Hepatitis B virus e antigen downregulates cytokine production in human hepatoma cell lines

Disease activities of hepatitis B are affected by the status of hepatitis B e antigen (HBeAg). The function of the hepatitis B virus (HBV) precore or HBeAg is unknown. We assumed that HBeAg blocks aberrant immune responses, although HBeAg is not required for viral assembly, infection, or replication. We examined the interaction of HBeAg and the immune system, including cytokine production. The inflammatory cytokine TNF, IL-6, IL-8, IL-12A, IFN-α1, and IFN-? mRNA were downregulated in HBeAg-positive HepG2, which stably expresses HBeAg, compared to HBeAg-negative HepG2 cells. The results of real-time RT-PCR-based cytokine-related gene arrays showed the downregulation of cytokine and IFN production. We also observed inhibition of the activation of NF-κB- and IFN-?-promoter in HBeAg-positive HepG2, as well as inhibition of IFN and IL-6 production in HBeAg-positive HepG2 cell culture fluids. HBeAg might modify disease progression by inhibiting inflammatory cytokine and IFN gene expression, while simultaneously suppressing NF-κB-signaling- and IFN?-promoter activation.     

Nuclear antigen detected in hepatoma cell lines containing integrated hepatitis B virus DNA.

We identified, by anticomplement immunofluorescence, a nuclear antigen (hepatitis B virus-associated nuclear antigen [HBNA]) in two human hepatoma cell lines containing integrated hepatitis B virus DNA but not in three hepatoma cell lines lacking it. The antigen resembled neoantigens associated with the oncogenesis of certain papovaviruses, adenoviruses, and herpesviruses. Antibody to the antigen (anti-HBNA) was found in 7.3% of hepatitis B surface antigen-positive sera from patients with hepatocellular carcinoma but not in surface antigen-negative sera. The staining of HBNA was characterized by two patterns, reticular nuclear fluorescence and nucleolar fluorescence. The expression of HBNA did not parallel the production of extracellular hepatitis B surface antigen. Treatment of cells with proteinase K, RNase, DNase, or cycloheximide significantly diminished the staining of HBNA.     

Propagation of human hepatitis A virus in conventional cell lines

Fecal extracts of hepatitis A (HA) patients were selected for the presence of hepatitis A virus (HAV) by radioimmunoassay (RIA) and immune electron microscopy (IEM). When FL and Vero cells were inoculated with fecal extracts containing HAV, development of hepatitis A antigen (HAAg) was evident in the cytoplasm of the two cell lines by the indirect immunofluorescence (IF) test. The antigen was detectable in the cells 12 hr postinoculation (pi), and reached a plateau within two days pi. FL cell cultures inoculated with a specimen containing HAV were harvested and passaged four times. During the passages, efficient production of HAAg was confirmed in the infected cultures by three different serological tests: The indirect IF test, RIA using fixed cells, and RIA by the sandwich method. At the second and fourth passages, HAV particles were recovered in abundance from infected FL cell cultures by IEM. Throughout these experiments, no cytopathic effect (CPE) was discernible in the cultures.     

HCV核心基因插入突变体编码蛋白对人肝癌细胞系(Huh-7)细胞基因表达的影响

目的用基因表达分析法鉴定丙型肝炎病毒(HCV)核心(Core，C)区基因插入突变体编码蛋白在人肝癌细胞系(Huh-7)表达，探讨该蛋白生物学功能及其基因表达改变与致病的关系。方法构建HCV-1bc基因插入突变体编码蛋白重组表达质粒，建立表达c基因插入突变体编码蛋白Huh-7细胞系，按Affymetrix公司实验程序制备探针、再与该公司H0u133A和Hg-u133b芯片杂交。对基因表达上调或下调≥3倍的基因，用NetAflk作进一步分析。并用半定量RT-PCR对其中3个上调基因进行鉴定。结果Microarray分析显示，HCV-1b C基因插入突变体编码蛋白比c蛋白引起更多的基因表达改变，主要集中在信号传导、蛋白酶活性、分子转运、免疫反应等，特别是免疫反应基因表达更加显著。C基因插入突变体编码蛋白表达可同时导致凋亡基因／抗凋亡基因表达上调或下调及致癌基因上调。半定量RT-PCR对有趣的致癌基因FHL2、抗凋亡基因PRKCZ和凋亡基因LGALSI的鉴定结果表明，FHL2、PRKCZ和LGALSI基因的表达比空载体转染对照组相同基因明显上调。结论Hcvc基因插入突变体编码蛋白在Huh-7细胞表达对其基因表达有很大影响，其中对免疫反应基因的影响更明显，这一结果对理解HCV C基因插入突变体编码蛋白在HCV致病过程中的作用及其研制抗HCV药物均有重大的参考价值。     

