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HEPATOLOGY

Table of Contents for July 2003 · Volume 38 · Number 1

Viral Hepatitis

Liver transplantation with hepatitis C virus-infected graft: Interaction between donor and recipient viral strains (*Human Study*)
Xiaofeng Fan, Dorothy M. Lang, Yanjuan Xu, Andre C. Lyra, Karina Yusim, James E. Everhart, Bette T. M. Korber, Alan S. Perelson, Adrian M. Di Bisceglie
Superinfection of different viral strains within a single host provides an opportunity for studying host-virus and virus-virus interactions, including viral interference and genetic recombination, which cannot be studied in infections with single viral strains. Hepatitis C virus (HCV) is a positive single-strand RNA virus that establishes persistent infection in as many as 85% of infected individuals. However, there are few reports regarding coinfection or superinfection of HCV. Because of the lack of tissue culture systems and small animal models supporting efficient HCV replication, we explored these issues in the setting of liver transplantation where both recipient and donor were infected with different HCV strains and therefore represent a distinct model for HCV superinfection. Serial serum samples collected at multiple time points were obtained from 6 HCV-positive liver donor/recipient pairs from the National Institute of Diabetes and Digestive and Kidney Diseases liver transplantation database. At each time point, HCV genotype was determined by both restriction fragment length polymorphism analysis and phylogenetic analysis. Furthermore, we selectively sequenced 3 full-length HCV isolates at the earliest time points after liver transplantation, including both 5´ and 3´ ends. Detailed genetic analyses showed that only one strain of HCV could be identified at each time point in all 6 cases. Recipient HCV strains took over in 3 cases, whereas donor HCV strains dominated after liver transplantation in the remaining 3 cases. In conclusion, in all 6 cases studied, there was no genetic recombination detected among HCV quasispecies or between donor and recipient HCV strains. (HEPATOLOGY 2003;38:25-33.)

A model to predict severe HCV-related disease following liver transplantation (*Human Study*)
Marina Berenguer, Jeffrey Crippin, Robert Gish, Nathan Bass, Alan Bostrom, George Netto, Judy Alonzo, Richard Garcia-Kennedy, Jose-Miguel Rayón, Teresa L. Wright
Post-transplantation recurrence is increasing in patients with HCV. Early antiviral therapy may be of benefit in this setting. Thus, accurate and early prediction of progression may help select candidates for treatment. We developed a model based on pre- and/or early post-transplantation variables, which may predict progression to severe disease. Clinical and histologic outcomes were assessed in 554 liver recipients. A total of 1,353 biopsy specimens obtained after 1 year (median of 2 biopsies per patient; range, 1-8) were scored. Two outcome measures were used: cumulative probability of developing severe disease (fibrosis 3 and 4) within 5 years and actual progression to severe disease in 2 years. We used Cox proportional hazard survival analysis for the whole cohort. Predictors analyzed included HCV genotype and recipient, donor, and transplant-related variables. The cumulative risk of progressing to fibrosis 3 and 4 was significantly greater in patients transplanted recently (P < .001) and was present in all centers. Factors increasing this risk were genotype, induction with mycophenolate, donor age, short course of azathioprine, and prednisone (<12 months). To create a model of prediction, 285 patients with 2-year follow-up were used to create a logistic regression analysis. The estimated probability of being at high risk for severe disease was calculated from a formula that included donor age and recipient therapy as critical variables. In conclusion, we have developed a model that uses early post-transplantation variables to predict severe HCV recurrence. Accuracy of the model is not perfect (c-statistic 0.80), probably reflecting the complexity of HCV in the liver transplant setting. (HEPATOLOGY 2003;38:34-41.)

Moderate alcohol consumption increases oxidative stress in patients with chronic hepatitis C (*Human Study*)
Cristina Rigamonti, Elisa Mottaran, Emanuela Reale, Roberta Rolla, Valentina Cipriani, Francesca Capelli, Renzo Boldorini, Matteo Vidali, Massimo Sartori, Emanuele Albano
The mechanisms by which alcohol consumption worsens the evolution of chronic hepatitis C (CHC) are poorly understood. We have investigated the possible interaction between hepatitis C virus (HCV) and ethanol in promoting oxidative stress. Circulating IgG against human serum albumin (HSA) adducted with malondialdehyde (MDA-HSA), 4-hydroxynonenal (HNE-HSA), or arachidonic acid hydroperoxide (AAHP-HSA) and against oxidized cardiolipin (Ox-CL) were evaluated as markers of oxidative stress in 145 CHC patients with different alcohol consumption, 20 HCV-free heavy drinkers (HD) without liver disease, and 50 healthy controls. Anti-MDA IgG was increased in CHC patients irrespective of alcohol intake as well as in the HD group. CHC patients with moderate alcohol intake (<50 g ethanol/d), but not HD, also had significantly higher values of anti-AAHP-HSA, anti-HNE-HSA, and anti-Ox-CL IgG (P < .05) than controls. A further elevation (P < .001) of these antibodies was evident in CHC patients with heavy alcohol intake (>50 g ethanol/d). Anti-AAHP and anti-Ox-CL IgG above the 95th percentile in the controls were observed in 24% to 26% of moderate and 58% to 63% of heavy drinkers but only in 6% to 9% of the abstainers. The risk of developing oxidative stress during CHC was increased 3-fold by moderate and 13- to 24-fold by heavy alcohol consumption. Heavy drinking CHC patients had significantly more piecemeal necrosis and fibrosis than abstainers. Diffuse piecemeal necrosis was 4-fold more frequent among alcohol-consuming patients with lipid peroxidation-related antibodies than among those without these antibodies. In conclusion, even moderate alcohol consumption promotes oxidative stress in CHC patients, suggesting a role for oxidative injury in the worsening of CHC evolution by alcohol. (HEPATOLOGY 2003;38:42-49.)

Hepatitis C virus infection and incident type 2 diabetes (*Human Study*)
Shruti H. Mehta, Frederick L. Brancati, Steffanie A. Strathdee, James S. Pankow, Dale Netski, Josef Coresh, Moyses Szklo, David L. Thomas
Although hepatitis C virus (HCV) infection is more common among adults with type 2 diabetes, it is uncertain whether HCV precedes the development of diabetes. Thus, we performed a prospective (case-cohort) analysis to examine if persons who acquired type 2 diabetes were more likely to have had antecedent HCV infection when enrolled in a community-based cohort of men and women between the ages of 44 and 65 in the United States (Atherosclerosis Risk in Communities Study [ARIC]). Among 1,084 adults free of diabetes at baseline, 548 developed diabetes over 9 years of follow-up evaluation. Incident cases of diabetes were identified by using fasting glucose and medical history and HCV antibodies were assessed at baseline. A priori, persons were categorized as low-risk or high-risk for diabetes based on their age and body mass index, factors that appeared to modify the type 2 diabetes-HCV infection incidence estimates. The overall prevalence of HCV in this population was 0.8%. Among those at high risk for diabetes, persons with HCV infection were more than 11 times as likely as those without HCV infection to develop diabetes (relative hazard, 11.58; 95% confidence interval, 1.39-96.6). Among those at low risk, no increased incidence of diabetes was detected among HCV-infected persons (relative hazard, 0.48; 95% confidence interval, 0.05-4.40). In conclusion, pre-existing HCV infection may increase the risk for type 2 diabetes in persons with recognized diabetes risk factors. Additional larger prospective evaluations are needed to confirm these preliminary findings. (HEPATOLOGY 2003;38:50-56.)

Alcohol potentiates hepatitis C virus replicon expression
Ting Zhang, Yuan Li, Jian-Ping Lai, Steven D. Douglas, David S. Metzger, Charles P. O'Brien, Wen-Zhe Ho
Although hepatitis C virus (HCV) infection is more common among adults with type 2 diabetes, it is uncertain whether HCV precedes the development of diabetes. Thus, we performed a prospective (case-cohort) analysis to examine if persons who acquired type 2 diabetes were more likely to have had antecedent HCV infection when enrolled in a community-based cohort of men and women between the ages of 44 and 65 in the United States (Atherosclerosis Risk in Communities Study [ARIC]). Among 1,084 adults free of diabetes at baseline, 548 developed diabetes over 9 years of follow-up evaluation. Incident cases of diabetes were identified by using fasting glucose and medical history and HCV antibodies were assessed at baseline. A priori, persons were categorized as low-risk or high-risk for diabetes based on their age and body mass index, factors that appeared to modify the type 2 diabetes-HCV infection incidence estimates. The overall prevalence of HCV in this population was 0.8%. Among those at high risk for diabetes, persons with HCV infection were more than 11 times as likely as those without HCV infection to develop diabetes (relative hazard, 11.58; 95% confidence interval, 1.39-96.6). Among those at low risk, no increased incidence of diabetes was detected among HCV-infected persons (relative hazard, 0.48; 95% confidence interval, 0.05-4.40). In conclusion, pre-existing HCV infection may increase the risk for type 2 diabetes in persons with recognized diabetes risk factors. Additional larger prospective evaluations are needed to confirm these preliminary findings. (HEPATOLOGY 2003;38:50-56.)

Maintenance therapy with ribavirin in patients with chronic hepatitis C who fail to respond to combination therapy with interferon alfa and ribavirin (*Human Study*)
Jay H. Hoofnagle, Marc G. Ghany, David E. Kleiner, Edward Doo, Theo Heller, Kittichai Promrat, Janus Ong, Farooq Khokhar, Alejandro Soza, David Herion, Yoon Park, James E. Everhart, T. Jake Liang
To assess the efficacy and safety of maintenance therapy with ribavirin alone in chronic hepatitis C, 108 patients were treated with the combination of interferon alfa and ribavirin for 24 weeks; those who failed to have a virologic response were offered enrollment in a randomized, double-blind, controlled trial of ribavirin (1,000-1,200 mg daily) versus placebo for the subsequent 48 weeks. Patients were monitored at regular intervals with symptom questionnaires, serum aminotransferase levels, hepatitis C virus (HCV) RNA levels, and complete blood counts and underwent liver biopsy at the completion of therapy. Among 108 patients, 50 were still HCV RNA positive after 24 weeks of treatment, of whom 34 agreed to be randomized to continue either ribavirin monotherapy or placebo. Among 17 patients who received placebo, there was no overall improvement in symptoms, serum alanine aminotransferase (ALT) levels, HCV RNA levels, or hepatic histology. Among the 17 patients who received ribavirin, serum ALT levels and necroinflammatory features of liver histology were improved, whereas symptoms, HCV RNA levels, and hepatic fibrosis scores were not changed significantly from baseline. Responses to ribavirin seemed to be categorical, such that 8 patients (47%) had definite improvement in liver histology. Patients with improved histology had improvements in serum ALT levels both on combination therapy and after switching to ribavirin monotherapy. In conclusion, continuation of ribavirin monotherapy may maintain serum biochemical improvements that occur during interferon-ribavirin combination therapy in some patients and that these improvements are often associated with decreases in necroinflammatory changes in the liver. Whether these improvements will ultimately result in prevention of progression of hepatitis C requires further study. (HEPATOLOGY 2003;38:66-74.)

Effect of treatment with peginterferon or interferon alfa-2b and ribavirin on steatosis in patients infected with hepatitis C (*Human Study*)
Thierry Poynard, Vlad Ratziu, John McHutchison, Michael Manns, Zachary Goodman, Stefan Zeuzem, Zobair Younossi, Janice Albrecht
It has been suggested that hepatitis C virus (HCV) and especially genotype 3 is associated with steatosis. We assess the effect of treatment with peginterferon or interferon alfa-2b and ribavirin on steatosis. We analyzed 1,428 naïve patients included in a randomized trial. A single pathologist scored steatosis at baseline and 24 weeks after the treatment. At baseline, steatosis was present in 935 of 1,428 patients (65%), including 175 (83%) of 210 patients with genotype 3 versus 760 (62%) of 1,218 with other genotypes (P < .001). The variables associated with steatosis in logistic regression were genotype 3 (P < .001), triglycerides greater than 1.7 mmol/L (P < .001), body mass index greater than 27 (P < .04), age greater than 40 years (P < .001), and septal fibrosis (P = .007). In genotype 3-infected patients, steatosis was associated with high viral load and with lower serum cholesterol. Steatosis was associated with lower sustained response rate, even after taking into account other factors (P < .001). Among virologic responders, steatosis was much improved in genotype 3, improvement of at least 1 grade in 77%, and disappearance in 46% compared with other genotypes, 46% and 29%, respectively (P < .001 both comparisons). In genotype 3 responders, the baseline low serum cholesterol was corrected by treatment (P < .001). Steatosis was associated with HCV genotype 3, triglycerides, high body mass index, age, fibrosis stage, and lower virologic response to treatment. In conclusion, sustained disappearance of the virus is associated with reduction of steatosis in genotype 3 as well as a correction of baseline low serum cholesterol. (HEPATOLOGY 2003;38:75-85.)

HBV DNA persistence 10 years after liver transplantation despite successful anti-HBS passive immunoprophylaxis (*Human Study*)
Bruno Roche, Cyrille Feray, Michele Gigou, Anne Marie Roque-Afonso, Jean Louis Arulnaden, Valerie Delvart, Elisabeth Dussaix, Catherine Guettier, Henri Bismuth, Didier Samuel
Long-term immunoprophylaxis with hepatitis B immune globulin (HBIG) is widely accepted for the prevention of recurrent hepatitis B virus (HBV) infection after liver transplantation in HBV-infected patients without viral replication. We report long-term results of HBIG administration in 284 hepatitis B surface antigen (HBsAg)-positive transplant patients. In protocol 1, 259 patients were given HBIG with the goal of maintaining the anti-HBs antibody (Ab) titer over 100 IU/L. After December 1993, 25 HBV DNA-positive patients received HBIG, with a target anti-HBs Ab titer over 500 IU/L, combined with posttransplantation antiviral therapy (protocol 2). At 10 years, 44 patients without recurrence were tested for the presence of HBV DNA in serum using real-time polymerase chain reaction (PCR); 28 were also tested in liver and peripheral blood mononuclear cells (PBMC). The overall 5- and 10-year posttransplantation actuarial rates of HBV recurrence were 24.2% and 25.4%, respectively. The 5-year recurrence rate in protocol 2 patients was 11.8%. On multivariate analysis, predictors of lower HBV recurrence risk were absence of serum HBV DNA before transplantation (P < .0001), acute liver disease (P = .0037), HDV superinfection (P = .012), and protocol 2 therapy (P < .0001). Low-level HBV DNA was detected by PCR in 45.4% of patients without HBV recurrence at 10 years. Overall actuarial 10-year survival was 74.4%. In conclusion, we confirm the efficacy of long-term HBIG immunoprophylaxis. Combination prophylaxis with HBIG and antiviral therapy is effective in patients with viral replication. Although there were only a few cases of HBV recurrence after 5 years, HBV DNA remained present in 45% of patients at 10 years. (HEPATOLOGY 2003;38:86-95.)

