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Revue de Presse du Mois Mois de Mars 2003

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HEPATOLOGY

Table of Contents for March 2003 · Volume 37 · Number 3

Genetic polymorphisms and the progression of liver fibrosis: A critical appraisal
Ramón Bataller, Kari E. North, David A. Brenner
Liver fibrosis is a highly dynamic process in which multiple genes interact with environmental factors. Recent human epidemiologic studies have identified possible polymorphisms in a number of candidate genes that influence the progression of liver fibrosis. These genetic factors could explain the broad spectrum of responses to the same etiologic agent found in patients with chronic liver diseases. Polymorphisms in genes encoding immunoregulatory proteins, proinflammatory cytokines, and fibrogenic factors may influence disease progression in patients with alcohol-induced liver disease, primary biliary cirrhosis, or chronic hepatitis C. However, some of the studies have yielded contradictory results. For example, conflicting results have been obtained in studies assessing the role of mutations in the hemochromatosis gene on fibrosis progression in patients with chronic hepatitis C. Large-scale, well-designed studies are required to clarify the actual role of this factor and other genetic variants in liver fibrosis. (HEPATOLOGY 2003;37:493-503.)

Liver Failure and Liver Disease

Arterial and portal circulation and parenchymal changes in Budd-Chiari syndrome: A study in 17 explanted livers (*Human Study*)
Dominique Cazals-Hatem, Valérie Vilgrain, Pascal Genin, Marie-Hélène Denninger, François Durand, Jacques Belghiti, Dominique Valla, Claude Degott
Hepatic parenchymal changes associated with Budd-Chiari syndrome (BCS) have been tentatively explained by combined arterial and portal perfusion disturbances in addition to the complete occlusion of hepatic veins. The aim of this study was to correlate pretransplant course and vascular imaging with pathologic findings in livers explanted for BCS. Seventeen consecutive white patients who underwent transplantation for severe classic BCS were retrospectively analyzed. Pretransplant course was 1 year or less in 8 patients and more than 1 year in 9 patients. Thrombophilia was found in 16 patients (94%). Imaging showed decreased portal perfusion in 16 patients (94%) and increased arterial perfusion in 9 patients. Histology showed obstructive portal venopathy and nodular regenerative hyperplasia (NRH) aspects in all cases, large regenerative nodules resembling focal nodular hyperplasia (FNH) in 9 cases, and cirrhosis in 2 cases. Patients with increased arterial inflow had large regenerative nodules and a protracted pretransplant course. Patients with acute thrombi in portal veins had parenchymal infarcts (2 cases) and a short pretransplant course. In conclusion, patients with severe BCS have a constant impaired perfusion inflow unrelated to progression of cirrhosis but related to the outcome. An early decrease in portal perfusion is observed in the short term and is responsible for NRH or infarcts if complicated with large thrombi. An increase in arterial perfusion compensates impaired portal flow in chronic BCS. Arterial hyperemia contributes to the development of large regenerative nodules that are FNH-like. This pathologic situation offers an interesting vascular model to further understand the parenchymal response to changes in hepatic blood flow. (HEPATOLOGY 2003;37:510-519.)

Prospective analysis of risk factors for hepatocellular carcinoma in patients with liver cirrhosis (*Human Study*)
Rosario F. Velázquez, Manuel Rodríguez, Carmen A. Navascués, Antonio Linares, Ramón Pérez, Nieves G. Sotorríos, Isabel Martínez, Luis Rodrigo
Better knowledge of the risk factors associated with the appearance of hepatocellular carcinoma (HCC) could improve the efficacy of surveillance programs. A total of 463 patients aged 40 to 65 years with liver cirrhosis in Child-Pugh class A or B were included in a program of early diagnosis. The predictive value of different risk factors was evaluated using the Kaplan-Meier method and Cox regression model. Thirty-eight patients developed HCC. In the multivariate analysis, 4 variables showed an independent predictive value for the development of HCC: age 55 years or older, antibody to hepatitis C virus (anti-HCV) positivity, prothrombin activity 75% or less, and platelet count less than 75 ¥ 103/mm3. According to the contribution of each of these factors to the final model, a score ranging between 0 and 4.71 points was constructed to allow the division of patients into 2 different risk groups. The low-risk group included those with a score of 2.33 points or less (n = 270; 4 with HCC; cumulative incidence of HCC at 4 years, 2.3%), and the high-risk group included those with a score greater than 2.33 (n = 193; 34 with HCC; cumulative incidence of HCC at 4 years, 30.1%) (P = .0001). In conclusion, a simple score made up of 4 clinical and biological variables allowed us to distinguish 2 groups of cirrhotic patients at high and low risk for the development of HCC. We believe this score can be useful in establishing a subset of cirrhotic patients in whom a surveillance program for early detection of HCC could be unjustified. (HEPATOLOGY 2003;37:520-527.)

Overexpression of orphan G-protein­coupled receptor, Gpr49, in human hepatocellular carcinomas with -catenin mutations (*Human Study*)
Yoshiya Yamamoto, Michiie Sakamoto, Gen Fujii, Hitomi Tsuiji, Kengo Kanetaka, Masahiro Asaka, Setsuo Hirohashi
To identify the genes responsible for carcinogenesis and progression of hepatocellular carcinoma (HCC), we screened differentially expressed genes in several human HCC cell lines. Among these genes, Gpr49 was up-regulated in PLC/PRF/5 and HepG2. Gpr49 is a member of the glycoprotein hormone receptor subfamily, which includes the thyroid-stimulating hormone receptor (TSHR). However, Gpr49 remains to be an orphan G-protein-coupled receptor. By real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis, overexpression (>3-fold increase compared with the corresponding noncancerous liver tissue) of Gpr49 mRNA was observed in 18 of 38 (47%) HCCs compared with corresponding noncancerous livers. Clinicopathologically, overexpression of Gpr49 was frequently observed in HCC with mutation in -catenin exon 3 (14 of 16 cases, 87.5%). Moreover, introduction of mutant -catenin into mouse hepatocytes in culture caused up-regulation of the Gpr49 mouse homologue. Therefore, Gpr49 is likely to be a target gene activated by Wnt-signaling in HCC. In conclusion, although much is still unknown, Gpr49 may be critically involved in the development of HCCs with -catenin mutations and has the potential to be a new therapeutic target in the treatment of HCC. (HEPATOLOGY 2003;37:528-533.)

Cyclins and cyclin-dependent kinases: Comparative study of hepatocellular carcinoma versus cirrhosis (*Human Study*)
Tsutomu Masaki, Yasushi Shiratori, William Rengifo, Kouichi Igarashi, Michiko Yamagata, Kazutaka Kurokohchi, Naohito Uchida, Yoshiaki Miyauchi, Hitoshi Yoshiji, Seishiro Watanabe, Masao Omata, Shigeki Kuriyama
Increasing evidence has indicated that perturbation of cyclins is one of the major factors leading to cancer. The aim of this study was not only to investigate various cell cycle-related kinase activities in hepatocellular carcinoma (HCC), but also to analyze the difference of cell cycle-related kinase activity levels between hepatitis C virus (HCV)-induced HCC and HCV-induced cirrhosis. The protein levels of cyclins D1, E, A, and H, and of cyclin dependent kinase 1 (Cdk1), Cdk2, Cdk4, Cdk6, and Cdk7 in HCC and in surrounding nontumorous cirrhosis were determined by Western blot. The enzymatic activities of cyclins D1, E, A, Cdk1, Cdk4, Cdk6, Cdk7, and Wee1 were measured using in vitro kinase assays. Protein levels and kinase activities of cyclin D1, Cdk4, cyclin E, cyclin A, and Wee1 were significantly elevated in HCC compared with surrounding cirrhotic tissues. The enhanced cyclin D1-related kinase activity in HCC was accompanied by the up-regulation of Cdk4 activity, but not Cdk6 activity. The kinase activities of Cdk6, Cdk7, and Cdk1 did not differ between HCC and surrounding cirrhotic tissues. In addition, the protein levels and kinase activities of cyclin D1, Cdk4, and cyclin E were higher in poorly differentiated HCC and advanced HCC. In conclusion, the increases of cyclin D1, Cdk4, cyclin E, cyclin A, and Wee1 play an important role in the development of HCC from cirrhosis. Cyclin D1, Cdk4, and cyclin E activation may be closely related to the histopathologic grade and progression of HCC. (HEPATOLOGY 2003;37:534-543.)

Hepatic cytochrome P450 2E1 activity in nondiabetic patients with nonalcoholic steatohepatitis (*Human Study*)
Naga Chalasani, J. Christopher Gorski, Maleeha S. Asghar, Ali Asghar, Brian Foresman, Stephen D. Hall, David W. Crabb
Cytochrome P450 2E1 (CYP2E1) plays an important role in the pathogenesis of nonalcoholic steatohepatitis (NASH) in animal models, but its role in the pathogenesis of human NASH is unclear. Therefore, we measured hepatic CYP2E1 activity and its correlates in a cohort of nondiabetic patients with NASH (NDN) and controls to explore its role in the pathogenesis of human NASH. Hepatic CYP2E1 activity was assessed using the oral clearance (CLPO) of chlorzoxazone (CHZ) in 20 NDN and 17 age, gender, and body mass index (BMI)-matched controls. The relationship between hepatic CYP2E1 activity and demographic and anthropometric variables; fasting levels of insulin, glucose, lipids, and -OH butyrate; insulin resistance; and nocturnal hypoxemia was assessed. Furthermore, expression of CYP2E1 in the peripheral lymphocytes was assessed using reverse transcription-polymerase chain reaction (RT-PCR). The CLPO of CHZ was significantly (P = .03) greater in NDN (41 ± 12 L/h) compared with controls (33 ± 16 L/h). Lymphocyte CYP2E1 messenger RNA was significantly higher in NDN compared with controls (11.5 ¥ 103 ± 10 ¥ 103 vs. 2.6 ¥ 103 ± 1.2 ¥ 103 molecules/µg total RNA, respectively, P < .001). On univariate analysis, BMI, respiratory quotient, high-density lipoprotein, triglycerides, insulin, insulin resistance, hypoxemia, and -OH butyrate significantly correlated with hepatic CYP2E1 activity. However, on stepwise regression analysis, only nocturnal hypoxemia (r = 0.50, P = .009) and -OH butyrate (r = 0.37, P = .04) were independent predictors of hepatic CYP2E1 activity. In conclusion, hepatic CYP2E1 activity and lymphocyte CYP2E1 expression are enhanced in NDN. The significant correlations noted between CYP2E1 and hypoxemia and -OH butyrate suggest that these factors play a role in increased CYP2E1 activity that is seen in patients with NASH. (HEPATOLOGY 2003;37:544-550.)

Oral bile acids reduce bacterial overgrowth, bacterial translocation, and endotoxemia in cirrhotic rats
Vicente Lorenzo-Zúñiga, Ramón Bartolí, Ramón Planas, Alan F. Hofmann, Belén Viñado, Lee R. Hagey, José Ma Hernández, Josep Mañé, Marco A. Alvarez, Vicente Ausina, Miquel Angel Gassull
Experiments were performed to test whether conjugated bile acid administration would decrease bacterial overgrowth, bacterial translocation, and endotoxemia in ascitic cirrhotic rats. Cholylsarcosine, a deconjugation-dehydroxylation resistant and cholylglycine, a deconjugation-dehydroxylation susceptible bile acid were used. Rats with CCl4-induced cirrhosis and ascites were fed cholylsarcosine, cholylglycine (both at 70 mg/kg/d), or placebo for 2 weeks. Healthy rats, as controls, were treated similarly. In cirrhotic rats receiving placebo, bile secretion from an acute biliary fistula was lower than in healthy rats (27.2 ± 6.5 vs. 53.0 ± 3.1 µL/kg/min; mean ± SE, P< .05). The administration of conjugated bile acids to cirrhotic rats normalized bile secretion (cholylsarcosine, 51.8 ± 6.29; cholylglycine, 52.72 ± 8.9 µL/kg/min). Total ileal bacterial content was 6-fold higher in ascitic cirrhotic rats than in healthy rats. Conjugated bile acid administration reduced bacterial content to normal levels. Bacterial translocation was less in cirrhotic animals receiving conjugated bile acids (cholylsarcosine, 33%; cholylglycine, 26%) than in animals receiving placebo (66%). Endotoxemia was decreased in cirrhotic rats by conjugated bile acid feeding (cholylsarcosine, 0.098 ± 0.002; cholylglycine 0.101 ± 0.007 EU/mL) compared with placebo (0.282 ± 0.124, P < .001). Survival was greater in animals receiving conjugated bile acids (cholylsarcosine, 10/15; cholylglycine, 11/15; placebo, 5/15). In conclusion, the administration of conjugated bile acids to ascitic cirrhotic rats increased bile acid secretion, eliminated intestinal bacterial overgrowth, decreased bacterial translocation, decreased endotoxemia, and increased survival. Oral conjugated bile acids may be useful in preventing bacterial translocation, endotoxemia, and spontaneous bacterial perotonitis in cirrhotic patients. (HEPATOLOGY 2003;37:551-557.)

