Development of an infectious surrogate hepatitis C virus based on a recombinant vesicular stomatitis virus expressing hepatitis C virus envelope glycoproteins and green fluorescent protein.

To develop surrogate viruses for hepatitis C virus (HCV), we previously produced recombinant vesicular stomatitis viruses (rVSVs) lacking glycoprotein G but instead expressing chimeric HCV E1/E2 fused to G. These rVSVs were not infectious in HCV-susceptible hepatoma cells. In this study, to develop an infectious surrogate HCV based on an rVSV (vesicular stomatitis virus [VSV]/HCV), we generated a novel rVSV encoding the native E1/E2 (H77 strain) and green fluorescent protein (GFP) instead of G. Here, we showed that this VSV/HCV efficiently infected human hepatoma cells, including Huh7 human hepatoma cells, expressed GFP in these cells, and propagated, but did not do so in nonsusceptible BHK-21 cells. The infectivity of VSV/HCV, measured as the number of foci of GFP-positive cells, was specifically reduced by the addition of chimpanzee anti-HCV serum, anti-E2 antibody, or anti-CD81 antibody to the cultures. When sera obtained from HCV-infected or uninfected patients were added, infection was selectively inhibited only by the sera of HCV-infected patients. These data together suggest that this infectious GFP-expressing VSV/HCV could be a useful tool for studying the mechanisms of HCV entry into cells and for assessing potential inhibitors of viral entry, including neutralizing antibodies.

B-cell-intrinsic hepatitis C virus expression leads to B-cell-lymphomagenesis and induction of NF-κB signalling.

Hepatitis C virus (HCV) infection leads to the development of hepatic diseases, as well as extrahepatic disorders such as B-cell non-Hodgkin's lymphoma (B-NHL). To reveal the molecular signalling pathways responsible for HCV-associated B-NHL development, we utilised transgenic (Tg) mice that express the full-length HCV genome specifically in B cells and develop non-Hodgkin type B-cell lymphomas (BCLs). The gene expression profiles in B cells from BCL-developing HCV-Tg mice, from BCL-non-developing HCV-Tg mice, and from BCL-non-developing HCV-negative mice were analysed by genome-wide microarray. In BCLs from HCV-Tg mice, the expression of various genes was modified, and for some genes, expression was influenced by the gender of the animals. Markedly modified genes such as Fos, C3, LTßR, A20, NF-κB and miR-26b in BCLs were further characterised using specific assays. We propose that activation of both canonical and alternative NF-κB signalling pathways and down-regulation of miR-26b contribute to the development of HCV-associated B-NHL.

Japanese reference panel of blood specimens for evaluation of hepatitis C virus RNA and core antigen quantitative assays.

An accurate and reliable quantitative assay for hepatitis C virus (HCV) is essential for measuring viral propagation and the efficacy of antiviral therapy. There is a growing need for domestic reference panels for evaluation of clinical assay kits because the performance of these kits may vary with region-specific genotypes or polymorphisms. In this study, we established a reference panel by selecting 80 donated blood specimens in Japan that tested positive for HCV. Using this panel, we quantified HCV viral loads using two HCV RNA kits and five core antigen (Ag) kits currently available in Japan. The data from the two HCV RNA assay kits showed excellent correlation. All RNA titers were distributed evenly across a range from 3 to 7 log IU/ml. Although the data from the five core Ag kits also correlated with RNA titers, the sensitivities of individual kits were not sufficient to quantify viral load in all samples. As calculated by the correlation with RNA titers, the theoretical lower limits of detection by these core Ag assays were higher than those for the detection of RNA. Moreover, in several samples in our panel, core Ag levels were underestimated compared to RNA titers. Sequence analysis in the HCV core region suggested that polymorphisms at amino acids 47 to 49 of the core Ag were responsible for this underestimation. The panel established in this study will be useful for estimating the quality of currently available and upcoming HCV assay kits; such quality control is essential for clinical usage of these kits.

HCV infection and B-cell lymphomagenesis.

