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Serum and liver tissues from hepatitis B surface antigen‐negative/anti‐hepatitis C virus (HCV)‐negative (non-B, non-C) hepatocellular carcinoma (HCC) patients in Japan were examined for the presence of hepatitis B virus (HBV), HCV, and TT virus (TTV) by polymerase chain reaction. The studies evaluated the contribution of these viruses to pathogenesis of HCC. HBV DNA was detected in the sera of 20 (47.6%) of 42 non-B, non-C HCC patients, which was significantly higher than in age-matched controls without liver disease ( ). P ! .001 In 8 of 12 patients with liver tissues available, HBV DNA was detected in cancerous and adjacent noncancerous liver tissues. No HCV RNA was detected. The positivity for TTV DNA was not significantly different between HCC patients and controls. These results indicate that HBV is associated with a substantial proportion of non-B, non-C HCC cases in Japan. The role of HBV in hepatocarcinogenesis in such patients needs to be clarified.

Objective To assess the efficacy of low-dose oral human interferon-α (LDO-IFN) (MR-22A) in the treatment of chronic viral hepatitis type B. Methods One of 4 doses (single tablet: 50 IU, 150 IU, 450 IU, 900 IU) of MR-22A or a single tablet of placebo was administered through the oral mucosa once daily for 24 weeks. Results At the end of the administration period, the proportion of patients who had become hepatitis B virus (HBV)-DNA negative was 6 (20.0%) of 30 in the placebo group, 4 (12.9%) of 31 in the 50-IU group, 6 (18.8%) of 32 in the 150-IU group, 2 (6.9%) of 29 in the 450-IU group, and 4 (16.0%) of 25 in the 900-IU group. The proportion of patients who had become hepatitis B e antigen (HBeAg) negative was 5 (17.9%) of 28, 4 (14.8%) of 27, 5 (16.7%) of 30, 4 (14.8%) of 27, and 1 (5.3%) of 19, respectively. None of the differences were statistically significant between the treatment and placebo groups in the proportion of patients who became HBV-DNA negative or HBeAg negative. No statistically significant differences were observed in patients given LDO-IFN or placebo in improvement of liver function. Adverse drug reactions were observed in 4(12.5%) of 32 patients in the placebo group, 8 (24.2%) of 33 in the 50-IU group, 10 (30.3%) of 33 in the 150-IU group, 10 (29.4%) of 34 in the 450-IU group, and 7 (21.9%) of 32 in the 900-IU group. One patient in the 50-IU group experienced moderate urticaria; all other adverse events were mild. Conclusion LDO-IFN was not shown to be clinically effective in the treatment of chronic viral hepatitis type B with the route of administration, dosing levels, and methods of assessment used in this study.

症例は77歳, 男性. 平成5年, 近医にてAIHと診断され無治療で経過観察されていた. 平成9年5月, 肝細胞癌 (HCC) とその骨転移を指摘され, 当院内科に紹介入院となる. 入院加療によっても, 腫瘍の縮小は認められず外来での経過観察となり, 同年9月死亡し剖検となった. 血清および肝組織にHBV-DNA, HCV-RNAが検出されず, 高齢男性の非硬変期AIH例に合併したHCCの1剖検例を経験したので報告する.

We studied the mutation patterns of hepatitis C virus (HCV) and GB virus C/hepatitis G virus (HGV). Although the mutation patterns of the two viruses were similar to each other, they were quite different from that of HIV. In particular, the similarity of the patterns between HCV or HGV and human nuclear pseudogenes was statistically significant whereas there was no similarity between HIV and human nuclear pseudogenes. This finding suggests that the mutation patterns of HCV and HGV are similar to the patterns of spontaneous substitution mutations of human genes, implying that nucleotide analogues which are effective against HCV and HGV may have a side effect on the normal cells of humans.

GB virus C (GBV-C) is related to hepatitis C virus (HCV) and has a similar genomic structure. Some predictors for the efficacy of interferon (IFN) therapy on HCV have been reported: genotype, viral load, IFN dose, and the amino acid substitutions in the NS5A region, designated as the interferon sensitivity determining region (ISDR). To evaluate the correlation between the amino acid substitutions in the GBV-C NS5A region and the response to IFN therapy, single-strand conformation polymorphism (SSCP) analysis was performed in the 12 concomitantly GBV-C–and HCV–infected patients who received IFN therapy at three time points: before, endpoint, and after the IFN therapy. The region in the GBV-C NS5A studied includes the amino acids that exhibit some homology to the ISDR and the various substitutions. By SSCP analysis, amplicons were separated into 1–4 bands, which indicated the existence of heterogeneity in each host. However, the deduced amino acid sequences in these bands exhibited no characteristic differences among these strains irrespective of response to IFN therapy. Of the 32 strains separated by SSCP, 7 strains were responders, and 25 were nonresponders. The mean amino acid substitution, compared with the consensus sequence of nonresponders, was 1.00 ± 0.93 among responders, and 1.40 ± 0.85 among nonresponders (P = NS). No correlation between the amino acid sequence in the GBV-C NS5A region and response to IFN therapy was found, indicating that the GBV-C NS5A region dose not act as the ISDR. J. Med. Virol. 57:376–382, 1999. © 1999 Wiley-Liss, Inc.

We have developed a sensitive and quantitative assay using transcription-mediated amplification and hybridization protection assay for the detection of hepatitis B virus (HBV) DNA in serum. The transcription-mediated amplification was carried out in a single tube. The hybridization protection assay was carried out in a microtiter plate with two probes with different specific activities to obtain a broad detection range. As a result, the assay had a detection range of 5 × 10 3 to 5 × 10 8 genome equivalents (GE)/ml and good quantitative accuracy on a logarithmic scale. A moderately sized manual assay run can be completed within 5 h. Measurements of the amounts of HBV DNA in clinical samples by the assay showed the amounts under various disease conditions to be widely distributed (more than 5 logs, from approximately 5 × 10 3 to 5 × 10 8 GE/ml). It was also shown that the amount of HBV DNA in one chronic hepatitis patient varied widely, with a range of more than 5 logs during long-term monitoring. Our assay has the potential to be used to monitor and determine the prognosis of HBV patients and carriers, especially during interferon treatment.

