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Revue de Presse du Mois Mois de Décembre 2002

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HEPATOLOGY

Table of Contents for December 2002 · Volume 36 · Number 6

Liver Failure and Liver Disease

Cerebral microdialysis in patients with fulminant hepatic failure (*Human Study*)
Flemming Tofteng, Linda Jorgensen, Bent Adel Hansen, Peter Ott, Jens Kondrup, Fin Stolze Larsen
Fulminant hepatic failure (FHF) is often complicated by high intracranial pressure (ICP) and fatal brain damage. In this study, we determined if a rise in [glutamate]ec and [lactate]ec preceded surges of high ICP in patients with FHF (median age, 42; range, 20-55 years; 7 women; 3 men) by inserting a microdialysis catheter into the brain-cortex together with an ICP catheter. The microdialysis catheter was perfused with artificial cerebrospinal-fluid at a rate of 0.3 µL/min. Dialysate was collected approximately every 30 minutes or when ICP increased. A total of 352 microdialysis samples were collected during a median of 3 days and allowed for ~1,760 bedside analyses of the collected dialysate. In 5 patients that later developed surges of high ICP, the initial values of [glutamate]ec and [lactate]ec were 2 to 5 times higher compared with patients with normal ICP. [Glutamate]ec then tended to vanish with time in both groups of patients. An increase in [glutamate]ec did not precede high ICP in any of the cases. In contrast, [lactate]ec was high throughout the study in the high ICP group and increased further before surges of high ICP. We conclude that in patients with FHF, cerebral [glutamate]ec and [lactate]ec are elevated. However, the elevated [glutamate]ec is not correlated to high ICP. In contrast, elevations in [lactate]ec preceded surges of high ICP. In conclusion, accelerated glycolysis with lactate accumulation is implicated in vasodilatation and high ICP in patients with FHF. The data suggest that bedside cerebral microdialysis is a valuable tool in monitoring patients with FHF and severe hyperammonemia. (HEPATOLOGY 2002;36:1333-1340.)

A phylogenetic analysis identifies heterogeneity among hepatocellular carcinomas (*Human Study*)
Katherine A. McGlynn, Michael N. Edmonson, Rita A. Michielli, W. Thomas London, Wen Yao Lin, Gong-Chao Chen, Fu-Min Shen, Kenneth H. Buetow
Primary hepatocellular carcinoma (HCC) is a significant cause of cancer morbidity and mortality on the global scale. Although epidemiologic studies have identified major risk factors for HCC, the sequence of oncogenic events at the molecular level remains poorly understood. While genetic allele loss appears to be a common event, the significance of the loss is not clear. In order to determine whether allele loss appears to be a random event among HCCs or whether patterns of loss cluster in groups of tumors, a phylogenetic approach was used to examine 32 tumors for genome-wide loss of heterozygosity employing 391 markers. Clusters identified by the phylogenetic analysis were then contrasted to compare candidate locus variation among individuals and to determine whether certain clusters exhibited higher loss rates than other clusters. The analysis found that 3 major and 1 minor cluster of loss could be identified and, further, these clusters were distinguished by variable rates of loss (cluster 1, 29%; cluster 2, 21%; cluster 3, 16%). The analyses also indicated that the allele loss rates in HCC were not insignificant and that the patterns of allele loss were complex. In addition, the results indicated that an individual's constitutional genotype at the EPHX1 locus may be a critical factor in determining the path of tumor evolution. In conclusion, it appears that in HCC, allele loss is not random, but clusters into definable groups that are characterized by distinctive rates of loss. (HEPATOLOGY 2002;36:1341-1348.)

NAFLD may be a common underlying liver disease in patients with hepatocellular carcinoma in the United States (*Human Study*)
Jorge A. Marrero, Robert J. Fontana, Grace L. Su, Hari S. Conjeevaram, Dawn M. Emick, Anna S. Lok
The incidence of hepatocellular carcinoma (HCC) in the United States is increasing, but the clinical characteristics of American patients with HCC have not been well described. The aims of this study were to determine the etiology of liver disease and short-term outcome among HCC patients presenting to a single center in the United States. One hundred five consecutive patients with HCC were studied; mean age was 59 years, 67% were men, and 76% were non-Hispanic white. The most common etiology of liver disease was hepatitis C (51%) and cryptogenic cirrhosis (29%). Half of the patients with cryptogenic cirrhosis had histologic or clinical features associated with nonalcoholic fatty liver disease (NAFLD). Fifty-three (50%) patients had HCC detected during surveillance (group I), whereas the remaining patients had symptomatic tumors (group II). Group I patients had smaller tumors (P = .01), were more likely to be eligible for surgical treatment (P = .005), and had a better median survival compared with patients in group II (P = .001). Patients with cryptogenic cirrhosis were less likely to have undergone HCC surveillance and had larger tumors at diagnosis. In conclusion, hepatitis C and cryptogenic liver disease are the most common etiologies of diseases in our patients with HCC. NAFLD accounted for at least 13% of the cases. Patients who underwent surveillance had smaller tumors and were more likely to be candidates for surgical or local ablative therapies. Because of the increasing incidence of NAFLD, further studies are needed to determine the risk of HCC in patients with NAFLD. (HEPATOLOGY 2002;36:1349-1354.)

Valine-alanine manganese superoxide dismutase polymorphism is not associated with alcohol-induced oxidative stress or liver fibrosis (*Human Study*)
Stephen F. Stewart, Julian B. Leathart, Yuanneng Chen, Ann K. Daly, Roberta Rolla, Daria Vay, Elisa Mottaran, Matteo Vidali, Emanuele Albano, Chris P. Day
The role of genetic factors in the pathogenesis of alcohol-induced liver disease (ALD) is receiving increasing attention. Recently, it has been reported that homozygosity for a valine to alanine substitution in the mitochondrial targeting sequence of manganese superoxide dismutase (Mn-SOD) represents a risk factor for severe ALD. Because this mutation is postulated to modify enzyme transport into mitochondria, we have sought confirmatory evidence of this association in a larger group of patients and investigated whether this polymorphism might influence alcohol-induced oxidative stress. Genotyping for the valine-alanine (Val-Ala) polymorphism of the Mn-SOD gene in 281 patients with advanced ALD (cirrhosis/fibrosis) and 218 drinkers without liver disease showed no differences in either the heterozygote (55% vs. 50%) or the homozygote (19% vs. 23%) frequency for the alanine allele. By measuring the titers of circulating antibodies against oxidized cardiolipin (OX-CL) and malondialdehyde (MDA) or hydroxy-ethyl radical (HER) adducts as markers of oxidative stress, we found a significant increase in ALD patients compared with healthy controls. However, the carriers of the alanine Mn-SOD allele had titers of anti-MDA, anti-HER, and anti-OX-CL IgG comparable with heterozygotes and patients homozygous for the valine allele. Similarly, the frequency of subjects with antibody titers above the 95th percentile of controls was not increased among homozygotes for the alanine Mn-SOD allele. In conclusion, in our population Val-Ala polymorphism in Mn-SOD influences neither susceptibility to alcohol-induced liver fibrosis nor alcohol-induced oxidative stress. (HEPATOLOGY 2002;36:1355-1360.)

"A La Carte" treatment of portal hypertension: Adapting medical therapy to hemodynamic response for the prevention of bleeding (*Human Study*)
Christophe Bureau, Jean-Marie Péron, Laurent Alric, Joséphine Morales, Jérôme Sanchez, Karl Barange, Jean-Louis Payen, Jean-Pierre Vinel
We report the results of adapting medical therapy to the monitoring of hemodynamic response in the prevention of a first variceal bleeding or rebleeding in patients with cirrhosis. Hepatic venous pressure gradient (HVPG) was measured before and after propranolol was initiated. The patients were considered responders if HVPG decreased below 12 mm Hg or at least 20% as compared with baseline value. If patients were not responders, isosorbide-5 mononitrate (I-5MN) was added, and a third hemodynamic study was performed. Thereafter, the patients were followed for a mean of 28 months. Thirty-four consecutive patients were treated to prevent a first bleeding episode in 20 patients and a rebleeding in 14 patients. HVPG value was initially 19.8 ± 4.6 mm Hg and decreased to 17.6 ± 5.7 mm Hg (P < .05) after propranolol alone. Thirteen patients (38%) were responders to propranolol. I-5MN improved hemodynamic response in 7 cases. Among these 20 (59%) hemodynamic responders, only 2 (10%) experienced variceal bleeding, as compared with 9 of 14 (64%) nonresponders (P < .05). Using multivariate analysis, only hemodynamic response was found to have an independent predictive value for the risk of variceal bleeding. In conclusion, hemodynamic response to drug therapy identifies patients who are efficiently protected from variceal bleeding as well as nonresponders in whom an alternative treatment should be considered. (HEPATOLOGY 2002;36:1361-1366.)

Randomized comparison of long-term carvedilol and propranolol administration in the treatment of portal hypertension in cirrhosis (*Human Study*)
Rafael Bañares, Eduardo Moitinho, Ana Matilla, Juan Carlos García-Pagán, José Luis Lampreave, Carlos Piera, Juan G. Abraldes, Alejandro De Diego, Agustín Albillos, Jaime Bosch
Short-term carvedilol administration is more powerful than propranolol in decreasing hepatic venous pressure gradient (HVPG) in cirrhotic patients, but induces arterial hypotension that may prevent its long-term use in portal hypertensive patients. This study compared the HVPG reduction and safety of long-term carvedilol and propranolol. Fifty-one cirrhotic patients were randomly assigned to receive carvedilol (n = 26) and propranolol (n = 25). Hemodynamic measurements and renal function were assessed at baseline and after 11.1 ± 4.1 weeks. Carvedilol caused a greater decrease in HVPG than popranolol (­19 ± 2% vs. ­12 ± 2%; P < .001). The proportion of patients achieving an HVPG reduction 20% or 12 mm Hg was greater after carvedilol (54% vs. 23%; P < .05). Carvedilol, but not propranolol caused a significant decrease in mean arterial pressure (MAP) (­11 ± 1% vs. ­5 ± 3%; P = .05) and a significant increase in plasma volume (PV) and body weight (11 ± 5% and 2 ± 1%, respectively; P < .05). Glomerular filtration rate (GFR) was unchanged with either drug, but the dose of diuretics was increased more frequently after carvedilol (27% vs. 8%; P = .07). Adverse events requiring discontinuation of treatment occurred in 2 patients receiving carvedilol and in 3 receiving propranolol. In conclusion, carvedilol has a greater portal hypotensive effect than propranolol in patients with cirrhosis. However, its clinical applicability may be limited by its systemic hypotensive effects. Further trials are needed to confirm the therapeutic potential of carvedilol. (HEPATOLOGY 2002;36:1367-1373.)

Liver disease in cystic fibrosis: A prospective study on incidence, risk factors, and outcome (*Human Study*)
Carla Colombo, Pier Maria Battezzati, Andrea Crosignani, Alberto Morabito, Diana Costantini, Rita Padoan, Annamaria Giunta
Incidence of liver disease (LD) associated with cystic fibrosis (CF) and its clinical characterization still is unsettled. We have assessed prospectively the incidence and risk factors of this complication, and its impact on the clinical course of CF. Between 1980 and 1990, we enrolled 177 CF patients without LD in a systematic clinical, laboratory, ultrasonography screening program of at least a 10-year duration. During a 14-year median follow-up (2,432 patient-years), 48 patients developed LD, with cirrhosis already present in 5. Incidence rate (number of cases per 100 patient-years) was 1.8% (95% confidence interval: 1.3-2.4), with sharp decline after the age of 10 years and higher risk in patients with a history of meconium ileus (incidence rate ratio, 5.5; 2.7-11), male sex (2.5; 1.3-4.9), or severe mutations (2.4; 1.2-4.8) at multivariate analysis. Incidence of cirrhosis was 4.5% (2.3-7.8) during a median period of 5 years from diagnosis of liver disease. Among the 17 cirrhotic patients, 13 developed portal hypertension, 4 developed esophageal varices, 1 developed liver decompensation requiring liver transplantation. Development of LD did not condition different mortality (death rate ratio, 0.4; 0.1-1.5) or higher incidence of other clinically relevant outcomes. In conclusion, LD is a relatively frequent and early complication of CF, whose detection should be focused at the first life decade in patients with history of meconium ileus, male sex, or severe genotype. Although LD does not condition a different clinical course of CF, in some patients it may progress rapidly and require liver transplantation. (HEPATOLOGY 2002;36:1374-1382.)

Characterization of recombinant monoclonal IgA anti­PDC-E2 autoantibodies derived from patients with PBC (*Human Study*)
Nobuyoshi Fukushima, Greg Nalbandian, Judy Van de Water, Kandra White, Aftab A. Ansari, Patrick Leung, Thomas Kenny, Shizuo G. Kamita, Bruce D. Hammock, Ross L. Coppel, Freida Stevenson, Hiromi Ishibashi, M. Eric Gershwin
Primary biliary cirrhosis (PBC) is an autoimmune liver disease characterized by the presence of autoantibodies to mitochondria (AMA). Recent evidence suggests that PBC develops after a locally driven response in the mucosa, where immunoglobulin A (IgA) is the dominant antibody isotype. In this study, we produced recombinant pyruvate dehydrogenase complex (PDC-E2)­specific dimeric human IgA monoclonal antibodies (mAbs) in a baculovirus expression system. By using 2 anti­PDC-E2 IgG mAbs derived from patients with PBC, we constructed 2 recombinant baculoviruses, each containing heavy chains with the C constant region. These were simultaneously co-infected into Sf9 insect cells with recombinant baculovirus containing the J chain. A sodium dodecyl sulfate­polyacrylamide gel electrophoresis (SDS-PAGE) immunoblotting profile of the IgA using a 6% nonreducing gel verified the dimeric nature of the autoantibodies. Both recombinants retained their original specificity for PDC-E2. In addition, the antibody showed a mitochondrial staining pattern in HEp2 cells and apically stained the biliary epithelial cells (BECs) in the liver of a patient with PBC but not a normal patient. Transcytosis experiments performed using human polymeric immunoglobulin receptor (pIgR) expressing Madine-Darby canine kidney (MDCK) cells showed that one of the recombinants showed a high degree of colocalization with PDC-E2. In conclusion, these data provide further support of the hypothesis that PDC-E2­specific IgA may enter biliary epithelial cells of PBC patients via the pIgR and complex with PDC-E2, thereby potentially contributing to the pathology of BECs. Moreover, this recombinant PDC-E2­specific mAb provides a tool for further determination of the role of anti-PDC-E2 IgA in the pathogenesis of PBC. (HEPATOLOGY 2002;36:1383-1392.)

Evolution of autoimmune hepatitis to primary sclerosing cholangitis: A sequential syndrome (*Human Study*)
Ayman A. Abdo, Vincent G. Bain, Krikor Kichian, Samuel S. Lee
Recently, the autoimmune hepatitis (AIH)/primary sclerosing cholangitis (PSC) overlap syndrome has been reported increasingly. In this syndrome, patients present with features of both AIH and PSC. It has been suggested that the 2 diseases may be sequential in their occurrence, whereby patients have features of AIH and then after a number of years develop features of PSC, but clear confirmation of evolution has not been documented in adults. We describe 6 adult cases in which PSC was diagnosed many years after well-established AIH. Six patients are described in whom AIH definitely was diagnosed at presentation. No evidence of biliary disease was noted on the initial liver biopsy or endoscopic retrograde cholangiography (ERCP). All patients responded well to immunosuppressive therapy. After an average duration of follow-up of 4.6 years they became resistant to immunosuppression, and developed clear features of PSC, which was confirmed by ERCP in all patients. The average age of the patients at first presentation was 31.3 years, 2 were women and 4 were men, and 3 had ulcerative colitis. We found no specific features at presentation that could predict this evolutionary outcome. In conclusion, patients with well-established AIH can, after variable duration of follow-up, develop PSC. In patients with AIH who become resistant to immunosuppression or develop significant cholestasis, PSC should be ruled out by ERCP. (HEPATOLOGY 2002;36:1393-1399.)

