Complete analysis of the B-cell response to a protein antigen, from in vivo germinal centre formation to 3-D modelling of affinity maturation

Somatic hypermutation of immunoglobulin variable region genes occurs within germinal centres (GCs) and is the process responsible for affinity maturation of antibodies during an immune response. Previous studies have focused almost exclusively on the immune response to haptens, which may be unrepresentative of epitopes on protein antigens. In this study, we have exploited a model system that uses transgenic B and CD4<sup>+</sup> T cells specific for hen egg lysozyme (HEL) and a chicken ovalbumin peptide, respectively, to investigate a tightly synchronized immune response to protein antigens of widely differing affinities, thus allowing us to track many facets of the development of an antibody response at the antigen-specific B cell level in an integrated system <i>in</i> <i>vivo</i>. Somatic hypermutation of immunoglobulin variable genes was analysed in clones of transgenic B cells proliferating in individual GCs in response to HEL or the cross-reactive low-affinity antigen, duck egg lysozyme (DEL). Molecular modelling of the antibody–antigen interface demonstrates that recurring mutations in the antigen-binding site, selected in GCs, enhance interactions of the antibody with DEL. The effects of these mutations on affinity maturation are demonstrated by a shift of transgenic serum antibodies towards higher affinity for DEL in DEL-cOVA immunized mice. The results show that B cells with high affinity antigen receptors can revise their specificity by somatic hypermutation and antigen selection in response to a low-affinity, cross-reactive antigen. These observations shed further light on the nature of the immune response to pathogens and autoimmunity and demonstrate the utility of this novel model for studies of the mechanisms of somatic hypermutation

A novel targeted/untargeted GC-Orbitrap metabolomics methodology applied to Candida albicans and Staphylococcus aureus biofilms

Introduction: Combined infections from Candida albicans and Staphylococcus aureus are a leading cause of death in the developed world. Evidence suggests that Candida enhances the virulence of Staphylococcus—hyphae penetrate through tissue barriers, while S. aureus tightly associates with the hyphae to obtain entry to the host organism. Indeed, in a biofilm state, C. albicans enhances the antimicrobial resistance characteristics of S. aureus. The association of these microorganisms is also associated with significantly increased morbidity and mortality. Due to this tight association we hypothesised that metabolic effects were also in evidence. Objectives: To explore the interaction, we used a novel GC-Orbitrap-based mass spectrometer, the Q Exactive GC, which combines the high peak capacity and chromatographic resolution of gas chromatography with the sub-ppm mass accuracy of an Orbitrap system. This allows the capability to leverage the widely available electron ionisation libraries for untargeted applications, along with expanding accurate mass libraries and targeted matches based around authentic standards. Methods: Optimised C. albicans and S. aureus mono- and co-cultured biofilms were analysed using the new instrument in addition to the fresh and spent bacterial growth media. Results: The targeted analysis experiment was based around 36 sugars and sugar phosphates, 22 amino acids and five organic acids. Untargeted analysis resulted in the detection of 465 features from fresh and spent medium and 405 from biofilm samples. Three significantly changing compounds that matched to high scoring library fragment patterns were chosen for validation. Conclusion: Evaluation of the results demonstrates that the Q Exactive GC is suitable for metabolomics analysis using a targeted/untargeted methodology. Many of the results were as expected: e.g. rapid consumption of glucose and fructose from the medium regardless of the cell type. Modulation of sugar-phosphate levels also suggest that the pentose phosphate pathway could be enhanced in the cells from co-cultured biofilms. Untargeted metabolomics results suggested significant production of cell-wall biosynthesis components and the consumption of non-proteinaceous amino-acids

Pemetrexed Induced Thymidylate Synthase Inhibition in Non-Small Cell Lung Cancer Patients: A Pilot Study with 3 '-Deoxy-3 '-[F-18]fluorothymidine Positron Emission Tomography

