancy. Future prospective studies will be discussed including evaluation of early counseling and support to this group of women.
In Botswana, over 30% of pregnant women are HIV-positive. The 2007 Botswana Family Health Survey showed that 51% of women used some form of contraception, predominantly condoms. Preventing unintended pregnancies is the second prong of the WHO four-prong approach to a comprehensive prevention of mother-to-child transmission (PMTCT) program. We used data from the 2011 Botswana Antenatal Clinic Sentinel Surveillance to determine the number of, and factors associated with unplanned pregnancies.
Pregnant women aged 15–49 years presenting for the first time to one of 262 selected antenatal clinics in all 24 health districts from August 1 through October 28, 2011 were included. Blood routinely drawn for routine antenatal care was tested for HIV using a two-test algorithm. A one-page form was completed for all participants that included demographic information, gravidity, HIV testing history, and planned pregnancy (“Was this pregnancy planned?”). Logistic regression analysis was used to determine factors associated with unplanned pregnancy, adjusted for linear and quadratic effects of age and for health district.
Of the 6745 participating women, 6667 (98.8%) responded to the planned pregnancy question. Of these, 3383 (51%) reported that the current pregnancy was unplanned. Women aged 15–19 years and 40–49 years reported the highest rates of unplanned pregnancy (68% and 67%, respectively). Unplanned pregnancies were higher among HIV-positive (56%) than HIV-negative women (49%). In a multivariable model, unplanned pregnancy was associated with unemployment (odds ratio (OR=1.29, 95% confidence interval (CI)=1.15–1.44), being unmarried (OR=2.17, 95% CI=1.81–2.60), having two or more previous pregnancies (OR=1.83, 95% CI=1.60–2.10), and having a previous positive test for HIV (OR=1.73, 95%CI=1.51–1.99).
The higher proportion of unplanned pregnancies among those who knew they were HIV-positive prior to the survey and among multigravid women suggest that current family planning services need to be strengthened as part of the Botswana PMTCT program.
Cryptococcal meningitis (CM) is a leading cause of death in AIDS patients in sub-Saharan Africa. Cryptococcal antigen (CRAG) can be detected weeks before symptom onset, and those who are asymptomatic but CRAG+ have a high risk of subsequent CM and mortality. A new CRAG point-of-care immunochromatographic lateral flow assay (LFA) is available that is remarkably easy to administer without laboratory infrastructure or expertise and has excellent sensitivity and specificity.
We assessed the cost-benefit of targeted CRAG screening for patients with CD4 <100 cells/mcL using the LFA ($2.50 total/screen) and preemptive fluconazole therapy in persons entering into HIV care in sub-Saharan Africa.
Based on published CRAG+ prevalence rates of 8.8% among HIV-infected persons with CD4 <100 cells/mcL in Kampala, Uganda, the cost of detecting one person with asymptomatic antigenemia with the LFA would be $28.37, and the cost of saving one life would be $39.73 (95% CI: $28 to $60). Assuming an average increase in life expectancy of 18 years for a 30 year old Ugandan initiating antiretroviral therapy (ART) with a CD4 count <100 cells/mcL, this equates to $1.57 per quality-adjusted life year (QALY) saved. Based on CRAG+ prevalence of between 4–12% in sub-Saharan Africa, the cost to save one life ranges from $37–$114 in persons with CD4 <100 cells/mcL. In contrast, the cost of hospitalization and treatment with amphotericin-based therapy for symptomatic CM is ~$414 per episode in Uganda. Thus, for the cost of treating a single episode of CM, one could screen 165 persons using the LFA screening protocol, which remains cost-effective to 1% CRAG+ prevalence.
Screening and preemptive fluconazole therapy is cost-saving to healthcare systems and should be integrated into routine HIV care, targeting those with CD4 counts <100 cells/mcL. Better understanding of the implementation science is needed to determine how best to scale up CRAG screening.
A significant body of work has examined the impacts of laws criminalizing commercial sex on sex workers’ health and safety. However, the impacts of laws criminalizing clients and third parties (i.e. managers, receptionists, cleaning staff …) on sex workers have hardly been documented. In Canada, sex work is criminalized through provisions against being found in a bawdy-house, running a bawdy-house, living on the avails of prostitution and communication for the purposes of prostitution. These are used to arrest sex workers, clients and third parties.
Stella is an organization by and for sex workers. The analysis of the impact of the different legal provisions criminalizing commercial sex on the health and safety of female and transgender sex workers specifically based on the street and in drug venues (i.e. crack houses, shooting galleries...) arises from available documentary data, reports on violent incidents through our aggressors’ list and also from our observations and personal experience.
The criminalization of clients under the ‘communicating’ law places sex workers based on the street and in drug venues at greater health and safety risks by limiting their choice of clients and ability to negotiate safer sex, and causing displacement to dangerous and isolated areas far from HIV prevention and outreach services. The provisions against bawdy- houses create a greater risk of homelessness as it is grounds for eviction, including from social housing.
Often justified as “protecting” sex workers, the laws against clients and third parties actually place sex workers at greater risk, especially as they preclude the opening of safe indoor spaces. Take note that drug use legislations also increase these risks when discriminating sex workers.
Ugandan guidelines recommend 6 months of exclusive breastfeeding (EBF) for HIV-infected women with continued breastfeeding (BF) for at least 1 year. Food insecurity has been posited to be a barrier to adherence to infant feeding recommendations. We therefore explored if greater food insecurity was associated with sub-optimal breastfeeding (BF) practices.
Data on food insecurity and BF practices and beliefs were collected among HIV-infected pregnant and BF women on antiretroviral therapy (ART) participating in the PROMOTE trial (NCT00993031) in Tororo, Uganda. Food insecurity was assessed using the Household Food Insecurity Access Scale (HFIAS). BF practices were determined using monthly maternal reports from a prospective cohort of 143 PROMOTE infants born between March 6, 2010 and October 9, 2011. Beliefs about food insecurity and BF were assessed using a purposive sample of 32 BF PROMOTE subjects who participated in in-depth interviews (IDIs) in July and August 2011.
Food insecurity was very high; 82.5% were from severely, 14.7% moderately, and 2.1% mildly food insecure households. Median (inter-quartile range) HFIAS score was 17 (12–22). The prevalence of EBF was 89.5% and 66.9% at 3 and 6 months respectively; the prevalence of BF at 12 months was 84.1%. In multivariate logistic regression adjusting for socio-demographics and HIV-stage, the odds (95% confidence interval) of EBF at 6 months was 0.16 (0.03–0.86) for women in severely food insecure households compared to those in non-severely food insecure households. Consistent with this, IDIs revealed that BF participants associated insufficient nutritional intake with difficulties adhering to infant feeding recommendations: 75.0% were concerned about adequate breast milk production and 46.9% reported experiencing problems while EBF, e.g. unsatisfied baby.
Severe food insecurity is associated with suboptimal EBF. The mitigation of food insecurity may increase the duration of EBF among HIV+ women in rural Uganda.
In resource limited settings (RSL) such as Haiti, people living with HIV/AIDS face great challenges in maintaining adequate nutrition.The evaluation of the benefits of nutritional supplementation(NS) programs on HIV clinic attendance among HIV infected subjects in developing countries is limited.This abstract focuses on the assessment of the rate of timely antiretroviral therapy (ART) visit attendance for a cohort of patients before food assistance, while receiving food assistance, and after receiving food assistance by using electronic pharmacy visit data.
Our study population included 2654 patients that received at least 6 months of ART prior to receiving NS between October 2007 and January 2011 at the GHESKIO Centers. Using pharmacy visit data, we calculated the number of late visits (at least 6 days after the scheduled visit date) during the 6 months before receiving NS from the World Food Program (WFP), during the approximate 6 months of WFP supplementation , and the 6 months after the receipt of WFP supplementation.
Among 2654 patients, from a total of 4098 scheduled visits for the period : November 2007- December 2011: there were 1625 late visits during the 6 months period prior to NS which indicates that 39% of the scheduled visits were late visits , there were 1291 late visits during the 6 months of NS ie 31% of the scheduled visits were late visits, and 1135 late visits during the 6 months after NS ie 27% of the scheduled visits were late visits.
Our findings demonstrate that food assistance plays a key role in the regularity of the visits to the pharmacy, which is associated with ART adherence. NS should be part of the package of care and support offered to all patients on ART, particularly those with advanced immunodepression who are so desperately in need.
UNHCR and its partners aim to ensure that refugees have similar access to HIV prevention, care and treatment services to that of local surrounding populations, and that HIV status is not used to discriminate against refugee rights to asylum, protection and assistance. HIV-related information is monitored systematically to ensure adequate protection and assistance to refugees.
HIV indicators collected routinely at the health facility level through UNHCR's Health Information System implemented in 21 countries where refugees live in camps, as well as review of indicators collected annually through HIV focal points at field level were analysed from 2008–2011.
By 2011, 81% of pregnant women had access to PMTCT programmes compared with 53% in 2008. 89% of countries had camps with satisfactory universal precautions. 78% of countries were implementing interventions addressing at least one high risk group. The six UNHCR programmes directly managing a blood bank reported 100% of blood screened before transfusion. Fifteen countries reported implementing clinical management of rape survivors; 77% of survivors received PEP within 72 hours. The proportion of countries in Africa, Asia and South America where refugees have similar access to ART as host populations reached 91% by end of 2010. Advocacy against mandatory testing for HIV and for inclusion of refugees in country HIV National Strategic Plans remained stable.
Access to protection, prevention, care and treatment has increased in most of the 21 countries hosting refugees in camps. HIV programming in emergency and protracted situations is possible and effective.
Given the importance of adherence to HAART for treatment success, our objective was to evaluate if refugees and the local host community attending a rural refugee camp clinic had acceptable adherence and virologic outcomes.
We conducted a cross-sectional survey among refugee and host community ART clients (≥18 years) in a rural camp-based clinic in Kakuma, north-western Kenya. Data sources included a structured questionnaire, a pharmacy-based measure of HAART prescription (Rx) refills over 24 months prior to study start, and HIV viral loads measured on dried-blood spots. Key outcomes were unsuppressed viral load (≥1000 copies/mL) and optimal adherence (≥95% Rx refill rate).
In refugees and host clients, 85% (n=73) and 86% (n=86), respectively, of all clients not lost to follow-up (≥6 months without HAART Rx refill) participated. Median age (years, IQR) was 36 (31–41) vs. 32 (27–38) (p=0.02),% females was 67 vs. 66 (p=0.91), median time on HAART (weeks, IQR) was 156 (43–264) vs. 139 (34–225) (p=0.43), first available CD4 (IQR) was 196 (136–320), n=61 vs. 198 (119–289), n=76 (p=0.28). Most clients (63%) were taking zidovudine, lamivudine, and nevirapine (twice-daily). Similar proportions of refugee and host clients on treatment for≥25 weeks had unsuppressed viral load (88% vs. 89%, p=0.92; 89% overall). The proportion optimally adhering to pharmacy claim schedule was 79% overall (85% vs. 75%, p=0.14). In multivariable analyses, refugee status was not independently associated with unsuppressed viral load. A longer time since HIV diagnosis (ORadj=6.81, 95% CI 1.20–38.58, p=0.02) and≥8 household members (ORadj=0.10, 95% CI 0.02–0.55, p=0.01) were independent risk factors.
When available, virologic measures are valuable for monitoring program effectiveness. No differences in treatment outcomes were detected between refugee and host clients; however, levels of viral suppression were unacceptable. Possible reasons include past adherence lapses. Detailed investigations exploring reasons for the poor virologic outcomes are underway.
There are few data evaluating adherence to HAART and virologic outcomes in clinics attended by urban refugees. Our primary objective was to evaluate if urban refugee and host community clients were maintaining acceptable treatment outcomes.
We conducted a cross-sectional survey among adult clients (≥18 years) receiving HAART from a public clinic in Kuala Lumpur, Malaysia. Data sources included a structured questionnaire with self-reported adherence measures, a pharmacy-based measure of HAART prescription (Rx) refills over 24 months prior to study start, and HIV viral loads. Key outcomes were unsuppressed viral load (cut-off<40 copies/mL) and optimal adherence (≥95Rx refill rate).
We recruited 90% of all eligible refugees (n=153) appearing on a UNHCR database and 81% (n=148) of serially-recruited host community clients. Refugees were younger (median age 35y IQR 31–39 vs. 40y IQR 35-48; p<0.001), more likely to be female (36% vs. 21%, p=0.004), to have been on HAART for less time (61weeks IQR 35–108 vs. 153weeks IQR 63-298; p<0.001),and to have a lower routine CD4 (278 cells/µL, IQR 182–423 vs. 350 IQR 202–486; p=0.03). Similar proportions of those on treatment for ≥25 weeks from both groups had achieved viral suppression (81% vs. 84%, p=0.54) and optimal adherence as measured by Rx (74% vs. 66%, p=0.15), and by self-reported one-month recall (72% vs. 70%, p=0.79). Refugee status was not independently associated with the outcome (ORadj=1.62, 95% CI 0.64–4.09; p=0.31). Independent risk factors were female sex (ORadj=0.39, 95% CI 0.14–1.05;p=0.05), optimal adherence to Rx (ORadj=0.42, 95% CI 0.24–0.73; p=0.002), travel in the past year (ORadj=3.64, 95% CI 1.43–9.30; p=0.008), and 25–50 weeks between diagnosis and treatment start (ORadj=1.66, 95% CI 0.56–4.96; p=0.01).
Refugees′ adherence to HAART and viral suppression was similar to the host community and at acceptable levels. Our findings support a policy of equal HAART provision and support to refugees and the host community in this urban setting.
For HIV-positive persons, optimal adherence (≥95% of tablets taken as prescribed) to highly active antiretroviral therapy (HAART) is required to prevent development of drug resistance and treatment failure. Forcibly displaced populations such as refugees and internally-displaced persons may face unique challenges with respect to adherence and succeeding on HAART. To this end, we performed a systematic review of the literature on adherence to HAART and treatment outcomes in conflict-affected and forcibly displaced populations, assessed the quality of the evidence, and make suggestions for future research.
MEDLINE, EMBASE, and Global Health databases for 1995–2011 were searched using the OVID platform. A backwards citation review of subsequent work that had cited the OVID results was performed using the Web of Science database. ReliefWeb and MSF websites were searched for additional grey literature.
We screened 297 records and identified 17 reports covering 15 quantitative and two qualitative studies. Three-quarters (11/15) of the quantitative studies were retrospective studies based on chart review; five included <100 clients. The geographic distribution was limited, and studies in North America and Africa were disproportionately represented. The range of optimal adherence prevalence was 87-99.5% and good treatment outcomes were also reported. The qualitative studies suggested that food security, community-based volunteers, health workers and social networks were important for maintaining adherence.
Results to date have been encouraging, though most studies had relatively weak designs. Given the diversity of settings where forcibly displaced persons are diagnosed with HIV and treated with HAART, further studies on adherence and treatment outcomes are needed to support HAART scale-up and justifications for inclusion of these vulnerable groups in national treatment plans. There is also a need for systematic and replicable measurement of adherence in future studies among these groups.
HIV has multidimensional effects that pose unique challenges to development in Cambodia. One effect is a large number of orphans and vulnerable children (OVC). Many OVC are less likely to access regular schooling and have low levels of social, financial and psychological support as well as wider community support. KHANA provides a program of community based care and support including social welfare, nutrition and schooling support for these children.
KHANA collected data among OVC reached by community based care and support to assess changes in nutrition and schooling as well as care and community support between 2009 and 2011. The sample size was 194 in 2009 and 316 in 2011. Both surveys used cluster sampling with a take-all approach, using health center catchment areas as clusters and the same data collection tools. Chi-square test was used to detect changes in schooling, nutrition and community support.
There was a significant increase in OVC regular access to schooling (83.1% vs 96.2%, p-value<0.001). The level of change was larger for girls (77.8% vs. 95.9%, p value< 0.001) than boys (87.5% vs. 96.6%, p-value<0.006) reaching similar outcomes. Reports of daily meal reduction due to not enough food decreased from 53.1% to 39.2% (p-value=0.002). Reports of periods of inadequate food supply during the year dropped from 36.1% to 16.5%. There were increases in community support to OVC, with psychological and schooling supports both increasing significantly by 32% and 45% respectively.
The findings highlight positive changes in some aspects of social support among OVC. The children reached with community based care and support demonstrated increased psychological support and access to regular schooling. Socio economic status as measured by secure access to food also improved significantly. These improvements are likely due to contributions from KHANA program as well as increased community and family support.
HIV is the strongest risk factor for progressing from latent
HIV+ persons ≥2 years old who were either tuberculin skin test positive or close contacts of TB cases were randomized to 3HP or 9H. Persons could not receive antiretroviral therapy (ART) for 90 days after enrollment. Participants were enrolled from the U.S., Brazil, Spain, Peru, and Canada between June 2001 and December 2010. Follow-up for TB continues through 2013.
Of 4,128 participants enrolled with known HIV status and who received ≥1 dose of study therapy, 393 were HIV +: 207 in the 3HP and 186 in the 9H arm. In the MITT analysis (enrolled participants who were eligible), 178/201 (89%) HIV+ persons completed 3HP vs. 125/193 (65%) on 9H (P< 0.001). The proportion of participants with a serious adverse event (SAE), ≥1 AE, or hepatotoxicity was lower in 3HP than 9H (4 vs. 11%; P=0.006; 22 vs. 40%; P=0.004; 2 vs. 6%; P=0.03). Compared to 1,888 HIV-negative participants treated with 3HP, HIV+ persons were less likely to permanently discontinue treatment for any reason (11 vs. 20%; P<0.001) or to have possible drug hypersensitivity (1 vs. 5%; P=0.003), and there were no significant differences in the proportion with SAE, ≥1 AE, or hepatotoxicity.
Among HIV+ persons not receiving ART, 3HP was better tolerated and had higher treatment completion rates than 9H for treatment of latent
HIV and TB are threats to pregnant women and infants. Treatment with rifampin can reduce ART concentrations and increase risk of treatment failure and vertical transmission. We describe the pharmacokinetics (PK) and pharmacodynamics of EFV among pregnant HIV-infected women.
Prospective cohort of HIV-infected pregnant women with and without TB in Soweto. Women taking ART with EFV 600 mg had PK sampling at 37 weeks’ gestation or at delivery and then six weeks post-partum. EFV concentrations were measured in cord blood at delivery and in infants at 7 days. Post-hoc Bayesian estimates of PK parameters from nonlinear mixed-effects modeling with allometric scaling are reported.
Among 41 HIV-infected pregnant women taking EFV ART, 19 received rifampin (TB/HIV) and 22 ART alone. Median age and weight were 29 years and 70 kg. For 35 women with pre-/peripartum EFV PK, median (IQR) estimated EFV trough (Cmin) was 1.31 (0.84, 1.86)mg/L, apparent oral clearance (CL/F) 13.62 (10.67, 18.44)L/h, and volume of distribution (Vd/F) 516 (440, 591)L. 31% had Cmin<1 mg/L. Predicted median Cmin by
TB treatment did not significantly reduce EFV Cmin, but pregnancy may lower EFV concentrations. Although ~30% of pregnant women had EFV Cmin< 1mg/L at standard doses, EFV-containing ART suppressed viral load in most and there were no vertical transmissions.
The pre-existence of antiretroviral (ARV) drug resistant virus is strongly associated with treatment failure in naive treatment patients, for that the knowledge of transmitted drug resistance (TDR) in newly diagnosed patients constitutes a fundamental premise in the epidemiological surveillance. The aim of this study was to analyze the profile of TDR in newly diagnosed patients from Havana city.
One hundred and twenty-seven HIV-1 infected patients from Havana and diagnosed between 2009 and 2012 were included in the study. The viral RNA was isolated from plasma and used as target to amplify the pol gene by RT-nested PCR. PCR products were sequenced and the data generated used to determine the viral subtype by phylogenetic analysis. The TDR were detected by HIVdb v6.1.1 and CPR tool v 6.0, according the 2009 surveillance drug resistance mutations list.
The majority of patient was male (87.4%), 72.4% of the infections was acquired by homosexual transmission and 13.4% were recent infection. As high as 39.4% of the analyzed samples corresponded to the subtype B, followed by CRF19_cpx (26%), CRF 20-23-24_BG (18.9%), CRF18_cpx (7.9%), URF (5.5%), C (0.8%), G (0.8%), CRF05_DF (0.8%). Overall, 22.8% (29/127) had evidences of TDR. 6.3% presented mutations to NRTI, 4.7% to NNRTI and 3.1% to PI. Drug resistance mutations against both NRTI and NNRTI were observed in 7.9%, whereas triple class resistance was found in only 0.8% of the studied samples. The most common mutations were M184V (34.5%) and G190A (31%) for the NRTIs and NNRTIs, respectively. In patients with TDR virus, the first line HAART may be effective in 17.2%, partially effective in 44.8% and ineffective in 38%.
This study confirmed the high HIV-1 genetic diversity in newly diagnosed patients from Havana. The increment of TDR in the HIV-infected people from Havana indicates the importance of maintaining the epidemiological surveillance in the patients of recently diagnosed due to its possible implications in the effectiveness the first- line regimens prescribed in Cuba.
Since 2001, Cuba achieved universal antiretroviral therapy with domestically manufactured antiretroviral (ARV) for patients meeting international clinical criteria and has resulted in a decrease in AIDS mortality rate and incidence of opportunistic infections. However, the emergency of HIV-1 drug resistance is strongly associated with treatment failure. The aim of this study was to analyze the associated mutations to ARV resistance and the resistance levels in a group of patients that attended to the infectious diseases outpatients department of Hermanos Ameijeiras Hospital, Havana City with treatment failure during the year 2012.
During 2012, were collected 25 plasma samples from HIV-1 patients with treatment failure of 157 HIV patients that attended to the infectious diseases outpatients department of Hermanos Ameijeiras Hospital. The viral RNA was used as target to amplified and sequenced pol gene. The viral subtype and the drug resistance mutations and levels were determined. The time between the appearance of resistance and the beginning the last therapy was considered.
Most of the patients were MSM (96%) and 52% had only received first-line HAART. Subtype B was the most prevalent subtype (52%) followed by non-B subtypes (CRF19_cpx, CRF 20-23-24_BG, URF and CRF18_cpx). Drug-resistant mutations were detected in 21 patients (84%). Of them, 61.9% presented mutations against both NRTI and NNRTI, 19.1% to the combination PI + NRTI, 9.5% to NRTI, and 9.5% to NNRTI. The most frequent mutation for NRTI was M184V (56%), while for NNRTI were Y181C/I/V (24%) and K103N (20%). As high as 64% presented high resistance to all or some of the drugs used as first-line HAART in Cuba (AZT/D4T + 3TC + NVP). The average of years in the appearance of resistance after beginning the last therapy was of 2.3 years. The therapeutic failure of 16% of the patients that presented susceptible virus was due to the poor adherence to HAART.
This study evidenced the high resistance levels to the first-line regimens prescribed in Cuba, as well as the fast appearance of resistant variants after beginning the therapy. These results emphasize the necessity of the monitoring of the resistance in those patients that will begin a new therapeutic regimen.
Residual HIV-1 replication among individuals under antiretroviral therapy is an obstacle towards reduction of chronic HIV related micro-inflammation. To determine the levels of residual viral replication of HIV-1 in distinct subgroups of patients inferred by quantification of episomal HIV DNA, quantification of total HIV DNA, and quantification of HIV-1 specific antibodies.
A total of 109 patients with undetectable viral load were divided into five groups: first suppressive therapy with efavirenz (26), first suppressive therapy with boosted protease inhibitors (PI) (25), salvage therapy using boosted PI (27), salvage therapy with raltegravir (15) and virological failure (16). Quantification of episomal and total DNA was performed by real-time PCR. Specific antibody quantification was performed using enzyme immunoassay capture.
Episomal DNA amplification was positive in 36 out of 109 patients’ samples (33%) and quantification of total DNA was obtained in 94 patients’ samples (86.3%). Individuals on salvage therapy using Raltegravir presented lower prevalence and lower quantitation of epissomal DNA as compared to other treatment groups (
Duration of treatment, CD4, CD8 counts, and raltegravir-based regimens relate to decreased residual viral replication (epissomal DNA). The relationship between episomal DNA and total DNA suggests replenishment of proviral reservoir with potential impact on HIV persistence. Lower antibodies levels among patients with PI-based initial treatment may suggest a more effective HIV suppression of these regimens, with higher capacity of decreasing the HIV antigenic component.
The determination of biological characteristics of the HIV strains is essential for the clinical handling in recently infected patients and to optimize your selection before starting treatment. The aim of this study was to relate the viral genotype and phenotype of the isolation from recently infected patient without treatment with the clinical-epidemiologic characteristics.
Peripheral blood mononuclear cells and plasma of Cuban patients HIV-1 recently infected, obtained at least eight months from the diagnostic date, were used for the isolation and capacity of syncytium-inducing (SI) and determination the subtype and resistance-associated mutations (RAMs) by sequence and analysis of the
We included in the study 38 treatment-naive HIV-1 infected patients, five women and 33 men. In male patients, 25 are men who have sex with men (MSM). The means values (range) of age and CD4+ cells were 36.7 years (18–68) and 386.8 cells/mm3 (53–1260), prevailing the clinical status from A1 to A3 (31/38). We detected 13 subtypes B, 15 circulating recombinant forms (CRF) (9 CRF 20-23-24_BG, 6 CRF_19cpx and 2 CRF_18cpx), 3 unique recombinant forms (URF) (2 URF B/18cpx and 1 URF B/19cpx) and five non-amplified. Five viruses (13.2%) presented RAMs, one with multiresistance (subtype B) and other 4 for protease inhibitors (one subtype B and three recombinant forms). The virus was isolated from 63.2% (24/38) of the patients, who were related with the conditions MSM, 40–50 years, clinical status A3 or superior, CD4+≤250 cells/mm3 and infection with CRF; but not with the presence of RAMs. Only 25% of the strains were IS. Recombinant forms without RAMs were detected in all women, while in men there was no relation between sexual conduct and viral genotype. The presence of recombinant form was also associated with CD4+ counts less than 500 cells/mm3.
The results show that in Cuban patients HIV-1 recently infected treatment-naïve predominate non-SI strains and recombinant forms with RAMs.
The protease inhibitor, lopinavir, co-formulated with ritonavir (LPV/r) plus two nucleoside reverse transcriptase inhibitors is recommended by the U.S. Department of Health and Human Services Perinatal Guidelines as a preferred regimen for HIV treatment in pregnant women. Pharmacokinetic studies suggest that the LPV/r standard dose of 400/100 mg twice-daily during pregnancy results in reduced plasma LPV exposure in the third trimester; however, these declines do not appear to impact maternal clinical outcomes or MTCT rates. Pharmacokinetic studies are not designed nor powered to assess clinical outcomes.
We conducted a systematic review to assess maternal and infant clinical and safety outcomes in pregnant women treated with LPV/r-based regimens. PubMed, EMBASE, and HIV congresses were searched for studies published through 31 May, 2012. Studies were selected if they included HIV-1-infected pregnant women treated with LPV/r-based therapy and reported maternal and infant outcomes as a primary objective. Data were extracted from publications and tabulated for analysis.
A total of 13 publications/presentations describing 9 studies were identified. These studies included 2,675 women treated with LPV/r: 1,618 received LPV/r 800/200 mg/day, 70 received >800/200 mg/day, and 987 received an unknown LPV/r dose. Overall, >80% of women (64–97%) achieved viral suppression below the threshold for the study in which they enrolled. There was no significant difference in the number of women with HIV-1 RNA
This systematic review of 2,675 pregnant women suggests no unique safety or efficacy concerns with the use of standard dose LPV/r-based ART in pregnant women. Although high dose LPV/r use was limited to one study in this analysis, the safety and efficacy outcomes reported, including MTCT rates, were similar to those obtained with standard dose.
Social factors increasing vulnerability to HIV in women hinder healthcare and early HIV diagnosis. Prevention efforts in countries with concentrated epidemics focus mainly in MSM and do not reach women at risk. We evaluated a sample of recently diagnosed HIV Mexican women to describe diagnosis circumstances, frequency of late diagnosis and factors associated with vulnerability.
Interviews were conducted on Mexican women from 2 large HIV care centers in Mexico City between January 2009 and November 2012. Information on socio-demographic data, partners, risk behaviour, history of physical/sexual violence, HIV diagnosis circumstances and access to medical and prenatal care was obtained. Clinical information was taken from medical records.
152 women were included (24% of total diagnosed in the period in both centers). Interviews were done to 122 women. Median age at diagnosis was 33 (17–75). Maximum level of education was primary school in 42 (30%), 14(9%) were illiterate. A median of 3 lifetime sexual partners (1–300) was reported. A third had their first pregnancy as teenagers. History of physical violence and sexual abuse was reported by 52% & 38%, respectively. In 85 (70%), likely source of infection was a stable partner. HIV diagnosis was made after a partner/child diagnosis in 40%; in 37% because women were symptomatic. Only 15% were diagnosed by screening, including during pregnancy (7.5%). 39% had sought medical care a median of 4 times because of symptoms related to HIV without being offered an HIV test. Median CD4 count at diagnosis was 154 (1–1341). CD4 <200 at diagnosis was present in 58%, and <100 CD4 in 34%. Lower CD4 were found in women with lower education levels, women diagnosed with symptoms, and those who sought medical care before being diagnosed. 87% of women with previous pregnancies reported never being offered an HIV test.
Women living with HIV in this sample had low socio-economic background, high prevalence of physical/sexual violence, and late stage disease at diagnosis. Missed opportunities for early testing (prenatal/primary care) were identified. In Mexico, HIV screening strategies are focused on selected groups that do not include women, unless they are pregnant. Women are diagnosed mostly when they become sick or after a family member is detected. Preventive policies should also address women; screening strategies need to be reinforced in primary care to increase early HIV detection in women.
Chile, a 16.6 million-people upper middle-income country has a 0.3×105 rate of HIV infection in adults, mostly cared by the Public Health System (PHS) through 34 AIDS care centers (ACC). Expanded access program (EAP) to HAART was begun in 2001 and there is now guaranteed free access to antiretrovirals and monitoring (CD4 count, viral load, and genotype) in the PHS (with low co-payment, also guaranteed for the privately insured). First-line HAART are assigned at the ACC but rescue therapies are approved centrally. Standards for professional staffing at ACC are based by norm on patient load. 32/34 ACC have enrolled their patient on HAART without exclusions in the Chilean AIDS Cohort (>15,000 patients) for homogeneous management and evaluation of the EAP. In 5,000 treatment naïve patients, 45% initiated HAART in AIDS stage and 50% had baseline CD4 count < 100 cell/mL, (earlier treatment is more frequent lately). In this treatment, naïve population 5-year survival and AIDS-free survival were 90% and 70%, respectively. Three-year mortality decreased from 20.8% for those initiating HAART in 2001 to 3.4% in those doing it from 2008 onward. Cross-sectional study showed 73% of patients with undetectable viral load; median CD4 count increased from baseline of 164 to 358 cell/mL at last count. The expanded access to HAART guaranteed by law and the Chilean AIDS Cohort has been a successful synergistic initiative for the implementation of treatment and its evaluation in Chile.
The Specialized Condesa Clinic of Mexico City is a detection, treatment and follow up centre for people with HIV/AIDS and sexually transmitted infections (STI), with a care program of HIV and STI for victims of sexual violence since 2008. According to research, almost one in four women can be a victim of sexual violence and one in three female adolescents informs that her first sexual experience was forced. Sexual violence is associated with sexual health problems later on and thus, considered a public health problem.
Cross-sectional study of data from female patients who attended the sexual violence program. The data collection tool used was a seroepidemiologic questionnaire which collected the clinical history along with blood tests from January 2, 2012 through December 31, 2012. The study sample was of 1,071 female patients, all received antibody HIV 1-2, surface antigen HBV, antibodies for HCV and Syphilis antibodies testing. All tests were performed during the initial visit and during each follow up visit at three and six months. All patients who procured services within 72 hours from the event received post exposure prophylaxis treatment for HIV, Gonorrhea, Syphilis, Trychomoniasis, Chlamydia and emergency contraception.
Of the 1,071 patients 44 were under 12 years (4.1% of total); 150 were 12–14 year olds and 354 were 15–19 year olds; for a total of 504 adolescent patients (47% of total). The average age of patients was 22.2 years (range of 8 to 82). Four-hundred and one patients (37.4%) accessed the service within 72 hours after the sexual violence event, while 670 patients (62.5%) accessed services afterwards. Five-hundred and thirty six patients received emergency contraception. Four-hundred one patients received HIV PEP for 28 days. They also received prophylaxis for STI. During initial screening two patients (0.18%) had a positive HIV result, and two (0.18%) had a positive HCV result. These infections were not acquired during the sexual violence event. Nine patients had pregnancies related to the sexual violence event, six terminated their pregnancies legally, two refused interruption and one was too late to perform the interruption. During the follow-up visits, no patients showed with HIV or STI infection.
Medical care to victims of sexual violence is a priority since it prevents HIV, STI and unwanted pregnancies. The time after the sexual violence event is crucial since if care is not rendered in time it could result in negative lasting consequences.
Two phase 3 studies of elvitegravir/cobicistat/emtricitabine/tenofovir DF (STB) in treatment-naive subjects are ongoing (GS-US-236-0102 and -0103). The W96 responses were high, durable and comparable for STB vs. efavirenz (EFV/FTC/TDF [ATR]) (84 vs. 82%) and STB vs. ritonavir-boosted atazanavir (ATV/r) + FTC/TDF (ATV/r + Truvada [TVD])(83 vs. 82%). Resistance analyses through week 96 are presented.
HIV-1 genotypes (protease and reverse transcriptase [RT]) were analyzed at screening (GeneSeq, Monogram Biosciences); subjects with resistance to study drugs were excluded. Retrospective integrase (IN) genotyping was conducted on a large set of STB baseline samples. The resistance analysis population had genotypic/phenotypic analyses at failure confirmation and baseline for PR, RT, and IN using PhenoSenseGT, IN GeneSeq, IN PhenoSense, PRIme (Monogram) or IN GenoSure (Labcorp).
STB-treated subjects with baseline PI (N = 18) and NNRTI mutations (N = 95) had high treatment responses through W96. Of the 95 subjects that had pre-existing NNRTI-associated mutations, including 27 with K103N, 87 and 85% maintained virological suppression (VS) at W96, respectively; among those with viral rebound in this group, none developed STB resistance. Eighteen subjects with pre-existing PI mutations had a VS rate of 89%. Fifty-two subjects with primary NRTI mutations had a VS rate of 85%. Baseline primary INSTI mutations were rare (N = 4/337; T97A [N = 3] and Y143H [N = 1]) and interestingly, all four subjects carrying these pre-existing INSTI mutations had VS. HIV-1 subtype B was the predominant subtype and all STB virologic failures (VF) occurred in subtype B. In the STB group through W96, 16 subjects (2.3%; 16/701) developed primary INSTI and/or NRTI resistance mutations and had phenotypic resistance to STB: 13 subjects in year 1 and 3 subjects in year 2. Emergent mutations were E92Q (N = 9), N155H (N = 5), Q148R (N = 3) and T66I (N = 2) in IN in combination with M184V/I (N = 15) and K65R (N = 5) in RT. STB VF retained susceptibility against NNRTIs, PIs and many NRTIs. In the ATR group, 10 subjects (2.9%; 10/352) developed RT resistance to ATR: 8 subjects in year 1 and 2 subjects in year 2. Resistance was most commonly K103N (N = 9) with M184V/I plus K65R (N = 3). In the ATV/r + TVD group, 16 subjects were analyzed and none developed resistance.
STB achieved durable high rates of VS in HIV-1 treatment-naive subjects, including subjects with pre-existing NNRTI, PI and NRTI resistance.
Antiretroviral simplification improves quality of life and adherence while reducing the risk of HIV virologic failure (VF). Carrying a transmitted K103N RT mutation is not rare [
SPIRIT is a phase 3b, randomized, open-label, multicentre, international, 48-week study to evaluate the safety and efficacy of switching from PI + RTV + 2NRTI regimens to RPV/FTC/TDF in virologically suppressed HIV-1 infected subjects. Subjects were randomized 2:1 to switch to RPV/FTC/TDF at baseline or maintain their current PI + RTV + 2NRTI regimen with a delayed switch to RPV/FTC/TDF at W24. The primary endpoint was non-inferiority (12% margin) of RPV/FTC/TDF relative to PI + RTV + 2NRTI regimens in maintaining plasma HIV-1 RNA <50 c/mL at W24 by FDA snapshot analysis. Inclusion criteria allowed enrolment of patients with evidence of a K103N mutation in their historical genotype obtained prior to first line therapy.
Four hundred seventy-six subjects were randomized. At W24 virologic suppression (VS) was achieved by 94% of subjects in the RPV/FTC/TDF arm vs.90% in the PI + RTV + 2NRTI arm; (difference 3.8%, 95% CI: [−1.6–9.1]). Through W48, 89% of subjects switching to RPV/FTC/TDF at baseline maintained VS. The rate of VS at W48 for the 152 subjects who switched to RPV/FTC/TDF at W24 was comparable to the rate of VS at W24 for those who switched to RPV/FTC/TDF at baseline (Delayed Switch to RPV/FTC/TDF 92%). VF was lower in the switch arm (0.9%) compared to remaining on PI + RTV + 2NRTIs (5%) at W24. VF was also lower in the delayed switch arm (1.3% from W24-48). At week 48, patients in the immediate switch arm had few VFs (1.1%; 4/317) and resistance development was infrequent after switching to RPV/FTC/TDF. There were 24 subjects with pre-existing K103N mutations: 18 in the immediate switch arm and six in the delayed switch arm. Twenty three out of twenty four subjects successfully maintained suppression after switching to RPV/FTC/TDF. One patient had viral rebound with resistance: showing pre-existing K103N and V179I with emergent M184V, E138K and V108V/I mutations in the RT.
In the W48 analysis, VS was maintained regardless of whether subjects switched to RPV/FTC/TDF at baseline or at W24. RPV/FTC/TDF-treated subjects with pre-existing K103N mutations had a high response rate.
Cobicistat increases serum creatinine (Cr) by inhibiting tubule Cr secretion, and tenofovir DF (TDF) may cause renal toxicity. We examined the renal safety profile of elvitegravir/cobicistat/emtricitabine (FTC)/TDF (STB) through week 96 in two phase 3, randomized, double-blind studies to determine whether co-administration of COBI and TDF increases the rate of TDF-associated renal toxicity.
Pooled data from Study 103 (STB vs. ritonavir-boosted atazanavir [ATV/r] + FTC/TDF [TVD]) and Study 102 (STB vs. FTC/TDF/efavirenz [ATR]) were analyzed for renal safety including renal discontinuations through week 96, and for subclinical renal toxicity defined as having abnormalities in at least two out of four renal laboratory parameters (tubular abnormalities [hypophosphatemia, proteinuria or normoglycemic glycosuria] or increases in serum Cr).
Median exposures to STB (
The rate and type of renal discontinuations in the STB group were similar to those in the ATV/r + TVD group and consistent with published historical rates. No STB patient developed PRT after week 24. The risk of PRT with STB is low, monitorable and reversible upon discontinuation, which is similar to a regimen containing TDF and boosted ATV.
Studies about the prevalence of diabetes mellitus in HIV-positive patients on HAART therapy show contradictory conclusions. This study aimed to find the prevalence of diabetes and whether specific antiretroviral treatment is associated with an increment amongst HIV patients on treatment.
A retrospective study was performed. HIV-positive patients on HAART whose first visit to Clínica Condesa was in 2009 to 2011 and are still active (last clinical visit within 2012) were included. General demographic information, antiretroviral therapy, CD4 and viral load were obtained from a clinical database; the general lab database provided fasting glucose levels. Diabetes mellitus was defined as doctor reported in the lab request and/or fasting glucose levels above 126 mg/dL. For analysis, SPSS 20 package was used.
In this study, 4,950 patients were included; the population was composed of 542 (11%) women, 4,288 (87%) men and 127 (3%) transgender men. The mean age was 39 years and average glucose was 90 mg/dL SD±23. Diabetes was found in 2.5% (ICI 2% ICS 3%) of the patients; of this, 15% were women, 83% men, and 2% transgender men.
The prevalence of diabetes mellitus in Clínica Condesa was low relative to general Mexican population (7.34%).[1] There are several factors yet to be studied that may influence this prevalence, but this result is a hint that Mexican HIV population behaves differently.
There is an increasing need to identify alternative antiretroviral (ARV) combinations for HIV (+) patients that avoid Nucleoside/Nucleotide Reverse Transcriptase Inhibitors (NRTI) to minimize long-term organ toxicity. The PROGRESS study (Reynes J, et al. AIDS Research and Human Retroviruses 2013; 29:256) compared the novel NRTI-sparing regimen LPV/r + raltegravir (RAL) to LPV/r + TDF and emtricitabine (FTC) in ARV-naïve patients. Through 48 weeks of follow up, LPV/r + RAL was noninferior in efficacy and exhibited comparable safety and tolerability profiles compared to LPV/r + TDF/FTC. As a component of a secondary endpoint, we evaluated the renal safety profile associated with each ARV regimen during the 96 weeks of follow up time.
Creatinine clearance (CrCl) was estimated by the Cockroft–Gault equation, and changes from baseline (BL) were assessed by one way ANOVA. Subjects were followed through 96 weeks or until discontinuation from the study based on the investigator's best clinical judgment.
Study participants (n = 172, LPV/r + TDF/FTC = 90) were 39.9±10.7 years old, 88.4% White, 11.6% women, BL CrCl of 120.3±34.9 ml/min, and 28.9% had a diagnosis of hypertension. There were no differences in BL demographics between arms. Mean change from BL to week 96 in CrCl was statistically significantly greater with LPV/r + TDF/FTC compared to LPV/r + RAL (Δ−7.3 versus −1.5 ml/min,
| Age in years | 33 | 42 | 52 | 55 |
|---|---|---|---|---|
| Gender | M | F | M | M |
| Race | W | B | W | B |
| ARV regimen | LT | LR | LT | LT |
| Renal diagnosis | Obstructive stone | Acute kidney injury | Fanconi syndrome | Acute kidney injury with tubulointerstitial nephritis (biopsy) |
| Outcome | Ureteral stent placed | Dialysis dependent | Persistent | Persistent |
M=Male, F=Female, W=White, B=Black, LR: LPV/r+RAL, LT: LPV/r+TDF/FTC
At week 96, the majority of patients had a CrCl in the normal range. Decline in CrCl was larger with LPV/r + TDF/FTC. Renal SAEs were temporally related to exposure to non ARV meds, except for one Fanconi case considered by the investigator to be related to LPV/r + TDF/FTC exposure.
Chronic diarrhoea has been historically a common problem within the HIV-infected population. Currently, we do not know the accurate prevalence in the clinical setting. The objective of the DIACRO study is to identify the presence of chronic diarrhoea within the HIV-infected patients and to outline its characteristics, as well as to profile its cause and to know the clinician perception about its frequency and repercussion.
A cross-sectional study was undertaken in 40 Spanish hospitals. HIV-infected patients that attended the HIV clinic during the study period (10 consecutive days from 7 May 2012) and signed informed consent went through a five question screening test. Those patients who marked positively having ≥2 stools/day and bowel movement disturbance >1 month plus another positive question of five (increase in bowel volume/frequency within the last month, weakness faeces or to abandon activities urgently to defecate within the last 15 days) were considered as chronic diarrhoea patients. These patients went on further evaluation and their socio-demographic and clinical characteristics were registered.
The analysis included 2,658 patients that fulfilled inclusion criteria. Eight hundred ninety-four (34%) of them had ≥2 stools/day. According to the study, chronic diarrhoea definition, the prevalence of these symptoms was 10.3% (273 patients). Within this population, 62.3% (170) of the patients did not report it proactively to their physicians. Of the patients, 28.8% (77) had sometimes self-medicated to try to mitigate it. Seventy-two percent (196) of them had to abandon urgently their activities to defecate in the last 15 days. Of the chronic diarrhoea patients, 17.6% declared having ≥4 stools/day and 76.2% (208) suffered from bowel movement disturbance >6 months. Physicians related the appearance of these symptoms to the ARV therapy in 50% of the cases. Regarding investigators perception, 47.5% of them believed that chronic diarrhoea is frequent within the HIV-infected population. Of them, 72.5% stated to ask proactively to patients about this disorder in their clinical practice.
Chronic diarrhoea is still a current reality in our HIV-infected patient setting. A lot of them do not communicate proactively its presence to the clinicians. A simple medical/case history could help to identify the underlying chronic diarrhoea, although there is not an established criteria about its cut-off point or definition.
Neglected infectious diseases (NID) overlap geographically and epidemiologically with HIV. There is growing interest about the mutual impact of the biological and clinical interactions and the potential for integrate the control of these diseases. The objective of this study was to describe the burden of co-infection of HIV and NID in Latin America and the Caribbean (LAC).
A systematic bibliographic review from Pubmed, Lilacs, and available conferences proceedings from relevant scientific meetings (ASTMH, IAS, CROI) between 1980 and 1 May 2011 was performed.
One hundred and forty-seven eligible articles were identified. The majority addressed co-infection between HIV and either Chagas’ disease, soil-transmitted helminthes (STH) or leishmaniasis, with little information about other NID (n = 18) and most of them were case-series (n = 113). Prevalence of co-infection ranges between 1.2 and 9.9% for
Despite the relevance of these diseases, information about prevalence, incidence, and interactions between specific NID and HIV is very limited and the possibility of integrating the control of these diseases is difficult. Prospective prevalence studies of co-infection in endemic areas for NID should be encouraged to better describe the current situation. This bibliographic review was started during an internship at Pan American Health Organization in May/June 2011, with support of a scholarship of HIV Research Trust to MES.
Yellow fever (YF) is prevalent from northwest to southeast of São Paulo State. YF vaccine is a live attenuated virus vaccine with an efficacy of 99% in one month. It is reasonably safe, with an incidence of mild adverse effects of 2 to 5%. More severe adverse events (encephalitis, immediate hypersensitivity reactions, viscerotropic disease) are rare (4.7 cases per 100,000 doses of vaccine administered) [
Clinical trial of vaccination against YF in HIV-infected patients. Participants were HIV-infected adults followed at the Centro Municipal de Especialidades de São Carlos (CEME), without severe immunosuppression (CD4 T-lymphocyte count >200 cells/mm3) living in São Carlos city, area where there is a risk of YF transmission. Data were collected on age, gender, mode of HIV transmission, CDC/08 classification, antiretroviral drugs, comorbities, coinfections and immunization charts. Patients received YF vaccine if not vaccinated in the past 10 years. Blood samples were taken before, one week and one month after vaccination. Clinical adverse events, liver enzymes and HIV infection markers were monitored.
Up to now, 35 HIV-infected patients were invited to the study. We excluded six subjects who presented CD4 count < 200 cells/mm3, seven patients who have recently received YF vaccine, and three individuals who have refused informed consent. The remaining 19 patients formed the basis for this study, 57.9% male, mean age 45.5 years (29–63). In relation to the mode of HIV transmission, 26.3% were MSM, 63.2% heterosexual, 5.26% IDU and 5.26% unknown. CDC classification: 47.4% A, 36.8% B and 15.8% C. HCV coinfection was found in three patients (15.8%). Most patients were on use of antiretroviral schemes including protease inhibitors (63.2%), with median CD4 count of 693 cells/mm3 and undetectable HIV plasmatic load (63.2%). Mild local side effects were referred by 10.5% of patients. No patient presented increase in serum transaminase values after vaccination.
YF vaccine seems to be safe in HIV-infected patients.
Options for antiretroviral therapy (ART) in patients receiving rifampin-containing anti-tuberculosis treatment (RIF anti-TB) are limited to EFV and more recently Raltegravir-containing regimens. In ART experienced subjects, few alternatives exist. The aim of this study was to describe a case series of co-infected patients who were treated with four nucleosides (4 NUCS) while on RIF anti-TB because of EFV resistance or intolerance.
Retrospective, chart review study was conducted in two large HIV treatment centres in Mexico. We included all patients treated with a 4 NUCS combination comprised by Abacavir, Tenofovir, zidovudine and 3TC/FTC while receiving RIF anti-TB for active tuberculosis.
From 1998 to 2012, 12 patients were included. Eleven (92%) were male. Median CD4+ nadir cell count at baseline was 65 cell/mm3 (IQR 43-105). Only two (17%) patients were naive to ART. The median time of ART exposure before TB diagnosis was 23 (IQR 13.5–23) months. Seven patients (58%) had used protease inhibitors before, and one patient (8.3%) used lopinavir + ritonavir 800/ mg/d during RIF anti-TB before the 4 NUCS regimen. Three patients (25%) had history of liver disease at baseline. The reason for not using EFV was previous failure in nine (75%), and intolerance or contraindications for its use in the rest. Four of 10 treatment-experienced subjects had a genotype before the ARV change was made. Median time of 4 NUCS treatment was 6.5 (IQR 5–17.5) months. Viral load <400 copies/ml was achieved in 58% of the patients. There was a median increase in CD4 cells of 81 cell/mm3 (IQR 41-250) during the 4 NUCS treatment. One subject (8.3%) discontinued treatment because of intolerance. GIII-IV alkaline phosphatase elevation occurred in three (25%) subjects. Eight (67%) patients are alive and continue in follow-up.
Selection of ART remains a challenge for ART-experienced and EFV-intolerant patients who receive RIF anti-TB. In this case series a four nucleoside regimen, used mostly in patients with previous virological failure, was well tolerated and the majority of the patients achieved virological suppression.
Antiretroviral therapy (ART) can sustain suppression of plasma viremia for years. However, if therapy is interrupted, there is a rapid resumption in viremia. Therefore, HIV-1 has the ability to persist in the face of potent ART. Identifying the mechanism through which HIV-1 persists in infected individuals on suppressive therapy is central to the goal of curing infection in these individuals. For this reason, research is focused on establishing tools with which to probe the reservoirs that persist in aviremic individuals as well as clinical protocols with which to perturb those reservoirs. How we measure viral reservoirs in aviremic patients and, as a consequence, how we gauge the impact of treatments on those reservoirs is a contentious issue. Most attention has focused on the role of latently infected T-cells in viral persistence and clinical strategies are geared towards purging of these reservoirs. However, other factors may be contributing to viral persistence including residual viral replication and establishment of non-T-cell reservoirs. In addition, although ART significantly improves health outcomes for the patient, several markers of immunopathogenicity persist in the face of effective viral suppression and drive comorbidities. The underlying cause of ongoing pathogenicity in the face of effective ART is unclear. Studies examining the impact of treatment intensification on markers of viral pathogenicity will be discussed.
The efficiency of transmission of HIV depends on the infectiousness of the index case and the susceptibility of those exposed. Infectiousness is dictated by the concentration of HIV-1 in relevant fluids (regardless of route of transmission) and the viral genotype and phenotype. People newly infected with HIV-1 (i.e. acute infection) and those with STD co-infections excrete such a large concentration of virus as to be “hyperinfectious”. The actual transmission of HIV likely occurs in the first few hours after exposure. The probability of transmission may be as low as 1/10,000 episodes of intercourse or 1/10 sexual exposures when anal intercourse is practiced. The transmission of HIV is generally limited to one or a small number of founder variants which themselves may be “hyperinfectious”. Synergistic behavioural and biologic HIV prevention strategies have been developed and implemented. Safer sex includes limiting the number of sexual partners, use of male latex condoms, and structural interventions to reduce exposure. These strategies appear to have contributed to reduced HIV incidence in many countries. Biological interventions have proven catalytic: these include treatment of inflammatory cofactors, voluntary male circumcision, and use of antiviral agents either for infected people (who can be rendered remarkably less contagious) or as pre- and post-exposure prophylaxis (PrEP and PEP). Ecologic evidence suggests that broader, earlier antiviral treatment of HIV may be reducing incidence in some (but not all) populations. However, maximal benefit of HIV “treatment for prevention” and application of PrEP will likely require a program of universal “test and treat”, where many more infected patients are identified, linked to care, and treated very early in disease and for life. Community randomized trials designed to support this approach are underway in Africa. The “test and treat” prevention strategy is resource intensive and serves to emphasize research that searches for a cure for HIV infection, so that HIV infected people can eventually reduce or stop treatment. Likewise, success in HIV prevention emphasizes the importance of development of an HIV vaccine, which remains focused on agents that may evoke CTL responses, antibody-dependent cytotoxicity, and (perhaps most important) broad neutralizing antibodies. A human clinical trial (RV144) and animal experiments have provided hope, excitement, and a roadmap for development of an HIV vaccine.
Latin America is globally recognized for achieving high coverage of antiretroviral treatment. Virtually all countries in the region have been able to achieve this important goal by offering universal access to antiretroviral drugs through their public health systems, despite the fact that antiretrovirals are available for these countries at relatively high prices, compared to other low- and middle-income countries. However, even though it would be fair to say that the access problem has been solved in the region, there are other important challenges that need to be strategically addressed. Both treatment and prevention programmes in our countries urgently need to be optimized, which means that although the infrastructure and capacity to implement highly effective programmes already exist, but the challenge now is to increase quality and efficiency. During this presentation, I will address some of the aspects, both programmatic and economic, that appear to be the most pressing ones requiring our attention, and also discuss some of the alternatives available to us toward improving the performance of our programmes. On the treatment side, I propose that there are likely large potential gains in improving timeliness of treatment initiation and efficiency of treatment regimes. On the prevention side, significantly improving testing efforts and increasing their coverage is probably a good measure toward increasing the health gains of our efforts. Using evidence to address these issues is key, and I wish to put to you that not only have we not done it enough in the past but that we also urgently need to generate evidence upon which to base our decisions. Most of the scientific evidence on the prevention realm comes from Africa. We need to be more creative and innovative and rigorously evaluate approaches to improve access, quality and efficiency.
Once a retrovirus infects a eukaryotic cell and integrates within chromosomal DNA, it becomes part of its genome and can be activated/transcribed/translated to produce viral proteins and/or new viral particles. Over the years, some of these retroviruses may lose their pathogenicity and become adapted to the new host. Under these conditions, retroviruses can paradoxically ameliorate the functional portfolio of the infected cell, thus potentially increasing its functionality and/or chances of survival in a difficult environment. Individuals whose cells are infected by retroviruses have acquired, in the last millions of years, innovative functions that, once transmitted to the new generation through germinal cells, have become essential for their homeostasis, or even for their survival. This is the case of some endogenous retroviruses, whose products are mandatory for the proper vascularization of human placenta. To our knowledge, there are no natural means able to selectively eliminate retroviral genes from an infected cell or an individual. Therefore, chances of getting naturally cured by retroviruses, once infection is set and viral genomes are spread into the body, are minimal or absent. HIV is a retrovirus that behaves as all other retroviruses that interacted with humans in the past millennia. Its fate is to remain forever within the infected body. For these reasons, chances of getting rid of HIV infection and being biologically cured (that is, eliminating all viral genomes from the body) are very limited if we consider current knowledge, biotechnology, and available medical tools (yet it cannot be fully excluded in very peculiar cases). The option offered by the so called “functional cure” is different. In this case, medical manipulation may create conditions whereby viral genomes, decreased in number and function by proper therapies/vaccines, are no longer able to harm the host for an indefinite period of time. Patients do remain infected, but viral replicative cycles are absent, and progression of the disease is interrupted. This latter clinical approach may be suitable, and this is where the clinical research is directing its efforts. If achievable, infected persons should cope with the virus and keep it under control for decades, without support of chronic antiviral therapy. In conclusion, the proper knowledge of the biological characteristics of HIV helps in selecting the best strategies aimed at obtaining the maximum achievable clinical result.
The collaborative international effort to provide universal access to HIV care and effective antiretroviral therapy (ART) has reached a critical time point. The global economic downturn, changing donor priorities and competing priorities in the health sector threaten the capacity of various agencies to maintain support for the continued scale-up of access toward the UN General Assembly-agreed target of 15 million people with HIV/AIDS receiving ART by 2015. We now know that treatment acts as prevention by reducing the infectiousness of treated individuals. It is now necessary to review the elements of the successes and challenges to date, in order to be able to best use finite resources. These elements include efforts to optimize the delivery of HIV care, including ART, in low- and middle-income countries (LMIC) and the emergence of new agents and drug classes, which have simplified HIV treatment and made broader successful management more achievable. Recent studies have indicated that earlier commencement of HIV therapy is beneficial, leading to changes in the recommended ART initiation threshold in LMIC to <350 CD4 T cells. Forthcoming revised WHO guidelines are anticipated to raise this threshold. Studies currently under way are investigating approaches to second-line ART in LMIC. The results from these studies will better inform the roll-out of effective second-line therapy. In addition, the financial cost of ART makes optimization of dosing an important consideration in LMIC, in order to maximize effectiveness while limiting costs. ART monitoring is also an important priority in LMIC. Efforts to develop simple and reliable technologies that can provide rapid results in the field are underway. In the meantime, a number of recent studies in adults and children point to the way forward and the best use of resources. The final priority is operational optimization, to ensure adequate service delivery. Although the challenges in LMIC are substantial and evolving, considerable inroads have been and are being made into optimizing HIV treatment and its delivery, which is crucial in reducing the global impact of the disease. This abstract is modified from an abstract provided by David Cooper, University of New South Wales, Sydney, Australia, when presented as the Lock Lecture at HIV Drug Therapy in Glasgow, November 2012.
There currently are 27 antiretroviral drugs approved for the treatment of HIV infection. In choosing the optimal initial regimen, a number of factors should be considered: antiretroviral activity (HIV RNA, CD4 cell, and clinical responses); durability of responses; baseline drug resistance; tolerability and both acute and chronic side effects; convenience (number of pills, dosing interval, food/fasting requirements); preserving future treatment options; patient's stage of HIV disease, concomitant illnesses and medications (and drug-drug interactions); access; and cost. The optimal initial regimen is one that is individualized to the specific patient's situation. Current preferred initial regimens include two nucleoside analogues (NRTI) combined with either a non-nucleoside analogue (NNRTI), a boosted protease inhibitor (PI), or an integrase inhibitor (II). One-pill, once-daily combination regimens have proven particularly popular with patients and providers alike. The most common initial regimen worldwide is two NRTI + an NNRTI. In the event of virologic failure of an initial regimen, multiple causes of failure should be assessed, including: adherence; drug resistance; use of less potent antiretroviral regimens; drug levels and drug-drug interactions; and theoretically, tissue reservoir penetration (central nervous system, genital tract). Of course, any or all of these reasons may be important and the challenge is to address the probable cause(s) of failure and address them in choosing the next regimen, to ensure success. Genotypic drug resistance testing is indicated in the assessment of first (or second) virologic failure and can help optimize the selection of the next regimen. Genotypic and phenotypic testing is recommended following failure of multiple regimens. A common sequence of regimens is two NRTI and an NNRTI, changing to two NRTI + a boosted PI. The availability of drugs with activity against drug-resistant viruses (e.g. newer NNRTIs and PIs) and new classes of drugs (e.g. integrase inhibitors, CCR5 antagonists) offers additional options for constructing successful subsequent ART regimens.
HIV resistance to ARV therapy is an unwanted consequence of HAART as it limits future options. The emergence and spread of high levels of HIV-1 drug resistance in resource-limited settings where combination antiretroviral treatment has been scaled up, without proper treatment monitoring and drug availability, could compromise the effectiveness of national HIV treatment programmes. Compared to developed countries, most of Latin America do not perform resistance assays before starting ARV treatment and after first line failure, having more resistance as a consequence in some cases and the need for more expensive salvage treatments. Some countries in the region do not have access to resistance tests due to cost and lack of infrastructure. The presentation will include data on the limited increase of transmitted resistance over time in the region compared to other developing countries, especially in Africa, as well as the prevalence of secondary resistance in first line failure and multiexperienced patients, with remark on specific issues with non-B subtypes. The elevated cross-resistance to the entire nucleoside analogue group in countries still using thymidine compounds in first line treatment will be stressed. Moreover, it will include the big limitations for resistance assay interpretations and performance in low level viraemia in Latin America as well as the excellent results of using resistance committees in the optimization of available assays as in the prescription of newer drugs.
Access to highly active antiretroviral therapy (ART) is expanding in resource limited settings (RLS), and ART regimens are frequently changed. Outcomes after changes in the first regimen (ART-1) have not been well studied in RLS. CCASAnet is a consortium of adult HIV clinics from seven countries (Argentina, Brazil, Chile, Haiti, Honduras, Mexico, and Peru).
Retrospective observational cohort study, assessing rates of death, virologic failure (VF), and change in second ART regimen (ART-2) after modification/changes of ART-1. Rates were computed using Kaplan-Meier methods and hazard ratios (HR)—adjusted for sex, age, CD4, clinical stage, calendar year, time between starting regimens, reasons for changing ART-1, and type of regimen change—were computed using Cox models stratified by site.
A total of 3,384 patients (25% of total initiating HAART) changed ART-1 by at least one drug and were included in this study, with a median follow-up of 2.8 years from start of ART-2. Median age at start of ART-2 was 37 years, 60% were male, and median CD4 was 181 cells/µL; toxicity was the most common reason for changing ART-1 (48%) and the median time from start of ART-1 was 275 days. After starting ART-2, 319 patients (9.4%) died; the probability of death at years 1, 3, and 5 after ART-2 was 0.06, 0.10, and 0.12, respectively. Risk factors for death were older age (HR = 1.24 for 50 vs. 40 years; p = 0.052), clinical AIDS at first visit (HR 1.30;
In a multi-centre resource-limited setting cohort of patients modifying their first ART regimen, rates of subsequent ART regimen modifications, mortality, and virologic failure were high. Access to expanded ART formularies, especially with newer, safer-profile drugs, is needed.
PA-824 (Pa) and bedaquiline (B) (TMC) are novel compounds in phase 2 development with established Early Bactericidal Activity (EBA) over 14 days. The study presented is an EBA study that evaluated these drugs alone or in combination with each other and with moxifloxacin (M) and pyrazinamide (Z) to identify a regimen with the potential to shorten treatment of TB in patients without the use of rifamycins or other drugs that interact adversely with antiretroviral Therapy (ART).
83 participants enrolled (26% F, 74% M, including 6 HIV+) as five cohorts of 15 TB patients, each who received daily dosages of B alone, B with Z, B with Pa, Pa with Z and Pa with M and Z. A cohort of 8 patients received daily standard TB treatment (isoniazid, rifampin, Z and ethambutol: HRZE) as a control for the EBA quantitative mycobacteriology. The primary efficacy endpoint was the rate of change in number of colony forming units (CFU) of
All cohorts had decreases in logCFU counts/ml of sputum from Days 0 to 14 that ranged from 1.2–2.7 over 14 days. While Z potentiated the activity of both B and Pa and compared favorably with the HRZE standard regimen, the cohort with the combination Pa-M-Z had numerically the greatest effect on CFU reduction.
The combination regimen of Pa-M-Z has potent bactericidal activity over 14 days in patients with pulmonary TB and has the potential to treat both Drug Sensitive- and Drug Resistant-TB (contains no INH or rifampicin) without adverse clinical interactions with ART. This regimen has been taken into an 8 week trial to treat DS- and DR-TB in patients with and without HIV infection.
The impact of HIV on MDR-TB treatment outcomes in sub-Saharan Africa remains unclear where extensive rollout of highly active antiretroviral therapy (HAART) has occurred. We therefore compared the time to initial culture conversion among patients with and without HIV infection in a setting of individualized, ambulatory MDR-TB care in Botswana.
We performed a prospective cohort study of MDR-TB patients receiving ambulatory care at two public clinics in Botswana. The time to culture conversion and proportion converting were compared by HIV status using Cox proportional hazard ratios (HRs).
40 HIV-infected and 30 HIV-uninfected patients with MDR-TB and follow up cultures were identified. The median CD4+T-lymphocyte count of those with HIV was 215 cells/mm3 (IQR 129–347), and 36 (90%) were on HAART. 85% of HIV-infected and 83% of HIV-uninfected achieved culture clearance. The median time to initial culture conversion was 78 days (IQR 42–186) for HIV-infected and 95 days (IQR 70–133)for HIV-uninfected individuals [log rank p=0.62; unadjusted HR=0.9 (95% CI: 0.5 to 1.5)]. Adjusting for age, gender, TB treatment history and number of active antitubercular drugs used did not change this result [adjusted HR=0.8 (95% CI: 0.4 to 1.4)]. Toxicity was frequent in all subjects: ototoxicity occurred in 53% and 70%, neuropathy in 40% and 10%, and nephropathy in 25% and 7% of HIV-infected and uninfected patients, respectively. Neuropathy (p=0.005) & nephropathy (p=0.044) were significantly associated with HIV infection.
We found no difference in the proportion or time to initial sputum culture conversion between an HIV-infected and non-infected cohort of MDR-TB patients in Botswana. These results suggest that microbiologic outcomes among those with HIV can be comparable to those without HIV in similar settings with access to individualized TB treatment and HAART.
In Nigeria, the progressive loss of patients at every stage in antiretroviral therapy (ART) programs especially at the point of uptake of CD4 monitoring poses a threat to the achievement of treatment targets. Long waiting time coupled with the burden of traveling long distances for blood draw and receipt of CD4 results has led to attrition in the number of clients who test positive to HIV versus the number who eventually commence ART.
In order to increase uptake of CD4 monitoring and reduce loss to follow up (LTFU) at three USAID/MSH supported HIV care and treatment clinics, the following interventions were instituted.
1. Task shifting to data clerks to fill laboratory request forms for CD4 monitoring instead of clinicians who complain of heavy workload.
2. Strengthened escort services from the point of enrollment to the laboratory to ensure that clients access laboratory investigations on the same day.
3. Task shifting to laboratory technicians on the use of automated CD4 equipments after consistent onsite training and supervision.
4. Established point of care sample collection for CD4 estimation.
5. We commenced daily CD4 investigations (Monday to Friday) ensuring that clients attending clinics from long distances access CD4 monitoring on any day of the week.
6. Commenced 24 hours turnaround time for release of CD4 result to ensure rapid initiation of eligible clients on ART.
After 12 months, number of clients accessing CD4 investigations increased from 53.8% to 93.3% while number of clients LTFU reduced from 58.7% to 10.7%. Turnaround time for CD4 results decreased from 7 days to 24 hours. Average client waiting time reduced from 4 hours to 1hour 30mins.
Strengthening laboratory systems helps increase uptake of CD4 investigations, shorten client waiting time and ultimately reduces LTFU especially among clients attending clinics from hard to reach communities with difficult terrains.
Despite expanded access to HIV testing, most newly-diagnosed South Africans present with severe immunosuppression. We sought to determine the risk factors for presentation with late-stage HIV disease and the perceived barriers to presenting earlier for care.
We enrolled and surveyed adults prior to HIV testing at four outpatient clinics in urban and peri-urban areas of Durban from August 2010 to November 2011. Late-stage HIV disease was defined as a CD4 count<100 cells/mm3. We used multivariate logistic regression models to determine the effects of sex, emotional health, social support, distance from the clinic, employment, perceived barriers to receiving healthcare, and foregoing healthcare to use money for food, clothing, or housing (“competing needs to healthcare”) on presentation for care with late-stage HIV disease.
Among 3,669 adults screened, 830 (22.6%) were enrolled, newly-diagnosed HIV-infected, and obtained a CD4 result. Among those, 279 (33.6%) presented with late-stage HIV disease. In multivariate analyses, participants who lived =5 kilometers away (OR 2.8, 95% CI 1.7–4.7), had reported competing needs to healthcare (OR 1.7, 95% CI 1.2–2.4), were male (OR 1.7, 95% CI 1.2–2.3), worked outside the home (OR 1.5, 95% CI 1.1–2.1), perceived health service delivery barriers (OR 1.5, 95% CI 1.1–2.1), and had poor emotional health (OR 1.4, 95% CI 1.0–1.9) had higher odds of late-stage HIV disease presentation.
In Durban, South Africa, the strongest independent risk factors for presentation with late-stage HIV disease were living further from the clinic, being male, and having competing needs to healthcare. Self-reported barriers related to personal illness, costs of care, and poor perceived service delivery were also significantly associated with late-stage disease presentation. Future studies should examine whether use of mobile units and financial assistance may reduce presentation with late-stage HIV disease in resource-poor settings.
Xpert® MTB/RIF is a new molecular diagnostic tool, developed to increase detection and shorten time to diagnosis of sputum-smear-negative (SSN) tuberculosis (TB). In April 2011, Médecins Sans Frontières (MSF) in collaboration with the Zimbabwean Ministry of Health and Child welfare implemented two Xpert® MTB/RIF systems in a rural district in Zimbabwe serving two hospitals and 26 decentralised primary care clinics.
From May to October 2011, parallel testing with both smear microscopy and Xpert® MTB/RIF was performed on specimens from all TB suspects. We used information abstracted from clinical and laboratory records to compare the number of laboratory-confirmed TB cases, number of TB notifications, and the time to diagnosis among HIV/TB co-infected patients with sputum-smear-negative TB during 6 months before and after Xpert® MTB/RIF implementation.
A total of 1672 sputum specimens were processed, of which 184 (11.0%) were smear-positive. Mycobacterium tuberculosis was detected by Xpert® MTB/RIF in 116 (7.8%) of the 1488 remaining smear-negative specimens. Comparing the period after implementing Xpert® with the period before, the proportion of TB notifications that were smear positive (33% versus 27%), smear-negative (48% versus 49%), sputum not tested (11% versus 12%), and extra-pulmonary (8% versus 12%) was unchanged. The median time to TB treatment initiation among HIV/TB co-infected patients with sputum-smear-negative TB, decreased at decentralised sites (from 18.5 days to 7 days), but remained constant at the hospital level (5.5 days before and 6 days after).
Xpert® MTB/RIF increased the number of laboratory-confirmed TB cases in rural Zimbabwe, enabling further task shifting of TB management. In settings where access to chest X-Ray and trained doctors is lacking the impact on TB notifications may be greater. Time-to-initiation of TB treatment at the decentralized clinics was reduced, which has the potential to reduce morbidity in individuals and reduce the risk of TB transmission to others.
Recently, WHO recommended that GeneXpert MTB/RIF be used as first line diagnostic to test for TB in HIV positive individuals. Most patients initiating ART lack the classical symptoms for TB resulting in missed diagnosis. The role of symptom screen in predicting a positive GeneXpert result among pre-ART patients was studied.
This was a nested cohort study within a GeneXpert impact evaluation trial in pre-ART patients. TB symptomatic and asymptomatic adults (>18 yrs) at an ART initiation clinic in Harare were recruited between October 2011 and February 2012. For each patient, two spot sputum samples were collected and induction with 6% hypertonic NaCl performed in those who could not expectorate. Sputum samples were tested with GeneXpert (Cepheid) Test. Participants were followed-up for 3months.
150 participants were recruited and 126 produced specimens and were tested for TB using GeneXpert. Median CD4 count was 165cells/ul (IQR 79–256). Fifty-four percent of the participants had a cough (68/126). Induction was carried out in 19 participants and of these, 47% (9/19) were coughers and 53% (10/19) non-coughers. TB was diagnosed in 10% of participants (13/126; 95% CI 4–16); with an additional 2 cases diagnosed on second GeneXpert test. Significant predictors of disease were cough of any duration (p=0.019), night sweats (p=0.03) and weight loss (p=0.04). Of those induced, 16% (3/19) had a positive GeneXpert result. Notably, induced samples accounted for 23% (3/13) of the TB cases detected. Three percent (2/58) of the non-coughers were GeneXpert positive. Seven participants (5%) with negative GeneXpert results were started on TB treatment based on clinical suspicion.
TB testing using GeneXpert in pre-ART patients, with sputum induction, should be carried out routinely regardless of patient TB symptom status. A two step screening test and Xpert testing algorithm is needed for scale-up of universal TB testing in pre-ART patients.
South Africa has an estimated 5.6 million people living with HIV, representing a quarter of the disease burden in sub-Sahara Africa and one fifth of the global disease burden. The National HCT Campaign was launched in April 2010 and ended in June 2011, coordinated by the National Nerve Centre.
The Campaign was launched at national, provincial and district levels and targeted everyone between the ages of 12–60 years, not exclusive of younger or older persons. Confidentiality and informed consent was required to test and every site was linked to a referral facility providing ART, care and support. HCT was done at health facilities, work places and community outreach sites.
South Africa committed a significant budget of US$3.5 billion to support the HCT Campaign and Treatment Expansion. By June 2011 a record 13,269,746 HIV tests were conducted and 2,155,312 (16%) people tested positive of whom 48% had CD4 counts above 350. Over 400,000 patients were initiated on ART, of whom 57,000 were pregnant women. National Laboratory DNA PCR results indicated that mother to child transmission rates at 8 weeks reduced to 5% compared to 11% in 2009. Over 8 million people were screened for TB and 185 million male condoms and 524,000 female condoms were distributed. 237 males were medically circumcised, exceeding the Campaign target of 100,000. The procurement system achieved a 53% ARV price reduction through competitive tenders and 3,686 (80%) health facilities were capacitated to deliver ART through task shifting and training of 10,542 nurses.
South Africa has succeeded in implementing a massive HCT campaign linked to a comprehensive HIV prevention model that optimizes treatment for prevention and reduction of new HIV infections. The Campaign created a shift from testing for treatment to testing for prevention and promoted combination prevention to turn the AIDS epidemic tide for South Africa.
Recent estimates indicate that 40% of the HIV infections in the Netherlands are undiagnosed. Earlier diagnosis of HIV will improve individual health outcomes and reduce further spread. Self tests for HIV have become available, which allow individuals to test at home without involvement of health care professionals. Although self tests may help to increase HIV test uptake, there are concerns about test quality, counseling and medical follow up. The Amsterdam Public Health Service has initiated the HivTest@Home project to develop and evaluate a service that provides reliable HIV self tests using oral fluid in combination with an Internet counseling strategy for individuals at high risk for HIV, especially MSM and migrants from HIV endemic countries.
A website and logistic infrastructure will be developed that provides an intake with personal advice about HIV self testing, and enable individuals to purchase the HIV self test we provide online. With the test, they will receive a code to access a pre-test trajectory including step-by-step instructions, counseling, and low-threshold contact options with health care professionals. For those who test positive, a follow-up procedure will be developed to motivate them to access regular health care for confirmation testing and referral to an HIV clinic.
The service will be launched in 2013, accompanied by a media campaign targeting high risk groups. We aim to distribute 2,000 tests within a 12-month period, assuming an HIV prevalence of newly diagnosed individuals of 2.5-5.0%. Using data collected online from participants, web statistics, interviews with participants, and clinical follow-up data, we will evaluate the feasibility and acceptability of the service and its effectiveness in identifying undiagnosed HIV infections.
If feasible and effective this new method can create another reliable low-threshold testing option for different risk populations -an option hardly needed to break the cycle of low-testing numbers.
The effect of chronic hepatitis B (HBV) on HIV outcomes is relatively unknown in sub-Saharan Africa (SSA) where a high burden of HIV-HBV co-infection exists.
Clinical and immunologic outcomes in response to ART were compared longitudinally between HIV mono- (HIV+) and HIV-HBV co-infected (HIV&HBV+) adults enrolled between November 2004-December 2010 at 18 Management and Development for Health (MDH)-PEPFAR supported HIV clinics in Tanzania. Inclusion criteria were: tested ≥×1 for HbsAg (DIMA), age ≥15, no prior ART.
The prevalence of HBV was 837/13,107 (6.4%).Compared to HIV+ patients, HIV&HBV+ patients were more likely to be male, older, have lower median CD4+ cell counts, and higher ALT's (p values <0.05) prior to ART initiation. Mean follow up time on ART was 9.8 (SD 7.8) and 10.1 (SD 7.5) months in HIV&HBV+ and HIV+ patients respectively. In multivariate analyses, there were no significant differences in overall mortality [HR 1.17 (95% CI 0.87,1.33); p=0.52] between HIV&HBV+ and HIV+patients. CD4+ count response also did not significantly differ between the 2 patient groups (p=0.11). HIV&HBV+ patients had a significantly higher risk of ALT>120IU/L [38/813 (4.7%) vs. 303/12,136 (2.5%); HR 1.76 (1.25, 2.49), p<0.01] and ALT>200IU/l [20/831 (2.4%) vs. 102/12,236 (0.8%); HR 2.74, (1.66, 4.05), p<0.01]. HBV+ vs. negative status was associated with a significantly lower mortality among patients on tenofovir (TDF) [HR 0.40 (95% CI -0.19–0.85; p<0.02)]; but higher mortality among patients on ART not containing TDF [HR 1.70 (95% CI -1.34–2.15; p<0.01)]; [interaction p-value (<0.01)].
Co-infection with HBV did not significantly impact mortality or immunologic outcomes in HIV-infected patients on ART in this SSA setting. However, routine monitoring of ALT levels after ART initiation in co-infected individuals is recommended. TDF should be included in initial ART for HIV&HBV co-infected individuals, given the significant mortality benefit observed.
Few studies have examined the natural history of chronic hepatitis C virus (HCV) infection among HIV-infected persons in the era of combination antiretroviral therapy (cART). Our objectives were to: 1) compare the incidence of hepatic decompensation between cART-treated HIV/HCV-coinfected and HCV-monoinfected patients, and 2) evaluate determinants of decompensation among coinfected patients on cART.
We performed a cohort study among 4,286 cART-treated HIV/HCV-coinfected and 6,639 HCV-monoinfected patients in the Veterans Aging Cohort Study Virtual Cohort (1997–2010). All patients had HCV viremia and were HCV treatment-naïve. Coinfected patients received cART for at least one year and had an HIV RNA result >500 copies/mL within 180 days prior to starting cART (to identify new cART initiators). Hepatic decompensation events (defined by diagnoses of ascites, spontaneous bacterial peritonitis, variceal hemorrhage, or hepatocellular carcinoma) and death were evaluated. Cox regression was used to determine the adjusted hazard ratio (aHR) of hepatic decompensation associated with cART-treated coinfection and evaluated baseline risk factors for decompensation (alcohol abuse, non-black race, diabetes mellitus, FIB-4 >3.25, hemoglobin<10 g/dL, and pre-cART CD4 count) in coinfected patients on cART.
Compared to HCV-monoinfected patients, cART-treated HIV/HCV-coinfected had a higher cumulative incidence and risk of hepatic decompensation (303/4,286 [7.1%] versus 370/6,639 [5.7%]; aHR=1.76 [1.50–2.06]) and hepatocellular carcinoma (50/4,286 [1.2%] versus 60/6,639 [0.9%]; aHR=1.69 [95% CI=1.14−2.49]). After decompensation, mortality was higher in coinfected patients (228/303 [75.2%] vs. 210/370 [56.8%]; p< 0.001). Non-black race (aHR=1.96 [1.53−2.49]), baseline FIB-4 >3.25 (aHR=7.18 [5.12−10.07]), and baseline hemoglobin<10 g/dL (aHR=2.86 [1.62−5.07]) were associated with decompensation among coinfected patients.
Despite cART, HIV/HCV-coinfected patients had a higher risk of hepatic decompensation and death compared to HCV-monoinfected individuals. Risk of decompensation was higher for coinfected patients with advanced liver fibrosis, severe anemia, and non-black race.
A large scale HIV prevention programs among long distance trucker drivers was started in 2003 across 36 Transhipment locations (TSLs) alongside the national highways. Program was re-designed in the year 2006 by reducing number intervention locations to 17 high performing TSLs. The current research assesses the effectiveness of program after the re-design process.
Program monitoring data over seven years and two rounds of cross-sectional behavioral survey conducted in January 2008 (N=1402) and July 2009 (N=1407) were used. Indicators on program outreach and service utilization were examined. Multivariate logistic regression models were used to assess if the increase from first survey to next was significant.
Coverage of program increased from 43535 in 2004 to 311667 in 2010, though there was a decline in number of TSLs. Along with this, there was a significant increase in number of truckers utilizing clinical services. Analysis from survey data showed that there was a seven-fold increase in clinic visit in last 12 months from 2008 to 2009 (21% vs. 63%, P< 0.001). Significant level of increase was also observed in percent of truckers who watched street plays, participation in health exhibitions. Furthermore, increase from round-1 to round-4 was significant in the following indicators: received condom (13% to 22%, P< 0.001), one-one counseling (15% to 21%, P< 0.01). Treatment seeking for sexually transmitted infection (STI)-related symptoms from STI clinics also increased six times during this time (16% vs. 50%, P< 0.001).
The re-designing of the intervention has increased the program coverage and service utilization. Implementing truckers program in limited number of high-impact locations after proper planning can help in saturating the coverage and optimum utilization of the available resources.
Current funding levels from PEPFAR/Global Fund may be too low to ensure Universal Access to antiretrovirals (ARVs) in the long-term. Additional, sustainable sources of funding are required.
For the 20 countries with the largest HIV epidemics, the additional costs required to achieve Universal Access were calculated, using WHO 2011 estimates of patient numbers requiring ARV treatment, combined with Clinton Foundation prices of ARVs, and PEPFAR estimates of cost of care/diagnostics. WHO estimates of adult population size, annual alcohol and tobacco consumption (commercially supplied) were used to estimate annual revenues from a “Global Health Charge” of 1 US cent per 10mL unit of alcohol, and 10c per 20 cigarettes.
In the 20 countries with largest HIV epidemics, 5.2/11.3 million eligible patients were receiving antiretrovirals (coverage 46%). The minimum cost of care was $861 per patient-year (antiretrovirals, $406, medical $300, diagnostics $155). Ten of the 20 countries (Botswana, Brazil, China, India, Nigeria, Thailand, Russia, Uganda, Ukraine and Vietnam), could fund 100% of Universal Access costs from National revenue using the “Global Health Charge”: $2.57 of the total $17.97 billion of revenue collected per year would cover treatment of 3.0 million eligible patients in these countries. In the other 10 countries (Cameroun, Cote d'Ivoire, DR Congo, Kenya, Malawi, Mozambique, Tanzania, South Africa, Zambia, Zimbabwe) $937 million could be collected annually with the Global Health Charge: sufficient to treat 1.1 million eligible patients (35% of the additional $2.67 billion budget required for Universal Access).
A “Global Health Charge” of 1 US cent per unit of alcohol, and 10 cents per 20 cigarettes, collected and spent at a National level, would be sufficient to fund an additional 4.1 million patients per year with antiretroviral treatment and care; Universal Access could be acheived in 10 of the 20 countries with this system.
HIV and TB treatment is provided free of charge in the public sector in South Africa. However, patients may lose time and incur expenses due to utilizing these services. We measure time and financial costs of utilizing HIV and TB services, and examine in how far they lead to financial distress (patients’ self-report of either borrowing money or selling assets to finance healthcare).
We randomly selected patients in a two-stage-sampling scheme and collected data in patient exit interviews: 300 patients on antiretroviral treatment (ART), 200 enrolled in a pre-ART programme, and 300 receiving TB treatment in rural KwaZulu-Natal, South Africa. We assess factors associated with financial distress in multiple regressions, controlling for sex, age and employment.
Most patients utilizing healthcare were women: 79% (in pre-ART), 62% (ART), 53% (TB). The average times patients spent utilizing care at the last clinic visit was: 3.5 (pre-ART), 2.8 (ART), and 1.1 hours (TB). The average total costs of utilizing healthcare during the last visit were: 15 (pre-ART), 25 (ART), and 20 South African Rand (ZAR) (TB). Transport was the single largest cost component in all three patient categories. 39% (ART), 31% (pre-ART), and 41% (TB) of patients reported financial distress due to healthcare utilization. For each additional hour spent utilizing healthcare, the odds of financial distress increased by 3% (pre-ART), 21% (ART) and 64% (TB). For every additional 10 ZAR spent on utilizing healthcare, the odds of financial distress increased by 25% (pre-ART), 9% (ART), and 6% (TB).
In this poor, rural community, very high proportions of patients utilizing HIV or TB services report financial distress due to healthcare utilization. Frequent, free transport to HIV and TB clinics would likely substantially reduce the time and financial burdens of healthcare, as well as the prevalence of financial distress.
ART rollout may have important indirect effects on households and communities. We assess household spillover effects of ART on economic outcomes, in a region of rural South Africa with high HIV prevalence.
Longitudinal socioeconomic data from a large population surveillance system (n>100,000) were linked with clinical records from the government ART program serving the area (n=6964). We first describe the extent of household-level exposure to ART among residents in the community. We then employ panel data techniques to evaluate the impact of ART on household assets, food insecurity, and perceived financial status; household composition; and the labor market participation and school enrollment of other household members. We assess trends in these economic outcomes, relative to the dates when household members accessed the ART program and/or initiated ART. As a point of contrast, we examine similar trends relative to dates of death due to HIV in the era before the public sector ART rollout.
By 2010, two-fifths of area residents lived with someone who had sought care in the government ART program. Prior to widespread availability of ART, households in the region experiencing an HIV-related death faced large economic costs. In contrast, we find little evidence that households of ART initiators experience significant economic hardship due to HIV illness.
South Africa′s public sector ART rollout has provided substantial social protection for households against the economic costs formerly associated with HIV illness and death. Through indirect exposure to ART via household living arrangements, large populations now enjoy the economic benefits of ART.
Antiretroviral therapy (ART) reduces the risk of tuberculosis, but the incidence still exceeds that in HIV-uninfected people. Retrospective cohort studies suggest an additive benefit of isoniazid preventive therapy (IPT) in patients on ART, but there are no controlled data on the efficacy and safety of IPT for patients on ART.
Using a pragmatic randomized double-blind placebo-controlled study design, we evaluated the efficacy of IPT among HIV-infected participants established on ART or newly starting ART in Khayelitsha, South Africa. Participants were randomized to daily isoniazid or matching placebo for twelve months, and followed for up to four years. Tuberculosis was excluded at screening by sputum culture. Development of incident tuberculosis was the primary endpoint. Secondary endpoints included toxicities and deaths.
1,329 participants contributed 3227 person-years (PY) of follow-up in the modified intention to treat analysis; 662 on placebo and 667 on IPT, with comparable CD4+count and proportion starting ART. Overall there were 95 incident tuberculosis cases: 3.6 (95% CI 2.8–4.7) versus 2.3 (95% CI 1.6–3.1) per 100 PY in the placebo and IPT arms respectively (HR 0.63, 95% CI 0.41–0.94, p=0.026). Study drug was discontinued due to pre-specified toxicity in 2.5% in the placebo arm and 4.1% in the IPT arm (logrank p=0.13). The number of deaths was similar between arms (3.0% and 2.1 respectively, logrank p=0.29).
Under field conditions, twelve months of IPT reduced the incidence of TB without causing excess harm in HIV-infected individuals established on ART or newly starting ART. It is feasible to implement IPT in busy ART clinics in settings with high HIV/TB co-morbidity.
With improved survival of HIV-infected children, neurocognition is an important area to address. We examined the effects of HIV infection on cognitive, neurological, and behavioral functioning on perinatally-infected children.
HIV-infected children (4–15yrs) were recruited from a tertiary-care center in India, along with age-gender-and-income-matched HIV-negative children. Assessment tools included (i) soft neurological signs: Physical and Neurological Examination for Soft Signs (PANESS); (ii) neurocognition: culturally-adapted Wechsler Preschool and Primary Scales of Intelligence (WPPSI), Wechsler Intelligence Scale for Children (WISC-III); (iii) adaptive behaviour: Vineland Adaptive Behaviour Scales (VABS).
We studied 167 children, (82 HIV-infected, 85 HIV-uninfected) with 56% males and mean age 8.6 yrs. Total IQ scores were lower among HIV-infected children compared to HIV-uninfected children (74.9 12.9 versus 87.915.4, p<0.001). Both domains of verbal IQ and performance IQ were uniformly affected. Severely immunosuppressed children (CD4<20%) had lower total IQ scores (70.712.3) compared to those with CD4>20%, (IQ 77.112.8, p=0.03). Viral load and ART status has no effect on IQ scores. Multivariate regression revealed that HIV status, weight-for-age Z-score and hemoglobin were independent factors that affected IQ scores (adjusted r2=0.25, p=0.006). The presence of HIV infection independently decreased IQ scores by 9.22 units. PANESS scores were higher among HIV-infected children compared to uninfected children (HIV-positive: 7.5, [3, 13.3]; HIV-negative: 4, [1.5, 9.5], p=0.02) suggesting higher degree of subtle neurological abnormalities in this group. Adaptive behaviour scores were similar for both HIV-infected and uninfected children irrespective of age and sex.
HIV-infected children had lower IQ scores and higher prevalence of soft neurological signs compared to HIV-uninfected children, indicating that subtle neurocognitive impairment is an important feature of perinatally-acquired HIV infection, particularly those with poor nutritional status. We recommend routine neurocognitive assessment and suggest that early intervention with initiation of ART before the onset of severe immunosuppression may improve outcomes in these children.
HIV infected patients receiving combination antiretroviral therapy are at higher risk of cardiovascular morbidity and have accelerated aging notably of cognitive functions. The link between cardiovascular risk factors and cognition has rarely been investigated in HIV-infected cohorts. In a large hospital-based cohort, we explored whether cardiovascular risk factors are associated with cognitive performances.
The ANRS-CO3 Aquitaine Cohort recruits patients through a hospital-based information system on HIV-1 infection in the Bordeaux University Hospital in the Aquitaine region, South Western France. Between 2007 and 2009, 403 patients participated in a sub-study and had a thorough assessment of several cognitive domains. Cognitive performances were analyzed using both the raw test scores and the presence of neurocognitive impairment (NCI), based on the most recent definition of HIV-associated neurocognitive disorders. Selected cardiovascular risk factors were type 2 diabetes, hypertension, hypercholesterolemia, smoking status and BMI.
Covariance analyses were computed to investigate the association between cardiovascular risk factors and raw cognitive test scores, adjusting for potential confounders. Logistic regression with the same covariates was used to analyse NCI as dependent variable.
Mean age was 47.3 years and 79% were male. The prevalence of cardiovascular risk factors ranged from 9.7% for diabetes to 49.6% for current smoking, and 37.7% of participants had NCI.
Lower performances in all cognitive tests were related to older age and lower education. Among cardiovascular risk factors, diabetes was significantly associated with lower performances in all cognitive tests after adjusting for potential confounders. By contrast, no such consistent associations were noted for any other cardiovascular risk factors. Diabetes prevalence did not significantly differ by NCI status (p=0.44).
In this hospital-based cohort, diabetes, but not the other cardiovascular risk factors, is associated with lower performances in all assessed cognitive domains. The mechanisms underlying our findings remain to be clarified but could involve inflammation and microcirculation.
Since the introduction of antiretroviral therapy (ART) in low-resource settings the use of laboratory monitoring for HIV infection has been the subject of great debate. This study aimed to assess the cost and cost-effectiveness of clinical plus laboratory monitoring (LAB) compared with clinical monitoring alone (CLIN).
A randomized non-inferiority trial was performed in nine rural district hospitals in Cameroon. HIV-infected, ART-naive adults with WHO stage 3-4 disease were enrolled and followed-up for 2 years. Participants were randomly assigned to CLIN (quarterly clinical monitoring) or LAB (six-monthly HIV viral loads and CD4 cell counts plus quarterly clinical monitoring) by a computer-generated list. Clinical outcomes and related-costs were calculated among patients followed-up for at least six months excluding the period unrelated to ART monitoring. Incremental cost-effectiveness ratios (ICER; US$ per life-years saved) were computed based on two HIV-RNA test costs (Abbot molecular technology, $67 and in house ANRS test, $35). One-way and multivariate sensitivity analyses were performed.
Analyses were conducted among 188 and 197 patients in the LAB and CLIN groups respectively. With the Abbott test, results per 100 individuals showed that LAB compared with CLIN increased costs by $49,800 and saved 10.6 life-years (ICER=$4678 per life-year saved). With the ANRS test, the incremental cost is $34,900 per 100 individuals, i.e. an ICER of $3276 per life-year saved. 50 000 trial Monte Carlo simulation showed ICERs (IQR) from $3536-$5136 and $2553-$3642 per life-year saved with the first and second tests respectively. Considering a cost-effectiveness threshold of $3800 (i.e. three times the Cameroonian capita gross domestic product), LAB is cost-effective when the cost of HIV-RNA plus CD4 cell count are lower than $75.
Monitoring of HIV viral load and CD4 cell count to guide switching to second-line ART is cost-effective in resource-limited settings when using less costly generic tests.
Expanding HIV testing in healthcare settings needs more economic evidence in China with concentrated HIV epidemic. This study was aimed to examine the costs and benefits of strategies to improve HIV testing and promote case finding.
Cost-effectiveness analysis based on a Markov model was applied to compare three HIV testing strategies for patients in outpatient departments: Strategy A=targeted testing based on risk behavior and symptoms, Strategy B=non-targeted testing all male adult patients with blood withdrawn for any clinical reasons, Strategy C=non-targeted testing all adult patients with blood withdrawn. Unidentified HIV prevalence, number of testing, number of newly identified cases, and related costs were derived from a controlled trial, which compared the effectiveness of strategies above in areas with concentrated epidemic in China. Long-term costs and health outcomes were derived from the literature. Incremental cost utility was evaluated from a societal perspective using a lifetime horizon. The benefit from reduced HIV transmission was not considered.
With 0.3% of the undiagnosed outpatient HIV prevalence in study areas, the cost per new HIV infection identified was $146.1, $258.4 and $350.9 under strategy A, B, C, respectively. Strategy A resulted in per-patient lifetime discounted costs of $11,349 and benefits of 6.21 quality-adjusted life-year (QALY) gained. Strategy B increased mean QALY by 0.018, with an incremental cost-effectiveness of ?5,710/QALY, and Strategy C increased mean QALY by 0.017 than strategy B, with an incremental cost-effectiveness of $6,592/QALY. Sensitivity analysis showed that even if the undiagnosed HIV prevalence was as low as 0.113%, annual implementation of strategy C would still be cost-effective, with a cost of $14,059 to save a QALY, which was just 3 times of GDP per capital in China.
In concentrated epidemic area, non-targeted HIV testing in healthcare setting is cost-effectiveness and justified for sustainable investment.
Maraviroc (MVC) is a CCR5 antagonist approved for the treatment of CCR5-tropic (R5) HIV-1. This study evaluated a once-daily (QD), dual-therapy regimen of MVC plus atazanavir/ritonavir (ATV/r) in treatment-naïve patients; 96-week outcomes are presented.
In this Phase 2b, randomized, open-label study, 121 R5 HIV-1-infected patients received either MVC 150 mg QD (n=60) or tenofovir/emtricitabine (TDF/FTC) 200/300 mg QD (n=61) with ATV/r 300/100 mg QD for 48 weeks, later extended to 96 weeks. The primary endpoint was the proportion of patients with HIV-1 RNA<50 copies/mL at Week 48.
At Week 96, the proportion of patients with HIV-1 RNA<50 and<400 copies/mL was 67.8% (40/59) and 78.0% (46/59), respectively, for MVC versus 82.0% (50/61) and 83.6% (51/61), respectively, for TDF/FTC. Protocol-defined treatment failure occurred in 5 patients (MVC: n=3; TDF/FTC: n=2). Median change from baseline in CD4+ count was similar for both arms (MVC: 269 cells/mm3; TDF/FTC: 305 cells/mm3). Median change from baseline in creatinine clearance was −5.5 mL/min for MVC and −18 mL/min for TDF/FTC. Five patients (MVC: n=4; TDF/FTC: n=1) had plasma HIV-1 RNA >500 copies/mL at failure or study discontinuation; virologic analyses detected no resistance, change in tropism or loss of susceptibility relevant to treatment in either arm. At Week 48, there was a greater reduction in immune activation on CD4+ cells in patients receiving MVC versus TDF/FTC. Markers of bone formation were significantly different between arms at both 48 and 96 weeks.
Durable virologic activity of MVC 150 mg QD+ATV/r was demonstrated through 96 weeks, with no differences between the arms in the rates of virologic failure, no resistance or change in tropism seen, and with most of the treatment difference due to low-level transient viremia. Differences between the arms in immune activation and bone markers require further investigation.
Nonhuman primate (NHP) models are needed for evaluation of proposed but unproven, and potentially dangerous strategies targeting residual virus and latent reservoirs in AIDS virus-infected subjects receiving suppressive antiretroviral drug treatment (ART), but such models have proven challenging to develop.
We treated a cohort of 6 Indian rhesus macaques with a novel three class (NRTI, PI, IN-STI) six drug (PMPA/FTC/DRV-RTV/L-870812/L-870564) ART regimen beginning at 4 weeks post-infection with SIVmac239. Peripheral blood CD4+ T cells from ART-treated animals with suppressed viremia were evaluated ex vivo for responses to SAHA, including changes in histone acetylation patterns and induction of expression of SIV. Beginning approximately 26 weeks post infection, animals received four 21 day courses of daily treatment with SAHA, with each course of SAHA separated by an approximately 3 week interval, with continuous ART throughout and longitudinal sampling of blood and lymph nodes for immunological, virological, and pharmacodynamic evaluations. Animals were euthanized and necropsied after the final SAHA dose, while still on ART, and tissues studied virologically.
The ART regimen was feasible, safe and well tolerated over one year of treatment and allowed suppression of plasma viremia to<30 copy Eq/mL. Ex vivo SAHA treatment of CD4+ T cells from ART-suppressed macaques increased histone acetylation and induced SIV expression. SAHA treatment of macaques was safe and well tolerated, and induced measurable in vivo changes in histone acetylation in CD4+ T cells but did not reproducibly impact plasma viremia. Analysis of cell associated viral DNA and RNA levels from blood and tissues is in progress and will be presented.
This study demonstrates the feasibility of developing and applying NHP models for studying AIDS virus reservoirs and eradication strategies, along with the in vivo safety of SAHA treatment at pharmacologically active doses.
The “Berlin patient” is the first patient functionally cured of HIV. He received stem cell transplantation from a homozygote CCR5-Δ32 donor. The reconstituted CD4+ T-cell population should be susceptible to infection with CXCR4-using viruses. According to gp120-V3 deep sequencing analysis of plasma-derived variants present before transplantation, the patient harbored a minority (2.9%) of viruses predicted to be CXCR4-tropic (geno2phenocoreceptor FPR 10%). It remains puzzling why these variants failed to emerge post-transplant. We hypothesize that these CXCR4-predicted variants depend on CCR5 for replication.
Patient-derived viral constructs were generated by cloning V3-sequences of the CXCR4-predicted viruses (pX1-pX7) and the dominant CCR5-predicted strain (pR5) into HXB2-ΔV3. As controls V3-sequences of HXB2 (cHXB2; CXCR4-tropic) and BaL (cBaL; CCR5-tropic) were cloned. Co-receptor preference was investigated in U-373-MAGI-cells expressing CD4+CCR5+ or CD4+CXCR4+, PBMCs from healthy donors and patient-derived post-transplant CCR5-Δ32 PBMCs.
Three pre-transplant CXCR4-predicted strains had an amino acid substitution in the V3 glycosylation-motif and one had a lysine at position 25, all associated with CXCR4-tropism. Five of the 7 viral clones were infectious. As expected cHXB2 infected CD4+CXCR4+-MAGI-cells and was inhibited by AMD-3100 (CXCR4-inhibitor) in donor PBMCs. Remarkably, the CXCR4-predicted viruses (FPR 2.7–9.3) depended on CCR5 for replication in MAGI-cells and were inhibited by maraviroc (CCR5-inhibitor) in donor PBMCs similar to pR5 and cBaL. As an ultimate proof it was shown that CXCR4-predicted strains could not replicate in the post-transplant derived CCR5-Δ32 PBMCs, whereas cHXB2 replication was observed.
The minority population of CXCR4-predicted viral strains which the patient harbored pre-transplant were fully dependent on CCR5 for replication in vitro. This could explain lack of rebound after treatment discontinuation. This provides a strong rationale for the further development of CCR5-targeted gene therapy and suggests that successful reconstitution of CCR5-depleted immune system may work, even if there is some evidence of CXCR4-predicted variants.
Although HIV-infected individuals can suppress plasma viremia to undetectable levels with antiretroviral therapy, infected cells remain in the body and can contribute to viremia when therapy is discontinued. Macaque models allow investigators to more easily characterize viral reservoirs.
Twelve male macaques were infected with RT-SHIV, an SIV virus containing HIV-1 reverse transcriptase, and monitored for plasma viremia and CD4 counts. After 10-14 weeks post-infection, 6 animals were not treated and 6 animals were treated for 17–20 weeks with 3 drugs (tenofovir, lamivudine, and efavirenz) or 4 drugs (tenofovir, lamivudine, efavirenz, and an integrase inhibitor). Viral RNA and viral DNA were measured longitudinally in the blood and at necropsy in over 20 different tissues by quantitative PCR and normalized for cellular RNA and DNA.
In untreated and treated animals, RT-SHIV DNA was highest in lymphoid and gastrointestinal tissues and very low to absent in the brain, genital tract, and kidney. The amount of viral DNA detected in multiple lymphoid tissues correlated with the level of plasma viremia 1 week post-infection. RT-SHIV RNA was abundant in the lymphoid tissues of untreated macaques with detectable viremia, but was detected variably in different regions of the gastrointestinal tract. Little or no viral RNA was detected in the tissues from animals after 17-20 weeks of therapy. There was no obvious difference in RT-SHIV RNA levels between animals treated with 3 or 4 drugs.
Our results suggest that the majority of virally-infected cells are located in lymphoid tissues with variable levels in the gastrointestinal tract. The number of infected cells in these reservoirs correlates with viremia one week after infection, suggesting that viral reservoirs are seeded within days of infection. Little viral RNA is evident in tissue after suppressive therapy with either 3 or 4 antiretroviral drugs.
The role of ongoing virus replication in HIV persistence during long-term antiretroviral therapy is unknown. Since residual replication should result in detectable evolution, we investigated the degree of sequence evolution in blood-derived and rectal tissue-derived CD4+ T cells.
Using single-genome and single-proviral sequencing techniques, we obtained 20-50 single viral genomes from pre-therapy plasma samples from 5 subjects who initiated therapy during acute infection and 3 subjects who initiated therapy during chronic infection. Pre-therapy plasma viral sequences were compared to single proviral HIV-1 genomes derived from HIV-1-infected T-cells (naïve, memory, central- and effector-memory) from peripheral blood (PB) and gut-associated lymphoid tissue (GALT) samples collected after 4-12 years of suppressive therapy. Maximum likelihood phylogenetic trees were constructed using the general time reversible model incorporating rate variation among sites. Evolutionary divergence was explored using root-to-tip analysis (Path-O-Gen).
The geometric mean infection frequency of memory and naïve CD4+ T-cells in the PB was 13- and 24-fold higher respectively in subjects treated during chronic compared to acute infection. This was also true for effector memory CD4+ T-cells from the GALT (6-fold higher). Phylogenetic analysis revealed clear evidence against any substantial evolution between the pre-therapy plasma-derived HIV RNA sequences and on-therapy intracellular HIV DNA sequences. Numerous intracellular HIV sequences identified after long-term therapy contained replication-incompetent virus. One patient had a predominant intracellular clone in both memory and effector memory T-cells containing a 380bp deletion after >9 years of therapy.
Early initiation of effective therapy results in substantially lower reservoir size in blood and gut. The lack of HIV-1 genetic evolution in the HIV-1 infected CD4+ T-cell populations after years of therapy argues against virus replication as a major cause of persistence in these cell populations. The role of replication in other tissues and cell types however remains to be defined.
HIV-1-infected individuals who control viremia to below the limit of detection without antiviral therapy have been termed elite controllers (EC). Functional attenuation of some HIV-1 proteins has been reported in EC. However, little is known about role of the HIV-1 accessory protein Vif function in EC, which enhances HIV-1 infectivity through APOBEC3G degradation. In this study, the anti-APOBEC3G function of Vif was compared between EC, chronic progressors (CP) and individuals with acute infection (AI).
Forty-nine EC, 49 CP and 44 AI were studied.
Anti-APOBEC3G activity of Vif from EC was significantly reduced compared to those from CP or AI (
Anti-APOBEC3G activity of Vif proteins derived from EC was reduced. This reduced activity was independent of presence or absence of known protective HLA alleles. Common Vif mutations in EC unlikely explain the observed reduction; rather it might be attributable to unique mutations to each EC Vif protein.
Comparison of anti-APOBEC3G activity of Vif From AI, CP and EC patients.
Each dot represents mean RLU of each patient.
Repressive epigenetic modifications have been shown to induce and maintain HIV latency; however underlying molecular mechanisms are not yet clear. We have previously demonstrated the critical role of CBF-1 (Latency-C-promoter binding factor 1) in establishing repressive chromatin structures at HIV LTR during latency establishment. The knockdown of CBF-1 results in the reactivation of latent proviruses and overexpression of CBF-1 facilitates latency establishment. Here we extend these studies to show that multiple repressive epigenetic modifications that CBF-1 induces are the result of recruitment of Polycomb Group (PcG) corepressor complex at HIV LTR by CBF-1.
Both transformed and primary T cells were infected with lentiviral vectors expressing Tat
In this study, we demonstrate that CBF-1 induces repressive chromatin structures at HIV LTR by recruiting Polycomb Group (PcG) corepressor complex at HIV LTR. The knockdown of endogenous CBF-1 results in the dissociation of PcG complex components from HIV LTR. Furthermore, knockdown of the individual components of PcG complex leads to the reactivation of latent HIV proviruses demonstrating the direct role of PcG complex in establishing HIV latency. Overall our results demonstrate that the CBF-1 induced various epigenetic modifications are the result of recruitment of Polycomb Group (PcG) corepressor complex at HIV LTR, which carry a variety of epigenetic factors that repress HIV gene expression via generating several layers of repressive epigenetic modifications.
We have established that analogous to the transformed T cell lines in primary T cells CBF-1 induced repressive chromatin structures play important role in establishing HIV latency. Additionally by recruiting PcG corepressor complex at LTR, CBF-1 not only facilitates HIV latency establishment also play critical role in maintaining and stabilizing the latent proviruses.
Virological and Immunological Studies in CONtrollers after Treatment Interruption (VISCONTI) are required to understand the benefits of an early treatment at acute HIV-1 infection on the HIV reservoir. We studied the distribution, magnitude and inducibility of the HIV reservoir in VISCONTI patients.
The prospective VISCONTI study included twelve patients controlling HIV for a median of 76[IQR:67.5–84.5] months after interruption of a 3[IQR:1.7–5.9] years long HAART initiated within 10 weeks post-infection. Circulating resting CD25-CD69-HLADR- CD4+T cell subsets were sorted as naive (TN), central-memory (TCM), transitional-memory (TTM) and effector-memory cells (TEM) for further cell-associated HIV-DNA quantification by ultrasensitive real-time-PCR, and viral inducibility by culture with anti-CD3/anti-CD28/IL-2/IL-7. Reservoir distribution was compared to the one observed in 8 untreated Elite-Controllers for whom 90% of HIV-RNA measures was undetectable (below 200 copies) over 12[9–14] years.
In the VISCONTI group, activated CD4+T cells had significantly higher HIV-DNA levels than resting ones (median 2.7[IQR:2.4–3.4] and 2[IQR:1.8–2.5] log copies/million cells, p=0.005). HIV-DNA was detected in all subsets from all patients except for 8 out of 12 TN-sorted cells, which were 10 fold less infected than all memory subsets (median TN:1.5[IQR:1.2–1.6], TCM:2.5[IQR:1.8–2.9], TTM:2.6[IQR:2.2–2.8] and TEM:2.4[IQR:2–2.8] log copies/million cells, p< 0.007). TTM was the major subset contributing to 56% of this reservoir. The same HIV reservoir characteristics were observed in Elite-Controllers in term of magnitude and distribution, except that both TCM and TTM equally contributed to the Elite-Controllers HIV reservoir. The VISCONTI HIV reservoir was inducible after TCR-stimulation in all sorted memory subsets from all patients, except in TN where no virus was recovered in 6 out of 8 patients.
In VISCONTI patients, treatment initiated at primary HIV-1 infection leads, after treatment interruption, to a low -but inducible- durable HIV reservoir distributed mainly in short-lived memory CD4+T cells that mimicks the natural distribution observed in Elite-Controllers.
A major hurdle toward curing HIV is the establishment of long-lived latent reservoirs such as the CNS. Using an SIV model of HIV infection we examined the effect of combination antiretroviral therapy (cART) on immune and inflammatory gene expression in the periphery and CNS that leads to virus downregulation.
To examine the effect of cART on acute and long-term systemic and CNS immunopathogenesis, groups of SIV-infected macaques were untreated and euthanized at 21 postinoculation (dpi) or end stage disease, or treated with cART starting at 4 or 12 dpi and euthanized at 21 or 175 dpi, respectively. RNAs for immune and inflammatory genes were quantified in the spleen and brain by non-amplification Nanostring technology or by qRT-PCR. SIV replication and viral DNA were also measured.
cART initiation at 4 or 12 dpi did not prevent SIV seeding of the brain; brain viral DNA levels were the same as in untreated animals. cART treatment initiated at 4 dpi had little effect on peripheral and CNS immune and inflammatory gene expression profiles as compared to responses mounted in untreated macaques after acute infection, as indicated by similar levels of IL17A, IL17F, and CCL5 in the periphery and IL-6, IFNß, and TNFa in the CNS.
The CNS is a latent reservoir for SIV that is seeded early after infection regardless of cART initiation at 4 or 12 dpi. The innate and adaptive immune responses are nearly as effective as early cART treatment at returning the host to peripheral and CNS immune homeostasis by 21 dpi. However, at the same time those responses likely promote the establishment of latent reservoirs by suppressing viral replication, not eliminating the reservoir.
Toll-like receptors (TLRs) are critical proteins of the innate immune system. We evaluated association of single nucleotide polymorphisms (SNPs) in 6
HIV-outcomes were assessed from birth to 1-year of age among infants from a Kenyan perinatal cohort in which HIV-infected women were enrolled during pregnancy and received short-course zidovudine. Infants were genotyped for 6 candidate and 118 haplotype-tagging polymorphisms in
Among 368 mother-infant pairs, 56 (15%) infants acquired HIV-1 by month 1 and 17 (4.6%) between 1 and 12 months. Infants with the
This study is the first to evaluate the association between
The Toll-like receptor (TLR) genes mediate the innate response to viral infections and may impact HIV-1 pathogenesis. We evaluated TLR polymorphisms for association with plasma HIV-1 RNA set-point in HIV-1 infected individuals from East and Southern Africa.
Analyses included prospective data and DNA from 500 Africans with heterosexually-acquired HIV-1 (125 incident, 375 prevalent). For incident HIV-1, set-point was defined as the median plasma HIV-1 RNA level ≥4 months after the estimated infection date. For prevalent HIV-1, set-point was the average of ≥2 consecutive plasma HIV-1 RNA measurements before ART initiation or CD4 decline to<200 cells/mm3. Genotyping was performed for 124 single nucleotide polymorphisms (SNPs) from 6 TLR and 2 TLR-associated signaling genes (TIRAP and MYD88) and 144 ancestral informative markers. These included 8 candidate SNPs previously associated with HIV-1, and 115 haplotype tagging SNPs (tagSNPs) representing common variation across TLR genes. Associations were determined using linear regression with adjustment for sex, age, and population stratification, and Bonferroni correction.
Among 492 HIV-1 infected individuals who passed quality control, the median HIV-1 set-point was 4.6 (IQR: 3.8–5.0) log10 copies/mL and did not differ between seroprevalent and seroincident participants.
These are the first associations between TLR polymorphisms and plasma HIV-1 RNA level reported among African populations.
Human cyclophilin A (CypA) encoded by peptidyl prolyl isomerase A gene (PPIA), is an important cellular co-factor for efficient human immunodeficiency virus type 1 (HIV-1) infection. In this study we investigated the effect of genetic variation in the regulatory region of PPIA on HIV-1 disease progression and CypA mRNA expression levels in HIV-1 South African cohorts.
A total of 603 black South African participants from these cohorts were genotyped for single nucleotide polymorphism (SNP) A1650G in the regulatory region of CypA using PCR-RFLP. 247 (195 HIV-1 seronegative participants [SNs] and 52 primary infected participants [SPs]) participants were from the CAPRISA acute infection (AI) 002 cohort and 356 HIV-1 chronically infected participants were from the Sinikithemba cohort. CypA mRNA expression was quantified in 30 SNs and 28 SPs from the CAPRISA AI 002 cohort by real-time RT-PCR. Lastly, we assessed the effect of SNPA1650G on viral (NL4.3) replication in PBMCs isolated from HIV-1 negative individuals.
The minor allele (G) of SNP A1650G (referred to as 1650G) was significantly associated with higher viral load (p<0.01) and lower CD4+ T cell count (p< 0.01) during primary HIV-1 infection. Interestingly, the1650G was associated with rapid CD4+ T cell decline during chronic infection (p=0.01). The 1650G was also significantly associated with higher CypA mRNA expression levels (p<0.01). PBMCs isolated from participants harboring the 1650G supported higher levels of NL4.3 replication ex vivo.
Our results suggest that higher expression levels of CypA mRNA enhance HIV-1 replication in a South African population. The results demonstrate the clinical relevance of CypA and provide additional in vivo validation of the CypA as a pertinent target for therapeutic intervention. This study supports the development of small molecule inhibitors against CypA and HIV-1 interaction.
Class I Human Leukocyte Antigens (HLA) play an important role in the adaptive immune response by presenting antigens to CD8+ T-cells. Previous studies have reported multiple HLA associations with rates of disease progression in HIV infected individuals, while few class I associations with resistance or susceptibility to HIV-1 infection have been reported.
HLA-A, -B, and -C were typed for more than 1000 women enrolled in the Pumwani sex worker cohort using a sequence-based typing method. Kaplan-Meier analysis was used to identify alleles influencing seroconversion and disease progression to AIDS (CD4+ decline to<200/mm3).
A*01 (
Understanding why these HLA class I alleles are associated with protection/susceptibility to HIV-1 acquisition and disease progression could contribute to the development of effective prophylactic and therapeutic vaccines for HIV-1.
The UNAIDS Investment Framework highlights ‘'critical enablers’‘ that influence the success of HIV programmes; and synergies with other development sectors. Although the need to address the structural drivers of HIV vulnerability is well recognised, there is a danger that these interventions are not prioritised in HIV budgets, given the limited body of evidence on their cost-effectiveness. A key challenge to economists evaluating such interventions is how to assess the wide range of costs and consequences of these comprehensive structural interventions. We use two methods to estimate the cost-effectiveness of a conditional cash transfer in Malawi to keep girls in school.
We model national costs based on full coverage of girls of secondary school age (15–19 years) living on less than US$1.25 a day. Based on trial evidence, we model a 64% reduction in HIV incidence among the target group to estimate economic benefits. We use two economic evaluation approaches, one that apportions the total costs of the intervention according to the HIV-related proportion of total economic benefit and another based on the WHO cost-effectiveness threshold.
We find that about 30% of estimated economic benefits in Malawi are related to HIV. The trial estimated a cost per infection averted (IA) of US$12,500, which we translate into a cost per DALY of US$711. Allocating total costs according to the proportion of HIV-related economic benefit reduces the cost per IA to US$3,750 and the cost per DALY to US$213. Alternatively, if the HIV programme wishes to achieve cost-effectiveness at the WHO threshold of GDP per capita (US$310) then it would achieve value for money by funding 44% of total costs.
HIV programmes are recommended to allocate funds to structural interventions based on proven effectiveness and according to the threshold at which the intervention becomes cost-effective from an HIV perspective, rather than estimating conventional cost-effectiveness ratios.
PLHA have decreased economic productivity both due to direct and indirect causes. Data from developed countries have shown that at the societal level, high costs ART are offset by increased productivity. However, these data have to be substantiated in developing countries where the expenditure for ART largely borne by individuals. We
We performed a comprehensive SES evaluation pre/post ART initiation using an ambispective cohort study design. Indian household-specific SES validated tool” (Aggarwal OP 2005), with score of <15 being indigent to >76 being affluent was used prospectively, along with clinical, ART adherence data at 6 &18 months post ART, compared using paired t-tests.
Among 140 started on ART, 118 had pre ART SES data, median f/u was 22 months, of these: 57% were women; median age 38 years; 67% married; 89 (78%) heterosexual sex as HIV risk; 40 (34%) had major OI and/or TB at presentation. Reported self-occupation was: skilled laborers 35%; 10% unskilled laborers; 23% housewives; 22% were professionals/ blue collar job;1 CSW, 1 student, 10 unemployed. The median pre-post ART CD4 cell counts were: 187& 454 (p<0.01); median body weight pre-post ART was 54 & 57 kg (p<0.01); 97% were 100% adherent. The mean Pre-ART total SES score was 37.06 (+/− 10.2); and Post-ART SES score 40.62 (±10.1 P<0.001) and these results were sustained over time, remained significant when only monthly income included on exclusion of fixed assets. Delta change in SES was directly correlated to increase in CD4 and weight, indirectly to being housewife/unemployed and having a dead spouse.
Our data show a significant impact of ART on SES in a sustained manner in a developing world setting, which has policy level implications.
This study assessed the cost-effectiveness of maternal triple-drug ART or infant nevirapine to reduce HIV transmission during breastfeeding from HIV-infected Malawian mothers, not meeting treatment guidelines for their own health. Strategies were evaluated at two entry points into the health system: (1) time of delivery, for mothers who have not accessed ART antenatally, and (2) antenatally.
Cost-effectiveness was estimated from the healthcare perspective, using nationally representative unit costs, and was evaluated in terms of HIV transmissions averted, life-years gained, quality adjusted life-years (QALYs), and disability adjusted life-years (DALYs). The risk of HIV transmission associated with the antenatal and postnatal strategies were taken from the Kesho Bora and BAN studies, respectively. Antenatal regimens were (1) AZT and (2) AZT, 3TC, lopinavir/ritonavir. The postnatal interventions were (1) maternal ART (AZT, 3TC, lopinavir/ritonavir); (2) infant NVP; and (3) no extended post-natal prophylaxis. Scenario analyses evaluated alternative drug combinations and prices, 2nd trimester initiation, and varying paediatric ART coverage.
For entry into the health system at delivery, infant nevirapine throughout breastfeeding was cost effective. With antenatal entry to the health system, antenatal AZT plus infant nevirapine during breastfeeding was cost effective. The incremental cost-effectiveness ratios for these strategies were $616.28 and $1,031 per HIV transmission averted, $31.84 and $53.33 per life-year saved, $34.25 and $57.20 per QALY and $32.47and $54.42 per DALY averted, respectively. The recommended strategies did not change in the alternative scenario analyses.
For pregnant, HIV-1 infected women presenting to the health system at delivery and not requiring ART for their own health, infant nevirapine throughout breastfeeding is a highly cost-effective method for postpartum PMTCT. For those presenting antenatally, antenatal AZT followed by infant nevirapine throughout breastfeeding (World Health Organization Option A) is the most cost-effective PMTCT strategy.
The integrase inhibitor, Dolutegravir (DTG; S/GSK1349572), has shown rapid and durable antiviral response, with a favorable tolerability profile.
In this multicenter, double-dummy-blinded, Phase III, non-inferiority study, HIV-1 infected ART-naive adults with HIV-1 RNA ≥1000 c/mL and no evidence of viral resistance were randomized 1:1 to receive DTG 50 mg QD or RAL 400 mg BID, in addition to investigator-selected backbone NRTIs of either TDF/FTC or ABC/3TC. Subjects were stratified by screening HIV-1 RNA (≤ and >100,000 c/mL) and backbone NRTI selection. The primary endpoint was proportion of subjects with HIV-1 RNA<50 c/mL through Week 48 (FDA “snapshot” algorithm).
827 subjects were randomized (DTG n=413, RAL n=414). At baseline: median age 36 years, 14% female, 15% non-white, 28% HIV-1 RNA >100,000 c/mL, 41% ABC/3TC. Proportion of subjects meeting the primary endpoint was 88% for DTG and 85% for RAL; difference (2.5%; 95% CI: −2.2% to 7.1%) met 10% non-inferiority criteria. For subjects with HIV-1 RNA >100,000 c/mL, response rate was 82% for DTG vs 75% for RAL. Secondary analyses supported non-inferiority: HIV-1 RNA<50 c/mL per-protocol (DTG 90% vs RAL 88%), treatment-related discontinuation=failure (93% vs 92%) and virologic non-response (5% vs 8%). Median CD4 increases were similar (230 cells/mm3 each). Most commonly reported (≥10%) adverse events (AEs) were nausea (DTG 14%, RAL 13%), headache (12% each), nasopharyngitis (11%, 12%) and diarrhea (11% each). Discontinuation due to AEs was 2% in each group. At virologic failure, there was no genotypic integrase or NRTI resistance in the DTG group vs 1 subject and 4 subjects, respectively, in the RAL group.
At week 48, once-daily DTG was non-inferior to twice-daily RAL in treatment-naive HIV-1 infected subjects, with no evidence of emergent resistance to DTG in virologic failure. DTG plus NRTIs could be an option for first-line HIV treatment.
AZT/3TC/NVP and TDF/3TC/NVP BID have been recommended 1st-line ART regimens in Rwanda. TDF/3TC/NVP is the least well studied of the WHO-recommended 1st-line regimens. We compared the efficacy of this regimen with AZT/3TC/NVP.
Between 2009 and 2011, we enrolled ART-naive patients. CD4 counts (cells/ul) and viral load (VL) were collected before ART and at 26 and 52 weeks. The primary endpoint was a VL <200 copies/ml by week 52 using an ITT analysis. Genotypic resistance testing (GRT) was performed on samples with VL >1000 copies/ml.
1,072 HIV+ ART-naive patients were enrolled: 521 (48.6%) received AZT/3TC/NVP (AZT), 551 (51.4%) received TDF/3TC/NVP (TDF). Median age was 37; 64% were women. Median baseline CD4 count was similar 260, VL was >100,000 copies/ml in 43% (AZT) vs. 32% (TDF) (p< 0.001). The AZT versus TDF, 5.4% vs. 3.8% transferred to others health facilities, 3.6% vs. 4.0% were lost to follow-up, and 1.9% vs. 2.7% died. 10%(AZT) vs 4%(TDF) of discontinued therapy due to adverse effects (p=0.001). The primary endpoint were: 80% (441/551) TDF and 78% (410/521) receiving AZT attained a VL < 200 copies/ml by week 52. The median CD4 count increase was 88 in the AZT and 50 in the TDF groups (p=0.034). However, in patients with baseline VL >100,000 copies/ml, 44% (133/351) in the TDF vs. 56% (170/351) in the AZT group attained the primary endpoint (p= 0.001). 11.8% (TDF) and 7.7% (AZT) underwent GRT. 58% had >=1 NNRTI-resistance mutation: most commonly Y181C (34%) and K103N (16%). 53% had >=1 NRTI-resistance mutation: most commonly M184V (48%) and K65R (29%). K65R emerged exclusively in the TDF group.
TDF/3TC/NVP was as effective as AZT/3TC/NVP at attaining <200 copies/ml by week 52. However, it was less effective in patients with higher baseline VL and was associated with a lesser CD4 count increase.
In 2009, South Africa's National AIDS Council recommended TDF access to HIV+ adults and ABC to HIV+ infants/children. We analyzed the effects of these guideline changes on NRTI-resistance mutations in ART virological failure (VF) and analyzed the effect of the cumulative second-line LPV/r use on emerging PI resistance.
HIV RT and PR sequences were obtained from plasma samples submitted for genotypic resistance testing to the Tygerberg National Health Service Laboratory between 2006 and 2011 from patients experiencing ART VF. Demographic and ART treatment data were obtained from the physicians submitting samples.
Between 2006 and 2011, 1,525 plasma samples were obtained from 1,293 patients, of whom 57% were female and 42% <15 years old. 99% of viruses were subtype C. TDF use increased from <2% of patients between 2006–2008 to 40% in 2011 and ABC use from <10% between 2006–2008 to 41% in 2011. K65R occurred in 33 (31%) of 105 TDF/3TC/EFV recipients, 7 (88%) of 8 TDF/3TC/NVP recipients, and 2 (7%) of 28 TDF/3TC/LPV/r recipients. L74V occurred in 22 (44%) of 50 ABC/3TC/EFV recipients, 2 (50%) of 4 ABC/3TC/NVP recipients, and 4 (6%) of 71 ABC/3TC/LPV/r recipients. Of 439 patients receiving an LPV/r-containing regimen, 42 (10%) had >=1 of the following major PI-resistance mutations: V32I, M46I, I47A, I50V, I54V, L76V, V82A/F, I84V, and L90M. 17 (4%) of 42 LPV/r recipients had major DRV/r resistance mutations (V32I, I50V, and L76V).
The increased use of TDF and ABC since 2009 has been associated with a markedly increased frequency of TDF-resistance (K65R) and ABC-resistance (L74V). Compared with TDF/3TC/EFV recipients, the risk of developing K65R was higher in patients with TDF/3TC/NVP VF (88% vs 31%; p=0.002) and lower in patients with TDF/3TC/LPV/r VF (7% vs 31%; p=0.008). Among 439 LPV/r recipients, 42 (10%) had LPV/r resistance and 17(4%) DRV/r cross-resistance.
Since the availability of viral load (VL) assay with a threshold of 20 copies/mL, some patients display VL values between 20 and 50 copies/mL. The aims of our study were to: (i) identify factors associated with low level viremia (LLV) in patients receiving stable suppressive antiretroviral therapy (cART); and (ii) assess virological outcome during the year following LLV detection.
Retrospective study among the 4820 patients followed in our institution fulfilling the inclusion criteria: (i) stable cART for at least 6 months; (ii) all VL <50 copies/mL; and (iii) at least 3 VL determinations during a one-year period. We compared patients with all VL <20 copies/mL (Group LLV-) and patients with at least 2 VL between 20 and 50 copies/mL (Group LLV+). “Blip Ratio” was defined as: (number of VL >50 copies/mL)/(number of VL determinations) before study inclusion.
Among the 656 patients included, 5.8% were in group LLV+. The nature of the ongoing cART did not differ between LLV- and LLV+ groups. In the multivariate analysis, only CDC clinical stage B/C at study inclusion (OR=2.9; 95% CI=1.4–5.9; P=0.003) and a higher “Blip Ratio” before study inclusion (OR=0.9; 95% CI=0.9–1.0; P=0.001) were independently associated with LLV. During the follow-up, the proportion of patients experiencing virological failure (2 consecutive VL >50 copies/mL) was not different between LLV- and LLV+ groups (4% vs 8%, respectively; P=0.32); and 40% of patients shifted from LLV+ to LLV- status.
LLV was infrequent in our series and the one-year follow-up did not evidence a higher rate of virological failure than in patients always fully-suppressed. LLV seems to be a transient phenomenon that might be driven by residual ongoing viral replication and/or viral release and/or accuracy of VL assay in lower values.
Alternatives to EFV for the treatment of HIV-infection in patients with TB are warranted. Rifampin decreases RAL exposure in healthy volunteers. We estimated the safety and efficacy of two doses of RAL and EFV in HIV-1-infected adults receiving rifampin for TB.
Multicentre, open-label, randomized, phase II trial. Antiretroviral naïve HIV-1-infected adults were randomized to receive RAL (400 or 800 mg bid) or EFV (600 mg qd), in combination with TDF and 3TC, after starting rifampin. The primary efficacy end-point was the proportion of patients with plasma HIV-RNA level<50 cp/ml at week 24 using a mITT TLVOR algorithm. The sample size was calculated to compare success rate in each arm to 70%. Safety was assessed by the report of adverse events using the ANRS grading scale.
From July 2009 to June 2011, 179 patients were screened and 155 randomized to RAL 800 (n=52), RAL 400 (n=51) and EFV (n=52). At baseline, 73% were male, mean age: 38 yrs, median HIV-RNA level: 4.9 log10 cp/ml and median CD4 cell count:140cells/mm3. At week 24 success rates were 78% [95% CI, 67–90], 76%[95% CI, 65–88] and 63% [95% CI, 49–76] in RAL800, RAL400 and EFV arms, respectively(mITT TLOVR).Plasma HIV RNA>50cp/ml was the main reason for failure and occurred in 6, 11 and 16 patients in RAL800, RAL400 and EFV, respectively. There was a trend towards more RAL, 3TC, and TDF resistance in the RAL400 than RAL800 arm. Safety of the three regimens was good with only 1, 1 and 3 grade 3/4 ALT elevations in RAL800, RAL400 and EFV arms, respectively.
At week 24, RAL800 mg bid provided the highest success rate in HIV-1-infected patients receiving a rifampin-based therapy for TB and should be considered for further evaluation.
Supply chains are essential to securing availability of life-saving commodities for patients. Unfortunately, logistics training is often absent from the professional education of staff operating the supply chain. In-service training, while traditionally used to teach supply chain skills, takes personnel away from their work, is expensive to implement and often relies heavily on external technical assistance. USAID is investing in pre-service training (PST) as a cost-effective and sustainable alternative to build a competent health workforce in supply chain management (SCM).
The Supply Chain Management System (SCMS), a project under PEPFAR administered by USAID, works with local universities to incorporate SCM courses into pharmacy and nursing programs and build the capacity of university staff to plan, teach and manage those courses. Course content is based on local context and existing supply chain systems and prepares health workers for day-to-day supply chain tasks they will encounter post-graduation.
As a result of SCMS PST initiatives, the Namibian School of Pharmacy now includes pharmaceutical management and procurement courses in its curriculum. In Zimbabwe, with support from the USAID ∣ DELIVER PROJECT, three pharmacy programs offer SCM courses, with nursing programs to follow suit. Two national universities in Rwanda and Zambia require laboratory logistics courses for laboratory science degree programs. Since the Zambia course's introduction, SCMS in-service training costs have been reduced by 62%; 80% of the 272 students trained currently work in MOH facilities.
SCMS PST activities build local capacity in health supply chain and develop a sense of ownership for local training institutions in creating a skilled SCM workforce. Future health workers graduate with a fundamental understanding of SCM, enabling them to make an immediate contribution to the health system. Savings from reduced reliance on in-service training can be reinvested into national HIV programs, allowing for expanded services and potentially improved health outcomes.
High HIV and TB rates, poor health services, overcrowding and lack of human and financial resources are common in prisons of sub-Saharan African countries, including Zambia. Severe staff shortages and the need for inmate buy-in led to recruitment and training of inmates to assist in the implementation of prison-based HIV testing and TB screening. We describe the inmate peer educator program implemented during a TB REACH-funded collaboration between the Zambia Prisons Service (ZPS) and the Centre for Infectious Disease Research in Zambia.
Eligible peers were chosen by prison officers, and received 5 days of training to complete symptom assessments, refer inmates for TB/HIV screening, collect sputum, assist with enrollment into National HIV and TB treatment programs, and provide educational outreach and counseling. Inmate drama members were also mentored to convey health messages through acting, music, and dance.
We trained 74 peer educators and 57 drama members and found that they were enthusiastic and committed to the screening program. Between November 2010 and September 2011, peers led 6,436 inmates (average of 32 inmates screened per day), through HIV testing and TB screening. Challenges included: requests for incentives, concerns over TB exposure, and difficulty maintaining adequate numbers of peers because of inmate release and transfer. Solutions included: promotions within inmate hierarchies, provision of N95 respirators, and additional training to maintain peer numbers and establish peer-to-peer mentorship.
Scale-up of prison health services in sub-Saharan Africa is an urgent priority. This inmate peer educator program was a low-cost, effective intervention that was critical to the successful implementation of this prison-based HIV and TB program. Ongoing support and ownership by ZPS and prisoners is needed to ensure long-term program sustainability.
Nevirapine (NVP) is metabolized by cytochrome P450 (CYP) 2B6. We investigated associations between single nucleotide polymorphisms (SNPs), haplotypes, and pharmacokinetics (PK) following SD NVP to prevent mother-to-child transmission (MTCT).
Protocol A5207 evaluated strategies to prevent NVP resistance following intrapartum SD NVP. At onset of labor, participants received SD NVP (200 mg) and were randomized to lamivudine/zidovudine, emtricitabine/tenofovir, or lopinavir/ritonavir (LPV/r), for 7 or 21 days. Plasma for NVP assay was obtained at post-partum day 1 and week 1, 3 and 5. Derived PK parameters included the NVP elimination constant estimated using linear mixed effect models based on natural logarithm of NVP measured between day 1 and week 3. We assayed 214 SNPs in
In A5207, 422 women in Haiti, India, Malawi, South Africa, Tanzania, and Uganda received SD NVP at onset of labor. This analysis includes 304 women (217 and 87 of African and Indian descent, respectively) with suitable NVP assay and genotype data. Among individuals of African descent,
Slow metabolizer
Pre-antiretroviral therapy for HIV (pre-ART) is increasingly recognized as an entry point to the continuum of care for HIV to facilitate timely initiation of antiretroviral therapy (ART). In this study we assess a public pre-ART program at primary health care level in rural Uganda.
This retrospective analysis used a file and register audit at all four governmental health centres (HC) providing ART and pre-ART care in Iganga district, Uganda. All HIV patients registered from February 2005 to August 2009 were included. We report rates of retention and attrition for patients in pre-ART care and facility performance indicators.
Out of 2357 registered HIV-patients, 2024 (85.9%) were still not initiated on ART by the time of our study. The overall retention in care of these patients was 19.3% after 4.6 years since introduction of the program with 1634 patients lost to follow up (LTFU) and 390 retained in care. At one HC, the retention rate was 3.6% (22 patients retained out of 603). The probability of retention in care was 0.75 [CI 0.72-0.78] at the first month, 0.46 [CI 0.42-0.49] at 6 months, 0.36 [CI 0.32-0.39] at 12 months and 0.26 [CI 0.23-0.30] at 24 months after enrollment and significantly lower than among patients who initiated ART. Of the patients alive and attending the pre-ART program, 20.8% were eligible to ART according to national guidelines but not yet initiated. Stock-outs of Cotrimoxazole occurred at all HCs. At two HCs, more than one third of the individual patient files of pre-ART patients were not available.
The pre-ART program at HC-level in the district under investigation faces challenges in high rates of LTFU early after enrollment of the patients. To assure timely initiation of ART, better quality and new strategies to retain patients in pre-ART care programs are needed.
As essential services for PMTCT are increasingly decentralized to antenatal care (ANC) sites, the consequences of shifting services from dedicated HIV care and treatment (C&T) clinics remain incompletely explored. We compared service delivery at ANC and C&T clinics in Kinshasa, DRC, a low HIV prevalence, resource-deprived setting.
In 10/2010, an enhanced standard of care was introduced at 44 ANC sites: personnel were retrained to implement the 2010 WHO PMTCT guidelines including Option A and co-located post-delivery care, and were provided with new individual-level tracking tools and supportive supervision. Women were encouraged to enroll at either of two affiliated C&T sites for continued PMTCT and HIV care but could opt to receive AZT-based prophylaxis at ANC sites when it became available alongside CD4 testing in 2011. Antiretroviral therapy was available only at C&T sites.
Of 1,233 HIV-infected women tracked between 10/2010 and 12/2011, 926 (75.1%) were newly diagnosed; 306/926 (33.0%) enrolled in C&T. Newly diagnosed women were more likely to receive CD4 testing (RR=2.2; 95% CI 1.9-2.6) and a WHO-recommended regimen (RR=1.6; 95% CI 1.4-1.8) if they enrolled in C&T than if they remained at an ANC site. Infants were more likely to receive a package of extended NVP, cotrimoxazole and DNA PCR testing at C&T than at an ANC site (RR=1.9; 95% CI 1.6–2.3). At ANC sites, 116 women received AZT-based prophylaxis and 91 received CD4 testing; 95 infants received the postnatal package.
Individual-level tracking of mothers and infants was feasible in Kinshasa and revealed that PMTCT services were delivered less effectively at sites historically focused on ANC rather than HIV C&T. While decentralization increased care access, its potential to further reduce vertical transmission cannot be fully realized without sustained training and supervisory support to ensure optimal quality of service delivery throughout the entire PMTCT cascade.
Since 1988, Nepal has evolved from a “low HIV prevalence” to a “concentrated epidemic”. In 2005, the Government initiated PMTCT services in hospitals, but they were found to be least accessible by most disadvantaged pregnant women living in remote areas. In 2009, UNICEF and Government in collaboration with CBOs introduced a community based PMTCT service integrated with MNCH, in one of the highest HIV burden districts of Nepal
The CB-PMTCT model uses the government's existing MNCH structures. Trained Volunteers provide HIV information to pregnant women and refer them for ANC services. During ANC visits pregnant women are encouraged to take HTC services. If positive, the pregnant woman is referred for further treatment and support. During the pregnancy she is provided with counselling on delivery preparedness and treatment adherence. HIV-positive women are encouraged for institutional delivery
The CB-PMTCT intervention increased the ANC coverage from 78.7% in 2009 to 82.2% in 2011. HTC uptake increased from nationally 9% in 2008 to 40% in 2011 in the intervention areas. In 2011, 82% positive pregnant women received ARV prophylaxis compared to 50% in 2008, and infant ARV coverage reached 85% from 57% in 2009. Institutional delivery increased from 7.5% to 16.1%.
Utilization of PMTCT by pregnant women dramatically increased by taking services at the community level. The volunteers and WLHIV created demand for PMTCT services and care practices. The integration of PMTCT in MNCH services is an efficient, cost effective and sustainable approach. Because of the proven efficacy of the intervention Government is keen to scale up the model in 7 districts with Global Fund funding. In order to improve the service utilization, HTC services should be decentralized up to the community level. It is also imperative to address stigma and discrimination and change social norms to ensure equitable access to services by KAP.
We hypothesized that APOBEC3G (A3G) was associated with provirus burden in resting memory CD4+ T cells, and infectivity of HIV produced from them.
Cells from antiretroviral-naïve, long-term non-progressor (LTNP, n=7) and HAART-suppressed (HS, n=11) subjects were negatively selected from PBMCs (Robo-Sep). Sorting (FACS Aria, BD) separated activated cells (CD25+, CD69+, CD38+ and HLA-DR+) from resting central memory (Tcm: CCR7+, CD45RO+), resting effector memory (Tem: CCR7-, CD45RO+, which includes resting transitional memory) and resting naïve (CCR7+, CD45RO) T cells. A3G was quantified by immunoblotting (Odyssey, Li-Cor). Provirus was quantified by
Tcm and Tem from LTNP had less provirus
Resting central and effector memory CD4+ T cells from LTNP had less provirus and higher levels of A3G protein than the same cell types from HS subjects. Infectivity of HIV reactivated
Community based organizations (CBOs) and non-governmental organizations (NGOs) have generally proven to be effective instruments for sustainable implementation of HIV prevention programs among female sex Workers (FSW). Affinity and integrity with FSWs have enabled them to expand coverage and medical service delivery by transferring responsibility to FSWs and fostering demand driven approach.
To identify and understand factors that enhance performance of programs with FSWs, Technical Support Unit (TSU) of the Karnataka State AIDS Prevention Society (KSAPS) Government of Karnataka, conducted case studies of two programs implemented by FSWs collective (CBO) and an NGO. Both programs have achieved high standards of service delivery.
Focused group discussions (FGD) were conducted with the following: board members of CBO and NGO 2 FGDs (11 participants), outreach team 2 FGDs (18 participants), FSW peer educators 2 FGDs (27 participants), Female Sex Workers 2 FGDs: (17 participants). FGDs were conducted with the KSAPS and TSU whose mandate is to provide technical assistance to the prevention programs.
Factors that contributed significantly to improved performance were that these projects are components of broader, holistic agenda that puts women at its centre, addressing women's needs, including, but not limited to, HIV prevention.
Programs generate evidence-based plans from program data. Site-based peer plans are used, which plan for individual FSW and tailor services according to individual profiles, have improved TI performance. This has helped Peer Educators to spend quality time in the field, while also improving their productivity. Supportive hand holding by Technical Support Unit has helped in diagnosing problems and its resolution.
Embedding HIV prevention within broader programs that respond to women's needs, and in which FSWs play a deciding role, increases likelihood of success of the programs. As NACP-III nears its end and the nation prepares for NACP-IV, these findings indicate promising direction for future.
The prevalence of HIV infection in Ukraine is 1.33% (adult). In Europe it is one of the highest rates. The highest concentration of-among IDUs.
Pharmacy interventions started in Ukraine in 2007. The clients of pharmacy interventions are mainly belong to IDUs, including female IDUs, and commercial sex workers (CSWs).
Currently 143 pharmacies are implementing pharmacy-based prevention projects in cooperation with 25 NGOs in 13 out of 27 regions of Ukraine.
Organization of the project activities: an NGO and a pharmacy sign a cooperation agreement.
NGO: transfers consumables to the pharmacy, trains pharmacy workers, informs the target group about the project, pays for the pharmacy services within the project and reports to the donor agency funding the project (ICF “International HIV/AIDS Alliance in Ukraine”, funded by the Global Fund).
Pharmacy: provides free of charge services to the program clients, provides the program client cards to the new clients (IDUs and FSWs), and keeps record of the clients and distributed products.
The basic package at the pharmacies includes: syringe distribution/exchange (3–5), alcohol swabs, condoms, information products, delivery of the harm reduction program client cards to the new clients. Counseling on safe behavior, referral of the clients to NGOs to receive other services (testing for HIV, STIs, counseling by specialists) are also provided.
Achievements for 2011: General year coverage: 27 435 clients, who made more than 387 000 visits to the pharmacies.11 714 new clients (IDUs, CSWs) became the participants and were issued à Participant card of the pharmacy programme. Syringes exchange organized in 32 pharmacies (out of 143)- permission for such exchange obtained from the local controlling institutions.
Approximately 20.5 % of the over-all number of new clients (57 143) was involved into the HIV prevention programs of Alliance in Ukraine through the pharmacies in 2011.
Semen, the most common vector for HIV transmission, enhances HIV infection
Seminal fluid liquefied for different amounts of time were assayed for viral enhancing activity and semenogelin levels in the absence or presence of PSA inhibitors. We also tested the effect of purified PSA on the activity of amyloids formed from chemically-synthesized semenogelin peptides.
The viral enhancing activity of semen decreases with increasing liquefaction time in a manner that parallels the degradation of semenogelins. Semenogelin degradation and loss of viral enhancing activity in semen are both prevented in the presence of a PSA inhibitor. Finally, PSA specifically cleaves and inhibits the viral enhancing activity of semenogelin fibrils.
Seminal fluid's viral enhancing activity is retained for several hours and then progressively decreases during prolonged liquefaction. We found a correlation between activity and semenogelin levels during liquefaction. These findings underscore the importance of semenogelins for the viral enhancing activity of semen. We also provide evidence that PSA directly regulates the activity of semenogelin fibrils, suggesting that these amyloids are regulated by liquefaction. As such, mechanisms to enhance the natural liquefaction process may be a useful approach to limit the ability of semen to enhance viral transmission.
Roan et al. Figure.
The second-line ART was rolled out in India in 2009 at 10 centers. Patients meeting immunologic/clinical failure criteria were evaluated by an expert panel and underwent viral load testing. Those found to have a confirmed virologic failure (VL> 5,000c/mL) were started on second-line ART (zidovudine/tenofovir/lamivudine/ lopinavir/ritonavir). We evaluated 18-month outcomes of patients started on second-line treatment.
Patients seen monthly and CD4 was performed every 6 months. VL testing was conducted 6 months after second-line ART initiation. We performed multivariable logistic regression modeling to determine factors associated with 6-month virologic suppression (<400 c/mL) and mortality at 12 months.
Between January and June 2010, 411 patients initiated second-line ART in the national programme. At treatment switch, median CD4 count was 82 cells/mm3, and median VL was 83,000 c/mL. After 6 months of second-line ART, 374 (91%) patients remained alive and on treatment, and of survivors, 79% achieved virologic suppression. At 12 and 18 months after second-line ART initiation, 362 (88%) and 306 (75%) patients remained alive and in care, respectively. Among survivors, median CD4 count increase at 18 months was 227 cells/mm3. Male sex (adjusted odds ratio (aOR) 2.8, 95% confidence interval (CI) 1.1–7.2), and baseline hemoglobin<10 g/dL (aOR 4.0, 95% CI 2.0–8.2) were associated with failure to achieve virologic suppression at 6 months. The presence of WHO clinical failure criteria at treatment switch (adjusted odds ratio (aOR) 3.6, 95% confidence interval 1.4 -9.2) was associated with increased risk for death at 12 months.
Despite advanced disease at the time of treatment switch, patients achieved good immunologic and survival outcomes after 18 months of second-line ART. Patients with clinical failure were at increased risk for death, highlighting the importance of routine immunologic and if feasible, virologic monitoring in first-line ART for earlier detection of treatment failure.
Lipoprotein Lipase (LPL) is a key enzyme in lipid metabolism, especially for plasma circulating triglycerides (TG). Genetic variants of LPL have been associated to lipid levels in healthy individuals, cardiovascular disease, obesity and diabetes. Our aim was to evaluate the influence of three polymorphisms:
52 children (28 girls and 24 boys) diagnosed with HIV-1 between 2005 and 2009, were retrospectively selected with at least one plasma TG level assessment. Also, 86 seronegative blood donors were randomly selected to estimate allelic frequencies in Argentinean population. TG levels were examined before and after one-year of HAART. Hypertriglyceridemia was defined as TG>150 mg/dL.
Allelic frequencies for HIV-1 infected children were: H-,0.21 P-, 0.53 and X: 0.05, with no significant difference to controls. After one year of HAART, median TG levels were significantly lower in P+/P− (144 mg/dL) and P−/P− (95 mg/dL) compared to P+/P+ (180 mg/dL) (p=0.03 and p= 0.0002, respectively). A gene dose-dependent effect was observed for P- allele, and its presence was associated with a 7-fold lower risk of hypertriglyceridemia. Additionally, when H-is accompanying P-, the risk diminished to 15-fold (p=0.008, OR=0.06, 95% CI=<0.01–0.63).
Our findings suggest a protective effect of LPL polymorphisms against hypertriglyceridemia in children after one-year of HAART. A long-term effect of these variants are under studied. These results could endorse a prompt nutritional or pharmacological intervention in patients lacking the P- allele.
Semen is the main carrier of sexually transmitted viruses, including HIV-1. However, semen of HIV-infected men is not merely a passive transporter of HIV-1 but, because of its richness in biologically-active compounds, including chemokines, may facilitate HIV-1 transmission. To test this hypothesis and to study HIV-1 transmission under controlled conditions, we evaluated HIV-1 loads and the cytokine milieu in semen and blood from infected men as well as, the role of these cytokines in HIV transmission to cervicovaginal tissue
We measured 21 cytokines/chemokines with a multiplex bead assay and evaluated the loads of HIV-1 in seminal and blood plasmas from 50 HIV-1-infected and 28 uninfected Indian men.
We found that semen and blood are two separate immunological compartments, in which concentrations of cytokines and loads of coinfecting herpesviruses are profoundly different. Upon HIV infection, the levels of blood and semen cytokines were significantly altered, thus facilitating cytokine network compartmentalization. HIV-1 infection changes the seminal cytokine spectrum by upregulating 16 of the 21 measured cytokines, while in blood 2 cytokines were downregulated and 7 were upregulated. One of the most prominent cytokine in semen was IL-7, which was significantly upregulated in semen of HIV-1-infected individuals. IL-7 in concentrations similar to that in semen of HIV-1-infected individuals facilitates HIV-1 infection in cervicovaginal tissue
HIV-1 infection results in an aberrant production of cytokines, changing the seminal cytokine network. The altered seminal milieu is an important determinant of HIV-1 sexual transmission: Cytokines altered by seminal infection facilitate HIV-1 transmission to cervicovaginal tissue
In the DRC, traditionally stigmatized groups such as MSM are also those most at risk of HIV infection. In the first activity of its kind in Kinshasa,
MSM were identified through risk mapping conducted at 15 sites in Kinshasa in 2010. Teams worked with groups of MSM peer educators from both the community and PSSP to provide HCT services using the rapid finger-prick method. Four mobile HCT teams, each including two laboratory technicians and a supervisor, conducted two moonlight HCT clinics per week. Attendance at the mobile HCT was carefully monitored, and observations on the strategy's successes were solicited from program staff.
MSM attendance was initially low, with only 20 MSM accessing HCT services per night; over the course of the pilot, however, this number grew to an average of 80 clients per night. A seroprevalence of 30 percent was found among MSM ages 20 to 30. Involving MSM peer counselors in this intervention played an important role in the feasibility and effectiveness of this pilot.
Rapid moonlight HIV testing for MSM led to increased service utilization because services were more easily accessible; clients were pleased to receive prompt, quality services without the perception of negative judgment. Staff reported that involving MSM peer counselors in this intervention plays an important role in these outcomes. Nighttime testing for MSM under ProVIC has since been expanded to two additional provinces in 2012.
Post-electoral unrest in Ivory Coast led to major population displacement. Looting, lack of personnel, shortage of drugs, difficult physical accessibility and insecurity challenged access to HIV services. Given the individual and public health impact of antiretroviral therapy (ART) interruption, addressing continuity of treatment was defined as a priority since up to 14,500 Ivorians under treatment could be affected.
As such, UNHCR ensured that all partners integrate HIV and AIDS related Identification, Communication and Case Management in the overall relief response. Out reach and registration of refugees and internally displaced persons were used as opportunities to link up those in need of ART. Messages delivered during gatherings encouraged PLWH to contact focal points for care and treatment.
HIV Joint assessment missions highlighted lack of ART-related inputs conflict affected areas.
In Ghana and Liberia, among 258 refugees living with HIV registered, 142 were receiving ART. 60% of PLWH receiving ART in western Ivory Coast and 83% in Abidjan had interrupted their treatment for more than 3 weeks at time of identification. Language barriers (refugees), fear for stigmatisation and lack of ART services in refugees and IDP hosting areas were main reasons for treatment interruption.
People in need of ART were referred to closest treatment centres. Cross-border arrangements were made between Liberia and Ivory Coast to overcome a difference in second line therapy. UNHCR procured portable CD4 machines in conflict-affected areas to improve treatment quality.
Early identification of cases and communication through different strategies was essential to limit treatment disruption. Cross-border coordination and contingency planning for drug procurement and distribution enables continuity of treatment. The importance of continuity of HIV-related business continuity including treatment need to be further emphasised to patients, as well as planners and relief assistance managers.
Media should be cautious when talking about HIV status of a displaced population.
Post-election crisis in 2010 in Ivory Coast caused militaries conflicts in several cities, disturbing health centres activities. NGO Aconda tried to support provision of care to PLWHA. To describe strategies implemented by Aconda to ensure continuum of care and support to PLWHA in health centers located in conflict zones.
It is a prospective study which describes activities of Aconda in health centres located at conflict area during war in Ivory Coast. Data has been collected from monthly reports.
Strategies were based on reduction of HIV activities package to an absolute minimum: supplying ARV to health centres; providing ART to patients in follow; continuing PMTCT and providing ARV prophylaxis to pregnant women. Communities based Organizations made active research of patients lost to follow and referred them to health facilities for care. Pharmacists gave systematically at least 3 monthly ARV provision to patients coming to visits. Counselling and testing Activities, biology and ART initiation were stopped.
From January to June 2011, 05 health regions: Abidjan, Duékoué, Guiglo, Bloléquin and Toulépleu were located conflict zones. We noted 45/68 centres were functional: 40 in Abidjan, 3 in Guiglo and 2 in Duékoué. We note that 11 centres have been looted; 7 completely and 4 partially. Because of non accessibility for drug's vehicle, 32 sites had failures in delivering ART. In 28 sites, less than 50% of medical staff was present. In 12 centres where caregivers were absent, the provision of ART to patients has been ensured by counsellors and data managers. In the 3 months, 17,471 PLWHA (8.7% of children) received ART and 475 pregnant women, the ART prophylaxis.
Militaries conflicts cause a dislocation of health systems. Aconda managed to maintain care and support to PLWHA in health centres in war area, by reducing to absolute minimum services.
P1093, is an ongoing, Phase 1/2 open-label PK, safety dose finding study of DTG plus optimized background regimen (children 6 wks to <18 yrs) in age defined cohorts. Selected pediatric doses will be those providing comparable PK exposure to those observed at 50mg once daily in adults with acceptable pediatric safety/tolerability.
Children ≥12 to <18 yrs were enrolled to evaluate DTG weight-based fixed doses at ~1.0 mg/kg once daily. Intensive PK evaluation, over 24 hours, following observed dose (Days 5–10) after DTG was added to stable, failing ARV regimen (or started as monotherapy, those not currently taking ARV). Background therapies were optimized immediately following completion of intensive PK. Safety, tolerability, HIV RNA assessments were performed (Week 4 and every 4 weeks throughout). Target PK exposures were AUC(0-24) range of 37–67 mg*h/mL (primary) and C24 range 0.77–2.26 mg/ml (secondary).
Ten adolescents (7 female) with mean (SD) age 14 yrs (1.89) and weight 57.3 kg (17.7) were enrolled. Nine subjects received DTG 50mg and 1 subject received DTG 35mg daily. Median Baseline (BL) CD4+ cell% and HIV-1 RNA log10 were 21.5% (IQR:18.4–26) and 4.40 log10 copies/mL (IQR:4.17–4.84), respectively. DTG demonstrated moderate intersubject PK variability; geometric mean (CV%) AUC(0-24) and C24 were 46.0 (43%) mg*h/ml and 0.90 (58%)mg/mL, respectively. HIV-1 RNA<40 c/mL was achieved (7/10 subjects (70%)) after 4 weeks of dosing; median change from BL was −2.8log10copies/mL (95% CI: −3.1, −2.6). DTG was generally well tolerated, with one Grade 3, no Grade 4 AEs, no discontinuations due to AEs, no trends in lab abnormalities.
Preliminary data suggest DTG achieved target mean AUC(0-24) and C24 in children ≥12 to <18 years. DTG plus OBT was generally well tolerated (Week 4). Data support further DTG investigation at selected dose, and younger pediatric cohort initiation.
Highly exposed seronegative (HESN) individuals have had repeated exposures to HIV-1 yet remain virus and antibody negative. We evaluated HIV-1 specific NK cell responses in men who have sex with men (MSM) defined as HESN based on high-risk sexual activities engaged in the early 1980's.
Fresh, whole blood samples from HIV-1 seropositives on ART (HIVpos), HIV-1 seronegatives (HIVneg), and HESN subjects in the Multicenter AIDS Cohort Study were cultured with overlapping 15-mer peptide pools representing consensus sequences of HIV-1 Gag, Env, and Reg (Tat, Rev, Vif, Vpu, Vpr), or peptide diluent (Tiemessen, et al., JID 2010). The cultures were analyzed for CD3-CD56+ NK cell and CD3+CD8+ T cell responses by flow cytometry.
HIV-1 peptide-specific NK cell IFN-g and TNF-a responses were present in 19/23 (83%) HIVpos, 6/13 (46%) HESN and 4/21 (19%) HIVneg (P< 0.001 and P=0.07 compared to HIVpos and HESN, respectively). The IFN-g response magnitudes ranged from 1% to 20% of all NK cells: HIVpos=5.6±1.1%, HESN=1.5±0.7%, and HIVneg=0.47±0.1% (P< 0.001 and P=0.065 compared to HIVpos and HESN, respectively). HIVpos NK cells predominately responded to Env peptides, whereas HESN NK cells responded to both Env and Reg peptides. While both HIVpos and HESN demonstrated CD8+ T-cell responses to Env and Reg, only HESN had CD8+ T-cell IFN-g reactivity to Reg in association with NK cell responses.
We show for the first time that contemporaneous blood samples from MSM defined as HESN exhibit relatively robust, innate NK cell immunity, and less common CD8+ T cell immunity, specific for HIV-1 proteins. These presumably long lasting, NK cell responses to Env and Reg peptides could be due to prior exposure to HIV-1, or to an inherited genetic resistance. In-depth assessment of these virus-specific NK cell responses could be important in designing effective HIV-1 therapeutics and vaccines.
Marked differences are seen in individuals’ susceptibility to HIV infection. Inflammation and immune activation are critical factors contributing to initial infection with HIV. The objective of this study was to define serum levels of cytokines and biomarkers of inflammation in participants in the Multicenter AIDS Cohort Study (MACS) who were at high risk for acquiring HIV infection but remained HIV seronegative (HESN), and in those at lower risk who did (LRSC), or did not (LRSN), subsequently undergo HIV seroconversion.
Serum levels of cytokines, and biomarkers for inflammation were quantified using Luminex multiplexed assays, in 188 HESN, 125 LRSC, and 197 LRSN. LRSC were tested at a study visit preceding HIV seroconversion. HESN were defined as persistently seronegative participants who were multiply-exposed (>45 anal sexual partners in the 2.5 years prior to MACS visit 2). The LRSC and LRSN groups had<20 anal partners during this same period. CCR5D32 homozygotes were excluded from the HESN group. Age-adjusted left-censored generalized gamma regression models were used to compare levels and logistic regression models for detectability across groups.
HESN men demonstrated significantly lower age-adjusted serum levels of APO-A1 (P=0.036) and higher levels of sTNFR2 (P=0.022) than LRSC. Additionally, HESN men demonstrated significantly higher serum levels of sTNFR2 (P=0.014), sCD27 (P=0.035), sCD14 (P=0.014), and IL-8 (P< 0.004), and lower serum levels of CXCL13 (P=0.034) and IFNg (P=0.22), than LRSN. The likelihood of having detectable IL-2, IL-4, and IL-12 was higher in LRSN vs HESN, but detection of these biomarkers was similar between LRSC and HESN men.
HESN men displayed lower age-adjusted serum levels of an anti-inflammatory molecule, APO-A1, and higher levels of a molecule associated with enhanced TNF responses (sTNF-R2) than did LRSC. These results are consistent with enhanced immune responsiveness and inflammation being associated with resistance to persistent infection with HIV.
Mother-to-infant transmission (MTIT) of HIV results in ~400,000 infected children each year, with a transmission rate of 35–40%. In contrast, nonhuman primate species that are naturally infected with SIV in the wild (“natural hosts”, including sooty mangabeys, SMs) rarely transmit SIV from mothers to infants. The mechanisms underlying this protection are unknown. In this study we tested the hypothesis that limited target cell availability protects SMs from MTIT.
The availability of target cells (CD4+CCR5+ and CD4+Ki67+ T-cells) for SIV infection in seven uninfected SM infants was measured by flow cytometry among naïve and memory T-cell subsets obtained from tissues (lymph nodes, spleen, tonsil, and multiple sites along the gastrointestinal tract) at necropsy.
We found that, in infant SMs, the median percentage of CD4+Ki67+ T-cells ranged from 3.2%-10.3% depending on the anatomic site analyzed, with lower values found in CD95-CD28+ naïve CD4+ T-cells. In contrast, the CD95+CD28+CCR7+ central memory and CD95+CD28+CCR7- transitional memory CD4+ T-cells displayed higher median levels of Ki67 (10.4%-49.3%). The percentage of Ki67+CD95+CD28-CCR7- effector memory CD4+ T-cells tended to be between that of the naïve cells and other memory subsets. Despite this increased level of proliferation (compared to adult SMs), CCR5+T-cells comprised<10% (and, in most cases,<5%) of the CD4+ lymphocyte population at all sites and within all T cell subsets, thus revealing restricted expression of the SIV coreceptor in infant SMs.
We have shown that MTIT is substantially less frequent in SIV-infected SMs than in HIV-infected humans. Here we demonstrate that while robust T-cell proliferation is present in infant SMs, SIV target cells (CD4+CCR5+ T-cells) are extremely limited in multiple tissues. This finding reveals an additional, previously unrecognized feature of the evolutionary adaptation to reduce the risk of MTIT in SIV-infected SMs.
Current HIV-1 integrase inhibitors target the catalytic activity, which is vital for sustained viral infection. Integrase mediates the critical step of proviral DNA integration. Efficient integration also requires a crucial cellular co-factor of HIV-1 integrase, LEDGF/p75, that tethers the viral DNA to the host chromatin. LEDGINs, small molecules designed to bind to the LEDGF/p75 interaction site on IN and to disrupt the interaction, have recently been shown to act as potent inhibitors of HIV replication in cell culture.
We have now analyzed the detailed mode of action of LEDGINs, dissecting the allosteric nature of inhibition
Biochemical evaluation of LEDGINs demonstrates in addition to a potent inhibition of the integrase-LEDGF/p75 interaction, a block of the catalytic integrase activities. This allosteric inhibition is promoted by the stabilization of the dimer-interface of IN upon LEDGIN binding most likely by affecting interaction with viral DNA. These properties of LEDGINs result in potent inhibition of HIV replication in both MT2 and PBMC cells. Moreover, LEDGINs are active across a broad range of HIV clades. LEDGINs retained full activity against a panel of viruses containing mutations that confer resistance to integrase strand transfer inhibitors. Combining LEDGINs with strand transfer inhibitors demonstrates a synergistic effect of these classes of integration inhibitors.
The biochemical data together with the lack of cross resistance and the observed synergistic effects of LEDGINs in combination with strand transfer inhibitors support the potential for combined use of LEDGINs with strand transfer inhibitors in HIV therapy.
HIV-1 integrates into the host chromosome and persists as a provirus flanked by long terminal repeats (LTR). To date, treatment regimens primarily target the virus enzymes or virus entry, but not the integrated provirus. Therefore, HAART requires lifelong treatment which is frequently accompanied by the occurrence of substantial side effects and/or the development of drug-resistant viruses. Previously, we engineered a LTR-specific recombinase (Tre-recombinase) that effectively excises integrated HIV-1 proviral DNA from infected human cell cultures, suggesting that customized enzymes might someday help to eradicate HIV-1 from the body. Therefore, we here analyzed the potential of Tre-recombinase to reverse HIV-1 infection in vivo.
We constructed an advanced lentiviral self-inactivating (SIN) vector that expresses Tre-recombinase conditionally in HIV-infected cells. We monitored Tre functionality and potential Tre-related cytopathic effects over time in tissue cultures. Moreover, the effect of Tre activity on HIV-1 infection was investigated in humanized mice.
It is shown that Tre-recombinase is efficiently delivered into cells and accurately excises HIV-1 proviral DNA from chromosomal integration sites. Apparently, prolonged overexpression of Tre-recombinase does not induce undesired cytopathic effects in the transduced cells. Finally, we demonstrate pronounced antiviral activity of Tre-recombinase in HIV-1 infected Rag2−/−?c−/− mice, which were either engrafted with Tre-transduced human CD4+ T cells or with Tre-transduced human CD34+ hematopoietic stem cells (HSC).
The presented data suggest that Tre-recombinase may be a valuable component of future antiretroviral therapies of the post HAART era that aim at virus eradication, thereby providing a cure for AIDS.
In HIV infection, the HIV-specific cytotoxic T lymphocyte (CTL) response is a critical component in controlling viral replication that ultimately fails in its ability to eradicate the virus from the body. Our primary aim is the development of a way to enhance the HIV-specific CTL response to allow long-term viral suppression or viral clearance.
In our approach, we sought to genetically manipulate human hematopoietic stem cells (HSCs) such that they differentiate into mature CTLs that will kill HIV infected cells. To perform this, we utilized molecularly cloned HIV-specific T cell receptors (TCRs) derived from CD8+ T cells. These TCRs were used to genetically transduce HSCs that were introduced into a humanized mouse and were allowed to differentiate into mature human CD8+ CTLs. Mice expressing the transgenic HIV-specific TCR and, separately, control mice were then infected with HIV-1 and functional cellular responses, viral suppression, and viral and T cell dynamics were assessed.
We found that genetic modification of human HSCs with a cloned TCR allows proper differentiation of the cells to occur
We have developed a system to closely characterize the engineering of antiviral immunity and HIV-specific CTL responses. Our results strongly suggest that stem cell based gene therapy may be a feasible approach in the treatment of chronic viral infections and provide a foundation towards the development of this type of strategy.
Treatment of HIV infection by antiretroviral therapy is effective but costly and often associated with numerous side effects. The key to a permanent treatment to chronic HIV infections is to elicit potent host resistance to viral infection and to restore immune functions. The prolonged incubation period of HIV-1 provides a good opportunity for applying non-conventional interventions such as gene therapy. For HIV gene therapy to be effective, the combination of an efficient gene transfer vector and a powerful anti-HIV strategy is necessary.
HIV resistance will be established in patients′ hematopoietic stem cells (HSCs) by lentivector (LV) transduction of (i) a microRNA to block endogenous CCR5 expression, (ii) a sequence-modified CCR5Δ32 gene to interfere with the function of native CCR5 and CXCR4 and (iii) effective multiple anti-HIV shRNAs to target viral RNAs.
We generated LVs encoding the native and a codon-optimized CCR5Δ32 gene. Ectopic expression of CCR5Δ32 in HOS-R5 and Magi-R5 cells established protection against R5-HIV-1 infection. Unexpectedly, we observed severe cytotoxicity in HOS-R5 cells and primary CD4 T cells when CCR5Δ32 was expressed. In a second approach, we generated a LV expressing an H1-promoter driven CCR5 miRNA and demonstrated marked protection against R5-HIV-1 infection. In a third approach, we generated a novel LV expressing three miRNA intronic cassettes (miR155-19a-30a) targeting HIV-1 pol, int and vpu, respectively, and demonstrated marked protection against HIV-1 infection. LV transduction of adult CD34+ HSCs had no adverse effect on hemopoiesis for dendritic cell development but T cell development appeared to be impaired based on an
We conclude that ectopic expression of CCR5Δ32 in adult CD34+ HSCs using a constitutive expression promoter is cytotoxic because the CCR5Δ32 transgene can activate uncontrollable intracellular T cell signaling. However, miRNAs targeting endogenous CCR5 and multiple HIV sequences is highly effective against HIV infection without cytotoxicity.
The transition from native to receptor-bound and eventually to the fusion-competent conformation of HIV-1 envelope glycoprotein (Env) Env requires a tightly choreographed interaction among highly conserved structures within the viral envelope glycoprotein. Recent structural information has revealed that these conformational transitions are regulated by three conserved but highly mobile layers stacked between the receptor-binding domain (gp120) and the fusion arm (gp41) of Env. We hypothesized that limiting the mobility of these layers by artificially insertion of covalent bonds will capture Env in a conformation where conserved sites are stably exposed.
We scanned residues that lie at the interface of gp120 layers (layers 1, 2 and 3) and identified targets that are predicted to form disulfide bonds if substituted with paired cysteines. We substituted a pair of residues between layers 1 and 2 with cysteine & expressed and purified the disulfide-stabilized gp120 (and gp140) antigens and analyzed them using Surface Plasmon Resonance (SPR) assay. Following in vitro analysis, we immunized rabbits with both the wild-type and disulfide-stabilized gp120 (and gp140) antigens, adjuvanted with Carbopol+MF59, and evaluated serum antibodies for binding, neutralization and epitope-specificity.
A single disulfide bond inserted between the highly conserved layers 1 and 2 led to enhanced stability of the receptor bound conformation of gp120. This was revealed by lower dissociation constant (Kd) of the mutant gp120 binding to 17b antibody, which recognized a conserved CD4 induced (CD4i)-epitope on gp120. Upon immunization in rabbits, the disulfide-stabilized gp120 (and gp140) antigens, in comparison to wild-type antigens, elicited higher level of antibodies directed to CD4-binding site and CD4i-site.
We demonstrate that structure guided stabilization of inter-layer interactions within Env can be used to improve and stabilize the exposure of conserved epitopes on the antigen and elicit improved antibody response, with the aid of a potent adjuvant, upon immunization.
The limited success of vaccines targeting the MPER of HIV-1 gp41, we hypothesize, may in part reflect the difficulty of mimicking its neutralization-competent structure (NCS). We have developed DNA-vaccine candidates meant to emulate the NCS of the MPER, and report on their ability to elicit MPER-specific, neutralizing (Nt) antibodies (Abs).
DNA vaccines encoding various gp41 ectodomain fragments, and the transmembrane region (TM) of either the platelet-derived growth factor receptor (PGDFR), or that of gp41 were engineered, transiently expressed in COS-7 cells, and tested for antigenicity. Rabbits were immunized with plasmid DNA of select candidates; sera were collected and tested for MPER reactivity.
Work with the protein products of these vaccines has shown they mimic the NCS of the MPER by several criteria, including the ability to be bound tightly by well-characterized Nt MAbs, but weakly by their non-neutralizing mutant-MAb counterparts. Immunizations with plasmids expressing the MPER tethered to the PDGFR-TM elicited MPER-specific Abs that targeted the epitope of the 2F5 NtAb. Immunization with DNA vaccines encoding the MPER and gp41 TM, elicited low-titre Abs that cross-reacted weakly with the MPER, and strongly with regions outside the MPER. Both sets of immunizations failed to produce Abs that cross-reacted with the 4E10 epitope, or neutralized pseudoviruses bearing HIV-1 Env. We found that the presence of the PGDFR-TM significantly reduced MPER-binding to 4E10 MAb, but not by 2F5. Putative models suggest that in the PDGFR-TM fusions, the 4E10 epitope faces into the lipid bilayer, thereby altering its exposure.
Our work reveals key structural features involved in promoting the NCS of the MPER. While the gp41 TM is vital in properly exposing neutralizing epitopes on the MPER, it was also found to elicit Abs against sites outside the MPER. Current work is focused on engineering the gp41 TM to optimally expose MPER epitopes.
The classical vaccine approach for combating other viruses has failed so far in dealing with HIV-1, a virus infecting a key component of immune system and with greater diversity and rapid mutation. New approaches are needed to develop a preventative vaccine.
The protease of HIV-1 is a small 99-amino acid aspartic enzyme mediating the cleavage of Gag, Gag-Pol and Nef precursor polyproteins. The process is highly specific, temporally regulated and essential for the production of infectious virions. A total of 12 proteolytic reactions are required to generate a viable virion. Therefore, a vaccine targeting the 12 protease cleavage sites(PCS) could be effective. The PCS of HIV-1 are highly conserved, direct immune responses against these sites would yield several advantages. First, the immune response could destroy the virus before its establishment in the host. Second, it could force the virus to accumulate mutations eliminating the normal function of the HIV protease. Third, restricting the immune responses to these sites can avoid distracting immune responses that often generate unwanted inflammatory responses, induce excess immune activation, and attract more targets for HIV-1 infection, establishment and spread.
We conducted a pilot study to investigate the feasibility and effectiveness of this approach. The recombinant VSV-peptides were used to immunize cynomolgus macaques and nanopackaged peptides were used to boost the immune response to the 12 PCS of SIVmac239. The controls and immunized macaques were repeatedly challenged intrarectally with an increased dosage of SIVmac239.
Antibody and T cell responses to the 12 PCS can protect macaques against higher dosage of SIVmac239 challenge (p=0.0005, R=0.8005) and the vaccine group maintains significantly higher CD4+ counts (p=0.0002) than the controls weeks after being infected. Population coverage analysis showed that this approach can be applied to >95% populations in the world.
Targeting 12 PCS of HIV-1 is a viable approach.
A strategy combining Canarypox based vaccine, ALVAC-HIV with gp120 protein has resulted in limited but significant protection from HIV infection in The RV144 vaccine efficacy trial. We have previously tested a similar strategy in the non-human primate model of HIV infection obtaining a similar efficacy to what observed in humans.
To study the contribution of innate immune responses as correlate of protection, we have performed microarray analysis in the blood collected from 6 macaques at 16, 24 48 and 72 hours after the first two immunizations with ALVAC (V1 and V2 respectively) and the 3rd immunization with ALVAC/gp120 protein (V3).
Our results show that 1) 24h after the 1st immunization with ALVAC-SIV (V1) genes with antiviral activity were up regulated (MX1, HERC-5, CD79b), but interestingly inflammatory genes where down regulated (IL1, IL18RAP, IFNR1), suggesting a reciprocal regulation of genes for IFN type I and II; 2) similar patterns of gene expression where observed earlier, at 16h from V2, suggesting the presence of "memory -innate" anti viral responses; 3) the 3rd boost with ALVAC, given simultaneously to the gp120 protein adjuvanted in Alum (V3), resulted in significant changes in the gene expression profile when compared to the first two vaccinations. At 24h from this immunization there were a far less number of IFN-related genes that were significantly up regulated (V3=3) when compared to the first and second immunization (V1= 17; V2=27), and the IFN-responses still up regulated were associated with NK cells, B cell- (CD79B) and T cell- (PKC, CD28 TCR) responses.
These results suggest that each component of vaccination could have contributed to the protection from infection underscored the ALVA/gp120 strategy. Understanding how to induce different types of immune responses that are protective for HIV may be relevant for the generation of more effective vaccine strategies.
One of the most frequent complications associated with antiretroviral therapy (ART) in HIV-tuberculosis (TB) co-infected patients is the Immune Reconstitution Inflammatory Syndrome (IRIS). While monocytes/macrophages play a major role in both HIV- and TB-infection individually, the putative contribution of monocytes to the development of TB-IRIS remains uncharacterized. We therefore applied a parallel approach of genome-wide microarray analysis and focused gene expression profiling in monocytes from Ugandan HIV-TB co-infected patients before and shortly after ART initiation, to investigate the possible functional contribution of monocytes to the development of IRIS.
Monocyte gene expression of TB-IRIS patients and non-TB-IRIS patients was analyzed by Affymetrix GeneChip® Human Gene 1.0 ST Arrays and was confirmed using the nCounter system; datasets were analyzed for overrepresented pathways using Ingenuity Pathway Analysis. Expression levels of and enzymatic activities of proteins of interest were characterized in the isolated monocyte fractions and in the plasma of IRIS patients.
Pathway analysis indicated that the complement system was significantly modulated in monocytes of TB-IRIS patients, both before initiation of therapy (baseline) and after two weeks of therapy. At baseline, expression of both C1q and C1-inhibitor was higher in TB-IRIS patients. After two weeks, the C1q mRNA levels in the majority of TB-IRIS patients increased, whereas C1-inhibitor mRNA levels decreased pronouncedly. Additionally, the inhibitory activity of C1-inhibitor was significantly higher in TB-IRIS patients compared with non-TB-IRIS patients at baseline but reduced to the level of C1-inhibitor activity in non-TB-IRIS patients at week 2.
For the first time, we provide evidence that monocytes at least partially contribute to the development of TB-IRIS, probably through the complement system response. An intriguing possibility is that the relative balance between C1q and C1-inhibitor may be affecting the inflammatory function of C1q in the complement cascade.
In 2009, the WHO recommended the transition from stavudine (d4t) containing regimens due to side effects in favor of regimens containing zidovudine (ZDV) and tenofovir (TDF). We describe the transitions from stavudine to preferred regimens and the current purchasing trends.
We analyzed 17,819 adult antiretroviral purchases between 2009 and 2011, as reported to the World Health Organization
Stavudine's total market value declined from 99.85 million USD in 2009 to 29.7 million USD in 2011. Purchases of 3-in-1 fixed dose combinations (FDC) containing d4t represented 1.2 million people who could be treated in a year (person-years) in 2009, increasing to 1.3 million person-years, before dropping to 472,000 person-years in 2011. Purchases of TDF 3-in-1 FDCs increased from 41,000 to 145,000 person-years from 2009 to 2011. Zidovudine 3-in-1 FDC purchases increased from 1.18 to 1.74 million person-years between 2009 and 2010, but then dropped in 2011. Of the 74 countries purchasing stavudine in 2009, 24% did not purchase stavudine in 2010 or 2011, while an additional 24% did not purchase stavudine in 2011. In all, 52% of countries continue to purchase stavudine products.
The median annual price of the 3-in-1 FDC containing d4t dropped from 77 to 62 USD per year from 2009 to 2011. ZDV median prices were 137 and 113 USD in 2009 and 2011, respectively. TDF 3-in-1 with emtricitabine exhibited the smallest change, from 250 USD/year in 2009 to 242 USD in 2011. The 3-in-1 with TDF and lamivudine held the lowest median price, at 174 USD in 2011.
Transition from stavudine has been slow, though some countries have exhibited rapid transition. Price remains a critical concern in transition from stavudine to ZDV or TDF regimens.
Approximately 25% of Zimbabwe's children are estimated to be orphaned, with the majority orphaned due to AIDS-related illnesses. HIV-affected households often face severe financial constraints making them unable to meet their basic needs. Financial stress can negatively impact overall mental health, life satisfaction, and wellbeing in numerous ways. Perceived control over one's economic situation and sense of self can mediate financial distress.
Over a one year period, 2000 adolescents participated in community-based savings and lending groups (SLG), and 50% also received life skills education (LSE). A quantitative survey was administered to a randomly selected sub-sample of the project population matched with a sample of adolescents from a non-intervention control ward. Surveys were administered in Shona and back translated into English. Data were entered into SPSS and analyzed using univariate and bivariate measures.
160 adolescents participated in the survey. Average age was 15-years-old with an average household size of seven members. Adolescent SLG group members (n=139) reported statistically significant higher scores on self-efficacy (p<.001), self-esteem (p<.01), and hope (p<.01) than adolescents from the control group (n=21). There were no statistically significant differences between adolescents who received SLG only compared to those who received SLG plus LSE, although most of the random sample received both interventions. While 75% of adolescents reported that caregivers decided how general household income was spent, 45% of adolescents reported caregivers decided how their SLG funds would be spent; 33%made independent decisions regarding their funds. Hope scale scores positively correlated with increased decision making power over funds.
Adolescents affected by HIV face many challenges including poverty and unknown futures. Economic empowerment may provide an option for responding to both immediate financial concerns and also building mental health resilience and outcomes for the future.
Despite growing evidence that socio-economic outcomes among HIV-infected adults show improvement after antiretroviral therapy (ART) initiation, little is known about the variation in these outcomes among a population that also includes individuals with high CD4 counts and those not enrolled in care. We examined associations between CD4 count and socio-economic outcomes among adults participating in a community-wide health campaign in a rural Ugandan parish.
A one-week community health campaign offering diagnostic and treatment services for HIV and other infectious and non-communicable diseases was conducted in May 2011. Data on campaign participants′ employment were collected, and a detailed household socio-economic survey was conducted among a random subset of participants. Multivariable regression was used to assess relationships between CD4 count and employment and educational outcomes.
2,323 adults (74% of the community) participated in the campaign. 179 adults (7.8%) tested HIV-positive and 46% were newly diagnosed. HIV-positive adult participants with CD4 >550 and 350–550 worked 4.8 and 5.3 more days during the past month than those with CD4<200 (p<0.05). No differences in work patterns were found between participants with CD4 200-350 and<200. The association was similar among those on ART and not on ART. Children's school enrollment was also associated with adults' CD4 counts. Children in households of adults with CD4 >350 had 20% higher school enrollment rates than children in households of adults with CD4<200 (p< 0.05). Finally, socio-economic outcomes of HIV-participants with high CD4 counts resembled those of HIV-negative participants.
Outcomes of HIV-positive adults with high CD4 counts are not only better than those of adults with low CD4 counts, they also resemble those of HIV-negative adults. Early initiation of ART could generate economic benefits by preventing a decline in employment and education outcomes and maintaining them at levels seen among HIV-negative peers.
Peripheral neuropathy is a well-recognised and common condition in HIV-infected adults and may be related to use of antiretroviral therapy (ART) as well as be directly caused by HIV infection. Data on the prevalence, manifestations and risk factors of neuropathy in children are limited. Only few tools are available for clinical screening for peripheral neuropathy in children. We used the neuropathy symptom score (NSS) and neuropathy disability score (NDS) to screen for peripheral neuropathy in a cohort of children on ART.
In this cross-sectional study we included 182 children aged 5-15 years attending to healthcare facilities for ART collection in rural Mopani District, South Africa. Subjective and objective assessment of neuropathy was done using the NSS respectively NDS. These scores are feasible for resource-poor and skills-limited settings and only require a reflex hammer, cotton butt, tooth pick, and cold water. A definite diagnosis of peripheral neuropathy was defined by NSS≥3 or NDS≥ 2.
Neuropathy screening was completed for 174/182 (96%) of children as 8 children did not fully cooperate. Median age was 9 years old and time on ART 2.0 years (2 months-6.4 years) with 86% on a stavudine-containing regimen. Symptoms related to neuropathy were reported by 49 children (27%) while NDS was positive for 25 children (14%). Forty-one (24%) of children fulfilled the criteria of peripheral neuropathy. Co-trimoxazole use was negatively associated with neuropathy presentation (OR 0.42, 95% CI 0.20–0.88; p=0.019) while there were tendencies for peripheral neuropathy to be associated with older age (p=0.09) and longer time on ART (p=0.06).
Peripheral neuropathy is a common condition in children collecting ART at healthcare facilities in rural Mopani District. The NSS and NDS can be used to screen for this condition in resource-poor settings.
Antiretroviral (ARV) administration to HIV positive pregnant women and neonates reduces perinatal HIV transmission to less than 2% worldwide. However, concerns have been raised about potential toxicity in some neonates following gestational ARV exposure. Precise quantification of ARV exposure by history is difficult. Quantitative meconium analysis may better reflect fetal exposure during the third and perhaps second trimesters than history alone. Therefore, we developed and validated the first liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for ARVs and metabolites in meconium.
Blank meconium (0.25g) was fortified with 16 ARVs and 4 metabolites, chosen based on prevalence of use by HIV-infected mothers in the SMARTT (Surveillance Monitoring of ART Toxicities) Study of PHACS. Samples were homogenized in methanol and subjected to solid phase extraction prior to quantification by LC-MS/MS. Tenofovir (TDF), lamivudine (3TC), emtricitabine (FTC), abacavir (ABC) and its carboxylate (CABC) and glucuronide (GABC) metabolites, nevirapine (NVP), raltegravir (RAL), saquinavir (SQV), amprenavir (AMP), darunavir (DRV), atazanavir (ATV), ritonavir (RTV), lopinavir (LPV), nelfinavir (NFV) and its bioactive hydroxyl (M8) metabolite were quantified with positive ionization; stavudine (d4T), efavirenz (EFV), zidovudine (AZT) and its glucuronide (GAZT) metabolite were quantified with negative ionization.
Chromatographic separation was achieved with gradient elution; two injections were required due to the need for both positive (35 min) and negative (18 min) ionization modes. Extraction efficiencies were greater than 60% for all analytes except GABC (30%), and TDF, 3TC, GAZT and CABC (50%). Linear calibration curves employing 1/x2 weighting ranged from 10–2500ng/g (TDF, FTC, ABC, GABC, NVP, RAL, SQV, ATV, RTV, LPV, NFV, and M8), 50–2500ng/g (3TC), 75–2500ng/g (CABC), 100–25,000ng/g (AMP, DRV, AZT, EFV) and 500–25,000ng/g (d4T, and GAZT).
We developed a selective and sensitive LC-MS/MS method to detect antiretroviral medications and metabolites in meconium, which may be useful in quantifying the
Mechanisms for increased cardiovascular risk in HIV-infected adults are incompletely understood, but heighted inflammation leading to a pro-thrombotic state has been proposed as a major contributor. In vitro platelet aggregation has been studied as a robust biological marker of coronary events and mortality.
We studied platelet aggregation in 25 HIV-infected subjects on ART with undetectable plasma HIV-1 RNA, median CD4 537 cells/mm3 (73.9%men) and 29 healthy HIV seronegative controls (44.4%men) in response to submaximal adenosine diphosphate (ADP, 0.4uM), arachidonic acid (AA, 0.15mM), or without agonist (spontaneous platelet aggregation [SPA]). The effects of one week of aspirin 81mg daily on activation markers, as measured by flow cytometry, and platelet aggregation were investigated. Two-tailed paired
Compared to controls, HIV subjects on ART had increased platelet aggregation in response to ADP (10.8% [6.5, 42.3] vs 7.6% [3.3, 10.2], p=0.02), AA (54.9% [8.7, 89.9] vs 11% [2.5, 77.6], p<0.05), and without agonist (SPA) 7.5% [4.7, 11.4] vs 5% [2.9, 9.1], p<0.05). Following aspirin therapy, percent aggregation in response to ADP and AA decreased significantly (p< 0.01 for each comparison). Compared to controls, HIV subjects on ART had increased %HLADR+CD38+CD4+ Tcells(8.3%[4.1, 12.1] vs 3.9%[1.4, 6.1], p=0.01) and %HLADR+CD38+ CD8+ Tcells (0.46%[0.21, 0.58] vs 0.21%[0.18, 0.34], p=0.01). Following aspirin therapy there was a significant decrease in % HLADR+CD38+ CD4+ Tcells, p<0.01 and a trend in decreased % HLADR+CD38+ CD8+ Tcells, p=0.08, in HIV subjects but no significant change was noted in controls.
Platelet activity is increased in HIV-infected subjects on suppressive ART, which may contribute to their heightened cardiovascular risk. One week of 81mg of aspirin attenuated platelet activation and immune activation in HIV-infected subjects on suppressive ART.
Unsafe sex is the major cause of transmission of HIV in developing countries like India. Condom use is, therefore, seen as the most effective instrument against the transmission of HIV. However, use of condoms in commercial sex is still nowhere universal. Part of the reason could lie in the evidence that sex workers lose income when they enforce the use of condoms among their clients. This paper re-examines how condom use affect the price for commercial sex.
A survey of 5498 female sex workers from four high HIV prevalence states of India was analyzed using a simultaneous equations model using three-stage least squares method, to identify the causality between condom use and price for commercial sex.
The data indicated that 77 percent of the sex workers are consistent condom user. The analysis shows that there both price of sex and condom use influences each other. Also, results indicate that all types of sex workers (Road, Brothel, Hotel and Bar) except brothel-based workers received 35 percent higher earnings when they have protected sex than unprotected sex. Brothel based sex workers earned 24 percent less when they enforced the use of condom. The other exogenous factors that affect the price are negotiating power, level of education, type of sex workers (other sources of income, number of client visit per day, contract under pimp etc.). Hotel and bar based sex worker getting comparatively higher price.
The market for sex work has changed in favour of sex workers and condom use generally no longer has a negative premium for most of the sex workers. It is, therefore, possible to step up the intensity of behavioral intervention for enhancing condom use among the sex workers without worrying about its impact on their earnings.
Given recent international commitments to the prevention of HIV among young people, we sought to assess the national response to prevent new infections among 10-24 year olds in countries with high HIV prevalence.
For 20 countries with generalized HIV epidemics, current national strategic plans and progress reports were reviewed to assess the national response in terms of: planning, measured by the inclusion of youth-specific strategies within national AIDS plans; and implementation, or the extent to which prevention activities reach the intended audience.
All 20 countries include youth-specific strategies in their current national AIDS plan, and school-based HIV prevention was the youth prevention strategy most often included. Governments of all 20 countries report that school-based HIV education is reaching the majority of people in need, and included in primary, secondary, and teacher training curricula. The proportion of schools providing life-skills based HIV education varies from 2% to 100%. Programmes for out-of-school youth, behaviour change communication, and condom promotion were commonly included in national strategies, however, their content, quality and coverage were generally not reported. In UNGASS country progress reports, few countries disaggregate by age and sex the UNGASS indicators relevant to young people, or report comparable statistics over time.
In 20 high-prevalence countries, HIV prevention among young people is considered a priority in national plans, and the most widely implemented intervention for youth is school-based prevention. Monitoring youth-focused programmes should be improved to assess coverage, quality and delivery through comparable data over time. At a minimum, reporting UNGASS indicators by age and sex will improve the usefulness of national progress reports in tracking prevention efforts for youth. As a priority, systems are needed to report planning and programmes to promote condoms and HIV testing among young people, given the scarcity of data at the national level.
The incidence of HPV-associated lesions is higher in HIV-infected than in HIV-uninfected individuals. Oral and anogenital mucosal epithelia of HIV/AIDS-positive individuals contain infiltrating HIV-infected immune cells that express viral tat and gp120, and proinflammatory cytokines TNF-a and IFN-g. These proteins may disrupt tight junctions (TJ) of mucosal epithelium, facilitating HPV penetration. Our aims were to investigate how HIV-associated disruption of mucosal epithelium promotes HPV infection.
Polarized oral and cervical epithelial cells and tissue explants from HIV-uninfected individuals were treated with recombinant HIV-1 tat, gp120, TNF-a and IFN-g independently and together. The cells and tissue explants were exposed to HPV 16 pseudovirions labeled with fluorescent dyes. Paracellular HPV penetration through disrupted epithelium was evaluated by confocal microscopy.
Treatment of oral and cervical epithelial cells with tat, gp120, TNF-a, or IFN-g independently for 24 h did not induce significant disruption of TJ but the combination of all 4 proteins caused disruption of TJ in about 90% of cells. Prolonged treatment of cells with these proteins independently for 5 days also induced substantial disruption of TJ. Epithelial disruption mediated by these proteins facilitated paracellular PsV passage through polarized cells-30-45% of apically applied virions were detected in the basolateral compartment. Treatment of oral epithelial explants with HIV tat, gp120, TNF-a, and IFN-g led to disruption of epithelial TJ with paracellular HPV penetration into epithelium and entry of HPV into basal cells.
Our data indicate that HIV tat, gp120, TNF-a, and/or IFN-g in the epithelial microenvironment disrupt epithelial TJ in a time-dependent manner. Alone or together, they potentiate HPV penetration into basal epithelial cells where HPV infection is initiated. Interference with the effects of these proteins may be useful to reduce the risk of HPV infection when applied topically to at-risk genital mucosal epithelium prior to sexual exposure.
TT virus is genetically variable and widespread among the general population without apparent pathological effects. It has been detected in the peripheral blood and a variety of body fluids such as semen and cervical fluids. Studies from North America and Europe have reported that TTV infection is more prevalent in HIV-1-infected individuals than in healthy control, but its prognostic significance has been controversial. No study has been reported for TTV co-infection in HIV-1-infected Asian people. This study was aimed to demonstrate prevalence, genotypic variability and prognostic significance of TTV coinfection in northern Thailand HIV cohort.
A total of 756 HIV-1-infected adults were enrolled in the Lampang HIV cohort in northern Thailand, and their blood samples were collected. HIV-1 plasma viral loads and CD4 counts were measured at enrollment, and their clinical courses had been monitored. 40 healthy Japanese adults were also tested as controls. DNA of 5 TTV genogroups (G1 to G5) was detected by PCR using genogroup-specific primer pairs.
753 (99.9%) of 754 HIV-1-infected adults were infected with any genogroup of TTV: G1 (700/754, 93%), G2 (0/754, 0%), G3a (740/754, 98%), G3b (732/754, 97%), G4 (601/754, 80%) and G5 (562/754, 75%). On the other hand, 38 (95%) of 40 healthy Japanese adults were infected with any TTV genogroup: G1 (63%), G2 (3%), G3a (70%), G3b (68%), G4 (63%) and G5 (20%). Infection with more than one genogroup is more common in HIV-1-infected adults (99%) than in controls (68%). 62% HIV-1-infected and only 13% healthy adults were infected with all of G1, G3a, G3b, G4 and G5.
TTV infection is highly prevalent in both HIV-1-infected and uninfected Asian adults; however, mixed genogroups were much more common in the former. Correlations between certain TTV genogroup or TTV loads and CD4 counts or HIV-1 load will be determined.
Secreted proteins play an important role in intercellular interactions, especially between cells of the immune system. Infection of human lymphoid tissues by HIV-1 may alter secretion patterns. Here, we describe a novel, easy, inexpensive, and versatile method, which allows the identification and isolation of a living cell actually secreting any protein of interest.
We coupled carboxylated magnetic iron oxyde nanoparticles (IONPs) or quantum dots to polyclonal goat anti-mouse IgG(H+L) antibodies (GAM) and used them as a platform to bind a cell-specific antibody, conferring cell targeting, and an antibody specific for a secreted protein. Purified complexed IONPs form an affinity matrix on the cell surface and capture the cell-secreted product, which can then be detected on the surface of the secreting cell by another secreted-protein-specific labeled antibody.
GAM-IONPs complexed with anti-CD45 antibody and either anti-IL-2 or anti-IFNgamma. This capture assay was as efficient as a commercial assay and intracellular cytokine staining in identifying cells that secrete IL-2 and IFNg. Respectively, these assays detected IL-2 secretion on 16.9±4%, 14.7±1.8% and 16.3±1.4% of T cells (N=6, P<0.88) in activated PBMC cultures. Similarly, IFNg secretion was detected equally by these three assays. Quantum dots can be used in place of IONPs and allowed similar detection of cytokines as well as fluorescent targeting of cells. The capture assay also detected the secretion of MIP-1a, MIP-1b and RANTES for which no commercial assays are available, and levels were comparable to intracellular staining. This method is amenable to multiplexing and several cytokines may be detected on the same cells.
We have developed a versatile secretion capture assay, which allows the detection of any secreted protein of interest, and does not compromise cell viability. This method is useful in understanding modifications to cytokine secretion induced by viruses such as HIV-1.
Although prices for first-line drugs fell during the last years, affordability remains a critical issue in developing countries. Failure to first-line drug treatment increases the number of people living with HIV/AIDS (PLWHA) that require second-line treatment. The aim of this study is to analyze the global antiretroviral prices between 2003 and 2011 and associated factors, particularly differences between innovator/generic drugs, and the innovator prices evolution as the patents get closer to expiration.
Retrospective transactional data for antiretroviral procurements between 2003 and 2011 from the Global Price Reporting Mechanism (GPRM/WHO) was used. For every transaction, price, quantity, supplier and drug characteristics were recorded. Descriptive analyses were based on daily dose prices and quantities (PDD, QDD). Logarithm of PDD was regressed on transaction year, log(QDD), therapeutic class, years before patent expiration, formulation, and innovator/generic indicator.
Mean PDD decreased from US$1.1 in 2003 to US$0.48 in 2011. In this period, mean PDD of generic drugs fell about 40.8%, while mean PDD of innovator drugs increased about 22%. Innovator firms sold 7% of purchased quantity (20% of total expenditure in antiretrovirals). Econometric analysis demonstrates price sensitivity to therapeutic classes, formulation, and quantities. Innovator prices are 35% (p< 0.001) higher than generics. In addition, the estimation suggests that innovator prices increase when initial patents arrive to expiration: 6.3% (p< 0.001) per year. Although first-line prices are in average 131% (p< 0.001) lower than second-line, innovator prices for first-line drugs are 33% (p< 0.001) higher than generic drugs.
Procurement policies should pay attention not only to higher prices practiced by innovator firms for drugs recommended in first-line regimens, but also to drugs with patents arriving to expiration whose prices are higher than drugs with patents expiring later. Where patent protection is concerned, high prices may impair the delivery of higher quality treatment in developing countries.
In Kenya, about 47,000 (9%) of the 520,000 people on antiretroviral therapy (ART) are children. Use of virologic measurement to monitor response to ART is not routinely provided due to cost and accessibility; however, viral load (VL) testing is being offered in limited settings to confirm treatment failure.
We analysed demographic and clinical data from pediatric patients (<15 years) meeting national criteria for treatment failure who submitted samples for VL testing from 68 clinical sites in Nyanza Province. Immunologic criteria included CD4 count or percent non-response (rise<50 cells/mm3 or persistent CD4<100 cells/mm3) or CD4 fall >50% of peak, to baseline, or below threshold value for age. Clinical criteria included new/recurrent WHO stage 3/4 conditions, poor growth, or recurrent infections. We evaluated the positive predictive value (PPV) of using immunologic and clinical criteria to diagnose virologic failure (VL>1,000 copies/ml, after>6 months of ART).
Between Sept 2008-June 2011 336 pediatric samples were tested; patient median age 10 years (range 1–14) and 143 (43%) were female. 94% were on a first-line nevirapine/efavirenez-based regimen, median duration on ART was 2.6 years, 213 (63%) patients met >1 immunologic criteria, 51 (15%) >1 clinical criteria, and 71 (22%) met both. Despite 99% of clinicians reporting satisfactory patient ART adherence, 287 (85%) samples indicated virologic failure. The PPV of immunologic criteria was 87% (95% Confidence Intervals [CI] 84%–90%) compared to 62% PPV (95% CI 50%–73%) for clinical criteria. The PPV of both criteria present was 96% (95% CI 88%–98%).
Virologic failure was confirmed in 85% of pediatric ART patients with suspected treatment failure. Immunologic criteria in combination with clinical criteria were more useful in correctly identifying patients requiring regimen changes and reduced the number of false positives compared to using clinical criteria or immunologic alone.
Highly active antiretroviral therapy (HAART) among HIV-infected women during pregnancy and breastfeeding reduces the risk of HIV transmission. Maternal HIV viral load (VL) is an important predictor of transmission risk, and VL monitoring during pregnancy could be useful to optimize clinical management. However, routine VL monitoring is not widely available in Kenya. We assessed routine VL monitoring for pregnant HIV-infected women on HAART to determine the proportion of women who were not reaching treatment and prophylaxis goals
We initiated a pilot in September 2008 in Nyanza at 66 clinical sites offering VL testing for patients identified as meeting clinical and immunologic criteria for treatment failure or who were pregnant. Frequencies, proportions, and interquartile ranges (IQR) for demographic and clinical data were calculated. Bivariate analysis examined potential correlates of detectable VL (>400 copies)
Between 2008 and June 2011, pregnant women, without evidence of clinical or immunological failure, constituted 683 (8%) of 8073 patient samples submitted for VL testing. Median age was 29 years (IQR 26–33) and median baseline CD4 count was 190cells/ mm3 (IQR 102–279). The median duration of HAART was 2 years (IQR 1–3) and 99% were on a first-line regimen (zidovudine/stavudine+lamivudine+nevirapine/efavirenz). 161 (24%) of samples yielded a detectable VL. Women with a baseline CD4<200 were more likely to have a detectable VL (OR=1.79, 95% CI 1.24–2.57) compared to those with a CD4>200. Age and duration of HAART were not associated with a detectable VL
One in four pregnant women on HAART, without current indications of treatment failure, had a detectable VL. Pregnant women, especially those with low baseline CD4 counts, should receive enhanced ART adherence counseling and close clinical monitoring. Routine use of VL testing during pregnancy can identify women requiring additional intervention to maximize care and prevention outcomes.
The content and views in this abstract are solely the responsibility of the authors and do not necessarily represent the official views of the affiliated organizations, United States Government, or Government of Kenya.
Transition to CD4 monitoring for pre-ART assessment and viral load (VL) monitoring for ART response assessment is planned in Malawi. We assessed how often clinicians ordered these tests as compared to current guidelines, and whether the samples were successfully and promptly processed.
We conducted a retrospective review from electronic medical records of all patients seen from August 2010 through July 2011, in two urban HIV-care clinics that utilize 6-monthly CD4 monitoring regardless of ART status. We calculated the percentage of patients on whom clinicians ordered CD4 or VL analysis for routine care, using blood-draw as a proxy for ordering. For all CD4 and VL samples sent to the adjacent Ministry of Health central laboratory, we determined rates of successful lab-processing, and mean time to returned results.
Of 22,488 patients seen, 8147 (35%) had at least one blood draw for CD4 count. Of all CD4 samples sent, 8061/8754 (92%) samples were returned as successfully processed. Of those, mean time to processing was 1.6 days (s.d 1.5) but mean time to results being available to clinician was 9.7 days (s.d. 3.5). Regarding VL, 189 patients (1% of the 18,330 on ART) had a blood draw and only 118/213 (55%) samples were returned as successfully processed. Mean processing time was 39.9 days (s.d. 24.6); mean time to results being available to clinician was 43.9 days (s.d. 28.8). During the one-year evaluated, there were multiple lapses in processing VL samples for up to 2 months; no samples were processed after May 2011.
Clinicians grossly underutilize CD4 and VL as diagnostic and monitoring tools. Laboratory processing failures and turnaround times are unacceptably high, especially for viral load analysis. Alternative processing techniques or results reporting strategies, such as point-of-care devices or SMS result reporting, may offer considerable improvement before successfully adopting these tests as standard-of-care.
Treatment-eligible HIV-positive pregnant women have the highest risk (>75%) of transmitting HIV to their infants, but only a small proportion of women are being initiated on antiretroviral therapy (ART) during pregnancy in Zimbabwe, partially due to limited access to CD4 testing to determine treatment eligibility. We assessed whether introducing point-of-care (POC) CD4 machines at 43 high-volume, Elizabeth Glaser Pediatric AIDS Foundation supported, PMTCT sites in Zimbabwe increased the proportion of HIV-positive pregnant women assessed for ART eligibility and subsequently initiated on ART.
A quasi-experimental before and after study design was conducted, with 43 high-volume PMTCT sites selected based on number of HIV-positive pregnant women seen. POC CD4 machines were deployed to all 43 sites in June 2011 following health worker trainings on usage of machines and tools (registers, summary sheets). Data were collected before (April–June 2011) and after (July–September 2011) deployment of POC CD4 machines (intervention). Data were analyzed using SPSS v15.0. Differences between proportions were tested using Wilcoxon signed rank test.
Before introduction of the POC machines, 617 (51%) of 1,210 HIV-positive pregnant women received a CD4 test at the 43 sites. After the machines were introduced, 890 (81%) of 1,100 women received a CD4 test. There was a significance difference between the proportion of women tested for CD4 count before and after the intervention (P=0.023) and between the proportion initiated on ART before and after the introduction of the CD4 machines (9% [104] before versus 25% [276] after; (P=0.001).
Deployment of POC CD4 machines was associated with increased CD4 testing and ART initiation for HIV-positive pregnant women at the 43 intervention sites. Based on these early results, expansion of POC CD4 machines to all high volume PMTCT sites in Zimbabwe is recommended to increase access to ART eligibility towards elimination of new HIV infections in children by 2015.
Evidence has emerged linking the lack of readily available accurate information of the patent status of key ARVs with high prices in some countries. Despite their similar socio-economic circumstances, it shows that countries often pay significantly different prices for the same ARVs. Greater cost savings can be achieved if organizations and officials involved in the national procurement have more transparent information regarding the patent status of key ARVs.
UNDP has co-sponsored the development of a methodology written by Barbara Milani and Cecilia Oh that can be used by officials involved in procuring ARVs and other essential medicines in low and middle income countries to determine their patent status and ascertain whether and when generics can be freely imported into their country.
The methodology was preliminarily tested in Canada, China, India, and South Africa in 2006 and proved to be effective in determining the patent status of ARVs. An updated and expanded version of the methodology is being finalized and will be launched before the AIDS Conference in July 2012.
In light of the decrease in AIDS funding and growing need for countries to lower treatment cost, the methodology can be used by officials and organizations involved in procurement to ensure that generic ARVs are imported legally, and by low and middle income countries to facilitate the use of other flexibilities, where necessary, if valid patents for key ARVs are hampering the procurement of more affordable equivalents of comparable quality.
Voluntary licensing has gained increased attention as a potential policy measure to improve access to generic versions of widely-patented antiretroviral (ARV) medicines in developing countries, particularly in relation to the Medicines Patent Pool, an international voluntary licensing mechanism. This study offers a description of “baseline” voluntary licensing practices before UNITAID's decision to establish the Pool in 2009, analyzes the evolution of licensing practices since then, and highlights the types of license provisions with the most important implications for public health.
We compiled data on all publicly-announced voluntary licenses between major ARV patent-holders and generic firms, and analyzed various provisions covering: geographic scope for production and sales; degree of competition enabled for end products and active pharmaceutical ingredients; royalty rates; freedom to co-formulate into fixed-dose-combinations and pediatric formulations; technology transfer; regulatory data; and various other provisions with public health relevance.
There is wide variation in voluntary licensing practices, with geographic scope ranging from one to 112 countries; number of licensees ranging from one to unlimited; royalty rates ranging from 0% to 5% of the generic price; freedom to co-formulate ranging from none to unlimited; and access to technology transfer and regulatory data ranging from minimal to extensive. Other terms and conditions may have important public health impact, but the lack of transparency on the text of licenses limits the scope of the analysis.
Voluntary licenses offer one potential route to improving access to low-cost, generic ARVs in a predictable, sustainable manner. However, attention must be paid to license provisions that can limit access to generic medicines, restrict competition, create onerous burdens, or otherwise reduce the public health benefits of licensing. Increased transparency in licensing practices, critical analyses of license provisions, and improved license terms are necessary to maximize the potential public health benefit of this policy tool.
In Brazil the number of patents and patent applications has increased, making it difficult to place generic drugs in the market. At the same time, it is fundamental that the pharmaceutical companies-either innovators or generic-register their drugs, formulations or combinations in the National Agency Sanitary Surveillance-ANVISA (acronym in Portuguese) so that the general population to access is their sanitary registration.
This study was conducted between April 2010 and April 2011 and it was funded by the Brazilian Ministry of Health. The research resulted in two methodological outcomes: 1) an ARV patent and sanitary registration database; 2) research methodology aimed at overcoming one key difficulty faced in Brazil in this area of investigation, which is that patents are not automatically linked to their corresponding final products. The methodology was designed for Brazil, but it may be adapted to other countries. Regarding to sanitary registration of ARVs, ANVISA provides an online database of granted and not granted sanitary registrations.
Patents and registration for 23 single-drug medicines and 8 combinations were reviewed. The study identified 96 patents or patent applications related to these drugs and combinations, which account for more than 3 patents/drug. One study result is that the granting of a secondary patent delays the entry of generic medication in the market. In the case of darunavir, the first patent will expire in 2016, but if the last patent application is granted (for Tibotec), the patent applicant, will benefit from a monopoly until 2025.
The results of the research led to the following policy recommendations: 1: Wide application of the Bolar Exception and rigid standards for patent examinations; 2: Incentives for pre and post-grant oppositions; 3: A possible solution for the non-transparent nature of the patent system in Brazil is the compulsory publication of granted patents.
Access to HIV programmes for refugees and internally displaced persons (IDPs) is a human rights issue and a public health priority for affected and host populations. Primary source of funding for HIV programmes for many countries is the Global Fund. This article analyses the current HIV National Strategic Plans (NSPs) and Global Fund approved proposals from rounds 1–8 for countries in Africa hosting populations with refugees and/or IDPs to document their inclusion.
The review was limited to countries in Africa as they constitute the highest caseload of refugees and IDPs affected by HIV. Only countries with refugee and/or IDP populations of =10,000 persons were included. NSPs were retrieved from primary and secondary sources while approved Global Fund proposals were obtained from the organisation's website.
Majority of countries did not mention IDPs (57%) compared with 48% for refugees in their HIV NSPs. A minority (21–29%) of HIV NSPs referenced and included activities for refugees and IDPs. The majority of countries with =10,000 refugees and IDPs did not include these groups in their approved HIV proposals (61%–83%).
Besides legal obligations, Governments have a public health imperative to include these groups in NSPs and funding proposals. Governments may wish to add a component for refugees that is additional to the needs for their own citizens. The inclusion of forcibly displaced persons in funding proposals may have positive direct effects for host populations as international and UN agencies often have strong logistical capabilities that could benefit both populations. Given recent developments in the Global Fund and HIV funding in general, the HIV funding situation for forcibly displaced persons may worsen. It is essential for their inclusion to occur if we are to reach the stated goal of universal access and the Millennium Development Goals.
The President's Emergency fund for AIDS Relief (PEPFAR) is the largest donor in Southern Africa, spending over US $6 billion since 2004 and supporting ARV treatment for 1.5 million people. However, since the introduction of PEPFAR conflicting narratives have appeared regularly. These conflicting narratives often tend to differently describe and interpret the content, intent, impact and outcome of PEPFAR policies, particularly with regards to prostitution.
This analysis utilized a competing narrative approach to identify recurrent and conflicting narratives relating to PEPFAR and its implementation among organizations and populations in southern Africa. A competing narrative methodology allows the identification and comparison of differing interpretations of phenomena. Here narratives found in published, ethnographic and interview sources from sex work and public policy communities in Southern Africa and elsewhere were analyzed and similarities and differences in these communities' interpretations of the PEPFAR policy compared.
The presentation and discussion of PEPFAR's anti-prostitution clause vary widely, and have varied over time. Sex worker communities and organizations have spoken of PEPFAR's "dark ripple effect"-the incremental phase-out of sex worker-accessed services, the increasing isolation of sex workers, and fears about disseminating and sharing information about sex work programs and funding. In contrast, public policy communities have been slow to acknowledge, publicly, the policy's suggested failures, or to provide clarifying guidance or response to proposed shortcomings.
Among the two often-opposing communities, conflicting narratives about PEPFAR and its implementation were found. Evident were narratives of PEPFAR's suggested harms and the lack of response to it. This analysis builds on earlier work to suggest that fostering improved dialogue between sex work and US funding communities would help to better broad understandings of PEPFAR's impacts, while potentially translating to improved HIV service access for sex workers.
The President′s Emergency Plan for AIDS Relief (PEPFAR) in its second phase (2009–14) has an increased emphasis on promoting the sustainability of the HIV/AIDS response. Such sustainability is related to greater country ownership, local capacity, and strengthened health systems. Investments promoting sustainability can compete for resources with the rapid delivery of services. There is need to measure the costs associated with an increased emphasis on sustainability.
The USAID HPI Costing Task Order evaluated the cost of PEPFAR support to service delivery and sustainability for the PMTCT program in Kenya. Data were analyzed from two implementing partners: FHI360 and the Elizabeth Glaser Pediatric AIDS Foundation. Data sources included implementation and financial records as well as interviews with partner staff. From this data, unit costs of PEPFAR support per mother receiving ARV-based prophylaxis and per infection averted were calculated for the study period, 2005–2010.
The average unit cost across EGPAF and FHI360 of PEPFAR support (excluding commodity costs) per HIV-positive mother provided antiretroviral-based prophylaxis declined by 52% from $567 to $271. PEPFAR support for commodities and supply chain added $48 to the latter cost in 2010. The unit cost of PEPFAR support per averted mother-to-child infection declined by 66% from $7,117 to $2,440, excluding commodity costs. The sustainability-related proportion of the unit cost of support to PMTCT increased from 12 to 33% over the study period. Investment into sustainability has grown in proportion and complexity while overall unit costs of support have declined.
Investing in sustainability did not inhibit the expansion of PMTCT services. Such investments build long-term viability and enhance the prospect of a transition from reliance on donor support. We do not see a trade-off between scaling-up services and investing in sustainability. Rather, investment in sustainability is critical to service delivery scale-up and an effective, affordable use of PEPFAR resources.
Of late, donors adjusting their funding policies and companies and agencies working on treatment access initiatives have included World Bank set economic criteria in their decision making. What has been the impact of these new parameters in HIV funding and treatment access in so-called "middle income" countries?
The analysis looks at 1) Changes in Global Fund funding criteria; 2) Changes in price discounts by MNCs on ARVs and 3) The coverage of the Medicines Patent Pool licence announced in 2011. The situations of India, China, Argentina, Botswana and Namibia are examined in particular.
The study finds that in all three cases, economic criteria are now playing a key role in determining the level and extent of assistance in relation to funding and treatment access. While the Global Fund funding criteria attempts to balance the economic status of a developing country with its disease burden, in the other two situations, the use of economic criteria appears motivated only by private interests in profits.
The use of GDP to determine access to HIV funding and treatment has gained acceptance in part because of the impression that the "middle income" status of a country indicates the ability of the country or its people to fund HIV programmes or HIV treatment. The GDP does not however reflect actual situations in country or the fact that several HIV programmes with marginalized communities are only in existence because of neutral, evidence based funding from the Global Fund. Such an approach undermines the very basis of Universal Access and is likely to be counterproductive both in the short term (with countries hesitating from adopting evidence of treatment as prevention into policies) and in the long term (as HIV programmes in these countries shrink or are shaped by national priorities and prejudices including in relation to marginalized groups).
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This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
This is an open-access article whereby the authors retain copyright of the work. The article is distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.
Sir,
We read with interest the recent review by Santiago Munné entitled, “Preimplantation Genetic Diagnosis for Aneuploidy and Translocations Using Array Comparative Genomic Hybridization” (1). As part of the review of array comparative genome hybridization (aCGH), the author provides additional information on other 24-chromosome preimplantation genetic diagnosis/screening (PGD/PGS) techniques. As a commercial lab that offers single nucleotide polymorphism (SNP) microarray analysis for PGD/PGS, we would like to comment on a few claims that were made within this article regarding SNP microarrays.
(Table
We also question a number of statements the author makes about aCGH and SNP microarray within the body of the paper. The author acknowledges that aCGH cannot detect haploidy or polyploidy but claims that this is a small limitation, as the majority of the haploid or polyploid embryos tested (7.7%) had additional detectable abnormalities; however, these additional abnormalities are not named. It is our experience that other abnormalities are not typically found with 69,XXX. Next, the author credits SNP microarray with the ability to detect uniparental disomy (UPD) but then goes on to use the incidence of UPD of chromosome 15 (UPD-15) to say that UPD in general is a very rare event. Chromosome15 is only one of six imprinted chromosomes (6, 7, 11, 14, 15, and 16), which if UPD is present, could lead to the birth of a baby with a severe genetic syndrome (4). We feel that detection of UPD prior to embryo transfer decisions is highly beneficial to, and desired by, couples undergoing IVF with PGD/PGS. Furthermore, in regards to PGD/PGS for reciprocal and Robertsonian translocations, we would like to clarify that not all SNP microarray approaches can differentiate between normal and balanced (carrier) embryos. Last, the author correctly points out that SNP microarray approaches require parental DNA analysis prior to the embryo sample analysis. However, our lab does not charge a cancellation fee for this parental analysis when IVF cycles are cancelled, thus patients do not pay for unnecessary parental testing.
We appreciate that the author mentions SNP microarray analysis that employs a qualitative/quantitative approach will avoid many of the limitations inherent to the purely qualitative or quantitative approaches. However, the review references our article (Johnson DS
New methods should always be validated against more established ones, but given the errors rates reported with FISH PGD/PGS (as stated by the author at the beginning of the paper), we strongly disagree that validation studies using FISH for reanalysis should be considered the gold standard (2, 5). Natera thus supports the creation of an oversight body to administer proficiency testing for laboratories offering 24-chromosome PGD/PGS.
Sincerely,
Pars plana vitrectomy (PPV) was introduced almost 40 years ago.
Herein, we review the advantages and disadvantages of small-gauge vitreous surgery.
TSV consists of a 23-G or 25-G microcannular system and a wide array of vitreoretinal instruments specifically designed for this operating system. The microcannula consists of a thin-walled tube, 4 mm in length. A collar is present at the extraocular portion, which can be grasped with a forceps to manipulate the microcannula. The insertion trocar has a sharp tip that forms a continuous bevel with the microcannula, allowing easy entry through the conjunctiva into the eye (
Small gauge vitrectomy is usually performed with the patient under local anesthesia. General anesthesia is only performed in selected cases (children or uncooperative adults). After appropriate anesthesia, the operative field is prepared using antiseptic solutions. Preoperatively, the eyelash margins are scrubbed with povidone-iodine solution. The microcannulas are inserted through the conjunctiva into the eye by means of a trocar. Insertion is accomplished by first displacing the conjunctiva laterally by approximately 2 mm. An initially oblique, then perpendicular tunnel is made parallel to the limbus through the conjunctiva and sclera creating a self-sealing wound (
The 23-G system is a variation of the 25-G TSV system. 23-G vitreous cutters have been improved by placing the cutter opening nearer to the end of the probe allowing a closer vitreous shave. This increases safety near the retina. At the end of vitrectomy, adequate gas/air tamponade must be performed which avoids significant postoperative leakage in most cases. However, in some cases leakage may occur and the sclerotomy site should be closed with a single 7–0 or 8–0 vycril suture. In addition, sclerotomy sites are to be closed if silicone oil is used. The microcannulas can be simply removed by grabbing the external collar with a forceps at the end of the procedure. The last microcannula to be removed should be the one with the infusion line (
In general, TSV seems to be particularly advantageous for procedures that do not require extensive intraocular tissue dissection or manipulation. Experience has shown that 25-gauge surgery is ideal for vitreous and preretinal hemorrhages in proliferative diabetic retinopathy (
Another advantage of TSV becomes apparent in pediatric cases. Typically, newborn and premature eyes are significantly smaller than adult eyes and the use of standard vitreoretinal instruments may be technically difficult.
One disadvantage of this system is the learning curve required to achieve maximum efficiency. However, this curve is short enough for the adaptable surgeon.
Due to its smaller fiberoptic size, illumination is also reduced with 25-gauge surgery. However, current systems provide adequate illumination in most cases. A noticeable difference of 25-G instruments is their marked flexibility.
There are some potential complications specifically related to the 25-gauge system, the most obvious being hypotony and a higher incidence of endophthalmitis.
For a surgeon used to performing 20-G vitrectomy, transition to 23-G is easier than 25-G. With the 23-G system, rigidity, flow and aspiration of the vitreous cutter are similar to the 20-G system, and lighting is comparable. The instruments have stiffness similar to 20-G. However the sclerotomies must be precisely formed, with tunnel or angular incisions to reduce complications.
The lower rigidity of instruments is a problem with 25-G, these instruments are more pliable and more damageable, furthermore manipulation of the globe can become cumbersome. This is not an issue with 23-G, as rigidity is similar to 20-G (
Initially, available instruments were limited to small-gauge forceps, however nowadays a full armamentarium of instruments is available in small-gauge, including extrusion cannulas for silicone oil injection and removal, scissors, dual-bore cannulas for perfluorocarbon injection, diathermy probes, multidirectional laser probes, chandeliers, and 40-G cannulas for subretinal injections. In essence, at present, the same range of instruments used in 20-G, is available in 23- and 25-G, with the exception of a fragmatome. Nevertheless, several companies are working on developing a 23-G fragmatome to address dislocated nuclei, and DORC (Zuidland, The Netherlands) has recently released the 23-gauge Rayes Fragmentation Needle.
Since the number of light fibers is reduced, particularly with 25-G, brighter light sources are needed, such as Photon (Synergetics Inc., O’Fallon, MO, USA) and Xenon (Alcon Laboratories, Fort Worth, TX, USA). The new Alcon Constellation system has much brighter light than the actual Xenon. Bausch & Lomb (Rochester, NY, USA) uses the Photon. With these recent modifications, illumination is not much of an issue anymore.
Slow vitreous removal is a potential problem with 25-G, this issue has been addressed in the new Alcon Constellation machine. The new 25-G probe has a bigger opening and a longer duty cycle (the amount of time the port is open); these alterations allow an increased aspiration rate while maintaining high-speed cutting rates. The 23-G system will benefit from the same duty cycle improvement, but the probe is different and does not have a spring mechanism, so it can stay open longer during each cut, allowing greater aspiration. Thus the rapidity of vitreous removal with 23- and 25-G will be markedly improved. Cutting rates of up to 5000 cuts/min are available, allowing for shaving of the vitreous base and safer vitrectomy, even in detached retinas.
Wound architecture is the most important aspect of TSV; complications such as endophthalmitis and retinal breaks are associated with badly fashioned wounds. Initially, wounds for 25-G vitrectomy were made by direct entry, this could have been the cause of hypotony and increased endophthalmitis rates among other complications. Displacement of the conjunctiva and a two-plane wound with fluid-air exchange at the end of the procedure, reduce wound leaks and decrease the risk of endophthalmitis and hypotony. The DORC and new Alcon 23-G TSV systems have a flat blade trocar system which produces a slit wound that closes better than the actual chevron wound made by the round trocar blade system. Wound construction in our view is the most important aspect of TSV and the key point in the learning curve. One should always keep in mind that if any doubt exists regarding leakage, the surgeon should suture the sclerotomy site. Our threshold for tends to be lower in complicated cases where silicone oil needs to be used.
Other benefits of TSV include astigmatism-neutral surgery, shortened operative time, less inflammation and reduced patient discomfort. We truly believe that the reportedly increased incidence of endophthalmitis is technique-dependent. With adequate preoperative povidone-iodine preparation, good wound construction in two planes, a partial or total fluid-air exchange at the end of the procedure, and subconjunctival antibiotics, the risk of hypotony and endophthalmitis can be reduced.
The learning curve is mainly due to wound construction and for 25-G, the current lack of rigidity. The TSV 23-G system has less of a learning curve, as the instruments have similar rigidity as 20-G. The 23-G system has been more user-friendly due to the illumination, rigidity, and increased flow, which are all similar to 20-G. With improvements in 25-G systems, at least with the Constellation system, 25-G surgery will feel more like 23-G, as rigidity, aspiration, and illumination are increased significantly.
Cataract surgery was revolutionized by the introduction of phacoemulsification and foldable IOLs. This enabled a reduction in the size of the incision and avoided the necessity of using sutures. This transition shortened operative time, reduced complications, and increased patient satisfaction and comfort.
The same transition is occurring in vitreous surgery. Both vitrectomy techniques, either 25-G or 23-G, have been improving with time, experience, and the introduction of better instruments. Surgical indications are expanding with experience and the availability of new technology. Performing minimally invasive surgery has many advantages for both the surgeon and the patient. The reduced surgical time improves efficiency and also reduces complications and surgical trauma. Postoperative recovery is more rapid, since there is less inflammation. Technological developments, more efficient vitreous cutters, and a variety of 23-G and 25-G instruments have made small-gauge vitrectomy the gold standard and it is here to stay.
Dr Arevalo and Dr Arias: None. Dr Berrocal is a speaker for Alcon.
This paper was supported in part by the Arevalo-Coutinho Foundation for Research in Ophthalmology, Caracas, Venezuela.
Some of the contents of this discussion have been previously published in: “Arevalo JF, Berrocal MH. Overcoming Myths and Misconceptions in Small-gauge Vitreoretinal Surgery.
Insertion of 23-gauge trocars and microcannulas. The microcannulas are inserted through the conjunctiva into the eye by means of a trocar. Insertion is accomplished by first displacing the conjunctiva laterally by approximately 2 mm. An initially oblique, then perpendicular tunnel is made parallel to the limbus through the conjunctiva and sclera, thus creating a self-sealing wound.
After withdrawal of the cannula, a cotton tip applicator can be used to misalign the outer and inner aspects of the sclerotomies thereby reducing the risk of leakage. subsided due to recent studies revealing a low incidence of endophthalmitis in a large series of patients undergoing small-gauge sutureless surgery.
Intraoperative photographs of a diabetic eye with vitreous and preretinal hemorrhages managed with 23-gauge instrumentation.
Intraoperative photograph of macular epiretinal membrane treated with 23-gauge instrumentation.
The microcannulas are inserted through the conjunctiva into the eye and 23-gauge instruments are in place at the sclerotomy sites.
Coronal shear fractures of distal humerus have received much attention in recent years (
We also agree with Giannicola et al. that a poor osteosynthesis leads to prolonged immobilization of elbow and limits the functional results. We also understand that stable fixation is sometimes difficult to achieve but most of capitellar fractures can be fixed reasonably well to allow early mobilization (
Supplementing stable fixations with external fixator not only increases surgical time but also subjects the patients to increased risks of complications of added surgery. We would like to ask the authors to comment on the increased cost of treatment associated with fixator used.
The high rates of reoperative procedures and the discordant results for these fractures are found in largest series (28 patients) of
Authors report extrusion of Herbert screws occurred in 2 cases. What could be the reason for this fixation failure in spite of being supplemented by external fixator?
Though we disagree on external fixation supplementation in all types of cases, we do congratulate the authors for extending the concept of supplementation of fixation to capitellar fractures. Further studies would be required to evaluate the efficacy and laying out specific indications for this kind of supplementation.
In 1997, it was decided at the Medical Faculty of Uppsala University that the number of departments should be reduced from over forty to about ten. One rationale for this decision was that some departments were too small to be able to carry the responsibilities with regard to economy, administration and long-term care of employees. Another rationale was the hope that larger departments would facilitate contacts between different research groups with beneficial effects on research. Since there were several departments with a strong inclination towards research within the field of neuroscience, eleven previously independent departments were merged into one Department of Neuroscience. This new department had the unusual feature of being a mixture of former clinical departments, such as Psychiatry, Neurology, Ophthalmology etc. with former preclinical departments such as Pharmacology, Medical Developmental Biology, part of Physiology etc. The reorganization took place in 1998 and this issue of the Uppsala Journal of Medical Science (UJMS) has been created in order to celebrate the recent ten-year jubilee. It is hoped that this issue of UJMS will demonstrate the wide-spread research areas within the department, as well as the fact that UJMS is open to publications of clinical, as well as preclinical papers within all areas of medicine.
Recently, 2 studies, published in prestigious journals, have presented widely different relative risk values for atypical femoral fractures with the use of bisphosphonates: 47 by
I think these remarkably disparate results merit a public discussion and Acta Orthopaedica is therefore publishing the Letter by Schilcher and colleagues that was originally sent to JBMR.
Sir—I write this letter to point out a grave error in the paper entitled “Outcome of the cementless Taperloc stem: a comprehensive literature review including arthroplasty register data” (
The paper incorrectly identifies Richard Rothman as the designer of the Taperloc femoral component. In fact, the developer of the Taperloc stem was William Kennedy at the hip and knee center in Oshkosh, WI. Therefore, the paper's conclusion that “the excellent results published by the developer's clinic are generally not reproducible by other surgeons” is invalid. The 4 articles authored by McLaughlin and Lee referenced in this paper present the rate of revision of the Taperloc stem from the center where the Taperloc originated. The only 4 articles authored by a Taperloc developer are the 4 from McLaughlin et al. The differences in revision rates for the Taperloc as published by McLaughlin and Lee versus the other (non-developing) authors, as well as for McLaughlin and Lee versus the registry data are both within a factor 3 and thus are not clinically relevant per the criterion used by Labek et al. to define outlier datasets. If this paper had correctly identified the designer of the Taperloc their conclusion would have been: Studies originating from the center where the Taperloc stem was developed accurately reflect the data published from other centers as well as registry data.
Sir—The work of Sigmundsson and colleagues (2011) represents a very good analysis of a cohort of persons subjected to surgery for spinal stenosis. However, the discussion of the disease itself far over-reaches any conclusion that can be drawn from this highly selected population that contains no control group. The Michigan Spinal Stenosis Study, a controlled study including asymptomatic persons and persons with back pain, addresses the same issues with contradictory findings. We found that, of 14 different measures of the lumbar vertebrae, including the thecal sac area and ‘smallest two area’ measures similar to those used in their study, only two measures (spinal canal diameter and smallest two spinal canal diameters) had any relationship to the clinical diagnosis of stenosis, and these two had no discriminant value (
The decision of a surgeon to operate and of a patient to accept an operation is a complex one that goes beyond objectivity on the part of both parties (
Clinical spinal stenosis remains a complex and poorly defined, and poorly validated syndrome. There may well be some relationship between spinal measures and disability. Despite the well intentioned work of Sigmundsson et al. modern research is showing that a surgical convenience sample does little to clarify the syndrome.
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This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Re-use of this article is permitted in accordance with the Creative Commons Deed, Attributed 2.5, which does not permit commercial exploitation.
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Re-use of this article is permitted in accordance with the Creative Commons Deed, Attribution 2.5 which does not permit commercial exploitation.
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Re-use of this article is permitted in accordance with the Creative Commons Deed, Attribution 2.5, which does not permit commercial exploitation
Re-use of this article is permitted in accordance with the Creative Commons Deed, Attribution 2.5, which dose not permit commercial exploitation.
Re-use of this article is permitted in accordance with the Creative Commons Deed, attribution 2.5, which does not permit commercial exploitation.
Re-use of this article is permitted in accordance with the Creative, Commons Deed, Attribution 2.5, which does not permit commercial exploitation.
Re-use of this article is permitted in accordance with the creative Commons Deed, Attribution 2.5, which does not permit commercial exploitation.
Reuse of this article is permitted in accordance with the Creative Commons Deed, Attribution 2.5, which does not permit commercial exploitation.
Reuse of this article is permitted in accordance with the Creative Commons Deed, Attribution 2.5, which does not permit commercial exploitation
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Antiretroviral therapies have proved life-saving in HIV infection, dramatically reducing morbidity and mortality. With longer survival, morbidities and mortalities in HIV infection are increasingly similar to the morbidities and mortalities associated with ageing. In treated HIV infection, the risk of these morbidities and mortalities is linked to immune activation, inflammation and coagulation indices. And in persons with treated HIV infection, failure to restore circulating CD4 T cell numbers is associated with a greater risk of morbidities and mortalities as well as to heightened levels of inflammation and coagulation. The drivers of immune activation, inflammation and coagulation in treated HIV infection are incompletely defined and could be related to sustained low levels of viral replication in tissues, to translocation of microbial products across a damaged gut mucosa, to replication of co-pathogens such as cytomegalovirus, to increased levels of inflammatory lipids, or to homeostatic responses to lymphocytopenia that may drive expansion of CD8 T cell numbers. We present here models that link inflammation and coagulation to morbid outcomes as well as to the pathogenesis of CD4 T cell restoration failure and CD8 T cell expansion.
Although the performance of HIV prevention and treatment programs varies considerably among countries, the variability within any one country is typically much greater than is the variability among countries. Even within an individual health system with relatively homogeneous conditions for its clinics (resource constraints, human resource system, professional training standards, compensation, incentives, etc.), there will typically be very heterogeneous performance across clinics. We have the opportunity to analyse existing heterogeneity to both learn about what is driving good and poor performance as well as to create incentives for clinics and providers to learn from and emulate the top performers. We will use examples from Mexico and Africa to show how much heterogeneity exists, discuss cases in which revealing that heterogeneity has driven performance improvement and speculate on how such approaches could be implemented more broadly in Latin America.
Among a number of HIV prevention approaches, pre-exposure prophylaxis (PrEP) is the strategy of administering antiretroviral drug(s) to HIV-positive people at risk for HIV infection to decrease their risk of acquiring HIV infection. With 28 approved antiretroviral drugs, tenofovir with or without emtricitabine emerged as the leading candidate(s) for PrEP, with demonstrated potency, safety, tolerability and convenience. Randomized, placebo-controlled studies of daily PrEP with tenofovir/emtricitabine in gay men (iPREX) and tenofovir with or without emtricitabine in the negative member of serodiscordant couples (Partners PrEP) demonstrated efficacy in reducing the risk of HIV infection and led to the US Food and Drug Administration approval of tenofovir/emtricitabine for PrEP; this was supported by additional data from the TDF-2 study. Randomized studies in British gay men and Thai injection drug users also demonstrated the efficacy of daily PrEP. Two follow-up studies in African women (FEM-PrEP and Voice) failed to show a benefit, but adherence rates were less than 40%. In all of these studies, adverse event rates were low, drug resistance was very rare and there was no evidence of behavioural disinhibition. More recently, the Ipergay study showed PrEP was effective when given “on demand” – before and after sex, rather than daily. Additional studies of intermittent PrEP dosing are underway. Also, additional antiretroviral agents for PrEP currently are in clinical trials including maraviroc and the long-acting parenteral formulations of rilpivirine and cabotegravir. Current US and WHO guidelines recommend PrEP for people at high risk for HIV infection, including gay men, heterosexuals and injection drug users.
In the past four years, unprecedented breakthroughs in HIV prevention, treatment and research have occurred. Particularly notable are the results of HPTN 052 which demonstrated that effective antiretroviral therapy of HIV-positive individuals dramatically cuts HIV transmission and the results of several studies demonstrating the impact on HIV acquisition of pre-exposure prophylaxis. These advances have made many realize that we can end the AIDS epidemic by 2030. However, a myriad of challenges will need to be overcome in order to achieve this goal. The first challenge is financial; although the mobilization of resources to treat HIV has been unprecedented, we are far from the necessary resources needed to treat over 90% of those infected. The second challenge is in implementation, in most of the world weak healthcare systems that are understaffed struggle to manage their current patient load, let alone the millions of additional patients who will require treatment. In addition, in order to ensure timely linkage to care after diagnosis and retention in care, healthcare systems will require to be reengineered and novel interventions will need to be developed. Furthermore, laboratory monitoring continues to be limited in most countries. The third challenge is persistent stigma and discrimination that has prevented many who are at risk or infected from accessing care. Finally, there is the challenge of political and societal will. It is theoretically possible to end the AIDS epidemic, but it will happen unless we dramatically change the way we are currently doing things.
Key populations share high burdens of HIV infection and low access to HIV services, and include people who inject drugs (PWID), sex workers of all genders, gay and other men who have sex with men (MSM), transgender women who have sex with men, and prisoners and detainees. These communities now account for an estimated 50% of new HIV infections worldwide and are at the centre of the HIV epidemic zones where HIV is still expanding: Eastern Europe and Central Asia, and the Middle East and North Africa. These populations have lower HIV treatment access in many settings and are excluded, or exclude themselves, from life-saving treatment and related services. Little data is available on the continuum of care for key populations, but it will be essential to better understand the barriers to successful testing, linkage, retention and successful viral suppression for these individuals and communities if HIV control is to be achieved. Advances and approaches which could improve the continuum of care for key populations will be reviewed.
Transgender people have specific health problems and challenges related to stigma, hormonal administration, sexual risk behaviour and application of body-modifying substances. There is limited experience on the provision of integral health care services for transgender people, particularly in developing countries. We describe the experience providing health care services for transgender people at Clínica Especializada Condesa (CEC) in Mexico City.
The Health Care Center for Transgender People Program started in 2009. It is the first specialized Clinic in Transgender Health in Mexico. We describe the model of integral health care for transgender people and basal information on the beneficiary population.
Primary health care is provided by one endocrinologist aided by two nurse practitioners, three psychiatrist, three psychologists, one gynaecologist, one radiologist and a specialised clinical laboratory. Internal Medicine specialists provide HIV/AIDS care for those HIV-positive. All patients receive Voluntary Counselling and Testing (VCT) and mental health care services. These comprise an extensive evaluation for gender life-experience, drug abuse and psychopathologies. Cross-sexual hormonal therapy (HT) is provided for free in selected individuals, according to pre-established criteria based in life experience as their cross-sexual role, mental health, age, co-morbidities and HIV status and antiretroviral therapy (ART) adherence. There are 1187 patients registered in the clinic, 889 whom actively are currently receiving care. The mean age is 31 (16–68) years old. There are 1004 male-to-female (MtoF) (84.58%) persons, and 65.63% (
A total of 191 (64%) of HIV-positive patients concomitantly receive HT and ART. Among those HIV-positive and receiving ART (
The provision of integral health care services for transgender people in an urban setting of middle-income country is feasible. Limited resources and personnel rotation are the main challenges. Provision of free HT in selected cases appears to be safe and may contribute to retention in care.
| Infection | Number of patients tested | Number of patients with reactive test | Prevalence (%) |
|---|---|---|---|
| HIV | 615 | 297 | 48 |
| HCV | 197 | 6 | 3 |
| HBV | 232 | 15 | 6.5 |
| Syphilis | 230 | 45 | 19.6 |
Global prevalence of HIV-1 is estimated at 35 million; >50% of those infected are women. Despite availability of effective antiretroviral therapy (ART), many HIV-positive individuals are not in care; women have greater difficulty accessing HIV care than men. The ELLA study is a cross-sectional, multi-country, non-interventional epidemiological study to investigate the population, disease characteristics, barriers to care, quality of life (QOL) and reproductive choices for women living with HIV. We describe results of women included in ELLA from the Latin American region (LA).
HIV-1-positive females ≥18 years were enrolled using a non-random sequential sampling frame in four global geographic regions: Western Europe and Canada, LA, Central/Eastern Europe and Asia. Eligible women completed self-administered Barriers to Access to Care Scale (BACS) and other health status questionnaires. Patients rated each of the 12 BACS items using a four-point Likert scale (1=“No problem at all” to 4=“Major problem”). Questionnaires with ≥6 items completed were included in the analysis, mean score >2.0 was considered a significant barrier to access to care.
A total of 519 women participated in ELLA from the LA region (total
The majority of HIV+ women in LA included in ELLA were receiving ART, approximately half reported to have undetectable viral loads and normal CD4 cell counts. Barriers to access to care remain high, particularly community stigma and personal resources. Special attention on reproductive health counselling, should be considered.
Despite the advance that highly active antiretroviral therapy (HAART) represents for the prognosis of HIV infection, opportunistic infections (OIs) continue to be a significant cause of morbidity and mortality in HIV-positive patients. Lack of access, late diagnosis, leaks in the HIV cascade of care, lack of treatment adherence and failure put patients at risk of developing OIs and dying from AIDS.
Nevertheless, HAART introduced changes in the prevalence of some IOs. Currently, the frequency of severe cytomegalovirus (CMV) retinal disease,
The global epidemics of tuberculosis and HIV are closely linked. Among persons with latent
Since 2009, earlier initiation of highly active antiretroviral therapy (HAART) after an opportunistic infection (OI) has been recommended based on lower risks of death and AIDS-related progression found in clinical trials. Delay in HAART initiation after OIs may be an important barrier to successful outcomes in patients with advanced disease. Timing of HAART initiation after an OI in “real life” settings in Latin America has not been evaluated.
All CCASAnet patients >18 years of age at enrolment, from 2001 to 2013, who had an OI before HAART initiation were included. Patients were divided in an Early HAART group (those initiating within four weeks of an OI) and a Delayed HAART group those initiating more than four weeks after an OI. Patients with cancer or cryptococcal infection were excluded. Patients with more than one OI were included using the first OI reported only. Calendar trends in the proportion of patients in the Early HAART group (before and after 2009) were estimated by site and for the whole cohort. Factors associated with Early HAART initiation were estimated with logistic regression.
A total of 20,148 patients were included; 1558 patients had an OI before HAART initiation and were included in the analysis: 207 from Argentina, 746 from Brazil, 322 from Chile, 112 from Honduras and 171 from Mexico. The proportion of patients who started treatment within four weeks of the OI was statistically different between sites (
The time from diagnosis of an OI to HAART initiation has decreased in this Latin American Cohort coinciding with the publication of evidence of its benefit. We found important heterogeneity between sites which may reflect differences in clinical practices, local guidelines and access to HAART. The impact of the timing of HAART initiation after OI on patient survival on this “real life” context needs further evaluation.
Median time since all OIs and non-TB OIs.
Combination antiretroviral therapy (ART) has reduced dramatically morbidity and mortality from HIV/AIDS worldwide and has the potential to halt HIV transmission. The availability of potent, well-tolerated, convenient regimens – several of which are available as single-tablet regimens – and the demonstration that ART substantially reduces the risk of transmission to uninfected partners has greatly increased enthusiasm for ART. Novel regimens include those based on newer agents approved over the last few years, including the non-nucleoside reverse transcriptase inhibitor (NNRTI) rilpvirine, and the novel integrase strand-transfer inhibitors (INSTIs) elvitegravir and dolutegravir. In addition, the development of cobicistat as an alternative to ritonavir for boosting protease inhibitors is leading to a greater number of fixed-dose combinations of boosted protease inhibitors. An alternative pro-drug of tenofovir, tenofovir alafenamide, achieves higher intracellular concentrations of the active moiety, tenofovir diphosphate, with substantially lower plasma levels of tenofovir, allowing the delivery of more active drug with lower dosing. Several novel agents within existing drug classes have entered clinical trials; drugs directed against novel targets are also in development. Long-acting injectable agents currently in development have the potential to transform treatment and make directly observed therapy an achievable goal. In addition, broadly neutralizing antibodies and a CD4-Ig/CCR5 peptide fusion protein may have a role in HIV therapeutics.
The Haitian Study Group on Kaposi Sarcoma and Opportunistic Infections (GHESKIO) in Port-au-Prince, Haiti, is the first AIDS prevention and treatment centre in the world. It is also one of the largest AIDS care centres in the Americas. Highly active antiretroviral therapy (ART) was made available in Haiti in 2003 and 2004 with the support respectively of the Global Fund against AIDS, TB and Malaria (GATM) and PEPFAR. From February 2003 to February 2015, 17,029 patients, 13 years old and older, have been placed on ART at GHESKIO-INLR, one of the two main GHESKIO centres in Port-au-Prince. GHESKIO conducted an observational cohort study on the outcomes of a non-research cohort of 1463 patients 13 years old and older starting their first ART regimen and followed for 10 years from February 6, 2013, to February 5, 2015. The primary endpoint was all-cause mortality. The probability of mortality after ART initiation was estimated using Kaplan-Meir methods, censoring subjects lost to follow-up at the time of their last visit. Median age for the entire cohort was 38 years old (IQT 32–45); 54% were females; the median entry CD4 counts was 130/mm3 (51–217); and 40% had AIDS by WHO criteria. The overall probability of survival at one year, five years and ten years was respectively 82, 72 and 50%. Mortality was 23% during the period. It was highest during the first three months and lowest from 5 to 10 years. The lost to follow-up rate during the 10-year period was 15% and was more evenly distributed. Factors associated with mortality and lost to follow-up will be presented.
A significant proportion of patients (approximately 25% in five years according to recent data from the region) require a second-line regimen because of failure of a first one. WHO guidelines suggest using lopinavir/ritonavir plus tenofovir/emtricitabine after first-line failure of an NNRTI-based regimen. Clinical trials exploring the efficacy and safety of the WHO recommended regimen compared to nucleoside-sparing regimens using integrase inhibitors have been conducted in recent years. The second-line study demonstrated non-inferiority of raltegravir/lopinavir/r compared to the standard WHO regimen at 96 weeks. In the Earnest study, a composite endpoint defined as “good HIV disease control” was reached in a similar proportion of patients treated with the WHO recommended regimen and those treated with raltegravir plus lopinavir/r. In both studies, the frequency of tenofovir use in the initial regimen was low (approximately 20%). Nucleoside-sparing regimens were associated with lower bone loss and higher limb fat gain in the second line trial. Other studies using the newer integrase inhibitor dolutegravir with or without nucleos(t)ides are currently being conducted or planned. The long-term efficacy, tolerability and cost-effectiveness of different second-line regimens as well as predictors of success both in clinical trials and “real life situations” remain to be evaluated. The efficacy of newer regimens, including single tablet combinations, the role of resistance testing to guide second-line treatment and second-line regimens used after a first-line boosted PI regimen are unanswered questions.
Antiretroviral exposure is critical to achieve viral suppression in HIV-positive patients and to prevent transmission to HIV-negative individuals. It is directly linked to individual host factors which include age, weight, diet and genetics. However, the main factor impacting long-term drug exposure is drug adherence. Although modern regimens are more forgiving to lower levels of adherence due to their higher potency and more favourable pharmacokinetics, adherence remains the major predictor of HIV outcomes. Unfortunately, quantifying adherence is difficult due to the inherent limitations of the adherence measures currently available in routine clinical practice and research settings. This indicates the need for new and objective measures of antiretroviral exposure that can integrate adherence and pharmacokinetics and provide additional information to what we obtain from the currently available methods. This presentation will focus on some of the new measures that have been recently developed to measure and monitor adherence to antiretrovirals and on the potential approaches that could lead to their utilization in the clinical and research settings.
Malignancies were hallmarks of the AIDS epidemic. Kaposi's sarcoma (KS), the first to be identified, was first described among MSM in 1981 as an enigmatic and unpredictable disease and its management has created great controversy. The human herpes virus-8 (HHV-8), discovered in 1994, is present in all KS lesions, later demonstrated that level of HHV-8 viremia correlated with the severity of the disease and identification of high levels of inflammatory cytokines. Additionally, as many as 80% of KS patients achieve complete remission solely with highly active antiretroviral therapy (HAART), and the occurrence of immune recovery syndrome with exacerbation of lesion in patients starting HAART and the polyclonality of lesions create the need to understand KS as an angioproliferative disease caused by an infectious agent mediated by cytokines. In its management, recovery of immunosuppression is essential as is the treatment of co-infections and avoidance of myelosuppressive therapy. Non-Hodgkin lymphoma has become the most frequent malignancy seen in HIV patients; it requires standard chemotherapy regimens and HAART along with prophylaxis for opportunistic infections while on chemotherapy. The HPV-associated neoplasia, incorporated as an AIDS-defining event in 1993, constitutes a major burden of morbidity in HIV women in countries with high prevalence of oncogenic HPV virus infection. In Mexico, 21% have been diagnosed with a high-grade lesion and 14% with cervical cancer. Recently, higher risk for vulvar and vaginal cancer has also been recognized with the increase survival of women on HAART, and thus the need for colposcopic evaluation of the genital tract and not only PAP smear screening for HPV lesions is required.
HIV infection is rapidly becoming an affliction of older individuals. It is estimated that sometime in 2015, over 50% of HIV-positive individuals in the United States will be over 50 years of age, and this phenomenon is occurring all over the world. It is a function of both patients living longer and older individuals becoming infected more frequently than in the past. Multiple factors are associated with the widely held misconception by the general public and healthcare workers that HIV continues to be a disease of younger people. Thus, more aggressive efforts need to be directed at testing, diagnosing and bringing into care older patients. However, we also need to develop more effective educational messages for those uninfected but at-risk individuals who mistakenly assume that they are protected from HIV infection because they are “old.” With this ageing of the population, it is becoming clear that specific challenges have arisen. While older patients frequently exhibit better adherence to antiretroviral therapy and respond quite well to it, their immune recovery is slower, they are at higher risk of progressing to AIDS and their mortality is higher. In addition to this, the normal ageing process seems to be accelerated by HIV infection itself, and it has become clear that the morbidities associated with advancing age are both more frequent and advance faster than in non-infected, age-matched individuals. Thus, more effective screening, prevention and management programs are needed for cardiovascular, metabolic, neurocognitive, renal, hepatic, hematologic and oncologic diseases of an ageing HIV-positive population.
It has been an important year for pre-exposure prophylaxis (PrEP). Further analysis of the Pre-exposure Prophylaxis Initiative (iPrEx) study showed that taking PrEP of at least four tablets weekly provides almost complete protection against HIV infection. Then came the PROUD and Ipergay studies showing 86% protection in high-risk men who have sex with men (MSM) who took continuous or intermittent (on demand) truvada. Now the issue will be about implementation of such successful strategies worldwide. For those patients who cannot take efavirenz first line what is the best strategy? A study of other initial therapies compared raltegravir vs boosted darunavir vs boosted atazanavir (ACTG 5257). Overall, using intent-to-treat (ITT), all regimens were of similar efficacy but if switching for tolerability or toxicity was taken into account, then raltegravir was better than the boosted proteases. Nucleoside-free or -limiting approaches were also prominent with the 96-week data from NEAT 001 study of darunavir and ritonavir plus either raltegravir or nukes. The combination did not perform well when the CD4 was <200 cells/mm. The 48-week GARDEL study of the two drug regimen of lopinavir and ritonavir plus lamivudine showed similar efficacy as triple therapy. This same nuke-limiting strategy was used in maintenance treatment with the SALT study, atazanavir and ritonavir plus lamivudine and the open label extension (OLE) study of lopinavir and ritonavir plus lamivudine. A nearly twofold increased risk for suicidality in efavirenz-treated patients was found by a
Countries in the Americas committed to eliminating mother to child transmission (MTCT) of HIV and syphilis by 2015. Ambitious goals were set for virtual elimination of both conditions by 2015 through a robust public health approach. The Pan American Health Organization (PAHO) monitors country and regional progress towards elimination. The analysis presented may assist policy-makers and healthcare workers in their efforts to achieve elimination of MTCT of HIV and syphilis in the Americas.
The presentation will summarize progress towards elimination goals from 2010, using data reported by countries to PAHO on sexual and reproductive health, policies and provision of services, and outcomes regarding paediatric HIV and congenital syphilis cases in the Americas.
The coverage of HIV testing among pregnant women in Latin America and the Caribbean has increased from 62% in 2010 to 74% in 2013, and the estimated coverage of ARV coverage among HIV-positive pregnant women has increased from 59% in 2010 to 93% in 2013. The calculated regional vertical HIV transmission rate has decreased from 18% (14–25%) in 2010 to 5% (2–23%) in 2013. Progress in the area of syphilis has been less pronounced, with the coverage of syphilis testing among pregnant women remaining stable in 20 reporting countries at around 80%. Syphilis treatment coverage for pregnant women remains largely unreported. Based on 2013 data, at least seven countries and territories may have achieved the dual EMTCT targets, with at least another eight approaching the targets. Around 21 countries had insufficient information to ascertain progress, of which several may also have achieved dual elimination.
The progress in the region of the Americas illustrates how political and programmatic efforts can result in rapid progress and major public health gains.
HIV epidemics in MSM are expanding in countries of all incomes and in many incidences of new infections is also rising. This talk will consider HIV epidemics in MSM and analyze the current and potential future roles of ART and condom use in limiting new infections. The analyses will be mainly based specifically on the UK epidemic as a typical example of an epidemic in MSM that has not been declining over time. The aim will be to discuss findings from modelling analyses in a manner that is readily understandable to non-modellers. The UK provides a good setting to use as an example due to the fact that there are multiple sources of data on various aspects of the epidemic, including on sexual HIV testing behaviour, as well as detailed data on HIV-infected populations. The link between the “cascade of care” and incidence of new infections will be made clear, which emphasizes the critical role of the HIV treatment and care system in controlling the epidemic. Adoption of effective policies to increase HIV testing rates and a change in ART initiation threshold to initiate ART at diagnosis (should this be supported by results from trials which are currently on-going) could potentially have a very high positive impact on HIV incidence. However, ART adherence and retention and condom-less sex are other key determinants of incidence and these need to be dealt with or the impact of higher testing and earlier ART will be lost.
In the Americas, the HIV epidemic is concentrated among men who have sex with men (MSM) and transgender women. In Latin America these groups continue to be hidden, stigmatized and discriminated against, even in health care facilities. The fight against the HIV epidemic in these populations requires tailored research on prevention strategies, structural changes, community participation and political willingness. After disappointing behavioural, vaccine and community level intervention trials, pre-exposure prophylaxis (PREP) has shown efficacy in preventing HIV acquisition in different populations including MSM (iPrEx study). Originally designed to be carried out in Latin America (LA), the iPrEx study was a multicenter clinical trial implemented in 11 sites from nine countries in four different continents. Finally, 74% of the 2499 participants came from either Brazil or the Andean Region. Despite such evidence, only Brazil has moved into the next steps to continue generating evidence to eventually make an informed decision about the potential role of PREP in LA. Implementation of PREP requires operational research to determine a variety of aspects such as priority target sub-populations, delivery systems, cost-effectiveness and public health impact based on “real-world” uptake and adherence data (including facilitators and barriers). Demonstration projects aimed to address these and other relevant factors are on-going in the US and Brazil. In spite of the proven efficacy of antiretrovirals in preventing HIV transmission, it is widely accepted that not a single strategy will be sufficient to curb the HIV epidemic in MSM. New promising interventions, such as rectal microbicides and HIV vaccines, are being evaluated; however, combined strategies will be needed to reach the goal of an AIDS-free generation. The next step in the HIV prevention research agenda for MSM would need to include the evaluation of acceptability, uptake of and adherence to behavioural, biological and structural components of a combined prevention package.
This year's CROI meeting had important new data on cure, prevention and resistance. While a cure is not yet on the horizon, a number of advances provide clues about how HIV might be eradicated. At 2013 CROI, the case of a baby that started ART 30 hours after birth was presented. After 18 months, treatment was stopped persisting with undetectable viral load despite being off antiretrovirals. This year an update of the case reported that despite being off treatment for two years, this infant still has undetectable blood viral load and extensive testing has not found HIV in her PBMCs or other reservoirs, suggesting that very early therapy for infants may lead to a “functional cure.” In another cohort study, proviral reservoirs were significantly reduced in perinatally infected patients with virologic control on ART by one year of age vs. patients with later virologic control. Prevention efforts have more interesting options. In female macaques, GSK744 long-acting injections every four weeks provide prophylaxis against vaginal SHIV challenge. The most important presentation on prevention (PARTNER study) demonstrated zero HIV transmissions between serodiscordant couples with HIV-infected partner on suppressive ART, despite on-going condom-less sex, stressing the importance of early start on ART, while some other options as gene therapy with cells that do not express CCR5, that showed promising results are ready for human use. Transmitted resistance is still a problem despite higher virological control in treated individuals. Using enhanced sensitivity mutation-specific polymerase chain reaction assay a 70% higher prevalence of key reverse transcriptase mutations was detected, being higher among youths aged 13–19 years vs. older age groups and lower among Hispanic individuals vs. black or white persons. However, the clinical significance of these mutants needs to be demonstrated. Looking for resistance-associated mutations to integrase strand transfer inhibitors, a retrospective study (2000–2013) showed that are very few 1/1617 integrase sequences evaluated (T66T/I mixture).
In the last year, the HIV/AIDS research field remained active, and so, several good quality papers were produced and new data were presented at the IAS conference in Kuala Lumpur, the European Conference (EACS) in Brussels and the Retrovirus meeting (CROI) in Boston, among other meetings and conferences. New data provide stronger evidence supporting the initiation of antiretroviral therapy in HIV-1-infected individuals regardless of their CD4 count. Also, treatment as prevention has now more data supporting the evidence for the effectiveness of this strategy, as shown in MSM serodiscordant couples. Morbidity and mortality from non-AIDS-defining illness does not differ from that of the general population if CD4 counts above 500 cells/ml are achieved in HIV-infected people. The new WHO guidelines reflect this situation, by adopting the 500 CD4 threshold for treatment initiation, as well as recommending treatment in several situations, regardless of the CD4 cell count. The aim of achieving a functional cure has received further impulse, provided by reports suggesting that if ARV therapy is started very early during acute infection, functional cure may be achievable in some patients. New drugs, with better tolerability profile, “user friendly” in terms of dosing and with high antiretroviral potency have been approved by regulatory authorities in high-income and some middle-income countries. Last but not least, class-sparing strategies have been tested successfully, challenging the triple-drug paradigm. While the vaccine field has failed to move forward, ARV therapy is now simple, frequently based in fixed-dose combinations and easier to comply with. In this presentation, the state of the art on ARV therapy will be discussed.
Approximately half of all patients living with HIV/AIDS (PLWHA) have underlying substance use disorders (SUDs), including problematic alcohol and drug use. In addition to the direct negative medical and psychiatric consequences associated with SUDs, they in turn are associated with a number of negative HIV treatment outcomes, including increased HIV risk behaviours, delayed HIV diagnosis, delayed linkage and retention in HIV care, decreased likelihood of being prescribed antiretroviral therapy (ART), decreased ART adherence and decreased likelihood of achieving viral suppression. As a result, PLWHA with underlying SUDs should be targeted using evidence-based interventions and provision of adequate support in order to achieve targets set forth for HIV treatment as prevention (TasP) to be optimized. In this presentation, a review of types of SUDs will be presented along with the negative medical and psychiatric consequences and their impact on HIV treatment outcomes. In addition, practical solutions for screening, brief interventions and referral to treatment (SBIRT) as well as evidence-based interventions that promote improved engagement in the HIV continuum of care will be covered to help guide HIV prevention and treatment strategies.
Hepatitis in the context of HIV infection is rapidly changing. The majority of individuals living with hepatitis B and HIV will be fully suppressed on a regimen including tenofovir and as yet there have been no confirmed reports of resistance developing to this drug in vivo. However, there will be some individuals who are not able to receive tenofovir principally due to renal dysfunction, and other strategies including dose reduction and the use of alternative agents may be required. The major revolution in the field of co-infection has been the development of direct acting antivirals to treat hepatitis C. Both in clinical trials and clinical practice the results in the co-infected mirror, or are even better, than that in the mono-infected population and HIV should not be a barrier to individuals receiving such agents. The first direct acting antivirals boceprevir and telaprevir have already been surpassed in efficacy results within clinical trials, by newer protease inhibitors including faldaprevir and somepravir. The results of the use of the polymerase show potentially even higher rates of treatment success in the HIV population when combined with Interferon and ribavarin. The recent results of the PHOTON study show HIV-infected individuals will also respond to Interferon sparring approaches, with similar success rates as in the monoinfected. Results of studies of combinations of oral DAAs in the monoinfected population have shown efficacy rates approaching 100%, with little drug associated toxicity. The results of the SYNGERY study have suggested that individuals receiving such combinations may only need to receive these drugs for six weeks. However, there will remain barriers; these will include access to medications, the cost of the medications and the increasing reports of re-infection within the HIV population. Although treatment strategies are changing dramatically so will the need for the education of the co-infected population to prevent both initial and re-infection.
Advances in antiretroviral therapy and increased access have brought enormous benefits to affected populations; however, these benefits have been limited by the impact of other chronic conditions among which Hepatitis C occupies a significant place. Chronic liver disease due to Hepatitis C affects a large number of HIV-infected patients in Latin America and currently poses important challenges for the health systems in the region. Treatment for Hepatitis C infection in monoinfected and dually infected patients has improved dramatically in the past decade. New direct acting antiviral agents are substantially increasing the rate of sustained virological responses observed previously with Peg interferon plus ribavirin regimens specially in difficult to treat patients. Regimens free of Peg interferon more likely will replace the current standard of more toxic and less efficacious options, albeit at costs (if no major changes occur) that are unreachable to most governments. Even though anti-HCV treatment has a limited duration and its goal is viral eradication – a concept not yet existent for HIV – access to anti-Hepatitis C agents has not followed the same path as antiretroviral treatment in the region. In fact, the majority of countries in Latin America do not have specific programs for Hepatitis C treatment access as the ones created to treat HIV infection more than a decade ago. The current era of Hepatitis C treatment in some ways resembles the time in which HAART first became available. Communities, academia, governments and business from the region should learn from the experiences derived from that era in order to face the challenges that Hepatitis C treatment represent. Delaying or negating access now based on purely monetary reasons is not a wise option. Creative solutions to improve access should be sought and implemented in a joint fashion among the different actors.
Transmitted drug resistance (TDR) has become an epidemiological and practical issue in any region where that there is widespread use of antiretrovirals. The World Health Organization (WHO) defines low TDR prevalence at a level below 5% and high-prevalence levels above 15%. There is a universal trend to maintain prevalence of NRTI resistance over time, increase NNRTI resistance and decrease PI resistance as a function of viral fitness and increasing use of boosted PIs. Emerging data confirm the relationship between TDR and virologic failure, thus emphasizing the importance of pre-treatment resistance determination. More sensitive methods to determine acquired drug resistance (ADR) using next-generation sequencing are promising and are helping to understand mechanisms of resistance, and also offering a window of opportunity to increase salvage therapy efficacy. Resistance mutants may be selected among low-level viraemic patients on antiretroviral treatment, especially for low genetic barrier drugs. Finally, in the test and treat era, treatment for longer periods of time may lead to long-term toxicity and to treatment interruptions. In this sense, the future points to personalized medicine where genetic tests targeting hosts may help to mitigate chronic side effects of antiretrovirals at an individual level.
The search for drugs that are efficacious but have very few side effects, do not induce resistance and are simple in their administration is still a priority in HIV research. The integrase inhibitors have excellent tolerability and low rates of serious adverse events. Recently, the newest integrase inhibitor dolutegravir showed superiority over efavirenz and darunavir/r in randomized naïve studies mainly based on its excellent side effect profile. New drugs in the pipeline include an alternative to tenofovir, as tenofovir alafenamide fumarate (TAF), which may have less bone and renal toxicity. The pharmacokinetic booster cobicistat is used at the moment in Stribild to boost elvitegravir. It is also being co-formulated with darunavir and atazanavir as a single pill to simplify PI therapy. There are prospects of having a single pill “boosted PI regimen.” New drugs in later stages of development with recent data include the attachment inhibitor BMS-663068 from BMS and the long-acting integrase S/GSK744 from ViiV. The experimental entry inhibitor cenicriviroc, a. gne therapy is also being piloted. Nucleoside sparing strategies are still being pursued and a large trial of darunavir/r, and raltegravir in naïve patients (NEAT 001) was non-inferior to standard of care except in those patients with low CD4s and high viral loads. Another smaller study in naïve patients has shown that at least in the short-term, lopinavir plus 3TC was non-inferior to lopinavir plus 2 nucleosides. The use of Maroviroc with DRV/rI has not proven to be efficacious and the MODERN study had to be terminated early.
This presentation will share the perspective of the Pan American Health Organization (PAHO) on the impact generic antiretrovirals (ARVs) have made in improving access to affordable, quality, safe and efficacious treatment to HIV in Latin America. Increased use of generic medicines, in particular ARVs has demonstrated to be a cost-effective approach and can result in significant reductions in expenditures on ARVs, which has allowed PAHO Member States to accelerate efforts to scale-up treatment towards sustained universal access to ARVs. Examples of how procurement of generic ARVs through the Regional Revolving Fund for Strategic Public Health Supplies (Strategic Fund) has resulted in significant cost savings in Latin America will be discussed. Additionally, the presentation will explore current barriers to accessing generic ARVs, focusing on current patents and licensing agreements between ARV manufacturers that limit the sale of generic ARVs Latin America.
Acute HIV infection is defined as the period of time when the HIV RNA and/or p24 antigen is detectable, but before HIV antibodies are detectable. Early HIV infection is the period of time within six months after infection when HIV antibodies are detectable. The vast majority of HIV infection occurs with R5 virus. The virus crosses a mucosal barrier over 2–6 hours, infects target cells (CD4 lymphocytes, dendritic cells, tissue macrophages) locally and then spreads to local lymph nodes over 3–6 days. After 6–25 days, the infection spreads systemically and peak viraemia occurs; this is associated with a substantial loss of mucosal CD4 cells and the greatest risk of onward transmission. The clinical presentation of acute HIV infections is symptomatic in ~75–90% of patients with non-specific symptoms of fever, myalgias and night sweats; rashes may be present in ~50% and oral ulcers in up to 10–20% of patients. The diagnosis of acute HIV is made with a combination of tests including HIV RNA, p24 antigen (included as part of the 4th generation ELISA test), and HIV antibody testing. Clinical trials of acute HIV infection suggest early antiretroviral treatment is associated with decreased clinical symptoms and signs, enhanced CD4 recovery and possibly a decrease in viral set point and/or HIV reservoirs. Recent studies suggest very early treatment of infection can lead to eventual virologic control of antiretrovirals or possibly even cure in some cases. Current guidelines support treatment, though differ in the strength of the recommendation. With transmitted drug resistance in mind, the optimal treatment choice is a protease inhibitor- (or integrase inhibitor-) based regimen that is started right away and not stopped. Drug resistance testing should be obtained and used to adjust the regimen later, when the results return. Diagnosing and treating acute HIV infection is an effective way to reduce onward HIV transmission.
Research towards a HIV cure has emerged as a major goal of investigators throughout the world, with significant resources being mobilized by national funding agencies and foundations. This renewed sense of purpose is driven in part by the demonstration that cure (or “sustained HIV remission”) may be possible, as illustrated by the Berlin patient and the Mississippi baby. A number of important steps forward in cure research have been reported. The report of a second baby positive at birth but in whom HIV cannot now be detected generated considerable interest at CROI 2014. Because this baby remains on antiretroviral therapy (ART), it is too soon to say whether ART-free remission has been achieved. Preliminary data from an on-going study of the histone deacetylase (HDAC) inhibitor panobinostat show that multiple cycles of the drug resulted in repeated stimulation of HIV expression, extending the results originally reported with vorinostat. In addition, studies in the macaque model of SHIV infection showed that broadly neutralizing monoclonal antibodies reduce the level of viraemia and decrease proviral DNA, suggesting that such antibodies may have a role in combination strategies for reducing the viral reservoir. Treatment interruption in two HIV-infected adults who had received allogeneic stem cell transplants for haematological malignancies and in whom no evidence of viral persistence could be detected in peripheral blood or rectal tissue resulted in virologic rebound after three and eight months. Although disappointing, these results nevertheless suggest an important role for graft-versus-host reaction in eliminating latently infected cells. Important questions that remain to be answered include identifying the site of viral persistence in these patients and determining whether additional interventions can prolong the time to viral relapse.
There have been significant improvements in the Central America region in HIV policy and care in three areas: (1) access to treatment, (2) reduction of stigma among health care providers and (3) the existence of national strategic plans. But, despite the existence of laws, norms, strategic plans and international agreements to respond to the HIV epidemic in the region, there are important gaps in the implementation, dissemination and monitoring of these policies. Stigma and discrimination against key populations disproportionately affected with HIV is considered a major hurdle in the implementation of HIV policies and still persists in the general population. The lack of political leadership to implement human rights laws for the protection and equality for key populations, and an appropriate response to gender-based violence are two other significant deficiencies that negatively impact the HIV policy environment. A fragmented civil society effort produces lack of cooperation and even competition between the different non-governmental organizations compromising the attainment of common goals. In many countries in the region, a lack of sufficiently strong national authority to solicit resources and coordinate the response has also been identified. In summary, despite advancement in certain areas of HIV policy and care in Central America, there are still many problems and obstacles that need to be addressed in order to improve this region's response to the HIV epidemic.
Combination antiretroviral therapy (cART) can successfully prevent traditional HIV-associated morbidity and mortality, thereby allowing patients with HIV to survive and become increasingly older. Not surprisingly, ageing-associated co-morbidities are increasingly being observed and importantly influence clinical management and care. The risk of some of these co-morbidities has been demonstrated to be increased in the context of uncontrolled infection, but even those with suppressed viraemia on cART seem to be at increased risk of the development of ageing-associated non-communicable co-morbidities, including cardiovascular, chronic kidney, liver and pulmonary disease, diabetes mellitus, osteoporosis, non-AIDS associated malignancies and neurocognitive impairment. The underlying pathogenesis is multifactorial and, next to traditional (often lifestyle-related) risk factors, seems to include sustained immune activation and inflammation, both systemically and within particular body compartments such as the central nervous system. In addition, circumscript toxicities resulting from both past and present exposure to particular antiretrovirals may also contribute to various co-morbidities such as, for example, chronic kidney disease, osteoporosis and cardiovascular disease. Whether chronic inflammation and cART exposure may also affect and potentially accelerate the biology of ageing and thereby enhance the premature onset of ageing-associated co-morbidities is an area of active investigation.
