1: Cell Tissue Res. 2008 May 14. [Epub ahead of print] Hepatoblast-like cells enriched from mouse embryonic stem cells in medium without glucose, pyruvate, arginine, and tyrosine. Tomizawa M, Toyama Y, Ito C, Toshimori K, Iwase K, Takiguchi M, Saisho H, Yokosuka O. Department of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, 1–8–1 Inohana, Chuo-ku, Chiba City, Chiba, 260–8670, Japan, nihminor-cib@umin.ac.jp. In order to enrich hepatocytes differentiated from embryonic stem cells, we developed a novel medium. Since only hepatocytes have the activity of ornithine transcarbamylase, phenylalanine hydroxylase, galactokinase, and glycerol kinase, we expected that hepatocytes would be enriched in a medium without arginine, tyrosine, glucose, and pyruvate, but supplemented with ornithine, phenylanaline, galactose, and glycerol (hepatocyte-selection medium, HSM). Embryoid bodies were transferred onto dishes coated with gelatin in HSM after 4 days of culture. At 18 days after embryoid body formation, a single type of polygonal cell survived with an enlarged intercellular space and micorvilli. These cells were positive for indocyanine green uptake and for mRNAs of albumin, transthyretin, and alpha-feto protein, but negative for mRNAs of tyrosine aminotransferase, alpha1-antitrypsin, glucose-6-phosphatase, and phosphoenol pyruvate carboxykinase. Since cells in HSM were positive for cytokeratin (CK)8 and CK18 (hepatocyte markers) and for CK19 (a marker of bile duct epithelial cells), we concluded that they were hepatoblasts. They showed weaker expression of CCAAT/enhancer-binding protein (C/EBP)alpha than fetal liver (18.5 days of gestation) and expression of C/EBPbeta at a similar level to that of fetal liver. These data support our conclusion that HSM allows the selection of hepatoblast-like cells. PMID: 18478268 [PubMed - as supplied by publisher] 2: World J Gastroenterol. 2008 Mar 21;14(11):1710-9. Current role of ultrasound for the management of hepatocellular carcinoma. Maruyama H, Yoshikawa M, Yokosuka O. Department of Medicine and Clinical Oncology, Chiba University Graduate School of Medicine 1-8-1, Inohana, Chuou-ku, Chiba 260-8670, Japan. maru-cib@umin.ac.jp. Hepatocellular carcinoma (HCC) has a decisive influence on the prognosis of cirrhotic patients. Although alpha-fetoprotein (AFP) is a known and specific tumor maker for HCC, it is not suitable for the screening and surveillance of HCC because of its poor predictive value and low sensitivity. The use of imaging modalities is essential for the screening, diagnosis and treatment of HCC. Ultrasound (US) plays a major role among them, because it provides real-time and non-invasive observation by a simple and easy technique. In addition, US-guided needle puncture methods are frequently required for the diagnosis and/or treatment process of HCC. The development of digital technology has led to the detection of blood flow by color Doppler US, and the sensitivity for detecting tumor vascularity has shown remarkable improvement with the introduction of microbubble contrast agents. Moreover, near real-time 3-dimensional US images are now available. As for the treatment of HCC, high intensity focused ultrasound (HIFU) was developed as a novel technology that provides transcutaneous ablation effect without needle puncture. These advancements in the US field have led to rapid progress in HCC management, and continuing advances are expected. This article reviews the current application of US for HCC in clinical practice. PMID: 18350602 [PubMed - in process] 3: Liver Int. 2008 Mar;28(3):355-62. Prevalence of diabetes mellitus and insulin resistance in patients with chronic hepatitis C: comparison with hepatitis B virus-infected and hepatitis C virus-cleared patients. Imazeki F, Yokosuka O, Fukai K, Kanda T, Kojima H, Saisho H. Department of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, Chiba, Japan. imazekif@faculty.chiba-u.jp BACKGROUND/AIMS: Our aim was to evaluate the relationship between hepatitis C virus (HCV) infection and development of diabetes mellitus (DM) or insulin resistance (IR) in comparison with hepatitis B virus (HBV) infection and eradication of HCV infection by interferon treatment. METHODS: This study consisted of 952 outpatients, including 544 HCV-infected (HCV+chronic), 286 HBV-infected (HBV+chronic) and 122 patients whose HCV was cleared by interferon treatment (HCV+cleared) (diabetes study). Among 849 without overt DM, IR was assessed in 423 patients, including 232 HCV-infected (HCV+chronic), 135 HBV-infected (HBV+chronic) and 56 HCV-eradicated patients (HCV+cleared) (IR substudy). RESULTS: The prevalence of DM in the HBV+chronic, HCV+chronic and HCV+cleared groups was 6.3, 13.6 and 9.0%, respectively (HBV+chronic vs HCV+chronic, P<0.005), in the diabetes study, and the prevalence of IR in the HCV+chronic group (54.3%) was also higher than that in the HBV+chronic (36.3%) (P<0.005) and HCV+cleared groups (35.7%) (P<0.05) in the IR substudy. However, HCV infection was not shown to be independently associated with DM development [odds ratio (OR) 1.669; P=0.0936] and with IR (OR 1.531; P=0.2154) by multivariate analysis in comparison with HBV infection as control. CONCLUSIONS: HCV-infected patients showed a higher prevalence of DM and IR than those with HBV infection. However, in Japan, other confounding factors appeared to be more important risk factors for the development of disturbance in glucose metabolism. Publication Types: Comparative Study PMID: 18290778 [PubMed - indexed for MEDLINE] 4: JNMA J Nepal Med Assoc. 2007 Jul-Sep;46(167):143-50. Current management of gastric varices. Maruyama H, Yokosuka O. Department of Medicine and Clinical Oncology, Chiba University Graduate School of Medicine, Japan. maru-cib@umin.ac.jp Gastric varices are a major hemodynamic feature in patients with portal hypertension. Its incidence as well as bleeding rate is lower than that of esophageal varices. However, rupture of gastric varices sometimes results in very serious consequences in the clinical course and its management is very important. Recent developments in imaging technology have made it possible to evaluate the portal hemodynamics in detail by less invasive procedure. Furthermore, advancements in medical instruments and technologies have led to the development of endoscopic ligation devices, interventional radiology techniques like transjugular intrahepatic portosystemic shunt and balloon-occluded retrograde transvenous obliteration, and skills in surgical treatments. This overview will focus on the classification, pathophysiology, current management of gastric varices, and treatments in patient with portal hypertension. Publication Types: Review PMID: 18274572 [PubMed - indexed for MEDLINE] 5: Hepatogastroenterology. 2007 Dec;54(80):2212-5. Diagnostic value of MRCP and indications for ERCP. Sakai Y, Tsuyuguchi T, Tsuchiya S, Sugiyama H, Miyakawa K, Ebara M, Saisho H, Yokosuka O. Department of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, Japan. BACKGROUND/AIMS: To assess the diagnostic value of magnetic resonance cholangiopancreatography (MRCP) and examine the indications for endoscopic retrograde cholangiopancreatography (ERCP). METHODOLOGY: MRCP was performed in 185 patients with hepatobiliary disease in whom abdominal ultrasonography (US) had not been of diagnostic value. These patients were selected for MRCP in view of their abdominal symptoms, high levels of hepatobiliary enzymes, and pancreatic/bile duct dilatation found by abdominal US. Based on MRCP findings, 75 patients were selected for ERCP. RESULTS: ERCP provided new findings in 14 (18.%) patients. In 110 patients subjected to only MRCP and follow-up as well as in 75 patients with MRCP followed by ERCP, MRCP-based diagnosis corresponded with the final diagnosis. In our study, patients who would have conventionally required ERCP, such as those with natural passed choledocholithiasis and postoperative bile duct dilatation, could be followed up without ERCP. These results the importance of considering indications for ERCP. CONCLUSIONS: MRCP can be an alternative to ERCP at least for diagnosis. PMID: 18265635 [PubMed - indexed for MEDLINE] 6: J Gastroenterol Hepatol. 2008 May;23(5):736-40. Epub 2007 Dec 5. Diagnostic value of magnetic resonance cholangiopancreatography for clinically suspicious spontaneous passage of bile duct stones. Sakai Y, Tsuyuguchi T, Yukisawa S, Tsuchiya S, Sugiyama H, Miyakawa K, Ohara T, Ebara M, Miyazaki M, Yokosuka O. Departments of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, Chiba, Japan. BACKGROUND AND AIM: We investigated the usefulness of magnetic resonance cholangiopancreatography (MRCP) and the need for endoscopic retrograde cholangiopancreatography (ERCP) in patients with clinically suspicious spontaneous passage of bile duct stones. METHODS: The study population consisted of 113 patients suspected of having common duct bile stones. Of them, 50 patients were clinically suspected of spontaneous passage of bile duct stones based on the presence of gallbladder stones on ultrasound examination or a history of common bile duct stones after cholecystectomy, clinical symptoms including abdominal pain and fever associated with inflammatory reaction and marked rise of hepatobiliary enzymes which resolved or normalized after conservative treatment without evidence of stones in the common bile duct on MRCP. These 50 patients were prospectively followed up for a median of 10.2 months. RESULTS: All patients except for one had had no symptoms related to cholangitis. Only one patient received ERCP due to recurrence of symptoms after 6 months. CONCLUSION: When clinical symptoms improve, hematological parameters normalize, and MRCP indicates that there are no stones in the common bile duct, it can be considered that the stones have passed naturally. PMID: 18070008 [PubMed - in process] 7: Hepatogastroenterology. 