Thrombopoietin in acute liver failure (*Human Study*)
Frank V. Schiødt, Jody Balko, Michael Schilsky, M. Edwyn Harrison, Annelise Thornton, William M. Lee, The Acute Liver Failure Study Group
Thrombopoietin (TPO) is the primary regulator of platelet production. TPO is produced in the liver and levels are low in patients with cirrhosis. Because thrombocytopenia is common in patients with acute liver failure (ALF), we measured TPO concentrations (normal TPO range, 31 to 136 pg/mL) in 51 patients with ALF to determine if low levels were associated with thrombocytopenia. TPO levels from hospital day 2 were elevated in 43% of patients, normal in 47%, and decreased in 10% of patients. Levels were higher in acetaminophen-induced than in non­acetaminophen-induced ALF, 160 (12 to 549) pg/mL versus 73 (18 to 563) pg/mL, respectively, P = .031. TPO levels did not correlate with platelet count and were not related with survival or infection. We analyzed daily TPO levels for the first week of hospitalization in 12 patients with acetaminophen-induced ALF and observed a gradual increase from a median admission level of 50 (5 to 339) pg/mL to a median peak level of 406 (125 to 1,081) pg/mL occurring on day 5 (3 to 6). Platelets were reduced in 11 of the 12 patients with a nadir platelet count of 52 (19 to 156) ¥ 109 cells/L occurring on day 5.5 (1 to 6). The peak TPO level did not correlate with the nadir platelet count (P = .43). In conclusion, the normal inverse relationship between platelet count and TPO levels was not observed in ALF. Despite severe hepatic dysfunction, serum TPO levels were initially normal and increased during hospitalization in acetaminophen-induced ALF, but did not prevent the development of thrombocytopenia. (HEPATOLOGY 2003;37:558-561.)

Significance of hepatitis B genotype in acute exacerbation, HBeAg seroconversion, cirrhosis-related complications, and hepatocellular carcinoma (*Human Study*)
Man-Fung Yuen, Erwin Sablon, He-Jun Yuan, Danny Ka-Ho Wong, Chee-Kin Hui, Benjamin Chun-Yu Wong, Annie On-On Chan, Ching Lung Lai
The pathologic role of hepatitis B virus (HBV) genotype in Chinese patients with HBV infection is largely unknown. We examined the relationship between HBV genotypes, and hepatitis B e antigen (HBeAg) seroconversion, acute exacerbation, cirrhosis-related complications, and precore/core promoter mutations. Three hundred forty-three HBV patients (288 were asymptomatic, 55 presented with cirrhosis-related complications) were recruited. HBV genotypes and precore/core promoter mutations were determined by line probe assays. Genotypes B and C were the 2 most common genotypes, contributing 28% and 60%, respectively. The median age of HBeAg seroconversion for patients with genotype B was 9 years earlier than patients with genotype C (P = .011). There were no differences in the liver biochemistry, HBV DNA level, and cumulative risk of acute exacerbation (defined as increased alanine aminotransferase level 1.5 ¥ upper limit of normal) between patients with genotypes B and C. There was a trend for patients with genotype B to have a higher cumulative rate of HBeAg seroconversion compared with patients with genotype C at the initial follow-up of 6 years (P = .053), but this difference became insignificant during subsequent follow-up. The prevalence of both genotypes was the same in patients with and without cirrhosis-related complications and/or hepatocellular carcinoma. Genotype B was associated with precore mutations (P < .0001), whereas genotype C was associated with core promoter mutations (P < .0001). In conclusion, although patients with genotype B had earlier HBeAg seroconversion, there was no significant reduction in the risk of development of complications. Genotypes B and C are associated with high prevalence of precore and core promoter mutations, respectively. (HEPATOLOGY 2003;37:562-567.)

Ribavirin and interferon is effective for hepatitis C virus clearance in hepatitis B and C dually infected patients (*Human Study*)
Chun-Jen Liu, Pei-Jer Chen, Ming-Yang Lai, Jia-Horng Kao, Yung-Ming Jeng, Ding-Shinn Chen
Ribavirin and interferon (IFN) are an effective treatment in 30% to 60% of patients with chronic hepatitis C. Whether they are also effective in dually infected patients with hepatitis B and C is unknown. Twenty-four patients with chronic hepatitis seropositive for both hepatitis B surface antigen and antibody to HCV received ribavirin 1,200 mg daily for 6 months, together with 6 million units (MU) IFN- 2a thrice weekly for 12 weeks and then 3 MU for another 12 weeks. Serum HCV RNA was positive in 21 patients (group I, serum HBV DNA positive in 17 patients) and negative in 3 patients (group II, all HBV DNA positive) by Amplicor (Cobas Amplicor Monitor, Roche Diagnostics, Branchburg, NJ). Serum alanine aminotransferase (ALT), HCV RNA, and hepatitis B virus (HBV) DNA were monitored regularly for 12 months. Another 30 patients with chronic hepatitis C alone receiving the same regimen, served as controls. The serum HCV clearance rate in group I patients (43%) was comparable with that in controls (60%, P = .63) 24 weeks posttreatment. The serum ALT normalization rate in group I and group II patients was 43% and 0%, respectively, 24 weeks posttreatment. After treatment, resurgence of HBV and HCV was encountered in 4 group I patients and 1 group II patient, respectively. In conclusion, in hepatitis B and C dually infected patients, combination of IFN with ribavirin can achieve a sustained HCV clearance rate comparable with hepatitis C alone. In dually infected patients, the treatment may alter the dominant, ruling hepatitis virus. (HEPATOLOGY 2003;37:568-576.)

Novel CD4+ and CD8+ T-cell determinants within the NS3 protein in subjects with spontaneously resolved HCV infection (*Human Study*)
Anne M. Wertheimer, Camette Miner, David M. Lewinsohn, Anna W. Sasaki, Ezra Kaufman, Hugo R. Rosen
Spontaneous resolution of hepatitis C virus (HCV) infection is a relatively infrequent event, and these individuals provide a unique opportunity to characterize correlates of protective immunity as an important first step in the development of vaccine candidates. The aim of this study was to directly and comprehensively enumerate HCV-nonstructural protein 3 (NS3) specific CD4+ and CD8+ T cells ex vivo from HLA diverse individuals who had been successful in spontaneously resolving HCV infection. We measured interferon gamma (IFN-) production with an ELISPOT assay using magnetic bead-separated CD4+ or CD8+ T cells in response to autologous DCs that had been pulsed with 15mer per peptides overlapping by 11 amino acids and spanning all of the NS3 protein (150 total peptides). All subjects with spontaneously recovered HCV infection demonstrated vigorous and multispecific CD4+ T-cell responses to NS3 peptides, and 6 of 10 subjects demonstrated CD8+ T-cell responses. More importantly, we identified novel, previously unpredicted antigenic regions, which in most cases elicited high frequencies within a given individual. In conclusion, subjects who have spontaneously eradicated HCV infection up to 35 years earlier demonstrate persistent CD4+ and CD8+ T-cell responses specific to NS3. By providing a comprehensive screening of all potential T-cell epitopes contained in the NS3 region, our strategy defines the breadth of the T-cell response and identifies novel, unpredicted specificities. (HEPATOLOGY 2003;37:577-589.)

Influence of ethnicity in the outcome of hepatitis C virus infection and cellular immune response (*Human Study*)
Kazushi Sugimoto, Jason Stadanlick, Fusao Ikeda, Colleen Brensinger, Emma E. Furth, Harvey J. Alter, Kyong-Mi Chang
This study was performed to examine the immunologic basis for the apparent ethnic difference in clinical outcome of hepatitis C virus (HCV) infection between African Americans (AA) and Caucasian Americans (CA). To this end, we recruited 99 chronically HCV-infected and 31 spontaneously HCV-cleared subjects for clinical, virologic, and immunologic analysis. In particular, CD4-proliferative T-cell response to genotype 1­derived HCV antigens (core, NS3-NS5) was examined in 82 patients chronically infected with genotype 1 (54 AA, 28 CA) and in all HCV-cleared subjects (14 AA, 17 CA). HCV-specific Th1 response also was examined in 52 chronic and 13 recovered subjects. Our results showed that HCV clearance was associated with a vigorous HCV-specific Th1 response irrespective of ethnic origin. Although the HCV-specific CD4 T-cell response clearly was weaker during chronic infection, AA ethnicity in this setting was associated with a significantly greater CD4-proliferative T-cell response to HCV, particularly to the nonstructural antigens (22% AA vs. 0% CA, P = .007) as well as better clinical parameters of liver disease. Interestingly, most HCV-specific CD4 T-cell proliferative responses in AA patients were unaccompanied by concurrent interferon (IFN-) production, suggesting a dysregulated virus-specific, CD4 T-cell effector function during chronic HCV infection. In conclusion, our results suggest that host ethnicity does influence the clinical outcome and antiviral T-cell response during HCV infection. AA ethnicity is associated with a more robust antiviral CD4 T-cell response than CA ethnicity, although these T cells are limited in direct virus or disease control due to their dysfunctional nature. (HEPATOLOGY 2003;37:590-599.)

Prediction of treatment outcome in patients with chronic hepatitis C: Significance of baseline parameters and viral dynamics during therapy (*Human Study*)
Thomas Berg, Christoph Sarrazin, Eva Herrmann, Holger Hinrichsen, Tilman Gerlach, Reinhart Zachoval, Bertram Wiedenmann, Uwe Hopf, Stefan Zeuzem
In patients with chronic hepatitis C virus (HCV) infection scheduled for a 48-week treatment period, premature discontinuation of treatment was previously recommended if HCV-RNA levels remained detectable at week 24 of therapy. Considering the number of side effects and treatment costs, measurement of initial viral decline during therapy may identify virologic nonresponse earlier than 24 weeks. We retrospectively analyzed 260 European patients treated with standard or pegylated interferon alfa (IFN-) and ribavirin for 24 to 48 weeks. Early prediction of virologic response by HCV-RNA decline at weeks 4 and 12 (Versant Quantitative [branched DNA (bDNA) 3.0]; Bayer Diagnostics, Emeryville, CA; and Qualitative [transcription-mediated amplification (TMA)] HCV RNA assay; Bayer Diagnostics) as well as clinical, biochemical, virologic, and histologic baseline parameters were analyzed by logistic regression and receiver operating characteristic (ROC) curves. A viral load at treatment week 4 above 450,000 IU/mL and at week 12 above 30,000 IU/mL was 100% predictive for virologic nonresponse in all patients. From multivariate logistic regression analysis of all patients, independent predictors for sustained virologic response were: genotypes 2 and 3 (P < .0001), a low baseline gamma-glutamyltransferase (GGT) level (P < .0001), a high baseline alanine aminotransferase level (P = .002), and a low baseline viral load (P = .04). None of the latter 3 factors were predictive for sustained virologic response when analysis was restricted to the subgroup of genotypes 2­ and 3­infected patients. In conclusion, virologic nonresponse can be predicted early at week 12 of treatment independent from the applied therapeutic regimen based on a cutoff level for HCV RNA of 30,000 IU/mL. This algorithm recognizes 53.7% of nonresponders previously identified at week 24 of treatment. (HEPATOLOGY 2003;37:600-609.)

