94 research outputs found

    Morbillivirus Glycoprotein Expression Induces ER Stress, Alters Ca2+ Homeostasis and Results in the Release of Vasostatin

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    Although the pathology of Morbillivirus in the central nervous system (CNS) is well described, the molecular basis of neurodegenerative events still remains poorly understood. As a model to explore Morbillivirus-mediated CNS dysfunctions, we used canine distemper virus (CDV) that we inoculated into two different cell systems: a monkey cell line (Vero) and rat primary hippocampal neurons. Importantly, the recombinant CDV used in these studies not only efficiently infects both cell types but recapitulates the uncommon, non-cytolytic cell-to-cell spread mediated by virulent CDVs in brain of dogs. Here, we demonstrated that both CDV surface glycoproteins (F and H) markedly accumulated in the endoplasmic reticulum (ER). This accumulation triggered an ER stress, characterized by increased expression of the ER resident chaperon calnexin and the proapoptotic transcription factor CHOP/GADD 153. The expression of calreticulin (CRT), another ER resident chaperon critically involved in the response to misfolded proteins and in Ca2+ homeostasis, was also upregulated. Transient expression of recombinant CDV F and H surface glycoproteins in Vero cells and primary hippocampal neurons further confirmed a correlation between their accumulation in the ER, CRT upregulation, ER stress and disruption of ER Ca2+ homeostasis. Furthermore, CDV infection induced CRT fragmentation with re-localisation of a CRT amino-terminal fragment, also known as vasostatin, on the surface of infected and neighbouring non-infected cells. Altogether, these results suggest that ER stress, CRT fragmentation and re-localization on the cell surface may contribute to cytotoxic effects and ensuing cell dysfunctions triggered by Morbillivirus, a mechanism that might potentially be relevant for other neurotropic viruses

    Cold-preservation of human adult hepatocytes for liver cell therapy

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    Hepatocyte transplantation is a promising alternative therapy for the treatment of hepatic failure, hepatocellular deficiency, and genetic metabolic disorders. Hypothermic preservation of isolated human hepatocytes is potentially a simple and convenient strategy to provide on-demand hepatocytes in sufficient quantity and of the quality required for biotherapy. In this study, first we assessed how cold storage in three clinically safe preservative solutions (UW, HTS-FRS, and IGL-1) affects the viability and in vitro functionality of human hepatocytes. Then we evaluated whether such cold-preserved human hepatocytes could engraft and repopulate damaged livers in a mouse model of liver failure. Human hepatocytes showed comparable viabilities after cold preservation in the three solutions. The ability of fresh and cold-stored hepatocytes to attach to a collagen substratum and to synthesize and secrete albumin, coagulation factor VII, and urea in the medium after 3 days in culture was also equally preserved. Cold-stored hepatocytes were then transplanted in the spleen of immunodeficient mice previously infected with adenoviruses containing a thymidine kinase construct and treated with a single dose of ganciclovir to induce liver injury. Engraftment and liver repopulation were monitored over time by measuring the blood level of human albumin and by assessing the expression of specific human hepatic mRNAs and proteins in the recipient livers by RT-PCR and immunohistochemistry, respectively. Our findings show that cold-stored human hepatocytes in IGL-1 and HTS-FRS preservative solutions can survive, engraft, and proliferate in a damaged mouse liver. These results demonstrate the usefulness of human hepatocyte hypothermic preservation for cell transplantation

    MicroRNA-155 Deficiency Attenuates Liver Steatosis and Fibrosis without Reducing Inflammation in a Mouse Model of Steatohepatitis

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    BACKGROUND & AIM: MicroRNAs (miRs) regulate hepatic steatosis, inflammation and fibrosis. Fibrosis is the consequence of chronic tissue damage and inflammation. We hypothesized that deficiency of miR-155, a master regulator of inflammation, attenuates steatohepatitis and fibrosis. METHODS: Wild type (WT) and miR-155-deficient (KO) mice were fed methionine-choline-deficient (MCD) or -supplemented (MCS) control diet for 5 weeks. Liver injury, inflammation, steatosis and fibrosis were assessed. RESULTS: MCD diet resulted in steatohepatitis and increased miR-155 expression in total liver, hepatocytes and Kupffer cells. Steatosis and expression of genes involved in fatty acid metabolism were attenuated in miR-155 KO mice after MCD feeding. In contrast, miR-155 deficiency failed to attenuate inflammatory cell infiltration, nuclear factor kappa beta (NF-kappaB) activation and enhanced the expression of the pro-inflammatory cytokines tumor necrosis factor alpha (TNFalpha) and monocyte chemoattractant protein-1 (MCP1) in MCD diet-fed mice. We found a significant attenuation of apoptosis (cleaved caspase-3) and reduction in collagen and alpha smooth muscle actin (alphaSMA) levels in miR-155 KO mice compared to WTs on MCD diet. In addition, we found attenuation of platelet derived growth factor (PDGF), a pro-fibrotic cytokine; SMAD family member 3 (Smad3), a protein involved in transforming growth factor-beta (TGFbeta) signal transduction and vimentin, a mesenchymal marker and indirect indicator of epithelial-to-mesenchymal transition (EMT) in miR-155 KO mice. Nuclear binding of CCAAT enhancer binding protein beta (C/EBPbeta) a miR-155 target involved in EMT was significantly increased in miR-155 KO compared to WT mice. CONCLUSIONS: Our novel data demonstrate that miR-155 deficiency can reduce steatosis and fibrosis without decreasing inflammation in steatohepatitis