Viral Hepatitis

Cellular vacuolization and apoptosis induced by hepatitis B virus large surface protein
Ngee-Chih Foo, Byung Y. Ahn, Xiaohong Ma, William Hyun, T. S. Benedict Yen
Fibrosing cholestatic hepatitis (FCH) is a rapidly progressive form of viral hepatitis B that occurs in severely immunosuppressed patients. Pathologically, the liver in FCH is characterized by widespread hepatocyte vacuolization and apoptosis, which, in contrast to more common forms of hepatitis B, is only rarely associated with significant inflammation. Therefore, it has been proposed that, in FCH, hepatocytes may be injured by a direct cytopathic effect of the virus rather than by the host immune response. In support of this hypothesis, we present evidence that cultured hepatoma cells that had been transfected with a plasmid selectively expressing the viral large surface protein form numerous large vacuoles and undergo apoptosis. The similarity of the cytopathology in FCH in vivo and in these transfected cells in vitro strongly implicates the large surface protein as the direct cause of this acute liver disease. This conclusion is further supported by the published demonstration that hepatocytes tend to accumulate large surface protein in FCH, which may reflect its overexpression by the virus. In conclusion, our data implicate the large surface protein as a major cause of hepatocyte injury in fibrosing cholestatic hepatitis. (HEPATOLOGY 2002;36:1400-1407.)

Quantitative serum HBV DNA levels during different stages of chronic hepatitis B infection (*Human Study*)
Chi-Jen Chu, Munira Hussain, Anna S. F. Lok
The goals of this retrospective study were to determine whether there is a threshold hepatitis B virus (HBV) DNA value associated with spontaneous or antiviral therapy­related hepatitis B e antigen (HBeAg) clearance. We also investigated whether there is an HBV DNA value that can be used for differentiating inactive carriers from patients with HBeAg-negative chronic hepatitis B. HBV DNA levels in sequential serum samples of 165 Chinese patients with different stages of chronic HBV infection were quantified by a polymerase chain reaction (PCR)­based assay. Our results showed that almost all of the patients (89%) who remained HBeAg-positive had HBV DNA levels that were persistently above 105 copies/mL. Serum HBV DNA levels decreased by a mean of 3 log10 in patients with HBeAg loss, but 51% had levels above 105 copies/mL at the time HBeAg first became undetectable. Mean serum HBV DNA levels were significantly lower in HBeAg-negative patients. HBV DNA value above 105 copies/mL would exclude all inactive carriers, but 45% of patients with HBeAg-negative chronic hepatitis would also be excluded if testing were only performed at presentation and 30% would be excluded if testing were performed on 3 occasions. In conclusion, serum HBV DNA levels decreased significantly in patients with HBeAg loss, but there was no threshold HBV DNA level associated with HBeAg clearance. Given the fluctuating course of HBeAg-negative chronic hepatitis, it is not possible to define a single cutoff HBV DNA value for differentiating inactive carriers from patients with HBeAg-negative chronic hepatitis.
(HEPATOLOGY 2002;36:1408-1415.)

Genetic polymorphisms in interferon pathway and response to interferon treatment in hepatitis B patients: A pilot study (*Human Study*)
Jennifer K. King, Shiou-Hwei Yeh, Ming-Wei Lin, Chun-Jen Liu, Ming-Yang Lai, Jia-Horng Kao, Ding-Shinn Chen, Pei-Jer Chen
Interferon alfa (IFN-) therapy remains a mainstay of treatment in active hepatitis B. However, sustained remission rates remain relatively low, and the search for factors important for response to therapy continues. Our study aimed to identify the host single nucleotide polymorphisms (SNPs) that predict IFN response in hepatitis B patients. We selected genes in the IFN pathway involved in antiviral and signaling activities and sequenced 22 SNPs for each of our 82 patients. Our results identified 2 SNPs in the antiviral pathway that may influence IFN response. One SNP in the regulatory region of the eIF-2 gene revealed A/G alleles. The rate of A/G heterozygotes is 22% in nonresponders (NR) and 2% in sustained responders (R), with an odds ratio (OR) of 12.82 (95% CI: 1.52-107.85, P = .009). After adjustment for age, sex, and HBV DNA level, the OR reaches 14.94 (95% CI: 1.45-153.71, P = .023). This marker revealed greater significance than HBV DNA levels (OR: 5, 95% CI: 1.01-2.43, P = .033) as a marker for IFN response, suggesting its potential advantage over conventional predictors. In addition, borderline significance for the SNP in MxA gene promoter at nt ­88 revealed G/T alleles, with the G/T heterozygote rate being 19% in nonresponders and 43% in sustained R (P = .061), concurring with a previous study involving hepatitis C patients. In conclusion, this pilot identified SNPs as potential markers that could predict hepatitis B patient response. These observations may help guide future large-scale studies in examining host SNPs for their clinical utility in predicting IFN response. (HEPATOLOGY 2002;36:1416-1424.)

HBV genotype B is associated with better response to interferon therapy in HBeAg(+) chronic hepatitis than genotype C (*Human Study*)
Chun Tao Wai, Chi-Jen Chu, Munira Hussain, Anna S. F. Lok
Hepatitis B virus (HBV) genotype and precore/core promoter mutations have been implicated in spontaneous and interferon alfa (IFN-)­related hepatitis B e antigen (HBeAg) seroconversion. We performed a retrospective analysis of a previously reported randomized controlled trial to determine the effects of HBV genotype and precore/core promoter mutations on IFN- response in patients with HBeAg-positive chronic hepatitis. Clinical data and stored sera from 109 (95%) patients in the original trial were analyzed. Seventy-three patients received IFN- and 34 received no treatment (controls). Almost all patients had HBV genotypes B (38%) and C (60%). Antiviral response was achieved in 39% and 17% of IFN-­treated patients (P = .03) and in 10% and 8% of untreated controls (P = .88) with HBV genotype B and C, respectively. Multivariate analysis identified HBV genotype B, elevated pretreatment alanine aminotransferase (ALT) levels, and low pretreatment HBV-DNA levels but not IFN- treatment as independent factors associated with antiviral response. Among the 66 patients with elevated pretreatment ALT level, antiviral response was achieved in 57% and 21% of IFN-­treated patients (P = .019), and in 25% and 8% of untreated controls (P = .45) with HBV genotype B and C, respectively. Multivariate analysis showed that genotype B and low pretreatment HBV-DNA levels were independent predictors of antiviral response. In conclusion, our data showed that HBV genotype B was associated with a higher rate of IFN-induced HBeAg clearance compared with genotype C. Stratification for HBV genotypes should be considered in future clinical trials of antiviral therapy of chronic hepatitis B. (HEPATOLOGY 2002;36:1425-1430.)

Expression profiling of liver cell lines expressing entire or parts of hepatitis C virus open reading frame
Hideki Aizaki, Takashi Harada, Motoyuki Otsuka, Naohiko Seki, Mami Matsuda, Yue Wei Li, Hayato Kawakami, Yoshiharu Matsuura, Tatsuo Miyamura, Tetsuro Suzuki
Although hepatitis C virus (HCV) is a causative agent of liver diseases, its mechanism of pathogenesis is still unclear, mainly because of the lack of adequate cell culture systems to support HCV infection and replication. In this report, we describe development and characterization of human hepatoma cell lines constitutively expressing entire (Hep394) or parts (Hep352, Hep3294) of the HCV open reading frame (ORF). The viral and cellular proteolytic machinery involved in the viral precursor processing was consistently functional, and processed HCV proteins were synthesized in these established cell lines. By using a cDNA microarray analysis coupled with semiquantitative reverse-transcription polymerase chain reaction (RT-PCR), we identified 12 genes up-regulated and 4 genes down-regulated in Hep394 cells. With regard to genes related to cell growth regulation, we found up-regulation of forkhead transcription factor FREAC-1, poly (A) binding protein PABP2, and Ras suppressor Rsu-1. Another category of changes in gene expression includes MHC antigens, which play an important role in the T-cell-mediated immune reaction in the liver. In conclusion, functional genomic approaches comparing expression among the different cell lines expressing parts of the HCV genome may promote our understanding of the molecular basis of pathogenicity of HCV infection. (HEPATOLOGY 2002;36:1431-1438.)

Extrahepatic manifestations of hepatitis C among United States male veterans (*Human Study*)
Hashem B. El-Serag, Howard Hampel, Christine Yeh, Linda Rabeneck
Hepatitis C virus (HCV) has been associated with several extrahepatic conditions. To date, most studies assessing these associations involved small numbers of patients and lacked a control group. Using the computerized databases of the Department of Veterans Affairs, we carried out a hospital-based case-control study that examined all cases of HCV-infected patients hospitalized during 1992 to 1999 (n = 34,204) and randomly chosen control subjects without HCV (n = 136,816) matched with cases on the year of admission. The inpatient and outpatient files were searched for several disorders involving the skin (porphyria cutanea tarda [PCT], vitiligo, and lichen planus); renal (membranous glomerulonephritis [GN] and membranoproliferative glomerulonephritis); hematologic (cryoglobulin, Hodgkin's and non-Hodgkin's lymphoma [NHL]); endocrine (diabetes, thyroiditis); and rheumatologic (Sjögren's syndrome). The association between HCV and these disorders was examined in multivariate analyses that controlled for age, gender, ethnicity, and period of military service. Patients in the case group were younger in age (45 vs. 57 years), were more frequently nonwhite (39.6% vs. 26.3%), and were more frequently male (98.1% vs. 97.0%). A significantly greater proportion of HCV-infected patients had PCT, vitiligo, lichen planus, and cryoglobulinemia. There was a greater prevalence of membranoproliferative GN among patients with HCV but not membranous GN. There was no significant difference in the prevalence of thyroiditis, Sjögren's syndrome, or Hodgkin's or NHL. However, NHL became significant after age adjustment. Diabetes was more prevalent in controls than cases, but no statistically significant association was found after adjustment for age. In conclusion, we found a significant association between HCV infection and PCT, lichen planus, vitiligo, cryoglobulinemia, membranoproliferative GN, and NHL. Patients presenting with these disorders should be tested for HCV infection. (HEPATOLOGY 2002;36:1439-1445.)

Oral lichen planus pathogenesis: A role for the HCV-specific cellular immune response (*Human Study*)
Massimo Pilli, Amalia Penna, Alessandro Zerbini, Paolo Vescovi, Maddalena Manfredi, Francesco Negro, Marco Carrozzo, Cristina Mori, Tiziana Giuberti, Carlo Ferrari, Gabriele Missale
Hepatitis C virus infection can be associated with different extrahepatic manifestations, including lichen planus; however, no clear role for HCV in their pathogenesis has been established. T cells were isolated from lichen biopsy specimens of 7 HCV positive patients with oral lichen planus. HCV-specific CD4+ T-cell lines were obtained in 4 patients from lichen lesions but only in 2 of them from the peripheral blood. Different clonal populations were found in oral tissue and peripheral blood of individual patients, as shown by TCR-V analysis of antigen-specific T cells. Frequency of HCV-specific CD8+ cells tested with 4 different HCV tetramers was significantly higher in the lichen tissue than in the circulation; moreover, lichen-derived HCV-specific CD8+ T cells showed the phenotype of recently activated T cells because most of them were CD69+ and produced interferon gamma (IFN-) but expanded poorly in vitro upon antigen stimulation. The specificity of HCV-reactive T-cell recruitment into the lichen tissue was further confirmed by the absence of HBV-specific T cells within lichen lesions in 3 additional patients with lichen planus associated with HBV infection. Our study shows HCV-specific T-cell responses at the site of the lesions of an HCV-associated dermatologic disease, sustained by HCV-specific T cells with phenotypic and functional characteristics of terminally differentiated effector cells. In conclusion, this finding and the detection of HCV RNA strands in the lichen tissue strongly suggest a role for HCV-specific T-cell responses in the pathogenesis of oral lichen planus associated with HCV infection. (HEPATOLOGY 2002;36:1446-1452.)

Liver Biology and Pathobiology

Endothelin-1 and heme oxygenase-1 as modulators of sinusoidal tone in the stress-exposed rat liver
Hauke Rensing, Inge Bauer, Jian X. Zhang, Markus Paxian, Benedikt H. J. Pannen, Yukihiro Yokoyama, Mark G. Clemens, Michael Bauer
Heme oxygenase (HO)-1 is up-regulated after ischemia/reperfusion and contributes to maintenance of hepatic perfusion and integrity. Blockade of HO-1 leads to an increased portal pressor response in the stress-exposed liver. We tested whether the increase in portal pressure reflects unmasking of a concomitant up-regulation of the vasoconstrictor endothelin (ET)-1. Hemorrhagic shock induced messenger RNAs encoding HO-1 (16-fold) and ET-1 (9-fold) with a similar time course in the liver. At maximum induction of both mediators, rats received either vehicle or the endothelin ETA/B antagonist bosentan (10 mg/kg intravenously). Subsequently, the HO pathway was blocked in all animals by tin-protoporphyrin (SnPP)-IX (50 µmol/kg intravenously). Portal and sinusoidal hemodynamics were measured using microflow probes and intravital microscopy, respectively. Blockade of the HO pathway led to a significant increase in portal resistance (sham/SnPP-IX, 0.17 ± 0.046 mm Hg · min · mL­1; shock/vehicle/SnPP-IX, 0.57 ± 0.148 mm Hg · min · mL­1; P < .05) and a decrease in sinusoids conducting flow (shock/vehicle/SnPP-IX: baseline, 28.3 ± 0.85 sinusoids/mm; 10 minutes after SnPP-IX, 23.1 ± 1.09 sinusoids/mm; P < .05). Intravital microscopy showed narrowing of failing sinusoids colocalizing with stellate cells after blockade of the HO pathway. Blockade of ETA/B receptors attenuated the increase in portal resistance (shock/bosentan/SnPP-IX, 0.29 ± 0.051 mm Hg · min · mL­1) and prevented sinusoidal perfusion failure (shock/bosentan/SnPP-IX: baseline, 28.2 ± 0.97 sinusoids/mm; 10 minutes after SnPP-IX, 28.8 ± 1.18 sinusoids/mm) as well as sinusoidal narrowing. In conclusion, a functional interaction of the up-regulated vasodilatory HO system and the vasoconstrictor ET-1 on the sinusoidal level exists under stress conditions. Both mediator systems affect sinusoidal diameter via direct action on hepatic stellate cells in vivo. (HEPATOLOGY 2002;36:1453-1465.)

Endogenous interferon protects against cholestatic liver injury in mice
Miguel E. Sewnath, Tom Van Der Poll, Cornelis J. F. Van Noorden, Fiebo J. W. Ten Kate, Dirk J. Gouma
Cholestatic patients suffer from high perioperative morbidity and mortality, but the pathophysiology is still unknown. Interferon (IFN-) may play a role during cholestasis. Therefore, bile duct ligation (BDL) was induced in IFN- -chain receptor­deficient (IFN-R1­/­) and wild-type (IFN-R1+/+) mice. BDL elicited increased IFN- messenger RNA and protein levels in the liver. One week after BDL, IFN-R1+/+ mice showed less severe jaundice and liver injury than IFN-R1­/­ mice, as reflected by lower bilirubin and liver enzyme levels. In accordance, livers of IFN-R1+/+ mice displayed smaller areas of necrosis by two-thirds than IFN-R1­/­ mice on histopathologic examination (P < .05), whereas mitotic activity and proliferating cell nuclear antigen (PCNA) labeling index was more than twice as high in IFN-R1+/+ mice (P < .05). Livers of IFN-R1+/+ mice displayed higher rates of apoptosis as indicated by DNA fragmentation rate, the number of apoptotic bodies, and poly ADP-ribose polymerase (PARP) immunostaining. BDL was not associated with lethality in IFN-R1+/+ mice; IFN-R1­/­ mice, however, died from 10 days onward and survival after 2 weeks was 62% (10 of 16). In conclusion, these data suggest that IFN- protects against liver injury during extrahepatic cholestasis by stimulation of apoptosis and subsequent proliferation of hepatocytes, leading to elegant removal of damaged hepatocytes, thus preventing necrosis and concomitant inflammatory responses. (HEPATOLOGY 2002;36:1466-1477.)