Tuberculosis (TB) has declined more rapidly in the Americas than in other regions of the world: from 1990 to 2012, TB prevalence and mortality decreased respectively from 103/100,000 to 40/100,000 and from 5.9 to 1.9/100,000. Similarly the incidence of TB has been reduced from 59 to 29/100,000. In 2012, only 3% of the TB cases worldwide occurred in the Americas. Haiti, Bolivia and Guyana have the highest TB incidence (Haiti 213; Bolivia 127; Guyana 109; per 100,000). Haiti and Bolivia are also the only two countries in the region for which the WHO is unable to estimate TB mortality using vital registration measurements. TB prevalence in Haiti has increased after the 2010 earthquake with 13% more cases nationwide in 2012. TB treatment success rates in the Americas are relatively low, with a 75% cure/completion rate (84% in Haiti) vs. 87% for the world. Outcomes are particularly poor for retreatment cases, with a cure/completion rate of only 62% (72% in Haiti); 10% of retreatment cases died, 4% failed and 24% defaulted. An estimated 2.2% of new cases and 14% of retreatment cases in the Americas have MDR-TB. Yet, in 2012 only 5481 of 23,811 (23%) retreatment cases were tested for MDR-TB. The management of HIV-TB co-infection is also challenging, with 16% of TB patients co-infected with HIV. Only 56% of TB patients have been tested for HIV (81% in Haiti), and of the 20,355 known to be co-infected, only 13,699 (76%) are on antiretroviral therapy, though it is universally recommended. Despite great success in reducing TB burden in the Americas, with much effort to be accomplished by Haiti, Bolivia and Guyana, there are gaps in care that must be bridged for the entire region.
In 2012, the Pan American Health Organization estimated 280,000 new tuberculosis cases in the Americas, with an estimated incidence of 29/100,000 individuals, 16% associated with HIV. Brazil, Peru, Mexico, Haiti, Colombia and Bolivia account for 72% of the cases in the region. In large countries, tuberculosis is concentrated in certain populations, in particular among urban high-risk groups including HIV individuals, immigrants from TB high-incidence countries and underserved populations such as homeless and those with a history of drug and alcohol abuse. At the country level, elimination of TB requires the implementation of multiple measures focused on different layers of the population. The particular complexities of big cities, such as population density and social structure, create specific opportunities for transmission, but also enable targeted TB control interventions. The presentation will describe the current TB situation in the Americas, the most affected populations, and the general and specific social, educational, operational, legal and monitoring TB control interventions required for the control, as well as providing some recommendations with particular emphasis on HIV-related TB. In addition, a summary of new tools for prevention and control, including new technologies, vaccines and new drugs will be presented.
Twenty years ago, PACTG 076 demonstrated that administration of zidovudine during pregnancy, labour and to the newborn for six weeks reduced perinatal transmission by nearly 70%, providing the first demonstration of “treatment as prevention.” In the United States, a comprehensive national response resulted in rapid implementation of the PACTG 076 regimen, with decreases in perinatal transmission from 25% before 1994 to ~5% within two years. Currently, in well-resourced health systems like the United States, where 98% of women receive prenatal care primarily starting in the first trimester, with implementation of opt-out universal HIV testing of pregnant women, the provision of combination antiretroviral therapy (cART) regimens during pregnancy, with individualized, laboratory-guided management; elective caesarean delivery if HIV RNA is >1000 copies/ml near delivery; infant antiretroviral prophylaxis; and avoidance of breastfeeding, the risk of perinatal transmission has been reduced to about 1%, and elimination of new perinatal infections is now possible. Current United States guidelines for use of antiretroviral drugs for maternal health and reduction of perinatal transmission will be discussed, as well as potential challenges to the elimination of new perinatal infections. Such challenges include continuing new infections in women, late/no prenatal care, lack of identification of HIV status and late or no antiretroviral prophylaxis. Additionally, with the dramatic decline in perinatal infections, there are now thousands of HIV-exposed infants who are now happily uninfected, but who have in utero exposure to multiple drugs with limited data on long-term safety. A major challenge – but critical need and ethical obligation – will be to evaluate potential long-term effects of antiretroviral exposures in uninfected children.
In high-income countries, MTCT transmission has been virtually eliminated through universal HIV testing and counselling, access to effective antiretroviral prophylaxis and treatment, safer delivery practices, family planning and safe use of breast-milk substitute. Scientific and programmatic evidence shows that PMTCT interventions can reduce the risk to less than 5% even in breastfeeding populations. Latin America has a prevalence of 1.4 million HIV cases, with 6300 (3600–9600) children newly infected per year. The estimated global rate of MTCT in 2011 for LA was 14.2% [5.8–18.5] (9.2% without breastfeeding). Key targets for 2015 include reducing the MTCT rate to <2% and having less than 0.3 new infant HIV infections per 1000 live births and less than 0.5 congenital syphilis cases per 1000 live births. Increasing antenatal access to medical care, incorporating educational strategies to increase accurate perception of personal risk and including rapid assays to meet the demand, where HIV testing is the main barrier to PMTCT, are ways to improve counselling and testing. The main challenges are to strengthen health systems, and service delivery models that integrate prenatal care, HIV and sexual and reproductive health, and to promote early initiation of prenatal care and improving the quality of prenatal care. The regional coverage of HIV testing in pregnant women increased from 29% in 2008 to 66% in 2011. Calculated coverage of antiretroviral therapy for HIV-infected pregnant women was 70% in 2011. Many countries in the region are close to reaching the goals of elimination of MTCT of HIV. Early diagnosis and treatment saves children's lives. Expanding the availability of early infant diagnostic testing is a critical need. Primary prevention should be re-enforced. There is a need to identify populations that are still not reached and the reasons why they have no access or are not using available services, and to eliminate the gaps of inequity, especially in young women, aborigines and other women in vulnerable situations.
From the 127 HIV perinatally infected girls aged 14 years or older followed by Instituto de Infectologia Emílio Ribas, 30 became pregnant (23.6%), making a total of 39 pregnancies and 35 babies (four miscarriages). Two adolescents got pregnant three times and four got pregnant twice. The gestational age varied from 32 to 39 weeks (mean 36.8 weeks) with three cases of premature birth. Twenty-six adolescent mothers had a fixed sexual partner from which 24 were aware of the adolescent's HIV status. Two adolescents got pregnant through domestic artificial insemination. Three adolescents mentioned the use of illicit drugs while pregnant. Only 50% of the adolescents showed undetectable viral load results by the end of the pregnancy, while the remaining showed results varying from 260 to 67,127 copies/mm3 (mean 20,555 copies/mm3). Ritonavir boosted lopinavir was part of the antiretroviral treatment to prevent mother-to-child transmission in 82.6% of the cases and all newborns received oral AZT syrup prophylaxis. Caesarean section was the mode of delivery of choice in 32 pregnancies, while vaginal delivery was applied to three cases. Just one of the newborns was HIV infected. The weight at birth ranged from 1900 to 3280 g (mean 2546 g) and the height at birth varied from 42 to 49 cm (mean 45.5 cm). This group of HIV vertically infected adolescents, despite being educated since childhood concerning their HIV status, the risk of HIV transmission through sexual relationships, the care with their sexual partners and the risk of an undesirable pregnancy, showed a high rate of pregnancies.
HIV drug resistance (DR) surveillance in antiretroviral treatment (ART)-naïve individuals (pre-ART DR, PDR) is key to maximizing the long-term effectiveness of ART regimens and to ensure sustainability of ART programs. We assessed HIV PDR in Mexico and Central America.
ART-naïve individuals from Mexico (
Belize showed the highest PDR prevalence (17.9%), followed by Nicaragua (12.7%), Panama (12.6%), Honduras (11.7%), Mexico (8.5%,
PDR prevalence in Mexico, Guatemala, Panama, Nicaragua and Honduras remains at the intermediate level but is high in Belize. Different epidemiologic scenarios can be observed in different Mesoamerican countries warranting local HIV molecular epidemiology and TDR surveillance studies.
We describe a model of HIV/AIDS control and prevention programme in an urban context developed for Mexico City.
The programme started implementation in 2008 with the aim of increasing the access to Voluntary Counselling and Testing (VCT) services, reducing the time between HIV diagnosis, incorporation to clinical services and antiretroviral therapies (ART) initiation, and improving the effectiveness of the ART universal access programme. The main components of the model include: 1) articulation between VCT services, specialized laboratory, and provision of clinical services; 2) co-ordination between federal and local government stakeholders with civil society organizations for people living with HIV (PLWH), vulnerable populations and human rights defence; 3) outreach programmes for vulnerable populations; and 4) strengthening of clinical services provision. These services are located or centrally co-ordinated at the Clínica Especializada Condesa, a primary care clinic for HIV care.
Mexico City HIV/AIDS Programme integrated the provision of preventive and care services in a primary care HIV/AIDS Clinic in 2008. Annual number of HIV tests increased from 2691 to 29,799 in 2007–2014. Time between pre-VCT session and delivery of results in post-VCT session decreased from 14 days to 36 hours (median). The number of patients receiving care at Clínica Especializada Condesa increased from 3870 to 10,064 in 2008–2014. The time from access to care to ART initiation decreased from four months to two weeks. The median time to achieve undetectable HIV-VL after ART initiation decreased from 8.2 to 3.3 months in 2008 to 2012. The programme now provides services for inmates, male and female sex street-workers, people living in the street, drug users and juvenile detainees and also provides specialized clinical care for trans-gender women, acutely HIV-positive patients, STDs, and reproductive and sexual health services for teenagers and sexual violence victims.
Mexico City HIV/AIDS Programme has implemented a multi-disciplinary approach for the prevention and control of the HIV-epidemic in an urban context in a middle-income country. The continuum between preventive, clinical care and supportive services and the cooperation of federal and local government instances with civil society organizations have been paramount for the programme's success.
We aimed to describe the frequency of CD4 and VL monitoring for HIV-positive patients (HIV + ) receiving antiretroviral therapy (ART) in Latin America and the Caribbean, assess adherence to recommendations in clinical guidelines, identify factors associated with lower frequency of monitoring and describe costs of monitoring patients on ART.
We used retrospective cohort data from all CCASAnet sites during 2000–2012. Patients initiating ART were included. Follow-up time after ART initiation was divided into 180-day periods. The primary outcome was the proportion of 180-day periods with adequate monitoring, defined as having at least one CD4 and one VL measurement. In secondary analyses, adequate monitoring was based only on CD4. Factors associated with monitoring frequency were assessed using multivariable Poisson regression models, with the number of 180-day periods with adequate monitoring as outcome. Median cost for CD4 and VL measurements per patient/year were estimated.
A total of 14,476 patients were included. Median follow-up time was 50.4 months (IQR: 27–82.3). CD4 were monitored from a median of 2.6 measurements/year for CMH-Argentina to 1.0/year for GHESKIO-Haiti; VL measurements ranged from 2.6/year for CMH-Argentina and INCMNSZ-Mexico to 0.9/year for IHSS/HE-Honduras; VL was not regularly measured at GHESKIO-Haiti. The mean proportion of periods with adequate CD4 and VL monitoring was 61.7% (95% CI: 52.3–72.8), ranging from 85.6% at INCMNSZ-Mexico to 25.6% at IHSS/HE-Honduras. The mean proportion of 180-day periods with adequate CD4 monitoring alone was 68.6% (95% CI: 57.5–81.8%). Rates ranged from 86.5% at INCMNSZ-Mexico to 48.2% at GHESKIO-Haiti. We will show in figures in the presented data the factors associated with adequate monitoring expressed in OR: increased age, and more recent initiation of ART, which were consistently positively associated with adequate monitoring. The costs of monitoring per patient/year were US$38 for CD4 and US$140 for VL but varied substantially across the region.
We observed that the adherence to recommendations of CD4 and VL monitoring for patients receiving ART is heterogeneous across the centres in CCASAnet, but on average is low. Costs of laboratory tests were also highly variable across sites. Our results are of use to national and international public health and policy organizations involved with HIV guideline development and implementation, and ART program cost planning.
Optimal adherence is critical to achieve the benefits of the antiretroviral (ARV) treatment and minimizes the risk of ARV resistance. Multiple aspects are involved in adherence in children and adolescents. Although, published evidence about strategies to improve it is scarce in our setting [
A prospective study was conducted in a cohort of HIV infected patients less than 25 years old. Patients taking ARV were evaluated to establish suboptimal adherence (SOA). Inclusion criteria were: HIV infection, taking ARV, viral load (VL) >1000 copies/ml, SOA reported by the primary physician, use of a mobile device. The intervention was based on mobile generic contact twice a month through any of the applications the patient chose (WhatsApp, Facebook, text message, etc.) during an eight month period. If the patient or parent required additional information, a feedback phone contact was generated. VL was performed before and after the intervention as an outcome measure of adherence.
Twenty-five of forty-seven patients identified as SOA were able to be contacted. One refused to participate and two have no mobile. Twenty-two patients were enrolled. Median age was 17.2 years old (range: 6–25); 15 (68%) were female; median baseline VL was 25,100 copies/ml (range: 500,000–1020 copies/ml), median log was 4.3 log (range: 3–5.7 log). Seven of twenty-two were contacted through their parents. Ten (45%) preferred to be contacted by WhatsApp, 8 (36%) by text message, 4 (18%) by Facebook and others. Each participant received a total of 16 contacts, 84% (296) were answered by the patient. Sixty-five percent (189) of the contacts generated additional requests about medications, appointments or symptoms. After eight months of strategy implementation 20/22 VL results were available. Thirteen of twenty (65%) were undetectable, 14/20 (70%) had VL <1000 copies/ml. Six of twenty (30%) VL had no changes.
The use of mobile technology improved adherence to treatment evaluated through VL measurement. The strategy is feasible in our setting. The reminder messages trigger additional contacts between patients and provider and may lead to better engagement with HIV care. Longer follow up time is needed to evaluate the effects of this intervention in the long term.
Chikungunya virus (CHIKV) is an alpha virus causing acute febrile illness characterized by crippling arthralgia [
During the most recent outbreak of CHIKV in the Caribbean, we selected randomly from two outpatients centres in the capital city Santo Domingo, Dominican Republic. The sample consisted of 100 participants meeting the CDC case definition for CHIKV infection. Random distribution of cases (HIV negative) and controls (HIV positive) were allocated based on HIV known status at the moment of presenting to care. After informed consent IgM antibodies were identified to differentiate dengue from CHIKV infections. CD4+ T lymphocytes and HIV viral load, and HIV medications were reviewed. Epidemiological data was analyzed with Epi Info™7.
A total of 100 participants fulfilled inclusion criteria. Distribution of cases and controls was 1:1. Men constituted 23% (
This study comparing clinical manifestations in HIV-negative and -positive patients during the acute phase of Chikungunya did not reveal any major differences in acute phase symptoms. The effect of lower CD4+ T cells on acute symptoms must be studied in patients with more advanced disease.
Clinical findings of CHIKV infection in HIV-positive and HIV-negative cohorts
| Signs and symptoms | VIH (+) (%) | % (95% CI) | VIH (–) (%) | % (95% CI) | Odds ( |
|---|---|---|---|---|---|
| Fever | 48 (94.11) | (87.65–100%) | 49 (100) | (100–100%) | 0.1400 ( |
| Headache | 35 (68.62) | (55.88–81.36%) | 37 (75.51) | (63.47–87.55%) | 0.7095 ( |
| Myalgia | 48 (94.11) | (87.65–100%) | 46 (93.87) | (87.15–100.59%) | 1.0435 ( |
| Upper limb arthralgia | 47 (92.15) | (84.77–99.53%) | 47 (95.91) | (90.36–101.46%) | 0.6667 ( |
| Lower limb arthralgia | 47 (92.15) | (84.77–99.53%) | 49 (100) | (100–100%) | 0.1371 ( |
| Arthritis | 36 (70.58) | (58.07–83.09%) | 38 (77.55) | (65.87–89.23%) | 0.6947 ( |
| Rash | 37 (72.54) | (60.29–84.79%) | 35 (71.42) | (58.77–84.07%) | 0.9600 ( |
| Nauseas | 15 (29.41) | (16.91–41.91%) | 24 (48.97) | (34.97–62.97%) | 0.4340 ( |
| Vomiting | 9 (17.64) | (7.18–28.1%) | 12 (24.48) | (12.44–36.52%) | 0.6607 ( |
| Diarrhoea | 17 (33.33) | (20.39–46.27%) | 17 (34.69) | (21.36–48.02%) | 0.9412 ( |
| Abdominal pain | 21 (41.17) | (27.66–54.68%) | 23 (46.93) | (32.96–60.9%) | 0.7913 ( |
| Others (pneumonia, encephalitis, Guillain-Barre) | 16 (31.38) | (18.64–44.12%) | 29 (59.18) | (45.42–72.94%) | 0.3153 ( |
By April 2013, the first autochthonous transmission of chikungunya virus (CHIKV) in the Dominican Republic was confirmed [
During the peak of incidence of cases, patients attending an outpatient clinic for HIV-positive individuals in Santo Domingo with signs and symptoms suggestive of CHIKV infection (fever>38°C, severe arthralgia or arthritis) were asked to participate in this study. After informed consent was collected a blood sample was obtained for CHIKV-IgM detection and analyzed using reagents provided by Aria CHIKV IgM detection (CTK Biotech, CA).
A total of 100 cases were tested. Women represented 73% (
This study suggests that immunological status is not affecting earlier inflammatory markers against CHIKV infection in HIV(+) individuals. Noteworthy, persistence of specific CHIKV IgM was detected after four months of onset of symptoms in HIV(+). Comparison of CHIKV-IgM in acute onset with an immune competent cohort demonstrates that rapid diagnostic test may be useful among immunosuppressed individuals to differentiate Dengue virus infection and CHIKV in the Americas where both pathogens co-circulate due to the presence of competent Aedes vectors.
CHIKV specific IgM detection in HIV (−) and HIV (+) individuals after onset of symptoms, Dominican Republic 2014.
CHIKV IgM detection in HIV (+) and HIV (–) individuals, Dominican Republic 2014
| HIV(+) | HIV (–) | ||||
|---|---|---|---|---|---|
| ( | (%) | ( | (%) | ||
| CHIKV IgM (+) | 11 | 21.57 | 15 | 30.61 | |
| CHIKV IgM (–) | 40 | 78.43 | 34 | 69.39 |
In 2011, Centers for Disease Control and Prevention (CDC) published the first HIV treatment cascade with data from 2008 and sets the basis to implement health policy in the areas of opportunity to stop the epidemic [
It is a retrospective study. We used data from the National System for Logistics Administration and Surveillance of ARV in Mexico (SALVAR in Spanish) up to the 31 December 2014, the database of the CIENI/CENSIDA and the database of the HIV Prison Programme in Mexico City. Criteria for inclusion: Incarcerated male patients with HIV infection in Mexico City during 2014 in Santa Martha Acatitla Penitentiary.
We started 2014 with 184 patients, adding 60 new patients throughout the year, 13 recidivists, 67 were freed and 9 died. At 31 December 2014, 206 HIV patients remained incarcerated, of which 92.2% (190) are linked and retained to health care (concentrated in the prison of Santa Martha Acatitla), of which 87.4% are on HAART, with 72.8% under virology control (VL<200) and 63.1% undetectable (VL<40) (
It is necessary to strengthen the diagnosis of HIV in prison settings. Linkage and retention in medical care is covered in this model (HIV Programme in Prisons), alongside working with improving adherence to HAART in order to increase the levels of undetectability. The model of supervised daily dosage has given partial effective results given that the ARV is provided daily but it does not guarantee that the patients swallow the pills. Another important problem is how to link 100% of patients to ambulatory care once they obtain their freedom. The HIV Programme in Prisons of Mexico City is effective and can be replicated in different penitentiary systems in other states and countries, but not in the general population.
Data from the National System for Logistics Administration and Surveillance of ARV in Mexico (SALVAR) 31 of December, 2014. Database of the CIENI/CENSIDA, 2014. Database of the HIV Prison Programme in Mexico City 2014.
We test conditional economic incentives (CEI) to motivate self-protection and health-care-seeking behaviours among male sex workers (MSW) in Mexico City. We present baseline and follow-up results for MSW, aged 18–30, recruited in Mexico City (
Participants were randomized into four groups. In Treatment 1 (
Higher incentives were associated with higher participation rates. Incidence of syphilis was lower (AOR: 0.4; CI: 0.1–1.0) among participants in the high-CEI group compared to the control group. Greater condom use self-efficacy was found among participants of the UEI group (AOR: 5.4; CI: 1.1–26.6) compared to the control group; and significant reduction in the number of non-commercial sexual partners was found in the medium-CEI group (coefficient: −1.6; CI: −3.3–0.0).
CEI and UEI are feasible and acceptable among MSW in Mexico City. CEI and UEI seem to help to reduce risk behaviours. Inconvenience fees (transport compensation) help increase retention and linkage to care. A larger randomized controlled trial is justified to test CEI and UEI using a fully-powered sample size for primary and secondary outcomes.
Early diagnosis of HIV infection is crucial for early treatment, which can lead to better individual prognosis and some benefits in public health, such as reduction of the HIV transmission. One of the proposed strategies to improve the opportunity of early diagnosis is to widespread the HIV screening to different settings, including health care units. It has been shown that routine HIV screening could be justified in cost-effectiveness terms in settings where the prevalence of undiagnosed HIV is 0.1% or greater. The aim of the present study was to determine the prevalence of undiagnosed HIV infection at the emergency room in a city considered to have a low prevalence of HIV in its population [
A cross-sectional study was performed at the Hospital General Regional de Leon (HGRL). Leon is the biggest city in Guanajuato, which is located at the centre in Mexico and is considered one of the states with lowest prevalence of HIV in the country. During a 10-month period, HIV rapid testing was offered to all patients attended in the emergency room area, counselling was given to all patients and those who accepted to participate signed an informed consent. Patients were excluded from HIV testing if they (1) were unable to provide consent for HIV testing as determined by the clinical staff (e.g. altered mental status or critical illness); (2) were detainees or prisoners; or (3) self-identified as being infected with HIV. Rapid HIV test was made using the NEOGEN® test. If the first test was negative no further tests were made, and the result was reported as non-reactive. If the first test was positive, confirmation was made by RT-PCR. Results were analyzed and presented using descriptive statistics.
During the study period 1823 rapid tests were made, 17 of them were reactive and 15 were confirmed as positive by RT-PCR, for a prevalence of undiagnosed HIV infection of 0.8% (95% CI: 0.4–1.2). Acceptance rate was 96%.
Considering the high acceptance for HIV rapid testing and the prevalence of undiagnosed HIV infection found, we can conclude that widespread screening of HIV in health care settings should be considered even in regions where the assumed prevalence is low.
Antiretroviral therapy (ART) has reduced morbidity and mortality related to human immunodeficiency virus (HIV) infection, but in spite of this advance, HIV mutations decrease antiretroviral susceptibility, thus contributing to treatment failure in patients [
This cross-sectional study of a non-probabilistic convenience sample was based on biological samples provided by DUs attended at Central Laboratory and STD/AIDS Reference Unit of the State of Piauí. In total, 107 DUs were recruited in Teresina, Northeast Brazil [
Primary drug resistance mutations ranged from 10% (IAS-USA) to 14% (SDRM). High level resistance to at least one antiretroviral drug was observed. T215D/S revertant mutations were identified in 8/107 patients. HIV-1 subtype B represented 84.1%, subtype F1 7.5%, subtype C 3.7%, B/F1 2.8% and two samples was a F1/C/B mosaic. HIV-1 subtype C sequences formed a monophyletic cluster with other Brazilian subtype C sequences.
Our HIV-1 pol sequences from Piuaí include the important inland HIV-1 subtype C sequences and help compose the molecular epidemiology map of HIV-1 in Brazil. This data also show that a significant proportion of DUs presented important drug resistance mutations. Therefore DUs from this setting may benefit from pre-treatment genotypic testing to optimize the choice of antiretroviral drugs and to help control HIV-1 transmission.
A high prevalence of HIV infection in prisons has been recognized as a significant health problem in developed countries. To this point, it is important to evaluate our prison health-system to see if the model of care is adequate or not and to look for new care strategies to achieve good HIV-control into prisons.
Cross-sectional, retrospective study of HIV-positive prisoners. To describe the main risk behaviours for HIV-infection, its control, the prevalence of comorbidities and other co-infections in prisoners who were treated in the HIV-Unit of Hospital Civil de Guadalajara, Mexico. All HIV-infected prisoners who were receiving medical care at our HIV-unit from May 1st 2013 to May 31st 2014 were included. Data were obtained from an electronic database of medical records.
A total of 88 patients were included, 95% were male, with a median age of 36 years, all Hispanic. The principal risk factors to HIV-infection were unprotected heterosexual intercourse (55%), following of homo/bisexual intercourse, intravenous drug use (45 and 33%, respectively). The 71% of patients were in an advanced stage of HIV infection and 72% of the patients on HIV-treatment, reached HIV control with undetectable viral load. The 21% had Syphilis. We found a serologic evidence of Hepatitis C co-infection in 33%, the majority was genotype 1a and none received HCV treatment, moreover, the 8% of patients had serologic markers for hepatitis B. Regarding opportunistic infections; pulmonary tuberculosis was the most frequent (36%), followed by disseminated histoplasmosis (10%). The principal comorbidity was dyslipidaemia in 42 and 16% had metabolic syndrome. The 89% used to smoke daily, 76% used any kind of drug (principally, marijuana and cocaine) and 85% were alcohol consumers.
Efficiency of highly active antiretroviral therapy (HAART) among our prisons is higher compared to other cohorts [
Determine HIV and sexually transmitted infections (STI) prevalence and respective sociodemographic.
Cross-sectional retrospective study performed at Condesa Specialized Clinic of Public Health Services of the Federal District. The sample was made up of female adolescents in juvenile correctional facilities in Mexico City. All patients have a clinical history, physical exam, cervical cytology, colposcopy, lab exams for HIV, HBV, HCV and Syphilis.
Seventy-two patients were studied. The average age was 16.5 years. Education was: 45.8% primary, 40.2% secondary and 8.33% without schooling. The average age for onset of sexual activity was 14 years and the average number of sexual partners was 4.4. Reported frequency of condom use was 70.8%; 55.5% were nulligravid and 38.8% had been pregnant once (two adolescents were pregnant during physical examinations), 2.7% had two previous pregnancies and 2.7% has three previous pregnancies and 8.3% had at least one abortion. All adolescents that had previous pregnancies had only primary education. 11.1% reported incidents of sexual violence during childhood and 81.9% reported some form of drug use, with an average age of onset for drug use at 12 years. The drugs reported with more use were: inhalants (55.5%), alcohol (43%), tobacco (37.5%) and cannabis (31.9%). Most of the drug using adolescents only had primary education. STIs were found in 33.3% of the population, the more frequent being: acuminated condiloma and low grade intraepithelial lesions (6.9% each). In second place Molluscum Contagiosum and high grade intraepithelial lesions (5.5% each), Human Papilloma Virus and Syphilis (4.1% each). HIV was 1.3%. In the group of patients with an STI, 12.5% had two or more. The age of onset for sexual activity for the adolescents with STI was 13.8 years and had an average of about five sexual partners at the time of the medical exploration. About half of adolescents with an STI (54%) had primary education and the other half (46%) had secondary education.
HIV prevalence in this group of adolescent females was high. Drug use and pregnancy were related to educational levels attained while early age of onset of sexual activity was more related to having an STI. Sexual education interventions are needed for adolescents in correctional facilities for further prevention of STI and unwanted pregnancies.
The infection of the human papilloma virus (HPV) of high risk (HR) is a necessary factor for cervical lesions and invasive cervical cancer (ICC) [
Learn the HPV (HR) and cervical lesions prevalence, describing the distribution of different genotypes, and the epidemiologic clinical characteristics of women co-infected with HIV and HPV (HR). Identify the cytological characteristics in HIV-positive women that frequent the Condesa Specialised Clinic in Mexico City. The cohort was made up of 401 HIV-positive women. The women had a gynaecological exploration, cervical cytology, colposcopy and HPV typfication, and biopsy if necessary. The data was obtained through questionnaires with socio-demographic, behavioural and clinical variables.
HPV (HR) infection prevalence was 34.41%. ASCUS, LSIL and HSIL prevalence was 11.85, 29.63 and 17.78% respectively. With negative genotyping ASCUS was 4.94%, LSIL was 25.1% and HSIL was 4.56%. The more frequent genotypes were HPV NO-16 NO-18 (63.04%), HPV16 (16.67%) and HPV18 (13.04%). Factors associated to HPV (HR) were: age, last PAP abnormal. Factors associated to cytological alterations were: first sexual relation <18 years, last PAP abnormal, CD4 count <200 cells/ml and VL >10,000 copies/ml.
Known factors associated with HIV infection such as low CD4 count and a high viral load are predictors of cytological alterations in this group of patients. There is a high prevalence of HPV (HR) infections and genotypes with a high oncological risk in the study group. Behavioural factors such as age of onset for sexual activity may play a role in these conditions.
Tuberculosis (TB) continues to be a major cause of morbidity and mortality in HIV patients globally, and this is particularly true in Peru. Our objectives are to describe the frequency, clinical characteristics and outcomes of tuberculosis in HIV/TB co-infected patients at a tertiary care hospital in Lima, and compare these findings among patients receiving highly active antiretroviral therapy (HAART) chronically (Group 1), recently started (Group 2) and not receiving HAART (Group 3).
Retrospective review of medical records of HIV patients who developed TB during 2014 at Guillermo Almenara Hospital in Lima, Peru. Group 1 included patients receiving HAART for >180 days. Short-term mortality was defined as death during hospitalization or within 30 days post-discharge.
There were 23 cases of TB during the period of study. Affected patients were 87% male with a mean age of 37.9 years and a mean CD4 count of 62. Thirteen cases were pulmonary (56.5%). Extra-pulmonary presentations (10/23, 43.5%) included one CNS involvement (4%), one gastro-intestinal disease (4%) and three multi-organ involvements (13.04%). Twenty-one diagnoses were confirmed microbiologically, 34.7% was MDR and one case was XDR. There were seven (30.4%) patients in Group 1, four (17.4%) in Group 2 – one associated to immune reconstitution – and 11 (47.8%) in Group 3 – associated with recent HIV/AIDS diagnoses. Short-term mortality was significant only in Group 3 (27.3%). Time to TB diagnosis averaged 18.8 days, and diagnoses delays were related to complicated or atypical presentation or lack of access to microbiological confirmation.
TB significantly affected HIV-positive patients. Short-term mortality was high in patients not receiving HAART. Although a relatively low proportion of cases had microbiologically confirmed diagnoses, drug resistance was documented for a high number of cases and higher than other Latin-American countries.
Determining the genotype of a virus is important for the development of diagnostics, prophylaxis and effective antiviral treatment. HIV-1 has high degree of diversity and variability: four groups, nine subtypes and seventy-two circulating recombinant forms (CRFs) [
A descriptive, retrospective study, which included sequences of genes protease (PR) and reverse transcriptase (RT) of HIV-1 was performed. Testing was conducted at the Laboratory of Molecular Biology Dinamica IPS Colombia (June 2011 to October 2014). For genotype sequences RT-PR of HIV-1, the TRUGENE HIV-1 Genotyping Assay (Siemens) using the DNA GeneObjects Sequencing System 4.1 software was used [
We genotyped and analyzed a total of 301 samples from HIV-1 positive Colombian patients, with failure to antiretroviral therapy. Geographical distribution of the samples: 23 (7.6%) from Bogotá, 123 (40.9%) from Medellin, 135 (44.9%) from Cali and 20 (6.6%) from Barranquilla. In Colombia subtype B was found in 245 (81.4%) of the cases. The recombinant subtype D/B was identified in two cases (0.66%) and six different CRFs from 54 patients were distributed as: 1 (0.3%) case carrying CRF02_AG, 11 (3.65%) cases with CRF03_AB, 2 (0.6%) cases with CRF05_DF, 25 (8.3%) cases with CRF07_BC,7 (2.33%) with CRF08_BC, in 8 (2.66%) cases CRF12_BF was found.
In Colombia subtype B predominates in more than 80%, similar to other South American countries [
Histoplasmosis is the second most frequently diagnosed systemic fungal infection in Argentina. Among people living with HIV (PLWH) and low CD4 counts (i.e. <100 cells/mm3), it presents as a disseminated disease. Microscopic examination and culture remain today as the reference methods for diagnosis. However, their sensitivity is limited and culture might take some weeks. In this context the detection of urinary antigen (UAg) for
Between April and December 2014, PLWH with ≤100 CD4/µl on follow-up in a major public HIV centre in Buenos Aires, Argentina, underwent routine screening of UAg through of an antigen capture enzyme-linked immunosorbent assay (ELISA, IMMY). A cut-off of 0.5 units was considered positive for histoplasmosis. In addition, lysis-centrifugation fungal blood culture and antibodies detection using immunodiffusion were performed. For patients with skin lesions direct examination and culture were also performed.
In total, 114 patients were included: 63.1% were male, the majority was currently off ART (78.9%) and the median CD4 count was 44 cells/µl (IQR 18–81). With the standard algorithm, seven patients were diagnosed as having histoplasmosis (three had positive blood culture plus histopathological diagnosis, two had only histopathological diagnosis while the other two had only blood culture diagnosis). Only one sample had positive serology. Four additional patients were identified with UAg positive. Two of them were symptomatic and improved with itraconazole, the remaining two were lost to follow up. When we included UAg positive samples as cases the prevalence of histoplasmosis increased from 6.1 to 9.6%. Sensitivity, specificity, VPP and VPN were 71, 96, 63 and 98%, respectively.
UAg increased the diagnosis rate of histoplasmosis and has higher sensitivity than other serological tests based on antibodies. These preliminary findings suggest that screening for histoplasmosis infection among PLWH and advanced disease in our setting might be an effective strategy to improve clinical care of PLWH.
TDF/FTC/RPV has advantages among STR including greater convenience, tolerability and lower drug-drug CYP3A interactions. Main drawbacks are need for food intake, concomitant PPI contra-indication and compromised antiviral activity when various NNRTI mutations are present. We evaluated TDF/FTC/RPV in a real life setting with focus on clinical and virological vigilance.
OCEAN II is a prospective, two-centre observational study. From September 2012 to December 2013, all antiretroviral naïve patients with HIV RNA <100,000 copies/ml or wish to switch for simplification were considered for treatment with TDF/FTC/RPV. A systematic review of potential obstacles to TDF/FTC/RPV administration was undertaken, including food requirement, PPI co-administration, physician's issues regarding adherence, and in case of undetectable plasma HIV RNA, DNA genotyping to detect archived RPV and/or NRTI-associated resistance mutations.
TDF/FTC/RPV was discussed in 498 patients: TDF/FTC/RPV was not offered to 194 patients (39%), mainly for NNRTI or NRTI resistance on genotypic testing (historical RNA or current proviral DNA) and/or history of virologic failure on dual NRTI therapy or NNRTI-containing regimen (
TDF/FTC/RPV is suitable for most patients. Strict clinical and virologic screening was associated with a low risk of virologic failure. In this real-life experience, 22% of patients stopped therapy during two years of follow-up, most frequently for minor events.
The introduction of highly active antiretroviral therapy (HAART) has significantly improved life expectancy on HIV-infected subjects and the population is becoming older. The CDC considers elderly HIV-patients at the age ≥50 years due to the impact of HIV-infection
Patients were retrospectively analyzed at the HIV-unit in Hospital Civil de Guadalajara. Patients ≥50 years currently being followed-up at the site were included. Data were obtained from an electronic database. Significance level was established ≤5%.
A total of 393 patients were included which represent the 17% of the total of patients currently followed-up at the site. Eighty-two percent were male, and age ranged from 50 to 90. Women have a higher Body Mass Index (BMI) than men and are mostly classified as pre-obesity. The 37% of patients were diagnosed at an age ≥50 years, and age at diagnosis was higher in women (mean 47 vs 49,
Contrary to the reports on young women, it seems that old women reached a good virologic response and immune reconstitution percentages as well as old men. The higher BMI and the association of the VACS Index to markers of chronic inflammation and hypercoagulability [
After the introduction of effective antiretroviral therapy (highly active antiretroviral therapy (HAART)), the average life expectancy of patients with HIV infection have approached the general population.
Analyze the causes of death in HIV patients according to the treatment regimen used.
Retrospective analysis of medical records of HIV patients who died in our service since 1985. For analysis, patients were divided into three periods, depending on the type of treatment carried out – from 1985 to 1996 (pre-HAART), 1997 to 1999 (partial use of HAART) and 2000 to 2013 (HAART).
Between 1985 and 2013 were observed in our service 4962 HIV-infected patients, of which 1056 died (21%). Of the deaths, 157 (15%) occurred before 1997, 235 (22%) in the period 1997–1999 and 664 (63%) between 2000 and 2013. The mortality rate was, respectively, 19, 12 and 13%. Of the deaths, 86% were male in the pre-HAART period, maintaining a similar proportion in the interim and HAART period (83 and 86%). Considering the average age at the date of death, this was 35 years in the pre-HAART era, 33 in partial use of HAART and 40 years in the HAART period. The average length of knowledge of HIV infection was 20 months, 25 months and 64 months, respectively. The mean nadir CD4 lymphocytes was similar in the three periods: 128, 112 and 96 cells/mm3; since the average value of CD4 to the date of death increased from 98 in the pre-HAART period, to 101 in the partial use of HAART and 130 cells/mm3 in the HAART era. Only 7% of patients presented suppressed on the death of the height in the pre-HAART era, a percentage that has remained by 13% in the HAART era.
It was possible to identify the causes of death in 889 of the cases (167 in the cause remained undetermined). The characterization is summarized in the table below:
There was a significant fall in the mortality rate of the first and the last years of HIV infection as a result of more effective therapeutic use. The introduction of HAART has led to a change in the cause of death, with relative increase in mortality related with chronic hepatic disease, not opportunistic infections and tumours.
| Death cause | Pre-HAART (%) | Partial use of HAART (%) | HAART (%) |
|---|---|---|---|
| AIDS defining illness | 63.9 | 71.1 | 51 |
| Non-opportunistic infections | 21.1 | 17.1 | 28.3 |
| Chronic hepatic disease decompensation | 4.5 | 5.9 | 12.6 |
| AIDS non-defining tumour | 0 | 0.7 | 3.5 |
| Others | 10.5 | 5.3 | 4.6 |
Mental disorders are more prevalent in people with HIV [
The sample was patients recently diagnosed with HIV at the Condesa Specialized Clinic from 2013 to 2014. A psychiatrist or clinical psychologist, based on the clinical interview and ICD-10 criteria, made the diagnosis of the mental disorder for each patient. Impulsivity and depressive symptoms were measured with adapted self-administered short versions of the State Impulsiveness Scale (SIS) and the Beck Depression Inventory (BDI). We performed ANOVA analysis to compare age, education, viral load, CD4 count, SIS and BDI score between groups.
We obtained a sample of 1350 patients, of whom 89.1% were male, 9.0% were female and 1.9% were transgender women. The mean age was 31.9 (±9.3) years, and the mean of education was 11.9 (±3.6) years. The mean viral load was 268,017 (±934,431) copies/ml, and the count of CD4 was 333 (±255) cells/µl. A total of 40.8% of the sample had no mental disorder, 31.8% adjustment disorder, 13.0% major depressive episode, 4.8% general anxiety disorder, 5.4% ethanol misuse, 2.6% cocaine misuse and 1.6% cannabis misuse. The mean difference of age (
This study suggests that HIV patients with high levels of impulsivity and depressive symptoms are aligned with their clinical mental diagnosis. This could be helpful in clinical practice to identify those HIV patients with impulsivity and depressive symptoms that are at greater risk to have unprotected sex or poor HAART adherence.
Lipid abnormalities in patients on HAART have been associated with use of boosted protease inhibitor (bPI), but some of them, such as atazanavir, are considered more lipid-friendly. Our aim was to investigate the effects of boosted atazanavir (ATV/r) and lopinavir (LPV/r) on lipids, and the proportion of patients who need lipid-lowering agents (LLA), in Mexican HIV-positive patients.
We conducted a retrospective cohort study in patients followed at the HIV clinic of the INCMNSZ in Mexico City. Patients receiving LPV/r or ATV/r for at least one year, and having a complete lipid profile, were included. History of antiretroviral therapy (ART), use of LLA and triglycerides (TG), total cholesterol (TC), HDL and LDL were collected at baseline and after one year of the start of PI/r. The primary endpoint was changed in TC, LDL and HDL and TG, and proportion of patients who needed to use LLA. We considered severe dyslipidemia (SD) TC values ≥240 and/or TG≥500 mg/dl.
A total of 101 (14.8% female, median age 44 [21–75]) patients were evaluated: 48 (47.52%) received ATV/r and 53 (52.47%) LPV/r. Sixteen and fourteen patients received ATV/r and LPV/r, respectively, as first line treatment, whereas 32 and 39 had ATV/r and LPV/r as salvage. At baseline, in patients with ATV/r, 16 (33%) were using LLA and 14/48 (29%) had SD, while in those on LPV/r, 14 (26%) were using LLA and 4/53 (7.5%) had SD. Thirty (29%) patients started LLA during treatment, with a median duration of 6.76 mo., most of them fibrates (83%); 16 (53%) belonged to ATV/r, and 14 (46%) to LPV/r (
The main lipid alteration seen in our study was hyper TG, related to the use of ritonavir and not different between the two bPI studied. No significant differences in lipid profiles, use of LLA nor in presence of SD were found between both ATV/r and LPV/r, especially in cases with dyslipidemia before treatment. For cases with lipid abnormalities before treatment, other lipid-friendly drugs should be preferred.
Ultra-deep sequencing (UDS) allows the detection of HIV-1 drug resistance mutations with prevalence <20%. However, there are conflicting data on the utility and interpretation of reporting low frequency mutations. Using UDS with two different cut-offs compared to standard capillary sequencing, the aim of this study was to evaluate the changes in HIV genotypic test reports and their potential impact on patient care.
Over a 12 month period, 68 plasma samples from in-patients were collected and extracted on a QiaSymphony and amplified using an in-house polymerase chain reaction (PCR)-based assay. DNA was cleaned up and quantified prior to UDS (Illumina MiSeq®). Data was analyzed and a report generated using a bespoke pipeline, reporting drug resistance mutations (DRMs) at cut-offs of 1 to 1.99%, 2 to 19.9% and >20%. Clinical details were obtained with consent from in-patient test request forms. Virus strains included 30 subtype B, 24 subtype C and 14 non B or C subtypes; patients were drug-naïve (
Overall, a significant increase in resistance to antiretroviral therapy (ART) was detected using the more sensitive sequencing technology. A>2% cut-off resulted in a change to the resistance report from susceptible to resistant for 5/68 patients; a>1% cut-off increased this number to 22/68. Using established criteria, transmitted drug resistance was 19% with the 2% cut-off and 41% with the 1% cut-off, compared to 14% with traditional Sanger sequencing (20% cut-off). DRMs were more frequent in patients for whom ART therapy was failing. All classes of ART had a 10 to 13% increase in DRM detected.
The UDS assay performed well across a wide range of subtypes, viral loads and different populations of patients, increasing the detection of minority variants, especially DRMs at a 1 to 1.99% cut-off. This increase in detected DRMs led overall to a change in 22% of HIV genotyping reports. Taking this into account, it is suggested that a 1% cut-off should be routinely utilized. The clinical implications of reporting the variants will be discussed; however, further studies are needed in order to assess what the full impact of reporting these minority variants will be on treatment efficacy.
EVG/COBI/FTC/tenofovir alafenamide (TAF) (E/C/F/TAF) is an integrase-inhibitor-based single-tablet regimen in clinical development for use in HIV-positive adolescents. Pharmacokinetics, safety and efficacy from a planned interim analysis of the first clinical trial of E/C/F/TAF in adolescents are reported.
Treatment-naïve 12 to <18 year-olds weighing ≥35 kg with HIV-1 RNA >1000 copies/ml (c/ml), CD4 >100 cells/µl and eGFR >90 ml/min/1.73 m2 received E/C/F/TAF once daily in a prospective, two-part, 48-week, single-arm, open-label trial. Steady-state pharmacokinetic (PK) parameters were compared to an adult reference population by ANOVA and related to the range of exposures associated with antiviral activity in adults. Adverse events (AEs), laboratory tests and the proportion of subjects with HIV-1 RNA<50 c/ml were assessed through Week 24. Bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry.
The trial enrolled 48 adolescents with a median age of 15 years, median weight of 52 kg, 58% female, 88% Black, 13% Asian, 67% vertically infected, 35% with HIV-1 RNA >100,000 c/ml, median CD4 count 468 cells/µl and median serum creatinine (sCr) 0.57 mg/dl. TAF, TFV, EVG, COBI and FTC PK profiles of adolescents were consistent in adults. Of 23 subjects followed to Week 24, 21 (91%) had HIV-1 RNA <50 c/ml (
Therapeutic plasma concentrations of all components of E/C/F/TAF were achieved, consistent with potent antiviral activity of the regimen. Treatment was generally well tolerated through 24 weeks with a favourable renal and bone safety profile. These promising findings support E/C/F/TAF's eventual use in adolescents and its further evaluation in other paediatric populations.
E/C/F/TAF in adolescents: virologic success and CD4 recovery.
In children with HIV vertical transmission infection, successful response to antiretroviral therapy (ART) depends on the previous susceptibility to antiretroviral drugs. In consequence, the virological failure (VF) may emerge due to the presence of drug-resistant viruses and clinical outcome could be deleterious. In low-resource countries, the causes of VF are not routinely assessed by genotyping assays, resulting in a continuous failure to different ART combinations. The aim of the study was to describe the emergence of resistance mutations (RMs) during the follow-up of HIV-1-infected children with VF, compared to patients with successful treatment.
Longitudinal study including plasma samples from 37 vertically infected HIV children collected between 1998 and 2011. Eighteen basal samples were obtained before starting treatment from patients that responded to ART, and 57 samples were obtained from 19 patients with VF in different time points during the follow-up. The samples were stored at −70°C, until the genotypic analysis was performed. A nested RT-PCR was utilized to amplify a fragment of 1084 bp, including protease and reverse transcriptase (RT) sequence. The resistance genotype was determined using a Stanford Genotypic resistance interpretation algorithm.
In basal samples, none of the successfully treated patients had protease inhibitor (PI) drug resistance mutations (DRM) compared to patients with VF (26% had PI DRM). With respect to RT inhibitors (NRTI/NNRTI), 44% of the responders had RT DRM (NRTI (28%), and NNRTI (16%)), and for the VF group, 47% (NRTI (32%) NNRTI (16%)). During the follow-up of the patients with VF, 63% acquired new PI DRM (I54V (58%), V82A (53%), M46I (23%), I84V (21.1%)). Only 16% did not have acquired RT DRM, but 84% developed NRTI DRM (M184V (74%), T215 (42%) D67N (37%), K70R (32%), K219Q (26%)) and 37% developed NNRTI DRM (Y181C, K103N and K101E, 10% each one). Average time to PI DRM emergency was 11 to 34 months, for NNRTI was 2 to 26 months, and for NRTI was 12 to 37 months.
HIV-infected children with transmitted PI DRM had a higher risk to developed VF (OR 8.88). Transmission of RT DRM was not associated to the VF (OR 0.91). During the follow-up, more new PI DRMs were observed in those patients with transmitted PI DRM from the infected mother. Genotyping assay is recommended in naïve paediatric patients to reduce risk of treatment failure.
Genotyping tests were developed to help attenuate the impact of viral resistance. These tests can detect mutations associated with phenotypic resistance of HIV to antiretrovirals [
We conducted a retrospective cohort study, which included 16 children with virological failure and triple-class drug HIV resistance HIV-1 infection. All patients had protease and retrotranscriptase genotype, and they were evaluated using the Stanford HIV database for resistance mutation interpretation. Switch of antiretroviral (ARV) regimen was based on genotyping data, evaluated by an experts group in antiretroviral drugs resistance. The primary end point was virologic suppression (<50 copies/ml) and immunologic improvement at Week 24. Median and interquartile range (IQR) were used in descriptive analysis.
A total of 16 children were enrolled. The children's median age was 14.5 (IQR 11 to 16.5). Baseline median CD4+ cell count was 382 cells/mm3 (IQR 281 to 687) and median plasma HIV-RNA viral load was 15,855 copies/ml (IQR 2952 to 77,089). New drugs such as darunavir (13/16), tipranavir (3/16), raltegravir (13/16) and etravirine (3/16) were included in the new regimen. Median increased in CD4+ cells count to 640 cells/mm3 after 24 weeks of regimen based on genotyping data (
Well tolerated and effective in our patients, DRV/r and RAL provide potentially good options for heavily pre-treated HIV-infected children. Regimens based on genotyping data were effective for children who had virological failure with multidrug-resistant HIV-1 infection. We observed a sustained antiviral response and improved immunologic indices in multidrug-resistant paediatric patients. Long-term follow-up is necessary to warrant the ARVT feasibility and sustainability.
Tenofovir (TFV) is renally eliminated, and the prodrug, tenofovir disoproxil fumarate (TDF), has been associated with renal toxicity and reduced bone mineral density (BMD) and must be dose adjusted in patients with estimated glomerular filtration rate (eGFR) <50 ml/min. Tenofovir alafenamide (TAF) is a novel prodrug of tenofovir (TFV), that is not renally eliminated and at clinical doses results in 90% lower plasma TFV levels as compared to TDF. The safety and efficacy of a once-daily single-tablet regimen of elvitegravir, cobicistat, emtricitabine and TAF (E/C/F/TAF) was assessed in HIV-1-positive patients with mild to moderate renal impairment.
Virologically suppressed adults with stable eGFRC-G (Cockroft Gault) of 30 to 69 ml/min had their treatment switched from both TDF- and non-TDF-containing regimens to open-label E/C/F/TAF. Efficacy and safety data of Week 24 are described, including tests of renal function and BMD. Actual GFR (aGFR) was assessed with iohexol clearance in a subset of subjects.
Of 242 subjects enrolled and dosed, mean age was 58 years (range: 24 to 82), 18% were Black, 39% had hypertension and 14% had diabetes. Sixty-five percent were taking TDF-containing regimens prior to switch. At baseline, median eGFRC-G was 55.6 ml/min (33% eGFRC-G 30 to 49 ml/min). Ninety-five percent of subjects maintained HIV-1 VL <50 c/ml at Week 24 (FDA Snapshot). At Week 24, the median (Q1, Q3) change from baseline eGFRC-G was 0.4 (−4.7, 4.5) ml/min, eGFR-cystatin C 3.8 (−4.8, 11.2) ml/min/1.73 m2, and aGFR (
These 24-week data support the virologic efficacy and renal and bone safety of once-daily single-tablet E/C/F/TAF for use in HIV+ patients with mild and moderate renal impairment (eGFR 30 to 69 ml/min). Switch to E/C/F/TAF was associated with no change in aGFR and with reductions in proteinuria.
Off-target renal and bone side effects may occur with tenofovir disoproxil fumarate (TDF) use. Compared with TDF, tenofovir alafenamide (TAF) results in significantly reduced plasma tenofovir (TFV) and may have less renal and bone toxicity.
Treatment-naïve HIV-1+ adults were randomized 1:1 to a single-tablet regimen of E/C/F/TAF or E/C/F/TDF once daily in two double-blind studies. Assessments for all subjects included measures of glomerular and proximal renal tubular function, and bone mineral density (BMD). Four pre-specified secondary safety endpoints were tested: serum creatinine, treatment-emergent proteinuria, spine and hip BMD; fasting lipid parameters were also collected. Week 48 off-target side effects data from both studies are described.
Combined, the two studies randomized and treated 1733 subjects. Plasma TFV was >90% lower (mean (% CV) AUCtau 297 (20) vs 3410 (25) ng*hr/ml) in the E/C/F/TAF arm, compared to the E/C/F/TDF arm. Serum creatinine (mean (SD) change: +0.08 (0.124) vs +0.11 (0.217) mg/dL,
Through 48 weeks, subjects receiving E/C/F/TAF had significantly better outcomes related to renal and bone health than those treated with E/C/F/TDF; lipid outcomes favoured E/C/F/TDF. Collectively these data demonstrate important safety benefits of TAF relative to TDF, especially given the ageing of the HIV population and the need for long-term treatment.
Tenofovir alafenamide (TAF) is a novel tenofovir (TFV) prodrug that, when administered in the single-tablet regimen elvitegravir/cobicistat/emtricitabine/TAF (E/C/F/TAF), has >4-fold increase in intracellular TFV diphosphate and >90% lower plasma TFV levels compared to tenofovir disoproxil fumarate (TDF). Two phase 3 studies of identical design were conducted in distinct geographic areas comparing two single-tablet regimens, E/C/F/TAF and E/C/F/TDF, in treatment-naïve HIV-1+ adults.
Patients were randomized 1:1 to receive a single-tablet regimen of E/C/F/TAF or E/C/F/TDF once daily in two phase 3 double-blind studies. Primary end point was Week 48 virologic response by FDA Snapshot algorithm in a pre-specified analysis of the combined studies.
A total of 1733 subjects were randomized and treated: 15% women, 43% non-White, 23% viral load ≥100,000 copies/ml. Median baseline characteristics were: age 34 years, VL 4.58 log10 c/ml, and CD4 count 427 cells/µl. The primary objective was met, as E/C/F/TAF was non-inferior to E/C/F/TDF with 92 and 90%, respectively, having HIV RNA <50 copies/ml at Week 48 (difference +2%, 95% CI −0.7% to +4.7%,
Through 48 weeks of treatment, high virologic response rates were seen in patients receiving E/C/F/TAF or E/C/F/TDF, and similar responses were seen across subgroups evaluated. Drug resistance was <1%. Both regimens were well tolerated, and no unique AEs associated with TAF occurred. These data support the use of E/C/F/TAF, the first TAF-based single-tablet regimen, as a potential new regimen for initial treatment of patients with HIV-1 infection.
By 2015, up to 50% of patients linked to care may be over 50 years old in some countries. Limited research has explored this issue in Latin American countries. The Latin American Study Group is a collaborative workshop of 19 HIV care centres from six countries including data of 30,524 HIV-positive patients. We compared age and gender distribution in HIV-positive population in Latin American countries and the impact of HIV acquisition at older ages.
A cross-sectional study was done between September 2014 and January 2015 in Peru, Argentina, Chile, Colombia, Ecuador and Dominican Republic. Data from 24,384 out of 30,524 patients with at least one visit in the last 12 months are presented. Age and gender distribution in newly diagnosed patients with HIV infection was also collected. Descriptive and analytical statistics were used to compare differences in age and gender distribution among centres in patients in active care and in those recently diagnosed.
Of 24,384 patients, 5662 (23.2%) were women; 60.3% of active patients are 30 to 49 years old, 17.4% are younger than 30 and only 22.3% are older than 50 years (1.4% older than 70) without gender differences in age distribution. Nevertheless, patients older than 50 years ranged from 16.3% in Ecuador to 33.7% in Dominican Republic (
To the best of our knowledge, this is the largest report on HIV and ageing in Latin America. HIV-positive patients in active control are younger than those reported in Europe and USA. Less than 25% of patients linked to care and only one out of six newly diagnosed patients are older than 50 years old without differences in ageing by gender but with regional differences that warrant further research.
This study aims to assess the virologic and immunologic effects of a ritonavir-boosted darunavir (DRV/r)-containing salvage regimens recommended to physicians by an antiretroviral (ARV) therapy peer-advisory committee, in patients with extensive ARV treatment experience and multiple treatment failures.
Nationwide, HIV-clinic-based cohort study was conducted in Mexico. Eligible patients were HIV-positive adults who had experienced the virologic failure of at least two prior ARV regimens and had detectable viremia while currently being treated; their physicians had received a therapeutic advice regarding the DRV/r-containing salvage regimen, by a panel of experts (intervention). Median (Md) follow-up time was 47 months (interquartile range (IQR)=38 to 57 months) with periodical plasma HIV-RNA level (pVL) and blood CD4+ T-cell count (CD4+) measurements. Outcomes were cumulative incidence (Kaplan-Meier survival analysis) and risk factors (Cox proportional hazards regression modelling) of loss of virologic response (LVR) (pVL of less than 200 copies per ml, followed by levels above this threshold) and change of CD4+.
A total of 380 patients were followed up. Md ARV therapy exposure=12 years; Md prior regimens=4; Md major protease inhibitor-resistance-associated mutations (mPI-RAM)=3; Md DRV-RAM=1. The probabilities of LVR were 4.5%, 5.6%, 6.7%, 8% and 11.7% at the 12-, 24-, 36-, 48- and 60-month follow-up assessments, respectively. Of the 346 patients who achieved virologic response (VR), Md increase in CD4+ was 206 cell/ml (IQR=79 to 341 cells/ml);
Our intervention aimed at avoiding functional monotherapy with DRV/r in deep salvage therapy led to a high rate of long-lasting VR and immune reconstitution in heavily ARV-experienced patients. LVR is associated with young age (as a possible surrogate of lack of adherence) and is independent of nadir of CD4+, basal mPI- and DRV-RAM. It seems that, under optimal patient's compliance, a DRV/r-containing regimen with at least 2.5 fully active drugs is highly effective in routine clinical practice.
This is an open access article distributed under the terms of the Creative Commons Attribution License (
This is an open access article distributed under the terms of the Creative Commons Attribution License (
This is an open access article distributed under the terms of the Creative Commons Attribution License (
This is an open access article distributed under the terms of the Creative Commons Attribution License (
This is an open access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (
On October 9, 2014, the second SCEM symposium on psychological traumatization was organized in Amsterdam, the Netherlands, chaired by Robbert-Jan Verkes, MD, PhD, and Anton van Balkom MD, PhD. The symposium was attended by over 100 participants: psychiatrists, medical doctors, psychologists, and psychiatric nurses.
This second symposium on the consequences of psychotrauma was organized because the first one focusing on the aspecific relationship between psychotrauma and psychopathology had been very successful. Apparently there is large interest in psychotrauma in the Netherlands (Olff & Vermetten,
In this second symposium, we focused on several topics relevant to both research and clinical practice. The first lecture addressed diagnostic issues of PTSD in recent history (Vermetten,
Both prolonged exposure (PE) and eye movement desensitization reprocessing (EMDR) are recommended in international guidelines as first choice treatments in PTSD patients. Its efficacy is showed in many randomized controlled trials and several meta-analytic reviews (Powers, Halpern, Ferenschak, Gillihan, & Foa,
In DSM-5, the classification of posttraumatic stress disorder (PTSD) has broadened its scope. The stressor criterion is more explicit with regard to events that can be qualified as a traumatic experience. Moreover, the specifier “with dissociative symptoms” was introduced for individuals experiencing depersonalization or derealization. However, this extension does not fully cover the more profound symptomatic expressions and developmental trajectories of severe and lasting (childhood) abuse and neglect, nor their impact on the development of self and interpersonal functioning as described for personality disorders (as in the alternative DSM-5 model in Section III). During the last decades a number of concepts have been suggested to describe these more profound phenomena: type II disorder (Terr, 1991), complex PTSD (Herman, 1992), enduring personality change after catastrophic experience (WHO, 1992), PTSD related to childhood abuse (Cloitre, Koenen, Cohen, & Han, 2002), complex posttraumatic self-dysregulation (Ford, Courtois, van der Hart, & Nijenhuis, 2005), disorders of extreme stress, not otherwise specified (DESNOS; van der Kolk, Roth, Pelcovitz,Sunday, & Spinazzola, 2005), and developmental trauma disorder (van der Kolk, 2005). However, from a developmental perspective, the etiological role of trauma in complex PTSD is anything but simple. Pre-trauma factors are significant (genetic vulnerability) and what happens after the trauma has the biggest impact (context)! Unfortunately, none of these concepts were incorporated within the new version of the official APA classification system.
With respect to the treatment of PTSD, cognitive behavioral therapies currently dominate the field of treatment approaches. Imaginary exposure and Eye Movement Desensitization and Reprocessing (EMDR) are straightforward evidence-based interventions, widely available to diminish symptoms like intrusive distressing memories, hyper arousal, flash backs, dreams, and nightmares. The aim is to reduce these symptoms within a short period of time.
The majority of PTSD patients (Type I) are characterized by failed prefrontal inhibition of limbic activity. In contrast, however, in the dissociative subtype (feeling zoned out; detached from body) PTSD high prefrontal activation in conjunction with inhibited limbic activation was found.
In complex PTSD, clinicians and patients struggle with long-standing and multifaceted problems (especially suffering from personality disorders, dissociative disorders, mood disorders, and somatic symptom-related disorders), more eclectic approaches are needed integrating emotion regulation and interpersonal functioning strategies as well as psychodynamic understanding. Especially when childhood sexual or physical abuse was long lasting, repetitive, and induced by attachment figures, and when emotional support was lacking, the impact on personality development can be so devastating that other treatment efforts are necessary to establish a trustful therapeutic relation as a starting point for cautious exploration. Evidence-based psychotherapeutic models for (borderline) personality disorders all describe efforts to treat PTSD symptoms within such a specific psychotherapeutic frame of reference. Most of these treatments are intensive (minimal once or twice a week) and long lasting (years). In dialectical behavior therapy (DBT), the initial phase of treatment explicitly focuses on stabilizing by diminishing para-suicidal and self-destructive behaviors, as well as treatment-interfering behaviors. Individual cognitive treatment is combined with skill training to improve emotion regulation. As soon as the patient is skilled and stabilized (most of the time after an initial first year of treatment), DBT will next focus on the treatment of PTSD, using cognitive behavioral approaches like imaginary exposure. During transference focused psychotherapy (TFP), in the mid-phase of treatment, the theme of abuse will be activated within the transference (split self and object representations centered around aggression and hatred). Identification with both victim and perpetrator is elaborated, and sexual and aggressive impulses should be disentangled, with the purpose of resolving inner conflicts, fostering identity integration, and enhancing adaptive functioning. Also in schema-focused therapy (SFT), the treatment of trauma and PTSD is not part of the initial phase of treatment. The first phase of treatment involves identifying maladaptive schemas and building up adaptive capacities (healthy adult mode). Enough social support is a prerequisite to focus on specific former traumatic experiences. SFT describes specific strategies for treating traumatic experiences, like experiential techniques and imagination with rescripting. In mentalization-based treatment (MBT), the concept of mentalizing is used to broaden the perspective on trauma treatment. Mentalizing goes offline when defensive (fight–flight–freeze) responses become activated, promoting rapid responses to imminent danger. In particular, the impact of “attachment trauma” on emotion regulation and mentalizing reflects a dual liability (extreme distress plus impaired development emotion regulation capacities). Later in life, trauma-triggered hyperactivation of the attachment system, and the failure to mentalize, induces primitive modes of thought: psychic equivalence mode, pretend mode, and teleological mode. According to the MBT treatment, model treatment involves far more than processing traumatic memories. Treatment helps the patient to help the self to mentalize trauma and relationship conflicts, in order to develop more secure attachments. The patient uses the therapist as a mirror to understand the self: a “surrogate prefrontal cortex (PFC)”; it provides a buffer between feeling and action: a “pause button”, an opportunity for the patient to reconstruct his or her narrative within a safe and containing environment (first priority), and grounded in reality. In this way, the therapist often becomes the “object of hope.”
What these evidence-based treatments for (borderline) personality disorder have in common is their clear contract setting, their supportive common factors, their efforts to first stabilize their patients before exposing them to traumatic memories, their focus on maintaining a trustworthy therapeutic relationship, and their efforts to tailor psychotherapeutic strategies toward specific vulnerabilities and capabilities to regulate emotions and control (self-destructive) impulses. So, in complex PTSS and personality disorders, the central therapeutic task is NOT specifically to work with the content of traumatic events, but rather involves supporting a mentalizing stance in relation to the meaning and effect of trauma. The focus is primarily on the patients’ mind, not on the event, on the process rather than the content. Over and beyond the holding environment of the therapeutic relation, phase specific and carefully tailored, symptom-focused approaches like exposure and EMDR can reduce typical PTSD symptoms.
Child abuse is widely prevalent in the Netherlands, and the strong social and political focus now put on the issue is fully warranted. Prevalence studies have estimated that around 3% of all Dutch children experience some form of abuse or neglect (Alink et al.,
Maltreatment leads to serious problems in children's psychosocial functioning. Mental health problems may include posttraumatic stress disorder, other anxiety disorders, depressive disorder, attachment disorder, and conduct disorder. Such conditions are detrimental to a child's development and may culminate in problems in the family, problems at school, and problems with other children (Jonkman, Verlinden, Bolle, Boer, & Lindauer,
The first objective of treatment is to create safety within the family. An end must be put to the traumatizing situation. That requires a multidisciplinary individual and systemic strategy that focuses both on the victim or victims and the perpetrator.
In forms of maltreatment such as violence or sexual abuse within the family, treatment must focus both on ensuring safety in the family and on assessing the childrearing capabilities of the parent or parents. Some questions that need to be asked are: (1) Do the parents acknowledge that their child has problems? (2) Do they acknowledge the part that they themselves have played in the onset of the problems? (3) Are they motivated in any way to try to change the situation? (4) Are the parents capable of recognizing experts as people that can truly help? (5) Do the parents themselves have mental health problems that complicate the treatment? Some situations are simply too unsafe, and some parents are utterly incapable of bringing up their child or children. That necessitates the placement of children away from home.
Once a safe environment has been created, the next question is whether the traumatized child should receive trauma-focused treatment right away or whether the first priority should be to ensure a degree of stability. There is considerable debate about this in the adult literature (e.g., De Jongh & ten Broeke,
In 2011, we received a large grant (ZonMW TOP) to perform a series of functional neuroimaging studies on the effects of a single intranasal administration of the neuropeptide oxytocin on neural emotional and reward processing in PTSD patients versus healthy traumatized controls, and in recently traumatized individuals at high risk for PTSD due to high levels of distress acutely after trauma.
These studies were based on literature showing that intranasal oxytocin impacted a variety of the behavioral, neural, and neuroendocrine dysregulations observed in PTSD patients and individuals vulnerable for PTSD. Thus, intranasal oxytocin appeared to be a promising candidate for PTSD prevention and augmentation of treatment response (Olff,
Our results show that oxytocin administration dampened amygdala reactivity toward emotional faces in PTSD patients, but increased amygdala reactivity toward (negative) emotional faces in both recently traumatized individuals with high levels of distress as well as highly traumatized individuals without psychopathology. Additionally, we found opposing effects of oxytocin administration on functional connectivity of the amygdala with brain areas involved in emotion regulation and salience processing between our study populations. In rest, male PTSD patients showed increased connectivity within the emotion regulation network after oxytocin, while female PTSD patients showed decreased connectivity within the salience network. In contrast, oxytocin administration resulted in decreased resting state connectivity within the emotion regulation network and increased connectivity within the salience network in recently traumatized individuals after exposure to trauma-related script-driven imagery. Regarding neural effects on reward sensitivity, oxytocin administration increased striatal responses to both reward and loss anticipation in PTSD patients and highly traumatized controls.
Our findings on the neural effects of a single oxytocin administration in PTSD patients are in line with our hypotheses. Thus, oxytocin is not a miracle panacea, but our results do support the notion that intranasal oxytocin administration is a promising candidate for augmentation of PTSD treatment response. As a next step, the potential of intranasal oxytocin administration as an adjunctive agent during psychotherapy for PTSD, such as before exposure-based therapies, should be further investigated in a clinical setting, preferably in a randomized placebo-controlled trial.
The study is supported by grants from ZonMw (Top Grant), the Netherlands organization for Health Research and Development (grant no. 40-00812-98-10041) and the Academic Medical Center Research Council (110614).
All authors declare that they have no biomedical financial interests and no potential conflicts of interest.
Substance use disorders (SUDs) and traumas frequently co-occur. A recent German study found that 66% of the women with a diagnosis of opioid dependence also reported sexual abuse, whereas the figure for men was 11% (Schäfer et al.,
In the general population, approximately 10% of women and 5% of men meet criteria for a lifetime diagnosis of PTSD. However, for patients with alcohol dependence, the figures are approximately 25% (OR 3.6) and 10% (OR 3.2), respectively. Conversely, about 35% of people diagnosed with PTSD also have an alcohol use disorder (Kessler, Sonnega, Bromet, Hughes, & Nelson,
Among treatment-seeking SUD patients, 20–50% have a lifetime diagnosis of PTSD, whereas 15–40% met criteria for PTSD in the last year (Brady, Killeen, Brewerton, & Lucerini,
There are multiple theories about the reasons for the high co-occurrence of SUD and PTSD. Some believe that PTSD precedes SUD and that SUD is the consequence of attempts to self-medicate PTSD symptoms. Others believe that SUD occurs first with substance-use-related traumas as an adult resulting in comorbid PTSD. Finally, some consider that both SUD and PTSD are the result of a shared vulnerability due to some genetic predisposition or early childhood trauma. Research shows that PTSD precedes SUD far more frequently than the other way around, that PTSD and SUD are both very often preceded by trauma in early childhood, and that there is some genetic overlap for the two disorders (Kessler et al.,
A large number of studies have looked at the effect of psychotherapeutic treatments of SUD patients with comorbid PTSD. The results of these studies have been summarised in two recent reviews (Najavits & Hien,
There are various different effective medications available for the treatment of nicotine, heroine, alcohol, and cannabis dependence, and there are some promising medications for the treatment of cocaine and other stimulant use disorders (Van den Brink,
An important problem that needs more attention is that most interventions have no effect on nightmares or other sleeping problems associated with either PTSD or SUD. A recent review (Nappi, Drummond, & Hall,
Based on this overview of the literature, the author recommends: 1) all treatment-seeking SUD patients should been screened for PTSD; 2) all treatment-seeking PTSD patients should be questioned about their alcohol and drug use; 3) all patients with PTSD and/or SUD should be screened for the presence of early childhood trauma; 4) the presence childhood trauma should always be validated and possibly treated; 5) the relationship between PTSD symptoms and substance use should be assessed and monitored; 6) the patient's preference for abstinence or controlled use should be taken into account because both drug use and withdrawal can worsen the course of PTSD symptoms; 7) all patients with PTSD and SUD should be screened for sleeping disorders; 8) it is preferable to treat PTSD and SUD simultaneously either with one or two different therapists; 9) cognitive behavioural therapy with exposure is preferred (including eye movement desensitisation and reprocessing), possibly in combination with pharmacotherapy for addiction and/or PTSD; 10) there is no role for benzodiazepines or tricyclic antidepressants in the treatment of patients with SUD and PTSD; and 11) there might be a role for topiramate, prazosin, or gabapentin in the treatment of nightmares in patients with PTSD and SUD.
On a neuronal level, this transfer is achieved through a process called replay during which the same hippocampal-cortical firing patterns that represent the stored memory during awake state are being played back to the cortex while asleep (Skaggs & McNaughton,
In case of emotional memory, current theories suggest that memory consolidation additionally serves to preserve and solidify the declarative, factual aspect of an emotional memory trace, while at the same time depotentiating its affective charge (Walker & Van der Helm,
Although often less accessible for therapeutic intervention, more research efforts should be directed towards intervening directly during post-trauma sleep, for instance, in emergency rooms or combat situations. The opportunity to influence the first-time transfer of potentially traumatic memory traces, either by administering drugs or just generally improving sleep quality, seems especially valuable.
A promising, but experimental, avenue for treatment is the selective targeting of replay to boost consolidation of trauma memory. Sound or smell stimuli that are linked to original trauma acquisition could be presented time-locked to different sleep stages or events. This patient-specific, trauma-tailored reactivation during sleep could by itself open up a new reconsolidation window, in which again facilitative or disruptive interventions can be applied.
So far, none of these interventions are available for clinical practice, but with a growing interest of clinical researchers in cognitive neuroscience, this is likely to happen sooner rather than later. A critical remaining question in PTSD pertains to whether any intervention aims to facilitate or speed up the (re)consolidation process thereby decoupling memory content from its affective charge (for instance, using DCS in first post-trauma sleep); or alternatively disrupt (re)consolidation of the traumatic memory trace thereby preventing its restorage (for instance, using propranolol or more invasive techniques such as ECT).
The recent havoc caused by typhoon Haiyan in central Philippines provides an example of the grim reality of the impact of disasters in the Association of South-East Asian Nations (ASEAN) region. This is personal to me as many of my relatives and friends died and many more had their properties all washed away. You see, I was born in Tacloban and grew up in the city. It pained me to see my 87-year-old grandmother, a veteran of many typhoons, struggling to survive amid the stench of death and destruction. No doubt, the President of the Philippines was prompted to echo the need to strengthen the framework of cooperation in managing disasters among the ASEAN countries, being one of the best approaches to address urgent issues and to build resilience.
Needless to say, many efforts have already been initiated and continued in reducing the vulnerability of the region to the risk of disasters in the context of sustainable development. This includes the establishment of the ASEAN Committee on Disaster Management (ACDM) in 2003, which the ASEAN body elevated to a full-fledged committee. The ASEAN also promulgated the Agreement on Disaster Management and Response (AADMER) which is a legal instrument that binds all member countries in promoting regional cooperation and collaboration so as to lessen disaster losses and having a joint emergency response to disasters. This document is a manifestation of the commitment of the ASEAN to the Hyogo Framework for Action 2005–2015 supported by 168 countries (
Many observers, including the Secretary-General of the ASEAN, believe that the tipping point in the vigorous supranational policy approach on disaster management was instigated by the Indian Ocean tsunami disaster in 2004. The scale of the devastation of the tsunami was so massive that people, not only from the region, but even those from beyond, realized that disasters are realities that could strike anytime, anywhere. ASEAN's rhetoric was hinged on six Rs – reduce disaster risks, rebound quickly, reinvigorate leadership, renovate the plan, respond better, and revive the ASEAN's sense of community. Many of these narratives have been echoed time and time again. This echo gets louder as disaster strikes. What is thought provoking in this rhetoric is the idea of constant reflection; thus, the key terms – ‘renovate’, ‘reinvigorate’, ‘better response’, and ‘revive’. If there is a constant need for changing interventions and approaches, does this mean that previous actions have remained insufficient?
This is probably the main impetus to push the discussions in the ASEAN and go beyond the level of consensus building and move vigorously away from rhetoric and pronouncements. As urgent actions are required in disasters, readily available resources and decisions should be at the disposal at the supranational level. This beckons for the need to have a strong coordinating body that can easily deploy immediate interventions at any geographical location. A substantial amount of relief fund should be readily available for immediate disposal and disbursement anywhere. This would wean away the region from too much reliance on donors that normally arrive after the critical period of search and rescue phase and comes in with their own philosophies and approaches. A cooperative framework would definitely benefit the countries that need most help, which apparently are the countries most affected by disasters. A good framework is also imperative for a regional relief fund to make it substantially sufficient to be significant in delivering impact.
A supranational framework and body would, however, face many challenges, as ASEAN countries are diverse in many different ways. The ASEAN, however, is cognizant of the disparities in economic and financial capabilities of the countries necessary to build and sustain the activities in building disaster management capabilities. To bring the lesser-equipped countries on par with the others, the organization included in their 2009–2015 strategic framework the assistance of countries such as Cambodia, Laos, Myanmar, and Vietnam in enhancing their capabilities in disaster responses, and search and rescue by organizing training courses and workshop; provision of support through equipment and infrastructure for search and rescue and disaster responses; and providing more capacity building in disaster management and emergency response.
History has shown that time and time again, the ASEAN region will see more disasters. The region is prone to numerous hazards, big and small, that result in the accumulation of many losses of lives and properties. Its geographical location makes it vulnerable being placed in between two big oceans – the Pacific and Indian oceans – resulting in many typhoons, floods, landslides, and storm surges. Earthquakes, tsunamis, and volcanic eruptions are common occurrences as the region lies in between a number of tectonic plates. There are also forest fires and a number of much-publicized epidemics such as SARS, H1N1, and H9N7 that have caused havoc and hardships among the populations affected (
According to the report of the ASEAN Disaster Risk Management Initiative (
For example, the recent Haiyan disaster in the Philippines on 8 November 2013 resulted in more than 6,000 deaths, with 1,700 still missing, and 27,000 injured. The devastation has affected 14 million people, including some 5 million children. A total of 3.9 million people were forced from their homes. The United Nations and aid groups called to raise US$791 million to assist those affected (
The ASEAN region also faces the challenges of ‘emerging’ disasters. These new ‘forms’ undoubtedly beg for a cooperative approach. For example, Malaysia Airlines Flight 370 may have directly affected few lives; however, its psychological impact on tourists and travelers was quite significant. The search for the plane and the people on board was an example why a multi-country effort was essential. Then Malaysia Airlines Flight 17 was another ‘global disaster’ that provided an impetus for a strong ASEAN stand. Albeit political, a unified voice of 10 countries with its innocent citizens killed can push swift actions at the international level. The same is true with political conflicts that resemble disaster scenarios such as the riots in Vietnam against Chinese nationals, the civil strife in Thailand, insurgency in southern Philippines, and the minority issues in Myanmar where political solutions become imperative.
Nuclear disasters such as Chernobyl and Fukushima may become a major issue in the future as countries such as Vietnam are planning four power reactors with the Philippines, Indonesia, Malaysia, Vietnam, and Thailand having research reactors (
Most of all, the impact of climate change should be seriously factored in as a transnational issue. As temperature increases sea level rises and drastic weather becomes more frequent resulting in more calamities (
Yet, time and time again, we always see hope amid all this misery. Economies rebound and the populations recover despite the upheaval brought about by the havoc of calamities. Resilience is always a good story and is shown to be an enduring phenomenon. No matter how long the impact affects the populace, they become survivors and not victims; they pick up the pieces and move on with their lives. Yes, resilience is an area that is never focused on. Not even in the field of disaster research. This is why research becomes imperative and should be a major component of the approach in a unified disaster management framework. Documentation and research should be central in a supranational effort, as there is much learning that is yet to be discovered.
There are no arguments that counter the need for a strong and firm cooperative ASEAN effort in developing resilience against disasters – vulnerability is high, disasters are getting stronger and more frequent, and uniting small countries is prudent in pooling resources. An ASEAN disaster body with a strong mandate from the member governments in the context of an integrated ASEAN may be the impetus toward innovative and novel approaches in disaster preparedness and prevention and in cooperating to protect civilians. This might be the answer to the chaos that transpired during the Haiyan disaster in the Philippines. Hopefully, my grandmother would live to see the fruition of immediate and rapid responses during calamities. She already had enough with 87 years of disasters. And time is of the essence.
The authors thank the internal reviewers of HealthSpace.asia and all the anonymous reviewers in Global Health Action for their constructive inputs in the revision of the paper.
The publication of this paper is funded by HealthScape.asia with the support from the Rockefeller Foundation and Thailand Research Center for Health Service System (TRC-HS).
Responsible Editor: Peter Byass, Umeå University, Sweden.
Responsible Editor: Peter Byass, Umeå University, Sweden.
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This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
This is an open-access article subject to an exclusive license agreement between the authors and the Frontiers Research Foundation, which permits unrestricted use, distribution and reproduction in any medium, provided the original authors and source are credited.
This is an open-access article subject to an exclusive license agreement between the authors and the Frontiers Research Foundation, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are credited.
This is an open-access publication subject to an exclusive license agreement between the authors and the Frontiers Research Foundation, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are credited.
This is an openaccess article subject to an exclusive license agreement between the authors and the Frontiers Research Foundation, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are credited.
Several common neuropsychiatric disorders (e.g., obsessive-compulsive disorder, Tourette syndrome (TS), autistic spectrum disorder) are associated with unpleasant bodily sensations that are perceived as an urge for action. Similarly, many of our everyday behaviors are also characterized by bodily sensations that we experience as urges for action. Where do these urges originate? In this paper, we consider the nature and the functional anatomy of “urges-for-action,” both in the context of everyday behaviors such as yawning, swallowing, and micturition, and in relation to clinical disorders in which the urge-for-action is considered pathological and substantially interferes with activities of daily living (e.g., TS). We review previous frameworks for thinking about behavioral urges and demonstrate that there is considerable overlap between the functional anatomy of urges associated with everyday behaviors such as swallowing, yawning, and micturition, and those urges associated with the generation of tics in TS. Specifically, we show that the limbic sensory and motor regions—insula and mid-cingulate cortex—are common to all of these behaviors, and we argue that this “motivation-for-action” network should be considered distinct from an “intentional action” network, associated with regions of premotor and parietal cortex, which may be responsible for the perception of “willed intention” during the execution of goal-directed actions.
Many of our everyday behaviors are characterized by bodily sensations that we experience either as an
In common usage, the terms “urge” and “desire” are frequently encountered as both a verb (as in to “
Nevertheless, it has been suggested that there is an important distinction to be made between urges and desires (e.g., Cameron, 2002; Davenport, 2008). Thus, Cameron (2002), when discussing interoception, and the relationship between conscious awareness and visceral events, makes a distinction between “detection,” which is an organism's reflexive response based solely upon afferent physiological information, and “perception,” which refers to an organism's response based upon all information available to the organism (which might include learned information and expectations that might be generated as a result of learning). Similarly, Davenport, when discussing mechanisms associated with the urge-to-cough, defines an
The motivation-for-action model proposed by Davenport to account for the urge to cough (adapted from Davenport, Sapienza, & Boiser, 2002).
Combining these two ideas we might conclude that an urge—as in a drive for action—need not enter conscious awareness, but that we are always aware of our desires. An example might help make this distinction clearer. Imagine that you are driving your car and you suddenly become aware of an uncomfortable sensation that your bladder is full, which you experience as the
As outlined above, urges are often defined as the drives or impulses that impel us to act. Nevertheless, it is argued that actions can, and frequently do, occur in the absence of any awareness of such drivers, as in the case where one finds oneself yawning without previously being aware of either the desire to yawn or of any bodily sensation that might reasonably be identified as giving rise to the yawn. Instead, one simply finds oneself yawning. In this case, it might be argued that this is a
One possible distinguishing feature of urges, as distinct from reflexes, may be that urges are chiefly associated with actions that cannot be realized immediately and must be held in check until an appropriate time when they might be released. For instance, when we become aware of having a full bladder, we experience an “urge-to-void” because we do not simply void our bladder, but instead employ a coordinated set of central, autonomic, and peripheral neural mechanisms to withhold micturition until we are in an appropriate behavioral context. Similarly, in the case of yawning, we might define the urge-to-yawn as arising in circumstances where we are forced to try to stifle the yawn rather than in the situation where we find that we are yawning.
One factor that may determine whether an urge enters awareness is the intensity of the physiological afferent. It has been demonstrated in the context of the urge-to-cough that the perceived strength of the urge that is experienced is related to the intensity of stimulation. Specifically, when capsaicin is added to the breathing circuit, it results in a sensation that is perceived as an urge-to-cough. Furthermore, increasing capsaicin levels leads to a reliable increase in estimates of this urge (Davenport, Sapienza, & Bolser, 2002). Similarly, in our own unpublished studies of the effects of oro-pharyngeal stimulation on the urge-to-swallow, we have found that oropharyngeal stimulation using pulses of air produces both an urge-to-swallow and overt swallowing (for similar findings, see also Lowell et al., 2008). More importantly, we have found that increasing the intensity of oropharyngeal stimulation leads to an increase in the strength of the perceived urge-to-swallow. An important question therefore concerns the role of awareness, and by implication “desire,” in the initiation of urge-related actions.
According to the motivation-to-action model represented in
If we accept the premise outlined above that urges-for-action are often accompanied by bodily sensations, then it strikes us that an important distinction can be drawn between being aware of a bodily sensation and being aware of an urge-for-action. This distinction can be best illustrated by considering the following examples. In the case of an itch, we may be aware of experiencing both an itch (bodily sensation) and an urge to scratch the itch (urge-for-action). By contrast, while we may become aware of an urge-to-yawn, it is not entirely clear that we are ever aware of the bodily sensation that gives rise to the urge to yawn. Furthermore, this becomes more important if we accept the argument outlined above that urges occur primarily in circumstances in which actions may need to be suppressed or their execution deferred. In such circumstances, we might distinguish between suppression of the
The kinds of actions that we have considered as representative of urges-for-action are highly automatic, habitual responses that occur primarily in response to sensory stimulation. These might include brushing an insect off your arm, scratching an itch, yawning when tired, coughing in response to a tickle in your throat, etc. While such actions can, in some circumstances, be executed with little or no awareness of the sensory stimulation that triggered the action, as when one finds oneself yawning or coughing, we have argued that a key characteristic of urges-for-action is that they involve the suppression or deferment of an action. Such actions might therefore be contrasted to intentional, goal-directed, forms of action.
The circumstances in which the “willed intention” to execute an action can be shown to follow the brain processes involved in the preparation for action were famously studied by Benjamin Libet (Libet, Gleason, Wright, & Pearl, 1983), and more recently by Patrick Haggard and colleagues (e.g., Haggard, 2005; Haggard & Eimer, 1999; Sirigu et al., 2004). In Libet's task, participants fixated on a time-varying, rotating visual spot and were instructed to make a voluntary hand movement whenever they felt the “urge” to do so. Participants were asked to indicate the location occupied by the moving spot when they had first felt the urge to move their hand. Libet showed that this “W judgment” occurred some 200 ms prior to movement onset, but, more importantly, he also showed that the preparatory brain activity that precedes voluntary action, the so-called “readiness potential,” itself preceded the “W judgment” by several hundred milliseconds. Such readiness potentials arise in the premotor regions of cortex, including both the supplementary motor area (SMA) and the presupplementary motor area (pre-SMA), regions that have been linked to the planning and preparation of intentional, goal-directed, actions and sequences of actions (Deecke & Kornhuber, 1978).
Haggard has argued that conscious awareness of our intention to act arises during the preparatory processes that precede an action, and is linked to the joint activity of premotor and parietal brain areas (Haggard, 2005). In support of this view, he has shown that patients with damage to the parietal cortex show a specific impairment in reporting when they became aware of their intention to move (i.e., Libet's “W judgment”). The proposal that the parietal cortex may maintain a dynamically updated state estimate of the current postural configuration of the body (the body schema) is well supported by neuropsychological (e.g., Wolpert, Goodbody, & Husain, 1998) and recent fMRI studies (e.g., Parkinson, Condon, & Jackson, 2010; Pellijeff, Bonilha, Morgan, McKenzie, & Jackson, 2006).
While it is clearly the case that in such experiments individuals can report when they first perceived themselves to have formed an “intention” to move, it is another thing entirely to argue that such conscious “intentions” typically precede everyday actions. Thus, when I am sitting at my desk typing and I break off to reach for my coffee cup I am not aware of forming an intention prior to each keystroke that I make or of forming an intention to reach for my coffee. Instead, I am aware of the actions I am making. Similarly, while it is clearly demonstrated that our actions are preceded by neural activity, as discussed by Libet and by Haggard and colleagues, it is currently unclear how these activations relate to the phenomenology of intention.
Haggard and others (e.g., Blakemore, Wolpert, & Frith, 2002; Haggard, 2005) have argued that the sense-of-agency that typically accompanies the execution of voluntary movements arises as a result of internal forward sensory models that generate a prediction of the sensory consequences of an action that is then matched against afferent sensory signals. It has been proposed that in cases where the link between these sensory predictions and confirmatory sensory input is broken, neurological syndromes such as anosagnosia (lack of awareness of injury) or somatoparaphrenia (denial of limb ownership) may occur (Tsakiris, 2010). Interestingly, both of these disorders have been linked to damage of the anterior insular cortex of the right hemisphere (Baier & Karnath, 2008; Karnath, Baier, & Nagele, 2005). However, it is important to note that this sense of agency may in fact have a significant postdictive or reconstructive component (Moore, Lagnado, Deal, & Haggard, 2009; Wegner, 2002) and thus is not necessarily an unambiguous index of intentionality.
Tics are involuntary, repetitive, stereotyped behaviors that occur with a limited duration. Motor tics can be simple or complex in appearance, ranging from repetitive movements to coordinated action sequences (Leckman, 2002). Verbal tics can consist of repeating words or utterances (palilalia), producing inappropriate or obscene utterances (coprolalia), or the repetition of another's words (echolalia).
Tics occur in bouts, typically many times in a single day, and are the most common form of movement disorder in children, with a prevalence of 1–29% depending upon the precise characteristics of the study population, the diagnostic criteria used, and the study design and methods employed (Leckman, 2002). Tics include a continuum of disorders: transient tic disorder (TTD), chronic tic disorder (CTD), nonspecific tic disorder (NSTD), and Tourette syndrome (TS). The etiology of tics is poorly understood and probably involves a complex interaction between genetic and environmental factors that exert an influence over brain development.
TS is a developmental neuropsychiatric disorder that lies at the extreme of the tic disorder spectrum and is characterized by the presence of chronic vocal and motor tics (Leckman, 2002). The neurological basis of TS is unclear; however, it is agreed that the basal ganglia, including circuits that link the striatum to the frontal lobes, are dysfunctional (Albin & Mink, 2006). A specific model of basal ganglia dysregulation in TS has been proposed as follows. Subsets of striatal neurons (matrisomes) are thought to become abnormally active in inappropriate contexts, leading to the disinhibition of thalamocortical circuits that in turn lead to tics. Activity-dependent dopamine inappropriately reinforces such activity, leading to stereotyped repetition of behavior (Albin & Mink, 2006). Brain-imaging and postmortem studies provide general support for the view that cortical–striatal–thalamocortical pathways are dysfunctional in TS (Gerard & Peterson, 2003). Furthermore, deep-brain stimulation of the globus pallidus or the thalamus has been shown to be effective in suppressing tics in individuals with TS (e.g., Ackermans et al., 2011).
The occurrence of repetitive, stereotypical behaviors in TS has been linked to operation of the brain “reward” and “habit” systems, and tics have been likened to an inappropriate overextension of habit learning (Graybiel, 2008). Key characteristics of habitual behaviors are that they are largely learnt, occur repeatedly, are performed almost automatically, and often involve stereotypical, ordered, action sequences (Graybiel, 2008). In this context, it is important to note that many individuals with TS report that their tics are often preceded by “premonitory sensory phenomena” (PSPs), which are described as the presence of uncomfortable cognitive or bodily sensations (e.g., tension, pressure, tickle), that precede the execution of a tic, and are experienced as a strong
Several attempts have been made to investigate the brain regions associated with the occurrence of tics in TS using human neuroimaging techniques. These studies have indicated that the neural mechanisms responsible for triggering of tics may in fact differ from those involved in voluntary movements (Bohlhalter et al. 2006). One notable study was that reported by Hallett and colleagues, which used functional magnetic resonance imaging (fMRI) to examine brain areas activated immediately preceding the spontaneous occurrence of motor and/or vocal tics, and thus likely to be associated with the urge to tic (Bohlhalter et al., 2006). This study identified a network of brain areas that were activated immediately prior to tic onset, and, most importantly, identified the insular cortex, the anterior cingulate cortex, and the parietal operculum, which includes the secondary somatosensory cortex (SII) (Eickhoff, Amunts, Mohlberg, & Zilles, 2006), as the most likely anatomic regions responsible for the uncomfortable feelings associated with premonitory urges to tic (Bohlhalter et al., 2006). Consistent with this proposal, electrical stimulation of the insular cortex or the parietal operculum can elicit unpleasant somatosensory or visceral sensations (Augustine, 1996; Ostrowsky et al., 2002; Penfield & Faulk, 1955). By contrast, electrical stimulation of the medial frontal lobes produces motor outputs in the face and upper limbs comparable to tics (Bancaud et al., 1976; Lim et al., 1994; Talairach et al., 1973).
The proposal that the insular and cingulate cortices are associated with the uncomfortable feelings associated with the urge to tic in TS is consistent with the putative role of these areas in the neural representation of bodily states more generally (interoception), and the initiation of behaviors associated with these bodily representations (for reviews, see Craig, 2002, 2009; Naqvi & Bechara, 2008). Thus, Craig suggests that these two regions are linked functionally and can be thought of as the limbic sensory and motor areas. He has proposed that these two areas form part of a functional brain system that is associated with the
It is important to note that Craig's particular view of interoception includes a representation of all body states relevant to homeostasis, including pain, temperature, taste, visceral sensation, inflammation, itch, and many aspects of touch and proprioception that are often viewed as part of an “exteroceptive” somatosensory system (Craig, 2003). Consistent with this view, recent functional brain-imaging studies have demonstrated that punctate somatosensory stimulation of the upper limbs produces significant increases in brain activity—blood oxygen level-dependent (BOLD) response)—bilaterally within the insular cortex (e.g., Jackson, Parkinson, Pears, & Nam, 2011; Parkinson et al., 2011). However, neurophysiological studies suggest that the insular cortex may play a particularly important role in representing the emotional significance of somatosensory signals. Thus, it has been shown that a neural pathway, consisting of unmyelinated fibers, projects to the insula (Olausson et al., 2002; Vallbo, Olausson, & Wessberg, 1999), and that these fibers are associated with affective or sensual touch (e.g., pleasant touch sensation).
Brain-imaging and neurological studies also indicate that the posterior and mid-insular cortex may play an important role in body ownership and our sense of agency (control) over our body. Thus, a positron emission tomography (PET) imaging study reported by Tsakiris, Hesse, Boy, Haggard, and Fink (2007) reported that body ownership, as indexed by the strength of the rubber hand illusion, was associated with activation increases within the right posterior insula and right frontal operculum. Similarly, Karnath and colleagues have shown that lesions involving the right insula impair body awareness, and the sense of limb ownership (e.g., Baier & Karnath, 2008; Karnath, Baier, & Nagele, 2005).
Arguably, the most direct neuropsychological evidence that the insular cortex is key to the experience of urges for action comes from a study that investigated the effect of insula lesions on the urge to smoke in those addicted to smoking (Naqvi, Rudrauf, Damasio, & Bechara, 2007). This study compared smokers who had sustained damage involving the insula with a group of smokers whose damage involved other brain areas, but spared the insula. The study investigated changes in smoking behavior post-stroke and demonstrated that smokers whose brain damage involved the insula were significantly more likely than smokers with lesions sparing the insula to exhibit a “disruption of smoking addiction.” Importantly, individuals described this disruption of addiction as like their body “forgetting the urge to smoke” (Naqvi et al., 2007).
Functional brain-imaging studies using fMRI have become central to cognitive neuroscience; however, it should be recognized that such studies have limitations. Many of these are well known and relate to the constraints imposed by hemodynamic signals (Logothetis, 2008). A discussion of these limitations is generally beyond the scope of this paper; however, below, we briefly outline some issues that specifically relate to difficulties in interpreting fMRI activations associated with the urge to tic in individuals with TS.
First, one obvious difficulty associated with interpreting the meaning of the patterns of BOLD activity reported in fMRI studies that have sought to identify brain regions associated with the occurrence of tics in TS, is that individuals with TS are instructed to remain still in the MRI scanner and to suppress their tics. Thus, the regions activated can reflect brain areas associated with the generation of tics or brain areas linked to their active suppression. Second, recent evidence suggests that individuals with neurodevelopmental disorders may follow unique developmental trajectories whereby they undergo compensatory, neuroplastic changes in brain structure and function that help them gain control over their symptoms (Jackson, Parkinson, Jung, et al., 2011). As a consequence, individuals with TS may exhibit differences in functional anatomy, compared to typically developing individuals, even when they are performing an identical behavioral task with comparable levels of task performance. Third, the functional anatomy of the urge to tic in TS may differ from the functional anatomy of other forms of the urge for action. In fact, a related limitation of fMRI studies is that it is very often difficult to carry out the range of control studies that might be conducted when using non-imaging experimental techniques. Finally, an important limitation of individual fMRI studies is that it is very often difficult to compare across studies. Thus, a comparison of individual fMRI studies of, for example, the urge to urinate and the urge to swallow, might reveal differences in functional anatomy because these behaviors have different underlying neural circuitry, or because of differences in the following factors: the behavioral paradigms used, the scanner hardware and imaging protocols adopted, analysis protocols and statistical thresholds, etc.
This last limitation can be overcome, however, through the use of quantitative meta-analytic studies. Such studies permit an estimation of the fMRI BOLD response associated with different behaviors by drawing upon the entire body of published studies within a particular behavioral domain. One method that has proven popular recently has been the activation likelihood estimation (ALE) method developed by Turkeltaub, Eden, Jones, and Zeffiro (2002) and modified by Eickhoff and colleagues (Eickhoff et al., 2009).
Here we report the use of the ALE method to directly compare brain activity associated with the urge to urinate (micturition) and the urge to swallow.
Functional neuroimaging studies were retrieved via searches in the PubMed, ISI Web of Knowledge, and Scopus databases, as well as identified by reference tracing and through reviews. Experiments reported in these papers that corresponded to the core behavioral contrasts under consideration, such as contrast of urine withholding against a resting baseline, urine voiding against baseline, and volitional swallowing against baseline, were included in the meta-analyses if they fulfilled the following criteria: analyses must be computed across the whole brain and not restricted by partial coverage or regions of interest analyses; coordinates must be reported in an XYZ format, either in MNI or Talairach space; only experiments that investigated differences between stimulation conditions in healthy control population were included; and experiments focusing on between-group differences were excluded. No selection was made on the basis of the applied statistical threshold, as all studies were obtained from peer-reviewed journals. Additional methodological information is provided in detail in the online supplementary material, available via the ‘Supplementary’ tab on the article's online page (
Experiments investigating the functional anatomy of
The quantitative comparison of ALEs across studies of swallowing revealed widespread clusters of activation that exceeded the conservative statistical threshold (
(A) Main results of an ALE meta-analysis of neuroimaging studies of swallowing. This analysis revealed a number of activation foci that survived conservative statistical correction (
To determine whether areas of overlap exist between brain regions activated during swallowing, or when individuals have an urge to swallow, and regions activated when individuals urinate, or have an urge to urinate, we carried out a conjunction analysis between the swallowing and micturition ALE maps. This analysis confirmed that, of the widespread regions activated in both studies, only two areas survived statistical comparison (
To further investigate this issue, we sought to investigate the functional anatomy of the urge to yawn. As there are insufficient neuroimaging studies currently published to permit an ALE meta-analysis, we carried out an fMRI study of the urge to yawn. Full methodological information is provided in detail in the online supplementary material.
The results of a statistical “conjunction” analysis between the ALE meta-analyses of swallowing and micturition illustrated in
While being scanned, participants observed a sequence of video clips, each approximately 12 s in length, which illustrated an actor yawning. These stimuli had been previously shown to induce high levels of spontaneous yawning in a previous fMRI study (Schüermann et al., 2005). In the current study, participants were instructed to suppress their yawns (which they were able to do successfully) but were required to report periods during which they experienced a strong urge to yawn.
Anatomic images were transformed into the Talairach coordinate system and co-registered with each fMRI data set. Regional activation maps were obtained with a single-subject GLM (general linear model) for each individual. We defined a single predictor that modeled the periods that participants reported experiencing the urge to yawn. All 10 individuals included in the analyses reported successfully suppressing their yawns, and inspection of the motion data confirmed this. Second-level analyses involved calculating three-dimensional statistical parametric maps with separate-subject predictors for the group, using a random effects GLM (RFX). The resulting fMRI activity maps were thresholded at a Z value of 3.29 corresponding
Details of statistically significant fMRI BOLD activations associated with the urge to yawn are presented in
Regions exhibiting a statistically significant increase in blood oxygen level-dependent (BOLD) signal corresponding to the self-reported urge to yawn in an fMRI study of yawning. Again this analysis revealed statistically significant foci of activation within the insular cortex and the dorsal mid-cingulate cortex. CM A: cingulate motor area.
Coordinates for center-of-gravity and peak activations for statistically significant clusters of activation associated with the urge to yawnRight parietal cortex 51.2 −40.8 30.0 50 −32 33 2386 5.85 .00000 Right insular cortex 48.2 4.4 4.9 47 4 −3 3663 6.23 .00000 Cingulate cortex 0.25 −5.0 44.4 −11 −8 30 3361 6.86 .00000 Left parietal cortex −47.4 −43.5 29.6 −43 −44 27 1713 5.95 .00000 Left insular cortex −52.3 −7.4 14.5 −43 −20 24 3133 5.89 .00000
To examine further whether the coordinates of the yawning activations overlapped with the activation foci observed for the ALE analyses of swallowing and micturition reported above, we obtained ALEs for the peak activations of the yawning CMA and insula activations so that they might be compared directly with the results of the ALE results for swallowing and micturition. Furthermore, we also obtained ALEs for the peak activations associated with the urge to tic in individuals with TS reported by Bohlhalter et al. (2006). These data are presented in
Picard and Strick (2001) review the location and functional anatomy of the motor areas located on the medial surface of the human brain. In addition to identifying the supplementary motor area (SMA) and pre-SMA, these authors identify three separable areas within the human cingulate cortex: a caudal cingulate zone (CCZ) situated ventral to the SMA, and a rostral cingulate zone (RCZ) that is further subdivided into anterior and posterior subregions. It is noteworthy that the cingulate fMRI BOLD activations associated with the urge for action in three everyday behaviors (i.e., swallowing, micturition, and yawning), and with the urge to tic in TS, are each located in a region of cingulate cortex that corresponds closely to the CCZ. While the RCZ is associated with conflict detection, attention and arousal processes, and the selection of action, the CCZ is, by contrast, associated with the execution of simple movements and is also activated in response to bodily stimulation such as the delivery of painful cutaneous heat and cold stimuli (Picard & Strick, 2001). It should be noted that whereas the SMA has itself been linked to the conscious intention to move (see Desmurget & Sirigu, 2009), and while the CCZ and SMA may be co-activated during movement execution, the SMA and CCA should be considered functionally distinct from one another (Picard & Strick, 2001).
Regions of overlap between ALE meta-analytic studies of swallowing and micturition and fMRI studies of the urge to tic in individuals with TS (Bohlhalter et al., 2006) and the urge to yawn in neurologically normal adults. Again these analyses reveal regions of overlap within the insular cortex (CX) of the right hemisphere and the mid-cingulate cortex bilaterally.
The insula and anterior cingulate cortex have been considered to be the input and output regions of a functional system that is engaged in cognitive, affective, and behavioral contexts (Craig, 2009; Medford & Critchley, 2010). Consistent with this proposal there is now considerable evidence that these regions are jointly active across a wide range of experimental conditions (for a recent review, see Medford & Critchley, 2010). In addition, functional connectivity analyses of resting state fMRI BOLD, using seed regions located within the insula, have shown that (1) the anterior insula is connected functionally with the anterior and mid-cingulate cortex; and (2) the mid- and posterior regions of the insula are connected only with the posterior region of the mid-cingulate cortex (Taylor, Seminowicz, & Davis, 2009).
To investigate the effective, or directional, connectivity of the anterior cingulate and insular cortices, we used the Granger causality mapping (GCM) technique. GCM implements a statistical concept of causality that is based on temporal prediction. Unlike some other methods used to examine effective connectivity, GCM makes no
Results of GCM analysis of effective connectivity for mid-cingulate cortex and right hemisphere insula “seed” regionsCenter-of-gravity in Talairach coordinates Mid-cingulate cortex 0.25 −5.0 44.4 Left anterior insular cortex −30 20 7 Right anterior insular cortex 30 20 −2 Left thalamus: anterior region −9 −16 −2 Right thalamus: anterior region 12 −17 1 Left mid-insular cortex and inferior frontal lobe −55 8 2 Right mid-insular cortex and inferior frontal lobe 52 11 −2 Left thalamus: centromedial region −2 −4 7 Right thalamus: centromedial region 3 −4 6 Right insular cortex 48.2 4.4 4.9 Left anterior insular cortex −42 8 5 Mid-cingulate cortex −1 3 44 Left thalamus: centromedial region −11 −19 5 Right thalamus: centromedial region 11 −17 5 Left thalamus: ventral lateral region −14 −14 16 Right thalamus: ventral lateral region 15 −11 17 Left thalamus: dorsal medial region −1 −17 9 Right thalamus: dorsal medial region 3 −17 8
The GCM analyses revealed a number of brain areas with effective connectivity values that exceeded statistical threshold (
In addition to revealing patterns of effective connectivity between the thalamus and the cingulate motor areas, the GCM confirmed that the cingulate motor region is influenced by the anterior portion of the insular cortex (i.e., the BOLD response in the anterior insular cortex predicts the BOLD response in the cingulate motor region). This finding is entirely consistent with the view of Craig (2002, 2009) that the insular and cingulate cortices form the input and output regions of a functional system, and can be characterized as the limbic sensory and motor areas. Importantly, the GCM analysis confirms that the cingulate motor region also exerts an influence over the activation of a different region of the insular cortex; specifically the mid-insular cortex. This can be clearly seen in
For the right hemisphere insular cortex seed region, the GCM analysis identified four brain areas that likely
(A) Results of the effective connectivity analyses, based upon Granger causality mapping (GCM), of the yawning fMRI study. In this case, the “seed” region for the GCM has been defined as the region of the mid-cingulate cortex significantly activated during the urge to yawn. The GCM analysis revealed that regions of the anterior insula bilaterally exert a significant influence over the seed region (blue), whereas regions of the mid-insula and inferior frontal lobe bilaterally are influenced by the seed area (pink). TRA: transverse; IFG: inferior frontal gyrus. (B) Further results of the GCM analysis based upon the mid-cingulate “seed” region. The analysis also revealed that regions of the centromedial thalamus bilaterally exert a significant influence over the seed region (blue), and bilateral regions of the anterior thalamus are influenced by the seed area (pink). CMA: cingulate motor area.
(A) Results of the effective connectivity analyses, based upon Granger causality mapping (GCM), of the yawning fMRI study. In this case, the “seed” region for the GCM has been defined as the region of the insular cortex of the right hemisphere that was significantly activated during the urge to yawn. The GCM analysis revealed that corresponding regions of the insular cortex within the left hemisphere, and the mid-cingulate cortex bilaterally, each exert a significant influence over the seed region (blue). CMA: cingulate motor area; TRA: transverse. (B) Further results of the GCM analysis based upon the right insula “seed” region. The analysis also revealed that regions of the centromedial thalamus bilaterally exert a significant influence over the seed region (blue), and that bilateral regions of the ventral-lateral and dorsomedial thalamus are influenced by the seed area (pink).
It is important to keep in mind that the patterns of effective connectivity revealed by the GCM analyses do not necessarily reflect anatomic connections. Nevertheless, the patterns of connectivity revealed by the GCM are consistent with known anatomy and function of specific thalamocortical connections. For instance, the anterior thalamic nuclei receive their primary input from the hypothalamus, project to the cin-gulate cortex, and are associated with visceral and emotional processing. In the current study, the GCM analysis confirms that the activity of the anterior thalamus exerts an influence over the cingulate cortex and thus provides a route by which emotional and visceral signals might influence the selection of motor responses.
The centromedian nuclei of the thalamus are a primary source of thalamostriatal projections and play a key role in motor function. In the current study, the GCM analyses confirm that the centromedian region of the thalamus is
The GCM analysis also revealed that the right hemisphere insular cortex “seed” area is influenced by the BOLD activation within the ventral lateral and dorsal medial regions of the thalamus. The ventral lateral nuclei of the thalamus receive their input from the basal ganglia and cerebellum and send outputs to motor regions of cortex. As illustrated in
Our everyday movements often involve interactions between our body and physical objects located within our environment, and engage multiple sensorimotor systems acting in concert. Action selection mechanisms must therefore take account of information about the current state of the motor apparatus (our body) and also of the behavior of objects within our immediate environment. Recent computational neuroscience approaches to the selection and control of movement (e.g., Wolpert, 1997; Wolpert & Ghahramani, 2000) and to reinforcement learning (e.g., Dayan & Niv, 2008) have emphasized the importance of prediction mechanisms to these processes.
It has been argued that efficient motor behavior relies to a large extent upon
Reinforcement learning also involves the evaluation of the outcomes that follow an action. Computational models of reinforcement learning propose that learning can be based upon internal models of the state transitions and action outcomes within an environment, or based upon model-free learning mechanisms (Dayan & Niv, 2008). Model-free reinforcement learning involves learning to estimate or predict the likely outcome (value or reward) of a given action (state) given an appropriate action-selection policy. This kind of learning is associated with the formation of “habitual” responses (Graybiel, 2008) and has been particularly linked to the operation of the ventral striatum, the neu-rotransmitter dopamine acting as a reinforcement signal that codes for reward “prediction error” (Schultz, Dayan, & Montague, 1997). By contrast, model-based reinforcement learning has been associated with goal-directed action in which the efficacy of candidate actions is evaluated with reference to an internal model of the task or state space. A key concept associated with this type of learning is that the selection of an appropriate action may involve a “mental simulation” of potential outcomes (Dayan & Niv, 2008). We suggest that the formation of “urges-for-action” is probably linked to the operation of the habit-learning system, whereas the formation of “desires-for-action” is probably associated with goal-directed, action-planning mechanisms.
In this paper, we have speculated that the insular cortex may accumulate evidence on the outcomes of an action and determine whether the conditions giving rise to the urge for action have been resolved. Within the computational framework offered by the forward model literature, this process might involve a comparison of a “next state estimate” (the output of a forward “sensory” model that provides an estimate of the sensory consequences of a planned action) with afferent sensory information that signals the sensory outcomes of the executed action. Within the computational framework offered by model-free reinforcement learning, this process would involve predicting or representing the likely outcome (value or reward) of an action.
To examine whether the insular cortex plays a key role in the representation of the reward estimates for planned actions, we carried out an ALE meta-analysis of functional brain-imaging studies that have investigated the anticipation or expectation of rewards. Full methodological information is provided in detail in the online supplementary material.
Studies were initially chosen by the following keywords: “reward,” “prediction,” “prediction error,” “fMRI” and “prediction,” “prediction error,” “expectation,” and “anticipation.” The reward modalities used in the studies reported here were monetary or pleasurable taste rewards. Studies were further refined according to the following criteria.
First, events used in the fMRI contrasts should occur
Second, the contrasts should reflect events in which participants hold an expectation of reward versus a control event such as the expectation of non-rewarding stimuli, or the expectation of loss. The study might include, however, contrasts of high reward probability versus low reward probability. Thus, results that reveal activations by association with the reward probability were explicitly included in the analysis.
Third, participants should expect primary rewards or secondary rewards. Studies that investigated the prediction of non-rewarding events were excluded from this analysis. As a result of these selection criteria, eight studies were included in the meta-analysis. Full details of these studies are provided in the online supplementary material.
The results of the ALE meta-analysis are illustrated in
The above analyses implicate the input and output nuclei of the basal ganglia together with the anterior insula and the dorsal cingulate cortex in the representation of reward estimates. These findings are consistent with recent proposals that the basal ganglia nuclei modulate movement execution according to motivational factors, specifically context-specific cost/reward estimates (Turner & Desmurget, 2010); that the anterior insular cortex may represent expectations of both positive and negative action outcomes (e.g., Preuschoff, Quartz, & Bossaerts, 2008); and that the anterior cingulate cortex plays a fundamental role in relating actions to their outcomes, and functions, together with the ventral striatum, to mediate cost–benefit decisions over the selection of action (Behrens, Woolrich, Walton, & Rushworth, 2007; Rushworth, Mars, & Summerfield, 2009; Rushworth, Walton, Kennerley, & Bannerman, 2004).
These findings are also consistent with the proposal being advanced here that the anterior insular cortex and the dorsal cingulate motor areas, along with the ventral striatum and thalamus, form core nodes in a motivation-for-action system. Specifically, we propose that pleasant or unpleasant somatosensory or visceral sensations—for instance, the premonitionary sensations that precede the occurrence of tics in TS—are represented within the SII, and in the posterior and mid-insular cortex, and that these sensations will often elicit habitual, overlearned, actions. We also propose that awareness of these bodily sensations, often perceived as an
Main results of an ALE meta-analysis of neuroimaging studies of reward expectation (see the online supplementary material for additional information). This analysis revealed a number of activation foci that survived conservative statistical correction (
Finally, as illustrated in
In this paper, we have begun to consider the nature and the functional anatomy of “urges-for-action,” both in the context of everyday behaviors such as yawning, swallowing, and micturition, and in relation to clinical disorders such as TS, where urges-for-action are considered pathological, and interfere with activities of daily living. We began by reviewing previous frameworks for thinking about behavioral urges, such as Davenport, Sapienza, and Bolser's (2002) motivation-for-action framework that had been formulated in the context of the urge to cough. While it contains several important insights, we took the view that a core aspect of this framework—that actions depend upon the conversion of an urge-for-action into a conscious desire-for-action—was very likely incorrect. We felt that this was particularly true in the case of individuals with TS, in whom urges-for-action were associated with the occurrence of motor and vocal tics that the individuals found both embarrassing and distressing. In this instance, it is difficult to reconcile such actions with the notion that they spring from a conscious desire for action. Instead we took the view that such actions may be habitual, and as such were highly overlearned and automatic, and could in some cases be executed with very little or no conscious awareness of the sensory stimulation that triggered the action. Importantly, we distinguished between reflex actions and the urge-for-action, which, we argued, occurred when bodily signals gave rise to an action that must be suppressed or deferred.
(A) GCM effective connectivity analyses of fMRI BOLD activations of yawning revealed a reciprocal pattern of effective connectivity between the anterior ínsula, the cingulate motor area, and the mid-insula. (B) On the likely function of these connections, we propose that the anterior insula may represent the urge-for-action, that the cingulate motor region may participate in the selection of an appropriate action following a cost-benefit analysis based upon the organism's past action-outcome history, and that the mid-insular cortex may evaluate, based upon a prediction of the likely outcome of the selected action, whether the conditions giving rise to the urge have been resolved, and, if appropriate, may generate a sense that the urge-for-action has been satisfied. CMA: cingulate motor area.
Then, using quantitative ALE meta-analytic techniques, we investigated the functional anatomy of the urge-for-action in the context of swallowing and micturition, and demonstrated that brain activations associated with these behaviors overlapped in two regions of the brain; the right insula and the dorsal anterior cingulate cortex. Furthermore, we showed that functional activations associated with the urge to tic in individuals with TS and the urge to yawn in neurologically healthy individuals also overlapped within these same two brain areas. These two areas have been conceptualized as the limbic sensory and motor areas respectively (Craig, 2002, 2009), and based upon the effective connectivity analyses of our yawning fMRI data, we proposed that they are central in representing the urge-for-action, and together form a neural circuit that represents bodily sensations, generates an urge-for-action (which may or may not reach awareness), selects a particular action based upon a cost–benefit analysis of the likely “value” of that action, accumulates evidence on the outcomes of that action, determines whether the conditions giving rise to the urge have been resolved, and, if appropriate, generates a sense that the urge has been satisfied. Finally, we argue that this circuit is anatomically separate and largely independent of the neural system responsible for the preparation and execution of intentional, goal-directed, actions, although it is possible that these systems may overlap partially with respect to the sense of agency that accompanies intentional action, which has been associated with the activity of the insular cortex of the right hemisphere.
Jackson and coauthors state that motor reflexes (as in recoiling from fire) are different from urge-for-action in that they cannot be cortically inhibited (or temporally postponed). They also characterize urges for actions as different from desires because of their highly implicit and not overly conscious nature. These characterizations are insightful and correct in principle, but the authors focus on very specific and peculiar urge-for-action types, such as the urge of urination, and of yawning and scratching, which are far from homogeneous.
The urge to urinate is one of the many signals that originate in the brain upon hypothalamic impulses and that are involved in the homeostatic regulation of physiological parameters (such as blood oxygen, delta sleep-inducing peptide levels, and plasma concentration of glucose (Burdakov, Luckman, & Verkhratsky, 2005). In this view, the urge to eat in a person with very low glucose levels cannot be considered a reflex (it can be cortically guided, inhibited, or deferred) and is not necessarily a conscious desire (e.g., as with someone who is working and is very concentrated); therefore, it can be legitimately considered an urge-for-action. Similarly, the urge to breathe when there is a lack of oxygen (at high altitude, or in badly air-conditioned places), or, again, the urge to lie down when cerebral blood pressure is low and we feel faint; the urge to quickly undress when it is too hot, or to wrap up when it is freezing; the urge to move arms and legs when we are drowning; and the urge to cough if we are suffocating can all legitimately be considered types of urges-for-action.
We are not sure that insula and mid-cingulate cortex are crucially involved in all these behaviors. It is possible that contagious yawning might share more similarities with other human behaviors reflecting the processing of social information (such as contagious crying and laughing). Indeed, we know that both insula and cingulate cortex play a very important role in empathy, in response to crying (Lang, Yu, Markl, Mü & Kotchoubey, 2011; Sander, Frome, & Scheich, 2007) or to the sight of suffering people (Proverbio, Adorni, Zani, & Trestianu, 2009). Possibly, these structures are responsible for other urges-for-actions such as those regulating instinctual parental behavior (catching a baby falling from a table, for example), aimed at increasing the survival rate of individuals (brushing an insect away from a child) or of the species.
Finally, I would make a distinction between spontaneous yawning and contagious yawning. Spontaneous yawning is thought to have the important function of increasing oxygen blood levels, by expanding the lungs and stretching the muscles (Thompson, 2011). It is also known to lower brain temperature, thus favoring increase in sleepiness and passage to the sleep stage (Gallup & Gallup, 2007). Overall, yawning is probably involved in the circadian and homeostatic control of sleep and wakefulness. Contagious yawning, on the other hand, is thought to be based on the activity of mirror neurons, with the purpose of synchronizing and regulating the behavior of cohabitant individuals, similarly to contagious laughing and crying (Geangu, Benga, Stahl, & Striano, 2010). In this view, the urge to yawn when another does so would stimulate regions involved in theory of mind and empathy, such as the cingulate cortex (Platek, Mohamed, & Gallup, 2005), as was found by Jackson and coauthors.
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Understanding the anatomical and physiological basis of “urges” is important not only because they occur in disease states, such as Tourette syndrome, but also because they are closely related to impulses that, when uncontrolled, can wreak havoc on individuals and societies. Problems with impulse control include substance abuse, domestic violence, sexual deviancy, and pathological gambling. In this context, it is important to clearly define what is meant by “urge,” to delineate what distinguishes normal from pathological urges, and to clarify how urges differ from other modes of action selection. Study of the underlying functional anatomy, as reviewed by Jackson et al., can then lead to a greater understanding and better treatment of impulse-control disorders.
The elementary components of brain function can be divided into sensory input, processing, and motor output. Urge is one mode of processing. Although it exists within a continuum of modes including reflex, desire, and intention, in which boundaries are somewhat artificial, defining the phenomenon helps unify research efforts. Sensory input that leads to urge can come from within the body (interoceptive) or from outside the body (exteroceptive). We characterize urge based on examples of common bodily urges: to urinate, yawn, cough, blink, and sleep. In each, sensory input can trigger the action out of awareness, in which case there is no urge and the action is perceived as occurring involuntarily. If, however, the action is delayed, the sensation reaches awareness and the urge to act develops. Urge is separate from the sensation. The sensation that our bladder is full is distinct from the
In many ways, Tourette patients experience a parallel series of events (Bliss, 1980). Their tics can occur completely out of awareness, and, in these instances, would be described as involuntary. Often, however, patients describe an uncomfortable bodily sensation that triggers the urge to tic: a feeling that the action
However, there are some key distinctions between normal interoception-related urges and those associated with tics. First, we do not yet know what type of sensory input generates the uncomfortable bodily sensations that trigger the urge to tic. Although patients sometimes describe these sensations as coming from muscles or joints, it has never been demonstrated experimentally that sensory activity initiated in the periphery is the origin of their sensory experience. Second, tics are not key to survival. It is an important aspect of the pathology in Tourette syndrome that patients experience such distress in relation to actions that are not only nonessential but also nonproductive. We do not yet understand when or how this association is made. Third, following the tic, the bothersome sensation is not eliminated, but only reduced in intensity. Finally it is not clear why tics must be performed in a particular way in order to achieve relief and diminishment of the bothersome sensations. Continued research into the circuitry that mediates both normal and pathological urges, as described by Jackson et al., will help our understanding and treatment of Tourette syndrome and other impulse-control disorders.
Features of normal and tic-related urgesSensory input from bodily organs Yes ? Sensation reaches awareness when action delayed Yes Yes Sensation is uncomfortable Yes Yes Experience urgency and need to take action Yes Yes Action can be suppressed (minutes), and then must Yes Yes occur Relief from discomfort with action taken Yes Some Action is necessary to organism survival Yes No
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Jackson et al. have proposed a novel model of urge-for-action. This model considers the nature and functional anatomy of urge-for-action in the context of normal life and clinical disorders. Through a meta-analysis, the authors show that there is an overlap between limbic sensory and motor neural circuits related to urges of everyday behaviors such as swallowing and tics in Tourette syndrome. The primary merit of this work is unquestionable; the authors propose an empirical and theoretical model of urge-for-action incorporating actions that do not necessarily require conscious awareness of the sensory stimulation that triggered them. Nevertheless, there are important issues that this model does not explicitly incorporate.
First, it is not clear how urge-for-action, as defined in this paper, can be a fully unconscious phenomenon. If the momentary inhibition of the action is an integral part of the definition of these urges, then that inhibition should be unconscious as well. In this paper, we find a lack of evidence on this topic, perhaps reflecting more than a mere lack of interest on this matter. Having searched the literature on unconscious inhibition of action, we found several instances in which willed intention is involved (e.g., go/no go tests in Eimer & Schlaghecken, 2002, and van Gaal, Ridderinkhof, van den Wildenberg, & Lamme, 2009). It is hard to imagine a fully unconscious inhibition of the kinds of actions considered to be representative of urge-for-action (coughing, swallowing, yawning, etc.). As long as that inhibition plays an important role in this process, we should be consciously aware of our urge-for-action as opposed to our reflexes.
The second issue concerns the nature of the relationship between urges and conscious awareness. Jackson et al. explained that the intensity of physiological afferent stimulation relates directly to the awareness of the urge's strength during a phenomenon such as swallowing, but it is not clear how this phenomenon could be explained by their proposed model.
Despite the straightforward relationship between insular cortex and interoceptive conscious awareness (e.g., Ibáñez, Gleichgerrcht, & Manes, 2010), this fact is not explicitly taken into account in terms of the neuronal activity of the cortical and subcortical regions considered in this model. More importantly, even if we consider that urge-for-action could be an unconscious phenomenon, it remains unclear how this model could explain the transition between urges the subject is not conscious of and those of which the subject is consciously aware. Along these lines, is it the strength of activation of the right insular cortex, the anterior cin-gulate cortex (ACC), or the circuit between these regions that is responsible for setting the threshold between unconscious and conscious awareness of urges? This point is far from being addressed in Jackson et al.'s model.
Finally, how could the urge-for-action model of Jackson et al. be integrated with more general models of consciousness? Current models have determined the activation of widespread cortical regions during goal-directed visual awareness (e.g., Dehaene et al., 2001) and have specified some neuronal markers for reaching the threshold of conscious perception (e.g., Del Cul, Baillet, & Dehaene, 2007). How does this urge-for-action circuit interact with more general circuits of conscious perception? Interoceptive awareness is conceptualized as the capacity of being aware of some specific
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The notion of “urge” has come to play a pivotal role in current conceptions of voluntary action. Curiously, it has done so without detailed exploration of the kinds of actions—involuntary or atypically voluntary—where the need to invoke it is arguably much greater, and where an understanding of its neural basis is far more plausibly within reach. And when one analyzes such actions—as Jackson and his colleagues lucidly and comprehensively do—the notion of urge that emerges is radically at odds with one that gives it a critical role in voluntary action. This is just as important a conclusion as the independent clarification of the neural substrate of urges itself.
It is surely right that urges must be dissociated from the sensations that in some cases prompt them. The urge to yawn, for example, is not plausibly any kind of sensation: One can only describe it with reference to the action it compels. What is minimally constitutive of the notion of an urge is that it is an urge
Indeed, it is striking that the actions commonly associated with urges—both in the normal and in the pathological state—are usually of the kind that cannot be actively
An urge to act, then, cannot be empirically dissociated from the inhibition of the action, and presumably also from the conflict between action and inaction: this is so not because we lack the experimental tools but because inhibition is constitutive of the notion of an urge. This implies that the neural substrate Jackson and his colleague identify must also subserve these processes. More importantly, it casts further doubt on the notion that an urge is a signature of the phenomenology that is argued to precede voluntary action and to take part in its self-ascription. For if an urge signifies the inhibition of an action, it can hardly be thought of as the “idea” driving it. This also explains why urges are often reported during macro stimulation of the dorsomedial frontal cortex: The set of multiple neuronal pools inevitably severally activated at that scale of stimulation is bound to contain units that inhibit as well as drive active movement.
There are, of course, numerous unanswerable arguments against the ideomotor theories of action popularized by Libet's work, but keeping the lid on that coffin is so extraordinarily difficult another nail can never go amiss.
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Jackson et al. develop a convincing case for the existence of a neuroanatomical system that is responsible for the “urge-for-action.” The role of the (right) insula and the dorsal anterior cingulate cortex is inferred on the basis of a number of separate meta-analyses looking at associated brain activations in response to specific stimulation—in this case different triggers of urges. The idea that there are separate systems for “willed” or planned actions and “urged” actions makes sense and is clearly supported by the clinical and neuroimaging data.
The definition of “urges-for-action” proves more complicated. The beginning is relatively straightforward. These urges involve a limited set of actions that are habitual, standardized routines, such as scratching, yawning, or swallowing. They typically do not require feedback, and they are functional in that they alleviate specific negative or unpleasant bodily states. Things become tricky when we are offered the suggestion that we have conscious and unconscious urges (cf. Dijkerman & De Haan, 2007). We are told that urges and desires are not synonymous, and the main reason for this is that urges may also remain unaware to the person who “experiences” them. Indeed, we may find ourselves swallowing or yawning without having had a conscious urge, but it is not clear to me why this behavior cannot be classified as “reflexive.” Indeed, the Davenport, Sapienza, and Bolser (2002) study seems to indicate that we are dealing with a system where the intensity of the (negative) stimulation is directly related to strength of the perceived urge. This makes sense, the limbic system including the insula, registers the amount of bodily discomfort, and depending on the severity of this discomfort, a more or less “urgent” urge is felt.
I also agree with the authors that what distinguishes a reflex from an urge is the fact that a reflex—by definition—is immediate and proceeds without conscious interference. An urge, however, may need to be postponed as the execution of the required action is socially or emotionally compromising. Here I would like to suggest a different interpretation from the one put forward by Jackson et al. that an urge may be construed as an interrupted reflex. The reflex entails a complex sequence of detecting a specific negative bodily state coupled to a proven alleviating motor action. The fact that this motor action is not socially or emotionally acceptable is in essence culturally defined, and therefore, is a learned response.
Thus, and this is my central thesis, the defining aspect of an urge is a learned interruption of a reflexive sequence. The result of this interruption is a conscious awareness of the pending action
Jackson et al. state that it is “difficult to reconcile the concept of a ‘desire,’ which is often defined as ‘a longing or craving for something that brings satisfaction or enjoyment,’ with unwanted actions, the execution of which is experienced as unpleasant and distressing” (p. 229). I find this surprising and I cannot subscribe to this “difficulty” felt by the authors, as in my experience this is often exactly what patients with obsessive compulsive disorder or Gilles de la Tourette syndrome report. The simultaneous experience of the urge to carry out an action and the concurrent realization that this is not acceptable, socially or emotionally, could be posited as the hallmark of obsessive compulsive disorder. In fact, after the act has been performed, there is again a simultaneous experience but now one of relief (because the act has—somewhat—alleviated the negative bodily state) and shame (because the executed act was socially or emotionally unacceptable). Interestingly, when I was reading the article for the first time, I thought that, toward the end, the authors would propose a role for the right insula in becoming aware of an urge. They did not do so, and I am glad they did not, because it is not important for their main conclusion.
This review provides strong evidence for a separate network in the brain involved in the detection of well-defined negative bodily states and a set of habitual, standardized routines to alleviate the discomfort. When the required action is consciously flagged because of learned associations with negative social or emotional connotations, the execution of these actions is— temporarily—inhibited, and an “urge” is experienced.
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In their nice and elaborated review, Jackson et al. provide a functional basis for a distinction between reflexive behaviors and actions that come from urges helpful in daily life (such as the urge to yawn, urinate, and cough) and those possibly interfering with daily life (such as the urge to tic). They argue that the latter are associated with
Examining the properties of the urge in GTS, and already before Leckmann's seminal paper (Leckmann, Walker, & Cohen, 1993) on the subject, Bliss, a clinician and GTS patient himself, published (Bliss, 1980) a thorough description of his own premonitory sensations and, as a result, argued for the voluntariness of tics. In the following years, systematic studies showed that more than 90% of patients with GTS report these urges and consider tics as, if at all, only partial involuntary. It has been consistently found that the development of the urge is reported with a lag of about 3 years after the onset of tics (Banashewski, Woerner, & Rothenberger, 2003; Kwak, Dat Vuong, & Jankovic, 2003; Leckmann et al., 1993). The delayed appearance of this phenomenon has led to a series of hypotheses: Is the association of urges and tics a compensatory evolutionary development as a basis for the option to suppress these phenomena, or are urges and tic suppressibility co-existing phenomena, as suggested by recent tic-suppression studies? (Banashewski et al., 2003; Conelea & Woods, 2008).
As for the neurophysiology of tic generation, the zeitgeist led to the study of the correlates of the voluntariness of movement disorders, with the pioneering work of Obeso, Rothwell, and Marsden (1981), followed 14 years later by another work (Karp, Porter, Toro, & Hallett, 1996), which examined the presence of the Bereitschaftspotential (BP) in tics. The results were equivocal, with the first study showing that tics were not preceded by the BP, and the second demonstrating its presence in 2 out of 5 patients. This led to the hypothesis (Kwak et al., 2003) that only tics associated with a premonitory sensation—and therefore consciously perceived and voluntarily initiated—are preceded by the BP. This has been shown in three patients in the only study to date that addressed this question (Duggal & Nizamie, 2002). Additionally, and in accordance with the latter study, which, surprisingly, found a shorter than normal BP, Moretto showed that patients with GTS have a delayed experience of volition (Moretto, Schwingenschuh, Katschnig, Bhatia, & Haggard, 2011). This would imply that not only the formation of tics but also the formation of normal movements would necessitate an altered pattern of motor organization through fronto-striato-thalamo-cortical pathways, as supported by current findings (Heise et al., 2010; Roessner et al., 2011). Furthermore, in the absence of direct evidence to support the notion that the awareness of urges correlates positively with the ability to suppress tics—a suppression, which, according to Jackson et al., would lead to the propagation of the urge and further activation of the anterior insula—the role of frontomesial networks of volitional inhibition (Kühn, Haggard, & Brass, 2009) and tic suppression has to be addressed in detail. These networks presumably influence the pre-supplementary motor area activity in a top-down fashion, as supported by a recent EEG experiment showing elevated interregional interactions between these and sensorimotor and prefrontal areas during tic inhibition, paralleled by an increasing urge to tic (Serrien, Orth, Evans, Lees, & Brown, 2005).
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This is an Open Access article distributed under the terms of the Creative Commons Attribution License (
This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits distribution, and reproduction in any medium, provided the original author and source are credited. This licence does not permit commercial exploitation or the creation of derivative works without specific permission.
This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits distribution, and reproduction in any medium, provided the original author and source are credited. This license does not permit commercial exploitation or the creation of derivative works without specific permission.
This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits distribution, and reproduction in any medium, provided the original author and source are credited.This license does not permit commercial exploitation or the creation of derivative works without specific permission.
This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits distribution, and reproduction in any medium, provided the original author and source are credited. This licence does not permit commercial exploitation or the creation of derivative works without specific permission.
This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits distribution, and reproduction in any medium, provided the original author and source are credited. This license does not permit commercial exploitation or the creation of derivative works without specific permission.
This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits distribution, and reproduction in any medium, provided the original author and source are credited. This license does not permit commercial exploitation or the creation of derivative works without specific permission.
This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits distribution and reproduction in any medium, provided the original author and source are credited. This licence does not permit commercial exploitation or the creation of derivative works without specific permission.
This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits distribution and reproduction in any medium, provided the original author and source are credited. This licence does not permit commercial exploitation or the creation of derivative works without specific permission.
This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits distribution and reproduction in any medium, provided the original author and source are credited. This license does not permit commercial exploitation or the creation of derivative works without specific permission.
This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits distribution, and reproduction in any medium, provided the original author and source are credited. This license does not permit commercial exploitation or the creation of derivative works without specific permission.
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This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits distribution and reproduction in any medium, provided the original author and source are credited. Creation of derivative works is permitted but the resulting work may be distributed only under the same or similar licence to this one. This licence does not permit commercial exploitation without specific permission.
This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits distribution, and reproduction in any medium, provided the original author and source are credited. This licence does not permit commercial exploitation without specific permission.
This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits distribution, and reproduction in any medium, provided the original author and source are credited. This license does not permit commercial exploitation without specific permission.
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This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
A 53-year-old male, formerly employed as a construction worker, was referred for massage therapy for treatment of debilitating low back pain. He has pain in all areas of the back; although he sometimes experiences cervical pain and/or thoracic pain, he reports the majority of his pain is in the lumbar area. His objective for treatment is to cut down on pain medication, acetaminophen and oxycodone (Percocet), currently prescribed at 7.5 mg four times daily. The subject states he does not like to take it that frequently because it makes him feel lethargic and disoriented. The massage used was a combination of Swedish strokes (effleurage, petrissage, friction), muscle stripping, and myofascial release, techniques chosen based on the practitioner’s 14 years of experience in working with clients who are in pain. A treatment plan of six weekly visits was agreed upon with re-evaluation after the sixth visit. The subject is dependent on only his disability income and is unable to commit to more frequent visits. Progress was measured using the Oswestry Low Back Pain Scale and by subjective statements on his decreased pain level and decreased need for medication, and positive effects on his activities of daily living. The massage therapy intervention was so successful that the subject switched to monthly maintenance care after four sessions instead of six as originally planned. The success of this intervention for this subject suggests that massage therapy can be an effective intervention for chronic low back pain.
To present research data representing short- and long-term stress reduction in a child with an autism spectrum disorder using zen shiatsu as a complementary and alternative treatment.
A seven-year-old male with a diagnosis of autism was given 20-min zen shiatsu sessions weekly for six consecutive weeks. Using a five-point stress scale that utilized facial expressions with corresponding stress values, the client indicated his stress level prior to the session, as well as afterwards. Measurement comparing the levels demonstrated a decrease in stress within the 20-min period. In addition, the parent was given the PEDS QL 4.0 Young Child Questionnaire to determine the child’s HRQoL (Health Related Quality of Life) prior to the six-week zen shiatsu treatment to establish a baseline. The parent completed the same questionnaire after the six weeks of sessions, to compare results.
Stress levels decreased in the client after receiving zen shiatsu after all six sessions. The PEDS QL 4.0 showed higher HRQoL scores in all domains, indicating that the child’s overall quality of life improved within the six weeks of receiving zen shiatsu.
Zen shiatsu, a form of traditional Chinese medicine, has the potential to be a viable treatment for stress reduction in children with autism spectrum disorders, thereby improving overall quality of life.
Reduced flexibility and balance are associated with aging and increased fall risk. The purpose of this study was to determine the effects a single active muscle therapy treatment on ankle flexibility and balance in adults aged 50–65 years.
Thirty-one volunteers (26 women, 5 men; 58.5 ± 4.6 yrs; 84.6 ± 22.7 kg; 166.1 ± 8.2 cm; mean ± SD) had their balance measured with the FDA-approved Sway Balance TM mobile application (Sway Medical, Tulsa, OK) which uses the built-in tri-axial accelerometer within an iPhone or iPod Touch to measure postural sway. Participants held the device against the chest while standing with feet together, in tandem, and on one foot for 10 sec each. Sway measures from each stance were compiled into a single score with 100 being perfect (no sway). In addition, ankle dorsi and plantar flexion flexibility were measured using a digital inclinometer. Participants then underwent a 2.5 min warm-up massage to each foot, ankle, and lower leg; a 2.5 min stripping massage (7/10 on a verbal pressure scale) on each tibialis anterior muscle while eccentrically resisting an elastic resistance band; and a similar 2.5 min massage with resistance on the gastrocnemius/soleus groups. After the 15-min massage intervention, balance and flexibility measures were repeated.
Balance scores increased (
Results indicate that combining eccentric resistance with massage improves balance and ankle flexibility in adults aged 50–65 yrs immediately postintervention. Future research is needed to determine the effects in older populations, the long-term effects of this treatment, the effects of multiple treatments, and the effects on fall incidence.
Knee osteoarthritis (OA) is a highly common and disabling condition among Department of Veterans Affairs (VA) health care users, and effective, conservative treatment options are limited. Massage therapy holds promise for improving knee symptoms, but there are limited data to date, especially for VA health care users, who tend to have more severe OA and comorbidity than the general population.
This pilot study assessed the feasibility and efficacy of massage among VA health care users with knee OA.
Participants were 25 VA health care users with symptomatic knee OA. Participants received eight weekly 1-hr sessions of full-body Swedish massage. Measures completed at baseline and eight weeks included the Western Ontario and McMaster University Osteoarthritis Index (WOMAC), pain interference (PROMIS-PI), and global pain (visual analog scale: VAS).
Participants’ mean age was 57 years (12 SD), 68% were male, and 52% were African American. The mean baseline total WOMAC score was 62.9 (16.1 SD). Following the eight-week intervention, mean total WOMAC scores decreased to 45.4 (22.6 SD,
Results of this pilot study offer support for the efficacy and feasibility of Swedish massage for improving symptoms among VA health care users with knee OA. If results are confirmed in a larger randomized trial, massage could be an important component of regular care for these patients.
Patients on dialysis can experience pain caused by muscle cramping that may result in shortened treatment times that have been linked to worse outcomes. It is estimated that up to 88% of dialysis patients experience cramping, and one study reported that 15% of patients with shortened treatment cited muscle cramping as reason. Over the past ten years, research studies using massage in cancer patients have shown decreases in pain, inflammation, and feelings of anxiety. Although there is limited evidence available about massage in dialysis patients, it may be an effective treatment modality for hemodialysis-related lower extremity cramping.
To determine the effectiveness of intradialytic massage on the frequency of cramping among hemodialysis patients prone to lower extremity cramping.
Our study protocol, approved by the Institutional Review Board at MetroHealth Medical Center included a two-month training period during which licensed massage therapists were trained in massage techniques and tested for intertherapist consistency. Thirty-two (16 intervention, 16 control) hemodialysis patients with frequent lower extremity cramps were enrolled in our study. Frequent cramping during dialysis treatments was defined as one or more episodes of lower extremity cramps, during or after dialysis over the previous two weeks. The intervention group received a 20-minute massage of the lower extremities during each treatment (three times per week) for two weeks. The control group received usual care by dialysis center staff.
Patient reported cramping at home decreased by 2.5 in the intervention group, compared to 0.3 in the control group (
Intradialytic massage appears to be an effective way to address muscle cramping. Larger studies with longer duration should be conducted to test for similar results.
Students of massage therapy programs are required to develop a rigorous blend of cognitive, psychomotor, and affective skills. From a pedagogical perspective, honing these skills can involve a multifaceted approach, frequently involving the adoption of learning technology.
The objective of this research was to evaluate the potential use of human patient simulation (HPS) technology to facilitate training of massage therapy students. The ability of HPS to assist in developing skills within the cognitive, psychomotor, and affective learning domains was central in this research. Relatable massage tasks investigated included the assessment and training of therapist biomechanics, communication skills, documentation, treatment planning, hygiene, vital sign assessment, draping procedures, reflection, awareness of treatment contraindications, and advising patients about self-care practices.
The chosen methodology for this inquiry involved a systematic review and meta-analysis. The studies were organized and critically assessed based on the Cochrane organizational matrix which facilitated the analysis of data contained within each study. A total of 16 studies published between the years 2000 and 2012, with an average sample size of 100, met the inclusion criteria. Inclusion criteria involved studies that directly related to the use of a health-related medium or high-fidelity HPS, and related to the learning domains for massage therapy. Studies that involved a great degree of interpretation by the author and opinion were excluded from this study. An effort by the researcher to ascertain a broad spectrum of literature has been made.
The literature review and subsequent synthesis has revealed a strong likelihood that HPS can effectively facilitate teaching and learning by addressing learning domains and entry-to-practice competencies. Some benefits cited included engaging critical thinking, developing biomechanics, improving clinical reasoning skills, and increasing student practitioner confidence.
The critical evaluation of current research shows strong potential for the use of HPS in the training of massage therapists.
Functional constipation, one of the most common somatic complaints encountered by practitioners, both negatively affects quality of life and leads to increased health care costs. Craniosacral therapy (CST) is a form of bodywork that addresses micro strain patterns which accumulate to create macro dysfunction. The core physiological intent of CST is to free restrictions in the cranial membrane and the dural tube to enhance central nervous system function. CST can elicit the release the somatic memories and residual effects of past injuries and negative experiences.
There is a paucity of data in the literature on the use of CST in the treatment of somatic complaints, such as gastrointestinal distress. This case report highlights the effect of CST on functional constipation in a trauma survivor.
A 49-year-old Caucasian female with a history of childhood abuse and domestic violence presented with a chief complaint of difficulty with elimination. She was treated with CST twice per week for four weeks and once per week for four weeks. The Constipation Assessment Scale (CAS) rates the severity of constipation on a scale of 0–16, with 16 being most severe. The CAS was administered before the start of treatment and again at four and eight weeks.
After eight weeks, a reduction in the severity of constipation was noted. The initial CAS score of 8 was reduced to 5 at four weeks and 2 at eight weeks.
CST facilitated the release of adverse mechanical strain patterns associated with somatic memories. Constipation decreased in severity. In this case study, CST was a useful tool in the treatment of constipation, underscoring the need for further evidence-based research in the use of CST to treat somatic complaints.
Investigate the use of myofascial massage therapy as treatment for longstanding contractures.
Contractures develop when the soft tissues in the muscle and surrounding fascia are replaced by inelastic, fibrotic tissue, making it hard to stretch the area, and prevents normal movement. Contractures are a concern for patients with strokes, spinal cord injuries, and other debilitating diseases. Severely limited range of motion inhibits many activities of daily living. The usual care for contractures include surgery, medication, braces, and botulinum toxin. The prevalence of contractures varies widely among patient population. The prevalence of contractures among multiple sclerosis patients was found to 56%. In one study, contractures were a complication for 73% of stroke survivors at the one year mark.
Subject was 23-year-old male massage student with history of contractures of the left arm and wrist since age six, when he suffered three left arm injuries over a one-year period. A series of six myofascial massages was applied over the course of two months, focusing on eliminating fascial adhesions and restrictions. Direct fascial strokes were applied including compartment separation, fascial wringing and spreading, cross-fiber friction, muscle energy techniques, and compression of myofascial trigger points. Approximately 80% of the strokes were applied to anterior and posterior forearm, with the balance applied to upper arm and pectoral regions.
Range of motion increased from severely limited supination, starting at approximately 70°, to normal range of 182° following the last massage. Other measures focus on daily living, such as an ability to play the banjo without pain, have more fluid movements while massaging, and more successfully complete small tasks, such as using the brakes on his bike and open doorknobs. This significant result warrants a more thorough investigation of massage therapy as a treatment for contractures.
Adequacy of treatment in patients with Wilson Disease (WD) was monitored by measuring 24-hour urinary copper excretion while on treatment with D-penicillamine. The major effect of the drug is to promote the urinary excretion of copper. Values between 3–8 μmol/24h (200–500 μg)
The aim of the study was to assess and compare the 6-hour and 24-hour copper urinary excretion in patients with WD treated with D-penicillamine. The data used ware from 42 measurements in 32 patients with proven WD in University Hospital St. Ivan Rilski, Medical University-Sofia. The average dose D-penicillamine was 800–1000 mg
The copper urine excretion in 6-hour, 18-hour and in 24-hour urine was respectively x = 4.12 ± 2.26 μmol / 6h, x = 5.75 ± 3.42 μmol / 18h and x = 9.89 ± 4.7 μmol / 24h. Copper excretion in 6-hour urine was 41.7% of 24-hour cupriuria.
About half of copper urine excretion was up to 6-hour diuresis. Reducing the time of urine collection could reduce the errors in preanalytical stage of examination. It is necessary the 6-hour urine copper excretion to be standardized and validated to use it as criteria of adequacy of therapy.
EDTA contamination of serum samples is one of pre-analytical errors occurring in daily work. EDTA may be present in serum due to improper order of tubes (EDTA tube being drawn before serum tube – possibility of backflow of EDTA into the collection device and carryover of additive into serum tube) or manual transfusion of EDTA blood into serum tube. EDTA causes hypocalcemia, hypomagnesiemia (due to chelation) and subsequently decreased activity of alkaline phosphatase. A case of high potassium value in non-hemolyzed serum due to EDTA contamination is reported.
61 years old male presented to hospital with hip fracture, serum potassium 10.44 mmol/L, normal sodium and chloride results, and calcium result below detection limit of the method. Suspecting pre-analytical error two subsequent samples were drawn in 2 and 4 hours. All samples were retested for potassium, sodium and chloride using three different methods: Beckman Coulter Synchron CX9 (USA), Abbott Architect ci8200 (USA), Radiometer ABL800 (Denmark). Total calcium concentration and alkaline phosphatase activity were measured by first two methods. Hospital administration organized audit on work of phlebotomists.
Mean of first sample (elevated potassium) was 10.31 mmol/L (SD = 0.27, N = 3). Means of other samples (normal potassium) were 5.14 mmol/L (SD = 0.07, N = 3) and 4.85 mmol/L (SD = 0.04, N = 3) respectively. Alkaline phosphatase activity (58
Irresponsible behavior of nurses may produce erroneous biochemical data, lead to incorrect diagnosis and unnecessary treatment. Education of medical and nursing staff regarding proper blood collection techniques is needed to prevent sample contamination.
The aim of this research is to study the influence of the lapsed time between the extraction and the determination procedure in the case of free beta human chorionic gonadotropin (free beta hCG) and pregnancy-associated plasma protein A (PAPP-A), and its effect on the calculation of risk of chromosomal abnormalities. Free beta hCG and PAPP-A increases over time, but it is to be determined how long it needs to cause a significant difference on the values and on the associated risk for the prenatal screening.
A group of 38 women in the first trimester of pregnancy were included in the study, blood samples were taken during the routine check-up. Blood samples were stored and transported to Virgen del Rocío Laboratories in the same conditions (uncentrifuged and in isothermal iceboxes). Once in the laboratory the samples were centrifuged and analyzed using the electrochemiluminescence immunoassay “ECLIA” on cobas 8000 (Roche Diagnostics).
Both beta hCG and PAPPA increase over time. There were significant changes among the determinations corresponding to the samples extracted four, seven, and nine hours before the analysis (P = 0.007). Both beta hCG and PAPPA have a linear ascending trend as the statistics and the profile graphs indicate (P < 0.001). Comparing two to two (the different levels of the factor -time elapsed since the extraction-), the critical levels associated to every comparison show that there are relevant differences among all the combinations of the different levels for both variables (P < 0.001). The associated risk has notable difference among the determinations carried out at different times from the extraction (P < 0.001). Such a difference, however, has no influence on the screening results.
Normal handling of samples is likely to increase the results of the determination of free beta hCG y PAPP-A as time till their processing increases. However, in most cases, these variations do not affect the screening results (positive or negative screening).
Intensive exercising may significantly damage muscles which is reflected in pain, fatigue and the increase of muscle proteins concentrations in blood such are creatinin kinase (CK), lactic dehydrogenase (LD), myoglobin (MB) and other biochemical parameters including urea serum concentration (SU). Biochemical markers vary with age, sex, race, muscle mass, physical activity and climate conditions. They also assist us in determining the limit between the capacity for adaptation to given training process which results in supercomepensation and in condition of overtraining (OT), in the case of load that exceeds the physiologic potential of regeneration. Concerning the problem of diagnosis and explanation of the symptoms of overtraining, markers that can apply reliably and with sufficient sensitivity and simplicity of interpretation in the praxis are sought. It is critical to take into account difference among individuals and groups that could hamper the interpretation.
The most frequently used biomarkers that provide us with the information on physical activity and on the amount of load through exercise are CK, SU and LD. Level of serum retaining kinas has been measured and interpreted for years as part of different scientific and professional investigations and presents one of basic parameters for determining the level of muscle damage. It reaches maximal concentration of the fourth day of exercising which depends on the type of exercise and the nature of stress triggered by exercise but also on individual characteristics.
The level of serum urea presents marker of nitric compounds metabolism and is the principle chemical substance in the urine of mammals. It is thus possible to draw a parallel between the increases of serum urea concentration on increased degradations of proteins. Significant fall of serum amino acid levels occurs after sixty to seventy minutes of exercising with the increase of urea and free tyrosine and these changes have high correlation with the duration and intensity of.
LD changes are important index of well-trained sportsmen and their capability to withstand the pace and force during strain in the training process. The level of LD is a good index of exercise intensity and marker of metabolic exchange in tissues whose concentration in serum is dependent of cell damage.
There is not a single, unique parameter that would provide enough valuable information for the estimation of the quality of exercising, amount of load and identification of overtraining. Delayed measurement of biomarkers is far from ideal, but it is obvious that the amount of stress/ load in training is the most important factor for the development of state of overtraining. Daily body weight control, diet, biochemical indices values and the input of water should be known and standardized before measurements. For the most of parameters determination of basal levels are needed in specific populations for more accurate interpretation and evaluation of results. The sampling process itself should be under the most strict conditions of standardization by repeating measurement at least every third day. Dependence of mentioned parameters (SU, CK, LD) on exercise intensity varies among individuals and without these additional measurements and subpopulation evaluations it is difficult to come to conclusions with certainty as well as to come to conclusions on causative correlations of training load and dynamic in biochemical parameters.
Pneumatic tube systems (PTSs) are widely used in hospitals for convenience and reduced turnaround times. The aim of this study was to investigate the effects of PTS on routine biochemistry and intact parathyroid hormone (iPTH) analysis.
In this study, we used blood samples from 20 healthy volunteers. Three vacutainer tubes of blood were taken from each volunteer. These samples were sent to the central laboratory from two different locations by PTS, and a human porter. Maximum distance location (Group I) and the minimum distance location (Group II)
The bicarbonate and lactate dehydrogenase (LD) levels were significantly higher in samples transported
The transport of non-centrifuged samples
To the Editor:
We applaud Vinson and Hoehn for eloquently demonstrating that the performance of sedation assisted procedures in the emergency department (ED) does not necessarily require a 2 physician team. From a Canadian perspective, where single physician coverage in smaller EDs is common, this has important implications in terms of efficiency of patient care, reduction in the need for patient transfer and decreasing the time to definitive treatment for ED patients. We would like to draw attention to a model of care practiced in Halifax, Nova Scotia for over 15 years, using a team consisting of an advanced care paramedic (ACP) and a single physician, the former to conduct the sedation, and the latter to do the procedure.
To the editor:
We were interested to read Gamble and Hess’s study finding that the daily incidence of violent crime in Dallas increased with temperatures up to 90°F (32.2°C), but decreased above this threshold. On this basis, their abstract surprisingly concludes that “higher ambient temperatures expected with climate change…. are not likely to be accompanied by markedly higher rates of violent crime” (p .239). This conclusion contrasts with the findings of previous studies.
Unfortunately, the authors did not attempt to actually estimate the magnitude of future warming that would be sufficient to bring about a decrease in aggregate annual violent crime, which will differ from the inflection point of the relationship between daily temperature and violent crime. We therefore used the piecewise regression model reported by the authors in order to investigate how annual aggravated assault incidence in Dallas is likely to be affected by changes in mean temperature. We focus on aggravated assault given that this was the crime for which a marked effect of temperature was reported. Temperature data for Dallas International Airport in 1999 was collected from the NCDC
The simulation was conducted as follows. For each of a range of hypothetical annual temperature anomalies from −5 to +20°F, the annual anomaly was added to the actual mean temperature in each day of 1999 to obtain an annual series of daily temperatures. The piecewise regression model was then used to obtain the predicted number of assaults for each day of the series. These were then summed over the course of the year.
Our simulation suggests that the mean temperature in Dallas would have to increase by around 13°F (7.2°C) before subsequent temperature increases would begin to reduce annual aggravated assaults. At this point, the model predicts an extra 146 annual aggravated assaults per 100,000 population in comparison to a world with zero warming. Before this point, temperature increases would continue to increase assaults. Notably, a temperature increase of 13°F would be substantially greater than the warming likely by the end of the 21st century on the basis of regional climate projections for central North America.
However, it is important to note that this analysis provides only
To the Editor:
French et al should be congratulated for reporting their study of the effects of an educational intervention on prehospital care management of pain.
First of all the intervention was quite substantial and multifaceted. However it is interesting to ask what facet of the intervention caused the positive outcomes. Could a shorter (1 or 2 hour) intervention resulted in a similar outcome? Could the same outcome have been achieved as a result of an e-learning intervention or print-based learning materials? Is it possible that the surveys themselves had an effect on the changed management? These questions cannot be answered from the current results as all learners received the same intervention. Perhaps a further follow up study might be conducted where different groups of learners receive different interventions. In this way more effective and more efficient interventions might be uncovered.
Secondly the educational intervention was not costed; nor indeed was any cost utility assigned to the outcomes. Low cost educational interventions that result in more efficient care and as a result lower cost care are obviously the interventions most sought after by educators and educational providers alike. However this is only possible when interventions and their outcomes are properly and thoroughly costed.
Dear Colleagues,
We are proud and honored to launch the inaugural issue of our new online publication – The
Maimonides, known as the
In the best interests of the medical and scientific community,
The editorial policy of
Sincerely Yours,
I am honored to be an Associate Editor of the newly launched
Rabbi Moshe ben Maimon – the
This activity has been planned and implemented in accordance with the Essential Areas, Elements and Policies of the ACCME. The Islamic Medical Association of North America (IMANA) is accredited by the ACCME to provide continuing medical education for physicians.
The Islamic Medical Association of North America designates this educational activity for a maximum of 12 AMA PRA Category 1 Credit(s)TM. Physicians should claim only credit commensurate with the extent of their participation in the activity.
Conference Schedule
| Each presenter will have 40 minutes for presentation with five minutes Q & A period. Conflict of interest declaration should be announced. | |
| 7:45–8:00 a.m. | Introduction |
| Abida K. Haque, MD, IMANA President, and Ayaz M. Samadani, MD, Chairman of the CME Committee | |
| 8:00–8:45 a.m. | The Epidemic of Obesity-related Complications and Measures for Prevention |
| Abida K. Haque, MD | |
| 8:45–9:30 a.m. | What Exactly is Public Health? Some Multiple Perspectives |
| Ayaz M. Samadani, MD, DCH, DTM&H, FAAFP | |
| 9:30–10:15 a.m. | Spontaneous Tumor Regression in Two Commonly Used Rat Tumor Models of Liver Cancer |
| Labiq Hossain Syed, M.D. | |
| 10:15–10:30 a.m. | Coffee and tea in the room but no formal break |
| 10:30–11:15 a.m. | Inpatient Hyperglycemia: Controversies in Management and New Guidelines |
| Shahid Athar, MD, FACP, FACE | |
| 11:15 a.m. -noon | Panel Discussion |
| 8:00 a.m -noon | CPR Training - Kanwal Shazia Chaudry, MD |
| 7:45–8:00 a.m. | Introduction |
| 8:00–8:45 a.m. | Advances in the Assessment and Treatment of Adult ADHD Syed Ahmed, MD |
| 8:45–9:30 a.m. | End-of-life Concerns for American Muslims |
| Shahid Athar, MD, FACP, FACE | |
| 9:30–10:15 a.m. | Immune Rejuvenation in People 55 years or Older: Immunotherapy/Comprehensive Medicine and Holistic Medicine Approach |
| Naima Abdel-Ghany, MD, PhD, FACPM | |
| 10:15–10:30 a.m. | Coffee and tea in the room but no formal break |
| 10:30–11:15 a.m. | Advanced Pain Management |
| Mohammad Nadeemullah, MD | |
| 11:15 a.m.-noon | Panel Discussion |
| 8:00 a.m -noon | Conscious Sedation Workshop - Ismail Mehr, MD |
| 7:45–8:00 a.m. | Introduction |
| 8:00–8:45 a.m. | A New You |
| Khalique Zahir, MD | |
| 8:45–9:30 a.m. | Unusual Cases of Diarrhea |
| Mohammad H. Bawani, MD, FACG, FACP | |
| 9:30–10:15 a.m. | Successful Use of Erythropoietin (EPO) and Intravenous Iron Therapy in Pregnant Hemodialysis (HD) Patients and Long-term Effects on Children |
| Mohammad Akmal, MD | |
| 10:15–10:30 a.m. | Coffee and tea in the room but no formal break |
| 10:30–11:15 a.m. | Nutritional Guidelines for School Lunch Programs |
| Abida K. Haque, MD | |
| 11:15 a.m.-noon | Panel Discussion |
| 8:00 a.m.-noon | Skin Rejuvenation Workshop - Khalique Zahir, MD |
To study the prevalence of cardiac and pulmonary and other organ system diseases in obese patients. For the past several decades, the global prevalence of obesity has been rising, with the greatest increase seen in the United States. Recent data indicate that 190 million Americans are either obese or overweight. The treatment of obesity-related diseases is estimated to be $147 billion a year. Between 280,000 and 325,000 deaths annually are attributable to obesity. Left ventricular dysfunction, atherosclerotic heart disease, obstructive sleep apnea, pulmonary hypertension, and venous thromboembolism are clinically recognized cardiovascular complications of obesity. We designed an autopsy study to examine the organ system involvement in obesity
A search of the autopsy database at a university medical school over a 10-year period (1993–2003) yielded 76 individuals with a body mass inde (BMI) > 30 kg/m2. Institutional Review Board approval was obtained for the study. Clinical and autopsy records were reviewed and data on demographics, body weight, height, cause of death, and comorbidities were collected. For the control group, 46 autopsied age-matched patients with BMI < 30 kg/m2 were randomly selected. Statistical analysis was performed by a biostatistician using multiple procedures including chi-square, Mantel-Hanszel chi-square, phi coefficient, contingency coefficient, probability table and two-sided paired t test. Significance was defined as p=<0.05.
The obese group had 76 individuals with a mean BMI of 52. Seventy-three percent were noted to have a BMI >40 kg/m2 (morbidly obese). There were 41 men and 35 women with a mean age of 50 (age range 14–84 years). The nonobese control group had 17 women and 29 men with a mean age 48 (range 20–84 years). There was no difference in the incidence of obesity between men and women or between races. The incidence of diabetes mellitus was greater for the obese group compared to the nonobese group; 39% and 11%, respectively (p=0.0007). Systemic hypertension was greater in obese compared to non obese individuals; 53.3% and 21.7%, respectively, (p=0.0006). In the obese group, seven were diagnosed ante mortem with obstructive sleep apnea, and nine with chronic obstructive pulmonary disease. The major causes of mortality in the obese group were pulmonary diseases (35%), cardiac disease (28%), and sepsis (11%). The pulmonary causes of death included large/saddle pulmonary embolus, bronchopneumonia, diffuse alveolar damage and pulmonary edema. The cardiac causes of death included acute myocardial infarction, sudden death, and congestive heart failure. Other causes of death were sepsis, acute pancreatitis, and heat stroke.
The major findings in this study were pulmonary hypertensive changes in 65% and cardiomegaly in 100% of obese patients. Investigations focusing on pathogenesis of obesity have identified the obesity gene and the protein product leptin secreted from the white adipose tissue, which acts on the brain to regulate food intake, energy expenditure, and neuroendocrine function. Obese rodents and humans have high circulating levels of leptin, thus demonstrating “leptin resistance.” Further research in this area may help develop targeted- therapeutic strategies for control of obesity. Prevention and treatment of obesity is a major health-care issue and requires a multidisciplinary approach with emphasis on reduction of caloric intake and increased physical activity.
Abstract Not Available
Abstract not available.
Abstract not available.
Abstract not available.
To review the evaluation of patients presenting with diarrhea.
How should a patient be evaluated once the usual evaluation methods i.e. stool studies for ova, parasites, cultures, C diff toxin, have turned out to be negative?
Additional testing should include thyroid status and serology for celiac disease. Endoscopic evaluation is an important tool in assessment of diarrheal illness. Neoplastic processes, aggressive peptic ulcer disease, and microscopic colitis may be detected with endoscopy. However unusual and uncommon causes of diarrhea such as carcinoid syndrome, hypergastrinemia, and hyper-parathyroidism should be considered.
Persistence and perseverance in determining the cause of diarrhea may at times yield results that are surprising and worth the effort.
Attention deficit hyperactivity disorder (ADHD) in adults is increasingly recognized, and more and more adults are seeking treatment. This presentation explores the new assessment and treatment strategies developed during the past 10 years to treat ADHD in adults.
Review of articles on new research on adult ADHD.
To highlight the historical time line of diagnostic approaches for ADHD in adults.
Pharmacotherapy of adult ADHD.
Assessment of ADHD is primarily based on clinical history. Validated quantitative measures can assist in the process.
During the past five years there has been considerable expansion and innovation in the pharmacotherapy of ADHD, although this innovation has focused primarily on novel delivery system for established agents. Stimulants remain the major first-line treatment for ADHD for the foreseeable future, but novel agents will be available in coming years.
ADHD is common among adults and children. It is a disorder of executive functioning. It is heritable and causes significant impairment. Review of recently published research clearly indicates the diverse presentations of ADHD in adults. Choosing medications for treating adult ADHD is to be based on the unique aspects of adult patients with ADHD.
To determine the status of lunch programs in Islamic schools in the United States and develop recommendations for improving them.
The Islamic Medical Association of North America (IMANA) conducted a survey of lunch programs by mailing questionnaires to 100 Islamic schools in the United States.
Fourty-eight Islamic schools responded to the survey. Only 20 schools follow guidelines, and only six have dietitians advising on menu planning. Based on this survey, IMANA, with the assistance of Muslims in Dietetics and Nutrition (MIDAN), a Member Interest Group of the American Dietetic Association, has developed a summary of guidelines that schools could follow. It includes sample menus in American and ethnic foods and sources for funding.
IMANA and MIDAN, recognizing the religious and scientific relevance of nutritious diet, have developed these recommendations. This information is provided to aid Islamic schools in implementing guidelines for nutritional school lunch menus and creating a culture of healthful lifestyle.
The following abstracts were accepted but will not be presented.
Hysterectomy is one of the most common gynecologic procedures performed for benign indications. To date more than 80% of hysterectomies are still performed via laparaotomy. Minimally invasive, laparoscopic and vaginal hysterectomy procedures were limited by uterine size, complex pathology, patient body mass index (BMI) and history of previous surgeries. The advent of robotic surgery using the da Vinci Surgical System, developed by Intuitive Surgical, has given laparoscopic surgeons the ability to approach more complex cases with minimum invasiveness, thus decreasing the need for laparotomy. Since its approval by the United States Food and Drug Administration for use in gynecologic procedures in 2005, the adoption curve for da Vinci Hysterectomy has been growing exponentially.
To review available data and studies conducted, assess the risks and/or benefits of robotic hysterectomy and evaluate possible advantages and disadvantages of its adoption as compared to traditional approaches to hysterectomy.
A Medline and Ovid literature search of all publications with key words “robotic hysterectomy” was performed. Search results were then screened to exclude assessments of surgery performed in the setting of cancer diagnoses. Outcome comparisons were made in terms of findings including differences in blood loss, complication rates, surgical time, and length of stay in the hospital for both routine hysterectomy and those with complex pathology.
Da Vinci hysterectomy has been shown to be safe and effective with low morbidity and fewer conversions to laparotomy than the traditional laparoscopic approach to hysterectomy. Blood loss and length of hospital stay are comparable to a laparoscopic hysterectomy. Although operative time is initially increased, this decreases significantly with increasing surgeon experience and becomes comparable to laparoscopic hysterectomy towards the end of the learning curve.
Since its approval for use in gynecologic procedures, robotic-assisted hysterectomy with the Da Vinci System, has allowed the performance of complex hysterectomies with the same advantages of traditional laparoscopic surgeries without increasing complication rates or conversions to laparotomy. Thus, its continued adoption and expansion of usage in hysterectomy is likely.
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The publishers would like to apologize for an error in the references. Reference 14 (Gangal
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This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commerical use, distribution, and reproduction in any medium, provided the original work is properly cited.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/>) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commerical use, distribution, and reproduction in any medium, provided the original work is properly cited.
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The prevalence of unidentified or untreated unhealthy alcohol use remains high. With the advent of pharmacotherapy and models of counseling appropriate for use in primary care settings as well as in specialty care, clinicians have new tools to manage the range of alcohol problems across the spectrum of health care settings. By extending treatment to primary care, many people who do not currently receive specialty care may have increased access to treatment. In addition, primary care providers, by virtue of their ongoing relationship with patients, may be able to provide continuing treatment over time. Extending the spectrum of care to hazardous drinkers who may not be alcohol dependent could result in earlier intervention and reduce the consequences of excessive drinking.
Unhealthy alcohol use, which includes the spectrum of drinking behaviors and consequences ranging from risky use to problem drinking, along with alcohol abuse and alcohol dependence (
Research has demonstrated that a variety of treatment approaches can help individuals with unhealthy alcohol use decrease their alcohol intake and thus avoid the many consequences described above. Counseling interventions have been designed to address the full spectrum of unhealthy alcohol use from brief interventions for risky use to more complex and rigorous counseling strategies for individuals with alcohol dependence. In addition, beginning with disulfiram in the late 1940s and more recently with naltrexone and acamprosate along with newer medications “in the pipeline,” pharmacotherapy has been demonstrated to be a useful adjunct to behavioral therapies for many people with unhealthy alcohol use, particularly those with alcohol dependence.
The spectrum of unhealthy alcohol use can be addressed in a variety of health care settings, including primary care, specialty practice, and alcohol treatment programs. Although complex behavioral strategies have been developed primarily for specialty settings and treatment programs where they can be effectively delivered, screening and brief intervention counseling has been developed for use in primary care settings, with a focus on treatment referral when necessary. Medication use in these nonspecialized settings and in a spectrum of patients including nondependent individuals is a recent phenomenon.
Research is needed to address the optimal use of medication therapy for the treatment of alcohol use disorders and for treating the broader spectrum of unhealthy alcohol use, from non-dependent risky drinking to alcohol dependence. This is especially true given the major scientific advances in pharmacotherapy that have been made over the past 60 years. To improve access to effective medication therapy, research also should explore the use of these medications in a range of health care settings. To optimize medication treatment outcomes, practitioners need to assess both the appropriate level of counseling (from minimal to more intensive) and the appropriate methods to enhance medication adherence for individual patients. The development of medications to address the spectrum of unhealthy alcohol use across the broad range of health care settings has the potential to maximize benefits for future patients.
After reviewing the medications currently approved for alcohol dependence and new medications being investigated, this article will outline ways to optimize treatment outcomes through patient–treatment matching and increased treatment adherence and review potential uses of medications for nondependent hazardous drinkers, including the use of medications in primary care settings.
The Food and Drug Administration (FDA) has approved four medications for the treatment of alcohol dependence: disulfiram (Antabuse®), oral naltrexone, extended-release naltrexone (Vivitrol®), and acamprosate (Campral®). Topiramate, a medication used to treat epilepsy and migraine, has demonstrated evidence in two clinical trials of alcohol dependence, and a number of other promising medications are being studied. For detailed information about mechanisms, risks, and benefits of approved medications and those on the horizon, please see
Disulfiram, the first drug approved for the treatment of alcohol dependence, and still one of the most commonly used agents, produces an aversive interaction with alcohol by interfering with the metabolism of alcohol. During alcohol metabolism, alcohol is converted to acetaldehyde, which then is broken down by the enzyme aldehyde dehydrogenease. Disulfiram inhibits this later step, leading to a build up of acetalydehyde and results in aversive effects such as nausea, vomiting, palpitations, and headache. Ordinarily, the negative consequences of alcohol consumption (e.g., health problems) are delayed and are uncertain (e.g., your significant other may or may not become angry with you; the police may not apprehend you for drunk driving). The knowledge of the potential disulfiram alcohol interaction, however, can make the consequences of drinking certain and immediate and thereby support a person’s motivation to avoid drinking, and the actual reaction may limit the amount consumed if abstinence is violated. Medication compliance can be a problem, however, and disulfiram is most effective when provided with supervised administration by a significant other or health care provider (
Naltrexone is an opiate antagonist that primarily blocks μ-receptors with more variable occupancy of δ-receptors at the standard dose of 50 mg daily (
Extended-release naltrexone, a formulation that only requires a monthly injection, holds the potential to minimize problems with medication adherence. In a 6-month trial, 64 percent of participants received all 6 months of double-blind medication, translating into daily coverage for the entire treatment period (
The primary adverse effects of naltrexone, whether oral or injectable, are nausea followed by headache and dizziness. Patients with significant liver disease are not candidates for naltrexone nor are patients who require opiate medications for pain control. Acute pain control requires alternatives to opioids. To avoid precipitating an opioid-withdrawal syndrome, patients should be free of opioids for 7 to 10 days before beginning naltrexone. If extended-release naltrexone is administered subcutaneously rather than as an intramuscular gluteal injection, the likelihood of severe injection-site reactions may increase (
Acamprosate, available in oral delayed-release tablets (Campral®), was approved for use in the treatment of alcoholism in the United States in 2004, following extensive use in many other countries. Acamprosate is believed to normalize the balance between excitatory and inhibitory pathways altered by chronic alcohol consumption (
One of the strengths of acamprosate is its side-effect profile; the most common side effects are gastrointestinal in nature. Acamprosate can be used in patients with moderate liver disease but is contraindicated in patients with severe renal impairment, and dose reductions are recommended for those with mild-to-moderate levels of renal impairment.
Topiramate, an anticonvulsant, is hypothesized to have beneficial effects on drinking by facilitating functioning of the neurotransmitter γ-aminobutyric acid (GABA) and antagonizing glutamate activity. Two placebo-controlled trials (
Topiramate requires very gradual dose escalation. The most common adverse events include cognitive dysfunction, abnormal sensations (e.g., numbness, tingling), and anorexia and taste abnormalities. Additional rarer serious adverse events have been identified, such as metabolic acidosis, acute myopia, and secondary narrow-angle glaucoma. The optimal dose for alcohol dependence has yet to be established and may be lower than that the target dose of 300 mg per day tested in prior research.
Currently available pharmacotherapies only have modest effects, which has spurred efforts to identify treatment responders, new medications, treatment combinations, and methods to enhance adherence. As reviewed by
Numerous studies have tested selective serotonin reuptake inhibitors (approved for depression), often with disappointing results including counter-therapeutic effects among patients with early-onset alcoholism. However, studies show that these medications (e.g., sertraline) may be efficacious among individuals with later-onset alcoholism (Kranzler et al. 1996; Pettinati et al. 2000) or in combination with naltrexone for patients with major depression (
Medications targeting GABA and glutamate systems show promise as treatments for acute and protracted alcohol withdrawal and for relapse prevention. Treatment with baclofen (a GABAb receptor agonist [i.e., it binds with the GABAb receptor] used for muscle spasticity) has been found to reduce symptoms of alcohol withdrawal, and a placebo-controlled study of 84 alcohol-dependent patients with cirrhosis yielded promising results (
Given the role of dopamine in the maintenance of alcohol dependence, drugs that have direct effects on dopamine through either partial agonism (e.g., aripiprazole) or through antagonist effects (e.g., olanzapine, quetiapine) have been investigated as candidates for alcoholism treatment. A multisite study did not find an overall advantage of the atypical antipsychotic aripiprazole over placebo on the primary outcomes, although some secondary outcomes suggested that studies at lower doses would be worthwhile (
There has been considerable enthusiasm about the potential of rimonabant, a cannabinoid receptor 1 antagonist, based on preclinical research showing that it reduced alcohol drinking. However, psychiatric adverse events noted in obese patients, a negative human alcohol self-administration study (George et al. 2009), and a negative clinical trial in individuals with alcohol dependence (
Many alcohol-dependent individuals also smoke cigarettes, and researchers have investigated the potential role of the nicotinic acetylcholine receptor (nAChR) system as a factor in both addictive behaviors (for a review, see
Researchers also are studying agents that may address the relationship between stress and alcohol consumption. Prazosin, an α-1 adrenergic antagonist that is effective in treating posttraumatic stress disorder (PTSD), has shown preliminary efficacy in a small pilot study with 24 alcohol-dependent patients without PTSD (
Research is being done in an attempt to identify predictors of patient response to FDA–approved treatments. In a secondary analysis of a U.S. acamprosate trial, patients with a strong commitment to abstinence benefited from acamprosate (
Poor adherence to prescribed medications c limit an a treatment’s effectiveness. As a result, research has investigated predictors of adherence and methods for enhancing adherence. One of the best predictors of future behavior is past behavior. In the case of medication compliance, self-reported problems with adherence characterized as purposeful nonadherence (e.g., stopping medication early due to either feeling better or worse) predict medication compliance and treatment outcome (
Researchers also have developed brief interventions to support adherence to alcoholism medications. Common features of these interventions include emphasizing the importance of adherence, providing positive feedback for good adherence, and problem-solving difficulties with adherence. The Medication Management Intervention (
Currently, research has evaluated alcoholism medications primarily in alcohol-dependent populations. Many individuals, however, drink at harmful levels but do not meet the criteria for dependence and may benefit from medications to augment counseling approaches used with this subgroup of drinkers.
Because young adults are less interested in quitting drinking than in reducing their drinking, interventions to help them moderate their alcohol consumption may be particularly useful (
Regardless of age, many individuals with alcohol dependence, particularly those with less severe problems, would prefer to reduce their drinking rather than seek total abstinence, and low severity of alcohol dependence is one of the characteristics that predicts recovery from alcohol problems, as evidenced by moderate drinking (
The rapid progress in the development of medications to treat alcohol dependence, although impressive, has resulted in a relatively slow adaptation of these new treatments. In 2007, the percentage of Veterans Administration patients with alcohol use disorders who received pharmacotherapy was 3 percent (
Primary care providers are well suited to address a wide variety of behavioral problems in their patients and routinely manage chronic diseases with a combination of counseling and medication management. Using primary care–based prevention strategies to address behaviors such as overeating and smoking, practitioners already routinely screen for conditions such as high cholesterol, hypertension, and cancer and treat a full range of chronic conditions such as diabetes and asthma. Similar clinical management strategies for unhealthy alcohol use and alcohol use disorders have been developed. However, despite convincing data supporting the value of evidence-based screening techniques, brief interventions, and medication approaches, primary care settings rarely use these tools (
With regard to alcohol, a single question about how often the patient exceeds the daily maximum drinking limits (i.e., more than four drinks for men and more than three drinks for women) in the prior year can be effectively used to screen for unhealthy alcohol use (
Evidence supporting the potential use of alcoholism medications in primary care settings derives from studies conducted in such settings and studies that compared specialty care with primary care models of counseling. These studies provide clues to the nature and amount of behavioral counseling needed to accompany pharmacotherapy. Some studies address both of these questions (or do not separate the questions), whereas others address one or the other. Most studies have not enrolled primary care patients but have evaluated primary care models of treatment provided by medical providers who are not alcoholism specialists in research settings.
In an initial study examining the effectiveness of naltrexone in combination with a primary care model of care, 197 alcohol-dependent participants were treated with naltrexone for 10 weeks in combination with cognitive–behavioral therapy (CBT) provided by an alcoholism specialist or in combination with primary care management (PCM) provided by a primary care practitioner (
The COMBINE Study (
The medical management intervention consisted of up to nine sessions over 16 weeks with a health care professional (e.g., a nurse, physician’s associate, nurse practitioner, or physician). Following an initial 45-minute interview, subsequent appointments were approximately 15 minutes long. The approach included monitoring of drinking, support and encouragement, establishing a plan for medication adherence, monitoring and problem solving adherence issues, and advice to attend support groups (e.g., Alcoholics Anonymous).
The results indicated that patients who received naltrexone plus medical management, CBI plus medical management and placebos, or naltrexone and CBI plus medical management had higher percentages of days abstinent (80.6, 79.2, and 77.1 percent, respectively) than those receiving placebos and medical management only (75.1 percent). Naltrexone also reduced the risk of having a heavy-drinking day, but this effect was most evident in those receiving medical management but not CBI. Acamprosate showed no significant effect on drinking versus placebo, either by itself or with any combination of naltrexone, CBI, or both. These results suggest that health care providers could use a primary care model of counseling with pharmacotherapy to improve treatment outcomes.
Consistent with the findings in COMBINE,
The results favored CBT compared with the two other less intensive treatments. The differences between the BRENDA and the doctors-only groups were not significantly different. The effect of naltrexone was not significant and did not vary by the type of psychosocial intervention, although the sample size was too small to detect anything other than very large interaction effects. Medication adherence was relatively low (50 percent took medication on 80 percent of days over the 24 weeks of therapy) and may have been related to the relatively longer duration of the study and the use of the 100-mg dose. Medication adherence was associated with better outcomes, irrespective of medication condition.
Extended-release naltrexone appears to be well suited for use in primary care settings. Skilled medical personnel are required to administer extended-release naltrexone with an intramuscular gluteal injection; many specialty programs do not have access to needed medical care providers. Moreover, the efficacy studies of extended-release naltrexone used BRENDA counseling, albeit the frequency of appointments may have exceeded that likely to occur in primary care. Future studies should evaluate the efficacy of once-a-month extended-release naltrexone with less frequent counseling and in patients recruited through primary care sites.
As reviewed by
One of the few medication trials actually conducted in primary care sites (
Recent studies of continuing-care interventions suggest that interventions of a year or longer and treatments that are less burdensome can promote sustained engagement and positive effects (
In summary, the research literature supports the effectiveness of medications, such as naltrexone, in combination with models of care that primary care providers or medical professionals associated with specialty alcohol programs could use. Several published manuals (NIAAA 2009;
In the management of both acute and chronic conditions, physicians and other medical professionals often need to consider carefully when to suggest medication treatment to individual patients. Typically, the decision to recommend medication treatment relies on a combination of an assessment of the evidence to support a particular therapy for a specific condition and clinical judgment concerning whether an individual patient is appropriate for that treatment based on a variety of patient-and disease-specific features. Clearly, such decisions are best arrived at using a patient-centered approach involving patient education, preferences, and mutual decisionmaking. Even when medication therapy has a clear evidence base in a given clinical situation, patients and their providers may identify a variety of reasons why a specific therapy may or may not be used. Beyond this, research often demonstrates that there are certain patient subgroups for whom a specific therapy may or may not be particularly effective. These subgroups may be identifiable based on clinical, demographic, genetic, or social features that all may play a major role in the decision process regarding medication use. With the availability of several FDA-approved medications, a provider may recommend a trial with a new medication should an individual patient not respond to the first medication tried.
The implementation and widespread use of medications to treat alcohol problems faces a unique set of barriers in primary care. Although primary care providers are proficient at prescribing a wide variety of medications, they generally are unfamiliar with medications for treating alcohol problems other than those used to treat alcohol withdrawal. Indeed, a growing body of research to support basic screening methods, brief interventions, and especially medication therapy has yet to have a major impact on how primary care providers care for individuals at risk for or with alcohol problems (
Identifying and treating people with alcohol use disorders remains a challenge. With the advent of pharmacotherapy and models of counseling appropriate for use in primary care settings as well as in specialty care, clinicians have new tools to manage the spectrum of alcohol problems across the spectrum of patients and settings. By extending the continuum of care to primary care settings, many people who do not currently receive specialty care may have increased access to treatment. In addition, primary care providers, by virtue of their ongoing relationships with patients may be able to provide continuing care interventions. Medication use with hazardous drinkers who may not be alcohol dependent may promote reduced drinking and likely will reduce the burden of excessive drinking.
The framework for BRENDA is based in the biopsychosocial model of addiction and consists of six stages: the clinician’s (1) biopsychosocial evaluation of the patient, (2) report of that assessment back to the patient, (3) empathy for the patient’s situation, (4) needs identification by both patient and clinician, (5) direct advice to the patient on how to meet those needs, and (6) assessment of the patient’s reaction to that advice as well as any necessary adjustments to the treatment plan.
The authors declare that they have no competing financial interests.
D
The workplace offers advantages as a setting for interventions that result in primary prevention of alcohol abuse. Such programs have the potential to reach broad audiences and populations that would otherwise not receive prevention programs and, thereby, benefit both the employee and employer. Researchers have implemented and evaluated a variety of workplace alcohol problem prevention efforts in recent years, including programs focused on health promotion, social health promotion, brief interventions, and changing the work environment. Although some studies reported significant reductions in alcohol use outcomes, additional research with a stronger and integrated methodological approach is needed. The field of workplace alcohol prevention also might benefit from a guiding framework, such as the one proposed in this article.
Workplace programs designed to prevent and reduce alcohol problems can potentially benefit the employee, the employer, and society in general. In 2007, 8.8 percent of full-time workers overall reported heavy alcohol use (i.e., they consumed five or more drinks on the same occasion on each of 5 or more days in the past 30 days), and 30.2 percent reported binge drinking (i.e., consuming five or more drinks on the same occasion on at least 1 day in the past 30 days) (
Substance abuse is associated with multiple negative workplace outcomes, including absences from work, accidents, turnover, arguments and fighting at work, sleeping on the job, and other sources of productivity loss (
The estimated costs of alcohol abuse for 1998 (the last year for which costs were estimated for the United States) were $184.6 billion, more than 70 percent of which was attributed to lost productivity ($134.2 billion), including losses of $87.6 billion from alcohol-related illness (
The workplace offers many advantages as a setting for preventing alcohol problems. For example, full-time employees spend a significant proportion of their time at work, increasing the possibility of exposure to preventive messages or programs offered through the workplace. Workplace interventions can access specific groups that would otherwise be difficult to reach and, because most people are employed, reach large populations. Employers have a vested interest in keeping their employees healthy and productive. They can therefore use their influence to encourage employees to participate in prevention programs. Many employers offer employee assistance programs (EAPs) to help employees deal with personal problems, including substance abuse, that might adversely affect their work performance, health, and well-being. EAPs generally include assessment, short-term counseling, and referral services for employees and their household members. Although EAPs primarily are treatment oriented, a survey of employee assistance professionals found that most believed that prevention should have a larger role in such programs (
It should be noted that small businesses (less than100 workers) tend to be the least likely to have an EAP or health plan (
This article will present findings from several recent workplace prevention–intervention studies and will focus on those intervention efforts that target all employees, regardless of level of alcohol consumption and problems (i.e., primary prevention strategies).
Well-developed programs for primary prevention of alcohol abuse in the workplace are more the exception than the rule. However, recent reports suggest that some promising approaches are being developed and implemented. The approaches reviewed here include strategies based in health promotion, social health promotion, and brief intervention, including Web-based feedback interventions, all of which focus on changing individual behavior, as well as environmental interventions, which seek to reduce risk factors by changing the work environment. As the state of the science for workplace prevention is relatively young, the authors present a model that gives equal weight to different approaches until comparatives studies examine their relative effectiveness. This broad suggestion is given to counter tendencies to favor any one approach. For example, health insurance plans currently overly focus on brief interventions for screening and counseling (e.g.,
Through lifestyle campaigns, employers can encourage workers to reduce stress, improve nutrition and exercise, and reduce risky behaviors, such as drinking, smoking, and other drug use. For example, in a study among insurance company workers,
All participants reported positive changes on measures of stress and healthy eating after the program. All the changes in the stress measures and some of the changes in healthy eating were maintained 8 months later. Importantly, participants in both groups showed similar, significant improvement regardless of the presence of the substance abuse prevention program. For example, the stress-management participants showed significant improvement on the three attitude/perception measures of substance abuse and significant decreases in alcohol and other drug use, regardless of whether they received the substance abuse program. The control group did not show these improvements. The study’s findings suggest that workers can change important attitudes, perceptions, and practices regarding substance abuse if they are exposed to stress-management sessions, regardless of whether explicit substance abuse prevention materials are presented.
In another study (
Several studies by Bennett and associates (
A fourth study (
In a more recent pair of studies (
Brief interventions typically involve personal assessment of an individual’s drinking rates and related problems as well as feedback about health risks (Bien et al. 1993). These interventions typically have been studied in medical settings and found to be effective there (e.g.,
Brief therapies tend to be physician oriented, whereby an employee, showing signs of alcohol abuse, is more likely to receive screening from their primary care physician (
Brief interventions also can include alcohol education and motivational-enhancement techniques to stimulate behavior change. A few studies have evaluated brief interventions (consisting of one to three sessions) in the work-place. In a study of 155 employees at a food and retail service company,
Strategic Brief Interventions or Strategic Brief Intervention and Referral to Treatment (SBIRT) (
Five studies have evaluated the effectiveness of interventions delivered via the Internet to adult workers. Such interventions have the advantage of allowing employees to access the intervention anytime they want and in private to avoid disclosing a potential alcohol problem.
The U.S. Department of Defense has evaluated a Web-based alcohol intervention called Program for Alcohol Training, Research, and Online Learning (PATROL) among active-duty military personnel. Two Web-based alcohol interventions were adapted for use in the military and tested at eight military installations. Volunteer participants completed a baseline assessment of alcohol use and associated problems and were then assigned to one of four intervention groups: (1) Alcohol Savvy (AS); (2) Drinker’s Check-Up (DCU); (3) the “risk level” condition, where high-risk drinkers were assigned to the DCU and low-risk drinkers were assigned to the AS; or (4) control. Across the installations, 3,912 participants completed the baseline survey and 1,371 completed the 1-month follow-up survey. Results showed that participants who completed one of the programs (i.e., either AS or DCU) had significant reductions on multiple measures of alcohol use compared with control participants. Initial analyses suggested no significant difference in the relative effectiveness of the three program conditions (
Two recent studies also support the use of Web-based programs for reducing alcohol risks for adults. In the first,
The environmental approach to research and prevention of workplace drinking problems considers the differences between individual and occupational influences on drinking behavior.
However, it should be noted that strengthening policy language and enforcement is complex, and reluctance to make changes in policy is more a factor of the organizational structure (e.g., management and/or union) than employee resistance. Using the Ames model as a guide, another study that surveyed 7,255 supervisors across seven corporations provided evidence that managers perceive personal, interpersonal, and organizational barriers to enforcing alcohol policy. The lower the manager is in the hierarchy, the more likely he/she is to perceive barriers; furthermore, women managers and first-line supervisors encountered the most barriers (
On the basis of this review of relatively recent research, it seems that approaches aimed at preventing alcohol problems and evaluations of interventions in the workplace have met with some, if not limited, success, and still are in developmental stages. Several studies that integrated alcohol interventions into health promotion programs using a combination of educational, counseling, and brief intervention strategies reported marginally successful results. Drinking rates overall were not significantly reduced; one of these studies reported reduced number of drinks by women only, and another a stated desire by some binge drinkers to reduce alcohol use but saw no actual reduction. The studies that fell into the social health promotion category evaluated skill-building intervention strategies. One successful approach was Bennett and colleagues’ “Team Awareness” program (
Evaluation of the environmental approach with a natural intervention between two large plants in a Fortune 500 industry used strategies that incorporate changing of identified risk factors embedded in the work culture rather than strategies that are targeted exclusively toward changing individual behavior. The results showed significant differences (28 percent compared with 3 percent) in work-related drinking rates in the plant that made changes in the work culture from the one that did not make changes, and with that outcome, it could be considered a highly successful approach.
All of the strategies that focused on changing individual risk factors and, thereafter, individual drinking behavior, showed some measure of success. As demonstrated in this review, the educational technique, when combined with the brief intervention program, has perhaps the most potential. In terms of improvement, the educational approach, by far the most prevalent program in most work organizations, might benefit from comparison with all of the current intervention approaches, instruments, and results and from there move on to a united effort for an improved design. However, the downfall to these approaches will occur eventually if the programs are not sustained over time and if obstacles (e.g., lack of organizational support and cooperation in implementing follow-up interventions) are not overcome.
Although the approach that focuses on changing the work environment as a means of changing work-related drinking behavior was clearly successful, the limitation here is that only work-related drinking was affected, rather than overall drinking rates, which were similar between the two plants. For purposes of primary prevention, this approach might attain more expansive results if it was blended with one or more of the programs that focus on individual attributes, selecting from among health promotion, social health promotion, and brief interventions. Furthermore, as strongly recommended by
Importantly, evaluations of the various strategies reviewed here—health promotion, social health promotion, brief interventions, Web-based interventions, and environment strategies—have proceeded in a piecemeal basis without consideration that each strategy varies in its potential reach and overlapping levels of intervention. Such consideration is important because programs that target individual-level behavioral change (e.g., SBIRT) may occur in the context of group processes (e.g., Team Awareness) or wider environmental strategies (e.g., policy enforcement). Likewise, factors like Internet access, health insurance, and EAP programming vary greatly from one organization to another. In short, there is lack of a contextual framework that could guide studies comparing the effects of different interventions.
A framework is recommended for guiding thought about future prevention efforts, potentially coordinating evaluation studies, integrating approaches, and ultimately determining which types of programs are most effective (see
Second, programs vary in their potential reach such that they overlap in different ways for different workplaces. Environmental programs have the potential to reach the widest group of individuals (the workforce) with the least or greatest effort, depending on the level of cooperation from management and/or unions. However, brief interventions, in addition to prevention, may reach more individuals who have serious alcohol problems on an in-depth or one-to-one basis. However, brief interventions do not happen in a vacuum and may be facilitated by a social health or health promotion approach, just as these approaches may be most effective in conjunction with an environmental strategy. Although it would require a major effort, a multisite or cross-site study with a cluster-randomized methodology (
The authors provide a framework (see
Third,
In light of the above, research from the workplace stress literature suggests that “systemic” prevention interventions—that use employee involvement and also integrate individual with organizational change—are more effective than either strategy alone (
Finally, the guiding framework may be applied to the study of prevention in high-risk populations. This includes the military (see sidebar), especially for those deployed or returning from deployment, as well as young adult and mature workers about to retire. For example, a recent multisite study of young workers (
The authors declare that they have no competing financial interests.
Past-month heavy alcohol use among full-time workers aged 18 to 64, by industry categories: 2002−2004 combined.
SOURCE: SAMHSA Analytic Series: A-29. No permission required.
Larson, S. L.; Eyerman, J.; Foster, M.S. and Gfroerer, J.C. Worker Substance Use and Workplace Policies and Programs (DHHS Publication No. SMA 07–4273, Analytic Series A–29). Rockville MD: SAMHSA, Office of Applied Studies, 2007.
Hypothetical framework for comparing and integrating strategies.
Framework for Generating Research Designs to Assess Effects of Four Types of Strategies
| WE vs. Control Group | (WE + other) vs. Control | (WE+HP+SH+B) vs. (HP+SH+B1) | Comparing organizations with different combinations of interventions vs. control | |
| HP vs. Control Group | (HP + other) vs. Control | (WE+HP+SH+BI) vs. (WE+SH+BI) | ||
| SH vs. Control Group | (SH + other) vs. Control | (WE+HP+SH+BI) vs. (WE+SH+BI) | ||
| BI vs. Control Group | (BI + other) vs. Control | (WE+HP+SH+BI) vs. (WE+HP+SH) | e.g., (WE+HP) vs (SH = BI) vs. Control |
NOTE: This framework is provided as a guide for developing and coordinating specific strategies rather than a recommendation for a full factorial design. Researchers are encouraged to consider how any one type of intervention occurs in the context of other strategic elements.
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Nanomedicine is a rapidly evolving field, for which polymer building blocks are proving useful for the construction of sophisticated devices that provide enhanced diagnostic imaging and treatment of disease, known as theranostics. These well-defined nanoscopic objects have high loading capacities, can protect embedded therapeutic cargo, and offer control over the conditions and rates of release. Theranostics also offer external surface area for the conjugation of ligands to impart stealth characteristics and/or direct their interactions with biological receptors and provide a framework for conjugation of imaging agents to track delivery to diseased site(s). The nanoscopic dimensions allow for extensive biological circulation. The incorporation of such multiple functions is complicated, requiring exquisite chemical control during production and rigorous characterization studies to confirm the compositions, structures, properties, and performance.
We are particularly interested in the study of nanoscopic objects designed for treatment of lung infections and acute lung injury, urinary tract infections, and cancer. This Account highlights our work over several years to tune the assembly of unique nanostructures. We provide examples of how the composition, structure, dimensions, and morphology of theranostic devices can tune their performance as drug delivery agents for the treatment of infectious diseases and cancer.
The evolution of nanostructured materials from relatively simple overall shapes and internal morphologies to those of increasing complexity is driving the development of synthetic methodologies for the preparation of increasingly complex nanomedicine devices. Our nanomedicine devices are derived from macromolecules that have well-defined compositions, structures, and topologies, which provide a framework for their programmed assembly into nanostructures with controlled sizes, shapes, and morphologies. The inclusion of functional units within selective compartments/domains allows us to create (multi)functional materials. We employ combinations of controlled radical and ring-opening polymerizations, chemical transformations, and supramolecular assembly to construct such materials as functional entities. The use of multifunctional monomers with selective polymerization chemistries affords regiochemically functionalized polymers. Further supramolecular assembly processes in water with further chemical transformations provide discrete nanoscopic objects within aqueous solutions. This approach echoes processes in nature, whereby small molecules (amino acids, nucleic acids, saccharides) are linked into polymers (proteins, DNA/RNA, polysaccharides, respectively) and then those polymers fold into three-dimensional conformations that can lead to nanoscopic functional entities.
Well-defined nanoscopic objects that can be embedded with therapeutics have great potential to protect the therapeutic cargo with high capacity for loading and control over the conditions and rates of release, while also offering external surface area for conjugation of ligands to impart stealth characteristics and/or direct their interactions with biological receptors. The nanoscopic dimensions allow for extended biological circulation and provide a framework for conjugation of imaging agents to track delivery to diseased site(s). Incorporating such multiple functions is complicated, requiring exquisite chemical control during production and rigorous characterization studies to confirm the compositions, structures, properties, and performance. Direct linkage to a particular disease is necessary to tune the chemical, physical, and morphological features to gain access to the disease site and deliver the cargo appropriately, for example, extravascular, intracellular, and so forth.
Nanomedicine is a rapidly evolving field, for which polymer building blocks are proving useful for the construction of sophisticated devices, theranostics, that provide enhanced diagnostic imaging and treatment of disease. We have special interests in the study of nanoscopic objects that are designed for treatment of lung infectious diseases, urinary tract infections, acute lung injury, and cancer. Our nanomedicine devices are derived from macromolecules that have well-defined compositions, structures, and topologies, allowing for programmed assembly into nanostructures having controlled sizes, shapes, and morphologies. The evolution of nanostructured materials from relatively simple overall shapes and internal morphologies to those of increasing complexity is driving the development of synthetic methodologies that allow for the preparation of increasingly complex nanomedicine devices. Moreover, the inclusion of functional units within selective compartments/domains is of great importance to create (multi)functional materials. Combinations of controlled radical and ring-opening polymerizations, chemical transformations, and supramolecular assembly are employed to construct such materials as functional entities. We employ a variety of multifunctional monomers, together with selective polymerization chemistries, to afford regiochemically functionalized polymers, and then further conduct supramolecular assembly processes in water, often together with chemical transformations, to afford discrete nanoscopic objects within aqueous solutions. This approach has similarities to the processes of nature, whereby small molecules (amino acids, nucleic acids, saccharides) are linked into polymers (proteins, DNA/RNA, polysaccharides, respectively) and then those polymers fold into three-dimensional conformations that can lead to nanoscopic functional entities. This Account will highlight our work over several years to tune the assembly of unique nanostructures, with examples of how their compositions, structures, dimensions, and morphologies can tune their performance as theranostic agents for the treatment of infectious diseases and cancer.
As indicated by their name, theranostic nanoparticle systems (TNPs) are designed to fulfill two objectives: therapeutic delivery and diagnostic imaging. Parameters such as the biodistribution (BioD), pharmacokinetics (PK), and pharmacodynamics (PD) are inherently different between drug delivery systems (DDS) and imaging systems. Nanoparticles for drug delivery typically exhibit extended blood circulation times in order to increase plasma concentration of the pharmacologically active substance, and also to exploit fully the enhanced permeation and retention (EPR) effect or ligand-mediated targeting, to reach further increases in local drug concentrations at disease sites. On the other hand, nanoparticulate imaging agents often require a different set of BioD, PK, and PD properties to operate with optimized signal to background. Nanoscopic imaging agents are administered to increase the signal-to-noise ratio in a specific tissue and allow for a diagnostic image to be obtained. In the case where the nanoparticle imaging agent is combined with an “active” targeting ligand, the circulation half-life of the construct becomes a critical parameter. In this setting, a longer circulation will allow for accumulation (via more circulation passes) at the target, but will require that the nanoparticle ultimately clears in order to reduce background (concentration) in the circulating blood to provide contrast. For successful realization of functional TNPs, this means that these parameters of the device must compromise between the optimum for drug delivery and that for diagnostic imaging, which in turn requires flexible chemical synthesis to systematically tailor the size, shape, charge, placement of targeting ligands, degradation, drug loading and release kinetics, and so forth.
In this context, shell cross-linked knedel-like nanoparticles (SCKs) are versatile constructs for which a substantial body of work has been performed to establish protocols for controlling such parameters that are fundamental for nanomedical research, in general, and theranostic applications, in particular. In 2008, the Wooley lab developed a protocol for designing SCK nanoparticles that exhibited tailored PK and BioD.(
SCKs having tailored in vivo behavior involve coupling onto polymer precursors to provide for chemical control and detailed characterization.
The concept of targeting TNPs to specific tissue or disease of choice is the fundamental premise of creating nanomedicines with high efficacy. The concept is not new; Paul Ehrlish postulated the magic bullet concept in the early 1900s, and it is still a highly appealing idea.(
Ligand-mediated targeting of nanoparticles, or “active” targeting, utilizes biomacromolecules, such as antibodies, proteins, peptides, aptamers, or small molecular entities attached to the surface of the nanoconstruct.(
In the case of SCKs of varying shapes, the inherent carboxylic acid functionality of the hydrophilic poly(acrylic acid) shell layer has been utilized via the formation of amide bonds for cross-linking and the attachment of imaging agents, PEGs for circulation enhancement, and targeting components. Given that the SCK nanostructure contains thousands of carboxylic acids or activated esters,(
SCKs having fluorescent labels and Tat peptides were prepared by conjugation of the active units onto pre-established SCKs.
The folate receptor (FR), or membrane-associated folate-binding protein, is one of the cellular transport systems that binds folates with high affinity in the nanomolar range and undergoes receptor-mediated endocytosis. Because the folate receptor is well characterized and is overexpressed in several cancer types with limited expression in normal tissue, the FR is an attractive target for nanoparticles to treat tumors.(
In order to explore the use of diverse and biologically complex targeting ligands, new conjugation methodologies have been developed for SCKs. One such example is the utilization of protected thiols, placed on the termini of PEG grafts presented topologically on the outer surface of the SCK nanoparticle, that can be deprotected/activated for further conjugation with thiol- or maleimide-containing targeting groups to form reversible disulfide or thioether linkages, respectively.(
In our experience with SCK nanoparticles, it has been clear that achieving efficient and quantifiable targeting (including results not published) is indeed difficult. Our hypothesis is that the ligand presentation is not optimum in many targeted NPs and that the chemical design of the carriers must be more sophisticated. If the ligand is placed on a spacer unit such as PEG, the remaining PEG (necessary for circulation enhancement) must be of sufficiently shorter length in order not to interfere with binding to the receptor. In addition, covalent attachment of the ligand to the spacer molecule must be such that the binding characteristics are not hampered. In the antibody, antibody fragment or protein case, this requires site specific modification reactions, and the employment of rapid, efficient and orthogonal (REO) ligation methods for covalent attachment to NPs.(
Control over the route of administration of the nanoconstruct is a highly attractive means by which to exploit the advantage that the specific organ provides combined with selected targeting approaches. For example, localized therapy of urinary tract or lung infections may be achieved via catheter delivery to the bladder or inhalation delivery of aerosols to the lung, respectively. We are currently devoting significant efforts toward the treatment of infectious diseases by the direct administration of antimicrobial-loaded SCKs(
A directed approach to actively target tissues in vivo, without the use of targeting ligands, utilizes superparamagnetic nanoparticles together with an external magnetic field to localize the drug carrier to the tissue of choice.(
Positron emission tomography is a highly sensitive imaging modality, with which we have had great success in tracking SCKs in vivo. As with the PEGylation of SCKs, it was found that conjugation of chelators for the PET-active radionuclides was best performed onto the polymer precursors to allow for efficient coupling and accurate quantification and characterization studies to be performed.(
Magnetic resonance imaging (MRI) is powerful in its ability to visualize soft tissues. Nanoparticles for MRI include both T1 (positive contrast) based agents, such as NPs containing rare earth metals, Gd3+, and others, as relaxation aids, or superparamagnetic NPs, such as SPIONs for T2 (negative contrast) weighted imaging. Apart from these standard MRI applications, NPs for T1 imaging of other natural isotopes such as 19F, 31P, 13C, 14N, and others have been developed. Of these isotopes, 19F nuclei are attractive due to the lack of background in normal tissue (only low levels in teeth and bone), good biocompatibility, sensitive chemical shift, high gyromagnetic ratio, and high natural abundance. We have explored the use of nanoscale 19F-MRI agents capable of both chemotherapeutic delivery and 19F-MRI either in the form of hyperbranched polymer micelles
SCKs have been investigated as carriers of therapeutics directed against three main applications: cancer,
In one case, we probed effects of amorphous versus semicrystalline core domains, with the intention being to trap doxorubicin, an anthracycline antibiotic and a very common chemotherapeutic agent used in the treatment of many cancer types, within the semicrystalline core at room temperature and promote their release at physiological temperature.
We also conducted an in-depth study of the effects of the particle dimensions, including overall diameter, core diameter, and shell thickness, on the drug release kinetics. Effects of the nanoparticle composition were held constant, by employing SCKs constructed from a single type of amphiphilic diblock copolymer (Figure
SCKs as carriers of doxorubicin, with the ability to tune the compositions and dimensions of the core and shell domains independently.
We have also investigated the SCK scaffold for the delivery of silver-based antimicrobial agents, which are of interest for treating bacterial infections in the lung and bladder. Silver-based agents are interesting as antibacterial therapeutics because toxicity to human tissues is low and bacteria rarely develop resistance against silver-based agents. The SCKs were either loaded with Ag+ within the framework of the poly(acrylic acid) shell or silver carbene complexes that were expected to reside near the core–shell interface (Figure
Loading of silver-based antimicrobial agents within the shell or core–shell interface of SCK nanoparticles.
Using nature as a guide, SCKs have been prepared as synthetic analogs of histones,(
For SCKs described so far, degradability has not been regarded as the primary objective, mainly because the physiochemical, in vitro, and in vivo studies have been time-consuming, and a rapidly degradable NP system would pose challenges with study prior to the disintegration of the controlled morphology. However, to successfully utilize nanoparticle constructs for therapeutic delivery in the clinic, clearance of the NP or its building blocks must be assured, while also avoiding toxic effects from the particles and degradation products. The metabolic fate of the nanoparticle and the single and multiple dose toxicity must be investigated, along with many other factors such as clearance routes.
For SCKs, both degradability of the cross-links stabilizing the micelles(
SCKs containing degradable cross-links.
Cholesterol and its relatives possessing the 1,2-cyclopentanoperhydrophenanthrene ring system (Figure
Common tetracyclic steroid frame containing the 1,2-cyclopentanoperhydrophenanthrene ring skeleton.
The first known sterol, cholesterol, was discovered by French chemists as a crystalline component of human gallstones over 230 years ago. In 1789, Francois Poulletier de La Salle observed an alcohol-soluble portion of bile stones, which 10 years later was reported by De Fourcroy to be identical to a waxy material in the fat of putrefied corpses referred to as adipocire.(
Sterol research changed in the mid- to late 20th century and centered on biomimetic chemistry, tracer work, enzymology, and structure determination using high-field NMR and X-ray diffraction methods, culminating with a broad outline for cholesterol synthesis and the partial or complete purification of all the microsomal-bound enzymes that act on sterols between lanosterol and cholesterol.
Central to the advances of the past two decades is the development of molecular genetic approaches that have witnessed the cloning, primary amino acid sequences, and functional characterization of a large number of enzymes that act on sterol and revealed unexpected inborn errors of cholesterol metabolism. The full exploitation of these genes lies in medical diagnostics, treatment, and the ability to ultimately engineer phytosterol pathways to generate plants with tailored sterol profiles for commercial production. Another spectacular discovery involving 13C-isotopically labeled compounds supplied to microorganisms and plants was the demonstration that the classic acetate–mevalonate pathway to animal cholesterol can be replaced in the biosynthesis of algal sterols and other isoprenoids by a mevalonate-independent pathway.
Sterols are amphipathic compounds that originate in isoprenoid biosynthesis with the main frame composed of a nucleus and side chain (Figure
Perspective drawings of the cholesterol molecule showing four domains of functional importance (left) and the flat elongated structure presumed to form in the membrane (right).
The earliest chemical definition for a sterol was provided by Fieser and Fieser. These substances will possess the characteristic perhydro-1,2-cyclopentanophenanthrene ring skeleton (Figure
Two systems recognized for numbering of carbon atoms of the sterol nucleus and side chain. The conventional system, which incorporates the α/β-chiral descriptors in nucleus and side chain is based on the Fieser and Fieser book
Because sterols are derived from the C30 squalene, they are a class of triterpenoids. These tetracycles are generated from the linear combinations of the C5-isoprenoid building block, isopentyl diphosphate. In order to conform to the empirical isoprene rule, triterpenes (C30) are the products of joining 6 × 5 C5-units.(
The cyclization of squalene-2,3-oxide (i) folded in either the chair–chair–chair–boat–unfolded (ii) or chair–boat–chair–boat–unfolded (ii) conformations to yield a cation (ii), which can stabilize to produce the dammarane, tirucallane, euphane, and cucurbitane skeletons (via path a) or cycloartane and lanostane skeletons (via path b). Generally, the cyclization products contain a 3β-OH group and a Δ8-bond or in the case of cucurbitacin a Δ5-bond.
Principal differences in cholesterol and related phytosterols are in the side chain (C20–C26/C27), which has different degrees of substitution and unsaturation. Chemical surveys of the sterol composition of prokaryotes, eukaryotes, and sedimentary organic matter show that there are at least 250 sterols and related steranes; in corn, 60 different sterols have been characterized.
According to the currently accepted hypothesis, the formation of steroids proceeds by a cationic cyclization process. This theory has it roots in the biogenetic isoprene rule of Ruzicka and his associates in Zurich who considered biosynthesis of triterpenoids was initiated by an electrophilic attack on a double bond of a linear polyprenoid substrate forming a cyclic (or polycyclic) intermediate cation, which, in turn, can then undergo various transformations and rearrangements.(
The prenyl transferase reaction and formation of farnesyl diphosphate (FPP). Prenyl (C5) units composed of isopentyl diphosphates (IPP) are assembled in a head to tail fusion to yield C10 (geranyl diphosphate, GPP) and in repeat fashion to C15 (FPP) isoprenoids. Alternate isoprene unit assemblies yield a variety of different structures notable in terpene metabolism. Enz = enzyme and M++ can be Mn2+ or Mg2+ ions.
Proposed mechanism for the coupling of farnesyl diphosphate (FPP) to form squalene.
Rahier and co-workers have drawn attention to ionic processes that generate sterol cations concomitant with allylic rearrangements, double bond C-methylations, or reduction reactions in the postsqualene segment of sitosterol biosynthesis (Table
The usual pathway to the C30 primary sterols proceeds along the well-established isoprenoid trail that leads from the “active isoprene unit”, isopentenyl diphosphate, to the C30 triterpenoid squalene. The biosynthesis of C30 sterols from squalene and thence to cholesterol can be outlined in three major stages as envisioned by Bloch:(
In the early phase of cholesterol research, it was not immediately apparent that the C27 structure of cholesterol was related to lanosterol, since it failed to be divisible by C5 units. To establish isoprenoid character, several groups incubated [1-14C]acetate and [2-14C]acetate with liver slices affording a decisive pattern in the distribution of acetate carbon atoms in the labeled cholesterol. As shown in Figure
Distribution of acetate carbon atoms found in cholesterol; a repeating pattern of five carbon atoms (isoprene unit), surrounded by dotted lines, is recognizable in three places in the molecule.
Since the late 1960s, it has been known that two major cyclization pathways exist for the conversion of oxidosqualene to steroidal tetracycles; lanosterol is formed in organisms of a nonphotsynthetic lineage, and cycloartenol is formed in organisms of a photosynthetic lineage by independent synthase enzymes.
By carrying out experiments with doubly labeled [2-14C(4
Interpretation of the mechanism of squalene-2,3-oxide cyclization to lanosterol and cycloartenol according to refs (
The different molecular libraries that constitute isoprenoid–sterol metabolomes across Kingdoms are organized through a series of discrete assemblies of enzymatic reactions, which are characterized compartmentally. The acetate–MVA pathway to squalene oxide is considered to be the main route to the production of steroidal backbones. Recent international efforts have resulted in the complete sequencing of the model plant
At least two alternative biosynthetic pathways to Δ2-(dimethyl allyl pyrophosphate) and Δ3-isopentenyl diphosphate (formerly pyrophosphate) have been shown, by biosynthetic labeling studies, that can supply prenyl units to squalene, which in turn is converted to sterols (Figure
Overview of compartmentalized isoprenoid–sterol biosynthesis pathways. Feeding studies of [2-13C]leucine or [1-13C]glucose distinguish the leucine–HMGCoA, acetate–mevalonic acid (MVA) or the 2-C-methyl-
It has been possible to establish the contribution of these variant isoprenoid pathways to sterol biosynthesis in plants and microorganisms by the retrobiosynthetic approach. When, for example, [1-13C]glucose, [2-13C]acetate,
By far the best studied of the enzymes involved in sterol biosynthesis are the lanosterol and cycloartenol synthases. For cyclization, the reaction requires (3
The cyclization of squalene-2,3-oxide (
The postulated mechanism of cyclization of squalene oxide into cycloartenol (and parkeol) is essentially the same as that for lanosterol, except for the final 9β,19 cyclopropane ring closure with the 9β-H atom migrating to C8 instead of C9α proton elimination. Nes found that the solid state and solution studies unambiguously show that cycloartenol is “flat” of A/B/C-rings in the chair/half-chair/twist-chair conformer (Figure
Structure of “flat” cycloartenol established by NMR (left) and X-ray (right) measurements.
Cloning and mutagenesis studies of cycloartenol and lanosterol squalene oxide (= oxidosqualene) synthases [OSS] suggest that the native enzymes in plants and fungi are catalytically and structurally similar to the human oxidosqualene–lanosterol and bacterial squalene–hopene cyclases.
Although cycloartenol is the first cyclized product in plants, lanosterol has been considered as an intermediate in phytosterol biosynthesis. Several reports involving plant sterol biosynthesis show similar rates of [2-3H] (or 14C)-lanosterol and [2-3H] (or 14C)-cycloartenol incorporation into phytosterols suggesting that cycloartenol might transform to lanosterol via a 9β,19-cyclopropane to Δ8-isomerase catalyzed reaction as a preliminary step to phytosterol formation.
Three different approaches have been used to study the nature and sequence of steps involved with sterol biosynthesis: organic, enzymatic, and genetic approaches. The enzymatic approach to understand and control formation of the sterol structure was hampered by the low abundance of sterol enzymes in cell-free preparations, as well as difficulties associated with purifying microsomal proteins to homogeneity. By the early 1980s, it was evident primarily from the pioneering efforts of Gaylor and his co-workers at cataloging the properties of enzymes that act on sterols that an accurate sequence of 10 enzymatic reactions performed by nine distinct enzymes can be written for the lanosterol conversion to cholesterol as outlined in Table
Steps 3 to 4 and 5 to 6 involve two discrete enzymatic reactions, 4-SMO and 4-SDC; see text.
Cholesterol synthesis requires one molecule of C30-4,4,14-trimethyl sterol converted to C27-4,4,14-trisdesmethyl sterol followed by the formation of a saturated isooctyl side chain and Δ5-bond in the final product. The stoichiometry of cholesterol synthesis from lanosterol is therefore lanosterol + 15NADPH + 4H+ + 10O2 → cholesterol + 2CO2 + HCOOH + 15NADP+. This process can be divided into two stages: stage 1 (reactions 1–6) represents the nuclear demethylation reactions that fashion the lanosterol frame into the cholesterol structure. The product of reaction 6 lacks methyl groups at C4 and C14 and contains a 3β-OH group that is distinct from the one generated by the cyclization of squalene-2,3-oxide to lanosterol. During these conversions of the C4-sterol to a C4-desmethyl sterol, a stable 3-keto sterol intermediate is formed. For stage 1, the process consumes 12 NADPH, 2 NAD+, 9 O2, and 1 H+. Reactions 3 and 4 are enzymically coupled and repeated with the resulting C4 methyl product catalyzed in reactions 5 and 6; in both sets of reactions two distinct enzymes, sterol C4-methyl oxidase (4-SMO) and sterol C4-decarboxylase (4-SDC, also referred to as 3β-hydroxysteroid dehydrogenase/C4-decarboxylase), are utilized to carry out the overall conversion of the sterol C4-demethylation reaction. Key to this series of reactions is that the equatorial (in the plane of the sterol nucleus) methyl group of the 4,4-dimethyl and 4-monomethyl substrates is recognized for catalysis. To maintain stereochemical consistency in 4,4-dimethyl substrate recognition by 4-SDC, during decarboxylation the methyl group that occupies the 4β-(axial) position of the C4-dimethyl sterol epimerizes to the more stable 4α-(equatorial) position in the 4-methyl 3-ketosteroid product and, in doing so, re-establishes the C4α configuration. Thus, the natural occurrence of C4β-methyl sterols seems unlikely. Stage 2 (reactions 7–10) involves the rearrangement of the Δ8-bond to the Δ5-position and saturation of the side chain double bond. Reactions in stage 2 consume 3 H+, 3 NADPH, and 1 O2, and the enzymes act sequentially to convert the ring structure from a Δ8 to Δ7 to Δ5,7 to Δ5. Two enzymatic studies completed on the cloned human sterol C8–C7 isomerase and microsomal rat C24(25)-reductase after Gaylor postulated a lanosterol–cholesterol pathway clarified the later sequence of reactions to cholesterol.
The nine catalysts that convert lanosterol to cholesterol may be considered the core enzymes of Δ5-sterol biosynthesis. As more enzymes have been isolated from an increasing number of sources, it has become clear that they fall into only a small number of reaction types and that the chemical names give varied indication of this. In an attempt to rationalize this situation, Table
In the case of cholesterol biosynthesis from lanosterol, two intersecting routes have been postulated. The choice of pathway is determined by the stage at which the double bond at C24 in the sterol side chain is reduced. If C24 double bond reduction is retained until the last reaction, cholesterol synthesis proceeds via cholesta-5,24-dienol (desmosterol) (Bloch pathway). On the other hand, early Δ24-reduction involving lanosterol can proceed to cholesta-5,7-dienol (7-dehydrocholesterol) and cholesterol (Kandutsch–Russell pathway). A common interpretation regarding which pathway is utilized involves the positioning of the Δ24-reductase in cholesterol biosynthesis such that skin and intestines, which have higher sterol C24-reductase activities than liver or brain, proceed via the C24–C25-terminal intermediates.
The relevant committed step that distinguishes sterol from isoprenoid–triterpenoid biosynthesis occurs at the cyclization of oxidosqualene. Major control points in sterol biosynthesis may arise in the primary pathway before squalene formation at hydroxymethyl-glutaryl-CoA reductase (HMGR) (coarse control)(
Early attempts to deduce sterol biosynthetic pathways in systems other than animals were based largely on indirect approaches that included structural and stereochemical correlations of co-occurring metabolites and
A generalized route from cycloartenol
The sterol alkylation reaction pathways operating in different organisms; routes 1 in fungi, protozoa, and plants to form C9 and C10 side chains and routes 2 and 3 to form C11 extended side chains in marine organisms.
In organisms that live in the marine world, sterol methylation patterns vary greatly and reflect the complexity of mixtures of sterols arising through the food chain. The 24-SMT of these organisms catalyze branched and highly alkylated side chains of distinct stereo- and regiochemistry, including tris- or quadruple alkylation of the C11 side chain, not typically observed in the sterol methyltransferase catalyzed products of terrestrial organisms that produce C8 and C9 side chains (Figure
In reviewing the action of enzymes that catalyze sterol formation, it has been found convenient to divide topics according to the properties of the enzyme that include its specificity, mechanism, inhibition, and where evidence is available results of mutagenesis experiments. We start with the enzymatic C24-methylation reaction, where progress has been made in identifying the genes and catalytic properties of the corresponding proteins. Four different sterol C24-methyltransferase enzymes (24-SMT) have been detected and classified according to the substrate favored by the enzyme for catalysis. Plants synthesize two 24-SMTs, SMT1, which prefers cycloartenol ((EC 2.1.1.142), and SMT2, which prefers 24(28)-methylene lophenol (EC 2.1.1.143); some fungi and protozoa synthesize 24-SMT1 of substrate preference for zymosterol (EC 2.1.1.410), and other fungi also synthesize 24-SMT with a substrate preference for lanosterol.
So far as is known, 24-SMTs vary in sequence identity (49–77%), the whole family containing only about 63 completely conserved residues (less than 20% of an average of
The structure of 24-SMT has not been established. In the absence of atomic coordinates, the general characteristics of the 24-SMT active site have been probed by means of photoaffinity labeling using AdoMet, suicide substrates using 26,27-dehydrozymosterol and site-directed mutagenesis experiments. These studies, focused on the
The 24β-chirality in algal ergosterol and the 24α-chirality of vascular plant campesterol and sitosterol is considered to arise from direct C24-alkylation or through a Δ24(28)-sterol intermediate, respectively. To account for the product diversity, ionic and X– group C24-alkylation mechanisms have been proposed as shown in Figure
Mechanisms for methyl transfer to Δ24(25)-substrate in the action of sterol C24-methyltransferase; dot shows to 13C-labeled carbon. In mechanisms A and C, the carbocationic intermediates are discrete species, and an important difference between the two mechanisms involves the SN2 (
In the ionic mechanisms, illustrated in paths a and c (see Figure
Arigoni and colleagues reported the preparation of [1H2H3H]AdoMet (only one enantiomer of the chiral methyl group was present) and have used it to probe the stereochemical course of a methyl transfer to carbon in fungal production of ergosterol. The results obtained indicate that the chirality of the acetate derived from the methyl group at C24 in ergosterol was the same as that of the methyl group of methionine but with a reduction in chiral purity.(
Yagi et al. investigated the second C1-transfer reaction by feeding a set of [26-13C], [27-13C], and [26,27-13C2]desmosterols (cholesta-5,24-dienol) to cultures of
C1-Transfer mechanisms in the conversion of Δ24(28)-substrate into 24-ethyl(idene) products. In each case, the isotopically labeled substrate contains a 13C-27 atom (dot) and a
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71.50
We would like to comment on a recent review article published in Sensors by Razak
The authors stated that this system was developed by our group, however, as stated in our article [
There are several papers which outline the existing technologies [
We felt compelled to write this note so that: (1) it is not misinterpreted that we developed this system and (2) our research findings are not inaccurately reported.
A recent review by Salvo et al published in
In their review of the efficacy of dapagliflozin, Salvo et al have provided little or no evidence that dapagliflozin reduces blood pressure. Dapagliflozin monotherapy was initially suggested to reduce blood pressure by a small amount at 24 weeks.
Secondly, Salvo et al do provide some evidence of weight loss with dapagliflozin, of up to 4.65 kg,
Also, Salvo et al suggest that the weight loss with dapagliflozin is an advantage over other oral medications in treating subjects with type 2 diabetes.
Thirdly, Salvo et al have suggested that dapagliflozin has a lesser ability to cause hypoglycemia than other oral anti-diabetes medicines, and provided evidence that dapagliflozin has a low potential to cause hypoglycemia compared to the sulfonylureas (glipazide, glimepride),
The authors did conclude that “long-term clinical trials and post-marketing studies are needed to further investigate dapagliflozin’s cardiovascular profile, and its impact on morbidity and mortality”,
The multicenter trial that will eventually be able to evaluate the cardiovascular safety of dapagliflozin, is the effect of dapagliflozin on the incidence of cardiovascular events (DECLARE-TIMI58).
In conclusion, dapagliflozin does not have a noted ability to reduce blood pressure. However, dapagliflozin does cause weight loss and have a low potential to cause hypoglycemia, but this is not necessarily an advantage, as other medicines available for the treatment of type 2 diabetes also cause weight loss and have a low propensity to cause hypoglycemia. Most importantly, the cardiovascular clinical outcome study DECLARE-TIMI58 being undertaken with dapagliflozin will not report for 5 years, and thus dapagliflozin will be available for the treatment of type 2 diabetes for 5 years without proven cardiovascular efficacy or safety.
The author reports no conflicts of interest in this communication.
Agmon et al
First, in the abstract, the authors claim to select randomized and nonrandomized controlled studies. In contrast with the stated study selection, Table 1 shows that not all of the 22 studies included are in compliance with these design requirements; several uncontrolled studies were examined, including an earlier uncontrolled study by the authors.
Second, at least one randomized controlled trial that fulfills the selection and inclusion criteria has not been considered in the review. Our randomized controlled trial examined the effects of a Feldenkrais intervention on balance, gait, and single-task and dual-task mobility.
After completion of the Feldenkrais intervention, participants improved significantly on balance (
The authors of the review underline in their discussion the prominence of dual-task training for improving dual-task performance. However, an important outcome of our publication is that a broad spectrum of activities that are part of the Feldenkrais method can result in improvements in both single-task performances and dual-task performance. From our perspective, this is an important piece of the puzzle and should be noted if we want to design evidence-based interventions based on current scientific knowledge.
The authors report no conflicts of interest in this communication.
Bledsoe et al presents an interesting study examining the disparities between radiologic and pathologic diagnoses of asbestosis in cases referred for consultation in pulmonary malignancy.
The authors have no conflicts of interest in this communication.
The authors have no conflicts of interest in this communication.
Determination of prognosis of exacerbation of chronic obstructive pulmonary disease (AECOPD) is a key cornerstone for health cost, organization, systems, and influences determinations of rationale in the use of non-invasive ventilation (NIV).
In an interesting study, Haja Mydin et al determined that anemia, and performance status based on World Health Organization performance status (WHO-PS) were independent prognostic markers in acute hypercapnic respiratory failure (AHRF) due to AECOPD.
We believe that the study makes a useful contribution to the establishment of predictors of poor prognosis in patients with AECOPD and it may help to make appropriate decisions for rational use of NIV. However, there are some concerns related to this study.
Firstly, the mechanism of anemia in chronic obstructive pulmonary disease (COPD) and its impact on survival are still unclear; principally it may be associated with acute systemic inflammation in COPD. Nevertheless, anemia prevalence shows a wide range among studies (7.5%–34%) and many confounding factors associated to comorbidities may exist.
Secondly, a high frequency of hospital admissions for AECOPD had no prognostic significance in this study, but it was not clarified if previous episodes needed NIV or endotracheal intubation.
Thirdly, the authors consider that the APACHE II score
Low performance status is an established prognostic factor for poor outcomes for patients with AECOPD, but this information was well known before the widespread use of NIV in general hospitals.
Finally, the patient population included in this study may be too low with not enough representation to perform a suitable search for independent prognostic markers of a varied clinical condition such as AECOPD. A previous study
In conclusion, we agree that anemia and a low performance status could have a negative impact on the outcome of patients with AECOPD, but possible patient selection bias and the lack of a control group make the interpretation of the results less firm for Haja Mydin et al,
The authors report no conflicts of interest in this communication.
I have read with great interest the article “Epidemiology of dengue: past, present and future prospects” by Murray et al.
Despite the impact of climate change, I strongly believe more factors have contributed to the increase of dengue disease: social and environmental factors; the increase of urbanization; the lack of and/or poor health services; and the expansion of international travel and trade are all linked to the increase of dengue fever.
The World Health Organization (WHO) described dengue as a “neglected” tropical disease in its Dengue Prevention and Control Strategy 2012–2020, due to the lack of global coordination efforts, researches and political will.
In conclusion, this letter addresses the impact of climate change on dengue disease and discusses other contributing factors that influence the vulnerability of populations to the health impact of climate change.
I agree that the expansion of dengue is multifactorial; climate change is one of the factors, but not the dominant one, since other factors may variously contribute, depending on the situation, and where and how the disease exists. The major factors may include: the unprecedented population growth associated with the urbanization increase in tropical, developing countries; substandard housing; overcrowding; and water and sewer quality.
The author reports no conflicts of interest in this communication.
I have read with interest the review by Rodriguez-Wallberg and Oktay.
The authors claim that “Experimental data from prepubertal and adult mouse models treated with cyclophosphamide do not support the notion that a prepubertal stage would be protective for the primordial follicles”, citing their own abstract
The use of GnRH agonists should be considered in women of reproductive age receiving chemotherapy. Intramuscular or subcutaneous GnRH analogs seem to be effective in protecting ovaries during chemotherapy and should be given before or during treatment […]
Furthermore, opposite to the authors’ opinion and declaration regarding fertility,
The author has no conflicts of interest in this communication.
I read with great interest the recently published article by Diao and Lee
However it should be stressed that dermatology, as well as the other medical branches and specialties, has faced many progresses in recent years, thanks to the technological advancements (the so-called “teledermatology”)
The relationship between the patient and physician has changed, shifting from paternalism to a new model of shared medicine: the so-called P6 model (where the six Ps stand for personalized, preventive, predictive, participatory, psycho-cognitive, and public).
However, in some cases, information available on the Internet is not always of high quality, complete, accurate, and reliable. The existence of a digital gap or divide could be a further obstacle in open access to these websites and in the adoption of proper preventive and protective behaviors.
General practitioners and dermatologists should be aware of this phenomenon, investigating the attitudes of their patients to surf the web and to provide them with authoritative and reputed information.
In conclusion, a new array of sun-protective behaviors is emerging, termed as “public skin health”, and understanding the drivers and the psychological variables of accessing online resources and adopting web-based sun-protective activities, in order to implement them and properly guiding the patients, is of extreme clinical interest.
The author reports no conflicts of interest in this communication.
I read with interest Mainz et al’s article in the February issue of your journal.
It is well known that isotonic saline solution nasal washing facilitates nasal drainage and cleans the airway of any postnasal discharge (including allergens); furthermore, it can be effective when applied appropriately.
As a result, nasal saline, administered with the correct technique, can be as effective as nasal drugs; therefore, it cannot be used as a placebo in a rhinosinusitis study.
The author reports no conflicts of interest in this communication.
I read with interest the article recently published in
The author reports no conflicts of interest in this communication.
I would like to make some comments and clarifications regarding the article “Intradermal air pouch leukocytosis as an in vivo test for nanoparticles” recently published in the
We have been using the murine air pouch model in our laboratory for more than 12 years. In 2001, we were the first to successfully use this model in an immunotoxicogical study to demonstrate that a chemical of environmental concern, the insecticide dieldrin, was found to induce an inflammatory response in vivo, as evidenced by a neutrophilic infiltration into the air pouches.
More recently, in 2011, we used the murine air pouch model to demonstrate for the first time that a given NP, namely titanium dioxide (TiO2), was pro-inflammatory, as evidenced by a rapid recruitment of leukocytes into the air pouch, where >80% of cells were neutrophils.
The other important point that has to be discussed is the characterization of NPs. Whether this is performed using dynamic light scattering analysis or other methods, it has to be carried out as closely as possible to the same experimental conditions that the NPs would be administered in in vivo (and in vitro) experiments, and not in pure water, as is frequently observed in the literature. One can easily understand that an NP will display completely different characteristics under water conditions than dispersed in culture medium with the presence or absence of serum, or in other biological fluids, for example, in regards to the phenomenon known as the corona of the NPs.
Temperature is another condition that must be taken into account for this characterization, since when the NPs are administered in vivo, body temperature is ~37°C, which differs from so-called “room temperature” (~23°C). Recently, we performed the characterization at 37°C (the temperature used in the in vitro study) and at room temperature, and we observed important differences using dynamic light scattering (Goncalves and Girard, unpublished data, 2014).
Although Vandooren et al
More recently, using the murine air pouch model, we have demonstrated that even if a given NP is not pro-inflammatory by itself, it can act by accelerating/amplifying the response of another agent,
In summary, I agree with the conclusion of Vandooren et al
The author reports no conflicts of interest in this communication.
Imashuku et al
Second, because the authors erroneously considered this a case with a normal anion gap metabolic acidosis, they mistakenly considered the high urine anion gap of 48 mmol/L to be indicative of a distal renal tubular acidosis due to multiple myeloma. However, the urine anion gap may be useful in evaluating the renal ammonium excretion only in a normal anion gap metabolic acidosis. In respiratory alkalosis, the urine anion gap has no added value in this context.
Third, a low anion gap is a feature of multiple myeloma, particularly of IgG myeloma, because the paraprotein is a cation.
The author reports no conflicts of interest in this communication.
In a recent review in
PERindopril–Thrombosis, InflammatioN, Endothelial dysfunction and Neurohormonal activation Trial (PERTINENT), a sub study of EUROPA, also showed that perindopril improves endothelial function, which the authors concluded may have provided the benefit and not blood pressure reduction per se.
The authors go on to state that,
The beyond-BP-lowering CV-protective benefits of telmisartan were demonstrated in the active-controlled ONTARGET (ONgoing Telmisartan Alone and in combination with Ramipril Global Endpoint Trial) trial.
However, ambulatory and night-time systolic blood pressure was significantly lower with telmisartan versus ramipril (−3.1 mmHg and −4.1 mmHg, respectively).
With respect to blood pressure-independent benefits of telmisartan, in Telmisartan Randomized Assessment Study in ACE intolerant (TRANSCEND), telmisartan as compared to placebo in patients with cardiovascular disease, or high-risk diabetes and without heart failure, who were intolerant to angiotensin-converting enzyme inhibitors (ACE-Is), did not reduce cardiovascular death, myocardial infarction, stroke or hospitalization for heart failure, despite a significant reduction in blood pressure.
Another point that we take issue with is,
ACE inhibitors and ARBs have been shown in head-to-head comparison trials to have comparable CV protective effects.
In Diabetics Exposed to Telmisartan and Enalapril (DETAIL), telmisartan (80 mg daily) was directly compared to enalapril (20 mg daily) in a randomized, multicenter, double-blind, 5 year study.
In regards to the ONTARGET trial, myocardial infarctions were numerically lower on ramipril (4.8%) versus telmisartan (5.2%) as were hospitalizations for heart failure (4.1% versus 4.6%, respectively).
In a direct comparison trial in hypertensive patients, perindopril, but not telmisartan, significantly improved endothelial function (eg, decreased von Willebrand factor), provided anti-platelet effects (eg, decreased soluble P- selectin and soluble glycoprotein V), and produced profibrinolytic activity (eg, decrease in plasminogen activator inhibitor type 1 and tissue type plasminogen activator antigens).
In regards to “ARBs having fewer adverse effects and better patient compliance,”
Regarding comparison trials for blood pressure lowering, in the only head-to-head randomized study comparing full-dose therapy of the two agents, perindopril (10 mg) as compared to telmisartan (80 mg) significantly reduced 24 hour systolic blood pressure (−22 versus −15 mmHg) and central aortic blood pressure (30% versus 14%).
In summary, as compared to placebo, perindopril but not telmisartan has been shown to reduce major cardiovascular events. A broad amount of data support the blood-pressure independent benefits of ACE-Is, especially in regards to perindopril. Numerous meta-analyses now confirm that ACE-Is, but not ARBs, reduce the rate of all-cause mortality and myocardial infarctions. As compared to other ACE-Is, perindopril has been shown to have fewer adverse side effects, including lower rates of cough and first-dose hypotension. Due to perindopril’s long history of cardiovascular protection and enhanced tolerability, it should be a first-choice ACE-I. Moreover, ACE-Is, as compared to ARBs, should be considered first-line antihypertensives, with telmisartan being no exception.
Dr DiNicolantonio received honorarium from Servier for preparation of this manuscript. Dr O’Keefe declares no conflicts of interest. Servier provided
Clinical trials testing perindopril and telmisartan
| Trial | Treatment | Comparator | Baseline SBP | SBP difference between arms | Total mortality benefit | CI |
|---|---|---|---|---|---|---|
| ADVANCE | Perindopril (+ indapamide) | Placebo | 145 mmHg | −5.6 mmHg | −14% | (−25; −2) |
| ASCOT | Perindopril (+ amlodipine) | Atenolol + thiazide | 164 mmHg | −2.7 mmHg | −11% | (−9; −1) |
| EUROPA | Perindopril | Placebo | 137 mmHg | −5 mmHg | −11% | (−22; +2) |
| ONTARGET | Telmisartan (+ ramipril) | Ramipril | 142 mmHg | −2.4 mmHg | +7% | (−2; +16) |
| PROFESS | Telmisartan | Placebo | 144 mmHg | −4 mmHg | +3% | (−7; +14) |
| TRANSCEND | Telmisartan | Placebo | 141 mmHg | −4 mmHg | +5% | (−9; +22) |
In the article “Management of adult diabetic ketoacidosis” by Gosmanov et al
On page 259, the authors write, “The use of bicarbonate in severe DKA is controversial […]”. On the same page, the authors also write that “severe acidosis […] can lead to impairment in sensorium […]”. Severe “impairment in sensorium” is a life-threatening coma. If intravenous sodium bicarbonate is also included in the treatment, however, the lethality of coma in DKA is zero.
The author reports no conflicts of interest in this communication.
It is with great interest that we read the publication entitled “Critical appraisal of ranibizumab in the treatment of diabetic macular edema” by Stewart.
In April 2013, the National Institute for Health and Care Excellence (NICE) of the UK approved the use of ranibizumab as a treatment option to treat diabetic macular edema of the eye if it has a central macular thickness (CMT) of 400 μm or more at the beginning of the treatment.
In light of this hypothesis, we conducted a preliminary analysis of 24 patients (48 eyes) with suspected DME who had OCT scans performed on the same day using both 3D OCT-1000 (Topcon; Itabashi, Tokyo, Japan) and Spectralis OCT. Matched macular-centered scans were obtained in 42 eyes; scans were not possible in 6 eyes due to media opacity or problems with patient fixation. The mean (standard deviation) CMT in this cohort was 282.0 (89.0) μm with a range of 191–689 μm using the Topcon OCT, and 312.4 (88.8) μm with a range of 224–719 μm using the Spectralis OCT (
Recognizing this issue is important for all those involved in care of patients in countries or institutions where the entry to treatment is limited by a defined CMT level. In the specific example considered here, this finding has a direct clinical impact on patients who have DME with a central macular thickness of 390–410 μm. In our small cohort of matched scans from 42 eyes, there were three whose CMT was >400 μm on the Topcon and four whose CMT was >400 μm on the Spectralis: ie, even in this small study, a patient’s eligibility for treatment depended on which scan was used. “Real-world” studies of OCT will become increasingly important if defined CMT levels are to be used as the “gate-keeper” for treatment, and should include repeatability and inter-instrument variability in defined patient cohorts.
Dr Keane is funded by the Department of Health’s NIHR Biomedical Research Centre for Ophthalmology at Moorfields Eye Hospital and UCL Institute of Ophthalmology. The views expressed in the publication are those of the author and not necessarily those of the Department of Health. None of the authors have any other conflicts of interest to declare.
Comparison of CMT measurements acquired on Topcon 3D OCT-1000 versus Spectralis® OCT for patients with DME. Direct comparison (
Kuptniratsaikul et al report the efficacy of Curcuma domestica extracts in the treatment of knee osteoarthritis.
The author has no conflicts of interest to disclose.
Miller et al
Due to its potency
NB-B, EM, GD, MW, and MS are employees of Pfizer Inc. The authors report no other conflicts of interest in this work.
Nowadays non-invasive ventilation (NIV) is a common therapeutic option in chronic hypercapnic respiratory failure, especially in chronic obstructive pulmonary disease (COPD).
Parnell et al used modafinil, out of label, in a very small heterogeneous group of hypercapnic respiratory failure patients unwilling to carry out NIV, with good results (benefic effects in blood gases, exacerbation admissions, with no adverse effects).
This pilot study open a new door to control hypercapnic respiratory failure in noncompliance patients with NIV. We understand limitations of this brief study, but consider, however, some key aspects need to take into account to a proper clinical extrapolation.
First, mechanism of acute and chronic hypercapnic are numerous and complex interaction, not only during daytime and night time period, muscular weakness play a central role in this pathways and respiratory central breathing could be influenced to chemical and mechanical properties of lung and upper airways.
Second, regarding limits of hypercapnic and how monitoring response to modafinil therapy is a key cornerstone.
Third, other co morbidity as chronic renal failure or neurologic conditions influence by hypercapnic could difficult interaction of modafinil as coincident psychiatric drugs like antidepressive or anxiolytic, that in some cases are associated in chronic respiratory insufficiency.
In our opinion, the good results shown in these six patients justify the conduct of extended studies on the action of modafinil in hypercapnic respiratory failure. However, if this drug may improve the quality of life, reduce health costs and safe alternative to non-invasive mechanical ventilation are still open questions.
The author have no conflicts of interest in this communication.
Rahman et al
The authors decided to use a cross-sectional study design, limiting knowledge of the temporal outcome. This was further limited by a 2-hour time frame for data collection, meaning cohort inclusion was representative of a time of day rather than whole population. Data would be ideally collected over a week or month, for 24 hours each day to ensure adequate recruitment. The study would have been further reinforced by grouping of study participants, giving the reader further insight into smoking characteristics. The authors acknowledge smokers are less likely to have taken part in the study than non-smokers, also impacting on result reliability. This bias may even be more pronounced in health professionals due to potential embarrassment.
Limitations aside, this paper is of interest, with hospital staff less likely to smoke (7% compared to 16% in general population).
The author reports no conflicts of interest in this communication.
Roskell et al present a systematic review comparing the long-acting beta2 agonists (LABA), olodaterol, and indacaterol in the July edition of the
The findings of this systematic review were questionable for several reasons, which were mostly related to missing data, the difference in the concomitant medication allowed between the trials of olodaterol and indacaterol, and the differences in COPD severity in the patient populations of the trials of the two LABAs. It should also be noted that four of the eight olodaterol trials were of a 6-week duration only,
Regarding data sources, this systematic review was limited to articles published from 1 January 1990 through to 5 August 2011. However, data from several indacaterol studies published in this period were not included in the analysis. The excluded data were: TDI data from the INSIST trial;
The authors state that olodaterol and indacaterol were compared under “similar trial conditions”; however the difference in the use of concomitant medication between the trials of the two LABAs makes this statement also questionable. Concomitant maintenance bronchodilator use was allowed in most of the olodaterol trials. In six out of eight trials,
While some subgroup analyses were carried out to account for concomitant LAMA medication, these were limited to two subgroup analyses: one subgroup analysis of change from baseline of trough FEV1 in patients who were “LAMA-free”; and another of patients who were allowed “LAMA add-on therapy”. Moreover, data were missing from these analyses; the LAMA-free analysis only included studies 1222.39 and 1222.40 of olodaterol
Data from the studies INTENSITY,
The differences found in disease severity between the trial populations of olodaterol and indacaterol were also a concern. Patients with COPD disease severity ranging from moderate to very severe COPD (Global initiative for chronic Obstructive Lung Disease [GOLD] stages II–IV)
The Methods section in the Abstract states that the systematic review was carried out to evaluate the efficacy and safety of olodaterol and indacaterol; however, no safety data were presented.
To conclude, the extent of the missing data from these analyses, the variation in concomitant medication allowed within the trials of the two LABAs, as well as the difference in disease severity between the trial populations, make it difficult to draw any meaningful conclusions regarding the relative efficacy of olodaterol and indacaterol.
The author reports no conflicts of interest in this correspondence.
The pooled analysis from Clemens et al
However, a recent meta-analysis from Artang et al
On these grounds, we think it could be useful to share some considerations to gain an in-depth understanding of a complex and undefined phenomenon in the absence of specific randomized trials addressing MI. Firstly, the pre-specified criteria of MI were erratically declared in the studies included in any meta-analysis, thus representing a confounding factor in terms of homogeneity and ultimately in the identification of CV outcomes. Moreover, there is no doubt that ST elevation and non-ST elevation MI are two distinct pathophysiological entities, whereas the latter may be frequently not related to coronary atherosclerosis. Unfortunately, this issue was not specified in randomized trials involving direct thrombin inhibitors.
Another pitfall may be attributed to lack of evaluation of the concomitant pre-existing antiplatelet medication throughout the studies, with potential confounding effects on CV outcomes. To our knowledge there are no published data regarding this issue (with the exception of bleeding events), and this may be the reason for which the authors did not consider concomitant antiplatelet therapy as a variable in the analysis. In a similar fashion, Artang et al
In this unclear scenario we are of the opinion that craving prospective data instead of additional confusing and contradictory meta-analyses may lead to more convincing and sound results for everyday clinical practice. Recently the FDA has proposed a study that will use data from the FDA Mini-Sentinel Distributed Database (MSDD) including nearly 100 million patients to assess systematically the rates of bleeding and thromboembolic outcomes associated with the use of dabigatran and warfarin for patients with atrial fibrillation, and that will furnish additional information regarding MIs (
The authors report no conflicts of interest in this correspondence.
The review by Posadas Salas and Srinivas of the clinical utility of once-daily tacrolimus formulations in the management of transplant patients
To date, two Phase III non-inferiority studies have been published evaluating the efficacy of LCP-Tacro in kidney transplant recipients.
Posadas Salas and Srinivas report results of a study that demonstrated rejection episodes requiring anti-lymphocyte therapy were more commonly seen among de novo kidney transplant patients treated with the MR-4 once-daily formulation of tacrolimus compared to twice-daily tacrolimus. They go on to note that about one-third of patients who received MR-4 had trough levels below target during the early post-transplant period (day 3 post-transplant). While the difference in rejection rate did not reach statistical significance, the authors note a trend toward higher mean tacrolimus levels in the twice-daily tacrolimus group who did not experience BPAR compared to those who developed BPAR. These data stand in contrast to data not presented by Posadas Salas and Srinivas, which demonstrate that in de novo kidney transplant patients treated with the LCP-Tacro once-daily formulation, therapeutic tacrolimus concentrations were achieved rapidly – with 66.5% of patients having a serum tacrolimus trough concentration of at least 6 ng/mL 24 hours after their first dose of LCP-Tacro.
Furthermore, pharmacokinetic (PK) differences are noteworthy between the two once-daily tacrolimus formulations. In contrast to data demonstrating the need for potentially higher total daily doses of tacrolimus when the MR-4 formulation is used,
Based on the PK data presented in this response, the greater bioavailability and lower dose requirement are clinically relevant PK differences of the LCP-Tacro formulation compared with MR-4. The authors put forth the message that the PK profiles of once- and twice-daily tacrolimus suggest bioequivalence, yet available data for LCP-Tacro do not support this claim versus twice-daily or the MR-4 once-daily tacrolimus formulations. While no direct Phase III data comparing the two once-daily formulations have been published, Phase I data in healthy volunteers demonstrate that LCP-Tacro was 50% more orally bioavailable compared to the MR-4 once-daily formulation.
Without clarity on the specific once-daily tacrolimus formulation evaluated in each of the trials discussed in the review by Posadas Salas and Srinivas, it is difficult to reconcile potentially conflicting data.
A more complete review of the data regarding once-daily formulations clearly shows important differences between the MR-4 and LCP-Tacro once-daily tacrolimus formulations. These differences may have important clinical implications when selecting a once-daily tacrolimus to manage immunosuppression in transplant recipients.
The author has no conflicts of interest to disclose.
The authors have no conflicts of interest to disclose.
This letter is in response to the article by Williams et al titled “Empathy levels among first year Malaysian medical students: An observational study” published in
This observational study addresses the issue of educating medical students about empathy as an attribute – a matter which is often not given enough importance – and including the topic in the academic curriculum, in view of the declining empathy levels observed in clinical practice. We would like to raise some concerns regarding the methods used for data analysis in the paper. The authors have analyzed the data using descriptive statistics such as means and standard deviation (SD) that are not appropriate parameters since the instrument used a seven-point Likert scoring scale. It would have been better if they had used median with minimum and maximum rather than the mean and SD.
Paired
Our query is also applicable to the comparison of the empathy scores before and after among the males and the females.
The authors have used Cronbach alpha to measure the reliability of Jefferson Scale of Physician Empathy–Student Version (JSPE-S), as mentioned in the Results and Discussion sections (α=0.70, before workshop and α=0.83, after workshop). The Cronbach alpha is used to determine the reliability of the tool during its construction: Why the authors have calculated the same before and after the interaction is not clear.
In Table 2, the confidence interval (CI) and its
The authors declare no conflicts of interest in this communication.
We correspond in response to the article entitled, “Contrast-enhanced MR imaging of atherosclerosis using citrate-coated superparamagnetic iron oxide nanoparticles: calcifying microvesicles as imaging target for plaque characterization” by Wagner et al.
Scientific advances in imaging technology offer many enticing prospects, including detection of early events that accelerate the progression of inflammatory lesions and atherosclerotic plaques,
One of the most interesting results of this study is the identification of “calcifying microvesicles” – a term used for the first time in the literature. Such particles were significantly related to atherosclerosis and calcification.
In the literature, the terms microvesicles and microparticles have been used interchangeably. A forum hosted by the International Society of Thrombosis and Haemostasis, however, provided a consensus definition of plasma microvesicles as vesicles of less than 1 μm in diameter which bear at least half of the surface protein and/or receptors of their cells of origin.
The observations of Wagner et al raise several important questions. First, this study provides only a preliminary account of the presence of calcifying microvesicles. Second, without knowing the lipid composition of the endosomal membrane, one cannot conclude that calcifying microvesicles originate from a specific membrane domain. Is this the case or are they functionally distinct features of endosomes that produce different intraluminal vesicles?
The authors report no conflicts of interest in this communication.
We have read the original research entitled “Altered diastolic function and aortic stiffness in Alzheimer’s disease” by Çalık et al
Firstly, when clinical and demographic characteristics were evaluated, it was stated that the study and control groups included eleven (37%) and 14 (58%) hypertensive (
Secondly, the status of the patients with regard to lipid profile and statin use is also not mentioned in the study. The lipid profile of patients in the study and control groups and status of statin use are important and should be stated in the article, because statins (HMG-CoA reductase inhibitors) provide additional protective/beneficial (pleiotropic) effects on the cardiovascular system in addition to their basic action in reducing low-density lipoprotein cholesterol. Although conflicting reports exist, statins are thought to improve arterial stiffness directly.
Lastly, the association between coronary atherosclerosis and aortic stiffness had already been well identified.
The authors have no conflicts of interest in this communication.
We have read with great interest the paper of de Oliveira et al, in which the authors report the case of a 76-year-old woman who presented with increased abdominal girth and dyspnea for 2 weeks.
All these investigations were used by our colleagues who reported that their case had an absence of leukocytosis, with neutrophilia and an increased value for C-reactive protein. In the current letter, we would like to point out that hematologic parameters associated with pseudomyxoma peritonei are not necessarily the ones described in the current case report as we have experience with a similar tumor with bicytopenia, confirmed by a bone marrow aspiration. As we have no clear reason for the clinical evolution, we can classify the bicytopenia as a paraneoplastic syndrome instead of pseudomyxoma peritonei.
This paper was published under the frame of European Social Fund, Human Resources Development Operational Programme 2007–2013, Project number POSDRU 159/1.5/138776.
The authors report no conflicts of interest in this work.
We have read with great interest the recent paper by Hassan et al
Measuring quality of life is complex due to the lack of consensus about what it really means; but in an academic setting, it is a tool commonly used to allow comparisons and evaluation of interventions.
This study has strengths. First, the authors have used two examiners calibrated for analysis of the dental health component using the index of orthodontic treatment needs, which gives reliability to the results. The second strength of this study is that it is the first study to use the Michigan OHRQoL scale to assess the association between OHRQoL and index of orthodontic treatment needs; also, in the present study, children with no/little need, children with borderline need, and children who need treatment were included. However, despite important results and widespread discussion, this study presents some limitations. The original Michigan OHRQoL scale was designed to investigate the effects of early childhood caries on children’s OHRQoL, and is usually applicable to younger children.
The authors report no conflicts of interest in this communication.
We have read with great interest the recent paper by Szaflarski et al
The treatment indicated for TBI may involve multiple medications and/or surgical procedures. However, none of these treatments has as its purpose to prevent the occurrence of late posttraumatic epilepsy. Antiepileptic drugs currently in clinical use are indicated for the control of seizures that may occur but not to prevent progression to epilepsy. A strong point of the paper refers to future directions in the treatment, including new insights into epileptogenesis. However, despite widespread discussion of treatments in this paper some considerations were not included. An important consideration should be the mechanisms involved in the genesis of posttraumatic epilepsy, including some evidence of a genetic component and posttraumatic epilepsy,
The authors report no conflicts of interest in this communication.
We have read with interest the recently published comparative study regarding the accelerated versus conventional corneal collagen cross-linking (CXL) in the treatment of mild keratoconus. We would like to underline the importance of the CXL treatment time reduction in this time consuming operation.
We already proved, by measuring (with the use of anterior segment optical coherence tomography) the demarcation line depth at 1 month postoperatively after CXL, that the treatment depth was significantly different (significantly shallower in accelerated protocol) between the 10 minute accelerated protocol with ultraviolet-A (UV-A) irradiation intensity of 9 mW/cm2 and the 30 minute standard Dresden protocol with UV-A of 3 mW/cm2.
Consequently, we have proceeded with relevant modifications for the UV-A time settings of the currently proposed accelerated CXL protocol (10 minutes) by increasing the UV-A irradiation time to 14 minutes (40% increase) and we achieved a treatment effect comparable with the Dresden protocol (as indicated by the demarcation line depth).
In the recently published article by Sherif, it is presumed that the accelerated group of patients received CXL treatment according to Bunsen-Roscoe law which should have been 3 minutes of UV-A with irradiation intensity of 30 mW/cm2 corresponding to 5.4 J/cm2 total energy dose delivered to the cornea.
As we previously published, an indirect and non-contact method to measure the effectiveness of CXL treatment is the demarcation line depth provided with anterior segment optical coherence tomography; our results were the same with the reliable but more invasive (due to contact with the treated cornea) confocal microscopy.
In conclusion we believe that accelerated CXL treatment settings in agreement to Bunsen-Roscoe law of reciprocity should be revised thoroughly and new modifications have to be carefully made regarding time and irradiation intensity adjustments keeping the safety aspect of CXL treatment as the first priority.
The authors report no conflicts of interest in this communication.
We note with surprise a recent paper entitled “The effects of an 8-week multicomponent inpatient treatment program on body composition and anaerobic fitness in overweight and obese children and adolescents” by Karner-Rezek et al
The authors of the paper were not the same as those who designed and performed the study. Only two of the authors were directly involved in assessing specific data for body composition (dual-energy X-ray absorptiometry) and anaerobic performance (Wingate test). Moreover, the other authors were never involved with any of the study participants nor were they part of the scientific team at the Alpine Children’s Hospital Davos. Further, the original scientific team was never informed that their study has been submitted for publication. Since the authors were only marginally involved in the study they published, we feel obliged to clarify relevant inaccuracies relating to the methods and their consequence on the data and the clinical conclusions reported in this paper.
The data reported by Karner-Rezek et al
Unfortunately, the selection of patients in the study reported by Karner-Rezek et al
Karner-Rezek et al
Karner-Rezek et al
In conclusion, without clarification of the unresolved issues raised in this letter, the study results reported by Karner-Rezek et al
The authors report no conflicts of interest in this work.
We read with great interest James et al’s article “Prevention and treatment of venous thromboembolism in pregnancy in patients with hereditary antithrombin deficiency.” The authors reported a case series of six women with antithrombin (AT) deficiency treated with plasma derived antithrombin concentrate (pdAT; Thrombate III®, Grifols Therapeutics, Clayton, NC, USA).
Patient 1 is a 31-year-old female with a history of unprovoked pulmonary embolism at age 20. Thrombophilia testing was completed due to her young age and a family history of pulmonary embolism in her brother. The patient was found to be AT deficient with an AT activity of 37%. She was maintained on long term anticoagulation with warfarin. Her other past medical history was significant for supraventricular tachycardia, rheumatoid arthritis, endometriosis, pyelonephritis, Crohn’s disease, and gastroesophageal reflux disease. The patient became pregnant in 2010 and was switched to enoxaparin at 4 weeks gestation. Her pregnancy was complicated by gestational diabetes, which was controlled by diet and exercise. Additionally, she had one admission due to supraventricular tachycardia and was found to have irregular uterine contractions. Due to the AT deficiency, anticoagulation was monitored using anti-Xa levels with goal peak level >0.6 IU/mL. At the highest dose, she required enoxaparin 120 units (~1.4 units/kg) subcutaneously every 8 hours (
Patient 1 was admitted at 35 weeks and 4 days due to concerns for preterm labor. She was continued on enoxaparin until the evening of hospital day (HD) 2. She started rhAT on HD3 with a loading dose of 48.5 IU/kg (calculated from [100-baseline activity]/1.3) and maintenance dose of 11 IU/kg/hour (calculated from [100-baseline activity]/5.4). She underwent induction of labor via cervical ripening catheter and oxytocin when the AT level was in the normal range. Amniotomy was performed on HD5, and the patient vaginally delivered a healthy infant weighing 2,268 grams on HD6. Apgar scores were 7 and 9 at 1 and 5 minutes, respectively. The estimated blood loss was 250 mL. The rhAT infusion did not require dose modifications and the patient’s AT levels remained between 90%–108% (
Patient 2 is a 40-year-old female with a past medical history significant for viral encephalitis and a left leg superficial thrombosis at age 32 while taking oral contraceptive pills. Her oral contraceptive pills were discontinued and she was treated with anti-inflammatory medication. At age 40, patient 2 was 8 weeks pregnant with her third child and sought evaluation for right calf and popliteal pain. She was diagnosed with a non-occlusive deep vein thrombosis of the distal femoral, popliteal, and posterior tibial vein. A hypercoagulable evaluation showed an antithrombin activity of 38%–41%. Her sister, father, and daughter were subsequently diagnosed with antithrombin deficiency but have not experienced thrombosis. Patient 2 was initially treated with enoxaparin at 1.5 mg/kg daily, and was switched to twice daily dosing after the antithrombin deficiency was diagnosed (
Patient 3 is a 27-year-old female with a history of pulmonary embolism 4 days after cesarean delivery of her first child. She was treated with weight-based enoxaparin without monitoring of anti-Xa levels. The patient sought hematology evaluation when she was 8 weeks pregnant with her second child. A hypercoagulable work-up was notable for an antithrombin level of 63%. Repeat testing showed antithrombin activity levels of 64%–73% with normal antigen levels. The patient had no family history of thrombosis. Due to the discrepancy between the amount of clinical heparin resistance and her antithrombin activity, sequencing of the
In contrast to the Phase III trial of rhAT at delivery, the presented three patients did not require dose modifications to maintain therapeutic levels.
We would like to thank Kerry Hansen, RN, for assistance with data abstraction.
Lisa M Baumann Kreuziger and Tracy L Prosen have no interests which might be perceived as posing a conflict or bias. Mark T Reding began serving as an advisory board member for GTC Biotherapeutics after the above cases were managed. Funding for Lisa Baumann Kreuziger’s time to complete this work was supported by a NIH T32 training grant (5T32HL00706).
Daily dose of enoxaparin required to maintain therapeutic anti-Xa levels.
Antithrombin levels during treatment with recombinant human antithrombin concentrate. Normal antithrombin levels between 80%–125% at our institution.
We read with great interest the multicenter, prospective, comparative cohort study by Zhang et al
α1-blockers are the most frequently prescribed medical therapy in the treatment of BPH/LUTS. A number of α1-blockers (alfuzosin, doxazosin, terazosin, tamsulosin, naftopidil, silodosin) have been approved for the treatment of BPH throughout the world; however, they exhibit different selectivity toward α1-adrenoceptor (AR) subtypes. Three types of α1-AR subtypes (α1A, α1B, and α1D) are found in human tissue. The α1A subtype is located in the human prostate, bladder base, bladder neck, prostatic capsule, and prostatic urethra, and mediates contraction of the smooth muscle in these tissues. In addition to α1A-ARs, α1D-ARs are also present to a significant extent in the human prostate, and α1B-ARs are thought to mediate contraction of human arteries.
The early α1-blockers (alfuzosin, doxazosin, terazosin) were nonselective for subtype and were associated with blood pressure-related adverse effects, such as orthostatic hypotension.
With respect to the indications for α1-blockers, doxazosin and terazosin are currently indicated for the treatment of both hypertension and BPH/LUTS, and are more likely to impair safety-relevant physiological blood pressure control in normotensives with LUTS than are tamsulosin and silodosin.
This work was supported by the Zhejiang Provincial Bureau of Health (2012KYA090).
The authors report no conflicts of interest in this work.
We read with great interest the recent study by Ślusarz et al
A strong point of the paper is the instrument used to evaluate the patient expectations with respect to the service provided by the staff in several areas, which helps in identifying the areas of strengths and weaknesses of the quality of care. However, despite the questionnaire covers five areas, it is unspecific to assess the quality of nursing care. We believe that future studies are necessary to evaluate the quality of nursing care especially in the special population with neurosurgical diseases, and to identify the factors that influence the quality of assistance.
Other important points of concern are that the authors do not mention details about sample size calculation and distribution of the sample in different institutions, which could ensure higher degrees of internal and external validity. The authors have presented results from a multicenter medical service-quality project executed by four university medical centers. Hence, information about distribution of the sample could enable comparison of the responses of patients regarding the evaluation of nursing care between different institutions, including number of patients and systematization of care provided by the nursing teams. These are some questions; however, these do not exclude the relevance of the results of this interesting paper.
The authors report no conflicts of interest in this work.
We read with great interest the recent study by Lozano et al
Although some studies have described experimental drugs which may eventually have neuroprotective effects in patients with TBI,
A strong point of the paper
Inflammatory process, as reported by the authors, is a double-edged sword, good toward neuroregeneration and bad toward enhancing brain damage. The comprehension of the mechanisms that rule this subtle switch to one side or the other should be the goal of this group and others working in this challenging domain.
The authors have no conflicts of interest to disclose.
We read with interest the article by Drs Thöne and Ellrichmann entitled “Oral available agents in the treatment of relapsing remitting multiple sclerosis: an overview of merits and culprits” recently published in
In reading the article, however, we did note a number of errors pertaining to teriflunomide, a once-daily oral immunomodulator approved in several countries for the treatment of relapsing forms of MS (RMS) and relapsing-remitting MS (RRMS). The most significant error pertains to a statement made within the safety section, which states: “Serious adverse effects (AEs) included pathological liver function, neutropenia, and trigeminal neuralgia as well as one case of progressive multifocal leukoencephalopathy (PML) in a patient with systemic lupus erythematosus.” We would like to draw the authors’ attention to the fact that this case of PML pertains to the use of the related drug, leflunomide, and not teriflunomide as suggested. It is important to note that leflunomide is licensed to treat active rheumatoid arthritis in adults, and has not been evaluated or approved for the treatment of MS; as such it is inappropriate to extrapolate this observation to the use of teriflunomide. Furthermore, the case of PML cited in the article is complicated by the fact that the patient received prior multiple immunosuppressant therapies before leflunomide (ie, prednisone, azathioprine, chloroquine, danazol, cyclosporin A and methotrexate), which may have contributed to the development of PML.
Based on leflunomide exposure now reaching approximately 2.5 million patient-years (data on file, Genzyme Corporation) and a background incidence of PML estimated at 1 in 100,000 patient-years in the rheumatoid arthritis population,
We also noted a number of other instances within the teriflunomide section of the review where the distinction between leflunomide and teriflunomide becomes blurred. For example, in the final paragraph of the safety section, leflunomide evidence and labelling are cited in relation to use in pregnancy, but (out of date) pregnancy outcomes data are provided from the teriflunomide clinical development program.
We thank the editor for providing us with the opportunity to bring these points to the attention of the authors and the readers of
The author is an employee of Genzyme Corporation. The author reports no other conflicts of interest in this communication.
We read with interest the article by Ruiz-Montero et al, in which the authors used a before-and-after study design to examine changes in body composition (fat mass and lean body mass) related to an aerobic-Pilates program in elderly Serbian women.
The authors report no conflicts of interest in this communication.
We read with interest the article entitled “Anterior capsulotomy improves persistent developmental stuttering with a psychiatric disorder: a case report and literature review” published in
The authors fail to indicate the patient’s maternal language. Given that the team is Chinese, we can assume that it was Mandarin; however, the most widely used variety of Chinese spoken in Sichuan is Sichuanese, which is the lingua franca in Sichuan, Chongqing, and part of Tibet. Either way, for the neuropsychological evaluation scales, it is surprising that the authors refer to articles that have validated these scales in English but not in Chinese. It would have been important for the authors to evaluate the patient’s personality before and after the intervention.
Concerning the surgical technique, it is unfortunate that no computed tomography scan was performed to correct for the inherent deformations associated with magnetic resonance imaging (MRI), especially when it is a high-field coregistered 3T MRI. The authors mention that a “test stimulation generated by the Elekta neurostimulator at both high frequency (130 Hz) and low frequency (5 Hz) was then carried out to verify the target of the electrode”, but they do not indicate the clinical signs that were sought or obtained during stimulation. It is unfortunate that we have no information on the quality of verbal fluency during high and low frequency stimulation. It would have been pertinent for the authors to include the voltage utilized during stimulation. We are not informed whether or not a speech therapist was present perioperatively during this evaluation.
Furthermore, one must question whether a lesional technique was appropriate in this case. The authors’ motivation for selecting a bilateral anterior capsulotomy over medical therapy or deep brain stimulation (DBS) was because “both the patient and his family opted for capsulotomy for financial reasons”. It would be interesting to know the reasons justifying the bias of burdening the patient with the cost of this supposed biomedical research protocol. Also, when the research is unpublished and founded on weak scientific evidence, a prudent approach is warranted so as to offer the technique associated with minimum risk and maximal benefits. In a research context where, like the authors remind us, “the evidence for surgical treatment of persistent developmental stuttering and associated psychiatric disorders is limited”, a DBS technique seems justified given that its effects are, contrary to lesional techniques, both reversible and adjustable. From a scientific standpoint, this technique provides the advantage of completing double-blind evaluations and excludes any potential placebo effect contributing to observed improvements. DBS would also have allowed us to verify with which plot and parameters the therapeutic effect was obtained, so as to better understand the underlying pathophysiological mechanisms. Finally, DBS would have permitted electrical recording for several days before connecting the neurostimulator.
The progress made in functional neurosurgery and the emergence of DBS offer an important hope for cure in patients suffering from medically refractory psychiatric illness. We must remain very vigilant regarding the methodology of these studies so that psychosurgery does not, once again, become a matter of controversy in such a way that it compromises its development.
The authors report no conflicts of interest in this communication.
We read with interest the article entitled “Effects of tafluprost treatment for 3 years in patients with normal-tension glaucoma” by Inoue et al.
The intraocular pressure (IOP)-lowering efficacy of tafluprost in NTG has been recently reported by Mizoguchi et al
Whereas tafluprost is available in Europe in a preservative-free formulation, the three aforementioned studies
Recent literature reported BAK-related ocular surface inflammation and epithelial damage,
As the presence of preservative may play a role in the IOP-lowering effect, we believe it is of interest to report the efficacy of BAK-free formulations in NTG.
In a previous study by our group, BAK-free tafluprost showed a comparable efficacy to other prostaglandin analogs, with a safe profile in patients with primary open-angle glaucoma.
To be included, patients had to have unilateral or bilateral NTG. Diagnosis of NTG was made on the basis of optic-disk abnormalities and visual-field defects judged by a trained glaucoma specialist to be characteristic of glaucoma with IOP <21 mmHg at any time points of the diurnal IOP curve (8 am, 11 am, 2 pm, 5 pm).
All patients were followed in our hospital (San Giuseppe Hospital) from January 2011 to December 2013. Inclusion criteria were: at least 3 months of treatment with BAK-preserved latanoprost followed by at least 3 months of treatment with unpreserved tafluprost; switch to tafluprost due to ocular discomfort; and availability of data from three diurnal IOP curves (untreated, treated with latanoprost, and after switch to tafluprost).
Both latanoprost and tafluprost significantly reduced the diurnal mean IOP compared to baseline (12.7±1.7 mmHg versus 14.9±1.9 mmHg, −15%,
The retrospective nature of this study, the absence of a wash-out period, and the short follow-up are all limitations of this report and we are aware that our results need to be confirmed by powered, prospective, randomized clinical trials. However, to the best of our knowledge, this is the first report on the efficacy of BAK-free tafluprost in NTG.
In our study, the percentage IOP reduction from untreated baseline for both latanoprost (15%) and tafluprost (18%) was similar to that reported in other studies investigating the efficacy of prostaglandin analogs in NTG and ranging from 16%
This study suggests the effectiveness of preservative-free tafluprost in NTG, showing results comparable to those obtained in Japanese patients
The authors report no conflicts of interest in this work.
Intraocular pressure at baseline, with latanoprost, and after switch from latanoprost to tafluprost.
We read with interest the results of the study from Chang et al
Closer examination of the data raises some concerns regarding the study as the results presented are not in agreement with several previous studies using MG-63 cells and curcumin. Chang et al
In 2008 Walters et al
In conclusion, we wish to thank the authors for bringing this apparent lower toxicity of curcumin in healthy osteoblasts to our attention. However, their observations are not in agreement with the literature on MG-63 cells. Clearly further studies are required in this area to establish the optimal physiological concentration at which curcumin may exert cytotoxic effects on osteosarcoma cells without cytotoxic sequelae over healthy osteoblasts. In addition it would be beneficial to compare the effects of curcumin on osteosarcoma cell viability across a number of different cell lines.
The authors report no conflicts of interest in this communication.
We recently read the paper “Evaluation of in vitro glistening formation in hydrophobic acrylic intraocular lenses” by Thomes and Callaghan.
