Search

Background Chronic liver injury triggers a progenitor cell repair response, and liver fibrosis occurs when repair becomes deregulated. Previously, we reported that reactivation of the hedgehog pathway promotes fibrogenic liver repair. Osteopontin (OPN) is a hedgehogtarget, and a cytokine that is highly upregulated in fibrotic tissues, and regulates stem-cell fate. Thus, we hypothesised that OPN may modulate liver progenitor cell response, and thereby, modulate fibrotic outcomes. We further evaluated the impact of OPN-neutralisation on murine liver fibrosis. Methods Liver progenitors (603B and bipotential mouse oval liver) were treated with OPN-neutralising aptamers in the presence or absence of transforming growth factor (TGF)-ß, to determine if (and how) OPN modulates liver progenitor function. Effects of OPNneutralisation (using OPN-aptamers or OPN-neutralising antibodies) on liver progenitor cell response and fibrogenesis were assessed in three models of liver fibrosis (carbon tetrachloride, methionine-choline deficient diet, 3,5,-diethoxycarbonyl-1,4-dihydrocollidine diet) by quantitative real time (qRT) PCR, Sirius-Red staining, hydroxyproline assay, and semiquantitative doubleimmunohistochemistry. Finally, OPN expression and liver progenitor response were corroborated in liver tissues obtained from patients with chronic liver disease. Results OPN is overexpressed by liver progenitors in humans and mice. In cultured progenitors, OPN enhances viability and wound healing by modulating TGF-â signalling. In vivo, OPN-neutralisation attenuates the liver progenitor cell response, reverses epithelial-mesenchymaltransition in Sox9+ cells, and abrogates liver fibrogenesis. Conclusions OPN upregulation during liver injury is a conserved repair response, and influences liver progenitor cell function. OPN-neutralisation abrogates the liver progenitor cell response and fibrogenesis in mouse models of liver fibrosis. [ABSTRACT FROM AUTHOR]

Hepatitis GB virus-C (HGBV-C)/hepatitis G virus (HGV) infection was investigated in 106 children with liver disease (54 boys and 52 girls, mean age 7.3 years); 12 with chronic hepatitis C virus infection, 29 with positive hepatitis B surface antigen, nine with idiopathic fulminant hepatic failure, seven with graft dysfunction after liver transplantation associated with autoimmune features, 20 with cryptogenic liver disease, and 29 with autoimmune liver disease. HGV RNA detected by reverse transcription polymerase chain reaction was found to be positive in 4/106 patients (3.8%). Risk factors were identified in three patients, including blood transfusion and/or medical treatment in Eastern Europe. The prevalence was higher than that of blood donors but lower than that of 2 adult patients with liver disease. HGV is not associated with any specific disease group and does not seem to be a major aetiological agent of liver disease in childhood in the UK. [ABSTRACT FROM AUTHOR]

Introduction Hepatitis B virus employs a variety of strategies aimed at overwhelming, evading or neutralising the host immune response to infection resulting in chronicity. We have previously shown that the Programmed Cell Death Pathway (PD-1/PD-L1) is an inhibitory T-cell pathway implicated in the homeostasis of immune responses and the balance between cytolytic and non-cytolytic CD8+ T-cell effector functions. Aim The aim of this study was to investigate the impact of hepatitis B virus (HBV) infection on hepatocytic PD-L1 expression. Method A human hepatoma cell line that constitutively expresses HBV-DNA (HepG2215), its parent cell line (HepG2) were cultured. A human hepatoma cell line (Huh7) was transfected with a plasmid containing an HBV head-to-tail dimer using Fugene 6 reagent. We also cultured a further HepG2 cell line (AD38) that produces full infectious virus under the control of a tetracycline (Tet)-responsive promoter. HBV-DNA and PD-L1 were quantitated longitudinally. Intracellular and secreted HBV-DNA was quantified with qRT-PCR. PD-1/PD-L1 expression was assessed by FACS and qRT-PCR. Co-cultures between virus-specific CD8+ T-cell lines and hepatocytes producing HBV were also established and analysis of T cell functions performed. Results The hepatoma cell lines which constitutively produce HBV virions (HepG2215) had significantly higher basal levels of PD-L1 expression compared with their parent cell line (HepG2) (p=0.01). A significant increase in intracellular and secreted HBV-DNA levels confirmed successful transfection of Hepatitis B virus. Following transfection there was a significant increase in PD-L1 levels (p=0.01) on infected hepatocytes, which was not observed following transfection with an empty vector. A significant correlation was observed between PD-L1 expression and both intracellular HBV-DNA (r=0.98, p=0.01) and secreted HBV-DNA (r=0.908, p=0.046) following transfection. Following activation of HBV-DNA expression in the AD38 cell line (-Tet), PD-L1 expression increased. Moreover, subsequent fluctuations in HBV-DNA in the absence/presence of Tet was temporally associated with the expression of PD-L1 (r=0.83, p<0.001). Hyperexpression of PD-L1 on hepatocytes was associated with a predominance of non-cytolytic T cell functions. Conclusion These results demonstrate that HBV-DNA drives PD-L1 expression on infected hepatocytes. As we have previously demonstrated, upregulation of PD-L1 impairs adaptive immune responses to HBV infection, and this novel function of HBV may reflect an important strategy by which hepatitis B virus extends the life-span of target hepatocytes and escapes an effective immune response contributing to the development of chronicity. [ABSTRACT FROM PUBLISHER]

