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To investigate the action of isothiafludine (NZ-4), a derivative of bis-heterocycle tandem pairs from the natural product leucamide A, on the replication cycle of hepatitis B virus (HBV) in vitro and in vivo. HBV replication cycle was monitored in HepG2.2.15 cells using qPCR, qRT-PCR, and Southern and Northern blotting. HBV protein expression and capsid assembly were detected using Western blotting and native agarose gel electrophoresis analysis. The interaction of pregenomic RNA (pgRNA) and the core protein was investigated by RNA immunoprecipitation. To evaluate the anti-HBV effect of NZ-4 in vivo, DHBV-infected ducks were orally administered NZ-4 (25, 50 or 100 mg·kg−1·d−1) for 15 d. NZ-4 suppressed intracellular HBV replication in HepG2.2.15 cells with an IC50 value of 1.33 μmol/L, whereas the compound inhibited the cell viability with an IC50 value of 50.4 μmol/L. Furthermore, NZ-4 was active against the replication of various drug-resistant HBV mutants, including 3TC/ETV-dual-resistant and ADV-resistant HBV mutants. NZ-4 (5, 10, 20 μmol/L) concentration-dependently reduced the encapsidated HBV pgRNA, resulting in the assembly of replication-deficient capsids in HepG2.2.15 cells. Oral administration of NZ-4 dose-dependently inhibited DHBV DNA replication in the DHBV-infected ducks. NZ-4 inhibits HBV replication by interfering with the interaction between pgRNA and HBcAg in the capsid assembly process, thus increasing the replication-deficient HBV capsids. Such mechanism of action might provide a new therapeutic strategy to combat HBV infection.

Long-term compliance with regular surveillance is important for the prevention and timely management of chronic hepatitis B (CHB). However, there are no researches focusing on the compliance of hepatitis B virus infected patients in regular surveillance so far. The purpose of our study was to investigate the outpatient compliance with long-term regular surveillance in China. Data of 3257 CHB outpatients was pooled and analyzed to assess the outpatient's compliance with the long-term regular surveillance plan. In all outpatients, the non-follow-up and the follow-up group accounted for 73.2% and 26.8%, respectively. Among the follow-up outpatient's, only 48.9% received ongoing-follow-up and 51.1% were finally lost to follow-up; the median length of visiting duration was 25 months; and the predictive 1-, 2-, 3-, 4- and 5-year ongoing follow-up rate was 72.7%, 52.5%, 42.4%, 33.8%, and 26.3%, respectively. In conclusion, our survey proved that the regular long-term surveillance on Chinese chronic HBV carrier is difficult to be fully implemented. A large proportion of outpatients do not receive routine follow-up and are at risk of treatment delay due to various social reasons.

The pathogenesis of HBsAg (+)/HBsAb (+) double positive hepatitis B virus infection was investigated by simulating HBsAg/HBsAb coexistence in vitro and establishing HBsAg/HBsAb double positive model in vivo. Eukaryotic expression plasmids PCI-SY, PCI-adw, PCI-adr, PCI-ayw, which expressed S gene product of different serotypes, were constructed and transfected into HepG2 cells. Recombinant proteins were purified from the transfected cells. At the same time, HBsAg mouse antiserum was obtained by immunizing mice with PCI-SY plasmid. HBsAg/HBsAb coexistence was simulated using these antigens and antiserum. Furthermore, the expression plasmids expressing different serotypes of S gene product including PCI-adw, PCI-adr, and PCI-ayw were injected into mice via tail vein. HBsAg and HBsAb in mice sera were tested at the first and 7th day respectively after antigen plasmids injection. Both in vitro simulation and in vivo animal models demonstrated that HBsAg antigen and HBsAb of the same serotypes could not coexist, but HBsAg antigen and HBsAb of different serotype could coexist. HBsAg/HBsAb double positive hepatitis B virus infection could be due to infection of viruses of different serotypes.

