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Revue de Presse du Mois Mois d'octobre 2002

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HEPATOLOGY

Table of Contents for October 2002 · Volume 36 · Number 4

Concise Review in Mechanisms of Disease

Epidemiology of the American Indians' burden and its likely genetic origins
Martin C. Carey, Beverly Paigen
It was not known until recently whether the endemic of cholesterol gallstones among certain southwestern American Indian tribes was unique among this ethnic group. With use of ultrasonography of the gallbladder and standard diagnostic criteria, gallstones are now found in epidemic proportions in 13 diverse American Indian tribes and communities living in Arizona, Oklahoma, and the Dakotas. We speculate that this predisposition is polygenic involving "thrifty" genes that conferred survival advantages when Paleo-Indians migrated from present-day Siberia to the Americas during the last Great Ice Age approximately 50,000 to 10,000 years ago. A reasonable hypothesis is that functioning of these genes promoted more efficient calorie utilization and storage in the form of adipose tissue. Beneficial results would have been operative during the isolation of Paleo-Indians in the Bering Strait land bridge (Beringia) when thrifty genes would have ensured sufficient fat reserves for survival of prolonged winters, successful pregnancy outcomes, and extended lactation periods. The authors' conjoint work on genetics of experimental cholesterol cholelithiasis in inbred mice promises help in pinpointing orthologous genetic loci (LITH genes) in the human genome. Moreover, the shared environments and homogeneity of American Indian tribes and communities should facilitate discovery of the ensembles of their common and rarer cholesterol gallstone genes. It is anticipated that knowledge of expression, polymorphisms, and functionality of LITH genes will help resolve the molecular mechanisms of this complex heterogeneous trait and thereby provide targets for novel therapies to prevent cholesterol cholelithiasis worldwide. (HEPATOLOGY 2002;36:781-791.)

Liver Biology and Pathobiology

Inducible differentiation and morphogenesis of bipotential liver cell lines from wild-type mouse embryos
Hélène Strick-Marchand, Mary C. Weiss
This work shows that hepatic cell lines reproducibly can be derived from E14 embryos of many mouse inbred strains. These bipotential mouse embryonic liver (BMEL) cell lines present a mixed morphology, containing both epithelial and palmate-like cells, and an uncoupled phenotype, expressing hepatocyte transcription factors (HNF1, HNF4, GATA4) but not functions (apolipoproteins, albumin). BMEL cells are bipotential: under inducing conditions they express hepatocyte and bile duct functions. In addition, they can undergo morphogenesis in Matrigel culture to form bile duct units. When returned to basal culture conditions, the differentiated cells revert, within a few days, to an undifferentiated state. The ensemble of markers expressed by BMEL cells implies that they originate from hepatoblasts, the endodermal precursors of the liver. In conclusion, the establishment of a simple and reproducible method to isolate from any mouse embryo bipotential hepatic cell lines that exhibit the properties of transit stem cells provides a novel paradigm for investigation of hepatic cell lineage relationships. (HEPATOLOGY 2002;36:794-804.)

Uroporphyria in the uroporphyrinogen decarboxylase-deficient mouse: Interplay with siderosis and polychlorinated biphenyl exposure
Michael R. Franklin, John D. Phillips, James P. Kushner
Several methods have been used to develop rodent models with the hepatic manifestations of porphyria cutanea tarda (PCT). Acute iron administration or mutations of the hemochromatosis gene (Hfe) have been used to generate hepatic siderosis, a nearly uniform finding in PCT. Heterozygosity for a null mutation at the uroporphyrinogen decarboxylase (Uro-D+/­) locus has been developed to mimic familial PCT in humans. This study examines the interplay of these 2 genetic risk factors and their influence, alone and combined with polychlorinated-biphenyl exposure. Neither an Hfe-null mutation nor iron-dextran administration alone or in combination with polychlorinated biphenyl exposure was porphyrinogenic in a 3-week model using mice wild-type at the Uro-D locus. Homozygosity for an Hfe-null mutation significantly elevated hepatic iron but not to the extent seen with parenteral iron-dextran administration. Homozygosity for an Hfe-null mutation but not iron-dextran administration was porphyrinogenic in animals heterozygous for the Uro-D mutation. Polychlorinated biphenyls were also porphyrinogenic in these animals. Uroporphyria in Uro-D+/­ animals was exacerbated by combinations of the homozygous Hfe-null mutation and polychlorinated biphenyls and iron-dextran and polychlorinated biphenyls. In all cases in which uroporphyria developed, a greater degree of experimental uroporphyria was seen in female animals. All elevated hepatic uroporphyrin concentrations were accompanied by depressed uroporphyrinogen decarboxylase activity and the presence of a factor in cytosol that inhibits recombinant human uroporphyrinogen decarboxylase. In conclusion, the expression of the uroporphyric phenotype, dependent on the susceptibility imparted by a genetic mutation, provides a uniquely facile model for dissecting the molecular pathogenesis of the disease. (HEPATOLOGY 2002;36:805-811.)

Cold ischemia decreases liver regeneration after partial liver transplantation in the rat: A TNF-/IL-6­dependent mechanism
Nazia Selzner, Markus Selzner, Yinghua Tian, Zakiyah Kadry, Pierre-Alain Clavien
New strategies of partial liver transplantation such as cadaveric split or living related liver transplantation have been developed to overcome organ shortage. Thus, studies on the ability of small partial grafts to regenerate to normal size while maintaining adequate function have become important. Here, we evaluated the effects of cold preservation on hepatocyte proliferation and function in a novel model of partial liver transplantation in rats. Lewis rats subjected to 70% liver resection (control) were compared with rats that underwent total hepatectomy and 30% partial liver transplantation (recipient). Livers were preserved at 4°C for 30 minutes, 10 hours, or 16 hours in University of Wisconsin solution. Seventy percent liver resection was associated with 100% survival, whereas 30-minute, 10-hour, and 16-hour preservation before 30% transplantation resulted in 80%, 40%, and 20% animal survival, respectively. Prolonged time of cold preservation (10 and 16 hours) was associated with a dramatic decrease of all markers of regeneration (P < .05). Tumor necrosis factor (TNF-) and interleukin 6 (IL-6) levels were also significantly decreased in recipient rats compared with the control group. Finally, pretreatment of recipients with recombinant IL-6 (rIL-6) normalized all markers of regeneration and significantly improved survival in the 10-hour group (90% vs. 40%; P < .05). In conclusion, sustained periods of cold preservation significantly impaired TNF- and IL-6 production, the regenerative ability of the liver, and animal survival. rIL-6 reversed impaired regeneration in the 10-hour cold ischemia group and suggests a primary role of nonparenchymal cells in modulating hepatocyte proliferation in the ischemic liver. (HEPATOLOGY 2002;36:812-818.)

Relevance of Niemann-Pick type C1 protein expression in controlling plasma cholesterol and biliary lipid secretion in mice
Ludwig Amigo, Hegaly Mendoza, Juan Castro, Verónica Quiñones, Juan Francisco Miquel, Silvana Zanlungo
Receptor-mediated endocytosis is one of the major mechanisms for uptake of lipoprotein cholesterol in the liver. Because Niemann-Pick C1 (NPC1) protein is a key component in the intracellular distribution of cholesterol obtained from lipoproteins by the endocytic pathway, it may play a critical role in controlling plasma lipoprotein cholesterol and its biliary secretion. A murine model of Niemann-Pick type C disease (NPC), the NPC1-deficient [NPC1 (­/­)] mouse, was used to evaluate the relevance of hepatic NPC1 expression in regulating plasma lipoprotein cholesterol profile and biliary lipid secretion under chow and high-cholesterol diets. Total plasma cholesterol concentrations were increased in NPC1 (­/­) mice compared with wild-type mice when both mouse strains were fed chow or high-cholesterol diets. The increased plasma cholesterol levels found in NPC1 (­/­) mice were mostly due to elevated cholesterol content in larger and more heterogeneous HDL particles. On the chow diet, biliary lipid secretion was not impaired by NPC1 deficiency. Furthermore, chow-fed NPC1 (­/­) mice showed a small, but significant, increase in biliary cholesterol secretion. On the high-cholesterol diet, wild-type mice increased biliary cholesterol output, whereas NPC1 (­/­) mice did not. Finally, hepatic NPC1 overexpression by adenovirus-mediated gene transfer increased biliary cholesterol secretion by 100% to 150% in both wild-type mice and cholesterol-fed NPC1 (­/­) mice. In conclusion, hepatic NPC1 expression is an important factor for regulating plasma HDL cholesterol levels and biliary cholesterol secretion in mice. (HEPATOLOGY 2002;36:819-828.)

Prevention of bile acid­induced apoptosis by betaine in rat liver
Dirk Graf, Anna Kordelia Kurz, Roland Reinehr, Richard Fischer, Gerald Kircheis, Dieter Häussinger
Bile acid­induced apoptosis plays an important role in the pathogenesis of cholestatic liver disease, and its prevention is of therapeutic interest. The effects of betaine were studied on taurolithocholate 3-sulfate (TLCS) and glycochenodeoxycholate (GCDC)-induced apoptosis in rat hepatocytes in vitro and in vivo. Hepatocyte apoptosis, caspase activation, and poly (ADP-ribose) polymerase (PARP) cleavage, which are normally observed in response to both bile acids, were largely prevented after preincubation of hepatocytes with betaine. Betaine uptake was required for this protective effect, which was already observed at betaine concentrations of 1 mmol/L. Betaine did not affect the TLCS-induced membrane trafficking of CD95 and tumor necrosis factor­related apoptosis inducing ligand (TRAIL) receptor 2 to the plasma membrane or the TLCS-induced recruitment of Fas-associated death domain (FADD) and caspase 8 to the CD95 receptor. However, betaine largely prevented cytochrome c release and oxidative stress exerted otherwise by TLCS. Inhibition of caspase 9 strongly blunted TLCS-induced caspase-8 activation. Further betaine did not prevent the TLCS-induced c-Jun N-terminal kinase (JNK), extracellular signal­regulated kinase (Erk), and p38 mitogen-activated protein kinase (p38MAPK) activation or TLCS-induced protein kinase B (PKB) dephosphorylation. The protective betaine effect was insensitive to inhibition of Erks by PD089059, of p38MAPK by SB203580, or of phosphatidylinositol 3-kinase (PI3-kinase) by LY294002. Betaine supplementation in the drinking water significantly ameliorated in vivo hepatocyte apoptosis following bile duct ligation. In conclusion, this study identifies betaine as a potent protectant against bile acid­induced apoptosis in vivo and in vitro, and its antiapoptotic action largely resides on an inhibition of the proapoptotic mitochondrial pathway. (HEPATOLOGY 2002;36:829-839.)

Fractal dimension can distinguish models and pharmacologic changes in liver fibrosis in rats
Frédéric Moal, Daniel Chappard, Jianhua Wang, Eric Vuillemin, Sophie Michalak-Provost, Marie Christine Rousselet, Frédéric Oberti, Paul Calès
Fractal analysis measures the complexity of geometric structures. The aim of this study was to evaluate the feasibility and accuracy of fractal analysis in liver fibrosis. A total of 77 rats were included: 10 sham, 46 with fibrosis secondary to bile duct ligation (BDL), and 21 with fibrosis due to CCl4 intoxication. Measurements included the fractal dimension of Kolmogorov (Dk), histologic lesions, the area of fibrosis by image analysis, liver hydroxyproline content, messenger RNA fibronectin, serum hyaluronate level, and portal pressure. Fibrotic rats were given placebo, octreotide, or O2-vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (V-PYRRO/NO). Intraobserver agreement of Dk was excellent with the intraclass (ic) correlation coefficient ric = 0.91 (P < .0001) as well as the interobserver agreement with ric = 0.88 (P < .001). Dk was correlated with other measurements or markers of fibrosis: the area of fibrosis (r = 0.75; P < .0001), hydroxyproline content (r = 0.51; P < .001), serum hyaluronate level (r = 0.52; P < .001), and portal pressure (r = 0.52; P < .01). Dk was significantly different between the 2 models of fibrosis (P < .0001), unlike the area of fibrosis, and this relationship was independent of other histologic lesions. The significant decrease in fibrosis observed with octreotide or V-PYRRO/NO was similarly reflected by Dk or the area of fibrosis. The diagnostic accuracy for the fibrosis model was 97% with the 5 main measurements or markers of fibrosis studied, with Dk isolated at the first step by stepwise analysis. In conclusion, fractal analysis is suitable for analyzing liver fibrosis and has excellent reproducibility. This is the only quantitative morphometric method that can discriminate among the models of fibrosis and is sensitive enough to detect pharmacologically induced changes in liver fibrosis. (HEPATOLOGY 2002;36:840-849.)

Tissue inhibitor of metalloproteinases-1 attenuates spontaneous liver fibrosis resolution in the transgenic mouse
Hitoshi Yoshiji, Shigeki Kuriyama, Junichi Yoshii, Yasuhide Ikenaka, Ryuichi Noguchi, Toshiya Nakatani, Hirohisa Tsujinoue, Koji Yanase, Tadashi Namisaki, Hiroo Imazu, Hiroshi Fukui
It has been suggested that the tissue inhibitor of metalloproteinases-1 (TIMP-1) is involved in spontaneous resolution of liver fibrosis. The aim of this study was to investigate whether TIMP-1 altered spontaneous resolution of liver fibrosis in conjunction with matrix metalloproteinases (MMP) inhibition and hepatic stellate cell (HSC) activation. The livers of liver-targeted TIMP-1 transgenic (TIMP-Tg) and control hybrid (Cont) mice were harvested at 0, 3, 7, and 28 days following spontaneous recovery from CCl4-induced liver fibrosis. The extent of fibrosis resolution, MMP expression, -smooth-muscle actin (-SMA) positive cells, and procollagen-(I) messenger RNA (mRNA) in the liver were assessed at the respective periods in both groups. We also examined the effect of TIMP-1 on HSC apoptosis. The TIMP-Tg mice showed significantly attenuated resolution of spontaneous liver fibrosis compared with the Cont mice. The hydroxyproline content, number of -SMA positive cells, and procollagen-(I) mRNA rapidly decreased with time in the Cont mice, whereas these markers were little changed in TIMP-Tg mice. The level of the active form of metalloproteinases-2 (MMP-2) in the TIMP-Tg mice was less than that in the Cont mice. TIMP-1 markedly decreased the nonparenchyma apoptotic cells in the liver fibrosis resolution model, and it also inhibited HSC apoptosis associated with suppression of caspase-3 activity in vitro. In conclusion, TIMP-1 significantly attenuated spontaneous resolution of liver fibrosis by the combination of a net reduction of the MMP activity and suppression of apoptosis in HSC. (HEPATOLOGY 2002;36:850-860.)

Regulation of endothelin-A receptor sensitivity by cyclic adenosine monophosphate in rat hepatic stellate cells
Roland Reinehr, Richard Fischer, Dieter Häussinger
Sensitization of the endothelin-A receptor (ETA) occurs during HSC transdifferentiation, but the underlying mechanisms remained unclear. Sensitization of ETA was studied in quiescent and activated hepatic stellate cells (HSC) at the levels of receptor phosphorylation, localization, endothelin (ET)-1-induced Ca2+ signals, and cell contraction. The endothelin-1 (ET-1) concentrations required to obtain an ETA-mediated Ca2+ signal in 50% of HSC cultured for 1 to 2 or 10 days were approximately 1.2 and 0.012 nmol/L, respectively. This transdifferentiation-dependent sensitization of ETA was accompanied by receptor translocation to the plasma membrane. Cyclic AMP rapidly desensitized ETA in activated HSC and shifted their ET-1 responsiveness from picomolar to nanomolar concentrations with respect to Ca2+ signals and HSC contraction. ETA desensitization also occurred in response to prostaglandin E2, adenosine, or ETB stimulation. Desensitization by cAMP in activated HSC was accompanied by an increased Ser/Thr phosphorylation of ETA and their rapid internalization. Quiescent HSC exhibited Ser/Thr phosphorylation of the ETA protein, which was not affected by cAMP. In conclusion, the ETA response in HSC is regulated by protein kinase A (PKA)-dependent receptor phosphorylation and internalization. This may explain the transdifferentiation-dependent sensitization of HSC towards ET-1 and its reversal by cAMP and ETB activation. (HEPATOLOGY 2002;36:861-873.)

Reversal of drug resistance of hepatocellular carcinoma cells by adenoviral delivery of anti-MDR1 ribozymes
Matthes Huesker, Yvonne Folmer, Michaela Schneider, Christine Fulda, Hubert E. Blum, Peter Hafkemeyer
Human cancers, including hepatocellular carcinoma (HCC), are characterized by a high degree of drug resistance. The multidrug resistance (MDR) transporters MDR1-P-glycoprotein and MRP2 (multidrug-associated protein 2) are expressed in almost 50% of human cancers, including HCCs. In this study, we analyzed the effect of anti-MDR1 ribozymes, especially AFP promoter-driven anti-MDR1 ribozymes, to specifically chemosensitize HCC cells. Epirubicin-selected HB8065/R cells were used as MDR1-P-glycoprotein-overexpressing cells. Adenoviral vectors were constructed to allow an efficient gene transfer of anti-MDR1 ribozyme constructs. AFP promoter-driven anti-MDR1 ribozymes reduced the IC50 30-fold for epirubicin in HCC cells, whereas human colorectal cancer cells were unaffected. Target sequences were either the translational start site or codon 196 of the human MDR1 gene. Adenoviral delivery of CMV promoter-driven anti-MDR1 ribozymes resulted in a reduced IC50 for epirubicin and doxorubicin (60- and 20-fold, respectively). They completely restored chemosensitivity in stably transfected anti-MDR1 ribozyme-expressing HCC cells as well as in HCC cells transduced with adenoviruses expressing wild-type anti-MDR1 ribozymes. Adenoviral delivery of ribozymes was so efficient that chemosensitization of HCC cells could be demonstrated in cell cultures without further selection of transduced cells for single anti-MDR1 ribozyme-expressing HCC cell clones. Northern blots showed a decreased MDR1 mRNA expression, and fluorescence-activated cell sorting (FACS) analysis revealed a significantly reduced expression of MDR1-P-glycoprotein on the cell surface of HB8065/R cells after transduction with the anti-MDR1 ribozymes. In conclusion, our data demonstrate that adenoviral delivery of ribozymes can chemosensitize HCC cells and that chemosensitization can be specifically achieved by ribozymes driven by an AFP promoter directed against human MDR1. (HEPATOLOGY 2002;36:874-884.)