Interferon alfa regulated gene expression in patients initiating interferon treatment for chronic hepatitis C (*Human Study*)
Xuhuai Ji, Ramsey Cheung, Stewart Cooper, Qingqin Li, Harry B. Greenberg, Xiao-Song He
Interferon alfa (IFN-) is an approved therapeutic agent for chronic hepatitis C. To directly characterize the effects of IFN- in humans, we used microarrays to profile gene expression in peripheral blood mononuclear cells (PBMCs) from hepatitis C patients treated with IFN-. Seven patients were studied using two strategies: (1) in vivo: PBMCs were collected immediately before the first dose of IFN-, and 3 and 6 hours after the dose; (2) ex vivo: PBMCs that were collected before the first IFN- dose were incubated with IFN- for 3 and 6 hours. The microarray datasets were analyzed with significance analysis of microarrays (SAM) to identify genes regulated by IFN-. We identified 516 named genes up-regulated at least 2-fold, at a false discovery rate (FDR) of less than 1%. In vivo and ex vivo studies generated similar results. No genes were identified as regulated differently between these 2 experimental conditions. The up-regulated genes belonged to a broad range of functional pathways and included multiple genes thought to be involved in the direct antiviral effect of IFN-. Of particular interest, 88 genes directly relating to functions of immune cells were up-regulated, including genes involved in antigen processing and presentation, T-cell activation, lymphocyte trafficking, and effector functions, suggesting that IFN- up-regulates multiple genes involving different aspects of immune responses to enhance immunity against hepatitis C virus. In conclusion, IFN-­inducible genes can be identified in human PBMCs in vivo as well as ex vivo. Signature changes associated with different treatment outcomes may be found among these genes. (HEPATOLOGY 2003;37:610-621.)

Liver Biology and Pathology

Hepatocyte nuclear factor 1: A key mediator of the effect of bile acids on gene expression
Diana Jung, Gerd A. Kullak-Ublick
Bile acids regulate the expression of genes involved in cholesterol homeostasis. They are ligands of the farnesoid X receptor, which induces small heterodimer partner (SHP)-1, a transcriptional repressor of bile acid synthetic enzymes. In cholestatic liver disease, hepatic bile acid concentrations are elevated and expression of the major Na+­independent bile acid uptake system, organic anion transporting polypeptide (OATP)-C (solute carrier gene family SLC21A6), is markedly decreased. Because the OATP-C gene is transcriptionally dependent on the hepatocyte nuclear factor (HNF) 1, we hypothesized that bile acids decrease OATP-C expression through direct repression of HNF1. To test this hypothesis, we studied the regulation of the human HNF1 gene by bile acids. HNF1 expression in cultured hepatoma cells was decreased ~50% after 12 hours' exposure to 100 µmol/L chenodeoxycholic acid (CDCA). Characterization of the human HNF1 gene promoter identified a consensus bile acid response element that binds and is activated by HNF4. Mutagenesis of the HNF4 site abolished baseline HNF1 promoter activity. The central mechanism by which bile acids repress HNF1 is decreased activation by HNF4. SHP directly inhibits HNF4-mediated transactivation of the HNF1 promoter in cotransfection assays. In addition, HNF4 nuclear binding activity is decreased by CDCA and the human HNF4 gene promoter is repressed by CDCA through an SHP-independent mechanism. In conclusion, we show that repression of HNF1 is an important new mechanism by which bile acids regulate the expression of HNF1-dependent genes in man. This explains the suppressive effect of bile acids on the OATP-C gene promoter, leading to decreased expression in cholestatic liver disease. (HEPATOLOGY 2003;37:622-631.)

Mouse A6­positive hepatic oval cells also express several hematopoietic stem cell markers
Bryon E. Petersen, Brian Grossbard, Heather Hatch, Liya Pi, Jie Deng, Edward W. Scott
Hepatic oval cells (HOC) are thought to be a type of facultative stem cell that arises as a result of certain forms of hepatic injury. A new and more efficient model has been established to activate the oval cell compartment in mice by incorporating 3,5-diethoxycarbonyl-1,4-dihydro-collidine (DDC) in a standard chow at a concentration of 0.1%. At the present time, very few markers exist for the mouse oval cells. One accepted marker is A6, an uncharacterized epitope recognized by mouse hepatic oval cells and it is accepted to be an oval cell marker. Sca-1 is a cell surface marker used to identify hematopoietic stem cells in conjunction with Thy-1+, CD34+, and lineage-specific markers. Both the CD34 and Sca-1 antigens are not normally expressed in adult liver, but are expressed in fetal liver, presumably on the hematopoietic cells. We report herein that mouse oval cells express high levels of Sca-1 and CD34, as well as CD45 surface proteins. Immunohistochemistry revealed that the cells expressing Sca-1/CD34/CD45 were indeed oval cells because they co-expressed the oval cell­specific marker A6 (94.57% ± 0.033%), as well as alpha-fetoprotein (AFP) (75.92% ± 0.071%). By using Sca-1 antibody in conjunction with magnetic activated cell sorting (MACS), followed with a flow cytometric cell sorting (FACS) method for CD34 and CD45, we have developed a rapid oval cell isolation protocol with high yields of greater than 90%. In conclusion, we have an efficient murine model for the production and isolation of large numbers of highly purified oval cells. Our system works with most strains of mouse, which will facilitate both in vivo and in vitro studies of mouse hepatic oval cells. (HEPATOLOGY 2003;37:632-640.)

GM-CSF expands dendritic cells and their progenitors in mouse liver
Venu G. Pillarisetty, George Miller, Alaap B. Shah, Ronald P. DeMatteo
Dendritic cells (DCs) are rare but ubiquitous antigen-presenting cells situated in lymphoid and nonlymphoid organs throughout the body. The study of DCs located in the liver has been restricted by their relative scarcity and the difficulty of their isolation. Because granulocyte-macrophage colony­stimulating factor (GM-CSF) is a critical growth factor for DCs in vitro, we postulated that it would expand hepatic DCs in vivo. We found that adenoviral-mediated GM-CSF overexpression in normal mice increased the number of liver DCs 400-fold to more than 100 million cells. GM-CSF­recruited DCs were CD11c+DEC205­ and had high expression of major histocompatibility complex (MHC) class II, CD54, and CD80 but low CD40 and CD86 staining. Further maturation occurred after overnight culture. In addition to CD11c+DEC205­ DCs, a population of CD11c­DEC205low/­ cells resembling DC progenitors described previously in normal mice was expanded as serum GM-CSF levels increased. GM-CSF­recruited CD11c+DEC205­ DCs and CD11c­DEC205low/­ cells had different functional capabilities. CD11c+DEC205­ DCs captured far more protein antigen in vivo, produced higher amounts of interleukin (IL)-6 and tumor necrosis factor (TNF)-, and induced greater allogeneic and antigen-specific T-cell stimulation. A proportion of CD11c­DEC205low/­ cells differentiated into CD11c+ cells and gained T-cell stimulatory ability when cultured in the presence of GM-CSF. In conclusion, our findings show that GM-CSF can profoundly influence recruitment and development of DCs in murine liver. (HEPATOLOGY 2003;37:641-652.)

Replicative senescence of activated human hepatic stellate cells is accompanied by a pronounced inflammatory but less fibrogenic phenotype
Bernd Schnabl, Carrie A. Purbeck, Youkyung Hwang Choi, Curt H. Hagedorn, David A. Brenner
Limited proliferative capacity is a characteristic of most normal human cells and results in a growth-arrested state, called replicative senescence. Functional expression of the telomerase catalytic subunit (human telomerase reverse transcriptase; hTERT) in human activated hepatic stellate cells (HSCs) rescues them from death with immortalization and maintains an activated HSC phenotype. The aim of this study was to evaluate alterations in gene and protein expression of in vitro aged human activated HSCs and to define the pathway by which senescent-activated HSCs are eliminated in culture. Altered patterns of gene expression in senescent human HSCs were assessed using DNA microarray analysis and compared with early passage HSCs or hTERT immortalized HSCs. Senescent HSCs showed higher expression of inflammation and stress-associated genes as compared with early passage HSCs. Senescent HSCs expressed reduced levels of extracellular matrix proteins, including collagens, tenascin, and fibronectin. TUNEL staining of senescent HSCs showed approximately 21% positive cells, indicating DNA fragmentation and apoptosis. Apoptosis involved the mitochondrial pathway with decreased levels of Bcl-2 and Bcl-xL protein, release of cytochrome c, and increased caspase-3 activity. In contrast, 4% to 5% of early activated HSCs or telomerase positive HSCs were TUNEL positive. In conclusion, cultured human HSCs undergo a switch from a fibrogenic to an inflammatory phenotype, suggesting that senescent human HSCs might modulate chronic wound healing processes. Maintenance of telomere length represents an important survival factor for activated human HSCs. (HEPATOLOGY 2003;37:653-664.)

CYP3A4 inducible model for in vitro analysis of human drug metabolism using a bioartificial liver
Tohru Iwahori, Tomokazu Matsuura, Haruka Maehashi, Ken Sugo, Masaya Saito, Masakiyo Hosokawa, Kan Chiba, Takahiro Masaki, Hideki Aizaki, Kiyoshi Ohkawa, Tetsuro Suzuki
CYP3A is responsible for approximately 50% of the therapeutic drug-metabolizing activity in the liver. The present study was undertaken to establish the CYP3A4 inducible model for analysis of human drug metabolism using a bioartificial liver composed of the functional hepatocellular carcinoma cell (HCC) line FLC-5. A radial-flow bioreactor (RFB), which is a carrier-filled type bioreactor, was used for 3-dimensional perfusion culture of FLC-5 cells. The CYP3A4 messenger RNA (mRNA) expression level 48 hours after rifampicin treatment in the RBF was approximately 100 times higher than that in a monolayer culture. Western blot analysis also demonstrated an increase in expression of the CYP3A protein. When testosterone, a substrate for CYP3A4, was added to the rifampicin-treated cell culture, 6-hydroxy testosterone as a metabolite was formed. Electrophoretic mobility shift assay (EMSA) with a CYP3A4 ER6 probe demonstrated that relatively high molecular weight complex containing pregnane X receptor (PXR)/retinoid X receptor(RXR), compared with that in the monolayer culture, is possibly generated in the RFB culture of FLC-5 treated with rifampicin. Similarly, the assay with a probe of HNF-4-binding motif indicated the formation of a large protein complex in the RFB culture. Because it is known that PXR transactivates CYP3A4 gene via its response element and expression of PXR is regulated by HNF-4, the large complexes binding to response elements of PXR or HNF-4 in the RFB culture may contribute to up-regulation of CYP3A4 mRNA. In conclusion, the bioartificial liver composed of human functional HCC cell line was useful in studying drug interactions during induction of human CYP3A4. (HEPATOLOGY 2003;37:665-673.)

Proangiogenic role of tumor-activated hepatic stellate cells in experimental melanoma metastasis
Elvira Olaso, Clarisa Salado, Eider Egilegor, Virginia Gutierrez, Aitor Santisteban, Paul Sancho-Bru, Scott L. Friedman, Fernando Vidal-Vanaclocha
Myofibroblasts infiltrate malignant liver tumors, although their pathogenic implications are unclear. Immunohistochemical detection of -smooth muscle actin, glial fibrillary acidic protein (GFAP), and CD31 and CD34 expression was used to analyze the contribution of myofibroblasts to angiogenesis in hepatic metastasis produced by intrasplenically-injected B16 melanoma (B16M). Because activated hepatic stellate cells (HSCs) are oxygen-sensing myofibroblasts producing vascular endothelial growth factor (VEGF), the effect of B16M and human A375 melanoma supernatants on VEGF production by immortalized rat HSC line T6 and primary cultured human HSCs also was studied under an hypoxic atmosphere mimicking a tumor microenvironment. Myofibroblast infiltration preceded endothelium recruitment in avascular micrometastasis and generated specific stroma for sinusoidal-type and portal-type angiogeneses. Thereafter, myofibroblasts and endothelial cells colocalized within both angiogenic patterns and their numerical densities correlated with metastasis development. Myofibroblasts often were GFAP-positive, suggesting an HSC origin. Melanoma supernatants stimulated VEGF messenger RNA and protein synthesis by HSCs. These effects were potentiated by hypoxia. VEGF up-regulation was accompanied by increased expression of cyclooxygenase type 2 (COX-2) and PGE2 synthesis. HSC production of VEGF decreased under COX-2 inhibition, whereas it was increased by exogenous PGE2. The high VEGF expression in HSCs induced by melanoma factors and hypoxia resulted in mitogenic, antiapoptotic, and motogenic stimulation of both murine hepatic sinusoidal endothelium and human umbilical vein endothelium. In conclusion, temporal and positional relationships evolve between myofibroblast and endothelium recruitment during metastasis development. Mechanistically, hypoxic induction of VEGF in tumor-activated HSCs may create a proangiogenic microenvironment, facilitating endothelial cell recruitment and survival during hepatic metastasis transition from an avascular to a vascular stage. (HEPATOLOGY 2003;37:674-685.)

