Inducible system in human hepatoma cell lines for hepatitis C virus production

We cloned the complete complementary DNA of an isolate of the hepatitis C virus, HCV-S1, into a tetracycline-inducible expression vector and stably transfected it into two human hepatoma cell lines, Huh7 and HepG2. Twenty-six Huh7 and two HepG2-positive clones were obtained after preliminary screening. Two Huh7 (SH-7 and -9) and one HepG2 (G-19) clones were chosen for further characterisation. Expression of HCV proteins in these cells accumulated from 6 h to 4 days posttreatment. Full-length viral plus-strand RNA was detected by Northern analyses. Using RT-PCR and ribonuclease protection assay, we also detected the synthesis of minus-strand HCV RNA. Plus- and minus-strand viral RNA was still detected after treatment with actinomycin D. Indirect immunofluorescence staining with anti-E2, NS4B, and NS5A revealed that these proteins were mostly localised to the endoplasmic reticulum (ER). Culture media from tet-induced SH-9 cells was separated on sucrose density gradients and analysed for the presence of HCV RNA. Viral RNA levels peaked at two separate ranges, one with a buoyant density of 1.08 g/ml and another from 1.17 to 1.39 g/ml. Electron microscopy demonstrated the presence of subviral-like particles (approximately 20-25 nm in diameter) in the cytoplasm of SH-9 and G-19 cells, which were positively labelled by anti-HCV core antibodies. Anti-E2 antibodies strongly labelled cytoplasmic vesicular structures and some viral-like particles. Complete viral particles of about 50 nm which reacted with anti-E2 antibodies were observed in the culture media of tet-induced SH-9 cells following negative staining. Supernatant from tet-treated SH-9 cells was found to infect nai;ve Huh7 and stable Huh7-human CD81 cells.     

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.     

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.     

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.     

Hepatitis B virus genotypes and spontaneous hepatitis B e antigen seroconversion in Taiwanese hepatitis B carriers

Hepatitis B virus (HBV) is classified into eight genotypes (A-H), and genotype C is associated with more aggressive liver disease compared to genotype B. However, the mechanisms responsible for the clinical differences remain unclear. To test whether genotype C patients had with lower rates of spontaneous hepatitis B ge antigen (HBeAg) seroconversion than genotype B patients, stored serum samples from 146 Taiwanese adult HBeAg-positive hepatitis B carriers followed-up for a mean of 52 months (range, 12-120 months) were tested for HBV genotype by a molecular method. Genotype C patients were significantly older than genotype B patients (mean age, 37 +/- 12 vs. 29 +/- 10 years, P < 0.001). During the follow-up period, genotype C patients had a significantly lower rate of spontaneous HBeAg seroconversion than genotype B patients (27 vs. 47%, P < 0.025). Spontaneous HBeAg seroconversion occurred one decade later in genotype C patients compared with genotype B patients. Multivariate analyses identified age < or =35 years (odds ratio: 2.08; 95% confidence interval [CI], 1.07-4.0; P < 0.05), high baseline serum alanine aminotransferase level (odds ratio: 2.34; 95%CI, 1.39-4.09; P < 0.005), and HBV genotype B (odds ratio: 1.94; 95%CI, 1.03-3.63; P < 0.05) as independent factors associated with spontaneous HBeAg seroconversion. In conclusion, genotype C patients, compared to genotype B patients, have a delayed HBeAg seroconversion in the immune clearance phase of chronic HBV infection, which may contribute to a more progressive liver disease and more refractory to antiviral therapy.     

Hepatitis B virus e antigen (HBeAg) may have a negative effect on dendritic cell generation

Hepatitis B virus (HBV) continues to be a serious worldwide health problem despite the use of protective HBV vaccines and therapeutic regimens against chronic HBV infection. Chronic HBV patients cannot induce sufficient immune responses against the virus. HBV and its antigens are believed to suppress immune responses during chronic infection. Hence, studying the role of HBV in immune suppression is very important for the development of alternative therapeutic strategies for HBV infections.     