2007 Sep;54(78):1758-60. Body mass index in Japanese patients with autoimmune liver disease: overweight patients with primary biliary cirrhosis tend to be asymptomatic. Kanda T, Yokosuka O, Imazeki F, Hirasawa Y, Ikeuchi T, Mikata R, Zhang KY, Kurihara T, Arai M, Fukai K, Saisho H, Mikami S, Azemoto R, Ito Y. Department of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, Japan. kandat@aol.com BACKGROUND/AIMS: The aim of this study was to investigate the effects of being overweight on autoimmune hepatitis (AIH) and primary biliary cirrhosis (PBC) patients. METHODOLOGY/RESULTS: 44 AIH and 95 PBC patients were enrolled in this study. Body weight and body mass index (BMI) of AIH (57.6 +/- 10.4 kg and 23.8 +/- 2.9 kgm(-2), respectively) were higher than those of PBC (51.6 +/- 7.0 kg and 22.0 +/- 2.6 kgm(-2), respectively) (P < 0.001). The prevalence of overweight patients in AIH was also higher than those in PBC (P < 0.005). Being overweight and having 25 < or = BMI < 30 did not affect the progression of hepatic fibrosis in AIH and PBC. In comparison with the non-overweight with PBC, overweight patients with PBC tended not to be symptomatic, such as having itching or fatigue (P = 0.027). CONCLUSIONS: Clinicians should be aware that not only non-alcoholic fatty liver disease but also PBC patients might be included among the overweight hepatic disease patients with unknown etiology. PMID: 18019712 [PubMed - indexed for MEDLINE] 8: J Gastroenterol Hepatol. 2007 Oct 17. [Epub ahead of print] How to characterize non-hypervascular hepatic nodules on contrast-enhanced computed tomography in chronic liver disease: Feasibility of contrast-enhanced ultrasound with a microbubble contrast agent. Yoshizumi H, Maruyama H, Okugawa H, Kobayashi S, Akiike T, Yoshikawa M, Ebara M, Yokosuka O, Matsutani S, Kondo F, Kamiyama N. Department of Medicine and Clinical Oncology, Chiba University Graduate School of Medicine, Chiba, Japan. Background and Aim: Although hypervascular appearance is characteristic in hepatocellular carcinoma (HCC), hepatic nodules without hypervascular appearance are sometimes found in patients with chronic liver disease (CLD). The aim of the present study was to clarify the efficacy of contrast-enhanced ultrasound (CEUS) with Levovist to characterize small, non-hypervascular hepatic nodules on contrast-enhanced computed tomography (CECT) in patients with CLD. Methods: The subject was 41 hepatic nodules (<30 mm, 18.5 +/- 5.6 mm) which showed non-hypervascular appearance on CECT in 35 patients with CLD; their histological results were 31 HCC (15 well, 14 moderate, and two poor) and 10 regenerative nodules (RN). CEUS with Levovist was performed under intermittent scanning (1-s interval) using APLIO at the early phase and the liver-specific phase, and the contrast enhancement of the nodule was assessed in comparison to that of the surrounding liver parenchyma. The contrast-enhanced findings with the time-intensity analysis were compared with the histological results. Results: Twelve nodules with weak enhancement in the liver-specific phase were HCC, regardless of their early-phase appearances. The other 29 nodules with equivalent or weak enhancement in the early phase and equivalent enhancement in the liver-specific phase were 19 HCC and 10 RN. Among them, the maximum-intensity ratio of tumor to non-tumor in the early phase was significantly higher in HCC than in RN (P < 0.01, n = 16), and the receiver-operating characteristic analysis showed a sensitivity of 1.0 and a specificity of 0.83 for their characterization. Conclusion: CEUS with Levovist may be an alternative to biopsy to characterize small, non-hypervascular hepatic nodules on CECT in patients with CLD. PMID: 17944882 [PubMed - as supplied by publisher] 9: Aliment Pharmacol Ther. 2008 Jan 1;27(1):66-71. Epub 2007 Oct 8. Four-day continuous gastric pH monitoring following anti-acid secretory drug administration: cross-over test to assess the early effects. Suzuki T, Yamaguchi T, Odaka T, Kobayashi M, Seza A, Kouzu T, Yokosuka O. Department of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, Chiba, Japan. suzuki4511@yahoo.co.jp BACKGROUND: There have been few reports that compare the effect of histamine H2-receptor antagonists (H2RAs) and proton pump inhibitors (PPIs) using continuous gastric pH monitoring for a long duration. AIM: To assess the early effects of both drugs on gastric pH using a wireless pH monitoring system. METHODS: The test was conducted by a cross-over test: 10 healthy male volunteers were administered famotidine 20 mg twice a day and lansoprazole 30 mg once a day. Monitoring of gastric pH over four consecutive days was performed using a unique method we have developed that is an elaboration of the Bravo system. RESULTS: The time to reach a pH level of 3 or more with famotidine was significantly shorter than that for lansoprazole. The pH3 holding time ratio of famotidine during the first 4 h of administration was significantly higher than that of lansoprazole. The pH3 holding time ratio on each day from day 1 to day 3 was significantly higher following lansoprazole administration compared with famotidine administration. CONCLUSIONS: Famotidine was shown to act quickly in elevating gastric pH; however, lansoprazole was superior to famotidine in terms of its ability to elevate gastric pH for a long duration. PMID: 17922801 [PubMed - indexed for MEDLINE] 10: Medinfo. 2007;12(Pt 2):1304-8. Temporal abstraction and data mining with visualization of laboratory data. Takabayashi K, Ho TB, Yokoi H, Nguyen TD, Kawasaki S, Le SQ, Suzuki T, Yokosuka O. Division for Medical Informatics and Management, Chiba University Hospital, Inohana, Chuou-ku, Chiba, 260-8677 Japan. To analyze the laboratory data by data mining, user-centered universal tools have not been available in medicine. We analyzed 1,565,877 laboratory data of 771 patients with viral hepatitis in order to find the difference of the temporal changes in laboratory test data between Hepatitis B and Hepatitis C by the combination of temporal abstraction and data mining. The data for one patient is temporal for more than 5 years. After pretreatment the data was converted to abstract patterns and then selected into sets of data combination and rules to identify Hepatitis B or C by D2MS and LUPC which were originally produced by ourselves. Not only data pattern, but also temporal relations were considered as a part of the rules. In the course of evaluating the results by domain experts, even though there were not so remarkable hypotheses, visualization tools made it easier for them to understand the relations of the complicated rules. PMID: 17911925 [PubMed - indexed for MEDLINE] 11: Cancer Sci. 2007 Dec;98(12):1921-9. Epub 2007 Sep 19. Analysis of the complete hepatitis B virus genome in patients with genotype C chronic hepatitis and hepatocellular carcinoma. Zhang KY, Imazeki F, Fukai K, Arai M, Kanda T, Mikata R, Yokosuka O. Department of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-Ward, Chiba City, Chiba 260-8670, Japan. Hepatitis B virus (HBV) genotype C and the basic core promoter (BCP) mutations were reported to be associated with the development of hepatocellular carcinoma (HCC). In this study the full sequences of HBV genomes were analyzed in order to find the other predictors of HCC development. We determined the full sequences of HBV genomes in 24 genotype C carriers who developed HCC (HCC group) at the beginning of follow-up and at the time of HCC diagnosis, and 20 patients who did not develop HCC (non-HCC group) served as a control. The number of nucleotide and amino acid substitutions in most regions was higher in the HCC group than in the non-HCC group, and the following substitutions and deletions were found more frequently in the HCC group than in the non-HCC group: G1317A and T1341C/A/G in the X promoter region were detected in 13 and six of the HCC cases, four and none of the non-HCC cases, respectively; and pre-S2 deletion was detected in eight HCC and none of the non-HCC cases. Compared with the wild type X promoter, the mutant type X promoters, M1 (G1317A), M2 (T1341C), and M4 (T1341G) showed increases in activity of 2.3, 3.8, and 1.4 times, respectively, in HepG2 cells. Substitutions and deletion of nucleotides of the HBV genome, especially the pre-S2 deletion and G1317A and T1341C/A/G mutations may be useful markers for predicting the development of HCC. PMID: 17888035 [PubMed - indexed for MEDLINE] 12: J Viral Hepat. 