Week 48 resistance surveillance in two phase 3 clinical studies of adefovir dipivoxil for chronic hepatitis B (*Human Study*)
Christopher E. Westland, Huiling Yang, William E. Delaney, IV, Craig S. Gibbs, Michael D. Miller, Michael Wulfsohn, John Fry, Carol L. Brosgart, Shelly Xiong, 437 and 438 Study Teams
Seven hundred nucleoside treatment-naive patients were enrolled in two phase 3 trials of adefovir dipivoxil (ADV) for the treatment of chronic hepatitis B. To monitor for the emergence of potential adefovir resistance mutations over the first 48 weeks, all intent-to-treat patients (467 ADV-treated and 228 placebo patients) were included in a prospectively defined, treatment-blinded, virology substudy. The study protocol mandated genotypic analysis for all patients with detectable hepatitis B virus (HBV) DNA by Roche Amplicor polymerase chain reaction (PCR) at baseline and week 48, and in vitro phenotypic analyses for patients with conserved site substitutions in HBV polymerase or 1.0 log10 or greater increase in HBV DNA from nadir. Paired sequences of the entire HBV reverse transcriptase were obtained for 271 ADV-treated and 227 placebo patients by using a sequencing method that detects down to 30% of minor species present within mixtures. Four substitutions (rtS119A, rtH133L, rtV214A, and rtH234Q) developed once each at conserved sites in HBV polymerase in 4 ADV-treated patients. Seven conserved site substitutions developed in 6 placebo patients. HBV mutants encoding the 4 substitutions that emerged in ADV-treated patients remained fully susceptible to adefovir in vitro. Furthermore, these 4 ADV-treated patients had HBV-DNA reductions of 3.3 to 5.9 log10 copies/mL by week 48 with no rebound. All other substitutions occurred at very low frequencies (<1.6%) at polymorphic sites and were not associated with HBV-DNA increases in patients or adefovir resistance in vitro. In conclusion, no adefovir resistance mutations were identified in a large group of chronic hepatitis B patients treated with ADV for 48 weeks. (HEPATOLOGY 2003;38:96-103.)

Liver Biology and Pathobiology

Salivary gland progenitor cells induced by duct ligation differentiate into hepatic and pancreatic lineages
Kenji Okumura, Kimitoshi Nakamura, Yuichiro Hisatomi, Koji Nagano, Yasuhiko Tanaka, Kunihiko Terada, Toshihiro Sugiyama, Kazuhiro Umeyama, Kozo Matsumoto, Tetsuro Yamamoto, Fumio Endo
Tissue damage can be assessed based on regenerative responses, including progenitor cell proliferation. In the salivary gland, tissue damage induced by ligation of main ducts leads to the disappearance of acinar cells and to marked proliferation of ductal cells. Reopening of the ducts leads to repopulation of acinar cells within 1 to 2 weeks, which suggests activation of tissue progenitor cells in a damaged state. Because submandibular glands derive from the endoderm and ectoderm, we investigated the possibility of the presence of endodermal progenitor cells. We cultured cells obtained from the ligated salivary gland and identified colonies of epithelium-like cells. We singled out and purified the cells by limited dilution, and one of the cells designated SGP-1 was used for further experiments. The SGP-1 expresses both 61 integrin and cytoplasmic laminin. The hematopoietic stem cell marker CD34 and hepatic oval cell markers such as albumin, -fetoprotein (AFP), and cytokeratin 19 are all negative. However, when SGP-1 cells were transplanted into the liver via the portal vein, these cells were integrated into hepatic trabecula and produced albumin. When SGP-1 cells formed clusters on type I collagen-coated dishes, they differentiated into endodermal lineage and 2 major types of clusters appeared: one contained cells positive for AFP and/or albumin (hepatic cluster) and the other positive for glucagon and/or insulin (pancreatic cluster). On laminin-coated dishes, SGP-1 selectively differentiated into hepatic-type cells. In conclusion, the multipotent progenitor cells isolated from the rat salivary gland have characteristics of tissue stem cells and can differentiate into cells of endodermal lineages. (HEPATOLOGY 2003;38:104-113.)

A highly efficient, stable, and rapid approach for ex vivo human liver gene therapy via a FLAP lentiviral vector (*Human Study*)
Carlo Giannini, Serban Morosan, J. Guilherme Tralhao, Jacques Emmanuel Guidotti, Serena Battaglia, Karine Mollier, Laurent Hannoun, Dina Kremsdorf, Helene Gilgenkrantz, Pierre Charneau
Allogenic hepatocyte transplantation or autologous transplantation of genetically modified hepatocytes has been used successfully to correct congenital or acquired liver diseases and can be considered as an alternative to orthotopic liver transplantation. However, hepatocytes are neither easily maintained in culture nor efficiently genetically modified and are very sensitive to dissociation before their reimplantation into the recipient. These difficulties have greatly limited the use of an ex vivo approach in clinical trials. In the present study, we have shown that primary human and rat hepatocytes can be efficiently transduced with a FLAP lentiviral vector without the need for plating and culture. Efficient transduction of nonadherent primary hepatocytes was achieved with a short period of contact with vector particles, without modifying hepatocyte viability, and using reduced amounts of vector. We also showed that the presence of the DNA FLAP in the vector construct was essential to reach high levels of transduction. Moreover, transplanted into uPA/SCID mouse liver, lentivirally transduced primary human hepatocytes extensively repopulated their liver and maintained a differentiated and functional phenotype as assessed by the stable detection of human albumin and antitrypsin in the serum of the animals for months. In conclusion, the use of FLAP lentiviral vectors allows, in a short period of time, a high transduction efficiency of human functional and reimplantable hepatocytes. This work therefore opens new perspectives for the development of human clinical trials based on liver-directed ex vivo gene therapy. (HEPATOLOGY 2003;38:114-122.)

Central role of PPAR-dependent hepatic lipid turnover in dietary steatohepatitis in mice
Emilia Ip, Geoffrey C. Farrell, Graham Robertson, Pauline Hall, Richard Kirsch, Isabelle Leclercq
We have proposed that steatohepatitis results from reactive oxygen species (ROS) acting on accumulated fatty acids to form proinflammatory lipoperoxides. Cytochrome P450 4a (Cyp4a) and Cyp2e1 are potential hepatic sources of ROS. We tested the hypothesis that increasing Cyp4a through activation of peroxisome proliferator-activated receptor (PPAR) should aggravate steatohepatitis produced by feeding a methionine and choline deficient (MCD) diet. Conversely, we assessed dietary steatohepatitis in PPAR/ mice that cannot up-regulate Cyp4a. Male wild type (wt) or PPAR/ mice (C57BL6 background) were fed the MCD diet with or without Wy-14,643 (0.1% wt/wt), a potent PPAR agonist. Controls were fed the same diet supplemented with methionine and choline. After 5 weeks, wt mice fed the MCD diet developed moderate steatohepatitis and alanine aminotransferase (ALT) levels were increased. Wy-14,643 prevented rather than increased liver injury; ALT levels were only mildly elevated whereas steatohepatitis was absent. Wy-14,643 up-regulated mRNA for liver fatty acid binding protein and peroxisomal -oxidation enzymes (acyl-CoA oxidase, bifunctional enzyme, and ketothiolase), thereby reducing hepatic triglycerides and preventing steatosis. In wt mice, dietary feeding up-regulated Cyp4a14 mRNA 2.7-fold and increased hepatic lipoperoxides compared with controls. Wy-14,643 prevented hepatic lipoperoxides from accumulating despite an 18-fold increase in both Cyp4a10 and Cyp4a14 mRNA. PPAR/ mice fed the MCD diet developed more severe steatohepatitis than wt mice, and were unaffected by Wy-14,643. In conclusion, PPAR activation both increases Cyp4a expression and enhances hepatic lipid turnover; the latter effect removes fatty acids as substrate for lipid peroxidation and is sufficiently powerful to prevent the development of dietary steatohepatitis. HEPATOLOGY 2003;38:123-132.)

Inhibition of microsomal triglyceride transfer protein: Another mechanism for drug-induced steatosis in mice
Philippe Lettéron, Angela Sutton, Abdellah Mansouri, Bernard Fromenty, Dominique Pessayre
Although many steatogenic drugs inhibit mitochondrial fatty acid -oxidation, limited information is available on possible effects on hepatic lipoprotein secretion. In the endoplasmic reticulum (ER) lumen, microsomal triglyceride transfer protein (MTP) lipidates apolipoprotein B (Apo B), to form triglyceride (TG)-rich very low density lipoprotein (VLDL) particles, which follow vesicular flow to the plasma membrane to be secreted, whereas incompletely lipidated Apo B particles are partly degraded. We studied hepatic MTP activity, the lipoproteins present in the ER lumen, and hepatic lipoprotein secretion 4 hours after administration of a single dose of amineptine (1 mmol/kg), amiodarone (1 mmol/kg), doxycycline (0.25 mmol/kg), tetracycline (0.25 mmol/kg), tianeptine (0.5 mmol/kg), or pirprofen (2 mmol/kg) in mice. These various doses have been shown previously to markedly inhibit fatty acid oxidation after a single dose, and to trigger steatosis either after repeated doses (doxycycline) or a single dose (other compounds) in mice. In the present study, amineptine, amiodarone, pirprofen, tetracycline, and tianeptine, but not doxycycline, inhibited MTP activity in vitro, decreased ex vivo MTP activity in the hepatic homogenate of treated mice, decreased TG in the luminal VLDL fraction of hepatic microsomes of treated mice, and decreased in vivo hepatic lipoprotein secretion (TG and Apo B). In conclusion, several steatogenic drugs inhibit not only mitochondrial -oxidation, as previously shown, but also MTP activity, Apo B lipidation into TG-rich VLDL particles, and hepatic lipoprotein secretion. Drugs with these dual effects may be more steatogenic than drugs acting only on -oxidation or only MTP. (HEPATOLOGY 2003;38:133-140.)

Chronic alcohol consumption increases the sensitivity of rat liver mitochondrial respiration to inhibition by nitric oxide
Aparna Venkatraman, Sruti Shiva, Ashley J. Davis, Shannon M. Bailey, Paul S. Brookes, Victor M. Darley-Usmar
Chronic alcohol consumption is a well-known risk factor for hepatic injury, and mitochondrial damage plays a significant role in this process. Nitric oxide (NO) is an important modulator of mitochondrial function and is known to inhibit mitochondrial respiration. However, the impact of chronic alcohol consumption on NO-dependent control of liver mitochondrial function is unknown. This study examines the effect of alcohol exposure on liver mitochondria in a rat model and explores the interaction of NO and mitochondrial respiration in this context. Mitochondria were isolated from the liver of both control and ethanol-fed rats after 5 to 6 weeks of alcohol consumption. Mitochondria isolated from ethanol-treated rats showed a significant decrease in state 3 respiration and respiratory control ratio that was accompanied by an increased sensitivity to NO-dependent inhibition of respiration. In conclusion, we show that chronic alcohol consumption leads to increased sensitivity to the inhibition of respiration by NO. We propose that this results in a greater vulnerability to hypoxia and the development of alcohol-induced hepatotoxicity. (HEPATOLOGY 2003;38:141-147.)

Expression of Wilms' tumor suppressor in the liver with cirrhosis: Relation to hepatocyte nuclear factor 4 and hepatocellular function (*Human Study*)
Carmen Berasain, José-Ignacio Herrero, Elena R. García-Trevijano, Matías A. Avila, Juan Ignacio Esteban, José M. Mato, Jesús Prieto
The Wilms' tumor suppressor WT1 is a transcriptional regulator present in the fetal but not in the mature liver. Its expression and functional role in liver diseases remains unexplored. In this study, we analyzed WT1 expression by reverse-transcription polymerase chain reaction (RT-PCR) and by immunohistochemistry in normal and diseased livers. In addition, we performed in vitro studies in isolated rat hepatocytes to investigate WT1 regulation and function. We detected WT1 messenger RNA (mRNA) in 18% of normal livers, 17% of chronic hepatitis with minimal fibrosis, 49% of chronic hepatitis with bridging fibrosis, and 71% of cirrhotic livers. In cirrhosis, WT1 immunoreactivity was localized to the nucleus of hepatocytes. WT1 mRNA abundance correlated inversely with prothrombin time (P = .04) and directly with serum bilirubin (P = .002) and with the MELD score (P = .001) of disease severity. In rats, WT1 expression was present in fetal hepatocytes and in the cirrhotic liver but not in normal hepatic tissue. In vitro studies showed that isolated primary hepatocytes express WT1 when stimulated with transforming growth factor (TGF-) or when the cells undergo dedifferentiation in culture. Moreover, we found that WT1 down-regulates hepatocyte nuclear factor 4 (HNF-4), a factor that is essential to maintain liver function and metabolic regulation in the mature organ. Hepatic expression of HNF-4 was impaired in advanced human cirrhosis and negatively correlated with WT1 mRNA levels (P = .001). In conclusion, we show that WT1 is induced by TGF- and down-regulates HNF-4 in liver cells. WT1 is reexpressed in the cirrhotic liver in relation to disease progression and may play a role in the development of hepatic insufficiency in cirrhosis. (HEPATOLOGY 2003;38:148-157.)

Translational regulation by p38 mitogen-activated protein kinase signaling during human cholangiocarcinoma growth
Yoko Yamagiwa, Carla Marienfeld, Laura Tadlock, Tushar Patel
The p38 mitogen-activated protein kinase (MAPK) signaling pathway is aberrantly expressed and maintains transformed cell growth in malignant human cholangiocytes. Because cell growth requires and is intimately related to protein synthesis, our aims were to assess the effect of p38 MAPK signaling on protein synthesis during growth of malignant human cholangiocytes. Inhibition of p38 MAPK activity during mitogenic stimulation decreased protein synthesis rates and tumor cell xenograft growth in nude mice. Altered protein synthesis resulted from decreased translational efficiency with impaired initiation of translation. Mitogenic stimulation resulted in phosphorylation of the eukaryotic initiation factor (eIF)-4E. Inhibition of p38 MAPK signaling or functional dysregulation of translation by small interfering double-stranded RNA (siRNA) to eIF-4E decreased anchorage-independent growth of malignant cholangiocytes. In conclusion, these studies identify a relationship between p38 MAPK activity and the regulation of protein synthesis during human cholangiocarcinoma growth. As protein synthesis is intimately linked to cell growth, dysregulation of translation initiation is a mechanism by which cellular p38 MAPK signaling participates in growth regulation of malignant cholangiocytes. (HEPATOLOGY 2003;38:158-166.)

PPAR ligands inhibit cholangiocarcinoma cell growth through p53-dependent GADD45 and p21WAF1/Cip1 pathway
Chang Han, A. Jake Demetris, George K. Michalopoulos, Qimin Zhan, James H. Shelhamer, Tong Wu
Ligands of peroxisome proliferator-activated receptor- (PPAR) induce differentiation and growth inhibition in several human cancers. However, the role of PPAR ligands in the growth control of human cholangiocarcinoma cells remains unknown. This study was designed to investigate the biological functions and molecular mechanisms of PPAR ligands in the growth regulation of human cholangiocarcinoma cells. Western blot analysis showed that PPAR is expressed in all of the three human cholangiocarcinoma cell lines used in this study (SG231, CC-LP-1, and HuCCT1). Transient transfection assays using a peroxisome proliferator response element (PPRE) reporter construct showed that the PPAR expressed in human cholangiocarcinoma cells is functional as a transcription activator. Exposure of SG231, CC-LP-1, and HuCCT1 cells to PPAR ligands 15-deoxy-12, 14-prostaglandin J2 (15d-PGJ2) and troglitazone for 24 to 96 hours resulted in a dose-dependent inhibition of cell growth. Flow cytometry analysis showed that 15d-PGJ2 and troglitazone-induced cell cycle arrest at the G2/M checkpoint. Consistent with these findings, both 15d-PGJ2 and troglitazone significantly inhibited the G2/M cyclin-dependent kinase (CDK) Cdc2 activity. Furthermore, cells treated with 15d-PGJ2 and troglitazone showed elevated expression of p53 and two p53-controlled downstream genes, GADD45 and p21WAF1/Cip1. Dominant negative inhibition of p53 in SG231 cells significantly blocked the 15d-PGJ2 and troglitazone-induced growth inhibition, G2/M arrest, and GADD45/p21 induction. 15d-PGJ2 and troglitazone failed to directly inhibit Cdc2 activity in a cell-free system in spite of direct association between GADD45 and PPAR proteins. In conclusion, these results show a novel p53-dependent mechanism in the PPAR ligand-mediated inhibition of cholangiocarcinoma growth and suggest a potential therapeutic role of PPAR ligands in the treatment of human cholangiocarcinoma. (HEPATOLOGY 2003;38:167-177.)

