Common variants of the TCF7L2 gene are associated with increased risk of type 2 diabetes mellitus in a UK-resident South Asian population

Background Recent studies have implicated variants of the transcription factor 7-like 2 (TCF7L2) gene in genetic susceptibility to type 2 diabetes mellitus in several different populations. The aim of this study was to determine whether variants of this gene are also risk factors for type 2 diabetes development in a UK-resident South Asian cohort of Punjabi ancestry. Methods We genotyped four single nucleotide polymorphisms (SNPs) of TCF7L2 (rs7901695, rs7903146, rs11196205 and rs12255372) in 831 subjects with diabetes and 437 control subjects. Results The minor allele of each variant was significantly associated with type 2 diabetes; the greatest risk of developing the disease was conferred by rs7903146, with an allelic odds ratio (OR) of 1.31 (95% CI: 1.11 – 1.56, p = 1.96 × 10-3). For each variant, disease risk associated with homozygosity for the minor allele was greater than that for heterozygotes, with the exception of rs12255372. To determine the effect on the observed associations of including young control subjects in our data set, we reanalysed the data using subsets of the control group defined by different minimum age thresholds. Increasing the minimum age of our control subjects resulted in a corresponding increase in OR for all variants of the gene (p ≤ 1.04 × 10-7). Conclusion Our results support recent findings that TCF7L2 is an important genetic risk factor for the development of type 2 diabetes in multiple ethnic groups

Harmonization of the intracellular cytokine staining assay

Active immunotherapy for cancer is an accepted treatment modality aiming to reinforce the T-cell response to cancer. T-cell reactivity is measured by various assays and used to guide the clinical development of immunotherapeutics. However, data obtained across different institutions may vary substantially making comparative conclusions difficult. The Cancer Immunotherapy Immunoguiding Program organizes proficiency panels to identify key parameters influencing the outcome of commonly used T-cell assays followed by harmonization. Our successes with IFNγ-ELISPOT and peptide HLA multimer analysis have led to the current study on intracellular cytokine staining (ICS). We report the results of three successive panels evaluating this assay. At the beginning, 3 out of 9 participants (33 %) were able to detect >6 out of 8 known virus-specific T-cell responses in peripheral blood of healthy individuals. This increased to 50 % of the laboratories in the second phase. The reported percentages of cytokine-producing T cells by the different laboratories were highly variable with coefficients of variation well over 60 %. Variability could partially be explained by protocol-related differences in background cytokine production leading to sub-optimal signal-to-noise ratios. The large number of protocol variables prohibited identification of prime guidelines to harmonize the assays. In addition, the gating strategy used to identify reactive T cells had a major impact on assay outcome. Subsequent harmonization of the gating strategy considerably reduced the variability within the group of participants. In conclusion, we propose that first basic guidelines should be applied for gating in ICS experiments before harmonizing assay protocol variables

Modeling flow cytometry data for cancer vaccine immune monitoring

Flow cytometry (FCM) is widely used in cancer research for diagnosis, detection of minimal residual disease, as well as immune monitoring and profiling following immunotherapy. In all these applications, the challenge is to detect extremely rare cell subsets while avoiding spurious positive events. To achieve this objective, it helps to be able to analyze FCM data using multiple markers simultaneously, since the additional information provided often helps to minimize the number of false positive and false negative events, hence increasing both sensitivity and specificity. However, with manual gating, at most two markers can be examined in a single dot plot, and a sequential strategy is often used. As the sequential strategy discards events that fall outside preceding gates at each stage, the effectiveness of the strategy is difficult to evaluate without laborious and painstaking back-gating. Model-based analysis is a promising computational technique that works using information from all marker dimensions simultaneously, and offers an alternative approach to flow analysis that can usefully complement manual gating in the design of optimal gating strategies. Results from model-based analysis will be illustrated with examples from FCM assays commonly used in cancer immunotherapy laboratories

