Association 1901  |  Plan du site  |  Partenariat  |  Contacts  |+++++++++++
|  Accueil-News  |  Revue de presse  |  Pda du médecin  |  Hepatobase  |  Anatomobase  |  Diététique  |  Forums  |  DES  |  Laboratoires  |

Téléchargement sur votre Agenda de Poche avec Hepatoweb Edition Mobile

Revue de Presse du Mois Mois de Novembre 2002

Archives depuis le 01/09/00

---

HEPATOLOGY

Table of Contents for November 2002 · Volume 36 · Number 5

Liver Biology and Pathobiology

Deletion of the Ron receptor tyrosine kinase domain in mice provides protection from endotoxin-induced acute liver failure
Mike A. Leonis, Kenya Toney-Earley, Sandra J. F. Degen, Susan E. Waltz
The targeted deletion of the cytoplasmic domain of the Ron receptor tyrosine kinase (TK) in mice leads to exaggerated responses to injury in several murine models of inflammation as well as increased lethality in response to endotoxin (lipopolysaccharide [LPS]). Using a well-characterized model of LPS-induced acute liver failure (ALF) in galactosamine (GalN)-sensitized mice, we show that Ron TK­/­ mice display marked protection compared with control Ron TK+/+ mice. Whereas control mice have profound elevation of serum aminotransferase levels (a marker of hepatocyte injury) and hemorrhagic necrosis of the liver, in dramatic contrast, Ron TK­/­ mice have mild elevation of aminotransferase levels and relatively normal liver histology. These findings are associated with a reduction in the number of liver cells undergoing apoptosis in Ron TK­/­ mice. Paradoxically, treatment of Ron TK­/­ mice with LPS/GalN leads to markedly elevated (3.5-fold) serum levels of tumor necrosis factor (TNF) , a key inflammatory mediator in this liver injury model, as well as reduced amounts of interleukin (IL) 10 (a suppressor of TNF- production) and interferon (IFN)- (a TNF- sensitizer). These results show that ablation of the TK activity of the Ron receptor leads to protection from the development of hepatocellular apoptosis in response to treatment with LPS/GalN, even in the presence of excessive levels of serum TNF-. In conclusion, our studies show that the Ron receptor TK plays a critical role in modulating the response of the liver to endotoxin. (HEPATOLOGY 2002;36:1053-1060.)

In vivo regulation of inducible NO ssynthase in immune-mediated liver injury in mice
Kerstin Koerber, Gabriele Sass, Alexandra K. Kiemer, Angelika M. Vollmar, Gisa Tiegs
Inducible nitric oxide synthase (iNOS) has been shown to play an important role in the development of liver injury. iNOS deficiency protects mice from hemorrhage/resuscitation as well as from cytokine-mediated liver injury, for example, after administration of concanavalin A (con A). Here we investigated the in vivo effects of tumor necrosis factor (TNF)- and/or interferon (IFN)-, two mediators of con A­induced liver injury, the TNF receptor (TNFR) usage leading to iNOS expression, and its connection with nuclear factor B (NF-B) activation. In conclusion, iNOS expression in vivo is dependent on both TNF- and IFN-. Although con A­induced liver injury depends on both TNFR1 and TNFR2, TNF-dependent iNOS expression is mediated exclusively by TNFR1 and requires NF-B activation. (HEPATOLOGY 2002;36:1061-1069.)

Terlipressin inhibits in vivo aortic iNOS expression induced by lipopolysaccharide in rats with biliary cirrhosis
Richard Moreau, Eric Barrière, Khalid A. Tazi, Bernard Lardeux, Delphine Dargère, Waldemar Urbanowicz, Odile Poirel, Laurence Chauvelot-Moachon, Marie-Christine Guimont, Dominique Bernuau, Didier Lebrec
In cirrhosis, lipopolysaccharide (LPS, a product of Gram-negative bacteria) in the blood may cause septic shock. LPS-elicited induction of arterial inducible nitric oxide synthase (iNOS) results in nitric oxide (NO)-induced vasodilation, which causes arterial hypotension and hyporeactivity to 1-adrenergic constrictors. In vitro studies have suggested that vasopressin inhibits iNOS expression in cultured vascular smooth muscle cells exposed to LPS. Thus, the aim of this study was to investigate the effects of terlipressin administration (a vasopressin analog) on in vivo LPS-induced aortic iNOS in rats with cirrhosis. LPS (1 mg/kg, intravenously) was administered followed by the intravenous administration of terlipressin (0.05 mg/kg, intravenously) or placebo 1 hour later. Arterial pressure was measured, and contractions to phenylephrine (an 1-adrenoceptor agonist), iNOS activity, and iNOS expressions (mRNA and protein) were investigated in isolated aortas. LPS-induced arterial hypotension and aortic hyporeactivity to phenylephrine were abolished in rats that received terlipressin. LPS-induced aortic iNOS activity and expression were suppressed in terlipressin-treated rats. In conclusion, in LPS-challenged rats with cirrhosis, terlipressin administration inhibits in vivo LPS-induced aortic iNOS expression. Terlipressin administration may be a novel approach for the treatment of arterial hypotension and hyporeactivity to 1-adrenergic constrictors in patients with cirrhosis and septic shock. (HEPATOLOGY 2002;36:1070-1078.)