A marked increase in the rate of eradication of hepatitis C virus (HCV) has been achieved by the combination of interferon-α2b and ribavirin when compared with interferon-α2b alone. However, even with combination therapy, hepatitis persists in more than half of the patients with chronic herpatitis C and progresses to liver cirrhosis and hepatocellular carcinoma with time. What needs to be kept in mind is that, whether by its natural course or by therapy to suppress liver inflammation or by interferon therapy, the rate of development of hepatocellular carcinoma is reduced if ALT is maintained at low levels. Attention should therefore be focused on the development of drugs which enhance the effect of interferon as well as drugs which suppress liver inflammation.

A questionnaire survey was conducted of members of the NANB Hepatitis Research Group of the Japanese Ministry of Health and Welfare on the incidence of GBV-C/HGV RNA-positivity among HCV, HBV, and non-B-C infected patients to clarify the involvement of the newly discovered GBV-C/HGV in hepatic diseases in Japan. As a result, GBV-C/HGV RNA was detected in about 10% of HCV-infected patients, 5% of HBV-infected patients, and 5% of patients with non-B non-C hepatitis. With HBs antigen-positive patients, the GBV-C/HGV positivity rate increased with the progression of liver disease from 3.9% with chronic hepatitis, 5.4% with liver cirrhosis, and 9.1% with hepatocellular carcinoma. This increase was not observed with HCV RNA-positive and non-B, non-C patients. Also no GBV-C/HGV RNA-positive patients were seen among HCV RNA-positive fulminant hepatitis patients having no history of use of blood or blood products. These results suggest that although GBV-C/HGV may possibly be involved as a causative virus in some non-A-E hepatitis in Japan, it is not a main cause. If HBV-infected patients are excluded, it is almost possible to conclude that GBV-C/HGV is not involved in the progression of chronic liver disease. Although a very slight possibility exists that GBV-C/HGV infection may be a cause of fulminant hepatitis, the data suggest that it is not a major cause.

No analysis has been done of the ambisense of GB virus C (GBV-C). When the anti-genomes of 16 reported sequences of GBV-C were analyzed, nucleotide codons 1758 and 1402 within the anti-genome were conserved initiation and stop codons, respectively. Nucleotide sequences were also determined within the same region of 22 GBV-C strains. The anti-genomes of 38 sequences were translated and a consensus sequence was determined. In accordance with the consensus sequence, overlapping peptides were synthesized and used for the detection of anti-synthetic peptide antibodies by ELISA. The positivity of antibodies among sera with GBV-C RNA was significantly higher than among sera without GBV-C RNA (66.7% vs. 15.6%), regardless of the simultaneous presence of hepatitis B surface antigen or antibodies to hepatitis C virus (P < .05). These results indicated that a novel protein associated with GBV-C might be expressed from the ambisense of this virus.

GB virus C/hepatitis G virus (GBV-C/HGV) is related distantly to hepatitis C virus (HCV). HCV has a hypervariable region (HVR), and exists as quasispecies in vivo. Although GBV-C/HGV also has replaceable amino acids in the presumed antigenic region, the existence and fluctuation of population of heterogeneous virus have not been evaluated. In this study, the heterogeneity of GBV-C/HGV and HCV was investigated by the single-strand conformation polymorphism (SSCP) analysis in six concomitantly infected patients. Two patients were observed for 4 years without any treatment, and four were treated with interferon-α (IFN). By SSCP analysis, amplicons of GBV-C/HGV RNA were separated into 1–5 bands on gels for each patient. The amplicons had different nucleotide but the same amino acid sequences in the presumed antigenic region. The amplicons of HCV RNA, separated into 1–4 bands, had different nucleotide and amino acid sequences in the HVR. In the two patients without treatment, the predominant strain of GBV-C/HGV was unchanged for the 4 years. In the four patients administered IFN, some strains of GBV-C/HGV disappeared after IFN therapy, whereas other strains persisted. The mean genetic distance among GBV-C/HGV strains represented by SSCP analysis was significantly lower than that of HCV (P < 0.05). The data indicate that: 1) GBV-C/HGV can be devoid of antigenic drift unlike HCV; 2) GBV-C/HGV has no HVR as seen in HCV in the presumed antigenic region; and 3) the sensitivity to IFN differs among GBV-C/HGV strains in the same hosts, as with HCV. J. Med. Virol. 55:109–117, 1998. © 1998 Wiley-Liss, Inc.

To define the duration of viremia in the course of acute hepatitis B, we semiquantitatively determined the levels of hepatitis B virus (HBV) DNA in the sera, using polymerase chain reaction (PCR) coupled with Southern blotting, of non-immunocompromised patients with self-limited acute hepatitis B. In the sera of 10 of 11 patients, HBV DNA, which was presumably coated with viral proteins, was detected for a long period after recovery, even at the final observation times, which ranged from 6 to 19 months after disease onset. To characterize the mode of HBV that was present in serum, we immunoprecipitated immune complexes in sera by the addition of anti-human immunoglobulin G (IgG) and determined the levels of HBV DNA separately in the supernatants and pellets. In the acute phase of hepatitis B, high levels of HBV DNA were detected both in the supernatants and pellets at comparative levels. After the convalescent phase, the amount of HBV DNA in the supernatant decreased with respect to that in the pellets. It is notable that, in most cases, serum HBV persisted as a form of immune complex even after the seroconversion to antibody to hepatitis B surface antigen (anti-HBs). These data suggest that the replication of HBV may persist in some organs, most likely in the liver or peripheral blood cells, for a long period after recovery from acute hepatitis B, and the data indicate the possible transmission of HBV from organ transplantation donors who exhibit serological markers of past infection only.

症例は35歳男性. 平成6年11月の検診にて肝機能異常を指摘され当科外来を受診, ALTの高値変動とHCV-RNA陽性を認めた. 平成7年4月腹腔鏡下肝生検を施行, C型慢性活動性肝炎 (F2/A2) と診断した. 同年5月20日よりIFN-α (天然型) 10MUの2週連投後3回/週を22週, 計24週の治療を開始した. 投与開始13週後頃より, 両側乳房の腫脹および乳頭の疹痛を自覚, 他覚的にも女性化乳房が出現した. 投与終了時に自他覚所見は最大となり, 以後次第に軽快し10週後に完全消失した. IFN投与中の血中総コレステロールは低値を示し, 症状出現時の血中プロゲステロンは1.2ng/mlと軽度高値を認めたが, 卵胞刺激ホルモン (FSH), 黄体化ホルモン (LH), エストロゲンは, 正常範囲内であった. 経過中, 新たな薬剤の使用はなく, 女性化乳房はIFN投与によるものと考えられた.