Proapoptotic and antiproliferative potential of selective cyclooxygenase-2 inhibitors in human liver tumor cells (*Human Study*)
Michael André Kern, Dominic Schubert, Dina Sahi, Mirja Mareike Schöneweiß, Ilona Moll, Anke Maria Haugg, Hans Peter Dienes, Kai Breuhahn, Peter Schirmacher
Recent studies have shown increased levels of cyclooxygenase-2 (COX-2) in a variety of human malignancies, including hepatocellular carcinoma (HCC), but so far it is unknown whether COX-2 contributes to the malignant growth and whether inhibition of COX-2 function modifies the malignant potential of liver tumors. COX-1 and COX-2 expression was determined in 4 liver tumor cell lines (Hep 3B, HuH-7, Hep G2, Sk-hep1) by Northern hybridization and Western immunoblot. The functional effects of the nonselective inhibitor sulindac sulfide and the COX-2 selective inhibitors SC-58635 and meloxicam were examined by 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide (MTT)-assays and BrdU uptake, morphology, and TUNEL analysis of apoptosis. Apoptosis regulating proteins were analyzed by Western immunoblot. COX-1 and COX-2 expression was demonstrable in all tested liver tumor cell lines. Sulindac sulfide (50 to 400 µmol/L), SC-58635 (6,25 to 400 µmol/L), and meloxicam (6.25 to 400 µmol/L) led to a significant time- and dose-dependent reduction of cell numbers of up to 80% (P < .05). At equimolar concentrations the effect was more pronounced when COX-2 was selectively blocked. COX-2 inhibition induced apoptosis and reduced tumor cell proliferation. Apoptosis after COX-2 inhibition with SC-58635 (50 µmol/L) was independent of BCL-2, BAX, and the phosphorylation status of AKT/PKB and BAD, but correlated with activation of caspase-9, caspase-3, and caspase-6. In conclusion, selective inhibition of COX-2 leads to a marked growth inhibition of human liver tumor cells, based on the induction of apoptosis and inhibition of proliferation and, thus, may offer therapeutic and preventive potential in human hepatocarcinogenesis. (HEPATOLOGY 2002;36:885-894.)

Liver Failure and Liver Disease

Bone metabolism in advanced cholestatic liver disease: Analysis by bone histomorphometry (*Human Study*)
Maureen M. J. Guichelaar, Michael Malinchoc, Jean Sibonga, Bart L. Clarke, J. Eileen Hay
Despite the clinical importance of cholestatic osteopenia, little is known about its pathophysiologic mechanism. By tetracycline-labeled histomorphometric analysis of bone biopsies taken at the time of liver transplantation, we prospectively evaluated bone resorption and formation in 50 consecutive patients with advanced primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC). Histomorphometric analysis confirmed low bone volume parameters, consistent with the mean T-score of the lumbar spine of ­1.9 by dual energy x-ray absorptiometry. Dynamic (bone formation rates, adjusted apposition rates) and static (osteoid markers, osteoblast number) parameters of bone formation were decreased in cholestatic patients with no abnormalities in mineralization. Increased osteoclast numbers and increased eroded surface areas suggested increased bone resorption, and this was supported in female patients by increased trabecular separation and decreased trabecular number. Male cholestatic patients, however, did not have significant increases in resorption parameters, although they were as osteopenic as female patients and had low bone formation markers. Bone histomorphometric changes were similar in PBC and PSC, suggesting an etiologic effect of chronic cholestasis rather than the individual diseases. Cancellous bone volume and osteoid markers correlated with bone mineral density measurements but no correlations were found between histomorphometric parameters and biochemical markers of bone metabolism. In conclusion, cholestatic osteopenia appears to result from a combination of decreased bone formation and increased resorption, especially in female patients, but the relative importance of these two abnormalities and their actual etiology remain to be elucidated. (HEPATOLOGY 2002;36:895-903.)

The significance of human jagged 1 mutations detected in severe cases of extrahepatic biliary atresia (*Human Study*)
Takao Kohsaka, Zeng-rong Yuan, Shu-xia Guo, Manabu Tagawa, Akio Nakamura, Miwako Nakano, Hideo Kawasasaki, Yukihiro Inomata, Koichi Tanaka, Jun Miyauchi
Mutations of human jagged 1 (JAG1) gene are responsible for Alagille Syndrome (AGS), whose 2 main symptoms are intrahepatic bile duct hypoplasia and pulmonary stenosis. We examined the JAG1 mutation in extrahepatic biliary atresia (EHBA), which is similar in phenotype to AGS, although a different pathogenesis is suggested. In 102 cases of EHBA, 9 missense mutations were detected, including 2 intrafamilial expressions in the propositus and an aunt of one family. These mutations were all missense and sporadic except for those of this particular family. The JAG1 gene mutations were generally found in severely ill patients subjected to liver transplantation at less than 5 years of age. None of the 9 cases of EHBA revealed any of the 5 major symptoms of AGS nor any identical pathological findings after 3 years of follow-up. Our cases were clearly separated from AGS by pathological findings and clinical features, and could be diagnosed as EHBA and not as atypical AGS. The increase of interleukin 8 (IL-8) production induced by tumor necrosis factor (TNF-) in Huh 7 cells was suppressed by the coexistence of JAG1 protein. We examined the different influences between wild-type cells and the 3 kinds of mutants detected in EHBA on Huh 7 cells and found that 2 of 3 mutants showed about half of the repressed activity compared with that of wild type. In conclusion, these results suggest that the JAG1 gene abnormality may be an aggravating factor in EHBA. (HEPATOLOGY 2002;36:904-912.)

Liver disease in pediatric patients with cystic fibrosis is associated with glutathione S-transferase P1 polymorphism (*Human Study*)
Alexandra Henrion-Caude, Cyril Flamant, Michel Roussey, Chantal Housset, Antoine Flahault, Anthony A. Fryer, Katarina Chadelat, Richard C. Strange, Annick Clement
Liver disease in patients with cystic fibrosis (CF) is inconstant and has not yet been clearly related to any specific risk factor. While the expression of cystic fibrosis transmembrane conductance regulator (CFTR) is restricted to the biliary epithelium in the liver, recent findings indicate that CFTR modulates reduced glutathione (GSH) transport and that CFTR dysfunction creates an imbalance in the antioxidant defense. Among liver detoxifying enzymes, the glutathione S-transferases (GSTs) play a key role in the protection against oxidative stress. Because oxidative injury contributes to the development of liver disease, we hypothesized that 2 members of the GST superfamily, GSTM1 and GSTP1, which are expressed in the biliary epithelium, could influence the hepatic status in patients with CF. The potential impact of GSTM1 and GSTP1 gene polymorphisms was assessed in 106 children with CF (mean age, 11.5 years). Based on polymerase chain reaction/restriction fragment length polymorphism analysis, we found that the frequency of GSTP1-Ile105/Ile105 genotype was significantly higher in patients with CF with liver disease than in those without (P < .03). Among the youngest patients, aged 6 years, GSTP1-Ile105/Ile105 genotype was associated with a 8-fold increase in the risk of liver disease compared with other GSTP1 genotypes (P = .002). No association between the GSTM1 genotype and liver status was documented. In conclusion, GSTP1-Ile105-encoding allele contributes to hepatic dysfunction in CF. Identification of this polymorphism may have prognostic value and prompt early treatment in patients with CF with an increased risk of liver disease. (HEPATOLOGY 2002;36:913-917.)

Analysis of TCR antagonism and molecular mimicry of an HLA-A*0201­restricted CTL epitope in primary biliary cirrhosis (*Human Study*)
Hiroto Kita, Shuji Matsumura, Xiao-Song He, Aftab A. Ansari, Zhe-Xiong Lian, Judy Van de Water, Ross L. Coppel, Marshall M. Kaplan, M. Eric Gershwin
Although the etiology and mechanism of primary biliary cirrhosis (PBC) is unknown, growing evidence suggests a major role for T cells. We have recently identified the first CD8 T-cell epitope, amino acid 159-167 of the E2 component of pyruvate dehydrogenase complexes (PDC-E2). To seek for analogue peptide-antagonizing effector function of CTLs specific for this autoantigen, we examined the effector functions of the PDC-E2-specific CTLs against alanine substituted peptides. Furthermore, because molecular mimicry has been postulated as a possible cause of initiating PBC, we carried out studies aimed at identifying naturally occurring peptides for the 159-167 peptide of PDC-E2 that may serve as agonists. An alanine substitution at position 5 of this epitope significantly reduced peptide-specific effector functions of CTLs. Moreover, this analogue peptide inhibited effector functions of the CTLs to the prototype peptide, including cytotoxicity and IFN- production. We also identified a peptide derived from Pseudomonas aeruginosa, which showed a higher binding affinity to the HLA-A*0201 than the prototype peptide. This homologous peptide was recognized by CTLs specific for the prototype epitope on PDC-E2. In conclusion, a modification of the immunodominant autoepitope can be utilized to manipulate the CD8 T-cell responses against the autoantigen PDC-E2. Our finding also supports the thesis that molecular mimicry may be implicated in the initiation of the autoreactive CD8 T-cell responses and has implications for the use of such peptides for immunotherapy. (HEPATOLOGY 2002;36:918-926.)

p53 gene and Wnt signaling in benign neoplasms: -catenin mutations in hepatic adenoma but not in focal nodular hyperplasia (*Human Study*)
Ya-Wen Chen, Yung-Ming Jeng, Shiou-Hwei Yeh, Pei-Jer Chen
Hepatocellular adenoma (HA) and focal nodular hyperplasia (FNH) are 2 rare, benign liver neoplasms that often are discovered incidentally. To date, few genetic changes have been found in these 2 benign lesions. However, the 2 pathways of p53 and Wnt signaling, which may be the most common molecular targets involved in liver tumorgenesis, were studied in HA and FNH. Ten HAs and 11 FNHs were analyzed for loss of heterozygosity (LOH) and sequencing analysis of mutation hot spots in exons 5 to 8 of the p53 gene. No LOH or mutant sequences were identified, indicating that p53 was not associated with these benign lesions. Genes in the Wnt signaling pathway, including -catenin, axin, and adenomatous polyposis coli (APC), also were studied. Polymerase chain reaction (PCR) amplification and direct sequencing of all samples of HA and FNH displayed no mutations in exon 3 of the -catenin gene. However, 3 HAs (30%) contained interstitial deletions from exon 3 to exon 4. Truncated forms of -catenin detected by Western blot and immunohistochemical analyses showed they had accumulated in the cytoplasm and nuclei. However, for the axin and APC genes, no genetic changes, including allelic loss, interstitial deletions and point mutations, were detected in any of the HAs and FNHs. In conclusion, -catenin, which participates in the Wnt signaling pathway, might play a more important role in the formation of HA than in that of FNH, but p53 is not associated with the development of either HA or FNH. (HEPATOLOGY 2002;36:927-935.)

Increasing intra-abdominal pressure increases pressure, volume, and wall tension in esophageal varices (*Human Study*)
Angels Escorsell, Angels Ginès, Josep Llach, Joan C. García-Pagán, Josep M. Bordas, Jaume Bosch, Joan Rodés
Many daily activities cause acute elevations of intra-abdominal pressure (IAP). In portal hypertensive cirrhotic patients, increased IAP increases absolute portal pressure and azygos blood flow, suggesting that it may have detrimental consequences at the esophageal varices. The aim of this study was to investigate the effects of increased IAP on variceal pressure, size, and wall tension. Endosonography and a noninvasive endoscopic pressure gauge were used to measure variceal pressure, radius, wall tension, and volume in baseline conditions and after increasing IAP by 10 mm Hg using an inflatable girdle in 14 patients with cirrhosis and esophageal varices. Increasing IAP markedly increased variceal pressure (from 13.3 ± 4.2 to 17.4 ± 4.6 mm Hg; P = .0001) and radius (from 2.9 ± 1.0 to 3.9 ± 1.1 mm; P = .0001). Consequently, wall tension dramatically increased (from 38.7 ± 13.6 to 65.9 ± 23.8 mm Hg · mm, +78%; P = .0001). Variceal volume increased significantly from 1,264 ± 759 to 2,025 ± 1,129 mm3 (P = .0001). In conclusion, in portal hypertensive cirrhotic patients, increases in IAP have deleterious effects on variceal hemodynamics, markedly increasing the volume, pressure, and wall tension of the varices. Increases in IAP may contribute to the progressive dilatation that precedes the rupture of the varices in portal hypertension. (HEPATOLOGY 2002;36:936-940.)

Terlipressin therapy with and without albumin for patients with hepatorenal syndrome: Results of a prospective, nonrandomized study (*Human Study*)
Rolando Ortega, Pere Ginès, Juan Uriz, Andrés Cárdenas, Blas Calahorra, Dara De Las Heras, Mónica Guevara, Ramón Bataller, Wladimiro Jiménez, Vicente Arroyo, Juan Rodés
Vasopressin analogues associated with albumin improve renal function in hepatorenal syndrome (HRS). The current study was aimed at assessing the efficacy of the treatment, predictive factors of response, recurrence of HRS, and survival after therapy. Twenty-one consecutive patients with HRS (16 with type 1 HRS, 5 with type 2 HRS) received terlipressin (0.5-2 mg/4 hours intravenously) until complete response was achieved (serum creatinine level < 1.5 mg/dL) or for 15 days; 13 patients received intravenous albumin together with terlipressin. Twelve of the 21 patients (57%) showed complete response. Albumin administration was the only predictive factor of complete response (77% in patients receiving terlipressin and albumin vs. 25% in those receiving terlipressin alone, P = .03). Treatment with terlipressin and albumin was associated with a remarkable decrease in serum creatinine level, increase in arterial pressure, and suppression of the renin-aldosterone system. By contrast, no significant changes in these parameters were found in patients treated with terlipressin alone. Only 1 patient showed ischemic adverse effects. Recurrence of HRS occurred in 17% of patients with complete response. The occurrence of complete response was associated with an improved survival. In conclusion, terlipressin therapy reverses HRS in a high proportion of patients. Recurrence rate after treatment withdrawal is uncommon. Albumin appears to improve markedly the beneficial effects of terlipressin. (HEPATOLOGY 2002;36:941-948.)

Albumin dialysis in cirrhosis with superimposed acute liver injury: A prospective, controlled study (*Human Study*)
Uwe Heemann, Ulrich Treichel, Jan Loock, Thomas Philipp, Guido Gerken, Massimo Malago, Sebastian Klammt, Matthias Loehr, Stephan Liebe, Steffen Mitzner, Reinhardt Schmidt, Jan Stange
Patients with liver cirrhosis and a superimposed acute injury with progressive hyperbilirubinemia have a high mortality. A prospective, controlled study was performed to test whether hyperbilirubinemia, 30-day survival, and encephalopathy would be improved by extracorporeal albumin dialysis (ECAD). Twenty-four patients were studied; 23 patients had cirrhosis; 1 had a prolonged cholestatic drug reaction and was excluded from per protocol (PP) analysis. Patients had a plasma bilirubin greater than 20 mg/dL and had not responded to prior standard medical therapy (SMT). Patients were randomized to receive SMT with ECAD or without (control). ECAD was performed with an extracorporeal device that dialyzes blood in a hollow fiber dialyzer (MW cutoff < 60 kd) against 15% albumin. Albumin-bound molecules transfer to dialysate albumin that is regenerated continuously by passage through a charcoal and anion exchange column and a conventional dialyzer. ECAD was associated with improved 30-day survival (PP, 11 of 12 ECAD, 6 of 11 controls; log rank P < .05). Plasma bile acids and bilirubin decreased on average by 43% and 29%, respectively, in the ECAD group after 1 week of treatment, but not in the control group. Renal dysfunction and hepatic encephalopathy improved in the ECAD group, but worsened significantly in the control group. ECAD was safe, with adverse events being rare and identical in both groups. In conclusion, ECAD appears to be effective and safe for the short-term treatment of patients with cirrhosis and superimposed acute injury associated with progressive hyperbilirubinemia and may be useful for increasing survival in such patients awaiting liver transplantation. (HEPATOLOGY 2002;36:949-958.)

