A cohort study of chronic liver disease in an HCV hyperendemic area of Japan: a prospective analysis for 12 years.

A mass screening in 1990 of H town in Japan demonstrated a high prevalence of hepatitis C virus (HCV) infection in our previous studies. The purpose of the present study was to evaluate the prognosis and natural history of liver disease among the same residents after 12 years. Of 509 residents, 69 people had died, and 55 people had moved to other regions. In all, 139 persons of the remaining 385 residing in H town were examined for liver function tests, antibodies to HCV (anti-HCV), serum HCV RNA, and hepatitis B virus surface antigen (HBsAg). The data of 14 of these 385 people were collected from medical records. The cause of death of the 69 individuals was investigated. The prognosis of liver disease could be clarified after 12 years in 222 of the 509 residents. Most of the residents with liver disease had an advanced stage of disease. Of the 69 persons who died, the mortality rate caused by liver cirrhosis or hepatocellular carcinoma (HCC) was 44 and 53%, respectively, among 25 persons with positive anti-HCV, and 19 with positive HCV RNA. One person with positive HBsAg died of HCC. Persons with chronic HCV or HBV infection had significantly higher mortality rates from liver cirrhosis and HCC than those without infection (P<0.00001). The present study suggests that early detection and treatment for HCC should be carried out as HCV carriers age. Furthermore, persistent HCV carriers should receive therapy for suppression of the development of HCC. The eradication of HCC should be considered a national goal.

High prevalence of anticardiolipin antibodies in patients with HCV-associated oral lichen planus.

Hepatitis C virus (HCV) has been linked to extrahepatic manifestations such as oral lichen planus (OLP). In addition, anticardiolipin antibodies (aCL) and cryoglobulin have been demonstrated in chronic hepatitis C. The aim of this study was to investigate these prevalences in patients with HCV-associated OLP. The prospective study investigated the role of these factors in 133 subjects: 28 with OLP-HCV(+) (group 1), 22 with OLP-HCV(-) (group 2), 33 without OLP-HCV(+) (group 3), and 50 healthy volunteers matched for age and sex served as control group (group 4). Levels of immunoglobulin G (IgG) and IgM aCL antibodies, and cryoglobulin in serum were evaluated by enzyme-linked immunosorbent assay. The prevalence of aCL in groups 1, 2, 3, and 4 were 32.1, 18, 36.3, and 8%, respectively. The positive rate of aCL was significantly higher in groups 1 and 3 than that in the control group (group 1; p=0.02 vs. the control group, group 3; p<0.01 vs. the control group). There were no significant differences in cryoglobulin among the groups. The findings of the present study showed a high prevalence of IgG and IgM aCL in the serum of patients with HCV infectious diseases. A positive factor for aCL was determined by age, sex, the presence of OLP, and HCV infection.

Laminin deposition to type IV collagen enhances haptotaxis, chemokinesis, and adhesion of hepatoma cells through beta1-integrins.

BACKGROUND/AIMS: In hepatocellular carcinoma, laminin deposition to type IV collagen along the sinusoids is observed with the development of arterial network, coinciding with intrahepatic metastasis. We investigated the influence of laminin deposition to type IV collagen on hepatoma cell adhesion, motility and secretion of matrix metalloproteinases (MMPs), which are indispensable behaviors for tumor metastasis. METHODS: Hepatoma cell lines (KYN-1, -2 and -3) were used. The expression of integrin subunit mRNAs in hepatoma cells was confirmed by RT-PCR. The influence of laminin addition to type IV collagen on the adhesion, chemokinesis, and migration of KYN-1, -2 and -3 was evaluated by the haptotactic migration, phagokinetic track motility, and cell adhesion assays. The effects of integrin subunits on these activities were evaluated using the function-blocking antibodies for integrins. Phosphorylation of MEK1/2 and secretion of MMPs were investigated by Western blotting and gelatin zymography. RESULTS: Integrin alpha1, alpha2, alpha3, alpha6 and beta1 subunit mRNAs were detected. The combination of type IV collagen and laminin enhanced the migration, chemokinesis, and adhesion of hepatoma cells compared to that of type IV collagen when used alone. The enhanced activity was significantly suppressed by function-blocking antibodies for integrin alpha1, alpha2, alpha3, alpha6 and beta1 subunits. Hepatoma cells cultured on the combination of type IV collagen and laminin showed phosphorylation of MEK1/2 and increased secretion of MMPs. CONCLUSIONS: The addition of laminin to type IV collagen enhances hepatoma cell adhesion and motility through beta1-integrins.