2007 Sep;14(9):661-6. Association between lamivudine sensitivity and the number of substitutions in the reverse transcriptase region of the hepatitis B virus polymerase. Fukai K, Zhang KY, Imazeki F, Kurihara T, Mikata R, Yokosuka O. Department of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, Inohana, Chuo-ku, Chiba, Japan. This study aimed to identify the viral factors responsible for poor sensitivity to lamivudine (LAM). We analyzed 49 LAM-treated chronic hepatitis B patients infected with hepatitis B virus (HBV) genotype C. Serum HBV DNA reached a level below the detection limit of the sensitive PCR assay in 31 (63.3%) within the first 24 weeks of LAM therapy (good responder group). Of the patients who did not achieve undetectable levels of HBV DNA within 24 weeks (poor responder group), 15 (83.3%) experienced virological breakthrough, whilst only four patients in the good responder group (12.9%) experienced virological breakthrough. Multivariate analysis revealed that failure to achieve a reduction in viral load to undetectable levels within 24 weeks was independently associated with the occurrence of virological breakthrough. Sequence analysis of the HBV genome revealed that point mutations in the precore region (G1896A) and enhancer I (A1287G/C) were observed more frequently in the good responder group than in the poor responder group (P = 0.002 and 0.019 respectively), and the number of substitutions in the reverse transcriptase domain of the polymerase was significantly higher in the good responders than in the poor responders (P = 0.026). In conclusion, determining the sequence of preexisting HBV, especially for enhancer I, the precore region, and the RT domain of the polymerase region, may be useful in predicting sensitivity to LAM therapy. PMID: 17697019 [PubMed - indexed for MEDLINE] 13: Gastroenterology. 2007 Sep;133(3):937-50. Epub 2007 Jun 20. Enhanced self-renewal capability in hepatic stem/progenitor cells drives cancer initiation. Chiba T, Zheng YW, Kita K, Yokosuka O, Saisho H, Onodera M, Miyoshi H, Nakano M, Zen Y, Nakanuma Y, Nakauchi H, Iwama A, Taniguchi H. Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan. BACKGROUND & AIMS: Transformed hematopoietic stem/progenitor cells with an enhanced or acquired self-renewal capability function as leukemic stem cells. In a variety of solid cancers, stem/progenitor cells could be also targets of carcinogenesis. However, it remains unclear whether disruption of stem cell function directly contributes to cancer initiation. We sought to elucidate the mechanisms of self-renewal in hepatic stem/progenitor cells and the relation between stem cell function and hepatocarcinogenesis. METHODS: Functional analyses of polycomb-group protein Bmi1 and Wnt/beta-catenin, the molecules that are responsible for the self-renewal capability of many types of stem cells, were conducted in c-Kit(-)CD29(+)CD49f(+/low)CD45(-)Ter-119(-) hepatic stem/progenitor cells using retrovirus- or lentivirus-mediated gene transfer. The tumorigenicity of these cells transduced with the indicated retroviruses was also assessed by transplantation into nonobese diabetic/severe combined immunodeficient mice. RESULTS: Forced expression of Bmi1 and constitutively active beta-catenin mutant similarly promoted the self-renewal of hepatic stem/progenitor cells. The transplantation of Bmi1- or beta-catenin-transduced cells clonally expanded from single hepatic stem/progenitor cells produced tumors, which exhibited the histologic features of combined hepatocellular and cholangiocarcinoma. CONCLUSIONS: These observations imply that the dysregulated self-renewal of hepatic stem/progenitor cells serves as an early event in hepatocarcinogenesis, and they highlight the important roles of Bmi1 and the Wnt/beta-catenin pathway in regulating the self-renewal of normal or cancer stem cells in liver. Publication Types: Research Support, Non-U.S. Gov't PMID: 17673212 [PubMed - indexed for MEDLINE] 14: J Gastroenterol. 2007 Jul;42(7):560-6. Epub 2007 Jul 25. Erratum in: J Gastroenterol. 2008 Feb;43(2):179. Analysis of hepatitis A virus protein 2B in sera of hepatitis A of various severities. Fujiwara K, Yokosuka O, Imazeki F, Miki M, Suzuki K, Okita K, Tanaka E, Omata M. Department of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. BACKGROUND: In our recent study of the full-length hepatitis A virus (HAV) genome from some patients with fulminant hepatitis and acute hepatitis, possible associations were suggested between the severity of hepatitis A and the amino acid substitutions in the nonstructural protein 2B. We therefore analyzed HAV 2B from many patients with various clinical disease severities. METHODS: Serum samples from 30 Japanese patients with sporadic hepatitis A from five widely separated regions of Japan, comprising nine patients with fulminant hepatitis (FH), six with severe acute hepatitis (AHs), and 15 with acute hepatitis (AH), were examined for HAV RNA. The entire sequences of HAV 2B were analyzed. RESULTS: Compared with the sequence of the wild-type HAV strain GBM, nucleotide sequences of 2B had homology of 94.5 +/- 1.0% in FH, 95.2 +/- 1.2% in AHs, and 95.1 +/- 1.8% in AH. Deduced amino acid sequences had homology of 97.5 +/- 2.1% in FH, 97.9 +/- 2.4% in AHs, and 98.5 +/- 1.3% in AH. Differences were not statistically significant among the three groups. The average number of amino acid mutations between amino acids 100 and 200 was 5.0 +/- 5.2 per case in FH, 4.0 +/- 6.0 in AHs, and 1.9 +/- 2.9 in AH. The differences between FH and AH, AHs and AH, and between severe cases (FH and AHs) and nonsevere cases (AH) were not statistically significant (P = 0.13, P = 0.45, and P = 0.10, respectively). CONCLUSIONS: There were no obvious differences in the sequences among FH, AHs, and AH throughout the 2B region, but there seemed to be more mutations in the strains obtained from FH and AHs patients than in those obtained from AH patients in the central part of HAV 2B. PMID: 17653652 [PubMed - indexed for MEDLINE] 15: Basic Clin Pharmacol Toxicol. 2007 Aug;101(2):121-6. Regionally differential effects of sennoside A on spontaneous contractions of colon in mice. Kobayashi M, Yamaguchi T, Odaka T, Nakamura T, Tsuchiya S, Yokosuka O, Yano S. Department of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, Chiba, Japan. rinko_kobayashi@yahoo.co.jp Sennosides, the most popular irritant laxatives, cause purgative actions in the intestine through biotransformation to rhein anthrone; however, the underlying mechanisms remain unclear. The purpose of this study was to define colonic motor actions of sennoside A with special reference to purgative action. Mice received a single oral dose of 30 mg/kg sennoside A, and the colon was removed about 6 hr later. Contractions of longitudinal and circular muscles were recorded using an isometric force transducer and a pressure transducer, respectively. In longitudinal muscle preparations, spontaneous contractions were augmented in distal colon compared to control. In circular muscle preparations, contractions were reduced in the proximal colon, but increased in the distal colon. Particularly in the proximal colon, the frequency of high-amplitude contraction was reduced. In the control group, non-adrenergic, non-cholinergic treatment decreased the amplitude of contractions in the proximal colon, but not in the distal colon. In the sennoside A group, non-adrenergic, non-cholinergic treatment only slightly depressed the amplitude of contractions in the proximal and distal colon. To confirm a causal relationship between luminal prostaglandin level and purgative action of sennoside A, the mice were treated with indomethacin. Significant changes induced by sennoside A were attenuated by indomethacin treatment. The present study indicates that spontaneous motility is inhibited by sennoside A in the proximal colon, but accelerated in the distal colon, and that effects are associated with luminal prostanoid level and only partially with cholinergic nerve mediation. PMID: 17651314 [PubMed - indexed for MEDLINE] 16: Hepatogastroenterology. 2007 Apr-May;54(75):871-7. Genetic analysis of hepatitis A virus protein 2C in sera from patients with fulminant and self-limited hepatitis A. Fujiwara K, Yokosuka O, Imazeki F, Miki M, Suzuki K, Okita K, Tanaka E, Omat M. Department of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. BACKGROUND/AIMS: To examine whether genetic differences in hepatitis A virus (HAV) are responsible for the range of clinical severities, we analyzed the HAV 2C genome, whose mutations have previously been shown to be important for enhanced replication in cell culture systems and to be related to virulence in simians. METHODOLOGY: Serum samples from 45 Japanese patients with sporadic hepatitis A, comprising 9 patients with fulminant hepatitis (FH), 10 with severe acute hepatitis (AHs), and 26 with self-limited acute hepatitis (AH), were examined for HAV RNA. RESULTS: Compared with the sequence of wild-type HAV strain HM-175, the nucleotide sequences of 2C had homology of 89.0 +/- 0.6% in FH, 88.6 +/- 0.9% in AHs, and 89.0 +/- 1.6% in AH. Differences were not statistically significant among the three groups. Deduced amino acid sequences had homology of 97.6 +/- 0.4% in FH, 96.5 +/- 1.9% in AHs, and 96.8 +/- 1.7% in AH. The difference between FH and AH was statistically significant (p < 0.05), although there were no specific nucleotide or amino acid substitutions. CONCLUSIONS: Fulminant hepatitis patients had fewer amino acid substitutions in 2C, indicating the association between severity of hepatitis A and amino acid variations in 2C of HAV. PMID: 17591082 [PubMed - indexed for MEDLINE] 17: J Med Virol. 