OBJECTIVES: Pemetrexed is a thymidylate synthase (TS) inhibitor and is effective in non-small cell lung cancer (NSCLC). 3'-deoxy-3'-[¹⁸F]fluorothymidine (¹⁸F-FLT), a proliferation marker, could potentially identify tumor specific TS-inhibition. The aim of this study was to investigate the effect of pemetrexed-induced TS-inhibition on ¹⁸F-FLT uptake 4 hours after pemetrexed administration in metastatic NSCLC patients. METHODS: Fourteen NSCLC patients underwent dynamic ¹⁸F-FLT positron emission tomography (PET) scans at baseline and 4 hours after the first dose of pemetrexed. Volumes of interest were defined with a 41%, 50% and 70% threshold of the maximum pixel. Kinetic analysis and simplified measures were performed. At one, two, four and six hours after pemetrexed, plasma deoxyuridine was measured as systemic indicator of TS-inhibition. Tumor response measured with response evaluation criteria in solid tumors (RECIST), time to progression (TTP) and overall survival (OS) were determined. RESULTS: Eleven patients had evaluable ¹⁸F-FLT PET scans at baseline and 4 hours after pemetrexed. Two patients had increased ¹⁸F-FLT uptake of 35% and 31% after pemetrexed, whereas two other patients had decreased uptake of 31%. In the remaining seven patients ¹⁸F-FLT uptake did not change beyond test-retest borders. In all patients deoxyuridine levels raised after administration of pemetrexed, implicating pemetrexed-induced TS-inhibition. ¹⁸F-FLT uptake in bone marrow was significantly increased 4 hours after pemetrexed administration. Six weeks after the start of treatment 5 patients had partial response, 4 stable disease and 2 progressive disease. Median TTP was 4.2 months (range 3.0-7.4 months); median OS was 13.0 months (range 5.1-30.8 months). Changes in ¹⁸F-FLT uptake were not predictive for tumor response, TTP or OS. CONCLUSIONS: Measuring TS-inhibition in a clinical setting 4 hours after pemetrexed revealed a non-systematic change in ¹⁸F-FLT uptake within the tumor. No significant association with tumor response, TTP or OS was observed

Understanding and using comparative healthcare information; the effect of the amount of information and consumer characteristics and skills

<p>Abstract</p> <p>Background</p> <p>Consumers are increasingly exposed to comparative healthcare information (information about the quality of different healthcare providers). Partly because of its complexity, the use of this information has been limited. The objective of this study was to examine how the amount of presented information influences the comprehension and use of comparative healthcare information when important consumer characteristics and skills are taken into account.</p> <p>Methods</p> <p>In this randomized controlled experiment, comparative information on total hip or knee surgery was used as a test case. An online survey was distributed among 800 members of the NIVEL Insurants Panel and 76 hip- or knee surgery patients. Participants were assigned to one of four subgroups, who were shown 3, 7, 11 or 15 quality aspects of three hospitals. We conducted Kruskall-Wallis tests, Chi-square tests and hierarchical multiple linear regression analyses to examine relationships between the amount of information and consumer characteristics and skills (literacy, numeracy, active choice behaviour) on one hand, and outcome measures related to effectively using information (comprehension, perceived usefulness of information, hospital choice, ease of making a choice) on the other hand.</p> <p>Results</p> <p>414 people (47%) participated. Regression analysis showed that the amount of information slightly influenced the comprehension and the perceived usefulness of comparative healthcare information. It did not affect consumers’ hospital choice and ease of making this choice. Consumer characteristics (especially age) and skills (especially literacy) were the most important factors affecting the comprehension of information and the ease of making a hospital choice. For the perceived usefulness of comparative information, active choice behaviour was the most influencing factor.</p> <p>Conclusion</p> <p>The effects of the amount of information were not unambiguous. It remains unclear what the ideal amount of quality information to be presented would be. Reducing the amount of information will probably not automatically result in more effective use of comparative healthcare information by consumers. More important, consumer characteristics and skills appeared to be more influential factors contributing to information comprehension and use. Consequently, we would suggest that more emphasis on improving consumers’ skills is needed to enhance the use of comparative healthcare information.</p

