Hepatitis B virus-mediated effects on host expression of the proprotein convertase Furin / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the effects of hepatitis B virus (HBV) infection on the expression of Furin, an important proprotein convertase, in liver cells to provide insights towards its potential as a therapeutic target for improved antiviral efficacy.</p><p><b>METHODS</b>Furin expression was measured in human liver specimens (infected tissues from patients with chronic HBV hepatitis vs. normal tissues from healthy donors) and in hepatoma cell lines (HBV-infected HepG2.2.15 cells vs. uninfected parental cell lines HepG2) using quantitative real-time RT-PCR (for mRNA), western blotting and immunohistochemistry (for protein).</p><p><b>RESULTS</b>Compared to the uninfected tissues and cells, the HBV-infected tissue and cells showed down-regulated expression of furin at both the mRNA and protein levels. In particular, the HepG2.2.15 cells showed -50% less furin mRNA expression than the HepG2 cells and the difference was statistically significant (P less than 0.05).</p><p><b>CONCLUSION</b>HBV may suppress the host cell's expression of furin, possibly to benefit its survival and replication in the host cell.</p>

Study on a putative, proprotein convertase-cleaved product of HBV core protein in vitro / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the cleavage of HBV core protein in vivo by proprotein convertase furin or its family members and observe the intracellular localization of the putative cleaved product.</p><p><b>METHODS</b>Recombinant HBV core protein was incubated with furin under different conditions in vitro, and the reaction was checked with Western blotting. The recombinant vectors expressed the putative cleaved fragment and intact core protein (serves as control) were constructed. The stable expression cell lines were established by transfecting constructs into HepG2 cell line, for which indirect immunofluorescence staining was used by monoclonal anti-HBc against the region shared by core protein and its cleaved product .The confocal microscopy was carried out to observe the intracellular distribution.</p><p><b>RESULTS</b>HBV core protein was cleaved by furin in vitro under different tested conditions. The molecular weight of the major cleaved product just about 15,000 was in concordance with the expectation. The expressed cleaved fragment could react to the monoclonal antibody against core protein, and mainly located in cytosol in particle style just like the intact core protein.</p><p><b>CONCLUSION</b>HBV core protein can be cleaved by furin in vitro. The major cleaved product has similar antigenicity and subcellular distribution to core protein. These data suggest that proprotein convertase furin or its family members play important roles in HBV replication regulation, and the cleaved product may be involved in antiviral immunity of HBV infection. Further investigations are imperative.</p>

Relationship between SNP rs10774671 on OAS-1 gene and spontaneous HBeAg seroconversion in chronic HBV infection / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the relationship between the SNP rs10774671 on OAS-1 gene and spontaneous HBeAg seroconversion in chronic HBV infection.</p><p><b>METHODS</b>Blood samples were collected from 58 HBeAg positive, 68 anti-HBe positive patients with chronic HBV infection, and 72 normal control cases without HBV infection. Chromosomal DNA was extracted and OAS-1 gene was amplified. SNP genotyping was performed with the competitively differentiated polymerase chain reaction and enzyme immunoassay.</p><p><b>RESULTS</b>In HBeAg positive group, frequencies of genotype GG plus GA and allele G were 31.0% and 16.4%. They were 48.5% and 29.4% in anti-HBe positive group, and 50.0% and 28.4% in normal control group respectively. Differences between HBeAg positive group and anti-HBe positive group or normal control group were statistically significant. But they weren't between anti-HBe positive group and normal control group.</p><p><b>CONCLUSION</b>Allele G on SNP rs10774671 of OAS-1 gene maybe benefits patients with chronic HBV infection to achieve spontaneous HBeAg seroconversion. Genotyping on this SNP may be predicting valuable for interferon therapy for chronic HBV infection.</p>