PI3K-FRAP/mTOR pathway is critical for hepatocyte proliferation whereas MEK/ERK supports both proliferation and survival
Alexandre Coutant, Claude Rescan, David Gilot, Pascal Loyer, Christiane Guguen-Guillouzo, Georges Baffet
Growth factors are known to favor both proliferation and survival of hepatocytes. In this work, we investigated the role of 2 main signaling pathways, phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MEK)/extracellular signal­regulated kinase (ERK), in these processes. First, evidence was provided that the PI3K cascade as well as the MEK/ERK cascade is a key transduction pathway controlling hepatocyte proliferation, as ascertained by arrest of DNA synthesis in the presence of LY294002, a specific PI3K inhibitor. Inhibition of FRAP/mTOR by rapamycin also abrogated DNA replication and protein synthesis induced by growth factor. We showed that expression of cyclin D1 at messenger RNA (mRNA) and protein levels was regulated by this pathway. We highlighted that 4E-BP1 phosphorylation was not activated by epidermal growth factor (EGF) but was under an insulin-regulation mechanism through a PI3K-FRAP/mTOR activation that could account for the permissive role of insulin on hepatocyte proliferation. No interference between the MEK/ERK pathway and 4E-BP1 phosphorylation was detected, whereas p70S6K phosphorylation induced by EGF was under a U0126-sensitive regulation. Last, we established that the antiapoptotic function of EGF was dependent on MEK, whereas LY294002 and rapamycin had no direct effect on cell survival. Taken together, these data highlight the regulation and the role of 2 pathways that mediate growth-related response by acting onto distinct steps. In conclusion, hepatocyte progression in late G1 phase induced by EGF generates survival signals depending on MEK activation, whereas PI3K and MEK/ERK cascades are both necessary for hepatocyte replication. (HEPATOLOGY 2002;36:1079-1088.)

Insulin-like growth factor I is a comitogen for hepatocyte growth factor in a rat model of hepatocellular carcinoma
Julie A. Price, Stephen J. Kovach, Timothy Johnson, Leonidas G. Koniaris, Paul A. Cahill, James V. Sitzmann, Iain H. McKillop
Hepatocyte growth factor­scatter factor (HGF-SF) is a potent hepatic mitogen yet inhibits hepatocellular carcinoma (HCC) cell growth in vitro. Insulin-like growth factor I (IGF-I) is a pleiotropic growth factor shown to be important in cell growth and differentiation in other tumors. We hypothesized that IGF-I may play a role in regulating HGF-SF activity and HCC progression. Using an in vivo model of HCC, we showed elevated IGF-I messenger RNA (mRNA) expression in normal liver from tumor-burdened animals in the absence of changes in circulating IGF-I levels. Analysis of IGF-I receptor (IGF-IR) and HGF-SF (c-met) receptor expression showed significantly higher expression of both receptors in normal liver compared with an HCC specimen. Using cultured HCC cells from this model, we next showed that treatment with IGF-I led to significant increases in mitogen-activated protein kinase (MAPK) activity. Furthermore, we observed significant time-dependent increases in the expression of the c-fos and c-jun proto-oncogenes after addition of IGF-I (n = 5 per group, P < .05). Despite activation of a MAPK pathway and increased proto-oncogene expression, IGF-I failed to significantly affect cell mitogenesis. In contrast, HGF significantly inhibited cell mitogenesis in HCC lines (68.4% ± 9.4% vs. control, n = 4, P < .05). Pretreatment of HCC cells with IGF-I (60 minutes) led to significant HGF-SF stimulation of total cell mitogenesis dependent on both IGF-I and HGF-SF dose (194% ± 8% increase vs. control, n = 4, P < .05). In conclusion, tumor burden is important in altering intrahepatic growth factor synthesis. Signal cooperation between multiple cytokine pathways is an important factor in the progression of HCC. (HEPATOLOGY 2002;36:1089-1097.)

Loss of cyclin D1 does not inhibit the proliferative response of mouse liver to mitogenic stimuli
Giovanna M. Ledda-Columbano, Monica Pibiri, Danilo Concas, Costanza Cossu, Marco Tripodi, Amedeo Columbano
Cyclin D1 is considered to play a critical role in the progression from G1 to S phase of the cell cycle, and its overexpression is seen in many human tumors. However, previous studies in cell lines have shown that cyclin D1 is not sufficient to trigger cell replication. To directly test the role of cyclin D1 in the progression of the cell cycle, we have examined the proliferative response of hepatocytes to the hepatomitogen 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) in mice with homozygous disruption of the cyclin D1 gene. We found that 24 hours after administration of TCPOBOP, the number of bromodeoxyuridine (BrdU)-positive hepatocytes was significantly reduced in cyclin D1­/­ (labeling index was 1.9% in knockout mice vs. 9.7% of heterozygous mice); however, no difference in the number of proliferating hepatocytes was found 36 or 72 hours after treatment (labeling index was 16% and 43% in cyclin D1­/­ mice vs. 20% and 41% of heterozygous mice), indicating that lack of cyclin D1 may transiently delay entry into S phase but is not sufficient to inhibit the response of hepatocytes to mitogenic stimuli. The results also show that although there was no difference in hepatic protein levels of cyclin D2 and D3 between untreated cyclin D1­/­ and cyclin D1+/­ mice, messenger RNA (mRNA) and protein levels of cyclin E were much higher in the former. In conclusion, our results show that cyclin D1 is not essential for liver development and hepatocyte proliferation induced by mitogenic stimuli and suggest that overexpression of cyclin E may compensate for the lack of cyclin D1. (HEPATOLOGY 2002;36:1098-1105.)