Environmentally Realistic Exposure to the Herbicide Atrazine Alters Some Sexually Selected Traits in Male Guppies

Male mating signals, including ornaments and courtship displays, and other sexually selected traits, like male-male aggression, are largely controlled by sex hormones. Environmental pollutants, notably endocrine disrupting compounds, can interfere with the proper functioning of hormones, thereby impacting the expression of hormonally regulated traits. Atrazine, one of the most widely used herbicides, can alter sex hormone levels in exposed animals. I tested the effects of environmentally relevant atrazine exposures on mating signals and behaviors in male guppies, a sexually dimorphic freshwater fish. Prolonged atrazine exposure reduced the expression of two honest signals: the area of orange spots (ornaments) and the number of courtship displays performed. Atrazine exposure also reduced aggression towards competing males in the context of mate competition. In the wild, exposure levels vary among individuals because of differential distribution of the pollutants across habitats; hence, differently impacted males often compete for the same mates. Disrupted mating signals can reduce reproductive success as females avoid mating with perceptibly suboptimal males. Less aggressive males are at a competitive disadvantage and lose access to females. This study highlights the effects of atrazine on ecologically relevant mating signals and behaviors in exposed wildlife. Altered reproductive traits have important implications for population dynamics, evolutionary patterns, and conservation of wildlife species

Benefits and barriers among volunteer teaching faculty: comparison between those who precept and those who do not in the core pediatrics clerkship

Background: Community-based outpatient experiences are a core component of the clinical years in medical school. Central to the success of this experience is the recruitment and retention of volunteer faculty from the community. Prior studies have identified reasons why some preceptors volunteer their time however, there is a paucity of data comparing those who volunteer from those who do not. Methods: A survey was developed following a review of previous studies addressing perceptions of community-based preceptors. A non-parametric, Mann&#x2013;Whitney U test was used to compare active preceptors (APs) and inactive preceptors (IPs) and all data were analyzed in SPSS 20.0. Results: There was a 28% response rate. Preceptors showed similar demographic characteristics, valued intrinsic over extrinsic benefits, and appreciated Continuing Medical Education (CME)/Maintenance of Certification (MOC) opportunities as the highest extrinsic reward. APs were more likely to also precept at the M1/M2 level and value recognition and faculty development opportunities (p&#x3C;0.05). IPs denoted time as the most significant barrier and, in comparison to APs, rated financial compensation as more important (p&#x3C;0.05). Conclusions: Community preceptors are motivated by intrinsic benefits of teaching. Efforts to recruit should initially focus on promoting awareness of teaching opportunities and offering CME/MOC opportunities. Increasing the pool of preceptors may require financial compensation

Self-reported pediatricians' management of the well-appearing young child with fever without a source: first survey in an European country in the anti-pneumococcal vaccine era

<p>Abstract</p> <p>Background</p> <p>Recent studies suggest a substantially reduced risk of invasive bacterial infection in children vaccinated with heptavalent pneumococcal conjugate vaccine (PCV). To investigate whether the introduction of PCV might affect clinical decision making, we conducted a cross-sectional survey aimed at Italian Pediatric physicians.</p> <p>Results</p> <p>The study included 348 (46.5%) primary care pediatricians; 251 (36.4%) hospital pediatricians, and 139 (20.1%) pediatric residents. In an hypothetical scenario, a well-appearing 12-month-old child with fever without source would be sent home with no therapy by 60.7% (419/690) of physicians if the child was not vaccinated with PCV. The proportion increased to 74.2% (512/690) if the child had received PCV (P < 0.0001). Also, physicians would obtain blood tests less frequently in the vaccinated than in unvaccinated children (139/690 [20.1%] <it>vs</it>. 205/690 [29.7%]; P < 0.0001), and started empiric antibiotic therapy less frequently (3.0% <it>vs</it>. 7.5%; P < 0.0001). In the hypothetical event that white blood cell count was 17,500/μL, a significantly lower proportion of physicians would ask for erythrocyte sedimentation rate (P < 0.017), C reactive protein (P < 0.0001), blood culture (P = 0.022), and urine analysis or dipstick (P = 0.028), if the child had received PCV. Only one third of participants routinely recommended PCV.</p> <p>Conclusion</p> <p>Our data suggest that implementation of educational programs regarding the proper management of the febrile child is needed.</p