Introduction Efforts to identify the immune-correlates responsible for resolution of hepatitis C virus (HCV) infection are fundamental to develop new treatment strategies and an effective vaccine against HCV. We have previously shown that imbalanced natural killer (NK)-cell subsets, with hyper-expression of co-inhibitory markers, are associated with chronic HCV infection. Aim In this study we aim to assess the role of NK-cells in determining the outcome of acute HCV infection in a cohort of well characterised patients. Method We analysed 12 patients with acute HCV infection who met the following criteria: ALT>10xULN, exposure to HCV within previous 4 months and HCV-RNA(+). Viral load was determined by qPCR. Peripheral blood mononuclear cells collected at 3 time-points (baseline, BL; month 1, M1; month 6, M6) were stained with fluorochrome-labelled antibodies to NK-cells (CD3/CD56/CD16). Proportions of CD56dim(CD16bright) and CD56bright(CD16dim) subsets and expression of PD-1/PD-L1 were evaluated by 6-colour flow cytometry and correlated with HCV-RNA and ALT at each time-point and over-time. Supernatants from cell-cultures in the presence of HCV-antigens were collected for cytokine analysis and quantification. Results Six patients resolved HCV spontaneously (Resolvers), whilst six developed chronic infection (Chronics). At presentation, mean viraemia and ALT levels did not differ between Resolved and Chronic patients. In Resolvers HCV-RNA became undetectable at M3, which was then followed by ALT normalisation. Overall, Resolvers had higher proportions of total NKs than Chronics (p=0.023). Cytotoxic CD56dim NK cells were also higher in Resolvers (p=0.001), and became progressively predominant within their total NK pool, as shown by their progressive increase of the CD56dim/CD56bright ratio, which differentiated Resolvers from Chronics after M1 (p=0.01). In Chronic patients’ cytotoxic CD56dim NK cells had, however, an overall greater expression of the cytotoxicity marker CD16 (p=0.008). Chronic patients also had higher proportions of both subsets of NK cells expressing the immunoinhibitory marker PD-1 (p=0.02), and stronger per-cell expression of the immunoinhibitory ligand PD-L1 on CD56dim NK cells (p=0.001). Analysis of clinical parameters revealed that in Resolvers the progressive increase of CD56dim/CD56bright ratio correlated with the decline of HCV-RNA over-time (r=−0.997, p=0.042), due to reduction of CD56bright (r=0.999, p=0.011) and expansion of CD56dim (r=−0.997, p=0.041). At M1, Resolvers’ CD56dim were positively correlated with ALT (r=0.820, p=0.046). Conclusion In patients with acute HCV infection the proportions and evolution of the two functionally distinct NK cell subsets differ in patients that resolve the infection compared to those who become chronically infected. A favourable outcome of infection is associated with the establishment of a defined NK profile, with predominance of CD56dim (cytotoxic) NK cells with low expression of the immunoinhibitory markers PD-1 and PD-L1, which may be linked to an improved early clearance of virus-infected hepatocytes, as shown by the correlation of this subset with serum HCV-RNA and ALT decline. [ABSTRACT FROM PUBLISHER]