Biological and technical variables affecting immunoassay recovery of cytokines from human serum and simulated vaginal fluid: A multicenter study

The increase of proinflammatory cytokines in vaginal secretions may serve as a surrogate marker of unwanted inflammatory reaction to microbicide products topically applied for the prevention of sexually transmitted diseases, including HIV-1. Interleukin (IL)-1β and IL-6 have been proposed as indicators of inflammation and increased risk of HIV-1 transmission; however, the lack of information regarding detection platforms optimal for vaginal fluids and interlaboratory variation limit their use for microbicide evaluation and other clinical applications. This study examines fluid matrix variants relevant to vaginal sampling techniques and proposes a model for interlaboratory comparisons across current cytokine detection technologies. IL-1β and IL-6 standards were measured by 12 laboratories in four countries, using 14 immunoassays and four detection platforms based on absorbance, chemiluminescence, electrochemiluminescence, and fluorescence. International reference preparations of cytokines with defined biological activity were spiked into (1) a defined medium simulating the composition of human vaginal fluid at pH 4.5 and 7.2, (2) physiologic salt solutions (phosphate-buffered saline and saline) commonly used for vaginal lavage sampling in clinical studies of cytokines, and (3) human blood serum. Assays were assessed for reproducibility, linearity, accuracy, and significantly detectable fold difference in cytokine level. Factors with significant impact on cytokine recovery were determined by Kruskal−Wallis analysis of variance with Dunn’s multiple comparison test and multiple regression models. All assays showed acceptable intra-assay reproducibility; however, most were associated with significant interlaboratory variation. The smallest reliably detectable cytokine differences (P < 0.05) derived from pooled interlaboratory data varied from 1.5- to 26-fold depending on assay, cytokine, and matrix type. IL-6 but not IL-1β determinations were lower in both saline and phosphate-buffered saline as compared to vaginal fluid matrix, with no significant effect of pH. The (electro)chemiluminescence-based assays were most discriminative and consistently detected <2-fold differences within each matrix type. The Luminex-based assays were less discriminative with lower reproducibility between laboratories. These results suggest the need for uniform vaginal sampling techniques and a better understanding of immunoassay platform differences and cross-validation before the biological significance of cytokine variations can be validated in clinical trials. This investigation provides the first standardized analytic approach for assessing differences in mucosal cytokine levels and may improve strategies for monitoring immune responses at the vaginal mucosal interface

The influence of glucose-lowering therapies on cancer risk in type 2 diabetes

AIMS/HYPOTHESIS: The risk of developing a range of solid tumours is increased in type 2 diabetes, and may be influenced by glucose-lowering therapies. We examined the risk of development of solid tumours in relation to treatment with oral agents, human insulin and insulin analogues. METHODS: This was a retrospective cohort study of people treated in UK general practices. Those included in the analysis developed diabetes >40 years of age, and started treatment with oral agents or insulin after 2000. A total of 62,809 patients were divided into four groups according to whether they received monotherapy with metformin or sulfonylurea, combined therapy (metformin plus sulfonylurea), or insulin. Insulin users were grouped according to treatment with insulin glargine, long-acting human insulin, biphasic analogue and human biphasic insulin. The outcome measures were progression to any solid tumour, or cancer of the breast, colon, pancreas or prostate. Confounding factors were accounted for using Cox proportional hazards models. RESULTS: Metformin monotherapy carried the lowest risk of cancer. In comparison, the adjusted HR was 1.08 (95% CI 0.96-1.21) for metformin plus sulfonylurea, 1.36 (95% CI 1.19-1.54) for sulfonylurea monotherapy, and 1.42 (95% CI 1.27-1.60) for insulin-based regimens. Adding metformin to insulin reduced progression to cancer (HR 0.54, 95% CI 0.43-0.66). The risk for those on basal human insulin alone vs insulin glargine alone was 1.24 (95% CI 0.90-1.70). Compared with metformin, insulin therapy increased the risk of colorectal (HR 1.69, 95% CI 1.23-2.33) or pancreatic cancer (HR 4.63, 95% CI 2.64-8.10), but did not influence the risk of breast or prostate cancer. Sulfonylureas were associated with a similar pattern of risk as insulin. CONCLUSIONS/INTERPRETATION: Those on insulin or insulin secretagogues were more likely to develop solid cancers than those on metformin, and combination with metformin abolished most of this excess risk. Metformin use was associated with lower risk of cancer of the colon or pancreas, but did not affect the risk of breast or prostate cancer. Use of insulin analogues was not associated with increased cancer risk as compared with human insulin

