The rat mast cell line RBL-2H3.1 contains an 85-kDa cytosolic phospholipase A2 (cPLA2) that is very likely involved in liberating arachidonate from membrane phospholipid for the synthesis of eicosanoids following stimulation with either calcium ionophore or IgE/antigen. In this study, the intracellular location of cPLA2 was determined using immunofluorescence microscopy and immuno-gold electron microscopy. In nonstimulated cells, cPLA2 is distributed throughout the cytosol and is excluded from the nucleoplasm. Following cell activation with calcium ionophore, most of the cPLA2 translocates to the nuclear envelope, and the enzyme remains there during the entire period that ionophore is present. With IgE/antigen stimulation for 5 min, approximately 20-30% of the cPLA2 translocates to the nuclear envelope, and after 30 min of stimulation, most of the enzyme returns to the cytosol. Measurement of intracellular calcium using the dye Fura-2/AM shows that the level of calcium rises immediately after antigen is added, remains high for about 30 s, and then declines back to resting levels. Activation with calcium ionophore produces a 10-fold larger release of arachidonate than does stimulation with IgE/antigen. Thus, the results suggest that the extent of membrane binding of cPLA2 correlates with the release of arachidonate and that the site of arachidonate liberation is the nuclear envelope where many of the enzymes that oxygenate this fatty acid are located.

Background: Characteristics of seed-competent Tau are unknown.Results: Native Tau aggregates have a higher seeding potency than recombinant Tau aggregates. Recombinant Tau acquires the conformation and potency of native Tau aggregates by seeded assembly.Conclusion: Conformation determines the seeding potencies of Tau aggregates.Significance: Understanding the properties of seed-competent Tau gives insight into disease mechanisms.

SummaryBackgroundStaphylococcus aureus bacteraemia is a common cause of severe community-acquired and hospital-acquired infection worldwide. We tested the hypothesis that adjunctive rifampicin would reduce bacteriologically confirmed treatment failure or disease recurrence, or death, by enhancing early S aureus killing, sterilising infected foci and blood faster, and reducing risks of dissemination and metastatic infection.MethodsIn this multicentre, randomised, double-blind, placebo-controlled trial, adults (≥18 years) with S aureus bacteraemia who had received ≤96 h of active antibiotic therapy were recruited from 29 UK hospitals. Patients were randomly assigned (1:1) via a computer-generated sequential randomisation list to receive 2 weeks of adjunctive rifampicin (600 mg or 900 mg per day according to weight, oral or intravenous) versus identical placebo, together with standard antibiotic therapy. Randomisation was stratified by centre. Patients, investigators, and those caring for the patients were masked to group allocation. The primary outcome was time to bacteriologically confirmed treatment failure or disease recurrence, or death (all-cause), from randomisation to 12 weeks, adjudicated by an independent review committee masked to the treatment. Analysis was intention to treat. This trial was registered, number ISRCTN37666216, and is closed to new participants.FindingsBetween Dec 10, 2012, and Oct 25, 2016, 758 eligible participants were randomly assigned: 370 to rifampicin and 388 to placebo. 485 (64%) participants had community-acquired S aureus infections, and 132 (17%) had nosocomial S aureus infections. 47 (6%) had meticillin-resistant infections. 301 (40%) participants had an initial deep infection focus. Standard antibiotics were given for 29 (IQR 18–45) days; 619 (82%) participants received flucloxacillin. By week 12, 62 (17%) of participants who received rifampicin versus 71 (18%) who received placebo experienced treatment failure or disease recurrence, or died (absolute risk difference −1·4%, 95% CI −7·0 to 4·3; hazard ratio 0·96, 0·68–1·35, p=0·81). From randomisation to 12 weeks, no evidence of differences in serious (p=0·17) or grade 3–4 (p=0·36) adverse events were observed; however, 63 (17%) participants in the rifampicin group versus 39 (10%) in the placebo group had antibiotic or trial drug-modifying adverse events (p=0·004), and 24 (6%) versus six (2%) had drug interactions (p=0·0005).InterpretationAdjunctive rifampicin provided no overall benefit over standard antibiotic therapy in adults with S aureus bacteraemia.FundingUK National Institute for Health Research Health Technology Assessment.

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Robust reference intervals are needed for the interpretation of bone turnover markers in large phase III fracture trials. The objectives of the study were to (1) estimate reference intervals for serum bone alkaline phosphatase (bone ALP), serum procollagen type I N propeptide (PINP), serum b cross-linked C-telopeptides of type I collagen (S-bCTX), and urinary cross-linked N-telopeptides of type I collagen (U-NTX) in healthy young premenopausal women; (2) examine geographical differences on bone turnover markers; and (3) assess factors known to influence bone turnover and test whether these explain any regional differences. We studied 637 eligible women from four countries that participated in the Horizon-PFT study (United Kingdom, France, Belgium, United States). The women were 30-39 yr of age (mean, 34.6 yr), with regular cyclic menses. Subjects completed a medical and lifestyle questionnaire. Two-sided 95% reference intervals were estimated on transformed values and transformed back to the original scale using the proposed methodology of the International Federation of Clinical Chemistry. S-bCTX was significantly higher in France relative to the United Kingdom (p = 0.01), and PINP was higher in France (p < 0.001) and Belgium (p = 0.02) relative to the United Kingdom and significantly higher in France relative to the United States (p < 0.01) by ANOVA. Overall, one could associate low bone turnover markers with nonsmoking, use of a contraceptive pill, exercise, being close to the time of ovulation, and having high 25-hydroxyvitamin D levels. Countries differed by these characteristics, and once allowed for in the statistical model, any country differences were attenuated or removed.

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BackgroundMeasurement of CD4+ T-lymphocytes (CD4) is a crucial parameter in the management of HIV patients, particularly in determining eligibility to initiate antiretroviral treatment (ART). A number of technologies exist for CD4 enumeration, with considerable variation in cost, complexity, and operational requirements. We conducted a systematic review of the performance of technologies for CD4 enumeration.Methods and FindingsStudies were identified by searching electronic databases MEDLINE and EMBASE using a pre-defined search strategy. Data on test accuracy and precision included bias and limits of agreement with a reference standard, and misclassification probabilities around CD4 thresholds of 200 and 350 cells/μl over a clinically relevant range. The secondary outcome measure was test imprecision, expressed as % coefficient of variation. Thirty-two studies evaluating 15 CD4 technologies were included, of which less than half presented data on bias and misclassification compared to the same reference technology. At CD4 counts <350 cells/μl, bias ranged from -35.2 to +13.1 cells/μl while at counts >350 cells/μl, bias ranged from -70.7 to +47 cells/μl, compared to the BD FACSCount as a reference technology. Misclassification around the threshold of 350 cells/μl ranged from 1-29% for upward classification, resulting in under-treatment, and 7-68% for downward classification resulting in overtreatment. Less than half of these studies reported within laboratory precision or reproducibility of the CD4 values obtained.ConclusionsA wide range of bias and percent misclassification around treatment thresholds were reported on the CD4 enumeration technologies included in this review, with few studies reporting assay precision. The lack of standardised methodology on test evaluation, including the use of different reference standards, is a barrier to assessing relative assay performance and could hinder the introduction of new point-of-care assays in countries where they are most needed.