The effect of ethanol on asialoglycoprotein receptor­mediated phagocytosis of apoptotic cells by rat hepatocytes
Benita L. McVicker, Dean J. Tuma, Jacy A. Kubik, Agnes M. Hindemith, Cheryl R. Baldwin, Carol A. Casey
Apoptotic cell death is a well-defined process that is controlled by intrinsic cellular mechanisms followed by the generation of apoptotic bodies and their subsequent rapid elimination through the action of phagocytic cells. Within the liver, the asialoglycoprotein receptor (ASGP-R) has been shown to be involved in the phagocytosis of apoptotic hepatocytes, as well as altered cellular endocytic events after ethanol administration. The goal of the present study was to further clarify the capacity of ASGP-R to phagocytose apoptotic cells in relationship to the damaging events that occur with alcohol consumption. For these experiments, we used an in vitro suspension assay coupled with flow cytometry to measure apoptotic cell engulfment by rat hepatocytes after chronic ethanol administration. The results of this assay indicated that the phagocytosis of apoptotic cells was decreased significantly (30% to 42%, P < .05) in the presence of antibody specific for ASGP-R as well as the introduction of competing sugars in the media. In addition, uptake of apoptotic cells was impaired by 40% to 60% (P < .05) in cells obtained from ethanol-fed animals as compared with controls. In conclusion, the ASGP-R is involved in the recognition and uptake of apoptotic cells and this process is altered significantly by ethanol treatment. These findings may play a role in a better understanding of the clinical manifestations of alcohol-induced liver injury as altered uptake of apoptotic cells via ASGP-R may result in the release of proinflammatory mediators, the introduction of autoimmune responses, and inflammatory injury to the tissue. (HEPATOLOGY 2002;36:1478-1487.)

Establishment of a highly efficient gene transfer system for mouse fetal hepatic progenitor cells
Kentaro Yasuchika, Tetsuro Hirose, Hideaki Fujii, Shoshiro Oe, Koichi Hasegawa, Takahisa Fujikawa, Hisaya Azuma, Yoshio Yamaoka
Because of a donor shortage problem in liver transplantation, cell transplantation has been anticipated as a useful bridge or substitute therapy, and has necessitated the development of cell sources other than donated organs. Therefore, the use of fetal hepatic progenitor cells (HPCs) is now being focused on. In this study, we intended to establish an efficient ex vivo nonviral gene-transfer system using a newly developed isolation and culture system for mouse fetal HPCs. Fetal HPCs, characterized using immunocytochemistry and reverse-transcription polymerase chain reaction (RT-PCR) for lineage markers, were collected from E13.5 Balb/c mice using change in size because of cell aggregation by their homophilic cell-to-cell binding occurring during suspension culture. Optimal conditions for culture and ex vivo gene transfection for fetal HPCs were determined by 3H-thymidine incorporation and the expression efficacy of transfected red fluorescent protein (DsRed) gene in different culture media. The optimum timing for gene transfection was also evaluated. To evaluate the in vivo expression of the transferred gene, DsRed-transferred fetal HPCs were transplanted into 70% partially hepatectomized allogenic mice. The highest efficacy of DsRed gene transfection into fetal HPCs in vitro (45% ± 12.3%) was achieved with culture media, which also enabled the highest 3H-thymidine incorporation, containing the deleted form of hepatocyte growth factor (dHGF) and insulin, and when transfection was performed immediately after isolation. In vivo DsRed expression in fetal HPCs was maintained concomitantly with albumin expression even after HPC transplantation. In conclusion, we established a highly efficient in vitro gene transfer system for mouse fetal HPCs using a newly developed isolation and culture system. (HEPATOLOGY 2002;36:1488-1497.)

TRAIL-mediated apoptosis requires NF-B inhibition and the mitochondrial permeability transition in human hepatoma cells (*Human Study*)
Young-Soo Kim, Robert F. Schwabe, Ting Qian, John J. Lemasters, David A. Brenner
Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induces apoptosis in a wide range of malignant cells. However, several cancers, including human hepatoma, are resistant to TRAIL. In this study, we analyzed TRAIL-induced pro- and antiapoptotic signaling pathways in human hepatoma cells. Nuclear factor B (NF-B) was found to be a critical TRAIL-induced antiapoptotic factor in the PLC/PRF/5, HepG2, and Hep3B cell lines. TRAIL-induced NF-B activation was preceded by IB kinase (IKK) activation and IB degradation and depended on TRAF2, NF-B-inducing kinase (NIK), IKK1, and IKK2. Accordingly, inhibition of NF-B by adenoviral dominant negative (dn) TRAF2, NIKdn, IKK1dn, IKK2dn, or IBsr sensitized PLC/PRF/5 cells to rhTRAIL, resulting in 40% to 50% cell death after 48 hours as compared with <10% with rhTRAIL alone. Agonistic anti-TRAIL receptor 1 and anti-TRAIL receptor 2 antibodies or combinations of both were equally efficient in inducing apoptosis as rhTRAIL, indicating that decoy receptors did not contribute to resistance toward TRAIL under the conditions of our study. TRAIL-mediated apoptosis depended on FADD, caspase 8 and 3 as demonstrated by the ability of FADDdn, CrmA, and pharmacologic caspase inhibitors to prevent apoptosis. Confocal microscopy showed the onset of the mitochondrial permeability transition (MPT) 5 hours after rhTRAIL plus actinomycin D, which was followed by cytochrome c release. The MPT was critical for TRAIL-induced apoptosis as demonstrated by the ability of pharmacologic MPT inhibitors to completely protect PLC/PRF/5 cells. In conclusion, NF-B prevents TRAIL-induced apoptosis in human hepatoma through a TRAIL-activated TRAF2-NIK-IKK pathway. Inhibition of NF-B unmasks a TRAIL-induced apoptotic signaling cascade that involves FADD, caspase 8, the MPT, and caspase 3. (HEPATOLOGY 2002;36:1498-1508.)

Epidermal growth factor­induced activation of the insulin-like growth factor I receptor in rat hepatocytes
Hazem Hallak, Giesla Moehren, Jei Tang, Mohamad Kaou, Mouhamad Addas, Jan B. Hoek, Raphael Rubin
Insulin-like growth factor I (IGF-I) plays a critical role in the induction of cell cycle progression and survival in many cell types. However, there is minimal IGF-I binding to hepatocytes, and a role for IGF-I in hepatocyte signaling has not been elucidated. The dynamics of IGF-I receptor (IGF-IR) activation were examined in freshly isolated rat hepatocytes. IGF-I did not activate the IGF-IR. However, des(1-3)IGF-I, which weakly binds IGF binding protein-3 (IGFBP-3), induced IGF-IR phosphorylation. IGFBP-3 surface coating was identified by confocal immunofluorescence microscopy. In contrast with the inactivity of IGF-I, epidermal growth factor (EGF) induced the tyrosine phosphorylation of the IGF-IR in parallel with EGF receptor phosphorylation. Transactivation of the IGF-IR by EGF was inhibited by tyrphostin I-Ome-AG538, a tyrosine kinase inhibitor with high specificity for the IGF-IR. Src kinase inhibitors pyrazolopyrimidine PP-1 and PP-2 inhibited transactivation of the IGF-IR by EGF. EGF stimulated the tyrosine phosphorylation of Src, and induced its association with the IGF-IR. EGF-induced phosphorylations of insulin-related substrate (IRS)-1, IRS-2, Akt, and p42/44 mitogen-activated protein kinases (MAPKs) were inhibited variably by I-Ome-AG538. In conclusion, the data show an EGF- and Src-mediated transactivation pathway for IGF-IR activation in hepatocytes, and indicate a role for the IGF-IR in hepatocyte intracellular signaling. The findings also show a role for IGFBP-3 in the inhibition of IGF-I signaling in hepatocytes. (HEPATOLOGY 2002;36:1509-1518.)

Hepatoma-derived growth factor is highly expressed in developing liver and promotes fetal hepatocyte proliferation
Hirayuki Enomoto, Kenya Yoshida, Yoshihiko Kishima, Taisei Kinoshita, Mitsunari Yamamoto, Allen D. Everett, Atsushi Miyajima, Hideji Nakamura
Hepatoma-derived growth factor (HDGF) is a heparin-binding protein, which has been purified from the conditioned media of HuH-7 hepatoma cells. Recent studies have suggested the involvement of HDGF in development of the kidney and cardiovascular systems. In the present study, we investigated the possibility that HDGF was also involved in liver development. Northern blot and immunostaining revealed unique expression patterns of HDGF in liver development. HDGF expression was strongly detected in the fetal liver of the midgestation stage and was markedly decreased near birth. Its expression was mainly detected in stromal cells, including immature hepatocytes. Expression in hepatocytes decreased with differentiation. Administration of recombinant HDGF enhanced the growth of primary cultured fetal hepatocytes significantly, although the effect was small. The effect of exogenous HDGF on the proliferation of neonatal hepatocytes was also small and significant only at one point, despite the lower expression of endogenous HDGF, suggesting that the differences exist between fetal and neonatal hepatocytes. However, adenoviral introduction of HDGF antisense cDNA into the fetal hepatocytes significantly suppressed their proliferation, and the inhibitory effect of HDGF antisense virus was reversed by exogenous HDGF. In conclusion, HDGF helps regulate the hepatocyte proliferation in liver development. (HEPATOLOGY 2002;36:1519-1527.)

Dysregulation of glycogen synthase kinase-3 signaling in hepatocellular carcinoma cells
Christèle Desbois-Mouthon, Marie-José Blivet-Van Eggelpoël, Eléonore Beurel, Mathieu Boissan, Roland Delélo, Axelle Cadoret, Jacqueline Capeau
It has been reported that upstream components of the insulin-like growth factor (IGF) signaling axis could be overexpressed during hepatocarcinogenesis in humans and rodents. However, the signal transduction pathways activated downstream have been poorly studied. Here, we examined whether glycogen synthase kinase-3 (GSK-3) could be a target in human hepatoma cell lines and transgenic ASV mice with hepatic expression of the SV40 large T antigen. In HuH7, Mahlavu, and Hep3B cells, basal levels of GSK-3Ser9 phosphorylation were strongly elevated, indicating that GSK-3 was inhibited. GSK-3 phosphorylation was insensitive to exogenous IGFs and was blocked with an IGF-1 receptor­neutralizing antibody in Mahlavu and Hep3B cells. By using LY294002 and ML-9, which act as phosphatidylinositol 3-kinase (PI3-K) and Akt inhibitors, respectively, we showed that GSK-3 phosphorylation required PI3-K activation in both cell lines whereas downstream Akt activation was required only in Mahlavu cells. However, in the 2 cell lines, GSK-3Ser9 phosphorylation was controlled by protein kinase C (PKC) because it was blocked by an inhibitory PKC peptide. The blockage of GSK-3 phosphorylation markedly inhibited glycogen synthesis and decreased -catenin expression. In addition, the overexpression of a constitutively active GSK-3 reduced AP-1­mediated gene transcription in Hep3B cells. Finally, we observed that reexpression of IGF-2 in tumoral livers from ASV mice was associated with a marked phosphorylation of GSK-3. In conclusion, our results identify GSK-3 as a molecular target of the constitutive activation of the IGF axis in in vitro and in vivo models of hepatocarcinogenesis. Persistent phosphorylation of GSK-3 could be critical for regulation of glycogen metabolism and cell growth in hepatoma cells. (HEPATOLOGY 2002;36:1528-1536.)

GASTROENTEROLOGY

Table of Contents for december 2002 · Volume 123 · Number 6

Clinical­Alimentary Tract

A randomized comparison of quadruple and triple therapies for Helicobacter pylori eradication: The QUADRATE study
P. H. Katelaris, G. M. Forbes, N. J. Talley, B. Crotty, for the Australian Pantoprazole H. pylori Study Group
Background & Aims: Direct comparisons of bismuth and proton pump inhibitor (PPI)-based triple and quadruple therapies for Helicobacter pylori eradication are lacking. To address this, a randomized study was conducted.
Methods: Infected dyspeptic patients received pantoprazole 40 mg, amoxicillin 1000 mg, and clarithromycin 500 mg, all twice daily, for 7 days (PAC7); or pantoprazole 40 mg twice daily, bismuth subcitrate 108 mg, and tetracycline 500 mg, both 4 times daily, and metronidazole 200 mg 3 times daily and 400 mg at night for 7 days (PBTM7); bismuth subcitrate 108 mg and tetracycline 500 mg, both 4 times daily, and metronidazole 200 mg 3 times daily and 400 mg at night for 14 days (BTM14). Outcome was assessed with 13C-urea breath test.
Results: Eradication rates (intention to treat [n = 405]/per protocol [n = 320]) were similar for PAC7 (78%/82%) and PBTM7 (82%/88%); the latter significantly superior to BTM14 (69%/74%; P < 0.01). Pretreatment metronidazole resistance (MR) was 53% and clarithromycin resistance was 8%. Eradication rates for primary metronidazole sensitive/resistant isolates were 74%/87% with PAC7 and 80%/81% for PBTM7, compared with 76%/55% (P < 0.02) for BTM14. Noncompliance was greater with BTM14 (15%; P < 0.001) than PAC7 (3%) or PBTM7 (6%). Moderate-severe adverse events were more common with BTM14 (45%; P < 0.001), than PAC7 (23%) or PBTM7 (25%) with more discontinuations (9%, 2%, 3%, respectively).
Conclusions: One-week PPI triple therapy is well tolerated and effective. The addition of PPI to bismuth triple therapy allows reduction of treatment duration with improved efficacy and tolerability, despite a high rate of MR. Quadruple therapy appears to overcome pretreatment MR in most cases. Two-week bismuth triple therapy is significantly inferior to quadruple therapy and less well tolerated than both 1-week therapies.

Nonsteroidal anti-inflammatory drugs, apoptosis, and colorectal adenomas
C. Martin, A. Connelly, T. O. Keku, S. B. Mountcastle, J. Galanko, J. T. Woosley, B. Schliebe, P. K. Lund, R. S. Sandler
Background & Aims: Observational studies indicate that nonsteroidal anti-inflammatory drugs (NSAIDs) reduce the risk of colorectal neoplasia. The mechanism of this effect could be via modification of apoptotic activity in colonic mucosa. We examined grossly normal rectal mucosa in patients with adenomas and adenoma-free controls to assess the associations between NSAID use, adenomatous polyps, and apoptosis.
Methods: Study participants were drawn from consecutive patients who underwent colonoscopy between August, 1998, and February, 2000. Biopsy specimens were taken from normal-appearing rectal mucosa 10 cm from the anal verge. Apoptosis was scored from coded, H&E-stained sections using morphologic methods. Proliferation was scored using whole crypt mitotic counts. Univariate and multivariate regression analyses were conducted to estimate crude and adjusted odds ratios (ORs).
Results: There were 226 patients with adenomas and 493 adenoma-free controls. After adjusting for sex, age, race, and body mass index (BMI), individuals in the highest tertile of regular NSAID use were substantially less likely to have adenomas (OR 0.4; 95% CI: 0.2­0.7) compared with occasional or nonusers. Similarly, compared with the lowest tertile, persons in the highest tertile of rectal mucosal apoptotic activity were much less likely to have adenomas (OR 0.12; 95% CI: 0.07­0.20). NSAID use and apoptotic activity were not correlated (r = 0.10). Mucosal proliferation was not related to adenomas or NSAID use.
Conclusions: Our observations suggest that NSAID use and higher levels of mucosal apoptosis are independently associated with a lower prevalence of adenomas. The study shows a strong field effect for apoptosis.

