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Revue de Presse du Mois Mois de Septembre 2002

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HEPATOLOGY

Table of Contents for September 2002 · Volume 36 · Number 3

Ursodeoxycholic acid in cholestatic liver disease: Mechanisms of action and therapeutic use revisited
Gustav Paumgartner, Ulrich Beuers
Ursodeoxycholic acid (UCDA) is increasingly used for the treatment of cholestatic liver diseases. Experimental evidence suggests three major mechanisms of action: (1) protection of cholangiocytes against cytotoxicity of hydrophobic bile acids, resulting from modulation of the composition of mixed phospholipid-rich micelles, reduction of bile acid cytotoxicity of bile and, possibly, decrease of the concentration of hydrophobic bile acids in the cholangiocytes; (2) stimulation of hepatobiliary secretion, putatively via Ca2+- and protein kinase C-­dependent mechanisms and/or activation of p38MAPK and extracellular signal-regulated kinases (Erk) resulting in insertion of transporter molecules (e.g., bile salt export pump, BSEP, and conjugate export pump, MRP2) into the canalicular membrane of the hepatocyte and, possibly, activation of inserted carriers; (3) protection of hepatocytes against bile acid­induced apoptosis, involving inhibition of mitochondrial membrane permeability transition (MMPT), and possibly, stimulation of a survival pathway. In primary biliary cirrhosis, UDCA (13-15 mg/kg/d) improves serum liver chemistries, may delay disease progression to severe fibrosis or cirrhosis, and may prolong transplant-free survival. In primary sclerosing cholangitis, UDCA (13-20 mg/kg/d) improves serum liver chemistries and surrogate markers of prognosis, but effects on disease progression must be further evaluated. Anticholestatic effects of UDCA have also been reported in intrahepatic cholestasis of pregnancy, liver disease of cystic fibrosis, progressive familial intrahepatic cholestasis, and chronic graft-versus-host disease. Future efforts will focus on definition of additional clinical uses of UDCA, on optimized dosage regimens, as well as on further elucidation of mechanisms of action of UDCA at the molecular level. (HEPATOLOGY 2002;36:525-531.)

Liver Biology and Pathobiology

Differentiated properties of hepatocytes induced from pancreatic cells
David Tosh, Chia-Ning Shen, Jonathan M. W. Slack
Transdifferentiation of pancreas to liver is a well-recognized phenomenon and has been described in animal experiments and human pathology. We recently produced an in vitro model for the transdifferentiation (or conversion) of the pancreatic cell line AR42J-B13 to hepatocytes based on culture with dexamethasone (Dex). To determine whether the hepatocytes express markers of hepatic intermediary metabolism and detoxification, we investigated the patterns of expression of glucokinase, cytochrome P450s CYP3A1 and CYP2B1/2, testosterone/4-nitrophenol uridine diphosphate glucuronosyltransferase (UDPGT), and aryl sulfotransferase. All were expressed. We also determined the expression of 2 enzymes involved in ammonia detoxification: carbamoylphosphate synthetase I (CPS I) and glutamine synthetase (GS). These enzymes are normally strictly compartmentalized in liver in a wide periportal pattern and the last downstream perivenous hepatocytes, respectively. Following culture with Dex, CPS I and GS are expressed in 2 different cell populations, suggesting that both periportal and perivenous hepatocytes are induced. We also produced a reporter assay based on the activation of green fluorescent protein (GFP) by the transthyretin (TTR) promoter or glucose-6-phosphatase (G6Pase) promoter. After culture with Dex, transfected cells begin to express GFP, showing that hepatic promoters are activated in concert with the induction of the hepatocyte phenotype. Lastly, we examined the stability of the hepatic phenotype and found that some cells still express liver markers (transferrin or albumin) up to 14 days after removal of Dex. In conclusion, these results suggest that pancreatic hepatocytes produced by this method may offer an alternative model to primary cultures of hepatocytes for the study of liver function. (HEPATOLOGY 2002;36:534-543.)

Delayed liver regeneration in peroxisome proliferator-activated receptor--null mice
Steven P. Anderson, Lawrence Yoon, Erika B. Richard, Corrie S. Dunn, Russell C. Cattley, J. Christopher Corton
Peroxisome proliferator chemicals, acting via the peroxisome proliferator-activated receptor- (Ppar), are potent hepatic mitogens and carcinogens in mice and rats. To test whether Ppar is required for hepatic growth in response to other stimuli, we studied liver regeneration and hepatic gene expression following partial hepatectomy (PH) of wild-type and Ppar-null mice. Ppar-null mice had a 12- to 24-hour delay in liver regeneration associated with a delayed onset and lower peak magnitude of hepatocellular DNA synthesis. Furthermore, these mice had a 24-hour lag in the hepatic expression of the G1/S checkpoint regulator genes Ccnd1 and cMyc and increased expression of the IL-1 cytokine gene. Hepatic expression of Ccnd1, cMyc, IL-1r1, and IL-6r was induced in wild-type mice, but not Ppar-null mice, after acute exposure to the potent Ppar agonist Wy-14,643, indicating a role for Ppar in regulating the expression of these genes. Expression of the fatty acid -hydroxylase gene Cyp4a14, a commonly used indicator gene for Ppar activation, was strongly induced in wild-type mice after hepatectomy, suggesting that altered hepatocyte lipid processing may also contribute to the impaired regeneration in mice lacking the Ppar gene. In conclusion, liver regeneration in Ppar-null mice is transiently impaired and is associated with altered expression of genes involved in cell cycle control, cytokine signaling, and fat metabolism. (HEPATOLOGY 2002;36:544-554.)

Pretreatment with FK506 up-regulates insulin receptors in regenerating rat liver
Oscar Escribano, María Dolores Fernández-Moreno, María Jesús Piña, Jesús Fueyo, César Menor, Irene Dolores Román, Luis G. Guijarro
This report examines the effect of FK506 pretreatment on liver insulin receptor expression in partially (70%) hepatectomized rats. FK506 pretreatment led to an increased insulin receptor number 24 hours after hepatectomy, detected by means of insulin binding and cross-linking procedures. This increase was related to enhanced insulin receptor expression determined by in vitro mRNA translation and Western blot techniques. We also tested the functionality of the expressed insulin receptors by [3H] thymidine incorporation into DNA in insulin-stimulated hepatocytes. The results show that FK506 pretreatment elicits an increase in the amount of insulin receptor ­subunits as measured by Western blot. Maximum -subunit expression recorded 24 hours after surgery was preceded by increased insulin receptor mRNA levels, which were detected 6 hours after hepatectomy. Moreover, in FK506­pretreated rat hepatocytes, obtained from remnant livers 24 hours after partial hepatectomy (PH), the increase in insulin receptor number was associated with improved sensitivity to the hormone. However, in both experimental groups (FK506-pretreated and nonpretreated rats), the sensitivity of hepatocytes toward epidermal growth factor (EGF) showed no significant change, which suggests a specific effect of FK506 on insulin receptor expression. In conclusion, our findings suggest that FK506 pretreatment induces insulin receptor expression in regenerating rat liver and promotes liver regeneration in hepatectomized rats. (HEPATOLOGY 2002;36:555-561.)

Preconditioning protects liver and lung damage in rat liver transplantation: Role of xanthine/xanthine oxidase
Leticia Fernández, Nicolás Heredia, Luis Grande, Gloria Gómez, Antonio Rimola, Alberto Marco, Emilio Gelpí, Joan Roselló-Catafau, Carmen Peralta
This study was designed to evaluate whether ischemic preconditioning could confer protection against liver and lung damage associated with liver transplantation. The effect of preconditioning on the xanthine/xanthine oxidase (XOD) system in liver grafts subjected to 8 and 16 hours of cold ischemia was also evaluated. Increased xanthine levels and marked conversion of xanthine dehydrogenase (XDH) to XOD were observed after hepatic cold ischemia. Xanthine/XOD could play a role in the liver and lung damage associated with liver transplantation. This assumption is based on the observation that inhibition of XOD reduced postischemic reactive oxygen species (ROS) generation and hepatic injury as well as ensuing lung inflammatory damage, including neutrophil accumulation, oxidative stress, and edema formation. Ischemic preconditioning reduced xanthine accumulation and conversion of XDH to XOD in liver grafts during cold ischemia. This could diminish liver and lung damage following liver transplantation. In the liver, preconditioning prevented postischemic ROS generation and hepatic injury as well as the injurious effects in the lung following liver transplantation. Administration of xanthine and XOD to preconditioned rats led to hepatic ROS and transaminase levels similar to those found after reperfusion and abolished the protective effect of preconditioning on the lung inflammatory damage. In conclusion, ischemic preconditioning reduces both liver and lung damage following liver transplantation. This endogenous protective mechanism is capable of blocking xanthine/XOD generation in liver grafts during cold ischemia. (HEPATOLOGY 2002;36:562-572.)

Blockade of the L-arginine/NO synthase pathway worsens hepatic apoptosis and liver transplant preservation injury
Gautam P. Yagnik, Yoshihito Takahashi, George Tsoulfas, Kaye Reid, Noriko Murase, David A. Geller
Organ graft preservation injury is a major problem complicating liver transplantation. The L-arginine/nitric oxide pathway has protective effects in several models of liver injury. The purpose of this study was to evaluate the role of the L-arginine/NO synthase (NOS) pathway on liver preservation injury and to characterize endogenous inducible NOS (iNOS) expression. Orthotopic liver transplantation was performed with 18-hour University of Wisconsin preservation solution in syngeneic rats. Recipient rats were either untreated or treated with L-arginine, D-arginine, nonspecific NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME), or iNOS selective inhibitor L-N6-(1-imino-ethyl)lysine (L-NIL) after revascularization. As early as 1 hour following reperfusion, circulating arginine levels decreased 10-fold and ornithine levels increased 4-fold. A corresponding increase in arginase I protein was detected in serum. To address the profound arginine deficiency, we supplemented recipients with arginine after transplantation. L-arginine (but not D-arginine) supplementation significantly reduced preservation injury 12 hours after reperfusion, suggesting that the protective effect of L-arginine was mediated through the generation of NO. iNOS protein expression peaked in the liver 6 to 12 hours following reperfusion. Blockade of the L-arginine/NO pathway with L-NAME significantly increased necrotic and apoptotic cell death in the transplanted graft. Addition of the iNOS selective inhibitor L-NIL mildly increased liver transaminase levels and also increased apoptosis in the liver graft. In conclusion, transplant recipients are profoundly arginine deficient postreperfusion due to arginase release. L-Arginine supplementation and NO synthesis decrease necrotic and apoptotic cell death and ameliorate liver transplant preservation injury. (HEPATOLOGY 2002;36:573-581.)

Cell adhesion regulates platelet-derived growth factor­induced MAP kinase and PI-3 kinase activation in stellate cells (*Human Study*)
Vinicio Carloni, Raffaella M. S. DeFranco, Alessandra Caligiuri, Alessandra Gentilini, Silvia Cappadona Sciammetta, Elisabetta Baldi, Benedetta Lottini, Paolo Gentilini, Massimo Pinzani
The biologic effects of growth factors are dependent on cell adhesion, and a cross talk occurs between growth factors and adhesion complexes. The aim of the present study was to evaluate the influence of cell adhesion on the major intracellular signaling pathways elicited by platelet-derived growth factor (PDGF) in hepatic stellate cells (HSC). PDGF signaling was investigated in an experimental condition characterized by lack of cell adhesion for different intervals of time. Basal and PDGF-induced focal adhesion kinase (FAK) tyrosine phosphorylation was maintained in a condition of cell suspension for 2, 4, and 6 hours, whereas it was completely lost after 12 and 24 hours. We examined MAP kinase activity at 2 and 24 hours, corresponding to the higher and lower levels of FAK phosphorylation. In these experiments, MAP kinase activity correlated with FAK phosphorylation. Stimulation with PDGF was able to cause Ras-GTP loading only in adherent cells. The ability of PDGF to induce phosphatidylinositol 3-kinase (PI 3-K) activity was abrogated in cells maintained in suspension. The Ser473 phosphorylation of Akt was only marginally affected by the lack of cell adhesion. We then evaluated the association of FAK with c-Src. This association was found to be cell adhesion dependent, and it did not appear to be dependent from phosphorylated FAK. These changes in PDGF-induced intracellular signaling were associated with a remarkable reduction of PDGF-proliferative potential in nonadherent cells, although no marked differences in the apoptotic rate were observed. In conclusion, these results suggest that cell adhesion differentially regulates major signaling pathways activated by PDGF in HSC. (HEPATOLOGY 2002;36:582-591.)