Hepatocyte growth factor activates CCAAT enhancer binding protein and cell replication via PI3-kinase pathway
Min Kyung Cho, Sang Geon Kim
Hepatocyte growth factor (HGF), a ligand of c-Met receptor, stimulates activation of cellular kinases via phosphatidylinositol 3-kinase (PI3-kinase). CCAAT/enhancer binding protein (C/EBP) controls cell cycle progression. The present study was designed to determine whether HGF activates C/EBP in association with the S-phase entrance for cell replication and whether PI3-kinase contributes to the activation of C/EBP. Treatment of H4IIE cells, a hepatocyte-derived cell line, with HGF increased protein binding to the C/EBP binding site at an early time. Immunodepletion, subcellular fractionation, and confocal microscopic analyses showed that the HGF-induced C/EBP DNA binding activity depended on nuclear translocation of C/EBP. Whereas stable transfection of the p110 catalytic subunit of PI3-kinase enhanced HGF-mediated nuclear translocation of C/EBP and DNA binding, stable transfection of p85 subunit or chemical inhibition of PI3-kinase completely blocked C/EBP activation. HGF increased luciferase reporter activity in cells transfected with a mammalian cell expression vector containing ­1.65 kilobase rGSTA2 promoter comprising C/EBP response element (pGL-1651). Whereas transfection with pCMV500, a control vector, allowed pGL-1651 to respond to HGF, expression of dominant negative mutant C/EBP completely inhibited the ability of HGF to stimulate the reporter gene expression. Flow cytometric analysis showed that HGF caused an increase in the area of S phase with a reciprocal decrease in that of G1 phase, suggesting that HGF promoted cell cycle progression to S phase. In conclusion, HGF induces nuclear translocation of C/EBP via the PI3-kinase pathway and stimulates C/EBP DNA binding and gene transcription and that the PI3-kinase­mediated C/EBP activation by HGF may contribute to cell replication. (HEPATOLOGY 2003;37:686-695.)

Antiangiogenic gene therapy for hepatocellular carcinoma using angiostatin gene
Hiroki Ishikawa, Kazuhiko Nakao, Kojiro Matsumoto, Tatsuki Ichikawa, Keisuke Hamasaki, Keisuke Nakata, Katsumi Eguchi
Recent studies have reported that antiangiogenic gene delivery into cancer cells inhibits growth of certain tumors in vivo. Hepatocellular carcinoma (HCC) is a hypervascular cancer, and antiangiogenic gene therapy might be suitable for HCC. In the present study, we investigated the antiangiogenic effects of angiostatin gene transduction into HCC both in vitro and in vivo. Angiostatin gene was cloned into a pSecTag2B mammalian expression vector to construct pSecTag2B-ANG. pSecTag2B or pSecTag2B-ANG were transfected into an HCC cell line, PLC/PRF/5, and then stable transfectants were obtained by Zeocin selection. pSecTag2B or pSecTag2B-ANG transfection did not alter the expression of vascular endothelial growth factor (VEGF), a potent angiogenic stimulator, or pigment epithelium-derived factor (PEDF), an angiogenic inhibitor, in PLC/PRF/5 cells. However, conditioned media (CM) derived from pSecTag2B-ANG­transfected PLC/PRF/5 cells (CM-ANG) suppressed the proliferation and migration of human umbilical vein endothelial cells (HUVEC) by 35% and 50%, respectively, relative to their effects on nontransfected cells. In in vivo experiments, pSecTag2B-ANG stable transfected (CM-Mock) and nontransfected cells (CM-N) were mixed at various proportions and the mixed cells were subcutaneously implanted into athymic mice. Suppression of tumor growth was noted in mice implanted with angiostatin gene­transfected cells, and such suppression was proportional with the percentage of transfected cells. Analysis of the vascular density in these tumors showed that the tumor growth suppression effect of angiostatin gene correlated with suppression of tumor vascularity. In conclusion, antiangiogenic gene therapy using angiostatin gene is potentially suitable for the treatment of patients with HCC. (HEPATOLOGY 2003;37:696-704.)

GASTROENTEROLOGY

Table of Contents for March 2003 · Volume 124 · Number 3

Clinical­Alimentary Tract

Gastrointestinal safety of NO-aspirin (NCX-4016) in healthy human volunteers: A proof of concept endoscopic study
S. Fiorucci, L. Santucci, P. Gresele, R. M. Faccino, P. del Soldato, A. Morelli
Background & Aims: NCX-4016 is a nitric oxide­releasing derivative of aspirin with antiplatelet activity. The aim of this study was to investigate the effect of NCX-4016 on gastrointestinal mucosa and platelet functions in healthy human volunteers.
Methods: This was a parallel-group, double-blind, placebo-controlled study. Forty healthy subjects were randomly allocated to receive 7 days of treatment with NCX-4016 (400 and 800 mg twice daily), equimolar doses of aspirin (200 and 420 mg twice daily), or placebo. Upper endoscopies were performed before and at the end of the treatment period, and gastroduodenal lesions were graded using a predefined scoring system. Basal and posttreatment platelet aggregation in response to arachidonic acid (AA) and serum thromboxane (TX) B2 and AA-stimulated platelet TXB2 production were investigated.
Results: Mucosal endoscopic injury score on day 7 was 0.63 ± 0.16 in the placebo group and 11.0 ± 3.0 and 16.1 ± 1.6 in healthy volunteers treated with 200 and 420 mg aspirin twice daily (P < 0.0001 vs. placebo). NCX-4016 was virtually devoid of gastric and duodenal toxicity, resulting in a total gastric and duodenal endoscopic score of 1.38 ± 0.3 and 1.25 ± 0.5 (P < 0.0001 vs. aspirin, not significant vs. placebo). NCX-4016 inhibited AA-induced platelet aggregation as well as serum TXB2 and platelet TXB2 generation induced by AA to the same extent as aspirin (not significant vs. aspirin).
Conclusions: In this study, we have proven the concept that addition of an NO-donating moiety to aspirin results in a new chemical entity that maintains cyclooxygenase-1 and platelet inhibitory activity while nearly avoiding gastrointestinal damage.

Screening for colorectal cancer in Chinese: Comparison of fecal occult blood test, flexible sigmoidoscopy, and colonoscopy
J. J. Y. Sung, F. K. L. Chan, W. K. Leung, J. C. Y. Wu, J. Y. W. Lau, J. Ching, K. F. To, Y. T. Lee, Y. W. Luk, N. N. S. Kung, S. P. Y. Kwok, M. K. W. Li, S. C. Sydney Chung
Background & Aims: Fecal occult blood testing (FOBT), flexible sigmoidoscopy (FS), and colonoscopy are the most commonly recommended screening tests for colorectal cancer. The aim of this study was to compare the accuracy and safety of these 3 screening procedures in a general population of ethnic Chinese.
Methods: Asymptomatic adults older than 50 years were recruited from the general public through health exhibitions. All enrolled subjects were offered FOBT and full colonoscopy under sedation. Advanced colonic lesions (defined as adenoma 10 mm, villous adenoma, adenoma with moderate or severe dysplasia, or invasive cancer) were recorded. Lesions at the distal 40 cm in the left colon and rectum were taken as findings of FS.
Results: A total of 505 subjects (56% women; mean age ± SD, 56.5 ± 5.4 years) were enrolled, and 476 (94.3%) had a complete colonoscopy. Advanced colonic neoplasms were documented in 63 subjects (12.5%), of which 45 had lesions in the distal colon and 26 in the proximal colon. Among the 385 subjects with a normal distal colon, 14 (3.6%) had advanced lesions in the proximal colon that would be missed by FS alone. The sensitivity and specificity of FOBT for advanced colonic lesions were 14.3% and 79.2% and the sensitivity and specificity of FS were 77.8% and 83.9%, respectively. Combining FOBT with FS would not significantly improve the results of FS alone. Among these 505 subjects who underwent colonoscopy and 148 who underwent polypectomy, there was no perforation and only one occurrence of postpolypectomy bleeding recorded.
Conclusions: Colonoscopy is a safe and accurate method for the screening of colorectal neoplasms in Chinese subjects.

Gastrin induces proliferation in Barrett's metaplasia through activation of the CCK2 receptor
C. R. Haigh, S. E. A. Attwood, D. G. Thompson, J. A. Jankowski, C. M. Kirton, D. M. Pritchard, A. Varro, R. Dimaline
Background & Aims: Factors associated with the development and malignant progression of Barrett's esophagus are poorly understood. Gastrin is a mitogen capable of inducing growth in normal and malignant gastrointestinal mucosa. It is unknown whether gastrin can influence cellular events in the esophagus in Barrett's.
Methods: Reverse-transcription polymerase chain reaction (RT-PCR) and northern analysis for the cholecystokinin (CCK2) receptor were performed on normal, inflamed, metaplastic, and malignant esophageal mucosa. Real-time PCR quantified expression of the receptor. [125I]-G17-autoradiography localized the CCK2 receptor in mucosal sections. [3H]-thymidine and bromodeoxyuridine (BrdU) incorporation determined proliferation in response to G17 in biopsy specimens incubated ex vivo. Proliferation and signaling studies were performed on OE33(E) cells transfected with the CCK2 receptor.
Results: RT-PCR identified receptor expression in 3 of 9 controls, 5 of 7 patients with esophagitis, 10 of 10 patients with Barrett's metaplasia, and 7 of 12 esophageal adenocarcinomas. Real-time PCR quantified expression in 10 patients with Barrett's showing a level of expression 2 orders of magnitude higher than in 12 control patients. [125I]-G17 bound to epithelia within glandular regions of Barrett's mucosa. Ten nmol/L G17 induced a 2-fold (n = 7, P = 0.0257, t test) increase in [3H]-thymidine incorporation in mucosal biopsy specimens, abolished by the addition of the CCK2 receptor antagonist L-740, 093. One nmol/L G17 induced a 1.94­ ± 0.13-fold (n = 6, t test, P = 0.001) increase in [3H]-thymidine incorporation in OE33(E)GR cells, abolished by L-740, 093.
Conclusions: Gastrin induces proliferation via the CCK2 receptor in Barrett's mucosa. This may have implications for the management of patients with Barrett's esophagus in whom gastrin is elevated by acid-suppression therapy.

p14 methylation in human colon cancer is associated with microsatellite instability and wild-type p53
L. Shen, Y. Kondo, S. R. Hamilton, A. Rashid, J.­P. J. Issa
Background & Aims: Colorectal cancers with high levels of microsatellite instability (MSI-H) have an unexplained low rate of p53 gene mutations. Most such cancers have the CpG island methylator phenotype (CIMP+) with methylation and transcriptional silencing of the mismatch repair gene MLH1. The p14 (ARF) gene on chromosome 9p is deleted and/or silenced by hypermethylation in a subset of human malignancies. There is evidence suggesting that p14 suppresses tumorigenicity by stabilizing the p53 protein.
Methods: We investigated the role of p14 in colorectal cancer by determining its methylation status in cancers that were studied previously for microsatellite instability, CIMP, and mutations of p53 and K-RAS.
Results: p14 methylation was present in 21 of 94 cases overall (22%) and was frequent particularly in the subgroups with MSI-H (52% [11 of 21] vs. 14% [10 of 72], P = 0.004), in CIMP+ cases (40% [19 of 48] vs. 4% [2 of 46], P < 0.001), and in cases without p53 alterations (36% [17 of 47] vs. 7% [3 of 44], P = 0.004). Of 91 fully characterized cases, 41 (45%) had p53 mutations alone, 17 (19%) had p14 methylation alone, 30 (33%) had neither, but only 3 (3%) had both p53 mutations and p14 methylation. p14 methylation is an early event in colorectal carcinogenesis, being detectable in normal aging epithelium by using sensitive assays.
Conclusions: In colorectal cancer, p14 methylation is associated with the presence of microsatellite instability and with absence of p53 mutations. The results provide a possible explanation for the paucity of p53 mutations in colon cancers with microsatellite instability.