Hepatitis B virus (HBV)-specific cytotoxic T lymphocytes (CTLs) are believed to play a major role in viral clearance and disease pathogenesis during HBV infection. To clarify the differences in host immune responses between self-limited and chronic HBV infections, we constructed three HLA-A*0201/HBV tetramers with immunodominant epitopes of core18-27, polymerase 575-583 and envelope 335-343, and analyzed the HBV-specific CTLs in peripheral blood mononuclear cells (PBMCs) from patients infected with HBV. The frequencies and expansion ability of HBV-specific CD8(+) T cells in most self-limited HBV infected individuals were higher than those in chronic HBV-infected patients. HBV-specific CD8(+) T cells could be induced by in vitro peptide stimulation from chronic patients with a low level of serum HBV-DNA but not from those with a high level of serum HBV-DNA. In chronic infection, no significant correlation was found either between the frequencies of HBV-specific CD8(+) T cells and the viral load, or between the frequencies and the levels of alanine transaminase. Our results suggested that the frequencies of HBV-specific CTLs are not the main determinant of immune-mediated protection in chronic HBV infection and immunotherapeutic approaches should be aimed at not only boosting a HBV-specific CD8(+) T response but also improving its function.

Previous studies have shown that expression of the interferon-sensitive gene (ISG)15 protease UBP43 is increased in the liver biopsy specimens of patients who do not respond to interferon (IFN)-α therapy. We hypothesized that UBP43 might hinder the ability of IFN to inhibit HBV replication. In this study, we investigated whether vector-based siRNA promoted by H1 (psiUBP43) could enhance IFN inhibiting HBV replication in cell culture. UBP43 was specifically silenced using shRNA. In HepG2.2.15 cells, the HBeAg and HBV DNA levels were significantly reduced by IFN after transfection of shRNA, imphicated that vector-based siRNA promoted by H1 (psiUBP43) could enhance IFN inhibiting HBV replication in cell culture. These data suggest that UBP43 modulates the anti-HBV type I IFN response, and is a possible therapeutic target for the treatment of HBV infection.

Immune-mediated inflammatory injury is an important feature of the disease aggravation of hepatitis B virus-related acute-on-chronic liver failure (ACLF). Toll-like receptors (TLRs) have been shown previously to play a pivotal role in the activation of innate immunity. The purpose of this study was to characterize the TLR4 expression in peripheral blood mononuclear cells (PBMCs) of ACLF patients and its possible role in the disease aggravation. Twelve healthy subjects, 15 chronic HBV-infected (CHB) patients and 15 ACLF patients were enrolled in this study. The TLR4 expression in PBMCs and T cells of all subjects was examined by real-time PCR and flow cytometry. The correlation of TLR4 expression on T cells with the markers of disease aggravation was evaluated in ACLF patients. The ability of TLR4 ligands stimulation to induce inflammatory cytokine production in ACLF patients was analyzed by flow cytometry. The results showed that TLR4 mRNA level was upregulated in PBMCs of ACLF patients compared to that in the healthy subjects and the CHB patients. Specifically, the expression of TLR4 on CD4(+) and CD8(+) T cells of PBMCs was significantly increased in ACLF patients. The TLR4 levels on CD4(+) and CD8(+) T cells were positively correlated with serum total bilirubin (TBIL), direct bilirubin (DBIL), international normalized ratio (INR) levels and white blood cells (WBCs), and negatively correlated with serum albumin (ALB) levels in the HBV-infected patients, indicating TLR4 pathway may play a role in the disease aggravation of ACLF. In vitro TLR4 ligand stimulation on PBMCs of ACLF patients induced a strong TNF-α production by CD4(+) T cells, which was also positively correlated with the serum markers for liver injury severity. It was concluded that TLR4 expression is upregulated on T cells in PBMCs, which is associated with the aggravation of ACLF.

AIM: To study the mutation in a determinant of HBV S gene, to explore how it influenced the biologic characteristics of S gene. METHODS: Through site-mutation PCR, we constructed series variant clones (T126S, M133L, T144A) of HBV S gene ‘a’ determinant according to epidemiologic data. RESULTS: After analysis of sequence and expression of cells, sequences of mutation clone were correct and the mutation of different positions in ‘a’ determinant could influence antigenicity of expressed HBsAg.