Hepatitis C virus (HCV) has been recognized as a major cause of chronic liver diseases worldwide. It has been suggested that HCV infects not only hepatocytes but also mononuclear lymphocytes including B cells that express the CD81 molecule, a putative HCV receptor. HCV infection of B cells is the likely cause of B-cell dysregulation disorders such as mixed cryoglobulinemia, rheumatoid factor production, and B-cell lymphoproliferative disorders that may evolve into non-Hodgkin's lymphoma (NHL). Epidemiological data indicate an association between HCV chronic infection and the occurrence of B-cell NHL, suggesting that chronic HCV infection is associated at least in part with B-cell lymphomagenesis. In this paper, we aim to provide an overview of recent literature, including our own, to elucidate a possible role of HCV chronic infection in B-cell lymphomagenesis.

Peripheral blood memory B cells are resistant to apoptosis in chronic hepatitis C patients.

Our recent study indicated that peripheral B cells in chronic hepatitis C (CHC) patients were infected with hepatitis C virus (HCV). It was also demonstrated that the frequency of CD27(+) B cells, i.e. memory phenotype, was significantly reduced in the peripheral blood of CHC patients. An assumption was made by these findings that the CD27(+) B cells are susceptible to apoptosis when infected with HCV. Therefore, in this study, the susceptibility of CD27(+) B cells to apoptosis in CHC patients was analyzed. Contrary to our assumption, it was found that CD27(+) B cells are more resistant to apoptosis than the counterpart subset, i.e. CD27(-) B cells. The rationale for this finding is discussed with regard to the possible role for memory B cells as an HCV reservoir for persistent infection in CHC patients.

Peripheral B cells may serve as a reservoir for persistent hepatitis C virus infection.

A recent study by our group indicated that peripheral B cells in chronic hepatitis C (CHC) patients are infected with hepatitis C virus (HCV). This raised the logical question of how HCV circumvents the antiviral immune responses of B cells. Because type I interferon (IFN) plays a critical role in the innate antiviral immune response, IFNß expression levels in peripheral B cells from CHC patients were analyzed, and these levels were found to be comparable to those in normal B cells, which suggested that HCV infection failed to trigger antiviral immune responses in B cells. Sensing mechanisms for invading viruses in host immune cells involve Toll-like receptor-mediated and retinoic acid-inducible gene-I (RIG-I)-mediated pathways. Both pathways culminate in IFN regulatory factor-3 (IRF-3) translocation into the nucleus for IFNß gene transcription. Although the expression levels of RIG-I and its adaptor molecule, IFN promoter-stimulator-1, were substantially enhanced in CHC B cells, dimerization and subsequent nuclear translocation of IRF-3 were not detectable. TANK-binding kinase-1 (TBK1) and IκB kinase ε (IKKε) are essential for IRF-3 phosphorylation. Constitutive expression of both kinases was markedly enhanced in CHC B cells. However, reduced expression of heat shock protein of 90 kDa, a TBK1 stabilizer, and enhanced expression of SIKE, an IKKε suppressor, were observed in CHC B cells, which might suppress the kinase activity of TBK1/IKKε for IRF-3 phosphorylation. In addition, the expression of vesicle-associated membrane protein-associated protein-C, a putative inhibitor of HCV replication, was negligible in B cells. These results strongly suggest that HCV utilizes B cells as a reservoir for persistent infection.

Possible recruitment of peripheral blood CXCR3+ CD27+ CD19+ B cells to the liver of chronic hepatitis C patients.

It has been suggested that hepatitis C virus (HCV) infects not only hepatocytes but also immune cells, including B cells. HCV infection of B cells is the likely cause of B-cell dysregulation disorders such as mixed cryoglobulinemia, rheumatoid factor production, and B-cell lymphoproliferative disorders that may evolve into non-Hodgkin's lymphoma. To clarify the effects of chronic HCV infection on B-cell dynamics, peripheral B cells from chronic hepatitis C patients (CHC) were characterized. We found that the frequency of CD27(+) B cells, that is memory phenotype, was significantly reduced in the peripheral blood of CHC. At the same time, the amount of IFN-gamma-inducible protein-10 (IP-10), a CXCR3 ligand, was markedly elevated in the plasma of CHC. Furthermore, the CD27(+) B-cell population was found to highly express CXCR3 in CHC, thus suggesting that the CD27(+) B-cell population was recruited from peripheral blood to the inflammatory site of the liver of CHC, where IP-10 is produced. Immunohistochemical analyses of intrahepatic lymphocytes indicated that CXCR3(+) B cells were infiltrated in the liver of CHC. Our results thus offer new insight into the role of memory B cells in the HCV pathogenesis.