Long-term improvement in functional dyspepsia using hypnotherapy
E. L. Calvert, L. A. Houghton, P. Cooper, J. Morris, P. J. Whorwell
Background & Aims: We have shown hypnotherapy (HT) to be effective in irritable bowel syndrome, with long-term improvements in symptomatology and quality of life (QOL). This study aimed to assess the efficacy of HT in functional dyspepsia (FD).
Methods: A total of 126 FD patients were randomized to HT, supportive therapy plus placebo medication, or medical treatment for 16 weeks. Percentage change in symptomatology from baseline was assessed after the 16-week treatment phase (short-term) and after 56 weeks (long-term) with 26 HT, 24 supportive therapy, and 29 medical treatment patients completing all phases of the study. QOL was measured as a secondary outcome.
Results: Short-term symptom scores improved more in the HT group (median, 59%) than in the supportive (41%; P = 0.01) or medical treatment (33%; P = 0.057) groups. HT also benefited QOL (42%) compared with either supportive therapy (10% [P < 0.001]) or medical treatment (11% [P < 0.001]). Long-term, HT significantly improved symptoms (73%) compared with supportive therapy (34% [P < 0.02]) or medical treatment (43% [P < 0.01]). QOL improved significantly more with HT (44%) than with medical treatment (20% [P < 0.001]). QOL did improve in the supportive therapy (43%) group, but 5 of these patients commenced taking antidepressants during follow-up. A total of 90% of the patients in the medical treatment group and 82% of the patients in the supportive therapy group commenced medication during follow-up, whereas none in the HT group did so (P < 0.001). Those in the HT group visited their general practitioner or gastroenterologist significantly less (median, 1) than did those in the supportive therapy (median, 4) and medical treatment (median, 4) groups during follow-up (P < 0.001).
Conclusions: HT is highly effective in the long-term management of FD. Furthermore, the dramatic reduction in medication use and consultation rate provide major economic advantages.

Complications of screening flexible sigmoidoscopy
T. R. Levin, C. Conell, J. A. Shapiro, S. G. Chazan, M. R. Nadel, J. V. Selby
Background & Aims: Flexible sigmoidoscopy (FS) is recommended for mass screening for colorectal cancer (CRC), yet little is known about the risk of adverse events when FS is used in general clinical practice. We aimed to determine the incidence of gastrointestinal complications and acute myocardial infarction (MI) after screening FS.
Methods: Northern California Kaiser Permanente Medical Care Program members of average risk for CRC (n = 107,704) who underwent screening FS during 1994 to 1996 (109,534 FS), as part of the Colorectal Cancer Prevention (CoCaP) program. The main outcome measure was hospitalization for gastrointestinal complications or acute MI within 4 weeks of FS.
Results: The mean age of subjects was 61 years, and 48.8% were female. Nongastroenterologist physicians, nurses, or physician assistants performed 72% of FS. Overall, 24 persons were hospitalized for a gastrointestinal complication. Of these, 7 were serious (2 perforations, 2 episodes of diverticulitis requiring surgery, 2 cases of bleeding requiring transfusion, and 1 episode of unexplained colitis). In multivariate models, complications were significantly more common in men than in women (odds ratio, 3.34; 95% confidence interval [CI], 1.34­10.13). MI occurred in 33 persons within 4 weeks of FS, but the incidence for this period was similar to that in the subsequent 48 weeks (rate ratio, 0.8; 95% CI, 0.6­1.2).
Conclusions: The risk of serious complications after screening FS in this setting appears to be modest. Although MI occurs after FS, the risk during the 4 weeks after the procedure appears to be similar to expectations for persons of screening age.

Effect of interleukin 1 polymorphisms on gastric mucosal interleukin 1 production in Helicobacter pylori infection
I.-R. Hwang, T. Kodama, S. Kikuchi, K. Sakai, L. E. Peterson, D. Y. Graham, Y. Yamaoka
Background & Aims: Although epidemiological studies suggest that interleukin (IL)-1 genetic polymorphisms are involved in Helicobacter pylori­related gastric carcinogenesis, the data are conflicting regarding the effects of these polymorphisms on IL-1 production.
Methods: IL-1B-511 polymorphism was genotyped by polymerase chain reaction (PCR)­restriction fragment length polymorphism, and IL-1RN variable number of tandem repeats was determined by PCR. Mucosal IL-1 levels were measured by enzyme-linked immunosorbent assay. To determine which factors influence mucosal IL-1 levels, gastric inflammation, and atrophy, multiple regression analyses were performed.
Results: We studied 117 H. pylori­infected Japanese patients. Carriers of the IL-1B-511T/T genotype or IL-1RN*2 allele had higher mucosal IL-1 levels than noncarriers (partial regression coefficient [PRC] ± SE), TT versus CC: 37.6 ± 6 [antrum] and 32.1 ± 6 [corpus] pg/mg protein (P < 0.001 for each), *1/*2 versus *1/*1: 24 ± 8 [antrum] (P <0.01) and 36.5 ± 7 [corpus] (P <0.001). Simultaneous carriers of IL-1B-511T/T genotype and IL-1RN*2 allele had the highest IL-1 levels (82.9 ± 12 [antrum] and 87.2 ± 11 [corpus]) and showed a synergistic effect between 2 loci. The *1/*2 carriers were closely related to atrophy (PRC ± SE; 0.87 ± 0.4 [antrum] and 0.93 ± 0.4 [corpus], P < 0.05), whereas being a carrier of the -511T/T genotype was related to severe gastric inflammation.
Conclusions: IL-1 genetic polymorphisms influenced H. pylori­related gastric mucosal IL-1 levels and were related to gastric inflammation and atrophy, factors thought to be important in gastric carcinogenesis.

Evaluation of tumor microsatellite instability using five quasimonomorphic mononucleotide repeats and pentaplex PCR
N. Suraweera, A. Duval, M. Reperant, C. Vaury, D. Furlan, K. Leroy, R. Seruca, B. Iacopetta, R. Hamelin
Background & Aims: The microsatellite instability (MSI) phenotype is a characteristic of the hereditary nonpolyposis colorectal cancer syndrome as well as approximately 15% of sporadic colon and gastric tumors. It is a valuable diagnostic marker for the identification of hereditary nonpolyposis colorectal cancer cases and may be a molecular predictive marker for the identification of colon cancer patients who benefit from chemotherapy. To evaluate MSI, a reference panel was proposed at an international consensus meeting, comprised of 2 mononucleotide (BAT-25, BAT-26) and 3 dinucleotide repeats. Analysis of BAT-26 is sufficient for detecting the MSI phenotype in most, but not all, cases. Additional results with dinucleotide markers can sometimes lead to incorrect classification of MSI tumors.
Methods: We describe here a single fluorescent multiplex system comprising 5 quasimonomorphic mononucleotide repeats for the detection of MSI tumors.
Results: None of 184 germline DNA samples, including 56 from African subjects, was found to contain allelic size variations in more than 2 of these markers. In contrast, all MSI tumors showed allelic size variations in 3 or more of the microsatellites. Using this assay, we confirmed (or reclassified in 6 cases) the MSI status of 124 colon and 50 gastric primary tumors and 16 colon cell lines.
Conclusions: We propose that using a pentaplex polymerase chain reaction system allows accurate evaluation of tumor MSI status of DNA with 100% sensitivity and specificity without the need to match normal DNA. This assay is simpler to use than those involving dinucleotides and is more specific than using BAT-26 alone.

Clinical­Liver, Pancreas, and Biliary Tract

Influence of HIV infection on the response to interferon therapy and the long-term outcome of chronic hepatitis B
V. Di Martino, T. Thevenot, J.-F. Colin, N. Boyer, M. Martinot, F. Degos, J.-P. Coulaud, J.-L. Vilde, F. Vachon, C. Degott, D. Valla, P. Marcellin
Background & Aims: The outcome of chronic hepatitis B and the efficacy of interferon alfa (IFN-) remain controversial in human immunodeficiency virus (HIV)-positive patients. We analyzed the influence of HIV coinfection on the response to IFN- therapy, long-term virologic status, progression to cirrhosis, and mortality.
Methods: This was a retrospective follow-up cohort study of 141 consecutive hepatitis B e antigen­positive patients (69 HIV positive) followed up for 45 months.
Results: The short-term response to IFN- therapy was not significantly different in HIV-positive and HIV-negative patients (28% vs. 51%; P = 0.06) but was poorer in cases of low CD4 cell count (P = 0.038). The hepatitis B virus (HBV) reactivation rate was higher in HIV-positive patients (P = 0.033) and was associated with low CD4 cell count. The risk of cirrhosis was higher in HIV-positive patients with a CD4 cell count <200/mm3 (relative risk [RR], 4.57; P = 0.007), in IFN-­untreated patients (RR, 2.63; P = 0.041), in patients older than 33 years (RR, 4.59; P = 0.008), and in cases of high necroinflammatory score at baseline (RR, 1.27; P = 0.010). Cirrhosis-related death was more frequent in HIV-positive patients with low CD4 cell count at baseline (P = 0.041), in alcohol consumers (P = 0.001), in IFN-­untreated patients (P = 0.052), and in patients with high histology activity index at baseline (P = 0.005).
Conclusions: HIV coinfection was associated with poorer response to IFN- therapy, more frequent HBV reactivations, and increased incidence of cirrhosis and cirrhosis-related death in cases of low CD4 count. IFN- therapy decreased the incidence of HBV cirrhosis regardless of HIV status or serologic response.

Coffee intake is associated with lower risk of symptomatic gallstone disease in women
M. F. Leitzmann, M. J. Stampfer, W. C. Willett, D. Spiegelman, G. A. Colditz, E. L. Giovannucci
Background & Aims: Metabolic studies have shown that coffee affects several hepatobiliary processes that are involved in cholesterol lithogenesis. We previously showed that coffee drinking was associated with a lower risk of symptomatic gallstone disease in men.
Methods: We prospectively examined the association between coffee drinking and cholecystectomy, a surrogate of symptomatic gallstone disease, in a cohort of 80,898 women age 34­59 years in 1980 who had no history of gallstone disease. Coffee consumption and cholecystectomy were reported by participants on biennial mailed questionnaires.
Results: During 20 years of follow-up to the year 2000, 7,811 women reported a cholecystectomy. Compared with women who consistently reported consuming no caffeinated coffee, the multivariate relative risks (adjusting for risk factors for gallstone disease) of cholecystectomy comparing increasing categories of consistent intake of caffeinated coffee (0, 1, 2­3, and 4 cups/day) were 1.0, 0.91, 0.78, and 0.72 (95% confidence interval comparing extreme categories, 0.62­0.84; P value of test for trend < 0.0001). Caffeine intake from beverages and dietary sources was also inversely associated with risk of cholecystectomy. The multivariate relative risks comparing increasing categories of caffeine intake (25, 26­100, 101­200, 201­400, 401­800, and >800 mg/day) were 1.0, 1.03, 1.01, 0.94, 0.85, and 0.85 (95% confidence interval comparing extreme categories, 0.74­0.96; P value of test for trend < 0.0001). In contrast, decaffeinated coffee was not associated with risk.
Conclusions: These data suggest that consumption of caffeinated coffee may play a role in the prevention of symptomatic gallstone disease in women.

Entecavir is superior to lamivudine in reducing hepatitis B virus DNA in patients with chronic hepatitis B infection
C.-L. Lai, M. Rosmawati, J. Lao, H. Van Vlierberghe, F. H. Anderson, N. Thomas, D. Dehertogh
Background & Aims: Entecavir is a novel and selective nucleoside analogue with potent activity against hepatitis B virus (HBV).
Methods: In a 24-week, double-blind, randomized, multicenter, phase II clinical trial, the safety and efficacy of entecavir (0.01 mg/day, 0.1 mg/day, or 0.5 mg/day orally) were compared with lamivudine (100 mg/day orally). Patients (n = 169) chronically infected with HBV (hepatitis B e antigen [HBeAg]-positive and -negative) were evaluated for efficacy.
Results: Compared with lamivudine, entecavir reduced HBV DNA by an additional 0.97 log10 at the 0.1-mg/day dose and an additional 1.28 log10 at the 0.5-mg/day dose (P < 0.0001). A clear dose-response relationship was observed for entecavir with the higher doses showing significantly greater viral suppression. In patients treated with entecavir 0.5 mg/day, 83.7% had an HBV-DNA level below the lower limit of detection of the Quantiplex branched DNA (bDNA) assay (Bayer-Versant Diagnostics, formerly Chiron Diagnostics, Emeryville, CA), compared with 57.5% treated with 100 mg/day lamivudine (P = 0.008). In both treatment arms, very few patients achieved HBeAg loss and/or seroconversion by week 22. More patients treated with the 0.1-mg/day and 0.5-mg/day doses of entecavir had normalization of alanine transaminase (ALT) levels at week 22 compared with lamivudine (P = not significant). Entecavir was well tolerated; most adverse events were mild to moderate, transient, and comparable in all study arms.
Conclusions: This study showed that entecavir has potent antiviral activity against HBV at 0.1-mg/day and 0.5-mg/day doses, both of which were superior to lamivudine in chronically infected HBV patients.

Transjugular intrahepatic portosystemic shunting versus paracentesis plus albumin for refractory ascites in cirrhosis
P. Ginès, J. Uriz, B. Calahorra, G. Garcia-Tsao, P. S. Kamath, L. R. del Arbol, R. Planas, J. Bosch, V. Arroyo, J. Rodés, for the International Study Group on Refractory Ascites in Cirrhosis
Background & Aims: The transjugular intrahepatic portosystemic shunt (TIPS) has been shown to be more effective than repeated paracentesis plus albumin in the control of refractory ascites. However, its effect on survival and healthcare costs is still uncertain.
Methods: Seventy patients with cirrhosis and refractory ascites were randomly assigned to TIPS (35 patients) or repeated paracentesis plus intravenous albumin (35 patients). The primary endpoint was survival without liver transplantation. Secondary endpoints were complications of cirrhosis and costs.
Results: Twenty patients treated with TIPS and 18 treated with paracentesis died during the study period, whereas 7 patients in each group underwent liver transplantation (mean follow-up 282 ± 43 vs. 325 ± 61 days, respectively). The probability of survival without liver transplantation was 41% at 1 year and 26% at 2 years in the TIPS group, as compared with 35% and 30% in the paracentesis group (P = 0.51). In a multivariate analysis, only baseline blood urea nitrogen levels and Child-Pugh score were independently associated with survival. Recurrence of ascites and development of hepatorenal syndrome were lower in the TIPS group compared with the paracentesis group, whereas the frequency of severe hepatic encephalopathy was greater in the TIPS group. The calculated costs were higher in the TIPS group than in the paracentesis group.
Conclusions: In patients with refractory ascites, TIPS lowers the rate of ascites recurrence and the risk of developing hepatorenal syndrome. However, TIPS does not improve survival and is associated with an increased frequency of severe encephalopathy and higher costs compared with repeated paracentesis plus albumin.

Influence of hepatitis B virus genotype on the long-term outcome of chronic hepatitis B in Western patients
J. M. Sánchez-Tapias, J. Costa, A. Mas, M. Bruguera, J. Rodés
Background & Aims: The aim of this study was to investigate if the variable outcome of chronic hepatitis B may be related to hepatitis B virus (HBV) genotype.
Methods: The clinical and virologic events observed over prolonged follow-up in 258 Spanish patients with chronic hepatitis B infected with different genotypes of HBV were compared.
Results: The prevalence of genotype A, D, and F was 52%, 35%, and 7%, respectively. Concomitant sustained biochemical remission and clearance of HBV DNA occurred at a higher rate in genotype A­ than in genotype D­ (log-rank, 14.2; P = 0.002) or genotype F­infected patients (log-rank, 4.2; P = 0.03). The rate of hepatitis B surface antigen (HBsAg) clearance was higher in genotype A than in genotype D hepatitis (log-rank, 4.6; P = 0.03). Sustained remission and clearance of HBsAg were associated with infection with genotype A by Cox regression analysis. Seroconversion to antibody to hepatitis B e antigen (anti-HBe) was unrelated to HBV genotype, but the rate of sustained remission after seroconversion was higher in genotype A than in genotype D hepatitis both in patients who seroconverted to anti-HBe during follow-up (log-rank, 4.5; P = 0.03) and in patients with positive anti-HBe at baseline (log-rank, 6.66; P = 0.009). Death related to liver disease was more frequent in genotype F than in genotype A (P = 0.02) or genotype D (P = 0.002) hepatitis.
Conclusions: The long-term outcome of chronic hepatitis B is different in patients infected with HBV genotype A, D, or F.