Hepatitis B virus e antigen specific epitopes and limitations of commercial anti-HBe immunoassays

Current commercial hepatitis B virus (HBV) anti-HBe immunoassays are designed so that anti-HBe is detectable only in the absence of excess HBeAg. Recently, with the use of direct anti-HBe assays, anti-HBe was detected in individuals who had been seropositive for several years for HBeAg [Maruyama et al. (1993) J. Clin. Invest. 91:2586-2595]. Although anti-HBe seroconversion does not necessarily indicate subsequent HBeAg clearance, the ability to detect earlier anti-HBe seroconversion could have clinical significance for monitoring patients undergoing HBV immunotherapy (e.g., alpha interferon therapy). Because the HBeAg and the HBcAg share 149 amino acids, an anti-HBe assay must distinguish anti-HBe from anti-HBc antibodies. Although the HBV HBeAg and HBcAg display distinct immunogenic determinants, much remains unknown regarding the complete epitope spectrum specific to each antigen. The goal of this study was 3-fold. The first objective was to identify HBeAg specific linear epitopes. The second objective was to design an anti-HBe immunoassay capable of detecting anti-HBe specific antibody in the presence of excess HBeAg. The third objective was to characterize early anti-HBe seroconversion antibodies. The major linear epitope residing in the HBeAg amino acid sequence was mapped and 2 novel minor epitopes (delta, gamma) which appear to be HBeAg specific have been identified. An anti-HBe immunoassay capable of detecting anti-HBe specific antibody in the presence of excess HBeAg was designed. Finally, it was found that early anti-HBe seroconversion antibodies appear to be conformational, whereas later seroconversion, more typically associated with the clearance of HBeAg, is characterized by the presence of antibodies to the linear HBeAg epitopes.     

High level expression of apoptosis inhibitor in hepatoma cell line expressing Hepatitis B virus

The serious result of hepatitis B (HBV) virus infection is development of hepatocellular carcinoma (HCC). However, the reason of development of HCC in HBV infected patients is still unclear. Recently, the suppression of cell apoptosis is found to relate with the development of cell carcinogenesis, therefore, the expression of apoptosis inhibitor in the virus related cancer line such as hepatoma cell line HepG2.215 was investigated. There are at least six Human apoptosis inhibitors (IAP) have been identified now. They are cIAP1, cIAP2, XIAP, NAPI, survivin and pIAP. Using gene-assay technology, we have recently compared the expression of IAPs in the HepG2.215 cells that persistently expresses Hepatitis B virus by integrated HBV genome with its parent cell line HepG2. The results suggest that there was obviously increase of cIAP2 and cIAP1 in the HepG2.215 cells versus HepG2 cells. Those observations imply a possibility of long time HBV infection could induce the over-expressing apoptosis inhibitors, furthermore, causing the liver cancer. The high expression of cIAP1 and cIAP2 in HBV expressing cells was confirmed by RT-PCR and Northern blot analysis. However, we did not find the change of NIAP and suvivin in HepG2.215 cells. In contrast, the expression of XIAP was down in the HepG2.215 cells comparing with HepG2 cells. How HBV triggers the over-expression of apoptosis inhibitor is unclear. Transient transfection of HepG2 cells with the plasmids expressing different HBV proteins such as S, M, L, X and core proteins did not give a decisive conclusion. Further study is going on now.     

Hepatitis B virus propagated in a rat hepatoma cell line is infectious in a primate model

The human hepatitis B virus (HBV) produced by a rat hepatoma cell line through transfection with HBV DNA is infectious in the human primate model--chimpanzee. Since hepadnaviruses are known to have an extremely narrow host range, our results support the idea that the host species barrier of HBV infection resides on the penetration/adsorption step rather than any postpenetration intracellular event during the virus life cycle.     

Hepatitis B virus X protein upregulates survivin expression in hepatoma tissues

The hepatitis B virus X protein (HBx) plays an important role in the development of hepatocellular carcinoma (HCC). The relationship was examined between HBV antigens and IAP (inhibitor of apoptosis) family in development of HCC. The expression levels of HBV antigens (HBsAg, HBcAg, and HBxAg) and members of the IAP family (survivin, XIAP, cIAP-1, and cIAP-2) were detected immunohistochemically in tissues from 34 cases of HCC and 30 cases of liver cirrhosis. The positive rate of survivin was higher than these three molecules in all three tissue types (P < 0.05). The positive rates of HBxAg and survivin were high in HCC (76.5% and 88.2%), paratumor (85.3% and 91.2%), and liver cirrhosis (100% and 93.3%) tissues, with no significant differences between the survivin- and HBxAg-positive rates (each P > 0.05). To examine the effect of HBx on survivin expression, plasmid pCMV-X (encoding the HBx gene) was transfected transiently with or without plasmid pcDNA3-sur (encoding the survivin gene) into H7402 hepatoma cells and L-O2 human normal liver cells. Cells over-expressing HBx alone showed increased apoptosis along with a dose-dependent increase in survivin levels. However, co-expression of survivin inhibited the HBx-induced apoptosis. To examine the effect of HBx on survivin in hepatoma cells without apoptosis, plasmid pCMV-X was transfected stably into human hepatoma H7402 cells and L-O2 cells. These H7402-X and L-O2-X cells showed high-level expression of both HBx and survivin, but did not show apoptosis. The addition of pSilencer 3.0-X, an RNAi vector targeting the HBx gene, reduced the expression levels of survivin protein in H7402-X cells. Collectively, these data demonstrate that HBx upregulates survivin expression in hepatoma tissues, suggesting that HBx and survivin may both be involved in carcinogenesis of HCC.     

