Role of Gas6 and TAM Receptors in the Identification of Cardiopulmonary Involvement in Systemic Sclerosis and Scleroderma Spectrum Disorders

Background: Few biomarkers are available for early identification of pulmonary arterial hypertension (PAH) and interstitial lung disease (ILD) in systemic sclerosis (SS) and scleroderma spectrum disorders (SSD). Aims: To evaluate Gas6, sAxl, and sMer as biomarkers for cardiopulmonary complications of SS and SSD. Methods: In a cross-sectional observational study, we recruited 125 consecutive patients, affected by SS and SSD and referred to a tertiary-level pulmonary hypertension outpatient clinic. All patients underwent a comprehensive evaluation for identification of PAH and ILD. Gas6, sMer, and sAxl concentrations were measured with ELISA protocols, and concentrations were compared according to PAH or ILD. Results: Nineteen subjects had pulmonary hypertension (PH) (14 PAH), and 39 had ILD (6 severe). Plasma sMer was increased in PAH (18.6 ng/ml IQR [11.7-20.3]) with respect to the absence (12.4 [8.0-15.8]) or other form of pulmonary hypertension (9.6 [7.4-12.5]; K-W variance p < 0.04). Conversely, Gas6 and sAxl levels were slightly increased in mild ILD (25.8 ng/ml [19.5-32.1] and 24.6 [20.1-32.5]) and reduced in severe ILD (16.6 [15.0-22.1] and 15.5 [14.9-22.4]) in comparison to no evidence of ILD (23.4 [18.8-28.1] and 21.6 [18.1-28.4]; K-W, p 64 0.05). Plasma sMer 65 19 ng/ml has 50% sensitivity and 92% specificity in PAH identification (area under the ROC curve (AUC) 0.697, p < 0.03). Values of Gas6 64 24.5 ng/ml and of sAxl 64 15.5 ng/ml have 100% and 67% sensitivity and 47% and 86% specificity, respectively, in identifying severe ILD (Gas6 AUC 0.787, p < 0.001; sAxl AUC 0.705, p < 0.05). Conclusions: The assay of Gas6 sAxl and sMer may be useful to help in the identification of PAH and ILD in SS and SSD patients. The Gas6/TAM system seems to be relevant in cardiopulmonary complications of SS and SSD and merits further investigations

Hepatitis C Virus Protects Human B Lymphocytes from Fas-Mediated Apoptosis via E2-CD81 Engagement

HCV infection is often associated with B-cell regulatory control disturbance and delayed appearance of neutralizing antibodies. CD81 is a cellular receptor for HCV and can bind to HCV envelope protein 2 (E2). CD81 also participates to form a B cell costimulatory complex. To investigate whether HCV influences B cell activation and immune function through E2 -CD81 engagement, here, human Burkitt's lymphoma cell line Raji cells and primary human B lymphocytes (PHB) were treated with HCV E2 protein and cell culture produced HCV particles (HCVcc), and then the related cell phenotypes were assayed. The results showed that both E2 and HCVcc triggered phosphorylation of IκBα, enhanced the expression of anti-apoptosis Bcl-2 family proteins, and protected Raji cells and PHB cells from Fas-mediated death. In addition, both E2 protein and HCVcc increased the expression of costimulatory molecules CD80, CD86 and CD81 itself, and decreased the expression of complement receptor CD21. The effects were dependent on E2-CD81 interaction on the cell surface, since CD81-silenced Raji cells did not respond to both treatments; and an E2 mutant that lose the CD81 binding activity, could not trigger the responses of both Raji cells and PHB cells. The effects were not associated with HCV replication in cells, for HCV pseudoparticle (HCVpp) and HCVcc failed to infect Raji cells. Hence, E2-CD81 engagement may contribute to HCV-associated B cell lymphoproliferative disorders and insufficient neutralizing antibody production

Impact of gene variants on sex-specific regulation of human Scavenger receptor class B type 1 (SR-BI) expression in liver and association with lipid levels in a population-based study