2007 Jun;79(6):683-93. Analysis of the complete hepatitis B virus genome in patients with genotype C chronic hepatitis in relation to HBeAg and anti-HBe. Zhang K, Imazeki F, Fukai K, Arai M, Kanda T, Mikata R, Yokosuka O. Department of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, Chiba, Japan. To investigate the relationship between viral factors and the development of chronic hepatitis B, the entire hepatitis B virus (HBV) genome of chronic carriers at different disease stages were analyzed. Eighty genotype C HBV carriers including 12 hepatitis B e antigen (HBeAg) positive asymptomatic carriers (Group A), 49 HBeAg positive patients with chronic liver diseases (Group B) and 19 anti-HBe positive patients with chronic liver diseases (Group C) were studied. HBV nucleic acid from serum samples was sequenced directly and compared with GenBank reference sequences HBV X01587 and M12906. On phylogenetic analysis, 76 cases were genotype C2. Of the 76 genotype C2 cases, the nucleotide and amino acid substitution rates in the precore/core region were significantly higher in Groups B and C than in Group A, also in Group C than in Group B. The nucleotide substitution rates in the full genome and the core promoter region were significantly higher in Group C than in Group A, also in group C than in Group B. The nucleotide and amino acid substitution rates in the X region were significantly higher in Group C than in Group A. The amino acid substitution rate in the pre-S2 region was significantly higher in Group C than in Group B. Deletion mutations were found mainly in Groups B and C. This whole genome analysis of HBV chronic carriers suggested that the nucleotide substitutions and deletions in HBV were closely associated with the pathogenesis of chronic HBV infection. (c) 2007 Wiley-Liss, Inc. Publication Types: Comparative Study PMID: 17457922 [PubMed - indexed for MEDLINE] 18: Hepatogastroenterology. 2007 Jan-Feb;54(73):32-5. Inhibition of subgenomic hepatitis C virus RNA replication in HeLa cells. Kanda T, Yokosuka O, Mikata R, Zhang KY, Tanaka M, Tada M, Fukai K, Imazeki F, Saisho H. Department of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, Chiba, Japan. kandat-cib@umin.ac.jp BACKGROUND/AIMS: Antiviral therapy such as combination interferon and ribavirin can eradicate hepatitis C virus (HCV) RNA by up to 40-50%. However, many patients still remain non-responders to this treatment for various reasons. The aim of this study was to evaluate the effect of interferon or ribavirin treatment on subgenomic HCV RNA replication in 'non-hepatic' HeLa cells. METHODOLOGY: Huh-7 or HeLa cells harboring HCV replicon were constructed by using cellular RNA of Huh-7 harboring HCV replicon RNAs, named as C13-3 cells. We also tested whether interferon or ribavirin can suppress HCV RNA in HeLa cells. RESULTS: Huh-7 or HeLa cells harboring HCV replicon RNAs were constructed by using cellular RNA of C13-3 cells than using in vitro-transcribed RNA. Ribavirin at 1 microg/mL or 10 microg/mL did not suppress colony formation in HeLa cells, but at 100 microg/mL suppression was observed. Interferon-alpha 2b suppressed HCV replication even at 1 U/mL. CONCLUSIONS: HeLa cells harboring HCV replicon RNAs also might be useful for the development of antiviral drugs. Publication Types: Research Support, Non-U.S. Gov't PMID: 17419226 [PubMed - indexed for MEDLINE] 19: Oncology. 2006;71(1-2):77-85. Epub 2007 Mar 6. Methylation status of genes upregulated by demethylating agent 5-aza-2'-deoxycytidine in hepatocellular carcinoma. Hirasawa Y, Arai M, Imazeki F, Tada M, Mikata R, Fukai K, Miyazaki M, Ochiai T, Saisho H, Yokosuka O. Department of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, Chiba, Japan. BACKGROUND/AIMS: To determine the clinical significance of gene promoter methylation in hepatocellular carcinoma (HCC), we examined in clinical samples the methylation status of those promoters that showed elevated activity in hepatoma cell lines after 5-aza-2'-deoxycytidine treatment. METHODS: Regarding the genes with promoter hypermethylation in the cell lines, their expression levels and methylation status in HCC and non-HCC tissues were assessed by semiquantitive RT-PCR and methylation-specific PCR. To confirm the result, the expression levels and methylation status in 16 additional HCC and non-HCC tissues were assessed. RESULTS: The promoter regions of caveolin 1 (CAV1), cysteine and glycine-rich protein 1 (CSRP1), Kruppel-like factor 6 (KLF6), myosin (light polypeptide 9) (MYL9), and transgelin (TAGLN) were highly methylated in the cell lines. CAV1 and CSRP1 were methylated in HCC more frequently than in non-HCC. KLF6, MYL9, and TAGLN were fully methylated in both HCC and non-HCC. Using additional clinical samples, downregulation of CAV1 and CSRP1 was observed in 38 and 56%, respectively, of the 16 HCC samples and aberrant methylation of CAV1 and CSRP1 was observed in 56% of HCC in both cases. CONCLUSION: CAV1 and CSRP1 were inactivated in HCC by aberrant methylation and they may serve as important biomarkers of malignancy. PMID: 17341888 [PubMed - indexed for MEDLINE] 20: J Hepatol. 2006 Dec;45(6):805-12. Epub 2006 Sep 22. Nucleotide change of codon 38 in the X gene of hepatitis B virus genotype C is associated with an increased risk of hepatocellular carcinoma. Muroyama R, Kato N, Yoshida H, Otsuka M, Moriyama M, Wang Y, Shao RX, Dharel N, Tanaka Y, Ohta M, Tateishi R, Shiina S, Tatsukawa M, Fukai K, Imazeki F, Yokosuka O, Shiratori Y, Omata M. Department of Gastroenterology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan. BACKGROUND/AIMS: The hepatitis B virus (HBV) genotype C is associated with the development of hepatocellular carcinoma (HCC). In addition, the HBV X gene, which encodes the pleiotropic transactivator HBx, has also been associated with the development of HCC. In this study, we investigated whether nucleotide changes in the X gene of genotype C are associated with the development of HCC. METHODS/RESULTS: We sequenced the X gene in age- and sex-matched 39 HBV-infected patients with HCC and 36 HBV-infected patients without HCC. A novel nucleotide change that resulted in a proline to serine substitution at codon 38 in HBx (codon-38 change) was preferentially found in patients with HCC. Then, sera were collected from a new group of age- and sex-matched 52 patients with HCC and 51 patients without HCC. In this cohort also, the codon-38 change was associated with HCC. Multiple logistic regression analysis showed the prevalence of the codon-38 change was significantly associated with HCC in all patients (P=0.001, odds ratio: 4.89). CONCLUSION: The codon-38 change in genotype C is an independent risk factor for the development of HCC and may serve as a useful molecular marker for predicting the clinical outcomes in patients infected with HBV. Publication Types: Research Support, Non-U.S. Gov't PMID: 17050029 [PubMed - indexed for MEDLINE] 21: Med Mol Morphol. 2006 Sep;39(3):113-20. Molecular biology of hepatitis B virus: effect of nucleotide substitutions on the clinical features of chronic hepatitis B. Yokosuka O, Arai M. Department of Medicine and Clinical Oncology, K1, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chiba, 260-8670, Japan. yokosukao@faculty.chiba-u.jp Hepatitis B virus (HBV) is a major cause of liver disease worldwide. It is covered with envelope (surface antigen) proteins with the nucleocapsid (core antigen) inside. In the nucleocapsid, there is an incomplete double-stranded DNA and a DNA polymerase. Four genes, S, C, X, and P, are encoded, and these partially overlap. Mutations have been reported in each gene and in their promoter regions, and these mutations can change the efficiency of HBV replication and the clinical course of patients. In this article, we review the relationship between the molecular biology of HBV and its clinical outcome. Publication Types: Review PMID: 16998621 [PubMed - indexed for MEDLINE] 22: Cancer Sci. 2006 Oct;97(10):1105-10. Randomized, double-blind, placebo-controlled trial of bovine lactoferrin in patients with chronic hepatitis C. Ueno H, Sato T, Yamamoto S, Tanaka K, Ohkawa S, Takagi H, Yokosuka O, Furuse J, Saito H, Sawaki A, Kasugai H, Osaki Y, Fujiyama S, Sato K, Wakabayashi K, Okusaka T. Hepatobiliary and Pancreatic Oncology Division, National Cancer Center Hospital, Tokyo, Japan. hiueno@ncc.go.jp Several studies have suggested that lactoferrin administration may decrease the serum level of hepatitis C virus (HCV) RNA in patients with chronic hepatitis C. The aim of the present study was to confirm the efficacy of orally administered bovine lactoferrin (bLF) in patients with chronic hepatitis C. The patients with chronic hepatitis C randomly received either oral bLF at a dose of 1.8 g daily for 12 weeks, or an oral placebo. The primary endpoint was the virologic response, defined as a 50% or greater decrease in serum HCV RNA level at 12 weeks compared with the baseline. The secondary endpoint was the biochemical response, which was defined as a 50% or greater decrease in the serum alanine aminotransferase (ALT) level at 12 weeks compared with the baseline. One hundred and ninety-eight of 199 patients were evaluable for efficacy and safety. bLF treatment was well tolerated and no serious toxicities were observed. A virologic response was achieved in 14 of 97 patients (14.4%) in the bLF group, and 19 of 101 (18.8%) in the placebo group. There was no significant difference in virologic response rates between the two groups (-4.4%, 95% confidence interval -14.8, 6.1). In addition, bLF intake did not have any favorable effect on the serum ALT level. The virologic responses were not different between two groups in any subgroup analysis. In conclusion, orally administered bLF does not demonstrate any significant efficacy in patients with chronic hepatitis C. Publication Types: Randomized Controlled Trial Research Support, Non-U.S. Gov't PMID: 16984383 [PubMed - indexed for MEDLINE] 23: Nippon Rinsho. 