Objective To clarify the relationship between loss of expression of DPC4 proteins and pathogenesis of biliary tract carcinoma.

Background & aimsThe natural course of chronic hepatitis B virus (HBV) infection is characterized by different immune responses, ranging from immune tolerant (IT) to immune activated (IA) stages. In our study, we investigated the natural killer (NK) cells activity in patients at different immunological stages of chronic HBV infection.MethodsBlood samples obtained from 57 HBeAg positive patients with chronic hepatitis B (CHB), including 15 patients in the immune tolerant (IT) stage, 42 patients in the immune activated (IA) stage, and 18 healthy individuals (HI). The analyses included flow cytometry to detect NK cells, the determination of cytokine levels as well as of surface receptor expression and cytotoxicity.ResultsNK cells in peripheral blood were significantly lower in patients in the IA stage of CHB compared to HI (pConclusionNK cells activity was lower in CHB patients, especially in those in the IA stage.

The recombinant plasmid pCI-TSLC1 carrying TSLC1 gene was stably transfected into human hepatocellular carcinoma cell line HepG2. Cell proliferation was analyzed by MTT assay. The ability of migration was determined by transwell and FACSort flow cytometry was used to detect the cell cycle distribution and apoptosis. Western blotting revealed that H4 expressed higher amounts of TSLC1 protein than H15 and H0 did. The growth of TSLC1-transfected cells was significantly suppressed in vitro, and the ability of migration was reduced as well. The re-expression of TSLC1 could induce cell apoptosis. It was concluded that TSLC1 strongly inhibited the growth and ability of migration of HepG2 cell line in vitro and also induced apoptosis, suggesting that TSLC1 could reduce the tumorigenicity of human hepatocellular carcinoma cell line HepG2 in vitro, which provided a basis for further exploring the roles of TSLC1 in hepatocellular cellular carcinoma.

This report aims to investigate the Toll-like receptor (TLR) signaling pathways and induced antiviral activity in hepatocytes.We isolated primary hepatocytes from wild-type C57BL/6 mice and examined the expression of TLR by realtime RT-PCR. Hepatocytes were stimulated with TLR 1-9 agonists and the supernatants were harvested. The secretion of cytokines were tested by ELISA. The antiviral effectors in supernatants were assayed via virus protection assay (in EMCV system) and the control of HBV replication were assessed via Southern blotting (in HBV system).We demonstrated that hepatocytes expressed TLR1-9. In accordance with these TLR expression profiles, hepatocytes responded to all TLR ligands by producing inflammatory cytokines (TNF-α or IL-6), to TLR -1,-3,-7 and -9 ligands by producing type I IFN (IFN-α or IFN-β). Only TLR 3 and TLR 7 agonists could stimulate the production of high amounts of antiviral mediators by hepatocytes in virus protection assay. By contrast, supernatants from TLR1, -3 and -4 directly stimulated hepatocytes and TLR 3, -7 and -9 transfected hepatocytes were able to potently suppress HBV replication.Primary hepatocytes display a unique TLR signaling pathway and can control HBV replication after stimulation by TLR agonists in mice. It may be helpful for the development of TLR-based therapeutic approaches against hepatotropic virus.

This study investigated the expression profiles of IL-10 gene in three human hepatoma cell lines including Huh7, HepG2, and HepG2 transfected with a plasmid containing hepatitis B virus (HBV) named HepG2.2.15. RT-PCR analysis demonstrated that IL-10 message RNA was absent in HepG2 and Huh7 cells, whereas it was present in HepG2.2.15 cells, which was consistent with ELISA result. Furthermore, except for lamivudine other antiviral treatments did not significantly decrease the HBV DNA level in HepG2.2.15 cells, while they had different effects on the expression of IL-10 protein, although stimulation by LPS had no significant effect. In addition, except for poly(I:C), the other treatments decreased the expression of IL-10 protein to different degrees, but had no significant effects on the expression of NF-κB and MyD88. Meanwhile, all treatments we used had effect on the expression of STAT1. In conclusion, IL-10 was expressed in HepG2.2.15 cells and STAT1 pathway might be involved in the regulation of IL-10 expression in HepG2.2.15 cells, but it was not the sole pathway, the exact mechanism warrants further study.