EBP50, a -cateninassociating protein, enhances Wnt signaling and is over-expressed in hepatocellular carcinoma
Tatsuhiro Shibata, Makoto Chuma, Akiko Kokubu, Michiie Sakamoto, Setsuo Hirohashi
Wnt signaling mediated by -catenin plays crucial roles in the development of hepatocellular carcinoma and other cancers such as colorectal cancer. -Catenin associates with T-cell factor (TCF) transcription factors and functions as a transcriptional activator in the nucleus. By protein interaction screening, we identified EBP50, a cytoplasmic protein with 2 PDZ domains, as a -catenin-associating molecule. EBP50 interacted with -catenin through its carboxyl-PDZ domain in vitro and in vivo. Northern blot and RT-PCR analysis revealed an increase of EBP50 messenger RNA (mRNA) in hepatocellular carcinoma (HCC) cell lines and surgical specimens of human HCC. Over-expression of EBP50 protein with focal nuclear localization was detected in human HCC. In human HCC and colorectal cancer cell lines, EBP50 enhanced -catenin/TCF-dependent transcription in a dose-dependent manner. In an HCC cell line, over-expression of the carboxyl PDZ domain resulted in a decrease of endogenous -catenin/TCF transactivation. EBP50 promoted -catenin-mediated transactivation only in cells in which -catenin was already stabilized, suggesting that EBP50 may work with stabilized -catenin for transcriptional regulation. In conclusion, the EBP50/-catenin complex promotes Wnt signaling, and over-expression of EBP50 may work cooperatively with -catenin in the development of liver cancer. (HEPATOLOGY 2003;38:178-186.)

Regulation of rat organic anion transporters in bile salt-induced cholestatic hepatitis: Effect of ursodeoxycholate
Daniel Rost, Thomas Herrmann, Peter Sauer, Hans-Ludwig Schmidts, Bruno Stieger, Peter J. Meier, Wolfgang Stremmel, Adolf Stiehl
Hepatic uptake of organic anions, including bile salts, is mediated by members of the organic anion-transporting polypeptide (Oatp) family. In rat liver, Oatp1 (Slc21a1), Oatp2 (Slc21a5), and Oatp4 (Slca10) are expressed at the basolateral membrane of hepatocytes and may be differentially regulated under pathophysiologic conditions such as cholestasis. The aim of this study was to determine the effects of cholic acid (CA) and ursodeoxycholic acid (UDCA) on the expression of Oatp4 compared with Ntcp, Oatp1, and Oatp2. Wistar rats were fed with CA (0.5%) or both CA (0.5%) and UDCA (0.25%) for 3 weeks. Oatp expression was studied by Northern and Western blot analysis as well as immunofluorescence analysis. Transport function was compared measuring biliary secretion of 14C-CA and 14C-taurocholic acid (TCA). In CA-fed animals, biliary secretion of 14C-CA and 14C-TCA was markedly delayed over 40 minutes compared with controls. Accordingly, Oatp4 protein was significantly down-regulated in CA-fed animals together with Oatp1 and Ntcp. Cofeeding of CA plus UDCA prevented the impairment of 14C-CA and 14C-TCA secretion and the down-regulation of Oatp4. Oatp4 messenger RNA (mRNA) levels did not differ significantly between bile salt-fed groups, suggesting a posttranscriptional effect of CA on Oatp4 expression. In contrast to Oatp1 and Oatp4, Oatp2 protein expression was increased by CA feeding, indicating a differential regulation of Oatp transporters. In conclusion, we show that CA feeding may cause cholestasis associated with a posttranscriptional down-regulation of Oatp4. UDCA may prevent impairment of hepatic function by restoring hepatic transporter expression. (HEPATOLOGY 2003;38:187-195.)

Liver Failure and Liver Disease

Pharmacokinetics and pharmacodynamic action of budesonide in early- and late-stage primary biliary cirrhosis (*Human Study*)
Wolfgang Hempfling, Frank Grunhage, Karin Dilger, Christoph Reichel, Ulrich Beuers, Tilman Sauerbruch
Budesonide has been discussed as a potential treatment option in primary biliary cirrhosis (PBC). Therefore, we studied the pharmacokinetics and pharmacodynamics of budesonide in patients with PBC stage I/II and stage IV. Twelve patients with early PBC stage I/II and 7 patients with PBC stage IV under continuous treatment with ursodeoxycholic acid (UDCA) were enrolled in an exploratory trial. Each patient received oral budesonide for 3 weeks at weekly increasing dosages of 3 mg once to thrice per day. Budesonide and cortisol plasma levels, urinary cortisol excretion, serum liver tests, and immunoglobulins were determined on days 1, 7, and 21 of the study. Patients with PBC stage IV showed significantly higher peak plasma concentrations (4.9 ± 3.5 vs. 1.5 ± 0.4 ng/mL; P < .05) and areas under the plasma concentration-time curves (AUC) (23.2 ± 16.8 vs. 5.1 ± 1.4 hours · ng/mL, P < .01, total AUC extrapolated to infinity [AUC0-]) after a single dose of 3 mg budesonide when compared with patients with PBC stage I/II. Equally, AUC of budesonide were significantly increased under a multiple dose regimen on day 21 (14.0 ± 11.6 vs. 5.0 ± 1.9 hours · ng/mL, P < .01, AUC at steady state from dosing time to 8 hours [AUCss,0-8 h]). Higher levels of budesonide were related to a significant decrease in plasma cortisol and reduction of urinary cortisol excretion in patients with stage IV disease. Two patients with stage IV disease developed portal vein thrombosis (PVT). In conclusion, administration of budesonide leads to markedly elevated plasma levels in cirrhotic patients with PBC associated with serious adverse drug reactions. Thus, further evaluation of combined treatment with UDCA may be considered in early-stage PBC but not in cirrhotic patients with PBC. (HEPATOLOGY 2003;38:196-202.)

Ursodeoxycholic acid therapy and the risk of colorectal adenoma in patients with primary biliary cirrhosis: An observational study (*Human Study*)
Lawrence Serfaty, Antoine De Leusse, Olivier Rosmorduc, Benoit Desaint, Jean-Francois Flejou, Olivier Chazouilleres, Renée E. Poupon, Raoul Poupon
Ursodeoxycholic acid (UDCA) is the first-line treatment for primary biliary cirrhosis (PBC). The long-term administration of UDCA might indirectly favor colon carcinogenesis by increasing the fecal excretion of secondary bile acids or, in contrast, it might inhibit colon carcinogenesis, as demonstrated in animal models. In patients with PBC, we examined the effect of prolonged UDCA administration on the prevalence and recurrence of colorectal adenoma and on the proliferation of colon epithelial cells. One hundred fourteen patients (103 women, 11 men; mean age, 55 years) with PBC, were enrolled in a colonoscopic surveillance program. The prevalence of colon adenoma was compared in patients already treated with UDCA (mean duration 46 months) at the time of colonoscopy (treated group, n = 52) and in patients undergoing colonoscopy just prior to treatment initiation (untreated group, n = 62). The recurrence of adenoma following removal (mean follow-up, 35 months) was compared between UDCA-treated patients and appropriate age- and gender-matched controls (2/1) selected from a cohort of 205 patients undergoing polypectomy. Epithelial cell proliferation was assessed using anti-Ki67 antibodies on colon biopsies from both treated and untreated patients. Treated and untreated patients displayed similar demographic characteristics. The prevalence of colorectal adenomas was 13% in the treated group versus 24% in the untreated group (P = .16). The colon epithelial cell proliferation index was significantly lower in treated patients than in untreated patients (P = .001). Following removal of the adenoma, the probability of recurrence was significantly lower in patients treated with UDCA than in controls (7% vs. 28% at 3 years, P = .04). In conclusion, this study suggests that, in patients with PBC, the prolonged administration of UDCA (1) is not associated with an increased prevalence of colorectal adenomas, and (2) significantly decreases the probability of colorectal adenoma recurrence following removal. These results are strengthened by the significant reduction in colon epithelial cell proliferation seen in patients treated with UDCA. (HEPATOLOGY 2003;38:203-209.)

Primary sclerosing cholangitis in children: A long-term follow-up study (*Human Study*)
Ariel E. Feldstein, Jean Perrault, Mounif El-Youssif, Keith D. Lindor, Deborah K. Freese, Paul Angulo
Primary sclerosing cholangitis (PSC) is increasingly diagnosed in children and adolescents, but its long-term prognosis remains uncertain. The aim of this longitudinal, cohort study was to determine the long-term outcome of children with PSC. Fifty-two children with cholangiography-proven PSC (34 boys and 18 girls; mean age 13.8 ± 4.2 years; range, 1.5-19.6 years) who were seen at our institution over a 20-year period were followed-up for up to 16.7 years. Two thirds presented with symptoms and/or signs of PSC and 81% had concomitant inflammatory bowel disease (IBD). Twenty-five percent had total alkaline phosphatase activity within the normal range for the age group, but all of them had elevated -glutamyl transpeptidase levels. Autoimmune hepatitis overlapping with PSC was present in 35% of children. A positive but transient clinical and/or biochemical response occurred under therapy with ursodeoxycholic acid, alone or in combination with immunosuppressive medications. During follow-up, 11 children underwent liver transplantation for end-stage PSC and 1 child died. The median (50%) survival free of liver transplantation was 12.7 years. Compared with an age- and gender-matched U.S. population, survival was significantly shorter in children with PSC (P < .001). In a Cox regression model, lower platelet count, splenomegaly, and older age were associated with shorter survival. Presence of autoimmune hepatitis overlapping with PSC (P = .2) or medical therapy (P = .2) did not affect survival. In conclusion, PSC significantly decreases survival in this child population. Although pharmacologic therapy may improve symptoms and liver test results initially, it does not seem to impact the long-term outcome.(HEPATOLOGY 2003;38:210-217.)

Interleukin 6/gp130-dependent pathways are protective during chronic liver diseases (*Human Study*)
Konrad L. Streetz, Frank Tacke, Ludger Leifeld, Torsten Wüstefeld, Andrea Graw, Christian Klein, Kenjii Kamino, Ulrich Spengler, Hans Kreipe, Stefan Kubicka, Werner Müller, Michael P. Manns, Christian Trautwein
The contribution of the acute phase inducer interleukin 6 (IL-6) in the pathogenesis of liver diseases is yet unclear. Our analysis showed enhanced expression of IL-6 in livers derived from patients with acute and chronic liver diseases. Additionally, IL-6 plasma levels were significantly increased in patients with chronic liver diseases and showed an inverse correlation with biochemical markers of liver function and a positive correlation with inflammatory markers, signs of portal hypertension, and the degree of liver fibrosis. To prove the relevance of these clinical findings, we applied the tetrachlorcarbonide (CCl4) model to conditional knockout animals (Cre/loxP system) for gp130, the common signal transducer of IL-6 family cytokines. Cre recombinases were expressed through a hepatocyte (AlfpCre) and a ubiquitous (MxCre) control element. Gp130 deleted mice had a totally abolished STAT3 activation and acute phase response induction, but gp130 deletion had no effect on the degree of acute liver injury and subsequent hepatocyte proliferation. In contrast, during chronic liver injury induced by biweekly application of CCl4, deletion of the gp130 receptor in nonparenchymal liver cells and not hepatocytes resulted in fibrosis progression. In conclusion, our experiments indicate an involvement of IL-6 in the pathogenesis of liver diseases and suggest a protective role of IL-6/gp130-dependent pathways in nonparenchymal liver cells during fibrosis progression in chronic liver diseases. (HEPATOLOGY 2003;38:218-229).

Deficiency of thrombin activatable fibrinolysis inhibitor in cirrhosis is associated with increased plasma fibrinolysis (*Human Study*)
Mario Colucci, Bianca M. Binetti, Maria G. Branca, Carlo Clerici, Antonio Morelli, Nicola Semeraro, Paolo Gresele
Hyperfibrinolysis is thought to contribute to bleeding associated with advanced cirrhosis. Thrombin activatable fibrinolysis inhibitor (TAFI) is a plasma precursor of a carboxypeptidase (TAFIa) with antifibrinolytic activity and was recently shown to be reduced in cirrhosis. In this study, we evaluated the influence of TAFI deficiency on in vitro fibrinolysis in cirrhotic patients. Fifty-three patients with cirrhosis and 43 healthy controls were studied. TAFI antigen was measured by enzyme-linked immunosorbent assay and TAFI activity by chromogenic assay. Fibrinolysis was evaluated as tissue plasminogen activator-induced plasma clot lysis time in the absence and in the presence of a specific inhibitor of TAFIa. TAFI antigen and activity levels were markedly reduced in cirrhotic patients (P < .0001). In these patients, the lysis time of plasma clots was shorter than in controls (median, interquartile range: 25 minutes, 21-36 minutes vs. 48 minutes, 40-60 minutes, respectively; P < .0001) and was poorly influenced by the TAFIa inhibitor. Accordingly, TAFIa and thrombin activity, generated in cirrhotic samples during clot lysis, were significantly lower than in control samples. Addition of purified TAFI to cirrhotic plasma prolonged the lysis time and enhanced the response to TAFIa inhibitor in a dose-dependent manner. In conclusion, our results indicate that in vitro plasma hyperfibrinolysis in cirrhosis is largely due to a defective TAFIa generation resulting from low TAFI levels and probably from impaired thrombin generation. Impairment of the antifibrinolytic TAFI pathway might contribute to bleeding associated with this disease. (HEPATOLOGY 2003;38:230-237.)

Octreotide in hepatorenal syndrome: A randomized, double-blind, placebo-controlled, crossover study (*Human Study*)
Gilles Pomier-Layrargues, Sarto C. Paquin, Ziad Hassoun, Michel Lafortune, Albert Tran
The hepatorenal syndrome (HRS) is related to vasoconstriction of the renal cortex induced by systemic hypovolemia that follows splanchnic vasodilatation as the primary event in the cascade of hemodynamic changes associated with portal hypertension. We evaluated the effects of octreotide, a splanchnic vasoconstrictor, on HRS in cirrhotic patients. We compared the effects of octreotide infusion (50 µg/h) to placebo using a randomized, double-blind, cross-over design over 2, 4-day periods. Nineteen patients were included, and 14 patients could complete the 2 phases of the study (group 1: placebo first; n = 8 and group 2: octreotide first; n = 6) The end point of the study was to evaluate improvement in renal function as defined by a 20% decrease in serum creatinine value after a 4-day treatment as compared with baseline. In all the patients, a normal central venous pressure was maintained by daily intravenous administration of 2 units of albumin. The 2 groups were similar with regard to demographic data and liver and kidney function parameters at baseline. Improvement in renal function was observed in 2 patients after the placebo and 1 patient after octreotide infusion in group 1 and in 2 patients after octreotide infusion and 1 patient after placebo in group 2 (P = not significant). In addition, treatment with octreotide infusion did not result in significant changes in creatinine clearance, daily urinary sodium, plasma renin activity, plasma aldosterone and glucagon levels, or renal and mesenteric artery resistance indices as measured by Doppler ultrasonography. In conclusion, the present study demonstrates that, under our experimental conditions, octreotide infusion combined with albumin is not effective for the treatment of HRS in cirrhotic patients. (HEPATOLOGY 2003;38:238-243.)

