Cloning and expression of retinoic acid-induced gene-I and its effect on hepatitis C virus replication.

OBJECTIVE: To explore the influence of the retinoic acid indicible gene-I (RIG-I) on hepatitis C virus (HCV) replication and the molecular mechanism of action of RIG-I. METHODS: We constructed an RIG-I expression vector and co-transfected it into Huh-7 cells along with HCV-replicon RNA. We assayed HCV replication and NS5A protein synthesis via real-time polymerase chain reaction (RT-PCR) and western blotting. Also, we performed an enzyme-linked immunosorbent assay (ELISA) to measure the level of interferon (IFN)-alpha/-beta secretion. Additionally, we examined, via western blotting, the phosphorylation state of p38, Erk1/2, and nuclear factor (NF)-kappaB p65. RESULTS: Overexpression of RIG-1 in Huh-7 cells co-transfected with an HCV-replicon RNA significantly inhibited HCV replication and NS5A protein synthesis. Co-transfected cells had increased production of IFN-alpha/-beta production and had higher levels of phosphorylated p38, Erk1/2, and NF-kappaB p65. CONCLUSIONS: RIG-I significantly inhibits HCV replication and NS5A protein synthesis by inducing type I IFN production. The underlying molecular mechanism for this effect appears to be mediated by increased phosphorylation of NF-kappaB p65, p38-mitogen-activated protein kinases (MAPK), and Erk1/2.

The protective effect of intrasplenic transplantation of Ad-IL-18BP/IL-4 gene-modified fetal hepatocytes on ConA-induced hepatitis in mice.

BACKGROUND: Concanavalin A (ConA)-induced hepatitis is an experimental murine model mirroring the pathology of human autoimmune hepatitis. AIM: To investigate the effects of intrasplenically transplanted fetal hepatocytes (BNL.CL2) transfected with recombinant adenovirus vector expressing the IL-18 binding protein (IL-18BP) and IL-4 fusion protein on ConA-induced hepatitis in mice. METHODS: Ad-IL-18BP/IL-4 was used to infect BNL.CL2 cells. IL-4 and IL-18BP fusion protein expression were detected by ELISA and Western blotting. BNL.CL2 cells infected with Ad-IL-18BP/IL-4 were intrasplenically transplanted into mice. After 10 days, mice were injected with ConA (15 mg/kg), and sacrificed 18 hours later. Liver injury was assessed by serum transaminase and liver histology. TNF-α, IL-18, IL-4, IL-10, IL-12p70 and monocyte-chemoattracting protein (MCP)-1 levels in serum and liver homogenates were detected by ELISA. Signaling molecules in liver homogenates were analyzed by Western blotting. RESULTS: Ad-IL-18BP/IL-4 effectively expressed the IL-18BP/IL-4 fusion protein for more than 14 days in BNL.CL12 cells. Treatment of mice with Ad-IL-18BP/IL-4-BNL.CL2 before ConA injection significantly reduced the elevated plasma levels of transaminases compared with ConA control groups. TNF-α, IL-18, IL-12p70 and MCP-1 levels in serum and liver homogenates from mice transplanted with Ad-IL-18BP/IL-4-BNL.CL2 were lower and IL-4 and IL-10 levels were higher than control groups. Phosphorylation levels of NF-κB p65, AKT, p38 and JNK1/2 in liver homogenates were markedly suppressed by Ad-IL-18BP/IL-4. CONCLUSIONS: Ad-IL-18BP/IL-4 was effectively transfected into mouse BNL.CL2 cells. Intrasplenic transplantation of Ad-IL-18BP/IL-4-BNL.CL12 cells alleviated the severity of inflammation in ConA-induced experimental hepatitis and provides a useful basis for the targeted gene therapy of liver disease.

[An analysis of CD3+CD56+ lymphocytes and their subsets in the peripheral blood of patients with chronic hepatitis B].