2006 Jul;64(7):1368-73. [Devices for the safe performance of self-injection of interferon therapy for chronic hepatitis C] [Article in Japanese] Kurihara T, Mikata R, Yokosuka O. Department of Medicine and Clinical Oncology, Graduate School of Medicine Chiba University. Long-term interferon therapy is an alternative therapy for patients with chronic hepatitis C who can not tolerate to the combination of pegylated interferon and ribavirin. In 2005, self-injection of interferon for chronic hepatitis C was covered by public health insurance in Japan making the long-term interferon therapy easier to perform. For the safe performance of self-injection, education about knowledge on various adverse effects of interferon, training for actual performance of self-injection, and close contact between the patients and doctors are indispensable. In this review, we briefly summarized why self-injection is needed and how the training for safe self-injection of interferon should be performed. Publication Types: English Abstract Review PMID: 16838658 [PubMed - indexed for MEDLINE] 24: Hepatology. 2006 Jul;44(1):240-51. Comment in: Hepatology. 2006 Jul;44(1):23-6. Side population purified from hepatocellular carcinoma cells harbors cancer stem cell-like properties. Chiba T, Kita K, Zheng YW, Yokosuka O, Saisho H, Iwama A, Nakauchi H, Taniguchi H. Department of Regenerative Medicine, Graduate School of Medical Science, Yokohama City University, Yokohama, Japan. Recent advances in stem cell biology enable us to identify cancer stem cells in solid tumors as well as putative stem cells in normal solid organs. In this study, we applied side population (SP) cell analysis and sorting to established hepatocellular carcinoma (HCC) cell lines to detect subpopulations that function as cancer stem cells and to elucidate their roles in tumorigenesis. Among four cell lines analyzed, SP cells were detected in Huh7 (0.25%) and PLC/PRF/5 cells (0.80%), but not in HepG2 and Huh6 cells. SP cells demonstrated high proliferative potential and anti-apoptotic properties compared with those of non-SP cells. Immunocytochemistry examination showed that SP fractions contain a large number of cells presenting characteristics of both hepatocyte and cholangiocyte lineages. Non-obese diabetic/severe combined immunodeficiency (NOD/SCID) xenograft transplant experiments showed that only 1 x 10(3) SP cells were sufficient for tumor formation, whereas an injection of 1 x 10(6) non-SP cells did not initiate tumors. Re-analysis of SP cell-derived tumors showed that SP cells generated both SP and non-SP cells and tumor-initiating potential was maintained only in SP cells in serial transplantation. Microarray analysis discriminated a differential gene expression profile between SP and non-SP cells, and several so-called "stemness genes" were upregulated in SP cells in HCC cells. In conclusion, we propose that a minority population, detected as SP cells in HCC cells, possess extreme tumorigenic potential and provide heterogeneity to the cancer stem cell system characterized by distinct hierarchy. Publication Types: Comparative Study In Vitro Research Support, Non-U.S. Gov't PMID: 16799977 [PubMed - indexed for MEDLINE] 25: Liver Int. 2006 Jun;26(5):520-8. Twenty-four weeks of interferon alpha-2b in combination with ribavirin for Japanese hepatitis C patients: sufficient treatment period for patients with genotype 2 but not for patients with genotype 1. Fujiwara K, Yokosuka O, Komine F, Moriyama M, Kato N, Yoshida H, Tanaka N, Imazeki F, Shiratori Y, Arakawa Y, Omata M; Tokyo Hepatitis Network. Department of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, Chiba, Japan. BACKGROUND: Hepatitis C virus (HCV) RNA titer and HCV genotype are two major determinants of the outcome of interferon (IFN) monotherapy. To clarify the usefulness of combination therapy with IFN and ribavirin in Japanese hepatitis C patients, we treated patients with a relatively high dose of IFN in combination with ribavirin for 24 weeks and examined the effects in relation to the viral parameters. METHODS: Two hundred and ninety-five patients were enrolled in the study. The patients received either 6 or 10 million units (MU) of interferon alpha-2b every day for 2 weeks and then three times a week for 22 weeks with a daily dose of either 600 or 800 mg of ribavirin. The treatment response and safety of this treatment were examined. RESULTS: The sustained virologic response (SVR) rates were 26.8% in genotype 1 and 76.5% in genotype 2 (P < 0.001), and 36.1% with the 6 MU group and 45.8% with the 10 MU group (P = 0.09). Multivariate analysis indicated that SVR was associated with genotype 2, HCV RNA <500 kilointernational unit/ml (kIU/ml), and HCV RNA undetectability at week 8 of treatment. CONCLUSION: Our current study showed that a 24-week course of IFN plus ribavirin combination therapy was effective with respect to virologic response in Japanese hepatitis C patients, particularly in patients with HCV genotype 2. Publication Types: Clinical Trial PMID: 16761995 [PubMed - indexed for MEDLINE] 26: Rinsho Byori. 2006 Apr;54(4):408-12. [Hepatitis E virus infection in Japan] [Article in Japanese] Fukai K, Yokosuka O, Imazeki F, Mikata R, Kojima H, Saisho H. Department of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, Chiba 260-0856. Hepatitis E virus (HEV) is the major etiologic agent of enterically transmitted viral hepatitis in many developing countries. Epidemics are primarily waterborne in areas where water supplies are contaminated with HEV of human origin. There is increasing evidence, however, that HEV is also prevalent in very low numbers in non-endemic countries, including Japan. Although the source of HEV in these sporadic cases is unknown, a recently isolated swine virus is the best candidate for causing a zoonotic form of hepatitis E. The virus is serologically cross-reactive with human HEV and genetically very similar, and the human and swine strains seem to be cross-infective. Very recent evidence has also shown that swine HEV, and possibly a deer strain of HEV, may be related to avian HEV and HEV in other hosts and potential reservoirs. We examined the prevalence of anti-HEV IgM and IgG among patients serologically diagnosed with non-A, non-B, non-C acute hepatitis (n = 126) and compared with a matched control group of 76 individuals. Enzyme-linked immunosorbent assay revealed a significant difference in seroprevalence between the two groups for anti-HEV IgM (5.6% versus 0%), whereas there was no difference for anti-HEV IgG (21.4% versus 26.3%). For confirmed cases of anti-HEV IgM we also detected HEV RNA in sera by means of a sensitive polymerase chain reaction (PCR) assay. This study provides evidence of locally acquired hepatitis E in the Chiba area. Therefore, in cases of unexplained acute hepatitis, the diagnosis of hepatitis E should be considered even in the absence of foreign travel. Publication Types: English Abstract PMID: 16722461 [PubMed - indexed for MEDLINE] 27: Int J Cancer. 2006 Oct 1;119(7):1616-22. Analysis of genes upregulated by the demethylating agent 5-aza-2'-deoxycytidine in gastric cancer cell lines. Mikata R, Yokosuka O, Fukai K, Imazeki F, Arai M, Tada M, Kurihara T, Zhang K, Kanda T, Saisho H. Department of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, Chiba, Japan. In gastric cancer, increasing numbers of genes have been reported to be silenced by aberrant methylation. However, global analysis of epigenetic inactivation in cancer cells has rarely been performed. For screening the genes upregulated by the demethylating agent 5-aza-2'-deoxycytidine (DAC), cDNA microarray analysis (AceGene(R), containing 30,000 genes) was performed using gastric cancer cell lines (AGS, MKN74, MKN1, MKN45 and Kato3) treated with DAC. The candidate upregulated genes were confirmed by real-time PCR, and the methylation status of 5'CpG islands was determined by bisulfite DNA sequencing or methylation-specific PCR. Among the upregulated genes considered to have CpG island in their promoter regions, we selected 5 genes (BCL2L10, DKK1, DNAJD1, GAGED2 and NMU) that exhibited a greater than 3-fold increase in at least 2 cell lines. Of these, we could determine the methylation status of 5'CpG islands of BCL2L10, DKK1 and DNAJD1. 5'CpG of BCL2L10 and DNAJD1 was hypermethylated in 4 of 5 gastric cancer cell lines, whereas 5'CpG of DKK1 was hypermethylated in only 1 cell line. MSP analysis for BCL2L10 revealed that the CpG island was demethylated after DAC treatment. In addition, we observed that overexpression of BCL2L10 could promote apoptosis and growth-inhibitory effect in gastric cancer cell lines. In conclusion, some of the genes upregulated by DAC treatment may be transcriptionally repressed by promoter hypermethylation. These genes might be related to gastric carcinogenesis. In particular, the suppression of BCL2L10, which could induce apoptosis and inhibit proliferation of cancer cells, might be one of the underlying mechanisms for gastric carcinogenesis. Copyright 2006 Wiley-Liss, Inc. PMID: 16671088 [PubMed - indexed for MEDLINE] 28: Cancer. 