<p>Abstract</p> <p>Background</p> <p>Several studies have noted that genetic variants of <it>SCARB1</it>, a lipoprotein receptor involved in reverse cholesterol transport, are associated with serum lipid levels in a sex-dependent fashion. However, the mechanism underlying this gene by sex interaction has not been explored.</p> <p>Methods</p> <p>We utilized both epidemiological and molecular methods to study how estrogen and gene variants interact to influence <it>SCARB1 </it>expression and lipid levels. Interaction between 35 <it>SCARB1 </it>haplotype-tagged polymorphisms and endogenous estradiol levels was assessed in 498 postmenopausal Caucasian women from the population-based Rancho Bernardo Study. We further examined associated variants with overall and <it>SCARB1 </it>splice variant (SR-BI and SR-BII) expression in 91 human liver tissues using quantitative real-time PCR.</p> <p>Results</p> <p>Several variants on a haplotype block spanning intron 11 to intron 12 of <it>SCARB1 </it>showed significant gene by estradiol interaction affecting serum lipid levels, the strongest for rs838895 with HDL-cholesterol (p = 9.2 × 10^-4) and triglycerides (p = 1.3 × 10^-3) and the triglyceride:HDL cholesterol ratio (p = 2.7 × 10^-4). These same variants were associated with expression of the SR-BI isoform in a sex-specific fashion, with the strongest association found among liver tissue from 52 young women <45 years old (p = 0.002).</p> <p>Conclusions</p> <p>Estrogen and <it>SCARB1 </it>genotype may act synergistically to regulate expression of <it>SCARB1 </it>isoforms and impact serum levels of HDL cholesterol and triglycerides. This work highlights the importance of considering sex-dependent effects of gene variants on serum lipid levels.</p

Multiple effects of toxins isolated from Crotalus durissus terrificus on the hepatitis C virus life cycle

Hepatitis C virus (HCV) is one of the main causes of liver disease and transplantation worldwide. Current therapy is expensive, presents additional side effects and viral resistance has been described. Therefore, studies for developing more efficient antivirals against HCV are needed. Compounds isolated from animal venoms have shown antiviral activity against some viruses such as Dengue virus, Yellow fever virus and Measles virus. In this study, we evaluated the effect of the complex crotoxin (CX) and its subunits crotapotin (CP) and phospholipase A2 (PLA2-CB) isolated from the venom of Crotalus durissus terrificus on HCV life cycle. Huh 7.5 cells were infected with HCVcc JFH-1 strain in the presence or absence of these toxins and virus was titrated by focus formation units assay or by qPCR. Toxins were added to the cells at different time points depending on the stage of virus life cycle to be evaluated. The results showed that treatment with PLA2-CB inhibited HCV entry and replication but no effect on HCV release was observed. CX reduced virus entry and release but not replication. By treating cells with CP, an antiviral effect was observed on HCV release, the only stage inhibited by this compound. Our data demonstrated the multiple antiviral effects of toxins from animal venoms on HCV life cycle

Hepatitis C Virus Core Protein Induces Neuroimmune Activation and Potentiates Human Immunodeficiency Virus-1 Neurotoxicity

BACKGROUND: Hepatitis C virus (HCV) genomes and proteins are present in human brain tissues although the impact of HIV/HCV co-infection on neuropathogenesis remains unclear. Herein, we investigate HCV infectivity and effects on neuronal survival and neuroinflammation in conjunction with HIV infection. METHODOLOGY: Human microglia, astrocyte and neuron cultures were infected with cell culture-derived HCV or exposed to HCV core protein with or without HIV-1 infection or HIV-1 Viral Protein R (Vpr) exposure. Host immune gene expression and cell viability were measured. Patch-clamp studies of human neurons were performed in the presence or absence of HCV core protein. Neurobehavioral performance and neuropathology were examined in HIV-1 Vpr-transgenic mice in which stereotaxic intrastriatal implants of HCV core protein were performed. PRINCIPAL FINDINGS: HCV-encoded RNA as well as HCV core and non-structural 3 (NS3) proteins were detectable in human microglia and astrocytes infected with HCV. HCV core protein exposure induced expression of pro-inflammatory cytokines including interleukin-1β, interleukin-6 and tumor necrosis factor-α in microglia (p<0.05) but not in astrocytes while increased chemokine (e.g. CXCL10 and interleukin-8) expression was observed in both microglia and astrocytes (p<0.05). HCV core protein modulated neuronal membrane currents and reduced both β-III-tubulin and lipidated LC3-II expression (p<0.05). Neurons exposed to supernatants from HCV core-activated microglia exhibited reduced β-III-tubulin expression (p<0.05). HCV core protein neurotoxicity and interleukin-6 induction were potentiated by HIV-1 Vpr protein (p<0.05). HIV-1 Vpr transgenic mice implanted with HCV core protein showed gliosis, reduced neuronal counts together with diminished LC3 immunoreactivity. HCV core-implanted animals displayed neurobehavioral deficits at days 7 and 14 post-implantation (p<0.05). CONCLUSIONS: HCV core protein exposure caused neuronal injury through suppression of neuronal autophagy in addition to neuroimmune activation. The additive neurotoxic effects of HCV- and HIV-encoded proteins highlight extrahepatic mechanisms by which HCV infection worsens the disease course of HIV infection