The paper raises a number of concerns and questions. Firstly, the statement in the introduction, “Glistenings are typically observed within a few months of surgery and plateau approximately 1 year after surgical implantation of the [intraocular lens] IOL”
In the Introduction, the authors state that “These results are consistent with most studies regarding glistening formation in AcrySof® IOLs that found no impact on visual function.”
The authors state that continuous improvements “… include the implementation of advanced manufacturing equipment, improved environmental controls, and tightened process controls/specifications. Specific details of these improvements cannot be fully disclosed due to the proprietary nature of the IOL manufacturing process.”
Because contradictory information exists in the literature concerning glistenings it would be helpful, in order to understand the relevance of these measurements, to know when the authors feel that impactful changes were made to the manufacturing process of Acrysof lenses. In a recent paper by van der Mooren et al,
With respect to the Acrysof lenses from 2003, the following statement in the Methods section is not supported or referenced, “Internal studies on the glistening response and storage conditions with AcrySof have shown that glistening formation will not be induced if samples are stored within these temperature and relative humidity ranges.”
Although the authors reference a published method for accelerated microvacuole formation,
It would have been extremely relevant for the authors to include lenses of other materials in the assessment, similar to Tognetto et al,
In summary, the conclusions made by the authors cannot be substantiated as the methods used to draw these conclusions have not been fully validated in the paper. In spite of this, the concern of the authors and their employer, Alcon Research Ltd, for glistening formation is to be lauded and encouraged to continue until the lenses are truly glistening free.
The authors report no conflicts of interest in this communication.
We would like to congratulate Kaercher et al
It is known that contact lenses (CLs) are becoming increasingly popular and a lot of people have begun to use CLs as an optical correction every day. Unfortunately, it was found that up to 50.1% of CL wearers report a sensation of dryness.
The recent report from the Contact Lens Discomfort Workshop reported that tear film changes in CL wearers are responsible for CL discomfort.
The authors
The authors have no conflicts of interest to disclose.
We would like to respond to the paper “Childhood depression: a systematic review”, recently published in
1. In the sentence “However, cases of depressive disorders have increased not only among adults, but also among children”
2. In the sentence “In the short-term, depressive disorders might be a source of psychological suffering for these children, whereas in the long-term they can compromise social, cognitive, and emotional aspects of child development, […] becoming an important predictor of psychopathologies in adulthood”
3. In the results section, our study is listed as an “experimental [study]”
4. In Table 1 of Lima et al
5. In addition, Table 1 (p 1420) states that we found that ‘FRIENDS for Life’ is effective as an indicated school-based prevention program.
6. In the section “Prevention” (p 1422) the authors refer to the program we evaluated, ie, ‘FRIENDS for Life’. Since we did not develop the ‘FRIENDS for Life’ program we believe the authors should refer to the original program description, published in Australia in 2004 by PM Barrett.
We hope these comments are helpful and are looking forward to your reply.
The authors report no conflicts of interest in this communication.
When the screening strategy for iron deficiency makes use of mean corpuscular volume (MCV) to the exclusion of mean corpuscular hemoglobin (MCH), as was the case in the recent study by Radia et al
In David et al’s
In the scenario where hypochromia and/or microcytosis coexist with a normal serum ferritin level, the hematological response to a diagnostic trial of intravenous iron replacement therapy could be decisive in validating or refuting a provisional diagnosis of iron deficiency anemia with all that it implies in terms of future management, as depicted in Figure 3.
The author reports no conflicts of interest in this communication.
Zappacosta et al
In
The Anyplex II HPV28 is based on melting curve analysis using the TOCE™ technology. In contrast to conventional High Resolution Melt (HRM) analysis, the TOCE™ technology enables identification of multiple targets (in this case, HPV types) simultaneously in a single channel by controlling the catcher melting temperature (catcher-Tm). The catcher is an artificial fluorescently labeled template; it is designed in such a way that the tagging portion of “the pitcher”, which previously recognized and hybridized with the target sequence, hybridizes with the catcher. Because each HPV-specific pitcher will hybridize with a catcher of a distinctive length causing a temperature shift during the melting out, different HPV types will be detected in one single channel using one fluorophore. Hereupon, catchers are labeled with different fluorophores (FAM, HEX, Cal Red 610, Quasar 670, Quasar 705) which are detected using different channels allowing multiplexing of 28 different HPV genotypes. Anyplex II HPV28 detects and differentiates 19 high-risk (16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 69, 73, 82) and 9 low-risk HPV genotypes (6, 11, 40, 42, 43, 44, 54, 61, 70). The kit consists of two independent panels: Panel A with 14 high-risks (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68) and Panel B with 5 high-risks (26, 53, 69, 73, 82) and 9 low-risks HPV.
Zappacosta et al described in their article that urine samples were analyzed using Hybrid capture II HPV test (HC2 HPV test).
To prevent sample-to-sample contamination, the microtome blade and working space were cleaned with DNA Away™ (Thermo Fischer Scientific) after sectioning of each FFPE test sample. However, it is unclear whether a negative control was included in the experiments in order to exclude cross-contamination. Validation for cross-contamination could be performed beforehand, but this is not reported in the article. A negative control (a tissue that is shown negative for the tested HPV types) should be cut between the test samples ideally after a positive specimen to verify absence of cross-contamination.
In conclusion, based on the observation that the melting curve of the HPV type detected in the cervical cancer cells does not correspond with the melting curve for HPV53 plasmid DNA, it can be deduced that another HPV type might be involved in the cervical cancer cells. Hence, it cannot be concluded that HPV53 is solely responsible in oncogenesis in this case as previously suggested by Zappacosta et al.
All authors state that they have no conflicts of interest in this work.
Zhang et al
An analysis is good or bad based on how well its results align with the underlying reality of the situation. In a simulation study, we would know this reality. In actual trials, we do not. There is no gold standard. Moreover, there is only one trial being considered. This is most assuredly not the way to compare analysis techniques. It is worth noting, however, that ANCOVA relies on normality, among other assumptions, for its validity. Since the data are never actually normally distributed, the method is never technically valid.
The author reports no conflict of interest in this work.
A recent review by Panagoulias and Doupis, published in
Although these conclusions about saxagliptin are reasonable, in my opinion this review seems to have put too much emphasis on clinical utility and not enough emphasis on the clinical outcomes of cardiovascular safety and efficacy. For the subject with type 2 diabetes, the ultimate goal is to reduce cardiovascular outcomes, and in this, achieving glycemic control is just a surrogate endpoint. Metformin has been shown to improve cardiovascular outcomes in subjects with type 2 diabetes. However, saxagliptin alone, or in combination with metformin has not been shown to improve clinical outcomes.
For metformin, improved cardiovascular outcomes were shown in the UK Prospective Diabetes Study (UKPDS) 34.
Even for metformin, it is not clear whether the findings of UKPDS-34 apply to the dose and preparation of metformin being used in SAXA/MET FDC. Thus, the findings in UKPDS-34, with metformin, are with the conventional rather than the extended release preparation, and are only applicable to the dose used, which is not given. When metformin hydrochloride extended release is used alone, in Australia, the initial dose is 500 mg once daily, which can be increased up to 2 g. When metformin hydrochloride extended release is combined with saxagliptin, the doses available are 500, 850, or 1,000 mg/day. Without testing of these doses of extended release metformin in clinical trial, it is not known whether they are high enough to have the clinical benefits observed in UKPDS-34. Thus, it cannot be assumed that the doses of metformin extended release, which have been combined with saxagliptin, are improving clinical outcomes.
Most trials with saxagliptin have been comparator trials with other anti-diabetes medicines with surrogate endpoints such as HbA1c. Similarly, surrogate endpoints have been the major outcomes for trials on saxagliptin as add-on or combination treatment with metformin. These trials are not discussed as they do not have clinical outcomes.
The only trial to determine cardiovascular endpoints with saxagliptin is the cardiac safety trial: SAVOR-TIMI 53 (Saxagliptin Assessment of Vascular Outcomes Recorded in Patients with Diabetes Mellitus-Thrombolysis in Myocardial Infarction 53 trial). In SAVOR-TIMI 53, saxagliptin 5 mg was compared to placebo, in subjects with type 2 diabetes, 70% of whom were taking metformin.
SAVOR-TIMI 53 has recently been published in the
In conclusion, although clinical utility is important for medicines used in diabetes, it should not be forgotten that improved clinical outcomes are the ultimate goal. These have not been shown for saxagliptin alone, or in combination with metformin. It is also important to firmly establish the cardiovascular safety of saxagliptin, and this has not been achieved to date. In my opinion, these points should have been emphasized in the review by Panagoulias and Doupis.
The author reports no conflicts of interest in this communication.
Mehta et al recently reported removal of dystrophic calcification on the posterior surface of a silicone intraocular lens (IOL) in a patient with asteroid hyalosis.
We have recently tried unsuccessfully to use PPV to treat an 86 year old patient with calcification of a silicone IOL in the presence of asteroid hyalosis. We chose to avoid IOL exchange due to a history of Fuchs endothelial dystrophy and glaucoma in the left eye, and a failed corneal graft in a rubeotic eye on the right. Our patient did not have an intact posterior capsule having been treated with Nd:YAG capsulotomy 2 years previously, before the calcification occurred. A 25 gauge pars plana vitrectomy was performed after attempting to remove the deposits unsuccessfully from the posterior surface of the IOL with Nd:YAG laser. It was felt that PPV would be less likely to cause corneal decompensation than IOL exchange. In contrast to the experience of Mehta et al
Mehta et al reported that initially their patient underwent an unsuccessful Nd:YAG capsulotomy, however it was not made clear whether the posterior capsule remained intact.
A similar case of this condition has been published recently by Ullman and Gupta.
We believe that vitrectomy and surgical capsulectomy should be considered as an alternative to Nd:YAG capsulotomy where IOL calcification is not yet established. It is likely that opening a significantly calcified posterior capsule by Nd:YAG capsulotomy in the presence of asteroid hyalosis will lead to significant future IOL dystrophic calcification. In established calcification of a silicone IOL we feel that vitrectomy is of limited value and IOL exchange should remain the standard treatment.
Some of this content was presented as a poster at the European Society of Cataract and Refractive Surgeons, London September 14th 2014. The authors declare no financial or proprietary conflict of interest in this work.
Smudging of posterior surface of IOL on transillumination postoperatively.
Table 1
Data type: Leaf stability test results
Explanation note: Leaf stability test results from the post-burnin posterior tree distribution from two MrBayes runs that included the full complement of taxa. Taxa are ranked based on their positional stability estimated from the Maximum, which is an average of all the highest percentages from all possible quartet sets for a particular taxon, Difference, which is the difference between the highest and the second highest percentages from all possible quartet sets for a particular taxon, and Entropy, which is calculated as the normalized sum of logs for each quartet percentages (except the unresolved polygamy).
The list of samples used for combined
Data type: species data
Cranial measurements, range, and standard deviation
Data type: species measurements
Explanation note: Cranial measurements, including range and standard deviation (SD), for
NEXUS file containing DNA sequences for
Data type: NEXUS file with DNA data
Explanation note: This is a NEXUS file containing six matrices showing observed sequences for all of the specimens of
Images of maximum likelihood trees
Data type: images of phylogenetic trees
Explanation note: This file shows the maximum likelihood trees for the concatenated, seven-gene matrices as well as each individual gene. Each figure is labeled to indicate the nature of the data analyzed for that tree.
Images of maximum likelihood bootstrap trees
Data type: images of phylogenetic trees
Explanation note: This file shows the maximum likelihood bootstrap trees for the concatenated, 7-gene matrices as well as each individual gene. Each figure is labeled to indicate the nature of the data analyzed for that tree. Numbers on each branch are the frequencies of that clade in the bootstrap replicates, expressed as a percentage.
Alignment of three
Data type: Picture
Explanation note: The
Fifty percent majority-rule consensus tree based on 1000 bootstrap pseudoreplicates generated using the maximum parsimony phylogenetic optimality criterion.
Data type: Picture
Explanation note: Values at nodes indicate the proportion of bootstrap trees (>50%) for which a particular bipartition was recovered.
Data type: Collection localities data.
Explanation note: Collection localities data. Additional material, INBio.
Data type: NJtree (BOLD TaxonID Tree).
Fig. S2
Data type: Adobe PDF file
Table S1
Data type: Microsoft Word document
Explanation note: Species included in the present study.
Table S2
Data type: Microsoft Word document
Explanation note: Primers used in the present study to amplify partial sequences of F-reticulon 4, 16S rRNA, cytochrome oxidase subunit I (COI) and cytochrome B (CytB).
Table S3
Data type: Microsoft Word document
Explanation note: DEC models tested to estimate distribution ranges inherited by the descending lineages at each node of the tree. The differences between the models are in the rate of dispersal among adjacent and no adjacent areas. * Represent the model used in the analysis.
Key to the species of
Data type: Lucid SDD file: Key to the species of
Explanation note: This is a XML-based file structured using the internationally agreed SDD (Structure of Descriptive Data) Schema. This SDD file may be used to exchange the Lucid key with other SDD-compliant applications.
Data type: (DNA sequences)
Explanation note: This 16S rRNA data includes 46 individual sequences of the examined
The combined dataset of COI and 16S rRNA of
Data type: (DNA sequences)
Explanation note: There is the concatenated sequences of COI and 16S rRNA genes correspondence with each sample.
Specimen locality map
Data type: occurence
Explanation note: Specimens examined that either included GPS coordinates or were georeferenced are included and sorted by species group. Waypoint records include species locality data, type status, and museum code.
Electronic appendix 2: PDF literature references file
Data type: References list.
Explanation note: A PDF file with additional references to first published records is also provided. Cross-references to Hackman’s 1980 checklist are included. This file includes an index to genus- and species-level names.
Locality information for all collections of the three species described in this paper.
Data type: Occurences.
Explanation note: Database containing catalog numbers, numbers of specimens, and localities for the three new species of
All sequence and specimen information
Data type: specimens data
Explanation note: Excel file with complete information of the specimens with DNA barcodes.
Supplemental Video 3
Data type: MPEG video file
Explanation note: Two males of Liancalus similis showing the typical looping flight of
Habitus of
Data type: Image file.
Explanation note: This image correspond to Fig.
Habitus of
Data type: Image file.
Explanation note: This image correspond to Fig.
Habitus of
Data type: Image file.
Explanation note: This image correspond to Fig.
Habitus of
Data type: Image file.
Explanation note: This image correspond to Fig.
Habitus of
Data type: Image file.
Explanation note: This image correspond to Fig.
Male genital apparatus of
Data type: Image file.
Explanation note: This image correspond to Fig.
Male genital apparatus of
Data type: Image file.
Explanation note: This image correspond to Fig.
Bibliographic sources for the morphology matrix
Data type: References list.
Bibliographic sources for the morphology matrix of the species of
List of positive and negative character dependencies in each identification key
Data type: multimedia
Key to genera
Data type: multimedia
Explanation note: A set of multi-entry identification keys to African frugivorous flies (
Key to
Data type: multimedia
Explanation note: A set of multi-entry identification keys to African frugivorous flies (
Key to
Data type: multimedia
Explanation note: A set of multi-entry identification keys to African frugivorous flies (
Key to
Data type: multimedia
Explanation note: A set of multi-entry identification keys to African frugivorous flies (
Key to
Data type: multimedia
Explanation note: A set of multi-entry identification keys to African frugivorous flies (
Key to
Data type: multimedia
Explanation note: A set of multi-entry identification keys to African frugivorous flies (
Key to
Data type: multimedia
Explanation note: A set of multi-entry identification keys to African frugivorous flies (
Key to
Data type: multimedia
Explanation note: A set of multi-entry identification keys to African frugivorous flies (
Maximum likelihood model parameters
Data type: Adobe PDF file
Explanation note: Model parameters used in the maximum likelihood approach to phylogenetic reconstruction.
Nucleotide substitutions
Data type: Comma-separated-value file of measurements
Explanation note: Substitutions of nucleotides (transitions/transversions) for 28S rDNA (below the diagonal) and COI sequence data (above the diagonal).
Patterns of COI sequence variation
Data type: Adobe PDF file
Explanation note: Patterns of COI sequence variation. Graphs and discussion of patterns of nucleotide substitions in the COI data-set.
Recordings examined
Data type: tape recording
Explanation note: A list of tape recordings examined for this study.
Overview of barcoded specimens.
Data type: sample list.
Results of the dissections of 123 flowers from 82 specimens of
Data type: measurements
Multi-entry, matrix-based identification key to the Afrotropical genera of
Data type: multi-entry, matrix-based key in SDD format
Explanation note: Multi-entry, matrix-based identification key to the Afrotropical genera of
Multi-entry, matrix-based identification key to the Afrotropical subfamilies of
Data type: multi-entry, matrix-based key in SDD format
Explanation note: Multi-entry, matrix-based identification key to the Afrotropical genera of
Multi-entry, matrix-based identification key to the Afrotropical genera of
Data type: multi-entry, matrix-based key in SDD format
Explanation note: Multi-entry, matrix-based identification key to the Afrotropical genera of
Multi-entry, matrix-based identification key to the Afrotropical genera of
Data type: multi-entry, matrix-based key in SDD format
Explanation note: Multi-entry, matrix-based identification key to the Afrotropical genera of
Multi-entry, matrix-based identification key to the Afrotropical genera of
Data type: multi-entry, matrix-based key in SDD format
Explanation note: Multi-entry, matrix-based identification key to the Afrotropical genera of
Multi-entry, matrix-based identification key to the Afrotropical genera of
Data type: multi-entry, matrix-based key in SDD format
Explanation note: Multi-entry, matrix-based identification key to the Afrotropical genera of
Multi-entry, matrix-based identification key to the Afrotropical genera of
Data type: multi-entry, matrix-based key in SDD format
Explanation note: Multi-entry, matrix-based identification key to the Afrotropical genera of
Multi-entry, matrix-based identification key to the Afrotropical genera of
Data type: multi-entry, matrix-based key in SDD format
Explanation note: Multi-entry, matrix-based identification key to the Afrotropical genera of
Multi-entry, matrix-based identification key to the Afrotropical genera of
Data type: multi-entry, matrix-based key in SDD format
Explanation note: Multi-entry, matrix-based identification key to the Afrotropical genera of
Total species richness estimations for 68 taxonomic groups in French Guiana
Data type: Spreadsheet (xls file)
Explanation note: Estimated and extrapolated species richness for 68 taxonomic groups in French Guiana and comparative figures for two well-known faunas. These taxa are used to extrapolate the total number of species in French Guiana given in
Video demontration
Data type: MOV file.
Taxon-Character Matrix with characters ordered (TNT file).
Data type: Matrix.
Characters in common on the most parsimonious trees diagnosing the nodes on the strict consensus tree in Figure
Data type: Characters.
Explanation note: Characters in common on the most parsimonious trees diagnosing the selected nodes on the strict consensus tree resulting from the analysis run with characters ordered.
Temporal ranges of Cretaceous metatherian taxa used to calculate taxonomic richness of
Data type: Range.
Explanation note: Temporal ranges of Cretaceous metatherian taxa used to calculate taxonomic richness of
Data used to calculate taxonomic richness for Metatherian shown in Figure
Data type: Range.
Explanation note: Data used to calculate taxonomic richness for
Consensus Bayesian tree of the subgenus
Data type: image
Explanation note: Consensus Bayesian tree of the subgenus
Organised by the Aspirin Foundation
On 23 November 2010, the Aspirin Foundation held a conference at the Royal Society of Medicine in London to consider the latest evidence for the role of aspirin in cancer prevention. The conference heard from leading specialists in the fields of epidemiology, genetics and gastroenterology, who discussed the implications of recent studies and considered how the role of aspirin might develop in the near future.
The conference was chaired by Professor Peter Elwood, Honorary Professor of Epidemiology at the University of Cardiff, who published the first clinical trial evidence of aspirin’s preventative effects against cardiovascular disease [
Recent evidence linking aspirin use with a reduced risk of several cancers had brought medicine to ‘the brink of a breakthrough of enormous importance’, Professor Elwood added, and could change the balance of risk and benefit in favour of wider use of aspirin as prophylaxis. He suggested that aspirin use was now a question of personal responsibility for health. Evidence of the risks and benefits of taking aspirin should be presented to the public in a package of measures to preserve health, so that individuals could make an informed choice about managing their health for themselves.
Dr Morgan’s review of the evidence of aspirin and the risk of cancers other than colorectal cancer predated the recent analysis of cancer mortality in randomised trials of aspirin [
The first hint that aspirin use may reduce the risk of cancer was published in 1988: an Australian observational study found a significantly lower risk of colorectal cancer among men and women who reported aspirin use [
More recently, studies involving a wider range of cancers have been summarised in two reviews [
Twelve case–control and six cohort studies involving ∼30,000 cases suggest that aspirin is associated with a 25% lower risk of breast cancer. The Women’s Health Study, with a total of almost 40,000 participants, failed to show that aspirin 100 mg on alternate days reduced the risk of developing breast cancer [
In stomach cancer, three case–control studies (1,600 cases) suggest a possible risk reduction of 20–40% with aspirin; four cohort studies (1,400 cases) suggest a smaller benefit but with less certainty. For pancreatic cancer, one case–control study (200 cases) found no benefit whereas five cohort studies involving ∼5,000 cases reported a small risk reduction. In ovarian cancer, six case–control studies (3,000 cases) suggest that aspirin may reduce risk but the size of this effect is unclear; one cohort study (500 cases) found no evidence of a benefit.
Approximately 30 case–control studies including 25,000 cases provide no clear evidence that aspirin is associated with a lower risk of cancers of the prostate, bladder or kidney. By contrast, 10 cohort studies involving about 15,000 men with prostate cancer suggest a small reduction in the risk of prostate cancer. Five cohort studies (4,000 cases) have identified a small increased risk of kidney cancer associated with aspirin use. Six observational studies involving 2,000 cases of lymphoma found no change in risk associated with aspirin use.
These findings raise the question of how to interpret observational evidence of aspirin and the risk of various cancers in light of strong evidence that it reduces the risk of colorectal and lung cancer. Does aspirin block pathways involved in cancer development? If it does, what level of evidence is necessary to establish its role? In 2006, a citizen’s jury considered the available evidence and called for a health education campaign about the benefits and risks of aspirin in preventing cancer and cardiovascular disease [
Aspirin might also be helpful in other ways in the adjuvant treatment of cancer. In cancer patients with acute coronary syndrome and thrombocytopenia, aspirin users had a 90% survival rate after one week compared with 6% in nonaspirin users [
The aspirin metabolite salicylate induces programmed cell death in cancer cells. Aspirin may therefore be useful in combination with chemotherapy [
Finally, as the population ages and the effectiveness of cancer treatments improves, it is likely that more people in the future will be living with cancer. Prophylaxis with aspirin may be useful in this group, given that the risk of vascular events also increases with age.
Observational data show that aspirin or non-steroidal anti-inflammatory drug use is associated with a reduced risk of colorectal cancer. Analysis of 19 case–control studies involving a total of 20,815 cases and 56,369 age- and sex-matched controls shows that the risk is 20% lower for any use of these medicines [odds ratio (OR) 0.80, 95% CI 0.73–0.87) (
Several randomised trials have shown that aspirin [
In the UK, two large randomised trials have evaluated the effects of aspirin in preventing vascular events of aspirin have been carried out in the UK. The UK-TIA trial compared aspirin 1,200 or 300 mg/day with placebo in 2,449 men [
A median of 23 years’ follow-up is available for both studies. Using UK cancer registration and death certificates for case ascertainment, analysis of patient data from these studies showed a significant reduction in the risk of colorectal cancer among aspirin users (hazard ratio 0.65, 95% CI 0.49–0.87) but no significant risk reduction of other cancers. The analyses by intention to treat and excluding non-compliant patients are illustrated in
Long-term follow-up data (>20 years) are now available from three other large randomised trials of aspirin: the Thrombosis Prevention Trial (TPT;
Analysis of patient-level data from these trials and the two UK trials shows that aspirin reduced the risk of death from colorectal cancer by approximately one-third overall (OR 0.66, 95% CI 0.51–0.84) and by over 40% among participants who took low-dose aspirin for at least 2.5 years (OR 0.57, 95% CI 0.40–0.81). The effect of higher dose aspirin (500–1,200 mg/day) was of borderline statistical significance (OR 0.72, 95% CI 0.50–1.03).
Pooled analysis of the SALT, UK-TIA and TPT trials showed that low-dose aspirin reduced the incidence and mortality of colorectal cancer, with a more marked effect seen in participants treated for at least 5 years (
These data suggest that taking aspirin at a dose of 75 mg/day reduces the incidence and mortality due to sporadic colorectal cancer by up to 50% after a latent period of about 7–10 years. This is followed by a gradual catch-up of rates as the effects of aspirin diminish. Confining the analysis to patients who could have taken aspirin for at least 5 years suggests a larger effect. Latency will depend on the point of action of aspirin, which might differ between clinical situations, and the effect size will depend on the accuracy of screening procedures. Follow-up of other randomised trials is required to determine the effects of lower and less frequent doses.
Any use of aspirin or NSAID in cases of colorectal cancer versus age and sex matched controls: 19 case–control studies.
Maximum use of aspirin or NSAID in cases of colorectal cancer versus age- and sex-matched controls: 19 case–control studies.
Colorectal cancer in the pooled analysis. UK-TIA cases with at least 5 years scheduled trial treatment.
Delayed effect of aspirin on risk of colorectal cancer in the UK-TIA Trial and British Doctors Aspirin Trial in compliant patients with scheduled treatment ≥5 years.
Pooled analysis of the effect of low-dose (75–300 mg) aspirin (thick line) versus control (thin line) on subsequent incidence and mortality due to colorectal cancer in TPT, SALT and UK-TIA.
Pooled analysis of the effect of low-dose (75–300 mg) aspirin (thick line) versus control (thin line) on subsequent incidence of proximal colorectal cancer in BDAT, TPT, SALT and UK-TIA.
Pooled analysis of the effect of low-dose (75–300 mg) aspirin (thick line) versus control (thin line) on subsequent incidence of distal colon and rectal cancer in BDAT, TPT, SALT and UK-TIA.
People with familial adenomatous polyposis have APC mutations, the precursor of sporadic colon cancer, and are at high risk of developing colon cancer. They are a valuable group of patients for chemoprevention trials because they are a homogeneous population with a high incidence of disease, and they are highly motivated to participate and are already under surveillance due to their high incidence of disease. As most colorectal cancers are initiated by loss of APC function, these patients with an inherited defect are an ideal model system for sporadic cancers.
The first trial carried out by the Colorectal Adenoma/carcinoma Prevention Programme (CAPP1) evaluated the effect of aspirin and/or resistant starch to prevent polyp formation in several European centres [
133 patients completed at least one year’s treatment. There was no apparent effect on adenoma numbers but this endpoint was difficult to measure in a multi-centre trial and due to the large numbers of polyps that develop in these patients [
Patients with Lynch syndrome, caused by an hMSH2 mutation that reduces DNA repair, are also prone to develop colon cancer (and endometrial cancer) but who tend to be older and have a lower incidence of polyps than is the case with familial adenomatous polyposis. Patients undergo colonoscopy every 1–2 years and therefore form a valuable group in which to study the effects of chemoprevention. Lynch syndrome accounts for approximately 3% of all cases of colorectal cancer and is also a good model people with microsatellite unstable cancers with chromosome stability, who represent 12% of all colorectal cancers.
The CAPP2 trial repeated the CAPP1 design in 1,071 people with Lynch syndrome recruited from 41 centres worldwide. Treatment was administered as 600 mg enteric-coated aspirin and/or 30g of a proprietary resistant starch (Novelose) or placebo cornstarch. After follow-up of up to 4 years and a mean treatment duration of 29 months, there was no difference in the incidence of adenoma or cancer of the colon, irrespective of treatment arm [
Colorectal tumours in patients with Lynch syndrome are less likely to have increased expression of COX-2, raising the question of how aspirin is reducing the risk of cancer. Aspirin appears to have an indirect effect on cancer cells with a latency of several years before the effect becomes apparent. One possibility is that aspirin promotes apoptosis in aberrant stem cells in colon crypts, which is the mechanism by which salicylates protect plants in the presence of infection. Another hypothesis is that aspirin renders crypt stem cells more susceptible to the immune system via inhibition of interleukin 4. The latent period for most people appears to be about 10 years but for individuals with Lynch syndrome, where disease progression is much faster, the latent period may be as short as 4 years.
A third CAPP study is now being planned. This will be a dose-finding study of enteric-coated aspirin 100 versus 600 mg/day for 5 years in 2,500 MMR gene carriers, using web-based recruitment. Follow-up will be at least 10 years. DNA will banked to allow identification of modifiers of response.
CAPP1 trial: patients randomised to aspirin tended to have smaller ‘biggest polyps’ versus placebo, with a trend to significance in those treated for at least one year.
Post trial divergence of colorectal cancer incidence in the CAPP2 trial, aspirin versus placebo (
Lifetable analysis, time to first ‘Lynch Syndrome cancers’ (colorectal and endometrial) in the CAPP2 trial. Hazard ratio 0.62 (95% CI 0.41–0.96),
Understanding of how aspirin reduces the risk of cancer is incomplete. Possible mechanisms include inhibition of nuclear factor-κB, induction of p38 kinase and catabolism of polyamines but most research has focused on the inhibition of COX-2 and this has the strongest evidence base. COX-2 is over-expressed by some cancers, including colorectal cancer (
This hypothesis was tested by analysis of molecular markers in two large cohort studies providing long-term data on aspirin use: the Nurses’ Health Study (
Tumour specimens were collected from both cohorts from the late 1990s, representing 662 cases (58%) in the Nurses’ Health Study and 648 (76%) in the Health Professionals Follow-Up Study. Analysis was limited to 368 and 268 cases respectively for which samples of sufficient tumour tissue and adjacent normal tissue allowed comparison using positive and negative controls.
Overall, these two studies showed that regular aspirin use was associated with a lower risk of colorectal cancer (relative risk, RR, 0.73; 95% CI 0.62–0.86). However, this benefit was largely confined to individuals whose tumour stained positive for COX-2 (RR 0.64, 95% CI 0.52–0.78) and not those with COX-2 negative tumours (RR 0.96, 95% CI 0.73–1.26) [
Patients diagnosed with colorectal cancer are a high-risk group for whom aspirin therapy may be useful. In these studies, 1,279 participants (mean age 65) were diagnosed with stage I, II or III colorectal cancer and underwent curative resection. After a median follow-up of 11.8 years there were 480 total deaths and 222 deaths due to colorectal cancer. Aspirin use before diagnosis was not associated with colorectal cancer-specific survival (HR 1.05, 95% CI 0.80–1.37) or overall survival (HR 0.93, 95% CI 0.77–1.11) [
In these cohorts with ∼20 years of follow-up, 11 deaths occurred due to gastrointestinal bleeding. The relative risk of major gastrointestinal bleeding (requiring a blood transfusion or hospitalisation for bleeding) was lower than has been reported in randomised trials of aspirin for secondary prevention of cardiovascular events (
Expression of COX-2 by normal gastrointestinal cells, adenoma and colon cancer (adapted from Eberhart et al [
Immunostaining for COX-2 (dark brown areas) in colorectal tumours. Samples A and B are negative for COX-2; samples C and D are positive for COX-2.
Aspirin use after diagnosis and colorectal cancer-related and overall survival [
Dose of aspirin and risk of adenoma [
Dose of aspirin and risk of colorectal cancer in women [
Dose of aspirin and risk of colorectal cancer in men.
Relative risk of major gastrointestinal bleeding and dose of aspirin in the Nurses’ Health Study and Health Professionals Follow-up Study cohorts.
Plant sources of salicylates have been used for medicinal applications for thousands of years. The Egyptians used extracts of myrtle or willow leaves for joint pain and Hippocrates recommended chewing willow leaves to provide analgesia during childbirth. Edward Stone is credited with identifying salicylin as the active component of willow bark in the 18th century; willow bark is still used as a herbal medicine today. The concentration of salicylin is very high in willow, where it is presumed to act as an antifeedant [
Classically, salicylates have been associated with regulating defence against pathogens but they are now emerging as multifunctional hormones protecting plants against stress. They are involved in signalling and the plant’s response to stressors such as disease, cold and warming and in senescence. In disease, aalicylic acid plays a dual role: it facilitates the lethal effects of pathogens that kill the plant host in order to obtain nutrients (necrotrophs) but inhibits and confines the effects of pathogens that rely on plant survival (biotrophs) by promoting programmed cell death (PCD) as a means of confining the spread of disease. In plants PCD, is triggered by recognition of the pathogen which initiates a kinase-dependent signalling cascade leading to the generation of oxygen free radicals and nitric oxide. The synthesis of salicylic acid is increased within 4–5 hours of infection which promoting defence gene expression and PCD [
Additionally, a plant can evolve methyl salicylate into its environment and confer inter-plant resistance. Methyl salicylate is produced in response to an attack by predators such as greenfly; this attracts insects such as hover flies and ladybirds which feed on greenfly.
The generation of transgenic plants encoding the bacterial gene – salicylate hydroxylase (NahG) – which reduces salicylate levels has provided great insights have shown the importance of salicylate to other forms stress, for example, tolerance to heat and chilling is compromised in these plants. Salicylic acid increases production of heat shock proteins, which act as chaperone proteins to protect unfolded proteins from heat [
At a molecular level, NahG plants have indicated the salicylate regulates a plethora of plant genes but other features have direct relevance for salicylate action in humans. Specifically, salicylic acid (at microgram levels) acts to potentiate the activity of NADPH oxidase, which increase oxygen free radical generation and perturbs mitochondrial function [
Salicylate levels are higher in plants that are exposed to stresses such as infection and temperature change than those cultivated in protected environments like greenhouses. This suggests that much of the food we grow may have low levels of salicylates. It is conceivable that humans benefitted from the protective effects of plant-derived salicylates when their diet comprised wild plants. Conversely, it is likely that a modern diet relying mass-produced plants lacks salicylate and, possibly, is less beneficial. However, salicylates also have industrial applications: they may be added artificially to improve a plant’s resistance to the effects of chilling and some fungicides act partly by increasing plant salicylate levels.
Survival in patients with colorectal cancer has been increasing over the last 50 years (
Data from a faecal occult blood screening programme demonstrates the earlier detection of colorectal cancer (
Increased screening has resulted in a huge shift in the stage of disease at diagnosis and in outcome: 2-yearly faecal occult blood screening reduces colorectal cancer mortality by 15% and increases the proportion of cancers diagnosed at an early stage [
We now have the opportunity to prevent colorectal cancer. The recent UK Flexible Sigmoidoscopy trial demonstrated the benefits of detection and removal of adenomas [
Screening can define four groups according to their risk of developing colorectal cancer. Individuals with colorectal cancer and high-risk adenomas are at greatest risk. It is important to reduce the risk of recurrent tumours in these groups, for which aspirin has a role to play. Those with low-risk adenomas or no adenomas are at least risk, though within these groups are an unknown number of individuals at high risk but who cannot be detected. It may be possible to identify these individuals by genetic screening. Genome-wide association studies have shown that the risk of colorectal cancer increases with the number of known risk alleles present [
This evidence makes a strong case for introducing chemoprevention of adenoma and colorectal cancer in high-risk individuals. There are now four randomised trials of aspirin as chemoprevention, involving a total of almost 5,000 patients; all have demonstrated a reduction in adenoma of 20–35%. This is overwhelming evidence that aspirin will prevent the formation of adenomas. The fact that aspirin reduces the risk of colorectal cancer with a latency of 10 years [
It is feasible to conduct a trial to identify genetic and environmental interactions for colorectal risk assessment and prevention. A suitable design would be a cross-sectional cohort study involving 20,000 people embedded within the screening programme. This could define the effect of known and novel genomic factors associated with increased colorectal neoplasia risk through their influence on adenoma occurrence. Within this, a nested randomised controlled trial of 6,000 people could determine the combined impact of aspirin and nutritional supplements in reducing adenoma recurrence. A biosample collection programme would facilitate genomic studies.
An algorithm for this design is illustrated in
United Kingdom 1950–2003: males and females colorectal cancer mortality at ages 35–69.
Study design for evaluating the efficacy of aspirin plus adjunctive therapy in adenoma prevention and targeting therapy.
Faecal occult blood screening increases detection of early stage (Dukes’ A) colorectal cancer [
| A | 51% | 37% | 11% | 16% |
| B | 20% | 36% | 35% | 31% |
| C | 24% | 18% | 22% | 29% |
| D | 5% | 6% | 29% | 24% |
| Total (no.) | 83 | 132 | 400 | 249 |
It has been almost 30 years since a reduction in adenoma formation after administration of the non-steroidal anti-inflammatory drug (NSAID) sulindac was first reported [
Observational studies also show that aspirin and NSAIDs are associated with reduced mortality in patients with colorectal adenoma [
There is increasing evidence from case–control studies, though less strongly from cohort studies, that use of aspirin or NSAIDs over about 20 years is associated with a reduced risk of oesophageal cancer (
The traditional approach to phase I, II and III trials for evaluating new treatments is expensive (trials cost $10–100 million each) and may not be appropriate for evaluating preventative therapies. Instead, phase I (discovery) could rely on cohort studies and the identification of biomarkers and precursor lesions. Case–control studies are less reliable than cohort studies due to their retrospective nature and they should be carried out in carefully selected groups of patients.
Phase II studies for early validation should be improved. It is important that these studies are randomised because of the biases inherent in cohort studies. Their cost can be minimised by using biomarkers and precursor lesions as endpoints, and recruiting patients with cancer. The value of this approach is illustrated by studies of the prevention of contralateral breast tumours in women taking tamoxifen after diagnosis of breast cancer, which demonstrated a similar effect 15–20 years before it was shown in randomised trials. In the case of aromatase inhibitors, the effects on contralateral tumours suggest a 75% reduction in recurrence risk. If this strategy is shown to provide a reliable estimate of the outcomes of randomised trials, it may become the standard in the future.
Phase III studies (late definitive validation) take 10–20 years to complete and cost hundreds of millions of dollars. They are unlikely to be funded for inexpensive drugs such as aspirin, so any that are carried out must be carefully designed and use cancer mortality or incidence as an endpoint.
NSAIDs – oesophageal cancer [
NSAIDs – stomach cancer [
NSAIDs – lung cancer [
NSAIDs – breast cancer [
NSAIDs – ovarian cancer [
On November 23rd 2011, the Aspirin Foundation held a meeting at the Royal Society of Medicine in London to review current thinking on the potential role of aspirin in preventing cardiovascular disease and reducing the risk of cancer in older people. The meeting was supported by Bayer Pharma AG and Novacyl.
Organised by the Aspirin Foundation
Professor Peter Elwood, Honorary Professor in Primary Care and Director of Primary Care and Public Health, University of Cardiff, was one of the investigators in the first trial of aspirin for the prevention of cardiovascular events [
Society has a poor record of adopting risk reduction strategies. For example, US studies show that the five healthy behaviours (exercise, healthy diet, maintain low body weight, no smoking, modest alcohol use) can reduce heart disease by 80–85 per cent but adherence to these lifestyle measures (in the context of the trials) was only 3–4 per cent [
People do not adopt preventive measures which bring great benefit to the population because they offer little to each individual [
The question now facing society is, Who has responsibility for ensuring the appropriate use of aspirin? The treatment of disease has been delegated to health professionals, Professor Elwood said. His belief is that individuals should be given valid evidence on the balance of risks and benefits, and decide for themselves whether to take aspirin.
In 2009, an international consensus statement on the role of aspirin and NSAIDs for cancer prevention concluded that there was clear evidence of a chemopreventive effect on colorectal cancer and probably other cancer types but there were insufficient data on the risk-benefit profile; as a result, no definitive recommendations were made-(Professor Jack Cuzick, Professor of Epidemiology at Cancer Research UK) [
And lead author of that paper, said that much new evidence has since been published; the consensus statement is being updated and will be published in 2012.
Professor Cuzick described recent evidence from prospective trials that aspirin reduced cancer mortality as very important. This analysis, published in 2010, involved approximately 25,000 people and identified about 600 deaths [
The first evidence that NSAIDs might protect against colorectal cancer was a 1983 case series in which sulindac reduced the incidence of colorectal polyps in individuals with Gardner’s syndrome [
Observational studies have also found that that regular aspirin use was associated with 10–30 per cent reductions in the risk of cancers of the oesophagus, stomach and breast, and probably a lesser effect on the risk of cancers of the lung and prostate. The Nurse’s Health Study further showed that current aspirin use is associated with a 40–60 per cent lower risk of breast cancer recurrence [
Professor Cuzick noted that several questions about aspirin remain unanswered. Which is the appropriate dose – 75 mg/day or 300 mg/day? Prospective studies [
Delayed reduction in the risk of colorectal cancer in the CAPP2 trial [
Europacolon (
Mr Spencer said the public is aware that interesting claims are being made that aspirin may reduce cancer risk and prolong survival. Where will they get their information about it? GPs seem more concerned by the possible risks of aspirin than its potential benefits and they are not particularly well informed about recent evidence. Media coverage is transitory and usually adopts a sensational perspective angle, emphasising headlines at the expense of detail. The Internet, of course, is an easy way to do research but it is full of anecdotal comment. It has an excess of information, much of which is contradictory, not easily understandable and possibly even frightening. It is easy to come away with the impression that taking aspirin is dangerous, Mr Spencer noted.
The public generally do not know enough about the risks and benefits of aspirin, or they do not understand the issues, he continued. Relevant studies should be undertaken as soon as possible to determine the effective but safe dose. If the evidence continues to support the current balance of risk and benefit, the public should be educated about a personal health strategy. Information should be targeted at key groups of people, patients who might benefit from aspirin and health professionals. It is essential not to go over people’s heads. This information should be precise and categorical about the benefits and risks such as adverse effects.
Is aspirin a low-cost lifesaver, Mr Spencer asked? If it is, it will have substantial impact on health at a low cost compared with current spending on cancer prevention and treatment, and its associated social and economic costs. It is clearly a long-term strategy, he said, and not without risk. europacolon, as the voice of the patient, wants to make a start now on collating the evidence and presenting it to the public and health professionals on a massive scale. If this does not happen, confusion and uncertainty will persist.
Ms Sarah Lyness, Executive Director of Policy and Information, Cancer Research UK, described a survey in which members of the public were asked their views about taking a daily tablet to reduce cancer risk. They were open to the idea, she said, and were incredulous when they were told the tablet concerned was aspirin. They asked why these benefits were only becoming known now and they wanted to see evidence to substantiate the claimed benefit. People were more comfortable if aspirin use was supported by a strong endorser such as the Department of Health, Bayer, NICE and particularly Cancer Research UK. Most respondents were familiar with the adverse effects of aspirin and were concerned about them. Many said they would ask their doctor for information rather than a community pharmacist. GPs, on the other hand, are unfamiliar with the evidence for reducing cancer risk and want endorsement from NICE before supporting aspirin use.
Ms Sarah Porch, Director of Services at Bowel Cancer UK, agreed that people want the reassurance of an authoritative endorsement. She said that Bowel Cancer UK is aware of the confusion about aspirin from monitoring social media – the public didn’t understand the CAPP2 study, for example. There should be a single clear message from all organisations and it should be emphasised that a daily aspirin is not a substitute for a healthy lifestyle.
Professor Marcus Longley, Director, Welsh Institute for Health and Social Care, and Professor of Applied Health Policy, University of Glamorgan, pointed out that a healthy lifestyle carries no risk whereas aspirin can rarely cause stroke. Professor Cuzick said that the problems associated with aspirin occur early but its benefits come late. The public’s attention span is short and the message will have to be continually reinforced to encourage long term adherence.
Ms Porch said the public are used to making judgements about risk and benefit but they need clear information about the level of risk with aspirin. Professor Jane Armitage, Professor of Clinical Trials and Epidemiology at Oxford’s Clinical Trials Surveillance Unit, said the evidence is overwhelming for aspirin as secondary prevention of cardiovascular events but there is a lack of consensus about its role in cancer prevention. Professor Cuzick replied that there is a great deal of evidence but it needs to be synthesised and debated.
Tablets are the dose form everyone prefers but swallowing them is, for many patients, an unrecognised problem, said Professor Alan Perkins, Professor of Radiological and Imaging Sciences, University of Nottingham Queen’s Medical Centre.
It is known that children and some patient groups (eg, those with stroke, heart failure or diabetes, and frail elderly people) have difficulty swallowing tablets but a US study found that 40 per cent of adults had difficulty swallowing tablets and the problem was more common among women and men [
The way a dose form is taken has a strong influence on its transit time from the mouth to the stomach – shorter when sitting upright and if the dose is swallowed with water – and may influence its therapeutic effect. For example, the rate of onset of effect of aspirin is more rapid if a tablet is chewed than if it is swallowed in solution or as a whole tablet [
Tablet shape and coating are also important. Transit time is shorter for oblong tablets than round ones, and for film-coated tablets compared with uncoated tablets [
Imaging studies have shown that a dose unit can remain lodged in the oesophagus for 60 minutes after ingestion but the individual may be unaware of this (
Professor Perkins described a study using γ scintigraphy to quantify the oesophageal transit times of a film-coated tablet and gelatin capsule formulation of the bisphosphonate etidronate in 25 healthy volunteers. This technique is superior to a barium swallow, which is unphysiological, and to MRI because this poses practical difficulties in positioning the patient. Using a radio-labelled tablet, he showed that the transit time from the mouth to the stomach was significantly longer for the capsule (mean 24 seconds) than the tablet (mean 3.3 seconds [
The formulation can be manipulated to target tablet disintegration and drug release at specific sites in the gastrointestinal tract to reduce the risk of gastrointestinal bleeding – for example by enteric coating or pH-controlled release. γ scintigraphy can be used to provide visual confirmation of the site at which the drug is released and this can be correlated with blood levels.
Time to 50% inhibition of thromboxane synthesis after ingestion of aspirin by chewing a tablet, swallowing a solution or swallowing a whole tablet [
γ scintigraphy showing gelatin capsule lodged in the lower oesophagus for 60 minutes after ingestion.
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Farblose Kristalle FP 118–120 °C (wässr. Ethanol). MS (
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Probiotic microorganisms have been shown to provide specific health benefits when consumed as food supplements or as food components. The main problem of such products is the poor survival of the probiotic bacteria in the low pH of gastric fluid. However the use of synthetic excipients for enteric coating to prevent the exposure of microorganisms to gastric fluid is limited in food supplementary industry. Therefore the aim of this study was to develop an enteric coating formulation containing shellac as a natural polymer. Shellac possesses good resistance to gastric juice; the major disadvantage of this polymer is its low solubility in the intestinal fluid [
The trials showed that an increasing amount of plasticizer results in a decrease of Tm and ΔH of the films whereat glycerol had a superior plasticization effect to GTA. The compatibility of films made of water-soluble polymers and shellac was also concentration dependent. HPMC and PVP showed superior compatibility with shellac compared to sodium alginate, since films containing shellac and more than 10% [w/w] sodium alginate tended to separate into two phases. In the end five formulations containing shellac and either 5% [w/w] glycerol, 10% [w/w] PVP, 20% [w/w] PVP, 10% [w/w] HPMC, or 5% [w/w] sodium alginate emerged as feasible for enteric coating purposes.
It was the purpose of this study to develop an oral oligonucleotide delivery system based on thiolated polymer/ reduced glutathione (GSH) system providing protective effect towards nucleases, permeation enhancement and controlled drug release. Polycarbophil-cysteine conjugate (PCP-Cys) was synthesized by the covalent attachment of cysteine to polycarbophil via amide bond formation. Incubation of 30-mer phosphorothioate oligonucleotide with DNase I and freshly collected intestinal fluid were performed in order to evaluate the protective effect of the polymer. Based on PCP-Cys conjugate together with GSH, permeation studies were performed on Caco-2 monolayer cell culture and on freshly excised intestinal rat mucosa in Ussing chambers. Additional drug release studies of tablets containing PCP–Cys, reduced GSH, and the oligonucleotide were performed in 100 mM phosphate buffer pH 6.8.
PCP-Cys displayed 223±13.8 μmol thiol groups per gram polymer. After 4 h the unprotected ODNs, which were incubated with DNase I and intestinal fluid, were significantly degraded by 61 % and 80 %, respectively. In contrast, less than 41 % (in DNase I) and 60 % (in intestinal fluid) of the ODNs were degraded in the presence of 0.02 % (m/v) of PCP-Cys. Permeation studies demonstrated due to the addition of PCP-Cys/GSH an 8-fold and 10-fold increase in the apparent permeability coefficient (
This work was supported by a scholarship from the Austrian Federal Ministry for Education, Science, and Culture to RM.
Mupirocin-loaded microparticles (MP) were designed to control drug release at the skin surface assuring that drug remains localized at the application site and does not unnecessarily enter into the systemic circulation [
The goal of this research was to design controlled release MP with acrylic polymer using spray-drying technique and assess influence of feed composition (in terms of native drug/polymer physical form and solvent used) and preselected drug loadings (1:5 and 2:1 (w/w) drug:polymer proportion) on MP performance under the same processing conditions. Physicochemical properties of MP were evaluated using thermal (MDSC, TGA), spectroscopic (FT-IR) and X-ray analyses and correlated with encapsulation efficacy and
Spray-drying of feed dispersion has formed partially coated crystalline MP with reduced encapsulation efficacy, irregular morphology and poor ability to control drug release irrespective of drug loading. Conversely, solid dispersions prepared from spray-drying feed solution have shown that drug/polymer miscibility, morphology and
Acrylic-based solid dispersions were confirmed as suitable microcarriers for controlled drug release using simple and scaleable spray-drying technique.
Monoglycerides, Phytantriol and a few other lipophilic molecules self-assemble in bulk in presence of water to form well defined liquid crystalline phases. Their structure can be tuned by temperature variation and/or by addition of oils. This leads to gel-like or fluid systems with a large internal interface between water and oil domains with different bulk viscosities. These nanostructured phases can be dispersed in the excess water phase by addition of an external stabilizer and energy input leading to internally self-assembled particles, so-called ISAsomes [
The hierarchical structure can be extended to a next level by gellifying the continuous aqueous phase by the addition of polymers like κ–Carrageenan or Methylcellulose. This leads to a new type of hydrogel, loaded with ISAsomes [
Finally, we can use the oil-continuous nanostructured bulk phase to create concentrated, stable water in oil emulsions having a paste-like consistency and water content from 50% up to 90% by volume. No additional stabilizer is needed to create these systems. They have a great potential as delivery systems for functional molecules in very different fields like pharmaceutical and cosmetic applications, as well as in food science and agro-chemistry.
These authors equally contributed to this work.
Lactose intolerance is the inability to metabolize lactose because of the absence of the enzyme lactase. It is estimated that 75–90% of birth lactase levels are lost by most people after weaning. The prevalence of lactase deficiency ranges widely with the ethnic background from 2–15% among Northern Europeans to 95–100% among Asians [
Therefore, the present work is aimed to develop an innovative long-acting peroral formulation for the treatment of lactose intolerance.
Biodegradable and biocompatibale polymeric microcarriers (2.78±1.05μm in diameter) were manufactured from poly(D,L-lactide-coglycolide) (PLGA) using spray-drying. They were functionalized with β-galactosidase from
The highest particle-bound enzyme activity (1470 U ß-galactosidase per gram PLGA) was obtained with hexamethylene diamine as a spacer using carbodiimide method representing a 6-fold increase as compared to particles without spacer. Surface immobilisation of WGA enhanced considerably the particle binding to porcine mucin layer (mucoadhesion) and Caco-2 cell monolayers (cytoadhesion).
PLGA-microparticles, surface-modified with active ß-galactosidase as enzyme substitute and WGA as a targeter, are able to bind to enterocytes and thereby to prolong the intestinal residence time. It is a promising approach towards a promising approach towards a more convenient therapy of lactose deficiency and intolerance.
Skin is main target for UV-oxidative stress and their antioxidant defenses can be quickly overcome. The consequences are reflected at the molecular and cellular level. Here, most important organelle included in survival pathway are mitochondria. Recently significant activation of mitochondria-mediated signaling cascades by resveratrol (RSV) has been discovered [
RSV is a naturally occurring polyphenolic phytoalexin, which has many beneficial biological effects but on the other hand possess some limitations. Few of these are: poor water solubility, high metabolic rate and frequent dosage–dependent effect in the cellular environment [
The protective effects of RSV in solution or loaded into SLN (SLN-RSV) on radiated keratinocytes were studied comparing with the effects on non-radiated cells.
Cosequences of damaged effect of UV-radiation was slightly changed cell morphology, as some fragments of actin fibers appeared and mitochondria activity was weakened and they were dispersed over whole cytoplasm. However, only SLN with incorporated RSV at 100 μM preserved normal cell morphology and improved mitochondria activity, while the other samples (RSV at 10 or 100 μM and also SLN-RSV at 10 μM) showed any significant difference regarding radiated control cells. These effects are ascribed to the transfer of RSV by nanoparticles intracellularly – integrating among mitochondria, where RSV can best realize its “anti-stress” potential.
To conclude, the results clearly revealed the hypothesis that loading of RSV into SLN significantly improve bioavailability and diminish UV-related damage of keratinocytes, and thus providing better photoprotection of cells compared to the application of RSV in solution.
Cationic SLN formulations were developed and optimized in terms of cationic lipid/surfactant ratio and production parameters. 5wt% of Compritol 888 ATO (COM, Gattefosse) or Imwitor 900(IMW,BASF) were used, 0.4–1.0wt% of cetyltrimethylammonium bromide (CTAB, Sigma) and 0.1–1.0 wt% of Lutrol F68 (BASF) were tested. Formulations were produced in triplicate. SLN were prepared by modified microemulsion method [
Optimized formulation consisted of 5.0% solid lipid, 0.5% CTAB and 0.25% Lutrol F68, yielding particles with z-ave=159nm, PdI=0.34 and ZP=54.8 mV (IMW) and z-ave=180nm, PdI=0.272 and ZP=56.2 mV (COM). These parameters remained unchanged during 8 days of storage at 8°C. Samples dried without cryoprotectants yielded light powdery product which could be redispersed easily; freeze-dried samples with saccharides tested yielded solid products.
Cationic SLN formulations intended for gene delivery were designed and optimized. Particles with z-ave below 200nm and low PdI were produced. These SLN showed good stability of 15 days .Suitability of cationic SLN for freeze-drying without cryoprotectants was confirmed [
The authors whish to thank Dr. Fernando Nunes for cooperation in freeze-drying experiments. This work was supported by grant SFRH/BD/60552/2009 from FCT and by Nadacia Slovenskeho Plynarenskeho Priemyslu.
Pharmaceutical secondary manufacturing has long stood in stark contrast to the drug discovery end of the business, as well as to other sectors, when it comes to innovation such as continuous manufacturing. Is that about to change? The traditional business model is breaking down with consequent pressures on all parts of the pharma value chain. Manufacturing’s contribution to improving yield, reducing time, cost and waste is increasingly critical. Regulation which previously had insisted on batch testing is now moving to be much more supportive of real time product release and process analysis, heralding a future where the validation and establishment of continuous manufacturing will be easier.
Integration of each process stage is crucial for continuous manufacturing. However, pharma companies tend to work with ‘islands of automation’ where every unit of operation is more or less an independent from an integration point of view.
Another key barrier is that these changes require companies to work in a more multi-disciplinary way, crossing system worlds. For the first time, if you are in analytics you have to speak to people in process control and so on. Multi-disciplinary teams are an absolute must but not everyone is ready for that. The relatively slow uptake of process analytical technology (PAT), crucial for continuous manufacturing, following the FDA’s 2004 PAT initiative, highlights the challenge facing companies.
Mindset changes and internal culture changes will be key to the successful introduction of PAT as a first step towards continuous manufacturing. The companies that are first to overcome the barriers will not just reap the reward of increased competitiveness. Because of the stage of its introduction, they have the opportunity to implement the technology to a higher level than in industries where it is already established. From being a laggard, secondary manufacturing has the potential to become a trendsetter.
Granular flows are of paramount importance in industry and nature. To understand, e.g., the effect of various process parameters on the final product performance is critical. In industry, the flow in agitated mixing equipment basically consists of granular flow over a blade. While previous experimental and numerical work [
We performed excessive high-speed video imaging of dry and wet granular flow under a controlled atmosphere in a 3D mixer as well as in a novel 2D flow setup. We use particle image velocimetry (PIV) to measure the velocity of the granular bed from the top (3D system) as well as in a cross section of the blade. Discrete-Element-Method (DEM) simulations are used to help interpreting our experimental results. The ultimate goal was to quantify the mixing in bladed mixers (e. g., high-shear granulators).
We show results for the instantaneous, time-averaged as well as fluctuating velocity fields for the flow over a single blade. Our results indicate that the bed height in front of the blade has a significant effect on the flow pattern. Also, the flow situation changes when multiple blades are used and the blade-to-blade distance is changed. Finally, we quantify the mixing efficiency of a single-blade passage using image analysis.
Porous particles are present in various technical applications as well as in the field of medical treatment. The latter involves amongst others the production of highly porous and respirable particles to serve as solid inhalants and substitute intestinal medication. One promising way of manufacturing such biotechnological therapeutic agents is the lyophilisation of single drops or sprays – a complex technological approach that still requires research and process layout. Besides experiments, numerical flow simulations are capable of providing insight into the physics that influence the transport and mass and heat transfer related to porous particles. Predicting the corresponding multiphase flows requires knowledge of the behaviour of porous particles in comparison to solid ones. By performing fully-resolved direct numerical simulations (DNS) of single complex particles correlations between particle properties and their fluid dynamic behaviour are to be established.
In this work, the Lattice-Boltzmann method (LBM) is used to simulate the flow around virtual lyophilisates and to calculate drag, lift and torque coefficients [
This work was supported by Deutsche Forschungsgemeinschaft (DFG grant SO 204/36-1, SPP 1423).
Flow around a heterogeneous particle with shrinking second phase
Numerical methods such as Finite Element Analysis (FEA) or Computational Fluid Dynamics (CFD) are considered to be well established technologies in industrial design and engineering. While the methodology of numerical simulation is fully recognized in conventional industry (e.g. automotive, aviation, construction, machinery) even (especially) for critical application, pharma-industry and biotechnology have just started to carefully consider numerical simulation methods as valueable tool to create a sound understanding of their processes. The high level of regulation in this specific industry as well as the strong drive for evidence based validation (verfication) may have contributed to the fact that numerical simulation methods are hardly applied in pharma industry and biotech.
The paper in hand shall demonstrate on typical applications how the CFD approach can be applied to design and optimize processes, to create additional knowledge about specific process interactions, to provide better understanding about correlations between process parameters and product quality, to supply a sound data base for process validation and thus to increase process flexibility. In four different references various aspects of the CFD methodology are discussed. The operational performance of a fermenter is presented with specific focus on gas distribution (oxygen) depending on mixer and vessel design and on shear stress exposure of the cell culture. Also, a stirred and heated storage vessel for thermal sensitive emulsions is investigated. Especially the temperature distribution within the products related to the temperature of the heating jacket and stirrer parameters shall be analysed. A third application considers a very specific freeze technology for protein-buffer-solutions. The freeze and thaw process is analysed by CFD under specific consideration of temperature induced concentration shifts in the protein-buffer. Finally a standard vessel-agitator set-up is discussed and strategies for process optimizations are presented based on CFD. Based on this specific problem it shall be demonstrated how CFD can support process-upscaling from lab to industrial size.
This contribution describes the dynamic optimization of a pharmaceuticals batch dryer. The optimal control problem is based on the control vector discretization or single shooting method, thus a non-linear programming (NLP) problem is obtained. The target of the open loop optimization problem is the batch time minimization and the control variables are the On/Off intervals of the mixer and the mixer rotation speed. In order to implement a relevant scenario from practical point of view the following constraints are applied: the on and off intervals have same duration, the stirrer speed is constant during the entire batch drying process and the final moisture specification must be met. Finally, an upper limit on the fines fraction in the particle size distribution is set. The dryer model [
In the pharmaceutical industry spray coating is a frequently used method to apply a film layer on the surface of tablets or pellets. The function of coating ranges from the controlled release of active pharmaceutical ingredients (APIs) to taste masking and coloring. A widely used technique is drum coating, where tablets are placed in a rotating drum and the coating liquid is sprayed onto the moving tablet bed surface. One of the most central quality attributes is the uniformity of coating, both inter-tablet and intra-tablet. Besides experimental work [
Coater performance depends on a range of different parameters, which could be essential for the quality of the final product. In order to quantify the impact of different CPPs (critical process parameters) on the related CQAs (critical quality attributes), the combination of advanced numerical methods on different scales is necessary. In this work we present an innovative approach to analyze, understand and optimize important steps of the pharmaceutical tablet coating process by means of numerical simulations. The final outcome is the application of the developed methods to industry-scale processes.
Concerning our numerical multi-scale approach, the Discrete Element Method (DEM) is used for the simulation of the tablet bed mixing. Furthermore, a detailed numerical analysis of the interaction between spray droplets and tablet surface is performed with multiphase Computational Fluid Dynamics (CFD) methods [
Summarizing, the presented combination of simulation methods helps to gain a deeper understanding of the spray coating process, providing a further step away from trial-and-error towards Quality-by-Design approach.
The impact of liquid drops on wetted and dry surfaces is a complex two-phase flow phenomenon with high technical relevance especially in surface coating. It is also of increasing interest in the development of novel drug dosage forms in pharmaceutical engineering. The complex physics after the impact, which is still not understood in full detail, has been investigated in numerous experimental and analytical studies. Based on dimensional analysis and/or simplifying assumptions various semi-empirical correlations and analytical solutions have been derived to describe the individual stages of the motion of the liquid (see, e. g. [
Evolution of the liquid phase (denoted by red area) after drop impact on a wetted surface (We=203, Oh=0.005).
Magnetoliposomes (MLs) are phospholipid vesicles encapsulating magnetic nanoparticles utilised as contrast agents for targeted molecular Magnetic Resonance Imaging (MRI). There are different methods of preparation and characterization of liposomes containing ultra small paramagnetic iron oxide ions (USPIOs) i. e. lipid film hydration method [
Ultrasound active liposomes currently have gained a lot of interest as therapeutic agents for targeted drug delivery. A well known problem is their fast clearance from the circulation and thus the short time frame for drug release. The use of ultrasound as external trigger for drug release is attractive because it can be easily focused and only few adverse effects on the healthy tissue can be observed.
To prolong the blood circulation kinetics the liposome surface can be modified with poly(ethylene glycol) (PEG) derivatives to prepare so called ‘Stealth’ liposomes[
A new ultrasound contrast agent composed of dipalmitoylphosphatidylcholine or distearoylphosphatidylcholine and varying concentrations of polyethyleneglycol-40stearate was prepared using the thin-film hydration method. It was demonstrated that concentrations of 1 mol% and 10mol% polyethyleneglycol-40stearate show very good ultrasound activity. This result was unexpected due to the small size of only 100–200nm.
The aim of this study was to determine the physico-chemical properties and the structural behavior of these contrast agents. Therefore 31P-NMR measurements, AFM investigations and TEM images were correlated. A combination of these methods should enable us to distinguish between organisations such as micelles, SLN and liposomes.
The authors would like to thank the DFG “Forschergruppe Nanohale 695” for support.
Specially engineered drug particles can solve solubility and formulation problems, which are major challenges for the pharmaceutical industry. Particle size reduction of poorly water soluble drugs results in increased dissolution rate and higher bioavailability or better processability as demonstrated in previous studies [
This work was supported by TÁMOP-Hungary research project: Development of teranostics in cardiovascular, metabolics, and inflammatory diseases (TÁMOP-4.2.2-08/1-2008-0013).
The availability and use of appropriate Process Analytical Technology (PAT) is fundamental to achieving the transformed style of pharmaceutical manufacture that is envisaged by the regulators. Implementing the knowledge-driven, risk-based approach to development and manufacture described in ICH Q8, Q9 and Q10 Guides, and adopting more efficient, modern operating practices, depends on being able to reliably identify, analyze and control those parameters that dictate pharmaceutical performance. This paper examines the potential contribution of laser diffraction particle sizing technology within this context, focusing on the use of industrially proven on-line solutions for real-time measurement and automated process control.
Particle size has a controlling effect on the behaviour of many pharmaceutical products, often influencing critical to quality parameters such as dissolution rate, bioavailability and product stability. Where this is the case, the robust and proven technique of particle sizing by laser diffraction has for many become the method of choice. Non-destructive, with measurement rates sufficiently rapid for real-time monitoring, laser diffraction transitions easily, from the laboratory through to on-line measurement within the pilot plant, or in a manufacturing environment. With on-line analysis in place, the design, optimization and control of unit operations such as granulation, spray drying and, perhaps most importantly, milling becomes simpler and much more effective. Case studies illustrate this, the most compelling describing the use of on-line laser diffraction technology to fully automate control of a mill such that the particle size of the exiting active is maintained within specification despite variations in feed quality.
The aim of Process Analytical Technology (PAT) is to gain deeper insight in pharmaceutical manufacturing processes, replacing empirical approaches by knowledge-based procedures. A detailed understanding of the key parameters of a process and their impact on the product quality allows companies to “build in quality” instead of “testing it into the final product”. From this point of view, PAT has to be tailored for a direct implementation to manufacturing lines to measure in real-time the key parameters of the process.
Here, we present results on real-time quantitative in-line monitoring of powder mixing processes by near-infrared (NIR) spectroscopy [
Beside the batch processes monitored here with NIR spectroscopy, we also introduce hyper spectral imaging for monitoring fast continuous processes and non-invasive product quality analysis of solid dosage forms.
Near-infrared spectroscopy (NIRS) is a rapid and nondestructive analytical method. The use of near-infrared spectroscopy in the pharmaceutical industry has been rapidly increasing over the past decade, with particular interest in the analysis of solid dosage forms, such as tablets and capsules [
According to the obtained results, it was concluded that both models ensured a high coefficient of correlation as well as low level of error of the calibration model and prediction. It can be concluded that NIR spectroscopy, a rapid and nondestructive technique, is a powerful tool that can be easily implemented, with appropriate technical solutions, in the production process, thus ensuring its on-line or in-line monitoring.
The use of nano-sized materials (NMs) offers exciting new options in technical and medical applications. Adverse effects on cells and organs, however, have also been reported. It is known that NMs may interfere with conventional assays. It may be suspected that inter-cell line differences in the sensitivity to NMs also exist. NMs may also interact physicochemically with plasma membranes. Cells in suspension, where a relatively large area is exposed, may therefore react different from adherent cells. As NMs by interaction with the replicative machinery may inhibit proliferation, also the proliferation rate (length of the doubling time) may be an important parameter.
To assess the effect of the medium 20–1000 nm large carboxyl polystyrene particles (CPS) were tested in the presence of different amounts of fetal bovine serum. Potential interference was evaluated by testing in the cytotoxicity screening assays WST-1, MTT, MTS, Neutral Red, Sulforhodamin B, leucine incorporation and ATP content. IC50 concentrations were compared between 20 different cell lines.
The size of small (20–60 nm) CPS increased markedly from medium with 0% to 10% FBS; for larger CPS only minimal differences were seen. 20 nm CPS in medium with 0% FBS acted cytotoxic in all cell lines. 20 nm CPS in 5% and 10% FBS and larger CPS in all media were only minimally cytotoxic to nonphagocytic cells. In phagocytic cells also CPS of ≥ 500 nm acted cytotoxic. Half maximal inhibition concentrations (IC50) in a given cell line did not differ markedly between the screening assays but varied more than 10 times between the cell lines. IC50 values were significantly lower in suspension cells. Large cells and human cell lines with long doubling times were more resistant to the cytotoxic action of 20 nm CPS.
In addition to medium composition, growth characteristics, proliferation and cell size of the cell line used for testing may influence the IC50 values of NMs. Cells in suspension are especially sensitive to the cytotoxic action of small carboxyl polystyrene nanoparticles.
This work was supported by the FP6 European integrated project “NanoBioPharmaceutics”, NMP4-CT-2006-026723 and by the RPC project ‘Nano-structured Materials for Drug Targeting, Release and Imaging’.
Praziquantel (PZQ) is a drug active against all species of
This work was supported by CAPES.
Many current dermatological preparations, sunscreens and other cosmetics, contain nanosized particles (NPs), which were observed to be rapidly internalized by keratinocytes [
The oral cavity acts as a complex barrier and displays the first main hindrance against uncontrolled uptake of a variety of substances. The oral mucosa represents 60% of the total surface area within the oral cavity, offers a good opportunity for drugs to be absorbed and shows a 4 to 4.000 times greater permeability than the skin.The barrier function of this tissue is mainly guaranteed by i) the saliva, ii) the mucus layer, iii) the cell junctions in the epithelium and iv) the membrane coating granules. However, as the permeability of drugs through the oral mucosa is limited, new delivery carriers have to be developed. Nanostructured materials (NMs) are small enough to overcome this tissue. Within the development of such nano-carriers, the efficacy as well as the safety are important factors that cannot be neglected. Currently, no standardized physiological
NMs (polystyrene particles (PS), silver particles Ag)) were primarily characterized in terms of size and surface charge in physiological media. The penetration of the particles was investigated through excised buccal mucosa from pig. The
Independent from the material small (20–35 nm) NMs showed a much higher penetration than larger (150–200 nm) NMs.
Studies on nasal epithelial models are important to develop vehicles for systemic nasal drug delivery, and also for targeting drugs to brain via the nasal route. RPMI 2650 human nasal septum carcinoma cell line was used in our experiments as an
For toxicity tests cells were cultured in 96-well plates, and MTT dye conversion and lactate dehydrogenase release were determined after treatments. For permeability tests RPMI 2650 cells were cultured on collagen-coated Millipore CM inserts (hydrophilic PTFE membranes, pore size: 0.4 μm, surface 4.2 cm2) in 6-well plates. Fluorescein was selected as a marker of paracellular permeability, and TEER and Papp were measured.
RPMI 2650 cells passaged at high cell density grew as confluent multilayers on inserts. To induce barrier properties several treatments and culture conditions were tested. The effects of serum, hydrocortisone, cAMP and air-liquid interface on RPMI 2650 cell layers were examined. Hydrocortisone and cAMP increased the tightness of the nasal epithelial barrier, and induced the expression and junctional localization of claudin-1, claudin-4, ZO-1, and β-cathenin visualized by immunohistochemistry and confocal microscopy. The changes in cell and junctional morphology were confirmed by electron microscopy. The resistance of the monolayers reached 240 ± 13 Ωcm2, and Papp of 2.7 ± 0.2 10−6 for fluorescein indicating a barrier typical for nasal epithelium. The model was used to test the non-toxic nasal doses of absorption enhancers Tween 80, Cremophor RH40, Transcutol P, and water soluble sucrose esters and to determine their effects on paracellular permeability. Tween 80 and Cremophor RH40 decreased TEER by 50 % and significantly increased Papp values of RPMI 2650 layers.
We have successfully established a human
This work was supported by TÁMOP-Hungary research project: Development of teranostics in cardiovascular, metabolic, and inflammatory diseases (TÁMOP-4.2.2-08/1-2008-0013).
Nanotechnologies and Nanomedicine are promising fields of scientific research. Experts expect a huge economic and social impact from Nanotechnologies and Nanomedicine in the next decades. New properties associated with size smaller than 100 nm opens a new world of applications. At the same time, possible toxicological aspects associated with nanotechnologies are being discussed in the scientific community. Nanotoxicity, possibly resulting from altered chemical and physical behaviour of nanoparticles compared to bulk material, is a major concern nowadays. These phenomena are believed to be related to the extreme high specific surface area and altered electrochemical properties of nanoparticles. The investigation of nanomaterials in terms of their toxicological behaviour is an extremely multidisciplinary challenge. Toxicology of non-nano chemicals is a well established procedure. Unfortunately, Nanotoxicology is quite more complicated and standard protocols in Toxicology can not be used. Nanomaterials have to be investigated in detail for different parameters like size, size distribution, shape, charge, etc. Furthermore, interaction with different matrices like blood, interstitial fluid, buffers, etc. influences significantly nanomaterials like, agglomeration, size distribution, charge, etc. In consequence, nanotoxicology needs new standardised protocols which are able to deal with the extreme complexity of nanomaterials and their toxicological behaviour!
To build a national contact point for nanotoxicology, the BioNanoNet as a network company, has initiated the build up of the European Center of Nanotoxicology (
Powder mixing is crucial for many processing stages within the pharmaceutical, catalysis, food, cement, and mineral industries, to name a few. A significant problem hindering process design is the paucity of information about the effects of changing process parameters on mixing efficiency, especially in the case of continuous mixing.
The main target of this talk is to highlight continuous mixing and to examin the effects of different process and design parameters. Interestingly, continuous processing has been utilized extensively by petrochemical, food, and chemical manufacturing but has yet to reach the pharmaceutical industry to a meaningful extent. Recent research efforts indicate that a well-controlled continuous mixing process illustrates the capability of scale-up and ability to integrate on-line control ultimately enhancing productivity.
In this talk we will decribe the use of engineering methodologies to design continuous pharmaceutical manufacturing systems for solid dose products. We will examine the performance of gravimetric feeders and continuous mixers, and their integrated dynamics, and develop guidelies for the optimal design and operation of the integrated system. Variance components will be characterized, and the monitoring of performance using PAT tools will be discussed.
Xanthan (XAN) is a well known negatively charged biopolymer that adopts different conformations in media, which are still poorly understood. XAN tablets in contact with water hydrate, forming a gel layer that regulates the drug release rate [
To determine XAN molecular conformations, its solution in water at pH 7.0 or 1.2 was deposited on freshly cleaved mica, dried and images were taken in air using tapping mode
Our results reveal that XAN adopts single chain conformation in water at pH 7.0, whereas at 1.2 double stranded structures are formed. This was confirmed by AFM images as well as by calculated parameters: persistence length of XAN in water was 210.37 compared to 116.83 in pH 1.2. Young modulus of XAN film in water was lower (1.52 GPa) compared to pH 1.2 (1.94 GPa). Higher rigidity of XAN films at pH 1.2 was further confirmed by formation of XAN gels, where elastic G’ and loss G” moduli were higher in the whole deformation range. Double stranded molecules form more cohesive gels, while single stranded are more flexible, due to the higher hydration of negatively charged polymer. MRI results proved much higher hydration and consequently the swelling of XAN tablets at pH 7.0 than at 1.2 and the thicker gel layer as well. Surprisingly, drug release in pH 1.2 media was faster regardless to firmer gel structure.
High rigidity and low hydration of XAN matrix in pH 1.2 are the consequence of XAN molecular structure regulating drug diffusion in tablets.
Dry powder coating is a relatively new technique to coat substrates without the use of any organic solvent or water. The film forming polymer is applied as a powder to the cores to be coated leading to reduced process times [
Dry powder coating was performed in a rotary fluid bed (GPCG1.1, Glatt, Binzen, Germany). Polymer powder and liquid were fed simultaneously via a three-way nozzle to the fluid bed. Coating efficiency was calculated by dividing the mass of adherent coating material by the mass of applied coating material. Theophylline pellets were used as cores and ethylcellulose, hydroxypropylmethylcellulose acetate succinate (HPMCAS) or Eudragit RS® as coating polymer. Various liquids were characterized with respect to several properties, like viscosity, spreading behavior and droplet size. Coated pellets were investigated via dissolution testing and scanning electron microscopy before and after curing steps to evaluate whether film formation was achieved.
The spreading behavior of the liquids seems to be one key factor affecting the coating efficiency. It can be investigated by measuring contact angles of liquids on the polymer or predicted by calculating surface energies. However, there are still other parameters impacting the coating efficiency of the process like viscosity of the liquid, which influences on the one hand the kinetic of spreading as well as the droplet size generated by the spraying nozzle. The film forming properties of the polymers used were very different. Using ethylcellulose a curing step was required to obtain dense films whereas Eudragit RS® formed films without any curing.
Coated pellets offer several advantages related to safety and effectiveness of the medicinal product such as reproducibility of gastric emptying and of absorption, and predictable plasma levels with lower probability of dose dumping due to modified release [
The objective of this study was to investigate the effects of formulation and manufacturing parameters on the pellet quality after the layering and coating process applying 3 types of inert core materials (sugar, microcrystalline cellulose, and isomalt). An additional objective was to investigate and compare the effect of the core material on the in vitro drug release of water soluble and poorly water-soluble drugs when they were coated with a permeable polymer membrane.
The drug layering and coating process were followed by non-destructive tests such as image analysis and NIR spectroscopy. Flowability, hardness, friability, wettability and morphology of starting cores as well as of finished products were investigated.
According to the non-destructive analysis and physical tests, all pellets manufactured from different core materials demonstrated satisfactory quality attributes. Since the type of inert core material may significantly influence the dissolution profile, the solubility characteristics of both the drug and core material should be considered.
There is very often a great gap between the performance of oral drug delivery systems in vitro and in vivo. During the last decade modern high resolution and/or real time imaging techniques like Magnetic Marker Monitoring (MMM) [
Complexes of β-cylodextrin with five nucleotides of adenine (A), thymine (T) guanine (G), cytosine (C), and 5-methylcytosine have been investigated using Hatree-Fock (HF) and density functional theory (DFT) calculations of different quality.
This work was inspired by recent technological advances made in single-molecule sequencing of DNA. One molecule of single-stranded DNA can be sequenced by using nanopores made of α-hemolysin as the sequencing device. An exonuclease enzyme attached to the top of the pore cleaves bases from the DNA strand so they can traverse the pore one at a time. The different nucleotides then bind to cyclodextrin attached to the inside of the pore [
We calculated low energy conformations of complexes of β-cyclodextrin with the five different nucleotides mentioned above, using different methods (HF, DFT). The interaction energy with β-cyclodextrin was estimated from the energy difference between a complex and the molecules it consists of. The presented results show, that it is possible to discriminate all five nucleotides very clearly. Because one can easily distinguish between cytosine and 5-methyl-cytosine, this method can be used to sequence methylated DNA directly in one step which might be an advantage compared to other similar sequencing methods.
Cationic lipids spontaneously bind, condense and coat DNA resulting in the formation of lipid/DNA complexes, so-called lipoplexes [
This project aims to analyse nuclear transport of lipoplex-released pDNA as a potential transfection barrier in two cellular models, A10 and MDCK, which have been characterized regarding their endocytic profile and transfectability in previous work [
These studies reveal nuclear entry of the pDNA to represent a significant transfection barrier in MDCK cells. The investigated cellular models differ significantly in the amount of nuclear-accumulated complex DNA: nuclear transport is by far more efficient in A10 compared to MDCK cells.
Androgens and estrogens increase the number of cell division and the opportunity for random genetic errors and are thus involved in carcinogenesis of hormone related cancers. Pre-receptor regulatory enzymes interconvert the active forms of hormones with high affinities to corresponding receptors to their less active forms. They represent interesting targets for development of new drugs for prevention and treatment of conditions caused by disturbed hormone action. We have focused our attention to four hydroxysteroid dehydrogenases (HSDs). AKR1C1 converts potent progesterone to a weak 20α-hydroxyprogesterone; AKR1C2 inactivates potent androgen 5α-DHT, and AKR1C3 reduces a weak androgen androstenedione to a potent testosterone. Both AKR1C3 and 17β-HSD type 1 activate weak estrogen estrone to a potent estradiol [
This work was supported by a J3-9448 grant to T.L.R. and a young researcher grant to P.B., both from the Slovenian Research Agency.
Deamidation of proteins is probably one of the most common chemical degradation pathways in protein pharmaceuticals. The reaction rate for deamidation of human growth hormone (hGH) is particularly pH-dependent and occurs typically faster at neutral and alkaline pH [
Protamine from salmon is an extremely basic peptide due to its high arginine content. It is isolated from sperm cells of salmon where it naturally builds a complex with DNA during spermatogenesis due to electrostatic interactions [
This work was supported by FFG, Land Steiermark and SFG.
Protein denaturation processes involving aggregation are among the prime factors impeding the development of stable protein drug formulations. Not only does aggregation limit the shelf-life of protein pharmaceuticals and potentially decreases the overall efficacy of therapeutic, it may also cause unwanted side effects such as immune reactions. Denaturation kinetically coupled to aggregation can occur in all stages of the production process and its prevention constitutes a major effort in biopharmaceutical technology. A problem in designing rational strategies counteracting the aggregation is that a sound molecular basis underlying the denaturation process is usually not available. Dissecting the overall denaturation pathway into discrete kinetic steps would allow one to evaluate stabilizing effects of certain process conditions more systematically. Generally, understanding of aggregation during or induced by the manufacturing process is an important goal to be achieved by the pharmaceutical industry.
For our studies we used human growth hormone (hGH), which is a common therapeutic protein, and examined its aggregation mechanism under a range of process conditions. hGH is a single domain, globular protein consisting of 191 amino acids and has a molecular mass 22 kDa. Its tertiary structure shows 4 antiparallel α-helices and two disulfide bridges (53 to 165 and 182 to 189). Experiments were carried out under accelerated aggregation conditions. We show that the gas/liquid interface generated by aeration is strongly promoting aggregation. Stirring likewise resulted in aggregation. Results from SEC measurements and native or SDS PAGE of time-course experiments should help understanding aggregation and its kinetic pathway. This may help in developing more stable drug formulations.
This work was supported by FFG, Land Steiermark and Steirische Wirtschaftsförderung (SFG)
Unsaturated Monoglycerides (or Phytantriol) dispersed in water form particles having a cubic nanostructure over a broad temperature range. The cubic structured phase has a high viscosity which requires high energy input in order to be dispersed in water.
By the addition of lipophilic substances the structure can be changed to hexagonal, micellar cubic and micellar, respectively [
Those nanostructured dispersions have a high interfacial area, which allows them to be good carrier systems for amphiphilic substances. Due to their oil and water compartments they can also host hydrophilic as well as lipophilic molecules. Furthermore, if substances are incorporated in the phases their stability against degradation is enhanced. These properties make them interesting as possible drug delivery systems.
With the structural change not only the viscosity of the self assembled phase alters but also properties which can affect the release rate. The diffusion inside the phase is heavily dependent on the nanostructure.
The internal structure can further be expanded by the addition of lamellar phase forming amphiphiles such as Diglycerol Monooleate and Phosphatidylcholine [
In order to be able to use nanostructured dispersions as delivery vehicles it is important to gain information about the release mechanism and possible ways to control it. For this purpose the release from the self-assembled bulk as well as from the dispersed phases is studied.
In the present investigation, vancomycin was incorporated in liquid crystalline gel made of glyceryl monooleate and water, with the aim to achieve a sustained-release and to deliver locally sufficient concentrations of vancomycin to the eye, improving its efficacy against bacterial infections [
Technique for implants preparation was developed involving combining glyceryl monooleate and vancomycin solution by vortex mixing, centrifuging, preparing the shape of implants and lyophilization.
In vitro characterization of liquid crystalline gel/implants included structure analyses by light microscope with polariser and small angle X-ray scattering. Furthermore, the drug release from the implants was studied.
Structure analyses of samples with different percentage of water showed, that samples with 10% water show a lamellar phase, whereas samples with 23–30% of water have a cubic phase, Ia3d, and the samples with 40% of water show a mixture of two cubic phases, i. e. Ia3d and Pn3m.
Investigating release properties of implants with an initial water content of 30% slow release was found. Approx. 40% of vancomycin was released within 2 hours.
The in vitro characterization of the liquid crystalline gel formulation/implants containing vancomycin showed the possibility to obtain different phases, i. e. lamellar phase and cubic phase as well as the possibility to sustain the release of vancomycin from them. Therefore, the implants show a great potential for the local treatment of infections of the eye.
Semisolid products are 8–10% of dosage forms, but – in contrast with the solid dosage forms – we do not have any validated method for their drug release in any Pharmacopoeia. Kinetics of release process and its critical factors in case of 1% diclofenac sodium containing hydrogel, organogel, gelemulsion, o/w and w/o creams were observed under in vitro conditions. Comparison of results between Franz diffusion cell and paddle over disk method was made using synthetic cellulose acetate membrane soaked in buffer solution or in isopropyl myristate. In vivo studies were carried out on male Wistar rats; the carrageenan paw edema decreasing effect of 12 different formulations was measured in comparison with a control group. All products reduced paw edema in rats, although we found significant differences among them both
Gel emulsions represent special drug delivery systems. Up to present only some authors dealt with the formation and properties of these systems, although the gel emulsions assurance interesting and useful possibility as controlled drug delivers systems [
In our experiments water-soluble (polar) and water-insoluble (unpolar) active agents were applied. Composition of gel emulsions, e. g. the ratio of water- and oil phase, the concentration of surface active copolymers, addition of coemulsifiers was changed systematically.
The drug release from emulsions was investigated by Hanson SR8 plus equipment and Franz vertical diffusion cell. The rheological characteristics was measured by Paar Physica oscillation rheometer Optical studies were accomplished with Leica image analyzer, and thermal-analytic investigations were carried out with DSC 281. Mettler Toledo equipment.
Relationships were established between following parameters: i) drug release vs. mucoadhesion, ii) concentration of polymer emulsifying agents vs. drug release iii) presence of microgel layer surrounding oil droplets vs drug release, iv) concentration of oil phase vs drug release.
The topic of this article was to monitor the impact of excipients on viscosity of gels with local anaesthetic. In previous studies the optimal excipients from the group of polymers, enhancers and humectants were examinated during the formulation of chlorhexidine, trimecain and terbinafine to the medical form – hydrogel [
The reological properties were evaluated in the study of hydrogels prepared from various gel creatig compounds (Natrosol 250 HX, Natrosol 250 HHX, Natrosol 250 HR and Chitosan) with active ingredient of trimecaini hydrochloridum and gels without the active ingredient. Then the influence of propylenglycol and glycerol humectants in various concentrations on Mesocain gel were examinated. Solitary influence of excipients was evaluated using the analyses of reological measurement results.
This work was supported by the Grant VEGA No. 1/ 0320/ 08
Quercetin is a flavonol with antioxidant properties, exhibiting poor water solubility and transdermal permeation ability. The objective of this study was to formulate a new microemulsion for the transdermal delivery of quercetin based on the use of Transcutol® P as a solubilizing agent and absorption promoter.
Quercetin hydrated (>95%) was purchased from Sigma-Aldrich (Germany). All the excipients were kindly supplied by Gattefossé (France). The microemulsion was prepared by mixing the water and the lipidic phase under gently and continuous stirring at room temperature. Formulations were characterized for physical stability (centrifugation), pH, viscosity (viscosimeter Rheomat RM 180), internal phase dimensions (optical microscope, Leitz),
The solubility of quercetin was spectrophotometrically determined in different excipients and whose in which it was more soluble were selected.
Through a ternary diagram, the percentages of the following phases were determined: lipidic phase, water phase, ratio surfactant/co-surfactant. The best formulation was: 1% of quercetin, 16.67% of water phase with 40% of Transcutol® P, 16.67% of lipidic phase (Labrafil®), 66.67% S/coS (Labraso®l, Capryol® 90). The most stable microemulsion was an O/W microemulsion, appearing as a yellow transparent liquid, with an internal phase size of 10–50 nm and a pH of 5.0. The dynamic viscosity was 0.068±0.003 Pa*s. The quercetin diffusion, compared to other different formulation, was good. From this study it was possible to highlight the fundamental role of the Transcutol® P in increasing the quercetin solubility in the microemulsion and as absortion promoter through the membranes.
Due to its topical irritant effect on gastric mucosa naproxen is an ideal candidate for encapsulation in pH sensitive drug delivery systems such as alginate– chitosan microparticles. In this study we aimed to investigate the feasibility of these carriers for oral delivery of naproxen, and to establish the kinetic mechanisms of naproxen release from microparticles under conditions simulating gastrointestinal environment. Sixteen different microparticles formulations in range between 262.3±14.9 and 358.4±21.7 μm have been prepared by well-known two-stage procedure using custom made air-jet device. Differences among formulations were hardening time in chitosan solution, drug/polymer ratio, chitosan molecular weight and chitosan concentration. After 2 h in acidic media (pH 1.2), naproxen release was less then 6.18% for all investigated formulations due to non-porous surface and low swelling ability of chitosan–alginate gel network in acidic environment. When the pH of the surrounding media was raised to 6.8, swelling of microparticles occurred, leading to the rapid release of encapsulated drug. ANOVA analysis revealed that all investigated factors have statistically significant influence on drug release from microparticles (p<0.05). Furthermore, ANOVA analysis showed existence of statistically significant negative interaction between chitosan molecular weight and its concentration in hardening medium. This observation may be explained by influence of the hardening medium viscosity on the formation of chitosan-alginate gel network. Naproxen release data obtained during second phase of experiment (pH 6.8) showed best fit to the Korsmeyer-Peppas equation, which can be confirmed by comparing the values of the correlation coefficient with those of other investigated models. The values of the Korsmeyer–Peppas release exponent (n) determined for all investigated formulations varied in range from 1.62 to 3.73 suggesting Super Case II transport mechanism for the release of naproxen from microparticles. Super Case II transport occurred due to a large increase in osmotic pressure driving forces, followed by rapid relaxation and swelling of alginate chitosan gel network.
The results indicate that chitosan-alginate microparticles represent convenient gastroresistance delivery system for oral administration of naproxen due to release controlled by pH dependent swelling ability.
Alginate-based microparticles have been widely investigated for applications like enzyme immobilization, immunoisolation in cell transplantation and drug release systems. Using the custom made air-jet device, by varying processing as well as several formulation factors, we aimed to prepare and investigate alginate-chitosan microparticles loaded with phenytoin, a standard antiepileptic agent. The extended-release formulations of antiepileptics simplify treatment of this chronic condition. Such commercial formulations with phenytoin appear to be well designed, with one possible shortcoming being the potential for irregular absorption that appears to occur particularly in elderly patients [
Development of drug delivery systems is an indispensable strategy for successful transport of drug to its therapeutic site by the appropriate choice of carrier and route. Solid lipid nanoparticles (SLN) include the advantages of conventional carriers and are additionally utilized for protection of labile compounds, as well as in controlling of drug release, targeting and stability.
In general, physicochemical characteristics of SLN surface are those, which affect their behaviour both in vivo and in vitro [
SLN unlabelled and particles labelled with different coumarin based fluorescent dyes such as SPP 189 (SLN SPP-189) and 6-coumarine (SLN C), produced by melt-emulsification process were circular in shape. Photon correlation spectroscopy and AFM provided approximately the same particle size of investigated samples. Surface roughness of SLN SPP-189 was significantly elevated in comparison to unlabelled SLN, what should be the consequence of into surface incorporated fluorescent dye. Phase imaging and force spectroscopy measurement estimated the surface heterogeneity and local surface hardness. Phase contrast confirmed two different regions of SLN SPP- 189, which could be associated with surface heterogeneity caused by diverse nature of main constituents as well as fluorescent dye. Only these nanoparticles demonstrated light hallow in phase image, likely related to inhomogeneous distribution of lipophilic ingredients that also reflects differences in local surface hardness.
Current research will be additionally complemented with analysis of particles-cells interaction in order to improve the understanding of SLN composition involved in the cell uptake and in cell drug delivery.
The enhancement of dissolution rate is one of the most commonly used approaches to improve the bioavailability of drugs, since for a great extent of new drug substances their absorption is limited with their dissolution rate. The aim of this work was to prepare and optimize microparticles containing ketoprofen using spray-drying technology, which would enhance the dissolution rate of ketoprofen, which is poorly soluble at lower pH values. Different hydrophilic polymers with relatively low melting point (polyethylene glycol 4000 and 6000, poloxamer 188, Gelucire 44/14 and 50/13) were used to prepare microparticles by spray drying. Due to the low melting point of the polymers, spray drying parameters were carefully optimized to ensure reasonable yields. Of the process parameters, atomizing pressure was shown to be the most important factor influencing product yield. Product yield was also greatly impacted by the adhesion of the particles to the inside wall of the spray dryer due to the sticking of the carrier material. A positive correlation between the melting point of the polymers and the product yield was established - the lower the melting point of the polymer, the lower was the yield. Carriers with promising yield (polyethylene glycol 4000 and poloxamer 188 with the average yield of 34,4% and 43,0%, respectively) were chosen for the preparation of the drug loaded microparticles. A reference sample was prepared using lactose as a carrier. In all cases ketoprofen was not dissolved in the final mixture and ketoprofen suspensions in carrier polymer solutions were spray dried. During the preparation of drug loaded microparticles, the drug concentration in the suspension was shown to have a great impact on the overall yield. Due to extremely poor yield when polyethylene glycol 4000 was used as a carrier, only samples prepared with poloxamer 188 and lactose were further studied. Image analysis was used to determine the size and shape of microparticles, which were recorded by scanning electron microscope (SEM). The dissolution rate of ketoprofen from the microparticles prepared with poloxamer 188 was enhanced compared to microparticles prepared with lactose as a carrier. All prepared microparticles exhibited markedly increased dissolution rate compared to ketoprofen alone. The differential scanning calorimetry (DSC) confirmed a similar thermal behavior of ketoprofen microparticles and corresponding physical mixtures which indicates the absence of chemical interaction between ketoprofen and water soluble carrier. It was also shown that there was no significant conversion of crystalline ketoprofen to the amorphous form during the spray drying process.
In recent years there has been increasing attention given to the utility of self-microemulsifying systems (SMES) for improving the gastrointestinal absorption of drugs with inadequate biopharmaceutical properties. Microencapsulation of those systems into polymeric matrix is one of the solidification techniques that allow formulation of solid self-microemulsifing formulations.
Microcapsules with furosemide-loaded self-microemulsifying core that merge the advantages of SMES with those of solid dosage forms where produced by co-extrusion of liquid jet by vibrating nozzle device. The composition of self-microemulsifying core was optimized previously [
Best shaped microcapsules with highest encapsulation efficiency were obtained from the shell-forming phase with the Ca-alginate/pectinate ratio of 1/3 containing 5–10% lactose [
Egg lecithin is a complex mixture of phosphatides that consists mainly of phosphatidylcholine and phosphatidylethanolamine, combined with various amounts of other substances such as triglycerides and fatty acids [
The aim of the study was to evaluate the solubilizing properties of WLD (containing 5% or 10% of lecithin) using 9 different active substances, demonstrating low water solubility: cyclosporin A, sulfamethoxazole, metronidazole, dexamethasone, hydrocortisone, carbamazepine, prednisolone, theophylline and testosterone. The solubility in water and WLDs was studied for untreated and lyophilized substances.
For solubility determination, active substance was suspended in 10 ml of water, 5% or 10% WLD and the mixture was stirred for 24 h at room temperature. Then the dispersion was centrifuged for 15 min and the supernatant was filtered and analysed by means of spectrophotometric or HPLC method.
Results indicated, that the solubility of the tested drugs in WLD was at least few times higher than in water and the amount of dissolved substance increased with the increase of the lecithin concentration in WLD. The only exceptions were drugs with relatively high water solubility (metronidazole, theophylline), for which WLD did not increase solubility. The highest increase in solubility was observed for cyclosporin A, for which measured solubilities were as follows [mg/ml]: 0.03 (water), 3.9 (5% WLD) and 5.7 (10% WLD). Microscopic analysis indicated, that in freeze-drying process reduction of the drug particles size was achieved and this resulted in much faster dissolution of the compounds in WLD. Due to biocompatibility, WLD may be considered as a carrier for poorly soluble parenterally administered drugs.
In modern pharmaceutical technology a number of methods offer themselves to enhancement of dissolution and rate of dissolution. The insert to a micelle or an inclusion complex and conjugatum between active agent and a polymer molecule can increase both solubility and the rate of dissolution, but then the microencapsulation increases only the rate of dissolution [
Aim of our research was to increase of solubility and rate of dissolution of poorly water-soluble active agents. Seven active agents poorly, slightly and very slightly soluble in water were investigated. In the course of preformulation the wettability of these active agents with different polymer solutions was studied. The contact angle of wetting was measured by OCA 20 equipment (produced Dataphysics). On the grounds of contact angle values the polymers with best wettability was chosen, which – presumaby – is most suitable to prepare microcapsules. The microcapsules was produced with Büchi’s spray drying equipment.
The microcapsules was produced with Büchi’s spray drying equipment. Relationships were appointed between the technical parameters of spraying (e. g. rate of spraying, the temperature of incoming and outgoing air) and the properties of microcapsules (e. g. size distribution, morphology and surfaces, and dissolution rate).
Flow diagram
Diazepam is one of most frequently prescribed benzodiazepines. It is classified as a class I substance according to the Biopharmaceutics Classification Scheme (BCS). Diazepam tablets were prepared using different API raw-materials. Before the formulation, we measured particle size distribution on Malvern Instruments Ltd Mastersizer 2000. The tablets were prepared by employing conventional wet granulation. The formulations were evaluated for hardness, friability, content uniformity, in vitro disintegration time (DT), release profiles. The dissolution profile testing is performed on the two formulations with different particles size distribution.
Test parameters were: medium 0.1 mol / l hydrochloric acid, 900 ml, temperature 37°C ± 0.5°C, Mixing Speed 100 rpm, Number of tested tablets 12, Testing Cycle 10, 15, 20, 25 and 30 min. Instrument: Varian Van Kel VK7025, Varian Cary 50. We used the spectrophotometric method with absorbance measuring on 242 nm for quantification.
Results for particle size distribution for first Diazepam API particle distribution were d (0.10): 88.743 μm; d (0.50): 305.231 μm; d (0.90): 655.911 μm, and for the second raw-material of API was d (0.10):1.998 μm; d (0.50): 4.419 μm; d (0.90): 8.845 μm. The results of diazepam dissolution for two formulations and f2 value with reference drug are presented in
It was concluded on the basis of formulation tests that Formulation B, for which micronized API was used, has a similar dissolution profile for API as compared to the reference medicine (f2=99.94) in comparison to Formulation A (f2=68.15)
| 10 | 15 | 20 | 25 | 30 | 10 | 15 | 20 | 25 | 30 | |
|---|---|---|---|---|---|---|---|---|---|---|
| 85.34 | 99.05 | 100.79 | 100.39 | 99.88 | 99,08 | 98,49 | 97,90 | 97,36 | 96,89 | |
| 4.65 | 2.57 | 3.20 | 3.34 | 3.31 | 1,32 | 1,32 | 1,28 | 1,27 | 1,28 | |
| 5.45 | 2.60 | 3.17 | 3.32 | 3.31 | 1,33 | 1,34 | 1,31 | 1,31 | 1,32 | |
| 68,15 | 99,44 | |||||||||
Many drug substances exhibit low solubility in water, which results in their poor bioavailability. Among ophthalmic preparations, oil solutions, suspensions and ointments are formulations prepared for such compounds. Promising modern drug carries are self-emulsifying drug delivery systems (SEDDS) which are isotropic mixtures of oils and surfactants. Due to presence of surfactants improved solubility and transcorneal passage of the active substances can be expected [
The aim of this study was to compare the in vitro release of indomethacin (IND) and hydrocortisone (HC) from SEDDS and aqueous or oily suspensions. The release experiments were carried out for 6 h at 37°C using dialysis cellulose membrane and acceptor fluid imitating composition of lacrimal fluid.
Amount (mg) of the drug and the percentage of the dose delivered to the acceptor medium was measured and diffusion coefficients were calculated. After 6 h about 7 mg (40%) of IND was released from SEDDS and about 5 mg (30%) from 0,6% aqueous and oily suspensions. Only small percentage of HC was released (below 20%) from the aqueous and oily suspensions. However percentage of hydrocortisone released from SEDDS (containing 1% or 5% Cremophor EL) came to over 60%. The results suggest that increased solubility in SEDDS [
In vitro dissolution testing serves as an important tool for drug manufacturing process control and quality assurance. In certain cases it can serve also as an indicator of how the formulation will perform in vivo [
Cefaclor, cephalosporin antibacterial drug, was chosen as a model drug, classified in BCS class III (high soluble and low permeable) [
The aim of this study was to investigate the impact of experimental conditions on drug release from capsules having the same composition, but containing 250 mg (product A) and 500 mg (product B) of cefaclor. Solubility, the drug intrinsic dissolution rate and in vitro drug release tests were performed in water as well as in three EP buffer media: pH 1.2, pH 4.5 and pH 6.8.
It has been shown that the solubility of cefaclor was two times lower comparing to that observed in other tested media. The drug intrinsic dissolution decreased in the following order: pH 1.2>pH 6.8>water>pH 4.5. The dissolution media composition strongly, and to variable extent, influence cefaclor release from the investigated drug products. At pH 1.2 the drug dissolution was very fast with more than 85% of cefaclor dissolved within 15 and 30 minutes for product A and B, respectively. In the case of EP pH 4.5 buffer medium, the drug dissolution was notably slower and, consequently, more than 75% of the drug dissolution was accomplished after 60 minutes. The employment of EP pH 6.8 buffer medium resulted in slow and incomplete drug release. The differences between the release profiles obtained for the investigated drug products A and B in each tested dissolution medium were found to be statistically insignificant. In addition to the drug characteristics, it is evident that the formulation factors strongly affected cefaclor dissolution rate and must be taken into account in selecting a test method that will be sensitive enough to reflect its bioavailability.
Due to complexity and interplay of variable gastrointestinal conditions with matrix tablets which are predominantly erosion controlled it is often difficult to predict in vivo release profile [
Present study describes approaches to establish a biorelevant in vitro test for evaluation of two different matrix tablets containing a BCS class 2 model drug. Mechanism of release was also evaluated. Obtained results were compared with in vivo pharmacokinetic data (Cmax) and possibilities for IVIVC or IVIVR were explored.
The tablets were first tested with conventional dissolution tests. Secondly, a test that simulates mechanical stress potentially occuring during transition of tablets through GI tract was performed [
Obtained results were used to elaborate the mechanism of release from the tablets. One formulation was accelerated after applying the mechanical stress, while the other formulation exhibited no changes and followed a 0. order release. This was proven by calculating the Korsmeyer-Peppas release rate constants.
The release mechanism was further examined by inspecting the rates of dissolution ΔQ/Δt before and after mechanical stress manipulation. IVIVR with Cmax results was also shown, the dissolution profile when approximately 70% of the drug is released being relevant for in vivo.
The results from dissolution apparatus USP3 and plastic beads test were used to develop an IVIVC model. Based on this model the maximal plasma concentration (Cmax) was predicted and compared with the observed values from the in vivo BE study. The average absolute percent prediction value for each tested matrix tablet was less than 15%, which demonstrates a good internal predictability of the IVIVC model.
The holt-melt extrusion technique has proven to be an advantageous method for preparation of solid dosage forms. The aim of this work was to evaluate the release profiles of sustained release cylinders produced by hot-melt extrusion. The cylinders were composed of inner and outer part, both containing theophylline [
Due to their hydrophilic / lipophilic properties inner part of cylinder releases the drug fast while outer part represents extended release component. No influence of stirring rate on theophyline release was observed for 1cm cylinders while for 0.5cm cylinders the amount of released drug slightly increased with increase of stirring rate (
Percentage of released drug after 7h of release in dependence on stirring rate
Stereomicroscopic view of co-extrudates. Arrow shows void space between two parts of cylinder.
The aim of this research is to prepare Moxifloxacin hydrochloride immediate release tablets by wet granulation using experimental design technique. Moxifloxacin is a fourth-generation fluoroquinolone that has been shown to be effective against Gram-positive, Gram-negative, and atypical strains, as well as multi-drug resistant Streptococcus pneumoniae. [
Administration of pellets in fed stomach state can prolong the gastric emptying of pellets in comparison to the administration under fasting conditions [
A systematic literature search on evaluation of human gastric emptying of pellets based on the technique of gamma scintigraphy was performed in the MEDLINE database. Studies estimating the gastric emptying of pellets under fed conditions were focused. In addition to the caloric value of the meal, a special attention was made on other factors such as, pellets size and density, pellets formulation (filled into capsules or compressed into tablets), a time delay for pellets administration in relation to the time of meal completion, and refreshments provision. The majority of pellets gastric emptying data was presented in terms of pellets t50 values – a time when 50% of the administered pellets emptied the stomach. In order to evaluate the influence of the caloric value of the meal on pellets t50 values a linear regression model was built. In this model the caloric value was regarded as a categorical variable, which was recorded into dummy variables with the lowest caloric value set as a reference.
In total 17 studies were recovered, however, only 6 of them met the selection criteria: pellets density between 1.2 and 1.5 g/cm3, administration of the pellets at the time of meal completion, pellets filled into capsules, and pellets size in the range from 0.6 to 1.4 mm. In these studies 48 individual values of pellets t50 were collected (6 or 8 subjects per study) and the obtained caloric values were 1200, 1500, 2300, 2800, and 3600 kJ. Mean value of t50 of pellets administered right after the 1200 kJ meal was 136 min. In comparison to the 1200 kJ meal, the mean values of t50 of pellets administered right after 2800 and 3600 kJ meal were significantly longer; 212 min (p=0.031) and 263 min (p=0.003), respectively. No significant difference in terms of mean values of pellets t50 could be noted between the 1200 kJ meal and 1500 or 2300 kJ meal.
Our results suggest that gastric emptying of pellets can be more prolonged if administered along with the food with higher caloric value.
Pellets are an attractive dosage form in the gender specific and pediatric therapy, since they offer the possibility of patient adapted dosing. Additionally, they are easily swallowed and unpleasant taste masking by coating is facilitated due to their smooth and homogenous surface. The development of disintegrating pellets is a new and innovative technology. The main advantage of this type of dosage form is the circumvention of the hepatic and the first pass metabolism [
Powders were blended and wetted with different water/ethanol mixtures (i. e., 10% 20%, 30%, 40% and 50% ethanol). The wet mass was subsequently extruded through a 0.8 mm multihole die plate and spheronized at different speeds for 2–4 minutes. Finally pellets were dried using three different methods (i.e, desiccation, tray drying and fluidized bed technology). The disintegration behavior in water at 37 ± 0.5 °C was evaluated according to Pharm. Eu. 6.0 2.9.1. In addition, the mechanical characteristics (tensile strength, friability) and the pellet shape were investigated.
For pellet formulations containing 5% Kollidon® CL-SF a minimum ethanol fraction of 30% in the granulation fluid was required for successful extrusion/spheronization. At lower alcohol contents extensive liquid movement occurred during extrusion resulting in non-reproducible process conditions. Pellets prepared with 30% and 40% ethanol disintegrated within 1 minute. Using 50% ethanol, however, yielded in increased disintegration times (>1min). Pellets containing 10% crospovidone were successfully prepared with all granulation liquids under investigation. Disintegration times exceeded 1 minute when the ethanol fraction was higher than 20%. All pellet formulations exhibited sufficient mechanical stability and an aspect ratio below 1.2.
Ludiflash® in combination with either 5% or 10% Kollidon® CL-SF is a promising extrusion and spheronization aid for the preparation of pellets showing disintegration times below 1 minute.
The objective of this study was to develop solid self-emulsifying pellets to deliver the poorly bioavailable Milk Thistle extract (Silybum Marianum) [
The authors thank Indena for the kind gift of Sylibum Marianum dry extract.
In biomedical applications, it should be useful to have a system easy to administrate (the best would be a liquid injectable form), with structural properties in physiological conditions (the best would be a soft gel), able to resist to external stimulus (such us the erosion from body fluids, in this case the surface should be a hard gel). Such a system could be used as a drug delivery system, as well as a temporary scaffold.
From previous investigation, we found out that the acqueous solution of Pluronic F127 and alginate (18% F127 + 2% alginate), is liquid at low temperature (around 5°C), it gives a soft gel due to the Pluronic thermogelation at body temperature (around 37°C), and it gives a hard gel after the exposition at solutions of bivalent cations (reticulation, using Ca2+, Cu2+).
Prior of any other application, the system has to be thouroughly characterized. Therefore, 1) the mechanical properties of the solution were investigated by using rotational rheometry (monitoring the increase of solution moduli during heating); 2) the compressive behavior was investigated with impermeable and porous parallel plates (underlining the rule of the water to build up the properties of the gel); 3) the kinetics of cuprum diffusion and of hard gel formation were quantified (obtaining the thickness of reticulated layer – see the
The scope of the present work was to establish the role of the polymer charge on its ability to enhance the permeability of the urinary bladder wall.
Cationic poly-L-arginine and chitosan, anionic sodium carboxymethyl cellulose (NaCMC) and alginate as well as nonionic hydroxypropyl cellulose (HPC) and hydroxypropyl methylcellulose (HPMC) were tested. The polymers were tested in concentrations from 0.00005 to 1% (w/v) and a concentration of a model drug pipemidic acid (PPA) was 0.014% (w/v) in all experiments. All solutions were prepared in phosphate buffer and their pH was adjusted to 4.5. In diffusion cells the luminal side of isolated pig urinary bladder wall was exposed to tested solutions. In some experiments the tissue was exposed for 60 min to a polymer dispersion with PPA, while in other experiments the tissue was first exposed to a polymer dispersion for 45 min, rinsed and then the solution of PPA was applied for additional 45 min. At the end of the experiment the tissue was frozen and sectioned parallel to luminal surface. PPA was extracted from the tissue segments and its concentration was determined by HPLC.
We showed that the charge of the polymer affects its ability to enhance permeation of PPA into the urinary bladder wall, but to a limited extent. Positively charged chitosan and poly-L-arginine were established as the most promising absorption enhancer into the bladder wall. Compared to chitosan, higher concentrations of poly-L-arginine were needed to significantly increase the tissue permeability. Moreover, poly-L-arginine increased the permeability within 90 min constantly, while chitosan reached the plateau of its absorption enhancement activity after 60 min. In our previous study anionic polymer polycarbophil significantly increased permeation of PPA into the bladder wall [
We can conclude that positive charge has an important influence, but the selection of absorption enhancers for intravesical drug delivery systems can not be done exclusively on the basis of a polymer charge.
General anesthetic drugs were introduced more than 160 years ago and are indispensable in daily surgery at hospitals. Interestingly, their mode of action remains largely unresolved. At present, there are two schools, one favoring specific (direct) interactions of the drugs with proteins of the central nervous system and a second adhering to nonspecific mechanisms through a modulation of biophysical membrane properties. We have focused on the latter mechanism and studied the effects of R-(−), S-(+)-ketamine and propofol on the biophysical properties of lipid model membranes composed of palmitoyl oleoyl phosphatidylcholine by a combination of X-ray diffraction and all-atom molecular dynamics simulations. In agreement with several previous studies, we do not find significant changes to the overall membrane structure up to 8 mol % drug content. However, we observed that the insertion of drugs within the lipid/water interface caused significant changes of lateral pressures within the membrane [
Cartilage lesions observed in osteoarthritis (OA) are related to an important release of cytokines. Current treatments though oral or systemic administration are suboptimal and not curative. Intra-articular administration of drugs is often proposed for localized forms of OA. MAPK inhibitors target important cytokines pathways and show promise for the treatment of OA [
The aim of the present work was to formulate nano- and microparticles loaded with a p38 MAPK inhibitor (VX-745) and to test their
Particles of different sizes were produced by a solvent evaporation method using a solution of PLGA and of VX-745 in dichloromethane and a solution of PVAL. The particles were characterized by laser diffraction, dynamic light scattering, scanning electron microscopy and reverse-phase HPLC.
In vitro studies were conducted by incubating nano- and microparticles with subconfluent human synoviocytes culture obtained from OA synovial samples. The ability of the particles to release the drug and consequently to inhibit the IL-6 biosynthesis was quantified by ELISA (eBioscience, San Diego, CA).
Spherical particles had a smooth surface and mean diameter of 300 nm, 2.5 μm and 25 μm. After 24 h of incubation with synoviocytes, IL-6 production was inhibited by VX-745-loaded nanoparticles in a dose-dependent and size-dependent pattern. For instance, the release of VX-745 from nanoparticles loaded at 100 nM inhibited IL-6 release to 52% compare to control and to 71% of control for a drug concentration of 800 nM.
To conclude, VX-745-loaded particles display extended release properties and inhibit significantly the production of IL-6 from human synoviocytes.
This work was supported by the De Reuter Fondation, Geneva, Switzerland.
Both the active pharmaceutical ingredient and the vehicle physicochemical characteristics are retained to be the main features responsible for favorable topical bioavailability. Emulsion systems based on natural-origin alkyl polyglucoside (APG) emulsifier (cetearyl glucoside & cetearyl alcohol) present promising vehicles in contemporary pharmaceutical compounding [
In recent years, there has been an increasing interest in cocrystallization of active pharmaceutical ingredients. For example the physical stability can be enhanced by cocrystallizing [
The purpose of this work was to study whether teophylline and nicotinamide form cocrystals spontaneously. Theophylline and nicotinamide powders were gently mixed manually at 1:1 molar ratio and stored at different relative humidity conditions. The solid state of the samples was analysed by differential scanning calorimetry (DSC, Mettler Toledo DSC823) and Raman spectroscopy (Kaiser Rxn1 equipped with a PhAT probe). Three different variations of theophylline were used as starting materials, e. g., two size fractions of theophylline anhydrate (small: sieved through a 355 μm sieve; large: the fraction remaining on the sieve), and monohydrate (recrystallized from water). For reference, pure TP-NCT cocrystals were prepared by solid-state grinding with a Retsch MM400 mixer mill.
TP-NCT cocrystals can form spontaneously from manually mixed physical mixtures of TP and NCT during storage without any mechanical activation. For anhydrous samples, increasing storage humidity has an accelerating effect on the cocrystal formation. Particle size of the starting material was found to affect the cocrystal formation kinetics, especially at high relative humidity conditions. Polymorphism of the starting material (i. e. TP anhydrate vs. monohydrate) leads to complicated solid-state behavaviour during storage, particularly when TP anhydrate containing samples are stored at high humidity and vice versa.
Pharmaceutical powders intended to target the deeper regions of the lung have to consist of particles with aerodynamic diameters in the range of 0.5 μm - 5 μm. In such fine powders several forces, mainly Van der Waals and electrostatic forces, have strong influence on particle interactions. Particle interactions significantly determine material-loss during powder handling for example in mixing containers, flow-behavior of the powder and the amount of drug released from the inhaler. In addition to this, electrostatic charge affects the deposition of the powder in the lung. Since most pharmaceutical powders are insulators, electrostatic charge is acquired during every contact of the particles among themselves or with other surfaces. The phenomenon of the emergence of electrostatic charge in common powder handling processes has been investigated by several scientists such as Murtomaa [
The aim of this work is to extend these mixing studies with respect to powder blend composition and mixing conditions. The charging behavior is investigated in a T2F Turbula® mixer using a Faraday cup connected to a high resistance electrometer for charge measurement. When a powder is mixed the charge accumulates until a saturated level is reached. The sign and magnitude of the charge and the mixing time required to reach saturation is an important information in the evaluation of the charging behavior of the powder blend. The acquired charge is determined by several factors such as energy of mixing, mixing time, mixing ratio of the blend, relative humidity, particle size, material and wall-roughness of the mixing container. Mixing studies are carried out by systematically altering these factors in order to be able to perform statistical analysis. The results will be highly useful to improve the understanding of the emergence and the impact of electrostatic charge on the performance of pharmaceutical powders especially for pulmonary application.
Onychomycoses, fungal nail infections, are responsible for 50% of all nail disorders affecting up to 18% of general population in some countries [
We developed six formulations of nail lacquer containing 0,9% (w/v) fluconazole, Eudragit RS 100 and acetone. The formulations contain di-butyl phthalate, polyethylene glycol 400 or propylene glycol as plasticizers in two different concentrations. We characterized the developed formulations with regard to the drying time, fineness of formed film, fluconazole assay and viscosity.
The drying time for the formulations with lower plasticizer content was 25 s, and for those with higher plasticizer content was 30 s. All developed formulations gave homogenous, smooth, glossy and transparent films.
We developed simple derivative spectrophotometric method for fluconazole assay in all six formulations and the obtained results were in range of 97,5– 103,9% of the declared content.
Viscosity was measured using rotating viscometer in accordance to Ph.Eur.6.0 [
The obtained results comply with the standards for this type of preparations with regard to the drying time, film appearance, as well as good flow characteristics. The content of fluconazole is within usually prescribed limits of 100±5%. The results indicate good compatibility of the used ingredients in all six developed formulations.
The drug release from solid matrices systems, made of polymes and drugs, is a basic concept for studies on controlled drug release. The most interesting class of polymers in this application is given by hydrogels. Matrices based on hydrogels, once swallowed (during the in-vitro tests, once immersed in the solvent mimicking body fluids), start to absorb water from the surrounding (water up-take) and the water causes a number of phenomena: the hydrogel swelling, the polymer plasticization (the lowering of the glass transition temperature), the increase of the diffusion coefficients, the polymer erosion (due to polymer chains disentanglement). As a result, the drug previously embedded in the matrix can be released (by diffusion into the matrix and then by dissolution in the outer solvent)
In this work, a model for the drug release kinetics was pointed out. Basically, it consists in differential balances of drug and water in the swelling matrices. It takes into account all the relevant phenomena: the diffusion of drug and water in polymer (with diffusion coefficients that vary with water concentration in the solid phase), the swelling and the dissolution of the polymer (which causes the boundary to be moving surfaces). The resulting code reveals itself able to correctly quantify all the observed phenomena, in terms of water, drug and polymer residual masses in the device, mass fractions of the three species, dimensions and shape of the matrix. To our knowledge, such a complete ability in description was never attained before. The model, therefore, could be a powerful tool in designing novel controlled drug release systems.
Shape and hydration levels for the HPMC-TP 1:1 matrices subjected to the hydration. Snapshot of cut matrices (experimental) and superimposed simulated profiles (model) after 12 hours of hydration. The initial, non-swollen half-matrices shape is a rectangle (diameter 13 mm, semi-thickness 1.1 mm).
Drug liberation-release from the dosage form is a primary cause of the movement of a drug in the body. Pharmaceutical availability of the drug depends not only on the type of aditives but also on the selected concentration. In this study there are compared influence of the concentration of selected cationic surfactants on the liberation of antiseptic chlorhexidine from the hydrogels prepared on the chitosan basis.
– formulation of hydrogels based on chitosan with chlorhexidine dihydrochloride
– observation and evaluation of the influence of concentrations of two cationic surfactants on liberation of chlorhexidine dihydrochloride
– selection of an apropriate concentration of cationic surfactants
– determination of the pH value of hydrogels
– design of an optimal composition of hydrogels.
The drug released was determined by using semipermeable membrane on the permeating apparatus. the released amounts were determined by spectrophotomentric method at 254 nm from 15 min to 3 hours
– As to pH ranged within 5.44–6.14 the prepared hydrogels were suitable for application in stomatology and dermatology.
– The release of chlorhexidine dihydrochloride depended on the presence and type of cationic surfactant and its concentration.
– It was shown that drug liberation increases with increasing lenght of alkyl chain at the 0.1 % (w/w) concentration
– Ten times reduction of surfactant concentration incured average increse of the released amount of chlorhexidine dihydrochloride by 15 %(w/w)
– The greatest released amount of chlorhexidine dihydrochloride was released from hydrogel of the following composition: 0.1% chlorhexidine dihydrochloride + 2.5% chitosan in the environment of lactic acid + 0.01% tetradecyltrimethylammonium bromide + 10% glycerol. This hydrogel was chosen as the most appropriate.
This work was supported by the Grant VEGA No. 1/ 0320/ 08
The drugs in a dosage form is rarely alone. Usually the dosage form is a system of several adjuvances and these more or less influence physico-chemical properties and the drug effect.
From the reason of increasing drug solubility many drugs and adjuvances have amphiphilic structure and are able to create associates. Since the drug is effective exclusively in monomeric form for formulation of a dosage form we need to know concentration at which the associates are created – so called critical micellar concentration (CMC).
The objective of this study is to observe the effect of adjuvances on hydrogel – surfactant
1-ethoxy-
Both substances – the drug and surfactant have amphiphilic structures, thus create micelles. CMC of the both was evaluated from conductivity and spectrophotometric dependences [Septonex at the temperature 25–50 °C, CMC increases non linearly in interval (8.07–9.59)·10−4 mol dm−3 and CHH at 25 °C – CMC = 7.25·10−5 mol dm−3].
The partition coefficients in system octanol / water were determined for both substances at the temperature 25 °C. Release profiles were evaluated at the temperature 25 °C. The released amount of drug was determined spectrophotometrically at λ = 281 nm.
The presence the Septonex as an adjuvance in the prepared hydrogel increased the amount of released drug (after 180 minutes 98.79 %) as compared with hydrogel containing polymer and drug only (51.9 %).
Surfactant used in prepared hydrogels positive effected the drug release.
This work was supported by the Grant VEGA No. 1/ 0320/ 08
The pharmaceutical industry currently experiences a trend towards continuous manufacturing as there are many advantages, including better control of process conditions, effectively eliminating batch-to-batch variability, and the lack of scale-up problems, since even bench-top continuous systems can produce the desired quantities required in the pharmaceutical or fine-chemicals industry. We present the development of continuous processes for the synthesis of active pharmaceutical ingredients (APIs). The set-ups include heterogeneous organometallic catalysts (titanocenes and Pd-complexes) that are ideally suited for these continuous processes. The covalent bond between the catalysts and the solid support effectively prevents metal leaching into the product. For this purpose we developed novel methods for catalyst immobilization [
The application of the heterogeneous catalysts for the synthesis of chiral amines and substituted biphenyls [
This work was supported by the Austrian Science Fund (Project No. 19410)
The broad application of the tetrazole scaffold in different fields, such as general and coordination chemistry, material science, microbiological and medicinal chemistry in recent years demands robust and safe methods for their preparation on production scale. From the many developed methods for the synthesis of the tetrazole nucleus, the [3+2] cycloaddition of an azide partner to a nitrile is by far the most simple, atom efficient and inexpensive strategy. However, hydrazoic acid (HN3) and its salts are extremely toxic and many metal azides and hydrazoic acid itself are extremely explosive.
Great advances in the synthesis of substituted tetrazoles were achieved since the first reports dating back to the mid 1950’s. Today, a variety of protocols are available that allow the synthesis of tetrazoles from azides and nitriles. However, none of these routes are suitable for a genuinely scalable production route, as these batch protocols often involve toxic/explosive intermediates or explosive sublimates, the use of toxic and/or expensive reagents or stoichiometric amounts of inorganic salts, in addition to long reaction times.
Here we present a simple, safe, fast and inexpensive protocol for the synthesis of 5-substituted tetrazoles under continuous flow conditions. We have discovered that the reaction can be cleanly performed using in-situ formed hydrazoic acid at very high temperatures and comparatively short reaction times using a process intensification regime to yield tetrazole products in excellent yields. The details of our microreactor strategy will be discussed.
Continous flow processing is a key enabling technology when it comes to scale-up microwave synthesis protocols to production scale quantities [
Organic chemistry in microreactors, being generally characterized as chemistry in miniaturized reaction devices, comprising channel diameters below 1000 μm have many benefits. Mixing and heat transfer are exceptionally fast on the microscale, taking advantage of very short diffusion paths and a very high surface-to-volume ratio, which enables a very efficient heat transfer in both directions, flash heating, as well as excellent process control. Strongly exothermic reactions as well as transformations involving hazardous reactants or products can be handled safely on a large scale.
Very notably, microreactor techniques can be used to overcome the scale-up limitations inherent to high-speed microwave batch processing in sealed vessels. Vice versa, microwave reactors can be used for rapid reaction optimization for a given transformation conducted under continuous flow conditions, since the reaction time at a certain temperature obtained in a microwave reactor can be directly translated to a residence time in a flow reactor. An established flow process is readily scaleable (in contrast to a batch process) by either extending product collection time, increasing the length of microchannel (thereby allowing for a higher flow rate), parallel operation of many identical channels (“numbering up principle”), or by a combination of these principles [
We will present how continuous flow processing can be realized in meso-fluidic flow devices featuring stainless steel tubes, allowing for an exceptionally high operating window of up to 350 °C and 180 bar working pressure. Microwave reactors were advantageously used for rapid modification and optimization of the reactions conducted in flow reactors and directly transferred to continuous flow conditions.
Catalytic heterogeneous hydrogenation processes arguably belong to the most valuable synthetic transformations known. Current batch reactor technology is not only time consuming and difficult to set up but also catalyst addition and filtration is hazardous. Because of the aromatic character of many substrates, hydrogenation often requires the use of significant hydrogen pressures in combination with elevated temperatures and extended reaction times.
In this context, continuous flow hydrogenation technology presents an attractive alternative to batch processing and the recent introduction of the H-Cube™ – a continuous flow hydrogenation device incorporating
In this poster we investigate the hydrogenations of olefins and heterocyclic aromatic rings. In general, the hydrogenations proceeded smoothly independent from the choice of the supported precious metal catalyst (Pd/C, Pt/C or Rh/C). Using 30–80 bar of hydrogen pressure at 60–80 °C full conversion was typically achieved in all cases at a flow rate of 0.5 mL min−1 providing the corresponding piperidines in high yields. For disubstituted pyridines variations in stereoselectivity were observed depending on both the metal catalyst and on the temperature/pressure in the reaction. For ethyl nicotinate the selectivity between partial and full hydrogenation could be tuned depending on hydrogen pressure and the choice of the supported metal catalyst. Changing the hydrogen source from H2O to D2O allowed the preparation of deuterated derivatives [
Efficient and effective powder mixing is an essential part of pharmaceutical manufacture. Positron emitting isotopes enable particles to be followed singly or in groups during mixing processes, using Positron Emission Particle Tracking (PEPT) or Positron Emission Tomography (PET) respectively. This paper shows how the two techniques can be adapted to pharmaceutical mixing operations and describes their application to mixing in V-mixers and binblenders. PEPT enables particle trajectories to be determined and methods of using trajectory information to quantify mixing are described.
Solid particle mixing is an important unit operation in process industry. In pharmaceutical industry it is of particular importance because the Active Pharmaceutical Ingredient (API) has to be properly mixed which is normally in the quantity of few milligrams in the final dosage form, and if not mixed properly can have no or severe therapeutic effects depending on API quantity.
Particle mixing is usually perceived as a simple unit operation because it does not involve any high-tech and complicated equipment but this operation is still under spotlight of latest research to understand complexity of the underlying physical phenomena. Mixing of solid particles depends on several parameters like nature of the material of the particles, their size, shape, number, and loading/unloading profile. It also depends on the size, shape, and material of the mixer container and likewise the size, shape, and material of the blades, fill level, position of the blade from bottom of the mixer, its speed, acceleration, any external surfaces/obstacles e. g. baffles present, phases involved, nature of the fluid (Newtonian or non-Newtonian) and its physical conditions like viscosity, surface tension, temperature, and pressure etc. Scale-up of powder flows and mixing is also a big issue because even small changes e. g. in equipment material, may change the powder flow and hence the mixing. Because of the number of parameters involved, mixing processes become complicated and expensive to understand experimentally.
In these mixing simulations of solid particles, DEM (Discrete Element Method) is used which treats every particle discretely and solves for each particle’s position, velocity, and all the forces acting on it including contact forces, body forces, hydrodynamic forces and cohesive forces. In this presentation, some physical parameters affecting mixing like blade rake angles, size of particles, and their loading profile will be studied by setting up a number of simulation cases for dry particles. First, velocity profiles for these mixing simulations will be analyzed and then the effects of blade rake angles, size of particles and their loading profile on mixing will be studied by calculating mixing index for each case.
Diffuse reflectance near-infrared (NIR) spectroscopy is a non-invasive and widely used technology with the potential for a wide range of applications in the pharmaceutical industry, ranging from raw material identification to final product qualification. Especially powder blending processes, as one of the key unit operations for solid dosage forms can be monitored in real-time for the blend quality and blending time until uniformity. Common NIR systems enable single point measurements at dedicated positions of the blender, based on the assumption, that the sampled position represents the whole blend. This is a problematic task as the mixing characteristics of powders significantly change (e. g., segregation, agglomeration, etc.) with the process parameters.
Here we introduce, a new process analytical technology (PAT) setup for a spatially resolved quasi-simultaneous process monitoring for batch and continuous manufacturing processes. Therefore, an FT-NIR spectrometer was combined with an optical multiplexer, simultaneously switching the excitation and detection path of the bifurcated fibers, which can be connected to a blender via sapphire sampling ports at different positions. Different multi-component powder mixing processes were investigated and quantitatively analyzed with multivariate data analysis (MVDA) using software packages of CAMO and Umetrics, which also support real-time process monitoring.
The new flexible PAT setup presented here, enables non-invasive in-line and real-time monitoring of dynamic processes, especially where spatial deviations can be expected e. g., segregation in solids (i. e., powder blends) and liquids (i. e., sedimentation).
The application of process analytical technology (PAT) has opened the possibility for continuous process monitoring and quality control in the pharmaceutical industry.
Transporting and mixing of pharmaceutical powders is a challenging task and changes significantly with the mixer type and with the process scale up. Therefore knowledge about the critical process parameters (CPPs) with respect to the powder and their influence on the mixing behavior are essential.
Traditional powder tests (e. g., shear cell, flow through a funnel, etc.) often lack reproducibility and depend strongly on the operator. In contrast to these powder characterization methods, here, an FT4 powder rheometer (Freeman Technology) was applied to determine standardized and repeatible results for powder properties, e. g., shear properties, permeability, cohesion, compressibility, etc.
Based on design of experiment (DoE), an experimental design was developed, testing a recent amount of parameters, which were evaluated for their responses on forming a uniform blend in a mixing process. Continuous process monitoring of the blending processes with near-infrared (NIR) spectroscopy allowed a non-destructive investigation of blend quality and mixing dynamics. Evaluation of the spectral data was performed with qualitative and quantitative multivariate data analysis (MVDA) methods, in order to look for correlations between FT4 parameters and blend quality.
Fluid mixing and dissolution processes are still an area of ongoing research. Numerical simulations have proven to be a valuable tool in understanding and optimizing industrial mixing problems [
In our work the dissolution kinetics of solids in water were measured in a stirred tank reactor utilising inline Near Infrared (NIR) spectroscopy. The dissolution rate can thus be determined as a function of the powder and process properties such as particle size distribution, solution temperature, impeller speed, and concentrations of the solution.
A dissolution model was derived from the analytical investigations and implemented in the CFD code AVL FIRE. The dissolution test mentioned above was simulated with the CFD method in order to validate the dissolution model.
Due to the fact that real-time CFD simulations of mixing processes are very time–consuming, the dissolution rate of the particles was accelerated in the simulation process and the calculation time was thus reduced. The concentration of a passive scalar is used as a measure for the mass fraction of the dissolving solid particles. This approach allows an analysis of the variation of mixing parameters, and the respective effect on the mixing performance, respectively.
Finally industrial mixing and dissolution processes can be investigated and optimized by applying CFD simulations. In addition, NIR inline measurement techniques enable real-time analysis of the mixing status. Industrial mixing process can thus be controlled by Process Analytical Technology (PAT).
Almost alone among the process industries, the pharmaceutical industry has tended to rely on batch operations. This is now changing, driven by a need for better utilisation of resources, particularly clean space, and a demand for more closely controlled processes. Process Analytical Technology (PAT) demands that processes be controlled; this is much more easily engineered into continuous processes.
Wet granulation presents a particular problem, in that it has traditionally been carried out in a high-shear mixer-granulator and is not particularly well understood. What is known is that the mechanisms of nucleation, consolidation, growth and breakage occur more-or-less simultaneously in a high shear environment, and in a relatively uncontrolled way. In principle, it would be highly attractive to separate these basic mechanisms so that they occur within a continuous process in a controlled way.
This paper proposes the practical development of an overall process for continuous pharmaceutical manufacture, with consideration of process control, instrumentation, validation and real-time product release.
Most currently produced pharmaceutical formulations are based on a single molecular entity – the active pharmaceutical ingredient (API) – that is synthesised in a dedicated (bio)chemical plant and then has to be stable throughout the supply chain all the way to the patient. However, many pathogens that the API is supposed to fight use a different strategy: they produce toxins locally within the host and the timing, quantity and even composition of the toxins can vary from host to host and depending on local conditions.
The aim of our work is to design and synthesise structured microparticles called chemical robots that are inspired by the structure and function of single-cell organisms. A chemical robot consists of an outer semi-permeable shell and several internal separate compartments able to store and release chemical reagents on demand. A predefined set of chemical or enzymatic reactions take place within the body of the chemical robot and the reaction product is released to the outside environment at a predefined rate. The chemical robot can therefore be thought of as a miniature chemical reactor with internal supply of reactants and a mechanism for triggering their release.
The synthesis of chemical robots follows a bottom-up strategy. In the first step, the internal compartments (either based on liposomes or on hollow mesoporous silica particles) are synthesised and pre-filled with the required reagents. Compartments containing different reagents are then mixed in a colloidal suspension called “roboplasma” (like cytoplasma), which is then encapsulated into the external membrane by means of either inkjet printing technology [
This work is supported by the European Research Council through grant number 200580-Chobotix.
Inflammatory bowel diseases, such as Morbus Crohn or Colitis Ulcerosa, are painful for the patient and moreover difficult to treat due to the increased mucus production and the occurrence of diarrhea. We could demonstrate that the anti-inflammatory drug rolipram, when delivered by nanoparticles made of biodegradable PLGA, led to a prolonged alleviation of colitis syndromes in rats and a reduction of central nervous side effects, compared to the same dose of the drug administered as an aqueous solution [
With respect to skin drug delivery, there is an interesting new hypothesis that nanoparticles may penetrate along hair shafts and to thus accumulate in hair follicles [
Due to their large surface area and excellent blood supply, the lungs are an attractive alternative route for drug delivery, both for local as well as for systemic action. By escaping mucociliary or macrophage clearance, inhaled nanopharmaceuticals could perhaps be used as platform for pulmonary sustained release delivery systems. Finally, nanoplexes formed between biodegradable polymeric carriers and DNA/RNA-based drugs can be used to facilitate cellular transfection [6]. We are currently using this approach for the delivery of telomerase inhibiting antisense oligonucleotides to lung cancer cells [7, 8].
Nanoparticles represent useful drug delivery systems for the specific transport of drugs to target cells and tissues. Over the last 30 years a multitude of different nanoparticle systems based on several starting materials were described. Recently, albumin based nanoparticles were approved by the FDA and EMEA as drug delivery device for tumor therapy. Most often nanoparticles are developed with the aim of selectively transporting a drug to a diseased tissue or organ. To reach this goal, such drug carrier systems may be combined with targeting ligands, which enable a cell-specific accumulation of the nanoparticles.
Within the presentation the development of targeted nanoparticles for tumor therapy will be described. The preparation and characterisation of antibody-modified protein-based nanoparticles will be focused [
The results indicate that protein-based nanoparticles conjugated to an antibody against a specific cellular epitope hold promise as selective drug delivery systems for the treatment of cells and tissues expressing a specific cellular antigen.
Nanoparticles (NP) are increasingly used in a wide range of applications in science, technology and medicine. Since they are produced for specific purposes which cannot be met by larger particles and bulk material they are likely to be highly reactive, in particular, with biological systems. Direct routes of intake into the organism are (1) inhalation and deposition of NP in the respiratory tract and (2) oral intake of NP and ingestion. Recently there is evidence that nanoparticles can cross body membranes – such as the air-blood-barrier in lungs and the intestinal epithelium – reaching blood circulation and accumulating in secondary target organs. Therefore, direct intravenous administration of NP into circulation provides a powerful tool to shed light on the various interactions of crossing body membranes.
To quantitatively determine accumulated NP fractions in such organs the ultimate aim is to balance the NP fractions in all interesting organs and tissues including the remaining body and total excretion. Since these gross determinations of NP contents in organs and tissues do not provide microscopic information on the anatomical and cellular location of nanoparticles such studies are to be complemented by electron microscopy analysis as demonstrated for inhaled titanium dioxide nanoparticles.
Based on quantitative biokinetics after all three routes of administration in a rat model (lungs, blood, gastro-intestinal tract) we found small NP fractions (iridium, carbon, titanium dioxide, gold,) in all secondary organs studied including brain, heart and even in foetuses. Fractions per secondary organ were usually below 0.1% of the administered dose but depended strongly on particle size, material and surface modifications as well as on the route of intake.
The current knowledge on systemic translocation of NP and their accumulation in secondary target organs and tissues of man and animal models does not suggest to cause acute effects of translocated NP but chronic exposure may lead to elevated NP accumulations resulting eventually in adverse health effects.
In fact, there is growing evidence that ambient ultrafine particles and some of the engineered NP can induce acute adverse health effects in humans and in animal models not only in the respiratory tract but also in the cardio-vascular-system. Since NP translocation is so low these effects are likely to be triggered by mediators released in the organ of intake.
Understanding the intracellular localisation of biomedical nanoparticles (NPs), such as their co-localisation within cellular organells, e. g. endosomes, lysosomes, mitochondria or nuclei, or, alternatively free in the cytosol, can provide essential information in regard to the potential toxicity of NPs.
Polymer coated iron-platinum and gold NPs with a fluorescent dye embedded in the polymer shell were used to investigate their intracellular localization in lung cells, i. e. epithelial cells, macrophages as well as dendritic cells [
By laser scanning microscopy it was shown that the iron-platinum NP were taken up by all three cell types but macrophages and dendritic cells to a higher extent than epithelial cells. In both cell types of the defence system but not in epithelial cells, a particle dose-dependent increase of tumor necrosis factor-α is found. By comparing the iron-platinum- and the gold NPs as well as the shell only it was shown that the cores combined with the shells are responsible for the induction of inflammatory effects and not the shells alone. The quantitative analysis revealed a significant, non-random intracellular gold NP distribution. No particles were observed in the nucleus, mitochondria, endoplasmatic reticulum or golgi, and the cytosol was not the preferred NP compartment. A significant increased gold NP localization in large vesicles (lysosomes) was found with prolonged post-incubation times, indicating intracellular particle trafficking.
In conclusion, by using sophisticated cell culture and microscopic methods, it is possible to determine if NPs exposed to cultured lung cells can penetrate into these cells, inducing an effect and furthermore, in which intracellular compartments they are subsequently localized (trafficking).
This work was supported by the German Research Foundation (DFG SPP 1313), the Swiss National Foundation (Nr. 3100A0_118420), the Doerenkamp Zbinden Foundation and the AnimalFreeResearch.
Continuous processing offers many advantages in pharmaceutical manufacturing. Low capital investment from a small factory foot print, removal of scale-up issues during process development and support of agile /lean supply chain being key economic benefits.
Process analytical technology is a key enabler of continuous processing of pharmaceuticals. The ability of rapid on-line measurement technologies reduces the risk of continuous mixing systems, and can support a QbD approach to control and lead to adoption of Real Time Release strategies for the product.
This paper will describe the on-line PAT systems developed for, and installed in Pfizer’s first commercial scale continuous drug product manufacturing facility. The focus will be on design of sample interfaces and the measure capability related to the product specifications. The paper will also include a discussion on method validation philosophies for on-line real time technologies applied to pharmaceutical processes.
Different solid state characterization techniques have been widely used to gain a better understanding of the physical solid state characteristics of drug substances and drug formulations. In the past, physical characterization in the pharmaceutical industry mainly relied on x-ray powder diffraction, thermal analysis and microscopy. More recently, vibrational spectroscopic techniques such as infrared (IR), near-infrared (NIR), Raman and solid state nuclear magnetic resonance spectroscopy (SS-NMR) have attracted growing attention in both academia and industry. Even more recently, terahertz pulsed spectroscopy (TPS) has also been utilized to investigate pharmaceutical materials.
Spectroscopic techniques possess many advantages over other traditional analytical techniques, for instance the fact that they allow rapid, non-destructive measurements, suitable for use in the process analytical technology (PAT) setting. In this presentation recent examples of the use of spectroscopic techniques especially Raman spectroscopy, NIR and TPS on characterizing pharmaceutical compounds and formulations will be presented. Specific examples from our own work will be presented in this talk.
Using the example of the important anticonvulsant drug carbamazepine (CBZ), we will demonstrate that Raman spectroscopy combined with partial least squares (PLS) analyses can be used to quantify the conversion of CBZ polymorphs to the CBZ dihydrate in aqueous suspension. It was found that crystal morphology and polymorphic form have a large effect on the conversion, but that the conversion is not quantitative. An increased understanding of the influence of factors of this conversion such as defects, crystalline face differences, which are important to the physical stability of CBZ, can be obtained using a combination of spectroscopic and imaging techniques.
Coating of tablets is an important way to design properties of the final dosage form. The purposes for coating are manifold, e. g. taste masking, humidity protection, gastric resistance, modified release, active coating. In some cases the coating uniformity is critical for the intended use. Therefore, it is important to know coating uniformity of tablets can be achieved and analysed. Several aspects have to be taken into account, namely the intra-tablet coating uniformity, the inter-tablet coating uniformity, and the batch-to-batch coating uniformity.
The intra-tablet coating uniformity can be determined by different methods. One modern approach is the determination of coating thickness by Terahertz pulsed imaging. Several thousands measurements can be done to scan the total surface of a coated tablet. It turns out that the coating thickness is higher on the upper and lower surface compared with the central band. This has consequences for the modified drug release. From simulations the film distribution on the tablet surface can be estimated.
The inter-tablet coating uniformity can also be estimated from simulations. These simulations are the basis for machine and process optimization in order to minimize the tablet-to-tablet coating variability. In case of active coating the content uniformity is limiting the variability. While the inter-tablet coating uniformity is typically determined offline first attempts are made for inline measurements.
Not only the amount and distribution of coated material on the surface of a tablet can influence the product properties, but also the quality of the film. Tablets coated with the same amount of polymer can show different release profiles, because the film thickness differs due to different densities of the film. Thus, not only the film thickness is of importance but also the film structure.
The majority of active agents used in medicinal therapy belong to category BCS II, which means that they have poor dissolution and good absorption properties, thus their absorption can be controlled and promoted first of all with various formulation technologies. Melt granulation, which is a thermomechanical technology, is used more and more frequently for the formulation of these poorly soluble active agents.
It can be considered as a possible technological operation only if the active agent(s) and excipients to be used are not heat-sensitive, and if the binding material has a solid state at room temperature but can be melted between about 30–90 °C. During the operation the active agent is either melted or is aggregated with the melt of the excipient.
Our aim was to create an excipient system with melt granulation the thermoanalytical and physical properties of which correspond to the values required for further processing (tabletting, encapsulation), and in which active agents belonging to category BCS II can also be processed well.
Melt granulation was performed with the hydrophilic Gelucire 44/14 (Gattefossé) lipid system, Mg-Al-silicate (Neusilin US2, Fuji Chem. Ind.) was used as a vehicle. Granulates were made with ProCepT high sheer granulator. The particle size distribution and the sphericity of the granulates was examined with Camsizer (Retsch Technology), their flowability with Erweka GT, the physical parameters of the tablets (breaking hardness, friability, disintegration) with Pharmatest equipment, while the dissolution studies were made with a Hanson apparatus. The changes in the thermoanalytical properties of the granulates were followed with DSC and TG (Perkin Elmer). A drug reducing appetite was used as an active agent.
During our work we were successful in formulating melt granulates which contained a sufficient quantity of adsorbed lipids to enhance the solubility of BCS II active agents and also had appropriate physical properties necessary for tabletting.
It was found that the yield of the granulate increases with the increase in the concentration of Gelucire, in addition to which its density also increases and its thermal stability improves.
Variability among individuals that affects clinical outcome is still one of the major challenges in drug development and in the practice of medicine. No single drug is 100% efficacious in all patients. While some individuals obtain the desired effects, there can be no or little therapeutic response in others. Additionally, some patients might experience adverse effects. This interindividual variability is a consequence of myriad of factors, such as disease states, genetic factors, patient age, concomitant medications, and life style factors such as smoking. Most drugs undergo biotransformation and their disposition in the body may involve multiple transport proteins. In addition, they interact with diverse protein targets. This concerted action results in the multigenic nature of a majority of drug responses. Pharmacogenomics, in the future, may provide a complex and more precise set of tools for clinicians to use for diagnosis and treatment. Extensive pharmacometric expertise and model building enables personalization of therapies, which is far from trivial if one considers the complexities of designing the most effective dosage regimen of one or more drugs in conjunction with novel biomarkers. Population pharmacokinetic/pharmacodynamic methods, such as nonlinear mixed effects modeling are able to obtain relevant information in patients who are representative of the target population. They recognize sources of variability such as inter- and intraindividual as important drug characteristics, and seek to explain variability by identifying various covariates, including genetic factors. Additionally, they aim to quantitatively estimate the magnitude of the unexplained part of the variability, which is important because the efficacy and safety of a drug may decrease as unexplained variability increases. This presentation will demonstrate how pharmacogenetics and population pharmacokinetics can personalize treatment with warfarin, leflunomide [
Coated dosage forms in which the active pharmaceutical ingredient (API) is incorporated in the film layer in order to achieve rapid release have become very common in recent years. Our present aim was to develop a matrix pellet containing an API in the coated layer. The presence of insoluble particles in the coating liquid is a critical parameter in the preparation of films as such particles can modify the properties of the film formed [
In this work, films were prepared on a teflon surface. The coating liquid was prepared as reported previously [
The mechanical strength and the degree of deformation of the films were determined by the recording of deformation curves. Contact angles were measured to evaluate the surface free energy, solid surface free energy, liquid surface tension and polarity. Glass transition temperatures were detected by means of DSC. Positron annihilation spectroscopy was applied to study the free volume and track interactions between the polymer and the active ingredient. The size distributions of free volume holes were calculated from positron lifetime data for films of different compositions. The long-term stability of films was also studied.
An essential step for the development of probiotic products is the stabilisation (drying) of the microorganisms. The aim of this study was to compare the efficiency of drying cells by lyophilisation, a classical method for cell stabilisation [
Both methods were studied regarding their effect on the culturability and the cellular activity of
The addition of 50% skim milk, based on the wet mass of cells, had the best protective impact on
Ballistic powder injection is an emerging needle-free technology. Powder particles are accelerated towards the skin and penetrate the superficial layers. Biopharmaceuticals like vaccines can be placed into the epidermal layer of the skin, widely known to be an attractive site for immunization [
We considered poly(esteramide) hyperbranched polymers (trade names: Hybrane S1200 and Hybrane HA1690) as enhancers of glimepiride solubility and therewith associated dissolution rate. We prepared solid dispersions (SD) of glimepiride with hyperbranched polymers (HB) and compared their solubility with those of crystalline and amorphous glimepiride as well as with the solubility of SD of glimepridie with conventional linear polymers, i.ie. Poly(ethylene glycol) and Gelucire (stearoyl macroglyceride)s.
Since the solubility and dissolution rate depend also on glimepiride crystallinity we characterized the morphology of solid dispersions by X-ray powder diffraction (XRD) analysis. The nature of molecular interactions involved in complexation of glimepiride with HB was studied by infrared spectroscopy (FTIR).
The comparison of the results of
IR results indicated that glimepiride forms complexes with HB polymers through hydrogen bonds between the NH groups of glimepiride and carbonyls of ester (O)-C=O and amide (N)-C=O groups of HB polymers. Therefore, the improved glimepiride solubility was ascribed to complex formation between glimepiride and HB polymers.
The study demonstrates two approaches in formulation of solid self-emulsifying drug delivery systems (SSEDDS) of gliclazide (BCS class II drug). Self-emulsifying (SE) formulations can improve oral bioavaiability of poorly water-soluble and lipophilic drugs. Traditional SE formulations are liquids, having some disadvantages such as high production cost, low stability, low drug loading and few choices of dosage forms. To address these problems solid SEDDS are being investigated [
The aim of the study was formulation of solid oral self-emulsifying gliclazide preparations - hard capsules and tablets.
Twelve formulations have been prepared with varying type of solubilizer (Labrafil® M2130CS, Cremophor® RH40, Labrafil® M 1944CSCG, Labrafil® M2125CS), ratio gliclazide:solubilizer (1:1 to 1:20), presence of adsorbent (Neusilin® UFL2), as well as the ratio of gliclazide solution:adsorbent (1:1 and 2:1) and presence of disintegrator (Ludiflash®) in concentration of 10–50%. Formulations have been filled in hard capsules or compressed into tablets using excenter tablet press. Determination of gliclazide release from prepared capsules or tablets has been conducted in rotating basket apparatus (Erweka DT600, pH=7.5, 100 rpm, 900 ml, 45 minutes).
It has been demonstrated that gliclazide has the highest solubility in Cremophor® RH40, at ratio 1:20. Gliclazide release studies have demonstrated that capsules filled with gliclazide solution adsorbed on adsorbent, with ratio solution:adsorbent 1:1, show the fastest gliclazide release rate. Addition of disintegrator in formulations having ratio of solution:adsorbent 1:1, slows down gliclazide release rate. On the other hand, disintegrator enhances gliclazide release in formulations having ratio of solution:adsorbent 2:1. Gliclazide release studies from tablet formulations containing adsorbed solution (in ratios 1:1 and 2:1) and Ludiflash® demonstrate faster onset of drug release in first minutes followed by more sustained gliclazide release, in comparison to capsules. Obtained results demonstrate the possibility to formulate solid self-emulsifyng gliclazide formulations, once appropriate emulsifier, adsorbent and disintigator, in adequate ratio, are selected.
The study demonstrates two approaches in formulation of solid oral lipid-based diazepam preparations (BCS class II drug). The aim of the study was to formulate tablets and hard capsules, using emulsifier and adsorbent. Lipid-based preparations improve drug solubility and permeability, i. e. bioavailability. Solid silicate carriers are able to adsorb liquid lipid formulations, resulting in freely flowable and compressible powders [
Twelve formulations have been prepared, using emulsifier Labrafil®M2125CS (linoleoyl macrogolglycerides) to solubilize diazepam, Neusilin®UFL2 (magnesium aluminium silicate) as adsorbent, with the varying ratio of diazepam solution:adsorbent (1:1 to 4:1), and disintegrators Ac-Di-Sol® and Ludiflash® (with the ratio varying from 0–90 %). Formulations have been filled in hard capsules or compressed into tablets using excenter tablet press. Diazepam release study from prepared capsules or tablets has been conducted in rotating basket apparatus (ErwekaDT600, 0.1 M HCl, 100 rpm, 500 ml, 45 minutes).
Diazepam release studies have demonstrated that it is possible to formulate lipid-based solid oral preparations with immediate diazepam release. Capsules filled with diazepam solution adsorbed on adsorbent, with the ratio of solution:adsorbent 1:1, show the fastest diazepam release rate. Addition of 10% of disintegrator in capsule formulation enhanced diazepam release rate, but when the ratio of disintegrator is increased furthermore, diazepam release is sustained. It was observed that compression of powders in tablets also led to decrease in diazepam release rate. Addition of disintegrators further sustained diazepam release leading to conclusion that a physical interaction occurs between adsorbent and disintegrators. When adsorbent was excluded from the tablet formulations diazepam release rate was enhanced, but tableting properties of such powders were compromised.
Obtained results demonstrate the possibility to formulate diazepam immediate release formulations, with the necessity of careful selection of excipients and dosage forms.
Adsorption onto silica-based high surface area carriers is a long known method of improvement drug dissolution rate, which has been described in early 1970s [
Solid dispersions of carvedilol in porous silica (Sylysia 350) were prepared by solvent evaporation in a vacuum evaporator which ensures efficient pore filling procedure [
The authors acknowledge to KRKA, d.d., Novo mesto for support in the study. Present work was partly financed by the European Union, European Social Fund.
Taste-masking technologies are important for obtaining high-patient compliance and drug therapy efficiency, since many oral-delivery drugs have unpleasant qualities such as bitterness, sourness or saltiness.
Taste-masking technology includes two aspects: selection of appropriate taste-masking substances (polymers, flavourings, sweeteners, amino acids, etc.) and selection of appropriate taste-masking technique. Such taste-masked multiparticulate systems (granules, powders, dry syrups, or suspensions) represent further base for preparation of peroral formulations, such as dispersible tablets and oral liquids.
Selection of appropriate taste masking technique can strongly impact both, quality of taste masking and the process efficiency. There are many available techniques for taste masking developed today, including conventional granulation and polymer coating, but also more sophisticated is taste masking by encapsulation with lipids or cyclodextrins or by making multiple emulsions [
The aim of this study was to reduce or completely inhibit bitterness of model drug of defined physical-chemical characteristics. Fluid bed technology was used as the suitable method for taste masking. Drug particles were coated with the polymeric material, which ensured masking the bitter taste of the drug substance. Two different polymers, amino methacrylate copolymer (Eudragit® E PO) and ethylcellulose (Aquacoat ECD) were used. Influence of spray position and process parametrs on quality of coating of drug particles was studied. Physico-chemical properties of formed granules were characterised and compared in terms to ensure proper masking of drug bitterness and process efficiency [
This work was supported by scientifically assistance and knowledge share of Gordana Matijašević and Ivan Simčić in performing analytical tests.
The dispersion and emulsification in micro channels by high pressure have several characteristics which recommend these methods especially for pharmaceutical applications, e. g. the possibility to use very small educt batches, a narrow residence time distribution and a relatively accurate adjustment of the induced stresses with a good reproducibility. Due to the small dimensions “Cleaning In Place” (CIP) is easy to realize without a large loss of product or time and for high hygienic requirements even the possibility of disposable devices exists. Nevertheless deposits, which can lead to blockages, and the abrasion of the micro systems with the associated product contamination pose big challenges.
In order to determine the dispersion efficiency of different micro channel geometries, experiments with nanoparticle agglomerates were carried out. The results show that the dimensions as well as the geometries of the micro channels influence the dispersion efficiency, the appearance of blockages and the amount of abrasion. Especially areas with low velocities as behind rough edges or oversized in- and outlets run the risk of depositions. With computational fluid dynamics calculations (CFD) and flow measurements (μPIV) areas of low velocities or backflow which run the risk of depositions and the occurence of cavitation are identified and subsequently eliminated or minimized in optimized channel designs.
The CFD calculations are also conducted to get a better understanding of the stress field which leads to the dispersion or emulsification. The turbulent flow fields of the different micro channels were solved and used for a stationary particle tracking. Based on these particle pathes the impressed elongential, shear and turbulent stresses are calculated. The agglomerate size distribution measured after the dispersion experiments are compared to the calculated stresses to determine the mechanisms involved in the dispersion or emulsification.
The authors gratefully acknowledge the DFG for financial support within the DFG research group 856 “Mikrosysteme für partikuläre Life-Science-Produkte“ (mikroPART).
Shrinkage cracking during the deliquoring of compressible filter cakes by means of differential gas pressure is an undesired phenomenon of great practical importance for the pharmaceutical industry amongst others. Cracks are detrimental to the deliquoring process as they can lead to a channelling effect whereby the gas flows preferentially through the network of cracks rather than displacing the interstitial liquid, reducing the effectiveness of the deliquoring process. This has economic consequences of increased gas consumption, as well as higher residual moisture content, which would lead to an increased energy input in the later thermal drying stages.
The precise mechanism of crack formation is not fully understood at present. Crack formation is usually preceded by shrinkage of the filter cake, the driving potential for this shrinkage being the action of the capillary forces on particles forming the cake. When the stresses generated by the capillary forces exceed the tensile strength of the filter cake, the formation and propagation of cracks can be observed [
In this contribution we will present the results of an experimental parametric study on cake cracking. The cracking probability was investigated for a model system of calcium carbonate-water on a laboratory-scale pressure filtration rig. The effects of the following parameters on crack formation were studied in detail: particle size distribution, filtration and deliquoring pressure, cake height, concentration of slurry and filter medium. The results will be presented in the form of graphs and correlations allowing the estimation of both the probability and severity (expressed by an increase in cake permeability) of cracking. For selected cases the crack network structure will be characterised by digital image analysis and three-dimensional techniques such as x-ray microtomography.
This work was supported by the UK Engineering and Physical Sciences Research Council (CASE grant number 0900052) and by GlaxoSmithKline Ltd.
Metoprolol is a cardioselective β-blocker that is classified as a class I substance according to the Biopharmaceutics Classification Scheme BCS, meaning that it is highly soluble and highly permeable. Dissolution test was carried out according to EP 2.9.3. in medium 0.1N HCl, 900 ml, 100 r/min, sampling time 5 minutes. Quantification was spectrophotometric measurement on 275 nm.
The results of dissolution analysis are summarized in
Sample 3 has the highest f2 compared to reference drug, Samples 2 and 1 have values significantly different from the reference drug results. This study helps in revealing the effect of formulation processing variables on tablet properties.
| 5 | 10 | 15 | 20 | 25 | 30 | f2 | |
|---|---|---|---|---|---|---|---|
| 24,29 | 47,88 | 68,32 | 86,01 | 98,99 | 100,35 | ||
| 1,29 | 3,31 | 3,99 | 4,44 | 3,54 | 1,39 | ||
| 5,33 | 6,91 | 5,84 | 5,17 | 3,58 | 1,39 | 75.21 | |
| 21,42 | 43,00 | 62,35 | 78,86 | 91,72 | 97,85 | ||
| 2,57 | 4,07 | 5,07 | 5,69 | 4,95 | 1,99 | ||
| 11,98 | 9,46 | 8,13 | 7,22 | 5,40 | 2,04 | 78,22 | |
| 22,18 | 44,78 | 65,45 | 82,85 | 95,47 | 98,47 | ||
| 3,60 | 5,78 | 6,65 | 6,38 | 4,20 | 1,02 | ||
| 16,21 | 12,90 | 10,15 | 7,70 | 4,40 | 1,04 | 99.18 | |
| 22,28 | 44,96 | 65,41 | 82,66 | 94,84 | 98,52 | ||
| 4,00 | 8,20 | 9,90 | 12,10 | 11,10 | 6,41 | ||
| 17,95 | 18,25 | 15,13 | 14,64 | 11,70 | 6,51 | ||
The application of a coating layer on a tablet is a commonly used technique to selectively control tablet characteristics. Among the most important functions of the coating is the regulation of the release of active ingredient.
In the course of a typical coating process, the tablet passes a spray zone multiple times. Each time, a partial coating layer is applied. For each tablet, this is repeated numerous times until a film of desired thickness is achieved. The number of repetitions needed as well as the quality of the final coating depends strongly on the quality of the partial coating. Therefore, it is of great interest to study the outcome of a single coating event in detail.
In this work, we show the application of numerical simulations to the investigation of film formation on a tablet (see
Film formation on a single vertical tablet after 0.3 s in the spray zone.
Particle shape and size have an influence on the solubility of an API-particle and hence on the bioavailability of the substance. Thus, bulk properties such as the crystal size distribution (CSD) are important quality attributes of powders. Furthermore, downstream processes (e. g., filtering, washing, drying etc.) and the handling abilities (e. g., flowability, tabletability) of the particles are positively affected by narrow CSDs. In order to obtain product crystals with desired features it is important to control numerous parameters during a crystallization process.
We present a versatile, continuously operated tubular crystallizer system, which is based on the growth of seeds to product particles in a tube. Due to the tubular appearance and the small inner dimensions of the crystallizer in the few millimeter range, it is possible to adjust the temperatures along the tubing according to the needs of the crystallization. Thus, the product can be manipulated under controlled conditions [
The effects of various process parameters on product characteristics have been investigated. All experiments resulted in significant crystal growth despite short residence times in the few minute range. Moreover, crystal masses increased by a few g/min and steady-state conditions were obtained in less than 5 minutes. Suppression of nucleation events by avoiding rapid cooling, thus high levels of supersaturation allowed successful achievement of narrow CSDs.
Additionally, simulations regarding the temperature gradients and the crystal growth have been performed and compared to the experimental results.
The aim of this work is to investigate image processing and process monitoring technologies which can provide a systematic, robust and sensitive solution for nucleation detection using bulk video imaging (BVI). The external BVI method is based on a video camera and a capture hardware, which captures 25 frames of the crystallization bulk per second [
The third approach relies on the advantages offered by the multivariate image analysis [
Spray coating is an important unit operation in the pharmaceutical industry. The ultimate goal is to produce uniformly coated products with the desired amount of coating material, to guarantee controlled active pharmaceutical ingredient (API) release. The coating thickness and homogeneity can be determined with spectroscopic process analytical technology (PAT) tools, like near-infrared (NIR) and Raman spectroscopy.
Here, both spectroscopic techniques were applied for the analysis and characterization of tablet coatings. Due to the relatively small coating thickness with respect to the penetration depth of the excitation, both laser light and Raman scattering penetrate through the coated layer. Hence, Raman chemical mapping can also be used to analyse qualitatively the chemical composition (i. e. the distribution of (active) components) in tablets. A quantitative multivariate data analysis (MVDA) based model for the tablet coating thickness was developed on basis of tablets sampled from different stages of a coating process [
We investigate the compaction behavior of the most usual pharmaceutical excipients mixture (placebo) contained lactose monohydrate (LmH), microcrystalline cellulose (MCC), silicum dioxyd (SiO2) and magnesium stearate (MgSt) with combined small- and wide-angle X-ray scattering (SWAXS). In general, the method is becoming an increasingly important technique in pharmaceutical solid-state characterization [
Protein aggregation represents probably the most common and troubling manifestation of protein instability. Shelf life of therapeutic proteins is particularly impaired by aggregation, which can occur almost in all phases of protein drug development. Administration of protein aggregates may lead to reduced pharmacological activity and adverse drug reactions. Thus, prediction of protein aggregation is of major interest for pharmaceutical industry. Existing sequence based bioinformatic tools are barely able to predict the aggregation behavior of therapeutic proteins. To implement new approaches for this, detailed analysis of protein aggregation, its underlying mechanisms and its kinetics is necessary.
In our study we simulate process conditions for induction of aggregation of a common therapeutic protein (granulocyte colony stimulating factor, G-CSF). Size exclusion HPLC is performed to quantify protein aggregates and to enable kinetic pathway analysis by identification of different aggregation intermediates under certain process and formulation conditions over time. Further characterization is accomplished using intrinsic fluorescence, polyacrylamid gel electrophoresis and mass spectrometry.
This work was supported by FFG, Land Steiermark and Steirische Wirktschaftsförderung (SFG).
To investigate the nonlinear kinetics of
Uptake to rat hepatocytes was saturable and progressed according to Michaelis-Menten kinetics. The Km and Vmax of Pitvastatin were 2050 pmols/min/106 cells and 33 μM respectively, which was in in good agreement with other literature reports [
A nonlinear pharmacokinetic model has been derived to characterise the uptake process. A structural identifiablity analysis was performed on the model to establish that all unknown parameters could be identified from the experimental observations available. The model was then subsequently used for parameter estimation and model validation using the data collected. Sensitivity analysis and model robustness analyses were also performed. Once fully validated the model has the potential to perform robust, predictive simulations to ascertain optimal levels of uptake and the effects of the use of appropriate inhibitors.
This work was supported by AZ & TG was supported by AZ and the MRC Capacity Building initiative.
Stressful life style of modern human societies leads to many diseases and one of the growing indispositions related to this fact is hypertension. Thus, there is a strong need for novel effective drugs. One of the new categories of antihypertensive drugs consists of AT1 antagonists (SARTANs), whose action is based on blocking of the active site of the AT1 receptor. The aim of the present study is to contribute to a basic understanding of the their molecular mode of action on membranes. We studied influences on the binary mixtures of DMPC/cholesterol and POPC/cholesterol. Losartan led to a complete loss of positional correlations between adjacent bilayers for all single component model membranes. This can be explained by the negative surface charged conferred to the bilayers upon losartan insertion. The effect was however, counter-balanced upon the addition of cholesterol. Both, POPC and DMPC bilayers exhibited no positional correlations up to 5 mol% cholesterol, respectively. However, only POPC remained uncorrelated at 20 mol% and above, while DMPC/cholesterol bilayers exhibited multilamellar vesicles, i. e. a reentrant transition of the positionally uncorrelated bilayers into correlated ones. Our results may be understood in view of the different hydrocarbon chain packing densities in saturated versus unsaturated bilayers and their respective affinities to interaction with cholesterol. This shows that the insertion of losartan into the membrane may be overridden by a tighter bilayer interface and emphasizes the role of hydrocarbon chain composition in its mode of action. Additionally, we also the influence of the SARTANs (e. g. losartan, candesartan-cilexiril and valsartan) on the global structure of phospholipid bilayers composed of pure dimyristoyl phosphatidylcholine (DMPC), palmitoyl oleoyl phosphatidylcholine (POPC) and dipalmitoil phosphatidylcholine (DPPC) applying synchrotron small-angle x-ray scattering. Valsartan is the only one which shows a disruption of the neutral DPPC bilayer, more visible at higher drug concentrations. This could be attributed to the valsartan chemical structure, which poses a carbonyl acid (carboxyl) group providing higher polarity compared to the other two drugs in the neutral environment.
The interaction of active pharmaceutical ingredients with various surfaces and solutions are of high interest in pharmaceutical industry in particular with respect to development of new formulations, new packaging material [
This work was supported by the RCPE K1 Competence Center – Initiated by the Federal Ministry of Transport, Innovation & Technology (MMVIT) and the Federal Ministry of Economics & Labour (BMWA). Funded by FFG, Land Steiermark and Steirische Wirtschaftsförderung (SFG).
Amlodipine is a long-acting calcium channel blocker used in the treatment of hypertension and angina pectoris.
This work was supported by ALIMS B&H.
Fundamental scientific knowledge is required to develop and optimize pharmaceutical manufacturing processes that yield constantly high quality products and provide most cost efficient manufacturing strategies. In our work we present combined Quality-by-Design and computer simulation approaches (Discrete Element Method, DEM, and Computational Fluid Dynamics, CFD) to characterize three key unit operations of solid and liquid dosage form manufacturing, namely powder blending [
Understanding the variability of both material attributes and process parameters, as well as their overall impact on the unit operations are critical elements for QbD. In a first step, the QbD-methodology is systematically used to (1) establish the critical quality attributes representative for the selected dosage forms, (2) identify potentially critical input factors that may affect process and product quality and (3) risk-rank these factors to define activities for process characterization. Subsequently, computer simulation-based characterizations of the three unit operations are performed. For the blending process the concentration of the active pharmaceutical ingredient, as well as the mixing time are related to the blending uniformity. The film homogeneity on a single tablet is then analyzed as critical quality attribute for the coating unit operation. Finally, a functional design space (engineering design space) for a mixing tank is established. The computational data are used to map out three-dimensional knowledge spaces, leading finally to the definition of design spaces as subset of them. Based on these results, appropriate strategies for process control are presented as well.
The aim of the study was to present an example of experimental design application to set up the dissolution test conditions for the two immediate-release products of levothyroxine sodium (L-Na) with proven bioequivalence: the generic product A and the reference product B [
The most commonly used drug physicochemical property to access its in vivo performance is in vitro dissolution of a drug product. It is, therefore, important to define drug release methodology that would be predictive of its bioperformance and to establish quantitative in vitro-in vivo correlation (IVIVC).
The objective of this study was to: i) to develop drug-specific absorption models for the selected BCS Class II model compounds (nimesulide, glimepiride, gliclazide) using gastrointestinal simulation technology (GST), ii) to use the generated absorption models to provide the target in vivo dissolution profiles for IVIVC, iii) to identify biorelevant dissolution specifications for IR tablet forms of the tested model drugs.
GST [
The results obtained indicate that: i) PSA can aid to identify critical parameters affecting the rate and extent of drug absorption, ii) GST can be successfully used to predict drugs absorption profiles, iii) IVIVC in conjunction with GST can aid to identify biorelevant dissolution specifications for the in vitro assessment of the tested model compounds.
This work was done under the project Biopharmaceutical Characterization of the selected BCS Class II and III drugs: In Vitro and In Silico Methods Evaluation (TR-23015) supported by the Ministry of Science and Technological Development, Republic of Serbia.
Highly purified organic materials are essential in various fields, including the fine-chemical, pharmaceutical and food industry. A method widely used to purify, e. g., active pharmaceutical ingredients (APIs), is preparative chromatography. In this context, continuous processing is becoming increasingly important. The development of a novel prototype for
We present the development of stationary phases for this CAEC process. Our approaches include functionalized silica-based monolithic materials [
This work is supported by the 7th Research Framework Programme of the European Commission (Grant agreement number: NMP2-SL-2008-206707).
Pregabalin is a structural analogue of γ-aminobutyric acid approved for treatment of neuropathic pain, as adjunctive therapy for partial seizures, and in generalized anxiety disorder. It has no significant UV or visible absorption, therefore most often derivatization methods have to be employed for its determination. Previusly published methods for pregabalin determination in pharmaceutical dosage forms are either time-consuming [
Sodium naproxen (SN) exists in an anhydrous form and in four different hydrated forms: one monohydrate (MSN), two dehydrates (DSN and CSN) and one tetrahydrate (TSN). Mechanisms of dehydration have been previously determined by an isothermal method [
SN (B.P.) was kindly supplied by A.C.R.A.F. (Italy).
Thermogravimetric analysis (TGA) was performed by a Simultaneous Thermal Analyzer (STA 6000, Perkin Elmer, Inc., Waltham, MA, USA), under nitrogen atmosphere (20 mL/min) in a 0.07 ml open aluminium oxide pans. After calibration, samples (approximately 10 mg) were tested in quadruplicate by heating from 293 to 413 K at four different heating rates (5, 10, 20, 40 K/min).
For each hydrate form, the degre of conversion (% dehydration) was plotted versus temperature at any heating rate. The temperature relative to each conversion degree was then determined and plotted versus temperature. The slope of the regression lines permitted the calculation of the activation energy (
The poorly-soluble Vinpocetine was successfully coground with micronized crospovidone in a planetary mill, which allowed an enhancement of bioavailability through a solid-state activation. Drug-to-polymer weight ratio and milling time variables led to statistically significant effects on the activation/amorphysation of the product. An
The authors thank Linnea for the kind gift of Vinpocetine and Fondazione CRTrieste for funding.
The use of enzyme-catalyzed reactions for the synthesis of sugar substrates represents a more desirable approach in terms of “green chemistry”. As the azidonitratization reaction of cellobial furnishes the 2-azido-2-deoxycellobiose stereospecifically, we have prepared 2-azido-2-deoxyglucose from 2-azido-2-deoxycellobiose using recombinant cellobiose phosphorylase (CeP) from
2-Azido-2-deoxycellobiose is cleaved quantitatively by the action of cellobiose phosphorylase and is separated from glucose 1-phosphate by filtration through DEAE-cellulose. In this manner, we have demonstrated that the substitution of the hydroxyl group by an azido group in position 2 of the reducing cellobiose moiety is tolerated by the recombinant CeP.
The knowledge of surface charge and the isoelectric point is important for many biomedical applications. The zeta potential, formally defined as the electrical potential at the electrokinetic plane of shear, is an important property of charged solid/liquid interfaces and a descriptor of the actual surface charge of a solid immersed in a dielectric. For macroscopic solid surfaces, the zeta potential is commonly determined by the measurement of streaming potential and streaming current. The versatility of the streaming potential method allows handling of planar surfaces, cylindrical capillaries, and packed beds of granular or fibrous materials.
Besides the evaluation of the charging behavior of solid surfaces, the zeta potential is a valuable indicator for surface stability in the presence of various liquids, and for the adsorption of proteins, polypeptides, and other biomacromolecules on biomedical and biomaterial surfaces.
In this paper the application of the streaming potential method in the field of biomedical surface characterization is presented with different case studies.
The aim of the presented study is to propose the utilization of dynamic neural networks and fuzzy algorithms as
Existing concepts for personnel locks in clean rooms are characterized by cost and time consuming procedures. Clean room personnel have to change their clean room garment in personnel locks when moving from one clean room class to another. The changing process of clothes is a source of contamination due to human imperfection.
It is a fact that the major source for airborne microbes is the human body. The results of confocal laser scanning microscopy show that the microstructure of synthetic fibers is very complex and offers many niches for the attachment of microbes to the fiber. These niches drastically reduce the point of action for example of UV sterilization.
Clean room clothing is intended to hinder particles from the wearer’s body to contaminate the environment. There is a demand of the industries for the design of new textiles with new features and new concepts for innovative personnel lock systems. Innovation will take place either in the modification of the textile as well in the design of new cleaning systems for personnel locks. To overcome this huge challenge an interdisciplinary team will completely reconsider common processes to create new standards in clean room technology. This will be achieved by the screening for new bio-based antimicrobials, the usage of light-activated disinfection and the modeling of the behavior of particles on the textile as well as in clean room environment. The great challenge is to create an innovative concept with the basic requirement of maximum safety for the wearer’s body as well for the subsequent processes.
This work was supported by the FFG, SFG, Das Land Steiermark, Ortner Clean Rooms Unlimited and Dastex.
Mechanical stress caused by gastrointestinal motility might have a great impact on drug release from oral dosage forms. In our experiments different mechanical influences on the tablet during dissolution experiments were tested using extended release tablets containing diclofenac. Dissolution experiments were performed on the flow-through apparatus, developed on Faculty of Pharmacy, University of Ljubljana [
Our experiments showed that mechanical stress induced by increasing stirring rate or decreasing the amount of glass beads resulted in increased diclofenac release. The insertion of different types of barriers into the working vessel increased the release rate, however, some crushing of tablets appeared. Thus, no subsequent decrease of release was observed when the barrier was removed. Namely, such dissolution kinetics was desired as we wanted to develop a dissolution test to predict double peaks in plasma profiles after oral administration of diclofenac extended release tablets. It seems that only squeezing the tablet and not other types of mechanical influences, simulates well
The purpose of this work is two fold: it describes the proof of concept of the newly introduced bulk video imaging (BVI) method and it presents the nucleation detection in comparison with existing process analytical technologies (PAT) such as focused beam reflectance measurement (FBRM) and ultra violet/visible (UV/Vis) spectroscopy. While the latter two sample the system closely to the probe, the BVI approach monitors the entire crystallizer volume.
The external BVI (eBVI) method is based on a video camera and a capture hardware, which captures 25 frames or pictures of the crystallization bulk per second [
One of the key steps in the manufacturing of solid oral dosage formulations is the blending of several ingredients in powdered form which are then processed further to achieve the final dosage form. Up until this blending process, it is relatively straightforward to measure critical quality attributes such as particle size and/or shape of the input materials using well established techniques such as laser light scattering or particle image analysis. A much more difficult challenge is to be able to characterise the size and shape of the individual ingredients once they have been through the blending process. This may be especially important when trying to understand any possible impact of the blending process on the size and shape of the API and how this might impact the bioavailability or bioequivalence of the drug. The technique of NIR chemical imaging has been used to get some information of the homogeneity of powder blends [
In this paper we describe a novel approach using a hybrid technique of Morphologically directed Raman Spectroscopy to study the effects of processing on a laboratory scale of a model formulation consisting of ibuprofen, starch and lactose. The sample is first of all dispersed in its dry state, and then images are captured of the individual particles in the mixture in order to determine their size and shape. Each particle is then targeted in an automated manner with a Raman microprobe to obtain a chemical ID. Particle size and shape distributions can then be constructed for each of the ingredients and compared directly with measurements made before the blending process. Results for the model formulation are presented and implications for a number of different types of pharmaceutical formulations are discussed.
14N NQR is a new method in pharmacy that has a potential to establish itself as an additional and valuable analytical tool for characterizing solid state of a substance [
The objective of presented study was to use 14N NQR as a way to characterize qualitatively and quantitatively different solid state forms of sulfanilamide, nifedipine and piroxicam [
The possibility of accurately determining the mixture composition of different forms was also demonstrated. Mixtures of piroxicam forms in different ratios were prepared and then measured with 14N NQR. Determined mixture ratios were in close agreement with the known values.
For many biomedical applications of proteins the knowledge of the three-dimensional structure of proteins is of great importance. It is the key to understand their function in biological processes. Small-Angle X-ray Scattering (SAXS), in contrast to other methods e. g. Protein Crystallography and Cryo-TEM, allows investigating proteins in solution, i. e. in their native state and under biological conditions. Furthermore SAXS does not need any specific sample preparation which might change the protein sample.
SAXS enables to study structural changes upon changing the external conditions and is a valuable tool for determining several important parameters : (1) protein size and shape, (2) aggregated structures, (3) folding state, (4) subunit structures.
By knowing these parameters of the proteins in native, valuable conclusions can be derived in order to explain the proteins’ functionalities. In this contribution the application of laboratory-based small- angle x-ray scattering studies performed with the SAXSess mc2 system in the field of biomedical applications is presented and discussed.
Overcoming biological barriers, including the gastrointestinal epithelial barriers, is a great challenge for pharmaceutical research. There is a need for new absorption enhancers with more favorable profile. Sucrose esters are widely used in the food and cosmetic industry, and there are reports on their potential use in pharmaceutical formulations as excipients [
We tested three water soluble sugar esters having HLB value of 16, laurate (L-1695), myristate (M-1695), palmitate (P-1695) esters, with 12, 14 and 16 carbon fatty acid chains, respectively, on human Caco-2 epithelial monolayers, a widely used model for the human intestinal epithelial barrier. Cremophor RH40 and Tween 80 were used as reference absorption enhancers. For toxicity tests cells were cultured in 96-wells, and MTT dye conversion and lactate dehydrogenase relase were determined after treatments. For permeability tests cells were cultured on Millipore CM inserts (hydrophilic PTFE membranes, pore size: 0.4 μm, surface 4.2 cm2) in 6-well plates for 3 weeks to reach stationary phase and barrier properties. Fluorescein was selected as a marker of paracellular permeability. Untreated monolayers formed a good barrier as reflected by a transepithelial resistance of 722 ± 80 Ω cm2 and positive immunostaining for tight junction proteins claudin-4 and ZO-1. Sucrose esters showed 50 % cytotoxicity (1 h treatment) at 300 μg/ml, while cell death was measured at concentrations above 1 000 μg/ml. The reference molecules were only toxic at doses above 10 000 μg/ml. L-1695 in non-toxic concentrations reduced TEER and increased permeability for fluorescein in Caco-2 monolayers indicating an absorption enhancer property.
This work was supported by TÁMOP-Hungary research project: Development of teranostics in cardiovascular, metabolic, and inflammatory diseases (TÁMOP-4.2.2-08/1-2008-0013).
Delayed release dosage form development usually requires an effective intermediate layer for separation of acid liable drug and acidic gastric polymer. A case is presented where an effective intermediate layer was developed for duloxetine pellets. Duloxetine pellets were produced in Wurster process using suspension/solution layering technique. Neutral pellet cores were coated with duloxetine layer, intermediate layer and gastric layer. Intermediate layer usually comprises water soluble polymer (e. g. povidone, hypromellose); additionally talc may be added as filler and a processing aid (it reduces tackiness of coating dispersion). These kind of intermediate layer compositions provide simple separation of API and gastric layer and are usually sufficient for effective separation. In duloxetine pellets development, however, accelerated stability tests indicated poor long term stability. Diffusion of acidic species (e. g. phthalic acid is a hypromellose phthalate degradation product) across intermediate layer was suspected. Addition of small amounts of sodium chloride (4.8% wt./wt. relative to mass of intermediate layer, dissolved in aqueous coating dispersion) drastically improved stability of duloxetine pellets. Increase in coat thickness resulted in further decrease of duloxetine degradation products: this fact indicates that stabilization is induced by strengthening the diffusion barrier and thus minimizing diffusion of acidic species between the layers. Scanning electron microscope images were used to determine the structure of intermediate coat: talc particles are bonded with hypromellose: within hypromellose phase small crystals of sodium chloride (sub micrometer size range) were detected. Sodium chloride is dissolved in coating dispersion and once applied onto pellet surface it re-crystallizes in form of small crystals (due to fast water removal during coating). It is sugested that these finely dispersed sodium chloride crystals within hypromellose phase provide effective diffusional barrier which prevents migration of acidic species between layers and thus enabling stabilization of duloxetine. In order to assure long term stability of duloxetine pellets it was shown that an effective diffusional barrier is required.
Topotecan (TPT) is a semi-synthetic derivative of the natural extract camptothecin, which has been found to act as an inhibitor of the DNA enzyme topoisomerase I in a specific and reversible fashion. The drug undergoes reversible hydrolysis from the pharmacologically active parent lactone form (TPTL) to an inactive hydroxy acid form. In the cytoplasm the irreversible inactivation of TPTL is catalysed by the enzyme aldehyde dehydrogenase (ALDH). Over-expression of the human breast cancer resistance protein (BCRP/ABCG2) has been linked to high levels of resistance to the anti-cancer agent TPT by promoting an active efflux pump mechanism. The expressions of both ALDH and BCRP have been experimentally identified in a large number of solid tumours and thus play an important role in clinical drug resistance of cancers. To investigate the catalytic reaction and efflux pump mechanism, a compartmental model for the
MIA is funded by the University of Warwick Vice Chancellor’s PhD Scholarship.
In the light of the current trend for natural surfactants to be used as emulsifiers [
Anisotropic structures defined as distorted Maltese crosses were seen in PLM micrographs, as well as birefringence at the emulsion oil droplets border, indicating the liquid lamellar phase [
At least three enzymes (i. e., trypsin, chymotrypsin and neutrophil elastase) are responsible for the degradation of salmon calcitonin (sCT) in the human body [
Stability of sCT was studied in human respiratory epithelial cells. The presence of trypsin, chymotrypsin and neutrophil elastase in cell supernatant and homogenate was studied by Western blot. Enzymes’ activities were studied in cell supernatant by measuring UV absorption, caused by interaction with model substrates.
We observed that sCT concentrations remained unchanged over the period of 2 h, when incubated in supernatant or on cell monolayers. When cell homogenates were studied, sCT concentrations were reduced to 82.5% (hBEpC), 45.9% (Calu-3), 45.1% (16HBE14o-), 4.3% (A549) and 18.5% (Caco-2), respectively. sCT was also degraded when incubated with pure enzymes. Western blot revealed strong signals for all proteinases in the cell homogenates and weaker, varying expression in supernatants.
Calcitonin is rapidly degraded in homogenate, indicating high intracellular enzymatic activity. Epithelial proteases hence, appear to play a role in interactions of sCT with lung epithelium. Although trypsin, chymotrypsin and neutrophil elastase are secreted
This work was funded by a Strategic Research Cluster grant (07/SRC/B1154) under the National Development Plan co-funded by EU Structural Funds and Science Foundation Ireland and by an IRCSET Embark Postgraduate Scholarship.
Wound healing is a complex process with many potential factors that can delay healing. Imbalance between tissue deposition stimulated by growth factors and tissue destruction mediated by proteases causes nonhealing of chronic wounds [
Hydrogels of hydroxyethylcellulose (HEC) and polyvinyl alcohol (PVA) were made at different m/m ratios and their rheological behaviors were performed by rotational viscosimeter (Rheolab MC 100, Physica). For HEC effects of sterilization process and osmolarity of the medium and for PVA effects of freeze-thaw cycles were evaluated. Egg albumin as model protein was incorporated into the hydrogels and drug release studies were performed by developed chamber simulating conditions at wound place. To increase protein stability hydrogels were lyophilized and tested for rehydration ability, viscosity and protein release. Electrospinning, which is a nanofiber-forming process where polymer solution or melt is charged to high voltages, was used for producing nanofibers from the same polymers.
HEC hydrogel at 3w/w % has appropriate rheologic properties, regardless of the medium used (water, phosphate buffer) or sterilization process performed. Strenght and rigidity of PVA hydrogels increases with number of freeze/thaw cycles. Mild process conditions enable homogenous incorporation of protein compound into HEC and PVA hydrogel without deteoriation of its native structure. The release of protein is in accordance with Fick’s diffusion. Freeze-dryed HEC hydrogels were spontaneously rehydrated in 24 hours. Viscosity and thixothropy of rehydrated samples were reestablished and neither lyophilization nor freezing mode affects the protein structure or releases kinetics. Additionaly some data about nanofibers obtained by electro-spinning will be presented. The nanofibers potentially mimic many roles of extracellular matrix to promote tissue growth.
Hydrogels with incorporated drugs that actively interfere in healing process and nanofibers with their unique properties represent improvement for treatment of chronic wounds.
Multiple carbohydrate structures in the chondrocyte glycocalyx can serve as targets for biorecognitive lectins. Recently, we have outlined the applicability of selected plant lectins as mediators of bioadhesion in cartilage research [
Primary human chondrocytes were isolated from donors (n=5) and cultured as high density monolayers. Cells were treated with 10 ng/ml IL-1ß or 40 ng/ml TNF-α. The transcription of 19 glycosyltransferases was quantified using RT-qPCR. N- and O-glycan analysis was performed using LC-ESI-MS.
We found that both IL-1ß and TNF-α increased overall sialylation of N- and O-glycans and induced a major shift from α2,6-linked sialic acid residues towards α2,3-linked sialic acids. These results were supported by RT-qPCR showing increased expression of α2,3 sialyltransferases in treated cells. In this context, ST6Gal1 mRNA levels were markedly reduced 4.7-fold in case of IL-1ß and 3.3-fold in case of TNF-α, whereas the expression of ST3Gal4 was enhanced 2.8-fold and 3.2-fold, respectively. Moreover, we found that both cytokines induced a considerable shift from oligomannosidic glycans towards complex-type N-glycans, whereas core α1,6-fucosylation was found to be reduced particularly by TNF-α.
In conclusion, IL-1ß and TNF-α induce a range of specific alterations in chondrocyte glycoproteins, which might be of relevance for malfunctioning cell-matrix interactions in osteoarthritis. Interestingly, such specific changes in the chondrocyte glycome could also be exploited to target functionalized pharmaceutical delivery systems to osteoarthritic chondrocytes.
This work presents a thorough investigation of the interaction of the novel synthetic pyrrolidinone analog MMK3 and other SARTANS (losartan, valsartan and candesartan) with the model membrane system of dipalmitoylphosphatidylcholine (DPPC). SARTANS are designed to exert antihypertensive activity by functioning as an antagonist of the angiotensin II receptor of subtype 1 (AT1). Small angle X-ray scattering (SAXS) experiments on the interaction of SARTANS with DPPC bilayers were carried out and results demonstrate that all studied SARTANS are well incorporated into the membrane leaflets and furthermore cause partial bilayer interdigitation. Further structural as well as dynamical effects will be discussed, and compared to their overall efficiency as antihypertensive drug.
Density functional theory studies at the B3LYP/6-31G(d,p) and M06-2X/6-31G(d,p) levels have been used to determine the geometries and the enthalpies of formation of several methylated β-cyclodextrins.
Various degrees of methylation can be performed at the hydroxyl groups of β-cyclodextrin at the 2-, 3-, and 6-positions. The number of methyl groups and the position influence the physico-chemical properties of the derivatives as well as their inclusion ability. Large changes of the association constants with different guest molecules are accompanied with increase or decreased solubilities, as well as by a modification of the inclusion mechanisms. In this study three different types of methylated β-CDs were investigated: Heptakis(2-
During building up the various β-CD derivatives C7-symmetry was used throughout. By applying systematic B3LYP/6-31G(d,p) and M06-2X/6-31G(d,p) calculations, the oxygen-oxygen distances were scanned and all remaining geometry parameters were optimized. Three conformational minima were obtained. In the lowest energy conformation two homodromic hydrogen bond rings were formed, one with very short hydrogen bonds at the primary hydroxyls and a second at the secondary hydroxyls of the CD. Four orientations of the homodromic hydrogen bond rings, which are different in energy, have been taken into consideration: both hydrogen bond rims orientated counterclockwise (cccc) or clockwise (cwcw) and the primary rim clockwise and the secondary rim counterclockwise (cwcc) and vice versa (cccw). These four conformations were used to construct the methyl-derivatives of the β-CD molecules. For the optimization with both DFT methods no constraints were imposed on the molecules. To compare the calculated energies with related values obtained experimentally, the respective energies of five crystallographic structures have also been calculated.
The essential manufacturing step of probiotic products involves dehydration of microorganisms, resulting in cellular stabilization and improved formulation and storage characteristics. Spray drying is one of the most common dehydration techniques used for the preparation of pharmaceutical formulations containing probiotics. This method is less time- and cost- consuming as compared to other drying techniques such as lyophilization [
The aim of the present study was investigation of the impact of heat on the cellular activity and culturability of probiotic strains
Oral drug administration is mainly regulated by the step of intestinal absorption. For various substances uptake is strongly superimposed by an active efflux back into the lumen via ATP-driven pumps like the P-glycoprotein (P-gp), significantly decreasing their bioavailability.
P-gp inhibitory effects can be determined via the calcein accumulation assay (CAA) or transport studies in cellular systems. Hereby it was previously shown that particular phospholipids (PL) reduce the efflux of a common P-gp substrate [
The aim of this study is to identify further potent PL derivatives and their molecular mechanism of action and to investigate the influence of different experimental parameters.
CaCo2 and MDCKII-mdr1 cells were routinely maintained in supplemented DMEM. Cells were grown for either 21 days in Transwell® plates in case of transport studies, or for 8, resp. 4 days (MDCKII-mdr1) in 96-well plates in case of CAA.
Lipid derivatives were applied as liposomal formulations in HBSS.
CaCo2 cell layers were pre-incubated with lipid and digoxin (3H-labeled) was added apically for absorptive, resp. basolaterally for secretory studies. Monolayer integrity was determined via transepithelial electrical resistance (TEER) measurements and the ratio of the apparent permeability coefficients (Papp) of both directions displayed P-gp effects.
After pre-incubation with lipid or Verapamil as a positive control the intra-cellular accumulation of the fluorescent dye calcein indicated P-gp inhibition.
In the transport studies one achieved a concentration-dependent enhancement of netto drug absorption with C12-PG, C6-PS and various unsaturated symmetric and asymmetric PC lipids.CAA confirmed these findings in both celllines for C12-PG and other middle-chained saturated PC derivatives.
This work was financially supported by Phospholipid e.V., Heidelberg, Germany
The control of particulate processes and their understanding can be improved by modern measuring techniques. The paper describes the optical probe system Parsum IPP 70 as an example of the modern measuring techniques which is based on the spatial filtering technique (SFT). Measuring principles are the fibre-optical spatial filtering velocimetry and the fibre-optical spot scanning in order to determine simultaneously the size and the velocity of particles. Therefore the measured particle size distributions are chord length distributions which can be recalculated. A calibration is not necessary.
The Parsum IPP 70 is a compact and highly robust measuring system for application as an in-line particle size analyzer for processes involving larger particulate sizes up to 6000 μm. It allows a data rate up to 20,000 particles per second and the system can track continuously a large variety of process parameters. Different options of the measuring system are available: tube length up to 4 m, air supply system for dispersion and cleaning, comprehensive software support, Ex-Zones and pharma solutions.
The application is given for grinding/dosing, agglomeration, fluidized bed processes, mixing and coating, sieving, wet and dry granulation, spray drying. The Parsum IPP 70 is a powerful tool for process control of fluid bed granulation in pharmaceutical industry. The advantages are: increase of process transparency, short response time in the event of process disturbances, continuous control of product quality, full feedback control for automated solutions, no need of samples and laboratory analysis. Results are given by Petrak et al. [
To establish a micro fluidic system that covers the whole production process of drug-loaded solid lipid nanoparticles especially high pressure emulsification and dispersion processes must be taken into account. The advantages of micro fluidic systems in a continuous production imply a high surface-to-volume ratio (material and energy transfer) and a narrow residence time distribution (homogeneously defined stresses). Detailed investigation is needed for process understanding and to improve micro channel geometries.
The micro fluidic geometries were designed to selectively expose product flows to different main break-up mechanisms (shear stress, elongational stress, turbulent stress, particle-particle and particle-wall impacts). Within these groups structural modifications were carried out to evaluate their influence on emulsification and dispersion efficiency.
Two formulations (to simulate the dispersion and emulsification process, respectively) were exposed to the specified micro channel geometries (including channel height variation) at different pressures, optionally using multiple homogenisation cycles in addition.
The emulsification process in general shows a decrease in particle size by elevated pressure and higher number of cycles, as expected. Regarding the pressure drop, straight or bent channel geometries show a much lower breakup efficiency compared to either orifice-like or Y-shaped geometries. The latter two types over all show comparable efficiencies. Applying minor structural modifications to several geometries results in significant differences in particle size. As an example, a cascading quadruple orifice geometry shows significantly smaller particle sizes when the flow first interacts with the smallest orifice in comparison to passing the smallest orifice at the end.
The dispersion of solid particles (hydrophilic alumina Alu C) shows similar results although the drop in particle size is less pronounced in these experiments. This is due to the high binding forces between primary particles.
The authors gratefully acknowledge the DFG for financial support of the DFG research group 856 “Mikrosysteme für partikuläre Life-Science-Produkte“ (
A pharmacokinetic (PK) model describes the absorption, distribution, metabolism and excretion of a pharmaceutical, thus providing, once properly validated, quantitative predictions of the consequences of various dosing regimens. Pharmacological activity might be linked to properties of a time course for a particular compartment (such as half-life or area-under-curve, AUC), or directly modelled by coupling the PK model with a pharmacodynamic (PD) model of the physiological effect of the agent.
Due to the high replication and mutation rates of the human immunodeficiency virus (HIV), which ultimately results in strains resistant to any single agent, a multiple-agent strategy has to be adopted. Highly active anti-retroviral therapy (HAART) uses two or more agents that target different components of the HIV replication cycle, but these agents typically produce adverse side effects in patients. A number of authors have applied techniques from nonlinear control to this problem, but in general the explicit link between the drug dose and the physiological effect, via the PK, is neglected so that the controls derived are continuous in time. In [
In [
The iterative method is applied to the coupled PK-PD model from [
The formulation of solid oral dosage forms significantly determines the release rate of active pharmaceutical ingredients (APIs). The release characteristics of the API are controled by the excipients in the tablet and the tablet microstructure. Computational simulation of tablet dissolution enables tablet designs and formulations to be tested quickly without resorting to many experiments. Crucially, the model offers a method of mapping the available design space with respect to both tablet composition and tablet geometry enabling parametric sensitivity studies to be carried out. This also enables the inverse problem to be solved i. e. for a desired API release rate, the model calculates the tablet composition and/or tablet geometry which achieves this.
The presented model discretises tablets using a grid of volume elements (for non swelling polymers) or discrete element method (DEM) (for swelling polymers), enabling the tablet microstructure to be explicitly defined. Mass transfer between volume elements is carried out using Fick's law with per-component porosity dependent diffusion coefficients. A concentration boundary layer is situated around the tablet whose thickness is defined by the Sherwood number based on flow conditions. Material diffusing across the boundary is considered released and plotted over time to form component release curves.
Optimisation minimises the difference between the simulated API release curve and the target release curve using the mean squared error and the downhill simplex method. This method can be used to find pure component parameters from dissolution experiments or can be used to optimise tablet formulations by adjusting one or more parameters. In this work, the method was applied to optimise a tablet formulation such that it meets a specified API release time. The tablet formulation contained a disintegrant whose fraction was varied in order to change this time. Two release times were specified (fast and slow) and the resulting formulations were then tested with dissolution experiments to validate the simulation.
The model demonstrates that optimisation can be applied to tablet formulation, leading to better Quality by Design (QbD) and faster realisation of custom formulations.
This paper presents results of the simulation of unsteady mixing processes using a unique, mesh-less particle approach. Similar simulations, performed by Hörmann et.al [
The investigations presented focus on the reproduction flow phenomena in a mixing tank, including viscosity adaption due to particle dissolution and analysis of particle concentration using a Discrete Phase Model (DPM). Applying CFD packages, problems including moving boundaries require time consuming work-arounds, which often lead to errors or divergence of the simulation. Due to the used mesh-less method, only the physical and mechanical properties of the problem, like number of revolutions, filling height or initial fluid viscosity need to be defined.
The results of the simulation are compared with Particle Image Velocimetry (PIV) measurements, showing good agreement.
Process performance in the synthesis of API and its intermediates can be reached through new technologies and methods. Significant development time reduction, operating cost reduction by yield improvement, energy savings and a fundamental improvement of plant safety are some of the main results of process intensification concepts. The activities can be divided into 4 phases: 1. evaluation, 2. verification, 3. development and 4. realization. A riskbased approach according to ICH Q9 is used during the whole project.
First of all the identification of reaction performance potential of existing chemical plants needs to be analyzed ending with the formulation of a target for the verification (basic feasibility). The evaluation starts with a process capability check, followed by the generation of a cost flow analysis and ends with a technology evaluation and comparison. Microinnova compares the needs for a chemical reaction with technology profiles to find out the most suitable solution for a process.
In the second phase the intensification potential of the reaction needs to be verified. Typically a continuous plant setup is developed and modified according to the needs of the chemical process. An operating procedure will be generated and optimized. Finally, a rough parameter scan of a few key parameters will deliver a relevant overview for the verification.
Once the potential is proven the process development needs to be done, where the basic feasibility delivers a good basis for the work (phase 3). The reaction process will be optimized. New process options are used as well as new technology concepts like microreactors and flow chemistry to find the best possible process. A knowledge based approach according to ICH Q8 will be used for the development by applying methods like the critical parameter concept and cause effect chains. Engineering data necessary for the scale up will be generated in this part of the work.
Finally in the fourth phase the observed performance needs to be realized. The deep specific knowledge in process intensification equipment needs to be combined with process experience and competence. The best possible equipment solution needs to be selected and calculated under considerations regarding the common standards like ATEX or cGMP. The gap between continuous processing and multi-purpose flexibility can be bridged by modularization of continuous plants.
Nowadays, a considerable range of industrial applications (pharmaceutics, cosmetics, foods agriculture products) relies on microencapsulation of solids or liquids by polymer coating and entrapment into polymer matrices.
Production of these micro-systems doubtless represents one of the most important improvements in the development of functional and therapeutic products belonging to the industrial fields above reported.
Generally, microencapsulation is widely used in pharmaceutical and nutraceutical fields, mainly to improve stability or to modify active principles release. Smart micro-systems release active molecules in fuction of external stimuli (pH, temperature). Preparation of the microparticles is achieved by both physico-chemical and mechanical processes. Feasibility studies about a process of microencapsulation are achieved by manual procedures in a lab-scale. The path toward commercialization of micro-particles needs plants or automated equipments on lab- or pilot-scale first, eventually on industrial scale. However, the state-of-art about microencapsulation techniques displays several processes based on manual operations that often don’t find an applicative realization in industrial field. Moreover, equipments reported in literature, especially in lab-scale systems, are not versatile devices and are penalized by a high consumption of resources. This work is focused on the development of techniques able to overcome such limitations. A promising new technique with low energy consumption consists in the coupling between ultrasonic atomization and microwave drying to produce micro-systems through an intensified process. The variation of energy/frequency of ultrasounds gives the dimensional distribution more suitable for the final applications. The microwave drying modifies materials structure giving unique properties to the micro-particles.
The propylene glycol Liquorice, Marigold and Yarrow extracts (LE, ME, YE-respectively) were obtained by the repercolation method, in the plant:extract ratio 1:2. Its appearance, relative density, index of refraction, pH values and dry materials content were evaluated. The extracts were encapsulated into the instant liposome (Natipide® II, Nattermann Phospholipids, Germany). Liposomal encapsulation of LE, ME and YE extracts was done by mixing of instant liposomes and the extract (2:1) in an ultrasound bath for 10 min. The obtained dispersions were diluted with purified water, in the ratio 1:1 and homogenised during 15 min to get final dispersion LD, MD and YD, respectively. Average radius of the liposoms size in dispersions was determined spectrofotometrically. For obtaining the O/W emulsion-type creams-vehicle, applying the cold-cold method, we used 5%w/w Emulgin®VL75 emulsifier [Lauryl Glycoside (and) Polyglyceryl-2 Dipolyhydroxystearate (and) Glycerine] (Cognis, Germany). The composition of vehicle is the same as described previously [
This work was supported by Serbian Ministry of Science and Technological Development, project number TR 20137.
In order to reach the deep lung API (active pharmaceutical ingredient) particles must have an aerodynamic diameter of 1 μm to 5 μm. Powders of this size are rather cohesive and exhibit poor flowing properties. Due to the fact that dosing is done volumetrically sufficient flowability is crucial. One solution to cope with cohesivity is to mix the API with coarser carrier particles of sufficient flowability. On the one hand interparticular forces of such interactive mixtures must be high enough to guarantee powder uniformity during mixing and powder handling and on the other hand low enough to allow API detachement form the carrier upon inhalation. Since interparticular forces depend on the contact area between API and carrier particles and consequently surface morphology the variation of the carrier surface may lead to optimized dry powder inhaler (DPI) formulations. Experiments of Maas [
The aim of this work was to prepare spray dried mannitol carrier particles of variable surface roughness. Intended particle size was between 50 μm and 100 μm. Interactive mixtures of carrier particles with salbutamol were prepared and the respirable fraction was determined according to the European Pharmacopoeia using the next generation impactor (NGI).
The lipophilicity of a drug plays a key role for its distribution and accumulation in the human organism. The analysis of the distribution behaviour between buffer and a model membrane is most suitable to ascertain the partition coefficient between hydrophilic and biological membranes. Liposomes are common membrane models and ideal systems to characterize drug-membrane interactions [
We verified an indirect screening method to investigate the liposome-buffer partition coefficient using the fluorescent dye 6-Lauryl-2-dimethylaminonaphtalene (Laurdan). Laurdan offers a distinctive emission spectrum which is characterized by a shoulder at 434 nm and a maximum at 486 nm. The ratio of the fluorescence intensities at these two wavelengths is termed as the membrane state parameter (MSP). MSP changes depend on the polarity of the dye’s enviroment. After insertion of drug molecules into the liposomal membrane, the MSP value decreases as a function of the amount of inserted drug. The calculation of the MSP for different drug concentrations and various lipid amounts enables the determination of the drug’s partition coefficient [
This indirect method offers the possibility to analyze the partition behaviour of a broad range of different drug classes without requiring further operations like separation steps or specific drug detection.
In an initial study, we determined the membran partition behavior of bile salts as amphiphilic model drugs.
Rhabdomyosarcoma is the most common soft tissue sarcoma of childhood. Especially the alveolar rhabdomyosarcoma (ARMS) shows poor prognosis when metastases have developed.
The aim of this project is to downregulate the expression of several genes that are involved in the aggressive behaviour of ARMS by means of siRNA. Therefore, a liposomal delivery system, which is able to transport siRNA selectively to the ARMS cells, is being developed.
For enhancing the interaction of the delivery systems with the target cells and thus the effectivity, active targeting is intended. Therefore, the surfaces of the liposomes were modified with ligands binding to receptors expressed on the ARMS. Two different kinds of targeting devices were utilized: a RGD-peptide (arginine-glycine-aspartic acid tripeptide) recognizing integrins on ARMS as well as an antibody binding to the IGF1-receptor (insulin-like growth factor 1 receptor).
siRNA was encapsulated into the liposomes during liposome preparation by speedmixing (dual asymmetric centrifugation). This method is well suitable for efficient siRNA entrapment [
To modify liposomes for active targeting the ligands were coupled to the liposomal surface using the sterol-based post-insertion technique (SPIT) [
For optimization of the delivery systems, cellular interaction of fluorescently labeled liposomes with the ARMS cell line RH-30 and gene silencing of siRNA-loaded (anti-GFP siRNA) liposomes in stably GFP-transfected RH-30 cells were determined by flow cytometry.
RGD-peptide as well as anti-IGF1-receptor antibody modified liposomes showed significant interaction with RH-30 cells. Furthermore, specificity and extend of gene silencing was analyzed.
Blood vessel diseases like antherosclerosis lead to formation of blood clots with severe consequences like acute ischemic stroke or myocardial infarction [
A well-directed treatment of thrombotic events implies fast localization of the blood clot as well as fast and effective destruction with low doses of lytic agents to prevent the patient from severe damage. Standard procedure is application of high doses of thrombolytic agents with bleeding complications as negative side effects. Daffertshofer
The aim of this study was to proof the concept that ultrasound active liposomes, surface modified with antifibrin antibodies, can act as drug carriers. We focused our work on the development of novel ultrasound active liposomes which are stable under similar conditions as in human body like blood pressure, physiological pH and body temperature. They are composed of DPPC/PEG40-stearate or DSPC/PEG40-stearate and show a small diameter between 100-200 nm with a sharp polydispersity. In order to develop long-circulating immunoliposomes, which combine sterical stabilization with a superior targetability, we attached fibrin antibodies directly onto the distal ends of s synthesized DPPE-PEG derivative, which had been endgroup-functionalized with cyanuric chloride [
The authors would like to thank the DFG “Forschergruppe Nanohale 695” for support.
Recently, increasing attention has been paid to lecithin/chitosan nanoparticles as colloidal drug delivery system showing the great potential to improve the permeation of encapsulated drug across various biological barriers [
Before the lyophilisation process, lecithin/chitosan nanoparticles obtained were characterised by mean diameters ranging between 117.4 ± 3.9 and 328.5 ± 3.1 nm, and were significantly larger than comparable lecithin nanoparticles. The zeta potential was inverted from negative values for lecithin nanoparticles to positive values for lecithin/chitosan nanoparticles (up to 30.2 mV).The size and the surface charge of the nanoparticles increased with the increase in the chitosan content and the negative charge of lecithin used in the preparation. Compared to lecithin nanoparticles, lecithin/chitosan nanoparticles were characterised by significantly higher melatonin loadings (up to 7.1 %, w/w).
In the lyophilisation process it was shown that a minimal concentration of 2.5 % (w/v) of trehalose or glucose was needed for a suitable reconstitution of nanoparticles lyophilised at particle concentration of 2 mg/ml. No significant difference in the mean particle size, size distribution or zeta-potential of nanoparticles in suspension before and after freeze-drying and reconstitution were observed. No significant melatonin leakage caused by the stress of freezing and dehydration in the presence of cryoprotectants occurred.
This work was supported by Grant 006-0061117-1244 of the Ministry of Science, Education and Sports of the Republic of Croatia.
Atherosclerosis is the major cause of mortality in the western world today. The main objective for the use of nanotechnology is to address prospects of imaging atherosclerotic (AS) plaques, which lead to clinical endpoints. Today various biomarkers are known to be involved in the pathophysiologic scenario of AS-plaques. For the targeting the biomarker globular Adiponectin (gAd) [
The eye provides a variety of defense mechanisms, which protect the organ against numerous different substances. These mechanisms include the bony orbit, the eyelids and cilia, the tear film and nasolacrimal drainage, corneal and conjunctival epithelium, and a submucosal secretory immune defense. In order to overcome the strong physiological barriers, new drug delivery systems have been established, including the development of nanocarriers. However, it is known that nanostructured materials are able to invade cellular structures and trigger toxic reactions.
For the establishment of an
Size and surface charge of the particles differed according to the medium. Particles suspended in tear fluid showed reduced surface charges and diameters three times higher, compared to those, suspended in MQ-water and 0.9% NaCl. This increase in particle size can not only be explained by partial aggregation of the particles but also by the fact that in biological fluids, proteins attach to the nanoparticle surface, which leads to the so called ‘protein corona‘. Histopathological Analysis showed that corneal epithelium and endothelium detached during prolonged storage and incubation. Fluorescent particles were seen in the entire corneal stroma.
20 nm CPP can penetrate through Bowman′s and Descement membrane and the entire corneal stroma. Corneae with shorter culturing times will be used for the assessment of the epithelial barrier function.
In studying
The aim of our study was to compare in vitro release profiles of budesonide from NPs, which were obtained using four different methods for the assessment of drug release: membrane diffusion technique (dialysis or reverse dialysis) and sample and separate technique (filtration or ultracentrifugation).
Budesonid as a hydrophobic drug was incorporated into poly(lactic acid) NPs, which were prepared by the nanoprecipitation method using vibrating nozzle device [
No clear agreement exists on the suitability of the techniques reported to date for drug release determination from particles in the nanometer size range. Our results show that the comparison of
Ketoprofen is a potent non-steroidal anti-inflammatory drug and an excellent candidate for transdermal/dermal delivery because of its adverse side effects and short elimination half-time after oral administration [
Formulation development of nanostructured lipid carriers (NLC) aims physical stability in both particle size and crystalline state. Besides production parameters, the selection of appropriate stabilizers is the most crucial parameter in the development. Very often the influence of different stabilizers on the size and the physical stability is investigated. However, little is known about the influence of stabilizers on the crystalline state of lipid nanoparticles (LN). Thus, two surfactants of polyglycerol fatty acid ester type were screened for their ability to form physically stable NLC and for their influence on the crystalline state of the lipid matrix. The investigated surfactants are polyglycerol 6-distearate (PD) and polyglycerol 6-isostearate (Pl). Physical stability study was performed using light microscopy, dynamic and low angle static light scattering. DSC study was used to analyze the crystalline status of LN. Both surfactants in the concentration of 1.0 % (w/w) led to almost similar LN with a mean diameter between 180–200 nm. LN were stable over the period of 90 days. The absence of large particles was also confirmed by light microscopy and low angle static light scattering. In contrast to this significant differences were found within DSC thermograms. LN stabilized with PD show a melting event, whereas LN stabilized with PI show no melting event. Hence, PI inhibited the crystallization of the lipid matrix, leading to a non-crystalline LN instead of NLC. A possible explanation is that PD which has a solid lipid tail leads to a different interaction with lipid matrix, than the liquid lipid tail surfactant (PI). These interactions can lead either to an initiated crystallization or to the circumvention of crystallization. In conclusion: Stabilizers can influence the crystalline state of the lipid matrix of LN. Thus, for a successful development, this parameter needs to be investigated. Furthermore, it might be a helpful tool to develop NLC with a “tailor-made” crystalline state and release profile in the future.
The authors would like to acknowledge the financial support obtained from DAAD (Deutscher Akademischer Austauschdienst) for the present research.
Solid lipid nanoparticles (SLN) represent rapidly growing class of colloidal transport system, particularly interesting for pharmaceutical applications [
The advantages of the
Accordingly, we present an extremely sensitive tool to monitor the location of fluorescently labelled NPs by SPP-189 within respective organelles that should find a wide application in drug study.
Imaging of internalized SLN (marked with arrows) located next to the nuclei (rounded structure). A - SLN-SPP are seen distinctly as (blue) dots, while B - SLN-C are hardly recognized due to the broadly distributed fluorescence from 6-coumarin (green). Bar is 10 μm.
Nanoparticles composed of silica and its derivatives are currently in use both in basic scientific research and applied enginnering development. Precursor silica particles have been investigated for applications in microeletronics, chemical, biological sensors, as targeting devices and in drug delivery [
This work was supported by FCT, SFRH/BD/60640/2009 to T. Andreani and by Capes to A.L.R. Souza. Whe are thankyfull to Dr. L. Fernandes for TEM assistance.
Aspergillosis is a common fungal infection in falcons. Standard treatment, systemic application of Itraconazol, is limited due to hepatotoxicity. The aim of the present study was to develop Itraconazol-loaded Nanostructured Lipid Carriers (NLC) for pulmonary application. NLC are a nanoparticulate carrier system composed of a blend of solid lipid and liquid lipid, with a particle matrix being solid at body temperature. NLC have shown many advantages for pulmonary application such as deep lung deposition due to the small particle size, adhersive properties leading to an accumulation in the lung, prolonged release properties leading to a reduction of dosing frequency and low toxicity due to the use of well tolerated excipients.
NLC composed of 0.02% Itraconazol (Jai Rhades Sales, India), 4.5% Precirol ATO 5 (Gattefossé, Germany), 0.5% oleic acid (Croda, Germany), 2% Tween 20 (Cognis, Germany) and 92.81% Milli-Q water were prepared by hot-high pressure homogenization method (Panda K2, GEA Niro Soavi, Germany). The particle size of NLC was determined using a NanoSizer ZS (Malvern Instruments, UK). The melting point of NLC was assassed by differential scanning calorimetry using a DSC 204
Itraconazol-loaded NLC with an average particle size of 119 nm and a polydispersity index (PI) of 0.254 were obtained applying 5 homogenisation cycles at 500 bar. NLC showed a melting point of 45.1°C proving the solid state of the particle matrix of the carrier system. Nebulizing the NLC dispersion using a Nanonebulizer led to an aerosol with an average particle size of 265 ± 1.78 nm. The particle size of NLC in the aerosol was found to be of the same order of magnitude as before nebulization, which shows that after nebulization the favorable characteristics of the delivery system are maintained.
Nebulization of Itraconazol-loaded NLC using a Nanonebuizer by MedicActiv leads to an aerosol with favorable characteristics for pulmunar treatment of aspergilosis in falcons.
This work was supported by MedicActiv Vertriebs-GmbH (
In order to use API (active pharmaceutical ingredient) particles in a dry powder formulation, they have to exhibit an aerodynamic diameter of 1 μm – 5 μm. Only this order of magnitude guarantees a penetration to the deeper parts of the lungs. Particles of this scale are very cohesive and possess rather poor flow properties [
The aim of this work is to create nanoparticles via a novel spray drying technique. With the help of this apparatus, it is feasible to produce API particles in the nanometer range. The intended benefit is the absence of any excipient, because both, the drug itself (1 μm – 5 μm) and the nanoparticles, acting as flowability enhancers, are made of the same API substance. During a mixing process [
The major limitations for the use of small interfering RNA (siRNA) in vivo and in vitro are the relatively high molecular weight, the negative charge, and the susceptibility to nuclease degradation [
The authors would like to acknowledge the financial support of the study by FFG, Bridge-program: P810994.
Pharmaceutical drug formulations in the nanometer scale received significant interest during the last years as they provide the possibility to stabilize drugs and to transport them directly to their target. Biodegradable self-assembled nanoparticles consisting of protamine and oligonucleotides, so-called “proticles”, have already been successfully developed for different applications [
The project is supported by one-A engineering austria, Prager Elektronik and the Austrian NANO Initiative (Nano-HEALTH).
Ischemic stroke results from a cerebral vessel occlusion by a blood clot. Treatments are intended to restore the cerebral blood flow as soon as possible to avoid major brain damage. Intravenous administration of recombinant tissue plasminogen activator (rtPA) is the only thrombolytic drug approved for treatment of acute ischemic stroke [
The aim of the present work was to set up a robust
Practically, fresh whole human blood clots were formed after three hours at 37°C in 200 μL glass micropipettes through which a silk suture had been threaded. The clots were characterized by their diameter, by scanning electron microscopy (SEM) and by immunostaining. The clots were placed in a cell mounted with transparent Mylar® films, and filled with human plasma. The cell was then placed in a thermostated water bath (37°C) and the clot was exposed to different experimental conditions of ultrasound (0.65MHz), rtPA and microbubble suspensions in a continuous infusion. Every five minutes during 60 minutes photographs were taken and analysed to determine the mean diameter decrease of the clot.
The clots had an initial diameter of 0.89 ± 0.06 mm (mean diameter ± sd). In the presence of rtPA at 3 μg/mL, microbubbles and ultrasound, the mean clot diameter dropped by more than 0.5 mm in 60 min. Under SEM and with the immunostaining, sonothrombolysis effects were clearly observed.
These preliminary experiments are encouraging and will enable a thorough evaluation of the synergetic effects of microbubbles, ultrasound and rtPA.
The majority of topical ocular preparations available today are in the form of aqueous eye drops. This might be due to existing problems related to ocular drug delivery systems such as cost, bulk manufacturing, and patient compliance. Commercial eye drops are commonly used by patients, due to their ease in usage and low interference with vision. A homogenous dosage solution form offers many industrial advantages including the simplicity of large-scale manufacturing. At the ame time, commercial eye drops are often ineffective and require frequent application. Only 1–5% of the applied drug penetrates the cornea and goes into intraocular tissues [
Micelle systems composed of the polyoxyethylated nonionic surfactant Pluronic® F127 (F127) and cationic polyelectrolyte chitosan (CH) were prepared with dexamethasone (DEX) as a hydrophobic model drug. The F127/CH micelles were characterised by their hydrodynamic diameter and a zeta-potential ranging between 25.4 and 28.9 nm and +9.3 and +17.6 mV, respectively. The DEX loading was between 0.48% and 0.56%, and no significant influence of CH on DEX loading was observed.
This colloidal carrier was well tolerated in rabbit eyes, and no clinically abnormal signs in various ocular structures were observed. The increase in intraocular pressure (IOP) in rabbits was used to evaluate DEX ocular bioavailability. The AUC values showed a 1.7- and 2.4-fold increase in bioavailability with F127 and F127/0.015 w/v % CH micelle systems, respectively, as compared to a standard DEX suspension. These data indicate improved intraocular DEX absorption from the micelle systems, which can be ascribed to both F127 and CH corneal permeability enhancement.
This work was supported by Grant 006-0061117-1244 of the Ministry of Science, Education and Sports of the Republic of Croatia
Different promising colloidal delivery systems including microemulsions and liquid crystals could be obtained with suitable combination of oil, surfactants/cosurfactants and water. However, the main problem from a formulation point of view has been to choose the surfactants capable of forming such systems and be physiologically acceptable in the same time. To overcome the later shortcoming the use of less toxic non ionic or biocompatible naturally obtained surfactant is proposed [
The aim of this study was to develop and characterize colloidal systems for dermal delivery based on nontoxic components, i. e. lecithin and Tween 80 as surfactant mixture, isopropyl myristate as oily phase and water.
Pseudoternary phase diagrams were constructed using conventional titration technique with 1:1 and 2:1 mass ratios of Tween 80 to lecithin, respectively, isopropyl myristate and water. Clear samples of high viscosity were deemed to be lyotropic liquid crystals. Representative samples were further evaluated for their physical stability and rheological characteristics, while the structure of the samples was examined with a polarization microscope.
The region of clear and highly viscous mixtures was detected at higher concentrations of Tween 80/lecithin (at both mass ratios) that were further on confirmed as lamellar lyotropic liquid crystals with polarization microscopy due to characteristic “Maltese cross” textures The samples were physically stable at room temperature during 2 months and after centrifuge test. Determination of viscosity and flow curves showed shear-thinning characteristics and thixotropic behaviour of samples containing more than 35% of water, while samples with less than 30% of water revealed rheopectic properties. Additionally, the viscosity was decreasing with increasing temperature, and was affected by composition of surfactant mixture (higher content of Tween 80 induced higher viscosity) and water content (up to 40% water content increased viscosity due to hydration of lecithin polar head groups).
Lamellar lyotropic liquid systems were identified from phase diagrams at higher surfactant concentration. They show great potential for dermal application due to nontoxicity of surfactants, physical stability and moreover, convenient rheological properties.
Solid dispersions of active pharmaceutical ingredients (APIs) in freely water soluble carriers are often used in order to improve dissolution rate and/or solubility of poorly water soluble drugs [
There are several techniques that are used in order to either determine the state of dispersion of the drug in the carrier or to monitor storage induced changes. Conventional techniques include thermal analysis and wide angle X-ray diffraction, although the detection of very fine drug crystals in the carrier or the transition of an initial solid solution to a solid suspension at a very early stage is almost impossible.
We apply simultaneous small and wide-angle X-ray scattering, which is becoming more important for solid-state pharmaceutical analytics [
Hot-melt granulation techniques have received increasing attention due to the significant advantages compared to the conventional granulation methods. Fluidized hot melt granulation (FHMG) is recognized as promising technique, but there is still lack of information related to this topic [
Granulation process was performed in Mycrolab fluid bed processor, OYSTAR Hüttlin. Precirol ATO 5 (Gattefossé, France) and PEG 2000 (Fluka AG, Switzerland) were used as meltable binders that were initially present as discrete particles within fluidized bed. The effects of binder content, particle size of the binder, granulation time, and the inlet air speed on the granule flow properties (Carr index) and drug dissolution parameters (T40, T80) were investigated using factorial design.
Formulations with Precirol ATO 5 showed considerably slower drug release due to the forming of the lipid matrix. The binder content had significant influence on granule properties, as well as on drug release rate in the case of both formulations with Precirol ATO 5 and formulations with PEG 2000. Concerning the paracetamol dissolution from tablets prepared with Precirol ATO 5, particle size and granulation were also found to be significant factors. Furthermore, two-factor interaction between the binder content and the particle size was identified.
FHMG is simple and rapid granulation technique that enables a formulator to manipulate drug release rate depending on the nature and concentration of the meltable binder.
The Leistritz Micro Pelletizer (LMP) is a tool to cut extrudates from a twin-screw extruder to a defined shape and size. The purpose of this study was to evaluate the influence of the parameters to the granual properties in a wet granulation process. 33 experimental design was applied.
A formulation consisting of 40% microcrystalline cellulose (MCC M101, Pharmatrans Sanaq, Switzerland) and 60% lactose (Granulac 200, Meggle, Germany) was used. A preblend was transferred to the gravimetric powder feeder of the twin-screw extruder (18GL-40D, Leistritz, Germany). The extrusion took place at a constant powder feed rate of 25g/min and different liquid feed rates. Deionized water was used as granulation liquid. The Pelletizer (Leistritz Micro Pelletizer, Leistritz, Germany) cut the extrudates when emerging from the orifices of the die-head, using variable cutting blade speed. Image analysis was conducted by using the Camsizer (Retsch Technology, Germany).
The study dealt with the effect of 3 different process parameters – screw speed, liquid feed rate and cutting blade speed – on the pellet shape and size. These parameters were varied systematically in a 33 full factorial design. In addition, 3 experiments were performed at the center point in order to approximate the reproducibility.
The regression model was simplified by a backward regression. The pellet size and the pellet shape were characterized by equivalent diameter and aspect ratio, respectively. The most spherical pellets were obtained by using a liquid feed rate of 0.95kg/h and a cutting blade speed of 2500rpm. In the variance analysis, the cutting blade speed and the screw speed were insignificant, whereas the liquid feed rate had an effect to the pellet shape and size.
One of 3 investigated process parameters affected the pellet properties. Neither the screw speed nor the cutting blade speed showed an influence on the pellet shape and size. The liquid feed rate was crucial in the used experimental setup. The continuous granulation process using the Leistritz Micro Pelletizer (LMP) in combination with the twin-screw Extruder (Leistritz 18GL-40D) was robust and reliable.
Pellet manufacturing by extrusion/spheronisation is quite common in the pharmaceutical field because the obtained product is characterized by a high spericity as well as a narrow particle size distribution. After extrusion of the bulk material, the cylindrically shaped extrudates are transferred to the spheronizer, where they are plastically deformed into spherical pellets. The established mechanisms [
This study investigates methods for visualizing and understanding the interparticle interactions and a possible mass transfer during the spheronisation step. Therefore, two common pelletization aids (Microcrystalline cellulose (MCC), kappa – carrageen) were used in combination with lactose as a filler. To visualize the interparticle exchanges of mass during the spheronization process, different colored extrudates were spheronized simultaneously, and the color change of the different particles was monitored.
The data obtained indicated that mass transfer between particles must be considered in addition to plastic deformation in order to capture the spheronization mechanism. A material transfer between pellet particles was observed in all investigations (
In conclusion, the commonly espoused pelletization mechanisms need to be extended to account for material transfer between pellet particles, which has not been considered before.
Images of samples taken during spheronization after different for a pure MCC formulation
The problem emerging from particle coating is how to manufacture particles with just enough material deposited to achive the desired result [
Coated multiparticulate systems are increasingly popular on the market. Their manufacture in a fluid bed often requires polymer binders. Polymers have been widely studied as inhibitors of crystallization [
In our experiments we studied the thermal behaviour of diltiazem hydrochloride-layered pellets before and after coating. The multiparticulate pellet samples were prepared in a Strea-1 (Niro Aeromatic, Bubendorf, Switzerland) fluid bed Wurster chamber. Acryl-EZE dispersion (a fully formulated USP copolymer type C coating system) was prepared and applied following the guidelines provided by the manufacturer as a 20% aquous dispersion. The thermoanalytical examinations were carried out with a Mettler-Toledo 821e instrument with a dynamic method in the interval of 0–400°C, at a heating rate of 10°C/minute.
We found that the coated samples exhibited an exothermic peak between 90–110°C that wasn’t characteristic of any of the used materials. Further examinations suggested that the exothermic peak is the result of recrystallization; this theory was tested with XRD. Results showed that the uncoated drug-containing pellets contained only a relatively small amount of the crystalline drug. Coated samples behaved similarly. XRD examinations after heating the samples to 120°C (above the range of the exothermic peak) showed a definite increase in the crystallinity of the drug. Thermal examinations showed a correlation between the thickness of the coating and the extent of recrystallization.
One explanation of the above is that the water-soluable drug dissolves into the aqueous coating dispersion on the surface of the pellets during the coating and crystallizes only in a small extent as the film is dryed; most of the drug forms a molecular dispersion in the film. Crystallization is completed only on heating, as can also be proven by microscopic evidence.
This work was supported by “Stiftung Aktion Österreich-Ungarn“.
Flows encountered in processes involving turbulent bubbly flow are highly complex, especially when the gas holdup and gas-liquid interface mobility are such that coalescence and breakup of bubbles take place. These phenomena have an enormous impact on the performance and productivity of the processes. Attempts have been made by researchers to gain insight in the hydrodynamics of bubbly flows by detailed numerical simulation. However, resolving coalescence and breakup has often been ignored due to their large computational requirements.
The goal of this presentation is to assess the feasibility of using computational fluid dynamics (CFD) modeling as an engineering tool for analyzing, designing and scale-up of processes involving bubbly flow with coalescence and breakup of bubbles. The parallel and highly efficient modeling technique for bubbly flows described in the work of Sungkorn et al. [
Simulations of gas-liquid flow in a bubble column have been performed. The simulations provide detailed insight for the liquid flow field as well as the gas dispersion pattern. The accuracy of the simulations is demonstrated by comparing the predictions with experimental data. Additionally, excellent speedup and scalability on parallel computing platforms demonstrate the capability of the present modeling technique for simulations of large-scale reactors.
Granular flows are extremely important for the pharmaceutical and chemical industry, as well as for other scientific branches dealing with granular media. Thus, the understanding of the impact of particle size and related effects on the mean as well as on the fluctuating flow field in granular flows is critical for design and optimization of powder processing.
We use a specialized simulation tool written in C and CUDA, a massive parallelization technique which runs on the Graphics Processing Unit (GPU). It was chosen to be able to simulate significantly higher amounts of particles in acceptable runtime.
We focus on flow fields and wet-mixing properties obtained in the lower million particle range. In our talk, we present simulation results of a pitched blade mixer on a laboratory scale. Particles with a medium particle size of 365 micrometers are blended at 30–60 rpm for several turns (> 10 revolutions). The GPU implementation enable the DEM simulation of quartz powders in the real geometric resolution. In order to investigate the mixing process during the continuous spraying of a liquid, the DEM code was extended suitable. Liquid films and bridges implement the spread of the fluid in the aggregates. Depending on the wetness level on particle surfaces, capillary forces account for cohesion and agglomeration. The simulations will shed light on the optimum location and the type of fluid entry, and allow predictions about the time until a homogeneous moisture distribution in a blended bulk can be achieved.
Complex process engineering like drying of fluidized particles in hot air streams is a huge challenge for numerical simulations. Highly resolved two-phase flow of one million particles will be investigated. Here we focus on both, a gas-phase simulated using the Lattice Boltzmann Method (LBM) and a coupled solid-phase of discrete particles, simulated by using Discrete Element method (DEM). Subgrid scale two-way coupling of liquid and solid flow allows the investigation of residence time as well as other important process parameters and the prediction of an optimum operation regime.
Particle coating using fluid bed technology such as Wurster chamber is a common process in pharmaceutical technology. Coatings are used to achieve variety of functions, such as: taste masking, controlled release, increase of stability and others. Coating uniformity is one of the most important parameters of coated particles and can play an important role in the release of the active ingredient and is a result of number of process settings and material properties [
We have analysed the effect of coefficient of restitution on particle distribution and their velocity in the Wurster process chamber. Simulations were performed on a 2D axisymetric model using Ansys Fluent. Euler-Euler approach was used for simulation of gas and solid phase. The particle size was set at 1 mm and the coefficient of restitution was set from 0,1 to 1,0 in 0,1 steps. All other process parameters such as fluidizing and atomizing air flow were kept constant.10 s of two-phase flow was calculated in each simulation using timestep of 2.5*10−5 s, starting from the same initial condition.
The analysis of the simulation has established that in most cases higher values of coefficient of restitution increase the particle volume fraction in the coating region of the chamber. The lowest value of volume fraction of particles in the coating region was 6.82% and the highest was 9.41%. It was established that coefficient of restitution has effect on particle velocity and flow rate through the coating region, however far less pronounced as in the case of volume fraction, which is one of the main factors that affects uniformity of coating. Therefore, according to our findings elastic properties of the particles can affect the outcome of the coating process i. e. coating functionality, even when using the same process chamber and process parameters.
The aim of this study was to investigate the possibility of using Polyox® as the binding and/or coating agent for paracetamol sustained release formulation development.
Paracetamol granules were prepared in fluidized-bed apparatus, using different ratios of Polyox® as binding agent. Granules were coated with Polyox® solution by applying the bottom spray fluidized-bed technique. Tablets were prepared by compressing coated granulates with an excenter tablet press. For comparison purposes, sample tablets were also prepared by direct compression of the homogeneous mixture of paracetamol, Polyox® and microcrystalline cellulose. In vitro release studies were performed in the paddle dissolution apparatus, using USP phosphate buffer pH 5.8.
The rate of drug release from Polyox® based formulations was determined by swelling and diffusion through the gel layer formed. Dissolution study results of coated granules show that an increased Polyox® level in the formulation resulted in a markedly decreased drug release rate. Similarly, tablets made from coated granules exhibited slower and additionally decreased drug release compared to uncompressed granules [
The results obtained indicate the potential use of Polyox® polymer as the binding and coating agent in the sustained release tablet formulation.
This work was supported by the Ministry of Science and Technological Development, Republic of Serbia project TR23015.
In recent years, the interest in melt granulation has increased due to the advantages of this technique respect to traditional wet granulation; utilizable equipments for melt granulation are high-shear mixer and fluidised bed [
The aim of the study was to evaluate, by asymmetrical factorial design [
The experiments have been performed in a lab scale apparatus (Mini-Glatt), employing spray dried lactose monohydrate as filler. Six factors were studied at different levels: the binder type was evaluated at 2 levels (Lutrol F68 and Lutrol F127), while the percentage of the binder, the size of the binder particles, the inlet air temperature, the inlet air rate and the granulation time were studied at 3 levels. The 16 experiments were run randomly in duplicate. Five variables were selected as experimental responses: the total process time, the yield, the particle size (d50), the friability and the flowability of the produced granules.
The analysis of the results showed that the percentage of the binder, the inlet air temperature and the inlet air rate significantly influenced all the experimental responses, while the binder type, the size of the binder and the granulation time had minor “weight” on the entire process. Five additional tests allowed to assess the validity of the screening.
In conclusion the factorial design results permitted to identify the most important variables of the in situ melt granulation process in fluidised bed. These findings result crucial for the ongoing further optimisation phase of the process.
We were interested in the study published by Dr. Alizadeh Ghavidel et al.,
The left internal mammary artery (LIMA) is mostly used as the conduit of choice for myocardial revascularization. The LIMA confers superior graft patency and better long-term survival and causes fewer cardiac events than does the saphenous vein.
With respect to our experience, a 70-year-old male patient with kyphoscoliosis and chronic obstructive pulmonary disease (COPD) presented with post-myocardial infarction angina. He underwent emergent coronary artery bypass grafting (CABG), during which the LIMA was harvested without pleurotomy. A pericardial drain was fixed postoperatively for 6 days, after which time it was removed. It is noteworthy, however, that we ourselves tend to remove the pericardial drain after 2 days. Our patient had a good postoperative period and was discharged on the 10th postoperative day without pulmonary complications. Pre-discharge chest X-ray and echocardiography showed no pleural effusion or pericardial effusion, and post-CABG delayed pericardial effusion was ruled one month later via echocardiography.
We believe that LIMA harvesting without pleurotomy during CABG is associated with lower pulmonary complications, especially in patients with pulmonary comorbidities such as COPD and chest wall deformity. Nevertheless, intact pleura may cause more pericardial effusion and tamponade. We would, therefore, recommend that in these patients, pericardial and retrosternal drains be fixed and kept until pericardial space drainage has dropped to lower than 50 cc per day. Nonetheless, when pleurotomy is done, the drain should be fixed and kept until pericardial space drainage has reduced to lower than 150 cc per day.
I eagerly studied Sayadmansour’s article.
The author correctly refers to the necessity of establishing a relationship between neuroscience and theology and the point that this can lead to achieving new insights in the field of neuroscience and the manner of human exposure to the surrounding world as well as promoting theological perceptions; however, I believe that the article does not determine the position of neurotheology. We cannot figure out the limits of neurotheology and more importantly, unfair expectations from this newfound science after studying the article. In my opinion, only a historical outlook to neurotheology can answer this question.
Perhaps one of the primary objectives of neurotheology was demoting religious and mystic experiences to neurophysiologic activities. God Helmet made by Michael Persinger
To assess the association between medication use and the incidence of delirium among residents residing in long-term care facilities (LTCFs).
This study was completed as part of a larger multisite, prospective observational study conducted within 7 LTCFs in Quebec. Delirium was assessed via nurse interviews, chart review, and weekly resident assessments for up to 24 weeks using the Confusion Assessment Method. Data were collected pertaining to both molecule and dose of all prescribed medications taken by residents on each day of study. Drugs were categorized by level of cholinergic activity both categorically (anticholinergic drugs, drugs with anticholinergic side effects, agents with published evidence of association with delirium, medications whose indication is either for or associated with delirium) and by the Anticholinergic Cognitive Burden (ACB) Scale. The study cohort was analyzed using a nested-case control method, with each incident case of delirium (the date of which was assigned as the index date) matched to up to 33 controls by institution and study time. Residents were defined as being currently exposed to a drug if they had taken the medication in the 28 days leading up to the index date. The rate ratio (RR) of delirium associated with current and new use of medications was estimated using conditional logistic regression, adjusting for age, sex, dementia severity, time since institutionalization, Charlson Co-morbidity Index score, documentation of a new medical problem or hospital visit, number of drugs being taken, and the number of environmental risk factors for delirium exposed to at the index date.
The study cohort included 279 residents, of whom 83 were classified as having incident delirium. Cases were more likely than controls to have a chart diagnosis of dementia, have a lower Mini-Mental State Examination (MMSE) score, and to be exposed to more environmental risk factors. The adjusted risk of current medication use for the group of drugs categorized as having anticholinergic side effects was (RR, 3.16; 95% CI, 1.8–5.4). More specifically, current exposure to antidepressants and antipsychotic medications was associated with an elevated risk of delirium (RR, 2.53; 95% CI, 1.4–4.6) and (RR, 2.53; 95% CI, 1.6–4.1), respectively. The risk associated with specific antipsychotic drugs did not seem be related to their associated ACB score. Among antidepressant drugs, exclusive exposure to tricyclics, selective serotonin reuptake inhibitors, and venlafaxine (the only serotonin-norepinephrine reuptake inhibitor under study) was found to be associated with incident delirium (RR, 7.27; 95% CI, 1.2–44.4), (RR, 2.18; 95% CI, 1.1–4.3), and (RR, 6.88; 95% CI, 1.9–24.3), respectively. No association was found between incident delirium and use of medications within other drug categories. An overall trend showing a positive association between total ACB score for all medications currently being taken and delirium was found but it was not statistically significant.
Current use of antipsychotic medications and antidepressants appears to be associated with the incidence of delirium among residents residing in LTCFs after adjusting for the emergence of new medical conditions. These findings should be verified by future research.
Previous research suggests there are distinct neuroanatomical correlates of delusions in patients with dementia, with asymmetric grey matter atrophy more severe in the right hemisphere of the brain. Few studies have explored the morphological differences in grey matter between mild cognitive impairment (MCI) patients who develop delusions and patients who don’t. The Alzheimer’s Disease Neuroimaging Initiative (ADNI) is an international collaboration which provides access to curated structural neuroimaging data for research purposes. With a defined subset of the ADNI data, we conducted voxel-based morphometry (VBM) to characterize the atrophy patterns associated with the development of delusions in MCI. We hypothesized that MCI patients with delusions will have greater grey matter atrophy in the right hemisphere, specifically the right frontal areas since these areas are involved in non-verbal inhibitory executive control. Atrophy in these areas may compromise the inhibitory/monitoring function of the right hemisphere, and when combined with asymmetric neuropathology in the left hemisphere, will allow certain delusional subtypes to appear.
With data from ADNI, we conducted a cross-sectional analysis with 29 delusional MCI patients diagnosed at baseline, and 29 matched non-delusional MCI patients diagnosed at baseline. 1.5 Tesla T1 pre-processed MRI images from the central ADNI server were matched based on time since baseline psychiatric diagnosis of MCI, age, sex, education, and global cognitive functioning score. Registered images were segmented with standard tissue probability maps. Resulting grey matter images were processed through Diffeomorphic Anatomical Registration Through Exponentiated Lie Algebra (DARTEL) to create a study-specific template. Individual grey matter images were normalized in Montreal Neurological Institute space to this template. Images were smoothed with an isotropic Gaussian kernel at 5 mm full-width half maximum. The modulated images were entered into a general linear model for a two-sample
The majority of prominent grey matter differences, in decreasing levels of significance, were observed in the right hemispheric insula (cluster size = 39), precentral gyrus (10), and inferior frontal gyrus (13). Smaller significant differences were observed in the left hemisphere. Fifteen suprathreshold voxel clusters were detected in total at the
Consistent with our prediction, greater right frontal hemisphere grey matter atrophy was observed in MCI patients with delusions, compared to patients without delusions. This supports our hypothesis that delusional MCI patients may suffer from increased right grey matter atrophy, leading to loss of executive inhibition and consequent psychiatric symptoms. In the future, we plan to investigate subtypes of delusions and their neuroanatomical correlates, as well as longitudinal MCI analyses correlating delusions with rapidity of clinical deterioration.
Cognitive reserve in Alzheimer’s disease (AD) is characterized by the ability to withstand the cognitive deficits of the disease relative to the degree of pathological damage to the brain. Activities or practices that can add to this reserve should be encouraged. The effect of artistic ability on cognitive reserve has not been explored in AD patients.
Artistic AD/mild cognitive impairment (MCI) patients with similar cognitive performances to non-artistic AD/MCI patients will show greater deterioration of the hippocampus. This greater deterioration of the hippocampus will result in larger radial width of the temporal horn (rWTH) measurements despite similar cognitive performance. This pathological damage has been resisted by increased cognitive reserve from artistic backgrounds.
rWTH has been accurately and reliably found to be larger in AD and MCI patients in previous studies. Imaging from 13 AD/MCI patients with artistic occupations and 13 AD/MCI matched patients with non-artistic occupations was obtained from a retrospective hospital database. The patients were individually matched based on age, sex, years of education, and disease severity based on cognitive scores. The 26 AD/MCI patients had their rWTH compared from CTs/MRIs using Analyze 9.0.
For artistic AD/MCI patients the average rWTH was 5.9 mm and 6.6 mm for left and right rWTHs, respectively. For non-artistic AD/MCI patients the average rWTH was 4.9 mm and 4.55 mm for left and right rWTHs, respectively. In a one tailed paired
Since larger neuropathological deterioration occurred in the artistic AD/MCI patients despite similar cognitive scores, artistic ability/exposure can be considered a factor that increases cognitive reserve. Artistic activities should be recommended to AD/MCI patients, along with other current treatments, to enhance cognitive reserve and increase the quality of life for AD/MCI patients.
The quest to address institutional boredom is fundamental to the success of person-centred (PCC) long-term care (LTC). Highlighting key findings from my recently completed interdisciplinary doctoral dissertation (University of British Columbia, March 2013), this presentation will consider the practical implications of focusing on the therapeutic potential of everyday activities to better enhance residents’ sense of well-being and feelings of social inclusion.
The purpose of my ethnographic study was to obtain an on-the-ground understanding of a PCC philosophy of care in LTC. My study was particularly concerned with how the culture of care was experienced by residents and direct carers in a facility located in Western Canada.
To this end, I spent over 12 months in the field learning from residents, their families, staff, and administrators about the successes and challenges they encountered as the organization endeavoured to a create positive culture change, integrating a person-centred philosophy (theory) into everyday care (practice). After locating data generation and data analysis in the literatures of PCC and personhood theory (Kitwood; McLean; Sabat), my study undertook a critical ethnographic approach (Madison; Kincheloe & McLaren) that was informed by critical theory (Levinas; Foucault), feminist methodology (Bartlett & O’Connor; Twigg), and Foucauldian gerontology (Katz; Powell & Biggs). I employed multiple qualitative research methods—participant observation, individual interviews, group interviews, organizational and policy document reviews, dementia care mapping, and researcher-produced photographs—to access the experiences, values, and understandings of the social worlds of the people who live and work in Cedar Grove.
Significant findings revealed that residents maintain a deep desire to engage in meaningful everyday activities, activities they once enjoyed and which they perceive to give their life meaning. My study found that a lack of access to seemingly mundane, everyday activities significantly impeded residents’ social inclusion and impacted their overall well-being. Examples of this type of everyday activity differed according to a resident’s personal preferences and his or her physical and cognitive abilities and might range from reading the newspaper and chatting over coffee to sweeping the floor or enjoying the sensory appreciation of a vase of flowers. My presentation will address how an organization’s attempt to create a more holistic approach to care, one that continues to position the person at its centre, might be better supported by addressing the imperative to balance structured activities (crafts, music programs, etc.) and self-directed, unstructured activities of the type mentioned above. I will argue that the positive transformation of LTC, notably for residents of special care units, requires stakeholders to address and revalue the long periods of time between organized activity and care.
The Montreal Cognitive Assessment (MoCA) is a validated tool in clinic-based settings that can discriminate between normal cognition and mild cognitive impairment (MCI) or dementia (cut-off of 26 or higher, with a 1 point adjustment for low education). However, there are limited data on MoCA performance in a population-based setting. A previous population-based study from Texas (Rosetti
Between 1/2011–6/2012 we performed the MoCA in 829 community-dwelling, stroke- and dementia-free participants from the Prospective Urban Rural Epidemiological (PURE) study. Participants were 40–79 years old and were recruited from pre-specified postal code regions centred around four Canadian cities and adjacent rural regions: Vancouver, Ottawa, Hamilton, and Quebec City. Participants also completed questionnaires on memory and cognitive symptoms, and instrumental activities of daily living (IADL): finances, shopping, laundry, organizing and taking medications, and driving ability. After excluding eight participants who reported moderate or greater difficulties with IADL due to memory complaints, there were 821 for analysis. Linear mixed models were used to determine whether MoCA score varied by age, sex or education (as fixed effects), with recruitment site as a random effect. Education was dichotomized as post-secondary vs. no post-secondary.
Mean age was 58.3±8.0 years. The overall mean MoCA score was 26.8 (SD 2.4), incorporating a 1 point adjustment for post-secondary education, and 27% had a score of 25 points or lower. There was a linear decrease in MoCA with advancing age (
In a Canadian population-based setting we found that 27% of stroke- and dementia-free persons score below the previously validated cut-off for MCI or dementia; however, this prevalence was only half that previously reported from the southern U.S. These findings suggest that local normative data are needed to interpret MoCA performance. More Canadian data are needed in the elderly (≥ 80 years old). The clinical relevance of low MoCA performance in persons that are asymptomatic or have subjective cognitive decline requires further investigation.
As of 2011, approximately 747,000 Canadians suffer from some form of dementia; Alzheimer’s disease (AD) is one such form. AD is a neurodegenerative disease characterized by significant neuronal death. Neuronal death has been associated with two pathophysiological features: 1) neurofibrillary tangles within the neurons, and 2) amyloid beta plaque formation between neurons. Excessive production of these two features is manifested by severe cognitive impairment. One of the most extensively researched compounds, associated with these characteristics, is the amino acid, homocysteine, which has been found to be higher in blood plasma concentrations in patients with AD compared to healthy counterparts. Folate, vitamin B12, and vitamin B6 have been effective in reducing plasma homocysteine and this reduction has been associated with a reduction in amyloid beta and tau phosphorylation. However, this reduction in homocysteine has not resulted in improved cognitive performance. More recently, research focus has shifted to the universal methyl donor, S-adenosylmethionine (SAM), as a dietary supplement to treat both the pathophysiological features and cognitive impairment of the disease in mice and has shown promising results in alleviating both domains of the disease.
Here, a meta-analysis was conducted to evaluate the effect size for Y maze performance between two groups of mice, one receiving a SAM supplemented diet and the other group receiving a non-SAM supplemented diet. A thorough literature review was conducted and all studies that met the inclusion criteria were included in the analysis. For each study, both groups of mice were fed a folate and vitamin E deficient diet for 1 month with or without SAM supplementation.
The results of four mouse studies demonstrated a significant effect of SAM supplementation on cognitive performance as measured by the percent of spontaneous alternations made in the Y maze, thus illustrating the utility of this supplement in research concerning mental health.
To identify primary care doctors knowledge, practices, and obstacles with regard to the diagnosis and management of dementia.
Standardized questionnaires covering knowledge, practices, and obstacles were distributed among a random sample of primary care doctors in Kathmandu, Nepal. 380 physicians responded (response rate = 89%).
Knowledge of practitioners with regard to the diagnosis and management of dementia was unsatisfactory. Diagnosis and management barriers are presented with regard to GP factors, patient factors, systemic factors, and carer factors.
Specifically, the results address the following issues: time, communicating the diagnosis, negative views of dementia, difficulty diagnosing early stage dementia, acceptability of specialists and responsibility for extra issues, knowledge of dementia and ageing, less awareness of declining abilities and diminished resources to handle care, not specified guidelines, poor awareness of epidemiology, and less confidence to advise.
Demographic changes mean that dementia will represent a significant problem in the future. The following paper outlines the problems and solutions that the Nepalese medical community needs to adopt to deal effectively with its diagnosis, care, and management.
As the number of individuals with dementia grows, we are seeing associated caregiving challenges. In one program for persons with dementia, involving caregivers in the creation of an individual’s life story is an important step in the development of a person-centred approach to care by identifying important life events and their meaning. This narrative contributes to individualized interventions for care. At the same time, the use of technology and ‘simulated presence’ is being explored with some caregivers as an additional intervention.
This poster will demonstrate how ‘simulated presence’ can be an effective strategy to engage family or other significant caregivers and the interprofessional team in provision of a truly person-centred approach to care.
Clients and caregivers are involved in creation of a life story when the client is first admitted to the program. Caregivers are also invited to participate in making audio-tapes where they provide reassurance or narration of care steps. These audio recordings are played during care, in order to redirect or engage the person with dementia. Elements of the person’s life story, as well as how to implement ‘simulated presence’, are integrated into a behavioural intervention plan. Several of these situations have been videotaped and caregivers have viewed the videos and participated in individual interviews, to learn about their perspectives on the use of ‘simulated presence’ in the care of their family member.
Videotapes of care when ‘simulated presence’ is part of the intervention demonstrate engagement of the person and a reduction in unwanted behaviour. The impact on care providers is also evident. With simulated presence, the number of staff required to provide care has been observed to be fewer than without this intervention. Interviews with family reveal a variety of themes. While they may have doubts about their ability to contribute to care, or about the effectiveness of ‘simulated presence’ for their family member, they are eager to participate in finding solutions to reduce responsive behaviours. Over time, observing changes in their family member’s behaviour and feeling like their participation has an impact on client care, can be reassuring and rewarding.
Simulated Presence therapy is an intervention which uses recordings of a client’s family members to be played during care or at other times when responsive behaviours occur. This can be a meaningful way to engage caregivers and to enhance care for persons with dementia. Consideration should be given to who may or may not be appropriate for such an approach, and future research is warranted to further explore this unique element of a person-centred approach to care for persons with dementia.
Assessment of quality of life (QoL) of long-term care (LTC) residents presents significant challenges. People with dementia (PwD) may be unable to comprehend information being sought, lack insight into their own experiences, and be unable to formulate responses that express their perceptions of their own QoL. Yet they have been shown to be able to respond to such questions. Further, the perspectives of people with higher levels of cognition may not be well-served by instruments based predominantly on observation of behaviours and non-verbal indicators. Evaluation of the outcomes of care measures and performance improvement interventions is therefore challenging. A method of reliable and valid assessment of QoL of LTC residents is needed that is responsive to changes in clinical status, clinically feasible, an indicator of quality of care and performance, and a valid research measure.
To compare and contrast selected measures of QoL of LTC residents in people with cognitive impairment levels ranging from none to severe dementia. This pilot study assessed the feasibility of a proposed protocol.
Instruments validated to assess QoL in PwD were compared and contrasted for validity and feasibility across levels of cognition in LTC settings. Seven instruments were selected for further evaluation. Twelve resident/staff member dyads were randomly selected and stratified based on cognitive status of residents (unimpaired, mild, moderate, severe impairment). All seven tools were administered to staff members. Two instruments were designed to be administered directly to PwD. Mini-Mental State Examinations were administered to residents. Semi-structured interviews were conducted in which resident and staff member participants evaluated the instruments in terms of representativeness of their concepts of QoL, formats of instrument items, relevance, and clinical feasibility. Preliminary qualitative analysis (open coding) was conducted.
Internal instrument consistencies ranged from Cronbach’s α = 0.678–0.914 (one outlier 0.039). Further quantitative analysis will be conducted on the subsequent full sample. In interviews, residents and staff members reported that instruments addressed all relevant domains; no omissions were identified. Both residents and staff members asked for clarification of various items within the scales. Preferences were expressed for scales with emphasis on observable behaviours and simplicity. Number of response options was not a determining criterion. Discrepancies were identified between residents’ self-evaluations and staff evaluations. Duration of caregiver data collection ranged from 31–66 minutes for testing and 5–23 minutes for interviews. Duration of data collection with residents was 15–33 minutes and 3–12 minutes, respectively. Residents with no, mild, and moderate impairment were able to complete the two instruments administered directly to them; none of the three severely impaired residents was able to complete the instruments. Caregivers commented that they found the data collection process to be long.
Findings validated the feasibility of the proposed methodology. The number of instruments was reduced. Understanding of concepts and use of instruments was problematic. No single instrument was deemed appropriate across the cognitive range. Preliminary findings identify the need for a simple instrument in language easily understood by residents and staff with clearly expressed items based on objective observation of behaviours.
Low socioeconomic status (SES) has consistently been shown to increase the risk of developing Alzheimer’s disease (AD) and other dementias. Not surprisingly, a few studies have also linked low SES with an increased risk of mild cognitive impairment (MCI), a brain syndrome that often precedes dementia. However, it is not known what the relationship of SES is to the initial clinical presentation to a memory disorders clinic. We hypothesized that lower SES can lead to delayed medical attention and disease diagnosis and greater clinical severity at time of diagnosis, and be associated with reduced use of cognitive enhancers.
Data from 127 AD and 135 MCI patients seen at a memory disorders clinic based in a large urban centre were analyzed retrospectively. We examined the relationship between SES and 1) the diagnosis of either AD or MCI; 2) the age of patients when they present to clinic; 3) objective cognitive tests using the Mini-Mental State Exam (MMSE) and Behavioural Neurology Assessment (BNA) to indicate clinical severity; and 4) the use of cognitive enhancers in patients with AD. SES was measured using the Hollingshead 2-factor index of social position, which is a linear scale from 11 to 77 that incorporates educational and occupational attainments, and is negatively correlated with SES. Upper and middle class (scores of 11–43) were compared with lower class (scores of 44–77) individuals.
AD patients had significantly lower SES than MCI patients (
Individuals with lower SES presented more frequently with established dementia, while higher SES individuals presented more frequently with MCI. This, combined with the greater age found among low SES individuals, could indicate that low SES may lead to delayed referral to memory disorders clinics and delayed diagnosis of AD. Furthermore, higher SES is associated with better cognitive functioning in MCI patients and increased use of cognitive enhancers in AD patients, possibly because low SES patients come in too late to benefit from treatment. This has broad health policy implications in terms of developing strategies to engage patients with low SES in the early stages of dementia, perhaps through better identification of patients at the primary care level.
Home oxygen therapy is prescribed to people with various health conditions including lung and heart diseases, such as Chronic Obstructive Pulmonary Disease (COPD) and heart failure. Managing the use of oxygen can be difficult in patients with dementia who have cognitive and functional losses. Hypoxia can exacerbate confusion and worsen behavioural symptoms. Patients with cognitive impairment often have great difficulty to learn and remember how to use unfamiliar oxygen equipment properly. Mortality and readmission rate are high in this group of patients. The burden of symptoms significantly affects quality of life and health status of patients and caregivers. Older people with dementia who have medical co-morbidities require careful attention to minimize behavioural consequences and improve quality of life. Clinical management of these patients differs from the younger population and care professionals must adjust their management strategies to accommodate their special needs.
Despite the fact that provision of home oxygen therapy is required by some older adults with cognitive impairment or dementia, there is no literature which describes the specific challenges and offers guidance to the provision of oxygen therapy. This study aims to explore the main issues associated with preparing older patients going home with oxygen therapy by inquiring the care providers’ perspective.
A total of 10 participants, including Physician, Respiratory Therapist, Physiotherapists, Occupational Therapist, Nursing and Social Work, participated in two focus groups. The participants from one group work in a local community hospital; the others work in the community sector. The focus group discussions were one hour each. The discussions were audio-taped and transcribed verbatim. Thematic analysis was undertaken to identify important themes and subthemes to reveal the challenges and specific areas for improvement.
Three broad themes emerged as main issues associated with preparing patients going home with oxygen. The first theme, ‘Education’, explored subthemes of Knowledge, Resources, and Barriers. For care providers in hospital, knowledge of equipment available in the community is needed to select appropriate equipment to meet varying needs of patients. The biggest barrier is patient-related factors including decreased cognition, visual and physical deficits, and language barriers that affect the learning ability of patients. Under the second theme, ‘Safety’, there were subthemes that considered environmental challenges and equipment. Participants reported high risk for falls due to long oxygen tubing in their homes and the manoeuvring of equipment. Other hazards include smoking, fire risks with gas stoves, and inappropriate levels of oxygen. The third theme, ‘Discharge Process’, discussed the subthemes of team collaboration, time limit, and home oxygen assessment. Participants consistently highlighted the importance of effective communication of information about patient’s cognitive, physical, and functional abilities, as well as safety issues to community teams.
This study demonstrates that there is a need to improve current processes in order to provide patient-centred, safe, and efficient home oxygen therapy to geriatric patients, particularly to the group with cognitive impairment/dementia. Careful attention and adaptations are required to meet the special needs of this vulnerable population.
Environmental interventions are an untapped source of therapeutic potential. Given the fact that we have a burgeoning older population with dementia in acute hospitals, there can be great patient benefits and potential cost savings of utilizing environmental strategies to promote safe recovery, reduce loss of function, and avoid adverse events. Older adults with dementia have decreased ability to cope with environmental stressors; they are more sensitive to the impacts of environmental features. Research suggests that an environment that is safe, warm, and familiar not only supports cognitive and functional needs of older people with dementia, but may also contribute to improving quality and safety of care in patients of all ages. However, research on effective environmental interventions in the acute setting to support patients with dementia is lacking.
Our study aims to: 1) provide a review of the literature to identify relevant evidence-based environmental interventions that may contribute to positive experience in older adults in acute hospitals, and 2) investigate the physical environment of a geriatric psychiatry unit in a community hospital to understand how physical environment may play a role in meeting needs of patients with dementia or other mental health needs.
We conducted a focused ethnography method on a 16-bed geriatric psychiatry unit in a community hospital. We began with a review of literature, an environmental scan, and a survey of 18 staff from different disciplines, including nurses, occupational therapists, and care aides. These guided our subsequent focused observations and interviews with patients and families. The sample included 7 patients (four of whom were diagnosed with dementia and three with depression/schizoaffective disorder), and 4 family members. We used purposive sampling to ensure we had a variety of patients with different behavioural symptoms and functional and psychosocial needs. A thematic analysis was conducted.
Our results demonstrate that physical environment plays an important role in impacting the hospitalization experience of older adults with dementia or other mental health needs and their families. The four inter-related themes of environmental qualities central in promoting healing and coping are: therapeutic; supportive in functional independence; facilitative in social connections; and personal safety. Therapeutic means the unit offers pockets of home-like environment and provides quality sensory stimulations. Supportive of functional independence refers to the environmental features that make it easy for older adults to use the bathroom, wash, groom, mobilize, locate places/rooms, and store personal belongings. Facilitative of social connection indicates the provision of safe and comfortable social spaces for patient, family, and staff to interact/engage in meaningful activities. The feeling of personal safety involves having staff in close proximity and minimizing disruptions (e.g., physical or verbal) from confused patients.
The evidence indicates that physical environment plays an important role in making hospitals safe and supportive of healing for older adults with dementia and other mental health needs. Patients’ and families’ perspectives provide us with a better understanding of current challenges of the hospital environment and assist in identifying specific priorities and interventions to make improvement.
Alzheimer’s disease (AD) and depression share many common pathological features — for example, decrease in the number of synapses. The synapse forms an important communication unit between neurons to maintain neuronal viability and sustain whole brain functioning. Actin is the main cytoskeleton that forms the architecture of the synapse. Polymerization and depolymerization of actin allow actin filaments to constantly remodel and maintain synaptic plasticity. Furthermore, synaptic vesicle proteins involved in the docking and fusion of the vesicles to the membranes allowing for neurotransmitter release, including synaptophysin and synaptotagmin, are also important in maintaining synaptic function. Abnormalities in synaptic and cytoskeletal proteins have been observed in both depression and AD.
To investigate morphological and protein changes in the synapse after treatments with oligomeric beta-amyloid and corticosterone.
14-day-old hippocampal primary-cultured neurons were treated with either oligomeric beta-amyloid or corticosterone separately for 24 or 48 hours. Neurons were transfected with beta-actin to observe synaptic morphological changes. Immunocytochemical analysis was used to investigate changes in the vesicle proteins synaptophysin and synaptotagmin in neurons. FM4-64 dye was used to investigate functional changes. All of the above were imaged by multiphoton microscopy.
After treatments with oligomeric beta-amyloid or corticosterone, changes in beta-actin morphology were observed. Rod shaped actin began to form within the cell body, and also along and at the ends of dendrites. Oligomeric beta-amyloid significantly reduced the expressions of synaptic vesicle proteins, whereas corticosterone induced aggregation of these proteins. FM4-64 dye showed that the function of the neurons was compromised; more specifically, exocytosis appeared to be abnormal in the amyloid- or corticosterone-treated synapses.
Our results show that both oligomeric beta-amyloid and corticosterone affect presynaptic vesicle proteins, cytoskeletons, and neuronal functioning. This may help to explain the decrease in dendritic spine number and dendritic regression observed in depression and AD. Moreover, such resulting neurodysfunction likely forms the basis of cognitive impairment seen in depressed and demented individuals.
It is well established that persons with Alzheimer’s disease and their family care partners may hold differing views on how the disease has impacted various aspects of their lives. For example, previous research has identified discrepancies in care partner/receiver perceptions of depression, diagnosis, pain, values and care preferences, quality of life, and everyday functioning. One domain that has not been closely examined, yet which contributes to all other aspects of daily functioning, is a person’s ability to communicate. The present exploratory study investigated family care partner/receiver perceptions of the care receiver’s communication abilities in daily life.
Seven participant dyads (care partner/receiver) were interviewed separately using the CLIMAT interview scale. Questions were asked regarding the care receiver’s abilities across four major domains: Social, Everyday Functioning, Cognitive, and Behavioural. The care partner and receiver interview data were transcribed and imported into Atlas-ti for coding. Open coding was undertaken to identify participants’ recurring comments and themes related to language and communication abilities, such as word-finding difficulty, repeating oneself, comprehension, initiating conversation, and engaging in social interaction. These themes were further analyzed to determine whether there were discrepancies between the care partner’s and receiver’s perceptions of functioning in each domain.
The results indicate that discrepancies were most apparent in describing the care receiver’s abilities to have meaningful conversations about recent events and to engage in social interaction outside the home. The differing views reflected care receivers’ underestimation of the impact of AD on their communication functioning. Possible sources of the diverging care partner/receiver perceptions include awareness and/or protection of self and others, and attitudes about the functional impact of AD.
The differing views of care partners and receivers point to the need for them to have more open and ongoing dialogue about changes in communication ability and their potential impact on interpersonal interactions and quality of social life.
Preventing falls among older adults remains a focus of health professionals. While fall prevention and injury reduction initiatives involve many excellent, evidence-based strategies, these same strategies are not always applicable within a dementia population. Recent trends at a geriatric hospital reveal an increase in falls with critical injury with clients who have dementia and also exhibit responsive behaviours. This relationship between falls and behaviour indicates a need to explore possible interventions aimed at this population specifically. Clients who exhibit responsive behaviour often have underlying neurological conditions which may make traditional falls prevention strategies ineffective, as they are not aimed at the strengths of the client.
As part of a larger Falls Prevention Initiative, a geriatric hospital implemented a three-month pilot project on two specific units involving strategies that were developed with a focus on the unique characteristics of each population. On one behavioural dementia unit, two falls prevention strategies: consistent, universal provision of hip protectors and a visual tracking of falls and falls with critical injury, were implemented for a three-month period.
Preliminary results from this pilot indicate that there have been zero falls with critical injury during the three-month period, and the average rate of falls is no different from the average falls rate observed during the past year. A visual tracking system, located in a lounge area in front of the care station, has been available for staff, clients, and families to observe and follow. Different team members were required to take on the duty of tracking falls, encouraging interprofessional accountability. Use of hip protectors was offered to all clients; however, many barriers arose to limit family members and staff from continuously implementing wearing of hip protectors over the course of the pilot project. Some examples of barriers include hip protectors limiting clients’ abilities to toilet themselves, clients exhibiting behaviours which may limit the effectiveness of hip protectors (e.g., disrobing, fidgeting, etc.), and having hip protectors contribute to responsive behaviours (e.g., restlessness).
The pilot project has so far been successful, in that there have been no falls with critical injury observed on the unit and the number of falls has been regularly below the annual average. Having one universal strategy (hip protectors) has not been sustainable on this unit, due to individual differences within the patient population. The visual management strategy has engaged staff and families. While there is a trend in the number of falls in people who exhibit responsive behaviours, falls strategies need to be individualized as this population is highly heterogeneous. Taking a team approach, including team conferences and implementing collaborative interventions, helps to minimize the risk associated with falls in this population.
Older adults identify themselves by what they do and the activities that structure their lives (Laliberte-Rudman D,
A geriatric hospital aimed to identify critical components of a process and develop a prototype to ease care setting transitions of patients with severe cognitive impairment and behavioural issues. A pilot project on the behavioural neurology inpatient unit in collaboration with the hospital’s Innovation, Technology, and Design Lab explored use of video communication across care settings. To showcase six clients’ engagement to subsequent care providers, videos were created of each client, depicting personhood, behaviour mitigation, and approach to care. A participatory action framework, based on the knowledge to action cycle was utilized (Graham ID. 2006). Focus groups were held with care providers at the discharge destination.
A thematic analysis was completed by the behavioural neurology unit and the Innovation, Technology and Design Lab which revealed three themes: 1) Video communication is valued as a medium for sharing client information; 2) Communication needs to be catered towards the discharge destinations, considering workload, culture, and accessibility; and 3) Staff value preserving client identities, through maintaining daily routines, incorporating their life story, and building connections with clients.
The current process and lack of individualized care plans leave clients with unmet needs and decreases engagement. Care facilities value video, so long as it is tailored to the needs of the care setting and highlights the shared goal of preserving the client’s occupational identity. Enhancing communication through video technology is one strategy to help ease care transitions and support continuous meaningful engagement. Next steps include activating a Cloud—a portal that will allow staff in other institutions to access client information securely and enable better communication across settings.
Apathy and depression, two of the most prevalent behavioural disturbances in Alzheimer’s disease (AD), often contribute to decline in quality of life for patients and their caregivers. Symptoms of apathy and depression may be difficult to assess, particularly as cognition deteriorates. Our team developed the Visual Attention Scanning Technology (VAST), an eye-tracker which enables real-time measurements of attention patterns towards competing visual stimuli. Previous results suggest that VAST has the ability to distinguish between depressed patients without dementia and healthy controls. Using VAST in the AD population for the first time, we explored an objective method of assessing symptoms of apathy and depression that does not rely on patient verbal skills or caregiver reports.
This is a cross-sectional study of patients with mild to moderate AD (NINCDS-ADRDA criteria; Mini-Mental Status Examination, MMSE). Participants were screened for significant depression (DSM-IV-TR; Neuropsychiatric Inventory, NPI depression, and apathy (NPI apathy). On a computer screen, participants were presented a series of 16 slides, containing 4 images of different themes (2 neutral, 1 social, 1 dysphoric), interspersed with filler slides. Patients were allowed 10.5 seconds to view each slide for a total test time of 20 minutes. Interest was measured using the number of fixations within specific images on a slide. Groups were compared using analysis of variance (ANOVA) and associations were determined using Pearson correlation coefficients.
Of the 37 AD patients (19 females, age =77.1±8.7, MMSE = 22.1±3.5) included in this preliminary analysis, 19 had neuropsychiatric symptoms (NPS, 12 significant apathy, 7 significant depression) and 18 had neither of these symptoms (non-NPS). These patients had comparable age, though depressed patients scored lower on MMSE compared with apathetic and non-NPS patients. There was a significant difference in number of fixations on social images between groups (F2,34 = 4.01,
These preliminary findings suggest that interest in social stimuli using VAST can distinguish AD patients with different behavioural disturbances and is associated with severity of apathy. The results of this study will begin the development of a non-invasive and novel objective tool for evaluating apathy and depression severity in AD, which might also be a useful biomarker for predicting and monitoring treatment response.
Cholinesterase inhibitors (ChEIs) are considered the first line treatment for symptoms of Alzheimer’s disease (AD). Despite their modest efficacy, lack of data regarding long-term use, and potential for side effects, patients with moderate to severe AD on ChEIs tend to remain on these medications for long periods of time and often until death. This warrants the investigation of predictors of response to discontinuation of ChEI therapy to determine if, and for whom, it is appropriate.