Effect of tauroursodeoxycholic acid on endoplasmic reticulum stress­induced caspase-12 activation (*Human Study*)
Qing Xie, Vladimir I. Khaoustov, Charles C. Chung, Joohyun Sohn, Bhuvaneswari Krishnan, Dorothy E. Lewis, Boris Yoffe
Activation of death receptors and mitochondrial damage are well-described common apoptotic pathways. Recently, a novel pathway via endoplasmic reticulum (ER) stress has been reported. We assessed the role of tauroursodeoxycholic acid (TUDCA) in inhibition of caspase-12 activation and its effect on calcium homeostasis in an ER stress-induced model of apoptosis. The human liver-derived cell line, Huh7, was treated with thapsigargin (TG) to induce ER stress. Typical morphologic changes of ER stress preceded development of apoptotic changes, including DNA fragmentation and cleavage of poly (adenosine diphosphate-ribose) polymerase (PARP), as well as activation of caspase-3 and -7. Elevation of intracellular calcium levels without loss of mitochondrial membrane potential (MMP) was shown using Fluo-3/Fura-red labeling and flow cytometry, and confirmed by induction of Bip/GRP78, a calcium-dependent chaperon of ER lumen. These changes were accompanied by procaspase-12 processing. TUDCA abolished TG-induced markers of ER stress; reduced calcium efflux, induction of Bip/GRP78, and caspase-12 activation; and subsequently inhibited activation of effector caspases and apoptosis. In conclusion, we propose that mitochondria play a secondary role in ER-mediated apoptosis and that TUDCA prevents apoptosis by blocking a calcium-mediated apoptotic pathway as well as caspase-12 activation. This novel mechanism of TUDCA action suggests new intervention methods for ER stress-induced liver disease. (HEPATOLOGY 2002;36:592-601.)

Hyperosmolarity triggers CD95 membrane trafficking and sensitizes rat hepatocytes toward CD95L-induced apoptosis
Roland Reinehr, Dirk Graf, Richard Fischer, Freimut Schliess, Dieter Häussinger
The effect of hyperosmolarity on CD95 membrane targeting and CD95 ligand (CD95L)­induced apoptosis was studied in rat hepatocytes. CD95 showed a predominant intracellular localization in normoosmotically exposed rat hepatocytes, whereas hyperosmotic exposure induced, within 1 hour, CD95 trafficking to the plasma membrane followed by activation of caspase-3 and -8. Hyperosmotic CD95 membrane targeting was sensitive to inhibition of c-Jun-N-terminal kinase (JNK), protein kinase C (PKC), and cyclic adenosine monophosphate, but not to inhibition of extracellular regulated kinases (Erks) or p38 mitogen activated protein kinase (p38MAPK). Hyperosmotic CD95 targeting to the plasma membrane was dose-dependently diminished by glutamine or taurine, probably caused by an augmentation of volume regulatory increase. Despite CD95 trafficking to the plasma membrane and caspase activation, hyperosmolarity per se did not induce apoptosis. Hyperosmolarity, however, sensitized hepatocytes toward CD95L-induced apoptosis, as assessed by annexin V staining and terminal deoxynucleotidyl transferase-mediated X-dUTP nick-end labeling (TUNEL) assay. This sensitization was abolished when hyperosmotic CD95 membrane trafficking was prevented by cyclic adenosine monophosphate, PKC, or JNK inhibition, whereas these effectors had no effect on CD95L-induced apoptosis in normoosmotically exposed hepatocytes. CD95L addition under normoosmotic conditions caused CD95 membrane trafficking, which was sensitive to JNK inhibition, but not to cyclic adenosine monophosphate or inhibition of PKC, Erks, and p38MAPK. In conclusion, multiple signaling pathways are involved in CD95 membrane trafficking. Hyperosmotic hepatocyte shrinkage induces CD95 trafficking to the plasma membrane, which involves JNK-, PKA-, and PKC-dependent mechanisms and sensitizes hepatocytes toward CD95L-mediated apoptosis. (HEPATOLOGY 2002;36:602-614.)

Apoptosis of human hepatic myofibroblasts promotes activation of matrix metalloproteinase-2 (*Human Study*)
Anne-Marie Preaux, Marie-Pia D'Ortho, Marie-Pierre Bralet, Yannick Laperche, Philippe Mavier
Liver fibrosis is potentially reversible after removal of the injurious agent. Fibrosis resolution is characterized by apoptosis of hepatic myofibroblasts and degradation of extracellular matrix components. Matrix metalloproteinase-2 (MMP-2) is involved in matrix remodeling. In the liver, it is synthesized by myofibroblasts, secreted as a proenzyme, and activated by membrane type-MMPs (MT-MMP) such as MT1-MMP. The goal of this work was to determine whether apoptosis induction in human hepatic myofibroblasts modulates the gene expression of MMP-2 and/or its activation by MT1-MMP. Induction of apoptosis by cytochalasin D or C2-ceramide did not modulate MMP-2 mRNA expression. In contrast, apoptosis was associated with marked activation of pro-MMP-2, as shown by gelatin zymography, which revealed the presence of the 59-kd active form, whereas untreated cells only expressed the 66-kd proform. SB-203580, a specific inhibitor of p38 MAPK, selectively abrogated both C2-ceramide­induced apoptosis and pro-MMP-2 activation. Apoptosis-induced pro-MMP-2 activation was inhibited by the tissue inhibitors of metalloproteinases (TIMP)-2 but not by TIMP-1, implying involvement of an MT-MMP­mediated process. Induction of apoptosis by cytochalasin D and C2-ceramide upregulated MT1-MMP protein expression and MT1-MMP mRNA expression. In conclusion, apoptosis of hepatic myofibroblasts induces pro-MMP-2 activation through increased MT1-MMP expression. HEPATOLOGY 2002;36:615-622.)

Demonstration of direct lineage between hepatocytes and hepatocellular carcinoma in diethylnitrosamine-treated rats
Marie-Pierre Bralet, Virginie Pichard, Nicolas Ferry
The question whether hepatocellular carcinoma (HCC) arises from dedifferentiation of mature hepatocytes or from proliferation of liver stem cells is still debated. In the present study, we used retroviral-mediated genetic labeling to investigate the fate of mature hepatocytes in rats after administration of diethylnitrosamine (DEN). Mature hepatocytes were genetically labeled by intravenous injection of retroviral vectors containing the Escherichia coli -galactosidase gene coupled to a nuclear localization signal (nls-LacZ) 1 day after partial hepatectomy. Liver biopsies performed after completion of hepatic regeneration showed that 18.3% of hepatocytes expressed the nls-LacZ transgene. Rats were then treated with DEN in drinking water for 12 weeks and sacrificed between 98 and 151 days after the onset of DEN administration. Clones of -galactosidase positive cells were observed, half of which (53%) also expressed the placental form of glutathione-S-transferase (GSTp), a marker of preneoplastic cells. HCCs of various sizes expressing GSTp were present in all animals. Careful examination of 90 HCCs revealed that 16 (17.7%) also expressed nls-LacZ. This figure precisely matched the proportion of labeled hepatocytes before DEN treatment (18.3%). In conclusion, a random clonal origin of HCC from mature hepatocytes is seen in the DEN model of hepatocarcinogenesis. (HEPATOLOGY 2002;36:623-630.)

Expression and regulation of gap junctions in rat cholangiocytes
Hans-Peter Bode, LiFu Wang, Doris Cassio, M. Fatima Leite, Marie V. St-Pierre, Keiji Hirata, Keisuke Okazaki, Marvin L. Sears, Paolo Meda, Michael H. Nathanson, Jean-François Dufour
Hepatocytes and other digestive epithelia exchange second messengers and coordinate their functions by communicating through gap junctions. However, little is known about intercellular communication in cholangiocytes. The aim of this study was to examine expression and regulation of gap junctions in cholangiocytes. Connexin expression was determined by confocal immunofluorescence in rat bile ducts and in normal rat cholangiocyte (NRC) cells, a polarized cholangiocyte cell line. Intercellular Ca2+ signaling was monitored by fluorescent microscopy. Microinjection studies assessed regulation of gap junction permeability in NRC cells and in SKHep1 cells, a liver-derived cell line engineered to express connexin 43. Immunochemistry showed that cholangiocytes from normal rat liver as well as the NRC cells express connexin 43. Localization of apical, basolateral, and tight junction proteins confirmed that NRC cells are well polarized. Apical exposure to ATP induced Ca2+ oscillations that were coordinated among neighboring NRC cells, and inhibition of gap junction conductance desynchronized the Ca2+ oscillations. NRC cells transfected with a connexin 43 antisense were significantly less coupled. Transcellular dye spreading was inhibited by activation of protein kinase A or protein kinase C. The same was observed in transfected SKHep1 cells, which expressed only connexin 43. Rat cholangiocytes and NRC cells express connexin 43, which permits synchronization of Ca2+ signals among cells. Permeability of connexin 43-gap junctions is negatively regulated by protein kinases A and C. In conclusion, cholangiocytes have the capacity for intercellular communication of second messenger signals via gap junctions in a fashion that is under hormonal control. (HEPATOLOGY 2002;36:631-640.)

Insulin inhibits secretin-induced ductal secretion by activation of PKC alpha and inhibition of PKA activity
Gene D. LeSage, Luca Marucci, Domenico Alvaro, Shannon S. Glaser, Antonio Benedetti, Marco Marzioni, Tushar Patel, Heather Francis, Jo Lynne Phinizy, Gianfranco Alpini
Insulin stimulates canalicular bile flow by interaction with hepatocytes. Insulin regulates the function of a number of epithelia through activation and membrane translocation of Ca2+-dependent PKC isoforms. No information exists regarding insulin regulation of ductal bile secretion. The aim of the study was to determine the role and mechanisms of action of insulin in the regulation of cholangiocyte secretion in BDL rats. We determined the subcellular localization of insulin receptor in cholangiocytes. We measured the effect of insulin on (1) secretin-stimulated cAMP levels in cholangiocytes and duct expansion in intrahepatic bile duct units (IBDUs) in the absence or presence of BAPTA/AM, H7 or rottlerin and (2) bile flow. We evaluated (1) if insulin effects are associated with activation of PKC alpha and (2) if activation of PKC causes inhibition of secretin-stimulated cAMP levels and PKA activity. We found insulin receptors only in the apical domain of cholangiocytes. Insulin inhibited secretin-induced choleresis and secretin-stimulated cholangiocyte cAMP levels. Insulin inhibited secretin-induced secretion in IBDUs when applied at the basolateral membrane or microinjected into IBDU lumen. Insulin inhibitory effects on cholangiocyte secretion were blocked by BAPTA/AM and H7. Insulin induced activation of PKC alpha, which decreased secretin-stimulated cAMP and PKA activity. In conclusion, insulin inhibited secretin-induced ductal secretion of BDL rats through activation of PKC and inhibition of secretin-stimulated cAMP and PKA activity. In conclusion, insulin counter-regulates cholangiocyte secretory processes in the BDL model, which is characterized by cholangiocyte proliferation. (HEPATOLOGY 2002;36:641-651.)

Liver Failure and Liver Disease

Novel fibrinogen 375 ArgTrp mutation (fibrinogen aguadilla) causes hepatic endoplasmic reticulum storage and hypofibrinogenemia (*Human Study*)
Stephen O. Brennan, Ghassan Maghzal, Benjamin L. Shneider, Ronald Gordon, Margret S. Magid, Peter M. George
The proposita and her sister had chronically elevated liver function test results, and needle biopsy specimens showed scattered eosinophilic inclusions within the hepatocytes. On immunoperoxidase staining, the inclusions reacted strongly with anti-fibrinogen antisera; on electron-microscopic (EM) examination, the material appeared confined to the endoplasmic reticulum (ER) and was densely packed into tubular structures with a swirling fingerprint appearance. Coagulation investigations showed low functional and antigenic fibrinogen concentrations that were indicative of hypofibrinogenemia. Amplification and DNA sequencing showed a heterozygous CGGTGG mutation at codon 375 of the fibrinogen chain gene. This novel 375 ArgTrp substitution segregated with hypofibrinogenemia in 3 family members and was absent from 50 normal controls. When purified plasma fibrinogen chains were examined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, reverse-phase chromatography, electrospray ionization mass spectrometry, and isoelectric focusing, only normal chains were detected. In conclusion, we propose that this nonconservative mutation causes a conformational change in newly synthesized molecules and that this provokes aggregation within the ER and in turn causes the observed hypofibrinogenemia. Whereas the mutation site, 375, is located in the D domain at the jaws of the primary E-to-D polymerization site, purified plasma fibrinogen showed normal polymerization, supporting our contention that molecules with variant chains never reach the circulation but accumulate in the ER. (HEPATOLOGY 2002;36:652-658.)

Serum phosphate is an early predictor of outcome in severe acetaminophen-induced hepatotoxicity (*Human Study*)
Lars E. Schmidt, Kim Dalhoff
Hypophosphatemia is frequently observed in acetaminophen-induced hepatotoxicity and may be involved in the pathogenesis of hepatic failure. The aim of the study was to evaluate the prognostic value of serial measurements of serum phosphate in patients with severe acetaminophen poisoning. Prospectively, serial measurements of serum phosphate were performed in 125 patients with severe acetaminophen poisoning. The optimum threshold value of serum phosphate to discriminate nonsurvivors was identified. Prognostic value and speed of identification were compared with those of the King's College Hospital (KCH) criteria. Phosphate concentrations were significantly higher in nonsurvivors than in survivors at 48 to 72 hours after overdose (mean 2.65 ± 1.18 mmol/L vs. 0.68 ± 0.22 mmol/L, P < .001) as well as 72 to 96 hours after overdose (2.12 ± 0.22 mmol/L vs. 0.59 ± 0.23 mmol/L, P < .001). A threshold phosphate concentration of 1.2 mmol/L at 48 to 96 hours after overdose had sensitivity 89%, specificity 100%, accuracy 98%, positive predictive value 100%, and negative predictive value 98%. The phosphate criteria had higher sensitivity, accuracy, and positive and negative predictive values than the KCH criteria, and it identified patients significantly earlier after transferal [median 1 hour (range 1-38 hours) vs. 12 hours (2-192 hours), P < .05, respectively]. In nonsurvivors, the degree of hyperphosphatemia correlated with renal dysfunction (R = .55; P = .02). In conclusion, hyperphosphatemia after acetaminophen overdose is seen exclusively in nonsurvivors, which makes it a highly specific as well as sensitive predictor of nonsurvival. We propose that hyperphosphatemia is caused by renal dysfunction in the absence of hepatic regeneration, as the latter appears to be associated with lowering of serum phosphate. (HEPATOLOGY 2002;36:659-665.)