Viral Hepatitis

A randomized 4-arm multicenter study of interferon alfa-2b plus ribavirin in the treatment of patients with chronic hepatitis C relapsing after interferon monotherapy (*Human Study*)
Giorgio Saracco, Alda Olivero, Alessia Ciancio, Silvia Carenzi, Antonina Smedile, Giuseppe Cariti, Massimo Andreoni, Pier Giulio Orsi, Alberto Biglino, Marco Tabone, Luigi Roffi, Guido Croce, Aldo Manca, Gianfranco Tappero, Giovannino Ciccone, Mario Rizzetto, for the North West Italian Hepatologic Group
To determine whether a higher dosage of interferon (IFN) and/or a prolonged time of administration may improve the efficacy of combination therapy, we conducted a 4-arm randomized trial on patients with chronic hepatitis C relapsing after 1 or more previous treatment courses with IFN monotherapy. Group A (n = 70) received 3 MU IFN alfa-2b 3 times per week plus ribavirin 1,000 mg/d for 12 months; group B (n = 70) received 5 MU 3 times per week plus ribavirin for 12 months; group C (n = 82) received 3 MU 3 times per week plus ribavirin for 6 months, and group D (n = 73) received 5 MU 3 times per week plus ribavirin for 6 months. The primary end point was the clearance of viremia at the end of 6-month follow-up: test results for hepatitis C virus (HCV)-RNA were negative in 54% of group A, 56% of group B, 40% of group C, and 49% of group D patients (P = NS). Among patients with genotype 1 and 4, the sustained response was significantly higher in groups A and B than in group C (45%, 49% vs. 22%, P = .03; group D = 33%, P = NS). In patients with genotype 2 and 3, the sustained virologic response was not affected by the different regimens (group A = 69%, group B = 68%, group C = 62%, group D = 71%, P = NS). In conclusion, duration of therapy rather than IFN dosage is more important in increasing the sustained virologic rate among HCV-positive patients with genotype 1 and 4 relapsing after IFN monotherapy; patients with genotypes 2 and 3 can be effectively retreated with a 6-month course of combination therapy, avoiding unnecessary side effects and waste of resources. (HEPATOLOGY 2002;36:959-966.)

Hepatitis C in adults and adolescents with hemophilia: A randomized, controlled trial of interferon alfa-2b and ribavirin (*Human Study*)
Michael W. Fried, Joy Peter, Keith Hoots, Paul J. Gaglio, Donald Talbut, P. Charleton Davis, Nigel S. Key, Gilbert C. White, Lauren Lindblad, Frederick R. Rickles, Thomas C. Abshire, for the Hemophilia Hepatitis Therapy Group
Adolescents and adults with inherited disorders of coagulation have one of the highest prevalence rates of hepatitis C among known risk groups. Few data are available on the use of combination therapy with interferon and ribavirin in this population. Patients 13 years of age and older who were positive for hepatitis C virus (HCV) RNA by polymerase chain reaction and negative for human immunodeficiency virus were randomized to receive interferon alfa-2b (3 million units 3 times a week) plus ribavirin (1,000 mg/day) or interferon alfa-2b alone for 48 weeks with 24 weeks of posttreatment follow-up. Patients started on interferon alone who remained positive for HCV RNA at week 12 crossed over to treatment with interferon plus ribavirin. A total of 113 patients were treated. Thirty-seven patients were younger than 18 years. At the end of treatment, 18 of 56 (32%) treated with interferon plus ribavirin and 6 of 57 (11%) treated with interferon alone were negative for HCV RNA (P = .005). Sustained virologic response in the combination arm was 29% (16 of 56) compared with 7% (4 of 57) for those started on interferon alone (P = .027). Among adolescents younger than 18 years who were treated with combination therapy, 10 of 17 (59%) had sustained response compared with 6 of 39 (15%) of adult patients on the same regimen (P = .001). In conclusion, in this U.S. multicenter, randomized trial of therapy for HCV in patients with inherited bleeding disorders, sustained virologic response rate was significantly improved for patients treated with interferon and ribavirin compared with those started on interferon alone. Adolescents treated with combination therapy had a significantly higher sustained response than adults did on the same regimen. (HEPATOLOGY 2002;36:967-972.)

Predictive value of ALT levels for histologic findings in chronic hepatitis C: A European collaborative study (*Human Study*)
Pierre Pradat, Alfredo Alberti, Thierry Poynard, Juan-Ignacio Esteban, Ola Weiland, Patrick Marcellin, Salvatore Badalamenti, Christian Trépo
The aim of this retrospective study was to determine the predictive value of alanine aminotransferase (ALT) levels for histologic findings in patients with chronic hepatitis C virus (HCV) infection. Data on 864 HCV RNA­positive patients were collected. ALT values were obtained at the time of biopsy (before treatment), and normal ALT values were defined as normal values obtained at serial evaluations during a 6-month period. Histologic results were scored using the METAVIR system. Among all patients, 99% of those with elevated ALT levels had a score of at least F1 (positive predictive value [PPV], 99%) and 88% had a score greater than A1F1. Among patients with persistently normal ALT values, 65% had a score of at least F1 (negative predictive value [NPV], 35%) and 26% had a score greater than A1F1. The receiver operating characteristics analysis indicates that the ALT threshold for the best compromise sensitivity-specificity was about 2.25 times the upper limit of normal (ULN). In conclusion, almost all HCV RNA­positive patients with elevated ALT levels have some degree of fibrosis. However, an important proportion of patients with persistently normal ALT levels also show some histologic signs of fibrosis; the degree of fibrosis is usually mild but is sometimes more marked, and in rare cases cirrhosis may be present. In this subset of patients, the indication of liver biopsy and the potential benefit of therapy need to be further evaluated. These results suggest the need to revisit the algorithm for liver biopsy practice. (HEPATOLOGY 2002;36:973-977.)

Hepatitis C, cryoglobulinemia, and cirrhosis: A meta-analysis
Zeid Kayali, Victor E. Buckwold, Bridget Zimmerman, Warren N. Schmidt
Approximately 40% of patients with chronic hepatitis C virus (HCV) infection develop detectable serum cryoglobulins or cryoprecipitates (CP), although most do not show clinical or physical signs of syndromic cryoglobulinemia. Although association of HCV with the extrahepatic complications of cryoglobulinemia is widely recognized, the relationship of cryoglobulinemia with liver disease is unclear. We wished to study the relationship between CP and cirrhosis and to determine whether the development of CP is a true covariate for progressive liver disease or a confounding variable that impacts cirrhosis because of patient age, duration of disease, or differences in gender. We undertook a meta-analysis of 19 studies published between 1994 and 2001. The incidence of cirrhosis was compared in patients with and without CP after logistic regression adjustments for accepted risk factors for progressive liver disease, including age, gender, and estimated duration of disease (EDD). A total of 2,323 patients with chronic hepatitis C were identified, with 1,022 (44%) having detectable CP. Cirrhosis was present in 40% of patients with CP but only 17% of patients without CP (total 2 = 141.69, P < .001). After adjusting for age, gender, and estimated duration of disease by logistic regression, the combined odds ratio for incidence of cirrhosis in patients CP positive versus CP negative was 4.87, (95% CI: 3.32, 7.15), indicating a highly significant association between cirrhosis and cryoglobulinemia. In conclusion, cryoglobulins may be a useful prognostic indicator for increased risk of cirrhosis with chronic hepatitis C. (HEPATOLOGY 2002;36:978-985.)

Identification of chronic hepatitis C patients without hepatic fibrosis by a simple predictive model (*Human Study*)
Xavier Forns, Sergi Ampurdanès, Josep M. Llovet, John Aponte, Llorenç Quintó, Eva Martínez-Bauer, Miquel Bruguera, Jose Maria Sánchez-Tapias, Juan Rodés
Liver biopsy is required for staging hepatic fibrosis in patients with chronic hepatitis C, but it is an expensive procedure with occasional complications and poor patient acceptance. This cohort study was designed to assess the accuracy of a noninvasive method aimed to discriminate between patients with and without significant liver fibrosis (stages 2-4 versus 0-1). Clinically relevant variables were analyzed in a cohort of 476 consecutive untreated patients (estimation group, 351 patients; validation group, 125 patients) with chronic hepatitis C who underwent a liver biopsy. Multivariate analysis identified age, gamma glutamyl transpeptidase (GGT), cholesterol, platelet count, and prothrombin time as independent predictors of fibrosis. We constructed a model and a score system combining age, GGT, cholesterol, and platelet count that proved useful to identify patients without significant hepatic fibrosis. The area under the ROC curve was 0.86 for the estimation group and 0.81 for the validation group. Using the best cutoff score (less than 4.2), presence of significant fibrosis (F2 to F4) could be excluded with high accuracy (negative predictive value of 96%) in 125 (36%) of 351 patients. Similarly, it could be excluded with the same certainty in 49 (39%) of the 125 patients of the validation group. Only 2 patients with liver fibrosis stage 2 were incorrectly classified. In conclusion, a combination of easily accessible variables accurately predicts the absence of significant fibrosis and might render liver biopsy unnecessary in more than one third of patients with chronic hepatitis C. (HEPATOLOGY 2002;36:986-992.)

Outcome of an outbreak of acute hepatitis C among healthy volunteers participating in pharmacokinetics studies (*Human Study*)
Alberto Larghi, Massimo Zuin, Andrea Crosignani, Maria Lisa Ribero, Cristina Pipia, Pier Maria Battezzati, Giorgio Binelli, Francesco Donato, Alessandro Remo Zanetti, Mauro Podda, Alessandro Tagger
We identified 15 patients with acute hepatitis C (AHC) among 29 healthy volunteers participating in 2 consecutive pharmacokinetics studies. Molecular techniques were used to determine the relatedness of viral strains, whereas clinical and virologic follow-up was started to establish the course and outcome of the acute infection. After presentation, serum liver enzymes and HCV RNA were monitored weekly for 4 months, then monthly for at least 12 months. Liver biopsy was performed 6 to 12 months after AHC diagnosis. Phylogenetic analysis of coding regions for the envelope glycoproteins E1 and E2 was performed. At presentation, all 15 patients tested HCV RNA­positive and had HCV genotype 2c. Phylogenetic analysis indicated a common source of infection. Fourteen patients agreed to be followed prospectively. Infection resolved spontaneously in 8 patients, HCV RNA becoming undetectable by 4 to 5 months after the presumed time of infection in 5 of them and by 8, 13, and 24 months in the remaining 3. Six patients developed chronic infection. Liver biopsies performed in 9 subjects who were HCV RNA­positive 6 months after AHC diagnosis revealed that the prevalent histologic finding was lobular inflammation. In conclusion, our homogeneous cohort showed a wide spectrum of clinical, virologic and histologic features, and, more importantly, short-term outcome differed noticeably despite the common source of infection. (HEPATOLOGY 2002;36:993-1000.)

Imbalanced intrahepatic expression of interleukin 12, interferon gamma, and interleukin 10 in fulminant hepatitis B (*Human Study*)
Ludger Leifeld, Silvia Cheng, Jan Ramakers, Franz-Ludwig Dumoulin, Christian Trautwein, Tilman Sauerbruch, Ulrich Spengler
In murine models, overexpression of interleukin (IL)-12 and interferon (IFN)- can induce severe liver damage, whereas IL-10 has anti-inflammatory and hepatoprotective properties. To analyze the potential role of these cytokines in human fulminant hepatitis B, we used immunohistochemistry to study expression of IL-12, IFN-, and IL-10 in explant livers of 11 patients with fulminant hepatitis B, 5 patients with fulminant hepatitis due to other etiologies, 37 patients with chronic liver disease (CLD; hepatitis B virus, n = 15; hepatitis C virus, n = 10; primary biliary cirrhosis, n = 12), and 10 normal controls (NCs). Furthermore, cytokine messenger RNA (mRNA) levels were determined in the liver specimens by quantitative real-time polymerase chain reaction (PCR). In NCs, faint IL-12 expression was detected in only a few Kupffer cells, whereas sinusoidal endothelial cells, hepatic stellate cells, bile ducts, and lymphocytes expressed IL-12 in CLD and, more conspicuously, in fulminant hepatitis B. In contrast, expression of IFN- and IL-10 was restricted to lymphocytes and Kupffer cells, respectively. In fulminant hepatitis B, numbers of IL-12­ and IFN-­positive cells markedly exceeded those found in CLD and NCs. A close correlation existed between IL-12 and IFN- expression (r = 0.68; P < .001). In contrast, IL-10 expression was not significantly different in CLD and fulminant hepatitis. The quantitative differences in immunohistologic cytokine expression closely corresponded to the mRNA levels. In conclusion, our data indicate massive induction of the proinflammatory cytokines IL-12 and IFN- in fulminant hepatitis B, which is apparently not counterbalanced by the anti-inflammatory cytokine IL-10. This cytokine imbalance may play an important role in promoting inflammatory reactions leading to massive liver damage in fulminant hepatitis B. (HEPATOLOGY 2002;36:1001-1008.)

GASTROENTEROLOGY

Table of Contents for October 2002 · Volume 123 · Number 4

Clinical­Alimentary Tract

Condition-specific deactivation of brain regions by 5-HT3 receptor antagonist Alosetron
S. M. Berman, L. Chang, B. Suyenobu, S. W. Derbyshire, J. Stains, L. FitzGerald, M. Mandelkern, L. Hamm, B. Vogt, B. D. Naliboff, E. A. Mayer
Background & Aims: The 5-HT3 receptor (5-HT3R) antagonist Alosetron (Alos) reduces the symptoms of female patients with diarrhea-predominant irritable bowel syndrome (IBS); yet, the mechanism(s) underlying this effect remains incompletely understood. We determined the effect of Alos on regional cerebral blood flow (rCBF) in the absence and presence of rectal or sigmoid stimulation to evaluate 2 hypothesized mechanisms of therapeutic action: peripheral antinociception and inhibition of emotional motor system (EMS) regions in the brain.
Methods: Forty-nine nonconstipated irritable bowel syndrome (IBS) patients (26 female) received H215O positron emission tomography (PET) brain scans before a randomized, placebo-controlled, 3-week trial with Alos (1­4 mg twice daily). PET scans were repeated after treatment in 37 completers. We assessed rCBF during baseline, rectal distention, and anticipation of undelivered rectal distention. The 3 conditions were repeated after a series of noxious sigmoid distentions. Rectal (45 mm Hg) and sigmoid (60 mm Hg) distentions were performed with a computer-controlled barostat device.
Results: Alos treatment, as compared with placebo, improved IBS symptoms and reduced rCBF in 5-HT3R containing regions of the EMS, but not in areas activated by pain. Reduction of rCBF appeared greatest in the absence of visceral stimulation, and was partially reversed by rectal or sigmoid distention. Symptom improvement across sessions was significantly correlated with rCBF decreases in the 5-HT3R-rich amygdala, ventral striatum, and dorsal pons.
Conclusions: Reduction in IBS symptoms correlated with a drug-induced reduction in the activity of central autonomic networks mediating emotional expression that was maximal in the absence of nociceptive input.

Budesonide treatment for collagenous colitis: A randomized, double-blind, placebo-controlled, multicenter trial
S. Miehlke, P. Heymer, B. Bethke, E. Bästlein, E. Meier, H.-P. Bartram, G. Wilhelms, N. Lehn, G. Dorta, J. Delarive, A. Tromm, E. Bayerdörffer, M. Stolte
Background & Aims: Collagenous colitis is an idiopathic microscopic colitis characterized by chronic watery diarrhea, a typical subepithelial collagen layer, and lymphoplasmacellular infiltration. We investigated the effect of budesonide on symptoms and histology in patients with collagenous colitis in a randomized, double-blind, placebo-controlled multicenter trial.
Methods: Patients with chronic diarrhea and histologically proven collagenous colitis were randomized to receive either oral budesonide (Entocort capsules; AstraZeneca, Sodertalje, Sweden) 9 mg/day for 6 weeks or placebo. Complete colonoscopy was performed before and after treatment. Histopathology was assessed by a single pathologist blinded to the patients' treatment. Clinical symptoms were assessed by standardized questionnaires.
Results: Fifty-one patients were randomized; 45 patients were available for per protocol analysis. The rate of clinical remission was significantly higher (P < 0.001) in the budesonide group than in the placebo group (per protocol 86.9% vs. 13.6%, respectively; intention-to-treat 76.9% vs. 12.0%, respectively). Histologic improvement was observed in 14 patients of the budesonide group (60.9%) and in 1 patient of the placebo group (4.5%; P < 0.001). Two patients in the budesonide group (7.7%) and 1 patient in the placebo group (4.0%) discontinued treatment prematurely because of side effects.
Conclusions: Oral budesonide (Entocort capsules) is an effective and safe treatment modality for patients with collagenous colitis. Long-term follow-up of these patients is necessary to investigate whether clinical and histologic remission is sustained.

Total antioxidant potential of fruit and vegetables and risk of gastric cancer
M. Serafini, R. Bellocco, A. Wolk, A. M. Ekström
Background & Aims: Dietary antioxidants, with additive and synergistic effects, can mediate the observed inverse association between plant food intake and risk of gastric cancer. We investigated whether the total dietary antioxidant potential of fruit and vegetables is an appropriate means of estimating the antioxidant impact on gastric cancer risk in a large population-based study.
Methods: With a population-based case-control design, data were collected through face-to-face interviews with 505 newly diagnosed gastric adenocarcinoma patients and 1116 control subjects to assess dietary habits 20 years before interview. The total radical-trapping antioxidant potential (TRAP) of different plant foods was used to convert food frequency intake into antioxidant potential. Gastric cancer risk in groups exposed to higher levels of oxidative stress (smoking and Helicobacter pylori infection) was also examined.
Results: Intake of antioxidant equivalents was inversely associated with the risk of both cardia and distal gastric cancer (odds ratio [OR], 0.65; 95% confidence interval [CI], 0.48­0.89 for the highest quartile of TRAP). Controlling for smoking, the inverse relationship between TRAP values displayed a clearer dose-response pattern. Never-smokers with the highest antioxidant intake had the lowest risk of cancer, 0.44 (95% CI, 0.27­0.71). Among H. pylori­infected subjects, the ORs varied between 0.66 and 0.41 for increasing levels of antioxidant potential.
Conclusions: Our results suggest that dietary intake of antioxidants measured as total antioxidant potential is inversely associated with risk of both cardia and distal cancer. The innovative approach used in this study provides a new tool for investigating the relationship between dietary antioxidants and oxidative stress-related carcinogenesis.

