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In this study, the serum concentrations of human hepatocyte growth factor (HGF) were examined to clarify the relationship between HGF and interferon (IFN) therapy for hepatitis C. The subjects were 94 patients with chronic hepatitis C who underwent liver biopsy at our institution from 1994 through 1996. These patients were treated with natural IFN-α, IFNα2a and IFNα2b for periods varying from 12 to 26 weeks. Serum levels of HGF were determined in individual patients just before and after the administration of IFN and at 6 months and 1 year thereafter. The serum concentration of HGF was evaluated using enzyme-linked immunosorbent assay kits. The intra-hepatic location of HGF was explored using an immunoperoxidase-staining method. A positive correlation was found between the degree of HGF expression in the liver and the serum HGF concentrations. The degree of HGF expression in the liver decreased in the virologically sustained responders (SVRs) following IFN therapy. The serum HGF concentrations immediately after IFN therapy were lower than those before therapy in 83% of the patients. The concentrations gradually rose thereafter in about 45% of the non-responders, while it remained low or declined further in about half of the patients in this group. In the SVRs, the serum HGF concentrations declined in 88% of patients immediately after IFN therapy. Thereafter, it remained equally low or declined further in 60% of the SVRs. The serum HGF concentrations at 6 months and 1 year after IFN therapy were significantly lower in the SVRs than in the non-responders. In conclusions, serum HGF concentrations declined following IFN treatment regardless of the virological outcome of treatment. The decrease in serum HGF concentrations results from a decrease in the number of mesenchymal cells producing HGF. Consequently, evaluation of the serum HGF concentration is of clinical value for assessing changes in liver tissues after IFN therapy.

Objectives: The aims of this study were to determine the hepatitis B virus (HBV) genotypes in hepatitis delta virus (HDV) RNA-positive patients and to characterize the HBV nucleotide sequences that may be found on a distant island of Japan. Methods: This study included three patients with chronic hepatitis who were positive for hepatitis B surface antigen (enzyme-linked immunosorbent assay; ELISA), HDV antibody (ELISA) and HDV RNA by polymerase chain reaction (PCR). The HBV genotype was determined by nested PCR using type-specific primers. The first-round PCR products from two patients were sequenced, followed by an investigation of nucleotide homology. Results: Viruses from all three patients in this study were classified as HBV genotype B. Comparison with HBV isolates from geographically neighboring regions revealed that the two HBV isolates had 97.9–98.6% identity at the nucleotide level to a Chinese isolate, 98.3–98.6% identity to the Okinawa isolate and 98.6–98.8% identity to a Japanese isolate of genotype B. On phylogenetic analysis, the HBV isolates from the two patients were classified as HBV genotype B. The HBV isolates of cases 1 and 3 clustered in the same group as isolates from the Chinese mainland and Japanese mainland, which are geographically near Miyako Island. Conclusion: The HBV isolates coinfected with HDV found on Miyako Island were of genotype B. The PCR method based on genotype-specific primers was useful in determining HBV genotypes.

China is an area of high endemicity for viral hepatitis, and the molecular epidemiological investigation of TT virus (TTV) infection is of interest. In the present study, we investigated the epidemiology, clinical significance and molecular characteristics of TTV infection in patients with chronic hepatitis B and C in Yanbian City, China. Serum samples obtained from 74 patients with hepatitis B and hepatitis C who visited Yanbian Hospital, located in northeast China, were analyzed in this study. The study group included 22 cases of chronic hepatitis B (B-CH), 17 cases of liver cirrhosis B (B-LC), 7 cases of hepatocellular carcinoma (B-HCC), 16 cases of chronic hepatitis C (C-CH), 11 cases of liver cirrhosis C (C-LC) and 1 case of hepatocellular carcinoma (C-HCC). Detection of TTV DNA was performed as described by Nishizawa et al. The second-round PCR products from 7 subjects were sequenced, followed by investigation of nucleotide homology and phylogenetic analysis. TTV DNA was present in 18.2, 5.9, 28.6, 6.3, 9.1 and 0% of the patients with B-CH, B-LC, B-HCC, C-CH, C-LC and C-HCC, respectively. The highest prevalence of TTV infection was seen in the groups aged 40–50 and over 60 years. There was no significant correlation between the presence of TTV DNA and the clinical parameters in patients with hepatitis B and C. The various isolates showed 97.9–100% with isolates reported previously from Japan and 98.4–100% with isolates reported previously from China. Nucleotide sequence analysis revealed that the Yanbian isolates could be classified in the same group as the Japan and China isolates. We concluded that chronic coinfection with TTV did not affect the serological features of chronic hepatitis B and C in China, as found in Tokyo, Japan.

