Dynamic changes in PD-1, TLR3, and TLR4 surface expression on peripheral blood mononuclear cells in chronic hepatitis C patients undergoing peg-IFNalpha-2a plus ribavirin combination therapy / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the dynamic changes in expression of programmed death (PD)-1, Toll-like receptor (TLR)3, and TLR4 on the surface of peripheral blood mononuclear cells (PBMCs) in patients with chronic hepatitis C (CHC) that occur in response to pegylated-interferon alpha-2a (peg-IFNalpha-2a) plus ribavirin (RBV) combination therapy, and to analyze the relation to achievement of sustained virological response (SVR). METHODS Twenty-three CHC patients and 10 healthy controls were enrolled in the study. All CHC patients underwent 48 weeks of combination therapy with peg-IFNalpha-2a (180 microg, subcutaneous injection, once weekly) plus RBV (15 microg/kg, oral, once daily). Total PBMCs were isolated from both groups (CHC patients at treatment week 0, 12, 24, and 48 and post-treatment week 24; controls at enrollment) and subjected to flow cytometric analysis of PD-1, TLR3, and TLR4 surface expression. In addition, serum levels of alanine aminotransferase (ALT) and hepatitis C virus (HCV) RNA levels were analyzed by enzymatic assay and the AmpliPrep/COBAS (Roche) nucleic acid amplification test, respectively. SVR was defined as undetectable levels of HCV RNA at post-treatment week 24. Intergroup differences were assessed by one-way ANOVA.</p><p><b>RESULTS</b>The expression ratios of PD-1, TLR4 and PD-1: TLR4 on PBMCs were significantly higher in CHC patients before therapy than in the healthy controls (45.20 +/- 7.12% vs. 16.82 +/- 4.13%, 58.45 +/- 15.13% vs. 21.09 +/- 2.89%, and 35.54 +/- 7.69% vs. 14.12 +/- 2.89%; all P < 0.05). In contrast, the expression ratios of TLR3 and PD-1:TLR3 were slightly, but not significantly, higher in CHC patients before therapy than in the healthy controls (P > 0.05). During the course of peg-IFNalpha-2a plus RBV combination therapy, the expression ratios of PD-1 and TLR4 on PBMCs showed a decreasing trend, while TLR3 expression showed an increasing trend. Furthermore, CHB patients who achieved SVR at post-treatment week 24 had a significantly different expression ratio of PD-1 and TLR3 than those who did not achieve SVR (P < 0.05).</p><p><b>CONCLUSION</b>Surface expression of PD-1, TLR4, and PD-1:TLR4 is up-regulated in the total PBMCs of CHC patients. Peg-IFNalpha-2a plus RBV treatment-induced suppression of HCV replication results in a significant reduction in PD-1 and TLR4 expression on the surface of PBMCs, but a remarkably elevated level of TLR3 expression. The dynamic change in PD-1 and TLR3 expression on PBMCs that occurs during antiviral therapy may be related to achievement of SVR.</p>

Hepatitis C virus strain JFH1 down-regulates expression of growth arrest and DNA damage-inducible gene 45a in human hepatoma Huh7.5.1 cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the effect of hepatitis C virus (HCV) strain JFH1 on expression of the human gene, growth arrest and DNA damage-inducible gene 45 alpha (GADD45a), in infected hepatoma cells.</p><p><b>METHODS</b>HCV JFH1 RNA-containing supernatants were used to infect the human hepatoma cell line, Huh7.5.1; infection was confirmed by Western blot detection of the HCV-encoded non-structural 5A (NS5A) protein and core protein. Infection-induced changes in GADD45a mRNA and protein expressions were measured by real time PCR using SYBR Green and Western blotting, respectively. Significance of differences between the levels detected in JFH1-infected or uninfected Huh7.5.1 cells was analyzed by single factor analysis of variance testing.</p><p><b>RESULTS</b>The HCV infection system was successfully established, as evidenced by expression of NS5A protein and core protein. The GADD45a mRNA and protein levels were significantly down-regulated in JFH1-infected Huh7.5.1 cells, by 0.57+/-0.09 and 0.28+/-0.03, respectively, as compared to levels in uninfected Huh7.5.1 cells (F values were 75.407 and 560.04, respectively; P less than 0.01).</p><p><b>CONCLUSION</b>HCV inhibits the mRNA transcription and protein expression of host GADD45a, which may contribute to the pathogenesis of hepatocellular carcinoma caused by HCV infection.</p>

HCV NS5A protein down-regulates hepcidin gene expression and increases hepatic intracellular iron storage / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate whether the nonstructural protein 5A (NS5A) encoded by the hepatitis C virus RNA genome affects the expression of hepcidin gene.</p><p><b>METHODS</b>HCV NS5A expression plasmid (pCN5A) and pRc/CMV were transfected into QSG7701 cells individually, RT-PCR was employed to detect the HCV NS5A and hepcidin mRNA transcription. Western blot was used for detection of HCV NS5A and hepcidin proteins. Iron was stained to evaluate the intracellular iron level.</p><p><b>RESULTS</b>HCV NS5A plasmid was successfully transfected into QSG7701 cells, which was evidenced by HCV NS5A mRNA and protein from the transfected cells. The hepcidin mRNA relative quantification in untransfected cells, pRc/CMV transfected cells and pCNS5A transfected cells were 0.711+/-0.049, 0.718+/-0.052 and 0.264+/-0.030 respectively. The transcription of hepcidin mRNA decreased remarkably in the cells transfected with pCNS5A plasmid as compared to the untransfected cells and pRc/CMV transfected cells (P less than 0.01). The level of hepcidin protein expression was found also significantly lower in the pCN5A plasmid transfected cells as compared to the untransfected cells and pRc/CMV transfected cells. The intracellular iron staining was remarkably higher in the pcNS5A transfected cells than untransfected or pRc/CMV transfected cells.</p><p><b>CONCLUSIONS</b>HCV NS5A inhibits the transcription of hepcidin mRNA and expression of hepcidin protein, inducing hepatic intracellular iron storage.</p>