Clinical­Liver, Pancreas, and Biliary Tract

The North American Study for the Treatment of Refractory Ascites
A. J. Sanyal, C. Genning, K. R. Reddy, F. Wong, K. V. Kowdley, K. Benner, T. McCashland, The Nastra Group
Background & Aims: The clinical utility of transjugular intrahepatic portosystemic shunts (TIPS) vis-à-vis total paracentesis in the management of refractory ascites is unclear.
Methods: A multicenter, prospective, randomized clinical trial was performed in which 109 subjects with refractory ascites were randomized to either medical therapy (sodium restriction, diuretics, and total paracentesis) (n = 57) or medical therapy plus TIPS (n = 52). The principal end points were recurrence of tense symptomatic ascites and mortality.
Results: A technically adequate shunt was created in 49 of 52 subjects. TIPS plus medical therapy was significantly superior to medical therapy alone in preventing recurrence of ascites (P < 0.001). The total number of deaths in the 2 groups was identical (TIPS vs. medical therapy alone: 21 vs. 21). There were no significant differences in the 2 arms with respect to overall and transplant-free survival. There was a higher incidence of moderate to severe encephalopathy in the TIPS group (20 of 52 vs. 12 of 57; P = 0.058). There were no significant differences in the number of subjects who developed liver failure (7 vs. 3), variceal hemorrhage (5 vs. 8), or acute renal failure (3 vs. 2). There were also no significant differences between the 2 groups in the frequency of emergency-department visits, medically indicated hospitalizations, or quality of life.
Conclusions: Although TIPS plus medical therapy is superior to medical therapy alone for the control of ascites, it does not improve survival, affect hospitalization rates, or improve quality of life.

Interferon- 2b plus ribavirin in patients with chronic hepatitis C after liver transplantation: A randomized study
D. Samuel, T. Bizollon, C. Feray, B. Roche, S. N. S. Ahmed, C. Lemonnier, M. Cohard, M. Reynes, M. Chevallier, C. Ducerf, J. Baulieux, M. Geffner, J. K. Albrecht, H. Bismuth, C. Trepo
Background & Aims: Hepatitis C virus (HCV) reinfection after liver transplantation is frequent and leads to chronic hepatitis and cirrhosis. The use of antiviral therapy in this situation remains controversial. This study aimed to assess the safety and efficacy of interferon alfa-2b plus ribavirin for recurrent hepatitis C following liver transplantation.
Methods: Transplant recipients with recurrent chronic hepatitis C were randomized to receive either no treatment or therapy with interferon alfa-2b (3 MU 3 times a week) plus 1000­1200 mg/day ribavirin for 1 year. Patients were followed up for 6 months after the end of treatment. The primary end point was loss of HCV RNA 6 months after the end of treatment.
Results: Fifty-two patients were randomized (treatment, 28; placebo, 24). Sixteen patients were withdrawn from the study; 12 (43%) were from the treated group (mainly for anemia [7 patients]) and 4 (17%) from the control group. In the treated group, serum HCV RNA was undetectable in 9 patients (32%) at the end of treatment and 6 (21.4%) at the end of the follow-up period, whereas no patient in the control group lost HCV RNA at any point (P = 0.036 at the end of follow-up). However, there was no significant histologic improvement.
Conclusions: The combination of interferon alfa-2b plus ribavirin induced a sustained virologic response in 21% of transplant recipients with recurrent hepatitis C. However, 43% discontinued therapy due to adverse events (primarily severe anemia). Strategies to enable treatment with lower doses of ribavirin need to be explored.

A quantitative gene expression study suggests a role for angiopoietins in focal nodular hyperplasia
V. Paradis, I. Bièche, D. Dargère, I. Laurendeau, J. Nectoux, C. Degott, J. Belghiti, M. Vidaud, P. Bedossa
Background & Aims: Although the pathogenesis of focal nodular hyperplasia (FNH) of the liver remains unclear, a vascular mechanism has been suspected. To gain insight into the pathogenesis of FNH, we performed a large-scale quantitative study of gene expression in FNH.
Methods: Quantitative expression level of 209 selected genes was assessed using real-time reverse-transcription polymerase chain reaction in 14 cases of FNH and compared with their expression level in 13 cases of liver cirrhosis, 4 adenomas, and 15 hepatocellular carcinomas.
Results: Among the 7 genes, the expression of which was significantly up-regulated or down-regulated in FNH, the most informative markers for the diagnosis of FNH as assessed using the receiving operative curve and area under the curve (AUC) were angiopoietin-1 (Ang-1; AUC, 0.82) and angiopoietin-2 (Ang-2; AUC, 0.80). These 2 genes are involved in the regulation of vasculogenesis. In FNH, Ang-1 was significantly up-regulated, Ang-2 was down-regulated, and the Ang-1/Ang-2 ratio was highly and specifically increased in FNH compared with normal liver or other groups of lesions (FNH, 15.2-fold increase; HCC, 2.78; adenoma, 2.28; cirrhosis, 1.92; P < 0.01 for FNH vs. all groups, analysis of variance). Tie-2 messenger RNA, the receptor of Ang-1 and Ang-2, was detected at the same level in FNH as in normal liver. Ang-1 protein was detected on Western blot of FNH and expressed by endothelial cells of dystrophic vessels and sinusoids as shown by immunohistochemistry.
Conclusions: A specific increase of Ang-1/Ang-2 ratio in FNH, in the presence of the functional Tie-2 receptor, might be involved in the formation of hyperplastic and dystrophic vessels of FNH.

Basic­Alimentary Tract

Isolation-stress increases small intestinal sensitivity to chemotherapy in rats
M. Verburg, I. B. Renes, A. W. C. Einerhand, H. A. Büller, J. Dekker
Background & Aims: Severe gastrointestinal damage often complicates the use of chemotherapeutic agents such as methotrexate for anticancer treatment. Psychologic stress is known to be detrimental to normal intestinal physiology. We set out to determine if psychologic stress adds to the intestinal damage provoked by chemotherapy.
Methods: Rats were treated with various doses of methotrexate and housed either alone, which induces mental stress, or maintained in groups of 3 animals. Treatment was evaluated by (immuno)histologic parameters.
Results: Epithelial crypt damage, increased lysozyme expression, decreased sucrase-isomaltase and sodium/glucose transporter 1 expression, and pathologic changes in mucin and trefoil factor protein expression could be prevented by avoiding isolation. Enhanced cytotoxicity of methotrexate through isolation was about 2-fold and involved an augmented inhibition of proliferation, increased epithelial apoptosis, increased villus damage, and delayed recovery. We could not identify a role for mucosal mast cells in the increased epithelial damage under isolated conditions.
Conclusions: The clear beneficial effects of avoiding mental stress on the protection of the intestinal epithelium during cytostatic drug-treatment may be an important element for the treatment of cancer patients.

T helper type-2 cells induce ileal villus atrophy, goblet cell metaplasia, and wasting disease in T cell­deficient mice
T. Dohi, K. Fujihashi, T. Koga, Y. Shirai, Y. I. Kawamura, C. Ejima, R. Kato, K. Saitoh, J. R. McGhee
Background & Aims: T helper (Th) 1 and Th2 cell subsets significantly influence the pathological features of inflammation in the gastrointestinal tract in a distinct manner. It is now established that the transfer of CD4+CD45RBHi (RBHi) T cells to either severe combined immunodeficient (SCID) or recombinase activation gene 2­deficient (RAG­/­) mice results in a severe granulomatous hypertrophic colitis mediated by Th1 cells. We have modified this approach to address the role of Th2 cells.
Methods: RBHi T cells from wild-type (Wt) mice or mice genetically predisposed to Th2 responses (interferon-­defective [IFN-­/­]) with or without B cells were transferred to T cell receptor (TCR)- and -chain­defective (TCR­/­) or SCID mice.
Results: Transfer of Wt RBHi T cells induced wasting disease with severe colitis in the TCR­/­ mice. In contrast, IFN-­/­ RBHi T cells induced severe weight loss and hypoalbuminemia without significant inflammation in the colon. The small intestine of these mice exhibited villus atrophy, a decrease in brush-border enzymes, reduced enterocyte proliferation, and an increased number of goblet cells. The presence of B cells was necessary for these changes, because SCID recipients required cotransfer of B cells, together with IFN-­/­ RBHi T cells for ileal lesions to develop. Treatment of TCR­/­ recipients of IFN-­/­ RBHi T cells with anti­IL-4 mAb abrogated both the wasting disease and the villus atrophy.
Conclusions: Dysregulated Th2 cells cause atrophic changes and goblet cell transformation in the small intestinal epithelium and wasting disease mediated by excess interleukin-4 and B cells.

Leptin mediates Clostridium difficile toxin A­induced enteritis in mice
A. Mykoniatis, P. M. Anton, M. Wlk, C. C. Wang, L. Ungsunan, S. Blüher, M. Venihaki, S. Simeonidis, J. Zacks, D. Zhao, S. Sougioultzis, K. Karalis, C. Mantzoros, C. Pothoulakis
Background & Aims: Leptin regulates energy homeostasis and participates in the regulation of the hypothalamic-pituitary-adrenal axis. Although hyperleptinemia is described in experimental colitis, its role in the pathophysiology of enterotoxin-mediated diarrhea and inflammation remains unclear. We examined the role of leptin in the inflammatory diarrhea induced by toxin A from Clostridium difficile, the causative agent of antibiotic-related colitis.
Methods: Toxin A (10 µg) or buffer were administered in ileal loops of leptin-deficient (ob/ob), leptin-resistant (db/db), or wild-type mice and enterotoxic responses were measured.
Results: In toxin A­treated wild-type mice, circulating leptin and corticosterone levels were increased compared with buffer-injected animals. Toxin A also stimulated increased mucosal expression of the Ob-Rb at the messenger RNA (mRNA) and protein level. Ob/ob and db/db mice were partially protected against toxin A­induced intestinal secretion and inflammation, and this effect was reversed by leptin administration in ob/ob, but not db/db, mice. Basal- and toxin A­stimulated plasma corticosterone levels in ob/ob and db/db mice were higher compared with toxin A­treated wild-type mice. To assess whether the effect of leptin in intestinal inflammation is mediated by corticosteroids we performed adrenalectomy experiments in db/db and wild-type mice. Our results suggested that the diminished intestinal response to toxin A in db/db mice was related only in part to increased levels of corticosteroids.
Conclusions: Leptin plays an important role in regulating the severity of enterotoxin-mediated intestinal secretion and inflammation by activating both corticosteroid-dependent and -independent mechanisms.

Basic­Liver, Pancreas, and Biliary Tract

 ICAM-1 triggers liver regeneration through leukocyte recruitment and Kupffer cell­dependent release of TNF-/IL-6 in mice
N. Selzner, M. Selzner, B. Odermatt, Y. Tian, N. V. Rooijen, P.­A. Clavien
Background & Aims: Tumor necrosis factor (TNF)- and interleukin (IL)-6 mediate hepatocyte proliferation in vivo, suggesting that local and systemic inflammatory reactions may trigger hepatic regeneration after major tissue loss.
Methods: Wild-type, intercellular adhesion molecule (ICAM)-1­/­, and neutropenic-induced mice were subjected to 70% hepatectomy. Three different approaches to block and/or deplete liver macrophages (Kupffer cells) were used.
Results: We found that liver from ICAM-1­deficient mice exhibited impaired regeneration after partial hepatectomy. This finding is associated with dramatic decrease in leukocyte recruitment and tissue TNF- and IL-6 levels. All markers of hepatocyte proliferation were restored in ICAM­/­ mice by injections of recombinant IL-6. Neutropenic animals and liver macrophage (Kupffer cell) depletion resulted in similar failure of regeneration with low levels of TNF- and IL-6.
Conclusions: The data suggest a novel pathway in which ICAM-1 binds to leukocytes after hepatectomy, triggering hepatocyte proliferation through Kupffer cell­dependent release of TNF- and IL-6.

Liver regeneration in heparin-binding EGF-like growth factor transgenic mice after partial hepatectomy
S. Kiso, S. Kawata, S. Tamura, Y. Inui, Y. Yoshida, Y. Sawai, S. Umeki, N. Ito, A. Yamada, J.­I. Miyagawa, S. Higashiyama, T. Iwawaki, M. Saito, N. Taniguchi, Y. Matsuzawa, K. Kohno
Background & Aims: Heparin-binding epidermal growth factor­like growth factor (HB-EGF), a member of the EGF family, is synthesized in the form of a membrane-anchored precursor (proHB-EGF), which subsequently is processed proteolytically to mature HB-EGF. This study describes the effects of HB-EGF on liver regeneration after 70% partial hepatectomy in proHB-EGF transgenic mice with liver-specific expression.
Methods & Results: No significant differences in liver/body weight ratios and in bromodeoxyuridine (BrdU)-labeling index (the ratios of BrdU-positive hepatocyte nuclei) were found between adult transgenic and wild-type mice. However, in regenerating liver after partial hepatectomy, transgenic mice had higher liver/body weight ratios than wild-type mice and at 120 hours reached a level equal to that before partial hepatectomy. The BrdU-labeling index was about 5 times higher in the livers of transgenic mice compared with the wild type (51.5% vs. 10.2%, respectively; P < 0.01) at 48 hours after partial hepatectomy. Activation of microtubule-associated protein kinase after partial hepatectomy was higher and earlier in the transgenic mice as compared with the wild-type mice. Soluble HB-EGF was increased in the liver (at 8 min) after partial hepatectomy, indicating that the shedding of proHB-EGF occurred after partial hepatectomy.
Conclusions: The transgenic expression of HB-EGF accelerates the proliferation of hepatocytes after partial hepatectomy, suggesting that HB-EGF functions as a hepatotrophic factor in vivo.