Expression of hepatitis C virus cDNA in human hepatoma cell line mediated by a hybrid baculovirus-HCV vector

Although great progress has been made in the characterization of the biochemical and biological features of hepatitis C virus (HCV) gene expression, the elucidation of the HCV life cycle and the evaluation of novel antiviral strategies have been hindered by the lack of a suitable cell culture system. In this context, the development of an efficient HCV cDNA delivery method would contribute to the understanding of HCV replication. To assess the functionality of baculovirus mediated gene delivery for HCV expression, we have constructed recombinant baculoviruses encoding HCV cDNA under the control of the cytomegalovirus promoter. Transduction of the human hepatoma cell line Huh-7 with Bac-HCV vectors was efficient and HCV cDNA expression was enhanced by treatment of the infected cells with dexamethasone. HCV structural and nonstructural polypeptides were processed correctly and were found to localize in the cytoplasm in a pattern characteristic of the endoplasmic reticulum. The expression of the HCV proteins was detected for 49 days after infection. Thus, these results indicate that the recombinant Bac-HCV vectors are a useful tool for the delivery of HCV cDNA and can facilitate the analysis of structural and functional properties of the HCV proteins. In addition, the Bac-HCV vectors can provide important information on the evaluation of novel anti-HCV antiviral strategies.     

Binding of liver derived, low density hepatitis C virus to human hepatoma cells

HCV recovered from low density fractions of infected blood is associated with lipid and host apo-lipoproteins in lipo-viro-particles (LVP). It has been proposed that these particles are capable of binding and entering hepatocytes by viral glycoprotein independent mechanisms utilizing uptake pathways of normal host lipoproteins after binding to cell surface glycosaminoglycans (GAG), the low density lipoprotein receptor (LDL-r) or scavenger receptor B1 (SR-B1). In this study binding to human hepatoma cells of HCV low density RNA containing particles, semi-purified from macerates of infected human liver, is compared with that of normal host low density lipoprotein (LDL). Binding of both LDL and HCV low density RNA containing particles paralleled LDL-r but not SR-B1 expression on the recipient cells. Binding of both particle types was sensitive to suramin at 0 degrees C but less so at 37 degrees C suggesting that they both bind initially to GAG but, at 37 degrees C, are internalized or transferred to a suramin resistant receptor. Suramin resistant uptake of both particles was blocked in the presence of excess LDL or oxidized LDL. However, whilst LDL uptake was blocked by anti-apoB-100, HCV low density RNA uptake was enhanced by anti-apoB100 and further enhanced by a cocktail of anti-apo-B100 and anti-apoE. Pre-incubation of HCV low density RNA containing particles with antibodies to the E2 glycoprotein had little or no effect on uptake. These data indicate that whilst liver derived HCV RNA containing particles are taken up by HepG2 cells by a virus glycoprotein independent mechanism, the mechanism differs from that of LDL uptake.     

Modulation of production of hepatitis B surface antigen by a human hepatoma cell line

Three drugs were assayed for their capacity to inhibit hepatitis B surface antigen (HBsAg) production by the PLC/PRF/5 human hepatoma cell line. The effect on cell growth and HBsAg production of Cordycepin, 6-azauridine, and Hygromicin B is reported. Hygromicin B, a translation inhibitor unable to penetrate normal cells, greatly reduced HBsAg production by growing and confluent cells.     

A microcarrier cell culture system for large scale production of hepatitis A virus

Hepatitis A virus (HAV) was isolated from human faeces using a fetal rhesus monkey kidney cell line (Frhk-4). Infectious medium from passage 12 was used to inoculate a large (5000 cm2) microcarrier cell culture maintained in suspension. The microcarriers used were swollen, collagen-coated dextran beads on which it was easy to propagate Frhk-4 cells. Intra- and extra-cellular virus levels were assayed and compared with conventional cultures in 25 cm2 plastic flasks. The results show that virus production per cell was similar in both systems. The number of cells per area unit in confluent cultures was initially lower in the microcarrier culture but subsequently increased. Two to three weeks post inoculation the virus yield per area unit in the microcarrier system was half of that of the conventional culture. The lower cell density per area unit in the microcarrier system was compensated by the large growth area that could be maintained in a single vessel and the total production of virus was substantial. Weekly harvests of medium with HAV antigen titres around 10(-2) contained antigenic material sufficient for several thousands of anti-HAV IgM tests. Propagation of HAV in microcarrier cell cultures thus seems a safe and simple way to produce large amounts of HAV.     