2006 Jun 1;106(11):2514-25. Sequential gene expression changes in cancer cell lines after treatment with the demethylation agent 5-Aza-2'-deoxycytidine. Arai M, Yokosuka O, Hirasawa Y, Fukai K, Chiba T, Imazeki F, Kanda T, Yatomi M, Takiguchi Y, Seki N, Saisho H, Ochiai T. Department of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, Chiba, Japan. BACKGROUND: 5-Aza-2'-deoxycytidine (5-AzaC) is known well for its demethylation effect and is a promising anticancer agent. However, to the authors' knowledge, serial changes in gene expression over time after 5-AzaC treatment have not been studied to date. To clarify the categories of genes that are up-regulated or down-regulated after 5-AzaC treatment, the authors surveyed the genes that had expression levels changed by 5-AzaC treatment in 6 hepatoma cell lines (Hep3B, HLE, Huh7, HepG2, PLC/PRF/5, and Huh6). METHODS: Cell lines were grown in medium that contained 1 microM of 5-AzaC. Changes in messenger RNA levels were monitored from 24 hours up to 120 hours after 5-AzaC treatment using an in-house microarray that consisted of 4608 combinational DNAs. Using clustering analysis to identify the genes that had gradually changed expression levels and to exclude the substantial experimental noise by microarray analysis, the authors focused on 206 up-regulated genes and 248 down-regulated genes. RESULTS: According to their functional characterization, genes that were involved in the cytoskeleton and the extracellular matrix were enriched significantly in the up-regulated genes. Conversely, genes that were involved in metabolism were enriched significantly in the down-regulated genes. CONCLUSIONS: The current results demonstrated that 5-AzaC can regulate the expression of groups of genes with characteristic functions. Copyright (c) 2006 American Cancer Society. Publication Types: Comparative Study PMID: 16649225 [PubMed - indexed for MEDLINE] 29: J Gastroenterol Hepatol. 2006 Mar;21(3):625-6. Hepatitis A protein VP1-2A reduced cell viability in Huh-7 cells with hepatitis C virus subgenomic RNA replication. Kanda T, Yokosuka O, Imazeki F, Fujiwara K, Saisho H. Publication Types: Letter PMID: 16638117 [PubMed - indexed for MEDLINE] 30: Dig Dis Sci. 2006 Jan;51(1):45-6. Occurrence of autoimmune hepatitis during the course of primary biliary cirrhosis: report of two cases. Kanda T, Yokosuka O, Hirasawa Y, Imazeki F, Nagao K, Saisho H. First Department of Medicine, Chiba University School of Medicine, Chiba, Japan. kandat-cib@umin.ac.jp Publication Types: Case Reports PMID: 16416210 [PubMed - indexed for MEDLINE] 31: Scand J Gastroenterol. 2006 Jan;41(1):102-10. Serum osteopontin levels in patients with acute liver dysfunction. Arai M, Yokosuka O, Kanda T, Fukai K, Imazeki F, Muramatsu M, Seki N, Miyazaki M, Ochiai T, Hirasawa H, Saisho H. Department of Medicine and Clinical Oncology, Graduate School of Medicine Chiba University, Japan. OBJECTIVE: Fulminant hepatic failure (FHF) is a clinical syndrome of sudden and severe liver dysfunction accompanied by encephalopathy in a previously healthy person. In FHF, hepatocytes are severely damaged and ordinary liver regeneration is impaired. We demonstrated that the expression of osteopontin (OPN), a multifunctional cytokine, was up-regulated in mouse oval cell (a stem-cell progenitor) induction models. MATERIAL AND METHODS: Based on this finding, serum OPN levels were examined in 43 patients with FHF and in 45 patients with acute self-limited hepatitis (AH). To determine the cellular source of OPN, the expression of OPN was studied in a liver specimen derived from an FHF patient. RESULTS: The mean OPN level of patients with FHF was 2.80+/-0.48 ng/ml (log(10), +/-SD), which was significantly higher than that of the patients with AH (2.42+/-0.39 ng/ml) (p=0.003, unpaired t-test). Patients with elevated serum OPN levels had a significantly poorer prognosis than patients whose serum OPN levels were not elevated. In the FHF patient's liver, OPN protein was expressed not only in inflammatory cells but also in regenerating hepatocytes and bile ductular structures. CONCLUSIONS: Our current study indicates that serum OPN levels increased in patients with FHF and that OPN might play an important role in liver regeneration due to activation of hepatic stem cells. PMID: 16373283 [PubMed - indexed for MEDLINE] 32: Liver Int. 2005 Dec;25(6):1209-16. Methylation status of p14ARF, p15INK4b, and p16INK4a genes in human hepatocellular carcinoma. Fukai K, Yokosuka O, Imazeki F, Tada M, Mikata R, Miyazaki M, Ochiai T, Saisho H. Department of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, Chiba, Japan. BACKGROUND: The INK4 locus consisting of three genes involved in the regulation of cell cycle, p16INK4a, p15INK4b, and p14ARF is often disrupted in human neoplasms. METHODS: We analyzed the promoter methylation of each gene by methylation-specific PCR in hepatocellular carcinoma (HCC). RESULTS: The methylation of p16INK4a, p15INK4b, and p14ARF was found to occur in 27 (69.2%), seven (17.9%), and none out of 39 HCC tumors, respectively. Regarding corresponding nontumorous liver tissues, the promoter regions of p16INK4a, p15INK4b, and p14ARF were methylated in three (17.6%), three (17.6%), and none out of 17 samples, respectively. Analysis of mRNA expression revealed that loss of p16INK4a expression was frequently observed in HCC. In contrast, transcripts of p14ARF and p15INK4b were detected in 16 (88.9%) and 16 (88.9%) of 18 tumors, respectively. CONCLUSIONS: The frequent loss of transcription of p16INK4a with promoter methylation not only in the advanced but also in the early stages of HCC suggests that the epigenetic alteration of p16INK4a promoter is likely to be involved in hepatocarcinogenesis. Together with the result of RT-PCR analysis, the role of aberrant methylation of p14ARF or p15INK4a promoter in hepatocarcinogenesis is thought to be limited. PMID: 16343074 [PubMed - indexed for MEDLINE] 33: Liver Int. 2005 Dec;25(6):1136-41. Evaluation of clinical usefulness of second-generation HCV core antigen assay: comparison with COBAS AMPLICOR HCV MONITOR assay version 2.0. Yokosuka O, Kawai S, Suzuki Y, Fukai K, Imazeki F, Kanda T, Tada M, Mikata R, Hata A, Saisho H. Department of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, Chiba, Japan. yokosukao@faculty.chiba-u.jp BACKGROUND: Hepatitis C virus (HCV) is an important etiologic agent for chronic liver diseases. METHODS: The aim of this study was to evaluate the clinical usefulness of second-generation HCV core antigen assay by comparing the results of the assay with those of the COBAS AMPLICOR HCV MONITOR version 2.0 (COBAS v2.0). RESULTS: HCV core antigen was detectable by this assay in 142/149 (95.3%) of serotype 1 (3821+/-322 fmol/l; mean+/-SD), in 56/58 (96.6%) of serotype 2 (2589+/-449 fmol/l), and in 6/6 (100%) of serotypes 1+2 (1240+/-548 fmol/l). The HCV core antigen levels measured by this assay correlated well with the HCV RNA levels by COBAS v2.0 (r=0.848, P<0.0001). In relation to the outcome of interferon monotherapy, the pretreatment HCV core antigen levels of sustained and non-sustained virological responders were 659+/-189 and 4904+/-376 fmol/l in serotype 1, 1993+/-740 and 3145+/-519 fmol/l in serotype 2. The cutoff values with the best accuracy for HCV core Ag levels to discriminate between sustained and non-sustained virological response were 699 fmol/l for serotype 1 and 292 fmol/l for serotype 2, respectively, by receiver operating characteristic curve analysis. CONCLUSION: This new assay was considered to be useful in evaluating the HCV levels in patients with chronic hepatitis C. Publication Types: Comparative Study PMID: 16343063 [PubMed - indexed for MEDLINE] 34: Hepatogastroenterology. 2005 Nov-Dec;52(66):1849-53. Clinicopathological features in patients with hepatic graft-versus-host disease. Chiba T, Yokosuka O, Goto S, Fukai K, Imazeki F, Kohno Y, Nishimura M, Saito Y, Saisho H. Department of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, Japan. BACKGROUND/AIMS: Hepatic graft-versus-host disease (GVHD) is a frequent complication after bone marrow transplantation and often results in a fatal outcome. However, hepatic manifestation of chronic GVHD accompanied by unresolved acute GVHD has not been clarified so far. The present study was intended to examine clinicopathological features in patients in which acute GVHD appeared to progress gradually into chronic GVHD. METHODOLOGY: We evaluated laboratory data, pathological features and response to immunosuppression in 11 patients whose diseases were diagnosed as hepatic GVHD, retrospectively. The patients were classified into 3 groups: acute GVHD group (n=3), non-progressive chronic GVHD group (n=5) and progressive chronic GVHD group (n=3), which means continuous liver dysfunction beyond day 100 post-transplant. RESULTS: Patients with progressive chronic GVHD showed higher peaks of aminotransferase and biliary tract enzyme levels than patients with acute GVHD and non-progressive chronic GVHD. Their biopsy specimens demonstrated severer changes in lobular parenchyma, portal area and small interlobular bile ducts. They also showed marked loss of bile ducts. Despite administration of prednisolone or dose escalation of cyclosporin A, their liver function tests did not return to normal within one year. CONCLUSIONS: In cases of liver dysfunction that emerges within 100 days after transplantation, liver biopsy appears to be important to confirm diagnosis. Patients with acute hepatic GVHD need strong immunosuppression to prevent progression to chronic GVHD. Publication Types: Comparative Study PMID: 16334791 [PubMed - indexed for MEDLINE] 35: DNA Cell Biol. 2005 Dec;24(12):805-9. Enhanced sensitivity of human hepatoma cells to 5-fluorouracil by small interfering RNA targeting Bcl-2. Kanda T, Yokosuka