Acetaldehyde impairs mitochondrial glutathione transport in HepG2 cells through endoplasmic reticulum stress
J. M. Lluis, A. Colell, C. García­Ruiz, N. Kaplowitz, J. C. Fernández­Checa
Background & Aims: Ethanol impairs the mitochondrial transport of reduced glutathione (GSH), resulting in lower mitochondrial GSH (mGSH) levels. Our purpose was to evaluate the role of acetaldehyde on the regulation of mGSH in HepG2 cells.
Methods: mGSH levels and transport, mitochondrial membrane microviscosity, and lipid composition were determined in mitochondria isolated from acetaldehyde-treated HepG2 cells.
Results: The major ultrastructural changes of acetaldehyde-treated HepG2 cells included cytoplasmic lipid droplets and appearance of swollen mitochondria. Acetaldehyde depleted the mGSH pool size in a time- and dose-dependent fashion with spared cytosol GSH levels. Kinetics of GSH transport into isolated mitochondria from HepG2 cells showed 2 saturable, adenosine triphosphate­stimulated, high- and low-affinity components. Treatment with acetaldehyde increased the Michaelis constant for the high- and low-affinity components, with a greater impact on the former. These changes were due to increased mitochondrial microviscosity by enhanced cholesterol deposition because preincubation with the fluidizing agent, 2-(2-methoxyethoxy) ethyl 8-(cis-2-n-octylcyclopropyl) octanoate, normalized the initial transport rate of GSH into isolated mitochondria. Isolated mitochondria from rat liver enriched in free cholesterol reproduced the disturbing effects of acetaldehyde on GSH transport. The acetaldehyde-stimulated mitochondrial cholesterol content was preceded by increased levels of endoplasmic reticulum (ER)-responsive gene GADD153 and transcription factor sterol regulatory element-binding protein 1 and mimicked by the ER stress-inducing agents tunicamycin and homocysteine. Finally, the mGSH depletion induced by acetaldehyde sensitized HepG2 cells to tumor necrosis factor (TNF)-­induced apoptosis that was prevented by cyclosporin A, GSH ethyl ester, and lovastatin.
Conclusions: Acetaldehyde sensitizes HepG2 cells to TNF- by impairing mGSH transport through an ER stress-mediated increase in cholesterol.

Macrophage migration inhibitory factor is a critical mediator of severe acute pancreatitis
Y. Sakai, A. Masamune, A. Satoh, J. Nishihira, T. Yamagiwa, T. Shimosegawa
Background & Aims: Macrophage migration inhibitory factor (MIF), originally described as an inhibitor of the random migration of macrophages, has been shown recently to be involved in the pathogenesis of several inflammatory diseases such as sepsis. The aim of this study was to clarify the role of MIF in acute pancreatitis (AP).
Methods: Hemorrhagic necrotizing pancreatitis and edematous pancreatitis were induced by the injection of taurocholic acid (TCA pancreatitis) and cerulein (cerulein pancreatitis), respectively, on male Wistar rats. MIF levels in ascitic fluids, serum, and the organs were determined. The effects of anti-MIF antibody were examined on the prognosis of rats with TCA pancreatitis and of female CD-1 mice with choline-deficient, ethionine-supplemented, diet-induced model of severe AP. In addition, serum MIF levels in AP patients and in healthy controls were measured.
Results: Serum and ascitic MIF levels in TCA pancreatitis were increased rapidly and decreased gradually thereafter. Ascitic MIF levels were also increased in cerulein pancreatitis, but to a lesser degree. MIF level was increased in the lung in TCA pancreatitis, but not in the pancreas and the liver. Prophylactic (1 hour before and immediately after induction) administration of anti-MIF antibody significantly improved the survival rate of rats with TCA pancreatitis. The survival rate of mice with severe AP was also improved significantly by the antibody treatment. Serum MIF levels were higher in severe AP patients than mild AP patients and healthy controls.
Conclusions: These results suggest a role of MIF in the pathogenesis of severe AP.

Cytokine-stimulated nitric oxide production inhibits adenylyl cyclase and cAMP-dependent secretion in cholangiocytes
C. Spirlì, L. Fabris, E. Duner, R. Fiorotto, G. Ballardini, T. Roskams, N. F. Larusso, A. Sonzogni, L. Okolicsanyi, M. Strazzabosco
Background & Aims: The biliary epithelium is involved both in bile production and in the inflammatory/reparative response to liver damage. Recent data indicate that inflammatory aggression to intrahepatic bile ducts results in chronic progressive cholestasis.
Methods: To understand the effects of nitric oxide on cholangiocyte secretion and biliary tract pathophysiology we have investigated: (1) the effects of proinflammatory cytokines on NO production and expression of the inducible nitric oxide synthase (NOS2), (2) the effects of NO on cAMP-dependent secretory mechanisms, and (3) the immunohistochemical expression of NOS2 in a number of human chronic liver diseases.
Results: Our results show that: (1) tumor necrosis factor (TNF)-, and interferon (IFN)-, synergically stimulate NO production in cultured cholangiocytes through an increase in NOS2 gene and protein expression; (2) micromolar concentrations of NO inhibit forskolin-stimulated cAMP production by adenylyl cyclase (AC), cyclic adenosine monophosphate (cAMP)-dependent fluid secretion, and cAMP-dependent Cl­ and HCO3­ transport mediated by cystic fibrosis transmembrane conductance regulator (CFTR) and anion exchanger isoform 2, respectively; (3) cholestatic effects of NO and of proinflammatory cytokines are prevented by NOS-2 inhibitors and by agents (manganese(III)-tetrakis(4-benzoic acid)porphyrin [MnTBAP], urate, trolox) able to block the formation of reactive nitrogen oxide species (RNOS); (4) NOS2 expression is increased significantly in the biliary epithelium of patients with primary sclerosing cholangitis (PSC).
Conclusions: Our findings show that proinflammatory cytokines stimulate the biliary epithelium to generate NO, via NOS2 induction, and that NO causes ductular cholestasis by a RNOS-mediated inhibition of AC and of cAMP-dependent HCO3­ and Cl­ secretory mechanisms. This pathogenetic sequence may contribute to ductal cholestasis in inflammatory cholangiopathies.

Case Report

Alterations of brain activity associated with resolution of emotional distress and pain in a case of severe irritable bowel syndrome
D. A. Drossman, Y. Ringel, B. A. Vogt, J. Leserman, W. Lin, J. K. Smith, W. Whitehead
Background & Aims: The association of psychosocial disturbances with more severe irritable bowel syndrome (IBS) is well recognized. However, there is no evidence as to how these associations might be mediated. Functional magnetic resonance imaging (fMRI) offers an opportunity to study whether activation of the cingulate cortex, an area involved with the affective and pain intensity coding might be linked to poorer clinical status with IBS. In this case report, we found an association between the severity of a patient's clinical symptoms and psychosocial state, with activation of the cingulate cortex. We also found that clinical and psychosocial improvement was associated with reduced cingulate activation.
Methods: Observational case report of a young woman observed for 16 years with a history of sexual abuse, psychosocial distress, and functional GI complaints. Psychosocial, clinical, and fMRI assessment was performed when the patient experienced severe symptoms and again 8 months later when clinically improved.
Results: During severe illness, the patient had major psychosocial impairment, high life stress, a low visceral pain threshold, and activation of the midcingulate cortex (MCC), prefrontal area 6/44, and the somatosensory cortex, areas associated with pain intensity encoding. When clinically improved, there was resolution in activation of these 3 areas, and this was associated with psychosocial improvement and an increased threshold to rectal distention.
Conclusions: Activation of the MCC and related areas involved with visceral pain encoding are associated with poor clinical status in patients with severe IBS and psychosocial distress and appear to be responsive to clinical improvement.

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JOURNAL OF HEPATOLOGY

Table of Contents for Journal of Hepatology Volume 38, Issue S1, February 2003

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Therapy in Liver Diseases 2003

Review
Stephen F. Stewart and Christopher P. Day
The management of alcoholic liver disease
[Online Article] S2-S13

Review
Stephen A. Harrison and Bruce R. Bacon
Hereditary hemochromatosis: update for 2003
[Online Article] S14-S23

Review
Cynthia Levy and Keith D. Lindor
Current management of primary biliary cirrhosis and primary sclerosing cholangitis
[Online Article] S24-S37

Review
Scott L. Friedman
Liver fibrosis - from bench to bedside
[Online Article] S38-S53

Review
Jaime Bosch, Juan G. Abraldes and Roberto Groszmann
Current management of portal hypertension
[Online Article] S54-S68

Review
Vicente Arroyo and Jordi Colmenero
Ascites and hepatorenal syndrome in cirrhosis: pathophysiological basis of therapy and current management
[Online Article] S69-S89

Review
Hari S. Conjeevaram and Anna Suk-Fong Lok
Management of chronic hepatitis B
[Online Article] S90-S103

Review
Alfredo Alberti and Luisa Benvegnù
Management of hepatitis C
[Online Article] S104-S118

Review
Dieter C. Broering, Martina Sterneck and Xavier Rogiers
Living donor liver transplantation
[Online Article] S119-S135

Review
Josep M. Llovet and Michel Beaugrand
Hepatocellular carcinoma: present status and future prospects
[Online Article]S136-S149

Copyright © 2001-2003  European Association for the Study of the Liver. All rights reserved.

Table of Contents for Journal of Hepatology Volume 38, Issue 3, March 2003

Cell Biology, Metabolism and Transport

Cathy Royer et al.
A study of susceptibility of primary human Kupffer cells to hepatitis C virus
Background/Aims: Histological examination of a liver-biopsy from a patient with chronic hepatitis C shows activated Kupffer cells. In vitro infection of human Kupffer cells (KC) was performed to study their interaction with hepatitis C virus (HCV). Methods: KC, isolated by collagenase perfusion and centrifugal elutriation, were infected with various HCV positive sera. The presence of the viral genome was followed, at different times, quantitatively by a branched-DNA assay and qualitatively by reverse transcriptase-nested polymerase chain reaction. A strand-specific assay performed with the thermostable enzyme rTth was used to detect the synthesis of a negative replicative intermediate. Cytopathic effect was examined by electron microscopy. Production of cytokines and inducible nitric oxide synthase was evaluated in the supernatants. Results: Quantification of HCV-RNA showed that the level of viral RNA associated with KC after adsorption decreased rapidly. Genomic viral RNA disappeared within 5 days of infection. Negative-strand RNA was never detected in any of these experiments. No cytopathic effects could be detected at any time. KC did not produce inflammatory nor antiviral cytokines. Conclusions: Our results strongly suggest that primary cultures of KC are not permissive for HCV in vitro.

Chronic Liver Diseases

Thierry Poynard et al.
A comparison of fibrosis progression in chronic liver diseases
Background/Aims: No study has compared the liver fibrosis progression rates among chronic liver diseases and the risk factors in order to better organize screening strategies. Methods: A total of 4852 patients were retrospectively studied (chronic hepatitis C (HCV) [n=2313], human immunodeficiency virus (HIV)-HCV co-infection (HIV-HCV [n=180]), hepatitis B (HBV [n=777]), alcoholic liver disease (ALD [n=701]), primary biliary cirrhosis (PBC [n=406]), genetic hemochromatosis (GH [n=383]) auto-immune hepatitis (AIH [n=57]) and delta hepatitis (n=35). The fibrosis progression rates were estimated from birth and from the date of exposure, when known, to the first biopsy. Results: There were highly significant differences in the rates of fibrosis progression, the most rapid being HIV-HCV co-infection (50% cirrhosis percentile at 52 years of age) and the slowest being PBC (50% cirrhosis percentile at 81 years). There was an acceleration of fibrosis progression with aging. Fibrosis progression was slower in females compared with males for HCV, HBV, GH, and PBC. In contrast, in ALD, the fibrosis progression was more rapid in females. Conclusions: Rates of fibrosis progression differ markedly between the predominant causes of chronic liver disease, and according to age and gender. Patients with HIV-HCV co-infection are at particularly high risk of fibrosis progression.