Enhanced expression of lymphomagenesis-related genes in peripheral blood B cells of chronic hepatitis C patients.

Epidemiological data indicate a close relationship between chronic hepatitis C virus (HCV) infection and B-cell non-Hodgkin's lymphoma (B-NHL), suggesting that chronic HCV infection is, at least in part, associated with B-lymphomagenesis. However, experimental data concerning these conditions remains elusive. In this study, we confirmed that peripheral blood B cells of chronic hepatitis C (CHC) patients were infected with HCV. Expression levels of activation-induced cytidine deaminase (AID) which are thought to be associated with occurrence of B-NHL were analyzed in these CHC B cells. It was demonstrated that AID mRNA/protein levels in CHC B cells were dramatically increased compared with those of healthy subjects. Furthermore, expression levels of several previously reported prognostic B-NHL marker genes in the B cell subset of CHC patients were increased. These results suggest a possible relationship between chronic HCV infection and B-lymphomagenesis.

Autophagic compartments gain access to the MHC class II compartments in thymic epithelium.

The presentation of self-peptides in the context of MHC molecules by thymic epithelial cells (TECs) is essential for T cell repertoire selection in the thymus. However, the underlying mechanisms of this process have not been fully elucidated. To address whether autophagy, a catabolic process involving the degradation of a cell's components through the lysosomal machinery, intersects the MHC class II-restricted Ag presentation pathway in TECs, we investigated the colocalization of LC3, a peculiar autophagy marker molecule, with MHC class II compartments in in vitro-established TEC lines by immunofluorescence microscopy and Western blotting analyses. We found that in both cortical and medullary TEC lines, LC3 was colocalized with the H2-DM-positive lysosomal compartments, in which MHC class II plus class II-associated invariant chain peptides complexes are formed. Furthermore, our analysis of thymic cryosections from 1-day-old mice revealed that LC3 colocalizes with the H2-DM-positive compartments in TECs. These results strongly suggest that the cytoplasmic self-Ags gain access to the H2-DM-positive compartments via the autophagic process in the thymus.

Evaluation of hepatitis C virus core antigen assays in detecting recombinant viral antigens of various genotypes.

A single substitution within the hepatitis C virus core antigen sequence, A48T, which is observed in approximately 30% of individuals infected with genotype 2a virus, reduces the sensitivity of a commonly used chemiluminescence enzyme immunoassay. Quantitation of the antigen is improved by using a distinct anticore antibody with a different epitope.

Functional phenotypes and gene expression profiles of peripheral blood mononuclear cells in chronic hepatitis C patients who developed non-Hodgkin's B-cell lymphoma.

Epidemiological data have indicated a close relationship between chronic HCV infection and non-Hodgkin's B-cell lymphoma (B-NHL). In this study, functional phenotypes and gene expression profiles of PBMCs were analyzed in chronic hepatitis C (CHC) patients who developed B-NHL. The frequencies of effector CD8(+) T cells and cytotoxic natural killer cells increased in CHC patients with B-NHL compared to those in CHC patients without B-NHL. These phenotypic changes may reflect the host's immune response to neoplasia. The mRNA expression levels of several oncogenes increased in CHC patients without B-NHL, but were much higher in CHC patients with B-NHL, while mRNA levels of type I IFNs were decreased in CHC patients without B-NHL and were nearly negligible in CHC patients with B-NHL. Interestingly, the mRNA expression levels of activation-induced cytidine deaminase and caspase recruitment domain-containing proteins markedly increased in CHC patients without B-NHL but decreased in CHC patients with B-NHL. These results are discussed in view of the possible involvement of HCV infection in B-cell lymphomagenesis.

Differential susceptibility of peripheral blood CD5+ and CD5- B cells to apoptosis in chronic hepatitis C patients.