症例は23歳, 女性. 飲酒歴, 輸血歴および鍼灸治療歴はない. 常用薬剤なし. 感冒様症状に伴う食欲不振, 腹部不快感および2週間で約4kgの体重減少を主訴に近医を受診した. 生化学検査でtransaminaseの上昇を認め急性肝炎が疑われ当科紹介, 入院となった. 入院時現症では標準体重の-32%のるいそうを認め, 入院時検査所見では総蛋白, コリンエステラーゼの低下及びtransaminaseの上昇が認められた. しかし肝炎ウイルスマーカーはすべて陰性で, 抗核抗体および抗ミトコンドリア抗体も検出されず免疫グロブリンはいずれも正常ないし軽度低下を呈した. 腹部超音波およびCTでは著明な脂肪肝を認めた. 肝生検像では肝実質にacuteyellow collapsed cellを含む巣状壊死を散見し, 大脂肪滴沈着をzone 2～3に小葉の1/2以上に認め飲酒歴がないことからnon-alcoholic steatohepatitisと診断した. 本例は肥満, 耐糖能異常を伴わず, 薬剤服用歴もなく経過より急激な栄養障害による飢餓状態が原因と考えられた

Precore hepatitis B virus (HBV) mutants may gradually prevail during or after seroconversion (SC) from hepatitis B e antigen (HBeAg) to hepatitis B e antigen antibody (anti-HBe) status in many chronic hepatitis B (CH-B) patients. However, patients with CH-B still produce anti-HBe more than several years after SC, and the relationship between SC and genome conversion in the precore region has not been clarified. Therefore, in patients with CH-B who had a sustained loss of HBeAg and complete remission of hepatitis after SC, the precore region was sequenced in paired serum samples from 1 year before SC to 3 years after SC. Mutant precore defective HBV DNA was found in only 6 (19%) of 31 CH-B patients who had a complete remission of hepatitis after SC. Mixed-type HBV DNA (precore wild-type and mutant-type) was found in 4 (13%) patients. Wild-type HBV DNA was found in 21 (68%) CH-B patients after SC. Longer-term follow-up of 11 CH-B patients indicated that 3 of 11 patients experienced precore genome conversion 2 to 3 years after SC. E-plus DNA or e-minus DNA was semiquantitated by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) assays before and after SC. E-plus DNA levels decreased from 10(5.56+/-1.58) to 10(2.45+/-1.61). Similarly, e-minus DNA levels declined from 10(4.25+/-1.56) to 10(1.86+/-1.37). By dot-blot assay, serum HBV DNA became negative soon after SC, as did serum HBeAg. In contrast, HBeAg-containing immune complexes were still detected after SC. Anti-HBe antibody was produced throughout SC and thereafter, as determined by a sensitive experimental assay. Therefore, we conclude that genome-conversion in the precore region is a separate event from HBeAg/anti-HBe seroconversion.

The immunopathogenesis of autoimmune hepatitis (AIH), and the role of T cells in the onset and maintenance of this disease, are still unclear. Since T cells expand clonally after stimulation by an antigen, it is important to analyze the behavior of T cells at a clonal level. We have established recently a novel system, using reverse transcriptase-polymerase chain reaction (RT-PCR) and subsequent single-strand conformation polymorphism (SSCP) that allows the identification of clonal accumulation of T cells in a lymphocyte population. Using this system, we demonstrated that oligoclonal T cells were accumulated in the liver of patients with AIH, and that identical T-cell clonotypes were detected in two different regions of the liver, although these features were also observed in cases with viral hepatitis. Only in cases with AIH, however, nearly all identical T cells were found to belong to CD8+ subset and there were very few CD4+ T cells in this population. Our results suggest that common antigens presented to CD8+ T cells in the context of HLA class I molecule are distributed diffusely in the liver of AIH. These findings also suggest that antigens recognized by CD4+ T cells may be relatively heterogeneous in the liver with AIH.

The nucleic acids of the helical and coccoid forms of Helicobacter pylori were studied to determine if the coccoid forms are "viable (capable of growing) but nonculturable." Using a reference strain (NCTC 11638) and five clinical strains, the nucleic acid contents, DNA integrity, and results of PCR and reverse transcription-PCR (RT-PCR) were compared for helical H. pylori and coccoid forms induced using glycochenodeoxycholic acid or bismuth citrate. The DNA and RNA contents of the coccoid forms were respectively 6.8- and 8.1-fold lower than those of helical H. pylori after 3 days of induction and 11.5- and 14.7-fold lower after 7 days. Agarose gel electrophoresis of DNA extracted from the coccoid forms after 3 days of induction showed a smear pattern indicating DNA cleavage, whereas DNA from helical H. pylori showed a single band with a high molecular mass. After 12 days of induction, all RNA samples from 100% coccoid cultures were negative for the mRNA of urease A or the 26-kDa species-specific protein by RT-PCR. However, most RNA samples obtained after 3 or 7 days of induction were positive at low levels despite the lack of recovery from these cultures. These results suggest that the coccoid form of H. pylori has impaired genomic DNA and is in the process of cellular degeneration, thus being still alive but nonincreasable.

Of 112 patients with chronic hepatitis C, 12 (11%) tested positive for RNA of GB virus C (GBV-C) by reverse-transcription polymerase chain reaction with primers deduced from the 5′ untranslated region. RNAs of GBV-C and hepatitis C virus (HCV) were followed in the 12 patients before and after they received 19 trials of interferon (IFN) therapy. GBV-C RNA disappeared from serum in 11 (58%) trials on eight patients. However, it stayed negative at 6 months after IFN only in two patients. One of them regained GBV-C RNA at 12 months and kept it thereafter, while an additional patient who failed to clear it at the completion of IFN turned negative at 24 months after therapy. Thus, two patients (17%) became persistently negative for GBV-C RNA. HCV RNA disappeared from serum at the completion of ten trials (53%) on eight patients, and stayed negative in three patients (25%). Two of them did not lose GBV-C RNA but kept normal transaminase levels. These results indicate that GBV-C is susceptible to IFN with a sensitivity comparable to but independent of HCV, and that GBV-C by itself would not elevate transaminases in hepatitis C patients who respond to IFN.