Long-term follow-up of interferon-treated chronic hepatitis C and serum hepatic fibrosis markers.

BACKGROUND/AIMS: We investigated the clinical application of serum fibrosis markers in a long-term follow-up of patients with chronic hepatitis C treated with interferon-alpha. METHODOLOGY: This study included 52 patients treated with interferon-alpha (total: 480 MU) for 6 months. They each underwent liver biopsy before and after treatment. Twenty-eight patients who underwent liver biopsy less than 2 years after treatment were classified as group 1, and 24 patients as group 2. The two groups were subdivided into HCV RNA-negative responders and HCV RNA-positive nonresponders. Liver specimens were estimated using grading and staging scores. Serum hyaluronan, PIIIP, and type IV collagen levels were measured before and after treatment. RESULTS: In the responders of groups 1 and 2, grading score after treatment was significantly decreased compared with that before treatment. Staging score after treatment was significantly improved only in the responders of group 2. In the responders of group 2, serum hyaluronan level was significantly decreased compared with that before treatment. In group 2, the grading score was significantly correlated with serum PIIIP and type IV collagen levels, and the staging score was significantly correlated with only serum hyaluronan level. CONCLUSIONS: These findings indicate that the serum PIIIP and type IV collagen levels reflect the activity, and serum hyaluronan reflects the degree of fibrosis in liver specimens of HCV RNA-negative patients in a long-term follow-up of patients after interferon-alpha treatment.

Transcription-mediated amplification is more useful in the follow-up of patients with chronic hepatitis B treated with lamivudine.

Changes in the HBV DNA level during the treatment of patients with chronic hepatitis B with lamivudine were investigated by the transcription-mediated amplification (TMA) assay. Twenty-four patients treated with lamivudine (males:female= 20:4, age: 44.0+/-9.0 years, chronic hepatitis: 14, cirrhosis: 7, cirrhosis with hepatocellular carcinoma: 3) were investigated. The dosage of lamivudine was 75 mg/day in 3, 100 mg/day in 8, and 150 mg/day in 13 patients, and the administration period was 48+/-16 weeks (24-79 weeks). Sixteen patients were HBe antigen-positive before treatment, and the HBV DNA level was 7.4+/-1.2 (4.0- more than 8.7) LGE/ml. The HBV DNA level was measured every 1-6 months by the TMA assay and the branched DNA signal amplification technology (b-DNA assay). Serum HBV DNA disappeared in all patients by the b-DNA during the treatment period, while six patients had persistent HBV DNA by the TMA. The time of HBV DNA disappearance by the TMA in 18 patients was 2-5 months after initiation of treatment. The disappearance rate of HBV DNA was 3/8 (38%) in patients whose HBV DNA level before treatment was 8.0 LGE/ml or higher, 7/8 (88%) in those with 7-7.9 LGE/ml, and 8/8 (100%) in those with 6.9 LGE/ml or lower, showing that disappearance of HBV DNA became difficult when the HBV DNA level before treatment was high (P<0.01). In six patients, the HBV DNA level disappeared once, then increased thereafter. The present findings suggested that these increases in the HBV DNA level were due to an increase of YMDD mutant in three of these six patients, and due to a decrease in the dosage in two patients. In treatment with lamivudine, the TMA assay is more useful for understanding the changes in the HBV DNA level than b-DNA assay.