Hepatic gene expression in histologically progressive nonalcoholic steatohepatitis (*Human Study*)
Raghavakaimal Sreekumar, Barbara Rosado, Deborah Rasmussen, Michael Charlton
Although the molecular basis for the pathophysiology of nonalcoholic steatohepatitis (NASH) is poorly understood, insulin resistance and mitochondrial dysfunction are physiologic hallmarks of this condition. We sought evidence of a transcriptional or pretranscriptional basis for insulin resistance and mitochondrial dysfunction through measurement of hepatic gene expression (messenger RNA [mRNA]) using high-density synthetic oligonucleotide microarray analysis (Hu6800 GeneChip, Affymetrix, CA). Global hepatic gene expression was determined in snap-frozen liver biopsy specimens from 4 groups: (1) patients with cirrhotic-stage NASH (n = 6), (2) patients with cirrhosis caused by hepatitis C virus (HCV) (n = 6), (3) patients with cirrhosis secondary to primary biliary cirrhosis (PBC) (n = 6), and (4) healthy controls (n = 6). Genes were considered to be expressed differentially in NASH only if there was a greater than 2-fold difference in abundance of mRNA when compared with each of the control groups. Sixteen genes were uniquely differentially expressed (4 overexpressed and 12 underexpressed) in patients with cirrhotic-stage NASH. Genes that were significantly underexpressed included genes important for maintaining mitochondrial function (copper/zinc superoxide dismutase, aldehyde oxidase, and catalase). Glucose 6-phospatase, alcohol dehydrogenase, elongation factor-TU, methylglutaryl coenzyme A (CoA), acyl CoA synthetase, oxoacyl CoA thiolase, and ubiquitin also were underexpressed in NASH. Genes that were overexpressed in NASH included complement component C3 and hepatocyte-derived fibrinogen-related protein, potentially contributing to impaired insulin sensitivity. In conclusion, these studies provide evidence for a transcriptional or pretranscriptional basis for impaired mitochondrial function (attenuated capacity for the dismutation of reactive oxygen species) and diminished insulin sensitivity (increased acute phase reactants) in patients with histologically progressive NASH. Further studies are required to determine the mechanism and the physiologic significance of these findings. (HEPATOLOGY 2003;38:244-251.)

GASTROENTEROLOGY

Table of Contents for July 2003 · Volume 125 · Number 1

Rapid Communication

Inhibition of hepatitis B virus replication in vivo by nucleoside analogues and siRNA
C. Klein, C.T. Bock, H. Wedemeyer, T. Wüstefeld, S. Locarnini, H.P. Dienes, S. Kubicka, M.P. Manns, C. Trautwein
Background & aims: Hepatitis B virus (HBV) causes acute and chronic infections that may result in severe liver diseases. Animal models to study new treatment options in vivo have several drawbacks. Therefore, we were interested to establish a new small animal model in which HBV replication and especially new treatment options can be studied easily.

Methods: Naked DNA of an HBV replication competent vector was transferred via tail vein into NMRI mice. HBV replication was studied in serum and liver of the animals. HBV replication was modulated by treatment through siRNA and nucleoside analogues.

Results: Tail vein transfer of a HBV replication competent construct resulted in expression of HBV-specific transcripts in the liver, and up to 10% of hepatocytes became HBc- and HBsAg-positive. HBeAg, HBsAg, and viral DNA could be detected in the serum of the animals, followed by the induction of HBV-specific cellular immune responses. Nucleoside treatment of the mice resulted in reduced polymerase activity in the liver. Additionally, siRNA transfer in the animals led to a significant reduction of HBsAg and/or eventually HBeAg expression, which was dependent on the localization of the complementary sequence in the HBV genome.

Conclusions: We have established a mouse model to study HBV replication and to investigate new and existing treatment approaches in vivo. Interestingly, siRNA seems a promising innovative treatment option to inhibit specifically HBV replication in vivo.

Clinical-alimentary Tract

Cognitive-behavioral therapy versus education and desipramine versus placebo for moderate to severe functional bowel disorders
Douglas A. Drossman, Brenda B. Toner, William E. Whitehead, Nicholas E. Diamant, Chris B. Dalton, Susan Duncan, Shelagh Emmott, Valerie Proffitt, Donna Akman, Karen Frusciante, Terry Le, Kim Meyer, Barbara Bradshaw, Kristi Mikula, Carolyn B. Morris, Carlar J. Blackman, Yuming Hu, Huanguang Jia, Jim Z. Li, Gary G. Koch, Shrikant I. Bangdiwala
Background & aims: Studies of antidepressants and psychological treatments in functional bowel disorders (FBD) are methodologically limited. The aim of this study was to assess the clinical efficacy and safety of cognitive-behavioral therapy (CBT) against education (EDU) and desipramine (DES) against placebo (PLA) in female patients with moderate to severe FBD (irritable bowel syndrome, functional abdominal pain, painful constipation, and unspecified FBD). We also evaluated the amenability of clinically meaningful subgroups to these treatments.

Methods: This randomized, comparator-controlled, multicenter trial enrolled 431 adults from the University of North Carolina and the University of Toronto with moderate to severe symptoms of FBD. Participants received psychological (CBT vs. EDU) or antidepressant (DES vs. PLA) treatment for 12 weeks. Clinical, physiologic, and psychosocial assessments were performed before and at the end of treatment.

Results: The intention-to-treat analysis showed CBT as significantly more effective than EDU (P = 0.0001; responder rate, 70% CBT vs. 37% EDU; number needed to treat [NNT ], 3.1). DES did not show significant benefit over PLA in the intention-to-treat analysis (P = 0.16; responder rate, 60% DES vs. 47% PLA; NNT, 8.1) but did show a statistically significant benefit in the per-protocol analysis (P = 0.01; responder rate, 73% DES vs. 49% PLA; NNT, 5.2), especially when participants with nondetectable blood levels of DES were excluded (P = 0.002). Improvement was best gauged by satisfaction with treatment. Subgroup analyses showed that DES was beneficial over PLA for moderate more than severe symptoms, abuse history, no depression, and diarrhea-predominant symptoms; CBT was beneficial over EDU for all subgroups except for depression.

Conclusions: For female patients with moderate to severe FBD, CBT is effective and DES may be effective when taken adequately. Certain clinical subgroups are more or less amenable to these treatments.

Autoimmunity associated with anti-tumor necrosis factor treatment in Crohn's disease: A prospective cohort study
Severine Vermeire, Maja Noman, Gert Van Assche, Filip Baert, Kristel Van Steen, Nele Esters, Sofie Joossens, Xavier Bossuyt, Paul Rutgeerts
Background & aims: Infliximab therapy is an effective approach to treating Crohn's disease. Development of antinuclear antibodies has been described in patients treated, but the size of the problem and the relationship with autoimmunity have not been investigated. We investigated the occurrence of antinuclear antibodies in 125 consecutive Crohn's disease patients and studied the relationship with symptoms of autoimmunity.

Methods: Autoantibodies and clinical data were investigated before and 1, 2, and 3 months after infliximab infusion. If antinuclear antibodies were 1:80, further study of double-stranded DNA, single-stranded DNA, histones, and ENA was performed.

Results: Cumulative antinuclear antibody incidence at 24 months was 71 of 125 (56.8%). Almost half of these patients developed antinuclear antibodies after the first infusion, and >75% became antinuclear antibody positive after fewer than 3 infusions. So far, only 15 of 71 patients have become seronegative, after a median of 12 months. Of 43 antinuclear antibody-positive patients who were further subtyped, 14 of 43 (32.6%) had double-stranded DNA, 17 (39.5%) had single-stranded DNA, 9 (20.9%) had antihistone, and 0% were ENA positive. Two patients (both antihistone and double-stranded DNA positive) developed drug-induced lupus without major organ damage, and 1 developed autoimmune hemolytic anemia. Antinuclear antibodies were associated with the female sex (odds ratio, 3.166; 95% confidence interval, 1.1678.585; P = 0.024) and with papulosquamous or butterfly rash (odds ratio, 10.016; 95% confidence interval, 1.70858.725; P = 0.011).

Conclusions: The cumulative incidence of antinuclear antibodies was 56.8% after 24 months in this cohort of infliximab-treated Crohn's disease patients. Antinuclear antibodies persisted up to 1 year after the last infusion, and only a few patients became seronegative. Two patients developed drug-induced lupus erythematosus. Antinuclear antibodies were associated with the female sex and skin manifestations.

Recurrent partial trisomy 1q22-q44 in clonal intraepithelial lymphocytes in refractory celiac sprue
Virginie Verkarre, Serge-Pierrick Romana, Christophe Cellier, Vahid Asnafi, Jean-Jacques Mention, Ullah Barbe, Sylvie Nusbaum, Olivier Hermine, Elizabeth Macintyre, Nicole Brousse, Nadine Cerf-Bensussan, Isabelle Radford-Weiss
Background & aims: Refractory celiac sprue, a low-grade intraepithelial lymphoma characterized by expansion of clonal intraepithelial lymphocytes with intracellular CD3 but no surface CD3-T-cell receptor complexes, can be an intermediary step between celiac disease and overt T-cell lymphoma. To gain insight into the mechanisms of lymphomagenesis in celiac disease, we have performed the first cytogenetic study in refractory celiac sprue.

Methods: Karyotypes were performed on: (1) 7 cell lines derived from clonal intraepithelial lymphocytes of patients with refractory celiac sprue; (2) 14 control T-cell lines, either from 4 of 7 patients with refractory celiac sprue or from 10 patients with uncomplicated celiac disease; and (3) bone marrow and peripheral blood lymphocytes in 1 of 7 patients with refractory celiac sprue. Rearrangements were confirmed by in situ hybridization using whole-chromosome painting probes and by comparative genomic hybridization in one patient.

Results: A recurrent structural chromosomal aberration leading to partial trisomy of the long arm of chromosome 1 was found in 6 of 7 cell lines from patients with refractory celiac sprue but in none of the control T-cell lines. In one patient with circulating abnormal intraepithelial lymphocytes, the partial trisomy 1q was confirmed on cells freshly isolated from bone marrow and blood.

Conclusions: Refractory celiac sprue is strongly associated with partial trisomy of the 1q region. Gain of chromosome 1q, recently found in 16% of enteropathy-type T-cell lymphoma, may be an early event in lymphomagenesis related to celiac disease and provides a key to investigating molecular mechanisms of lymphoid transformation in this disease.

Crohn's disease and the NOD2 gene: A role for paneth cells
Sanjay Lala, Yasunori Ogura, Caroline Osborne, Sok Ying Hor, Annabel Bromfield, Susan Davies, Olagunju Ogunbiyi, Gabriel Nuñez, Satish Keshav
Background & Aims: The NOD2 gene, which is strongly associated with susceptibility to Crohn's disease (CD) of the terminal ileum, interacts with bacterial lipopolysaccharide (LPS), inducing cellular activation. However, the mechanisms by which NOD2 mutations cause terminal ileitis are unknown, and NOD2 is expressed most highly by peripheral blood monocytes, which are distributed ubiquitously and readily respond to LPS via cell-surface receptors. Paneth cells on the other hand, are most numerous in the terminal ileum, are critically important in enteric antibacterial defense, and respond to LPS through as yet undefined pathways. We therefore determined if these specialized intestinal epithelial cells also expressed the NOD2 gene.

Methods: In situ hybridization, immunohistochemistry, and laser-capture microdissection were used to determine RNA and protein expression in tissue sections, and real-time reverse-transcription polymerase chain reaction (RT-PCR) was used to quantitate gene expression in intestinal epithelial cells and peripheral blood mononuclear cells.

Results: NOD2 was detected readily in monocytes, but not in mature macrophages in the lamina propria or within granulomas, and levels declined as monocytes differentiated into macrophages in vitro, so that Caco-2 cells expressed more NOD2 mRNA than macrophages. NOD2 mRNA was enriched in crypts compared with villi, and in situ, Paneth cells were the most prominent cells expressing NOD2 in normal and CD-affected intestinal tissue, where they also strongly expressed tumor necrosis factor (TNF) RNA.

Conclusions: The NOD2 gene product is most abundant in Paneth cells in the terminal ileum, which could therefore play a critical and hitherto unrecognized role in the pathogenesis of NOD2-associated CD.

Acquired microvascular dysfunction in inflammatory bowel disease: Loss of nitric oxide-mediated vasodilation
Ossama A. Hatoum, David G. Binion, Mary F. Otterson, David D. Gutterman
Background & Aims: Inflammatory bowel disease (IBD; i.e., Crohn's disease, ulcerative colitis) is characterized by refractory inflammatory ulceration and damage to the intestine. Mechanisms underlying impaired healing are not defined. Because microvascular dysfunction resulting in diminished vasodilatory capacity and tissue hypoperfusion is associated with impaired wound healing, we hypothesized that microvascular dysfunction may also occur in chronic IBD.

Methods: Intact submucosal arterioles from control, involved, and uninvolved IBD specimens were assessed using in vitro videomicroscopy to assess endothelium-dependent vasodilation in response to acetylcholine (Ach) and fluorescence microscopy to detect oxyradicals.

Results: Normal microvessels dilated in a dose-dependent and endothelium-dependent manner to Ach (maximum, 82% ± 2%; n = 34). Inhibition of nitric oxide synthase with NG-nitro-L-arginine methyl ester (L-NAME) reduced maximal dilation to 54% ± 6% (P < 0.05, n = 7), and further reduction was observed after inhibiting cyclooxygenase (indomethacin; 23% ± 10%, n = 6). Chronically inflamed IBD microvessels showed significantly reduced Ach-induced vasodilation (maximum, 15% ± 2%; n = 33), with no effect of L-NAME. Indomethacin eliminated the remaining Ach-induced vasodilation, resulting in frank vasoconstriction (54% ± 9%, n = 6). Uninvolved IBD gut vessels and non-IBD inflammatory controls responded in a fashion similar to normal vessels. IBD-involved microvessels generated significantly higher levels of reactive oxygen species compared with control and uninvolved IBD vessels (P < 0.01).

Conclusions: Human intestinal microvessels from chronically inflamed IBD show microvascular endothelial dysfunction, characterized by loss of NO-dependent dilation that may contribute to reduced perfusion, poor wound healing, and maintenance of chronic inflammation.

Anti-HuD-induced neuronal apoptosis underlying paraneoplastic gut dysmotility
Roberto De giorgio, Monica Bovara, Giovanni Barbara, Marco Canossa, Giovanni Sarnelli, Fabrizio De ponti, Vincenzo Stanghellini, Marcello Tonini, Silvia Cappello, Eleonora Pagnotta, Eduardo Nobile-Orazio, Roberto Corinaldesi
Background & Aims: The role of autoimmunity underlying paraneoplastic gut dysmotility remains unsettled. Because anti-Hu antibodies may impair enteric neuronal function, we tested whether anti-HuD-positive sera from patients with paraneoplastic gut dysmotility or commercial anti-HuD antibodies activated the apoptotic cascade in a neuroblastoma cell line and cultured myenteric neurons.

Methods: Anti-HuD antibodies from patients with severe paraneoplastic gut dysmotility were characterized by immunofluorescence and immunoblot. SH-Sy5Y neuroblasts and cultured myenteric neurons were exposed to sera containing anti-HuD antibodies or 2 commercial anti-HuD antibodies. Cells were processed for terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) technique to evaluate apoptosis. Immunofluorescence was used to identify activated caspase-3 and apaf-1, along with microtubule-associated protein 2.