The prevalence of GB virus C/hepatitis G virus (GBV-CIHGV) infection among intravenous drug users (IVnDs), patients with liver diseases, and blood donors in Nanning, southern China was studied. GBV-C/HGV RNA was detected by reverse transcription polymerase chain reaction with primers derived from the 5'-untranslated region. GBV-CIHGV RNA was detected in 64 of 85 IVnDs, 20 of 80 persons with liver disease, and 1 of 50 blood donors. Among IVnDs, GBV-C/HGV infection was associated with antibodies to hepatis C virus (HCV) and with hepatitis B surface antigen (HBsAg). Eleven nucleotide sequences were determined and analyzed by molecular evolu­ tionary analysis. In a phylogenetic tree, the isolates were grouped in three clusters with GBV-C and HGV grouped in two clusters. These data indicate that GBV-CIHGV infection is common in China among IVnDs but uncommon among persons with liver disease without HBsAg or anti-HCV and that there is a new group of GBV-C/HGV.

大量飲酒歴のためにアルコール性肝障害として観察されていた原発性胆汁性肝硬変 (PBC) の一男性例を経験した.症例は69歳, 男性. 約6年前, 健康診断にて肝障害を指摘され, 推定積算飲酒量1.5tの飲酒歴からアルコール性肝障害として観察されていた. その後, 約2年の禁酒にもかかわらずγGTPの低下が乏しいため精査を行ったところ, PBCの診断を得た. 経過中, 抗核抗体およびIgGは高力価陽性, HLAクラスIIはDR4を呈すも抗ミトコンドリア抗体 (AMA) は陰性ないし低力価にとどまり, IgMも正常範囲にあった. しかし肝組織像ではPBCに一致する所見を示し, western blot法によるM2抗体の検索ではPDH-E2およびBCKD-E2が検出された. AMA測定の普及により無症候性PBCの診断は比較的容易になった. しかし本症例のように, γGTP高値でも男性かつ大量の飲酒歴を有し, かつAMA, IgMなども定型的でないところから, PBCの病態が想定しにくい症例も存在し, 興味深い一例と考え報告した.

Recently, a novel hepatitis virus, GB virus C/hepatitis G virus (GBV-C/HGV), has been isolated. To elucidate the seroprevalence of chronic GBV-C/HGV infection in Japan and the phylogenetic relationship between Japanese strains and the strains previously reported, serum GBV-C/HGV RNA was detected by reverse transcription polymerase chain reaction (RT-PCR) in 203 patients with chronic liver diseases and 200 samples of voluntary blood donors. RT-PCR was performed with primers derived from the 5'-untranslated region which were conserved between GBV-C and HGV and distant from other flaviviruses including hepatitis C virus (HCV). The nucleotide sequences were determined by the dideoxy chain termination method. The phylogenetic analysis was performed by the neighbor-joining method. In 10 (4.7%) of 203 patients with chronic liver diseases and in 1 (0.5%) of 200 blood donor samples, serum GBV-C/HGV RNA was detected. Of 10 patients, 9 patients were positive for anti-HCV and negative for HBsAg, and 1 patient was positive for HBsAg and negative for anti-HCV. The phylogenetic analysis indicated that there were three major groups which were group 1 (GBV-C), group 2 (HGV), and group 3 (a group of Japanese strains). These data indicated that (1) there was a low prevalence of GBV-C/HGV infection in Japanese patients with chronic liver diseases, (2) a high proportion of patients with GBV-C/HGV infection had chronic HCV infection however, and (3) there were at least three groups in strains of GBV-C/HGV.

In hepatitis A virus (HAV) infection, fecal excretion of the virus has been reported to cease shortly after symptoms occur. Although there have been several reports on detection of HAV in feces using polymerase chain reaction (PCR), the duration of fecal HAV shedding in human adult hepatitis A has not been well described. In the present study, we applied the reverse-transcription (RT)-PCR system to the detection of fecal HAV RNA in 10 patients with sporadic hepatitis A. The viral genomic RNA was detected in the stools from five patients after the onset of clinical symptoms. All stool samples collected within 10 days of onset of illness were HAV-RNA-positive, and the duration of positivity lasted from a few days to as long as 3 months. In four patients, HAV RNA was detected in the stool even after the serum alanine transaminase (ALT) levels had peaked, and in one patient well after ALT levels fell to normal. These results show that fecal shedding of HAV can last for months after resolution of symptoms and such patients could be a source of further spreading of the virus in the community.

A common-source outbreak of fulminant hepatitis B in hemodialysis patients induced by precore mutant. From September 9 to October 3, 1994, five patients on maintenance hemodialysis in a dialysis unit in Tokyo contracted hepatitis B virus (HBV) infection successively, and four of them died of fulminant hepatitis. The unit treated 181 patients three times a week on eight shifts, and all five afflicted patients were on the same shift along with 27 other patients. HBV DNA clones from the hepatitis patients had a point mutation converting codon 28 in the precore region to a stop codon, which aborts the synthesis and secretion of hepatitis B e antigen, and showed a sequence similarity of >99.5% within 645 base pairs covering the X gene and precore region. There were two HBV carriers with antibody to hepatitis B e antigen who were receiving hemodialysis on the same shift. HBV DNA clones from one of them had the stop codon 28 in the precore region, and a sequence similarity of >99.7% to those from the five patients. Based on these results, it was deduced that the fulminant HBV strain was transmitted from the carrier to five patients, and resulted in the death of four. The outbreak indicates that immunocompromised hosts like hemodialysis patients can develop fulminant hepatitis B if and when they are infected with extremely virulent HBV strains.