Autocrine motility factor enhances hepatoma cell invasion across the basement membrane through activation of beta1 integrins.

Autocrine motility factor/phosphohexose isomerase (AMF/PHI) is a cytokine that is linked to tumor invasion and metastasis. In hepatocellular carcinoma (HCC) tissues, hepatoma cells produce AMF/PHI and its receptor, Mr 78,000 glycoprotein (gp78), is strongly detected in hepatoma cells invading into the stroma and tumor thrombi in the portal vein. Here, we investigated the mechanism of hepatoma cell invasion through Matrigel induced by AMF/PHI using 3 hepatoma cell lines. Production of AMF/PHI, phosphorylation of MEK1/2, and Rho activity were investigated by immunoblotting. Expression of AMF/PHI and gp78 was observed by confocal fluorescence microscopy. The influence of AMF/PHI on activated integrin beta1 subunit expression was evaluated by flow cytometry. Changes in invasion, adhesion, and motility induced by AMF/PHI were evaluated using chemoinvasion, adhesion, and phagokinetic track motility assays. The effect of AMF/PHI on matrix metalloproteinase (MMP) secretion was evaluated by gelatin zymography. Hepatoma cells produced AMF/PHI and expressed gp78. Although AMF/PHI was ubiquitously detected, gp78 was strongly expressed in migrating cells. AMF/PHI induced up-regulation of activated integrin beta1 subunit expression. AMF/PHI stimulated hepatoma cell invasion through Matrigel, and stimulated the adhesion, motility, and MMP-2 secretion of hepatoma cells. The latter effects were suppressed by the function-blocking antibody for integrin beta1 subunit. AMF/PHI also enhanced Rho activity and the phosphorylation of MEK1 and MEK 2. Our results indicate that AMF/PHI enhances hepatoma cell invasion through Matrigel in an autocrine manner by stimulating the adhesion, motility, and MMP-2 secretion of these cells through activation of beta1 integrins.

[Study on the intensity of MMC and UFT in postoperative adjuvant chemotherapy for gastric cancer--study report of JFMTC Study No. 10].

The purpose of this study was to investigate the correlation between efficacy and dose intensity of postoperative adjuvant chemotherapy with MMC and UFT. A total of 1,410 patients from 180 institutions were allocated into a low-dose group and a high-dose group. The patients in the low-dose group received MMC at 8 mg/m2 on the day of surgery and 3 capsules of UFT (300 mg in tegafur) daily for 6 months. The patients in the high-dose group received MMC at 8 mg/m2 on the day of surgery, and in weeks 4, 10, 16, and 22 after surgery and 6 capsules of UFT (600 mg in tegafur) daily for 6 months. The patients in the high-dose group tended to exhibit higher survival rates than those in the low-dose group, although the difference was not significant. For the n(+)ps(-) patients, however, the survival rates were significantly higher in the high-dose group (p = 0.043). The recurrence-free rates showed a similar tendency. The incidence rates of adverse events were significantly higher in the high-dose group than in the low-dose group. Compliance was poorer in the high-dose group. Although the number of adverse events increases, a better prognosis can be expected with a high dose. These results confirmed a dose-dependency in adjuvant chemotherapy with MMC and UFT.

Second generation amplicor-HCV monitor assay: clinical features and predictors of the response to interferon.