OBJECTIVES: To investigate CD3+CD56+ lymphocytes and their subsets in the peripheral blood of chronic hepatitis B patients and to explore the relationship between these cells and the pathogenesis of their diseases. METHODS: Blood samples from 53 chronic hepatitis B patients, 17 from HBV asymptomatic carriers (ASC) and 19 from healthy controls (HC) were collected. CD3+CD56+ lymphocytes were detected by flow cytometry (FCM), then the CD3+CD56+ lymphocytes were gathered to analyze their expressions of CD4, CD8, TCR Valpha24, TCRalpha/beta and TCRgamma/delta. RESULTS: The number of CD3+CD56+ lymphocytes of chronic hepatitis B patients (7.4+/-4.6%) was more than those of ASC (4.5%+/-3.5%) and healthy controls (4.4%+/-3.7%). The expressions of TCR Valpha24 on CD3+CD56+ lymphocytes showed no significant differences among the three groups, but the expression of TCR Valpha24 on CD3-CD56+ lymphocytes of ASC ( 2.8%+/-1.4% ) was much more than that of the HC (1.7%+/-1.0%). For the subsets analysis, the CD8 and TCRalpha/beta subsets of CD3+CD56+ lymphocytes of chronic hepatitis B (61.9%+/-16.8% and 68.1%+/-16.9%) were significantly higher than those of the HC (49.2%+/-15.6% and 56.4%+/-17.9%), while the TCRgamma/delta subsets of chronic hepatitis B and ASC (29.6%+/-15.4% and 30.5%+/-14.8%) were decreased significantly than those of the HC (41.4%+/-19.4%). On the other hand, the CD8 and TCRalpha/beta subsets of CD3+CD56+ lymphocytes of severe chronic hepatitis B (69.0%+/-14.0% and 76.1%+/-12.9%) and CD8 subsets of moderate chronic hepatitis B patients (66.4%+/-14.9%) were significantly higher than those of the mild chronic hepatitis B patients (51.4%+/-16.2% and 62.1%+/-14.6%). CONCLUSION: The pathogenesis of chronic hepatitis B may positively relate to the high expression of CD8 on the CD3+CD56+ lymphocytes.

HBV genotype characterization and distribution in patients with HBV-related liver diseases in Zhejiang Province, P.R. China: possible association of co-infection with disease prevalence and severity.

BACKGROUND: There are 8 well-documented genotypes of hepatitis B virus (HBV) at this time point. Genotyping can be accomplished based on a partial sequence of hepatitis B virus (HBV) genome such as the pre-S or S gene. Several methods have been developed and used for HBV genotyping including direct sequencing, restriction fragment length polymorphism, line probe assay and enzyme-linked immunoassay. Recently, a novel, rapid and cost-effective genotyping method based on PCR amplification assay using type-specific primers that can identify all six major genotypes has been developed. This study was undertaken to characterise HBV genotypes and investigate the association between the prevalence of different genotypes and the severity of HBV-induced liver diseases. METHODS: Serum samples from carriers of HBV and patients with HBV-related liver diseases from Zhejiang Province were screened for viral serological markers using commercially available radioimmunoassay (RIA) and enzyme linked immunosorbent assay (ELISA) kits. Serum HBV DNA load was determined by real-time detection PCR. A type-specific primer based the nested-PCR method was employed in the HBV genotyping. The genotype results obtained were confirmed by direct sequencing of nested PCR amplicons of the pre-S region. Ten samples of each genotype (B and C) were sequenced. RESULTS: The survey on a cohort of 125 HBV carriers in and around Hangzhou City, Zhejiang Province showed the existence of HBV genotypes A (0.8%), B (48%), C (40.8%), D (0.8%), mixed B and C (9.6%) and an absence of E and F genotypes. Distribution of HBV genotypes in patients with liver diseases revealed a statistically insignificant higher prevalence of genotype B in mild chronic hepatitis (CH). Among the three genotypes B, C and mixed B/C infections 11 (73.3%), 3 (20%) and 1 (6.7%), (P<0.05), respectively in subjects with moderate CH, genotype B was significantly predominant. The infection patterns for genotypes B, C and B/C mixed in (i) liver cirrhosis (LC) 4 (23.5%), 10 (58.8%) and 3 (17.7%) and (ii) hepatocellular carcinoma (HCC) 2 (28.6%), 5 (71.4%) and 0 (0.0%) respectively revealed a marked association of C genotype with liver disease; however, the association was statistically insignificant (P>0.05). Differences in positive rate of HBeAg for the three genotypes B, 16 (30.8%), C, 27 (51.9%), and mixed B/C, 9 (17.3%) were significant (P<0.05), with genotype C showing predominance. CONCLUSIONS: These findings show an interesting distribution of HBV A-D genotypes in Zhejiang Province. Furthermore, our results indicate a novel and markedly high prevalence of mixed B/C genotype infections in subjects with severe CH and LC, and a possible association of mixed B/C infections with the severity of liver diseases in this region of Mainland China.