Minimal information about T cell assays: the process of reaching the community of T cell immunologists in cancer and beyond

Many assays to evaluate the nature, breadth, and quality of antigen-specific T cell responses are currently applied in human medicine. In most cases, assay-related protocols are developed on an individual laboratory basis, resulting in a large number of different protocols being applied worldwide. Together with the inherent complexity of cellular assays, this leads to unnecessary limitations in the ability to compare results generated across institutions. Over the past few years a number of critical assay parameters have been identified which influence test performance irrespective of protocol, material, and reagents used. Describing these critical factors as an integral part of any published report will both facilitate the comparison of data generated across institutions and lead to improvements in the assays themselves. To this end, the Minimal Information About T Cell Assays (MIATA) project was initiated. The objective of MIATA is to achieve a broad consensus on which T cell assay parameters should be reported in scientific publications and to propose a mechanism for reporting these in a systematic manner. To add maximum value for the scientific community, a step-wise, open, and field-spanning approach has been taken to achieve technical precision, user-friendliness, adequate incorporation of concerns, and high acceptance among peers. Here, we describe the past, present, and future perspectives of the MIATA project. We suggest that the approach taken can be generically applied to projects in which a broad consensus has to be reached among scientists working in fragmented fields, such as immunology. An additional objective of this undertaking is to engage the broader scientific community to comment on MIATA and to become an active participant in the project

Formation of regulatory modules by local sequence duplication

Turnover of regulatory sequence and function is an important part of molecular evolution. But what are the modes of sequence evolution leading to rapid formation and loss of regulatory sites? Here, we show that a large fraction of neighboring transcription factor binding sites in the fly genome have formed from a common sequence origin by local duplications. This mode of evolution is found to produce regulatory information: duplications can seed new sites in the neighborhood of existing sites. Duplicate seeds evolve subsequently by point mutations, often towards binding a different factor than their ancestral neighbor sites. These results are based on a statistical analysis of 346 cis-regulatory modules in the Drosophila melanogaster genome, and a comparison set of intergenic regulatory sequence in Saccharomyces cerevisiae. In fly regulatory modules, pairs of binding sites show significantly enhanced sequence similarity up to distances of about 50 bp. We analyze these data in terms of an evolutionary model with two distinct modes of site formation: (i) evolution from independent sequence origin and (ii) divergent evolution following duplication of a common ancestor sequence. Our results suggest that pervasive formation of binding sites by local sequence duplications distinguishes the complex regulatory architecture of higher eukaryotes from the simpler architecture of unicellular organisms

Long term benzodiazepine use for insomnia in patients over the age of 60: discordance of patient and physician perceptions

BACKGROUND: The aim of this study was to determine and compare patients' and physicians' perceptions of benefits and risks of long term benzodiazepine use for insomnia in the elderly. METHODS: A cross-sectional study (written survey) was conducted in an academic primary care group practice in Toronto, Canada. The participants were 93 patients over 60 years of age using a benzodiazepine for insomnia and 25 physicians comprising sleep specialists, family physicians, and family medicine residents. The main outcome measure was perception of benefit and risk scores calculated from the mean of responses (on a Likert scale of 1 to 5) to various items on the survey. RESULTS: The mean perception of benefit score was significantly higher in patients than physicians (3.85 vs. 2.84, p < 0.001, 95% CI 0.69, 1.32). The mean perception of risk score was significantly lower in patients than physicians (2.21 vs. 3.63, p < 0.001, 95% CI 1.07, 1.77). CONCLUSIONS: There is a significant discordance between older patients and their physicians regarding the perceptions of benefits and risks of using benzodiazepines for insomnia on a long term basis. The challenge is to openly discuss these perceptions in the context of the available evidence to make collaborative and informed decisions

Harmonization guidelines for HLA-peptide multimer assays derived from results of a large scale international proficiency panel of the Cancer Vaccine Consortium