Genotype and phenotype correlations in patients with cystic fibrosis and pancreatitis
C. Durno, M. Corey, J. Zielenski, E. Tullis, L.-C. Tsui, P. Durie
Background & Aims: Pancreatitis is known to occur in some patients with cystic fibrosis (CF), but the prevalence, natural history, and genotypic basis are unclear. We examined a well-defined cohort of patients with CF to answer these questions.
Methods: Patients with CF were identified from a computerized database (1966­1996). Chart audit identified all patients with CF and pancreatitis.
Results: Among 1075 patients with CF, 937 (87%) were pancreatic insufficient at diagnosis, 28 (3%) were pancreatic sufficient but developed pancreatic insufficiency after diagnosis, and 110 (10%) have remained pancreatic sufficient. No patients with pancreatic insufficiency developed pancreatitis. Nineteen patients (17.3%) with pancreatic sufficiency experienced one or more attacks of pancreatitis. The mean age at diagnosis of pancreatitis was 22.7 ± 10.3 years (range, 10­35 years), and pancreatitis was recognized before the diagnosis of CF in 6 patients (32%). The diagnosis of CF in pancreatic-sufficient patients, with and without pancreatitis, was established at a significantly older age than in those with pancreatic insufficiency (P < 0.0001). Genotyped patients with pancreatic insufficiency carried 2 severe mutant alleles. All genotyped patients with pancreatic sufficiency and pancreatitis carried at least one mild mutation. No specific genotype was predictive of pancreatitis.
Conclusions: Patients with CF with pancreatic sufficiency carry at least one mild mutant allele and are at a significant risk of developing pancreatitis. Symptoms of pancreatitis may precede the diagnosis of CF. Pancreatitis is associated with an otherwise mild CF phenotype.

Basic­Alimentary Tract

Prevention of colitis by interleukin 10­transduced T lymphocytes in the SCID mice transfer model
C. van Montfrans, M. S. Rodriguez Pena, I. Pronk, F. J. W. ten Kate, A. A. te Velde, S. J. H. van Deventer
Background & Aims: Regulatory CD4+ cells secreting the anti-inflammatory cytokine interleukin (IL)-10 play a key role in maintaining the immune balance in the intestinal mucosa. In this study we engineered primary CD4+ cells to express IL-10 and investigated the efficacy of this approach in offering protection against experimental colitis.
Methods: Spleen-derived CD4+ cells were transduced by using a retroviral vector to simultaneously express IL-10 and green fluorescent protein (GFP). The therapeutic benefit of CD4+ cells transduced with IL-10 GFP was studied in experimental colitis, induced by transfer of CD45RBhigh CD4+ cells to severe combined immunodeficient mice, and in acute trinitrobenzene sulfonic acid (TNBS)­induced colitis.
Results: Transferred engineered GFP fluorescent cells were detected for at least 15 weeks in peripheral blood, spleens, colon, and lymph nodes draining the intestine of recipient SCID mice. IL-10­GFP CD4+ cells prevented CD45RBhigh-induced transfer colitis effectively, whereas no effect was observed after transfer of nontransduced CD4+ cells. IL-10­GFP CD45RBhigh CD4+ cells lost the capacity to induce colitis. By contrast, no therapeutic benefit was observed in TNBS-induced colitis.
Conclusions: Primary murine CD4+ cells that were engineered to express IL-10 by retroviral transduction act as regulatory cells in CD45RBhigh-induced transfer colitis. This approach may induce long-term maintenance of mucosal immune homeostasis in Crohn's disease.

Generation of regulatory gut-homing human T lymphocytes using ex vivo interleukin 10 gene transfer
C. van Montfrans, E. Hooijberg, M. S. Rodriguez Pena, E. C. de Jong, H. Spits, A. A. te Velde, S. J. H. van Deventer
Background & Aims: Systemic treatment of Crohn's disease patients using recombinant interleukin (rIL)-10 has not resulted in significant therapeutic benefit presumably because of limited bioavailability and unexpected proinflammatory effects of high-dose rIL-10. Ex vivo gene transfer of the interleukin (IL)-10 gene to gut-homing CD4+ cells may lead to improved long-term management.
Methods: Peripheral blood mononuclear cells (PBMCs) were transduced with a retroviral vector containing the IL-10 and green fluorescent protein (GFP) gene or a control vector containing GFP only. Transduced CD4+ cells were sorted and maintained in culture for phenotypic and functional analysis.
Results: Stimulated IL-10­GFP CD4+ cells produced significantly higher levels of IL-10 than control cells for at least 4 months. The IL-10 transgene was biologically active and decreased proliferation of IL-10­GFP CD4+ cells as well as expression of major histocompatibility class (MHC) class II, proliferation of autologous responder cells, and IL-12 production by dendritic cells (DCs). The majority of transduced CD4+ cells had a gut-homing potential because they expressed the mucosal integrin 47, and displayed efficient binding to MAdCAM-1­expressing cells in vitro.
Conclusions: Transduction of peripheral blood CD4+ lymphocytes with IL-10 results in a regulatory phenotype. The use of regulatory gut-homing human CD4+ cells may provide a novel approach to local delivery of immunomodulatory signals to the intestine in Crohn's disease.

Gastric hyperplasia in mice with targeted disruption of the carbonic anhydrase gene Car9
M. Ortova Gut, S. Parkkila, Z. Vernerová, E. Rohde, J. Závada, M. Höcker, J. Pastorek, T. Karttunen, A. Gibadulinová, Z. Závadová, K.-P. Knobeloch, B. Wiedenmann, J. Svoboda, I. Horak, S. Pastoreková
Background & Aims: Carbonic anhydrase (CA) IX is a highly active enzyme with adhesion capacity that is functionally implicated in acid-base balance and intercellular communication. It is normally present in basolateral membranes of gastrointestinal epithelial cells and ectopically expressed in various carcinomas. To show its physiologic relevance, we have cloned the Car9 gene and generated CA IX­deficient mice.
Methods: The mice with null mutation of the Car9 gene were obtained by targeted gene disruption. Tissue architecture and expression of markers were determined by histochemical and immunohistochemical techniques.
Results: Mice homozygous for the mutation developed gastric hyperplasia of the glandular epithelium with numerous cysts. The first changes were observed in the newborn animals, and the hyperplasia became prominent at the end of gastric morphogenesis in 4-week-old mice. Loss of CA IX led to overproduction of mucus-secreting pit cells and depletion of pepsinogen-positive chief cells. The proportion of H+/K+-adenosine triphosphatase­positive parietal cells significantly decreased, but their absolute number was not reduced. Correspondingly, CA IX­deficient mice had normal gastric pH, acid secretion, and serum gastrin levels.
Conclusions: Phenotypic consequences of the Car9 null mutation show the important role of CA IX in morphogenesis and homeostasis of the glandular gastric epithelium via the control of cell proliferation and differentiation.

Intraileal capsaicin inhibits gastrointestinal contractions via a neural reflex in conscious dogs
C. Shibata, X.-L. Jin, H. Naito, S. Matsuno, I. Sasaki
Background & Aims: The aim of the present study was to determine the effect of intraileal administration of capsaicin on gastrointestinal motility.
Methods: Mongrel dogs equipped with strain gauge force transducers on the stomach, small intestine, and colon were used. We studied the effects of intraileal capsaicin on gastrointestinal contractions with or without pharmacologic antagonists. The effects of capsaicin administration into the lumen of innervated and extrinsically denervated ileal Thiry loops were also studied.
Results: Intraileal capsaicin dose dependently inhibited postprandial contractions at all sites and interdigestive contractions in the upper gastrointestinal tract. Intraileal capsaicin-induced inhibition of gastrointestinal contractions was partially reversed by a nitric oxide (NO) synthase inhibitor, a 5 hydroxytryptamine-3 receptor antagonist (5-HT3), and an opiate antagonist. Administration of capsaicin into the innervated ileal Thiry loop had inhibitory effects on gastrointestinal contractions, but gastrointesinal motor activity was not affected by capsaicin administered into the extrinsically denervated Thiry loop.
Conclusions: Stimulation of ileal afferent fibers by capsaicin inhibits gastrointestinal contractions via an extrinsic neural reflex.

Curcumin prevents and ameliorates trinitrobenzene sulfonic acid­induced colitis in mice
K. Sugimoto, H. Hanai, K. Tozawa, T. Aoshi, M. Uchijima, T. Nagata, Y. Koide
Background & Aims: Curcumin is known to have a variety of pharmacologic effects, including antitumor, anti-inflammatory, and anti-infectious activities. The pleiotropic effects of curcumin are attributable at least in part to inhibition of transcriptional factor nuclear factor B (NF-B). However, the effect of curcumin on intestinal inflammation has hitherto not been evaluated. The aim of this study was to determine whether treatment with curcumin prevents and ameliorates colonic inflammation in a mouse model of inflammatory bowel disease.
Methods: Mice with trinitrobenzene sulfonic acid (TNBS)-induced colitis were treated with 0.5%, 2.0%, or 5.0% curcumin in the diet, and changes in body weight together with histologic scores were evaluated. Colonic T-cell subsets were characterized, and NF-B in colonic mucosa was detected by immunohistochemistry. NF-B activity in the colonic mucosa was evaluated using electrophoretic mobility shift assay. Cytokine messenger RNA expression in colonic tissue was assessed by semiquantitative reverse-transcription polymerase chain reaction.
Results: Treatment of mice with curcumin prevented and improved both wasting and histopathologic signs of TNBS-induced colonic inflammation. Consistent with these findings, CD4+ T-cell infiltration and NF-B activation in colonic mucosa were suppressed in the curcumin-treated group. Suppression of proinflammatory cytokine messenger RNA expression in colonic mucosa was also observed.
Conclusions: This study has shown for the first time that treatment with curcumin can prevent and improve murine experimental colitis. This finding suggests that curcumin could be a potential therapeutic agent for the treatment of patients with inflammatory bowel disease.

Strain- and blood group­dependent binding of Helicobacter pylori to human gastric MUC5AC glycoforms
S. Lindén, H. Nordman, J. Hedenbro, M. Hurtig, T. Borén, I. Carlstedt
Background & Aims: In the stomach, Helicobacter pylori is found both in the mucus layer and adhering to the gastric epithelium. The aim of this study is to characterize the binding of H. pylori to human gastric mucins.
Methods: H. pylori strains that bind the Lewisb (Leb) structure (via the BabA adhesin) and/or sialylated structures, along with isogenic adhesion deletion mutants, were used to identify microbe-binding mucins. Gastric mucins from 5 healthy individuals, isolated by density-gradient centrifugation, were investigated for H. pylori binding at neutral pH using a microtiter-based technique.
Results: H. pylori strains that express the BabA adhesins were shown to bind to the MUC5AC mucin in individuals expressing the Leb antigen. Further fractionation with an ion-exchange chromatography revealed Leb-positive MUC5AC glycoforms that differed in their receptor properties for different H. pylori strains. None of the H. pylori strains studied bound to mucins from Leb-negative individuals. However, all strains bound to low-density, nonmucin, Leb-negative material on top of the gradients.
Conclusions: Binding of H. pylori to human gastric MUC5AC isolated from healthy individuals is BabA dependent and mediated by the Leb structure presented by the mucin. However, the BabA adhesins demonstrate strain-dependent preference in binding to MUC5AC glycoforms substituted with Leb, allowing for great interindividual variability in host­microbe interactions.

The high metabolic cost of a functional gut
S. R. D. van der Schoor, P. J. Reeds, B. Stoll, J. F. Henry, J. R. Rosenberger, D. G. Burrin, J. B. van Goudoever
Background & Aims: Animal studies have shown that more than half of the dietary protein intake is used by the gut and that a large proportion of this utilization is devoted to (glyco-)protein synthesis. Recycling of these secretions may play a critical role in the regulation of overall dietary amino acid bioavailability.
Methods: Four piglets (age 32 days, 8­10 kg) bearing portal, arterial, and duodenal catheters and a portal flow probe were infused with a complete diet via the duodenum for 12 hours, followed by 12 hours of fasting. The portal balance of glucose and amino acids was measured throughout the 24-hour period. The animals also received duodenal and intravenous infusions of different lysine and threonine tracers. Measurements of intestinal tracer utilization and reappearance in the portal blood were used to calculate intestinal amino acid utilization and recycling.
Results: From 0 to 6 hours, one third of the protein intake appeared in the portal blood. As feeding continued, the portal glucose balance (60% of intake) was constant, but the net amino acid portal balance became progressively more positive. Significant net amino acid absorption continued for at least 6 hours after the cessation of feeding. Over 24 hours, 52% of the dietary protein intake appeared in the circulation and one third of this derived from recycled intestinal secretions.
Conclusions: Intestinal recycling of amino acids contributes significantly to their systemic availability and may be a critical factor in amino acid nutrition.

Identification and isolation of candidate human colonic clonogenic cells based on cell surface integrin expression
K. Fujimoto, R. D. Beauchamp, R. H. Whitehead
Background & Aims: The surface epithelium of the colon is being replaced constantly with cells derived from the stem cells of the crypt. Although the location of the stem cells is known, there are no markers for these cells. This study tested the hypothesis that colonic stem cells might be isolated and cultured on the basis of specific integrin expression patterns in normal human colonic epithelium.
Methods: Integrin expression in normal human colonic mucosa was determined by using indirect immunofluorescence. Crypt cells were then isolated as single cells from normal colon tissues and the expression pattern of integrins was analyzed by flow cytometry. Based on the specific expression of integrin 1 in colonic crypts, the cells were sorted by using a flow cytometer, and colony assays in soft agar were performed to evaluate the clonogenicity of the sorted cells.
Results: By immunofluorescence, the cells located in the lower one third of crypts expressed higher levels of 1-integrin than the cells in the remainder of the crypt. When isolated crypt cells were stained with the 1-integrin antibody and examined in a flow cytometer, there were 2 peaks of fluorescence. Sorting of crypt cells based on staining with anti­1 integrin antibody produced a cell population with a significantly enhanced ability to form colonies.
Conclusions: 1-integrin is a candidate surface marker for the proliferative zone of the human colonic crypt. Our in vitro culture system for the clonal growth of a single colonic crypt cell suspension could facilitate the identification of other candidate stem cell markers.

Differential localization of colitogenic Th1 and Th2 cells monospecific to a microflora-associated antigen in mice
M. Yoshida, Y. Shirai, T. Watanabe, M. Yamori, Y. Iwakura, T. Chiba, T. Kita, Y. Wakatsuki
Background & Aims: Clonal expansion of T cells is associated with inflammatory bowel diseases, which indicates antigenic activation of the T cells. We investigated whether the introduction of CD4 T cells specific to a microflora would initiate colitis and assessed the cytokine requirements for colitogenic CD4 T cells.
Methods: Severe combined immunodeficiency disease (SCID) mice were reconstituted with CD4 T cells, which were either deficient in interleukin (IL)-4/interferon (IFN)- production or differentiated in vitro to T-helper (Th) 1/Th 2 and bearing a transgenic T-cell receptor (TCR) specific to ovalbumin (OVA), and then inoculated with an Escherichia coli­producing OVA (ECOVA). Clinical and histologic manifestations of colitis were assessed.
Results: Mice with ECOVA colonization and OVA-specific CD4 T cells developed colitis with histologic features of focal infiltration by mononuclear cells, destruction of crypts, and loss of goblet cells. Further, infiltration was initiated in pre-existing lymph follicles. Th1- and IL-4 deficient T cells were diffusely localized in the lamina propria and submucosa, whereas Th2- and IFN-­deficient T cells were localized preferentially in lymph follicles.
Conclusions: A microbe-associated antigen, non­cross-reactive to colonic tissue, can drive antigen-specific CD4 T cells to cause colitis in SCID mice. Although the presence of IFN- and IL-4 in the effector CD4 T cells was not an absolute requirement for the development of colitis, they seemed to regulate it in part by modulating migration of the effector T cells.