Isolation and propagation of hepatitis A virus in hepatoma cell cultures

Hepatitis A virus (HAV) was isolated directly from human faeces in PLC/PRF/5 cells. In the first passage cell-bound and supernatant viruses were found by immune electron microscopy and by enzymeimmunoassay. Serial passaging of HAV in PLC/PRF/5 cells resulted in its adaptation to the cell line and in reduction of the incubation time. HAV was still detectable after 10 cell passages. Cell-bound as well as supernatant HAV were employed as antigens in anti HAV IgM-enzymeimmunoassay.     

基因芯片筛选HepG2与HepG2.2.15细胞中的差异表达基因

目的 探讨HepG2细胞及HepG2.2.15细胞中差异表达的基因并对其基因表达谱进行生物学信息分析.方法 用Trizol一步法提取HepG2细胞及HepG2.2.15细胞的总RNA,并纯化mRNA,反转录合成荧光分子(Cy3/Cy5)标记的cDNA探针,与基因芯片杂交;采用LuxScan3.0图像分析软件对芯片图像进行分析,把图像信号转化为数字信号,最后以差异为2倍的标准来确定差异表达基因.结果 在54614个基因表达谱的筛选中,发现有4462个基因表达水平显著上调,2592个基因表达水平显著下调.结论 HBV基因组及其表达产物对于肝细胞基因表达谱有显著影响,可能参与了肝癌的发生发展.     

Generation of inducible hepatitis C virus transgenic mouse lines

Hepatitis C virus (HCV) is the causative agent of most cases of chronic liver disease, cirrhosis, and hepatocellular carcinoma (HCC) affecting more than 170 million people world-wide. Progress in elucidating the nature of HCV and the development of new therapeutic strategies is hampered fundamentally by the absence of adequate small animal models simulating natural HCV infection. The creation of conditional mouse lines with the tetracycline-controlled gene expression system holds new perspectives for simulation of wild-type HCV infection in a small animal model. Transgenic mice were established with tetracycline-inducible coexpression of HCV core or HCV open reading frame (ORF) and luciferase. In long-term induction experiments, mice were examined for immunopathological changes after expression of HCV proteins. Inducible and liver-specific expression of transgenes was detected by Western blot, immunoprecipitation, luciferase assay and in vivo imaging of bioluminescence of luciferase in genetically modified mice. Ectopic expression levels were determined quantitatively in the liver, kidney, heart and spleen of mice in the induced and non-induced state. During long-term induction an elevation of aminotransaminases (ALT) was observed only in HCV core/ORF-expressing mice, but HCV-specific immune response was not confirmed by in vitro immunological assays. The histology of liver sections provided evidence of steatosis, which was correlated with an inflammatory response. The inducible HCV-transgenic mouse lines provide further evidence of liver pathogenesis in the presence of inflammation during liver-specific expression of HCV proteins and offer new insights into the effects of temporally and spatially controlled protein expression of HCV.     

Inducible nitric oxide synthase (iNOS) activity could be responsible for resistance or sensitivity to IFN-gamma-induced apoptosis in several human hepatoma cell lines.

Response to interferon-gamma (IFN-gamma)-induced apoptosis of human hepatoma cell lines (HHCLs) is variable. We analyzed this different behavior in Hep3B, Chang-liver, HepG2, and HuH7 cells. We studied (1) IFN-gamma-induced apoptosis, (2) protein expression of Stat1, (3) binding of nuclear proteins to IFN-gamma activated sequence (GAS), (4) mRNA and expression of proteins acting in apoptosis, and (5) HuH7 sensitivity after inducible nitric oxide synthase (iNOS) siRNA transfection. IFN-gamma induced apoptosis in Hep3B and Chang-liver cells only. In all HHCLs, Stat1 protein increased. Binding of proteins and transactivation activity of GAS increased much more in HuH7. In all HHCLs, caspase activity and apoptotic proteins were not implicated in resistance or sensitivity. iNOS mRNA and protein expression increased in HuH7, disappeared in Hep3B, and remained unchanged in Chang-liver and HepG2. We compared the role of iNOS in Hep3B and HuH7. The iNOS inhibitor, L-NAME, sensitized HuH7 to IFN-gamma, Hep3B/HuH7 coculture partially inhibited Hep3B apoptosis, and HuH7 transfection with iNOS siRNA induced a 50% inhibition of iNOS protein and cell apoptosis. GAS activity and overexpression of iNOS in HuH7, but not in the other HHCLs, suggest that this enzyme could play an important role in the resistance of HuH7 to IFN-gamma-induced apoptosis, perhaps by the antiapoptotic action of NO.     