PURPOSE: The Cancer Vaccine Consortium of the Cancer Research Institute (CVC-CRI) conducted a multicenter HLA-peptide multimer proficiency panel (MPP) with a group of 27 laboratories to assess the performance of the assay. EXPERIMENTAL DESIGN: Participants used commercially available HLA-peptide multimers and a well characterized common source of peripheral blood mononuclear cells (PBMC). The frequency of CD8+ T cells specific for two HLA-A2-restricted model antigens was measured by flow cytometry. The panel design allowed for participants to use their preferred staining reagents and locally established protocols for both cell labeling, data acquisition and analysis. RESULTS: We observed significant differences in both the performance characteristics of the assay and the reported frequencies of specific T cells across laboratories. These results emphasize the need to identify the critical variables important for the observed variability to allow for harmonization of the technique across institutions. CONCLUSIONS: Three key recommendations emerged that would likely reduce assay variability and thus move toward harmonizing of this assay. (1) Use of more than two colors for the staining (2) collect at least 100,000 CD8 T cells, and (3) use of a background control sample to appropriately set the analytical gates. We also provide more insight into the limitations of the assay and identified additional protocol steps that potentially impact the quality of data generated and therefore should serve as primary targets for systematic analysis in future panels. Finally, we propose initial guidelines for harmonizing assay performance which include the introduction of standard operating protocols to allow for adequate training of technical staff and auditing of test analysis procedures

Lateral orbitofrontal cortex anticipates choices and integrates prior with current information

Adaptive behavior requires integrating prior with current information to anticipate upcoming events. Brain structures related to this computation should bring relevant signals from the recent past into the present. Here we report that rats can integrate the most recent prior information with sensory information, thereby improving behavior on a perceptual decision-making task with outcome-dependent past trial history. We find that anticipatory signals in the orbitofrontal cortex about upcoming choice increase over time and are even present before stimulus onset. These neuronal signals also represent the stimulus and relevant second-order combinations of past state variables. The encoding of choice, stimulus and second-order past state variables resides, up to movement onset, in overlapping populations. The neuronal representation of choice before stimulus onset and its build-up once the stimulus is presented suggest that orbitofrontal cortex plays a role in transforming immediate prior and stimulus information into choices using a compact state-space representation

Reasons of general practitioners for not prescribing lipid-lowering medication to patients with diabetes: a qualitative study

Background: Lipid-lowering medication remains underused, even in high-risk populations. The objective of this study was to determine factors underlying general practitioners' decisions not to prescribe such drugs to patients with type 2 diabetes. Methods: A qualitative study with semi-structured interviews using real cases was conducted to explore reasons for not prescribing lipid-lowering medication after a guideline was distributed that recommended the use of statins in most patients with type 2 diabetes. Seven interviews were conducted with general practitioners (GPs) in The Netherlands, and analysed using an analytic inductive approach. Results: Reasons for not-prescribing could be divided into patient and physician-attributed factors. According to the GPs, some patients do not follow-up on agreed medication and others object to taking lipid-lowering medication, partly for legitimate reasons such as expected or perceived side effects. Furthermore, the GPs themselves perceived reservations for prescribing lipid-lowering medication in patients with short life expectancy, expected compliance problems or near goal lipid levels. GPs sometimes postponed the start of treatment because of other priorities. Finally, barriers were seen in the GPs' practice organisation, and at the primary-secondary care interface. Conclusion: Some of the barriers mentioned by GPs seem to be valid reasons, showing that guideline non-adherence can be quite rational. On the other hand, treatment quality could improve by addressing issues, such as lack of knowledge or motivation of both the patient and the GP. More structured management in general practice may also lead to better treatment

Evaluation and Recommendations on Good Clinical Laboratory Practice Guidelines for Phase I–III Clinical Trials

Marcella Sarzotti-Kelsoe and colleagues harmonize various approaches to Good Clinical Laboratory Practice for clinical trials into a single set of recommendations