Results: In SH-Sy5Y cells, the percentage of TUNEL-positive nuclei observed after exposure to anti-HuD-positive sera (32% ± 7%) or anti-HuD antibodies (23% ± 2%) was significantly greater than that of control sera or fetal calf serum (P < 0.001). The time-course analysis showed a significantly greater number of apoptotic neuroblastoma cells evoked by the 2 commercial anti-HuD antibodies at 24, 48, and 72 hours versus controls. The number of TUNEL-positive myenteric neurons exposed to anti-HuD antibodies (60% ± 14%) was significantly greater than that of fetal calf serum (7% ± 2%; P < 0.001). Apaf-1 and caspase-3 immunolabeling showed intense cytoplasmic staining in a significantly greater proportion of cells exposed to anti-HuD-positive sera or to commercial anti-HuD antibodies compared with controls.

Conclusions: Anti-HuD antibodies evoked neuronal apoptosis that may contribute to enteric nervous system impairment underlying paraneoplastic gut dysmotility. Apaf-1 activation suggests participation of a mitochondria-dependent apoptotic pathway.

Clinical-liver Pancreas and Biliary Tract

Acute hepatitis C: High rate of both spontaneous and treatment-induced viral clearance
J. Tilman Gerlach, Helmut M. Diepolder, Reinhart Zachoval, Norbert H. Gruener, Maria-Christina Jung, Axel Ulsenheimer, Winfried W. Schraut, C. albrecht Schirren, M. Waechtler, M. Backmund, Gerd R. Pape
Background & Aims: Acute hepatitis C virus infection accounts for approximately 20% of cases of acute hepatitis today. The aim of this study was to define the natural course of the disease and to contribute to the development of treatment strategies for acute hepatitis C virus.

Methods: The diagnosis of acute hepatitis C virus in 60 patients was based on seroconversion to anti-hepatitis C virus antibodies or clinical and biochemical criteria and on the presence of hepatitis C virus RNA in the first serum sample.

Results: Fifty-one of 60 (85%) patients presented with symptomatic acute hepatitis C virus. In the natural (untreated) course of acute symptomatic hepatitis C (n = 46), spontaneous clearance was observed in 24 patients (52%), usually within 12 weeks after the onset of symptoms, whereas all asymptomatic patients (n = 9) developed chronic hepatitis C. The start of antiviral therapy (interferon- with or without ribavirin) beyond 3 months after the onset of acute hepatitis induced sustained viral clearance in 80% of treated patients.

Conclusions: The management of acute hepatitis C has to take into account the high rate of spontaneous viral clearance within 12 weeks after the onset of symptomatic disease. Treatment of only those patients who remain hepatitis C virus RNA positive for more than 3 months after the onset of disease led to an overall viral clearance (self-limited and treatment induced) in 91% of patients, and unnecessary treatment was avoided in those with spontaneous viral clearance. Patients with asymptomatic acute hepatitis C virus infection are unlikely to clear the infection spontaneously and should be treated as early as possible.

Glypican-3: A novel serum and histochemical marker for hepatocellular carcinoma
Mariana Capurro, Ian R. Wanless, Morris Sherman, Gerrit Deboer, Wen Shi, Eiji Miyoshi, Jorge Filmus
Background & aims: Early detection of hepatocellular carcinoma (HCC) is critical for successful treatment. However, the differential diagnosis between HCC and benign hepatic lesions is sometimes difficult and new biochemical markers for HCC are required. It has been reported that glypican-3 (GPC3) messenger RNA (mRNA) is significantly increased in most HCCs compared with benign liver lesions or normal liver. The goal of this study is to determine whether GPC3 is also overexpressed at the protein level and whether GPC3 is detectable in the serum of patients with HCC.

Methods: GPC3 was assessed in liver tissue sections by immunohistochemistry and in serum by enzyme-linked immunosorbent assay. Serum -fetoprotein (AFP) level was also measured in the same patients.

Results: Immunohistochemical studies showed that GPC3 is expressed in 72% of HCCs (21 of 29), whereas it is not detectable in hepatocytes from normal liver and benign liver diseases. Consistent with this, GPC3 was undetectable in the serum of healthy donors and patients with hepatitis, but its levels were significantly increased in 18 of 34 patients (53%) with HCC. In addition, only 1 of 20 patients with hepatitis plus liver cirrhosis displayed elevated levels of serum GPC3. Interestingly, in most cases, there was no correlation between GPC3 and AFP values. Thus, at least 1 of the 2 markers was elevated in 82% of the patients with HCC.

Conclusions: GPC3 is specifically overexpressed in most HCCs and is elevated in the serum of a large proportion of patients with HCC. The simultaneous determination of GPC3 and AFP may significantly increase the sensitivity for diagnosis of HCC.

Distribution of CCK1 and CCK2 receptors in normal and diseased human pancreatic tissue
Jean Claude Reubi, Beatrice Waser, Mathias Gugger, Helmut Friess, Jörg Kleeff, Hany Kayed, Markus W. Büchler, Jean A. Laissue
Background & Aims: The localization and functional role of cholecystokinin (CCK) receptor proteins in normal and diseased human pancreas, particularly in ductal pancreatic carcinomas, remain unclear.

Methods: Tissue samples of normal human pancreas, chronic pancreatitis, and ductal pancreatic carcinomas were investigated under carefully controlled conditions for expression of CCK1 and CCK2 receptor messenger RNA (mRNA) and proteins using in situ hybridization and in vitro CCK receptor autoradiography by means of subtype-selective analogues. Synaptophysin immunohistochemistry was used concomitantly for optimal identification of islets, nerves, and tumor areas with neuroendocrine features.

Results: CCK2 receptor mRNA and proteins were found abundantly in human pancreatic islets in normal pancreas and chronic pancreatitis. CCK1 receptor proteins were found occasionally in small-sized pancreatic nerves, whereas acini expressed a low density of CCK2 receptors in a few cases of chronic pancreatitis. Ductal pancreatic carcinomas rarely expressed CCK receptors; a few receptor-positive tumors, often characterized by neuroendocrine differentiation, expressed the CCK2 receptor at the mRNA or protein level. However, the main source of CCK receptors in the pancreatic tumor samples consisted of CCK2-expressing islets and/or CCK1-expressing nerves rather than neoplastic tissue.

Conclusions: These data indicate that the presence of CCK receptors in human ductal pancreatic tumor samples is mainly due to CCK2 expression in residual pancreatic islets and CCK1 in pancreatic nerves. Pancreatic acini and ductal pancreatic tumor cells very rarely express CCK2 receptors. These observations suggest that CCK analogues may not be of clinical use to target most of these cancers.

Hepatitis B virus genotypes and virologic response in 694 patients in phase III studies of adefovir dipivoxil
Chris Westland, William Delaney, IV, Huiling Yang, Shan-shan Chen, Patrick Marcellin, Stephanos Hadziyannis, Robert Gish, John Fry, Carol Brosgart, Craig Gibbs, Michael Miller, Shelly Xiong
Background & aims: Hepatitis B virus (HBV) genotype may influence disease progression and antiviral response. We therefore analyzed the frequency and distribution of genotypes in patients from 2 multinational phase III studies of adefovir dipivoxil. Antiviral efficacy of adefovir dipivoxil 10-mg therapy was examined with respect to HBV genotype, hepatitis B e antigen (HBeAg) serostatus, and race.

Methods: HBV genotypes were assigned by phylogenetic analyses of DNA sequences amplified from baseline serum samples (n = 694).

Results: Patients from Asia/Oceania were infected predominantly with genotypes B and C, whereas patients from Western European countries were infected predominantly with genotypes A and D. In Mediterranean countries, genotype D was dominant. The most common genotype in North America was C, followed by A, B, and D. Regardless of location, Asian patients were infected predominantly with genotypes B or C, whereas Caucasian patients were infected predominantly with A or D. There were significant differences in the baseline serum HBV-DNA levels of patients infected with different HBV genotypes regardless of HBeAg serostatus. Forty-eight weeks of adefovir dipivoxil 10-mg therapy resulted in potent reductions in serum HBV DNA with no significant differences based on genotype, HBeAg status, or race; similarly, there was no statistical difference in HBeAg seroconversion rates between genotypes in these patients.

Conclusions: HBV genotypes were distributed asymmetrically with respect to race, geography, and HBeAg status. Forty-eight weeks of adefovir dipivoxil therapy resulted in significant decreases in serum HBV-DNA levels in patients regardless of HBV genotype, HBeAg status, or race.

Activated human hepatic stellate cells express the renin-angiotensin system and synthesize angiotensin II
Ramón Bataller, Pau Sancho-bru, Pere Ginès, José M. Lora, Amal Al-garawi, Manel Solé, Jordi Colmenero, Josep M. Nicolás, Wladimiro Jiménez, Nadine Weich, José-carlos Gutiérrez-ramos, Vicente Arroyo, Juan Rodés
Background & aims: The renin-angiotensin system plays an important role in hepatic fibrogenesis. In other organs, myofibroblasts accumulated in damaged tissues generate angiotensin II, which promotes inflammation and extracellular matrix synthesis. It is unknown whether myofibroblastic hepatic stellate cells, the main hepatic fibrogenic cell type, express the renin-angiotensin system and synthesize angiotensin II. The aim of this study was to investigate whether quiescent and activated human hepatic stellate cells contain the components of the reninangiotensin system and synthesize angiotensin II.

Methods: Hepatic stellate cells were freshly isolated from normal human livers (quiescent hepatic stellate cells) and from human cirrhotic livers (in vivo activated hepatic stellate cells). Culture-activated hepatic stellate cells were used after a second passage of quiescent hepatic stellate cells. Angiotensinogen, renin, and angiotensin-converting enzyme were assessed by quantitative polymerase chain reaction. Angiotensin II production was assessed by enzyme-linked immunosorbent assay and immunohistochemistry.

Results: Quiescent hepatic stellate cells barely express the renin-angiotensin system components-angiotensinogen, renin, and angiotensin-converting enzyme-and do not secrete angiotensin II. In contrast, both in vivo activated hepatic stellate cells and culture-activated hepatic stellate cells highly express active renin and angiotensin-converting enzyme and secrete angiotensin II to the culture media. Mature angiotensin II protein is also detected in the cytoplasm of in vivo activated and culture-activated hepatic stellate cells. Growth factors (platelet-derived growth factor and epidermal growth factor) and vasoconstrictor substances (endothelin-1 and thrombin) stimulate angiotensin II synthesis, whereas transforming growth factor- and proinflammatory cytokines have no effect. Vasodilator substances markedly attenuate the effect of endothelin-1.

Conclusions: After activation, human hepatic stellate cells express the components of the renin-angiotensin system and synthesize angiotensin II. These results suggest that locally generated angiotensin II could participate in tissue remodeling in the human liver.

Bradykinin B2 receptors mediate contraction in the normal and inflamed human gallbladder in vitro
Marcello Trevisani, Silvia Amadesi, Fabien Schmidlin, Maria T. Poblete, Elisabetta Bardella, Barbara Maggiore, Selena Harrison, Carlos D. Figueroa, Michele Tognetto, Giuseppe Navarra, Alessandro Turini, Nigel W. Bunnett, Pierangelo Geppetti, Roberto De Giorgio
Background & aims: The components of the kinin system, including kinongens, kininogenases, and B2 and B1 receptors, are expressed and activated during inflammation. Here, we investigated the expression of the kinin B2 receptor messenger RNA, kininogen and kallikrein immunoreactivity, and the ability of kinins to contract control and inflamed gallbladders in vitro.

Methods: Human gallbladders, obtained from patients undergoing cholecystectomy either for acute cholecystitis secondary to gallstone disease or during elective gastro-entero-pancreatic surgery (controls), were processed for reverse-transcription polymerase chain reaction analysis, kallikrein and kininogen immunohistochemistry, binding studies, and in vitro contractility studies.

Results: Tissue expression of B2 receptor messenger RNA and specific binding of [3H]-bradykinin increased significantly in acute cholecystitis compared to controls. Kallikrein immunoreactivity was detected in the epithelium and infiltrating leukocytes, whereas kininogen immunoreactivity in the lumen of blood vessels and interstitial space. Bradykinin contracted isolated strips of control and acute cholecystitis gallbladders. In acute cholecystitis tissue, efficacy of bradykinin was higher than that of control gallbladders and similar to that of cholecystokinin. The contraction induced by bradykinin was significantly attenuated by B2 receptor antagonism but not by cyclooxygenase inhibition and B1, muscarinic, or tachykinin receptor antagonism.

Conclusions: All the components of the kinin system are expressed in the human gallbladder. Bradykinin is a powerful spasmogen via B2 receptor activation in the normal and, especially, in the inflamed human gallbladder.

Basic-alimentary Tract

GLP-2-mediated up-regulation of intestinal blood flow and glucose uptake is nitric oxide-dependent in TPN-fed piglets
Xinfu Guan, Barbara Stoll, Xiaofeng Lu, Kelly A. Tappenden, Jens J. Holst, Bolette Hartmann, Douglas G. Burrin
Background & aims: Our aim was to determine whether the intestinotrophic effects of GLP-2 are mediated by acute up-regulation of intestinal substrate utilization in TPN-fed piglets.

Methods: Twenty-four 12-day-old pigs, fitted with a portal flow probe and carotid, jugular and portal catheters, were fed by TPN for 7 days. On day 8, a group of pigs (n = 8) was infused intravenously with saline (control) for 4 hours and then with GLP-2 (500 pmol · kg1 · hour1, GLP-2) for 4 hours. 2H-glucose and 13C-phenylalanine were infused to estimate their kinetics and protein turnover. Another group (n = 8) received consecutive intravenous infusions of saline, GLP-2, and GLP-2 plus NG-Nitro-L-arginine methyl ester (L-NAME, 50 µmol · kg1 · hour1) for 4 hours each.

Results: GLP-2 acutely increased portal-drained visceral (PDV) blood flow rate (+25%) and intestinal blood volume (+51%) in TPN-fed piglets. GLP-2 also increased intestinal constitutive nitric oxide synthase (NOS) activity and endothelial NOS protein abundance. GLP-2 acutely increased PDV glucose uptake (+90%) and net lactate production (+79%). Co-infusion of GLP-2 plus L-NAME did not increase either PDV blood flow rate or glucose uptake. GLP-2 increased PDV indispensable amino acid uptake by 220% and protein synthesis by 125%, but did not decrease protein breakdown or phenylalanine oxidation.

Conclusions: We conclude that in TPN-fed neonatal pigs, GLP-2 acutely stimulates intestinal blood flow and glucose utilization, and this response is nitric oxide-dependent. These findings suggest that GLP-2 may play an important physiological role in the regulation of intestinal blood flow and that nitric oxide is involved in GLP-2 receptor function.

Colonic epithelial functional phenotype varies with type and phase of experimental colitis
Emiko Mizoguchi, Ramnik J. Xavier, Hans-christian Reinecker, Hirofumi Uchino, Atul K. Bhan, Daniel K. Podolsky, Atsushi Mizoguchi
Background & Aims : Colonic crypt elongation occurs during both chronic colitis and in the recovery phase of acute colitis. The impact of these alterations on epithelial cell functions is not fully defined.

Methods: DNA microarray analysis of freshly isolated colonic epithelial cells (CECs) from acute and chronic colitis was performed, and the results were confirmed by reverse transcription polymerase chain reaction. Localization of the selected molecules was examined by immunohistochemistry using newly generated antibodies. The function of selected molecules detected in this study was examined by administering the specific inhibitors in dextran sodium sulfate (DSS) colitis.