To better understand the effect of a new split variant of human asialoglycoprotein receptor (ASGPR H1b) on ASGPR ligands’ binding ability, we established a functional cell line which expresses ASGPR. The full lengths of ASGPRH1a and H2c fragments from human liver were amplified by reverse transcript PCR (RT-PCR) and inserted into eukaryotic expression vector pIRES2EGFP, pCDNA3.1 (Zeo+) respectively. The recombinants were co-transfected into HeLa cells. After selection by using Neocin and Zeocin, a stably transfected cell line was established, which was designated 4-1-6. The transcription and expression of ASGPRH1a and H2c in 4-1-6 were confirmed by RT-PCR, Western blotting and immunofluorescence. The endocytosis function of the artificial “ASGPR” on the surface of 4-1-6 was tested by FACS. It was found that the cell line 4-1-6 could bind ASGPR natural ligand molecular asialo-orosomucoid (ASOR). After the eukaryotic plasmid H1b/pCDNA3.1 (neo) was transfected into cell line 4-1-6, H1b did not down-regulate the ligand binding ability of ASGPR. The eukaryotic expression plasmid H1b/pcDNA3.1 (neo) and H2c/pcDNA3.1 (neo) were co-transfected transiently into Hela cell. Neither single H1b nor H1b and H2c could bind ASOR. In conclusion, a functional cell line of human asialoglycoprotein receptor (ASGPR) which expresses both H1a and H2c stably was established. The new split variant H1b has no effect on ASGPR binding to ASOR. ASGPRH1b alone can’t bind to ASOR, it yet can’t form functional complex with ASGPRH2c.

To study the effect of HCV core protein on the interferon-induced antiviral genes expression and its mechanisms. Methods HepG2 cells were transiently transfected with HCV core protein expression plasmid and the blank plasmid respectively. RT-PCR was used to analyze the effect of HCV core protein on PKR and 2'-5'OAS expression. The effect of HCV core protein on ISRE-medicated gene expression was detected by luciferase activity assay. Western-blot assay was performed to observe the change of mRNA and protein levels of SOCS3, STAT1 and p-STAT1 following HCV core expression. In the presence of HCV core protein, the transcription of PKR and 2'-5' OAS are down-regulated. ISRE-medicated reporter gene expression and STAT1 phosphorylation were inhibited. The transcription and expression of SOCS3 were induced compared with blank plasmid-transfected group. In HepG2 cells, HCV core protein can down-regulate the expression of some interferon-induced antiviral genes, which involves the induction of SOCS3 and the inhibition of STAT1 phosphorylation.

To construct two soluble HLA-A*2402-peptide tetramers and detect the HBV-specific cytotoxic T lymphocytes (CTLs) with the constructed HLA-A*2402-peptide tetramers.After proteins HLA-A*2402-BSP and beta2m were made an effective prokanyotical expression, the purified proteins were refolded into HLA-A*2402-beta2m-peptide complexes in the presence of two antigenic peptides (Hepatitis B virus Pol756-764 or Core117-125) with dilution method. Then the complexes were biotinylated by BirA enzyme and purified by gel-filtration chromatography. The tetramers were generated by mixing the complex with PE-Streptavidin in the molar ratio of 5:1. FCM can and cell quest software were used to analyze the stained PBMCs.Two biotinylated HBV-HLA-A*2402-peptide complexes were identified by Western blot and they were shown to have natural conformation by Dot-ELISA and ELISA.The constructed HLA-A*2402-peptide tetramers can detect the HBV-specific CTLs in the patients with self-limited acute HBV infection.