In an attempt to establish a model for hepatitis C virus (HCV) infection, we produced mice transgenic for the HCV envelope genes, E1 and E2, under the control of a regulatory element from hepatitis B virus. F1-generation mice from the established founders demonstrated expression of both E1 and E2 proteins as glycosylated forms in their organs including the liver. Immunostaining revealed the localization of envelope proteins principally in the cytoplasm of hepatocytes around the hepatic central veins. Furthermore, E1 and E2 proteins were shown by immunoprecipitation to be associated with each other in the mouse liver. There was no evidence of tissue pathology in the mouse liver up to 16 months of age, suggesting that E1 and E2 proteins per se may not have direct pathogenic effects. Our results demonstrate the first successful expression of HCV gene products in the mouse liver and the association of in vivo expressed HCV envelope proteins. This transgenic mouse would be a good model to study the virus-cell interactions of HCV such as intracellular transport and assembly of envelope proteins. Also it may be a good model system to determine the role of these proteins in the pathogenesis of hepatitis or extra-hepatic manifestations in HCV infection by the introduction of cytotoxic T lymphocytes specific for the envelope proteins.

Background. Among several genotypes of hepatitis C virus (HCV), genotypes 1b (or II) and 2a (or III) are predominant in Japan. Although it has been shown that the efficacy of interferon treatment of patients with chronic hepatitis C varies with the HCV genotype, the relationship between HCV genotype and the development of hepatocellular carcinoma (HCC) has not been well described. Methods. The genotypes and serum levels were determined for HCV-RNA in 72 patients with HCC and 131 patients without HCC, all of whom were positive for second-generation HCV antibody and HCV-RNA. In addition, clinical data from 34 patients with HCC who each had a history of blood transfusion were analyzed. Results. Fifty-seven (79.2%) of 72 patients with HCC had genotype 1b HCV, whereas 101 (77.1%) of 131 patients without HCC had genotype 1b, indicating that there was no significant difference in the prevalence of genotype 1b HCV between the patients with and without HCC. Furthermore, comparison of patients with HCC with genotype 1b HCV with those with genotype 2a who had a history of blood transfusion did not differ significantly in the number of years from blood transfusion to diagnosis of HCC. Levels of HCV-RNA were not significantly different among patients with liver diseases of various stages. Conclusions. Hepatocellular carcinoma develops in patients with either genotype 2a HCV or genotype 1b HCV. A difference in genotype is not likely to be responsible for the difference in development of HCC.

We determined quantitative values of serum hepatitis C virus RNA by branched DNA amplification assay in 52 consecutive patients with chronic hepatitis C immediately before high-dose treatment with interferon-alpha. Thirty-four out of 52 patients had >106.3 (≈2 × 106) equivalents/ml of viral genomes. Only three (8.8%) out of these 34 were long-term responders, while 16 (88.9%) out of 18 patients with ≤106.3 equivalents/ml of viral genomes were long-term responders (P 106.3 equivalents/ml was a long-term responder (P

The effect of interferon on hepatitis C virus RNA levels in the liver was studied by polymerase chain reaction in chronic hepatitis C patients who were enrolled into a pilot study with short-term interferon treatment. Among 17 patients treated with interferon α or β (168–560 M.U. in total), eight were ‘long-term responders’ as defined by sustained normal serum alanine aminotransferase levels for more than 12 months, while the other nine were ‘nonresponders’ with abnormal alanine aminotransferase levels. In eight ‘long-term responders’, both the plus and minus strands (replicative intermediate) of hepatitis C virus RNA could not be detected in the liver after interferon treatment whereas eight of nine ‘non-responders’ retained the minus strand or both strands of hepatitis C virus RNA in the liver. Serum hepatitis C virus RNA was no longer detected in seven of nine ‘non-responders’ or in all ‘long-term responders’ at the end of treatment, but it reappeared in ‘non-responders’ with the elevation of serum alanine aminotransferase levels, which excluded serum hepatitis C virus RNA as a prognostic marker for sustained alanine aminotransferase normalization after interferon treatment. Our results indicate that the disappearance of hepatitis C virus RNA including the minus strand RNA from the liver is a predictive marker for good prognosis in chronic hepatitis C patients after interferon treatment. It is of great use to determine the levels of hepatitis C virus RNA in the liver to know the prognosis of interferon-treated patients who maintain sustained normal serum alanine aminotransferase levels and undetectable serum hepatitis C virus RNA after interferon treatment.

The HBx gene of hepatitis B virus has been shown to induce hepatic tumors in transgenic mice and is implicated in hepatocarcinogenesis in human hepatitis B virus infection. To further characterize the role of HBx gene in carcinogenesis, we established mouse fibroblast cell lines in which the expression of HBx gene could be controlled by glucocorticoid hormone and examined the effect of HBx gene expression on cell growth in vitro. Along with the expression of HBx gene, most cells in the G0/G1 phase moved into the S phase in 24 h, and the cell cycle progressed further toward 48 h. Induction of DNA synthesis was also demonstrated by bromo-deoxyuridine labeling analysis. These results indicate that HBx gene has a function to trigger the synthesis of cellular DNA and suggest that HBx gene may play a role in hepatocarcinogenesis in human infection by driving deregulated cell cycle progression.

Ninety-three Japanese chronic hepatitis C patients treated with natural β interferon (IFN-β) in a 6-week protocol were investigated retrospectively for factors affecting responsiveness to the therapy. Normalization of serum ALT levels was found in patients with a decrease in serum HCV RNA titer. These patients (responders) had a significantly lower pre-treatment level of HCV RNA than others. Patients without such response (non-responders) were characterized by higher age, advanced liver histology, and higher frequency of type II (or 1b) HCV genotype. Of the four factors, HCV RNA titer, age, histology, and genotype, the most significant factor to differentiate between responders and non-responders was the pre-treatment level of HCV RNA. Our study did not clearly associate HCV genotype III (or 2a) with responders, although the results suggested that patients with genotype III have relatively lower HCV RNA titer and milder liver histology than those infected with genotype II.