Appendicitis: Should diagnostic imaging be performed if the clinical presentation is highly suggestive of the disease?
T. Rettenbacher, A. Hollerweger, N. Gritzmann, T. Gotwald, K. Schwamberger, H. Ulmer, D. zur Nedden
Background & Aims: Our aim was to investigate whether diagnostic imaging is required if the clinical presentation suggests acute appendicitis with high probability.
Methods: On the basis of clinical findings, 350 consecutive patients with clinical suspicion of acute appendicitis were prospectively divided into 3 groups as follows: low, intermediate, and high probability of having appendicitis. All patients then underwent diagnostic ultrasonography. The clinical likelihood of appendicitis and the ultrasonographic results were correlated with the definite diagnoses.
Results: In the patients with clinically low probability of having appendicitis, appendicitis was present in 10% (11 of 109 patients), and, in those with intermediate probability, appendicitis was present in 24% (23 of 97 patients). Patients with clinically high probability of having appendicitis had appendicitis in 65% (94 of 144 patients), an alternative diagnosis in 18% (26 of 144 patients), and no specific definitive diagnosis in 17% (24 of 144 patients). Ultrasonography diagnosed appendicitis and the differential diagnoses with a sensitivity of 98% and 97%, specificity of 98% and 100%, positive predictive value of 96% and 99%, negative predictive values of 99% and 99%, and accuracy of 98% and 99%, respectively.
Conclusions: Even in patients with clinically high probability of acute appendicitis, diagnostic imaging should be performed because it accurately depicts a high percentage of normal appendices and differential diagnoses.

A prospective trial comparing small bowel radiographs and video capsule endoscopy for suspected small bowel disease
G. Costamagna, S. K. Shah, M. E. Riccioni, F. Foschia, M. Mutignani, V. Perri, A. Vecchioli, M. G. Brizi, A. Picciocchi, P. Marano
Background & Aims: This study was undertaken to prospectively compare the clinical outcomes of small bowel radiographs with the wireless capsule endoscopy.
Methods: Twenty-two patients were selected consecutively because of suspected small bowel disease. Two patients were excluded owing to ileal stenosis. Thus, the results of barium follow-through and the Given M2A wireless video capsule (Given Imaging Ltd., Yoqneam, Israel) endoscopy were compared in 20 patients (13 men; mean age, 52.5 yr; range, 29­78 yr).
Results: Barium follow-through was normal in 17 patients and showed ileal nodularity in 3 patients. Capsule endoscopy was normal in 3 patients and showed positive findings in the remaining 17 patients. The barium study was considered diagnostic in 4 (20%) patients. The capsule endoscopy was considered diagnostic in 9 (45%) patients, suspicious in 8 (40%) patients, and failed in 3 (15%) patients. For obscure gastrointestinal (GI) bleeding, the diagnostic potential of barium follow-through was much worse as compared with the capsule endoscopy (5% vs. 31%, P < 0.05). Capsule endoscopy was well tolerated and better accepted by patients when compared with the most recently performed endoscopic procedure.
Conclusions: The video capsule endoscope was found to be superior to small bowel radiograph for evaluation of small bowel diseases. However, this novel wireless endoscope system needs further assessment because of limitations such as difficulties in interpretation of potentially nonspecific findings.

Stratifying the risk of NSAID-related upper gastrointestinal clinical events: Results of a double-blind outcomes study in patients with rheumatoid arthritis
L. Laine, C. Bombardier, C. J. Hawkey, B. Davis, D. Shapiro, C. Brett, A. Reicin
Background & Aims: Epidemiologic data indicate that the risk of nonsteroidal anti-inflammatory drug (NSAID)­related gastrointestinal (GI) clinical events varies based on patients' clinical characteristics. The authors determined risk factors for NSAID-related clinical upper GI events and the event rates, absolute risk reductions, and numbers needed to treat for individual risk factors for a nonselective NSAID and a selective cyclooxygenase 2 inhibitor in a double-blind outcomes trial.
Methods: Eight thousand seventy-six rheumatoid arthritis patients aged 50 years (or 40 on corticosteroid therapy) were randomly assigned to rofecoxib 50 mg daily or naproxen 500 mg twice daily for a median of 9 months. The development of clinical upper GI events (bleeding, perforation, obstruction, and symptomatic ulcer identified on clinically indicated work-up) was assessed.
Results: Significant risk factors included prior upper GI events, age 65, and severe rheumatoid arthritis (RR, 2.3­3.9). Patients administered naproxen who had prior upper GI complications or who were aged 75 years had 18.84 or 14.46 events per 100 patient-years, and the risk of events remained constant over time. The reduction in events with rofecoxib was similar in high- and low-risk subgroups (RR, 0.31­0.68). The number needed to treat with rofecoxib instead of naproxen to avert 1 GI event was 10­12 in highest risk patients (prior event, age 75 years, or severe rheumatoid arthritis), 17­33 in patients with other risk factors, and 42­106 in low-risk patients.
Conclusions: NSAID-related GI events increase dramatically with risk factors such as prior events or older age. Ten to twelve high-risk patients need to be treated with a protective strategy such as the selective cyclooxygenase 2 inhibitor, rofecoxib, to avert a clinical GI event.

Clinical­Liver, Pancreas, and Biliary Tract

A randomized, controlled trial of medical therapy versus endoscopic ligation for the prevention of variceal rebleeding in patients with cirrhosis
D. Patch, C. A. Sabin, J. Goulis, G. Gerunda, L. Greenslade, C. Merkel, A. K. Burroughs
Background & Aims: Patients who have had one variceal bleed are at high risk of rebleeding. Since its introduction, endoscopic variceal banding has been shown to be superior to needle sclerotherapy. Banding has not been compared with hepatic venous pressure-guided medical therapy (-blockers and nitrates).
Methods: One hundred two patients with cirrhosis and a recent esophageal variceal bleed were randomized to either endoscopic banding (51 patients) or medical therapy (51 patients). The hepatic venous pressure gradient was measured in all patients at baseline, at 3 months (drug therapy arm), and at yearly intervals (all patients). Primary end points were death or rebleeding.
Results: The 2 groups were well matched. Fifty-one percent were Pughs C, with a median Pughs score of 9.5. Nineteen patients rebled in the drug arm (median time, 24 days) and 27 patients in the banding arm (median time, 24 days). At 1 year, 43.7% of patients had bled in the drug arm compared with 53.8% in the banding arm (P = 0.25). Thirty-two percent of patients on medical therapy had died at 1 year, 22.5% on banding (P = 0.97).
Conclusions: In the prevention of variceal rebleeding, -blockers ± nitrates are as effective as endoscopic banding.

Tropical calcific pancreatitis: Strong association with SPINK1 trypsin inhibitor mutations
E. Bhatia, G. Choudhuri, S. S. Sikora, O. Landt, A. Kage, M. Becker, H. Witt
Background & Aims: Tropical calcific pancreatitis (TCP) is a chronic pancreatitis unique to developing countries in tropical regions. The cause of TCP is obscure. Whereas environmental factors, such as protein energy malnutrition and ingestion of cassava, have been implicated, a genetic predisposition to the disease also may be important. In the present study we report on mutations in the serine protease inhibitor, Kazal type 1 (SPINK1) gene in north Indian patients with TCP.
Methods: We studied 66 unrelated TCP patients (44 men, 49 with diabetes, and 6 with family history of TCP), 25 relatives, and 92 healthy control subjects. Samples were analyzed for SPINK1 variants (­53C>T, L14P, N34S, P55S, and 272T>C) and cationic trypsinogen (PRSS1) variants (A16V, K23R, N29I, and R122H) by melting curve analysis.
Results: Twenty-nine patients (44%) carried the N34S missense mutation, of whom 9 (14%) were homozygotes. In contrast, only 2 (2.2%) control subjects were N34S heterozygotes (prevalence ratio 20.2; 95% confidence interval 5.0­81.8; P < 0.0001 vs. TCP). The severity of pancreatitis did not differ between TCP patients with or without N34S, or among those heterozygous or homozygous for N34S. Among TCP patients with or without diabetes, the frequency of N34S carriers (43% vs. 47%) and N34S homozygotes (14% vs. 12%) was similar.
Conclusions: TCP is highly associated with the SPINK1 N34S mutation. The high prevalence of N34S in TCP patients with and without diabetes suggests that these 2 subtypes have a similar genetic predisposition. The genetic predisposition to TCP resembles, at least in part, the idiopathic chronic pancreatitis found in industrialized countries.

SPINK1/PSTI mutations are associated with tropical pancreatitis and type II diabetes mellitus in Bangladesh
A. Schneider, A. Suman, L. Rossi, M. M. Barmada, C. Beglinger, S. Parvin, S. Sattar, L. Ali, A. K. Azad Khan, N. Gyr, D. C. Whitcomb
Background & Aims: Tropical pancreatitis, including tropical calcific pancreatitis and fibrocalculous pancreatic diabetes, is endemic in parts of Asia and Africa. In a preliminary study, we found serine protease inhibitor, Kazal type 1 (SPINK1) mutations in 6 of 8 patients with fibrocalculous pancreatic diabetes in Bangladesh. A more extensive investigation of patients with pancreatic diseases in Bangladesh, including non­insulin-dependent diabetes mellitus, was undertaken.
Methods: Patients with fibrocalculous pancreatic diabetes (n = 22), tropical calcific pancreatitis (n = 15), and non­insulin-dependent diabetes mellitus (n = 43) and controls (n = 76) from Bangladesh were studied. DNA was extracted, and the SPINK1 gene was sequenced in all patients and 50 controls. Exon 3 was sequenced in an additional 26 controls.
Results: SPINK1 N34S mutations appeared in 1 of 76 controls (1.3%), 12 of 22 patients with fibrocalculous pancreatic diabetes (55%; odds ratio, 83; P < 0.00001), 3 of 15 with tropical calcific pancreatitis (20%; odds ratio, 11.2; P = 0.04), and 6 of 43 with non­insulin-dependent diabetes mellitus (14%; odds ratio, 11.9; P = 0.009). P55S was present in 2 of 76 controls (3%) and in 1 of 22 patients with fibrocalculous pancreatic diabetes (5%; P = not significant). A novel Y54H (160T>C) mutation was identified in 1 of 15 tropical calcific pancreatitis patients.
Conclusions: In Bangladesh, the SPINK1 N34S mutation increases the risk of several forms of pancreatic disease, including fibrocalculous pancreatic diabetes, tropical calcific pancreatitis, and non­insulin-dependent diabetes mellitus.

Quantitation and phenotypic analysis of natural killer T cells in primary biliary cirrhosis using a human CD1d tetramer
H. Kita, O. V. Naidenko, M. Kronenberg, A. A. Ansari, P. Rogers, X.-S He, F. Koning, T. Mikayama, J. Van de Water, R. L. Coppel, M. Kaplan, M. E. Gershwin
Background & Aims: Natural killer T (NKT) cells are a subset of lymphocytes incriminated in playing an important role in the modulation of the innate immune response and the development of autoimmunity. However, there have been only limited studies attempting to quantitate the number of NKT cells in autoimmune disease, particularly because of difficulties associated with definition of this subpopulation.
Methods: We used a human CD1d (hCD1d) tetramer produced by a baculovirus expressing recombinant CD1d protein complexed with -galactosylceramide (-GalCer) and quantitated hCD1d tetramer reactive cells in blood and liver from controls and patients with primary biliary cirrhosis (PBC).
Results: The majority of CD1d-GalCer-restricted NKT cells were positive for TCR V24 and V11. There was a distinct CD4­ CD8+ population within the CD1d-GalCer-restricted NKT cells in addition to the CD4­ CD8­ and CD4+ CD8­ population. The frequency of CD1d-GalCer-restricted NKT cells was similar between blood and liver in healthy individuals. In contrast, the frequency of CD1d-GalCer-restricted NKT cells in the liver was significantly higher than in the blood of PBC patients. The frequency of CD1d--GalCer-restricted NKT cells in the liver was also significantly higher in PBC patients than in healthy individuals.
Conclusions: The frequency and function of such cells should be studied not only in blood but also in the target organ of the autoimmune disease. Selective enrichment of CD1d-GalCer-restricted NKT cells at the site of inflammation is observed in PBC, suggesting a role of these cells in the development of PBC.

Survival and symptom progression in a geographically based cohort of patients with primary biliary cirrhosis: Follow-up for up to 28 years
M. Prince, A. Chetwynd, W. Newman, J. V. Metcalf, O. F. W. James
Background & Aims: Although several excellent studies have described the natural history of primary biliary cirrhosis, most were reported from tertiary referral centers. We examined the prognosis of primary biliary cirrhosis in a comprehensive geographically defined cohort.
Methods: We followed up 770 primary biliary cirrhosis patients prevalent between January 1987 and December 1994 until death, transplantation, or censor on January 1, 2000, by interview and review of case notes and death certificates. Analysis of survival data was performed with Kaplan­Meier methods and Cox regression.
Results: Median patient survival was 9.3 years from diagnosis. Patient age, alkaline phosphatase, albumin, and bilirubin at diagnosis independently predicted survival in Cox modeling. Prothrombin time and histologic stage did not independently affect survival. Observed survival was predicted well by this model and by the Mayo prognostic score (R2M = 0.37 and 0.18, respectively; R2M is a likelihood-based measure of the percentage information gain from the model due to covariates). Forty-two percent of deaths were caused by liver disease. Thirty-nine patients had liver transplantations by the censor date. Survival was much poorer than for an age- and sex-matched control population (standardized mortality ratio = 2.87 [1.73 excluding liver deaths]). The most common symptoms at diagnosis were pruritus (18.9%) and fatigue (21.0%). Twenty-six percent of patients developed liver failure by 10 years after diagnosis.
Conclusions: Although primary biliary cirrhosis is often now diagnosed at an early stage, the diagnosis still carries important prognostic implications. A significant proportion of patients develop liver failure, require transplantation, or die prematurely after this diagnosis.

Aberrant expression of MUC5AC and MUC6 gastric mucins and sialyl Tn antigen in intraepithelial neoplasms of the pancreas
G. E. Kim, H.-I. Bae, H.-U. Park, S.-F. Kuan, S. C. Crawley, J. J. L. Ho, Y. S. Kim
Background & Aims: It has recently been suggested that infiltrating adenocarcinoma of the pancreas arises from histologically well-defined precursor ductal lesions called pancreatic intraepithelial neoplasia (PanIN-1A, -1B, -2, and -3). This study examined alterations in the pattern and the level of expression of several mucin genes (MUC1, MUC2, MUC5AC, and MUC6) and mucin-associated tumor antigens (Nd2 and sialyl Tn) in these precursor lesions.
Methods: We examined 139 PanINs and 68 infiltrating ductal adenocarcinomas of the pancreas by using immunohistochemistry and in situ hybridization methods.
Results: Overexpression of MUC1, a pan-epithelial mucin, and MUC6, a pyloric-gland mucin, and de novo expression of MUC5AC, a gastric foveolar mucin, was observed in all stages of PanINs and invasive ductal adenocarcinoma. In contrast, the expression of mucin-associated carbohydrate antigen, sialyl Tn, was markedly increased only in PanlN-3 and invasive ductal adenocarcinoma. In addition, a decrease in the expression of these mucin-associated peptide and carbohydrate antigens was correlated with the degree of differentiation of the tumor.
Conclusions: Expression of both gastric-foveolar and pyloric-gland mucin in PanINs is an early event, whereas sialyl Tn expression is a late event in the recently defined progression model of pancreatic carcinogenesis. This altered mucin gene expression provides new insight into the role of cell lineage­associated metaplasia in pancreatic carcinogenesis.

Adherence to combination therapy enhances sustained response in genotype-1­infected patients with chronic hepatitis C
J. G. McHutchison, M. Manns, K. Patel, T. Poynard, K. L. Lindsay, C. Trepo, J. Dienstag, W. M. Lee, C. Mak, J.-J. Garaud, J. K. Albrecht, for the International Hepatitis Interventional Therapy Group
Background & Aims: Patient adherence to prescribed antiviral therapy in human immunodeficiency virus infection enhances response. We evaluated the impact of adherence to combination therapy with interferon or peginterferon plus ribavirin in chronic hepatitis C patients.
Methods: We assessed the effect of dose reduction on sustained virologic response (SVR) from prior trials with interferon -2b plus ribavirin (n = 1010) or peginterferon -2b 1.5 µg/kg/week plus ribavirin (n = 511). The actual treatment administered was verified from drug dispensing/return records and patient diaries. Two groups were defined: (1) patients who received 80% of both their total interferon and ribavirin doses for 80% of the expected duration of therapy and (2) patients who received reduced doses (<80% of one or both drugs for 80% of the expected duration of therapy). A statistical model provided comparative estimates of the response rates in compliant patients.
Results: Most patients were at least 80% compliant with interferon -2b/ribavirin or peginterferon -2b/ribavirin therapy and had SVR rates of 52% and 63%, respectively, for the 2 regimens. This was most apparent for HCV-1­infected patients. The impacts of adherence on efficacy from subgroup analysis and the statistical modeling approach were similar.
Conclusions: HCV-1­infected patients who can be maintained on >80% of their interferon or peginterferon -2b and ribavirin dosage for the duration of treatment in the setting of a clinical trial exhibit enhanced sustained response rates. Our results suggest that adherence will enhance the likelihood of achieving an initial virologic response. Adherence beyond 12­24 weeks will be advantageous only for those patients who have achieved such an early virologic response.