Specific systemic nonviral gene delivery to human hepatocellular carcinoma xenografts in SCID mice
Markus F. Wolschek, Christiane Thallinger, Malgorzata Kursa, Vanessa Rössler, Matthew Allen, Cornelia Lichtenberger, Ralf Kircheis, Trevor Lucas, Martin Willheim, Walter Reinisch, Alfred Gangl, Ernst Wagner, Burkhard Jansen
Systemic tumor-targeted gene delivery is attracting increasing attention as a promising alternative to conventional therapeutical strategies. To be considered as a viable option, however, the respective transgene has to be administered with high tumor specificity. Here, we describe novel polyethylenimine (PEI)-based DNA complexes, shielded by covalent attachment of polyethylene glycol (PEG), that make use of epidermal growth factor (EGF) as a ligand for targeting gene delivery to EGF receptor-expressing human hepatocellular carcinoma (HCC) cells. In vitro transfection of luciferase reporter DNA resulted in high levels of gene expression in the human HCC cell lines Huh-7 and HepG2. An excess of free EGF during transfection clearly reduced expression levels, indicating a specific EGF receptor-mediated uptake of the DNA particles. Following intravenous injection into human HCC xenograft-bearing SCID mice, luciferase expression was predominantly found in the tumor, with levels up to 2 logs higher than in the liver, which was the highest expressing major organ. Histologic investigation showed reporter gene expression (-galactosidase) localized to tumor cells. Assessing DNA distribution within the tumor by immunofluorescence microscopy, rhodamine-labelled transgene DNA was found to be mainly associated with HCC cells. In the liver, DNA was taken up almost exclusively by Kupffer cells and, as indicated by the low expression, subsequently degraded. In conclusion, we have shown that intravenous injection of PEGylated EGF-containing DNA/PEI complexes allows for highly specific expression of a transgene in human HCC tumors. (HEPATOLOGY 2002;36:1106-1114.)

Synergistic induction of apoptosis by acyclic retinoid and interferon- in human hepatocellular carcinoma cells (*Human Study*)
Akihiro Obora, Yoshimune Shiratori, Masataka Okuno, Seiji Adachi, Yukihiko Takano, Rie Matsushima-Nishiwaki, Ichiro Yasuda, Yasuhiro Yamada, Kuniharu Akita, Tetsuro Sano, Jun Shimada, Soichi Kojima, Yukio Okano, Scott L. Friedman, Hisataka Moriwaki
Acyclic retinoid, a synthetic retinoid analog, as well as interferon alfa (IFN-) and IFN- induce apoptosis in hepatocellular carcinoma (HCC) cells and are used clinically in the prevention of HCC. Here, we show that acyclic retinoid acts synergistically with IFNs in suppressing the growth and inducing apoptosis (as characterized by DNA fragmentation and chromatin condensation) in 5 human HCC cell lines (JHH7, HuH7, PLC/PRF/5, HLE, and HLF). This synergism was only observed when cells were pretreated with the acyclic retinoid, whereas natural retinoic acids (all-trans and 9-cis retinoic acid) were ineffective. This promotion may be due to up-regulation of type 1 IFN receptor (IFNR) expression by the retinoid. Accordingly, incubation with antitype 1 IFNR antibody abolished the synergy. Enhanced IFNR expression was accompanied by increased expression and DNA-binding activity of STAT1, an intracellular signal transducing molecule of IFNR, and increased induction of 2', 5'-oligoadenyl-5'-triphosphate synthetase, which is a target gene of STAT1. Acyclic retinoid did not have any effects on the growth of normal human hepatocytes (Hc) probably because of a lack of IFNR and STAT1 up-regulation. In conclusion, these results provide a rationale for combined biochemoprevention of HCC using acyclic retinoid and IFN-. (HEPATOLOGY 2002;36:1115-1124.)

Comprehensive mapping of HLA-A*0201­restricted CD8 T-cell epitopes on PDC-E2 in primary biliary cirrhosis (*Human Study*)
Shuji Matsumura, Hiroto Kita, Xiao-Song He, Aftab A. Ansari, Zhe-Xiong Lian, Judy van de Water, Kazuhide Yamamoto, Takao Tsuji, Ross L. Coppel, Marshall Kaplan, M. Eric Gershwin
Growing evidence has implicated the involvement of autoreactive T lymphocytes in the pathogenesis of primary biliary cirrhosis (PBC). We have recently taken advantage of motif prediction analysis of HLA-A*0201 and identified the first major histocompatibility complex (MHC) class I restricted epitope, amino acids 159 to 167 on E2 components of pyruvate dehydrogenase complexes (PDC-E2), the major mitochondrial antigens in PBC. The mechanisms involved in the selection of epitope peptide(s) that comprise the PDC-E2­specific autoreactive cytotoxic T lymphocytes (CTLs) are unknown and likely involve other epitopes on PDC-E2 restricted by MHC class I molecules. To address this issue, a comprehensive mapping of the CTL epitope repertoire on the PDC-E2 molecule that binds HLA-A*0201 was performed to provide further clues regarding the role of CTLs. We used the T2 cell line to screen 79 overlapping 15mer peptides, spanning the entire PDC-E2 molecule. Six of the 79 peptides exhibited significantly higher binding activity to HLA-A*0201 than the other 15mer peptides. Two of these 6 peptides induced CTL lines from patients with PBC. Fine mapping with N-terminus or C-terminus truncated peptides identified 10mer peptide, PDC-E2 amino acids 165 to 174, which is a novel CD8 epitope restricted by HLA-A*0201. In conclusion, using a combination of the 15mer peptide library screening with the T2 binding assay and also the induction of CTL lines with candidate peptides, we have defined a novel HLA-A*0201­restricted epitope PDC-E2 165 to 174 in patients with PBC. These data will become important in the development of altered peptide ligands to modulate disease activity. (HEPATOLOGY 2002;36:1125-1134.)