Helicobacter pylori CagA protein activates serum response element­driven transcription independently of tyrosine phosphorylation
Y. Hirata, S. Maeda, Y. Mitsuno, K. Tateishi, A. Yanai, M. Akanuma, H. Yoshida, T. Kawabe, Y. Shiratori, M. Omata
Background & Aims: Infection with Helicobacter pylori possessing the cag pathogenicity island (PAI) is associated with severe gastritis and gastric cancer. CagA protein, one of the products of cag PAI, is translocated into epithelial cells, where cytoskeletal rearrangements occur as a result of CagA tyrosine (Tyr) phosphorylation. Here we identify a new role for CagA protein as an activator of host cell signaling.
Methods: We transfected CagA into epithelial cells and analyzed its effect on transcription by reporter assays. The mechanism of reporter activation was assessed by electrophoretic mobility shift assays (EMSA) and immunoblots. Responsible regions of CagA for reporter activation were determined by truncation and mutagenesis of cagA gene.
Results: In HeLa cells, expression of CagA increased serum response element (SRE)-driven and serum response factor (SRF)-driven transcription by 40-fold and 3.3-fold, respectively, but did not affect nuclear factor B­ or AP-1­driven transcription. CagA-mediated SRE activation was also observed in gastric cell lines. Immunoblotting and EMSA revealed that transfection of CagA enhanced phosphorylation of and DNA binding by Elk1. Furthermore, involvement of Ras and MEK in CagA-mediated Elk1 phosphorylation was observed. SRE activation was dependent on several regions within the C-terminal portion of CagA (CagA873-1002), and independent of Tyr phosphorylation.
Conclusions: The C-terminal portion of CagA enhances SRE-driven transcription by activating an upstream signaling cascade without requiring CagA Tyr phosphorylation. This result suggests that translocated CagA regulates 2 distinct cellular responses: phosphorylation-dependent cytoskeletal rearrangement and phosphorylation-independent transcriptional activation.

Full-thickness biopsy of the jejunum reveals inflammation and enteric neuropathy in irritable bowel syndrome
H. Törnblom, G. Lindberg, B. Nyberg, B. Veress
Background & Aims: Irritable bowel syndrome (IBS) is regarded as a functional bowel disorder. Few studies have looked for histopathologic changes in the gut and only then in biopsy specimens from intestinal mucosa. Because bowel function is governed mainly by nerve plexuses in the bowel wall, we have investigated full-thickness bowel biopsy specimens in patients with severe IBS.
Methods: We used a laparoscopy-assisted technique to obtain full-thickness biopsy specimens from the proximal jejunum. Tissue specimens were investigated with light microscopy using routine stainings and immunohistochemical techniques. Horizontal sectioning was done to visualize large areas of the myenteric plexus. Fifteen autopsy specimens were used as controls regarding the myenteric plexus. Colorectal adenoma controls with terminal ileum biopsy specimens and full-thickness jejunal biopsy specimens from patients with degenerative enteric neuropathy were used as control groups for intraepithelial lymphocyte counts.
Results: Ten patients (2 males, 8 females) were studied. In 9 patients, we found low-grade infiltration of lymphocytes in the myenteric plexus. Lymphocytes had peri- and intraganglionic location. The mean number of lymphocytes per ganglion ranged from 1.9 to 7.1 per patient, with an overall mean of 3.4. No intraganglionic lymphocytes were found in the control group and only a few periganglionic lymphocytes (mean, 0.2). Four patients had concomitant intraepithelial lymphocytosis. Neuron degeneration was evident in 6 of 9 patients with and 1 patient without ganglionic lymphocyte infiltration.
Conclusions: Our findings indicate that inflammation and neuronal degeneration in the myenteric plexus are involved in the pathogenesis of IBS.

Differentiation state-selective roles of p38 isoforms in human intestinal epithelial cell anoikis
P. H. Vachon, C. Harnois, A. Grenier, G. Dufour, V. Bouchard, J. Han, J. Landry, J.-F. Beaulieu, A. Vézina, A. Bondo Dydensborg, R. Gauthier, A. Côté, J.-F. Drolet, F. Lareau
Background & Aims: Little is known of the signaling events implicated in the induction of human enterocytic anoikis. In the present study, we analyzed the role of the stress-activated protein kinase p38 in this process.
Methods: Anoikis was induced in undifferentiated and differentiated enterocytes by inhibition of focal adhesion kinase (Fak; pharmacologic inhibition or overexpression of a dominant negative form) or 1 integrins (antibody blocking), or by maintaining cells in suspension. Expression/activation parameters of p38 (isoforms , , , ) and of the Fak/phosphatidylinositol-3-kinase (PI3-K)/Akt anoikis-suppressing pathways were analyzed. Kinase activities of p38 isoforms also were blocked by pharmacologic inhibitors or by overexpression of dominant-negative forms.
Results: (1) p38 activation is sustained transiently after induction of anoikis in both undifferentiated and differentiated enterocytes; (2) such sustenance of p38 activation is associated with a down-regulation of the Fak/PI3-K/Akt pathway; (3) distinct profiles of p38 isoform expression are exhibited by undifferentiated (, , ) and differentiated (, , ) enterocytes; (4) none of the 4 known p38 isoforms was found to promote cell survival in either differentiation state; and (5) only p38 and p38 are required specifically for anoikis in undifferentiated and differentiated cells, respectively.
Conclusions: Distinct p38 isoforms play a major role in the induction of enterocytic anoikis and the regulation of such selective p38 isoform-mediated anoikis is linked with the state of cell differentiation. These data provide novel insights into the synchronized regulation of cell survival/death required in the epithelial renewal process along the human intestinal crypt-villus axis.

Helicobacter pylori infection in mice: Role of outer membrane proteins in colonization and inflammation
Y. Yamaoka, M. Kita, T. Kodama, S. Imamura, T. Ohno, N. Sawai, A. Ishimaru, J. Imanishi, D. Y. Graham
Background & Aims: Mouse models of Helicobacter pylori infection have yielded variable results with respect to colonization and inflammation. We examined whether outer membrane proteins (OMPs) or the cag pathogenicity island (PAI) was responsible for some of this variability.
Methods: C57BL/6 mice received clinical H. pylori isolates with different genotypes for the cag PAI and OMP gene switch status, as well as isogenic gene knockout mutants for cagE, oipA, babA2, hopZ, cagE/oipA, or oipA/hopZ. Bacterial density, histology, and mucosal cytokine/chemokine levels were measured after 4 and 12 weeks.
Results: oipA, hopZ, hopO, and hopP switch status influenced both H. pylori density and colonization ability in mice. When 2 or more of the genes were "off," colonization rates were markedly reduced compared with those for strains with all genes "on" (from 58% to 0% after 12 weeks). oipA knockout mutants caused reduced inflammation and decreased mucosal interleukin 6 messenger RNA and KC messenger RNA and protein levels. H. pylori density and colonization ability were reduced if hopO or hopP switch status was changed from "on" to "off." There was a close relationship (r > 0.7) between the H. pylori density of the gastric mucosa of humans and mice when using the same H. pylori strains.
Conclusions: Many of the differences reported with mouse models may reflect subtle unrecognized differences (e.g., switch status of an OMP gene), emphasizing the necessity of characterizing isolates before and after experiments. The mouse model may be suitable for investigating factors related to colonization ability, H. pylori density, and gastric mucosal inflammation.

Inflammatory-mediated repression of the rat ileal sodium-dependent bile acid transporter by c-fos nuclear translocation
F. Chen, L. Ma, R. B. Sartor, F. Li, H. Xiong, A.-Q. Sun, B. Shneider
Background & Aims: Ileal malabsorption of bile salts is observed in Crohn's ileitis. We define the transcriptional mechanisms involved in cytokine-mediated repression of the rat apical sodium-dependent bile acid transporter (ASBT).
Methods: ASBT regulation was studied in IL-1­treated IEC-6 and Caco-2 cells and in indomethacin-treated rats.
Results: Indomethacin-induced ileitis in Lewis rats leads to specific reductions in ileal ASBT messenger RNA and protein levels, whereas c-jun and c-fos are induced. The proinflammatory cytokines interleukin-1 and tumor necrosis factor repress the activity of the ASBT promoter in Caco-2 and intestinal epithelial cell-6 cells. This effect is blocked by the proteasome inhibitor, MG-132, or by the phosphatidyl inositol 3-kinase inhibitor, wortmannin. Indomethacin (in vivo) or proinflammatory cytokine (in vitro) treatment leads to serine phosphorylation and nuclear translocation of c-fos. Mutation of a 5' activated protein (AP)-1 site inactivates the ASBT promoter, whereas mutation of the 3' site abrogates the proinflammatory cytokine­mediated repression. The 5' site binds a c-jun homodimer, whereas the 3' site binds a c-jun/c-fos heterodimer. c-Jun overexpression enhances ASBT promoter activity, whereas a dominant negative c-jun construct inactivates the promoter. c-Fos overexpression represses promoter activity. A 27 base pair cis-element from the 3' site in the ASBT promoter imparts cytokine-mediated down-regulation to a heterologous SV40 promoter construct.
Conclusions: The ASBT promoter contains 2 distinct cis AP-1 elements; the 5' element binds homodimeric c-jun and mediates basal transcription. Inflammation is associated with up-regulation, phosphorylation, and nuclear translocation of c-fos, which then represses ASBT promoter activity via binding of the 3' AP-1 element by a c-fos/c-jun heterodimer.

Basic­Liver, Pancreas, and Biliary Tract

Diverse expression of ErbB receptor proteins during rat liver development and regeneration
R. S. Carver, M. C. Stevenson, L. A. Scheving, W. E. Russell
Background & Aims: The protein expression and interactions of the ErbB receptors were examined in different liver proliferation models in vivo and in vitro, including ontogeny and regeneration following partial hepatectomy.
Methods: Expression and tyrosine phosphorylation status of specific ErbB proteins were studied by immunologic methods.
Results: The epidermal growth factor receptor, ErbB2, and ErbB3 were the only ErbB proteins detected in the liver parenchyma on embryonic day 19. ErbB2 disappeared by the third week after birth and could not be appreciably induced in the adult animal by partial hepatectomy. ErbB2 was also detected in multipotent stem (RLE) and hepatoma (H4IIe) cell lines as well as in fetal, but not adult, hepatocyte cultures. Only epidermal growth factor receptor and ErbB3 were detected in adult liver, and both showed circadian variation in protein expression. ErbB4 was not detected in any model. Patterns of ligand-induced ErbB phosphorylation differed between fetal and adult hepatocytes.
Conclusions: Complex and independent programs regulate the ErbB receptors, with implications for differential cell signaling in hepatic development and regeneration.

Plasticity of electrical pacemaking by interstitial cells of Cajal and gastric dysrhythmias in W/WV mutant mice
T. Ördög, M. Baldo, R. Danko, K. M. Sanders
Background & Aims: Interstitial cells of Cajal (ICC) generate and propagate slow waves in the stomach. Gastric peristalsis depends on a proximal-to-distal gradient in slow wave frequency. We tested whether the gastric frequency gradient was an intrinsic property of ICC and whether dysrhythmias result from disruptions of ICC networks.
Methods: We studied wild-type (WT) and W/WV mice, which have only myenteric (pacemaker) ICC in the stomach. ICC distributions were analyzed by Kit immunofluorescence. Pacemaking in tissues was studied by intracellular electrophysiologic recording and in cultured ICC by monitoring mitochondrial [Ca2+] oscillations with rhod-2 fluorescence or membrane potential with DiBAC4(3) fluorescence.
Results: Slow wave frequencies were constant throughout WT gastric muscle sheets containing corpus and antrum. Separating the antrum from the corpus caused a significant drop in antral slow wave frequency. ICC from WT antrums also displayed significantly slower pacemaker frequencies than corpus ICC, but the corpus pacemaker frequency dominated in cocultures of corpus and antrum ICC. Myenteric ICC networks were reduced in W/WV mice, particularly in the corpus. In W/WV mice, separating the antrum from the corpus failed to reduce antral slow wave frequency. Antral pacemaker frequency in ICC from W/WV stomachs was the same as in corpus ICC.
Conclusions: The proximal-to-distal slow wave frequency gradient and entrainment of distal electrical activity by proximal pacemakers are fundamental properties of gastric ICC. Chronic depletion of ICC networks disrupts the proximal-to-distal frequency gradient, and emergence of ectopic pacemakers in the antrum may be caused by "reprogramming" of the ICC pacemaker apparatus.

Genome-wide analysis of hepatic fibrosis in inbred mice identifies the susceptibility locus Hfib1 on chromosome 15
S. Hillebrandt, C. Goos, S. Matern, F. Lammert
Background & Aims: Host genetic factors are likely to contribute to the variable course of hepatic fibrosis in response to chronic liver injury. Similarly, the fibrotic response differs among inbred mouse strains after challenge with CCl4. Our aim was to identify unknown susceptibility loci for hepatic fibrosis in a cross between fibrosis-susceptible and -resistant inbred mice.
Methods: Seven inbred mouse strains were treated with CCl4, and hepatic fibrosis was phenotypically characterized by histology, hepatic hydroxyproline levels, and serum surrogate markers. F1 hybrids of susceptible BALB/cJ and resistant A/J inbred strains were intercrossed to obtain 358 F2 progeny. Quantitative trait loci (QTL) that determine hepatic fibrosis were identified by genome-wide interval mapping and haplotype analysis.
Results: In this model, marked strain differences in fibrosis susceptibility exist, with BALB/c inbred mice being most susceptible. The hydroxyproline levels of F1 mice resemble the resistant parental strains, indicating that fibrosis susceptibility is a recessive trait. QTL analysis identifies a susceptibility locus on chromosome 15 that significantly affects the stage of fibrosis and hydroxyproline levels. According to standard nomenclature, this locus is called Hfib1 (hepatic fibrogenic gene 1). Hfib1 is defined by genetic markers D15Mit26 and D15Mit122. A suggestive QTL on chromosome 2 colocalizes with the complement factor 5 gene, known to be mutated in the resistant strain A.
Conclusions: The set of inbred strains provides a framework for systematic analysis of fibrogenic genes. QTL mapping is useful to identify genetic susceptibility loci for hepatic fibrosis that might harbor new molecular targets for antifibrotic drug design.

Stat3 and NF-B activation prevents apoptosis in pancreatic carcinogenesis
F. R. Greten, C. K. Weber, T. F. Greten, G. Schneider, M. Wagner, G. Adler, R. M. Schmid
Background & Aims: Human pancreatic adenocarcinoma has an overall poor prognosis. Therapeutic efforts are often ineffective because of late diagnosis and a high degree of chemoresistance. Overexpression of transforming growth factor in the pancreas of transgenic mice causes the formation of premalignant ductal lesions and the development of invasive ductal adenocarcinoma. The aim of the present study was to explore regulation of proapoptotic and antiapoptotic signals during pancreatic tumor development in mice.
Methods: EL-TGF-hGH transgenic mice crossbred to p53-deficient mice develop ductal pancreatic adenocarcinoma resembling the human disease. During the multistep carcinogenesis up-regulation of Bcl-xL is evident early and persists throughout tumorigenesis as detected by Real Time PCR, Western blot analysis, and immunofluorescence.
Results: Up-regulation of Bcl-xL is evident early in tumor development and persists throughout tumorigenesis. The transcription factors Stat3 and NF-B induce increased Bcl-xL expression in the premalignant lesions and tumor cells. Inhibition of either transcription factor alone leads to Bcl-xL down-regulation in transient transfection assays. Functional analysis shows that blocking of both Stat3 and NF-B together induces programmed cell death in murine pancreatic tumor cells.
Conclusions: These findings indicate that apoptosis resistance precedes formation of invasive pancreatic cancer. Therefore, combined inhibition of Stat3 and NF-B might represent a novel strategy for tumor prevention and therapy.