O, Imazeki F, Arai M, Saisho H. Department of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, Japan. kandat-cib@umin.ac.jp This study was designed to reveal whether the apoptosis induced in human hepatocellular carcinoma (HCC) cell lines by 5-fluorouracil (5-FU) could be enhanced by transfecting Bcl-2 small interfering RNA (siRNA). Bcl-2 siRNA and control siRNA were transfected into cells following treatment with or without 5-FU. Suppression of Bcl-2 expression was confirmed by Western blotting; cell viability was evaluated by MTS assay, and the occurrence of apoptosis in cells was evaluated by apoptosis assay. Expression of Bcl-2 protein after transfection of 20 nM Bcl-2 siRNA was significantly lower than that of control. Incubation of all cell lines with Bcl-2 siRNA reduced cell viability 96 h after 5-FU treatment compared with all other controls: Huh-7 (P < 0.01), Huh-7 with hepatitis C replicon (P < 0.01), HepG2 (P < 0.01), HLE (P < 0.05). Moreover, the proportion of apoptosis in control siRNA, Bcl-2 siRNA, control siRNA prior to 5-FU treatment, and Bcl-2 siRNA prior to 5-FU treatment groups were (4.6 +/- 2.3)%, (7.5 +/- 0.5)%, (6.0 +/- 2.1)%, and (19.5 +/- 0.86)%, respectively. The Bcl-2 siRNA prior to 5-FU treatment group showed the strongest effect of inducing apoptosis. In conclusion, the combination Bcl-2 siRNA and 5-FU might represent a new therapeutic option for HCC. Publication Types: Comparative Study PMID: 16332177 [PubMed - indexed for MEDLINE] 36: Oncogene. 2006 May 11;25(20):2950-2. Absence of PIK3CA hotspot mutations in hepatocellular carcinoma in Japanese patients. Tanaka Y, Kanai F, Tada M, Asaoka Y, Guleng B, Jazag A, Ohta M, Ikenoue T, Tateishi K, Obi S, Kawabe T, Yokosuka O, Omata M. Department of Gastroenterology, Graduate School of Medicine, University of Tokyo, Japan. A recent study revealed that the p110alpha (PIK3CA), catalytic subunit of phosphatidylinositol 3-kinase (PI3K), is somatically mutated in many types of cancer. For example, PIK3CA is mutated in an estimated 35.6% of hepatocellular carcinoma (HCC) cases. To measure the frequency of PIK3CA hotspot mutations in Japanese HCC patients, exons 9 and 20 of the PIK3CA gene were sequenced in 47 clinical HCC samples. Contrary to expectations, no hotspot mutations were found any of the HCC samples. In addition, we found abnormally migrating waves near the end of exon 9 in the PCR chromatograms from 13 of the 47 samples. PCR amplification and subsequent cloning and sequencing revealed that these chromatograms contained two distinct sequences, the wild-type p110alpha sequence and a different sequence found on human chromosome 22q11.2, the Cat Eye Syndrome region, which contains a putative pseudogene of PIK3CA. These abnormally migrating waves were also found in noncancerous liver tissue, indicating that this was not a result of HCC-associated mutations. Therefore, it is likely that the percentage of hotspot mutations in the PIK3CA gene of Japanese HCC patients is lower than was previously reported. Publication Types: Research Support, Non-U.S. Gov't PMID: 16331247 [PubMed - indexed for MEDLINE] 37: Oncol Rep. 2005 Oct;14(4):975-9. 5-aza-2'-deoxycytidine sensitizes hepatoma and pancreatic cancer cell lines. Kanda T, Tada M, Imazeki F, Yokosuka O, Nagao K, Saisho H. The Department of Medicine and Clinical Oncology, Graduate School of Medicine Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-0856, Japan. kandat-cib@umin.ac.jp Hepatocellular carcinoma (HCC) and pancreatic cancer are at the forefront of chemotherapy-resistant tumors with poor prognosis. Even with innovative treatment regimens, response rates remain low and the duration of response is short. We examined whether the suppression of DNA methylation was capable of enhancing the sensitivity of hepatoma and pancreatic cancer cell lines to 5-fluorouracil (5-FU). 5-aza-2'-deoxycytidine (5-aza-dC) at 2 microM, a specific DNA methylation inhibitor, did not induce cell death in Huh-7 with or without HCV, HLE, HepG2 and MIA PaCa-2 cells. However, a combination of 5-aza-dC with 5-FU showed a reduction in cell viability and induction of apoptosis in these cell lines to a greater degree than with 5-FU only. These findings underline the fact that DNA methylation plays a key role in conferring chemoresistance to hepatoma and pancreatic cancer, and the combination of DNA methylation inhibitor with chemotherapy could be a novel and highly effective tool for future targeted therapy of chemoresistant tumors. PMID: 16142360 [PubMed - indexed for MEDLINE] 38: Nippon Rinsho. 2005 Jul;63 Suppl 7:401-3. [Diagnostic tests: Serum anti-hepatitis A virus antibody and hepatitis A virus RNA] [Article in Japanese] Fujiwara K, Yokosuka O. Department of Internal Medicine, Kawatetsu Chiba Hospital. Publication Types: Review PMID: 16111285 [PubMed - indexed for MEDLINE] 39: Leuk Lymphoma. 2005 Jun;46(6):915-7. Lamivudine treatment in a patient with hepatitis B virus reactivation after allogenic peripheral bone marrow transplantation. Imamura T, Yokosuka O, Chiba T, Kanda T, Kojima H, Fukai K, Imazeki F, Nishimura M, Saito Y, Saisho H. Department of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, Japan. Taimamura@aol.com We report the case of a 52-year-old woman with hepatitis B virus (HBV) reactivation during treatment for chronic graft vs. host disease (GVHD) after peripheral bone marrow transplantation (PBSCT) to treat chronic myelocytic leukemia. She was given cyclosporine and prednisolone orally to treat chronic GVHD after PBSCT. Liver dysfunction first developed 25 months after transplantation with the appearance of hepatitis B s antigen (HBsAg), hepatitis B e antigen (HBeAg), and elevation of HBV-DNA up to 4.5 log copies/ml. Retrospective examination of her serum before PBSCT proved negative for HBsAg and HBeAg, and positive for anti-HBsAg, anti-HBeAg, anti-hepatitis B core antigen, and HBV-DNA (2.7 log copies/ml), showing that she was in a state of occult HBV infection. Nucleotide sequences of the HBV genome obtained from her serum showed no core promoter mutations at nt 1762 and 1764 and no pre-core mutation at nt 1896. Polymerase chain reaction-restriction fragment length polymorphism showed that she was infected with HBV genotype B. The administration of lamivudine, a nucleoside analog, improved her liver function and reduced HBV-DNA replication. We conclude that antiviral agents, such as lamivudine, are effective for treating hepatitis B reactivation during immunosuppressive treatment, such as for GVHD. The administration of a nucleoside analog before transplantation should also be considered in the light of HBV genotypes and mutations, even if HBsAg was negative and the viral load was low before transplantation. PMID: 16019538 [PubMed - in process] 40: Hepatogastroenterology. 2005 Jul-Aug;52(64):1233-5. Acute-onset autoimmune hepatitis resembling acute hepatitis: a case report and review of reported cases. Kanda T, Yokosuka O, Hirasawa Y, Imazeki F, Nagao K, Suzuki Y, Saisho H. First Department of Internal Medicine, Chiba University School of Medicine, Chiba, Japan. kandat-cib@umin.ac.jp We report a 54-year-old Japanese man, whose ALT level was 1689 IU/L, without increased gamma-globulin level or autoantibodies. He could not be diagnosed as autoimmune hepatitis (AIH) using scoring systems, and his liver function became normalized after steroid treatment. Recently, AIH with acute presentation of disease and acute-onset AIH without bridging fibrosis have been increasingly reported but cases without the character of increasing gamma-globulin level or autoantibodies before immunosuppressive treatment are extremely rare. This is the third such case report in the literature. Publication Types: Case Reports Review PMID: 16001669 [PubMed - indexed for MEDLINE] 41: Liver Int. 2005 Aug;25(4):772-8. Lower incidence of hepatic failure than hepatocellular carcinoma in Japanese patients with chronic hepatitis C. Imazeki F, Yokosuka O, Fukai K, Kawai S, Kanda T, Kojima H, Saisho H. Department of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, Chiba, Japan. imazekif@faculty.chiba-u.jp BACKGROUND: Previous studies have shown that the development of hepatic failure was found more frequently than that of hepatocellular carcinoma (HCC) in patients with chronic hepatitis C in the United States and European countries. We investigated the status in Japan in a retrospective cohort study. METHODS: The incidences of HCC and hepatic failure were accessed in 459 patients with biopsy-proven C-viral chronic liver disease with a mean follow-up period of 8.9+/-3.2 years and the cause of death was also analyzed in the cohort. RESULTS: HCC developed in 63 patients, 46 of 355 interferon (IFN)-treated and 17 of 104 untreated patients. In contrast, the development of hepatic failure was found in 18 patients, 12 of 355 IFN-treated and six of 104 untreated patients. HCC developed in four of 116 with sustained virological response (SVR), and hepatic failure developed in one of them. Thirty-two of 63 patients developing HCC and eight of 18 patients developing hepatic failure died. CONCLUSIONS: Development of hepatic failure was less frequent than that of HCC in Japan. It is important for a favorable prognosis of patients with C viral chronic liver disease to achieve a higher SVR and thus inhibit the development of HCC in Japan. Publication Types: Research Support, Non-U.S. Gov't PMID: 15998428 [PubMed - indexed for MEDLINE] 42: World J Gastroenterol. 