To study the effects of tumor suppressor in lung cancer-1 (TSLC1) on human hepatocyte carcinoma cell line HepG2.A full length of TSLC1 cDNA was amplified from RNA of normal human liver cells by RT-PCR, and it was cloned into a pCI-neo expression vector and transfected into human hepatocellular carcinoma cell line HepG2. The HepG2 cells transfected with this plasmid (experimental group) and those treated with pCI-neo vector (control group) and without any treatment (blank group) were compared. Cell morphology was studied microscopically and cell growth was analyzed with MTT assay. FACSort flow cytometry analysis was performed to assess the cell cycle distribution and apoptosis.A stable cell line expressing TSLC1 protein was successfully established. Morphologically, cells of the experimental group were tightly aggregated when compared with those of the control and blank groups. The growth of TSLC1-transfected cells was significantly suppressed in vitro compared with those of the control and blank groups. The amount of G0-G1 cells was 63.66%+/-3.83% (P less than 0.01) in the experimental group, while those of the control and blank groups were 47.45%+/-0.91% and 54.47%+/-0.96% respectively. The amount of S phase cells in the experimental group, 22.90%+/-6.04%, was significantly lower (P less than 0.05) than that of the control group (36.58%+/-0.61%) and the blank group (33.61%+/-2.99%), which suggested a G0-G1 cell cycle arresting. The number of cells in early and late phase apoptosis (17.09%+/-0.20% and 16.11%+/-0.40% respectively) were significantly higher than those of the control and blank groups (P less than 0.01).TSLC1 strongly inhibits the growth of HepG2 cells in vitro and induces apoptosis of the cells, suggesting that TSLC1 may have a tumor suppressor function in HCC.

AIM: To investigate the effect of human apolipoprotein B mRNA-editing enzyme catalytic-polypeptide 3G (APOBEC3G) and its N-terminal or C-terminal cytosine deaminase domain-mediated antiviral activity against hepatitis B virus (HBV) in vitro and in vivo. METHODS: The mammalian hepatoma cells HepG2 and HuH7 were cotransfected with APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain expression vector and 1.3-fold-overlength HBV DNA as well as the linear monomeric HBV of genotype B and C. For in vivo study, an HBV vector-based mouse model was used in which APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain expression vectors were co-delivered with 1.3-fold-overlength HBV DNA via high-volume tail vein injection. Levels of hepatitis B virus surface antigen (HBsAg) and hepatitis B virus e antigen (HBeAg) in the media of the transfected cells and in the sera of mice were determined by ELISA. The expression of hepatitis B virus core antigen (HBcAg) in the transfected cells was determined by Western blot analysis. Core-associated HBV DNA was examined by Southern blot analysis. Levels of HBV DNA in the sera of mice as well as HBV core-associated RNA in the liver of mice were determined by quantitative PCR and quantitative RT-PCR analysis, respectively. RESULTS: Human APOBEC3G exerted an anti-HBV activity in a dose-dependent manner in HepG2 cells, and comparable suppressive effects were observed on genotype B and C as that of genotype A. Interestingly, the N-terminal or C-terminal cytosine deaminase domain alone could also inhibit HBV replication in HepG2 cells as well as Huh7 cells. Consistent with in vitro results, the levels of HBsAg in the sera of mice were dramatically decreased, with more than 50 times decrease in the levels of serum HBV DNA and core-associated RNA in the liver of mice treated with APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain as compared to the controls. CONCLUSION: Our findings provide probably the first evidence showing that APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain could suppress HBV replication in vitro and in vivo.

Human ISG20 gene was cloned and the effect of its anti-HBV was primarily studied. The ISG20 gene was amplified from HeLa cells by RT-PCR and recombinant vector expressing ISG20 was constructed by genetic engineering. The overexpression of ISG20 in HepG2 cells was detected by Western blot and the levels of secretion of HBs antigen and HBe antigen tested by ELISA. The results showed that: (1) Sequence of ISG20 cloned was consistent to that published in Genebank; (2) Recombinant vector expressing ISG20 could be expressed in HepG2 cells by transfection; (3) The overexpression of ISG20 protein could reduce the levels of the secretion of HBs antigen and HBe antigen in transfected HepG2 cells. It was suggested that the overexpression of recombinant ISG20 in culture cells could reduce the synthesis of HBV proteins.