Optimization of cultivation methods of MSCs from hepatitis B patients and investigation of their biological characteristics / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To optimize cultivation methods of bone marrow mesenchymal stem cells (MSCs) from hepatitis B patients and to investigate their biological characteristics.</p><p><b>METHODS</b>Growth curves of hepatitis B patients MSCs cultivated with five culture media and two inoculation methods were compared; the shapes, appearances, surface markers and bionomics of the cultivated MSCs were studied.</p><p><b>RESULTS</b>Inoculating the cells obtained directly from bone marrow aspirations was not as successful as using the marrow cells after their density gradient centrifugations (76% vs 88%), but the differences in the results were not statistically significant (P more than 0.05). The successful cultivation rates using five culture media were different and the differences were statistically significant (P less than 0.01). The autoserum medium was most successful, fatal bovine serum (FBS) medium was next successful and the non-patient serum medium was the least successful. The growth curves of the cultivations using the different media were parallel to this. Changing the whole culture media every 2 or 3 days was better than changing half of the media. The shapes, appearances, surface markers and the growth characteristics of MSCs from the hepatitis B patients were almost the same as MSCs from the normal adult.</p><p><b>CONCLUSION</b>The best cultivation method of MSCs from hepatitis B patients is: separating marrow cells using density gradient centrifugal separation, cultivating them using an autoserum culture medium, and completely changing the medium every 2-3 days. The biological characteristics of MSCs from the hepatitis B patients using the above methods are almost the same as those from normal adults.</p>

MxA gene-88 G/T polymorphism influences the outcomes of HBV infection / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the relationship between a G/T substitution at position -88 of myxovirus resistance-1 gene (MxA) and the self-limiting or chronic infection of HBV.</p><p><b>METHODS</b>Blood samples from 100 patients with self-limiting HBV infection (positive anti-HBs and anti-HBc) and from 340 patients with chronic HBV infection were collected. MxA-88 G/T polymorphism was typed using a protocol based on competitively differentiated-polymerase chain reaction. For statistical analysis, odds ratio and chi-square test were used.</p><p><b>RESULTS</b>The detective rate of G/G genotype (low expression genotype) of MxA-88 G/T was 50.2% (221/440), those of T/T genotype (high expression genotype) and G/T heterozygous genotype were 5.5% (24/440) and 44.3% (195/440). Compared to patients with chronic infection, patients with self-limiting infection had lower frequency of G/G genotype (41.0% vs 52.9%, P < 0.05) or G allele (62.5% vs 75.9%, P < 0.01) and had higher frequency of T/T genotype (16.0% vs 2.4%, P < 0.01) or T allele (37.5% vs 24.1%, P < 0.01), but there was no significant difference in the G/T heterozygous genotype.</p><p><b>CONCLUSIONS</b>MxA gene -88 G/T polymorphism influences the natural outcomes of HBV infection to some extent. This SNP of MxA gene may be used as a clinical prognostic marker of HBV infection.</p>

Changes of antigen-specific cytotoxic T lymphocytes during acute flare-ups in chronic hepatitis B patients / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To detect HBV antigen specific cytotoxic T lymphocyte (CTL) changes in patients during acute flare-ups and to study their association with flare-ups and aggravations into grave hepatitis by quantitative analysis of HLA-A2* restricted HBcAg-specific CTL cells.</p><p><b>METHODS</b>The frequency of HBcAg-specific CTL cells in the peripheral blood mononuclear cells (PBMC) from 29 patients with persistent infection with HBV were quantified by flow cytometry using one HLA-A2*HBV peptide pentamers complex (Pro5TM MHC Pentamers).</p><p><b>RESULTS</b>There was a statistical difference of HBcAg specific CTLs between the patients with acute exacerbations (1.4%+/-0.8%) and the patients with immune tolerance (0.6%+/-0.4%) (t = 2.180, P = 0.01-0.05); There was no significant difference between the grave hepatitis group (1.3%+/-1.0%) and the chronic hepatitis group (1.4%+/-0.8%) regarding frequencies of antigen specific CTL (t = 0.215, P = 0.833-0.05). The level of antigen specific CTLs in PBMC in the 6 cases of chronic hepatitis B with acute exacerbations maintained a relatively high level (more than 0.7%) within the 12 week follow-up period.</p><p><b>CONCLUSION</b>HBcAg-specific CTLs may play an important role in hepatic flare-ups in patients with chronic HBV infection, but there was no direct relationship between antigen- specific CTLs and grave hepatitis.</p>