    Selective blockade of interferon-α and -β reveals their non-redundant functions in a mouse model of West Nile virus infection

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    Although type I interferons (IFNs) were first described almost 60 years ago, the ability to monitor and modulate the functional activities of the individual IFN subtypes that comprise this family has been hindered by a lack of reagents. The major type I IFNs, IFN-β and the multiple subtypes of IFN-α, are expressed widely and induce their effects on cells by interacting with a shared heterodimeric receptor (IFNAR). In the mouse, the physiologic actions of IFN-α and IFN-β have been defined using polyclonal anti-type I IFN sera, by targeting IFNAR using monoclonal antibodies or knockout mice, or using Ifnb-/- mice. However, the corresponding analysis of IFN-α has been difficult because of its polygenic nature. Herein, we describe two monoclonal antibodies (mAbs) that differentially neutralize murine IFN-β or multiple subtypes of murine IFN-α. Using these mAbs, we distinguish specific contributions of IFN-β versus IFN-α in restricting viral pathogenesis and identify IFN-α as the key mediator of the antiviral response in mice infected with West Nile virus. This study thus suggests the utility of these new reagents in dissecting the antiviral and immunomodulatory roles of IFN-β versus IFN-α in murine models of infection, immunity, and autoimmunity

    Health Behaviours, Socioeconomic Status, and Mortality: Further Analyses of the British Whitehall II and the French GAZEL Prospective Cohorts

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    Background: Differences in morbidity and mortality between socioeconomic groups constitute one of the most consistent findings of epidemiologic research. However, research on social inequalities in health has yet to provide a comprehensive understanding of the mechanisms underlying this association. In recent analysis, we showed health behaviours, assessed longitudinally over the follow-up, to explain a major proportion of the association of socioeconomic status (SES) with mortality in the British Whitehall II study. However, whether health behaviours are equally important mediators of the SES-mortality association in different cultural settings remains unknown. In the present paper, we examine this issue in Whitehall II and another prospective European cohort, the French GAZEL study.Methods and Findings: We included 9,771 participants from the Whitehall II study and 17,760 from the GAZEL study. Over the follow-up (mean 19.5 y in Whitehall II and 16.5 y in GAZEL), health behaviours (smoking, alcohol consumption, diet, and physical activity), were assessed longitudinally. Occupation (in the main analysis), education, and income (supplementary analysis) were the markers of SES. The socioeconomic gradient in smoking was greater (p < 0.001) in Whitehall II (odds ratio [OR] = 3.68, 95% confidence interval [CI] 3.11-4.36) than in GAZEL (OR = 1.33, 95% CI 1.18-1.49); this was also true for unhealthy diet (OR = 7.42, 95% CI 5.19-10.60 in Whitehall II and OR = 1.31, 95% CI 1.15-1.49 in GAZEL, p < 0.001). Socioeconomic differences in mortality were similar in the two cohorts, a hazard ratio of 1.62 (95% CI 1.28-2.05) in Whitehall II and 1.94 in GAZEL (95% CI 1.58-2.39) for lowest versus highest occupational position. Health behaviours attenuated the association of SES with mortality by 75% (95% CI 44%-149%) in Whitehall II but only by 19% (95% CI 13%-29%) in GAZEL. Analysis using education and income yielded similar results.Conclusions: Health behaviours were strong predictors of mortality in both cohorts but their association with SES was remarkably different. Thus, health behaviours are likely to be major contributors of socioeconomic differences in health only in contexts with a marked social characterisation of health behaviours

    Estimation of local and external contributions of biomass burning to PM2.5 in an industrial zone included in a large urban settlement

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    A total of 85 PM2.5 samples were collected at a site located in a large industrial zone (Porto Marghera, Venice, Italy) during a 1-year-long sampling campaign. Samples were analyzed to determine water-soluble inorganic ions, elemental and organic carbon, and levoglucosan, and results were processed to investigate the seasonal patterns, the relationship between the analyzed species, and the most probable sources by using a set of tools, including (i) conditional probability function (CPF), (ii) conditional bivariate probability function (CBPF), (iii) concentration weighted trajectory (CWT), and (iv) potential source contribution function (PSCF) analyses. Furthermore, the importance of biomass combustions to PM2.5 was also estimated. Average PM2.5 concentrations ranged between 54 and 16 μg m−3 in the cold and warm period, respectively. The mean value of total ions was 11 μg m−3 (range 1–46 μg m−3): The most abundant ion was nitrate with a share of 44 % followed by sulfate (29 %), ammonium (14 %), potassium (4 %), and chloride (4 %). Levoglucosan accounted for 1.2 % of the PM2.5 mass, and its concentration ranged from few ng m−3 in warm periods to 2.66 μg m−3 during winter. Average concentrations of levoglucosan during the cold period were higher than those found in other European urban sites. This result may indicate a great influence of biomass combustions on particulate matter pollution. Elemental and organic carbon (EC, OC) showed similar behavior, with the highest contributions during cold periods and lower during summer. The ratios between biomass burning indicators (K+, Cl−, NO3−, SO42−, levoglucosan, EC, and OC) were used as proxy for the biomass burning estimation, and the contribution to the OC and PM2.5 was also calculated by using the levoglucosan (LG)/OC and LG/PM2.5 ratios and was estimated to be 29 and 18 %, respectively
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