Regulation of JC virus expression in B lymphocytes.

The etiologic agent of progressive multifocal leukoencephalopathy, a subacute demyelinating disease of the central nervous system, is the human polyomavirus JC virus (JCV), which causes a lytic infection of myelin-producing oligodendrocytes. In infected individuals the JCV genome can be detected in brain tissue and B lymphocytes isolated from the blood, bone marrow, or lymph nodes. Using mobility shift assays and a radiolabeled oligonucleotide from the JCV promoter-enhancer region (JCV bp 130 to 160), referred to as domain B, we were able to detect specific bands of the same mobility in nuclear extracts from human fetal glial cells, U-251 glioma cells, different B-cell lines, and in vitro-activated tonsillar B lymphocytes but not from T cells. In addition, a specific shift was detected when using nuclear extracts from freshly isolated tonsillar or lymph node B cells from five AIDS patients, two of whom later developed progressive multifocal leukoencephalopathy. Somewhat surprisingly, the above gel shift was partially inhibited by unlabeled oligonucleotides containing a kappa E2-binding site. UV cross-linking of the protein-DNA complex from either B cells or glial cells and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of a 46-kDa band. Transient transfection of a reporter plasmid constructed by fusing a trimer of the domain B sequence to a minimal promoter revealed activity in B lymphocytes and glial cells but not in T cells. Mutational analysis of this region demonstrated that the core TGGC repeat was essential for enhancer activity. Thus, a similar protein in B lymphocytes and glial cells may account for the preferential replication of JCV in these two cell types.     

Targeting hepatitis B virus-infected cells with a T-cell receptor-like antibody

Virus-specific CD8 T cells are activated when their T-cell receptors (TCRs) recognize the specific viral peptide/major histocompatibility complex (MHC) class I (pMHC) complexes present on the surface of infected cells. Antibodies able to recognize the specific pMHC can mimic TCR specificity and both represent a valuable biological tool to visualize pMHC complexes on infected cells and serve as a delivery system for highly targeted therapies. To evaluate these possibilities, we created a monoclonal antibody able to specifically recognize a hepatitis B virus (HBV) envelope epitope (Env at positions 183 to 91 [Env183-91]) presented by the HLA-A201 molecule, and we tested its ability to recognize HBV-infected hepatocytes and to deliver a cargo to a specific target. We demonstrate that this antibody detects and visualizes the processed product of HBV proteins produced in naturally HBV-infected cells, is not inhibited by soluble HBV proteins present in patient sera, and mediates the intracellular delivery of a fluorescent molecule to target cells. Additionally, compared to CD8 T cells specific for the same HBV epitope, the TCR-like antibody has both a superior sensitivity and a specificity focused on distinct amino acids within the epitope. These data demonstrate that a T-cell receptor-like antibody can be used to determine the quantitative relationship between HBV replication and specific antigen presentation to CD8 T cells and serves as a novel therapeutic delivery platform for personalized health care for HBV-infected patients.     

Fcgamma receptor-like activity of hepatitis C virus core protein

We have previously demonstrated that viral particles with the properties of nonenveloped hepatitis C virus (HCV) nucleocapsids occur in the serum of HCV-infected individuals (1). We show here that nucleocapsids purified directly from serum or isolated from HCV virions have FcgammaR-like activity and bind "nonimmune" IgG via its Fcgamma domain. HCV core proteins produced in Escherichia coli and in the baculovirus expression system also bound "nonimmune" IgG and their Fcgamma fragments. Folded conformation was required for IgG binding because the FcgammaR-like site of the core protein was inactive in denaturing conditions. Studies with synthetic core peptides showed that the region spanning amino acids 3-75 was essential for formation of the IgG-binding site. The interaction between the HCV core and human IgG is more efficient in acidic (pH 6.0) than in neutral conditions. The core protein-binding site on the IgG molecule differs from those for C1q, FcgammaRII (CD32), and FcgammaRIII (CD16) but overlaps with that for soluble protein A from Staphylococcus aureus (SpA), which is located in the CH2-CH3 interface of IgG. These characteristics of the core-IgG interaction are very similar to those of the neonatal FcRn. Surface plasmon resonance studies suggested that the binding of an anti-core antibody to HCV core protein might be "bipolar" through its paratope to the corresponding epitope and by its Fcgamma region to the FcgammaR-like motif on this protein. These features of HCV nucleocapsids and HCV core protein may confer an advantage for HCV in terms of survival by interfering with host defense mechanisms mediated by the Fcgamma part of IgG.     