Institutionalized patients with moderate to severe AD (Mini-Mental Status Exam 2 years ChEI use were randomized, double-blind to ChEI continuation or placebo (with 2-week taper) for 8 weeks. Vitals: weight (kg), Clinician’s Global Impression (CGI), neuropsychiatric symptoms (Neuropsychiatric Inventory/Nursing Home Version [NPI-NH]), cognition (Severe Impairment Battery [SIB] and the MMSE), and safety (standardized symptom checklist) were monitored biweekly. Demographic and clinical characteristics were investigated at baseline.
To date, 25 patients (72% male, mean age 87.9±3.0, mean MMSE 6.8±5.2, mean NPI 17.6±13.6, mean CGI 3.8±0.7 at baseline) have been enrolled. Based on un-blinded results, patients were classified into two groups to determine whether baseline measures of vitals (blood pressure, pulse rate), weight, cognition (MMSE and SIB), and behaviour (NPI) were objective predictors of change in CGI status. When patients were grouped based on CGI status at study endpoint, a total of 8 (32%) patients worsened, while 16 (64%) showed no change and 1 (4%) had improvement. Preliminary data indicates that vitals (χ2 (3) = 4.642, R2 = .169,
Thus far, there have been no baseline predictors of worsening. Once the recruitment goal of 60 patients is met and study treatment allocation revealed, placebo and ChEI continuation groups will be compared and predictors of response will be determined. Further assessment of predictors of improvement following ChEI discontinuation will provide data for guidelines for ChEI discontinuation.
Gait and cognition are interrelated. However, it is still unknown if there is a “motor signature” associated with cognitive dysfunction. Previous studies assessing older people with normal cognition, mild cognitive impairment (MCI), and with dementia have found that executive dysfunction is consistently associated with a slower gait. However, associations between episodic memory dysfunction and gait performance are inconsistent, and it is unknown if memory dysfunction, which is cardinal sign in MCI, is specifically associated with the gait disturbances seen in MCI.
To determine whether gait performance in older adults with MCI differs based on their cognitive subtyping classification: amnestic type (aMCI) or non-amnestic type (na-MCI).
Older adults (≥ 65 years) with MCI from the “Gait and Brain Study” were included in this analysis. Global cognition was evaluated using the MMSE and the MoCA. Specific cognitive domains were evaluated using a battery of neurocognitive tests: Trail Making Tests A and B, Rey Auditory Verbal Learning Test, Digit Span Test, and Letter Number Sequence Test. Gait performance was evaluated with the GaitRITE mat under usual and dual-task walking conditions (walking while naming animals out loud and walking while doing serials subtractions by 7). Participants were divided in aMCI and naMCI based on their episodic memory assessment performance. The relationship between cognitive group (aMCI vs. na-MCI) and gait variables was evaluated with linear regression modeling.
Sixty-four participants, mean age 77±6 years and 57.6% female were included. Forty-three were aMCI and 21 were na-MCI. Groups were similar in age, co-morbidities, level of physical activity, and history of previous falls. aMCI participants walked slower than na-MCI (98.5 vs. 112.1 cm/sec,
Memory dysfunction, specifically episodic memory impairment, was associated with poor gait performance, particularly under dual-task test conditions. Associations were maintained even after adjustments for potential confounders in the multivariate logistic regression. Our findings suggest that there is a motor signature in aMCI characterized by slowing gait under dual-tasking and higher variability, which seems to be independent of executive dysfunction.
Extensive research in behavioural neuroscience has established that the hippocampus and the medial temporal lobe (MTL) systems are required to form new long-term declarative memory until slow consolidation processes allow neocortical networks to represent memory independently. Sharon
Examine older adults’ ability to learn through FM, and the impact of dementia on such learning.
Healthy older adults (OA), mild cognitive impairment (MCI), and Alzheimer’s disease (AD) patients performed an FM task. On each trial, participants saw pictures of two items—an unknown (e.g., umbretta) and a well-known (e.g., duck) item. They had to make a perceptual decision (e.g., “Is the umbretta’s beak purple?”) that required an inference about the association between novel labels and novel items. Sixteen new items were incidentally encoded in this way. Memory was tested using a 3-alternative-choice associative recognition task after 10 minutes and again after 1 week. A matched Explicit Encoding (EE) task was also used in which participants were simply asked to “remember the Caracara”, and testing was the same.
Similar to previous studies with young and middle-aged adults, OA perform better on EE than FM, but in addition they displayed moderate reductions in FM performance. AD and MCI patients demonstrated equivalent performance to OA when tested after 10 minutes following FM encoding, despite significant impairment on the EE task. By contrast, when tested after a week, FM gains were lost in AD, but not in OA or MCI. Brain behaviour correlations in AD and MCI patients showed that EE scores were correlated with hippocampal volumes and with clinical tests of episodic memory. By contrast, FM scores were correlated with neocortical regions such as ATL and specific frontal and parietal regions, and with semantic memory tasks.
Our study concurs with that of Sharon and colleagues, that MCI and AD patients were able to learn new associations through FM despite an impaired episodic memory system. However, AD patients also demonstrated accelerated forgetting over a week. Interestingly, the pattern of correlations with brain volumes suggests FM is less sensitive to hippocampal atrophy and more sensitive to anterior and posterior neocortical degeneration that is also part of AD. These findings are in line with previous patient research that demonstrated learning through FM depends on the ATL probably due to its role in representing semantic associative networks. The data are consistent with the idea that acquisition of semantic information through FM and EE rely on distinct neural systems.
Dementia is a major predictor of the need for long-term home care and becomes increasingly common with greater age. Retirement living is an alternative residential option available to seniors that offers some support (e.g., cleaning, cooking, medical support) but is independent of provincial health services. Retirement living may facilitate physical and social activity among older adults by reducing health, social, and environmental barriers. Since regular physical activity is associated with slower cognitive decline, an increase in physical activity in retirement living may slow cognitive decline with age.
The objective of this study is to (1) quantify changes in physical activity over the transition from community living to retirement living, and (2) describe the association between these changes and cognitive function.
Older adults living in and on the wait-lists for retirement living were recruited for this study. Physical activity was assessed objectively with a tri-axial actigraph activity monitor and was self-reported using the CHAMPS questionnaire. Cognitive function was assessed using the MoCA and a 30 minute cognitive battery based on the vascular cognitive impairment harmonization standards, which assess cognitive domains including memory, executive function, and attention. Current residents participated in one assessment in which they reported current and past (prior to retirement living) physical activity; current activity was objectively measured. Wait-list participants reported physical activity and had both physical activity and cognitive function measured prior to and after their transition to retirement living.
Physical activity in retirement living will be compared to physical activity in the community using paired t-tests. The relationship between physical activity changes and cognitive function will be assessed with correlational analysis. Results: Sixty-seven percent of current residents increased weekly participation in purposeful exercise (e.g., aerobic classes, use of fitness equipment in the facility, and walking groups). Four residents reported beginning purposeful exercise activities only after the transition to retirement living. Conversely, the frequency of physical activity related to activities of daily living decreased among all residents. At this time, 10 wait-list residents have completed pre-transition assessments and will have post-transition assessments completed in the fall. These results will also be presented at the Canadian Dementia Conference.
This study investigates the impact of a residential choice and alternative health care option (retirement living) on physical activity patterns and cognitive function. It is possible that this alternative care model may improve physical activity and thereby decrease cognitive decline and dementia among older Canadians.
In 2038, there will be 257,800 new cases of Alzheimer’s disease or a related dementia in Canada, equaling 756 million hours of informal care, and a projected economic burden of $153 billion for that year (
This paper focuses on the relationship between individuals with dementia and their environments. Specifically, it concentrates on improving quality of life for those with dementia and increasing the capacity of the existing urban spaces through safety, sense of community, equality of access and opportunity, and enabling independence.
The impact of public spaces on those affected by dementia is often overlooked in the academic literature and, more seriously, in public policy formulation. To help address the shortage of material on dementia-friendly public spaces, a review of the literature on dementia-friendly communities is included to produce recommendations for “best practices” addressing dementia, with special emphasis on dementia-friendly public environments. The paper then employs Penny McCourt’s ‘Dementia Policy Lens Toolkit’ to assess the new ‘dementia-friendly’ approaches in York, England in the context of the identified “best practices”. Addressing the questions: “How can we make our urban public spaces more dementia-friendly?’’ and ‘’What are the health implications of ‘dementia-friendly’ urban spaces?’’, the paper concludes with recommendations on implementing these best-practices in Canadian settings.
Recent prospective studies have shown that high Aβ amyloid is associated with a faster rate of memory decline in healthy older adults and adults with mild cognitive impairment (MCI). However, because these studies were conducted over shorter durations (i.e., 18 months), longer prospective studies are required to determine if Aβ-related memory decline is unremitting.
Healthy older adults (n = 177), and adults with MCI (n = 48) underwent positron emission tomography (PET) neuroimaging using Pittsburgh Compound B (PiB) for Aβ amyloid, APOE ε4 genotyping, and cognitive assessment using Cognigram as part of their baseline assessment in the Australian Imaging, Biomarkers, and Lifestyle (AIBL) study. Cognitive function was reassessed 18 and 36 months later.
Compared to healthy older adults with low Aβ amyloid, healthy older adults and adults with MCI with high Aβ amyloid showed a moderate decline across 36 months on the Cognigram learning working memory composite. In contrast, adults with MCI and low Aβ amyloid showed a slight improvement on the Cognigram learning/working memory and psychomotor/attention composites across the 36 months. APOE ε4 carriage did not moderate the relationship between Aβ amyloid and cognitive decline.
The results of this study suggest that in healthy older adults, high Aβ amyloid most likely indicates that AD-related neurodegeneration has begun. They also support the hypothesis that adults with MCI and high Aβ amyloid is indicative of incipient AD, while MCI with low Aβ amyloid may reflect the presence of other neurodegenerative or psychiatric processes. Once commenced, the rate of decline in cognitive function remains constant across the preclinical and prodromal stages of AD. Finally, the results indicate the sensitivity of the Cognigram learning and working memory composite to the effects of Aβ amyloid in non-demented adults.
Prospective studies show that in healthy older adults and adults with mild cognitive impairment (MCI), high levels of Aβ amyloid are associated with cognitive decline and more rapid progression to the next clinical disease stage. However, as yet single cognitive assessments or cognitive screening has not been able to differentiate non-demented individuals with low and high Aβ amyloid.
Healthy older adults (n = 288) and adults with amnestic MCI (n = 56) enrolled in the Australian Imaging, Biomarkers and Lifestyle (AIBL) study, underwent positron emission tomography (PET) neuroimaging using Pittsburgh Compound B (PiB) for Aβ amyloid, and completed the Cognigram cognitive screen.
In healthy adults, performance on the attention/psychomotor function (d = 0.16) and learning working memory (d = 0.23) composites were equivalent between low and high Aβ amyloid groups. In MCI, performance on the attention/psychomotor function composite was equivalent between low and high Aβ amyloid groups (d = 0.21); however, performance on the learning working memory composite was significantly worse in the MCI high amyloid group compared to the MCI low Aβ amyloid group (d = 0.69).
The data indicate that in MCI high Aβ is associated with more severe impairment in learning and working memory. In MCI, Aβ amyloid levels do not influence attention and psychomotor function. In healthy adults, cognitive screening is not sensitive to elevated amyloid levels. These data suggest that prospective cognitive screening may be necessary to identify high Aβ amyloid in healthy adults. However, in MCI more severe memory impairment can indicate that Aβ amyloid levels are abnormally high.
The objective of this study was to determine the role of music in promoting an enhanced brain reserve capacity in Franz Schubert and Maurice Ravel, two professional musicians who suffered from neurological disorders.
We consulted medical journals, reports, historical reviews, memoirs, and books written in English describing the life of each composer, the progression of their disease, and its effects on their musical faculties.
Schubert suffered from a brain infection, most likely tertiary syphilis. In 1822, he experienced hair loss, skin rashes, ulcers in the mouth and throat, bone pain, and headaches—all characteristics of second stage syphilis. During this time however, Schubert composed numerous pieces, including “Die Schöne Müllerin”, which was written while being treated in the hospital. In the final year of his life, Schubert’s disease progressed to a tertiary phase where his brain was affected, resulting in chronic headaches, dizziness, paranoia, memory deficits, and eventually delirium. Despite the cognitive and physical deterioration, Schubert’s musical composition output remained intact with the completion of his last three piano sonatas just weeks before his death. In fact, Schubert was reported to have made corrections to Part II of his piece “Winterreise” a day before he died. Likewise, Ravel first exhibited symptoms of primary progressive aphasia with underlying corticobasal degeneration as early as 1927, about the same time as he composed his famous work,
The literature on the preservation of musical competency in famous artists affected by various brain diseases such as frontotemporal dementia and Alzheimer’s disease is supported by our review on the musical integrity in Schubert and Ravel. We raise the hypothesis that the neural pathways recruited in composing and understanding music at a professional level are separate from those used in daily activities. These networks are unique in that they are resistant to neurodegenerative diseases. Therefore, music may serve as another basis for enhanced brain reserve capacity in artists.
Behavioural and psychiatric symptoms of dementia (BPSD) may disable a patient from performing activities independently; the reverse may be true in that losing independence may affect mood and behaviour. The purpose of this study is to investigate the association between function and severity of neuropsychiatric disturbance in the context of dementia.
We analyzed data from a longitudinal study of caregiver informants responding to the Functional Rating Scale (FRS), Clinical Dementia Rating Scale modified for frontotemporal dementia (CDR-FTLD), Frontal Behavioural Inventory (FBI), and Neuropsychiatric Inventory (NPI). Participants granted 2–3 telephone sessions separated by at least one year. We performed bivariate correlations for the FRS and CDR-FTLD against the behavioural inventories and compared patterns among 3 subtypes of dementia and Mild Cognitive Impairment (MCI) who converted to Alzheimer’s disease (AD).
The dataset includes 20 sessions regarding 9 MCI converters, 194 sessions for 94 AD patients, 63 sessions for 28 behavioural variant frontotemporal dementia (bvFTD) patients, and 32 sessions for 14 primary progressive aphasia (PPA). For the total sample, we found positive correlations for FRS and FBI: r from .682 to .870,
This study indicates links between functional ability and severity of neuropsychiatric symptoms across several types of cognitive impairment. Further study will explore causality in the association, as well as seeking the relative roles of additional covariates, such as educational level or duration of illness.
Neuropsychiatric symptoms (NPS) are common in patients with dementia including Alzheimer’s disease (AD), vascular dementia (VaD), and mixed AD and VaD. The most common NPS encountered in dementia are apathy, irritability, agitation, depression, delusions, hallucinations, anxiety, disinhibition, and eating abnormalities (Cummings JL; 1997). These symptoms contribute to patients’ distress, caregiver burden and institutionalization. Different neurode-generative diseases may be associated with certain NPS, thus impacting treatment and care. Moreover, frontal lobe injury is often associated with development of NPS (Damasio A. In:
The aim of this study was to compare NPS in patients with AD, VaD and mixed AD and VaD, and to evaluate the differences in incidence of NPS in relation to frontal white matter hyperintensities (WMH).
This was a retrospective chart review of 510 patients who presented to the Toronto Western Hospital Memory Clinic with cognitive complaints. Ninety-three patients with AD (McKhann GM,
There were no significant differences in gender, education or MMSE (AD 21.7; VaD 23.8; mixed 23.8) between patients with AD, VaD, and mixed, but there was a significant difference in age with mixed being older (mixed 82.3±6.6, AD 76.6±10.2; VaD 75.3±10.2;
NPS were prevalent in AD, VaD, and mixed AD and VaD, but their frequencies varied amongst the different dementia causes. Agitation, depression, sleep disturbances, and aberrant motor behaviour were most prevalent in VaD. Volumetric analysis revealed significantly more left, right, and total frontal WMH in patients with delusions and hallucinations versus those without these NPS. These differences are likely related to underlying pathology and warrant further study, as they have implications for treatment.
The ongoing pilot implementation of remote monitoring devices for dementia patients is facing impending dangers. The government perceives the initiative as an IT-based therapy for complementing pharmaceutical and non-pharmaceutical therapies, while health policy formulators are promoting the initiative because of its usefulness for tracking dementia patients. However, misconceptions are building up by the families of dementia patients and carers who will administer the surveillance therapy whenever it goes live regarding its compliance with best clinical practices in the areas of legal, data sharing, privacy, and security issues. Usually, experience shows that lack of acceptability and design flaws are central to the failures of most health service initiatives at the implementation and post-implementation stages over the years. Therefore, this paper investigates the aforementioned issues from the perspectives of families of dementia patients and carers. The results obtained suggest strategies for improving the success of the remote surveillance initiatives after implementation.
This study examined the perspectives of families of dementia patients and carers on resiliency, privacy, legal, and security of smart devices for tracking dementia patients. Tracking of vulnerable patients involves police and ambulance system. Thus, this study further seeks to proffer strategies for reducing the growing cost of managing dementia patients.
Thirty-six mental health and admiral nurses in UK and abroad participated in the survey. We introduce smart devices to them as knowledge-based systems for tracking dementia patients who are vulnerable to self-discharge. The inclusion and exclusion criteria are respondents that have experiences with patients officially diagnosed for early-onset dementia or late-onset dementia and with the following three characteristics: 1) acute dementia patients (ADP) are disorientated and confused patients, vulnerable to wandering, lost or putting family members, friends and carers into distress situations; 2) strong-minded dementia patients (SDP) are aggressive patients who discharge themselves against medical advices without referring to Mental Health Review Tribunal (MHRT) or certified by doctors; 3) isolated dementia patients (ISP) are patients that live alone and take care of themselves without recourse to relatives, friends or carers.
The degree of resiliency of smart devices if they are suddenly compromised by hackers is unanimous affirmed as an important issue to be investigated thoroughly. The results demonstrate that 86.10% of participants agree that some of the information regarding the patients can be adapted to many uses, while 44.40% believe that smart devices may have false positives detection rate. The results reveal correlations between IT-based therapy and continuous training, while 50.40% say patient’s health records are indirectly transferred to vendors of smart devices to manage.
Continuous education and development of operational policies to cover privacy and security issues in the administration of smart devices are strategies to improve perceptions of mental health nurse, carers, and families. There is need to strengthen mental health laws to protect carers who will generally administer IT-based therapies. Unlawful accessibility to the devices and alerts they generate must be prevented using suitable Intrusion Detection and Prevention Procedures (IDPP) in accordance with best clinical standards.
The discovery of dementia sickness which often results into sudden declination or deterioration in the memory functionality and social functions of affected persons is a central problem in the social health-care services over the years. Several research findings have supported doll therapy in a recent decade. However, the methodology for applying doll therapy suffers moral criticisms in social care setting across the globe despite the benefits that are associated with the therapy whenever it is compared with pharmaceutical interventions. Firstly, critics are of the view that modelling specially loved personalities in the form of pets are deliberate attempts to reduce the dignity, worth, efforts, and invaluable contributions that the affected patients have done to the society during their active years. Secondly, conventional doll therapy is seen as dehumanizing and harmful to the mind of aspiring and productive youths. Thirdly, doll therapy is applied in fragments to patients without compliance to the best clinical practices. Consequently, this study proposes automated doll therapy for treating dementia patients in order to lessen the above issues. The results show that automated doll therapy has positive effects on society, patients, families, friends, and carers of dementia patients. Further analysis suggests that automated doll therapy is compliance to best clinical practices.
The study reviews methodology for applying doll therapy against best clinical practices.
Thirty-four mental health practitioners, and 26 friends and family members of dementia patients volunteered to participate in the survey. The sample population were selected based on their experiences in in-patient wards in North, East, West or South of England. Participants were exposed to methods, strengths, and weaknesses of conventional and computer aided devices (CAD) methods for applying dolls to a group of dementia patients in a multimedia room within an in-patient. Thereafter, participants were interviewed on their perceptions on both methods. Their responses transcribed immediately. The perceptions of the respondents were repeated clarifications to improve data reliability and validity, and the results obtained were statistically analyzed.
Analysis of the results underpinned four hypotheses: 1) The perception that automated doll therapy will be better than the conventional method for managing dementia patients is high; 2) Automated doll therapy shows possibility of stabilizing emotions of patients with mild dementia problems to a certain degree; 3) Automated doll therapy suggests potential improvements in the perceptions of families, friends, and carers of dementia patients; 4) Automated doll therapy suggests positive impacts on social interactions among dementia patients in all age range of dementia patients.
This survey suggests strategy for improving the efficacy and perception of families, friends, and carers of dementia patients on doll therapy irrespective of the ages of the patients. More so, 73.33% of the population sample agree that automated doll therapy is indicative of compliance to best clinical practices for treating dementia patients. Approximately 58.33% of the respondents elaborate health and safety issues, maintenance cost, training, and suitable space to set up a media room to implement the therapy as major barriers to the implementation of this framework.
Selective attention, the ability to maintain mental focus, declines across normal aging. This decline is exaggerated in Alzheimer’s disease (AD), which is reflected by increased reaction times and error rates on the Stroop task, a classic measure of selective attention. While it has been well established that impairment in selective attention is a common symptom of AD, often occurring early on in the disease, the neural correlates underlying these deficits remain elusive. The default mode network (DMN), a collection of functionally related brain areas, normally exhibits task-induced deactivation. However, this pattern of activation is altered in AD. We hypothesized that less DMN deactivation may contribute to errors in selective attention, especially in the AD group.
Using an event-related Stroop task in a functional MRI paradigm, we tested 10 patients with mild Alzheimer’s disease (mean age 73.9±8.4) and 10 healthy elderly (HC) (mean age 63.6±7.8). To analyze failures of selective attention, we assessed the differences in neural activity preceding an incongruent error between HC and AD.
The AD group had significantly slower reaction time for incongruent stimuli compared to the HC group t(18) = −3.85,
While it is not surprising that the ACC was activated in both groups since its involvement in conflict detection, activation of different areas within these relatively large structures suggests that the ACC and precuneus are differentially affected by the disease. Thus the AD group showed more default mode network activity and the HC group showed more frontal activity preceding errors in the Stroop task. This result suggests that the neural correlates underlying errors of selective attention are different in AD than in HC.
Attentional lapses can occur on a daily basis and disrupt the completion of goal-oriented tasks. While the neural correlates of attentional lapses have been studied in young adults, it is unclear whether the mechanisms behind this phenomenon change with age.
We scanned healthy young (n = 12) and older (n = 28) adults with functional magnetic resonance imaging while participants performed a trial-by-trial attention task, the Stroop task, where we measured the response time to each stimulus. We defined an attentional lapse as a longer response time relative to the average response time, and a fast reaction as a faster response time relative to the average.
Young and older adults performed equivalently on all behavioural measures, such as reaction time and accuracy (both
Our results support previous research that activity in frontal and parietal regions of the attentional and default mode networks contribute to lapses of attention. Our results also suggest that the neural correlates of attentional lapses change with healthy aging, reinforcing the idea of functional plasticity to maintain high cognitive function throughout the lifespan.
We investigated the relationship between the precuneus volumes (a component of the Default Mode Network or DMN) and cognitive scores, including scores of verbal memory, in older and younger adults to assess possible functional differences among age groups.
A high-resolution anatomical scan was acquired with a T1-weighted, 3D MP-RAGE sequence in 30 older adults (21 cognitively normal and 9 patients with mild cognitive impairment or MCI); mean age 71.5 years and in 12 younger adults, mean age 23 years. Full cognitive testing had been administered to all subjects within 1–3 weeks of the MRI. The cognitive testing included the California Verbal Learning Test (CVLT), the Montreal Cognitive Assessment (MoCA), the Mini-Mental State Exam (MMSE), and the Stroop test. Manual precuneus segmentation followed previously described anatomical guidelines, marked in the sagittal plane and then segmented in the coronal plane. Precuneus volumes were normalized by total intracranial volume (ICV). Pearson correlations were used to analyze the relation between precuneus volumes and cognitive scores.
Patients with MCI had significantly lower cognitive scores and precuneus volumes compared to the cognitively normal older control group and to the younger group. Among the 30 older participants there were highly significant correlations between the right precuneus and the CVLT short and long delay free and cued recall scores (SDFR r = 0.636,
The volume of the right precuneus appears to be related to scores of verbal memory in older adults but not in younger adults. Selective attention and scores of general cognitive function are also related to right and left precuneus volumes in older adults but not in younger adults. This may explain lack of deactivation of the precuneus during task performance among older adults.
We wanted to investigate whether amateur musical training and leisure playing can protect Alzheimer’s patients from degenerating their episodic memory for music, and to compare these effects with the deficits produced by Alzheimer’s disease (AD) using conventional memory measures.
We recruited an amateur piano player with a 10-year history of studying music and the DSM IV diagnosis of probable AD. The patient was visited at his home each day for a week to conduct logical memory testing, as well as episodic memory testing specific to music. The logical memory section from the Wechsler Test was conducted at 1 and 15 minutes. Similarly, the patient was first shown the piece “A Winter Scene” and asked to sight-read the first 8 bars and then play the 8 bars with both hands from memory at 1 and 15 minutes.
The patient’s performance on the memory test was very poor at onset and showed no improvement over the course of the study. His ability to sight-read the 8 bars of music on the first day was intact and accurate. His immediate recall of the music on the first day showed accurate performance of the first 3–4 bars of music with notes played in both hands. From the second to the fifth day, the patient demonstrated difficulties remembering the melody line, especially in the left hand. The patient, however, was able to recall the right hand melody for the first 3–4 bars correctly on most days. Despite minimal improvements within the first five days, the patient’s performance on the sixth and seventh days reveals nominal improvements in musical expression. On the sixth day, he was able to recall four full bars of music with no errors in the right hand. On the last day, he accurately performed the four bars with both hands for the first time. Even when playing incorrectly, the patient remained within the music’s A-minor key.
Our case study reveals differences in the way AD affects logical memory and episodic memory for music. The patient had deteriorated in logical memory, but was able to retain musical literacy, memory of music, music sensibility and, most importantly, the ability to learn music after repeated trials. Like our previous work on professional artists, our findings here suggest that exposure to music training and performance at an amateur level can preserve the brain’s memory networks involved in musical expression when faced with neurodegenerative disease.
Dementia is a highly prevalent condition among elderly residents in long-term care (LTC) facilities. BPSD can significantly increase both residents’ mortality risk and the burden on the health-care system. A large body of research has identified the importance of BPSD in the management of dementia in LTC. Yet very few studies have assessed the prevalence of BPSD in LTC as a function of the time of day during which symptoms are evaluated (i.e., day vs. evening vs. night). This is an important knowledge gap to be addressed, given that some symptoms may occur more frequently at or after dusk than during daylight hours, and that their emergence at a specific time of day may be associated with different risk factors.
To characterize and compare the prevalence of BPSD in a LTC setting as evaluated by front-line staff who work during the day, evening, and night shifts.
As part of a larger study examining BPSD prevalence and incidence, we assessed neuropsychiatric symptoms of LTC residents over a 3-month period. Frequency and severity of symptoms over a 2-week window were assessed using the Neuropsychiatric Inventory Nursing Home Version (NPI-NH) during the day shift (07:00–15:00), the evening shift (15:00–23:00), and at night (23:00–07:00). The Cohen-Mansfield Agitation Inventory and the Pain Assessment Checklist for Seniors with Limited Ability to Communicate were also administered for all study residents.
A total of 72 residents were evaluated: 56 during the day, 44 during the evening, and 46 at night. Twenty-three residents were evaluated by staff from all three shifts, 24 by staff from two shifts and 25 by staff from one shift only. The prevalence of BPSD was 62.5% during the day, 68.2% during the evening, and 39.1% at night. Among residents who were awake at night, the proportion exhibiting BPSD was 50%. The percentage of residents identified as having more than 4 clinically significant BPSD symptoms increased significantly from 10.7% during the day to 34.1% in the evening (χ2 = 8.12, df = 1,
Our findings are consistent with data reported in previous studies which found BPSD prevalence in LTC as being above 60%, with agitation/aggression and irritability being the most common symptoms. We found evidence of an increased BSPD symptom load during the evening (sundowning) as compared with daytime, and a decrease in BPSD prevalence at night. Our results highlight the importance of considering the time of day during which BPSD symptoms are evaluated in LTC residents.
Older adults diagnosed with mild cognitive impairment (MCI) are considered as a high-risk population for progression to Alzheimer’s Dementia (AD) (e.g., Gauthier
Our study aimed to replicate the results of Belleville and colleagues (2006) with some modifications: 1) a bigger sample size, 2) inclusion of a wider range of MCI sub-types (not only aMCI), 3) inclusion of a Lifestyle Training control group to account for placebo effects and the impact of psychosocial interactions on cognition, and 4) the use objective primary outcome variables from CANTAB (Cambridge Neuropsychological Test Automated Battery) and neuropsychological tests.
We conducted a pseudo-randomized clinical trial in which a treatment group (TR, N = 24, male =9) and a life-style training control group (Control, N = 20, male = 10) underwent a combined Relaxation/Tai Chi Therapy training for 3 weeks and a 6-week training using a modified MEMO method, while the Control group received 6 weeks’ health and lifestyle training program (e.g., discussing factors contributing to diabetes, the importance of exercise, how to prevent falls). All participants were older Francophone adults (age = 69.23±8.78 years, education = 15.50±4.34 years) referred to DMHUI Memory Clinic and having a diagnosis of one of the four subtypes of MCI. Significant medical, psychiatric, neurological or cognitive co-morbidities were ruled out. Participants were tested before and after intervention with cognitive screeners, computerized cognitive tasks, neuropsychological testing, and questionnaires about mood and subjective judgment of memory.
Preliminary results on Repeated Measure ANOVA controlling for age and education indicate a significant treatment (pre/post) effect (F(1,40) = 4.22;
Older persons with advanced Alzheimer’s disease or related disorders (ADR) receive numerous medications to treat an average of 21 health conditions. This is problematic given that the likelihood of drug–drug interactions and adverse drug events increase with the number of medications prescribed. Emergence of symptoms such as agitation, depression, constipation, and pain may in fact be due to adverse events caused by medications originally prescribed for the purposes of long-term prevention strategies. However, as ADR progresses, the objectives of care should shift from a curative to a palliative approach, and medication regimens should be revised and adjusted to reflect this change. There is limited research providing evidence with regard to the risk-benefit profiles of many medications for this specific patient population. Research evaluating interventions in which medication profiles are reviewed and adjusted in ADR patients is even more lacking, thereby underlining the need for new evidence-based guidance.
A scoping review of the literature and an ensuing Delphi panel were conducted to answer the following questions: 1. What criteria exist to determine whether a medication is still appropriate in patients with advanced ADR? 2. Which medications may be considered inappropriate for these patients? 3. Do interventions to optimize medication use in these patients currently exist?
Phase I consisted of a scoping review (NICE, Cochrane Collaboration, Arksey and Levac). Thirteen scientific databases and websites of scientific and gray literature were searched in order to select articles for inclusion using an iterative process. Identified studies were analyzed by two independent reviewers. Studies were included if they were a guideline, review or a primary study, focusing on patients with ADR, at end-of-life, or the elderly, in either a palliative care, long-term care facility (LTCF), or unspecified setting. Letters, editorials, meeting abstracts or studies taking place in a hospital or ambulatory setting were excluded. In Phase II, a Delphi panel following the RAND approach sought consensus from 15 expert clinicians (family physicians, geriatricians, nurses, pharmacists, social workers, and an ethicist) to identify medications deemed inappropriate within the Quebec clinical care context. Interventions judged as promising and applicable were also identified.
The search strategy identified 6,186 references, of which 356 were retained after double screening. Forty articles were identified as being specifically relevant to the research questions at hand, among which 25 intervention studies provided evidence of small but significant reductions in potentially inappropriate medications, adverse events or medication load without associated consequences to morbidity or mortality. The Delphi panel produced three lists of medications: medications always appropriate, medications mostly appropriate, and medications rarely appropriate in these patients. The panel also identified promising key elements of a complex intervention to optimize medication use in this patient population.
Medications frequently prescribed for patients with advanced ADR in LTCFs were categorized as being either always, mostly or rarely appropriate. Several key elements of multidisciplinary interventions involving patients, families, and care teams appear promising for improving medication use among this vulnerable patient population: a pilot study for such an intervention is under way.
Psychotic symptoms in dementia are associated with several negative outcomes, such as earlier institutionalization and increased caregiver stress. Most studies of psychosis in dementia have involved patients with moderate to severe cognitive impairment. Few have examined development of psychotic symptoms in patients who were non-psychotic at baseline. Knowledge of psychosis risk factors at the mild cognitive impairment or early dementia stage is important for understanding the mechanisms underlying psychosis in dementia, and for developing effective prevention and treatment strategies. Our objective was to examine factors associated with the development of delusions and hallucinations in a large sample of patients with an initial diagnosis of amnestic mild cognitive impairment (aMCI, CDR = 0.5) or early stage probable Alzheimer’s disease (AD, CDR = 1.0) who were non-psychotic at baseline.
ADNI data for participants with aMCI (n = 397) or AD (n = 193) at baseline were examined. Individuals with psychosis at baseline were excluded, as were those who developed both delusions and hallucinations as their initial presentation of psychosis, resulting in a sample of 473 never psychotic and 79 who developed delusions (n = 56) or hallucinations (n = 23) as their initial psychotic symptom. The presence of delusions and hallucinations was ascertained from informant ratings on the Neuro-psychiatric Inventory Questionnaire (NPI-Q). Patients with/without delusions or hallucinations were compared with respect to demographic, genetic, and vascular risk factors (history of hypertension, baseline smoking) using chi-squared tests and
A small minority of participants with an initial diagnosis of aMCI developed psychosis. Most of these (55.8%) had progressed to AD by the visit at which symptoms were first reported, with onset of psychosis typically occurring more than 6 months after dementia diagnosis (7.5 months for delusions, 13.2 months for hallucinations). In the combined aMCI/AD sample, more patients developed delusions (10.1%) than hallucinations (4.2%). Age, race (white vs. non-white), gender, baseline smoking status, history of stroke, and presence or number of ApoE-E4 alleles were unrelated to development of psychosis. Having a history of hypertension was associated with development of delusions, while patients who developed hallucinations had a lower level of education and lower baseline MMSE score (
Psychotic symptoms affect a significant minority of patients with early-stage AD. Hypertension was identified as a potentially modifiable risk factor for delusions in dementia. Participants who developed hallucinations had less education than those who were never psychotic, although the average person in both groups had some post-secondary schooling. Lower education is well-recognized as a risk factor for dementia in general, but the possibility of a relationship to hallucinations requires further evaluation. The finding of differing risk factors for delusions and hallucinations suggests that dementia with psychosis is not a unitary construct, and future studies should examine these symptoms separately.
The treatment and care methods used for Alzheimer’s disease (AD) operate within the perceptions of our culture; thus, developing care models for AD individuals is influenced by popular language and attitudes. Critical reflection of our culture and its influences unveils how it impacts beliefs and behaviours; from a health perspective, cultural biases could translate into certain diagnoses and treatment options prescribed by practitioners. Negative misconceptions about people with AD cause unhelpful behaviours that focus on the symptoms of dementia rather than the remaining abilities of the people affected. These misconceptions are reflected in the language associated with AD, which is consistent with the terminology used for “zombies” in media, reflecting our society’s negative views of aging with memory loss. This association is important since 81% of adult Canadians felt they would be treated and viewed differently if others knew they had received an AD diagnosis (Werner & Davidson; 2004).
This paper explores how references to zombies may limit care in North America by framing an individual as ‘dead’ rather than building upon treatments involving social approaches. This paper does not intend to imply causality, rather to associate the perception of AD individuals as zombified with the dominant care approach in North America. In contrast, Danish perceptions driving care are documented to highlight differing perspectives.
Conducting a review of the zombie trope, its impact on stigma, identity, and care of those with AD, it was it was found that the ‘living-dead’ language was thematic throughout both lay and academic literature. Some examples that illustrate this theme include ‘living dead,’ ‘undead,’ and ‘death in slow motion’. Using this zombie language is not conducive to improving quality of life for those with AD.
Though attention for AD is increasing, it often propels a negative view through terms such as ‘living-death’. The presentation of AD in this way focuses on the fear of falling ill, rather than on the way persons with dementia are making the best of their abilities. The ‘living-death’ stigma correlates with the inhibition of developing social care approaches to AD treatment. Unfamiliarity and lack of knowledge incite fear about the illness, and if AD is continuously pushed away with negative stereotyping, we may never truly hear the voices of those with AD. The ‘living-dead’ perception of AD requires scrutiny because this popularised assumption shapes views, and continues to burden current AD practices despite the changes that we see occurring; discrimination and dehumanisation still need to be challenged.
Treatment of dementia varies around the world, and through this exploration I propose that being reflective of our perceptions can lead to better quality of care for those with AD. All individuals exist within a dynamic web of relationships that form who we are and how we behave in the world; awareness of this interconnection reveals how our culture impacts AD.
The cognitive and neuropsychiatric symptoms associated with Parkinson’s disease dementia (PDD) and dementia with Lewy bodies (DLB), collectively known as Lewy Body Disease (LBD), are primarily treated with cholinesterase inhibitors (ChEIs). However, there is significant variability in adverse effects and response among LBD patients taking ChEIs.
To examine the efficacy of ChEIs in treating cognitive and neuropsychiatric symptoms of LBD longitudinally using brain perfusion SPECT.
53 patients diagnosed with PDD or DLB according to standard criteria were initiated on ChEI therapy and prospectively assessed for efficacy and adverse effects. A standardized neuropsychological battery, the Neuropsychiatric Inventory (NPI), and brain ECD-SPECT were ascertained at baseline (no treatment) and at 24 weeks. A repeated measures ANOVA design was used to determine change over time in these measures.
LBD patients treated with ChEIs showed significant improvements in visuospatial, attention, and phonemic fluency tasks (
Treatment with ChEIs was found to be effective in reducing visual hallucinations and improving cognitive function. The negative correlation found above suggests that perfusion in the occipital region could be developed as a bio-marker for use in distinguishing between LBD responders and non-responders to ChEIs in terms of visual hallucinations.
Retirement/residential homes (RHs) are a generally underappreciated component of the health-care system. A prevalence of > 70% dementia is recognized in long-term care homes, but the prevalence in RHs has not been established. The average age in both types of homes is approximately 86, and chronic geriatric conditions are common in both. In Ottawa, Ontario there are far more RH places (8,500) than long-term care beds (5,500). A previous study in an Ottawa RH showed recognized dementia in 40% and dementia screening was positive in an additional 32%. This study in the Prince of Wales Manor (POW) goes one step further in that specially trained nurses did a comprehensive cognitive assessment and, with geriatrician review, a diagnosis of normal cognition, mild cognitive impairment (MCI) or dementia was established.
After resident/family consent,73 POW residents underwent: 1) chart review to establish residents with a diagnosis of MCI or dementia; 2) screening (Cognitive Quickscreen:CGS) of all residents without a diagnosis of MCI or dementia (the CQS was three-item: recall, clock drawing, and animal fluency); 3) cognitive assessment for those failing the CGS by trained nurse assessors (see assessment guide); and 4) diagnostic review by a geriatrician and resident attending physician to establish a cognitive diagnosis.
Chart review showed 30 residents with dementia (41%), 2 residents with MCI (3%), and 41 residents with neither (56%). The CQS results in the 41 remaining residents revealed 73% failure, 12% pass in all 3 items, and 15% refusal. The 30 residents failing the CQS and the 2 residents with MCI had comprehensive cognitive assessment, and provisional diagnoses were that 15 residents had dementia (22% of the original sample of 73 residents minus the 6 refusers). Additionally, 8 residents were felt to have MCI. Overall 45 of 67 residents (67%) were felt to have dementia, 8 (12%) had MCI, and only 14 (21%) were felt to be cognitively normal.
Retirement/residential homes have a very high prevalence of dementia (67% in this study) with approximately 1 out of every 3 cases of dementia being unrecognized (15 out of 45 total). A cognitive screening and assessment program using a structured dementia assessment guide can be utilized in a time- and resource-efficient manner to address this important health-care issue. RH residents without a diagnosis of dementia or MCI should be screened for dementia at admission and regularly after admission.
The time required to complete a comprehensive geriatric assessment is significant, and the demand for specialized geriatric services is increasing through the current demographic shift in the Canadian population. Within a specialized memory clinic, more time is required to dictate detailed comprehensive geriatric assessment clinic visit reports. A timely report to the hospital electronic record is an essential element of good specialist care. To reduce report production time, an innovative solution was designed and implemented in the clinic’s new longitudinal research registry and documentation system. A novel approach to the automatic generation of a smart narrated synoptic report was developed allowing for reports to be autopopulated with the required patient data.
Using computer-programming semantics, a report template was created for the initial assessment. The smart report template employed the use of algorithms to determine: 1) what clinical information will be reported; 2) whether the report will be in short or long form; 3) the specific places in the report where information from the clinical database would be injected; and 4) how the information is visually presented in the report. The wording generated in the smart report is determined through the logical analysis of the patient data collected. For example, depending on the context of the pronoun use, the gender (male/female) stored in the database indicates which version of the pronoun should be used.
These reporting algorithms can potentially have various steps of decision-making, or nodes, each with increasing complexity. When using a smart report template to generate a clinic report, each algorithm in the report is automatically evaluated by the system. The generated report shows the cumulative results from the algorithms as a complete, finished report. The user is presented with an automatically generated report that can then be modified and customized to meet any special needs for that particular report. The user may edit the final report by traditional keyboard, via dictation, or by inserting a report snippet. A report snippet is a predetermined piece of code which can represent a standard report element (e.g., a standard page letterhead), a data element extracted from the system (e.g., the patient’s birth date or referring physician’s name, etc.), or an automatically evaluated logic statement (as in the example of pronoun use above). Once inserted into the report, the report snippet is fully rendered, displaying the final report content which can be manually edited by the user.
The use of the customized smart synoptic reporting solution has anticipated benefits on geriatric clinical practice. Since most of the report is automatically generated from a customized template, the amount of dictation time required is reduced. The use of this solution can improve report accuracy through the use of a standardized template, which can then be customized and sent to multiple recipients in short or long form. Smart reports can increase the timeliness of new reports, as reports can be generated at the end of the visit in short form with recommendations and instructions for patients and caregivers.
Current registries, information systems, and family practice electronic medical record systems are generic in nature and are not tailored to a specialist’s workflow or clinic needs. The use of an efficient data collection system, along with the application of an effective workflow model, can lead to significant improvements in the quality of health care provided to patients. We have developed a unique longitudinal web-based tracking system for patient treatment, outcome management, clinical reporting, and research.
The system’s design was informed by health-care providers, a review of existing systems, and published literature. Several modules have been established to address the concerns of the adopting clinic, including: patient demographics, course of symptoms, co-morbid illnesses, medications, cognitive testing, physical and neurological examination, diagnoses, synoptic and detailed reporting, and data analysis for both clinical and research purposes. Conditional patient access provides an interactive model of care to the clinic and allows for future expansion with a patient portal. In a traditional system, baseline information is typically collected manually by a health-care provider using a paper-based form. These forms are transcribed or coded into an electronic database of patient record. This method of retrospective data entry leads to various quality control issues. To address this challenge, our platform was designed to be used not only with web-capable desktop and tablet devices, but with kiosk systems, as well. Various techniques were established to improve quality control and to ensure the accuracy of patient records. Demographic data can be imported or entered directly into the database by the patient, registration clerk or clinician. Furthermore, field validation is used to ensure that no data are missing or incorrectly filled. All data collection forms are clinical workflow oriented, with built-in secondary form validation, autocomplete, autosave, and dropdowns to facilitate fewer entry mistakes.
The resulting increase in quality control ensures accurate patient record entry—the more accurate the data, the more accurate the statistical analysis. The platform has been customized to provide the following system-wide features: side-by-side comparison of past and current information; real-time data entry collaboration between interdisciplinary team members; forms are modular in design to allow for easy expansion; look-up lists (e.g., national medication repository, clinic staff, past occupations, hobbies, standardized diagnostic codes from the US National Alzheimer’s Coordinating Centre); digital capture of cognitive tests; synoptic reporting; interactive progress notes and comments; simplified web-based modelling and statistical analysis tools.
The benefits of utilizing such a registry platform can significantly increase both the quality of care provided to patients and the efficiency of clinical practice. Our registry can allow for the measurement of the quality of care indicators, reduce clinical and data-entry errors, and facilitate research since all clinical data can be analyzed statistically in real time.
Brain disorders that lead to aberrations of affect, cognition, and behaviour (ABC) constitute a growing and resource-demanding health crisis in Canadian society. The number of individuals with dementia, which is an important disorder of ABC, is rapidly increasing. Specialist input is often sought in the diagnosis and treatment of dementia. However, several specialties and subspecialties manage dementia, including geriatric medicine, neurology, geriatric psychiatry, and Care of the Elderly family practice. Other specialists, including neurosurgeons, are becoming involved in the management of dementia. Many of these specialists have either “learned on the job” or taken informal additional training to acquire special competency in the area, without standardization or formal recognition of that training. Creating a standardized training program for physicians wanting to acquire additional competency in disorders of ABC would assure quality of care and attract more physicians to this area, which will be needed to cope with the increased burden that will be posed by ABC disorders. Such training would gather specialists into a more harmonized community of practice in disorders of ABC, and could lead to the emergence of transdisciplinary knowledge and competencies that will allow trained physicians to better cope with these conditions, especially dementia.
The Department of Psychiatry at the University of Toronto recently sponsored a meeting for a “grass-roots” group of specialists from across Canada, who all deal with disorders of ABC to a substantial degree in their practice. They explored the creation of a Royal College of Physicians and Surgeons of Canada (RCPSC) Diploma program in disorders of ABC. This presentation highlights the results of that meeting and forthcoming efforts.
There was broad consensus that such a Diploma program would be useful. The precise name of this field of training is still being debated, although the preliminary frontrunner is “Integrative Brain Medicine”. A consensus definition for this field of study was agreed upon. A “core” training program for the Diploma was proposed, to be accompanied by additional specific “streams” that trainees could choose to focus on, including one in dementia.
A transdisciplinary team of medical educators, with the support of RCPSC, is developing a new Diploma training program to formally recognize training in disorders of ABC, including dementia, and to boost the number of physicians undertaking this training. This is a meaningful step to stem the “rising tide” of these disorders. The Diploma program proposed at the Toronto meeting is being refined with further input from interested stakeholders being sought and warmly welcomed, with the goal of presenting a full proposal to the RCPSC in the spring of 2014. Please contact the first author, Dr. Alex Henri-Bhargava, at
Traditionally physicians have viewed mild memory complaints in older people to be benign. However, subjective memory complaints in people who have “normal” cognition on testing is termed Subjective Cognitive Impairment (SCI), a pre-MCI stage, and may last up to 15 years. Memory loss as a self-observed complaint is more easily identified than changes in executive function. Identifying people with MCI who are at increased risk for dementia/Alzheimer’s disease, and arranging for follow-up is the current best practice recommendation from the Third Canadian Consensus Conference on the Diagnosis and Treatment of Dementia (CCCDTD3). For the last five years, we have advertised during Alzheimer Awareness Month (January) and Senior’s Awareness Month (June) for people 55 years and over who have memory concerns, who are interested in research, and who have not had a stroke.
1) To classify the clinical suspicion of memory complaints in respondents and offer follow-up. 2) To complete clinical assessments in those with a clinical suspicion of MCI or dementia on case finding, confirm a clinical diagnosis, and offer research studies for which they may be eligible.
Over the last 5 years a total of 166 people 55 years and over responded to newspaper advertisements with self-reported memory concerns. Participants received cognitive screening tests using the standardized MMSE, the MoCA, the 15-point GDS, the AD8, the Cornell Scale for Depression in Dementia, and the Lawton Brody Activities of Daily Living Scale. The test results were case-conferenced with a geriatrician and a clinical suspicion of SCI, MCI, depressive symptoms, mixed picture, possible dementia or other was given. All participants agreed for their test results to be sent to their family physician. Fifty-eight individuals have repeat measures on these tests from 2009 to 2013.
Of the 58 follow-up subjects, 45 returned for follow-up after one year and 29 returned for follow-up after two years. In 2013, of those 58 follow-up participants, 54% (31) had no change on their cognitive tests. However 33% (21) had declined over the 5 years and 10% (6) had improved. Of those who were given the clinical suspicion of SCI in 2009 or 2010, 39% had progressed to amnestic MCI or multiple-domain MCI. Those individuals who reported depressive symptoms in 2009 (32%) tended to have lower scores on the GDS and Cornell on follow-up visits. Individuals who declined follow-up appointments maintained that their memory was ‘fine’ and no longer wished to be followed.
Of those who returned for follow-up, 33% progressed to MCI within 5 years; however, they only represent 35% of the total sample. Therefore a conservative estimate would be 12% of the participants progressed to MCI. It is uncertain whether those who declined follow-up represent individuals who have reverted to ‘normal.’ Limitations: 1) The participants are drawn from those who have insight to changes in their memory, therefore it may understate the total number; 2) 65% to date have elected not to return for follow-up.
A growing number of middle-aged individuals presenting with concerns of memory loss and decreased mental efficiency are being diagnosed with previously unrecognized attention deficit/hyperactivity disorder (ADHD). However, specific neuropsychological tools to differentiate adult ADHD from prodromal Alzheimer’s disease or mild cognitive impairment (MCI) are lacking. One of the core deficits that have been consistently associated with both childhood and adult ADHD is impairment in inhibitory control, as commonly measured using the Stop Signal Task (SST). One study has found mild differences in inhibitory control between MCI and normal controls (NC), but this is still being investigated. Deficits in visual working memory (VWM) have also been reported in both ADHD and MCI. These deficits can be examined using a task that specifically distinguishes random errors from errors due to the inability to divert attention from non-target objects to target objects during visual encoding. No previous studies have yet examined performance on these specific measures in adult ADHD and MCI.
The aim of the present study is to compare performance on both the SST and this VWM task between individuals with ADHD and MCI and examine potential correlations with regional grey matter volumes. We hypothesize that deficits in inhibitory control and VWM errors due to non-target responses will discriminate ADHD from MCI. Our second hypothesis is that ADHD subjects will show increased medial and lateral prefrontal cortical thinning and lower putamen and caudate volumes than both MCI and NC.
25 ADHD and 25 single and multi-domain amnestic MCI participants will be recruited from the memory clinic at Sunnybrook Health Sciences Centre. All participants will be assessed using the Adult ADHD Self-Report Scale-V1.1 and Connors’ Adult ADHD Rating Scale-S:L. The Albert and Peterson Criteria will be used to diagnose MCI. The SST will be administered to obtain measures of inhibitory control, response latency and variability, and error monitoring. Intra-individual variability will be studied using ex-Gaussian fitting, and error monitoring will be assessed based on the extent to which participants slow their response following inhibition failures. A previously described VWM task will be administered in which multiple items are presented in the visual field and the subject must recall the colour of a probed item. The proportions of target responses, non-target responses, and random errors will be calculated for each participant.
Results will be compared between groups using Analysis of Covariance (ANCOVA), correcting for age and education. Assessments of memory, attention, and executive function will be obtained through standard neuropsychological testing. Cortical thickness and grey matter volumes of targeted structures will be measured from structural 3D T1 MRI using a previously published semi-automatic pipeline. Partial correlations, controlling for age and education, will be used to assess the relationship between neuropsychological measures and brain volumetrics.
This study will explore the utility of neuropsychological tools to differentiate ADHD among middle-aged patients presenting with memory complaints from MCI. This study will also provide the foundation for a larger project aimed at examining the relationship between ADHD and Alzheimer’s disease in the baby boomer population.
Vitamin D (25OHD) insufficiency has been associated with cognitive decline and dementia. In addition to comparatively worse global cognitive performance, individuals with deficient or insufficient vitamin D levels (less than 75 nmol/L) tend to perform worse on tasks of executive functioning. It remains unclear if “supratherapeutic” levels (100 nmol/L or greater) are associated with even better cognitive performance than sufficient levels. The present study sought to address this question, hypothesizing that executive functioning tasks would be most associated with vitamin D insufficiency (less than 75 nmol/L) and that cognitive performance would not differ significantly between those with sufficient and supratherapeutic levels.
Healthy adults, at least 20 yrs of age participated in the winter phases of the D-COG (Nov. 2010–March 2011) and D-COG2 (Nov. 2011–March 2012) studies. Cognitive testing consisted of the Symbol Digit Modalities Test, Verbal (phonemic) Fluency, Digit Span, and CANTAB computerized battery. Body mass index (BMI) and mood (i.e., Beck Depression Inventory-II) were also assessed. Participants were also asked about vascular risk factors and physical activity. Serum vitamin D (25OHD) levels were analyzed via liquid chromatography/mass spectrometry. PTH, phosphorous, and ionized calcium levels were also obtained.
Data from the D-COG (n = 43) and D-COG2 (n = 99) were pooled due to identical study protocols. The 142 participants were 56.3±14 yrs old with 14.9±4 yrs of education and 71.8% female. They were categorized into the following three groups depending on vitamin D levels: Insufficient (less than 75 nmol/L; n = 73); Sufficient (75–99.9 nmol/L; n = 36), and Supratherapeutic (> 100 nmol/L; n = 33). Vitamin D levels were significantly correlated with performance on Verbal Fluency (partial correlation corrected for age, education, r = .23,
Vitamin D levels were positively and linearly associated with performance on verbal fluency, a task that assesses executive functioning and language. Surprisingly, Supratherapeutic levels were associated with even better performance than sufficient levels on this task. Importantly, however, these sufficiency categories are based on bone health guidelines and the optimal level of vitamin D for cognition is not known. This study suggests that levels exceeding 100 nmol/L may be optimal for at least some aspects of cognition, including executive functioning and perhaps language. What effects vitamin D supplementation has on these and other cognitive domains is not known, but is currently being tested in a randomized supplementation study.
Impaired memory is a core component of Alzheimer’s disease (AD), and patients with AD have been shown to have increased impairments in working memory. Along with this loss in memory, patients also often experience difficulties in attention and, in fact, studies have posited that it is the attentional impairments that underlie many of the deficits in cognition and function seen in patients with AD. Our team has developed the Visual Attention Scanning Tool (VAST), an eye-tracker which enables real-time measurements of attention patterns towards competing visual stimuli. The objective of the present analysis is to observe the spontaneous visual scanning patterns of AD patients in the presence of novel and repeated stimuli using a modified n-back paradigm in order to explore working memory in a naturalistic setting.
This is cross-sectional study of patients with mild to moderate Alzheimer’s disease (probable AD by NINCDSARDRA criteria, with a Mini-Mental State Examination score > 10). Visual attention was assessed using the VAST system. Patients were presented with 48 slides, each containing four images simultaneously presented. All four images have similar complexity, valance, and arousal. Two images on each slide were novel and two were repeats of images that were shown previously—repeats of one slide back (n = 1) and 2 slides back (n = 2). Images on each slide were arranged 2 by 2, with the position of the novel stimuli and previously shown stimuli randomly intermixed. Comparisons between and within groups were conducted using two way ANOVA.
61 patients have been recruited to date (37 AD, 24 controls). Overall, the average age was 74.6±9.2 years, with patients with AD being older than controls (77.1 vs. 70.7 years). The average Mini-Mental State Examination score was 24.4±4.2, with AD patients having a lower score (22.1 vs. 28.0). There was a significant main effects of disease (F1,118 = 23.5,
These preliminary data for our n-back paradigm of working memory suggest that the orientation towards novel stimuli observed in cognitively intact subjects was not observed in AD patients. These findings suggest that working memory deficits can be detected in AD patients without requiring verbal communication.
Neuropsychiatric symptoms associated with dementia present significant challenges to family caregivers and health providers, yet data illustrating variation in the prevalence and correlates of symptoms across care settings or by sex are scarce. We sought to estimate the prevalence and associated correlates of neuropsychiatric symptoms across home care (HC), long-term care (LTC), and complex continuing care (CCC) settings and by sex.
Cross-sectional study of all HC clients (n = 470,183), LTC residents (n = 127,285), and CCC residents (n = 93,206) aged 50+ years assessed with the Resident Assessment Instrument (RAI-HC or RAI 2.0) in Ontario, Canada from 2004 to 2010. Multivariable logistic regression models were used to identify correlates of neuropsychiatric symptoms across care settings, for total samples and stratified by sex.
There were 100,500 (21.4%, 95% CI 21.3–21.5%) HC clients, 72,732 (57.1%, 95% CI 56.9– −57.4%) LTC residents, and 23,459 (25.2%, 95% CI 24.9–25.4%) CCC residents with a diagnosis of dementia. The severity of impairment associated with dementia generally increased from HC to LTC to CCC; however, there were important differences across care settings. LTC residents with dementia were significantly older, more likely to be women, to exhibit depression and aggressive behaviours, and to be receiving 1+ antipsychotics and/or antidepressants, whereas those with dementia in CCC (despite showing comparable levels of cognitive impairment to LTC residents with dementia) were more likely to be functionally dependent, to have significant health instability, and to have a recent decline in mood, apathy, anxiety (and use of 1+ anxiolytics), and loss of appetite. The proportion of persons with dementia exhibiting 1+ neuropsychiatric symptom(s) was higher in LTC and CCC (∼ 98%) than in HC (∼ 61%). Adjusting for age, cognitive and functional status, women with dementia were significantly more likely to exhibit depression and anxiety, appetite/eating issues, delusions (HC & LTC), and night-time behaviours (LTC). Conversely, men with dementia were significantly more likely to exhibit agitation/aggression/disinhibition, apathy (LTC & CCC), irritability, motor disturbance (CCC), and hallucinations (HC). The percentage of HC clients with a distressed caregiver was higher among males with dementia and for both men and women, increased with number of neuropsychiatric symptoms. The associations between age, functional and cognitive impairment levels, and selected neuropsychiatric symptoms were generally similar for females and males with dementia, although there were some notable differences. For example, female HC clients with dementia showed stronger associations between increasing cognitive impairment and agitation/aggression/disinhibition and irritability, whereas male HC clients with dementia showed stronger associations between increasing cognitive impairment and anxiety.
We observed significant differences in the profile of neuropsychiatric symptoms among persons with dementia across care settings and by sex. These differences suggest the need for more targeted care planning and interventions to better prevent and manage select neuropsychiatric symptoms across the care continuum.
Alzheimer’s disease (AD) leads to cognitive declines in language, memory, and executive function, affecting an individual’s ability to complete activities of daily living (ADLs) independently. At the moderate and severe stages of AD, there is a need for formal caregivers (e.g., a nurse, personal support worker) to assist residents with AD during the completion of self-care tasks (e.g., grooming and washing). Unfortunately, breakdowns in communication commonly occur between formal caregivers and residents with AD during ADLs, leading to strained communication interactions and task completion difficulties. The systematic examination of which verbal and nonverbal task-focused communication strategies caregivers’ use to support residents with AD during task completion has been done. However, there is a need for the systematic examination of (1) which communication strategies contribute to fewer communication breakdowns during daily tasks, and (2) which communication strategies effectively repair communication breakdowns when they do occur. This systematic observational comparison study aims to examine which task-focused communication strategies formal caregivers’ use to repair communication breakdowns that occur while assisting residents with moderate and severe AD during the completion of a basic ADL: teeth-brushing.
Fifteen (15) formal caregivers (personal support worker = 14; nurse = 1) and thirteen (13) residents with a confirmed diagnosis of AD (moderate = 6; severe = 7) participated in this study. Participating caregivers and residents with AD were recruited from two different community-based, long-term care facilities. Established caregiver–resident dyads were observed during the completion of six separate teeth-brushing sessions (78 teeth-brushing sessions in total). Each teeth-brushing session was transcribed verbatim into the Systematic Analysis of Language Transcripts (SALT), a language analysis software program. Next, utilizing conversation analysis (CA) method and the trouble source-repair (TSR) sequence paradigm, communication breakdowns were identified. In addition to the identification of communication breakdown and repairs, instances of no trouble source-repair (NTSR) sequences were identified. Finally, the TSR sequences (i.e., trouble source, repair signal, repair type, and resolution) and the NTSR sequences will be coded. Descriptive statistics will be used to analyze the relative frequency of task focused communication strategies occurring during TSR sequences and NTSR sequences as a function of disease severity. Correlation analysis will be used to examine the relationships between the resolution of repair strategy (outcome) and the relative frequency of communication strategies as a function of disease severity.
Across 78 observed teeth-brushing sessions, 215 TSR sequences and 150 NTSR sequences were identified. Agreement analysis was performed on 20% of the transcripts using occurrence percent agreement. Two raters showed 92% agreement for the identification of TSR sequences and 92.4% agreement for the identification of NTSRs. The complete analysis of the TSR sequences and the NTSR sequences is currently underway.
We will present results and conclusions at the 7th CCD. Findings from this study will help to understand further which communication strategies are most effective when assisting residents with AD during daily activities. Moreover, findings from this study will be used to help inform the development of evidence-based communication guidelines for caregivers assisting individuals with AD.
The NIMH-Provisional Diagnostic Criteria for depression of Alzheimer’s Disease (PDC-dAD) have been proposed over a decade ago. However only few studies examined the validity of depression scales, including the Cornell Scale for Depression in Dementia (CSDD) and the Montgomery-Ãsberg Depression Rating Scale (MADRS), for this novel diagnostic approach to depression of AD (dAD). The validity of brief self-report scales with a parallel version for informant to provide collateral input for assessment of depression of AD has not been examined.
To study the validity of the Geriatric Depression Scale (GDS-30) developed for older adults and validated for the DSM [Major Depressive Disorder (MDD)] in detecting dAD, and to compare the subject (GDS-30) to the informant scale (GDS-IF-30).
Subjects with AD and their informants, recruited at the UBCH-CARD (Clinic for Alzheimer Disease and Related Disorders) completed the GDS-30 and GDSIF-30, Neuropsychiatric Inventory (NPI) (informants), Quality of Life in AD (QoL-AD), and Montreal Cognitive Assessment (MoCA) (subjects). Subjects were assessed by a UBCH-CARD clinician for dAD according to the NIMH-PDC. Inclusion criteria were: a) subject meets possible or probable AD criteria (Mini Mental State Examination (MMSE) = 10 to 26); b) is able to communicate in English; c) has a knowledgeable informant who has contact at least 3–4 times/week. To examine concurrent validity, we performed ROC analyses on the accuracy of GDS scores in detecting a dAD diagnosis. To examine convergent validity, we computed correlations between GDS, NPI depression item scores, and QOL-AD. To examine discriminant validity, we performed correlations between GDS and MoCA scores.
The sample consisted of 21 subject/informant dyads (subject mean age = 71.33; mean education = 14.67; mean MMSE score = 22.2; 11/21 (53%) were men). Six subjects were found to have dAD (mean age = 69.33; mean education = 14; mean MMSE = 23.5; 50% were men) and 15 were non-dAD (mean age = 72.13; mean education = 14.93; mean MMSE = 21.6 (n = 14); 53% were men). The AUC for GDS-30 was 0.79 (
The Geriatric Depression Scale based on subject and informant showed good accuracy in detecting dAD. The cut-off scores for dAD were lower than those reported for DSM-MDD. The correlation between GDS-30, GDSIF-30, NPI-depression item, and QOL-AD support the depression scales convergent validity. The lack of correlation between GDS-30 and GDSIF-30 and MoCA supports the depression scales discriminant validity. Overall, the study provides validity of inference for GDS-30 and GDSIF-30 with a limited sample of 21 dAD and non-dAD.
“Poster cortical atrophy (PCA) is a neurode-generative syndrome that is characterized by progressive decline in visuospatial, visuoperceptual, literacy, and praxic skills. The progressive neurodegeneration affecting parietal, occipital, and occipitotemporal cortices that underlies PCA is attributable to Alzheimer’s disease in most patients.”(Crutch
A brief review of the clinical features and subsequent safety concerns of PCA will be provided, as well as the limited options for pharmacotherapy and non-pharmacologic therapy management. The OT role and general intervention strategies for patients with PCA will be presented, including a recently developed set of recommendations for OT intervention for use with patients experiencing AD-related visual dysfunction. The process of developing an OT specific resource for clinicians providing direct and consultative services for patients with AD-related visual dysfunction will be discussed. The interprofessional context of the tool and the tool itself will be reviewed with recommendations for its use, including practical visual aid interventions and adaptations that address 7 main areas of concern in relation to visual dysfunction in dementia. A brief description of an early stage, international systematic study looking at the effectiveness of visual compensatory strategies for this population will be discussed.
While prevalence and incidence of PCA are currently unknown, with the rapidly expanding older population and forecasted increase in dementia in the coming decades, it is evident that the incidence of PCA will expand and subsequently the demand for OT services to optimize the independence and safety of this population at home. Occupational therapists who are experts in the analysis of function that are aware of the issues regarding PCA play a vital role in the management of this patient population for which no other management currently exists. While there is considerable research demonstrating the impact of visual impairment on ADL and IADL performance in the older adult population and the research examining the effect of OT in this area is growing, further research is required to measure the unique contributions of OT, especially for people with PCA, for which no research current exists.
Corticobasal syndrome (CBS) is a progressive, neurodegenerative condition typified by asymmetric motor symptoms (dystonia, rigidity, akinesia, myoclonus) in the setting of cortical sensory impairment, apraxia, and in prototypic cases, alien limb phenomenon. A diversity of pathologies including Alzheimer’s disease (AD), Lewy body disease (LBD), and cerebrovascular disease have been associated with CBS. Similarly, AD is itself associated with significant phenotypic variation and may result from an array of genetic mutations, in particular in presenilin-1 (PS1), presenilin-2, and amyloid precursor protein, all producing a highly aggressive, early-onset phenotype. PS1 in particular has been described in association with a heterogeneous phenotypic array, although not as CBS. Here we describe the first known association between a novel PS1 mutation and CBS in two brothers, one with right-predominant CBS, and the other with left-predominant CBS. These cases illustrate not only remarkable phenotypic mimicry, with an AD gene resulting in CBS, but also the phenotypic heterogeneity that may result even when the same causative mutation is present.
Two brothers were assessed at the Sunnybrook Health Sciences Centre, Toronto, Canada between October 2008 and June 2010 (Brother RP: follow-up 11 months with 3 visits; Brother LP: 19 months with 4 visits). Both underwent detailed neurologic assessment including physical examination, screening blood work, detailed conventional neuropsychological testing, MRI (1.5 T), and SPECT (T99 ECD). Both brothers consented to and underwent post-mortem pathologic assessment, as well as genetic analysis by deep gene sequencing for PSEN1 mutations.
Right Predominant (Brother RP). RP was a 55 y.o. dentist with right arm myoclonus, dystonia, and mild rigidity for about 1 year prior to initial presentation. His wife also noticed word-finding difficulties, poor comprehension, and empty speech for 2 years, with significant apathy and depression emerging more recently. On initial examination he had impaired stereognosis and graphesthesia, subsequently developing significant apraxia. Based on these findings RP met criteria for probable CBS2. Post-mortem confirmed Braak stage VI/VI Alzheimer’s pathology. Genetic analysis demonstrated a PSEN1 mutation of phenylalanine to leucine at codon 283 (F283L). Case 2: Left Predominant (LP). LP was a 56 y.o. urban planner with left arm myoclonus and apraxia at initial presentation and left predominant akinesia and rigidity emerging 1 year later. Initial examination demonstrated impaired stereognosis and graphesthesia on the left. At last follow-up, he additionally had left arm and leg weakness, left facial droop, and left tongue fasciculations. Mood or behaviour was normal. LP’s speech was slowed at onset, eventually becoming nonsensical. Based on these findings, RP met criteria for probable CBS2. Post-mortem confirmed Braak stage VI/VI Alzheimer’s pathology. As with PR, LP demonstrated the same F283L mutation of PSEN1. Position-specific independent counts (PSIC) analysis yielded a score of 2.5, suggesting good likelihood of protein dysfunction resulting from this mutation.
This is, to our knowledge, the first description of an autosomal dominant case of AD resulting in the CBS phenotype, caused by a novel F283L mutation in PSEN1. Further, these cases, presenting on opposite sides of the body, illustrate how phenotypic heterogeneity can occur despite identical genotype.
Elderly nursing home residents often have multiple medical co-morbidities and are prescribed numerous medications. With the use of more medications comes the risk of adverse drug reactions due to pharmacokinetic and pharmacodynamic changes, as well as drug interactions. Previous studies have found a relation between polypharmacy and a higher number of care problems (falls, pain, or constipation). There are various criteria regarding medications that are potentially inappropriate in the geriatric population, such as the Beers criteria; however, there seems to be less known about the use of medications and nutritional supplements which are generally not considered harmful, but may no longer be providing benefit, and which may be worsening quality of life, particularly in late dementia.
After appropriate ethics approval, we conducted a chart review on nursing home residents with advanced dementia (Fast Stage 7) living on dementia units at 4 nursing homes. De-identified data were sent to a clinical advisory team consisting of a pharmacist, a specialist in the use of nutrient supplements, a family physician with expertise in the care of the elderly, and a geriatric psychiatrist. The advisory team members completed standardized questionnaires regarding the appropriateness and potential problems with each medication and nutritional supplement, taking into consideration a clinical summary (prepared by the first author) on each study participant. A follow-up meeting with the advisory team reviewed and debated the results of the questionnaires and attempted to come to consensus decisions about the use of each medication based on the clinical context of each patient. Results were summarized by the first author.
Consensus was achieved on many, but not all, of the individual medications prescribed, with differences related to the clinical experiences and specialty of the advisory team member. Many vitamins were prescribed at excessive doses, while other recommended vitamins were not prescribed at adequate doses, or frequency. Reasons for administration of PRN medications were often not specified, contributing to the risk of prescribing of those medications for inappropriate reasons (such as using antihistamines for sleep or behavioural problems). Medications with a long time to benefit and significant adverse effects (such as statins) were prescribed in some patients, even those with short anticipated life expectancy and challenges with oral medication administration.
In end-stage dementia there are many factors to consider when determining which medications may or may not be appropriate. Determining medication appropriateness is simpler when a particular medication is known to have significant adverse effects with little benefit. Choosing appropriate medications is more complicated when the medication has few or mild side effects, but a long time to benefit. Many of these patients have swallowing difficulties and medications can contribute to overall burden of illness.
The opinions expressed in the abstracts are those of the authors and are not to be construed as the opinion of the publisher (Canadian Geriatrics Society) or the organizers of the 33rd Annual Scientific Meeting of the Canadian Geriatrics Society. Although the publisher (Canadian Geriatrics Society) has made every effort to accurately reproduce the abstracts, the Canadian Geriatrics Society and the 33rd Annual Scientific Meeting of the Canadian Geriatrics Society assumes no responsibility and/or liability for any errors and/or omissions in any abstract as published.
The 85+-year-old population – the “oldest old” – is now the fastest growing age segment in Canada. Although existing research demonstrates high health services utilization and prescribed medications in this population, little epidemiological evidence is available to guide care for this age group.
To describe the epidemiological characteristics of common health conditions and medication prescriptions in the “oldest old”.
We conducted a retrospective chart review of all family practice patients aged ≥ 85 (N = 564; 209M:355F) at Sunnybrook Health Sciences Centre, Toronto. Electronic medical records were reviewed for all current chronic conditions and medication prescriptions, and then stratified by sex and age subgroup (85–−89, 90–94, 95+) for descriptive analysis.
On average, patients experienced 6.4 concurrent chronic conditions and took 6.8 medications. Most conditions were related to cardiovascular (79%) and bone health (65%). Hypertension (65%) was the most common condition. Bone-related conditions (e.g., osteoarthritis, osteoporosis) and hypothyroidism predominantly affected women (
Multimorbidity and polypharmacy are highly prevalent in patients over 85. Most clinical concerns in this population relate to cardiovascular and bone health; medications predominantly treat risk factors. In the absence of epidemiological data to guide clinical decision-making, this study provides a first look at the common health concerns and medication profiles in this population.
Although a serious public health concern, very little is known about the neurocognitive basis of suicidal behaviour in the elderly. Here we aimed at: 1) assessing alterations in cognitive inhibition in suicidal depressed elderly people, and 2) reviewing the literature on cognitive inhibition and decision-making in elderly suicidal behaviour.
First, we compared 20 currently depressed patients, aged 65 and older, who had recently attempted suicide to 20 elderly subjects with a current depression but no personal history of suicide attempts and 20 elderly controls. Different aspects of cognitive inhibition were examined: access to relevant information (reading with distraction task), suppression of no longer relevant information (Trail Making Test, Rule Shift Cards), and restraint of cognitive resources to relevant information (Stroop Test, Hayling Sentence Completion Test, Go/No-Go task). Second, systematic MEDLINE literature search was performed on neurocognitive deficits in suicidal behaviour. References from our research group’s online database were also selected (
After adjustment for age, depression intensity, Mini-Mental Status Examination score, and speed of information processing, suicidal depressed elderly people showed significant impairments in all three domains of cognitive inhibition in comparison to the affective and healthy control groups. Moreover, the results of a meta-analysis study will also be presented.
Cognitive inhibition deficits and impaired decision-making appear to be part of a series of cognitive deficits and may impair the patient’s capacity to respond adequately to stressful situations, which could subsequently lead to an increased risk of suicidal behaviour during late-life depression. Suicide prevention interventions may be developed to specifically target cognitive impairment in depressed elderly people.
Anticholinergic drugs may induce cognitive decline in older adults, but data are conflicting. One research challenge is ascertaining the effect of different exposure & outcome definitions on measures of association.
Using baseline and 1-year follow-up data from 131 community patients aged 60+, we applied 4 measures of anticholinergic drug exposure (the Anticholinergic Drug Burden Index (DBI), ACB, ADS & ARS, and 4 definitions of cognitive decline (neuropsychological test raw change scores, the RCI, the standardized regression based change score (SRB), and the clinical diagnosis of a new mild neurocognitive disorder according to DSM-5 criteria). The frequency of classification for each patient and the number needed to harm (NNH) was calculated according to each exposure & outcome definition. The consistency of associations between drug exposure & cognitive decline was examined using logistic regression models for each definition.
The Anticholinergic DBI identified the smallest number of patients with an increase in anticholinergic exposure (n = 18) and the ACB identified the largest number (n = 23). The RCI identified cognitive decline in only 6 patients; 12 patients were diagnosed with a new mild neurocognitive disorder, 44 had changes in raw neuropsychological test scores, and 99 had changes on the SRB measure. The NNH ranged from 0–100. A significant association between increased anticholinergic drug exposure & cognitive decline was found in only one model that used the Anticholinergic DBI and the SRB measure of cognitive decline on the Trail B test (OR 2.2; 95% CI −1.1–8.06).
The choice of definition by which to classify drug exposure and cognitive decline has a significant effect on the results of causal association studies.
Few studies in long-term care (LTC) have examined the feasibility and acceptability of knowledge translation (KT) programs. We conducted a qualitative evaluation of LTC professionals’ experience with a multifaceted, interdisciplinary KT intervention.
We invited Medical Directors, Directors of Care (Nursing), and Consultant Pharmacists who participated in the Vitamin D and Osteoporosis Study (ViD
Overall, 4 Directors of Care, 7 Consultant Pharmacists, and 2 Medical Directors participated. Medical Directors were not included in group comparisons due to the limited sample size. Most respondents (10/13) attended all sessions and thought it was a valuable experience. The on-site involvement of an expert opinion leader was seen as most useful by all participant groups. Perceived utility of the other KT components varied by group: Directors of Care highly valued audit & feedback, whereas Consultant Pharmacists highly valued small-group learning and internally nominated champions. Common themes for improvement were ready-touse educational fact sheets and having expert opinion leaders attend in person or via video conference.
The ViD
Older men receiving ADT for prostate cancer have a 5–10 fold increased rate of bone loss and up to 20% fracture risk by 5 years of treatment. Guidelines exist for bone-loss management in this population, but adherence is poor. We assessed the knowledge and current practices regarding bone-loss management in a sample of Canadian prostate cancer (PC) specialists.
Using Dillman’s tailored design method, a questionnaire was distributed to Canadian PC specialists through three major specialty organizations.
156 PC specialists completed the questionnaire. Awareness of recommendations for frequency of repeat bone mineral density (BMD) scans (76.3%) and vitamin D use (70.3%) was relatively high, but lower for calcium intake (53.2%) and amount of weekly exercise (20.7%). A minority were aware of the true prevalence of osteoporosis in otherwise healthy 60-year-old males (27.3%), the risk of developing osteoporosis after 1 year of continuous ADT (37.8%), and the excess fracture risk after 5 years on ADT (14.7%). 34.4% of respondents reported routinely ordering BMD tests pre-ADT treatment and 36.6% ordered routine BMD tests after initiating ADT. Most reported routinely recommending exercise, calcium, and supplemental vitamin D. The most significant barriers to implementing the recommendations were lack of time to counsel patients and lack of supporting structures (e.g., patient education).
Participants were fairly knowledgeable regarding recommendations for managing bone loss in men on ADT. However, there were gaps in knowledge regarding risk of developing osteoporosis and in clinical surveillance and risk assessment. These findings suggest the need for knowledge translation strategies and tools to address this gap between evidence and clinical practice.
An audit was conducted on the recorded reason for invasive treatments in older patients. According to the British Geriatric Society and NICE guidelines catheterisation and regular sedation should be avoided in elderly patients especially those with delirium. Additionally, many studies have been conducted showing a link between sedation and delirium. The aim of the study was to discern whether invasive treatments such as the use of catheters, cannulas, intravenous antibiotics, and the provision of sedatives is justified, as these procedures have associated risks including delirium.
Data were collected data from three Geriatric Medicine wards, looking at the first 48 hours of care. Data were assembled on patient demographics, patient’s AMT score, invasive procedures conducted, and the reason for the procedure. The gold standard for this audit is that 100% of procedures are provided with a reason in the notes.
72% of patients were Caucasians, the mean age 84.6 ± 8.0 (SD), and 50% of patients in the audit were classed as delirious. The findings show that 98% of invasive procedures were not clearly justified in the notes, regardless of whether the patient was suffering from delirium. 97% of cannulas inserted were not justified in the notes and was the most common invasive procedure.
These results are in agreement with the hypothesis that the majority of procedures will not have a clear justification in the notes. A justification column could be added in order to make doctors think twice about their reasoning for providing these treatments and thus prompt doctors to provide a reason for these invasive procedures.
The management of multimorbidity in the oldest old (aged ≥ 85) is recognized as one of the most pressing challenges facing clinicians. Given the increasing prevalence of T2DM in this population, a more precise understanding of the epidemiology of co-existing chronic illnesses is necessary to guide therapy.
To characterize co-morbidity in T2DM patients aged ≥ 85 in primary care.
We conducted a retrospective chart review of family practice patients aged ≥ 85 at Sunnybrook Health Sciences Centre, Toronto. Electronic medical records were reviewed for all chronic conditions. For all T2DM patients, each condition was coded as “concordant/discordant” with diabetic care (whether it is related to its pathophysiologic risk profile or management complications), “symptomatic/ asymptomatic” (whether it causes symptoms noticeable to the patient), and “clinically dominant/not dominant” (complex or serious enough to eclipse the management of all other health conditions). We recorded the total number of co-morbid conditions (other than diabetes) in each category for each patient.
T2DM patients comprised 16% (n = 91; 42M:49F) of all patients aged ≥ 85. On average, each patient experienced 6.8 co-morbid conditions other than diabetes (range: 2–16); patients generally had discordant and symptomatic co-morbidities (
Co-morbidity is highly prevalent in very old type 2 diabetic patients. Most co-morbid chronic conditions are symptomatic and discordant with diabetes care. A significant proportion of these patients also suffer from clinically dominant conditions. In the absence of evidence-based care guidelines for this age group, it may be beneficial to focus therapy on the management of symptoms and functional limitations rather than aggressively pursuing risk factor modification.
Men receiving androgen deprivation therapy for prostate cancer have low knowledge of osteoporosis (OP) and are engaging in few healthy bone behaviours (HBBs). A multi-component intervention was piloted in this population, and changes in OP knowledge, self-efficacy, health beliefs, and engagement in HBBs were evaluated.
A pre–post pilot study was performed in a convenience sample of men recruited from the genitourinary clinics at Princess Margaret Hospital. Men were sent personalized letters explaining their dual X-ray absorptiometry (DXA) results and fracture risk assessment with an OP-related education booklet. Participants completed questionnaires assessing OP knowledge, self-efficacy, health beliefs, and current engagement in HBBs at baseline (T1) and 3 months post-intervention (T2). Paired
A total of 148 men (median age 72) completed the study. There was an increase in OP knowledge (9.7 ± 4.3 to 11.4 ± 3.3,
Our results provide preliminary evidence that a multi-component intervention such as the one described can lead to increased knowledge, feelings of susceptibility regarding OP, and uptake of some HBBs.
Fitness-to-drive guidelines recommend employing the Trailmaking B test (a.k.a. Trails B), but do not provide guidance regarding cut-off scores. There is ongoing debate regarding the optimal cut-off score on the Trails B test.
To address this controversy by systematically reviewing the evidence for specific Trails B cut-off scores (e.g., cut-offs in both time to completion and number of errors) with respect to fitness-to-drive.
Systematic review of all prospective cohort, retrospective cohort, case-control, correlation, and cross-sectional studies reporting the ability of the Trails B to predict driving safety that were published in English-language, peer reviewed journals.
47 articles were reviewed. None of the articles justified sample sizes via formal calculations. Cut-off scores reported based on research include: 90 seconds, 147 seconds, 180 seconds, and < 3 errors.
There is support for the previously published Trails B cut-offs of 3 minutes or 3 errors (the
The Geriatric Medicine (GM) academic half-day (AHD) at the University of Toronto is targeted to structured teaching of the CanMEDS roles. This seminar series must fulfill learners’ needs, GM program mandates, and the RCPSC standards for structured education. Given that the University of Toronto has the largest GM program in Canada, the aim is to produce a competency-based AHD framework that can be translated to other Canadian GM programs.
The RCPSC CanMEDS framework for educational design was utilized. A literature review and a national needs assessment surveying the trainees were conducted. Subsequently, an audit and blueprint of the current AHD curriculum at the University of Toronto were completed. Those domains that were less emphasized were the focus of improvement. Suggestions were made through an educational consultation to improve the structured teaching.
The literature review found no publications related to a Canadian GM AHD curriculum. The needs assessment demonstrated satisfaction in training of all domains, but lesser satisfaction in three areas: the sciences of aging, ethical and legal issues, and formal teaching of the Manager role. The four most formally taught GM specific enabling competencies were Medical Expert 2.1, Manager 1.3, Scholar 3.2, and Medical Expert 3.1. An educational consultation provided practical suggestions for improvement.
The AHD at the University of Toronto is one example of structured teaching, but as a 2-year, weekly seminar series, GM residents invest a great amount of time in this formal education. Peer-reviewed educational tools are available to further enhance the AHD teaching. Improvements to meet the needs of the learner, program, and RCPSC are currently being implemented.
Medication-related problems are common, costly, associated with poor outcomes, and are potentially preventable in older adults. Older adults with cognitive impairment are at higher risk of adverse drug reactions. The retirement home (RH) setting is a prime opportunity to intervene to screen for cognitive impairment and for medication review.
This project is a two-phase project taking place in a RH setting. The first phase included resident chart review for diagnosis of dementia or MCI, then cognitive screening using the Dementia Quick Screen (Mini-Cog & animal fluency). Screen failure lead to full assessment. The second phase included an intervention with Medchecks by pharmacist using the anticholinergic load scale and the Ottawa Top Ten Tool (OTTT). OTTT was developed after a thorough review of the literature/available tools with subsequent geriatrician panel review for the Top 10 higher risk drug classes with practical recommendations. All were sent to the treating physician for review. 3-month follow-up was done to identify physician acceptance of recommendations. Barriers to acceptance will be reviewed.
75 residents were included in study. Per chart, 45 had normal cognition so were included in the memory screen: 32 (71%) failed screening. Medchecks were done on 48 residents (16 with dementia). Total of 78 recommendations (range 0–5 & mean 1.6 per resident) were made. 11 (14%) anticholinergic-related, 11 (14%) OTTT-related, and 56(72%) were other pharmacist recommendations. 31(40%) recommendations were accepted by treating physician; 4 (5%) were rejected; 43 (55%) pending.
Cognitive screen and Med-checks using the new OTTT & anticholinergic load scale should be incorporated in RH setting to improve care of this aging population.
The loss of muscle mass, sarcopenia, in older adults is an important marker of frailty due to the association with mobility decline, falls, fractures, and mortality. However, dynapenia, the loss of muscle strength, has been shown to manifest earlier than sarcopenia, and is more consistently associated with disability and mortality. It is unknown whether dynapenia is associated with early gait disturbances, specifically gait variability. Gait variability is a measure of gait regulation, and high gait variability has been proposed as an early marker of mobility decline and a predictor of falls. Therefore, our aim was to determine if dynapenia in community older adults is associated with poorer gait performance, specifically high gait variability.
In 184 community-dwelling older adults (aged ≥ 75), muscle weakness was assessed by measuring the average grip strength in the dominant hand using a handheld dynamometer. Gait variables were assessed under “usual” and “fast” pace conditions using an electronic walkway. Relative risk analysis evaluated the association of muscle weakness to each of the gait parameters.
Older male adults in the lowest quartile of grip strength (< 20.67 kg) had slower gait velocity [mean %CoV (SD) = 82.93 (34.51)] [RR (95% CI) = 1.53(0.58,4.06)], and increased stride time variability [mean %CoV (SD) = 5.81(1.94)] [RR (95%CI) = 1.71(0.82,3.57)], then those in the highest quartile of grip strength (≥ 32.33 kg). Results were similar in female participants.
Our findings have interesting clinical implications because muscle strength assessments can be used in the clinic as an early screening tool to detect those with high gait instability, risk of falls, and mobility decline.
The purpose of this study is to investigate the integration of two non-intrusive approaches to monitoring home care clients’ activity level, along with access to best practice guidelines for clinicians at the point of care. A prototype Remote Activity Monitoring and Guidelines System has been developed that uses a GPS-equipped Blackberry to monitor an elderly client’s mobility outside the home. The System includes a pressure-sensitive mat that is placed under a regular bed mattress and can monitor sleep disturbances, and how long it takes to enter and exit the bed.
A proxy client who is over the age of 65 with chronic health issues was invited to carry a Blackberry and to use a pressure sensitive mat to collect data about the client’s physical activity. After a period of 7 days, 4 different nurses made home visits to the proxy client, where a research member observed clinicians interacting with the prototype System in the client’s home.
The findings indicated the value of the mobility-related data to gerontological clinicians when they plan care to address the aging needs of their home care clients. The results also suggested the usefulness and placement of the Best Practice Guidelines in the electronic user interface. The observational data generated information about the clinicians’ needs and interaction with the prototype in actual home care setting.
This study provides important implications about the value of remote monitoring technology in providing clinical support to assist gerontological clinicians’ decision-making process when planning care for seniors in home care settings.
Receiving rehabilitation enables geriatric patients to regain their function prior to return home. However, long waiting times associated with access to rehabilitation are detrimental to the quality of care for geriatric patients.
Geriatrics consults at the Royal Victoria Hospital and the Montreal General Hospital for 1 year were examined. Relevant information in the consultations was extracted. OACIS was consulted to determine the date of discharge to rehabilitation or home and the number of medications prescribed. The admission/discharge logbooks of the Geriatrics Units were also examined to determine the date of transfer to these units. Statistical analysis was performed on these data using SPSS. A survey of geriatric health professionals determined the reasonable waiting time.
The mean waiting time was 11.4 days for outside rehabilitation or home and 4.3 days for the Geriatrics Units. A theoretical reasonable waiting time of 48 hours was defined from a survey of health professionals. Only 7.5% of patients were admitted within this theoretical time frame to outside facilities or home and 44% to a Geriatrics Unit. We didn’t find any patient characteristics (age and number of medications) that contributed to explain the waiting time for rehabilitation.
This study documents the long waiting time for patients who were recommended for rehabilitation by the Geriatric Consultation Team. The percentage of patients whose waiting time was lower or equal to the reasonable waiting time set by geriatrics health professionals was quite low. However, the waiting times for the Geriatrics Units were significantly lower than those for outside facilities or home. Discharging efficiently to rehabilitation could decrease length of stay and improve patient turnover.
Hip fracture is the leading cause of transfer to acute care for long-term care (LTC) residents. Osteoporosis and falls put LTC residents at a high risk for fractures that lead to pain, loss of mobility, heavy costs to patients their families and to the health-care system, and increased mortality. Effective prevention includes pharmaceutical and non-pharmacological interventions to decrease fractures.
We conducted a pilot telephone survey of LTC residents’ family members to inform investigators who are adapting the
91% of the 11 respondents supported osteoporosis medication use if indicated, while expressing concerns about potential side effects and polypharmacy issues. All respondents supported Vitamin D supplements without any concerns; 82% supported calcium + vitamin D use, but worried about swallowing difficulties. Participants felt pain prevention and preserving quality of life were among the most important outcomes for their family members, and extending life was among the least important outcomes.
Results from this pilot survey indicate family members support pharmaceutical interventions, per recommendations in the
Inappropriate prescribing in the elderly population is associated with adverse drug events and increased hospitalization, ultimately reducing quality of life and increasing mortality rates. The aim was to measure the prevalence of inappropriate prescribing in elderly patients with dementia at Ayrfield Medical Practice in Kilkenny, Ireland. The 2012 Beers Criteria was the standard used for comparison. The Beers Criteria was developed to improve the quality of care for elderly patients and provide physicians with a guideline for safe prescribing.
Medical charts of 80 patients ≥ 65 years old with documented dementia at a primary care centre were studied. The range of age was between 65 and 98 years of age, the mean age was 83.5 years (± SD 9.3). Of the patients studied, 67.5% were female. Patients’ current diagnoses and medications were documented and the Beers Criteria was applied to measure the prevalence of inappropriate prescribing.
The mean number of diagnoses per patient was 3.3 (± SD 1.8) and the mean number of medications prescribed per patient was 9.3, ranging from 0–22. Beer’s criteria identified a total of 129 inappropriate medications in 72.5% (58) of patients. Medications with strong anticholinergic properties (antidepressants, antihistamines, anti-parkinson agents, skeletal muscle relaxants, antipsychotics, antimuscarinics, and antispasmodics) accounted for the majority of inappropriate medications and were prescribed to 66.3% (53) of patients.
Potentially inappropriate drug prescribing is widespread among elderly patients. Regular review of medications by a primary care team and cessation of inappropriate medications should be incorporated into intervention strategies to reduce the number of inappropriately prescribed medications and associated adverse outcomes.
Medication-related adverse events are a significant cause of morbidity and mortality in the geriatric population. With the percentage of Canadians over age 65 expected to double within the next 20 years, educating medical trainees about appropriate prescribing of medications for geriatric patients is becoming increasingly important. Using the internal medicine teaching ward, this study explored the teaching discussions that occur with respect to prescribing, and the use of potentially inappropriate medications (PIMs).
Four admission histories for elderly patients were scripted to include learning opportunities regarding geriatric prescribing. A simulated student orally presented 1–3 admission histories to each of 24 internal medicine ward attending physicians (12 geriatricians and 12 internists) who were instructed to respond as they normally would during morning rounds. Semi-structured interviews following the case discussions explored how attending physicians chose the topics they talked about. Transcribed audio-recordings of 66 case review discussions were analyzed using template and inductive analysis for teaching scripts pertaining to PIMs.
Twenty of 24 interviews involving the review of 54 patient cases have been completed. Geriatrician and non-geriatrician attendings varied in terms of their degree of uptake of the geriatric prescribing teaching and learning opportunities built into the cases. Preliminary analysis of 20 completed interviews will be presented.
Teaching about geriatric prescribing and PIMs on the internal medicine clinical teaching wards can play a crucial role for the care of geriatric patients. Our study is the first to examine the teaching discussions around the prescription of medications for older adults in the context of the general medicine ward.
Children born to mothers of advanced or adolescent ages face increased risk for multiple health problems. In this study, we investigated whether individuals born to mothers of these ages were more likely to experience frailty later in life.
This was a retrospective cohort study of the Health and Retirement Survey, including 3,080 Americans age 50+ (mean = 58.2 ± 5.5 years, 54% women) for whom maternal age data were collected. Frailty was assessed using a 33-item frailty index; participants with scores 0.25 were considered frail. Maternal age at participants’ time of birth was categorized as older (35 years) or younger (< 20 years), compared to a reference group (20–34 years).
Mean maternal age was 22.7 ± 6.5 years. After controlling for participants’ age, gender, and education level, being born to an older mother was associated with higher risk for frailty compared to the reference group (OR = 1.61, 95% CI = 1.05–2.48), as was being born to a younger mother (OR = 1.40, 95% CI = 1.15–1.71). When maternal education level was added to the regression model, being born to an older mother was no longer associated with higher risk for frailty (OR = 1.52, 95% CI = 0.98–2.34), but being born to a younger mother remained associated with higher risk, at a similar level (OR = 1.41, 95% CI = 1.15–1.72).
Among middle-aged and older Americans, being born to an older mother is not associated with greater risk for frailty once maternal education is taken into account. However, being born to an adolescent mother is associated with higher risk for frailty later in life, regardless of maternal education level.
Social vulnerability has been shown to be associated with mortality in Canadian populations. The purpose of this study was to investigate whether social vulnerability can predict mortality in middle-aged and older Europeans, after considering frailty.
This was a secondary analysis of the first wave of SHARE (Survey of Health and Retirement in Europe), which began in 2004 and included a probability-based sample of non-institutionalized participants aged 50+ from 11 European countries. We used the deficit accumulation approach to create a frailty index and a social vulnerability index. The frailty index included 70 health deficits from the physical health, behavioural risks, cognitive function, and mental health sections of the main questionnaire. The social vulnerability index included 29 social factors from the drop-off questionnaire. For each index, an individual’s score reflects the proportion of deficits present out of the total possible deficits.
18,289 participants were included in the analysis (age 65.0 ± 9.67, 45.9% male). The mean frailty index score was 0.15 ± 0.11 and the mean social vulnerability index score was 0.32 ± 0.09. Social vulnerability significantly predicted 5-year mortality when controlling for age and sex (adjusted hazard ratio = 1.33, confidence interval 1.25–1.42,
Similarly to Canadian populations, social vulnerability appears to be an important component for mortality risk stratification in middle-aged and older Europeans. Future investigations are needed to focus on the clinical implications of social vulnerability in older patients.
The relationship between increased arterial stiffness and cardiovascular mortality is well-established in older adults. Short-term vigorous exercise interventions have been shown to reduce arterial stiffness in older adults with T2DM. We examined whether training type (aerobic training versus strength training) influences the improvement in arterial compliance in older adults with Type 2 diabetes complicated by co-morbid hypertension and hyperlipidemia.
A total of 45 older adults (mean age 72.3 ± 0.7 years) with diet-controlled or oral hypoglycemic-controlled T2DM, hypertension, and hypercholesterolemia were recruited. Subjects were randomly assigned to one of three groups: an aerobic group (6 months vigorous aerobic exercise, AT group, n = 20), a strength training group (6 months strength training, ST group, n = 15), and a control group (no training, C group, n = 10). Exercise sessions were supervised by a certified exercise trainer three times per week. Arterial stiffness was measured as pulse-wave velocity (PWV) using the Complior device.
There was a significant difference in the response to training (group × time) between the AT and NA groups for both radial (
Our findings indicate that in older adults with T2DM, long-term strength training resulted in no improvement in measures of arterial stiffness, while aerobic exercise resulted in short-term improvements in arterial stiffness that became attenuated over the long term.
Our program aims to provide physicians with Enhanced Skills in Care of the Elderly training. The program has undergone significant educational changes in the last year.
The COE Program was established at the University of Alberta in 1993. To date, 51 residents have completed the program. Program description: 6 months to 1 year Enhanced Skills Diploma Program with core program requirements including geriatric inpatient, geriatric psychiatry, ambulatory, continuing care, and outreach. There is a longitudinal clinic component and a research project requirement. The program is designed to cover 85 core competencies encompassing the CanMEDS-Family Medicine Roles.
With the increased complexity of the frail elderly we are expanding the program to a 1-year program for the majority of residents, with an exit exam upon completion. This exit exam is comprised of MCQ and geriatric assessment observation with patient encounter. We have been able to increase our positions to four 1-year positions from four 6-month positions. With the increase of the program to 1 year, we have added new rotations in Palliative Care and significantly developed the community experience with rotations in Continuing Care which includes Supportive Living and a Home Living rotation. We have also introduced the electronic Competency Based Achievement System to give formative feedback to our residents.
There is a need for Care of the Elderly physicians to provide clinical care, as well as educational, administrative, and research roles to meet the health-care needs of medically complex seniors. We have made changes to our program to prepare residents for these roles.
Post-operative delirium is associated with pain but also from the use of analgesics. Gabapentin has an opioid sparing effect and reduces pain in the acute post-operative period. The study objective was to determine the treatment effect of perioperative gabapentin on the incidence of post-operative delirium among elective total knee arthroplasty (TKA) patients.
161 patients with American Society of Anesthesiology (ASA) physical status class I–III scheduled for elective total knee arthroplasty at an orthopedic centre were randomized to receive gabapentin 200 mg (n = 83) or placebo (n = 78) before surgery and up to 3 days post-operatively. Incident delirium in the post-operative period was determined by a validated chart abstraction tool. A subset of charts was abstracted by two independent reviewers to determine inter-rater reliability. Data abstractors and patients were blinded to the study drug allocation.
Inter–rater agreement was good (κ = 0.83). Baseline characteristics, co-morbidities, type of anesthesia and analgesia, and previous exposure to gabapentin between the 2 groups were similar. Incident delirium in gabapentin (12%) and placebo (9%) groups was not significantly different (
Perioperative gabapentin was not effective for the prevention of post-operative delirium in elective TKA patients nor did gabapentin have an effect on delirium duration.
The objectives are: to describe factors associated with multi-morbidity in community-dwelling older adults; and to determine if a simple measure of multi-morbidity predicts death over 5 years.
Analysis of an existing population-based cohort study.
Multi-morbidity was more prevalent in women, older age groups, those with lower education levels, lower MMSE scores, more depressive symptoms, and higher levels of disability. Multi-morbidity was a strong predictor of mortality in unadjusted models—the Hazard Ratio (HR) and 95% confidence interval (95% CI) was 1.09 (1.05, 1.12). In models adjusting for age, gender, education, marital status, living arrangement, the CES-D, and the MMSE, this effect persisted: the HR and 95% CI was 1.04 (1.00, 1.08). However, after adjusting for functional status, the effect of multi-morbidity was no longer significant.
Multi-morbidity strongly predicts 5-year mortality, and the effect may be mediated by disability. The cumulative effect of health problems, however minor, is associated with poor outcomes. Guidelines and clinical care models must consider multi-morbidity.
Older patients often pose a challenge to physicians who must determine which patients are good candidates for invasive cardiac procedures, a decision often left to clinical gestalt. The concept of frailty, a multidimensional approach to stratify older patients by physiology and function rather than age, has been associated with poor outcomes. However, due to the lack of consensus on significant measures and the increased time and personnel required, routine frailty assessments are not often used.
A retrospective chart review was completed on 171 consecutive patients over the age of 85 who underwent PCI between 2007 and 2010. Four outcomes were evaluated: major adverse cardiac event, in-hospital death, increase in creatinine by > 25%, or any in-hospital complication. Sixteen demographic, clinical and frailty variables were studied.
The univariate analysis, using chi square for categorical and
Our study found that patients over the age of 85, who were unable to walk without an aid or assistance, were 3.9 times more likely to have a post-PCI in-hospital complication. Asking a patient this simple question about their mobility is both quick and straightforward. A larger prospective study will need to assess whether this type of question could be used as a bedside screening tool to predict poor outcomes in older adults undergoing PCI.
There is paucity of information concerning the epidemiology of multimorbidity in the frail elderly in Alberta. Four rehabilitation wards at a Rehabilitation Hospital have collected data from 2003–2012 for each admission. The de-identified data include ICD-10 diagnosis codes, length of stay (LOS), admission and discharge dates, admission and discharge Functional Independence Measure (FIM) scores, and age of patients. The objective is to begin analyzing and characterizing multi-morbidity in the geriatric population of Alberta.
Data for 2010–12 were separated. A list of all present ICD-10 codes was formed. ICD-10 codes were put into diagnosis groups, which were then counted. The number of ICD-10 codes per patient was counted. The rate of FIM change (FIM efficiency) was calculated according to the equation: (Discharge FIM-Admission FIM)/Length of Stay). Regression analysis was performed to compare the relatedness between FIM Efficiency and Admit FIM, Length of Stay, and Number of Diagnosis Codes.
Initial analysis of codes of interest showed that 0% of this geriatric population had a code for chronic obstructive pulmonary disease, congestive heart failure, or urinary tract infection. Regression analysis revealed that Admission FIM and LOS are significant with FIM Efficiency, but Number of Diagnosis Codes is not.
The ICD-10 codes do not reflect expected prevalence for major chronic diseases. This may be a result of codes forming a present problem/treatment list, rather than a list of all diagnoses. There is a need for another study to fully describe the epidemiology of multi-morbidity in this population.
Wounds, such as diabetic, venous ulcers, pressure ulcers, and surgical wounds, present a significant economic burden on health-care systems. High-quality cost-effectiveness evidence may play a role in considering resource allocation. We conducted a systematic review of cost-effectiveness analyses (CEAs) of wound care interventions to evaluate the methodological quality and cost-effectiveness of this evidence-base.
Potentially relevant material was identified through searching MEDLINE, EMBASE and the Cochrane Library. Inclusion criteria included CEAs assessing any type of intervention for treating wounds in adults. Two reviewers independently screened search results and abstracted data from relevant articles in duplicate. The methodological quality of the included CEAs was appraised using the Drummond tool.
6199 titles and abstracts and 421 full-text articles were screened for inclusion. Of these, 35 CEAs (including 12 cost-utility analyses) were included. The majority of the included CEAs (69%) focused on elderly patients. Only 12 CEAs were deemed to be high-quality (including one from Canada). Seven high-quality CEAs found the following interventions were cost-effective: pentoxifylline plus usual care versus standard compression with external treatment, 4-layer high-compression bandages versus short-stretch high-compression bandages, multi-disciplinary community wound care teams versus usual nursing care, hyperbaric oxygen therapy plus standard care versus standard care alone, becaplermin gel containing recombinant human platelet-derived growth factor plus standard care versus usual wound care alone, and ertapenem versus piperacillin/tazobactam.
We identified a large research gap in CEAs of wound care interventions, and the quality of the evidence is limited.
Older adults have multiple chronic health and social conditions, requiring expertise from different health-care professionals. With the proportion of older adults increasing, it’s important for these professionals to work together effectively. Interprofessional education (IPE) (when two or more professionals learn with, from, and about each other to improve collaboration and quality of care) has been incorporated into policy, systems, and curricula globally. However, the impact of IPE remains unclear. An updated systematic review was performed to assess the effectiveness of IPE interventions on professional practice and health outcomes.
We searched MEDLINE, CINAHL, and the Cochrane EPOC Register from 2007 to 2010. Additional articles were identified through reference lists and discussion with experts. Randomised controlled trials (RCTs), controlled before and after (CBA), and interrupted time series (ITS) studies of IPE interventions reporting objectively measured or self-reported (validated instrument) patient and/or health-care process outcomes were included. Two reviewers independently assessed potential study eligibility, performed data abstraction, and quality assessments.
Three studies met inclusion criteria. The CBA study reported improvements in presurgical procedure briefings and teamwork behaviours in the operating room of a community hospital. One RCT showed mixed results with no change in adverse patient outcomes, but a reduction in process outcomes (time from decision to perform a caesarean section to incision) in a labour and delivery team. Another RCT did not demonstrate an impact on primary care management of asthma.
Although studies suggest some positive impact, the difficulty of drawing conclusions about the effectiveness of IPE remains. Due to the heterogeneous and small number of studies with methodological limitations, further rigorous study designs are warranted.
Cognitive impairment can affect driving performance among older drivers. The objective of this study was to examine the association between selected cognitive measures and self-reported driving comfort, abilities, and behaviours.
We conducted a cross-sectional analysis of data from the first year of the Candrive II prospective cohort study, a 5-year longitudinal study of healthy drivers aged 70+ from seven Canadian cities. Cognitive assessment tools included: the Montreal Cognitive Assessment (MoCA) and Trail Making Tests, parts A and B. Driver perceptions were assessed using the Day and Night Driving Comfort scales and the Perceived Driving Abilities scale, while driving practices were captured by the Situational Driving Frequency and Avoidance scales and the Driving Habits and Intentions Questionnaire.
A total of 928 drivers, 62.2% male, with a mean age of 76.2 ± 4.8, were recruited. Univariate regression analyses revealed that the times to complete Trails A and B were modestly associated with self-reported driving avoidance, day and night driving comfort, and perceived driving abilities (
Time to complete Trails A and B was statistically significant, but only modest predictors of self-reported driving comfort, abilities, and behaviours in this cross-sectional analysis. Results from the prospective follow-up of this cohort of older drivers will help clarify the relationship between cognitive performance and self-reported driver perceptions and driving restrictions.
Hospital malnutrition is prevalent in elderly and contributes to loss of functional status, increases morbidity, mortality, length of stay and cost of care. Nutritional interventions improves outcomes in hospitalized elderly. Systematic service of diet based solely on age is not customary in adult acute care settings.
As part of the OPTIMAH approach of care, we analyzed the protein and caloric content of every available diet at the Montreal University Hospital and compared it to metabolic requirements of hospitalized elders (75 yrs).
Most diets did not meet increased metabolic needs of the hospitalized elder population. Thus, we elaborated a menu that fulfills nutritional requirements and preferences of elders. New enrichment processes were developed to minimize cost. We modified the distribution process to ensure automatic serving of the OPTIMAH diet to this population. Nutritionists, diet technicians, and nurses on ward received a short training to inform them of the new diet and process of distribution. Nurses and physicians were sensitized to avoid prescribing restricted diets unless part of immediate essential medical treatment. Six months after the availability of the OPTIMAH diet throughout the 3 sites of the Montreal University Hospital, 74% of elder hospitalized patients were receiving this adapted diet.
The new OPTIMAH diet more closely fulfills the metabolic needs of elders in acute care. It is the first step to prevent in-hospital malnutrition. Adult acute care services should, like pediatric health services, offer adapted diets according to age. Government norms and correspondent financing should be readjusted to meet elders’ metabolic requirements to prevent costly complications related to hospital-acquired malnutrition.
Education is an important component of dementia treatment for patients and their support networks. To compliment recommending education available from the Alzheimer Society of Canada, practical booklets were developed to improve the written educational material available regarding dementia.
Hobbs, Hurley and Rhynold wrote three booklets:
As of September 2012, six sites in New Brunswick have ordered a total of 4151 booklets. Survey results averaged > 9/10 on the visual analog scale with a score of 10 indicating “very helpful”. Email feedback was positive with some suggestions for improvements. By survey, clinic attendees often indicated they were not interested in reading the material online.
Future directions: The writing team has always made the free distribution of these booklets their priority. Now that these booklets are available, the focus has shifted to increase distribution through written and online versions.
Osteoporosis Canada’s Clinical Practice Guidelines for the Diagnosis and Management of Osteoporosis (OCG) provide guidance for the management of individuals 50 years and older at risk for fractures. However, OCG cannot benefit long-term care (LTC) residents if physicians perceive barriers to their application. Our objectives are to explore current practices to fracture risk assessment by LTC physicians, and describe barriers to applying OCG for fracture assessment and prevention in LTC.
A cross-sectional survey was conducted with the Ontario Long-Term Care Physicians Association using an online questionnaire. Closed-ended responses were analyzed using descriptive statistics and thematic framework analysis for open-ended responses.
We contacted 347 LTC physicians; 88 submitted completed surveys (81% men, mean age 60 years (SD 11), average 32 [SD 11] years in practice). 87% of LTC physicians considered the prevention of fragility fractures important, but a minority (34%) reported using validated fracture risk assessment tools, while 33% did not use any. Clinical risk factors recommended by OCG for assessing fracture risk considered applicable included: glucocorticoid use (99%), fall history (93%), age (92%), and fracture history (90%). Recommended clinical measurements considered applicable included: weight (84%), TSH (78%), creatinine (73%), height (61%), and Get-Up-and-Go (60%). Perceived barriers to assessing fracture risk included difficulty acquiring necessary information (35%) due to lack of access to tests (bone mineral density, X-rays) or obtaining medical history; resource constraints (30%); and a sentiment that assessing fracture risk is futile in this population because of short life expectancy and polypharmacy (28%).
These findings highlight the necessity to adapt the OC guidelines so they are evidence-based and applicable to LTC, and to disseminate them to LTC physicians.
We conducted a systematic review examining the comparative safety and efficacy of cognitive enhancers for patients with Alzheimer’s disease (AD). Numerous outcomes to assess AD were identified but selecting optimal ones for inclusion in our systematic review remained unclear. We compiled the identified outcomes and surveyed decision-makers to identify relevant outcome measures for inclusion in our systematic review.
A systematic review was conducted on cognitive enhancers for AD by searching MEDLINE, EMBASE, and the Cochrane Library. Subsequently, two reviewers independently abstracted outcome measures used to assess cognition, function, behaviour, and global status. The identified outcome measures were compiled and sent to 36 clinicians (geriatricians from the Divisions of Geriatric Medicine at the University of Toronto and McMaster University) and 17 health policy-makers (from Health Canada) using FluidSurvey. Participants voted on the utility of 72 cognition measures, 29 function measures, 13 behavioural measures, and 12 global status measures using a 7-point Likert scale ranging from not important to most important. The scores for each scale were averaged to obtain a rating per scale.
60% of invitees completed the survey. The average ratings per scale ranged from 6.50 to 2.97. The top-rated scale for cognition was the Trail Making test (average score 5.80), for function was the Activities of Daily Living (6.50), for behaviour was the Brief Neuropsychiatric Inventory (5.53), and for global status was the Clinician Interview-based Impressions of Change plus Caregiver Input (6.10). These results were used to inform data abstraction for our systematic review.
Our results can inform clinicians and researchers about relevant outcomes to assess patients with AD.
In Canada, cognitive enhancers such as donepezil, rivastigmine, galantamine, and memantine have been approved for use in AD. Our objective was to examine the comparative efficacy and safety of these agents through network meta-analysis (NMA). NMA is an extension of traditional meta-analysis, and covers both indirect treatment comparison and mixed treatment comparison.
Experimental and observational studies were identified through searching electronic databases (e.g., MEDLINE, AgeLine) from inception to 2011. Studies reporting on adverse events, cognition (e.g., Alzheimer’s disease assessment scale – cognitive subscale [ADAS-cog]), function, behaviour or global status were included. Reviewers independently screened search results and abstracted data from relevant articles in duplicate. Methodological quality was appraised using the Cochrane Risk of Bias for experimental studies and the Newcastle Ottawa scale for observational studies. Random effects and network meta-analyses were conducted.
A total of 132 randomized controlled trials, 4 non-randomized clinical trials, 2 controlled before-after studies, and 44 cohort studies were included after screening 15,676 titles and abstracts and 964 full-text articles. Preliminary results from the NMA indicate the following drugs performed better than others on cognition as per the ADAS-cog scale (listed in descending order): donepezil 10 mg, donepezil 5 mg, galantamine 16–24 mg, and memantine 20 mg. For nausea, use of the following drugs resulted in lower proportions of patients experiencing nausea (listed in descending order): memantine 20 mg, rivastigmine patch 9.5 mg, placebo, and donepezil 5 mg.
Donepezil 5–10 mg was most effective at improving cognition for patients with AD. The cognitive enhancer with the lowest risk of nausea was memantine 20 mg. These results can be used by patients and clinicians to tailor their AD treatment by specific cognitive enhancers.
Individuals with mild cognitive impairment (MCI) suffer from memory problems without significant limitations in activities of daily living (ADL). Cognitive enhancers are used to treat dementia, but their effectiveness for MCI is unclear. We conducted a systematic review to examine the comparative efficacy and safety of cognitive enhancers for patients with MCI.
Experimental studies were identified through searching electronic databases (e.g., MEDLINE, EMBASE). Studies examining cognitive enhancers in MCI and reporting on adverse events, cognition (Mini-Mental State Exam [MMSE], Alzheimer’s disease assessment scale – cognitive subscale [ADAS-cog]) or function (Alzheimer’s disease cooperative study: ADL inventory [ADCS-ADL]) were included. Two reviewers independently screened search results, abstracted data, and appraised risk of bias using the Cochrane risk of bias tool. Random effects meta-analysis was conducted.
Nine randomized controlled trials were included after screening 15,676 titles and abstracts and 964 full-text articles. No significant findings were observed for impact on cognition (MMSE: 3 RCTs, mean difference [MD] 0.14, 95% confidence interval [CI] −0.22, 0.50, ADAS-cog 5 RCTs, MD −0.52, 95% CI −1.09, 0.06), although there was a trend towards favouring cognitive enhancers. Similarly, function was not significantly impacted (ADCS-ADL, 2 RCTs, MD 0.30, 95% CI −0.26, 0.86) and no trend was observed. Cognitive enhancers were associated with a higher risk of nausea (5 RCTs, relative risk [RR] 2.95, 95% CI 2.48, 3.52), diarrhea (5 RCTs, RR 2.71, 95% CI 1.90, 3.85), and vomiting (3 RCTs, RR 4.40, 95% CI 3.21, 6.03).
Cognitive enhancers did not improve cognition or function among patients with MCI and were associated with a greater risk of nausea, diarrhea, and vomiting.
Venlafaxine is a commonly prescribed antidepressant, but it is unknown whether its noradrenergic effects impart an increased cardiovascular risk.
To examine the cardiac safety of venlafaxine relative to sertraline in older patients.
We conducted a retrospective cohort study using administrative health-care databases in Ontario, Canada. We included all patients aged 66 years or older who commenced treatment with either venlafaxine or sertraline between April 1, 2000 and March 31, 2009. We used inverse probability of treatment weighting (IPTW) with the propensity score to account for observed systematic differences between the two treatment groups. The primary outcome was a composite of death or hospitalization for acute myocardial infarction or congestive heart failure within the first year of therapy. In secondary analyses, each outcome was examined separately.
We studied 48,876 patients initiated on venlafaxine and 41,238 patients initiated on sertraline. Of these, 4259 (8.7%) and 3459 (8.4%) experienced the primary outcome, respectively. We found no significant difference in the risk of adverse cardiac events with venlafaxine relative to sertraline (hazard ratio 0.97; 95% confidence interval 0.94 to 1.02). Secondary analyses revealed no differences in the risk of death or acute myocardial infarction between the two drugs, but the risk of heart failure was unexpectedly lower among patients treated with venlafaxine (hazard ratio 0.87; 95% CI 0.80 to 0.95). We found consistent results after stratification according to pre-existing cardiovascular disease.
As compared with sertraline, venlafaxine is not associated with an increased risk of adverse cardiac events in older patients.
Frequent users of emergency departments, clinics and hospitals utilize a disproportionately large amount of health-care resources, thereby reducing efficiency and decreasing overall quality of care. As such, efforts have been made to implement quality improvement (QI) strategies targeting this population. Our systematic review aims to identify effective care coordination QI strategies for frequent users.
We searched multiple databases (e.g., Cochrane Library, EMBASE, MEDLINE) from earliest date to March 2012. Additional citations were identified by scanning the reference lists of included studies. Citations and full-text articles were screened by two independent reviewers and relevant studies were abstracted and appraised for quality in duplicate using the Cochrane Effective Practice and Organization of Care tool. Random effects meta-analyses were conducted using data from randomized clinical trials (RCTs).
We screened 9564 citations and 132 full-text articles resulting in the inclusion of 44 relevant studies, including 36 RCTs. The three most commonly examined QI strategies were case management, self-management, and team changes. Nineteen studies included only patients with mental illness, while 25 included patients with other chronic illnesses. Our overall meta-analyses identified that QI strategies were effective in reducing the mean length of stay in all patients. In studies including patients with chronic illness, QI strategies effectively reduced the number of patients with emergency visits and the number hospitalized. QI strategies did not significantly reduce clinic visits or the number of patients hospitalized in studies including patients with mental illness.
QI strategies can reduce utilization in patients with chronic conditions. However, patients with mental illness may be more difficult to target with these QI strategies.
Acute and chronic wounds result in substantial costs to our health-care system and significantly impact quality of life. Although a number of interventions are available to treat wounds, optimal strategies for wound care remain unclear. Our objective was to identify effective wound care interventions from high-quality systematic reviews in the literature.
A search was conducted using MEDLINE, EMBASE and the Cochrane Library. Citations and full-text articles were screened in duplicate to include systematic reviews of adult patients receiving wound care. Two reviewers independently abstracted study characteristic and outcome data from the included reviews and appraised review quality using the AMSTAR tool.
From the 6199 titles and abstracts and 421 full-texts screened for inclusion, 110 systematic reviews were included. Fifty-seven reviews included meta-analyses and approximately 40% were rated as high-quality. From the highest quality meta-analyses, we identified a number of effective interventions across 5 wound types: 2-layer stockings, high-compression stockings, 4-layer bandages, elastic bandages, multi-layer high-compression, elastic high-compression, Pentoxifylline with or without compression, Cadexomer iodine, and engineered skin in patients with leg ulcers; air-fluidized beds, foam mattresses, hydrocolloid dressing, nutritional support and electrotherapy for pressure ulcers; granulocyte-colony stimulating factor, hydrogel dressing, hyaluronic acid, low-frequency/high-frequency ultrasound, and skin grafts for patients with diabetic ulcers; skin grafts and silver dressing for mixed chronic wounds; and honey for patients with burns.
Our results can be used by clinicians and patients to tailor treatment by wound type. Further analysis of this data through network; meta-analysis will be of utility to decision makers, as it will allow ranking of the effectiveness of all wound care interventions in the literature.
Osteoporosis affects over 200 million people worldwide at a high cost to health care. Guidelines are available, but many patients are not receiving appropriate care. We developed an osteoporosis knowledge translation (Op-KT) tool to support clinical decision making: a tablet-initiated risk assessment questionnaire (RAQ), which generates best practice recommendations for physicians; and a customized education sheet for patients. We evaluated its impact on the initiation of appropriate osteoporosis disease management in primary care.
Following an implementation plan in 3 family practices in Hamilton, Ontario that included workflow analysis, the Op-KT tool was evaluated using an interrupted time series design. This involved multiple assessments 12 months before (baseline) and 12 months after introducing the tool. Analysis included segmented linear regression models and analysis of variance.
Five family physicians from 3 practices participated; 2840 patients (mean age 67 years; 79% women) were eligible (31% of the practice population). Time series regression models showed an increase from baseline in the initiation of bone mineral density testing (3.2%;
Our multi-component Op-KT tool significantly increased osteoporosis investigations in 3 family practices. The study highlights the potential of using decision support tools at the point of care in busy, short-visit practices to facilitate patient self-management.
Almost 30,000 patients annually experience a hip fracture in Canada. They tend to be older, frail with multiple chronic illnesses, including a high incidence of dementia and delirium. For many, the hip fracture results in poor outcomes including loss of function and use of ALC (alternate level of care) beds. In 2011 Bone and Joint Canada (BJC) worked with health-care professionals from across the country to develop a National Hip Fracture Toolkit, which was based on available evidence and a consensus building approach, to provide clinical and system best practices to better manage these patients and return them home.
A knowledge translation approach was used to assist provinces to review their performance and to facilitate uptake of best practices. Identified barriers to care included the management of frail patients and their co-morbidities, access to rehabilitation, weight bearing, and patient education.
All provinces participated in the KT strategy at a national level, as well as hosting provincial and local events to measure their performance against the Toolkits recommendations. Care maps were implemented at a provincial level, and local improvement initiatives were undertaken in all provinces in 2012/2013. Surgeon practices to promote weight bearing were investigated and patient education materials were developed. In Ontario, recommendations on best practice were made for future funding of hip fracture patients.
The National Toolkit provides a system and clinical practice information on pre-operative, surgical, and post-operative care. It uses a multidisciplinary and multi-faceted approach to the clinical and operational management of older hip fracture patients and has improved care across the country.
The UK Commissioning for Quality and Innovation Dementia (CQUIN) framework (2012) aims to facilitate early identification of patients with dementia during their inpatient stay and ensure they are referred to appropriate services. Step 1 is to find all patients over age 75 years meeting the inclusion criteria. Step 2 is assessment using a screening questionnaire, AMTS, collateral history, examination, and investigations, in order to stratify all patients as suspected dementia, known dementia or no cognitive impairment. Step 3 is referral to memory services or GP for further follow-up. We aimed for rapid assessment of all patients over 75 years of age admitted acutely to achieve 90% compliance in Steps 1–3.
All patients over 75 were identified on a daily basis. A pro forma was developed and available in all wards. Junior physicians in elderly care wards assessed patients during daily rounds. The roles of the memory nurses were changed to supervise data collection and review patients in other wards.
The total number of patients assessed in September was 341/377 (90.5%) and October 2012 465/494 (97%). Of these patients with a diagnosis of delirium or who scored positively on the screening question, 113/113 (100%) and 192/198 (97%), respectively, had a dementia diagnostic assessment. In September 54/55 (98.2%) and October 133/133 (100%) of patients with suspected dementia were referred for further follow-up who might have been missed with standard care.
Implementation of the UK Dementia (CQUIN) framework is achievable through staff education, change in working practice, and clear implementation of protocols, with little extra resources. Early recognition of suspected dementia increases early access to appropriate support for patients and their careers.
Our objective was to examine the impact of specialized palliative care (PC) (defined as a physician consultation focusing on PC needs, lasting at least 40 minutes) for adults 70 and older on: (a) use of chemotherapy within 14 days of death, (b) more than one emergency department (ED) visit, (c) more than one hospitalization, and (d) at least one intensive care unit (ICU) admission, all within 30 days of death.
A retrospective population-based cohort study using linked administrative databases in Ontario was conducted with patients diagnosed with advanced pancreatic cancer from 1 Jan 2005 to 31 Dec 2010. Multivariable logistic regression analyses were performed with the above quality indicators as the outcomes of interest and PC as the exposure, adjusting for other variables (age, sex, comorbidity, rurality, and health region).
Of 6,076 patients with advanced pancreatic cancer, 58% were age 70 or older, and 5,381 had died at last follow-up. 57% (1251/2187) of those younger than 70 and 49% (1565/3194) of those 70 and older received a PC consultation (
In patients with advanced pancreatic cancer, PC is associated with less frequent aggressive care across all age groups, but PC consultation and aggressive care were both less likely in older patients (70+).
To meet the challenges of population aging and increasing multimorbidity, significant reform to health-care systems is underway. New models of care include the patient-centred medical home and interprofessional team-based approaches; however, there has been limited exploration of the effectiveness of such interventions for patients with multimorbidity. To evaluate both the clinical-effectiveness and cost-effectiveness of a team-based model of primary care specifically designed for older patients with multimorbidity.
Multi-site randomized controlled trial of the IMPACT clinic (Interprofessional Model of Practice for Aging and Complex Treatments). Inclusion criteria: patients aged 65+, three or more chronic diseases requiring monitoring and treatment, five or more long-term medications, and minimum of one functional ADL limitation. Exclusion criteria: home-bound or institutionalized patients, or deemed unsuitable by the usual family physician. The IMPACT team comprises family physicians, specialist physicians, visiting nurse, pharmacist, community social worker, occupational therapist, physiotherapist, dietitian, and care navigator. IMPACT patients are scheduled for extended visits (1.5 to 2 hours) during which the full team works collaboratively with the patient and family caregiver(s) to address current medical, functional, and psycho-social issues. During the visit, a pro-active interprofessional care plan is developed, a comprehensive medication review is conducted, and a discussion of anticipatory care planning is initiated.
Primary outcomes include Emergency Department visits, hospitalizations, and physician visits. Secondary outcomes include patient satisfaction, family caregiver satisfaction, provider satisfaction, quality of life, caregiver strain, and interprofessional team function.
This RCT will contribute much-needed evidence on the effectiveness of a team-based primary care intervention for older patients with multimorbidity.
Cancer is a disease that mostly affects older adults. A geriatric assessment (GA) has been recommended for older adults to assist with treatment decision-making. The aims of review: 1) to provide an overview of the use of GA in oncology; 2) to examine feasibility and psychometric properties; 3) to systematically evaluate the effectiveness of GA in predicting/modifying outcomes.
A systematic review of literature published between November 2010 and July 2012. Articles published in 5 databases in English, Dutch, French, and German were included. Articles were selected and reviewed by 2 independent reviewers.
34 manuscripts reporting on 33 studies were identified. The quality of most studies was moderate to good. Of all studies, 14 were prospective, 8 cross-sectional, 5 retrospective and 7 others (mostly phase II trials of a new treatment regimen). A GA was shown to be feasible, the time needed to complete ranged between 5 and 120 minutes, were mostly conducted in the outpatient oncology setting by nurses, and most often included the domains activities of daily living, co-morbities, cognitive function, depression, medications, and fall risk assessment. Four studies examined psychometric properties of the GA with satisfactory results, and 18 studies examined the predictive ability of the GA and showed that components of the GA predicted treatment toxicity and overall survival.
Although the studies showed that GA was feasible and had predictive validity, there has not been a randomized controlled trial showing the effectiveness of the GA in improving outcomes for older adults with cancer.
The capacity for bone repair and regeneration diminishes with age. This prolongs fracture healing time and, in some instances, results in non-union, requiring extensive surgery. The mechanism behind this is not known; however, studies thus far have assumed the reason to be a decrease in the capacity of bone marrow mesenchymal stem cells (MSCs) to differentiate into bone-forming cells (osteoblasts). We found that old MSCs can be “rescued” to behave like young MSCs when cultured in media pre-treated by young cells. These findings implicate the presence of a “youth factor” that is secreted by young bone marrow cells and is able to rescue the aged phenotype of old cells. Thus, the purpose of this study is to determine the cell type responsible for rescue of osteoblast differentiation in old cells, and to determine its effects on fracture repair in old mice.
Bone marrow cells were isolated from young and old mice. Osteoblast differentiation in culture was determined by quantifying colony forming unit-osteoblast. Fracture repair was assessed using a tibial fracture mouse model.
Co-culture of old cells with young hematopoietic stem cells (HSCs) promoted osteoblast differentiation of these old cells. Interestingly, an adherent F4/80+ cell population (a marker of monocyte–macrophage cell lineage) was identified in young, but not old, HSC cultures. In culture, exposing old MSCs to media pre-treated by young macrophages induced osteoblast differentiation of these cells. Furthermore, bone marrow transplantation of young F4/80+ cells into old mice resulted in improved fracture repair.
This study demonstrates that young macrophages secrete soluble factors that can rescue osteoblast differentiation and improve fracture repair in older animals.
The Canadian Institute of Health Information (CIHI) designates patients who remain in hospital after their acute care phase is completed as Alternate Level of Care (ALC) patients. Understanding who the ALC population is in hospital is needed.
All ALC patients Horizon Health Network (HHN) as of Feb 9, 2012 were identified. A data collection tool, designed for the study, was used to review charts.
There were 413 ALC patients identified, occupying 25.2% of all hospital beds within HHN. A stratified random sample from 7 hospitals comprised the sample of 223. Two were excluded due to long length of stays, giving a sample of 221.The mean age was 78.4 years. Prior to admission, 51 (23.1%) were living in a care institution in the community. Dementia was a diagnosis in 53.9%. The overall mean length of stay until data collection was 293.4 days. Six months later, 81 (36.7%) were still in hospital, 32 (14.5%) had died, and 65(29.4%) were discharged to nursing home. For those discharged to a nursing home, the mean length of stay was 262.8 days. For those still in hospital, the mean length of stay was 683.9 days.
The majority of ALC patients are elderly with dementia. Six months after data collection, the majority remain in hospital with a mean length of stay of almost 2 years. Even those who went to a nursing home, the length of stay was almost 9 months.
Identifying measures to predict short-term toxicities in patients undergoing intensive chemotherapy (IC) for acute myeloid leukemia (AML) is needed. Emerging data suggest that quality of life (QOL) assessment and/or physical performance measures (PPMs) may predict outcomes in oncology, although there are no data in AML patients.
We conducted a prospective, longitudinal study of adults (age 18–59) and older (age 60+) AML patients undergoing IC. Prior to starting IC, patients completed the EORTC QLQ-C30 and FACT-Fatigue, in addition to PPMs (grip strength, timed chair stands, and 2-minute walk test). Outcomes included 60-day mortality, intensive care unit (ICU) admission, and achievement of complete remission (CR). Logistic regression was used to evaluate each outcome.
Of the 243 patients (median age 57.5 yrs), 56.7% were male, and 96 (40%) were older. 60-day mortality, ICU admission, and CR occurred in 9 (3.4%), 15 (6.2%), and 171 (70.4%), respectively. In univariate regressions, neither QOL nor PPMs were predictive of 60-day mortality (all
Baseline QOL and PPMs were not associated with short-term mortality, ICU admission, or achievement of CR after the 1st cycle of chemotherapy for AML.
Self-rated health (SRH) has been shown to predict functional status in older adults, but this has less often been examined for older cancer patients. The aim of this study was to determine the association between SRH and functional status, comorbidity, toxicity of treatment, and mortality in older newly diagnosed cancer patients.
Patients aged 65 and over, newly diagnosed with cancer, recruited at the Jewish General Hospital. SRH was evaluated prior to treatment, and at 3, 6, and 12 months. Functional status (Instrumental Activities of Daily Living (IADL), Basic Activities of Daily Living (ADL), ECOG Performance Status (ECOG PS), and frailty markers (low grip strength, mobility impairment, physical inactivity, cognitive impairment, mood impairment, and poor nutritional status) were measured at baseline, 3, and 6 months. Treatment toxicity and mortality were abstracted from the chart. Chi-square tests and
There were 112 participants, median age 74.1. At baseline, 74 (66.1%) had a good SRH and 38 (33.9%) had poor SRH and those had more co-morbidities, more frailty markers present, lower ECOG PS and IADL impairments. We found no association between SRH and toxicity or mortality.
There was a moderate correlation between SRH and the number of frailty markers, IADL disability, and co-morbidities, but SRH did not predict toxicity or mortality.
People over the age of 60 account for 60% of paramedic responses in Canada. Many of these calls are not life-threatening or time-sensitive. Paramedics have a unique opportunity to engage people in their homes and they often see people in vulnerable circumstances. The field of Community Paramedicine is growing in Ontario and across Canada due to its potential to provide alternative patient care pathways. In 2006, Toronto Emergency Medical Services (EMS) established its Community Referrals by EMS (CREMS) program to link patients with Community Care Access Centres (CCAC). With a simple phone, call paramedics identify and connect patients with community support services.
To determine the effectiveness of the Community Paramedicine program, 904 patients referred to CCACs in 2011 were evaluated for improved outcomes and reduced reliance on EMS. The 6-month period prior to initiating the CCAC referral was compared to the 6 months post-CCAC referral.
The total calls to EMS were reduced from 2,715 to 1,340 for this patient group. Transports to emergency departments also decreased from 1654 to 582. Paramedics spent less time overall with these patients, reduced from 4597.28 hours to 1898.87 hours. Based on the decreased transports and time spent assessing and treating these patients, cost savings have been estimated to be as high as $321,600.00 for the 6-month post-referral period.
Community Paramedicine offers an innovative, cost effective opportunity to improve the health care of elders wishing to age and live at home independently. Future directions will include exploring an expanded scope of clinical, practice for paramedics, and a more systematic evaluation of the CREMS program with an eye toward broader implementation.
Men with PCa on ADT are at risk of decreased bone mineral density (BMD) and osteoporosis. Guidelines recommend referral to specialized clinics, but the quality of care in osteoporosis clinics and benefits to patient have never been reported.
Charts for 67 men (mean age 74.2 yrs) on ADT referred to an academic osteoporosis clinic between 2010 and 2011 were reviewed. The following quality of care issues were examined: (a) services provided to PCa patients receiving ADT (e.g., screening, preventing, and treating osteoporosis); (b) use of Canadian guidelines to target appropriate therapies.
56 (83.6%) received continuous ADT for a mean of 27.4 ± 30.7 months at the baseline visit. 37 (55.2%) had osteopenia and 15 (22.4%) had osteoporosis. At initial consultation, 55.2% were taking 1000 mg calcium daily from all sources, while 26.9% were taking more than 1200 mg; 22.4% were taking Vitamin D 3 months. For lifestyle recommendations, 71.4% of sedentary patients were advised to increase exercise. Of the 39 (70%) and 24 (39.2%) patients who were not taking appropriate amounts of calcium and vitamin D, respectively, 100% were recommended to adjust their intake to guideline levels.
The osteoporosis clinic performed a comprehensive assessment and recommended guideline-based bone health care for the vast majority of men on ADT, suggesting a systematic approach to assessing bone health is associated with high rates of guideline-adherent care.
Metastatic castration-resistant prostate cancer (mCRPC) is characterized as disease progression despite adequate androgen deprivation therapy (ADT). Although chemotherapy for mCRPC prolongs survival, whether its impact on elderly-relevant outcomes and toxicity differ by frailty status is not known.
Men aged 65+ with mCRPC who were starting first-line chemotherapy were enrolled in this longitudinal prospective pilot study. Elderly-relevant information was collected at baseline and before the start of each chemotherapy cycle. Frailty was assessed by the Vulnerable Elders Survey (VES-13), functional status by OARS-IADL, social activities limitation and support by MOS measures, and FACT-G and FACT-P for general and prostate-specific quality of life (QOL), respectively. Physical function was assessed by timed up and go (TUG), timed chair stands, and grip strength. Changes in outcomes were analyzed between frail vs. non-frail patients using Student’s
21 patients (mean age 74), of whom 11 were frail (VES-13 3), were assessed. Generally, at baseline frail patients were slightly older and scored lower than non-frail patients in QOL, functional status, physical function, and social support and activities. However, frail patients improved more than non-frail patients in all domains, except TUG. 18% of frail patients died during the course of therapy compared to no deaths in non-frail patients.
Frail patients, as determined by VES-13 3, with mCRPC may represent a heterogeneous population; one group destined to die soon and the other who may do well with chemotherapy. Further research and patient recruitment is needed to determine whether a subset of frail older patients would benefit from first-line chemotherapy treatment.
The Clock Drawing Test (CDT) is a screening tool used by physicians for detecting dementia in the clinical setting and is commonly used for identifying drivers with a dementia whose driving skills may have declined to an unsafe level. However, the accuracy of the CDT for detecting declines in driving due to a dementia is not well-established and is confounded by the presence of multiple scoring systems. The purpose of the study was to examine the
50 cognitively impaired and cognitively intact participants completed the CDT and an on-road assessment. A novice scorer and a trained clinical geriatric specialist scored the clocks using 4 CDT scoring systems (Rouleau, Shulman, Freund, and MoCA).
The intra-rater reliability of the novice scorer across the four scoring schemes was high (Pearson’s
Although there was good intraand inter-rater reliability for the scoring systems tested, none of the CDTs examined were significantly associated with on-road outcomes, indicating that use of CDT scores is most likely to result in erroneous driving decisions for cognitively impaired patients.
In 2010, Baycrest implemented a Slow Stream Rehabilitation Program (SSR) to deliver a low-intensity long-duration rehab for frail seniors’ post-acute hospitalization. To examine the change in function, length of stay, and discharge destination of patients admitted to SSR.
Psychosocial and functional measures were administered to patients on admission and discharge to the SSR Unit.
Over a period of 15 months, 105 patients (70% of all admissions) were recruited; mean age was 82, mean stay in acute care was 32 days, and the mean LOS in SSR was 88 days. On admission, 85% had mild/moderate to severe cognitive impairment (MoCA: 26) and 78.5% were dependent with transfers with or without devices. Mean admission FIM: 51 and discharge FIM: 74; admission Berg Balance Scale (BBS): 10 and discharge BBS: 19.7. On admission 51% could ambulate 10 steps with a device and 80.4% on discharge. Upon discharge, 68% were discharged home or to other community residences; 24% to Long-Term Care (LTC) and 9% went to acute care.
This study confirms that the SSR population is a frail elderly group admitted after a mean of 32 days in acute care. With low functional ability on admission, this group was able to achieve over 80% ambulation with or without a device and had a mean discharge FIM of 74. After 88 days of low-intensity rehab, 68% were able to return to community living. After a long acute hospital stay, frail older adults with cognitive impairment can benefit from slow stream rehabilitation to prepare them for living in the community rather than going to LTC.
Residents who are international medical graduates (IMGs) are a heterogeneous group of learners with distinct backgrounds of ethnicity, religion, and culture. They came from various countries with differing medical education standards, societal values, and professional codes of conduct. When training and working in Canada, IMG residents may experience trans-cultural challenges. The purpose of this study is to identify cultural strengths and challenges that IMG family medicine residents encounter when working and training within the Canadian medical context, and to identify the values, behaviours, and codes of conduct expected of family physicians working in Canada.
Focus group with seven academic/community preceptors who teach residents. Qualitative data were transcribed and analyzed for emerging themes.
Distinctive Canadian socio-medico-cultural values were identified in six theme areas – communication, gender, cultural awareness, ethics, medical knowledge, and social hierarchy. IMG residents were noted to possess
Cultural gaps appear to be present when IMG residents interface within the Canadian medical context. Identification of trans-cultural challenges will assist in the development of teaching resources for use in IMG resident training.
Limited research has suggested that rural residents are at increased risk of dementia.
The objective of this study is to determine if rural residence is associated with dementia using both cross-sectional and prospective analyses.
In 1991, 1763 community-dwelling adults aged 65+ participated in the Manitoba Study of Health and Aging (MSHA), which sampled all regions of the province of Manitoba. Baseline measures included age, gender, years of education, the Modified Mini-Mental State Examination (3MS), and rural/urban status. Rural was defined as a census subdivision with a population <20,000 and urban as 20,000+. Dementia was diagnosed in participants with a 3MS score <78 and clinical examination applying DSMIIIR criteria at baseline and at follow-up five years later. All other participants were categorized as not demented. Two analyses were conducted: 1) a cross-sectional analysis (
Forty per cent (
Rural residents had significantly lower educational attainment and 3MS scores than urban residents. However, rural residence was not associated with dementia in either cross-sectional or prospective analyses. Further study into heterogeneity between rural areas is warranted.
The MoCA is a widely accepted screening tool which tests multiple cognitive domains. It is currently being used to diagnose degree and types of cognitive impairment.
The aim of our study is to identify and measure test variability between two or more tests among a group of patients diagnosed with mild cognitive impairment (MCI).
A retrospective study was performed on a sample of patients attending the Memory Clinic of Jewish General Hospital between January 2005 and December 2012. Inclusion criteria included: aged 60 and over who had a minimum of two visits and were diagnosed with MCI and who remained clinically stable over follow-up. The data were analyzed using SPSS, then a repeated ANOVA model was performed to test the variation in the mean between each group. The groups were analyzed according to age gender, education level, and MoCA score.
345 MCI individuals met these criteria. Visits range from 2–5 (mean of 2.8) with MoCA testing over 2–7 years of follow-up. Mean MoCA score was initially 23.49 (SD of 2.95). The study showed no significant changes in MoCA score over time. Evaluation of 37 patients who had 5 visits showed a variance ranging from 1–11 points during the study period. 35% showed a score range of 4 points, 29.7% showed a variance greater than 4 points (max 11), while 35% showed only 1–3 points score difference over the follow-up period.
Even in MCI patients who did not progress, the MoCA usually varies by 4 points during follow-up visits. A change in MoCA score from one visit to the next should not be taken as inevitable evidence of progression.
Individuals with diabetes are at an increased risk of cognitive decline and dementia compared to age-matched, non-diabetic controls. Recent population cohort studies further suggest that higher blood glucose levels among individuals with diabetes incrementally increased the risks for developing dementia even after controlling for major cardiovascular risk factors. Thus, improving glycemic control in adults with diabetes has been postulated as a potential modifiable risk factor for preventing cognitive impairment and dementia.
Database searches of CENTRAL, Embase, MED-LINE, and Ovid HealthSTAR were conducted from 1966 to December 2013, and supplemented with searches of conference proceedings and manual reviews of bibliographies of retrieved articles. Randomized controlled trials (RCTs) examining the risk of dementia, global cognition, and psychomotor efficiency among individuals achieving intensive glycemic control (i.e., HbA1c ≤ 7.0%) compared to standard glycemic control (i.e., HbA1c > 7.0%) were included.
Seven RCTs were included in the systematic review and meta-analysis. Achieving intensive glycemic control was not associated with benefits in global cognition (SMD: 0.07, 95% CI: −0.29 to 0.42) or psychomotor efficiency (SMD: 0.55, 95% CI: −0.08 to 3.24). One RCT examined the association between intensive glycemic control and dementia, and no statistically significant reduction in the relative risk was observed.
There is currently insufficient evidence to support the hypothesis that achieving intensive glycemic control is associated with benefits in cognition among adults with type 2 diabetes. Furthermore, these estimates of effect need to be interpreted with caution given the presence of statistical heterogeneity.
Antipsychotics are commonly used in behavioural and psychological symptoms of dementia (BPSD). However, due to safety profile, the medical literature supports their use only in severe cases. Currently, there is a paucity of data regarding their use in short-stay units. Our primary objective was to determine which antipsychotics were most frequently used in the short-stay behavioural unit of the Montreal General Hospital. Our secondary objective was to determine their efficacy and incidence of side effects.
This is a retrospective chart-review study of 88 patients admitted between May 2009 and July 2012 with an average age of 80 years old. BPSD were divided in categories inspired by the “Neuropsychiatric Inventory”, including agitation, paranoid ideas, etc.
Only 13 patients (14.8%) did not receive antipsychotics. At admission, 30 patients (34.1%) were already on an antipsychotic; however, 51 patients (58%) were discharged on an antipsychotic. Regularly administered Quetiapine was the most commonly prescribed first line agent (28.4%), followed by Haloperidol as needed (26.1%). A second antipsychotic was needed in 54 patients (61.3%). The most common BPSD encountered was agitation (29.2%) and 37 side effects were noted, including sedation (12.5%) and falls (10.2%). Furthermore, there was a significant loss of autonomy in that 39 patients newly required long-term placement and there was more than a 20% decline observed in the independence for ADLs (activities of daily living).
Antipsychotics were frequently used in our unit and were associated with significant side effects, including sedation and loss of autonomy. We believe that such a high use of antipsychotics was associated with the observed functional decline. Quetiapine was the most commonly used, possibly because of the desired sedation effect.
The evidence on the use of ‘as required’ or prn medications in the older population is limited.
To determine the appropriate use of opioid analgesics, benzodiazepines, antipsychotics, antiemetics, and non-steroidal anti-inflammatory drugs (NSAIDs) for patients aged 75 years and older.
A list of patients aged 75 years and older discharged from medical and acute geriatric medical units within a consecutive four-week period were generated. Patients with a change of care and those deceased were excluded. The remainder were investigated for the presence of prn medications in their discharge summary. The demographics included age, gender, and residence. Indications for the prn medications and geriatric syndromes (falls, delirium, and dementia) were also obtained.
A total of 24 prn medications were present in 23 of the 104 discharged patients. Of these, 8 were new and 16 were pre-existing. Only 10 of the latter were used during admission. Seventy per cent were discharged home. Half of the prn medications were opioid analgesics, followed by benzodiazepines (21%), antiemetics and antipsychotics (both 13%), and NSAIDs (4%). Eleven of the 23 patients had one or more of the geriatric syndromes.
Twenty-two per cent of older patients discharged in a four-week period have a prn medication documented in the discharge medication list. Majority of these patients are home-dwelling and nearly half of the patients have one or more of the geriatric syndromes. As such these patients are more prone to adverse effects. Over one-third of the preexisting prn medication were not used during the admission, but documented in the discharge medication list.
Prn medications should be reviewed during admission and be judiciously documented in the discharged medication list.
With the growing immigrant population in Canada, an accurate assessment tool is needed for those who are from culturally and linguistically diverse backgrounds. Many common cognitive screening tools are not ideal for these populations due to the dependency on English proficiency. In 2004, the Rowland Universal Dementia Assessment Scale (RUDAS) was created to address cognitive screening in culturally and linguistically diverse populations. A systematic review of the literature is warranted to determine whether the RUDAS has been validated in a diversity of populations as this may provide another easily administered and freely available cognitive screening tool for clinicians.
A literature search was performed in MEDLINE, Embase, PsycINFO, CINAHL, and other relevant databases from date of onset to January 2014. Reference lists of articles were hand-searched and authors were contacted for further relevant studies. All studies comparing the RUDAS with other cognitive testing or a neuropsychological assessment were included. Abstracts and full-text of articles were reviewed independently by two authors. The data were extracted using a standardized protocol and the quality of studies was evaluated using the QUADAS-2 tool.
Ten studies meeting the pre-specified inclusion criteria were found from five different countries. A total of 3,194 patients were analyzed with varying levels of cognitive impairment. Results indicate that the sensitivity and specificity of the RUDAS is superior to the MMSE. Furthermore studies indicate that the RUDAS is more useful in those with lower education and those from linguistically diverse backgrounds. There was stronger clinician preference for RUDAS compared to the MMSE.
The RUDAS is a useful short cognitive screening tool, particularly for individuals from linguistically diverse backgrounds.
There are two types of hip fractures: intertrochanteric and subcapital. Both types can have associated vertebral fractures. In this study, we explored the nature of vertebral fractures in the two hip fracture populations in an effort to gain insight into their etiology.
This was a retrospective analysis of 120 patients: 40 with subcapital fractures and vertebral fractures, 40 with intertrochanteric fractures and vertebral fractures, and 40 with vertebral fractures only. Based on Genant’s semiquantitative assessment method, the distribution, type, and severity of each patient’s vertebral fractures were described.
Patients with subcapital fractures had significantly fewer total vertebral fractures (
The distribution of vertebral fractures among patients with subcapital fractures differed from the other fracture groups. Patients with subcapital fractures were more likely to have only a single vertebral fracture, while patients with intertrochanteric fractures and those with only vertebral fractures were more likely to have multiple vertebral fractures throughout the thoracolumbar spine. The subcapital and vertebral fractures of some patients may be a consequence of trauma and not osteoporosis.
Over 35 million people worldwide currently live with dementia. A phenomenal proportion of the care provided to these individuals is provided by unpaid, family caregivers. These caregivers are often overwhelmed by the numerous stresses imposed on them, resulting in deterioration of their own health, social isolation, loss of income, and distress. Caregiver interventions have been shown to improve caregiver mood and morale, to reduce caregiver strain and to delay transition of the person with dementia to long-term care. Due to the nature and intensity of care that is required, flexible and user-friendly methods of providing interventions to caregivers have been explored.
The purpose of this review was to evaluate the evidence regarding Web-based resources as an avenue for providing support to caregivers.
We performed a systematic review of the literature using search terms such as “dementia”, “caregiver”, “support”, and “interventions”. We searched Ovid Medline, PubMed, Embase, and CINAHL to October 2013. The abstracts were reviewed and full text articles were included. Studies were included if they were in English and were systematic reviews, randomized controlled trials or other intervention studies that examined internet or Web-based interventions for caregivers of persons with dementia.
Only 13 studies met all eligibility criteria. Positive outcomes from included studies of internet caregiver resources demonstrated increased caregiver confidence in decision-making, increased self-efficacy, improved perceptions of the positive aspects of caregiving, and significant reductions in stress, strain, anxiety, and depression in caregivers.
Family caregivers are an integral, yet increasingly overburdened, part of the health care system. Given the many demands placed on family caregivers of persons with dementia, the potential for caregivers to access support services from the convenience of their own home, such as Internet resources, is attractive.
There is a strong predilection for REM sleep behavioural disorder (RBD) to occur in synucleinopathies, a group of neurodegenerative disorders that includes Parkinson’s Dementia and Dementia with Lewy Bodies (DLB). RBD often precedes the onset of Parkinsonism and degenerative dementia in both Parkinson’s disease and Dementia with Lewy Bodies. This suggests an interconnection between RBD and degenerative dementias.
To identify the interplay between the presence of RBD and the progression of DLB.
In this single centre, retrospective study, the medical charts of 47 patients with clinically suspected DLB were reviewed. Baseline demographic data were collected with respect to presenting cognitive and functional scores, age of dementia onset, DLB clinical characteristics, gender, ethnicity, education, co-morbidities, use of medication, and RBD age of onset/symptomology/treatment when applicable. Progression is defined by the average change over a three-year period in cognitive (3MS) and activities of daily living (Lawton Scale) scores. Chi square tests were performed between the control (patients with DLB without REM sleep behaviour disorder) and experimental (patients with DLB and REM sleep behavioural disorder) groups with respect to changes in cognitive (3MS) and activities of daily living (Lawton Scale) scores over time.
Chi square tests demonstrate no significant difference between the progression of DLB with or without RBD (
An association cannot be made between RBD and DLB progression. The frequent tendency of RBD to occur in synucleinopathies and rarely in tauopathies supports the concept of a selective vulnerability in key neuronal networks. This selective vulnerability may result in RBD development in, but not progression of, RBD.
Historically, identification rates of mental health disorders in seniors presenting in primary care settings are low. There have been repeated calls for standardized ‘screening’ of mental health disorders for seniors, with the goal of enhancing detection and more timely intervention.
The primary objective of this grant-funded project was to develop a standardized, user-friendly toolkit for the early identification of mental health issues in seniors for use in both rural and urban primary care settings in Alberta.
Systematic reviews of the literature initially were conducted for anxiety, dementia, depression, and substance use disorder. Studies meeting inclusionary criteria (e.g., assessment of predictive properties, used a gold standard for diagnosis, targeted the population of interest, etc.) were included. Following the systematic reviews, an external Expert Panel provided validation of the selected psycho-metrically sound tools for use in the toolkit. Health-care professionals in both rural and urban primary care settings then provided input on the feasibility of use of the selected tools in their setting.
Screening tools were identified for each of the four disorders, with all tools meeting a number of criteria, including a high degree of accuracy in identifying those with and without the disorder, ease of administration (e.g., short, easy to score), and non-proprietary.
The development of a standardized, user-friendly toolkit for the early identification of mental health issues in seniors for use in both rural and urban primary care settings in Alberta represents an important and foundational step toward increasing rates of early detection and improving treatment of mental disorders. To facilitate uptake, Web-based and print versions of the toolkit will be developed and available to health-care professionals.
Day centre programs, such as CHOICE, help support older people who are experiencing multiple ongoing health problems, with the goal of allowing them to remain living independently in their own homes longer, and to reduce their use of in-patient and emergency unit services. Since CHOICE’s inception, there have been changes to the health-care system, including an increased orientation to ‘aging in the right place’. Goals of this research were to describe the characteristics of CHOICE clients and to determine their use of other segments of the health-care system (e.g., Emergency room visits).
Retrospective chart review of 195 clients at two Edmonton CHOICE Day Care sites.
Mean age was 79.91 (SD=8.09); 56% were female; with an average of 6.78 (SD=2.58) co-morbidities; 45% of the clients had a dementia. Clients scored below the cutoffs for impairment on all cognitive (MMSE/MoCA) and functional (FAB/Berg/Tinetti) tests, except the Tinetti on admission. Emergency room visits (1.26 vs. 0.52) and hospitalizations (1.26 vs. 0.52) decreased significantly in the one-year period pre- vs. post-CHOICE admission. Incidence of falls declined by 80% from pre-admission to one-year post-admission.
CHOICE client profiles are consistent with the program’s mandate. Attendance at the CHOICE program was found to be associated with significant decreases in emergency room visits, hospitalizations, and incidence of falls. Day programs appear effective in not only reducing the use of acute care costs by medically complex community dwelling seniors, but also in allowing CHOICE attendees to remain living independently in their own homes longer.
In the absence of an advance care plan (ACP), patients with end-stage serious illnesses or injuries who can no longer speak for themselves may be subjected to unwanted invasive treatments or interventions. Hospitalist physicians can contribute to the quality and dignity of end-of-life care by eliciting advance care preferences with patients and families while the patient is still capable of consenting to, or refusing, treatment or other care.
The purpose of this study was to determine whether physician-to-physician conversation about their own advance care preferences increases the likelihood that they may talk with patients about the patient’s ACP.
Over a two-week period, the hospitalist study lead engaged 107 physician colleagues in informal conversations about their own advance care planning. After responding to two standard questions, the physician received an “Advance Care Planning Matters!” button and information card. A follow-up survey was delivered two weeks later.
72% of survey respondents (
Physicians who journey through their own ACP may be more likely to talk with patients about ACP, and physician-to-physician conversations can increase their comfort in initiating these talks.
In a series of four case studies, frail seniors recently discharged from an acute care admission and referred to geriatric outreach teams were asked to identify the ‘team’ helping to keep them independent at home and to rate their perceptions of the relationships between each provider. Providers were then contacted and their perceptions of the inter-relationships were rated.
The patient and provider inter-relationship ratings were then analyzed using social network analysis.
The analysis reveals the complexity and diversity of home and community-based ‘teams’, compares the perceptions of patients and providers, and indicates the ‘centrality’ of outreach teams in these post-discharge situations.
In recent years there has been an increase in pharmacist-managed anticoagulation programs. However, anticoagulation management services (AMS) for patients in supportive living have not been examined.
This study examines pharmacist-managed versus comprehensive multidisciplinary physician-managed anticoagulation monitoring.
We conducted a retrospective review of patients enrolled in a seniors’ day program (physician-managed) vs. Pharmacare’s supportive living AMS (pharmacist-managed) using case control methodology. The primary outcome was time in therapeutic range (TTR), with TTR determined by using both the fraction of International Normalized Ratio (INR) values method and Rosendaal’s linear interpolation method. Secondary outcome measures were frequency of INR testing and adverse events, including major bleeding and thromboembolic events.
The pharmacist-managed AMS had a TTR of 69.8% vs. 64.7% (
Pharmacist-managed AMS demonstrated non-inferiority compared to the multidisciplinary physician-managed AMS. Differences in TTR varied as a function of methodology (e.g., use of fraction of INRs or the Rosendaal linear interpolation method), with these differences confounded by practice settings and access to INR testing.
There is extensive evidence that exercise, even moderate amounts, improves health. The literature also shows that decreased activity or inactivity is detrimental to health and wellness. This study examined the feasibility of measuring physical activity of in-patients in a geriatric rehabilitation setting using a custom-built, tri-axial accelerometer, in preparation for a study of the effectiveness of a mobility program.
A tri-axial accelerometer housed in a custom-built package was used to track the ambulatory movement of patients in a tertiary rehabilitation hospital. Patients were asked to participate once they were assessed as ‘Independent’ or ‘Supervised’ by a physical therapist. Those who agreed to participate were asked to wear the device until discharge. The device was secured to the lower leg. Each accelerometer was individually calibrated using a standardized distance and validated throughout the trial. Ambulation was determined using the horizontal and vertical components of the acceleration data. A thermometer was used to confirm that the device was being worn.
13 patients (8F 5M) agreed to take part. The average age was 83 yrs. Six were classified as ‘Independent’ walkers and 7 as ‘Supervised’. Ten patients completed the study; one withdrew and two were stopped due to changes in their medical condition. Total distance walked in the 6 days prior to discharge averaged 6,529 m (+ 4,313 m), 4,152 m (+ 1,331 m) for females and 8,917 m (+ 5,126 m) for males. Thermometer data confirmed the wear time indicated by the accelerometer measure.
Measuring physical activity in an older population presents challenges. This study demonstrated that measuring ambulation with a custom-designed/built device is feasible in an older in-patient population.
In the past, persons with MS died younger than the general population. Since the 1990s, important treatment-related changes, including disease modifying drugs, have been introduced which might influence MS outcomes. This study examined whether such changes are reflected in Canadian MS mortality rates.
Statistics Canada provided data on deaths due to MS from 1975 to 2009 and population statistics. Average annual MS mortality rates per 100,000 population were calculated for each 5-year period within this time span, along with rates for each year and for the entire 35-year period, by gender and age.
The 35-year Canadian MS mortality rate was 1.23; trend analysis indicated that annual rates were stable over this time span. The 35-year rate for females was higher (1.45) than for males (0.99), with both female and male rates remaining stable over time. Regardless of gender, trend analysis showed a significant decrease in mortality rates for persons with MS under age 40 and a significant increase for persons over 60. The mortality rate for females under 40 dropped from 0.11 in 1975–79 to 0.05 in 2005–09 and rose from 0.51 to 1.00 for those over 60. The mortality rate for males under 40 dropped from 0.06 to 0.03 and rose from 0.34 to 0.52 for those over 60.
More persons with MS still die under age 60 than members of the general population. However, there has been a shift to later age at death, according to Canadian MS mortality rates, that has especially benefited women. The fact that persons with MS are living longer suggests that more health and social services will be required to meet the particular needs of those aging with MS.
What is the adherence to guidelines in Hospital-acquired pneumonia? What is the adherence to guidelines in aspiration pneumonia?
Ethics and a site approval Chart Review from Grey Nuns Community Hospital. 207 charts were pulled; 108 charts with diagnostic code hospital acquired pneumonia were pulled, from 10/1/2008 to 09/30/2011. 227 charts with diagnostic code aspiration pneumonia were found, from 10/1/2008 to 09/30/2011; 99 charts were randomly selected out of those 227 charts. Information on demographic, comorbidities, antibiotics used and length of therapy, mortality, ICU admission, mortality, and admitting services was gathered. Information on whether or not swallowing assessment was done, and on chest X-ray, MRSA positive, and VRE positive was also gathered from the charts.
Hospital-acquired pneumonia group, guidelines were followed in 44.9% of the time Aspiration pneumonia group, guidelines were followed 64.5% of the time.
This study showed that the guidelines were commonly noted when it came to hospital-acquired pneumonia. The dose of levaquin was often followed n too low, which could be explained by the fact that Alberta Health has a pathway for community-acquired pneumonia but nothing for hospital-acquired pneumonia. Mortality rates were higher in the group where guidelines were followed, which could be explained by the fact that the sicker patients were more likely to have guidelines followed, and the sicker patients were more likely to pass away.
A revised standard order sheet for hospital pneumonia would be beneficial.
Edmonton’s Home Living Geriatric Consult Team (GCT) is a community-based interdisciplinary team that provides in-home comprehensive geriatric assessment and interventions for unstable elders receiving home care (HC) support. It was designed with the goal of reducing avoidable emergency department visits, hospital admissions, and early institutionalization through the early identification of high-risk elders and immediate intervention.
The purpose of this project was to evaluate the effectiveness of the GCT based on the experiences of its stakeholders: geriatric patients, their caregivers, HC case managers, and family physicians.
Data from family physicians were collected through anonymous surveys while semi-structured interviews were conducted for HC case managers, patients, and their caregivers. Both quantitative and qualitative analysis of data will be performed. Ethical considerations have been supported by ARECCI guidelines.
Results to date include interview responses from six patients and eight caregivers. Overall, patients and caregivers reported that: 1) They felt involved in GCT discussions and decisions about care; 2) They believed GCT involvement was necessary and played a direct role in allowing patients to remain at home; and that 3) GCT involvement lessened the burden of being a caregiver. Results from family physicians and HC case managers are pending and will be incorporated once complete.
Given its unique position in primary care, the GCT has the ability to facilitate collaboration between HC case managers and family physicians in the parallel care of complex geriatric patients, thus bridging the gap between these two major care providers. An understanding of GCT stakeholders’ experiences and perceptions is fundamental to program success.
Although the primary intention of this project was for GCT quality improvement, our findings may also have useful implications for future innovation in geriatric care.
Type 2 diabetes is a common condition in hospitalized geriatric patients. We modified the Canadian Diabetes Association (CDA) flow sheet for use on a geriatric in-patient rehabilitation unit to evaluate our performance on indicators of quality care and feasibility of using this tool to optimize diabetes management.
The flow sheet was piloted from October 2011 to October 2012. The care of diabetic patients was guided by the flow sheet, but was otherwise “as usual” care with no other formal interventions.
28 diabetic flow sheets were collected, with completion rates for each item ranging from 61% to 100%. House staff required reminders to complete the flow sheet and its use varied with different attending physicians. 89% of patients had an A1c ordered or available, 32% had an ACR done in hospital, and 64% a documented sensory foot exam. 71% were on a statin, 64% on ASA, and 50% on an ARB/ACEi. 25% of patients had an annual eye exam arranged and 18% had nail care arranged. 43% had the influenza, and 21% the pneumococcal vaccines.
House staff regularly checked A1c and ensured patients were on appropriate medications. However, clarification of the status of interventions usually done as outpatients (eye exams, community nail care, and vaccinations) was less commonly completed.
Use of a modified CDA flow sheet with geriatric in-patients is feasible to audit and encourage guideline-directed diabetes care. Better integration into the patient’s chart, use within an electronic patient record, and house staff training would improve its utilization and effectiveness. The diabetes flow sheet may have a role as a communication tool with primary care physicians to identify items requiring follow-up upon discharge.
Benzodiazepines are frequently used for extended periods of time and are associated with significant morbidity in older adults.
The purpose of this presentation is to describe the contributions from health professionals in the development and implementation of an interprovincial benzodiazepine de-prescribing guideline.
Canadian Institute of Health Research funding was received to conduct an expert and stakeholder 1.5 day planning meeting regarding interprofessional approaches to benzodiazepine tapering. Objectives were to discuss challenges related to benzodiazepines, critique a proposed interprofessional model for de-prescribing benzodiazepines, and identify interventions and evaluation strategies for implementing the model. Discussions and presentations were recorded and transcribed. Activities and outcomes are presented descriptively.
Nineteen local service providers and 14 stakeholders, in addition to the 5 members of the research team, participated in the meeting. Key messages identified by the participants were: adaptability of the de-prescribing model, the need to address the challenges of implementing changes in a structured and highly regulated environment such as long-term care, the impact or liability of de-prescribing on the prescriber and other health professionals, and the challenge of changing practice amongst established health professionals. Development and implementation of a benzodiazepine ‘deprescribing guideline’ was discussed. The RE-AIM (reach, effectiveness, adoption, implementation, maintenance) model (a 5-dimensional framework that increases chances of an intervention being successful in “real world” settings) was chosen to frame further intervention and evaluation work.
Health professionals are supportive of guidelines for benzodiazepine de-prescribing. Evidence to support positive impact of benzodiazepine tapering is available to guide development of a de-prescribing guideline. The primary concern is related to usability of such a guideline and implementing the practice into a culture of prescribing.
The Queen’s Geriatric Interest Group (QGIG) is a student-based initiative developed to foster interest in the field of geriatric medicine, with goals of increasing overall knowledge of geriatric care and to increase recruitment to geriatric-focused fields. Pre-clerkship observerships have been documented as valuable methods in increasing exposure and interest to a given specialty of medicine. QGIG leaders collaborated with the Division of Geriatric Medicine to arrange scheduled observerships at St. Mary’s of the Lake Hospital, a health-care centre with a focus on specialized geriatrics and complex continuing care.
Given challenges of student and preceptor schedules, four-hour observerships were organized for weekend mornings. Students participated in “on-call rounds” on the geriatric rehabilitation unit under supervision of the resident and/or attending physician. Students were given small roles including patient assessment, appropriate chart documentation, and reviewing medication lists. After each experience, students were asked to comment on strengths, weaknesses, and recommendations for the experience. The UCLA Geriatric Attitude Survey (GAS) was used pre- and post-observership to determine changes in participant attitudes towards geriatric medicine.
A total of 50 students participated in the observer-ship program between February 2013 and January 2014. Students viewed the observerships as providing excellent role models for geriatric care and subjectively increased their interest in the specialty. Analysis of student attitudes towards geriatrics pre- and post-observership is ongoing.
Current electronic medical record systems are not tailored to the workflow of a specialized geriatric clinic. Systems that can be adapted to meet the needs of a geriatric clinic rarely allow the collected data to be analyzed for research purposes. Also, mobile touch-based computers have become popular amongst health-care workers, but this technology has not been quickly supported by health-care organizations. Many software solutions are not practical and lack usability due to the usage of small buttons, fonts, complex menu systems, and challenging user interfaces.
We have developed a Web-based geriatric patient registry system that is usable on both desktop and tablet computers. The system was designed to allow for the capture of all data points commonly collected in a comprehensive geriatric assessment, including patient demographics, referral information, histories, review of symptoms, physical assessment, differential diagnosis, and plans for future care.
The system allows for the user to digitally capture tests including the SMMSE, GDS, BNA, ADL, FAQ, and Zarit Burden Interview, among others. From collected data, the system generates clinic notes and reports, potentially reducing, or in some instances eliminating, dictation time. Lastly, the system has been designed for use in clinical research, with data anonimization and analytical tools built into the application. Data can also be analyzed using statistical analysis software such as SPSS and SASS.
Through the use of this system, we anticipate an increase both the quality of care provided to patients and efficiency of clinical practice. Although this system is in its “beta testing” stage, we anticipate this system will decrease visit length and dictation time, facilitate clinical research, and accurately measure quality of care indicators.
As the number of Canadians aged 65 years and older continues to rise, more attention has been given to home-based health care. Homebound seniors have higher rates of diseases, chronic medication use, emergency department visits, hospitalizations, and challenges in accessing care. Despite this growing concern, the number of physicians participating in house calls is declining. Family Medicine residents have generally perceived lack of training as a significant factor limiting their likelihood of pursuing house calls in the future. Many academic centres have looked into instituting a structured homebound seniors program as part of residency training to improve resident knowledge, skills, attitudes, and confidence in performing house calls.
A survey was distributed to Family Medicine residents from all 15 teaching sites at the University of Toronto. Sites having either a structured or non-structured homebound seniors program implemented in the residency curriculum were compared to assess if there is a difference in resident perception of house calls. A needs assessment of resident perspective on improving the house call curriculum was also performed.
The study demonstrated with strong statistical significance that structured programs compared to non-structured programs increase resident exposure, positive attitudes, confidence, and plans of pursuing house calls in their future practice. Similarly, a strong correlation of increasing exposure to house calls was associated with higher resident satisfaction. The needs assessment demonstrated that training on billing, procedures, having increased supervision, and greater exposure to house calls would improve their experience during residency.
There are positive implications of this study for the health-care system, medical education system, practitioners, patients, and families in improving and sustaining care for homebound seniors, which can be implemented at a national level.
The use of psychotropic and analgesic medications is common in older adults and may be associated with drug–drug interactions (DDI) and adverse effects. DDI can be based on Phase 1 metabolism (oxidation/ reduction, including the cytochrome p 450 system) or phase 2 metabolism (conjugation or the UGT and ABC transporters).
The aim is to assess the prevalence of possible metabolic drug interactions with psychotropic and analgesic medications in octogenarians.
Retrospective chart review of all consecutive patients seen at the outpatients Senior’s Clinic at the University of Alberta in 2012. Each subject’s medication list was analyzed and coded, regarding substrate and inhibitor of enzymes in Phase I and Phase II systems and for polypharmacy (use of 5 or more prescribed medications). Psychotropic and pain medications were analyzed for interaction with the phases of metabolism. Simple descriptive statistics, chi-square and logistic regression analysis were done using SPSS.
Mean age was 85.9 years (SD 4.09) and 259 (69%) were females. Polypharmacy was seen in 253 (68%) subjects. Out of 372 subjects, 90 subjects (24%) were found to have potential drug interactions within their list of medications. Fifteen subjects (4%) who were taking pain medications and 75 subjects (20%) who were taking psychotrophic medications had possible metabolic drug interactions. Polypharmacy was associated with a 3.5 fold increase in the risk of metabolic drug interactions, OR 4.46, 95% CI 2.27–8.74 (
In this study, a significant proportion of octogenarians who were on pain and psychotrophic medications had possible metabolic drug interactions within their list of daily medications. Increasing awareness and knowledge of metabolic drug interactions is important to choose safe drug combinations and to prevent toxicity.
Fragility fractures secondary to osteoporosis are associated with excess mortality and disability among older individuals. Studies have consistently reported low rates of osteoporosis screening; however, few have examined which patient and physician factors influence screening uptake and pharmacotherapy for osteoporosis.
A retrospective chart review was conducted among 455 individuals (281 women and 174 men) age 65 or greater with at least one clinic visit between 2011 to October 2013. Components of the Fracture Risk Assessment Tool (FRAX), Charlson Comorbidity Index, Osteoporosis Self-Assessment Tool (OST) score, and patient and physician sociodemographic variables were extracted using a standardized data extraction form. The outcomes of interest were the proportions of individuals who received bone mineral density screening and subsequent bisphosphonate therapy.
Three hundred seventy-one patients (69.7%) had received at least one bone mineral density screening. Multivariate analysis revealed that women compared to men were more likely to receive osteoporosis screening (OR=4.17, 95% CI: 2.47–7.02). Furthermore, individuals with a female physician (OR=3.87, 95% CI: 2.20–6.81), previous fragility fracture (OR=14.92, 95% CI: 3.41–65.22), previous breast or prostate cancer (OR=4.43, 95% CI: 1.17–16.87), and a positive screen on the OST (OR=2.40, 95% CI: 1.32–4.362.20–6.81) were more likely to receive bone mineral density test. Male and female physicians were equally as likely to prescribe bisphosphonates for individuals with osteoporosis. However, individuals with an active solid or hematological malignancy were less likely to receive bisphosphonate therapy (OR=0.49, 95% CI: 0.07–0.75).
Compared to women, men continue to be underscreened for osteoporosis. Screening and pharmacotherapy for osteoporosis is multifactorial and influenced by patients’ current co-morbidity burden and fracture risk.
Inflammation is a risk factor for osteoporosis. Treating inflammation may improve bone mineral density (BMD).
This study aimed to examine if anti-rheumatic drugs for rheumatoid arthritis (RA), psoriatic arthritis (PsA), psoriasis (PSO), and ankylosing spondylitis (AS) improve BMD.
MEDLINE, Embase, and Cochrane were searched from 1960 to present for English-language randomized controlled trials conducted in adults. Studies were grouped based on disease, treatment type, and site of BMD measurement. Differences in change of BMD (ΔBMD) between treatment and control were standardized across included studies to yield standardized mean difference (SMD).
13 studies were eligible (11 RA, 0 PsA, 0 PSO, 2 AS). For RA, less hand bone loss was seen with TNF inhibitors (TNFi) (SMD ΔBMD = 0.33, 95% CI 0.13–0.53,
Based on our RA analysis, TNFi yielded less hand bone loss, where synovitis is often present, but had no effect on LS and hip BMD. Corticosteroids also yielded less hand bone loss, but it had negative effect in LS and not the hip. For AS, TNFi increased BMD.
Geriatric patients are at high risk for in-hospital deconditioning, malnutrition, and poly-pharmacy, resulting in delayed discharge and iatrogenic complications. Parameters for improving the care of geriatric patients on the Clinical Teaching Unit (CTU) were proposed by internal medicine residents during academic half-day. The parameters include: 1) obtaining patient’s body weight (admission & weekly); 2) supplementing all patients with daily oral Calcium and Vitamin D, and weekly bisphosphonate, where appropriate; and 3) reviewing patient’s diagnoses and medications weekly in writing.
Reminders in person/in writing to all (1st intervention) and in email to senior residents (2nd intervention) were implemented to improve compliance with the above measures. Electronic progress forms were initiated as a 3rd intervention. During the second year of the study, we developed a pre-printed CTU admission order set that included multiple “elderly-friendly” measures (4th intervention). Retrospective chart review was performed of all geriatric patients discharged from CTU at Royal University Hospital in Saskatoon from September 2012 to April, 2014.
In the first year 317 charts were reviewed. Our study found that the 1st intervention resulted in improvement in ordering of weights during admission (from 33% to 38%), Ca/Vitamin D supplementation (from 33% to 56% and from 45% to 65%, respectively), and medication reconciliation (from 20% to 35%). The 2nd intervention was found to be less effective, with medication reconciliation dropping to 32%; returning to 34% after initiating the 3rd intervention. The 3rd intervention was paradoxically associated with decreased ordering of weights. Final evaluation of the impact of the pre-printed CTU admission orders is pending.
Education and active involvement of residents can improve the implementation of “elderly-friendly” basic health measures on a provincial CTU.
To determine whether bilingualism is associated with dementia in cross-sectional or prospective analyses of older adults.
In 1991, 1616 community-living older adults were assessed and followed five years later. Measures included age, gender, education, subjective memory loss (SML), and the modified Mini-Mental State Examination (3MS). Dementia was determined by clinical examination in those who scored below the cut point on the 3MS. Language status was categorized based upon self-report into three groups: Monolingual English, Bilingual English, and English as a Second Language (ESL).
The ESL category had lower education, lower 3MS scores, more SML, and were more likely to be diagnosed with Cognitive Impairment, No Dementia (CIND) at both time 1 and time 2, compared to English-speakers. There was no association between being bilingual (ESL and English bilingual vs. Monolingual) and having dementia at time 1 in bivariate (OR (95% CI)=0.76 (0.41, 1.43)) or multivariate analyses (OR (95% CI)=0.84 (0.77, 0.92)). In those who were cognitively intact at time 1, there was no association between being bilingual and having dementia at time 2 in bivariate (OR (95%)=0.99 (0.61, 1.59) or multivariate analyses (OR (95%)=0.94 (0.88, 1.01).
We did not find any association between speaking more than one language and dementia.
The association between benzodiazepines and the risk of falls in the elderly is well described. It remains unknown whether urinary incontinence mediates this risk.
The aim of this study was to assess the impact of urinary symptoms on the risk of falls among chronic benzodiazepine users.
A cross-sectional analysis was conducted using baseline data from 303 older benzodiazepine users enrolled in a randomized trial. A history of falls was ascertained by self-report. The presence of incontinence was defined as a score ≥ 1 on the International Consultation on Incontinence Questionnaire (ICIQ). Medication history, including daily benzodiazepine dose (converted to lorazepam equivalents), was obtained from pharmacy renewal profiles. The Montreal Cognitive Assessment (MoCA) was used to ascertain cognitive status. The prevalence of falls in participants with and without incontinence was compared with descriptive statistics; the magnitude of association was ascertained using univariate logistic regression.
Of 303 chronic benzodiazepine users (mean duration of use = 10 years), 85 (28%) reported a history of falls and 66 (21.8%) reported incontinence. Participants reporting incontinence were twice as likely to report a history of falls than continent individuals (40.9% vs. 24.5%, respectively,
Urinary incontinence was significantly associated with a history of falls in this older cohort of chronic benzodiazepine users. Further investigation is required to elucidate the temporal relationship between “rushing to the bathroom” and falling, as well as the causal contribution of other functional/mobility impairments.
Geriatric assessment and treatment units (GATUs) in acute care hospitals that electively admit frail older adults have all but disappeared.
The study objective is to describe the characteristics and outcomes of patients admitted to a GATU located in a chronic hospital/rehabilitation setting, which primarily admits patients from the community, distinguishing it from Acute Care for Elders units.
A retrospective chart review was conducted on patients discharged from the GATU between April 9, 2012 and June 3, 2013. Descriptive statistics were used to characterize the patient sample. Statistical differences were explored using paired
102 patients were included, representing 111 admissions. The mean age was 82.0 (±7.9) years. Most patients were female (81.4%), widowed (61.8%), and admitted from the community (76.5%). The most common reasons for admission were pain management, falls, and deconditioning. The average number of co-morbidities per patient was 7.9 (range 2–19). On admission, the average Berg Balance Scale score was 28.5 (±13.0) and 74.2% were at medium or high risk for falls. Patients stayed on the unit an average of 28.3 (±12.0) days. On discharge, 72.1% were independent in ambulation vs. only 47.1% on admission (
The majority of patients on the GATU improved in mobility and functional status and returned to the community, demonstrating that frail patients with complex needs can benefit from a GATU in a non-acute care setting.
Rapid detection and relief of pain is challenging when caring for older patients in the Emergency Department (ED). Fractures, a common reason for ED visits cause acute pain, which if relieved rapidly, improves well-being and reduces adverse events. The aim of this quality improvement initiative was to improve pain management and reduce adverse events including delirium, length of stay, and return visits in patients 75 years and older presenting with a fracture at any of three hospital EDs in Montreal, Canada.
A multifaceted intervention was developed based on data collected from electronic medical records and surveys of ED professionals and patients. A stepped-wedge design was used to implement the intervention sequentially; data are collected each time a site begins the intervention. Primary outcome is time to optimal pain management.
Prior to intervention, we identified 95 patients (67% women, mean age 84 6 years) with 102 fractures over a two-month period. Median length of stay was 11 (IQR: 7.1–23.3) hours. Pain score was documented during the ED stay in 44% of patients with median time to documentation of 5 (IQR: 2.2–7.2) hours; 25% had no score documented. The median time to first analgesic was 3.4 (IQR: 1.8–5.5) hours, 27% did not receive any analgesia. Surveys distributed to 81 physicians and 198 nurses identified barriers to optimal pain management: time constraints, lack of resources and monitoring and potential adverse events. Results post-implementation at two EDs will be presented.
There is an important care gap in documentation and timely management of pain. Anticipated contributions of the PAINFREE initiative will be the development of tools to facilitate best practices for pain control and consequently improve outcomes.
Population aging has contributed to increasing falls in the older population. Fall detection has been widely studied, but most studies have not addressed possible physiological causes. This study examined the feasibility of simultaneously measuring stability and vital signs in real time to better understand the possible linkages.
The tilt angle of the body was logged while walking. Detection of a “near fall” or “fall” event was defined as a tilt angle greater than 45° and an impact acceleration > 2g, respectively. Oxygen saturation, heart rate, and body temperature were sampled once per minute and synchronized with the fall detector.
Devices were tested on 7 healthy subjects performing activities of daily living between 16 to 40 hours over 5 days. The mean tilt angle was 24°±15°. The false-positive rate was 0.11%. The mean heart rate, oxygen saturation, and skin temperature were 72.2±10.0 heart bpm, 97±2% and 29.7±1.8°C, respectively. An 84-year-old woman volunteered to test the device at home for 6 hours: average tilt angle was 28°±17°; heart rate, oxygen saturation, and body temperature averaged 75.4±6.9 bpm, 94.6±1.7%, and 33.0±0.8°C, respectively.
This study supported the feasibility of combining fall detection with real-time physiological monitoring. Measurements taken by the physiological monitor agreed with expected values from the literature. These results were based on a one-day trial and further study is required to correlate the vital signs and the risk of falls.
To review the impact of provider-based, organizational strategies in acute care settings to improve pneumococcal vaccination rates among patients at risk of pneumococcal disease (i.e., those over 65 years, and 2–64 years of age with high-risk medical conditions).
A search was conducted using MEDLINE, Scopus, CINAHL, and Web of Science databases for hospital-based, in-patient studies which evaluated strategies to improve pneumococcal vaccination rates. Studies published in English from 1983–2013 were included.
A total of 34 studies were included; 14 studies evaluated physician reminders such as chart or paper reminders, pre-printed orders (PPOs), and computerized reminders, and 26 standing orders programs (SOPs). Pre/post design was the most common study design; only 7 studies had a control group. Overall, 31 studies showed improvements in the rate of pneumococcal vaccination following intervention, of which 18 were statistically significant. Physician reminders resulted in 29%–67% immunization rate, PPCO 5%–42%, and SOPs 3%–78%.
Although this review found higher immunizations rates with SOPs, the impact on immunization rates in eligible patients varied significantly. The quality and design of the studies makes interpretation of the best approach challenging. High-quality, randomized-controlled studies are required to determine the true effect of each type of institutional immunization strategy.
Hospital-based interventions improve pneumococcal vaccination in older adults and younger individuals at risk. Further research is required to determine the ideal intervention.
The Canadian Association of Interns and Residents (CAIR) Specialist Forum Project Proposal September 2013 was aimed to create a health human resources platform in Canada by providing residents with reliable information on physician employment opportunities in Canada.
1. Determine number of Specialist Geriatricians (SpGrtn) in geriatrics; 2. Estimate the societal need; 3. Project the number of SpGrtns physicians retiring in geriatrics within the next 5 to 10 years.
In the absence of an available benchmark, we used a physician/population ratio of 1.25 SpGrtns/10,000 people 65+2 or 1 SpGrtn/ 4,000 people 75+3, and 2011 Canadian Census data (med population projections 65+ or 75+) over ten years. We estimated the anticipated retirement of present Canadian SpGrtns as 40 years beyond their medical degree (MD).
In January 2014, there were 285 practicing SpGrtns and 134 Care of the Elderly (CoE) trained physicians, an increase of 11% and 30%, respectively, from the 2011 estimate. The calculated need in 2014 is 445 SpGrtns (1.25/10,000 65+) or 610 (1/4,000 75+). The calculated need for SpGrtns in 2021 is 567 (1.25/10,000 65+) or 756 (1/4,000 75+). Across Canada, at least 10 SpGrtns are trained annually (100 in 10 years). Between 2012 and 2021, approximately 95 of the existing SpGrtns will have practiced 40 years.
As a preliminary response to the CAIR initiative, we have estimated that in 2021 there will be 290 SpGrtns (285−95+100) resulting in a shortfall 277 Sp-Grtns (567-290) in Canada. This is sufficient justification for continued efforts to attract Canadian medical trainees to the field of Geriatric Medicine and to recruit geriatric specialists to Canada. Caveat: Not all SpGrtns and Coe MDs are working full time in geriatrics, so the short fall will be even worse.
Cognitive impairments are prevalent in heart failure and have been mechanistically linked to cerebral hypoperfusion. This relationship has been based solely on measurements of cerebral blood flow (CBF) in the supine position; however, upright postures common to daily living may pose an additional challenge.
The purpose of this study was to examine CBF in response to upright sitting in heart failure patients and healthy controls.
Twenty-two heart failure patients (age=69±9 years, ejection fraction=33±11%) were age- and sex-matched to twenty-two healthy controls (age=70±9 years). Participants were administered the Montreal Cognitive Assessment (MoCA) to assess global cognition. Gait speed was calculated by utilizing an 8 m usual speed walking test. The right internal carotid artery diameter and mean flow velocity was obtained with ultrasound when supine and seated to provide a quantitative measure of CBF.
Heart failure patients scored lower on the MoCA (24±3) than their healthy counterparts (28±1;
Importantly, this is the first time CBF has been measured in an upright position in a heart failure population. The found reduction from supine may have important clinical implications on cognition.
Previous studies using Rasch analysis in Brazilian, US, and Chinese samples have yielded mixed conclusions regarding the unidimensionality of the GDS-15, suggesting that differences in test language or sample characteristics may influence psychometric properties.
To estimate the psychometric properties of the GDS-15, including the extent to which they are influenced by test language and cognitive ability among Canadian elders referred to a university-based geriatric outpatient clinic.
Patient data were obtained through retrospective analysis of a clinical database in two geriatric outpatient clinics in Montréal, Québec. GDS data (
A significant item-trait interaction indicated poor fit of the GDS-15 to a unidimensional Rasch model (chi2=83.5,
Fear about the future contributes more to severity of depressive symptoms among more cognitively intact patients. Revisiting the French translation of item “feeling happy” may further improve the validity of this tool. Clinicians should consider administering a 12-item GDS to obtain the most psychometrically valid measure of depressive symptoms in geriatric outpatient settings.
The perception that gait is an automatic motor task, requiring minimal cognitive processes, is too simplistic. It is well established that at least one cognitive domain, executive function (EF), plays an important role in controlling gait, its dysfunction and falls. Nonetheless, the role of additional cognitive domains, including memory, remains unknown. Individuals with mild cognitive impairment (MCI) are at greater risk for falls, mobility decline and, as expected, cognitive deterioration—representing an ideal target population to explore this relationship.
Our aim is to identify associations between deficits in specific cognitive domains and gait variability (GV), an accepted marker of gait control and future falls.
Older adults with MCI were cognitively assessed for EF (Trail Making A&B), attention (Digit Span), language (Boston Naming), working (Letter Number Sequencing), and episodic memory (Rey Auditory Verbal Learning). Gait assessments performed under usual gait (UG) and dual-tasking (DT) conditions using an electronic walkway (GaitRITE®). Gait variability was evaluated using the co-efficient of variation.
Sixty-four MCI participants (Mean age: 76.0±6.7 and 57% males) were included. Multi-variable linear regression analysis (adjusted for potential confounders) indicated under both walking conditions EF, attention, working memory, and episodic memory were significantly associated with GV (
Deficits in cognitive domains beyond EF, including working and episodic memory, are associated with poorer gait performance. These associations suggest gait control shares similar neural brain circuits as memory and language. Our result s may help to understand the role of cognition in fall risk of cognitively impaired older adults.
Frail elderly home care patients are at high risk of drug-related problems (DRPs). While the involvement of a pharmacist has been shown to reduce polypharmacy in the geriatric population, the role of the pharmacist in home care remains poorly defined.
To determine the frequency of DRPs in the elderly home care population and the uptake of medication modification recommendations made by pharmacists to physicians after a home visit.
In this cross-sectional observational study, the pharmacist performed a medication assessment by chart review for 81 home care patients (age ≥ 65). The Pharmaceutical Care Network Europe Drug Related Problems (PCNE) classification system and the 2012 Beers List of drugs to avoid in the elderly were systematically applied to patients’ medication profiles by a pharmacist to determine the frequency of DRPs. Forty-one patients additionally received pharmacist home visits. The uptake of pharmacist recommendations by the treating physician was recorded.
Pharmacists identified 213 DRPs (mean 2.63±1.9 per patient), with 41% of patients receiving at least one inappropriate Beers list medication. Home visits significantly increased the detection of DRPs from 1.5±1.2 DRPs prior to the visit, to 2.9±1.8 after the home visit (
Home visit by a pharmacist facilitate the detection of DRPs among high-risk home care patients. This is the first study to use the PCNE classification to evaluate DRPs in the home care setting.
The results support the inclusion of home-based medication assessment by pharmacists for high-risk home care patients.
Despite guidelines recommending against the use of benzodiazepine drugs for older adults aged 65 years and older, their use is still highly prevalent in Canadian elders and may be responsible for hundreds of millions of dollars in extra medical expenditures. The effect of direct patient education to catalyze collaborative care for reducing inappropriate prescriptions remains unknown.
The objective of this study was to compare the effect of a direct-to-consumer educational intervention against usual care on benzodiazepine discontinuation in community-dwelling older adults.
This was a cluster-randomized trial set in the general community. A total of 303 chronic benzodiazepine users, aged 65–95 years, were recruited from 30 community pharmacies. The active arm received a de-prescribing patient empowerment intervention describing the risks of benzodiazepine use and a step-wise tapering protocol, while the control arm received usual care. The main outcome consisted of benzodiazepine discontinuation at 6 months post-randomization, ascertained by pharmacy medication renewal profiles. General estimating equations were used to account for possible clustering.
Two hundred and sixty-one participants (86%) completed the 6-month follow-up. Sixty-two per cent of recipients in the intervention group initiated conversation about benzodiazepine cessation with a physician and/or pharmacist. At 6 months, 27% of the intervention group had discontinued benzodiazepine use compared to 5% of controls (risk difference 23%, 95% CI 14–32%, ICC 0.008, NNT=4). Dose reduction occurred in an additional 11% (95% confidence intervals 6–16%). Neither age greater than 80, sex, duration of use, indication for use, dose, previous attempt to taper nor concomitant polypharmacy (≤ 10 drugs/day) had a significant interaction effect with benzodiazepine discontinuation in multivariate sub-analyses.
Direct-to-consumer education effectively elicits de-prescribing of benzodiazepines in older adults.
Previous studies have demonstrated that aerobic exercise interventions have a positive impact on sleep quality in older adults. Little work has been done however, on the impact of sedentary behaviour (such as sitting, watching television, etc.) on sleep efficacy.
54 community-dwelling men and women > 65 years of age living in Whistler, British Columbia (mean 71.5 years) were enrolled in this cross-sectional observational study. Subjects were in good health and free of known diabetes. Measures of sleep efficiency, as well as average waking sedentary (ST), light (LT), and moderate (MT) activity, were recorded with SenseWear accelerometers worn continuously for 7 days. An initial univariate analysis of activity measures, alcohol consumption, sleep efficiency, age, and gender was performed and significant variables (
From the univariate regression analysis, there was no association between sleep efficiency and the predictors LT and MT. There was a small negative association between ST and sleep efficiency that remained significant in our multivariate regression model containing alcohol consumption, age, and gender as covariates. (Standardized Beta Correlation Coefficient −0.322,
While light and moderate physical activity had no association with sleep efficiency, sedentary behaviour had a statistically significant, but clinically small, negative association with sleep efficiency. This suggests that interventions to reduce sedentary time will have a beneficial, but small, impact on quality of sleep in older adults
Early motor changes associated with later adult aging predicts cognitive decline, suggesting that a “motor signature” can be detected in pre-dementia states.
Our aim was to determine whether gait performance in older adults with mild cognitive impairment (MCI) differs based on their cognitive subtype classification: amnestic (a-MCI) or non-amnestic type (na-MCI).
Older adults with MCI and cognitively healthy controls (CHC) from the “Gait and Brain Study” were assessed for global cognition and specific cognitive domains with a neurocognitive test battery. Mean gait velocity and stride time variability were evaluated with the GaitRITE® under usual and three dual-task conditions. The relationship between cognitive group (a-MCI vs. na-MCI) and gait parameters, including velocity and variability, was evaluated with linear regression models and adjusted for potential confounders.
Ninety-eight older participants, 56 MCI (mean age 76.3±7.2 years and 50.9% female) and 42 CHC (mean age 71.2±4.50 and 73% female) were included. Thirty-eight participants were a-MCI and 18 were na-MCI. Groups were similar in age, co-morbidities, and history of falls. Amnestic-MCI participants walked slower than na-MCI in all test conditions (
Participants with a-MCI, specifically with episodic memory impairment had poor gait performance, particularly under dual-task. Our findings suggest that slow gait and higher stride time variability is a distinct ‘motor signature” in a MCI.
Many age-related health problems have been associated with dementia, leading to the hypothesis that late-life dementia may be determined less by specific risk factors and more by the operation of multiple health problems in the aggregate. Our study addressed: 1) how the predictive value of dementia risk varies by the number of deficits considered, and 2) how traditional and nontraditional risk factors compare in their predictive ability.
Older adults in the Canadian Study of Health and Aging who were cognitively healthy at baseline were analyzed (men = 2,902, women = 4,337). Over 10 years, 44.8% men and 33.4% women died; 10.2% men and 9.1% women developed dementia. Forty-two self-rated health problems, including, but not restricted to, dementia risk factors were coded as deficit present/absent. Variable numbers of potential deficits were randomly selected to construct an index of proportional presentation of the deficits.
Age-adjusted odds ratios per additional deficit were 1.27 (95% CI 1.23–1.34) in men and 1.16 (1.12–1.19) in women in relation to death, and 1.18 (1.12–1.25) in men and 1.08 (1.04–1.11) in women in relation to dementia. The index’s predictive value increased with the number of deficits considered, regardless whether they were known dementia risks. The index constructed using all the available deficit measures in the dataset best predicted death and dementia in both men and women (C-statistics: 0.71±0.02 for death; 0.67±0.03 for dementia).
The variety of items associated with dementia suggests that some part of the risk might relate more to aberrant repair process, than to specifically toxic results.
Older adults admitted to the intensive care unit (ICU) are seriously ill, but many are also frail. Here we evaluated the frailty of older ICU patients using a Frailty Index (FI) based on health deficit accumulation. We examined the FI in relation to short-term survival in a specialized geriatric ICU, in comparison with several prognostic ICU scores.
Geriatric ICU patients (aged 65+ years) at the Liuhuaqiao Hospital, Guangzhou, China, admitted between July and December 2011, were studied (
In a culturally diverse and ageing society like Canada, the development of cultural competency skills has become a necessity for health professionals. Research in cultural studies has given rise to effective methods and practices for working with ethnicities.
To develop communication and learning tools to assist health professionals to provide culturally competent care through effective communication with their patients.
Based on ethno-cultural and multicultural theoretical perspectives, we used communication tools in the published literature and developed new self-assessment and cultural understanding tools for cultural competency skill development. Eleven teaching videos based on our research in ageing ethnic communities were also incorporated.
This work resulted in an eight-module, structured course on cultural competency skill development. The tools utilized for cultural competency skill development include: 1) the Health Professionals Self-Assessment of Cultural Competency (HPSACC) Questionnaire—used for self-assessment of one’s own beliefs, values, and attitudes; 2) cultural understanding and insight into patients values, beliefs and attitudes—gained through the BRIDGES model; 3) the LEARN Model—facilitates intercultural communication; and 4) strategies for life-long learning are identified. The range of issues addressed by the videos include: (a) traditional roles of family care; (b) cultural issues in obtaining consent; (c) cultural issues in compliance; (d) language diversity in health care; (e) generational views on personal directives; (f) cultural issues in end-of-life; (g) challenging cultural norms; and (h) cultural influence in family decisionmaking. An evaluation component is also included.
The tools can be applied to a wide range of health professionals’ learning and provide effective methods for working with ageing populations.
The development of cultural competency skills is a life-long process that requires ongoing training and practice.
Family doctors play a central role in looking after the complex medical needs of growing population of seniors in Canada. The University of Alberta Family Medicine Residency training program is expanding its options for training a new generation of family physicians competent in providing care to the elderly in line with the College of Family Physicians of Canada Triple C curriculum and the mandate for more integrated experiences.
The traditional Care of the Elderly rotation is offered to the Family Medicine residents on their second year of training (PGY-2).The rotation includes 4 weeks at one of the acute care sites and provides residents with the combination of acute care and ambulatory experience under the preceptorship of the Care of the Elderly physicians. In 2011, a formalized Integrated Care of the Elderly option became available for PGY-1 residents. This option provides a longitudinal experience under the supervision of a family medicine preceptor and a Care of the Elderly mentor. It is enhanced by long-term care, community, and specialty clinic experiences during PGY-1 and PGY-2 training.
The enrolment: 2011–2012: 5 (8%), 2012–2013: 8 (13%), 2013–2014: 8 (13%). The study will compare the educational, clinical, and academic activities offered within the 4-week and integrated learning options.
In summary, there is an obvious interest in Integrated Care of the Elderly as a viable alternative to the more traditional 4-week rotation in geriatrics. More research is needed to assess the strengths and areas for improvement for both models to ensure that future family physicians have adequate skills and knowledge to look after Canada’s aging population.
Mild cognitive impairment is a growing public health concern, affecting up to 19% of the population aged 65 and older. Approximately 10% of those with MCI will convert to dementia every year. Thus, there is a focus on identifying effective strategies to prevent its progression to dementia and limit associated morbidity. Our systematic review aims to evaluate high-quality evidence on interventions for MCI.
We included randomized clinical trials (RCTs) and systematic reviews evaluating pharmacologic or nonpharmacologic interventions on patients 65 years of age or older with MCI. Studies were eligible if they were published in English, included a comparison group, and evaluated cognitive, functional, quality of life or safety outcomes. Studies that examined patients of normal cognition or dementia were included if outcomes of MCI patients were reported separately. Literature was obtained by performing a comprehensive search of MEDLINE, Embase, CINAHL, and Cochrane Central Register of Controlled Trials. Two reviewers independently assessed 6,078 titles and abstracts and 237 full-text articles for eligibility.
Thirty-two articles met our inclusion criteria and have been included for data-extraction and quality assessment using the Cochrane Effective Practice and Organization of Care (EPOC) risk of bias tool. Eight of the studies were systematic reviews and 23 were RCTs. Twenty studies investigated acetylcholinesterase inhibitors, including two that studied acetylcholinesterase inhibitors combined with antidepressants and one trial with a Vitamin E treatment arm. Two studies investigated other pharmaceuticals (Piribedil, transdermal nicotine). Five studies investigated natural supplements, vitamins or Chinese herbal remedies. Five studies investigated non-pharmaceutical interventions including brain stimulation, dietary interventions, and cognitive-training programs.
Various strategies for MCI have been evaluated. Data synthesis is underway and results will be available by March.
A gap exists between research and clinical practice in long-term care (LTC). While there is ample literature discussing interventions for dealing with behavioural and psychological symptoms of dementia (BPSDs), not all of these strategies are practical or easy to implement in a clinical environment. BPSDs are expressed differently in each person, which makes predicting problematic behaviours and finding appropriate solutions difficult. Sensory integration is one promising area of therapeutic interventions for BPSDs. This annotated literature review will provide staff with a useful summary of new and current interventions for addressing BPSDs in LTC.
A literature review of sensory integrative interventions designed to reduce BPSDs will be completed. Sensory systems that will be studied include proprioception, vestibular sensation, tactile senses, audition, and vision. Staff members with different clinical roles will also be interviewed. They will judge the utility of the interventions and be asked to comment or revise the approaches. Staff interviews will also uncover what are perceived to be barriers to implementing interventions in clinical practice. This project will expose new interventions and confirm the use of current interventions for ameliorating BPSDs.
Results are forthcoming. This will include a staff annotated literature review which will provide an up-to-date summary of practical interventions for use in LTC.
The transmission gap between evidenced-based interventions and clinical implementations in LTC needs to be addressed. This review will help staff make more informed care decisions and improve the quality of care they are able to provide. This review will also uncover areas where an organization can better support staff willing to implement evidence-based interventions.
The objective of the ViDOS cluster randomized trial was to evaluate the feasibility and effectiveness of a knowledge translation (KT) intervention aimed at integrating evidence-based osteoporosis/fracture prevention strategies in long-term care (LTC) homes. The target audience was interdisciplinary care teams (physicians, nurses, pharmacists, dietitians).
We randomized 40 LTC homes in Ontario, Canada to intervention (
At baseline, 5,478 residents, mean age 84.4 (SD 10.9) years, 71% female, resided in 40 LTC homes, mean size=137 beds (SD 76.7). Post-randomization, seven LTC homes declined to participate. Over 12-months, the mean home-level prescribing change for vitamin D ≥ 800 IU/day was 22.2% in the intervention arm vs. 7.5% in the control arm (between group difference = 14.7%, 95% CI: 13.1, 16.2). Mean home-level prescribing change for calcium ≥ 500 mg/ day was 8.8% in the intervention arm vs. 1.8% in the control arm (between group difference = 7.0%, 95% CI: 6.2, 7.9). There was no significant difference in prescribing between arms for osteoporosis medications.
Our KT intervention significantly improved prescribing of vitamin D and calcium, and is a model that could potentially be applied to other topics requiring quality improvement.
Vitamin D status is low in the elderly and especially in long-term care (LTC) residents. Low Vitamin D is associated with increased risk for osteoporotic fracture. Osteoporosis Canada recommends Vitamin D supplementation in all patients at high risk of fracture.
The purpose of this study is to evaluate the prevalence of Vitamin D supplementation in LTC residents, and the characteristics of those with and without supplements.
Ethics approval was obtained. Subjects were recruited from five LTC facilities in Edmonton, Alberta. After consent, chart review, clinical evaluation, and blood work was completed.
100 subjects (29 men, 71 women) were recruited with an average age of 81 years (range 59–93). Vitamin D levels ranged from 13–243 nmol/l, with an average of 75.1 nmol/l. 29% had levels below 50 nmol/l and 63% below 80 nmol/l. The rate of Vitamin D supplementation was 63% in total, but 48% in men versus 69% in women. Age, history of falls, history of fractures, MMSE, BMI, and calcaneal ultrasound were comparable in the two groups. The diagnosis of osteoporosis was higher in the supplement group (57% vs. 18%). The level of 25(OH)D was 113.5 nmol/l (75–243) in the supplement group and 46.7 nmol/l (13–69) in the non-supplemented group.
This study documents the high prevalence of Vitamin D insufficiency in this group of LTC residents. Men had lower Vitamin D status than women, and a lower level of supplementation. The risk of falls was high in those on and off supplements. This study highlights the need for further education on Vitamin D supplementation.
The Team Standardized Assessment of a Clinical Encounter Report (StACER) was designed for Geriatric Medicine residency programs to evaluate Communicator and Collaborator competencies. There are no studies of validity, reliability or acceptability in spite of its mandatory use. It is unknown whether a geriatrician’s assessment reflects the observations made by the interdisciplinary team.
To determine the inter-rater reliability of the items on the Team StACER among interdisciplinary team members and between geriatricians. Other objectives were to determine the face validity, discriminatory power, and acceptability of the instrument as a feedback tool.
Postgraduate trainees in Family, Internal, and Geriatric Medicine at the University of Toronto were recruited from July 2010 to November 2013. The Team StACER was completed by two geriatricians and interdisciplinary team members based on observations during a geriatric medicine team meeting. Raters completed a survey that was previously administered to Canadian geriatricians to assess face validity. Trainees completed a survey to determine the usefulness of this instrument as a feedback tool. Inter-rater reliability was determined using the Prevalence Adjusted Bias Adjusted Kappa (PABAK). Comments were reviewed by thematic analysis by two reviewers.
30 postgraduate trainees from three sites participated. A mean of 5.67 Team StACERs were completed per trainee, with 93% completed by two geriatricians. The PABAK range for Communicator and Collaborator items were 0.87–1.00 and 0.86–1.00, respectively. The instrument lacked discriminatory power, as all trainees scored “meets requirements” in the overall assessment. Face validity was limited by dichotomous choices. 93% and 86% of trainees found feedback based on the instrument useful for developing Communicator and Collaborator competencies, respectively.
The Team StACER has adequate inter-rater reliability, but poor discriminatory power. Trainees felt it provided useful feedback on Collaborator and Communicator competencies.
Medication use and prescribing in older adults can be challenging due to lack of geriatric-specific health information. Health professionals refer to product monographs for this information; however, there are situations where there is little information regarding older adults.
The purpose of this study was to examine the geriatrics-specific content in product monographs for products introduced to the Canadian market.
Products approved since 1994 were identified through Health Canada. Medications or biological products that could be used by older adults were included. Information related to the geriatric population was abstracted from the standard monograph categories: general information, pharmacokinetics, pharmacodynamics, precautions, warnings, adverse effects/events, dosage information, clinical trial inclusion, and geriatric specific clinical trials. Data were analyzed descriptively.
A total of 296 drug monographs were evaluated from 75 different drug manufacturing companies. Antineoplastic agents had the highest proportion of drugs studied, 36 out of 296 (12%). Two hundred eighty-two (95%) included general information on the geriatric population; 147 (50%) had information on dosing in the geriatric population. Inclusion of reference to geriatrics in the section on clinical trials was noted at 173 (58%), whereas only 11 (4%) included specific details about geriatric-focused clinical trials. The majority (200 [67.6%]) included geriatric pharmacokinetics content, and 6 (2%) included pharmacodynamics content. The adverse effects information specific to geriatrics was found in only 31 (10%) of the product monographs.
Many product monographs include general precautions about drug use in the elderly. However, disclaimers and encouraging prescribers to use caution, or noting that information is not available, is inadequate to support safe and effective drug use in seniors.
Most drug product monographs in Canada have limited information that is specific to the elderly population.
Two commonly used cognitive screening tools in Canada are the Folstein Mini-Mental State Examination (MMSE) and the Montreal Cognitive Assessment (MoCA). In 2009, Brown et al. created a new cognitive screening test called the Test Your Memory (TYM), which is unique in the fact that it is a patient self-administered exam. In a system where family physicians and other specialists are pressed for time, the TYM offers a potential to save screening time.
This study aimed to determine the validity of the TYM tool in comparison to the traditional MMSE and the MoCA in a Canadian Geriatric Assessment clinic setting.
Patients aged 65 and older attending a regularly scheduled appointment at one of three geriatric clinics in Edmonton, Alberta were invited to participate in the study. Participants had to complete the self-administered Test Your Memory tool in addition to the MMSE ± the MoCA as part of their geriatric assessment.
A total of 36 participants completed the study. The Pearson correlation coefficient between the TYM and MMSE is R2 = .689 and the coefficient between the TYM and the MoCA is R2 = .508. Scores for the TYM had statistically significant correlation to the MMSE and MoCA, but sensitivity, specificity, and positive predictive value of the TYM was poor.
This study was not able to validate the TYM as a good clinical cognitive screening tool. The greatest value of the TYM may be in its negative predictive value.
The proactive geriatric trauma consultation service started at St. Michael’s Hospital (SMH) in September 2007. Patients aged ≥ 65 years admitted to the Trauma service are referred to the GTCS for a comprehensive geriatric assessment within 72 hours. The GTCS includes a geriatrics nurse specialist and geriatrician. Recommendations focus on early involvement in prevention and management of age-specific complications, and discharge planning. In a previously published before and after case series, we demonstrated a significant decrease in delirium, consultations to Internal Medicine and Psychiatry, and discharge to long-term care.
To determine the sustainability of impact of a proactive GTCS.
Patients aged ≥ 65 years admitted to the Trauma service from July 2012 to December 2013 were prospectively recruited for this sustainability case series and received a proactive geriatric trauma consultation. Geriatric specific in-hospital complications, trauma quality indicators, discharge destination, rate of recommendation adherence, and consultations were determined. Proportions were compared using the Fisher exact test.
138 patients were identified and 82 (59.4%) patients consented to participate. 66 charts have been reviewed thus far. Compared to the post-intervention phase of the case series, there was no difference in the sustainability phase in Internal Medicine consultation (
Decreases in delirium, consultations to Internal Medicine and Psychiatry, and discharge to long-term care were sustained outcomes in this proactive geriatric trauma consultation model. A prospective interrupted time-series study is underway at a second site, Sunnybrook Health Sciences Centre, and will guide the feasibility and generaliz-ability of impact on quality outcomes.
Understanding the awareness of fall risk factors by adults is an important start to educational interventions. Since its development in 2002, the Falls Risk Assessment Questionnaire (FRAQ) has been administered in older adults to assess awareness and perception of falls risk.
The objective of this pre/post study was to further validate the FRAQ as being responsive to change.
Seniors (≥ 65 years) who attended one of six standardized 1-hour falls education programs in the community offered by Alberta Centre for Injury Control and Research from Sep-Dec 2013 were recruited. The presentation focused on fall risks and prevention strategies. Each participant completed the FRAQ before and after the presentation. The FRAQ is a 22-item questionnaire that assesses the knowledge of risk factors (medical, environmental, pharmacologic, physical) for falling, as well as demographics, medical, and fall history, and risk factors. A score of 1 was given for each correct answer, with a summative score of 32.
Of 46 seniors who completed the survey, 36 completed both the pre- and post- measures. The mean age was 84.7±6.6 years; 83.7% (36) female. Arthritis or rheumatism was the most commonly reported condition (55.8%,
Initial results indicate responsiveness of the FRAQ in assessing falls risk awareness is congruent with increased knowledge and awareness.
Baycrest, the Toronto Central and Central Community Care Access Centres and North York General Hospital (NYGH), together with the Regional Geriatric Program, have established a model of integrated care for frail, older adults who are at high risk for ED visits, hospitalization, and institutionalization. The model, “The Integrated Community Care Team (ICCT)”, connects older adults living in north Toronto to a dedicated, inter-professional team consisting of primary, community, and specialty care resources. The ICCT model is unique in that it is not only client/family caregiver-centred, but it also tailors its services to the specific needs of community primary care physicians through a consultation, shared care or transfer of care approach. The ICCT model is further enhanced through a link with NYGH and Baycrest’s in-patient specialty services to ensure that major components of the patient journey across the continuum of care are integrated as part of the intervention.
Its dual aim is to support medically frail older adults to experience care from one team and to support community primary care physicians with an integrated interdisciplinary team.
An early phase mixed methods formative evaluation of the ICCT model is under way. The design emphasizes a quality improvement approach with the aim to provide continuous rapid-cycle improvement to the team. The quantitative evaluation will include data from the Resident Assessment Instrument–Home Care to describe the patient population, process measures to document service provision, the Zarit Burden Interview to capture caregiver burden, and the Dimensions of Teamwork Survey to evaluate inter-professional team function. The qualitative evaluation includes interviews with patients and their caregivers, primary care physicians, and team members to document the implementation experience and satisfaction with the model.
Dr. David S. Gold-bloom was born in Montreal and raised in Quebec and Nova Scotia. He completed an honours degree, majoring in Government, at Harvard University and then attended the University of Oxford as a Rhodes Scholar where he obtained an M.A. in Physiological Sciences. He trained in medicine and psychiatry at McGill University and is a Professor of Psychiatry at the University of Toronto. Dr. Goldbloom’s activities have been recognized and awarded by his peers and students. He has authored numerous scientific articles and book chapters and has provided talks and lectures to student, professional, and public audiences. He maintains an active clinical and teaching role at the Centre for Addiction and Mental Health where he serves as Senior Medical Advisor. He is also Chair of the Mental Health Commission of Canada. In addition to his professional activities, Dr. Goldbloom is the immediate past Chair of the Board of Governors of the Stratford Shakespeare Festival of Canada.
To become familiar with the mission, structure, and work of the Mental Health Commission of Canada (MHCC).
To understand the specific initiatives of the MHCC with regard to seniors.
To consider how attendees can contribute to these mental health reform efforts.
Dr. Lenze is Professor in the Department of Psychiatry at Washington University School of Medicine. Dr. Lenze completed medical school and psychiatry residency at Washington University School of Medicine. He trained in a geriatric psychiatry fellowship at University of Pittsburgh, and then underwent a research fellowship at the Late-life mood disorders center at University of Pittsburgh.
Dr. Lenze’s research focuses on treatment development and testing in anxiety disorders and depression. He has led numerous clinical trials and treatment development studies of pharmacological and behavioral treatments for depression and anxiety disorders. He is author on over 130 peer-reviewed articles, reviews, and book chapters. Dr. Lenze is also a practicing geriatric psychiatrist who focuses on the management of anxious and depressed patients.
Dr. Joel Sadavoy is professor of psychiatry and member of the Institute of Medical Sciences and Neurosciences program at the University of Toronto. He is head of Geriatric and Ethno-cultural Community Psychiatry, and founder and Director of the Reitman Centre for Alzheimer’s Support and Training at Mount Sinai Hospital where he is the inaugural holder of the Pencer Family Chair in Applied General Psychiatry and the immediate past Psychiatrist-in-Chief. His key academic interests have been in service development research and implementation, most recently in the area of family care-givers of persons with dementia, ethno-cultural psychiatry, and psychotherapy of the elderly. He is the founding past president of the Canadian Academy of Geriatric Psychiatry, and a past president of the International Psychogeriatric Association.
Dr. Rapoport is an associate professor in the department of psychiatry at the University of Toronto, and is an associate scientist at Sunnybrook Research Institute. He is also a member of the Canadian Driving Research Initiative for Vehicular Safety in the Elderly (CanDRIVE), and an associate editor of the
This symposium application highlights the development of novel, cutting-edge biomarkers using neurophysiological and neuroimaging approaches that are relevant for geriatric populations. The presenters will show how these novel biomarkers have been developed in older populations. The practical utility of these biomarkers to track symptom or disease progression, understand treatment effects, and predict treatment response will also be highlighted.
First, Dr. Daniel Blumberger will introduce transcranial magnetic stimulation (TMS), which is a non-invasive form of brain stimulation that capitalizes on the ability of time-varying magnetic fields to induce electrical currents in biological tissue via the principle of electromagnetic induction. TMS can measure dysfunctional cortical inhibition (CI) and neuroplasticity, which have been postulated as a neuro-physiological mechanisms involved in treatment resistant depression (TRD). These paradigms also have potential to predict response to existing psychopharmacological treatments. Neurophysiological data from three groups will be presented (i.e., from non-depressed older adults, depressed older adults without treatment resistance and depressed older adults with treatment resistance.) Neurophysiological predictors of response to treatment will also be shown, highlighting the utility of TMS neurophysiology paradigms as biological predictors of treatment response and illness severity.
Second, Dr. Tarek Rajji will discuss deficits in neuroplasticity in patients with schizophrenia across the adult lifespan. Deficits in working memory performance are a core feature of people with schizophrenia across adult life. Modulation of gamma oscillations amplitude by theta oscillations phase (“theta–gamma coupling”) is thought to mediate working memory in the dorsolateral prefrontal cortex (DLPFC). Dr. Rajji will describe the results of a study which assessed deficits in coupling in response to a brain stimulation intervention delivered to the DLPFC in healthy people and people with schizophrenia, using a paired associative stimulation (PAS) paradigm. He will show that people with schizophrenia are impaired compared to the healthy subjects who also received PAS on PAS-induced potentiation of cortical evoked activity or coupling, compared with a PAS control intervention. Dr. Rajji will then describe how PAS paradigms can be used to improve brain plasticity and working memory.
Third, Dr. Aristotle Voineskos will describe a novel diffusion tensor imaging (DTI)-based approach that can index biomarkers of normal and pathological aging. While DTI studies have shed light on white matter microstructural abnormalities associated with aging, the focus has been on deep white matter (DWM) tracts, the long-range connections between brain regions. We now have a method to measure superficial white matter (SWM), which can index local brain connectivity, and may be important for cognitive function. Dr. Voineskos will describe results from a large study of normal aging individuals across the adult lifespan who underwent DTI and a full cognitive battery. Areas of SWM susceptible to aging and areas predicting cognitive performance will be described. The results of this study show that SWM declines in integrity with age, and could be a key neural substrate for age-related cognitive decline. SWM should be considered as an early outcome biomarker in clinical intervention studies aimed at improving or preserving cognitive performance in late-life.
Finally, Dr. Benoit Mulsant, the symposium discussant, will provide perspective regarding biomarker utilization in clinical trials and interventions, particularly in light of new position statements from major funding agencies expecting the use of biomarkers in such studies.
The DSM-5 was published in May 2013 after six years of literature reviews, input from experts, field trials, secondary data analyses, and public commentary. Changes were guided by clinical utility (e.g., decreasing diagnosis of over-inclusive diagnoses; drawing attention to under-recognized conditions; reducing stigma); research evidence; maintaining continuity with DSM-IV; and harmonization with ICD-11. The DSM-5 promotes a lifespan perspectives with chapters ordered based on when disorders manifest in life. Changes that impact older individuals include eliminating the 2-month wait before diagnosing a major depressive disorder following bereavement; listing hoarding disorder as a new disorder and persistent complex bereavement disorder as a condition requiring further study; classifying agoraphobia separately from panic disorder; and reorganizing the cognitive disorders, now called neurocognitive disorders (NCD). To decrease stigma, dementias and MCI are called major and mild NCD. Both are subtyped based on 13 probable or possible etiologies and associated with specifiers indicating behavioural disturbances (e.g., psychotic symptoms, mood disturbances, agitation, or apathy) and severity (i.e., functional dependence). The DSM-5 acknowledges that: boundaries between normal cognition, mild NCD, and major NCD are arbitrary; substantial decline in only one cognitive domain is needed to diagnose major NCD, making it broader than dementia; the importance of neuropsychological assessment; and the potential utility of biomarkers (e.g., genetics, neuroimaging).
Transcranial direct current stimulation (tDCS) has been investigated in psychiatry since the 1960s. The last ten years have seen a resurgence of interest in this modality as a potential treatment for depression. Recent data on the efficacy of the tDCS in depression will be presented and the implications for this treatment in late-life depression and cognitive impairment will be discussed. Electroconvulsive therapy (ECT) remains the most effective treatment for severe and refractory depression. Recent advances in parameters of ECT will be presented as a background to a newer form of convulsive therapy. Magnetic seizure therapy (MST) has been investigated in small studies as a potential alternative to ECT as there is a significantly better cognitive adverse effect profile. Data on the efficacy and cognitive adverse effect profile of MST will be presented from a series of older adults with depression.
The goal of this interactive solution-based workshop is to enhance knowledge, confidence, and skills of practitioners to better prepare for and manage the disclosure of a diagnosis of dementia to older persons and their family caregivers.
The aim of this interactive workshop is to provide practical and evidence-based recommendations for a person-centred, progressive, and comprehensive approach to dementia diagnosis disclosure to assist practitioners to adequately prepare and manage this critical encounter.
Dementia is one of the most feared diagnoses by older adults, and the diagnostic process is one of the most fundamental elements in the experience of dementia. Many practitioners admit having difficulty disclosing a diagnosis of dementia, and identify communication about the diagnosis as one of the most difficult aspects of dementia care.
The disclosure of a diagnosis of dementia is an evolving and dynamic process, involving: a) pre-disclosure evaluation and preparation, b) timely, individualized, honest, and sensitive disclosure, and c) post-disclosure follow-up educational and supportive interventions. Practical tips will be provided to meet the two intertwined key needs of patients/caregivers at each step during this process: their information/educational needs and their emotional/support needs. These include recommendations to develop rapport, explore perceptions, help gain insight, maximize comprehension, respond to emotional reactions, and foster a sense of hope and meaning. There will be opportunity for the participants to: a) work on a case study; b) reflect on their practice; c) relate the content to their realities; d) exchange ideas and experiences; e) solve problems; f) develop communication strategies. Handouts and educational resources will be provided.
It is hoped that this approach will help practitioners better prepare for this encounter in order to optimize their patients’/caregivers’ responses.
To provide a practical overview of the clinical and research utility of neuroimaging in cognitive disorders.
This is an interactive workshop that provides an overview of the current evidence for the clinical and research utility of key neuroimaging modalities in patients with cognitive impairment in old age.
Speakers in this workshop were among the neuroimaging expert group that reviewed and summarized the evidence and made recommendations to the CCCDTD4 regarding the clinical and research utility of various neuro-imaging modalities in cognitive disorders. This work was accepted for publication in two review papers which the audience can access for more details.
To optimize the outcomes of pharmacotherapy when treating older patients with late-life depression.
Depression is the most prevalent treatable psychiatric disorder in late life. An increasing number of older persons are being treated for depression, and pharmacotherapy with antidepressants is the main modality used by psychiatrists and by family physicians. More than 70 placebo-controlled trials and meta-analyses of these trials support the efficacy of these medications guidelines based on this body of evidence have been published and disseminated. However, up to half of older depressed patients do not receive adequate pharmacotherapy and most do not benefit from it. In this workshop, the panelists will discuss the clinical relevance and applicability of the evidence supporting the benefits and the risks of antidepressant and other psychotropic medications in the treatment of late-life depression. They will also discuss the relative merits of a systematic (“algorithmic”) versus an individualized approach to the pharmacotherapy of late-life depression. The audience will be invited to participate in these discussions. At the conclusion of the workshop, participants will have improved their ability to optimize outcomes of pharmacotherapy when treating patients with late-life depression.
To learn an evidence-based approach to the prevention and management of medical effects associated with psychotropic medication use in late-life mood disorders.
In this session, we will review the available literature regarding medical effects of psychotropic medications commonly used in older adults with mood disorders. We will also present data from Canadian studies, including some of our own work in this area.
To evaluate the advantage of artificial intelligence fall detection in conjunction with traditional clinical monitoring.
This presentation will summarize risk event and falls data, and describe the role of artificial intelligence in detecting and monitoring falls in this population.
Due to multiple intrinsic and extrinsic risk factors, patients in psychogeriatric hospitals are frequently at higher risk for falls. Regardless of whether or not an injury occurs, once a patient has experienced a fall, the care team, as well as the individual, fear recurring falls which then can lead to decreased activity and loss of strength and mobility, resulting in increased risk of falling and reduced quality of life.
The study goal was to evaluate the impact of the HELPER (Health Evaluation Logging and Personal Emergency Response) system in a tertiary-care psychiatric hospital in London, Ontario, Canada. Developed by Dr. Alex Mihailidis and collaborators at Toronto Rehabilitation Institute, HELPER (
To facilitate the ease of setting up a clinical geriatric psychiatry telemedicine service.
This workshop is intended to be a practically oriented look at issues and considerations involved in setting up a geriatric psychiatry telemedicine service. It is aimed at anyone considering involvement on either side of the consultation process—specialists, family doctors, primary care staff, and those responsible for commissioning such services.
Canada has many rural and remote communities where accessing specialist services is a major barrier to health-care provision. Telemedicine offers a solution to this by allowing such communities to have synchronous contact with specialists at a remote site, removing or reducing significantly the need for patient or specialist to travel. Recent literature has demonstrated the effectiveness and tolerability of such services to users and providers.
The workshop will use a didactic introduction and literature review, plus significant time in small group discussions of issues in the planning and operational stages of service development, with review and summary in the larger group. The evolution of the Waterloo-Wellington Geriatric Psychiatry clinic will be used as a framework to stimulate discussion.
To selectively review international experience of psychiatrists with matters of euthanasia and physician-assisted suicide. To appreciate the challenges posed to psychiatrists by the possible legalization of “medical aid in dying” in Québec, and other recent legal developments in Canada.
To selectively review international experience of psychiatrists with matters of physician-assisted suicide/euthanasia, and to appreciate challenges posed to psychiatrists by the discussed legalization of “medical aid in dying” in Québec.
Euthanasia and physician-assisted suicide have been legalized or tolerated in a few jurisdictions over the past half-century. This impacts predominantly patients of geriatric age. The introduction of “medical aid in dying” legislation in Québec was discussed in a National Assembly Select Committee, which released its report in 2012. A bill implementing “medical aid in dying” was presented in June 2013 to the Québec National Assembly. The reports published in Québec recommend that psychiatrists be consulted to assess patients requesting “medical aid in dying” when competency is debated, or when the impact of mental illness or cognitive impairment on patients’ decision-making is unclear.
We selectively review international psychiatric experience with assessment of patients requesting assisted dying. We review challenges pertaining to the assessment of competency in this population. We also review ethical challenges these assessments pose to psychiatrists.
The involvement of psychiatrists in other jurisdictions has been rather limited. The proposed implementation of “medical aid in dying” will present challenging issues to psychiatrists, particularly in assessing competency in patients requesting assisted dying.
Improve knowledge about teleconsultation programs for BPSD in order to stimulate implantation of that kind of service in Canada and increase accessibility to experts in this domain.
In 2011, it was estimated that about half a million Canadians were suffering from dementia, about 60% of Alzheimer’s disease. Behavioural and psychological symptoms of dementia (BPSD) are highly prevalent, to the point that about 80–97% of patients with dementia will present some kind of BPSD in their evolution. However, patients and families are still confronted with difficulties to access expert’s opinions and services. Caregivers have a lack of education in this field and are often not skilled to intervene appropriately when BPSD arise. This situation is associated with suboptimal care and overuse of medication, mostly antipsychotics, even though the risks linked to this medication are well known. Numerous studies indicate the feasibility of assessing the elderly people’s cognition via teleconsultation and establish the efficacy of teleconsultation on behaviour of patients with BPSD.
With this in mind, the BPSD team at Institut Universitaire de gériatrie de Montréal (IUGM) developed a program of telepsychogeriatry (teleformation and teleconsultation) for BPSD with two partners located in distant areas of the province of Québec. We will describe the operating procedures and some results.
This kind of program is efficient in educating caregivers about BPSD and in increasing quality of care for these patients. We believe that it is easily transferable to other partners, and is an easy way to increase accessibility to expert opinion in the evaluation and management of BPSD.
To present four years of data collected from 2009 to 2013 on patients over ninety years of age who presented to Senior’s Mental Health in Thunder Bay. The patients come from seven different points of referral, have the full spectrum of psychiatric diagnoses, and may be growing faster than other age groups heralding a greater need for geriatric services not previously seen.
The North West LIHN is Ontario’s largest LIHN geographically, covering 45% of Ontario’s land mass and home to a quarter of a million inhabitants, many living on remote reserves north of the largest city, Thunder Bay (population 120,000). The North West LIHN projections for people over the age of sixty-five in 2010 were 35,000 individuals, of whom 1600 were over ninety. Data exist on the numbers that presented to Senior’s Psychiatry, point of referral, and diagnosis from the 2009 to 2013.
We wish to present information comparing the general rise (i.e., over sixty-five) in referral rates to the specific rise in the over-ninety-years-of-age group and explore the future implications in terms of resources and expertise issues. Furthermore, we wish to explore the range and categories of diagnoses across the over-ninety age group of presenting seniors in that same four-year span. By 2030, the number of individuals over ninety will almost double in North Western Ontario LIHN. We are particularly interested in determining if the percentage of presenting patients over ninety will remain the same or stabilize at some percentage, and the impact and demands on Senior’s Mental Health. What is unknown is what other LIHNs are experiencing in terms of the oldest of the old and the wider view of this as an emerging concept on its own. In addition, it is increasingly apparent that there is considerable risk in providing pharmacological treatment to this group, even in the absence of dementia.
To disseminate recent findings from a novel study examining knowledge translation of dementia best practices in a primary care setting.
Alzheimer’s disease (AD) and related forms of dementia are common in primary care settings. The quality of care (QoC) provided by primary care providers (PCP) to older adults with AD could be improved through adherence to best practices identified in dementia guidelines. This project evaluates the effects of a guideline knowledge translation intervention with PCPs to improve the QoC of older adults with AD in primary care.
PCPs in seven locations in south-east Ontario are participating in the Primary Care—Dementia Assessment and Treatment Algorithm Project (PC-DATA). The intervention consists of an educational session introducing the DATA tool and Web-based resource supported by a dementia care manager to optimize adherence and uptake of the DATA protocols. A booster session will occur six months following the initial education session. The primary outcome for the study will be the change in the number of QoC process quality indicators achieved by PCP in the three years preceding the intervention, compared to the year following the intervention, using chart audit reviews. Selection of content for the DATA tool and website has been informed by literature reviews and focus groups with PCP, and persons with dementia and their caregivers to identify and address knowledge gaps in the current system of dementia care. The evaluation of education sessions will be presented. The roles and activities of the dementia care manager during the initial implementation will be described, along with a profile of individuals evaluated by the dementia care managers. The PC-DATA project is a novel collaborative approach to the provision of care for the growing population of older adults with AD. Through this project we hope to improve the QoC for older adults with AD, and provide greater access to supports and services for PCPs caring for this population.
To examine the CPAP-ADH in elderly patients in a mental health (MH) and non-mental health (NMH) setting and elucidate possible contributing factors associated with CPAP adherence.
Obstructive sleep apnea (OSA) is a common sleep disorder with significant medical and psychological consequences. The prevalence of OSA is high in adults over sixty, up to 37.5–62%. Continuous positive airway pressure (CPAP) therapy is an effective first-line treatment in adults with OSA. However, a number of studies have reported CPAP adherence (CPAP-ADH) as a problem in both younger and older adults.
Demographic, baseline polysomnography and CPAP-ADH data from 2009–2012 were retrospectively collected for 464 patients (389 from NMH and 75 from MH setting), sixty-five years of age and older who have been referred from Ottawa area sleep disorders clinics. Adherence data has been downloaded from CPAP machines loaned to patients during their initial 30 day trial.
Preliminary analysis using Spearman Correlation Coefficients showed that in NMH setting, age is negatively correlated to CPAP-ADH (cumulative usage
This suggests that different factors contribute in CPAP-ADH in the elderly patients referred from a MH versus NMH setting. We speculate that underlying co-morbid medical and mental health issues and other psychosocial factors (e.g., marital status, living arrangements) play a key role in CPAP-ADH.
Cognitive deficits are among the strongest prediction of function in patients with schizophrenia. There have been no pharmacological interventions that have been shown to improve cognitive function in patients with schizophrenia. Age-related deficits only serve to compound cognitive challenges. A number of studies have shown benefit from cognitive remediation (CR) in improving cognitive function in patients with schizophrenia. To date, the efficacy of cognitive remediation in the geriatric patient population with schizophrenia remains to be studied. We have been adapting cognitive remediation to focus on the cognitive and functional aspects of schizophrenia that are particularly impaired and relevant in late life. The ultimate goal is not only to improve cognition but also to facilitate or “bridge” this improvement in the patients’ daily lives.
Our work targets improving executive function and social cognition using an open-label, non-controlled study of 20 older patients with schizophrenia, aged sixty or above. Techniques used include weekly two-hour group treatment sessions including computerized drills, in-class strategic monitoring, with an emphasis on bridging cognitive skills practiced in-session to everyday life. Subjects work on homework drills throughout the week to consolidate the concepts and skills practiced during the weekly sessions. Baseline and follow-up clinical, cognitive, and functional assessments are conducted before and after the intervention.
Baseline and follow-up clinical, cognitive, and functional assessments are conducted before and after the intervention. We have just completed our first patient cohort.
The goal of cognitive remediation is to improve basic and higher order aspects of cognition, such as attention (including its subcomponents of selective attention and sustained attention), working memory, processing speed, declarative learning, and executive functions. The ultimate goal is not only to improve cognition but also to facilitate or “bridge” this improvement in the patients’ daily lives. To date, the efficacy of cognitive remediation in the geriatric patient population with schizophrenia remains to be studied. Should this pilot project prove to be successful, we plan on testing CR in a randomized controlled design.
To decrease the burden associated with mental/cognitive illness, and to encourage seniors to maintain a sense of meaning when facing challenging end-of-life issues
As Nova Scotia has the highest provincial percentage of seniors per capita in Canada, the potential for increased demands on the health-care system has reached unprecedented levels. In order to mitigate the impending repercussions of the “silver tsunami”, there remains a distinct paucity of primary prevention programs targeted towards the elderly. The Fountain of Health is an initiative currently in the second phase of a ten-year project targeted towards health promotion for seniors. The aim is to provide support in reducing their risk of long-term illness and disability through the dissemination of six key evidence-based messages throughout Nova Scotia. The goal is to increase awareness in the elderly and pre-senior population of concrete strategies to improve their own health outcomes, in addition to enhancing collaboration and communication among caregivers.
Six key evidence-based messages were identified for dissemination including positive aging, social activity, anti-ageism, physical activity, mental health, and lifelong learning, with the author playing a role in message development and data analysis of surveys released at three points during the year-long phase. Current literature on knowledge translation using a Seniors Mental Health network was reviewed, and a strategy for information dissemination to health-care providers has been developed, with the intention of completing a quality assurance initiative in conjunction with the educational component of the Fountain of Health.
Identify medications that may no longer be appropriate in end-stage dementia. Identify nutritional supplements that may be contributing to overall health burden in end-stage dementia.
Elderly nursing home residents often have multiple medical comorbidities and are prescribed numerous medications. With the use of more medications comes the risk of adverse drug reactions due to pharmacokinetic and pharmacodynamic changes, as well as drug interactions. Previous studies have found a relation between polypharmacy and a higher number of care problems (falls, pain, etc.). There are various criteria regarding medications that are potentially inappropriate in the geriatric population, such as the Beers criteria; however, there is less known about the use of medications and nutritional supplements, which are generally not considered harmful but may no longer be providing benefit, and which may be worsening quality of life.
After appropriate ethics approval, we conducted a chart review on nursing home residents with advanced dementia (Fast Stage 7) living on dementia units at four nursing homes. De-identified data were sent to a clinical advisory team consisting of a pharmacist, a specialist in the use of nutrient supplements, a family physician with expertise in the care of the elderly, and a geriatric psychiatrist. The advisory team members completed standardized questionnaires regarding the appropriateness and potential problems with each medication and nutritional supplement, taking into consideration a clinical summary (prepared by the first author) on each study participant. Results were summarized by the first author.
Many vitamins were prescribed at excessive doses, while other recommended vitamins were not prescribed at adequate doses, or frequency. Reasons for administration of PRN medications were often not specified, contributing to the risk of prescribing of those medications for inappropriate reasons.
To provide an update on the current literature concerning the possible effects of antidepressants on driving in senior populations.
The potential effects of psychoactive drugs on motor vehicle operation have been investigated perfunctorily in recent literature, but many studies focus on a young or middle-aged population. Many seniors are diagnosed with depressive disorders, for which antidepressant drugs are commonly prescribed medications. The increasing number of senior drivers makes the possible consequences of antidepressant use on driving skills in a senior population a pressing issue.
A systematic review was conducted using MED-LINE, targeting articles specifically pertaining to antidepressants and driving. Relevant articles were dichotomized into epidemiological or experimental categories. These were subsequently filtered for articles focusing on an elderly population or subgroup (≥ 55 years of age).
An initial search yielded 233 references, thirty-six of which attended to the effects of antidepressants on driving. Of these thirty-six, one was experimental and six were epidemiological studies which assessed a senior population. The experimental study focused on imipramine and nefazodone, concluding that imipramine was detrimental to standard deviation of lateral position for highway driving. The epidemiological studies assessed a wide range of antidepressant drugs—often in addition to other psychoactive medications—and generally indicate that most of the antidepressants investigated have significant negative effects on driving performance.
Results of this review highlighted a dearth of knowledge concerning the effects of antidepressants on driving performance in the elderly. Few conclusive statements are made in the existing literature, and there is a growing need for further research in this area.
Older adults frequently present with co-morbid mood and cognitive symptoms, posing a diagnostic challenge for clinicians to differentiate late-life depression (LLD) and amnestic mild cognitive impairment (aMCI), a prodrome of Alzheimer’s disease. This study compared functional connectivity (FC) between LLD and aMCI using resting-state functional magnetic resonance imaging (rsfMRI).
Eleven non-depressed aMCI, six LLD, and nine healthy comparison subjects (HC) (age M = 70 years, SD = 5; ten males, sixteen females), all free of psychotropic medications and neurological disorders, were scanned at 3T for 10 minutes at rest (eyes closed, alert state). Neuroimaging data were analyzed by Partial Least Squares (PLS) with a left posterior cingulate cortex (PCC) seed to identify patterns of FC common and distinct across groups. The Kruskal-Wallis test was used to test differences in the patterns of FC to the PCC amongst groups.
Seed PLS identified a common pattern of FC to the PCC that included the right medial prefrontal cortex and right inferior temporal gyrus (
The common pattern suggests similarities in default network connectivity across groups. Greater connectivity to the OFC in LLD relative to aMCI or HC suggests that the affective network in LLD may be active even at rest, possibly accounting for emotion dysregulation in depression. Larger sample sizes are needed to confirm these initial observations and the clinical utility of rsfMRI.
Numerous studies have documented that family members taking care of individuals with Alzheimer’s disease and related dementia (ADRD) experience high levels of stress, which are disproportionately associated with risk of depression and various physical symptoms and disorders.
In this study, the focus group methodology was employed to obtain information directly from five adult children and five spouses about their role as caregivers for family members with ADRD.
Upon framework analysis of the transcribed focus group discussions, seven major themes were identified: Meaning of Caregiving and Caregiver Self-Definitions, Attempts to Fulfill Self-Expectations and Perceptions, Making Sense of Changes in the Relationship, Satisfactions Experienced by Caregivers, Frustrations Experienced by Caregivers, Needs of Caregivers, and Support Available to Caregivers. These themes were organized into a dimensional matrix that highlights their interconnectedness and dynamic nature.
The rich narrative gave researchers, clinicians, and policy-makers insights into the emotional and psychological challenges that caregivers encounter both in their relationships with the ADRD individuals, and their experiences and engagement with supportive services and community programs. This information may be utilized to help guide the design and delivery of appropriate programs to support them meaningfully in the task.
Collaborate with other educators to research regarding psychoeducation.
Psychoeducation is an extremely valuable tool in our interaction with patients. In geriatric psychiatry, psychoeducation is a valuable and cost-effective intervention for treatment of psychiatric illness. Given the increased population of geriatric patients, it is essential that we empower those who provide care. Over the course of the next two years of our subspecialty training, we will provide wider access to standardized psychoeducation modules targeted to groups who care for geriatric psychiatric patients.
We wish to design appropriate seminar series to multiple groups: family physicians, family members, and staff of long-term care facilities. The University of British Columbia Psychiatry program has three current training sites for subspecialty residents (Vancouver Coastal Health, Fraser Health Authority, and Vancouver Island Health Authority). Our project will connect these three health regions in order to provide wider access to psychoeducation in British Columbian long-term care facilities. The Royal College CanMEDS roles are the cornerstone of resident education. CanMEDS roles we will specifically fulfill are: advocate, communicator, manager, and collaborator. Being able to distill information (communicator) in a succinct manner to caregivers while advocating (advocate) for better care of these patients is our main goal. We strive to provide a service that will provide more efficiency to the burdened health-care system (manager). Through this CAGP poster presentation, we hope to collaborate (collaborator) with others in Canada who have similar interests in psychoeducation and gather insight from their experiences.
To describe how we successfully treated five patients with disruptive vocalization using ECT.
There is emerging evidence that electro-convulsive therapy (ECT) can help with the behavioural and psychological symptoms of dementia. One of the most distressing behavioural symptoms of dementia is disruptive vocalization. Previous small case series have suggested that antidepressants and ECT can be beneficial for this distressing condition.
A retrospective chart review of the five patients with dementia of mixed etiologies was conducted comparing pre- and post-treatment using the Cohen Mansfield Agitation Inventory. All five patients had unsuccessful treatments with non-pharmacologic methods and pharmacotherapy including antidepressants.
After completion of a series of ECT, the reduction in the verbal agitation score went from 6.8 ± 0.5 to 2.3 ± 1.0 (
The diagnosis of dementia is important although cognitive tests can be time consuming, which may serve as a barrier to timely assessment of cognitive complaints. The Mini-Cog has been proposed as a quick and accurate cognitive test that may be suitable in a range of health-care settings. In the current study, we evaluated the diagnostic test accuracy (DTA) of the Mini-cog in community, primary care, and tertiary care settings.
Electronic databases were searched through the Cochrane Dementia and Cognitive Improvement group. We included all studies that evaluated the Mini-Cog when compared to reference standard dementia diagnostic criteria. Information on sensitivity, specificity, predictive values, and likelihood ratios were derived from individual studies. Random effects bivariate meta-analysis was used to determine pooled sensitivity and specificity. The quality of studies was assessed using QUADAS 2 criteria.
A total of 11 studies (three community setting, four primary care, and four tertiary care) were identified, including a total of 4,622 participants. In meta-analysis combining all study settings, the pooled sensitivity of the Mini-Cog was 87.6% (95% CI): 71.3% to 94.9%,
The Mini-Cog is a relatively accurate brief cognitive test that may be useful in a number of health-care settings. Further information is required in order to compare accuracy of the Mini-Cog to other brief cognitive tests.
To report findings of a study that aims at determining which clinical factors predict rehabilitation success.
More than half of community-dwelling seniors will experience at least one disabling medical event, such as hip fracture or stroke, within a four-year period. Given that successful inpatient rehabilitation is a major determinant of long-term functional outcome, knowledge of factors predictive of rehabilitation success is critical. In the current study, we examined mood, and cognitive and physical factors at admission, as well as patients’ level of participation during rehabilitation, and their impact on functional outcome.
Patients (N = 50, 31 F, M age = 82 years, SD = 10.2) admitted to a four-week geriatric high-tolerance rehabilitation unit were assessed at admission using the Geriatric Depression Scale (GDS), State Trait Anxiety Inventory (STAI), Montreal Cognitive Assessment (MoCA), Visual Analogue Scale for Pain (VAS-pain), gait speed, Berg balance, and Instrumental Activities of Daily Living (IADL). Effort during physiotherapy sessions was rated using the Pittsburgh Participation Measure (PPM). Outcome was assessed using the Functional Independence Measure (FIM). Linear regression models were used to determine predictors of discharge FIM and PPM.
A model which included admission FIM, GDS, STAI, VAS-pain, IADL, and PPM as significant predictors, accounted for 79% of discharge FIM. MoCA, VAS-pain, gait speed, and Berg balance were significant predictors in a model which accounted for 61% of PPM.
These findings suggest that, while mood and anxiety impact overall rehabilitation outcome, cognition specifically predicts level of participation during physiotherapy, an important mediator of outcome. Future work should examine how cognition impedes effort during physiotherapy, and focus on developing interventions to improve functional status in patients with cognitive difficulties.
Delirium is an acute confusional state characterized by inattention, disorganized thinking, and perceptual disturbances. Previous research has shown that hospitalized elderly patients on a general medicine ward were more likely to develop incident delirium if they had baseline cognitive impairment, vision impairment, dehydration, and/or severe illness. Environmental factors likely play a role in delirium development. The primary study objective was to determine if room changes are associated with an increased incidence of delirium per patient days in elderly patients on a general medicine ward after controlling for baseline risk factors. Secondary objectives were (1) to determine if room changes increase the length of delirium in patients who had delirium at admission, (2) to determine if room changes increase length of hospital stay, and (3) to determine if bed-spacing and room characteristics affect these outcomes. Our study sample consists of patients 70 years of age or older who were admitted to the general medicine service at St. Michael’s Hospital between October 2009 and September 2010. A total of 1,384 patients met these criteria. A validated chart abstract abstraction technique was used to identify patients with delirium, and Decision Support data was used to identify room changes and bed spacing. So far, 1,354 patient charts have been abstracted. A total of 388 patients (28.7%) had delirium at admission, and 140 (14.5%) of the remaining patients developed delirium during their first week of hospital stay. We are expecting to complete data abstraction and analysis by the end of February 2012.
Primary Author:
Language of the presentation: Eng
In preparation for the 2012 Canadian Consensus Conference on Dementia, background papers are being written on 8 topics in order to make recommendations for clinical practice. Rapidly Progressive Dementia (RPD) is an uncommon condition with numerous possible causes, for which there is no universally accepted definition. We conducted a systematic review to make recommendations about [1] definitions for RPD in (a) dementia developing in previously healthy individuals, and (b) individuals with an existing dementia who experience unusually rapid cognitive decline; [2] a logical diagnostic approach based upon the prevalence of conditions which cause RPD. The initial search identified over 900 articles. Each abstract was assessed for relevance (to [1] and [2] above) by two independent reviewers. If either reviewer deemed an article relevant or possibly relevant, it was fully reviewed for quality against pre-agreed criteria; if assessed of good quality, data were extracted. In the example of a report of a case series, a good article described patient population (and referral bias if any), diagnostic criteria for dementia, and definition of RPD. We describe the process of conducting the review, proposing criteria for standard definitions, and the iterative process leading to a recommended diagnostic approach.
Primary Author:
Language of the presentation: Eng
Increasing incidence and prevalence of dementia and staff time constraints have created the need for an improved and streamlined system of care for dementia patients in primary care. The objective of this study was to develop a collaborative model of dementia care in partnership with and endorsed by staff members and stakeholders at a Primary Care Network (PCN) in Alberta. Phase 1 involved a retrospective chart review with Phase 2 involving focus groups and structured questionnaires that were distributed to staff members to assess their perspectives on dementia care. Phase 3 involved the creation of a preliminary care model for patients with dementia, followed by feedback on the model from staff members using consensus based methodology. Phase 4 of the project will focus on the implementation of the model in the PCN, with process and formative evaluation of the model planned. In this presentation, we provide a comprehensive overview of our model, components of the model, and resources that are foundational to successful implementation.
Primary Author:
Language of the presentation: Eng
Falls are a common condition that had important impacts in elderly patients. Previous study suggested that falls lead to limitation of activities due to fear.
To report impacts of falls, expectations on Thai health-care system and fall events in falling elderly patients with chronic disease.
Qualitative in-depth interviews, using an interview guide, were conducted with 18 participants who were referred from primary care clinic, geriatrics clinic and home health care unit. Content analysis was performed for analysis.
Falls were not found to be related to chronic disease in elderly patients. The most common reaction was fear, particularly fear of being dependent and burden to family members. Chronic pain was the most common illness developed after fall. Patients tended to be more careful, walking slowly, decrease activities, decrease traveling, and use gait aid more regularly. Most patients eventually told family member’s about their falls. Family’s reaction to patient’s fall included concern of patient’s condition, distrust, sarcastic comments. Doctors did not take falls into account by not asking patients about their falls. In addition, patient did not mention their falls events to doctors particularly, specialist doctors. Patients focused more on results of falls compared to causes of falls. Accident was the most common cause in fall event.
Falls affected patients not only physical aspect, but also psychological status, behavior and their families. Health care providers should pay more attention to elicit causes of falls in elders.
Primary Author:
Language of the presentation: Eng
Our symposium will focus on mental health issues which impact on outcomes after hip fracture. Dr. Seitz will present his epidemiological research on the impact of dementia in outcomes for older adults with hip fractures. Dr. McGilton will discuss the implementation of models of care for supporting older adults with dementia in rehabilitation. Dr. Iaboni will discuss mood disturbance and other psychiatric symptoms after hip fracture, and will present data about the initiation of antidepressants and other psychotropic medications after hip fracture. The symposium will conclude with a panel discussion about optimizing care for older adults after hip fracture, from the perspective of mental health.
The opinions expressed in the abstracts are those of the authors and are not to be construed as the opinion of the publisher (Canadian Geriatrics Society) or the organizers of the 32nd Annual Scientific Meeting of the Canadian Geriatrics Society. Although the publisher (Canadian Geriatrics Society) has made every effort to accurately reproduce the abstracts, the Canadian Geriatrics Society and the 32nd Annual Scientific Meeting of the Canadian Geriatrics Society assumes no responsibility and/or liability for any errors and/or omissions in any abstract as published.
Primary Author:
Language of the presentation: Eng
To identify current practices and care gaps for elderly patients admitted following a hip fracture, and to characterize patients’ patterns of functional recovery over 1-year. Relevance Increased awareness of existing gaps and improving our understanding of patients’ recovery can help optimize patients’ outcomes.
Forty community-dwelling participants with an osteoporotic hip fracture (≥ 65 years) were recruited and followed over 1 year. Patients were divided according to their pre-fracture mobility: low, medium, and high. Recovery was defined in two ways: “traditional definition” based on return to pre-fracture mobility, and “acceptable” based on ability to do stairs. Statistical analysis: Single-subject design approach for analyzing small samples was used to identify sources of variability in recovery over time.
Some gaps in services received during hospitalization and at the time of discharge were: (i) 63% had a surgical delay > 48 hours; (ii) > 75% had inadequate osteoporosis management; and (iii) only 35% had a home visit within 1 week of returning home. Using the traditional definition for recovery: 80%, 52%, 33% recovered from the low, medium, and high baseline groups, respectively; 40%, 43%, 33% maintained this recovery up to 1 year. Using the definition for acceptable recovery, 20%, 43%, 71% recovered, respectively, and 10%, 38%, 57% maintained the recovery. Patients generally lost functional improvement between 6–12 months, following waning of rehabilitation services.
Despite the plethora of guidelines specifically for osteoporosis management following hip fracture, gaps exist in care practices across the continuum. The extent of recovery depended on the definition however, after initial improvement, the majority of patients deteriorated after 6-months. A booster rehabilitation program is indicated.
Primary Author:
Language of the presentation: Eng
While much knowledge is gained from quantitative health research, illness itself is subjective. By appreciating the experience of failing health and its impact on outcomes for individual patients, it is hoped that healthcare providers will be able to practice more humanely and effectively. Falls are a common and serious health problem experienced by older persons. How they perceive and interpret the experience of falling can influence the long-term consequences of the event. Other than work done with fear of falling, to date this has not been rigorously studied. Our primary objective in this pilot study was to explore whether there was additional value in obtaining a patient’s narrative as part of the assessment of older persons who had fallen. We interviewed a convenience sample of 5 patients referred to the Calgary Fall Prevention Clinic (CFPC) using the Narrative Interview technique proposed by Jovchelovitch and Bauer. These narratives and the CFPC assessments underwent separate analyses for themes and patterns. Phenomena generated from narratives were determined through several readings of the transcript, using original audio recordings and field notes to help provide context. A comparison between phenomena found in the narrative analyses and the CFPC assessments was performed to highlight commonalities and gaps. Our findings will be presented to a focus group consisting of members of the CFPC who will discuss the potential usefulness of narratives in care planning for these patients. These deliberations will inform further research on the use of narratives in the assessment of patients referred to the CFPC.
Primary Author:
Language of the presentation: Eng
The growing number of elderly patients with multiple chronic conditions presents a pressing challenge to the Canadian health-care system. Current practice models are not well suited to this challenge. Our primary objective was to design and evaluate a new interprofessional care model for community-dwelling seniors with complex health-care needs. A secondary objective was to explore the potential of the new model as an interprofessional training opportunity.
The IMPACT clinic (
Observed benefits of the IMPACT clinic include: significantly more time and “space” for the patient and family to discuss current concerns; reduction in repeat visits and multiple referrals; enhanced real-time information-sharing; improved professional understanding of other disciplines; greater satisfaction among health-care providers; and enhanced interprofessional learning among clinical trainees. Challenges included: extended length of visits proved exhausting for some frail patients; interprofessional team-based models perhaps not optimal for patients with sensory impairments or severe mental health concerns; and scheduling issues sometimes arose owing to the number of clinicians involved.
Evaluation of the IMPACT clinic is encouraging with positive feedback from patients/ families, team members, and clinical trainees. Interprofessional care models hold great promise for meeting the challenge of complex chronic disease in the elderly. Further evaluation is underway.
Medical Directors in LTC homes in Ontario are increasingly being faced with adminstrative needs of a more complex patient population and in an environment of increased legislative and regulatory oversight. There are roles identified within the LTC Homes Act, as well as key roles outlined in Medical Director Contracts agreed to by MOHLTC and the OMA. The Ontario Long Term Care Physicians is a non-profit organization with close to 300 members who are physicians working in LTC homes in Ontario. The organization runs a clinically focused conference each fall and increasingly is aware of administrative skills and expertise for which many members may not have received formal training. In addition, we hear from members challenges they face with being informed of important system changes and new programs being implemented. The purpose of the survey was to identify perceived and unperceived learning needs of physicians working in Long Term Care to explore future educational initiatives.
Unrestricted grant received from Pfizer to develop a LTC physician survey and begin developing educational initiatives based on outcomes of the survey. Survey questionnaire developed with input from OLTCP board member working group. Survey was circulated via OLTCP database. Survey results then analysed and presented to OLTCP board and membership.
Survey identified perceived and unperceived learning needs in areas of legislative requirements, quality improvement, program management, high-risk clinical areas, and working with teams. Barriers to involvement in areas of administration included time and knowledge, not lack of interest. The details of these results will be shared in the poster format.
Survey identified key learning needs that are facing medical directors in LTC homes that are integral to the role of Medical Director. The OLTCP has explored training programs and conferences in North America and has determined that the content areas of the Core Curriculum on Medical Direction in LTC run by the American Medical Directors Association in the United States best matches the learning needs we have identified. We have now developed goals and objectives for an equivalency curriculum, and are in the process of developing the curriculum to address medical direction and leadership skills required to be an effective medical director in LTC.
As the life expectancy and chronicity of health conditions affecting Canadians continues to rise, the assessment of autonomous decision-making capacity becomes an issue of increasing importance. Adults with diseases and disabilities are at particular risk in this regard. Comprehensive assessments and realistic interventions that employ the least intrusive and least restrictive measures possible have been determined to be the most ethical and desirable.
The inter-disciplinary DMC Model was developed based on a literature search, environmental scan, needs assessment, surveys, and discussions with inter-disciplinary groups at various health-care sites within Covenant Health in 2006. An iterative process was used to formulate a model, which was then implemented in the Covenant Health and AHS sites, Edmonton zone, from 2007–2012.
This model was “provincialized” through the AHS Seniors Health Cognitive Strategic Planning Committee and has been made available for use provincially. It includes a care map, worksheets, and staff training workshops and in-services, and an inventory of educational materials. Staff trained in the assessment of decision-making capacity and use of the model (e.g., physicians, psychologist, nurses, nurse practitioners, social workers, occupational therapists, care co-ordinators) effectively implemented the DMC Model in Edmonton and Calgary zones, and to varying degrees in the other zones from 2010–2012.
The DMC Model offers a holistic inter-disciplinary approach to capacity assessment that maximizes client autonomy, offers the least restrictive and intrusive solutions, and facilitates inter-disciplinary and inter-organization collaboration.
In a re-analysis of data from the Canadian Study of Health and Aging, non-traditional risk factors, which were not typically associated with dementia, were found to impact an individual’s level of frailty and subsequently their risk of Alzheimer’s disease (AD). We examined whether an index consisting of such factors could predict future reports of incident AD and dementia, as well as mortality, in a similar manner to traditional risk factors, in a larger, multinational cohort.
Secondary analyses were conducted on data from the Survey of Health, Ageing, and Retirement in Europe and consisted of cognitively healthy individuals 50 years or over, from 12 European countries (N = 11,817). Three AD risk factor indices (RFIs) were constructed to predict a ∼ 4-year risk for a self or informant report of AD, dementia, and survival; a 31-item non-traditional RFI, a 6-item traditional RFI, and a 37-item combined RFI.
After adjusting our risk model for age, sex, education, and traditional risk factors for AD, the non-traditional RFI significantly predicted the risk of dementia (OR = 1.49, 95% CI 1.34–1.67), and mortality (OR = 1.53, 95% CI 1.19–1.96) after an average of 4.3 years. The combined RFI exhibited the strongest prediction of dementia (OR = 1.79, 95% CI 1.38–2.32) and mortality (OR = 1.68, 95% CI = 1.50–1.89).
The typically small impact of health deficits that are not traditionally associated with AD can significantly increase one’s risk of both dementia and mortality when combined. Health professionals should place greater importance on the examination of overall health decline, rather than solely assessing traditional risk factors for illness.
Based on clinical trials, treatment of metastatic castration-resistant prostate cancer (mCRPC) with chemotherapy is seen to improve disease control and survival in older men (age 65+). Its effects, though, on the daily functioning, physical performance, and quality of life (QOL) in elderly men outside the clinical trial setting are not well understood.
Men aged 65+ with mCRPC starting first-line chemotherapy at a tertiary cancer centre were enrolled in this prospective observational pilot study. Physical function was assessed with the timed up and go (TUG) test, Timed Chair Stands, and grip strength. Functional status was measured using the OARS-IADL questionnaire, in addition to social activities limitations and social support (MOS measures). Patients completed the FACT-P and FACT-G to measure prostate-specific QOL and general QOL, respectively. Assessments were completed before each cycle of chemotherapy. Pre–post within-group comparisons were done using Student’s
25 patients (mean age 75) receiving Docetaxel + Prednisone were enrolled, 3 of whom died and 2 dropped out. Both general and prostate-specific QOL improved over a median of 6 cycles. Patients’ instrumental activities of daily living (IADL) scores remained stable over time. On average, grip strength was stable, and lower extremity function improved on both the TUG and Timed Chair Stands.
Contrary to our hypotheses, QOL improved in this frail elderly cohort, and IADL function remained stable. Although physical function remained stable or improved during first-line chemotherapy, there was significant variability among individual patients. Older men with mCRPC appear to tolerate first-line chemotherapy fairly well in terms of QOL and geriatric domains.
Despite treatment of the associated condition delirious persons do not always recover, for unknown reasons. We sought to derive and validate a prognostic model to predict poor recovery after an episode of delirium based on early admission characteristics.
This prospective cohort study consecutively enrolled older medical in-patients (admitted to London Health Sciences Centre) from the community. Participants were screened for delirium. Delirious (by the Confusion Assessment Method) patients were followed in hospital and after discharge.The primary outcome was poor recovery, in delirious patients, defined by death, institutionalization or functional decline (decreased activities of daily living), at discharge or 3 months after discharge, elicited from the medical chart or post-discharge caregiver telephone interviews.
1235 medical in-patients (mean age 82.6 years, 42% male) were screened. Delirium occurred in 355 (or 29%) and recovery status was known in 342 (96%). Fifty-four patients (15%) died in hospital and 24% (
Results suggest that poor recovery after delirium is common, and is associated with certain characteristics available on admission.
The
Two surveys were held among physiotherapists and physical rehabilitation therapists working at a GAU unit (
Overall, professionals from 36 GAU responded to one or both surveys. The most frequent scales used by the participants are Berg Balance Scale (BBS)–97%; Timed Up and Go Test (TUG)–80%; and walking speed test–57%. Those tests (BBS, TUG, and walking speed test) were also the most frequently recommended by the participants for assessing a patient with moderate-to-severe gait and balance disorder.
The mobility committee of the RUSHGQ recommends that the assessment of gait and balance disorders should include at least the Berg Balance Scale, the Timed Up and Go Test, and a walking speed test.
Traditionally physicians have viewed Subjective Cognitive Impairment (SCI) in older people to be benign and related to age-associated memory loss. However, research in this field suggests that people who self-report memory problems, but score normal on cognitive testing, have a higher rate of progressing to mild cognitive impairment (MCI).
Over the last 4 years a total of 165 people over 55 responded to newspaper advertisements with self-reported memory loss. Participants received cognitive screening tests using the standardized MMSE, the MoCA, the 15-point GDS, the AD8, the Cornell Scale for Depression in Dementia, and the Lawton Brody Activities of Daily Living Scale. The test results were case conferenced with a geriatrician, and a clinical suspicion of normal, SCI, MCI, depressive symptoms/mixed picture, possible dementia or other was given. 46 individuals have repeat measures on these tests from 2009 to 2012.
In 2012, of those 46 follow-up participants, 54% had no change on their cognitive tests. However 33% had declined over the 4 years and 9% had improved. Of those who were given the clinical impression of SCI in 2009 or 2010, 39% had declined to amnestic MCI or multiple-domain MCI. Those individuals who reported depressive symptoms in 2009 (32%) tended to have lower scores on the GDS and Cornell on follow-up visits.
In studies published on SCI, those who self-report memory problems compared to normal health controls are at greater risk of declining to MCI.
Our study captured this trend as 39% of those with SCI had declined to MCI within 4 years.
Those with depressive symptoms may have improved with non-drug/drug approaches.
Many older adults are prescribed benzodiazepines despite their association with cognitive decline, postural instability, falls, hip fractures, and a five-fold risk of hospitalization after a motor vehicle collision. Yet, 16% to 33% of elderly, community-dwellers use benzodiazepines, and 54% use them daily. In this review, we address the approach to discontinuation and effective alternative options.
MEDLINE (1946–2012), EMBASE (1980–2012), and the Cochrane Database of Systematic Reviews (2005– 2012) were searched. The following key search terms were used: MeSH & EMBASE terms for benzodiazepines, sleep initiation and maintenance disorders, drug withdrawal and abuse terms, and keywords for sleep, addiction, dependence, and insomnia, as well as specific drug names and terms for taper, withdrawal, and alternative therapies.
Chronic benzodiazepine use is associated with many adverse outcomes. Hospitalization may play a pivotal role in both the initiation and discontinuation of sedative hypnotics. There is a paucity of long-term data for the use of non-benzodiazepine sedative hypnotics. Cognitive behavioural therapy, brief behavioural interventions, and benzodiazepine tapering protocols have shown proven benefit in benzodiazepine discontinuation.
There may be evidence for non-benzodiazepine sedative hypnotics; however, there is a paucity of long-term, placebo-controlled studies to support their safety, and some evidence to suggest harm in the frail older adult. Cognitive behavioural therapy and/or the use of a taper protocol may increase the success of withdrawal and improve sleep parameters. Exercise, sleep education, massage, and brief behavioural intervention are excellent non-pharmacological options for managing insomnia and for aiding discontinuation. Lastly, it is important to be cognizant of the impact that prescribing sedative hypnotics in hospital can have on long-term use.
Clinical practice guidelines are intended to improve patient care. Clinicians may not be able to implement guideline recommendations because of time pressures, which are particularly challenging in primary care. We aimed to quantify the time required to implement guideline recommendations regarding the most common chronic diseases in older adults, including hypertension, diabetes, dyslipidemia, asthma, chronic obstructive pulmonary disease, and chronic kidney disease.
We determined the time required to apply national guidelines to a cohort of primary care patients. Eight Canadian clinical practice guidelines addressing management of chronic diseases in adults were reviewed. Their recommended interventions, along with the indications for each intervention, were identified. Three primary care physicians reviewed each recommendation and identified the time required to perform it on an average patient. A cohort of 160 randomly selected patients aged 55 years from a university-affiliated primary care clinic was analyzed to determine how often each intervention should be applied to these patients. These data were used to estimate how much time it would take a clinician to apply guideline recommendations to his or her practice.
103 different interventions from 8 clinical practice guidelines were identified. The total time required to apply these interventions to the selected cohort of patients was 340 hours (SD ± 189). Extrapolating this value to a clinical roster of 1000 patients, 266 working days would be required each year to implement the recommended interventions.
The implementation of chronic disease guideline recommendations in primary care requires a prohibitive amount of time. Guideline developers should consider the time required to implement their recommendations when drafting clinical practice guidelines.
Quebec will face accelerated aging of its population in the years to come. Its health-care system will have to adapt to this situation in order to assure efficiency and relevance of interventions to meet the growing needs. The model of care of the Geriatric Evaluation and Management Unit (GEMU) is a well-known hospital-based mode of organization of geriatric services, and its efficiency has been proven. However, the implementation of this model of care within various Quebec hospitals has brought a noticeable heterogeneity in the care practices among GEMUs. We then want to provide hospital managers with a tool which would define the processes and framework needed to efficiently run GEMUs. This tool would direct the evaluation and development of these services with a strong scientific basis.
We first did a worldwide literature review and identified two recent meta-analyses on the efficiency of GEMUs. The studies included in the two meta-analyses were rigorously selected and both were analyzed. We also included in our review a Quebec Delphi study on selection criteria applicable to the GEMUs in Quebec.
We extracted and categorized all the process of care items from the studies including: patient selection, type of ward, type of health centre, composition of the geriatric team, and evaluation and treatment processes.
This tool will allow the decision makers and hospital managers to conduct evaluation and development of GEMUs in Quebec and elsewhere.
Studies have shown increased adverse outcomes are related to hospital admissions from Long-Term Care (LTC) homes, often for etiologies that could be safely treated in the facility. We examined the reasons for transfer and outcomes of LTC residents admitted to Hamilton Health Sciences (HHS) hospitals.
Patient matched hospital and LTC home charts were retrospectively reviewed for all HHS hospital admissions transferred from LTC homes during 4 non-consecutive months in 2011. We considered patient demographics, events leading to transfer, diagnosis, and course during admission to hospital. Data presented within are limited to the analysis of hospital medical charts.
A total of 201 charts were reviewed. Altered level of consciousness (21%), dyspnea (18%), and fever (9%) were the most frequent events leading to transfers from LTC homes. Most patients (33%) transferred for altered LOC were diagnosed with either a urinary tract infection (UTI) or pneumonia. A total of 47 patients experienced an adverse event(s) while hospitalized. Fifteen patients were transferred despite a “do not hospitalize” order. Advanced directives were not documented in 34 patients on arrival to the hospital.
The rate of adverse events in patients transferred from LTC homes to hospitals is high. An intervention aimed at identifying early signs of altered level of consciousness, as well as treating frequent causes, such as UTI’s and pneumonia in the LTC homes, may prevent avoidable transfers to hospitals. There is a need to improve discussions and documentation of advanced directives, as well as a system to ensure these are followed.
Hyponatremia has been associated with increased mortality and length of stay (LOS) in hospitalized patients. However, other adverse associations such as falls or syncope, fractures, unplanned readmission, need for inpatient rehabilitation, and change in discharge destination to a
This is a retrospective case control study of patients admitted with hyponatremia (serum Na ≤ 134 mEq/l) under the General Internal Medicine Unit during a 6-month period. The relevant data were collected by explicit medical record review and analyzed in univariate and multivariate models. Data from 3 months in patients aged 65 years are presented.
The prevalence of hyponatremia was 21%. Hyponatremia had a significant univariate association with LOS (OR 1.03
The study confirms the association between hyponatremia and falls or syncope. Among the adverse outcomes of hospitalization, hyponatremia was independently associated with only unplanned readmission within 30 days. Falls or syncope at presentation and admission diagnosis of metabolic disorders appear to have a greater association with LOS than hyponatremia. The study was probably underpowered to assess other outcomes.
In 2011, the Memorial University Family Medicine (FM) Residency Program introduced a Care of the Elderly (COE) rotation to enhance residents’ skills in managing the complex health issues of the elderly population. The purpose of this project was to understand FM residents’ perceived needs in COE training and to evaluate the COE rotation with respect to these needs.
Survey methodology was used with the pre-rotation survey designed to evaluate perceived needs in COE training and the post-rotation survey designed to assess whether learning needs where addressed.
The pre-rotation survey was sent to 57 FM residents with a response rate of 40%. The majority of students indicated a need for further training in COE topics. Students identified that in certain areas further training was
Memorial University FM residents recognize the need for COE training. With the growth of the elderly population, newly trained family physicians must be prepared to provide these patients with appropriate care. This COE rotation addresses most learning needs. However, results from our survey indicate that there is room for improvement.
The most common cognitive screening tool used by family physicians is the Folstein Mini-Mental State Examination (MMSE). In 2009, Brown
Patients aged 65 and older attending a regularly scheduled appointment in two family physician offices in New Brunswick were invited to participate in the study. Participants had to complete the self-administered Test Your Memory tool and complete a MMSE.
A total of 52 participants completed the study. The mean TYM score was 44.7/50 (SD 2.4) and the mean MMSE score was 27.8 (SD 5.6). The Pearson correlation coefficient between the TYM and MMSE is R2 = .58. This is a significant correlation with a p-value of .01. A score of ≤ 42/50 on the TYM had a 100% specificity for picking up patients who will score < 24 on the MMSE. The sensitivity of the TYM was 100% and the specificity was 81.6%.
This study validates the TYM test as a screening tool in a Canadian primary care population. However, the strength of the TYM test is in its negative predictive value in participants who score above 42.
Sedentary behaviour has been proposed as an independent cardiometabolic risk factor, even in adults who are otherwise physically active through leisure-time recreational activities. Because little is known about the metabolic effects of sedentary behaviour in seniors, we examined the relationship between sedentary behaviour and cardiometabolic risk in physically active older adults.
54 community-dwelling men and women 65 years of age (mean 71.5 years) were enrolled in this cross-sectional observational study. Subjects were in good health and free of known diabetes. Activity levels (sedentary, light activity, moderate activity, and vigorous activity time per day) were recorded with accelerometers worn continuously for 7 days. Cardiometabolic risk factors measured consisted of the American Heart Association diagnostic criteria for metabolic syndrome (waist circumference, triglycerides, high-density lipoprotein (HDL), systolic blood pressure, fasting glucose), as well as low-density lipoprotein (LDL). The relationships between activity measures and cardiometabolic risk factors were examined. Significant variables were entered into a multivariate regression model.