The ecto-nucleoside triphosphate diphosphohydrolase NTPDase2/CD39L1 is expressed in a novel functional compartment within the liver
Jonathan A. Dranoff, Emma A. Kruglov, Simon C. Robson, Norbert Braun, Herbert Zimmermann, Jean Sévigny
Extracellular nucleotides regulate diverse biological functions and are important in the regulation of liver metabolism, hepatic blood flow, and bile secretion. Ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) hydrolyze extracellular nucleotides and are therefore potential regulators of nucleotide-mediated signaling. To examine this, we have contrasted the structural and functional distributions of the 2 characterized membrane-bound NTPDases NTPDase1 and NTPDase2 within the rat liver. Hepatic expression of NTPDase2 was determined and contrasted to NTPDase1 using confocal immunofluorescence, immunoelectron microscopy, reverse-transcription polymerase chain reaction, Northern blot analysis, Western blot analysis, and functional assays. NTPDase2 was expressed in the periportal region surrounding intrahepatic bile ducts, whereas NTPDase1 was found in hepatic arteries, portal veins, and hepatic central veins, consistent with its known vascular distribution. Functional and molecular expression of NTPDase2 was shown in portal fibroblasts near basolateral membranes of bile duct epithelia. In conclusion, NTPDase2 is expressed in a novel cellular compartment surrounding intrahepatic bile ducts, namely portal fibroblasts. This distribution may represent a previously unrecognized mechanism for regulation of nucleotide signaling in bile ducts and other epithelia. (HEPATOLOGY 2002;36:1135-1144.)

Lith genes control mucin accumulation, cholesterol crystallization, and gallstone formation in A/J and AKR/J inbred mice
Frank Lammert, David Q.-H. Wang, Henning Wittenburg, Guylaine Bouchard, Sonja Hillebrandt, Bärbel Taenzler, Martin C. Carey, Beverly Paigen
We recently identified 2 Lith genes that determine cholesterol gallstone formation in C57L/J inbred mice, which show a gallstone prevalence of ~80% on feeding 1.0% cholesterol and 0.5% cholic acid. The aim of this study was to explore if the same Lith loci contribute to the variation in gallstone susceptibility in a new experimental cross. After 12 weeks of feeding the lithogenic diet to inbred mice of strains A/J and AKR/J as well as their F1 progeny, we used microscopy of bile to assess mucin accumulation, crystallization pathways, and stone formation. Backcross progeny (n = 225) were phenotyped and genotyped selectively for microsatellite markers spanning the genome. Quantitative trait loci (QTL) affecting gallstone phenotypes were identified by linkage analysis. Both inbred strains showed accumulation of mucin gel and cholesterol supersaturation. However, only strain AKR developed gallstones (prevalence of 20%), whereas strain A showed a stable liquid crystalline state and no stones. QTL analysis identified a gallstone locus on chromosome 17 (Lith3). A second gene locus on chromosome 15 that controls mucin accumulation harbors the mucin gene Glycam1, which was shown to be expressed in gallbladder epithelia by immunohistochemistry. Gallstone and mucin loci colocalized with potential QTLs affecting the formation of cholesterol crystals. In conclusion, QTL analysis identified specific gene loci determining mucin accumulation, cholesterol crystallization, and gallstone formation. Characterization of the pathophysiologic roles of Lith3 and the new biliary mucin gene Glycam1 might provide insights into primary defects of human cholelithiasis and lead to new therapeutic strategies for prestone intervention. (HEPATOLOGY 2002;36:1145-1154.)

Liver Failure and Liver Disease

Region selective alterations of soluble guanylate cyclase content and modulation in brain of cirrhotic patients (*Human Study*)
Regina Corbalán, Nicolas Chatauret, Sönke Behrends, Roger F. Butterworth, Vicente Felipo
Modulation of soluble guanylate cyclase (sGC) by nitric oxide (NO) is altered in brain from experimental animals with hyperammonemia with or without liver failure. The aim of this work was to assess the content and modulation of sGC in brain in chronic liver failure in humans. Expression of the -1, -2, and -1 subunits of sGC was measured by immunoblotting in autopsied frontal cortex and cerebellum from cirrhotic patients and controls. The contents of -1 and -2 subunits of guanylate cyclase was increased both in cortex and cerebellum, whereas the -1 subunit was not affected. Addition of the NO-generating agent S-nitroso-N-acetyl-penicillamine (SNAP) to homogenates of frontal cortex from controls increased the activity of sGC 87-fold, whereas, in homogenates from cirrhotic patients, the increase was significantly higher (183-fold). In contrast, in cerebellum, activation of guanylate cyclase by NO was significantly lower in patients (156-fold) than in controls (248-fold). A similar regional difference was found in rats with portacaval anastomosis. In conclusion, these findings show that the NO-guanylate cyclase signal transduction pathway is strongly altered in brain in patients with chronic liver failure and that the effects are different in different brain areas. Given that activation of sGC by NO in brain is involved in the modulation of important cerebral processes such as intercellular communication, learning and memory, and the sleep-wake cycle, these changes could be implicated in the pathogenesis of hepatic encephalopathy in these patients. (HEPATOLOGY 2002;36:1155-1162.)