Ca2+ Extrusion by NCX Is Compromised in Olfactory Sensory Neurons of OMP−/− Mice

The role of olfactory marker protein (OMP), a hallmark of mature olfactory sensory neurons (OSNs), has been poorly understood since its discovery. The electrophysiological and behavioral phenotypes of OMP knockout mice indicated that OMP influences olfactory signal transduction. However, the mechanism by which this occurs remained unknown.We used intact olfactory epithelium obtained from WT and OMP(-/-) mice to monitor the Ca(2+) dynamics induced by the activation of cyclic nucleotide-gated channels, voltage-operated Ca(2+) channels, or Ca(2+) stores in single dendritic knobs of OSNs. Our data suggested that OMP could act to modulate the Ca(2+)-homeostasis in these neurons by influencing the activity of the plasma membrane Na(+)/Ca(2+)-exchanger (NCX). Immunohistochemistry verifies colocalization of NCX1 and OMP in the cilia and knobs of OSNs. To test the role of NCX activity, we compared the kinetics of Ca(2+) elevation by stimulating the reverse mode of NCX in both WT and OMP(-/-) mice. The resulting Ca(2+) responses indicate that OMP facilitates NCX activity and allows rapid Ca(2+) extrusion from OSN knobs. To address the mechanism by which OMP influences NCX activity in OSNs we studied protein-peptide interactions in real-time using surface plasmon resonance technology. We demonstrate the direct interaction of the XIP regulatory-peptide of NCX with calmodulin (CaM).Since CaM also binds to the Bex protein, an interacting protein partner of OMP, these observations strongly suggest that OMP can influence CaM efficacy and thus alters NCX activity by a series of protein-protein interactions

Optical Coherence Tomography in the UK Biobank Study – Rapid Automated Analysis of Retinal Thickness for Large Population-Based Studies

Purpose: To describe an approach to the use of optical coherence tomography (OCT) imaging in large, population-based studies, including methods for OCT image acquisition, storage, and the remote, rapid, automated analysis of retinal thickness. Methods: In UK Biobank, OCT images were acquired between 2009 and 2010 using a commercially available “spectral domain” OCT device (3D OCT-1000, Topcon). Images were obtained using a raster scan protocol, 6 mm x 6 mm in area, and consisting of 128 B-scans. OCT image sets were stored on UK Biobank servers in a central repository, adjacent to high performance computers. Rapid, automated analysis of retinal thickness was performed using custom image segmentation software developed by the Topcon Advanced Biomedical Imaging Laboratory (TABIL). This software employs dual-scale gradient information to allow for automated segmentation of nine intraretinal boundaries in a rapid fashion. Results: 67,321 participants (134,642 eyes) in UK Biobank underwent OCT imaging of both eyes as part of the ocular module. 134,611 images were successfully processed with 31 images failing segmentation analysis due to corrupted OCT files or withdrawal of subject consent for UKBB study participation. Average time taken to call up an image from the database and complete segmentation analysis was approximately 120 seconds per data set per login, and analysis of the entire dataset was completed in approximately 28 days. Conclusions: We report an approach to the rapid, automated measurement of retinal thickness from nearly 140,000 OCT image sets from the UK Biobank. In the near future, these measurements will be publically available for utilization by researchers around the world, and thus for correlation with the wealth of other data collected in UK Biobank. The automated analysis approaches we describe may be of utility for future large population-based epidemiological studies, clinical trials, and screening programs that employ OCT imaging

The RNA Chaperone Hfq Is Important for Growth and Stress Tolerance in Francisella novicida

The RNA-binding protein Hfq is recognized as an important regulatory factor in a variety of cellular processes, including stress resistance and pathogenesis. Hfq has been shown in several bacteria to interact with small regulatory RNAs and act as a post-transcriptional regulator of mRNA stability and translation. Here we examined the impact of Hfq on growth, stress tolerance, and gene expression in the intracellular pathogen Francisella novicida. We present evidence of Hfq involvement in the ability of F. novicida to tolerate several cellular stresses, including heat-shock and oxidative stresses, and alterations in hfq gene expression under these conditions. Furthermore, expression of numerous genes, including several associated with virulence, is altered in a hfq mutant strain suggesting they are regulated directly or indirectly by Hfq. Strikingly, we observed a delayed entry into stationary phase and increased biofilm formation in the hfq mutant. Together, these data demonstrate a critical role for Hfq in F. novicida growth and survival

Dengue Incidence in Urban and Rural Cambodia: Results from Population-Based Active Fever Surveillance, 2006–2008