Peginterferon alone or with ribavirin enhances HCV-specific CD4+ T-helper 1 responses in patients with chronic hepatitis C
S. M. Kamal, J. Fehr, B. Roesler, T. Peters, J. W. Rasenack
Background & Aims: Pegylated interferons (IFNs) with or without ribavirin were shown in several studies to improve sustained virologic response compared with standard IFN -2 therapy. This study investigated if the greater efficacy of pegylated IFNs might be related to modulation of immunologic responses.
Methods: Hepatitis C virus (HCV)-specific CD4+ T-cell responses and cytokine production to various HCV proteins (Elispot assay) in peripheral blood were prospectively assessed in 42 patients receiving IFN -2a monotherapy, peginterferon (PEG IFN) -2a monotherapy, or PEG IFN -2a plus ribavirin and correlated to the outcome of therapy.
Results: The sustained virologic response rate was significantly higher in the PEG IFN groups (42% in PEG IFN -2a monotherapy and 57% in PEG IFN -2a/ribavirin combination) than in the standard IFN -2a group (14%). The sustained response was 48% in HCV genotype 1 patients treated with PEG IFN -2a/ribavirin therapy. Pretreatment HCV-specific CD4+ responses were either weak or absent. PEG IFN alone or combined with ribavirin induced significant increase in the frequency, strength, and breadth of HCV-specific CD4+ T-cell responses with type 1 predominance; whereas interferon -2a monotherapy was associated with lower, fluctuating, short-lived responses. Sustained responders maintained multispecific HCV-specific CD4+ T-cell responses with enhanced IFN- production. Relapsers and partial responders initially displayed significant HCV-specific CD4+ T-cell responses that waned or were lost.
Conclusions: The efficacy of PEG IFN -2a alone or in combination with ribavirin in inducing high rates of sustained virologic response may be owing to the higher efficacy of PEG IFN in induction and maintenance of significant multispecific HCV-specific CD4+ T-helper 1 responses.

Prognosis following spontaneous HBsAg seroclearance in chronic hepatitis B patients with or without concurrent infection
Y.-C. Chen, I.-S. Sheen, C.-M. Chu, Y.-F. Liaw
Background & Aims: Spontaneous hepatitis B surface antigen (HBsAg) seroclearance is a rare event in patients with chronic hepatitis B virus infection. The aim of this study was to clarify the controversy on long-term prognosis following spontaneous HBsAg seroclearance using a large series of patients.
Methods: A total of 218 patients (172 men and 46 women) who had undergone spontaneous HBsAg seroclearance were followed up for 12­179 months (median, 61.7 months; mean, 63.4 ± 38.5 months) with liver biochemistry, serology, measurement of -fetoprotein level, and abdominal ultrasonography every 6 months or every 3 months for the 29 patients who had developed cirrhosis at the time of HBsAg seroclearance.
Results: Of the 189 patients who were noncirrhotic at the time of HBsAg clearance, 3 (1.6%) developed cirrhosis, 2 (1.1%) developed hepatocellular carcinoma (HCC), and 1 died of HCC. These complications all developed in patients with concurrent hepatitis C virus or hepatitis delta virus infection (P < 0.001). The prognosis of the noncirrhotic patients without concurrent infection was significantly better than that of the matched control group (elevation of alanine aminotransferase level, 11.6% vs. 0%, P < 0.001; development of cirrhosis/HCC, 4% vs. 0%, P = 0.004). In contrast, of the 29 patients who had developed liver cirrhosis, 4 (13.8%) had hepatic decompensation and one died of HCC.
Conclusions: The prognosis following spontaneous HBsAg seroclearance is excellent, except in patients with cirrhosis or those with concurrent hepatitis C virus or hepatitis delta virus infection.

p16INK4a Promoter mutations are frequent in primary sclerosing cholangitis (PSC) and PSC-associated cholangiocarcinoma
M. Taniai, H. Higuchi, L. J. Burgart, G. J. Gores
Background & Aims: Primary sclerosing cholangitis (PSC) predisposes individuals to cholangiocarcinoma; however, the molecular mechanisms involved in the carcinogenesis process remain unclear. Because p16INK4a inactivation has been implicated in cholangiocarcinoma, our aims were to examine PSC cholangiocytes for p16INK4a gene mutations.
Methods: We studied 4 patient groups: PSC patients without cholangiocarcinoma (n = 10), patients with PSC-associated cholangiocarcinoma (n = 10), non-PSC controls (n = 10), and disease controls with primary biliary cirrhosis (n = 10). Cholangiocytes and hepatocytes were isolated from tissue sections using laser capture microdissection. Genomic DNA was extracted, and the promoter region and the 3 exons for p16INK4a were amplified by PCR and directly sequenced.
Results: In the promoter region, 8-point mutations in 5 PSC cases and 14 mutations in 8 cholangiocarcinoma cases were observed. In exon 1, 1 PSC patient and 3 cholangiocarcinoma patients had point mutations. In contrast, no case had a mutation in exon 2 or 3. Mutations were not detected in cholangiocytes from control patients or primary biliary cirrhosis patients nor in hepatocytes from any of the groups; these data indicate that the observed base changes were disease specific and not genetic polymorphisms. Several of the promoter mutations (4 of 8) dramatically decreased promoter activity (>50% reduction in luciferase activity) in a reporter gene assay.
Conclusions: The results show that functional point mutations in the p16INK4a promoter region likely contribute to the initiation/progression of cholangiocarcinoma in PSC. Promoter mutations in CpG islands may function as a methylation equivalent phenomenon resulting in gene inactivation.

Basic­Alimentary Tract

Chronic stress induces mast cell­dependent bacterial adherence and initiates mucosal inflammation in rat intestine
J. D. Söderholm, P.-C. Yang, P. Ceponis, A. Vohra, R. Riddell, P. M. Sherman, M. H. Perdue
Background & Aims: Chronic psychological stress is an important factor in relapses of intestinal disorders, but it remains unclear if stress can induce primary gut inflammation in a previously healthy host.
Methods: Mast cell-deficient (Ws/Ws) rats and wild-type control (+/+) rats were submitted to water avoidance stress or sham stress (1 h/day) for 10 consecutive days, as a model of ongoing life stress.
Results: Both rat groups had similar systemic responses to stress, as assessed by changes in weight, corticosterone levels, and defecation. In +/+ rats, chronic stress induced barrier dysfunction in the ileum and colon (increased macromolecular permeability and depletion of mucus) and ultrastructural changes in epithelial cells (enlarged mitochondria and presence of autophagosomes) associated with bacterial adhesion and penetration into enterocytes. Moreover, hyperplasia and activation of mast cells, infiltration of neutrophils and mononuclear cells, and increased myeloperoxidase (MPO) activity were documented in the mucosa. In intestine of Ws/Ws rats, epithelial function and morphology were unchanged by chronic stress, bacterial-epithelial cell interaction was not demonstrated, and there was no evidence of inflammatory cell infiltration.
Conclusions: These findings suggest that chronic psychological stress can be an initiating factor in intestinal inflammation by impairing mucosal defenses against luminal bacteria and highlight the importance of mast cells in this process.

Colorectal adenoma to carcinoma progression follows multiple pathways of chromosomal instability
M. Hermsen, C. Postma, J. Baak, M. Weiss, A. Rapallo, A. Sciutto, G. Roemen, J.-W. Arends, R. Williams, W. Giaretti, A. de Goeij, G. Meijer
Background & Aims: Current models of colorectal adenoma to carcinoma progression do not fully reflect the genetic heterogeneity and complexity of the disease. The aim of the present study was to identify genetic changes discriminating adenomas that have progressed to carcinoma from adenomas that have not progressed, and to refine the current genetic models of colorectal adenoma to carcinoma progression, based on a genome-wide analysis of chromosomal aberrations.
Methods: Sixty-six nonprogressed colorectal adenomas, 46 progressed adenomas (malignant polyps), and 36 colorectal carcinomas were screened for chromosomal aberrations by comparative genomic hybridization, and for mutations in the adenomatous polyposis coli (APC) and K-ras gene. Data analysis focused on cancer-associated genetic changes in adenomas.
Results: Accumulation of losses in 8p21-pter, 15q11-q21, 17p12-13, and 18q12-21, and gains in 8q23-qter, 13q14-31, and 20q13 were strongly associated with adenoma-to-carcinoma progression, independent of the degree of dysplasia. Hierarchic cluster analysis demonstrated the presence of 3 distinct subgroups of adenomas, characterized by unique combinations of genetic aberrations in the adenomas (17p loss and K-ras mutation, 8q and 13q gain, and 18q loss and 20q gain, respectively).
Conclusions: The presence of 2 or more of the aforementioned 7 chromosomal changes was associated with progressed colorectal adenomas and colorectal cancer. In addition, evidence was found that these chromosomal abnormalities occurred in specific combinations of a few abnormalities rather than as a mere accumulation of events, indicating the existence of multiple independent chromosomal instability pathways of colorectal cancer progression.

The role of the gastric afferent vagal nerve in ghrelin-induced feeding and growth hormone secretion in rats
Y. Date, N. Murakami, K. Toshinai, S. Matsukura, A. Niijima, H. Matsuo, K. Kangawa, M. Nakazato
Background & Aims: Visceral sensory information is transmitted to the brain through the afferent vagus nerve. Ghrelin, a peptide primarily produced in the stomach, stimulates both feeding and growth hormone (GH) secretion. How stomach-derived ghrelin exerts these central actions is still unknown. Here we determined the role of the gastric afferent vagal nerve in ghrelin's functions.
Methods: Food intake and GH secretion were examined after an administration of ghrelin intravenously (IV) to rats with vagotomy or perivagal application of capsaicin, a specific afferent neurotoxin. We investigated Fos expression in neuropeptide Y (NPY)-producing and growth hormone­releasing hormone (GHRH)-producing neurons by immunohistochemistry after administration IV of ghrelin to these rats. The presence of the ghrelin receptor in vagal afferent neurons was assessed by using reverse-transcription polymerase chain reaction and in situ hybridization histochemistry. A binding study on the vagus nerve by 125I-ghrelin was performed to determine the transport of the ghrelin receptor from vagus afferent neurons to the periphery. We recorded the electric discharge of gastric vagal afferent induced by ghrelin and compared it with that by cholecystokinin (CCK), an anorectic gut peptide.
Results: Blockade of the gastric vagal afferent abolished ghrelin-induced feeding, GH secretion, and activation of NPY-producing and GHRH-producing neurons. Ghrelin receptors were synthesized in vagal afferent neurons and transported to the afferent terminals. Ghrelin suppressed firing of the vagal afferent, whereas CCK stimulated it.
Conclusions: This study indicated that the gastric vagal afferent is the major pathway conveying ghrelin's signals for starvation and GH secretion to the brain.

Cannabinoid receptor agonism inhibits transient lower esophageal sphincter relaxations and reflux in dogs
A. Lehmann, L. A. Blackshaw, L. Brändén, A. Carlsson, J. Jensen, E. Nygren, S. D. Smid
Background & Aims: Transient lower esophageal sphincter relaxations (TLESRs) are the major cause of gastroesophageal acid reflux, and are triggered by postprandial gastric distention. Stimulation of GABAB receptors potently inhibits triggering of TLESR by gastric loads. The functional similarity between GABAB and cannabinoid receptors (CBRs) prompted us to study the role of CBRs on mechanisms of gastric distention-induced TLESRs.
Methods: Gastric nutrient infusion and air insufflation was performed during gastroesophageal manometry in conscious dogs. The effects of the CBR agonist WIN 55,212-2 were assessed alone and in combination with the CBR1 antagonist SR141716A or the CBR2 antagonist SR144528. The effects of WIN 55,212-2 were also studied on firing of gastric vagal mechanosensitive afferents in an isolated preparation of ferret stomach.
Results: WIN 55,212-2 (57 nmol/kg) inhibited the occurrence of TLESR after gastric loads by 80% (P < 0.01). The latency to the first TLESR after the load was prolonged (P < 0.001), and the occurrence of swallowing was reduced (P < 0.05). The CBR1 antagonist SR141716A reversed the effects of WIN 55,212-2, whereas the CBR2 antagonist SR144528 did not. The CBR1 antagonist alone increased occurrence of TLESR (P < 0.05). The responses of gastric vagal mechanoreceptors to distention were unaffected by WIN 55,212-2 at a concentration of 3 µmol/L.
Conclusions: Exogenous and endogenous activation of the CBR1 receptor inhibits TLESRs. The effects of CBR1 are not mediated peripherally on gastric vagal afferents, and therefore are most likely in the brain stem.

Neurotensin receptor-1 and -3 complex modulates the cellular signaling of neurotensin in the HT29 cell line
S. Martin, V. Navarro, J. P. Vincent, J. Mazella
Background & Aims: The neuropeptide neurotensin (NT) exerts its intracellular effect by interacting with 3 different receptors. Two of these receptors (NTR1 and NTR2) belong to the G protein­coupled receptor family, whereas the third one (NTR3) is a type I receptor with a single transmembrane domain. We recently showed that the 2 structurally different receptors NTR1 and NTR3 were coexpressed in several human cancer cells on which NT exerts proliferative effects.
Methods: Here, by an immunoprecipitation approach, we provide biochemical evidence for an endogenous heterodimerization of the G protein­coupled receptor NTR1 with the NTR3 in the human adenocarcinoma cell line HT29.
Results: We show that both receptors are expressed and colocalized within the cell surface of HT29 cells where they already interact to form a heterodimer. The NTR1-NTR3 complex is then internalized on NT stimulation.
Conclusions: The complex formed between these 2 structurally unrelated NT receptors modulates both the NT-induced phosphorylation of mitogen-activated protein kinases and the phosphoinositide (PI) turnover mediated by the NTR1.

Antibody-mediated gastrointestinal dysmotility in scleroderma
F. Goldblatt, T. P. Gordon, S. A. Waterman
Background & Aims: Defects in enteric excitatory neurotransmission have been proposed to underlie the gastrointestinal dysmotility associated with scleroderma (systemic sclerosis). This study investigated whether patients with scleroderma produce antibodies that inhibit M3-muscarinic or neurokinin receptor­mediated intestinal contractions, either directly or via an effect on L-type voltage-gated calcium channels (VGCCs).
Methods: Responses of mouse colon longitudinal muscle to stimulation by the muscarinic agonist carbachol (1­300 µmol/L) and neurokinin-1 and -2 receptor agonists were measured in the absence and presence of serum (2%) or immunoglobulin G (IgG) (0.3­1.0 mg/mL) from patients with scleroderma, those with other autoimmune disorders, and healthy controls. The role of L-type VGCCs in carbachol- and tachykinin-evoked contractions was assessed using nicardipine.
Results: M3-muscarinic receptor­mediated contractions were inhibited by Ig fractions from 7 of 9 patients with scleroderma (limited and diffuse forms), 4 of 4 patients with primary Sjögren's syndrome, and 3 of 3 patients with secondary Sjögren's syndrome. Ig fractions from healthy controls did not inhibit the M3-muscarinic receptor­mediated contractions. Inhibition by Ig was concentration-dependent; a maximum inhibition of approximately 40% occurred at 0.6 mg/mL IgG. Both M3-muscarinic and neurokinin receptor­mediated contractions were L-type VGCC dependent. Patient sera had no effect on responses to neurokinin receptor stimulation, demonstrating the lack of antibodies inhibiting L-type VGCCs.
Conclusions: Functional antibodies specifically inhibiting M3-muscarinic receptor­mediated enteric cholinergic neurotransmission may provide a pathogenic mechanism for the gastrointestinal dysfunction seen in patients with scleroderma.

A novel human fibroblast growth factor treats experimental intestinal inflammation
M. Jeffers, W. F. McDonald, R. A. Chillakuru, M. Yang, H. Nakase, L. L. Deegler, E. D. Sylander, B. Rittman, A. Bendele, R. B. Sartor, H. S. Lichenstein
Background & Aims: We recently identified a novel member of the human fibroblast growth factor (FGF) family of signaling molecules, designated FGF-20. In the present study, we examined the activity of this protein in 2 animal models of acute intestinal inflammation and in mechanistic studies in vitro.
Methods: In vivo experiments consisted of a murine dextran sulfate sodium (DSS) model of colitis and a rat indomethacin model of small intestinal ulceration/inflammation. Cell growth, restitution, gene expression (cyclooxygenase-2 [COX-2] and intestinal trefoil factor [ITF]), and prostaglandin E2 (PGE2) levels were examined in vitro.
Results: In the DSS-colitis model, prophylactic administration of FGF-20 significantly reduced the severity and extent of mucosal damage as indicated by a 55%­93% reduction in luminal blood loss, distal colonic edema, histologic inflammation, and epithelial cell loss relative to animals administered vehicle control. No toxicity was noted during administration of FGF-20 to normal controls. In addition, therapeutic administration of FGF-20 enhanced survival in this model. In the indomethacin­small bowel ulceration/inflammation model, administration of FGF-20 reduced small intestinal weight gain, necrosis, inflammation, and weight loss (36%­53% relative to vehicle control). In vitro studies demonstrated that FGF-20 stimulates growth, restitution, mRNA expression of COX-2 and ITF, and PGE2 levels in human intestinal epithelial cells and enhances the growth of human intestinal fibroblasts.
Conclusions: FGF-20, having demonstrated therapeutic activity in 2 experimental models of intestinal inflammation, represents a promising new candidate for the treatment of human inflammatory bowel disease.