Mouse hepatitis virus 3 replication in T and B lymphocytes correlate with viral pathogenicity

Viral pathogenicity may be regulated by host defense mechanisms at the virus-immune cell interaction level. The immune system plays an important role in the outcome of acute disease induced by the mouse hepatitis virus type 3 (MHV3) virus. The lymphoid cells act as effectors in the virus elimination as well as targets for viral replication. In order to demonstrate a correlation between MHV3 pathogenicity and viral replication in lymphocytes, genetically-determined resistant A/J and susceptible C57BL/6 mice were infected with pathogenic (L2-MHV3) or nonpathogenic (YAC-MHV3) viral strains. Pathogenicity and histopathologic studies have revealed that lymphoid organs such as thymus and spleen, showed injuries or atrophy in susceptible mice infected with L2-MHV3. No histopathologic lesions in the lymphoid organs occurred in C57BL/6 mice infected with YAC-MHV3 or A/J mice infected with both viruses. The mechanisms involved in the lymphoid injuries were studied regarding viral replication in the lymphoid organs and cells in infected mice. Results indicate that cell depletion in lymphoid organs is caused by a complete viral replication in lymphoid cells. Thy1.2+ and surface IgM+ lymphoid cells from susceptible C57BL/6 mice infected with L2-MHV3 were permissive to viral replication and to subsequent cell lysis. No cell lysis, however, occurred in lymphoid cells from C57BL/6 mice infected with YAC-MHV3 and A/J mice infected with both virus strains. In vitro studies, with purified T and B cell populations were performed to determine the mechanism effecting susceptibility or resistance to viral-induced cell lysis occurring in such cells. A blockade, probably occurring at the viral RNA polymerase activity level, prevents viral replication in resistant cells between the stages of fixation of the virus at the cell-surface receptor and the viral protein translation. These experiments indicate that an intrinsic virus-specific resistant mechanism occurs in lymphoid cells that plays a major role in the viral pathogenicity.     

Reduction in mitochondrial respiratory capacity in Saccharomyces cerevisiae induced by expression of hepatitis B virus surface antigen

Transformants of Saccharomyces cerevisiae strain TL154 (MATalpha, trp1, leu2) expressing hepatitis B virus surface antigen showed reduced rates of cell growth compared with those of nontransformed cells. The rates of phosphorylative, nonphosphorylative, and uncoupled respiration in mitochondria isolated from the transformants were reduced relative to those of mitochondria derived from nontransformed cells, regardless of whether the cells were cultured in rich or minimal medium. The electrophoretic protein profiles of cell and mitochondrial extracts did not differ substantially between transformed and nontransformed cells. These results suggest that the reduced rate of mitochondrial respiration in the transformants may be due to impairment of metabolic function rather than to inhibition of the expression of components of the respiratory chain.     

Direct injection of hepatitis B virus DNA into liver induced hepatitis in adult rats

Hepatitis B virus (HBV) surface antigen (HBsAg) genes were injected directly into the liver of adult rats with non-histone chromosomal protein high mobility group 1 by the hemagglutinating B virus of Japan (Sendai virus)-liposome method (Kato, K., Nakanishi, M., Kaneda, Y., Uchida, T., and Okada, Y. (1991) J. Biol. Chem. 266, 3361-3364). Immunohistochemical analysis showed that HBV surface antigen was expressed by the hepatocytes in vivo. On successive injections of the HBsAg genes, the antibody to HBV surface polypeptides was produced in the rats, and characteristic pathological changes of lymphocytic focal necrosis and denaturation of hepatic cells were observed in the liver of all the rats. We conclude that hepatitis is caused by the direct injection of HBsAg genes.     

Soluble CD40 ligand-activated B cells from patients with chronic hepatitis B virus infection as antigen presenting cells to induce hepatitis B virus specific cytotoxic T lymphocytes

Chronic hepatitis B virus (HBV) infection is the result of an inadequate antiviral immune response to the virus. In this study, we aimed to investigate whether the soluble CD40 ligand-activated B (CD40-B) cells could present antigen and induce specific cytotoxic T lymphocytes (CTLs) in patients with chronic HBV infection. We observed that after activated by sCD40L, the expression of CD80, CD86, major histocompatibility complex (MHC) I and II molecules on the CD40-B cells was significantly increased. Cytometry and fluorescence microscopy showed that more than 41.34% CD40-B cells were loaded by the HBcAg peptide. Furthermore, after been activated and HBcAg18–27 antigen peptide pulsed, B cells obtained from patients with chronic HBV infection could induce HBcAg18–27 specific CTLs in vitro. Taken together, our results show that B cells from patients with chronic HBV infection can be activated by sCD40L and may function as antigen presenting cells and induce HBV-specific CTLs.     