Endoscopic ligation compared with sclerotherapy for bleeding esophageal varices in children with extrahepatic portal venous obstruction (*Human Study*)
Showkat Ali Zargar, Gul Javid, Bashir Ahmad Khan, Ghulam Nabi Yattoo, Altaf Hussain Shah, Ghulam Mohammad Gulzar, Jaswinder Singh, Bilal-ul Rehman, Ziaud Din
Endoscopic sclerotherapy is an effective treatment for bleeding esophageal varices, but it is associated with significant complications. Endoscopic ligation, a new form of endoscopic treatment for bleeding varices, has been shown to be superior to sclerotherapy in adult patients with cirrhosis. To determine the efficacy and safety of endoscopic sclerotherapy and ligation, the 2 methods were compared in a randomized control trial in 49 children with extrahepatic portal venous obstruction who had proven bleeding from esophageal varices. Twenty-four patients were treated with sclerotherapy and 25 with band ligation. No significant differences were found between the sclerotherapy and ligation groups in arresting active index bleeding (100% each) and achieving variceal eradication (91.7% vs. 96%, P = .61). Band ligation eradicated varices in fewer endoscopic sessions than did sclerotherapy (3.9 ± 1.1 vs. 6.1 ± 1.7, respectively, P < .0001). The rebleeding rate was significantly higher in the sclerotherapy group (25% vs. 4%, P = .049), as was the rate of major complications (25% vs. 4%, P = .049). After eradication, esophageal variceal recurrence was not significantly different in patients treated by ligation than by sclerotherapy (17.4% vs. 10%, P = .67). In conclusion, variceal band ligation in children is a safe and effective technique that achieves variceal eradication more quickly, with a lower rebleeding rate and fewer complications compared with sclerotherapy. (HEPATOLOGY 2002;36:666-672.)

Noninvasive prediction of cirrhosis in C282Y-linked hemochromatosis (*Human Study*)
Melanie Beaton, Dominique Guyader, Yves Deugnier, Romain Moirand, Subrata Chakrabarti, Paul Adams
The aim of the present study was to examine the predictive accuracy of noninvasive clinical and biochemical variables associated with cirrhosis among patients with C282Y homozygous hemochromatosis. Sixteen clinical and laboratory variables were recorded at the time of diagnosis in 193 Canadian C282Y homozygous patients. All patients underwent percutaneous liver biopsy and 27 (14%) had biopsy specimen­proven cirrhosis. Prediction of cirrhosis was assessed first by univariate regression analysis. Variables significantly related to cirrhosis were then evaluated by stepwise linear multivariate regression. Receiver operating characteristic curve analysis of the most informative variables from multivariate analysis was then used to devise a clinically applicable index for the noninvasive prediction of cirrhosis. This index was then validated in 162 C282Y homozygous patients in France. Ferritin, blood platelets, and aspartate transaminase (AST) level were selected for the clinical index. The combination of ferritin levels of 1,000 µg/L or greater, platelet levels of 200 ¥ 109/L or less, and AST levels above the upper limit of normal led to a correct diagnosis of cirrhosis in 77% of Canadian patients. In the French patients, this led to a correct diagnosis of cirrhosis in 90%. In conclusion, in C282Y homozygous patients, a combination of easily measured laboratory variables (ferritin, platelets, AST) can be used to make the diagnosis of cirrhosis in approximately 81% of cases, reducing the need for liver biopsy. (HEPATOLOGY 2002;36:673-678.)

Bacterial motif DNA as an adjuvant for the breakdown of immune self-tolerance to pyruvate dehydrogenase complex
David E. J. Jones, Jeremy M. Palmer, Alastair D. Burt, Claire Walker, Amanda J. Robe, John A. Kirby
Bacterial DNA containing unmethylated CpG dinucleotide motifs is immunostimulatory to mammals, skewing CD4+ T-cell responses toward the Th1 phenotype. Autoreactive T-cell responses seen in primary biliary cirrhosis (PBC) are typically of the Th1 phenotype, raising the possibility that bacterial DNA might play a role in the generation of pathologic autoimmunity. We therefore studied the effects of CpG motif-containing oligodeoxynucleotides (ODN) on responses to pyruvate dehydrogenase complex (PDC, the autoantigen in PBC) in a murine model. Sensitization of SJL/J mice with non­self-PDC has been shown to result in induction of autoreactive T-cell responses to PDC sharing characteristics with those seen in patients with PBC. Administration of CpG ODN to SJL/J mice at the time of sensitization with PDC resulted in a significant skewing of splenic T-cell response to self-PDC, with significant augmentation of the Th1 cytokine response (interleukin [IL] 2 and interferon [IFN] gamma) and reduction of the Th2 response (IL-4 and IL-10). In fact, CpG ODN seemed to be more effective at biasing the response phenotype and as effective at inducing liver histologic change as complete Freund's adjuvant (CFA), the standard adjuvant used for induction of Th1 responses in murine autoimmune and infectious immunity models. In conclusion, our findings raise the possibility that bacteria play a role in the development of autoimmunity (in PBC at least) through the potential of their DNA to shift the T-cell responses toward the phenotype associated with autoimmune damage. Moreover, this study suggests caution in the therapeutic use of CpG ODN as vaccine adjuvants. (HEPATOLOGY 2002;36:679-686.)

A randomized placebo-controlled study of long-acting octreotide for the treatment of advanced hepatocellular carcinoma (*Human Study*)
Man-Fung Yuen, Ronnie Tung-Ping Poon, Ching-Lung Lai, Sheung-Tat Fan, Chung-Mau Lo, Ka-Wah Wong, Wai Man Wong, Benjamin Chun-Yu Wong
Although various types of treatment of hepatocellular carcinoma (HCC) have been tried, the prognosis remains dismal, especially in patients with advanced stage of the disease. Somatostatin analogues exert antitumor effects. HCC have been shown to exhibit somatostatin receptors. The present randomized placebo-controlled study aimed at examining the efficacy of long-acting octreotide (Sandostatin LAR) for the treatment of advanced HCC. Seventy patients were randomized to receive a 2-week course of 250 µg short-acting octreotide twice daily followed by Sandostatin LAR 30 mg injection once every 4 weeks for 6 doses (n = 35) or placebo (control group) (n = 35). The clinical and laboratory parameters were monitored. There was no difference in the cumulative survival between the Sandostatin LAR-treated group compared with the control group [median survival 1.93 months vs. 1.97 months, respectively, P = NS (log-rank test)]. There was no tumor regression and no reduction of -fetoprotein (AFP) levels in patients receiving Sandostatin LAR treatment. There was no improvement of quality of life assessed by Karnofsky performance score. In conclusion, Sandostatin LAR monotherapy did not have survival benefit in our selected group of patients with advanced HCC. Further studies should be performed in patients with less advanced disease and/or different etiology to evaluate its benefit. (HEPATOLOGY 2002;36:687-691.)

Altered expression of E-cadherin in hepatocellular carcinoma: Correlations with genetic alterations, -catenin expression, and clinical features (*Human Study*)
Yu Wei, Jeanne Tran Van Nhieu, Sylvie Prigent, Petcharin Srivatanakul, Pierre Tiollais, Marie-Annick Buendia
E-cadherin is a key cell adhesion protein implicated as a tumor/invasion suppressor in human carcinomas and a binding partner of -catenin, which plays a critical role in Wnt signaling and in tumorigenesis. Here we report genetic and expression studies of E-cadherin and -catenin in hepatocellular carcinoma (HCC). Immunohistochemical analysis of E-cadherin expression in 37 HCCs and adjacent nontumor tissues revealed important variations among tumor samples, ranging from complete or heterogeneous down-regulation in 35% of cases to marked overexpression in 40% of tumors. Loss of E-cadherin expression was closely associated with loss of heterozygosity (LOH) at the E-cadherin locus and methylation of CpG islands in the promoter region (P < .002), predominantly in hepatitis B virus (HBV)-related tumors (P < .005). No mutation of the E-cadherin gene could be detected in the tumors examined, suggesting the requirement for reversible mechanisms of E-cadherin down-regulation. In most HCCs, including E-cadherin­positive and ­negative cases, -catenin was strongly expressed at the cell membrane and nuclear accumulation of the protein was correlated with the presence of mutations in the -catenin gene itself, but not with E-cadherin loss. At difference with a number of epithelial cancers, vascular invasion was frequently noted in HCCs showing enforced expression of the membranous E-cadherin/-catenin complex. In conclusion, these data support the notion that E-cadherin might play diverse and seemingly paradoxic roles in HCC, reflecting specific requirements for tumor growth and spread in the liver environment. (HEPATOLOGY 2002;36:692-701.)

Viral Hepatitis

Preemptive use of lamivudine reduces hepatitis B exacerbation after allogeneic hematopoietic cell transplantation (*Human Study*)
George K. K. Lau, Ming-Liang He, Daniel Y. T. Fong, Angeline Bartholomeusz, Wing-yan Au, Albert K. W. Lie, Stephen Locarnini, Raymond Liang
Exacerbation of hepatitis B virus (HBV) is a serious cause of morbidity and mortality in hepatitis B surface antigen (HBsAg)-positive patients undergoing transplantation. Our aim was to evaluate the effectiveness of lamivudine to prevent hepatitis due to exacerbation of HBV in HBsAg-positive patients treated with allogeneic hematopoietic cell transplantation. We studied 20 consecutive HBsAg-positive recipients of allogeneic hematopoietic cell transplantation who received lamivudine 100 mg daily starting one week before transplantation until week 52 after transplantation (group 1). Serial serum alanine aminotransferase and HBV DNA levels were measured before and after transplantation at 4- to 8-week intervals for the first year and then 4- to 12-week intervals. Their virologic and clinical outcomes were compared with 20 case-matched recipients who did not receive any antiviral therapy to HBV (anti-HBV) before and after hematopoietic cell transplantation (group 2). After transplantation, 9 patients (45%) in group 2 and one patient (5%) in group 1 had hepatitis due to exacerbation of HBV (P < .008), with 3 hepatic failures in group 2 and none in group 1. The one-year actuarial probability of survival without hepatitis due to exacerbation of HBV was higher in group 1 than group 2 (94.1% vs. 54.3%, P = .002). By multivariate Cox analysis, preemptive use of lamivudine effectively reduced hepatitis due to exacerbation of HBV (adjusted hazards ratio, 0.09; P = .021). In conclusion, preemptive lamivudine reduced HBV exacerbation. The use of lamivudine with other immunosuppressive regimens to prevent exacerbation of HBV should be further explored. HEPATOLOGY 2002;36:702-709.)

Inhibitory activity of dioxolane purine analogs on wild-type and lamivudine-resistant mutants of hepadnaviruses
Béatrice Seignères, Christian Pichoud, Perrine Martin, Philip Furman, Christian Trépo, Fabien Zoulim
To design combination strategies for chronic hepatitis B therapy, we evaluated in vitro the inhibitory activity of 4 nucleoside analogs, (­)FTC, L-FMAU, DXG, and DAPD, in comparison with lamivudine (3TC) and PMEA. In a cell-free assay for the expression of wild-type duck hepatitis B virus (DHBV) reverse transcriptase, DAPD-TP was found to be the most active on viral minus strand DNA synthesis, including the priming reaction, followed by 3TC-TP, (­)FTC-TP, and DXG-TP, whereas L-FMAU-TP was a weak inhibitor. In cell culture experiments, important differences in drug concentration allowing a 50% inhibition of viral replication or polymerase activity (IC50s) were observed depending on the cell type used, showing that antiviral effect of nucleoside analogs may depend on their intracellular metabolism. IC50s obtained for wild-type DHBV replication in primary duck hepatocytes were much lower than with DHBV transfected LMH cells. IC50s were also significantly lower in the 2.2.1.5 and HepG2 cells compared with HBV transfected HuH7 cells. Moreover, L-FMAU inhibited preferentially HBV plus strand DNA synthesis in these cell lines. The antiviral effect of these inhibitors was also evaluated against 3TC-resistant mutants of the DHBV and HBV polymerases. These mutants were found to be cross resistant to (­)FTC. By contrast, the double DHBV polymerase mutant was sensitive to DXG-TP and DAPD-TP. Moreover, both purine analogs remained active against DHBV and HBV 3TC-resistant mutants in transfected LMH and HepG2 cells, respectively. In conclusion, the unique mechanism of action of these new inhibitors warrants further evaluation in experimental models to determine their capacity to delay or prevent the selection of drug resistant mutants. (HEPATOLOGY 2002;36:710-722.)