Interorgan ammonia and amino acid metabolism in metabolically stable patients with cirrhosis and a TIPSS (*Human Study*)
Steven W. M. Olde Damink, Rajiv Jalan, Doris N. Redhead, Peter C. Hayes, Nicolaas E. P. Deutz, Peter B. Soeters
Ammonia is central to the pathogenesis of hepatic encephalopathy. This study was designed to determine the quantitative dynamics of ammonia metabolism in patients with cirrhosis and previous treatment with a transjugular intrahepatic portosystemic stent shunt (TIPSS). We studied 24 patients with cirrhosis who underwent TIPSS portography. Blood was sampled and blood flows were measured across portal drained viscera, leg, kidney, and liver, and arteriovenous differences across the spleen and the inferior and superior mesenteric veins. The highest amount of ammonia was produced by the portal drained viscera. The kidneys also produced ammonia in amounts that equaled total hepatosplanchnic area production. Skeletal muscle removed more ammonia than the cirrhotic liver. The amount of nitrogen that was taken up by muscle in the form of ammonia was less than the glutamine that was released. The portal drained viscera consumed glutamine and produced ammonia, alanine, and citrulline. Urea was released in the splenic and superior mesenteric vein, contributing to whole-body ureagenesis in these cirrhotic patients. In conclusion, hyperammonemia in metabolically stable, overnight-fasted patients with cirrhosis of the liver and a TIPSS results from portosystemic shunting and renal ammonia production. Skeletal muscle removes more ammonia from the circulation than the cirrhotic liver. Muscle releases excessive amounts of the nontoxic nitrogen carrier glutamine, which can lead to ammonia production in the portal drained viscera (PDV) and kidneys. Urinary ammonia excretion and urea synthesis appear to be the only way to remove ammonia from the body. (HEPATOLOGY 2002;36:1163-1171.)

Hypoxia is an inducer of vasodilator agents in peritoneal macrophages of cirrhotic patients (*Human Study*)
Pilar Cejudo-Martín, Manuel Morales-Ruiz, Josefa Ros, Miguel Navasa, Guillermo Fernández-Varo, Josep Fuster, Francisca Rivera, Vicente Arroyo, Juan Rodés, Wladimiro Jiménez
The aim of the investigation was to assess whether hypoxia induces the production of endogenous vasoactive peptides in macrophages of cirrhotic patients with ascites because low tissue oxygenation is a relatively frequent event in these patients. Peritoneal macrophages were isolated from ascites, seeded on well plates, and cultured at different times under hypoxic (5% O2) or normoxic conditions (21% O2). Then, accumulation of vasoactive peptides sensitive to hypoxia including endothelin-1 (ET-1), vascular endothelial growth factor (VEGF), and adrenomedullin (ADM) was measured. Only VEGF and ADM were constitutively secreted, and hypoxia further stimulated the release of these vasodilator peptides. In concordance, increased messenger RNA (mRNA) levels of VEGF and ADM were found at culturing macrophages in hypoxia. This characteristic response was not observed in circulating monocytes of either cirrhotic patients or healthy subjects. Next the expression of the transcription factor, hypoxia inducible factor 1 (HIF-1), was analyzed. Expression of HIF-1 and HIF-1 messengers and HIF-1 protein subunit remained unchanged regardless of O2 tension, whereas HIF-1 protein subunit was overexpressed under hypoxic conditions. Moreover, conditioned medium from macrophages cultured under hypoxic conditions promoted a larger nitric oxide (NO) release in endothelial cells than that of normoxic macrophages. In conclusion, these data indicate that hypoxia induces the synthesis of VEGF and ADM in macrophages of cirrhotic patients, likely through HIF-1­enhanced transcriptional activity. These data suggest that a local reduction in O2 tension could enhance the synthesis of macrophage-derived vasodilators, thus aggravating the circulatory disturbance of these patients. (HEPATOLOGY 2002;36:1172-1179.)

Quantitative evaluation of altered hepatic spaces and membrane transport in fibrotic rat liver
Daniel Y. Hung, Ping Chang, Kee Cheung, Clay Winterford, Michael S. Roberts
Four animal models were used to quantitatively evaluate hepatic alterations in this study: (1) a carbon tetrachloride control group (phenobarbital treatment only), (2) a CCl4-treated group (phenobarbital with CCl4 treatment), (3) an alcohol-treated group (liquid diet with alcohol treatment), and (4) a pair-fed alcohol control group (liquid diet only). At the end of induction, single-pass perfused livers were used to conduct multiple indicator dilution (MID) studies. Hepatic spaces (vascular space, extravascular albumin space, extravascular sucrose space, and cellular distribution volume) and water hepatocyte permeability/surface area product were estimated from nonlinear regression of outflow concentration versus time profile data. The hepatic extraction ratio of 3H-taurocholate was determined by the nonparametric moments method. Livers were then dissected for histopathologic analyses (e.g., fibrosis index, number of fenestrae). In these 4 models, CCl4-treated rats were found to have the smallest vascular space, extravascular albumin space, 3H-taurocholate extraction, and water hepatocyte permeability/surface area product but the largest extravascular sucrose space and cellular distribution volume. In addition, a linear relationship was found to exist between histopathologic analyses (fibrosis index or number of fenestrae) and hepatic spaces. The hepatic extraction ratio of 3H-taurocholate and water hepatocyte permeability/surface area product also correlated to the severity of fibrosis as defined by the fibrosis index. In conclusion, the multiple indicator dilution data obtained from the in situ perfused rat liver can be directly related to histopathologic analyses. (HEPATOLOGY 2002;36:1180-1189.)