All but 1 subject met Canada Health guidelines for an active “fit” adult. Despite this, the average proportion of time spent at a sedentary activity level each day was 72.7%. From the regression analysis, the only significant association found was between LDL and sedentary time, with LDL detrimentally associated with average sedentary time per day (Standardized Beta Correlation Coefficient 0.302,
Sedentary behaviour is associated with an adverse metabolic effect on LDL in older adults, even those who meet Canada Health guidelines for an active “fit” adult. Emphasizing activities that reduce sitting (e.g., standing desks, less television) may be a practical recommendation to reduce sedentary behaviour in older adults.
Post-operative delirium in older adults is a common complication of surgery with significant consequences. Delirium often portends poorer clinical outcomes including increased mortality, length of stay, and increased likelihood of discharge to a facility. The role of antipsychotics to prevent post-operative delirium has not been well-established. We therefore wished to determine the effectiveness of antipsychotics in preventing postoperative delirium.
We searched online literature databases and registers for randomized controlled trials (RCTs) of adults undergoing surgery who were given antipsychotics to prevent post-operative delirium, using a placebo as the comparator. Two researchers independently reviewed citations and abstracts, selecting those meeting inclusion criteria. Quality was assessed via the Cochrane risk of bias tool. Random effects meta-analysis and meta-regression were conducted. Q-statistics and I2 were used for assessment of heterogeneity.
We evaluated 4340 citations from our initial search and from this reviewed 32 full-text articles. Five randomized controlled trials met criteria for inclusion. Antipsychotics were found to reduce post-operative delirium [OR: 0.41; 95% CI: 0.235 to 0.744]. The effect-size estimate was heterogeneous [Q-value: 15;
Within the limits of few RCT’s available, antipsychotics appeared to reduce the incidence of post-operative delirium in a variety of surgical settings. Larger, well-designed RCTs are needed to help confirm our findings.
Patients with mild cognitive impairment (MCI) and significant amyloid burden on PiB PET imaging manifest impaired performance on episodic memory tasks when compared to MCI patients with lower amyloid burden. This association has yet to be defined with regards to non-episodic memory tasks. Therefore, we sought to further characterize the cognitive profile of subjects with MCI who underwent PiB PET imaging.
Forty-six subjects aged 60–90 with a clinical diagnosis of MCI underwent neurospychological evaluation. PiB PET images were obtained within 8 months of a subject’s cognitive assessment. Subjects were matched for age and education and classified as PiB− (SUV < 1.5; n = 22) or PiB+ (SUV > 1.5; n = 24). The results from the neuropsychological evaluation were compared between groups and correlated with amyloid burden. A regression analysis was conducted to determine whether amyloid burden was a predictor of cognitive performance.
There were no significant group differences on global cognitive measures. There was considerable overlap between PiB+ and PiB− subjects on all cognitive domains, but the PiB+ subjects performed significantly worse than PiB− subjects on tasks of episodic memory and executive functioning. Regression analysis showed that amyloid beta deposition was a significant predictor of performance on episodic memory and inhibition.
These preliminary results suggest that MCI patients who are considered to be prodromal Alzheimer’s disease may be distinguishable by the presence of impairment in both episodic memory and inhibition. Future studies may be useful for addressing whether a specific neuro-psychological battery can aid in early diagnosis of dementia.
Frail elderly adults are particularly vulnerable to medication errors when transitioning from hospital to home. The objective of this study is to describe the prevalence and causes of medication discrepancies (MDs) in geriatric community-dwelling adults during this transition period.
A descriptive study was carried out from a community hospital setting in British Columbia, Canada. The study population consists of patients 70 years and older who met selection criteria for home visits within 24–72 hours after hospital discharge by a Geriatric Transition Nurse (GTN) between November 2011 to May 2012. Using the Medication Discrepancy Tool, the GTN performed medication reconciliation between discharge medications and medications individuals were taking at home. Patient-level and system-level factors contributing to the MDs were identified.
Out of the 100 patients seen by the GTN, 65% were female and 85% were on five or more medications at the time of discharge. 72% of patients had five or more co-morbid chronic conditions. Medication reconciliation identified 46% of patients with at least one medication discrepancy. More than half of MDs were caused by patient-level factors and the remainder were caused by system-level factors. The most common reported patient-level factors were: non-intentional non-adherence and intentional non-adherence. The most frequently seen system-level factors were: incomplete/inaccurate/illegible discharge instructions and not recognizing patient’s lack of support. In some instances both types of factors contributed to the occurrence of a medication discrepancy.
Medication discrepancies in the frail elderly are common when transitioning from hospital to home. Identifying common patient-level and system-level factors may serve as starting points when designing quality improvement efforts with the aim to decrease medication discrepancies.
In 2010, Osteoporosis Canada developed guidelines for the diagnosis and management of osteoporosis for people > 50 at high risk of fragility fractures. These guidelines did not address frail elderly where access to diagnostic technology, such as bone mineral density, and research is limited.
We used the GRADE process to develop guidelines applicable to frail elderly with over 50 stakeholders, including resident/family representatives of long-term care, interdisciplinary health professionals, and program managers. We surveyed the panel to determine questions and outcomes most relevant for this population. We searched the literature for baseline risks of fractures and intervention effects. When making recommendations, we discussed benefits/harms, strength of evidence, values/preferences, and resources.
In addition to outcomes from the 2010 guidelines, this panel identified mobility, pain, and quality of life as important in this population. However, few studies reported these outcomes. To make recommendations, the panel considered absolute risk differences in outcomes with or without treatment, which are calculated from baseline risks. It was critical that the panel agreed on baseline risks which can vary between low- and high-risk groups. Agreement was challenging, but the process was enlightening to recognize gaps/uncertainties in existing research. When evidence in frail elderly was lacking, the panel assessed the applicability of effects found in other populations to make recommendations. The GRADE process incorporated values/preferences, particularly of families and residents, which was uniquely challenging in view of life expectancy, multiple co-morbidities, and serious consequences of fractures.
The GRADE process helped identify gaps in the literature for important outcomes, the impact of baseline risks, and the importance of balancing benefits and harms, and their value and consequences in this population.
Since 2006, the Ontario Osteoporosis Strategy for Long-Term Care has engaged in outreach activities to increase uptake of evidence-based osteoporosis/fracture prevention strategies (
In August 2012, de-identified medication/demographic data were downloaded from Medical Pharmacies, a pharmacy provider for approximately one-third of Ontario LTC homes. After excluding 40 LTC homes participating in a targeted intervention (ViD
The analysis cohort was 21,699 residents, mean age 83.5 (SD: 10.7) years, 70% women. 57% of LTC homes were for-profit, 45% affiliated with a corporate chain, 61% had age-100 residents. Mean LTC home prescribing rates were 59.9% (95% CI: 57.2, 62.6) for vitamin D, 32.2% (95% CI: 30.2, 34.2) for calcium, and 18.5% (95% CI: 17.4, 19.7) for osteoporosis medications. Prescribing rates were normally distributed and ranged from 22.3%–94.9% (vitamin D), 1.6%–78.4% (calcium), and 0%–55.9% (osteoporosis medications).
Although there was a range in prescribing between LTC homes, our results indicate that wide-scale implementation of outreach activities resulted in uptake by many LTC homes, particularly for Vitamin D, with half the homes prescribing at approximately 60% or better. Currently, osteoporosis consensus guidelines for LTC are being developed.
Currently far too many seniors (∼ 20%) consume inappropriate benzodiazepines, which increase the risk of adverse drug reactions and unnecessary hospitalizations among community-dwelling elders. As of 2012, the new Beers criteria lists all benzodiazepines as drugs to avoid in the elderly no matter the half-life.
A written educational tool was mailed to 144 benzodiazepine consumers aged 65 years recruited from community pharmacies. Knowledge and beliefs about inappropriate prescriptions were queried prior to and 1-week after the intervention. Primary outcome was a change in risk perception. Explanatory variables were a change in knowledge and beliefs about medications, as well as cognitive dissonance occurrence. Self-efficacy for tapering and intent to discuss discontinuation were also measured.
Post-intervention, 65 (45.1%) of chronic benzodiazepine consumers (mean duration use 10.5 years, SD 8.2 years) perceived increased risk. Increased risk perceptions were explained by better knowledge acquisition (mean change score 0.9, 95% CI (0.5, 1.3)), and a change in beliefs (BMQ differential mean change score −5.03, 95% CI (−6.4, −3.6), suggesting elicitation of cognitive dissonance. Experience of cognitive dissonance was associated with a 6-fold higher likelihood of patients reporting increased risk perception (OR = 6.61 95% CI (3.2, 13.8)). Intent to discuss discontinuation of benzodiazepines with a doctor (83.1% vs. 44.3%,
Risk perception on benzodiazepines can be altered through direct delivery of an educational tool to aging consumers. Results suggest patients could potentially be targeted directly with information to catalyze discontinuation of inappropriate prescriptions.
Gait and cognition are interrelated. Executive dysfunction is associated with slower gait. It is unknown if memory dysfunction, a cardinal sign in MCI, is associated with the gait disturbances seen in MCI. The objective was to determine if gait in older adults with MCI varies by subtype: amnestic (a-MCI) or non-amnestic (na-MCI) type.
Older adults with MCI from the “Gait and Brain Study” were included. Cognition was evaluated using MMSE, MoCA, Trails Making Test A and B, Rey Auditory Verbal Learning Test, Digit Span Test, and Letter Number Sequence Test. Gait performance (velocity and gait variability) was evaluated with the GaitRITE® mat under usual walking and three dual-task conditions (walking while: naming animals out loud, serial subtractions by 1s and serial subtractions by 7). Participants were divided into a-MCI and na-MCI by episodic memory test. The relationship between cognitive subtype and gait was evaluated with multivariable linear regression.
Fifty-six participants (mean age 76.3 ± 7.2 years, 50.9% female) were included. Thirty-eight were a-MCI and 18 were na-MCI. Groups were similar in age, co-morbidities, and history of previous falls. The a-MCI participants walked slower than na-MCI (98.5 vs. 112.2 cm/sec,
Episodic memory impairment was associated with poor gait performance, in particular under dual-task conditions. This suggests slow gait and higher variability under dual-task testing is a motor feature in a-MCI independent of executive dysfunction.
Assessing frailty should be an essential part of the care of older adults. Several scales have been proposed to quantify frailty and the operational criteria of each scale vary. The purpose of this study was to compare the prevalence of frailty in community-dwelling, middle-aged and older Europeans as estimated by eight scales and to examine the agreement among scales in classifying participants as frail.
27,527 participants aged 50+ years (mean age 65.3 ± 10.5, 54.8% women) from the 11 countries (Austria, Belgium, Denmark, France, Germany, Greece, Italy, Netherlands, Spain, Sweden, Switzerland) which participated in the first wave of the Survey of Health, Ageing and Retirement in Europe comprised the study sample. Frailty was operationalized, based on eight scales: frailty phenotype, a 70-item Frailty Index, a 44-item Frailty Index based on a Comprehensive Geriatric Assessment, Clinical Frailty Scale, Edmonton Frail Scale, Groningen Frailty Indicator, Tilburg Frailty Indicator, and “FRAIL” scale. A score threshold was assigned for each scale to represent the frailty state, based on the relevant literature.
The prevalence of frailty ranged from 44% (Groningen scale) to 6% (FRAIL scale). About half of participants were categorized differently between scales. 49.3% of participants were categorized as non-frail by all scales, and 2.5% were categorized identically as frail by all scales.
Frailty scales capture related but distinct groups of individuals, and each scale provides different estimates of frailty prevalence. Future studies should compare various scales using data from clinical settings.
Hip fracture patients are at high risk for recurrence. Appropriate pharmacotherapy reduces this risk and is associated with reduced mortality after hip fracture, but a care gap exists for fracture prevention in these patients. This evaluation determined rates of osteoporosis treatment and bone mineral density (BMD) testing in hip fracture patients following discharge from a rehabilitation unit.
A prospective cohort study of hip fracture patients aged 50 on an inpatient rehabilitation unit in 2008 and 2011. Patients were seen by a nurse specialist, and encouraged to see their family physician for further assessment and treatment. Physicians were sent a letter indicating the need to follow up with their patient. Patients were contacted following discharge from hospital to determine treatment rates.
Of 310 eligible hip fracture patients admitted to the rehabilitation unit in the years studied, 207 patients were reached post-discharge and provided data. Of patients who were not previously taking osteoporosis medication, 50% of patients had osteoporosis treatment initiated by 6 months following discharge. By 2 months following discharge, 46% of patients in the 2008 cohort had a new BMD performed or scheduled, while this was true for 14% of patients from the 2011 cohort. 35% of patients in 2011 had not seen their family physician by 2 months following discharge.
Rates for osteoporosis treatment and BMD were higher than those reported in the literature for patients not enrolled in case manager programs. BMD testing declined from 2008 to 2011. Lower treatment rates may be due to concerns regarding bisphophonates. There remains room for improvement for follow-up with family physicians.
Assessing fitness to drive in patients with dementia is challenging. The SIMARD was developed as a tool to assist with assessing fitness to drive. This study compares the clinical decision made by a geriatrician regarding driving with the score on the SIMARD.
Patients seen by geriatricians with a diagnosis of dementia or mild cognitive impairment, who had had a SIMARD test completed after the clinical decision regarding driving was made, were included in the sample. Charts were reviewed to gather diagnosis, driving status and history, cognitive and functional information.
Sixty-three patients were identified and 57 met the inclusion criteria. The mean age was 77.07 years. Alzheimer’s dementia in 22 (38.6%) patients was the most common diagnosis. The mean MMSE was 24.85 (SD 3.34) and the MoCA was 19.85 (SD3.58). The mean SIMARD score was 37.16 (SD 19.54). Twenty-four patients had a SIMARD score below 31, 28 scored between 31–70, and 5 scored greater than 70. Of those scoring less than 31, 8 patients continued to drive, 3 of whom had passed a driving test performed by the Department of Public Safety of New Brunswick. In the 5 patients who scored greater than 70, 2 had their licenses revoked by the geriatrician.
There did not appear to be a clear association between the SIMARD score and the clinical decision made by the geriatrician.
Cancer survivorship programs often focus on modifiable behaviours such as smoking and alcohol use and physical activity. Whether these behaviours differ among elderly survivors and whether special considerations should be given to these elderly cancer survivors (age 65+) is unclear.
616 adult cancer survivors (23% elderly) across multiple solid and haematologic malignancies and treatment trajectories were surveyed about smoking, alcohol, physical activity, and attitudes and knowledge about effects of these habits on cancer outcomes. Multivariate logistic regression models evaluated the effect of age on these factors.
9.0% of elderly survivors were current smokers; 35.7% had been binge drinkers recently or in the past (5 or more standard drinks per day for male; 4 or more for female); 24.0% were not meeting exercise guidelines (150 minutes of moderate-to-vigorous intensity activity per week). Compared to younger survivors, elderly were one-third as likely to be current smokers (
Elderly cancer survivors have different smoking, alcohol, and exercise characteristics from younger survivors. Survivorship programs may need to tailor counseling by age group.
Indwelling urinary catheterization is a ubiquitous procedure in the inpatient setting: between 16% and 25% of hospitalized patients will receive an in-dwelling catheter at some point during their stay. While sometimes medically indicated, previous studies have shown that between 21% and 52% of catheters are used unnecessarily, exposing patients to significant morbidity and mortality, including increased risk of urinary tract infection and bacteremia. Here we present the results of a multi-modal educational intervention directed at reducing the overuse of catheters in a large teaching hospital.
The multi-modal intervention targeted nurses and used a variety of approaches to improve catheter use, including small group meetings, educational posters, and modifications to the patient chart. The study patient population included all admitted patients to internal medicine, surgery, and orthopedic surgery, as well as the GIM/ACE Unit from 1 September 2009 to 1 October 2011. Data were structured and analyzed as an interrupted time series using a segmented regression approach.
A total of 14,531 patients, 1,878 of whom were catheterized, were included in this study. A decrease in mean catheter days per patient of between 5.8 and 9.7 days (
A multi-modal educational intervention using nurse education and process changes resulted in a significant reduction in catheter days per patient and the proportion of patients catheterized.
Life course influences on health may be most evident at older ages. In a large sample of middle-aged and older Europeans, we compared grip strength, cognitive performance, and walking speed between native-born participants, immigrants who were born in low- and middle-income countries (LMICs), and immigrants who were born in high-income countries (HICs).
This is a retrospective cohort study of the Survey of Health, Ageing, and Retirement in Europe, including 33,745 participants age 50+ in 14 countries (mean age = 64.9 ± 10.2 years; 54% women). Four performance-based measures were assessed: grip strength, delayed recall, and verbal fluency were measured in all participants, while walking speed was measured only in individuals age 75+. Analyses were divided by participants’ current residence in either relatively wealthier Northern/Western or relatively poorer Southern/Eastern Europe, and adjusted for age, gender, and education.
About 7% of participants (n = 2,369) were immigrants. In Northern/Western Europe, compared to native-born participants, LMIC-born immigrants demonstrated weaker grip strength (mean 32.8 kg vs. 35.7 kg,
Middle-aged and older immigrants demonstrated worse physical function and cognitive performance than native-born Europeans in Northern/ Western Europe, but not in Southern/Eastern Europe. Country of birth and current country of residence were each associated with these performance-based measures of age-related health.
Although the principle goal of hip fracture management is a return to pre-event functional level, most survivors fail to regain their former autonomy. One of the most effective strategies to mitigate the fracture’s consequences is exercise.
To review the reported effect of an extended exercise rehabilitation program offered beyond the regular rehabilitation period on improving physical functioning for patients with hip fractures.
Sources: The Cochrane Bone, Joint and Muscle Trauma Group, the Cochrane Central, PubMed, CINAHL, PEDro, EMBASE, and reference lists of articles were searched from inception to October, 2010. Study Selection: Included were all randomized controlled trials comparing extended exercise programs to usual care for community dwelling after hip fracture. Data Extraction and Synthesis: Two reviewers conducted each step independently. The data from included studies were summarized and then pooled estimates were calculated for nine functional outcomes.
Ten articles were included in the review and eight in the meta-analysis. The extended exercise program showed small–modest effect sizes which reached significance for knee-extension strength for affected and non-affected sides 0.46 (CI 95%: 0.2–0.6) and 0.45 (CI 95%: 0.16–0.74), respectively, balance 0.29 (CI 95%: 0.7–0.51), fast gait speed 0.52 (CI 95%: 0.18–0.85
To our knowledge this is the first meta-analysis to provide evidence that an extended exercise rehabilitation program for patients with hip fractures has a significant impact on various functional abilities. The focus of future research should go beyond just effectiveness and study cost-effectiveness of extended programs.
Primary Author:
Language of the presentation: Eng
By 2050, the proportion of seniors is estimated to increase to 27% from 14% currently. In 2011, there were only 238 Canadian specialists certified in Geriatric Medicine. Beyond the expansion of geriatric specialists, an improvement in physicians’ attitudes, knowledge and skills in geriatrics is important regardless of the specialty.
This study aimed to identify changes in attitudes of preclerkship University of Toronto (UofT) medical students towards geriatric care after participating in an interdisciplinary Geriatric Clinical Skills Day (GCSD) organized by UofT’s Geriatrics Interest Group.Methods. This was a before and after study. First and second year UofT medical students registered for the GCSD participated in this study.
A questionnaire, including the validated UCLA Geriatrics Attitudes Scale, was administered before and after the GCSD. Both a one-sample t-test and the signed rank non-parametric test were used to determine any changes in attitudes.
Of 19 study participants, four students did not complete the post-test questionnaire. 42.1% indicated an interest in Geriatric Medicine, 26.3% in Geriatric Psychiatry, and 63.2% in working with elderly patients. Both pre- and postmean scores were greater than 3 (neutral), indicating a positive attitude before and after the intervention (
There is an overall positive attitude towards geriatrics among study participants. However, a one day GCSD did not alter attitudes towards geriatric care. This small study warrants further investigation in a larger multicentred trial.
Primary Author:
Language of the presentation: Eng
Canada’s population is aging and research has shown that primary care physicians find it difficult to care for elderly patients. Canadian family physicians have appreciated need for geriatrics continuing medical education (CME) and based on the expert opinions of experienced care of the elderly family physicians, geriatric knowledge and skills felt necessary for a family physician caring for the elderly, were put into a curriculum based on the 5 weekend program style. The University of Toronto Department of Family & Community Medicine developed a 5 weekend leadership program in the mid 1990’s and this format allowed community physicians to train without giving up regular clinical time. The Five Weekend Care of the Elderly Certificate Course used discussion in small groups of four as per Malcolm Knowles’ theory of andragogy and adult learning. These discussions were directed carefully as per Dave Davis’ research on effective CME. Donald Schon’s theory of reflective practice shaped the course homework assignments. These homework assignments were created to allow immediate «reflection in action» with real life patient experiences and «reflection on action» later during presentation of their written essays to the entire class. Participants were asked to complete a survey regarding their self rated knowledge of curriculum topics before and after the course. The results showed improved family physician self-reported knowledge of the curriculum topics. Favourable response to small group discussion and debriefing of assignments showed that there is interest amongst family physicians to these types of interactive learning.
Primary Author:
Language of the presentation: Eng
Carotid sinus hypersensitivity (CSH) is a common cause of fainting and falls in older adults and is diagnosed by carotid sinus massage (CSM). Previous work has suggested that age-related stiffening of blood vessels reduces afferent input from the carotid sinus leading to central upregulation of the overall arterial baroreflex response. A potential intevention to reduce carotid sinus hypersensitivity is aerobic training.
We examined whether aerobic exercise could reverse carotid sinus hypersensitivity in older adults with Type 2 diabetes complicated by co-morbid hypertension and hyperlipidemia.
15 older adults (mean age 72.2±0.7) with diet-controlled or oral hypoglycemic-controlled Type 2 diabetes, hypertension, and hypercholesterolemia were recruited. Subjects were randomized to each of 2 groups: an aerobic group (AT, 3 months vigorous aerobic exercise), and a nonaerobic (NA, no aerobic exercise) group. Exercise sessions were supervised by a certified exercise trainer 3 times per week, and utilized a combination of cycle ergometers and treadmills. Arterial stiffness was measured using the Complior device.
Although aerobic exercise significantly increased arterial compliance as measured by both radial (
Although aerobic training can reverse arterial stiffness, there is no evidence for a corresponding reduction in carotid sinus hypersensitivity in older adults with diabetes.
Primary Author:
Language of the presentation: Eng
Despite the importance of self-care, evidence suggests that people with heart failure (HF) do not consistently engage in such behaviours. One possible reason for poor self-care may be the presence of underlying and undetected mild cognitive deficits (MCD)
This study is prospectively evaluating whether MCD measured with the MoCA in HF patients aged ≥60 years at hospital discharge is associated with impaired ability to self-care (measured with the Self-Care Heart Failure Index (SCHFI – 3 subscales: self-maintenance, self-management, self-confidence).
Exclusion criteria: no caregiver, not English speaking, living in a long term care (LTC) facility, documented cognitive impairment, visual or hearing impairment, or life expectancy.
Primary Author:
Language of the presentation: Eng
Disadvantaged seniors living in non-family situations in Toronto are more likely than seniors living in family situations to have less economic security, less social support, and less choice in housing. Seniors who live in poverty, and are precariously housed, are more likely to be chronically ill, to live with multiple illnesses, to have poor nutrition, high stress and loneliness, all of which are strongly associated with the determinant of health social exclusion.
To understand how support services for income, housing, food security, social support, and health care mitigate the effects of social exclusion, we interviewed 15 male seniors at the Good Neighbours Club in downtown Toronto. The semi-structured interview is designed to assess barriers to, utility of, and perceived impact of support services available to disadvantaged seniors living in the central core of Southeast Toronto.
Results suggest support services play a vital role in not only mitigating the effects of social exclusion, support services reduce the level of social isolation experienced by these seniors.
Primary Author:
Language of the presentation: Fr
Considering the psychosocial factors at play, the management of elderly patients requires an interdisciplinary approach centered on the patient and his/her caregivers. An effective communication between the professionals is nevertheless an important asset in the client’s management. The Individualized Interdisciplinary Intervention Plan (IIIP) is a tool aimed at documenting and communicating information discussed during team meetings. Optimization of the IIIP is necessary to facilitate access to its information, to respect confidentiality and to integrate with existing computerized system.
To devise a computerized IIIP intent on optimizing quality of care and access to patient information.
Modification of the pre-existing IIIP was done based on literature review, integration of the geriatric vital signs (AINÉES), the OPTIMAH (OPTIMisation des soins aux personnes Âgées à l’Hôpital) approach, and training in Project management using the Interprofessional Collaborative Approach. A demo session with team members of the two geriatric assessment units was organized prior to conducting a 6-month trial. A survey was created in order to gather feedback from users in both units.
An updated version of the IIIP was developed. Analysis of the survey is underway and the tool will be modified accordingly.
The updated version of the computerized IIIP assures optimal management of elderly hospitalized patients and their caregivers. Not only is the IIIP accessible and easily integrated in existing computerized system, but it also respects the confidentiality code of conduct. It allows effective communication between interprofessional team members during current or future hospital stays, which is at the core of quality care.
Primary Author:
Language of the presentation: Eng
Given the growing proportion of older people, the prevention of cognitive decline is an important issue for patients, clinicians, and policy makers. There is significant interest in finding the “magic bullet” which will keep us cognitively intact for as long as possible.
To complete a systematic review of the literature to determine the effectiveness of pharmacological therapies for preventing cognitive decline in healthy older adults and in those older adults with mild cognitive impairment.
We searched Medline, EMBASE, and the Cochrane Central Register of Controlled Trials from date of onset to August 2011. No restrictions were placed on date of publication. Publications were excluded if they were not randomized control trials or systematic reviews, were not examining older adults (age > 65) with normal cognition or mild cognitive impairment, if they did not list adverse outcomes of their interventions, or if they were published in a language other than English. Two investigators independently completed study selection, quality assessment, and data abstraction. Quality assessment of articles was conducted using Cochrane Risk of Bias. Our initial search yielded 3,882 potential articles. An abstract review by two independent reviewers narrowed search results to 226 articles that met inclusion criteria. Further assessment of full-text articles resulted in 45 articles for data abstraction and analysis. Data synthesis is underway and will be completed by April 2012.
While final results of the systematic review are currently pending, it is evident from our preliminary results that there are very few high-quality studies that demonstrate any successful interventions to prevent cognitive decline in older adults.
Primary Author:
Language of the presentation: Eng
In Canada, 93% of older adults live at home and a substantial proportion of this population has complex and inter-related health and social problems. This sometimes renders them frail and homebound and poorly-served by predominantly office-based primary care delivery models. Several comprehensive and ongoing home-based primary care models have emerged internationally in order to address access-to-care deficiencies, postpone adverse health trajectories, and reduce overall costs for homebound elders.
To identify the successful operational components of home-based primary care programs.
We completed a systematic review of studies investigating home-based primary care programs for community-dwelling older adults that measured at least one of: hospitalizations, emergency department visits or long-term care admissions as an outcome of their intervention. Using the Cochrane, PubMed, and MEDLINE databases, 322 articles were identified and seven met our criteria for review.
The seven reviewed interventions were all based in the United States, with four emerging from the Veteran Affairs System. All seven programs demonstrated substantial effect on at least one of our inclusion outcomes, with four programs effecting two outcomes. All interventions were characterized by three common design principles: 1) house calls are made by the ongoing primary care provider, 2) the primary care provider leads an interprofessional care team, and 3) the program provides after-hours support.
Specifically designed home-based primary care programs can substantially affect patient, caregiver, and systems outcomes. Adherence to the core design principles identified in this review could help guide the development and spread of these programs in Canada.
Primary Author:
Language of the presentation: Eng
Smoking is common in China, where the population is aging rapidly. This study evaluates the relationship between smoking and frailty and their joint impact on survival in older Chinese adults.
Data come from the Beijing Longitudinal Study of Aging. Community-dwelling people (n=3257) aged 55+ years at baseline were followed between 1992–2007, during which time 51% died. A frailty index (FI) was constructed from 27 self-reported health deficits.
Nearly half (45.6%) of the participants reported smoking (66.8% men, 25.3% women). On average, male smokers were frailer (FI=0.18±0.15) than male nonsmokers (FI =0.14±0.10;
Primary Author:
Language of the presentation: Fr
Benign prostatic hyperplasia (BPH) with bladder outlet obstruction (BOO) can result in lower urinary tract symptoms (LUTS). Early, accurate diagnosis may reduce pain and complications.
To systematically review the evidence on the diagnostic accuracy of office-based tests for BPH with BOO in males with LUTS.
Search of MEDLINE and EMBASE (1950 to August 12, 2010), Cochrane Central Register of Controlled Trials via Ovid, and references of retrieved articles. Data selection: Prospective studies comparing at least one diagnostic test, feasible in a clinical setting and readily available to non-specialist clinicians, to the gold standard reference test, invasive urodynamics.
There were 6692 unique citations identified with 9 prospectively conducted studies (N=1217 patients) meeting inclusion criteria and describing use of 2 symptom questionnaires as well as individual symptom(s). The best constellation of symptoms suggesting BPH with BOO was ‘poor stream and frequency and/or nocturia’ (positive LR, 1.76; 95% CI, 1.17–2.64). The most useful symptom for which the absence made a diagnosis of BPH with BOO less likely, was nocturia (negative LR, 0.19, 95% CI, CI 0.05–0.79). The best symptom questionnaire to support or rule out a diagnosis of BPH with BOO was the International Prostatic Symptom Score (I-PSS) at a cut-off of 8 (summary positive LR, 1.34; 95% CI, 1.06–1.70; summary negative LR, 0.28, 95% CI, CI 0.12–0.70).
Although urodynamic testing is the gold standard for diagnosis of BPH with BOO, symptoms obtained through history may be useful. The best evidence supports asking about nocturia, stream and frequency.
Primary Author:
Language of the presentation: Eng
Le rôle des unités de courte durée gériatriques (UCDG) est d’offrir des soins spécialisés dans le continuum des soins et services de santé offerts à la personne âgée. Les professionnels de ces programmes doivent maintenir leurs compétences cliniques, et les gestionnaires mettre en place des processus organisationnels efficaces. Un besoin d’échange et d’actions spécifiques au niveau national a été exprimé par la majorité des responsables d’UCDG.
Améliorer de façon continue la qualité des soins dans les services hospitaliers de gériatrie, généraliser de hauts standards de pratique afin d’y traiter des patients aux situations cliniques complexes et agir comme milieu de référence. Méthodes : 1) Création d’un comité exécutif composé de médecins et gestionnaires provenant des diverses régions du Québec; 2) Embauche d’une coordonnatrice; 3) Développement d’un site internet (
60% des centres hospitaliers ont adhéré au RUSGHQ. Les activités en cours sont : 1) Circonscrire la population cible des UCDG; 2) Harmoniser les mécanismes d’évaluation et d’intervention cliniques sur la base des meilleures pratiques; 3) Mettre à la disposition des membres une « boîte à outils » clinique et de gestion pertinente; 4) Établir les ratios de ressources professionnelles nécessaires à un fonctionnement optimal; 5) Offrir des activités de développement professionnel continu.
Une communauté de pratique en gériatrie a été mise sur pied facilitant réflexions et apprentissages collectifs des professionnels de la santé et des gestionnaires travaillant en milieu hospitalier.
Primary Author:
Language of the presentation: Eng
Mild cognitive impairment (MCI) is a heterogeneous condition affecting up to 40% of seniors. Almost a third with MCI will progress to dementia. Similarly, gait abnormalities, depressive symptoms, and executive dysfunction are commonly found in seniors, and this “triad” has been linked with brain ischemic lesions. To date, the presence of such a “triad” and its relationship with vascular risk factors (VRF) has not been described in MCI. We hypothesized that seniors with MCI who have high VRFs will be more likely to exhibit the “triad” of gait abnormalities, depressive symptoms, and executive dysfunction.
Baseline data from 62 participants of the “Gait and Brain Study”, an ongoing prospective cohort of seniors with MCI at London, Ontario, was used for this project. Biannual assessments include executive function test (Clock Drawing and TMT B), quantitative gait analysis (velocity), and depression ratings (Geriatric Depression Scale), among other evaluations. VRFs were assessed at baseline using a modified Vascular Risk Factor Index which ranges from 1 to 7.
Forty-four percent of the participants had at least one VRF. There was a significant association between the number of VRFs and the presence of the triad (MANOVA, F(3,36) = 3.41,
VRF were prevalent in our MCI cohort. VRFs were associated with the specified triad. A future prospective analysis of this cohort should elucidate causal mechanisms for this relationship. VRFs may play an important role in the development of cognitive, mobility, and mood dysfunction in people with MCI.
Primary Author:
Language of the presentation: Eng
TAVI decreases mortality and morbidity in older patients who are deemed inoperable or at high risk for surgical aortic valve replacement. Premorbid functional status and rates of geriatric-specific postoperative complications have not been well described. This study aimed to clarify these issues.
Data collection occurred through the Division of Cardiology at St. Paul’s Hospital in Vancouver, Canada. Information on activities of daily living (ADLs), instrumental activities of daily living (IADLs), clinical frailty score (CFS), timed up and go (TUG), and a mini-mental state examination were collected prospectively by a study nurse. Patient charts were reviewed for medical co-morbidities, cardiac-specific metrics, pre-specified delirium criteria, complications, and discharge disposition.
Twenty-six cases were reviewed. The average patient age was 80 years and average Charlson Co-morbidity Index score was 3.5. Despite the advanced age and presence of significant co-morbidities, the incidence of delirium was low at 8% (2/26), with only 15.5% (4/26) receiving psychotropic medications during the hospitalization. All patients with available functional data were independent for ADLs at baseline (18/18), with 89% (16/18) requiring assistance with 2 IADLs or less. The mean scores on the CFS, TUG, and MMSE were 4, 12.8 seconds, and 27.9, respectively. Ninety-two percent (16/18) of patients were discharged home, with two patients going to a rehabilitation institution and eventually being discharged home.
Appropriately selected older adults, with the functional and cognitive attributes noted above, appear to tolerate this procedure very well from a geriatrics point of view. Studies involving larger patient populations are warranted.
Primary Author:
Language of the presentation: Eng
Socio-economic status is related to health both at the individual and country level. The health status of the older population of each country can be monitored by measuring its frailty status.
To examine the relationship between the Frailty Index (FI) and national economic indicators.
30,025 participants aged 50+ years (13,700 men, 16,325 women) from 12 countries (Austria, Belgium, Denmark, France, Germany, Greece, Israel, Italy, Netherlands, Spain, Sweden, Switzerland) which participated in the Survey of Health, Ageing and Retirement in Europe comprised the study sample. Following a standard procedure, an FI was constructed from 71 items. The economic indicators used for cross-country comparison were: gross domestic product (GDP), gross national income (GNI), health expenditure, and an inequality measure.
Across countries, the mean FI increased with age and was higher in women. Between countries, the mean FI ranged from 0.11 (Switzerland) to 0.21 (Israel). GDP, GNI, and health expenditure were negatively correlated with both the mean (r = GDP −0.85; GNI −0.86; health expenditure −0.86).
Primary Author:
Language of the presentation: Fr
The training of Specialist Geriatricians (SpGrtn) within Canada has not kept pace with the aging of the population over the last 15 years. The anticipated retirement of existing SpGrtns in Canada will exacerbate the shortfall for specialized geriatric services (SGS) across the country.
1. To document the existing number of SpGrtns and practicing Care of the Elderly (CofE) trained Family Physicians practicing in SGS. 2. To project the anticipated number of SpGrtns that will retire over the next 15 and 30 years. 3. To calculate the ideal number of Geriatricians in Canada, based on published ratios.1,2
Using the ratio of 1.25 SpGrtns: 10,000 people 65+1 or 1 SpGrtns: 4,000 people 75+2 and 2006 Canadian Census data (low, med. and high pop. projections 65+ or 75+) over the next 30 years, the need for SpGrtns was identified. The anticipated retirement of present Canadian SpGrtns 40 years beyond their medical degree (MD) was determined.
In 2011, there were 256 SpGrtns in Canada and 93 CofE physicians. The calculated need in 2011 is 613 SpGrtns (1.25:10,000 65+) or 688 (1:4,000 75+). The calculated need for SpGrtns in 2026 is 969 (±27 (1.25:10,000 65+). Across Canada, 10 SpGrtns are trained annually (150 in 15 years). Over the next 15 years, 105 of the existing SpGrtns will have practiced 40 years beyond the date of their MD.
In 2026 there will be 301 SpGrtns (256- 105+150) resulting in a shortfall of 668 SpGrtns (969–301) in Canada.
Primary Author:
Language of the presentation: Eng
Décrire les différents volets du programme Participe présent qui vise à promouvoir la participation communautaire des aînés vivant avec un problème de santé mentale.
Relater les principales étapes de la démarche de co-construction d’innovations sociales utilisée pour développer ce programme de promotion de la santé.
Les problèmes de santé mentale (PSM) sont associés à la stigmatisation des aînés, à des répercussions sur leur santé et à plusieurs facteurs de risque (pauvreté, itinérance, longévité réduite) (Fleury et al., 2012). Cette présentation décrit un programme visant à promouvoir la participation sociale des aînés vivant avec des PSM. Les étapes de développement du programme et le résultat de sa validation par un comité consultatif seront exposés.
Une démarche collaborative a sous-tendu le développement du programme. Les principales étapes sont: formation d’un comité consultatif, étude des besoins, recension d’écrits, élaboration du modèle logique, conception des outils du programme, validation des outils et mise à l’essai auprès de la population cible (en cours). La validation du programme a impliqué des intervenants, des chercheurs, des organismes communautaires et des personnes qui vivent avec des PSM.
Le programme Participe-présent vise à renforcer les compétences personnelles en matière de participation sociale et à créer des conditions favorables dans la communauté. Il comporte quatre volets: 1) une entrevue individuelle inspirée de l’entretien motivationnel, 2) un atelier éducatif de groupe (7 rencontres); 3) un projet collectif de communication médiatique et 4) des visites d’organismes communautaires. Le programme peut être offert par des professionnels de la santé, des intervenants du milieu communautaire ou des pairs aid-ants, à des groupes de six à dix aînés vivant avec des PSM.
Après sa mise à l’essai, Participe-présent pourra être diffusé largement afin de mieux outiller les intervenants à promouvoir la participation sociale des aînés qui vivent avec un PSM.
Author Presentations: 9:15 AM – 10:15 AM
Author Presentations: 1:30 PM – 2:30 PM
The 2014 abstracts are organized by both content and pedagogy to help participants navigate more easily through the poster session. The content themes are based upon the ASM Recommended Curriculum Guidelines for Undergraduate Microbiology Education (
In May 2012, a Perspectives article published in the
For the purposes of ASMCUE, a seventh concept, advancing STEM education and research, has been added to the abstracts in order to identify authors working in this broader-scoped area. The seven concepts are therefore evolution, structure and function, pathways, information flow, systems, the impact of microorganisms, and advancing STEM education and research.
The pedagogy themes are organized into five categories: course design, hands-on projects, student learning, teaching approaches, and teaching tools.
Each abstract is assigned to both content and pedagogy themes. These assignments, designated by the submitting author, are indicated below the abstract. A quick reference index listing abstracts by content is available on p. 79, and by pedagogy on p. 79-80.
University of Calgary, Calgary, Alberta, Canada.
The University of North Carolina at Charlotte, Charlotte, NC.
San Diego State University, San Diego, CA.
American Academy of Microbiology Topic: The Human Microbiome
Spring Hill College, Mobile, AL.
Clayton State University, Morrow, GA.
Grinnell College, Grinnell, IA.
University of California San Diego, La Jolla, CA.
1City College of the City University of New York, New York, NY; 2University of Texas, Austin, Austin, TX.
North Carolina State University, Raleigh, NC.
Mount Mary University, Milwaukee, WI.
University of the Incarnate Word, San Antonio, TX.
University of California Los Angeles, Los Angeles, CA.
Duquesne University, Pittsburgh, PA.
North Carolina State University, Raleigh, NC.
Southern Oregon University, Ashland, OR.
Salisbury University, Salisbury, MD.
Johnson County Community College, Overland Park, KS.
Washington & Jefferson College, Washington, PA.
Washington & Jefferson College, Washington, PA.
University of Maryland, College Park, MD.
John Hopkins University, Baltimore, MD
Monash University, Melbourne, Australia.
St. John Fisher College, Rochester, NY.
Hypothesis testing is central not only to the scientific method but also to understanding the nature of scientific knowledge. Although it is widely appreciated that students should develop hypothesis-testing skills earlier in their undergraduate careers, there are many challenges preventing students from engaging in scientific inquiry in large-enrollment classes (500–1000 students). The hypothesis of this study is that by combining collaborative in-class activities with problem-based computer simulation software, an effective environment can be created to foster both content acquisition and the development of scientific inquiry skills such as hypothesis testing. Four inquiry-based modules were introduced in winter 2012 to cohort A, a class of 500 students in introductory biochemistry. Each module consisted of a podcast reviewing essential concepts, an in-class peer-learning activity based on formative assessment principles and an interactive student-centered JAVA computer simulation in which each student is provided with the opportunity to design a virtual experiment, formulate a hypothesis, and record and interpret the results of the simulation. To assess the impact of these modules on learning, three approaches were developed. First, the ability of students in cohort A to solve problems that required hypothesis testing was compared with that of students who completed the course prior to the introduction of the four modules (cohort B). Whereas 90% of students in cohort A correctly solved these problems, only 50% of students in cohort B were successful (
The literature is clear that the non-science majors’ population is largely dissatisfied with their experiences in college science courses and that relying solely on lecturing techniques is not the most effective way to reach students. We teach non-majors biology to multiple sections of 200 students, many of whom feel unprepared for and uninterested in college science courses. Our goal was to redesign our course to be more engaging and to enhance student success and achievement as measured by end of semester grades and feedback provided by students. A complication was that there were neither financial nor human resources provided for implementation of the redesigned course. In order to work around these constraints, undergraduate students were recruited to serve as learning assistants (LAs) in our course. The LAs were high-achieving former students who showed a strong interest in science and peer leadership. The LAs were unpaid but received credit for their work by attending a seminar course we designed to discuss activities, brainstorm implementation, review the pedagogy behind activities, and consider relevant literature. In the non-majors course, the LAs provided frequent individualized interactions with students while facilitating group work. We noted that students who felt uncomfortable talking to their instructor seemed more comfortable voicing questions and concerns to a peer LA. Overall, we were able to redesign our course so that it actively engages students, encourages collaborative learning, and provides frequent formative assessment and feedback to students. Prior to implementation of the redesigned course, the proportion of students (N=1186) earning a grade of D or F in the course was 25.92% with an average grade of 2.28 (on a 4.0 point scale). For the three semesters following the implementation of the redesigned course, the proportion of students (N=995) earning a grade of D or F in the course was reduced to 19.82% with an average course grade of 2.51. Additionally, feedback related to student satisfaction in the course was very positive. We expect to see a further reduction of D and F grades as we refine our redesign and continue working with LAs.
The revolution in DNA sequencing technology continues to dramatically change microbiology. The training and recruitment of the next generation of researchers who are able to use these new technologies is vital, but lagging. We developed a cross-disciplinary class that provided practical experience in using next generation sequencing instruments, covering every step from DNA extraction, through sequencing, to genome annotation. The class has been taught 3 times and the students have sequenced approximately 40 microbial genomes, 60 metagenomes, and the California Sea Lion.
The student body was culturally diverse, and included Junior and Senior undergraduates, MSc and PhD graduates. The course brought together biologists and computer science students. By having the two groups of students work side by side, the computer scientists learned biology and the biologists learned computational constraints. Both groups of students learned research techniques.
We hypothesized that students would learn about DNA sequencing and increase their ability to conduct science. Students’ perceived ability was measured in 1) the scientific process, 2) genomic sequencing and annotation, 3) biology, and 4) computer science. A 66-item pre- and post-test was conducted. The surveys used a Likert scale and reliability was tested using Cronbach’s alpha. Significance was tested using matched paired
This K–12 collaboration examines the influence of biology community service on academic at-risk freshman biology majors. The first semester biology course at Spring Hill College has a history of 27% of the students not succeeding (W/D/F). Analysis of the data since 2007 showed that more than 60% of the freshman biology majors with ACT scores of 21–23 did not succeed in this course (W/D/F). Freshman students with ACT scores 24 and higher were likely to succeed in their first biology course. The population of students with ACT scores below 21 rarely earned a grade of C or better. The goal of this study was to determine if service learning in the form of teaching biology to 7th graders would increase the success rate of the academic at-risk college freshman with ACT scores of 21 to 23. It was also hoped that academic success in the first semester biology course would increase the retention of biology majors. The community service opportunity was offered to freshman biology majors with ACT scores of 21–23 in the fall semesters of 2012 and 2013. The college students created several lab activities plus weekly fun review activities for a 7th grade biology class at an under-served middle school (98% of the students at this middle school are on the free lunch program). One hundred percent of students who completed the freshman biology course earned a C or better (
Applied microbiology laboratory activities are outstanding additions to undergraduate microbiology courses since they encourage students to apply their knowledge of basic microbiology to an interesting topic that is relevant to their lives. Our proposed hypothesis is that an inquiry-based laboratory activity that covers the microbiology and biochemistry of cheese production will result in a high level of student engagement and will improve the students’ abilities to meet learning outcomes related to cheese production and cheese biology. During this activity, students designed experiments to answer a research question related to the effects of different types of rennet on aspects of cheese production. Six learning outcomes linked to skills and knowledge of cheese production, the microbiology/biochemistry of cheese, and the process of science were assessed using pre- and post-quizzes, laboratory reports, and surveys. On the surveys, students rated their skills and knowledge related to cheese production after completing the activity. The average score on this survey was 4.2 out of 5. The average grade (66.6%) on the pre-quiz was significantly lower than the average (77.8%) on the post-quiz (student
There is interest in using investigative learning in courses to generate the benefits of research experiences, in a way that is more broadly available and less resource intensive. In this study, we look at whether a single introductory biology course can provide students with comparable learning gains to a summer research experience. Grinnell College’s BIO150 Introduction to Biological Inquiry course introduces students to how biologists pose questions, design experiments, analyze data, and communicate. Individual sections have different topics, but all sections involve intensive student-directed investigation and are taught in a workshop format. Since the course design was modeled on research, it is expected that outcomes from such a course would be comparable to those of an undergraduate summer research experience. Learning gains reported from the CURE (Classroom Undergraduate Research Experience) survey by students from two sections of BIO150 are compared to those reported on the SURE (Survey of Undergraduate Research Experiences) by students who recently completed a summer undergraduate research experience. Mean student responses (
Authentic research experiences increase student engagement in science. But do these experiences affect every student equally? We examined two factors that could influence the effect of research on student attitudes about science: gender and previous research experience.
We have incorporated an authentic research module into several large courses at UC San Diego. Students are using DNA barcoding to create a species inventory of the Scripps Coastal Reserve. Students are gaining skills in molecular biology and bioinformatics while adding novel data to the Barcode of Life Database and establishing baseline ecological data for future conservation efforts.
We used a pre/post survey design to quantify how student attitudes changed over the quarter that included authentic research. The survey was developed by the authors to address the specific goals of this project. A priori, we categorized questions into 5 topics: excitement about research, interest in collaboration, tolerance of ambiguity, environmental engagement, and students’ perception of themselves as scientists.
We determined that across all questions, the attitudes of female students changed more than the attitudes of male students (F(1,25)=15.2,
In conclusion, students do vary in the way that research alters their attitudes. These results indicate that certain groups of students may benefit more from research than previously recognized. We are currently conducting focus groups to gain a more qualitative understanding of this variation.
There is general agreement that traditional classroom approaches fall short in supporting learning, but student resistance to change may discourage innovation, especially for faculty assessed only by student post-course evaluation. Concerns about negative student reaction may thus affect faculty willingness to apply evidence-based nontraditional strategies.
We use intensive analysis of primary literature to demystify and humanize science through the CREATE (Consider, Read, Elucidate hypotheses, Analyze/interpret data, and Think of the next Experiment) strategy. In a 2007 expansion of CREATE we trained NY/NJ/PA college faculty who subsequently (2008) taught their own courses using CREATE. We hypothesized that students of these first-time CREATE implementers (CIs) would have an overall positive reaction to CREATE. Such reaction could support students’ learning by influencing their motivation, while also facilitating expanded use of this nontraditional strategy on the implementers’ campuses.
We tested the hypothesis by including a survey of student reaction to CREATE as one of a suite of cognitive/affective assessments used by the PIs and the study’s Outside Evaluator (OE). We also followed up with CIs 5 years later regarding potential expanded use of CREATE on their campuses. The OE collected 104 open-ended written student responses to the prompt: “How do you feel about the CREATE method of instruction and your learning in this course?” A total of 79% of responses were positive (range: 70–100% on individual campuses). Positive comments fell into 14 categories. Negative comments were seen in three categories, each of which (e.g. concept mapping) was also mentioned by other students as a positive. Thus in all seven implementations, students’ overall response to CREATE was positive. CIs continue to use CREATE and, on multiple campuses, additional faculty now use the strategy for other courses. We conclude that student reactions to their first exposure to nontraditional CREATE teaching are overall positive, potentially supporting both learning and the adoption of CREATE strategies by additional faculty. We thank NSF for support through DUE 0618536.
At North Carolina State University, the General Microbiology lecture was a large-enrollment (>1000 students/year), critical-path course taught year-round in multiple sections by different instructors. Large lecture halls with one instructor delivering a passive lecture were the standard model of instruction. As expected, this environment leads to passive learners; students receive information and internalize it by memorization. In order to improve the student classroom experience, reduce course drift and drop/failure/withdrawal (DFW) rates, a large course redesign was undertaken. The intention of the redesign is to ultimately improve student engagement and learning outcomes by moving students into a learner-centered model of instruction. The hypothesis is that a redesign that incorporates such a model would lead to student retention, increased interest, and deeper understanding.
To achieve this model, we adopted the Student-Centered Active Learning Environment with Upside-Down Pedagogies (SCALE-UP) approach. The basic idea of SCALE-UP is to give students something interesting to investigate in order to construct new knowledge. Students sit in three groups of three students at round tables with networked laptops. They work in collaborative teams on case studies based on engaging real world problems while the instructor roams around the classroom providing short explanations and asking leading questions. In order for students to be prepared to learn in this format, they are expected to listen to and view pre-recorded lectures prior to class. This “flipped” aspect of the classroom is intended to free up in-class time. Preliminary analysis of the final course grades indicates a higher course average, lower DFW rate, and increased class attendance when compared to traditional lecture classes. Analysis of student responses to critical thinking questions indicates increased conceptual understanding and better problem-solving skills. In conclusion, the redesign of the in-class environment utilizing the SCALE-UP approach has positively impacted student learning gains, and has the potential to influence future course redesigns.
This study examines the effect of explicit instruction on improving problem-solving skills and students’ confidence in their problem-solving ability. Problem solving is a key component in the process of science. The average score on complex problems presented as homework questions in an upper level microbiology course was 75% across 2009–2011. Student evaluations and informal interviews indicated students did not have the skills or the confidence to approach and successfully solve complex problems. In 2012, I implemented a new assessment approach using individualized feedback and targeted interventions for each homework problem. The average score on the same homework problems was 80%, with an average improvement of 4% over the semester (
While recent reports suggest that authentic research improves STEM education, classroom-based research projects are difficult to implement and their effectiveness for improving student learning in specific disciplines needs further study. Our hypothesis is that students who first experience a classroom-based authentic research project (ARP) retain knowledge of genetics concepts better than students who first experience a traditional laboratory project (TLP). Study participants were enrolled in different sections of an upper division Genetics & Lab course. Study participants were assigned to two treatment groups. Treatment A students (
Studies have shown that science majors enter into their introductory science coursework with misconceptions about evolution and natural selection, which may affect their ability to understand and apply evolutionary concepts to their daily life and education. Teaching this topic over a single chapter or course module generally does very little to mitigate student misconceptions. We tested the hypothesis that repeated exposure to the process of natural selection over the duration of an introductory course will increase students’ conceptual understanding of evolution and their mastery of related key concepts pertaining to natural selection. A pedagogical intervention consisting of question progressions relevant to evolutionary concepts was designed to help students apply and integrate evolution as a governing principle across the course. The intervention was assessed by administration of the Conceptual Inventory of Natural Selection (CINS) Pre/Post course. Students made significant (
For many doctor of pharmacy (PharmD) students, basic sciences and clinical pharmacy often appear disconnected. In the infectious disease field, PharmD students additionally struggle with mastering the diversity of microorganisms and the corresponding variety of therapies. The objective of this study was to design an interdisciplinary project that would integrate fundamental microbiology with clinical research and decision-making skills. The Emerging Microbe Project guided students through patient cases from the first stages of microbe identification via sequence analysis to defending a therapeutic strategy for an infected patient in a case study. The unknown pathogens were selected because they had not been seen in the classroom or in the students’ clinical practice sites, so pathogens and corresponding therapies were new to the students. We hypothesized that the students would develop a better understanding of how microbiology fit into clinical practice and that they would gain confidence and skill in independently selecting appropriate anti-microbial therapies for a new disease state. We demonstrate that the Emerging Microbe Project significantly improved student learning through multiple assessment strategies and increased student confidence in their clinical infectious disease skills. Direct evidence of student learning was assayed through a microbiology report, a care plan, and through exam questions before and after the project. Students demonstrated substantial improvement in mastering learning objectives (average individualized learning gain=0.38,
In an effort to provide the student population of North Carolina State University with exposure to flow cytometry and ELISA, we designed a novel half-semester course that focused on introducing students to applications, experimental design, and data analysis aspects of these techniques. We hypothesized that the course student learning outcomes (SLO), which included higher-order skills defined by Bloom’s Revised Taxonomy (BT), would be attained upon completion of this course. The SLOs for this course were to: identify applications of flow cytometry and ELISA and compare to other techniques (BT analyze), describe how a flow cytometer operates, design and troubleshoot flow cytometry experiments (BT evaluate and create), analyze flow cytometry data (BT analyze), compare different types of ELISA (BT analyze), design and troubleshoot ELISA experiments (BT evaluate and create), and analyze ELISA data (BT analyze). Undergraduate (
Significant research experience is a desirable, and often mandatory feature of undergraduate Biology and Microbiology degree programs. It is difficult to provide all students with research experience at universities that have high student-faculty ratios or limited facilities. At Southern Oregon University, community partners are leveraged to provide undergraduates with applied research projects. Partnering with the State Department of Environmental Quality (DEQ) and local government officials has resulted in a win-win-win situation for students, faculty, and the community. Students gained valuable research experience, the faculty burden of supervising multiple student research projects was lessened, and the DEQ completed community projects that would have been impossible without volunteer student researchers. I investigated whether students who completed DEQ research projects were able to demonstrate proficiency in experimental design, data collection, data analysis, and presentation as well as students who worked on non-community oriented projects. The quality of student DEQ research projects was assessed and compared to the quality of student research projects carried out under Biology faculty mentors without involvement of a community partner. An assessment rubric was used to score 23 student presentations of on-campus research projects and 8 presentations of DEQ-based projects. To reduce bias, each presentation was scored by an average of 5 different faculty members. Mean scores were only slightly higher for students who did DEQ research and statistical tests failed to reveal significant differences between the two groups of students. The assessment indicated that students who completed DEQ projects gained effective research experience. Results from projects such as identification of microbial pollution sources, effects of irrigation water on stream quality, and monitoring of water recreation sites were highly beneficial to the community as they were used to guide local watershed management decisions.
Student drawings have been recognized as an important learning strategy in science (e.g. Ainsworth et al., 2011, NAS 2011). For example, drawings can promote model-based reasoning, which is one of the six Vision and Change core competencies (AAAS, 2009). Does the creation of visual models by the learner promote learning because it is a form of active learning, because it is a visual process, or both? This study tested the hypothesis that visual modeling is effective because it is both active and visual. Introductory biology students (
Scientific writing fuels the dissemination of knowledge in the field of biology, yet many students struggle with articulating the scientific method through writing. A course was developed to introduce students to scientific literature and guide them through reading, analyzing, and synthesizing information to produce an original scientific paper. Students developed an original question via a guided exploration of literature, and authored a scientific report based on a central hypothesis, integration of primary literature, and original data sets. A student-centered approach that utilized peer critiquing and assessment in a group setting was implemented throughout the writing and revision process. It was hypothesized that if students were trained to evaluate the work of others using a standardized rubric they would gain a better understanding of how writing is assessed, which would lead to improvements in their own approach to writing. Each student also submitted a justification for assigning a particular grade, and these justifications were assessed by the instructors. Outcomes were measured using student self-assessments, weekly reflections and course performance. Data indicated that students gained significant knowledge in key skill and content areas including writing the sections of a scientific report, reading and analyzing scientific literature, use of a standardized rubric, and peer assessment of scientific writing (from little knowledge to very knowledgeable, Likert scale, self-reported by students). In addition, students felt that working in a group facilitated multiple perspectives, helped develop strategies for solving conflicts, aided in identifying weaknesses within their own writing, and increased confidence in public speaking and justification of arguments. A clear trend in writing quality was observed through grade analysis, and average assessment grades improved by a full letter by the end of the course (from C+ to B+ average). In conclusion, peer assessment resulted in improved writing skills, increased self-awareness during the writing process, and an enhanced understanding of scientific literature.
The Boston College Biology Department has a three-credit sophomore-level laboratory course, which we have engineered to engage large numbers of undergraduate students in authentic research projects. For the scientific project, students study the phylogenetic conservation of the enzymes involved in methionine biosynthesis. Conservation of MET gene function is tested by cross-species plasmid complementation of
Pre- and post-course evaluation instruments include concept tests and student self-assessed confidence and learning gains. Comparison of pre- and post-course scores of over 350 students on a 20-question concept test showed an average increase of 6.35 correct answers (8.7/20 to 14.9/20), with consistency across multiple semesters. These post-course scores correlated with final grades. Interestingly, self-assessed confidence gains showed no correlation to final course grades. We found that adoption of a semester-long project narrative, emphasizing scientific communication, correlated with significant increases in core knowledge. This research project was deliberately designed to have a flexible format that could be easily adopted for metabolic pathways in other genetically-tractable organisms with sequenced genomes. Course materials are freely available on a website.
The flipped classroom has been shown to be an effective way to engage students and deliver active learning. The flipped classroom is described as delivering the majority of course content outside of class and using class time to develop higher levels of understanding of the content and spend time solving problems in the course. Since introducing the flipped model into a pre-allied health microbiology course, course evaluations have been mixed. The purpose of this study was to understand why a large number of students in the course had a negative attitude about the flipped course method. Students who have been previously successful in traditional classroom environments seemed to have the most problems with the flipped classroom. Therefore my hypothesis was that students with higher GPA coming into the course would have a more negative attitude about the course format than students with a lower GPA. To explore attitudes about the course, students (
To go from ‘Vision’ to ‘Change’, an inquiry-based laboratory curriculum with a semester-long research project was implemented in an introductory biology course for science majors. Students characterized soil bacteria at a Field Station, proximal to a Marcellus shale gas drilling pad. Students worked in small groups and were involved in all stages of the project – from constructing the hypothesis to poster presentation. It was hypothesized that this lab would result in greater student engagement while also allowing for conceptual mastery. After two years of implementation (
The intricacies of DNA replication in prokaryotes and eukaryotes are difficult and yet crucial for students to master. To achieve better student learning and engagement, flipping the instruction is an option. But, would selective ‘flipping’ of difficult topics be conducive to student learning? To investigate this, flipped instruction of DNA replication was implemented in an introductory biology course taught to science majors. It was hypothesized that flipping the classroom, by substituting lecturing with problem solving, would improve student learning. To test this, DNA replication was taught to two sections of students using the traditional lecture style [
The Host Pathogen Interactions Concept Inventory (HPI CI) consists of 18 multiple-choice questions validated through an iterative process (Marbach-Ad et al. 2009, 2010). After students answer each question, they are asked to write an explanation for their response. We suggest that reviewing students’ open-ended explanations to HPI CI responses will allow definition of student alternative conceptions (ideas that differ from valid scientific explanations) that will serve to inform faculty involved in curriculum development. The HPI teaching team (20 research and teaching faculty) have developed a method to mine HPI CI data for alternative conceptions. This process involves working in teams to read student responses, developing statements that define common problems in student thinking, grouping these together to define a set of codes and then using that code book to quantify the prevalence of the codes in the data set. We will report on the analysis of one question that targets student understanding of antibiotic resistance. Comparing analysis of student explanations to CI responses on pre-course vs. post-course implementation of the CI (∼800 responses from students in introductory microbiology) we defined 20 alternative conception codes. We quantified the prevalence of the codes in the data set and determined the codes that were most frequent. We then collapsed the coding into larger categories. Categories most resistant to change from our teaching efforts included “Antibiotics cannot be degraded by bacteria – not a possible mechanism for resistance” (17.9% of responses pre-course to 15.6% post-course); “Gene expression cannot change in response to antibiotics or other environmental factors” (17.9% of responses pre-course to 26.6 % post-course); Selective growth is not necessary for evolution (7.1% of responses pre-course to 7.4% post-course). Our approach allows us to uncover student alternate conceptions and serves as a “bridging the gap” exercise that informs faculty about the distinctions in their way of thinking in comparison to student or novice thinking.
Due to the clear benefits of collaborative learning, we encourage students in our large biochemistry lecture course to study in groups. However, our anecdotal observations indicated that students use traditional study methods instead of more active methods, including group studying. We hypothesized that in-class group problem sessions would increase the use of and/or attitudes about group studying. In fall 2013 we implemented two in-class problem sessions with 265 students randomly assigned to groups of 5. We evaluated studying behavior and attitudes using a survey of study habits given to students at the beginning (pre-) and end (post-) of the semester and to alumni (2012) who did not participate in the in-class problem sessions.
Consistent with our previous observations, less than half of students regularly study with others. Of the 2012 students surveyed (
In conclusion, ∼40% of biochemistry students regularly use group studying. Interestingly, 10–15% of students don’t regularly participate in group studying, but think it is effective. Although student responses to the in-class problem sessions were quite positive, the problem sessions did not significantly affect short-term behavior or attitudes on group studying. These results will inform our efforts to revise the biochemistry course to encourage collaborative learning inside and outside of class.
Despite requiring skills in critically interpreting and evaluating presented scientific data, many students skip the results section of a journal article and instead rely solely on the authors’ conclusions. This study examined methods to re-focus final-year biological science students onto the results of a paper, rather than the discussion. Two activities were introduced into the unit/subject. 1. Journal club presentations (un-assessed) given by the lecturers. 2. Interactive tutorials in which students discussed and presented their interpretation of the results from journal articles. The tutorials were directly assessed in questions on the final exam. The hypotheses for this study were that these activities would improve students’ 1) attitudes towards scientific data, 2) confidence in interpretation of data, and 3) skills in data interpretation, by repeated exposure to results interpretation, opportunities for practice, and the motivation of directly aligned assessment. Their attitudes and confidence were assessed in pre- and post-surveys and their skills assessed with embedded pre- and post-tests on simple molecular genetics data interpretation. The students showed no significant changes in either their confidence in reading journal articles or interpreting data, but did improve in their attitudes toward the fallibility of published scientific research (
Gordon, Catchpole and Baker
Anecdotal evidence suggests these prescribing tests have not been popular with students, and a recent poll (by the authors of this letter) of students whom had taken this test in 2013, added credence to this with 63 out of 76 (83%) stating they found the prescribing test of no benefit.
Unfortunately, prescribing tests seem to be a last ditch attempt by medical educators to convey the message to the public that medical students have been trained and examined in prescribing, whilst arguably not being not the case. The authors would argue, that the first group of students to take this will have more than likely received no formal training, tutorials or lectures regarding the skills of prescribing – the skills will have been expected to have been picked up on already busy placements. If students are to be expected to take an exam in prescribing then some provision must be made within the curriculum to allow them to learn the skills needed to pass this exam. To reduce iatrogenic medication errors and have competent medical undergraduates then prescribing skills must be instigated from an early stage in the undergraduate curriculum.
Comparison can be made to communication skills which have only recently been given a central role in the UK undergraduate curriculum, perhaps prescribing is next.
The authors report no conflicts of interest in this correspondence.
One of the most surprising and, at the same time, most frustrating aspects of the continual rise in the use of botulinum toxin type A (BoNT-A), particularly in aesthetic applications, is the sheer number of reviews currently being published. So far in 2013, there have been seven single or joint reviews of BoNT products focusing on facial aesthetics.
The frustrating aspects of these reviews cover two areas: Firstly, they inevitably speculate about why there are “apparent differences” between the products. They attempt to use the science of BoNT-A to explain these differences. This speculation is both inappropriate and weak. In fact, the majority of differences between the products seen clinically are, by far, due to simple dose differences used in studies, especially when two or more products are being compared.
Secondly, these reviews usually have tables of data about the products which contain, according to the authors, “clinically relevant” information. They are claimed as “pharmacologic differences”. Again inevitably, they are not. The data are mostly irrelevant to clinical practice and, by far the worst situation, they are actually incorrect. Readers are presented with data that are sometimes not even referenced and clearly have not been cross-checked with the appropriate sources – such as the manufacturers’ own product literature.
The latest review by Prager
The table of product data presented by Prager (Table 2)
To start with (and also applicable even to the title of the paper), the United States approved names for the products (incobotulinumtoxinA, onabotulinumtoxinA, abobotulinumtoxinA) have been used. Why? These names are only applicable to the United States and have no relevance in Europe. They are not generic drug names since there is no such thing as a “generic biologic” product of BoNT-A. The BoNT-A products are each considered very individual and the units of potency are not interchangeable, as required by the regulatory authorities worldwide. Dr Prager is a German clinician, working in Germany, the writers of the article are based in the UK, the sponsor of the article is a German company. Europe has managed perfectly well with the original product trade names for 25 years, so why are these names used by Prager?
There are six full lines of Table 2
In particular, the continuing and highly confusing information being supplied in reviews about the so-called “toxin complex size” is given by Prager, yet again.
Prager has additionally failed to recognize that Galderma is the company marketing Azzalure® within Europe, not Ipsen or Medicis, which is very strange given that Galderma and Azzalure® are strongly represented in Germany. Previous comments in an earlier review by Prager et al,
Finally, if the storage temperature of Xeomin®/Bocouture® (Merz Pharma GmbH & Co. KGaA, Frankfurt, Germany) does rise above 25°C, then refrigeration of those products will be required. A statement “no refrigeration required” cannot be made, as in Table 2 of Prager.
All of these errors in the Prager review
However what is the final and perhaps most strange aspect of this review is that, given the Merz sponsorship, there should be no errors and no speculation about the BoNT-A products. There is no place for such failures in the world of evidence-based product assessments today.
The author is Director and Founder of Toxin Science Limited, Wrexham, UK and Adjunct Professor at the Botulinum Research Center, UMASS Dartmouth, Dartmouth, USA. He is also Head of Development for Q-Med AB, a Galderma Division.
The opinions and comments expressed in this article are those of the author and Toxin Science Limited alone.
Dear editor
Firstly we would like to thank Upatising et al for undertaking an interesting and relevant study.
With shifting society, the attitudes and expectations of health care are changing significantly. With increased pressures on waiting list times, drug provision/administration, and service provision, patients become more critical and budgets become tighter. Outside of health care the technological advancement is astonishing. Smartphones, robotics, tablets, the internet has truly revolutionized health care provision and training in countless ways.
The proposed union of this technological advancement with ‘older adults,’ as a mechanism to prevent hospitalisation through access to health care via telemonitoring is one which can help reduce the pressure on the health service. However more development and research needs to be undertaken to expand this proposal. Through the clinical use of telemedicine, frail individuals would be kept out of the hospital system, in which they often can become ‘trapped;’ allowing them to stay at home, where many wish to be. This equates to additional bed space, more nurse availability (as often these patients need more care than others), and a lesser reduction in mobility (very often frail patients in hospital become quickly immobile, which is unlikely to have occurred if they had still been at home).
An issue however surrounds the ever changing systems in health care, such as ObamaCare and the National Health Service in Great Britain. In these organizations with significant financial and political input from their governments, amendments and additions such as implementing telemedicine centrally, would be problematic and likely to have as many opponents as proponents given the results of this study.
This trial
The authors report no conflicts of interest in this communication.
We would like to applaud the authors of the article “The worldwide epidemiology of acute rheumatic fever (ARF) and rheumatic heart disease (RHD)”
The authors state that: the evidence of serologic infection is based on one of “a pharyngeal swab culture positive for GABHS, positive rapid GABHS antigen test or rising serologic antibody titers”. Very few countries in highly affected regions of the world utilize routine rapid antigen or microbiological testing due to the cost involved. Furthermore, the delay involved in repeat serological testing before a diagnosis can be made, will result in missed diagnoses and tragic consequences. We recommend therefore that elevated serological markers of previous GABHS infection, together with major and minor criteria, are sufficient for a diagnosis of ARF.
Another contentious issue is echocardiography and the diagnosis of ARF. Although the American Heart Association guidelines do not recommend that evidence of subclinical regurgitation be used as a major or minor criterion, readers should be cognizant of recommendations by the Heart Foundations of Australia and New Zealand developed in response to an extremely high prevalence of RHD in their indigenous populations.
The third controversy stems from the treatment of GAS pharyngitis. Evidence for recommending oral penicillin as first line treatment is scant. Given that the few trials testing the efficacy of penicillin for preventing ARF were confined to intramuscular (as opposed to oral) penicillin,
A final point relates to the use of aspirin. Aspirin is, indeed, particularly effective at rapidly resolving joint manifestations of ARF – so much so, that in areas of high endemicity, indiscriminate use of aspirin without due consideration of the diagnosis of ARF could lead to dramatic underestimation of the incidence of ARF.
In conclusion, we suggest that this review would have been greatly improved by a more robust systematic review design, incorporating other databases, as well as foreign language and unpublished data, in the search.
An interesting study recently reported by Johnston et al
Using a method that measured the interblink interval achieved exactly what the authors expected, and enabled better discrimination between normal and dry eye subjects. As the authors state, monitor usage may have impaired their data slightly, and hence a comparative study of the same cohort but using a fixed visual stare would have been both interesting and relevant. The variability seen, as the authors discuss, could be related to an integral homeostatic mechanism. Taylor et al, who investigated and published an interesting paper relating to dopamine’s role in blink rate, suggested the ventromedial part of the caudate nucleus in particular is critically involved.
We thank Johnston et al for reporting this enlightening study. Should it be repeated or augmented, a comparative study of fixed visual stare to monitor usage in the same participants and also comments on the relationship with dopamine levels would be interesting.
The authors report no conflicts of interest in this correspondence.
Recently I communicated with the Editor regarding Drs Naryshkin and Austins’ article entitled “Limitations of widely used high-risk human papillomavirus laboratory-developed testing in cervical carcinoma screening.”
In their response to my letter they noted that a 10% false negative rate for women with invasive cervical carcinoma tested by the HC2 method using US Food and Drug Administration (FDA)-approved collection media tested within one year of the cancer diagnosis is the norm and is consistent with previous estimates of achievable HPV test sensitivity. This is most likely due to a low HPV viral load in the negative samples. They also correctly pointed out that such data is available for only a limited number of cases tested using HC2 on SurePath™ samples. They then reiterated their call for a nationwide data collection effort to document the likelihood of false negative hrHPV test results for all HPV testing methods over the 5–10 year period preceding histopathologic cervical cancer diagnoses.
In this communication I respond to their call. Our laboratory uses the FDA-approved HC2 hrHPV method on SurePath™ Pap media which does not have FDA-approval for hrHPV testing. This testing methodology is used based on the results of an in-house validation performed under Clinical Laboratory Improvement Amendments (CLIA) guidelines and has enjoyed excellent College of American Pathologists (CAP) hrHPV proficiency testing results. This retrospective study focuses on the sensitivity of this testing for invasive cervical squamous carcinoma in our low risk patient population. Our methodology included identifying all histologically confirmed cervical squamous carcinoma cases at our institution from 2002–2012. Prior Pap test and hrHPV test records were reviewed for each patient. The results include 48 cases of carcinoma. Thirty-one of the 48 (65%) had Pap tests and seven of 48 (15%) had hrHPV tests within 5 years of diagnosis. All seven hrHPV tests were performed by HC2 on SurePath™ samples. All seven (100%) were positive. During the study period the laboratory performed 496,859 Pap tests with an overall ASC-H (atypical squamous cells - cannot exclude high grade squamous intraepithelial lesion) or higher Pap diagnosis rate of 0.5%. hrHPV tests were performed on 63,726 of the Pap tests (12.8%). The percentage of Pap tests with concurrent hrHPV tests increased from 0.65% in 2002 to 42.7% in 2012. In conclusion, based on a limited number of cases, no false negative hrHPV tests performed on SurePath™ samples from patients with cervical carcinoma were identified at our institution. Since current cervical cancer screening guidelines recommend increasing screening intervals which are dependent upon hrHPV test results, it is essential that the testing methodology is reliable. A large-scale national or international study, as suggested by Drs Naryshkin and Austin, is recommended in order to establish false negative rates for all hrHPV testing methods and sample types.
I read with great interest the article by Andukuri et al in a recent issue of your journal.
For instance, alogliptin decreases plasma nonesterified fatty acid levels by 11%.
Interestingly, these hypolipidemic effects of alogliptin are markedly enhanced when used in combination with pioglitazone. For instance, alogliptin with adjunctive pioglitazone therapy decreases plasma nonesterified fatty acid levels by 25%–48% and serum triglyceride levels by 67%–77%.
This book is presented to its reader as “a concise atlas and text for the practicing dermatologist, pathologist, dermatopathologist, endocrinologist, and internist.” In this context, what should we expect from it?
Certainly an
Therefore, I have retained three criteria to evaluate in my review of this book: completeness, image quality, and logic and clarity of the approach presented.
Concerning completeness, suffice it to say that an atlas pertaining to, among other things, metabolic diseases with cutaneous manifestations should include lysosomal diseases (mucolipidoses, glycogenoses, sphingolipidoses, cystinosis, etc.) that do not appear in this book but can be found in other textbooks [
As for the quality of the images, great efforts have been made to include numerous clinical photographs and histological microphotographs. The quality of these images, however, is very unequal. Some are crystal clear but, unfortunately, others are suboptimal and look as if they had been taken through filters of various colors: blue, green, yellow, pink, etc. With the technology available today, it should have been relatively easy to standardize the quality of the images. One wonders, by the way, why the photograph on page 185 occupies the whole page and is the only photograph in the whole book to do so.
Concerning the logic of the approach, things start badly, as no introduction is offered to define the various categories of diseases discussed in the book. One may argue that everybody knows what endocrine, metabolic, nutritional, and deposition diseases are, but the authors themselves admit rightly that the categories as they use it are not exclusive, therefore questioning the pertinence of their enterprise. Moreover, the warning placed in the preface that some diseases will appear in more than one category made this reader uneasy and suggests that some confusion exists in the organization of this book. For me, this was confirmed by the reading of it, which I found very hard to complete. The authors suggest that that hormones and nutrition influence metabolism profoundly and any “deposition” is the result of faulty metabolism, the incapacity to catabolize the material deposited. Therefore, the inclusion of the entities discussed in one or another category can only be arbitrary, and it is.
One of the consequences that results from classifying into undefined and overlapping categories is that some diseases are classified in a manner that appears simply nonsensical to a naïve reader. If hyperlipidemia is easily understood as a metabolic disease resulting from inherited or toxic faulty metabolism of lipids, it is hard to understand how obesity can be included in the same group of diseases and not considered nutritional! Isn’t obesity the stereotypical example of nutritional disease, or does it result, as in the mind of the authors, from a faulty metabolism?
And what about glucagonoma syndrome (GS) and necrolytic migratory erythema (NME)? Glucagonoma is a neoplasm and, therefore, logically, GS and NME, the physiopathology of which is unknown, are classical paraneoplastic syndromes as the authors themselves admit in the very first sentence of their chapter 16. So why do they discuss it as a nutritional disease? And why isn’t glucagonoma discussed in the chapter on endocrine or pancreatic diseases or, if need be, as a “metabolic” disease?
This book raises more problems than it offers solutions, and many, many other questions could be enumerated here, but this is only a book review and no place for such lengthy development.
In this work, the authors place the Latin phrase,
Lastly, the very title of this book sounds bizarre:
Our main goal was not to write a complete and exhaustive compendium. Rather, it was to write just a concise atlas-text to transfer to the practicing dermatologist, pathologist, endocrinologist, and internist all of our experience and our photographic and graphic materials on some topics that are often covered scantily, receive little emphasis, or are found scattered in different sections of the general textbooks of dermatology or dermatopathology.
This is a first edition where, of course, there is ample room for improvement and all criticisms are welcome and really appreciated. Milette’s criticisms are best divided into constructive and irrelevant.
The criticism for not including lysosomal disorders is valid. However, these diseases belong largely to the pediatric sphere that is not a strict part of our expertise. We understand that all this does not necessarily follow from the title of the volume, and actually may leave some readers unsatisfied. In fact, we have discussed adding “in adults” to the title (which is already perceived as long). If a next edition is taken into consideration, it will be our first concern to add a chapter devoted to lysosomal storage diseases.
We agree that some pictures are not of outstanding quality. It is not always easy to get good clinical and histological photographs from uncommon diseases. Some pictures were old, difficult to reproduce, and supplied from the archives of the departments; the individuals who wrote the chapters supplied some others. Of course, we know that is Editor’s task to ensure that the image quality is optimal. We will take heed of this suggestion for the future.
Classification: There is no perfect classification on which all can agree. Classification, however, is a fundamental process and a necessary starting point in the study of the natural history of disease. We think that there is nothing wrong to classify obesity as a metabolic disease, and we are not alone in this position [
Concerning the repetition of just a few disease in different chapters, this is not a matter of redundancy or arbitrary and incomplete classification of rare diseases, but, on the contrary, it reflects how metabolic, endocrinologic, nutritional, and deposition diseases may have overlapping features presenting with the same skin manifestation. For instance, pretibial myxedema can be considered both as a skin manifestation of thyroid disease and also classified among the primary cutaneous mucinoses; the same is true for macular or lichen amyloidosis that can be considered both as a skin manifestation of MEN 2A and also classified among the primary cutaneous amyloidoses. We think that there is nothing obscure or confusing to discuss a disease that is always the same but occurs in different backgrounds and eventually from distinct perspectives. Of course, the more the reader is familiar with this field, the higher and clearer is the perception and the understanding of the different diseases. This book, in fact, is not for absolute beginner.
The criticism of the words “skin diseases” in the title and the suggestion that they should be replaced by “cutaneous manifestations” is an example of an irrelevant criticism without any practical consequence. “Disease” as paraphrased according to the Merriam-Webster dictionary is a condition of the living animal or of one of its parts that impairs normal functioning and is typically manifested by distinguishing signs and symptoms. Thus, we don’t understand why the term cannot be applied in our setting. Many of the illustrated skin conditions are “disease” per se and not only skin manifestations of systemic conditions. Incidentally, we consider cutaneous mucinoses and cutaneous calcifying disorders not as simple histological patterns but as a group of different deposition disorders that are heterogeneous. An accurate knowledge of their clinicopathologic features is essential in order not to miss some important underlying association (scleromyxedema and monoclonal gammopathy, calciphylaxis in renal disease, etc).
In conclusion, we believe constructive criticism is welcome and worth voicing and may contribute to the improvement of our book. Spurious criticism goes nowhere and sometimes may mask an underlying lack of robust arguments.
Serial dermatoscopic monitoring of pigmented skin lesions may introduce a delay in diagnosis of melanoma. Conversely it is argued that feature-poor melanomas may not otherwise be diagnosed without a large increase in biopsy rates. This study compared average Breslow thickness of melanomas that have been serially monitored with those that have not.
A retrospective analysis of a clinical audit of all histopathologically confirmed melanomas detected over a two-year period in a primary care skin cancer clinic was performed. Data collected included patient age and sex, date of first monitoring, date of decision to biopsy, Breslow thickness, and histopathologic diagnosis. Lesions judged to be dermatoscopically borderline for melanoma were serially imaged using a MoleMax I (Derma Instruments, Austria). Monitored lesions were reviewed at three months and then on subsequent patient review throughout the normal course of their care. Incidences of in-situ lesions and averages of Breslow thickness of monitored and unmonitored lesions were calculated and compared.
A total of 90 melanomas were detected. 26% were invasive, with an average Breslow thickness of 0.9 millimetres. 33 melanomas had been monitored and 27% of these were invasive with an average Breslow thickness of 0.33 millimetres. 57 melanomas were not monitored and 26% of these were invasive with an average Breslow thickness of 1.2 millimetres. There was no significant difference in the proportion of in-situ lesions detected. Monitored lesions which were invasive were significantly thinner (p <0.04; z=−1.743; Two Independent Sample Wilcoxon Rank Sum Test). Only 7/33 (21%) of monitored melanomas showed change at three months.
In a small single centre study, the use of monitoring to detect melanomas resulted in a similar proportion of in-situ melanomas and did not result in an increase in Breslow thickness of invasive melanomas at time of excision, compared with unmonitored lesions. This may be because borderline lesions are detected earlier in their evolution, or may be inherently slower growing. A monitoring interval of 3 months may not be sufficient to detect change in borderline lesions. A further larger study is required to confirm these findings.
Accurate identification of malignant lesions is important for patient safety as well as reducing the number of benign lesions removed and thus reducing costs and work-load. By quantifying malignant to benign biopsy ratios at this point in time we can see if current methods have improved the accuracy of diagnosis. This also helps effectively assess the impact of new vectors in skin cancer diagnosis when they are introduced.
6546 biopsies/excisions were performed in an 18-month period from July 2010 until December 2011 in the Dermatology OPD at a Tertiary Teaching Hospital. Dermatology Registrars and Consultants were involved in assessing lesions for biopsy.
The Biopsy to Treatment Ratio (BTR) was calculated as the total number of biopsies divided by the number of non-melanoma skin cancers identified. The BTR of 1.97 indicated about one in two biopsies identified a skin cancer. The ratio of melanoma to nevi was 1:6.4 (55 melanomas were diagnosed). The ratio of melanoma to benign pigmented lesion was 1:14.7 giving a Number Needed to Treat (NNT) of 15.
A previous study
Skin cancer surgery is usually performed under either local or general anaesthetic. In our hands the use of intravenous sedation combined with local anaesthetic has both greatly increased the acceptability of local anaesthetic as well as decreased the need for general anaesthetic. This paper describes the accreditation requirements for the use of sedation and local anaesthetic in skin cancer surgery, our set up, technique, and monitoring for nurse sedation as well as staff training and approximate set up costs.
10-year retrospective audit of skin cancer surgery intravenous sedation cases in a private clinic. All sedation cases had a ‘Sedation Audit Form’ completed
During the last 10 years we have performed over 3000 operations for skin cancer with 28% done under local, 68% sedation & local and only 2.6% under general anaesthetic. Sedation is usually used for multiple and more complex procedures with 80% of our flaps and 85% of skin grafts being done under sedation. There were no deaths, no respiratory arrests, no cardiac arrests, no patients needing transfer to an overnight facility and a minor complication rate of less than 0.5%. Most patients were recovered and discharged within an hour of their procedure’s completion with less than 0.1% requiring more than two hours of recovery for sedation reasons. Adding sedation to local increases the cost of surgery with us by approximately $275 but is $1100– 1500 less expensive than the same procedure under GA.
Properly carried out, intravenous sedation is a safe and cost effective procedure in the ambulatory surgical management of skin cancer.
Non pigmented eccrine poroma is a benign tumor that can have dermoscopic features mimicking malign neoplasias. The characteristic vascular pattern of this tumor hasn’t been established. We found a scarcely reported vascular pattern, which can be useful to distinguish this tumor from malignant ones and propose a new nomenclature to these vessels due their similarity with the common calla flower and cherry blossoms tree.
We study 10 proven Mexican cases of eccrine poroma and nearly half of them presented irregular linear and branched vessels with semi-elliptical, circular or semicircular endings, we called them “chalice-form” and “cherry-blossoms” vessels. The structureless pink-white areas were the most common finding and some vascular patterns reported in other studies were barely found.
Due to the variability in the dermoscopic patterns found in non pigmented eccrine poroma, further studies are required to establish the specificity of these vessels, however they hasn’t been reported in other benign or malign neoplasias so, if seen, they can be an useful key leading to the diagnostic of eccrine poroma.
Basal cell carcinoma is a common tumour, which presents with various subtypes. These subtypes usually display vascular features, which are readily identified using dermoscopy.
Dermoscopy vascular features of superficial, superficial and nodular, nodular and combined aggressive subtypes were compared for diagnostic discrimination.
Dermoscopy vascular features were recorded in vivo for 1098 consecutive BCC. Cases with potential confounding from previous intervention were excluded. These vascular features included branching, serpentine, dot, glomerular, loop and linear vessels, the proportions of pink, central verses peripheral vessel distribution and the presence of large vessels. Kappa values were calculated for each defined vascular feature.
Different subtypes of BCC have distinctive vascular features. Superficial BCC (n=284) have more than half the tumour area pink (85%) and absent large vessels (93%), CI 85%—95%. Nodular BCC (n=230) are characterized by large vessels (46%, CI 39%–52%, P<0.001) compared to other subtypes, as well as less dot, coil and loop vessels. Aggressive BCC (n=213) display a tumour area with no pink (12%) or less than half the area pink (27%) and absent vessels in the central tumour area (22%, P<0.001) compared to other subtypes.
Kappa values for all recorded features ranged from 0.48 to 1.0.
Aggressive subtypes within the combined aggressive group were not assessed separately. All data was recorded using non polarized dermoscopy.
Diagnostic discrimination between different subtypes of BCC is facilitated by vascular feature assessment. The lower limb has different variation in BCC vessel features, compared to other anatomic sites.
Dermoscopic features of nail apparatus melanoma are different from those of other sites. Ronger et al. described seven dermoscopic features of melanonychia.
Features of dermoscopic examination of 31 patients with nail apparatus melanomas were reviewed retrospectively.
A brownish discoloration of the background and irregular lines were the most common feature, followed by and (micro-) Huchinson’s sign. Dots/globules were seen in 8 of 31 cases (25.8%).
Dermoscopy was very useful in the diagnosis of nail-apparatus melanoma, and dots/globules might be the one of features of nail apparatus melanoma.
In spite of the fact that frequency of melanoma makes 3–5 % of all primary malignant tumors of skin, it is the main reason of death of patients in oncodermatology. Over the last 6 years complex dermatoscopy examination has been intensively used in clinical practice of Moscow Oncological Hospital no. 62 for the detection and characterization of melanoma and other pigmented skin lesions.
Complex dermatoscopy diagnostics include digital photo, Zoom-photo, standard and microdermatoscopy, fluorescent dermatoscopy. At the first stage, we take digital photos and perform computer mapping of the patient’s skin. At the second stage, zoom-photo with a 10-fold enlargement is taken for each suspicious lesion. At the third stage, we perform a standard dermatoscopy with a 10-fold enlargement, microdermoscopy with a 120-fold enlargement and a fluorescent dermatoscopy with 5-ALA (Alasens). Applying the complex method of dermatoscopy diagnostics we studied the reliability of characteristics describing malignant and benign pigmented skin lesions in 497 patients with 1735 pigmented skin lesions (280 non-melanocytic, 1271 melanocytic lesions (65 melanoma, 259 dysplastic nevi)). The data of the complex dermatoscopic investigation were compared to the results of morphological investigation of surgery samples.
Sensitivity and specificity dermatoscopy diagnostics of melanoma has made 92 % and 72 % accordingly. Consider high efficiency, non-invasive character of method of complex dermatoscopy diagnostics of melanoma of skin it should be used first of all for examination in groups of high risk of melanoma. This scientific trial is supported by Moscow.
Circumscribed palmar hypokeratosis (CPH) is a rare skin disorder that typically develops on the palms of middle-aged women. CPH is clinically characterized by well-demarcated, slightly scaling erythema that is sometimes difficult to differentiate from Bowen’s disease and porokeratosis of Mibelli. CPH is usually diagnosed by its clinical and histopathological features. We present a 61-year-old Japanese female presented with a 10-year history of a well-demarcated, 6×6-mm asymptomatic erythema on the right palm. We show an apparent correspondence between the dermoscopic findings and histopathological features of the lesional skin, which indicates that the pathogenesis of CPH is associated not only with abnormal keratinization but also with hyper vascular formation.
Melanocytic naevi have been observed to undergo morphological changes following exposure to narrowband ultraviolet B (NBUVB). We aimed to analyse changes occurring in naevi exposed to NBUVB in a large cohort.
51 subjects referred for phototherapy had macro and dermoscopic images of prominent melanocytic naevi taken immediately prior to NBUVB treatment; after 10 exposures; after 30 exposures or at the end of the treatment course if earlier; and 3 months after discontinuing treatment.
Four dermatologists, by consensus, examined each naevi for specific clinical and dermoscopic features, at each time point. The size (area) of each naevus was determined by plenimetry.
36 of 51 patients had complete sets of images. The most common global dermoscopic pattern in the 440 naevi examined, were reticular (50%) and globular (32%).
Following NBUVB exposure, 45% of reticular naevi displayed changes in local features with blurring or merging of lines. Increase in colour intensity and in the number of dots/globules was observed in 63% of globular naevi.
167 naevi (40%) underwent change in size following UV exposure. Of these, 54% (91/167) decreased in size, with median area reduction of 8% (0.9%–42%); whilst 46% (76/167) increased in size, with median area increase of 9% (1%–76%). The trend was for these naevi to return to their pre-treatment size after phototherapy. Of the 440 naevi reviewed, none displayed changes suspicious of malignancy.
Around half of exposed naevi undergo size/morphological changes following a course of NBUVB. Size changes tended to revert to pre-treatment values 3 months after discontinuing phototherapy.
Distinguishing between recurrence of lentigo maligna (LM) and postinflammatory hyperpigmentation after treatment may be challenging. Reflectance confocal microscopy (RCM) and dermoscopy might be of help in this clinical setting.
We performed dermoscopy and RCM in 7 patients that showed pigmentation in areas previously treated for LM. Previous treatments were radiotherapy (4), cryotherapy (2) and surgical excision (1). Correlation between RCM findings and histological/immunohistochemical features was evaluated.
Two cases treated with radiotherapy exhibited blue-gray granules around hair follicle openings under dermoscopy. RCM exam correlated these structures with widespread plump cells in the upper dermis without other features suggesting recurrence of LM, definitely excluded in the biopsy. In 3 cases (2 radiotherapy, 1 surgery) dermoscopy showed irregular brown pigmentation, areas with fingerprint-like structures and focal grayish dots. In these cases RCM demonstrated widespread dendritic bright cells at basal and suprabasal layers. No evidence of LM was detected on histology, but immunohistochemistry showed positive Langerhans’ cells at suprabasal layers and dendritic HMB-45-positive melanocytes at the basal layer. The two cases that had been treated with cryotherapy did not show clear-cut dermoscopic criteria for LM. However, RCM was highly suggestive of melanoma that was histologically confirmed.
RCM can be useful in the monitoring of LM after treatment. In photodamaged skin, the visualization of widespread dendritic cells in basal and suprabasal layers by RCM may mimic a recurrence of LM. These structures correlate with activated nonmalignant HMB45-positive melanocytes and Langerhans’ cells and may represent a pitfall in the confocal evaluation of these lesions.
Dermatoscopy is one of several non-invasive approaches that use to improve the diagnostic accuracy. It has been applied in the diagnosis of skin tumors such as Basal cell carcinoma (BCC).
One hundred lesions from 39 patients (28 men and 11 women) were included in this study. All patients had a past history of childhood radiation therapy, primarily for treatment of tinea capitis. They presented with lesions that were morphologically highly suspicious to be pigmented BCC. Using a digital dermatoscope, all lesions were photographed and evaluated for the presence of Menzies BCC criteria and other features. All lesions were subsequently, underwent biopsy.
When combined with clinical diagnosis, Menzies dermatoscopic criteria for BCC diagnosis have 100% sensitivity and 90.9% specificity. Tree like vessels, maple leaf-like structures and spoke wheel patterns were only seen in BCC lesions. Large gray-blue globules were seen in 76.4%, blue gray ovoid nest in 66.3%, arborizing vessels in 63%, maple leaf like pattern in 45%, ulceration in 43.8% and spoke wheel pattern in 28% of BCC lesions. The results confirmed the usefulness of dermatoscopy for better decision-making in the clinically suspected BCC lesions after radiation.
It is well known that when mice are engineered with mutations in molecular pathways that drive human malignant melanoma (MM) development, they too develop the disease. Little attention has been paid, however, to the natural history of naevi and melanomas developing in mouse models, and how this relates to the particular mutation the animal carries. Gaining the knowledge of the behaviour of each individual mouse lesion provides a relevant link for translation of information obtained from the mouse model to the corresponding clinical condition. The ultimate goal of the mouse MM model is to gain understanding of how certain mutations influence lesion dynamics, and to provide appropriate models for preclinical evaluation of melanoma therapies. We recently reported the development melanocytic lesions, along the spectrum of naevus to MM, in Cdk4R24C/R24C::Tyr-NrasQ61K mice. We followed the development of lesions over time using digital photography and dermoscopy to correlate the clinical appearance with histopathologic features of melanocytic lesions developing in this model. We have identified essentially two types of lesions and studied their respective growth patterns. We developed a staging system, based on the level of extension in to the dermis, which offers a practical linkage between murine MM models and standard clinical diagnosis. We will discuss how we have used these methods to help us understand the development of melanomas in mice carrying other mutations (e.g., in the p53 and Arf genes) that result in a very different mode of tumour progression than we see in the original model.
Individuals with increased quantities of clinically dysplastic nevi (DN), such as those with the Atypical Mole Syndrome (AMS), have an elevated melanoma risk. Little is known, however, about the influence of qualitative mole phenotypic factors on melanoma risk, such as anatomic distribution and dermoscopic appearance of DN.
To compare the anatomic distribution and dermoscopic appearance of DN among AMS patients with or without a personal history of melanoma.
We conducted a retrospective case-control study of clinical and dermoscopic images from 22 AMS patients with or without a personal history of melanoma (11 cases and 11 controls, respectively) using MoleMap software. The anatomic location and dermoscopic pattern for each DN was analyzed.
676 of 1822 lesions met criteria for DN. 391of 676 (58%) DN were located on cases compared to 285/676 (42%) located on controls. Cases had more DN on the legs than controls (29% vs. 15%, p = 0.009), and fewer DN on the chest (7% vs. 14%, p = 0.009). Disorganized globular DN and homogeneous DN, were more common among cases than controls (18% vs. 11%, p = 0.0126; 18% vs. 6%, p = 0.001, respectively). DN exhibiting peripheral network with central globules were more frequent in controls (20% vs. 3%, p = 0.002).
These data suggest that DN exhibit significantly different anatomic distributions and dermoscopic patterns among AMS patients with or without a melanoma history. These differences may be helpful in improving melanoma risk prediction among high-risk individuals with increased quantities of DN.
Genital warts may mimic a variety of conditions, thus complicating their diagnosis and treatment. The recognition of early flat lesions presents a diagnostic challenge.
We sought to describe the dermatoscopic features of genital warts, unveiling the possibility of their diagnosis by dermatoscopy.
Dermatoscopic patterns of 61 genital warts from 48 consecutively enrolled male patients were identified with their frequencies being used as main outcome measures.
The lesions were examined dermatoscopically and further classified according to their dermatoscopic pattern. The most frequent finding was an unspecific pattern, which was found in 15/61 (24.6%) lesions; a fingerlike pattern was observed in 7 (11.5%), a mosaic pattern in 6 (9.8%), and a knoblike pattern in 3 (4.9%) cases. In almost half of the lesions, pattern combinations were seen, of which a fingerlike/knoblike pattern was the most common, observed in 11/61 (18.0%) cases. Among the vascular features, glomerular, hairpin/dotted, and glomerular/dotted vessels were the most frequent finding seen in 22 (36.0%), 15 (24.6%), and 10 (16.4%) of the 61 cases, respectively. In 10 (16.4%) lesions no vessels were detected. Hairpin vessels were more often seen in fingerlike (x2 = 39.31, P = .000) and glomerular/dotted vessels in knoblike/mosaic (x2 = 9.97, P = .008) pattern zones; vessels were frequently missing in unspecified (x2 = 8.54, P = .014) areas.
There is a correlation between dermatoscopic patterns and vascular features reflecting the life stages of genital warts; dermatoscopy may be useful in the diagnosis of early-stage lesions.
The aim of this study was to compare the specificity and sensitivity of the dermoscopic findings of alopecia areata (AA), androgenetic alopecia (AGA) and cicatricial alopecia.
A total of 99 patients with alopecia (37 AA, 39 AGA, 23 cicatricial alopecia) were admitted to our study. An informed consent form was signed by the patients and volunteers, followed by dermoscopic examination and photography.
Yellow dots, exclamation mark, broken hairs, black dots were more specific for AA; reduced follicular ostia, white patches, white dots, blue-grey dots, red dots, keratin plugs, branching capillaries, tufted follicle and perifollicular scales were more frequently observed in cicatricial alopecia. Hair diameter diversity, perifollicular pigmentation and peripilar erythema were mostly observed in AGA. Yellow dots with short vellus hairs were observed both in AA and AGA. Reduced hair diameter was observed in significant ratio both in AA and cicatricial alopecia. Unlike previous studies, “yellow dots” were observed nearly equally both in AA and AGA. “Short vellus hair” was observed frequently both in AGA and AA. Moreover, we revealed “reduced hair diameter” in AA and “honeycomb-like erythema” in cicatricial alopecia, which have never been reported before in previous dermoscopic and trichoscopic studies that were carried in different alopecia types.
We think that our study is unique and important not only because it is the first study in literature to compare the clinical and dermoscopic findings of AA, AGA, and cicatricial alopecia, but also evaluating the specificity and sensitivity of these findings.
The incidence of multiple primary melanoma ranges from 2–12% in various studies, with the majority of second primary melanomas detected within the first three years. In particular, many patients in this group have more than one melanoma at presentation (synchronous melanoma).
Clinicopathological studies of patients with multiple primary melanomas have found that most subsequent melanomas are less invasive than the initial melanoma, however many do not have the typical clinical or dermoscopic appearances of melanoma.
Patients with multiple benign naevi frequently show a predilection for certain dermoscopic patterns across all of their naevi (the concept of ‘moles breed true’). Furthermore, dysplastic naevi or melanomas are frequently first detected due to their difference from surrounding benign naevi: the ‘ugly duckling sign.’
We sought to determine whether synchronous melanomas in individual patients had similar dermoscopic profiles. Therefore, the dermoscopic appearances of synchronous melanomas arising in patients attending a tertiary skin cancer clinic in the United Kingdom from 2005–2010 were reviewed.
We found that patients with synchronous melanomas may not show a predilection for any particular dermoscopic pattern. Therefore, unlike benign naevi, multiple primary melanomas do not ‘breed true’; these ‘ugly ducklings’ need to be assessed, clinically and dermoscopically, on an individual basis.
Dermoscopy is a non-invasive instrumental method for the in-vivo study of pigmented skin lesions. This technique allows for viewing the parameters that would otherwise be invisible to the naked eye. Over recent years dermoscopy has proved to be extremely useful even on dermatological pathologies of an inflammatory origin and skin parasitosis. The introduction of the new laser and intense pulsed light systems has made it possible to cure pathologies difficult to treat with the instruments available to doctors ten years ago.
The use of dermoscopy has proved to be particularly important immediately after treatment for observing positive or negative results, any side effects, or early signs of relapses. In this way the patient and the doctor know with good approximation what they can expect from a specific treatment. The constant application of dermoscopy to laser and IPL treatment can also be useful for educational purposes in order to better understand how to interpret the dynamics of specific pathologies and the results which the different types of lesions and skin may have when crossed by a laser beam or luminous energy. The use of dermoscopy before surgical excision of a given melanocytic lesion with CO2 laser is also indispensable for medical—legal purposes. The dermoscopic image of the lesion immediately after treatment is important for demonstrating the absence or residual presence of pigment as well as the depth reached and the absence of any thermal damages. Dermoscopy is a particularly useful method for predicting the therapeutic success of the vascular treatments. The dermoscopic exam performed before treatment makes it possible to quantify the number and gauge of the vessels to be treated and the presence of any photodamage. The reaction of the vessel immediately after IPL or Nd-Yag laser treatment can be of two types: coagulation of the vessel with colour change from red to blue or contraction of the vessel with disappearance or reduction in size. These epiphenomena are clearly visible with the help of dermoscopy.
In our experience, dermoscopy has demonstrated to be an extraordinary instrument for determining any possible adverse events immediately after laser or IPL treatment. This allows physicians to provide suitable therapy in advance and inform the patient about any possible undesirable effects.
Our results demonstrated that dermoscopy can definitely be considered a first choice exam of great validity, above all due to the exactness and extreme accuracy in identifying the target to be hit. In most cases this has made it possible to immediately predict the treatment results. The dermoscopic exam performed some time after treatment demonstrated that it is indispensable for verifying the final results, especially in the cases of epithelial tumours treated with PDT. On concluding this study we suggest that the dermoscopic exam should be an integral part of all laser and IPL treatments.
Visual language refers to the integration of shapes (symbols or signals), images (codes or objects) and words (text and speech) into a single communication unit.
The broad concept of shape can be subdivided into the basic elements of visual language: dot, line, shape and colour. These are the raw materials of all visual information. Dermoscopic images are made up of these basic elements.
Reading these elements abstractly can help both the learner and expert in dermoscopic diagnosis.
The use of dermoscopy has led to an improvement in diagnosing malignant melanoma (MM), which is important for the prognosis of the patient. Sunless tanning agents containing dihydroxyacetone (DHA) could lead to a decrease in UV exposure decreasing the risk for MM. Importantly, DHA has been reported to change the dermoscopic image and could thus endanger the diagnostic improvement of dermoscopy.
To investigate if the use of DHA can lead to changes that simulate a real, clinically relevant dermoscopic change suggesting malignant transformation in facial solar lentigo/initial seborrheic keratosis (SL/ISK) or in nevi on the body.
Seven patients with 25 pigmented skin lesions (PLS) were photographed resulting in 38 dermoscopic images. Photographs were taken before, one week after and 1–2 months after the use of DHA. Two dermatologists separately evaluated the dermoscopic features according to Menzies’ method (lesions on the body) or Pattern analysis (facial lesions).
In facial PSLs unclear dermoscopic lesions were registered by both evaluators significantly more often after DHA use than before (42 vs.12 %, p=0,021 and 69 vs.19 %, p=0.001). Furthermore, follicular pigmentation that partly mimics that of lentigo maligna was also seen significantly more often after DHA use than before (80 vs.12%, p<0.001 and 69 vs. 15%, p<0.001) and in these instances the evaluators recommended a biopsy. Unclear lesions in nevi on the body were not significantly increased after DHA use.
Dermatologists that come across unclear dermoscopic findings, especially in facial PLS, should ask about the use of DHA.
A 2002 survey of fellows of the American Academy of Dermatology found substantial variation in the management of dysplastic nevi and attitudes toward their biological significance.
To assess the attitudes and practices of newly graduated US dermatologists with respect to dysplastic nevi and compare these findings with those from the 2002 survey.
An online survey was sent to 139 chief residents of US Dermatology Training Programs.
A 59% response rate was achieved. All residents accept the dysplastic nevus as a clinical entity, and all report access to a dermatoscope in clinic. 98% agree that dysplastic nevi mark persons at increased risk for melanoma, compared to 59% of AAD fellows in 2002. 94% of chief residents use dermoscopy to manage pigmented lesions, compared to 23% of AAD fellows in 2002. Although 92% of chief residents report receiving dermoscopy training, only 48% train with a pigmented lesion specialist. Among those training without a specialist, less than half receive classroom or bedside teaching compared to 77% of those who train with a specialist. Of those who train with a specialist, 77% agree that dermoscopy can help differentiate melanoma from benign lesions, compared to 46.5% of those who train without a specialist (p=0.0067).
Residents are receiving a more unified message regarding the biological significance of dysplastic nevi and nearly all use dermoscopy as a diagnostic tool. Additionally, specialists can provide dedicated instruction to trainees fostering greater confidence in the utility of dermoscopy for the management of pigmented lesions.
Little is known about dermoscopy in dark-skinned people and whether it can influence diagnostic performance. Conditions as diverse as inflammatory and infective dermatoses, tumours and pigmented and non-pigmented skin lesions have been documented in white population but few studies report dermoscopic features in dark skin. Moreover, clinical diagnosis in skin of darker color is made difficult by unusual presentations, annular patterns and absence of erythema.
The aim of this study is to assess whether dermoscopy can be a useful diagnostic tool in dark skinned population and compare dermoscopic features in the different photo-types.
Migrants of ethnic skin attending the dermatology department of NIHMP in Rome affected by inflammatory and infective dermatoses, tumours and pigmented skin lesions underwent dermoscopic examination after clinical evaluation.
We present a case series and document how the routine use of dermoscopy can guide the dermatologist in diagnosing skin lesions of difficult interpretation and accurately classify pigmented lesions.
The aetiology of seborrheic keratoses (SK), the most common benign epithelial tumours, and any relationship with malignancy are not yet known. As a protective anti-cancer mechanism, apoptosis reflects cellular loss as a reaction to proliferative activity.
The objective of this study was to quantify apoptosis in different SK types (acanthotic, hyperkeratotic, reticulated, irritated, and clonal) and correlate the dermoscopic picture with apoptosis rate.
After a qualitative and quantitative analysis of dermoscopic images, we defined a new quantitative dermoscopic score (C3V2F, crypts, millia cysts, colours, hairpin vessels, irregular vessels, fissures) from 0 to 22, which enabled us to establish cut-offs correlating with apoptosis rates.
All five SK forms were associated with lower apoptosis rates compared to normal skin. A C3V2F score >10 and greater number of crypts and colours reflected a higher apoptosis rate, which implies a benign character of evolution. In contrast, the presence of irregular vessels on more than 50% of the lesion surface implied a lower rate of apoptosis and probably associated with a risk of malignant transformation. Based on the dermoscopic information, we used multiple regression to establish a model for estimating the rate of apoptosis with a 0.7 prediction interval (approximately 1 standard deviation).
The new C3V2F score could be valuable for the clinical evaluation of possible SK prognosis and decisions regarding excision.
Usage of the diagnostic two-step method in teaching dermatoscopy is widely accepted and was approved in a consensus meeting in 2003. The differentiation of melanocytic and non-melanocytic lesions (“the first step”) contains the risk of misclassification and can therefore lead to wrong diagnoses. This risk is inherent especially when applied by dermatoscopists at a beginning level, the most frequent users of published algorithms.
The present study aimed to evaluate the frequency of misclassifications according to the first step. We included 707 consecutive cases from 553 patients (mean age: 54.7 years, SD: ±18.1 years) with (n=331) and without (n=222) chronically sun-damaged skin. The cases were collected from a tertiary referral center at a University hospital in Europe and from a Primary Care Skin Cancer Clinic in Brisbane (Australia). Dermatoscopic images were evaluated in a blinded fashion for the presence of features described in the 2-step algorithm to determine their melanocytic or non-melanocytic origin. Mucosal, genital and non-pigmented lesions were excluded. The sensitivity of the first step was 97.1% for patients with chronic sun-damage and 96.8% for patients without or moderate sun damaged skin. The specificity was 33.6% for patients with chronic sun-damage and 67.9% for patients without or moderate sun-damaged skin. The most common reasons for misclassification were a pigmented network in 69 cases and an absence of any given features in 74 cases.
Pigmented actinic keratoses play a difficult role in the differential diagnosis of lentigo maligna on the face. However some helpful hints as neighborhood sign, rough appearance, abrupt interruption of pigment pseudo network could help in such diagnosis. The authors present a visual set of lesions illustrating this concept.
While Australia has the highest worldwide melanoma incidence, certain subpopulations are at extreme risk and early melanoma diagnosis is crucial. Knowledge of the role of clinical imaging techniques in this group is somewhat limited.
To determine for extreme high risk patients the natural history and role of total body photography (TBP) and sequential digital dermoscopy imaging (SDDI) in early primary melanoma detection.
312 patients at extreme melanoma risk were monitored six monthly after baseline TBP over a 5 year period. Inclusion criteria were ≥1 of: (1) CDKN2A or CDK4 mutation; (2) ≥3 first/second degree relatives with previous melanoma and a personal melanoma history; (3) Dysplastic Naevus Syndrome and a personal melanoma history; (4) History of ≥2 primary melanomas.
All patients were screened against TBP at each visit with SDDI short term (3 months) and long term (≥6 months) monitoring employed following established criteria and atypical lesions excised.
The median follow-up time was 3.5 years (IQR: 2.4–4.2 years). 79 primary melanomas were detected, 16 at baseline visit and 63 subsequently. Median Breslow thickness was 0.12 mm (IQR: in situ-0.60mm).
38.1% were detected using TBP and 39.7% with SDDI. Five melanomas, including three desmoplastic melanomas, were ≥1mm Breslow thickness (including three desmoplastic and one scalp melanoma). 142 BCCs and 64 SCCs were excised and the overall benign to malignant excision ratio was 1.4:1 and 4.2:1 for melanocytic lesions.
Monitoring extreme risk patients with TBP and SDDI assisted with the effective early diagnosis of primary melanoma. Hyper-vigilance for difficult to detect thick melanoma subtypes is crucial.
With recent advances in skin imaging technologies there has been an increasing demand for image processing techniques in computer aided-diagnosis of pigmented skin lesions using dermoscopy images. Our work follows a relatively new trend in clinical dermatology to identify specific ‘dermoscopic structures’ such as streaks, scale, and pigment network, which are used to aid the diagnosis of malig nant melanoma.
Irregular pigment network and streaks are two important positive features of melanoma and scale seems to be a negative feature based on our studies on the dry polarized contact dermoscopy. We present a novel approach to detect, segment, analyze and visualize pigment network, streaks, and scale in dermoscopic images, according to their clinical definitions. Three important dermoscopic structures (pigment network, streaks, and scale) are modeled based on the structural (shape), geometric (spatial arrangement), chromatic and textural features defined by experts in dermoscopy. Our approach has several steps; pre-processing that includes image enhancement and automatic skin lesion segmentation, object detection using morphologic techniques, and feature extraction and classification into irregular or regular structures. The presence and absence of these structures can be used for malignancy detection in skin lesions.
Our results over 945 images show an accuracy of 92% on pigment network detection, 82% on typical-atypical pigment network classification, 78.5% on streaks detection, 83.5% on regular-irregular streaks classification and 84% on scale detection.
High melanoma-related mortality rates remain a problem in Poland. The aim of the study was to evaluate melanoma invasiveness depending on dermoscopy screenings, preventive behavior and clinical findings.
A retrospective study based on revised medical records between years 2004–2011 and completed questionnaires of patients diagnosed with melanoma.
In the analyzed timeframe 176 patients were diagnosed in our department with melanoma (57%-F, 43%-M). A total of 38,4% were diagnosed as melanomas in situ. Among invasive melanomas mean Breslow thickness was 1.03 mm. About 80% of melanoma patients were phototype I–II, what corresponds to usual numbers in Polish population. A history of sun burns recorded 83,6% of patients with melanoma and 69,3% of healthy control. About 60% of patients had more than 50 nevi. In this group mean Breslow thickness was 1,32 mm and 46,6% of melanomas were in situ. In patients with less than 50 nevi mean Breslow thickness was 2,25 mm and 32,7% of melanomas were in situ. About 60% of patients were never screened for melanoma. In this group 35% of melanoma were in situ, and mean Breslow thickness was 1,77mm (1,35 mm—F, 2,77 mm—M). Within patients screened for melanoma at least once 49% of melanomas were in situ and mean Breslow thickness was 0,89 mm -F and 1,18 mm—M patients.
Thickness of melanoma (in Breslow scale) is significantly lower in patients who had a history of at least one dermoscopy screening prior to diagnosis and in patients who have multiple nevi.
The use of dermoscopy is rapidly expanding. The dermatoscope is now used in everyday practice. We sought to investigate the use of dermoscopy in Australian dermatology trainees.
An invitation to complete a web-based survey was sent via e-mail. The survey was composed of a combination of questions from a standardized survey of the International Dermoscopy Society, a previous published dermoscopy survey of Australian consultant dermatologists and questions posed by us. Two-sided Fisher’s exact tests, chi-square tests and exact chi-square tests for trend were used to assess differences between Australian consultant dermatologists (n=99) and trainee dermatologists (n=44). A significance level of 0.05 was assumed. The statistical analysis was conducted using SPSS release 18 (IBM SPSS Inc, Chicago, IL).
The response rate was 55% (44 out of 88 trainees). There were 31.8% (n=14) male and 68.2% (n=30) female respondents. The mean age was 33 years (SD=5.41). All respondents used dermoscopy with the majority (54.5%, n=24) using a dermatoscope for 3–5 years. When asked whether a dermatoscope was an essential tool for a trainee dermatologist, 95.5% (n=42) responded yes. When comparing consultants and trainees, there was a significant difference in their answers when questions concerning identifying melanoma early in the curable stage, use in non-pigmented tumours, helping to improve record keeping, documentation for medical liability and anticipation for future use of dermoscopy were asked (p<0.05).
Comparing diagnostic skills for identifying malignant melanoma strongly depends on characteristics of the tumors analyzed. For benchmarking diagnostic studies on melanoma the average tumor thickness is neither representing clinical nor dermoscopic difficulty of diagnosis. It is evident that an investigator encountering only large, dark and asymmetric lesions has to face less diagnostic problems as compared to another one excising small, symmetric and possibly hypopigmented melanoma, too. Most important for cost efficiency in health care systems is the benign-malignant ratio of excisions with suspect of melanoma. Comparing the ratios of different investigators cannot be made without taking diagnostic difficulty of the lesions into account.
A two-step procedure is presented including clinical as well as dermoscopic features contributing to the diagnostic difficulty of a given lesion. First, a lesion must be considered worthy of examination by dermoscopy. An index of clinical diagnostic difficulty characterizes these lesions. In a second step, certain dermoscopic criteria must be present to raise suspicion of malignancy. A score of features is presented to define diagnostic difficulty of melanoma for both steps. The index of diagnostic difficulty ranges from zero to infinite and thus comprises easy-to-diagnose classical melanoma as well as so-called featureless melanoma, fairly impossible to spot in the skin. Examples are demonstrated.
In future studies, the average index of difficulty of melanoma with the study population should be calculated in order to compare benchmark results of various centres.
Regressive cutaneous lesions are a significant diagnostic challenge as they lack distinct clinical and dermoscopic features. Reflectance confocal microscopy (RCM) is a tool for non-invasive diagnosis of melanocytic skin lesions that has been shown to be useful in diagnosis of non-pigmented skin lesions. Little is known, however, about the value of RCM in diagnosis of regressive lesions. We will present a case series of regressive lesions including clinical, dermoscopic and RCM images as well as histopathological diagnoses, and discuss specific features that, in our view, might be of use in diagnosis of these challenging lesions.
Melanoma is a malign tumor of the skin. Malign transformation in the already existing nevus is the most seen etiological factor for melanoma. This study was done to determine the clinical and dermatoscopic characteristics of melanocytic nevi in Turkish young people with 18–25 years old.
A total of 688 young people from the patients and students of Gülhane Military Medical Academy, School of Medicine, and Department of Dermatology were included in the study. A questionnaire was applied including age, sex, sunblock use, and sunburn history. On clinical examination, we evaluated number of nevi, location of nevi and skin type. Nevi that are equal or greater than 3mm were examined dermatoscopically, recorded and scored by using pattern analysis, ABCD total dermoscopic score, 7 point checklist, Menzies algorithm, 3 point checklist and CASH algorithm.
Totally 668 young people were physically examined in this study (268 of which were women and 400 of which were men). The most common skin phototype in both sexes was type 3 (91.6% of women, 85.25% of men). A total of 453 nevi were examined dermatoscopically. The most common localization of nevi was on the head and neck region (n=144; 31.8%), followed by anterior trunk and abdomen (n=128; 28.3%), back (n=122; 26.9%) and extremities (n=45; 9.9%). The most common dermatoscopic global feature was the globular pattern (n=135; 29.8%), followed by reticular pattern (n=88; 19.4%), and cobblestone pattern (n=64; 8.8%).
This is the first study about the characteristics of melanocytic nevi at this age group and lays the foundation for future studies that will elucidate the relationship between nevi, dermatoscopic pattern and the other factors in a population-based cohort.
Alopecia neoplastica is a rare disorder that in most cases represents metastatic breast cancer. Dermoscopy might be a useful tool in clinical evaluation.
A 72 year-old woman reported asymptomatic localized hair loss with a progressive course during the past 4 months. She presented a past history of breast adenocarcinoma diagnosed 5 years ago. On physical examination there was a mildly indurated, slightly erythematous plaque of alopecia on parietal scalp. Dermoscopy reveals only overlying telangiectasias diffusely distributed. Biopsy proved a metastatic carcinoma compatible with a mammary origin.
In woman, breast cancer is the most common malignancy that metastasizes to the skin. Alopecia neoplastica is a rare asymptomatic manifestation of cutaneous metastases and it might present as single or multiple slightly erythematous round plaques of alopecia, usually with peripheral telangiectasias. The clinical picture may mimic several dermatoses and therefore the diagnosis might be delayed. Dermoscopy might be an important diagnostic tool, mainly on the exclusion of other dermatoses with already described dermoscopy patterns such as alopecia areata, which presents for example short “exclamation mark” hairs and yellow dots, discoid lupus erythematous, that shows follicular plugs and red dots, and tinea capitis, which exhibits broken and comma hairs. In conclusion, alopecia neoplastica is an uncommon cutaneous disease. Although it lacks a characteristic dermoscopic pattern, dermoscopy might be helpful in its evaluation especially when dermoscopic features of other common dermatoses are not present and should lead dermatologists to perform histologic examination in such cases.
Acral melanomas (AM) can be misdiagnosed in early stages and it has postulated that the delay in the detection could be the main cause of a poor prognosis.
Retrospective clinical-prognostic, dermoscopic and histopathologic review of AM in a referral unit from 1986 to 2010.
275 AM were included (61% women; mean age of 56y, range 12–96). The most frequent location was on feet (83%), 60% were ulcerated 20% achromic. Dermoscopically (68 cases) multicomponent global pattern was the most frequent (35%), parallel ridge pattern could be identified in up to 50% of cases. More than 40% presented blue whitish veil and streaks. 60% of achromic tumours showed milky red areas and/or atypical vessels, and were correlated to higher Breslow index. At the time of consultation in our Unit all showed dermoscopic clues of malignancy. Histopathologically they consisted in acral lentiginous melanoma (ALM) 57%, superficial spreading (SSM) 30% (75% women), and nodular (NM) 6%. 24% were in situ melanomas whereas the mean Breslow thickness of invasive cases was 3.02mm. In a mean follow-up of 55.16 months 27% died due to melanoma. Prognostic factors by multivariate analysis were age at diagnosis, Breslow, and histopathologic subtype. ALM and NM presented a poorer outcome than SSM (OR 10.95, p0.02).
Diagnosis of AM in our population is delayed and dermoscopy could help to identify difficult lesions, especially achromic tumours. In addition to misdiagnosis, subtypes of ALM and NM presented a poorer prognosis after been adjusted by age and Breslow.
Skin inflammation of the face can be caused by different factors such as infections, allergies, physical and chemical agents and reactions due to medications. Skin inflammatory diseases are often accompanied by patches, blisters, dryness of the skin, burning and itching, which affect the appearance and texture of the skin. Differential diagnosis may be difficult. We investigated, whether dermoscopy may aid differential diagnosis of inflammatory skin diseases of the face.
A total of 50 patients with nummular lesions on the face were included into the study (pemphigus vulgaris, pemphigus foliaceous, discoid lupus erythematosus, subacute cutaneous lupus erythematosus, atopic dermatitis, psoriasis, seborrheic dermatitis, rosacea, tinea, pityrosporum folliculitis, sycosis). Videodermoscopy was performed with Fotofinder 2.
In dermoscopy of pemphigus vulgaris and pemphigus foliaceous we observed: bloody-red, sharply demarcated, polygonal areas, glomerular vessels and peripherally attached linear scales. In discoid lupus erythematosus most characteristic dermoscopy features were: thick arborizing vessels, fine scaling and large, keratotic plugs. In seborrheic dermatitis fine arborizing vessels and yellowish scaling were most prominent. Dermoscopy of psoriasis showed regularly distributed globular vessels. UV-enhanced dermoscopy (UVED) may be beneficial in differential diagnosis of
In conclusion, dermoscopy may be applied in differential diagnosis of inflammatory diseases of the face.
Cutaneous metastases of melanoma can be confused with other skin lesions. Dermoscopy could be helpful in the differential diagnosis.
We described the distinctive dermoscopic patterns of CMMM assessing their sensitivity and specificity performing a retrospective study of 146 dermoscopic images of CMMM from 42 patients attended in our institution since 2002 to 2009. First, two investigators established six dermoscopic patterns of CMMM. The correlation of 75 dermoscopic images with their distinctive patterns was assessed by 4 independent dermatologists. Finally, a pool of 163 dermoscopic images including CMMM and non-metastatic lesions were evaluated by the same four dermatologists in order to assess the diagnostic utility of the dermoscopic patterns to recognize CMMMs.
The six dermoscopic patterns of CMMM were blue nevus-like, nevus-like globular and non-globular, angioma-like, vascular and unspecific. When CMMM were classified accordingly to these patterns, agreement between the investigators and the four dermatologists ranged from ⊠ = 0.56 to 0.7. The interobserver agreement was good (> 80% for angioma-like, nevus-like and blue nevus-like patterns).
71 CMMM, 16 angiomas, 22 blue nevus, 15 malignant melanoma (unspecific or globular pattern), 11 seborrheic keratosis, 15 melanocytic nevus with globular pattern and 13 pink lesions with vascular pattern were evaluated by 4 dermatologists showing an overall sensitivity of 68% (between 54.9–76%) and specificity of 80% (between 68.5–93.5) for the diagnosis of CMMM that varies according to the experience of the observer and point out the difficulty in the identification of some metastases. Nevertheless, the majority of the lesions were correctly identified as CMMMs.
Pennsylvania is a very rural state within the United States with many health care concerns. Of the 67 counties within the Commonwealth, 52 are designated as primary care shortage areas with significant concerns of access to primary health care services. The economic backbone of this state is agriculture, lumbering and mining, which require long hours of exposure to the environment. Health indices of the Commonwealth demonstrate an increase in skin cancer and melanoma incidence. With a minimum of six months or greater to schedule an appointment with a dermatologist, the Department of Family Medicine and The Penn State Hershey Melanoma Center developed an education and training program for primary care physicians in dermoscopy to:
Develop an educated primary care physician workforce in rural and underserved areas skilled in the use of dermoscopy
Increase access to screening and early detection interventions for skin lesions through primary care to prevent progression of the disease
Decrease cost to health care interventions through early detection and better diagnosis accuracy
This workshop describes the educational content, instructional methods and evaluative processes used to train primary care physicians to become competent and skilled in the use of dermoscopy and confident in the delivery of primary and secondary interventions. The facilitator also explore the lessons learned in the pilot dermoscopy training session implemented in the Department of Family and Community Medicine at Penn State College of Medicine which served to frame the changes in curriculum and instructional media applied in the first training series.
New mobile phones, so-called smartphones, with built-in digital cameras used in combination with customized dermoscopes can hopefully be used to carry out teledermoscopic evaluations of suspicious skin lesions. In this study, we included 69 melanocytic and non-melanocytic lesions in which malignancy was suspected upon clinical and dermoscopic examination by a dermatologist during a face-to-face visit. Prior to biopsy or excision, clinical and dermoscopic digital photographs were taken with a smartphone and a dermoscope that could be fitted directly onto the smartphone. The suspected diagnosis, the level of diagnostic difficulty and the management decision was provided and later compared to the histopathological report. Furthermore, the image quality was assessed. These same parameters were also provided by two experienced dermoscopists who independently reviewed the clinical and dermoscopic photographs together with relevant clinical information but without seeing the patient and without knowledge of the histopathological report. We will report on the diagnostic accuracy of the dermatologist meeting the patient face-to-face compared to histopathology, the diagnostic accuracy of the two teledermatologists, the interobserver agreement between the two teledermatologists and between the teledermatologists and the dermatologist meeting the patient face-to-face. We will also present the image quality of this innovative dermoscopic technology and discuss the potential of using this method for teledermoscopy between primary care physicians and dermatologists.
Teledermoscopy is a rapidly developing field of dermatology and teledermatology. Many studies have described its advantages and disadvantages with special emphasis to shorten waiting lists, make dermatologist consultation easy accessible and in reducing the costs of examination of pigmented skin lesions (PSL). A non-commercial teledermatology network based on store-and-forward operation was established in Serbia in 2009.
Six dermatologists, two surgeons and three general practitioners trained in dermoscopy from five towns in Serbia equipped with Teleskin Teledermoscopy System® obtained clinical and dermoscopic images of the suspicious PSL. The images were sent using store-and-forward system in order to obtain expert second opinion and recommendation concerning excision of the PSL.
Total of 2528 patients with 3153 PSL was enrolled into the study from July 1st 2009.—December 1st 2011. Dermoscopical diagnoses were: 1800 melanocytic nevi, 84 melanomas, 697 seborrheic keratoses, 122 angiomas, 87 dermatofibromas and 341 basocellular carcinomas. Interobserver agreement between dermatologists and experts was a perfect agreement (first and second opinion) for melanoma K value > 0.89 (0,79—0,92), for melanocytic nevus K value > 0,92 (0,85—0,94), for seborrheic keratosis K value > 0,89 (0,75—0,92), for angioma K value > 0,93 (0,78—0,96), for dermatofibroma K value > 0,86 (0,49—0,94) and for basal cell carcinoma K value > 0,95 (0,84—0,98 ). Between general practitioners and experts, moderate to perfect (0,60—0,92) agreement was obtained for most of the lesions. And at the end, between surgeons and experts, K value was substantial to perfect (0,67—0,83) for all lesions.
In our experience, this large teledermoscopy study provided data that teledermoscopy is more reliable concerning melanocytic lesions vs. non-melanocytic lesions. Apart from its teaching potential, the use of teledermoscopy as a triage tool offers the potential to improve the healthcare access and delivery, both in general practice and on specialist level.
Poromas are often misdiagnosed as skin cancers.
To describe clinical and dermoscopic features of biopsy-proven poromas.
Biopsy-proven poromas were retrospectively collected from image-database of four hospitals. Data collected included patient’s demographics and anatomic location of lesions. Clinical and dermoscopic images were analyzed.
In all, 19 patients (10 males, mean age 64, range 35–90 years) contributed 19 biopsy-proven poromas. Nine lesions (47%) were on the foot and 10 (53%) on leg, thigh, trunk or face. Mean size was 7.8 mm; plantar poromas were larger than poromas elsewhere on the body (10 versus 5 mm, p<0.01). On dermoscopy, common vascular patterns were glomerular vessels in 10 cases (53%), looped in 9 (47%) and leaf and flower-like vessels in 8 (42%). Additional dermoscopic features included structureless areas in 15 cases (79%) and interlacing white cords in 9 (47%). In retrospective search through image-database of 5200 excised lesions, interlacing white cords were found in 2of 201 melanomas (2%).
Among biopsied poromas, about half arose on non-acral skin. Leaf and flower-like vessels may be a unique dermoscopic feature of poromas. While interlacing white cords are commonly seen in poromas, they are rarely seen in melanoma.
The dermatoscopic structures, which are suggestive for melanoma, are in general well defined and some of them have a stronger diagnostic value. The blue or grey-blue colour can be found in some melanomas but also in some melanocytic nevi.
We evaluated the dermoscopies of 152 melanomas and 123 atypical nevi recommended for excision. The melanoma group was divided in four subgroups in accordance with the thickness of melanoma: in situ, under 1 mm, between 1–2 mm and more than 2 mm. We registered the following dermatoscopic structures: grey-blue areas with different patterns (globules, net with lines and holes, structureless, grey dots with peppering aspect), blue-whitish veil and white areas. We analyzed the frequency of every structure in each group of lesions.
We found that grey-blue areas are high suggestive for melanoma in all the four types of patterns: the reticular ones are most frequently encountered in thin melanomas and the structureless blue areas in thicker melanomas. The whitish-blue veil was most frequently found in the thicker melanomas. The blue areas were also found in dysplastic nevi but in a lower degree in comparison with melanomas.
The grey-blue structures are relevant for melanoma in high degree, even for very thin melanomas.
Regular dermatological examination for patients with dysplastic nevi is indicated. However, the literature on whether those patients should also be examined by ophthalmologists or not regarding a relation between ocular melanoma and cutaneous dysplastic nevi is limited. In this study we aimed to screen the eyes of our patients who had been followed-up with the diagnosis of cutaneous dysplastic nevi.
We examined the eyes of 110 patients with dysplastic nevi (47 had the diagnosis of dysplastic nevus syndrome type A, B, C, D1 or D2) for any lesion and compared the results with a control group consisted of 110 sex, age and skin-type matched patients without a diagnosis of dysplastic nevi or melanoma.
No ocular melanoma was detected in any of the groups. The frequency of the conjunctival nevi, iris nevi, choroidal nevi and conjunctival acquired melanosis were similar in both groups. Iris freckles were detected more frequently in the study group. Conjunctival racial hyperpigmentation was detected more frequently in the control group (p<0.05).
Dermatoscopic features of facial lentigo maligna (LM) and acral lentiginous melanoma are well described. Lentiginous growth pattern (LGP) melanoma, including LM, is increasing in diagnostic incidence but the dermatoscopic features of non-acral non-facial LGP melanomas are not yet described. Early recognition of LGP melanomas is important, as these tend to lack BRAF mutations that are a target of therapies for metastatic melanoma.
A 12-month case series of melanomas detected in a primary care skin cancer clinic was imaged clinically and dermatoscopically before biopsy. Dermatoscopic images were assessed for proposed clues to LGP melanoma, including: lentigo-like pigment patterns associated with a lack of lentigo-like border; atypical follicular pigmentation patterns; geometric/polygonal pigment patterns and grey structures. The results of this were compared for statistical significance between groups of non-facial non-acral melanomas categorised by the following histologically reported growth patterns: LGP, mixed lentiginous and nested growth pattern, and superficial spreading melanoma (SSM).
66 melanomas (12 invasive) were diagnosed in 59 patients: 11 facial, 1 acral, 54 non-facial/non-acral (23 LGP, 13 mixed pattern, 14 SSM, 1 desmoplastic, 3 not specified). The following were associated with non-facial non-acral LGP and mixed pattern melanomas over SSM: a disorganized lentigo-like pigment pattern lacking a lentigo-like border (p<0.001); atypical follicular pigmentation patterns (p<0.05); and rhomboidal shapes (p<0.05). Mixed pattern melanomas and SSM were more likely to have structureless pink areas than were LGP melanomas (P<0.05). SSM were more likely to be invasive at diagnosis (p<0.05).
LGP melanomas may have a specific pattern of dermatoscopic features. Further multicentre study is required.
Clear-cell acanthoma is a rare benign epidermal tumour. The dermoscopic features appear fairly unique.
To delineate the key features of clear-cell acanthoma on dermoscopy.
We reviewed the dermoscopic features of all published cases of clear-cell acanthoma in the literature to date and report 4 new cases of our own.
All cases featured scattered pinpoint dotted or larger red globules. The majority of cases showed the typical ‘string of pearls’ or linear arrangement. This characteristic linear arrangement of red globules is ultimately reticular and strikingly symmetric when fully developed. In some cases, the pattern is incomplete or partly developed but still recognisable. Other features such as crusting, hyperkeratosis or a peripheral collarette may be present.
Clear cell acanthoma has distinctive dermoscopic features that help in reaching a confident clinical diagnosis and minimizing the need for biopsy.
Merkel cell carcinoma (MCC) is an aggressive cutaneous malignancy with a high mortality rate. Diagnosis is often delayed. No case series analysing the dermoscopic features of MCC have been published. Clinical and dermoscopic images of biopsy proven MCCs were analysed in a retrospective manner with existing dermoscopic criteria being scored independently by three dermatologist. The most frequent clinical features were cherry red colour, shiny and well-circumscribed nodules. Significant dermoscopic features include linear irregular and poorly focused vessels, milky pink areas, white areas and architectural disorder. Pigmented structures were absent for all lesions. The dermoscopic features described here should help achieve earlier diagnosis of MCC, which facilitates timely treatment.
Cutaneous metastases might be the initial presentation of internal malignancies in 22% of patients. 1 Up to 10% of all visceral malignant tumors develop cutaneous metastases, which represent 2% of all skin tumors. However, skin metastases from signet ring cell gastric carcinoma are uncommon. We present the dermoscopic aspects of a patient with gastric adenocarcinoma and multiple skin nodules.
A 55-year-old man presented with a progressive generalized cutaneous eruption. Examination revealed asymptomatic small erythematous firm nodules. Dermoscopy showed a polymorphous vascular pattern. One year prior to this event, this patient had been submitted to a partial gastrectomy due to a poorly differentiated, diffuse type, signet-ring cell gastric adenocarcinoma. Histologic and immunohistochemical studies of the lesions confirmed progression of the neoplasia to the skin.
Few reports have described the dermoscopic features of non-melanoma cutaneous metastases. An atypical vascular pattern was observed in one report of skin metastases of thyroid carcinoma and this feature was considered pathognomonic for neoplastic neovascularization, both benign and malign, concluding that such lesions should be biopsied. To our knowledge, no reports analyzed the dermoscopic features of gastric carcinoma metastases. We observed a prominent atypical vascular pattern, demonstrating that this feature might also be observed in nodular lesions of cutaneous metastases of gastric carcinoma. This report emphasizes that cutaneous metastases of internal malignancies might have its initial presentation on the skin and that dermoscopy is an important diagnostic tool in evaluating such lesions.
Lichen sclerosus (LS) is an unusual chronic inflammatory skin disease. Although it may affect all areas of the body, only 15% compromises the extragenital area. Initial lesions are porcelain white papules, plaques or atrophic patches. We present the dermoscopy aspects of two patients with extragenital LE.
Patient 1: A 19-year-old woman, presented with a 2-year history of two pruritic whitish plaques on her left upper back. Dermoscopy showed a whitish-pink background, surrounded by linear vascular structures and hairpin-like vessels. Yellowish areas were also visualized.
Patient 2: A 59-year-old woman, presented with a 5-month history of asymptomatic hyperchromic plaques on her axillas. Dermoscopy showed irregularly distributed black granules, predominantly over whitish areas. Discrete follicular plugging was also observed.
Dermoscopy of extragenital lichen sclerosus has been described in previous reports. Garrido-Ríos et al studied four women with extragenital LS and described, as major dermoscopic findings, whitish areas with comedo-like openings in the center of the lesions, which corresponded to follicular plugging and atrophy of the epidermis. Moreover, Kimura et al reported one case, with comedo-like openings, telangiectasias on a whitish-pink background and red-violet to brown-red perifollicular ovoid structures, corresponding to follicular plugging.
In our patients, we observed linear vascular structures, hairpin-like vessels and irregularly distributed black granules, predominantly over whitish areas. Contrasting with the prior descriptions, follicular plugging was discrete. In conclusion, we present a different dermoscopic pattern of extragenital LE, which may aid the clinical diagnosis of this unusual skin disorder.
Teledermoscopy, with all its pros and cons, allows for a grand entrance of new perspectives, and not only for Pigmented Skin Lesions (PSL). Regardless of good interobserver concordance, literature still lacks the data about the comparison of teledermoscopic diagnoses to histopathologic (HP) ones.
5 experts from different centers in Serbia, Bosnia and Herzegovina, and Macedonia are included in the study. During a two-month period, they will establish clinical and dermoscopic diagnoses on 1000 patients with 1000 histologically verified Pigmented Skin Lesions, through use of The Teledermoscopy Network. 1000 PSL contain: 130 melanomas, 230 basal cell carcinomas, 450 melanocytic lesions, 150 seborrheic keratoses, 20 angiomas and 20 dermatofibromas. Each patient has, other than personal, the following data: age, sex, skin phototype and melanoma risk factors. Each lesion has: a clinical image, anatomic localization, ABCDE clinical information and a dermoscopic image.
In the course of 2011 Jadran Bandic has selected the material for the study from the ORS Hospital database in Belgrade. During the period spanning February 1 to April 1 2012, Marijana Bandic will include 20 new cases daily (100 per week, over the course of 10 weeks with week 11 reserved for cases that get left behind for any reason) into The Teledermoscopy Network. Clinical diagnosis will be the one to be established first, with the dermoscopic image being made available for review and diagnosis after the clinical diagnosis has been made. Clinical diagnosis will not be made available for correction.
The study will show results of accordance amongst experts in relation to HP diagnosis, for every PSL type and not only the relation between dermoscopic vs. HP diagnosis, but also relation between clinical and HP diagnosis.
We believe that the right path for teledermoscopy is to be directed towards primary healthcare. In order for that path to be realised it is necessary for experts to provide background education and quick an efficient control, until the arrival of an automated process. The validity of this opinion will be pointed out by the expected results.
We present a case of a 49-year-old man who presented with a solitary atypical pigmented lesion with a surrounding halo of dermatitis. Dermoscopy showed a pigment network at the periphery with areas of scar-like depigmentation, negative pigment network and erythema. The lesion was treated preoperatively with a potent topical corticosteroid resulting in a reduction of inflammation. Histology showed an early Clark level 1 melanoma arising within a severely dysplastic compound melanocytic naevus. There was an adjacent perivascular chronic inflammatory cell infiltrate with occasional eosinophils. Minimal, though definite spongiosis with parakeratosis was also present. The scar was subsequently re-excised achieving appropriate excision margins for melanoma in situ. Six months later, there was recurrence of dermatitis at the scar with no evidence of recurrent melanoma. To our knowledge, melanoma with Meyerson phenomenon has not been reported in the literature. This case highlights that all lesions should be evaluated on clinical and dermoscopic grounds regardless of the presence or absence of eczema. Our case adds yet another entity that may display Meyerson phenomenon and consequently a halo of eczema cannot be considered a reassuring sign when evaluating melanocytic lesions.
CDKN2A and CK4 are high penetrance genes associated to high risk of melanoma. CDKN2A is a tumor suppressor gene located in 9p21, it is composed by 4 exons, which code for p14 and p16. Isoform p16 acts as a tumour suppressor gene, which binds to CDK4 and CDK6, inhibiting linking with cycline D1, thus avoiding both formation of CDK/cycline d1 complex and cell cycle. CDKN2A is inactivated by some different ways: intragenic mutations, homocygotic deletion and CpG islands hypermetilation on its promoter causing some different neoplasms such as pancreatic adenocarcinoma and malignant melanoma.
To identify alelic variants which could (or could not) influence risk (predisposition) of MM in Mexican patients.
Genomic DNA was obtained from peripheral blood samples. Alpha isoform codifying region of CDKN2A was amplified, after that automatized sequenciation in a ABI310 sequencer was performed.
Twenty-eight patients with MM Analysis of codifying and intronic regions of p16 showed 2 polymorphous heterocygotic variants: c.-2G>A in 4 patients with MM and p.I49T in 11 patients with MM. Both variants were also identified in healthy controls.
Our study found 2 different variants in melanoma patients: c.-2G>A which-–to our knowledge—has not been reported and p.I49T which partially influences in the binding to cyclin D1-CDK4/6 complex, and inhibition of cellular growth and proliferation. As well as presence of other alelic variants in genes involved in melanoma development, considering also studying influencing environmental factors.
In the context of melanocytic tumours white shiny streaks (SWS) or chrysalides have been related to malignant melanoma. It still remains unclear the biological significance of this dermoscopic criterion.
Systematic study of SWS in 125 melanomas (56% in situ; 44% invasive with mean Breslow 1.7 mm (48%<1mm)) and 305 melanocytic nevi consecutively excised in a Melanoma Unit of a referral Hospital in Barcelona.
SWS were present in 5 nevi (4.67%) compared to 41 melanomas (38,32%) (24 SSMM (58,5%), 11 LMM (26.8%), 3 NM (7.4%), 3 others (7.3%)). The presence of SWS correlated with a 10.33 fold risk of harboring a diagnosis of invasive melanomas when compared to in situ melanomas (OR: 10.33, IC 95% 3.812–28.014, p<0.005). Among invasive melanoma, SWS had 4.46 fold risk to be thick melanomas (Breslow>1mm) (OR 4.46, IC95% 1.444–13.792 p=0.009). SWS were also observed more frequently in MM with black (p<0.05), gray, white or red colours (p<0.001); structureless area, blue whitish veil, regression, atypical blotch, multicomponent (all p<0.001) or unspecific pattern (p<0,05), polymorphic vessels and milky red globules (both p<0.001) but not with dotted vessels (p =0.792). The mean TDS score for melanomas with SWS was 6.61 and without SWS 5.62 (p<0.05). SWS were also observed in 3 cases with TDS <4.75 (3.8%).
SWS in the context of a melanocytic tumour is associated to malignancy, and to invasive melanoma, with a higher Breslow thickness and higher TDS. In some few cases the presence of SWS was seen in MM with TDS of benignity.
Extramammary Paget’s disease (EMPD) is a rare, usually non-invasive intraepithelial adenocarcinoma, preferentially localized on genitals in postmenopausal women in their sixth-seventh decade of life. EMPD may or may not be associated with an underlying malignancy. The diagnosis is obtained by histopathology and immunohistochemistry. In the last few years, dermoscopy, besides pigmented lesions, has been increasingly employed also for the evaluation of non-pigmented skin tumours and inflammatory diseases, such as for assessment after therapies. We report five cases of EMPD: 4 women and 1 man, aged from 65 to 79 years old, with lesions localized on the vulva, perianal region and glans penis, which we evaluated by dermoscopy. In 5/5 cases, dermoscopic examination revealed a repetitive pattern characterized by a diffuse pinkish background mottled with crimson and bright white irregular structures, grossly mixed together, occasionally forming a sort of thick reticulation; this picture was reminiscent for us of a raspberry slush. In all 5 cases histopathology subsequently confirmed the diagnosis of EMPD. Whereas there is no standard treatment, as alternative to surgery, we treated our patients by superficial brachytherapy. This new therapy, successfully utilized for non-melanocytic skin tumors, basically consists in a superficial high dose brachytherapy, characterized by the use of a radioactive beta-emitting isotope, rhenium188, incorporated in a specially formulated inert synthetic resin. An histological examination confirmed the clinical-dermoscopic healing of our patient’s lesions. Thanks to dermoscopy, we could better identify the extent of the tumor and follow up our patient after therapy.
A naevus spilus, or speckled lentiginous naevus (SLN), is characterised by darkly pigmented macules or papules on a background of light-brown pigmentation. It is usually present at birth as a “café-au-lait” macule, with often widespread darker pigmented macules often developing years to decades later. The importance of close follow-up is underlined by case reports of melanoma developing within naevus spili.
A 54-year-old lady with no prior melanoma history presented with a pigmented lesion on her lateral thigh present since birth. Clinical examination revealed a 4×5cm café-au-lait macule with superimposed maculopapular speckles, consistent with naevus spilus. Dermoscopy showed a darker focus within the lesion though no classic melanoma features.
In-vivo reflectance confocal microscopy demonstrated multiple atypical bright large cells with upward migration and a disarranged epidermis consistent with melanoma-in-situ. The remainder of the lesion contained only monomorphous small bright cells organised around a very regular papillae ring. Histopathology of a targeted biopsy taken from the atypical area noted could not exclude a melanoma-in-situ.
Malignant melanoma arising in a naevus spilus is a rare event. The café-au-lait macule is often present at birth and the darker pigmented speckles that develop subsequently in number and size over many years are challenging to monitor.
In-vivo reflectance confocal microscopy is a well-proven laser imaging technique for pigmented lesions, allowing non-invasive examination of the epidermis. We propose its use in identifying areas of dynamic change in clinically and dermoscopically equivocal lesions, thereby assisting in the early detection of melanoma arising in a naevus spilus.
Australia has the highest worldwide incidence of cutaneous melanoma. Certain subpopulations are at extreme risk and early detection in this group is critical to reducing disease mortality.
Clinical imaging techniques permit early melanoma diagnosis and improved patient prognosis. Regular patient and doctor examination of total body photography (TBP) baseline images enables identification of changing or new skin lesions.
Sequential digital dermoscopy imaging (SDDI) additionally permits the capture and assessment of successive dermoscopic images over short term (average 3 months) or long-term (≥6 months) intervals to assess for morphological change and permits detection of still featureless incipient melanomas.
A melanoma case series of extreme risk patients diagnosed with melanoma through SDDI monitoring is presented from a 312 patient cohort managed in the Sydney Melanoma Diagnostic Centre high-risk melanoma clinic since 2006.
No classic dermoscopic features were present initially and only minimal changes were noted on SDDI monitoring. These included subtle lesion enlargement, regression and focal change, with the cases representing key diagnostic lessons from this extreme risk patient group.
Early melanoma diagnosis is crucial though can be challenged by the absence of classic diagnostic criteria. This case series reinforces the importance of the role of TBP and SDDI in monitoring patients at extreme risk of melanoma.
Melanoma thickness is a key determinant in long-term prognosis. Thick melanomas (≥1mm Breslow thickness) confer an especially poor prognosis, especially in patients already at extreme melanoma risk.
Increased knowledge regarding the characteristics of thick melanomas diagnosed in this subgroup is crucial in enabling earlier detection and increased survival.
To evaluate thick (≥1mm) melanomas detected during a five-year study for patients at extreme melanoma risk.
312 patients at extreme melanoma risk were examined six monthly using baseline total body photography for 5 years. Inclusion criteria were ≥1 of: (1) CDKN2A or CDK4 mutation; (2) ≥3 first/second degree relatives with a previous melanoma and a personal melanoma history; (3) Dysplastic Naevus Syndrome (DNS) and a personal melanoma history; (4) History of ≥2 primary melanomas.
Short term (3 months) and long term (≥6 months) sequential digital dermoscopic imaging were employed following established protocols. Atypical lesions were excised and ≥1mm melanomas were analysed.
79 primary melanomas were detected, 16 at baseline visit and 63 subsequently. 5 melanomas had a Breslow thickness of ≥1mm. These included three desmoplastic melanomas (1.6mm, 8.5mm and 21mm) and a 2.3 mm nodular melanoma and a 1.39 mm superficial spreading melanoma with nodular component.
Early diagnosis of desmoplastic melanoma vital and especially challenging in patients at extreme risk of melanoma. A high vigilance for light or tan coloured amelanotic lesions and early biopsy of longstanding scar-like or atypical lesions may assist in reducing the incidence of thick primary melanomas in patients at extreme melanoma risk.
The first clinical signs of melanoma are sometimes difficult to be diagnosed. Due to dermoscopy we can make very often an early diagnosis. The general wish is to make an in situ melanoma diagnosis as often as possible in the patients with melanoma.
We analyzed 26 dermoscopies of 12 cases with in situ melanoma and 14 cases with melanoma thickness under 1 mm. We registered for each dermoscopy the following structures: irregular pigmented network, irregular brown globules, irregular distributed black or grey dots, pseudopods and radial streaming, grey-blue areas with different patterns, atypical vascular pattern, white scar-like areas and regression of the structures which were fading.
We found that irregular pigment network and radial streaming are of great help for diagnosis of thin melanomas. Regression of dermatoscopic structures, grey-blue areas and irregular grey or black dots are often found in the in situ melanomas. In the followed up lesions, the changes in the pigment network, a small white or pinkish structure, new minimal vessel structures and few uniform grey, black dots or globules in the periphery indicate the change to malignant state.
From our few cases we can conclude that the grey-blue structures and the fading of the structures could be important dermatoscopic changes that could announce the onset of melanoma.
Jellyfish are free-life members of the phylum Cnidaria, who share the presence of a type of venomous stinging cell (cnidocyte, cnidoblast, or nematocyte) mostly located in the tentacles and around the mouth.
Diagnosis of jellyfish stings is mainly made on clinical grounds, but the finding of cnidioblasts in skin scraping or biopsy may be helpful in some cases. We evaluated the usefulness of dermoscopy in the diagnosis of jellyfish stings.
We reviewed retrospectively the clinical and dermoscopic pictures of 8 patients (6 females and two males) diagnosed of jellyfish stings in the last 5 years at Mallorca, a Spanish island in the Mediterranean Sea.
Depending on the clinical stage of the lesions, there were dotted and telangiectatic vessels and reticular pigmentation. However, the most conspicuous image on the acute stage was the presence of brown pinpoint crusts, 0.1mm in diameter on average, almost evenly spaced following a lineal distribution. This feature corresponds to the entry points of the harpoon-like structures contained in the nematocysts that are responsible for injecting the toxin into the skin. To the best of our knowledge, these findings have not been previously reported in the literature.
Pyoderma gangrenosum (PG) is a chronic skin disease, characterized by neutrophilic infiltration and destruction of tissue. It occurs most commonly in association with a systemic disease, especially chronic colitis. The main clinical finding is ulcer. Differential diagnosis includes infectious ulceration and skin cancer. We tried to use dermoscopy for make precise diagnosis.
A 24-year-old white man presented with a 3-month history of a quickly growing painful ulcer on his left groin. The patient had a history of Crohn disease for 18 years. He took oral glucocorticoids or sulfasalazine from time to time. Clinically, the lesion had irregular shape 4–6 cm with dusky-red or purple sharply borders. The base of the ulcer was purulent with hemorrhagic exudate, partially covered by necrotic eschar with granulation tissue and scars areas. On dermoscopy diffuse white to red shiny areas with ulceration, arborizing vessels and short fine telangiectasia, multiple structures like blue-gray ovoid nests was found in the lesion. It’s a typical dermoscopic picture of basal cell carcinoma (BCC). Biopsy excluded any skin cancer. Serology, cultural investigation tests helped rule out infectious etiology of the ulcer. Histopathological findings were non-specific and corresponded to PG. The lesion healed during some weeks after treatment by oral sulfasalazine and topical corticosteroids.
PG may simulate BCC clinically and dermoscopically. Diagnosis of PG, just as any other lesions, should base on the integrated assessment of the detailed history, physical examination, dermoscopy and skin biopsy.
Clinical photography is a useful tool for dermatologists particularly when monitoring skin lesions. Clinicians using photography should be aware of potential image artifacts that may blur the clinical picture. Dust contamination for instance can mimic a regressing melanocytic lesion as exemplified by our recent experience. Regular cleaning of photographic hardware, ideally by professionals, will help to minimise this problem.
Skin staining as a result of exposure to silver is known as localized argyria. This is a rare phenomenon caused by the direct application of silver and is clinically manifested by blue-grey discoloration.
We report the case of a 30-year old patient, bearer of a clinical condition of localized argyria in the ear lobes, caused by an earring screw, who had remained asymptomatic for over 20 years. Clinically, the patient manifested a well-demarcated, dark blue grey a papule of 1 cm in diameter similar to a blue nevus.
In the dermoscopy we observed blue gray, granular and annular structures around the eccrine ostium, and little linear structures. These structures are not observed in blue nevis. We also observed a scar, not visible clinically.
The histopathology showed the presence of fine, brownish to blackish particles throughout the upper and deep dermis prone to concentrating around the elastic collagenic, vascular vessels and eccrine glands.
To our knowledge, dermoscopy features have not reported in argyria.
Clinical and epidemiological characteristics of melanocytic vulvar lesions are different from those of other body sites. Vulvar melanoma typically occurs in postmenopausal women as solitary or multifocal disease; a small subset of vulvar nevi, known as atypical melanocytic nevi of the genital type (AMNGT), are commonly associated with a younger age compared with melanoma and common nevi and show histological features that may overlap with melanoma. We report the dermoscopic features of 42 melanocytic lesions that include 29 common nevi, 8 AMNGT and 5 melanomas collected at the Melanoma Unit of San Gallicano Dermatological Institute in Rome. All nevi appeared as solitary flat or palpable tumors. On dermoscopy, a globular pattern, a mixed pattern and homogeneous brown-gray pigmentation were the most prevalent patterns among nevi. The mixed pattern, defined as the combination of 2 or more dermoscopic patterns in the absence of melanoma-specific features, and characterized by the combination of parallel structures with globules or a homogeneous brown-gray pigmentation, was the most frequently pattern observed in AMNGT. All melanomas showed a multicomponent pattern, and, in most cases, reticular depigmentation and blue-white veil as dermoscopic local features. Based on our results, we propose to perform a dermoscopic follow-up for solitary flat or palpable lesions showing a mixed pattern in child to teenager. Surgical excision or a punch biopsy are recommended when a multicomponent pattern, reticular depigmentation or blue-white veil are seen, independent of the clinical features of the lesion, to rule out a melanoma.
Dermatofibroma is a skin neoplasm that is usually easy to be diagnosed clinically and dermoscopically, but in some cases its differentiation from other tumors may be difficult. Different morphologic faces of dermatofibromas may be dependent to various evolutive stages, but also to special histopathologic variants or special locations of these tumors. We report the results of a dermoscopic study of 130 cases of dermatofibromas that were consecutively collected at the Melanoma Unit of San Gallicano Dermatological Institute in Rome. “Central white scar-like patch and peripheral thin pigment network” was the most frequently observed pattern. In particular we described additional patterns defined by us, namely, the non-dermatofibroma-like patterns that were found in 17.7% of cases. These patterns were characterized by a combination of features reminiscent of melanoma (melanoma-like pattern) in 7.7% of cases or other neoplasms (such as a melanocytic nevus, a vascular tumor, or a basal cell carcinoma) in 10% of cases. Although we were not allowed to define a specific dermoscopic profile for each histopathologic variant of dermatofibroma for the low number of these variants, we found a significant association with locations, global dermoscopic patterns and local features. The knowledge of all these variables could represent a further aid for the diagnosis. However, a full surgical removal is always recommended in all doubtful cases, especially in high-risk patients and/or with a history of recent onset or changes.
A 25-year-old woman presented with non-palpable pigmented reticulated pigmentation with discrete brown macules on her dorsal surface of hands and feet. She had noted those hyperpigmentation from a childhood. The pigmented macules gradually increased in intensity and extent and, slight pigmentation also appeared on her nasal wings five years ago. Physical examination revealed multiple, discrete, coalescing faint brown macules and small brown spots distributed symmetrically on the dorsa of the hands and feet, the bilateral extensor surfaces of the forearms and the lower legs, and the nasal wings. The patient was otherwise healthy and taking no medication. Her older sister has also deeply-pigmented macules of her extremities. Dermoscopy showed coarse reticular pigmentation pattern with color heterogeneity and blurred margin. Skin biopsy from the left lower leg showed discontinuous basal melanosis in the epidermis and melanin-laden cells in the upper dermis. Immunohistochemical analysis revealed the presence of S-100 positive dermal melanocytes and CD68 positive melanophages. These clinicopathological features were consistent with acquired dermal melanocytosis of the face and extremities (ADMFE), which was proposed by Hidano in 1991. ADMFE is characterized by the development of multiple coalescing brown spots of the extremities and the nasal wings. Differentiation from dyschromatosis symmetrica hereditaria (DSH) is necessary. Unlike DSH, there was no psuedopigment network in ADMFE suggesting that brown pigmentation may be due to dermal melanocytes and melanophages, but not basal melanosis. We report here the first dermoscopic description of a Japanese patient with ADMFE.
We elaborated dermoscopy modules with acrylic tubes and plates that can be combined with a high-vision video camera and a macro lens.
To design a narrow tipped module that can fit onto interdigital or periorbital concave skin surfaces and to design a tilting module for oblique view dermoscopy.
We examined melanocytic skin lesions on the concave skin areas such as interdigital and periorbital skin using a narrow dermoscopy module and soles of the feet using a tilting dermoscopy module.
All the dermoscopy images taken with these dermoscopy modules were clear with high resolution. A narrow dermoscopy module allowed clear dermoscopy images of melanocytic nevi on the interdigital or periorbital areas. A tilting dermoscopy module enabled fibrillar pattern to be observed as original parallel furrow pattern.
Subungual melanoma in situ (early melanoma of nail apparatus) is a relatively rare subtype of malignant melanoma. The clinical resemblance of this type to benign melanonichia striata makes it difficult to differentiate between these nail disorders. Detection of early lesions of subungual melanoma is beneficial for the improvement of prognosis. In this paper, we examined 3 patients with subungual melanoma in situ and analysed the characteristics of their pigmentation of the nail apparatus with the use of a dermatoscope. Case 1 is a 33 year-old woman with melanonychia of her right thumb. Case 2 is a 44 year-old woman with narrow pigment line on her right little fingernail with 2 years of evolution. Case 3 is a 61 year-old man showing pigment striae on his index finger nail. Excisional biopsy was performed, and pathological examination revealed all 3 cases as melanoma in situ. Two of 3 cases had multiple pigment lines of nail plate. Only one presented irregular lines with variegation in colours. Though all the cases showed no Hutchinson’s sign, only one case showed pseudo-Hutchinson’s sign. Micro-Hutchinson’s sign was detected in 2 of 3 cases. Our results suggest that precise dermoscopic examination can be an easier and more reliable procedure for the detection of early subungual malignant melanoma.
Differential diagnosis of pigmented lesions of the nail has special importance. Dermoscopic features of various melanocytic and nonmelanocytic pigmented lesions of the nail are available in the literature, however the data on dermoscopy of fungal melanonychia is lacking. In this study, we aimed to define dermoscopic features observed in cases of fungal melanonychia.
We reviewed the cases of fungal melanonychia that had been seen at our dermoscopy unit within the past year. Specimen for mycologic examination was obtained by the curettage of the pigmented portion of the nail. The pigmented part was totally removed by curettage in order to see the nail bed to exclude any melanocytic lesion.
Twenty lesions in 13 cases (10 male, age ranged 34–80 years) were observed. All were located on toes and all were gray-black in color. Clinically, 2 of them were difficult to differentiate from a melanocytic lesion. On dermoscopy, melanonychia was mostly observed as multicolored (brown, gray, black, and in some cases red indicating hemorrhage) pigmentation (19/20). The multicolored pigmentation was homogeneous in 9 of the cases and, gray-black pigment aggregates, which may be called as pigmented clusters, accompanied the multicolored homogeneous pigmentation in 10 lesions. In 1 of the cases, the pigmentation was homogeneous and was observed only in gray-black color.
Basal cell carcinoma (BCC) is a slowly growing malignant epithelial tumor and world’s most common cancer. Clinicopathologic appearances of BCCs are classified as non-aggressive types, which include nodular, superficial, and adenoid variants, and aggressive types, which consist of morpheaform, micronodular, and infiltrative variants.
Dermoscopy is a noninvasive diagnostic tool, which is helpful in differential diagnosis between benign and malignant pigmented skin lesions. Though numerous studies have reported dermoscopic patterns of BCCs, there was a lack of study for dermoscopic features of BCC according to their histopathologic subtypes. In this study, we conducted retrospective histopathologic and dermoscopic analysis of 128 BCCs (91 with non-aggressive type and 37 with aggressive type) and compared dermoscopic differences according to histopathologic subtypes.
Epidermal cysts are one of the most common types of benign skin tumors. Although they are frequently encountered in the daily dermatological practice, the differential diagnosis of epidermal cysts is very broad. Therefore, they often become a complex diagnostic challenge for the clinicians. An epidermal cyst exhibits as a well-defined dermal nodule and is characterized by a central punctum that represents the plugged pilosebaceous unit. Though observation of a punctum can be used as a clue for the diagnosis of an epidermal cyst, a study regarding it was hardly reported. To our knowledge, there was only a single study conducted in 1980, had reported that a punctum was visible to the naked eye in 15 out of 34 epidermal cysts (42.1%). Therefore, we evaluated the presence or not of punctum and punctum-like structure of the skin lesion of which the first clinical diagnosis was epidermal cyst. And, we examined dermoscopic features of punctum and punctum-like structure, because dermoscopy provides clinicians with magnified in vivo observation of the morphologic features of the skin that are often imperceptible to the naked eye. We thought it could be used as an adjuvant tool in the diagnosis of epidermal cysts. Herein, we report the probability to find punctum in epidermal cyst and punctum-like structures in other dermatoses. In addition, other various skin tumors or cysts such as pilomatricoma, lipoma, pilar cyst, neurofibroma, and so on were examined to compare the dermoscopic differences of punctum in epidermal cyst and punctum-like structures in other dermatoses.
Miyazaki et al. reported that regular fibrillar pattern of an acral nevus was changed into parallel furrow pattern by horizontally moving the cornified layer with the probe of a dermoscope. Maumi et al also reported that regular fibrillar pattern changed into parallel furrow pattern by oblique view dermoscopy.
We observed an irregular fibrillar pattern in acral melanoma in situ of a 54-year-old Japanese woman by oblique view dermoscopy. The slanting angle of the melanin columns in the cornified layer was confirmed by use of DermLite DL-100 (3Gen, Dana Point, Calif., USA). After the direction of viewing was fixed so that the parallel ridge pattern was observed, pictures were taken with the Derma9500 (Derma Medical, Yokohama, Japan) and K-Y Jelly (Johnson and Johnson, New Brunswick, N.J., USA).
While a picture taken by ordinary dermoscopy showed an irregular fibrillar pattern, a picture by oblique view dermoscopy demonstrated a parallel ridge pattern.
A 75-year-old Japanese woman was referred with a 17-year history of a solitary brown macule on the right elbow. Physical examination revealed a 6-mm smooth light brown macule. Dermoscopic examination demonstrated central hypopigmentation area surrounding homogeneous yellowish light brown structureless area and radially arranged light brown leaf-like areas at the periphery. Dark brown pigment network and spoke-wheel areas were also seen in the leaf-like areas. Clinical and dermoscopic findings suggested superficial basal cell carcinoma with a differential diagnosis of early seborrheic keratosis. Histopathological diagnosis was reticulated acanthoma with sebaceous differentiation (RASD).
An 89-year-old Japanese man was referred with a 1-year history of a solitary brown nodule on the left neck. Physical examination revealed a 9.2 × 6.7 mm multi-lobulated brown nodule. Dermoscopic examination demonstrated numerous grayish to blue-gray blotches with a non-pigmented area containing multiple linear and hairpin vessels. Peripheral radially arranged light brown leaf-like areas and comedo-like openings were also noted. Clinical and dermoscopic findings suggested seborrheic keratosis with a differential diagnosis of pigmented eccrine poroma. Histopathological diagnosis was RASD.
It is well known that follow-up observation is important to detect melanoma at an early stage. However, to our best knowledge, such capabilities have been beyond the scope of specifications for automated melanoma detection systems.
To investigate whether temporal changes of the objective discrimination index previously proposed by our group help us to detect early nail apparatus melanoma.
Dermoscopic images of five lesions of longitudinal melanonychia seen in Japanese adult patients were used. Two lesions were clinically and dermoscopically diagnosed as benign longitudinal melanonychia and 3 lesions showed equivocal dermoscopic features, suspected to be evolving lesions of melanoma in situ. The size of each image was adequately reduced to ensure that the spatial resolution of each image was the same. Nail plate pigmentation excluding artifact bubbles was analyzed. The index representing variegation in color was automatically calculated from RGB values contained in each pixel according to the previously proposed method. Temporal changes were evaluated at most for 31.6 months.
In the 2 benign longitudinal melanonychia, the indices were always below the threshold value, mostly constant or decreasing monotonically. The suspicious 3 lesions were finally biopsied. Histopathologically, one lesion was diagnosed as benign melanonychia since no proliferation of atypical melanocytes was detected. In this lesion, the indices were always below the threshold and decreased monotonically during the course. The remaining2 lesions were histopathologically diagnosed as melanoma in situ. In one lesion, the index remained above the threshold, though it slightly decreased with time. The other lesion was characterized by a rapidly increasing index, though the value remained under the threshold. In conclusion, temporal changes of the index, which reflects activity of melanocytes, surely help us to identify early evolving lesions of nail apparatus melanoma.
Vascular structures are a most important tool for dermoscopic diagnosis of (hypo)-pigmented lesions. Especially basal cell carcinoma (BCC) displays a distinctive pattern, so-called arborizing vessels. However, similar vascular structures can be found in various other tumors. As the descriptive term “arborizing vessels” is somewhat subjective and there is a considerable interobserver variability in perception we felt it was desirable to achieve a precise and quantitative characterization of morphological details.
Images of 18 basal cell carcinoma, 4 malignant melanoma, 3 hyperplastic sebaceous glands and 3 blue nevi showing vascular structures resembling “arborizing vessels” were used for morphometric analysis. Vascular patterns in facial skin were analyzed as well, serving as a pattern of reference. Analysis was performed with established methods used in geography for characterization of fluvial systems. The following parameters were recorded: Branching of the vessels, determining the number (“order”) of bifurcating vessels from the stem vessel. Diameter of vessels in all sub-branches and distances between each fork was measured. The angle within each fork and the curve radius of winding vessels were determined.
Further analysis of the data permitted calculation of parameters such as number of bifurcations and number of curves along the course of a winding vessel. Analysis was performed using standard statistical procedures.
Arborizing vessels in non-BCC-tumors can be clearly distinguished from BCC vessels using these parameters. The data may be helpful for developing imaging systems diagnosing BCC by means of the vascular pattern or for follow-up of alterations of the vascular supply under therapy.
Pemphigus is an autoimmune bullous disease affecting the skin and mucous membranes. There are two main types of pemphigus: pemphigus vulgaris (PV) and pemphigus foliaceus (PF). The aim of the study was to evaluate the characteristic features of PV and PF in videodermoscopy.
Twenty-four lesions of 15 patients with PV and twenty six lesions of 11 patients with PF were examined using Fotofinder 2 videodermoscope.
Pink-red areas were present in 58% (14/24) of PV and in 61% (16/26) of PF lesions. Glomerular vessels were observed in 50% (12/24) of PV and in 69% (18/24) of PF lesions. In the proximity of skin lesions multiple, irregular elongated blood vessels were visible in 67% (16/24)of PV and in 54% (14/26) of PF cases. Videodermoscopy of skin lesions also showed yellowish areas in 25% (6/24) of PV and in 23% (6/26) of PF lesions. Moreover yellow dots with whitish halo were visible in 42% (10/24) of PV and 46% (12/26) PF lesions. Videodermoscopy also revealed the presence of two type of scaling: scaling like broken ice floe visible in 12% (3/24) of PV and in 8% (2/26) of PF lesions and scaling like a wave hitting the shore visible in 25% (6/24) of PV and in 38% (10/26) of PF lesions.
These data show the most characteristic feature of pemphigus in videodermoscopy are pink-red areas and glomerular vessels within the lesions and multiple, irregular elongated blood vessels in the proximity of skin lesions.
We conducted a survey to evaluate the prevalence of dermoscopy use among Greek dermatologists, the method by which they learned to use it, how often they use it and whether they believe it is effective.
A questionnaire was handed over to all participants attending an annual dermoscopy seminar in Thessaloniki, Greece, in November 2011. Four hundred thirty nine attendants completed the survey.
Three hundred participants (68.5%) had been previously trained in dermoscopy. The majority (73.6%) first learned how to use dermoscopy by attending a seminar. Two hundred and forty-six (56.1%) dermatologists claimed to use dermoscopy more than once daily and 20 (5%) never had used dermoscopy. The most popular algorithm used for the evaluation of pigmented skin lesions was the ABCD algorithm (70.8%) followed by pattern analysis. The most common response (33.8%) as to why dermoscopy is effective was that it detects melanoma earlier. One third of those who found dermoscopy ineffective thought so, because it needs excessive training. About a minute was the time most attendants (53%) needed to evaluate a skin lesion by dermoscopy; less than a minute was reported by 33.8%. Most dermatologists (76.8%) used dermoscopy not only for evaluating pigmented skin lesions. For 25.8% of the participants it was the first time they attended a dermoscopy seminar and 98.7% would like to attend more such seminars. Nearly half of the attendants (41.7%) were dermatologists with less than 10 years of clinical experience and 23.8% were residents.
Acquired nevi often present in childhood and increase in number and size during early and middle life. As they represent important risk factors for melanoma, the knowledge of their epidemiology, especially in young age, is essential. In our study, that is currently ongoing, we intend to determine the dermoscopic features and prevalence of dermoscopic patterns of nevi and their association with environmental factors and skin types, using cross-sectional data from a population-based cohort of children and adolescents.
The study population is going to include all students, aged 6–18 years, from 6 different schools in Thessaloniki, Greece, whose parents are going to consent to a total body clinical and dermoscopic examination of their nevi. For each participant a questionnaire is completed, which includes data on age, sex, pigment phenotype, sun sensitivity, sun exposure, sunblock use, previous sunburn history and family history of skin cancer. The total number of nevi is recorded, distributed on head and neck, anterior trunk, back, upper and lower extremities, palms and soles. Dermoscopic patterns of all nevi are recorded as globular, reticular, homogeneous, mixed and unspecified.
To date, only 388 adolescents, aged 15–18 years, and no children have been examined. A total of 17759 nevi have been recorded with a reticular predominant dermoscopic pattern (74.25%). Globular pattern was seen in 16.08% of nevi, homogeneous pattern in 3.77% and mixed pattern in 5.9% of nevi. This is the first, still ongoing, population-based study evaluating clinical and dermoscopic features of nevi in Greek children and adolescents.
Angiokeratomas are benign vascular lesions that histopathologically consist of dilated subepidermal vessels and in most cases are associated with acanthosis or hyperkeratosis. The prevalence of angiokeratomas is estimated to be approximately 0,16% among the general population. Five clinical types are recognized: angiokeratoma of Mibelli, angiokeratoma of Fordyce (angiokeratoma scroti), angiokeratoma corporis diffusum, angiokeratoma circum-scriptum neviforme and solitary angiokeratoma.
Angiokeratomas of Fordyce are typically asymptomatic, soft, solitary or multiple blue-to-red papules, plaques or nodule with a diameter of 2 to 10 mm located on the scrotum, shaft of penis, labia majora, inner thigh or lower abdomen. The pathophysiology of angiokeratoma remains unknown, although increased venous pressure may contribute to their formation. Other causative factors include acute or chronic trauma and nevoid or vascular malformations. The precise incidence of angiokeratomas of Fordyce is unknown, but they are considered common, especially with increasing age and male gender. Usually, they do not require treatment.
We performed clinical, videodermoscopic, ultrasound and histopathological examination of Fordyce angiokeratoma. Ultrasound examination was performed with 30MHz ultrasound transducer with 0,1mm resolution and 7mm penetration. Fotofinder 2 system was used for videodermoscopy.
We present a 36-year-old man with asymptomatic red-violaceous papules, 2–5 mm in diameter on the scrotum. Videodermoscopy revealed red-blue lacunae and whitish veil that corresponded to hyperkeratosis and acanthosis. Ultrasound scan showed hypoechogenic and mixed echogenicity lesions with indistinct margins. Histopathology was characteristic of angiokeratoma.
In conclusion, Fordyce angiokeratoma shows red-blue lacunae and whitish veil in videodermoscopy. In ultrasonography the lesions are hypoechogenic or show mixed echogenicity.
Becker nevus is a late-onset of
Meyerson nevus (halo dermatitis, halo eczema, Meyerson’s phenomenon) occurs when a focal and transitory eczematous eruption arises around melanocytic lesions. It appears to occur more commonly in young males (average age 30 years). It most often occurs in healthy individuals but also observed in patients with eczema or other atopic conditions. It usually develops as a single itchy patch.
Videodermoscopy was performed with a Fotofinder 2 system at magnifications 20× and 70×.
We present a 4-year-old boy with Becker nevus localized on right arm and a 1-week history of a pruriginous and erythematous halo with blistered yellowish rash which partially surrounded proximal part of hairy, symmetrical Becker nevus of 3×5cm in diameter. Videodermoscopy revealed regular melanocytic pattern with yellowish vesicles in the proximal pole. After one week of topical anti-inflammatory and antibacterial treatment Meyerson nevus disappear and control videodermoscopy revealed only pigment network.
Meyerson phenomenon does not seem to alter significantly dermoscopic features of Becker nevus.
Essential for increasing the chance of detecting melanoma are: the health education, regular skin examination by the patient and by the dermatologists and improving diagnostic methods in medicine. Dermoscopy has developed into a standard method in diagnosing melanoma. This method may provide some insight into the tumor thickness, but no precise measurement is possible. The aim of our study was to evaluate whether ultrasonography may provide important pre-excision information about the tumor and its thickness.
A total of 40 common nevi and 25 cases of cutaneous melanoma were evaluated clinically, by videodermoscopy, and 30 MHz ultrasonography. In ultrasonography assessment of echogenicity, depth of the lesion and vascularity were performed. All images were analyzed in the context of histopathology results.
In ultrasonography melanoma has been visible as hypoechogenic area clearly separated from the environment, usually with uneven borders. Dimensions of lesions were adequate to those seen in histopatology. We found that ultrasound evaluation involved a risk of mistake in evaluation of lesion size related to various causes: presence of hair follicles, sweat glands and sebaceous glands or the inflammatory infiltrates. In 24/25 cases videodermoscopy showed features characteristic of melanoma.
In conclusion, we believe that skin ultrasonography allows evaluation of melanoma thickness prior to surgery and is an important accessory tool to dermoscopy or videodermoscopy, which serve as diagnostic aids to establish the diagnosis.
Beard folliculitis (folliculitis simplex barbae, sycosis barbae) is characterized by pustules penetrated by hairs, with inflammation of the hair follicles localized in the bearded area and on the upper lip. Infection can be spread through contaminated shaving tools. Laboratory examinations are typically not obtained because diagnosis is usually made based on history and clinical examination alone. In cases resistant to standard therapy, cultures, Gram stain, potassium chloride preparation and biopsy are the diagnostic tests of choice. Histologically sycosis barbae is defined as the presence of inflammatory cells within the wall and ostia of the hair follicle, creating a follicular-based pustule.
Videodermoscopy was performed with Fotofinder 2. RCM was performed with Vivascope 1500
We report the case of a 43-year-old man with an acute onset of small pustules surrounded by erythema that were pierced by a central hair easily extracted from the follicle localized on the upper lip and chin. Pustules ruptured and leaved a yellow crust. Deeper changes manifest as erythematous, fluctuant nodules. Skin lesions were associated with discomfort and suppurative drainage. In videodermoscopy we observed: dilated linear vessels around hair follicle, in some areas absence of follicular units with yellow dots and in some dystrophy of hair shaft (hair contraction), scaling and white-yellowish lacunas correspond to pustules.
RCM images revealed the presence of groups of inflammatory cells inside hair follicles, inflammatory cell infiltration at the level of the spinous and granular layers and increased vascularity.
In conclusion, dermoscopy and RCM may be helpful tools in diagnosing beard folliculitis of atypical clinical presentation.
The association of Spitz nevus with deep penetrating nevus is a tumor of rare cancers combined, especially in puberty.
The authors report a case of a 11-year-old girl with a pigmented lesion of about 1×08 cm, centered by a bluish papule on the skin of 0.7×06 detected with sharp margins. The appearance was characterized by a composite pattern with all the features of melanoma. The authors discuss the complexity of the case. Furthermore, the authors discuss aspects dermoscopy and histological features of the tumor combined.
Macular amyloidosis (MA) is a common variant of primary localized cutaneous amyloidosis. It is secondary to amyloid deposits on dermis, but the etiopathogenic mechanism is still unknown. Clinical presentation consists of grayish-brown pigmented and pruritic macules located on the upper back or arms. Nowadays, a cutaneous biopsy may be performed for a final diagnosis. The amyloid deposits are within the dermal papillae and they are usually globular, resembling colloid bodies, but sometimes they are too small and escape to detection. Congo red stain shows apple-green birefringence under polarizing light. Differential diagnoses of poikiloderma of Civatte and postinflammatory hyperpigmentation could be established. So, the description of dermoscopy patterns for MA could be useful to diagnose and to differentiate it from other entities. We present a series of 15 cases of MA with histological confirmation. First, we describe the main dermoscopic findings in MA. We analyze the results and compare MA dermoscopic features with dermoscopic findings in other entities as postinflammatory hyperpigmentation.
Urticarial vasculitis (UV) is a subset of vasculitis characterized clinically by urticarial skin lesions and histologically by leukocytoclastic vasculitis. The cutaneous lesions are urticarial in appearance, but usually last 24–72 hours and may have residual changes of purpura, scaling and hyperpigmentation. However, it is difficult to find out clinical differentiation between common urticaria and early urticarial lesions of urticarial vasculitis.
Polarized dermoscopes (PD) use the properties of cross-polarized light to view deeper skin structures, not visible to the unaided eye, and they allow better visualization of blood vessels and reddish hue associated with some lesions. In this respect, through PD, clinical, histological and dermoscopic changes over time in urticarial vasculitis were observed. Although clinically hard to distinguish early urticarial lesions in urticarial vasculitis from common urticaria, but histological diagnosis was available to show the early phase of leukocytoclastic vasculitis characterized by prominent dermal edema, focal fibrinoid vascular change and a sparse infiltrate composed of lymphocytes and eosinophils. Although not prominent, dermoscopically purpuric red dots and globules were seen in early lesions. Urticarial lesions faded, made it easier to distinguish between common urticaria and urticarial vasculitis. Purpura or hyperpigmentation lesions showed dermoscopically purpuric dots or globules in a patchy orange-brown background. These structures were associated with fibrin deposits, nuclear dust, and extravasation and degradation of red blood cells, demonstrating fully developed LCV. In conclusion, PD faithfully reflect clinicohistopathologic transition from early to late stages in UV and appear to be helpful in differentiating early lesions of UV from common urticaria.
We describe novel dermoscopic features observed on the pebbly surface of pseudoxanthoma elasticum (PXE) in two adult cases. Clinically, a 45-year-old and a 60-year-old woman showed typical ‘chicken skin’ appearance on the neck and axillary regions. The lesional skin showed hypotrichosis and concomitant comedones. Histopathologically the affected areas showed degenerated collagen fibers and calcium deposits in the dermis. Notably, dermoscopy clearly revealed that the hypotrichosis was associated with remnant or broken hairs whose follicles were accentuated as brownish dots. In addition, remnant hair follicles were distributed around the yellow pebbly surface, probably corresponding to dermal calcium deposits. Furthermore, abnormal hair follicles were located in gaps in the yellow net. Consistent with these dermoscopic findings, histopathology showed immature hair follicles that were displaced by degenerated collagen fibers and keratotic plugging. Dots in the yellow net pattern would be characteristic dermoscopic feature observed in ‘chicken skin’ regions of PXE.
Access to health care services takes on a new definition when scheduling visits with specialists in urban areas is a six-month wait listing. This is the case for dermatology consults in Pennsylvania. With the rural nature of Pennsylvania and exposure to the environment through farming, lumbering and mining, the Departments of Family Medicine and the Melanoma Center developed a pilot training program for primary care physicians in the use of dermoscopy. Physicians were required to complete workshops in real time as well as extensive self-directed activities that developed the knowledge and skills in dermoscopy and the self-confidence and self-efficacy in its application to patient care. This study looked at the learning process as well as the implication to practice transformation, increasing patient access, early diagnosis and avoidance of unnecessary biopsies.
Basal cell carcinoma is the most frequent cutaneous neoplasm. Today all known treatments are effective on the primary stages of BCC, hence it is very important early detection of the tumor. Dermatoscopy has proven to be a helpful tool in diagnosing of BCC.
Dermatoscopic characteristics were analyzed in 57 patients at 42–90 years of age. We observed 73 BCCs. Multiple BCCs were included. All diagnoses were confirmed by the cytologic or pathologic examination.
Among numerous variations in clinical presentation of BCC, such as superficial BCC, nodular BCC, ulcerating BCC, pigmented BCC, sclerosing BCC most common dermatoscopic features were ulceration in 49 BCCs (67%); shiny white to red structureless areas in 27 (36%); blue/gray ovoid nests in 13 BCCs (17%); leaflike areas in 12 BCCs (16%). The vessels were presented by arborizing telangiectasia in 33 BCCs (45%), which were more frequent in nodular BCCs; hairpin vessels in 33 BCCs (45%), short fine telangiectasia in 18 BCCs (24%) that was more frequent in superficial BCCs. Less frequent we observed glomerular vessels, dotted, comma vessels, linear irregular vessels. The large amount of the dermatoscopic features observed proves that the dermatoscope increases our chances of detecting BCC in early stages.
Mibelli’s angiokeratoma is a rare benign condition. The lesion is a 2- to 8-mm hyperkeratotic or verrucoid nodule that is blue-red or grey and may have a central haemorrhagic crust. It is most often seen on the dorsa of the toes and fingers, knees, elbows, feet, and lateral lower quadrants of the breasts. When extensive thromboses develop in an angiokeratoma, this nodular bluish-black lesion may clinically simulate a nodular melanoma. A 13-year-old girl presented to our outpatient clinic with a one-year history of a dark red-black lesion 0.6×0.8 cm in size on the medial side of her right femoral region. She had a history of bleeding of the lesion following traumas that most of its surface was covered by firm haemorrhagic crusts. Dermoscopic examination revealed dark red-black, sharply demarcated haemorrhagic crusts in different sizes, under which red-blue lagoons could be distinguished. Moreover, some hairpin vessels supporting malignant melanoma were detected at the periphery of the lesion. Finally the lesion was totally extirpated, and based on dermoscopic and histopathologic findings, it was diagnosed as trombosed solitary angiokeratoma of Mibelli. In conclusion, dermoscopy seems to represent an effective and reliable method in differential diagnosis between pigmented non-melanocytic (angiokeratoma Mibelli) and melanocytic lesions (malignant melanoma).
Shiny white streaks (SWS) are a new der-moscopic criteria detected under polarized light that have been associated to melanoma and other skin tumours.
Analysis of 800 dermoscopic images for the evaluation of the presence of SWS in the diagnosis of malignant skin tumors. The data set comprised 125 melanomas, 133 basal cell carcinomas (BCC), 305 melanocytic nevi, 49 seborrheic keratosis, 36 actinic keratosis, 21 squamous cell carcinomas (SCC), 19 solar lentigos, 11 dermatofibromas, 9 lichenoid keratosis, 1 neuroendocrine carcinoma, and 44 benign tumors (hemangiomas, benign keratomas).
SWS were observed in 107 tumors (13.37%); 41 melanomas (38,32%), 41 BCC (38.32%), 5 nevi (4.67%), 6 dermatofibromas (56,1%), 4 actinic keratosis (3.74%), 3 SCC (2.80%), 2 lichenoid keratosis (1.87%), 2 solar lentigos (1.87%), 2 seborrhreic keratosis (1.87%), 1 neuroendocrine carcinoma (0.93%). Only 1,6% of all nevi presented SWS. The presence of SWS suggested a 10-fold risk of malignancy (melanomas, BCC, SCC, neuroendocrine carcinoma) (OR: 10.534 IC 95% 6.357- 17.455 p<0.0005).
Concerning the subtype of BCC data set (133 lesions) they were; 65 superficial (48.87%), 20 infiltrating (15.03%), 35 nodular (26.31%), 9 tricholemmal (6.76%), 3 unspecified (2.25%), 1 pigmented BCC (0.75%). Among all BCC 30.8% presented SWS and there was no significant difference regarding histopathological subtype. Interestingly, SWS were observed more frequently in ulcerated BCCs (p<0.005).
The presence of SWS in a skin tumor is associated to malignancy. Except in the case of dermato-fibromas, this criterion is rarely seen in benign cutaneous tumours.
Basal cell carcinoma (BCC) presents with variable thickness in a variety of histopathological subtypes. Dermoscopy features of BCC include large diameter blood vessels.
To investigate if dermoscopy identified large diameter blood vessels correlate with: (1) thicker tumours and (2) BCC subtype histopathology.
Consecutive BCC cases (n=1098) were assessed in vivo for large diameter blood vessels within the tumour “footprint” prior to full excision. The histopathological identified subtype categories of (1) superficial plus nodular, (2) nodular and (3) aggressive subtypes were compared for the presence of large blood vessels. Large vessels were defined as any vessel with a diameter larger than the largest background vessel out to 10mm from the tumour margin. Tumours with known previous intervention were excluded. Ultrasound gel was applied between the tumour surface and glass plate of the dermatoscopes to avoid vessel compression. Data validation was assessed by two observers (n=108) by a Cohen Kappa value of 0.96 for agreement on the presence of large vessels.
All 3 BCC categories recorded a higher incidence of thicker tumours with large diameter blood vessels. Nodular BCC consistently has the highest incidence of large diameter vessels compared to the other subtypes (p<0.005).
Cases were not categorized by tumour horizontal size. Recurrent tumours were excluded from the study.
Thicker tumours do tend to display larger vessels, although the difference in thickness is relatively small. Large diameter vessels add the features used to discriminate BCC subtypes.
Squamous cell carcinoma management and prognosis is influenced by the grade of tumour differentiation. Dermoscopy features associated with different grades of differentiation in SCC have not been previously reported.
Compare well differentiated (n=255) to combined moderate and poorly differentiated SCC (n=39) with an emphasis on dermoscopy features.
A prospective study of 294 consecutive cases of histopathology confirmed invasive SCC was conducted to compare the dermoscopy features of well with moderate or poorly differentiated SCC.
Dermoscopy vascular features were recorded in vivo and included: branching, serpentine, dot, hairpin, glomerular and linear vessels, the proportion of pink in the tumour and the number of distinct vessel types. These features were identified using a Heine Delta 20 dermatoscope. Photographic images were recorded by a Dermlite foto dermatoscope coupled to a Canon EOS 550D camera.
Vascular feature validation was assessed for inter-observer agreement using Kappa values, which ranged from 0.66 to 1.00.
Moderate and poorly differentiated SCC, display branching (28%, P<0.001) and serpentine (62%, P<0.005) blood vessels are more frequent than well differentiated tumours. The proportion of pink areas in the tumour varies between differentiation grades. Moderate and poorly differentiated tumours display larger numbers of vessel types. Increasing tumour depth from 1 to greater than 4mm was found to be associated with an increased proportion of moderate or poorly differentiated SCC.
All data was generated using non-polarized light dermoscopy. Data was not categorized by anatomic site or tumour horizontal diameter.
Dermoscopy enhances the identification of the grade of tumour differentiation in SCC.
Detecting changes in lesions is the most sensitive clinical sign for the early detection of melanoma. Thus, some diagnostic procedures incorporated some years ago the ‘evolving’ factor on their methods. This ‘Evolving’ parameter measures changes in colour, shape and/or size of pigmented lesions as an early sign of melanoma. Total Body Photography (TBP) has been reported in the literature describing its advantages on diagnosing skin diseases, combining images acquired with high-resolution digital cameras and baseline photography. These images are referenced by patient and date of acquisition and then compared over time with other explorations using specific software to automatically detect changes or by a physician that visually compares them.
TBP systems acquire nowadays around 20 images in standardized patient positions covering the face, neck, area behind the ears, scalp (in bald individuals), anterior and posterior trunk, and the extremities (including palms and soles). Our proposed method acquires 800 images covering the full body with a much higher resolution (in the order of 25 pixels per millimetre). Also the system incorporates a cross-polarized lighting in order to both reduce the reflexions on the images and also to see deeper pigmentation.
Such volume of images will be mapped to a standard reference system, which encodes a 3D representation of the body of the patient. This allows the registering of the spatial position of each lesion, automatically managing duplicated views of the same lesion from different perspective points. Further works will allow lesion segmentation, characterization and automatic comparison.
Pigmented basal cell Carcinoma (BCC) can be clinically confused with melanoma, dermoscopy being a useful tool in differential diagnosis by identifying criteria that favor one or the other. We present two cutaneous melanoma-simulating lesions to the naked-eye, but with significant BCC dermoscopic features, emphasizing the usefulness of this diagnostic method in the approach of pigmented lesions.
CCPL, white, 19-year-old, male, student, presenting a dark, papular lesion with irregular pigmentation and asymmetric borders on his left arm deltoid region, four years of progress, and a “change-in-aspect” in the last 12 months. Dermoscopy revealed ovoid nests, spokewheel-like pigmented structures, and maple leaf-like pigmentation, typical of pigmented BCC. Histopathology: BCC
FFA, white, 43-year-old, female, doctor, presenting a 1cm pigmented lesion on her right paravertebral region, crusted surface with a pearly, pigmented edge. Der-moscopy revealed arborizing vessels, ovoid nests, specific for pigmented basal cell carcinoma, and a greyish blue veil, more common in melanoma, directing us to a dermoscopic high score, characteristic of lesions at high risk of malignancy. Lesion excised. Histopathology: BCC.
Dermoscopy can assist in directing the conduct in cases of cutaneous melanoma simulating lesions, and is an essential semiological method for these lesions, once it allows the identification of several criteria for melanocytic and non-melanocytic lesions. In INCA, dermoscopy is used as a screening exam for dermathological surgery, allowing faster surgery of melanocytic lesions suggesting melanoma and elective resection of BCC.
There are a number of published algorithms developed to assist in the diagnosis of malignancy in pigmented skin lesions. Many of these algorithms require mathematical calculations making application to routine practice laborious.
We present an algorithm based on ‘Revised pattern Analysis’ known as ‘Chaos and Clues.’
Structures are clearly defined and named with simple geometric, rather than metaphoric, terminology. There is no need to make a presumptuous decision about melanocytic status as a first step. There are no mathematical calculations and the method is designed to integrate seamlessly into routine skin examination.
‘Chaos and Clues’ was evaluated in a study on 463 consecutively treated pigmented skin lesions, including 29 melanomas, in a primary care practice in Australia and was shown to diagnose pigmented skin malignancy (including melanoma, pigmented basal cell carcinoma and pigmented squamous cell carcinoma in situ) with a sensitivity of 90.6% and a specificity of 62.7%.
The algorithm is presented on a poster complete with an algorithmic flowchart, a step-by-step explanation of the method illustrated with high resolution dermatoscopy images, and a summary of validation studies.
New dermoscopes come with a polarization feature that allows the user to reduce the effect of reflections and glare. However, the built-in white LEDs used for polarization cause a special redial lighting artifact resulting in separate areas of over and under-exposure that must be removed before any image analysis. For example, the consistent lighting is an extremely important feature in segmenting lesions from normal skin.
We have proposed a new approach that finds intrinsic images by a fast entropy minimization and subtraction. First, two images with different lightings (polarized and non-polarized) are captured from the same skin surface. Pixels are transformed from 3D RGB triples into a 2D colour space G/R, B/R, and then logarithms are taken. The values across different lightings tend to fall on straight lines in 2D and change of illumination simply amounts to movement along such lines. Therefore, it is straightforward to devise a 1D illumination-invariant image by projecting the 2D chromaticity points into a direction perpendicular to all such lines. We find the projection angle such that minimizes the entropy. This intrinsic images plus a calibration image of the normal skin is used to find the artifact pattern to be subtracted from dermoscopy images to recover the image that portrays only the inherent reflectance properties of skin.
The qualitative results of our experiments show that the proposed method can significantly improve the quality of images and our quantitative results show 11.5% improvement in skin lesion segmentation accuracy in illumination corrected data sets.
Inverted follicular keratosis is almost always a solitary lesion, occurring mainly in adult life. About 85 % of these lesions are found on the face. The duration of the lesion, when known, has varied between six weeks and three years. Most of the lesions are between 3 and 8 mm in maximum diameter. They are generally asymptomatic, firm, pinkish papules. Clinically they are often considered to be viral warts, basal cell carcinoma or a variety of benign lesions. They are thought to originate specifically from the infun-dibulum of the hair follicle. Differential diagnosis includes “irritated” seborrheic keratosis, keratoacanthoma, viral warts, and squamous carcinoma, An 85-year-old female patient admitted our out-patient clinics with a complaint of itchy papular lesion on her right cheek for 2 months. Dermatologic examination revealed centrally located grey-blue papular lesion 0.5 cm in size, with a peripheral hyperpig-mented macular component 1.5 cm in size. Dermoscopically, macular part of the lesion was consisting of pseudo-pigment network. Central papular lesion was white-grey in colour, where hairpin vessels, linear and glomerular vessels in radial distribution could be seen. On the papular part of the lesion 3 crypt-like structures brown in colour with different sizes were seen. Histopathologic examination revealed multilobu-lated cells with narrow cytoplasm, and slight pleomorphism; focal keratinizations and squamous nests inside the lesion. There was a chronic inflammatory response at the periphery of the lesion consisting of foreign body type giant cells. PAS stain was negative. Where some immunohistochemical stainings including HMB-45, cytokeratin-7, and S-100 were negative, EMA and HMWCK were found to be positive. According to these histopathologic and dermoscopic findings our case was diagnosed as inverted follicular keratosis. We present this case to draw attention to the importance of dermoscopy and histopathology in the diagnosis of inverted follicular keratosis.
Tufted hair folliculitis which affects the scalp, is a rare, progressive pattern of scarring alopecia. The presence of groups of 10–15 hairs emerging from a single follicular opening in adults is its characteristic feature. Tufts of hair associated with scars have been described in association with several other forms of alopecia. Staphylococcal organisms frequently are cultured from lesions of tufted hair folliculitis, but their role in pathogenesis is unclear. Patients with tufted hair folliculitis report slowly developing hair loss. Pain or swelling of the affected scalp frequently accompanies the hair loss. Crust and scales adherent to the scalp and hair are frequently seen. The ability to express pus from the follicular orifice is a constant finding. This process usually is limited to a single area of the scalp that enlarges gradually. The most prominent feature of this disorder is the presence of tufts of 8–15 hairs that appear to emerge from a single follicular orifice in a “doll’s hair” pattern. Adjacent to and intermingled with the tufts are areas of scarring alopecia, with complete loss of follicles. The area of tufts and scarring is somewhat well circumscribed and may be accompanied by varying degrees of edema, erythema, and tenderness. Tufts of hair amid areas of scarring, giving the classic appearance of tufted hair folliculitis, have been described in patients with a number of different disorders, including scars from surgery or trauma, acne keloidalis, folliculitis decalvans, dissecting cellulitis of the scalp, lichen planus, and pemphigus vulgaris. A 37-year-old male patient admitted to our out- patient clinics with complaint of itchy areas on his scalp for 8 years. Clinical examination revealed scattered cicatricial alopecia areas and tufted hairs limited to his parietal region. There were crusts and scales around hairs and scalp in trichoscopy. Tufts of 8–15 hairs that appear to emerge from a single follicular orifice were seen. Perifollicular teleangiectasic erythema was detected in cicatricial areas. We present this rare case to demonstrate and discuss the trichoscopic, dermoscopic, and histopathologic findings of tufted hair folliculitis.
Dermatologists often come up against a problem of treatment for longitudinal melanonychia in children. Clinical, dermatoscopic and pathological criteria that permit clear differentiation between benign melanocytic activation and proliferation from nail matrix melanoma have not been established for children. The clinical and dermato-scopic features that are considered to be possible indicators of nail unit melanoma in adults are sometimes observed in benign melanocytic activation and hyperplasia in children.
Nail melanoma in children is very rare. Thus, there is still a controversy whether a single band of LM with clinical and dermoscopic features that suggest melanocyte hyperplasia in a child should be excised or not.
We aim to provide more insight into the diagnosis of longitudinal melanonychia in children.
In the present study, we examined the characteristics of longitudinal melanonychia in children, which have been followed in our institute.
In some LM lesions in children, color irregularity, irregular lines and triangular pigmentation were seen at first sight. In addition, pigmentation of the periungual skin was sometimes observed. These features were suggestive of malignant melanoma in situ. However, further dermoscopic examinations often revealed features indicating benign nature of the lesions, including regular lines in the LM and parallel furrow, fibrillar, lattice-like and globular patterns in pigmentation on the periungual skin (pseudo-Hutchinson’s sign).
We often encounter embarrassing LM cases in children. However, even in such cases, thorough dermo-scopic examinations might give us clues to deny malignant melanoma.
Dermoscopy is a portable tool for the diagnosis of pigmented skin lesions. The aim of this prospective study was to evaluate how many malignant melanomas might not be excised based on clinical examination alone, but would be removed if dermoscopy were used. The secondary aim was to describe the morphologic characteristics of these misdiagnosed (false negative) MMs.
All patients coming for a routine check-up underwent a total body clinical examination, followed by dermoscopic evaluation. Lesions found to be suspicious on either clinical or dermoscopic evaluation were excised for histopathologic examination. Prior to surgical removal, the clinical and the dermoscopic index of suspicion was scored, categorized on a scale of: 0- not suspicious for MM, 0.5- low index of suspicion 1- high index of suspicion for MM, and clinical and dermoscopic imaging was performed. For all biopsy-proven MMs, the clinical and dermo-scopic scores were retrospectively evaluated and proportions of true positive MMs (clinical and dermoscopic scores >0) and false negative MMs (clinical or dermoscopic scores =0) were compared using chi square test. Images of those that received a clinical or dermoscopic score of 0 were evaluated and the morphologic characteristics of those false negative MMs were described.
Between august 2008 and august 2010, 5700 patients came for routine examination. Fifty-nine biopsy-proven MMs (1%) were identified. The proportion of MMs receiving a clinical score of 0 was 27%, 0.5 was 33% and 1 was 40%; dermoscopy was more accurate in identifying true positive MMs, the proportion of MMs receiving a der-moscopic score being 0 was 15%, 0.5 was 3% and 1 was 82% (p< 0.01). Among these 59 MMs, 16 (27%) of MM received a clinical score of 0 (false negative MMs); 3 of them (18.7%), would still have been excised because of clinical suspicion for BCC, but other 12 lesions (81.3%), would not have been excised on clinical grounds alone. The false negative MMs were morphologically characterized as being significantly smaller in size and having a light color or being amelanotic higher than true positive MMs.
Dermoscopy enable the clinician to correctly diagnose a subset of MMs that would be false negative MMs on clinical examination alone. Especially, dermoscopy is helpful above and beyond clinical examination for smaller and lightly pigmented MMs.
Since dermoscopy had been introduced and popularized many authors quote the ration of 4 to 1 (benign to malignant pigmented lesion ratio- nevus to MM) at the highly skilled pigmented lesion clinics, to be a magic number reflecting the efficiency of dermoscopy in reducing unnecessary skin lesion excision. But in true life plastic surgeons and dermatologist are excising a great variety of other skin lesion. In this prospective study we are evaluating the work of one plastic surgeon and calculating the ratios between the different types of excised skin tumors with and without dermoscopy.
From August of 2008 until July 2010 all cutaneous lesions that were excised by a single plastic surgeon were first examined by dermoscopy and then the lesions were photographed with the dermoscope. All excised cutaneous lesions were recorded in the surgeon logbook and in a computerized database according to the pathology report in a standardized manner; this was defined as the study group. The control group compared of all patients that were treated by the same surgeon between October 2001 to December of 2003 in which the entire log book with the clinical evaluation and pathology reports was available, and at that time patients had only clinical evaluation without dermoscopy examination. Once the data was gathered, analysis using excel program was used in order to evaluate the exact ratios of each type of lesion excised as part of the total excised skin lesion, the total malignant to benign ratio was determined and the ratio of benign to malignant ratio of the pigmented lesion was calculated as well (malignant melanoma to nevus). The proportion of the different types of malignant tumors within the total malignant lesions, and the proportion of the different types of benign lesions within the benign lesions group was calculated as well
In the study group 1817 lesions were excised, there were 1491 (82%) benign lesions and 329 (18%) malignant lesions. In the dermoscopy group 2162 lesion were excised, there were significantly less benign lesions excised 1278 (59%), and significantly more malignant lesions 884 (41%) excised. The ratio of nevus to melanoma was 1:34 and it has been decreased significantly to 1:12 on the der-moscopy group. The ratios between the different malignant lesions had an increase in the BCC and MM excision in the dermoscopy group. The overall MM excision has doubled from 1:90 to 1:44 from all excisions.
Dermoscopy has proved to be an efficient tool to prevent unnecessary skin biopsies. It has increasing significantly the proportion of malignant lesions excised, and doubled the amount of MM excision. It has a significant impact on morbidity reduction (reducing the amount of unnecessary surgeries), and great economic saving impact by the same manner.
Desmoplastic melanoma (DM) has been shown to have adverse features including deeper invasion, propensity for perineural spread, and higher rates of local recurrence and distant metastasis. Current literature has focused on the subclassification of DM into pure (pDM) and mixed (mDM) subtypes and their differing capacity for nodal spread and recurrence. We report a single institution’s experience of DM, examining the clinical behavior of the two histologic subtypes and implications for management.
A 10-year review of all patients diagnosed with DM at the Peter MacCallum Cancer Centre was undertaken. Cases were divided into 2 groups based on histologic classification: pDM and mDM.
Sixty-five patients were reviewed (42 pDM and 23 mDM patients). The majority of both subtypes arose in the head and neck with presentation as a pigmented lesion more frequent for mDM (60.9% vs 38.1%). Breslow thickness was greater in the pDM than in the mDM group (mean 7.0 vs 3.5mm). One of 11 pDM and none of 5 mixed DM patients had positive sentinel lymph node bopsy (SLNB). Disease recurrence occurred in 26 of 61 patients (43%) overall (14 mDM and 12 pDM patients). Regional LN metastasis was seen in 18% of patients with a higher rate seen in the mDM group.
Clinicopathologic features of pDM and mDM subtypes differ. Despite a low rate of positive SLNB, a significant number of both pDM and mDM patients developed regional nodal disease. These factors should be taken into account when managing the different subtypes of DM.
Dysplastic nevus has got a definite set of clinical, dermatoscopy and morphological features and shows a high risk of skin melanoma occurrence. Statistical research data reveal that from 7 to 18% of the total population are patients with dysplastic nevus. However, despite the revealed clear correlation between a dysplastic nevus and high risk of skin melanoma occurrence, the referential data demonstrate that the majority of the dysplastic nevi never developed into a melanoma.
In Moscow Oncological Hospital ⊠ 62, the dermatoscopy follow-up examination was carried out from 2004 through 2008 to monitor the dynamics in structure of melanocytic nevi in 44 patients with high risk of melanoma. The total number of investigated clinically atypical nevi was 223. The final diagnosis was based on histopathological examination.
A definitely clear change in the dermatoscopy structure was recorded in 10.3% of the total clinically atypical melanocytic nevi. All the changed nevi had the features of the melanocytic dysplasia of different categories. In 75% of clinically diagnosed atypical nevi, there was no evidence of the melanocytic dysplasia and there was no change in the nevi dimension or structure for the follow-up period of 6 to 24 months.
The obtained data of dynamic dermatoscopy examination demonstrate that the noninvasive method in question should be applied to detect the skin melanoma at its early stages, dysplastic nevus and to reduce the number of excisions of clinically atypical benign melanocytic skin lesions.
It is important to evaluate subclinical extension of morphea-like basal cell carcinoma (BCC) before planning surgery because recurrence may be caused by incomplete tumor excision. Dermoscopy and/or ultrasonography sometimes fails to identify the extent of subcutaneous involvement.
To verify the usefulness of hyperspectral images of BCC in identifying the extent of subcutaneous involvement and setting the margin required for complete tumor excision.
The patient was a 64-year-old Japa-nese female. The pigmented tumor was located on the nose. Dermoscopic and ultrasonographic images were taken and hyperspectral data were measured using a hyperspectral imager (MSI-03: Mitaka Kohki, Japan). The hyperspectral data were analyzed and converted into several images using an original spectral analysis algorithm. The resultant images were compared with the dermoscopic and ultrasonographic images and, furthermore, with histopathological findings.
An image constructed from the hyperspectral data satisfactorily represented subclinical extension of morphea-like BCC, while dermoscopic images could not. The radial size was consistent with the tumor size estimated based on the histopathological examination. With respect to the ability to predict tumor size, hyperspec-tral images are superior or at least comparable to ultrasonographic images. The hyperspectral imaging technique presented here is considered to have considerable promise in setting the margin required for complete tumor excision.
Dermoscopy of hair-trichoscopy allows exploration of the hair at 10 to 800× and to observe precisely the types of hair, follicular openings, the peripilar signs and to follow up the evolution of the disease or the treatment efficacy prior to naked eye clinical observation. I studied 84 adults with 143 plaques of alopecia areata by trichoscopy before and after 3 months of treatment. 71,3% of plaques had regularly distributed yellow dots, corresponding to hyperkera-totic plugs in hair follicle. 51,7% had exclamation mark hair. 46,1% had dystrophic–broken hair; 27,9% had cadaverised hairs, black dots in the hair follicles. 8,8% had short pseudo regrowing hairs that are apparently regrowing but they are atrophic hairs and are a sign of activity of alopecia areata. They mostly disappear at 3 months trichoscopic follow up. 13,2 % had corkscrew hairs; 4,8% had circle hairs; 4,1% had vellus hairs-0,03 mm or less in thickness; 3,5% had white dots-feature of fibrosis; they have extensive persistent alopecia areata. I did not find any pseudo moniletrix hairs. The most frequent pattern is the presence of regular yellow dots (71,3 %), the second the presence of exclamation mark hairs (51,7%), and the third is the presence of dystrophic-broken hairs (46,1 %). The presence of pseudo regrowing hairs is a sign of the activity of alopecia areata. They are thin hairs that differ from normal thick, real regrowing hairs, sign of treatment efficacy.
The typical dermoscopic features of basal cell carcinomas have been well known. Besides them, some other dermoscopic features such as multiple brown to black dots and globules, blue/white veil-like structures, and non-arborizing vessels have also been described. Recently, “diffuse blue-white areas” has been reported as a dermoscopic pattern in some pigmented BCCs, namely “blue-white variant.”
We aimed to evaluate our cases with blue-white variant of basal cell carcinoma seen at the dermoscopy unit between 2003 and 2011. We reviewed the patient files; and for the cases with blue-white variant of basal cell carcinoma, the clinical and dermoscopic images and histopathological sections were re-evaluated.
Eleven cases were detected. Three of them were excluded because of poor image quality. Eight cases (2 females, age ranged 45–66 years) were included. On der-moscopy, diffuse blue-white areas were observed together with arborizing telangiectasia, non-arborising vessels, focal areas of ulceration, milia-like cysts or thin scales. In all of them, melanoma was considered in the differential diagnosis. Histopathologically all of the lesions were compatible with nodular or nodulo-ulcerative type of pigmented BCC. Diffuse blue–white areas corresponded to aggregates of basaloid cells together with the diffusely distributed pigmented melanophages in the stroma. Although this type of pigmented BCC is rare, it needs a special attention mimicking a melanoma.
Linear porokeratosis (LP) is a rare form of porokeratosis characterized by linear hyperkeratotic papules and annular plaques arranged along the Blaschko lines. Here, we report a case and describe the dermoscopic features of LP for the first time.
A twenty-two-year-old-woman had red-brown hyper-keratotic linear and annular plaques with elevated borders extending from the right mammary to the right axilla along the Blaschko lines.
The dermoscopic features observed in the case were varying according to the age of the lesions. The “early” papular lesions exhibited peripheral, thin whitish-yellow “thread-like structure” together with brown-black dots at the inner side. The early plaques revealed the same peripheral thread-like structure, however the dots were at the outer side and the ones in the inner part coalesced to form a gray-brown “network-like appearance.” The early larger plaques showed linear arrangement of these dots both in the inner and the outer side of this thread-like peripheral structure, thus appeared as a “whitish-yellow track” together with a network-like appearance at the center again. The “mature” plaques showed the peripheral whitish-yellow track together with a central “reddish vascular network” instead of gray-brown network-like structure. Finally in the oldest lesions central “pinkish- white scar like area” accompanied the features of the mature plaques.
Dermoscopic features of other types of porokeratoses have been defined in a few case reports in the literature. However, to our knowledge, the dermoscopic features of LP are described for the first time. In addition, we point out “the variation of the dermoscopic findings in relevance to the age” and a new dermoscopic feature, “the gray-brown pigment network-like appearance,” which may help improve the clinical and differential diagnosis.
Porokeratosis was named based on Mibelli’s concept that the column of parakeratosis, the cornoid lamella, emerges only from ostia of eccrine ducts. However, in 1970, Reed et al. proposed the cornoid lamella was not originated from the ostia of eccrine duct. Although this hypothesis has been generally accepted, some researchers have reported that cor-noid lamella is sometimes seen in infundibular and eccrine ductal epithelium as well as epidermis. Several dermoscopic findings of porokeratosis have been reported, including the whitish peripheral rim, the brown globules and/or dots, red dots/red lines and scar like structures in the center of the lesions. We studied 5 cases of porokeratosis of Mibelli der-moscopically. In all cases, dermoscopic findings showed the small shining white or brown spots inside the lesions. The staining of the skin surface by white board marker (furrow ink test) visualized more clearly multiple open pores with plugs and some of which corresponded to hair. Pathological findings showed that keratotic column was seen in the part corresponding to hair and sweat pore. Open pores might be an important sign suggesting porokeratosis. The staining method by white board marker, furrow ink test, could allow us to better visualize the texture of skin.
A 37-year-old Japanese male presented with a 4-month history of a solitary dark brown plaque located on the right angle of the mouth. There was no significant medical history, and he was otherwise healthy. On the physical examination, there was a well-demarcated, solitary dark brown plaque that measured 7.8 × 5.6 mm in size.
Dermoscopic examination demonstrated slate-blue dots/globules and irregular blue-white network in the center of the lesion. Atypical vascular pattern and multiple gray to brown dots in lines were observed at the periphery. Glomer-ular vessels were not observed. We suspected a pigmented skin lesion and performed the excisional biopsy.
The histopathology showed the psoriasiform pattern with regular acanthosis with thickening of the rete ridges and overlying hyperkeratosis. Epidermis showed full-thickness involvement with an atypical keratinocytes. Atypical mitoses were also observed. Basal hyperpigmentation was observed. Dermal papillae are elongated upward and filled with melanin pigment. There was no evidence of dermal invasion. Inflammation was not observed in the dermis. In the papillary and reticular dermis, numbers of dilated vessels were increased. Considering these findings, the diagnosis of pigmented Bowen’s disease was made and the local wide resection was performed by the plastic surgeon.
Bowen’s disease on the lip is exceedingly rare and this is the first reported case featuring the dermoscopic findings as far as we know. We discuss the dermoscopic/dermatopatho-logic correlation and conclude that Bowen’s disease should be considered in the differential diagnoses of mucosal pigmented lesions.
Dermoscopy is a non-invasive imaging technique that improves accuracy in the diagnosis of melanoma. It has been associated with a reduction in the false-positive detection rate and a subsequent decrease in unnecessary excisions.
The number-needed-to-treat (NNT) ratio is an effective method for measuring accuracy in melanoma detection. The aim of the present study was to assess the impact of RCM analysis on the number of dermoscopically equivocal pigmented lesions excised for every melanoma, in a clinical setting.
Three hundred and forty-three consecutive patients presenting with dermoscopically equivocal lesions, assumed to be melanocytic neoplasms based on clinical and dermoscopic features, were prospectively enrolled. Dermoscopy and confocal microscopy diagnosis were made by dermatologists with expertise in both techniques. Histopathological assessment was considered as the reference standard. The main outcome was NNT, calculated as the proportion of dermoscopically and RCM equivocal lesions excised for every melanoma. Secondary outcomes included sensitivity, specificity, positive predictive value and negative predictive value of each technique for diagnosing melanoma.
Dermoscopy alone obtained a hypothetical NNT of 3.73; the combination of dermoscopy and RCM identified 264 equivocal lesions that qualified for excision, 92 of which were confirmed to be a melanoma, resulting in an NNT of 2.87, whereas the analysis of RCM images classified 103 lesions as melanoma, with a consequent NNT of 1.12. The difference in NNT was statistically significant between the three groups (P < 00001). There was no significant improvement in sensitivity when comparing the combination of dermoscopy and RCM with RCM alone (94.6% vs. 97.8%; P = 0.043). However, the differences between specificities were statistically significant (P < 1×106), favouring RCM alone.
The addition of RCM analysis to dermoscopy reduces unnecessary excisions with high diagnostic accuracy and could be a mean for reducing the economic impact associated with the management of skin cancer.
We present 8 cases of an unusual presentation of a fixed drug eruption (FDE) mimicking a clinical lentigo on the face. Since Brocq’s first and very detailed description of FDE, we have not found any description in the literature of the special form of FDE described herein. The sudden appearance of a lentigo on the face after the ingestion of NSAIDs can mimic other forms of lentigo and can easily be misdiagnosed. Most of our patients presenting with this form of post inflammatory lentigo-like reaction (PILLR) have a clinical lentigo like appearance on the face from the beginning. The appearance of these PILLR usually lack strong local symptoms such as burning and itching, demonstrating that this is mainly a subclinical form of FDE that later became a clinical PILLR. Only after specific questioning about mild burning or itching, required for the medical records, did patients remember having these symptoms.
All of them had a history of previous NSAIDs intake, specifically of ibuprofen or ketoprofen.
The majority of the patients had a PILLR with different intensities of brown clinical color upon first clinical examination; with the exception of two patients whose PILLR had also a pink color in the acute phase (Fig 1). The clinical appearance of a PILLR generally persisted for more than six months.
Under dermatoscopy, the lesions were generally asymmetric and the borders were ill-defined. All cases presented a brown uniform background color of different shades—from light brown to dark brown—that produced a pseudonetwork. In all patients a light pink area due to vessels arranged as red dots or short telangiectatic vessels accompanied the brown pseudonetwork. Brown and/or brown-gray dots also distributed randomly or in focal areas of the lesion generated a dermatoscopic granular pattern. In those patients with darker skin, an annular granular pattern was observed in focal areas along symmetric follicular openings.
The pink color present under dermoscopy (as the result of vessels associated with inflammation) is a strong indicator for a PILLR diagnosis and is not found in the dermatoscopy of other forms of lentigo. The histopathologic analysis of PILLR displays features of a mild form of FDE.
Most skin cancers originate at and spread from the dermal epidermal junction. Currently, biopsy followed by histology is the “gold standard.” However, this is an invasive procedure, which is painful, costly and time consuming. Moreover, statistics show that, depending on the setting, up to 80% of these biopsies may turn out to be benign. With the aid of new imaging technologies, lesions may be diagnosed non-invasively and the number of the biopsies reduced [
In this study, the development of tools such as automated dermal epidermal junction delineation and video-mosaicing are reported. In the first scenario, the problem of finding the dermal epidermal junction (DEJ), which is a trivial task in H&E stained histology but not in RCM images is addressed. This problem is particularly important because, most of the time, clinicians make their diagnostic decision using mosaics of RCM images collected at or around the DEJ level. In current practice, DEJ level is determined by the clinicians in a subjective manner, by going back and forth between the RCM images collected at different depths and examining them. Standardization of this procedure by automatically delineating the DEJ level in a quantitative manner will lead to the accuracy and repeatability of both image acquisition procedure and RCM based diagnosis. In order to automate DEJ delineation, we developed 2 algorithms, one for highly-pigmented (type > III) and another for lightly pigmented (type < III) skin types, as they have different reflectance characteristics due to their varying melanin content. In stacks of dark skin, the algorithm aims to locate highly reflective basal cells at the DEJ level using an order statistics based filtering approach. In lightly pigmented skin, as the pigmentation level is lower, the basal cells are not bright enough to the detected reliably. In this case, we benefit from the fact that, blurring occurs in RCM images of deeper levels of skin, especially below the DEJ (due to optical aberrations). We model this phenomenon using a multi scale entropy filtering based method, and delineate the DEJ using this model. We tested the proposed DEJ delineation algorithms on 16 highly pigmented and 12 lightly pigmented skin stacks and compared our results against manual segmentation of expert readers. The algorithms can delineate DEJ with a mean ±std of 7.5±5 um in highly pigmented skin and 29±5.4 um in lightly pigmented skin.
In another scenario, we showed the feasibility of creating mosaics out of RCM videos collected at a given level of skin, so called video-mosaicing6. Video-mosaicing provides a tool for rapidly and adaptively imaging over large areas of skin which can be useful for examination of larger areas around dermal-epidermal and various other application such as delineating margins of lentigo maligna melanoma to guide surgical excision or non-melanoma skin cancer margins to guide Mohs surgery. In our study, we showed that if (i) there is 25–50% overlap between consecutive frames and (ii) the imaging depth is kept constant, it is possible to convert these videos into high-quality mosaics of the imaged area. For this purpose we developed a computer program that can first find the identification tag in the video, crop out that region automatically and extract individual frames of the RCM video. Then, we use freely available software (ICE, Microsoft) to stitch and blend the consecutive frames into a mosaic. We evaluated the proposed algorithm on videos of non-melanoma margins in Mohs surgical wounds, lentigo maligna margins, malignant melanoma in situ, seborrheic keratosis, and benign lesions. A sample case of “malignant melanoma in situ” is presented in Figure 1. Several diagnostically-significant structures, such as atypical epidermal cells, epidermal disarray, perifollicular infiltration, roundish, dendritic, and pleomorphic pagetoid cells, dilated blood vessels and meshwork pattern with atypical cells can be clearly be identified in the resulting videomosaic. The results of this study suggest that in principle, a coverage rate of ∼240–360 mm2/min is within reach for any confocal microscope with configuration similar to that (1 mm × 1mm FOV, ∼ 8 frames/ second) of the Vivascope 3000. Thus, video-mosaicing technology has the potential for much faster imaging of large areas compared to the current commercially-available RCM mosaicing approach, which typically images at ∼14 mm2/ min. Moreover, it enables the clinician to cover areas with any desired shape and along any desired path that may be determined real-time during acquisition.
Following the first descriptions of the dermatoscopic pattern of basal cell carcinoma (BCC) that go back to the very early years of dermatoscopy, the list of dermatoscopic criteria associated with BCC has been several times updated and renewed. Up to date, the usefulness of dermatoscopy in differentiating pigmented and non-pigmented BCC from other skin tumors has been extensively demonstrated. In addition to its well-documented value in improving the diagnosis, dermatoscopy continuously gains an essential role in the management of BCC.
Dermatoscopy for choosing the appropriate treatment modality
In our era, the therapeutic armamentarium of clinicians for BCC includes several surgical methods as well as non-surgical modalities. The choice of the appropriate treatment depends on several factors including the histopathologic subtype, the presence of pigmentation or ulceration, the tumor depth, the anatomical site and the presence of residual disease or recurrence. Dermatoscopy has been shown to provide valuable information for several of the aforementioned parameters.
The histopathologic subtype is the most crucial factor influencing the treatment choice for BCC. In the recent years, sBCC has been shown to respond perfectly to non-ablative treatments such as imiquimod or photodynamic therapy, prompting experts to recommend the latter modalities as first-line therapeutic options for this subtype. In contrast, conventional or Mohs surgery is considered the choice treatment for nodular, infiltrative and sclerodermiform subtypes, while non-surgical treatments are much less effective.
A recent study investigated the accuracy of dermatoscopic criteria for discriminating superficial from the other subtypes of BCC. This is particularly relevant in clinical practice, since the possible misinterpretation of a nodular or infiltrate tumor as superficial BCC could lead the clinician to the inappropriate choice of a non-surgical treatment modality. According to the results of the latter study, the presence of short fine telangiectasia, multiple small erosions and structures corresponding to dermo-epidermal pigmentation predict the superficial subtype. In contrast, detection of ovoid nests should lead clinicians to exclude the diagnosis of superficial BCC, while arborizing vessels and large ulcerations are also suggestive of nodular, sclerodermiform or infiltrative tumors.
The presence of pigmentation is not routinely reported in histopathologic reports, since in the past it was not considered to influence the management and prognosis of the tumor. However, the use of PDT in BCC treatment restored the importance of pigmentation, since its presence was shown to influence the tumor’s response. In detail, case series studies reported a poor response of pigmented BCC to PDT, compared to non-pigmented variants (14% versus 62–100%). The low efficacy of PDT in pigmented tumors has been attributed to melanin, which appears to act as a competitive light-absorbing pigment, decreasing response rates.
Effectively, the presence of clinically undetectable pigmentation might represent a diagnostic pitfall for clinicians, forcing them to apply an ineffective treatment on a subset of BCCs. This problem seems to be, at least partially, solved by the application of dermatoscopy, which was recently shown to reveal clinically undetectable pigmentation in approximately 30% of macroscopically non-pigmented BCCs, enhancing clinicians to better select tumors potentially sensitive to PDT and minimizing treatment failures.
Dermatoscopy for assessing excision margins
Dermatoscopy, by providing a more accurate assessment of the true extension of the tumor, allows a more precise estimation of the required surgical margins, helping to minimize the recurrence rate. The discrimination of BCC vessels from the dermal plexus vasculature of the surrounding healthy skin can be based on the blurred appearance and dark red-to-purple color of the surrounding sun-damaged skin, in contrast to the bright-red and focused vessels of the tumor. However, while the diagnostic significance of pigmented structures is unquestionable, the usefulness of vascular structures in defining the surgical margins is controversial. It has been suggested that arborizing vessels do not directly correspond to BCC cells, but represent feeding vessels of the tumor and may extend also to the perilesional skin. Subsequently, if the extension of vessels is used to define the excision margins, there is the risk of unnecessarily removing healthy skin surrounding the BCC. Although the latter hypothesis seems reasonable, it was supported by only one published case and, accordingly, the question whether vascular structures should be considered for defining surgical margins of BCC remains to be further elucidated.
Dermatoscopy for monitoring response to non-ablative treatments
A common problem associated with non-ablative modalities is the post-treatment evaluation, since at the end of a treatment cycle, the clinical morphology of the lesion often does not allow a reliable estimation of the possible presence of residual disease.
Dermatoscopy has the potential to improve the post-treatment evaluation of BCC following non-ablative procedures, minimizing therefore the risks of under- or over-treatment of BCC. Specifically, the disappearance of the dermatoscopic criteria of BCC after treatment has been shown to accurately predict histopathologic clearance, while the persistence of some BCC criteria correlates well with the presence of residual disease. According to the results of a recent study, the presence of arborizing vessels, ulceration or pigmented structures (e.g., blue-gray ovoid nests and maple leaf-like areas) accurately predicts residual disease, and should prompt the clinician to continue the treatment. Instead, red/white structureless areas and/or superficial fine telangiectasia might represent equivocal features, since they do not always correspond to residual disease. Effectively, detection of the latter criteria warrants close monitoring to recognize a possible recurrence of the BCC.
In usual practice, the diagnosis of a suspected basal cell carcinoma (BCC) is typically confirmed by biopsy of the suspected lesion, followed by the histopatho-logic evaluation of tissue specimens via light microscopy, prior to referral to surgery. A physician’s presumptive diagnosis of BCC can be enhanced by identifying dermoscopic features typical for BCC. Reflectance confocal microscopy (RCM) is also useful for diagnosis of BCC. It is conceivable a presumptive diagnosis of BCC supported by dermoscopic and RCM features could circumvent the need for biopsy prior to surgery, with confirmation of the diagnosis performed on the surgical specimen.
To investigate the feasibility of bypassing biopsy of suspected BCC and proceeding directly to excision or Mohs surgery based on dermoscopic features alone, as well as when combined with RCM. In addition, we sought to compare the diagnostic accuracy for BCC based on prior RCM experience.
Potential study subjects were identified during the routine office visit by the presence of one or more lesions for which BCC was suspected, thus warranting a biopsy. Clinical, dermoscopic and RCM images were obtained prior to biopsy. Eight clinicians with varying levels of expertise, each blinded to the histopathologic findings, individually interpreted the clinical, dermoscopic and RCM images. Based on these interpretations, the clinicians chose between 4 hypothetical options: definite BCC (willing to send for definitive treatment without biopsy), other malignancy (perform biopsy for diagnosis), uncertain diagnosis (perform biopsy), and benign (do not biopsy). The choice decisions were made based on dermoscopy alone and subsequently on dermoscopy supplemented by RCM.
Of the 100 suspected lesions enrolled, 90 were verified as BCC on histopathology. The pooled sensitivity for direct referral to definitive surgery without biopsy was 67.59% for dermoscopy alone. Adding RCM imaging increased the pooled sensitivity for direct referral to surgery to 76.53%. For the diagnostic decision to refer for treatment without biopsy, the pooled positive predictive value was 96.97% for dermoscopy alone and 98.64% for dermoscopy plus RCM. The sensitivities were generally higher for dermoscopy alone as the level of experience increased, although with a sacrifice to specificity.
Actual patient management was not affected in this study. Physician behavior might be different if bona fide referrals were actually being made. The interpretations were made on image evaluation alone rather than at bedside, which might enhance accuracy.
Dermoscopy provides a high positive predictive value for BCC diagnosis. The addition of RCM to dermoscopy increases diagnostic sensitivity. Based on this study, from the standpoint of cost-effectiveness and patient convenience, dermoscopy and dermoscopy with RCM could be acceptable for direct-referral-to-surgery clinical decisions.
The term lentigo maligna (LM) refers to melanoma in situ arising on chronically sun-damaged skin with a flattened dermal-epidermal junction. It most commonly arises on the face although it is not limited to this anatomic region. Little is currently known about the influence of age, gender and topography on the clinical and dermoscopic variability of LM. Therefore, we retrospectively collected all consecutive cases of histopathologically proven facial and extra-facial LM diagnosed between January 2012 and January 2013 at 4 academic skin cancer clinics in France, Italy, Serbia and USA. The frequency of clinical and dermoscopic features of 201 cases of LM from 200 patients were assessed in correlation to specific anatomic sub-sites, patient’s age and gender. Most cases were located on the face, with the cheeks being the most commonly affected sub-site. Location on the cheek was significantly associated with female gender compared to all other sub-sites. Dermoscopically, gray color irrespective of a specific pattern, was the most prevalent finding. The knowledge about the age, gender and site-related clinical features of LM associated with dermoscopicgray colour may enhance the clinical recognition of LM.
Dear colleagues,
Maimonides, known as the
This Supplement of Rambam Maimonides Medical Journal presents the abstracts from the Eleventh Rambam Research Day. These abstracts represent the newest basic and clinical research coming out of Rambam Health Care Campus—research that is the oxygen for education and development of today’s generation of physicians. Hence, the research presented on Rambam Research Day is a foundation for future generations to understand patient needs and improve treatment modalities. Bringing research from the bench to the bedside and from the bedside to the community is at the heart of Maimonides’ scholarly and ethical legacy.
We hope you will appreciate the potential represented in these abstracts, which touch on nearly every aspect of clinical practice.
I would also like to thank Ms. Deborah Hemstreet, for the invaluable and professional editorial work that went into making this special issue of Rambam Maimonides Med J possible.
Rambam Maimonides Medical Journal would be pleased to publish other original basic and clinical research articles, and related abstracts, which will subsequently be applied to patient care. In addition, we are open to publishing such abstracts from meetings, seminars, or conferences. For more information on seeing your event’s abstracts published here, please contact our editorial board.
Sincerely Yours,
We are proud to introduce you to the Eleventh Rambam Research Day, now established as a key annual event at Rambam Health Care Campus (Rambam), reflecting the diverse research activities here.
Integral to Rambam’s research activities are two competitive research funding programs that Rambam administers: The “Ofakim” program, which provides start-up and bridge research funding for young staff physicians with strong research credentials and plans, and the “Atidim” program, which is directed towards research training support for our residents, or as we call them in Israel “Mitmachim.” The Atidim program continues to receive wide recognition, and serves as an especially important example of Rambam’s dedication to medical trainees and the development of physician scientists.
We would like to acknowledge the excellent collaboration and the fruitful research ties between Rambam Health Care Campus and the Technion-Israel Institute of Technology, in particular The Ruth & Bruce Rappaport Faculty of Medicine. We would also like to thank Professor Chaim (Howard) Cedar from the Hebrew University of Jerusalem as the Chair of our Scientific Advisory Board.
We are particularly proud of the scientific abstracts that were presented at the meeting, and which compose this Supplement to
We would also like to recognize Rambam’s Research Division, including the Institutional Review Board (IRB), and the Office for Promotion of Research; together they are responsible for the coordination and oversight of all research activities at Rambam, including clinical research, applied research, competitive grants management, patent protection, institutional review board ethical approvals, and bringing forward innovative ideas to clinical practice. We value collaboration with industry, which serves as an integral part of health care progress; over the years, several Rambam-originated research projects have led to remarkable success in Israel, with practical application to health care.
We are particularly pleased to see this second Supplement to
Director Division of Psychiatry, Head Research Division
Director, Department of Neonatology, and Editor-in-Chief, Rambam Maimonides Medical Journal
Director, Department of Cardiac Surgery
Director, Department of Hematology and Bone Marrow Transplantation
Nursing Research Coordinator
Director, Department of Nuclear Medicine
Director, Gastroenterology Institute
Director, Pain Research Unit of the Pain Medicine Institute
Oncology Institute
Rambam Health Care Campus, Haifa, Israel
07.30
08.00
General Director, Rambam Health Care Campus
Dean, The Ruth & Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology (IIT), Haifa, Israel
Director, Rappaport Research Institute, Technion-Israel Institute of Technology, Director of Medical and Research Development Rambam Health Care Campus
Director, The Clinical Research Institute at Rambam (CRIR), at Rambam Health Care Campus
Chairman, Research Division, Rambam Health Care Campus
08.30
Professor of Molecular Neuroscience The Edmond and Lily Safra Center for Brain Sciences Department of Biological Chemistry and The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel.
09.15
Professor Rafael Beyar, Director, Rambam Health Care Campus
Professor Naim Ziv, Vice Dean for Research, The Ruth & Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology
Professor Gadi Shooster, Executive VP for Academic Affairs, Technion-Israel Institute of Technology
Professor Hermona Soreq, The Hebrew University
Dr. Ruth Perets, Division of Oncology, Rambam Health Care Campus
09.45
Director, Department Pediatrics A, Head, Pediatric Diabetes and Obesity Clinic, Rambam Health Care Campus
10.05
Thrombosis and Hemostasis Unit, Department of Hematology and Bone Marrow Transplantation, Rambam Health Care Campus, and The Ruth & Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel
11.00
Bertoldo Badler Chair, Department of Physics, Technion-Israel Institute of Technology, Haifa, Israel
11.45
Laboratory of Applied Cancer Science, Clinical Research Institute at Rambam (CRIR), Rambam Health Care Campus
Department of Internal Medicine C, and Hematology Institute, Thrombosis and Haemostasis Unit, Rambam Health Care Campus
Cognitive Neurology Institute, Rambam Health Care Campus
12.15
XXX, Rambam Health Care Campus
12.35
Director of Nursing, Rambam Health Care Campus
14.15
Concluding Remarks
Awards for Rambam Research Day Poster Presentations
Awards for Rambam Atidim Graduates
Awards for Rambam Ofakim Graduates
Announcement of Rambam Atidim 2014 Recipients Rambam Ofakim 2014 Recipients
Announcement of Rambam Innovation Fund (Nitzoz) 2014 Awardees
One hundred years after the discovery of acetylcholine (ACh) by Otto Lowei, ACh receptors, transporters and synthesizing and degrading enzymes became well-recognized contributors to cognition, neuromuscular, metabolic and immune processes. However, newer technologies identified unexpected molecular controllers over ACh signaling, including the SLEEPLESS, Isl1 and Lynx1 genes. These regulators are responsible, among other effects to the fine-tuned identity, functioning modes, dynamics and inter-cellular interactions of cholinergic cell types in and out of the brain, changing our understanding of ACh’s roles in human health and wellbeing. Furthermore, Genome-Wide Association Studies identify new disease-associated mutations and single nucleotide polymorphisms in coding and non-coding sequences within these genes. These discoveries add autism, amyotrophic lateral sclerosis, acute cardiac events, narcolepsy and obesity to the established acquired and inherited neuromuscular, stress-induced, dementia and epilepsy disorders that were traditionally associated with impaired ACh functioning. At the molecular level, cholinergic signaling involves both up- and down-regulation events of transcription, epigenetic modulations, alternative splicing and microRNA suppression that together coordinate the multi-targeted ACh signaling in brain and body functions and are also responsible to the reactions of patients to anti-cholinesterase therapeutics of Alzheimer’s disease as well as to global exposure to agricultural pesticides and to individual tendencies for nicotine addiction, calling for new basic and translational research venues for regulating ACh signaling. Integrating these molecular ACh regulators into every discussion of cholinergic issues, should incorporate data obtained by clinicians and molecular geneticists, neuroscientists and structural biochemists over the past decades into a refreshed look at the intricate checks and balances over cholinergic signaling. Our understanding of the cholinergic regulators is incomplete, but time is ripe to summarize the recent reports on checks and balances of cholinergic signaling and their implications in health and disease.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 6.
There are millions of microvesicles (MVs) in the circulation of healthy persons, and their level may increase in a variety of pathologies. Microvesicles may be divided into sub-groups, i.e., exosomes, microparticles, and apoptotic bodies that are shed from both normal and malignant cells upon cell activation or apoptosis. Microvesicles promote clot formation, mediate pro-inflammatory processes, facilitate cell-to-cell interactions, transfer proteins and miRNA to cells, and induce cell signaling.
Over the last 10 years, we have studied the role of MVs in a variety of diseases including gestational vascular complications, diabetic vascular complications, cancer, i.e., solid tumors (breast, colon) and hematological malignancies (AML, myeloma, ALL) and in thalassemia. We have characterized MV structure, expression of membrane antigen, content of proteins and DNA/RNA, and evaluated their effects on cell cultures, including MV cell interaction, apoptosis/survival, migration, invasion, angiogenesis via signal transduction. The data acquired in these studies suggested that MVs may be used for wound healing and they may also serve as biomarkers for prothrombotic state and as a source for genetic diagnosis.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 6–7.
Solid tumor associated nonneoplastic stromal cells also include mesenchymal stem cells (MSC) which are recruited by epithelial cancer cells from either the bone marrow or from adjacent tissue and become an integral cellular component of the tumor microenvironment where they modulate tumor progression. Nevertheless, there is insufficient information about MSC derived from human malignant tumors and the modulation of these cells by cancer cells. MSC can differentiate into multiple lineages, including adipocytes, chondrocytes and osteoblasts and they express specific cell surface markers such as CD29, CD73, CD90 and CD105. MSC tropism into tumor sites is mediated by extrinsic signals released into the tumor microenvironment and in turn promotes further cell recruitment through secretion of chemokines, cytokines and growth factors.
To elucidate the mechanism whereby non-neoplastic stromal cells elicit their functional impact on tumor progression and modulate the behavior of the epithelial cancer cells, we have harvested cells from various anonymous patient tumor samples. In addition to the epithelial cancer cells, the harvested tumor-derived cells included stromal cells, identified as non-neoplastic tumor-derived MSC by their multi-potential capacity to differentiate
While the LC2 cancer cell growth is independent of their LC-MSC, the GSC1 cancer cell growth is critically dependent on the presence of their counterpart GSC-MSC or their conditioned medium (CM). The fact that none of the various other TD-MSC were able to restore the specific effect of GSC-MSC on GSC1 cancer cell growth suggests specificity of TD-MSC, which is specifically recruited and ‘educated’/reprogrammed by the cancer cells to support tumor growth. Using cytokine array analysis, we were able to demonstrate that GSC1 cell growth is mediated through HGF/c-MET signaling pathway which is activated exclusively by HGF secreted from GSC-MSC. An innovative approach demonstrates GSC1-mediated specific tropism of ‘naïve’ MSC from the adjacent tissue a tumor specific manner to support tumor progression.
The results suggest that specific tumor tropic ‘naïve’ MSC are reprogrammed in a tumor-specific manner to support gastric tumor progression. Understanding the mechanisms involved in the interactions of the tumor cancer cells and TD-MSC will constitute the basis for developing multimodal anti-cancer therapeutic strategies that will also take into account the specific tumor tropism properties of MSC and their reprogramming.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 7.
This study examines the care transition experience of cancer patients and assesses barriers to effective transitions.
Participants were adult Hebrew, Arabic, or Russian speaking oncology patients at Rambam’s Oncology Center. The study used qualitative and quantitative methods. Qualitative information was obtained from 33 healthcare providers (physicians, nurses, social workers, and administrative managers) from the hospital and community settings and 25 hospitalized patients (with their families)—all of which participated in focus groups and/or in-depth interviews. Quantitative information was obtained from 422 patients via validated questionnaire that assessed patients’ perceptions of the quality of their primary care using the Primary Care Assessment Survey (PCAS), patients’ care transition experience using the Care Transition Measure (CTM), as well as structured observations of the discharge explanations and language used during the hospital discharge process.
Qualitative analysis showed that patients face a complex and fragmented system with multiple providers and services. Difficulties in navigating the healthcare system are exacerbated for minorities with language and cultural barriers. Mechanisms to overcome barriers included undertaking of informal routes such as personal relationships, coordinating roles by nurse coordinators, and of the patients’ general practitioners (GPs). The involvement of GPs was found to be the most significant variable affecting the quality of the transition process as rated on the CTM (p<0.001).
Our findings point to the important interpersonal role of the oncology nurses in coordinating and facilitating the care transition process. Interventions targeted towards supporting the care transition process should emphasize ongoing counseling throughout the course of a patient’s care during and after hospitalization.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 7–8.
Gradual bone lengthening using distraction osteogenesis principles is the gold standard for the treatment of hypoplastic facial bones. However, the long treatment time is a major disadvantage of the lengthening procedures.
the aim of the present study is to review the current literature and summarize the cellular and molecular events occurring during membranous craniofacial distraction osteogenesis.
Mechanical stimulation by distraction induces biological responses of skeletal regeneration that is accomplished by a cascade of biologic processes that may include differentiation of pluripotential tissue, angiogenesis, osteogenesis, mineralization, and remodeling. There are complex interactions between bone-forming osteoblasts and other cells present within the bone microenvironment, particularly vascular endothelial cells that may be pivotal members of a complex interactive communication network in bone. Studies have implicated number of cytokines that are intimately involved in the regulation of bone synthesis and turnover. The gene regulation of numerous cytokines (transforming growth factor-b, bone morphogenetic proteins, insulin-like growth factor-1, fibroblast growth factor-2) and extracellular matrix proteins (osteonectin, osteopontin) during distraction osteogenesis has been best characterized and are discussed. Understanding the bio-molecular mechanisms that mediate membranous distraction osteogenesis may guide the development of targeted strategies designed to improve distraction osteogenesis and accelerate bone regeneration that may lead to shorten the treatment duration.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 9.
Numerous studies have shown that metastases formation depends on the ability of tumor cells to invade basement membranes and tissue barriers in a process involving enzymes capable of degrading extracellular matrix (ECM) components. One of these enzymes is heparanase, an endoglycosidase that degrades heparan sulfate.
In the past we have shown that the nuclear localization of heparanase is found in all oral verrucous carcinomas, i.e., very well differentiated non-metastesizing tumors, as opposed to only 28% nuclear localization detected in oral squamous cell carcinomas. Heparanase expression level also significantly correlated with the degree of tumor differentiation. Moreover, while cytoplasmic localization of heparanase was associated with high grade carcinomas, nuclear localization of the enzyme was found primarily in low grade, well differentiated tumors. The aim of the present study was to use an in vivo model to study the role of heparanase in cancer formation and progression.
4NQO is a water soluble carcinogen that induces tumors, predominantly in the oral cavity. It results in all the stages of oral carcinogenesis and evidence suggests that similar histological as well as molecular changes are observed. We used this carcinogen to establish a mouse model of oral tongue SCC (squamous cell carcinoma). Heparanase expression and activity was evaluated using immunohistochemical analysis and real time PCR (for protein and mRNA expression levels). Heparanase enzymatic activity was evaluated for both cytosolic and nuclear fractions of total proteins using a radio-labeled ECM (extracellular matrix) and a scintillation counter for the degradation products.
Heparanase was over expressed in tongue cancer from the hyperkeratosis stage through to invasive SCC. Heparanase enzymatic activity was significantly higher in the nucleus of normal tongue tissues as opposed to developing cancer, which exhibited a higher cytoplasmic activity of the enzyme.
Heparanase nuclear expression and enzymatic activity is higher in normal tongue tissue. When carcinogenesis is initiated and progresses, heparanase translocates from the nucleus to the cytoplasm. The overall expression of heparanase increases as the cancer progress from the pre-malignant stage to invasive SCC. Hence, the expression level and cellular localization of heparanase could serve as reliable predictive indicators of oral carcinoma development, metastatic potential, and patient prognosis.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 9–10.
Aortic valve stenosis caused by valvular calcification, a form of extra-skeletal ossification, is the most prevalent indication for valves replacement and is associated with high morbidity. Recent studies have shown that extra-skeletal ossification is a highly regulated process that involves the differentiation of mature vascular and stromal cells into osteochondrogenic cells and recapitulates embryonic endochondral ossification. Histone deacetylases (HDAC’s) are enzymes that are known to play a major role in chondrocytes and osteoblasts differentiation and maturation yet the role of HDACs in extra-skeletal ossification is still unknown.
MOVAS cells, originated from mouse vascular aortic smooth muscle, were transfected with pcDNA plasmids encoding different HDAC’s (1, 3, 4, 7). RNA was extracted from the cells and RT-qPCR was performed to detect an increase in several known ossification markers. In addition, MOVAS and HeLa cells were transfected with pGFP plasmid for over expression of HDAC4 and observed under florescent microscope to assess the subcellular localization of HDAC4 in both cell types.
MOVAS cells were previously induced to ossify in culture. Gene expression analysis showed that the expression levels of HDAC4 were up-regulated, while HDAC’s 1, 3 and 7 levels did not change significantly. Over expression of HDAC4 in an aortic smooth muscle cells model (MOVAS cells) showed a significant increase in ossification markers (especially the transcription factor Osterix) at the mRNA level. HDAC4 is known to shuttle between the nucleus and cytoplasm and is presumed to function in the nucleus is deacetylating histone tails. Assessment of subcellular localization of HDAC4 in control HeLa cells demonstrated presence of the protein both in the cytoplasm and nuclear whereas in MOVAS cells HDAC4 was located almost exclusively in the cytoplasm.
We showed that in aortic smooth muscle cells HDAC4 is up-regulated during ossification and positively contributes to the ossification process. Despite the believed role of HDAC4 as histone deacetlylase in the nucleus, we show that in aortic smooth muscle cells HDAC4 is found almost exclusively in the cytoplasm, and presumably deactylates cytoplasmic proteins. HDAC4 may be a target for future therapies aiming to limit valve and vascular calcifications.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 10.
Chronic lymphocytic leukemia (CLL) patients demonstrate impaired effector T-cell activity and a general exhausted T-cell phenotype. The mechanisms by which CLL cells inflict these changes are a subject of active investigation. We have previously shown that TLR9-activated CLL cells can inhibit autologous CD4+ T-cell proliferation and induce regulatory T-cells. Moreover, this activation induces PD1 and PDL1 expression on CLL B-cells, along with IL10, CD25, CD38, and CD23, markers characterizing regulatory B-cells (Bregs). The current study aimed to determine whether PD1/PDL1 increased expression contributes to the cells’ inhibitory activity.
B-cells were isolated from peripheral blood mononuclear cells of untreated CLL patients. Cells were stimulated with ODN (TLR9 agonist) and co-cultured with autologous CD4+ T cells. T-cell proliferation was evaluated using the CFSE method following stimulation with anti-CD3/CD28 antibodies and IL2. Involvement of the PD1/PDL1 axis was examined by incubating B-cells with antiPD1 neutralizing antibodies. PD1 blockage effects on ODN regulated gene expression were tested by Real-time PCR. Inhibition of TLR9 signaling was accomplished by adding an IRAK4 (critical downstream kinase) inhibitor to B-cell cultures.
ODN-activated CLL cells inhibit autologous T-cell proliferation in 82% of patients (p<0.001), however in the presence of neutralizing PD1 antibodies inhibition is observed in only 71% of patients, with loss of statistical significance (p=0.128) [N=7]. Blocking the PD1 signal on CLL cells reverses the TLR9 mediated gene expression attenuation of CD5 and TLR9 itself, [fold 2.35± 0.58, fold 1.65±0.28, respectively, N=7]. Among all proteins upregulated by ODN activation, only IL10 and PDL1 show reduced expression [fold 0.78±0.1, fold 0.72±0.2, respectively, N=7] following IRAK4 inhibition.
CLL cells “acquire” Breg characteristics, elicited by TLR9 activation, most probably promoting immune regulation and cancer evasion. The involvement of B-cell-associated PD1 in the inhibition of T-cell proliferation and in transcription of TLR9 regulated genes implies that TLR9 regulated effects depend on proper PD1 signaling. The role of the PD1/PDL1 axis and its cooperation with TLR9-elicited signals is further emphasized by the attenuation of IL10 and PDL1 expression by IRAK4inhibition, demonstrating the importance of these specific factors in promoting regulatory activity. The current study underscores the proficiency of CLL cells to recruit separate pathways operating cooperatively to support immune modulation and promote survival.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 11.
Infection is a common complication of orthopedic implant procedures. The average overall infection rate in the US for fracture fixation and joint prostheses is estimated at 5%, but may be as high as 30% for open fracture reductions. Local delivery of antibiotics to the implant site is considered advantageous over systemic treatments, since increased drug concentrations can be delivered to the infected tissues while avoiding systemic side-effects. Release of antimicrobial agents from bone-healing devices can dramatically reduce the risk of implant-associated infection. We report on the fabrication and antimicrobial activity of a multifunctional load-bearing bioresorbable material that provides mechanical support to healing bone tissue along with slow release of an antibiotic drug.
This study evaluated the effect on antibiotic activity of high pressure consolidation of high-strength beta-tricalcium phosphate (β-TCP)-polylactic acid (PLA) nanocomposites, with incorporated vancomycin at room temperature and at 120°C.
Dense beta-tricalcium phosphate (β-TCP)-40 vol.% polylactic acid (PLA) nanocomposite containing 1 wt.% vancomycin (VH) was high pressure consolidated at 2.5 GPa, at room temperature, or at 120°C.
Over the course of 5 weeks in TRIS solution, the β-TCP-PLA-VH nanocomposite released approximately 90% of its drug load. Specimens consolidated at 120°C had the highest initial mechanical properties, maintaining 85% of their compressive strength and 30% of their bending strength after 5 weeks of drug release. Study of
The results indicate that the developed high strength antibiotic-eluting β-TCPPLA nanocomposite may be a promising material for orthopedic surgical devices.
Citation: Rambam Maimonides Med J 2015;1 Suppl:11–12.
The current non-inferiority, prospective, randomized study compared the efficacy and safety of the third generation, starch-based volume expander tetraspan (TS) to human albumin solution (ALB). Following the FDA’s declaration doubting the safety of starch solutions used as volume expanders in critically ill patients (2013), we discontinued recruiting patients and performed a data analysis. Postoperative bleeding during the first 48 hours was defined as the primary outcome measure.
Patients admitted for open-heart surgery were prospectively randomized to perioperative volume replacement therapy with TS or ALB. Preoperative variables were demographics, aspirin and plavix use, baseline creatinine, and procedure type. Pre- and postoperative variables were coagulation profiles, thromboelastography (TEG) and creatinine. Intraoperatively, bypass time and minimal temperature were monitored. Intra- and postoperative volumes of used TS and ALB were recorded. Postoperative bleeding, RBC, and blood products use during ICU stay, acute kidney injury, hospitalization length, and 30-day in-hospital mortality were assessed.
Thirty patients fulfilled the study protocol (TS group: n=18; ALB group: n=12). Perioperative TS and ALB volumes were comparable (5095±288cc vs. 5174±332cc, respectively; P= 0.923). The TS group demonstrated excess postoperative bleeding compared to the ALB group (median [25th–75th percentiles]: 965 [560–1312.5] vs. 610 [488.7–937] cc, respectively; P=0.038). Both groups were comparable in terms of all other variables (Ps>0.1). No renal replacement therapy or mortality cases were recorded.
Increased bleeding was detected in patients treated with TS as compared to ALB after open heart surgery, although used volumes were comparable. This justifies vigilance before resumption of starch-based solutions ALB volume expanders in patients undergoing open-heart surgery.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 12.
Stroke is a major cause of morbidity and mortality during open-heart surgery. Up to 60% of intraoperative cerebral events are emboli induced. This randomized, controlled, multicenter trial is the first human study evaluating the safety and efficacy of a novel aortic cannula producing simultaneous forward flow and backward suction for extracting solid and gaseous emboli from the ascending aorta and aortic arch upon their intraoperative release.
Sixty-six patients (25 females; 68±10 years) undergoing elective aortic valve replacement surgery, with or without coronary artery bypass graft surgery, were randomized to the use of the CardioGard (CardioGard Medical, Or-Yehuda, Israel) Emboli Protection cannula (“treatment”) or a standard (“control”) aortic cannula. The primary endpoint was the volume of new brain lesions measured by diffusion-weighted magnetic resonance imaging (DW-MRI), performed preoperatively and postoperatively. Device safety was investigated by comparisons of complications rate, namely neurologic events, stroke, renal insufficiency and death.
Of 66 patients (34 in the treatment group), 51 completed the presurgery and post-surgery MRI (27 in the treatment group). The volume of new brain lesion for the treatment group was (mean±standard error of the mean) 44.00± 64.00 versus 126.56±28.74 mm3 in the control group (p=0.004). Of the treatment group, 41% demonstrated new postoperative lesions versus 66% in the control group (p=0.03). The complication rate was comparable in both groups.
The CardioGard cannula is safe and efficient in use during open-heart surgery. Efficacy was demonstrated by the removal of a substantial amount of emboli, a significant reduction in the volume of new brain lesions, and the percentage of patients experiencing new brain lesions.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 13.
Postoperative delirium constitutes a range of neuropsychological disturbances with incidence of up to 57% of cardiac surgery patients. Lack of natural daylight exposure in the intensive-care setting induces a circadian rhythm disturbance, which may result in delirium. We evaluated the effect of lack of daylight exposure on the prevalence, intensity, and duration of postoperative delirium.
Altogether, 942 patients were enrolled and constituted two groups: (i) Group D (Dark) of 485 patients, operated from October 10, 2010, to October 30, 2011, while the Department of Cardiac Surgery resided in the hospital’s underground floor, with no daylight exposure; (ii) Group L (Light) of 457 patients, operated from November 1, 2011, to October 1, 2012, while the department resided on the hospital’s 6th floor, with normal daylight exposure. The prevalence, intensity, and duration of delirium were retrospectively assessed by means of (i) haloperidol prescribed [yes/no]; (ii) its amount [mg]; and (iii) administration length [days], respectively.
During both periods, 190 patients developed delirium: 88 (19.2%) were from group L and 102 (21%) from group D (P=NS). Among females, delirium in group D lasted longer (3.7±4.2 vs. 2.4±3.9 days; P=0.028). The average haloperidol dose was larger in group D both in males (19.1±19.1 vs. 13.6±3.5; P=0.021) and females (19.0±14.4 vs. 10.5±3.9, P=0.004). The average postoperative length of stay was longer in group D (15.4±11.9 vs. 13.6±6.9 days; P=0.003).
The study demonstrates that among patients with lack of daylight exposure: (i) both females and males experienced more intense delirium; (ii) females also suffered of longer delirium; and (iii) length of stay was longer. These findings emphasize the importance of daylight exposure for maintaining circadian rhythm and thus reducing the risk of developing postoperative delirium among cardiac patients.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 13–14.
Cardiac angiography, using the radial access compared to the femoral approach, is associated with reduced complication rate and improved patient comfort but has significantly increased radiation dose. Improvements in radiation protection are needed. We aimed to determine the efficacy of a 0.5-mm lead apron across the patient’s abdomen in addition to standard operator protection for the reduction of scatter radiation on operator and patient radiation exposure.
We randomly assigned 332 patients undergoing coronary angiography to a group with pelvic lead shielding and a group without. In each procedure 8 digital dosimeters were used to measure operator radiation dose (under the lead apron, outside the thyroid shield, and at the left side of the head), patient dose at the level of the umbilicus (above and beneath the lead apron), and two on the acrylic shielding and one on the image receptor to measure scattered radiation.
Both groups were similar in BMI, procedures performed, and number of sequences. Usage of lead shielding statistically significantly reduced the radiation dose of the operator at all three sites measured: under lead apron (all in μSv): 0.53±1.4 Vs. 0.17±0.6, on thyroid collar 5.9±7.7 vs. 2.9±3.4 and left side of head 3.3±3.4 vs. 2.1±2.2, p<0.001. However, the radiation to the patient doubled from 15.4±24.1 to 28.9±81.1, p=0.04.
The use of a pelvic lead shield during radial angiography reduced operator radiation exposure at multiple measurement sites. However there was an increased exposure to the patient. This balance has to be further investigated and taken into account before the widespread use of this method.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 14.
Acute myeloid leukemia (AML) patients are treated with intensive chemotherapeutic protocols, suffer from protracted pancytopenias, and need prolonged transfusion support. Long-term transfusion dependency exposes these patients to alloimmunization to both leukocyte (HLA) and platelet (HPA) antigens, expressed by immune platelet transfusion refractoriness, a major risk factor for bleeding-associated morbidity and mortality. The published incidence of platelet refractoriness in patients with hematologic malignancies ranges between 7% and 34%. There is no data regarding the incidence of platelet refractoriness in the Israeli population. The aims of the study were: to estimate the prevalence of platelet transfusion refractoriness among Israeli AML patients, to define the “timing” of alloimmunization during treatment protocol, and to evaluate weekly antibody screening as a tool to predict the risk for immune transfusion refractoriness.
One hundred newly diagnosed AML patients (55 males and 45 females) between 25–60 years of age, from of various ethnic origins were included. The newly diagnosed AML patients were screened for the presence of anti-platelet antibodies and anti-HLA antibodies using flow cytometry. Antibodies were identified by the MAIPA assay. All patients were followed using weekly antibody screening. Clinical parameters of patients were consecutively registered. Patients received platelet transfusions according to the local transfusion policy with continuous PLT increment monitoring. Patient blood counts and platelet transfusion requirements were followed weekly, platelet refractoriness was determined when no increment was documented on two consecutive platelet transfusions.
Platelet refractoriness was found in 50 (50%) patients (21 males and 29 females; 44 patients (44%) developed new anti HLA and/or anti HPA antibodies, 32/44 (73%) were females. Immune platelet refractoriness was defined in 34/50 (68%) refractory patients (26 females and 8 males). For patients with antibodies, 34/44 (77%) developed refractoriness; 13/34 had anti-HPA with (11) or without (2) anti-HLA antibodies. Only anti-HLA antibodies were found in 21/34 patients. Average time to antibody production was 28 days from the beginning of treatment protocol.
Our results demonstrate that anti-HPA antibodies are strongly correlated with the appearance of immune platelet refractoriness and that the prevalence of immune platelet refractoriness in Israeli AML patients is higher than expected according to published data.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 29–30.
Lymphoma is the most common hematological cancer reported during pregnancy. Recent data suggest that unlike lymphoma occurring outside of pregnancy, pregnancy-associated lymphoma is characterized by an excessive involvement of reproductive organs, advanced disease stage at diagnosis and an aggressive course, potentially leading to a high death rate of mothers.
This study was aimed at determining the role of estrogen and progesterone in lymphoma progression
Several human lymphoma cell lines were analyzed by FACS for expression of estrogen (α & β) and progesterone receptors. Human breast cancer cell line Mfc-7 was used as a positive control. CFSE-stained BL2 cells, were treated with E2 (25μM and 100μM) for 24h and 48h. Cells were harvested and analyzed by FACS for determining cell proliferation. Ramos cells were treated with E2 (5–25μM) for 72h. Cells were harvested and stained with Annexin-PI for determining live/necrotic/apoptotic cell proportion. The BL2 cells, either seeded alone or co-cultured with HK cells for 24h, were treated with E2 (25μM & 100μM) for 24h. The proportion of live, necrotic, and apoptotic cells was also analyzed.
Lymphoma cell lines express both estrogen receptors but not progesterone receptors. Estrogen-treated lymphoma cells demonstrated a reduced proliferation rate in a concentration-dependent manner. Furthermore, apoptosis and necrosis of lymphoma cells were significantly increased with concentration-dependent estrogen treatment. Progesterone had no effect on lymphoma cells proliferation, apoptosis or necrosis. The negative effect of estrogen on lymphoma cells was reversed in the presence of lymph node stromal cells. Estrogen treated lymphoma cells that were cultured on lymph node stromal cells demonstrated significantly less apoptosis and necrosis rates relative to non-cultured cells.
Lymphoma cells express estrogen but not progesterone receptors. Estrogen induces lymphoma cell necrosis and apoptosis and reduces proliferation. Lymph node stromal cells protect lymphoma cells from estrogen mediated effects. The direct effect of estrogen on lymphoma cells does not support lymphoma progression. Other mechanisms should be sought to explain the aggressive nature of lymphoma diagnosed during pregnancy.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 30–31.
Myeloid leukemia cell population is heterogeneous and composed of different immunophenotypically sub-populations. These leukemic sub-populations may also vary in their sensitivity to chemotherapy. During treatment, inherent chemo-resistant sub-populations survive and are becoming a dominant clone.
It has shown that soluble HLA (sHLA) -bound peptides molecules present in the plasma of cancer patient are mostly derived from the disease cells and thus allow the identification of sHLA peptidomes.
Here we analyzed and compared differences in the expression of sHLA peptides between boon marrow (BM) plasma at: diagnosis, during chemotherapy induction and remission, as well as normal healthy population, to find a peptide model which will be used to measure the response of each patient to the treatment and map out disease-specific peptide spectrum, clarify the possible pathogenesis, resistance mechanism, and disease prognosis.
Plasma from BM aspirates samples derived from acute myeloid leukemia (AML) patients will be extracted during the follow time points: at diagnosis (D1), fifth day (D5) and day fourteen (D14) from induction therapy. sHLA and peptides purification from plasma: samples are loaded sHLA class I molecules with their bound peptides loaded on pretreated monoclonal antibody W6/32. Elution is done with Trifluoroacetic acid and the sHLA is separated from their peptides on C18 Micro TipColumn with acetonitrile. The peptides fraction is concentrated by vacuum centrifugation until complete evaporation and are examined in liquid chromatography coupled mass spectrometry (LC-MS/MS).
Peptides repertoire bound to sHLA-plasma were identified by LC-MS. The peptides from diagnosis (D1), during chemotherapy treatment (D5) and remission (D14) was compared in eleven AML patients. We found that during chemotherapy (D5) there was an elevation of 25 peptides related to a variety of cancers genes and to proteins involved in different cell biological processes. These peptides were correlated with patients who did not respond to chemotherapy treatment.
In this study we found peptides unique to AML which did not exist in healthy donors, and also found a difference in the appearance of those peptides during chemotherapy treatment compared with pre-treatment. Furthermore, we found a correlation between specific peptides and patient responses to treatment.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 31.
Hepatitis C virus (HCV) is a leading cause of cirrhosis and hepatocellular carcinoma worldwide. Several viral and host factors related to viral response have been reported. Morphometry is a field that investigates changes in shape, size and orientation of objects and allows for the quantification of these parameters, which can highlight areas with significant differences. This study had two aims: 1. Quantification of histological findings from patients with chronic HCV using computerized morphometrics; and 2. Prediction of response to medical treatment of chronic HCV using baseline histomorphometric findings.
Fifty-eight patients with chronic HCV infection with a Metavir score F1 and F2, and followed in our Liver Unit were selected and grouped according to treatment response: sustained viral response (SVR) and non-SVR. Histomorphometric analysis was blinded to patient identification or previous histological information.
Slides were scanned using the dot slide virtual microscopy (Olympus) system. The entire slide was manually scanned and 3–4 representative images were recorded from each one. Each biopsy contained 6–8 representative portal spaces in average. The Imagepro Plus 0.0 (MediaCybernetics, Rockville, MD, USA) software was used to analyze and quantify collagen fibers, inflammatory cells, and liver architecture. MATLAB (MathWorks®, Natick, MA, USA) software was used to analyze fractal and lacunar dimension, giving an indication of the architectural distortion in the liver parenchyma.
Clinical parameters, including age, white blood cell count and hemoglobin concentration, and histomorphometric variables, including the density of collagen fibers, the number of inflammatory cells in the portal space, and textural parameters were found to be statistically significant and could be used together as a formula to predict response to treatment in HCV patients, with 93% sensitivity and 100% specificity.
The histomorphometric method is promising and may contribute to developing an expert guided automatic system predicting response to treatment in chronic HCV patients. Morphometry may be used in the future to investigate liver diseases due to different etiologies.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 32.
Atypical hemolytic-uremic syndrome (aHUS) is a rare thrombotic microangiopathy (TMA), attributed to complement dysregulation. Recently, loss-of-function mutations in Diacylglycerol Kinase, Epsilon (
Three males with early-onset aHUS and 16 of their close relatives were clinically and genetically evaluated. Genetic analysis was performed using whole exome sequencing, followed by conventional Sanger sequencing for mutation verification.
Patients presented at infancy with normocomplementemic aHUS, nephrotic syndrome and hypertension. Renal biopsies demonstrated glomerular migroangiopathy. Patient 1, currently 27 years-old, was not managed with plasma infusions, and developed end stage renal disease (ESRD) at 20 years of age. Patients 2 and 3, currently 8- and 6-years-old, respectively, demonstrated clinical response to plasma exchange/infusions during aHUS relapses, and maintained prolonged remission under prophylactic plasma infusions. Temporary cessation of prophylactic plasma infusions and switch to eculizumab therapy in patient 3, resulted in aHUS relapses, which resolved after re-introduction of monthly plasma infusions. Exome sequencing excluded disruption of all currently known genes involved in aHUS, but revealed a novel homozygous nonsense mutation in
Our findings confirm that
Citation: Rambam Maimonides Med J 2015;1 Suppl: 32–33.
Human telomeric regions are packaged as constitutive heterochromatin, characterized by extensive subtelomeric DNA methylation and specific histone modifications. Immunodeficiency, centromeric instability, facial anomalies (ICF) syndrome type I patients carry mutations in DNA methyltransferase 3B (DNMT3B) that methylates de novo repetitive sequences during early embryonic development. The fibroblasts of ICF type I patients display hypomethylated subtelomeres, abnormally short telomeres, and premature senescence.
In order to study the molecular mechanism by which the failure to de novo methylate subtelomeres results in accelerated telomere shortening, we generated induced pluripotent stem cells (iPSCs) from three ICF type I patients.
Telomeres were elongated in ICF-iPSCs during reprogramming, and the senescence phenotype was abolished despite sustained subtelomeric hypomethylation and high TERRA levels. Fibroblast-like cells isolated from differentiated ICF-iPSCs maintained abnormally high TERRA levels, and telomeres in these cells shortened at an accelerated rate, leading to early senescence, thus recapitulating the telomeric phenotype of the parental fibroblasts.
These findings demonstrate that the abnormal telomere phenotype associated with subtelomeric hypomethylation is overridden in cells expressing telomerase, therefore excluding telomerase inhibition by TERRA as a central mechanism responsible for telomere shortening in ICF syndrome. The data in the current study lend support to the use of ICF-iPSCs for modeling of phenotypic and molecular defects in ICF syndrome and for unraveling the mechanism whereby subtelomeric hypomethylation is linked to accelerated telomeric loss in this syndrome.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 33–34.
Cell free DNA (cfDNA) has emerged over the last year as an alternative for amniocentesis for diagnosis of the common aneuploidies looking at trisomy 21, 13, 18, sex chromosomes and triploidy.
We present our experience of the first 300 Panorama™ tests sent from Israel. This method is based on massively multiplexed PCR amplification of cfDNA isolated from maternal plasma, targeting 19,488 SNPs, followed by high-throughput sequencing. The fetal fraction is determined. The SNP pattern of maternal DNA (from buffy coat) is compared to the SNP pattern of free DNA from maternal plasma, which contains maternal and fetal DNA. Paternal genomic samples, when available, were included in the analysis; in the absence of a paternal sample, the algorithm considers population allele frequencies. Combining the maximum likelihood ratio with
The results of the first 300 sequential tests performed in Israel were analyzed. Fifteen samples necessitated redraw, two samples failed analysis. Four samples yielded high risk scores: two cases for trisomy 21, one for Kleifelter syndrome (KS) (47,XXY), and one for trisomy 18. Confirmation of both trisomy 21 and one KS were done by CVS or amniocentesis. The mother of suspected trisomy 18 was not interested in invasive testing in view of normal ultrasound scans and delivered a healthy baby. There are no known false negative results.
Panorama™ test is a reliable tool for identification of pregnancies at high risk for fetuses with the common aneuploidies with a high success rate. We recommend confirmation of the diagnosis for high risk scores pregnancies using invasive tests.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 34.
A 45-day old infant, the first child to parents who are first degree cousins of Muslim origin, was referred to the Genetics Institute at Rambam Health Care Campus due to multiple defects. The infant presented with hypertonicity, seizures, bilateral absent patellae, femoral epiphysiolysis, contractures of the lower extremities and retrognathia. Mild expansion of the lateral cerebral ventricles and mild hydronephrosis were also observed. No genital anomalies were noted. Follow up showed severe psychomotor delay. Genitopatellar syndrome (GPS) was suggested as the primary differential diagnosis.
Genitopatellar syndrome is a rare disorder, characterized by skeletal defects, most prominently hypoplastic or absent patellae, combined with urogenital anomalies, such as hypoplastic scrotum and micropenis in males or clitoral hypertrophy in females. Microcephaly, severe intellectual disability, characteristic craniofacial dysmorphism, and flexion contractures of the lower extremities had also been described. This is an autosomal dominant syndrome that is caused by de novo heterozygous mutation in
Following informed consent by the parents, whole-exome sequencing was conducted on the child’s genomic DNA. Initial analysis included inspection of variants found in
Exome analysis revealed a heterozygous missense variant, caused by a G-to-T alteration in the first nucleotide of exon 16 of
When attempting genetic diagnosis of cases with an unclear clinical presentation, we are facing a dilemma whether to Sanger sequence candidate genes or directly apply whole-exome analysis. The growing availability and lowering prices of whole-exome sequencing are directing the global trend to make it the diagnostic method of choice, both in research and in clinical settings. Nevertheless, a full clinical assessment is critical in guiding data analysis towards the causative variant.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 34–35.