The aims of this study were to compare the amplicor-HCV monitor assay versions 1.0 and 2.0, and to investigate the clinical usefulness of this assay in patients with chronic hepatitis C. We retrospectively analyzed 154 patients, and 133 of these patients received interferon therapy. Sixty-nine patients were complete responders (CR), and 64 were non-responders. Serum HCV RNA levels of version 1.0 and version 2.0 and HCV genotypes were determined in all patients. There was a good correlation between versions 1.0 and 2.0 in both genotype 1b and 2a, 2b (r=0.907 and 0.726, respectively). In genotype 1b, the mean HCV RNA level obtained by version 1.0 was 384+/-547 kcopies/ml and that obtained by version 2.0 was 488+/-825 kI.U./ml. In genotype 2a/2b, the mean level obtained by version 1.0 was 170+/-369 kcopies/ml and that obtained by version 2.0 was 340+/-402 kI.U./ml. Discriminant analysis revealed that the discriminating points of IFN response were 168 kcopies/ml (genotype 1b, version 1.0), 106 kcopies/ml (genotype 2a and 2b, version 1.0), 102 kI.U./ml (genotype 1b, version 2.0), and 277 kI.U./ml (genotype 2a and 2b, version 2.0). When the patients were stratified according to the discriminating points, the CR rate below the discriminating points were 73.8 and 86.2% in versions 1.0 and 2.0, respectively, in genotype 1b, and the rates were 73.2 and 82.3% in genotype 2a/2b. In addition, receiver-operating characteristic analysis revealed that version 2.0 had significantly better discriminative ability in patients with genotype 1b. We conclude that the second version of the amplicor-HCV monitor assay measures HCV RNA levels with the same precision as version 1.0 and is more useful for the prediction of interferon response than version 1.0.

Bright loop appearance; a characteristic ultrasonography sign of early hepatocellular carcinoma.

Ultrasonography (US) and computed tomography (CT) are the most effective screening methodologies for hepatocellular carcinoma (HCC). In our US screening, 20% of small HCC nodules less than 20 mm in diameter were detected as hyperechoic tumors. Among these hyperechoic HCC nodules, we have often observed (BL) which is defined as hypoechoic nodules in the hyperechoic tumor. In this study, we report that the BL is a sign of dedifferentiation of early stage of HCC with fatty change by US. From 1994 to 1998, we performed tumor targeting needle biopsy in 938 hepatic nodular lesions. Among them, 284 nodules <20 mm in diameter, histologically diagnosed as HCC, were studied. BL is defined as a hyperechoic tumor containing a hypoechoic nodule >4 mm in diameter by US. Among 284 nodules, well, moderately and poorly differentiated HCC were 183 (64.4%), 100 (35.2%) and 1 (0.4%), respectively. On US, hypoechoic, isoechoic, and hyperechoic nodules were 188 (66.2%), 32 (11.3%) and 64 (22.5%), respectively. Forty-seven nodules of 64 hyperechoic HCC nodules <20 mm in diameter, 47 nodules (73.4%) showed fatty changes. Of 64 hyperechoic HCC nodules, we recognized 22 nodules (34.4%) as BL. The proportion of BL type hyperechoic nodules increased with the tumor size. Two hyperechoic nodules followed by US changed to BL with tumor enlargement. Histologic examination of a resected HCC with BL showed that hyperechoic HCC nodule represented well-differentiated HCC with fatty change and inner hypoechoic lesion represented moderately differentiated HCC without fatty change. In US screening for HCC, BL was often observed in HCC nodules from 11 to 20 mm in diameter. Histologic examination revealed that BL of HCC on US was associated with tumor progression and indicated dedifferentiation showing moderately differentiated HCC in well-differentiated HCC with fatty change.

Effect of interferon treatment on serum 2',5'-oligoadenylate synthetase levels in hepatitis C-infected patients.

Interferon (IFN) is widely used for patients with hepatitis C. Less than half of treated patients respond to IFN therapy, however, and increased resistance to IFN is particularly observed in genotype 1b patients. Recently, genotype 1b patients with the wild type sequence in the NS5A gene were shown to be resistant to therapy, suggesting that the NS5A protein may be involved to IFN resistance. Thus, we investigated the serum 2',5'-oligoadenylate synthetase (2',5'-OAS) levels before and during IFN treatment. In addition, other biochemical markers and NS5A mutations were also examined in 30 HCV genotype 1b-positive patients. Before IFN treatment, 2',5'-OAS activity in sera was significantly lower in wild type patients than in mutant type patients. All patients were subsequently enrolled in IFN therapy, and 2',5'-OAS activity was elevated both in wild and mutant type patients, irrespective of the number of mutations in NS5A. Logistic regression analysis revealed that clearance of serum HCV RNA was independently related to the pretreatment viral load and NS5A mutations, but not to serum 2',5'-OAS activity. We concluded that the NS5A protein, that is associated with the outcome of IFN therapy, affects the kinetics of IFN-induced molecules, such as 2', 5'-OAS. 2',5'-OAS activity does not, however, seem to be related to long-term virological response to IFN therapy.