Clinical trial of sequential antiviral therapy for patients with chronic hepatitis B in China / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To establish a sequential antiviral regime and evaluate its efficacy in patients with chronic hepatitis B using a controlled trial.</p><p><b>METHODS</b>Seventy-four patients with chronic hepatitis B were divided into 3 groups: 30 cases were enrolled in the sequential antiviral group in which patients received eight-week treatment with thymosin alpha1 (1.6 mg/time, subcutaneous injection, 2 times/week), six-month treatment with interferon (500 MU/ times, muscle inject, every other day) begun in the fifth week of the therapeutic course, and lamivudine treatment (100 mg/days) begun 2 months later after HBeAg seroconversion or just after the withdrawal of interferon to more than eighteen months. Fourteen cases were enrolled in combination group in which patients received six-month treatment with interferon and thymosin alpha1 simultaneously in the same manner as in sequential antiviral group. Thirty cases were enrolled in lamivudine group in which patients received more than eighteen-month treatment with lamivudine.</p><p><b>RESULTS</b>The temporary rates of HBeAg seroconversion and normalization of alanine aminotransferase (effective rate) in sequential antiviral group, combination group and lamivudine group were 76.7%, 78.6% and 13.3%, respectively. The effective rates of sequential group and combination group were very similar, and significantly higher than that of lamivudine group (P less than 0.01). Long-term efficacy rates were 76.7%, 57.1% and 16.7% among the three groups, respectively. The long-term effective rate of sequential group was relatively higher. The rate of liver damage sensitive period in sequential antiviral group and combination group was 47.7%. The time of onset was from 2 to 8 weeks after the treatment begun, earlier than that from 6 to 8 weeks after the beginning of interferon alone in the literature.</p><p><b>CONCLUSION</b>Sequential antiviral therapy had much higher rates of long-term HBeAg seroconversion, undetectable HBV DNA and normalization of alanine aminotransferase with good cost-effectiveness. Its mechanism to promote the antiviral effect might be dependent on the immunoregulatory action of thymosin alpha1 in the earlier period and the specific inhibition of HBV DNA replication by lamivudine in the later period of the therapeutic course.</p>

Initiative factors of the damage sensitive stage of hepatocytes induced by interferon in patients with chronic hepatitis B / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To probe into the initiative factors of the damage sensitive stage of hepatocytes induced by interferon in patients with chronic hepatitis B (CHB).</p><p><b>METHODS</b>Forty-four CHB patients with positive HBeAg and HBV DNA were treated with interferon. Serum ALT and viral markers levels of HBsAg, HBeAg, anti-HBc and HBV DNA were examined regularly. Liver biopsy was carried out just before the treatment.</p><p><b>RESULTS</b>The rate of HBeAg seroconversion was 75% at the sixth month, and 68.2% after one year of follow up. The rate of damage sensitive stage of hepatocytes was 47.7%. The average onset time was (3.14+-1.49) weeks after the treatment, and lasted for (8.24+-3.52) weeks. The ALT level raised (1.73+-1.13) times. The occurrence of damage sensitive stage of hepatocytes was indicator for good curative effect (Fisher exact probability, P=0.028). Damage sensitive stage of hepatocytes was more often developed in patients with moderate inflammation, overexpression of HBcAg in liver and higher level of HBeAg in blood stream before treatment. HBeAg and anti-HBc levels in peripheral blood decreased in the onset period of damage sensitive stage of hepatocytes.</p><p><b>CONCLUSIONS</b>The initiative factors of the damage sensitive stage of hepatocytes may be: HBeAg decreasing in peripheral blood induced by interferon may dismiss immune lutation of HBeAg and anti-HBc to cytotoxic T lymphocyte (CTL), which recognize HBcAg as target, thus activates the cytotoxicity of HBV-infected hepatocytes mediated by CTL.</p>