A meta-analytic review of stand-alone interventions to improve body image

Objective Numerous stand-alone interventions to improve body image have been developed. The present review used meta-analysis to estimate the effectiveness of such interventions, and to identify the specific change techniques that lead to improvement in body image. Methods The inclusion criteria were that (a) the intervention was stand-alone (i.e., solely focused on improving body image), (b) a control group was used, (c) participants were randomly assigned to conditions, and (d) at least one pretest and one posttest measure of body image was taken. Effect sizes were meta-analysed and moderator analyses were conducted. A taxonomy of 48 change techniques used in interventions targeted at body image was developed; all interventions were coded using this taxonomy. Results The literature search identified 62 tests of interventions (N = 3,846). Interventions produced a small-to-medium improvement in body image (d+ = 0.38), a small-to-medium reduction in beauty ideal internalisation (d+ = -0.37), and a large reduction in social comparison tendencies (d+ = -0.72). However, the effect size for body image was inflated by bias both within and across studies, and was reliable but of small magnitude once corrections for bias were applied. Effect sizes for the other outcomes were no longer reliable once corrections for bias were applied. Several features of the sample, intervention, and methodology moderated intervention effects. Twelve change techniques were associated with improvements in body image, and three techniques were contra-indicated. Conclusions The findings show that interventions engender only small improvements in body image, and underline the need for large-scale, high-quality trials in this area. The review identifies effective techniques that could be deployed in future interventions

A comprehensive 1000 Genomes-based genome-wide association meta-analysis of coronary artery disease

Existing knowledge of genetic variants affecting risk of coronary artery disease (CAD) is largely based on genome-wide association studies (GWAS) analysis of common SNPs. Leveraging phased haplotypes from the 1000 Genomes Project, we report a GWAS meta-analysis of 185 thousand CAD cases and controls, interrogating 6.7 million common (MAF>0.05) as well as 2.7 million low frequency (0.005<MAF<0.05) variants. In addition to confirmation of most known CAD loci, we identified 10 novel loci, eight additive and two recessive, that contain candidate genes that newly implicate biological processes in vessel walls. We observed intra-locus allelic heterogeneity but little evidence of low frequency variants with larger effects and no evidence of synthetic association. Our analysis provides a comprehensive survey of the fine genetic architecture of CAD showing that genetic susceptibility to this common disease is largely determined by common SNPs of small effect siz

Dupuytren's contracture: a retrospective database analysis to determine hospitalizations in the Netherlands

Background: Dupuytren's contracture is a condition of the palmar fascia involving contractures of the fascia and skin in the hand. Current treatment for Dupuytren's contracture is mainly limited to surgery. In the Netherlands, little is known about the prevalence of Dupuytren's contracture. In this study we determined the prevalence of patients with a hospitalization for Dupuytren's contracture in the Netherlands and characterized their (re)hospitalizations. Methods. From the PHARMO database, which consists of multiple observational databases linked on a patient level, all patients hospitalized for Dupuytren's contracture between 2004 and 2007 were included in the source population (ICD-9-CM code 728.6). Numbers from this source population were used to provide estimates of hospitalizations for Dupuytren's contracture in the Netherlands. Patients with a medical history in the PHARMO database of at least 12 months before their hospitalization were included in the study cohort and followed until end of data collection, death, or end of study period, whichever occurred first. Type of admission, length of stay, recorded procedures, treating specialty, number of rehospitalizations for Dupuytren's contracture, and time to first rehospitalization were assessed. Results: Of 3, 126 patients included in the source population, 3, 040 were included in the study population. The overall prevalence of patients with a hospitalization for Dupuytren'