Copper does not alter the intracellular distribution of ATP7B, a copper-transporting ATPase.

Wilson's disease is a genetic disorder characterized by the accumulation of copper in the body due to a defect of biliary copper excretion. However, the mechanism of biliary copper excretion has not been fully clarified. We examined the effect of copper on the intracellular localization of the Wilson disease gene product (ATP7B) and green fluorescent protein (GFP)-tagged ATP7B in a human hepatoma cell line (Huh7). The intracellular organelles were visualized by fluorescence microscopy. GFP-ATP7B colocalized with late endosome markers, but not with endoplasmic reticulum, Golgi, or lysosome markers in both the steady and copper-loaded states. ATP7B mainly localized at the perinuclear regions in both states. These results suggest that the main localization of ATP7B is in the late endosomes in both the steady and copper-loaded states. ATP7B seems to translocate copper from the cytosol to the late endosomal lumen, thus participating in biliary copper excretion via lysosomes.

Two-year follow-up study after treatment with lamivudine for chronic hepatitis B: seven cases reported.

2 times baseline) and returned to seropositive for HBV DNA and HBeAg after lamivudine cessation. One of these five patients died of liver failure 3 months after treatment. However, in two of five patients whose alanine aminotransferase (ALT) had rebounded, HBV DNA became undetectable, and the ALT levels markedly decreased 2 years after the end of therapy. Since the disappearance of HBV DNA and stabilization of the ALT level were observed within two patients by 2 years after cessation of treatment, the patients whose ALT had rebounded should be followed up for a long-term period. To confirm the effect of lamivudine, long-term follow-up in many patients is necessary.

Clinical characteristics of patients in their 40s with HCV antibody-positive hepatocellular carcinoma.

BACKGROUND: We investigated the clinical characteristics of hepatitis C virus (HCV) antibody-positive hepatocellular carcinoma (HCC) patients who developed HCC at a relatively young age. METHODS: Clinical characteristics of patients in their 40s were investigated and were compared with those of patients 50 years and older. The subjects were 648 HCC patients, 469 men (72%) and 179 women (28%), who were treated at our hospital between 1991 and 1997. RESULTS: No patient was under 40 years of age. Eighteen patients (3%) were in their 40s, 137 patients (21%) were in their 50s, 338 patients (52%) were in their 60s, 143 patients (22%) were in their 70s, and 12 patients (2%) were in their 80s. Fifteen of the patients (83%) in their 40s were male. The proportion of men in their 40s was higher than that of all men. Eight of the 15 men in their 40s (53%) were heavy drinkers, and 2 (14%) were habitual drinkers. Three of the 15 men (20%) were HBV carriers, and these 3 HBV carriers were not drinkers. The proportion of heavy drinkers and HBV carriers was significantly higher among the patients in their 40s than in the 60 patients randomly sampled from the patients 50 years of age and older. The mean ages of male patients with HCC who were heavy drinkers, habitual drinkers, occasional drinkers, or nondrinkers were 52.3, 58.9, 62.0, and 61.7 years, respectively. HCC occurred significantly earlier in heavy drinkers than in the other 3 groups. We compared laboratory data of the patients in their 40s with data of all of the patients of 50 years and older. Serum total bilirubin, prothrombin time, and platelet counts were significantly worse in the patients in their 40s. CONCLUSIONS: Logistic regression analysis revealed that heavy drinking and presence of HBV infection were independently related to HCV antibody-positive HCC development at a younger age.