2005 Jun 28;11(24):3791-3. Prolonged acute hepatitis A mimicking autoimmune hepatitis. Mikata R, Yokosuka O, Imazeki F, Fukai K, Kanda T, Saisho H. Department of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan. AIM: We report a case with a prolonged course of hepatitis A, with alanine aminotransferase (ALT) higher than 500 IU/L for more than 2 mo. METHODS: A middle-aged woman had an elevated IgG level of more than 2,000 mg/dL, positive anti-nuclear antibodies (ANA) and anti-smooth muscle antibodies (ASMA), but no evidence of persistent hepatitis A virus (HAV) infection. Liver biopsy findings were compatible with prolonged acute hepatitis, although acute onset of autoimmune hepatitis could not be ruled out. RESULTS: It was assumed that she developed a course of hepatitis similar to autoimmune hepatitis triggered by HAV infection. Ursodeoxycholic acid (UDCA) treatment was initiated and a favorable outcome was obtained. CONCLUSION: We describe a case of a middle-aged woman who showed a prolonged course of acute hepatitis A mimicking autoimmune hepatitis. Treatment with UDCA proved to be effective. Publication Types: Case Reports PMID: 15968741 [PubMed - indexed for MEDLINE] 43: J Infect Dis. 2005 Jul 15;192(2):266-75. Epub 2005 Jun 13. Hepatitis C virus core protein and hepatitis activity are associated through transactivation of interleukin-8. Hoshida Y, Kato N, Yoshida H, Wang Y, Tanaka M, Goto T, Otsuka M, Taniguchi H, Moriyama M, Imazeki F, Yokosuka O, Kawabe T, Shiratori Y, Omata M. Department of Gastroenterology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan. BACKGROUND: We evaluated the association between variations in hepatitis C virus (HCV) core protein and hepatitis severity in patients with chronic HCV infection who achieved remission without viral eradication and had a biochemical response to interferon (IFN) therapy, to evaluate the effect of HCV core sequence in the absence of the influence of host factors. METHODS. Using serum from 10 patients with a biochemical response and 10 patients with no response, we measured serum levels of interleukin (IL)-1 beta , IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IFN- gamma , and tumor necrosis factor- alpha before and after IFN therapy. Expression vectors with the core region were transfected into Huh7 cells, and cytokine induction was evaluated by reporter assay. RESULTS: In biochemical responders, only IL-8 levels decreased after IFN therapy (P=.04). Changes in the C-terminal hydrophobic region were observed more frequently in biochemical responders. Activation of the IL-8 promoter by HCV core protein was significantly decreased in biochemical responders after IFN therapy (P=.04). When 69 C-terminal amino acids from before IFN therapy were replaced with those from after IFN therapy in 3 biochemical responders, their ability to transactivate IL-8 decreased. CONCLUSIONS: Differences in amino acids in the HCV core protein correlates with hepatitis activity through the modulation of IL-8 induction in HCV-infected patients. Publication Types: Research Support, Non-U.S. Gov't PMID: 15962221 [PubMed - indexed for MEDLINE] 44: World J Gastroenterol. 2005 Jun 14;11(22):3346-50. Effect of lamivudine in HBeAg-positive chronic hepatitis B: discordant effect on HBeAg and HBV DNA according to pretreatment ALT level. Kurihara T, Imazeki F, Yokosuka O, Fukai K, Kanda T, Kawai S, Saisho H. Department of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuou Ward, Chiba 260-8670, Japan. AIM: To clarify differences in antiviral effect of the drug in patients with different ALT levels, we examined the changes in HBV markers in patients with high or low ALT levels with or without lamivudine treatment. METHODS: Thirty-seven HBeAg-positive patients were studied. Ten patients with ALT levels higher than 200 IU/L (group 1) and 8 patients with ALT below 200 IU/L (group 2) were treated orally with 100 mg/d of lamivudine. As untreated control, 9 patients with ALT above 200 IU/L (group 3) and 10 patients with ALT below 200 IU/L (group 4) were examined. ALT level, HBeAg/HBeAb status, and HBV DNA level were examined monthly for 11.9+/-0.4 mo. RESULTS: The ALT level normalized in all 10 patients of group 1, 7/8 of group 2, 4/9 of group 3, and 1/10 of group 4 within 6 mo (groups 1 vs 2, P = NS; groups 1 vs 3, P = 0.002; groups 1 vs 4, P<0.0001). HBV DNA fell below the detection limit in all 10 patients of group 1, 7/8 of group 2, 0/9 of group 3, and 0/10 of group 4 within 6 mo (groups 1 vs 2, P = NS). HBeAg became seronegative in 7/10 patients of group 1, 1/8 of group 2, 3/9 of group 3, and 0/10 of group 4 within 12 mo (groups 1 vs 2, P = 0.02; groups 1 vs 3, P = NS). CONCLUSION: Our data suggest that HBeAg-positive patients with higher ALT levels can be considered good candidates for lamivudine therapy, probably because lamivudine accelerates the natural seroconversion of HBeAg, accompanied by HBV DNA loss, in these patients. Publication Types: Clinical Trial Controlled Clinical Trial PMID: 15948237 [PubMed - indexed for MEDLINE] 45: Am J Gastroenterol. 2005 Jun;100(6):1322-9. Fas polymorphisms influence susceptibility to autoimmune hepatitis. Hiraide A, Imazeki F, Yokosuka O, Kanda T, Kojima H, Fukai K, Suzuki Y, Hata A, Saisho H. Department of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, Chiba City, Japan. BACKGROUND AND AIMS: Genetic factors associated with autoimmune hepatitis (AIH) and primary biliary cirrhosis (PBC), immune-mediated chronic inflammatory liver diseases of unknown etiology, remain to be elucidated. Polymorphisms of the gene encoding Fas have been linked to a variety of autoimmune diseases. We hypothesized that Fas gene polymorphisms might be genetic markers for AIH and PBC. METHODS: To determine the frequency and significance of Fas polymorphisms in patients with AIH and PBC, 74 Japanese AIH patients, 98 Japanese PBC patients, and 132 ethnically matched control subjects were investigated by the use of the Taqman assay. RESULTS: We found significant differences between AIH patients and controls in allele frequencies of Fas-670 (p=0.009), Fas IVS (intervening sequence) 2nt176 (p=0.018), Fas IVS3nt46 (p=0.031), and Fas IVS5nt82 (p=0.013) polymorphisms. Haplotype analysis revealed that one of the haplotypes, GATGC, was associated with increased AIH prevalence. On the other hand, we found no statistically significant differences between PBC patients and controls in allele frequencies of the Fas polymorphisms genotyped in this study. CONCLUSIONS: These results indicate a genetic link of Fas polymorphisms to the development of AIH. Further studies are needed to determine the genetic factors contributing to the development of AIH. Publication Types: Comparative Study PMID: 15929764 [PubMed - indexed for MEDLINE] 46: Cancer Lett. 2006 Mar 28;234(2):143-8. State of hepatitis C viral replication enhances activation of NF-kB- and AP-1-signaling induced by hepatitis B virus X. Kanda T, Yokosuka O, Nagao K, Saisho H. Safety and Health Organization, Chiba University, 1-33 Yayoicho, Inage-ku, Chiba 263-8522, Japan. kandat-cib@umin.ac.jp We examined the effect of hepatitis B virus X (HBx) on NF-kB- and AP-1- mediated transcription in human hepatocellular carcinoma cell lines, Huh-7 with or without subgenomic hepatitis C virus (HCV) RNA. Expression of HBx in Huh-7 cells with HCV resulted in 4.9 times increased NF-kB-activation and 3.8 times AP-1-activation whereas that without HCV resulted in 2.4 times increased NF-kB-activation and 2.3 times AP-1-activation. Interestingly, the expression of the matured form of HCV core protein, Core173, did not activate NF-kB- or AP-1-transcription in either Huh7 with or without HCV replicon. HBx protein might play an important role in HCV-related hepatocarcinogenesis. Publication Types: Research Support, Non-U.S. Gov't PMID: 15885885 [PubMed - indexed for MEDLINE] 47: Hepatol Res. 2005 Jul;32(3):141-3. Prevention of hepatocellular carcinoma in chronic hepatitis B. Yokosuka O. Department of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-Ward, Chiba City, Chiba 260-8670, Japan. Publication Types: Editorial PMID: 15862782 [PubMed - in process] 48: Biochem Biophys Res Commun. 2005 Jun 3;331(2):621-9. Amantadine inhibits hepatitis A virus internal ribosomal entry site-mediated translation in human hepatoma cells. Kanda T, Yokosuka O, Imazeki F, Fujiwara K, Nagao K, Saisho H. Safety and Health Organization, Chiba University, 1-33 Yayoicho, Inage-ku, Chiba 263-8522, Japan. kandat-cib@umin.ac.jp The effect of six drugs (amantadine, glycyrrhizin, ribavirin, ursodeoxycholic acid, alcohol, and IFN) on HAV RNA translation from the HAV internal ribosomal entry site (IRES) was investigated using a bicistronic reporter construct containing HAV IRES as intragenic spacer. Huh-7 cells and derivatives were transfected with in vitro transcripts, and the reporter gene activity was determined. IFN suppressed both cap-dependent and HAV IRES-dependent translation, while amantadine specifically inhibited HAV IRES-dependent translation. In contrast to IFN, by reporter assay, amantadine did not activate the interferon-stimulated response element (ISRE) or interferon gamma-activated sequence (GAS)-associated pathways. Immunoblot analysis revealed that amantadine had no effect on PKR and on IFN-regulatory factor-1 (IRF-1) expression. These findings demonstrated a novel antiviral effect of amantadine against HAV with or without HCV infection. Publication Types: Research Support, Non-U.S. Gov't PMID: 15850805 [PubMed - indexed for MEDLINE]