Constitutive production of B cell differentiation factor-like activity by human T and B cell lines

A lymphokine demonstrating human B cell differentiation factor (BCDF)-like activity was isolated from immature (MOLT-4f, CCRF-CEM and CCRF-HSDB-2) and mature (HUT-78) malignant human T lymphoid cell lines and from human B lymphoblastoid cell lines (BJAB and ALL-7031-B). All the cell lines were grown long term in serum-free medium. This BCDF-like activity has a molecular mass in the range of 40-60 kDa and stimulates immunoglobulin synthesis of cell lines capable of producing IgA (GM-1056), IgG (GM-1500 and CESS) and IgM (CBL#3). It was not produced by a myeloid cell line. We were only able to identify the differentiation activity produced by the T and B cell lines by using appropriate molecular mass fractions from the serum-free medium as controls. This BCDF-like activity is different from that of the human BCDF so far described as it has a higher molecular mass and is constitutively produced by malignant T lymphoid cell lines which are human T cell leukemia virus-I negative and by B lymphoblastoid cell lines.     

Impaired T cell activation and cytokine production by calcitriol-primed human B cells

The biologically active form of vitamin D₃, 1, 25-dihydroxyvitamin D₃ (calcitriol), is a potent modulator of the immune response. We have shown previously that calcitriol modulates the immunoglobulin response in vitro and in vivo in mice and humans. To analyse the underlying molecular mechanisms we studied whether calcitriol-primed B cells modulate T cell activation and function. Human B cells were stimulated with anti-CD40 and interleukin (IL)-4 in the presence of increasing concentrations of calcitriol. After removal of calcitriol, primed B cells were co-cultured with autologous CD4⁺ T cells; the B cell phenotype T cell activation and their consecutive cytokine production were also assessed. Naive T cells co-cultured with calcitriol-primed naive B cells showed a reduced expansion, nuclear factor of activated T cells, cytoplasmic 2 (NFATc2) expression and cytokine production upon restimulation. CD86 expression on B cells after calcitriol priming was identified as an underlying mechanism, as T cell activation and expansion was rescued by activating anti-CD28 antibodies. Our data indicate that calcitriol-primed B cells display an impaired capacity to activate T cells. Taken together, we identified a novel B cell-dependent vitamin D immune regulatory mechanism, namely by decreased co-stimulation of calcitriol-primed B cells.     

Hepatitis B virus surface antigen impairs myeloid dendritic cell function: a possible immune escape mechanism of hepatitis B virus

Chronic hepatitis B virus (HBV) infection is the result of an inadequate immune response towards the virus. Myeloid dendritic cells (mDC) of patients with chronic HBV are impaired in their maturation and function, resulting in more tolerogenic rather than immunogenic responses, which may contribute to viral persistence. The mechanism responsible for altered mDC function remains unclear. The HBV-infected patients display large amounts of HBV particles and viral proteins in their circulation, especially the surface antigen HBsAg, which allows multiple interactions between the virus, its viral proteins and DC. To assess whether HBV directly influences mDC function, the effects of HBV and HBsAg on human mDC maturation and function were investigated in vitro. As already described for internalization of HBV by DC, the present study shows that peripheral blood-derived mDC of healthy controls also actively take up HBsAg in a time-dependent manner. Cytokine-induced maturation in the presence of HBV or HBsAg resulted in a significantly more tolerogenic mDC phenotype as demonstrated by a diminished up-regulation of costimulatory molecules and a decreased T-cell stimulatory capacity, as assessed by T-cell proliferation and interferon-gamma production. In addition, the presence of HBV significantly reduced interleukin-12 production by mDC. These results show that both HBV particles and purified HBsAg have an immune modulatory capacity and may directly contribute to the dysfunction of mDC in patients with chronic HBV. The direct immune regulatory effect of HBV and circulating HBsAg particles on the function of DC can be considered as part of the mechanism by which HBV escapes immunity.