The organ where the GB virus (GBV)-C/hepatitis G virus (HGV) localizes and proliferates is not known. We examined the digestive organs for HGV RNA to determine the localization of the HGV. Two cases of patients with serum-positive HGV RNA were investigated. We embedded surgically excised materials and digestive secretion materials from cases 1 and 2 in paraffin blocks. The tissue specimens investigated included lymph nodes No. 201 and 202, ascending colon (nontumor and tumor area), ileocecum, appendix, liver (nontumor and tumor area) and gall bladder. We made cDNA after extraction of total RNA from thin tissue sections and detected HGV RNA with a reverse transcription polymerase chain reaction method. No HGV RNA was detected in liver, colon and gall bladder tissues. HGV RNA was only detected in the appendix tissue. Comparison of nucleotide sequences of PCR products from serum and appendix was almost the same. Homology between US type (PNF2161) and the serum and appendix PCR products was 92.6 and 93.6%, respectively. These results suggest that HGV proliferates in the appendix and is carried by the portal blood flow to the liver, and may cause a hepatitis reaction in the liver.

We determined hepatocyte growth factor (HGF) levels in the serum and liver of patients with hepatitis C and assessed the relationship to histological findings of the liver and hepatitis C virus-related markers in the serum in patients with type C liver diseases. The subjects were 108 patients with chronic hepatitis C (CH), 70 patients with liver cirrhosis C (LC), 38 patients with hepatocellular carcinoma (HCC) and 20 patients with acute hepatitis (AH). As normal controls 20 subjects were studied. The serum HGF levels were measured using an enzyme-linked immunosorbent assay kit. Intrahepatic HGF was investigated by immunoperoxidase staining using monoclonal HGF antibody. The serum HGF level was highest in patients with AH. The serum HGF levels tended to be higher in patients with LC and HCC than those with CH. Further, the serum HGF level was related to the degree of intrahepatic inflammatory cell infiltration and fibrosis, and intrahepatic HGF was noted primarily in the cell membrane of mesenchymal cells in focal necrosis. The degree of intrahepatic HGF expression tended to be higher in patients with high serum HGF levels. In patients with HCC, however, HGF showed little localization in cancer cells, but was noted in infiltrating mesenchymal cells in both cancerous and noncancerous regions. In conclusion, the measurement of serum HGF levels may be useful for estimating the degree of intrahepatic inflammatory reaction and fibrosis. Although further study is necessary, the high serum level of HGF revealed high carcinogenic states in chronic hepatitis and liver cirrhosis type C.

Hepatitis B virus (HBV) DNA has been detected in the sera of hepatitis patients who are negative for hepatitis B surface antigen (HBsAg) by polymerase chain reaction (PCR). The purpose of the present study was to clarify the clinical characteristics of patients with chronic hepatitis C who are negative for serum HBsAg and positive for HBV DNA. The subjects included 49 patients with chronic hepatitis C who were negative for serum HBsAg and 119 blood donors who served as healthy controls. Serum samples were tested for the presence of HBV DNA by the nested PCR method. Serum HBV DNA was detected in 18 (37.7%) of the 49 chronic hepatitis C patients and in none (0%) of the 119 blood donors. Among the hepatitis C patients, HBV DNA was detected in 20.7% of those who were negative for all HBV-associated markers and in 57.1% of those who were positive for one or more HBV-associated marker. The HBV DNA-positive rate among those in each F stage did not significantly differ. The liver function parameters of the HBV DNA-positive and the HBV DNA-negative chronic hepatitis C patients did not significantly differ. These results suggest that hepatitis C virus is frequently coinfected with serum HBsAg-negative HBV, and that the incidence of HBV infection in blood donors is low. However, it is considered that HBsAg-negative HBV infection does not modify the blood biochemical features of chronic hepatitis C.