Cirrhosis and its Complications

Manuela Merli et al.
Incidence and natural history of small esophageal varices in cirrhotic patients
Background/Aims: The incidence and natural history of small esophageal varices (EV) in cirrhotics may influence the frequency of endoscopies and the decision to start a pharmacological treatment in these patients. Methods: We prospectively evaluated 206 cirrhotics, 113 without varices and 93 with small EV, during a mean follow-up of 37±22 months. Patients with previous gastrointestinal bleeding or receiving any treatment for portal hypertension were excluded. Endoscopy was performed every 12 months. Results: The rate of incidence of EV was 5% (95%CI: 0.8-8.2%) at 1 year and 28% (21.0-35.0%) at 3 years. The rate of EV progression was 12% (5.6-18.4%) at 1 year and 31% (21.2-40.8%) at 3 years. Post-alcoholic origin of cirrhosis, Child-Pugh's class (B or C) and the finding of red wale marks at first examination were predictors for the variceal progression. The two-years risk of bleeding from EV was higher in patients with small varices upon enrolment than in those without varices: 12% (95% CI: 5.2-18.8%) vs. 2% (0.1-4.1%); (P<0.01). Predictor for bleeding was the presence of red wale marks at first endoscopy. Conclusions: In patients with no or small EV, endoscopy surveillance should be planned taking into account cause and degree of liver dysfunction.

E. Michael Joebges et al.
Bradykinesia in minimal hepatic encephalopathy is due to disturbances in movement initiation
Background: One of the predominant symptoms of early stages of hepatic encephalopathy (HE) is bradykinesia. Aims: To further analyze the pathophysiology of bradykinesia in HE. Methods: A three-dimensional computer-assisted movement analysis was performed in 36 cirrhotics with grade 0-I HE compared to 18 controls selected with regard to sex and age and 16 patients with Parkinson's disease (PD). Four types of movement were studied: finger tapping, hand tapping, pronation/supination of the forearm and flexion/extension in the hip joint. Results: The patients with PD presented with a decrease of the maximal movement velocity (VMAX) and a prolongation of the time needed to reach VMAX (VTIME). In patients with minimal or grade I HE, the VMAX of all movements was unchanged compared to controls while the VTIME was significantly prolonged. This was caused by a delay before the beginning of each new part of the diadochokinetic movement cycle. Conclusions: The data suggest an impairment of movement initiation as main cause of bradykinesia in early HE.

Inflammation and Fibrosis

James J. Potter, Lynda Rennie-Tankesley and Esteban Mezey
Influence of leptin in the development of hepatic fibrosis produced in mice by Schistosoma mansoni infection and by chronic carbon tetrachloride administration
Background/Aims: Leptin, a product of the obese (ob) gene is present in activated stellate cells. This study investigated whether leptin is essential for the development of hepatic fibrosis caused by various agents. Methods: Control and ob/ob mice were infected with Schistosoma mansoni or were administered chronic carbon tetrachloride to cause hepatic fibrosis. Results: Fibrosis developed in both ob/ob and control mice. However, the amount of histologically detectable fibrosis and the increase in liver hydroxyproline content was significantly greater in both models of fibrosis for treated controls than for treated ob/ob mice. Fibrosis was associated with higher secretion of TGF1 from spleen cells of treated control than treated ob/ob mice. Chronic leptin administration in ob/ob mice infected with Schistosoma mansoni resulted in an increase in the amount of fibrosis caused by Schistosoma mansoni, eliminating any significant differences in the amount of fibrosis between infected ob/ob mice and control mice. It also eliminated any significant difference in TGF1 secretion between the infected ob/ob and infected control mice. Conclusions: This study shows that leptin deficiency decreases but does not eliminate hepatic fibrosis produced by Schistosoma mansoni and carbon tetrachloride administration. The effect of leptin in potentiating fibrogenesis is most likely mediated by TGF1.

Alexandra K. Kiemer, Thomas Hartung, Christian Huber and Angelika M. Vollmar
Phyllanthus amarus has anti-inflammatory potential by inhibition of iNOS, COX-2, and cytokines via the NF-B pathway
Background/Aims: Phyllanthus amarus is a herbal medicine traditionally applied in the treatment of viral hepatitis. Aim of this study was to investigate potential anti-inflammatory properties of standardized P. amarus extracts concerning a potential influence of P. amarus on endotoxin-induced nitric oxide synthase (iNOS), cyclooxygenase (COX-2), and cytokine production in vivo and in vitro. Methods: Investigations were performed in rat Kupffer cells (KC), in RAW264.7 macrophages, in human whole blood, and in mice. Cells were stimulated with lipopolysaccharides (LPS) in the presence or absence of P. amarus extracts (hexane, EtOH/H2O), mice were treated with galactosamine/LPS as a model for acute toxic hepatitis. Nitrite was measured by Griess assay, prostaglandin E2 (PGE2) by radioimmunoassay, and cytokines by enzyme-linked immunosorbent assay. iNOS and COX-2 were determined by Western blot, activation of NF-B and AP-1 by EMSA. Results: P. amarus EtOH/H2O and hexane extracts showed an inhibition of LPS-induced production of NO and PGE2 in KC and in RAW264.7. The extracts also attenuated the LPS-induced secretion of tumor necrosis factor (TNF-) in RAW264.7 as well as in human whole blood. Both extracts reduced expression of iNOS and COX-2 and inhibited activation of NF-B, but not of AP-1. P. amarus inhibited induction of interleukin (IL)-1, IL-10, and interferon- in human whole blood and reduced TNF- production in vivo. Conclusions: This work shows that standardized extracts of P. amarus inhibit the induction of iNOS, COX-2, and TNF-. Therefore, we report for the first time an anti-inflammatory potential of this traditionally employed herbal medicine both in vitro and in vivo.

Liver Growth and Cancer

Nathalie Wong et al.
Positional mapping for amplified DNA sequences on 1q21-q22 in hepatocellular carcinoma indicates candidate genes over-expression
Background/Aims: Comparative genomic hybridization analysis on hepatocellular carcinoma (HCC) indicated frequent gains of 1q and an amplicon at 1q21-q22. Current cytogenetic evidences confer much importance on 1q21-q22, where a role in drug resistance, tumor metastasis and shorter patient survival had been implicated. Methods: Using positional mapping by interphase cytogenetics, we investigated the amplicon 1q21-q22 in five HCC cases. Three amplification maxima represented by yeast artificial chromosomes (YACs) 955E11, 876B11 and 945D5 that mapped to regions 1q21.1, 1q21.2 and 1q22, respectively, were indicated. We further investigated candidate genes expression in the mapped YACs by quantitative reverse-transcription-polymerase chain reaction. A panel of genes encoding protein transcripts involved in apoptosis, cell cycle progression, calcium binding and jumping translocation was studied. Results: Among ten HCC cases with the amplicon 1q21-q22 examined, we found a significant gene expression level of JTB, SHC1, CCT3 and COPA in the tumors than the paired adjacent non-malignant liver tissues (P0.04). Conclusions: Our interphase findings on 1q21-q22 pinpointed three affected loci between D1S305 and D1S2369. Up-regulation of candidate genes identified within these over-represented regions may represent targets in the progression of HCC and may carry prognostic significance.

Viral Hepatitis

Jean-Pierre Zarski et al.
Rate of natural disease progression in patients with chronic hepatitis C
Background/Aims: The interval at which liver biopsy should be repeated in untreated patients with chronic hepatitis C is not defined. We examined fibrosis change by METAVIR scoring in these patients in whom two or more liver biopsies were available.Methods: One hundred and eighty patients with histologically proven chronic hepatitis C were studied. Mean delay between biopsies was 3.67±2.69 years and 3.08±1.43 in the 16 patients having three biopsies. Univariate and multivariate analyses were performed to determine factors associated with liver fibrosis progression. Results: Median rate of fibrosis progression per year was 0.04 (0.00-0.55) to first biopsy, 0.00 (0.84-1.02) between first and second biopsy (NS), and 0.17 (0.00-1.50) between second and third biopsy (P<0.05). In multivariate analysis, only age at first biopsy >40 years (OR=5) (2-12) and alcohol consumption of 1-50 g per day (OR=4) (2-12) and more than 50 g per day (OR=8) (3-23) were associated with severe fibrosis. The number of patients who increased in fibrosis stage was significantly higher after 4 years (P<0.02). Conclusions: An interval of at least 4-5 years is needed between liver biopsies to measure change in patients with mild liver disease.

Norio Akuta et al.
The influence of hepatitis B virus genotype on the development of lamivudine resistance during long-term treatment
Background/Aims: Genotype-dependent development of lamivudine resistance in hepatitis B virus (HBV) has not been reported. Methods: We determined the cumulative rate of emergence of YMDD motif mutant in 213 Japanese patients with chronic hepatitis B infected with genotype A, B, or C and treated with lamivudine for more than 1 year. Results: The emergence rate of lamivudine resistance was independent of the genotype (A, B, and C). In contrast, the emergence rate was significantly higher in the Ba (`a' stands for Asia) subgroup of HBV than in Bj (`j' for Japan) subgroup (P<0.05). For genotype C (HBV/C), the emergence rate in hepatitis B e antigen (HBeAg)-positive was significantly higher than in HBV/C-HBeAg-negative (P<0.05), but the rate in HBV/B-HBeAg-positive was similar to HBV/B-HBeAg-negative. Only four patients with HBV/C developed severe acute exacerbation of hepatitis accompanied by emergence of YMDD mutant, but none of the patients with other genotypes developed such complication. Conclusions: Our results suggest that lamivudine resistance in HBV does not seem to depend on the genotype but rather on the subgroup of HBV/B. The results also suggest that lamivudine resistance according to HBeAg state might be different between HBV/B and HBV/C. Large-scale prospective studies of each genotype should be conducted in the future to confirm these findings.

Rong-Nan Chien, Chia-Hsien Lin and Yun-Fan Liaw
The effect of lamivudine therapy in hepatic decompensation during acute exacerbation of chronic hepatitis B
Background/Aims: Severe acute exacerbation (AE) of chronic hepatitis B (CHB) can lead to hepatic decompensation and death. The aim of this study was to investigate the effect of lamivudine therapy in hepatic decompensation during such AEs. Methods: In a 10-month period, a total of 60 consecutive AE patients with jaundice and prolonged prothrombin time over 3s were treated with lamivudine 150mg daily. As a historical control, another 31 CHB patients with AE resulting in hepatic decompensation hospitalized in an immediate past 6-month period were enrolled for comparison. Results: Patients in both groups were comparable in clinical and biochemical features. After a median treatment period of 6 weeks (range 1-48 weeks), all of the 25 patients with pretherapy bilirubin level <20mg/dl in the treatment group survived, while five (25%) of 20 patients in the control group died (P=0.013; odds ratios, 2.667; 95% confidence interval, 1.787-3.979). However, the mortality rate was similar in patients with pretherapy bilirubin level 20mg/dl in both groups. Conclusions: These results suggest that lamivudine may prevent fatality in CHB patients with hepatic decompensation if therapy starts early enough or before serum bilirubin level rise over 20mg/dl, but helps little if serum level already rised over that level.

Franck Le Guerhier et al.
Antiviral effect of adefovir in combination with a DNA vaccine in the duck hepatitis B virus infection model
Background/Aims: Combination of antiviral drugs with immunotherapeutic approaches may be a promising approach for the treatment of chronic hepatitis B. We used the duck HBV (DHBV) infection model to evaluate the efficacy of the combination of adefovir with DNA-immunization by comparison with the respective monotherapies. Methods: Pekin ducks chronically infected with DHBV received adefovir treatment alone or in association with intramuscular immunization with a plasmid (pCI-preS/S) expressing the DHBV large envelope protein. Ducks immunized with pCI-preS/S plasmid alone and two control groups receiving empty plasmid injections or no treatment were followed in parallel.Results: All animals treated with adefovir showed a marked drop in viremia titers during drug administration, followed by a rebound of viral replication after drug withdrawal. Eight weeks after the third DNA boost, the median of viremia within the duck group receiving the combination therapy tended to be lower compared to that of the other groups. In addition, our results suggest a trend to an additive effect of adefovir and DNA vaccine since a 51% decrease in DHBV DNA was observed in autopsy liver samples from combination therapy group, whereas pCI-preS/S or adefovir monotherapies decreased intrahepatic viral DNA by 38 and 14%, respectively. This effect was sustained since it was observed 12 weeks after the end of therapy. Conclusions: Our results suggest that combination of adefovir with DNA-vaccine may be able to induce a sustained antiviral effect in vivo.