Background/Aims: Differentiating between an acute hepatitis B (AH-B) infection and an acute exacerbation of a chronic hepatitis B (CH-B) infection can present a problem for the clinician. The only current serological method of distinguishing between acute and symptomatic chronic hepatitis B virus (HBV) infection is the immunoglobulin M antibody to hepatitis B core antigen (anti-HBc) assay, which can be problematic. Therefore, in an attempt to better distinguish between acute and chronic HBV infection, sera from 26 patients with AH-B and 53 patients with CH-B were compared in a variety of experimental immunoassays. Methods: Experimental assays have been designed to detect free antibody to hepatitis B e antigen (anti-HBe), hepatitis B e antigen (HBeAg)/anti-HBe immune complexes (ICs), and hepatitis B surface antigen (HBsAg)/antibody to hepatitis B surface antigen (anti-HBs) in the presence of excess antigen. An additional assay was developed to detect a novel anti-HBc specificity, designated antibody to woodchuck hepatitis virus (anti-HBc w ), which cross-reacts with the core antigen of the woodchuck hepatitis virus. Results: Sera from patients with CH-B showed significantly higher levels of free anti-HBe, HBeAg/anti-HBe ICs, and HBsAg/anti-HBs ICs compared with AH-B patient sera. Furthermore, patients with CH-B consistently produced high titer anti-HBc w , whereas patients with AH-B produced little or no anti-HBc w antibody. Conclusions: The serology of AH-B infection and symptomatic CH-B infection can be distinguished using a variety of experimental immunoassays in addition to the immunoglobulin M anti-HBc assay.

We studied the development of liver tumors in male HBx gene transgenic mice. Of two lineages studied, in the lineage with the lowest HBx gene expression liver tumors developed only in an incidence comparable with that in normal CD-1 strain, whereas 84% of male mice with a high level of the HBx gene product succumbed to liver neoplasia, indicating that continued HBx gene expression higher than a certain threshold level may be necessary for the development of hepatic neoplasia. Sixty-five mice from a lineage with a high level of HBx expression were then followed throughout their 24-mo lifespan. The livers of transgenic mice showed foci of cellular alteration with cytoplasmic vacuolations around the central veins from the age of 2 mo, but these foci did not expand progressively by the age of 12 mo. Immunostaining demonstrated such hepatocytes had higher expression of HBx protein than surrounding cells. Neoplastic lesions including liver cell adenomas and hepatocellular carcinomas developed from the age of 13 mo. By bromodeoxyuridine labeling analysis, hepatocytes in altered foci were found to have increased DNA synthesis, whereas no labeling was observed in age- and sex-matched nontransgenic littermate controls. Furthermore, DNA content analysis revealed the existence of several small aneuploid peaks in the transgenic liver before the age of tumor development. These results suggest that the continued expression of HBx gene may initiate a complex process to hepatocellular carcinoma by inducing DNA synthesis and placing large numbers of hepatocytes subjective to secondary events for transformation.

Interferon induces remission in about 50% of patients with chronic hepatitis C, but it is difficult to predict which patients will respond. Host and viral factors were evaluated for correlation with response to interferon in patients with chronic hepatitis C. Recombinant interferon alpha-2b with a total dose of 480-560 million units was given to 136 patients, of whom 74 (54%) responded. Genotypes of hepatitis C virus (HCV) in sera, I, II, III, IV, and V, were determined by polymerase chain reaction (PCR) with type-specific primers. In 72 patients, pretreatment levels of HCV RNA were titrated by PCR in serial tenfold dilutions of RNA extracted from serum. Response to interferon occurred in 34 (40%) of 85 patients infected with HCV of genotype II, less frequently than in 22 (85%) of 26 with genotype III (P0.001) or in 7 (70%) of 10 with genotype IV. Of 51 patients with genotype II HCV, 6 of 8 (75%) with HCV RNA titers10(6) responded, more frequently than 4 of 43 (9%) with titersor = 10(6) (P0.001). Responders were younger than non-responders (45.7 +/- 11.7 vs. 50.3 +/- 9.6 yr) and had received transfusions less frequently (26/74 or 35% vs. 37/62 or 60%, P0.01). Response to interferon correlated inversely with the severity of liver histopathology. These results indicate that response to interferon is influenced by HCV genotypes and pretreatment levels of HCV RNA in serum.

B型肝炎ウイルス(HBV)マーカーにおける最近の進歩はHBVのpre-C変異株の発見とその臨床的意義,また, S遺伝子変異にともなう, HBワクチン回避HBVの発見である.これらHBV遺伝子分析の結果で得られたことから従来のウイルスマーカーの意義と解釈に多少の修正を要するところがあるが,大筋では多くのマーカーの意義はほとんど変るところはない.ここでは新しい考え方を取り入れたHBVマーカーの解説を行った.

The current status of interferon (IFN) therapy in chronic hepatitis C is presented, focusing on the results of studies in Japan. Depending on the IFN treatment regimen used in chronic hepatitis C, it is possible to eradicate HCV in a relatively high percentage of patients (about 40%) and to achieve a cure for chronic hepatitis C. The objective of treatment should therefore be the eradication of HCV. The efficacy of IFN in chronic hepatitis C is dependent on the dosage of IFN, duration of treatment, liver histology findings, serum HCV-RNA levels, and HCV genotype. Besides a flu-like syndrome, many adverse reactions are associated with high-dose, long-term IFN treatment. However, as a rule, full recovery or improvement follows prompt withdrawal of IFN. By modification of the IFN, it will be possible to reduce adverse reactions and to create a more effective IFN to further enhance the effect of IFN in chronic hepatitis C.

We employed two different methods of 131I treatment for Graves' disease in 285 patients and compared the results between the two. (We also analyzed the factors affecting the treatment outcome.) A single dose of 131I adjusted to the patients' thyroid weight was administered to 180 patients in group 1, while a relatively lower dose of 131I (approximately 30Gy) was given repeatedly to 105 patients in group 2. A 5-year follow-up showed that in group 1, 34% of the patients were euthyroid, 11% hypothyroid, 11% subclinical hypothyroid and 44% still remained hyperthyroid. In group 2, 43% of the patients were euthyroid, 5% hypothyroid, 35% subclinical hypothyroid and 17% hyperthyroid. The factors affecting the outcome of the treatment in group 1 patients were their thyroid weight, the duration of the disease and TRAb levels. No significant correlation was observed between the efficacy of 131I treatment and the patients' sex, age, 24hr 131I-uptake, effective half life of administered 131I or titers of antithyroid antibodies. We conclude that the repeated low dose administration of 131I provides the best outcome in a 5-year follow-up. However, we suggest that an adjusted dose of 131I in relation to the patients' thyroid weight should be employed to obtain a faster therapeutic response.