Rapid detection of hepatitis B virus mutations using real-time PCR and melting curve analysis (*Human Study*)
Manna Zhang, Yuewen Gong, Carla Osiowy, Gerald Y. Minuk
Current methods of detecting hepatitis B virus (HBV) mutations are time consuming, labor intensive, and not suitable for screening large numbers of samples. In the present study, we documented the advantages of a system that exploits differences in thermal stability between perfect match and mismatch hybrids, and thereby distinguishes between wild-type and mutants. Hybridization probes were designed complementary to specific wild-type HBV sequences in surface (S), precore, and basal core promoter (BCP) regions of the HBV genome (nt 587, 1896, and 1762/1764, respectively). Two probes were designed for each mutation: anchor probes were 3' labeled with fluorescein and sensor probes, 5' labeled with LC-Red 640, and 3' phosphorylated. Temperatures for each probe melted from amplification products were then determined in a melting program. Sera from 12 patients, each containing identified HBV mutants (6 S-escape, 1 precore, 1 BCP, and 4 mixed precore and BCP), and 5 control sera from patients with wild-type virus were analyzed. Genomic sequences of mutant and wild-type viruses were confirmed by direct sequencing. Real-time polymerase chain reaction (PCR) with fluorescent hybridization probes accurately identified each mutant and wild-type genome. Melting temperatures obtained from probe-product duplexes for the 3 mutants were distinguished from wild-type (>4.0°C, minimal) within 45 minutes. The sensitivity of the system was 100 copies/mL and as few as 5% of mutant among wild-type virus were detected. In conclusion, real-time PCR with fluorescent hybridization probes is a specific, sensitive, quantitative, and rapid means of detecting clinically relevant HBV mutants. (HEPATOLOGY 2002;36:723-728.)

Steatosis in chronic hepatitis C: Relative contributions of obesity, diabetes mellitus, and alcohol (*Human Study*)
Alexander Monto, Judy Alonzo, Jessica J. Watson, Carl Grunfeld, Teresa L. Wright
Steatosis has emerged as a histologic finding of importance to the progression of hepatitis C virus (HCV)-associated liver disease. However, most studies of HCV-associated steatosis have excluded alcohol drinkers and individuals with diabetes and thus have not addressed the relative contribution of known causes of steatosis to liver injury in HCV-associated disease. To address this issue, we studied 297 consecutive patients with HCV who met inclusion criteria. Alcohol consumption, demographics, and serologic tests were correlated with degrees of steatosis and fibrosis on liver biopsy. Liver biopsy specimens were also examined for evidence of significant alcohol or nonalcoholic steatohepatitis (NASH) injury. In univariate analysis, steatosis correlated with type 2 diabetes mellitus (P = .005) and body mass index (BMI) (P = .0001) but not with the intensity of alcohol intake (in grams per day). In multivariate analysis, BMI (P = .0002) and genotype 3a infection (P = .02) were independent predictors of steatosis. When patients with risk factors for NASH were excluded, genotype 3a infection was the only independent predictor of steatosis. Steatosis (P = .04) and inflammation (P < .0001) scores on liver biopsy were the only independent predictors of fibrosis. Significant alcohol or NASH injury was found in only 6% of biopsy specimens. In conclusion, steatosis in HCV infection is associated with risk factors for NASH, particularly obesity, rather than alcohol consumption. (HEPATOLOGY 2002;36:729-736.)

Opportunities for prevention: Hepatitis C prevalence and incidence in a cohort of young injection drug users (*Human Study*)
Cari L. Miller, Caitlin Johnston, Patricia M. Spittal, Kathy Li, Nancy LaLiberté, Julio S. G. Montaner, Martin T. Schechter
The objective of this study was to compare sociodemographic, drug, and sexual risk characteristics between hepatitis C virus (HCV) baseline positive and negative young (13-24 years) injection drug users (IDUs) and to determine prospective risk factors for HCV seroconversion among the youth. Data were collected through the Vancouver Injection Drug Users Study (VIDUS). To date, more than 1,400 Vancouver-area IDUs have been enrolled and followed up; 234 were aged 24 years and younger. Semiannually, participants have completed an interviewer-administered questionnaire and have undergone serologic testing for human immunodeficiency virus (HIV) and HCV. Univariate and multivariate logistic regression analyses were undertaken to investigate predictors of baseline HCV positivity. In the multivariate analyses, Cox regression models with time-dependent covariates were used to identify predictors of HCV seroconversion. Of the 232 young injectors, 107 (46%) were HCV positive at baseline and a further 37 HCV seroconverted during the study period for an incidence rate of 37.3 per 100 person-years. Baseline positivity was associated with Aboriginal ancestry, older age, greater number of years injecting drugs, recent incarceration, sex trade work, more than 100 lifetime sexual partners, a previous sexually transmitted disease, living in the IDU epicenter, and injection more than once per day of heroin, cocaine, and speedball. Factors independently associated with HCV seroconversion were having a partner who uses injection drugs, requiring help to inject, and injection of cocaine more than once daily. In conclusion, unlike older IDUs, more than one half of young injectors were HCV negative at recruitment. Thus, there is a window of opportunity for prevention. However, the incidence rate of HCV among these young IDUs is alarming, suggesting that the opportunity to intervene is exceedingly small. (HEPATOLOGY 2002;36:737-742.)

The dynamics of T-lymphocyte responses during combination therapy for chronic hepatitis C virus infection (*Human Study*)
Eleanor Barnes, Gillian Harcourt, Dave Brown, Michaela Lucas, Rodney Phillips, Geoffrey Dusheiko, Paul Klenerman
Hepatitis C virus (HCV) readily sets up a persistent infection and is a major cause of liver disease worldwide. Interferon alfa and ribavirin therapy lead to sustained clearance of virus in 31% to 64% of patients with type 1 and non­type 1 genotypes, respectively. It is not clear to what extent these drugs act directly to reduce HCV replication, or indirectly via host immune responses, and what evoked immune responses are associated with clinical outcome. We have examined prospectively 15 patients with chronic HCV infection before, during, and after combination therapy. Quantitative assays for HCV antigen­specific CD4+ and CD8+ T-cell responses, and flow cytometric assays for analysis of the phenotype of T cells, in addition to viral sequencing of core protein, were performed throughout the treatment and follow-up period over 18 months. We found enhancement of proliferative T-cell responses during therapy. Proliferative responses are strikingly heterogeneous in terms of specificity, kinetics, and magnitude. Proliferative responses are often not associated with interferon- release. T-cell responses are rarely sustained irrespective of treatment outcome and this is not due to the evolution of new immune escape variants. T-cell responses tend to peak late in the course of treatment. In conclusion, combination therapy for HCV has a transient effect on host virus­specific T cells in the blood. Induction of sustained T-cell responses may require additional immune modulation later in therapy. (HEPATOLOGY 2002;36:743-754.)

GASTROENTEROLOGY

Table of Contents for September 2002 · Volume 123 · Number 3

Clinical­Alimentary Tract

Paradoxical coexpression of proinflammatory and down-regulatory cytokines in intestinal T cells in childhood celiac disease
G. Forsberg, O. Hernell, S. Melgar, A. Israelsson, S. Hammarström, M.-L. Hammarström
Background & Aims: Specific T-lymphocyte reactions are central in the pathogenesis of celiac disease, an inflammatory small-bowel enteropathy caused by a permanent intolerance to gluten. To delineate local T-lymphocyte responses to gluten, the cytokine expression in jejunal T lymphocytes of pediatric celiac patients with active disease, i.e., untreated and gluten-challenged celiac patients, was determined and compared with that of treated, symptom-free celiac patients and controls.
Methods: Biopsy samples were collected from celiac patients and controls. Intraepithelial and lamina propria T lymphocytes were isolated separately, and the cytokine messenger RNA levels were determined by using quantitative real-time reverse-transcription polymerase chain reaction. Interferon (IFN)- and interleukin (IL)-10 were determined at the protein level by immunohistochemistry.
Results: Active celiac disease was characterized by distortions in cytokine expression by T lymphocytes, with highly significant increases of IFN- and IL-10 but no concomitant increases in tumor necrosis factor , transforming growth factor 1, or IL-2 and no induction of IL-4. A marked shift of IFN- and IL-10 production from the lamina propria to the epithelium was characteristic of active celiac disease, and as many as one fourth of the intraepithelial lymphocytes expressed IFN-. Intraepithelial T lymphocytes in treated, symptom-free celiac patients still had increased IFN- levels compared with controls.
Conclusions: In celiac patients, gluten intake seems to cause an overreaction in intraepithelial T lymphocytes, with uncontrolled production of IFN- and IL-10. This may cause both recruitment of intraepithelial lymphocytes and a leaky epithelium, leading to a vicious circle with amplified immune activity and establishment of intestinal lesions

Mutations in NOD2 are associated with fibrostenosing disease in patients with Crohn's disease
M. T. Abreu, K. D. Taylor, Y.-C. Lin, T. Hang, J. Gaiennie, C. J. Landers, E. A. Vasiliauskas, L. Y. Kam, M. Rojany, K. A. Papadakis, J. I. Rotter, S. R. Targan, H. Yang
Background & Aims: The clinical manifestations of Crohn's disease (CD) are diverse, ranging from fibrostenosing small-bowel disease to colon-predominant inflammation. These distinctions may represent genetic, immunologic, and microbial heterogeneity. NOD2 gene mutations in CD have been described recently and may alter innate immune responses. We hypothesized that NOD2 mutations may be associated with distinct phenotypic expressions of CD.
Methods: Two cohorts of consecutively identified patients referred to an inflammatory bowel disease center (n = 142 collected between 1993 and 1996; n = 59 collected between 1999 and 2001) were genotyped for 3 single nucleotide variants of NOD2-R675W, G881R, and 3020insC-and phenotyped for disease behavior, disease location, and serum immune markers.
Results: Univariate analysis showed that CD-associated NOD2 variants were significantly associated with fibrostenosing disease in each cohort (P = 0.049 and P = 0.002, respectively). When both cohorts were analyzed together, the association between NOD2 variants and fibrostenosing disease was more significant (P = 0.001). These relationships were observed in both Jews and non-Jews. Forty-six percent of patients with fibrostenosing disease carried at least 1 of these alleles, compared with only 23.5% of patients without fibrostenosing disease (odds ratio, 2.8; 95% confidence interval, 1.6­5.2). Multivariate and conditioning analyses showed a primary association between NOD2 allelic variants and fibrostenosing disease, but not with small-bowel disease.
Conclusions: In this description of a genotype/phenotype correlation in CD patients and NOD2 variants, data suggest that variation in this gene contributes to the occurrence of fibrostenotic CD of the small bowel.

Selected loss of tolerance evidenced by Crohn's disease­associated immune responses to auto- and microbial antigens
C. J. Landers, O. Cohavy, R. Misra, H. Yang, Y.-C. Lin, J. Braun, S. R. Targan
Background & Aims: Previous studies in Crohn's disease suggest global loss of tolerance with sonicated bacteria preparations containing hundreds of antigens. Monoassociation studies show that a solitary bacterium can induce colitis in one animal model, whereas another is responsible in other models. Among patients with Crohn's disease, serum responses have been documented to microbial and autoantigens (antibodies to the Escherichia coli outer-membrane porin C and the Pseudomonas fluorescens-associated sequence I2, antisaccharomyces cerevisiae antibody (ASCA), and perinuclear antineutrophil cytoplasmic antibodies). Our aim was to determine whether there are heterogeneous responses to these specific antigens.
Methods: Sera from 330 Crohn's patients were analyzed. Immunoglobulin A enzyme-linked immunosorbent assays to ASCA, outer-membrane porin C, or I2 and immunoglobulin G enzyme-linked immunosorbent assay to ASCA and ANCA determined the presence and level of antibodies. Perinuclear antineutrophil cytoplasmic antibodies were determined by immunofluorescence.
Results: ASCA was detected in 56% of patients; 55% were seroreactive to outer-membrane porin C, 50% were seroreactive to I2, and 23% were perinuclear antineutrophil cytoplasmic antibody positive. Eighty-five percent responded to at least 1 antigen; only 4% responded to all 4. Among microbial antigens, 78% responded to at least 1, and 57% were double positive, but only 26% responded to all 3. The level of response was stable over time and with change in disease activity. Among patients with the same qualitative antigen-response profiles, quantitative response differed. Cluster analysis of these antibody responses yielded 4 groups: ASCA, outer-membrane porin C/I2, perinuclear antineutrophil cytoplasmic antibodies, or no/low response.
Conclusions: Rather than global loss of tolerance, there seem to be patient subsets with differing responses to selected microbial and autoantigens.

Lipid-induced intestinal gas retention in irritable bowel syndrome
J. Serra, B. Salvioli, F. Azpiroz, J.-R. Malagelada
Background & Aims: We hypothesized that lipids, which induce various motor and sensory effects on the gut, modulate intestinal gas dynamics and that alteration of this regulatory mechanism may result in impaired gas transit in patients with irritable bowel syndrome (IBS).
Methods: In 45 healthy subjects and 30 patients with IBS, evacuation of gas infused into the jejunum (at 12 mL/min) was measured for 2 hours. The effect of simultaneous duodenal perfusion of lipids at 0 kcal/min (saline), 0.5 kcal/min, and 1 kcal/min was tested in groups of 15 subjects each.
Results: In healthy subjects, duodenal lipids at 1 kcal/min but not at 0 kcal/min or 0.5 kcal/min produced significant gas retention (281 ± 53 mL vs. 22 ± 64 mL at 0 kcal/min and ­65 ± 72 mL at 0.5 kcal/min; P < 0.05 for both). Patients with IBS exhibited gas retention during saline perfusion (259 ± 85 mL at 0 kcal/min; P < 0.05 vs. healthy subjects) and were hypersensitive to duodenal lipids (505 ± 61 mL retention at 0.5 kcal/min; P < 0.05 vs. saline and vs. healthy subjects). The "gas plus lipids" challenge test discriminated patients with 100% sensitivity and 93% specificity.
Conclusions: Physiologic concentrations of intestinal lipids exert an inhibitory control on intestinal gas transit, and this mechanism is up-regulated in patients with IBS. Hence, impaired gas propulsion, shown by the gas challenge test, may be useful as a diagnostic test if replicated in a larger series of patients.