A body of evidence has suggested a close link between chronic hepatitis C virus (HCV) infection and B cell abnormalities, including mixed cryoglobulinemia, rheumatoid factor (RF) production, and lymphoproliferative disorders that may develop into non-Hodgkin's lymphoma. Recent studies have demonstrated the expansion of CD5(+) B cells in the peripheral blood of chronic hepatitis C patients (CHC). As CD5(+) B cells, which are capable of producing autoantibodies and RF, are apparently crucial for the development of HCV-associated pathogenesis, the fate of both the CD5(+) and CD5(-) B cell subsets upon chronic HCV infection is of interest. In this study, the degree to which chronic HCV infection induces apoptosis in each B cell subset was investigated. Our results demonstrated that peripheral CD5(-) B cells were more susceptible to apoptosis than CD5(+) B cells in CHC. Furthermore, plasma levels of IL-4, IL-10, and IL-12 were significantly elevated in CHC, thus suggesting that these interleukins protect CD5(+) B cells from apoptosis. The rationale for the differential susceptibility of distinct B cell subsets in CHC is also discussed with regard to extrahepatic manifestations associated with chronic HCV infection.

Derivation, culture, and characterization of thymic epithelial cell lines.

The major histocompatibility complex (MHC)-restricted presentation of self-peptides, generated from tissue-specific antigens, by thymic epithelial cells (TECs) is essential for development of central tolerance and for generation of the regulatory T-cell repertoire in the thymus. However, the mechanisms by which self-peptides are generated in and presented by TECs have not been well defined. To elucidate the processes involved in MHC class II-restricted presentation of self-peptides by TECs, cortical and medullary TEC lines may be established from C57BL/6 mouse thymi. Localization of a variety of molecules, including the MHC class II molecules critically involved in the presentation of antigen by TECs, may be investigated by both immunofluores-cence microscopy and Western blotting analyses. Our own studies using these approaches have demonstrated that such molecules are localized in the H2-DM+ lysosomal compartments isolated from both cortical and medullary TECs.

Evaluation of 10 commercial diagnostic kits for in vitro expressed hepatitis B virus (HBV) surface antigens encoded by HBV of genotypes A to H.

Genetic variability of the hepatitis B virus (HBV) constitutes one of the major challenges for diagnosis of HBV infection. It is plausible that amino acid substitutions in the "a" determinant of the HBV surface antigen (HBsAg) that affect antigenic sites, whether originating from genetic diversity or from mutations in the HBV strain itself, will affect the sensitivity of some diagnostic kits. In fact, recent studies have indicated that some diagnostic kits had false negative results with particular HBsAg mutants. There have been, however, few substantial studies evaluating sensitivities of diagnostic kits to the HBsAg encoded by different HBV genotypes. Our recent study found that 10 diagnostic kits available in Japan were able to detect HBsAg irrespective of whether it originated from HBV genotypes A, B or C, with the latter two genotypes being the dominant species in East Asia. In this study, we extended our previous efforts by assessing the ability of diagnostic kits to detect recombinant HBsAg derived from HBV genotypes A to H. Our results demonstrated that 9 out of 10 diagnostic kits evaluated were able to detect as low as 0.2 International Units (IU)/ml HBsAg, irrespective of HBV genotype. The genotypic differences in the HBV family thus appear to have little impact on the sensitivity of currently available HBsAg diagnostic kits.

Reactivity of genotypically distinct hepatitis B virus surface antigens in 10 commercial diagnostic kits available in Japan.

Hepatitis B virus (HBV) surface antigen (HBsAg) is one of the most important serological markers of current HBV infection. However, there are significant antigenic variations of HBsAg caused by genotypic diversity as well as mutation of the HBV genome. It is predictable that amino acid substitutions occurring in the HBsAg "a" determinant of a particular HBV genotype will affect the sensitivity of some diagnostic kits, since all the diagnostic kits currently available utilize monoclonal and/or polyclonal antibodies against the "a" determinant. One possible concern is that there may be a significant variation in the sensitivity of HBsAg diagnostic kits to HBsAg encoded by HBV of different genotypes, which might result in a failure to detect HBsAg of a particular HBV genotype. In this study, we assessed the reactivity of HBsAg specimens derived from three different HBV genotypes (A, B, and C) that are prevalent in Japan by 10 commercially available EIA (enzyme immunoassay), CLIA (chemiluminescent immunoassay), and CLEIA (chemiluminescent enzyme immunoassay) diagnostic kits. Specimens included both clinical samples and recombinant HBsAg. Our results showed that all the diagnostic kits evaluated were able to detect HBsAg irrespective of HBV genotypes. At the same time, it is apparent that some, but not all of the kits showed clear differences in sensitivity to the three HBV genotypes.