We tested the sera of patients with non-A, non-B, non-C, non-G (non-A to -G) hepatocellular carcinoma (HCC) for the presence of TT virus (TTV) DNA and clinicopathologically elucidated the relationship between TTV infection and hepatocarcinogenesis. The study cohort consisted of 19 patients with non-A to -G HCC. Detection of TTV DNA was performed by the nested polymerase chain reaction according to a previously published method. TTV DNA was detected in the sera of 9 (47.4%) of the 19 patients with non-A to -G HCC. The clinical background factors and blood biochemical parameters of the TTV-DNA-positive and -negative HCC patients did not significantly differ. Three TTV-DNA-positive and 2 TTV-DNA-negative patients underwent surgical resection of the HCC. The histological findings in the non-cancerous liver tissue of the TTV-DNA-positive and -negative patients did not significantly differ. In conclusion, TTV infection does not affect the features of non-A to -G HCC.

Although human hepatocellular carcinoma (HCC) is one of the most common types of tumors in the world, the molecular mechanisms underlying hepatitis-C-related human hepatocarcinogenesis are still not clear. HCC is accompanied by virus infections in most cases, and it is suggested that hepatitis B virus and hepatitis C virus (HCV) significantly influence the oncogenic process. The persistence of inflammation following HCV infection is reportedly related to carcinogenesis, and the mechanism of chronic inflammation has been approached by taking viral, immunologic, cytokine and apoptotic responses into consideration. With the progress made in molecular biology, the functional abnormality of oncogenes/tumor suppressor genes has been identified and, apart from the p53 gene, involvement of the IGF-II gene has also been described recently. Furthermore, it has been suggested that uncontrolled proliferation of cancer cells might be based on abnormal regulation of intracellular signal transduction pathways. Here we review the cutting edge of molecular hepatitis C virology in terms of virus-cell interactions, which may contribute to the development of human HCCs. We also discuss the recent progress made in the molecular and cell biology of human hepatocarcinogenesis.

With the evolution of confirmatory assays for hepatitis C viral infections, bulk populations of unknown hepatitis were diagnosed as hepatitis C virus (HCV)-mediated chronic liver dysfunction throughout the world. More recently, several systems for molecular diagnosis of hepatitis C viral infections were developed. These are commonly performed for efficient antiviral therapy using interferon. Now we are at the crossroads studying hepatitis virology for the development of sensitive and powerful treatment against HCV-mediated chronic liver dysfunction. Here we introduce the current strategy for the advanced diagnosis of hepatitis C viral infection and discuss the exploration of novel hepatitis viruses.

Objective: We measured the concentrations of serum intercellular adhesion molecule 1 (sICAM-1) in patients with C-viral chronic liver diseases and started prospective studies immediately thereafter, in order to determine whether the concentration of sICAM-1 is useful for predicting the occurrence of hepatocellular carcinoma (HCC) following C-viral chronic hepatitis (CH) and liver cirrhosis (LC). Methods: We studied 74 patients with CH, 18 with LC, and 28 patients with HCC who visited our institute from 1993 through 1996. All were positive for hepatitis C virus RNA in the blood. The concentrations of sICAM-1 were measured using enzyme-linked immunosorbent assay kits. The expression of ICAM-1 in the liver was detected by indirect immunoperoxidase staining. Results: The concentrations of sICAM-1 were significantly higher in patients with LC and HCC than in patients with CH. The sICAM-1 concentrations were high in patients whose platelet counts were low. ICAM-1 in the liver was localized to the endoplasmic reticulum or the membrane of cancer cells. The cumulative rate of occurrence of HCC from CH or LC was significantly higher in the high-sICAM-1 group (>400 ng/ml) than in the low-sICAM-1 group. Multivariate analysis revealed that elevation of the sICAM-1 concentration is a significant risk factor for the occurrence of HCC. Conclusion: Evaluation of the sICAM-1 concentration is useful for prediction of the occurrence of HCC in patients with C-viral CH or LC.