Special Reports and Reviews

Suppressors of cytokine signaling: Relevance to gastrointestinal function and disease
C. J. Greenhalgh, M. E. Miller, D. J. Hilton, P. K. Lund
Background & Aims: The suppressor of cytokine signaling (SOCS) proteins are a family of Src homology 2 domain-containing proteins. Currently, there are 8 members of the SOCS family, of which a number have been implicated strongly in the negative regulation of cytokine signal transduction pathways.
Methods: This review focuses on recent discoveries about 4 SOCS family members, SOCS-1, -2, and -3, and cytokine-inducible SH2-domain containing (CIS), and provides more limited information about other SOCS family members.
Results: A large number of cytokines and growth factors are now known to induce SOCS proteins. In turn, SOCS inhibit the actions of a growing number of cytokines and growth factors in vitro or in vivo. SOCS proteins exert their inhibitory effects at the level of activation of janus kinases (JAKs) or by competing with transcription factors for binding sites on activated cytokine receptors. SOCS proteins also may mediate the ubiquitination and subsequent degradation of the SOCS protein and its bound signaling complex. Genetic modification of SOCS genes in mice has revealed crucial roles in the negative regulation of a number of important physiologic parameters including interferon activity, growth, blood cell production, and placental development.
Conclusions: Information about SOCS action in gastrointestinal function and disease is only just emerging, but available data indicate a role in growth of gastrointestinal tissues, inflammatory bowel disease, and cancer.

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JOURNAL OF HEPATOLOGY

Volume 37, Issue 6, December 2002

Biliary Tract and Cholestasis

Rubén Terg, Emma Coronel, Juan Sordá, Alberto E. Muñoz and Jorge Findor
Efficacy and safety of oral naltrexone treatment for pruritus of cholestasis, a crossover, double blind, placebo-controlled study

Background/Aims: To assess the efficacy and safety of naltrexone for the short and long term treatment of pruritus of cholestasis. Methods: Twenty patients with pruritus and cholestasis were included. A baseline pruritus score was obtained over 1 week. Patients were then randomized to receive 50 mg/day of naltrexone or placebo for 2 weeks. Subsequently, a 1-week washout period ensued and patients were crossed over to the other therapy for 2 additional weeks. Pruritus was assessed daily with a visual analogue scale (VAS) from 0 to 10. Patients whose pruritus decreased >50% of basal with naltrexone received naltrexone 50 mg/day for 2 additional months. Results: Mean basal VAS was similar in both groups. VAS showed greater and more significant changes with naltrexone than with placebo (P<0.0003). In nine out of 20 patients (45%) receiving naltrexone, pruritus decreased >50% compared to basal value, including five whose pruritus disappeared completely. No significant changes were observed in serum biochemistry. Most of the adverse events that occurred during the first 48 h of naltrexone therapy were consistent with opioid withdrawal-like phenomena and spontaneously disappeared 2 days after starting treatment. Conclusions: Naltrexone can be considered as an alternative option to treat pruritus of cholestasis. In the current study, side effects were transient and did not require specific medication.

Paloma Sanz-Cameno et al.
Enhanced intrahepatic inducible nitric oxide synthase expression and nitrotyrosine accumulation in primary biliary cirrhosis and autoimmune hepatitis

Background/Aims: Nitrosative stress resulting from increased nitric oxide (NO) synthesis contributes to the pathogenesis of chronic inflammatory diseases, including chronic viral hepatitis. Our goal was to assess the expression of inducible nitric oxide synthase (iNOS) and the formation of nitrotyrosine (NTY), as a marker of nitrosative stress, in liver biopsies from primary biliary cirrhosis (PBC) and autoimmune hepatitis (AIH) patients. Methods: Intrahepatic expression of iNOS and NTY was measured immunohistochemically and compared to histological scores of the severity of liver disease. Results: Hepatocellular iNOS expression was observed in liver sections from PBC patients (with a diffuse lobular distribution) and from AIH patients (marked staining in areas of pronounced inflammation and necrosis), but not in control liver sections, including non-autoimmune cholestatic liver disease. Liver samples from PBC and AIH patients, but not from controls, showed NTY accumulation in clusters of hepatocytes and Kupffer cells. Increased iNOS expression and NTY accumulation correlated with the histological severity of PBC or AIH, especially with the degree of inflammation. Conclusions: Patients with PBC and AIH showed an enhanced intrahepatic iNOS expression and NTY accumulation, related to the histological severity of liver disease, consistent with NO-mediated nitration of hepatocellular proteins contributing to liver damage in both diseases.

Cell Biology, Metabolism and Transport

Géraldine Gouysse et al.
Relationship between vascular development and vascular differentiation during liver organogenesis in humans

Backgrounds/Aims: The complex vascular architecture characteristic of the normal adult liver is progressively acquired during the fetal life. In this study, we aimed to evaluate the relationship between angiogenesis and vascular differentiation during liver organogenesis. Methods: We studied, in 51 fetuses of different gestational ages, the expression of markers of endothelial cell differentiation, integrins, pro- and anti-angiogenic extracellular matrix components, vascular endothelial growth factor (VEGF) and its receptors. Results: Three main stages in the development of the vascular architecture of the liver were identified: (a) from 5 to 10 gestation weeks (GW), no evidence of de novo angiogenesis was detected; the vessels present in the liver primordium were the precursors of portal veins and sinusoids, deriving from preexisting vessels; (b) from 10 to 25 GW, angiogenesis and vasculogenesis resulted in the development of, respectively, arteries and intra-portal capillaries, while portal veins and hepatic sinusoids followed a differentiation process; (c) after 25 GW, little changes were detected in the various vascular compartments. The maximal expression of VEGF and its receptors was from 5 to 25 GW.

Chronic Liver Diseases

Christoph R. Meier, Stephan Krähenbühl, Raymond G. Schlienger and Hershel Jick
Association between body mass index and liver disorders: an epidemiological study

Background/Aims: Cross-sectional studies showed an association between obesity and liver disorders. Information on incidence rates from epidemiological studies is scarce. Methods: To explore the association between body mass index (BMI) and incident liver disorders, we conducted a follow-up study with a nested case-control analysis using the UK-based General Practice Research Database. The study population encompassed normal weight (BMI <25 kg/m2), pre-obese (BMI 25-29.9 kg/m2) or obese (BMI 30 kg/m2) subjects aged 20-79 years, matched on age, sex and general practice attended. Results: Within the study population of 207,630 subjects, we identified 2718 cases of newly diagnosed liver disorder, of whom 1524 (56.1%) had known predisposing conditions. The incidence rates were 2.48 (95% CI 2.31-2.66)/1000 person-years (py) in normal weight, 2.91 (95% CI 2.72-3.11)/1000 py in pre-obese, and 3.83 (95% CI 3.62-4.07)/1000 py in obese subjects. In the nested case-control analysis, the adjusted odds ratio (OR) for obese, as compared to normal weight subjects, was 1.2 (95% CI 1.1-1.4). The OR for idiopathic cases (without known risk factors for liver disorders) was 1.3 (95% CI 1.1-1.6).

Jean-François Dufour, Matthias Zimmermann and Jürg Reichen
Severe autoimmune hepatitis in patients with previous spontaneous recovery of a flare

Background/Aims: Autoimmune hepatitis can present acutely and pursues a variable, usually progressive course. Spontaneous remissions have been described, but their influence on the course of subsequent attacks has not yet been studied. Methods: Charts from 1980 to 2000 were reviewed. Cases of autoimmune hepatitis were further examined to identify those with prior episodes of transient elevation of the serum aminotransferase activities of unknown etiology. Results: Fifty-one patients with autoimmune hepatitis were identified, nine of whom (18%) had experienced a transient episode of elevated aminotransferase activities which had resolved spontaneously. The median period of time separating the two flares was 10 months (range: 4 months-23 years). The median age at diagnosis was 41 years in both groups. More than 75% of the patients in either group suffered from a type 1 autoimmune hepatitis. Importantly, patients with spontaneous recovery of a previous episode had a higher serum alanine transaminase activity (P<0.02), were less likely to respond to immunosuppressive therapy (78 versus 98%, P=0.077) and required transplantation more frequently (22 versus 0%, P=0.028). Histologically, these patients did not have more fibrosis than patients without previous flare. Conclusions: More than 10% of autoimmune hepatitis may begin with a spontaneously resolving episode. However, the spontaneous recovery of a previous attack should not falsely reassure: a subsequent flare of autoimmune hepatitis can still be severe.

Giovanna Fabio et al.
Peripheral lymphocytes and intracellular cytokines in C282Y homozygous hemochromatosis patients

Background/Aims: Several abnormalities in the immune status of hereditary hemochromatosis patients have been reported. We evaluated the peripheral blood lymphocytes phenotype and cytokine profile of CD8+ and CD4+ T cells in C282Y homozygous hereditary hemochromatosis patients compared to control subjects. Methods: Peripheral blood lymphocytes from 17 asymptomatic patients and 14 control subjects were analyzed. We determined the distribution of lymphocyte subsets and investigated at single-cell level by flow-cytometry the potential of cytokines production. The frequency of cytokine (interferon gamma, tumor necrosis factor alpha, interleukin 2 (IL-2), IL-4, IL-5, IL-10 and IL-13) producing cells was assessed in total T-lymphocytes, CD3+CD8+ and CD3+CD4+ subsets. Results: The patients studied showed a significant decrease of total lymphocyte count, T CD4+CD3+, CD28+, CD8+CD28+ lymphocytes and natural killer (NK) CD56+CD16+CD3 cells. The reduction of CD28+ and CD8+CD28+ lymphocyte count was inversely related to transferrin saturation index. An increase in the ability of T-cells to produce all the cytokines studied and a major increase in IL-4 and IL-10 production in the CD3+CD8+ subset was found. Our results demonstrate that activated Th1 and Th2 lymphocytes coexist in the peripheral blood of hereditary hemochromatosis patients and that T-cytotoxic (Tc) 2 subset is more expanded than in control population. Conclusions: The association of a decreased number of T CD8+ cytotoxic lymphocytes and NK cells, and the development of Tc2 cells in asymptomatic C282Y homozygous patients represents an imbalance in their immune function that might contribute to the high incidence of hepatocarcinoma.

Cirrhosis and its Complications

Luigi Pagliaro, Linda Pasta, Gennaro D'Amico, Salvatore Madonia and Giada Pietrosi
Familial clustering of (mostly) HCV-related cirrhosis. A case-control study

Background: Increased prevalence of cirrhosis in cirrhotics' families in previous studies. Aims: To compare the prevalence of cirrhosis in cirrhotics' families with that in families of patients with non-hepatic diseases. Methods: Case-control study including 500 index cases with cirrhosis and 500 pair-matched controls with chronic non-hepatic diseases, interviewed about cirrhosis in first-degree relatives and spouses using standardized forms. Results: Ninety-three index cases (88% anti-hepatitis C virus (HCV)-positive) and 13 controls had one or more cirrhotics among first-degree relatives and/or spouses (odds ratio (OR) 7.38, 95% confidence interval (CI) 4.21-12.9); cirrhosis was found in 123/4485 first-degree relatives of the index cases and in 16/4086 of controls (OR 7.17 95% CI 4.25-12.09), in 14/467 spouses of the index cases and in 1/416 spouses of controls (OR 12.8, 95% CI 1.67-97.96). The percentage of secondary cases in the families of 440 anti-HCV-positive and 60 anti-HCV-negative index cases was similar (18.8 and 21.6%, respectively). Almost all the secondary cases in families of anti-HCV-positive index cases and none in families of anti-HCV-negative index cases were anti-HCV-positive. Conclusions: Cirrhosis is significantly more frequent among first-degree relatives and spouses of patients with cirrhosis, mostly HCV-related, than among first-degree relatives and spouses of controls.

Jörg Heller et al.
Increased urotensin II plasma levels in patients with cirrhosis and portal hypertension

Background/Aims: Vasodilatation - despite activation of endogenous vasoconstrictors - is pronounced in portal hypertension. We therefore investigated the role of Urotensin II (U II), a newly described peptide reported to be a vasoconstrictor in the central arterial compartment and a vasodilator in the splanchnic vasculature. Methods: U II immunoreactivity was measured in 50 patients with cirrhosis and in 15 healthy controls. U II levels were compared in portal venous and central venous blood of 30 patients immediately before transjugular intrahepatic porto-systemic stent shunt implantation. Results: U II levels (median, range, ng/ml) were significantly increased in cirrhotics (12.3, 1.6-41.4) compared to controls (3.6, 0.1-12.0; P<0.001). In patients with cirrhosis, U II levels were significantly higher in central venous (12.9, 2.5-41.4) than in portal venous blood (11.0, 0.6-31.9; P<0.005). U II levels were higher in ascitic than in non-ascitic patients (P<0.02). They correlated positively with the wedged hepatic venous pressure gradient (=0.34, P<0.005) and negatively with the mean arterial pressure (=0.41; P<0.001). Conclusions: Urotensin II formation is upregulated in patients with cirrhosis and portal hypertension. The transhepatic gradient suggests a hepatic production of this peptide.

Vincent Croquet et al.
Hemodynamic and antifibrotic effects of losartan in rats with liver fibrosis and/or portal hypertension

Background/Aims: To assess the effects of the early and chronic administration of losartan -a specific angiotensin II receptor antagonist- in the prevention of hepatic fibrosis and portal hypertension. Methods/Results: (1) In CCl4 rats, losartan at 5 and 10 mg/kg per day significantly decreased portal pressure (11, 18%, respectively), splenorenal shunt blood flow (60, 80%) and liver fibrosis (liver hydroxyproline and area of fibrosis) without significant changes in mortality and mean arterial pressure (MAP). (2) In bile duct ligated (BDL) rats, losartan at 5 mg/kg per day significantly decreased portal pressure (14%), splenorenal shunt blood flow (70%) and liver fibrosis. Losartan at 10 mg/kg per day significantly worsened liver and renal functions, mortality and liver fibrosis, without significant changes in portal pressure and splenorenal shunt blood flow. Losartan at 5 and 10 mg/kg per day significantly decreased MAP (24, 30%). (3) In portal vein ligated (PVL) rats, losartan significantly decreased MAP (12%) but did not change portal pressure or splenorenal shunt blood flow. Conclusions: In BDL and CCl4 rats, losartan has beneficial effects on splanchnic hemodynamics and liver fibrosis. Losartan might decrease hepatic resistances in fibrotic liver. Losartan decreased MAP except in CCl4 rats. Higher dosage of losartan had deleterious effects in BDL rats.

Manuel Romero-Gómez et al.
Altered response to oral glutamine challenge as prognostic factor for overt episodes in patients with minimal hepatic encephalopathy

Background/Aims: We assessed the usefulness of oral glutamine challenge (OGC) and minimal hepatic encephalopathy in evaluating risk of overt hepatic encephalopathy in cirrhotic patients. Methods: Minimal hepatic encephalopathy (MHE) was inferred using neuro-psychological tests. Venous ammonia concentrations were measured pre- and post-60 min (NH3-60m) of a 10 g oral glutamine load. Receiver-operating-characteristic curve analysis indicated a pathological glutamine tolerance cut-off value of NH3-60m >128 µg/dl. Results: In healthy control subjects (n=10) ammonia concentrations remained unchanged but increased significantly in cirrhotic patients (from 70.41±45.2 to 127.43±78.6; P<0.001). In multiple logistic regression analysis, altered OGC was related to Child-Pugh (odds ratio, OR=7.69; 95% confidence interval, CI=1.72-33.3; P<0.01) and MHE (OR=5.45; 95% CI=1.17-25.4; P<0.05). In the follow-up 11 patients (15%) developed overt hepatic encephalopathy (HE). In multivariate analysis OGC (OR=14.5; 95% CI=1.26-126.3) and MHE (OR=1.56; 95% CI=1.02-21.9) were independently related with HE in the follow-up. Patients with MHE and altered OGC showed significantly higher risk of overt HE in the follow-up (60%) than patients without MHE and normal OGC (2.8%) (Log rank test=21.60; P<0.0001). Conclusions: A pathological OGC in patients with MHE appears to be a prognostic factor for the development of overt hepatic encephalopathy, whereas a normal OGC in patients without MHE could exclude risk of overt HE.