Patients under anesthesia cannot communicate pain; hence an objective monitoring system is needed to assess intra-operative nociception and guide proper treatment. Based on a combination of physiological parameters, the Nociception Level (NoL) index was developed (0–100 scale). Random Forest Regression, capable of dealing with large numbers of input parameters arising from the complexity of the autonomous nervous system and their time derivatives, was applied to reveal their non-linear relations to the nociceptive state.
The aim of this study was to validate the NoL index during surgery and anesthesia and to compare its performance to other currently used nociception related monitors.
The study recruited 69 adults scheduled for surgery under general anesthesia (GA). After induction of GA (Entropy<60), all study subjects were subjected to two identical tetanic stimulations, first without analgesia (TET1) and again after 2mcg/kg fentanyl (TET2). All subjects were subjected to intubation (TP1) under the same analgesic conditions. After intubation study subjects were randomly divided into two groups of remifentanil TCI: 2 and 4 ng/ml. TET1, TET2, intubation (TP1), skin incision/first trocar insertion (TP2), and no pain (TNP) were annotated for analysis. Parameters recorded for analysis were NoL, heart rate (HR), plethysmograph amplitude (PPGA), and surgical plethysmograph index (SPI). Three values were analyzed for each parameter: pre-stimulus, post-stimulus, and reaction median values. Positive study outcomes were a significant reaction to noxious stimuli and an insignificant reaction to non-noxious stimuli; discrimination between noxious and non-noxious stimuli with AUC>0.8; grading of response to stimuli as expected (TET1>TET2; TP1>TP2> TNP); and reflection of different analgesic dosing (p-value<0.00625 after Bonferroni correction).
Fifty-eight subjects were valid for analysis. The NoL changed with statistical significance in response to both TET1 and TET2 (p=0.0006, p=0.0051, respectively). The SPI failed to identify TET1 but changed with statistical significance in response to TET2 (p=0.0025). The HR and PPGA failed to identify both TET1 and TET2. All parameters graded correctly the intensity of response to tetanic stimuli as: tet1>tet2. All parameters identified administration of fentanyl prior to TET2. All parameters reacted with a statistical significance to TP1 and TP2 except for the HR, which failed to respond to TP2. The only parameter to grade correctly TP1>TP2>TNP was the NoL. In ROC analysis for the ability of parameters to discriminate noxious (TP1, TP2) from non-noxious stimuli (TNP) NoL reached an AUC of 0.93 (CI 0.89–0.97), outperforming all other parameters. At a given working point of 90% specificity, NoL reached a sensitivity of 86.67% (CI: 78.64%–92.51%). The NoL post-stimulus median value was the only parameter to reflect a different basal level of remifentanil TCI during noxious stimulus (p<0.0069). The NoL reaction to TP2 reflected the different analgesic states of subjects in the high and low dose groups, with a significant reaction in the low dose group, and an insignificant reaction in the high dose group; PPGA, NIBP and SPI behaved similarly. The HR did not react with statistical significance for both low and high remifentanil doses around TP2.
Study results validated the NoL index, demonstrating its ability to correctly identify the response to various noxious and non-noxious stimuli and to discriminate, with high sensitivity and specificity, between the two entities. Moreover, the NoL index was proved able to grade the response to noxious stimuli as expected by the intensity of stimulus and the analgesic state of the patient. The NoL index was found to be superior in comparison to all other physiological parameters and indices tested in this study (HR, PPGA, NIBP and SPI) and currently in use within the OR to monitor patients under GA, or referenced in the literature as nociception related. Furthermore, the NoL index algorithm, based on artificial intelligence learning, coupled with nonlinear integration of several physiological parameters and their time derivatives, proved to be better adapted for representing the analgesic and nociceptive states of patients under general anesthesia than any other monitor currently used.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 35–36.
Intrapulmonary Percussive Ventilation (IPV) was introduced to Rambam Health Care Campus in September 2010. It was used as an adjunct to conventional ventilation in patients with severe respiratory failure where standard measures failed to improve their status.
In an 18-bed medical-surgical Intensive Care Unit (ICU), IPV was superimposed on Pressure Regulated Volume Controlled Ventilation (PRVC) as a rescue therapy. All patients had severe Acute Respiratory Distress (ARDS) with refractory hypoxemia andor extreme hypercapnia, and failed maximal therapeutic efforts. These included muscle paralysis, Inverse-Ratio ventilation, Nitric Oxide inhalation, and prone position. Both IPV and PRVC were delivered in parallel to the airway via a special connector. The resulting pressure waveform was combined of slow squares representing PRVC with super-imposed rapid short rises representing IPV. The Fraction of Inspired Oxygen (FiO2) was set to maintain arterial saturation above 90%. Weaning from IPV was possible when respiratory parameters allowed returning to PRVC ventilation alone.
Over 2.5 years, 81 patients, 51 (63%) males, were placed on IPV. Nitric oxide inhalation was given to 75 (93%) patients; prone position was performed in 28 (35%). Median values (range): Age 50 (14–80); APACHE-II score 28.2 (11–48); ICU days 20.5 (1–163); ventilator days 18 (1–163); IPV days 3 (1–33); admission to IPV interval 4 days (0–35); and pre-IPV lung compliance 26 (10–63). Tracheostomy was performed in 42(52%). SAPS-2 median predicted (all cause) mortality was 53%; survival to ICU discharge, 43 patients (46.9% mortality). Immediate pre-IPV data and best results within the first 24 hours of IPV were prospectively collected and retrospectively analyzed (
Mean and Standard Deviation (SD) of Pre- and Post-IPV Measurements.
| Respiratory Rate, Breath/min | 21 (5.1) | 13.9 (3) | 0.0001 |
| Tidal Volume, cc | 513.9 (117.9) | 314 (92.1) | 0.0001 |
| Minute Ventilation Liter/min | 9.47 (2.35) | 32.5 (8.37) | 0.0001 |
| Mean Airway Pressure, cc H2O | 23.3 (4.9) | 20.05 (4.8) | 0.0001 |
| PaO2/FiO2 Ratio | 97.2 (51.9) | 225 (108) | 0.0001 |
| PaCO2 | 57.4 (22.9) | 38.2 (10.1) | 0.0001 |
| Oxygen Index | 30.87(15.5) | 12.67 (9.9) | 0.0001 |
| Mean Arterial Pressure, mmHg | 74.2 (16.6) | 91.66 (15) | 0.0001 |
| Mean Urine Flow, ml/hour | 72.9 (46) | 182.9 (97.9) | 0.0001 |
Legend: Oxygen Index = (Mean airway pressure*FiO2*100)/ PaO2.
Combining IPV with PRVC improved oxygenation and CO2 removal in these extremely ill patients with severe, refractory ARDS. Hemodynamic status was improved, as witnessed by increased blood pressure and urine flow. In order to evaluate the effect of combined IPV-PRVC ventilation on the outcome of patients with acute respiratory failure, a prospective randomized study is needed.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 37–36.
Sepsis is a clinical syndrome that complicates severe infection. Systemic inflammatory response syndrome (SIRS) is an identical clinical syndrome that complicates a noninfectious insult (e.g., acute pancreatitis, pulmonary contusion). Current theories about the onset and progression of sepsis and SIRS focus on dysregulation of the inflammatory response, including uncontrolled release of proinflammatory mediators with subsequent multiple organ dysfunction syndrome (MODS), which is the cause of the high mortality associated with these syndromes.
B-type natriuretic peptide (BNP) is a neuro-hormone that has been isolated first in the porcine brain and later in human ventricular cardiomyocytes. Both BNP and N-terminal pro-BNP are used for the early diagnosis of heart failure (HF) in patients presenting to the emergency department with dyspnea. Recently, elevated BNP levels have been measured in patients with septic shock and have been attributed to myocardial dysfunction due to sepsis. Because BNP synthesis is also induced by endotoxin and inflammatory mediators, the mechanisms leading to elevated BNP levels in patients with sepsis remain unclear. Little is known concerning N-terminal pro-BNP levels in patients with critical illness, especially with sepsis.
We prospectively studied 259 patients with sepsis in the absence of heart failure. The BNP levels were obtained for all patients. The relationship between BNP and clinical outcomes was tested using multivariable analysis models.
Eighty-two patients died during the 90-day follow-up (31.66%), 53 died in the current hospitalization (20.5%), and 80 patients were re-admitted (30.9%). By bivariable logistic regression analysis of demographic data, risk factors and laboratory tests, a statistically significant correlation was found between BNP mortality and morbidity. According to multivariable analysis models, there was a clear increase in mortality and morbidity in the population of patients who had elevated BNP values.
We have shown in a population of hospitalized patients with SEPSIS that BNP is a strong independent predictor of greater morbidity and mortality.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 37–38.
Peritoneal dialysis (PD) is the preferred mode of renal replacement therapy (RRT) in infants with end stage renal disease (ESRD). Hemodialysis (HD) is seldom used in neonates and infants because of its major complications in the very young.
We analyzed demographic, clinical, laboratory and imaging data on all infants younger than 12 months with ESRD who received HD therapy in our Pediatric Dialysis Unit between January 1997 and June 2013.
Eighteen infants (M:F 6/12; Arabs/Jews 14/4) with ESRD (median age, 94 days; median weight, 4.06 kg) received HD through a central venous catheter (CVC) for a total of 16,292 days. Seven (39%) were neonates (<1 month of age; group 1) and 11 (61%) were infants (1–12 months; group 2) who received HD for a cumulative 7141 and 9151 days, respectively. In 6 of the patients, the initial mode of RRT was PD for 1–3 months. Five (28%) of the patients had serious comorbidities. Thirty-eight CVCs were inserted (34 angiographically). There were five episodes of CVC infection—a rate of 0.3/1000 CVC days. Median catheter survival time was 320 days. Five (28%) of the infants underwent renal transplantation and 10-year graft survival was 80%. Seven (39%) of the patients died. Most infants had good oral intake and only 4 (22%) required gastric tube. Thirteen (72%) of the infants displayed normal growth. Eight (44%) of the patients (5 in group 1) had delayed psychomotor development.
Long term HD in neonates and infants with ESRD is technically feasible, can be implemented without major complications, carries a very low rate of CVC infection and malfunction, and results in good nutrition, growth and relatively good survival. Future efforts should aim to improve neurodevelopmental outcome and lower mortality rate in these infants.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 38.
Inborn errors of metabolism (IEOM) such as urea cycle defects (UCD) and organic acidemias (OA) can cause rapid, devastating and irreversible damage to the neonatal brain if not promptly treated. The most efficient therapy for IEOM-induced life-threatening metabolic crisis is hemodialysis (HD), which rapidly removes accumulated toxic compounds from the circulation. Data on the use of HD in IEOM, in particular in the neonatal period, is scarce.
This study summarizes our experience with acute HD in neonates with IEOM-induced metabolic/clinical crisis and describes the outcome of these neonates.
We analyzed the demographic, clinical, and biochemical data of all neonates with IEOM who were admitted to our Pediatric Intensive Care Unit between January 2004 and September 2013, and presenting with a metabolic crisis necessitating HD therapy.
Sixteen neonates (M:F 8/8) with IEOM (6, UCD; 6, maple syrup urine disease (MSUD); 2, mitochondrial cytopathies (MC), and 2, other OA) had uneventful deliveries and were admitted because of metabolic/neurological deterioration starting at 48h–14 days of age. Median age and weight of the infants at initiation of HD was 6.7 days and 2944 g, respectively. Hemodialysis was performed through double-lumen, 6.5-French (Gambro, Lund, Sweden) acute catheter inserted blindly or under sonographic guidance into the internal jugular vein; AK200 (Gambro) dialysis machine and tubing, and FXpaed (Fresenius, Bad Homburg, Germany) filters were used. A total of 34 dialysis sessions (3 hours each; at least two sessions per patient) were performed. The first HD session markedly decreased (91.5%) mean ammonia level in UCD and MC patients from 1071 to 86 μmol/L (nl: 53–90), and markedly decreased (88%) mean leucine level in MSUD patients from 2381 to 284 μmol/L (nl: 00–240). The second dialysis session decreased the rebounded mean ammonia and leucine levels from 433 μmol/L and 1064 μmol/L to 69 μmol/L and 136 μmol/L, respectively. The HD procedure was free of complications, resulted in marked clinical improvement in 11 patients, and enabled the initiation of the appropriate dietary/pharmacological therapy in all patients. Thirteen (81.2%) of the infants survived, and three (18.7%) succumbed to their metabolic disease in the neonatal period.
Hemodialysis in neonates with IEOM is safe, very efficient, and life-saving when performed by skilled personnel and in the appropriate setting.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 38–39.
During midlife and aging, subjective reports of cognitive problems become common. Conditions such as age associated cognitive impairment, mild cognitive impairment and dementia, increase in prevalence and are the diagnoses most often decided on. Background medical conditions and risk factors are often regarded as contributing to cognitive decline. The contribution of prior undiagnosed attention deficit hyperactivity disorder (ADHD) is seldom taken into account. The aim of the current study is to examine whether childhood or adult ADHD should be considered relevant in the differential diagnosis of cognitive complaints during midlife and aging.
Neuropsychological evaluation consisted of the Conners’ Adult ADHD Rating Scale-SL, Beck Depression Inventory, Sleep Quality Questionnaire and the following cognitive tests: logical memory subscale (LM-WMS), California Verbal Learning Test (short form), CANTAB (PAL, IED), and the Test of Variables Attention (T.O.V.A.). The study was conducted at the Cognitive Neurology Clinic, at Rambam Health Care Campus and granted the approval of the local IRB (Helsinki committee).
Participants included 36 people, aged 50–70, diagnosed with probable ADHD (pADHD) and a control group of 29. The pADHD group included 12 individuals self-referred due to complaints of cognitive decline or memory impairment, previously undiagnosed with ADHD (ADHD-A) but with lifelong symptomatology of ADHD and fulfilling ADHD criteria, and 24 individuals who are the parents of diagnosed ADHD children and reporting ADHD symptoms (ADHD-B), but without complaints regarding recent cognitive decline.
The ADHD-A patients were impaired on attention parameters while memory and executive functions were intact. The ADHD-B patients did not present measurable attention or other neuropsychological deficits as compared to the control group. Neither group fulfilled criteria for mild cognitive impairment or dementia.
Attention deficit hyperactivity disorder should be considered as a new/additional entity in the differential diagnosis of subjective cognitive complaints among middle-aged and older persons. Recognition of the specific cognitive and behavioral profiles of patients with ADHD should contribute to the ability to reach optimal differentiation from pre-dementia conditions in order to tailor appropriate therapies. The pathophysiology and future trajectory of the emerging ADHD symptomatology in older patients fulfilling lifelong ADHD symptomatology remains to be clarified. When examining older adults, ADHD should be considered as a differential diagnosis.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 39–40.
It is currently accepted that complex behavior and mental disorders result from a combination of biological susceptibility and exposure to environmental stimuli. Previously we showed that transient early in life interference with the expression of multiple genes by mithramycin followed by exposure to chronic stress, led to a “daring” and novelty seeking behavior in rats. In this study we used these rats to explore the molecular changes that may contribute to the behavioral alteration.
Monoamines and amino acids levels were analyzed by HPLC in brain and serum. The RNA was extracted from the right prefrontal cortex for whole genome cDNA array analysis. Various
Data from the
This study suggests that gene and environmental factors modulate both peripheral and central physiological processes, which converge and reciprocally interact to induce specific behavioral patterns Peripheral intervention can serve as a simple and novel add-on treatment for behavioral abnormalities.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 40.
Dysregulation of apoptosis has been increasingly studied as a potential causative mechanism in schizophrenia; however, the data are still controversial. Although multiple pathways can induce apoptosis, the mitochondrial pathway has been most frequently implicated in CNS apoptosis. The apoptotic prototypical interaction occurs in the mitochondrial membrane between pro-apoptotic Bax and anti-apoptotic Bcl-2, finally leading to DNA fragmentation (the hallmark of active apoptosis). Previous studies found an impaired mitochondrial function in schizophrenia. We hypothesize that transfer of isolated active normal mitochondria (IAN-MIT) into patient cells may improve their impaired apoptosis.
An attractive model for studying apoptosis in schizophrenia is to reprogram somatic cells into induced pluripotent stem cells (iPSCs) and to study their differentiation into neurons. We analyzed spontaneous and H2O2-induced Bcl-2/Bax ratio and DNA fragmentation levels in patient-derived iPSCs and their differentiated glutamatergic neurons from two healthy controls and two patients with schizophrenia. We first studied the apoptosis parameters. We then tried to correct the impaired apoptosis by transferring IAN-MIT into the cells.
In iPSCs, the spontaneous Bcl-2/Bax ratio was significantly higher in patients as compared to the controls (P<0.001). However, patient-iPSCs and controls showed similar sensitivity to induced apoptosis by H2O2, as the Bcl-2/Bax ratio was reduced by 67.42±7.36% in both groups. Spontaneous DNA fragmentation as observed by the TUNEL reaction was about four-fold lower (P<0.006) in patient-iPSCs than in control cells, while their sensitivity to H2O2 was higher by six-fold. The spontaneous Bcl-2/Bax ratio was higher (30% P<0.029) in patient differentiated neurons as compared with controls, similar to our findings in iPSCs. However, patient neurons showed lower sensitivity to induction of apoptosis by H2O2, as Bcl-2/Bax ratio was reduced by 93± 4.36% in control cells, whereas only by 56±1.98% in patient neurons. Spontaneous DNA fragmentation was about five-fold lower (P<0.021) in patient differentiated neurons than in control cells. Despite the similar sensitivity of control and patient neurons to H2O2, DNA fragmentation in the presence of H2O2 was significantly lower in patient neurons (P<0.009). The transfer of IAN-MIT had no significant effect on spontaneous or H2O2-induced Bcl-2/BAX ratio and DNA fragmentation in patient neurons.
Our results point to an impaired apoptotic process in schizophrenia patient-derived cells, which further supports the impaired neurodevelopment hypothesis in schizophrenia.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 40–41.
A neurology department patient population is composed partially of patients with what should be a ‘clear cut’ diagnosis, receiving treatment and/or going through procedures aimed at secondary prevention, and of patients who are admitted with a complaint or one or more symptoms requiring diagnostic procedures and the appropriate treatment. We evaluated the initial diagnostic accuracy and the diagnostic yield in a 29-bed neurology department at an academic tertiary hospital.
All patients admitted and discharged at the neurology department during the month of August 2014 were included; excluded were patients hospitalized in the EEG Video Telemetry Unit. The admission diagnoses were compared with the discharge diagnoses for agreement.
In all, 121 patients (62 males, 59 females, average age 57.3 years) were admitted and discharged during the study. A total of 124 consecutive admission and discharge documents were analyzed (three patients were admitted twice). The overall degree of agreement between initial and discharge diagnoses was 61.2%. Of the 48 changed diagnoses 7 (14.5%) of the cases were due to incorrect classification of the primary diagnosis at admission or discharge (for example three cases were coded as ‘headache’ instead of ‘meningitis’ on admission, although diagnosis of meningitis was made in the emergency department), and 36 (75%) were attributed to diagnostic work performed during hospitalization (a 48-year-old man suffering from convulsions was found to have a symptomatic cavernoma; a 79-year-old man admitted for diplopia was diagnosed with myasthenia gravis, etc.). For 5 (10.4%) of the cases, there was no diagnosis noted in the admission documents. Average duration of hospital stay for all study patients was 4 days.
A correct admission diagnosis of neurologic patients is crucial as it determines initial treatment and subsequent patient management. Nevertheless, the main role of many of neurology department hospitalizations is evaluation of symptoms and syndromes using specific specialized clinical skills, laboratory tests, and imaging, to arrive at the correct diagnosis. Emergency department assessment enabled a correct diagnosis in most cases. However, many cases were impossible to resolve without further investigation. Study of this patient series showed that despite the high patient turn-over and relatively short in-house stay, neurology department hospitalizations have a high diagnostic yield.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 41–42.
Selected for Oral Presentation
Aging has long been suggested to be accompanied by cognitive decline, even in the absence of dementia or other neurological insults. Decreases in gray and white matter volume and integrity parameters have been postulated to underlie this decline. More recently, late life cognitive impairment has also been linked to decreased functional connectivity within large-scale brain networks, particularly the default mode network (DMN). However, the exact time course of connectivity reduction along the adult lifespan and network-specific differences have not been addressed.
We studied resting-state functional connectivity MRI (rs-fcMRI) data of 845 healthy subjects aged 21 to 83 years. Functional and anatomical data were obtained from the online dataset of the International Neuroimaging Data-sharing Initiative. Seed-based analysis was applied to analyze four high-order cognitive networks (DMN, salience network, dorsal attention network and fronto-parietal control network), and three primary sensory and motor networks. Node pair correlation matrices were computed for three age groups: young (Y, 21–40 yrs), middle-aged (M, 41–60 yrs) and old (O, 61–83 yrs). Difference matrices were calculated for the Y vs. M and M vs. O groups. Additionally, averaged
Lifetime decline of functional connectivity in high-order cognitive networks was nonlinear. It was characterized by an early reduction (reaching its nadir at the fifth to sixth decades), followed by a plateau. In contrast, a motor network showed increased connectivity in middle adulthood and a subsequent decline. Moreover, alterations in internetwork interactions were noticeable primarily in the transition between young and middle adulthood.
These results confirm the hypothesis that brain aging involves adaptive reorganization of large-scale brain networks. Moreover, it stresses that most alterations, principally those related to high-order cognitive networks, occur relatively early in the adult lifespan.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 42–43.
Spinal cord stimulation (SCS) is often use in the treatment of neuropathic pain, although the precise mechanisms by which it produces analgesia are not fully understood. Evidence from animal studies has shown that electrical stimulation at the dorsal columns attenuated the ‘wind up’ phenomenon in dorsal horn neurons in nerve-injured rats. The present study was aimed to test the effect of SCS on temporal summation (TS), the human clinical correlate of the ‘wind up’ phenomenon, in patients with radicular leg pain.
Eighteen patients with SCS implants were tested both 30 minutes following SCS activation (‘ON’) and 2 hours after turning it off (‘OFF’), in a random order. Temporal summation was evaluated in the most painful site in the affected leg and in a corresponding area in the contra lateral leg, by applying tonic painful heat stimulus (46.5°; 120 seconds) and simultaneous recording of the perceived heat pain intensity. Patients were also requested to report their clinical pain intensity (0–100 numerical pain scale) during SCS ‘ON’ and ‘OFF’. Wilcoxon Signed Ranks test was used in the comparisons between SCS ‘ON’ and ‘OFF’.
The SCS activation significantly attenuated clinical pain intensity (from 66±18 to 27±31, p<0.001). In the non-painful leg SCS activation failed to produce an effect on TS (24±20 vs. 21±24 in SCS ‘OFF’ and ‘ON’, respectively; p=0.277). In contrast, a significant decrease in the magnitude of TS in the affected leg was observed in response to SCS activation (from 32±33 to 19±24; p=0.017).
These results suggest that attenuation of TS, which likely represents suppression of hyperexcitability in spinal cord neurons, is a possible mechanism underlying SCS analgesia in patients with neuropathic pain.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 43.
Opioid analgesia is mediated by spinal and supra-spinal mechanisms. At the spinal level opioids attenuate the electrophysiological ‘wind-up’ phenomenon. The aims of the present prospective study were to test the effects of the short-acting μ-agonist remifentanil on temporal summation (TS), the clinical correlate of ‘wind up’ in patients with neuropathic pain (NP) and to correlate it with remifentanil’s analgesic effect on the clinical pain.
Following obtaining a written informed consent fourteen patients (5F/9M) with chronic NP (sciatica), received an infusion of placebo (normal saline) followed by an infusion of remifentanil (target plasma concentration of 2 ng/ml) for a duration of 40 minutes each, in a single-blinded fashion. Temporal summation, spontaneous clinical pain intensity (0–100 numerical pain scale, NPS), and evoked pain (straight leg raise=SLR) were assessed 20 minutes after the initiation of each infusion. The TS was evaluated by a paradigm of tonic heat stimulation (46.5°) for 120 seconds and pain intensity was simultaneously recorded on a Computerized Visual Analogue Scale (COVAS, 0–100).
The magnitude of TS during saline and remifentanil infusions significantly differed from each other (33.4±29 and 13.1±23.39, respectively; Wilcoxon Signed Ranks test, p=0.033). During remifentanil infusion, compared to placebo, clinical pain was lower (47.5±23.9 and 23.9±18.7, p=0.002) whereas SLR was significantly higher (34o±20.3 and 25.8o±15.6, p=0.004). Significant correlation was found between the difference in TS (remifentanul vs. placebo) and the difference in the intensity of the clinical pain under these two conditions (r=0.538, p=0.039). A similar trend towards a significant correlation between the difference in TS and the difference in SLR was also observed (r= −0.474, p=0.074).
These results suggest that remifentanil reduces TS and chronic NP as evident by improvement in both patients’ reports of pain intensity and in SLR. In addition, the correlation between the reduction in TS and the reduction in NP intensity suggests that opioids exhibit their analgesic effect via reducing excitability at the spinal cord level.
This research was supported by an unrestricted grant from the Israeli Pain Association (IPA).
Citation: Rambam Maimonides Med J 2015;1 Suppl: 43–44.
We recently showed that a combination of multiple autonomic parameters accurately differentiates between experimental pain intensities in healthy subjects. However, this technology has not been tested so far in patients with chronic pain. Spinal cord stimulation (SCS) is widely used in the treatment of pain conditions when conservative or less invasive techniques are ineffective. The present study was aimed to test the ability of the combination of autonomic parameters to objectively detect effectiveness of SCS treatment.
Thirty-three patients with chronic radicular (neuropathic) pain in one lower extremity and permanent SCS participated in the study. Patients were requested to rate the intensity of their pain on numerical pain scale (NPS, 0–100) twice, at a random order: 30 minutes after turning the SCS on and 2 hours after turning it off. For the purpose of this study, a difference of 15 NPS points or greater between the two ratings (stimulator “on” and “off”) was regarded as an “effective SCS.”
Photoplethysmogram (PPG) and skin conductance (SC) were recorded twice, along with the subjective pain ratings, each time for 120 sec, using the PMD-100™ (Medasense Biometrics, Ramat Yishai, Israel). The following autonomic parameters and their derivatives were extracted: PPG wave amplitude, PPG wave amplitude variation, pulse rate (PR) interval, PR variability and SC fluctuations. The parameters were combined using a non-linear classifier. The accuracy, sensitivity, specificity, positive and negative predicted values (PPV and NPV) of each parameter alone and their combination were calculated. Paired t-test was used for statistical analyses.
Effective SCS was found in 18 patients and ineffective in 15 (ΔNPS: 40±10 vs. 8.5±0, respectively; p<0.05). The combination of the parameters outperformed each of the parameters alone in the detection of the SCS effectiveness, with regards to all classification performance criteria (accuracy of the combination was 85% and that of each parameter ranged from 52% to 70%; sensitivity 94% vs 53–82%; specificity 75% vs 38–73%; PPV 76% vs 64–76%; NPV 92% vs 46–79%). The study is still ongoing.
These findings suggest that autonomic-based multi-parameter assessment may be used for objective measurement of SCS effectiveness.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 44.
Pro-nociceptive pain modulation profile (PMP) characterized by less-efficient descending inhibition and enhanced ascending facilitation was observed in various chronic pain syndromes including neuropathic pain. However, the characteristics of a PMP in painful diabetic neuropathy (PDN) have not yet been fully explored. This study aimed to: (1) illuminate the response pattern to experimentally-induced pain for ‘static’ and ‘dynamic’ psychophysical measures in PDN, (2) to investigate the relationships between PDN-related clinical parameters (pain intensity and neuropathy duration) and pain modulation characteristics, and (3) to evaluate the role of anxiety and pain catastrophizing levels on the PMP and clinical pain parameters.
Heat pain threshold, pain scores to pinprick mechanical stimulation, mechanical temporal summation, temporal changes along tonic painful heat stimulation and conditioned pain modulation (CPM) were tested at non-affected regions, and compared between 28 PDN patients and 33 healthy controls.
The PDN patients demonstrated less-efficient CPM responses (3.9±17.7 vs. −7.6±18.1; P=0.019), deficient adaptation to the tonic heat stimulation (51.3±14.3 to 50.9±24.1 vs. 60.3±14.1 to 47.3±25.0, numerical pain scale (NPS); P=0.013) and higher scores for the mechanical pinprick pain (25.6±24.6 vs. 13.5±12.6, NPS; P=0.017). Patients also had higher levels of pain catastrophizing (26.9±10.7 vs. 13.8±9.7; p<0.001).
The pro-nociceptive PMP was most evident for patients with higher intensity of ongoing pain and shorter duration of neuropathy. These findings indicate the pro-nociceptive PMP of PDN patients, demonstrating the precise alteration of mechanisms involved in their pain processing. Categorizing neuropathic pain patients based on pain modulation characteristics and neuropathy duration may be considered in determining treatment approach.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 45.
Theta-burst transcranial magnetic stimulation (TBS) has been shown to induce potent and long lasting effects on cortical excitability. In a previous open study, we demonstrated safety, tolerability, and antidepressant properties of continuous TBS (cTBS) in major depression (MD). The present study was aimed to evaluate the therapeutic efficacy of cTBS in depressed patients using a double-blind, sham-controlled design
Twenty-nine patients with MD were randomized to receive either active cTBS to the right dorsolateral prefrontal cortex (n=15) or sham cTBS (n=14) for 10 consecutive work days. After the tenth session, patients who received sham TBS were crossed over to active cTBS which consisted of 10 daily sessions. Patients who received active cTBS continued with the same treatment protocol for an additional 10 treatments. Each treatment session consisted of 3600 stimuli at an intensity of 100% of the active motor threshold. Severity of depression was assessed weekly.
Overall, there was no significant difference in the degree of clinical improvement between active and sham cTBS groups. However, in patients whose medication status remained unchanged before the trial (n=8) and in those who were medication-free (n=3), active cTBS resulted in a significantly greater reduction of Hamilton depression scores as compared to sham cTBS.
Our results suggest that the antidepressant effect of cTBS is modest, yet it might be beneficial to patients nonresponsive to ongoing pharmacological treatment. A direct comparison between cTBS and conventional rTMS protocols is warranted.
Citation: Rambam Maimonides Med J 2015;1 Suppl:45–46.
This study examined whether professional training of inexperienced mothers of 4-month-old infants improves feeding relationship and the eating habits of infants at 12 months.
First-time mothers were recruited 4–6 months after delivery for a training program (intervention group). Training was in small workshop groups, once a week for the duration of a month. Internet-based support was continued until infants reached 12 months by an experienced pediatric dietitian and social worker. The control group received customary support given at municipal mother-child health clinics. Mealtime interactions were evaluated at 12 months using blinded viewing of videotaped feeding interaction in the home setting by the Chatoor Feeding Scale. Infants eating habits were self-reported using questionnaires.
The training program was completed by 86 mothers. Controls of 42 mother-infant dyads were recruited. Mothers were 30 (+2.6) years old, with 16 (+2.2) years of education, all normative according to personality questionnaires, and without clinical eating disorders. Mealtime interactions according to the Chatoor Feeding Scale revealed statistically significant differences in favor of the intervention group (IG) for four of the five dimensions: dyadic conflict (IG 4.69 vs. 8.38 control), talk and distraction (3.75 vs. 4.9), struggle for control (2.3 vs. 4.88), and maternal non-contingency (1.61 vs. 2.75). This indicated more positive mother-infant interactions during mealtime, and a better response to the infant’s cues. The eating habits self-report indicated IG infants consumed less snacks at 12 months (1.08 vs. 1.38), less sweets (1.44 vs 2.07), and fewer sweet-drinks (1.03 vs 1.27). The intervention group mothers reported lower frequency of vomiting or spiting-up during mealtime (1.89 vs 2.41). The intervention group infants were also less distracted during mealtime (2.83 vs 3.29).
This research evaluated very early maternal training as a means of developing a positive feeding relationship at infancy. Results suggest that parental guidance can support the establishing of a more positive feeding interaction of 12-month-old infants. This may contribute to better and healthier eating habits with preserved internal eating cues based on hunger and satiety. We postulate this may prevent future eating disorders and obesity. Long-term follow-up and variation of groups is necessary to further examine these affects and optimize training programs to better accommodate specific target populations.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 46.
Feeding interaction is an essential part of parenting, especially in infancy. Errors in the early stages of feeding such as maternal control of feeding practice by restriction or pressure to eat may cause problems in feeding and eating patterns for infants in the present and future. These feeding interaction errors often originate with a mother’s pathology associated with eating disorders.
The aim of this study was to examine the relationship between maternal pathology associated with eating disorders and the observed feeding interactions with their one-year-old infants. In addition, we examined whether mother-child interaction group counseling for mothers of 4-month-old infants affects this relationship.
We performed a cross-sectional study comprised of 128 mothers aged 23–35, after their first birth, and living in Haifa and its surroundings. Eighty-six mothers underwent group counseling on feeding relationship when their infants were between the ages of 4–6 months. The rest of the mothers did not undergo counseling. The feeding relationship was evaluated by videotaping mothers and their 12–month-old infants during mealtime and then coded using the Chatoor questionnaire. In addition, mothers were asked to complete a questionnaire to examine their pathology associated with eating disorders (Eat 26, EDI).
We found an association between the mother’s pathology associated with eating disorders and the feeding interaction with their one year old infants in some of the measurements. The score on the distractions dimension was higher among mothers who reported having more eating psychopathology and with a higher tendency for drive for thinness. It was also found that mothers who reported having more eating psychopathology had a higher tendency for control during mealtime. Counseling mothers about feeding interaction did not affect this relationship.
The tendency for control and use of distractions during mealtime are associated with a maternal pathology associated with eating disorders. This may be one of the mechanisms that contribute to the effect of the intergenerational transmission of eating pathology. Therefore, finding methods of treatment and proper feeding relationship counseling for mothers with pathology associated with eating disorders may reduce and possibly eliminate this risk.
Citation: Rambam Maimonides Med J 2015;1 Suppl:46–47.
Bulimia nervosa (BN) and Binge eating disorder (BED) are two eating disorders (EDs) which involve binge eating behavior as a primary symptom. Night eating syndrome (NES) is conceptualized as a delay in the circadian pattern of food intake, manifested by two core criteria: evening hyperphagia and/or nocturnal ingestions. It is not entirely clear whether NES among persons with BED or BN may represent a separate entity, or a variant of BED or BN, in which the binge episodes occurs mainly in the evening. Specifically, it is unclear whether the frequency of binge episodes, the amount of calories consumed, and the macronutrient composition are similar between subjects with BED or BN with and without NES.
This cross-sectional study included 56 women, aged 18–60, recruited from the outpatient ED clinic of Rambam Health Care Campus, Haifa, Israel. The participants completed food diaries, a demographic questionnaire, the Eating Disorder Examination-Questionnaire, and the Beck Depression Inventory. A linear regression model was fitted for each dependent variable. Potential confounders were included in each model. For each model in which the study group variable was the only significant variable, an independent samples t-test was performed.
Of 56 participants, 33 (58.9%) were diagnosed with BED or BN (BE), and 23 (41.1%) were diagnosed with BED or BN, in addition to NES (BE+NES). The BE+NES group reported a significantly higher frequency of binge days (3.34±2.34 vs. 1.85±2.06, p=0.016) and binge episodes (4.55±4.39 vs. 2.23±2.71, p=0.024) during the week, as well as higher energy consumption (2,643.5±1,374.9 vs. 1,868.6±851.3, p=0.014) and fat consumption (36.9%±4.3% vs. 32.2%±4.8% of daily intake, p=0.001) than the BE group.
Our findings indicated that patients who have NES in addition to BED or BN have higher levels of eating pathology than patients who have BED or BN alone. In light of these results, we suggest that NES among patients with BED or BN may not represent just a variant of BED or BN, but rather a separate entity, which leads to a more severe disorder.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 47–48.
This study evaluated the lower uterine segment (LUS) and cervical length (CL), measured at 16–25 weeks, as predictors for preterm birth in twin pregnancies.
Both LUS thickness and CL were measured in women with twin gestation between 16–25 weeks of gestation. Demographic data, time, and cause of delivery were documented. Etiologies for PTD were divided into premature contractions, premature rupture of membranes (PPROM), and iatrogenic (sever PET, abnormal fetal well-being, IUGR, etc). Statistical analysis was performed to correlate between CL and LUS thickness and the different etiologies of PTD.
Inclusion criteria were met by 243 women with twin gestation; 41% delivered at term (>37 weeks); 26% delivered before 35 weeks; and 8% delivered before 32 weeks of gestation. Overall, both CL and LUS thickness were significantly decreased in women with PTD (
The LUS thickness and CL measured during the 2nd trimester of pregnancy are both predictors of PTD in twin pregnancies. Moreover, they may predict different etiologies of PTD which may indicate different preventive treatment modalities.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 48–49.
The objective of this study was to identify risk factors for obtaining an abnormal oral glucose tolarance test (75 gr OGTT) in women with gestational diabetes mellitus (GDM).
Included in this study were 538 patients with GDM that were treated in the HMO high risk clinics (MACCABI). For all participants relevant data was gathered including: maternal age, parity, family history of diabetes, gestational week at diagnosis, spontaneous or ART pregnancy, BMI score at the beginning of pregnancy, at time of diagnosis of GDM, and at the end of pregnancy, fasting plasma glucose levels at the beginning of the pregnancy, the 100 gr OGTT results, GDM treatment (insulin, oral medication, or diet therapy), gestational age and mode of delivery, fetal weight, and complications. The OGTT (75 gr. Glucose) was perfomed 6–12 weeks after delivery. Statistical analysis was performed in order to compare between an abnormal OGTT 75 gr. (over 140 mg/dl) 6–12 weeks post-delivery and the variables presented.
Thirty patients (5.6%) had abnormal 75 gr OGTT results; 508 (94.4%) were normal. Only the fasting plasma glucose level either at the beginning of pregnancy (91.7gr±9.2 vs 95.7gr±8.7; p=0.023) or during the 100 gr OGTT (105.2gr± 40.85 vs 127.5gr±51.3; p=0.002) correlated with abnormal 75 gr OGTT performed 6–12 weeks after delivery. Neither maternal age, BMI, weight gain during pregnancy, maternal or fetal outcome, mode of treatment (Insulin or diet), or other variables assessed in this study correlated with the 75 gr OGTT.
We suggest that the recommendation for early post-delivery OGTT should be limited to gestational diabetic patients with additional risk factors.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 49.
The aim of the present study was to compare fetal blood flow patterns in the cerebral and pulmonary arteries in women with low- and high-risk pregnancies, prior to and following maternal hyperoxygenation.
Ten pregnant women with normal- and ten women with high-risk pregnancy participated in the study. All had a singleton gestation with mean gestational age of 35.2±3.5 and 35.6±3.4 weeks, respectively. Doppler blood flow velocity waveforms were obtained from the middle cerebral artery (MCA), the main pulmonary artery, and the proximal right pulmonary artery. Pulsatility indices (PI) were calculated for the pulmonary arteries and MCA. Following baseline measurements, each woman received 70% humidified oxygen for ten minutes. Doppler measurements were then repeated.
Maternal hyperoxygenation caused a significant and similar decrease in PI values in the main and the right pulmonary artery in both groups. The PI value in the MCA did not change significantly following maternal hyperoxygenation in the low risk group (pre-O2 1.64±0.31 and post-O2 1.60±0.41). However, in the high risk group it increased significantly from 1.44±0.27 to 1.73±0.51, respectively; p<0.02.
This study shows that in both normal and high risk pregnancies hyperoxygenation abolishes the relative vasoconstriction in fetal pulmonary vessels. While no significant change was observed in the MCA in the low risk group, the relative brain sparing phenomena that was observed in the high risk group was completely abolished following O2 in the high risk group. These findings may be useful in monitoring high-risk pregnancies and suggest that maternal hyperoxygenation may be beneficial to the fetus in high-risk pregnancies.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 49–50.
Prenatal infection with gram negative bacteria is common during pregnancy. We have shown previously that acute maternal LPS exposure at E18 programs significant decrease the offspring innate immune response. The adaptive immune response is composed of B and T lymphocytes. T helper (CD4+, CD8-) and T Cytotoxic (CD8+, CD4-) compose the majority of T cells, and are essential for adequate immune responses. While T helper cells play an important role in establishing and maximizing the capabilities of the immune system, T Cytotoxic destroy virally infected cells and tumor cells. We sought to determine whether the adaptive immune response is programmed by maternal inflammation.
Pregnant Sprague Dawley rats (n=4) at 18 days gestation received intraperitoneal (i.p.) injections of LPS (500 μg/kg) or saline (control). Male and female pups were delivered spontaneously (e21) and allowed to mature until postnatal day 24 (p24). CD+4 and CD+8 lymphocytes, granulocytes, and macrophages expressing Toll-like receptor 4 (TLR4) were characterized in the blood, spleen, and thymus of offspring pups by flow cytometry.
Pups of LPS-treated dams had significantly lower blood T Cytotoxic lymphocytes than pups of saline treated dams, though the same number of T helper lymphocytes. Pups of LPS dams had significantly higher counts of blood CD4+ TLR4+ and CD8+ TLR4+ lymphocytes, TLR4+ granulocytes, and a trend towards higher TLR4+ macrophages. The same pattern was demonstrated in the spleen and thymus.
The decrease in the T Cytotoxic lymphocyte in the Offspring of LPS exposed dams may increase their ability to cope with viral infection and potential tumor development. The increase in CD4+ TLR4 + lymphocytes may aid in anti-inflammatory responses and may explain the decrease in the pro-inflammatory cytokines previously demonstrated in these offspring following exposure to LPS. These results suggest that maternal inflammation during the antenatal period may induce long term sequelae in the offspring innate immune response which may predispose to adult disease.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 50.
Maternal magnesium sulphate (Mg) may protect the preterm fetus from inflammation-associated brain injury. Leukemia inhibitory factor (LIF) is a pleiotropic cytokine essential for central nervous system development. Maternal LIF stimulates placental ACTH secretion, which subsequently induces fetal LIF secretion from nRBCs, ultimately promoting fetal brain development. Thus, transmission of maternal LIF signal to the fetus is mediated by placental ACTH. We hypothesized that maternal infection/inflammation may influence placental ACTH/fetal brain LIF signal transduction pathway and these effects may be prevented with Mg.
Pregnant rats at day 18 (n=6) received intraperitoneal injections of lipopolysaccharide (LPS) or saline (SAL) at time 0 and randomized to receive subcutaneous Mg or SAL for 2 hours prior to and following LPS or SAL injections. At 4 hours after LPS administration, dams were sacrificed and fetal brain and placenta were collected for determination of ACTH, LIF, and LIF-R protein levels in each group (LPS/SAL, LPS/Mg, SAL/SAL, SAL/Mg). Relative band density was determined after normalization to ß-actin levels.
Administration of LPS/SAL significantly increased ACTH, LIF and LIF-R protein levels in the placenta compared to SAL/SAL group (0.89± 0.04 vs 0.12±0.015 u, 1.0±0.17 vs 0.12+0.02 u, 0.46±0.07 vs 0.1±0.08 u, respectively, p<0.05). Magnesium sulphate treatment to LPS dams (LPS/Mg) significantly decreased placental ACTH and LIF-R compared to LPS dams (0.27± 0.07 vs 0.89±0.04 u, 0.25±0.05 vs 0.46±0.07 u, respectively). In the fetal brain, LPS/SAL significantly increased LIF levels compared to SAL/SAL but did not change LIF-R levels. Magnesium sulphate treatment to LPS dams decreased significantly LIF-R levels compared to LPS dams.
Lipopolysaccharide-simulated infection during pregnancy may attenuate fetal brain development and induce brain injury through increased placental ACTH and brain LIF. Magnesium sulphate may protect the fetus by suppressing both placental secretion of ACTH and fetal brain LIF-R levels.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 50–51.
Maternal infection/inflammation may induce fetal inflammatory responses that have been associated with long-term offspring cerebral injury. We previously demonstrated that prophylactic N-acetyl-cysteine (NAC), administered prior to and following maternal lipopolysaccharide (LPS), reduced the extent of cerebral injury measured by MRI after birth. This study examines the effect of therapeutic NAC on neonatal brain injury following maternal LPS-induced inflammation, as measured by MRI.
Pregnant Sprague Dawley dams (n=5) at 18 days gestation received an intraperitoneal injection of either LPS (500 μg/kg) or saline at time 0. Animals were randomized to receive an intravenous injection (tail vein) of NAC (300 mg/kg) or saline (time +30 minutes). Pups were delivered spontaneously and allowed to mature until postnatal day-25. The male offspring (5–8 per group) were examined by MRI and analyzed using voxel-based analysis. The apparent diffusion coeffient (ADC) was determined using diffusion tensor imaging to assess white and gray matter injury. The extent of injury was analyzed in three dimensions: The medial coefficient (MD), axial diffusivity (AD) and radial diffusivity (RD).
The offspring of LPS-treated dams exhibited significantly increased MD, AD and RD levels in the white and gray matter (e.g., thalamus, amygdale, visual cortex, corpus callosum, and hypothalamus), consistent with cerebral injury. In contrast, the offspring of NAC-treated LPS dams demonstrated, in most regions reduced MD, AD and RD levels, similar to the saline group.
Maternal NAC treatment following maternal inflammation significantly reduced evidence of neonatal brain injury. These studies suggest that maternal NAC therapy may be effective in human deliveries associated with maternal/fetal inflammation such as preterm rupture of the membranes and chorioamnionitis.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 51–52.
The rate of repeat cesarean delivery (CD) is above 90%. This increase is mainly attributed to a decrease in vaginal births after CD. In the last years several models have been proposed to predict the vaginal birth after cesarean section (VBAC) success rate, none of which included cervical length (CL). The aim of this study is to examine the accuracy of a cervical length model to predict VBAC success.
Pregnant women with singleton pregnancies, after 30 week of gestation interested in trail of labor after cesarean section (TOLAC) in our institution from April 2013 through March 2014 were included. Patients underwent transvaginal measurement of CL by one physician. Women in labor or with uterine contractions at presentation were excluded. The CL measurement was recorded and blinded from the attending physician. Clinical and demographic data were collected. After delivery logistic regression was used based on cervical length to predict successful VBAC.
After 30 weeks of gestation (79 patients >36 weeks), 91 pregnant women agreed to participate and were interested in TOLAC. The mean cervical length for women who had successful VBAC was 2.7cm±0.94 compared to 3.6 cm±1.05 for the women who failed VBAC and had repeat CD (p<0.0001). Multivariate logistic regression found that only a short CL and previous vaginal delivery were associated with an increased rate for VBAC. For each reduction of 10 mm in the cervical length, the odds ratio for vaginal delivery was 3.3 (p<0.0001;
Multivariate Model to Predict Successful Vaginal Delivery.
| Cervical length | 3.3 (1.6–6.6) | 0.001 |
| Previous vaginal birth | 5.2 (1.3–20) | 0.018 |
| BMI<30 | 0.97 (0.3–3) | 0.95 |
| Age<30 | 0.56 (0.18–1.7) | 0.31 |
| Birth weight | 0.99 (0.99–1) | 0.15 |
Cervical length measurement after 36 weeks of gestation has high predictive accuracy for VBAC. Further larger studies, should set up a combined model that includes cervical length and other parameters, to predict successful VBAC.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 52.
Selected for Oral Presentation
Pancreatic ductal adenocarcinoma (PDAC) ranks fourth among cancer-related deaths in the US. Gemcitabine (Gemzar. Eli Lilly) is the drug of choice for PDAC patients, despite its modest efficacy. The disappointing effectiveness of the drug is attributed largely to Gemcitabine resistance, which develops within weeks of initiation of therapy. Our group has recently demonstrated that tumor-associated macrophages cause gemcitabine resistance by inducing the level of cytidine deaminase (CDA), the enzyme that metabolizes Gemcitabine to its inactive form in the PDAC cell. The ligand-receptor interaction that mediated this phenomenon, however, could not be identified.
Exosomes were collected from conditioned media of IL-4 induced tumor associated macrophages. Exosomes were applied to PDAC cells in the presence of gemcitabine, and cell proliferation was assessed by XTT. Resistance was assessed following exosome treatment with RNase and DNase. Exosomes stained with PKH-67, a membrane incorporating dye, were applied on PDAC cells; internalization to PDAC cells was assessed by confocal microscopy. Exosome RNA content was screened for potential miR targets by qRT-PCR. Finally, Gemcitabine resistance in the presence of exosomes was assessed in PDAC cells treated with antagomiR (miR inhibitor), which acted as a ‘miR-trap.’
Exosome treatment led to Gemcitabine resistance in a dose dependent manner, inducing 28% resistance in comparison to non-treated cells (p<0.05). RNase treatment on exosomes, but not DNase, reversed Gemcitabine resistance (p<0.05, p=0.3 respectively). Stained exosomes were found to be internalized to the PDAC cell, 89% of which were found in the cytoplasm and 11% in the PDAC cell nucleus. Tumor-associated macrophage-derived exosomal RNA content was found to be enriched in specific miRs, as compared to the exosomal content of resting macrophages (p<0.05). AntagomiRs abolished exosome mediated gemcitabine resistance in PDAC cells (p<0.05).
Our results demonstrate that gemcitabine resistance is mediated, at least partially, by macrophage-derived exosomes. This mechanism for inter-cellular communication is supported by our finding that Exosomes that are secreted by macrophages are internalized to cancer cells. This inter-cellular communication is mediated by shuttling of miRNA’s between the macrophages and tumor cells.
AntagomiRs augment chemotherapy efficacy and might be a novel treatment for PDAC patients.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 52–53.
Perineural invasion of cancer cells (CPNI) is found in most patients with pancreatic adenocarcinomas (PDAs). Although the presence of M2 macrophages in the perineural environment has been associated with shortened survival rates in PDA patients, the mechanism underlying this phenomenon has not been elucidated. It has been suggested that secretion of the RET ligand glial-derived neurotrophic factor (GDNF) by M2 macrophages causes cancerous RET-expressing cells to migrate along affected nerves. This concept, however, has not been demonstrated
Immunohistochemical analysis of PDA specimens of a novel Pdx-1-Cre/KrasG12D /p53R172H (KPC) mouse model, revealed that CD163+ (M2) endoneurial macrophages (EMΦs) predominate the endoneural space of invaded nerves. In a bone marrow transplant (BMT) from
Taken together, our results suggest a paracrine response between bone marrow-derived tumor-associated EMΦs and PDA cells. This loop probably orchestrates the formation of cancer nerve invasion. Further studies using novel transgenic animal models aimed at specifying the specific molecular signals that induce disease invasion and proliferation in the perineural micro-environment are needed.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 53–54.
Head and neck tumors of mesenchymal origin (mainly sarcomas), have a very poor prognosis and most of the patients are subjected to surgical morbidity and eventually mortality. The complex anatomy of the head and neck usually prevents a surgical resection with clear margins. As a result an increased risk of recurrences is documented.
In this study we aimed to examine the expression levels of three genes known to be associated with biological development and tumor progression, and to correlate the staining levels of the protein expression with our head and neck sarcoma patient outcomes.
A retrospective immunohistochemical study was performed for heparanase, tuftelin, and FANCF in 20 head and neck sarcomas and 20 mesenchymal origin controls.
Heparanase and Tuftelin were found to be overexpressed in head and neck sarcomas compared with control. Almost 70% of the sarcoma tissues were stained positive for heparanase compared to only 35% of the control tissues. For Tuftelin, 50% of the sarcoma tissues were stained positive while only 10.5% of the control samples. No significant difference was detected between the sarcoma and control tissues in the FANCF staining levels; 15% of the sarcoma tissues were stained positive to FANCF and 10.5% of the control tissues. When we tried to correlate the staining levels to patient outcomes (5-year survival rate), Tuftelin overexpression was correlated with poor prognosis of our patients. Heparanase staining levels exhibited no significant benefit in the survival of sarcoma patients. All of the FANCF-positive patients did not survive more than one year following their diagnosis while (61%) of sarcoma FANCF-negative survived 5 years after diagnosis.
Currently, The Oral Cancer Research Laboratory at Rambam is investigating the mechanism of expression, possible roles, and the quantitative PCR analysis of these genes, which may help in the future understanding of head and neck sarcomas. The preliminary results presented in this work highlight the poor prognosis of sarcoma and the importance of large scale studies to help establish molecular markers for these aggressive tumors of head and neck mesenchymal origin.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 54.
Human papillomavirus (HPV) has been confirmed as the primary etiological factor that transforms cervical epithelia into cancer. The presence of HPV in oral cancers suggests that HPV may play a similar role in transforming the oral epithelia. A high degree of variability in the prevalence of HPV in oral cancers has been found, however, raising questions regarding its role in the transformation and development of oral cancer. The aim of the present study is to analyze, using tissue array technology and PCR (Polymerase Chain Reaction), the possible influence of viral infection on the carcinogenesis of oral carcinomas.
The study included 59 biopsies of oral neoplasms. Immunostaining of formalin fixed, paraffin-embedded 5 microns tissue array sections was performed. Slides were incubated with the primary antibody, LMP1 for EBV, pp65 for CMV, and P16 for HPV. Slides were scored as 0=no staining, 1=weak staining, and 2=strong staining, and the specific intracellular localization was recorded. Fresh tissue DNA was extracted (TRIZOL). The PCR Primers for specific viral DNA were fabricated. The total DNA was evaluated using PCR.
The ages of the patients in the experimental group ranged from 38 to 92 years (mean 63 years). A predominant number of the OSCC (Oral Squamous Cell Carcinoma) patients had reported tobacco use (47%). A much smaller portion reported consumption of alcohol (6%). Immunoreactivity for CMV and EBV was not significant, while HPV antibody expression was found in 32% of the cases. In 40% of the OSCC patients, HPV DNA was detected using PCR, While in control normal tissues of the same patient HPV genotype was expressed only in two tissue specimens (9%), one of which had positive margins for OSCC.
A correlation between HPV and OSCC was found in this study. In 40% of the tissues, HPV DNA was expressed exclusively in the tumor tissues and not in the same patients’ normal contra lateral epithelium. These finding support specific HPV infection and not generalized oral epithelium infection as the possible etiological factor for HPV induced oral cancer. We also found that cancer patients who were HPV-positive were significantly younger and had better prognoses than patients who were negative for HPV infection.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 54–55.
Non-Thermal Plasma (NTP), with ion temperature close to room temperature has tremendous applications in biomedical engineering, and can potentially offer a surgical option for cancer cell elimination without influencing the whole tissue. The aim of our study was to evaluate the safety of NTP treatment, and its effect on melanoma cells both
Our team has developed a prototype of NTP delivery device that can be used both
(
Grey color indicates non emitting skin area, resulting from melanocyte coverage.
We explored the effect of our novel NTP delivery devise on cancer progression, both
Citation: Rambam Maimonides Med J 2015;1 Suppl: 55–56.
This study aimed at creating a reliable and reproducible orthotopic mouse model of laryngeal malignancy that recapitulates its biologic behavior, local invasiveness, and spread as seen in patients.
The first mouse Laryngeal Speculum design was hand-crafted of a silicon tube with different shapes attached to an adult ear speculum. The selected prototype was printed using a three dimensional printer. Using this speculum it was possible to view mouse larynxes. Using direct laryngoscopy, human squamous cell carcinoma line FaDu (ATCC® HTB-43™) was implanted in the larynxes (supraglottis and glottis) of nu/nu mice (n=31) (
We performed 31 successful direct mouse laryngoscopies. Supraglottic and glottis tumor uptake was 100% and 25%, respectively. Median survival for the animals with supraglottic tumors was 35 days. Histopathologic evaluation revealed pre-epiglottic extension, paraglottic extension, thyroid cartilage invasion, and lymph node metastasis.
We describe the first orthotopic model for laryngeal cancer. Our model faithfully recapitulates the phenotype and malignant behavior that reproduces its natural biologic behavior as seen in laryngeal cancer patients. This model offers an opportunity to identify and specifically target therapy for larynx squamous cell carcinoma.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 56–57.
Tuftelin was originally identified in the enamel extracellular matrix and is expressed in the epithelial ameloblasts at an early stage of amelogenesis. Tuftelin cDNA sequences were detected in morula, embryonic stem cells, and different mesenchymal mineralizing and soft tissues, normal as well as cancerous. Recently we found that during mouse embryonic development, the dynamic spatio-temporal expression pattern of tuftelin seem to shift from the cytoplasm to the nucleus with developmental progression. Tuftelin mRNA and protein were increased during hypoxia (1% O2), through induction of HIF1α, in the neuronal PC12 cell model and in mesenchymal stem cells. Down-regulation of HIF1α mRNA in PC12 cells reduced hypoxia-induced tuftelin expression by 89%.
Indirect immunohistochemiostry was performed using Histostain®-SP kit (Zymed®, San Francisco, CA, USA). Polyclonal anti tuftelin antibodies raised against tuftelin synthetic peptides were used. Total RNA was extracted using TRI-reagent. Reverse transcription was performed using high-capacity cDNA reverse transcription kit. Real-time quantitative PCR was performed using TaqMan® Assays-On-Demand (Applied Biosystems®, Foster City, CA, USA).
Examination of human oral squamous cell carcinoma (SCC), ameloblastoma, and verrucous carcinoma, revealed strong staining for tuftelin protein in these types of tumors as compared to control tissues. In addition, tuftelin protein staining intensity in head and neck sarcomas was significantly higher in cancer samples as compared to normal, non-cancerous samples from the same individuals. Real-time PCR showed a 24-fold elevation of tuftelin mRNA expression in oral cancer tissues and 77-fold elevation in verrucous carcinoma, as compared to normal, non-cancerous samples from the same individuals.
Our previous results on tuftelin involvement in hypoxia, and its nuclear localization during late embryonic development and post-natally, indicates that tuftelin is involved in the adaptive mechanisms, regulating protective processes, possibly by regulating gene expression. These results can explain tuftelin’s expression in SCC and sracomas of the head and neck. Tuftelin expression in these tumors can also be explained by preliminary results of our lab, in which tuftelin has been shown to be involved in cell proliferation. Tuftelin’s involvement in cancer progression needs to be further investigated. Tuftelin should also be investigated as a possible marker for SCC and sarcomas.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 57–58.
The aim of the present study was to review the published data regarding the management of polymorphous low-grade adenocarcinoma (PLGA) and present our experience in the treatment of patients diagnosed with PLGA.
We performed a review of the published data of the treatment possibilities for PLGA, and report on a case series of four patients diagnosed with palatal PLGA.
The mean age at diagnosis was 61.75 years. All cases were localized on the hard palate. The male/female ratio was 1:3. One patient had an ulcerative painful lump. In correlation with the published data, most of our patients presented initially with a diagnosis that was not conclusive for the presence of PLGA. The most common initial diagnostic findings were adenoid cystic carcinoma and pleomorphic adenoma. The treatment of choice was surgical excision of the tumor with safe margins of 1.5 cm. One patient had a positive cervical lymph node metastasis and underwent an elective neck dissection followed by radiotherapy. Local recurrence was diagnosed in 1 patient 6 years after the surgical management. Most cases were reconstructed using an obturator, except for 1 patient who underwent reconstruction using the temporalis myofascial flap.
PLGA is a slow-growing, distinct, uncommon neoplasm of the minor salivary glands. Because it is characterized by a diverse morphologic pattern that resembles adenoid cystic carcinoma or pleomorphic adenoma, the initial diagnosis is usually inconclusive. Until a final diagnosis has been made, we believe that the reconstruction possibilities should be mainly obturators to allow a good visualization of the surgical site. The use of microvascular free flaps or rotational flaps should be reserved to patients with clearer pathologic diagnosis.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 58.
Febrile neutropenia (FN) is a common complication among children undergoing chemotherapy for hematologic malignancies. A microbial agent is identified in 15–30% of these episodes. Recently, several studies reported a high incidence (25%–60%) of respiratory viral infections (RVI) in children with FN. Implementation of RVI workup in children with FN was suggested, in order to minimize the overuse of antibiotics. However, data on the incidence of RVI in children with persistent FN (PFN) is meager. In this setting, invasive fungal infection (IFI) is a major concern, although most children who present with PFN rarely develop IFI. Yet, most international guidelines indicate empiric initiation of antifungal therapy after 4–7 days of FN.
Based on published data, we hypothesized that RVI is a significant cause of PFN in children with hematologic malignancies, and that IFI is the cause only in minority of cases. We further aimed to investigate whether detection of RVI in these children, would affect their risk of IFI.
Charts of children with hematologic malignancies, presented to our department during 2007–2013 with PFN (>38°c, for >96 hours and ≤500ANC/μl) and documented PCR of RVI, were analyzed. Patients were considered IFI positive if they had ‘possible,’ ‘probable,’ or ‘proven’ infection according to revised EORTC criteria. Respiratory viral infectons were detected by PCR and included: RSV, Influenza, Parainfluenza, HMPV, Adenovirus, and HHV-6.
A total of 75 PFN episodes were evaluated. Of these, there were 31 RVI episodes (41.3%). There were 16 possible (21%), 2 probable (2.6%), and 2 proven IFI episodes. There were only 3 episodes of IFI and RVI co-infections. Multivariate analysis revealed that RVI was significantly associated with reduced risk for IFI – O.R of 7.47 (p<0.0082, 95% CI −1.6–50.4). Total days of neutropenia, and antibiotic therapy were independent risk-factors for IFI (p<0.0109; P< 0.0034 respectively).
Children with PFN and RVI have a significantly lower risk for IFI. Further studies are needed in order to establish whether RVI-status can be implemented in the workup algorithm of PFN in children with hematologic malignancies.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 58–59.
Obstructive sleep apnea (OSA) is characterized by pharyngeal obstructions during sleep. We have recently demonstrated that during sleep and mild anesthesia, upper airway collapse is not explained solely by sleep-induced decline in pharyngeal dilator muscle activation, as dilator muscles EMG activity often increases during obstructive apneas and hypopneas, but fails to improve airflow. The present study was undertaken to evaluate the hypothesis that failure of intense activation of tongue protrudors to prevent pharyngeal obstruction may be associated with sleep-induced changes in tongue muscle coordination.
Esophageal pressure (Pes), airflow, and intramuscular EMG of the genioglossus (GG) and tongue retractors (TR) were recorded in 15 OSA patients. While awake, EMG’s of tongue protrusion and retraction were compared to biceps and triceps EMG flexion and extension. EMG/Pes was assessed while patients breathed through variable resistors, to estimate the magnitude of EMG required to prevent pharyngeal collapse. During sleep, EMG/Pes was evaluated during flow limitation. Pre-arousal peak inspiratory EMG (sleepEMG) was compared to peak EMG at the same Pes during wakefulness (awakeEMG). SleepEMG/awakeEMG >1 indicated a level of EMG expected to be sufficient to prevent pharyngeal collapse.
Patients were 47.7 ± 10.7 years old, with Apnea/Hypopnea Index of 61.8 ± 16.1/hr. During wakefulness, the pattern of antagonist tongue muscle activation differed considerably from that of the arm antagonist muscles, as both GG and TR were activated similarly during all tongue movements. During sleep, the levels of GG-EMG before arousal were significantly larger than those observed during wakefulness at equal Pes (125±69%, p<0.01). TR-EMG levels, however, were significantly lower than those recorded during wakefulness (52±21%, p<0.05).
In OSA patients during sleep, augmentation of GG-EMG parallels respiratory efforts during obstructed breathing, but fails to improve airflow. This mechanical inefficiency is not explained by high inspiratory suction pressures, as GG-EMG may increase to levels by far higher than those required to prevent pharyngeal collapse during wakefulness. Unlike the similar pattern of activation of GG and TR during wakefulness, co-activation of TRs is deficient during sleep. This alteration in the pattern of tongue muscle activation may be involved in the pathogenesis of pharyngeal obstruction during sleep.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 59.
SM-art MEDicine is a project of the Division of Internal Medicine at Rambam Health Care Campus. The project was started in the beginning of 2014 as the result of a Rambam-Intel collaboration program and was also inspired by the American Choosing Wisely® campaign. Healthcare worldwide has undergone many changes over the past years, mainly due to the introduction of new technologies, imaging modalities, blood tests, and procedures. This new reality presents a huge challenge to medical teams with regard to intelligently managing the medical diagnosis process, choosing the right diagnostic tests, and perhaps most important, to know when a supporting test is
Our aim is to add order and logic to this process by making common medical practices within the Division more exact, focused, and clinically based. This supports a few goals such as minimizing waste of medical resources, reducing the number of patient days, reducing the workload of supporting staff, and providing safer patient care.
The project is an educational campaign managed by senior doctors of the six internal medicine wards at Rambam Health Care Campus. This forum meets once every three weeks and discusses day to day practice topics (with the supporting literature), together with an external content expert and two Intel engineers. At the end of the meeting the team writes up a set of recommendations that are communicated to the doctors through various channels. The primary purpose of the recommendations is to refine, clarify and shed light on problematic areas in the overall work and management of internal medicine cases mainly pertaining to the uneducated use of hospital resources. The team also implements structural changes to the indicated process. Finally, the project impact is measured by quantifiable data from blood and imaging tests, where applicable.
A significant drops in various tests was observed after the recommendations were published throughout the departments and the structural changes were implemented. There was a 30% reduction (P<0.0001) in BNP tests, a 25% drop (P<0.0001) in Troponin tests, and and LDH testing was reduced by more than 50% (not yet statistically tested).
The SM-art MEDicine campaign has led to a marked reduction in the administration of diagnostic tests by improving the management of medical investigations and more intelligent use of medical resources.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 60.
A subset of patients does not react to anti-TNF agents or lose response. Increasing the dose has economic implications and switching to another biological may be suitable for rheumatoid arthritis (RA), but problematic for other autoimmune diseases in which the therapeutic “arsenal” is limited. This study was aimed at assessing the role of measuring the drug and antibody (ab) levels in the management of patients (pts) with autoimmune diseases, treated with monoclonal anti-TNF agents.
Trough serum levels and antibodies (abs) to infliximab (INF) and adalimumab (ADA) were measured by ELISA in pts treated at the ambulatory day care of Rheumatology of Rambam Health Care Campus. Anti-TNF abs were identified by anti-human lambda chain ab. Treated pts were classified into responders and non-responders based on disease activity.
One hundred and thirty-one serum tests for INF and 48 for ADA were performed in 101 pts (mean age 50.2 and disease duration 9.9 years). Among responders 51 pts received INF and 16 pts received ADA. Among non-responders 34 pts had no response and 10 pts lost response. Levels of INF and ADA in responders were significantly higher than in non-responders; levels of anti-INFAb and anti-ADA-Ab in responders were significantly lower than in non-responders. High levels of anti-drug Abs predicted treatment discontinuation. In all non-responders with low INF/ADA levels and low anti-INF-Ab/anti-ADA-Ab the shortening of retreatment intervals lead to significant improvement (
Outcome of Non-responding Patients to Anti-TNF Therapy.
| Low INF/ADA levels, | 6/6 | 3: allergic reaction | 3: shortening intervals |
| High anti-INF abs/anti-ADA abs | 6: inefficiency | 2: methotrexate renewal | |
| Low INF/ADA levels, | 12/5 | 1: allergic reaction | 15: shortening intervals |
| Low anti-INFabs/anti-ADA abs | 1: inefficiency | ||
| High INF/ADA levels, | 6/9 | 2: inefficiency | 13: improved |
| Low anti-INF abs/anti-ADA abs |
INF-infliximab, ADA-adalimumab, abs-antibodies.
Assessment of immunogenicity of anti-TNF monoclonal antibodies provided useful information for guiding the therapy in auto-immune diseases with suboptimal clinical response. Patients with low INF/ADA levels and low levels of corresponding Ab may benefit from increasing the drug dose or decreasing of retreatment intervals. In pts with negligible serum levels of INF/ADA and high levels of corresponding Ab the therapy should be switched to a different drug.
Citation: Rambam Maimonides Med J 2015;1 Suppl:60– 61.
The gastrointestinal tract is involved in nearly all patients with systemic sclerosis (SSc) and is a source of significant morbidity and even mortality.
This study was aimed at assessing whether there is a correlation between upper gastrointestinal (UGI) endoscopy findings and mortality in SSc patients.
The records of 256 SSc patients seen in our rheumathologic clinic between 2003 and 2013 were reviewed. Of these, 140 patients who had at least one detailed upper endoscopy report and at least 6 months follow-up were included in the study. Patient data included demographics, type of SSc, disease duration, modified Rodnan skin score (mRSS), lung, cardiac, renal or musculoskeletal involvement, hemoglobin at endoscopy and type of antibodies. Endoscopic findings that were included in the analysis were esophagitis, ulcerations, tumors, gastric antral vascular ectasia (GAVE), gastric erosions, submucosal hemorrhages and lumenal blood. The statistical methods used included descriptive statistics, T test, bivariable analysis, and cox regression analysis.
Forty-seven patients (16 diffuse SSc) had evidence of GAVE or antral erosions and hemorrhage. The mortality rate in this group during follow-up was 37% vs. 25% in the group of 93 (39 diffuse) SSc patients without GAVE or UGI bleeding (p=0.001). There were no statistical differences between the groups regarding mean age (55) or Hb (10.87 in the group with the UGI bleeding vs 11.77). The mean mRSS score was higher in the group with UGI bleeding 8 vs.5.6 (p=0.019). Mean (median) disease duration was 6.9 (4.5) years in the group with UGI bleeding vs 10.4 (10) (p< 0.001). Esophagitis was found in 90% of patients, despite use of PPI. Co-morbidity of myositis had a negative impact on survival. The mortality hazard ratio (95% CI) for UGI bleeding, myositis and interstitial lung disease were 5.9 (2.7–13.2), 4.9 (2–12.4) and 2.7 (1.3–5.8) respectively.
A diagnosis of GAVE or UGI bleeding on upper endoscopy was associated with higher mortality in our cohort of SSc patients. Myositis was associated also with increased mortality. The long-term survival of patients with GAVE/UGI bleeding was similar to the patients with myositis that were free of such GI complications. The patients with both myositis and GI bleeding had a very poor prognosis.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 61–62.
During the last decade the number of robotic devices and medical procedures utilizing them has increased significantly worldwide.
This study aimed at evaluating the implementation of robotic surgeries in Israel in various surgical disciplines.
A retrospective study was performed by accessing information about the annual purchases of surgical robots, the number of physicians trained for their use, and the number of robotic surgeries performed each year, according to indications of surgery and the disciplines of the operating medical staff. The data were taken from the database of Intuitive Surgical Inc. (Sunnyvale, CA, USA).
Six robots were purchased by six medical centers in Israel between 2008 and 2013. There are currently 150 doctors trained to use the robot in one of the simulators of Intuitive Surgical Inc. Of them, 104 doctors are listed as active robotic surgeons. Most are urologists, gynecologists, or general surgeons. The number of robotic surgeries increased each year in all fields in which it had been implemented. In 2013, 975 robotic surgeries were performed in Israel. Of them, 52% were performed by urologists; 80% of which were radical prostatectomies.
The use of robotic surgery increased considerably in Israel over recent years, in urology, gynecology, general surgery, and otolaryngology. Despite the lack of conclusive evidence of the advantages of robotic surgery over the laparoscopic approach, the market power and the desire to be at the technological forefront is driving many medical centers to purchase surgical robots and to train physicians in their use.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 62–63.
Severely atrophic posterior maxilla usually requires lateral wall sinus augmentation to enable proper placement of dental implants. The kinetics of the grafting material resorption or contraction, following lateral wall sinus augmentation, is yet to be fully understood. The aim of the present study is to evaluate changes in sinus dimensions immediately post-op and 1 year later using a composite bone graft of Xenograft and autogenous bone (90:10 ratio) for lateral bone augmentation of the maxillary sinus.
Patients that underwent lateral wall sinus augmentation procedure (without concomitant implant placement) were considered for this retrospective analysis if they had three consecutive panoramic radiographs: pre-treatment (T0), immediately after lateral wall sinus augmentation (T1) and approximately one year post-op (T2). Included in the study were 18 subjects, ages 47–69. Surgical protocol included bone grafting with a composite bovine derived Xenograft and autogenous bone graft (9/1 ratio). Digital panoramic radiographs were transferred into measurement software and the following measurements performed: (i) Vertical distance base of the alveolar ridge to the height of the augmented material; (ii) Sagittal area of the grafted area; and (iii) Vertical distance from the base of the ridge to the roof of the maxillary sinus. All measurements were performed by one examiner. Double measurements were performed in nine patients at T2 and the calculated Cronbach’s alpha statistics was high (0.9658–0.9882).
The mean overall sinus sagittal area was 1330±481 mm2 prior to the grafting procedure which decreased to 858.2±436 mm2 (380.7–2134 mm2, range) immediately post-op (p=0.0035) and stayed stable at one year (mean 882.2±384 mm2, p>0.05). Similarly, the overall area of the grafted material at the time of surgery was 468.3±208 mm2 and was only slightly reduced at 1 year (422.2±193 mm2). The mid-sagittal grafts height at T1 (mean 20.67±4.5 mm) was insignificant and minimal reduction at 1 year (mean 19.35 ± 4.9).
The linear dimension of composite BDX/autogenous bone grafts following lateral wall sinus augmentation exhibited minimal changes during the 8–12 months healing period.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 63.
Diabetes mellitus (DM) is a metabolic disease which may lead to diabetic nephropathy (DN). Pneumoperitoneum is known to induce adverse influence on the normal functions of the heart, lung and kidneys. Clinical and experimental studies in healthy subjects or animal models have shown a decrease in urine flow in laparoscopic surgery as compared with open approach. This study was designed to examine the effects of incremental increases in intra-abdominal pressure (IAP) from 0 to 14 mmHg on and renal function and acute kidney injury (AKI) in rats with DM as compared with healthy animals.
Following IAP of 0 (baseline), rats with DM induced by streptozocin (60 mg/kg, intra-abdominally) one week prior to operation or healthy controls were subject to IAP of 7, 10 or 14 mmHg, over 45 min for each pressure, followed by deflation period of 60 min (recovery). Urine flow rate (V), Na+ excretion (UNaV), glomerular filtration rate (GFR), and renal plasma flow (RPF), were determined throughout the experiment. Urinary levels of NGAL, a novel biomarker of AKI, were determined in both groups.
There were no significant changes in V, UNaV, GFR, and RPF during 7 mmHg insufflation. Reductions in these parameters were observed in control rats during 10 and 14 mmHg. Baseline values of renal hemodynamics were significantly lower in diabetic rats. Diabetic rats subjected to 10 and 14 mmHg exhibited aggravated declines in V, UNaV, GFR, and RPF. Elevation of IAP induced a pressure dependent increase in urinary NGAL in control rats. Similarly, pneumoperitoneum provoked an increase in urinary NGAL in diabetic animals; however, this increase was more profound than that obtained in non-diabetic rats.
Adverse renal function/perfusion of IAP of 10 and 14 mmHg were observed in healthy controls and to a larger extent in diabetic animals. Moreover, diabetic rats are more susceptible to the adverse real effects of elevated IAP as was evident by enhanced urinary excretion of NGAL.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 63–64.
The trauma service at Rambam Health Care Campus is the busiest in Israel, and handles a particularly heavy load of patients with severe brain injuries (SHI). Since 2007, Magen David Adom (MDA) has used helicopters to supplement transport of casualties to Rambam, thereby reducing the proportion of hospital land referrals to Rambam from 62% (prior to 2008) to 40%.
From 2008–2013, 1,112 patients were admitted with SHI (comatose and typical CT findings). We recorded pertinent information and verified mortality data at 6-month of injury with correlation to core+CT-IMPACT death probability (based on international data). Three modes of evacuation from the site of injury were assessed: paramedic staffed ambulances (Direct, DTR), helicopter (HEMS), and via referral from another hospital (TRANS). Hospital care was classified as neurosurgical mass lesion evacuation (EM), other neurosurgical surgery (Not-EM), and no neurosurgery performed. The differences between individual IMPACT mortality risk and actual deaths in each subgroup were assessed for significance.
The following distributions were noted: 71% of all SHI patients underwent neurosurgery. Before 2008, only 6% were evacuated by HEMS as compared to 29% today. The mortality of the three groups of patients was significantly different: 31.4% mortality for the EM group as opposed 13.8% for the Not-EM group, and 17% for patients who did not undergo neurosurgery (one half of whom were designated “Do Not Resuscitate”). The modern SHI-specific mortality predictor was a good benchmark. The most significant reduction in mortality was achieved by HEMS evacuation of patients needing neurosurgery—a 34.8% improvement over the predicted mortality for mass lesions, and a 46.2% improvement for patients needing neurosurgical procedures. This represented a marked improvement as compared to other groups. Other differences such as the evacuation distance, speed of transfer, and resuscitative measures were also examined; these data pointed to a marked benefit of HEMS for casualties in distant locations.
Patient transfer by HEMS facilitated advanced care, as determined by the mortality risk factors, resulting in increased survival—without any other marked improvement in medical treatment. The greatest advantage was noted for patients evacuated by helicopter when comparing risk and actual mortality, particularly in comatose patients in urgent need of neurosurgery. Regional improvement is based on local data that can readily support decisions for continuous improvement, and to achieve results higher than the international benchmark.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 64–65.
Heparanase is implicated in angiogenesis and tumor progression. We have demonstrated previously that heparanase may also affect the hemostatic system in a nonenzymatic manner. It forms a complex and enhances the activity of the blood coagulation initiator tissue factor (TF). Peptides inhibiting heparanase procoagulant activity, generated by us, were recently demonstrated to be effective in heparanase injected and sepsis mice models. In the present work we studied the peptides effect in a tumor model.
B16 mouse melanoma, EMT-6 mouse breast cancer, and MDA-231 human breast cancer were injected subcutaneously (s.c.). Inhibitory peptides 5, 6 and 7 were injected s.c. at a dose of 1–3 mg/kg, every other day, starting 4 days after tumor cell injection. The experiments ended after 3 weeks. In order to evaluate the effect on tumor relapse, the tumor was left to grow to 1–1.5 cm, and then extracted following peptides injections for 3 weeks. Tumor, organs, and plasma were studied.
Peptides 5, 6 and 7 inhibited tumor size and vascularization in a dose-dependent manner reaching a 2/3 reduction, compared to control tumors, using the three tumor cell lines. Additionally, survival advantage (P<0.05) and reduced plasma thrombin-antithrombin complex (P<0.05) were observed in the treatment groups. Peptides completely inhibited tumor relapse after extraction of the primary tumor.
Peptides inhibiting heparanase procoagulant activity significantly inhibited tumor growth, vascularization, and relapse. The effect of peptides in cancer patients should be further investigated.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 65.
Urodynamic studies serve as an objective method for evaluating lower urinary tract function. Urodynamic parameters are the basis for characterization of urinary disorders of both the filling and/or voiding phase. One limitation of urodynamic testing is that the unnatural environment for urination may negatively affect the text results and not accurately represent actual lower urinary tract function. We examined the feasibility of using the International Prostate Symptom Score (IPSS), specifically its sub-categorical questions, as a reliable diagnostic tool for evaluation of voiding disorders caused by bladder outlet obstruction.
Eighty-nine adult patients who underwent urodynamic testing completed the IPSS questionnaire, which included four questions regarding bladder emptying (voiding phase) and three questions regarding bladder filling (filling phase). We compared the objective parameters measured during the urodynamic pressure/flow study (the standard measurement for assessing the degree of outflow obstruction) with the degree of voiding difficulty as scored by the IPSS sub-category questions. In an attempt to find a score that accurately reflected a state of obstruction, we correlated the bladder voiding and filling phase questions to the pressure flow measurements.
Out of 89 patients (mean age 49, 30 men, 59 women), 43 (48.3 %) had a higher score in the voiding phase vs the filling phase (ratio score > 1), suggesting that this cohort of patients had more emptying difficulties than storage ones. In contrast, according to the objective urodynamic pressure/flow study only 10 out of 89 (11.2 %) subjects fell under the category of obstructive voiding disorder. The mean total score for the emptying disorder questions in this sub-group was 12.9 (range 5–20), which was of high statistical significance for obstruction (p=0.017). Seventy-nine (88.7 %) of the subjects were not obstructed according to the pressure/flow study; their mean total scores on questions dealing with emptying phase was 7.8 (range 0–20). Statistical analysis showed that a score of 9.27 or higher for the voiding phase, as expressed in the pressure flow study, indicated an obstructive disorder (p=0.05), while a score of 8 and below characterized patients with voiding difficulties but without an obstruction (p<0.05).
Based on our findings, a mean score above 12.9 for the emptying phase questions clearly indicates outflow obstructive disorder as seen in the pressure/flow study, while a score below 8 is typical of patients with no obstructive voiding. Our study indicates that this simple 7-question questionnaire could be easily used as a clinical tool for assessment of urinary outlet obstruction.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 65–66.
The beneficial short-term effect of low-intensity shockwave treatment for erectile dysfunction has been previously studied, but its long-term efficacy has not been fully evaluated. The objective of this study is to examine if patients that responded well to shockwave treatment maintain their response after two years.
Patients who had responded satisfactorily to shockwave treatment in our department and had a minimal period of two years follow-up were included in the study. For inclusion, their positive initial response was defined according to the improvement in the IIEFEF domain questionnaire (Rosen minimal change clinical improvement), showing a significant improvement in their erectile function at three months post-treatment (second follow-up visit). These men were prospectively observed after 6, 12, 18, and 24 months and their erectile function was evaluated by an interview (face-to-face, telephone, or an e-mail) that included IIEF-EF domain questionnaires and the CGIC (Clinical Global Impression of Change).
Out of 192 treated patients who had surpassed the two-year follow-up, 113 (58.8%) met the inclusion criteria. Their median age was 60 years (27–78); 37 patients (33%) had diabetes mellitus and 36 patients (32%) had cardiovascular disease. Forty-seven patients (42%) had severe ED (IIEF-ED score between 1–10) before starting shockwave therapy. In the entire group, 85%, 66%, 62%, and 52% of the patients maintained their initial success after 6, 12, 18, and 24 months, respectively. The success rate after two years was not significantly different between diabetic and cardiovascular patients, but patients with mild-moderate ED succeeded more than those with severe ED (29% vs. 62%; P<0.01).
The positive effect of shockwave treatment for erectile dysfunction declines gradually over two years in about half of the patients. This downtrend is more profound in severe ED patients. Further research is needed to develop modifications to the treatment protocol that will make the effect of shockwave therapy last longer.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 66–67.
Chronic pain is associated with disability in various daily activities, which is well documented. In contrast, there is paucity of reports regarding the prevalence and characteristics of sexual dysfunction (SD) in these patients.
The aim of the present study was to evaluate the prevalence and characteristics of SD in a cohort of patients suffering from chronic pain.
The study population consisted of patients with chronic pain who agreed to participate in a study on SD. They completed questionnaires assessing demographics, pain-intensity (Visual Analogue Scale=VAS), pain qualities (Short-form McGill Pain questionnaire=SFMPQ), disability (Ostwestry Disability Index= ODI), depression (Beck depression inventory= BDI) and SD, including the Female Sexual Function Index (FSFI) and the International Index of Erectile Function (IIEF) for men. Chi Square and T-Test were used to compare between different sub-groups within the cohort. Spearman’s coefficient test was used to test correlations between pain and SD parameters.
Four-hundred and four out of 709 responding patients (58%) reported pain related SD, 249 (61.6%) were men and 155 (38.4%) women. No differences in age, weight, years of marriage, gender, and education were found between those with and without SD. In contrast, patients with SD exhibited significantly higher scores in the following pain and disability questionnaires [mean±STD, (median)]; VAS: 70±23 (72) vs. 50±27 (47), p<0.001; SF-MPQ: 16.5±10 (15) vs. 12.3±8 (10), p<0.001; ODI: 23.5±8.6 (24) vs. 16.4±8.5 (15), p<0.001. Patients with SD consumed significantly higher doses of pain medications than those without SD. Sexual dysfunction in women (FSFI) was 50.7 (out of 90 points) and in men (IIEF)-15, indicating moderate SD in both genders. On both numerical (0–100) and categorical (0–4=no to extreme SD) self-report scales of SD severity, women scored significantly higher than men (73.8±24.2 vs. 67.8±22.9; p=0.024 and 3.1±0.9 vs. 2.8±0.9; p=0.016 respectively). A significant correlation was found between pain intensity and severity of SD (r=0.349, p<0.001).
A significant number of patients with chronic pain suffer from SD. Pain intensity correlated to SD severity. Although SD in this population is more prevalent in men, its self-reported severity is higher in women. These findings highlight the importance of increased awareness to SD in patients with chronic pain.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 67.
Hemodynamic overload induces hypertrophy in cardiomyocytes that ultimately leads to cardiac decompensation and heart failure. The activation of several genes, termed the ‘fetal genes’ plays a key role in the process. Transcriptional control by enhancers involves the establishment of physical connections with promoters by genome folding. We hypothesize that the expression of the fetal genes, is governed by distal enhancers and aim to identify them. We further hypothesize that a shift in chromatin folding during hypertrophy allows different enhancers to interact with induced fetal genes promoters.
To explore the molecular mechanism of the activation of this coordinated gene program during cardiac hypertrophy we used primary cultures of both quiescence and hypertrophied neonatal rat ventricular myocytes. Hypertrophy is induced by incubating cardiomyocytes under serum-free conditions in the presence of phenylephrine. We used the multiplexed 4C sequencing to detect of long-range interactions of the fetal gene promoters. The main steps of 4C-seq include treating cells with formaldehyde to cross-link protein-DNA interactions, digesting chromatin with a primary restriction enzyme, and religation under dilute conditions. In our 4C-seq application, the known fragment designed to be part of the fetal gene promoter and the unknown fragment is suspected as enhancer. The last step is high-throughput sequencing to determine the frequency of ligation events between genomic regions of interest, and this provided information about the folding of the genome.
We performed multiplexed 4C-seq using the promoters of the ‘fetal genes’ nppb, acta1, myh6, myh7, and cacna1g. We identified multiple in-cis (on the same chromosome as the promoter) and in-trans (on a different chromosome than the promoter) interactions and characterized them. Most of the genome-genome interactions between the ‘fetal gene’ promoters and distal sites were present both during quiescence and during hypertrophy. However about 35%–40% of the interactions differed between the two states. These experiments show that cardiac hypertrophy is consistently associated with changes in both in-cis and in-trans associations.
We identified multiple in-cis and in-trans genome-genome interactions between the ‘fetal gene’ promoters and distal sites and characterized them. We also show for the first time that cardiac hypertrophy is consistently associated with changes in both in-cis and in-trans associations.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 15–16.
Brugada syndrome, an inherited primary electrical heart disease, disposes to sudden cardiac death. Arrhythmic risk stratification in patients with Brugada syndrome remains challenging and new tools are needed. The objective of our work was to investigate whether nonsense mutations in the SCN5A gene, which result in truncated, non-functional protein are associated with a virulent clinical course.
The study population was drawn from the database of Fondazione Salvatore Maugeri, IRCCS, Pavia, Italy. Subjects were included if they had a clinical diagnosis of Brugada syndrome and carried a SCN5A mutation. Family members, silent SCN5A mutation carriers were also included.
The study included 360 patients (mean age 38±17 years, 231 males) with Brugada syndrome and family members who carried 141 different SCN5A mutations. During mean follow–up (7.1±3 years), 59 patients experienced cardiac events (16%), 25 had severe cardiac events (7%), 19 experienced cardiac arrest (5%), and 3 died (0.8%). In multivariate logistic regression analysis, nonsense mutation was a significant risk factor for severe events (95%CI 1.01–8.95, OR=3, p=0.04), cardiac arrest (95%CI 1.08–10.7, OR=3.4, p=0.03), and a risk factor for cardiac arrest as the first manifestation of the disease (95%CI 1.08–12.2, OR=3.6, p=0.03). The increased risk in patients with nonsense mutation was independent of type 1 spontaneous pattern. The Kaplan-Meier curve plotted for first event cardiac arrest free survival revealed a significant difference between patients with and without nonsense mutation (95% CI 0.96–8.6, HR=2.8, p=0.05).
In Brugada syndrome, patients with nonsense mutations were associated with higher risk for severe cardiac events, cardiac arrest, and higher risk to have cardiac arrest as the first manifestation of the disease, as compared to the Brugada syndrome patients without nonsense mutations. Genetic data might be an additional tool for risk stratification in BrS.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 16.
Cardiac hypertrophy is the heart’s maladaptive response to elevated stress on the heart. Initially, the hypertrophic process normalizes wall tension; however, prolonged hypertrophy is associated with arrhythmias and development of heart failure. Despite extensive research, the molecular mechanisms involved in cardiac hypertrophy remain uncertain. The three best characterized Rho GTPases in the realm of cardiac hypertrophy are RhoA, Rac1, and Cdc42. While RhoA and Rac1 exerted
To determine the effects of RhoJ on cardiac myocyte hypertrophy we isolated neonatal rat ventricular myocytes and infected them with adenoviral vectors encoding for RhoJ wildtype, RhoJ dominant negative (T17N mutant), RhoJ constitutive active (Q79L mutant) or lacz (control) and stimulated to cardiac hypertrophy with serum free medium containing phenylephrine. Hypertrophy was determined by qPCR assessment of hypertrophy markers (ACTA1, NPPA, NPPB) and histology.
(
Myocytes were infected with control adenovirus Ad-Lacz or adenoviruses encoding for RhoJ WT, RhoJ-DN, and RhoJ CA. Fold expression levels (normalized to GAPDH) are shown, demonstrating suppression of hypertrophy with RhoJ-WT and CA, and no suppression with RhoJ-DN mutant. Serum free medium PE-untreated cells infected with lacz served as control.
Our results suggest that RhoJ is a cardiac hypertrophy suppressor and acts directly on cardiomyocytes.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 16–17.
Acute Decompensated Heart Failure (ADHF) is the leading cause of hospital admissions in patients age 65 and above. Identifying risk factors associated with increased morbidity and mortality may improve treatment, and clinical outcomes. Recently, B-Natriuretic Peptide (BNP), a protein secreted by the ventricular myocardium in response to an increased mechanical load and wall stretch, is being used as a diagnostic and prognostic marker in heart failure. The decrease in renal perfusion in patients with heart failure is due to the decrease in contraction and cardiac output. As a result, nephrons reduce the glomerular filtration and increase the re-absorption of salt and fluid, leading to a decrease in excretion of urea in the urine, and increased blood urea nitrogen (BUN).
This analytical, observational and retrospective study, collected demographic, laboratory, and echocardiography data from the medical records of 542 ADHF patients who had BNP blood tests, and were hospitalized at Rambam Health Care Campus, regardless of the reason for admission. Seventy-five percent of the patients were over 70 years old. The mortality rate was the highest in the fourth quartile of BUN.
Blood urea nitrogen is an independent predictor of increased morbidity and mortality in patients with decompensated heart failure.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 17–18.
Successful bone tissue engineering relies on early neovascularization in order to supply oxygen and nutrition to the graft. The aim of this study was to enhance neovascularization at bone regeneration sites, by local transplantation of human blood derived endothelial progenitor cells (hEPC).
Human blood derived endothelial progenitor cells were isolated from adults, expanded and characterized. Expanded hEPC (5×105 cells) were mixed βTCP and transplanted in guided bone regeneration (GBR) model that was established by fixing a gold dome to nude rat calvarium (n=12). Domes filled with βTCP alone served as control (n=12). Rats were sacrificed after 3 or 5 months. Histomorphometry was used to analyze blood-vessel density (Bv/mm2) and immuohistochemistry was used to trace the transplanted cells in the newly regenerated bone.
Expanded hEPC presented a polygonal morphology, formed homogenous monolayer cells and replicated rapidly. Two days after seeding on Matrigel, cells spontaneously formed capillary-like structures. The FACS analysis demonstrated that >95% of the cells were positive for CD31 (endothelial marker), but negative to CD14, ruling out their monocytic origin. Local transplantation of hEPC significantly increased vascularization and stimulated bone formation. Three months post transplantation, blood vessel density was elevated by ∼700% in the hEPC transplanted group, as compared to the control group (1.08±0.29 vs. 7.52±1.2; Bv/mm2; p≤0.0001; control vs. hEPC, respectively). At 5 months, blood vessel density was still significantly higher in the hEPC transplanted group (1.06±0.19 vs. 9.52±0.69; Bv/mm2; p≤0.0001; control vs. hEPC, respectively). Transplanted hEPC were localized in the newly formed bone using immuno-staining with human-specific CD31 antibodies that do not react with rat CD31. Transplanted cells were found lining blood vessel walls in the newly regenerated tissue.
Transplanted hEPC increased neovascularization at bone regeneration sites. The presence of hEPC in vessels walls implies on their direct role in vasculogenesis.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 18.
Busulfan (BU) is commonly used in conditioning regimens for hematopoietic stem cell transplantation. As a narrow-therapeutic-index drug with highly variable pharmacokinetics (PK), its therapeutic drug monitoring (TDM) is recommended. The area under the BU plasma concentration (Cpl)-time curve (AUC) serves as a measure of exposure, and sub- and supra-therapeutic AUCs are associated with graft failure and toxicity, respectively. A relatively expensive and burdensome intense sampling strategy (ISS) of 9–10 blood samples is traditionally used (including by us). Limited sampling strategies (LSS) of 4–5 samples have been reported but have not been tested in Israeli patients. Our aim was to test the validity of LSS in this patient population.
Our database containing PK BU data (analysis of ∼1800 samples collected by ISS, ∼200 intravenously-dosed patients) was used to calculate several PK parameters for each patient: AUCISS was calculated by the trapezoidal rule (TR) using Cpl measured separately in all 9 ISS samples. AUCcombo was estimated by analyzing one single sample composed of aliquots from these ISS samples (aliquot volumes defined by an algorithm previously developed in our laboratory). LSS parameters: AUC5, AUC4 and AUC3 were estimated by TR and multiple linear regressions (MLR) using Cpl of 5, 4 or 3 blood samples. These were used to estimate statistics such as percentage of individual parameters deviating from AUCISS by > 10% (PERDEV) and AUCISS/parameter ± SD.
The results are shown in
Study Results.
| PK Parameter | |||||
|---|---|---|---|---|---|
| Method | Statistic | AUCcombo | AUC3 | AUC4 | AUC5 |
| TR | PERDEV | 14 | 17 | 17 | 5 |
| Mean (AUCISS/Parameter) ± SD | 1.00±0.12 | 1.03±0.13 | 1.01±0.10 | 1.00±0.06 | |
| MLR | PERDEV | - | 17 | 5 | 6 |
| Mean (AUCISS/Parameter)± SD | - | 1.02±0.07 | 0.99±0.05 | 1.00±0.06 | |
Deviation by >10% of AUC5 from AUCISS in only 5–6% of patients in this preliminary analysis suggests that a refined and reliable LSS (an ongoing project) for Israeli patients is feasible. Characterization of patients suitable for LSS will contribute to BU TDM personalization. Limited sampling strategies will reduce patient discomfort, clinical staff workload, and TDM costs. These will allow repeated TDM after dose adjustments, further improving personalized treatment. Improvement of the “combo” method may offer a rapid laboratory response for dose adjustments.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 19.
Liquid chromatography with mass spectrometry (LC-MS/MS) is the method of choice for the determination of everolimus whole blood concentrations, but is not routinely available. Therefore, immunoassays have been developed for clinical monitoring of everolimus. In previous studies, the QMS immunoassay had a positive bias compared to LC-MS/MS, but was judged acceptable, although clinical agreement (e.g. 95% limits of agreement) was not reported. The objective of this study was to assess whether the agreement between the QMS assay and the reference LC-MS/MS method was clinically acceptable for using them interchangeably in therapeutic everolimus monitoring.
Whole blood samples from organ transplanted patients on everolimus therapy were analyzed by both QMS (on Architect ci4100 analyzer) and LC-MS/MS. Paired results were compared using paired Student’s t-test, Bland-Altman plots, and Deming regression analysis. The proportion of falsely supra- and sub-therapeutic results on the QMS assay compared to the reference LC-MS/MS were calculated.
Among 250 samples (169 patients), mean everolimus concentrations determined by LC-MS/MS and QMS assays were 4.8±2.1 and 6.3±2.1 ng/mL, respectively (p<0.001), with 95% lines of agreement between −2.1 to 5.2 ng/mL, a range corresponding to 152% of the mean concentration. When stratified by type of transplant, a similar positive bias was found in each subgroup (all p < 0.014). 69% of the samples yielding supra-therapeutic concentrations on the QMS assay were within the therapeutic range on the LC-MS/MS.
The everolimus QMS immunoassay, using the Architect ci4100 analyzer, had a significant positive bias compared to LC-MS/MS, with a wide range between the limits of agreement. The lack of agreement may result in inadequate everolimus dose adjustments, suggesting that the QMS assay cannot be used interchangeably with the LC-MS/MS method for therapeutic everolimus monitoring in organ transplanted patients.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 19–20.
We performed a retrospective assessment of the tooth survival rate and its association with patient and oral variables in patients who were followed for up to 18 years.
A private periodontal office provided anonymous data for their patients. Patient data were divided into three groups: baseline (T0), reevaluation after cause-related therapy (TRe), and reevaluation at the last recorded visit 8 to 18 years later (TF). Inclusion criteria were receipt of periodontal therapy and supportive periodontal therapy (SPT). General health, plaque scores (PI), probing depth (PD), and bleeding on probing (BOP) at six points/tooth, extractions, and the number of SPT visits were also obtained at T0, TRe, and TF. For each patient, mean PI, PD, and BOP indices, and SPT/year were calculated at different time points. Teeth were classified as multi-rooted for molars and single-rooted for others. Descriptive statistics and Cox regression analysis were performed for assessing the putative factors affecting tooth survival.
The inclusion criteria were met by 50 patients. There were 1301 teeth between all patients (mean 26±4 teeth/patient). There were 20 extractions before TRe and 129 after TRe. Of these, 96 were extracted for periodontal reasons. The annual extraction rate per patient was 0.16. The Proportional Hazards Model indicated that PD>7mm at TRe (HR=17.7, 95%CI 8.6, 36.6), over 60 years of age (HR=3.3, 95%CI 1.5, 7.2), multi-rooted teeth (HR= 1.9, 95%CI 1.2, 3.1), and SPT<3 times per year (HR=1.8, 95%CI 1.1, 2.9), correlated with increased tooth loss during follow-up (P<0.05, Cox regression analysis). A continuous, statistically significant reduction was observed in mean PD among teeth that survived the follow-up period (4.3±1.8, 3.5±1.4, 3.2±1.3, at T0, TRe, and TF respectively; p<0.001, repeated measures test).
Active periodontal treatment followed by regular SPT results in relatively low tooth loss rates and a continuous reduction in probing depth. Probing depth after the initial phase of therapy, over 60 years old at admittance, multi-rooted tooth type, and infrequent SPT were found to be strong predictors for long-term tooth survival among periodontally maintained patients.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 20–21.
Dyslipidemia has been associated with an increased risk for developing cancer. To explore the role of dyslipidemia in breast cancer growth and metastasis, we used the apolipo-protein E (ApoE) knockout mice (ApoE −/−), which exhibit marked dyslipidemia, with elevated circulating cholesterol and triglyceride levels in the setting of normal glucose homeostasis and insulin sensitivity. We previously showed that Mvt-1 mouse mammary cancer cells developed larger tumors and more metastasis in ApoE −/− compared to WT mice. Here we wanted to extend our research with the human mammary cancer cell line MDA-MB-231.
ApoE −/− mice were crossed with immunodeficient Rag1 −/− mice. The control Rag1 −/− and Rag/ApoE −/− mice were fed a high fat diet (HFD) for 10 weeks after which the human mammary cancer cell line MDA-MB-231 were injected to the fourth mammary fat pad. Mice were followed for their weight gain and tumor volume.
Rag/ApoE −/− mice gained more weight when fed a HFD compared with Rag/WT mice, ensuring these mice retained their ApoE −/− phenotype. Rag/ApoE −/− mice injected with these cells into the fourth mammary fat pad developed larger tumors compared to Rag/WT mice when fed a HFD.
These results suggest that hypercholesterolemia enhances the growth of human breast cancer and may explain the association seen in patients and their responsiveness to statin therapy.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 21.
Several recent meta-analyses including over 10 RCT concluded that GnRHa co-treatment along chemotherapy significantly decreased premature ovarian failure (POF) rate. However, cyclic ovarian function is not equivalent to fertility (pregnancies). Therefore, we evaluated the pregnancy rate (PR) after exposure to gonadotoxic chemotherapy+GnRHa vs controls.
GnRH-a was administered to 281 patients in parallel with gonadotoxic chemotherapy, and compared to 173 patients of a comparative age, who were similarly treated, but without GnRH-a (control group). Patients who had not visited our clinic for more than a year were interviewed to verify the PR and outcome. The study was approved by the Institutional Review Board (Helsinki) Committee.
Less than 12% of the GnRHa group developed POF, vs 50% in controls (P<0.05). The remaining resumed cyclic ovarian function; 81 patients conceived 161 times and were delivered of 119 healthy neonates. In the control group 30 patients spontaneously conceived 49 times (P<0.05). In the GnRHa group, 88% resumed cyclic ovarian function (COF) vs only 50% of the controls; the rest suffered POF (P=0.003). Of the survivors in the GnRHa group 62% conceived, vs 42% of the controls (P=0.033). Spontaneous pregnancies occurred in 57.7% of the survivors in the GnRHa group (up to 6 pregnancies/patient), vs 34.9% of the controls (P=0.009). The age at chemotherapy for those who spontaneously conceived was 14–38 years in the GnRHa group, vs. 14–28 years in the control group, suggesting a possible prolongation of the “Fertile window” by 10 years. One patient in the GnRHa group spontaneously conceived three times and was delivered of three healthy neonates, despite two stem cell transplantations (SCT) 11 years apart. GnRH-a cotreatment significantly decreased POF rate, not only in conventional chemotherapy, but also in lymphoma patients undergoing aggressive conditioning before SCT.
GnRHa co-treatment in parallel to chemotherapy is beneficial in minimizing POF rate and increasing pregnancy rate. Therefore, young women facing gonadotoxic chemotherapy should be offered the options of fertility preservation by GnRHa in addition to IVF and cryopreservation of embrya, ova, and ovarian tissue.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 21–22.
Acromegaly, excess growth hormone (GH) production secondary to a pituitary adenoma, has important metabolic effects. The two most significant effects of GH on metabolism in adipose tissue are insulin resistance and lipolysis. The objective of this study was to determine the effect of chronic excess growth hormone in acromegaly on gene expression in adipose tissue in humans.
To study the effect of chronic excess growth hormone on adipose tissue we performed RNA sequencing in adipose tissue biopsies from patients with acromegaly (n=9) or non-functioning pituitary adenomas (n=11). The patients underwent preoperative clinical and metabolic profiling including assessment of HOMA-IR. Explants of adipose tissue were assayed
Patients with acromegaly had higher glucose, higher insulin levels and higher HOMAIR score. There was a trend for enhanced
We have identified the acromegaly gene expression signature in human adipose tissue. The significance of altered expression of specific transcripts will enhance our understanding of the metabolic and proliferative changes associated with acromegaly.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 22.
In clinical trials, treatment with Zoledronic acid (ZOL) was reported to induce acute phase reaction in about 15% of patients after the first infusion. This study was aimed at evaluating the incidence of adverse events (AE) when using ZOL in the regular clinical setting.
The study included 215 consecutive female patients with an average age of 69.7±9.59, (45–100); 151 (70%) had been previously treated with Alendronate 65 pts (43%); Raloxifene 9 (5.96%); Risedronate −31 (20.5%); Teriparatide 23 (15.2%); Livial-1 (0.7%); calcium supplements133 (61.9%); Vitamin D 147 (68%) All patients received IV ZOL 5 mg; of these, 52 received a second infusion of ZOL. Patients were provided with a detailed form to complete and were asked to report adverse events over the ten days following ZOL infusion.
Adverse events were reported in 136 (63.3%) of the patients after the first ZOL dose, and in 32 (61.5%) after the second dose. The AEs experienced were: fever 69 (50.7%) and 10 (31.3%), p=0.024; bone/joint pain/headache 129 (94.8%) and 0, p<0.0001; weakness 109 (80.1%) 20 (62.5%), p=0.05; muscles pain 106 (77.9%) and 10 (31.3%), p=0.04; and uveitis 2 (1.47%) and 0, after the first and second doses of ZOL, respectively. The duration of AEs was 2–5 days, with no correlation to prior treatments. It is worth noting that previous factures were reported in 114 (53%) patients. Of these, there were 5 (2.3%) new fractures: 3 to the hip; 2 of the ramus pubis.
The incidence of acute phase reaction in this study was higher than previously reported, with a significant decrease after the second dose of ZOL.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 23.
Twelve years ago we evaluated 29 liver transplant patients. Nineteen (65.5%) had decreased bone mass, 11 (37.8%) were osteoporotic, 28 (96.5%) had 25-hydroxyvitamin D (25-OHD) serum levels <20 ng/ml, mean 12.52±3.19, mean parathyroid hormone (PTH) 59.67±29.78 pg/ml. None of the patients were treated with calcium or vitamin D supplements, in spite of their low calcium intake and low or suboptimal 25-OHD serum level. All patients received letters containing their diagnoses and treatment recommendations.
This study aimed at evaluating the metabolic status of the same patients 12 years later.
The patient’s charts were re-evaluated. Nineteen patients remained under follow-up at the Liver Unit: aged 58.8±15.8, 9 men and 10 postmenopausal women. Ten patients had died, none of them had fractures. Twelve fractures were reported in the surviving patients following transplantation, 10 (83.3%) within the first year, 2 in the following years, both were traumatic.
Repeat bone mineral density (BMD) results were available for 8 (42%) patients: 2 (25%) were osteoporotic and 6 (75%) were osteopenic. Thirteen patients (68.4%) received 600 mg of elemental calcium, 400–1600 IU of vitamin D daily, 2 (10.5%) received bisphosphonates.
Plasma PTH ranged from 32–71 pg/ml, mean 61.9±26.42; 25-OHD range was 14–22, mean 24.72±9.49 ng/ml; p=0.068. These results represented an increase from their first assessment 12 years ago: Plasma PTH 59.67±29.78 pg/ml; 25-OHD 12.52±3.19 ng/ml; p=0.003.
Since the first evaluation, 20 new post-liver transplant patients were admitted to the Rambam Liver Unit. They were not evaluated in the Bone Metabolism Unit. According to the information retrieved from their charts, 7 underwent BMD evaluation. Of these, one woman was in the osteoporotic range and 6 were osteopenic, with PTH of 82.83±67.01 pg/ml and 25-OHD of 23.95±8.7 ng/ml.
Nine (45%) of the 20 are currently being treated with calcium 600 mg/day and vitamin D, 400–2000 IU/day, and two are receiving oral bisphosphonates. No fractures have been reported by these patients since the time of transplantation.
Overall, the PTH was 71.82±49.64 pg/ml and the 25-OHD was 25.11±9.31ng/ml for the entire group of patients (39).
Osteoporotic fractures in liver transplant patient occurred during the first post-transplantation year. No osteoporotic fractures were observed during subsequent long-term follow-up. Vitamin D deficiency and low calcium intake were actively treated and secondary hyperparathyroidism was not observed during long term follow-up. A BMD follow-up was not routinely performed in all patients. No significant change in BMD was observed during long-term follow-up in the tested group.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 23–24.
The risk of additional osteoporotic fractures in patients who have undergone fragility fractures is at least six times higher than in age and gender-matched adults. The study purpose was to define the anti-osteoporotic treatment status in these high risk patients. Treatment strategies should be directed to provide intensive fracture prevention treatment to patients with osteoporotic fractures
A collaborative (orthopedics and bone metabolism) fracture prevention program for patients hospitalized with osteoporotic fractures was started at Rambam Health Care Campus in 2008. All patients hospitalized with fractures were offered fracture-prevention treatment after surgical fracture repair. The study population included 1,647 patients who enrolled in the program between 2008 and 2013: median age 78 (range 23–103); 1,165 (71%) women with a median age of 78 (range 33–100) and 482 (29%) men with a median age of 77 (range 23–103).
Proximal femoral fractures were sustained by 1,115 (68%) of the patients: 769 (66%) women. 346 (72%) men. During the study period 256 (16%) patients died: 152 (13%) women and 104 (22%) men; hip fracture occurred in 212 patients: 123 (16%) women and 89 (26%) men. The relative risk (RR) for death in men following a hip fracture was 1.61 (95% CI 1.26–2.05) as compared to women. The major predictors of death in women were greater age, number of medications, higher creatinine level and lower albumin; in men, the risk factors were greater age, number of medications, and higher creatinine level. Prior to admission 264 (23%) women and 10 (2%) men were treated for osteoporosis, and hip fractures had occurred in 171 (22%) women and 6 (2%) men. After discharge 404 (35%) women and 42 (9%) men received anti-osteoporosis treatment; 247 (32%) of women and 31 (9%) of men received treatment for subsequent hip fractures. While no factors were related to an increased likelihood of men being treated, women were more likely to be treated if they had any previous fractures, previous anti-osteoporosis treatment, higher vitamin D or albumin, and lower creatinine. Treatment distribution for all patients was: 319 (19.3%) oral bisphosphonates; 73 (4.45%) IV bisphosphonates (Zoledronate); 2 (0.12%) pamidronate); 47 (2.87%) teriparatide; and 5 (0.31%) raloxifen.
We conclude that in spite of a marked increase in the rate of patients treated after an index fracture, in a hospital initiated collaborative treatment program, this treatment rate is still unsatisfactory. In Israel, the use of IV bisphosphonates as the first line of therapy after a hip fracture remains quite low, even though it is an officially recognized and available treatment.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 24–25.
25-25
About 60% of infliximab (IFX) treated inflammatory bowel disease (IBD) patients develop anti-infliximab antibodies (ATI) with a sizable population suffering from loss of response (LOR). However, these antibodies are heterogeneous, some of which have no therapeutic consequences. Defining their neutralizing potential has marked therapeutic significance.
A biological assay in which TNF-α induced IL-8 secretion from HT-29 cells followed by application of IFX and control pooled IBD sera, or ATI-containing sera was developed and IL-8 amplification ratio was determined relative to controls. An immune test, in which the IFX concentration was determined before and after its incubation with sera compared to control, was also developed.
(
LOR, loss of response.
Two assays for detection of neutralizing ATI were developed and compared. No advantage was found for the more complex bioassay. A predictive potential of the test was identified allowing identification of patients prone to develop immunogenic LOR. These findings may assist in optimizing infliximab therapy in IBD patients.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 25.
Despite advances in understanding the etiopathogenesis of acute pancreatitis (AP), the mechanisms underlying this acute inflammatory disease have not been fully determined. In the majority of cases AP a self-limiting process, yet 20% of patients develop a severe form of necrosis with multi-organ complications and a high mortality rate. Heparanase (Hpa), an endoglycosidase which cleaves heparan sulfate, participates in degradation and remodeling of the extracellular matrix. Heparanase is preferentially expressed in human tumors, including pancreatic adenocarcinoma. Besides its role in cancer, Hpa plays an important role in the pathogenesis of several inflammatory disorders, such as lung injury, rheumatoid arthritis, and chronic colitis. The involvement of Hpa in AP pathogenesis has not yet been addressed. Therefore, the current study uses an experimental model to examine whether Hpa is involved in the pathogenesis of cerulein-induced AP in mice.
Heparanase over-expressing transgenic mice (
Cerulein-induced pancreatitis in wild type mice was associated with significant rises in the serum levels of amylase and lipase. These increases were characterized by enhancement of Hpa activity and pancreatic inflammation. The elevation in amylase and lipase as well as pancreatic edema/inflammation responses to administration of cerulein were profoundly exaggerated in
The
Citation: Rambam Maimonides Med J 2015;1 Suppl: 26.
Aging of the population is associated with a marked rise in the number of older people with cognitive impairment. Pharmacological interventions for cognitive decline have limited efficacy and are, at best, symptomatic. Psychosocial and lifestyle interventions, which aim primarily to relieve cognitive and behavioral symptoms, also promote well-being and provide caregiver support. Increasingly, computerized systems are being designed for the treatment of patients with dementia, including web sites for providing information, computerized tools for monitoring, assistive technology and computer systems offering emotion-oriented treatments in dementia care. We evaluated two computer-supported interventions for patients with cognitive impairment, namely personalized computerized reminiscence therapy and computerized cognitive training.
A total of 167 older subjects with cognitive impairment or dementia, residing in the community, were recruited for the study. All underwent a preliminary assessment to determine their cognitive status. Following initial screening 95 suitable subjects were randomly assigned to one of the following three groups: (1) Personal computer-based reminiscence therapy; (2) Computer-based cognitive training; and (3) Control group. All subjects participated in two sessions a week, each of 30-minutes duration, supervised by a mediator, for a period of 3 months, and controls continued with usual activities. Assessments were performed at baseline, at one month, and at 3 months. They included an evaluation of cognitive function using the Neurotrax computerized testing battery, psychological and behavioral well-being using Quality of Life in Alzheimer’s Disease (QoL-AD), Will to Live (WTL) and Neuropsychiatric Inventory (NPI) questionnaires, and caregiver burden using the short version of Zarit Caregiver Burden Interview.
A total of 85 participants completed at least one follow-up evaluation. No differences between groups were found at baseline. For the reminiscence group, beneficial effects were found for the global cognitive score, QoL-AD-patient, and Will To Live. For the cognitive training group modest effects were found for orientation, verbal function, and Go-No-Go reaction time.
This study found positive results for both computer-based reminiscence and cognitive training in older subjects with cognitive impairment. Due to the limited sample size the effects demonstrated were not adequate to draw firm conclusions. We suggest conducting additional randomized controlled trials with greater sample sizes and longer evaluation periods.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 26–27.
Selected for Oral Presentation
Microparticles (MPs) are membrane vesicles shed from various cells and may express antigens that reflect their cellular origin. Tissue factor (TF)-bearing MPs plays a major role in the pathogenesis of the prothrombotic state observed among patients with malignant disease. Acute myeloid leukemia (AML) is characterized by rapid growth of abnormal blast cells. Patients with AML can develop venous thromboembolism despite thrombocytopenia. We hypothesize that circulating MPs may serve as biomarker, reflect the change in blood cell population, and predict the thrombogenic state of AML patients at diagnosis and at remission.
Blood samples were collected from healthy controls and from patients with newly diagnosed AML at three time points: diagnosis, at nadir, and at remission achievement. The cell origin of MPs was characterized by specific fluorescent antibodies and analyzed by FACS. The MPs were labeled for various antigens including CD34, CD33 –blast leukemic cells markers. To determine the procoagulant and anticoagulant potential, each sample was labeled with florescent antibodies against TF and tissue factor pathway inhibitor (TFPI). The procoagulant activity of MPs was also evaluated by FXa chromogenic assay.
Forty-two patients with AML were enrolled in the study. Platelet markers were significantly higher in controls compared to rates at diagnosis of AML (30.7% vs. 10.3%; p<0.05), with no change in the markers for activated platelets. CD34 as a blast cell marker was significantly higher in patients at diagnosis as compared to controls (4.4% vs. 1.4%; p<0.001) and to patient MPs at remission (4.4% vs. 1.7%; p<0.05). CD33 as another blast cell marker was also found to be significantly higher in patients at diagnosis as compared to controls (34.9% vs. 15.3%; p<0.05) and to remission (34.9% vs. 15.7%; p<0.01). Procoagulant activity was significantly higher in patients at diagnosis and at remission compared to healthy controls (100.5 AU vs. 58.5 AU; p<0.05, and 135 AU vs. 58.5 AU; p<0.001, respectively).
The MPs of AML patients at diagnosis express blast cell markers and may serve as a biomarker for disease and for remission. Increase of endothelial MPs at AML diagnosis may point to vascular injury, which may also result in an increase of TF and a reduction of TFPI, with an overall increase in patient thrombogenicity. The AML-MPs reflect a patient’s disease and my serve as a “red flag” for a hypercoagulable state.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 27–28.
Acute Myeloid leukemia (AML) is associated with poor outcome mainly due to relapse. There is substantial evidence that AML, like other cancers, is a heterogeneous disease composed of different subclones. Intraclonal genetic diversity reflects natural selection which may lead to clonal evolution, disease progression, relapse, or metastasis. Delineation of the tumoral genomic landscape is mandatory for demonstrating the presence of heterogeneous leukemic populations. We hypothesized that AML heterogeneity may contribute to relapse through several possible mechanisms such as: clonal evolution, presence of dormant cells escaping treatment and/or abnormal interaction with the micro-environment (ME) supporting leukemic subclones expansion.
Most studies use bulk DNA for genotyping the tumor; however, this approach can miss rare subclones which could eventually lead to relapse. Therefore, in order to investigate the role of different subclones in disease progression, we studied AML associated mutations using single cell analysis (SCA).
Using this unique approach, we were able to show that patients, considered negative for the FLT3-ITD AML mutation, when tested by bulk DNA, actually possessed a small fraction of this mutation. The presence of a minor clone carrying FLT3-ITD strongly suggests that this lesion is a common late event in leukemogenesis. Additionally, further clonal evolution was shown in the FLT3 gene by the accumulation of different mutations in the same patient and loss of heterozygosity of wild type allele in 25–100% of tested cases.
Combining single leukemic cell genetic analysis at diagnosis and relapse with clonal hierarchy as determined by the mutation rate in 200 micro-satellites of single cells, we were able to identify the presence of dormant cells that may evade chemotherapy due to their dormancy, as well as the appearance of mutations in mismatch repair genes contributing to relapse.
Subclones derived from single cells were subjected to various combinations of drugs and differential chemosensitivity was observed, further supporting the significance of tumor heterogeneity.
This comprehensive study suggests that relapse mechanisms interplay between intrinsic genetic properties of various clones, together with micro-environmental alterations supporting tumor expansion. Understanding mechanisms of disease relapse may promote targeted therapy and also serve as a model for investigating progression of other cancers.
Citation: Rambam Maimonides Med J 2015;1 Suppl: 28.
President / Président :
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Treasurer / Trésorière :
Secretary / Secrétaire :
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Track A: Basic Sciences / Volet A : Sciences fondamentales :
Track B: Clinical Sciences / Volet B : Sciences cliniques :
Track C: Epidemiology and Public Health Sciences / Volet C : Épidémiologie et sciences de la santé publique :
Track D: Social Sciences / Volet D : Sciences sociales :
Community Representative / Représentant communautaire :
Dr Trevor Hart, Dr Darrell Tan
Track A: Basic Sciences / Volet A : Sciences fondamentales
Dr Richard Harrigan, Dr Charu Kaushic
Track B: Clinical Sciences / Volet B : Sciences cliniques
Dr Jason Brophy, Dr Cécile Tremblay
Track C: Epidemiology and Public Health Sciences / Volet C : Épidémiologie et sciences de la santé publique
Dr Greta Bauer, Dr Sharmistha Mishra
Track D: Social Sciences / Volet D : Sciences sociales
Dr Martin Blais, Dr Daniel Grace
Community Representative / Représentant communautaire
Wangari Tharao
Family Physician / Médecin de famille
Dr Gordon Arbess
Conference Advisory Members / Membres du comité consultatif du congrès
Dr Robert Hogg, Andrew Matejcic
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Welcome to the 24th Annual Canadian Conference on HIV/AIDS Research (CAHR 2015).
Dr Robert Hogg
The Canadian Association for HIV Research (CAHR) is proud to be part of the community of researchers and community groups working tirelessly in the global fight against HIV. With a membership of more than 2,000 researchers and others interested in HIV research, CAHR is the leading organization of HIV/AIDS researchers in Canada. The annual CAHR conference is the premier gathering in Canada for those working in the field of HIV, as well as policy makers, persons living with HIV and other individuals committed to ending the pandemic. It is a chance to assess where we are, evaluate recent scientific developments, and together chart a course forward.
I congratulate the members of the 2015 Scientific Program Committee for developing such a strong and thematic programme that will present new scientific knowledge and offer many opportunities for structured dialogue on the major issues facing the global response to HIV. A variety of sessions such as abstract-driven presentations, symposia, and plenary sessions will meet the needs of various participants. Other related activities, including ancillary meetings and training workshops, will contribute to an exceptional opportunity for professional development and networking.
CAHR 2015 will be a tremendous opportunity for researchers and community members from coast to coast to share the latest scientific advances in the field, learn from one another’s expertise, and develop new ways to treat and prevent HIV. I hope you enjoy the conference, find it to be a worthwhile learning experience, and thank you in advance for your contributions, participation and continued support.
Bienvenue au 24e Congrès annuel canadien de recherche sur le VIH/sida (congrès de l’ACRV 2015).
L’Association canadienne de recherche sur le VIH (ACRV) est fière de travailler avec le milieu des chercheurs et les groupes communautaires qui œuvrent sans relâche à la lutte mondiale contre le VIH. Comptant plus de 2 000 chercheurs et d’autres personnes s’intéressant à la recherche sur le VIH –, l’ACRV un organisme canadien de premier plan se consacrant à la recherche sur le VIH/sida. Le congrès annuel de l’ACRV est l’événement le plus important au pays s’adressant aux personnes travaillant dans le domaine du VIH, aux décideurs, aux personnes vivant avec le VIH et aux autres personnes déterminées à enrayer la pandémie. Il offre l’occasion de faire le point sur la situation, d’évaluer les récentes percées scientifiques et d’établir conjointement un plan d’action.
Je tiens à féliciter les membres du comité du programme scientifique de 2015, qui ont su créer un riche programme thématique présentant les nouvelles connaissances scientifiques et offrant de nombreuses occasions de dialogue structuré sur les principales difficultés rencontrées dans le cadre de la lutte mondiale contre le VIH. Un large éventail de séances, dont des présentations d’abrégés, des colloques et des séances plénières, est prévu afin de répondre aux différents besoins des participants. Des activités connexes, notamment des réunions auxiliaires et des ateliers de formation, s’avéreront d’excellentes occasions de perfectionnement professionnel et de réseautage.
Le congrès de l’ACRV 2015 permettra aux chercheurs et aux membres des collectivités d’un océan à l’autre d’échanger sur les derniers développements scientifiques dans le domaine du VIH, d’enrichir leurs connaissances au contact des autres participants et d’établir de nouvelles stratégies de traitement et de prévention du VIH. Nous espérons que le congrès vous sera agréable et qu’il s’avérera pour vous une expérience d’apprentissage utile. Nous vous remercions d’avance de votre contribution, de votre participation et de votre appuiconstant.
We are delighted to welcome you to Toronto for the 24th Annual Canadian Conference on HIV/AIDS Research. In recent years, there has been an increasing focus on work across disciplines to understand and fight the HIV epidemic. Added to this are recent data showing the benefits of a combination approach to HIV prevention, analogous to the continuing successes of combination antiretroviral therapy. At CAHR 2015, we will showcase the benefits of combining treatment, prevention, and research across methodologies, research tracks, and disciplines to combat the HIV epidemic. This combined approach can help us to improve our successes in reaching all those living with HIV, and the diverse marginalized populations across Canada and beyond who are at higher risk for HIV.
Dr Trevor Hart
Dr Darrell Tan
This year’s program was designed from over 400 abstracts covering diverse disciplines and topics within HIV research. We also have a large number of Special Sessions and Ancillary Events this year. These sessions will discuss HIV vaccine collaborations and outcomes; Hepatitis C and co-infections among women, trans people, and girls; sociocultural and ethical issues in Treatment as Prevention; program science; drug policy; HIV and its transmission in criminal law; needle and exchange programs in Canadian prisons; Casey House, an HIV/AIDS care facility in Toronto; HIV and immigration; pre-exposure prophylaxis and more. We are also pleased to introduce a special abstract session on community practices in HIV prevention and care, highlighting the important research contributions of community members and organizations in the response to the epidemic.
Nous sommes heureux de vous accueillir à Toronto à l’occasion du 24e Congrès annuel canadien de recherche sur le VIH/sida. Nous avons constaté ces dernières années une concertation accrue interdisciplinaire afin de comprendre et de contrer l’épidémie de VIH. De plus, les données récentes mettent en évidence les avantages des approches combinées à la prévention du VIH, ce qui nous rappelle les succès non démentis des thérapies antirétrovirales combinées. L’ACRV 2015 fera ressortir les avantages des traitements associés, de la prévention et de la recherche entre méthodologies, des volets de la recherche et des disciplines où l’on lutte contre l’épidémie de VIH. Cette approche combinée peut nous aider à relever le taux de succès en établissant le contact avec toutes les personnes vivant avec le VIH et les diverses populations marginalisées au Canada et ailleurs, qui courent plus de risque de contracter le VIH.
Le programme de cette année s’articule autour de plus de 400 abrégés portant sur nombre de thèmes et de disciplines de la recherche sur le VIH. Vous y trouverez aussi nombre de séances spéciales et d’événements connexes, portant par exemple sur les initiatives de collaboration dans la recherche d’un vaccin contre le VIH et les résultats; l’hépatite C et les co-infections chez les femmes, les personnes trans et les filles; questions déontologiques et socioculturelles dans le traitement à titre préventif; science des programmes; politique sur les drogues; le VIH et sa transmission en droit pénal; programmes d’échange de seringues dans les prisons canadiennes; Casey House, établissement de soins de santé spécialisé sur le VIH/sida à Toronto; le IH et l’immigration; prophylaxie pré-exposition et plus encore. Nous avons aussi le plaisir d’annoncer qu’il y ayra une séance spéciale d’abrégés sur les pratiques communautaires en prévention et traitement du VIH, faisant ressortir l’apport important, en matière de recherche, des membres et organismes de la collectivité en réponse à l’épidémie.
The C-type lectin CD161 is expressed by CD4+ and CD8+ T cells sharing conserved transcriptional and functional signature. CD4+ T cells with type-17 profiles derived from CD161+ precursors and CD161-expressing CD8+ T cells share the same differentiation profile and include the unique anti-bacterial CD161++ mucosal-associated invariant T cells (MAIT) expressing invariant TCR Vα7.2. During HIV infection, circulating cells harbouring the signature of CD161+ cells are impaired in blood and mucosal compartments. In the female genital tract (FGT), portal of entry for HIV infection, the pattern of CD161 expression on CD4+ and CD8+ subsets and how HIV infection impacts these compartments remain unknown. Here, we explore these questions by characterizing CD161-expressing cells in the FGT of chronically HIV-infected (n=16), HIV-negative newly practising sex work (n=36) and highly HIV-exposed seronegative (HESN) (n=33) female sex workers (FSW) from Nairobi, Kenya.
When compared to blood, CD161+CD4+ T cells were enriched in the FGT of HIV-negatives. CD161+CD8+ T cells were also enriched in the FGT of new FSW but not in HESN. Circulating and cervical CD161-expressing cells harboured a more activated profile with tissue-homing properties (CD69, CCR5 and β7) on both CD4+ and CD8+ subsets. The CD161++ subsets included MAIT. Stimulated CD161++ and CD161+CD8+ cells expressed IL-22 at higher frequency than CD161- cells. Expression of IL-17A and IL-22 was enriched in FGT, but independently of CD161 expression in cervical CD4+ cells. CD161++CD8+ T cells were depleted in HIV-positives compared to new FSW in both blood and cervix, but depletion of CD161+CD8+ subset was only observed in cervix.
Impairment of the IL-17A/IL-22-enriched CD161+ +CD8+ and CD161+ subsets was observed for the first time in the FGT of HIV-infected FSW. Also, the absence of cervical enrichment of CD161+CD8+ cells in HESN agrees with a protective reduction of cervical inflammation. In contrast to blood, CD161 expression did not efficiently describe type-17 CD4+ T cells in the FGT. The FGT unique environment may importantly impact T cells plasticity promoting Th17 differentiation independently of CD161 expression.
HIV persistence in anatomical reservoirs is a major hurdle in HIV eradication. Testicular tissue represents one of the main viral anatomical reservoirs as it constitutes an immunological privileged tissue. Here, we assessed T-cell distribution in testicular tissue versus blood in ART-treated individuals.
Testicular tissue and blood samples were collected from ART-treated and virally suppressed HIV-infected (n=6, plasma viral load <50 copies/mL for at least 6 months prior to surgery) and uninfected men (n=6) who underwent elective orchiectomy for gender reassignment. T-cells were purified using CD3 microbeads from freshly isolated testicular interstitial cell suspensions. T-cell subsets, CCR5 expression, T-cell activation and frequency of regulatory T-cells (Tregs) were assessed using multicolor flow cytometry.
Lower proportions of CD4 T-cells were found in testis versus blood, in both HIV− and HIV+ subjects (40.5 ± 8.9% vs. 81±9.5% and 29.2±7,4% vs. 73.2±11.5%; p<0.001). A substantial decrease in naïve and a notable increase in effector-memory T-cell subsets were observed in testis compared to PBMCs in both groups (p<0.001 for all comparisons). Importantly, a remarkable increase was observed in the expression of CCR5 on testicular CD4 and CD8 T-cells when compared to blood (CD4 HIV−: p<0.0001, CD4 HIV+: p=0.003, CD8 HIV−: p=0.015, CD8 HIV+: p=NS). Increased T-cell immune activation (CD38/HLA-DR co-expression) in testis was observed in HIV+ individuals. A higher expression of immunosuppressive CD39+ Tregs was found in testis of both HIV− and HIV+ subjects compared to blood (63.2±26% vs. 37±12%, p=0.007 and 62.9±11,5%, vs. 42.6±10%, p<0.001).
For the first time, our results indicate an increase in the frequency of effector memory T-cells, CCR5 and CD38/HLA-DR expression on T-cells and higher CD39+ Tregs in testicular tissue when compared to blood. Collectively, these findings demonstrate the potential contribution of distinctive T-cells distribution in testicular tissue as anatomical reservoir for HIV persistence.
Interleukin (IL)-7 is an essential non-redundant cytokine for T-cell differentiation, proliferation, survival and function. We previously reported a significant down-regulation of the IL-7 receptor (IL-7R)-alpha-chain (CD127) on CD8 T-cells in HIV+ patients, mediated by soluble HIV Tat protein and IL-7, both of which are elevated during HIV infection. We now characterize the detailed mechanisms by which IL-7 suppresses CD127 transcription and protein expression.
Primary human CD8 T-cells isolated from healthy volunteers were treated with IL-7 and levels of c-Myb, SOCS1-7 and CIS transcripts and protein were examined by qPCR and Western. The interaction of SOCS proteins with CD127 was examined by co-IP and confocal microscopy. Candidate transcriptional repressors were identified using DNA microarrays, PCR-arrays and knockdowns. Nuclear run-on assays and ChIP were used to measure rates of CD127 transcription.
Upon binding IL-7, surface CD127 is rapidly phosphorylated and internalized while activation of the JAK/STAT5 pathway induces production of CIS proteins. CIS binds directly to CD127 and co-localizes with both CD127 and early endosomal marker EEA1. Subsequent proteasomal degradation of CD127 and CIS is dependent on an E3 ligase. IL-7 also suppresses CD127 transcription via JAK/STAT5 signaling which up regulates expression of c-Myb. Using siRNA-mediated knockdowns and ChIP, we identified c-Myb as a repressor of CD127 transcription.
IL-7 is currently being investigated as a potential therapy in HIV+ individuals with poor immunological response to antiretroviral therapy. In order to optimize the use of IL-7 in therapeutic settings, it is crucial to understand how expression of the IL-7 receptor is regulated. We show here that IL-7 suppresses CD127 expression by two mechanisms: transcriptional which is dependent on c-Myb and at the protein level by inducing expression of CIS protein which in turn binds to CD127 in early endosomes and shuttles the receptor complex to the proteasome for degradation.
HIV-1 infection leads to numerous B-cell abnormalities, including hypergammaglobulinemia, non-specific B-cell activation, non-specific class switching, increased cell turn-over, breakage of tolerance, increased immature/transitional B-cells, B-cell malignancies as well as a loss of capacity to generate and maintain memory, all of which contribute to a global impairment of the immune humoral compartment. Several cytokines and soluble factors, which are increased in sera of HIV-1-infected individuals, have been suggested to directly or indirectly contribute to these B-cell dysfunctions and one of these is the B-cell-activating factor (BAFF). We provide evidence that HIV-1 (X4- and R5-tropic) up-regulates BAFF expression and secretion by human monocytes. Moreover, we show that the virus-mediated production of BAFF by monocytes relies on a type-I interferon (IFN) response by a small percentage of plasmacytoid dendritic cells (pDCs) present in the monocyte cultures. HIV-1-induced type-I IFN by pDCs triggers BAFF production in both classical and intermediate monocytes but not in non-classical monocytes, which nonetheless display a very strong basal BAFF production. We demonstrate also that basal BAFF secretion is higher in monocytes obtained from females compared to those from male donors. This study provides a novel mechanistic explanation for the increased BAFF levels observed during HIV-1 infection and highlights the importance of pDC/monocyte crosstalk to drive BAFF secretion.
Increased HIV susceptibility has been associated with inflammatory cytokines at mucosal surfaces, but the underlying mechanisms remain unclear. This is likely a function of the general complexity of immune systems. Here we used a global proteomics approach, coupled to multivariate modeling, to examine the impact of cytokine-associated inflammation upon female genital tract mucosa in order to elucidate processes with potential to enhance HIV transmission.
Cervicovaginal lavage samples collected from HIV-uninfected Kenyan women were classified as ‘inflammation+’ (n=28) or ‘inflammation−’ (n=68) based on the elevation (upper quartile) of at least 3 of 7 inflammatory cytokines (MIP-3α, RANTES, IL-8, MIP-1β, IL-1β, IL-1α, and TNF-α), and analyzed by a combination of tandem mass spectrometry, hierarchical clustering, gene set enrichment analysis (GSEA), and multivariate modeling (Lasso algorithm).
Of 455 human proteins measured, 53 were significantly associated with inflammation at a 5% FDR threshold. Hierarchical clustering revealed two distinct branches of upregulated (60%) and downregulated (40%) protein groups. Functional enrichment analysis of upregulated clusters included endopeptidases, cell motility, and actin cytoskeleton factors (p<0.01), several of which critical to leukocyte migration. In contrast endopeptidase inhibitors, epidermal cell differentiation, and cornified envelope (barrier proteins) pathways were downregulated in women with inflammation (p<0.0001). Multivariate analysis identified an optimal signature of 16 proteins that distinguished inflammation with 88% accuracy, including neutrophil-associated proteases, which correlated with IL-1β and MIP-3α levels (p<0.0001). Furthermore, GSEA implicated activated immune cell signatures (NES: −2.10; p<0.0001). Finally, this type of inflammation was accompanied by a two-fold higher frequency of endocervical CD4+ T cells (p<0.001).
This data proposes new hypotheses and potential mechanism of enhanced HIV transmission during cytokine-associated inflammation, which includes protease-mediated disruption of mucosal barrier function and an accumulation of HIV target cells. Further exploration of these processes at mucosal surfaces is needed to understand the role of these factors in HIV infection, and may provide new avenues for HIV prevention technologies.
HIV transmission events occur more frequently during the luteal phase of the menstrual cycle based on data from non-human primate models, but the underpinning mechanism is not understood. We hypothesize that the immunological environment generated during the luteal phase may be more conducive to successful HIV infection. We performed a comprehensive proteomic analysis of cervicovaginal secretions to better understand mucosal events occurring in the follicular and luteal phases of the menstrual cycle.
Secretions were collected from 19 STI-uninfected, premenopausal women, and characterized as follicular or luteal phase samples based on days since last menstrual period. Samples were analyzed by tandem-mass spectrometry and data evaluated using hierarchical clustering, pathway and gene set enrichment analysis. Data-driven multivariate modeling was also performed to select a minimal set of proteins that specifically distinguish each phase.
Of the 384 proteins identified, 39 were differentially abundant between phases (p<0.05, ≥2 fold change). Pathway and biofunctional analysis indicated that cell-cell adhesion proteins and protease inhibitors were reduced, and neutrophil recruitment factors (IL-8 pathway, p=1.99E-5) and proteases involved in leukocyte extravasation (p=6.65E-4) were elevated during the luteal phase. A LASSO/PLSDA multivariate model identified 18 factors able to classify the phases with 100% accuracy (93% CV). Eight of these factors positively associated with the luteal phase including cytoskeletal elements and proteases, known to be involved in cellular infiltration. Lastly, a comparison to experimentally-derived data sets by gene set enrichment analysis indicated CD4+ T cell and neutrophil gene set signatures were significantly enriched in the luteal phase (p<0.05).
This data suggests that multiple events important for HIV transmission are enhanced during the luteal phase, including epithelial barrier remodeling, the activation of HIV target cell recruitment pathways and neutrophil activity. These processes should be examined further as a potential link to better understanding increased susceptibility to HIV infection in young women.
A recent study from the US CHARTER Cohort demonstrated that ANI is associated with a 2- to 6-fold increased risk for the development of symptomatic HAND [mild neurocognitive impairment (MND) or HIV-associated dementia (HAD)]. The objective of this study is to replicate and extend these results in a Canadian sample.
Study sample included 575 adults (82% men, 64% Caucasian, 86% on cART, 73% with undetectable HIV viral load) who were either normal on neuropsychological (NP) testing (NP-Normal; n=299) or had ANI (n=276) at baseline. NP testing was done annually (median follow-up time = 30 months) with brief NP battery that included measures of processing speed, attention/working memory, and learning/memory. Cognitive complaints were assessed with four-item Medical Outcomes Study Cognitive Functioning scale. HAND status was assigned according to established Antinori et al., (2007) criteria. We used proportional hazards regression modelling to estimate risk ratios for progression to symptomatic HAND.
Over follow-up period, 99 individuals (39 NP-Normal and 60 with ANI at baseline) showed progression to symptomatic HAND. Participants with ANI had shorter time of progression than those who were NP-Normal at baseline, after adjusting for baseline and time-varying predictors: adjusted hazards ratio of 1.85 (95% confidence interval: 1.12–2.82; p=0.005). Among covariates examined, depression and current smoking were associated with higher risk of progression; whereas undetectable plasma HIV viral load was associated with lower risk of progression to symptomatic HAND. Low nadir CD4 (<200 cells/mm3) was not a significant predictor of progression in adjusted analyses although it was associated with higher risk in bivariate analyses.
Asymptomatic Neurocognitive Impairment is associated with almost a two-fold increased risk of progression to symptomatic HAND. Early treatment with cART and addressing medical and mental health comorbidities may delay or lower the risk for the development and progression of symptomatic HAND.
HIV infection is characterized by reduced mucosal Th22 and Th17 cell numbers and function, which contribute to microbial translocation and inflammation. Standard antiretroviral therapy (ART) is slow to reverse these mucosal defects, and the resulting persistent inflammation is linked to serious non-AIDS illnesses (SNAs). We examined whether ART intensification with maraviroc and raltegravir during early HIV infection would accelerate the resolution of gut immune dysfunction, microbial translocation, and SNA biomarkers.
ART-naïve men with early HIV infection were randomized in a double-blind manner to receive standard ART (emtricitabine/tenofovir + lopinavir/ritonavir) with either raltegravir and maraviroc, or placebo, for 48 weeks [NCT01154673]. In a predefined substudy, paired blood and sigmoid biopsies were collected from participants at baseline and week 48, and from HIV-uninfected controls. Mucosal CD4 T cell immunology (Th1, Th17 and Th22 cells), and blood markers of microbial translocation (LPS), immune activation (sCD14) and SNA (IL-6 and D-dimer) were assessed. Twenty-two participants documented to have acquired HIV a median of 4 months ago were enrolled. Prior to ART initiation, gut Th22 cell numbers and Th17 polyfunctionality were reduced compared to controls, and plasma LPS and D-dimer levels were elevated. At 48 weeks after ART initiation, overall gut Th22 cell numbers were restored, but plasma LPS levels and gut Th17 function were unchanged, and blood D-dimer levels had actually increased. ART intensification had no impact on gut CD4 T cell immune subsets (Th1, Th17 and Th22 cells), microbial translocation (LPS), or SNA biomarkers (D-dimer and IL-6); there was a trend to reduced plasma sCD14 in the intensified arm.
Early HIV infection was associated with substantial gut mucosal immune dysfunction, bacterial translocation and systemic inflammation. Regardless of intensification with raltegravir and maraviroc, one year of ART had a limited impact on mucosal immune reconstitution or blood markers of microbial translocation, inflammation, and SNAs.
Suppressor of tumorigenicity 2 (ST2), a member of the IL-1 receptor family, can be soluble or membrane-bound. The soluble form of ST2 (sST2) binds IL-33 as a decoy receptor to negate its effects and has been recently validated as a marker of intestinal inflammatory diseases and GVHD/transplant-related mortality. The importance of sST2 in HIV infection, characterized by gut mucosal damage, remains unknown. Here, we assessed sST2 plasma levels in comparison with validated markers of gut mucosal integrity, microbial translocation, and T-cell activation in patients with different clinical outcomes.
Longitudinal plasma and PBMCs were collected from 41 untreated patients during primary HIV infection (PHI, <180 days) and 1 year later when 24 remained untreated while 17 initiated ART, as well as elite controllers (EC, n=12) and healthy subjects (HS, n=12). Markers of gut mucosal damage (I-FABP) and microbial translocation (LPS) were measured in addition to sST2. Immune activation was assessed in plasma by Tryptophan (Trp) and its catabolite Kyneurenine (Kyn) and in PBMCs by HLADR/CD38 co-expression on CD4 and CD8 T-cells by FACS.
Following PHI, sST2 was elevated compared to HS but did not normalize with early ART. This contrasts with T-cell activation and tryptophan metabolism (Kyn and Kyn/Trp ratio) normalization post-treatment. However, gut markers of integrity (I-FABP) and microbial translocation (LPS) remained elevated echoing the trend seen in sST2. Furthermore, sST2 was associated with tryptophan metabolism only during PHI (Kyn/Trp ratio: p=0.045, Kyn: p=0.039). Notably, sST2 was equally elevated in EC when compared to PHI.
sST2 can now be considered as a link between tissue damage and inflammation, representing a new marker of gut damage as it mirrors I-FABP and LPS during HIV infection. Insights into sST2 biology in the gut mucosa should be further investigated and may lead to therapeutic interventions in the sST2/IL-33 axis.
To determine prevalence and awareness of cardiovascular disease (CVD) risk factors and potential barriers to reducing CVD risk in HIV-infected patients.
Patients ≥40 years of age and engaged in HIV care for ≥6 months were approached at their clinic appointment to participate in a telephone survey. Patients were excluded if they did not speak English or did not have a phone. Survey questions were based on the Canadian Heart Health Survey. Demographic and medical information were collected from the patient’s chart. Outcomes evaluated were prevalence and knowledge of CVD risk factors, as well as barriers to reducing CVD risk. “Poor” CVD risk factor awareness was defined as the ability to identify <3 risk factors.
Sixty-four patients completed the survey. The majority (73%) were male. Mean age was 52 years (standard deviation 7.7) and 50% had less than secondary school education. Using the Framingham risk score (FRS), 27% were considered high risk (>20% risk of a vascular event in 10 years). Smoking (58%), hypertension (23%) and dyslipidemia (22%) were the most common CVD risk factors. Patients less commonly named CVD risk factors such as dyslipidemia and hypertension, as compared to smoking and poor diet. Overall, the mean number of CVD risk factors identified by participants was 2.2. Participants who smoked were more aware smoking was a risk factor whereas participants with hypertension or dyslipidemia were less aware of their CVD risk. The most common reported barriers to reducing CVD risk were lack of motivation (31%), chronic disease or physical disability (17%), and lack of energy (14%).
Overall, the prevalence of CVD risk factors was high while the awareness of risk factors was low. Consideration of barriers to CVD risk reduction is necessary when planning interventions to reduce CVD risk in this patient population.
Advances in HIV therapies have transformed HIV from a progressive, fatal condition to a manageable chronic disease. Accordingly, trends in reasons for hospital admission among people living with HIV may have shifted over time. This study describes causes of admission to the HIV/AIDS ward at St. Paul’s Hospital (SPH).
This study is a retrospective review of the SPH HIV/AIDS ward database between July 1, 2005–June 30, 2014. Primary discharge diagnoses were manually categorized by related condition and reviewed by two physicians. Data were analysed in twelve-month intervals beginning on July 1. Trends were fitted using generalized estimating equations.
3919 admission for 1595 participants were included in the analysis. 77.0% identified as male, 63.6% had a history of injection drug use and 61.8% were hepatitis C co-infected. Most common reasons for admission included respiratory tract infections (including pneumonia, influenza and upper respiratory infection, but excluding tuberculosis), cellulitis and gastroenteritis, accounting for 18.4%, 6.6% and 6.3% of admissions respectively. The most common opportunistic infections accounted for 6.9% of admissions, including mycobacterial disease (2.6%), Pneumocystis jirovecii pneumonia (2.3%), and cryptococcal infection (1.9%). The proportion of admissions attributable to those opportunistic infections declined consistently from 2005–2014 (risk ratio 0.823; 95% confidence interval 0.755–0.897), accounting for 14.6% of admissions in 2005–2006 and 2.6% of admissions in 2013–2014. There were 497 deaths, 322 of which occurred in hospital. 19.3% of deaths were related to HIV-associated infections, 17.3% malignancy-related, 15.3% HIV-related but non-infectious or unspecified and 2.4% related to other infections.
In the era of combination antiretroviral therapy, non-opportunistic respiratory tract infections were the most common cause of admission to the HIV/AIDS ward at SPH. This highlights the need for preventative measures such as pneumococcal and influenza vaccination.
Timely HIV diagnosis is fundamental for optimal linkage to care, and improved health outcomes. Our objective was to quantify trends in late diagnosis as indicators of delays in care engagement.
We obtained data from medical chart reviews and patient interviews at 10 HIV specialty clinics across Ontario. We defined “late diagnosis” as CD4<350 Using CD4 cell counts within 12 months of diagnosis or presence of an AIDS-defining condition in the 5 years preceding or 12 months following diagnosis; “very late diagnosis” was defined as CD4<200 or ADC. We used multivariable logistic regression to identify trends over calendar time and risk factors for very late diagnosis.
Among the 3073 participants, the majority were male, reported sex with men as an HIV risk factor, lived in Toronto, and were White. Mean age and CD4 at diagnosis were 36.0 years (SD 10.0) and 227.1 (SD 173.3), respectively, and 15.9% had an ADC. In total, 54.4% had “late” and 35.0% had “very late” diagnosis; this ranged from 58.7% and 39.3% in 1996–99 to 52.6% and 32.6% in 2010–13. In 1996–2005, the proportion with very late diagnosis declined annually (OR=0.94, 95% CI 0.91–0.97); no time trend was observed in 2006–2013 (OR=0.98, 95% CI 0.94–1.01). Independent risk factors for very late diagnosis were older age (OR=1.5, 95% CI 1.4–1.6 per decade), being Indigenous (OR=1.7, 95% CI 1.3–2.2 versus White), being a female (OR=1.6, 95% CI 1.2–1.9) or heterosexual male (OR=1.9, 95% CI 1.6–2.4) versus MSM, being IDU (OR=0.7, 95% CI 0.6–0.9). We observed no independent association with other ethnicities, immigration, or region of Ontario.
We observed a decline in the proportion with late or very late diagnosis before 2006, and no change thereafter. The cohort likely under-represents those diagnosed very late who may not have survived to volunteer for research. Nevertheless, our results suggest that there is room for improvement to facilitate early diagnoses in Ontario.
Between 2003 and 2011, repeated cross-sectional surveys among people who inject drugs (PWID) were conducted in Toronto (4 surveys), Sudbury (4), Kingston (2), and Thunder Bay (2) as part of the I-Track behavioural surveillance system sponsored by the Public Health Agency of Canada (PHAC). This analysis describes trends over time in key variables from these surveys.
Participants were recruited from needle exchange programs in each city using comparable methods in each survey. Interviewers administered questionnaires and collected dried blood spot samples using coded linkage to maintain anonymity.
Toronto recruitment numbers ranged from 221–262 per survey; Sudbury 147–169; Kingston 200–224; and Thunder Bay 139–149. All cities except Thunder Bay showed an upward trend in proportion of participants self-reporting as aboriginal; Thunder Bay had the highest proportion of aboriginal participants. Toronto showed an upward trend in reports of stable housing, and a downward trend in injecting with used needles/syringes & injecting in public places; other cities showed no significant change. Trends in reported patterns of drugs used varied across cities for both injected and non-injected drugs. Toronto and Sudbury showed significant upward trends in reports of having an HIV test in the previous year; for Thunder Bay this declined. Toronto showed an upward trend in reporting of HCV testing. Kingston & Thunder Bay reported increases in proportions under physician care for HIV, and proportions of those receiving HIV meds. Sudbury was the only city showing a downward trend in reporting of needle exchange access.
Although Ontario has a province-wide program supporting needle exchange provision, these results suggest that specific contexts in different cities may affect key aspects of HIV risk among PWID. Trends must be considered with caution and interpreted using local knowledge, since although recruitment methods are constant, samples are relatively small and may be affected by unrecognized selection and volunteer bias. The lack of downward trend in injecting risk behaviours in most cities suggests consideration of additional strategies such as supervised injection facilities.
Understanding testing patterns of sexually transmitted infections (STI) and previous STI history of HIV positive individuals, prior to their diagnosis, can inform earlier diagnosis and engagement and prevention strategies.
This was a population-based, retrospective analysis of prior testing and history of bacterial STIs among HIV-positive individuals presenting to care between 2007–2011 to the Manitoba HIV program (MHP). HIV cases were age-, sex- and region-matched to HIV-negative controls at a 1:5 ratio. Clinical information was linked to testing data from Cadham Provincial Laboratory (CPL) as well as STI databases at Manitoba Health. CPL data were used to calculate rates of chlamydia (CT) and gonorrhea (GC) testing. In Manitoba, CT and GC tests are performed concomitantly, thus testing rates are for CT/GC combined. Person-days prior to entry into MHP care (case date) was used as the denominator in rate calculations. Infection history for CT and GC was derived from Manitoba Health’s STI databases. Testing and CT/GC infections incident in the 6-month period prior to case date were not included. Stratified Poisson regression models were used to compare testing rates, while conditional logistic regression was used to compare CT and GC infections between cases and controls. Where appropriate, relative rates (RRs), odds ratios (ORs), and their 95% confidence intervals (95% CI) are reported.
A total of 181 cases and 1,129 controls were included. In the 5 years prior to HIV diagnosis, CT/GC testing rates for HIV cases were approximately threefold higher than their HIV-negative controls (RR: 2.9, 95% CI: 2.5–3.4). HIV cases had higher odds of having had CT (OR: 3.1, 95% CI: 1.6–5.9) and GC (OR: 11.8, 95% CI: 4.9–28.5) infections.
HIV cases had higher STI testing rates, as well as being more likely to have had a history of a bacterial STI, suggesting significant gaps and missed opportunities for prevention.
Abduction by the Lords Resistance Army has had a profound impact on the physical and psychosocial well-being of well being of people who have survived the conflict in Northern Uganda. Despite the pervasiveness of the conflict into every level of society, relief and recovery efforts often target perceived ‘at-risk’ groups and lack a sound evidence base.
The Cango Lyec Project is a prospective cohort study of 2500 participants age 13–49 in three districts of Northern Uganda. Participants consented to participate in the study, completing trauma (HTQ), depression (HSCL-25) sociodemographic surveys, and providing blood samples for HIV testing. Three separate multivariable logistic regression models examined a history of abduction and the likelihood of HIV positivity, PTSD, and Depression.
Of 2388 participants in the study, 27.4% of men (n=271) and 22.9% of women (n=319) experienced abduction during the conflict. Abducted women reported higher proportions of HIV risk factors than non-abducted women including lifetime sexual abuse (34.2% vs. 7.15%), participation in sex work (2.2% vs. 0.5%), active syphilis (7.2% vs. 4.4%), and experiencing 12 or more traumatic events (32.9% vs. 1.8%). Among men, abductees were more likely to report inconsistent condom use (6.3% vs. 15.6%), to have experienced 12 or more traumatic events (33.1% vs. 1.84%), and to have abused their current sexual partner (19.6% vs. 8.9%). After adjustment, history of abduction was significantly associated with depression (AOR 1.89; 95% CI 1.43–2.49) and PTSD (AOR 2.10; 95% CI 1.56–2.83), but was not significantly associated with HIV infection (AOR 1.08, 95% CI 0.80–1.47).
HIV remains a crucial issue and is not confined to easily defined groups in Northern Uganda, requiring a rethinking of treatment and prevention programs. Former abductees face much higher levels of both PTSD and depression. Trauma informed HIV prevention and culturally safe mental health care initiatives are urgently required.
Despite increased risks associated with socio-structural barriers to care and clinical comorbidities, the prevalence and risk factors associated with adverse obstetrical outcomes among women living with HIV (WLWH) are not well measured. The objectives of this study were: 1) investigate the prevalence and correlates of adverse obstetrical outcomes among WLWH over a 20-year time span and 2) compare it to that of the general obstetrical population in the Ottawa area.
This 20-year (1990–2010) clinical case series assessed adverse obstetrical outcomes among pregnant WLWH receiving care at The Ottawa Hospital (TOH). General estimating equation (GEE) modeling was used to identify factors most strongly associated with adverse obstetrical outcomes, while controlling for year of childbirth clustering. Control group data were abstracted from the Better Outcomes Registry & Network (BORN) Ontario to compare the prevalence of obstetrical outcomes among WLWH to the general obstetrical population.
A total of 145 pregnancies among 94 WLWH were recorded at TOH between 1990–2010 – 17.32% were premature, 7.09% were low birth weight, 9.45% were small for gestational age and 3.14% were still birth. For each additional year, the odds of adverse obstetrical outcomes increased by 15% (OR 1.15, 95% CI 1.03–1.30). In GEE analyses, psychiatric history (AOR 2.64, 95% CI 1.12–6.24), teen pregnancy (AOR 3.35, 95% CI 1.04–1.46), and recent immigrant status (AOR 7.24, 95% CI 1.30–40.28) were the strongest predictors of adverse obstetrical outcomes. Compared to the general obstetrical population, WLWH had significantly higher odds of premature birth (OR 2.06, 95% CI 1.29–3.21) and stillbirth (OR 6.00, 95% CI 2.13–16.83).
This study shows an increasing proportion of adverse obstetrical outcomes among pregnant WLWH over the past 20 years, and significantly higher adverse outcomes compared to the general population. It is a public health imperative to ensure WLWH achieve the best possible obstetrical outcomes for optimal maternal and child health.
Chronic kidney disease (CKD) screening guidelines have recommended monitoring of estimated glomerular filtration rates (eGFR) for patients on stable ART. We sought to determine the risk of prevalent and incident CKD associated with cocaine injection use, a known nephrotoxin, among HCV-HIV co-infected patients receiving care.
The Canadian Co-infection Cohort is a multicentre, prospective cohort study of 1,321 co-infected patients with visits scheduled every 6 months. Ever and cocaine injection use since the last visit was obtained by self-report. CKD was defined as a confirmed (two measurements ≥3 months apart) eGFR of ≤70 mL/min/1.73 m2 for patients with a baseline eGFR >70 mL/min/1.73 m2. eGFR was calculated using the 2009 serum creatinine-based CKD-EPI equation. Risk ratios (RR) for prevalent CKD were calculated using modified Poisson regression. Hazard ratios (HR) for incident CKD were calculated using a discrete-time proportional hazards model.
At baseline, 94 patients (7%) had prevalent CKD and 882 (67%) reported any past cocaine injection exposure. Ever cocaine injection users were younger, more likely to be female and aboriginal, and have been on atazanavir in the past. After adjusting for these covariates, past cocaine injection was associated with prevalent CKD (RR 1.87, 95% CI 1.09–3.21). During follow-up, 407 (39%) of 1,035 patients free of CKD reported using injection cocaine at least once and 122 patients (12%) developed incident CKD. After adjusting for current age, gender, aboriginal ethnicity, current CD4+ count, hypertension, and tenofovir and protease inhibitor use, recent cocaine injection increased the risk of incident CKD (HR 1.48, 95% CI 0.94–2.31). This effect was largest among those who injected ≥ 3 days/week (HR 2.98, 95% CI 1.07–8.03).
Past cocaine injection was associated with prevalent CKD and recent heavy use increased the risk of incident CKD independent of traditional risk factors.
The incidence rate of HCV infection is estimated at 26/100 py among Montreal IDU. Though HCV reinfection has been reported in IDU and MSM patients, the extent to which it occurs is unknown. Given the high treatment costs, HCV reinfection in cured patients may limit future access to treatment. We therefore aimed to evaluate the incidence of reinfection in a clinical cohort of HCV treated patients.
HCV patients with a sustained virological response (SVR) were included. Censoring date was the date of HCV reinfection or the date of the last negative HCV RNA test. Reinfection was defined as detectable HCV RNA. The rate of reinfection was calculated using the number of person-years of observation after the end of treatment (EoT). Time from SVR to reinfection was estimated using Kaplan-Meier analyses.
338 patients were included. The sample was 77% male; mean age was 46 years; and the main risk factor for HCV infection was IDU (n=275, 82%). Patients were followed for a median of 2.7 years after EoT (IQR=1.7–4.8), for a total of 1175 person-years. 316 (94%) patients remained HCV-negative, while 22 (6%) became reinfected during follow-up with an overall reinfection rate of 1.7/100py (95% CI 1.07–2.58). Median time to reinfection was 14.7 years (95% CI 13.6–15.7). Cumulative incidence of seroconversion within 2 years of SVR was 4% (9/210) and 11% (10/88) within 5 years. When controlling for drug use, the incidence rate of HCV reinfection was 0.43/100py (95% CI 0.02–0.11) for non-IDU; 1.90/100py (95% CI 1.13–3.14) for past IDU and 3.60/100py (95% CI 1.44–7.39) for present IDU.
HCV reinfection after successful treatment in our cohort is low. Although the rate of HCV reinfection is higher in IDU than non-IDU, it is much lower than the overall incidence rate of the first HCV infection among IDU in Montreal.
In recent years, the HIV prevention strategies used by MSM have become more complex. Participants at SPOT were asked about the risk-reduction strategies they used in the previous 3 months.
Identify patterns in the use of risk-reduction strategies and key characteristics of these patterns.
Cross-sectional data from 360 MSM tested for HIV at SPOT between August 2013 and December 2014 were used to perform a latent class analysis, integrating scales for four risk-reduction strategies: 1) communicating with sexual partners about HIV and STI status; 2) avoiding anal sex without condoms (ASWC); 3) avoiding sex with HIV-positive partners; 4) opting only for very low-risk sexual practices. Univariate comparisons of classes were performed using sociodemographic, behavioral, and sociosexual indicators.
Six classes were identified: Class 1: total avoidance of sex with HIV-positive partners and strong propensity to communicate about HIV status (45%); Class 2: little use of any strategy (16%); Class 3: strong propensity to avoid ASWC and little use of other strategies (13%); Class 4: strong propensity to opt for very low-risk practices and high overall use of other strategies (12%). Class 5: strong propensity to avoid HIV-positive partners and little use of other strategies (11%). Class 6: No communication about HIV status and high use of other strategies (3%). Differences among classes were observed for various indicators: attitudes toward prevention, barriers to condom use, recreational drug use, number of partners in the previous 3 months, testing habits, etc.
These results point to patterns of diversification in prevention strategies, shaped by sexual lifestyle and other characteristics. Serosorting and avoidance of ASWC are predominant, but most participants use a combination of strategies. However, nearly a third have difficulty using an effective strategy. A better understanding of the individual, social, and structural factors that influence these patterns is required.
Analyses of gay/bisexual men’s group sex events (GSE) report high levels of poly-substance use and condomless sex. Yet recent studies also note harm reduction practices in these risk environments. We analyzed Vancouver Momentum Health Study data to compare characteristics of men who attended GSE in the past 6 months to those who did not.
Momentum participants were recruited from February 2012 – February 2014 using respondent-driven sampling (119 seeds, 16 waves); all analyses use RDS weights. Three separate logistic regression models compared GSE attendees (n=180) to non-attendees (n=531) with respect to: 1) substance use patterns, 2) psychosocial characteristics & sexual behaviour and, 3) sero-adaptive strategies. Variables with p<0.05 from initial univariable models were selected for inclusion in subsequent multivariable models, while the Akaike Information Criterion determined the final models.
The first model’s results showed that GSE attendees featured significantly higher use of erectile dysfunction drugs (AOR=2.86, 95% CI=1.66–4.92) and poppers (AOR=3.76, 95% CI=2.49–5.70), and were more likely to have >15 drinks/week (AOR=2.39, 95% CI=1.40–4.08). For the second model GSE attendees were significantly more likely to report unprotected anal sex with a sero-discordant or unknown sero-status partner (AOR=2.35, 95% CI=1.52–3.64), fisting (AOR=2.29, 95% CI=1.17–4.49) and sex toy use (AOR=2.49, 95% CI=1.59–3.89), and scored higher on the Sensation Seeking Scale (AOR=1.10, 95% CI=1.04–1.15). In the third model GSE attendees featured differing sero-adaptive strategies for condomless anal sex, as HIV-positive men reported sero-sorting with sero-concordant partners (AOR=3.20, 95% CI=1.37–7.44), while HIV-negative men sought HIV-positive partners on treatment and/or with low viral loads (AOR=4.92, 95% CI=2.49–9.74).
Despite higher substance use and adventurous sex practices, GSE attendees reported distinctive sexual harm reduction practices. For condomless anal sex, HIV-positive men reported sero-sorting and HIV-negative men favored a Treatment as Prevention strategy by seeking HIV-positive partners on treatment and/or with a lowered viral load. Results have potential for promoting safer GSE participation.
To optimize combination HIV prevention, we sought to examine how an existing strategy (condom use) was differentially reasoned by MISM in Ontario.
Data were drawn from the province-wide community-based Cruising Counts study of MISM aged 16+ who completed a 15-minute anonymous online questionnaire. Participants described their last anal sex encounter (event-level factors) and qualitatively described reasons a condom was used or not. Qualitative responses were coded non-exclusively and differences in terms of event-level and individual-level factors (e.g., age in years, sexual identity, race/ethnicity) were determined quantitatively using manual backward stepwise multivariable logistic regression.
Among 1,830 participants, 883 (79.8%) reported a recent anal sex event, of whom 60.5% used condoms. Reasons for condom use included protection/safety (82.4%), norm (30.5%), and combination prevention (6.2%). Reasons for non-condom use were grouped non-exclusively as intentional non-use (43.2%), trust (28.1%), unintentional non-use (25.4%), and other risk reduction (20.0%). Event-level substance use was associated with all non-use reasonings: e.g., participants who used substances were more likely to reason non-use as unintentional (AOR=1.70, 95% CI 1.04–2.78) and partner substance use was negatively associated with trust as the reason for non-use (AOR=0.53, 95% CI 0.31–0.90). Participants were more likely to explain condom non-use with partners met online (versus not) as intentional (AOR=2.43, 95% CI 1.62–3.67) and less likely due to trust (AOR=0.25, 95% CI 0.15–0.40). Younger men were less likely to reason non-use due to other risk reduction practices (AOR=1.03, 95% CI 1.003–1.05) and also less likely to explain condom use as a norm (AOR=1.01, 95% CI 1.001–1.03). Bisexual men and men who didn’t self-identify as White were also less likely to explain condom use as a norm (AOR=0.59, 95% CI 0.40–0.87 and AOR=0.62, 95% CI 0.42–0.92, respectively). Participant-partner sero-concordance was an important predictor across all condom use and non-use reasons: e.g., discordant partnerships were more likely to explain condom use as part of combination prevention compared with negative concordant partnerships (AOR=13.40, 95% CI 5.09–35.32).
MISM described various reasons for using condoms or not. This understanding supports the need for targeted, culturally appropriate health promotion to optimise combination HIV prevention.
The Internet has increasingly become the most common tool for gay and bisexual men, and other men who have sex with men (MSM) to find sexual partners. As a result some AIDS Service Organizations (ASOs) provide online outreach on different socio-sexual networking websites and mobile apps. Little is known about the challenges and opportunities this online outreach provides. The aim of this analysis was to investigate the experiences of ASOs and other community-based organizations (CBOs) across Ontario in the planning and implementation of online outreach.
From November 2013 to January 2014, ASO/CBO online outreach providers and managers (n = 22) were recruited to complete a 1-hour in-person/telephone interview to explore in-depth their experiences with, and perspectives on, delivering online outreach services for MSM in Ontario. Participants were asked to identify the organizational structures, policies, or programs that are supportive of this work and were asked to comment on the capacities, opportunities, barriers, and challenges for providing online outreach. Participants also commented on their evaluation practices and the parameters they used to indicate success. Interviews were digitally recorded and transcribed verbatim. Thematic analyses were conducted using NVivo10.
The findings indicate that service providers consider online outreach a vital tool for HIV prevention. Providers highlighted the strengths and advantages (anonymity, engaging hard-to-reach MSM) of online outreach over face-to-face outreach. Across interviews, the following themes were identified as barriers to providing effective online outreach services: staff capacity and training, confidentiality and privacy of online interactions, safety of participants and staff, uncertainty of best practices, and lack of updated HIV prevention tools.
The findings highlight the barriers to and advantages of providing online outreach for MSM. These findings can inform ASOs/CBOs on how to increase the effectiveness of online outreach to MSM communities.
Micro-cultural contexts, such as party and play (PnP), BDSM, and the leather scene, have received attention as contexts for potential HIV infection among men who have sex with men (MSM). In this analysis, using Latent Class Analysis (LCA) approach, we examined patterns of scene participation and psychosocial factors associated with these patterns.
Using data from 470 MSM recruited from Toronto, we calculated posterior probability of being in a class from participation in 9 separate scenes. Simultaneously we fit a multinomial regression model to differentiate MSM within each class by personal, psychological and behavioral characteristics. We used Entropy, the Bayesian information criterion and the Lo-Mendel-Rubin likelihood ratio test to identify the best fit model.
Fit indices suggested a four class solution. Half (51%) of the MSM reported Minimal participation in any scene; 28% reported higher probability of participating in dance club scene; 14% reported higher probability of participating in BDSM (0.53), Bear (0.46) and in Leather (0.71) scenes and 6% reported higher probability of participating in circuit (0.64), PnP (1.00) and in sex party (0.58) scenes. Compared to Minimal scene participants, BDSM-Leather scene participating MSM were more likely to be older, White, to report higher sexual self-esteem and to engage in condomless sex; Party scene participants were more likely to be men of color and to use drugs before/during sex, whereas MSM participating in Dance Club scene were more like to be younger, and to report lower self-efficacy but higher hope.
LCA allowed us to identify distinct social-niches/micro-cultures of participating MSM and factors characterizing these micro-cultures. Distinct scene participating MSM differ in their risk for HIV and STIs as their characteristics. Tailored interventions are needed that focus on reducing HIV risk and promoting sexual health in specific sexual contexts such as the BDSM-Leather and Party scenes.
The quadrivalent human papillomavirus vaccine (HPV4) is 90% effective in preventing HPV-types associated with genital warts, and anal, penile and oral cancers among men. Among gay and other men who have sex with men (MSM), the high prevalence of anal HPV is associated with 44-times (among HIV-positive MSM 60-times) higher incidence of anal cancer than the general population. Yet, 11 of 13 Canadian provinces/territories only include girls in publicly-funded HPV vaccination. We examined rates and correlates of HPV vaccine acceptability, and ramifications of HPV vaccine policy for MSM in Canada.
We conducted a systematic search across multiple electronic databases to locate empirical studies that examined rates and/or correlates of HPV4 acceptability among men, including MSM. We performed meta-analysis on studies examining similar correlates of HPV4 acceptability and calculated effect sizes using a random-effects model, following PRISMA guidelines. We critically reviewed policies that exclude boys from publicly funded HPV vaccination.
Of 301 identified studies, 29 were included. Weighted mean HPV vaccine acceptability was 50.4 (SD=21.5) (100-point scale) across 22 studies (n=8360); in nine studies reporting sexual orientation, acceptability was 58.44 (SD=16.76) among MSM (n=986) and 50.98 (SD=19.67) among heterosexuals (n=1713) (t (2699)=0.24, p=0.81). In meta-analyses, perceived HPV4 benefits, anticipatory regret, and healthcare provider (HCP) recommendation had medium effect sizes on acceptability. Concerns about cost-effectiveness, health equity, and benefits of universal versus targeted vaccination emerged as central to HPV4 policy.
Given disproportionate HPV-associated disease burden among MSM, the unfeasibility of widespread targeted vaccination of young MSM before sexual debut, gender-based health equity concerns, and the suboptimal HPV4 coverage among boys and girls in Canada support extending publicly-funded HPV4 programs to boys/young men. Targeted messaging for young MSM to support perceived HPV4 benefits and of HCP to promote HPV4 recommendation for boys may further support HPV4 coverage among MSM.
The capacity of HIV to rapidly establish latent infection is a major obstacle to finding a cure for HIV infection. A pivotal step controlling latency is the activation of HIV transcription by host cell proteins in concert with the HIV trans-activating protein Tat. Tat trans-activation requires the well-studied TAR RNA of the nascent viral transcript to recruit P-TEFb, as well as a less-well understood DNA element within the HIV core promoter, termed TASHET. Our team has focused on the identification and characterization of host cell pre-initiation complexes (PICH) that bind TASHET to regulate HIV transcription, since these represent potential targets to control HIV latency. Using DNA affinity chromatography followed by mass spectrometry analysis, we have identified a novel series of host cell proteins that recognize TASHET. PICH-130 and PICH-35 are components of a novel HDAC complex and the silencing of their expression via siRNA reduces HIV transcription in cellulo. We have recently shown that PICH-35 binds directly and specifically to CTGC motifs within TASHET. We therefore propose that PICH-35 nucleates the formation of host cell complexes upon the HIV core promoter. Our data reveal that another host cell protein termed PICH-115 interacts directly with the core domain of HIV Tat and can bind TAR RNA in vitro in concert with Tat. Silencing of PICH-115 expression by siRNA strongly inhibits HIV transcription, suggesting it can act as a novel cofactor for Tat trans-activation. Our ongoing characterization of PICH components should provide new mechanistic information into the control of HIV latency.
During infection, the HIV genome incorporates itself into the cellular genome through integration process, leading to life-long infection that is typically accompanied by a period of latency. The potential for a single virion to restart infection despite therapy means that total elimination of all latently-infected cells is necessary for curing infection. Several findings suggest that integration location has the potential to confound anti-latency treatments, necessitating a greater understanding of integration site selection and its effect on latency. We discovered that non-B DNA motifs (nBDMs) attract integration of HIV genome. nBDMs form structures that assume higher energy state conformations and are facilitated at specific sequence motifs by the free energy generated from negative super-coiling, arising from processes such as transcription and protein binding. We performed bioinformatic analyses of murine leukemia virus (MLV), avian sarcoma leucosis virus (ASV) and human T-lymphotropic virus (HTLV) integration site datasets and observed that these integration sites are also enriched in or near nBDMs, showing that the preference for nBDMs is not specific to HIV and plays an important role in the integration of diverse retroviruses. We also identified a strong nBDM consensus sequence flanking integration sites. Interestingly, the simple retroviruses ASV and MLV exhibited a strong preference for integration directly within nBDMs, whereas the complex retroviruses HIV and HTLV preferred to integrate near nBDMs rather than within the motifs themselves. We also compared the integration site profiles of acute and latent models of HIV infections. Integration sites in both datasets were enriched near nBDMs. Remarkably, integration sites in latently-infected cells were substantially enriched near specific nBDMs, up to 84% of total integration sites. This is important since nBDMs can inhibit expression of nearby genes and possibly proviruses. Our data will help inform the design of experiments in HIV eradication research and better retroviral gene therapy vectors.
Upon entry into the body through mucosal surfaces, HIV-1 disseminates to remote lymphoid and non-lymphoid tissues, leading to a chronic infection of the immune system. Previous in vitro studies have shown that specialized virological synapse (VS) formations between infected and uninfected cells can greatly enhance viral transmission and dissemination. However, it is currently unclear whether such cell-to-cell contacts remain sufficiently stable within lymphoid tissues where immune cells are constantly undergoing migration. Here, we utilized intravital microscopy techniques to visualize the dynamic behavior of HIV-infected T cells in the lymph node of humanized mice using infectious, GFP-reporter HIV. Surprisingly, we found that most productively infected T cells migrated robustly, albeit at reduced speeds, compared to uninfected T cells. We now show that the reduction in motility speeds is due to the expression of Nef and its ability to disrupt the actin cytoskeletal machinery by binding to Pak2. Prolonged cell-to-cell interactions were observed in the presence of functional Nef, suggesting that the formation of virological synapse was modulated by its expression. We will discuss how alterations in cellular motility impacts on HIV pathogenesis in vivo.
Today, there are 35 million people living with HIV infection worldwide. Nearly 2 million people have died of HIV/AIDS in 2012 alone. Anti-retroviral therapy (ART) is used to prevent infection by targeting multiple facets of the viral replication cycle. However, the biggest barrier to HIV eradication comes from the fact that the virus quickly establishes a latent reservoir by successfully integrating its genome into its host’s DNA. Unfortunately, due to the fact that ART has considerable side effects, unreasonable costs and limited accessibility by most of the infected population globally, it is imperative that a cure is found for HIV/AIDS. In order to tackle the problem of latency it will be essential to specifically control HIV transcription of latent proviruses. We have recently shown that a sequence of nucleotides within the core promoter of HIV, named TASHET, plays a pivotal role in the formation of a pre-initiation complex of HIV (PICH). We have successfully been able to identify cellular components of this complex that render it specific to the HIV promoter compared to the canonical AdMLP. We have shown that one of these components, PICH115, a cell host factor with a molecular weight of 115KDa, can bind specifically and directly the HIV trans-activating protein, Tat in vitro. Moreover, the silencing of endogenous PICH-115 by siRNA results in strongly reduced HIV transcription in HeLa cells. Together our results suggest that it may be the bridge between TASHET and the Tat-TAR trans-activation complex and reveal PICH-115 as a novel therapeutic target to control HIV latency. Our imminent goals will be to study the functions of PICH-115 involved in the regulation of the HIV promoter activity and in its interaction with Tat.
The majority of the productively HIV-1 infected CD4+T cells undergo programmed cell death. Multiple factors including induction of pro-apoptotic HIV-1 proteins, microbial translocation across the breached gastrointestinal tract and activation induced T cell death drive apoptosis during HIV infections. In this study, we investigate the pro-apoptotic role played by the HIV-1 accessory protein Nef and explore the cell signalling pathways hijacked by Nef to modulate cell survival.
Apoptosis was quantified by live cell staining of SupT1 cells infected with the replication incompetent pNL4-3 pseudovirus or the corresponding nef deletion mutant. The levels of apoptotic marker expression were 2.3 fold lower (p=0.0011) in the nef deletion mutant and were partially dependent on two Nef motifs (P72xxP75 and W113). In addition, Nef induced a two-fold increase in activated caspase-3/7 activity. The serine/threonine kinase Akt is a vital protein in controlling cell survival and is activated by growth factors and cytokines. To gain insight into the signalling pathways contributing to Nef-induced apoptosis, we measured the activation status of Akt protein using phospho-specific flow cytometry. Strikingly, Akt phoshorylation (Thr308 and Ser473) was increased in the absence of Nef. Consistent with this observation, increased phosphorylation of proteins associated with the Akt pathway was noticeable for phosphatidylinositol 3-kinase, the PTEN c-terminal tail and mTOR. Mechanistic studies further indicated that the nef deletion mutant maintained increased phosphorylation of the pro-apoptotic protein BAD reducing its mitochondrial accessibility and higher expression levels of the anti-apoptotic protein Bcl-xL.
HIV-1 Nef hijacks the Akt pathway to induce apoptosis. Elucidating the mechanistic details of this pathway enhances our understanding of the strategy used by HIV-1 for inducing cell death.
The Dynein is a microtubule (MT) associated protein complex that mediates retrograde transport of macromolecules. Although Dynein has also been implicated in viral uncoating and retrograde transportation of HIV-1 preintegration complex (PIC) for successful entry into nucleus, the cellular and/or viral factors that mediate HIV-1 PIC-Dynein targeting and their contribution to early stage HIV-1 replication is still unknown.
In the present study, we have investigated the potential interactions between HIV-1 PIC associated viral proteins and cargo adopter proteins of dynein, and their role in early stage HIV-1 replication. By using co-immunoprecipitation assay, we have examined the interaction of HIV-1 Integrase (IN), reverse transcriptase (RT), Matrix, and Capsid proteins with Dynein adopter proteins such as Dynein light chain 1 (DYNLL1), Dynein light chain Tctex-type 1 (Tctex1), and Dynactin p150 in 293 T cells. Results showed that only IN interacted with DYNLL1. This interaction was further confirmed in HIV-1 infected C8166 T cells and by in vitro binding assay. To evaluate the functional significance of DYNLL1/IN interaction, we used a gene knockdown (KD) approach and found that down regulation of DYNLL1 expression has impaired HIV-1 replication at reverse transcription step. Furthermore, by mutational analysis of putative DYNLL1 interaction motifs in IN, we identified the motifs “52GQVD” and “250VIQD” in IN as essential for DYNLL1 interaction. The DYNLL1 interaction-defective IN mutant HIV-1 (HIV-1INQ53A/Q252A) exhibited impaired reverse transcription. Significantly, we have found a relatively faster HIV-1 uncoating in DYNLL1-KD cells or in infections with HIV-1INQ53A/Q252A mutant virus, suggesting that IN and DYNLL1 interaction is required for the proper uncoating of HIV-1 and contributes to efficient reverse transcription.
Overall, our study demonstrates the novel interaction between HIV-1 IN and cellular DYNLL1 proteins and the requirement of this viral-cellular interaction for the early stage of HIV-1 replication, including the proper uncoating and reverse transcription.
Follicular T helper cells (Tfh), a subset of CD4 T lymphocytes, are essential for B cell activation and provide help to B cells in the production of antigen-specific antibody. Although several studies have analyzed the dynamics of Tfh cells in the context of AIDS by analyzing peripheral blood and LNs of HIV-infected patients. Paradoxically, none of these studies in HIV/SIV infection have addressed the role of Tfh cells in the primary organ of B cell activation, the spleen.
To address the dynamic of splenic Tfh cells, we infected rhesus macaques with SIVmac251 (20 AID50). Animals were sacrificed at different time points post infection and lymphoid organs were recovered. Tfh cells (PD-1highCXCR5+) and CD4+ T cell subsets were monitored by flow cytometry. Concomitantly B cell subsets were also analyzed. CD4 T cell subsets were sorted and SIV DNA was quantified by RT-PCR.
Herein, we demonstrated for the first time that the percentages and numbers of splenic Tfh cells decrease early during the acute phase in macaques infected with SIV. This profound loss and abnormal differentiation of Tfh is also associated with the loss of memory B cell subsets. Moreover, SIV DNA is detected in splenic Tfh cells early after infection. Finally, our results showed at the chronic phase that the frequency of splenic Tfh and memory B cells are higher in non-progressors compared to progressors RMs. Altogether, our results demonstrate the drastic depletion of splenic activated and tissue memory B cells which might be related to the loss of fully maturated Tfh cells.
This work was supported by CIHR (Canadian Institute of Health Research) to JE. JE thanks the Canada Research Chair program for financial assistance.
Since its emergence in Central Africa, the Human Immunodeficiency Virus type 1 (HIV-1) has undergone continuous selective pressure leading to a staggering degree of genetic diversity. Indeed, the ability of HIV-1 to withstand rapid and extensive mutations to its genome is one of the reasons the HIV/AIDS epidemic has been so devastating. Currently, there are over 10 major subtypes of HIV-1 that are responsible for more than 90% of worldwide infections, however, research has been limited to only a few of these subtypes. We aimed to study the impact of this genetic variability in the HIV-1 protein Nef, which plays a key role in the ability of HIV-1 to evade immune surveillance, modulate T cell activation and increase viral replication by downregulating cell surface receptors MHC I, CD28 and CD4, respectively. Cell surface MHC I and CD28 levels were measured in HIV-1 pseudoinfected human T cells expressing Nef proteins from 10 major HIV-1 subtypes (A1, A2, B, C, F1, F2, G, H, J, and K). CD4 cell surface levels were measured by transfecting CD4+ HeLa cells with expression plasmids encoding select Nef-GFP fusion proteins. MHC I, CD28 and CD4 were labeled using fluorescently conjugated antibodies and downregulation efficiency was measured by flow cytometry. Our results demonstrate that MHC I, CD28 and CD4 are differentially downregulated between subtypes. Differences in downregulation efficiency were attributed to variations in Nef protein expression, which was determined by western blot analysis and confirmed by flow cytometry. Our study represents a comprehensive analysis of Nef function among HIV-1 subtypes and provides increased relevancy as the use of an HIV-1 based expression system provides results in the context of a viral infection. These findings support further study of all major HIV-1 subtypes and emphasize the need to consider subtype differences when developing alternative treatment options.
Historically HIV/HCV was considered a negative predictor of response to interferon/ribavirin (IFN/RBV). For sofosbuvir-based regimens, HIV/HCV patients have achieved similar SVR rates as HCV monoinfected patients. We evaluated the safety and efficacy of LDV/SOF in HCV genotype 1 or 4 patients HIV/HCV in the Phase 3 ION-4 study.
HCV TN and TE HIV/HCV patients on stable, approved antiretroviral (ARV) regimens received LDV/SOF (90mg/400mg) OD for 12weeks. Patients with compensated cirrhosis were eligible. Permitted ARVs included TDF+FTC, with RAL, EFV or RPV. Safety evaluations included AE and laboratory parameter monitoring in addition to enhanced renal toxicity monitoring, CD4 count and HIV-1 RNA levels. The primary endpoint was SVR12.
335 patients with GT1a (75%), GT1b (23%) and GT4 (2%) were enrolled; 82% were male, 61% were white, mean age was 52 (range 26–72), mean baseline HCV RNA was 6.7 log10 IU/mL (range 4.1–7.8), median baseline CD4 count was 662 cells/μL (Q1, Q3=469, 823), 20% had cirrhosis, 24% were IL28B CC genotype and 55% had failed prior HCV treatment. Patients were taking EFV (48%) or RAL (44%) or RPV (9%).
LDV/SOF administered once daily for 12 weeks is highly effective and well tolerated in treatment-naïve/experienced, GT1/4 HCV-infected patients with HIV-1 co-infection, including cirrhosis.
SVR12 by HIV ARV regimen and Overall
| (N=160) | (N=146) | (N=29) | (N=335) | |
| SVR12, n (%) | 151 (94) | 141 (97) | 28 (97) | 320 (96) |
| On-Treatment | 1 (<1) | 0 | 1 (3) | 2 (<1) |
| Failure, n (%) | ||||
| Relapse, n (%) | 8 (5) | 2 (1) | 0 | 10 (3) |
| Other, n (%) | 0 | 3 (2) | 0 | 3 (<1) |
Liver fibrosis progression is faster in HIV-HCV co-infected individuals due to an elevated inflammatory profile. Interferon Lambda-3 (IFNλ-3), encoded by the IFNL3 gene (formerly IL28B), has antiviral and pro-inflammatory properties, though reports of its association with liver fibrosis are inconsistent. Homozygous recessive SNPs (rs12979860CC, rs8099917TT) in this gene are linked to spontaneous HCV clearance and better treatment response, possibly via functional variant rs8103142, which causes a lysine-arginine substitution at position 70(K70R). We examined the relationship between IFNL3 genotypes and significant liver fibrosis (AST-to-platelet ratio index (APRI) ≥1.5) in HIV-HCV co-infected Canadians.
From the prospective Canadian Co-infection Cohort (n=1176), HCV RNA-positive participants free of fibrosis, end-stage liver disease and chronic Hepatitis B at baseline (n=612) were included. Cases (n=126) developed an APRI≥1.5 over follow-up. Cox proportional hazards was used, adjusting for sex, ethnicity, alcohol use, age, CD4 count (≤350 vs. 350+), HCV genotype 3 vs. non-3, baseline APRI, and a product term between rs8099917 and rs8103142. Multiple imputation accounted for missing data. Haplotype analysis was performed, adjusted for ethnicity.
Overall 69% were male with median HCV duration=17 years. 126 participants developed fibrosis over 1346 person-years of risk (event rate= 9.40/100 person-years, 95% CI=7.90, 11.20/100 p-y). Beneficial genotypes at rs8099917 and rs8103142 individually increase the risk of significant liver fibrosis, HR (95% CI) = 1.96 (1.18, 3.26) and 3.51 (1.15, 10.70), respectively. When present together, each genotype modifies the other (product term p-value=0.03), resulting in a protective effect, verified by haplotype analysis. Inheritance of TCT, a haplotype with beneficial alleles at all 3 SNPs, is protective (OR=0.52, 95% CI=0.39, 0.70).
Our results suggest that rs8099917 and rs81013142 are individually linked to a higher rate of liver fibrosis among HIV-HCV co-infected Canadians. When present together, these SNPs reduce fibrosis risk. Larger studies in other populations are needed to confirm these effects as well as functional studies to examine any potential biological interactions.
HCV-related disease is associated with duration of infection. Co-infected individuals may also have longer duration of HIV infection and increased risk for virologic failure on ART. Presence of HIV drug resistance may affect HCV antiviral therapy due to ART interactions with direct-acting antiviral agents (DAA’s). We evaluated the association between advanced liver fibrosis and presence of HIV drug resistance in co-infected patients in British Columbia, Canada.
Data were analyzed from a cohort of HIV-infected individuals followed through the Provincial HIV Drug Treatment Program between January 1, 1992 and December 31, 2013. Co-infection was defined as the presence of HCV antibody/RNA. Advanced fibrosis was defined on the basis of APRI ratio >1.5 or history of end-stage liver disease event. Factors associated with presence of NRTI/NNRTI resistance mutations were assessed in co-infected individuals using multivariable logistic regression adjusted for age at ART initiation, gender, HIV risk group (PWID vs. MSM), duration of ART use, and presence of advanced fibrosis.
Overall 8318 individuals were included, 42% were co-infected and 30% had evidence of prior NRTI/NNRTI resistance. Co-infected individuals were more likely to have history of IDU (39% vs. 2%, p<0.001), have evidence of resistance (34% vs. 29%, p<0.001) and be on a PI-based regimen (48% vs. 38%, p<0.001) than mono-infected individuals. Amongst co-infected individuals, those with advanced fibrosis (n=488, 14%) were more likely to have resistance (45% vs. 32%, p<0.001) compared to those without advanced disease. In multivariable analysis presence of resistance was associated with advanced fibrosis (odds ratio [OR] 1.34; 95% CI 1.08 – 1.65), age at ART start (0.88; 95% CI 0.80 – 0.96) and duration of ART use (OR 1.15; 95% CI 1.13 – 1.16).
HIV/HCV co-infected individuals with advanced fibrosis have higher burden of HIV drug resistance compared to those with less advanced liver disease. Presence of HIV drug resistance may limit HCV DAA options due to restriction of potential HIV regimen switches.
Currently there is little information on what is preventing high risk vulnerable populations from engaging in HCV care. The aim of this study was to survey this population using a targeted questionnaire and to identify barriers to HCV care. This was administered during community portable pop-up clinics (PPCs) at specific locations frequented by people who inject drugs (PWID).
Participants were recruited at PPCs held at different community-based centers in Vancouver’s Downtown East Side. During these PPCs OraQuick HCV Rapid Antibody point of care testing was offered. Participants who were tested were then offered to complete a questionnaire while they waited for test results.
Between January and December 2014, 581 individuals completed the questionnaire (123 female, 53.9% Caucasian, 32.0% First Nation; mean age: 45.2). Amongst all participants, 392 reported prior HCV testing (67.5%), 118 identified sharing needles (20.3%), 210 injected drugs (36.1%), 301 were previously incarcerated (51.8%), 299 were aware of a HCV cure (51.5%) and 452 stated they would consider treatment if they had HCV (77.8%). Among HCV positive individuals, 139 of 165 participants reported prior HCV testing (84.2%), 107 were aware of their positive status (64.9%), 113 were previously incarcerated (68.5%) and 114 would consider treatment (69.1%). With regards to HCV treatment, 39.2% of participants believed that they did not need treatment, 18.6% did not know where to go for treatment, 26.3% expressed concern regarding side effects of treatment, 19.5% did not believe they can financially afford it and 11.5% reported not wanting to consult a physician to receive treatment.
Barriers such as inaccessible medical care, unfamiliarity with available resources, and concerns regarding treatment side effects have been identified. Organized and targeted community events such as PPCs increase likelihood of reaching out to high risk inner city populations to address these barriers in a systematic way.
Anal infection with oncogenic, high-risk HPV (HR-HPV) is a key step in anal cancer pathogenesis, with HPV-16 and -18 being the types most commonly implicated. This is a significant health concern for MSM, and a malignancy disproportionately affecting those who are HIV-positive. With no large-scale anal cancer screening or HPV vaccination programs for MSM, and the suboptimal performance of cytology in predicting high-grade dysplasia, there is an urgent need to add to the evidence base to optimize management strategies for precancerous lesions.
HIV-positive and HIV-negative MSM were recruited from a large primary care clinic in Toronto. Participants completed a sociobehavioural questionnaire and self-collected anal swabs for HPV-DNA testing using a Luminex-based assay detecting 46 types, including 13 high-risk types. We compared the proportions with detectable HPV-DNA using Chi-square tests.
We recruited 442 MSM; 294 were HIV-positive and 148 HIV-negative. Median age was 45 and 44 years, respectively. The majority of HIV-positive and HIV-negative MSM had had ≥50 lifetime sexual partners (80.6% and 72.3%, respectively), and half of the men in each group reported anal sex in the preceding 6 months. The presence of any HPV type was more common in HIV-positive MSM (88.4% vs. 77.9%; P=0.0043), as was the presence of any HR-HPV (67.6% vs. 51.7%; P=0.0013) and ≥2 HR-HPV types (29.2% vs. 13.8%; P=0.0004). HR-HPV types were common among HIV-positive men; these differences were statistically-significant (P <0.05) for HPV-16 (34.9% vs. 23.5%) and HPV-18 (16.9% vs. 8.3%).
In this sexually-experienced group of MSM, anal HPV infection is very common, and a majority in both populations have infection with HR-HPV. These burdens are amplified for HIV-positive MSM. Our results support the need for HPV vaccination in MSM, and highlight the importance of further investigating the incorporation of HPV testing – particularly for HPV-16/-18 – along with cytology, into anal cancer screening algorithms for MSM.
HPV infection and cervical dysplasia are common among HIV positive women and progression to cervical cancer is more rapid. HPV vaccines have demonstrated high immune response and effectiveness in preventing HPV infection and cervical dysplasia in HIV-negative women, but data is limited in HIV-positive women.
Demographic, clinical and HIV data was collected as part of an open labeled, multi-centered study of seroresponsiveness to the quadravalent HPV vaccine in HIV-positive females. Participants received 3 doses (0,2,6 months) of the qHPV (types 6,11,16,18) vaccine. Cervical sampling was conducted using liquid based cytology (months 0,6,12,18,24) reported by Bethesda criteria, and HPV testing was done concurrently with the Linear array assay (Roche diagnostics).
Baseline cytology in 290 women was: 77% normal, 4% AS-CUS, 13% LSIL, 1% ASC-H, and 5% HSIL. Of the 170 women with month 18 or 24 post vaccine cytology, 97% received all three doses of the vaccine. These vaccinated participants had a median age of 40 years (IQR: 34–46; range: 16–66), median years since HIV diagnosis of 9 years (IQR: 5–12), median CD4 at first vaccination of 496/mm3 (IQR:383–684), and 71% had HIV RNA<50/mL. Of women with a normal cytology at baseline, 15(5.1%) progressed to ASCUS, LSIL or HSIL over 370 person-years of follow-up for this cohort. The incidence rate for abnormal cytologies was 4.1/100 person years (95% CI: (2.3–6.7)). None of the following risk factors for progression of cervical dyskariosis were statistically significant: age, CD4, nadir CD4, presence of oncogenic HPV, HIV viral load status.
Of the 15 women who progressed to cervical dyskariosis, none had evidence of new infection with HPV 6, 11, 16 or 18, suggesting the qHPV vaccine is protective against HPV-induced lesions caused by vaccine types. Even in a cohort of highly HPV exposed women.
Cervical cancer caused by oncogenic types of the human papillomavirus (HPV) is of concern among HIV-positive women due to impairment of immune responses required to control HPV infection. Fortunately, this cancer is preventable with Pap cytology screening followed by management of detected dysplasia. We calculated annual rates of cytology testing in 2008–2013 among screen-eligible women in Ontario and compared these by HIV status. We hypothesized that HIV-positive women would be more likely to undergo testing due to guideline recommendations and higher burden of disease.
We conducted a retrospective, population-based cohort study using health administrative data housed at the Institute for Clinical Evaluative Sciences. For each year in 2008–2013, eligible individuals were female, aged 21 to 70 years, alive, and residents of Ontario with no prior record of hysterectomy or cervical cancer diagnosis. HIV-positive women were identified using a validated algorithm (Antoniou PLoS One 2011). We report the annual proportions of women undergoing at least one cytologic test.
A total of 2,600 HIV-positive and 4,732,223 HIV-negative women were included in findings presented in the
| 2008 | 36.0 (33.5–38.5) | 37.1 (37.1–37.2) | 0.94 (0.88–1.01) |
| 2009 | 36.6 (34.2–39.1) | 37.4 (37.4–37.5) | 0.95 (0.89–1.02) |
| 2010 | 35.8 (33.5–38.2) | 36.5 (36.4–36.5) | 0.96 (0.90–1.02) |
| 2011 | 35.6 (33.3–37.9) | 36.0 (35.9–36.1) | 0.97 (0.91–1.03) |
| 2012 | 34.0 (31.9–36.2) | 31.7 (31.6–31.7) | 1.05 (0.99–1.12) |
| 2013 | 30.3 (28.3–32.3) | 19.8 (19.8–19.8) | 1.50 (1.40–1.60) |
Annual testing rates were low for HIV-positive women and, for most years, they were no more likely to undergo testing than HIV-negative women despite heightened cancer risk. There was a concerning drop among HIV-positive women following the 2012–2013 release of new guidelines for less frequent screening among HIV-negative women although annual screening continues to be recommended for HIV-positive women. Further research is needed to identify health system, provider, and individual barriers for screening to inform targeted interventions.
In order to effectively assess prevention and treatment of a communicable disease, we need to understand the perceptions that community members hold about that illness. Saskatchewan has had an disproportionately high incidence HIV rate for the last ten years, which the health sector has reduced from more than three times higher than the national average to two times higher (i.e., 16.2/100,000 vs. 5.9/100,000 nationally; SK Ministry of Health, 2012). In Saskatoon, which provides health support to northern SK communities, the incidence rate is slightly higher (i.e., 17.0/100,000) and accounts for more than 40% of new diagnoses (compared to Regina at 24% and Prince Albert at 12%; SHR, 2014). One could question the effectiveness of local HIV primary prevention practices and interventions methods, but many barriers contributed to the local HIV epidemic (e.g. limited access/engagement, service deficiencies, discrimination; Lang et al., 2013a). Community organizations have struggled to provide support, with HIV education as the minimum level of intervention. Health education is a common prevention strategy, which relies on the hypothesis that the right knowledge will lead individuals to make rationale choices to reduce risk (Younge et al., 2010). However, research with Saskatoon’s low-income communities most at risk for HIV has found that many of these community members possess atypical beliefs about health. This helps explain the low engagement in health behaviours, higher risk taking, and poorer health outcomes (Lang et al., 2013b). Given the health education hypothesis and the high HIV incidence rates found in these communities, one could deduce that these community members possess low factual knowledge about HIV. Working with community organizations to examine the impact of their organizational HIV intervention messaging, our study indicated that community members possess a relatively accurate knowledge of HIV. These results and the impact for HIV programming for low-income communities will be discussed.
Northern British Columbia is a region that comprises more than half the province – at approximately 500,000 km sq, it is twice the size of the United Kingdom. The Northern Health Authority (NHA), the northernmost regional health authority in BC, is tasked with providing healthcare services, including HIV prevention, treatment and care to 285,300 residents, dispersed in a range of urban centres, rural areas, and in some instances very remote locations. The challenges associated with healthcare delivery within such an expansive and diverse geographic area are multiple, including, for example, travel distances and costs and the limited number of healthcare professionals working on HIV/AIDS in BC’s North.
The purpose of this presentation is to share some of the key findings of the Moving Mountains project. The Moving Mountains HIV/AIDS Community Based Research (CBR) Conference held in Prince George, BC, in June, 2014, was born out of a desire to bring people together to review HIV/AIDS research underway, build the capacity to participate in CBR, and to identify gaps in information and priorities for future research – all with the goal of strengthening the regional response to HIV/AIDS.
The conference participants identified three intersecting priorities: (1) the impact of the natural resource development industries on the regional HIV/AIDS epidemic; and, (2) methods to reduce stigma and discrimination; (3) and improve HIV prevention, treatment and care.
Following the release of a community report and using the substantial interest generated by the event, the core research group sent an open call to participants of the conference to become part of a Research Working Group (RWG). This RWG has since convened with the purpose to further develop a research project(s) based on one or more of the identified research priority themes, and to design, develop and submit a grant application or applications to continue this work.
Sex Workers encounter social-structural barriers to accessing affordable housing (e.g., stigma, legal concerns), which compromise their capacity to negotiate safety when exchanging sex and enact sexual risk reduction. Meanwhile, social housing programs often perpetuate similar risks by requiring compliance with policies (e.g., curfews, guest policies) that undermine access to environmental supports, including indoor environments in which to exchange sex. Improvements to housing policies and interventions for sex workers are urgently needed. To this end, we undertook this community-based study to generate housing recommendations aligned with the lived experiences of sex workers.
We conducted a focus group with twenty-five sex workers using a modified World Café approach, focusing on the development of housing recommendations. Participants were recruited from among the membership of a peer-driven sex worker organization operating in Vancouver, BC. Recommendations were documented by the focus group facilitator, and other team members recorded field notes documenting the focus group discussion.
Participants underscored the need for increased availability of safe and affordable housing, and housing policies and interventions accounting for the lived experiences of sex workers. Participant emphasized the urgent need to: (1) reorient social housing policies to facilitate low-threshold access to housing (e.g., removing curfews) and indoor sex work environments (e.g., changing guest policies); (2) promote cultural safety within social housing for sex workers, including the adoption of peer-driven and cooperative housing models; and, (3) provide housing as part of integrated and peer-driven service delivery models that include comprehensive environmental supports (e.g., safer sex work spaces). Participants emphasized how the implementation of these recommendations would likely foster conditions that promote safety, sexual risk reduction, and well-being.
In addition to highlighting the urgent need for increased investment in affordable housing, this study demonstrates the need to align social housing models with the lived experience of sex workers in order to promote safety and sexual risk reduction.
Shared measurement systems require all programs or initiatives operating within a field to report on the same measures, using identical indicators and methodologies (Kramer, Vaidyanathan, & Parkhurst, 2009). Such systems have the potential to increase efficiency, learning and impact within any given field by accurately documenting the work that is collectively being conducted.
Led by the Pacific AIDS Network (PAN) − a network of over 50 community-based member organizations in British Columbia − and funded by the Provincial Health Services Authority, the Community HIV/HCV Evaluation and Reporting Tool (CHERT) is an online survey that collects annual data from community-based HIV/AIDS and/or Hepatitis C (HCV) organizations operating in BC. CHERT questions explore both process and outcome level indicators related to the programs’ prevention, testing, treatment and support services. One of the central objectives of the CHERT is to demonstrate the collective contribution community programs are making to the success of the provincial HIV/AIDS strategy. This presentation will explore the successes, challenges and lessons learned from the process of establishing this shared measurement system in BC. The CHERT has had many successes, for example to-date the CHERT has collected three years of data, with approximately 70% of PAN member organizations participating each year. This allows the sector to monitor performance and to track progress towards common goals. But there have also been some challenges and lessons learned. For instance, while CHERT respondents have a shared vision for change their approaches and structures are different, which makes the establishment of shared measures (i.e. indicators) challenging. It is also challenging to integrate different perspectives and needs into this type of tool – i.e. a community-based organization vs. a funding body such as a health authority.
From 2011–2014 CATIE developed an Ethnocultural Hepatitis C Outreach project that produced in-language hepatitis C resources and a media campaign for four major immigrant communities living in Ontario: Pakistani, Punjabi, Chinese and Filipino.
Of all the hepatitis C infections reported in Canada, 35 per cent are estimated to be among immigrants (PHAC, 2014). Immigrants also face cultural and linguistic barriers to healthcare, and in Canada immigrant health is shown to decline over time. In-language resources and community development are required to engage these communities in health issues.
The project was developed through partnership and community consultation including community advisory councils. The project implemented a multi-level strategy including education, outreach and social marketing. The education component included facilitator training, a multilingual website, in-language workshops and brochures. Educational tools developed include puzzles, case studies and curriculum. The outreach component included community partnerships and event outreach. The social marketing component included a media campaign across four communities, in print, radio and online. Evaluation activities included collecting key performance indicators (to measure the type and quantity of work performed) and in-person evaluation forms for workshops.
The project has partnered with 20 organizations. Event outreach each year reaches over 2000 people.
Three waves of a hepatitis C awareness campaign have run in up to 26 ethnocultural print, radio and online outlets. Editorial content was also produced including articles, radio interviews and tv programs.
Meaningful partnership, community engagement and a strong interest to raise awareness of hepatitis C within immigrant communities facilitated the project’s success and reach.
Funder: The Hepatitis C Secretariat, AIDS Bureau, Ontario Ministry of Health and Long Term Care.
An estimated 332,414 people in Canada are anti-HCV positive, some people will/ have cleared the infection while others will go onto chronic infection. Without treatment, chronic infection can lead to severe liver damage, liver cancer and liver failure. There is a lack of Canadian data on the information needs of people living with hepatitis C in order to help them manage their health.
CATIE developed a national needs assessment of patients engaged in hepatitis C care to inform the development of educational resources and services.
Through this national needs assessment we will learn about the types of information people with hepatitis C need and how they would like to receive it.
Twenty medical clinics across Canada serving people living with hepatitis C have been approached to request their help in recruiting people with hepatitis C who are receiving care to complete an online needs assessment. The needs assessment is available in both English and French through Fluid Survey. To date, eight clinics have agreed and 226 participants have been recruited. The aim is to recruit 250 participants by February 2015.
An analysis will be presented that includes information on demographics (age, gender, family background/place or origin) health and care (year of diagnosis, discussion of hepatitis C treatment options with physician; current hepatitis C treatment experience, involvement in decision making around hepatitis C care), knowledge needs (rating of current knowledge of hepatitis C, rating of need for hepatitis C information, importance of certain topic areas for hepatitis C information), and preferred formats to receive information on hepatitis C.
The information will be shared broadly through various knowledge exchange mechanisms in order to help strengthen programming for people with hepatitis C. The information will also be used to inform CATIE’s work in hepatitis C.
CATIE champions and supports innovation and excellence in knowledge exchange (KE) by collaborating with and building the capacity of front line organizations to use knowledge effectively to respond to HIV and hepatitis C. CATIE uses a variety of KE activities to strengthen the frontline response which are tailored according to the target audience, the content of the knowledge and the purpose of the knowledge for the target audience. Many of these approaches have been evaluated and provide important information and insight regarding effective KE strategies
Various approaches to evaluation of KE activities included online surveys and in-person evaluation forms. Standard evaluation indicators were used to assess key knowledge exchange outcomes, including relevance, usefulness, increased knowledge, and application of knowledge.
The evaluation results of five different methods of knowledge exchange were analyzed and compared. These methods were: 1) a deliberative dialogue on policy and programming implications of emerging biomedical research; 2) an online bulletin highlighting new prevention research and demonstrating how research findings can be integrated into frontline programming; 3) learning institutes that support frontline workers to learn from research conferences and share findings with their communities; 4) a health magazine written by and for people living with HIV and 5) a website providing comprehensive information on HIV and hepatitis C.
Based on the participant/user evaluations, these methods of knowledge exchange were relevant (96% to 100%) and useful (87% to 100%). They resulted in increased knowledge (94% to 98%) that was applied to the frontline response (94% to 100%). The evaluations also provided insight into the factors that contribute to successful knowledge exchange.
KE methods that are tailored to the audience, content and purpose for KE, can strengthen the frontline response to HIV and hepatitis C.
Scientific evidence indicates that harm reduction services prevent the spread of HIV/AIDS and other diseases, reduce the risk of overdose death, and connect people who use illicit drugs to addiction treatment and other health and social services. Under an instrumental-rational view of health policy making, a large evidence base supporting the cost-effectiveness of harm reduction interventions should translate into unproblematic policy support for the uptake of this approach as a routine component of health services. However, Canadian provinces and territories exhibit wide variability with respect to implementation of these services, and the approach remains highly contentious. Unfortunately, little research to date has explored factors that underlie the diversity of these services across Canada. To address this gap we have assembled a team of academics and national and provincial-level knowledge users to implement a mixed-method, multiple case, policy analysis study. Drawing on four provincial/territorial data sources: policy documents, media articles, key informant interviews, and a national public opinion survey we will 1) systematically document and compare the strength of each province and territory’s harm reduction policy framework (including funding commitments, governance structures, etc.) and 2) explore relationships between the comprehensiveness of these policy frameworks and the way media, stakeholders, and the public frame these services.
Funding for this project has been secured and implementation is underway. This presentation will describe our novel methodology and introduce this project to potential key informants and additional interested knowledge users. Our findings are expected to contribute to academic scholarship on contentious health interventions for socially marginalized target populations, and will generate timely data to assist knowledge users in their efforts to advocate for equitable evidence-based policy supporting the expansion of harm reduction services across Canada.
Despite successes in preventing and treating HIV infection in British Columbia in recent years, young Aboriginal people continue to face disproportionately higher risk of infection, HIV-related mortality and challenges accessing ART treatment. Disparities in rates of infection and access to treatment between Aboriginal and non-Aboriginal people remain among people who use injection drugs.
Since 2003, The Cedar Project has investigated HIV and HCV vulnerabilities among young Aboriginal people who use drugs in British Columbia. The Cedar Project mHealth study was initiated to explore whether the WelTel mHealth intervention, shown effective in improving HIV health outcomes in Sub-Saharan Africa, has the potential to improve HIV prevention and treatment among young Aboriginal people who use illicit drugs. Initial cross-sectional surveys were conducted to understand current mobile phone use patterns and interest in an mHealth program among Cedar Project participants.
Of 82 participants who responded to an initial feasibility survey, 43 (52%) do not currently own a phone. Of these, 8 people shared a mobile phone with someone else, including a family member or regular partner. Twenty-seven (69%) participants with phones reported using the Internet on their devices. A majority of people surveyed (95%) felt that using a mobile phone for health would be helpful to them. Commonly cited reasons for wanting the mHealth intervention include increased safety, access to services, and connection with family. Three participants had concerns about using a mobile phone for their health, including challenges with technology and privacy concerns.
Lack of access to phones among young Aboriginal people who use drugs in Vancouver and Prince George, British Columbia is a key obstacle in initiating an mHealth intervention focused on improving HIV prevention and treatment. However, young people are eager to have access to this technology as a means to improve their health.
To respond to the lack of HIV prevention interventions for positive gay men in Colombia, we sought to further adapt a positive prevention intervention already adapted for positive Latino gay men living in Canada.
During March 2014, the Spanish language version of Gay Poz Sex (GPS) was adapted and tested using pilot qualitative and quantitative data gathered from 5 gay men living with HIV in Cali, Colombia. Two facilitators were trained after the adaptation process. The facilitations delivered GPS to one group of 3 HIV positive gay men. A psychologist followed the sessions and assessed the facilitators regarding compliance with the intervention objectives and motivational interviewing counselling (MI). Qualitative interviews with the recipients of GPS were processed and summarized in order to improve the further implementations of GPS.
The adapted GPS was highly acceptable to Colombian HIV-positive gay men. Minimum adaptation of materials was needed and included language and local cultural specificities (i.e. more information about treatment, Colombian laws regarding HIV and sexual rights). The use of sexual diaries, the fact that the facilitators were gay and HIV positive, and the level of knowledge of the facilitators were positive aspects of the implementation. Participants felt that they still need to be more conformtable using condoms in oral sex, but all achieved close 100% use of condoms in their anal sex. In addition, they reported GPS helped them to increase 1) HIV knowledge, 2) confidence in having an stable partner, and 3) self-esteem. Facilitators met the objectives and very little extra training was needed as a way to improve their MI skills. Participants suggested changes to GPS implementation including increasing time of each session, more audiovisual tools, bigger groups of men of similar ages, and additional peer support after the intervention.
The result of this process is an adapted positive intervention of gay men in Colombia, with training materials and very acceptable for local communities. Further results of current implementation will help us to assess effectiveness of GPS.
A strategy that incorporates peer facilitation and RDS recruitment for offering HIV/Syphilis counselling and testing is being currently conducted. The strategy impact on engaging most-at-risk populations, and linking to care those who result positive, is preliminarily assessed.
Leaders belonging to target communities (Men who have sex with men, transgender women and afro-Colombian minorities) were trained as counsellors. They selected a number of “seeds” for starting a RDS recruitment network based the seeds connections with the community. Seeds and all subsequent participants were directed to ask three people, whom they considered at risk, to participate in the study.
The intervention included: a) pre and post- test HIV/STI counselling; b) a structured questionnaire; c) HIV/Syphilis rapid testing; d) collection of samples for confirmatory tests and referral to medical care, if the HIV rapid test was reactive; and e) referral for complementary testing and medical care, if the syphilis rapid test was reactive.
Since July 2014, 174 participants have been recruited from 17 seeds. The rate of HIV+ cases for both screening and complementary tests was 8.2%, the rate of return for confirmatory HIV tests results was 71.4%. 64.3% of HIV positive cases were linked to care, 80% of which had a CD4 count over 500 cells/mm3. 16.1% rapid tests for syphilis were positive; 57% returned for complementary tests and 46.4% returned for medical attention.
These results suggest that the proposed recruitment strategy has been effective in reaching at risk population, reflected in the high frequencies of reactive HIV and syphilis rapid tests. A high rate of return for HIV results has been achieved, possibly attributable to peer facilitation. Once the sample size is achieved, these findings will be further explored; also, a qualitative assessment of the RDS methodology will be performed, and the strategy impact will benchmarked against other initiatives.
This research examines a pilot project for HIV Point of Care (POC) testing in four pharmacies across Vancouver and Vancouver Island, Canada. The pilot is a joint collaboration between Vancouver Coastal Health, Island Health, Pharmacy BC and Medicine Shoppe pharmacy owners. The pilot aims to provide HIV testing to individuals who may not have obtained a test through more conventional methods such as via their family physician or hospital emergency department.
The development of AIDS shortly after diagnosis indicates late testing (CDC, 2010). By providing an alternative option to obtain testing, the ultimate goal of this pilot is to ensure that individuals are diagnosed with HIV in the early onset of the disease.
The pilot was initiated in July 2014 and extends into 2015. Clients volunteered to obtain free POC testing, offered by pharmacists, visiting four pharmacies in Vancouver, Victoria and Nanaimo. Quantitative data was collected using Point of Care testing screeners administered by the pharmacists.
Between July and November 2014, 404 individuals obtained an HIV POC at the Vancouver pharmacies. 68 tests were conducted at the Vancouver Island sites between launch and October 2014.The pilot succeeded in its goal to test hard to reach clients as well as unsuspected population: in Vancouver, the majority of individuals who tested were first time testers (74%), and Asian ethnicity (60%). Island sites saw the majority of individuals identifying as Caucasian (88%) and male (56%).
The pharmacies attracted a large number of first time testers. The results of this short pilot suggests that offering access to Point of Care testing within low-barrier settings such as community-based pharmacies can be effective in reaching populations that tend to avoid testing in more traditional environments.
To test the efficacy of a brief motivational interviewing (MI) intervention to reduce high-risk injection behaviours over a 6-month period compared with a brief educational intervention (EI).
A single site 2-group parallel randomized controlled trial was carried out. People who injected drugs (PWID) and had shared drug injection equipment or shared drugs by backloading or frontloading in the month prior to recruitment were randomized to either the MI or EI group. The interventions (at baseline) and interviews (baseline and 3 and 6 months later) took place at the study office located in downtown Montréal, close to the community-based harm reduction programs where PWID were recruited. The primary outcome was defined as reporting any of the following risk behaviours in the month prior to interview: having shared syringes, containers, filters or water to inject drugs, and backloading/frontloading; each behaviour was examined separately, as secondary outcomes. Intent-to-treat analyses were carried out using generalized linear mixed-effects model with an underlying logistic distribution for dichotomous outcomes.
The final sample consisted of 219 participants (mean age 37.85 years; 82.2% male) including 108 and 111 participants in the EI and the MI group respectively. The probability of reporting a risk injection behaviour decreased over the study period in both groups. Further, after 6 months of follow-up, participants who reported any risk behaviour were 50% (OR=0.50; CI: 0.13–0.87) less likely to be in the MI group than in the EI group as well as those who reported sharing containers (OR=0.50; CI:0.09–0.90). PWID who reported sharing equipment excluding syringes were 53% less likely to be in the MI group (OR = 0.47; CI: 0.11–0.84).
A brief motivational interviewing intervention was more effective than a brief educational intervention in reducing some high risk injecting behaviours up in the subsequent 6 months.
Annual cervical cancer screening is recommended in Canada for women living with HIV (WLWH). We assessed the prevalence of annual Pap tests and measured socio-spatial disparities to Pap testing among WLWH across three Canadian provinces.
Baseline survey data were analyzed among WLWH (≥16 years) enrolled in the Canadian HIV Women’s Sexual and Reproductive Health Cohort Study (CHIWOS), a longitudinal, community-based research study in British Columbia (BC), Ontario (ON) and Quebec (QC). The questionnaire assessed medical history, use of clinical and social services (including Pap testing), health outcomes, and socio-spatial determinants of health. For this analysis, eligibility criteria included having a cervix and a uterus. Multivariable logistic regression identified independent correlates of annual Pap testing.
Among 946 women, 24% were from BC, 55% from ON and 21% from QC. The median age was 41 [IQR: 35–49.5] and 22% reported being partnered or married. In the sample, 37% identified as Caucasian, 31% as African/Caribbean/Black, and 23% as Aboriginal. Overall, 71% of WLWH in the study reported receipt of Pap test in the previous year, with significant variation by province (BC: 82%, ON: 65%, QC: 77%) and the majority (73%) receiving Pap tests at their HIV clinic. There were no significant differences in spatial variables (distance to HIV care, length of travel time, location of clinic) between women who did and did not report annual Pap testing. In the multivariable logistic regression model, women who identified as African/Caribbean/Black (Adjusted Odds Ratio [AOR]=2.02, 95% Confidence Interval (CI): 1.3–3.14) and had a female primary HIV doctor (AOR=2.27, 95% CI: 1.59–3.25) were more likely to report annual Pap testing.
In this analysis, receipt of Pap testing was associated with having a female primary HIV doctor and identifying as African/Caribbean/Black among a cohort of WLWH with relatively high rates of cervical cancer screening. Significant social disparities exist in Pap testing practices among WLWH in Canada. Experiences of where and how WLWH access cervical cancer screening will be further investigated.
The BC Harm Reduction Supplies and Services (HRSS) committee coordinates harm reduction supply and service delivery in partnership with the province’s Health Authorities (HAs), including Vancouver Coastal Health (VCH). VCH has over 150 registered harm reduction supply distribution sites, which provide services to reduce harms related to substance use and/or sexual practices. HIV prevention is central to the provision of harm reduction supplies and delivery of harm reduction services. Ongoing program evaluation and quality improvements are necessary to ensure harm reduction services are effective and meeting the needs of those most at risk for substance use / sexual health harms, including HIV. In 2014 BC HRSS partnered with VCH to conduct over 600 Harm Reduction Site Surveys at 20 sites across VCH. These surveys capture information on demographics, drug use trends, site usage, access to supplies, overdose experiences, potentially harmful practices, use of methadose, and need for supervised and/or assisted injection. Participants received a $5 gift card.
BC HRSS Survey responses are entered and SPSS analysis has begun. In January 2015, initial findings will be examined by the VCH Harm Reduction Coordinating Committee. Findings will be used to prioritize improvements to the delivery of harm reduction services at each site surveyed, and will guide quality improvements across the VCH region.
Quality improvements will address gaps in policies and practices, including in harm reduction supply availability, educational materials, overdose prevention and intervention services, and other services aimed at preventing HIV and other substance use / sexual health harms.
Despite effective therapies to treat HIV, a substantial proportion of newly-diagnosed HIV positive individuals continue to present at advanced stages of their disease. Understanding utilization patterns of HIV positive individuals prior to diagnosis can inform earlier diagnosis and engagement strategies. We examined rates of ambulatory care visits and hospitalizations among HIV cases and compared them to HIV negative controls.
Data were from a retrospective cohort from Manitoba, Canada. Participants included HIV-positive individuals presenting to care between 2007–2011 to the Manitoba HIV program (MHP), and HIV-negative controls, matched (1:5) by age, sex and region. Clinical information was linked to population-based administrative databases, including physician visits and hospitalizations. Rates were calculated with utilizations as the numerator, and the number of days registered with the single insurance provider, prior to case date, as the denominator. Case date was defined as first clinic visit with the MHP. Diagnoses associated with physician visits were classified according to International Classification of Diseases chapters. Stratified Poisson regression models were used to compare rates between cases/controls, with relative rates (RRs) and their 95% confidence intervals (95% CI) are reported.
A total of 164 cases and 809 controls were included. Physician visits and hospitalizations were higher for cases, compared to controls. In the 5 years prior to case date, compared to their HIV-negative controls, cases were more likely to be diagnosed with “Infectious and Parasitic Conditions” (RR: 4.7, 95% CI: 4.3–5.1; p<0.0001) and “Blood and Blood Disorders (RR: 3.1, 95% CI: 2.6–3.7; p<0.0001). Similarly, hospitalization rates were higher among cases (RR: 2.5, 95% CI: 2.1–2.9; p<0.0001).
The higher use of health services by those subsequently diagnosed with HIV present lost opportunities for screening, diagnosis and treatment. This research was conducted in partnership with program managers and policy makers and these results will provide important information for both prevention and care services within the MHP and Manitoba Health.
HIV and STI rates are very high among female sex workers (FSWs) in Ukraine. Despite active HIV/STI prevention programmes, very little is known about their reproductive health needs. In 2012, UNICEF Ukraine, the University of Manitoba, and the Ukrainian Institute for Social Research conducted epidemiological mapping in Zaporizhzhya, Ukraine to characterise the local HIV epidemic, with specific focus on FSWs. The project also focused on understanding their reproductive health needs.
Behavioural surveys were conducted by members of local, key populations who were trained as field researchers. Epidemiological mapping identified “hotspots” in Zaporizhzhya where FSWs congregate. Multistage cluster sampling was used to recruit participants from these hotspots. Descriptive statistical analyses are presented.
Of 124 self-identified FSWs, 34% reported seeing ≥10 clients in the past week, while 56% had seen ≥3 clients in the last day. Despite this high risk, only 40.2% of participants had ever been tested for HIV. Nearly 60% of participants reported ever being pregnant and 53.3% had received abortion services in the past. However, utilisation of reproductive health services was low. Only 16.2% had ever visited a health facility for obstetrical/gynaecological services, 10.8% for STI-related services, and less than one-third of participants had ever had a Pap test. Over 97% of women reported ever using contraceptives, but when asked about family planning strategies, 100% mentioned solely using condoms and only 18.3% had used oral contraceptives, while 14.2% had relied on coitus interruptus at some point.
Dual methods of protection are emphasized for FSWs. Our findings highlight the need for healthcare facilities and programmes to focus on enhancing accessibility of reproductive health services. Condoms and other modern, non-barrier methods of contraception must be made easily available to women involved in sex work, in addition to conventional HIV prevention services. As well, further research is needed to understand potential barriers to utilisation of dual methods of protection among FSWs in Ukraine.
The Canadian HIV Vaccine Initiative (CHVI) is a partnership between the Government of Canada and the Bill and Melinda Gates Foundation with the goal of developing a safe, affordable, effective and globally accessible HIV vaccine. Given that several major vaccine clinical trials are targeted for HIV endemic regions of the word such as Sub-Saharan Africa, Health Canada’s role within the CHVI is to assist in strengthening regulatory capacity building in developing National Regulatory Authorities (NRAs) in collaboration with the World Health Organization and other international organizations. The objective is to help protect the ethical and scientific integrity of the vaccine clinical trials and speed the development and approval of HIV vaccines.
The regulatory needs of the NRAs were identified using surveys, face-to face meetings and teleconferences. In cooperation with the NRAs, Health Canada regulatory experts develop training materials and conduct workshops using case studies and interactive approaches. Training sessions included topics such as clinical trial application review for both clinical and quality elements, as well as the role of risk benefit assessment in regulatory decisions.
Since 2010, Health Canada has trained over 200 participants from more than 40 countries under the CHVI. This includes the establishment and continued mentorship with regulatory agencies in Nigeria and Malawi and regional training in Southern and Eastern Africa. Health Canada also organises annual International Regulatory Forum and sponsors participants from different NRAs to provide learning opportunities and encourage the exchange of best regulatory practices.
Health Canada has been successfully implementing capacity building activities in collaboration with NRAs to enhance their regulation of vaccines. Moving forward, training efforts will utilize a regional approach in order to maximize training of regulatory staff in countries with similar training needs.
This study was aimed at examining the association between the age of women at first marriage and uptake of contraceptive services among women of reproductive age (15–49 years) in Nigeria.
This study involved a re-analysis of 2008 Nigeria demographic and health survey (NDHS) data set. This is national surveys and the design used was a cross-sectional population based study design. The primary data obtained from the survey were collected with interviewer administered questionnaires. Data was summarized as proportion and graph was used in representing current contraceptive use by age at first marriage. A chi square was used to assess association between variables. Level of significant was set at 5%.
The total numbers of respondents studied were 13,678. Result showed that only 14.7% of the respondents were currently using a method, majority of the respondents (40.5%) were between the age group 25–34, 47.4% had no formal education, 43.2% married at age (15–19). The following characteristics of respondents were found to be associated with contraceptive uptake: being 25 and above years [O.R = 2.25; 95%C.I=1.94–2.61] and [OR= 2.50; 95% CI=2.15–2.90] for women aged 25–34 and 35–49 respectively, having at least a form of formal education [OR= 4.96; 95% CI= 4.24–5.79], [OR= 8.47; 95% CI = 7.32–9.81] and [OR=13.53; 95% CI=11.37–16.11] for primary, secondary and tertiary education respectively, being married at the age 14 and below [OR= 0.23; 95% CI= 0.20–0.27]. Findings from this study showed that a high proportions of sexually active women aged 15–49 years in Nigeria are currently not using a method of contraceptive despite widespread of the knowledge of the services. There is an urgent need to address this through improved political will, promotion of girl child education, empowerment of female so as to achieve millennium development goals.
Strategies to improve access to healthcare for people living with HIV (PLWH) have demonstrated limited success. Whereas previous approaches have been informed by the views of health providers and decision-makers, it is believed that incorporating patient perspectives into the ways we design and evaluate healthcare programs will lead to improved access and connections to healthcare services.
To map the literature on the perspectives of PLWH concerning access to healthcare services; identify gaps in evidence, and to produce an evidence-informed research action plan to guide LHIV program of research.
This scoping review includes peer-reviewed and grey literature from 1946 to May 2014 using double extraction. Variations of the search terms “HIV“, “patient satisfaction“ and “health services accessibility” were used to include relevant literature. The search strategy was developed in consultation with content experts, review methodologists and a librarian, and validated using “gold standard” studies identified by those stakeholders. Inclusion criteria were: 1) study includes the perspectives of PLWH 2) study design includes qualitative, quantitative, and mixed methods, and 3) outcome measures are limited to patient satisfaction, their implied needs, beliefs and desires in relation to access to healthcare. The papers were extracted by two independent reviewers, and quality of extracted papers was assessed. Data was collated, summarized and thematically analyzed.
Of the 20,694 references retrieved, 326 documents were identified as eligible in pre-screening and 67 articles met the inclusion criteria (54% qualitative studies, 39% quantitative studies and 7% mixed-method studies). Only four studies were conducted in Canada. Data extraction and synthesis in progress (ready for presentation at conference).
This scoping review recorded and characterized the extensive body of literature on perspectives of PLWH regarding access to healthcare. A literature repository was developed to assist stakeholders, decision-makers, and PLWH in developing and implementing patient-oriented healthcare programs.
Patient-centred perspectives are important for determining what people living with HIV (PLWH) value when making choices. We used a discrete choice experiment to elicit preferences from PLWH about the relative importance of attributes of antiretroviral drugs.
Participants selected their preferred antiretroviral regimen based on six attributes: efficacy in suppressing viral load, cardiovascular risk, wrist and hip fracture risk, mood effects, pill burden, and potential for future resistance. Each choice set consisted of 3 hypothetical regimens characterized by varying levels of these attributes. Participants completed 16 sets and a consistency test; we excluded participants with inconsistent responses. We recruited participants from clinics and AIDS service organizations, analyzed responses using mixed logit regression, and expressed results as how much inefficacy (increased risk of virologic failure) participants would be willing to accept to avoid negative attributes.
We analyzed data from 127 of 135 participants. Participants were willing to accept some inefficacy (risk of virologic failure) to avoid less convenient dosing, viral resistance, and all toxicities except wrist fracture. Participants were willing to accept a regimen that was 38.4% less effective (95% confidence interval [95CI] 25.6–51.1) but without cardiovascular toxicity than one with a greatly increased heart attack risk. To avoid a regimen with a high chance of future resistance (compared to almost zero), participants were willing to accept regimens that were 38.3% less effective (95CI 25.6–50.9). Participants were willing to accept one pill once daily regimens that were up to 11.5% (95CI 5.6–17.4) less effective than regimens with greater pill burdens. The greatest heterogeneity of responses was for preferences about cardiovascular risk, mood, and risk of resistance.
PLWH are willing to accept regimens that are significantly less effective to avoid major toxicities and many are concerned about resistance. The heterogeneity of preferences indicates the need for multiple treatment options.
The Patient Assessment of Chronic Illness Care (PACIC) survey assesses patient-centredness of care delivered through the Chronic Care Model. The PACIC provides an overall score as well as subscale scores for five areas of care (patient activation, decision support, goal setting, problem-solving, and follow-up), and has been validated in many chronic diseases including diabetes, cardiovascular disease and mental health, but not for HIV.
This pilot study aimed to investigate the validity of PACIC in chronic HIV care.
English-speaking HIV+ adults attending the provincial HIV clinic between January and July 2014 (n=95).
A telephone survey was administered to consenting, eligible patients. The survey included items from the PACIC, socio-demographic characteristics, and additional questions assessing satisfaction with care. The survey was pretested for face and content validity. Results presented are for PACIC items only. Internal consistency was evaluated using Cronbach’s alpha. Inter-item correlation between overall PACIC score and subscales was assessed using Pearson correlation. Factorial structure was examined by factor analyses of complete cases. Score associations with so-ciodemographic characteristics were evaluated using T-test and ANOVA.
Response rate was 52% (49/95 patients). Overall mean (sd) PACIC score was 3.11 (0.83) out of 5, with subscale scores ranging between 2.91 (1.13) and 3.97 (1.11). The Pearson correlation between overall score and the subscales ranged from 0.57 to 0.86. Cronbach’s alpha was 0.87 (range: 0.61 and 0.77 for subscales). Exploratory factor analyses for PACIC and subscales distinguished a 5 factor model, explaining 80% of the variation. Scores were not associated with age, sex, years living with HIV, living in rural area or type of clinic attended (P >0.05).
The PACIC has good face and construct validity in chronic HIV care. Validation in a larger population of HIV+ patients is warranted.
Matched cohort studies are increasingly common across health disciplines. The unique socio-demographic characteristics of people living with HIV make generalized comparisons to population data challenging. Ensuring that comparisons between data collected from people living with HIV and ‘control’ data do not overlook possible confounders is tantamount to a well-designed study.
This abstract draws on recent discussions regarding the development of matching criteria to form matched cohorts of HIV negative women for the HIV Mothering Study and the Angiogenesis and Adverse Pregnancy Outcomes (AAPH) studies in Ontario, Canada.
Our analysis focuses on the methodological and logistical challenges of developing matched cohorts that permit robust comparison analyses, while balancing feasibility challenges associated with matching. These challenges relate to: identifying requisite matching criteria to ensure reliability of the matched results, particularly in the field of basic science; selecting matching criteria that are specific, such that the comparative results will be reliable, but not prohibitive in terms of finding a suitable match; considering what comparators will be of interest for analysis and excluding these criteria for matching; and exploring the suitability of recruitment sites for matched cohorts to optimize recruitment success. Furthermore, in reflecting on our decision making processes, ethical challenges associated with matching diverse groups of women who are marginalized on a range of issues, including the matching criteria that were selected, have also emerged as an important considerations.
The need for matched cohorts in HIV research will continue to grow. Tackling the challenges associated with this methodology, and developing a strong knowledge base in the experience of matching, will facilitate improved research processes and successful outcomes. Both methodological and ethical tensions must be fully addressed in order to do so.
This study describes the sociodemographic, clinical and social attributes of persons living with HIV/AIDS (PHA) in home and institutional care settings in Ontario. Population aging and successful drug therapy in HIV management mean that more people are living longer with HIV presenting an emerging public health trend. As these individuals age, they become more at risk of developing other chronic health conditions which can have implications for disease management. Aging PHA may require more care in both the home and institutional settings.
This quantitative analysis used secondary data collected from the interRAI home care and minimum data set instruments in Ontario between 2003 and 2011. The total sample included 837,652 individuals. Descriptive analysis was used to outline key attributes of people living with and without HIV in long-term care (LTC), complex continuing care (CCC), and home care (HC) settings. Differences between PHA across the three settings were compared using Chi-square analysis.
1,091 PHA were identified (4.4%, 32.6% and 63.0% in LTC, CCC and HC, respectively). PHA were predominantly male and much younger than others in all three settings; in CCC, more than 70% of PHA were younger than 54 years. PHA were less likely to be married, had less comorbidity and physical impairment and poorer self-rated health than other populations in the same care settings. PHA also exhibited more social isolation, had more conflicts with others and received more psychotropic medications.
Aging PHA represent a complex population with distinctive health needs. A better understanding of these needs will help to develop strategies to provide better care across care settings and improve the quality of life of this group. InterRAI standardized assessment instruments may be important tools for benchmarking, evaluating, and quality measurement. As a continuation of this project, a larger analysis across Canada is underway.
Housing instability is a complex issue and important determinant of health for Aboriginal people affected by HIV and AIDS across Canada. Aboriginal people often live in housing that incorporates Eurocentric constructs of family and home in the design. Stable Homes, Strong Families (SHSF) is a community-based research (CBR) project that aims to develop cultural understandings of housing and home amongst Aboriginal people to influence housing programs and policies.
Digital Storytelling (DS) was chosen as a medium to explore cultural understandings and meanings of home because of its participatory, arts-based approach that increases community involvement in the research process. Five workshops were held with 22 participants across Canada from June 2013 to November 2014.
DS is an engaging methodology to explore cultural understandings of housing and other issues that intersect with meanings of home and family amongst Aboriginal peoples across Canada. The DS workshops highlighted a number of important issues when conducting CBR with Aboriginal communities. Sufficient time must be invested for local promotion and recruitment, especially to understand the needs of workshop participants, and for the planning committee to adapt the workshop template to the local context. Early engagement with an Elder and community supports is essential to creating a safe space for all involved and to ensure local Aboriginal cultural protocols are respected. Although there is tremendous opportunity to integrate ceremony throughout the workshop, our team recognizes the tension between the heavy reliance on technology for DS and our commitment to Aboriginal oral traditions and decolonizing methodologies.
DS workshops are more than an arts-based methodology to collect data and must be carefully planned and executed. DS is complimentary to Aboriginal oral traditions, however the tensions and limitations of employing DS as a decolonizing research approach will continue to be discussed amongst the SHSF team.
While there is much research regarding HIV testing in aboriginal and general populations, less information exists regarding knowledge of HIV testing. This study was conducted to investigate differences in HIV testing knowledge among groups of aboriginal Canadians.
In 2012, PHAC conducted a national telephone survey to examine HIV attitudes and knowledge among the general Canadian population (including First Nations Canadians living on reserves). Interviews were conducted at EKOS call centres in Ottawa. Up to eight call-backs were made to unresponsive contacts, with follow-up calls made on successive days at varying times (respondents also had the option to reschedule). Daily records were kept of all calls made, whether successful (completed or rescheduled interviews) or not. Data were extracted via the Open Data portal of the Government of Canada, with Pearson’s chi-squared and Fisher’s exact used to examine differences (n=411).
Overall, knowledge of HIV testing via blood test varied significantly between aboriginal groups (p<0.001). More than 90% of First Nations Canadians correctly knew that HIV could be diagnosed by a blood test. While on-reserve First Nations individuals were slightly more knowledgeable (with 96.1% answering correctly) than those living off-reserve (91.4%), the difference between these groups was not significant. In addition, Métis individuals also demonstrated high levels of knowledge, with 95.1% answering correctly. However, among Inuit Canadians, knowledge was particularly low. Just 80.4% of respondents knew that HIV could be diagnosed by blood test.
Though knowledge was high among other aboriginal groups surveyed, one in five Inuit Canadians were unaware that HIV could be diagnosed by a blood test. This finding may have been influenced by cultural or language barriers, as well as limited by small sample sizes. Results (while limited by small sample sizes) demonstrate a need to re-evaluate knowledge translation and health promotion efforts among this population.
CATIE champions and supports innovation and excellence in HIV knowledge exchange by collaborating with and building the capacity of front line organizations to use knowledge effectively to respond to the HIV epidemic; supporting and connecting people with HIV, other individuals, and organizations to develop, synthesize, share and apply HIV knowledge; and acting as a central contact point for the flow of comprehensive, accurate, unbiased, timely and accessible HIV information and community-based knowledge.
In 2014, a national survey of frontline workers was conducted to assess the impact of CATIE’s complement of programs and services. Frequency descriptives from the 369 front-line workers who responded to the survey have been complied.
Based on the responses, CATIE is doing a good job at reaching its intended audiences. Respondents came from the diverse array of frontline organizations working in HIV, hepatitis C and sexually transmitted infections (STIs) – most of whom (>83%) work from an integrated STBBI approach. Collectively, these organizations provide a full range of HIV and HCV services; target a diversity of populations; and provide services across Canada.
The information provided by CATIE is useful to frontline workers (95%) and their clients (91%). The diverse channels used by CATIE for information dissemination (web, print, email, in-person) were all highly rated, indicating the strength of CATIE’s model for accessible knowledge exchange. CATIE’s work is having a significant impact on the national response to HIV and hepatitis C. CATIE has been successful at building frontline workers’ knowledge of HIV (96%) and hepatitis C (97%). Frontline workers report they can use this knowledge in their work (97%). Almost all respondents (94%) reported using information from CATIE to educate or inform others. In addition, 76% of frontline workers had used information from CATIE to change work practices and/or to change programming.
According to front line workers, CATIE’s services and resources are effective vehicles for knowledge exchange.
Despite decades of HIV prevention efforts, new infections continue in Canada. Access to and ease of uptake of HIV testing remains an important element in both HIV prevention and treatment. As it currently stands, HIV testing in Nova Scotia requires access to a health care provider for a testing requisition or accessing 1 of 2 anonymous HIV testing sites in the province. Although HIV point-of-care-testing (HIV POCT) was first introduced in Canada in 2000, and now available in most jurisdictions, it is not available in Nova Scotia.
This 8-month pilot project sought to determine both the acceptability and uptake of rapid HIV point-of-care testing (POCT) among a sample of hard-to-access populations in Halifax who may be insecurely housed, street-involved, currently using injection drugs, or involved in methadone maintenance therapy. Four currently established mobile (n=2) and fixed (n=2) outreach services in Halifax were used to offer HIV POCT in partnership with a street outreach nurse.
A total of 305 individuals accessing these various outreach services were offered HIV POCT, and of these, 67 individuals accepted. The majority of those offered HIV POCT asked for HCV POCT and declined HIV POCT when told HCV POCT testing was not offered as part of the pilot project. All HIV POCT tests were found nonreactive, and were confirmed through laboratory testing.
The overall low uptake rate found among the participants of this HIV POCT pilot project reflects, in part, the need to continue to address the basic determinants of health and related social needs of hard-to-access populations, including the need for stable housing and access to nutritious food. As evidenced by the low uptake of stand-alone HIV POCT among this population, greater emphasis on testing innovation such as HCV/HIV multiplex POCT in both fixed and mobile outreach settings, and increased awareness of testing options is warranted.
Our community-based research study sought to investigate the needs and interests of gay, bisexual, and other men who have sex with men (MSM) in Ontario’s Simcoe-Muskoka region.
MSM aged 18+ were recruited via online sociosexual websites (e.g., Squirt) and in-person LGTBQ-targetted events/services from June–November 2014 to complete an anonymous 20–30 minute online questionnaire detailing their community and sexual health wellness as well as their demographics. Multivariable logistic regression (p<0.05 significant) was used to identify factors associated with particular interests, service use, and characteristics of our sample.
A total of 230 eligible participants at least partially completed the questionnaire (57.8% completion rate). Our sample was 61.0% gay-identified, 58.7% raised locally in Simcoe-Muskoka, median age 43 (range 18–82), and predominantly HIV-negative (73.1%) or unsure of their status (23.4%). Locally-raised men were less likely to travel to Toronto for sex with other men (OR=0.42, 95% CI:0.24–0.72) and HIV-positive men were more likely to travel to Toronto for care (OR=19.4, 95% CI:2.17–173.61). Interest in a coming out support group was less likely for men who didn’t know their HIV status compared with those who were HIV-negative (AOR=0.26, 95% CI:0.08–0.79), but more likely for younger men (AOR=0.95, 95% CI:0.93–0.98). Younger men were also more likely to want an LGBTQ sports teams (OR=0.97, 95% CI:0.94–0.99). Men who were partnered with a woman were significantly less likely to participate in local LGTBQ-identified events (OR=0.17, 95% CI:0.06–0.47); one-quarter of these participants were out to their female partner (23.3%, n=7/30). Half of participants were out to their doctor (48.9%), which was negatively associated with being a parent (AOR=0.18, 95% CI:0.04–0.92) and being unsure of their HIV status versus identifying as HIV-negative (AOR=0.19, 95% CI:0.07–0.50). Participants were most comfortable getting an HIV test from sexual health clinics (37.7%) or their family doctor (30.5%) although some felt most comfortable with home testing (12.0%). Men partnered with a women were less likely to have ever been tested for HIV (OR=0.41, 95% CI:0.17–0.96).
Community-grounded, culturally-sensitive approaches to HIV prevention, care, and support benefit from these diverse individuals’ involvement.
The training of community based research (CBR) teams from the ground up including the GIPA based participation of people living with HIV is done piecemeal in Canada. Current literature on peer training evidences a lack of consensus on what a HIV CBR curriculum should include, what the goals are and how learning can be evaluated. To meet this need, a partnership was formed in 2011 between the Ontario HIV Treatment Network (OHTN), the Canadian Association for HIV Research (CAHR) and the Canadian Institutes of Health Research (CIHR) to create The Learning Place for Community Based Research in HIV/AIDS (TLP -
We use a) qualitative reports of the application of the Modules contained in TLP and the effectiveness of its educational approach, b) aggregates of individual Module use, and c) audited the process of forming the TLP partnership and of incorporating the academic, policy and community perspectives into the learning components of TLP.
Since 2011, over 50 collaborators across HIV research sectors and disciplines contributed with over 100 hours of work toward building the TLP curriculum. 10 CBR teams provided positive qualitative and quantitative reports of having integrated TLP materials into their team training; the online TLP Modules were used 70 times in the 2014 period; and over 100 persons attended the events sponsored by TLP (e.g. online program “What’s Hot with PRAs in Canada>). TLP users highlight the plain language, appeal, and adaptability features of the materials.
Further educational research is needed to evaluate the experiential and specific content learning of all those involved in HIV CBR, especially of PRAs. Interdisciplinary collaboration and in-kind contribution from all stakeholders have been significant in designing and implementing the TLP curriculum on HIV CBR.
In HIV community-based research (CBR), compensating people with lived experience to participate actively in the research process is a common practice. Peer research associates (PRAs) can take on diverse roles within CBR studies, and compensating PRAs for their time, skills, and expertise is critical; these types of payments are intended to support inclusion and the effective and equitable participation in research processes. Further, having clear compensation policies and procedures can be helpful in clarifying expectations and responsibilities relating to people’s involvement. Yet, few resources exist to provide guidance on developing compensation structures.
Our CBR Approach: Based on consultation with members of the British Columbia HIV CBR Quarterly Working Group (comprised of PRAs, research coordinators, community-based organization representatives, and academics collectively involved in numerous studies) we captured tips, best practices, and guidelines to consider when developing compensation plans for PRAs. Additionally, we captured and analyzed case examples of practices implemented within existing studies and conducted a brief review of regional social assistance earning exemption policies.
We found that developing a compensation plan with PRAs can be a complex task and many factors should be considered, such as the financial administration rules of your institution and funders and the potential impact that receiving financial compensation may have on a PRA, including their eligibility for other forms of social assistance, such as disability benefits. Findings were used to develop a “CBR Tips” knowledge translation document, which outlines important considerations to take into account when developing compensation procedures.
“CBR Tips” is a valuable toolkit for informing compensation plans for CBR studies. Importantly though, every research project is unique, as are the financial realities of PRAs, therefore, while “CBR Tips” offers important lessons, we recommend that each study team develop their own approach to compensation in consultation with involved PRAs.
The prevalence of HIV infection among heterosexual men has increased over the past two decades. Consequently, the need for health and support services for this group is likely to increase. The purpose of this scoping review is to provide an overview of the evidence related to the health and health service use of heterosexual men with HIV related to domains of interest identified by the community.
We searched 6 databases from inception to August 2014. We included all English-language qualitative and quantitative studies examining the health and health service use of heterosexual men with HIV. Two reviewers independently screened titles and abstracts for inclusion in the review, and disagreements were settled by a third reviewer. We extracted data regarding study characteristics (i.e. country of study, design, participant demographics, comparison groups, main findings, and limitations), and used content and thematic analysis to summarize the findings.
Our search strategy yielded 2344 references, of which 87 were included in the scoping review. We summarized the research into the following domains: treatment of HIV and its complications (n=7), health and social support services utilization (n=20), social determinants of health (n=8), prevention (n=17), family planning (n=8), and psychosocial research (n=29). Key findings include difficulties accessing care, poor mental health-related well-being and self-reported functional health, over-representation among ‘late presenters’ to care, greater fear of disclosure relative to gay men, being recast as violent and monstrous by mainstream media, and a lack of support regarding family planning and fatherhood.
This is the first comprehensive review of the literature regarding heterosexual men with HIV. The review supports the need for multi-sector collaboration (medical, community) to develop programming and support for these patients.
Effective HIV treatment and the promotion of greater and meaningful involvement of people living with HIV (PHAs) (GIPA/MIPA) have facilitated increased participation of PHAs in the workforce. Unfortunately, research shows that as PHAs transition into service-provider roles, they experience loss of peer support networks, barriers in accessing services, and increased work related stress. To address these needs, the CIHR Centre for REACH in HIV/AIDS Interventions Program of Research team organized a national think tank to engage concerned stakeholders to share strategies and co-develop a national research agenda to bridge policy and service gaps.
Prior to the think tank, a literature review and program scan on interventions used to support peer engagement in service roles in HIV and related sectors were conducted. The two-day think tank included: a presentation of key concerns identified by PHA delegates; presentations of the literature review, program scans and cross country program initiatives; small group work to brainstorm key concerns, review resources and gaps, and identify key priorities for research project development.
Forty-seven delegates participated in the think tank including representatives from all national AIDS organizations. Sixty percent of the delegates were PHAs in varied service provision roles. Five research priority projects were identified: (1) Develop and evaluate a third party counselling support mechanism for PHA service providers; (2) Develop and evaluate a role transition preparedness training resource; (3) Assess the impact of wise practices of informal peer support; (4) Develop a measurable framework to guide GIPA/MIPA practices; and (5) Evaluate ASO implementation of GIPA/MIPA and its resultant impact on PHAs.
The think tank facilitated the building of a national research network that has since received official working group status from the CIHR CBR Collaborative to support further stakeholder engagement to advance the identified research agenda. Lessons learned and key outcomes will be highlighted.
In April 2014, CPHA launched a project focused on increasing the capacity of health care, social service and public health professionals, and their organizations, to deliver non-stigmatizing STBBI prevention services in Canada.
Identify the capacity building opportunities currently available to front-line professionals, and their organizations, that increase knowledge and skills and facilitate the provision of non-stigmatizing services for persons living with or at risk of STBBIs in Canada.
Explore the ways in which stigma impacts STBBI prevention efforts in Canada and identify the promising practice approaches for the development and dissemination of capacity building opportunities.
Capacity building opportunities were identified through a search of the grey literature, a scan of the websites of health and social service agencies in Canada as well as the websites of relevant professional associations, and based on suggestions from key informants.
Twenty semi-structured telephone interviews were conducted with professionals working in sexual health, STBBI prevention and/or harm reduction.
Over 100 capacity building opportunities were retrieved and several gaps were identified, including limited opportunities in rural areas and limited opportunities with explicit focus on stigma.
Key informants discussed perceived and enacted stigma, as well as the structural drivers of stigma as problematic to STBBI prevention efforts. With respect to the development and dissemination of capacity building opportunities, several best practices were identified (e.g., self-reflection, inclusion of individuals with lived experience).
The key informant interviews and the environmental scan identified several gaps as well as several promising opportunities. Capacity building opportunities that facilitate self-reflection and that also address the structural drivers of stigma are needed. The findings will be used to inform the development and dissemination of capacity building opportunities that are responsive to front-line providers’ needs and that challenge the drivers of STBBI-related stigma.
People living with HIV (PLHIV) who had been disengaged from the workforce for long periods face several personal and structural barriers to re-entry. In addition to more common barriers, including momentum and professional experience and skills, PLHIV experience additional anxiety regarding HIV-related stigma, and bouts of illness and fatigue forcing disclosure to employers and co-workers. Punitive policies that govern social assistance compound these concerns and serve as additional disincentives to work-force re-entry.
At Home At Howe (AHAH) is a community-based study that examines the impact of a supportive housing complex on the health of its residents. We conducted a review of field notes kept since the study’s launch to identify the mechanisms and processes through which employment barriers were alleviated in the process of hiring two Peer Research Associates (PRAs) who provide research support for this study.
The study team developed a low-barrier hiring process that included an open information session where interested candidates could ask questions prior to being invited to an interview. No formal CVs were required of candidates. The interviewers were respectful, straightforward and communicated openly.
When the job formally started, the research team was clearly committed to cultivating a fulfilling and low-barrier workplace experience. We participated in a robust and accessible training program and our knowledge and input were clearly valued by the study team. For example, opportunities were provided to contribute to all aspects of the project, meet senior members of the investigative team, and participate in national networking meetings. Involvement in this project has provided a boost in self-esteem, strong sense of belonging and purpose.
Researchers interested in hiring PRAs must be as low barrier as possible in their approach to recruiting, hiring and training. Even the most low-barrier approaches could exclude people who lack the experience and momentum. Researchers should endeavour to use accessible language, ensure communication is open and honest, and work with peers to cultivate a flexible and empathetic work environment.
Despite the high prevalence of HIV-associated neurocognitive disorder (HAND) and many clinical assessment tools, limited qualitative information exists on how people with HAND view, cope with, and speak about cognitive impairments. HEADS UP> is investigating the lived experience of HAND using participatory ethnographic methods at Casey House and St. Michael’s Hospital in Ontario and at The Dr. Peter Centre in BC. This study is funded by a CIHR catalyst grant and a REACH grant.
We audited the steps to bringing experience and expertise together in an interdisciplinary team and the implementation of a CBR strategy that is inclusive of people living with HIV and sensitive to the ecology of community-based organizations. These steps included a) a critical reading of the HAND and qualitative methods literature informed by community, clinical, and academic perspectives; b) the design of an in-depth interview schedule; c) recruitment and training of Peer Research Associates (PRAs); d) the inclusion of clinical screening for HAND; and e) an ongoing KTE strategy.
A critical reading of the literature has softened the disciplinarian edges of the research team and allowed people with HIV to challenge the boundaries of clinical assessments and practices regarding cognitive difficulties. It has positively impacted the collaborative construction of a) a recruitment strategy that includes clinical screening for HAND but remains flexible to the organizational environment (e.g., increases awareness but not alarm amongst clinicians and participants); b) an open-ended interview schedule that incorporates the constructs regarding memory, attention and thought processes of people living with HIV; c) the training of six peer researchers from Ontario and BC led by a PRA with lived experience of HAND; and d) multimedia HIV/HAND information resources and café scientifique.
We expect that these CBR steps will effectively engage communities in several aspects of the study to maximize results and sharpen the grounded theory analysis of 24 in-depth interviews with participants living with HIV and HAND.
There is a paucity of literature exploring the role of Peer-Research Associates (PRAs) in survey administration in the context of Community-Based Research (CBR) studies. As part of a multi-year CBR study evaluating the Dr. Peter Centre (DPC), an integrated health care facility in Vancouver, BC for people living with HIV, two PRAs were hired to administer baseline and follow-up quantitative surveys to study participants.
Two PRAs (one DPC client, one external) were hired as DPC staff members to administer a quantitative survey as part of the DPC study. The PRAs are people living with HIV who identify as having shared backgrounds with DPC clients, including current and past experience with mental health challenges, homelessness, and illicit substance use.
Following hiring, PRAs participated in a comprehensive 3-day training program led by a Peer Mentor in collaboration with the DPC on-site PRA supervisor and the study coordinator. Over a 12-month period, the PRAs received ongoing support from this team to effectively administer the survey. They also became acquainted with: principles of CBR; confidentiality and privacy; organizational culture as an employee of an HIV health care organization; and strategies around personal boundaries when living the dual role of an employee and a peer.
The peer-led survey administration process improved the quality of participant responses, and PRAs were able to develop diverse skills through the PRA-led interview process. Participants gained by being able to talk openly with another peer about their experiences outside of the typical power structures inherent in researcher/subject relationships. This approach also fostered connections between PRAs and a diverse range of stakeholders working in the area of HIV/AIDS, potentially leading to future paid employment and/or volunteer opportunities. This process may be useful to academics and organizations looking to promote the Greater Involvement of People Living with HIV/AIDS (GIPA) in research.
HIV community-based research (CBR) in Canada involves engaging people living with HIV (PLHIV) and affected communities. CBR targeted funding often requires formal engagement, such as community advisory committees (CAC) and “peer research assistants/associates”. However, scholarship that evaluates community engagement is lacking, and current models may not be suitable or desirable for all PLHIV, particularly those with complex medical and social needs, those who experience poverty, have psychosocial challenges or use substances. This study compares two models for engaging PLHIV who use substances – a traditional CAC and an activity-based series called “Research Rec(reation)” (RR), a flexible drop-in approach that uses creative and participatory methods to promote inclusion. We used mixed methods to compare approaches with participants who were recruited and self-selected into Research Rec or traditional CAC. Intake interviews and participant evaluation surveys were administered at three sessions to assess motives for joining, level of participation and enjoyment of each session. Data were analyzed from 21 Research Rec and 11 CAC evaluations (from 13 RR and 5 CAC participants). Most participants were male (72.2%), White (77.8%) and rated their health as ‘fair’ or ‘poor’ (61.1%). Fewer than 50% of each group were employed in the past 12 months (23.1% RR, 40% CAC) and many rated their mental health as ‘fair’ or ‘poor’ (38.9% RR, 40% CAC). Less than half of the Research Rec participants had advised on research previously, compared to 80% of the CAC participants. Across all three sessions of both groups, most participants rated session productivity as ‘good’ or ‘excellent’ (66.7%–100% RR, 75%–100% CAC). Research Rec participants consistently rated their participation level as ‘good’ or ‘excellent’ (80–100%) and most (66.7%–100%) rated their enjoyment of each session as ‘good’ or ‘excellent’. Across all three unique activity-based sessions, more than 80% of Research Rec participants said the activity format was ‘good’ or ‘excellent’ for discussing the topic. Our preliminary results support the development and implementation of flexible activity-based methods to engage a wider group of PLHIV in CBR.
Community-based research (CBR) has become a prominent form of HIV/AIDS research across key populations, and there has been a proliferation of guidelines for ethical conduct of CBR. However, few publications address CBR research ethics with people who use drugs. Our aims were (a) to describe the best practices and challenges when conducting CBR with people who use drugs and (b) to produce a print resource for people who use drugs regarding their rights as participants in CBR studies.
Searching for peer-reviewed and grey literature focused on substance use, CBR, and ethics, we identified 884 English-language articles published from 1985 to 2013. Employing a two-level screening process, 25 references met inclusion criteria and were summarized for main themes relating to ethical issues. Thematic summaries and three group consultations with people who use drugs informed the development of a resource for community members.
Five overarching themes were most commonly raised in reviewed publications: participant compensation; the perspectives of people who use drugs on CBR; responsible conduct of CBR; capacity building; and pragmatic issues regarding inclusion within CBR projects. These themes were reflected in the community resource, which aims to both provide basic information about participant rights (e.g., regarding payment, participation while intoxicated, and confidentiality) and to empower potential participants to raise ethical questions with research teams.
This scoping review and community resource complement recent academic and community-based work focused on the experiences of peer researchers in CBR. Incorporating a scoping review and community consultations, we were able to identify themes in CBR ethics for people who use drugs from multiple perspectives, including those of CBR-experienced and less-experienced community members. We recommend the development of pragmatic ethical guidelines for researchers conducting CBR with diverse communities of people who use drugs.
People living with HIV (PHAs) are increasingly recognized as experts in their own health and in HIV. In addition, patients are being engaged in medical education to provide experiential learning opportunities for students. Joining these concepts, we developed a Simulated Clinical Encounter (SCE) in which medical students provided pre- and post-test counselling and a point-of-care HIV test for trained PHA-Patient Instructors (PHA-PIs). Our primary objectives were to decrease HIV-related stigma among medical students and to increase interest and comfort in providing HIV-related medical care.
67 second-year medical students were recruited from the University of Toronto to participate in 10 SCEs from September 2012 to March 2013 facilitated by 16 PHA-PIs and 22 Clinical Preceptors. Students completed the Healthcare Provider HIV/AIDS Stigma Scale (HPASS) before and after the SCE. This scale has been previously validated in Canadian trainees. It contains thirty 6-level Likert-scale questions around the themes of stigma, stereotyping, discrimination and prejudice. Higher scores represented higher levels of stigma. An additional 5-level Likert item assessed interest in providing HIV-related care. Results of paired t-tests are presented.
62 of 67 medical students completed paired pre- and post- SCE surveys. Following the SCE, students demonstrated a decrease in overall stigma (68.74 vs. 61.81, p<0.0001), stereotyping (27.61 vs. 26.15, p=0.0456), discrimination (13.65 vs. 12.37, p=0.0007) and prejudice (27.48 vs. 23.29, p<0.0001). Participants also reported increased interest (4.01 vs. 4.25, p=0.0052) in providing HIV-related care.
Involving PHAs as patient instructors for HIV counselling and testing significantly reduced HIV-related stigma and increased interest in HIV-related care among medical students. Actively engaging PHAs in the delivery of HIV-related medical education can have beneficial outcomes for both learners and PHAs. Additional studies on the potential for improved HIV care are warranted.
Involving people living with HIV (PHAs) in clinical research has been an essential facet of the response to HIV since the declaration of the Greater Involvement of People with HIV/AIDS (GIPA) Principles in 1994. In undergraduate medical education, PHAs are increasingly being recognized for their HIV expertise grounded in lived experience. We employed community-based participatory research (CBPR) methodology to develop a Simulated Clinical Encounter (SCE) in which medical students provided pre- and post-test counselling and a point-of-care HIV test for trained PHA-Patient Instructors (PHA-PIs). Here, we describe the formation and processes of an innovative Patient Instructor Steering Committee (PISC), formed to guide SCE design, development and implementation.
The PISC’s primary goals were to ensure that the SCE was relevant, acceptable, and effective in reducing stigma and increasing interest in HIV-care among medical trainees, but also relevant, acceptable, and empowering for PHA-PIs. PISC membership drew from research team members, PHAs, standardized patient educators, patient advocates, and medical educators. The PISC met to collectively address PHA-PIs training and support needs, develop recruitment strategies, and clarify SCE logistics and critique and improve our research processes. Decisions were reached through consensus, with quorum defined as two-thirds of PISC members.
The PISC evolved from a purely advisory committee to a participatory means of fostering community ownership and empowerment through power sharing, capacity-building, mentoring, and learning exchanges, throughout each phase of the research project. All members of the PISC were involved in co-creating knowledge and teaching one another, with considerable time spent on developing common understandings of the broader determinants of health for PHAs.
The PISC is a robust model of CBPR methodology for involving PHAs in medical education and HIV research, and can provide useful insights for other educational initiatives involving patient groups.
HIV stigma impedes effective HIV responses. It creates fear that discourages HIV disclosure, testing, and care, resulting in silence and denial. Community Champions HIV Advocates Mobilization Project (CHAMP) is a community-based intervention study undertaken to promote HIV championship among PHAs and non-PHA faith-based, media, and social justice leaders in Toronto’s African, Caribbean, Asian, and Latino communities.
CHAMP drew on the strengths of mixed methods and health promotion evaluation strategies. It piloted two interventions: Acceptance Commitment Training (ACT) to promote psychological flexibility and Social Justice Capacity Building (SJCB) to promote collective empowerment. CHAMP engaged both PHAs and non-PHAs from diverse community sectors. Pre-, Immediate-Post, and 9-month-Post surveys, focus groups, and individual interviews were used to assess the impact of the interventions. Post-intervention monthly activity logs over 9 months and two structured networking sessions were used to document collective empowerment.
A total of 31 non-PHA and 35 PHA participated in CHAMP. Results showed significant reduction in internalized and enacted stigma in all intervention groups. Participants reported 1090 HIV championship activities over 9 months post-intervention. This presentation will specifically focus on an analysis of the unique impact of each of the three key design elements in CHAMP: (1) the combination of psychological interventions with collective empowerment strategies to reduce stigma; (2) the promotion of empathy and collaboration among PHA and non-PHA leader across sectors to advance championship; and (3) the embedding of a collective sustainable process to identify next-step strategies to further promote resilience and advance social justice.
CHAMP has demonstrated the positive impact of community-campus partnership that taps into grassroots knowledge and scientific evidence to address stigma, promote community resilience and advance social change. Expanded partnerships and evaluation of scale-up adaption of CHAMP interventions in real-life setting are needed to assess sustainable impact.
This study aimed to explore the social factors associated with the experience of food insecurity (FI) among those with HIV-HCV co-infection.
Qualitative interviews were conducted with a convenience sample of 40 food insecure persons of the CCC attending clinics in Vancouver, Toronto, and Windsor in 2014. A mixed-methods approach to coding interviews was applied. Participants’ experience of FI (lack of access to food) was examined based on a framework of vulnerability to FI in which the physical, social, and livelihood aspects of a person’s life are organized along three categories of factors (structural, contextual, proximal) that are more or less amenable to modification.
Multiple vulnerabilities were associated with participants’ FI. From a structural perspective, participants appeared vulnerable by both their physical characteristics (e.g., co-infected and living with addiction) and livelihood (e.g., dependence on supplementary sources of income). Drugs including cannabis (n=25) were a major expense. In addition to disability pensions, wages and odd jobs (n=11) and hustling (selling sex, drugs; n=11) were key contributors to income. Among the contextual factors, availability of food sources (n=37) when money runs out and food buying strategies and preparation skills (n=35) were a concern to participants. The majority of them cared about having good food (n=28). Social factors (n=25) were also closely linked to their experience. For example, being in a relationship was positive for food security (FS) while the street drug environment influenced FS positively (supplementary income to buy food) or negatively (facilitating continued drug use). Proximally, the loss of a partner and effects of new medications could precipitate FI.
The perspective of those who experience FI and co-infection showed that access to food is a complex problem. Measures to improve FS in this population must consider competing financial priorities (e.g., drug use), risky social environments, and health concerns.
Sexual education in families with mothers living with HIV (MLHIV) is often focused on unplanned pregnancy and HIV prevention, with limited discussion of emotional, social and legal issues. This paper aims to examine 1) the prevalence of mother-child communication about sexuality, from the youth perspective and 2) the ease of mothers to talk about sexuality.
A mixed-method, which combine quantitative and qualitative methods, was used. First, 55 youth (12–28 years; M = 16.2 years) and their mothers (n = 33; 33–49 years; M = 40.9 years) completed a face-to-face questionnaire. A total of 35 youth knew their mother’s diagnosis and 11 were infected through vertical transmission. Descriptive analyses and a multivariate regression model (MRM) were performed controlling for gender and age of children, ethno-cultural origin, country of birth, and mothers’ religiosity. Eleven mothers also took part in a semi-structured interview.
Prevention and contraception are the most common themes discussed. Children who are aware of their mother’s diagnosis are more likely to have talked about contraception, condoms and STIs, and consider their mothers as being comfortable, informed and empowered to discuss sexuality. The MRM indicates that knowing the mother’s diagnosis plays a significant role only when it comes to talking about STIs. Data analysis indicates that mothers feel responsible to protect their children from STIs and unwanted pregnancies. However, several barriers to sex education are raised by these women like a lack of knowledge about sex
The results highlight the importance of targeting parental attitudes regarding discussions about sexuality, in respect for cultural values and beliefs. Interventions should consider the HIV status of the child and the willingness of the mother to disclose their HIV status.
Reports from the United States indicate that HIV discrimination is a barrier to adoption. This study aimed to address the gap in academic documentation regarding organizational level policies and to identify structural and social facilitators and barriers for people with HIV who are navigating the adoption system. A list of adoption service providers recognized by the Ontario government was compiled using online searches (total of 181, including agencies, practitioners, and licensees). Services were pre-scanned using available websites, and all were then contacted via telephone to address missing data.
Telephone surveys were completed with 75 adoption service providers and online data collection was possible for an additional 2. Most providers did not have formal policies prohibiting individuals with HIV from adopting (n=68/77). International adoption agencies (n=6/53 total agencies) had the most detailed policies regarding eligibility criteria for prospective parents, ranging from full prohibition of people with HIV adopting to some restrictions apply. Domestic private adoptions were also noted as problematic as health status is disclosed at the request of the birth parents (noted by 7 service providers). Private adoptions (international and domestic) posed significant, if not insurmountable, barriers to adoption for prospective parents with HIV. Some of the domestic adoption service providers (n=17) lacked clarity in terms of admissibility, admitting they were unsure if people with HIV were eligible to adopt. However, a small portion (n=6) of service providers confirmed they were aware of successful adoptions to people with HIV. Incidental findings during the telephone surveys included that some service providers were unaware of the medical advances in the field of HIV and believed that HIV was a terminal illness that hastens death. People with HIV are exploring various ways to become parents. Although less commonly considered, adoption may be a viable option for individuals living with HIV.
Depression has been linked with both substance use and sexual risk behaviours among gay, bisexual, and other men who have sex with men (GBM). However, less is known about how depression affects unprotected anal intercourse (UAI) when conducted in the context of substance use among HIV-negative GBM.
Using baseline data from the Protective Factors Study (n=470), we examined three separate HIV risk behaviors: number of casual sex partners, UAI with a casual partner, and UAI with HIV-positive or unknown partners in the last 90 days. We fit a zero-inflated negative binomial model for count data and log binomial models for binary outcomes.
Most of the sample was younger (≤35 years), White, well-educated and Canadian-born. Around 22% reported having UAI with a casual partner and 45% reported depressive symptoms (a score ≥16 on CES-D). Higher self-reported mean depression scores were associated with any substance use (RR=1.26, 95% CI:1.05–1.51) and substance use before or during sex (RR=1.42, 95% CI1.17–1.70) in the last 90 days, but not with alcohol use. In addition, when substance use measures were controlled, depression was associated with UAI with a casual partner (RR=1.39, 95% CI:1.07–1.83) and UAI with a casual partner whose HIV status was HIV-positive or unknown (RR=1.68, 95% CI:1.28–2.22). Depression was not associated with more sexual partners in multivariable analyses.
In this sample of HIV-negative GBM, depression and substance use were associated with UAI with casual partners, including partners who were HIV-positive. Though temporality between depression and substance use and reasons for substance use during sex are unknown, there was high substance use in sexual contexts and engagement in risky sexual behaviors by GBM with depressive symptoms. HIV prevention interventions for HIV-negative GBM should also consider syndemic psychosocial problems that may increase risk of HIV contraction in this key population.
Biomedical knowledge of HIV transmission, risk, and prevention has greatly evolved over the last 10 years.
We conducted focus groups and interviews in Vancouver, Toronto and Montreal with 4 subgroups of gay men (n=87), including those: connected to the HIV sector; in serodiscordant relationships; sexually active and HIV-positive; and, HIV-negative and ‘at risk’. We used mock online dating/hook-up profiles and scenarios, and real media headlines to elicit their reactions and understandings. Using an interpretive description approach, we conducted thematic analysis of the data, supplemented by field and debrief notes, and a quantitative intake survey.
The biomedical concepts discussed in the focus groups (e.g., undetectable viral load, PrEP, condoms, seroadaptive behaviours) resonated differently among gay men, ranging from enthusiastic uptake (e.g., “undetectable is the new negative”), misconstrued or partial integration (e.g., “you can’t get it if you only top”), to resistance and suspicion of any prevention messages/strategies other than condom use (e.g., “Truvada whores” and “condoms are non negotiable”).
