Norio Hayashi

Detection of Hepatitis C Virus RNA in Chronic Non-A, Non-B Liver Disease

Serum samples were tested for detection of hepatitis C virus (HCV) RNA from 156 patients with chronic non-A, non-B liver disease. HCV RNA was detected in 121 (93.8%) of 129 patients positive for anti-C100-3 but was also found in 15 (55.6%) of 27 patients negative for anti-C100-3. The rate of positivity for HCV RNA was not significantly different among various stages of liver diseases. These results showed that HCV continues to replicate even in advanced liver disease and that it seems to be related to half of the cases of chronic non-A, non-B liver disease negative for anti-C100-3.

Detection of hepatitis C virus RNA in liver tissues by an in situ hybridization technique

Hepatitis C virus (HCV) infection of cells in liver tissues was determined by detecting HCV RNA by an in situ hybridization technique using synthetic oligonucleotide probes derived from the 5-non-coding and core regions of HCV genome. Aggregated silver grains indicating hybridization with HCV RNA were observed over the nuclei as well as the cytoplasm of hepatocytes with none on non-parenchymal cells. The specificity of the hybridization was confirmed by absence of autoradiographic signals after ribonuclease predigestion, addition of an excess of non-labeled probes, or application of an M 13 probe. The hepatocytes with HCV RNA-positive signals were scattered in the periportal and mediolobular zones of liver lobules rather than in the pericentral zones. Fifteen out of 33 biopsy specimens from patients with chronic HCV infection studied had the HCV RNA-positive hepatocytes. These cells were more frequently detected in specimens with advanced periportal, bridging and intralobular necrosis but showed no correlation with the extent of inflammatory cell infiltration. These findings suggest a close correlation between the detection of HCV RNA in hepatocytes and advanced necrosis of the specimens.

Hepatitis B virus markers and antibodies to hepatitis C virus in Japanese patients with hepatocellular carcinoma

Sera from Japanese patients with chronic liver disease were tested for hepatitis B virus (HBV) markers and antibodies to hepatitis C virus (anti-HCV), and the results were correlated to the presence of hepatocellular carcinoma. In chronic non-A, non-B liver disease, anti-HCV prevalence was high both in patients with hepatocellular carcinoma (78/89, 88%) and without it (66/84, 79%), while previous HBV infection was more common in patients with hepatocellular carcinoma (65/89, 73%) than in those without it (46/84, 55%) (P<0.05). Coexistence of anti-HCV and antibodies to HBV was observed frequently in patients with hepatocellular carcinoma (56/89, 63%) compared with patients without it (39/84, 46%) (P<0.05). In chronic HBV carriers, anti-HCV was more common in patients with hepatocellular carcinoma (12/38, 32%) than in those without it (3/62, 5%) (P<0.01). These results suggest that infection with the two viruses may be a risk factor for more serious liver disease.

Expression of MBP-HCV NS1/E2 fusion protein in E. coli and detection of anti-NS1/E2 antibody in type c chronic liver disease

To characterize the putative NS1/E2 (non-structural protein 1/envelope 2) domain of HCV (hepatitis C virus), we expressed the hydrophilic three-quarters of this domain in a form of MBP (maltose binding protein) fusions in Escherichia coli. When we checked the positive frequency of antibody to this fusion protein, 17% of patients with type C chronic liver disease had this antibody. However, they were all positive for HCV-RNA in sera. These results suggest that the appearance of anti-NS1/E2 antibody does not serve as evidence of viral clearance.