A recombinant yeast-derived pre-S2 + S-containing hepatitis B vaccine (TGP-943) was clinically evaluated through three phases of testing in a total of 2137 volunteers. We observed the immunogenic purity of TGP-943 (phase 1), the inter-lot reproducibility of both safety and immunogenicity (phase 2), no significant side-effects, a high capability of inducing both anti-HBs and anti-pre-S2 antibodies (phases 1, 2 and 3), and an ability to induce seroconversion in the majority of vaccines who had been non-responsive to conventional hepatitis B vaccines (phases 2 and 3). In conclusion, TGP-943 is a safe and tolerable vaccine, with special merits: the ability to induce an early anti-pre-S2 response that circumvents the problem of delayed appearance of anti-HBs, and efficacy in non-responders to previous vaccination.

Chronological measurements of various HCV markers were conducted to clarify the course and prognosis of acute hepatitis C. Among 49 patients with acute non-A, non-B hepatitis, 32 (65.3%) were diagnosed as having acute hepatitis C by these markers. Twenty-four (82.8%) of 29 patients with posttransfusion hepatitis were type C, while only eight (40.0%) of 20 patients with sporadic hepatitis were type C. Patients were also divided into those who returned to normal within one year based on changes in s-ALT levels and unresolved cases. Anti-HCV was present in 11 (44.4%) of 25 resolved cases and in 21 (87.1%) of 24 unresolved cases. Only one case was continuously positive for HCV-RNA although s-ALT levels returned to normal. In addition, quantitative determinations were conducted on those positive for anti-HCVs. Anti-second generation tested positive in all HCV-RNA-positive cases and positive rates were highest from the onset of hepatitis. In unresolved patients with continuous HCV infection, anti-core increased and titers at 12 months were 10 units or more in all cases. On the other hand, in eight of 10 resolved cases, titers declined gradually after initial seroconversion and titers were 10 units or less at 12 months. From these results, second generation anti-HCV was considered most useful in early diagnosis of acute hepatitis C and anti-core titer was considered most useful in predicting prognosis of acute hepatitis C.

Background: Liver injury in many patients with chronic hepatitis B is sporadic and is often characterized by acute exacerbations alternating with relatively normal liver function. The aim of this study was to perform detailed serological and biochemical analysis during periods of active liver disease to better understand the mechanisms responsible for the cyclic nature of liver injury. Methods: A series of serum samples from 19 hepatitis B e antigen (HBeAg)-positive patients were analyzed for alterations in serum hepatitis B virus (HBV) DNA, HBeAg, anti-HBe production, and HBeAg-specific immune complex formation before, during, and after spontaneous acute exacerbations of liver injury. Results: This analysis revealed significant correlations between increasing levels of serum HBV DNA, HBeAg, HBeAg-specific immune complexes, and liver injury. These results suggest that increases in viral replication and accumulation of viral proteins in the serum and intracellularly and the subsequent immune response play an important role in initiating acute exacerbations of liver injury in chronic hepatitis B infection. Conclusions: We propose that production of the secreted and cellular forms of the nucleoprotein reaches a threshold level and elicits specific immune responses, which mediate liver injury.

A comparative study of three different high-dose regimens of interferon-alpha-2b (IFN) was conducted in patients with chronic hepatitis C to determine which was better at obtaining a sustained remission. A total of 126 patients were assigned randomly to one of three groups: group A was given 10 million international units (MIU) of IFN six times a week for eight weeks; group B was given 10 MIU IFN six times a week for four weeks followed by three times a week for an additional eight weeks; group C was given 10 MIU IFN six times a week for two weeks followed by three times a week for 12 weeks. The total dose administered to each group was 480 MIU/patient. Only the dosing schedule varied among the three groups. Among 98 efficacy-evaluable patients, a sustained alanine aminotransferase (ALT) response, defined as persistent normalization of the ALT for greater than six months after the termination of treatment, was achieved in 21.2% (7/33) of group A, 42.3% (11/26) of group B, and 54.5% (18/33) of group C patients. Similarly, a sustained loss of measurable serum hepatitis C virus RNA was observed in 28.6% (8/28) of group A, 40.9% (9/22) of group B, and 48.3% (14/29) of group C patients. Based upon these data, it can be concluded that 10 MIU of IFN administered six days a week for two weeks followed by three times a week for an additional 12 weeks produces the highest rate of both biochemical and virological responses to IFN therapy in patients with chronic HCV.

C100-3抗体陽性献血者178例の追跡調査ならびに臨床病理学的検討により,約50%が6ヵ月以上ALTが持続正常で,これらのうち約1/3はHCV-RNA陽性で,臨床的に無症候性HCVキャリアーと考えられた.一方,ALT異常を呈する例は,HCV-RNA陽性で肝組織学的検索からもC型慢性肝疾患と考えられた.C100-3抗体陽性献血者において,HCV core抗体の有無は,HCV-RNAの有無とよく一致した.また,C100-3抗体陽性無症候性HCVキャリアーの肝組織所見は,いずれも慢性肝炎像を有しており,肝組織が正常肝であるHCV healthy carrierは存在しないものと推察された.

Diabetic nephropathy is the most important complication of diabetes, because it is a major cause of morbidity and mortality for diabetic subjects. Since not all subjects with diabetes are at risk of developing this complication, we conducted a study to determine if heredity might be a possible risk factor for diabetic nephropathy in non-insulin dependent diabetes. Twenty-one factors including inheritance of nephropathy and hypertension were investigated in 109 individuals with NIDDM: 50 patients without proteinuria (Group I), 20 patients with intermittent proteinuria (Group II), and 39 patients with continuous proteinuria (Group III) matched for age and duration of diabetes. Of those patients, 55 patients with inheritance of diabetes were also divided into three groups: 29 patients without proteinuria (Group I), 9 patients with intermittent proteinuria (Group II), and 17 patients with continuous proteinuria (Group III). Individuals in Groups II and III has significantly higher frequency of inheritance of diabetic nephropathy than those in Group I, and also individuals with inheritance of diabetic nephropathy had significantly higher frequency of diabetic nephropathy than those without it. Frequency of hypertension, retinopathy and body mass index in the past were significantly higher in subjects in Groups II or Group III than in those in Group I. There were no significant differences between subjects in Groups II and III. These findings suggest that susceptibility to diabetic nephropathy in NIDDM may be hereditary, although hypertension and obesity may also be important risk factors for diabetic nephropathy.