A Novel Small Molecule Inhibitor of Hepatitis C Virus Entry

Small molecule inhibitors of hepatitis C virus (HCV) are being developed to complement or replace treatments with pegylated interferons and ribavirin, which have poor response rates and significant side effects. Resistance to these inhibitors emerges rapidly in the clinic, suggesting that successful therapy will involve combination therapy with multiple inhibitors of different targets. The entry process of HCV into hepatocytes represents another series of potential targets for therapeutic intervention, involving viral structural proteins that have not been extensively explored due to experimental limitations. To discover HCV entry inhibitors, we utilized HCV pseudoparticles (HCVpp) incorporating E1-E2 envelope proteins from a genotype 1b clinical isolate. Screening of a small molecule library identified a potent HCV-specific triazine inhibitor, EI-1. A series of HCVpp with E1-E2 sequences from various HCV isolates was used to show activity against all genotype 1a and 1b HCVpp tested, with median EC50 values of 0.134 and 0.027 µM, respectively. Time-of-addition experiments demonstrated a block in HCVpp entry, downstream of initial attachment to the cell surface, and prior to or concomitant with bafilomycin inhibition of endosomal acidification. EI-1 was equally active against cell-culture adapted HCV (HCVcc), blocking both cell-free entry and cell-to-cell transmission of virus. HCVcc with high-level resistance to EI-1 was selected by sequential passage in the presence of inhibitor, and resistance was shown to be conferred by changes to residue 719 in the carboxy-terminal transmembrane anchor region of E2, implicating this envelope protein in EI-1 susceptibility. Combinations of EI-1 with interferon, or inhibitors of NS3 or NS5A, resulted in additive to synergistic activity. These results suggest that inhibitors of HCV entry could be added to replication inhibitors and interferons already in development

Evaluation of the red cell distribution width as a biomarker of early mortality in hepatocellular carcinoma

Background: The red cell distribution width is a biomarker of early mortality across various disease states. Aim: To verify whether it may refine estimates of survival in hepatocellular carcinoma. Methods: The red cell distribution width measured at diagnosis was analyzed in relationship to mortality by any cause both in a retrospective training cohort (N = 208), and in an independent prospectively collected validation cohort (N = 106) of patients with hepatocellular carcinoma. Based on Cox proportional hazards modelling, a prognostic index was validated. Results: In the training and the validation cohort, median survival time was respectively 1026 and 868 days in patients with red cell distribution width ≤14.6%, vs. 282 and 340 days in patients with red cell distribution width >14.6%; the corresponding hazard ratios were 0.43 (95% CI: 0.31–0.60), p < 0.0001 and 0.28 (95% CI: 0.17–0.47), p < 0.0001. At multivariate analysis, the red cell distribution width remained an independent predictor of survival (p < 0.001) in a Cox model including other widely accepted prognostic factors. Applying to the validation dataset the prognostic index derived from the training dataset, the ability of the model to discriminate the survival probabilities of patients was confirmed (Harrell’s C = 0.769). Conclusions: The red cell distribution width is a novel, reproducible, prospectively validated predictor of survival in patients with hepatocellular carcinoma