NS1 Specific CD8(+) T-Cells with Effector Function and TRBV11 Dominance in a Patient with Parvovirus B19 Associated Inflammatory Cardiomyopathy

Background: Parvovirus B19 (B19V) is the most commonly detected virus in endomyocardial biopsies (EMBs) from patients with inflammatory cardiomyopathy (DCMi). Despite the importance of T-cells in antiviral defense, little is known about the role of B19V specific T-cells in this entity. Methodology and Principal Findings: An exceptionally high B19V viral load in EMBs (115,091 viral copies/mg nucleic acids), peripheral blood mononuclear cells (PBMCs) and serum was measured in a DCMi patient at initial presentation, suggesting B19V viremia. The B19V viral load in EMBs had decreased substantially 6 and 12 months afterwards, and was not traceable in PBMCs and the serum at these times. Using pools of overlapping peptides spanning the whole B19V proteome, strong CD8(+) T-cell responses were elicited to the 10-amico-acid peptides SALKLAIYKA (19.7% of all CD8(+) cells) and QSALKLAIYK (10%) and additional weaker responses to GLCPHCINVG (0.71%) and LLHTDFEQVM (0.06%). Real-time RT-PCR of IFN gamma secretion-assay-enriched T-cells responding to the peptides, SALKLAIYKA and GLCPHCINVG, revealed a disproportionately high T-cell receptor Vbeta (TRBV) 11 expression in this population. Furthermore, dominant expression of type-1 (IFN gamma, IL2, IL27 and Tbet) and of cytotoxic T-cell markers (Perforin and Granzyme B) was found, whereas gene expression indicating type-2 (IL4, GATA3) and regulatory T-cells (FoxP3) was low. Conclusions: Our results indicate that B19V Ag-specific CD8(+) T-cells with effector function are involved in B19V associated DCMi. In particular, a dominant role of TRBV11 and type-1/CTL effector cells in the T-cell mediated antiviral immune response is suggested. The persistence of B19V in the endomyocardium is a likely antigen source for the maintenance of CD8(+) T-cell responses to the identified epitopes

The metabolic significance of octulose phosphates in the photosynthetic carbon reduction cycle in spinach

(14)C-Labelled octulose phosphates were formed during photosynthetic (14)CO(2) fixation and were measured in spinach leaves and chloroplasts. Because mono- and bisphosphates of d-glycero-d-ido-octulose are the active 8-carbon ketosugar intermediates of the L-type pentose pathway, it was proposed that they may also be reactants in a modified Calvin–Benson–Bassham pathway reaction scheme. This investigation therefore initially focussed only on the ido-epimer of the octulose phosphates even though (14)C-labelled d-glycero-d-altro-octulose mono- and bisphosphates were also identified in chloroplasts and leaves. (14)CO(2) predominantly labelled positions 5 and 6 of d-glycero-d-ido-octulose 1,8-P(2) consistent with labelling predictions of the modified scheme. The kinetics of (14)CO(2) incorporation into ido-octulose was similar to its incorporation into some traditional intermediates of the path of carbon, while subsequent exposure to (12)CO(2) rapidly displaced the (14)C isotope label from octulose with the same kinetics of label loss as some of the confirmed Calvin pathway intermediates. This is consistent with octulose phosphates having the role of cyclic intermediates rather than synthesized storage products. (Storage products don’t rapidly exchange isotopically labelled carbons with unlabelled CO(2).) A spinach chloroplast extract, designated stromal enzyme preparation (SEP), catalysed and was used to measure rates of CO(2) assimilation with Calvin cycle intermediates and octulose and arabinose phosphates. Only pentose (but not arabinose) phosphates and sedoheptulose 7-phosphate supported CO(2) fixation at rates in excess of 120 μmol h(−1) mg(−1) Chl. Rates for octulose, sedoheptulose and fructose bisphosphates, octulose, hexose and triose monophosphates were all notably less than the above rate and arabinose 5-phosphate was inactive. Altro-octulose phosphates were more active than phosphate esters of the ido-epimer. The modified scheme proposed a specific phosphotransferase and SEP unequivocally catalysed reversible phosphate transfer between sedoheptulose bisphosphate and d-glycero-d-ido-octulose 8-phosphate. It was also initially hypothesized that arabinose 5-phosphate, an L-Type pentose pathway reactant, may have a role in a modified Calvin pathway. Arabinose 5-phosphate is present in spinach chloroplasts and leaves. Radiochromatography showed that (14)C-arabinose 5-phosphate with SEP, but only in the presence of an excess of unlabelled ribose 5-phosphate, lightly labelled ribulose 5-phosphate and more heavily labelled hexose and sedoheptulose mono- and bisphosphates. However, failure to demonstrate any CO(2) fixation by arabinose 5-phosphate as sole substrate suggested that the above labelling may have no metabolic significance. Despite this arabinose and ribose 5-phosphates are shown to exhibit active roles as enzyme co-factors in transaldolase and aldolase exchange reactions that catalyse the epimeric interconversions of the phosphate esters of ido- and altro-octulose. Arabinose 5-phosphate is presented as playing this role in a New Reaction Scheme for the path of carbon, where it is concluded that slow reacting ido-octulose 1,8 bisphosphate has no role. The more reactive altro-octulose phosphates, which are independent of the need for phosphotransferase processing, are presented as intermediates in the new scheme. Moreover, using the estimates of phosphotransferase activity with altro-octulose monophosphate as substrate allowed calculation of the contributions of the new scheme, that ranged from 11% based on the intact chloroplast carboxylation rate to 80% using the carboxylation rate required for the support of octulose phosphate synthesis and its role in the phosphotransferase reaction

