Thrombospondin-1 Contributes to Mortality in Murine Sepsis through Effects on Innate Immunity

BACKGROUND:Thrombospondin-1 (TSP-1) is involved in many biological processes, including immune and tissue injury response, but its role in sepsis is unknown. Cell surface expression of TSP-1 on platelets is increased in sepsis and could activate the anti-inflammatory cytokine transforming growth factor beta (TGFβ1) affecting outcome. Because of these observations we sought to determine the importance of TSP-1 in sepsis. METHODOLOGY/PRINCIPAL FINDINGS:We performed studies on TSP-1 null and wild type (WT) C57BL/6J mice to determine the importance of TSP-1 in sepsis. We utilized the cecal ligation puncture (CLP) and intraperitoneal E. coli injection (i.p. E. coli) models of peritoneal sepsis. Additionally, bone-marrow-derived macrophages (BMMs) were used to determine phagocytic activity. TSP-1-/- animals experienced lower mortality than WT mice after CLP. Tissue and peritoneal lavage TGFβ1 levels were unchanged between animals of each genotype. In addition, there is no difference between the levels of major innate cytokines between the two groups of animals. PLF from WT mice contained a greater bacterial load than TSP-1-/- mice after CLP. The survival advantage for TSP-1-/- animals persisted when i.p. E. coli injections were performed. TSP-1-/- BMMs had increased phagocytic capacity compared to WT. CONCLUSIONS:TSP-1 deficiency was protective in two murine models of peritoneal sepsis, independent of TGFβ1 activation. Our studies suggest TSP-1 expression is associated with decreased phagocytosis and possibly bacterial clearance, leading to increased peritoneal inflammation and mortality in WT mice. These data support the contention that TSP-1 should be more fully explored in the human condition

Developing a collaborative agenda for humanities and social scientific research on laboratory animal science and welfare.

Improving laboratory animal science and welfare requires both new scientific research and insights from enquiry in the humanities and social sciences. Whilst scientific research provides evidence to replace, reduce and refine procedures involving laboratory animals (the ‘3Rs’), work in the humanities and social sciences can help understand the social, economic and cultural processes that enhance or impede humane ways of knowing and working with laboratory animals. However, communication across these disciplinary perspectives is currently limited, and they frame questions, generate results, engage users, and seek to influence policy in different ways. To facilitate dialogue and future research at this interface, we convened an interdisciplinary group of 45 life scientists, social scientists, humanities scholars, non-governmental organisations and policy-makers to generate a collaborative research agenda. This drew on other agenda-setting exercises in science policy, using a collaborative and deliberative approach for the identification of research priorities. Participants were recruited from across the community, invited to submit research questions and vote on their priorities. They then met at an interactive workshop in the UK, discussed all 136 questions submitted, and collectively defined the 30 most important issues for the group. The output is a collaborative future agenda for research in the humanities and social sciences on laboratory animal science and welfare. The questions indicate a demand for new research in the humanities and social sciences to inform emerging discussions and priorities on the governance and practice of laboratory animal research, including around: international harmonisation, openness and public engagement, ‘cultures of care’, harm-benefit analysis and the future of the 3Rs. The process underlines the value of interdisciplinary exchange for improving mutual understanding of different research cultures and identifies ways of enhancing the effectiveness of future research at the interface between the humanities, social sciences, science and science policy

Maternal exposure to polychlorinated biphenyls and the secondary sex ratio: an occupational cohort study

Though commercial production of polychlorinated biphenyls was banned in the United States in 1977, exposure continues due to their environmental persistence. Several studies have examined the association between environmental polychlorinated biphenyl exposure and modulations of the secondary sex ratio, with conflicting results. Our objective was to evaluate the association between maternal preconceptional occupational polychlorinated biphenyl exposure and the secondary sex ratio. We examined primipara singleton births of 2595 women, who worked in three capacitor plants at least one year during the period polychlorinated biphenyls were used. Cumulative estimated maternal occupational polychlorinated biphenyl exposure at the time of the infant's conception was calculated from plant-specific job-exposure matrices. A logistic regression analysis was used to evaluate the association between maternal polychlorinated biphenyl exposure and male sex at birth (yes/no). Maternal body mass index at age 20, smoking status, and race did not vary between those occupationally exposed and those unexposed before the child's conception. Polychlorinated biphenyl-exposed mothers were, however, more likely to have used oral contraceptives and to have been older at the birth of their first child than non-occupationally exposed women. Among 1506 infants liveborn to polychlorinated biphenyl-exposed primiparous women, 49.8% were male; compared to 49.9% among those not exposed (n = 1089). Multivariate analyses controlling for mother's age and year of birth found no significant association between the odds of a male birth and mother's cumulative estimated polychlorinated biphenyl exposure to time of conception. Based on these data, we find no evidence of altered sex ratio among children born to primiparous polychlorinated biphenyl-exposed female workers