Murine macrophages produce secretory leukocyte protease inhibitor during clearance of apoptotic cells: implications for resolution of the inflammatory response.

Macrophage-derived secretory leukocyte protease inhibitor (SLPI) can be induced locally as well as systemically in response to microbial products such as LPS and lipotechoic acid. It is not known whether phagocytosis of apoptotic cells, an essential function of macrophages, can regulate expression and secretion of SLPI. In this study, we report that exposure of peritoneal macrophages of BALB/c mice or murine macrophage cell lines RAW264.7 and J774.1 to apoptotic target cells induced an elevation in SLPI secretion. Secreted SLPI retained its antichymotrypsin activity. SLPI expression in thymuses from BALB/c mice that had been injected with anti-CD3 Ab to induce apoptosis of thymocytes was also elevated both at the mRNA and protein levels. Colchicine, a microtubular inhibitor, blocked the internalization of apoptotic cells by macrophages but not SLPI secretion, suggesting that surface recognition of apoptotic cells is sufficient for the induction of SLPI. Exposure of RAW264.7 cells to apoptotic CTLL-2 cells induced both SLPI and TNF-alpha, and addition of IFN-gamma inhibited SLPI but augmented TNF-alpha production. Transfection of either the secreted or a nonsecreted form of SLPI into RAW264.7 cells led to suppression of TNF-alpha production in response to apoptotic cells. Thus, macrophages secrete an increased amount of SLPI when encountering apoptotic cells, which may help to attenuate potential inflammation during clearance of these cells.

Studies on the generation and maintenance of mucosal cytotoxic T lymphocytes against human immunodeficiency virus type 1 Gag in mice.

Aspects of the generation and maintenance of mucosal immunity against human immunodeficiency virus type 1 (HIV-1) were examined. Mice were immunized either intranasally or intrarectally with recombinant HIV-1 Gag p24 protein plus cholera toxin. Nasal immunization generated strong nasal IgA responses but low vaginal IgA, whereas rectal immunization yielded good vaginal IgA responses but poor nasal responses. Nasal immunization resulted in strong Gag-specific cytotoxic T lymphocyte (CTL) activity in nasal-associated lymphoid tissue (NALT), posterior cervical lymph nodes (pCLNs), and the spleen, but not in mesenteric lymph nodes (MLNs). Rectal immunization induced weak Gag-specific CTLs in the MLNs only, indicating distinct compartmentalization of the upper and lower mucosa. Combining nasal and rectal immunizations overcame their respective deficiencies. CTL memory after the third nasal immunization was found to persist for up to 6 months in the draining pCLNs, but was gradually lost from the NALT induction site. Analysis of the T cell receptor Vbeta usage of Gag-specific CD8(+) T cells in lymphoid tissues of intranasally immunized mice indicated that the memory CTLs in the pCLNs are generated from a few clones in NALT. The memory CTL clones also appear to be poor killers whereas the NALT clones from which the pCLN clones appear to originate are potent killers. Our results support the view that CTL activity is determined by the level and duration of antigen stimulation and that in NALT, CTLs develop as effector memory T cells with high avidity, whereas the pCLNs sequester the memory T cells with low avidity but longer survival.

Murine T cells expressing high activity of prolyl endopeptidase are susceptible to activation-induced cell death.

Prolyl endopeptidase (PEP) is widely distributed and thought to play an important role in the degradation of peptide hormones and neuropeptides, but its biological role is totally unknown. In this study, we examined PEP activity in subpopulations of murine T cells and found that PEP activity was significantly higher in immature thymocytes than in mature thymocytes or in peripheral T cells. Stimulation of murine peripheral T cells time-dependently increased PEP activity. Although murine T cell hybridomas exhibited high PEP activity, the PEP activity was fully inhibited by treatment with PEP inhibitor. The pretreated T cells were found to be resistant to activation-induced cell death (AICD). Similar results were obtained in murine thymocytes as well as in activated peripheral T cells. PEP activity in T cell hybridomas remained unchanged during AICD. These results suggest that T cells expressing high PEP activity are susceptible to ACID.