Association between TRAIL expression on peripheral blood lymphocytes and liver damage in chronic hepatitis B.

AIM: To explore a novel mechanism for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), upregulation of CD4(+) and CD8(+) T lymphocytes participating in the patho-physiological process of chronic hepatitis B (CHB). METHODS: The levels of serum soluble TRAIL (sTRAIL), serum IFN-gamma and membrane-bound TRAIL expression on peripheral leucocytes from 58 CHB patients were examined by ELISA and flow cytometry respectively. The levels of TRAIL were compared with the baseline levels of 17 healthy controls, and correlation analysis was performed between ALT, TBIL, PT, morphological change in hepatic tissues, and serum IFN-gamma. RESULTS: The results showed that TRAIL levels on membranes of CD4(+), CD8(+) T cells in CHB patients were much higher than those in healthy controls (P<0.001), and were correlated with serum TBIL (r = 0.354, P = 0.008 for CD4(+) and r = 0.522, P = 0.000 for CD8(+), respectively), ALT (r = 0.393, P = 0.003 for CD8(+)), PT (r = 0.385, P = 0.004 for CD8(+)) and serum IFN-gamma level (r = 0.302, P = 0.011 for CD4(+) and r = 0.307, P = 0.009 for CD8(+)). On the contrary to membrane-bound TRAIL expression, serum level of sTRAIL was not correlated with that of TBIL and PT, though it was higher than that of the normal population and was positively correlated with serum HBeAg expression (r = 0.695, P = 0.001). CONCLUSION: The expression level of TRAIL on the membrane of lymphocytes was upregulated and associated with the liver injury in CHB patients. These findings suggest that upregulation of TRAIL expression may be induced by virus antigen and inflammatory cytokine IFN-gamma.

[Involvement of TRAIL up-regulation of CD4+, CD8+ T cells in liver injury in chronic hepatitis B].

OBJECTIVE: To describe a novel mechanism for TRAIL up-regulation of CD4+, CD8+ T cells to participate in the pathophysiological process in patients with chronic hepatitis B (CHB). METHODS: The serum levels of soluble TRAIL (sTRAIL), IFN-gamma and membrane bound TRAIL expression on peripheral leucocytes from 58 CHB patients were examined by ELISA and flow cytometry respectively. The levels of TRAIL were compared with the baseline levels of 15 healthy controls, and correlation analysis were performed between ALT, TBil and PT, morphological change in hepatic tissues. RESULTS: The results showed that TRAIL levels on membranes of CD4+, CD8+ T cells in CHB patients were much higher than the healthy controls (P < 0.001), which of CD4+ T cells positively correlated with serum TBil (r=0.354, P = 0.008), Serum IFN-gamma level (r=0.302, P = 0.011) and which of CD8+ T cells positively correlated with serum TBil (r=0.522, P = 0.000), ALT (r=0.393, P = 0.003), PT (r=0.385, P = 0.004), serum IFN-gamma level (r=0.307, P = 0.009). The serum levels of soluble TRAIL only correlated with serum HBeAg expression (r=0.695, P = 0.001). CONCLUSION: These findings suggest that the expression of TRAIL on the membranes of lymphocytes was up-regulated, which may take part in the immunopathogenesis in CHB patients. TRAIL expression can be induced either by virus-specific protein expression or by inflammation cytokine IFN-gamma

[Influence of HGV super-infected with HIV or HCV on the virus replication].