Predictors of response to infliximab in patients with Crohn's disease
M. A. Parsi, J.-P. Achkar, S. Richardson, J. Katz, J. P. Hammel, B. A. Lashner, A. Brzezinski
Background & Aims: Identifying predictors of response to infliximab in Crohn's disease may lead to better selection of patients for this therapy.
Methods: One hundred patients with either inflammatory or fistulous Crohn's disease and at least 3 months of follow-up after infliximab infusion were evaluated. Clinical response and duration of response were the primary outcome measures.
Results: For inflammatory disease, 73% of nonsmokers, compared with 22% of smokers, responded to infliximab (P < 0.001). Among patients taking concurrent immunosuppressives, 74% responded to infliximab compared with 39% not taking any immunosuppressives (P = 0.007). Prolonged response (duration >2 months) was achieved in 59% of nonsmokers compared with 6% of smokers (P < 0.001) and in 65% of patients on immunosuppressives compared with 18% not on immunosuppressives (P < 0.001). For fistulous disease, overall response rates were not different between nonsmokers and smokers, but nonsmokers had a longer duration of response (P = 0.046). Concurrent use of immunosuppressive medications had no effect on rate or duration of response. Multivariable logistic regression analysis confirmed the harmful effect of smoking and the beneficial effect of immunosuppressive use on response in patients with inflammatory disease. The same analysis for fistulous disease did not show an association between smoking or concurrent immunosuppressive use and response to infliximab.
Conclusions: In patients with inflammatory disease, nonsmoking and concurrent immunosuppressive use are associated with higher rates of response and longer duration of response to infliximab. In patients with fistulous Crohn's disease, nonsmoking is associated with longer duration of response to infliximab.

Uveitis and erythema nodosum in inflammatory bowel disease: Clinical features and the role of HLA genes
T. R. Orchard, C. N. Chua, T. Ahmad, H. Cheng, K. I. Welsh, D. P. Jewell
Background & Aims: There are few systematic studies on the natural history or immunogenetic associations of erythema nodosum (EN) or ocular inflammation in inflammatory bowel disease (IBD), but they are reportedly more common in patients with other extraintestinal manifestations (EIMs), particularly arthritis. Immunogenetic associations have previously been described in IBD arthritis and in EN associated with sarcoidosis. This study examined the clinical features and HLA-B, DR, and tumor necrosis factor (TNF-) associations of ocular inflammation and EN and their clinical and immunogenetic relationship to arthritis in IBD.
Methods: Details of EN and ocular inflammation were gathered by case-note review and questionnaire in 976 ulcerative colitis patients and 483 Crohn's patients. Sequence-specific PCR typing for polymorphisms in HLA-B, DR, and TNF- was performed in 39 EN and 40 ocular patients. Results were compared with 490 IBD controls without EIMs, 38 patients with type 1 and 31 with type 2 peripheral arthritis, and 16 AS patients.
Results: EN and ocular inflammation were more common in women, were associated with IBD relapse, and recurred in approximately 30% of patients. They occurred more commonly with arthritis and AS than expected by chance. Ocular inflammation was strongly associated with HLA-B*27, B*58, and HLA-DRB1*0103. There is a weak association between EN and HLA-B*15 but a strong association with the ­1031 TNF-.
Conclusions: EN, uveitis, and arthritis associated with IBD occur together commonly. They are associated with genes in the HLA region, and linkage disequilibrium between these genes may account for the clinical picture of overlapping but independent clinical manifestations.

Clinical­Liver, Pancreas, and Biliary Tract

Determinants of early mortality in patients with decompensated chronic hepatitis B treated with antiviral therapy
R. J. Fontana, H.-W. L. Hann, R. P. Perrillo, J. M. Vierling, T. Wright, J. Rakela, G. Anschuetz, R. Davis, S. D. Gardner, N. A. Brown
Background & Aims: Chronic hepatitis B is a leading cause of death worldwide. To identify patients who might require urgent liver transplantation despite antiviral therapy, we investigated the determinants of early mortality in a large cohort of patients with decompensated chronic hepatitis B treated with lamivudine.
Methods: One hundred fifty-four North American patients with decompensated chronic hepatitis B received lamivudine for a median of 16 months. Univariate and multivariate Cox regression modeling was used to develop a model of 6-month mortality.
Results: A biphasic survival pattern was observed, with most deaths occurring within the first 6 months of treatment (25 of 32, 78%) because of complications of liver failure. The estimated actuarial 3-year survival of patients who survived at least 6 months was 88% on continued treatment. In multivariate modeling, elevated pretreatment serum bilirubin and creatinine levels as well as the presence of detectable hepatitis B virus (HBV) DNA (by the bDNA assay) pretreatment were significantly associated with 6-month mortality. An equation approximating the probability of early mortality was developed from these variables.
Conclusions: Our data demonstrate a distinct alteration in the slope of the survival curve after 6 months of lamivudine treatment for decompensated chronic hepatitis B. An equation consisting of 3 widely available pretreatment laboratory parameters was developed that can be used to predict the likelihood of early death in patients receiving lamivudine for decompensated chronic hepatitis B. These observations may help identify patients who can be stabilized with suppressive antiviral therapy vs. those who require urgent liver transplantation.

Banding ligation versus nadolol and isosorbide mononitrate for the prevention of esophageal variceal rebleeding
G.-H. Lo, W.-C. Chen, M.-H. Chen, P.-I. Hsu, C.-K. Lin, W.-L. Tsai, K.-H. Lai
Background & Aims: -blockers and banding ligation are effective in the prevention of variceal rebleeding. However, the relative efficacy and safety remains unresolved.
Methods: One hundred twenty-one patients with a history of esophageal variceal bleeding were enrolled. Patients were randomized to undergo regular endoscopic variceal ligation (EVL group, 60 patients) until variceal obliteration, or drug therapy by using nadolol plus isosorbide mononitrate (N+I group, 61 patients) during the study period to prevent rebleeding.
Results: After a median follow-up period of 25 months, recurrent upper gastrointestinal bleeding developed in 23 patients in the EVL group and 35 patients in the N+I group (P = 0.10). Recurrent bleeding from esophageal varices occurred in 12 patients (20%) in the EVL group and 26 patients (42%) in the N+I group (relative risk = 0.45; 95% confidence interval, 0.24­0.85). The actuarial probability of rebleeding from esophageal varices was lower in the EVL group (P = 0.01). The multivariate Cox analysis indicated that the treatment was the only factor predictive of rebleeding. Treatment failure occurred in 8 patients (13%) in the EVL group and 17 patients (28%) in the N+I group (P = 0.01). Fifteen patients in the EVL group and 8 patients of the N+I group died (P = 0.06). Complications occurred in 17% of the EVL group and in 19% of the N+I group (P = 0.6).
Conclusions: Our trial showed that ligation was more effective than nadolol plus isosorbide-5-mononitrate in the prevention of variceal rebleeding, with similar complications in both treatment modalities. However, there is no significant difference in the survival rate between the 2 groups.

Primary prophylaxis of variceal hemorrhage: A randomized controlled trial comparing band ligation, propranolol, and isosorbide mononitrate
H. F. Lui, A. J. Stanley, E. H. Forrest, R. Jalan, W. S. Hislop, P. R. Mills, N. D. C. Finlayson, A. J. MacGilchrist, P. C. Hayes
Background & Aims: This randomized controlled trial compared variceal band ligation (VBL), propranolol (PPL), and isosorbide-5-mononitrate (ISMN) in the prevention of first esophageal variceal bleed.
Methods: Over a 6-year period, 172 patients with cirrhosis, grade II or III esophageal varices that had never bled, were recruited; 44 into VBL, 66 into PPL, and 62 into ISMN. Baseline patient characteristics: age, 55 ± 11 years; Child­Pugh score, 8 ± 2; 65% alcohol-induced cirrhosis; follow-up period, 19.7 ± 17.6 months (range, 0.13­72.1 months), were comparable in the 3 groups.
Results: On intention-to-treat analysis, variceal bleeding occurred in 7% of patients randomized to VBL, 14% to PPL, and 23% to ISMN. The 2-year actuarial risks for first variceal bleed were 6.2% (95% confidence interval [CI], 0.0%­15.0%) for VBL, 19.4% (95% CI, 0.1%­32.4%) for PPL, and 27.7% (95% CI, 14.2%­41.2%) for ISMN. A significant number of patients reported side effects with drug treatment (45% PPL and 42% ISMN vs. 2% VBL; P = 0.00), resulting in withdrawal from treatment in 30% of PPL and 21% of ISMN patients. There were no statistically significant differences in mortality rates in the 3 groups. In as-treated analysis, there was a statistically significant difference in actuarial risk for bleeding at 2 years between VBL and ISMN (7.5%, 95% CI, 2.5%­10.6% vs. 33.0%, 95% CI, 15%­49%, respectively, log rank test P = 0.03) but not between VBL and PPL.
Conclusions: VBL was equivalent to PPL and superior to ISMN in preventing first variceal bleed. The side-effect profile for pharmacotherapy was considerable.

The utility of radiological imaging in nonalcoholic fatty liver disease
S. Saadeh, Z. M. Younossi, E. M. Remer, T. Gramlich, J. P. Ong, M. Hurley, K. D. Mullen, J. N. Cooper, M. J. Sheridan
Background & Aims: This prospective study evaluates the role of radiological modalities in establishing the diagnosis of nonalcoholic steatohepatitis (NASH).
Methods: Consecutive patients with biopsy-proven nonalcoholic fatty liver disease (NAFLD) were enrolled (2000­2001). Patients with other liver diseases and significant alcohol consumption (>20 g/day) were excluded. Clinicodemographic data were gathered at the time of liver biopsy. Each biopsy specimen was assessed by a hepatopathologist. Each patient underwent a limited abdominal ultrasonography (US), computerized tomography (CT), and magnetic resonance imaging (MRI). Films were interpreted by a radiologist who used a predetermined radiological protocol. Each radiological study was reread by the same radiologist and a second radiologist.
Results: Patients with NASH had greater aspartate aminotransferase levels (P = 0.03), greater ferritin levels (P = 0.05), more hepatocyte ballooning (P < 0.0001), and more fibrosis (P = 0.002). None of the radiological features distinguished between NASH and other types of NAFLD. No radiological modality detected the presence of hepatocyte ballooning, Mallory's hyaline, or fibrosis, which are important features in the diagnosis of NASH. The presence of >33% fat on liver biopsy was optimal for detecting steatosis on radiological imaging.
Conclusions: Differences between NASH and nonprogressive NAFLD were not apparent with any radiological modality. Of the pathologic features important for establishing the diagnosis of NASH, only the severity of steatosis was reflected in these radiological modalities. Good intraobserver agreement was evident for each modality (US, CT, and MRI) that was superior to interobserver agreement.
Basic­Alimentary Tract

The APC tumor suppressor controls entry into S-phase through its ability to regulate the cyclin D/RB pathway
C. D. Heinen, K. Heppner Goss, J. R. Cornelius, G. F. Babcock, E. S. Knudsen, T. Kowalik, J. Groden
Editorial, page 935
Background & Aims: APC gene mutation is an early alteration in most colorectal tumors. In an attempt to determine its role in tumor development, we asked whether reintroducing wild-type APC into colorectal cancer cells with mutant APC affected cell cycle progression.
Methods: Using transient transfection, a plasmid containing the APC complementary DNA and DNA encoding the green fluorescent protein was expressed in SW480 cells. In addition, several other constructs were co-expressed with APC to determine their combined effects.
Results: We report that colorectal cancer cell lines transfected with wild-type APC arrest in the G1- phase of the cell cycle and that this arrest is abrogated by cotransfecting constitutively active -catenin or cyclin D1 and cMYC together. This APC-induced cell cycle arrest involves the disruption of -catenin­mediated transcription and depends on components of the G1/S regulatory machinery, as overexpression of E1a or E2F-1, -2, or -3 overrides the G1 arrest. Consistent with this, APC transfection inhibits RB phosphorylation and reduces levels of cyclin D1.
Conclusions: Our results suggest that APC functions upstream of RB in the G1/S regulatory pathway, cyclin D1 and cMYC affect APC-mediated arrest equivalently to oncogenic -catenin, and most colon tumors disrupt control of G1/S progression by APC mutation.