PTEN and TNF- regulation of the intestinal-specific Cdx-2 homeobox gene through a PI3K, PKB/Akt, and NF-B­dependent pathway
S. Kim, C. Domon-Dell, Q. Wang, D. H. Chung, A. Di Cristofano, P. P. Pandolfi, J.-N. Freund, B. M. Evers
Background & Aims: PTEN (phosphatase and tensin homologue deleted from chromosome 10) is a dual-specificity phosphatase implicated in embryonic development, intestinal cell proliferation and differentiation, and tumor suppression. The transcription factor Cdx-2 is critical in intestinal development and homeostasis, and its expression is altered in colorectal cancers. However, the regulation of the Cdx-2 gene has not been entirely elucidated. Here, we hypothesize that Cdx-2 may be a target of PTEN signaling in the intestine.
Methods: The expression patterns for Cdx-2 and PTEN along wild-type mouse colon, as well as in colon tumors occurring in Pten+/­ mice, were examined. The effect of PTEN or phosphatidylinositol 3-kinase inhibition and tumor necrosis factor on Cdx-2 messenger RNA and protein expression, Cdx-2 DNA binding activity, and the promoter activity of the Cdx-2 gene was analyzed in human colon cancer cell lines.
Results: Cdx-2 expression correlates with PTEN along the length of the murine colon and in colonic polyps that develop in Pten+/­ mice. In colon cancer cells, PTEN stimulates Cdx-2 protein expression and the transcriptional activity of the Cdx-2 promoter. Phosphatidylinositol 3-kinase inhibition by wortmannin or by a dominant-negative phosphatidylinositol 3-kinase mimics the Cdx-2 stimulation by PTEN. Inversely, cell treatment by tumor necrosis factor decreases Cdx-2 expression. Phosphatidylinositol 3-kinase inhibition by PTEN or wortmannin has an inverse effect compared with tumor necrosis factor on the balance between the p50 and p65 subunits of nuclear factor B. p65 inhibits the activity of the Cdx-2 promoter, whereas p50 prevents p65 action.
Conclusions: Our results suggest that the intestinal Cdx-2 homeobox gene is a target of PTEN/phosphatidylinositol 3-kinase signaling and tumor necrosis factor signaling via nuclear factor B­dependent pathways.

Intestinal infection with Giardia spp. reduces epithelial barrier function in a myosin light chain kinase­dependent fashion
K. G.-E. Scott, J. B. Meddings, D. R. Kirk, S. P. Lees-Miller, A. G. Buret
Background & Aims: Giardiasis causes malabsorptive diarrhea, and symptoms can be present in the absence of any significant morphologic injury to the intestinal mucosa. The effects of giardiasis on epithelial permeability in vivo remain unknown, and the role of T cells and myosin light chain kinase (MLCK) in altered intestinal barrier function is unclear. This study was conducted to determine whether Giardia spp. alters intestinal permeability in vivo, to assess whether these abnormalities are dependent on T cells, and to assess the role of MLCK in altered epithelial barrier function.
Methods: Immunocompetent and isogenic athymic mice were inoculated with axenic Giardia muris trophozoites or sterile vehicle (control), then assessed for trophozoite colonization and gastrointestinal permeability. Mechanistic studies using nontransformed human duodenal epithelial monolayers (SCBN) determined the effects of Giardia on myosin light chain (MLC) phosphorylation, transepithelial fluorescein isothiocyanate­dextran fluxes, cytoskeletal F-actin, tight junctional zonula occludens-1 (ZO-1), and MLCK.
Results: Giardia infection caused a significant increase in small intestinal, but not gastric or colonic, permeability that correlated with trophozoite colonization in both immunocompetent and athymic mice. In vitro, Giardia increased permeability and phosphorylation of MLC and reorganized F-actin and ZO-1. These alterations were abolished with an MLCK inhibitor.
Conclusions: Disruption of small intestinal barrier function is T cell independent, disappears on parasite clearance, and correlates with reorganization of cytoskeletal F-actin and tight junctional ZO-1 in an MLCK-dependent fashion.

Effects of genetic blockade of the insulin-like growth factor receptor in human colon cancer cell lines
Y. Adachi, C.-T. Lee, K. Coffee, N. Yamagata, J. E. Ohm, K.-H. Park, M. M. Dikov, S. R. Nadaf, C. L. Arteaga, D. P. Carbone
Background & Aims: Insulin-like growth factor (IGF)-I receptor (IGF-Ir) signaling is required for maintenance of growth and tumorigenicity of several tumor types. We have previously shown successful therapy in a lung cancer xenograft model using an adenovirus expressing antisense IGF-Ir. In this study, we sought to better dissect the mechanism and develop potentially more effective IGF-Ir­targeted therapeutics by developing and testing tetracycline-regulated and recombinant adenoviruses expressing dominant negative receptors.
Methods: Truncated IGF-I receptors (IGF-Ir/tf; 482 and 950 amino acids long, respectively [IGF-Ir/482st and IGF-Ir/950st]) were cloned into tetracycline-regulated vectors and recombinant adenoviruses and then studied in colorectal cancer cells. We assessed the effect of IGF-Ir/tf on signaling blockade, colony formation, stress response (serum starvation and heat), chemotherapy-induced apoptosis, and in vivo therapeutic efficacy in xenografts.
Results: Activation of IGF-Ir/tf expression by withdrawal of tetracycline suppressed tumorigenicity both in vitro and in vivo and up-regulated stressor-induced apoptosis. It effectively blocked both IGF-I­ and IGF-II­induced activation of Akt-1. IGF-Ir/tf expression increased chemotherapy-induced apoptosis, and this combination therapy was very effective against tumors in mice. These findings were confirmed in a therapy model against established tumors using adenoviruses expressing IGF-Ir/tf. Moreover, IGF-Ir/482st was more effective than IGF-Ir/950st because of its bystander effect.
Conclusions: Anti-tumor activity of IGF-Ir/tf is mediated through inhibition of Akt-1 and enhances the efficacy of chemotherapy. Adenovirus IGF-Ir/482st may be a useful anticancer therapeutic for colorectal carcinoma.

Immune-mediated neural dysfunction in a murine model of chronic Helicobacter pylori infection
P. Berík, R. De Giorgio, P. Blennerhassett, E. F. Verdú, G. Barbara, S. M. Collins
Background & Aims: Neuromuscular changes producing dysmotility and hyperalgesia may underlie symptom generation in functional gastrointestinal disorders. We investigated whether chronic Helicobacter pylori­induced gastritis causes neuromuscular dysfunction.
Methods: In vitro muscle contractility and acetylcholine release were evaluated in mice before and after H. pylori eradication. H. pylori colonization and gastritis were graded histologically. Substance P (SP)-, vasoactive intestinal polypeptide (VIP)-, and calcitonin gene-related peptide (CGRP) immunoreactivity (IR) and macrophages were studied by immunohistochemistry.
Results: In Balb/c mice, chronic H. pylori infection did not affect muscle function but augmented antral relaxation after nerve electric field stimulation. Infected mice had lower acetylcholine release by electric field stimulation and had higher density of SP-, CGRP-, and VIP-IR nerves in the stomach and of SP- and CGRP-IR in the spinal cord. Cholinergic nerve dysfunction worsened progressively and was associated with increasing macrophage and mononuclear but not polymorphonuclear infiltrate or bacterial colonization. SCID mice had unchanged acetylcholine release despite high H. pylori colonization and macrophage infiltration. Eradication of H. pylori normalized functional and morphologic abnormalities except for increased density of gastric SP- and CGRP-IR nerves.
Conclusions: H. pylori infection induces functional and morphologic changes in the gastric neural circuitry that are progressive and lymphocyte dependent, and some persist after H. pylori eradication. The data have direct implications regarding the role of H. pylori infection in functional dyspepsia.

Long-lasting changes in central nervous system responsivity to colonic distention after stress in rats
R. Stam, K. Ekkelenkamp, A. C. Frankhuijzen, A. W. Bruijnzeel, L. M. A. Akkermans, V. M. Wiegant
Background & Aims: The highly prevalent functional gastrointestinal disorders involve visceral pain and disturbed bowel habit and are associated with preceding stressful experiences, although causality and biological mechanisms remain unclear. The aim of the present study was to establish whether stress can directly and lastingly alter central nervous system responsivity to colonic distention in the rat as well as which neural pathways are likely to be involved. Methods: Rats were treated with a brief session of stressful foot shocks known to induce long-term behavioral and autonomic sensitization. Two weeks later, after induction of inhalation anesthesia, a balloon catheter was inserted in the distal colon and repeatedly inflated with brief, constant-pressure air pulses. Results: Reflex decreases in blood pressure and heart rate indicative of visceral afferent activation were greater in previously shocked rats than in controls. Colonic distention increased the expression of Fos, a marker of neuronal activation, in the sacral spinal cord and caudal brain stem. In the central amygdala and several cortical areas (prelimbic, infralimbic, agranular insular, cingulate), previously shocked rats showed reduced Fos expression following colonic distention compared with relevant controls. Conclusions: The results indicate that a brief but intense stressful experience causes long-lasting alterations in higher-order central nervous system responsivity to colonic distention even in the absence of conscious affective responses, pointing to basic alterations in the neural pathways involved.

Basic­Liver, Pancreas, and Biliary Tract

Bile acid depletion and repletion regulate cholangiocyte growth and secretion by a phosphatidylinositol 3-kinase­dependent pathway in rats
G. Alpini, S. Glaser, D. Alvaro, Y. Ueno, M. Marzioni, H. Francis, L. Baiocchi, T. Stati, B. Barbaro, J. L. Phinizy, J. Mauldin, G. LeSage
Background & Aims: We tested the hypothesis that during bile duct obstruction, increased biliary bile acids trigger cholangiocyte proliferation and secretion by a phosphatidylinositol 3-kinase (PI3-K)­dependent pathway.
Methods: In bile duct­incannulated (BDI) rats, bile duct obstruction present for 7 days was relieved for 24 hours by external bile drainage. During the 24-hour drainage period, animals received either Krebs Ringer Henseleit (the bile-depleted group), or sodium taurocholate (the bile-depleted, taurocholate-infused group). We evaluated cholangiocyte proliferation and secretin-stimulated ductal secretion. Apical bile acid transporter (ABAT) expression and bile acid transport activity was determined. In pure preparations of cholangiocytes, we examined the effect of taurocholate (in the absence or presence of wortmannin or PI 3,4-bisphosphate the lipid product of PI3-K) on cholangiocyte proliferation and secretin-stimulated cyclic adenosine 3',5'-monophosphate (cAMP) levels.
Results: Bile depletion reduced cholangiocyte proliferation and secretin-stimulated ductal secretion and ABAT expression and bile acid transport activity compared with 1-week BDI control rats. In bile-depleted, taurocholate-infused rats, cholangiocyte proliferation and secretion and ABAT expression and bile acid transport activity were maintained at levels similar to those seen in BDI control rats. In vitro, taurocholate stimulation of DNA replication and secretin-stimulated cAMP levels was blocked by wortmannin. The inhibitory effect of wortmannin on taurocholate stimulation of cholangiocyte proliferation and secretion was prevented by PI 3,4-bisphosphate.
Conclusions: Bile acid uptake by ABAT and the PI3-K pathway are important for bile acids to signal cholangiocyte proliferation. In bile duct obstruction, increased biliary bile acid concentration and ABAT expression initiate increased cholangiocyte proliferation and secretion.

Ursodeoxycholic acid aggravates bile infarcts in bile duct­ligated and Mdr2 knockout mice via disruption of cholangioles
P. Fickert, G. Zollner, A. Fuchsbichler, C. Stumptner, A. H. Weiglein, F. Lammert, H.-U. Marschall, O. Tsybrovskyy, K. Zatloukal, H. Denk, M. Trauner
Background & Aims: The effects of ursodeoxycholic acid (UDCA) in biliary obstruction are unclear. We aimed to determine the effects of UDCA in bile duct­ligated and in Mdr2 knockout (Mdr2­/­) mice with biliary strictures.
Methods: Mice fed UDCA (0.5% wt/wt) or a control diet were subjected to common bile duct ligation (CBDL), selective bile duct ligation (SBDL), or sham operation. UDCA was also fed to 2-month-old Mdr2­/­ mice. Serum biochemistry, liver histology, and mortality rates were investigated. The biliary tract was studied by plastination, India ink injection, and electron microscopy. The effects of UDCA on biliary pressure were determined by cholangiomanometry.
Results: UDCA feeding in CBDL mice increased biliary pressure, with subsequent rupture of cholangioles and aggravation of hepatocyte necroses, resulting in significantly increased mortality. UDCA feeding in SBDL mice aggravated liver injury exclusively in the ligated lobe. Mdr2­/­ mice developed liver lesions resembling sclerosing cholangitis characterized by biliary strictures and dilatations. UDCA induced bile infarcts in these animals.
Conclusions: UDCA aggravates bile infarcts and hepatocyte necroses in mice with biliary obstruction via disruption of cholangioles as a result of increased biliary pressure caused by its choleretic action.

Cytokine-dependent bystander hepatitis due to intrahepatic murine CD8+ T-cell activation by bone marrow­derived cells
D. G. Bowen, A. Warren, T. Davis, M. W. Hoffmann, G. W. McCaughan, B. Fazekas de St. Groth, P. Bertolino
Background & Aims: Intrahepatic accumulation of CD8+ T cells following antigen-specific activation has been demonstrated in a number of transgenic models and also in extrahepatic viral infections. In some transgenic models, intrahepatic accumulation of cytotoxic T lymphocytes is associated with hepatitis. This observation suggests that hepatocellular damage may occur in some forms of immune-mediated hepatitis on the basis of a "bystander injury," whereby cytotoxic T lymphocytes accumulating in the liver mediate injury to hepatocytes in a nonspecific manner. Mouse transgenic models were therefore developed to investigate whether bystander damage to non­antigen-bearing hepatocytes occurs in vivo.
Methods: T cell receptor transgenic T cells were adoptively transferred into transgenic mice ubiquitously expressing the specific antigen, or into bone marrow radiation chimeras in which hepatocytes did not express the antigen.
Results: Selective accumulation of transgenic CD8+ T cells in the liver of intact recipients could be detected within 2 hours of transfer, despite ubiquitous antigenic expression. T cells retained in the liver were activated and induced hepatitis. Similar results were obtained using bone marrow chimeras, suggesting that antigen expression by hepatocytes was not required either for intrahepatic accumulation or for subsequent hepatitis. This "bystander hepatitis" was dependent on tumor necrosis factor and interferon .
Conclusions: Intrahepatic accumulation of activated CD8+ T cells and subsequent hepatitis can result from primary activation of CD8+ T cells by liver resident bone marrow­derived cells, inducing bystander damage to non­antigen-bearing hepatocytes. This mechanism may play a role in some forms of biologically significant hepatitis, including autoimmune hepatitis and hepatitis associated with extrahepatic diseases.

Toll-like receptor 2 contributes to liver injury by Salmonella infection through Fas ligand expression on NKT cells in mice
H. Shimizu, T. Matsuguchi, Y. Fukuda, I. Nakano, T. Hayakawa, O. Takeuchi, S. Akira, M. Umemura, T. Suda, Y. Yoshikai
Background & Aims: Toll-like receptors (TLRs) for bacterial constitutes are expressed not only by phagocytes but also by some subsets of T cells. We previously reported that natural killer T cells (NKT cells) play an important role in liver injury induced by Salmonella infection. In the present study, we investigated whether TLRs on NKT cells are involved in Salmonella-induced liver injury.
Methods: Gene expression of TLR2 was examined in sorted natural killer, NKT, and T cells from livers of naive mice by the reverse-transcription polymerase chain reaction method. Serum alanine aminotransferase level and FasL expression on liver lymphocytes were examined in TLR2-deficient (TLR2­/­) and FasL-deficient gld/gld mice before and after intraperitoneal inoculation of Salmonella choleraesuis 31N-1 using an enzyme-linked immunosorbent assay and flow cytometry.
Results: TLR2 gene was abundantly expressed by NKT cells freshly isolated from naive mice. FasL expression on liver NKT cells increased in TLR2+/­ mice but not in TLR2­/­ mice after Salmonella infection. Serum alanine aminotransferase level was significantly lower in the TLR2­/­ and gld/gld mice than in the control mice after infection.
Conclusions: TLR2 may contribute to liver injury induced by Salmonella infection via FasL induction on liver NKT cells.

Impaired adaptive resynthesis and prolonged depletion of hepatic mitochondrial DNA after repeated alcohol binges in mice
C. Demeilliers, C. Maisonneuve, A. Grodet, A. Mansouri, R. Nguyen, M. Tinel, P. Lettéron, C. Degott, G. Feldmann, D. Pessayre, B. Fromenty
Background & Aims: A single dose of alcohol causes transient hepatic mitochondrial DNA (mtDNA) depletion in mice followed by increased mtDNA synthesis and an overshoot of mtDNA levels. We determined the effect of repeated alcohol binges on hepatic mtDNA in mice.
Methods: Ethanol (5 g/kg) was administered by gastric intubation daily for 4 days, and mtDNA levels, synthesis, and integrity were assessed by slot blot hybridization, in organello [3H]deoxythymidine triphosphate incorporation, and long polymerase chain reaction analysis, respectively.
Results: mtDNA levels were decreased for 48 hours after the last dose, with no overshoot phenomenon later on. Two and 24 hours after the fourth dose, long polymerase chain reaction experiments showed DNA lesions that blocked the progress of the polymerases and in organello mtDNA synthesis was decreased, although DNA polymerase activity was unchanged with synthetic templates. Mitochondria exhibited ultrastructural abnormalities, and respiration was impaired 2 and 24 hours after the fourth binge. Cytochrome P450 2E1, mitochondrial generation of peroxides, thiobarbituric acid reactants, and ethane exhalation were increased.
Conclusions: After repeated doses of ethanol, the accumulation of unrepaired mtDNA lesions (possibly involving lipid peroxidation-induced adducts) blocks the progress of polymerase on mtDNA and prevents adaptive mtDNA resynthesis, causing prolonged hepatic mtDNA depletion.