Remethylation and transsulfuration of methionine in cirrhosis: Studies with L-[2H3-methyl-1-13C]methionine (*Human Study*)
Stefan Russmann, Edith Junker, Bernhard H. Lauterburg
Disturbances of the methionine cycle may result in liver injury. Patients with alcohol-induced liver disease often exhibit hypermethioninemia and a delayed clearance (CL) of methionine, but the extent to which transsulfuration and remethylation pathways of the cyclic methionine metabolism are affected is unknown. Methionine turnover was determined in 7 healthy volunteers and 6 patients with alcohol-induced cirrhosis after oral administration of 2 mg/kg [2H3-methyl-1-13C]methionine, which permitted us to follow transsulfuration by its decarboxylation to 13CO2 and remethylation by replacement of the labeled methyl group by an unlabeled one. Basal plasma concentrations of endogenous methionine (50 ± 5 vs. 25 ± 2 µmol/L, mean ± SEM, P < .001) were significantly higher in patients with cirrhosis and its CL was significantly decreased (774 ± 103 vs. 2,050 ± 141 mL/min, P < .001). Methionine turnover amounted to 42 ± 4 vs. 27 ± 3 µmol/kg/h (P < .05) in controls and patients with cirrhosis, respectively. The fraction of administered methionine undergoing remethylation was lower in patients with cirrhosis (7.6 ± 1.5 vs. 14.1 ± 1.1%, P < .005). However, because of the larger pool of circulating methionine, the total flux of methionine through the remethylation pathway was similar in both groups. A significantly lower fraction of the administered dose appeared in the form of 13CO2 in breath in patients with cirrhosis (2.2 ± 0.4 vs. 11.0 ± 0.8%, P < .001). In conclusion, the data indicate that the liver with cirrhosis compensates for a decreased activity of remethylating enzymes by operating at higher concentrations of methionine. In contrast, transsulfuration is impaired in patients with alcohol-induced cirrhosis such that an assessment of transsulfuration by a simple breath test may provide a clinically useful estimate of hepatic function. (HEPATOLOGY 2002;36:1190-1196.)

Pharmacodynamic effects of a nonpeptide antidiuretic hormone V2 antagonist in cirrhotic patients with ascites (*Human Study*)
Dominique Guyader, Alain Patat, Evelyn J. Ellis-Grosse, Gayle P. Orczyk
Water retention and dilutional hyponatremia, mainly attributable to an impairment of free water excretion and increased vasopressin activity, are well-documented complications in cirrhotic patients with ascites. VPA-985 is a selective, nonpeptide, orally active, vasopressin-2­receptor antagonist. The aim of this study was to determine the pharmacodynamics, safety, and pharmacokinetics of ascending single doses (25, 50, 100, 200, and 300 mg) in cirrhotic patients with ascites in a randomized, double-blind, placebo-controlled trial. Each dose level was studied in 5 patients (4 active and 1 placebo). After an overnight fast and fluid restriction (continued for 4 hours after dose administration), all patients were given placebo on baseline day and an oral suspension of VPA or placebo on the following day. VPA produced a significant dose-related increase in daily urine output (1,454 ± 858 mL to 4,568 ± 4,385 mL with VPA 300 mg) and a dose-related decrease in urine osmolality. The free water clearance reached greater than 3 mL/min for doses 100 mg or greater. Simultaneously, significant increases in serum osmolality, sodium, and vasopressin levels were found. There was a significant increase in sodium urine excretion. VPA was rapidly absorbed and maximum serum concentrations were achieved within 1 hour after administration. Elimination half-life ranged from 9.0 hours after 100 mg to 22.6 hours after 200 mg. In conclusion, VPA induced a dose-related aquaretic response, suggesting a therapeutic potential in managing water retention in patients with liver cirrhosis with ascites. (HEPATOLOGY 2002;36:1197-1205.)

Risk factors for hepatocellular carcinoma: Synergism of alcohol with viral hepatitis and diabetes mellitus (*Human Study*)
Manal M. Hassan, Lu-Yu Hwang, Chiq J. Hatten, Mark Swaim, Donghui Li, James L. Abbruzzese, Palmer Beasley, Yehuda Z. Patt
Risk factors associated with hepatocellular carcinoma (HCC) are well documented, but the synergisms between these risk factors are not well examined. We conducted a hospital-based, case-control study among 115 HCC patients and 230 non­liver cancer controls. Cases and controls were pathologically diagnosed at The University of Texas M. D. Anderson Cancer Center and were matched by 5-year age groups, sex, and year of diagnosis. Information on risk factors was collected by personal interview and medical records review. Blood samples were tested for the presence of antibodies to hepatitis C virus antigen (anti-HCV), hepatitis B surface antigen (HBsAg), and antibodies to hepatitis B core antigen (anti-HBc). Conditional logistic regression was used to determine odds ratios (ORs) by the maximum likelihood method. Multivariate ORs and 95% confidence intervals (CIs) were 15.3 (4.3-54.4), 12.6 (2.5-63.1), 4.5 (1.4-14.8), and 4.3 (1.9-9.9) for anti-HCV, HBsAg, heavy alcohol consumption (80 mL ethanol/d), and diabetes mellitus, respectively. Synergistic interactions on the additive model were observed between heavy alcohol consumption and chronic hepatitis virus infection (OR, 53.9; 95% CI, 7.0-415.7) and diabetes mellitus (OR, 9.9; 95% CI, 2.5-39.3). Independent of the effect of HCV, HBV, and diabetes mellitus, heavy alcohol consumption contributes to the majority of HCC cases (32%), whereas 22%, 16%, and 20% were explained by HCV, HBV, and diabetes mellitus, respectively. In conclusion, the significant synergy between heavy alcohol consumption, hepatitis virus infection, and diabetes mellitus may suggest a common pathway for hepatocarcinogenesis. Exploring the underlying mechanisms for such synergisms may indicate new HCC prevention strategies in high-risk individuals. (HEPATOLOGY 2002;36:1206-1213.)