Serodiagnosis of chronic hepatitis C in Japan by second-generation recombinant immunoblot assay

The seroprevalence of antibodies to hepatitis C virus (HCV) by a second-generation recombinant immunoblot assay (RIBA-2) was tested using 4 recombinant antigens. The results were correlated with those of C100-3 enzyme-linked immunosorbent assay (ELISA) and with the detection of HCV-RNA sequences by the polymerase chain reaction. Sera were obtained from 27 C100-3 ELISA-positive Japanese patients with chronic non-A, non-B liver disease and from 29 C100-3 ELISA-negative patients. All C100-3 ELISA-positive patients and 19 (66%) out of 29 C100-3 ELISA-negative patients reacted to two or more RIBA-2 antigens (reactive by RIBA-2). Of the remaining 10 C100-3 ELISA-negative patients, one patient reacted to just one antigen (indeterminate by RIBA-2), while 9 reacted to none of the 4 antigens (non-reactive by RIBA-2). Forty-three (93%) of the 46 RIBA-2-reactive patients and the single RIBA-2-indeterminate patient tested positive for HCV-RNA sequences, whereas only one (11%) of the 9 RIBA-2-non-reactive patients tested positive. These findings indicate that HCV infection is more prevalent than expected from the results of C100-3 ELISA, and that RIBA-2 is a more sensitive assay for estimating the presence of HCV infection in chronic liver disease.

HCV RNA and antibody to HCV core protein in Japanese patients with chronic liver disease

We evaluated the prevalence of hepatitis C virus (HCV) RNA and antibody (anti-HCVcore) to the putative HCV core protein in Japanese patients with chronic liver disease. Sera were screened by solid-phase enzyme immunoassay with a recombinant HCV core protein and by the reverse transcription-polymerase chain reaction (RT-PCR) test which directly detects the HCV genome. Anti-HCVcore was detected with high titers in 95% (69/73) of chronic non-A, non-B hepatitis, and 94% (65/69) of anti-HCVcore-positive patients had the genome. Anti-HCVcore was also found with lower titers in 24% (10/41) of chronic hepatitis B virus carriers, and three of them had the genome. Only one (3%) of the 35 patients negative for anti-HCVcore tested positive to RT-PCR. These findings indicate the overwhelming prevalence of HCV infection in Japanese patients with chronic non-A, non-B hepatitis and a close relationship between the presence of anti-HCVcore and chronic hepatitis C in this population.

Two-dimensional flow cytometric analysis of intrahepatic lymphocyte subsets from patients with chronic hepatitis

Peripheral and intrahepatic lymphocyte subsets were analyzed in 22 patients with chronic hepatitis by two-dimensional flow cytometry. Activated T cells in the liver significantly increased compared with those in the peripheral blood. Helper T cells increased, but the CD4+ cells decreased due to a marked decrease of supppressor inducer T cells. CD8+ cells increased due to a increase of both cytotoxic T and suppressor T cells. Fc-receptor-positive cells, which increased significantly, were not NK cells but Fc-receptor-bearing T cells. In comparison with immunohistochemical methods, flow cytometric analysis enables more objective quantitation and simpler two-color staining of intrahepatic lymphocytes. Our findings using this method suggest hepatocellular injury may be generated not only by cytotoxic T cells but also by Fc-receptor-bearing T cells.

Three cases of posttransfusion hepatitis C treated with interferon-alpha. Confirmation of a carrier state by detection of hepatitis C virus RNA after interferon therapy.

SummaryInterferon therapy is useful for decreasing the serum ALT level and improving liver histology in patients with chronic non-A, non-B hepatitis. This study examined the effect of interferon therapy in acute cases of posttransfusion hepatitis C. we report on three cases in which interferon alpha was administered at 100–220.5 million units. HCV RNA became undetectable during interferon administration and the ALT level declined to the normal range. However, after the cessation of the therapy, the ALT level began to fluctuate and HCV RNA reappeared in two patients. We concluded that interferon therapy for the acute phase of posttransfusion hepatitis is useful for suppressing viral replication and quickly improving the ALT level, but it can not always prevent the development of chronic hepatitis. Furthermore, there was a close correlation between the profile of HCV RNA and that of the ALT level, indicating that the replication of HCV plays an important role in liver injury.