Observation and study of baryonic B decays: B -> D(*) p pbar, D(*) p pbar pi, and D(*) p pbar pi pi

We present a study of ten B-meson decays to a D(*), a proton-antiproton pair, and a system of up to two pions using BaBar's data set of 455x10^6 BBbar pairs. Four of the modes (B0bar -> D0 p anti-p, B0bar -> D*0 p anti-p, B0bar -> D+ p anti-p pi-, B0bar -> D*+ p anti-p pi-) are studied with improved statistics compared to previous measurements; six of the modes (B- -> D0 p anti-p pi-, B- -> D*0 p anti-p pi-, B0bar -> D0 p anti-p pi- pi+, B0bar -> D*0 p anti-p pi- pi+, B- -> D+ p anti-p pi- pi-, B- -> D*+ p anti-p pi- pi-) are first observations. The branching fractions for 3- and 5-body decays are suppressed compared to 4-body decays. Kinematic distributions for 3-body decays show non-overlapping threshold enhancements in m(p anti-p) and m(D(*)0 p) in the Dalitz plots. For 4-body decays, m(p pi-) mass projections show a narrow peak with mass and full width of (1497.4 +- 3.0 +- 0.9) MeV/c2, and (47 +- 12 +- 4) MeV/c2, respectively, where the first (second) errors are statistical (systematic). For 5-body decays, mass projections are similar to phase space expectations. All results are preliminary.Comment: 28 pages, 90 postscript figures, submitted to LP0

Differential Roles of HOW in Male and Female Drosophila Germline Differentiation

The adult gonads in both male and female Drosophila melanogaster produce gametes that originate from a regenerative pool of germline stem cells (GSCs). The differentiation programme that produces gametes must be co-ordinated with GSC maintenance and proliferation in order to regulate tissue regeneration. The HOW RNA-binding protein has been shown to maintain mitotic progression of male GSCs and their daughters by maintenance of Cyclin B expression as well as suppressing accumulation of the differentiation factor Bam. Loss of HOW function in the male germline results in loss of GSCs due to a delay in G2 and subsequent apoptosis. Here we show that female how mutant GSCs do not have any cell cycle defects although HOW continues to bind bam mRNA and suppress Bam expression. The role of HOW in suppressing germ cell Bam expression appears to be conserved between sexes, leading to different cellular outcomes in how mutants due to the different functions of Bam. In addition the role in maintaining Cyclin B expression has not been conserved so female how GSCs differentiate rather than arrest

How Long and Low Can You Go? Effect of Conformation on the Risk of Thoracolumbar Intervertebral Disc Extrusion in Domestic Dogs

Intervertebral disc extrusion (IVDE) is a common neurological disorder in certain dog breeds, resulting in spinal cord compression and injury that can cause pain and neurological deficits. Most disc extrusions are reported in chondrodystrophic breeds (e.g. Dachshunds, Basset Hounds, Pekingese), where selection for ‘long and low’ morphologies is linked with intervertebral discs abnormalities that predispose dogs to IVDE. The aim of this study was to quantify the relationship between relative thoracolumbar vertebral column length and IVDE risk in diverse breeds. A 14 month cross-sectional study of dogs entering a UK small animal referral hospital for diverse disorders including IVDE was carried out. Dogs were measured on breed-defining morphometrics, including back length (BL) and height at the withers (HW). Of 700 dogs recruited from this referral population, measured and clinically examined, 79 were diagnosed with thoracolumbar IVDE following diagnostic imaging ± surgery. The BL:HW ratio was positively associated with IVDE risk, indicating that relatively longer dogs were at increased risk, e.g. the probability of IVDE was 0.30 for Miniature Dachshunds when BL:HW ratio equalled 1.1, compared to 0.68 when BL:HW ratio equalled 1.5. Additionally, both being overweight and skeletally smaller significantly increased IVDE risk. Therefore, selection for longer backs and miniaturisation should be discouraged in high-risk breeds to reduce IVDE risk. In higher risk individuals, maintaining a lean body shape is particularly important to reduce the risk of IVDE. Results are reported as probabilities to aid decision-making regarding breed standards and screening programmes reflecting the degree of risk acceptable to stakeholders

Genome-wide association analysis of diverticular disease points towards neuromuscular, connective tissue and epithelial pathomechanisms.