Erythropoietic protoporphyria with fatal liver failure.

A 33-year-old woman with a history of photosensitivity, persistent abdominal pain, and liver dysfunction was admitted to our department because of abdominal pain and progression of liver dysfunction. On admission, levels of protoporphyrin and coproporphyrin within erythrocytes were markedly increased. Autofluorescent erythrocytes were also detected, leading to a diagnosis of erythropoietic protoporphyria. A liver biopsy specimen revealed cirrhosis with dark brown granules filling hepatocytes, bile canaliculi, and bile ductules. Transfusion of washed erythrocytes, hemodialysis, and administration of cholestyramine and beta-carotene transiently improved levels of porphyrins and liver function. The patient died of rupture of esophageal varices followed by multiple organ failure. However, the treatments were believed to have extended survival.

Mutations in the NS5A gene predict response to interferon therapy in Japanese patients with chronic hepatitis C and cirrhosis.

The virus genotype, serum HCV-RNA level and liver histology are reported to be important factors in the response to interferon therapy. Recent studies have revealed that HCV NS5A 2209-2248 amino acid changes affect the response to interferon therapy of genotype 1b chronic hepatitis C. In contrast, some studies done in western countries have reported no such correlation. In the present study, interferon therapy was given to 58 Japanese patients, including 15 liver cirrhosis patients. NS5A 2209-2248 changes, the serum HCV level, ALT level, age and histology were examined in relation to the interferon effect. Twenty-four of the 58 patients (41%) showed a sustained virological response to the therapy. The responses to interferon therapy were significantly correlated with NS5A 2209-2248 changes (p < 0.0001), the HCV-RNA level (p < 0.0001) and histology (p < 0.0060). Among 15 liver cirrhosis patients, 3 of 6 mutant type patients showed a sustained virological response; 5 intermediate and 4 with wild type virus infected patients showed no responses. In conclusion, NS5A 2209-2248 changes may be a useful predictive marker of response to interferon therapy in addition to the serum HCV RNA level even in histologically advanced patients.

The incidence of hepatocellular carcinoma in patients with chronic hepatitis C after interferon treatment.

To determine whether serum hepatitis C virus (HCV) RNA disappearance after interferon (IFN) treatment prevents development of hepatocellular carcinoma (HCC), we evaluated retrospectively the incidence of HCC in patients with chronic hepatitis C. A total of 213 patients were monitored for more than 6 months after completion of IFN treatment. Sixty-three of the 213 patients (29.6%) achieved a complete response (CR) to treatment and 150 (70.4%) had no response (NR). HCC developed in 12 (5.6%), all of whom were NR. Logistic analysis showed age, alpha -fetoprotein, and staging of histological finding before IFN treatment were independent factors to development of HCC. The fact that there was no HCC development from CR provides a basis for IFN treatment in chronic HCV infection.

In vitro study of neutrophil apoptosis in liver cirrhosis.

Cirrhotic patients frequently manifest neutropenia and are predisposed to bacterial infections. We examined neutrophil apoptosis to determine if neutrophil survival in cirrhotic patients is shortened. Neutrophils isolated from 10 cirrhotic patients and 10 healthy volunteers were cultured for 24 hours. The time course of neutrophil viability was assessed by the trypan blue dye exclusion test and apoptosis was determined morphologically by light and electron microscopy. Apoptotic cells were also confirmed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick and labeling (TUNEL) and DNA gel electrophoresis. Fas expression of neutrophils was examined by flow cytometry. Viabilities were significantly decreased in liver cirrhosis (p<0.0001). Neutrophils from cirrhotic patients exhibited significantly greater apoptosis. Fas expression of neutrophils was significantly reduced for cirrhotic patients (p=0.0001). Neutrophils from cirrhotic patients exhibited markedly accelerated apoptosis in vitro. Shortening of neutrophil survival via apoptosis may explain in part the mechanism of neutropenia in cirrhotic patients.