Atrial natriuretic peptide attenuates Ca2+ oscillations and modulates plasma membrane Ca2+ fluxes in rat hepatocytes
A. K. Green, O. Zolle, A. W. M. Simpson
Background & Aims: Oscillations in cytosolic free Ca2+ concentration are a fundamental mechanism of intracellular signaling in hepatocytes. The aim of this study was to examine the effects of atrial natriuretic peptide (ANP) on cytosolic Ca2+ oscillations in rat hepatocytes.
Methods: Cyclic guanosine monophosphate (cGMP) was measured by enzyme immunoassay. Cytosolic Ca2+ oscillations were recorded from single aequorin-injected hepatocytes. Ca2+ efflux from hepatocyte populations was measured by using extracellular fura-2. Ca2+ influx was estimated by Mn2+ quench of fluorescence of fura-2 dextran injected into single hepatocytes.
Results: ANP attenuated cytosolic Ca2+ oscillations through a decrease in their frequency. In addition, ANP dramatically stimulated plasma membrane Ca2+ efflux and modestly inhibited basal Ca2+ influx. All of the observed effects of ANP were mimicked by the cGMP analogue 8-bromo-cGMP (8-Br-cGMP), and were prevented by inhibition of protein kinase G. In contrast, activation of cytosolic guanylyl cyclase by sodium nitroprusside had no effect on Ca2+ efflux, Ca2+ influx, or Ca2+ oscillations.
Conclusions: ANP decreases the frequency of Ca2+ oscillations and modulates plasma membrane Ca2+ fluxes in rat hepatocytes. Attenuation of oscillatory Ca2+ signaling in hepatocytes may represent a key role for ANP in vivo.

Murine leptin deficiency alters Kupffer cell production of cytokines that regulate the innate immune system
Z. Li, H. Lin, S. Yang, A. M. Diehl
Background & Aims: ob/ob mice are used to study the mechanisms that regulate the progression from steatosis to nonalcoholic steatohepatitis. The livers of ob/ob mice are depleted of CD4-positive natural killer cells, components of the innate immune system that induce anti-inflammatory cytokines. Although this may explain the sensitivity of fatty livers to lipopolysaccharide, why such hepatic CD4-positive natural killer cell depletion occurs is uncertain. Because leptin regulates macrophages, our hypothesis is that leptin deficiency alters Kupffer cell production of cytokines that inhibit (e.g., interleukin [IL]-12) or enhance (e.g., IL-15) hepatic CD4-positive natural killer cell viability.
Methods: Kupffer cell cytokine production and the hepatic content of CD4-positive natural killer cells were compared in ob/ob and lean mice. ob/ob mice were then treated with IL-15 or leptin to determine whether either factor improved their immunologic abnormalities.
Results: Compared with control Kupffer cells, ob/ob Kupffer cells produced less IL-15 basally and more IL-12 after lipopolysaccharide stimulation. Treatment of ob/ob mice with IL-15 for 1 week normalizes their hepatic CD4-positive natural killer cell content. Leptin increases the hepatic expression of IL-15 in ob/ob mice and partially replenishes their hepatic CD4-positive natural killer cells.
Conclusions: Leptin deficiency increases hepatic IL-12 and reduces hepatic IL-15 expression. The abnormal production of these Kupffer cell factors promotes hepatic CD4-positive natural killer cell depletion in ob/ob livers.

Cyclooxygenase-2 gene disruption attenuates the severity of acute pancreatitis and pancreatitis-associated lung injury
R. T. Ethridge, D. H. Chung, M. Slogoff, R. A. Ehlers, M. R. Hellmich, S. Rajaraman, H. Saito, T. Uchida, B. M. Evers
Background & Aims: Cyclooxygenase (COX) catalyzes the rate-limiting step in prostaglandin production; the inducible isoform, COX-2, has been implicated in a variety of inflammatory processes. The role of COX in acute pancreatitis and pancreatitis-associated lung injury is not known.
Methods: Acute pancreatitis was induced in Swiss Webster mice or mice deficient in the COX-2 (Ptgs2) or the COX-1 (Ptgs1) genes. Pancreata and lungs were harvested, and histologic sections of these tissues were scored. COX-2 expression, myeloperoxidase activity (a measurement of neutrophil sequestration), and serum amylase levels were determined.
Results: Acute pancreatitis was associated with induction of COX-2 expression. Treatment with NS-398 (a COX-2 inhibitor) significantly decreased the severity of pancreatitis. Furthermore, Ptgs2-deficient mice showed minimal histologic evidence of pancreatitis, a marked attenuation in the severity of lung injury, and a significant reduction in myeloperoxidase activity. In contrast, Ptgs1-deficient mice had pancreatitis and pulmonary inflammation, which was as severe or, in some instances, more severe than in the wild-type mice.
Conclusions: Inhibition of COX-2 by either pharmacologic inhibition or selective genetic deletion markedly attenuated the severity of acute pancreatitis. Our findings identify the COX-2 isoform as an important regulator of the severity of acute pancreatitis and pancreatitis-associated lung injury.

Fas enhances fibrogenesis in the bile duct ligated mouse: A link between apoptosis and fibrosis
A. Canbay, H. Higuchi, S. F. Bronk, M. Taniai, T. J. Sebo, G. J. Gores
Background & Aims: Hepatocyte apoptosis and fibrosis are both features of liver injury. However, the potential mechanistic link between these 2 processes remains obscure. Our aim was to ascertain if Fas-mediated hepatocyte apoptosis promotes liver fibrogenesis during extrahepatic cholestasis.
Methods: Wild-type and Fas-deficient lymphoproliferation (lpr) mice underwent bile duct ligation. Liver injury was assessed by quantitating hepatocyte apoptosis with the terminal deoxynucleotide transferase­mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay and determining serum ALT values. mRNA expression was quantitated using real-time polymerase chain reaction technology. Liver fibrosis was assessed by digital image analysis of Sirius red­stained sections.
Results: In 3-day bile duct ligated (BDL) animals, TUNEL-positive hepatocytes and serum ALT values were reduced in lpr versus wild-type animals. Likewise, hepatic mRNA transcripts for -smooth muscle actin and platelet-derived growth factor receptor- (initiation phase of stellate cell activation) and transforming growth factor 1 mRNA, collagen 1, and tissue inhibitor of matrix metalloproteinases (perpetuation phase of stellate cell activation) were also reduced in 3-day BDL wild-type mice compared with lpr mice. Finally, in 3-week BDL mice, immunoreactivity for -smooth muscle actin and Sirius red staining for collagen were significantly less in lpr compared with wild-type animals.
Conclusion: Fas-mediated hepatocyte injury is mechanistically linked to liver fibrogenesis. These observations suggest that inhibition of Fas-mediated apoptosis may be a therapeutic antifibrogenic strategy in cholestatic liver diseases.

Retinoid signaling controls mouse pancreatic exocrine lineage selection through epithelial-mesenchymal interactions
H. Kobayashi, T. L. Spilde, A. M. Bhatia, R. B. Buckingham, M. J. Hembree, K. Prasadan, B. L. Preuett, M. Imamura, G. K. Gittes
Background & Aims: The early embryonic pancreas gives rise to exocrine (ducts and acini) and endocrine lineages. Control of exocrine differentiation is poorly understood, but may be a critical avenue through which to manipulate pancreatic ductal carcinoma. Retinoids have been shown to change the character of pancreatic ductal cancer cells to a less malignant phenotype. We have shown that 9-cis retinoic acid (9cRA) inhibits acinar differentiation in the developing pancreas, in favor of ducts, and we wanted to determine the role of retinoids in duct versus acinar differentiation.
Methods: We used multiple culture systems for the 11-day embryonic mouse pancreas.
Results: Retinoic acid receptor (RAR)-selective agonists mimicked the acinar suppressive effect of 9cRA, suggesting that RAR-RXR heterodimers were critical to ductal differentiation. RAR was only expressed in mesenchyme, whereas RXR was expressed in epithelium and mesenchyme. Retinaldehyde dehydrogenase 2, a critical enzyme in retinoid synthesis, was expressed only in pancreatic epithelium. 9cRA did not induce ductal differentiation in the absence of mesenchyme, implicating a requirement for mesenchyme in 9cRA effects. Mesenchymal laminin is necessary for duct differentiation, and retinoids are known to enhance laminin expression. In 9cRA-treated pancreas, immunohistochemistry for laminin showed a strong band of staining around ducts, and blockage of laminin signaling blocked all 9cRA effects. Western blot and RT-PCR of pancreatic mesenchyme showed laminin-1 protein and mRNA induction by 9cRA.

Case Reports

Syngeneic living-donor liver transplantation without the use of immunosuppression
L. U. Liu, T. D. Schiano, A. D. Min, L. Kim­Schluger, M. E. Schwartz, S. Emre, T. M. Fishbein, H. C. Bodenheimer, Jr., C. M. Miller
Transplantation between monozygotic twins has been successfully performed using the kidney, small intestine, and pancreas. Identical HLA matching has enabled these individuals to be transplanted without the need for immunosuppressive medication. Liver transplantation without immunosuppression would lessen the risk of recurrent viral hepatitis and eliminate much of the morbidity associated with long-term use of immunosuppressive medication. Living-donor liver transplantation (LDLT) has been performed with increasing success in recent years without an opportunity arising to use a monozygotic twin as a donor. We report 2 cases of LDLT between identical twins wherein perfect haploidentity has allowed these recipients to be transplanted without the need for immunosuppression. Although HLA matched genotypically, there may be differences in anatomy between donor and recipient. Mild liver chemistry test abnormalities may occur after transplant despite the absence of immunosuppression.

A family with gastrointestinal amyloidosis associated with variant lysozyme
B. Granel, J. Serratrice, S. Valleix, G. Grateau, D. Droz, J. Lafon, M.-C. Sault, B. Chaudier, P. Disdier, R. Laugier, M. Delpech, P.-J. Weiller
Hereditary nonneuropathic systemic lysozyme amyloidosis is a very rare form of amyloidosis, and only 4 families with this condition have been detailed until now in the literature. Clinical manifestations of lysozyme amyloidosis observed until now mainly concerned the kidneys, liver, and digestive tract. We report here a new family with hereditary lysozyme amyloidosis who presented predominantly with gastrointestinal involvement. The proband, a middle-aged woman, underwent partial gastrectomy for a hemorrhagic "gastric peptic ulcer" in 1984. Gastrointestinal amyloidosis was diagnosed in 1998 on biopsies performed on the gastroduodenal anastomosis, which appeared to be very congestive at presentation. Immunohistochemical stainings in tissue sections were positive for lysozyme. Amyloid was also observed in the colonic mucosa. The patient had a mutation in the lysozyme gene characterized by substitution of the amino acid at position 64 in the mature protein from tryptophan to arginine, previously described in only 1 French family with prominent nephropathy. It is interesting to note that her father had died many years before with an uncharacterized digestive amyloidosis. Our observation shows that a search for gastrointestinal amyloidosis is important, particularly when physicians are faced with congestive mucosa, unexplained abdominal hemorrhage, or abdominal symptoms. When gastrointestinal amyloidosis is diagnosed, it is important to determine with precision the nature of the amyloid fibril proteins, because various types of amyloidosis can involve the gastrointestinal tract.

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JOURNAL OF HEPATOLOGY

Volume 37, Issue 4, October 2002

Cell Biology, Metabolism and Transport

Lori A. Gustafson et al.
Clofibrate improves glucose tolerance in fat-fed rats but decreases hepatic glucose consumption capacity
Background/Aims: High-fat (HF) diets cause glucose intolerance. Fibrates improve glucose tolerance. We have tried to obtain information on possible hepatic mechanisms contributing to this effect. Methods: Rats were fed a HF diet, isocaloric with the control diet, for 3 weeks without or with clofibrate. Several parameters related to liver glucose and glycogen metabolism were measured. Results: Clofibrate prevented the induction of glucose intolerance by 3 weeks HF feeding. Improved glucose tolerance by clofibrate was not due to increases in glucose phosphorylation or glycolysis in the liver, since both the HF diet and clofibrate suppressed glucokinase and pyruvate kinase activities with no effect on glucose 6-phosphatase. Clofibrate decreased glycogen storage in both control and HF rats. Clofibrate, with and without HF feeding, inhibited weight gain during the experimental period. Body temperature was significantly elevated by clofibrate, indicative of an increased basal metabolic rate. The capacity of liver mitochondria to oxidize long-chain fatty acids increased by clofibrate treatment. Mitochondria did not show uncoupling. Conclusions: Clofibrate does not improve glucose tolerance by improving hepatic glucose or glycogen metabolism. Peripheral glucose oxidation may be facilitated by increased energy dissipation.
Keywords: Glycogen; Glucose; Clofibrate; High-fat diet; Insulin

Harald H. Klein et al.
Differential modulation of insulin actions by dexamethasone: studies in primary cultures of adult rat hepatocytes
Background/Aims: Steroid diabetes is associated with hepatic insulin resistance; in hepatic cell models, however, mainly insulin-permissive effects have been described. Here we investigate modulation by dexamethasone of a larger number of insulin actions. Methods: Adult rat hepatocytes were cultured±dexamethasone for 48 h; insulin actions were studied subsequently. Results: Stimulation of glycolysis by insulin but not by glucose required culture with dexamethasone. Activation of glycogen synthesis by insulin or glucose was strongly enhanced by dexamethasone, the insulin effects on glycogenolysis and amino acid uptake were not modulated. When dexamethasone was omitted from the culture, insulin was incapable to activate glycogen synthase, inactivate glycogen phosphorylase or elevate the level of fructose 2,6-bisphosphate. Dexamethasone did not alter insulin binding, insulin receptor number or kinase activity, insulin receptor substrate-1 and Akt protein expression/phosphorylation. Insulin-stimulated association of phosphatidylinositol 3-kinase with insulin receptor substrates-1 and -2 was increased with dexamethasone, the increased association with IRS-2 may, at least partially, be explained by higher IRS-2 protein expression. Conclusions: The steroid does not cause hepatic resistance in vitro. The differential attenuation under steroid deprivation points to defects in branches of the insulin signal chain and/or loss of hormonal regulation at the level of target enzymes.
Keywords: Steroid; Dexamethasone; Insulin; Rat hepatocyte culture

Chronic Liver Diseases

Peter G. Langley, James Underhill, J. Michael Tredger, Suzanne Norris and Ian G. McFarlane
Thiopurine methyltransferase phenotype and genotype in relation to azathioprine therapy in autoimmune hepatitis
Background/Aims: Toxicity and efficacy of azathioprine is governed partly by the activity of thiopurine methyltransferase (TPMT). Azathioprine has been used for many years, with corticosteroids or alone, for the treatment of autoimmune hepatitis (AIH) but no studies of TPMT phenotype and genotype in relation to response to the drug in AIH have been published. Methods: Erythrocyte TPMT activities were measured by a radioincorporation assay in 72 consecutive outpatients with AIH, 53 of whom were genotyped for the commonest defective alleles in Europeans (TPMT*3A, *3B and *3C) by restriction fragment length polymorphism analysis. Results: TPMT activities were significantly lower in patients intolerant of azathioprine (group I, n=15) than in those who sustained remission on azathioprine alone (group II, n=28; P=0.003) and those who tolerated azathioprine but continued to require corticosteroids (group III, n=29; P<0.0001), and were higher in group III than in group II (P=0.034). Ten patients with defective alleles (all heterozygotes) had significantly lower TPMT activities (P=0.002). However, in 25% there was discordance between phenotype and/or genotype and response to azathioprine. Conclusions: TPMT phenotyping or genotyping may be advisable before institution of azathioprine therapy in AIH but neither approach invariably predicts response to the drug.
Keywords: Autoimmune hepatitis; Azathioprine; Thiopurine methyltransferase

Laurent Spahr et al.
Combination of steroids with infliximab or placebo in severe alcoholic hepatitis: a randomized controlled pilot study
Background/Aims: The aim of this study is to evaluate the tolerance and effects of infliximab combined with steroids in severe alcoholic hepatitis (AH). Methods: Twenty patients with biopsy-proven severe AH (Maddrey's score>32) received prednisone 40mg/day for 28 days and either infliximab 5mg/kg IV (group A) or placebo (group B) at day 0. Histology, plasma interleukin-6 (IL-6) and interleukin-8 (IL-8) were measured at baseline and at day 10. Results: Infliximab was well tolerated. Histology showed no significant changes. At day 28, Maddrey's score significantly improved in group A (39 (32-53) to 12 (7-52), P<0.05 vs. baseline) but not in group B (44 (33-50) to 22 (2-59), P=NS). At day 10, IL-6 and IL-8 decreased in group A (25pg/ml (10-85pg/ml) to 4.5pg/ml (2-25pg/ml); 301pg/ml (107-1207pg/ml) to 146pg/ml (25-252pg/ml), P<0.01, P<0.05 vs. baseline, respectively). In group B, changes were not significant (38pg/ml (13-116pg/ml) to 16pg/ml (4-128); 315pg/ml (26-1698pg/ml) to 110pg/ml (27-492pg/ml)). Conclusions: In severe AH, infliximab was well tolerated and associated with significant improvement in Maddrey's score at day 28. Although the size of this study does not allow comparison between groups, these promising results should encourage larger trials assessing the effects of this therapy on survival.
Keywords: Transvenous liver biopsy; Tolerance; Apoptosis; Cytokine; Maddrey's score