The influence of hepatitis B virus on antiviral treatment with interferon and ribavirin in Asian patients with hepatitis C virus/hepatitis B virus coinfection: a meta-analysis

ABSTRACT: BACKGROUND: Clinical and laboratory studies have indicated that coinfection with hepatitis B virus (HBV) and hepatitis C virus (HCV) can suppress one another, eliciting a dominant disease phenotype. To assess whether HBV can influence the antiviral effect of treatment on HCV, we performed a meta-analysis to comparatively analyze the response to interferon plus ribavirin treatment in patients with HBV/HCV coinfection and HCV mono-infection. METHODS: Published studies in the English-language medical literature that involved cohorts of HBV/HCV coinfection and HCV mono-infection were obtained by searching Medline, Cochrane and Embase databases. Studies that compared the efficacy of treatment with interferon plus ribavirin in HBV/HCV coinfection and HCV mono-infection were assessed. End-of-treatment virological response (ETVR), sustained virological response (SVR), HCV relapse rate, and alanine aminotransferase (ALT) normalization rate were compared between HBV/HCV coinfection and HCV mono-infection patients. RESULTS: Five trials involving 705 patients were analyzed. At the end of follow-up serum ALT normalization rates in patients with HCV mono-infection were significantly higher than in patients with HBV/HCV coinfection (odds ratio (OR) = 0.56, 95% confidence interval (CI): 0.40--0.80, P = 0.001). The ETVR and SVR achieved in HBV/HCV coinfection patients were comparable to those in HCV mono-infection patients (OR = 1.03, 95% CI: 0.37--2.82, P = 0.96 and OR = 0.87, 95% CI: 0.62--1.21, P = 0.38, respectively). The rate of relapse for HCV or HCV genotype 1 was not significantly different between HBV/HCV coinfection patients and HCV mono-infection patients (OR = 1.55, 95% CI: 0.98--2.47, P = 0.06; HCV genotype 1: OR = 2.4, 95% CI: 1.17--4.91, P = 0.19). CONCLUSIONS: Treatment with interferon and ribavirin achieves similar ETVR and SVR in HBV/HCV coinfection and HCV mono-infection. HBV/HCV coinfection patients had distinctively lower end of follow-up serum ALT normalization.     

Increased frequency of micronuclei in the lymphocytes of patients chronically infected with hepatitis B or hepatitis C virus

In this study, we analysed the frequency of micronuclei (MN), nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) and evaluated mutagen-induced sensitivity in the lymphocytes of patients chronically infected with hepatitis B virus (HBV) or hepatitis C virus (HCV). In total, 49 patients with chronic viral hepatitis (28 HBV-infected and 21 HCV-infected patients) and 33 healthy, non-infected blood donor controls were investigated. The frequencies (‰) of MN, NPBs and NBUDs in the controls were 4.41 ± 2.15, 1.15 ± 0.97 and 2.98 ± 1.31, respectively. The frequencies of MN and NPBs were significantly increased (p < 0.0001) in the patient group (7.01 ± 3.23 and 2.76 ± 2.08, respectively) compared with the control group. When considered separately, the HBV-infected patients (7.18 ± 3.57) and HCV-infected patients (3.27 ± 2.40) each had greater numbers of MN than did the controls (p < 0.0001). The HCV-infected patients displayed high numbers of NPBs (2.09 ± 1.33) and NBUDs (4.38 ± 3.28), but only the HBV-infected patients exhibited a significant difference (NPBs = 3.27 ± 2.40, p < 0.0001 and NBUDs = 4.71 ± 2.79, p = 0.03) in comparison with the controls. Similar results were obtained for males, but not for females, when all patients or the HBV-infected group was compared with the controls. The lymphocytes of the infected patients did not exhibit sensitivity to mutagen in comparison with the lymphocytes of the controls (p = 0.06). These results showed that the lymphocytes of patients who were chronically infected with HBV or HCV presented greater chromosomal instability.     

Granzyme H of cytotoxic lymphocytes is required for clearance of the hepatitis B virus through cleavage of the hepatitis B virus X protein

The granule exocytosis pathway of cytotoxic lymphocytes plays critical roles in eradication of intracellular viruses. However, how hepatitis B virus (HBV) is cleared has not been defined. To clarify immune mechanisms underlying inhibition of the HBV replication, the relationship between granzyme H (GzmH) and HBV clearance was investigated. In this study, we found that the granule exocytosis pathway can inhibit HBV replication without induction of cytolysis of the infected cells. GzmH is essential for HBV eradication. The HBx protein (HBx), required for the replication of HBV, is cleaved at Met(79) by GzmH. GzmH inhibitor can abolish GzmH- and lymphokine-activated killer cell-mediated HBx degradation and HBV clearance. An HBx-deficient HBV is resistant to GzmH- and lymphokine-activated killer cell-mediated viral clearance. Adoptive transfer of GzmH-overexpressing NK cells into HBV carrier mice facilitates in vivo HBV eradication. Importantly, low GzmH expression in cytotoxic lymphocytes of individuals is susceptible to HBV infection and hepatocellular carcinoma. These results indicate that GzmH might be detected as a potential parameter for diagnosis of HBV infection and hepatocellular carcinoma.     