JunD stabilization results in inhibition of normal intestinal epithelial cell growth through P21 after polyamine depletion
L. Li, L. Liu, J. N. Rao, A. Esmaili, E. D. Strauch, B. L. Bass, J.-Y. Wang
Background & Aims: Normal intestinal mucosal growth requires cellular polyamines that regulate expression of various genes involved in cell proliferation, growth arrest, and apoptosis. We have recently shown that growth inhibition after polyamine depletion is associated with an increase in JunD/AP-1 activity in normal intestinal epithelial cells (IEC-6 line). The current study tests the hypothesis that polyamine depletion-induced JunD/activator protein 1 (AP-1) activity results from the activation of junD gene expression and plays a critical role in regulation of intestinal epithelial cell growth.
Methods: The junD gene transcription was examined by nuclear run-on assays, and messenger RNA (mRNA) stability was measured by determination of JunD mRNA half-life. Functions of JunD were investigated by using JunD antisense oligodeoxyribonucleotides and transient transfection with the junD-expressing vector.
Results: Depletion of cellular polyamines by DL--difluoromethylornithine (DFMO) induced levels of JunD mRNA and protein, which was associated with an increase in G1 phase growth arrest. Polyamine depletion did not increase the rate of junD gene transcription but significantly increased the stability of JunD mRNA. Decreasing JunD protein by using JunD antisense oligomers promoted cell growth in polyamine-deficient cells. Growth arrest following polyamine depletion also was accompanied by increases in both p21 expression and its promoter activity. Treatment with JunD antisense oligomers inhibited the p21 promoter and prevented the increase in p21 expression in the presence of DFMO. Ectopic expression of the wild-type junD increased p21-promoter activity and inhibited epithelial cell growth.
Conclusions: Polyamines negatively regulate junD gene expression posttranscriptionally, and increased JunD/AP-1 inhibits intestinal epithelial cell proliferation at least partially through the activation of p21 promoter.

Fas/CD95 is required for gastric mucosal damage in autoimmune gastritis
A. C. J. Marshall, F. Alderuccio, B.-H. Toh
Background & Aims: Experimental autoimmune gastritis (EAG), characterized by a gastric mononuclear cell infiltrate, mucosal cell damage, and autoantibodies to parietal cell­associated H+/K+ adenosine triphosphatase, is a model for human autoimmune gastritis that leads to pernicious anemia. Previous in vitro studies have implicated Fas/CD95 in initiating damage to gastric mucosal cells in humans and an animal model of autoimmune gastritis. Here we used 2 in vivo animal models to examine the role of Fas in the development of mucosal cell damage in autoimmune gastritis.
Methods: We initiated EAG in BALB/cCrSlc mice by neonatal thymectomy and examined for Fas expression in the gastric mucosa by immunohistochemistry. To address the in vivo relevance of Fas in mucosal injury, we examined the stomachs and sera of BALB/cCrSlc lpr/lpr mice subjected to neonatal thymectomy and BALB/cCrSlc nu/nu lpr/lpr mice transferred with lymphocytes from gastritic BALB/cCrSlc mice.
Results: Fas expression was up-reguiated in parietal cells of mice with EAG. Neonatally thymectomized lpr/lpr mice were resistant to developing destructive gastritis compared with heterozygous and wild-type littermates. Nu/nu Fas-sufficient mice transferred with lymphocytes from thymectomized lpr/lpr mice developed destructive gastritis. Nu/nu lpr/lpr mice transferred with lymphocytes from gastritic mice developed a nondestructive gastritis.
Conclusions: The observations that Fas is up-regulated in gastric parietal cells of mice with EAG and that Fas-deficient mice are resistant to development of destructive gastritis provide compelling evidence that Fas is required in vivo for development of gastric mucosal cell damage in autoimmune gastritis.

HMGB1 B box increases the permeability of Caco-2 enterocytic monolayers and impairs intestinal barrier function in mice
P. L. Sappington, R. Yang, H. Yang, K. J. Tracey, R. L. Delude, M. P. Fink
Background & Aims: High mobility group (HMG) B1 is a nonhistone nuclear protein that was recently identified as a late-acting mediator of lipopolysaccharide-induced lethality in mice. The proinflammatory actions of HMGB1 have been localized to a region of the molecule called the B box.
Methods: To determine whether HMGB1 or B box are capable of causing derangements in intestinal barrier function, we incubated cultured Caco-2 human enterocytic monolayers with recombinant human HMGB1 or a 74-residue truncated form of the protein consisting of the B box domain.
Results: Both HMGB1 and B box increased the permeability of Caco-2 monolayers to fluorescein isothiocyanate­labeled dextran (FD4) in a time- and dose-dependent fashion. The increase in permeability was reversible following removal of the recombinant protein. Exposure of Caco-2 cells to B box resulted in increased expression of inducible nitric oxide synthase messenger RNA and increased production of NO. When we used various pharmacologic strategies to inhibit NO production or scavenge NO or peroxynitrite (ONOO­), we abrogated B box­induced hyperpermeability. Administration of B box to wild-type mice increased both ileal mucosal permeability to FD4 and bacterial translocation to mesenteric lymph nodes. These effects were not observed in inducible nitric oxide synthase knockout mice.
Conclusions: These data support the view that HMGB1 and B box are capable of causing alterations in gut barrier function via a mechanism that depends on the formation of NO and ONOO­.

Celiac lesion T cells recognize epitopes that cluster in regions of gliadins rich in proline residues
H. Arentz-Hansen, S. N. McAdam, Ø. Molberg, B. Fleckenstein, K. E. A. Lundin, T. J. D. Jørgensen, G. Jung, P. Roepstorff, L. M. Sollid
Background & Aims: Celiac disease is a gluten-induced enteropathy that shows a strong association with HLA-DQ2 and -DQ8. Gluten-specific T cells, invariably restricted by DQ2 or DQ8, can be isolated from celiac lesions. Such gut-derived T cells have a preference for recognition of gluten that has been specifically deamidated by tissue transglutaminase. Only a few gliadin T-cell epitopes have been identified by earlier work. The aim of this study was to perform a systematic characterization of DQ2-restricted T-cell epitopes in - and -gliadins.
Methods: Epitopes were identified by mass spectrometry analysis of peptide fragments of recombinant gliadins and by use of synthetic peptides.
Results: We identified several new -gliadin epitopes and an additional -gliadin epitope. Interestingly, these and the previously identified epitopes are not randomly scattered across the gliadins but cluster in regions of the proteins with high content of proline residues.
Conclusions: Several DQ2-restricted T-cell epitopes exist in gliadin that are located in regions rich in proline. This likely reflects epitope selection at the levels of digestive and antigen-presenting cell processing, transglutaminase-mediated deamidation, and/or peptide binding to DQ2.

The type of sodium-coupled solute modulates small bowel mucosal injury, transport function, and ATP after ischemia/reperfusion injury in rats
R. A. Kozar, S. G. Schultz, H. T. Hassoun, R. DeSoignie, N. W. Weisbrodt, M. M. Haber, F. A. Moore
Background & Aims: Gastrointestinal function may be impaired after severe injury, hampering tolerance to enteral nutrition. The purpose of this study was to determine how different sodium-coupled solutes modulate gut function after ischemia/reperfusion (I/R) in a rodent model.
Methods: At laparotomy, rats had jejunal sacs filled with (glucose + alanine), glucose, glutamine, alanine, or mannitol (osmotic control), followed by superior mesenteric artery clamping for 60 minutes and 30 minutes of reperfusion. After reperfusion, sacs were harvested for morphologic examination, adenosine triphosphate (ATP) assay, or mounted in an Ussing chamber.
Results: Small intestinal epithelial absorptive capacity, as assessed by changes in short-circuit current in response to glucose, after gut I/R, was decreased by alanine or (glucose + alanine) but not glucose or glutamine alone and correlated with changes in tissue ATP. Gut I/R caused a significant morphologic injury that was worsened by alanine or (glucose + alanine) but lessened by glucose or glutamine alone.
Conclusions: During gut I/R, alanine can enhance gut injury, deplete energy (ATP), and impair gut absorption, whereas glucose or glutamine is protective against injury and can maintain absorptive capacity and ATP. These results suggest that solutes (such as alanine), which further stress an already metabolically stressed bowel, should be cautiously administered to critically ill patients whereas those solutes that contribute to energy production (such as glucose and glutamine) may be safely continued.

Expression of human intestinal mucin is modulated by the -galactoside binding protein galectin-3 in colon cancer
S. P. Dudas, C. K. Yunker, L. R. Sternberg, J. C. Byrd, R. S. Bresalier
Background & Aims: The L-type Ca2+ channel is a major pathway for Ca2+ influx in colonic smooth muscle and is modulated by endogenous levels of nonreceptor tyrosine kinase, c-src. Tyrosine kinases are also activated by G-protein­coupled receptors (GPCR). This study determined whether muscarinic receptor couples to Ca2+ channels via c-src kinase.
Methods: Currents were measured in rabbit colonic smooth muscle cells and in transfected HEK293 cells by patch-clamp technique. Tyrosyl phosphorylated proteins were detected by Western blots and the interaction of c-src with the c-terminus of subunit of Ca2+ channel was determined by a GST pull-down assay.
Results: Methacholine (10 µmol/L) enhanced Ca2+ channel currents by 30% under conditions whereby the M3 receptor pathway was blocked by either 4-DAMP or by intracellular dialysis with anti-Gq antibody. Similar effects were observed by blocking intracellular Ca2+ release with heparin. Enhancement was abolished by intracellular anti-Gi antibody and by the c-src inhibitor, PP2 but unaffected by the inactive analog PP3. Immunoblot with anti-src antibody revealed increased src phosphorylation by muscarinic receptor stimulation. Purified c-src directly associated with the c-terminus of 1c subunit of the Ca2+ channel. In M2 receptor transfected HEK293 cells, currents were enhanced 2-fold by carbachol.
Conclusions: These studies demonstrate stimulation of Ca2+ current in colonic smooth muscle cells by M2 receptor coupled to Gi­G protein and c-src activation. They also suggest a central role of c-src kinase in the cross-talk between tyrosine kinase receptor and GPCR.

Coupling of M2 muscarinic receptor to L-type Ca2+ channel via c-src kinase in rabbit colonic circular smooth muscle
X. Jin, N. Morsy, F. Shoeb, J. Zavzavadjian, H. I. Akbarali
Background & Aims: The L-type Ca2+ channel is a major pathway for Ca2+ influx in colonic smooth muscle and is modulated by endogenous levels of nonreceptor tyrosine kinase, c-src. Tyrosine kinases are also activated by G-protein­coupled receptors (GPCR). This study determined whether muscarinic receptor couples to Ca2+ channels via c-src kinase.
Methods: Currents were measured in rabbit colonic smooth muscle cells and in transfected HEK293 cells by patch-clamp technique. Tyrosyl phosphorylated proteins were detected by Western blots and the interaction of c-src with the c-terminus of subunit of Ca2+ channel was determined by a GST pull-down assay.
Results: Methacholine (10 µmol/L) enhanced Ca2+ channel currents by 30% under conditions whereby the M3 receptor pathway was blocked by either 4-DAMP or by intracellular dialysis with anti-Gq antibody. Similar effects were observed by blocking intracellular Ca2+ release with heparin. Enhancement was abolished by intracellular anti-Gi antibody and by the c-src inhibitor, PP2 but unaffected by the inactive analog PP3. Immunoblot with anti-src antibody revealed increased src phosphorylation by muscarinic receptor stimulation. Purified c-src directly associated with the c-terminus of 1c subunit of the Ca2+ channel. In M2 receptor transfected HEK293 cells, currents were enhanced 2-fold by carbachol.
Conclusions: These studies demonstrate stimulation of Ca2+ current in colonic smooth muscle cells by M2 receptor coupled to Gi­G protein and c-src activation. They also suggest a central role of c-src kinase in the cross-talk between tyrosine kinase receptor and GPCR.

Basic­Liver, Pancreas, and Biliary Tract

Hepcidin expression inversely correlates with the expression of duodenal iron transporters and iron absorption in rats
D. M. Frazer, S. J. Wilkins, E. M. Becker, C. D. Vulpe, A. T. McKie, D. Trinder, G. J. Anderson
Background & Aims: Hepcidin is an antimicrobial peptide thought to be involved in the regulation of intestinal iron absorption. To further investigate its role in this process, we examined hepatic and duodenal gene expression in rats after the switch from a control diet to an iron-deficient diet.
Methods: Adult rats on an iron-replete diet were switched to an iron-deficient diet and the expression of iron homeostasis molecules in duodenal and liver tissue was studied over 14 days. Intestinal iron absorption was determined at these same time-points by measuring the retention of an oral dose of 59Fe.
Results: Iron absorption increased 2.7-fold within 6 days of switching to an iron-deficient diet and was accompanied by an increase in the duodenal expression of Dcytb, divalent metal transporter 1, and Ireg1. These changes precisely correlated with decreases in hepatic hepcidin expression and transferrin saturation. No change in iron stores or hematologic parameters was detected.
Conclusions: This study showed a close relationship between the expression of hepcidin, duodenal iron transporters, and iron absorption. Both hepcidin expression and iron absorption can be regulated before iron stores and erythropoiesis are affected, and transferrin saturation may signal such changes.