A Single Peroxisomal Targeting Signal Mediates Matrix Protein Import in Diatoms

Peroxisomes are single membrane bound compartments. They are thought to be present in almost all eukaryotic cells, although the bulk of our knowledge about peroxisomes has been generated from only a handful of model organisms. Peroxisomal matrix proteins are synthesized cytosolically and posttranslationally imported into the peroxisomal matrix. The import is generally thought to be mediated by two different targeting signals. These are respectively recognized by the two import receptor proteins Pex5 and Pex7, which facilitate transport across the peroxisomal membrane. Here, we show the first in vivo localization studies of peroxisomes in a representative organism of the ecologically relevant group of diatoms using fluorescence and transmission electron microscopy. By expression of various homologous and heterologous fusion proteins we demonstrate that targeting of Phaeodactylum tricornutum peroxisomal matrix proteins is mediated only by PTS1 targeting signals, also for proteins that are in other systems imported via a PTS2 mode of action. Additional in silico analyses suggest this surprising finding may also apply to further diatoms. Our data suggest that loss of the PTS2 peroxisomal import signal is not reserved to Caenorhabditis elegans as a single exception, but has also occurred in evolutionary divergent organisms. Obviously, targeting switching from PTS2 to PTS1 across different major eukaryotic groups might have occurred for different reasons. Thus, our findings question the widespread assumption that import of peroxisomal matrix proteins is generally mediated by two different targeting signals. Our results implicate that there apparently must have been an event causing the loss of one targeting signal even in the group of diatoms. Different possibilities are discussed that indicate multiple reasons for the detected targeting switching from PTS2 to PTS1

Identification of genetic variants associated with Huntington's disease progression: a genome-wide association study

Background Huntington's disease is caused by a CAG repeat expansion in the huntingtin gene, HTT. Age at onset has been used as a quantitative phenotype in genetic analysis looking for Huntington's disease modifiers, but is hard to define and not always available. Therefore, we aimed to generate a novel measure of disease progression and to identify genetic markers associated with this progression measure. Methods We generated a progression score on the basis of principal component analysis of prospectively acquired longitudinal changes in motor, cognitive, and imaging measures in the 218 indivduals in the TRACK-HD cohort of Huntington's disease gene mutation carriers (data collected 2008–11). We generated a parallel progression score using data from 1773 previously genotyped participants from the European Huntington's Disease Network REGISTRY study of Huntington's disease mutation carriers (data collected 2003–13). We did a genome-wide association analyses in terms of progression for 216 TRACK-HD participants and 1773 REGISTRY participants, then a meta-analysis of these results was undertaken. Findings Longitudinal motor, cognitive, and imaging scores were correlated with each other in TRACK-HD participants, justifying use of a single, cross-domain measure of disease progression in both studies. The TRACK-HD and REGISTRY progression measures were correlated with each other (r=0·674), and with age at onset (TRACK-HD, r=0·315; REGISTRY, r=0·234). The meta-analysis of progression in TRACK-HD and REGISTRY gave a genome-wide significant signal (p=1·12 × 10−10) on chromosome 5 spanning three genes: MSH3, DHFR, and MTRNR2L2. The genes in this locus were associated with progression in TRACK-HD (MSH3 p=2·94 × 10−8 DHFR p=8·37 × 10−7 MTRNR2L2 p=2·15 × 10−9) and to a lesser extent in REGISTRY (MSH3 p=9·36 × 10−4 DHFR p=8·45 × 10−4 MTRNR2L2 p=1·20 × 10−3). The lead single nucleotide polymorphism (SNP) in TRACK-HD (rs557874766) was genome-wide significant in the meta-analysis (p=1·58 × 10−8), and encodes an aminoacid change (Pro67Ala) in MSH3. In TRACK-HD, each copy of the minor allele at this SNP was associated with a 0·4 units per year (95% CI 0·16–0·66) reduction in the rate of change of the Unified Huntington's Disease Rating Scale (UHDRS) Total Motor Score, and a reduction of 0·12 units per year (95% CI 0·06–0·18) in the rate of change of UHDRS Total Functional Capacity score. These associations remained significant after adjusting for age of onset. Interpretation The multidomain progression measure in TRACK-HD was associated with a functional variant that was genome-wide significant in our meta-analysis. The association in only 216 participants implies that the progression measure is a sensitive reflection of disease burden, that the effect size at this locus is large, or both. Knockout of Msh3 reduces somatic expansion in Huntington's disease mouse models, suggesting this mechanism as an area for future therapeutic investigation

Serum pro-inflammatory cytokine analysis.