Effect of B cell stimulatory factor-1 (interleukin 4) on Fc epsilon and Fc gamma receptor expression on murine B lymphocytes and B cell lines

Culture of murine splenic B cells with interleukin 4 (IL-4) caused the up-regulation of the lymphocyte Fc receptor for immunoglobulin E (IgE) (Fc epsilon R) over a similar dose range as required for Ia up-regulation. However, the expression level of the Fc receptor for immunoglobulin G (Fc gamma R) did not increase, rather IL-4 caused a slight but consistent decrease in the Fc gamma R level on the B cells. Fc epsilon R+ B hybridoma cells also responded to IL-4 by exhibiting increased Fc epsilon R expression; with the hybridoma cells Fc gamma R levels were unaffected. IL-4 caused an increase in the number of Fc epsilon R per cell and the highest levels of expression were obtained by having both IgE and IL-4 present in the culture. The specificity of the increase was demonstrated by blocking IL-4-mediated actions with monoclonal anti-IL-4 (11B11). Experiments following the incorporation of [35S]methionine into the Fc epsilon R demonstrated that IL-4 increased the rate of Fc epsilon R biosynthesis; this provides an explanation for the IL-4-induced increase in Fc epsilon R expression. IL-4, unlike IgE, had no effect on the rate of degradation of the Fc epsilon R. Interferon-gamma (IFN-gamma) totally abrogated IL-4-mediated Fc epsilon R up-regulation; at the same concentration of IFN-gamma Ia up-regulation is also suppressed, although not as effectively. IFN-gamma was shown to directly suppress Fc epsilon R synthesis, thereby explaining the inhibitory action on Fc epsilon R levels. Finally, it was shown that 11B11 inhibited the increased expression of Fc epsilon R on B cells obtained from mice during the early, but not the late, stages of Nippostrongylus brasiliensis infection. This latter finding suggests that the high Fc epsilon R levels seen early in parasite infections are dependent upon IL-4. The results overall provide further insight into the biologic activities of IL-4.     

Characterization of B lymphocytes present in the demyelinating lesions induced by Theiler's virus

Theiler's virus, a murine picornavirus, persists in the central nervous system of SJL/J mice and causes inflammation and demyelination in the white matter of spinal cord. We isolated inflammatory cells from the central nervous system of infected animals and studied their functions in vitro. Flow microfluorimetry analysis showed the presence of all major lymphocyte subsets, namely CD4+ and CD8+ T cells as well as B lymphocytes. B lymphocytes were activated in vitro and the antigenic specificity of secreted Ig was determined by immunoblotting. Secreted Ig reacted strongly with viral capsid proteins VP1 and VP2 and had neutralizing activity. They reacted also with two nonviral white matter components which were present only in infected animals. Therefore, it is likely that Igs secreted at the site of infection play a role in limiting virus spread. It is also possible that virus induced autoreactive antibodies participate in demyelination.     

Prevalence of hepatitis B virus in chronic hepatitis B patients

The aim of the current study was to detect HBV by Real time - PCR in chronic hepatitis B patients. Fifty-eight sera of chronic hepatitis B patients were subjected during the period March 2009 to April 2010 in Ilam cities in West of Iran. Sera assayed by real-time PCR and ELISA methods. Twenty serum samples from healthy volunteers and non-hepatitis B patients and negative for hepatitis B seromarkers served as negative controls for the study. Among fifty-eight sera, ELISA showed fifty-five (94.8%) of the samples were positive for HBsAg and three (5.2%) negative results obtained while real-time PCR specified fifty-eight (100%) positive results in chronic hepatitis B patients. HBsAg status did not necessarily reflect HBV DNA level in the serum, as 5.2% of chronic Hepatitis B patients were positive for HBV DNA but negative for HBsAg. HBV DNA was not found to be positive amongst any of the negative controls. Real time - PCR is a sensitive and reproducible assay for HBV DNA quantization.     

Multiplication of hepatitis B virus in fulminant hepatitis B.

The presence in serum of hepatitis B e antigen (HBeAg) and hepatitis B virus DNA, which are each regarded as reflecting multiplication of hepatitis B virus, were looked for one to five days after the onset of hepatic encephalopathy in 64 patients with fulminant hepatitis B. HBeAg and hepatitis B virus DNA were found in the serum of only 24 (37%) and six (9%) patients, respectively. Hepatitis B virus DNA was absent from the serum in all 13 patients positive for anti-HBs. These findings indicate that replication of hepatitis B virus stopped after the onset of hepatic encephalopathy in most of the patients and support the view that an enhanced immune response stops the replication. Agents that inhibit viral multiplication would probably not have any effect at this stage of the disease.