This is the author accepted manuscript.OBJECTIVE: Diverticular disease is a common complex disorder characterised by mucosal outpouchings of the colonic wall that manifests through complications such as diverticulitis, perforation and bleeding. We report the to date largest genome-wide association study (GWAS) to identify genetic risk factors for diverticular disease. DESIGN: Discovery GWAS analysis was performed on UK Biobank imputed genotypes using 31 964 cases and 419 135 controls of European descent. Associations were replicated in a European sample of 3893 cases and 2829 diverticula-free controls and evaluated for risk contribution to diverticulitis and uncomplicated diverticulosis. Transcripts at top 20 replicating loci were analysed by real-time quatitative PCR in preparations of the mucosal, submucosal and muscular layer of colon. The localisation of expressed protein at selected loci was investigated by immunohistochemistry. RESULTS: We discovered 48 risk loci, of which 12 are novel, with genome-wide significance and consistent OR in the replication sample. Nominal replication (p<0.05) was observed for 27 loci, and additional 8 in meta-analysis with a population-based cohort. The most significant novel risk variant rs9960286 is located near CTAGE1 with a p value of 2.3×10-10 and 0.002 (ORallelic=1.14 (95% CI 1.05 to 1.24)) in the replication analysis. Four loci showed stronger effects for diverticulitis, PHGR1 (OR 1.32, 95% CI 1.12 to 1.56), FAM155A-2 (OR 1.21, 95% CI 1.04 to 1.42), CALCB (OR 1.17, 95% CI 1.03 to 1.33) and S100A10 (OR 1.17, 95% CI 1.03 to 1.33). CONCLUSION: In silico analyses point to diverticulosis primarily as a disorder of intestinal neuromuscular function and of impaired connective fibre support, while an additional diverticulitis risk might be conferred by epithelial dysfunction.German Research CouncilAustrian Science FundFaculty of Medicine, Saarland UniversityResearch Council of LithuaniaSwedish Research CouncilMedical Research Counci

Phosphoinositide Regulation of Integrin Trafficking Required for Muscle Attachment and Maintenance

Muscles must maintain cell compartmentalization when remodeled during development and use. How spatially restricted adhesions are regulated with muscle remodeling is largely unexplored. We show that the myotubularin (mtm) phosphoinositide phosphatase is required for integrin-mediated myofiber attachments in Drosophila melanogaster, and that mtm-depleted myofibers exhibit hallmarks of human XLMTM myopathy. Depletion of mtm leads to increased integrin turnover at the sarcolemma and an accumulation of integrin with PI(3)P on endosomal-related membrane inclusions, indicating a role for Mtm phosphatase activity in endocytic trafficking. The depletion of Class II, but not Class III, PI3-kinase rescued mtm-dependent defects, identifying an important pathway that regulates integrin recycling. Importantly, similar integrin localization defects found in human XLMTM myofibers signify conserved MTM1 function in muscle membrane trafficking. Our results indicate that regulation of distinct phosphoinositide pools plays a central role in maintaining cell compartmentalization and attachments during muscle remodeling, and they suggest involvement of Class II PI3-kinase in MTM-related disease

Preventive Antibacterial Therapy in Acute Ischemic Stroke: A Randomized Controlled Trial