Results: Several detoxification-associated molecules, which contribute to prevent inflammation by regulating physiological balance under normal conditions, were markedly down-regulated, and anti-inflammatory molecules, which are not normally expressed, were up-regulated in the CEC under the chronic colitis. Among the detoxification-associated molecules, carbonic anhydrase IV was specifically down-regulated in CEC of Th2- but not Th1-mediated colitis. Functionally, inhibition of carbonic anhydrase activity led to the enhancement of recovery from DSS-induced acute colitis by directly stimulating CEC proliferation. Increased expression of regeneration-associated molecules such as regenerating gene-III was detectable in the CEC of acute and chronic colitis but not in the recovery phase of colitis. The expression of this molecule was restricted in surface epithelium and upper crypts but not lower crypts.

Conclusions: These studies suggest that functional alterations, which result in either the exacerbation or the suppression of colitis, coexist in the CEC during chronic colitis. CEC functions are likely to be differentially regulated in the context of the stage and mechanism of colitis.

Bacteroides vulgatus protects against escherichia coli -induced colitis in gnotobiotic interleukin-2-deficient mice
Marc Waidmann, Oliver Bechtold, Julia-stefanie Frick, Hans-anton Lehr, Sören Schubert, Ulrich Dobrindt, Jürgen Loeffler, Erwin Bohn, Ingo B. Autenrieth
Background & Aims: The microflora plays a crucial role in inflammatory bowel diseases (IBDs). Specific pathogen-free (SPF), but not germ-free, interleukin (IL)-2-deficient (IL-2/) mice develop colitis. The colitogenicity of commensal bacteria was determined.

Methods: Gnotobiotic IL-2/ and IL-2+/+ mice were colonized with Escherichia coli mpk, Bacteroides vulgatus mpk, or both bacterial strains, or with E. coli strain Nissle 1917. DNA arrays were used to characterize E. coli mpk. Colitis was analyzed by histology and real-time reverse-transcription polymerase chain reaction (RT-PCR) for interferon (IFN)-, tumor necrosis factor (TNF)-, IL-10, and CD14 messenger RNA (mRNA) expression. Bacterial numbers in feces and bacterial localization in the colon was determined by culture and fluorescence in situ hybridization (FISH).

Results: IL-2/ but not IL-2+/+ mice monocolonized with E. coli mpk developed colitis, whereas mono-association with B. vulgatus mpk, or E. coli Nissle, or co-colonization with E. coli mpk and B. vulgatus mpk, did not induce colitis. DNA array experiments and cellular studies revealed that E. coli mpk is a nonpathogenic strain. FISH and culture methods revealed that the anticolitogenic effect of B. vulgatus mpk on E. coli mpk cannot be explained by a significant reduction in numbers of E. coli in the colon. E. coli mpk-induced colitis was associated with increased IFN-, TNF-, CD14, and IL-10 mRNA expression in the colon.

Conclusions: In IL-2/ mice, B. vulgatus mpk protects against E. coli mpk-triggered colitis by an unknown mechanism. E. coli Nissle does not induce colitis. Various bacterial species common to the microflora differ in their ability to trigger IBD.

Basic-liver, Pancreas, and Biliary Tract

Smad7 prevents activation of hepatic stellate cells and liver fibrosis in rats
Steven Dooley, Jafar Hamzavi, Katja Breitkopf, Eliza Wiercinska, Harun M. Said, Johann Lorenzen, Peter Ten Dijke, Axel M. Gressner
Background & aims: Numerous studies implicate transforming growth factor (TGF)- signaling in liver fibrogenesis. To perturb the TGF- pathway during this process, we overexpressed Smad7, an intracellular antagonist of TGF- signaling, in vivo and in primary-cultured hepatic stellate cells (HSCs).

Methods: Ligation of the common bile duct (BDL) was used to induce liver fibrosis in rats. Animals received injections of an adenovirus carrying Smad7 cDNA into the portal vein during surgery and via the tail vein at later stages. The effect of Smad7 on TGF- signaling and activation of HSC was further analyzed in primary-cultured cells.

Results: Smad7-overexpressing BDL rats displayed reduced collagen and -SMA expression and reduced hydroxyproline content in the liver, when compared with animals administered AdLacZ. Such a beneficial effect was also observed when Smad7 was expressed in animals with established fibrosis. Accordingly, Smad7 arrested transdifferentiation of primary-cultured HSCs. AdSmad7 infected cells remained in a quiescent stage and retained storage of vitamin A droplets. Smad7 expression totally blocked TGF- signal transduction, shown by inhibiting Smad2/3 phosphorylation, nuclear translocation of activated Smad complexes, and activation of (CAGA)9-MLP-Luc, resulting in decreased collagen I expression. Smad7 also abrogated TGF--dependent proliferation inhibition of HSC. Smad7 did not decrease expression of -SMA, but immunofluorescent staining with anti -SMA antibodies displayed destruction of the fibrillar organization of the actin cytoskeleton.

Conclusions: In summary, gene transfer of Smad7 inhibits experimental fibrogenesis in vivo. Studies with isolated HSC suggest that the underlying mechanisms involve inhibition of TGF- signaling and HSC transdifferentiation.

Protection against liver damage by cardiotrophin-1: A hepatocyte survival factor up-regulated in the regenerating liver in rats
Matilde Bustos, Naiara Beraza, Juan-Jose Lasarte, Elena Baixeras, Pilar Alzuguren, Thierry Bordet, Jesus Prieto
Background & Aims: Cardiotrophin-1 (CT-1) is a member of the interleukin 6 (IL-6) family of cytokines, which protect cardiac myocytes against thermal and ischemic insults. In this study, we investigated the expression of CT-1 by liver cells and its possible hepatoprotective properties.

Methods: We analyzed the production, signaling, and antiapoptotic properties of CT-1 in hepatocytes and the expression of this cytokine during liver regeneration. We also investigated whether CT-1 might exert protective effects in animal models of liver damage.

Results: We found that CT-1 is up-regulated during liver regeneration and exerts potent antiapoptotic effects on hepatocytic cells. Hepatocytes cultured under serum starvation or stimulated with the pro-apoptotic cytokine transforming growth factor (TGF-) produce CT-1, which behaves as an autocrine/paracrine survival factor. Treatment with an adenovirus encoding CT-1 efficiently protects rats against fulminant liver failure after subtotal hepatectomy, an intervention that causes 91% mortality in control animals whereas 54% of those receiving CT-1 gene therapy were long-term survivors. This protective effect was associated with reduced caspase-3 activity and activation of the antiapoptotic signaling cascades signal transducer and activator of transcription (Stat-3), extracellular regulated kinases (Erk) 1/2, and Akt in the remnant liver. Gene transfer of CT-1 to the liver also abrogated Concanavalin A (Con-A) liver injury and activated antiapoptotic pathways in the hepatic tissue. Similar protection was obtained by treating the animals with 5 µg of recombinant CT-1 given intravenously before Con-A administration.

Conclusions: We show that CT-1 is a hepatocyte survival factor that efficiently reduces hepatocellular damage in animal models of acute liver injury. Our data point to CT-1 as a new promising hepatoprotective therapy.

In vitro interleukin-6 treatment prevents mortality associated with fatty liver transplants in rats
Zhaoli Sun, Andrew S. Klein, Svetlana Radaeva, Feng Hong, Osama El-Assal, Hong-na Pan, Barbara Jaruga, Sandor Batkai, Sumito Hoshino, Zhigang Tian, George Kunos, Anna mae Diehl, Bin Gao
Background & aims: Orthotopic liver transplantation is currently the only curative therapy for chronic end-stage liver disease and acute liver failure. However, a scarcity of cadaveric donors has led to a critical shortage of organs available for transplant. This is further complicated by the prevalence of steatosis in about 13%50% of donor livers, which is associated with a high risk of dysfunction and primary nonfunction.

Methods: Steatotic Zucker rat livers and livers from alcohol-fed rats were transplanted into lean control rats. Liver injury, activation of survival signals, and hepatic microcirculation were compared in nontreated and interleukin-6 (IL-6)-treated steatotic isografts.

Results: IL-6 pretreatment of steatotic Zucker rat liver isografts dramatically reduces mortality and liver injury following transplantation. Reperfusion after transplantation induces significant sinusoidal endothelial cell necrapoptosis in steatotic Zucker rat liver isografts, which is prevented by in vitro IL-6 pretreatment. IL-6 treatment activates cell survival signal transducer and activator of transcription factor 3 (STAT3) in hepatocytes and sinusoidal endothelial cells. Laser Doppler imaging and microsphere analyses demonstrate that IL-6 treatment markedly improves hepatic microcirculation, which is impaired in steatotic Zucker rat liver transplants. Finally, in vitro IL-6 treatment of donor livers also markedly reduces mortality associated with fatty liver transplants from alcohol-fed rats.

Conclusions: IL-6 induces hepatoprotection of steatotic liver isografts via preventing sinusoidal endothelial cell necrapoptosis and consequent amelioration of hepatic microcirculation, and protecting against hepatocyte death. IL-6 pretreatment of steatotic livers may render such allografts useable for clinical transplantation.

Cryptosporidium parvum invasion of biliary epithelia requires host cell tyrosine phosphorylation of cortactin via c-Src
Xian-Ming Chen, Bing Q. Huang, Patrick L. Splinter, Hong Cao, Guan Zhu, Mark A. Mcniven, Nicholas F. Larusso
Background & aims: Cryptosporidium parvum invasion of epithelia requires polymerization of host cell actin at the attachment site. We analyzed the role of host cell c-Src, a cytoskeleton-associated protein tyrosine kinase, in C. parvum invasion of biliary epithelia.

Methods: In vitro models of biliary cryptosporidiosis using a human biliary epithelial cell line were used to assay the role of c-Src signaling pathway in C. parvum invasion.

Results: c-Src and cortactin, an actin-binding protein and a substrate for c-Src, were recruited to the parasite-host cell interface during C. parvum invasion. Tyrosine phosphorylation of cortactin in infected cells was also detected. Inhibition of host cell c-Src significantly blocked C. parvum -induced accumulation and tyrosine phosphorylation of cortactin and actin polymerization at the attachment sites, thereby inhibiting C. parvum invasion of biliary epithelial cells. A triple mutation of tyrosine of cortactin in the epithelia also diminished C. parvum invasion. In addition, proteins originating from the parasite were detected within infected cells at the parasite-host cell interface. Antiserum against C. parvum membrane proteins blocked accumulation of c-Src and cortactin and significantly decreased C. parvum invasion. No accumulation of the endocytosis-related proteins, dynamin 2 and clathrin, was found at the parasite-host cell interface; also, inhibition of dynamin 2 did not block C. parvum invasion.

Conclusions: C. parvum invasion of biliary epithelial cells requires host cell tyrosine phosphorylation of cortactin by a c-Src-mediated signaling pathway to induce actin polymerization at the attachment site, a process associated with microbial secretion but independent of host cell endocytosis.

Case Report

Mesenteric inflammatory veno-occlusive disorder: A rare entity mimicking inflammatory bowel disorder
Kalyana Lavu, Anil Minocha
Mesenteric inflammatory veno-occlusive disorder (MIVOD) is a rare variety of inflammatory bowel disease. In addition to the case presentation, pathogenesis, and conflicting clinical, endoscopic, and computed tomography findings, we present for the first time the angiographic findings that would provide important clues to distinguish MIVOD from the chronic idiopathic variety of inflammatory bowel disease and confirm the diagnosis of MIVOD. Final diagnosis is made on full-thickness biopsy. Although medical treatment of MIVOD is unsuccessful, surgical resection of the involved segment results in resolution of symptoms.

Copyright 2003 Elsevier Science (USA). All rights reserved.

JOURNAL OF HEPATOLOGY

Table of Contents for Journal of Hepatology Volume 39, Issue 1, July 2003

Biliary Tract and Cholestasis

Biliary pronucleating proteins and apolipoprotein E in cholesterol and pigment stone patients
Karel J. van Erpecum, Piero Portincasa, Mehmet H. Dohlu, Gerard P. van Berge-Henegouwen and Dieter Jüngst
Background/Aims: Although cholesterol gallstone patients exhibit higher biliary cholesterol saturation than pigment stone patients, underlying mechanisms that affect stone type are unknown. We hypothesized that pronucleating proteins, hydrophobic bile salts or apolipoprotein E genotype affect stone type. We therefore compared these putative factors in cholesterol and pigment stone patients.

Methods: In 74 cholesterol and 12 pigment stone patients, bile lipids, various pronucleating proteins, crystallization and apolipoprotein E genotype were determined.

Results: Crystallization was enhanced, and cholesterol saturation higher in case of cholesterol stones, without any difference in bile salt composition. Concentrations of mucin (0.91±0.08 versus 0.31±0.06 mg/ml: P<0.0001), protein, IgM, IgG, IgA, haptoglobin, 1-acid glycoprotein and haptoglobin were 2-6-fold higher in cholesterol stone patients. Twenty cholesterol stone pts (27%) but only one pigment stone pt (8%) had at least one 4 allele. There was a significant difference in allele frequencies between both groups (cholesterol stones similar to Dutch population: 2 0.074, 3 0.770, 4 0.156: pigment stones: 2 0.250, 3 0.708, 4 0.042).

Conclusions: Various pronucleating biliary proteins are markedly higher in cholesterol than pigment stone patients. Also, apolipoprotein E genotype differs between cholesterol and pigment stone patients. These factors may affect gallstone type.
Keywords: Gallstone; Bile; Protein; Mucin; Apolipoprotein E

Combined analysis of the effect of treatment with ursodeoxycholic acid on histologic progression in primary biliary cirrhosis
Renée Eugénie Poupon et al.
Background/Aims: This study aimed at evaluating the effect of ursodeoxycholic acid (UDCA) treatment on histologic progression in primary biliary cirrhosis (PBC).

Methods: Using combined individual histologic findings from four clinical trials, we selected the patients in whom paired liver-biopsy specimens were available with a time interval of about 36 months between biopsies. A total of 367 patients were selected (UDCA: 200 vs. placebo: 167).

Results: Overall, there was no significant difference in the progression of the histologic stage between the two groups. By contrast, in the subgroup of patients with initial stages I-II (n=177) there was a significant decrease in the histologic stage progression in the UDCA group relative to the placebo group (P<0.03). Overall, there was a significant delay in the progression of periportal necroinflammatory lesions (P=0.03), and an improvement in the degree of ductular proliferation (P=0.02) in the UDCA group compared with the placebo group. There was no significant difference in the progression of other specific lesions.

Conclusions: A 2-year UDCA treatment reduces periportal necroinflammation and improves ductular proliferation, and when initiated at the earlier stages I-II of the disease also delays the progression of histologic stage. These data support the early initiation of the drug to prevent these histologic features of PBC.
Keywords: Primary biliary cirrhosis; Liver fibrosis; Histologic features; Histologic progression; Medical treatment

Cell Biology, Metabolism and Transport

Sera from liver failure patients and a demethylating agent stimulate transdifferentiation of murine bone marrow cells into hepatocytes in coculture with nonparenchymal liver cells
Shintaro Yamazaki et al.
Background/Aims: The plasticity of bone marrow cells (BMCs) is shown by their ability to differentiate into mesenchymal as well as endodermal and ectodermal lineages. Transdifferentiation of BMCs into hepatocytes has also been demonstrated, both in vitro and in vivo. In the present study we investigated the effects of liver nonparenchymal cells (NPCs) and sera from liver failure patients (HSLF) on the in vitro transdifferentiation of murine BMCs into hepatocytes.

Methods: Liver NPCs from wild-type mice, and 5-azacytidine-treated BMCs from green fluorescence protein transgenic mice, were cocultured in medium containing HSLF in combination with several cytokines. Hepatocyte-specific gene expression in BMCs was identified by immunocytochemistry and reverse transcription-polymerase chain reaction.