To investigate the effect of apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G) mediated antiviral activity against hepatitis B virus (HBV) and duck hepatitis B virus (DHBV).Total RNA was extracted from peripheral blood mononuclear cells (PBMCs), RT-PCR product was cloned into the EcoR I/Hind III restriction sites of the CMV-driven expression vector fused with a hemagglutinin fusion epitope tag at its carboxyl terminal. Replication competent 1.3 fold over-length HBV was constructed with full-length HBV of ayw subtype. The mammalian hepatoma cell HepG2 was cotransfected with the replication competent 1.3 fold over-length HBV and various amounts of CMV-driven expression vector encoding APOBEC3G-HA. Levels of HBsAg and HBeAg in the media of the transfected cells were determined by ELISA, HBV DNA. RNA from intracellular core particles was examined using Northern and Southern blot analyses. Chicken hepatoma cell LMH was cotransfected with head-to-tail dimer of an EcoR I monomer of DHBV and various amounts of CMV-driven expression vector encoding APOBEC3G-HA. DHBV DNA from intracellular core particles was examined using Southern blot analysis.CMV-driven expression vector encoding APOBEC3G-HA and replication competent 1.3 fold over-length HBV were constructed. There was a dose dependent decrease in the levels of intracellular core-associated viral (HBV and DHBV) DNA and extracellular production of HBsAg and HBeAg. Levels of intracellular core-associated viral RNA were also decreased, but the expression of HBcAg remained almost unchanged.APOBEC3G suppresses HBV and DHBV replication and also suppresses HBsAg and HBeAg expression.

To investigate the distribution of hepatitis B virus genotype in Hubei province (China) and its clinical significance.Serum samples from 190 HBV DNA positive patients with chronic HBV infection,including 52 asymptomatic HBV carriers (ASC), 56 chronic hepatitis (CH), 32 fulminant hepatic failure (FHF), 22 liver cirrhosis (LC), and 28 hepatocellular carcinoma (HCC) patients were collected and tested for HBV genotypes by type-specific primers.A simple and precise genotyping system based on PCR using type-specific primers was developed for the determination of genotypes of hepatitis B virus (HBV). Of the 190 patients, 140 (73.7%) were genotype B and 42 (22.1%) were genotype C. Genotype B was more prevalent in the FHF and HCC patients than in the ASC patients; the ALT value was significantly higher in genotype B than in genotype C patients. The rate of anti-HBe was significantly higher in genotype B than in genotype C except in the patients of the ASC group.The system we used seems to be a useful tool for the molecular diagnosis of HBV infection and for large-scale surveys. Genotype B, genotype C and BC combination exist in Hubei province, and genotype B is the major genotype in this area especially in FHF and HCC patients.

To clarify the relationship between loss of DPC4 gene expression and pathogenesis of pancreatobiliary carcinoma.75 slides of normal duct (20), hyperplasia (15), dysplasia (15), invasive carcinoma (25) from patients with pancreatic diseases including pancreatic carcinoma (25 patients), chronic pancreatitis (6), pancreas injury (2) and 71 slides of common bile duct (CBD) carcinoma (38), gallbladder carcinoma (18), hilar bile duct (HBD) carcinoma (15) from patients with primary biliary tract carcinoma were analyzed for the expression of DPC4 protein by immunohistochemical staining.All specimens from 20 cases of normal duct and 15 cases of hyperplasia showed marked expression of DPC4 protein. The frequency of loss expression of the DPC4 gene was 33% in dysplasia, and 48% in invasive carcinoma. There was a significant statistical difference between hyperplasia and dysplasia (P0.01) and in dysplasia vs invasive carcinoma (P0.05). The frequency of loss expression of the DPC4 gene was 47.3% in CBD carcinoma, 11% in gallbladder carcinoma, and 13% in HBD carcinoma. The frequency of loss expression of the DPC4 gene was significantly different in CBD carcinoma vs gallbladder carcinoma and HBD carcinoma (P0.01).Inactivation of the DPC4 gene occurs late in the neoplastic progression of pancreatic carcinoma. The frequency of DPC4 gene alternation was different in various locations of biliary tract carcinoma. In CBD carcinoma, this frequency is similar to that in pancreatic carcinoma, indicating their similar molecular alternations.