OBJECTIVE: To realize human immunodeficiency virus(HIV) and hepatitis C virus(HCV) super-infected with hepatitis G virus(HGV or GBV/C) and to probe into the mechanism of these virus infection in the body. METHODS: HIV and HCV load were tested by the quantitated RT-PCR in the HIV or HCV infected plasma samples respectively and the HGV RNA was detected in all of the samples. Then some of the HGV positive were sequenced. RESULTS: 123 of 317 HIV patients were positive for HGV, the positive rate was 38.8%. Among the 91 HCV patients, 19 were positive for HGV. The positive rate is 20.9% which was less than that of HIV patients. HIV load of the patients super-infected with HGV was less than that of those without HGV[(1.8+/-0.6)x10 copies/ml compared with (1.9+/-1.1)x10(2)copies/ml]; while HGV and HCV super-infection did not influence the HCV RNA load significantly [(1.5+/-0.6)x10(4) copies/ml compared with (5.4+/-1.8)x10(4)copies/ml]. The HGV sequences from HIV or HCV patients were compared and showed no difference markedly. CONCLUSION: The rate of the HIV and HGV super-infection is higher than that of HCV. HGV may inhibit HIV reproduction in the body while superinfection.

[The effect of mycophenolic acid on activation antigen expression and in vitro T lymphocytes proliferation in peripheral blood].

OBJECTIVE: To explore the effect of mycophenolic acid (MPA) on activation and proliferation of T lymphocytes in vitro. METHODS: Peripheral blood mononuclear cells (PBMNC) were prepared from normal donors, and incubated with phytohemagglutinin (PHA) with or without MPA and cyclosporin A (CSA) in vitro. After incubation, the expression of T lymphocytes activation markers such as CD(69), CD(25) and CD(3) on cell surface was assayed with flow cytometry. At the same time, incorporation rate of bromodeoxyuridine (BrdU), an analog of thymidine, was detected for T lymphocytes proliferation by flow cytometry. RESULTS: (1) Both MPA and CSA could significantly inhibit the expression of CD(3) on T lymphocyte surface. After 96 hours, the expression of CD(3) in MPA and CSA group was (37.60 +/- 7.89)% and (55.85 +/- 13.64)% respectively, while that of the control group was (74.20 +/- 7.51)%. (2) The expression of CD(3) was still significantly inhibited when MPA was added to PBMNC which was prior incubated with PHA for 72 hours. (3) MPA, CSA did not affect the expression of CD(69) on T lymphocyte surface after 24 hours. (4) Both MPA and CSA could significantly inhibit the expression of CD(25) on T lymphocyte surface. After 72 hours, the expression of CD(25) in MPA group and CSA group was (37.15 +/- 7.15)% and (62.47 +/- 12.50)% respectively, while that of the control group was (84.85 +/- 8.46)%. (5) MPA (10(-5) mol/L) significantly inhibited the incorporation of BrdU in PHA activated PBMNC (9.77 +/- 7.55)% VS (43.27 +/- 18.85)% in control (P = 0.046). Cell cycle analysis showed that MPA decreased especially the percentage of S phase cells, while the percentage of G(0)/G(1) phase cells was comparatively increased and that of G(2)/M phase cells was not changed. CONCLUSIONS: The expression of CD(3) and CD(25) was inhibited by MPA. The expression of CD(3) could be suppressed by MPA even if MPA was added to PBMNC after PHA. It is suggested that MPA could suppress T lymphocyte activation and could also reverse T lymphocyte activation. MPA could suppress T lymphocyte proliferation by down-regulating the DNA synthesis. MPA decreased especially the percentage of S phase cells decreased and increased comparatively the percentage of G(0)/G(1) phase and G(2)/M phase cells.