Results: Bone marrow cell-derived hepatocyte-like colonies appeared after several days of coculture in medium containing HSLF, oncostatin M (OSM) and hepatocyte growth factor (HGF). These colonies expressed hepatocyte-specific genes. Transdifferentiation was enhanced by 5-azacytidine treatment, and by HSLF, OSM and HGF. It did not take place when the BMCs were separated from the NPCs in a dual chamber dish, or cultured with other mesenchymal cells.

Conclusions: Direct interaction of murine BMCs with liver NPCs, as well as soluble factors in the HSLF and a demethylating agent, strongly stimulate transdifferentiation into hepatocytes.
Keywords: Transdifferentiation; Bone marrow cell; Nonparenchymal liver cell; Coculture; Hepatocyte growth factor; Oncostatin M; 5-Azacytidine; Liver failure; Cell fusion

Cirrhosis and its Complications

Plasma volume expansion by albumin in cirrhosis. Relation to blood volume distribution, arterial compliance and severity of disease
Kim Brinch, Søren Møller, Flemming Bendtsen, Ulrik Becker and Jens H. Henriksen
Background/Aims: The aim of the study was to investigate the effect of a standard albumin load on blood volume distribution, arterial compliance, and the renin-angiotensin-aldosterone system in patients with different degrees of cirrhosis.

Methods: 31 patients with cirrhosis (Child classes A/B/C=8/14/9) received an intravenous infusion of 40 g human serum albumin during a haemodynamic investigation.

Results: Whereas plasma- and blood volume increased by 23 and 15%, respectively (P<0.001), a significant increase in central blood volume was found only in class A patients (+8%, P<0.05), but not in class B or class C patients (+2.7%, not significant (n.s.)). In contrast, arterial compliance only increased significantly in class C patients (+18%, P<0.05), but not in class A or class B patients (+6%, n.s.). Plasma renin activity (PRA) decreased significantly in class C patients (31%, P<0.05). When all patients were compared, the change in arterial compliance was inversely correlated to the change in PRA (r=0.50, P<0.01).

Conclusions: Although infusion of albumin does not expand the central blood volume in patients with advanced cirrhosis, the results indicate a significant improvement in the low effective arterial blood volume of such patients, which may be important in the prevention of circulatory dysfunction.
Keywords: Albumin; Blood volume distribution; Central blood volume; Circulatory dysfunction; Effective arterial blood volume; Plasma volume expansion; Renin-angiotensin-aldosterone system

Inflammation and Fibrosis

Initial signaling of the fibronectin receptor (51 integrin) in hepatic stellate cells is independent of tyrosine phosphorylation
M.T. Milliano and B.A. Luxon
Background/Aims: Activation of hepatic stellate cells (HSC) plays an integral role in hepatic fibrosis. HSC activation increases fibronectin (51) receptor expression and interactions between 51 and the extracellular matrix increase collagen synthesis. It is unclear how signaling by the 51 receptor initiates these changes. We aimed to determine the signaling cascade after 51 stimulation in activated HSC.

Methods: HSC were isolated from male Sprague-Dawley rats. Activated HSC were exposed to beads coated with fibronectin (ligand for 51) or D-polylysine (inert control). HSC were stained with FTC-labeled antibodies against classes of signaling molecules. Tyrosine phosphorylation was blocked using genistein or herbimycin A. The fraction of beads with localized immunostaining (indicating accumulation of signaling protein) was determined.

Results: The majority of cytoskeletal proteins, Src substrates, Src kinases and members of the ERK and JNK signaling molecule families require actin cytoskeletal organization and tyrosine-kinase-mediated phosphorylation to accumulate. Several proteins (e.g. tensin, FAK) accumulated in the absence of tyrosine phosphorylation.

Conclusions: The 51 integrin-ligand interaction induces accumulation of cytoskeletal molecules, activating multiple kinase pathways. Initial integrin signaling by 51 are associated with cytoskeletal proteins and are independent of tyrosine phosphorylation. We suggest that there may be cytoskeletal changes that may be targeted to diminish HSC activation.
Keywords: Integrin; Hepatic stellate cell; Fibronectin receptor; Signaling

Gliotoxin-mediated apoptosis of activated human hepatic stellate cells
Young-Oh Kweon et al.
Background: Activated hepatic stellate cells (HSCs) play a central role in liver fibrogenesis, and apoptosis of activated HSCs might be essential to clear HSCs from injured liver. Gliotoxin induces apoptosis of activated human and rat HSCs by an unknown mechanism.

Aim: This study investigated the role of reactive oxygen species (ROS) and membrane permeability transition (MPT) in gliotoxin-induced apoptosis of activated human HSCs.

Methods: Primary and immortalized human HSCs were analyzed using confocal microscopy for ROS with dichlorodihdrofluorescence diacetate (DCFH-DA) fluorophore and for the mitochondrial membrane potential (MMP) using tetramethylrhodamine methylester (TMRM).

Results: Gliotoxin at higher concentrations (7.5 µM) markedly increased ROS formation, and ROS production was also evident at concentrations of gliotoxin causing necrotic cell death (32.5 µM). Gliotoxin rapidly (begins about 20 min at 1.5 µM and 10 min at 7.5 µM) disrupts MMP at a concentration as low as 300nM. MMP disruption was followed by cytochrome c release and caspase-3 activation. The MPT inhibitors, cyclosporine A (5 µM) plus trifluoperazine (12.5 µM), blocked depolarization of the mitochondrial membrane and release of cytochrome c, but did not block apoptosis in HSCs.

Conclusions: Gliotoxin (0.3-7.5 µM) induces apoptosis of activated human HSCs with induction of MPT, cytochrome c release and caspase-3 activation, whereas at higher doses (>32.5 µM), it induces necrosis. However, gliotoxin also activates a mitochondrial independent pathway.
Keywords: Hepatic stellate cells; Liver fibrosis; Gliotoxin; Apoptosis; Mitochondrial permeability transition

Liver Cell Injury and Liver Failure

Protective effect of central thyrotropin-releasing hormone on carbon tetrachloride-induced acute hepatocellular necrosis in rats
Yoichi Sato, Masashi Yoneda, Kimihide Nakamura, Isao Makino and Akira Terano
Background/Aims: Thyrotropin-releasing hormone (TRH) acts in the brain to stimulate hepatic proliferation and blood flow through vagal-muscarinic and prostaglandin-mediated pathways. Hepatic blood flow and prostaglandins are well recognized as cytoprotective factors for liver damage, and central TRH is known to play a role in gastric cytoprotection. The effect of central TRH on carbon tetrachloride (CCl4)-induced acute hepatocellular necrosis was investigated in rats.

Methods: Male fasted rats were injected with either TRH analog, RX 77368 (1-10 ng), or vehicle intracisternally, and CCl4 (2.0 ml/kg) was injected subcutaneously 60 min later. Acute hepatocellular necrosis was assessed by serum hepatic enzymes and histological changes 24 h after CCl4.

Results: Intracisternal TRH dose-dependently inhibited elevation of serum alanine aminotransferase level induced by CCl4. Intracisternal TRH reduced CCl4-induced hepatic histological changes. The cytoprotective effect of central TRH on CCl4-induced acute hepatocellular necrosis was abolished by hepatic branch vagotomy, atropine, indomethacin and NG-nitro-L-arginine methyl ester, but not by 6-hydroxydopamine. Intravenous TRH did not influence CCl4-induced acute hepatocellular necrosis.

Conclusions: These results suggest that the cytoprotective effect of central TRH on acute hepatocellular necrosis is mediated through vagal-muscarinic, and prostaglandin- and nitric oxide-dependent pathways.
Keywords: Neuropeptide; Autonomic nervous system; Central nervous system; Thyrotropin-releasing hormone; Hepatocellular necrosis; Nitric oxide; Prostaglandin; RX 77368

Ischemic preconditioning protects the steatotic mouse liver against reperfusion injury: an ATP dependent mechanism
Nazia Selzner, Markus Selzner, Wolfram Jochum and Pierre-Alain Clavien

Background/Aims: Hepatic steatosis is a major risk factor for liver surgery and transplantation. The increased susceptibility of fatty livers to ischemic injury is associated with a necrotic form of cell death as opposed to apoptosis in lean animals, and is possibly related to low contents of ATP. Ischemic preconditioning, a brief period of ischemia prior to a prolonged period, protects the lean liver against ischemia through anti-apoptotic properties. We evaluated whether ischemic preconditioning also confers protection in the fatty liver and whether it protects against the ATP loss.

Methods: The effect of ischemic preconditioning was tested in steatotic and lean livers subjected to 75 min of ischemia and 4 or 24 h of reperfusion. Tissue ATP contents were assessed at various times, and a model of low hepatic ATP contents (starvation) was studied to assess the type of injury following ischemia and the effects of preconditioning.

Results: Ischemic preconditioning protected steatotic livers against massive necrosis. ATP levels were significantly higher before and after reperfusion in liver subjected to preconditioning when compared to controls. Liver with low baseline ATP levels (starvation) were also associated with necrosis, and were protected by ischemic preconditioning.

Conclusions: Ischemic preconditioning mainly protects the fatty liver against necrosis possibly through preservation and restoration of tissue ATP contents.
Keywords: Ischemic preconditioning; Steatosis; Reperfusion injury; Adenosine triphosphate

Hepatitis induced by Kava (Piper methysticum rhizoma)
Felix Stickel et al.
Background/Aims: Botanical drugs are widely used and often contain highly active compounds. Kava root (Piper methysticum rhizoma), used frequently in Europe as a remedy against anxiety, contains kavapyrones with sedative effects. Seven case reports suggested the development of hepatitis after the intake of Kava.

Methods: We analyzed 29 novel cases of hepatitis along with Kava ingestion which occurred between 1990 and 2002 in addition to the seven already published case reports using a clinical diagnostic scale established for adverse hepatic drug reactions.

Results: Hepatic necrosis or cholestatic hepatitis were noticed with both alcoholic and acetonic Kava extracts. The majority of the 29 patients and the additional seven published reports were women (27 females, nine males). Both the cumulative dose and the latency to when the hepatotoxic reaction emerged were highly variable. Nine patients developed fulminant liver failure, of which eight patients underwent liver transplantation. Three patients died, two following unsuccessful liver transplantation and one without. In all other patients, a complete recovery was noticed after the withdrawal of Kava. Pathophysiologically, both immunoallergic and idiosyncratic factors may be responsible.

Conclusions: The present report emphasizes the potentially severe hepatotoxicity of Kava which has recently led to the retraction of Kava-containing drugs by the pharmacovigilance authorities in Germany.
Keywords: Drug-induced liver damage; Herbal medicine; Kava; Liver failure

Liver Cell Injury and Liver Failure

Chronic alcohol exposure sensitizes mice to galactosamine-induced liver injury through enhanced keratinocyte chemoattractant and defective IL-10 production
Filip Sermon et al.

Background/Aims: Alcohol sensitizes the liver to several injuries. The mechanisms leading to this sensitization are poorly defined. In the present study, we developed a mouse model of chronic exposure to alcohol vapours that sensitize mice to galactosamine (GAL) liver injury.

Methods: C57BL/6 mice were exposed to ethanol vapours for 10 days. Liver injury was induced by intraperitoneal injection of GAL (1 g/kg) and mice were killed 24 h later.

Results: GAL challenge after ethanol pre-treatment significantly raised serum alanine aminotransaminase (ALT) levels and enhanced liver inflammation when compared with the controls (GAL alone). Serum keratinocyte chemoattractant (KC) and monocyte chemoattractant protein-1 (MCP-1) levels were significantly increased in the GAL+ethanol group. On the contrary, serum interleukin 10 (IL-10) levels were lower than in controls. Anti-KC, anti-tumour necrosis factor antibodies and intestinal decontamination significantly protected mice from liver injury. In GAL+ethanol-treated mice, IL-10 treatment reduced ALT release, KC and MCP-1 serum and hepatic mRNA levels, and improved liver inflammation.

Conclusions: Enhancement of GAL-induced liver injury by ethanol is associated with an imbalance between proinflammatory cytokines and the anti-inflammatory cytokine IL-10 and depends on gut bacterial flora.
Keywords: Alcohol; Liver; Cytokines; Galactosamine; Mice

Liver Growth and Cancer

The identification of small nodules in liver adenomatosis
Sébastien Lepreux et al.
Background/Aims: Liver adenomatosis is characterized by the presence of multiple adenomas of various sizes in the liver. The aim of this study was to characterize the morphology of small nodules which can be difficult to identify.

Methods: Seven patients included in this study underwent surgery for the removal of one or several nodules. All, but one, were females. Three out of seven presented with acute bleeding. Five had six or more nodules at presentation, and two only one, who developed nodules later on during follow-up.

Results: All of the large nodules that were >2 cm, except one, were typical adenomas with or without hemorrhagic areas. Smaller nodules (1-2 cm), some of which discovered on the resected specimen were either typical adenomas or non-typical nodules. These non-typical nodules were characterized by a polylobulated aspect with steatotic zones, and in between, bands of non-steatotic hepatocytes with portal tracts-like structures containing occasional cytokeratin 7 and less often cytokeratin 19 positive biliary cells. Numerous steatotic foci were also seen in four cases. They were isolated or grouped forming microadenomas or non-typical micronodules (<1 cm) containing biliary elements.

Our findings lead to the following hypothesis: adenomatosis is characterized by the simultaneous occurrence of multiple adenomas; if several adenomatous foci expand at the same time in the same area, they will form one polylobulated nodule containing non-adenomatous tissue with portal tracts in between areas of adenomatous tissue (non-typical micronodule). Such a small micronodule may in turn expand and join another micronodule to form a bigger one by the same process (non-typical nodule). As nodules grow, their non-adenomatous components including hepatocytic plates and portal tracts constituents will progressively disappear to end up with the classical aspect of an adenoma.

Conclusions: This hypothesis supports the concept that non-typical nodules/micronodules are adenomas precursors. However, they can be difficult to classify since they resemble focal nodular hyperplasia precursor.
Keywords: Adenoma; Adenomatosis; Focal nodular hyperplasia

Role of c-kit receptor tyrosine kinase-mediated signal transduction in proliferation of bile epithelial cells in young rats after ligation of bile duct: a study using Ws/Ws c-kit mutant rats
Makoto Satake et al.
Background/Aims: Recently, it has been shown that the c-kit receptor tyrosine kinase (KIT) is expressed in the liver of young rats, and its expression is up-regulated in bile epithelial cells (BEC) after ligation of the common bile duct (BDL). To clarify a role of KIT in BEC, we examined whether BEC of Ws/Ws rats, whose KIT kinase activity was severely impaired, could proliferate in response to bile stasis after BDL.

Methods/Results: When 2-week-old control normal (+/+) and Ws/Ws mutant rats underwent BDL, only a few BEC were found in the portal field of livers of Ws/Ws rats, whereas many BEC were found in that of +/+ rats. Furthermore, Ki-67 immunostaining showed that the proliferative activity of BEC in 2-week-old Ws/Ws rats was much lower than that in +/+ rats of the same age. In contrast, when 6-week-old +/+ and Ws/Ws rats underwent BDL, BEC similarly proliferated in the livers of +/+ and Ws/Ws rats, and the proliferative activity of BEC was comparable.

Conclusions: The mechanism whereby BEC proliferate in response to bile stasis after BDL may be different between 2- and 6-week-old rats, and KIT mediated-signal transduction plays a crucial role in the proliferation of immature BEC in young rats.
Keywords: c-kit receptor tyrosine kinase; Ws/Ws rats; Bile epithelial cells; Bile stasis; Oval cells; Liver regeneration

Spiral computed tomography versus ultrasound in the follow-up of cirrhotic patients previously treated for hepatocellular carcinoma: a prospective study
Stefano Colagrande et al.
Background/Aims: To assess the value of hepatic-arterial-phase computed tomography (HAP-CT) versus ultrasound (US) plus -fetoprotein (AFP) in the surveillance of cirrhotic patients with previously treated hepatocellular carcinoma (HCC).