Caspase 9­dependent killing of hepatic stellate cells by activated Kupffer cells
R. Fischer, A. Cariers, R. Reinehr, D. Häussinger
Background & Aims: Hepatic stellate cells play an important role in liver fibrogenesis, and hepatic stellate cell death may be involved in the termination of this response.
Methods: Molecular mechanisms of hepatic stellate cell killing were studied in hepatic stellate cell/Kupffer cell cocultures.
Results: Lipopolysaccharide stimulation of hepatic stellate cell/Kupffer cell cocultures, but not of hepatic stellate cell monocultures, induced profound alterations of hepatic stellate cell morphology and hepatic stellate cell death. Kupffer cell­induced hepatic stellate cell killing required hepatic stellate cell/Kupffer cell contacts and was prevented by dexamethasone, prostaglandin E2, tumor necrosis factor­related apoptosis-inducing ligand (TRAIL) receptor 2 antagonists, and down-regulation of receptor-interacting protein, but not by antioxidants, tumor necrosis factor receptor, or CD95 antagonists. Hepatic stellate cell death was characterized by activation of caspases 3, 8, and 9, terminal deoxynucleotidyl transferase­mediated deoxyuridine triphosphate nick-end labeling negativity, lack of gross calcium overload, and TRAIL trafficking to the plasma membrane. Inhibition of caspase 9, but not of caspases 3, 8, or 10, prevented hepatic stellate cell death. Lipopolysaccharide induced a dexamethasone- and prostaglandin E2­sensitive expression of TRAIL in Kupffer cells. TRAIL receptors 1 and 2, FLIP (caspase 8­inhibitory protein), and receptor-interacting protein were up-regulated during hepatic stellate cell transformation; however, TRAIL addition did not induce hepatic stellate cell death. Hepatic stellate cell susceptibility toward Kupffer cell­induced death paralleled receptor-interacting protein and TRAIL-receptor expression levels.
Conclusions: Activated Kupffer cell can effectively kill hepatic stellate cell by a caspase 9­ and receptor-interacting protein­dependent mechanism, possibly involving TRAIL. The data may suggest a novel form of hepatic stellate cell death.

Special Reports and Reviews

Emerging concepts in colorectal neoplasia
J. R. Jass, V. L. J. Whitehall, J. Young, B. A. Leggett
An understanding of the mechanisms that explain the initiation and early evolution of colorectal cancer should facilitate the development of new approaches to effective prevention and intervention. This review highlights deficiencies in the current model for colorectal neoplasia in which APC mutation is placed at the point of initiation. Other genes implicated in the regulation of apoptosis and DNA repair may underlie the early development of colorectal cancer. Inactivation of these genes may occur not by mutation or loss but through silencing mediated by methylation of the gene's promoter region. hMLH1 and MGMT are examples of DNA repair genes that are silenced by methylation. Loss of expression of hMLH1 and MGMT protein has been demonstrated immunohistochemically in serrated polyps. Multiple lines of evidence point to a "serrated" pathway of neoplasia that is driven by inhibition of apoptosis and the subsequent inactivation of DNA repair genes by promoter methylation. The earliest lesions in this pathway are aberrant crypt foci (ACF). These may develop into hyperplastic polyps or transform while still of microscopic size into admixed polyps, serrated adenomas, or traditional adenomas. Cancers developing from these lesions may show high- or low-level microsatellite instability (MSI-H and MSI-L, respectively) or may be microsatellite stable (MSS). The suggested clinical model for this alternative pathway is the condition hyperplastic polyposis. If colorectal cancer is a heterogeneous disease comprising discrete subsets that evolve through different pathways, it is evident that these subsets will need to be studied individually in the future.

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Journal of Hepatology

Volume 37, Issue 3, September 2002

Chronic Liver Diseases

Albert J. Czaja et al.
Clinical distinctions and pathogenic implications of type 1 autoimmune hepatitis in Brazil and the United States
Background/Aims: Type 1 autoimmune hepatitis has a strong genetic predisposition that varies among different ethnic groups. Our aims were to determine if the clinical manifestations differed between patients with type 1 autoimmune hepatitis from Brazil and the United States and if classical disease could be associated with region-specific susceptibility markers. Methods: The clinical manifestations and genetic risk factors of 161 patients from the United States were compared to those of 115 patients from Brazil. Results: The patients from Brazil had earlier disease onset, lower frequency of concurrent immune diseases, higher serum levels of aspartate aminotransferase and -globulin, greater occurrence of smooth muscle antibodies, and lower frequency of antinuclear antibodies than the patients from the United States. Human leukocyte antigen (HLA) DR13 and DRB1*1301 occurred more commonly in the Brazilian patients and HLA DR4 less often. Normal subjects from each country had similar frequencies of HLA DR13 and DR3. Conclusions: Type 1 autoimmune hepatitis in Brazil has different features at presentation than the disease in Caucasoid patients from the United States, and it is associated with HLA DR13. Background populations in each country have similar frequencies of HLA DR13 and DR3, and region-specific etiologic factors may determine the HLA association.

Peter W. Whiting et al.
Concordance of iron indices in homozygote and heterozygote sibling pairs in hemochromatosis families: implications for family screening
Background/Aims: Concordance of iron indices between same-sex siblings homozygous for the cysteine-to-tyrosine substitution at amino acid 282 (C282Y) mutation suggests that the variable phenotype in hereditary hemochromatosis is caused by genetic factors. Concordance of iron indices between same-sex heterozygous sibling pairs would provide further evidence of genetic modifiers of disease expression, and guidance for family screening strategies of subjects heterozygous for the C282Y mutation. Methods: We compared the iron indices of 35 C282Y homozygous and 35 C282Y heterozygous same-sex sibling pairs. To clarify whether concordance between siblings was due to environmental or genetic factors we compared the iron indices of 164 C282Y homozygous-normal, same-sex dizygotic twins. Results: Serum ferritin (r=0.50, P=0.003), hepatic iron concentration (r=0.61, P=0.025) and hepatic iron index (r=0.67, P=0.01) were highly concordant in C282Y homozygotes. Heterozygote siblings were concordant for serum ferritin (r=0.76, P=0.0001) and transferrin saturation (r=0.79, P=0.0001). Homozygote-normal same-sex dizygotic twins were concordant for serum ferritin (r=0.62, P=0.0001) but not for transferrin saturation. Conclusions: Concordance of iron indices exists in C282Y homozygote and heterozygote sibling pairs. Siblings of expressing C282Y heterozygotes require phenotypic assessment. These data provide evidence for modifying genes influencing disease expression in hemochromatosis.

Cirrhosis and its Complications

Gianmario Borroni et al.
Nadolol is superior to isosorbide mononitrate for the prevention of the first variceal bleeding in cirrhotic patients with ascites
Background/Aims: -blockers effectively prevent first variceal bleeding (FVB) in cirrhotic patients. In patients with ascites, however, their use might be precluded by a high rate of contraindications and side effects. We compared the efficacy and applicability of nadolol and isosorbide-mononitrate (IsMn) in preventing FVB in a population of cirrhotic patients at high risk of variceal bleeding with ascites, who can be frequently intolerant to -blockers. Methods: A total of 80 consecutive cirrhotic patients with ascites and esophageal varices (25% average risk of bleeding at 1 year) were considered, 28 were excluded due to contraindications and 52 were randomly assigned to receive nadolol (n=25) or IsMn (n=27). Results: Frequency of contraindications was greater for -blockers than IsMn (35 versus 0%, P=0.001). During 21.3±11.6 months of follow-up, side effects forced six patients taking nadolol and four taking IsMn to stop treatment. Bleeding occurred in two patients taking nadolol and ten taking IsMn. The probability of bleeding was significantly lower in the nadolol group (P<0.05), whereas overall survival was similar (seven patients on IsMn and eight on nadolol died, P=0.3). Conclusions: In patients with ascites IsMn is tolerated but ineffective while nadolol is effective but less tolerated.

Inflammation and Fibrosis

Antonio Mazzocca et al.
Expression of transmembrane 4 superfamily (TM4SF) proteins and their role in hepatic stellate cell motility and wound healing migration
Background/Aims: Migration of activated hepatic stellate cells (HSC) is a key event in the progression of liver fibrosis. Little is known about transmembrane proteins involved in HSC motility. Tetraspanins (TM4SF) have been implicated in cell development, differentiation, motility and tumor cell invasion. We evaluated the expression and function of four TM4SF, namely CD9, CD81, CD63 and CD151, and their involvement in HSC migration, adhesion, and proliferation. Methods/Results: All TM4SF investigated were highly expressed at the human HSC surface with different patterns of intracellular distribution. Monoclonal antibodies directed against the four TM4SF inhibited HSC migration induced by extracellular matrix proteins in both wound healing and haptotaxis assays. This inhibition was independent of the ECM substrates employed (collagen type I or IV, laminin), and was comparable to that obtained by incubating the cells with an anti-1 blocking mAb. Importantly, cell adhesion was unaffected by the incubation with the same antibodies. Co-immunoprecipitation studies revealed different patterns of association between the four TM4SF studied and 1 integrin. Finally, anti-TM4SF antibodies did not affect HSC growth. Conclusions: These findings provide the first characterization of tetraspanins expression and of their role in HSC migration, a key event in liver tissue wound healing and fibrogenesis.

Liver Growth and Cancer

Gadi Spira et al.
Halofuginone, a collagen type I inhibitor improves liver regeneration in cirrhotic rats
Background/Aims: Hepatic fibrosis involves excess deposition of extracellular connective tissue of which collagen type I fibers form the predominant component. Left untreated it develops into cirrhosis, often linked with hepatocellular carcinoma. Owing to the fact that cirrhotic liver regeneration is impaired, resection of hepatocellular carcinoma associated with cirrhosis is questionable. The aim of the present study was to determine the potential of halofuginone, a collagen type I inhibitor, in improving liver regeneration in cirrhotic rats. Methods: Partial hepatectomy (70%) was performed in thioacetamide-induced cirrhotic rats fed a halofuginone-containing diet. Liver regeneration was monitored by mass and proliferating cell nuclear antigen. The Ishak staging system and hydroxyproline content were used to evaluate the level of fibrosis. Results: Halofuginone administered prior to and following partial hepatectomy did not inhibit normal liver regeneration despite the reduced levels of collagen type I mRNA. When given to rats with established fibrosis, it caused a significant reduction in smooth muscle actin, TIMP-2, collagen type I gene expression and collagen deposition. Such animals demonstrated improved capacity for regeneration. Conclusions: Halofuginone may prove useful in improving survival of patients with hepatocellular carcinoma and cirrhosis undergoing surgical resection.

Chandrashekhar R. Gandhi et al.
Portacaval shunt causes apoptosis and liver atrophy in rats despite increases in endogenous levels of major hepatic growth factors
Background/Aims: The response to the liver damage caused by portacaval shunt (PCS) is characterized by low-grade hyperplasia and atrophy. To clarify mechanisms of this dissociation, we correlated the expression of `hepatotrophic factors' and the antihepatotrophic and proapoptotic peptide, transforming growth factor (TGF)-, with the pathologic changes caused by PCS in rats. Methods: PCS was created by side-to-side anastomosis between the portal vein and inferior vena cava, with ligation of the hilar portal vein. Hepatic growth mediators were measured to 2 months. Results: The decrease in the liver/body weight ratio during the first 7 days which stabilized by day 15, corresponded to parenchymal cell apoptosis and increases in hepatic TGF- concentration that peaked at 1.4¥baseline at 15 days before returning to control levels by day 30. Variable increases in the concentrations of growth promoters (hepatocyte growth factor, TGF- and augmenter of liver regeneration) also occurred during the period of hepatocellular apoptosis. Conclusions: The development of hepatic atrophy was associated with changes in TGF- concentration, and occurred despite increased expression of multiple putative growth promoters. The findings suggest that apoptosis set in motion by TGF- constrains the amount of hepatocyte proliferation independently from control of liver volume.

Viral Hepatitis

Ludwig Kramer et al.
Subclinical impairment of brain function in chronic hepatitis C infection
Background/Aims: Central nervous system abnormalities such as fatigue and depression occur more frequently in chronic hepatitis C virus (HCV) infection than in many other causes of chronic liver disease. The finding that fatigue is unrelated to activity of hepatitis or mode of infection could indicate an independent effect of HCV on brain function. This study tested the hypothesis of a subclinical cognitive dysfunction in HCV-infected patients. Methods: One-hundred untreated HCV-RNA positive biopsy-proven patients were investigated by P300 event-related potentials, a sensitive electrophysiologic test of cognitive processing. Health-related quality of life and fatigue were assessed using the SF-36 questionnaire and the Fatigue Impact Scale, respectively. Results: Cognitive brain function was subclinically impaired in the cohort of HCV-infected patients as indicated by significantly prolonged P300 latencies (P=0.01 for comparison to matched healthy subjects) and reduced P300 amplitudes (P<0.001, respectively). Seventeen of the 100 HCV-infected patients had P300 latencies outside the age-adjusted normal range. Abnormal P300 characteristics were not related to the degree of histologic or biochemical activity of hepatitis, severity of fatigue or mental health impairment. Conclusions: This study demonstrates that patients with HCV infection showed a slight but significant neurocognitive impairment, possibly indicating a further extrahepatic manifestation of chronic hepatitis C.

Hiroshi Yotsuyanagi et al.
Precore and core promoter mutations, hepatitis B virus DNA levels and progressive liver injury in chronic hepatitis B
Background/Aims: To elucidate the viral factors responsible for progressive liver injury in chronic hepatitis B. Methods: We analyzed 179 persistently infected patients (21 asymptomatic carriers, 126 with chronic hepatitis and 32 with cirrhosis) with genotype C hepatitis B virus (HBV). HBeAg/anti-HBe, levels of HBV DNA, mutations in the basic core promoter (BCP) region at nucleotides 1762/1764 and mutation in the precore (preC) region at nucleotide 1896 were determined. Serial samples from 18 patients also were analyzed. Results: HBeAg/anti-HBe and HBV DNA levels per se were not related to liver fibrosis. The frequency of BCP mutations increased with progression of liver fibrosis. Although the preC mutation was detected more often among the LC group, the role of this mutation in progression of fibrosis seems less than that of the BCP mutations. Sequential analysis showed that (1) rapidly progressing cases were positive continuously for double mutations in the BCP with a wild-type precore sequence, and (2) asymptomatic cases with anti-HBe acquired the preC mutation during their clinical course. Conclusions: Double mutations in the BCP region at nucleotide 1762/1764 are closely related to progression of chronic liver disease. Acquisition of mutation in the preC region at nucleotide 1896 may contribute to inactivation of chronic liver disease.