The Resonance Project has uncovered diverse ways in which gay men in Canada – individually and as communities – are making sense of, incorporating into their understanding, and modifying their behaviours based on the new biomedical knowledge of HIV. The next phase of the Project will identify the main messages, precautions and caveats which must be incorporated into communication about biomedical knowledge of HIV to be sensitive to the lived realities of gay men.
In Canada, young men who have sex with men (YMSM) are an under-researched population. They are also early adopters of new media technologies, including websites and GPS-based applications for seeking sexual partners. This research explores the motivations, experiences, and HIV risk behaviours of YMSM seeking sexual partners with these new technologies.
This is a descriptive cross-sectional study using data collected from self-completed questionnaires and one-on-one interviews. Self-identified YMSM participants between the ages of 16–28 were recruited from the area surrounding one of Canada’s largest universities. Ethical review was obtained. Interviews were audio-recorded and transcribed. Data analysis focuses on qualitative themes and quantitative associations.
While data collection is ongoing, this unique study of YMSM explores 1) the motivations participants have when using partner-seeking websites and applications; 2) the experiences and attitudes towards misrepresentation, both self-reported and perceived; and 3) the reported risk behaviours that occur during exchanges with partners met online compared to partners met offline. Results describe the interplay between motivation, HIV vulnerability and resilience, and online partner-seeking technologies among YMSM.
New web- and GPS-based partner-seeking technologies may facilitate a greater number of sexual partners, often between men who might not have otherwise encountered each other. Yet, the literature suggests that men’s motivations, behaviours, and interactions, rather than the technologies themselves, are more accurate indicators of the relationship between HIV risk and the online partner-seeking technologies. Understanding YMSM’s motivations in tandem with their experiences with these technologies informs key actors working within HIV prevention and influences the development of HIV prevention programs specifically targeted to YMSM.
The Simcoe Muskoka District Health Unit (SMDHU) uses social media to reach the hidden and hard to reach population of men having sex with men (MSM). The SMDHU INFOnurseX profile went live on
E-blast messages are another tool used to engage the online population. Approximately 2000 men receive our messages in Simcoe Muskoka and on average 300–400 people open and read our eblast message.
This is a new outreach environment for public health and we have seen a steady increase in the number of MSM accessing our clinic services since May 2012. This unique initiative provides an on-going, discreet opportunity for men to ask questions and receive information in a confidential online environment.
In 2014, SMDHU received the Judges Award for Innovation as part of Canada Health Infoway’s Public Health Social Media Challenge for this initiative.
Asian gay/bi men often face multiple forms of discrimination including racism, homophobia, and xenophobia. Those living with HIV face additional stigma and social exclusion. Existing research on related issues seldom adopts a strength-based approach. To bridge this knowledge gap, Asian Community AIDS Services undertook the Asian MSM Pathways to Resiliency (AMP2R) study to identify facilitating conditions and resilience strategies that helped Asian MSM address their life challenges.
Employing a community-based research methodology, a partnership of Asian gay/bi men, PHAs, service providers, policy-maker and researchers made up the investigative team. Six focus groups were conducted, including five groups of Asian gay/bi men (N=51) plus one group of service providers (N=13). One of the focus groups was PHA-specific and attended by 12 participants. Average age of PHA participants was 45 (range 29–60), and 8 were first generation immigrants who had been in Canada for an average of 20.6 years. Participants discussed their major life challenges, and their strategies for addressing them. Transcribed data were thematically coded and preliminary findings were presented back to participants to engage them to co-develop recommendations.
Participants identified key challenges with: managing health complications relating to long-term medication side effects and aging; social isolation relating to multiple losses; and, sexual exclusion due to criminalization of HIV non-disclosure. Positive moderators include access to culturally-appropriate services, supportive social network, intimate relationships, and stable income. Key resilience strategies include prioritizing and practicing self-care; drawing strengths from past challenges; overcoming self-blame for contracting HIV; accessing cultural strengths; and engaging in capacity building.
The study generates fresh insights into facilitating factors and resilience responses that help HIV-positive Asian gay/bi men overcome complex life challenges. Intervention seeking to promote their resilience should focus on helping them access cultural strength, engage in cross-generational mentorship, and apply resilience strategies across life challenges.
Biomedical knowledge of HIV transmission, risk, and prevention has greatly evolved over the last 10 years.
We conducted focus groups and interviews in Vancouver, Toronto and Montreal with 30 service providers who provide sexual health, counseling and HIV prevention services to gay men. Using an interpretive description approach, we conducted thematic analysis of the data, supplemented by field and debrief notes, and a quantitative intake survey.
Service providers face complex challenges in providing HIV prevention information, advice and counseling to their gay men clients/patients. They must handle: basic knowledge levels of HIV among some gay men; prevention in the context of the coming out process; difficult questions; feeling the need to err on the side of caution in prevention messages; diverse needs within ethnocultural communities; the complexity of research translation; silences around HIV in the community; the variety of physical and virtual spaces within which gay men connect; and lack of consensus within organizations and among service providers of the level of biomedical complexity that is appropriate.
The Resonance Project has uncovered the major challenges experienced by service providers and the key strategies they use to communicate biomedical aspects of HIV risk, transmission and prevention. Next steps for the Resonance Project are to develop KTE tools and messages that incorporate main messages, precautions and caveats that are sensitive to the lived realities of gay and bisexual men.
Although not currently approved in Canada, HIV self-testing could facilitate better access to testing, in particular for MSM who test frequently or experience barriers to testing. The acceptability of different types of self-tests (oral vs. fingerstick) and approaches to making self-testing available (supervised, community-based vs. unsupervised, over-the-counter) needs to be assessed.
Evaluate the acceptability of HIV self-testing (in terms of various factors including beliefs, preferences, concerns, facilitators / barriers to access) among MSM and service providers in Vancouver, Toronto, and Montreal.
In October and November 2014, focus groups were held in each city for community members who identify as MSM (n=16) and service providers (n=20). Participants also completed a questionnaire. Descriptive analyses of means and proportions were generated. Transcripts of the focus groups discussions were analyzed thematically.
Qualitative analysis showed that key concerns include accuracy of test result, cost, confidentiality, and ease of access. Among community members, 87.5% would be willing to use a self-test at home. All service providers (100%) were in favour of making self-testing available to MSM in Canada. A majority of both community members and service providers preferred the fingerstick self-test, respectively 68.8% and 60%. Half of community members and one third of service providers preferred a supervised, community-based approach to making self-testing available and learning how to use the test, compared to unsupervised, over-the-counter purchase in a pharmacy. However, a proportion of community members (18.9%) and service providers (25%) preferred a mixed approach.
HIV self-testing appears to be highly acceptable and could increase testing rates among MSM. A fingerstick test may be the best choice for introducing self-testing in Canada. These findings highlight the need to provide MSM with various options for accessing and learning how to do HIV self-testing, adapted to a range of personal profiles and contexts.
Despite the longstanding shift in conceptualizing HIV/AIDS as a site of chronic care, versus one of acute palliation (Seigel & Lekas, 2002), few attempts have been made to examine care-giving in the context of this change. The poster presented at this conference outlines a paper aimed at exploring the evolving nature of HIV/AIDS care-giving among sexual minorities. This paper, whose empirical analysis was based on a study originally designed to examine the care-giving experiences of gay men more broadly, compared the narratives of two partnered care-givers of HIV-positive gay men whose stories were located in disparate sociohistorical contexts. Whereas one care-giver provided support during an era in which HIV/AIDS was primarily conceptualized as acutely life-limiting, the other did so in the contemporary period of chronic HIV care. Although similarities were noted across the stories, distinctions were also apparent in how the care-givers encountered expressions of homophobia and HIV stigma, and negotiated these realities distinctly as functions of sociohistorical context. In particular, despite the salience of these systemic barriers across the two narratives, experiences with homophobia and HIV stigma appeared to occur more routinely and indefinitely in the context of chronic care. The poster lists implications of the presented paper’s analysis on theory, research, policy and practice, including importantly the need for programs that address and mitigate homophobia and HIV stigma across a range of professional contexts associated with the provision of practical and emotional support to carers of those living chronically with HIV.
The HiMMM Project is a community-based study of factors identified within the LGBT2SQ community of London-Middlesex, Ontario and their impact on HIV-related risk, HIV testing, and health services access. Canadian information on the health of gay, bisexual and other men who have sex with men (GB-MSM) and information on HIV are lacking outside of major metropolitan areas.
Over a six month period, 20 individual, in-depth interviews were completed with participants between the ages of 17 and 76. Using Nvivo 10 software, the data collected from these interviews were coded based on identified research themes (communication, homophobia, and isolation/social exclusion). Additional themes were identified by the team from the data. One of the predominant themes was ‘risk’ and how it impacted participants’ understanding of sexual health.
In general, participants felt that sex was an important component of their overall health, linked to mental and emotional health. Participants’ understandings of ‘risk’ and risk reduction strategies generally reflected the strong adoption of traditional messages about HIV prevention. Condom use and monogamous relationships were identified as risk reduction strategies for HIV. Participants frequently defined ‘risk’ in relation to sex and HIV transmission. Risk activity was identified as: multiple partners, physical abuse, condomless sex, and substance use. Participants associated specific spaces with these practices, including bathhouses and clubs. Often, participants reported the frequency of their HIV testing was related to their perceived level of risk.
Findings from this study highlight that participants engage with traditional HIV prevention strategies. However, the field of HIV prevention continues to evolve to include new prevention strategies and technologies that were not observed in this sample. Further data analysis will explore whether risk assessment differs according to select age cohorts. These results will be helpful in building targeted HIV prevention and testing efforts in London, Ontario.
We conducted a mixed-method analysis to explore HIV risk for transgender men in the Momentum Health Study, a bio-behavioural study of gay and other MSM.
We conducted bivariate analysis comparing trans-MSM (TMSM) with cisgender participants and performed semi-structured qualitative interviews with 11 of these participants. Interview transcripts were catalogued in the qualitative analysis software NVIVO and systematically reviewed to identify emergent themes within the data; individual accounts were grouped into discrete categories describing common themes of experience.
Of the 14 gay or queer transgender-identified men that met Momentum eligibility criterion “male-identified”, median age was 26 years [IQR:24–28], 86% identified as White and all were HIV uninfected. In bivariate analyses, compared with cisgender peers, TMSM engaged in fewer HIV sexual risk behaviours (e.g. anal sex) and greater use of some HIV risk reduction strategies (e.g. consistent condom use). Qualitatively, participants described engaging in a range of sexual activities, with transgender and cisgender partners. While some participants described participation in largely cisgender gay men’s communities, others perceived exclusion and lack of access to health education and services for gay men. Substance use, specifically alcohol, was presented as a facilitator of sex and dating. The ability to control trans-status disclosure encouraged seeking sexual partners online. Though less familiar with other forms of anti-retroviral therapy based HIV prevention, participants were largely familiar with PEP and several had considered using or used PEP.
While interview accounts uncovered the multi-faceted potential vulnerability of evolution for these TMSM into a socio-sexual arena with unique sexual health needs and risks, both qualitative and quantitative results framed HIV risk for these TMSM as low, consistent with participant perceptions. These findings inform trans-inclusive MSM research and highlight the need for tailored health services for TMSM not reached by existing gay men’s health promotion.
Research suggests that there is a dearth of updated HIV education and training materials and opportunities for social workers and students, particularly within the field of child welfare. This is important given that parents living with HIV perceive their relationships with child and family service workers (CAS) to be underpinned by HIV-related stigma and a lack of knowledge and understanding of the clinical, social and legal aspects of living with HIV. Drawing on findings from the Positive Parenting Pilot Project, this paper will present the HIV education and training needs of CAS workers and social work students in Ontario.
Focus groups were conducted with social workers from three Ontario Child and Family Services agencies (N=19) and students enrolled in child welfare and case management courses from three social work/social service worker programs in Ontario (N=44). Focus group questions explored HIV related knowledge and attitudes, and critical areas of training and skill development for supporting families affected by HIV.
CAS workers and students require training on HIV transmission, prevention and treatment; HIV related stigma and social and emotional health; and the criminalization of HIV and its impact on individuals and families. CAS workers expressed their need for agency based policies and resources that will guide their practice with parents living with HIV, while students view HIV education as an integral component of their social work training.
Providing HIV education and training for CAS workers and social work students has the potential to result in developing appropriate policies, practices and strategies for supporting parents living with HIV. These components must be viewed as an integral aspect of the Ontario Association of Child and Family Service commitment to anti-oppressive education and integrated into existing policies and compulsory professional development modules that social workers and social work students currently access.
The Dr. Peter Centre (DPC) is an HIV health care facility that provides support to some of Vancouver’s most vulnerable citizens who face poverty, homelessness, and mental health and addiction issues, in addition to HIV/AIDS. DPC services are designed to engage individuals in their health care with a specific goal of improved adherence to HIV treatment and overall improved health care. In 2008, the DPC introduced a nurse practitioner (NP) role to its programs, creating a unique care context in which the role of the NP has rarely been documented.
In 2013, following a scoping review, an MPH student developed a case study on the role of the NP at the DPC and identified six broad role elements of the NP in relation to harm reduction services (facilitation of access to services, case management, clinical services, patient education and support, support for multidisciplinary teams, and use of evidence-based practice). The six role elements complement each other effectively to form a complex and integrated approach to NP care, allowing the NP to address many significant barriers to care for DPC clients.
As care providers face evolving roles and contexts for prevention, treatment, and care of individuals with substance use disorders and comorbid complex illnesses, the NP role offers a promising contribution to future HIV/AIDS management. Particularly in the stable and chronic phases of illness, the NP role enables the delivery of effective, efficient, and consistently engaged care. Optimizing the use of the NP role is fundamental to creating sustainable solutions and increasing access to care for high-need priority populations in Canada.
While empirical evidence points to the positive impacts of integrating the NP role at the DPC, there is a need to identify end point goals through a robust evaluation to demonstrate the impacts that the NP’s role facilitates for clients.
AIDS Service Organizations (ASOs) play an important role in providing services to people living with HIV worldwide. Changes to the HIV service paradigm in Canada, such as the broadening mandate to address sexually transmitted and blood-borne infections and the shift towards service integration, will affect people living with HIV. These changes are significant and may end essential disease-specific programming that people living with HIV regularly access, including transportation support or support groups. My research uses qualitative data from semi-structured interviews with women living with HIV to document their experiences accessing health and social services in the Maritime Provinces. This study is part of a larger project that will conclude in 2015.
In total 71,300 people are living currently live with HIV in Canada; Atlantic Provinces account for the lowest HIV prevalence rate (2%). As a result of this low incidence, especially among women, provincial ASOs receive governmental funding that is only sufficient in covering management and administration expenses. Fundraising campaigns are necessary to fund priorities not covered by the government. My research uses grounded theory-based methodology to interpret the interviews of women living with HIV to assess the landscape of services women are accessing in their communities for maintaining their well-being. Preliminary results indicate a need for women HIV-centered care focusing on issues of reproduction, childcare, and food security. More effective service delivery strategies, such as online networks and the availability of mobile health clinic outreach, may help to meet the needs of women living with HIV. This paper aims to increase knowledge about health and social service usage among women living with HIV, the role ASOs currently play in their lives, and concludes with women’s views on the future direction of service delivery for ASOs in Canada.
Reliable access to combined antiretroviral therapy has turned HIV into a chronic, episodic illness for many in Canada. Since 1998, The Canadian Working Group on HIV and Rehabilitation (CWGHR) has been promoting access to rehabilitation services for people living with HIV (PLWHIV) to ensure that their quality of life is also sustained. CWGHR’s work has become increasingly challenging, and vital, in an environment of funding cutbacks which have limited access to rehabilitation services outside inpatient settings. This presentation will introduce a novel initiative which successfully situated occupational therapy (OT) practicum students in community-based HIV organizations (CBHOs) and highlight some of the lessons learned during the project pilot.
The objectives of this program are: 1) to increase access to OT by PLWHIV who access CBHOs; and 2) to familiarize OT students with the role of OT in the care of PLWHIV. During the pilot, nine students from three Canadian universities completed practicums in six CBHOs. Their activities included: resource and program development; program implementation; raising awareness about the role of OT, and clinical support for PLWHIV. These placements were considered “role-emerging” by the universities as students were placed in environments which did not have Occupational Therapists (OTs) on staff nor established roles for OTs.
During the pilot, project stakeholders (CWGHR staff, academic fieldwork coordinators, OT clinical supervisors, CBHO staff and students) engaged in ongoing dialogue and debriefing to clarify their roles in this cross-sectoral approach to training and service provision. When the pilot practicum placements were complete, an online survey conducted with students and their CBHO supervisors showed that working together had increased knowledge and capacity to deliver care for PLWHIV among both groups. This project exemplifies how a catalyst organization can foster intersectoral partnership-building between organizations and institutions with seemingly different goals and mandates, to their mutual benefit.
In 2013 the Canadian AIDS Society undertook a needs assessment of community-based organizations serving communities that are rural and remote in nature. Approximate 20% of Canadians live in rural and remote communities. While HIV is often perceived as a urban issue, there is an increasing amount of qualitative and anecdotal evidence that demonstrates that HIV strongly affects rural and remote populations in Canada. An advisory committee developed a series of questions to capture a glimpse of the realities of service provision in these communities and launched a national survey in the fall of 2013. In total we received 28 completed responses. Fluid Survey was used to disseminate the findings and conduct the analysis.
The findings of this survey are divided into the following categories: Characteristics of organizations. What makes Rural and Remote Communities Unique? Services and Gaps. Organizational needs. Barriers and Challenges. Stigma and Discrimination. Unmet needs. Partnerships. Funding. Two of the biggest challenges raised in this research are the impacts of transportation and of stigma and discrimination. Challenges related to transportation include the impact that having access to transport has on the daily life of a person living with or at risk for HIV, while stigma is cited as a problem that impacts confidentiality, housing, employment and access to services and programs.
A series of recommendations follow from the findings of this survey. These recommendations include the need for targeted anti-stigma campaigns, the need for peer-based programs, flexibility in funding, particularly when it comes to transportation (of clients as well as staff supports) and improved services for people who use drugs.
Over thirty years after the dawn of the HIV epidemic, arguments against the need for an exceptional response to HIV have gained popularity. Once widely regarded as a “death sentence,” bio-medical advances and great strides in the visibility and voices of people infected and affected by HIV have since made HIV a chronic disease in specific contexts and for specific populations. Proponents for arguments against HIV exceptionalism ignore the fact that HIV is a harbinger of social inequality and continues to disproportionately impact many marginalized communities around the world who do not have the political power and money needed to keep the issue a high priority on the public agenda. This oversight is also informed by a narrow recount of the history of the epidemic and the HIV movement, which is often thought to be historically and politically located in and synonymous with the White, gay men’s liberation movement in the west. However, this problematic meta-narrative that links advances in HIV to a singular victory for one community wreaks of the operations of power and invisibility of identities at the intersections of race, gender, sexual orientation, class, ability, geography and so on. It contributes to the erasure of less popularized acts of resistance from Indigenous communities, women and communities of colour around the world, deeming them agentless victims and passive recipients of HIV advances. This paper argues that locating the HIV movement in the struggles for Black liberation, Indigenous sovereignty, feminism and LGBT rights helps to legitimate the histories, narratives and make space for the voices of marginalized communities; legitimates the relevance of an exceptional response to HIV, which continues to have a devastating impact on disenfranchised communities; and sheds light on the fact that HIV thrives in environments prone to human rights violations and inequality along intersectional identities.
Pharmacists play a variety of roles in the interdisciplinary care of HIV-infected patients. The objective of this study was to describe how HIV healthcare professionals perceive the relative importance of pharmacist activities and compare pharmacists’ perception to the other disciplines.
A descriptive cross-sectional survey was developed and sent to Canadian HIV practitioners involved in interdisciplinary teams, including pharmacists, physicians, nurses, etc. Data was collected anonymously in Fluid Survey™, a secure online survey tool, using a snowball sampling technique.
Of the estimated 335 emails requesting participation, 95 participants completed the survey (response rate of 28%). Of the 53 criteria, 19 (36%) were characterized as “very important” by more than 50% of respondents. There was a high level of agreement between pharmacists, physicians and nurses on the top 5 most important pharmacist activities requiring patient referral: evaluation of patients on complex treatments, counselling for initiation in ARV therapy, assessment of drug interactions, counselling for change in ARV therapy and patient assessment for recommendations to change ARV therapy; the latter was considered less important by physicians (ranked 8th), whereas assistance in securing drug coverage was rated higher (ranked 3rd). When examining important patient characteristics, both pharmacists and nurses ranked compromised organ function, peri-organ transplant, malignancies requiring therapy and pregnancy as highest priority; pharmacists also identified paediatrics (ranked 1st) whereas nurses added the presence of multiple co-morbidities (ranked 4th). Physicians rated paediatrics, pregnancy, no legal status in Canada, refugee status and hospitalization as the 5 most important criteria.
A large variety of pharmacist activities were considered “very important” by the majority of participants. The different perceptions of the role of a pharmacist in the care of HIV patients warrants the development of a short, simple screening or referral tool to identify patients most likely to benefit from a pharmacist consult.
The objectives were to identify sexual risk profiles of male adolescents aged 14 to 22 years-old in the province of Quebec and to explore correlates of profile membership in terms of interpersonal victimization.
Participants were 3461 male adolescents aged 14 to 22 years-old, living in the province of Quebec and interviewed in school settings as part of the first wave of the Youths’ Romantic Relationships Survey. We used a person-centered approach to model sexual risk taking indicators (age at first sex, number of sexual partners, systematic condom use for anal or vaginal sex, non-relational sex, transactional sex, and events of pregnancy among partners) and compared profile membership according to exposure to various forms of interpersonal violence.
Four profiles were identified: Abstinent (64%), Normative debut-few sexual partners (15%), Early debut-Multiple partners (13%), and High risk (8%). The High risk profile showed the highest number of sexual partners combined with non-systematic condom use (59%), non-relational sex (33%), transactional sex (16%), and events of pregnancy among partners (9%). The High risk profile also involved the highest proportion of non-exclusively heterosexual participants. Membership in profiles with greater at-risk sexual behaviors was associated with a history childhood sexual abuse and dating victimization experiences (emotional, sexual and threats). Experience of physical dating violence, childhood physical abuse in the family or witnessing interparental violence failed to distinguish profiles.
The results show correlational evidence supporting a relationship between interpersonal violence and sexual risk-taking in male adolescents. STIs and HIV prevention programs should not assume a homogeneous population with respect to sexual risk taking among male youths. The psychosexual sequels of interpersonal victimization among adolescent male need to be efficiently addressed to decrease their engagement in at-risk sexual behaviors. Potential mediators such as posttraumatic stress symptoms, substance use and sense of hopelessness need to be explored in future investigations.
ASAAP is leading a Community-Based Research study to explore the effectiveness of storytelling as a culturally relevant and gender based model of HIV prevention and support among South Asian women. Funded by the Women’s College Hospital Research Institute, the study uses a mixed methods approach to assess storytelling tools and approaches developed and used in ASAAP’s current women’s programs.
The study design employs qualitative and quantitative tools to structure data collection and analysis. 12 focus groups are conducted with South Asian women across the GTA in which six (6) groups are provided with currently used storytelling tools and six (6) with traditional sexual health and HIV educational fact sheets. The benefits and limitations of storytelling and fact sheets are compared and explored through analysis of pre and post sessions questionnaires and recorded discussion.
As a community based and community driven research project, a large emphasis of the study is on sharing results, findings and on building community capacity so peers can meaningfully participate and lead research. The project employs eight (8) peer leaders who have been trained to deliver and conduct focus groups. To build capacity, peers have key roles in sharing sexual health knowledge and study results in multiple mediums through print, online and through video.
The results of the study are situated on key findings around the potential of storytelling to increase community uptake of sexual health and HIV/AIDS information, and to change attitudes and perceptions of people living with HIV/AIDS, thus tackling stigma. Consequently, these findings have significant implications for sexual health and HIV service provision among South Asian women. The tools assessed in the study are also easily available for scale-up or application within comparable communities of women or ethno-racial populations.
Considered the “house organ of the Canadian gay movement”, The Body Politic is a rich source of the history of activism during the emergence of the AIDS epidemic. Through a Foucauldian-informed content analysis of The Body Politic magazine’s coverage of HIV/AIDS from 1981 to 1987, this paper explores the idea that this serious health issue constituted a defining moment for the Canadian gay rights movement. It uses current, in-depth interviews with activists from the magazine to consider notions of health “from above” and “from below” by examining relations between the community and various levels of government, as well as confrontations with medicalization and the medical profession. The early struggle against the health inequities created by a deadly virus that disproportionately affected a marginalized community in Canada illuminates the intricate and complex power dynamics of the development of a community identity. The movement rose to the challenge of addressing health inequities based on homophobia through advocacy, political action, and even early service provision. I also examine how The Body Politic reported and negotiated issues of the community’s self-policing and “self-managed oppression” through efforts to promote “safer sex” and risk reduction, including the struggle over mandatory versus anonymous testing. Interviewees reflect on the scope of the movement as broader than a struggle for rights or identity, but rather as a liberation movement that sought to embrace feminism, alternative education, anti-racism, and alternative culture and human relations. Ultimately, this paper reflects on the impact of the AIDS era and neoliberalism on a “hard-fought gay liberation”.
Though we have learned a great deal about how to medically manage the progression of HIV, social stigma remains a challenge. It can compromise access to health and supportive community care and significantly influence the lives of women living with HIV (WLHIV). HIV-related stigma is intensified in women because they are women. Research has shown that for WLHIV, receiving and providing peer-driven services results in a range of benefits associated with care and positive effects on their overall health and well-being.
Positive Women’s Network (PWN) is the longest running women-specific HIV support and education organization in Canada. Since 2010, PWN has offered 5 annual peer support trainings with 48 WLHIV participants. The training includes a focus on experiences of stigma and how to cope with stigma.
Participants report that discussion helps distinguish between internal and external sources of stigma; internalized stigma can affect whether they see themselves as even deserving of care and external stigma in health care and community services can limit how and when women seek ongoing access to treatment and support. Participants developed a resource sheet on coping with stigma, written by WLHIV for WLHIV, including tips for physical wellness, psychological wellness, and steps women can take if they feel they are experiencing discrimination.
Personalized coping strategies do not address the larger systemic forces that cause HIV stigma to persist.
Supporting women to identify internal and external stigma messages/ experiences can help women improve self-care, access to health care, and community involvement. Education and campaigns addressing HIV stigma and gendered social and structural violence are proposed.
In Canada, there are 16,600 women living with HIV (WLHIV), 23% of the national total. There is a lack of women-specific HIV services available. WLHIV face specific challenges: biological vulnerability to infection; the invisibility of women with HIV; sexual stigma; power imbalances in relationship; women acting as caregivers with little time for themselves; the dangers of disclosure. Since 1991, Positive Women’s Network (PWN), a partnership of WLHIV and women affected by HIV, has provided a range of leadership, support, and health education. PWN is the only women-specific HIV organization in BC and the longest standing women-specific HIV organization in Canada. As demand grows for gender-specific approaches to HIV support, how can existing ASOs ensure physical safety, emotional safety, validation and connection for women living with HIV? In 2010, PWN conducted a needs assessment. 34 WLHIV from throughout the province were interviewed about their experiences seeking services; staff at ASOs were interviewed about providing services for WLHIV. In 2014, PWN conducted a member engagement survey; topics included: access to and evaluation of PWN programs and resources, feedback on program development. Survey was mailed/emailed to 301 members with a 12% return rate. Drawing on feedback provided from these 2 projects regarding improving services for WLHIV, suggestions for ASOs include: having a childcare policy and subsidies, offering individualized support, engaging WLHIV as peer workers, staff training, women-only safe spaces, developing a best practices procedure for supporting WLHIV. ASOs may be challenged to adopt these suggestions because of: apathy to engaging in gender-specific approaches because of sexism, lack of funding, lack of female clients, the under-representation of WLHIV at all organizational levels of HIV services. Promoting strategies to navigate and overcome the barriers WLHIV face to seeking and engaging with HIV support services is proposed.
Transgender individuals who use drugs often have higher rates of HIV infection compared to cisgender populations. Addiction treatment programs can act as an HIV prevention tool by contributing to reductions in drug use and by encouraging adherence to antiretroviral treatment. However, little information is available on the experiences of transgender individuals in addiction treatment. Therefore, we sought to qualitatively investigate the residential treatment experiences of transgender individuals who use illicit drugs in a Canadian setting.
In-depth semi-structured interviews were conducted with 34 transgender individuals in Vancouver, Canada between June 2012 and May 2013. Participants were recruited from three open prospective cohorts of individuals who use drugs, an open prospective cohort of sex workers, and through snowball sampling. Theory-driven and data-driven approaches were used to analyze the data and two transgender researchers aided with the coding and the interpretation of data.
Among the 34 participants in this study, 16 (47%) reported attending residential treatment in the past and their experiences varied according to whether their gender identity was accepted in the treatment programs. Participants reported a range of challenges associated with accessing and remaining in addiction treatment. Specifically, three themes emerged from the data that characterized individuals’ experiences in treatment settings: (1) gender-based barriers to entering treatment, (2) incidents of transphobia and stigmatization perpetuated by staff and clients, and (3) participants leaving treatment prematurely after negative encounters.
Participants reported a range of challenges associated with accessing and remaining in addiction treatment, which disrupted opportunities for HIV prevention. These findings illustrate the need for gender-based, anti-stigma policies and programs to be established within addiction treatment programs. Additionally, it is vital to expand treatment to include access to medications (e.g., buprenorphine) for transgender individuals who are not comfortable engaging with residential treatment.
People who use drugs (PUD) are increasingly included in committees related to policy, service delivery or research. Compared to the general population, PUD are disproportionately affected by HIV and hepatitis C, stigmatization and social exclusion. Such inequity is due in part to current drug policy of criminalization which thwarts harm reduction and health promotion efforts and creates barriers to access to services. The practice of inclusion aims to address these inequities by sharing power in decision-making. There is a gap in understanding whether this strategy works, and if so, how. This study sought to better understand power relations in committees where PUD are at the table.
In partnership with the Drug Users Advocacy League and the Society of Living Illicit Drugs Users, this participatory critical ethnographic inquiry explored power relations in four committees in Ontario and BC. Data were collected in 2013 through observations of meetings, individual interviews, demographics surveys and document reviews. Power relations were analysed using a framework which combined critical theory and transformative learning to identify factors that contribute to or hinder shared decision-making power.
Shared decision-making power was enabled by opportunities for PUD to be at the table, which were enhanced by the international movement of PUD, policies of inclusion, and leadership. Membership, number of seats for PUD and organizations’ commitment to inclusion enhanced shared power while limited organizational capacity and challenges with representation of PUD hindered it. Accommodating PUD’s economic realities, including remuneration, contributed to equitable participation. Negative views of PUD were still present. Power relations were improved when stigma was addressed both critically and with sensitivity. Social activities, negotiated relationships, democratic practices and skilled facilitation nurtured shared decision-making power.
Practice implications for more equitable inclusion and future research directions are offered.
Despite growing evidence on the critical role of sex work venue type on HIV prevention, few studies have sought to disentangle the influence of various workplace features on sex workers (SWs’) sexual and reproductive health practices, particularly in industrialized settings. This study therefore examined the associations between social cohesion, physical and policy workplace features on condom use for pregnancy prevention among indoor SWs.
Data were drawn from the AESHA project (An Evaluation of Sex Workers’ Health Access), a prospective open cohort of SWs from Metro Vancouver, aged 14+ years. Using factor analysis, a “Safer Indoor Work Environment Scale” was developed to measure the supportiveness of SWs’ venues, by cataloguing a range of indoor work environments including: safety policy and managerial practices (Cronbach alpha= 0.903); sexual/reproductive health services (Cronbach alpha= 0.960), drug harm reduction services (Cronbach alpha= 0.926), security features (Cronbach alpha=0.698), and social cohesion (Cronbach alpha=0.919). Six multivariable models were built using generalized estimating equations (GEE) to determine the association between indoor venue features and social cohesion on condom use.
Of 588 indoor SWs, 63.6% used condoms for pregnancy prevention in the last month. In multivariable GEE analysis, the following venue-based features were significantly correlated with condom use for pregnancy prevention: managerial practices and venue safety policies (Adjusted Odds Ratio (AOR)=1.09; 95% Confidence Interval (95% CI)1.01–1.17) access to sexual and reproductive health services/supplies (AOR=1.10; 95% CI 1.00–1.20) access to drug harm reduction (AOR=1.13; 95% CI 1.01–1.28), and social cohesion among workers (AOR=1.05; 95% CI 1.03–1.07). Security features access was marginally associated with condom use (AOR=1.13; 95% CI 0.99–1.29).
These findings suggest that a range of venue-based features and community interventions may enhance SWs’ ability to use condoms. There is a need to further explore the role of such venue- and community-based interventions in mitigating the expected harmful effects of Canada’s recently adopted anti-prostitution laws.
Despite global evidence that sex workers (SWs) are disproportionately impacted by HIV infection, data on HIV treatment outcomes among SWs remains sparse. This study therefore aimed to identify correlates of plasma viral load (PVL) suppression among HIV seropositive street- and off-street SWs on HAART in Vancouver, Canada.
Our analyses drew on 2010–2012 data from a longitudinal cohort of cis- and trans-gendered SWs known as AESHA (An Evaluation of Sex Workers Health Access), linked to the BC Centre for Excellence in HIV/AIDS’ Drug Treatment Program (DTP) data for HAART dispensation and disease monitoring. The analytic sample was restricted to 74 HIV seropositive SWs (14+ years) who had initiated HAART prior to baseline. Bivariate and multivariable logistic regression using generalized estimating equations (GEE), were employed to identify longitudinal correlates of PVL suppression, defined as plasma RNA viral load <50 copies/mL3.
The baseline median age of the sample was 35 years old (Inter-quartile range: 29.0–43.0), with 63.5% reporting Aboriginal ancestry. While 60.8% of women achieved PVL suppression, only 27.0% maintained PVL suppression from baseline. In multivariable GEE analyses, ≥ 95% adherence (AOR= 5.04; 95% CI 2.62–9.73) and older age (AOR=1.07; 95% CI 1.03–1.13) were positively correlated with PVL suppression. Having an intimate partner (AOR=0.46; 95% CI 0.22–0.96) and homelessness (AOR=0.44; 95% CI 0.26–0.74) were negatively correlated with PVL suppression.
Even after adjusting for adherence, having an intimate partner reduced the odds of PVL suppression, suggesting a need to more closely consider the gender dynamics surrounding serostatus disclosure with SWs’ intimate partners. This research highlights the need for increased attention to the social and structural factors shaping HIV treatment outcomes. Combination interventions, including youth and couple-level strategies remain critical, alongside access to stable housing women-centred approaches to HIV care for SWs.
Lesbian, bisexual, queer (LBQ) women and other women who have sex with women (WSW) are vulnerable to sexually transmitted infections (STI), including HIV. Yet structural drivers of HIV/STI among WSW, such as sexual stigma, are underexplored. Scant research has evaluated HIV/STI prevention interventions among this population. The Queer Women Conversations (QWC) study pilot tested a group-based HIV/STI prevention intervention with WSW in Toronto and Calgary, Canada.
We conducted a multicentre, non-randomised pragmatic cohort pilot study using a pre-test/post-test design with 6-week follow-up. The intervention was developed in collaboration with community-based organizations in Calgary and Toronto, informed by key informant interviews (n=6), and theoretically grounded in the social ecological model. The primary outcome was sexual risk practices, and secondary outcomes included intrapersonal (self-esteem, STI knowledge, resilient coping), interpersonal (safer sex self-efficacy), social (community connectedness, social support) and structural (sexual stigma, healthcare access) factors.
Forty-four women (mean age 28.7 years) participated in a weekend retreat consisting of six consecutive sessions tailored for LBQ women. Sessions covered topics addressing social and structural drivers of HIV/STI and sexual risk practices, including STI information, safer sex negotiation, healthcare access, and sexual stigma. Adjusted for socio-demographic characteristics, sexual risk practices (β2= −2.96, 95% CI −4.43, −1.50), safer sex self-efficacy (β2= 2.78, 95% CI 0.82, 4.78), STI knowledge (β2= 4.41, 95% CI 3.52, 5.30) and sexual stigma (β2= −2.62, 95% CI −3.48, −1.75) scores showed statistically significant changes 6 weeks post-intervention.
QWC is among the first studies to address social and structural drivers of HIV/STI among WSW. The QWC intervention was effective in impacting several inter/intra personal and structural factors. Initial increases in social support and community connectedness were not sustained, highlighting the need for alternative approaches to influence social factors. Study results may inform HIV/STI prevention interventions tailored for LBQ women.
The Dr. Peter Centre (DPC) is a low-threshold care facility for People Living with HIV in Vancouver who experience concurrent barriers to achieving optimal therapeutic outcomes. There is no documented evidence of the impact of the DPC’s health-equity approach on the health outcomes of clients. Here we report on the relationship with adherence to antiretroviral treatment (ART).
A longitudinal cohort of recently enrolled DPC clients provides the data frame for this analysis. Socio-demographic, relevant social determinants of health, and health and social service utilization data are ascertained through an interviewer-administered survey. Clinical variables are obtained through longitudinal linkages with the provincial Drug Treatment Program. The survey adapts a set of eight health-equity indicators (HEI) designed to measure clients’ perception of the DPC based on a likert response scale, which were then trichotomized as positive (always/most of the time), neutral (sometimes) and negative (not usually/never). Univariate analyses were conducted to measure the association between optimal ART adherence (≥95%) in the 12 months prior to interview date and salient explanatory variables, including positive responses to the HEI.
This analysis is based on the 74 out of 99 participants who completed the baseline interview between February 2012 and December 2014, and for whom we have an up-to-date clinical linkage. Median age was 46.5 (IQR 41–51), 18.9% were female, 31.1% identified as Aboriginal and 45.9% were optimally adherent to ART. The three HEI that were most frequently ranked positively were feeling: welcomed by staff (95.9%), respected by staff (95.9%), and cared for by staff (89.2%). In univariate analyses, only higher income was associated with optimal adherence (p=0.031). None of the HEI attained statistical significance.
The HEI fell short in explaining the disparity in ART adherence, potentially a reflection of the adherence measure being taken during a time period when the client was not engaged in DPC services. More in-depth consultation with clients is underway to refine the indicators and isolate the impact of a health-equity oriented approach to care.
Rates of sexually transmitted and blood borne infections (STB-BIs) among older adults in Canada have increased over the past decade due to a variety of interpersonal, community and environmental factors. The number of older adults living with chronic STBBIs such as HIV and hepatitis C also continues to rise. The Public Health Agency of Canada has developed a Questions and Answers resource to address the most commonly asked questions about the prevention of STBBIs among older adults.
This resource was developed through a synthesis of findings from a systematic review of the literature related to the key determinants of STBBI vulnerability among older adults
Individual, interpersonal, and community factors including: knowledge, risk perception and awareness, communication and relationships contribute to increased STBBIs rates among older adults. Ageism and discrimination, social connectedness, mental health, socio-economic status, and the intersection of aging with gender, culture and sexual orientation are key determinants of vulnerability to STBBIs among older adults. Findings suggest that: a) acknowledging the importance of sexual health for older adults; b) recognizing the influence of life events and transitions in people’s lives; c) establishing multiple points of service access; d) intersectoral collaboration; e) including older adults in program development and delivery; and f) incorporating age and culturally appropriate approaches, have the potential to improve health outcomes and prevent STBBIs among older adults.
Community organizations, health professionals and service providers are encouraged to use the findings in this resource to help guide the development and implementation of STBBI prevention interventions that address the needs of older adults living with or at risk of STBBIs. Increased inclusion of older adults in research addressing STBBI prevention is needed in order to build evidence on effective interventions among this population.
Although methadone maintenance treatment (MMT) has been found to reduce HIV risk behaviours and promote HIV treatment adherence among drug-using populations, little is known regarding how regulatory changes influence MMT engagement and related outcomes. In British Columbia (BC), provincial regulations governing MMT have recently been modified, including: replacing the existing methadone formulation with Methadose® (pre-mixed and 10 times more concentrated); prohibiting pharmacy delivery of methadone; and, prohibiting pharmacies incentives for methadone dispensation. We undertook this study to examine how these changes shaped MMT engagement and HIV risks and treatment adherence among a structurally vulnerable drug-using population enrolled in MMT in Vancouver, BC.
Qualitative interviews were conducted with 34 people enrolled in MMT and who were recruited from two ongoing observational prospective cohort studies comprised of drug-using individuals in the six-month period following the changes to the provincial methadone program. Interview transcripts were analyzed thematically, and by drawing on the concept of ‘structural vulnerability’.
These regulatory changes disrupted MMT treatment engagement, and thereby fostered HIV risks and treatment interruptions. The introduction of Methadose® precipitated increased withdrawal symptoms. The discontinuation of pharmacy delivery services led to interruptions in MMT and co-dispensed HIV medications due to constraints stemming from poverty and housing instability. Meanwhile, the loss of pharmacy incentives limited access to material supports utilized by participants to overcome barriers to MMT. Collectively, these changes functioned to compromise MMT engagement and increased structural vulnerability to HIV treatment interruptions and risks, including reinitiation of injection drug use and participation in high-risk income-generating strategies.
Greater attention to the impacts of structural inequities on MMT engagement and related HIV outcomes is needed when modifying MMT programs, especially with other provinces moving to introduce similar changes. Comprehensive environmental supports should also be provided to minimize adverse outcomes during transitional periods.
CACTUS Montreal has been offering harm reduction services to the downtown community for several years. While its’ fixed needle exchange program is well utilized, the need for another service delivery model was identified. The “Messages de rue” program was put in place in 2010, with the aim of increasing access to harm reduction supplies through street-based peer intervention. Since June 2014 we started a service called Kit Mobile. Kit Mobile was made possible through a prevention grant on HIV, hepatitis and other sexually transmissible infections in the Montreal area from the Director of Public Health at the Agence de la santé et des services sociaux de Montréal.
Kit Mobile is an injection material delivery service for injection drug users (UDI).
According to the SurvUDI 2010 study, sharing injection material is the main cause for the spread of STBBI among injection drug users. in order to decrease this spread a project involving 8 organizations identified the delivery of sterile injection material in communities as an innovative practice that would help reach injection drug users, whatever their life situation.
The Kit Mobile delivery service aims to reach people who do not use conventional outreach services.Teams from able partner organizations carry out deliveries in the areas needing the service.
CACTUS have the “Messagers de rue” team already in place which constitutes current or former drug users, and/or working in the sex industry. The Messagers are provided with training on a range of issues including HIV, hepatitis C, harm reduction and safe needle disposal. They are supported by a Program Coordinator and a Support Worker, who offers guidance while out on the streets.
In addition to providing clean works, the Messagers also act as harm reduction promotion agents. Meeting people “where they are” in their experience of addiction, the Messagers help their peers to better understand and reduce risks related to drug-use, and encourage them to seek testing and treatment for STBBIs.
The problematic consumption of psychoactive substances (PAS) constitutes a health problem for certain gay/bisexual men. The present qualitative descriptive study, inscribed in symbolic interactionist perspective, aims to: 1) describe and understand the interrelations between certain dimensions of the gay life course experience and the consumption trajectories of PAS among gay and bisexual men; 2) document their experience in relation to their use of addiction services; 3) establish links between their experiences and their gay life course experience; and, 4) identify their needs in matter of services. Thirty five semi-structured interviews were conducted with gay/bisexual men, who have a problematic consumption of PAS and have an average age of 46 years. The whole material has been analysed thematically.
The addiction trajectories and the homosexual experience intersect at the time of the discovery and acceptance of homoerotic desires, during sexual experimentation; when finding a sexual/emotional partner; and as part of the socialization in the gay community, particularly in sexualized spaces. Most participants have used substance abuse services but only about half of them have received a reference to such services. The increase in consumption often emerges when they have problems accepting their sexual orientation, during sexual experimentation or when they receive an HIV diagnosis. The increase of consumption associated to these life events sometimes leads the participants to question their consumption and to demand help from a specialized service. For certain participants their consumption has not been detected in a good moment. Moreover, sexuality is not addressed in the addiction services.
The results show the relevance of considering the different dimensions of the homosexual experience, including sexuality, in the prevention strategies for consumption as well as in the addiction therapies for this population. In addition, the detection of problematic consumption in this population represents a challenge.
Canada has had limited success in addressing HIV/AIDS among People Who Inject Drugs (PWID). This is of growing concern, particularly, in the northern regions of the western Canadian provinces where the rate of new infections has grown in opposition to stabilized or declining trends elsewhere and healthcare systems are struggling to manage the increased number of cases.
The purpose of this study was to determine the comprehensiveness of existing services for HIV prevention, treatment and care for PWID, specifically in northern BC, and recommend targets for additional services, and/or stepping-up or scaling-down of existing services.
This project, using a Community Based Research (CBR) model, was completed in four phases: (1) mapping of existing services in northern BC, based on a Comprehensive Package listed in the 2012 WHO Technical guide for countries to set targets for universal access to HIV prevention, treatment and care for injecting drug users; (2) an adaptation of the WHO Guide to the local northern BC context and the development of interview guides to be used in the next Phase, through individual and/or group discussions with a Community Advisory Committee (CAC); (3) the collection of primary information through key informant interviews with three distinct populations, including service users, frontline staff, and managers/decision-makers; and (4) reconvening with the CAC to discuss the most appropriate and effective way to translate the knowledge gained in this study.
Results from the project included a detailed overview of current services, as well as an assessment of service gaps and needs, based on information gathered through meetings with a 10 person CAC and interviews with 52 participants throughout northern BC. The final outcome of this exercise was a set of recommendations for implementing, monitoring, and stepping-up or scaling-down services for PWID in northern BC.
Increasing numbers of Ottawa youth who use drugs drove request from community for study to examine the HIV- and HCV-related risk environment experienced to inform the development of age- and gender-fluid harm reduction interventions. This paper uniquely examines factors associated with non-fatal overdose – an all too frequent occurrence among Ottawa youth.
November 2013 personal structured interviews with 125 young people aged 16–25 who were capable of informed consent and had either ever injected drugs or smoked crack or had used >2 hard drugs in the past 3 months. Dried blood spot samples collected for HIV and HCV testing.
HCV prevalence 16% among women (95% CI 5.3, 34.20) among men 14% (95% CI 8.5, 23.4).
The majority of women (53%) and men (71%) had seen someone else overdose and close to the majority of both women (47%) and men (49%) had themselves overdosed. Mean overdoses among men 3.6, range 1–20; among women 2.9, range 1–10. Youth with history of injecting drugs twice as likely to overdose (OR 2.4, 95% CI: 1.1, 4.8); who binged three or more times a month three times as likely to overdose (OR 3.4, 95% CI: 1.1, 10.5) as were youth who had tried unsuccessfully to get into drug treatment (OR 3.2, 95% CI: 1.2, 8.0); and youth who used opiates were six times as likely to overdose (OR 5.9, 1.9, 18.6).
Youth who use and inject drugs are an increasing demographic in Ottawa and are a population whose HIV- and HCV-related risk environment and prevention needs are not widely known or understood. Documenting factors associated with non-fatal overdose among this population is key to enabling community agencies to identify youth with substance use disorders at high risk of adverse outcomes and in driving the development of evidence-informed responses; particularly expansion of drug treatment programmes.
Despite a landmark Supreme Court ruling, Canada has not yet seen new supervised injection facility (SIF) implementation. Developing rules and policies at SIFs that are appropriate for local needs is challenging, and age restrictions represent a topic that is likely to be especially contentious in the Canadian context. People under age 18 who inject drugs represent a population with high needs for health and social services, including education on HIV and hepatitis C prevention. Internationally, age restrictions at harm reduction programs often formally exclude this population. We documented the perspectives of people who use drugs and other stakeholders regarding whether SIFs should have age restrictions. Interviews and focus groups were conducted with a total of 95 people who use drugs and 141 other stakeholders in Toronto and Ottawa. Consensus regarding SIF age restrictions was elusive. Some people who use drugs and other stakeholders did not specify minimum age(s) for SIF access, while others recommended minimums (e.g., 16, 19). For many participants, including people who use drugs, the thought of people under 18 injecting drugs generated great discomfort. This discomfort appeared to contribute to difficulty accepting SIFs as a potential response to injection drug use by young people. Participants invoked notions of vulnerability and recommended that it was more important that youth receive “help” than access to SIFs, suggesting that there is still opportunity to prevent youth from becoming adults who continue to use drugs. At the same time, participants stated that young people make their own decisions and said that those who continue using drugs would benefit from the safer environments that SIFs provide. This study contributes new empirical insights regarding youth access to important HIV prevention and health services. Such considerations may help establish optimal age-related policies for harm reduction programs.
Increasing numbers of Ottawa youth who use drugs drove request from community for study to examine the HIV- and HCV-related risk environment experienced to inform the development of age- and gender-fluid harm reduction interventions. This paper uniquely examines participant’s self-assessment of their mental health and impact on HIV- and HCV-related risk behaviors and practices.
November 2013 personal structured interviews with 125 young people aged 16–25 who were capable of informed consent and had either ever injected drugs or smoked crack or had used >2 hard drugs in the past 3 months. Dried blood spot samples collected for HIV and HCV testing.
HCV prevalence 16% among women (95% CI 5.3, 34.20) among men 14% (95% CI 8.5, 23.4).
The majority of women (65%) and nearly half of the men (46%) self-assessed their mental health as fair or poor.
Youth who reported non-consensual sex were significantly more likely to assess their mental health as poor or fair (OR 2.8 95% CI: 1.2, 6.8) as were: younger participants aged 16–21 (OR 2.9, 95% CI: 1.4, 6.1); those who reported any form of emotional or physical abuse or neglect (OR 3.8 95% CI: 1.3, 11.3); and youth who reported thoughts of suicide or hurting self (OR 5.5 95% CI: 2.5 12.1).
Of concern, among youth who responded to questions about sharing drug injection equipment, those who reported poor mental health were significantly more likely than those who reported better mental health to report sharing needles (p=0.004) and sharing other injecting equipment (p=0.07).
Youth who use and inject drugs are an increasing demographic in Canada. Documenting factors associated with self-assessment of poor mental health among this population is key to enabling community agencies to identify youth with substance use disorders at high risk of engagement in HIV- and HCV-related risk behaviors and practices.
In this narrative inquiry study, we focused on the lives of three African immigrants living with HIV in Alberta, Canada. Living alongside each participant over a period of a year, we attended to the phenomena of living with HIV as an African immigrant in Canadian society. We discovered that each participant had searched for narrative coherence across geographic, political and social spaces, and lived their lives in the midst of a continuous unfolding life narrative. That is, people’s lived experiences were located along the dimensions of time, place, and the personal and social. Participants’ lives continued to unfold against and within a familial, cultural, and social particularity that had not been left behind in their home countries. In this presentation, we will make visible the stories that participants carried across different spaces and examine how these stories shaped their experiences of living with HIV in their new host country of Canada. In this presentation we will also discuss notions of sacred and mundane stories, to show how time, context and place interconnect. These interconnections have profound effects on narrative coherence and the (dis)continuity of narratives in the life making of immigrants. We will highlight the social significance of participants’ experiences, attending particularly to their sacred and mundane stories, and present key considerations for support and intervention work with the African immigrant community affected by HIV in Canada.
African, Caribbean and Black (ACB) people carry a disproportion burden of HIV infection. This CIHR funded study examines the placement of “riskier sex” (RS) in the social and sexual scripts of ACB youth.
Between April, 2013 and March, 2014, 543 16–25 year old ACB participants living in the Windsor area of Ontario, recruited using Respondent Driven Sampling, completed a survey inquiring about sexual behaviours and social/living situations. Riskier sex (RS) was measured on a 7-point scale combining information about penile-vaginal (PVI) or anal (AI) intercourse, number of lifetime partners, partner concurrency, and condom use. Logistic regression examined social correlates of ever engaging in PVI and/or AI. OLS regression examined sexual and social correlates of level of risk on the RS scale.
Ever engaging in PVI or AI was significantly associated with being older, using more sexual health services, spending more leisure time with friends, and spending less leisure time in religious activities. African youth postponed PVI and AI longer than other groups. Riskier sex developed over time with those who were sexually active longer having significantly higher scores on the RS scale. It was embedded in sexual scripts that included non-relational sex. The social profile of youth who engaged in riskier sex included living in distressed neighborhoods, living independent of natal family, birth in Canada, and spending more leisure time playing sports or in gyms. There was no difference in RS scores across the ACB ethno-racial groups and predictors/correlates did not differ by sex.
Results contribute to knowledge about social and sexual correlates of riskier sex among ACB youth, an area of sparse research. They also provide guidance for prevention programming in terms of important social associated with (e.g., sports teams and gyms; distressed neighborhoods; independent living) and the sexual scripting of riskier sex in this population.
In Canada, since the late 80’s, there have been over 70 convictions and more than 90 HIV-positive people have been on trial for having unprotected sex, even when no HIV transmission occurred (Mykhalovskiy, 2010). Notably, the Cuerrier decision (1998) by the Supreme Court of Canada became a landmark case that ruled that a person living with HIV who does not disclose his or her HIV- positive status and exposes another person to a “significant risk” of HIV transmission, could be found guilty of aggravated assault (Symington, 2009). The majority of cases of persons convicted under HIV criminalization legislation in Canada were African/Black men (ACCHO, 2010). This presentation will examine the impact of criminalization of non-disclosure of HIV positive African/Black people living with HIV from a historical and contemporary transnational perspective. This presentation utilizes an intersectional anti-oppression, social determinant of health approach to examine African/Black communities affected by HIV/AIDS in the greater Toronto area as well as its international impact. Specifically, this presentation will discuss my research findings on the experiences and responses to the impact of criminalization of non-disclosure of HIV positive status and increased HIV stigma on African Diasporic communities from the perspectives of African/Black women and men living with HIV; service providers (therapist, case workers, social workers) and agency directors working in the HIV Sector; and the legal sector (politicians, lawyers and/or judges). This presentation is relevant to the conferences themes as it offers a critical look at an anti-oppression/anti-colonial analysis on criminalization of non-disclosure of HIV positive status and the impact on the health of African/Black communities.
In Canada, the majority of women living with HIV (WLHIV) were infected through heterosexual sex. Over the last decade, prosecutions for nondisclosure of HIV has increased. Many ASOs have developed position statements advocating against HIV criminalization, arguing that criminal law is ineffective and inappropriate to address HIV exposure. Positive Women’s Network (PWN), a partnership of WLHIV and women affected by HIV, is the only women-specific HIV organization in BC and currently has over 750 “members” (WLHIV). PWN is in a unique position since it must take into account the use of the criminal law from the perspective of a group with distinct concerns: infected and affected women.
From 2010–2014, PWN conducted 6 focus groups with a total of 60 WLHIV participants. Topics included level of knowledge about nondisclosure laws, level of comfort disclosing to sexual partners, family and friends, level of comfort sharing information about nondisclosure laws with peers. Diversity of participants is representative of PWN’s membership demographics.
Drawing on focus group discussions, there are mixed and contentious views: criminalization denounces and deters dishonest men who put women’s health at risk; criminalization puts vulnerable women, such as those in abusive relationships, at higher risk; criminalization deters women from reporting sexual assault because the perpetrator could counter with nondisclosure allegations, criminalization perpetuates HIV stigma; criminalization adds another barrier to disclosure and testing. The majority of participants did not know current nondisclosure legislation.
Emotional distress about the topic, language barriers, literacy issues, the complexity and ever changing nature of the legislation.
Our results indicate that women-specific HIV services face challenges to taking a position on the criminalization of HIV non-disclosure because of the complex realities of sexual practice and gender, gendered violence, and HIV stigma. Promoting in-person education about nondisclosure legislation for WLHIV is proposed.
In 2009, Johnson Aziga was found guilty of two counts of first-degree murder, along with multiple counts of aggravated sexual assault, and was subsequently declared a dangerous offender. Using the discussion surrounding this unique case, this paper explores claims about responsibility. This paper shows how the rhetoric of responsibility has shifted from one of mutual responsibility concerning safe sex practices to one that puts the onus on those who are aware of their HIV-positive status. The discussion surrounding the Aziga case has generated a dichotomization between ‘victims’ and ‘non-disclosers’, with understandings of responsibility at the core. This paper employs a social constructionist perspective as an analytical tool to better understand the creation and strategic uses of these categorizations. The paper also establishes the relevance of Talcott Parsons’ concept of the ‘sick-role’ in understanding how these categories and typifications work. With the criminalization of HIV non-disclosure on the rise, it becomes important to understand the rhetorical bases and justifications for taking a criminal, rather than solely a public health, approach to dealing with HIV transmission. Understanding this process will shed light not only on how those who do not disclose their HIV-positive status are perceived and treated, but may also provide insights into the repercussions on the HIV/AIDS community as a whole, including the exacerbation of HIV-related stigma.
Disclosure is linked to reductions in HIV transmission; adherence to medical regimens; access to support services; improved mental health status; and effective adaptation to living with HIV. Yet, it has remained a significant challenge for HIV/AIDS service providers to successfully incorporate into support and treatment programs. The HIV disclosure intervention developed in Ontario provides a systemic way to guide AIDS Service Organizations (ASOs) to support their clients’ disclosure efforts through a controlled and managed process. This study explores opportunities and challenges of adopting the intervention within ASOs in Ontario.
In 2012/14, we conducted an extensive literature review to identify existing guidelines on intervention integration and facilitators/inhibitors of organizational adoption of HIV disclosure intervention. We reviewed both published and ‘grey’ literature to ensure inclusion of a broad range of information including from community-based settings. We also conducted 4 focus groups with organizational management; peers/volunteers; support workers; and women living with HIV to identify unique possibilities and challenges of intervention integration.
The key identified facilitators of integration included: the use of focus groups and key informant interviews to guide the process; the incorporation of peers and support groups to implement intervention; identifying roles and responsibilities to allow for efficient and effective application of intervention; creation of supportive learning environments to share knowledge and build capacity; and organizational assessment/review to identify how interventions would impact service delivery.
Identified challenges included: mitigating clients’ internalized stigma and ensuring safety; social class contentions in small cities; issues of support for those being disclosure to; and limited resources to train staff and peers implementing intervention.
Disclosure is an effective HIV prevention strategy and when managed properly, it can be beneficial for both the person disclosing and the person being disclosed to. Our study highlights the significance of strengthening organizational infrastructures to support integration of the HIV disclosure as part of their overall service delivery.
A recent Canadian Association for HIV Research (CAHR) report calls for training initiatives that will produce trainees who are better equipped for careers in HIV research (Campbell, M., & Bisby, M., 2013). Universities Without Walls (UWW) a CIHR-funded HIV training program, bridges the gap between academic research and “on the ground” work in public health through an online and in-person curriculum of community-based research (CBR), research ethics, interdisciplinarity, and community service learning. Here, we assess the strengths and challenges of UWW through an analysis of the evaluation of five generations (2009–2014) of Fellows’ experience with, and knowledge of, core UWW components using a modified Kolb (1984) experiential learning scale.
Each UWW fellow completes a “retrospective post-then-pre” evaluation (Lam, T. C. & Bengo, P., 2013) and a brief written reflection of their fellowship. The evaluation team, co-led by UWW fellow and UWW core mentors, conducted thematic analysis on narratives from the 61 fellows and summarized the results according emerging themes.
Forty-six (75%) were graduate students, 8 (13%) were community fellows, 7 (12%) were health professionals and 9 (15%) disclosed they were HIV positive. Overall, 63% of fellows were in the top 10% of their previous cohort and nearly 75% of fellows had a multi-disciplinary background. Four key results emerged: 1) developed a “community of practice” with other researchers and mentors that break down geographic, disciplinary and academic-community barriers; 2) increased confidence in conducting inclusive, community-driven CBR; 3) enhanced professional skills to link research with community practice; 4) increased self-reported knowledge, skills and experience along the core UWW components from “novice/advance beginner” to “competent/proficient”, and 5) published 52 peer-reviewed articles of which 22 (42%) were in the top 5 journals in their field.
UWW encourages fellows’ participation in HIV research communities of practice, builds confidence in new researchers, encourages the retention of promising researchers in HIV and related research fields, and promotes the greater and meaningful inclusion of people living with HIV in research.
There is a growing body of empirical evidence that shows extremely high rates of childhood sexual and physical abuse among HIV positive adults. For women, the situation is particularly dire. Women are disproportionately affected by trauma and post traumatic stress disorder (PTSD). Research reveals that the rate of PTSD among HIV infected women is 30%, which is five times higher than the general female population. Rates of childhood sexual abuse are 39.35%, which is double that of the general population.
A total of 42 female patients with HIV on an inpatient unit have been asked to complete the Binghamton Childhood Sexual Abuse Survey as well as a demographic tool that looked at a number of variables including housing, drug and alcohol use, history of sex trade work, CD4 and viral load. The expected sample is 50.
An overwhelming majority of the women who have participated so far have screened positive for childhood sexual abuse and had a history of illicit drug use. Research is ongoing at this time although we anticipate a complete sample of 50 within the month. It appears that childhood sexual abuse is significantly associated with illicit drug use and non-adherence. Will need final results to confirm.
To explore the perspectives of rehabilitation providers (PTs and OTs) in Nyanza Province, Kenya, and Lusaka, Zambia, regarding their role in the care of people living with HIV (PHAs).
HIV prevalence is greater than 20% in both Nyanza Province and Lusaka. ART access is growing in these settings, resulting in many people living longer with HIV and the resulting comorbidities and disablement. Therefore, the need is great for HIV training across the health care continuum, including rehabilitation.
Pilot testing was conducted to assess an HIV teaching tool for rehabilitation providers in Sub-Saharan Africa in October–November, 2014. The tool was adapted from the Canadian “E-Module on Evidence-Informed HIV Rehabilitation” developed by CWGHR. Pilot testing was conducted in Kenya (n=32, 21 PT/11 OT) and Zambia (n=31, all PT) and included a demographic questionnaire, a survey on knowledge of HIV and rehabilitation, and focus groups (5 in Kenya, 5 in Zambia) to discuss the teaching tool. We present an interpretive analysis of focus group data regarding participants’ perspectives on rehabilitation for PHAs. Ethics approval was received from University of Toronto, University of Zambia, and KEMRI (Kenya).
This was the first exposure to the role of rehabilitation with PHAs for many participants, despite the high local HIV prevalence. Participants described rehabilitation as largely excluded from HIV trainings, policy meetings, and care (except end-of-life). They emphasized that rehabilitation will require engagement of community-based, non-professional providers. Participants noted that their new insights regarding HIV also broadened their perspectives on rehabilitation for other conditions.
Formal HIV policies (e.g., national strategic plans) in Sub-Saharan Africa now frequently recognize disability within the HIV continuum, yet rehabilitation providers remain marginalized if not excluded from the HIV response in many settings. Incorporating HIV training into undergraduate and continuing rehabilitation education is a crucial next step for the HIV response in Africa.
Indigenous leadership and residents of remote and rural areas in interior BC are concerned about lack of research that addresses sexual and drug related vulnerabilities among young Aboriginal people in their communities.
The Cedar Project is an ongoing prospective cohort study of young Aboriginal people (aged 14–30) who use drugs in Vancouver, Prince George, and Chase. Cross-sectional data was collected on drug use, resiliency and sexual risk. Participants completed the 28-item Childhood Trauma Questionnaire (CTQ) and the 25-item Connor-Davidson Resilience Questionnaire (CD-RISC) between 2011–2012.
Of the 340 participants in Prince George and 104 in Chase, many were taken away from their biological parents (63% and 44%, p<0.0001), and had parents who were in residential schools (46% and 37%, p=0.0762). Those in Chase scored higher on the resilience scale (64 vs. 60), and had lower frequency and severity of all abuse types: emotional abuse (43% vs. 28%), physical abuse (41% vs. 27%), and sexual abuse (45% vs. 22%). Injection drug use among Chase participants was much lower (52% vs. 11%, p<0.0001). Among participants who injected drugs, 2 of the 8 participants in Chase, and 13 of the 166 participants in Prince George tested positive for HIV (p=0.5374). 2 of the 8 participants in Chase and 96 of the 166 participants in Prince George tested positive for HCV (p<0.0001). While alcohol consumption was higher in Chase (91% vs. 82%, p=0.0212), more Prince George participants reported experiencing non-consensual sex (50% vs. 27%, p<0.0001; average age at first experience 7.7 years and 10.5 years) or engaging in sex work (38% vs. 18%, p<0.0001; average age at initiation 16.9 years and 19.3 years). There was no difference in sexual risk (63% vs. 64%).
Young Aboriginal people in Chase have lower levels of injection drug use but have elevated levels of alcohol consumption, and sexual vulnerability. Sexual health education and programming is urgently required in this rural community.
Combining behavioural, biomedical, and structural prevention strategies has shown promise as a way to reduce HIV incidence in the gay community. A prerequisite for combining these strategies is that they be accessible to community members.
Discuss barriers identified in the context of an exploratory study with respect to accessing and combining prevention strategies.
A intersectoral committee composed of 30 participants (clinicians, researchers, community and public health stakeholders) was formed to identify current challenges for HIV prevention in Montreal’s gay community and propose models for implementing a combination prevention approach. Using ethnographic methods, an analysis was undertaken of the accessibility of different prevention services from the perspective of a gay man wishing to access to them. A cartography of all available services was developed and an initial assessment was made from a systems perspective in relation to five dimensions of accessibility: approachability; acceptability; availability / accommodation; affordability; appropriateness / adequacy (Lévesque et al. 2013).
Committee discussions and results from the cartography showed that in theory, numerous prevention services are offered to gay men in Montreal, either separately or in combination. In reality, problems were identified in relation to all five dimensions of accessibility and particularly with respect to approachability, acceptability, and appropriateness / adequacy. Results from the cartography were subsequently used to develop indicators that will be useful for a more comprehensive assessment in the future.
In order for a combination HIV prevention approach to be operationalized, problems with the accessibility of programs and services must be addressed, in particular a systemic lack of integration and coordination among stakeholders working in different sectors. The conceptual framework and methodology developed for this study could be useful in other locations within Canada or elsewhere as part of broader-based reflection on the potential and challenges of combination prevention.
An integrated approach to sexually transmitted and blood borne infections (STBBIs) focuses on common transmission routes, risk behaviours and social and structural risk factors. However, the unique impact of specific infections on certain populations highlights a need for population- and disease-specific prevention within an integrated approach. Two examples are chlamydia infection among young women and syphilis infection among gay men and other men who have sex with men (MSM), where rates across Canada are on the rise.
A comprehensive review of existing evidence on chlamydia and syphilis and other STBBI prevention interventions was conducted to inform the development of two population-specific prevention resources.
Based on a review of the evidence, prevention strategies for both chlamydia and syphilis include the application of elements of the Information, Motivation, Behavioural Skills model to promote and support increased awareness and behaviour change, in addition to challenging STBBI-related stigma. Disease-specific prevention strategies for chlamydia among young women include using interactive strategies to deliver information and teach skills and enhancing personal perception of risk, and for syphilis among gay men and other MSM include increasing testing and innovative approaches to partner notification.
Within an integrated approach, effective STBBI prevention takes into account the specific impact of specific infections on certain populations. It also considers the social, structural and economic factors that increase vulnerability to STBBIs and includes a combination of upstream, primary, secondary, and tertiary prevention interventions. Expanding the scope and understanding of promising practices and identifying knowledge gaps through program evaluation are important for an effective response to chlamydia and syphilis infection.
Pre-exposure prophylaxis (PrEP) has generated considerable interest as a strategy for preventing HIV among gay, bisexual and other men who have sex with men (MSM). As its clinical delivery gets underway in Canada, further input from potential PrEP users and the broader MSM community is needed on how PrEP is perceived and how it should be delivered.
Working within a grounded theory and method framework, we conducted thirty qualitative interviews about PrEP with adult MSM from the Toronto area who self-identified as HIV-uninfected. Participants were recruited through a busy sexual health clinic largely servicing the gay community, and completed a brief questionnaire including a validated HIV risk index for MSM (HIRI-MSM) prior to the interview. Interviews were audio recorded and transcribed and averaged about one hour in length.
Median (interquartile range, IQR) age was 34.5 (27,44) years, 70% were of European descent, and all identified as gay (n=28) or bisexual (n=2). Participants reported a median (IQR) of 12 (6,30) male partners in the preceding 6 months and 73% scored >10 on the HIRI-MSM, indicating substantial risk of HIV seroconversion. Key issues discussed include 1) criteria for determining PrEP eligibility, 2) how to communicate with users about combining PrEP with condoms and other prevention methods, and 3) the potential for PrEP-related stigma. While data analysis is ongoing, preliminary results additionally include clinical concerns about PrEP (eg. adherence, side effects, risk of other sexually transmitted infections, financial cost), as well as variable perceptions of HIV risk, disclosure of both HIV status and PrEP use, and indirect benefits of PrEP use.
The input of potential PrEP users is valuable for PrEP delivery. Further work is needed to understand the opinions of key at-risk populations and how PrEP programming can best address their concerns.
Developing new qualitative methods created from communities for communities is a challenging process. HN is a community-based method that was evaluated in a focus group in order to understand the more efficient use of meaningful methodologies for developing HIV ethno-racial prevention efforts.
Program Science new approach in HIV requires a better use of resources, methodologies and methods. How to understand emerging community needs and how to better evaluate qualitative efforts has been an issue, particularly where immigration status and language barriers could be in the way of effectively communicating participant’s life experiences.
15 HN were conducted within the context of two CN groups (2013–2014). In HN the hands are used as metaphors for understanding intersectionalities, life events and sexual desires, pleasures and passions narratives. In order to gain more information, HN methodology was presented in CN (Oct–Nov 2014 group) for open discussion, themed identification and evaluation. (n=10: 7 Venezuelan, 1 Brazilian, 1 Salvadorian, 1 Colombian). The focus group was audio recorded (41’48”).
HN is an excellent method for sharing experiences related to sexuality, desires, life events-trajectories and intersectionality.
Allow participants naming and narrating their major issues such as: migration, “coming out”, falling in love, HIV infection, sexual pleasures and sexual health practices.
HN is appropriated for depicting personal sexual health narratives using a visual and colourful format.
The method allows an easy communication tool for ethno-racialized individuals (even the ones who do not speak official Languages) to communicate and inform sexual health policy making, research and programming, assuring the approach to Program Science guidelines.
As a community-based method in its early stages, many methodological and qualitative issues are related to this new format. However, HN’s major value is that the method was developed at the core of a community program, and it requires minimum economical resources.
In recent years, barebacking has become a popular focal point within the academic literature aimed are HIV prevention among gay men. From a poststructural standpoint, barebacking has come to signify a tension within the subjectivities of gay men because of the desire to both engage in this practice and to avoid HIV transmission. In addition, barebacking has become a way in which gay men can be classified as “at risk” within the broader HIV-prevention public health discourse.
This paper will present the findings of a qualitative research project in Nova Scotia aimed at examining the perspectives of HIV/AIDS activists on the way barebacking is conceptualized and discussed across various sectors including public health, ASOs, physicians and within the gay men’s communities. This research employed a Foucauldian discourse analysis to investigate how 17 HIV/AIDS activists experienced living and working in Nova Scotia during the HIV/AIDS movement.
Activists’ perspective on barebacking conceptually shifts the practice from an individual, isolated act to a discursive production. Specifically, what emerges in this interpretation is the normalizing effects of both silence and rendering visible barebacking practices and subjectivities in public and private spaces. The effect of silence is to draw the boundaries of discourse, while at the same time, rendering visible the discursive, prevention-focused spaces public health occupies.
Barebacking discourse is not a completely stable concept and cannot simply be reduced to convincing gay men to wear condoms. The depth of meaning associated with raw sex has evolved significantly during the decades of HIV/AIDS and, as barebacking discourse evolves within the gay male community, the public health and health promotion response must become more nuanced. This nuance involves understanding barebacking as the site where the competing discourses of condomless sex and HIV prevention play out in the lives of gay men.
As an Anthropology Master’s student, I am interested in how sexual health, HIV/AIDS, and risk discourses are constructed and circulated. I recently attended a World AIDS Day talk at a sexual health organization located in downtown Winnipeg, Manitoba; this event focused on the sexual education of newcomer adults and youth. I am interested in how the connection was drawn between a World AIDS Day talk and newcomer families. In particular, how are newcomer communities explicitly and implicitly linked to HIV/AIDS and risk discourses in sexual education settings? Throughout the various presentations I noticed the language of bureaucracy and bio-pedagogy (revealing the entanglement of citizenship and health); the context of this event was tailoring sexual education programs for particular “clientele”. Even when programs seek to be culturally-appropriate, there is still a sense of rigid institutionalization. This can be gleaned from the way in which sexual education is presented, as unquestionable; there is a right answer and a wrong answer. This was readily apparent in a game called “Risk Rater’ that was introduced to the group, which the programmers play with the youth. Every participant is given three pieces of construction paper (one red, one yellow and one green). The presenter would give a scenario, for example kissing, and then everyone had to raise the card that they thought represented the risk level of contracting HIV/AIDS/STI’s (respectively high, low, and no). This clearly illustrated how risk is conceptualized in a sexual education setting. There is no room for discussion and the answers are not up for debate. I propose that efforts aimed at the promotion of sexual health and HIV/AIDS prevention can be strengthened by critically reflecting on how these messages are enacted and how they come to target particular communities.
Globally, HIV and HCV associated with illicit drug use remain significant health and social care consequences. While harm reduction approaches are well established in Canada, research has begun to question whether current harm reduction practices are sufficient to address the ongoing HCV and HIV epidemics among people who use illicit drugs (PWUID), in particular, injecting drug users.
Applying a Foucauldian interpretation of policy and practice, this paper aims to: i)present an analysis of the developments surrounding harm reduction policies and practices in Canada; ii)articulate connections between elements of Canada’s harm reduction paradigm, and the marginalization of PWUID. To accomplish this, we outline the epidemiological context of HIV and HCV relative to a historical analysis of the development of harm reduction efforts in Canada. We examine national debates on harm reduction policy, and point out how particular features of Canada’s public policy context are based on attitudes towards harm reduction knowledge, as well as diverse cultural and socio-political contingencies (social movements, institutional interests, legislation, community practices, and anti-pleasure stigma).
We find that multiple historical junctures, rather than single causes of social exclusion engender the processes of marginalization of PWUID in Canada. While rhetorics of harm reduction are widespread, a bias towards abstinence and a failure to account for pleasure as a meaningful component of illicit drug use are evident within this discourse. Recommendations for individual, meso, and structural-level intervention strategies to counter the marginalization of PWUID within Canada’s harm reduction efforts are proposed.
Researchers, policy-makers and practitioners are encouraged to consider the utility of a critical Foucauldian stance when reflecting on the elements of a harm reduction approach. Doing so can translate to a renewed attention on the structural context and pragmatics of drug use in Canada, and its intersection with blood-borne infections like HIV and HCV.
A focus group evaluation was conducted as part of the HIV prevention program CN. The process was recorded. A home-made video was developed as an educational effort to depict a “puppet” show, which was presented to the CN group with the idea of collecting individuals feedback and ideas for future prevention efforts.
Spanish-speaking MSM continue to get infected with HIV in Canada due to the effect of the negative social determinants of health that increase personal vulnerabilities to infection. In Toronto, CN a Canadian HIV prevention intervention, evaluated the video that intended to involve communities, raise HIV prevention awareness (particularly promoting condom use during intercourse) and to adopt social media as health promotion tools.
An educational video called Tuturutu created with the idea of generating condom use promotion among MSM, gay and bisexual communities. Although, created for Spanish-speaking men, the video use a non-specific language allowing increase its impact by communicating the message to individuals from any language background. The video’s length is 1:03”. The video also promotes the different services available at HPP-CSSP. This project was evaluated using a focus group method with a cohort of CN, an HIV behavioural intervention. (n=10, 7 Venezuelan, 1 Brazilian, 1 from El Salvador, and 1 Colombian).
Participants had a 23’conversation. They expressed what they understood the message from the video to be, and they discerned the rationales of what they considered plausible from the video. Finally, they discussed things they would have done differently for increasing clarity and message scope.
All participants agreed that using social media, particularly “funny” videos depicting puppets is a good educational strategy to create opportunities for community dialogue.
Some participants talked about the need to put more emphasis in showing condoms and the benefits of using condoms to reduce HIV/ STI’s infections.
The video was posted on different social media networks on December 1st. World’s Aids Day.
Community-based research with Canadian Aboriginal communities seeks to map Indigenous worldviews and develop anticolonial research approaches. However, less attention has focused on how Indigenous knowledges might influence data collection and analysis. The Medicine Wheel (MW), a common North American Aboriginal symbol, represents an individual’s healing journey in balancing the four aspects of self (physical, emotional, mental and spiritual) with the interconnection to all things. This presentation focuses on how MW teachings were used for data collection, analysis, and knowledge translation to understand the resiliency perspectives of two-spirit males living long-term with HIV/AIDS in Ontario, Canada.
Aboriginal sharing circles (i.e., focus groups) were used to gather data in three locations (Hamilton, Toronto and Ottawa, Ontario, Canada). Here, the MW was used to solicit stories of participants’ healing journeys living with HIV/AIDS (n=14). Data analysis involved several stages: (1) Cross-cultural training on MW teachings and qualitative data analysis were provided; (2) once transcribed, all data expressing similar patterns were coded using NVivo 10; (3) using participatory analysis, selected key quotes were mapped to the MW in areas expressing similar meaning; (4) codes that expressed a relationship to one another were grouped and then labelled consistent with the teachings of the MW; and (5) data visualization was used to present findings using the MW. Validity was established by independently verifying coding work and utilizing member-checking of the tentative findings with our community advisory committee.
Culturally-bound symbols potentially provide attuned and structured ways to interpret and understand data and to develop knowledge consistent with Aboriginal worldviews. This potentially facilitates the goal of knowledge translation, exchange and uptake of research findings in communities of interest.
The analytic process described above potentially shifts the way researchers come to understand, write, speak about and present scholarly work focused on Aboriginal HIV/AIDS resiliency through the world views of those most directly affected.
Food insecurity has been associated with increased risk of HIV transmission, delayed treatment access, suboptimal adherence and poor responses to antiretroviral therapy. In order to better understand the prevalence and correlates of food insecurity in British Columbia (BC), we led a pioneering community based research (CBR) study involving AIDS Service Organizations (ASOs) and Peer Research Associates (PRAs). We describe the philosophy, operational process and lessons learned of this project for knowledge transfer to other CBR initiatives across Canada.
This study was implemented with the meaningful participation of community members affected by HIV and began in 2009 involving nine ASOs and ten PRAs who coordinated participant recruitment, administered questionnaires, managed honoraria and conducted interviews. Research processes aligned with scientific best practice and ethic approval was obtained from UBC/PHC and SFU.
This was the first national HIV CBR study to utilize highly trained PRAs. The study had many successes including: building strong capacity in a team of PRAs, gaining more honest and accurate information from respondents, and shifting the community’s perception of the value of research. One major challenge was with PRAs’ isolation given the large geographic area covered. The team had many key learnings, including the importance of: engaging PRAs at the beginning of the research process and building a strong and ongoing communication structure between all team members.
This study demonstrates the invaluable contribution PRA’s provide to the research process as well as the many issues that have to be taken into consideration when utilizing peers in research. This study provided the PRAs opportunities to learn new skills interact with their peers in different ways, cross education and de-stigmatization as well as opened up new career opportunities. The lessons collectively learned have informed other CBR projects such as CHIWOS and the Positive Living Positive Homes study.
Through ABRPO’s on-going community development initiative ‘Turning to One Another’ (TTOA), agency staff and peer facilitators from 13 Ontario ASOs continue to increase the capacity of ASOs to build effective working relationships between ASO staff and PHA in multiple roles through the development of training and organizational tools.
At the February 2014 Provincial Collaborative Practice Development Consultation, both PHA Peer Facilitator trainees and ASO frontline staff (N=37) responded to a two-page needs assessment survey entitled, “Taking the Pulse: TTOA 2014 Pilot Site Check-in”. Participants were asked to: describe one-three issues that they perceive are impacting PHAs; how their agency is dealing with these issues; rate their confidence in their ability to respond; and to rate their perception of their agency’s ability to respond.
The majority of respondents (N=25, 68%) were PHAs who were peer facilitators or employed at ASOs. The most common issue perceived as “impacting HIV+ people in your community” was aging, followed by equal listing of housing, stigma, and medication access issues. In total, 84 descriptions of how their agency is dealing with issues were provided, with a small number identifying a gap in local response. Next, respondents indicated that they were moderately, mostly or highly confident (N= 80, 88%) in their ability to respond to most issues that they identified. Lastly, for most issues identified respondents were moderately, mostly or highly confident (N= 68, 77%) in their agency’s ability to respond.
From the high levels of respondents’ confidence to address many issues, a strong affirmation of the success of local collaborations and responsiveness can be inferred. Further examination is warranted of areas of challenge identified by respondents who had little or no confidence (N= 20, 23%) in their agencies’ ability in addressing community/peer/MSM/PHA involvement, addictions/harm reduction, burnout of peers or leaders, isolation/loneliness, and housing.
Opportunities for health, social participation and security are requisites to guarantee quality of life as people age. In this study, we document physical and mental health experiences of aging people living with HIV/AIDS (PHAs) in order to inform AIDS service organizations (ASOs) about perceived needs.
Data was collected through a community-based participatory study with and for PHAs. Qualitative information on physical and mental health challenges over time was collected using in-depth interviews. Twenty two PHAs were interviewed. Nine of the participants were women, 10 came from rural areas, 14 reported previous or current drug addiction, and 8 had experienced previous incarceration. Trained peer-researchers performed the interviews in a local ASO. Analysis focused on finding common themes in the experiences reported by participants (prior and after HIV diagnosis, and changes over time) with special attention to similarities and differences across individual reports.
The majority of participants identified shifts in their physical health, from poor health at diagnosis to an improved current health. Antiretroviral medications, support from ASOs and connection with peers were related to those positive changes. As PHAs age, physical challenges were related to mobility problems, heart conditions and cancer rather than the HIV infection itself. In contrast, mental health challenges were common prior to and remained unchanged after HIV diagnosis. Depression, suicide ideation, and isolation were commonly experienced. Drug addiction negatively affects future health expectations. Participants identified lifestyle changes, drug addiction treatment, and more social support as key aspects to be addressed by ASOs.
Our findings highlight the importance of addressing mental health and drug addiction, as specific health issues related to PHAs. However, the experience of aging in PHAs suggests that the response also requires a non-HIV-related approach. Thus, ASO may need to look for linking PHAs to programs available to HIV-negative older adults.
Opportunities for health, social participation and security are requisites to healthy aging. In this study, we documented the experiences of participation and security of aging PHAs in order to inform AIDS service organizations about perceived needs.
Data was collected through a community-based participatory study with and for PHAs. Qualitative information on physical and mental health challenges over time was collected using in-depth interviews. Twenty two PHAs were interviewed. Nine of the participants were women, 10 came from rural areas, 14 reported previous or current drug addiction, and 8 had experienced previous incarceration. In-depth interviews asked questions on aspects related to social participation (i.e. working, education, peer support, spiritual, social inclusion) and security (i.e. physical, financial, social). Analyses were done by researchers and peers, and organized qualitative data in common themes and giving particular attention to similarities and differences across individuals.
Most participants consider that social participation (in AIDS and non-AIDS related organizations) and/or networking with family and friends positive experiences. However, for some, stigma and drug addiction continue to hinder social participation. Two key themes emerged in the security topic: fear of disclosure and having a safe place. Again, HIV-related stigma and the “Law” were the main factors related to fear of disclosure and therefore feelings of insecurity. Most participants, especially those who have experienced incarceration, felt that having a stable lodging, support from family, and the support of ASOs are key to keeping their security as they age.
Social participation emerged as a key aspect to promote healthy aging among PHAs. Our findings also highlight the important role of ASOs in in helping PHAs manage fear of disclosure, stigma and adequate housing. IDU and people with previous experience of incarceration are special groups to target.
A capacity building video series has been developed in order to prepare long term care (LTC) homes for an aging population of people living with HIV (PLHIV). A program evaluation has been completed to assess barriers and facilitators to HIV education in LTC in order to optimize uptake and foster an environment of inclusion for PLHIV. The HIV and LTC Video Series: Compassionate Care in a Changing Landscape was developed in partnership with a sub-acute HIV hospital (Casey House) and a LTC home (The Rekai Centres).
Four long-term care homes in Toronto were recruited to participate in a study to pilot two different approaches to disseminate video based HIV education and to identify barriers and facilitators to providing HIV education in long-term care. The homes were randomly assigned either as a control group to receive the educational videos in hard and soft copy formats to implement as part of their educational structure, or assigned to receive facilitated video training sessions with a registered nurse and a PL-HIV. All four homes were provided with a copy of two 20 minute videos, Bedside Care and Families and Networks of Support. Data were collected from evaluation forms completed (n=147) from LTC staff who viewed the videos and from interviews with LTC home education coordinators(n=4). The consolidated evaluation data from the videos showed that 81% of LTC staff in the control group and 86% in the facilitated training group “somewhat to strongly agreed” they had acquired new information. In over 95% of responses it was indicated that the videos were an effective way of having the information delivered. As well, there was strong agreement that having someone available after the video to answer questions would be very helpful; however, there were no significant differences in the change in staff comfort level providing care for people living with HIV between the two groups.
Through advances in HIV treatment, care and support over the past few decades, HIV is no longer a fatal illness. While we can rightly celebrate this success, our response must adapt to meet the new challenges associated with living with HIV as a chronic and potentially disabling condition. Recognizing the enabling potential of rehabilitation, the Canadian Working Group on HIV and Rehabilitation (CWGHR) is working to ensure equitable access to quality rehabilitation for people living with HIV.
Despite the promising potential, rehabilitation in the context of HIV remains under-recognized, undervalued, and underutilized component of the continuum of health care programs and services (CWGHR Discussion Paper, 2012). Cognisant of this, in 2013, CWGHR conducted a series of in-person and phone consultations to engage representatives from sectors which have a high stake in the rehabilitation agenda: HIV community (n=7), the Canadian Association of Occupational Therapy (n=8), the Canadian Physiotherapy Association (n=50). A reflexive iteration process was used to refine and interpret the consultation data which provided clear direction to the “Access to Rehabilitation Project” by identifying the challenges, opportunities and recommendations way forward.
Though participants from the three sectors addressed issues specific to their professions and contexts, they held common opinions about the major barriers and strategies to accessing rehabilitation by PHAs. Some of the key issues identified include the lack of coordination between the traditionally isolated worlds of the chronic illnesses, disability and rehabilitation sectors; little integration for seamless rehabilitation services in the continuum of health and social care; cost and coverage issues; government delisting of services and funding cut; impeding health and social policies; lack of enough research evidence and best practice models; unavailability and lack of awareness on specialized services and stigma.
Based on the findings and recommendations from these consultations, CWGHR, in collaboration with other partners, is currently coordinating a “Pan-Canadian Rehabilitation Network” to facilitate cross-sectoral collaboration in education, research, policy change and programming initiatives.
The ‘Community of practice’ concept (coined by Lave and Wenger in 1991) proposes that interaction among practitioners in the context of situated learning can lead to the development of new knowledge that responds to complex work-related issues. In this presentation, we will use community of practice concepts to understand and interpret the evolution, organization and impacts of ACT Research Day (ACTRD) in relation to local HIV service provision, research and policy.
ACTRD is a knowledge transfer and exchange event hosted by the AIDS Committee of Toronto (ACT) annually since 2003. The specific content of ACTRD varies from year to year, but it remains focused on presenting and discussing current research and the implications for HIV programs and policy among service providers, people living with HIV, researchers, policy makers and community members. The event also includes workshop-style segments that promote practical appreciation of emerging or critical issues. The one-day event attracts 80–100 participants annually.
This presentation draws on data collected from annual post-event evaluations, a multi-year review conducted in 2011 that included interviews and focus groups with multiple stakeholders, and our experiences organizing the event over several years. We interpret the ACTRD audience, participants and principal beneficiaries as constituting a community of practice. We then (a) review the extent to which the objectives of ACTRD have been met, (b) describe how features of ACTRD contribute to fostering mutual engagement among the community of practice involved in the response to HIV, (c) examine the role ACTRD plays in developing shared understandings of complex issues across a diverse range of stakeholders, and (d) explore the limitations of using community of practice concepts in relation to ACTRD.
In conclusion, we argue ACTRD demonstrates a community-based process of knowledge production, and illuminates the critical connection between research and practice in the response to HIV.
AIDS service organizations (ASOs) have long been the cornerstone of a community response to HIV/AIDS. These ASOs have evolved to respond to complex policy and funding environments, each developing services/programs relevant for their own local environment. With indications that federal funding will integrate HIV, HCV and STIs (collectively known as sexually transmitted infections and blood borne pathogens, STBBIs), service providers and people living with HIV/AIDS in Atlantic Canada and across Canada are abuzz over the implications for organizational structures, service delivery and client engagement.
The Landscapes Project is a collaborative investigation into the current state of communicable diseases, affected populations, and associated service delivery needs in Atlantic Canada. Through document review and extensive consultations with service providers, partner organizations and key client populations in four Atlantic provinces, we identified the various ways in which ASOs are already integrating their services, their anxiety and optimism around integration, and service delivery models and principles that must be preserved. A total of 14 organizations and projects across Atlantic Canada that receive operational or project funding from PHAC participated in this work. Of the 33 in-person interviews, 12 were with Executive Directors or project leads from funded groups, 8 were staff members from partner organizations, and 12 service users or clients who have accesses programs or services.
This presentation will provide both service provider and client perspectives about: 1) how organizations in Atlantic Canada have already been integrating STBBIs; 2) the experienced and anticipated impacts of these shifts in service delivery on both ASOs and people who access their services; 3) the challenges and successes of integration for individual ASOs and their partner organizations; and 4) future forecasting of collaborative models to meet the needs of those living with and those most affected by communicable diseases in the Atlantic region. This project’s results will not only guide ASOs and their partners in Atlantic Canada, but will have relevance to others outside of Atlantic Canada.
According to national surveillance data (1985–2011), young people, aged 15–29, make up 27% of all HIV nationally recorded HIV diagnoses. Sexual health curriculum is often not tailored to the diverse experiences, needs and identities of young people or the socio-structural and contextual factors (e.g., poverty, racism, homophobia) that influence HIV vulnerability. This may result in education that is often irrelevant and at its worst, detrimental to young peoples’ sense of self and identity. One strategy for improving the relevance of HIV programming is to include young people in the design and development of programs and research. But what does meaningful youth engagement look like in practice? While some literature exists on the meaningful involvement of youth in sexual health research, research exploring critical issues of youth participation and engagement in HIV programming is limited, particularly for youth who have experienced marginalization. Similarly, engagement is often discussed but rarely defined or unpacked. This community-based practice session comes out of 10 workshops with young people involved in Empower, a youth-led HIV prevention program housed at Central Toronto Community Health Centre that uses the arts to train youth to develop and deliver workshops in their own community. In partnership with Gendering Adolescent AIDS Prevention (New College, University of Toronto) Empower youth met over 4 months to design presentations for clinicians, researchers and policy makers on what they thought was most important in building sexual health and harm reduction programs. In this session, youth presenters critically reflect on their experiences and understandings of participating in harm reduction and HIV programming. How do we move beyond the transportation token? What does queer, trans and sex positivity have to do with it? How do we structure spaces so we can work across difference? What does harm reduction programming look like in practice? What does glitter have to do with it?
The advent of highly active antiretroviral therapy (HAART) has dramatically improved health outcomes for people living with HIV/AIDS (PHA). However, certain populations continue to experience higher rates of co-morbidities, poorer HIV-related health outcomes and increased mortality, even within a context of universal health care. This study aims to identify correlates of mortality among a sample of harder-to-reach PHA in British Columbia (BC), Canada.
Cross-sectional socio-demographic, behavioural and service utilization data from the Longitudinal Investigations into Supportive and Ancillary Health Services (LISA) study were utilized for this analysis. The LISA study is comprised of 1000 harder-to-reach PHA in BC who completed a comprehensive interviewer-administered survey between July 2007 and January 2010. Survey data has been linked to clinical data obtained from the provincial Drug Treatment Program at the BC Centre for Excellence in HIV/AIDS. Multivariable logistic regression was used to identify independent correlates of mortality.
Between July 2007 and June 2013, 95 (10%) of the 915 LISA participants were confirmed as deceased. Of the 95 cases of mortality, the median age was 48 (IQR: 41–54), 30 (32%) were female, 70 (74%) identified as heterosexual, 35 (37%) identified as Aboriginal, and 34 (36%) reported injection drug use at the time of interview. In the multivariable analysis, individuals who did not complete high school (AOR=1.746, 95% CI: 1.056-2.88), had annual income of less than $15,000 (AOR=2.873, 95% CI: 1.551–5.323), and had been incarcerated in the 6 months prior to the survey (AOR=2.397, 95% CI: 1.376–4.176) had a higher odds of mortality. Moreover, individuals who did not achieve optimal HAART adherence (≥95%)(AOR= 1.884, 95% CI: 1.179–3.010), those who were not virally suppressed (defined as <50 copies/mL) (AOR=3.961, 95% CI: 1.525–10.287), and older individuals (AOR=1.073, 95% CI: 1.043–1.104) had a higher odds of mortality.
Individuals who reported lower education, less income, recent history of incarceration and poorer clinical characteristics have a higher likelihood of mortality, demonstrating significant gaps in care for harder-to-reach individuals.
There is limited research on what constitutes women-centred HIV care and how it affects health status of women living with HIV. We measured differences in health utilities in women who perceived their HIV clinic as women-centred care (WCC) compared to those who did not (non-WCC). Ranging from 0 to 1, the utility values represent preference for a particular health state that is between worst health state (i.e., death, denoted by 0) and perfect health (denoted by 1).
Women with HIV aged 16 or older were enrolled in the Canadian HIV Women’s Sexual and Reproductive Health cohort study (CHIWOS) in British Columbia (BC), Ontario (ON), and Quebec (QC). Using a 5-point Likert scale, participants rated whether they perceived the care they received from their HIV clinic in the past year as women-centred. Health-related quality of life was measured using the Short Form-12 (SF-12), classified and converted into utilities using preference weights generated via standard gamble. Utilities and covariates between WCC and non-WCC were compared using t-test or chi-square test.
995 women were included in this analysis with a median age of 44 years (IQR: 36–51). Overall, 54% perceived their HIV clinic as women centred. The WCC group had a significantly higher mean utility value (0.64) compared to the non-WCC group (0.62, p<0.01), generating an absolute difference of 0.02. While some authors argue that utility values should have a minimum difference of 0.03, others propose that incremental cost per quality-adjust-life-years (QALY) is more important than the improvement in utility values alone.
Women-centred care can have a meaningful and positive effect on patients’ health. To better inform resource allocation decisions, these findings will be used to generate QALYs alongside measurement of costs, as part of a cost-utility analysis of health services use by women with HIV.
The civil war in Northern Uganda resulted in countless deaths, child abductions, and the displacement of the majority of the population. In the aftermath of the war, conditions are rife for the spread of HIV and other STIs and HIV epidemiological data are scarce. This prospective cohort study affords a unique opportunity to assess other STIs in post conflict Northern Uganda.
“The Cango Lyec Project” is a prospective cohort of conflict-affected populations in three districts of Northern Uganda. Participants completed sociodemographic surveys, mental health screening tools (HTQ, HSCL), and provided blood samples for HIV and syphilis testing. Syphilis serology (RPR with TPHA conformation) was conducted at each study visit, and seropositive participants were treated. Gender stratified multivariable logistic regressions examined the relationship between syphilis and HIV infection and vulnerability.
Of 2388 participants, 792 men (80.0%) and 1163 women (83.2%) were sexually active and included in the analysis. Overall prevalence of syphilis for men (3.91%) and women (4.82%). The highest prevalence of active syphilis was in Amuru district (5.7%), and the highest prevalence was 10.99% in one rural community. Syphilis was significantly associated with HIV in both men (Odds Ratio [OR]: 2.97; 95% Confidence Interval [CI]: 1.15–6.82) and women (OR: 2.88; 95% CI:1.60–5.04). Among women, syphilis was also associated with a history of abduction (OR: 2.04; 95% CI: 1.16–3.51), depression (OR: 1.80; 95%C: 1.00–3.16), being >30 years of age (OR: 1.99; 95% CI: 1.16–3.46), and more lifetime sexual partners (OR: 1.84; 95% CI: 1.07–3.18). Multivariate models included women only due to sample size. Adjusted factors associated with syphilis among women included: HIV infection (AOR: 2.75; 95% CI:1.53–4.83) and a history of abduction (OR: 1.92; 95% CI: 1.09–3.33).
Women are disproportionately impacted by both HIV infection and syphilis in this post conflict-affected population. Trauma informed HIV and STI care is urgently needed in this population.
There is limited recent information regarding HIV testing among aboriginal and non-aboriginal IDU in Ontario. This analysis was conducted to examine differences in HIV testing between aboriginal and non-aboriginal IDU in order to identify the need for aboriginal specific approaches to encourage HIV testing.
Data were collected in 2011 as part of PHAC’s I-Track surveillance system. Interviewer-administered surveys and blood spot collection (for HIV and HCV testing) were conducted at service sites in Kingston (n=197), London (n=202), Sudbury (n=146), Thunder Bay (n=136) and Toronto (n=260). Participants were at least 16 years old and injected drugs in the previous six months. Differences were tested using Pearson’s chi-square and Fisher’s exact.
Lifetime testing was highest in Kingston (97.0%), Toronto (95.8%) and Sudbury (91.8%), and comparatively lower in London (86.1%) and Thunder Bay (82.4%). No differences between aboriginal and non-aboriginal IDU were observed. In Kingston and Toronto, 100% of aboriginal participants reported previous testing, compared to 96.2% and 94.7%, respectively, of non-aboriginals. In Thunder Bay, just 80.0% of aboriginal participants reported testing compared to 87.0% of others. In terms of recent testing, differences were noted in Sudbury and Toronto. In Sudbury, 84.0% of non-aboriginal participants had an HIV test in the previous year while 67.9% of aboriginal participants did so (p=0.030). In Toronto, 63.8% of non-aboriginal participants tested in the last year compared to 73.9% of aboriginal participants (p=0.047).
While there were few differences in HIV testing between aboriginal and non-aboriginal IDU, results were limited by small sample sizes (as these data can be expensive and logistically challenging to collect). More research is required to determine any substantial differences. Of concern is the lower lifetime testing prevalence in London and Thunder Bay, where more than one in ten IDU reported never having an HIV test in their lifetime.
Incarcerated individuals represent a population at risk for HIV and its complications. Factors associated with incarceration amongst HIV infected persons within the Ontario HIV Treatment Network Cohort Study (OCS) were explored by cross sectional analysis. We used a proxy measure of incarceration, defined as having a history of at least one available lab result from any correctional facility in Ontario. As of December 31, 2013 a total of 6,408 persons had been enrolled – 5,585 were linked to Public Health Laboratory of whom 118 had at least one such test sent from a correctional facility. Differences were seen between incarcerated group vs. non-incarcerated: MSM 20% vs 67%; aboriginal 8% vs. 20%; East/North region of Ontario 43% vs. 18%; history of IDU 72% vs. 15%; HCV positive 78% vs. 17%; current smoker 83% vs. 37%; recent hazardous drinking 42% vs. 33%. Incarcerated persons were also more poor and less well educated. No clear differences in treatment uptake or outcome were evident – possibly related to insufficient numbers. Persons in OCS who are known to have been incarcerated are at risk for poor outcomes. Identifying barriers to access and interruptions in treatment cascade will be important for persons coping with HIV in prison.
Ukraine is experiencing Europe’s most rapidly growing HIV epidemic, driven primarily by injection drug use and unprotected sex between women involved in sex work and clients. In 2012, UNICEF Ukraine, the University of Manitoba, and the Ukrainian Institute for Social Research conducted an epidemiological mapping and epidemic appraisal project in Zaporizhzhya, Ukraine to assess and characterise its local HIV epidemic, with specific focus on women involved in sex work. As in many contexts, globally, sex workers are disproportionately burdened by Ukraine’s epidemic. This study examines overlapping “risk behaviours” reported by young women involved in sex work in Zaporizhzhya.
Behavioural surveys were conducted to characterise sexual- and drug-using behaviours of young women (14–25 years) self-reporting involvement in sex work. Members of local, key populations were trained as field researchers to collect data. Mapping identified “hotspots” in Zaporizhzhya where sex workers and other key populations congregate. Multistage cluster sampling was used to recruit participants from identified hotspots. Descriptive statistical analyses are presented to highlight frequency and concurrency of self-reported risk behaviours among participants.
Among 124 young women involved in sex work, the average age at entry into sex work was 15.7 years. Participants most commonly reported practicing street-based sex work, and on average, saw 10.3 clients/week. Consistent condom use was reported by 69% of women and anal sex with male partners and clients was very common – reported by three-quarters of participants. Over half (50.8%) of participants reported ever using drugs, while 47% reported ever injecting drugs, 12.5% admitted to sharing needles, and nearly two-thirds injected ≥2 times/day.
The complex array of vulnerabilities that are faced by women involved in sex work highlights the need for well-tailored interventions to address the emerging HIV epidemic in Zaporizhzhya. Importantly, given the extensive overlap of high-risk behaviours reported by participants, targeted interventions must not only focus on the traditional promotion of safe sexual practices, but also incorporate harm reduction strategies more broadly, through needle exchange and methadone programmes.
Within population health, program science offers a new approach to addressing the increasing complexity of HIV prevention and treatment (Blanchard and Aral, 2013) that could harness and democratize linkages between science and community-based practice. Yet few models have been developed to guide the use of this approach and most fail to specify the processes needed to produce and apply knowledge and mobilize stakeholders and communities throughout the design, implementation, and evaluation of interventions.
Outline and discuss a model for operationalizing and applying some principles of program science to address HIV and its impact at the population level.
A multiple-case study was conducted, focused on interventions with which the author has been involved over the course of 25 years of community-based HIV research, to analyze processes associated with the implementation of these interventions for various populations.
This model integrates four transversal and recurrent processes of knowledge and community mobilization: 1) partnership to bring stakeholders together (community members, researchers, knowledge users, decision makers) in an equitable manner; 2) co-production of knowledge, as a shared goal and specific to each stage of implementation; 3) knowledge mobilization and exchange to generate action; 4) capacity building to ensure stakeholders have the necessary resources to pursue and consolidate the translation of knowledge into action. These processes play a role at each stage of implementation: a) analysis of needs, strengths, and individual and community resources; b) piloting; c) scaling-up; and d) securing long-term sustainability.
This model emphasizes processes for mobilizing knowledge and communities and the identification of issues specific to each process. In order for community mobilization to truly contribute to the illumination of science and vice versa, and to avoid its intrumentalization for public health purposes at the expense of social justice, more attention to these processes is required within program science.
The aim was to understand why people practiced unprotected sex and substance use regarding the risk of infection by blood-borne viruses and STIs, and the factors influencing risky practices. The purpose of the study was to inform policy recommendations and improving prevention methods of BBVs/STIs among Egyptian substance users, particularly health/sex education in schools, media, peer groups, NGOs, and the criminal justice system.
A questionnaire survey of knowledge, attitude and practice with 410 substance users was conducted. Respondents were analyzed according to gender, age, education and intravenous injection. Chi square, student t-test, Mann-Whitney, Spearman correlation and multiple logistic regression tests were calculated, and p-value <0.05 was considered statistically significant. Three qualitative studies were conducted; they comprised four focus group discussions with 27 substance users, including female sex workers, MSM and IVDUs and 14 interviews with policy-makers.
Respondents with higher education had higher knowledge scores for safer sex. Positive correlation was shown between age and knowledge of safer sex. Older respondents were predicted to know more about safer sex. Females and IVDUs were predicted to have higher attitude score for safer sex. KAP of risky sexual behavior to infection with BBVs/STIs among substance users was low. More risky injection behavior was observed in females and non-IVDUs. The participants chose peer-to-peer outreach and health/sex education, as their preferred prevention method. Policy-makers found that harm reduction programs, like methadone-replacement, needle-exchange in prisons and police stations should be introduced.
The study described and explored the cultural influences on high risk behaviors among Egyptian substance users. Recommendations included further research on the substance users who are not engaged with drug rehabilitation programs, stigma in detail, evaluating the effectiveness and acceptability of prevention, e.g. randomized trials and qualitative process evaluations.
Women with HIV experience distinct stressors and greater depression than men with HIV that may interfere with antiretroviral therapy (ART) adherence. We investigated the relationship between stress, depression and adherence among women in care for HIV.
A cross-sectional study was conducted using data from clinical charts of 307 women from 3 sites in the Ontario HIV Treatment Network (OHTN) Cohort Study (OCS), and a standardized questionnaire completed at the last interview. Variables of interest were: stress (National Population Health Survey), depression (Center for Epidemiologic Studies Depression Scale), hazardous alcohol use (Alcohol Use Disorders Identification Test) and ART adherence (AIDS Clinical Trial Group Questionnaire). Adequate adherence was defined as missing ≤1 pill in 4 weeks (≥95% adherence). Logistic regression models were used: to identify covariates of adequate adherence and to quantify the relationship between stress and adherence and evidence of mediation via depression and moderation by employment status, ethnicity, age at interview, and living alone.
The median age of participants was 43 years and 60.7% (n=181) self-identified as African, Caribbean or Black. The majority (88.5%, n=192) had ≥200 CD4 cells/mm3 and an undetectable viral load (85.7%, n=186) despite 65.5% (n=201/307) having adequate adherence. No hazardous alcohol use (OR=2.24, 95% CI:1.14–4.40) and fewer stress events (OR=0.56, 95% CI:0.33–0.94, per 5 events) had increased likelihood of adequate (≥95%) adherence. The relationship between environmental stress (i.e., stress derived from area of residence) and adherence was attenuated among women ≤35 years (OR=0.73, 95% CI:0.56–0.94) compared to ≥45 years (OR=0.51, 95% CI:0.34–0.77) and was not significant for women aged 36–44 years (OR= 1.05, 95% CI:0.67–1.65).
Interventions for women accessing HIV care are needed to improve adherence to ≥95%. Interventions should aim to manage, adapt to or mitigate stress and depression and focus on education and adaptive coping strategies for hazardous alcohol use for improved HIV management and adherence.
Risky sexual behaviour can result in a high perceived risk, which can motivate precautionary behaviour thus decreasing perceived risk. However, if risky sexual behaviour doesn’t correlate with high perceived risk, it cannot initiate precautionary behaviour. Our goal was to determine the association between risky sexual behaviour and perceived risk.
Sexually active men who have sex with men (MSM) were recruited at a Toronto medical clinic in 2010–12. Participants completed a socio-behavioural questionnaire using ACASI. Actual risk was created using factor scores from principal component analysis (PCA). Perceived risk was measured by asking participants what the chances were that they would ever get HIV. Multivariable regression was performed using the Harrell method.
We recruited 150 HIV-negative MSM (median age 44.5 years [IQR 37–50]). Actual HIV risk comprised three components. 1) ‘casual sex’: number of casual partners and condom use during insertive or receptive anal intercourse (IRAI) with a casual partner. 2) ‘HIV-positive sex’: condom use during IRAI with a regular HIV-positive partner. 3) ‘HIV unknown status sex’: condom use during IRAI with a regular HIV unknown status partner. Twenty nine MSM (20%) perceived their HIV risk to be high. In multivariable analysis, those with low condom use on the ‘HIV-positive sex’ component were found to be 18 times more likely to have a high perceived risk (95% CI 1.71–189.80) compared to those with high condom use. Older age was associated with lower perceived risk but only age 40–49 compared to less than 30 was statistically significant (95% CI 0.015–0.70). Men who used poppers in the previous 6 months were 5.59 times more likely to have a high perceived risk compared to those who didn’t use poppers (95% CI 1.23–25.48).
Perceived HIV risk increased significantly as condom use with an HIV-positive partner decreased. However, perceived HIV risk was not significantly increased when condom use with casual partners or HIV unknown status regular partners decreased, even though these behaviours could be considered risky.
Our aim was to create an evidence-based set of user-friendly recommendations for Canadian harm reduction programs that provide service to people who use drugs and are at risk for HIV and other harms. This new document addresses practice issues not addressed in Part 1 (see
Development of a comprehensive cohort of people living with HIV (PLHIV) to help improve healthcare has long been the vision of researchers, clinicians and decision-makers. Cohort development is particularly difficult in regions that have dispersed populations and small numbers of PLHIV. With funding from the Community-based Primary Healthcare group (CBPH-CIHR) and the support of the NL Center for Health Information, provincial HIV clinic and Public Health Laboratory, a comprehensive cohort of PLHIV is being developed. The database includes longitudinal data from NL data sources to assess the epidemiology and management of HIV.
Present Phase One – To develop a timely process for compiling and updating the NL HIV-cohort database, while ensuring confidentiality and privacy.
A feasibility study on the development of a governance structure, approvals including ethics and data custodians, methodology for data extraction and linkage, development of a de-identified electronic database, and initial assessment of data quality and data-sharing strategies. A snowball approach identified relevant stakeholders. An iterative modified Delphi process and a member-checking survey is employed to document the process by which data are accessed, linked and refined, and to identify barriers and facilitators for compiling and updating. A logic model and evaluation framework guides more timely updates of the database.
The existing acquisition and aggregation of health-administrative and clinical data took over two years. Barriers include the use of dissimilar data formats and different technology systems, lack of alignment between approaches to ethics and health authorities’ legislation, staff training and the challenges of planning secure systems for controlled data-sharing. Enablers include ethically-approved data extraction which ensures privacy and confidentiality with minimum impact on data quality and analysis.
Evidenced-informed approaches to HIV policymaking are contingent upon a dynamic, population-based PLHIV database kept current through systematic and transparent processes.
People living with HIV are concerned about their memory, because as life expectancy increases, it is becoming clear that this chronic illness affects both cognition and mental health, even with excellent systemic viral control. Patients often report deteriorating cognition but what is relevant to the health care situation and what is related to natural aging or other factors like stress, anxiety, or depression is difficult to untangle. The overall aim of this study is to create an item bank reflecting the cognitive concerns expressed by people with HIV as a first step towards developing a measure.
The steps outlined by the FDA for developing a patient-reported outcome are being followed. Semi-qualitative interviews on an international sample of 234 people HIV+ was carried out in 3 independent waves using an anonymous web-based survey (Canada) and face-to-face interviews (International). The concerns were mapped to standard neurocognitive domains to identify content coverage and compared with those domains in existing cognitive questionnaires. A bilingual questionnaire has been developed and is being posted on HIV sites across Canada to obtain estimates of prevalence and importance. Rasch analysis will be used to calibrate the items and develop short forms to fit different purposes (screening, prevalence, change over time). Comparative values will be obtained from people without HIV.
136 areas of cognitive concern were identified covering all 15 neurocognitive domains plus emotional concerns and change. None of the generic or HIV specific measures came close to this extent of content coverage. Memory concerns were the most common (n=40) and covered prospective, episodic, semantic, immediate, procedural memory; 15 concerns related to attention; 12 concerns were identified for each of language and executive function; 4 and 3 concerns related to visuospatial and calculation domains.
The lack of relationship between self-reported cognitive concerns and results on neurocognitve testing may partially be explained by poor questions. Using all due diligence, this study will contribute optimal items so a fair comparison can be made between the two methods.
The purpose of ENGAGE is to identify socio-demographic factors associated with late initiation of antiretroviral therapy (ART) among people living with HIV in British Columbia.
People living with HIV and newly initiating ART (within the previous 6 months) were enrolled in ENGAGE, a prospective cohort study nested within the provincial Drug Treatment Program (DTP) at the BC Centre for Excellence in HIV/AIDS. Participants complete a 1-hour structured survey collecting demographics, ART attitudes and adherence behaviours, and use of healthcare and support services. CD4 count and plasma viral load were obtained via linkage to the DTP. The primary outcome, ‘late initiation of ART’, was defined as CD4 cell count <500 cells/μL at time of initiation. Bivariate analyses (Wilcoxon rank-sum and Fisher’s exact test) were used to test the association between late initiation and socio-demographic characteristics.
Since December 2013, 55 participants were enrolled in ENGAGE, representing 14% of the 380 eligible individuals. Enrollees were 15% female, median age of 40 years [IQR: 30 to 45], 28% reported Aboriginal ancestry, and had a median annual personal income of $13000 [IQR: 7800 to 27600]. In addition, 24% reported ever being incarcerated and 25% a history of injection drug use. The median CD4 cell count at time of ART initiation was 500 cells/μL [IQR: 310 to 640]. Overall, 55% of our participants were late ART initiators.
Higher personal income was the only variable found to be negatively associated with late initiation (Odds ratio (OR)=0.62 (95% CI 0.40 to 0.98) per thousand-dollar increase). Female gender was marginally associated with late initiation (0R: 7.30, 95% CI 0.83 to 62.50) with a greater proportion of women (88%) initiating late than men (49%).
In this analysis, over half of the individuals initiating ART in BC initiated late, with disparities observed by income level. Multivariate models are needed to adjust for confounders and clarify the interpretation.
Despite known disparities in access to HIV care and treatment in Canada, the exact number of physicians who provide HIV treatment is unknown. In addition, there is limited information available regarding the characteristics of physicians who provide HIV treatment. Knowing the profile of physicians who prescribe ARVs (hereon ARV Physicians) can provide a better understanding of educational and programmatic needs to improve and expand HIV care and treatment by primary care providers.
The characteristics of ARV Physicians in BC between January 1, 2013 and December 31, 2013 are described, based on the data from the BC Centre for Excellence in HIV/AIDS Drug Treatment Program.
In 2013, there were 894 ARV Physicians in BC, and among them 749 (83.8%) only refilled ARV during follow-up (i.e. did not initiate/change ARV regimen), 8 (0.9%) only initiated/changed ARV and 137 (15.3%) performed both initiation/change and refill. 93.4% of the ARV Physicians were general practitioners (GPs) (infectious diseases 2.8%, internal medicine 1.9%, other specialists 1.9%) and a mean of 7.8 HIV patients were seen per one ARV Physician in BC. There were 105 ARV Physicians practicing in rural areas (11.7%) (789 [88.3%] in urban areas) and among them there was only one specialist (internal medicine) in rural areas. More ARV Physicians in urban areas were experienced in HIV care (i.e. have treated more than 6 HIV patients in the past) compared to their rural counterparts (30.9% vs. 9.5%, p<0.05).
In 2013, the majority of ARV Physicians in BC did not initiate/change ARV regimen, but rather refilled an existing prescription. Also, ARV Physicians were predominantly GPs. ARV Physicians in urban areas were more experienced in HIV care compared to their rural counterparts. These results suggest the need to support and train GPs in HIV care and treatment, particularly in rural areas.
Many Canadians are diagnosed at relatively late stages of HIV infection, leading to poorer long-term health outcomes and increased probability of transmission. Understanding patterns of health service utilisation among individuals prior to diagnosis of HIV may be helpful to develop strategies facilitating earlier diagnosis and linkage into care. Using data from a population-based retrospective cohort, we examine rates of prescription drug utilisation among clients of the Manitoba HIV Program (MHP), prior to entry into HIV care.
We performed population-based case-control analyses, in which MHP clients (cases) were age-, sex-, and health region-matched (1:5) to HIV-negative controls. Clinical data from MHP was linked to an administrative database of Manitoba’s Drug Program Information Network. Rates of prescription drug utilisation were calculated for cases and controls, using total number of pills prescribed (normalised to a 7-day dispensation cycle) as numerator and person-days prior to HIV diagnosis as denominator. Dispensations were categorised based on drug-type: antibiotics, drugs for chronic conditions (hypertension, diabetes, etc.), and mental health conditions (anxiety, bipolar disorder, etc.). Stratified Poisson regression models were used to generate relative rates (RR) and 95% confidence intervals (95% CI).
193 MHP clients and 965 controls were included for analyses. In the one-year prior to entry into care, cases were significantly more likely than controls to use antibiotics (RR=3.2; 95% CI=2.8–3.7), anxiolytics (RR=2.2; 95% CI=2.0–2.5), diabetic medications (RR=1.2; 95% CI=1.1–1.3), and mood stabilisers (RR=1.4; 95% CI=1.2–1.7). During the same period, lower dispensation rates for drugs to treat hyperlipidemia (RR=0.7; 95% CI=0.6–0.8), hypertension (RR=0.6; 95% CI=0.6–0.7), and schizophrenia (RR=0.7; 95% CI=0.6–0.8) were observed among cases than controls.
Generally higher rates of prescription drug dispensation were observed among MHP clients, highlighting complex healthcare needs, even prior to entry into care. Greater dispensation of anxiolytics and mood stabilisers among cases may suggest a need for more diligent HIV testing among individuals using such medications. Notably, high rates of antibiotic utilisation prior to entry into care with MHP imply greater burden of opportunistic co-infections, and specifically highlight missed opportunities for earlier diagnosis of HIV.
Les pratiques de consommation de drogues qui peuvent entraîner la transmission du VIH, du VHC et d’autres méfaits sont des enjeux pressants qui affectent les communautés, aux quatre coins du Canada. Les recommandations de pratiques exemplaires qui existaient pour les programmes de seringues et d’aiguilles sont obsolètes. Notre équipe communautaire a impliqué des individus de partout au pays, représentant les personnes qui utilisent des drogues, les fournisseurs de services, les chercheurs et les responsables des politiques, dans la conception, la création et la dissémination de recommandations de pratiques exemplaires actualisées, à présent disponibles dans les deux langues officielles. À l’aide d’une méthode de synthèse narrative, nous avons examiné et résumé les meilleures données scientifiques disponibles, et avons récemment fait traduire de l’anglais vers le français l’ensemble résultant de recommandations de pratiques exemplaires pour la distribution de seringues; la distribution d’autres types de matériel pour l’injection; la distribution de matériel plus sécuritaire pour fumer le crack; l’élimination et la manipulation de tout matériel qui a servi à consommer des drogues; l’éducation sur une utilisation plus sécuritaire des drogues; et l’utilisation de la naloxone pour prévenir les surdoses. Au moyen d’une vaste stratégie de dissémination, nous joignons des publics anglophones et francophones qui utilisent le document, notamment des personnes qui utilisent des drogues et des fournisseurs de services, dans des programmes de réduction des méfaits à travers le Canada.
A process embedded with Indigenous methodology for using cohort data is lacking. Building Bridges (BB) aimed to develop and pilot a model to meaningfully engage Aboriginal leaders and peoples living with HIV (PHAs) in epidemiology research.
BB is a multi-site (Toronto and Vancouver) community-based research project using Canadian Observational Cohort (CANOC) collaboration data. In Phase I (model development), advisory committees of Aboriginal stakeholders were developed to guide subsequent phases and inform the application of Indigenous methodology to epidemiology. In Phases II and III (recruitment; research question development), Aboriginal PHAs collaborated with researchers and Indigenous leaders to identify epidemiology questions of interest. In Phase IV (data analysis), analysis of CANOC data was performed. In Phases V and VI (data interpretation; knowledge translation), participants, researchers and Indigenous leaders discussed findings and developed strategies for sharing them. For each phase, meals were provided at meetings, Traditional Leaders led ceremonies and discussions occurred in culturally safe gatherings.
Lessons learned: Consistent, frequent and respectful engagement through meetings, emails and teleconferences facilitated team building, however, limited email or phone access was a barrier. Mitigating barriers and clear communication of roles and project duration was important for recruitment. Identifying Indigenous leadership at community and research levels was pivotal to all phases. Team expertise in lived experiences, Indigenous culture, HIV research, epidemiology, services and support enabled multi-directional learning. Limitations included recruitment challenges and breaks in continuity during data analysis. CANOC limitations included low numbers of Aboriginal participants and the inability to address socio-behavioural questions. Involvement of allied academic researchers facilitated meaningful relationships with community through the advisory committee structure.
BB led to a collaborative Aboriginal epidemiology model and impactful research questions. Success hinged on ongoing engagement of Indigenous leaders, participants, service providers and researchers with diverse expertise and co-learning occurring among these groups.
This qualitative study explored the experiences of immigrant, refugee, and non-status men from the African, Caribbean, and Black (ACB) communities living with HIV/AIDS in the Greater Toronto Area (GTA). The purpose was to identify gaps in current HIV health equity resources in the GTA and propose appropriate solutions. The focus group was conducted at a local AIDS Service Organization (ASO) in Toronto with 8 men living with HIV/AIDS from the ACB communities who self-identified as either gay or heterosexual. Based on a modified Grounded Theory analyses of the transcripts and on thematic coding, a range of issues faced by the ACB men emerged that (1) increased the risk for HIV infection, (2) delayed seeking timely care, (3) created obstacles to HIV testing and equitable access to medical treatment, and (4) identified a lack of mental well-being services, especially those operating from an anti-oppressive framework. The themes that emerged from the group identified the need for more culturally specific support services, for greater involvement from ASO’s in facilitating discussion groups and social spaces for ACB men living with HIV/AIDS, greater sensitivity to and more recognition of the dangers for men having sex with men in their countries of origin from Canadian immigration officials, and the need for more specific guidelines regarding HIV disclosure for healthcare providers. In particular, in the area of interventions needed, participants noted that campaigns and initiatives were needed to address the prominence of HIV-related stigma in ACB communities. This stigma was cited as a reason that many ACB men decline to be tested for HIV, and that this stigma also decreases the willingness of men to disclose their HIV status to others, including their sexual partners. Following from the analyses of this focus group data, it is also clear that ASO’s and health care providers serving these communities must address the link between normative gender role expectations and hetero-normative assumptions concerning sexual behaviors that can expose ACB men to potential HIV infection.
HIV/AIDS researchers and theorists from the field of population and public health have yet to discuss how the generation of empirical data ought to be considered in the task of ethical inquiry. Nonetheless, various contexts are advocating for the generation of population and public health ethics- (PPHE-) relevant data in order to inform HIV/AIDS-related policy. Thus, the timing is ideal to engage in philosophical and methodological discussions about how PPHE can (and should) consider or generate data within the task of HIV/AIDS-related normative inquiry related to HIV/AIDS.
In this paper, I review the philosophical debates related to the ‘empirical turn’ in clinical bioethics. I also describe how population and public health ethics has engaged with the philosophical implications of using or generating empirical data. Finally, I provide examples for potential empirical PPHE studies pertaining to five HIV/AIDS-related issues.
Each issue pertaining to HIV/AIDS that I discuss differs from traditional empirical bioethical approaches in that it emphasizes: (1) concerns related to the population; (2) “upstream” policy-relevant health interventions – within and outside of the health care system; and (3) the prevention of illness and disease and the promotion of health and well-being. As I highlight, HIV/AIDS represents an ideal issue to begin an exploration into issues pertaining to empirical PPHE approaches, given that there are a variety of issues that arise within and outside of the clinic, provide opportunities for prevention and concern individual- and population-level outcomes and interests.
I advance a conceptualization of an empirical PPHE in order to open up new interdisciplinary ‘spaces’ in which empirical and normative approaches to ethical inquiry are transparently (and ethically) integrated in the realm of HIV/AIDS research. Critical approaches to empirical PPHE regarding HIV/AIDS can provide opportunities to develop more philosophical and empirical rigour within this field.
Migrant female sex workers experience unique risks and protective factors related to sexual health. Given the dearth of knowledge in high-income countries, we explored factors associated with inconsistent condom use by clients among migrant sex workers over time in Vancouver, BC.
Questionnaire and HIV/STI testing data from a longitudinal community-based cohort of female sex workers, AESHA, were collected from 2010–2013. Logistic regression using generalized estimating equations (GEE) was used to model correlates of inconsistent condom use by clients among international migrant sex workers over a 3-year study period.
Of 685 participants, analyses were restricted to 182 (27%) international migrant sex workers, whose primary country of origin was China. Of 401 observations, 28 (7%) events of inconsistent condom use by clients were reported over 3 years. In multivariate GEE analyses, difficulty accessing condoms (Adjusted Odds Ratio (AOR) 3.76, 95% Confidence Interval (CI) 1.13–12.47) was positively correlated with inconsistent condom use by clients, whereas servicing clients in indoor establishments (e.g., massage parlours) (AOR 0.34, 95% CI 0.15–0.77), and high school attainment (AOR 0.22, 95% CI 0.09–0.50) were negatively correlated with inconsistent condom use by clients.
Findings of this study highlight the persistent difficulties faced by migrant sex workers in terms of accessing and using condoms with clients. Given that criminalization and police raids often impede condom access for migrant sex workers in indoor work venues, a shift away from criminalization to support occupational health and rights for migrant sex workers remains critically needed. Unfortunately, Canada’s recent move towards increased criminalization of the sex industry (e.g., bill C-36 enacted in December 2014) may further exacerbate barriers to condom access for migrant sex workers. Laws, policies and programs promoting access to safer, decriminalized indoor work environments remain urgently needed to promote health, safety and human rights for migrant sex workers.
To help contribute to poz prevention resource allocation, this analysis examines prevalence of condomless anal sex (CAS) among male participants in the OHTN Cohort Study (OCS) who identified as gay or bisexual or ever reported having had sex with a male partner (MSM).
Multiple logistic regression of sociodemographic, substance use, and clinical data to identify factors associated with CAS.
Demographics: N=2,171 gay/MSM had eligible sexual risk behaviour data between 2010 and 2013. Of these, 88% per cent identified as gay, 7% as bisexual and 4% as heterosexual. Mean age was 49. Seventy percent had been diagnosed with HIV ≥10 years. Eight-seven percent reported an undetectable viral load (<50 copies/mL).
Forty-four percent reported no male partner in the previous three months, 25% reported having a regular partner, 14% a casual partner, and 17% both regular and casual partners. Overall, 13% reported CAS with an HIV-negative or unknown status partner in the previous three months. Bisexual or heterosexual identity was associated with less CAS (OR=0.45, CI=0.24–.85, p=0.01) as was a CD4 count <200 (OR=0.23, CI=0.10–0.56, p=0.001). Detectable viral load was associated with more CAS (OR=1.73, CI=1.01–2.96, p=0.04) as was non-medicinal drug use in the past six months (OR=3.71, CI=2.68–5.15, p=<0.0001). Overall 18% reported non-medicinal drug use, primarily cocaine, methamphetamines, and club drugs. Among those with ≥1 partner, N=1,225, CAS was associated with larger numbers of partners (5–9 partners, OR=2.0, CI=1.06–3.76, p=0.03; ≥10 partners, OR=4.82, CI=2.57–9.04, p=<0.0001).
OCS data suggest that condomless anal sex with non-positive partners is significantly associated with detectable viral load, five or more sexual partners, and using club drugs, crystal meth, and cocaine. Poz prevention resources would be best focused on people living with HIV with higher risk of CAS.
We sought to identify psychosocial and event-level factors associated with condom-use during anal intercourse among self-identified HIV-negative and HIV status unknown MSM in the Greater Vancouver Area.
We analyzed data from participants in the Momentum Health Study collected at enrollment on their most recent sexual encounter with each of up to five sexual partners in the past six months. Explanatory factors included event-level factors (substance use, partner’s HIV status, sexual history with partner, and sexual position), psychosocial scales (e.g., HAART Optimism, Sexual Altruism, Sexual Sensation Seeking (SSS), Cognitive Escape), and demographics. Of all sexual encounters where anal intercourse was reported, factors associated with condom-use versus not were determined using manual backward stepwise multivariable generalised linear mixed models.
The majority of HIV-negative or unknown status MSM reported at least one anal intercourse event in the past six months (85.0%, n=436/513). Two-thirds of all sexual encounters involved anal intercourse (64.1%, n=1196/1866) of which 56% used a condom. Condom-use was positively associated with higher sexual altruism community sub-scale scores (AOR=1.98, 95% CI: 1.46–2.68) and negatively associated with greater HAART optimism (AOR=0.95, 95% CI: 0.91–0.99), sexual sensation seeking (AOR=0.94, 95% CI:0.89–0.98), and cognitive escape (AOR=0.97, 95% CI: 0.94–0.99). At the event-level with that partner, longer time since first sex and higher frequency of recent anal sex were both negatively associated with condom-use (AOR=0.99, 95% CI: 0.99–0.99 and AOR=0.96, 95% CI: 0.95–0.98, respectively). Compared with men who didn’t know their partner’s HIV status, participants who were certain their partner was HIV-negative or HIV-positive were less likely to report condom-use (AOR=0.24, 95% CI: 0.08–0.72 and AOR=0.11, 95% CI: 0.03–0.39, respectively). Event-level substance use was associated with condom-use: participant alcohol use (AOR=1.43, 95% CI: 1.02–2.00) and partner crystal methamphetamine use (AOR=0.19, 95% CI: 0.07–0.55). Lower odds of condom-use were associated with annual income >$30,000 (AOR=0.66, 95% CI: 0.45–0.95) and being in a monogamous relationship vs. single (AOR=0.50, 95% CI: 0.30–0.82).
Health promotion for gay and other MSM must consider how event-level factors such as substance use, HAART optimism, partner familiarity, discussions of HIV serostatus, and psychosocial traits collectively affect condom-use decision-making.
Included in Statistics Canada’s largest geographical “peer group”, London, Ontario is typical of many mid-size Canadian cities. At a local health forum, community acceptance and homophobia were identified as key factors impacting LGBTQ health, with implications for gay and bisexual men. We studied these with regard to HIV-related sexual risk.
Survey data were collected from gay and bisexual men (n=202) in Middlesex-London, Ontario; 173 reported their HIV status as negative/unknown and were included in this analysis. Modified Poisson regression was used to model unadjusted and adjusted prevalence risk ratios (PRRs). A model was fit for non-modifiable sociodemographic and background factors, and then community factors added: social support; internalized homonegativity; perception of community acceptance of people like oneself (based on orientation, racialization, gender identity) from the broader Middlesex-London community, and separately from the gay community.
Older age was associated with decreasing risk. Other sociodemographic factors and childhood background factors (childhood homophobia, religious upbringing) were not. For each 10-year increase in age, prevalence of high-risk sex decreased by 24% (PRR=0.76; 95% CI: 0.60, 0.95). Controlling for age, we found an interaction between perception of broader community acceptance and gay community acceptance of people like oneself. Overall, as broader community acceptance increased, high-risk sex decreased. However, this effect varied depending upon perceptions of gay community acceptance, with men feeling most accepted within gay community having the smallest reductions in high-risk sex.
The interaction between broader community acceptance and gay community acceptance, independent of age, raises a series of questions. Among these: How do community norms and availability of partners shape sexual risk-taking? Are conventional “contextualized” measures of sexual risk sufficient, or do they miss important risk-mitigation strategies (undetectable viral load, strategic sexual positioning, or PEP/PrEP) used within gay communities?
We sought to examine differences by geographic region for HIV and STI testing prevalence, HIV treatment, and STI notification among a sample of Ontario MISM.
From December 2013 to January 2014, MISM aged 16 or older were recruited from Internet sites, mobile-apps, and ASOs across Ontario to complete a 15-minute anonymous online questionnaire, which included demographic and other questions on HIV and STI testing, treatment, and notification. Regional differences were determined using multivariable logistic regression controlling for significant confounders (i.e., age, sexual orientation, education, and race/ethnicity).
Of 1,830 participants, 146 reported being HIV-positive (8.1%), 217 unsure (12.0%), and 1,439 HIV-negative (79.9%). A greater proportion of HIV-positive respondents were from Toronto (12.7%) compared with all other regions (6.3%, p<0.001). Of HIV-positive men, the median year of diagnosis was 2004 and 90.3% reported being on ARV treatment. Of those on treatment, all but one reported an undetectable viral load, which did not vary by region (p=0.25), but was marginally associated with an earlier year of diagnosis (p=0.058). While 50.4% of HIV-negative men had received a negative test in the past six months, 9.2% reported never testing for HIV. Participants outside Toronto were marginally more likely to have never tested for HIV (AOR=1.43, 95% CI:0.98–2.07). For STI testing, 60.7% of participants had been tested in the past year and 18.3% of those tested received at least one positive diagnosis. Men from Toronto were more likely to have been tested in the past year (AOR=1.84, 95% CI:1.44–2.34) and, if tested, to have received a positive diagnosis (AOR=1.56, 95% CI:1.13–2.22). Of all participants, only 4.2% reported being notified by public health about an STI in the past year. After adjustment, Toronto men were no more likely to have been notified (6.1%) than men from other regions (3.5%, p=0.15). Participants qualitatively described their benefits, concerns, and challenges with STI notification experiences.
Differing HIV and STI testing and positivity prevalence should help inform targeted testing promotion to those MISM most susceptible to HIV and STI transmission.
Gay, bisexual, and other men who have sex with men (GB-MSM) remain the group most heavily affected by HIV in Canada. There is a paucity of research available outside of larger metropolitan areas regarding GB-MSM use of HIV testing.
The Health in Middlesex Men Matters (HiMMM) Project is a community-based research project examining health and HIV in GB-MSM in Middlesex County, Ontario. Respondents of an online, cross-sectional survey (n=202) were recruited through web-based, smart phone-based, and in-person promotion, and through social networks. Surveys were completed over 2011–2012 by adult GB-MSM living in Middlesex County, Ontario, Canada (n=202). Modified Poisson regression was utilized to calculate crude and adjusted prevalence ratios for whether participants had not been tested within the past 6 months. Additionally, reasons for not having been tested in the past 2 years are presented. Analyses were limited to HIV-negative individuals and those of unknown status (n=173).
Almost two thirds (64.1%) had not received an HIV test within the past 6 months. Factors significantly associated with reduced likelihood of being untested – controlling for other predisposing, enable and need factors – included greater social connection to GLBT communities, being less religious or spiritual than in childhood, and having a high school (versus postgraduate) education. Being unemployed was associated with greater likelihood of being untested. Primary reasons for not having accessed testing within the past two years included feeling at low risk for HIV (69.2%), always having safer sex (51.9%), not having had sex with an infected person (28.9%), and being in relationships (15.5%).
Knowledge of testing practices for GB-MSM subgroups in Middlesex County is important for directing competent, effective health promotion efforts, allowing local testing services to direct resources towards GB-MSM with lesser reported access testing services, depending on need.
Treatment as Prevention (TasP) is a critical public health strategy to reduce HIV morbidity, mortality, and transmission. We assessed awareness and knowledge of TasP among gay, bisexual, and other men who have sex with men (GBMSM) in Vancouver.
Data were analyzed for Momentum Health Study participants, recruited via Respondent Driven Sampling. Socio-demographic and behavioural variables were measured through a self-administered questionnaire, and clinical variables through nurse screening and linkage to the BC Centre for Excellence in HIV/AIDS. Stratified by HIV status, multivariable logistic regression identified covariates of TasP awareness (ever vs. never heard of TasP). Among those aware, TasP knowledge was explored through examination of short-answer definitions of TasP, risk perceptions, and information source. Analyses are RDS-adjusted.
Of 719 participants, 23% were HIV-positive and 46% heard of TasP. TasP awareness was higher among HIV-positive (69%) than HIV-negative GBMSM (41%, p<0.0001). Models showed HIV-positive GBMSM were more likely to have heard of TasP if they were Canadian born, unemployed, not using party drugs, and had higher CD4 counts. HIV-negative GBMSM were more likely to have heard of TasP if they were Caucasian (vs. Aboriginal), students, had higher education, a regular partner, and multiple sexual partners. Among those aware of TasP, 21% of HIV-positive and 13% of HIV-negative GBMSM (p<0.0001) demonstrated complete TasP definitions (with three factors identified: ARV use, viral suppression, and prevention of HIV transmission), and 64% and 41% respectively felt HIV treatment made the risk of transmitting or acquiring HIV ‘a lot lower’ (p=0.0020). The leading information source was doctors (44%) for HIV-positive GBMSM and gay media (34%) for HIV-negative GBMSM.
While TasP awareness was high among HIV-positive GBMSM, it was relatively low among HIV-negative GBMSM and varied by key socio-behavioural and clinical factors. Men’s articulation of their knowledge of TasP was poor, albeit better among HIV-positive men. For GBMSM to make use of TasP, they must understand it. Health communication strategies relevant to diverse communities of GBMSM are critical to advancing TasP health literacy.
HIV seroprevalence among transfeminine (i.e. male-to-female spectrum transgender) persons in high-income countries has been estimated at 21.6%, while 3.1% in Ontario self-reported being HIV-positive. This study is the first to identify correlates of HIV-related sexual risk in a Canadian transfeminine population.
The Trans PULSE Project surveyed trans Ontarians in 2009–2010 (n=433; transfeminine n=205), using respondent-driven sampling. Estimates were weighted based on network size using RDS II methods, and variances were adjusted for clustering by recruiter. Polytomous logistic regression models were built to identify associations of demographic, trans-specific, and psychosocial factors with both HIV-related sexual risk and abstinence (chosen or involuntary) in the past year, versus low-risk sex.
19% of transfeminine Ontarians reported past-year sexual behaviours consistent with high HIV risk (primarily as insertive partners in vaginal sex) while 51% reported no sex. Demographic and modifiable factors were associated with abstinence, including geographic location, childhood sexual abuse, and hormone and surgical status. Few intervenable factors were associated with high HIV risk. In bivariate analyses, those who had not socially transitioned gender had higher odds of sexual risk (OR=5.31, 95% CI: 1.12, 25.1). Sexual anxiety, body image worries, and lower sexual satisfaction were negatively associated. Adjusting for demographic and transition background, older age was associated with higher odds of sexual risk (OR for a 10-year increase= 1.79, 95% CI: 1.03, 3.13). Condom efficacy was associated with lower sexual risk.
Factors hypothesized to be associated with sexual risk were largely associated with abstinence. In this broad trans population sample, the relatively low prevalence of high HIV-related risk, coupled with the low fraction of such risk attributable to sex with non-trans men, posed challenges for identification of intervenable factors to reduce HIV vulnerability. Canadian studies that focus on the sub-group of trans women who have sex with men are needed.
Despite evidence suggesting emotion regulation difficulties are associated with high-risk sexual behaviour, no study to date has examined the use of emotion regulation strategies on high-risk sexual behaviour among gay, bisexual, and other men who have sex with men (GBM).
Using data from 470 HIV-negative sexually active GBM in Toronto, we examined the impact of two specific emotion regulation strategies: cognitive reappraisal, associated with better interpersonal functioning, and expressive suppression, associated with worse interpersonal functioning, on several indicators of high-risk sexual behaviour. Zero-inflated negative binomial models were fit for count data and log binomial models were fit for binary sexual behaviour outcomes.
Most participants (mean age=35, SD=12) were White (59%), born in Canada (60%), and self-identified as gay (86%). Participants reported on average 9 (8.4±21.1) casual sexual partners over the past 90 days, and approximately 22% reported engaging in unprotected anal intercourse (UAI) with a casual partner. After adjusting for age, income, ethnicity, country of birth, sexual orientation, substance use, and depressive symptoms, the use of reappraisal as an emotion regulation strategy was negatively associated with total number of casual partners (IRR=0.86, 95% CI:0.75–1.00), whereas suppression was not significantly associated with this outcome. Similarly, reappraisal was negatively associated with engaging in UAI with any serodiscordant partner, whether casual or regular (RR=0.86, 95% CI:0.75–0.99), whereas suppression was positively associated with this outcome (RR=1.12, 95% CI: 1.00–1.26).
Reappraisal was associated with reduced sexual risk behaviour, whereas suppression was associated with greater sexual risk behaviour, suggesting that not only are these two emotion regulation strategies differentially associated with interpersonal functioning, but they are also differentially associated with sexual behaviour. Future HIV prevention interventions should target emotion regulation strategies in order to reduce sexual risk behaviour among individuals at high risk for contracting HIV.
In the HIV literature, there is a focus on risk factors that put certain populations at greater risk for HIV, including gay, bisexual and other men who have sex with men (GBM). However, this traditional model does not adequately explain why many GBM do manage to practice consistently protected sex. This study examines the individual and community-level protective factors that are associated with reduced CAS among GBM.
Using data from 470 HIV-negative Toronto GBM, we examined if protective factors were associated with CAS with sero-discordant partners. Considering personal characteristics, risk factors (depression, substance use during sex, and childhood trauma) and protective factors (hope, social capital, and social support) as separate blocks, we fit log binomial models with a sandwich estimator calculating relative contribution of each factor as well as block of factors. Using Bayesian Information Criteria (BIC), Likelihood Ratio (LR) test and Tjur R2, we identified the best fit model and calculated effect of protective factors above and beyond the risk factors.
Approximately 18% reported CAS with a sero-discordant partner (regular/casual) and 15% reported CAS with a casual partner. The lowest BIC value and a significant LR test indicated that models with protective factors fit the data best. In predicting CAS with any (regular/casual) sero-discordant partner the protective-factor model had 73% more predictive power (%Δ in Tjur R2 =73.31%), and in predicting CAS with a casual sero-discordant partner the protective-factor model had 18% more predictive power (%Δ in Tjur R2 =17.59%) than the risk factor–only model (personal+risk factors).
Protective factors are associated with reduced CAS above and beyond both personal variables and risk factors that are typically assessed in empirical tests of syndemic theory. These findings show the benefits of examining protective factors against CAS in models of HIV transmission risk behaviours.
Although gender-based sexual violence has not been explored among Sri Lankan youth, it has been associated with increased population-level risk for HIV in high prevalence areas, including countries neighbouring Sri Lanka. This study examines support for gender-based sexual violence among Sri Lankan youth.
The YOU Study (initiated by the Grassrooted Trust) collected data from (n=398) older youth (18–24) across Sri Lanka. The sampling strategy used pre-specified goals of proportionally representative numbers from each district in Sri Lanka, using ratios calculated from the 2012 Census. Modified Poisson regression was used to calculate prevalence risk ratios (unadjusted/adjusted) with two violence-related outcomes (acceptability of a man hitting a woman who has disrespected him, and whether respondents had experienced sexual based violence).
Of all respondents, 24.4% agreed that it is acceptable for a man to hit a woman who has disrespected him. Further, of those answering the question, 10.3% had experienced physical violence from their partner during a sexual experience. Not earning an income, and increasing Double Standards (measuring acceptance of traditional sexual double standards) were associated with agreeing that it was acceptable for a man to hit a woman, both at bivariate levels and when modelled together. Inconsistent condom use was not significantly associated with this outcome. Those with lower education levels and who were married were more likely to have experienced physical violence during sex. During modeling, identifying with an ethnicity other than Sinhalese was significantly associated with a lower likelihood of experiencing violence, whereas lower education was significantly associated with a greater likelihood.
There was a high level of support for gender-based sexual violence and experience of actual sexual violence among Sri Lankan youth. Sexual and reproductive health education and services should work on changing attitudes toward gender-based sexual violence as a part of HIV prevention services.
Socio-historical factors (tendency towards later marriage, limited contraception access, emphasis on female virginity) combined with relatively low but steadily increasing HIV and STI rates contribute to a unique environment for providing sexual health education in Sri Lanka. Low levels of sexual health education for youth, and teachers who indicate discomfort with delivering sexuality education have been reported. We examined socio-demographic, psychosocial, and behavioural variables’ associations with knowledge of STI and HIV prevention in older Sri Lankan youth.
The YOU Study collected data from (n=398) older youth (18–24) across Sri Lanka. The sampling strategy used pre-specified goals of proportionally representative numbers from each district in Sri Lanka, using ratios calculated from the 2012 Census. Modified Poisson regression was used to calculate prevalence risk ratios (unadjusted/adjusted) with two knowledge outcomes (STI and HIV prevention knowledge). For modelling, backward elimination was used to retain variables at p=0.20.
Approximately one third of respondents were sexually active. For both STI and HIV prevention knowledge, only half (50.8%; 53.6%) were able to identify that condoms were the best way to protect against infection. Younger age, being female, not wishing to disclose or being unsure about their sexual orientation, not earning an income, having lower education were all associated (bivariate) with lower knowledge about STI protection and HIV prevention, as were not having experienced thigh, oral, or vaginal sex. In the final model, younger age, being female, and having never experienced sex were associated with lower STI protection and HIV prevention knowledge.
Given the steadily increasing STI and HIV rates in Sri Lanka, yet low STI and HIV knowledge, a need for accurate HIV and STI prevention information is becoming increasingly important. Our results highlight the need for an approach that considers sexuality education separately for males and females.
Sri Lanka’s HIV rates have historically been lower than its neighbours, but HIV incidence is rising. While information about sexual behaviours among older youth (18–24) is available, few studies have explored results obtained across the entire country. This study examines associations between sociodemographic variables and having experienced thigh, oral, vaginal, and anal sex (lifetime & within 12 months), and condom use for anal and vaginal sex in a cross-national study of older Sri Lankan youth.
In 2013, the YOU Study collected data from 398 older youth (18–24) across Sri Lanka. The sampling strategy used pre-specified goals of proportionally representative numbers from each district in Sri Lanka, using ratios calculated from the 2012 Census. Modified Poisson regression was used to calculate prevalence ratios (unadjusted/adjusted). For modeling purposes, backward elimination was used to retain variables at p=0.20.
Over one third of respondents (35.7%) reported any lifetime sexual activity (thigh, oral, vaginal, or anal sex), with 40.9%, 39.4%, 65.5%, and 20.4% reporting each, respectively, within the past 12 months. Within this timeframe, 55.1% and 73.0% reported never using a condom during vaginal and anal sex. In bivariate associations, respondents more likely to have ever had sex (thigh, oral, anal, or vaginal) were older, male, married, earning an income, identified as Sinhalese (versus Tamil), as Buddhist (versus Hindu), and identified as heterosexual. After backward elimination, only older age, male sex, and heterosexual orientation remained significant at p=0.05.
Given low condom use but increasing HIV incidence in Sri Lanka, comprehensive sexual health and HIV prevention education programs for youth are needed. Programs should attend to ethnic and religious diversity, encouraging condom use during vaginal and anal sex. Programs should incorporate culturally-relevant alternatives such as thigh sex, focusing on encouraging young heterosexual men to engage in HIV and STI prevention behaviours.
In Mexico and Central America, HIV is concentrated in several marginalized populations, including sex workers. Despite high population mobility in this region, evidence regarding the pathways by which migration/mobility experiences influence HIV and sexually transmitted infection (STI) prevention among sex workers remains limited. This study investigated the relationship and pathways by which migration/mobility shape HIV/STI risk and prevention among migrant sex workers at the Mexico-Guatemala border.
As part of a mixed-methods study, 142 female sex workers in the Mexico-Guatemala border region completed interviewer-administered questionnaires and HIV/STI testing. 47 migrant sex workers completed in-depth interviews regarding migration/mobility patterns and experiences, sex work history, HIV/STI prevention, and health care access.
Of 142 participants, 64.8% had travelled outside their birth country, 11.3% had travelled to the U.S., 23.2% had ever been deported, and 22.5% had engaged in sex work in another country in the prior year. Migrant sex workers’ narratives indicated that limited access to HIV/STI prevention (e.g., condoms), safety risks along transit routes (e.g., violence, isolation), and abuses by government authorities constrained HIV prevention and exacerbated risks, particularly upon arrival to the border region. However, working in supportive venues where condoms were promoted was critical in fostering risk mitigation, and condom use was often motivated by a desire to maintain good health to remittances to family in home countries. Additionally, many women accessed HIV/STI testing within the context of circular mobility to home countries.
Migration experiences and patterns can have heterogeneous impacts on exposure to HIV/STI risks and prevention for sex workers, especially for recent arrivals. To promote health and safety for migrant sex workers, the design and implementation of community-based interventions and policy changes to ensure access to supportive work environments and address human rights abuses remain critical.
Female sex workers in Sub-Saharan Africa face a disproportionate burden of HIV. Despite the critical role of structural determinants in shaping HIV epidemics, there remain major gaps in evidence among sex workers in Sub-Saharan Africa, with most research focused on behavioural and biological risks. Using questionnaire and serological data from a cross-sectional study conducted in partnership with The AIDS Service Organization (TASO) Gulu among female sex workers from 2011–2012, we used logistic regression to explore structural determinants of HIV among female sex workers in Gulu, Northern Uganda.
Of 400 female sex workers, 135 (33.75%) were HIV positive. Most (n=363, 90.75%) solicited clients in bars/clubs and almost a quarter (n=94, 23.50%) reported paying a third party (e.g., manager) in the prior six months. Almost a third of participants (27.75%) had been previously incarcerated, two-thirds (66.50%) had lived in an internally displaced person (IDP) camp, and one-third (n=129, 32.25%) had experienced abduction by the Lord’s Resistance Army.In a multivariate model, after adjusting for other factors, having children (AOR: 2.62, 95% CI: 1.45–4.70) and history of incarceration (AOR: 2.05, 95% CI: 1.25–3.43) were positively associated with HIV seroprevalence, whereas paying a third party such as a manager (AOR: 0.55, 95% CI: 0.31–0.97) appeared to have a protective influence.
The linkages between incarceration and HIV among sex workers in post-conflict Uganda highlight the significant harms of an increasing trend towards criminalization of key populations in Sub-Saharan Africa, which remains a critical impediment to scaling-up access to HIV prevention and care as well as sexual and reproductive health for populations most in need of these services. The protective role of paying a third party may reflect the role of managers or other third parties (e.g., “trainers”) in supporting condom use and access and mitigating occupational violence. Community-based, sex worker-led interventions to promote access to safe workspaces and policy shifts to move away from criminalization towards a human rights-based approach remain needed.
In Colombia, HIV testing remains low with only 20%–30% of the at-risk population having ever been tested. The Corporacion de Lucha Contra el SIDA (CLS) conducted HIV testing campaigns in low-income neighborhoods in Cali-Colombia and investigated factors associated with previous HIV testing (PHT).
Between 2012–2014, 939 participants were recruited through voluntary HIV counselling/testing campaigns tailored to reach socioeconomically disfavoured (SED) and high-risk groups (HRG) for HIV acquisition (MSM, Trans, sex workers). Behavioural surveillance indicators were inquired according to the UN Populations Fund questionnaire. Bivariate and multivariate Poisson regression model were used to calculate prevalence risk ratios and determine the factors associated with PHT.
Mean age of participants was 28.5±10.9 years, and 50% were male. There were 356 HRG participants (71% were male). No PHT ever was reported in 38% and 59% of participants in the HRG and SED group, respectively. Less than half of the HRG, and 20% of the SED participants had HIV testing in the previous three years. In both groups, being younger than 25 years and having had a previous doctor-diagnosed STI were strongly associated with PHT. In HRG, having no income was associated with absence of PHT, whilst consistent condom use, practice of commercial sex, and suspecting HIV+ status in a current/past partner were associated with PHT. In the SED group, female sex, being single were associated with PHT. Importantly, 40% of those in the HRG who practiced anal sex had never had PHT.
The frequency of HIV testing in HRG continues to be alarmingly low. HRG seemed slightly more aware of HIV-acquisition risk than the SED group. However, risk behaviours such as practice of anal sex, sex while using psychoactive substances are not resulting in higher rates of HIV testing. Education about these aspects needs to be enhanced in the HIV-testing campaigns.
High HIV incidence and prevalence among young men who have sex with men (MSM) and transgender persons (TG) in Thailand indicate the importance of expanded HIV prevention options; nevertheless availability does not ensure uptake. We examined the association of HIV-related stigma with uptake of current and emerging HIV preventive interventions among MSM and TG in two Thai cities.
From February–May 2014, we conducted a 30-minute cross-sectional survey among MSM and TG aged 18–30 years in Pattaya and Chiang Mai to assess HIV-related stigma, HIV risk, and HIV prevention uptake. Participants were recruited using venue-based sampling among gay entertainment venue (go-go bars, massage parlors) staff and MSM- and TG-focused community-based organization attendees. Tablet assisted self-interviewing (TASI) was administered in Thai. We conducted multiple logistic regression to assess correlations between HIV-related stigma (total, felt normative and vicarious) and HIV prevention uptake (condom use, HIV testing, rectal microbicide [RM] acceptability), and forced sex.
Across participants (n=389) in Chiang Mai (51%) and Pattaya (49%), 53% identified as gay, 25% transgender, 21% heterosexual/bisexual; median age, 25 years. Two-thirds (67%) reported >3 male partners (past month), 55% paid sex. 65% reported consistent condom use, 48% ever being tested for HIV, and 21% being forced to have sex against their will. Adjusting for sociodemographic variables, participants with higher total HIV-related stigma scores had significantly lower odds of HIV testing uptake (AOR=0.75, 95% CI: 0.60, 0.92), lower RM acceptability (AOR=0.59, 95% CI: 0.41, 0.87), and higher likelihood of forced sex (AOR=1.38, 95% CI: 1.07, 1.78). Both vicarious and felt-normative dimensions were associated with HIV testing and RM acceptability.
HIV preventive interventions and research among young MSM and transgender persons in Thailand, including roll-out of new prevention technologies, should address HIV-related stigma as a social determinant of risk, and reducing the risk of forced sex.
While HIV and HCV testing remains an important intervention for youth, many barriers to access and uptake remain. This study explored issues that influence testing among youth (ages 15–24) in Nova Scotia where there are limited options for anonymous testing and a general lack of youth-focused HIV and HCV prevention initiatives. This is particularly challenging in the context of Nova Scotia given the limited options for anonymous testing, the current lack of access to HIV point-of-care-testing (HIV POCT), the overall lack of youth-focused prevention policies, and the variable access to youth-inclusive HIV/HCV education strategies.
Data were collected from a policy scoping review, in-depth interviews and focus group discussions with a diverse sample of youth and adult HIV/HCV prevention stakeholders from across the Atlantic region on issues related to perceptions of and experiences with prevention interventions for youth. Our multidisciplinary research team, including policy trainees, analyzed the data using both a gender-based and a health equity lens to explore key themes related to how issues of access to testing and uptake of testing are understood and confronted.
Barriers at both the individual and structural level were identified as influencing youth’s reluctance to discuss or seek testing. Stigmatizing perceptions among the general public and negative attitudes about HIV/HCV, a lack of understanding regarding the importance of targeted testing among health care providers were seen as contributing to youths’ reluctance to discuss or seek testing, even where services were available.
Although HIV/HCV prevention in Nova Scotia is hampered by competition for limited funding for public health issues, greater awareness among health care providers and parents about the benefits of targeted testing for youth, the need to ensure confidentiality, and a reframing of current negative conceptualizations of HIV/HCV harm reduction policies and programs are needed to meet the prevention needs of youth.
Individuals who misuse substances and who are homeless (IMSH) are at higher risk of acquiring HIV, sexually transmitted infections and bloodborne infections and have greater medical needs than the general population; however, providing healthcare with informed consent is challenging as many IMSH have impaired cognition due to substance use. A Capacity Assessment Instrument for People who misuse Substances (CAIPS) has been developed to assist clinicians in deciding if clients lack capacity to consent to healthcare (CTC-HC).
Eleven items were identified by examining existing capacity assessment instruments and by interviewing nurses who deliver services to IMSH. A panel of experts assessed the items for construct and content validity and the items were revised as needed. A validation study was conducted that compared the CAIPS instrument to two gold standards: 1) a clinical assessment by a psychiatrist and 2) assessment with the MacArthur Assessment Tool for Treatment (MacCAT-T). Reliability was examined by calculating a Chronbach’s alpha. A confirmatory factor analysis was conducted to determine dimensionality. Sensitivity and specificity were determined using the composite CAIPS score compared to each gold standard.
The final instrument consists of items that address understanding, voluntariness, orientation, ability to communicate, sustained attention, distorted reality, appreciation, reasoning, expression of choice, decision making demands, and physical indication of substance use. A total of 302 individuals (182 [60.3%] male; 124 [41.1%] Caucasian) participated in the validation phase. The CAIPS instrument demonstrated good internal reliability (Cronbach’s alpha: 0.861 – 0.893) and inter-observer reliability (weighted kappa statistic of 0.657). Unidimensionality was confirmed. Sensitivity was 0.75 – 0.81 and a specificity was 0.63 – 0.51.
The CAIPS instrument is a reliable instrument with moderate validity and is the first validated capacity assessment instrument available to assess CTC-HC among IMSH. We hope that this new instrument will enable nurses to make informed decisions surrounding the capacity of their clients and, in turn, will result in increased health equity for IMSH. Future research is required to validate the instrument with other healthcare professionals.
The tradition of storytelling not only reflects a distinct cognitive framework; it also represents a fluid and open knowledge system in which storytellers as well as their audience can actively participate. In the context of HIV/AIDS, storytelling has been used as culturally viable and effective strategies to both manage HIV-related stigma and carry out HIV education, especially in non-western societies (e.g., Galavotti, 2005; Tafoya, 2000; Zeelen et al., 2010). Using a narrative approach, this paper examines the contents, storylines, perspectives, and meanings of some stories about HIV/AIDS selected from a CIHR-funded qualitative study of HIV risk faced by Indian immigrants in Canada.
Many participants in this study told stories – stories of other people whom they personally know or heard of – about this disease in response to our interview questions about their own knowledge about HIV/AIDS. Despite the diversity of the storylines, their stories represent some common understanding of this disease, such as perceived “risk groups” (e.g., drug drivers), transmission routes (e.g., extramarital heterosexual sex) and consequences (e.g., discrimination, further transmission, and death). While such understanding illustrates the overarching influence of the cultural notions of marriage, family, and morality on their story constructions, several specific gender dimensions are also identified. For some women, for instance, storytelling also played a role in giving voice to their silenced concerns about their spouses’ infidelity and related health risks, both of which they perceived to have little control over.
We contend that storytelling enabled these participants to somewhat transcend the heavy stigma associated with HIV/AIDS and thus to speak about some culturally taboo topics (e.g., sex and HIV/AIDS) that are otherwise unspeakable in the community. The disjuncture between their stories and sound HIV knowledge also suggests the urgent need for the development of culturally comprehensible and gender-sensitive approaches to HIV prevention for this community, however.
Studies suggest that people living with HIV/AIDS in high-income settings suffer high levels of food insecurity. However, limited evidence exists regarding the dietary intake of this population and sub-components driving food insecurity (i.e. food quantity, quality, safety or procurement). We therefore examined the prevalence and characteristics of food insecurity among HIV-positive people across British Columbia (BC), Canada.
This analysis was conducted within a national community–based research initiative involving 30 AIDS service organizations and four universities across Canada. Peer research associates led participant recruitment and survey / dietary recall administration. Food security was measured using the Health Canada Household Food Security Scale Module. Logistic regression was used to determine key factors associated with food insecurity, controlling for potential confounders.
Of 262 participants deemed eligible for analysis, 192 (73%) reported being food insecure. Sub-components associated with food insecurity in bivariate analysis included below daily recommended intake consumption of protein (p = 0.046); being sick from spoiled/unsafe food in the past six months (p=0.010); and using non-traditional means of food procurement (p<0.05). In multivariable analyses, factors significantly associated with food security included: procurement of food in using non-traditional means of food procurement [adjusted odds ratio (AOR)=11.11, 95% confidence interval (CI): 4.79–25.68, p=<0.001]; younger age [AOR=0.92, 95% CI: 0.86–0.96, p=<0.001]; unstable housing [AOR=4.46, 95% CI: 1.15–17.36, p=0.031]; household gross annual income [AOR=4.49, 95% CI: 1.74–11.60, p=0.002]; and symptoms of depression [AOR=2.73, 95% CI: 1.25–5.96, p=0.012].
Food insecurity among people living with HIV in BC appears to be defined by poor dietary diversity and food procurement methods. Notably, participants who reported using non-traditional means of food procurement were over 10 times more likely to be food insecure. These findings suggest a need for tailored food security and social support interventions in this setting.
Empirical research has pointed out the relationship of mental heath issues, adequate access to cultural sensitive services and HIV Prevention. On the ground, providing a marginalized community such as Spanish-speaking MSM with one-on-one counselling services can be challenging due to lack of adequate number of staff.
In 7 years I have provided counselling related to homophobia, lack of support, family rejection, underemployment/unemployment, trauma, violence, immigration issues, difficulties in understanding self sexual desires, sexual orientation, and how to deal in a Spanish-speaking marginalized environment in Toronto.
An innovating strategy of working off traditional hours from 9-5 was developed along with Skype and new technologies for hard to reach clients, allowing people who work on that schedule, having flexibility for accessing counselling.
Due to the diversity of issues that CSSP HIV/AIDS’s clients have, the role of the Counsellor has to be more flexible since clients need proper case managing and proper social work diligence.
Having an independent program’s office that is conveniently located and away from the other services that CSSP provides, helps to keep clients’ confidentiality and reduces stress, while promotes seeking for help.
Over time the program has provided counselling to over 7,000 clients.
Some clients require a longer treatment and different and deeper Counselling approaches.
Accessing services with referrals is also challenging for our clients due to long waiting list procedures, economic factors (too poor to access private services), and language barriers.
By comparing other HIV/AIDS services that are cultural specific for other groups, CSSP’s counselling services realizes the need of increasing the number of qualified staff that can take in the area of case management and social work.
Mental Health services should be an integral part of any HIV/AIDS structural intervention effort.
The epidemic and number of new HIV infections in Latino MSM has had a dramatically increase in the last 5 years.
Bringing together Latino PHA’s and non-PHA’s to encourage them to get more informed and transfer that knowledge into new prevention practices to peers and reduce the infection numbers went into practice with a new project.
Being and Outreach Worker providing services for Latino PHA’s and non-PHA’s, has been a challenge within the last 5 years. I can see guys getting infected almost every week because there’s a lack of communication, information in specific language or even specific cultural approach that can help them to understand the way of how sexual health plays the big role into their life.
We created a new graphic tool to provide information and address issues that Latino PHA’s and non-PHA’s were facing.
Discrimination and stigma between participants was an unfortunate factor to get them to participate in the project, as they were concerned about to disclose to the general Latino population because of cultural/social taboos.
4 focus groups were facilitated to get the sense of the most important issues among the participants including the confidentiality part.
There’re three “365 Days of Prevention Calendars” developed since 2010.
More than 95 Latino PHA’s and non PHA’s participated on the project either as advisory committee members or as a volunteer models.
Notorious learning and best sexual health practices were implemented by each of one of the participants.
There’s still collaboration with Hassle Free Clinic with same and new Latino MSM clients to evaluate the success of the project.
Conducting research with Aboriginal communities necessitates developing positive relationships within the community long before data collection begins. This process is important to maximize leadership within communities and organizations that are critical to supporting the research process. Stable Homes, Strong Families (SHSF) demonstrates the successes and challenges of relationship building in community-based research (CBR). SHSF is a national project that aims to develop cultural understandings of housing and home amongst Aboriginal peoples living with and affected by HIV and AIDS in order to influence housing policy and programs.
Relationships were developed between Aboriginal HIV service organizations, academic researchers and community leaders prior to grant submission. Following receipt of funding, a strategy to hire, train and support local Peer Research Associates (PRAs) to plan and lead digital storytelling (DS) workshops along with the research team was developed. Five workshops were held with 22 participants across Canada from June 2013 to November 2014.
Our CBR approach highlighted a number of important lessons for working in partnership with diverse Aboriginal communities across Canada and conducting research with PRAs who also bring diverse identities and experiences. Time must be invested to develop partnerships even before the grant is finalized and submitted. PRAs should be trained and supported in all aspects of the research. Early engagement with Elders, community supports and organizations is critical to understanding the needs of participants, to creating a safe space where the research will be conducted and ensuring local Aboriginal cultural protocols are respected.
Community engagement and partnership development are as important as generating data and are essential to effective, community-driven knowledge translation activities. Lessons learned regarding key considerations for partnerships between Aboriginal communities and researchers will critically inform our analysis of data from the DS workshops.
In 2012–2013, Asian Community AIDS Service (ACAS) conducted a community-based research to assess and identify contributing factors to HIV vulnerability of Asian Migrant Farm Workers (AMFWs) in Ontario by exploring their HIV/AIDS knowledge and sexual behaviours, personal and structural determinants of HIV/STI vulnerabilities, and resilience factors.
A Purposive Sampling was used to recruit 80 Thai and 20 Filipino MFWs to complete a written questionnaire. Twenty-four participants completing the survey component were invited to participate in one of the three follow-up focus groups to further explore the studied issues. Trained peer research assistants assisted with participants’ recruitment, survey and focus group administration.
Participants’ average age was 38 years with 53% male and 47% female. Overall, participants had a moderate knowledge about HIV/AIDS. 62% were sexually active in the past year and 55% of them had sex with a regular partner. Use of condoms was relatively low, with over 76% indicating they rarely or never used condoms with regular partners. 23% reported having difficulty negotiating safe-sex practices with partners. 4% were tested for HIV, 3% for STIs and 4% for both HIV and STIs. Structural issues: limited English proficiency and social support; transportation problems; long working hours etc., contribute greatly to their inadequate use of health services.
In the focus groups, participants discussed perceived risks and fear of being infected with HIV/STIs, particularly with a casual partner. The majority assumed sexual partners from the same country were free from HIV because they passed a work visa’s medical screening prior arriving Canada. Condom use is mainly for preventing pregnancy. All considered sexual health issues were private and did not necessary discuss the matters with co-workers.
Access to culturally and linguistically appropriate health, HIV, and labour rights, and how to use health and sexual health services information would increase their overall health and well being. AMFWs also recommended such activities as having a sexual health discussion among peers and an annual physical check up for all workers.
Transmission clustering is sustaining the MSM epidemic in Quebec in the post-antiretroviral therapy (ART) era. Phylogenetic, virological, and behavioural data were combined to describe factors contributing to large cluster outbreaks.
Phylogenetic analysis was performed on sequence datasets from 5.828 new diagnoses (2002–2013), first genotyped during primary or chronic stage infection, using nucleotide mixed-base call assays to estimate recency of infection. MSM infections (n=4027) were stratified as solitary transmissions (n=1755), 219 small (2–4, n=562), 66 large (6–9, n=426) and 52 X-large (10–116, n=1284) clusters. PHI cohort data (1998–2013) provided sequence, behavioural, and virological data on 379 MSM (n=210 untreated for 2 years). The SPOT site provided testing habit and behavioural data on 1790 MSM (2009–2011).
Disturbingly, half of onward spread of MSM epidemics could be ascribed to 2.5% of viral lineages (n=52), averaging 25 infections/cluster, expanding over 18 month intervals. These latter variants showed significantly lower quasispecies diversity than the 84% of lineages leading to dead-end transmissions (58% vs. 25% <0.44% diversity, median 0.13% vs. 0.40%, respectively). Variants belonging to large (n=98) and small (n=57) clusters elicited extended high viremia (median 4.7 log copies/mL) over two-year periods as compared to solitary group lineages (n=54) whose viremia fell to set-point nadirs after 6 months. Although 14%, 18%, 27% and 30% of persons in unique, small, large and X-large cluster groups were under 30 years, risk behaviours were similar with 0–4%, 70–74%, 7–10% and 10–14% of participants reporting 0, 1–4, 5–9, 10+ partners three months prior to infection. SPOT observational data show poor testing habits with half of persons untested in the last year (median 4 years).
Frequent testing and immediate ART initiation is needed to avert the selection of large cluster variants overcoming transmission bottlenecks. Studies are ongoing to characterize signature features allowing for their selection.
HIV prevalence differs substantially by race and ethnicity in Canada and in Quebec. We completed a survey to describe risk behaviours and HIV prevalence among Montrealers born in Sub-Saharan Africa (MSSA) and in the English-speaking Caribbean countries (MESC).
From June 2013 to February 2014, a voluntary, anonymous, cross-sectional survey of 1602 participants, 15 to 49 years of age (1105 MSSA and 497 MESC) was conducted. Recruitment strategies included social events (ethnic festivals, churches), neighborhood venues (malls) and fixed sites (associations). Participants provided a biological specimen tested for HIV, hepatitis C and syphilis antibodies and completed a computer-assisted questionnaire. Correlates of having ≥1 casual heterosexual partner (e.g., “one-night stand”; past 12 months) were ascertained using multivariable logistic regression.
Of 1958 eligible individuals solicited, 1602 (81.8%) participated with the majority providing a biological sample of sufficient quantity for testing (96.4%, (1545/1602). Among MSSA, the overall HIV prevalence was 1.7% (95% CI: 1.1–2.6%), 1.2% (0.5–2.4%) among males and 2.4% (1.2–4.2%) among females. Of the 18 HIV-positive MSSA, 38.9% were aware of their infection. Among MESC, overall HIV prevalence was 0.8% (0.3–2.1%), 0.9% (0.2–3.2%) among males and 0.8% (0.2–2.8%) among females. Of the 4 HIV-positive MESC, none were aware of their infection. Correct knowledge of modes of HIV transmission was significantly lower for MESC compared to MSSA. Six out of 10 (60.7%; 963/1587) did not have a family physician. Participants reporting ≥1 casual heterosexual partner (n=1106) were more likely to be male (AOR=5.0; p<0.0001), single (AOR=7.9; p<0.0001), unemployed (AOR=2.7; p=0.046), to have had sex in an HIV-endemic country (past 5 years; AOR=2.9; p=0.001), and HIV testing ≤2 years (AOR=2.2; p=0.034).
This successfully implemented first E-Track survey highlights the importance of potential HIV risks in these populations. HIV/AIDS prevention interventions must address their unique needs.
Globally, sex workers report substantially lower condom use with their intimate or non-paying partners (NPPs) than with commercial partners. To explore the role of non-paying partnerships in shaping HIV-related vulnerability, this study investigated interpersonal factors associated with inconsistent condom use among sex workers and their NPPs in Vancouver, Canada.
Baseline data (2010–2013) were drawn from an open prospective cohort of sex workers, AESHA (An Evaluation of Sex Workers’ Health Access), in Metro Vancouver. Multivariable logistic regression using generalized estimating equations was used to identify interpersonal factors associated with inconsistent condom use (i.e., <100% in the last six months) with up to three NPPs reported per sex worker. Adjusted odds ratios and 95% confidence intervals were reported (AOR [95% CI]).
Overall, 369 sex workers reported having at least one NPP (mean=2.2 NPPs per sex worker), with 70.1% of the study data reporting inconsistent condom use in the last six months. In the study, 11.2% and 14.3% of NPPs had sexual relationships with other sex workers or other non-commercial female partners, respectively, in the last six months. In multivariable analysis, factors significantly associated with increased odds of inconsistent condom use included: having a regular cohabiting NPP (5.49 [2.60–11.58]) or a regular non-cohabiting NPP (2.17 [1.14–4.11]) (versus casual NPP), providing drugs to a NPP (3.12 [1.54–6.31]), providing financial support to a NPP (2.45 [1.09–5.51]), and having a NPP provide physical safety (2.21 [1.20–4.09]). Non-injection drug use was significantly associated with reduced odds of inconsistent condom use (0.37 [0.20–0.67]).
Our study highlights the important role of interpersonal factors in shaping HIV risk pathways for sex workers and their intimate or NPPs, and potentially for NPPs’ other commercial or non-commercial sexual partners. Efforts to reduce HIV risk for sex workers and their noncommercial partners should incorporate gender-focused and empowerment-based strategies, as well as male- and couple-focused interventions.
Very little is understood regarding associations between concurrent trauma, addiction, psychological distress and HIV and HCV vulnerability among Indigenous men and women in Canada.
The Cedar Project is a cohort study of young Indigenous people aged 14–30 who used drugs in Vancouver, Prince George, and Chase, BC. Participants were eligible for this analysis if they completed a baseline Symptom Checklist-90-Revised (which provides an average measure for severity of psychological distress), returned for at least one follow-up visit between 2010–2012, and completed the Childhood Trauma Questionnaire. Adjusted linear mixed effects models (LME) estimated the effect size (B) and 95% confidence intervals (CI) of study variables on mean change in psychological distress scores separately for men and women.
In total, 202 participants were eligible; 53% were women, and the mean age was 28 years. Baseline psychological distress scores were significantly higher for women (1.25) than men (0.81), (p<0.001). In the LME models for men, factors associated with increased psychological distress included: emotional abuse (B=0.57; 95% CI: 0.21–0.93), physical abuse (B=0.48; 95% CI: 0.14–0.82), physical neglect (B=0.41; 95% CI: 0.01–0.82), blackouts from drinking (B=0.24; 95% CI: 0.05–0.44), and sex work (B=0.95; 95% CI: 0.44–1.47); while living by traditional culture was associated with reduced psychological distress (B=−0.20; 95% CI: −0.39, −0.01). In the LME models for women, factors associated with increased psychological distress included: emotional abuse (B=0.60; 95% CI: 0.29–0.91), physical abuse (B=0.37; 95% CI: 0.06–0.68), physical neglect (B=0.55; 95% CI: 0.26–0.83), blackouts from drinking (B=0.30; 95% CI; 0.05–0.55), sex work (B=0.32; 95% CI: 0.12–0.52), and sexual assault (B=0.50; 95% CI: 0.30–0.70); while having tried to quit using drugs was associated with reduced psychological distress (B=−0.25; 95% CI: −0.47, −0.02).
Public health providers must provide culturally-safe mental health supports to young Indigenous people who use drugs and address harmful coping patterns that may exacerbate psychological distress and increase vulnerability to HIV and HCV infection.
The importance of integrating health care and harm reduction services, including supervised injection services (SIS), for persons who use illicit drugs has been recognized for over 20 years in Europe and is becoming increasingly well recognized in Canada. Despite this, there is not a standard of best practices for clinical policies and procedures for SIS in integrated health care settings in Canada.
Since 2002, the Dr. Peter Centre (DPC) has integrated SIS within a broad range of health care services for people living with HIV in its Day Health Program and 24-hour Specialized Nursing Care Residence. The DPC started the service for its clients after the College of Registered Nurses of British Columbia (CRNBC) confirmed that it was within the scope of nursing practice to supervise injections for the purposes of preventing illness and promoting health. It has evolved to include the interface with other professional disciplines. DPC clinical policies and procedures for SIS include ones which address agreement for services, the parameters of nursing involvement, role of other professional disciplines, counselling and addiction treatment, dealing with emergencies, record-keeping and the disposal of biohazards and controlled substances.
While DPC clinical policies and protocols were prepared in consultation with the CRNBC to conform to the scope of nursing practice, developing clinical policies and procedures for SIS is a complex process in an environment that engages other professional disciplines in addition to nursing.
A review and evaluation of policies and procedures for health care organizations providing SIS in an integrated setting is needed for the Canadian health care context so that organizations interested in applying for an exemption under Section 56 of the Controlled Drugs and Substances Act (CDSA) can do so with a complete range of policies and procedures that meets best practice standards.
Our objectives were to identify factors associated with condom-use during anal intercourse among HIV-positive MSM in Vancouver and to determine what preventive attitudes and alternative strategies were employed by MSM who did not report using condoms.
We analyzed data from participants in the Momentum Health Study collected at enrollment on their most recent sexual encounter with each of up to five sexual partners in the past six months. Explanatory factors included psychosocial scales on HAART Optimism, Sexual Altruism, Sexual Sensation Seeking (SSS), and Cognitive Escape. Of all sexual encounters where anal intercourse was reported, factors associated with condom-use versus not were determined using manual backward stepwise multivariable generalised linear mixed models.
648 sexual encounters were reported by 184 self-identified HIV-positive participants. Most encounters included anal intercourse (72.4%), during which condoms were used 22.6% of the time. Lower odds of condom-use were associated with event-level GHB substance use (vs. not: AOR=0.12, 95% CI:0.02–0.77), being certain their partner was HIV-positive (vs. unknown status: AOR=0.28, 95% CI:0.11–0.73), reporting more anal sex events with this partner in the past 6 months (AOR=0.86, 95% CI:0.77–0.97), and higher scores on SSS (AOR=0.86, 95% CI:0.78–0.94) and Cognitive Escape (AOR=0.93, 95% CI:0.88–0.99). Higher Sexual Altruism community sub-scale scores were positively associated with condom-use (AOR=3.32, 95% CI:2.00–5.50). HAART Optimism was not associated with condom-use. HIV-positive men who reported condomless anal intercourse were also more likely to report asking partners their HIV status (AOR=3.43, 95% CI:1.71–6.91), only having sex with other HIV-positive men (AOR=3.64, 95% CI:1.78–7.43), and only having sex with men on treatment or with low viral loads (AOR=2.32, 95% CI:1.12–4.80). MSM who reported condomless anal intercourse were more likely to hold differing attitudes to those MSM who used condoms: e.g. more likely to agree that “knowing a sex partner’s viral load is just as important as knowing their HIV status” (AOR=2.39, 95% CI:1.09–5.21).
Psychosocial traits, attitudes, and substance use are important predictors of condom-use and HIV-positive men who report condomless anal intercourse employ various prevention strategies that consider HIV status and viral load.
Health agency websites are a central hub for providing and accessing HIV information. We aimed to scope information provided about HIV risk and prevention on Canadian agency websites relevant to MSM. Our scan examined topics covered, how information was displayed, and the reading grade level of online information.
Eligible sites provided information relevant for MSM on HIV risk or prevention, were from community or government agencies, and for the public. Sites were found by google search using French and English search terms, expert suggestion, and review of links. Eligibility and content for review was determined by two independent reviewers, with data collected by a single reviewer using a standardized form and entered into Epi-Data (final results based on dual review will be presented). Reading grade level and usability scores were assessed through Flesch-Kincaid and LIDO instruments, respectively, Analyses were conducted in SPSS.
Of 49 eligible sites 25% were campaigns, 21% and 78% were community or government agencies respectively, 26% were focused on MSM, 19% were French/bilingual. 49 sites included on average 7.9 of 20 topics on HIV risk, with sero-sorting (22%), viral load (15%) and acute HIV (15%) being least frequent. 46 sites included an average on average 6.4 of 24 topics on HIV prevention, with treatment as prevention (13%) and PrEP (9%) being least frequent. Almost all sites presented information by text, with a median reading grade level of 10. Less common were use of tables, images, or graphs to present information; only 4% used interactive or tailored features. On usability scoring, sites scored lower on functionality and engagement domains.
Overall we observed less information about emerging topics and a reliance on text with high reading requirements. Updating content, communicating information effectively, and engaging audiences appeared common challenges for assessed websites.
A key response to HIV has been the delivery of prevention programs by diverse AIDS Service Organizations (ASOs). While there is research that assesses the effectiveness of individual HIV prevention programs, challenges have been identified in the uptake of these programs. There is a lack of clarity on how ASOs measure success, and a deeper understanding of this could enhance monitoring and evaluation (M&E) practice in the sector.
In this presentation, I summarize the findings and conclusions of my doctoral research about HIV prevention program M&E at ASOs. Employing a constructivist approach, I carried out a qualitative case study of 2 ASOs, conducted in-depth interviews with 23 managers, staff, volunteers, and government funders; I reviewed approximately 100 documents. A community reference group which included practitioners, policymakers and people living with HIV provided advice on key stages of the project.
In this research, the complexity and plurality of HIV prevention programming and M&E emerged. M&E practices employed by these ASOs included one-time evaluations, routine monitoring, and tacit assessments. The ways in which these informed each other was complex. Tacit knowledge was drawn upon to make explicit information derived from M&E required by government funders meaningful. Accountability to government funders required M&E that was different than what was needed to maintain relationships with the communities these organizations served. Overall, there were differences in the ways that this community of practice understood success and built knowledge compared to research that evaluates the effectiveness of interventions.
A deeper understanding of M&E at ASOs can contribute to responses to the evidence-based practice movement. This research describes community-based discernment strategies that shed light on the knowledge-building processes in communities, which is an important part of practice-based evidence and program science. The lessons learned may be a resource for evaluators, policymakers, and other stakeholders in HIV prevention.
Most HIV research involving Indigenous people is based on deficit models of health and limited in cultural/practical relevance. Indigenous scholars have urged health researchers to identify factors related to cultural connectedness that may protect against HIV infection and buffer the effects of historical/lifetime trauma among Indigenous peoples.
The Cedar Project is a cohort study of young Indigenous people who use illicit drugs in Vancouver, Prince George, and Chase, British Columbia. Participants completed the Childhood Trauma Questionnaire and the Connor-Davidson Resilience Questionnaire between 2011–2012. Adjusted linear mixed effects models (LME) estimated the effect of the size (B) and 95% confidence intervals (CI) of study variables on mean change in resilience scores.
Overall, 191 participants were eligible; 51% were women, the mean age was 28.9 years, 48% had a parent who attended residential school, 71% had been in foster care. Only 8% of participants reported no childhood maltreatment, while 39% reported severe sexual abuse, 41% severe physical abuse, 33% severe emotional abuse, 20% severe emotional neglect, and 39% severe physical neglect. Factors associated with diminished mean resilience scores included severe emotional neglect (B=−13.34, 95% CI: −21.25, −5.42), smoking crack daily or more (B=−5.42, 95% CI: −10.66, −0.18), having been sexual assaulted (B=14.4, 95% CI: −28.09, −0.76), and having blackouts from drinking (B=−6.19, 95% CI: −11.62, −0.75). Factors associated with higher mean resilience scores included having a family that often/always lived by traditional culture (B=7.70, 95% CI: 2.53–12.86) or often/always spoke their traditional language (B=10.52, 95% CI: 5.72–15.33). Participants who were currently often/always living by traditional culture (B=6.50, 95% CI: 0.86–12.14) knew how to speak a traditional language (B=13.06, 95% CI: 4.85–21.26), and had sought drug/alcohol treatment (B=4.84, 95% CI: 0.35–9.33) also had significantly higher mean resilience scores.
In the aftermath of colonization, cultural foundations continue to function as buffers that protect young Indigenous people from severe health outcomes, including HIV infection.
Despite numerous obstacles, many two-spirit and/or gay/bisexual HIV-positive Aboriginal men (TS PHAs) who have been living with HIV for a long time report they are doing well. This study was designed to bring together a team of researchers comprised of Aboriginal and non-Aboriginal community members and academics to examine the ways in which two-spirit men with HIV understand the skills, resources, knowledge and practices that contribute to their resiliency and wellbeing.
Three Aboriginal sharing circles (focus groups) were conducted in Toronto, Hamilton, and Ottawa, Ontario, Canada. Participants were men (n=14) who identify as TS PHAs, who were living with HIV for 8 years or more. The sharing circles were modeled after a traditional Aboriginal sharing circle and involved symbol-based inquiry to stimulate dialogue about strengths, assets, and resiliency. Through the use of the Medicine Wheel (MW), participants were invited to discuss historical, biomedical, social, spiritual, sexual, and behavioural factors affecting their health, wellness and resiliency. Qualitative data were coded and analyzed using NVivo 10. A participatory analysis approach was used to extrapolate themes and relationships from participants’ quotations and mapped across the MW. Validity was established by verifying coding work and utilizing member-checking of the findings with our community advisory committee.
Through the analysis of participants’ discussion, the Seven Paths of Resiliency arose as integral components of their health, wellness, and resiliency. The Seven Paths of Resiliency are: (1) Worldview, (2) Finding One’s Strength, (3) Walking Towards Balance, (4) Recognizing True Power, (5) Mino-bimaadiziwin (living the way of a good life), (6) Self-care, and (7) Living Our Truth.
Through an active community/academic partnership with meaningful engagement to address the resiliency of long term HIV-positive TS PHAs, the results identify the ways in which TS PHAs understand their own resiliency. The methods and findings have broad implications for advancing the development of knowledge necessary to address the needs and concerns of those who have been living with HIV longer term.
Colonialism is at the heart of disproportionate HIV-related mortality and morbidity experienced by Aboriginal Peoples in Canada. Postcolonialism is a theoretical approach that enables healthcare professionals (HCPs) to better understand and address health inequities. Yet, the literature is silent on how to incorporate postcolonialism into healthcare training. This study explores strategies for including postcolonialism into the training of all HCPs in Canada.
This descriptive qualitative study involved in-depth, 45 to 60 minute semi-structured interviews with nineteen individuals with insight into postcolonialism and health in Canada, including academics, clinicians and Aboriginal Peoples. Interviews were transcribed verbatim. Data were analyzed collaboratively to identify, code and translate key emergent themes according to the six phases of the DEPICT method.
Postcolonialism was viewed as an essential tool for all Canadian HCPs to better address health inequities in Canada, including among Aboriginal Peoples living with HIV. Participants described content that should be included into training programmes, who should teach it, when it should be taught and how it should be delivered. In particular, participants emphasized that curricula related to postcolonialism and health should: (1) include a foundation in Canadian colonial history; (2) be integrated longitudinally using a variety of interactive teaching strategies; and (3) be developed in collaboration with local communities. Findings reinforce the importance of understanding health and health care as situated in social, political and historical contexts, often rooted in colonialism.
This is the first empirical study to provide Canadian healthcare educators with guidance for incorporating postcolonialism and health into healthcare training programs. This is a key step for improving access to and quality of care for people living with HIV.
African, Caribbean and Black (ACB) people carry a disproportion burden of HIV infection in Canada. This CIHR funded study examines social and sexual predictors of condom use at first and most recent penile-vaginal intercourse (PVI) among ACB youth.
Between April, 2013 and March, 2014, 543 16–25 year old ACB participants living in the Windsor area of Ontario, recruited using Respondent Driven Sampling, completed a survey inquiring about sexual behaviours and social/living situations. Logistic regression examined potential social, partner, and sexual predictors of condom use at first and most recent PVI.
Of the 273 participants reporting PVI, 22% reported condom use at first and 61% at most recent PVI. Those more likely to report condom use at first PVI were younger, students, and either Muslim or Christian (as compared to having no religious affiliation). Condom use at most recent PVI was more likely for Christians or those with no religious affiliation, students, those who reported consuming alcohol prior to PVI and those whose first partner was ACB. They were least likely to use a condom at most recent PVI if their partner was more than 4 years older than they and had a history of more sexual partners than they did.
Results contribute to a sparse body of knowledge about sociological and sexual correlates of condom use among ACB youth. Religious affiliation and student status influence condom use at first and most recent PVI. Alcohol use (short of drunkenness) does not appear to inhibit condom use at most recent sex, although having an older and more experienced partner does. This provides guidance for locations for interventions encouraging condom use (e.g., religious and educational institutions) and the contexts that need to be addressed (e.g., alcohol as part of sexual encounters and the age and experience of partners).
Although HIV-related stigma undermines effective HIV treatment and prevention strategies, there is little research on what primes HIV-related stigma, or on HIV-related stigma among priority populations in Canada.
This CIHR funded study uses Goffman’s stigma theory (1963) as elaborated by Link and Phelan (2001), combined with Henry’s low-status compensation theory (2009) to examine factors which influence expressions of stigmatizing attitudes against people living with HIV or AIDS among 16- to 25-year-old African, Caribbean, and Black (ACB) youth.
Between April, 2013 and March, 2014, 543 16–25 year old ACB participants living in the Windsor area of Ontario, recruited using Respondent Driven Sampling, completed a survey using ACASI methods. Structural equation modeling using responses of 519 youth estimated the direct influence of religiosity, religious denominations, experiences of discrimination, HIV test history, and sex on HIV/AIDS stigma, as well as the indirect influences of select variables on stigma through HIV/AIDS knowledge and sexual health service use.
Model estimation revealed significant negative associations between knowledge and religiosity, stigma and knowledge, and stigma and HIV testing. Muslim and male participants had significantly higher stigma scores than non-Muslim and female participants.
This is one of the few studies that examines contributors to stigmatizing attitudes related to HIV. Results support a focus on knowledge about HIV/AIDS as a potentially important inhibitor of stigma. The importance of culturally, religiously, and gender competent knowledge enhancing and stigma reducing initiatives is also highlighted in these results.
In the context of immigration, cultural ideals of family and marriage have remained important for South Asian groups living in the West (e.g., Dale & Ahmed, 2011; Grewal, et al., 2005). Although marriage as an institution does not suggest a static relationship to gender inequality, empirical studies in India have revealed married women’s salient HIV vulnerability as a result of their husbands’ extramarital sex (Newmann, et al., 2000; Chatterjee & Hosain, 2006). While immigrants from India represent one of the largest visible minority groups in Canada, we know little about the impacts of immigration on their intimate relationships, sexual practices, and responses to related sexual health risks within marriage.
This paper explores the gendered impacts of immigration on the HIV risk faced by Indian immigrants in Canada, paying closer attention to the role of marriage in mediating gender relations in their post-immigration lives. Our data was collected through qualitative interviews with 15 female and 12 male immigrants from the Punjabi community living in Ontario.
The post-immigration employment challenges led to a high concentration of these men in certain occupations (e.g., long-distance truck driving), which exposed them to health risks associated with commercial sex. Despite women’s increasing participation in employment outside the home, marriage, family, and community were perceived as integral to their cultural identities and social support networks. While men tended to take advantage of the newfound sexual freedom in Canada, many women were concerned about the HIV risk within marriage due to their constrained capacity to discuss and negotiate safer sex. The findings confirm the need for the Indo-Canadian community and families to play a creative yet crucial role in addressing the HIV risk faced by immigrant women and men through attending to both the silence surrounding the disease and the gender inequality embedded in HIV vulnerability.
African, Caribbean and Black (ACB) populations in Canada are over-represented in the HIV epidemic. Social drivers of HIV – including racism, sexual stigma, and HIV-related stigma – enhance HIV vulnerability among ACB populations. The Black Coalition for AIDS Prevention implements monthly peer support and psycho-education groups: (1) Rainbow Sistahs (RS), for lesbian, gay, bisexual, transgender and queer (LGBTQ) ACB women, and (2) Foreign Integration (FI), for LGBTQ ACB newcomers and refugees. We explored perceived benefits of social support group participation among LGBTQ ACB newcomers and refugees in Toronto, Canada.
We conducted three focus groups with ACB participants (n=29) who attended RS, FI or both. We conducted semi-structured individual interviews with healthcare and social service providers (n=5) from AIDS service organizations and community health centres serving ACB populations. Interviews and focus groups were recorded, transcribed verbatim, and analyzed using narrative thematic techniques.
Focus group participants (n=29; mean age: 30.5 years [SD 8.0]; cis-male: 51.7%, cis-female: 37.9%, transgender: 10.4%) attended FI (64.3%), RS (10.7%) and both RS and FI (25.0%). Participants identified as bisexual (42.9%), gay (32.1%), and lesbian (17.9%). All participants were born outside of Canada (70.4% Caribbean; 29.6% African) and most (79.3%) came as refugees. Findings revealed multi-level social and health benefits of social support group participation. Individual/micro level benefits included intrapersonal (e.g. HIV knowledge, self-acceptance, reduced depression) and interpersonal (e.g. friendships, networks) dimensions. Social/meso level benefits included community connectedness and reduced stigma (HIV-related, sexual). Structural/macro level benefits included increased access to employment, housing, immigration and refugee services, and health care.
Findings highlight the synergistic effects of social exclusion produced through multiple forms of marginalization, including sexuality and newcomer/refugee status. Social support groups tailored for LGBTQ ACB newcomers and refugees have the potential to address the complexity of social, legal, economic and health issues that elevate HIV vulnerability.
Interethnic and interracial intimate relationships are integral to an ethnoculturally diverse society. Between 2001 and 2006, Canada witnessed a rapid increase – at a rate of 30% – of mixed unions, five times higher than the growth for all couples (6.0%) (Milan, Maheux, & Chui, 2010). Based on a CIHR-funded transnational study of the recent generation of Chinese immigrants in Canada, this paper examines these individuals’ HIV vulnerability in the context of interracial intimate relationships, paying closer attention to the role of their transnational perspectives – such as their HIV knowledge, risk awareness, and social positioning across countries – in mediating their perceptions about and responses to HIV risk. The data was collected through in-depth interviews with 61 participants at four sites (Beijing, Shanghai, Toronto and Vancouver).
While the structural barriers (e.g., foreign credential recognition and employment) to successful settlement are often seen as challenging for individual immigrants to overcome, this study reveals that sex or intimate relationships (both heterosexual and same-sex) – especially with local white Canadians – were perceived by some participants as a venue to pursue personal freedom, sexual desires, relative sexual and gender equality, and/or accelerated integration. The asymmetrical yet fluid power relations embedded in the intimate encounters have compromised some participants’ abilities to negotiate safer sex on the one hand, and enabled others the room for a more autonomous relationship on the other hand. We contend that interracial intimate relationship is a site where the exemplified inequalities – perceived power differentials related to nation-state, language, culture, race/ethnicity, sexuality and gender – can be both accommodated and resisted, which has deferentially shaped those individuals’ capacities to respond to HIV risk. The findings suggest the need for researchers and service providers to “think transnationally” about the complex relations of power of agency when addressing immigrants’ HIV vulnerability in the context of interracial intimate relationships.
Human Immunodeficiency Virus (HIV) infects over 2.7 million people annually. More than half of the new infections occur in women, and the majority of these women are in developing countries where societal norms may prevent them from insisting on condom use. As such, new prevention options are urgently needed. We previously proposed the development of an HIV-1 specific microbicide using the S-layer mediated display capabilities of the non-pathogenic freshwater bacterium Caulobacter crescentus. We have successfully expressed 17 diverse anti-HIV proteins on the surface of C. crescentus, including MIP1alpha, CD4 decoy receptor, fusion inhibitors, and anti-viral lectins. Using an in vitro viral blocking assay, we have demonstrated that 12 of the recombinant C. crescentus are able to provide significant protection from infection with HIV-1, with protection levels reaching 74% with MIP1alpha. In vivo studies with immune-competent mice indicate that application of C. crescentus to the vaginal tract does not induce the production of inflammatory cytokines or recruitment of immune cells. We are continuing our in vivo studies using the humanized bone marrow-liver-thymus (BLT) mouse model of HIV-1 infection. We have combined the implantation of human fetal liver and thymus tissue with the intravenous injection of autologous CD34+ cells into NOD-scid IL2Rgnull mice to create BLT mice and we have demonstrated that the peripheral blood of these mice contains 40–50% human CD45+ cells, including CD4+ and CD8+ T cells, B cells, myeloid cells, and NK cells. Our initial data indicates that these mice are susceptible to intravaginal infection with HIV-1JR-CSF. Subsequent experiments have demonstrated significant protection from vaginal HIV-1JR-CSF infection when recombinant C. crescentus is applied intravaginally at the time of HIV-1 infection. Taken together these results suggest that a C. crescentus based microbicide might be a safe and effective method for HIV-1 prevention.
Current anti-HIV-1 treatments target viral components but are limited by drug resistant viruses. This study focuses on disrupting viral RNA processing, a stage where HIV-1 survival is dependent upon balanced host splicing factor activity to properly process its RNA. We demonstrated that modulation of the Cdc2-like kinase (CLK) family has a substantial impact on HIV-1 gene expression (GE). It was also observed that chlorhexidine, an inhibitor of CLKs 2, 3, and 4, potently suppresses HIV-1 replication whereas TG003, an inhibitor of CLKs 1, 2, and 4, had little effect on viral GE. Given the selectivity of chlorhexidine for CLK3, it is likely that CLK3 is an important regulator of HIV-1 GE and therefore could be modulated to control viral infection. Using short hairpin RNA targeting CLK 2 or 3, we observed that depletion of CLK3 suppresses HIV-1 structural protein expression (Gag/Env) while reduction of CLK2 has negligible effects on relative viral GE. From an assay of ∼60 RNA splicing modulators, we identified compound 191 as an inhibitor of CLK3 activity and HIV-1 structural [Env/Gag (IC90: 1.8 μM)] and regulatory (Rev/Tat) protein expression. We confirmed that 191 impairs viral GE/replication in a CD4+ T cell line (IC80: 4 μM) and patient PBMCs (IC80: ∼1 μM) infected with HIV. Furthermore, 191 has low impact on cell viability, splicing, translation, and Tat stability. However, analysis of HIV-1 mRNAs demonstrated that 191 treatment causes only a 60% reduction in both unspliced and singly spliced RNAs encoding Gag and Env, respectively. Examination of the subcellular distribution of unspliced RNAs revealed nuclear retention after 191 treatment, explaining that Rev depletion further impacts structural protein expression by blocking export of their RNAs. This study confirms that CLK3 is a key factor for efficient viral GE and suggests its targeting for the therapeutic control of HIV-1 infection.
The success of current anti-HIV treatment regimens targeting viral enzymes and host cell entry is limited by the growing emergence of drug-resistant strains of HIV. Therefore, drugs targeting different stages of the virus lifecycle are vital for continued success in combating HIV infection. Since HIV gene expression is critically dependent upon controlled splicing of the viral transcript, small molecule modulators of RNA processing hold tremendous promise as novel anti-HIV drugs. To identify such compounds, we screened a subset of splicing modulators for their effect on HIV-1 Gag protein expression. Of the sixty compounds examined, we identified four, 892, 791, 833 and 191, that strongly suppress HIV-1 gene expression as well as viral structural and regulatory proteins. Subsequent analysis of HIV-1 RNA abundance revealed that the compounds reduced levels of unspliced and singly spliced viral mRNAs, with limited effects on multiply spliced (MS) mRNAs. To explore the discrepancy between HIV-1 MS mRNA levels and the expression of encoded regulatory factors, Rev and Tat, the effect of these compounds on viral protein stability, HIV-1 mRNA localization, and protein synthesis were examined. Results indicate that the compounds do not enhance viral protein turnover and induce little to no alteration of cellular protein synthesis or alternative splicing of >9,000 host RNA splicing events. Subsequent studies have confirmed anti-HIV activity of these compounds in the context of CD4+ T cells. The distinct effects of these compounds from previously characterized HIV-1 RNA processing inhibitors Digoxin, 8-Azaguanine and 5350150, validate targeting this stage of the virus lifecycle as a novel therapy. Elucidating the mechanism by which these four compounds alter HIV-1 MS RNA expression is important for new drug development and holds key implications for novel therapeutic strategies that complement existing HIV-1 treatment options or as a second line of defense to combat drug-resistant strains of HIV.
The discovery of new antiviral drugs is necessary to enhance treatment options and to counter resistance. Here, we examined the mechanism of anti-HIV activity for a novel acylguanidine compound, SM111.
GFP-reporter CEM T cell assays were used to test the ability of SM111 to inhibit replication of WT NL4.3, NL4.3ΔVpu (lacking vpu), and recombinant NL4.3 strains encoding major resistance mutations in pol for RTIs, PIs and INIs. Cytotoxicity was evaluated using Via-Count (Millipore). WT NL4.3 was passaged in the presence of SM111 to select resistant mutants. Vpu-mediated downregulation of CD4 and BST-2/tetherin was monitored by flow cytometry.
SM111 exhibited a nontoxic dose-dependent inhibition of HIV replication; including >95% reduction of infected (GFP+) T cells on day 7 following inoculation with WT as well as RTI, PI and INI resistant strains. Three SM111-resistant viruses were selected in vitro and all encoded mutations in the transmembrane domain of Vpu. A 5AA deletion (strain A), a stop codon at highly conserved W22 (strain C) or a substitution (I17R) (strain H) impaired Vpu-mediated downregulation of CD4 and BST-2/Tetherin. Notably, SM111 was partially active against NL4.3ΔVpu and resistant strains (52%, 92%, 54%, and 16% reduction for NL4.3ΔVpu, strains A, C, and H, respectively).
SM111 inhibited replication of WT as well as RTI, PI and INI resistant HIV-1 strains. SM111 selected major mutations in Vpu; however these mutants and a ΔVpu strain remained partially sensitive to the drug. Together, these results indicate that SM111’s mechanism of action is unique from current antiretroviral drugs and suggest that SM111 targets an interaction between Vpu and an unknown host cell factor. More studies are necessary to explore this promising prototype. Funded by CIHR & the Michael Smith Foundation for Health Research
Host restriction factors APOBEC3F (A3F) and APOBEC3G (A3G) both has potential to inhibit human immunodeficiency virus type 1 (HIV-1) replication. HIV-1 primarily infects human T cells and macrophages, where both A3F and A3G are co-expressed in the absence of Vif. It is widely accepted that antiviral activities of A3F and A3G are predominantly mediated by deoxycytidine deamination, which causes lethal G to A mutations of the viral +DNA. Yet, some reports suggested that these proteins can also use deamination-independent mechanisms to inhibit viral replication, although the influence of this mechanism is debateable. therefore, we wanted to dissect the antiviral mechanisms of A3F and A3G. In single cycle infectivity assay with GFP expressing HIV vif, we found that compared to A3F, A3G is significantly more potent in preventing HIV-1 infection in 293T cell line. A3G inflicted large number of concentrated mutations in viral genome; whereas A3F induced mutations were less and dispersed. More than 95% of A3G induced mutations were lethal to HIV-1. In contrast, only 50% of A3F mediated mutations were able to inactivate virus. Interestingly, we found that in comparison to A3G, A3F could significantly block the reverse transcription of RNA to DNA, potentially by acting as a road-block for reverse transcriptase. These results together suggest that the mechanisms utilized by A3F and A3G to restrict HIV-1 viral replication are different.
Despite effective viral suppression on HAART, latent HIV reservoirs continue to present a major barrier to eradication. We propose a novel strategy to target this reservoir using a class of oncolytic viruses (OV) that include the Maraba (MG1) and Vesicular Stomatitis Virus (VSVΔ51). These recombinant OV target cancer cells by exploiting defects in type I interferon (IFN)-signaling. Similar alterations in IFN-mediated antiviral responses are also seen in HIV-infected cells, providing a crucial link between cancer cells and cells that constitute the HIV reservoir. We hypothesize that MG1 and VSVΔ51 selectively target and kill latently HIV-infected cells.
Latently HIV-infected myeloid (U1 and OM10.1), as well as their respective parental uninfected controls (U937 and HL60) were infected with GFP-expressing MG1 or VSVΔ51. Productive OV infection was quantified by flow cytometry. Viability was assessed by PI, MTT, and Alamar Blue assays. Type I IFN response to OV infection was characterized by measuring IFNα secretion by ELISA and PKR expression by Western blot.
The latently HIV-infected myeloid cell lines U1 and OM10.1 were significantly more susceptible to MG1 and VSVΔ51 infection and viral cytopathic effects than the HIV-uninfected controls in both a dose- and time-dependent manner. IFNα secretion in response to OV infection increased significantly in U937 and HL60 controls compared to the latently HIV-infected cells. In addition, PKR expression under basal conditions and in response to OV infection was significantly higher in U937 than in U1 cells.
Latently HIV-infected myeloid cell lines are preferentially targeted and killed by MG1 and VSVΔ51 when compared to their un-infected parent cells. In parallel, underlying defects in type I IFN response are present in latently HIV-infected cells, which may facilitate selective targeting by OV. Therefore, our results suggest that the use of OV may represent a novel approach to selective elimination of the HIV reservoir.
The past decade has witnessed the feminization of the HIV epidemic globally. Gender and structural inequities that result in vulnerability to HIV infection among women may also result in vulnerability to poor HIV-related health outcomes among women living with HIV, including higher prevalence of suboptimal antiretroviral therapy (ART) adherence. We undertook this study to examine gender differences in achieving optimal adherence with long-term ART use, while controlling for established confounders such as ethnicity and injection drug use.
The study sample consisted of HIV-positive adults (≥18) in British Columbia (BC) enrolled in the HAART Observation Medical Evaluation and Research (HOMER) cohort, with data collection between 2000 and 2011. Optimal ART adherence was defined as ≥95%, based on pharmacy refill compliance per six-month period from initiation of therapy onwards. Bivariate analyses and generalized linear mixed models with logistic regression were used to examine the role of gender in ART adherence. Sub-analyses compared men and women by injection drug use status and ethnicity.
Among the sample of 3,612 individuals followed for an average of 54 months, 718 (19%) of which were women, a significantly lower proportion of women achieved optimal ART adherence, within the entire sample (54.3% versus 76.3%; p<0.001) and per subgroup. In multivariate models, women maintained an association with suboptimal adherence, within the total study population (adjusted odds ratio [AOR]: 0.53; 95% confidence interval [CI]: 0.43 to 0.66) and among subgroups, including Aboriginal people who use injection drugs (AOR: 0.56; 95% CI: 0.39 to 0.80) and non-Aboriginal people who do not inject drugs (AOR: 0.41; 95% CI: 0.27 to 0.46).
Women living with HIV in BC, Canada, are at greater vulnerability to suboptimal ART adherence than men, overall and within subgroups. Emphasis should be placed on providing better access to women-centered HIV care and support services.
Following long-term antiretroviral therapy (ART) CD4/CD8 ratio is associated with AIDS and non-AIDS events, vaccine response, CD4 recovery, inflammation, immune activation and HIV reservoir size. However factors associated with normalization of CD4/CD8 ratio during long-term ART have not been elucidated.
A cross-sectional study was performed on a database of HIV-1 infected patients attending the McGill University Healthcare Centre (MUHC). Adult patients with undetectable viral load (<50 copies/mL) on ART for over 12 months were included. Contribution of age, gender, ethnicity, HIV clades, last available CD4 and CD8 T cell count and percentage was analysed according to CD4/CD8 ratio.
A total of 893 subjects were analyzed and displayed the following characteristics: mean age 49 (18–84), 69.3% male, 65.3% Caucasian, 8.8% Hispanic, 22.2% Black/African and 15.6% Black/Caribbean. The CD4+ T cell counts were 620±289 for males versus 656±267 cells/mm3 for females. The CD8+ T cell counts were 917±456 for males versus 793±383 cells/mm3 for females. A majority of patients (64.9%) reached optimal CD4 recovery (>500 cells/mm3, n=589) contrasting with only 28.7% (n=256) who normalized their CD4/CD8 ratio (>1.0). In total, 21.6% patients (n=210) continued to display a low CD4/CD8 ratio (<0.5). Patients with older than 60 had a lower CD4/CD8 ratio than those younger (p<0.001), and females had a significant higher CD4/CD8 ratio than males (0.94±0.45 versus 0.84±0.45, p<0.0001). Duration of ART therapy was similar no regardless of gender or age. No association between ethnicity, HIV clades and CD4/CD8 normalization was observed.
We report that normalization of CD4/CD8 ratio was infrequent and both gender and aging have impact on immune recovery in successfully treated patients. These study findings are pointing out the new challenge that consists of the understanding of factors associated with persistent CD8 T cell count in long-term treated patients.
The number of heterosexual men living with HIV has increased over time, but they remain an under-studied population. We report sociodemographic and clinical characteristics of heterosexual men living with HIV in Ontario.
We analyzed data from the OHTN Cohort Study, a cohort of people receiving HIV care in Ontario. Data were obtained from medical charts, interviews, and laboratory record linkage. Analysis was restricted to 552 self-identified heterosexual men interviewed in 2010, 2011, or 2012. Descriptive statistics, chi-square tests, and ANOVA were used to compare characteristics of heterosexual men with 2,023 gay and 171 bisexual men, and for within-group comparisons by injection drug use (IDU) history.
38.5% (n=205) of heterosexual men were born outside Canada (bisexual: n=54, 34.8%; gay: n=425, 22.4%; p<0.0001), and heterosexual men were more likely to be African/Caribbean/Black (n=146, 26.4%; bisexual: n=17, 9.9%; gay: n=124, 6.1%) or Aboriginal (n=75, 13.6%; bisexual: n=15, 8.8%; gay: n=148, 7.3%; p<0.0001). IDU history was most common among heterosexual men (n=175, 31.7%; bisexual: n=27, 15.8%; gay: n=215, 10.6%; p<0.0001). Very late diagnosis (CD4 count <200 cells/mm3 at diagnosis and/or AIDS-defining condition) was more frequent among heterosexuals (n=220, 40.1%) relative to gay (n=533, 26.6%) or bisexual men (n=49, 28.8%, p<0.0001). The proportion on antiretrovirals was similar across groups (∼95%), yet at last follow-up, heterosexual men had significantly lower CD4 counts (mean=501.5 cells/mm^3 cf gay men: 548.7 cells/mm3; p=0.001) and were more likely to have detectable viremia (n=78, 15.1% cf gay men: n=180, 9.5%; p<0.0001). Heterosexual men were also more likely than gay or bisexual men to have Hepatitis C, regardless of IDU history (p<0.0001).
Very late diagnosis, hepatitis C co-infection and suboptimal virologic suppression were more common among heterosexual men. Further exploration is needed to address heterosexual men’s unique challenges with timely HIV diagnosis and successful antiretroviral treatment outcomes.
Among women living with HIV (WLHIV) in Canada, we assessed whether health-related quality-of-life (QoL) differs by receipt of perceived women-centred HIV care (WCC).
Baseline survey data were analyzed for WLHIV (≥16 years) who received care in the last year, enrolled in the Canadian HIV Women’s Sexual and Reproductive Health Cohort Study (CHIWOS), a longitudinal, community-based study in British Columbia (BC), Ontario (ON), and Quebec (QC). Physical and mental health QoL was assessed by the SF-12 (scored from 0–100; higher scores indicating better QoL), and WCC by measuring perception of women-centredness of care received from their HIV clinic and HIV doctor in the last year. Multivariable linear regression analyses examined the relationship between perceived WCC and QoL, adjusting for confounders.
Of 981 participants, 27% were from BC, 50% ON, and 22% QC. Median age was 44 (IQR=36–51) and 38% identified as Caucasian, 29% as African, Caribbean, or Black, and 22% as Aboriginal. 53% and 57% perceived their HIV clinic and doctor’s care (respectively) to be women-centred. Overall, mean physical and mental health QoL scores were 43.9 (SD=14.4) and 41.6 (SD=13.5). Physical health scores were higher among women from ON [45.4 (SD=13.1)] and QC [45.5 (SD=14.7)] compared with BC [39.9 (SD=15.5)] (p<0.001); no variation was observed in mental health scores. Women were more likely to have lower physical and mental health scores if they were older, separated/divorced or widowed, and had an annual household income <$20,000. In adjusted analyses, receipt of perceived WCC from an HIV clinic [β=2.89 (95% CI:1.17–4.62); p=0.001] and HIV doctor [β=2.57 (95% CI:0.84–4.30); p=0.004] were associated with higher mental health QoL scores.
For WLHIV in this study, mental and physical QoL was significantly lower than estimates for the general population of Canadian women and other HIV populations. Receipt of perceived WCC was associated with higher mental health QoL. Through CHIWOS, we will investigate the potential of WCC to reduce known gendered inequities in health outcomes and improve health-related QoL for WLHIV in Canada.
To describe the number and type of comorbidities among adults living with HIV in Canada and compare younger to older adults.
We conducted a cross-sectional web-based online survey from October 2013–August 2014. We recruited adults living with HIV in Canada in collaboration with 27 knowledge user, community-based organizations, and clinics across Canada using electronic (emails, newsletters, websites, video) and on-site (posters, cards) strategies supplemented with snowball sampling. The survey included sections on external factors (stigma, social support), personal factors (mastery, coping, living strategies, demographic and disease characteristics), and an item asking whether participants were living with concurrent health conditions. We compared the median number and type of comorbidities for younger (<50 years) versus older (≥50 years) participants using chi-square and Mann-Whitney tests for nominal and continuous variables, respectively (p<0.05).
Of the 1850 individuals who accessed the survey, 1477 (80%) initiated and 1171 (79%) completed the survey, resulting in 941 complete and estimated valid responses. Of the 932 participants who provided age-related information (527 <50 years; 405 >50 years), the majority were men (79%), taking antiretroviral therapy (90%) and living with >2 concurrent health conditions (72%). Older participants had a higher median number of concurrent health conditions compared with younger participants (4 versus 2; p<0.001). Common comorbidities included mental health conditions (42% of sample), muscle pain (33%), joint pain (30%), high cholesterol (28%), addiction (26%), and neurocognitive decline (22%). A greater proportion of older participants reported living with joint pain, muscle pain, elevated triglycerides, high blood pressure, high cholesterol, bone and joint disorders, cardiovascular disease, neurocognitive decline, mental health condition, COPD, neuropathy, frailty, and liver disease (p<0.05). Compared with younger participants, older participants had an earlier mean year of diagnosis, were working less for pay, and had lower maladaptive coping and lower stigma scores (p<0.01).
In this sample, older adults with HIV reported living with a greater number of comorbidities compared with younger adults. The relationship between comorbidities and extrinsic and intrinsic contextual factors among adults with HIV needs further study.
Adult survivorship of childhood abuse (CA) is increasingly being recognized as been associated with deleterious psychosocial and health outcomes. The epidemiology and clinical associations of CA within the HIV community is not clear.
All patients attending a medical appointment for HIV in Southern Alberta between May 2009 and August 2014 were screened for domestic violence, including specifically for CA (abuse in the home before 16 years of age). Patients were offered same-day social work consultation and referral to community or in-house services as required. Clinical data was obtained through usual care and extracted from a database. Poisson regression adjusted for age, gender, sexual orientation, self-reported ethnicity, time since HIV diagnosis, and HIV risk factor.
320 (19.4%) of 1,653 patients screened disclosed a history of CA. Prevalence varied by ethnicity: Aboriginal (50.8%, n=124), white (22.1%, n=993), other (13.5%, n=148), black (4.2%, n=379), P<0.001. Gay/bisexual vs. heterosexual (adjusted odds ratio=2.5, 1.1–5.7) and female vs. male (1.7, 1.1–2.5) predicted a history of CA. Depression (2.3, 1.7–3.1), anxiety disorders (2.2, 1.6–3.2), previous psychiatry consultation (1.7, 1.2–2.5), and suicide attempts (1.8, 1.1–2.9) were more frequent among CA survivors. CA survivors were more likely to report intimate partner violence as an adult (4.9, 3.6–6.5), insecure housing (2.2, 1.3–3.8), and smoking (1.7, 1.2–2.4). Health-related quality of life was also lower: AOR=2.2 (1.5–3.3) for poor/fair vs. good/excellent.
HIV-related hospitalizations after HIV diagnosis were more frequent among CA survivors (1.4, 1.1–1.9). This is despite the fact that survivors of childhood abuse were less likely to have had a delayed HIV diagnosis, with a CD4 count <200 cells/mm3 (0.6, 0.4–0.9).
A history of CA is common among HIV-positive adult patients and is associated with poor social, psychiatric, and HIV outcomes. Efforts to address the sequelae of CA could lead to improved wellbeing in this population.
In Quebec, the proportion of new HIV diagnostics amongst 0–35 years old youth has continuously increased, from 33% (75/230) in 2002 to 45% (160/358) in 2013. For young teens, HIV may seem a distant problematic. As they get into their 20’s, the number of infected peers dramatically increases. Goal is to document new diagnosis numbers by age (20–35 years old) and inform new HIV prevention strategies towards youth.
Distributions of HIV data from surveillance program in Quebec were calculated based on new HIV diagnosis absolute numbers stratified per age and sex. Distributions were smoothed using average number of diagnostics over the last twelve years.
The average of new diagnostic numbers are shaped as geometric progression for both sexes from 20 to 30 years old. For males and females respectively, average new diagnostics per year are 0.17 and 0.17 diagnostics at 15 years old; 2.25 and 1.08 at 20 years old; 9.08 and 3.17 at 25; 11.00 and 4.08 at 30. After 30 years of age, the average number of new diagnosis remains stable from 31 to 35 years old (13.48 for males, 4.48 for females in average).
Teenage years are important for socialization and to shape adult life style, sex and drug practices. Developing and keeping safe practices need to be integrated while they explore new sexual practices. Although prevention tools have been developed to reach youth and respond to prevention needs regarding HIV infection, these tools may not be fully adapted to the realities and needs of young adults before and as they initiate their sexual life.
The acceleration of new cases between 20 and 30 years old suggests revisiting actual preventive actions regarding youth realities including their structural access to good sexual health conditions (information, condoms, physicians and professional advice being some of them).
Adolescents in sub-Saharan Africa represent an identified group with heightened vulnerabilities to HIV/AIDS, particularly in fragile states. Recovering from civil war, Sierra Leoneans have endured egregious human rights abuses, family separation, and politico-economic instability – all factors known to influence sexual risk. This analysis sought to examine the role of individual and social-structural factors on sexual risk among adolescents living in Sierra Leone.
Following extensive community-based collaborations established during an ethnographic research phase, adolescents were recruited through household surveys from 12 villages within the Moyamba and Bombali Districts of Sierra Leone. Generalized estimating equations (GEE) were used to examine factors independently associated with condom use at last sex while controlling for within village clustering using Adjusted Odds Ratios (AOR) and 95% confidence intervals.
From a total of 530 adolescents, 212 (40.0%) were sexually active and comprised the analytic sample. The median age was 18 years (interquartile range 17–18), 90 (42.45%) attended school in the last 12-months, 78 (36.79) reported living without a biological parent, 85 (40.10%) were single/double orphans. In the last 12-months, 80 (37.74%) had a pregnancy and 29 (13.68%) had engaged in transactional sex. Only 30 (14.15%) used a condom at last sex. In GEE analyses, living without a biological parent (AOR: 0.37, 95% CI: 0.13–1.08) and recent pregnancy (AOR: 0.16, 95% CI: 0.04–0.55) were negatively associated with condom use. High dwelling quality was positively associated with condom use (AOR: 2.71, 95% CI: 1.28–5.75).
This study provides data on the prevalence of adolescent sexual risk and the potential role of housing and family connectedness as structural factors influencing adolescent sexual health in Sierra Leone. The results speak to the need for on-going empirical investigations to inform the development of evidence-based programming to mitigate sexual risk and support families, households, and societies to ensure the well being of their adolescents.
HIV/HCV infections are a persistent concern among youth in the Atlantic Provinces. Despite a strong body of literature on the topic of youth engagement, youth are still often left out of HIV/HCV policy and program development. Understanding how programmers and policymakers can increase collaboration with youth was a major driving force behind this project.
Our Youth, Our Response was a three-year study that explored HIV/HCV prevention in the Atlantic Provinces, with the goal of developing evidence-based recommendations for policies and programs serving youth aged 15–24. Year 1 consisted of a policy scan of HIV/HCV and youth-related documents; year 2 included in-depth interviews with key informants from health, education, community, and corrections; and year 3 utilized focus group discussions with youth and youth-serving organizations. Using a thematic analysis approach, a multidisciplinary team analyzed the study transcripts.
Consultation and collaboration with young people to determine what HIV/HCV prevention approaches they need, how they wish to be engaged, and what formats will capture their interests must be a priority for all organizations providing youth services. Nevertheless, youth engagement remains low in the region. Organizations face difficulties recruiting and retaining youth, particularly youth from LGBTQ populations, youth from Indigenous communities, street-involved youth, and young people who use drugs. Potential strategies identified by participants for promoting youth engagement include: the provision of dedicated funding to support youth partnerships, capacity building with organizations and youth to support collaboration, and establishing a standing commitment to solicit youth input. Programs should focus on providing age-appropriate and youth-friendly outlets for inclusion, such as arts-based projects, peer mentorships, and social media or internet-based programs.
Increasing youth engagement in the development and implementation of HIV/HCV prevention policies and programs can increase their reach and uptake leading to services that better reflect the needs and preferences of youth.
Sexual and gender minority youth (SGMY) are vulnerable to HIV because of mental and sexual health risks. Yet, there is a lack of evidence-informed interventions that are grounded in contemporary service delivery. This study examined the preliminary effectiveness of a cognitive-behavioral affirmative coping skills training (AFFIRM) to increase sexual self-efficacy and reduce depression among SGMY in Toronto.
An open pilot study using a pre-post design was used to determine preliminary feasibility and acceptability. Following purposive and venue sampling, a pilot implementation of the 16 hour AFFIRM group intervention (August 2014) was delivered in a two day workshop format. SMGY completed reliable measures of health risk sexual behaviors, depression, sexual self-efficacy and coping at three time points. Repeated measures linear mixed modeling was used for analysis.
SGMY (n=30) between the ages of 15 – 18 completed AFFIRM. Participant identities (all non-mutually exclusive) included: sexual orientation (pansexual 29%; lesbian 25%; queer 21%; bisexual 18%; gay 11%; questioning 11%); gender identity (female 57%; non-binary 21%; male 18%; trans* 7%; two-spirit 4%); and racial/ethnic identity (Caucasian 59%; Asian 29%; Black 25%; Aboriginal 18%; Mixed 13%; Latino 7%). Seventy percent reported one immigrant parent. Risks included anxiety (79%); substance use (62%); homelessness (44%) and suicide attempts (31%).
AFFIRM participants experienced a significant reduction in depression scores on the Becks Depression Inventory-II (B=5.21; P<0.01). Significant improvements in sexual self-efficacy (B=1.56; P<0.05) and reflective coping (B=1.01; P<0.05) were found. Changes in knowledge or attitudes were not indicated.
This study is the first to demonstrate the potential of an affirmative intervention to address sexual self-efficacy and depression for a community sample of SGMY. These results indicate the utility of a community-engaged, inclusive model of HIV prevention for SGMY and warrant a larger study to determine AFFIRM’s effectiveness.
Despite the epidemiological and clinical rationale to expanding HIV testing, some have expressed concern that public health practices to enhance testing uptake may not sufficiently align with the values and principles from bioethics that emphasize patient autonomy and the limits of State paternalism. Alternatively, emerging theoretical work has argued that public health interventions ought to be subject to normative inquiry that considers ‘public’ values, including relational concepts such as solidarity, reciprocity and health equity. As yet, however, the extent to which ‘public’ values influence the ‘autonomous’ decisions of the public remains largely unexplored.
Drawing on qualitative interviews with 50 young men (ages 18–24) in Vancouver, Canada, this study employs a critical discourse analysis to examine participants’ decisions and motivations to voluntarily access HIV testing and/or to accept a routine HIV test offer.
The findings reveal that, while some young men position the individual-level act of testing as constituting relational and ‘public’ value, others choose to test for predominately individually-oriented reasons. Within a sub-set of interviews, a transactional discourse emerged in which the decision to test features an arrangement of ‘giving and receiving’ – between men and their clinicians and/or their sex partner(s). Another set of discourses related to notions of collective solidarity emphasize considerations of justice and positions testing as a ‘public’ act – an act that does not feature direct obligations but remains deeply ‘social’ and is implicated within collective efforts to advance justice (e.g., eliminate HIV transmission). Lastly, ‘individualistic’ discourses also emerged, focusing on individual-level considerations, with less concern for the broader public ‘good’.
These findings underscore how normative dimensions pertaining to men’s HIV testing practices are dialectically interrelated with the broader social and structural influences on individual and collective HIV-related behaviour, thereby suggesting a need to advance an explicit empirical-normative research agenda related to research regarding HIV-related health behaviour.
Young adults are at significant risk for sexually transmissible and blood-borne infections (STBBI) and may not perceive their need to seek for STBBI testing services or may face barriers to access those services. This study aims to describe key barriers to STBBI testing specific to sex among young adults.
PIXEL is a cross-sectional and descriptive study targeting 17–25 years old. It combines observations from self-administered questionnaires and biological samples. Between 2013 and 2014, participants were recruited according to a stratified multistage sample plan based on 3 geographical entities (Montreal, Quebec and periphery regions) and a variety of school settings (ex. vocational schools, colleges, universities). Using Levesque & al. (2013) Access to health care framework, barriers to STBBI testing were explored with Chi-square tests.
In the subgroup of 17 to 20 years old, 781 sexually active women and 508 sexually active men answered PIXEL questionnaire. Proportion of those participants who reported having sought for STBBI testing in the past 12 months was significantly lower among men than women (men: 14.5%; women: 33.5%; p<0.00). Among those participants, the most frequent barriers related to their ability to seek and reach for STBBI testing services were: “Not having enough time” (men: 40%; women: 27.5%; p <0.05), “Being scared of the results” (men: 38.8%; women: 40.9%; p=0.76), “Not knowing where to go” (men: 32.4%; women: 12.7%; p <0.00), “Having difficulties to get an appointment” (men: 25.0% women: 29.6%; p=0.47), “Being afraid people would find out” (men: 25.4%; women: 16.3%; p=0.08) and “Feeling uncomfortable having to talk with a nurse/doctor about sexuality” (men: 25.0%; women: 21.1%; p=0.49).
Results illustrate certain disparities between 17 to 20 years old men and women access to STBBI testing services. Through the Access to health care framework (Levesque & al., 2013), these findings provide guidance for prevention programs.
For People living with HIV/AIDS (PHAs) who experience instability related to social determinants of health, time in hospital can be a respite from the chaos of their lives. The structure of hospital can provide escape from precarious housing, chaotic substance use, lack of social support, stigma, and poverty. The transition period following a hospital discharge can be a high-stress time which is not often discussed by hospital staff nor well captured in research studies.
A qualitative case study approach was utilized to explore factors and multiple perspectives that impact the hospital discharge transition period for PHAs. Semi-structured interviews with nine clients being discharged from in-patient care at Casey House (a sub-acute care HIV hospital) at four timepoints (approximately one week pre-discharge to one month post-discharge) were conducted and data were abstracted from hospital charts and discharge plans. Data were analyzed using qualitative thematic analysis across time and cases to capture the lived experience at each timepoint.
The level of complexity of a client’s life affected the transition from hospital to community. Although the client and care team named HIV-related health priorities in the discharge plan, often these were not the priorities of the client post-discharge. Clients, upon discharge, often returned to chaotic living situations that promoted the poor health outcomes which had led to their recent hospitalization: a cycle demonstrated by the lived experience of all participants in this study.
The lived experience of PHAs during the transition period following hospitalization demonstrated that attempts to prioritize medical concerns, such as adherence, became secondary to client priorities related to daily living needs and other social determinants of health. The complexity and instability of the person’s life should be incorporated into hospital admission goals and discharge plan, thereby mitigating challenges which adversely impact their health and care needs.
As people living with HIV live longer lives, the healthcare they require is changing. The health system in Canada was originally organized around specialists physicians providing HIV medical care, but it must adapt to treating HIV as a complex chronic condition that requires co-management and integration across a variety of care providers. Shifting treatment protocols can have profound but unforeseen consequences on the experience of illness, as well as other aspects of life for people living with HIV. As part of a five-year, multi-centre, multi-provincial program of linked research-to-action projects, we are conducting an institutional ethnography of healthcare for people living with HIV at clinics in Newfoundland and Labrador (NL), Manitoba and Ontario. Our institutional ethnography involves interviews with people living with HIV and healthcare providers, observation of clinical encounters and text analysis of medical records. This presentation will describe our study design and present preliminary findings from our first site in NL. The organization of HIV healthcare in NL must take into account the geographic dispersion of people living with HIV, issues related to patient confidentiality and stigma, and the psychosocial needs of the diversity of people living with HIV in the province within a national context of heightened criminalization. Ethical issues that arise in providing healthcare to people living with HIV in this province will be explored.
Findings will provide new insights into the ethical concerns raised in the changing domain of HIV healthcare from the perspectives of providers and people living with HIV. As a follow-up to this project we intend to develop a toolkit for providers to help navigate ethical issues arising in HIV practice.
It is estimated that 1 in 8 Canadian households experience food insecurity. People living with HIV face greater challenges with food security due to lower household incomes, poorer health outcomes and experiences of stigma and discrimination. The primary purpose of this study was to understand the existence of food insecurity among Ontarians living with HIV and the impact this has on their daily lives.
Participants were recruited from community-based agencies throughout Ontario. Questionnaires were administered by Peer Research Associates. Food security was assessed using Canada’s Household Food Security Survey Module. Sociodemographic data and information on substance use, depressive symptoms and measures of physical and mental health-related quality of life were collected. Descriptive statistics were obtained for variables of interest. Univariate regression analyses were conducted to identify variables associated with food insecurity. Variables significant (p<0.10) in univariate analyses were incorporated into multivariate linear regression models.
649 people living with HIV were recruited. Mean age was 45 years. Two-thirds of participants were men. Half were heterosexual. Sixty-nine percent experienced food insecurity. Non-Caucasian ethnicity (AOR: 1.76, 95% CI: 1.11–2.81), living outside the GTA (AOR: 1.62, 95% CI: 1.03–2.54), unemployment (AOR: 2.18, 95% CI: 1.27–3.76), household income less than $20,000 (AOR: 2.11, 95% CI: 1.29–3.46), depression (AOR: 2.24, 95% CI: 1.36–3.60), substance use (AOR: 2.14, 95% CI: 1.30–3.51), injection drug use (AOR: 2.19, 95% CI: 1.30–3.70) and higher levels of stigma (AOR: 1.04, 95% CI: 1.02–1.06) were associated with food insecurity.
Compared to the general Canadian population, people living with HIV are significantly more likely to experience food insecurity. Our findings highlight the importance of developing holistic programs that address the underlying causes of food insecurity while building stronger, broader community partnerships to prevent food insecurity among people living with HIV.
During adolescence and young adulthood, youth with perinatally acquired HIV (PAHIV) face the question of disclosing their HIV status to significant others, such as peers and romantic partners. The objective of this study is to explore the management of HIV disclosure among youth with PAHIV.
Semi-structured interviews were performed with 18 youths with PAHIV (aged 13–22) recruited through the CHU Sainte-Justine (Montreal), using a qualitative typological analysis.
Three types of disclosure were identified: 1) Public disclosure where the vast majority of family, friends and community members are aware of the youth’s status. These youth feel well supported, perceive less stigmatization and do not feel like they are holding a secret. 2) Conditional disclosure where youth set conditions, such as trust or being in a serious relationships before disclosing their HIV status. Youth carefully select the people to whom they disclose, and disclosure is a multi-stage process. 3) Inconceivable disclosure where youth are overwhelmed with fear of stigmatization and cannot imagine disclosing their status to anyone. Fear of rejection and discrimination are dominant. These youth set unrealistic conditions to their disclosure, such as getting married, having children, or being cured. These conditions confine them to secrecy and constant fear of their HIV-status being discovered.
Anticipated and perceived stigma play a crucial role in management of HIV-disclosure. Implications of inconceivable disclosure issues for youth with PAHIV and their romantic and sexual partners should be taken into account for intervention. A peer intervention should be planned to help those who feel overwhelmed by stigma to enhance empowerment and decision-making skills about HIV-disclosure.
Since the beginning of the gay liberation movement in North America, gay identities have been contested as much as they have been employed as a tactic of political resistance. HIV/AIDS has a profound impact on the identities and communities associated with gay men. At the outset of the outbreak, HIV/AIDS created a focal point around which gay communities could rally. Despite its devastating impacts, HIV/AIDS began to shape and render visible the conception of the gay male subject. Today, HIV has been inextricably woven into the subjectivities of gay men.
This paper will discuss the findings of a qualitative research project in Nova Scotia aimed at examining the perspectives of HIV/AIDS activists on complex ways in which the disease influenced how we have come to know and understand gay men. To investigate these issues, 17 HIV/AIDS activists were interviewed about their experiential connection to the HIV/AIDS movement in Nova Scotia.
Employing a Foucauldian theoretical framework and a discourse analysis to these data, this research finds that HIV has given rise to sanctions against some sexual acts, the provision of instructions for ways to act on the risks and responsibilities entailed in sexual relations, and has also become the site of resistance to these very instructions often reinforced by public health HIV prevention rhetoric. The perspectives of HIV/AIDS activists provides insight into how this shaping of the gay male subject enters the decision-making processes for HIV policies and programming, further contributing to the discursive field of gay men’s subjectivities.
Considering the ways in which HIV prevention and stigma messaging has influenced gay men’s subjectivities can have significant implications for future public health and health promotion efforts, particularly in local contexts such as Nova Scotia.
The concept of “episodic disability” (i.e., fluctuations of wellness, illness and disablement) was developed in Canada to describe the experience of PHAs in the era of ART. Framing challenges in terms of “episodic disability” contributed to health care and employment policies and practices in Canada. However, episodic disability has not been explored in Sub-Saharan Africa despite increased access to ART in the region.
To describe experiences of episodic disability among women and men living with HIV in Lusaka, Zambia.
“Sepo II” is a qualitative longitudinal study involving in-depth, semi-structured interviews approximately 6 months apart with participants from public and private clinics in Lusaka, Zambia. Thirty-five participants were purposively recruited for variability by gender (17 men, 18 women), time on treatment (1–13 years), and SES. An interpretive analysis was conducted of the first two waves of interviews using the collaborative “DEPICT” method (Flicker & Nixon, 2014). The study was guided by the WHO “International Classification of Functioning, Disability and Health” (ICF), and O’Brien’s “Episodic Disability Framework”.
Participants did not perceive their health-related experiences as episodic. However, participants readily described fluctuations in their impairments, activity limitations and participant restrictions (i.e., disablement described in WHO ICF) in the time between wave 1 to wave 2 interviews. Four types of changes in disability were described: improvements, complete resolution, worsening, and new onset. Changes were greatest at the level of impairment.
This is the first study to demonstrate the experience of episodic disability among women and men living with HIV in a Sub-Saharan African context. Findings point to shortcomings in the dominant model of HIV care centred on ART, suggesting that services and policies in settings like Zambia also need to address the episodic health- and life-related impacts of living longer with HIV.
Coinfection with hepatitis C virus (HCV) and HIV-1 is common and worsens the prognosis of hepatitis C. HCV exists as quasispecies, and the bulk of its genetic variability is located within hypervariable regions of the E2 envelope glycoprotein. During pregnancy, selective pressures exerted on HCV target solvent-exposed regions of E2, suggesting the involvement of humoral immunity. The objectives of the study were to examine potential associations between HCV quasispecies evolution and maternal immune responses, and to characterize the impact of coinfection on these processes.
Sera from HCV-infected women (n=17) or women coinfected with HCV and HIV-1 (n=20) were collected during the first, second and third trimesters of pregnancy and in the early postpartum period. HCV viral RNA was extracted, amplified by RT-PCR, and sequenced on a Roche 454 GS-FLX next-generation sequencing system. Files were subjected to a k-mer based error correction algorithm prior to calculation of the frequencies of HVR1 amino acids variants. Variant spectra obtained for each time point were used to calculate UniFrac sample distances, Shannon entropy and Hamming distances. Clinical data were collected to identify potential differences between monoinfected and coinfected subjects.
Analysis of UniFrac distances revealed a faster rate of HCV quasispecies evolution in the monoinfected group compared to coinfected subjects (slope of UniFrac distance/time=0.0244 vs 0.0095). HCV viral load decreased following childbirth in monoinfected women but not in coinfected subjects (p=0.0485). Lower Shannon entropy values reflecting simpler composition of the variant spectra were associated with increased risk of mother-to-child HCV transmission in monoinfected women (p=0.0024).
Differential evolution rate and viral load dynamics suggest that maternal immune responses drive HCV evolution during pregnancy and that coinfection with HIV-1 impairs these responses. Low entropy values could reflect the presence of neutralization escape variants and/or high fitness variants that are more likely to be transmitted from the mother to her child.
The genetic diversification of HIV heralds significant challenges for the treatment and management of patients in Canada. Since HIV accumulates genetic changes on a contemporary time scale, phylogenetic tools can be used to reconstruct the recent history of HIV epidemics and to characterize the processes that have shaped the epidemic and the circulating HIV variants. In this study we integrate phylogenetic analyses of HIV sequence data with clinical attribute data from across Canada to identify the processes structuring the nationwide epidemic.
We assembled 39,477 anonymized HIV protease and reverse transcriptase sequences sampled from 15,749 unique patients from 5 provinces (British Columbia, Alberta, Saskatchewan, Ontario, and Quebec) within Canada between 1995 and 2014. Our core dataset consists of sequences sampled from patients in the Canadian Observational Cohort (CANOC) supplemented with data from clinics not currently included in CANOC. Sequences were aligned using MAFFT v.7.54b and visually inspected using AliView v.1.15. Codons associated with known drug resistance mutations were censored from the alignment prior to tree inference. We inferred a distribution of 1000 phylogenetic trees using FastTree2 under a general time reversible model of molecular evolution. We rooted the resulting phylogenetic trees under a molecular clock using a modified version of Path-O-Gen; node heights were re-estimated using a non-parametric rate smoothing method.
We used these time-scaled phylogenies reconstructed from the sequence data to quantify HIV incidence and prevalence in the Canadian epidemic with phylogenetic estimators. Results revealed expected patterns of within province diversification along local transmission networks, but also a surprising amount of interprovincial mixing. We observed substantial variation among provinces in the proportion of non-subtype B infections (P<0.05).
Overall, our analyses reveal temporal changes in the processes shaping the Canadian HIV epidemic that highlight the impacts of past public health and clinical interventions. Our study represents the first integrated, comprehensive nationwide evaluation of the HIV epidemic in Canada.
The protective HLA-B*13 allele selects escape mutations across HIV-1, but their effects on viral replication capacity (RC) and protein functions remain incompletely understood. We evaluated the impact of 10 described HLA-B*13 escape mutations located in Gag, Pol and Nef on viral RC. We also assessed the impact of Nef mutations on CD4 and HLA class I-downregulation, and the latter’s consequence for recognition of virus-infected cells by epitope-specific T-cells.
HLA-B*13 escape mutations in Gag (A146S, I147L, K436R, I437L), Pol (Protease-L63S; RT-Q334N, T369A, K374R) and Nef (E24Q, Q107R) were engineered alone and in biologically relevant combinations into HIV-1NL4-3. Viral RC was determined using a GFP-reporter T-cell assay. Nef-mediated CD4 and HLA-A*02 downregulation was measured by flow cytometry; recognition of infected-target cells by HIV-1-specific effector cells was assessed via co-culture with an NFAT-driven luciferase reporter T-cell line specific for the A*02-restricted Gag-FK10 peptide.
Of all mutations tested, only Gag I437L showed 14% reduced RC, alone and in combination with A146S and I147L. This defect was rescued to wild-type levels by K436R. A putative novel B*13 epitope was identified in p24Gag (GQMVHQAI, 140–147). Single Nef mutations exhibited no effect on CD4 or HLA-A*02-downregulation; however, the double mutant was impaired for the latter function [NL4-3, E24Q, Q107R (100%) > E24Q/Q107R (31%) > M20ANefNL4-3 (8%)]. Correspondingly, luciferase signal emitted by HIV-1-specific effector cells upon co-culture with HIV-1E24Q/Q107R infected-target cells was 2-fold higher than for cells infected with NL4-3 or single Nef mutants.
A minority of HLA-B*13-driven escape mutations modestly dampen HIV-1 RC or Nef function, which could contribute in part to B*13-associated protection from disease progression. The observation that a naturally-occurring (albeit rare; 4%) Nef double mutation impaired HLA downregulation and enhanced recognition of infected cells by HIV-1-specific T-cells suggests a novel escape-associated defect that ironically dampens a key viral immune evasion strategy.
HLA-driven HIV-1 immune escape mutations that persist following transmission could gradually spread in the viral population, compromising host antiviral immunity over time. We investigate the extent and correlates of escape mutation accumulation in HIV-1 Polymerase (Pol) in North America from 1979-present.
HIV-1 Pol and HLA class I genotyping was performed on 338 Historic (1979–1989) and 278 Modern (2001– 2011) specimens from Boston, New York, San Francisco and Vancouver. Ancestral reconstruction of the HIV-1 epidemic founder sequence was performed using BEAST and HyPhy. HLA-associated polymorphisms were defined according to published lists.
The estimated HIV-1 epidemic founder sequence dated to ∼1969 and was near-identical to the modern subtype B consensus, suggesting no selective sweeps have occurred since this time. However, pairwise sequence diversity of modern HIV-1 sequences was ∼twofold greater than historic sequences, with diversification predominating at known HLA-associated sites (p<0.0002). N=20 published HLA-associated escape mutations were investigated for potential spread over time. Overall, their median frequencies in individuals not expressing the restricting HLA were 6.6% vs 16.8% in the historic and modern eras respectively (p=0.0004). Escape mutation frequencies in reconstructed pre-1979 ancestral sequences were also consistent with gradual spread (p<0.01). Escape mutations restricted by protective HLA alleles appear to have increased to the greatest relative extent over time (p<0.05). Despite these increases, many escape mutations (eg: B*51-associated RT-I135T) remained consistent in frequency throughout the eras. Moreover, at the whole-sequence level, the median extent of adaptation of the typical circulating HIV-1 Pol sequence to the average North American host remains 0%, indicating the risk of acquiring HIV-1 immune escape mutations at transmission remains low.
Immune escape mutations in HIV-1 Pol have spread significantly in the population since the genesis of the North American epidemic, however these changes are unlikely to herald immediate consequences for host immunity on this continent.
During primary mucosal HIV-1 infection, only one or a limited number of infectious HIV-1 clones are transmitted from the donor to the recipient. Only little is known about the role phenotypic properties of the transmitted virus play in the genetic bottleneck selection process. Therefore we analyzed possible genotypic and phenotypic differences that may influence transmission between early and chronic HIV-1.
Using deep sequencing we compared the genetic diversity of HIV-1 isolated from the female genital tract to viruses isolated from blood. We further constructed chimeric viruses from envelop genes of early and chronic isolates and compared their pathogenic and transmission fitness measured by competitions in PBMCs, T cells and macrophages or in ex vivo human mucosal explant tissue respectively. We further evaluated mechanisms such as receptor affinity, entry efficiency or envelope glycosylation that might control pathogenic and transmission fitness.
Sequence analysis revealed that early HIV-1 isolates in the female genital tract had high genetic diversity while isolates from matched blood were genetically homogenous. We found that early and chronic isolates showed similar entry kinetics and replication fitness in PBMCs, T cells and macrophages. In contrast we observed that early virus clones penetrated human tissue and subsequently established infection of T cells more efficient than chronic HIV-1, which resided and replicated mainly in the tissue. Furthermore early HIV-1 isolates from the female genital tract had a higher number of N-linked glycans compared to virions from the blood.
The majority of heterosexually transmitted HIV-1 appears trapped in mucosal tissue, possibly due to high levels of soluble mannose binding lectins in tissue and lectins expressed on epithelial cells. As a result HIV-1 with fewer N-linked glycans is passively selected for transmission across mucosal tissues due to reduced lectin binding.
In Kenya, HIV incidence among female sex worker (FSWs) is five time higher than the general population. However, despite being at high risk of HIV infection a small group of FSW remain uninfected. Over the years, we have observed that HIV exposed seronegative FSW have a unique immune phenotype called Immune Quiescence (IQ). In this model, decreased levels of baseline T cell activation limit the number of target cells and prevent HIV infection. Interestingly, CAPRISA 004 showed that participants with prior immune activation had a higher likelihood of becoming HIV infected. We hypothesised that hydroxychloroquine (HCQ) and/or aspirin can mimic the IQ phenotype and help preventing HIV infection.
Non-FSW HIV uninfected women (n=105) from Kenya were enrolled and followed for three months. At month 1, systemic/mucosal baseline immune activation was assessed by flow cytometry. Participants were randomized into two arms (HCQ 200 mg/day or aspirin 81 mg/day) and followed for a 2 months period to assess changes in T cell immune activation (systemic and mucosal).
Preliminary data are indicating that both HCQ and aspirin can reduce T cell activation. A significant decrease of CD95 and CCR5 expression on CD4+ T cells (p<0.0001 and p=0.01 respectively) in the HCQ arm was observed in the blood and a reduction of the percentage of CD4+C69+ (p=0.01) and CD4+CCR5+ (p=0.03) T cells was observed in the aspirin arm. Analysis of the mucosal samples is currently ongoing.
The ability to modulate mucosal lymphocyte activation could represent a new avenue to prevent HIV infection.
Clinical and epidemiological studies have shown correlation between hormonal status of women and their susceptibility to HIV, but this has been difficult to test experimentally. Humanized mice (Hu-mice), a human-mouse chimera, where an immunocompromised mouse is reconstituted with a human immune system, has become an important pre-clinical tool for HIV studies. We have developed a NOD-RAG2 −/− gamma chain −/− Hu-mouse to study the hormonal conditions in which intravaginal (IVAG) HIV infection can occur successfully. Hu-mice were infected IVAG at different stages of the reproductive cycle, or following DMPA (a hormonal contraceptive) injection, with 10^7 infectious units/ml of NL4.3 Bal-Env virus and compared to mice infected intraperitoneally (IP) at the same dose. Mice infected IVAG during the diestrus (progesterone-high) stage and IP had high plasma viral loads (6.3×104 ± 2.6×104 RNA copies/mL) three weeks post-infection, while mice infected during the estrus (estradiol high) stage had no detectable plasma viral load. P24-positive cells and a noticeable decrease in CD3+ T cells was detected by immunohistochemistry in vaginal tracts of mice successfully infected with HIV. Time course experiments showed that there was a rapid replication of virus locally in the vaginal tract which peaked 1 week post IVAG infection, followed by a steady increase in plasma viral loads 3 and 5 weeks post-infection. DMPA treated mice infected IVAG showed a significant increase in plasma viral loads from 482.9 ± 943.8 RNA copies/mL at 1 week post-infection to 1.6×105±4.07×104 HIV RNA copies/mL 5 weeks post-infection (p-value = 0.0017), indicating that DMPA treatment may lead to increased viral replication and higher viral set-point. Overall, these studies show that the hormonal conditions may play a critical role in determining susceptibility to IVAG HIV infection and viral replication. The Hu-mouse model will be a useful tool to better understand HIV pathogenesis following sexual transmission.
FREM1 was a novel candidate gene in resistance to HIV-1 infection in the Pumwani sex worker cohort. Studies have shown that it can modulate TLR and IL-1R1 regulated inflammatory responses. However, the role of FREM1 in regulating genes in immune response is not clear. We analyzed FREM1 expression in cervico-vaginal tissure of Rhesus macaques during early SIV vaginal infection and its role in vaginal HIV transmission by gene expression analysis of FREM1 expression modified cell lines.
Immunohistochemistry(IHC) staining was conducted to analyze archived cervicovaginal tissue of Rhesus macaques during early phase of SIVmac251 infection. FREM1 was knocked down in 293F cells and overexpressed in HeLa cells. A custom PCR array consisting 84 genes was used for the gene expression analysis.
IHC staining detected high levels of FREM1 in epithelial cells and laminar propria of human ectocervical tissue. FREM1 is expressed in epithelial cells and CD3+ T-lymphocytes in the vaginal tissue of Rhesus macaques. FREM1 expression was increased in the epithelial cells and lamina propria of cervicovaginal mucosa days after SIV vaginal inoculation in Rhesus macaque, specificxally, in the inflammatory areas where the cellular infiltrations were apparent. Knocking down FREM1 in 293F cells increased the expressions of 14 genes including genes regulating cell cycle, transcription/transcription factors, TLR1 and TLR3. Whereas, the expression of 25 genes was decreased, including IL-1α and IL-1β, the genes are important in regulating immune and inflammatory response to infections. Increasing FREM1 expression in HeLa cells up-regulated the expression of 17 genes, including genes regulating transcription, expression, cell cycle, response to stress, extracellular matrix integrity and cell migration. Whereas the expression of 6 genes including STAT1, FOXO1, IRF1, NOS2, MMP2 and MMP13 were down regulated.
Our study showed that FREM1 is important in innate immune response to HIV vaginal infections.
Combination neonatal antiretroviral prophylaxis (cART) has been used in high-risk situations in Canada to prevent vertical transmission of HIV (VT), despite limited data supporting its use, and little consensus on optimal regimens and dosing. The objective of this study was to describe the use of cART as prophylaxis in neonates in Canada.
All HIV-exposed newborns born between 1997–2003 were identified from the Canadian Perinatal HIV Surveillance Program. Neonatal prophylaxis was categorized as single, 2 or 3-drug regimens and further sub-categorized by specific treatment type. Factors associated with prescription of cART (≥3 drugs) were determined by logistic regression.
Between 1997–2013 (n=2968), 63.7% of newborns received only zidovudine (ZDV), 17.6% received a two-drug combination (10.1% ZDV and Lamivudine (3TC), 7.3% ZDV and 1–3 doses of nevirapine (NVP), and 0.2% other), and 15.3% received cART (11.3% ZDV, 3TC and a protease inhibitor, 2.2% ZDV, 3TC and 4 weeks of continuous NVP, and 1.8% ZDV, 3TC and 1–3 doses of NVP). The highest proportion of cART prescriptions were from Quebec (45.3%) followed by Ontario (8.4%) and Alberta (7.3%), with the lowest from British Columbia (1%). Factors associated with the prescription of cART (2008–2013) included no maternal antenatal treatment vs. maternal cART (OR: 13.5, 95% CI 7.90–23.40), duration of maternal therapy ≤4 vs. >4 weeks (OR 24.5, 95% CI 12.0–50.0), suboptimal vs. excellent adherence (OR: 39.9, 95% CI 18.2–87.1), no-intrapartum vs. intrapartum ZDV (OR 2.95, 95% CI 1.83–4.75), and region (eastern vs. western Canada, OR 4.05, 95% CI 1.46–11.23). The VT rate in cART-treated infants was 4.07%.
While cART has been used for neonatal prophylaxis in Canada since 1998, there is considerable heterogeneity in regimens used, and prescribing practices across provinces. Further work is needed to identify the safest and most effective cART regimens for newborns at high risk of VT.
To describe the completeness of interventions to prevent vertical HIV transmission (VT) in HIV-infected women and their newborns in Canada during the combination antiretroviral therapy (cART) era based on data from the Canadian Perinatal HIV Surveillance Program (CPHSP).
All children born in Canada to HIV-infected mothers from 1997–2013 in the CPHSP database were reviewed. Completeness of antenatal cART (acART), intravenous zidovudine during labor and post-natal infant zidovudine were assessed.
114 children acquired HIV infection perinatally in Canada during the study period. Of these, 54 (47%) received no preventive interventions and were identified after 3 months of age (median 1.4 years; range 0.27–13.1); 81.5% of these MIP were from Ontario/Quebec, 57.4% were black and 85% had sexual exposure as maternal risk acquisition category. Amongst MIPs identified either antenatally or prior to 3 months of age, the rate of transmission was 2.1% (60/2875). Among the entire cohort of MIP (n=2929), the proportion of mothers who received no/≤4 weeks of acART declined over time, ranging from 40.7–91.3% (1997–2001) to 23.6–30.8% (2002–2005), 14.0–22.9% (2006–2009) and 7.3–9.4%, (2010–2013). Since 2010, ≥1 missed intervention (irrespective of infection status) was identified in 20% (168/853): no acART (4.2%; n=36); ≤4 weeks acART (4.2%; n=36); no intravenous zidovudine during labor (12.7%; n=108); no/incomplete infant therapy (2.8%; n=24). In multivariate analysis (2010–2013 period) receipt of no/≤4 weeks of acART differed by race (Aboriginal 12.9%, White 11.1%, Black 5.8%, other 7.1%; p=0.02); there was a trend for region of birth (AB/SK/MB 11.7%, eastern provinces 7.4%, BC/YT 4.5%; p=0.06). Of 12 babies infected since 2010, 3 were identified after 3 months of age.
While significant reductions in VT have been achieved, implementation of standard-of-care preventive interventions remains suboptimal. Further exploration of circumstances leading to these missed opportunities is urgently needed in order to ameliorate VT in Canada.
Increased tenofovir (TDF) prescribing for HIV+ adults, including pregnant women, has resulted in more women becoming pregnant on TDF or starting it during pregnancy. There is little data on fetal effects of TDF, but animal studies suggest risk for renal and bone toxicity in fetuses exposed during gestation. These toxicities stem from TDF’s potential to cause proximal renal tubular dysfunction. We present results of a prospective evaluation for nephrotoxicity in infants with in utero TDF exposure.
Infants >35 weeks gestation from two Canadian centres born to HIV+ women treated with TDF for at least one month in pregnancy were eligible for inclusion. Testing performed at 1, 6 and 18 months included: plasma creatinine, phosphate; urinary phosphate, creatinine, glucose, calcium, and alpha-1-microglobulin (A1M); and x-rays for rickets assessment.
Nineteen TDF-exposed infants 36–41 (median 39) weeks gestation were enrolled. Creatinine clearance was normal in all infants at 1 month (13/13), 6 months (13/13), and 18 months (11/11). Urinary A1M was normal in 10/10, 9/9, and 8/8 infants at 1, 6, and 18 months, respectively. Tubular reabsorption of phosphate (TRP) was normal (>85%) in all (11/11) infants tested at 1 month, 8/9 at 6 months, and 6/8 at 18 months; mild decreases in TRP were noted in the 3 children (range 80–84%) but with normal serum phosphate measurements. No infants were found to have glycosuria (among 11, 8, & 8 at 1, 6, & 18 months). No radiographic evidence of rickets was noted (14/14 at 1 month, 12/12 at 18 months).
In this prospective evaluation of renal toxicity among TDF-exposed infants, we demonstrated no impairment in overall renal function nor significant renal tubular dysfunction up to 18 months. Ongoing assessment of perinatal TDF renal and bone toxicity is required to better understand its safety in prevention of vertical transmission.
Raltegravir (RAL), though currently category C in pregnancy, and not recommended for use in newborns, has been used in exceptional cases for preventing mother-to-child-transmission (PMTCT). We report on drug levels in two infants exposed in utero to RAL, and in the first newborn to be treated with RAL for 6 weeks for PMTCT.
RAL levels in exposed newborns from the Centre Maternel et Infantile sur le Sida (CMIS) mother-child cohort were tested on the first available stored plasma sample after birth, and in the treated newborn, pre- and post-therapeutic drug monitoring was done at weekly intervals.
In RAL-exposed infants, RAL was given to mothers at standard dosing of 400 mg BID during pregnancy. RAL levels tested in two newborns at 16 and 30 hours of life were detectable at 0.9345 mg/L and 0.0381 mg/L, respectively. RAL granules for suspension (Merck, special access) were obtained for the treatment of a term newborn (39 weeks GA) from a mother with multidrug-resistant virus, and was started at 1.5 mg/kg BID, along with zidovudine and lamivudine at standard newborn doses. RAL levels were consistently above the targeted trough for treatment (0.02 mg/L) (
RAL in the newborn at doses of 1.3–1.6 mg/kg BID was well tolerated and resulted in therapeutic drug levels. Given detectable levels of RAL in the first 30 hours of life in exposed infants, the role of RAL in PMTCT should further be considered.
Raltegravir Levels in a Treated Newborn
| 6 | 3.115 | 5 mg BID | 1.61 | 11.67 | 0.36 | 1.97 | 0.87 | No |
| 9 | 3.220 | 5 mg BID | 1.55 | 11.25 | 0.75 | 1.25 | 0.15 | No |
| 20 | 3.565 | 5 mg BID | 1.40 | 12 | 0.07 | 1.17 | 0.33 | No |
| 27 | 3.835 | 5 mg BID | 1.30 | 11 | 0.06 | 1.15 | 0.02 | Increased to 6 mg BID |
| 40 | 4.275 | 6 mg BID | 1.40 | N/A | N/A | N/A | N/A | Stopped |
Since the introduction of combination antiretroviral therapy, patients with HIV have successfully kept their viral loads to an undetectable level and reduced mortality and morbidity. With this, pregnancy planning has become an important issue for HIV-positive individuals and couples. In 2007, a study surveying Canadian fertility clinics found a lack of access to fertility services for HIV-positive patients. Given the extensive efforts made to address this lack of services, a follow up assessment was warranted. We assessed the access to Canadian fertility clinics and services for HIV-positive individuals in 2014.
Surveys were sent to medical and laboratory directors of assisted reproductive technology (ART) clinics registered under the Canadian Fertility and Andrology Society (as such a sample size of convenience was applied). Main outcomes included: the proportion of fertility clinics in Canada willing to provide ART to HIV-positive individuals; the specific services offered, and; whether SOGC HIV Pregnancy Planning guidelines were implemented to inform practice.
Across Canadian provinces, 20/34 (59%) clinics completed the survey. 95% (19/20) of clinics accept HIV-positive patients for consultation; of these, 58% (11/19) have seen either HIV-positive men or women in the last twelve months. While clinics in every province offer infertility investigation and risk reduction techniques, full ART is only offered in 10/20 (50%) clinics in five provinces. Ten clinics (50%) in five provinces were aware that guidelines exist, half of which have read them and four report that they have implemented all of the guidelines in their practice.
Access to fertility clinics for people with HIV seems to have improved over time but is still regionally dependent and full ART is still limited. We will further analyze the comparison to 2007 data to confirm the improvement and tease out which areas could use further advocacy for improved access to fertility services for people with HIV in Canada.
Combination antiretroviral therapy (cART) has been linked to pregnancy complications. Previously we have shown that cART exposure was associated with decreased levels of progesterone (P4) mid-pregnancy in HIV+ cART-exposed women, and P4 levels correlated with birth weight percentiles. In mice, progesterone supplementation improved cART-induced fetal weight deficits. Here we investigate the molecular mechanisms leading to cART-associated P4 alterations.
Levels of enzymes of P4 synthesis and metabolism were assessed by qPCR on placenta tissue from HIV+ cART-exposed (Study group, N=33) and HIV-negative women (Control group N=15). Plasma P4 and human prolactin (hPL) levels were quantified at gestational week 33–37 by EIA. BeWo cells were treated with increasing doses of hPL and 20αHSD and P4 levels were measured by qPCR and EIA respectively. P4 levels in cART-exposed BeWo cells were assessed with or without 20αHSD inhibition.
P4 levels were significantly lower in the study group compared to the control group. Placental expression of most P4 metabolism enzymes was similar between groups. Only the P4-eliminating enzyme 20αHSD was significantly higher in the study group. hPL, the main regulatory hormone for 20αHSD, was significantly lower in the study group compared to controls. 20αHSD expression significantly correlated with hPL levels in women’s plasma at GW 33–37 (r=−0.822, p<0.0001). In BeWo cells, hPL down-regulated 20αHSD expression and P4 production in a dose-dependent manner. cART exposed BeWo cells produced significantly less P4 compared to controls, and P4 levels were restored by inhibiting 20αHSD activity.
Our data suggest that low P4 levels observed in cART-exposed HIV+ pregnant women could be the result of higher levels of 20αHSD induced by low hPL levels. We describe a new mechanism by which cART maybe influencing maternal hormone balance during pregnancy, and identify potential new therapeutic targets that may improve birth outcomes for HIV+ women on cART.
With the increase in pregnancy intention among people with and affected by HIV (PWAH) in Canada, the need to understand their decision-making is vital. The discrete choice experiment (DCE) method measures preferences for attributes (factors) in the face of multiple benefit-risk trade-offs. In this study, the decision was in regards to pregnancy-planning choices for PWAH.
The attributes were: different methods of conception, risk of vertical and horizontal HIV transmission or super-infection, chance of successfully conceiving, cost, and duration of travel. Split sample analysis using the nested logit regression approach was employed to analyze the affect of the attribute levels on respondents’ preferences. The relative ranking of the attributes were examined through an importance score, which calculates the relative importance of each attribute such that the importance values of the attributes add to 100%.
Thirteen respondents completed a HIV+woman/HIV−man DCE and 12 respondents completed a HIV+woman/HIV+man DCE. For HIV+women/HIV−men, the attributes: horizontal and vertical transmission risk, 5-hour travel time, and cost were statistically significantly associated with decreased utility. Respondents’ utility for an increased chance of a successful pregnancy was positive and statistically significant. For HIV+women/HIV+men, vertical transmission risk and cost were statistically significantly associated with decreased utility. Respondents’ utility for an increased chance of a successful pregnancy was positive and statistically significant. For HIV+women/HIV−men, the derived highest to lowest attribute rankings were: likelihood of successful conception method, method of conception, vertical transmission risk, cost, horizontal transmission risk, then travel time. For HIV+women/HIV+men, the ranking was: likelihood of successful conception method, vertical transmission risk, cost, methods of conception, time travel, then horizontal transmission risk.
We determined that while risk of HIV transmission and cost were associated with decreased utility, participants placed the highest importance and demonstrated increased utility related to the likelihood of successful conception. These findings require confirmation in a larger study.
Maternal combination antiretroviral therapy (cART) in pregnancy could have long-term consequences for HIV-exposed uninfected (HEU) children. Some antiretrovirals and HIV proteins inhibit telomerase. As leukocyte telomere length (LTL) is a marker of cellular aging and is linked to age-related morbidities, our objective was to compare HEU and HIV-unexposed uninfected (HUU) control infant LTL at birth and over the first three years of life, and investigate any relationship to cART exposure.
Of 324 HEU children aged 0–3y enrolled in the CARMA cohort study, most (n=215) had ≥2 blood samples collected. HUU controls (0–3y, n=308) had a single blood sample each. Relative LTL was measured via monochrome multiplex qPCR. A subset of 0–3y HEU and HUU children were age- and sex-matched 1:1. Factors associated with LTL were investigated using linear regression modeling.
In a cross-sectional analysis of LTL at birth (0–3d) in 115 HEU (56% male) and 91 HUU (54% male), male sex was associated with shorter LTL at birth (p=0.02). In a multivariable model, there was no discernible effect of maternal age or ethnicity on LTL but there was some trend toward higher birth weight being associated with shorter LTL (p=0.07). There was a significant (p<0.001) interaction between HEU/HUU status and maternal smoking in pregnancy (∼50% smoked), whereby maternal smoking was associated with significantly shorter LTL in HEU and longer LTL in HUU. Among HEU, neither duration of cART exposure in utero nor type of cART was related to birth LTL. Furthermore, among age and sex-matched children (n=214:214), LTL attrition rate was similar between groups (p=0.69).
These results further support that exposure to maternal HIV/cART in utero does not affect infant LTL, a reassuring finding. The opposite LTL effect associated with maternal smoking may relate to other factors such as fetal growth.
Saskatchewan has the highest HIV incidence among Canadian provinces, over twice the national average. As part of provincial and regional HIV control strategies, rapid point-of-care testing has been introduced to enhance case-finding. This study examines the characteristics and test results of populations accessing rapid and standard testing in Saskatoon Health Region (SHR).
Cross-sectional study examining data collected during delivery of rapid and standard HIV tests in SHR, May 2013–August 2014. Variables collected include age, gender, postal code of residence (transformed to neighbourhood deprivation quintile), test type and result. Chi-square testing and logistic regression analyses were performed.
SHR provided 1,890 rapid HIV tests and 17,890 standard HIV tests during the study period. Median age at testing was 26 years [IQR 21–35], with approximately 59% of testers male, 41% female and less than 1% transgender. 45.49% of tests were performed by residents of the two most deprived quintiles, 21.47% by residents of the middle quintile, and 33.04% by residents of the two least deprived quintiles. There was a significant association between receipt of testing and male gender, neighbourhood deprivation and age >25 years (p<0.05). Rapid testing was significantly more frequently completed than standard testing among residents of the most deprived neighbourhood quintile and among individuals >25 years of age (p<0.05). While the rate of HIV positivity was greater among rapid tests than among standard tests, this relationship was not statistically significant (1.9% of rapid tests and 1.2% of standard tests were positive, p=0.125).
Rapid HIV testing was completed by a population with differing composition than the population completing standard testing, in terms of neighbourhood deprivation quintile and age. Expansion of this testing modality should be considered in the study context, in order to enhance access to testing, case-finding, HIV prevention and treatment outcomes among all communities.
To encourage more MSM to test for HIV more often, some health promotion campaigns seek to educate them to recognize seroconversion symptoms. As part of a larger study of the social networks and circumstances of HIV seroconversion, newly diagnosed men were asked their perceptions of their own seroconversion symptoms.
51 men, drawn from 2 downtown Toronto clinics that record the highest rate of HIV positive tests in Ontario were interviewed within a year of their seroconversion concerning signs and symptoms experienced prior to testing using the question, “Did you experience flu-like symptoms, fevers, rashes, muscle and headaches, sore throats and diarrhoea over the last year?” Recent seroconversion was verified through biological markers, testing history, and self-report of risk behaviour and seroconversion symptoms.
11 of 51 report signs or symptoms (typically fever, rash, and/or diarrhoea) for which they sought medical attention and/or suspected were related to HIV seroconversion. Another 21 report symptoms that they perceived to be ambiguous, only possibly attributable to HIV, or categorizable as HIV-related only in retrospect. These were typically one or two flu or flu-like symptoms, malaise, swollen lymph glands, sweating, or exacerbation of pre-existing conditions. The remaining 19 could not recall any signs or symptoms at all.
This profile of perceptions of seroconversion signs and symptom poses challenges to HIV testing and prevention. Educating people on seroconversion symptoms is likely to have limited effect in encouraging HIV testing among the newly infected as just 22% of this set of new seroconverters perceived themselves as having signs or symptoms that could be attributed to HIV prior to testing positive. Most report symptoms that they perceived to be temporary or indistinguishable from stress or party-related symptoms or other viral infections.
Antiretroviral therapy has reduced the morbidity and mortality due to HIV. However, it only works if adherence is optimal. Text messaging interventions have been shown to improve adherence to ART, yet it is unclear if they are cost-effective.
To examine the cost effectiveness of text messaging interventions in comparison to standard of care.
We retrieved data from an overview of reviews to comprehend the scope of text-messaging interventions in impacting adherence. A systematic review was conducted to establish pooled estimates of EQ-5D utility values for the HIV/AIDS health states. A deterministic and probabilistic cost-utility analysis was conducted with a 50-year time horizon and 1-year cycle. The Ministry of Health and Long-term Care perspective was used to obtain measures for costs. The incremental costs and quality adjusted life years were calculated to estimate the cost-effectiveness of text messaging interventions in improving adherence to ART in people living with HIV.
The deterministic incremental cost-effectiveness ratio was found to be $22,247.97 per quality adjusted life year (QALY) and the probabilistic model based on 1000 simulations was found to be $21,659,21 per QALY. Based on a threshold of $50,000 per QALY the text messaging intervention is cost effective. One-way deterministic sensitivity analysis supported the cost-effectiveness of the intervention.
Text messaging interventions to improve adherence to ART in people living with HIV are cost effective. Further economic evaluations incorporating cost data from Canadian trials, and other outcomes are needed before the program can be taken to scale.
There is increased interest in the therapeutic potential of cannabis in recent decades. As many as one-third of people living with HIV/AIDS use cannabis to relieve symptoms. Canada, the Netherlands, Israel and some states in the United States have developed programs to allow access to cannabis for therapeutic purposes (CTP). In Canada, enrollment in the federal CTP program represented fewer than 5% of the estimated users of CTP. The discrepancy between the number of Canadians who reported using CTP and the rate of utilization of the federal CTP program suggests the existence of barriers to access to this program.