Methods: Thirty-six cirrhotic patients, treated for single nodular HCC <4cm with complete response and no evidence of other focal lesions, were enrolled in a prospective study and underwent simultaneous AFP/US/spiral-CT follow-up every 6 months. Focal lesions were considered recurrences when they appeared as globular enhancement areas (EA) at HAP-CT and increased in size during the follow-up.

Results: Fifteen of 36 patients showed at least one focal lesion for a total of 43 EA: 38/43 increased in size, four did not change and one disappeared. EA were first observed after a follow-up of 9±4 (range 6-18) months. At the same time, no patient had either nodular lesion at US examination or diagnostic levels of AFP. In 22 matched lesions, diagnosis by CT was 8.2±3.5 months earlier than by US. In 13 patients, one evolved EA was submitted to US-guided biopsy and histological examination showed HCC in all cases.

Conclusions: Periodical spiral-CT examination is more effective than US-AFP in early detection of HCC recurrence in cirrhotic patients successfully treated for HCC.
Keywords: -Fetoprotein; Cirrhosis; Diagnosis; Recurrent hepatocellular carcinoma; Spiral computed tomography; Ultrasonography

Retrorsine: a kinetic study of its influence on rat liver regeneration in the portal branch ligation model
Christian Picard et al.
Background: Retrorsine, a naturally occurring pyrrolizidine alkaloid, impairs liver regeneration after partial hepatectomy by mechanisms that are still unclear.

Aim: The aim of the study was to clarify the influence of retrorsine on cell cycle progression in the regenerating liver lobes of rats after portal branch ligation (PBL).

Methods: Liver weight, protein and DNA contents, DNA synthesis (5´-bromodeoxyuridine (BrdU) incorporation) and cellular levels of Cyclin E, CDK-2, CDK-4 and proliferating cell nuclear antigen (PCNA) were assessed before and 24, 48, 72 and 168 h after PBL.

Results: Before surgery, higher levels of cyclin E, CDK-2, CDK-4 and PCNA as well as BrdU incorporation were found in the liver of retrorsine-treated rats than in untreated rats. Liver weight gain, protein and DNA synthesis as well as induction of cell cycle related proteins were all strongly impaired by retrorsine in the regenerating lobes after PBL.

Conclusions: In conclusion, retrorsine impairs liver regeneration in the PBL model not only by an S or G2/M phase block, but also by a block located before the G1/S transition of the cell cycle.
Keywords: Retrorsine; Rat; Liver regeneration; Portal branch ligation; Pyrrolizidine alkaloids; Cell cycle; Cyclins

Viral Hepatitis

Twelve weeks of follow-up is sufficient for the determination of sustained virologic response in patients treated with interferon for chronic hepatitis C
Stefan Zeuzem et al.
Background/Aims: The current standard for the determination of sustained virologic response in patients treated for hepatitis C is undetectable hepatitis C virus (HCV) RNA 24 weeks following the completion of therapy. Sensitive molecular tests may permit earlier determination of sustained virologic response following the completion of therapy in end-of-treatment responders.

Methods: We examined this possibility in 1441 patients, who received 48 weeks of treatment with either standard or pegylated interferon -2a. HCV RNA was determined by polymerase chain reaction assay (Amplicor HCV Monitor vs. 2.0) at baseline and monitored at 4-week intervals throughout the treatment and 24-week post-treatment follow-up periods.

Results: End-of-treatment and sustained response were achieved in 624 and 342 patients, respectively. For all treatments, relapse was most frequent at weeks 52 and 56 and became rare following week 60. Only six patients out of 348 patients (2%) became HCV RNA positive between weeks 60 and 72. Analysis of baseline characteristics failed to identify a specific set of parameters associated with early relapse.

Conclusions: This finding suggests that determination of HCV RNA levels at 12 weeks of follow-up may be sufficient for making decisions related to the management of most patients treated with standard or pegylated interferon .
Keywords: Hepatitis C virus; Interferon ; Pegylated interferon ; Polymerase chain reaction; Virologic relapse

Abbreviations: HCV, hepatitis C virus; RT-PCR, reverse transcription-polymerase chain reaction; TMA, transcription-mediated amplification

BRITISH MEDICAL JOURNAL

19 July 2003 (Volume 327, Issue 7407)

Longevity and carrying the C282Y mutation for haemochromatosis on the HFE gene: case control study of 492 French centenarians
Hélène Coppin, research scientist1, M Bensaid, PhD student1, S Fruchon, PhD student1, N Borot, research scientist1, H Blanché, research scientist2, MP Roth, research scientist1

1 Unité de Physiopathologie Cellulaire et Moléculaire, CNRS UPR 2163, CHU Purpan, 31059 Toulouse Cedex 3, France, 2 Centre d'Etude du Polymorphisme Humain, Fondation Jean Dausset, 75010 Paris, France Correspondence to: M P Roth roth@cict.fr

Hereditary haemochromatosis is a common autosomal recessive disorder of iron metabolism. Most patients are homozygous for a C282Y mutation in the HFE gene. This mutation is frequent in northern Europe, where one in five to ten people are carriers. People who are heterozygous for the C282Y mutation have slightly but significantly higher values for serum iron and transferrin saturation and are less likely to have anaemia because of iron deficiency.1 2

Iron promotes the generation of free radicals, which leads to mutagenesis, atherosclerosis, inflammation, and bacterial growth. Therefore, genotypes that increase the concentrations of iron for transport and storage may be associated with an increased risk for common diseases, such as cancers and cardiovascular diseases, and for inflammatory and infectious conditions. Other studies, which investigated the associations of C282Y heterozygosity with morbidity, found conflicting results, and consensus has not been reached about whether C282Y is associated with the development of extrahepatic cancers, coronary heart disease, or diabetes.1 2

We hypothesised that people who are heterozygous for the C282Y mutation are under-represented in a centenarian population because many would have died younger from life threatening diseases which are more prevalent in C282Y heterozygotes.

5 July 2003 (Volume 327, Issue 7405)

Cohort study of hepatotoxicity associated with nimesulide and other non-steroidal anti-inflammatory drugs
Giuseppe Traversa, senior epidemiologist1, Clara Bianchi, statistician1, Roberto Da Cas, data manager1, Iosief Abraha, epidemiologist2, Francesca Menniti-Ippolito, senior epidemiologist1, Mauro Venegoni, head3

1 Department of Epidemiology, National Institute of Health, Viale Regina Elena 299, 00161 Rome, Italy, 2 Regional Health Authority of the Umbria Region, 06124 Perugia, Italy, 3 Department of Internal Medicine, Fatebenefratelli Hospital, 20121 Milan, Italy Correspondence to: G Traversa giuseppe.traversa@iss.it

Objective To estimate the risk of acute hepatotoxicity associated with nimesulide compared with other non-steroidal anti-inflammatory drugs.

Design Retrospective cohort and nested case-control study.

Setting Umbria region, Italy.

Participants 400 000 current, recent, and past users (almost 2 million prescriptions) of non-steroidal anti-inflammatory drugs between 1 January 1997 and 31 December 2001.

Main outcome measures Admissions to hospital for acute non-viral hepatitis and incidence of all hepatopathies and liver injury among users of nimesulide and other non-steroidal anti-inflammatory drugs.

Results Current use of non-steroidal anti-inflammatory drugs was associated with a 1.4 (95% confidence interval 1.0 to 2.1) increased risk of hepatopathy compared with past use. In current users of nimesulide the rate ratio for all hepatopathies and more severe liver injury was 1.3 (0.7 to 2.3) and 1.9 (1.1 to 3.8), respectively.

Conclusion The risk of liver injury in patients taking nimesulide and other non-steroidal anti-inflammatory drugs is small.

NEW ENGLAND JOURNAL

Volume 349:350-357 July 24, 200 Number 4

Epidermal Growth Factor Enemas with Oral Mesalamine for Mild-to-Moderate Left-Sided Ulcerative Colitis or Proctitis
Atul Sinha, M.R.C.P., Jeremy M.D. Nightingale, F.R.C.P., Kevin P. West, F.R.C.Path., Jorge Berlanga-Acosta, Ph.D., and Raymond J. Playford, F.R.C.P.

Background Epidermal growth factor (EGF) is a potent mitogenic peptide produced by salivary glands. We examined whether EGF enemas are an effective treatment for active left-sided ulcerative colitis and ulceration limited to the rectum (proctitis).

Methods In a randomized, double-blind clinical trial conducted at Leicester Royal Infirmary, 12 patients with mild-to-moderate left-sided ulcerative colitis received daily enemas of 5 µg of EGF in 100 ml of an inert carrier and 12 received daily enemas with carrier alone for 14 days. All also began to receive 1.2 g of oral mesalamine per day or had their dose increased by 1.2 g per day. Patients were assessed clinically at 0, 2, 4, and 12 weeks and by sigmoidoscopy and biopsy at 0, 2, and 4 weeks. The primary end point was disease remission (defined by a St. Marks score of 4 or less without sigmoidoscopic evidence of inflammation) at two weeks. Secondary end points were clinically significant improvements in disease activity (defined by a decrease of more than 3 points in the St. Marks score or the ulcerative colitis disease-activity index) at two and four weeks. Analyses were performed according to the intention-to-treat principle.

Results After two weeks, 10 of the 12 patients given EGF enemas were in remission, as compared with 1 of 12 in the control group (83 percent vs. 8 percent, P<0.001). At the 2-week assessment, disease-activity scores, sigmoidoscopic score, and histologic scores were all significantly better in the EGF group than in the placebo group (P<0.01 for all comparisons), and this benefit was maintained at 4 weeks and at 12 weeks.

Conclusions This study provides preliminary data suggesting that EGF enemas are an effective treatment for active left-sided ulcerative colitis.

Volume 349:247-257 July 17, 2003 Number 3

Tumor Microsatellite-Instability Status as a Predictor of Benefit from Fluorouracil-Based Adjuvant Chemotherapy for Colon Cancer
Christine M. Ribic, M.Sc., Daniel J. Sargent, Ph.D., Malcolm J. Moore, M.D., Stephen N. Thibodeau, Ph.D., Amy J. French, B.A., Richard M. Goldberg, M.D., Stanley R. Hamilton, M.D., Pierre Laurent-Puig, M.D., Ph.D., Robert Gryfe, M.D., Ph.D., Lois E. Shepherd, M.D., Dongsheng Tu, Ph.D., Mark Redston, M.D., and Steven Gallinger, M.D.

Background Colon cancers with high-frequency microsatellite instability have clinical and pathological features that distinguish them from microsatellite-stable tumors. We investigated the usefulness of microsatellite-instability status as a predictor of the benefit of adjuvant chemotherapy with fluorouracil in stage II and stage III colon cancer.

Methods Tumor specimens were collected from patients with colon cancer who were enrolled in randomized trials of fluorouracil-based adjuvant chemotherapy. Microsatellite instability was assessed with the use of mononucleotide and dinucleotide markers.

Results Of 570 tissue specimens, 95 (16.7 percent) exhibited high-frequency microsatellite instability. Among 287 patients who did not receive adjuvant therapy, those with tumors displaying high-frequency microsatellite instability had a better five-year rate of overall survival than patients with tumors exhibiting microsatellite stability or low-frequency instability (hazard ratio for death, 0.31 [95 percent confidence interval, 0.14 to 0.72]; P=0.004). Among patients who received adjuvant chemotherapy, high-frequency microsatellite instability was not correlated with increased overall survival (hazard ratio for death, 1.07 [95 percent confidence interval, 0.62 to 1.86]; P=0.80). The benefit of treatment differed significantly according to the microsatellite-instability status (P=0.01). Adjuvant chemotherapy improved overall survival among patients with microsatellite-stable tumors or tumors exhibiting low-frequency microsatellite instability, according to a multivariate analysis adjusted for stage and grade (hazard ratio for death, 0.72 [95 percent confidence interval, 0.53 to 0.99]; P=0.04). By contrast, there was no benefit of adjuvant chemotherapy in the group with high-frequency microsatellite instability.

Conclusions Fluorouracil-based adjuvant chemotherapy benefited patients with stage II or stage III colon cancer with microsatellite-stable tumors or tumors exhibiting low-frequency microsatellite instability but not those with tumors exhibiting high-frequency microsatellite instability.

LANCET

Volume 362, Number 9377 05 July 2003

Association between innate response to gliadin and activation of pathogenic T cells in coeliac disease
Luigi Maiuri, Carolina Ciacci, Ida Ricciardelli, Loredana Vacca, Valeria Raia, Salvatore Auricchio, Jean Picard, Mohamed Osman, Sonia Quaratino, Marco Londei

Background The adaptive immune system is central to the development of coeliac disease. Adaptive immune responses are, however, controlled by a preceding activation of the innate immune system. We investigated whether gliadin, a protein present in wheat flour, could activate an innate as well as an adaptive immune response in patients with coeliac disease.

Methods Duodenal biopsy samples from 42 patients with untreated coeliac disease, 37 treated patients, and 18 controls, were cultured in vitro for 3 h or 24 h, in the presence of either immunodominant gliadin epitopes (p-2 and p-9) or a non-immunodominant peptide (p31-43) known to induce small intestine damage in coeliac disease. We also incubated biopsy samples from nine untreated and six treated patients with a non-immunodominant peptide for 3 h, before incubation with immunodominant gliadin epitopes. Different combinations of interleukin-15 or signal transduction inhibitors were added to selected incubations.

Findings Only the non-immunodominant peptide induced rapid expression of interleukin-15, CD83, cyclo-oxygenase (COX)-2, and CD25 by CD3- cells (p=0·005 vs medium alone) and enterocyte apoptosis (p<0·0001). Only the non-immunodominant peptide induced p38 MAP kinase activation in CD3- cells. Pre-incubation with the non-immunodominant peptide enabled immunodominant epitopes to induce T-cell activation (p=0·001) and enterocyte apoptosis. Inhibition of interleukin-15 or of p38 MAP kinase controlled such activity.

Interpretation A gliadin fragment can activate the innate immune system, affecting the in situ T-cell recognition of dominant gliadin epitopes. Although our findings emphasise the key role of gliadin-specific T cells, they suggest a complex pathogenic situation, and show that inhibition of interleukin-15 or p38 MAP kinase might have the potential to control coeliac disease.

Lancet 2003; 362: 30-37

Autosomal recessive colorectal adenomatous polyposis due to inherited mutations of MYH
Julian R Sampson, Sunil Dolwani, Sian Jones, Diana Eccles, Anthony Ellis, D Gareth Evans, Ian Frayling, Sheila Jordan, Eamonn R Maher, Tony Mak, Julie Maynard, Francesca Pigatto, Joan Shaw, Jeremy P Cheadle

Familial adenomatous polyposis (FAP) and attenuated FAP are autosomal dominant disorders characterised by multiple colorectal adenomas and cancers. Both are caused by inherited mutations in the APC gene, and management includes genetic testing, colonoscopic surveillance, and prophylactic surgery for the relatives of index cases. Among 614 families recorded in six regional registers of polyposis in the UK, we identified 111 with neither dominant transmission nor evidence of APC mutation. Molecular genetic analysis showed that 25 had biallelic mutations of the MYH gene. Since our data show that MYH polyposis can be transmitted as an autosomal recessive trait, a change in genetic counselling, testing, and surveillance is needed.

Lancet 2003; 362: 39-41