<p>Data represent median (25^th–75^th percentile). n = 9 for WT or TSP deficient animals at 12 hours after CLP; all pg/ml except HMGB1 in ng/ml.</p

TSP-1 deficient mice are resistant to surgical sepsis-induced mortality, and have a lower peritoneal bacterial load following induction of surgical peritonitis.

<p>WT and TSP-1−/− mice were subjected to either CLP or sham surgery as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019654#s4" target="_blank">Materials and Methods</a> on Day 0. A. Mice were monitored for a course of 7 days for survival (p<0.001 by Kaplan-Meier, n = 20 WT CLP, n = 20 TSP-1−/− CLP). All mice receiving sham surgeries survived until study end (7 days) (n = 5 WT sham, n = 6 TSP-1−/−sham). B. Mice underwent CLP surgery at hour 0 and were euthanized at various time points after surgery. Sterile peritoneal lavage was performed and colony-forming unit counts were performed on the lavage fluid from mice 3, 6, 12, and 24 hours after the CLP procedure. Data represents 3 mice per time point, and error bars are +/−SEM (*p<0.05, for 6, 12 and 24 hours after CLP, ANOVA). C. Spleens were harvested under sterile conditions from WT and TSP-1−/−mice 3 hours post-CLP. Organs were then crushed under sterile conditions. Data represents colony-forming unit counts from total organ protein of 3 mice per genotype and error bars are +/−SEM (*p = 0.04).</p

TSP-1 deficient and WT mice exhibit similar amounts of wound healing markers after CLP.

<p>CLP surgery was performed on WT and TSP-1−/− mice, and cecums were harvested 3, 6, and 12 hours afterwards and immediately processed for protein and RNA assessment [CTGF (A) and VEGF (B) and collagen I (E)]. [For all panels, WT (black bars) and TSP-1−/− (white bars); error bars represent ±SEM] A. Cecal CTGF transcript is shown for each timepoint after surgery. Data represents n = 6 for WT and TSP-1−/− at each timepoint. B. Cecal VEGF transcript is shown for each timepoint after surgery. Data represents n = 5 for 3 hours, n = 6 for 6 hours and n = 3 for 12 hours. C. Cecal protein lysates were assessed via ELISA for active TGFβ1. Data represents active TGFβ1 in 50 µg total protein/sample (n = 6). D. Cecums were harvested at 12 and 24 hours after CLP and collagen measured via Sircoll Assay. Data represents n = 3 for all timepoints. E. Cecal collagen I transcript is shown for each timepoint after surgery. Data represents n = 6 for background and timepoint. F. WT and TSP-1 −/− fibroblasts were compared in their ability to close a mechanical scratch <i>in vitro</i> by scratch closure assay. The mechanical defect was photographed hourly for 48 hours, and unclosed scratch area (“wound” size) was determined using Photoshop. Data points represent the “wound” size at each time point for each background [n = 4 for WT (black squares) and n = 5 for TSP-1−/− (white squares)].</p

Mice containing functional TSP-1 protein have decreased phagocytic capacity as compared to mice lacking functional TSP-1, and show increased survival in a non-surgical model of sepsis.

<p>BMMs were co-incubated with IgG-opsonized, labeled sheep red blood cells and phagocytic index was determined via blinded counting of ingested red blood cells per 100 macrophages. Phase contrast images of BMMs overlayed with images of SRBCs ingested are shown for WT mice (A) and TSP-1−/− mice (B). C. The quantification of phagocytic capacity is indicated by phagocytic index. Images and Data represent n = 3 for WT <u>animals </u>and n = 3 for TSP-1−/− <u>animals</u>, and error bars are +/– SEM (*p<0.01 by student's t test). D. WT and TSP-1−/− mice were subjected to either <i>E.coli</i> or PBS injection as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019654#s4" target="_blank">Materials and Methods</a> on Day 0. Mice were monitored for a course of 7 days for survival (p<0.001 by Kaplan-Meier, n = 40 WT CLP, n = 40 TSP-1−/− <i>e.coli</i> injection). All mice receiving sham surgeries lived the course of 7 days (n = 5 WT PBS, n = 5 TSP-1−/− PBS).</p

Peritoneal lavage fluid cell counts after CLP.

<p>Data represent mean ± SEM. n = 3 for WT at 3,6,12 hours, n = 3 for TSP-1−/− at 3,6,12 hours.</p