Inflammation and Fibrosis

Krista Rombouts et al.
Actin filament formation, reorganization and migration are impaired in hepatic stellate cells under influence of trichostatin A, a histone deacetylase inhibitor

Background/Aims: Previously, trichostatin A (TSA), a histone deacetylase inhibitor, has been shown to exhibit strong antifibrotic characteristics in hepatic stellate cells (HSC), which are known to play a central role in chronic liver diseases. TSA retained a more quiescent phenotype in spite of culture conditions that favor transdifferentiation into activated HSC. Methods: To identify TSA-sensitive genes, differential mRNA display, Northern and Western blot analysis were used and genes were functionally validated by using contraction and motility assays. Results: TSA prevented new actin filament formation by down-regulation of two nucleating proteins, actin related protein 2 (Arp2) and Arp3, and by up-regulation of adducin like protein 70 (ADDL70) and gelsolin, two capping proteins. RhoA, a key mediator in the development of the actin cytoskeleton, decreased following TSA exposure. Expression of proteins of Class III intermediate filaments was affected by TSA. Furthermore, F-actin and G-actin were expressed heterogeneously under influence of TSA. Functionally, TSA treatment abrogated migration of quiescent HSC, while migration was reduced in transitional HSC. The endothelin-1-induced contractility properties of HSC was not affected by TSA. Conclusions: These data indicate that TSA affects the development of the actin cytoskeleton in quiescent HSC and thereby abrogates the process of HSC transdifferentiation.

Leonel García et al.
Pirfenidone effectively reverses experimental liver fibrosis

Background/Aims: Our group has been involved in searching for different strategies to ameliorate hepatic cirrhosis. The aim of this study was to evaluate the effect of Pirfenidone in the reversion or prevention of cirrhosis experimentally induced in rats by chronic administration of CCl4 and bile-duct ligation (BDL). Methods: Male cirrhotic Wistar rats (8 weeks of intoxication and then hepatotoxin was discontinued) received either oral saline or Pirfenidone at 500 mg/kg per day. Results: High levels of alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase decreased significantly (P<0.001) in animals treated with Pirfenidone (n=11) with regard to saline-administrated animals (n=9). Prothrombin activity and bilirubins were also reduced. Computerized fibrosis index demonstrated a 70% decrease (P<0.001) along with less hydroxyproline content, reduction in activated HSC and higher active cell regeneration. A rearrangement of the parenchyma was also noted and gene expression of collagens I, III and IV, transforming growth factor -1, Smad-7, TIMP-1 and PAI-1 decreased considerably in treated animals. Cirrhotic rats in which CCl4 was not discontinued displayed 40% liver fibrosis reduction. In a different cirrhosis model, 4-week BDL rats treated with the drug showed a significant 50% reduction in hepatic fibrosis (P<0.01). Conclusions: This new drug might be useful in healing human disease.

Liver Growth and Cancer

Shahid A. Khan et al.
Changing international trends in mortality rates for liver, biliary and pancreatic tumours

Background/Aims: The age-standardized mortality rate for hepatocellular carcinoma is increasing in several countries. However, in England and Wales we previously reported an increase in mortality rates from intrahepatic cholangiocarcinoma. Trends in cholangiocarcinoma in most other industrialized countries are unknown. To further study trends in hepatobiliary and pancreatic tumours, we analysed mortality data from the United States, Japan, Australia and Europe. Methods: Age-standardized mortality rates for men and women for subcategories of liver tumours, tumours of the gall bladder and extrahepatic biliary tree and pancreas from 1979 to 1998 were obtained from the World Health Organization mortality database. Results: We confirmed previously reported increases in hepatocellular carcinoma, but also found increases in other countries, particularly Australia (3-year average rise from 1.20 to 2.27, men). Mortality for intrahepatic cholangiocarcinoma increased in men in all countries studied, with the largest increases in Australia (from 0.10 to 0.70) and England and Wales (from 0.20 to 0.83). Conclusions: We present a hitherto unreported rise in age-standardized mortality rates from intrahepatic cholangiocarcinoma across four continents. The cause remains uncertain. An impact on the observed trends of improved diagnostic techniques and death certificate misclassification cannot be completely ruled out. Future research should include epidemiological studies to examine possible case-clustering and investigation of potential aetiological and host factors.

Transplantation and Surgery

Izumi Sakamoto et al.
Experimental study on hepatic reinnervation after orthotopic liver transplantation in rats

Background/Aims: The present study examined whether extrinsic hepatic reinnervation occurred after orthotopic liver transplantation (OLT) in rats. Methods: Inbred male Lewis rats were the recipients and females the donors. Tissue specimens were obtained postoperatively from the stump of a recipient's hepatoduodenal ligament (A), and the hepatic hilus (B) and peripheral parenchyma (C) of liver allografts, up to 6 months post-operation. Specimens were subjected to immunohistochemical examination using growth-associated protein (GAP)-43 as an axonal marker and transmission electron microscopy (TEM) for observing regenerating axons, as well as the polymerase chain reaction assay to detect the rat sex-determining region Y (SRY) protein gene of the regenerating nerves. Results: At site A, GAP-43-positive nerve axons were identified from day 1 to 1 month post-OLT and SRY protein genes were expressed at and after 3 days post-OLT. At site B, GAP-43-positive axons were observed between 3 days and 1 month, and SRY protein genes were detected at 1 month post-OLT and thereafter. TEM confirmed the presence of regenerating axons at and after 3 days post-OLT. Conclusions: The results demonstrated that regenerating nerve fibers originating from the recipients reinnervated liver allografts. This extrinsic innervation occurred shortly after OLT, and most likely terminated after about 3 months.
Keywords: Orthotopic liver transplantation; Rat; Hepatic reinnervation; Nerve axon; Schwann cell; Immunohistochemical examination; GAP-43; PGP 9.5; Polymerase chain reaction assay; Sex-determining region Y protein gene

Viral Hepatitis

Fumitaka Suzuki et al.
Mutations of polymerase, precore and core promoter gene in hepatitis B virus during 5-year lamivudine therapy

Background/Aims: The effects of long-term lamivudine therapy on changes in polymerase and precore/core promoter mutations are unknown. The aim of this study was to determine the changes in these regions in patients with chronic hepatitis B virus (HBV) infection treated with lamivudine for 5 years. Methods: Serum samples obtained from 16 patients at the beginning of and during therapy were polymerase chain reaction-amplified, and nucleotide sequences of HBV analyzed. Results: By the end of 5-year therapy, mutations in YMDD motif emerged in ten patients. Mutations in L526M, M550V and M550I in polymerase were found in seven, six and six patients, respectively. The L526M mutant was found at the time or after detection of M550V/I mutant in six of seven patients. At baseline, precore and core promoter mutations were found in eight and 12 of 16 patients, respectively. Mutants of precore and core promoter were replaced by wild-type virus in each of three patients infected with mutants at 1 year. However, at 5 years of treatment, precore and core promoter mutations reappeared in some patients. Conclusions: Our data showed that lamivudine initially selected from precore/core promoter mutants to wild-type virus, but precore mutation reappeared during prolonged therapy.

Norio Akuta et al.
Efficacy of interferon monotherapy to 394 consecutive naive cases infected with hepatitis C virus genotype 2a in Japan: therapy efficacy as consequence of tripartite interaction of viral, host and interferon treatment-related factors

Background/Aims: The mechanism of variable response to interferon (IFN) monotherapy in patients infected with HCV genotype 2a is still unclear. Here we investigated the response in a large group of patients infected with genotype 2a. Methods: We evaluated 394 consecutive non-cirrhotic naive patients infected with genotype 2a who received IFN monotherapy for 24 weeks, including initial aggressive induction therapy. Of these, 97 were also evaluated for early viral kinetics in serum and treatment efficacy. Results: The overall sustained response (SR) rate was 68.3% (viral load <1.0 Meq/ml (82.4%); 1.0 (52.4%)). Multivariate analysis identified five independent factors associated with SR; viral load <1.0 Meq/ml, total IFN dose 700 million units, hepatocyte steatosis none or mild, albumin 3.9 g/dl, and alanine aminotransferase 75 IU/l. The kinetic study showed that serum viral clearance at 1 week was the best predictor of SR, and persistence at 4 weeks was a predictor of non-SR. Conclusions: Our study suggests that viral, host and IFN treatment-related factors determine the response to IFN monotherapy in patients infected with HCV genotype 2a. Further, we report that IFN monotherapy is very effective for patients with genotype 2a, especially for those with low viral load; and that early viral kinetics is useful as a predictor of the response.

Johan Westin, Hans Nordlinder, Martin Lagging, Gunnar Norkrans and Rune Wejstål
Steatosis accelerates fibrosis development over time in hepatitis C virus genotype 3 infected patients

Background/Aims: Steatosis is common in patients with hepatitis C virus (HCV) infection. Its influence on disease progression is only partially understood. The aim of this study was to evaluate the impact of steatosis on fibrosis progression over time in relation to HCV genotype. Methods: We retrospectively analyzed 98 patients who underwent dual liver biopsies prior to antiviral treatment. The median follow-up time was 5.8 years. Biopsy specimens were assessed for necroinflammatory activity, fibrosis and steatosis. Results: The prevalence and grade of steatosis were strongly associated with HCV genotype 3, independent of sex, age, body mass index and alcohol consumption. Progressive fibrosis was more prevalent in patients whose initial biopsy showed steatosis, an effect seen mainly in genotype 3 infected patients. Low-grade steatosis was observed in overweight patients, but high-grade steatosis was associated with genotype 3, independent of body mass index. Conclusions: Our data confirm the association between HCV genotype 3 and steatosis. Furthermore, we showed that steatosis in genotype 3 infected patients is a risk factor for progression of fibrosis. Therefore, patients with genotype 3 and steatosis ought to be recommended for early therapeutic intervention.

Markus Cornberg et al.
Mycophenolate mofetil in combination with recombinant interferon alfa-2a in interferon-nonresponder patients with chronic hepatitis C

Background/Aims: Since ribavirin was able to improve the antiviral efficacy of interferon alfa in patients with chronic hepatitis C, several other adjuncts have been studied. It has been shown that mycophenolate mofetil (MMF) is a more potent inhibitor of the inosine 5´-monophosphate-dehydrogenase (IMPDH) than ribavirin. The present study is a pilot study evaluating the efficacy and safety of combination therapy with interferon alfa-2a and MMF in interferon alfa nonresponder patients. Methods: Thirty-eight adult patients with chronic hepatitis C who did not respond to a previous interferon alfa monotherapy were enrolled to receive 6 million units of interferon alfa-2a tiw in combination with MMF (1 week 500 mg/day, 1 week 1000 mg/day, 22 weeks 2000 mg/day). Results: An interim analysis of 29 patients after 12 weeks of therapy showed that only one patient had negative hepatitis C virus-RNA at this time point. There was no significant reduction of the viral load during therapy. Due to inefficacy the study was discontinued. Conclusions: Combination therapy of interferon alfa-2a and MMF is ineffective in improving virological response rates in nonresponder patients with chronic hepatitis C. These data suggest that inhibition of the IMPDH seems not to be the major mechanism of ribavirin in enhancing the antiviral effect of interferon alfa in chronic hepatitis C.

Herbert L. Bonkovsky et al.
Iron and HFE or TfR1 mutations as comorbid factors for development and progression of chronic hepatitis C

Background/Aims: Recent evidence implicates iron as a comorbid factor for development of non-hemochromatotic liver diseases. Mutations or polymorphisms in the HFE gene or the TfR1 gene may influence the accumulation of iron in the liver or other tissues or may influence chronic viral hepatitis apart from effects on iron homeostasis. The aim of this study was to assess the role of hepatic iron, HFE and TfR1 variations on development and progression of chronic hepatitis C infection. Methods: We studied 119 consecutive patients with chronic hepatitis C, correlating clinical, laboratory, histopathological, and genetic data. Frequencies of genetic variations were compared with local and national controls. Results: HFE mutations were more common in patients than controls (48% vs. 38%, P=0.04), and advanced degrees of fibrosis developed at younger ages in subjects with the C282Y mutation (38.6 vs. 46.5 years, P=0.03). Patients carrying C282Y had higher mean hepatic iron concentrations (P=0.02), hepatic iron indices (P<=0.0001), and hepatic fibrosis scores (P=0.01). Hepatic fibrosis was correlated with hepatic iron concentration (P=0.03). TfR1 polymorphisms bore no detectable relation to disease severity or response to therapy. Conclusions: Hepatic iron and HFE mutations are comorbid factors that increase development and progression of chronic hepatitis C.

Christian G. Schüttler et al.
Suppression of hepatitis B virus enhancer 1 and 2 by hepatitis C virus core protein

Background/Aims: Epidemiological studies have shown that coinfection or superinfection with hepatitis B virus (HBV) and C virus (HCV) frequently leads to the suppression of hepatitis B virus replication. The mechanism of this phenomenon is still unclear. Shih et al. [J Virol 1993;67:5823] reported a direct suppression of HBV replication by the core protein of HCV. The target structure of HCV core protein in this system remained unclear. Methods: As HCV core protein has been shown to influence expression from transcriptional elements, we studied whether HCV core protein altered the activity of the two HBV enhancers 1 and 2. Luciferase vectors for HBV enhancers 1 or 2 were cotransfected with expression constructs for HCV core protein in murine and human hepatocyte lines. Results: Full-length HCV core protein suppressed the HBV enhancer 1 up to 11-fold, the enhancer 2 3-4-fold. Suppression of HBV enhancer 1 by HCV core from genotype 1b was stronger than by HCV core of genotypes 3a or 1a. Carboxyterminally truncated core proteins had lower or no suppression activity. Conclusions: These data suggest that HCV core protein may directly repress transcription of the HBV RNAs. This trans-repression may contribute to suppression of HBV replication in patients coinfected with both viruses.

Case Report

Norbert H. Gruener et al.
Hepatitis C virus eradication associated with hepatitis B virus superinfection and development of a hepatitis B virus specific T cell response
Background/Aims: Specific T cell responses during acute hepatitis B and during chronic hepatitis C have been described in detail. However, the T cell responses during the rare setting of acute hepatitis B virus (HBV) infection in the course of chronic hepatitis C that eventually lead to clearance of both viruses are completely unknown. Methods: We analyzed the virus specific CD4+ and CD8+ T cell response during an acute HBV superinfection in a patient with chronic hepatitis C. Results: The patient eliminated hepatitis C virus (HCV)-RNA and HBV-DNA from serum soon after the clinical onset of acute hepatitis B. The HBV specific T cell response found in this patient corresponds to the typical response that has been described in acute hepatitis B without chronic HCV infection. In contrast the hepatitis C specific immune response was similar to that generally found in chronic hepatitis C despite the fact that the patient also eliminated HCV-RNA. Conclusions: We hypothesize that the acute HBV infection induced a HBV specific T cell response which was associated with elimination HBV DNA and HCV-RNA, the latter possibly by bystander mechanisms, e.g. via secretion of cytokines. If such a non-specific bystander mechanism which has proven to be effective in the experimental setting and which is formally described here for a single patient can be shown to be a more general phenomenon, it may support the approach with new antiviral strategies, e.g. the induction of non-specific defense mechanisms against HCV.

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