Dominant role of hepatitis B virus and cofactor role of aflatoxin in hepatocarcinogenesis in Qidong, China (*Human Study*)
Lihua Ming, Snorri S. Thorgeirsson, Mitchell H. Gail, Peixin Lu, Curtis C. Harris, Nengjin Wang, Yongfu Shao, Zhiyuan Wu, Guoting Liu, Xiaohong Wang, Zongtang Sun
We assessed the separate and combined effects of hepatitis B virus (HBV), hepatitis C virus (HCV), and aflatoxin in causing hepatocellular carcinoma (HCC) in Qidong, China. A consecutive series of 181 pathologic-diagnosed HCC cases were studied for hepatitis B surface antigen (HBsAg), anti-HBc, HBV X gene sequence, anti-HCV, the 249ser-p53 mutation, and chronic hepatitis pathology. Each of the 181 incident HCC cases had markers for HBV infection and hepatitis pathology; only 6 of 119 cases were coinfected with HCV. The 249ser-p53 mutation was found in 54% (97/181) of HCC cases and in all 7 cases with tissue for analysis from the hepatitis cohort but in none of 42 matched cases from Beijing. The estimated cumulative dose of aflatoxin B1 in these 7 cases ranged from 0.13 to 0.49 mg/kg. Follow-up data through 13.25 years on a cohort of 145 men with chronic HBV hepatitis showed that the relative risk from aflatoxin exposure was 3.5 (1.5-8.1). A similar relative risk was found using 249ser-p53 mutation as a marker for aflatoxin exposure. In conclusion, HBV hepatitis is ubiquitous in Qidong HCC cases, whereas HCV contributes little to its risk. The 249ser-p53 mutation appears to result from coexposure to aflatoxin and HBV infection. Even modest levels of aflatoxin exposure tripled the risk of HCC in HBV-infected men. (HEPATOLOGY 2002;36:1214-1220.)

High-dose tamoxifen in the treatment of inoperable hepatocellular carcinoma: A multicenter randomized controlled trial (*Human Study*)
Pierce K. H. Chow, Bee-Choo Tai, Chee-Kiat Tan, David Machin, Khin Maung Win, Phillip J. Johnson, Khee-Chee Soo, for the Asia-Pacific Hepatocellular Carcinoma Trials Group
In the Asia-Pacific region and elsewhere, almost 85% of patients with hepatocellular carcinoma (HCC) are inoperable at diagnosis and have a dismal prognosis. Tamoxifen (TMX) is believed to inhibit HCC positive for estrogen receptor (ER), but most HCCs are ER negative. Results of previous phase 3 trials in inoperable HCC have been conflicting and inconclusive. At higher doses, however, TMX inhibits HCC through ER-independent mechanisms. A multicenter randomized controlled trial was performed to assess the role of high-dose TMX versus placebo (P) in the treatment of patients with inoperable HCC with respect to survival and quality of life (QoL). A total of 329 patients from 10 centers in 9 countries in the Asia-Pacific region enrolled in a double-blind randomized controlled trial of TMX 120 mg/d (TMX120) against P as a control arm with an intermediate dosage of TMX 60 mg/d (TMX60) to assess possible dose response. An independent data monitoring committee reviewed all aspects of the trial. QoL was assessed using the European Organization for Research and Treatment of Cancer QLQ-C30 questionnaire. Three-month survival rates for the P, TMX60, and TMX120 groups were 44%, 41%, and 35%, respectively, with a statistically significant trend difference in survival across the 3 treatment regimens (P = .011). There was a significantly higher risk of death in the TMX120 group compared with the P group (hazard ratio, 1.39; 95% confidence interval, 1.07-1.81). Adverse drug reactions were reported in 3% (9 patients), and 8 patients were lost to follow-up. In conclusion, TMX does not prolong survival in patients with inoperable HCC and has an increasingly negative impact with increasing dose. No appreciable advantage to QoL with TMX was observed. (HEPATOLOGY 2002;36:1221-1226.)

Differential expression of intestinal trefoil factor in biliary epithelial cells of primary biliary cirrhosis (*Human Study*)
Yasuhiko Kimura, Patrick S. C. Leung, Thomas P. Kenny, Judy Van De Water, Mikio Nishioka, Andrew S. Giraud, James Neuberger, Gordon Benson, Rashmi Kaul, Aftab A. Ansari, Ross L. Coppel, M. Eric Gershwin
Intestinal trefoil factor (ITF) promotes epithelial cell migration and mucosal restitution during inflammation. We used real-time quantitative PCR, in situ nucleic acid hybridization, and immunohistochemistry to study the expression of the ITF gene and protein expression in the liver of primary biliary cirrhosis (PBC) and controls. There were significantly higher levels of ITF messenger RNA (mRNA) in PBC liver compared with primary sclerosing cholangitis (PSC) (P < .05) or normal controls (P < .001) and also higher in hepatitis C virus (HCV) liver (P < .05) and cryptogenic cirrhosis (P < .01) compared with normal controls. However, only in PBC was there a significant difference between small (interlobular and bile ductules) and large (intrahepatic and septal) bile ducts. Using in situ hybridization, the highest levels of ITF gene expression were localized to the large bile ducts in PBC. This differential expression of ITF was also noted at the protein level. Thus, in PBC, although 92% of large bile ducts expressed the ITF protein, only 2% of small bile ducts (P < .0001) expressed ITF. In contrast, in control livers, 34% of large bile ducts and 13% of small bile ducts expressed ITF. ITF protein is absent in small bile ducts in all stages of PBC. In conclusion, the expression of ITF may play an important role in bile duct damage. In small bile ducts, ITF production in response to damage is absent, making such cells vulnerable to damage and providing a thesis for the selective loss of small, but not large, bile ducts in PBC. (HEPATOLOGY 2002;36:1227-1235.)