The aims of this study were to determine the full-length genome sequences of hepatitis delta virus (HDV) in HDV RNA-positive subjects, and to elucidate the molecular specificity of the HDVs that are clustered on a distant island in Japan. This study included 3 subjects with chronic hepatitis who were positive for hepatitis B surface (HBs) antigen and HDV RNA, and who were admitted to the Okinawa Prefectural Miyako Hospital in 1998. The full-length genome sequence of HDV was determined by nested polymerase chain reaction (PCR) using four kinds of primer sets. The genomic length of HDV was 1,675, 1,679 and 1,681 base pairs, respectively. There was 90–92% nucleotide homology between each pair of isolates. In comparison with HDV isolates in geographically neighboring regions, the nucleotide homology of the 3 HDV isolates were 73–75% with the China isolate of genotype I; 77–78% with the Taiwan isolate of genotype I; 83–84% with a Japan isolate of genotype IIa; 85–87% with the Taiwan isolate of genotype IIa, and 87–88% with the Taiwan isolates of genotype IIb. Therefore, the Miyako Island isolates had high homology with the Taiwan isolate of genotype IIa and IIb. Phylogenetic analysis of the full-length genome sequences of HDV revealed that the two Miyako Island isolates were classified into a genotype IIb’. The other one was classified as genotype IIb. In conclusion, the HDV of Miyako Island isolates can be classified as a novel subgroup of genotype IIb, designated type IIb’, and genotype IIb.

Recently, a new method of extracorporeal granulocyte depletion apheresis has been developed to treat inflammatory systemic diseases using an Adacolumn (Japan Immunoresearch Laboratories, Takasaki, Japan) that is filled with acetate cellulose beads (G-1 beads) to adsorb the granulocytes. We examined whether hepatitis C virus (HCV) is adsorbed after incubation of the Adacolumn with the sera from patients with HCV-RNA-positive chronic hepatitis C. Patients and Methods: A total of 10 patients with chronic hepatitis C, whose levels of HCV RNA were greater than 800 kIU/ml were examined. The serum was incubated with 500 G-1 beads in a syringe at 37°C for 1 h. After removal of the serum, the beads were washed with RNase-free water. The G-1 beads were removed from the syringe after centrifugation. RNA was extracted from 200 µl of the wash waste and from 10, 50, 100 and 200 beads, respectively, using TRIZol regent. Detection of HCV RNA was performed using the nested PCR method. Results: HCV RNA was detected from as few as 10 G-1 beads. HCV RNA was not detected from waste fluid collected after the last wash from any of the patients. Further, HCV RNA was detected in the initial waste fluid after the 37°C incubation with serum in all of the patients. Since HCV RNA was detected on the G-1 beads, but not from the last washing solution in the current examination, these results suggest that the G-1 beads adsorbed HCV RNA. Conclusions: Our in vitro study confirmed that G-1 beads adsorbed HCV; therefore, apheresis using a column filled with G-1 beads may reduce the HCV RNA load in the blood of patients with chronic hepatitis C.

Objective: Because the determination of the stage of fibrosis depends on rather subjective judgment, more objective parameters are needed. In this study, we followed the long-term outcome, with monitoring of platelet counts, in patients with chronic hepatitis C or liver cirrhosis (LC) who had undergone interferon (IFN) therapy. Methods: 596 patients who were diagnosed at our institute from 1987 to 1998 with chronic hepatitis C and LC were treated with IFNs. A further 58 patients were not treated (NT). The annual rate of changes in platelet counts were calculated and compared for IFN-treated and NT patients. Results: The relationship between the efficacy of IFN therapy and the incidence of hepatocellular carcinoma (HCC) showed that the patients who were virologic sustained responders (VSR) had a significantly lower incidence of HCC than the nonresponders (NR) and NT patients. The change in platelet counts was +4,350/µl/year in the VSR, +1,010/µl/year in the biochemical sustained responders (BSR), –4,540/µl/year in the NR and –6,180/µl/year in the NT patients, indicating a significant platelet increase in the VSR, a decrease of the same magnitude in the NR and NT patients, and no change in the BSR. The cumulative probability of developing HCC and liver failure was significantly higher in groups with decreased platelet counts than in groups with increased platelet counts among patients who had undergone IFN therapy. Multivariate analyses revealed that a decrease in platelet counts was the cardinal risk factor for development of HCC and liver failure in chronic hepatitis C or LC patients. Conclusion: Investigation of platelet counts was useful for determining the long-term outcome of patients who had undergone IFN therapy and for predicting the development of HCC.