Andreas Erhardt et al.
HFE mutations and chronic hepatitis C: H63D and C282Y heterozygosity are independent risk factors for liver fibrosis and cirrhosis
Background/Aims: The impact of heterozygous HFE mutations on the course of chronic hepatitis C and iron indices was studied. Methods: Ferritin, transferrin saturation (TS), serum iron, C282Y and H63D mutations were determined in 401 patients with chronic hepatitis C virus (HCV) infection and 295 healthy controls. Liver histologies were available in 217 and HCV genotypes in 339 patients. Results: Allele frequencies of the C282Y and H63D mutation did not differ between HCV patients and healthy controls (6.95 vs. 6.2%; 14.75 vs. 16.4%; n.s.). HFE heterozygous HCV patients had higher ferritin (349±37 vs. 193±15µg/l; P<0.0005), TS (38±2 vs. 32±1%; P<0.0005), serum iron (144±6 vs. 121±3µg/dl; P<0.0005), semiquantitative liver iron staining (0.26±0.07 vs. 0.09±0.03; P<0.006) and fibrosis scores (1.9±0.2 vs. 1.4±0.1; P<0.003) compared to HFE wildtypes. By multivariate regression analysis odds ratios for liver cirrhosis were 5.9 (confidence interval (CI) 1.6-22.6; P<0.009) for C282Y heterozygotes and 2.9 (CI 1.0-8.4; P<0.05) for H63D heterozygotes compared to HFE wildtypes. Considering all HFE heterozygous HCV patients, odds ratios of 3.6 (CI 1.4-9.3; P<0.009) for cirrhosis and 3.1 (CI 1.3-7.3; P<0.009) for fibrosis were calculated. Conclusions: C282Y or H63D heterozygosity is an independent risk factor for liver fibrosis and cirrhosis in HCV infected individuals. Screening for HFE mutations should be considered in HCV infection.

Patrick Soussan et al.
The expression of hepatitis B spliced protein (HBSP) encoded by a spliced hepatitis B virus RNA is associated with viral replication and liver fibrosis
Background/Aims: We have previously demonstrated the in vivo expression of a new spliced hepatitis B virus (HBV) protein (HBSP) encoded by a singly spliced pregenomic RNA. The present study was designed to evaluate the impact of HBSP expression on the clinical status and liver pathology of HBV infection. Methods: Sera from 125 chronic HBV carriers were tested for the presence of HBSP antibodies by an indirect enzyme-linked immunosorbent assay test. The severity of liver damage was evaluated using the Knodell score. Results: Anti-HBSP antibody prevalence in HBV chronic carriers was 46%. We highlighted the concomitant expression of HBSP protein and anti-HBSP antibody. An association between anti-HBSP antibody detection and serum markers of HBV replication was demonstrated. With respect to HBV-related liver disease, an association was only observed with the severity of fibrosis. Furthermore, an elevation of secreted tumor necrosis factor (TNF), but not of soluble TNF receptor 75, was observed in anti-HBSP-antibody-positive patients. Multivariate analysis showed that anti-HBSP antibody detection was independently associated with viral replication, severity of fibrosis and elevated TNF secretion. Conclusions: Our data suggest the hypothesis that HBSP might play a role in the natural history of HBV infection and may be involved in the pathogenesis and/or persistence of HBV infection.

Anthony J. Freeman et al.
The presence of an intrahepatic cytotoxic T lymphocyte response is associated with low viral load in patients with chronic hepatitis C virus infection
Background/Aims: The role of cytotoxic T lymphocytes (CTL) in limiting viral replication and producing hepatocellular injury in patients with chronic hepatitis C virus (HCV) infection is controversial. Methods: Intrahepatic and peripheral blood HCV-specific CTL activity against the entire HCV polyprotein was assessed in 26 patients. CTL responses were assessed after effector lymphocytes were re-stimulated for 6 days in vitro using HCV-vaccinia virus-infected autologous cells expressing HCV antigens. Serum and hepatic viral loads were measured and immunohistochemistry for CD3 and CD8 was performed to localise and enumerate effector cells in liver. Results: A positive CTL response was detected in 39/52 (75%) of assays conducted with intrahepatic mononuclear cells and 21/52 (40%) of peripheral blood assays (P<0.001). The presence of an intrahepatic CTL response was associated with low hepatic viral load (P=0.004). Hepatic lobular infiltration by CD8+T cells correlated weakly with serum alanine aminotransferase levels (r=0.42, P=0.04) and no relationship was demonstrated between CTL activity and histological evidence of liver damage. Conclusions: HCV-specific CTL activity is found more commonly in liver than in blood. An inverse relationship between CTL responses and viral load supports the hypothesis that HCV-specific CTL limit viral replication in patients with chronic HCV infection.

BRITISH MEDICAL JOURNAL

NEW ENGLAND JOURNAL

Volume 348 March 6, 2003 Number 10

A Randomized Trial of Aspirin to Prevent Colorectal Adenomas in Patients with Previous Colorectal Cancer
Robert S. Sandler, M.D., M.P.H., Susan Halabi, Ph.D., John A. Baron, M.D., Susan Budinger, B.S., Electra Paskett, Ph.D., Roger Keresztes, M.D., Nicholas Petrelli, M.D., J. Marc Pipas, M.D., Daniel D. Karp, M.D., Charles L. Loprinzi, M.D., Gideon Steinbach, M.D., Ph.D., and Richard Schilsky, M.D.

Background Experimental studies in animals and observational studies in humans suggest that regular aspirin use may decrease the risk of colorectal adenomas, the precursors to most colorectal cancers.

Methods We conducted a randomized, double-blind trial to determine the effect of aspirin on the incidence of colorectal adenomas. We randomly assigned 635 patients with previous colorectal cancer to receive either 325 mg of aspirin per day or placebo. We determined the proportion of patients with adenomas, the number of recurrent adenomas, and the time to the development of adenoma between randomization and subsequent colonoscopic examinations. Relative risks were adjusted for age, sex, cancer stage, the number of colonoscopic examinations, and the time to a first colonoscopy. The study was terminated early by an independent data and safety monitoring board when statistically significant results were reported during a planned interim analysis.

Results A total of 517 randomized patients had at least one colonoscopic examination a median of 12.8 months after randomization. One or more adenomas were found in 17 percent of patients in the aspirin group and 27 percent of patients in the placebo group (P=0.004). The mean (±SD) number of adenomas was lower in the aspirin group than the placebo group (0.30±0.87 vs. 0.49±0.99, P=0.003 by the Wilcoxon test). The adjusted relative risk of any recurrent adenoma in the aspirin group, as compared with the placebo group, was 0.65 (95 percent confidence interval, 0.46 to 0.91). The time to the detection of a first adenoma was longer in the aspirin group than in the placebo group (hazard ratio for the detection of a new polyp, 0.64; 95 percent confidence interval, 0.43 to 0.94; P=0.022).

Conclusions Daily use of aspirin is associated with a significant reduction in the incidence of colorectal adenomas in patients with previous colorectal cancer.

Source Information

From the Departments of Medicine and Epidemiology, University of North Carolina, Chapel Hill (R.S.S.); Cancer and Leukemia Group B Statistical Center (S.H., S.B.) and the Department of Biostatistics and Bioinformatics (S.H.), Duke University Medical Center, Durham, N.C.; the Departments of Medicine and Community and Family Medicine (J.A.B.), Dartmouth Medical School (J.M.P.), Hanover, N.H.; Wake Forest University School of Medicine, Winston-Salem, N.C. (E.P.); Weill Medical College of Cornell University, New York (R.K.); Roswell Park Cancer Institute, Buffalo, N.Y. (N.P.); Eastern Cooperative Oncology Group (D.D.K.), M.D. Anderson Cancer Center (G.S.), Houston; North Central Cancer Treatment Group, Mayo Clinic, Rochester, Minn. (C.L.L.); and Cancer and Leukemia Group B, Central Office of the Chairman, Chicago (R.S.).

Address reprint requests to Dr. Sandler at CB# 7555, 4111 Bioinformatics Bldg., University of North Carolina, Chapel Hill, NC 27599-7555, or at rsandler@med.unc.edu.

A Randomized Trial of Aspirin to Prevent Colorectal Adenomas
John A. Baron, M.D., Bernard F. Cole, Ph.D., Robert S. Sandler, M.D., Robert W. Haile, Dr.Ph., Dennis Ahnen, M.D., Robert Bresalier, M.D., Gail McKeown-Eyssen, Ph.D., Robert W. Summers, M.D., Richard Rothstein, M.D., Carol A. Burke, M.D., Dale C. Snover, M.D., Timothy R. Church, Ph.D., John I. Allen, M.D., Michael Beach, M.D., Ph.D., Gerald J. Beck, Ph.D., John H. Bond, M.D., Tim Byers, M.D., E. Robert Greenberg, M.D., Jack S. Mandel, Ph.D., Norman Marcon, M.D., Leila A. Mott, M.S., Loretta Pearson, M.Phil., Fred Saibil, M.D., and Rosalind U. van Stolk, M.D.

Background Laboratory and epidemiologic data suggest that aspirin has an antineoplastic effect in the large bowel.

Methods We performed a randomized, double-blind trial of aspirin as a chemopreventive agent against colorectal adenomas. We randomly assigned 1121 patients with a recent history of histologically documented adenomas to receive placebo (372 patients), 81 mg of aspirin (377 patients), or 325 mg of aspirin (372 patients) daily. According to the protocol, follow-up colonoscopy was to be performed approximately three years after the qualifying endoscopy. We compared the groups with respect to the risk of one or more neoplasms (adenomas or colorectal cancer) at least one year after randomization using generalized linear models to compute risk ratios and 95 percent confidence intervals.

Results Reported adherence to study medications and avoidance of nonsteroidal antiinflammatory drugs were excellent. Follow-up colonoscopy was performed at least one year after randomization in 1084 patients (97 percent). The incidence of one or more adenomas was 47 percent in the placebo group, 38 percent in the group given 81 mg of aspirin per day, and 45 percent in the group given 325 mg of aspirin per day (global P=0.04). Unadjusted relative risks of any adenoma (as compared with the placebo group) were 0.81 in the 81-mg group (95 percent confidence interval, 0.69 to 0.96) and 0.96 in the 325-mg group (95 percent confidence interval, 0.81 to 1.13). For advanced neoplasms (adenomas measuring at least 1 cm in diameter or with tubulovillous or villous features, severe dysplasia, or invasive cancer), the respective relative risks were 0.59 (95 percent confidence interval, 0.38 to 0.92) and 0.83 (95 percent confidence interval, 0.55 to 1.23).

Conclusions Low-dose aspirin has a moderate chemopreventive effect on adenomas in the large bowel.

Source Information

From the Norris Cotton Cancer Center, Dartmouth­Hitchcock Medical Center, Lebanon, N.H. (J.A.B., B.F.C., E.R.G.); Dartmouth Medical School, Hanover, N.H. (J.A.B., R.R., M.B., E.R.G., L.A.M., L.P.); the University of North Carolina School of Medicine, Chapel Hill (R.S.S.); the University of Southern California School of Medicine, Los Angeles (R.W.H.); the Veterans Affairs Medical Center, Denver (D.A.); the University of Colorado School of Medicine, Denver (D.A., T.B.); Henry Ford Health Sciences Center, Detroit (R.B.); the University of Toronto, Toronto (G.M.-E., N.M., F.S.); the Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City (R.W.S.); the Cleveland Clinic Foundation, Cleveland (C.A.B., R.U.S., G.J.B.); Fairview Southdale Hospital, Minneapolis (D.C.S.); the University of Minnesota, Minneapolis (T.R.C.); the University of Minnesota School of Medicine, Minneapolis ( J.I.A., J.H.B.); Minnesota Gastroenterology, Minneapolis (J.I.A.); the Veterans Affairs Medical Center, Minneapolis ( J.H.B.); and the Rollins School of Public Health, Emory University, Atlanta (J.S.M.).

Address reprint requests to Dr. Baron at Biostatistics and Epidemiology, Evergreen Center, 46 Centerra Pkwy., Lebanon, NH 03766, or at john.a.baron@dartmouth.edu.

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