To investigate the mechanism of glucose intolerance in patients with Graves' disease, a 2-hour oral glucose tolerance test and euglycemic glucose clamp study using Biostator were performed in patients with Graves' disease and control subjects. 80 per cent of the patients showed impaired glucose tolerance. Insulinogenic index in the patients with borderline or diabetic glucose response was lower than that in subjects with normal glucose response. Insulinogenic index was inversely correlated with sigma PG during the test. Despite normal basal plasma glucose concentrations, basal plasma insulin levels in the patients with Graves' disease were higher than in the controls. Using the euglycemic glucose clamp technique, the glucose utilization rate (M value), the metabolic clearance rate of glucose (MCRG) and the insulin sensitivity index (M/I x 100) in the patients with Graves' disease were lower than in the controls. After treatment with antithyroid drug in 3 patients, glucose tolerance completely normalized, and there was a significant increase in the M value and the MCRG and a significant decrease in the metabolic clearance rate of insulin (MCRI) compared to the values before treatment. In the patients with Graves' disease, basal serum glucagon levels were higher than in the controls, and glucagon suppression during insulin infusion was found to be decreased. From these data, it is concluded that the decrease in glucose tolerance in patients with Graves' disease can be explained by 1) the impairment of early insulin release response to rapid intestinal glucose absorption, 2) increased insulin metabolic clearance and 3) hyperglucagonemia.

The safety and immunogenicity of a lyophilized inactivated hepatitis A vaccine was tested in healthy adult male volunteers. Thirty-six volunteers, all of whom were negative for antibody to HAV (anti-HAV), were divided into three dosage groups, 1.0, 0.5 and 0.25 micrograms of viral protein, respectively. Each group received a total course of three intramuscular injections at months 0, 1 and 6. Slight side effects were noted after 16 of 99 injections and the occurrence and degree were almost identical to those of other commercial vaccines. On the other hand, all subjects had measurable titres of serum anti-HAV neutralizing antibodies as early as 2 months after the first injection. The mean values of serum anti-HAV neutralizing antibodies at 7 months in the 1.0, 0.5 and 0.25 micrograms dose groups were 64-, 12-, and 9-fold higher, respectively, than those observed at 5 days in five recipients given 7.5 mg kg-1 body weight of immune serum globulin (ISG).

We report the results of our study on 8-week consecutive interferon (IFN) treatment for chronic non-A, non-B hepatitis. In the 6 MIU/day group, most cases had continuously normalized alanine-aminotransferase (ALT) levels even after IFN treatment was discontinued. In responders whose ALT levels remained normal, not only the degree of inflammation, as seen from HAI scores, but also liver histopathological features were improved. Moreover, antibody to hepatitis C virus (anti-HCV) titer in responders was continuously decreased. Antiviral effects of IFN were observed by detection of HCV-RNA using the PCR technique. HCV-RNA became negative with IFN treatment, however, in some patients who relapsed after discontinuation of IFN treatment, re-elevation of ALT levels was usually preceded by re-appearance of HCV-RNA.

In Japan, hepatocellular carcinoma (HCC) is one of the most prevalent cancers, with a reported fatality rate showing a consistent and significant increase in the last decade. At most, only 25% of HCC cases are positive for the hepatitis B surface antigen (HBsAg). To investigate a potential role for hepatitis C virus (HCV) in the development of HCC, sera from 105 HBsAg-negative HCC patients were collected from five districts of Japan and assayed for antibody to HCV antigen (HCVAb). A large number of these patients (76.2%) were found to be positive for the HCVAb in comparison with the reported prevalence in sera from blood donors (1.1%). A history of blood transfusion was found in 39.6% of the cases positive for HCVAb, which was significantly different to the lower rate (4.7%) observed in HCC patients who were both positive for HBsAg and negative for HCVAb (P less than 0.001).

Clinical studies were undertaken of three pairs of monozygotic twin sisters affected by Graves' disease. The evidence that the twins were monozygotic was established by similarity in physical appearance, identical blood-group antigens and identical HLA types. All pairs were female, and the evidence of Graves' disease in both members of the pair was observed in 2 out of 3 pairs. In the remaining pair the disease started in the elder sister, but the younger sister still remained in euthyroidism in spite of the presence of diffuse goiter. The disease occurred in the second decade in all the patients, and the interval between the occurrence of the disease in twin sisters was 1 to 4 years. No common specific type of HLA was found among all three pairs, but DR4 was common in two pairs of twin sisters showing positive MCHA test. Serum TRAb was positive in 2 pairs of twin sisters but negative in the remaining one. However, it became positive in the elder sister of the latter pair during the antithyroid treatment. The effect of antithyroid drug treatment was found to be similar in each of the twin sisters, and the serum TRAb moved almost in parallel with serum FT4 level. These results indicate that a genetic factor may be of great importance in the aetiology of Graves' disease, and there was a strong preponderance for it to occur in women and the second decade was the peak age for its occurrence. The common specific type of HLA for all Graves' patients was not found in this study. A strong relationship was observed between Grave's disease and TRAb.

妊婦および老年者に合併するバセドウ病はもっとも見逃され易いものの一つである.その理由は,妊婦においては妊娠自体によっておこる症状が甲状腺中毒症状に酷似するからであり,老年者においては甲状腺中毒症状が極めて軽度のことが多いからである.その上妊婦に認められる血中TBGの増加とアルブミンの減少は,甲状線ホルモン濃度に影響を与える.本稿では,妊婦と老年者におけるバセドウ病の特徴,診断,治療について述べた.

B型肝炎ウイルスは外被と芯の部分から成り立っている.外被に存在するマーカーは感染成立に関係するものであり,それらに対する抗体は防御抗体として働く.一方,芯に存在するマ-カーは増殖に直接関係するものであり,また,肝細胞破壊に関係している.多種存在し,複雑な変動を示すこれらマーカーも上記のように整理すると理解しやすい.