BACKGROUND: Pneumonia is a major risk factor of death after acute stroke. In a mouse model, preventive antibacterial therapy with moxifloxacin not only prevents the development of post-stroke infections, it also reduces mortality, and improves neurological outcome significantly. In this study we investigate whether this approach is effective in stroke patients. METHODS: Preventive ANtibacterial THERapy in acute Ischemic Stroke (PANTHERIS) is a randomized, double-blind, placebo-controlled trial in 80 patients with severe, non-lacunar, ischemic stroke (NIHSS>11) in the middle cerebral artery (MCA) territory. Patients received either intravenous moxifloxacin (400 mg daily) or placebo for 5 days starting within 36 hours after stroke onset. Primary endpoint was infection within 11 days. Secondary endpoints included neurological outcome, survival, development of stroke-induced immunodepression, and induction of bacterial resistance. FINDINGS: On intention-to treat analysis (79 patients), the infection rate at day 11 in the moxifloxacin treated group was 15.4% compared to 32.5% in the placebo treated group (p = 0.114). On per protocol analysis (n = 66), moxifloxacin significantly reduced infection rate from 41.9% to 17.1% (p = 0.032). Stroke associated infections were associated with a lower survival rate. In this study, neurological outcome and survival were not significantly influenced by treatment with moxifloxacin. Frequency of fluoroquinolone resistance in both treatment groups did not differ. On logistic regression analysis, treatment arm as well as the interaction between treatment arm and monocytic HLA-DR expression (a marker for immunodepression) at day 1 after stroke onset was independently and highly predictive for post-stroke infections. INTERPRETATION: PANTHERIS suggests that preventive administration of moxifloxacin is superior in reducing infections after severe non-lacunar ischemic stroke compared to placebo. In addition, the results emphasize the pivotal role of immunodepression in developing post-stroke infections. TRIAL REGISTRATION: Controlled-Trials.com ISRCTN74386719

Dysfunctional GABAergic inhibition in the prefrontal cortex leading to "psychotic" hyperactivation

<p>Abstract</p> <p>Background</p> <p>The GABAergic system in the brain seems to be dysfunctional in various psychiatric disorders. Many studies have suggested so far that, in schizophrenia patients, GABAergic inhibition is selectively but consistently reduced in the prefrontal cortex (PFC).</p> <p>Results</p> <p>This study used a computational model of the PFC to investigate the dynamics of the PFC circuit with and without chandelier cells and other GABAergic interneurons. The inhibition by GABAergic interneurons other than chandelier cells effectively regulated the PFC activity with rather low or modest levels of dopaminergic neurotransmission. This activity of the PFC is associated with normal cognitive functions and has an inverted-U shaped profile of dopaminergic modulation. In contrast, the chandelier cell-type inhibition affected only the PFC circuit dynamics in hyperdopaminergic conditions. Reduction of chandelier cell-type inhibition resulted in bistable dynamics of the PFC circuit, in which the upper stable state is associated with a hyperactive mode. When both types of inhibition were reduced, this hyperactive mode and the conventional inverted-U mode merged.</p> <p>Conclusion</p> <p>The results of our simulation suggest that, in schizophrenia, a reduction of GABAergic inhibition increases vulnerability to psychosis by (i) producing the hyperactive mode of the PFC with hyperdopaminergic neurotransmission by dysfunctional chandelier cells and (ii) increasing the probability of the transition to the hyperactive mode from the conventional inverted-U mode by dysfunctional GABAergic interneurons.</p

(R)-[11C]Verapamil PET studies to assess changes in P-glycoprotein expression and functionality in rat blood-brain barrier after exposure to kainate-induced status epilepticus

<p>Abstract</p> <p>Background</p> <p>Increased functionality of efflux transporters at the blood-brain barrier may contribute to decreased drug concentrations at the target site in CNS diseases like epilepsy. In the rat, pharmacoresistant epilepsy can be mimicked by inducing status epilepticus by intraperitoneal injection of kainate, which leads to development of spontaneous seizures after 3 weeks to 3 months. The aim of this study was to investigate potential changes in P-glycoprotein (P-gp) expression and functionality at an early stage after induction of status epilepticus by kainate.</p> <p>Methods</p> <p><it>(R)</it>-[¹¹C]verapamil, which is currently the most frequently used positron emission tomography (PET) ligand for determining P-gp functionality at the blood-brain barrier, was used in kainate and saline (control) treated rats, at 7 days after treatment. To investigate the effect of P-gp on <it>(R)</it>-[¹¹C]verapamil brain distribution, both groups were studied without or with co-administration of the P-gp inhibitor tariquidar. P-gp expression was determined using immunohistochemistry in post mortem brains. <it>(R)</it>-[¹¹C]verapamil kinetics were analyzed with approaches common in PET research (Logan analysis, and compartmental modelling of individual profiles) as well as by population mixed effects modelling (NONMEM).</p> <p>Results</p> <p>All data analysis approaches indicated only modest differences in brain distribution of <it>(R)</it>-[¹¹C]verapamil between saline and kainate treated rats, while tariquidar treatment in both groups resulted in a more than 10-fold increase. NONMEM provided most precise parameter estimates. P-gp expression was found to be similar for kainate and saline treated rats.</p> <p>Conclusions</p> <p>P-gp expression and functionality does not seem to change at early stage after induction of anticipated pharmacoresistant epilepsy by kainate.</p