Screening for hepatitis C virus in human immunodeficiency virus-infected individuals

Immunosuppression from human immunodeficiency virus (HIV) may impair antibody formation, and false-negative hepatitis C virus antibody (anti-HCV) tests have been reported in individuals coinfected with HIV and HCV. It is unknown if the frequency of false-negative tests is sufficiently high to change screening recommendations in this setting. Thus, the prevalence of false-negative results for anti-HCV by third-generation tests was determined with samples from HIV-infected individuals. Sera from 559 HIV-infected and 944 HIV-negative prospectively followed injection drug users were tested for anti-HCV by a third-generation enzyme immunoassay and for HCV RNA by using a branched DNA assay and the HCV COBAS AMPLICOR system. Of 559 HIV-infected participants, 547 (97.8%) were anti-HCV positive. One of the remaining 12 anti-HCV-negative participants was HCV RNA positive, and she later developed detectable anti-HCV. Of the 944 HIV-negative participants, 825 (87.4%) were anti-HCV positive. One of the remaining 119 anti-HCV-negative participants was HCV RNA positive, and she also developed detectable anti-HCV at a later visit. These data indicate that HIV infection does not alter the approach to hepatitis C virus screening, which should be performed with third-generation assays for anti-HCV unless acute infection is suspected.     

Spontaneous interferon production and Epstein-Barr virus antigen expression in human lymphoid cell lines.

Established human lymphoid cell lines, many of which spontaneously produce interferon, differ in the efficiency by which they allow expression of Epstein-Barr virus (EBV) lytic functions. Six EBV carrying lymphoid cell lines, selected to either be extremely susceptible or very refractory to EBV superinfection, were tested for spontaneous interferon production. Only the three cell lines which were poorly superinfectable with EBV were found to produce interferon. These same three lines could not be induced to express EBV-specific early antigens from intrinsic EBV genomes. It is suggested that interferon acts as a negative control factor affecting a cell's susceptibility to EBV.     

丙型肝炎病毒非结构区NS3基因在大肠埃希菌中的可诱导性表达

目的 构建丙型肝炎病毒（HCV）NS3基因的原核细胞表达载体。实现在大肠埃希菌中的可诱导性表达。方法 应用聚合酶链反应（PCR）技术，以美国HCV-H株全长cDNA质粒为模板，扩增获得NS3基因片段，克隆到原核表达载体pET-30C( )中，构建原核表达载体pET-NS3，转化BL21（DE3）宿主菌，以IPTG诱导，获得NS3蛋白的可诱导性表达，以HCVNS3的单链可变区抗体（ScFv)证实表达的NS3蛋白的特异性，结果 以HCVNS3基因序列特异性引物，PCR扩增获得1893bp的NS3DNA征段，插入pET-30C( )表达载体，转化BL21（DE3）受体菌，经培养，IPTG诱导，获得了重组HCVNS3蛋白的表达，以HCVNS3的ScFv证实了表达的重组蛋白HCVNS3的特异性。结论 以大肠埃希菌表达了HCVNS3的重组蛋白质。     

Recombinant human antibodies specific for hepatitis C virus proteins

Summary.  Human antibodies to hepatitis C virus core, NS4A and NS3 were cloned in a prokaryotic vector and expressed as soluble Fab fragments and as phage-displayed Fabs. The recombinant Fabs were shown to be a suitable tool for immunohistochemistry, since they recognize the cognate antigen expressed in mammalian cells. The nucleotide sequence of the cDNA for the variable domains of these antibodies was determined and the V-gene usage was derived. On the basis of the deduced amino acid sequence, a structural model of the V domains of the Fabs was constructed.