In the present study we employed a health services analytical framework to examine barriers to access to CTP among 628 self-selected current CTP users. We defined barriers to access as areas of poor fit between clients and services. We used five dimensions of accommodation, accessibility, availability, affordability, and acceptability to examine access to CTP.
Our findings revealed that it is difficult for Canadians to find a physician to support their application to access CTP. Accessing CTP from unauthorized sources was common; only 7% of respondents accessed CTP exclusively from authorized sources. Access to CTP was positively associated with the presence of medical cannabis dispensaries, which were not included in the regulatory regime. Access to CTP varied by medical condition and general quality of health. Affordability of CTP was a substantial barrier to access.
Strategies need to be developed to encourage scientific inquiry into CTP and address the barriers to access to CTP and the stigma and controversy that surround CTP and strain patient–physician relationships.
sVL is the goal of antiretroviral treatment (ART) and is critical for the success of Treatment as Prevention (TasP). We investigated effects of social determinants of health and substance use in a clinical cohort in Ontario.
The Ontario HIV Treatment Network Cohort Study (OCS) is a multi-site clinical cohort of people receiving HIV care. Data collection includes chart review, annual interviews, and record linkage with the provincial public health laboratory for viral load tests. Among participants interviewed between 2008 and 2011 and who were on ART, we estimated the annual proportions with sVL (<200 copies/mL) as of the last VL in the year. We calculated ratios of proportions according to time-updated socioeconomic and behaviour factors, adjusted for clinical characteristics, using multivariable generalized estimating equations with a log link function.
A total of 3,322 participants were followed for 7,309 person years. The mean annual proportion with sVL was 93.3%; this rose from 91.4% in 2008 to 94.8% in 2011. Independent prognostic factors for sVL are shown in
| Personal income (Canadian dollars) | <$20,000 | 90.4% | 0.97 (0.95, 0.99) |
| $20,000 to <$40,000 | 94.0% | 0.99 (0.97, 1.01) | |
| $40,000 to <$60,000 | 96.2% | 1.00 (0.99, 1.02) | |
| Over $60,000 | 96.6% | 1 | |
| Living alone | No | 94.2% | 1 |
| Yes | 92.2% | 0.98 (0.96, 1.00) | |
| Cigarette smoking | Never smoked | 94.7% | 1 |
| Former smoker | 96.1% | 1.01 (0.99, 1.02) | |
| Current smoker | 89.9% | 0.96 (0.94, 0.98) | |
| Occasional smoker | 91.6% | 0.98 (0.94, 1.01) | |
| Recent non-medicinal drug use (past 6 months) | No | 94.2% | 1 |
| Yes, IDU | 83.9% | 0.93 (0.87, 0.99) | |
| Yes, no IDU | 90.0% | 0.97 (0.95, 0.99) | |
| Cannabis use | Past 30 days | 94.3% | 1.04 (1.01, 1.07) |
| Past year, not past 30 days | 90.0% | 0.99 (0.96, 1.03) | |
| More than a a year ago | 93.6% | 1.01 (0.99, 1.04) | |
| Never | 93.0% | 1 |
Adjusted for all covariates shown and year, age, sex, ethnicity, region, immigration, years HIV+, time on ART, ART regimen, education, employment status, marital status, living with children, alcohol use, and clinic site.
Subset analysis
Despite being a setting with access to universal health care, poverty, social factors, tobacco and substance use were independently associated with unsuppressed VL, although disparities were slight and direction of effects varied. This should be taken into account when designing comprehensive TasP interventions.
Saskatchewan currently faces a unique HIV epidemic characterized by high rates of transmission through injection drug use. The Regina Qu’Appelle Health Region Infectious Diseases Clinic (RQHR IDC) is the tertiary referral site for all HIV-positive individuals in southern Saskatchewan. To address the need for epidemiologic and clinical information to characterize the provincial epidemic, as well as providing a framework for an eventual province-wide data set, the RQHR IDC developed a comprehensive database for its HIV-positive individuals, the Regina Qu’Appelle HIV Cohort (RQHC). The development of the RQHC and current demographic and clinical data from the cohort are discussed.
The cohort consists of all active HIV-positive individuals provided care by the RQHR IDC. Using a heavily customized electronic medical record, epidemiologic and clinical data is collected by clinic staff at every appointment and is standardized within the charting system for maximal data accuracy.
The RQHC consists of 429 patients. Aboriginal peoples account for 231 of 429 (53.8%) of the cohort. Injection drug use is the primary risk factor for 264 (61.5%) individuals, and 244 of 429 (56.9%) have a positive hepatitis C antibody. Of these 244 co-infected individuals, 61 individuals had undetectable hepatitis C viremia, for a spontaneous clearance rate of 25%. 328 of 429 (76.5%) patients are on antiretroviral therapy, and 279 are virologically suppressed (HIV viral load <= 200 copies/mL). Individuals who identified as Aboriginal had the lowest rates of virologic suppression compared to other ethnicities.
The RQHC informs the current HIV epidemic in Saskatchewan, manifested by high rates of injection drug use and hepatitis C co-infection, and disproportionate representation of Aboriginal peoples. The cohort will provide insight into successes and gaps in care provision in southern Saskatchewan and facilitate regional and provincial research and evaluation initiatives.
We evaluated physician factors associated with retention in HIV care in Ontario using population-based databases.
We identified people living with HIV in Ontario using health administrative data. We defined the date of engagement in care as the first date on which a physician billing included an HIV-related diagnostic code, an HIV-experienced provider as one who cared for at least 5 HIV-positive people in one year, and retention in care as not having a gap of more than 365 days without seeing an HIV-experienced provider. We used the Andersen-Gill recurrent event model, allowing us to study multiple episodes of non-retention in care over time. Time-varying covariates included seeing a specialist and physician HIV experience. We also evaluated multiple clinical and demographic covariates.
We included 12,162 individuals. In multivariable models, each additional episode of non-retention in care was a strong risk factor for future episodes of non-retention (relative rate [RR] 1.43, 95% confidence interval [95CI] 1.40–1.47). Seeing a specialist was associated with retention in care compared to seeing only a family physician (RR 0.92; 95CI 0.87–0.96), but seeing both was the strongest predictor of retention (RR 0.43; 95CI 0.41–0.46). Retention was higher if the first visit was with an HIV-experienced physician (RR 0.85; 95CI 0.78–0.92) or a physician in practice for fewer years (RR per decade 0.91; 95CI 0.89–0.93). Retention in care was also associated with receiving publicly funded drugs, being an immigrant or refugee, older age, being female, region of the province, several comorbidities, and having a previous mental health condition at baseline.
In Ontario, retention in care is strongly associated with care models in which family doctors and specialist physicians share care for people living with HIV. Retention in care is also associated with seeing an experienced provider at the time of engagement in care.
This study aims to examine the changing mortality rates and trends in causes of death over time among HIV-positive individuals in British Columbia (BC), and to compare these patterns of death to those observed in a 1% random sample of the BC population.
Mortality and cause of death data for individuals (≥19 years) in the Comparison of Outcomes And Service Utilization Trends (COAST) study at the BC-CfE were analyzed from 1996 to 2010. Deaths were identified through Population Data BC – which contains information on all registered deaths in BC (BC-Vital Statistics Agency dataset), and classified into categories using ICD 9/10 codes for the underlying cause of death. Crude mortality rates (per 1000 person-years) with 95% CIs were calculated assuming a Poisson distribution. The Cochran-Armitage trend test was used to assess how the distribution of deaths from specific causes has changed over time.
Overall, 2,033 (23.6%), 959 (23.3%), and 3935 (8.3%) individuals died during the follow-up period (1996–2010) among HIV-positive individuals ever on ART (n=8,620), HIV-positive individuals never on ART (n=4,110), and HIV-negative individuals (n=47,569), respectively. Mortality rate from HIV-related causes decreased by over 4-fold from 1996–1998 to 2008–2010 (p≤0.001 for test of trend) in the two HIV-positive cohorts, and remained over two times higher (1996–2010) among those who were never on ART compared to those who had received ART (16.4 vs 8.9). Variations in death from causes not typically considered to be HIV-related were however less stable and more inconsistent throughout the observation period.
Our result indicates that deaths from HIV-related causes have decreased markedly among HIV-positive individuals over time. Future analyses will examine mortality trends by causes adjusted by age, sex, time periods and CD4 counts and will also compare mortality rates between the HIV-positive cohorts and the general population (reference) using standardized mortality ratios.
Vancouver’s Downtown Eastside (DTES) neighbourhood is home to a robust informal economy, including drug and sex work scenes. While income-generating activity is an important determinant of health among people who use drugs (PWUD), the role of transitions between types of income generation within the informal economy in shaping vulnerability to violence and HIV risk remains poorly understood. This study examines how transitions in informal and illegal income-generating activities influence exposure to HIV risk and violence among PWUD in the DTES.
We conducted qualitative interviews with twenty-five individuals engaged in informal and illegal income-generating activities in the DTES. We analyzed interview transcripts thematically, focusing on relationships between income generation, violence, and HIV risk, and drew upon concepts of social violence when interpreting these themes.
Our findings highlight how informal and illegal income-generating activities in the DTES are characterized by structural and everyday violence, and transitions from ‘high risk’ (e.g., sex work, drug dealing) to perceived ‘low risk’ (e.g., recycling) activities represented attempts to reduce exposure to violence and HIV risks. Participant accounts underscored how policing strategies and poverty, alongside drug scene and gender-based violence, shaped ‘high-risk’ informal and illegal income-generating strategies and functioned to increase vulnerability to HIV risks (e.g., sexual assault, drug sharing). Participants sought out ‘low-risk’ informal income-generating activities (e.g., recycling) to limit exposure to violence and HIV risks. However, participants emphasized how these forms of income generation were nonetheless governed by cultural logics (e.g., ‘street codes’) shaped by everyday violence, and introduced new risks (e.g., infections resulting from cuts received while recycling, violent enforcement of recycling routes).
Our findings underscore the critical role of income generation in shaping exposure to violence and HIV risk, highlighting the need for low-threshold employment interventions targeting PWUD as a central component of structural HIV prevention strategies.
In October 2012, the Supreme Court of Canada (SCC) ruled that people living with HIV must disclose their HIV status to sexual partners prior to vaginal intercourse, unless they use a condom and have a low viral load, defined as <1500 copies/mL.
Using cross-sectional data from the AIDS Care Cohort to Evaluate Access to Survival Services (ACCESS) a prospective cohort of HIV-positive illicit drug users in Vancouver, we estimated the proportion of participants who would be legally obligated to disclose their HIV status to sexual partners based on the 2012 SCC ruling. Interviewer-administered surveys collected socio-behavioural data, which were linked with clinical data and de-identified. ACCESS participants interviewed since October 2012 and self-reporting vaginal intercourse within six months before interview were included in the analysis. Participants self-reporting 100% condom use and demonstrating viral load <1500 copies/mL at every test within six months before interview were deemed to satisfy the non-disclosure criteria. Multivariable logistic regression identified independent covariates of satisfying the non-disclosure criteria.
Our analytic sample included 176 participants, including 77 (44%) women. The median participant age was 45 (IQR: 40–51), and 42% were in a stable relationship at interview. Within six months before interview, 95% of participants had received ART for ≥1 day, 25% were employed, 12% were homeless, 16% had engaged in sex work, 66% had used injection drugs, and 6% had been incarcerated. Overall, 56% of participants satisfied the criteria for non-disclosure. Independent predictors of not satisfying non-disclosure criteria were female vs. male gender (aOR 0.43 [95% CI: 0.22–0.87]), having one recent sexual partner (vs. >1 partners) (aOR 0.35 [95% CI: 0.16–0.77]), recent incarceration (aOR 0.20 [95% CI: 0.05–0.99]), and being in a stable relationship (aOR 0.40 [95% CI: 0.20–0.80]).
Female and recently incarcerated participants were less likely to satisfy the non-disclosure criteria, and as such are more likely to face a legal obligation to disclose HIV status, irrespective of the challenges to disclosure within the highly criminalized environment in which they seek care.
Limited research has explored how participants describe the impact of their involvement in HIV research. We enrolled 166 gay and bisexual men who tested HIV-negative at a community sexual health clinic in Vancouver, British Columbia, into a year-long mixed methods study. Thirty-four of these participants who reported recent condomless anal intercourse, were purposively-recruited into an embedded qualitative study and completed two in-depth interviews to discuss experiences related to their sexual, social, mental and physical health. We developed semi-structured interview guides with our community partner to understand men’s lived experiences as sexually active gay and bisexual men. In addition to other key findings, thematic analysis of these interviews elicited rich accounts of the self-described impacts and knowledge gained from being a study participant in HIV research. Five related impacts of research involvement described by study participants upon their completion of engagement in the study emerged. These included (1) insights into the study processes and methods used (e.g., areas for improvement on research design, data quality and implications for analysis); (2) altruism and pride in contribution (e.g., as a rationale for enrollment and/or an outcome of participation); (3) experiencing research as a form of counseling (e.g., qualitative interviews were described as having a major therapeutic component to them and highlighted the lack of opportunities for many men to discuss their sexual and social health with peers and/or counselors); (4) the impact of study involvement on their testing knowledge and behaviours (e.g., some men described participation increased HIV testing consistency); and (5) the impacts of participation on sexual behaviours and feelings about sexuality (e.g., how one thinks about current/past sex and views on responsibility for HIV/STI prevention). We draw upon this narrative data to discuss implications for research ethics and practice. Our analysis elucidates the potentially complex emotional and behavioral impacts of study participation.
Psychological trauma is widely experienced, and can produce a myriad of psychological outcomes (Van Ameringen et al., 2008). Providers within a care system that competently and comfortably respond to individuals who have experienced trauma is essential (Ko et al., 2014). Multiple training models have been developed, and networks are beginning to form to create trauma-competent health care systems in certain jurisdictions (e.g., the U.S. National Child Traumatic Stress Network) (Ko et al., 2014). However, what do trauma-competent individuals and systems look like, and specifically when considering HIV and sexual health? This presentation will describe an on-going project with the Gay Men’s Sexual Health Alliance in Ontario to develop trauma-competent trainings for its members. This presentation will draw on best practices in developing trauma-competent approaches to care, and will specifically examine individual settings’ needs. The role of community and culture will specifically be examined, with an emphasis on the need to engage in an iterative, community-based process while implementing and developing training modules. The application of these principles with both clinicians and non-clinicians will be highlighted. The presentation will offer the opportunity for reflection regarding identifying provider needs when working with traumatized individuals. Barriers to such systems will be discussed.
Globally, transgender sex workers face an overwhelmingly increased burden of HIV. Access to health care services, such as hospitals and drop-in centres that distribute harm reduction supplies, are a vital part of HIV prevention and treatment efforts. However, little is known about the lived experiences of transgender sex workers and HIV-related services. As such, we conducted a qualitative investigation into experiences with HIV-related health care among transgender sex workers in a Canadian setting.
In-depth semi-structured interviews were conducted with 33 transgender sex workers in Vancouver, Canada between June 2012 and May 2013. Participants were recruited from three open prospective cohorts of individuals who use drugs, an open prospective cohort of sex workers, and through snowball sampling. Codes and themes were developed using theory-driven and data-driven approaches and two transgender researchers assisted with the analysis.
Of the 33 participants, 54.5% (n=18) were HIV positive and all participants reported previous encounters with HIV prevention services. When accessing services participants reported experiencing stigma related to drug use and sex work (e.g., quality of care was affected by being labeled as an individual who uses drugs), discrimination based on their gender presentation (e.g., access to services was dependent on perceptions of femininity), and feelings of not belonging (e.g., name calling by cisgender individuals). Participants reported avoiding hospitals and HIV prevention services due to these experiences.
Transgender sex workers in this setting reported experiencing a range of barriers to accessing essential HIV-related services. Drug use- and sex work-related stigma coupled with heteronormative practices suggest a critical need for the training of health professionals and HIV services that are welcoming to transgender sex workers. Models of peer sex worker and transgender inclusive HIV services and programs should be considered.
Sex workers (SW) in Toronto are, more than ever, accessing sexual and HIV-related health services in the context of a broader political landscape. Since Bedford v. Canada, and subsequent federal Bill C-36, SW have been at the crossroads of surveillance, criminalization, politics, community and visibility. This research explores sex work, HIV status, race, class, trans identity, employment, and homelessness as these intersect with sexual health services, and the national, provincial and local policies that structure standards of service provision, and effect accessibility and inclusion for SW.
An online survey that addresses service delivery to SW is being distributed to sexual health service providers and s SW in Toronto. Data collected explores perceived dynamics between service providers and SW, and the availability, form, quality and barriers to the provision. Analysis will focus on bivariate associations with an emphasis on intersections that impede quality health service interactions. This study has been reviewed for ethics by the University of Toronto.
Programs and services that seek to address the sexual and HIV health of SW are constrained by discrepancies between health client need, the limits of service provision, the occupational landscapes of sex work, and the stigma and discrimination encountered when accessing sexual and HIV health care.
These research findings can reveal discrepancies in HIV health and social care to SW within the current Canadian political landscape, allowing sexual and HIV health service groups to expand on strategies for more accessible care to SW. By better understanding the intentions of HIV and sexual health services, and the perceived effectiveness of these for people like SW, this research will highlight the lived experience of accessing care while addressing the intersections of diverse social and systematic realities that influence the structure of HIV and sexual health care delivery.
The legacies of colonization through the appropriation of Indigenous land and the import of African slave labor were integral for the colonial project, a project that unintentionally connected these groups throughout time and space. From this point onward, Indigenous and African diasporic communities would be connected through their shared, and distinct histories of resistance and oppression, which had ill consequences for their health as both communities are disproportionately impacted by HIV.
Data for this paper comes from my doctoral research project, in which I employed conventional qualitative methods (e.g. focus groups and interviews) with innovative feminist and arts-based approaches (digital storytelling and collaborative mural-making) to explore how Indigenous and ACB youth view alliance building for HIV prevention and health promotion in their communities. Paying homage to the social resistance that sparked the HIV movement in the 1980s, as well as Indigenous sovereignty and Black liberation struggles, in this project I explore the questions: In what ways did and didn’t the youth move beyond the colonial divide when undertaking this collaborative project? And how were the benefits and challenges of these partnerships represented artistically in the youth-led collaborative mural?
Youth leaders were very optimistic about the potential for co-resistance and collaboration between their communities for the sharing of Indigenous and alternative healing, cultural practices, health promotion messaging and HIV prevention approaches. However, the youth identified that such collaborations are also heavily fraught with tensions, contradictions and conflict, including the ‘oppression olympics’ that occur between different groups; settler of colour colonialism; anti-black racism; and the need for the decolonization of both groups. I take from anti-racist and Indigenous scholarship to unpack these research findings. This work is of great value as Indigenous and African Diasporic communities have younger populations than other groups, making youth contributions towards the health and wellbeing of these communities significant and transformative. These young people can be a part of health promotion strategies that have lasting generational consequences within their communities for generations to come.
Within Atlantic Canada unique challenges exist in the promotion of youth-oriented HIV and HCV prevention. Recognizing this, a 3-year study “Our Youth, Our Response” (OYOR) was initiated to support Atlantic wide HIV and HCV prevention policy and programmatic responses to meet the unique needs of Atlantic Canadian youth.
The OYOR research team conducted 3 iterative stages of data collection and analysis: A policy scan of existing HIV/HCV prevention documents (Year 1), in-depth interviews with key informants from education, health and corrections sectors (Year 2), and focus groups with youth and youth-serving health and social organizations (Year 3). Interview and focus group data were audio-taped and transcribed, and thematic analysis was conducted.
Many HIV/HCV prevention policies and programs within the region do not differentiate between the needs of youth and those of adult populations resulting in missed opportunities for providing youth-friendly HIV/HCV testing and referral services. School-based sexual health curricula are not standardized, nor are measures in place to ensure that youth are provided comprehensive, accurate, and relevant prevention information. In particular, little information is provided to youth regarding the types of services that are available within their communities. Prevailing stigma regarding youth sexuality, misconceptions related to HIV/HCV, and a lack of youth-focused harm reduction services further exacerbate these issues.
To address these challenges there is a need to mobilize existing expertise within each of the Atlantic Provinces, to share resources and expertise, and to reduce duplication of effort – all with an emphasis on increasing youth access to testing options, reframing harm reduction, and expanding education provided to youth, parents, and service providers. This can be achieved, in part, through multisectoral responses that incorporate youth as active partners. It is critical that these steps serve as the foundation for the next generation of youth-oriented HIV/HCV prevention innovation.
While the Greater Involvement of People Living with HIV/AIDS (GIPA) and the Meaningful Involvement of Women Living with HIV/AIDS (MIWA) are core principles in participatory research with Women Living with HIV (WLHIV), ethical tensions emerge in the application of these principles, especially within the context of HIV-stigma, barriers to disclosure, and intersecting inequities.
Peer, academic, and community researchers from the Canadian HIV Women’s Sexual and Reproductive Health Cohort Study (CHIWOS), a longitudinal, Community-Based Research study operating in British Columbia, Ontario, and Quebec, examined the question: what tensions emerge when applying GIPA/MIWA principles in research with WLHIV in Canada?
Reflecting on moving GIPA/MIWA from principle to practice, we identified the following tensions: (1) Participation: ensuring the meaningful participation of Peer Researchers must be met with adequate training, guidance and support, and appropriate compensation including payment for time, travel, and preparation, a challenge in projects with limited funds. Further, meaningful participation varies greatly depending on the time, capacity, and interest of every Peer Researcher. Particular attention must also be paid to bridging linguistic barriers in a bilingual, national study. (2) Recognition: crediting peer contributions and being transparent (for partners and participants) about the input of WLHIV within CHIWOS must be balanced with the need to protect HIV-confidentiality. (3) Well-being: though it is imperative to provide a safe and healthy work environment, Peer Researchers may be confronted with emotionally traumatizing information during interviews, and may find themselves in precarious situations when navigating insider/outsider roles on the research team and within their communities.
Various tensions emerge when moving GIPA/MIWA from principle to practice. Research teams must be flexible, communicative and creative in order to properly recognize and engage WLHIV, despite the current context of limited funding, HIV-stigma, barriers to disclosure and intersecting Social Determinants of Health which impinge on the full realization of GIPA/MIWA principles. The perspectives and resilience of WLHIV can provide rich insights into these challenges and their mitigating factors.
CHEO (Children’s Hospital of Eastern Ontario) and ACO (AIDS Committee of Ottawa) identified a lack of awareness about and engagement with support services amongst families living with HIV who access care at CHEO. Common goals and challenges between the two organizations in providing support were identified. A formal partnership was developed enabling sharing of experiences and expertise between clinicians, support workers and community. Program development, implementation and lessons learned are described.
Members of ACO’s Afro-Caribbean support group voiced concerns that some families in the Ottawa region were not engaged with support services yet had challenges that were not being addressed. The idea of offering ACO services within the CHEO HIV clinic was proposed, leading to a partnership agreement enabling the ACO worker to attend clinics and the clinic social worker to attend ACO support groups. Program objectives were to reduce stigma, enhance services and collaboration amongst organizations, and increase awareness and accessibility of clinic and community services. After six months, a program review revealed that 47 families who were not previously accessing community services had initial encounters in the clinic setting with 22 remaining engaged with ACO. Issues identified included immigration, medication access, system navigation, isolation, food security, housing and childcare. Services accessed included the food bank; legal, social and health service referrals; support groups; and harm reduction.
Meeting the support worker in a familiar clinic environment often led to engagement by women previously unable to connect with peers or acknowledge their diagnosis. The partnership allowed for meaningful family centered care by the clinic, supporting parents in ways that were not previously possible. The two roles (support worker and social worker) are complementary.
This unique institution-agency partnership enabled sharing of views, resources and information leading to improved client engagement in services, optimizing each organization’s capacity.
Increasingly, community-based organizations are employing Peer Research Associates (PRA) on HIV research projects. The PRA role creates space for meaningful and active participation of people living with HIV in research, and improves the quality of participant responses. Despite the growing use of PRAs, there are few published models or dynamic “how-to” guides for community-based organizations seeking to integrate PRAs on research projects.
To assist community-based organizations navigate the process of integrating PRAs in research projects, the Dr. Peter Centre (DPC, a not-for-profit community HIV health care organization) documented its experience of recruiting, hiring, training, orientating, supervising, and mentoring PRAs for a large-scale research project. To document the process, the DPC hired an independent PRA to interview the two PRAs hired for the large-scale research project as well as various parties who influenced the hiring process including: DPC staff, external researchers, and members of a Community Advisory Committee that includes DPC stakeholders and four DPC clients. Interview findings were synthesized, developed into six Prezi-based Learning Modules, and then disseminated to Canadian community-based organizations, researchers and policymakers in December 2014.
Initial responses to the PRA Learning Modules were extremely positive. As a knowledge translation strategy, using Prezi to communicate this model to other community-based organizations has several advantages: i) it is web-based and can be developed by multiple parties working from different locations, and then disseminated easily; ii) audio recordings for slide narration can be easily made and revised for single slides; iii) unlike academic papers with codified rules and language, this knowledge translation strategy allowed us to cover multiple topics relevant to community-based organizations while using accessible language.
Knowledge translation strategies that meet the unique requirements of community-based organizations are needed to effectively communicate models that can improve the functioning and services of those organizations.
PrEP is an emerging strategy for HIV prevention in Canada, particularly for gbMSM. While PrEP is generally considered a biomedical intervention, there may be important roles for CBOs to play in an HIV prevention landscape that includes PrEP.
We invited representatives from 26 Toronto-based organizations serving a range of culturally diverse gbMSM to participate in a series of Think Tanks exploring the needs & roles of CBOs in PrEP rollout. The first session was held on 10/Dec/2014; three will be held in 2015. Participants completed a brief survey and participated in small group discussions. Results will be used in subsequent sessions to co-develop an action agenda on CBO involvement in PrEP implementation.
Most organizations (n=22) agreed to participate; one declined and three did not respond. Seventeen organizations attended the first session; twenty completed the survey. Half the organizations were ASOs; the remainder included government (n=1), public health (n=1), and healthcare (n=3) and mental health service (n=3) organizations. 75% felt comfortable sharing PrEP information with clients but only 35% felt comfortable delivering PrEP-related services. Participants were generally supportive of PrEP because of its potential to improve HIV prevention and provide additional benefits, such as reduced HIV-related anxiety and stigma and increased self-efficacy and empowerment. However, participants expressed concerns related to toxicity, adherence, stigma, risk compensation and increased STI rates, access and the “medicalization” of HIV prevention. Possible roles in implementation identified included education, awareness raising, adherence support, referrals to prescribers, assistance covering medication costs, research and advocacy. Some were skeptical of their role due to access barriers for their clients or because PrEP was viewed as a medical intervention.
CBOs are generally supportive of PrEP and could play multiple roles in its implementation. However more effort is needed to meaningfully engage these important stakeholders in PrEP rollout.
In 2013, women of ELAN’s support group at ASAAP discussed how to address a lack of representation in the HIV/AIDS movement. Their discussions stemmed from feelings of frustration as they participated in research and focus groups yet, did not see their voices in larger dialogue. This was the impetus for “More Than Fiction”, an anthology of these 12 women’s personal narratives.
The project was developed not as a standard research endeavour, although one of its primary goals is to inspire research in narrative writing with a focus on its therapeutic and social outcomes. This project addresses the unique needs of people living with HIV/AIDS.
Eight (8) participants for the project were recruited from ELAN and attended workshops on creative writing and photo art led by two (2) skilled facilitators to help pen their stories for the anthology over a period of four (4) months from May to August 2014.
At the completion of the project in the fall of 2014, “More Than Fiction” was launched with literary and visual design content for presentation and distribution to POZ communities, partner agencies, and service providers. The anthology covers HIV disclosure, treatment adherence, support networks, education, family, sex and sexuality, gender identity, and spirituality. Its aim is to inform and improve service provision specifically tailored for racialized women living with HIV/AIDS. “More Than Fiction” received much praise from leaders in the HIV/AIDS service and research sectors after its launch. The authors of “More Than Fiction” participated in a focus group where they shared their insights and learning outcomes from the project and expressed the therapeutic and skills-development benefits of writing and sharing their stories.
To date, print copies of “More Than Fiction” have been widely distributed at various community events. An online version is in the works. ASAAP would like opportunities to present and share this unique narrative writing model with the greater HIV/AIDS service and research sectors to help foster positive change in HIV/AIDS service and support provision.
Due to multiple personal and contextual barriers, labour force participation remains a challenge for many people living with HIV (PHAs) in Canada. Low employment rates have frequently been reported among PHAs following HIV diagnosis. However, improved employment outcomes are associated with receiving effective vocational counselling. Currently, AIDS service organizations are those most involved in the provision of HIV-specific employment supports in Canada, and have been identified as an important resource for services related to vocational rehabilitation. The objectives of this community-initiated study were to explore the benefits and challenges of community vocational rehabilitation services in assisting PHAs to return to the workplace and maintain successful employment.
The study was conducted with the guidance of a community advisory committee that included PHAs and community service providers. Community-based research methods and grounded theory were used to explore the perspectives of PHAs in Canada who have sustained successful participation in the labour force. Purposive sampling was conducted in the provinces of Ontario, Alberta, and British Columbia. Qualitative, semi structured interviews were conducted with 31 PHAs who were successfully engaged in the labour force.
Study findings identified important factors in effective vocational rehabilitation and the role for community-based AIDS service organizations. The results emphasize a dual focus on supporting PHAs at the personal level while also addressing contextual issues. Nine thematic categories summarized the findings related to vocational rehabilitation for PHAs. These include the importance of the GIPA principles; social and emotional support; support groups; skill development; benefits counselling; anti-oppressive practice; peer support; outreach and advocacy; and HIV-specific services.
Study findings call attention to the vocational needs of PHAs in Canada and identify effective strategies that can guide HIV-related vocational service provision and community practice. They also contribute to a growing body of resources available to community service providers interested in addressing HIV and employment. Results will be of interest to those working in community-based AIDS service organizations and those interested in developing effective vocational programming and supportive public policies.
Effective clearance of hepatitis C virus (HCV) requires HCV-specific CD8+ T cells, yet their activity is impaired in chronic HCV (cHCV) infection. Generalized CD8+ T cell dysfunction is also observed, for unknown reasons, with potential effects extending beyond HCV infection. Reduced interleukin-7 (IL-7) receptor alpha (CD127) expression and IL-7 activity contribute to CD8+ T cell impairment in HIV infection, and this may also be the case in cHCV infection and HIV-HCV co-infection.
Treatment naïve, cHCV mono-infected and HIV-HCV co-infected individuals (HAART-treated, <50 copies HIV RNA/ul for >1yr) and HIV-, HCV- healthy controls were studied. In cHCV mono-infection, we observed fewer naïve CD8+ T cells (TN) and unchanged bulk-CD8+ T cell CD127 expression, yet reduced CD127 expression on central memory T cells (TCM) in blood compared to controls. Secondly, IL-7 activity was impaired, with lower levels of activated STAT5 (especially TN and TCM), and lower baseline and inducible Bcl-2 expression. This muted Bcl-2 response was associated with higher fibrosis scores. In HIV-HCV co-infection, we observed an increased proportion of effector memory cells in blood, and unchanged CD127 expression compared to controls or cHCV mono-infection. The activity of IL-7 was impaired as in cHCV mono-infection, and IL-7-mediated proliferation was reduced. Cytokine stimulation did not activate STAT5 in liver-CD8+ T cells in cHCV mono-infection and baseline Bcl-2 levels were lower relative to blood-CD8+ T cells in the same individual.
Generalized CD8+ T cell impairment in HCV infection, and its increased severity in HIV-HCV co-infection, is characterized by impaired cytokine signaling and survival, independent of CD127 expression, unlike in HIV infection. This impairment is pronounced in the liver, and fibrosis may contribute to this broad dysfunction. Identifying the underlying mechanisms will facilitate the design of novel immune therapies to complement existing antivirals and improve the health status of those with cHCV infection.
Combination antiretroviral therapy (cART), HIV proteins, and oxidative stress can affect telomerase activity and/or leukocyte telomere length (LTL), a marker of aging and lifespan predictor. We investigated the relationships between pregnancy, HIV, and cART on LTL in HIV+ and HIV− women. HIV+ (n=107) and HIV− (n=68) pregnant women were enrolled in a prospective cohort study. Relative LTL was assessed during pregnancy (13–23, >23–31, and >31–40 weeks of gestation), at delivery and at 6 weeks post-partum for HIV+ women. Mixed-effects regressions were used to examine the relationship between LTL and ethnicity, HIV status, hepatitis C virus (HCV), substance use throughout pregnancy, gestational age (GA) at visit, and preterm delivery. Predictors significantly associated with LTL were included in a multivariate model. Among HIV+ women, CD4 nadir, CD4 and HIV plasma viral load (pVL) at visit, peak pVL, on/off cART at visit, and duration of cART in pregnancy were also considered.
HIV+ and HIV− women were similar in age (31±6 vs. 31±5 years, p=0.49), but there were fewer Black/African Canadians and HCV+ in the HIV− group (p<0.001). 26% of HIV+ women started cART pre-conception, 74% during pregnancy, and 61% continued post-partum. Univariately, LTL was significantly shorter in HIV+ women (p=0.02) and smokers (p=0.01). Multivariate analysis suggested shorter LTL in smokers (p=0.02), and a significant interaction between HIV status and GA (p=0.01), with LTL unchanging in HIV− women, and increasing in HIV+ women over pregnancy. Among HIV+ women, smoking, higher pVL, and off cART at visit were associated with shorter LTL (all p≤0.001), and time of visit with increasing LTL (p<0.001). Multivariately, HIV+ women who smoked (p=0.005) and those off cART (p<0.001) had shorter LTL.
In the context of pregnancy, HIV+ status and smoking are associated with shorter LTL. Within HIV+ women cART initiation may increase LTL, possibly via reduced inflammation and oxidative stress.
HIV infection induces a chronic inflammatory state in the infected individuals. At the local level, proinflammatory cytokines disrupt the intestinal barrier function and enhance intestinal permeability. Several researchers have shown increased intestinal permeability and apoptosis of intestinal cells in HIV infected individuals. We and others have shown an imbalanced production of Interleukin (IL)-18 and IL-18 Binding Protein (IL-18BP) in HIV-infected individuals. In this abstract, we show that incubation of HIV-1 with HT29 (Human colon adenocarcinoma grade II cell line) induces IL-18 and reduces IL-18BP production. We tested the effect of IL18 in both HT29 and Caco2 intestinal cell lines. Interestingly we noticed very high destructive effects on HT29 cell cultures after 48h of contact with IL18 as compared with non IL18 treated cells. The cytokine induced death via apoptosis in both HT29 and Caco2 cells, which was dose dependent. This increase in apoptosis was preceded by a concomitant decrease in some intestinal tight junction proteins. IL18 also decreased adherent junction-associated proteins like beta-catenin and disturbed spatial arrangement of F- actin in Caco2 and HT29 cells. Caco2 cells showed a decrease in trans-epithelial electrical resistance (TEER) after treatment by IL18 for 24h. Interestingly, the serum IL-18 concentrations correlated with their LPS contents in HIV-infected individuals. Overall, our results suggest that HIV-induced IL-18 production from human intestinal epithelial cells plays a role in microbial translocation and chronic immune activation in HIV-infected individuals.
The decreased expression of IL-7 receptor alpha (CD127) and impaired IL-7 signaling in T-cells in chronic viral infections is well established, particularly in HIV infection. The soluble CD127 (sCD127) can be observed in the plasma, however its role in maintaining T cell function or dysfunction is not known. We previously found increased plasma sCD127 levels in untreated HIV infection that remained elevated despite subsequent viral control. The goal of this work was to determine if the immunorestorative effects of IL-7 therapy shown in HIV infection are associated with sCD127 expression.
The concentration of plasma sCD127 levels did no change during acute primary SIV infection or in response to antiretroviral (ART, 105 d.p.i) treatment. After the acute infection, macaques were administered recombinant IL-7 (Cytheris) treatments (3 clusters of: 3 weekly IL-7 injections + 2 week reprieve). Treatment with IL-7 frequently increased sCD127 levels in either a transient or persistent (i.e. up to 5 weeks) manner. Before ART, sCD127 levels correlated with increased CD8+ T-cell numbers. Following an IL-7 cluster treatment, sCD127 correlated with increasing CD4+ and CD8+ T-cell numbers. In the IL-7 clusters, as the number of activated (Ki67+) CD4+ or CD8+ T-cells increased, so too did sCD127 concentrations, particularly if sCD127 levels had fallen below assay detection between IL-7 treatment clusters. Finally, the higher levels of sCD127 in IL-7-treated macaques related to the greater retention of proliferating cells (i.e. BrdU uptake), compared to controls
These data confirm findings from our colleagues demonstrating that IL-7 promotes the release of sCD127. Furthermore, these data demonstrate a parallel relationship between higher levels of plasma sCD127 levels with IL-7-mediated T-cell numbers, activation and survival. This suggests that IL-7 therapy in SIV infection increases sCD127 release. We hypothesize that the endogenous sCD127 plays a role in the observed IL-7 activity, and contributes to viral control in vivo.
The innate immune system becomes dysfunctional during chronic HIV infection and fails to fully reconstitute even during suppressive ART. Restoring full activation and function of these cells during ART may be a crucial component of overcoming viral latency or supporting functional cure approaches. Contact between DCs and T cells expressing inhibitory markers such as PD-1 and Tim-3 can lead to reverse signaling through DC-expressed ligands (like PD-L1/L2) that alters DC maturation and phenotype. The goal of this project is to assess the role of another inhibitory marker, LAG-3, on innate lymphocyte function. The first step is to determine the kinetics and sources of LAG-3 expression.
A study of HIV infected and uninfected research participants from Nairobi Kenya were assessed for systemic and mucosal LAG-3 levels. LAG-3 is upregulated on T cells in HIV infection although expression is a low proportion of cells. Invariant NK T cells are innate lymphocytes and have elevated LAG-3 levels that are not recovered following ART. On T cells a kinetic assessment shows that LAG-3 expression occurs within 6 hours of stimulation of the cells. When de novo protein synthesis is blocked, LAG-3 expression still occurs within 6 hours.
LAG-3 is stored in pre-formed intracellular vesicles and is trafficked to the membrane following activation of the T cell. For the cell to invest in the production and storage of LAG-3, this may imply that its function is required very early following activation and that its role is not solely as a marker of exhaustion, rather, is involved in early regulation of the immune response.
∼1.4 million HIV-infected women give birth each year, including ∼200 in Canada. Antiretroviral therapy can prevent > 98% of mother-to-child transmissions. As a result, numbers of HIV-exposed un-infected (HEU) children are increasing worldwide. Several groups have reported increased morbidity and mortality in HEU children. Analysis of retrospective data from the CMIS Mother-Child Cohort (705 HEU children) revealed that at 2 months of age, HEU infants born to mothers with HIV-1 viral load >1,000 copies/mL had significantly higher CD19+ B cell frequencies compared with children born to mothers with undetectable viral load, suggestive of immunologic anomalies. The objective of this study was to characterize these abnormalities.
HIV-infected women were enrolled during the course of pregnancy. Phenotyping of B lymphocytes was performed by multiparametric flow cytometry (CD3/CD10/CD14/CD16/CD19/CD20/CD21/CD27/IgM) in HEU children using samples of umbilical cord blood (UCB; n=8) or venous blood (n=2) obtained at 4–6 months of age. Magnitude of vaccine-elicited antigen-specific B cell responses was estimated by staining with fluorescent tetanus toxoid (TT) oligomers.
Decreased frequencies of naïve B cells and increased frequencies of activated memory B cells and plasmablasts were observed between HEU UCB and 4–6 months of life (70.2%±0.07% vs. 58.9%±3.29%, 0.3%±0.0007% vs. 3.7%±1.05%, and 0.3%±0.0022% vs. 1.6%±1.12%). Conversely, frequencies of classical memory and atypical memory B cells (CD19+CD10-CD27-CD20+CD21-/low) were unchanged. TT-specific B cells were detected at 4–6 months of life but not in UCB. TT oligomer-positive cells represented 1.6% of class-switched plasmablasts and 0.2% of class-switched classical memory B cells, compatible with vaccine-elicited B cell responses in HEU children.
Results from this study provide a high-resolution portrait of antigen-specific B cell responses and immunocompetence in HEU children, which could significantly impact clinical management of these patients. Whether these responses are influenced by the clinical picture observed in the mother will need to be validated.
The interferon regulatory factor (IRF) family members are regulators of many biological processes such as cytokine signalling, immune response, and apoptosis. Both IRF-1 and IRF-7 have been shown to regulate anti-viral immune response via regulating the expression of type 1 and 2 interferons and interferon-stimulated-genes. We hypothesize that increased cellular expression of IRF-1 or IRF-7 will boost anti-viral responses and reduce viral replication in infected cells. Conversely, reducing cellular IRF-1 or IRF-7 will render the cells more susceptible to infection, with the exception of HIV-1.
IRF-1 and IRF-7 level were examined in defined immune subtypes in blood using multi-color flow-cytometry. IRF-1 and IRF-7 levels in ex-vivo human T cells and monocytes were modulated by transfection with either plasmids or siRNA. The outcomes were assessed by the efficiency of transactivating HIV-1 genes (p24 ELISA and Gag mRNA) and host cell anti-viral genes.
Without stimulation, preliminary data showed that IRF-1 was expressed in all cellular subtypes examined (CD-4, CD-8, B-cells, NK cells, monocytes, and dendritic cells) with the highest expression found in monocytes. In response to exogenous interferon-gamma (IFN-gamma) stimulation, IRF-1 protein level was increased by ∼2-fold. In contrast, IRF-7 was expressed at low levels in unstimulated cells, and its expression was augmented by ∼3-fold in IFNαA-treated cells. Our preliminary work showed that as little as 30% knockdown of endogenous IRF-1 level resulted in >90% reduction in transcription of HIV-1 genes and consequently impaired viral replication. Such modest IRF-1 knockdown had no effects on the transactivation of host anti-viral genes.
Fine-tuning the expression of immune regulators is critical, as modest reduction in IRF-1 expression significantly impaired the trans-activation of HIV genes, but not the regulation of host genes. Findings from this study will define the roles of IRF-1 and IRF-7 roles in anti-viral responses assisting in vaccine design.
The link between HIV infection and age-related immunosenescence is primarily supported by lymphocyte immunologic abnormalities. In untreated infection, these include shorter telomeres in the inflated senescent CD8+ T cell compartment, and increased CD4+/CD8+ ratio. This HIV-mediated immunosenescence may manifest in early-onset age-related comorbidities in cART-treated people. The relationship of telomere length (TL) in specific blood cell subsets to HIV immunosenescence is unclear. Our objective was to investigate TL in several lymphocyte subsets, and its relation to HIV clinical factors.
This pilot study involved 33 HIV+ subjects and 10 HIV-controls enrolled in the CARMA cohort. Live PBMCs were sorted for CD4+, senescent CD8+CD28-, and proliferative CD8+CD28+ T cells using FACS. QPCR was used to measure relative TL in each of the cell subset with sufficient count. Results were analyzed by two-tailed Mann-Whitney, Spearman’s correlation, and ANCOVA.
Consistent with previous research, the HIV+ group presented an expanded senescent CD8+CD28- compartment (n=43, 39 vs 17% of total T cells, p=0.02) and a decreased CD4+/CD8+ ratio (n=43, median 0.24 vs. 1.75, p<0.001) compared to the HIV− group. Overall, older age was associated with shorter TL in proliferative CD8+CD28+ (n=27, R=−0.47, p=0.01) but not senescent CD8+CD28- T cells (n=29, R=−0.06, p=0.77). Proliferative CD8+CD28+ T cells TL in the HIV+ group (n=19, median[IQR] 3.35[2.63–3.93]) was shorter than in HIV-individuals (n=8, 3.73[3.48–4.08]) and this difference was statistically significant after controlling for age (p=0.02). In contrast, TL in senescent CD8+CD28- T cells (n=22 HIV+ 2.48[2.18–3.16] and n=7 HIV− 2.32[2.15–2.84], p=0.56), or CD4+ T cells (n=18 HIV+ 3.76[3.21–4.14] and 8 HIV− 3.43[2.96–3.57], p=0.53) were not statistically different in both groups.
Our results suggest that proliferative CD8+CD28+ T cells TL are affected by age as well as HIV, highlighting the relevance of this compartment in studies of HIV-mediated immunosenescence.
HIV is linked with markers of cellular aging such as shorter leukocyte telomere length and an inflated senescent CD8+CD28- T cell subset. HIV and/or cART are also suggested to increase mitochondrial DNA (mtDNA) mutations. Oxidative stress leads to nuclear telomerase recruitment to mitochondria where it may protect mtDNA against oxidative damage and possibly mutation, linking the roles of telomerase and mitochondria in cellular aging. Our objective was to measure mtD-NA apparent oxidative damage (AOD) in sorted lymphocyte subsets from HIV+ and HIV− individuals, and explore its putative relationships with cART exposure, clinical, and demographic factors.
In this pilot study, live PBMCs were collected from 33 HIV+ subjects and 10 HIV− controls enrolled in the CARMA cohort. Proliferative CD8+ CD28+ and senescent CD8+ CD28- T cells, along with CD19+ B cells were separated by FACS. QPCR was used to measure mtDNA AOD in all subsets with adequate cell count. Data analysis was performed using Spearman’s correlation and two-tailed Mann-Whitney tests.
No significant relationship was seen between mtDNA AOD and HIV or age in the studied subsets. HIV viral load was associated with decreased mtDNA AOD in CD19+ B cells (n=16, R=−0.71, p=0.006). A possible positive relationship between mtDNA AOD and lifetime cART duration was seen in both proliferative CD8+CD28+ and senescent CD8+CD28- T cell subsets (n=22, R=0.53, p=0.01, and n=22, R=0.45, p=0.04).
These preliminary results may point toward turnover of CD19+ B cells with viremia-damaged mtDNA, potentially resulting in the selection of cells with less mtDNA damage. The association between lifetime cART duration and mtDNA AOD in both CD8+ subsets reinforces the concept that cART may play a role in exacerbating lymphocyte aging. Future work will involve measuring mtDNA AOD in HIV slow-progressors not on treatment to further evaluate the role of cART on mtDNA oxidative damage.
Carriage of certain NK cell receptors (NKR) and their HLA ligands is associated with slow time to AIDS in HIV+ subjects and protection from infection in HIV-exposed seronegative subjects, implicating NK cells in HIV control. NK cells acquire anti-viral functions through licensing, which requires signals from inhibitory NKR (iNKRs), such as NKG2A and KIR3DL1 (3DL1), engaging their ligands. NKG2A interacts with HLA-E presenting HLA-I leader peptides; 3DL1 interacts with Bw4+ HLA-A and B antigens. HIV infected cells can activate NK cells by down-regulating HLA-A/B ligands for some iNKR and upregulating ligands for activating NKR. We studied the impact of NKG2A and 3DL1 expression on NK function in response to autologous HIV-infected CD4 cells (iCD4). We hypothesized that the NKG2A+3DL1+ subset would have higher functional potential and greater responsiveness to iCD4.
We studied 24 HIV− subjects. The functional potential of NKG2A±3DL1± populations was assessed by simulating PBMCs with HLA-null cells. Responses to HIV were assessed by co-culture of NK cells with autologous iCD4. Flow cytometry was used to gate on NKG2A± 3DL1±populations with all possible combinations of CD107a, IFNγ, and CCL4 functions by boolean gating.
NKG2A+3DL1+ NK cell population had the highest functional potential (p<0.0001). iCD4 cells induced differential frequencies of total-responsiveness, tri-functional (CD107a+IFNγ+CCL4+), CD107a+IFNγ+ and CD107a+ responses in the NKG2A±3DL1±populations (p≤0.008). iCD4 stimulated a significantly higher frequency of NKG2A+3DL1- than NKG2A-3DL1+ or NKG2A-3DL1- NK cells with the CD107a+IFNγ+CCL4+, CD107a+IFNγ+ and CD107a+CCL4+ and total CD107a and IFNγ functional profiles (p≤0.03).
Detecting the highest functional potential in the NKG2A+3DL1+ population is in line with licensing through two iNKRs. Observing the greatest response to iCD4 cells in the NKG2A+3DL1- population may suggest that NKG2A+ NK cells have a role superior to those that are 3DL1+ in NK cell mediated anti-HIV immunity.
While constitutive IL-7 expression regulates T cell homeostasis, following infection IL-7 levels increase and contribute to CTL activity. As a result, decreased IL-7 receptor (CD127) expression on CD8 T-cells evident in HIV+ individuals may not only reduce cell survival but may also impair naïve CD8 T cell activation in response to foreign antigens. A better understanding of IL-7’s role in the immune response and its deregulation in HIV infection is essential to understanding HIV-induced immune suppression.
To investigate IL-7’s role in T cell activation, naïve CD8 T cells were isolated from PBMC of healthy donors and stimulated in vitro with anti-CD3/anti-CD28 beads ± IL-7. Although IL-7 alone had no effect on any surface marker measured with the exception of CD127, IL-7 significantly enhanced many TCR induced phenotypic changes including up regulation of CD25, CD56, CTLA4, CD69, HLADR, CCR7, CD8, TIM3, PD1 and PDL1. IL-7 also increased Ki-67 expression and enhanced TCR induced proliferation as measured by CFSE dilution over 7 days. Although IL-7 alone did not induce IFNγ or perforin in naïve CD8 T cells, IL-7 significantly enhanced production of these cytotoxic molecules in the presence of TCR stimulation. Interestingly while T-bet expression was only transiently induced by TCR stimulation, in the presence of IL-7 TCR-induced T-bet expression was sustained suggesting IL-7 may induce a specific subset of cytotoxic CD8 T cells. Of particular note, IL-7 induced expression of Bcl2 was completely blocked by TCR activation consistent with the apparent differentiation of pro-apoptotic effector cells.
Given the role IL-7 plays in driving differentiation of naive CD8 T cells to an effector phenotype, decreased IL-7R expression in HIV+ individuals most likely limits cell mediated immune responses to a variety of pathogens including HIV itself. Restored IL-7 signaling and recovered CTL activity may then be an important pathway to establishing a cure.
Myeloid cells including macrophages, monocytes, dendritic cells (DCs) and neutrophils contribute significantly to immune dysfunction in the context of treated HIV infection. Recently in long-term treated patients, the myeloid-associated inflammatory markers such as sCD14, CD16+ monocytes and tryptophan catabolizing enzyme IDO-1 activity better predict disease progression and mortality than T cell markers.
We systematically reviewed 81 publications indexed on PubMed database from 2009–2015 using the multiple combinations of key words: ‘HIV’ and ‘myeloid cells, monocytes, macrophages, dendritic cells and neutrophils’. We also summarized the ongoing clinical trials registered on
The role of myeloid cells in HIV infection is reviewed and depicted as an integrated vicious cycle of inflammation involving monocytes, macrophages, DCs, neutrophils and T cells. Functional polarization and plasticity of macrophages into M1/M2 has significant implications. Myeloid-derived suppressor cells open up new avenues of research on immune dysfunction.
Recent evidence indicates that innate immune response can be “trained” to become adaptive. Such changes are linked in myeloid cells to a shift of central glucose metabolism from oxidative phosphorylation to aerobic glycolysis (the “Warburg effect”) fulfilling the high energy demand for rapid proliferation in the context of cancer and viral infections. The interaction of programmed death-1 (PD-1), an important regulator of T cell function, with its ligand PD-L1 on myeloid cells has a major impact on immune dysfunction. The recent commercialization of inhibitors of PD-1 and CTLA-4 pathways in oncology is paving the way for immune-based therapies in treated HIV infection.
This systematic literature review indicates a very important link between metabolism and immune response driven by myeloid cells highlighting the immunotherapeutic targets. Further studies aimed to understand and modify myeloid cell immune check points will contribute to the development of novel interventions for viral eradication.
The activation state of natural killer (NK) cells, which function in antiviral immunity, is regulated by activating and inhibitory receptors. Previous work from our group found that carriage of the Killer Immunoglobulin-like Receptor (KIR) 3DS1 homozygous (3DS1h-mz) genotype and the combination of the high expression KIR3DL1 homozygous (*h/*y) genotype with its HLA-B*57 ligand (*h/*y+B*57) was associated with protection from HIV infection in a cohort of injection drug users (IDU). Additionally, others reported that alloreactive KIR/KIR-ligand mismatched combinations were associated with protection from heterosexual HIV transmission in HIV discordant couples. Here, we investigated whether these KIR/HLA genotype combinations maintain protection in the context of mother-to-child transmission (MTCT).
We studied 61 HIV-1 seroconcordant and 64 serodiscordant mother-infant pairs. DNA for HLA and KIR geno- and allotyping was extracted from dried blood spots. All samples were typed for HLA-C, while infants alone were typed for HLA-B, KIR3DL1 generic genotypes, and the presence of KIR2DL1, KIR2DL2, and KIR2DL3 genes. 4-position HLA typing was used to classify the HLA-B and -C antigens as Bw4/Bw6 and C1/C2. Bw4 antigens are ligands for KIR3DL1, C1 for KIR2DL2/3, and C2 for KIR2DL1.
The frequency of the protective 3DS1hmz and *h/*y+B*57 genotypes was not significantly different between infected (INF) and exposed uninfected (EU) children. Moreover, INF and EU children did not differ significantly in the frequency of KIR2DL1/KIR2DL2/KIR2DL3, HLA-C1/C2, or KIR/maternal KIR-ligand mismatched combinations.
Due to the ethnic composition of the MTCT cohort, the frequency of the 3DS1hmz and *h/*y+B*57 genotypes was low, reducing our power to observe significant inter-group differences had they existed. Several aspects of the biology of MTCT could account for the inability to translate the observed protective effects of certain KIR/HLA combinations. These findings highlight the unique characteristics of vertical HIV-1 transmission and the need for further research in this field.
Th17 cells maintain mucosal immunity against pathogens. Human Th17 cells were previously reported to express CCR6 and CCR4 (CCR4+Th17) or CXCR3 (CXCR3+Th1Th17). During HIV-1 infection, Th17 cells are depleted from the gut-associated lymphoid tissues and their restoration under ART is only partial. We identified two previously uncharacterized CCR6+ subsets: CCR4-CXCR3- (double negative; CCR6+DN) and CCR4+CXCR3+ (double positive, CCR6+DP) expressing the Th17-specific transcription factor RORC. In contrast to CCR4+Th17, CXCR3+Th1Th17 and CCR6+DP, the frequency of CCR6+DN was preserved in the peripheral blood of HIV-infected subjects on ART. We investigated functional and transcriptional differences between the four CCR6+ subsets and tested their permissiveness to HIV infection in vitro and in vivo.
Memory T-cell subsets were sorted by flow cytometry. Cytokine profiles were investigated upon TCR triggering in vitro. Antigenic specificity was determined by measuring T-cell proliferation. Genome-wide transcriptional profiling was performed using the Illumina technology. HIV integration was measured by real-time PCR.
CCR6+DN and CCR6+DP from HIV-uninfected subjects produced IL-17A similar to CCR4+Th17 and CXCR3+Th1Th17, respectively. CCR6+DN distinguished by their superior IL-17F, IL-21 and IL-8 production. Similar to CCR4+Th17, CCR6+DN proliferated in response to Candida albicans hyphae but not CMV. Although they shared known molecular Th17 markers, CCR6+DN, CCR6+DP and CCR4+Th17 expressed specific molecular signatures thus, reflecting the heterogeneity of memory Th17 cells in humans. Similar to CCR4+Th17 and CXCR3+Th1Th17, CCR6+DN and CCR6+DP were permissive to HIV in vitro and carried integrated HIV-DNA in HIV-infected subjects. CCR6+DN represented the most predominant Th17 subset in HIV-infected subjects on ART and uninfected controls.
Our results reveal the existence of four transcriptionally distinct Th17 subsets in humans and suggest a major contribution of CCR6+DN to HIV persistence under ART. Understanding mechanisms allowing the preservation of CCR6+DN in HIV-infected individuals will be critical for designing new intelligent cell-specific HIV eradication strategies.
Tenofovir alafenamide (TAF), a new prodrug of the HIV-1 NRTI, tenofovir (TFV), shows improved antiviral activity in monotherapy clinical studies, at lower doses than tenofovir disoproxil fumarate (TDF). TAF delivers TFV more efficiently than TDF to lymphoid cells with a 5-fold increase in intracellular TFV-diphosphate (TFV-DP) level and a 90% reduction in plasma TFV. To evaluate TFV-DP distribution among primary human CD4+ T-lymphocytes (CD4) subsets following TAF treatment, intracellular TFV-DP levels were evaluated in CD4 subsets at clinically relevant TAF concentrations.
TAF loading studies were conducted using primary cells from healthy human donors. PBMCs, total CD4, naïve, effector, central memory and effector memory CD4 T-cell subsets were evaluated. Loading was evaluated using a 2 h pulse incubation followed by washout and then incubation in drug-free media mimicking in vivo TAF exposure. Cell extracts were prepared and TFV, TFV-MP, and TFV-DP levels were measured by LC/MS/MS.
Cell loading studies in PBMCs demonstrated that a 2 h pulse and 22 h washout of 200–400 nM TAF achieved TFV-DP levels comparable to those observed in vivo following clinical TAF dosing. There was minimal variation in intracellular TAF metabolites between donors. Additionally, comparable TFV-DP levels were achieved after a 2 h pulse with either 200 or 400 nM TAF in total CD4, and CD4 subsets, with a trend for higher TFV-DP levels in memory cells compared to other CD4s subsets. For each CD4 subset evaluated, there was no significant decrease in TFV-DP levels at 24 h, indicating a long intracellular half-life in all cell populations.
The sustained levels of TFV-DP 24 h post-treatment suggest high levels of TFV-DP will be maintained across most CD4 cell subsets in patients receiving TAF. The higher levels in memory subsets may have implications for maintaining viral suppression in latent viral reservoirs and for future cure efforts.
The widespread use of antiretroviral (ARV) created the emergence of mutant strains resistant to treatment. Thus, the World Health Organization recommends epidemiological monitoring for newly infected patients with HIV. The objective of this work is to determine the genetic diversity of HIV Type 1 and the prevalence of mutations associated with resistance to ARV in treatment-naïve patients in Kinshasa.
One hundred fifty-three subjects diagnosed positive for HIV Type 1 by serology voluntarily participated in this work. They were recruited in different centers of Kinshasa. The inclusions were performed from August 2013 to February 2014. Five milliliters (5 mL) of blood were collected in a tube with EDTA anticoagulant. Plasma was sent for analysis to the AIDS Reference Laboratory of the University Hospital of Liège (CHU-Liège) in Belgium. RNA was extracted from plasma 140 μL using the QIAamp RNA Mini Kit QIAGEN®. A Reverse Transcriptase PCR and Nested PCR enabled amplification of regions of interest on the Protease and Reverse Transcriptase (RT) for subsequent sequencing.
The mean age of patients was 37 years, ranging from 18 to 65 years. The median values of Viral Loads (VL) and rate of CD4 lymphocytes were respectively 5.68 log10 RNA copies/mL and 180 cells/mL. Protease and RT were amplified and sequenced, respectively, for 130 (84.9%) and 145 (94.8%) patients. Subtype A was dominant with 35 cases (22.9%); followed by CRF02_AG (11.1%), C (9.8%), G (9.8%), K (9.8%), D (7.8%), H (7.8%) and J (5.0%).
The results of our study confirm the high diversity of HIV Type 1 in Kinshasa. It reveals the heterogeneity of the virus and the presence of transmitted resistance associated with antiretroviral drugs. Several minor and major resistances associated with Protease Inhibitors, as well as mutations associated with the Reverse Transcriptase Inhibitors have been detected in antiretroviral treatment-naive patients.
FREM1 transcript variant 2 (TILRR) is a novel regulatory component, which stimulates host defense against infection through binding of IL-1R1 and TLR complex and enhancing the recruitment of MY88 in the Ras-dependent NFKB signal transduction pathway. Our previous study has identified FREM1 as a novel candidate gene in resistance and susceptibility to HIV infection in the Pumwani Sex worker cohort. In this study we investigated the effect of TILRR on gene expression of several important signal transduction pathways by overexpressing it in the HeLa cells. TILRR was overexpressed in HeLa cells using eGFP tagged plasmid construct. Transfection efficiency was determined by fluorescence microscopy and flow cytometry. TILRR RNA overexpression was confirmed by qRT-PCR. The effect of TILRR on the expression of 252 genes in important signal transduction pathways was subsequently investigated by qRT-PCR with 3PCR arrays (Human signal transduction, extracellular matrix and transendothelium migration, and MAPKinase). Overexpression of TILRR significantly upregulated 64 genes, and downregulated 69 genes (p<0.001) in MAPKinase, transendothelium migration and NFKB pathways. These findings are novel. Pathway studio analysis showed that some of the most significant upregulated genes directly influence gene expression and inflammatory responses. Although how TILRR influence the expression of these genes need to be investigated, our study is the first to show that TILRR may direct influence gene expression in addition to its role in enhancing NFKB and inflammatory responses. Because transendothelium migration, NFKB and inflammatory response pathways are extremely important in HIV vaginal transmission, further study of the role of TILRR in gene regulation may identify novel targets and develop intervention technology against HIV-1 vaginal infection.
A putative transmission of CXCR4-using HIV from a CCR5wt/wt donor to a homozygous CCR5Δ32/Δ32 recipient was retrospectively identified in the Vancouver Injection Drug Users Study (VI-DUS) via phylogenetic analysis. We apply phylogenetic reconstruction methods to longitudinal intrahost deep-sequence data to characterize HIV transmission and evolution in this rare event.
Donor and recipient bulk plasma HIV gag, pol, nef, and env-V3 sequences exhibited the lowest overall pairwise genetic distances in VIDUS. The transmission date estimated from clinical data was Aug/01. Donor plasma/PBMC were available at −13, −7, −1, and +35 months from transmission; recipient plasma/PBMC were available +5, +6, and +12 months from transmission. Env-V3 from plasma-RNA and PBMC-DNA were triplicate amplified, pooled equally and deep-sequenced (Roche 454). BEAST and HyPhy were used to reconstruct phylogenies, estimate multiplicity of infection and reconstruct transmitted/founder (T/F) viruses from plasma-derived deep sequences from donor and recipient.
Despite infection with the same X4 HIV strain, the donor’s nadir CD4 count was 20 cells/mm3 within 1.5 years of infection whereas the recipient’s remained >270 cells/mm3;. Donor/recipient plasma viral loads were comparable (∼4.5 Log). All 10 ancestral reconstructions were consistent with transmission of a single X4 virus between May–Aug 2001. The estimated T/F virus sequence was identical to the co-dominant variant (36%) observed in the recipient’s first (+5 month) timepoint. This sequence was also observed in 0.16% of donor plasma and 33.5% of PBMC at month −1, suggesting minority variant transmission. In the donor, replacement of the dominant V3 sequence with a R25G variant, signifying a gradual reversion of X4 to R5 HIV-1. In contrast, the recipient’s dominant V3 sequence was quickly replaced by R25K and others, but remained consistently X4.
Results highlight the power of phylogenetic reconstruction applied to deep-sequence data to estimate the number and sequence of T/F viruses and characterize intrahost evolution in donor/recipient pairs. Differential CD4 depletion and V3 evolution of a near-identical X4 virus in genetically distinct individuals underscores the impact of host genetics on HIV evolution/pathogenesis.
Few epidemiological and clinical outcome data exist for HIV-1 intersubtype recombinants in rural African communities. The objective of this study is to estimate prevalence, examine time trends, and test for clinical correlates and outcomes associated with HIV-1 intersubtype recombination in Mbarara, Uganda, where HIV-1 subtypes A1 and D co-circulate.
Near-full-genome HIV-1 RNA population sequence data was collected using nested PCR targeting gag to nef as five amplicons followed by Sanger sequencing from n=504 treatment-naïve individuals enrolled between 2005–2010 in the Mbarara-based UARTO cohort, who then received PI or NNRTI-containing regimens and were monitored until 2013. HIV-1 subtypes were inferred by Los Alamos RIP 3.0 (window size 400). Statistical significance was defined as p=0.003 after Bonferroni correction.
When each genomic region was individually examined, intersubtype recombinants were detected most frequently in the vif-vpu region (24%), followed by GP41 (18%), gag (15%), prrt (10%), int (8%), nef (4%). Of the 200 patients that had sequence data for all seven genomic regions examined, prevalence of intersubtype recombination was 46%. The most frequently observed recombinant was A1–D (25%). Phylogenetic analysis by maximum-likelihood tree of the 200 near-full-genome sequences showed that A1–D recombinants did not share a common ancestor and suggested multiple recombination events. Stratification by year shows no temporal trend (all p>0.1). Subjects infected with non-recombinants versus recombinants were not significantly different in baseline viral load (p=0.7), baseline CD4 count (p=0.2), time to suppression <=400 copies/mL (p=0.03), time to post-suppression virologic rebound (p=0.1), and time to CD4 recovery defined as baseline+200 or above 350 cells/μL (p=0.6).
Intersubtype recombination is common in Uganda but it was not associated with baseline viral load nor CD4 count and did not impact treatment outcomes. HIV-subtyping for clinical records and/or association studies should be annotated with the name of the gene used for subtyping.
Next-generation sequencing (NGS) may offer a new resource for studying HIV adaptation within hosts by quantifying the prevalence of minority species. We evaluated the potential of this application using longitudinal samples from individuals in the British Columbia Drug Treatment Program.
HIV RNA was extracted from plasma samples from 43 individuals, one pre-therapy (viral load ≥5×104 copies/mL) and a second taken ≥90 days after therapy start. HLA genotypes were determined by sequence-based typing. Gag and nef were amplified by nested RT-PCR. Amplicons were processed by Nextera XT paired-end library preparation for the Illumina MiSeq. Short-read data were processed with a custom pipeline using Bowtie2 for iterative mapping to an adaptive reference. We used Pearson’s χ2 test to identify sites with significant changes in amino acid frequency distributions within hosts. “Known” HLA associations were taken from Brumme et al. (2009; PLoS ONE:e6687).
Median short-read coverage per sample was 7,236 and 9,181 for gag and nef, respectively. The number of significantly evolving amino acid sites (Y) was significantly higher in nef and was associated with time between samples (binomial regression, z=3.5, P=4.5×10–4) and mean viral load (z=3.1, P=0.002); this multivariate regression explained 16% of variation in Y. Median proportions of Y with known HLA associations were 56% (gag) and 85% (nef). Direction of amino acid frequency change was significantly associated with adaptive versus non-adaptive HLA associations for individuals with matching HLA alleles (odds ratio OR=3.4, P=4.7×10–11), but not significant for non-matching HLA alleles (P=0.2).
Trends in within-host HIV adaptation quantified by NGS correspond well to documented HIV-HLA associations from population-based studies. However, substantial numbers of evolving sites lacked “known” HLA associations; these are candidates for studying host-specific adaptation at the individual level, for example due to HLA alleles too uncommon for associations to be detected at the host population level.
The membrane-proximal external region (MPER) of HIV-1 gp41 harbors conserved epitopes recognized by HIV-1 broadly neutralizing monoclonal antibodies and is thus a target for vaccine development. However, the MPER has proven to be poorly immunogenic and MPER-specific neutralizing antibodies are difficult to elicit. Flagellin is a toll-like receptor 5 (TLR5) agonist under intensive scrutiny as a vaccine adjuvant. We investigated its potential as a gene-based vaccine adjuvant for gp41 MPER.
A flagellin/gp41 MPER fusion protein, encoded by a gene-based vaccine, will trigger TLR5 agonist activity, maintain gp41 MPER antigenicity and elicit a greater magnitude of gp41-specific humoral responses upon immunization relative to a vaccine expressing gp41 MPER alone.
We designed and produced flagellin/gp41 MPER fusion proteins using a mammalian expression system. Based on secretion, TLR5 agonist activity and gp41 MPER antigenicity, we selected a candidate fusion protein to be delivered by DNA vaccine and carried out mouse immunization studies.
We demonstrate that the insertion position of gp41 MPER within flagellin greatly affects the resulting fusion protein’s secretion, TLR5 agonist activity and gp41 MPER antigenicity. A DNA vaccine expressing one candidate fusion protein elicited a modest gp41 MPER-specific humoral response which was of at least ten-fold greater magnitude than that elicited by a DNA vaccine expressing gp41 MPER alone.
Flagellin can augment the immunogenicity of HIV-1 gp41 MPER when delivered as a flagellin/gp41 MPER fusion protein. Gene-based vaccines expressing flagellin/gp41 MPER fusion proteins are thus promising platforms for HIV vaccine development.
When treated with the new integrase inhibitor (INI) dolutegravir (DTG), patients previously treated with older INIs have lower response rates than other patient populations but are unable to develop the DTG resistance mutation R263K. We investigated whether the presence of classic INI resistance mutations preclude the emergence of R263K.
We grew HIV-1 with different classic INI resistance mutations in cord blood mononuclear cells over protracted periods, with the concentration of DTG being incrementally increased. We then assessed the activity of purified recombinant integrase (INB) biochemically and the infectivity of NL4.3 virus in tissue culture, both harbouring the classic mutations in combination with R263K.
Each combination reduced the strand transfer activity of INB relative to INBwt or INBR263K and only INBQ148R/R263K had increased resistance to DTG biochemically. The NL4.3IN(Y143R/R263K) and ¬NL4.3IN(Q148R/R263K) viruses grew very poorly in tissue culture, however the addition of N155H to R263K partially restored the infectious defect of the R263K mutant, while increasing DTG resistance compared to either single mutant. The NL4.3IN(E92Q/R263K) virus had increased DTG resistance relative to E92Q-containing virus alone, but the combination negatively impacted infectivity. Both the E92Q- and N155H-containing viruses were also able to select for R263K under DTG pressure.
On most accounts, the combination of classic INI resistance mutations with R263K did not lead to increased fitness or drug resistance. The combination of N155H and R263K, however, does represent a possible mechanism through which resistance may develop. This combination was recently identified in an INI-experienced patient failing an older INI, and the N155H pathway is increasingly being associated with DTG failure in the clinic. These results suggest that two prominent DTG resistance pathways are complementary in vitro. This will have significant implications for the treatment of HIV-positive individuals as DTG becomes more common in clinical settings.
Clinical studies have shown that integrase strand transfer inhibitors (INSTIs) can be used effectively against HIV-1 infection. To this day, no resistance mutation has been found in INSTI-naive patients who were treated with the second-generation inhibitor dolutegravir (DTG). In a recent selection study with DTG using a virus bearing the H51Y substitution in integrase, the emergence of an arginine to a lysine substitution at position 262 (R262K) was observed. We characterized the addition of R262K to H51Y with regard to integrase strand-transfer activity and resistance to DTG in vitro and in tissue culture.
Here we show that the addition of R262K to H51Y decreased recombinant integrase strand-transfer activity but improved integrase DNA binding affinity compared to the wild-type or H51Y enzymes. The defect in strand-transfer activity did not translate into a decrease in HIV-1 infectivity. The combination of H51Y and R262K substitutions slightly decreased susceptibility to DTG (FC=1.87) in cell-based resistance assays.
Although viral replication was not affected and enzyme efficiency was impaired by the combination of R262K with H51Y, we observed an overall increase in the level of drug resistance against DTG. Our findings suggest that arginine at position 262 plays an important role in DNA binding.
Soluble CD4 (sCD4) binds to the HIV envelope proteins and inhibits viral entry. In a small clinical trial, frequent administration of high doses of recombinant sCD4 completely neutralized cell-free HIV. However, continuous injection of purified proteins is not feasible for the long-term treatment of patients. Therefore, we used a lentiviral vector (LV) for the continuous secretion of sCD4 from human cells. sCD4 was secreted from gene-modified producer cells and bound to recombinant gp120 and to cells expressing gp160. Viral entry and gp160-mediated cell fusion were inhibited in the presence of sCD4. Infectivity of HIV produced from gene-modified cells expressing sCD4 was severely reduced.
To optimize sCD4 expression, we evaluated LVs with different promoters in myeloid and lymphoid cell lines. The elongation factor α (EF1α) promoter was active in all tested cell lines, while the cytomegalovirus promoter mediated a low expression in T cells, which was only modestly improved by the addition of a ubiquitous chromatin opening element. To further increase sCD4 secretion, we tested different signal peptides, showing that the human alpha-1 antitrypsin signal peptide fused to sCD4 (AAT-CD4) increased secretion in comparison to the native signal peptide.
The optimized vector with the EF1α promoter and AAT-CD4 was subsequently used to transduce CD4+ T cells and CD34+ hematopoietic stem/progenitor cells (HSPCs). Gene-modified T cells were protected from infection and culture supernatants from HSPCs inhibited viral entry by >90%. Gene-modified HSPCs differentiated into CD4+/CD8+ cells ex vivo, suggesting that sCD4 does not interfere with T cell development. The HSPCs were injected into immunodeficient mice and gave rise to gene-modified progeny cells. The humanized mice will be tested for serum levels of sCD4 and challenged with HIV.
Overall, relevant cell types can produce significant quantities of sCD4 and transplantation of gene-modified T cells or HSPCs has the potential to suppress HIV replication.
Monocyte-derived macrophages (MDM) from HIV-infected patients and MDM infected in vitro with HIV manifest inhibition of cytokines including IL12. IL27, an IL12 family cytokine, was shown to inhibit HIV replication in macrophages. Whether HIV infection or HIV accessory protein(s) impact IL27 production in macrophages remains unknown. Herein, we show that in vitro HIV infection as well as intracellular HIV and HIV-tat peptides inhibited LPS-induced IL27 production in MDM suggesting that HIV-tat inhibits IL27 production by impairing TLR-4 signalling. To study the mechanism governing HIV-tat-mediated inhibition of LPS-induced IL27 production, we first established that p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK), the phosphoinositide-3-kinase (PI3K), SRC homology region 2 domain-containing tyrosine phosphatase-1 (SHP-1) and Src kinases regulated LPS-induced IL27 production in MDM. HIV-tat caused TNF receptor associated factor (TRAF)-6 inhibition and consequent decreased phosphorylation of downstream PI3K, and p38 and JNK MAPKs implicated in LPS-induced IL27 production. However, SHP-1 and Src kinases were not involved in HIV-Tat-mediated inhibition of LPS-induced IL27 production. In contrast to HIV-tat, in vitro HIV infection of MDM inhibited LPS-induced p38 and JNK activation. Overall, HIV inhibits LPS-induced IL27 production via HIV tat through the inhibition of TRAF-6, and consequent inhibition of PI3K and p38 and JNK MAPKs in macrophages.
Chimigen® Platform Technology has been used to design a novel dendritic cell (DC) receptor-targeted HIV vaccine that incorporates multiple HIV-1 antigens and is capable of inducing antigen-specific cellular and humoral immune responses for prophylactic and early intervention therapeutic applications.
Chimigen® Vaccines are chimeric recombinant fusion proteins of selected antigen(s) and specific xenotypic (murine) antibody fragments including the Fc region. These chimeric molecules bind to specific receptors on DCs and other antigen presenting cells for antigen uptake. They are processed through both proteasomal and endosomal pathways and presented to T cells through MHC class I and class II molecules, stimulating cellular and humoral immune responses against the chosen antigens.
The Chimigen® HIV Vaccine, containing the HIV-1 Gag, Env, Tat, Rev, Vpr and Vpu antigens, was expressed in Sf9 insect cells and purified. DC-binding experiments and antigen presentation assays ex vivo using human peripheral blood mononuclear cell-derived DCs, T cells and B cells demonstrated that the Chimigen® HIV Vaccine binds to immature DCs in a dose-dependent manner, induces CD4+ and CD8+ T cell activation and proliferation, and promoted increased production of IFN-γ and TNF-α from both CD4+ and CD8+ T cells. Furthermore, B cells stimulated with vaccine-loaded DCs were found to produce antigen-specific IgM antibodies. Evaluation of Immune responses to the vaccine in vivo, in Sprague Dawley rats, confirmed that all antigenic components of the HIV vaccine are immunogenic and induce both HIV-specific cell-mediated and humoral immune responses. Th1 immune responses were predominant, with IFN-γ cytokine responses prevalent over IL-4 production in rat splenocytes, and IgG2A serum antibody titres dominant over IgG1 antibodies. This study established safety and “proof of concept” and therefore, shows potential for development as a prophylactic/early intervention therapeutic vaccine against HIV infections.
Financial support: National Research Council Canada–IRAP, CHTD and Alberta Innovates–Technology Futures.
The membrane proximal region of HIV-1 gp41 (MPER) is well-conserved and the target of several broadly neutralizing Abs. However, antibody (Ab) responses against the MPER are rarely observed in HIV+ individuals, or through vaccine strategies using MPER-based immunogens. Impediments to the design of vaccines targeting the MPER include an incomplete understanding of the MPER structure in the pre-fusogenic spike, the close association of the MPER with the plasma membrane, and restricted/exposure of key neutralizing sites. In a previous study, immunization with a MPER-based DNA vaccine encoding a non-native trans-membrane domain (TMD), required the use of the adjuvant, flagellin, to elicit marginal Ab responses against the MPER in rabbits; boosting with liposomes bearing MPER peptides moderately increased titres. Serum responses mapped to the 2F5 region of the MPER and affinity-purified Abs neutralized a Tier 2 HIV-1 envelope, but were not durable and of low titre. In assessing alternative vaccine strategies to increase Ab titres, we observed that co-immunization with DNA and liposome/peptide vaccines improved durability of titers against an MPER peptide (to ∼12 weeks); but titers remained low. Immunization of guinea pigs with a DNA-prime/liposome-boost elicited a negligible anti-MPER response, with detectable serum Abs being against flagellin and the hemagglutinin tag encoded by the DNA vaccine. To address the poor exposure of the 4E10 site by our original DNA vaccine, we further engineered our MPER vaccine to support a trimeric helical structure and to better expose key neutralizing sites. Our initial analyses indicate that DNA vaccination required boosting with a liposome/peptide vaccine to elicit moderate responses against a MPER peptide bearing the 2F5 sequence; analysis of the Ab response against the 4E10 site is currently ongoing. Our results suggest that MPER reactivity can be augmented with liposome/peptide boosts, and that the nature of the TMD affects MPER antigenicity and immunogenicity.
We previously showed that SIVmac239 is susceptible to raltegravir (RAL), elvitegravir (EVG) and dolutegravir (DTG) with IC50s in the nanomolar range, and integrase (IN) mutant SIV displayed similar resistance profiles to HIV. A long-acting form of a new IN strand transfer inhibitor (INSTI) termed S/GSK-1265744, a DTG analogue, was shown to protect macaques against repeated vaginal and rectal exposures of SHIV. These studies show that nonhuman primates can be utilized to investigate the potential role of INSTIs in HIV therapy, pathogenesis and transmission. Our objectives were to observe whether HIV and SIV share similar resistance pathways under INSTI pressure in selections and cell-free assays and to test the effects of HIV-1 IN resistance mutations on SIV IN activity.
Tissue culture selections were performed in rhesus macaque peripheral blood mononuclear cells infected with SIVmac239 in the presence of INSTIs. The SIVmac239 IN gene was cloned into a pET15b vector. Purified recombinant SIVmac239 WT, E92Q, T97A, G118R, Y143R, Q148R, N155H, R263K, E92Q/T97A, E92Q/Y143R, R263K/H51Y and G140S/Q148R IN enzymes were generated and integrase activities and INSTI inhibitory constants were assessed using cell-free assays.
Genotypic analysis of the IN coding region of SIVmac239 in tissue culture selections under EVG pressure yielded the E92Q mutation after 29 weeks, and a mixture including the 263R/K mutation after 21 weeks of DTG pressure. The G118R and G140S/Q148R substitutions diminished target DNA affinity (∼5.5 and 2-fold) and enzyme efficiency by 80% and 60%, respectively. G140S/Q148R negatively impacted strand transfer activity (70% of WT levels). RAL and EVG showed reduced activity against the Q148R, E92Q/Y143R and G140S/Q148R variant enzymes. The Q148R and G140S/Q148R enzymes showed moderate resistance to DTG.
This study supports the use of nonhuman primates to study HIV pathogenesis, therapy and transmission. SIVmac239 viruses treated with DTG led to the emergence of a R263R/K mixture, and the detection of the E92Q mutation in SIVmac239 viruses treated with EVG. This study further confirms that the same mutations associated with drug resistance in HIV display similar profiles in SIV.
Previous studies have reported that HIV-1 is capable of persistent infection in the testes, a proposed viral sanctuary site with potentially limited antiretroviral drug (ARV) penetration due to a naturally restrictive environment caused in part by the blood-testis barrier (BTB). This study aims to characterize drug transporters and metabolic enzymes in the human testes to gain insight on ARV disposition in this organ. Testicular tissue samples were obtained from uninfected men (N=8) and HIV-1 infected men on ARV therapy (plasma viral load < 50 copies/mL, N=5) who underwent elective orchiectomy for gender reassignment surgery. We selected 11 representative drug transporters and two drug metabolic enzymes to study in the testes based on their relevance to ARV disposition. We observed high gene expression of MRP1 and OATP2B1, moderate expression of BCRP, and low expression of P-gp, MRP2, OATP1A2, OATP1B1, OAT1, OCT1, CNT1, ENT2, CYP2D6 and CYP3A4. However, we detected protein expression for all transporters and metabolic enzymes analysed with the exception of OATP1A2 and OCT1. Overall, gene and protein expression levels displayed interindividual variations, but did not differ significantly between the two study groups. Our fluorescence microscopy results also showed that drug transporters and drug metabolic enzymes are not limited to BTB localization at the seminiferous epithelium, but can be found throughout the testicular tissue. Our data are the first to demonstrate protein expression and localization of key drug transporters and metabolic enzymes in the testes of ARV-treated HIV-1 infected men, and suggest the testes are a complex pharmacological compartment that has the potential to regulate the disposition of several ARVs. (Supported by CIHR and OGS)
HIV-1 protease mediates the cleavage of Gag, GagPol and Nef precursor polyproteins in a highly specific and temporally regulated manner. Because a total of 12 cleavage reactions are required to generate a mature virion, generating focused immune response targeting the sequences surrounding the protease cleavage sites(PCS) could drive viral mutations to its disadvantage. We have conducted a proof of concept study with Cynomolgus macaques and pathogenic SIVmac239 as a model and used a modified recombinant vesicular stomatitis vector and nanocarriers to deliver 12 20-amino acid antigens. We showed that a vaccine targeting the sequences surrounding the 12 protease cleavage sites is promising at prevention of HIV-1 infection and disease progression. In this study we systematically analyzed breakthrough viruses of vaccinated and control macaques.
The sequences surrounding the 12 PCS were amplified from plasma RNA of all SIVmac239 positive samples. The amplified PCR products were sequenced with 454 pyrosequencing technology. The amino acid and frame shift mutations were analyzed and correlated with viral load and CD4 counts by regression analysis. Multivariate analysis was conducted to determine amino acid mutations at multiple PCS sites on viral load reduction.
Extensive mutations were detected around PCS and both conserved and non-conserved mutations are correlated with lower viral load (p< 0.0001). The breakthrough viruses from the vaccinated macaques carry significantly higher mutations than the controls. Longitudinal analysis revealed that the high rate of non-conserved and conserved amino acid mutations along the sequences surrounding the PCS lead to the reduction and diminishing of viral load.
The pathogenic SIVmac239 is extremely vulnerable to any amino acid alternations surrounding PCS and focusing immune response to sequences surrounding the PCS of HIV-1 can drive amino acid mutations and lead to complete viral control.
Dolutegravir is the only antiretroviral drug that has not led to the emergence of HIV drug resistance mutations in clinical trials with treatment-naive participants. This is true not only for resistance mutations against dolutegravir itself but also for mutations against other drugs co-administered with it. Previous results suggested that this might be due to a decrease in viral replicative capacity associated with a R263K substitution in integrase that causes low-level resistance against dolutegravir and a diminished ability to generate resistance against reverse transcriptase inhibitors. Given the recent approval of a once-daily single pill regimen combining dolutegravir with lamivudine and abacavir, we investigated the effect of combining the dolutegravir-specific R263K integrase resistance substitution with either M184I or M184V, two reverse transcriptase substitutions that are associated with resistance against lamivudine and emtracitibine and that are frequently detected in individuals failing therapeutic regimens containing either of the latter agents. Our results show that the presence of R263K/M184I/V in a single virus resulted in further decreases in viral replicative capacity compared to the presence of single substitutions alone. This study supports the use of dolutegravir together with lamivudine or emtracitibine in HIV therapy.
APOBEC3 (A3) proteins are host intrinsic restriction factors that hinder retroviral replication and spread. The most potent members of the human A3 family against HIV-1 include APOBEC3D (A3D), APOBEC3F (A3F), APOBEC3G (A3G) and APOBEC3H (A3H). These enzymes mutate cytosines into uracils in the minus-strand viral cDNA during reverse transcription. Deamination by the A3 proteins occurs in a specific DNA context; A3G prefers to mutate 5’CC substrates whereas all other A3 target 5’TC motifs. Intense mutation of proviral DNA causes premature stop codons and protein malfunction resulting in the abortion of a productive infection. However, the viral infectivity factor (Vif) expressed by HIV gradually induces the degradation of A3 proteins as it accumulates over the course of the infection, thereby allowing for the generation of negligible amounts of mutations as A3 protein levels drop. This sublethal mutagenesis is believed to be beneficial for the virus through genetic diversification and emergence of more fit or drug-resistant variants. Here, since the generation of termination codons occurs by deamination opposite to TGG tryptophan (Trp) codons, we investigated how target site specificity of A3 proteins influences their gene inactivation potency. We found that A3G is extremely potent at introducing termination codons independently of the intensity of deamination as long as Trp codons are present in a gene. In contrast, A3F’s ability to inactivate gene function is highly dependent on the intensity of mutation rather than the generation of stop codons. In summary, our results support that the sublethal mutational burden produced by A3F, and presumably also by A3D and A3H, is likely to result in HIV-1 genetic diversification that is overall more beneficial for the virus than detrimental.
Previous studies have demonstrated that HIV-1 RNA processing can be altered to suppress virus replication by modulating the activity/abundance of the SR or hnRNP proteins of the host cell. Harmine has been shown to inhibit DYRK1a, a host kinase known to regulate RNA splicing. On the other hand, sudemycin c1 has been shown to inhibit SF3b, a host splicing factor. We observed that harmine and sudemycin c1 were potent inhibitors of HIV-1 gene expression, reducing levels of all viral proteins (Gag, Env, Tat) evaluated. In addition, both harmine and sudemycin c1 reduced the overall HIV-1 mRNA species at 3uM in vitro. Consistent with this response, harmine also reduced accumulation of all classes of HIV-1 RNAs and blocked export of viral genomic RNA to the cytoplasm. Finally, both harmine and sucemycin c1 inhibited HIV-1 replication in PBMCs, raising the prospect that this approach could offer an alternative to existing treatments for this infection.
Interestingly, parallel experiments evaluating the effect of DYRK1a depletion revealed that reduction of DYRK1a levels by >90% had little to no effect on HIV-1 gene expression nor did it alter the ability of harmine to repress viral protein synthesis.
Although harmine is also an inhibitor of monoamine oxidase, another inhibitor of this enzyme (moclobemide) failed to elicit a similar suppression of HIV-1 gene expression. Together, the data suggest that the action of harmine on HIV-1 replication is mediated by an unknown host factor and current efforts are directed at defining the host factor(s) mediating its effect. Finally, this study highlights modulating host mRNA splicing machinery as a previously under-appreciated strategy to supress HIV-1 replication.
Human Immunodeficiency Virus (HIV-I) produces the viral accessory protein Nef, and hijacks a variety of the membrane trafficking regulators to facilitate immune evasion. Specifically, the subversion of the phosphofurin acidic sorting cluster protein – 1 (PACS-1) has been demonstrated to be critical for the downregulation of cell surface Major Histocompatibility Complex class I (MHC-I) levels. Past research has largely focused on biochemical assays to dissect the role of Nef in MHC-I down regulation, rather than visualizing interactions within cells. In this report we describe a novel vector system which allows for the simultaneous expression of Nef and host cellular proteins from a single lentiviral vector. Insertion of an F2a cleavage site from the foot and mouth disease virus, enabled concurrent expression of MHC-I or PACS- 1 in addition to Nef. Furthermore, our lentiviral vector system allowed the efficient visualization of the PACS-1/Nef interaction in the endolysosomal network through bimolecular fluorescence complementation, a method used to visualize protein interactions in cells. Altogether, our vector system represents a new and novel way of studying Nef and its multiple functions at the cellular level.
Apoptosis has been proposed as one of the main mechanisms responsible for the CD4 T-cell depletion observed during HIV infection and AIDS. We identified a new compound as an inhibitor of spontaneous and activation-induced cell death of T cells from SIV-infected rhesus macaques (RMs). When injected in SIV-infected RMs during the acute phase of infection, this compound reduced the levels of T cell apoptosis and preserved memory CD4 T cells. This treatment reduced the levels of the inflammatory markers IL-18, CD95L, and sCD14. Although this treatment was limited to the acute phase, the levels of viral replication and SIV DNA remained low during the chronic phase. Most importantly, we prevented the development of AIDS.
This work was supported by ANRS to JE. JE thanks the Canada Research Chair program for financial assistance.
Viruses co-opt and disturb numerous cellular functions upon their entry into cells. RNA interference (RNAi) is a natural mechanism used by eukaryotes for regulating gene expression. This process is dependent upon 20–25-nt exogenous small interfering (si)RNAs or endogenous micro (mi)RNAs, which target mRNAs. siRNAs and miRNAs are incorporated into a complex called the RNA-induced Silencing Complex (RISC). In mammals, the minimal RISC is composed of the RNase Dicer, the TAR RNA binding protein (TRBP), an Argonaute protein and a si or miRNA. The RNAi pathway may restrict virus replication by targeting RNA and viruses have evolved with RNA silencing suppressors (RSS). The HIV Tat protein and the TAR RNA have been described as RSSs, but their activity has been questioned. We tested the RRE RNA for its RSS activity and evaluated its mechanism. We also evaluated if HIV-1 or a lentiviral vector harbouring TAR and RRE could act as RSSs.
We used a cellular reporter gene (RL or EGFP) assay based on miRNA Let7 activity to measure RNAi activity or its suppression. We observed that RRE and TAR, but not Tat, act as RSSs with no modification of the endogenous RISC. Using RNA-immunoprecipitation and gel mobility shift assays, we found that TAR and RRE RNAs displace siRNAs from TRBP. A lentiviral vector expressing TAR and RRE, also suppressed RNAi, but this function was abolished when Rev or GagPol were coexpressed. Because RNAi remained functional in cells replicating HIV-1, we concluded that TAR and RRE RSS activity is largely masked when viral proteins are expressed.
TAR and RRE compete with siRNAs and miRNAs for binding to TRBP, but HIV proteins mask this RSS function. A functional RNAi activity during viral replication validates the use of siRNAs or short hairpin RNAs against HIV as gene or drug therapy.
Measurement of Viral Load (VL) is the most reliable mean for evaluating virological monitoring of the Human Immunodeficiency Virus (HIV) infection. It allows determination of the amount of virus present in a given volume. Due to the constraints of costs, the VL is not often requested for patient’s follow-up in countries with limited resources. Hence the objective of this study is to implement an in-house Quantitative Real-Time PCR to assess the VL of HIV infected patients in Kinshasa.
One hundred and fifty five patients positive for HIV type 1, naive of Antiretroviral Therapy (ART) and eligible for treatment were included in the study. Five milliliter of blood was collected in a tube with anticoagulant. One milliliter of plasma was sent to the laboratory for analysis. After RNA extraction, a Quantitative Real time PCR was performed on a portion of the region of the Long Terminal Repeat (LTR) of the virus.
Of 155 samples received for determination of VL by Quantitative Real-Time PCR, 153 were successfully amplified according to the protocol. The median VL was 301,052.97 copies/mL or 5.48 log10.
The results of VL were used to assess the feasibility of the Real-Time Quantitative PCR. It turns a simple, reliable and less expensive alternative for the diagnosis and virological monitoring of HIV patients under ART.
Viral Load (VL), CD4 T cells count and clinical signs are significant parameters for the AntiRetroViral Treatment decision (ART). The objective of this work is to determine the profile of the Viral Load of eligible patients on treatment in the centers according to the algorithm used in Kinshasa and the DRC.
Our sample consisted of 153 HIV-positive patients naïve to ART. All patients aged over 18 years were included in the study without gender discrimination. The determination of the VL was done at the laboratory of Molecular Biology of the Faculty of Medicine of the University of Kinshasa using a previously described PCR assays.
Of the 153 patients included in the study, 92 (60.1%) were women. The age of the patients was in the range 18 to 65 years with a mean of 37 years. Most patients (91.5%) were in clinical stage 3, while the rest (8.5%) in clinical stage 4 for HIV infection. The rate of CD4 + T lymphocytes was between 8 and 915 cells/mm3 with a median value of 180 cells/mm3. Seventy nine patients (86.8%) had CD4 count under 500 cells/mm3. The median VL of patients included is 5.68 log10 RNA copies/mL. The minimum and maximum values are respectively 0.37 log10 and 7.95 log10 RNA copies/mL.
The majority of treatment-eligible patients (63.4%) in Kinshasa begin Antiretroviral Treatment with a poor prognosis. The Viral Loads are usually very high in these patients and CD4 collapsed.
It has been shown that neutrophil extracellular traps (NETs) can capture HIV-1 virions (Cell Host Microbe, 2012). Notably, it has also been determined that these NETs contain extracellular histones, which also help kill bacteria (Science, 2004). Previously, we identified histones, found in the plasma of the saltwater crocodile (Crocodylus porosus), as possible inhibitors of HIV-1 infection. We have now established the efficacy of histones as novel inhibitors for HIV-1 infection
Infections were conducted using HIV-1IIIB (X4) and p24 as readout and/or recombinant viruses, HXB2 (X4) or JR-FL (R5) and luciferase readout. Human histones H1, H2A, H2B, H3 and H4, or crocodile H2B, were tested for ability to inhibit HIV-1 infection.
Results using human recombinant histones H2B, H3 and H4 were equivocal as consistent inhibition was not obtainable. However, human recombinant histones H1 and H2A were able to inhibit R5 infection by 59% and 60% respectively. Using recombinant crocodile-derived H2B, we could significantly inhibit both X4 and R5 HIV-1, by 73% and 91% respectively. The IC50 for R5 HIV-1 inhibition using recombinant crocodile histone H2B was 0.767ug/mL.
We conclude that extracellular histones found in crocodile blood are significant inhibitors of HIV-1 infection in vitro. Differences between protein sequences of human and crocodile histones appear to affect efficacy for HIV-1 inhibition. Future studies, in vitro and in vivo using a humanized mouse model for HIV-1 infection, with crocodile H2B and with peptides derived from the crocodile H2B, as well as human histones, are planned to further evaluate the significance of extracellular histones in regulating HIV-1 viral infection. Finally, the release of extracellular histones through NETs may represent a novel resistance mechanism to HIV-1 infection.
Host restriction factors constitute an important domain of innate immunity. Some of the well-studied examples include APOBEC3G, TRIM5α, tetherin, IFITM and SAMHD1. A recent addition is the human myxovirus-resistant protein B (MxB, also called Mx2) that strongly inhibits HIV-1 infection. Results from several groups demonstrate that MxB impedes the nuclear import of HIV-1 DNA by interacting with viral core complex and likely delaying viral uncoating. In support of this mechanism of action, multiple mutations in HIV-1 capsid have been reported to resist MxB inhibition. It is noteworthy that these MxB-resistant capsid mutations reduce HIV-1 infectivity and thus are not found in HIV-1 isolates from the infected patients. In order to determine whether HIV-1 has naturally evolved to evade MxB restriction, we tested a group of transmitted funder (T/F) HIV-1 strains for their sensitivity to MxB. Two T/F strains were found refractory to MxB inhibition and this resistant phenotype was further mapped to two amino acid positions Q87 and A208 in viral capsid. The Q87 variant is located in the cyclophilin A (CypA) binding loop and has a prevalence of 21% in HIV-1 sequences registered in HIV database (
The existence of antisense transcription in HIV-1 has been suggested over the years and readdressed in several recent reports. Furthermore, this transcript has been proposed to encode a protein termed Antisense Protein (ASP). Using a codon-optimized ASP expression vector, we have previously detected ASP in various mammalian cell lines by Western blot (WB), flow cytometry and confocal microscopy analyses. Our results have further demonstrated that this protein forms multimers, is unstable and is capable of inducing autophagy, which likely accounts for difficulties in its detection in infected cells. The aim of the study was to further examine the pathway leading to induction of autophagy by ASP.
We first performed confocal microscopy analyses to search for potential co-localization between ASP and all Autophagy-related Genes (ATG). COS-7 and HeLa cells were transfected with an ASP expression vector and tested for its distribution with respect to p62 (SQSTM1) or LC3B, two well-known autophagy markers. Our analyses clearly demonstrated that these proteins co-localized with ASP in various cellular structures, some of which were autophagosome-like. Furthermore, co-Immunoprecipitation experiments confirmed the interaction between LC3B and ASP in COS-7 cells. We next analyzed various ASP deletion mutants by confocal microscopy and Western blot in COS-7 cells, and found that the proline-rich domain was needed for ASP multimerization, its cellular distribution, and induced autophagy. We have also initiated a series of experiments with ASP genes from various HIV-1 clades. Our preliminary results show that HIV-1 clade A ASP which lacks the first 25 amino acids, is more stable and presents a distinctive cellular distribution, when compared to other HIV-1 clades. We are currently assessing the capacity of the various clade-specific ASP to induce autophagy.
Overall, our results demonstrate that ASP-induced autophagy might be associated with various ATG proteins and that the extent of autophagy induction might be clade-dependent.
Splicing of HIV-1 viral transcript is highly controlled, and plays a crucial role in HIV-1 replication and gene expression. For generation of all mRNAs required, HIV-1 utilizes alternative splicing to produce three classes of viral RNAs (unspliced, singly spliced and multiply spliced RNA), totally over 40 mRNAs, from one single 9 kb pre-mRNA transcript. Manipulating this alternative splicing process will alter HIV-1 RNA splicing and further affect viral protein synthesis, resulting in the failure of infectious virion assembly. Given HIV-1 resistance to the standard highly active anti-retroviral therapies (HAART), we have shown that targeting viral RNA processing is a viable approach for HIV treatment.
HIV-1 mRNA processing requires the combinatory usage of four 5′ splice sites and eight suboptimal 3′ splice sites. Host splicing factor SR proteins were reported to be involved in the use of 3′ splice sites to regulate HIV RNA splicing. To identify host factors crucial for this process, the knockdown of various SR proteins was performed, and the effects on HIV-1 protein expression and viral RNA processing were examined. The knockdown of SRp20 showed the most significant effect in the expression of viral structural proteins Gag and Env. The reduction of SRp20 level led to significant increase of unspliced viral RNA accumulation. On the other hand, the expression of viral regulatory protein Tat was attenuated. Subsequent analysis also revealed that the depletion of SRp20 resulted in alteration of splicing site usage of tat mRNAs in both MS and SS viral RNAs. Together, these studies suggest SRp20 to be a key host regulator of HIV-1 RNA processing and replication, and might be used as the target to control HIV-1 infection.
In spite of the enormous effort to combat the human immunodeficiency virus type 1 (HIV-1), it remains largely unknown how this deadly pathogen thrives in the face of the host antiviral response. In contrast to many pathogenic viruses, HIV-1 relies mostly on a single gene product, Nef, to evade the immune response. The Nef protein mediates immune evasion in part by binding to host cellular proteins, including the membrane trafficking machinery, to induce endocytosis of the cell surface receptor major histocompatibility complex class I (MHC-I). MHC-I downregulation by Nef results in decreased cytotoxic T lymphocyte mediated killing of HIV-1 infected cells. However, the pathway utilized by Nef to mediate MHC-I endocytosis has yet to be fully elucidated. We hypothesized that this pathway utilizes membrane trafficking regulator proteins within the sorting nexin family. Using recombinant proteins, we demonstrate a novel interaction between the sorting nexin 18 (SNX18) protein and HIV-1 Nef. Furthermore, the Nef-SNX18 interaction is dependent on the SH3 domain of SNX18 and a polyproline motif on Nef. Ultimately, we propose that a Nef-SNX18 interaction plays a role in Nef-mediated MHC-I downregulation. The identification of novel protein interactions necessary for Nef-mediated immune evasion may contribute to the development of HIV-1 therapeutics targeting the activities of Nef.
APOBEC3 proteins are host intrinsic restriction factors that have a key role in protecting humans and other mammals against retroviruses, most notably HIV. These enzymes interfere with their replication and intensely mutate their DNA, thereby inactivating viral progeny and the spread of infection. Here we describe a new way that some retroviruses have evolved to defend themselves against the mutator activity of APOBEC3 proteins. We show that gammaretroviruses expressing an accessory protein called glyco-Gag, or gPr80, use the host’s posttranslational machinery, and more specifically N-linked glycosylation, as a way to regulate their sensitivity to APOBEC3 induced mutagenesis. By carefully governing the intensity of mutations caused by APOBEC3 proteins, gammaretroviruses can turn an otherwise restricting mutator activity to their advantage to help them evolve and persist. This new mechanism could potentially be exploited as a novel way to inhibit APOBEC3 protein activity in humans infected with HIV, which has been linked to immune viral escape and drug resistance.
With the increase of resistant HIV-1 strains worldwide the need for new drug targets has become increasingly urgent. One such focus is viral mRNA processing, where a delicate balance of host factors is required to regulate the generation of over 40 HIV-1 mRNAs required for complete viral protein expression. Host nuclear ribonucleoproteins (hnRNPs) are complexes of protein involved in post-transcriptional metabolism of RNAs to ensure optimal processing before export to the cytoplasm. Since disruption of HIV-1 RNA processing inhibits efficient replication, the effect of hnRNP knockdown on viral protein expression was examined. In this study, lentiviral vectors containing short hairpin RNAs (shR-NAs) targeting several hnRNP mRNAs were used in the context of a doxycycline-inducible HIV-1 provirus. Depletion of hnRNPs C, L, and M mRNA had negligible effect on HIV-1 mRNA and protein expression by qRT-PCR and western blot. In contrast, knockdown of hnRNP G mRNA resulted in a decrease of HIV-1 unspliced (US), singly-spliced (SS) and multiply-spliced (MS) RNAs (corresponding to Gag, p14 Tat and p16 Tat respectively). These results correlated with a decrease in Gag protein levels and an increase of p14 or p16 Tat. Depletion of hnRNP U resulted in an increase of all three HIV-1 RNAs, particularly MS, with only a 52% decrease p14 Tat and an overall increase of Gag and p16 Tat. Analysis of specific splice site usage is still ongoing, but the data emphasizes the influence of specific hnRNPs on HIV-1 RNA processing and their potential as new therapeutic targets.
Complete viral clearance in HIV-infected patients is significantly impeded by the formation of latent HIV reservoirs, which are unresponsive to current therapies. Oncolytic viruses (OVs) are currently being investigated as a novel approach to eliminating the HIV reservoir. However, for this to be a viable treatment option it is imperative that healthy cells are spared from cytopathic effects. Two OVs, Maraba (MG1) and Vesicular Stomatitis Virus (VSV-Δ51), have been designed to preferentially infect cancer cells lacking a robust Type I IFN response. Similar impairment of Type I IFN-mediated antiviral proteins has been described during HIV infection. Thus, we hypothesize that MG1 and VSV-Δ51 will spare healthy cells, while specifically infecting and killing HIV-infected cells.
Human CD4+ T cells and monocytes were isolated from healthy donors’ blood. CD4+ T cells (unstimulated or PHA/IL-2 stimulated), monocytes, and MDMs were infected with increasing MOIs of GFP-expressing MG1 or VSV-Δ51. At various time points, GFP expression and PI staining were measured by flow cytometry to quantify productive infection and cell viability, respectively. The ability of resting CD4+ T cells to express specific IFN-stimulated genes involved in viral clearance was also investigated. Cells were treated with IFN-α and expression of PKR was assessed by immunoblotting.
Primary monocytes, MDMs, and unstimulated CD4+ T cells were relatively resistant to OV infection and killing. However, PHA/IL-2 treated CD4+ T cells exhibited increased GFP expression in a dose dependent manner. Preliminary results demonstrate a loss in viability during VSV-Δ51, but not MG1 infection. Increased PKR expression following IFN-α treatment in both resting and PHA/IL-2 stimulated CD4+ T cells demonstrated a robust response to exogenous Type I IFN.
These results suggest that healthy immune cells are resistant to OV infection and cytopathic effects. Future experiments will investigate both specificity of OV infection and expression of Type I IFN-mediated antiviral proteins during HIV infection. This may lend feasibility to the eradication of the latent HIV reservoir by MG1 or VSV-Δ51 treatment.
In the gut-associated lymphatic tissues, Th17 cells maintain homeostasis and initiate inflammatory responses against pathogenic microbes. During HIV infection, Th17 cells become depleted for unknown reasons and do not become fully restored following HAART. IL-7 is a cytokine that promotes T cell survival and Th17 cell development. Although impaired IL-7 responses have been reported in HIV-infected CD4+ T cells, the effects of HIV on IL-7 responses in Th17 cells have yet to be investigated.
HIV inhibits Th17 cell survival by dysregulating IL-7 signalling.
CD4+CXCR3-CCR6+ memory T cells (436 cells), which represent a Th17-enriched population, were isolated from PBMCs from uninfected donors. To characterize IL-7 receptor expression on Th17 cells, 436 cells were stimulated with PMA/ionomycin and treated with Brefeldin A to induce expression of intracellular IL-17A, the main effector cytokine secreted by Th17 cells. Cells were stained for CD127, the unique IL-7 receptor α component, and IL-17A. To characterize IL-7 responses in Th17-enriched cells, 436 cells were stimulated with IL-7 and stained for phospho-STAT5 and the anti-apoptotic protein Bcl-2. Cell proliferation was measured by CFSE dilution assay.
CD127 was expressed on 50% of total 436 cells and 50% IL-17A+ 436 cells (Th17 cells). Less than 5% of the 436 cells expressed phospho-STAT5 under basal conditions, which increased to 80% following stimulation with IL-7 (0.1ng/mL). Roughly 15% unstimulated 436 cells expressed Bcl-2, while IL-7 (5ng/mL) stimulation increased the frequency of Bcl-2-expressing cells to 50%. Lastly, fewer than 5% 436 cells proliferated when cultured with media alone, whereas IL-7 (10 ng/mL) induced proliferation in 80% of the cells.
Th17-enriched cells are responsive to IL-7, suggesting an important role for IL-7 in promoting Th17 cell survival. Studying the effects of HIV on IL-7 signalling in 436 cells may provide insight into the mechanisms of Th17 cell depletion during infection.
During HIV infection, CD16+ monocytes representing a source of pro-inflammatory cytokines, are highly expanded (up to 50% in AIDS patients), and their frequency is not normalized under ART. Monocytes are precursors for dendritic cells (DC). Functional characteristics of CD16+ versus CD16- monocyte-derived DC (MDDC) during HIV infection remain unknown. To explore the contribution of these MDDC subsets to HIV pathogenesis, we investigated their transinfection ability and immunogenic potential in relationship with their transcriptome and autophagy markers.
Monocytes were isolated from PBMC of healthy donors by negative selection using magnetic beads (Miltenyi). CD16+ and CD16- monocytes were sorted by flow cytometry (BD-AriaII). MDDCs were obtained upon monocyte culture in presence of GM-CSF/IL-4. The trans-infection ability (intracellular HIV-p24, HIV-p24 ELISA, HIV-DNA PCR) and immunogenic potential (CFSE dilution assay) were evaluated by co-culture of MDDC with autologous CD4+ T-cells in the presence or absence of HIV (NL4.3BaL) and antigens (SEB, CMV, C. albicans, S aureus) or CD3/CD28 Abs. MDDC were pretreated with the autophagy activator rapamycin or inhibitors bafilomycin and 3-MA. Genome-wide transcriptional analysis (Affymetrix) and quantification (western blotting) of autophagy markers were performed in MDDC subsets.
CD16+ versus CD16- MDDC exhibit a superior ability to trans-infect CD4+ T-cells and a reduced ability to induce the antigen-specific proliferation of CD4+ T-cells and their subsequent production of Th17 cytokines. Rapamycin decreased the HIV trans-infection ability of total MDDC, while bafilomycin and 3-MA did not significantly affect this process. A molecular signature associated with pathogenicity on CD16+ MDDC was identified, together with alterations in the autophagy process.
These results emphasize the critical role played by CD16+ MDDC in HIV pathogenesis and suggest that autophagy alterations are linked to pathogenicity. New therapeutic strategies aimed at restoring autophagy in CD16+ MDDC may improve their immunogenic potential while limiting viral dissemination in HIV-infected subjects.
HIV-1, the etiological agent of the AIDS epidemic, can be categorized into evolutionarily distinct clades. The most prevalent of these clades is Group M (Main) which can be further subdivided into 9 subtypes: A–D, F–H, J and K. The predominant viral subtypes of the epidemic in Sub-Saharan Africa are A, D and C, with subtype C making up more than 50% of infections alone. Unfortunately, despite their importance in global health, these subtypes are studied far less frequently relative to subtype B, the predominant viral subtype in North America and Europe. This study sought to compare rates of pathogenesis between these understudied subtypes in a natural history cohort.
A cohort of HIV positive Zimbabwean and Ugandan women with known dates of infection had their CD4+ memory T-cell and viral loads monitored every three months post-infection. Subtype was determined via PCR of the viral envelope. All analyses were done using Generalized Estimating Equations (GEEs, n=302) and Generalized Linear Models (GLMs, n=68).
These data showed distinct patterns of T-cell subset decline between HIV subtypes. Infection with a subtype C virus shows a significantly slower rate of cell decline in both total CD4+ cells as well as in CD4+ memory subsets compared to subtypes A and D (p<0.01 and p<0.003 respectively). Additionally, subtype C infections demonstrate a significantly longer time to viral load set point with no difference in total viral load at set point relative to subtypes A and D (p=0.009 and p<0.001 respectively). Finally, acute early viruses (within 3 months of infection) were isolated from this cohort and their viral envelopes were cloned into a reporter virus for ongoing work on infection in human primary PBMCs.
Disease stemming from infection by an HIV subtype C virus progresses at a diminished rate compared to subtypes A and D.
HIV-1 resistance to current antiretroviral therapies requires the development of innovative strategies to control this infection. We aimed at developing novel inhibitors of HIV-1 replication that target viral RNA processing, a stage dependent on host splicing. We recently reported that the cardiac glycoside (CG) digoxin, a drug treating heart failure, is a potent inhibitor of this stage. CGs are known to bind the Na+/K+-ATPase and increase intracellular Ca2+ concentration ([Ca2+]i), leading to undesired toxicities and cardiac arrhythmias. However, since CGs were discovered to induce multiple signaling pathways, we hypothesized that CGs may alter HIV-1 RNA processing independent of [Ca2+]i changes. We identified that members of the cardenolide and bufadienolide classes block HIV-1 (Gag) gene expression (GE) without cytotoxicity compared to control. Suppression of HIV-1 GE occured even if [Ca2+]i influx via the reverse mode of the Na+/Ca+-exchanger—responsible for triggering arrhythmias—is blocked by KB-R7943. Digitoxin, digitoxigenin, and RIDK-34 potently inhibited viral replication of HIV clinical isolates like digoxin (IC50: 1 nM) at concentrations below those treating heart conditions (digitoxin: 26–46 nM). Similar to digoxin, all CGs dramatically suppress late phase expression of HIV-1 structural proteins (Gag/Env) and p14-Tat with marginal effects on the early phase factor p16-Tat. However, digoxin-like CGs (digoxin/digoxigenin/lanatoside-C/RIDK-34) drastically deplete Rev while other CGs (digitoxin/digitoxigenin/ouabain) had low/no effect on this factor. All CGs induce oversplicing of HIV-1 RNAs, reducing unspliced and singly spliced mRNAs encoding Gag and Env/p14-Tat, respectively. Like digoxin, changes in viral RNA levels were associated with hyperphosphorylation of SRp20 and modification of Tra2β, whereas digitoxigenin causes de-modification of these splicing factors. Moreover, digoxin/digitoxin/ouabain but not digitoxigenin inhibited HIV-1 GE through activation of MEK1/2 in a manner independent of canonical/arrhythmogenic mechanisms. Although all CGs supposedly modulate [Ca2+]i, this study suggests that modulation of the Src-EGFR-Ras-Raf-MEK1/2-ERK1/2 pathway could be used to control HIV-1 infection.
Interleukin-7 (IL-7) regulates the development, homeostasis and cytotoxic (CTL) activity of CD8+ T-cells. IL-7 downregulates the expression of the membrane bound IL-7 receptor α chain (mCD127) and induces the release of a soluble form (sCD127). sCD127 alters IL-7 activity and plasma concentrations are increased in the course of HIV infection. Despite the potential biological importance of sCD127, the mechanisms of its production and release have been only partially described.
Human thymocytes and blood-isolated CD8+ T cells were treated with IL-7 (10ng/mL) and TcR-stimulating antibodies (anti-CD3/CD28 1μg/ml each). Culture supernatants were collected every 24 hours over 96 hours and sCD127 concentration was measured by ELISA. To evaluate the contribution of shedding of mCD127, surface protein biotinylation assays were performed and analyzed through Western blots. To further characterize the signaling pathways leading to the shedding of sCD127, pharmacological inhibitors for JAK, STAT5 and PI3K were also used. Specific MMP2/9 and MMP3 inhibitors were used to evaluate the implication of proteolytic cleavage in sCD127 release. MMP9 and MMP2 levels where measured by specific ELISA to further determine their roles in the release.
Biotinylation assays revealed that shedding of mCD127 contributes to the release of sCD127. In combination with pharmacological inhibitors, biotinylation assays also revealed that JAK/STAT5 and PI3K pathways were both involved in the shedding of sCD127 by CD8+ T-cells. Proteolytic cleavage by MMP2 and 9, but not MMP-3 contributes to IL-7/TcR-induced sCD127 release by CD8+ T-cells. In human thymocytes both MMP2 and MMP9 were shown to be involved in the basal release of sCD127. MMP9 levels in supernatants decreased upon IL-7/TcR stimulation.
Our results demonstrated that sCD127 release can mediated by different mechanisms. Firstly, IL-7/TcR stimulation can induced direct shedding of mCD127 from the cell surface, which is mediated by the JAK/STAT5 and PI3K pathways. Secondly, a shedding-independent mechanism of sCD127 production relies on MMP2 and 9 activities. Furthermore, the role of mRNA splicing in the IL-7/TcR-induced release of sCD127 is under investigation.
HIV infected individuals are more prone to develop secondary bacterial infections as well as having elevated levels of bacterial endotoxin which contribute to a chronic state of inflammation. A potentiated inflammatory response occurs in the presence of co-infections due to dysregulated immune cells making them more sensitive to subsequent exposure to bacterial products. As infection- induced inflammation alters the expression of many drug transporters and metabolising enzymes, it is plausible that this may be potentiated in HIV infected individuals due to augmented inflammatory responses. Similar to humans the HIV-1 transgenic rat (HIV-Tg) develops immune disorders and AIDS associated conditions. Therefore, our objective was to examine the impact of endotoxin administration on hepatic gene expression of drug transporters in HIV-Tg rats. Three month old HIV-Tg male and female rats or wild-type littermates (WT) were treated with 5 mg/kg endotoxin or saline (n=4–9/group), animals sacrificed and tissues collected 18 hr later. Gene expression was measured in liver using qRT-PCR and cytokine levels were measured in serum using ELISA. An augmented inflammatory response was seen in the HIV-Tg group, and was associated with significantly greater endotoxin- mediated downregulation of Ent1/Slc29a1 in HIV-Tg rats. In addition, a pronounced endotoxin- mediated downregulation of Abcb1a, Abcc2, Abcg2, Abcb11, Slco1a2, Slco1a4, Slco1b2, Slc10a1, Slc22a1, Cyp3a2 gene expression was seen in the HIV-Tg rats which was similar to WT groups. As several hepatic transporters are involved in the transport of bile salts, we observed significantly higher total bile acid concentrations in the serum of endotoxin-treated HIV-Tg as compared to endotoxin-treated WT. Our results indicate that the endotoxin-mediated down-regulation of numerous hepatic transporters in HIV-Tg rats increases the possibility that heptobiliary drug clearances may be altered in the HIV population due to co-existing infections or inflammatory conditions. This may be important in identifying potential drug-disease interactions.
The in-house technical or experimental methods are increasingly recommended for their low cost reagents for the determination of the Viral Load (VL) in resource-limited settings. The objective of this study was to compare the determination of VL from HIV-1 non-B samples by an in-house technic with the COBAS Ampliprep/TaqMan version 2.0.
In this cross-sectional study, 39 plasma samples from patients infected with HIV type 1 non-B from N’Djamena and Kinshasa were used to determine the VL using the two techniques.
The mean values of VL are respectively 4.68 ± 1.26 and 4.58 ± 1.33 log10 copies of RNA/ml for the COBAS Ampliprep/Taq-Man assays and the in-house PCR assays. A good correlation (Spearman Correlation) was obtained, with a coefficient (R2) of 0.9452 (p <0.001).
This work demonstrates that there is no significant difference between the results of VL determined by the COBAS Ampliprep/TaqMan assays and the in-house assays used.
The prevalence of neurological complications in Human Immunodeficiency Virus-1 (HIV-1) infected patients is dramatically increasing with about 50% of patients developing HIV-associated neurocognitive disorders (HAND). Clinically, these patients appear to be aging prematurely; perhaps due to persistent low-level viral replication and associated inflammation in the brain leading to neuronal loss. The goal of this project is to implement an in vivo rat model of HIV-1 brain inflammation and neurocognitive dysfunction by intracerebroventricular (icv) administration of HIV-1 viral coat protein, gp120, in adult male Wistar rats and to investigate the effect of anti-inflammatory compounds on both the inflammatory/oxidative stress responses and prevention/improvement of neurocognitive deficits. Anesthetized rats were injected icv with a single dose of R5-tropic gp120¬ADA in both lateral ventricles (4ug/ventricle). Lipopolysaccharide (LPS), a bacterial endotoxin that has been reported to induce inflammation and behavioural abnormalities, was administered icv as a positive control. Real-time qPCR was used to assess gene expression of inflammatory/oxidative stress markers in different regions of the brain. Cognitive deficits in spatial learning and memory were characterised using the Morris Water Maze test. Immunoblotting of caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) will be used to examine extent of neuronal apoptosis and death. Our data demonstrate high levels of cytokines (IL-1b and TNFa) and oxidative stress marker (iNOS) transcripts in the brain post injection of gp120/LPS compared to control saline. Rats injected with a single dose of 50ug LPS displayed significantly impaired spatial learning/memory compared to saline controls. However, gp120 injection did not result in significant impairments, possibly due to insufficient neuronal apoptosis. Additional biochemical analyses are in-progress to characterise expression of the apoptotic marker caspase-3 and DNA fragmentation. Our gp120 and LPS-induced neuroinflammatory rat model could constitute a useful tool to examine the potential effect of anti-inflammatory drugs in reversing HAND.
Transporters expressed in the kidney can impact the renal clearance of many drugs including antiretroviral agents. Bacterial co-infections, low grade endotoxemia and immune activation are common in chronic HIV (+) patients. Inflammation due to endotoxin or HIV may influence the expression and activity of drug transporters in the kidney. The HIV-transgenic (HIV-Tg) rat develops immune dysfunction and AIDS associated conditions similar to humans. Thus, our objective was to study the effect of endotoxin on the renal expression of drug transporters in an HIV1-Tg rat model.
Five month male HIV1-Tg rats or wild-type (WT) littermates were treated with endotoxin (5 mg/Kg, i.p.) or saline (n=7/group). The mRNA expression of transporters and cytokines were measured 18 hours after treatment in kidney samples using qRT-PCR. Serum cytokine levels were measured by ELISA.
As compared to WT, basal expression of IL-1β, IL-6, Oat2 and Urat1 was significantly higher in HIV-Tg rats while OCT1 levels were decreased. Endotoxin induced serum levels of inflammatory cytokines in both HIV1-Tg and WT, however interferon induction was significantly diminished in HIV-Tg. Endotoxin significantly down-regulated the expression of Mdra1, Oct3, Oat2, Urat1, Mate1 and Pept2 in both HIV-Tg and WT. Endotoxin significantly up-regulated the expression of Oct1 and Pept1 in HIV1-Tg but not WT. Levels of Mdr1b, Mrp4 and Octn2 were not changed.
Our results demonstrate that HIV and endotoxin- induced inflammation imposes alterations in the expression of many clinically important drug transporters in the kidney. Therefore the renal clearance of drug substrates could be altered in patients with co-existing infections. This may provide new insight to potential drug-disease interactions.
The unprecedented Ebola outbreak in West Africa has infected 20,206 people and claimed 7,906 lives, destabilizing medical infrastructure and preventing the proper treatment of other infections such as HIV-1. Experimental nucleoside analogues that inhibit Ebola RNA replication, such as brincidofovir and favipiravir, are being fast-tracked for the potential treatment of Ebola virus disease (EVD). However, it remains untested whether potent nucleoside inhibitors with well established safety and efficacy in treating HIV-1 show effectiveness in inhibiting Ebola.
We infected 293 T cells with transcription and replication competent virus-like particles (trVLPs) related to
5uM of lamivudine, zidovudine or tenofovir inhibited the transcription and/or translation of Ebola by 49–62%, and combination of these three analogues caused an additive inhibition of Ebola replication by as much as 87%, and outperformed all other drugs tested. This increased to 97% by the second passage of cells. Maraviroc had no effect.
| uninfected cells | -- | 1.09% (± 0.61) | <0.001 |
| infected cells | -- | 100.00% (± 13.73) | -- |
| toremifene | 5 | 28.03% (± 12.39) | <0.01 |
| cidofovir | 100 | 19.73% (± 5.78) | <0.01 |
| favipiravir | 100 | 14.06% (± 3.16) | <0.001 |
| lamivudine + | 5 | 17.00% (± 5.39) | <0.01 |
| zidovudine + tenofovir |
Our results demonstrate that widely available nucleoside analogues clinically used to treat HIV-1 show activity against Ebola. Moreover lamivudine, zidovudine and tenofovir are on the WHO list of Essential Medicines and could be used in resource-limited settings. Thus combination antiretroviral therapy (cART), successful at treating HIV-1, may be effective for treating EVD.
Engagement in HIV care is critical to achieving and maintaining optimal health outcomes. We wished to determine factors associated with transitions among recommended and low engagement care states.
We analysed data from OHTN Cohort Study participants who had initiated cART and achieved virologic suppression, completed ≥1 extended questionnaire, and had ≥2 viral load (VL) results within 2 years of a questionnaire. Six states of HIV care were defined: (1) cART Initiation: first year after the first observed undetectable VL, (2) suppressed VL (VL<50 copies/mL), (3) engaged: unsuppressed VL, <6 months since last VL, (4) unengaged: unsuppressed VL, >6 months since last result, (5) loss to follow-up and (6) death. Multi-state models were used to determine hazard ratios (HR) of transitioning between states 2 and 3, and between states 3 and 4 associated with time-updated covariates.
1338 participants were included. At baseline, 81% were male, 9% were IDU, with median age of 40 (IQR 34–47), depression score of 20 (IQR 6–40), and total stress score of 5 (IQR 3–8). Transitions between engaged and unengaged states were more likely in later calendar years and less likely among women, relative to MSM. Older participants were more likely to maintain virologic suppression and re-achieve it after virologic failure. Higher levels of depression were associated with increased risk of virologic failure and higher levels of stress were associated with lower likelihoods of re-achieving virologic suppression.
| State2-State3 | State3-State2 | State3-State4 | State4-State3 | |
| HR (95% CI) | HR (95% CI) | HR (95% CI) | HR (95% CI) | |
| Calendar Year | 0.81 (0.77–0.86) | 1.00 (0.96–1.04) | 1.24 (1.08–1.42) | 1.39 (1.18–1.63) |
| Age (per 5 years) | 0.94 (0.89–0.99) | 1.08 (1.04–1.13) | 0.93 (0.84–1.03) | 0.98 (0.86–1.12) |
| Females (ref=MSM) | 1.02 (0.81–1.29) | 0.97 (0.79–1.19) | 0.45 (0.28–0.71) | 0.34 (0.20–0.60) |
| Heterosexual Male (ref=MSM) | 0.91 (0.70–1.19) | 0.90 (0.72–1.13) | 1.40 (0.62–3.13) | 1.32 (0.48–3.61) |
| Injection drug use | 1.03 (0.75–1.42) | 0.98 (0.75–1.28) | 0.87 (0.47–1.61) | 0.59 (0.28–1.25) |
| Depression (per 5 points) | 1.04 (1.02–1.06) | 1.01 (0.99–1.02) | 0.99 (0.94–1.03) | 1.00 (0.94–1.06) |
| Stress (per event) | 1.00 (0.98–1.03) | 0.96 (0.94–0.98) | 1.04 (0.98–1.10) | 0.98 (0.91–1.04) |
Management of stress and depression and understanding barriers to care among women may improve engagement in HIV care.
Building Bridges was a collaboration between Aboriginal and allied stakeholders and the Canadian Observational Cohort (CANOC) collaboration. Working with community and CANOC investigators, a review of treatment interruptions was identified as a proxy for health care engagement. Accordingly, this endpoint of interest was compared between Aboriginal and non-Aboriginal CANOC participants.
CANOC participants are antiretroviral treatment-naïve individuals who initiated combination antiretroviral therapy (cART) after January 1, 2000. Cox proportional hazard models were used to estimate the effect of ethnicity on time to treatment interruption. Treatment interruptions were defined as interruptions of all antiretroviral medications for at least 90 consecutive days. Models were adjusted for age, gender, injection drug use, calendar year of cART initiation and province of residence.
A total of 8260 participants, including 416 Aboriginal participants, were included in the analysis. 197 (47%) Aboriginal participants and 1498 (18%) non-Aboriginal participants had a treatment interruption. The median time to a first treatment interruption after treatment initiation was shorter among Aboriginal participants (0.55 years) than among participants of other ethnicities (1.07 years) (p<0.001). In Cox regression analysis, Aboriginal ethnicity (adjusted hazard ratio [aHR]=1.39, 95% CI: 1.19–1.63), residing in British Columbia (aHR=1.80, 95% CI: 1.61–2.01), injection drug use (aHR=2.43, 95% CI: 2.16–2.72), and female gender (aHR=1.59, 95% CI: 1.42–1.77) remained independent predictors of increased risk of treatment interruption. Treatment interruptions were less likely in later calendar years (aHR=0.67, 95% CI: 0.59–0.76 for 2003–05, aHR=0.48, 95% CI: 0.41–0.54 for 2006–08 and aHR=0.33, 95% CI: 0.28–0.38 for 2009–12 relative to 2000–02).
Among CANOC participants initiating cART, Aboriginal participants were found to have a shorter time to treatment interruption. Efforts to provide culturally safe care and facilitate continuity of care, particularly among mobile populations may be helpful in reducing treatment interruptions among Aboriginal people living with HIV, particularly women. Injection drug use was also found to be a significant predictor of treatment interruption and interventions are needed to support consistent ART use among populations who use injection drugs.
Continuity of care is well recognised within HIV care programs for effective HIV management but there is less focus on maintaining continuity between HIV care centers. We compared the clinical profiles of patients before and after transferring care to the Southern Alberta Clinic (SAC) in order to determine the clinical impact of disruptions in the continuity of care as a consequence of moving between HIV care centres.
HIV patients transferring to SAC from another care centre within Canada between 1/1/2006–1/1/2013 were reviewed. We determined the patient’s CD4, Viral Load, and ART status at last date of prior clinic contact and the first clinic contact date at SAC. We categorized patients as ‘continuous care patients’ (CCP) or ‘non-continuous patients’ (NCCP) based on the time gap between visits (±180 days respectively). We analyzed the impact on clinical factors between continuous versus non-continuous patients.
193 HIV-experienced individuals transferred care to SAC. The median time from last to initial SAC visit was 134 days [80–324]. 42% of patients were on ART at first SAC visit. Median CD4 count decreased from 410/mm3 to 371/mm3. 47% of all transfer patients had detectable viral loads (i.e. >400) at first SAC visit. NCCP had longer gaps in treatment (median-431 vs. 94 days), had steeper declines in median CD4 counts (460/mm3 to 272/mm3 vs. 400/mm3 vs. 407/mm3), and had higher proportions of detectable viral loads (61.2% vs. 41%) (all p<0.01) respectively.
Almost 50% of patients transferring care had detectable viral loads, and >25% had major gaps in care. Patients accessing care within 6 months, following recommended guidelines, had only minimal changes in health whereas patients delaying care had significant decreases in CD4 and higher rates of unsuppressed viremia. It is clinically important for patients to maintain HIV care when transferring between HIV care centers.
The ultimate goal of antiretroviral therapy (ART) is to control HIV replication while minimizing adverse drug reactions (ADRs). Despite their potency and efficacy, many ARVs cause adverse effects and can compromise patient adherence. This study aims to assess the burden of ADRs on treatment satisfaction and adherence among HIV patients.
During July and August 2014, we conducted a cross-sectional study on ADRs of ARVs at Clinique l’Actuel. All HIV-patients attending a follow-up visit received an auto-administered questionnaire about potential ADRs, ART satisfaction and self-reported adherence. Physicians also answered questions regarding their perception of the ADRs reported by the patients. The data was analysed using correlation and logistical regression analyses.
361 treated HIV-patients were included. Mean age was 50y. Patients were infected for 15 y and 114 (32%) were on their first-line treatment. 86% were MSM, 8% IDU and 4% from endemic regions. Globally, 81% were very satisfied with their ART, despite 95% reporting ADRs (73% CNS problems, 63% less energy, 61% sexual problems, 61% GI, 51% sleeping, 42% dermatological problems). Nevertheless, 81% were highly disturbed by the ADRs. The most disturbing ADRs were sleep (42%) and sexual (42%) problems. In the adjusted model, the presence, number and types of ADRs were not significantly associated with satisfaction. Only IDU were significantly unsatisfied with ARVs (OR=0.36;p=0.016). Moreover, adherence to treatment was higher in men (OR=6.22;p=0.005) and among those who were satisfied with their ARVs (OR=3.13;p=0.034) when controlling for type and number of ADRs, pill burden and other covariables. Physicians discussed the reported ADRs in 79% of cases and felt that ADRs were mostly unrelated to ART. Medication was changed in 19 patients (5%).
Our results underlined the high frequency and disturbance of ARV-related adverse drug reactions. However, ADRs did not preclude adherence to treatment for our patients.
Hepatitis C virus (HCV) is both a risk factor for and common comorbidity associated with HIV. Individuals with HIV/HCV co-infection face serious health risks including risk of end-stage liver disease and mortality. The purpose of this study was to compare socio-demographic and clinical characteristics between HIV/HCV co-infected and HIV mono-infected individuals and to determine covariates of optimal ART adherence among co-infected individuals enrolled in a large cohort of HIV-positive individuals in British Columbia, Canada.
The study utilizes survey data from the Longitudinal Investigations into Supportive and Ancillary Health Services (LISA) study collected between 2007 and 2010 by the British Columbia Centre for Excellence in HIV/AIDS. This cross-sectional data is linked with longitudinal clinical data through the provincial Drug Treatment Plan (DTP). HCV co-infection was determined through self-report. Optimal ART adherence was defined as ≥95% based on pharmacy refill compliance. Multivariable logistic regression models compared optimal adherence by HIV/HCV co-infection, as well as independent covariates of optimal ART adherence among co-infected individuals.
Of 912 individuals (28.2% women) included in this analysis, 536 (58.8%) were HIV/HCV co-infected. In adjusted multivariable analysis, co-infected individuals were significantly more likely to have a history of IDU (adjusted odds ratio [AOR]: 20.8; 95% confidence interval [CI]: 11.2 to 38.5) and incarceration (AOR: 2.52; 95% CI: 1.41 to 4.51), and less likely to be optimally adherent (AOR: 0.53; 95% CI: 0.28 to 0.99). Optimal adherence among HIV/HCV co-infected participants was associated with stable housing (AOR: 1.86; 95% CI: 1.14 to 3.05) and accessing an adherence support program (AOR: 4.76; 95% CI: 2.62 to 8.57).
HIV/HCV co-infected individuals exhibit significantly lower ART adherence than HIV mono-infected individuals, however, stable housing and adherence support services were associated with improved adherence within this population. The findings highlight the importance of integrating social services within treatment programs.
Retention in care is associated with improved clinical outcomes. Our objectives were to report the frequency of and risk factors for missing physician visits and having gaps in care, as measures of suboptimal retention.
Patients attending the Toronto General Hospital Immunodeficiency Clinic with ≥1 kept visit and ≥12 months of follow up between 2004 and 2014 were studied. A sub-analysis of OHTN Cohort Study (OCS) participants was conducted for questionnaire measures within 6 months of a scheduled visit. Multivariable generalized estimating equation models identified factors associated with (1) missed visits with HIV specialists and (2) inter-visit intervals ≥12 months.
1585 patients were included, of whom 602 were in the OCS. OCS participants were older and more likely to be white and MSM than non-OCS participants. 15% of visits were missed. 70% of patients missed ≥1 visit and the median (IQR) rate of missed visits per patient was 11% (0, 27%). 6% of inter-visit intervals were ≥12 months and 45% of patients had ≥1 12 month interval. Among all participants, factors associated with missed visits were younger age (OR=1.34 per 10 years, p<0.0001), black race (OR=1.34 vs. white, p<0.001), other non-white race (OR=1.21 vs. white, p=0.01), IDU (OR=1.94, p<0.0001), longer HIV duration (OR=1.02 per year, p<0.01), VL ≥ 50 (OR=1.11, p=0.03), and winter season (OR=1.12 vs. fall, p=0.01). In the OCS sub-analysis, participants without children living at home were more likely to miss a visit (OR=1.64 vs. no children, p<0.01). Notably, stress and depression were not independently associated with missing a visit. Factors associated with intervals ≥12 months were: younger age (OR=1.24 per 10 years, p<0.0001), white race (OR=1. 35 vs. other race, p<0.01), VL ≥ 50 (OR=1.17, p=0.03), and higher CD4 count (OR=1.05 per 100 cells/mm3, p<0.01).
Although attendance at clinic visits at this tertiary care center was high relative to other centers, continued efforts are needed to maximize visit attendance.
Once daily atazanavir/ritonavir (ATA/r) and darunavir/ritonavir (DRV/r) are both recommended as protease-inhibitor-based regimens for antiretroviral naïve patients. To date, only one study has compared these two options.
Participants of the Canadian Observational Cohort (CANOC) collaboration whose first regimen included either ATA/r or DRV/r were studied. Fine and Gray models accounting for the competing risks (CR) of death and discontinuation were used to examine differences between regimens with regard to (1) time to virologic suppression (viral load (VL) <50 copies/mL on two occasions ≥30 days apart), (2) time to virologic failure (VL >1000 copies/mL at or after week 16 or >200 copies/mL at or after week 24) and (3) time to discontinuation of DRV/r or ATV/r (CR of death only).
1847 participants started on ATA/r and 234 started on DRV/r. Patients on ATA/r were more likely to be female (19% vs 12%, p=0.01), IDU (29% vs 9%, p<0.0001), and from BC (68% vs 17%, p<0.0001). 109 participants died during the study period. After adjusting for age, gender, race, MSM, IDU, province, baseline CD4, baseline VL, calendar year and rate of VL measurement, the hazard ratio (HR) and 95% confidence interval (CI) of virologic suppression for patients on ATA/r relative to those on DRV/r was 1.02 (0.84, 1.23), p=0.86. After adjusting for the same covariates, the HR (95% CI) of virologic failure for patients on ATA/r relative to those on DRV/r was 1.46 (0.81, 2.64), p=0.21. After adjustment for covariates, the HR (95% CI) of treatment discontinuation for ATA/r vs DRV/r in BC, Ontario and Quebec were 1.84 (0.95, 3.57), 2.10 (1.28, 3.44) and 1.14 (0.76, 1.71) respectively. Sensitivity analyses including men only to avoid pregnancy-related regimen changes yielded similar results.
There was no significant difference in rates of virologic suppression or failure among naïve patients starting DRV/r and ATA/r. Differences in treatment discontinuation varied regionally.
In cohort studies for which antiretroviral (ARV) medication data is extracted from medical records, missing start and stop dates may prevent accurate determination of current and cumulative exposures to ARVs.
We developed an algorithm to impute missing or improbable start and stop ARV dates in consultation with HIV specialist physicians and pharmacists. Twelve rules (5 for NRTIs/7 for other classes) were developed using information on past history of ARVs, the formulation of the medication, class of ARV, number of other medications in that class, virologic suppression, and prescribing patterns for specific ARVs. The algorithm was applied to chart-abstracted ARV data of participants in the OHTN Cohort Study (OCS). We validated imputed dates via record linkage with Ontario Drug Benefit Program (ODBP) data. Before application of the algorithm, ARVs with missing stop dates were assumed to be active until the later of last follow-up or December 31, 2012.
4080 OCS participants had ≥1 ARV record in the ODBP. The algorithm imputed ≥1 ARV date for 37% of participants. Among 2354 participants who received ARVs through the ODBP for whom the algorithm did not change any dates, the 50th%ile, (75th%ile,90th%ile) of the difference between cumulative exposure to all ARVs as calculated using the OCS versus ODBP databases was 1.3 years (4.3, 9.4). For 1546 participants with ≥1 imputed date, these values were 6.9 (15.4, 27.8) before and 2.5 (7.9, 15.2) after the algorithm was applied. The ARVs with the greatest reduction in cumulative exposure were efavirenz, lamivudine and abacavir. The rule resulting in the most improvement applied a stop date to existing NRTIs on the date that 2 new NRTIs were started.
Application of the ARV algorithm to chart-abstracted data improved the accuracy of cumulative ARV exposure. The ODBP is not a gold standard comparison, however, since participants may access ARVs through multiple sources and since prescription data may overestimate ARV exposure.
Patients with HIV drug-resistant virus often require complex antiretroviral (ARV) regimens. Since the fixed dose combination tablet Stribild (TDF/FTC/EVG/cobi) contains the pharmacoenhancer cobicistat, it may be suitable for concomitant boosting of protease inhibitors such as darunavir (DRV), potentially providing a simple once daily regimen for patients with drug-resistance.
We reviewed all patients prescribed TDF/FTC/EVG/cobi+DRV at St.Michael’s Hospital Positive Care Clinic between 09/2013 and 11/2014, and summarized reasons for selecting this regimen, virologic and immunologic responses, tolerability, discontinuations, and drug concentrations where available.
Thirteen patients were evaluated. Median (interquartile range) age was 43 (38,48), duration of HIV-positivity 18 (6,22) years, and median baseline CD4-cell count 164 (32,462) cells/mm3. Median baseline viral load (VL) was 4703 (105,123137) copies/mL and 3 of these patients were virologically suppressed at time of switch. Eleven patients were treatment-experienced; this group had experienced a median of 5 (3,7) prior regimens, most recently including a median of 5 (4,7) ARV pills/day. Reasons for selecting TDF/FTC/EVG/cobi+DRV included simplification (n=10), non-adherence (n=6), virologic failure (n=3), and toxicity (n=1). Patients had a median of 3 NRTI, 1 NNRTI, 1 PI, and no INSTI resistance mutations at baseline. Median follow-up at time of writing was 15 (12,34) weeks, during which 7 patients achieved or maintained virologic suppression, 2 had ongoing viremia, 2 had results pending, 1 was lost to follow-up and another did not start treatment due to lack of drug coverage. There were no discontinuations for intolerance and no medication-associated serious adverse events. The DRV trough concentration in the single patient tested was low at 0.1 mg/L, despite VL<40 copies/mL, likely due to incorrect dosing prior to testing.
In this small observational sample, once daily TDF/FTC/EVG/cobi+DRV appears safe and effective. Further data are needed on this simple 2-tablet option for selected patients with HIV drug resistance.
Antiretroviral therapy options for treatment-experienced patients are more limited, and often consist of multiple tablets administered twice daily. The availability of single tablet regimens including integrase inhibitors may provide the rationale for simplified regimens in this population.
We identified treatment-experienced patients in whom a change in therapy was indicated to address questions of virologic failure, toxicity or regimen complexity. The combination of Stribild and Darunavir was selected as it could be administered as 2 tablets once daily, would be generally well tolerated and is currently being evaluated in clinical trials, despite a possible negative drug interaction profile that may decrease potency. Patients were followed prospectively to monitor the efficacy and toxicity of the new regimen.
To date, we have evaluated 11 patients, 9 male, 5 PWID, median age 56 years. Median baseline CD4 count and viral load were 580 cells/mm3 and < 40 copies/mL, with patients taking a median of 5 tablets (1–8)/day prior to the study. The median duration of follow-up on Stribild and Darunavir was 207 (59–299) days. At last follow-up, 9/11 had full virologic suppression, with low-level viremia (< 200 copies/mL) documented in the other 2 cases. The median CD4 count remained at 501 cells/mm3. No significant toxicity of the regimen has been observed, and 11/11 patients remain on the study regimen. Data on 2 additional subjects initiating study therapy will be presented.
The combination of Stribild and Darunavir offers a simple, well tolerated and effective regimen to be considered in treatment-experienced patients. The predicted negative pharmacokinetic interaction does not appear to translate into decreased potency. Additional studies are needed in larger homogeneous populations (such as patients with multi-drug resistant virus or those on more complex and poorly tolerated regimens) to define the usefulness of this novel regimen in clinical practice.
Preliminary data suggest a beneficial effect of Maraviroc (MVC)-based antiretroviral therapy on short-term increase in serum mediators of fibrogenesis and non-invasive markers of liver fibrosis in HIV/HCV co-infected patients. We sought to confirm these data in patients newly initiating MVC-based regimens and in whom ongoing evaluation of liver fibrosis was available.
We identified 50 patients receiving MVC at an inner city clinic. A retrospective chart review was undertaken to collect data on demographic and laboratory data and specific antiretroviral therapy. AST-to-Platelet Ratio Index (APRI) and Fibrosis-4 (Fib-4) scores were calculated based on laboratory results within 3 months before starting MVC-based therapy and at the most recent visit. The percentage change in these parameters was calculated and correlated with relevant variables (paired sample t-test): virologic suppression, HCV co-infection, NRTI backbone, recreational drug and alcohol use.
Subjects were predominantly male (41/50) and more frequently PWID (46%) with a mean age of 50.6 years. HCV co-infection was present in 20 patients (40%). APRI and Fib-4 scores were improved all in patient categories (0.07 & 0.12 respectively) with statistical significance reached in patients being on MVC for more than one year (0.14 & 0.23, p<0.05). Mean change of APRI and Fib-4 scores were higher in mono-infected (0.11 & 0.16) compared to co-infected (0.02 & 0.08) patients (p = NS). In patients receiving Truvada, mean changes in APRI (0.29) & Fib-4 (0.50) were the most significant.
MVC use was associated with improvement in liver fibrosis, an effect which seems to increase over time. Our data suggest that a prospective study should be designed to evaluate the long-term clinical significance of this effect in HIV and HIV/HCV infected populations, with a particular interest in serial FibroScan measurements and co-infected subjects with significant pre-existing fibrosis who are successfully treated for their HCV infection.
Dolutegravir (DTG) is a second-generation HIV-1 integrase inhibitor which is now approved for use in treatment-naïve and treatment-experienced individuals. Multiple phase III investigations and extensive clinical experience to date with DTG have failed to reveal evidence of the development of any drug-resistant mutations against DTG, suggesting that it may be unique amongst all antiretroviral agents in having a very high barrier to resistance. DTG may be a potential candidate for monotherapy studies, but limited clinical experience exists on long-term DTG monotherapy. We report a unique case of an individual who accidentally switched to DTG monotherapy for over eight months, but remained virologically suppressed.
A 49-year-old man with hypertension and dyslipidemia was diagnosed with HIV in 2012 and started on an antiretroviral regimen consisting of raltegravir (RAL) and tenofovir/emtricitabine (TDF/FTC). He quickly achieved virologic suppression and his adherence to medications was perfect. In March of 2014, he expressed a desire to simplify his regimen and RAL was switched to DTG to provide a once-daily regimen. Due to miscommunication, after this intended switch he discontinued TDF/FTC and continued on DTG monotherapy until December of 2014, a period of over eight months. During the monotherapy period, the patient reported perfect adherence to medications. Two HIV viral load results from late June and early December of 2014 both were less than 40 copies/mL. TDF/FTC was added back to his regimen after his viral load in December was confirmed as being undetectable.
To the best of our knowledge, this is the first clinical report of long-term DTG monotherapy in a patient with chronic HIV-1 infection. In our highly-adherent patient, an unintended switch to DTG monotherapy maintained virologic suppression for over eight months. DTG monotherapy may be feasible with its favorable tolerability, interaction, and resistance profiles, and warrants further clinical investigation.
Tenofovir alafenamide (TAF), a novel prodrug of the NtRTI tenofovir (TFV), more efficiently loads lymphocytes with TFV-diphosphate compared to the current prodrug tenofovir disoproxil fumarate (TDF). The fixed-dose combination of elvitegravir (EVG)/cobicistat (COBI)/emtricitabine (FTC)/TAF (E/C/F/TAF) has been evaluated in one Phase 2 and two Phase 3 randomized, double-blinded studies in treatment-naïve subjects, comparing E/C/F/TAF to E/C/F/TDF. The TAF-containing arm demonstrated non-inferiority to the TDF-containing comparator arm in both Phase 3 studies at Week 48 with >90% of patients achieving HIV-1 RNA <50 copies/mL. An integrated resistance analysis across these 3 studies was conducted.
HIV-1 resistance testing was conducted using commercial assays to assess PR/RT/IN susceptibility to study drugs. Patients with HIV-1 RNA >400 copies/mL at time of virologic failure were evaluated for resistance.
HIV-1 subtype B was found in 87% of the 1903 treated patients. Pre-existing primary resistance-associated mutations (RAMs) were detected at baseline: 7.5% had NRTI-RAMs, 18.2% had NNRTI-RAMs, and 3.4% had PI-RAMs. HIV-1 subtype or baseline RAMs did not influence treatment response at Week 48. In the E/C/F/TAF group,19 patients qualified for on-treatment resistance analyses (1.9%; 19/978). Seven patients (0.7%, 7/978) developed NRTI-RAMs (K65R, n=1; M184V/I, n=7), including 5 patients that developed primary INSTI-RAMs (T66A, n=2; E92Q, n=2; Q148R, n=1; N155H, n=1). In the E/C/F/TDF group, 22 patients qualified for on-treatment resistance analyses (2.4%; 22/925). Seven patients (0.8%, 7/925) developed NRTI-RAMs (K65R, n=2; K70K/E, n=1; M184V/I, n=7), including 4 patients that developed primary INSTI-RAMs (E92Q, n=3; Q148R, n=2). Similar patterns of emergent mutations were observed in each treatment group.
E/C/F/TAF achieved a high level of virologic suppression in HIV-1 treatment-naïve patients through 48 weeks of treatment. Presence of PI-, NNRTI-, or NRTI-RAMs at baseline did not affect treatment response to either regimen. Emergence of resistance was rare (<1%) and comparable between the 2 arms.
Minority drug resistant HIV-1 variants may eventually be selected under antiretroviral pressure, leading to therapy failure. In a recent study, we described that despite close monitoring and adherence to treatment (Kyeyune et al 2013 AIDS 27:1899), approximately 20% of HIV-infected individuals failing antiretroviral therapy in a Ugandan HIV/AIDS clinic had a susceptible HIV-1 genotype using a Sanger sequencing-based assay,.
In this study, we used a novel HIV-1 genotyping assay based on deep sequencing (DEEPGEN™HIV) to quantify minority HIV-1 drug resistant variants in 75 patients failing antiretroviral therapy in the absence (n=38) and presence (n=27) of mutations detected by Sanger sequencing, respectively.
Most of the patients were infected with subtype A (n=36) or D (n=28) HIV-1 strains. Not surprisingly, DEEPGEN™HIV was able to detect all mutations originally detected by Sanger sequencing in the 27 control samples as well as additional drug resistance mutations at frequencies >1% and <20% in most of these patients, e.g., M41L (15.8%), D67N (14%), K70R (14.4%), M184V (19.6%), G190A (9.6%), K103S (11.1%), V106A (2.2%), etc. Low-level drug resistance mutations were detected by DEEPGEN™HIV in 53% (20/38) of patients with susceptible HIV genotyping based on standard Sanger sequencing. Mutations associated with resistance to NRTI (e.g., M41L, D67N, M184V) and NNRTI (e.g. K103N, Y181C) were quantified, ranging from 1% to 17.6%.
Poor or intermittent adherence, which is not uncommon in sub-Saharan Africa, may lead to an increased prevalence of drug resistant virus, which “fades” from dominance in the intra-patient population in the absence of drug pressure. These low-abundance drug resistant variants detected in antiretroviral-experienced individuals failing treatment, may have significant consequences on current or future outcomes, especially if treatment is not modified based on a susceptible HIV-1 genotype (Sanger) report.
Limited access to HIV drug resistance testing in low- and middle-income countries (LMIC) continues to impede clinical decision-making and population resistance surveillance. We assessed the potential of next-generation sequencing to provide accurate HIV drug resistance genotypes for hundreds of samples simultaneously.
Plasma samples were collected from participants of a Ugandan cohort (N=349) or from Canadian patients (N=759). Amplicons spanning reverse transcriptase codons 90–234 were prepared for Sanger and MiSeq sequencing. Sanger data were analyzed by in-house software (RECall). MiSeq data were analyzed using an iterative mapping pipeline. Samples with <100-fold coverage at any position were excluded. Minority bases ≥20% were called as mixtures in MiSeq data.
Sequencing was successful in 881 (80%) and 892 (81%) samples by Sanger and MiSeq respectively with 832 samples having results by both methods. Samples failing MiSeq had a median pVL 2.7 (IQR=2.4–3.7) log 10 copies/mL. Median depth of MiSeq coverage was ∼9,900 (IQR=5,300–13,400) reads/sample. Overall 99.3% nucleotide concordance was observed between methods. The majority (>95%) of discordances were differences in mixture calls, with neither method overcalling mixtures relative to the other (κ=0.72). Resistance mutations (2013 IAS-USA List) were observed in 155 and 154 samples by Sanger and MiSeq respectively. MiSeq achieved 97% sensitivity and 99.4% specificity in detecting resistance mutations identified by Sanger. Technical replicates of a laboratory clone (N=9) and clinical isolate (N=5) indicated a high level of repeatability of the MiSeq results (100% and 98.5% nucleotide concordance). Phylogenetic analysis revealed clustering of samples by subtype and cohort, consistent with expected subtype prevalence in Africa and North America.
Approximately 20% of Canada’s annual HIV drug resistance testing was repeated in a single MiSeq run. Accurate MiSeq sequences were obtained for multiple subtypes, suggesting that routine individual testing or annual population resistance surveillance in LMIC could be performed with this strategy.
With advancements in pharmacotherapy, HIV is now considered a treatable chronic disease; however, many HIV-infected individuals are hospitalized for other co-morbidities. Due to the complexity of combination antiretroviral (ARV) therapy and the fact that non-specialized clinicians are often unfamiliar with HIV management, very high rates of ARV medication errors in this population have been reported in the literature. The objective of this project was to address the common types of ARV errors by creating an evidence-based educational guide for non-HIV clinicians.
The project consisted of two phases. Phase 1 involved a comprehensive literature review in order to characterize the types of drug error found in HIV-positive hospitalized patients. Phase 2 consisted of the development of an ARV assessment guide which addressed the types of drug error found in the literature. The guide was peer reviewed by several HIV pharmacists, an inpatient pharmacist focus group and clinical practice leaders for content, readability and applicability.
The guide included both a framework for patient assessment and a collection of useful resources, with the objective of facilitating clinical decisions and minimizing drug-related errors. A 3-step patient assessment framework addressed significant drug errors found on admission, internal unit transfer, and discharge. Hospital-focused patient assessment pointers regarding ARV indication, efficacy, safety, adherence and seamless care were included. Supplementary appendices included information on specific laboratory tests, drug interactions and toxicities, ARV dosing, web-based resources, and provincial HIV team contact information. The guide was posted on the institutional internet website. Implementation and interdisciplinary educational sessions are in progress.
An evidence-based ARV guide for inpatient assessment was developed for non-HIV clinicians in an effort to reduce drug error. This guide is adaptable to various institutions and practice settings and can serve as the basis for education of health care workers and students/residents.
Evidence suggests that pharmacist delivered cognitive services have a significant impact on improving HIV drug management. As the role of pharmacists evolves with expanded scopes of practice and new funding models it is imperative that pharmacists build a body of research to understand the impact of this transformation. This project will deploy a novel text based survey to better understand the impact pharmacist intervention has on drug therapy modifications.
To understand the frequency and types of pharmacist interventions that may directly or indirectly lead to a modification of drug therapy for HIV patients.
To evaluate the ease of administration, convenience, and utility of a novel text based survey system to collect practice level data.
A text based survey was sent to twelve participants on Mondays to Fridays for a period of 10 weeks. The first question, “Did you make an intervention today, that may change drug therapy in order to reduce pills, improve tolerability or avoid a DDI”, was answered with a y or n. Based on the first answer a second multiple choice question was sent to determine the type of intervention made.
1134 text questions were deployed; response rate of 95.1%. A total of 218 interventions on 49.0% of days were made. The most common intervention made by hospital pharmacists was to improve tolerability at 22%. The most common intervention for retail pharmacists was to reduce pill burden made on 29% of days at work. Of eight respondents to a post study evaluation, 100% agreed that texting was an acceptable method for clinical practice data collection.
Pharmacists in HIV care are critical members of the health team; delivering disease state management by making regular interventions to reduce pill burden, improve tolerability and avoid drug drug interactions.
Tenofovir disoproxil fumarate (TDF) has been associated with nephrotoxicity and reduced bone mineral density (BMD). Tenofovir alafenamide (TAF), a novel prodrug of tenofovir (TFV), results in 90% lower plasma TFV levels compared to TDF. Safety and efficacy of elvitegravir/cobicistat/emtricitabine/TAF (E/C/F/TAF) was assessed in HIV-1 infected patients with mild to moderate renal impairment.
Week 24 efficacy and safety data are described for virologically-suppressed adults with stable eGFRCG (Cockroft Gault) of 30 to 69 mL/min who switched from both TDF- and non-TDF-containing regimens to open-label E/C/F/TAF.
Of 242 subjects, mean age was 58 years (range: 24–82), 18% Black, 39% hypertension, and 14% diabetes. At baseline, median eG-FRC-G was 55.6 mL/min (33% eGFRC-G 30–49 mL/min). At Week 24, the median (Q1, Q3) change from baseline eGFRC-G was −0.4 (−4.7, 4.5) mL/min, eGFR-cystatin C 3.8 (−4.8, 11.2) mL/min/1.73m2, and aGFR (n=32, 68.8% TDF at baseline) was 0.1 (−4.3, 4.4) mL/min, indicating that GFR was not affected by E/C/F/TAF. Two subjects (0.8%) discontinued study drug for decreased GFR by eGFRC-G and eGFR-cystatin C, with no evidence of renal tubulopathy. Prevalence of clinically significant proteinuria (UPCR > 200 mg/g) and albuminuria (UACR ≥ 30 mg/g) decreased from 42% to 21% and 49% to 27%, respectively. Significant decreases in urine retinol binding protein to creatinine ratio, beta 2 microglobulin to creatinine ratio, and fractional excretion of uric acid were observed (p<0.001 for all). Hip and spine BMD percentage change from baseline to Week 24 was 0.74% (−0.71, 2.03) and 1.27% (−0.44, 3.83) (median, IQR), respectively.
These 24-week data support the virologic efficacy and renal and bone safety of once-daily single-tablet E/C/F/TAF for use in HIV+ patients with mild and moderate renal impairment (eGFR 30–69 mL/min). Switch to E/C/F/TAF was associated with no change in aGFR and with reductions in proteinuria.
We previously reported high incidence (4.3 per 100 person-years) of new syphilis diagnoses among HIV-positive MSM. Syphilis may potentiate HIV transmission due to rises in viral load (VL), as is well documented among the ART-naïve. We aimed to quantify VL at time of syphilis diagnosis according to past VL and ART status.
The OHTN Cohort Study is a multi-site clinical cohort of 6,408 people in HIV care across Ontario. Syphilis diagnoses and VLs were obtained via record linkage with databases at the Public Health Ontario Laboratory. We conducted a descriptive case-based analysis among MSM who experienced a new diagnosis of syphilis in 2006–2013 after follow-up for ≥12 months.
We analysed data from 193 new syphilis cases; the majority were gay (89%), white (73%), and in Toronto (87%). At time of syphilis diagnosis, mean age and CD4 cell count were 45 years and 500 cells/mm3, 33% had evidence of past syphilis, 88% were on ART for >1 year, and 10% were naïve. Ninety percent (173) had VL measurement within 90 days; of these, 78% had undetectable VL, 7% were <1000, and 15% were ≥1,000 copies/mL. Among men with detectable VL (≥50 copies/mL), 39% were ART-naïve or recent initiators, 79% had detectable VL 6–12 months ago, and, for men on ART, 57% reported ≥1 missed dose within 30 days. For men on ART with undetectable VL 6–12 months ago, only 5% had detectable VL at syphilis diagnosis compared to 44% and 92% of men on ART whose VL was <1000 or ≥1000 copies/mL 6–12 months ago, respectively (P<0.0001).
Among men on ART, plasma VL remained undetectable for the majority of new syphilis cases. Most with detectable VL were naïve, had previously unsuppressed VL, or were non-adherent. VL quantification in genital/rectal secretions is needed to fully establish whether modern suppressive ART limits HIV infectiousness even during syphilis co-infection.
In British Columbia, screening for LTBI is recommended at baseline for HIV positive individuals. Screening includes risk factor and symptom identification, chest X-ray and TST in all patients with a CD4+ cell count >200 c/μL. We sought to determine factors associated with patients having at least one TST.
We conducted a retrospective analysis of patient data from 2004–2014 at John Ruedy Immunodeficiency Clinic (IDC), a multidisciplinary HIV clinic. This data was linked to the BC Centre for Excellence in HIV/AIDS drug treatment database. We examined associations between having at least one TST using Pearson Chi-square Test and Wilcoxon rank-sum test. Multivariate logistical regression was used to determine factors related to TST screening.
There were 2007 patients included, 1141 (56%) with at least one TST. Males and individuals with higher current CD4+ counts were more likely to have TST (
LTBI screening at IDC was suboptimal according to present guidelines. Individuals with better engagement evidenced by excellent antiretroviral adherence, higher current CD4+ count, regular clinical visits and hepatitis B vaccination are more likely to be screened for LTBI. Socioeconomic variables did not predict low screening rates.
| Male sex (%) | 42.3 | 57.7 | 0.05 |
| Age median (IQR) | 42.80 (34.89–49.42) | 42.23 (35.13–49.49) | 0.982 |
| Nadir CD4 c/μL median (IQR) | 150.00 (50.00–280.00) | 160.00 (70.00–290.00) | 0.180 |
| Most recent CD4 c/μL median (IQR) | 440.00 (250.00–620.00) | 510.00 (350.00–690.00) | <0.001 |
HPV-associated anal cancers disproportionately impact HIV-positive MSM, with rates exceeding 100-fold those of the general population. Despite this, there are no established protocols for screening and treatment of anal cancer precursors, no universal coverage for HPV vaccine in males, and no rollout of organized screening programs. Further, there is growing epidemiologic evidence that HPV infection may enhance sexual transmission of HIV.
In early 2014, the HPV-SAVE research team was formed, and received CIHR funding in late 2014. It consists of a multidisciplinary group of Canadian experts from biostatistics, community-based organizations, clinical care, epidemiology, laboratory medicine, and social sciences. Our assembled team developed a plan of research, knowledge translation, capacity building, and mentorship aimed at contributing to the evidence base of HPV care in MSM. Additionally, we partnered with clinics providing HIV care from which participant recruitment will occur, and will establish a community advisory board to help direct our mandate.
We identified a number of knowledge gaps that translate into fundamental deficiencies in prevention and care of HPV-associated anal disease for HIV-positive MSM. The inter-related projects focus on: (1) Anal cancer screening and vaccination programs, including an examination of acceptability and barriers, and the validation of a triage algorithm using combination cytology and HPV testing; (2) a randomized controlled trial of anal dysplasia treatments, comparing different modalities to observation alone; and (3) elucidating the mucosal immune mechanisms that underpin the association of anal HPV infection with HIV transmission and susceptibility in MSM.
Our newly-funded CIHR team has initiated its five-year research program, with participant recruitment to start in late 2015. To our knowledge, this comprehensive HPV research program specifically focused on MSM is the first of its kind. With its broad-ranging objectives focusing on multiple facets of HPV management, it has the potential to significantly alter clinical practice and patient outcomes, as it attempts to answer key management questions for which there is a paucity of evidence.
With the availability of direct acting antiviral agents (DAAs), recent data have shown HCV therapies to be equally effective in the setting of HIV coinfection. However, this has not been fully evaluated in vulnerable inner city populations. Here we aimed to measure HCV treatment outcomes in monoinfected and HIV/HCV coinfected cohorts treated with DAAs in this population.
A retrospective observational study was conducted in patients treated with DAAs (Jun09–Jul14) or interferon (PR)-based regimens (Jan02–Jul14) at an inner city clinic in Vancouver. Data regarding HCV treatment, HIV coinfection status and lifestyle comorbidities were collected through chart review. The two-sample z-test was performed to compare the efficacy of DAA and PR-based regimens in mono- and HIV co-infected subpopulations, the treatment endpoint being a sustained virologic response (SVR) 12 weeks post treatment.
Among 372 treatment courses, 128 were completed with DAAs in 116 patients (97male), median age 53yrs, 87% genotype (GT)1, 66% treatment naïve, 13% compensated cirrhotic. Nineteen cases (15%) were HIV coinfected. Other baseline conditions: 76% PWID, 58% ethanol abuse, 18% methadone maintenance therapy. Treatment outcomes included 65% SVR, 13% relapse and 6% premature discontinuation due to toxicity. Factors associated with SVR: treatment naïve to DAAs and PR (p=0.001), pegINF-α free regimens (p=0.004), baseline HCVRNA≤106 IU/mL (p=0.04), rapid virologic response (p<0.001). The overall SVR rate attained with DAAs was significantly higher than the 55% with 244 PR courses (p=0.03). There was no statistically significant difference in SVR rates between mono- and co-infected cohorts in PR or DAA-based regimens.
With equivalent response rates observed in mono- and co-infected cohorts, the advent of DAA-based therapies has improved SVR rates in all target inner city populations. Going forward, the availability of safer, simpler HCV treatment modalities will allow us to treat a greater number of individuals from our “core transmitter” populations.
HIV/HCV co-infected individuals are at risk for faster progression of hepatic fibrosis. Large scale characterization of fibrosis stage in co-infected individuals is now possible through the use of non-invasive transient elastrography (TE). The prevalence of significant fibrosis (≥F2) in co-infected patients undergoing care at an urban HIV/HCV program in Vancouver was evaluated.
Fibrosis stage was assessed using TE in sequential HIV/HCV co-infected individuals referred between October 2013 and December 2014. Fibroscan was performed following recommended 4 hour fast. Prevalence of moderate fibrosis (F2) and cirrhosis (F4) was determined. Factors associated with F4 fibrosis were assessed using multivariate logistic regression models adjusted for age, gender, duration of HIV and HCV, CD4 nadir, prior AIDS illness, and use of alcohol.
Overall 144 individuals (82% male) underwent TE. The median age was 51years (interquartile range [IQR] 46 – 57 years), 60% reported history of injection drug use and 94% received ART at time of TE with median pVL < 40 c/mL. The median duration of HIV infection and HCV infection was 14 years (IQR 8 – 18) and 18 years (IQR 3 – 18) respectively. Overall the prevalence of F2 fibrosis was 10%, F3 fibrosis was 12% and 22% of individuals were found to have F4 fibrosis. Factors associated with F4 fibrosis included duration of HCV infection (adjusted odds ratio [aOR] 1.08; 95% CI 1.02 – 1.15), and prior history of AIDS defining-illness (aOR 3.08; 95% CI 1.05 – 8.59).
Significant fibrosis (≥F2) was common in 44% of individuals undergoing TE in an HIV/HCV clinic. Cirrhosis was associated with longer duration of HCV and prior advanced HIV with history of prior AIDS illness. Although referral bias must be considered, strategies to improve access to HCV therapy must be evaluated to prevent future end-stage liver disease.
We assessed the real-life effectiveness of pegylated interferon (peg-IFN) α-2b combined with ribavirin in a cohort of treatment-naïve patients with chronic genotype 2 (G2) or genotype 3 (G3) hepatitis C virus (HCV) infection, followed in Canadian routine clinical care, and identified indicators predicting sustained virologic response (SVR).
Post-hoc pooled analysis of two Canadian open-label, multi-center, observational studies, RediPEN and PoWer. A total of 1242 patients infected with G2 (n=468) and G3 (n=774) were included. The primary outcome was SVR. The secondary endpoints included early virologic response (EVR), end-of-treatment (EOT) response, and relapse. Multivariate logistic regression was used to identify independent predictors of treatment response among potential variables identified in univariate analyses.
The EVR rate in G2 and G3 infected patients was 18.2% and 16.9%, respectively. SVR in G2 and G3 was 74.4% and 63.6%. SVR for F0/F1, F2, F3 and F4 was 76.4%, 74.1%, 69.4% and 78.9% for G2 and 73.1%, 52.6%, 66% and 41.1% for G3. Relapse occurred in 12.7% and 19.1% of G2 and G3 infected patients achieving EOT response. Overall, G3 was found to be an independent predictor of reduced SVR (OR=0.196; P=0.007) and increased relapse (OR=6.841; P=0.022). Among G3 infected patients, increasing fibrosis score was the most important factor predicting reduced SVR [F2 vs. F0/F1 (OR: 0.409; P=0.009); F3 vs. F0/F1 (OR: 0.716; P=0.338); F4 vs. F0/F1 (OR: 0.265; P=0.001)]. Male gender (OR: 0.076; P=0.020) and higher fibrosis score [F2 vs. F0/F1 (OR: 9.718; P=0.016); F3/F4 vs. F0/F1 (OR: 4.234; P= 0.1130] were associated with increased relapse rate in G3 patients. No predictors of response were identified in G2 infected patients.
The results of this Canadian cohort analysis support the real-life effectiveness of peg-IFN α-2b plus ribavirin in HCV G2 and G3 infected patients which were comparable to the established clinical efficacy profile. Overall, genotype was identified as the most significant predictor of treatment outcome. Fibrosis score and gender were key predictors of outcome in the G3 infected population.
A clinical trial of adjuvanted influenza vaccine dosing strategies in HIV+ adults assessed immunogenicity by hemagglutination inhibition (HAI) assay. We subsequently evaluated a newly developed microbead array (BA) assay in this sub-study. The performance of both assays was compared.
A phase III, randomized, multi-centered, controlled, vaccine trial was conducted at 4 CTN sites. Two dosing strategies (standard dose vs standard dose plus day 21 booster) were assessed in 150 HIV+ adults during the H1N1 (2009) pandemic. A single antigen, inactivated split adjuvanted (AS03(A)) influenza vaccine (Arepanrix) was used. Immunogenicity was measured at baseline, days 21 and 42.
The sub-study population was similar between groups and to those in the original study: 83% male, 77% on ART, median CD4=567 cells/mm3, 83% with HIV RNA < 50 copies/mL. Baseline BA medians (IQR) were 1983 Mean Fluorescence Intensity (MFI) (238–7970) and 1362 (24–4670) for the 1 dose and 2 dose groups. HAI seroprotection results were 66% and 76% at visit 2 and 68% and 89% at visit 3 for the two groups. Using a BA seroreactivity cut-off of 500, results were 75% and 59% at visit 2 and 78% and 61% at visit 3 for the two groups. The raw agreement between HAI and BA tests was 59% at visit 2 and 57% at visit 3. Using a BA cut-off of 1000, results were identical at both visits, with the exception of the 1 dose group at visit 3, which fell to 70% seroreactive. Visit 3 raw agreement remained at 57%. There were no strong relationships between BA values and key baseline variables.
The BA assay does not appear to be an effective indicator of seroprotection in HIV+ adjuvanted vaccine recipients. The lack of agreement with the HAI results suggests poor assay utility.
Kaposi sarcoma (KS) is a vascular tumor caused by oncogenic human herpesvirus 8 (HHV-8) that often occurs with HIV-associated immunosuppression. Receptor tyrosine kinases such as c-kit and PDGFR have a role in pathogenesis of KS. Imatinib, a tyrosine kinase inhibitor, has resulted in partial regression of KS lesions in one-third of patients. Herein, we reported a case of a Caucasian male with recurrent KS despite well suppressed HIV infection, who received the imatinib therapy and showed clinically relevant tumor response.
A 38-year-old male presented with a notable history of HIV infection since 2005 whereupon he began antiretroviral therapy. Patient remained undetectable with a CD4 count >500cells/uL, when he developed cutaneous nodular KS on lower limbs in 2008. He was treated with multiple cycles of liposomal doxorubicine, paclitaxel and bortezomib, and failed to sustain response for more than 6 months post chemotherapy. In 2013, when referred to our centre patient developed left lower limb tumoral KS aggravated by prominent pain and swelling. He received 8 cycles of paclitaxel with the mTOR Inhibitor sirolimus for 1 year with transient and partial tumor control. Due to clinical failure, oral imatinib 400mg daily with liposomal doxorubicine was administrated. Patient tolerated the treatments well and major regression of lesions occurred. Chemotherapy was withheld after 2 cycles as his symptoms continued to improve on imatimib monotherapy, with nearly all KS lesions resolved at last visit.
Imatinib has activity in AIDS-KS, which makes it a promising treatment especially in refractory or recurrent cases. To our knowledge, this is the first ever reported case on the value of imatinib in control of KS in the context of long-lasting virological control with adequate quantitative CD4 recovery. KS occurrence despite apparently successful immune reconstitution warrants renewed investigation into what constitutes a true functional immune restoration.
Primary HIV-1 infection (PHI) has been regarded as a window of opportunity for strategies of prevention and early intervention. Understanding of the distinctive socio-demographic and virological/immunological characteristics of these patients will contribute to a rational management of primary infection and secondary transmission.
A prospective cohort of PHI has been established in Montreal in both private medical centres (PCs) and hospital-based centres (HCs). From May 1996 to Feb 2014, 326 PHI participants <6 month of infection were selected, with 166 from PCs and 160 from HCs. Socio-demographic and virological as well as immunological profiles between the two groups were compared.
Routes of infection differed significantly between participants recruited in different medical centres, with MSM accounting for 93.4% in PC participants v.s. 62.5% in HC participants, and IDU accounting for 4.2% in PC participants v.s. 22.5% in HC participants (p<0.001). Participants from PCs had a higher proportion of education beyond college/university levels (p<0.001) and a higher annual income (p<0.001) compared with those from HCs. Numbers of sexual partners in the last 6 months before enrollment were similar for MSMs in both PCs and HCs. However, in both PCs and HCs, MSMs with less education and lower income tended to have more sexual partners (p<0.05). No difference was observed in baseline HIV viral load or CD4+ T cell counts between the two groups.
Patients who participated in the Montreal PHI cohort differ in their socio-economic status and sexual behaviors, which needs to be considered in the implementation of early interventions, surveillance as well as prevention of secondary transmissions.
The ability of HIV-1 to establish and persist within cellular reservoirs as a transcriptionally silent provirus represents a substantial roadblock to cure research. Therapeutic interventions designed to eliminate this hidden reservoir have thus far failed, and may require bold and innovative strategies. We have designed an activator vaccine (ACT-VEC), based on autologous derived VLPs, which can specifically target the resting CD4 T cell reservoir, and induce latency reversal. Here we describe the safeguards we have implemented into our 2nd generation VLPs to render the vaccine formulations safe for future in vivo, pre-clinical testing as well as preliminary results from our in vitro latency reversal studies.
We have successfully created autologous ACT-VEC VLP constructs for all HIV-1 infected recruits. We report that our 2nd generation ACT-VEC VLPs have reduced packaging of genomic HIV-1 RNA (up to 221-fold), increasing its safety profile. This along with the PCR-generated AAH>RRK mutation in Integrase and deleted 5’ LTR renders our ACT-VEC incapable of reverse transcription, integration, or packaging of its RNA. Preliminary studies show that ACT-VEC share considerable genetic similarity to virus derived from patients. Of note, autologous ACT-VEC were able to stimulate the generation of 20 to 30-fold more HIV RNA from infected T cells than Flu/Tet/CMV stimulation and more than NL4-3 ACT-VEC control. Critically, our studies revealed ACT-VEC to outperform a number of clinically relevant latency reversal agents such as Romidepsin and Vorinostat as well as the T cell mitogen PHA.
Here we clearly demonstrate that our 2nd generation ACT-VEC formulations represent a safe vaccine platform for use as a therapeutic intervention and that ACT-VEC formulation may signify a promising strategy to purge the latent viral reservoir and facilitate cure.
Individuals living and aging with HIV may experience cognitive impairments in speed of processing, attention and memory performance. Our previous work has shown that subjective cognitive complaints may be overestimated relative to actual neurocognitive deficits when depression is present. To understand these relationships better, we investigated the role of personality style in the reporting of subjective cognitive complaints.
141 male participants from an ongoing study of HIV Associated Neurocognitive Disorders (HAND) at St. Michael’s Hospital completed an extensive neuropsychological evaluation, the NEO personality inventory, Beck Depression Inventory, and the Patient’s Assessment of Own Functioning to measure cognitive complaints. Profile analysis was used to visualize relationships between the five NEO personality dimensions and cognitive complaints scores, and then we selected two NEO scales of interest, neuroticism and extraversion for further analysis. Multilevel regression was performed in steps to explore how neuropsychological, demographics (age / education), HIV status (plasma viral load, nadir and recent CD4), depression and personality dimensions contribute to subjective cognitive complaints.
In the 4-level model, higher neuropsychological impairment (p<0.001) and neuroticism (p<0.05) significantly predicted higher cognitive complaints, while higher extraversion was negatively associated with complaints (p<0.05). However, inclusion of depression in the model (p<0.01) diminished the effect size of neuroticism and extraversion such that they were no longer significant. Testing for collinearity revealed a strong, positive correlation between NEO neuroticism score and depression (r=0.504, p<0.01) and moderate negative correlation (r= −0.384, p<0.01) between NEO extraversion score and depression.
Individuals with a high neuroticism style may report higher cognitive complaints. However, the presence of depression appears to be a more robust predictor of cognitive complaints and is recommended be included in an assessment of the etiology of cognitive complaints in HIV and HAND.
The experience of living with chronic HIV infection can threaten brain health, whether through direct effects of the infection or its treatment on the brain, or by affecting stress levels, coping skills, physical health, and social supports. Although the burden of poor brain health in HIV in Canada is unknown, it is likely to be high. The Brain Health Now project is a multi-site Canadian study using a cohort multiple randomised controlled trial design to meet goals of identifying, understanding and optimizing brain health in people living with HIV.
A cohort of 900 people HIV+ is being fully characterized for health-related quality of life with a specific focus on determinants of cognitive and emotional health and followed longitudinally to establish how brain health changes over time. Five interventions targeting cognitive ability through cognitive training, exercise, self-management and treatments for insomnia and depression will also be tested in sub-cohorts drawn from the main study.
Data from the first 123 people recruited from Montreal (85% men; mean age 53) indicated that most health aspects of quality of life were considerably below populations norms. In particular, 50% would be classified as at risk for clinical depression and 36% had low cognitive ability making them eligible for interventions. In addition, 30% are eligible for the insomnia intervention, 22% were sedentary and are eligible for an exercise intervention, and all are eligible for self-management. Overall, 40% expressed some degree of dissatisfaction with life, in comparison to only 10% for other Canadians.
This novel methodology combining observational and interventional approaches is an efficient and attractive way of conducting research on vulnerable populations.
Diagnosis of HIV-associated Neurocognitive Disorder (HAND) typically requires a neurocognitive assessment and interview by a trained clinician. This clinical determination of impairment is considered the “gold standard” for detecting neurocognitive impairment, however, it tends to be time-consuming, requires expertise, and has potential for subjectivity. An alternative, quantitative approach involves calculating a global deficit score (GDS) based on an algorithm that summarizes the number and severity of impairments using only neurocognitive test results. While this method may fail to account for important confounds or contextual details, comparing the results of clinical diagnosis and data-derived GDS can help to inform the clinical picture of HAND. Because GDS is used in HAND research as a quantitative measure of impairment, evaluating its concordance with clinical diagnosis would serve to validate the use of this measure, and could highlight potential caveats.
117 HIV+ patients were evaluated as part of an ongoing HAND research program at St. Michael’s Hospital in Toronto, Canada between 2009–2014. Patients were diagnosed as normal or impaired by a neuropsychologist using the gold standard clinical approach. GDS was calculated from the average of converted neuropsychological test T-scores, with cutoff for impairment set at GDS≥0.5. We observed 81.2% concordance between clinically diagnosed impairment and GDS-determined impairment, 82.8% sensitivity and 79.2% specificity.
Compared to the dually-impaired group, those found to be GDS-only impaired had significantly lower age (p=0.006), lower subjective cognitive complaints (p=0.002), and lower depression scores on the Beck Depression Inventory (p=0.011). Conversely, those found to be impaired only clinically had significantly higher scores on subjective cognitive complaints (p=0.004).
Overall, GDS appears to be a good proxy for determining impairment among HIV+ individuals. However, younger individuals, those with fewer cognitive complaints and depressive symptoms may have greater likelihood of scoring a false-positive for impairment; likewise, individuals with fewer or less severe neuropsychological deficits and higher cognitive complaints may be missed by GDS despite positive diagnosis of HAND by a clinician. These factors should be considered when using GDS to determine impairment status.
HIV-associated neurocognitive disorders (HAND) remain prevalent despite the availability of cART. Several reviews, including our own, have shown that most cognitive screning instruments are useful for more severe forms of HAND but less so for the milder forms, although there are numerous methodological issues that need to be addressed. Our CIHR-funded study assessed these isseus, and the relative and concurrent validity of four screening tests for HAND against the gold standard for HAND diagnosis.
104 adults (mean age: 51 years; 86% men) completed four screening tests: Cogstate Brief Battery (Cogstate), HIV Dementia Scale (HDS), Computer Assessment of Memory and Cognitive Impairment (CAMCI), Montreal Cognitive Assessment (MoCA), and comprehensive neuropsychological battery assessing processing speed, attention/working memory, learning/memory, and executive functions. HAND clinical diagnosis was made by a Neuropsychologist who was blind to results of screening tests. Results from screening tests were compared with clinical HAND diagnosis using Receiving Operator Characteristics (ROC) analysis; area under the ROC curve (AUC) was calculated to determine classification accuracy rates of the screening tests.
HAND was diagnosed in 60 participants (58%). Compared to clinical diagnosis, sensitivity [95% CI] estimates of screening tests were: adjusted HDS, 90% [80%–96%]; MoCA, 68% [55%–80%]; Cogstate, 67% [53%–78%]; and CAMCI, 27% [16%–40%]. Specificity estimates were: CAMCI, 100% [92%–100%], MoCA, 86% [73%–95%]; Cogstate, 80% [65%–90%]; and HDS, 36% [22%–52%]. AUC estimates indicated the MoCA test had the best accuracy (AUC=0.774), followed by Cogstate (AUC=0.731), CAMCI (AUC=0.633), and adjusted HDS (AUC=0.632). Combination of any two screening tests (test positive by either one or both tests) resulted in classification accuracy improvements (AUC ranges: 0.623–0.815).
Our preliminary results suggest that the MoCA and Cogstate screening tests have moderate classification accuracy for HAND in people living with HIV. Further work is underway to determine the clinical utility and generalizabiilty of these fundings.
Limited information is available on the predictors of neurocognitive decline in individuals with good virological control. Identification of modifiable risk factors that predict decline would support the development of targeted interventions aimed at minimizing neurocognitive decline in higher-risk individuals. The objective of this study was to identify baseline factors predicting decline over the subsequent 3 years in aviremic HIV+ individuals.
HIV+ individuals in the CNS HIV Anti-Retroviral Therapy Effects Research (CHARTER) study were assessed with 15 neuropsychological tests every 6 months. Patterns of neurocognitive change on each test were detected using group-based trajectory analysis. Deterioration in performance ≥ 0.5 standard deviation on 1 or more tests was considered neurocognitive decline. Multiple logistic regression was used to identify baseline sociodemographic and clinical characteristics predicting neurocognitive decline in aviremic patients.
Among 191 HIV+ patients who were aviremic throughout the first 3 years, 23 (12%) experienced neurocognitive decline during this period. Results from multiple logistic regression indicated predictors of decline were: low eGFR (OR: 6.80, 95% CI: 1.25, 34.23); low education (OR: 4.25, 95% CI: 1.45, 12.43); prolonged duration of HIV infection (OR: 5.45, 95% CI: 1.19, 25.02); and increased CSF protein levels (OR: 3.25, 95% CI: 1.13, 9.35). Several factors associated with cognitive impairment in cross-sectional studies (e.g. older age, low nadir CD4, CPE score, hepatitis C co-infection, diabetes, hypertension, smoking, obesity) did not reach statistical significance in this group.
Neurocognitive decline over 3 years was uncommon in this sample of aviremic HIV+ individuals. Results on eGFR, a known independent predictor of atherosclerotic vascular disease, suggest targeting additional cardiovascular risk factors could be a useful strategy for minimizing risk of neurocognitive decline. Potential interventions such as smoking cessation or obesity management could be applicable for up to 80% of the participants.
High-resolution peripheral quantitative computed tomography (HR-pQCT) is a novel, non-invasive technology that measures cortical and trabecular bone microarchitecture and volumetric bone mineral density (vBMD). Emerging data suggests HIV-infected individuals have differences in HR-pQCT parameters compared to uninfected controls. No prior study has compared HR-pQCT outcomes between HIV-infected persons with and without fracture.
Adults with a history of low-trauma fracture after HIV diagnosis (cases) were matched 1:1 with HIV-infected adults without prior fracture (controls) based on age, sex, race and smoking history. Participants underwent HR-pQCT at the distal radius and tibia, and dual energy X-ray absorptiometry (DXA) at the hip and lumbar spine. Parameters were compared between cases and controls using paired t-tests, with differences expressed as percentages of control group values.
23 matched pairs were included, with median (IQR) age 50 (46,56) years, 78% male, 78% white and 57% smokers. Median (IQR) duration of HIV was 19 (11,23) years for cases and 10 (7,18) years for controls. On DXA, case patients had significantly lower areal BMD at the hip (median difference =−4.3%, p=0.04), but not the lumbar spine (−3.47%, p=0.33). On HR-pQCT there was a trend towards lower total volumetric BMD at the tibia (−11.1%, p=0.10) but not the radius (−0.09%, p=0.80). Numerical differences were observed in cortical and trabecular parameters, with differences more pronounced at the tibia than radius. At the radius, the largest percentage differences were in cortical porosity (+13.4%, p=0.78), axial failure load (−10.0%, p=0.23), trabecular bone volume fracture (−6.03%, p=0.65) and trabecular vBMD (−5.83%, p=0.64). At the tibia, the largest percentage differences were in cortical thickness (−14.6%, p=0.06), tvBMD (as above), axial failure load (−10.4%, p=0.06) and ultimate stress (−10.2%, p=0.08).
Low-trauma fracture appears to be associated with differences in cortical and trabecular bone microarchitecture in HIV-infected adults. The association requires further study.
HIV-positive individuals represent a population that is at a higher risk of developing chronic obstructive pulmonary disease (COPD). In this study, we sought to determine the effects of smoking on respiratory symptoms and smoking related diseases among HIV-positive patients and to determine if symptomatic patients are being appropriately screened for COPD.
HIV-positive individuals were asked to complete a self-administered questionnaire regarding respiratory symptoms and diseases. The effects of smoking on respiratory symptoms and diseases were reported as estimates of odds ratio. The screening criteria used to determine at risk patients for COPD were adapted from the Canadian Thoracic Society (CTS) guidelines.
A total of 247 patients were recruited. The median age was 49 years; 75% were male and 92% were currently on combination anti-retroviral therapy. Current and former smokers represented 66% of the population. Smoking had a statistically significant effect on respiratory symptoms including wheeze (OR 4.8 [95% CI 1.6–14.2]), phlegm production (OR 4.9 [95% CI 2.2–10.5]), current cough (OR 7.0 [95% CI 3.0–16.2]), chronic cough ≥3 months (OR 5.2 [95% CI 2.3–11.8]) and dyspnea (OR 7.2 [95% CI 1.7–31.2]). Smoking also had a statistically significant effect on respiratory diseases including COPD (OR 4.9 [95% CI 1.1–21.9]), bronchitis (OR 3.8 [95% CI 1.9–7.7]), asthma (OR 6.0 [95% CI 2.0–17.7]) and pneumonia (OR 2.1 ([95% CI 1.2–4.0]). Among HIV-positive smokers, 40% met the CTS criteria for COPD screening, while only 12% of smokers self-reported a diagnosis of COPD and 9% reported use of inhaled puffers.
The burden of smoking in the HIV population is significant. HIV-positive smokers are more likely to report both respiratory symptoms and diseases than HIV-positive non-smokers. A discrepancy exists between patients who meet the CTS COPD screening guidelines and those who have been diagnosed with COPD, raising the concern for under-recognition and under-diagnosis of COPD in the HIV-positive population.
There is increasing evidence to support an association between antiretroviral exposure during pregnancy and adverse birth outcomes including, preterm birth and small birth weight. Raltegravir was the first integrase strand transfer inhibitor to be used for the treatment of HIV and is most commonly used when patients develop resistance to other drugs. The main objective of this study is to investigate the effects of raltegravir on birth outcomes and liver toxicity in a mouse model.
Pregnant mice were exposed to either water or human equivalent doses of lopinavir or raltegravir (in combination with the NRTI backbone Combivir) during the pre-implantation period (gestational day 1–5.5) and post-implantation period (gestational day 6–13.5). At day 5.5 and 13.5 mice were sacrificed and serum and tissue samples were collected for further analysis. Birth outcomes, including number of pregnant mice, number of fetuses, fetal viability, and fetal and placental weight were recorded. Livers were embedded in paraffin, sectioned and stained with hematoxylin and eosin.
There was no difference in birth outcomes or liver toxicity with pre-implantation drug exposure. Mice exposed to either lopinavir or raltegravir during the post-implantation period had a significant increase in non-viable pubs per litter and resorption levels compared with controls. Exposure to only raltegravir during the post-implantation period resulted in significantly lower fetal and placental weights. Finally, raltegravir but not lopinavir increased the lipid content in the liver of mice exposed post-implantation.
Results from these studies suggest that exposure to raltegravir in pregnant mice during the post-implantation period results in lower fetal viability, higher resorption levels, significantly decreased fetal and placental weight, as well as an increase in lipid accumulation in the liver. Further experiments will help identify the specific pathways that lead to these adverse effects during pregnancy.
Health-care providers play a major role in providing good quality care and in preventing psychological distress among women living with HIV (WLHIV). WLHIV face distinct reproductive concerns regarding fertility and parenthood that may not be adequately addressed by care providers. The objectives of this study are to explore the impact of health-care services and satisfaction with care providers on psychological distress in mothers living with HIV (MLHIV).
One hundred MLHIV were recruited from community and clinical settings in the province of Quebec (Canada). Participants were aged 40.8 years old on average, were diagnosed with HIV for a mean time of 11.5 years and reported a median number of two dependent children or grandchildren (range 1–5). Variables included sociodemographic and control variables, psychological distress, resilience, number of HIV care providers, quality of communication and satisfaction with care providers, barriers to care and HIV stigma. Prevalence estimation of clinical psychological distress and univariate and multivariable logistic regression models were performed to predict clinical psychological distress.
Forty-five percent of the participants reported clinical psychological distress. In the multivariable regression, the following variables were significantly associated with psychological distress while controlling for sociodemographic variables: resilience, quality of communication with the care providers, resources, and HIV disclosure concerns.
The multivariate results support the key role of personal, structural, and medical resources in understanding psychological distress among MLHIV. The findings show the importance of paying special attention to mothers with dependent children, as they face unique challenges regarding their family’s needs. By inquiring about the overall quality of life of their patients, care providers would learn more about their needs and be better able to direct them to the relevant resources.
HIV stigma may be the most significant challenge for HIV-positive women and is associated with decreased self-esteem, hopelessness and increased psychological distress. HIV services were not originally created by or for women, therefore, women’s unique needs have not been considered in program design. Furthermore, HIV-positive women may be prevented from accessing services because they fear public disclosure of their HIV status or because of financial and logistical barriers including transportation, employment and childcare. The Women’s HIV Empow-erment And Life Tools For Health (wHEALTH) Intervention was developed to respond to these barriers based on a Peer Case Management (PCM) approach, whereby a PCM works in partnership with the woman in an informal setting to assess different areas of daily living, housing, finances, social supports, education/training opportunities, health, leisure or meaningful activity, and coordinate essential health and social services. Women receiving this intervention experienced increased mental health-related quality of life, increased perceived social support, decreased symptoms of depression, and increased awareness of and access to community-based supports. The relationship between the woman and the PCM was a mutually empowering experience, facilitating a unique level of sharing. Normally, the support HIV-positive women receive in a group format results in anxiety about the potential for a breach of confidentiality. The introduction of internet-based platforms allows participation from home, thereby improving both convenience and privacy. However, there is limited research on web-based interventions in real time focusing on women living with HIV. The e-wHEALTH study will evaluate the applicability of the wHEALTH Intervention in a web-based format, facilitated through a videoconferencing platform, and compare its effectiveness to the face-to-face design, in order to extend support to women living in rural/remote areas and to those not ready to meet face-to-face.
To describe adherence to regional guidelines to prevent perinatal transmission in northern Alberta by analyzing antiretroviral therapy (ART) and mode of delivery in pregnant women and their infants.
Retrospective chart review of a cohort of HIV-infected pregnant women who delivered in our region March 2006 through October 2013.
121 women had 141 pregnancies resulting in 146 live born infants (5 sets of twins). 31 women (25.6%) were first diagnosed with HIV during pregnancy; 2 (1.7%) were diagnosed at delivery. Of the 141 pregnancies, treatment with ART was administered prior to delivery in 134 (95%). The maternal viral load (VL) closest to delivery was <200 copies/mL in 119 pregnancies (84%), 200–1000 copies/mL in 8 (6%) and > 1000 copies/mL in 14 (10%). Standard protocol of intravenous (IV) zidovudine during delivery was administered in 128 (91%); in 12 out of the 13 other cases IV zidovudine was not given maternal VL near delivery was < 400 copies/mL. All women with VL > 1000 copies/mL near delivery had a cesarean section except two where the previous VL was undetectable. All infants received oral zidovudine and 18 infants (12%) were initiated on 3 antiretroviral drugs due to high/unknown maternal VL near delivery. Two infants whose mothers had a VL > 1000 copies/mL did not receive triple therapy but in both cases maternal VL was < 2000 copies/mL. The single confirmed HIV-infected infant was born to a woman who was non-adherent to ART with a VL near delivery >50 000 copies/mL; she received IV zidovudine and delivered by emergency cesarean section and the infant received 3 antiretroviral drugs for 6 weeks.
Overall adherence to regional perinatal guidelines was excellent and perinatal transmission was low. This is likely due to efforts of a multidisciplinary team coordinating care for mother-infant pairs.
The frequency of pregnancies among mothers living with HIV is increasing as a result of recent advances in combination anti-retroviral therapy and the increasing proportion of women living with HIV (WLWH) of childbearing age. WLWH are also at increased risk of adverse obstetric outcomes. This study examines socio-demographic and clinical characteristics as correlates of infant prematurity and low birth weight.
The HIV Mothering Study is a longitudinal study of mothers living with HIV. Data during the 3rd trimester of pregnancy and at 3 months postpartum was obtained through surveys, medicals records, and narrative interviews. Stepwise logistic regression was used to create final multivariate models; for each of the two outcomes, covariates with p-values ≤ 0.20 were included, then a backward elimination of covariates was performed until a best-fit model was reached.
Of the seventy-seven women enrolled, eight deliveries were preterm and eleven were at low birth weight. Univariate logistic regression established that the best predictors of preterm birth were marital status (OR=2.05; p=0.080), education (OR=7.00; p=0.076), CD4 count (OR=4.91; p=0.014), viral load (OR=3.11; p=0.157), cardiovascular disease (OR=0.132; p=0.019), and immigration status (OR=4.68; p=0.127). Eligible correlates of low birth weight were race (OR=0.713; p=0.174), gestation at which ARV was started (OR=0.951; p=0.200), and depression (OR=0.202; p=0.020). Stepwise regression determined that the most significant predictors of preterm birth were CD4 count (OR=0.100; p=0.007); and cardiovascular disease (OR= 49.5; p=0.006); while low birth weight was significantly associated with past medical history of depression (OR=5.27; p=0.018).
Pregnant mothers with low CD4 counts, history of cardiovascular disease, and depression are at increased risk of adverse obstetric outcomes. To improve health trajectories for both mother and child, specific strategies to address clinical and emotional risk factors of the mother should be adopted prior to delivery.
cART is recommended during pregnancy to prevent HIV vertical transmission. However, cART has been associated with adverse birth outcomes through unknown mechanisms. Angiogenesis, the process of blood vessel formation, is crucial to fetal and placenta development. We have previously shown that cART-exposure in a mouse pregnancy model leads to reduced fetal and placenta weight. Here we investigate the effects of cART on placental angiogenesis in this mouse model.
Pregnant C57Bl/6 mice were exposed to human equivalent doses of lopinavir/ritonavir/AZT/3TC or vehicle throughout gestation. Pregnancy loss, fetal number, viability and weight, and placenta weight were recorded on gestational day 15. Angiogenic factors in placenta and maternal blood were analysed by q-PCR and ELISA. Some placentas were perfused with a casting agent into the arterial vasculature, and then scanned using a micro-CT.
cART exposure resulted in significantly lower fetal and placental weight compared to control. Expression of pro-angiogenic factors vascular endothelial growth factor (VEGF), VEGF receptor-2, and placenta growth factor did not differ between groups. Expression levels of the anti-angiogenic factor soluble VEGF receptor-1 (sVEGFR1) were significantly lower in the cART-exposed group. Placental vascular imaging revealed a significantly higher number of arterioles with shorter length in the cART-exposed group.
Our findings suggest that cART exposure alters the angiogenic balance during pregnancy. cART was associated with a pro-angiogenic state characterised by lower sVEGFR1 levels and greater branching of small blood vessels in the placenta. This state correlated with fetal growth restriction. These results address a gap in knowledge of HIV antiretroviral toxicity in pregnancy. We are currently investigating the mechanisms involved in cART-induced down-regulation of sVEGFR1 and are extending our findings to HIV+ pregnant women. Understanding the impact of cART on angiogenic processes during pregnancy may lead to better clinical management of HIV+ pregnant women.
To review the incidence of antenatal complications among a cohort of HIV-positive pregnant women cared for at one center over a ten-year period.
A retrospective review of all HIV-positive pregnant women cared for at an urban tertiary care center from March 2000–March 2010. Data collected included the presence of other infectious or medical conditions, genetic screening information, and the presence or absence of antenatal complications.
There were 142 singleton pregnancies during the study period. Almost 95% of women were on combination antiretroviral therapy during pregnancy and >90% had viral loads <1,000 at delivery. The presence of co-infections was low (syphilis 4/142 [3%], hepatitis B 8/142 [6%], hepatitis C 2/142 [1%], gonorrhea/chlamydia 3/142 [2%]), although 10/142 (7%) had a history of tuberculosis (tb). 41 (29%) women had other medical co-morbidities, with the most common being tb, asthma and hypertension. 104/142 (73%) of women had genetic screening in their pregnancies, with 4/104 (4%) results abnormal. One woman underwent amniocentesis, and there were no genetic abnormalities in any babies born to the entire cohort, including the 4 women with abnormal testing. With respect to other complications of pregnancy, 7/142 (5%) women had pregnancy-induced hypertension or preeclampsia, 10/142 (7%) had gestational diabetes (6 managed with diet control and 4 with insulin), and 5/142 (4%) had fetal growth restriction. The rate of group B streptococcus (GBS) positivity was 45/142 (32%).
Although some studies have found increased rates of adverse pregnancy outcomes, in this cohort of well-controlled HIV-positive pregnant women, the rates of co-infection and co-morbidities were low, and there were also low risks of genetic abnormalities and pregnancy complications. GBS rates were higher than among the general population.
Antibody detection beyond 18 months of age is diagnostic of pediatric HIV-infection. Absence of antibody beyond 18mo has recently been reported in vertically infected children treated with early, aggressive combination anti-retroviral therapy (cART) and is associated with markedly reduced latent HIV reservoirs and sustained viral remission.
We report two children with virologic evidence of HIV-1 infection but negative serology beyond 18 months of age. Case 1 (2yr male born in Lesotho) received nevirapine beginning at 2wk, then cART (zidovudine, lamivudine, lopinavir/ritonavir) from 3mo, based on two positive HIV DNA PCR results at 6 and 10 wk. He had chronic cough, failure to thrive, and was treated for presumed tuberculosis (age 5–11 months). He immigrated to Canada with his adoptive parents at age 14mo, at which time he was asymptomatic, with normal CD4 count, negative HIV-1/2 serology, and undetectable (<40 copies/mL) HIV RNA on cART. HIV-1/2 antibody and RNA remained negative/undetectable on repeated testing. HIV-1 pro-viral DNA was positive. Normal levels of total IgG and appropriate antibody response to live viral vaccines were demonstrated. Case 2 (4 yr male born in Vietnam) received cART from 3wk, had failure to thrive, and had a detectable HIV-1 viral load (78 copies/mL) at the time of immigration to Canada (11mo). His CD4 count remained in the normal range on zidovudine, lamivudine, lopinavir/ritonavir, with intermittently detectable HIV RNA (230 copies/mL at 14 mo, 90 copies/mL at 24 mo). HIV-1/2 antibody was indeterminate at 10mo and 16mo, then negative at 19 mo. HIV-1 proviral DNA was positive (subtype CRF34B01B). Antibody response to hepatitis B vaccine was adequate. Unlike recently reports of seroreversion following very early, effective cART, both seronegative international adoptees in this report has evidence of poorly controlled HIV replication in infancy. Further study (e.g., quantifying HIV reservoir) may illuminate seronegativity as a marker of sustained viral remission versus B-cell anergy.
The nucleoside reverse transcriptase inhibitors alter mitochondrial replication and function. Since brain development requires energy, medications affecting mitochondrial energy production during fetal life and infancy could affect brain development. Studies of infants and toddlers suggest no effect of pre- and peri-natal antiretroviral drug exposure (ARV) on early cognitive and adaptive function, but little is known about longer-term outcomes. We report here on neurodevelopmental outcomes of 3 to 6 year old HIV-exposed uninfected (HEU) children exposed in utero and perinatally to ARVs, in comparison to a sociodemographically matched control group.
HEU children underwent neurodevelopmental assessments as part of routine care in the SickKids Family-Centred HIV Clinic. A control group was recruited in the communities where the HEU children are known to live. Children were administered standardized tests of intelligence and the Vineland Adaptive Behaviour Scales. Children were divided into two groups: mean age 3.5 years and 5.5 years.
110 children (74 HEU, 37 controls) were assessed. Their families were from an ethnically diverse, largely immigrant background, with maternal country of origin being Africa (32% HEU, 24% Controls), Canada (28% HEU, 24% Control), the Caribbean (23% HEU, 27% Control), South/Southeast Asia (11% HEU, 16% Control); and South America (5% HEU, 8% Control). There were no group differences in birth weight, gestational age, and maternal education or employment status. At age 3.5, the HEU and control groups did not differ on IQ, but the HEU children had significantly lower adaptive function. The 5.5-year-olds differed from controls on IQ and adaptive function.
Adaptive skills were reduced at both ages in ARV-exposed children. Differences in intelligence emerge with increased age, highlighting the importance of examining long term development. It remains to be determined whether cognition worsens and whether other deficits appear with increased age. These findings need replication in larger samples.
Congenital Cytomegalovirus infection (cCMV) is the leading cause of congenital infection worldwide, and while HIV-infected women have a higher rate of transmission of cCMV, there are currently no recommendations for the screening of their newborns. We report on a pilot cCMV-screening program for HIV-exposed newborns at CHU Sainte Justine (HSJ).
A CMV screening protocol was implemented in the HSJ nursery for infants of HIV exposed mothers beginning in April 2013, and consisted of testing by salivary swab for cCMV using 1) PCR or 2) Standard culture/shell vial, within 3 days of birth. The medical records of all HIV exposed infants born between April 2013 and Nov 2014 were reviewed to determine adherence to the protocol.
Among 56 live births to 58 HIV infected mothers, 48 newborns were successfully tested for cCMV (12.5% by PCR alone, 64.5% by CMV shell vial/culture, and 23% by both PCR and shell vial/culture). PCR results were available within a mean of 4.72 (range 1–6) days after birth, and CMV shell vial results within 6.42 (range 2–22 days) after birth. 92% of the women had been tested for CMV during pregnancy, among whom 98% were CMV IgG positive – mean IgG immune status ratio was 3.20 (SD 1.16) with a positive cutoff >=1.10. Only one 1 case of cCMV infection was identified by screening PCR, and confirmed by positive urine culture at 13 days of life. The overall birth prevalence of cCMV in this cohort was 2.1%.
CMV screening by salivary swab in newborns is a feasible, practical and time sensitive method of screening for cCMV. Given the potential to improve childhood outcomes by early diagnosis and treatment, further work is necessary to determine the cost effectiveness of such a screening program in the general population of HIV exposed infants.
There have been no studies directly assessing the utility of therapeutic drug monitoring (TDM) in the pediatric HIV population. Routine TDM was implemented on a trial basis in the HIV Clinic at the Hospital for Sick Children in March 2014. The purpose of this project was to assess the utility of this strategy.
This was a prospective observational study of routine TDM for protease inhibitors (PIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs) and integrase inhibitors (IIs) in combination antiretroviral therapy (cART) treated HIV-infected children. Voluntary informed consent was required. Outcome measures included the proportion of serum antiretroviral medication (ARV) levels in the therapeutic range and correlation of levels with virologic control, adherence and toxicity.
Forty-eight of 64 cART treated children in the clinic were recruited (75%). Their median age, viral load (VL) and CD4 percent were 13 (3–18) years, <40 (<40–124) copies/mL and 37.4% (8.4–47.9%), respectively; 45.8% were female. VL was <40 copies/mL in 91.7%. Adherence was assessed as excellent (>95%) in 95.8%. Fifty baseline trough serum levels were taken, including 19 (38%) for PIs, 27 (54%) for NNRTIs, and 4 (8%) for IIs. Sixty-eight percent (n=34) of levels were within the therapeutic range, 10% (n=5) were subtherapeutic, and 22% (n=11) were supratherapeutic. The highest proportion of therapeutic levels were within the NNRTI class (77.8%) followed by PIs (52.6%) and IIs (50%) (p=0.047). There was no statistically significant correlation between serum ARV levels and demographic data, VL, CD4%, adverse event scores, or adherence. Only one dose adjustment was made for subtherapeutic raltegravir levels due to a presumed interaction with ritonavir.
This study does not support routine use of TDM in generally healthy, well-controlled cART-treated HIV-infected children. A more targeted strategy, such as when adherence is questioned or when there are suspected drug interactions, may be more appropriate.
Youth constitute 78% of Uganda’s population. HIV prevention programmes should pay special attention to this group to prevent devastating effects HIV can have on the country. This paper highlights the implications of low knowledge of the youth on HIV/AIDS in Jinja district.
To highlight the implications of low knowledge of youth on HIV/AIDS transmission and prevention to inform interventions
Lot Quality Assurance Sampling Methodology was adopted. Jinja district was divided into five supervision areas. Nineteen villages were selected from each Supervision Area. Data was collected from youth between 15 and 24 years. The youth were asked information on the following sub categories: HCT, PMTCT, HIV knowledge and sexual behavior. Data was analyzed in Ms. Excel.
Over 90% of the Youth sampled were above 19 years and 60% had partners. 42.1% were able to identify atleast three benefits of HIV counselling and testing. 42.1% had tested for HIV in the past twelve months and received their results. When asked information about PMTCT, 14.7% knew when HIV can be transmitted from an infected mother to her child. When asked about the knowledge on HIV and Sexually Transmitted Infections (STIs), 13.7% were able to correctly identify ways of preventing sexual transmission of HIV and reject major misconceptions about HIV transmission. 35.8% were able to identify atleast two symptoms of STIs in men while 37.9% were able to identify atleast two symptoms of STIs in women.
Majority of the youth do not understand the benefits of HIV counselling and testing and when transmission of HIV can occur from an infected mother to her baby. This has implications on whether they then will seek for HIV testing and prevent HIV infections. Therefore involvement of key stakeholders should be prioritized to design specific interventions to curb the increasing HIV infections.
Inmates identified as HIV-positive upon admission into a large remand facility in a northern Canadian city are seen by an infectious disease physician at bi-weekly onsite outreach HIV clinics. This facility has the ability to house up to 1,900 inmates with an average length of stay of 17 days, making the timely provision of HIV care challenging. Patients frequently reported delayed, missed or incomplete antiretroviral therapy (ART) due to the rapid turnover within the facility and the lack of onsite prescribers to promptly ensure ART continuance. The objective is to describe a pharmacist-led initiative aimed at reducing gaps in ART in this vulnerable population.
Since April 2013, this facility has been supported by clinically deployed pharmacists who provide comprehensive medication management. In accordance with provincial regulations, pharmacist scope of practice includes the ability to prescribe with varying degrees of independence, and all licensed pharmacists can adapt prescriptions for continuity of care based on their assessment. On admission, all HIV-positive patients are assessed by a pharmacist to ensure ART is continued promptly and appropriately and to initiate planning for release. The infectious disease physician is only involved in the initial management of these patients if concerns are identified, or if the pharmacist determines more urgent specialist consultation is required. To ensure issues arising during incarceration are managed appropriately, the need to have a designated pharmacist follow HIV patients was identified. Ongoing improvements include pharmacist training via formal education and informal mentorship with local HIV pharmacists.
Although further research is required to fully capture the impact of this initiative, preliminary feedback indicates that the incidence of missed ART doses has declined since pharmacists began continuing ART therapy upon admission to the facility. Through this collaborative interdisciplinary initiative, there has been improved care of HIV patients within this correctional facility.
The unique challenges of remoteness and geographic isolation faced by rurally located HIV-positive individuals can negatively affect their care outcomes. This study looked at whether rurality and/or the degree of rurality of antiretroviral therapy (ART)-naïve individuals impacts their Programmatic Compliance Score (PCS), a previously validated metric for all-cause mortality.
We determined the PCS score for all patients (≥19 years) initiating ART in British Columbia between 2000–2013. The PCS score is a sum of six outcome measures scored from 0–6, where zero is most compliant, and six is least compliant with established treatment guidelines. Rurality was determined for each individual at time of ART initiation using 1) a categorical Postal Code method; and 2) the General Practice Rurality Index (GPRI), a previously validated score representing their degree of rurality. Ordinal logistic regression modelling was used to assess the relationship between rurality and PCS score.
Of 4616 individuals with a PCS score, 176 were classified as rural, and 3512 as urban, using the Postal Code method (928 had an unknown postal code and were therefore excluded). GPRI score was calculated for 4544 individuals. After adjusting for age, gender, Hepatitis C status, Aboriginal ethnicity, baseline CD4 count, and baseline viral load, categorical rurality was not associated with a higher PCS score (AOR 1.02; 95% CI: 0.57–1.84). However, the degree of rurality (per 10 increase in GPRI score) was associated with a higher PCS score (AOR 1.09; 95% CI: 1.02–1.16).
For individuals initiating ART, degree of rurality was predictive of poorer PCS scores, while classifying individuals as “rural” or “urban” using a categorical method was not. Given that higher PCS scores are predictive of all-cause mortality, strategies to enhance access to care for rural individuals should be evaluated to improve HIV outcomes in British Columbia.
Literature in the United States has shown that uptake of new antiretroviral (ARV) medications is slower in rural locations. This study examined whether rurally-located antiretroviral therapy (ART)-experienced individuals in British Columbia had a longer time-to-uptake of new ARVs compared to their urban counterparts.
We included HIV-infected, ART-experienced individuals (≥19 years) who switched to regimens that included Atripla™ (efavirenz-emtricitabine-tenofovir), raltegravir, Complera™ (rilpivirine-emtricitabine-tenofovir), or Stribild™ (elvitegravir-cobicistat-emtricitabine-tenofovir) between October 2007 and June 2014. Individuals whose initial ART regimen included these ARVs were excluded. Time-to-uptake of each ARV was measured from the date of drug availability or ART-initiation date (whichever was later) to the new ARV switch-date. Rurality was determined at ARV switch-date using 1) a categorical Postal Code method; and 2) General Practice Rurality Index (GPRI), a previously validated score representing degree of rurality.
Among 3404 individuals ever prescribed Atripla™, raltegravir, Complera™, or Stribild™, 2166 were ART-experienced – 1083 had switched to Atripla™, 948 to raltegravir, 216 to Complera™, and 193 to Stribild™. Of these 2166 individuals, 40.2% (870) were co-infected with Hepatitis C, 36.7% (794) had used injection drugs, and 55.0% (1192) had ART adherence rates ≥95%. Using the Postal Code method, 97 new ARV-switches were classified as rural, and 2048 as urban (295 had an unknown postal code and were excluded). GPRI score was calculated for 2399 new ARV-switches. A Wilcoxon rank-sum test was used to assess the relationship between rurality and time-to-uptake of each ARV. Rurally-located individuals (compared to urban) had a longer median time-to-uptake for Atripla™ (9.1 versus 6.3 months, p=0.024) and raltegravir (27.3 versus 26.4 months, p=0.062), but not Complera™ (14.8 versus 7.0 months, p=0.194), or Stribild™ (1.3 versus 6.1 months, p=0.076).
Rurality is associated with longer time-to-uptake of some, but not all, new ARVs. Possible reasons for rural/urban prescribing differences requires further investigation.
Building Bridges was a collaborative community-based research project between Aboriginal and allied stakeholders and the Canadian Observational Cohort (CANOC) collaboration. The research team identified mortality as an endpoint of interest to compare Aboriginal and non-Aboriginal CANOC participants.
CANOC is a collaboration of 8 cohorts of treatment-naïve HIV-infected participants initiating combination antiretroviral therapy (cART) after 1/1/2000. Participants were followed from the date of cART initiation until death or date of last viral load (VL) test before 12/31/2012. Cox proportional hazard models were used to estimate the effect of ethnicity on time until death after adjusting for age, gender, injection drug use, being a man who has sex with men, province of origin, baseline VL and CD4 count, year of cART initiation and antiretroviral class.
9300 participants (498 Aboriginal Peoples, 2474 Caucasian, 789 African/Caribbean /Black (ACB), 629 other and 4910 unknown participants) were included in the analysis. 7608 (82%) were male and the median (interquartile range) age was 40 (33–46). During the study period, 794 (9%) participants died including 131 (26%) Aboriginal Peoples, 20 (3%) ACB, 185 (7%) Caucasian, 29 (5%) other ethnicity and 429 (9%) unknown ethnicity participants. Five year survival was lower for Aboriginal Peoples (68%) when compared to Caucasian (91%), ACB (97%), other ethnicity (93%) and unknown ethnicity (86%) participants (p<0.0001). In a proportional hazard model adjusted for confounding variables, Aboriginal Peoples were more likely to die than Caucasian participants (HR=2.39, p<0.0001) whereas mortality was similar between Caucasian, ACB (HR = 0.76, p=0.30) and other (HR = 1.02, p=0.94) participants.
The mortality rate for Aboriginal Peoples with HIV in CANOC was significantly higher than for other ethnic groups accessing cART. Addressing treatment challenges and identifying HIV-and non-HIV related causes for mortality among Aboriginal persons is required to optimize their clinical management.
Building Bridges was a collaborative community-based research project between Aboriginal and allied stakeholders and the Canadian Observational Cohort (CANOC) collaboration. The research team identified time to virologic suppression (VS) and virologic rebound (VR) as endpoints of interest to compare between Aboriginal and non-Aboriginal CANOC participants.
CANOC participants are treatment-naïve and have initiated combination antiretroviral therapy (cART) after 1/1/2000. Fine and Gray models were used to estimate the effect of ethnicity on 1) time to VS (two consecutive viral loads (VLs) <50 copies/mL at least three months apart) after adjusting for the competing risk of death and 2) time until VR (two consecutive VLs >200 copies/mL at least 3 months apart) following VS. Models were adjusted for age, gender, injection drug use and men who have sex with men status, province of residence, VL measurement rate, baseline VL and CD4 count, antiretroviral class and year of ART initiation.
9300 participants (498 Aboriginal Peoples, 8802 other race/ethnicity (e.g., African, Caribbean, or Black, Caucasians, unknown, other) were included in the analysis. 7608 (82%) were male and the median (interquartile range) age was 40 (33–46) years. VS was achieved in 7811 (84%) participants, and of these 1489 (16%) rebounded. 384 participants died before achieving VS, including 72 (14%) Aboriginal Peoples and 312 (4%) other participants. In the adjusted model, Aboriginal Peoples were less likely to achieve VS than Caucasian participants (Hazard Ratio=0.71, p<0.0001). In a model adjusted for the same covariates, VR (Hazard Ratio=1.10, p=0.39) was similar between Aboriginal Peoples and Caucasian participants.
Among CANOC participants initiating cART, Aboriginal Peoples were less likely to achieve VS, but had similar rates of VR as other ethnic groups. Further research is required to identify socio-demographic, clinical and psychosocial predictors of VS to target interventions, programming and services to improve HIV health outcomes.
The uptake of pre-exposure prophylaxis (PrEP) for HIV prevention remains low. We hypothesized that a high proportion of patients presenting for non-occupational post-exposure prophylaxis (nPEP) against HIV would be candidates for PrEP based on current CDC guidelines. We describe a novel, multidisciplinary comprehensive outpatient HIV prevention clinic and describe transition from nPEP to PrEP.
We evaluated all patients who attended the HIV Prevention Clinic for nPEP between January 1, 2013 and September 30, 2014. Each patient was evaluated for candidacy for PrEP objectively based on current CDC-guidelines and subjectively based on physician opinion. Patients were then evaluated for initiation of PrEP if they met guideline suggestions. Demographic, social, and behavioural factors were then analyzed with logistic regression for associations with PrEP candidacy and initiation.
99 individuals who attended the nPEP clinic were evaluated for PrEP. The average age was 32 years (range, 18–62), 83 (84%) were male, of whom 46 (55%) were men who had have sex with men (MSM). 31 (31%) met CDC guidelines for PrEP initiation, which had very good agreement with physician recommendation (kappa= 0.88, 0.78–0.98). Factors associated with PrEP candidacy included sexual exposure (100% of PrEP candidates vs. 72% of non-candidates), prior nPEP (adjusted odds ratio = 27.8, 3.1–250.0), and lack of drug insurance (AOR=2.9. 1.0–9.1).
Combining nPEP and PrEP services can lead to identification of PrEP candidates and facilitates PrEP uptake. There was very good agreement between physician recommendation and PrEP guidelines. Given the high cost for PrEP, it is concerning that patients who would benefit from PrEP were less likely to have medication insurance.
PREPARATORY-5 is Canada’s first demonstration project of daily TDF/FTC-based HIV pre-exposure prophylaxis (PrEP), among Toronto men who have sex with men (MSM). We hypothesized that individuals referred to the study by service providers would be at objectively higher risk of HIV acquisition than those who self-referred.
Trial participants were solicited through self-referrals in response to advertisements on the gay networking application Grindr and newspaper/website Xtra, and provider referrals from 13 Toronto-based community-based organizations (CBOs). Interested MSM were offered a screening visit involving testing for sexually transmitted infections and comprehensive questionnaires including a validated HIV risk index (HI-RI-MSM). A log-binomial model with sandwich estimators was fitted to examine factors associated with referral source.
By 31/12/2014, the Grindr and Xtra advertisements generated 1460 and 36 click-throughs respectively, and CBOs referred 115 individuals. 170 men inquired about the trial, of which 83 expressed interest in participating; 58 have thus far undergone screening, of which three withdrew. The remaining 55 included 42 (76%) self-referrals (27 Grindr, 3 Xtra, 12 other) and 13 (24%) provider referrals (11 clinician, 2 CBO). 96% had previously heard about PrEP, and 18% reported prior PrEP use. Prevalence of newly diagnosed syphilis was similar between groups (0/13 versus 1/42, p=0.57). Provider-referred participants were older than self-referred participants (median age 38 versus 31.5 years, p=0.02) but had similar HIV risk scores (median 28 versus 26.5, p=0.37). In multivariate analysis adjusting for sexual orientation, income, education and ethnicity, age remained marginally associated with provider referral source (RR=1.08 per year, 95% CI=0.99,1.17) while HIV risk score was not (RR=1.02, 95% CI=0.91,1.14).
These preliminary findings suggest that MSM self-referring for PrEP generally had a similar clinical and HIV risk profile compared to those referred by service providers. Online strategies warrant further study for their potential to reach at-risk, previously unengaged individuals.
HIV pre-exposure prophylaxis (PrEP) has not received regulatory approval for use in Canada. PREPARATORY-5, Canada’s first demonstration project of open-label tenofovir/emtricitabine based PrEP, began enrolling Toronto-based adult men who have sex with men (MSM) at high risk of HIV infection in October 2014.
Consenting MSM interested in participation attended a screening visit including questionnaires on socio-demographics and validated psychometric measures. The latter included the Center for Epidemiologic Studies-Depression (CES-D), Alcohol Use Disorder Identification Test (AUDIT), Drug Use Disorder Identification Test (DUDIT) and Sexual Compulsivity Scale (SCS). The HIV Incidence Risk Index for MSM (HIRI-MSM) quantified HIV risk, Multi-Axial Gay Men’s Inventory—Men’s Short Version (MAGI-MSV) screened for internalized homophobia, and Benefits of Barebacking Scale (BOBS) examined attitudes towards intentional unprotected anal intercourse.
As of December 11, 2014, 55 MSM completed the questionnaire. Median (interquartile range) age was 33 (28,41). All identified as gay (95%) or bisexual (6%). Most identified as Caucasian (75%), fewer as Asian (13%), Middle Eastern (6%), or Black (2%). Many had a college/undergraduate (53%) or graduate/professional degree (29%). Median income range was $50,000–$59,999. Median number of male sexual partners in the preceding six months was 20 (11,35). Almost all (98%) scored above the suggested HIRI-MSM cut-off for identifying MSM potentially appropriate for PrEP). Approximately 42% screened positive for depression, 9% for at-risk drinking, and 31% for sexual compulsivity, with 18% being heavily influenced by drugs at least once per week. The median MAGI-MSV score of 8.0 (2,13) suggested minimal internalized homophobia. The median BOBS score of 30 (23,36) suggested moderate value placed on barebacking.
Results suggest potential high prevalence of health concerns in MSM seeking PrEP in Toronto. The role of co-occurring health concerns (syndemics) in mediating HIV risk in PrEP users and their association with longer term health outcomes warrants further exploration.
Little is known about pre-exposure prophylaxis (PrEP) use and adherence in real-life settings and its impact on high-risk behaviors is unclear. We aimed to evaluate adherence to follow-up and treatment, and behavioral changes in a high-risk clinical population.
We prospectively assessed patients receiving PrEP (TDF-FTC) at our clinic from 2011–2014. After their initial visit, patients were seen at 3-month follow-up intervals (FU). Treatment adherence and behavioral data were measured by self-report at every FU visit. Adherence and behavioral changes were analyzed by χ2 and time to treatment discontinuation was estimated by Kaplan-Meier analysis.
112 patients were prescribed PrEP. The main indication for PrEP was regular unprotected anal intercourse (64%). Patients requesting PrEP were male (99%) and MSM (98%) with a mean age of 38 (Range=20–61y). The majority of patients had a history of STDs (80%) and 67% reported having more than 10 sexual partners. On average, condom use was 59% for receptive anal intercourse and 63% for insertive. Among the 87 patients with available FU data, median FU was 12 weeks. In the first 3 months after starting PrEP, 92% of patients attended a FU visit. Furthermore, 86% of patients reported taking PrEP daily, whereas 2% had adherence problems and 4% took PrEP intermittently. Overall, 18 patients (21%) stopped PrEP; 44% of which occurred in the first 3 months of FU. Five patients (5/18, 28%) discontinued due to adverse events: four patients had elevated creatinine and 1 patient suffered from nausea and vomiting. Increases in high-risk behavior following PrEP use were not observed. There was no difference among the reported number of sexual partners (p=0.557) and condom use (p=0.293).
Patients receiving PrEP seem adherent to treatment and to follow-up. However, one-fifth discontinued prophylaxis. PrEP does not promote an increase in high-risk behaviors.
Case Management (CM) improves the care of HIV-infected patients with substance use disorders (SUDs). Little is known about factors affecting CM implementation within existing clinical settings. We report on the feasibility, specifically, practicality (extent to which an idea can be implemented using existing resources) of developing and delivering a CM intervention: FEAT (Find, Engage And Treat).
Frameworks for designing and evaluating complex interventions (Craig et al. 2008) and feasibility studies (Bowen et al. 2009) were used to identify and analyze information. Data related to practicality (e.g., appointment dates, number confirmed SUDs) were collected and considered.
FEAT was developed over 11 months involving frequent consultations with clinic staff, selection of clinical and evaluation tools (e.g., e-version of the Addiction Severity Index, Teamwork.com), agreement on a mixed CM model (time divided between FEAT and regular patients), and training of personnel. Screening for SUDs identified 22 patients; 9 confirmed positive. The clinical team referred 23 patients with SUDs. Of the 32 patients confirmed with SUDs, 14 enrolled in FEAT and 13 received an individualized care plan. Reasons for non-participation included not interested and no financial incentive. Main difficulties related to the intervention (patient screening, finding instruments in Spanish, time required for assessments), the capacity to locate and reach some patients, and the clinical setting (space for privacy, clinic expansion). The intervention was refined to meet these challenges. The faster pace at which recently recruited patients go from screening to care plan and the interest expressed by other clinical staff to become case managers suggest an improved and acceptable intervention.
The development and delivery of a complex intervention requires frequent and diverse feedback to refine its components and related processes. Successful implementation of a mixed CM model is threatened in milieus that are under pressure because of changing resources.
Appropriate management of low-level viremia is an ongoing issue in patient care for HIV infection. We wished to examine whether there was evidence that some low level viral load measurements are simply assay “false positive” values, rather than resulting from other factors such as intermittent periods of non-adherence.
The distribution of all viral load values reported to the BC Centre for Excellence in HIV/AIDS in 2013 was examined (N=28,469 tests). During 2013, the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 Test v2.0 assay was used.
More than 77% of samples tested had a pVL <50 copies/mL. Assay results with low values >50 copies/mL were systematically overrepresented, with the prevalence decreasing monotonically with the reported value. For example, there were 64 observations of “51 copies/mL”, 54 observations of “52 copies/mL”, 42 observations of “53 copies/mL”, etc, up to ∼250 copies/mL. In contrast, 95% of individual viral load values between 500–1000 copies/mL had 0, 1, or 2 observations. A similar pattern was observed for other years examined.
Patients receiving constant therapy for >6 months with initial pVL in the 50–100 copies/mL range were >2.5-fold more likely to have a confirmatory pVL measurement (collected within 15 days) <50 copies/mL than those with initial pVL in the 500–1000 copies/mL range. (<20% vs >50%, respectively)
The suspicious distribution of pVL values suggest that low viral load measures may commonly represent assay false-positive results, and reinforces the fact that viral load measurements should not be used as an HIV diagnostic test. A similar analysis of large numbers of viral load measurements using the Abbott RealTime assay is in progress to establish whether this is a generic problem with viral load measurement.
Population-based studies describing rates of emergency department utilization by persons living with HIV are lacking. Because emergency room visits may be emblematic of poor linkage with primary care, particularly for non-urgent conditions, such data are required to optimize healthcare delivery to persons with HIV. Accordingly, we compared rates of emergency department visits between persons living with and without HIV in Ontario between April 1, 2011 and May 31, 2012.
We conducted a population-based study using Ontario’s health administrative data. Individuals living with HIV were frequency matched to 4 non-infected individuals on age, sex and Ontario census division. We used multivariable negative binomial regression with a random intercept to compare rates of emergency department visits and generalized estimating equations with a logit link function to examine the association of HIV infection with hospital admission following an emergency department visit. We used the Canadian Triage and Acuity Scale (CTAS) to classify emergency room visits according to severity of patient presentation.
We studied 14,534 individuals living with HIV and 58,136 matched control individuals. Following multivariable adjustment for demographic variables, comorbidity and primary care use, rates of emergency department utilization were higher for persons with HIV (67.3 vs. 31.2 per 100 person-years; adjusted rate ratio 1.58; 95% confidence interval 1.51 to 1.65). Similar results were observed following stratification by urgency of visit. Persons with HIV were also more likely to be admitted to hospital following an emergency department visit (adjusted odds ratio 1.55; 95% confidence interval 1.43 to 1.69), including for visits classified as non-urgent by CTAS score.
Persons with HIV have higher rates of emergency department visits and subsequent hospitalizations, including visits classified as less urgent by CTAS score. These results could reflect inadequate access to primary care.
To examine the safety and effect of aerobic exercise interventions on immunological and virological, cardiopulmonary, weight and body composition, strength, and psychological outcomes in adults living with HIV.
We conducted an update of a systematic review of literature on HIV and exercise according to Cochrane protocol searching databases up to April 2013. We included randomized controlled trials comparing aerobic exercise with no exercise or another intervention performed at least 3 times/week for at least 4 weeks among adults (18 years or older) living with HIV. Two reviewers independently determined study eligibility. Data were extracted from studies that met inclusion criteria using standardized forms. We assessed methodological quality using the Cochrane Risk of Bias Tool. Outcomes were analyzed as continuous and meta-analyses conducted using random effects models with RevMan5 computer software.
Twenty-four studies met inclusion criteria (n=956 participants); the majority were men (73%), taking combination antiretroviral therapy. Exercise included aerobic exercise alone (11 studies) or a combination of aerobic and resistive exercise (13 studies) ranging from 5 to 52 weeks. Sixty-eight meta-analyses were performed. Main results indicated statistically significant improvements in selected outcomes of cardiopulmonary status (maximum oxygen consumption, exercise time), body composition (lean body mass, percent body fat, leg muscle area), strength (chest press, knee flexion), depression symptoms, and quality of life (SF-36 questionnaire) among exercisers compared with non-exercisers. Greater improvements were found among participants exercising at greater intensity. No significant differences in change in CD4 count and viral load were found.
Performing aerobic exercise or a combination of aerobic and resistive exercise at least 3 times/week for at least 5 weeks is safe and can lead to improvements in cardiopulmonary fitness, body composition, strength and quality of life for adults with HIV. Findings are limited to participants who continued to exercise. Aerobic exercise is safe and beneficial for adults living with HIV who are medically stable.
Discharge from hospital is often framed as a clinical success. For people living with HIV/AIDS (PHAs) who experience psychosocial challenges and medical complexity, this transition period is a high-stress time, which can contribute to hospital re-admission. Research has left unexamined the congruency between the clinicians’ discharge plans and the clients’ experience of the execution.
A mixed-methods case study approach was utilized to explore factors that impact the discharge and transition period of PHAs who received in-patient care at Casey House (a sub-acute care HIV hospital). Demographic and medical data were abstracted from hospital charts and discharge planning documentation. Semi-structured interviews were conducted at four timepoints (approximately one week pre-discharge to one month post-discharge). Clinical data were analyzed and reported using descriptive statistics. Interview data were analyzed using qualitative thematic analysis across time and cases to capture the lived experience at each timepoint.
Data were collected from nine participants between October 2013 and June 2014. Participants presented with complex medical and psychosocial challenges, including substance use (n=8), psychiatric diagnoses (n=9) and a mean of 5 medical comorbidities (range 2–8). Participants were discharged with an average of 7 referrals and 15 medications. Discharge plans outlined medication adherence strategies and referrals. Analysis revealed significant discrepancies between “the plan” and post-discharge “reality” including minimal referral uptake, lack of social support, substance use relapse and poor medication adherence. Clinicians’ use and defining of terms such as ‘social support’ and ‘medication adherence’ differed from that of the participants and post hospitalization expectations of clinicians were not realistic given the context of participants’ lives.
This unique longitudinal study identified challenges experienced by complex clients in the execution of hospital discharge plans. The study findings have implications for future research, policy, and clinical practice in the delivery of hospital discharge services for PHAs.
GIS continue to be side-effects of HIV disease and ARV, associated with reduced quality of life and ARV non-adherence. Understanding up-to-date risk factors for GIS in a modern ARV era is important to inform clinical management.
The OHTN Cohort Study is a multi-site clinical cohort of persons in HIV care in Ontario. We analysed data from 1744 participants with ≥2 interviews between 2007–2013 and who completed the ACTG Symptom Distress Module. Measured GIS were nausea/vomiting, diarrhea, appetite loss, bloating, or weight loss in the past 30 days and their perceived level of distress. We tested trends in GIS scores (range 0–20, where a higher score indicated more symptoms with greater distress) using generalized linear mixed models with random effects.
At first questionnaire, most were male (78%), Canadian born (54%), white (53%), employed (41%), mean age 44.9 (SD 10.5) years, and median time since HIV diagnosis of 9.7 (IQR 3.9, 16.9) years. Majority (89%) were ARV experienced, mean CD4+ count 498 (SD 279) cells/μL and 1,365 (84%) virologically suppressed (<200 copies/mL) at baseline. Two-thirds (68%, 95% CI 64.7, 69.3) reported ≥1 GIS with median score of 2 (IQR 0, 6) and 522 (44%) indicated moderate to severe symptom distress. Among those reporting GIS, bloating (41%), diarrhea (39%), appetite loss (24%), nausea/vomiting (21%), and weight loss (21%) were reported. Participants had median of 2 (IQR 1, 4) questionnaires. In multivarable analyses, GIS score was significantly higher among those with depressive symptoms (≥16 score, CES-D: B=1.23, p<0.001), who had history of IDU (B=1.02, p<0.001), hepatitis C co-infection (B=0.74, p<0.001), and were born in Canada (B=0.44, p<0.001). On average, GIS scores decreased significantly with each year since HIV diagnosis (B=−0.55, p<0.001). Current ARV regimen and 30 day adherence (>95%) were not significant.
Although there is uncertainty regarding specific causes of GIS in this population, our findings suggest that clinical management of GIS is still relevant in the modern ARV era, particularly among those with depressive symptoms.
The International Classification of Function, Disability and Health (ICF) is an international language for describing health and disability. It can be used as a content analysis tool to understand the construct and dimensions of health-related quality of life (HQOL) measures. Content analysis is a standardized method of linking items from a specific measure to the ICF. It provides insight into the heterogeneity, precision and depth of each measure so that clinicians and researchers can choose the instrument most suitable for their needs. There are no content comparisons of HIV specific HQOL measures with the ICF. The purpose of the study is to conduct a content analysis of the items of the Medical Outcome Study-HIV (MOS-HIV), the Multidimentional Quality of Life -HIV (MQOL-HIV) and the HIV Disability Questionnaire (HDQ) by linking these to the ICF and its generic & disability core set.
Four raters individually linked 145 items from the measures based on standardized linking rules. Inter-rater agreement was determined. For items where there was no agreement among raters, the opinion of a fifth rater, an expert in applying the ICF was obtained.
The items were linked to 73 ICF categories across the three measures. Final inter-rater agreement was 83.3%. The content of all three measures was highly linkable with ICF. The HDQ had the best representation among the three measures. It was 56% linkable with the generic & disability core set; of these 70% were linked with disability codes.
The HDQ provides a more precise description of the disablement experienced by people living with HIV (PHA) by addressing the body function, activities, participation and environmental factors specific to PHAs. HDQ is thus recommended for those who are interested in measuring HIV-specific disabilities more precisely.
Prevention interventions targeted to female sex workers (FSWs) are a key component of HIV prevention programs globally, particularly in regions where commercial sex fuels and sustains heterosexual HIV epidemics. However, targeted preventive interventions (TIs) reach women after they self-identify as sex workers, thus raising concerns that HIV acquisition by women prior to, and within the very early stages of formal sex work, could mitigate the impact of TIs on reducing HIV incidence among FSWs and in the wider community. The overall goal of this study is to characterize HIV risk faced by young women during the transition period (from first sex until self-declared entry into sex work, including periods of casual sex and transactional sex if applicable) and during the access gap (the first two years of formal sex work, but prior to program engagement), and to examine the importance of this early HIV risk for HIV epidemic control. Mapping and estimating the population sizes of FSWs and young women during the transition period in Mombasa, Kenya and Dnipropetrovsk, Ukraine, are central to the Transitions Study. Unlike conventional mapping, which counts predefined risk groups, the approach used in this study geographically locates and profiles risk behaviors irrespective of occupation.
This presentation will 1) provide an overview on the mapping and enumeration methodology used for Transitions; 2) share some early mapping findings from Mombasa and Dnipropetrovsk, including types of venues where FSWs solicit clients and information on the extent to which these venues overlap with places where casual and/or transactional sex is sought; 3) provide an estimate on the population sizes of FSWs and young women aged 14–24 years who are in the transition period at the two study sites; and 4) highlight new analyses planned for the mapping data.
To describe geographic origin trends of HIV+ mother-infant pairs (MIP) in Canada from 1990 to 2013.
Data collected annually from 22 pediatric and HIV centres participating in the Canadian Perinatal HIV Surveillance Program were reviewed including: maternal country of origin and clinical characteristics, antiretroviral usage and infant outcome.
Among 3877 MIP from 1990–2013, 2089 (53.9%) mothers were foreign-born (FBM). Of 1481 (70.9%) African-born mothers, 30.7%, 20.1%, 17.7%, and 16.7% came from East, Central, Horn, and West Africa, respectively. Most mothers in Western Canada (971/1456, 66.7%) were Canadian-born, whereas FBM predominated in Ontario (945/1357, 69.6%; greatest proportion East African, 25.0%) and Quebec (713/1020, 69.9%; greatest proportion Caribbean, 36.2%). By individual country, the largest numbers of FBM originated from Haiti (261), Ethiopia (181), Congo (146), Zimbabwe (113), Nigeria (96), Burundi (89), Rwanda (82), and Sudan (81). Distribution of countries of origin changed over time; in the pre-cART era (1990–1996), Haiti contributed 29.9% (90/301) of FBM but this decreased to 13.0% (119/918) from 1997–2007, and 6.6% (52/782) in 2008–2013. Since 2008, Ethiopia (80/782, 10.2%), Congo (64/782, 8.2%), and Nigeria (62/782, 7.9%) predominated. The proportion of FBM diagnosed during/after pregnancy decreased from 84% (136/162) overall, 87.3% (55/63) from Africa, and 87.8% (72/82) from Haiti in the pre-cART era to 10.5% (82/778), 9.6% (58/606), and 11.5% (6/52) in 2008–2013. Of the 300 HIV-infected children with FBM, the greatest numbers came from Haiti (48, 16%), Ethiopia (33, 11%), Burundi (30, 10%), Congo (15, 5%), Rwanda (13, 4.3%), Jamaica (12, 4%), Zimbabwe (12, 4%) and Somalia (11, 3.7%).
Geographic origins of HIV+ FBM in Canada have changed over time, with a shift from predominantly Haitian in the pre-cART era to predominantly African more recently. Understanding cultural background and potential obstetrical/pediatric health issues associated with country of origin is important in providing optimal care.
To describe demographics of mother-infant pairs (MIP), treatment during pregnancy and vertical transmission (VT) rates in the Canadian perinatal HIV surveillance cohort of births to HIV+ mothers from 1990 to 2013.
Maternal and infant data are collected yearly from 22 Canadian HIV centres. VT rates are obtained from the prospective cohort defined as MIP delivered in Canada and identified within 3 months after birth. Data collected include maternal characteristics, antiretroviral therapy (ART) given, and infant outcome.
Of 200 identified HIV-positive women giving birth in Canada in 2013, 76% had acquired HIV heterosexually, 17% through injection drug use (IDU) and 2% perinatally; 53% of mothers were black and 23% were aboriginal. 37% of MIP were identified in Ontario, 19% in Alberta, 18% in Québec, 11% in Saskatchewan, 9% in BC and 7% in Manitoba. The proportion untreated has steadily decreased from 20.3% in 1997 to 3.0% in 2013. Aboriginal women (7%) continued to have a higher proportion untreated in 2013, although this has decreased from 2005–2009 when non-treatment was consistently above 20%. A similar trend in improvement in non-treatment (3%) was seen in IDU. Among 2914 MIP in the prospective cohort in the combination ART (cART) era (1997–2013), the overall VT rate was 2.1% but only 0.7% in MIP receiving antenatal cART and 0.1% in women receiving >4 weeks of antenatal cART. In 2013, seven (3.5%) women either had no antenatal cART or suboptimal treatment, the lowest annual number and percentage in the cART era. Two children were infected perinatally in 2013, one of whom was identified after 3 months of age.
Continued improvement in treatment access for pregnant HIV+ women was seen but perinatal transmission continues to occur. Aboriginal women remain at greater risk of inadequate treatment and risk of vertical transmission to their infants.
Treatment as Prevention (TasP) aims to reduce HIV burden by achieving viral suppression in people with HIV. Without laboratory data measuring viral load (VL), observational studies rely on self-report in questionnaires but the accuracy remains unclear. We assessed the validity of a self-reported measure of undetectable VL to assess viral suppression among women living with HIV (WLHIV).
We used linked questionnaire and laboratory data from WLHIV (≥16 years) in BC enrolled in the Canadian HIV Women’s Sexual and Reproductive Health Cohort Study (CHIWOS) and the BC Centre for Excellence in HIV/AIDS (a population-based registry capturing 100% of VL data in BC). Self-reported undetectable VL was assessed by the question: “What was your most recent VL, undetectable (i.e. below 50 copies/mL) or detectable (i.e. over 50 copies/mL)?” Laboratory measurements of VL <50 copies/mL (closest to/before study visit) were the criterion for validity analyses. We measured positive and negative predictive values (PPV, NPV) of self-reported undetectable VL.
Questionnaire data were linked to laboratory data for 99.7% of participants (n=285); 13 were excluded due to missing self-report data. Median age was 45 (IQR: 37–51). 47% identified as Aboriginal, 36% Caucasian, and 6% African, Caribbean, or Black. 31% and 44% reported recent injection drug use and sex work. 83% were currently on ART and 93% enrolled in HIV care. 84% self-reported having undetectable VL while 82% had laboratory data indicating suppression. Women reporting recent illicit drug use and a current CD4 count <350 cells/mm3 were significantly less likely to be virally suppressed. PPV of self-reported undetectable VL was 94% (95% CI: 89–96), showing 94% of women who self-reported being undetectable truly were. NPV was 81% (67–92), showing 81% of women who self-reported being detectable truly were.
A brief and simple self-reported measure assessing undetectable VL strongly predicted true viral suppression among a cohort of WLHIV in BC with a high prevalence of laboratory-confirmed viral suppression. This measure can be used in research settings without laboratory data to assess TasP-related goals.
We sought to describe temporal changes in the demographic and clinical profile of HIV-positive individuals initiating combination antiretroviral therapy (cART) in Canada from 2000–2011.
Participants of the Canadian Observational Cohort (CANOC) collaboration, a multisite cohort of HIV-positive individuals aged ≥18 years and initiating cART naively after 2000 in BC, Ontario, and Quebec, were included. Participants with <12 months of follow-up were excluded. Participants were grouped by era of cART initiation (2000–2002, 2003–2005, 2006–2008, 2009–2011). Demographic and clinical characteristics were compared by era using Pearson’s χ2 and Wilcoxon rank-sum tests. Cox proportional hazards models were used to estimate the effect of calendar period of cART initiation on virologic responses to cART, including time to viral load suppression (2 measures <50 copies/mL at least 30 days apart) and rebound (2 measures >200 copies/mL at least 30 days apart, after suppression).
Of 8006 participants, 1453 (18%) were female, 46% lived in BC, 33% in Ontario, and 19% in Quebec. The median baseline age at treatment initiation was 38 (IQR=33–45) in 2000–2002, compared to 40 (IQR=32–47) in 2009–2011 (p<0.001). The proportion of participants with IDU history decreased from 26% in 2000–2002 to 19% in 2009–2011 (p<0.001). After adjustments for age, sex, province, transmission risk category, Aboriginal ancestry, baseline CD4 count, baseline viral load, baseline third ARV class, and viral load testing rate, participants initiating cART in 2003–2005, 2006–2008, and 2009–2011 were more likely to achieve viral suppression than those in 2000–2002 (aHR=1.16 [95% CI=1.07–1.25], aHR=1.22 [95% CI=1.10–1.34], aHR=1.14 [95% CI=1.01–1.26], respectively). After adjusting for the same confounders, participants initiating cART in the later eras were significantly less likely to experience viral rebound than in 2000–2002 (aHR=0.68 [95% CI=0.59–0.79], aHR= 0.54 [95% CI=0.43–0.67], aHR=0.31 [95% CI=0.23–0.42], respectively).
Notable temporal changes in the demographic profile and improvements in virologic response to cART are evident among CANOC participants. Characterizing the demographic and clinical profile of people living with HIV supports the optimal delivery of clinical care for the evolving Canadian HIV epidemic.
Globally, sex workers face disproportionate health and social inequities, with those in sub-Saharan Africa facing a particularly high burden of HIV. While global evidence has shown that structural determinants (e.g., laws and policies, work environment, armed conflict, migration patterns) shape sex workers’ ability to protect themselves from HIV and other harms, there remains a paucity of data regarding access to contraception among sex workers in sub-Saharan Africa. The object of this analysis was to explore the structural determinants of dual contraceptive use (i.e., use of non-barrier contraceptives and male condoms for pregnancy prevention) amongst female sex workers in Gulu, northern Uganda.
We draw on questionnaire data from a cross-sectional study conducted in Gulu, northern Uganda from 2011–2012. Participants were recruited through extensive peer/sex worker and community-led outreach. We used logistic regression to assess correlates of dual contraceptive use, defined as the combined use of non-barrier contraceptives and male condoms for pregnancy prevention.
Among 400 female sex workers, 180 (45%) used dual contraceptives. Multivariable modeling revealed that older age (Adjusted odds ratio (AOR): 1.10; 95% Confidence Interval (CI): 1.04–1.15), prior unintended pregnancy (AOR 1.53; 95% CI, 1.01–2.34), and prior HIV testing (AOR 5.22; 95% CI: 1.75–15.57) significantly increased the odds of dual contraceptive use, whereas having to rush sexual negotiations due to police presence (OR 0.65; CI, 0.42–0.99) was negatively associated with dual contraceptive use.
This analysis has shown the utility of HIV testing as a critical entry point for sex workers to access sexual and reproductive health (SRH) care. However, the criminalization of sex work and limited access to safer work environments continues to undermine existing SRH programming for sex workers. There is an urgent need for rights-based programming and integration of sexual and reproductive health into HIV prevention and care programs for sex workers.
The HIV care “cascade” is a framework that depicts the degree to which people infected with HIV are diagnosed in a timely fashion, become engaged in HIV care, and ultimately are successfully treated with antiretrovirals (ART). Many jurisdictions are seeking to monitor the cascade to target and prioritize HIV testing, linkage to care, and ART support interventions. We aimed to quantify the cascade using existing data and recommend future directions for ongoing cascade monitoring in Ontario.
We identified existing data sources to derive high and low estimates for 4 indicators among all Ontarians with HIV: the proportions diagnosed, linked to care, retained in care (≥2 viral loads per year), and with undetectable viral load. These included published population-based estimates from the Public Health Agency of Canada (values for 2011) and the Ontario HIV Epidemiological Monitoring Unit (values between 2007 and 2011); and unpublished estimates from the OHTN Cohort Study (values for 2012), a multi-site clinical cohort of persons in specialty HIV care.
High and low estimates for each indicator of the HIV care cascade are shown in the
| Low Estimate | 100 | 65 | 52 | 43 | 27 |
| High Estimate | 100 | 75 | 65 | 55 | 40 |
Using existing information, populating the cascade for Ontario was possible with similar estimates for percentage undetectable compared to other North American jurisdictions. Estimates were challenged by varying years for published estimates. Updated estimates of the proportion diagnosed are urgently needed for Ontario as this parameter has the most uncertainty and the greatest influence. Revised Ontario-based mathematical models and linked population-based data sources for empirical estimates would be ideal and should be the focus for future monitoring.
Supervised injection services (SIS) are an important means for preventing HIV-related risk behaviours and overdose mortality amongst people who inject drugs (PWID). However, in Canada scale-up of this public health intervention has been slow due to consistent political opposition at the federal level. The Respect for Communities Act (recently passed by the House of Commons and expected to become law in 2015), outlines 26 onerous requirements SIS applicants must satisfy prior to receiving a federal exemption. In particular, the Act requires extensive epidemiological data on illicit drug use in a prospective jurisdiction. In Edmonton (as well as many other mid-sized Canadian cities) these data are not available. The present study was designed to address this gap, inform local SIS discussion and planning, and develop the scientific basis for a potential federal exemption application.
Adopting a community-based research approach, we conducted the largest-to-date survey of people who use illicit drugs in Edmonton. 324 participants were convenience-sampled from two community agencies with embedded needle exchange sites. Interviewers administered a structured questionnaire measuring illicit substance use, HIV-related risk behaviours and willingness to attend SIS. Descriptive and inferential statistics were used to describe characteristics of the sample.
279 participants reported recent injection drug use. Of these PWID, approximately 80% reported recent public injection; 47% reported difficulty accessing sterile syringes; 29% reported improper syringe disposal; 18% reported syringe sharing; and 22% reported experiencing a nonfatal overdose in the past 6 months. 90% of PWID were willing to attend SIS, if made available in Edmonton.
Relative to other Canadian jurisdictions, PWID in Edmonton engage in high rates of HIV-related risk behaviours and are at considerable risk for overdose-related morbidity and mortality. Supervised injection services should be made available in Edmonton in an effort to improve health and safety for PWID.
We measured HIV incidence among participants in a prospective cohort of MSM in Vancouver, British Columbia and explored characteristics that may distinguish new HIV seroconverters from MSM who remained HIV-negative.
The Momentum Health Study employs respondent-driven sampling to recruit participants into a longitudinal bio-behavioural cohort study with 6-monthly visits. At baseline, all participants completed a computer-assisted questionnaire and nurse-administered sexual health check-up including a point-of-care HIV test. Seroconverters were participants who tested HIV-negative at baseline and HIV-positive at a subsequent study visit or another testing source between visits. Behavioural data are drawn from their most recently completed questionnaire prior to their HIV diagnosis. Comparisons between HIV seroconverters and men who remained HIV-negative were made using non-parametric statistical tests (p<0.05).
As of December 7, 2014, 378 MSM who tested HIV-negative at baseline contributed a mean follow-up time of 1.27 years. The HIV incidence rate was 1.25 per 100 person-years (6 MSM seroconverted). Although not significantly different when compared with MSM who remained HIV negative, all seroconverters identified as gay, 5/6 as Caucasian, and 5/6 were aged ≤30 years. The HIV incidence rate for MSM aged ≤30 was 2.40 per 100 person-years. Compared with MSM who remained HIV-negative, MSM who seroconverted reported a greater median number of sexual partners in the past six months (15.5 vs 4.0, p=0.01), reported a greater median number of anal sex events with sexual partners in the past six months (47.5 vs 7.0, p=0.02), and felt at high risk for HIV (50.0% vs 8.0%, p<0.01). There were no significant differences in the proportion of participants reporting any condomless anal intercourse, other socio-demographics, substance use patterns, mental health diagnoses, or reported prevention / risk reduction practices.
Recent HIV seroconverters in our study were more likely to be younger MSM with frequent partner change and greater rates of anal intercourse, but appear to understand that they are at higher risk for HIV acquisition. These findings can help target further HIV prevention programs towards such individuals.
Post-Exposure Prophylaxis (PEP) is a strategy to reduce HIV infection in those with high-risk exposure. This study characterized PEP awareness amongst Vancouver MSM.
Momentum Health Study participants were recruited via respondent driven sampling and completed a self-administered computer-based interview. Multivariable logistic regression identified factors associated with PEP awareness, asked between November 2012 and February 2014.
Of 673 participants included in this analysis, 384 (57%) had heard of PEP, a proportion that did not significantly differ over the study period. Of those who had heard of PEP, 32% reported knowing “not much, or nothing at all” about PEP, 59% “a bit in general”, and 9% “a lot”. 131 reported talking about PEP in last 6 months. Of the 9 participants who had used PEP, 7 identified the BC nPEP pilot program as the source. Factors associated with greater odds of PEP awareness include being White (52%) compared with another racial/ethnic group (17.6%–42.8%) being a student (AOR=2.09, 95% CI:1.31–3.35), ≥10 lifetime insertive anal sex partners (AOR=1.81, 95% CI:1.15–2.83), having had any condomless anal intercourse with an unknown or discordant serostatus partner in the past six months (AOR=1.6, 95% CI:1.00–2.43), having been diagnosed with genital warts (AOR=1.76, 95% CI:1.04–2.98), and reporting use of other preventive strategies such as abstaining from anal intercourse (AOR=2.06, 95% CI:1.36–3.11) or sero-sorting for condomless anal intercourse (AOR=1.92, 95% CI:1.27–2.89) PEP aware HIV-positive participants were also more likely to ask their partner’s serostatus (AOR=3.86, 95% CI:1.61–9.25). PEP awareness was negatively associated with non-gay sexual orientation (AOR=0.47, 95% CI:0.28–0.81), high school education or less (AOR=0.60, 95% CI:0.38–0.94), higher Sexual Altruism-Personal sub-scale scores (AOR=0.56, 95% CI:0.39–0.79), and lesser agreement with the statement “I always have condoms when having sex” (agree versus strongly agree: AOR=0.47, 95% CI:0.29–0.76).
PEP awareness is associated with certain behaviours with greater potential for HIV transmission; further research is needed to investigate how best to incorporate this strategy within combination HIV prevention.
To compare testing, awareness of infection, and treatment (three key indicators along the cascade of HIV and HCV care) across regions and over time among injection drug users (IDUs) in the SurvUDI network.
Since 1995, IDUs (injection in the past 6 months) are recruited in harm reduction and health programs across Québec and in Ottawa. Participants provide consent, complete a questionnaire and give saliva samples for anti-HIV/anti-HCV antibody testing. Four regions were compared: Montréal, Québec City (QC), Ottawa/Outaouais (OO) and semi-urban sites (SUS) and one questionnaire per participant (and per region or period as required) was retained. Wald tests from GEE models were used for comparisons across regions and the bootstrap method for trend analysis.
From December 2003 to January 2013, 6,421 IDUs completed 12,146 interviews. Overall, 8.7% of participants were never tested for HIV. This proportion decreased from 2003 to 2013 (10.4% to 4.3%; p<0.001) and varied across regions (Montréal: 7.1%, QC: 8.0%, OO: 10.4%, SSU: 10.2%; p<0.001). Among HIV-infected participants, 19.0% were unaware of their infection. This proportion was similar in all regions (p=0.12) but decreased over time (22.7% to 8.8%; p<0.001). Among those aware of being HIV-infected, 63.3% were currently taking HIV medication. This proportion, similar in all regions (p=0.47), increased over time (60.9% to 81.6%; p<0.001). Regarding HCV, 10.2% were never tested, 20.9% were unaware of having anti-HCV antibodies and, among those knowing, 15.3% had ever taken medication. These proportions respectively decreased, decreased and increased over time.
The three studied indicators improved over the past decade for HIV and HCV. However, many IDUs remain untested and unaware of their infection. HIV treatment seems now more accessible to IDUs, but few have access to HCV treatment. The existing services must be strengthened and new approaches developed to better link infected IDUs to care.
Relatively few harm reduction programs focus on gender specific programming. This analysis assesses the extent of gender differences within Ontario PWID in order to consider the need for gender-specific HIV prevention.
Phase 3 of the I-Track behavioural surveillance surveys sponsored by the Public Health Agency of Canada (PHAC) recruited from needle exchange programs in five Ontario cities between 2010–12 (Kingston, London, Sudbury, Thunder Bay, Toronto). Interviewers administered questionnaires and collected dried blood spot samples using coded linkage to maintain anonymity.
A total of 1323 PWID participated – 68.3% male, 37.7% female. Males were more likely to report being under-housed in the previous 6 months (33% vs. 20%, p< 0.001). Females were more likely to have borrowed needles (20% vs. 12.2%, p=0.001), and to have lent needles (25.9% vs. 12.7%, p<0.001) in the previous 6 months. 47% of females reported borrowing other injecting equipment vs. 34% of males (p<0.001); 46% of females reported lending other injecting equipment vs. 34% of male respondents (p<0.001). Females were more likely to report borrowing equipment from regular sex partners (p<0.001), while men were more likely to report borrowing equipment from friends (p=0.002) and casual sex partners (p=0.05). Women reported greater frequency of borrowing equipment than men: of the 204 men who reported borrowing equipment, 10% borrowed frequently, and 8% always, while of the 145 women who reported borrowing equipment, 15% borrowed frequently, and 16% always (p=0.03). Men were more likely to have been in jail in their lifetime (89.1% vs. 70.6%, p<0.001), more likely to report injecting while incarcerated (19.6% of those who had been in jail, vs. 10.5% for women, p=0.002), with no significant gender difference in sharing needles/syringes among those who had injected while incarcerated. Being female was independently associated with being HIV+ and with being HCV+ in multivariate analyses.
There is ample epidemiological evidence of gender specific risks among PWID in Ontario suggesting a need for specific gender focused prevention programming.
To compare the characteristics of individuals diagnosed with HIV less than 12 before their AIDS diagnosis to those diagnosed 12 months or more before their AIDS diagnosis.
AIDS case surveillance data submitted to the PHAC from 1979 to 2013 were included in the analysis. Provinces that do not submit HIV diagnosis date with their AIDS cases were removed from the analysis. Of the remaining, 58.6% of AIDS cases had a valid HIV diagnosis date. Cases were classified as Late HIV Diagnosis (LD) (<12 months between HIV and AIDS diagnosis) or Not-late HIV Diagnosis (NLD) (>12 months). Univariate analyses were done to explore factors associated with LD versus NLD. Factors significantly associated with the outcomes (p<0.05) were examined in a multivariate logistic regression model.
Of the 5,304 cases included in the analysis, 38.3% (n=2,033) were classified as LD. In the univariate analysis, sex, age, ethnicity and exposure category were significantly associated with LD. Males had lower odds of LD compared to females (OR: 0.78, 0.67–0.915). Compared to the 20–29 age group, each successive 10-year age group showed an increase in the odds of LD: 30–39 (OR: 2.0, 1.71–2.34), 40–49 (OR: 3.1, 2.60–3.69), 50–59 (OR: 4.7, 3.72–5.96) and 60+ (OR: 6.8, 4.62–9.97). Compared to White ethnicity, all ethnic groups showed significantly higher odds of LD: Black ethnicity (OR: 2.5, 2.11–2.94), Latin American (OR: 3.0, 1.64–5.38), Asian/South Asian/West Asian/Arab (OR: 2.0, 1.15–3.41) and Aboriginal (1.3,1.00–1.59). Heterosexual contact exposure was associated with LD (OR=2.24, 1.94–2.57 compared to MSM exposure, p <0.0001).
Late HIV diagnosis limits the benefits of available treatment strategies and reduced risk behaviors. These analyses suggest that women, older persons, ethnic groups other than White, and risk groups such as heterosexuals would benefit from increased awareness of the risk of HIV transmission and improved testing programs.
Harm reduction (HR) uses strategies that aim to reduce the secondary consequences associated with risky activities (e.g. substance use), without strictly focusing on cessation of the activity itself. Many HR programs have strong empirical support. As such, there is increasing recognition of the need for the inclusion of HR strategies and services across the continuum of HIV care. However, research and evaluation of HR programs typically focuses on clients’ physical health outcomes and utilization of the HR services provided. The primary aim of this study is to expand our understanding of the impact and process of providing care with an HR approach by exploring health care providers’ views on the benefits and challenges of implementing HR strategies.
This study surveyed staff at two HIV/AIDS dedicated facilities in Canada (Casey House, Toronto and The Dr. Peter Centre, Vancouver) that implement an HR policy. A 20-question survey was informed by a literature review of HR research and developed by clinicians, researchers and administrators in both cities. The survey included open-ended questions and quantitative rating scales. The survey was administered online (through Fluid SurveyTM) and in paper format. Sixty-four health care providers (HCP) completed the survey in February and March, 2014. Results indicate that in addition to client physical and mental health as key endpoints when evaluating HR programs, engagement in the health care system and building positive client relationships are positive outcomes strongly valued by HCP. However, HCP highlighted personal and inter-team related challenges experienced while working within an HR framework. Respondents identified the importance of clear procedures and continued HCP support in the implementation of HR programming. The HCP perspective captured in this study has implications for program and staff development.
Social media has gained widespread popularity as an HIV prevention tool, in part due to its unique properties: It can support interaction between numerous individuals, and permit users the opportunity to shape their online environment. As such, social media may prove useful in the conduct of integrated HIV prevention interventions in Canada. However there is a lack of information regarding appropriate means of utilizing social media for effective HIV prevention.
Based on the findings of an NCCID-funded scoping review, this paper focuses on examining peer-reviewed research on interventions designed to prevent transmission of HIV and/or STBBIS, and to promote sexual health more broadly. Articles were gathered through iterative searches of PubMed and EBSCOhost, covering 21 databases. Analysis focused on identifying: 1) how social media were used as an intervention platform, 2) how they were used to effect change in knowledge, behavior, attitudes, or social environments, and 3) the evaluative methods and measures employed to measure intervention impact.
Of the 236 articles retrieved, twelve were included for analysis. Most describe the use of Facebook to disseminate HIV prevention information, and/or engage participants in interactive discussions regarding sexual health. Notable patterns were identified regarding the recruitment of participants or promotion of the intervention, and the use of randomized control trial designs. While self-report measures of behavior and attitudinal change were most common, several studies utilized epidemiologic data, Facebook analytics statistics, and qualitative analysis of comments to assess impact.
The results of this scoping review illustrate considerations for developing social media-based interventions, including how target audiences are engaged. Opportunities for integrated HIV prevention interventions are also discussed. Social media based sexual health approaches may be particularly well-suited to community-level interventions that target existing social networks. Further research is needed to examine existing grassroots social media campaigns to identify measurable and effective strategies.
Jack & Jacques (J&J) is a mobile website for the prevention of sexually transmitted infections (STI) developed by AIDS Community Care Montreal in partnership with the Montreal Public Health Department. Users may complete a quiz about sexual behaviours and be provided recommendations regarding HIV and STI testing. Also, information on local resources for prevention information and confidential testing is provided. This analysis describes how J&J is used through Google Analytics (GA) with a view to improve its uptake.
Information about J&J users’ behaviour was tracked using GA. Filters were applied to exclude non-informative sessions (spam and bounced) and to select sessions made from February 1st 2014 to December 8th 2014.
Over a 10-month period, 8560 sessions were recorded. Of these, 5.2% were spam, and 71.0% were bounced; 2036 sessions were included in this analysis. Traffic to the site was highly dependent on promotional events. Most sessions were from users in the Montreal area (73.2%) and made using a mobile phone (52.7%). The average session duration was 2.46 minutes. One-time visitors accounted for 1534 sessions and returning visitors for 502. Most sessions were direct traffic (i.e., entering URL, bookmark) (53.9%). Of all sessions, 45.8% trafficked through the quiz or resource page. Of the 690 sessions where the quiz was visited, 76.7% were of duration greater than 30 seconds, a time considered sufficient for completion. By December 8th, 69 people had subscribed for screening reminders via email.
GA facilitates the examination of a tremendous amount of indicators but is not without limitations. Because it does not distinguish a one-page session from a bounced session, some sessions where only the quiz or the resource page was visited were excluded. The use of customized filters is a necessary step in data cleaning.
Despite high HIV incidence among Latino MSM, Latino gay, bisexual, and other MSM men living with HIV in Canada have few confidential, safe spaces to talk openly about sex, sexual health and HIV disclosure, as they face cultural and language barriers and social isolation. Thus, our objective was to adapt Gay Poz Sex (GPS), an HIV/STI prevention intervention initially developed for English-speaking HIV-positive persons to make it linguistically and culturally appropriate for Spanish-speaking Latino gay men living with HIV in Canada.
From November 2010 to December 2013, a multiphase process was undertaken to identify an appropriate intervention, adapt the intervention, develop training materials, and test the adapted version. GPS was identified as a pertinent intervention for Latino positive men. Two community members of the Latino gay community were trained as facilitators for GPS. During the training, facilitators made minor revisions to linguistically and culturally adapt each session’s content. “GPS Latino” was tested in a group and the adaptation was further informed by pilot qualitative and quantitative data gathering from five Latino gay men living with HIV.
Culturally specific adaptation was needed, which included: language translation, introduction of role-play scenarios instead of videos, and more information about HIV and the law. Qualitative assessments during implementation of GPS demonstrated that all 8 sessions of GPS met the objectives in terms of increasing confidence in disclosure and improving self-perceptions of sexual health. Community members who received GPS suggested few changes, which included increasing the number of sessions, number of role-plays of sexually risky situations, and duration of each session.
GPS needed very few changes as to be adapted for Latino gay men living with HIV in Canada. This is consistent with previous linguistic adaptations of GPS. Testing of GPS in a larger group will help us to identify positive outcomes in terms of sexual well-being.
The Dr. Peter Centre (DPC) is an integrative health service for vulnerable people living with HIV with complex health and psychosocial care needs. This DPC study aims to describe service use and characteristics of DPC clients.
We administered a quantitative baseline questionnaire covering eight areas to individuals who have been clients of the DPC since 27 February, 2011. Baseline demographic and clinical participant responses were used for this report. Participant enrolment into the baseline survey is ongoing.
During the period between February and November 2014, 96 participants completed the DPC Study quantitative baseline survey. Out of 96 respondents, 85% are male, 59% are Caucasian, 46% are heterosexual, 52% have reported ever being incarcerated, and 76% have reported ever being homeless. The median age of clients is 46.5 years. Among DPC clients, 73% reported ever being diagnosed with substance use disorder, 73% reported diagnosis of depression, and 57% reported diagnosis of anxiety. Currently, the percentage of clients on HAART is 91%, while 71% report receiving treatment for various mental health conditions. Out of 34% of clients with current injecting drug use, 27% report current use of the DPC supervised injection site. Common drugs injected by participants include cocaine (61%), heroin (80%), and crystal meth (75%). Several DPC services are heavily utilized among DPC clients. Meals (breakfast and lunch) are ranked as the most important service. Consultation with the nurse practitioner and medication support are also ranked among the top three most important services offered at the DPC.
DPC clients experience a high level of vulnerability due to a multitude of overlapping complex heath issues and barriers to health services. Further research will delve into motivations behind service use based on demographic characteristics, social, structural, and behavioral factors.
To estimate HIV and HCV prevalence among injection drug users (IDUs) in the SurvUDI network and to examine trends in HIV and HCV incidence and use of syringes previously used by someone else (“used syringes”).
Since 1995, IDUs having injected recently (past 6 months) are recruited in harm reduction and health programs across Québec and in Ottawa. Participants provide informed consent, complete an interviewer-administered questionnaire and give saliva samples for antibody testing (anti-HIV: since 1995; anti-HCV: retrospectively for 1997–2003 and prospectively since). Through a unique identifier, multiple visits by a same IDU (repeater) are linked and incidence measured. The bootstrap method was used for trend analyses.
As of March 31, 2014, 13694 IDUs had completed 25899 interviews. Overall, 75.7% were males with a median age of 36 years (females: 29 years). At baseline (2009–2014), 72.2% had recently injected cocaine and 62.7% had recently injected prescription opioids. HIV prevalence (2003–2014) and incidence (1995–2014) were 14.2% [95% Confidence Interval (95% CI): 13.4–15.1%] and 2.2 per 100 person-years (PY) [95% CI: 1.9–2.4 per 100 PY; 319 seroconversions among 3443 repeaters initially HIV-negative]. HCV prevalence (2003–2014) and incidence (1997–2014) were 62.9% [95% CI: 61.7–64.1%] and 22.2 per 100 PY [95% CI: 20.3–24.1 per 100 PY; 531 seroconversions among 1141 repeaters initially HCV-negative]. HIV/HCV co-infection rate was 12.1% (2003–2014). Recent use of “used syringes” decreased significantly from 1995 to 2013 (43.4% to 16.8%; p<0.001). HIV incidence decreased significantly between 1995 and 2012 (5.1 to 0.9 per 100 PY p<0.001). HCV incidence decreased significantly between 1998 and 2012 (22.1 to 18.5 per 100 PY; p=0.0033).
Despite encouraging decreases in HIV incidence and use of “used syringes” and a slight decrease in HCV incidence, all remain unacceptably high. Epidemiological surveillance of that population continues to be essential to further develop and strengthen appropriate harm reduction programs.
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