Marco Carrozzo et al.
Molecular evidence that the hepatitis C virus replicates in the oral mucosa
Background/Aims: Patients infected with the hepatitis C virus (HCV) often have extrahepatic manifestations, which significantly contribute to HCV-related morbidity, but whose pathogenesis is largely unknown. Our aim was to evaluate the HCV replication in oral mucosa of chronic hepatitis C patients. Methods: We collected oral mucosa specimens from 17 anti-HCV-positive and four anti-HCV-negative patients. Fifteen had oral lichen (12 anti-HCV-positive). Total mucosa RNA was extracted and analyzed for presence and titer of genomic and negative-strand HCV RNA. Findings were compared with clinical and pathological features. Results: Genomic and negative-strand HCV RNA were detected, respectively, in 12 of 17 (70.6%) and four of 17 (23.5%) specimens from the chronic hepatitis C patients. No negative-strand HCV RNA was detected in five anti-HCV-positive patients without lichen (including three with normal mucosa). Presence and titer of the negative-strand HCV RNA were independent of HCV genotype, serum viral load, and histological diagnosis of liver lesions. The phylogenetic analysis of the envelope 2 region cloned from a normal mucosa and the corresponding serum further suggested that only lichen tissues appear to harbor replicating HCV. Conclusions: HCV may occasionally replicate in oral lichen tissue, possibly contributing to the pathogenesis of mucosa damage.

Rita De Vos et al.
Ultrastructural visualization of hepatitis C virus components in human and primate liver biopsies
Background/Aims: Molecular and structural studies of hepatitis C virus (HCV) replication and infection have been performed on cultured cells and on serum of infected patients. No conclusive studies were conducted yet on human liver biopsies. This paper describes the ultrastructural findings of hepatitis C virus components in liver biopsies. Methods: Liver specimens from acutely and chronically HCV-infected chimpanzees (five each) and 29 chronic hepatitis C patients were studied. Diagnosis of HCV infection was based on clinical, serological, light microscopic and immunohistochemical data and on HCV RNA polymerase chain reaction. Results: In HCV-infected chimpanzees, tubular aggregates were observed in the cytoplasm of a significant number of hepatocytes and proven by immuno-electron microscopy to contain HCV-E2 viral envelope material. Identical tubular aggregates were seen in hepatocytes of chronic HCV-infected patients, although in smaller quantity and less frequently. A few single enveloped virus-like particles of 50-60 nm in diameter were seen for the first time in the hyaloplasm of hepatocytes of HCV-infected chimpanzees and patients. Conclusions: For the first time, HCV envelope material was ultrastructurally identified in hepatocytes of HCV-infected chimpanzees and patients. Virus-like particles, although strongly suggestive for HCV, failed final confirmation at least by routinely used methods.
Keywords: Hepatitis C pathology; Electron microscopy; Liver section; Virus-like particle; Blood mononuclear cell

Mi-Ock Lee et al.
Hepatitis B virus X protein induced expression of interleukin 18 (IL-18): a potential mechanism for liver injury caused by hepatitis B virus (HBV) infection
Background/Aims: The hepatitis B virus X protein (HBx), a major viral transactivator, is implicated in hepatic inflammation, since it induces many pro-inflammatory cytokines at transcriptional level. The aim of this study was to investigate role of HBx in expression of interleukin 18 (IL-18), a newly identified cytokine that up-regulates Fas ligand (FasL) expression. Methods: Chang X-34 that expressing HBx under the control of a doxycycline-inducible promoter, and hepatitis B virus (HBV)-integrated hepatoma cell lines were examined for IL-18 expression by Northern and Western blotting analysis. To test the role of IL-18 produced by hepatoma cells, FasL expression was examined by flow cytometry after treatment with neutralizing anti-IL-18 antibodies. Further, IL-18 expression was examined in the liver tissues of HBx-transgenic mice. Results: Induction of IL-18 following HBx expression in Chang X-34 and the pattern of IL-18 expression in HBV-integrated cell lines, implicated that HBx transcriptionally induces IL-18 expression. Neutralizing anti-IL-18 antibodies blocked the expression of FasL, suggesting that IL-18 plays a critical role in FasL expression. Further, IL-18 expression in the HBx-transgenic liver, was correlated with the degree of hepatitis. Conclusions: Our results demonstrated that HBx induces IL-18 expression in liver, which may be associated with hepatic injury by amplifying FasL expression during HBV infection.

Thomas Höhler et al.
C4A deficiency and nonresponse to hepatitis B vaccination
Background/Aims: Hepatitis B vaccination failure has been linked to the presence of certain human leukocyte antigen class II alleles. However, the functional background of these associations has remained unclear. Complement component C 4 is encoded within the major histocompatibility complex and is essential for classical pathway activation. Methods: Healthy individuals (n=4269) were vaccinated in a prospective trial with Engerix B. Nonresponse was classified as anti-HBs<10 U/l after the last vaccination. Seventy-three nonresponders (NR) (1.7%) were identified. For comparison 53 responders (R) (anti-HBs>10 IU/l) were drawn randomly from the same cohort. C4 allotyping was carried out by high-voltage agarose gel electrophoresis and C4-chain typing using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. C4 gene deletions (C4Del) were studied by Southern blot. Results: C4AQ0 alleles were observed in 45/73 (62%) NR compared to 17/53 (32%) R (P=0.001). C4ADel was observed in 24/73 (33%) NR and in 6/52 (12%) R (P=0.006). C4AQO alleles were present in 21/49 (43%) NR without C4Del compared to 10/46 (22%) in R without C4Del (P=0.031). In a logistic regression with DRB1*0301, DRB1*07, DRB1*1301 and C4AQ0 all except for DRB1*0301 showed a significant association. Conclusions: C4AQ0 shows a DRB1*0301 independent association with vaccine failure. C4AQ0 alleles probably contribute to inefficient complement activation and failure of B cells to secrete anti-HBs.

BRITISH MEDICAL JOURNAL

14 September 2002 (Volume 325, Issue 7364)

Observational study of vaccine efficacy 14 years after trial of hepatitis B vaccination in Gambian children
Hilton Whittle, Shabbar Jaffar, Michael Wansbrough, Maimuna Mendy, Uga Dumpis, Andrew Collinson, and Andrew Hall
BMJ 2002; 325: 569. [Full text]

Objective: To determine the duration of protection from hepatitis B vaccine given in infancy and early childhood.
Design: Cross sectional serological study of hepatitis B virus infection in children of various ages 14 years after the start of a trial of vaccination regimens.
Setting: Two villages in the Gambia.
Participants: Children and adolescents given hepatitis B vaccine in infancy or early childhood: 232 were aged 1-5 years, 225 aged 5-9 years, 220 aged 10-14 years, and 175 aged 15-19 years.
Main outcome measures: Vaccine efficacy against infection and against chronic infection in the different age groups.
Results: Vaccine efficacy against chronic carriage of hepatitis B virus was 94% (95% confidence interval 89% to 97%), which did not vary significantly between the age groups. Efficacy against infection was 80% (76% to 84%). This was significantly lower in the oldest age group (65%, 56 to 73). Of the uninfected participants in this age group, 36% had no detectable hepatitis B virus surface antibody. Time since vaccination and a low peak antibody response were the most powerful risk factors for breakthrough infection (P<0.001 in each case). Low peak antibody response was also a risk factor for chronic carriage (odds ratio 95, 19 to 466).
Conclusions: Children vaccinated in infancy are at increased risk of hepatitis B virus infection in the late teens. The risk of chronic carriage after sexual exposure needs further assessment to determine if booster vaccines are necessary.

Alosetron for irritable bowel syndrome
Michel Lièvre
BMJ 2002; 325: 555-556. [Full text]

On 9 February 2000 alosetron (marketed as Lotronex by GlaxoSmithKline), a type 3 serotonin (5-HT3) receptor antagonist, was approved by the US Food and Drug Administration (FDA) for the treatment of patients with irritable bowel syndrome, a benign though unpleasant disorder that affects one in five adults in the industrialised world.1 By November 2000, the FDA had received 49 reports of ischaemic colitis and 21 of severe constipation related to the drug, resulting in 44 admissions to hospital, 10 surgical interventions, and 3 deaths.2 The drug was withdrawn from the market by its sponsor. Severe adverse events continued to be reported for some time, with a final total of 84 instances of ischaemic colitis, 113 of severe constipation, 143 admissions to hospital, and 7 deaths.3 On 7 June 2002, however, the FDA issued a supplemental new drug application that permits marketing of alosetron through a prescribing programme for treating women with irritable bowel syndrome whose main symptom is severe diarrhoea (5% of patients). Doctors will have to sign an attestation of qualification and acceptance of responsibilities. Patients will have to sign a patient-physician agreement attesting that they have been adequately informed of the risks and that they have the form of irritable bowel syndrome that may be treated with alosetron.4 This prescription programme is unlikely to prevent severe adverse reactions due to alosetron. In November 2000, the FDA's office of post-marketing drug risk assessment underlined that ischaemic colitis could not be predicted, some patients were not able to recognise the signs and symptoms of constipation, the reversibility of ischaemic colitis had not been established, and the signs and symptoms of these severe adverse effects were too similar to those of the disease being treated.2 The increasing number of severe adverse experiences reported after the "Dear Doctor" and "Dear Pharmacist" letters issued in June 2000 at the request of the FDA also suggests that a real and effective risk management policy is not possible. The FDA's decision to put alosetron back on the market was made despite strong opposition of an insider (read Paul Stolley's story, p 592), and dissent is now being voiced by members of the advisory committee (see p 561). According to the information given in the patient-physician agreement, severe constipation occurred in about 1 in 1000 patients treated for six months, and ischaemic colitis in 1 in 350. The present prescription plan would theoretically allow up to two million people in the United States to receive alosetron, which might result in 2000 cases of severe constipation, 5714 cases of ischaemic colitis, 1109 surgical interventions, and 329 deaths; 240 000 women would experience some relief of symptoms.5 The price to pay for this benefit looks very high.

Doctors' perceptions of drinking alcohol while on call: questionnaire survey
Tahir Ahmad, Jimmy Wallace, James Peterman, and Norman A Desbiens
BMJ 2002; 325: 579-580. [Full text]

At its monthly ethics conference in September 1999, the department of internal medicine considered consumption of alcohol by doctors. The conference discussed the case of a young doctor who saw a senior colleague drinking heavily at a party and overheard him prescribing a questionably large dose of medication over the telephone.1 We discussed whether doctors should drink any amount of alcohol while on call.  Previous studies have considered alcohol use that impairs doctors' judgment and whether doctors should attend an emergency if they have been drinking but are not on call. 2 3 Few studies have considered doctors' drinking while on call. We decided to survey doctors to test our hypothesis that doctors rarely drink alcohol while on call but that opinion would differ about usage, depending on doctors' specialty and age.

NEW ENGLAND JOURNAL

LANCET

Volume 360, Number 9335  7 September 2002

Wait-and-see policy or laparoscopic cholecystectomy after endoscopic sphincterotomy for bile-duct stones: a randomised trial
Djemila Boerma, Erik A J Rauws, Yolande C A Keulemans, Ignace M C Janssen, Clemens JM Bolwerk, Ron Timmer, Egge J Boerma, Huug Obertop, Kees Huibregtse, Dirk J Gouma

Background Patients who undergo endoscopic sphincterotomy for common bile-duct stones, who have residual gallbladder stones, are referred for laparoscopic cholecystectomy. However, only 10% of patients who do not have this operation are reported to develop recurrent biliary symptoms. We aimed to assess whether a wait-and-see policy is justified. Methods We did a prospective, randomised, multicentre trial in 120 patients (age 18-80 years) who underwent endoscopic sphincterotomy and stone extraction, with proven gallbladder stones. Patients were randomly allocated to wait and see (n=64) or laparoscopic cholecystectomy (56). Primary outcome was reoccurrence of at least one biliary event during 2-year follow-up, and secondary outcomes were complications of cholecystectomy and quality of life. Analysis was by intention to treat. Findings 12 patients were lost to follow-up immediately. Of 59 patients allocated to wait and see, 27 (47%) had recurrent biliary symptoms compared with one (2%) of 49 patients after laparoscopic cholecystectomy (relative risk 22·42, 95% CI 3·16-159·14, p<0·0001). 22 (81%) of 27 patients underwent cholecystectomy, mainly for biliary pain (n=13) or acute cholecystitis (7). Conversion rate to open surgery was 55% in patients allocated to wait and see who underwent cholecystectomy compared with 23% in those who were allocated laparoscopic cholecystectomy (p=0·0104). Morbidity was 32% versus 14% (p=0·1048), and median hospital stay was 9 versus 7 days. Quality of life returned to normal within 3 months after either treatment policy. Interpretation A wait-and-see policy after endoscopic sphincterotomy in combined cholecystodocholithiasis cannot be recommended as standard treatment, since 47% of expectantly managed patients developed at least one recurrent biliary event and 37% needed cholecystectomy. No major biliary complications arose, but conversion rate was high.

Lancet 2002; 360: 761-65

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