Cirrhosis and its Complications

Maite Chiva et al.
Effect of Lactobacillus johnsonii La1 and antioxidants on intestinal flora and bacterial translocation in rats with experimental cirrhosis
Background/Aims: Probiotics and antioxidants could be alternatives to antibiotics in the prevention of bacterial infections in cirrhosis. The aim of the present study was to determine the effect of Lactobacillus johnsonii La1 and antioxidants on intestinal flora, endotoxemia, and bacterial translocation in cirrhotic rats. Methods: Twenty-nine Sprague-Dawley rats with cirrhosis induced by CCl4 and ascites received Lactobacillus johnsonii La1 109cfu/day in vehicle (antioxidants: vitamin C+glutamate) (n=10), vehicle alone (n=11), or water (n=8) by gavage. Another eight non-cirrhotic rats formed the control group. After 10 days of treatment, a laparotomy was performed to determine microbiological study of ileal and cecal feces, bacterial translocation, endotoxemia, and intestinal malondialdehyde (MDA) levels as index of intestinal oxidative damage. Results: Intestinal enterobacteria and enterococci, bacterial translocation (0/11 and 0/10 vs. 5/8, P<0.01), and ileal MDA levels (P<0.01) were lower in cirrhotic rats treated with antioxidants alone or in combination with Lactobacillus johnsonii La1 compared to cirrhotic rats receiving water. Only rats treated with antioxidants and Lactobacillus johnsonii La1 showed a decrease in endotoxemia with respect to cirrhotic rats receiving water (P<0.05). Conclusions: Antioxidants alone or in combination with Lactobacillus johnsonii La1 can be useful in preventing bacterial translocation in cirrhosis.
Keywords: Experimental cirrhosis; Bacterial translocation; Antioxidants; Probiotics; Lactobacillus johnsonii; Spontaneous bacterial peritonitis

Paolo Montalto et al.
Bacterial infection in cirrhosis impairs coagulation by a heparin effect: a prospective study
Background: Bacterial infections have been postulated as a trigger for variceal bleeding in cirrhotic patients, and impair coagulation evaluated by thrombelastography (TEG). Endogenous heparinoids have been detected after variceal bleeding and during liver transplantation in some cirrhotics using heparinase-modified-TEG. Aim: To assess if bacterial infection is associated with endogenous heparinoids in cirrhotics, thus impairing coagulation. Methods: Native and heparinase-modified-TEG (cleavage of heparin and heparan-sulphate) was performed in 60 cirrhotics (Grade A, 2; B, 30; C, 28): 30 infected [septicaemia, 6 (culture positive); 6 (culture negative); spontaneous bacterial peritonitis, 10; chest infection, 4; others, 4], 30 not infected, and five infected patients without liver diseases, comparing TEG parameters r, , and ma. Eight cirrhotics were studied before and after infection. The diagnosis of presence and type of infection was based on international standard criteria. Results: A significant heparin effect was found only in infected cirrhotics (28 of 30) with significant changes in r (P=0.0003), (P<0.0001), and ma (P<0.0001), but in none of those not infected. This effect completely reversed in the eight evaluated after resolution of infection. There was no heparin effect in infected non-cirrhotics. Conclusions: A heparin effect was only found in cirrhotic patients with infection, further confirming that infection significantly modifies coagulation in cirrhotic patients.
Keywords: Cirrhosis; Infection; Coagulation; Heparin; Thrombelastography

Liver Growth and Cancer

Yaacov Baruch et al.
Von Willebrand factor in plasma and in liver tissue after partial hepatectomy in the rat
Background/Aims: Von Willebrand factor (vWf) is found in high levels in plasma of patients with acute and chronic liver disease. The role of vWf in liver injury and repair is unknown. We studied the effect of liver mass and remodeling on plasma and tissue vWf after partial hepatectomy. Methods: Rats were sacrificed postoperatively at intervals ranging from 60 min to 5 days, and vWf plasma levels were measured by enzyme-linked immunosorbent assay, using rabbit anti-human vWf, and by immunoperoxidase on cryosections, using rabbit anti-vWf/factor VIII. Northern blot hybridization was prepared with a complementary DNA specific to human vWf. Results: vWf plasma levels increased early after sham operation and after 70% partial hepatectomy. The highest levels were reached at 24 h, remaining high for 5 days. Immunostaining showed intense staining of sinusoidal lining cells 4 h after partial hepatectomy, remaining so for 5 days. Non-significant changes in overall liver messenger RNA expression of vWf were seen over 5 days in sham operation and partial hepatectomy. Conclusions: After partial hepatectomy, plasma vWf is increased, probably due to both acute-phase reaction and decreased degradation. An increase in sinusoidal vWf immunostaining may suggest a role for this factor in tissue remodeling.
Keywords: Von Willebrand factor; Partial hepatectomy; Liver regeneration; Extracellular matrix; Coagulation

Yanhua Li, Hans Jörg Hacker, Annette Kopp-Schneider, Ulrike Protzer and Peter Bannasch
Woodchuck hepatitis virus replication and antigen expression gradually decrease in preneoplastic hepatocellular lineages
Background/Aims: Hepatocellular carcinomas elicited in woodchucks by the woodchuck hepatitis virus (WHV) emerge gradually from parenchymal areas of minimal structural deviation via two predominant preneoplastic hepatocellular lineages, composed of either glycogenotic/basophilic or amphophilic/basophilic cell foci. In this study we analyzed WHV replication during neoplastic development in both lineages. Methods: In minimal deviation areas, preneoplastic hepatocellular foci, and hepatocellular neoplasms, developing in 16 WHV-carriers 31-38 months after WHV-inoculation, the proportion of hepatocytes containing WHV replicative intermediates (as detected by in situ hybridization for WHV DNA) and immunoreactive for WHV core and surface antigens was assessed. Results: Appearance of WHV replicative intermediates and expression of antigens were limited to the cytoplasm of hepatocytes and were strongly correlated (P<0.0001), both showing high levels in minimal deviation areas, but markedly reduced amounts in all types of preneoplastic hepatic focus (P<0.0001), and in hepatocellular adenomas. Most hepatocellular carcinomas were negative for WHV replicative intermediates and antigens. Conclusions: In both the glycogenotic-basophilic and the amphophilic-basophilic preneoplastic hepatocellular lineage, WHV replication and antigen expression gradually decrease early during the preneoplastic phase. The close correlation of these changes with metabolic aberrations characterizing preneoplastic hepatocellular lineages suggests that oncogenic effects mimicking insulin/glucagon imbalances may be responsible for the repression of hepadnaviral replication.
Keywords: Hepadnaviral hepatocarcinogenesis; Preneoplastic hepatocellular foci; Hepatocellular neoplasms; Woodchuck hepatitis virus DNA; Woodchuck hepatitis virus antigen expression; Insulin/glucagon imbalance

Marc Dubourdeau et al.
Infection of HepG2 cells with recombinant adenovirus encoding the HCV core protein induces p21WAF1 down-regulation - effect of transforming growth factor
Background/Aims: Chronic infection with hepatitis C virus leads to liver cirrhosis and hepatocellular carcinoma. Hepatocellular carcinoma is sometimes associated with p53 dysfunction and decreased p21WAF1 expression. The p21WAF1 gene is a major target of p53, and p21WAF1 protein regulates the activities of cyclin/CDK complexes involved in cell cycle control and tumor formation. Because core protein has oncogenic properties, we investigated the expression of p21WAF1 following core expression. Methods: We analyzed by Western blot, Northern blot and transfection the expression of p21WAF1 in HepG2 cell line under transient expression of Hepatitis C core protein by recombinant-adenoviral infection. Results and discussion: Infection of HepG2 with core-encoding viruses induced the down-regulation of p21WAF1 expression. This effect is due to a decrease in the p21WAF1 gene transcription and of the p21WAF1 protein half-life. These results support a role for Hepatitis C virus core protein in cell transformation. We also found also that the transforming growth factor can counteract the core-induced p21WAF1 down-regulation. The antagonist effect of TGF , or of other molecules, on p21WAF1 expression may be of particular interest for the treatment of HCV-positive hepatocellular carcinoma.
Keywords: Viral protein; p53 effector; Cytokine

Yoko Tamada et al.
Overexpression enhances interferon-mediated expression and activity of double-stranded RNA-dependent protein kinase in human hepatoma cells
Background/Aims: Double-stranded RNA-dependent protein kinase (PKR) is a key factor involved in interferon (IFN)-induced antiviral actions. Since p48, together with signal transducers and activators of transcription 1 and 2 (STAT1 and STAT2), is an indispensable mediator in IFN- signaling pathways, we investigated the effect of p48 gene transduction on PKR expression and its activity in HuH-7 human hepatoma cells. Methods: HuH-7 cells were infected or transfected with p48 gene expression adenoviral vector or plasmid vector, respectively, and incubated with or without IFN-, then PKR expression and phosphorylation of -subunit of eukaryotic protein synthesis initiation factor-2 (eIF2) in the cells were examined. In addition, PKR activity inhibiting protein translation was determined by the decrease of chloramphenicol acetyltransferase (CAT) gene translation or -fetoprotein secretion. Results: p48 overexpression itself could not stimulate PKR expression. However, p48 overexpression in combination with interferon- treatment caused a marked increase in PKR expression and augmented the phosphorylation of eIF2, by which the transfected CAT gene translation, as well as the endogenous -fetoprotein synthesis, was blocked without affecting their mRNA levels. Conclusions: These results suggest that p48 gene transduction may provide a strategy to enhance the IFN-mediated PKR expression and its activity in hepatocytes.
Keywords: RNA-dependent protein kinase; Interferon-; p48; Interferon-stimulated regulatory element; Eukaryotic protein synthesis initiation factor 2; Hepatitis C virus

Viral Hepatitis

Samuel S. Lee et al.
Prognostic factors and early predictability of sustained viral response with peginterferon alfa-2a (40KD)
Background/Aims: Baseline factors and early decline in serum hepatitis C virus RNA are predictive of sustained virological response to interferon therapy in patients with chronic hepatitis C. We evaluated the prognostic value of baseline factors and early viral RNA among patients treated with peginterferon alfa-2a (40KD). Methods: Data were pooled from three randomized trials involving 814 patients treated with peginterferon alfa-2a (40KD) (90, 135, or 180µg). Stepwise and multiple logistic regression identified independent baseline factors associated with response. Receiver operating characteristic curves for both absolute values and log10 decline in viral RNA at 4, 8, 12 and 24 weeks of therapy were created. Results: Independent prognostic factors for sustained virological response included viral genotype non-1, low pretreatment viral load, age (<40 years), no cirrhosis and body weight (<85kg). In addition, alanine aminotransferase quotient (>3) and histological activity index score (>10) were also independently prognostic. Receiver operating characteristic curves showed that detectable or less than 2-log10 decline in viral RNA at week 12 predicted sustained virological non-response (negative predictive value is 98%). Conclusions: In patients with chronic hepatitis C treated with peginterferon alfa-2a (40KD), the decision to continue or stop treatment can be made as early as week 12.
Keywords: Hepatitis C; Pegylated interferon; Peginterferon alfa-2a (40KD); Interferon alfa-2a; Prognostic factors; Predictability; Viral kinetics; Hepatitis C virus RNA

Jim E. van Steenbergen and the Working Group Vaccination High-risk Groups Hepatitis B for The Netherlands
Results of an enhanced-outreach programme of hepatitis B vaccination in the Netherlands (1998-2000) among men who have sex with men, hard drug users, sex workers and heterosexual persons with multiple partners
Background/Aims: The Dutch Ministry of Health funded a pilot vaccination project targeting groups at high risk for sex- and drug-related hepatitis B transmission. Methods: In seven Municipal Health Service (MHS) areas, three-part hepatitis B vaccination was offered free to men who have sex with men (MSM), drug users (DUs), and heterosexuals with multiple partners, including sex workers (SWs). Four intervention areas recruited participants through care-givers and opinion leaders and offered vaccination at non-MHS sites. Three control areas only used flyers to offer vaccination at MHS during regular hours. Results: Over 18 months, 13,808 persons enrolled for the first vaccination, representing 63% of the targeted population in the intervention areas and 23% in control areas. In intervention areas, only 19% of DUs enrolled, versus 4% in control areas. In both areas, enrollment of the targeted heterosexual population (64%) was satisfactory. MSM were most compliant in having the full series. Of vaccination sources, general practitioners (GPs) attained highest compliance (71%, odds ratio 1.82). Conclusions: Dutch MHS facilities can reach high-risk individuals, but DUs require additional outreach. Vaccine coverage was disappointing, but our experience will be deployed nationwide and successful strategies might be employed elsewhere in countries of low endemicity.
Keywords: Hepatitis B; Vaccination program; High-risk groups; Evaluation

Gabriella Pár et al.
Decrease in CD3-negative-CD8dim+ and V2/V9 TcR+ peripheral blood lymphocyte counts, low perforin expression and the impairment of natural killer cell activity is associated with chronic hepatitis C virus infection
Background/Aims: As chronic hepatitis C virus (HCV) infection is associated with impaired natural killer (NK) cell cytotoxicity, we examined the phenotypes and perforin expression of peripheral blood lymphocytes, as well as the effect of interferon-2b (IFN-2b) therapy. Methods: Thirty-three patients had chronic hepatitis C, and of them 12 had been on IFN-2b treatment. Eleven individuals had been treated earlier with IFN-2b and completely cured, and eight were HCV carriers with persistently normal serum alanine aminotransferase. Three-colour flow cytometry was used to measure the percentage of CD3+/CD8+, CD3+CD4+, TcR+, V2 TcR+, V9 TcR+, V1 TcR+, CD3CD16+, CD3CD56+, CD19+ and perforin-positive cells. NK cell activity was assessed by single cell cytotoxic and flow cytometric assay. Results: Patients with chronic hepatitis C showed an impaired NK cytotoxicity, decreased percentage of CD3-negative-CD8dim-positive (NK subtype) and V9/V2 TcR+ as well as perforin-positive T lymphocytes, compared to controls and to those who were cured from HCV infection. IFN-2b increased NK cell cytotoxicity and the percentage of perforin-positive lymphocytes. Conclusions: Our findings suggest that in chronic HCV infection a decreased percentage of CD3CD8+, V9/V2 TcR+ and perforin-positive T cells and simultaneous decreased peripheral NK activity may contribute to the impaired cellular immune response and the chronicity of the disease.
Keywords: Hepatitis C virus; CD3-negative-CD8+ lymphocytes; Gamma/delta T cells; Perforin; Natural killer cytotoxicity; Interferon

Case Report

Rajiv Jalan, Anthony Pollok, Syed H.A. Shah, Krishna K. Madhavan and Kenneth J. Simpson
Liver derived pro-inflammatory cytokines may be important in producing intracranial hypertension in acute liver failure
We describe a patient with paracetamol induced acute liver failure (ALF) who fulfilled criteria for poor prognosis and was waiting for a liver to become available for transplantation. Because of severe uncontrolled intracranial hypertension she underwent a hepatectomy that resulted in stabilization of her systemic and cerebral hemodynamics. She remained anhepatic for 14h and was successfully bridged to liver transplantation. The removal of the liver was associated with a sharp and sustained reduction in the circulating pro-inflammatory cytokine concentration suggesting that liver derived pro-inflammatory cytokines may be important in the pathogenesis of intracranial hypertension in patients with ALF.
Abbreviations: ALF, acute liver failure; CBF, cerebral blood flow; ICP, intracranial pressure; OLT, orthotopic liver transplantation; NO, nitric oxide

BRITISH MEDICAL JOURNAL

NEW ENGLAND JOURNAL

Volume 347:975-982 September 26, 2002 - Number 13

Peginterferon Alfa-2a plus Ribavirin for Chronic Hepatitis C Virus Infection
Michael W. Fried, M.D., Mitchell L. Shiffman, M.D., K. Rajender Reddy, M.D., Coleman Smith, M.D., George Marinos, M.D., Fernando L. Gonçales, Jr., M.D., Dieter Häussinger, M.D., Moises Diago, M.D., Giampiero Carosi, M.D., Daniel Dhumeaux, M.D., Antonio Craxi, M.D., Amy Lin, M.S., Joseph Hoffman, M.D., and Jian Yu, M.D., Ph.D.

Background Treatment with peginterferon alfa-2a alone produces significantly higher sustained virologic responses than treatment with interferon alfa-2a alone in patients with chronic hepatitis C virus (HCV) infection. We compared the efficacy and safety of peginterferon alfa-2a plus ribavirin, interferon alfa-2b plus ribavirin, and peginterferon alfa-2a alone in the initial treatment of chronic hepatitis C. Methods A total of 1121 patients were randomly assigned to treatment and received at least one dose of study medication, consisting of 180 µg of peginterferon alfa-2a once weekly plus daily ribavirin (1000 or 1200 mg, depending on body weight), weekly peginterferon alfa-2a plus daily placebo, or 3 million units of interferon alfa-2b thrice weekly plus daily ribavirin for 48 weeks. Results A significantly higher proportion of patients who received peginterferon alfa-2a plus ribavirin had a sustained virologic response (defined as the absence of detectable HCV RNA 24 weeks after cessation of therapy) than of patients who received interferon alfa-2b plus ribavirin (56 percent vs. 44 percent, P<0.001) or peginterferon alfa-2a alone (56 percent vs. 29 percent, P<0.001). The proportions of patients with HCV genotype 1 who had sustained virologic responses were 46 percent, 36 percent, and 21 percent, respectively, for the three regimens. Among patients with HCV genotype 1 and high base-line levels of HCV RNA, the proportions of those with sustained virologic responses were 41 percent, 33 percent, and 13 percent, respectively. The overall safety profiles of the three treatment regimens were similar; the incidence of influenza-like symptoms and depression was lower in the groups receiving peginterferon alfa-2a than in the group receiving interferon alfa-2b plus ribavirin. Conclusions In patients with chronic hepatitis C, once-weekly peginterferon alfa-2a plus ribavirin was tolerated as well as interferon alfa-2b plus ribavirin and produced significant improvements in the rate of sustained virologic response, as compared with interferon alfa-2b plus ribavirin or peginterferon alfa-2a alone.

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