Objective: We investigated the background clinical factors of patients with hepatocellular carcinoma (HCC) diagnosed at our institute and, from these results, determined those factors important for evaluation of a population at high risk of HCC. Methods: This study comprised 250 patients diagnosed with HCC from 1990 through 1995 in the Nihon University Itabashi Hospital. Background clinical factors, such as the results of blood chemistry at the time of the first angiography, were examined. Results: Markers of viral hepatitis in the 250 cases were as follows: type B (B-HCC), 29 (11.6%), type C (C-HCC), 201 (80.4%); type B+C, 3 (1.2%), and non-B, non-C (NBNC-HCC), 17 (6.8%). Approximately 35% of B-HCC and NBNC-HCC cases, but only 6% of C-HCC cases, exhibited platelet counts (PLT) equal to or more than 200,000/ml. On the other hand, 56.5% of the C-HCC cases exhibited PLT less than 100,000/ml, in contrast to less than 30% of the B-HCC and NBNC-HCC cases. Irrespective of the etiology of HCC, male sex and a history of smoking were characteristic risk factors. The percentages of abnormal results in combinations of tests in the B-HCC, C-HCC and NBNC-HCC subsets were: 69, 97 and 70% in the aspartate aminotransferase (AST) + PLT group (group A); 83, 98 and 70% in the group A + alanine aminotransferase (ALT) group (group B), and 86, 98 and 100% in the group B + γ-glutamyl transpeptidase (γ-GTP) group (group C), respectively. When hepatitis B surface antigen (HBsAg) and hepatitis C antibody (HCV-Ab) were also taken into account, abnormal results were found in every case in all of the HCC groups. Conclusion: C-HCC was found predominantly in cases with liver cirrhosis, whereas B-HCC and NBNC-HCC were observed more frequently in cases without liver cirrhosis. Testing for HBsAg and HCV-Ab, in addition to AST, ALT, PLT and γ-GTP, is considered necessary when screening for HCC.

A hepatitis B virus (HBV) carrier who is positive for hepatitis B surface (HBs) antigen but negative for hepatitis B core (HBc) antibody despite persistent HBV infection, is designated has having hepatitis B virus 2 (HBV2). HBV2 is reported to be induced by mild-grade hepatitis. Patients with HBV2 have been reported in Taiwan and Senegal. In the present study, we determined the nucleotide (nt) sequence of the precore/core gene coding region and X gene region of the HBV DNA sequence in 7 subjects who were positive for HBs antigen and negative for HBc antibody. HBV DNA was detected by nested polymerase chain reaction (PCR). Nested PCR was carried out to amplify the precore/core and X open reading frames (ORFs) of HBV DNA. The second PCR products were sequenced, followed by investigation of nt homology. There were no deletions nor insertions in the nt sequence of the precore/core and X ORFs in the HBV DNA of these 7 patients, and mutations were found only sporadically in the 7 patients. Also, there were no common amino acid substitutions in the examined regions of the amino acid sequence of HBV in the 7 patients, and we could not find a common mutation in the examined regions of HBV DNA that could potentially contribute to the development of negativity for HBc antibody. Thus, it is suggested that negativity for HBc antibody in patients with HBV2 is due to an immune response abnormality in the host.

Objective: To evaluate changes in serum immunoglobulin M (IgM) and G (IgG) hepatitis C virus (HCV) core antibodies after HCV infection in acute hepatitis C. Methods: Serum HCV RNA and IgM and IgG HCV core antibodies were investigated using sera sequentially sampled from three chimpanzees experimentally infected with HCV. Serum IgG HCV core antibody titer was measured using a JCC.2 enzyme-linked immunosorbent assay (ELISA) kit (Chemo-Sera-Therapeutic Research Center, Kumamoto, Japan). IgM core antibody titer was measured using horseradish peroxidase-labeled monoclonal anti-human IgM as the secondary antibody for the JCC.2 ELISA kit. Serum HCV RNA was detected using the 5′ noncoding region as the primer according to the reverse transcriptase (RT) nested polymerase chain reaction (PCR) and competitive RT-PCR method. Results: IgM JCC.2 antibody was detected when alanine aminotransferase (ALT) peaked, showing the closest correlation with the changes in ALT. A period during which IgM JCC.2 antibody was positive but HCV RNA as determined by RT-nested PCR was negative was observed after the elevation of ALT level. Conclusion: These results indicate the usefulness of detection of serum IgM JCC.2 antibody in making a definitive diagnosis of acute hepatitis C and the follow-up observation of hepatitis C.