Dengue is a major public health problem in South-East Asia. Several dengue vaccine candidates are now in late-stage development and are being evaluated in clinical trials. Accurate estimates of true dengue disease burden will become an important factor in the public-health decision-making process for endemic countries once safe and effective vaccines become available. However, estimates of the true disease incidence are difficult to make, because national surveillance systems suffer from disease under-recognition and reporting. Dengue is mainly reported among children, and in some countries, such as Cambodia, the national case definition only includes hospitalized children. This study used active, community-based surveillance of febrile illness coupled with laboratory testing for DENV infection to identify cases of dengue fever in rural and urban populations. We found a high burden of dengue in young children and late adolescents in both rural and urban communities at a magnitude greater than previously described. The study also confirmed the previously observed focal nature of dengue virus transmission

Investigating the Role of T-Cell Avidity and Killing Efficacy in Relation to Type 1 Diabetes Prediction

During the progression of the clinical onset of Type 1 Diabetes (T1D), high-risk individuals exhibit multiple islet autoantibodies and high-avidity T cells which progressively destroy beta cells causing overt T1D. In particular, novel autoantibodies, such as those against IA-2 epitopes (aa1-577), had a predictive rate of 100% in a 10-year follow up (rapid progressors), unlike conventional autoantibodies that required 15 years of follow up for a 74% predictive rate (slow progressors). The discrepancy between these two groups is thought to be associated with T-cell avidity, including CD8 and/or CD4 T cells. For this purpose, we build a series of mathematical models incorporating first one clone then multiple clones of islet-specific and pathogenic CD8 and/or CD4 T cells, together with B lymphocytes, to investigate the interaction of T-cell avidity with autoantibodies in predicting disease onset. These models are instrumental in examining several experimental observations associated with T-cell avidity, including the phenomenon of avidity maturation (increased average T-cell avidity over time), based on intra- and cross-clonal competition between T cells in high-risk human subjects. The model shows that the level and persistence of autoantibodies depends not only on the avidity of T cells, but also on the killing efficacy of these cells. Quantification and modeling of autoreactive T-cell avidities can thus determine the level of risk associated with each type of autoantibodies and the timing of T1D disease onset in individuals that have been tested positive for these autoantibodies. Such studies may lead to early diagnosis of the disease in high-risk individuals and thus potentially serve as a means of staging patients for clinical trials of preventive or interventional therapies far before disease onset

Colony Collapse Disorder: A Descriptive Study

BACKGROUND: Over the last two winters, there have been large-scale, unexplained losses of managed honey bee (Apis mellifera L.) colonies in the United States. In the absence of a known cause, this syndrome was named Colony Collapse Disorder (CCD) because the main trait was a rapid loss of adult worker bees. We initiated a descriptive epizootiological study in order to better characterize CCD and compare risk factor exposure between populations afflicted by and not afflicted by CCD. METHODS AND PRINCIPAL FINDINGS: Of 61 quantified variables (including adult bee physiology, pathogen loads, and pesticide levels), no single measure emerged as a most-likely cause of CCD. Bees in CCD colonies had higher pathogen loads and were co-infected with a greater number of pathogens than control populations, suggesting either an increased exposure to pathogens or a reduced resistance of bees toward pathogens. Levels of the synthetic acaricide coumaphos (used by beekeepers to control the parasitic mite Varroa destructor) were higher in control colonies than CCD-affected colonies. CONCLUSIONS/SIGNIFICANCE: This is the first comprehensive survey of CCD-affected bee populations that suggests CCD involves an interaction between pathogens and other stress factors. We present evidence that this condition is contagious or the result of exposure to a common risk factor. Potentially important areas for future hypothesis-driven research, including the possible legacy effect of mite parasitism and the role of honey bee resistance to pesticides, are highlighted

Is the ADA/EASD algorithm for the management of type 2 diabetes (January 2009) based on evidence or opinion? A critical analysis

The ADA and the EASD recently published a consensus statement for the medical management of hyperglycaemia in patients with type 2 diabetes. The authors advocate initial treatment with metformin monotherapy and lifestyle modification, followed by addition of basal insulin or a sulfonylurea if glycaemic goals are not met (tier 1 recommendations). All other glucose-lowering therapies are relegated to a secondary (tier 2) status and only recommended for selected clinical settings. In our view, this algorithm does not offer physicians and patients the appropriate selection of options to individualise and optimise care with a view to sustained control of blood glucose and reduction both of diabetes complications and cardiovascular risk. This paper critically assesses the basis of the ADA/EASD algorithm and the resulting tiers of treatment options