Trafficking and functional defects by mutations of the ATP-binding domains in MRP2 in patients with Dubin-Johnson syndrome (*Human Study*)
Kenkichi Hashimoto, Takeshi Uchiumi, Toshikazu Konno, Takuya Ebihara, Takanori Nakamura, Morimasa Wada, Shotaro Sakisaka, Fumio Maniwa, Teruo Amachi, Kazumitsu Ueda, Michihiko Kuwano
Dubin-Johnson syndrome (DJS) is a hereditary disease characterized by hyperbilirubinemia. We investigated the consequences of 2 missense mutations, R768W and Q1382R, of nucleotide-binding domains (NBDs) of the multidrug resistance protein 2 (MRP2; ABCC2) that were previously identified in patients with DJS. Pulse chase analysis revealed that the precursor form of the wild-type and Q1382R MRP2 were converted to the mature form, which is resistant to endoglycosidase H (Endo H) in about 60 minutes. However, the precursor form of the R768W MRP2, which is sensitive to endoglycosidase H, was degraded within 120 minutes and did not mature to the fully glycosylated form. Proteasome inhibitors inhibited the degradation of the precursor form of the R768W MRP2. Unlike the R768W MRP2, the Q1382R MRP2 was mainly localized on the apical membrane in the wild-type form. However, efflux of glutathione monochlorobimane (GS-MCLB) and ATP-dependent leukotriene C4 (LTC4) uptake into plasma membrane vesicles from cells expressing the Q1382R MRP2 were markedly reduced, suggesting that the Q1382R MRP2 on the apical membrane was nonfunctional. Vanadate-induced nucleotide trapping with 8-azido-[-32P]ATP in the wild-type MRP2 was stimulated by estradiol glucuronide (E217G) in a concentration-dependent manner but that in the Q1382R MRP2 was not. In conclusion, the R768W mutation causes deficient maturation and impaired sorting, and the Q1382R mutation does not affect maturation or sorting but impairs the substrate-induced ATP hydrolysis. (HEPATOLOGY 2002;36:1236-1245.)

Viral Hepatitis

Preemptive lamivudine therapy based on HBV DNA level in HBsAg-positive kidney allograft recipients (*Human Study*)
Tak Mao Chan, Guo Xiang Fang, Colin S. O. Tang, Ignatius K. P. Cheng, Kar Neng Lai, Stephen K. N. Ho
Hepatitis B surface antigen (HBsAg)-positive kidney transplant recipients have increased liver-related mortality. The impact of lamivudine treatment on patient survival, the optimal time to start treatment, and the feasibility of discontinuing treatment have not been determined. This study examined these issues with a novel management protocol. Serum hepatitis B virus (HBV) DNA levels were measured serially in HBsAg-positive kidney transplant recipients, and lamivudine was administered preemptively to patients with increasing HBV DNA levels with or without elevation of aminotransferase levels. Outcomes of patients who underwent transplantation before or after institution of this preemptive management strategy (in January 1996) were compared. Eleven de novo patients (91.7%) who underwent transplantation between 1996 and 2000 and 15 existing patients (39.5%) who underwent transplantation between 1983 and 1995 received preemptive lamivudine therapy for 32.6 ± 13.3 months. The treatment criteria were met by de novo patients at 8.4 ± 6.2 months (range, 1-18 months) after transplantation. Suppression of HBV DNA and normalization of aminotransferase levels were achieved in all treated patients, and 21.4% had hepatitis B e antigen (HBeAg) seroconversion. The survival of preemptively managed de novo transplant patients was similar to that of HBsAg-negative controls, whereas HBsAg-positive patients who underwent transplantation before January 1996 had inferior survival (relative risk of death, 9.7 [P < .001]; relative risk of liver-related mortality, 68.0 [P < .0001]). Eleven patients (40.7%) developed lamivudine resistance. Discontinuation of lamivudine was attempted in 12 low-risk patients after stabilization and was successful in 5 (41.7%). In conclusion, preemptive lamivudine therapy based on serial HBV DNA levels and clinical monitoring improved the survival of HBsAg-positive renal allograft recipients. Treatment can be discontinued safely in selected patients after stabilization to minimize the selection of drug-resistant HBV mutants. (HEPATOLOGY 2002;36:1246-1252.)

A pilot study of interferon alfa and ribavirin combination in liver transplant recipients with recurrent hepatitis C (*Human Study*)
A. Obaid Shakil, Brendon McGuire, Jeff Crippin, Lewis Teperman, A. Jake Demetris, Hari Conjeevaram, Robert Gish, Paul Kwo, Vijayan Balan, Teresa L. Wright, Clifford Brass, Jorge Rakela
Although interferon alfa (IFN-) and ribavirin are widely used in the treatment of hepatitis C, their role in the transplant recipient is unclear. We conducted a pilot study to determine the efficacy and safety of this therapy in transplant recipients with recurrent hepatitis C. Patients at least 6 months posttransplantation were treated with IFN- 3 million units 3 times a week subcutaneously and ribavirin 800 mg daily by mouth for 48 weeks followed by ribavirin monotherapy for 24 weeks. The primary end point was sustained virologic response, and secondary end points included biochemical, virologic, and histologic responses at the end of combination treatment. Thirty-eight patients initiated therapy but 16 withdrew due to adverse effects, including 2 with myocardial infarction. Median age was 50 years; 74% were men, and 91% had genotype 1. The median interval between transplantation and enrollment was 23 months. On an intention-to-treat basis, 7 patients (18%) had a biochemical and 5 (13%) had a virologic response at the end of combination treatment. Inflammatory activity did not change, but fibrosis worsened in virologic nonresponders. Ribavirin maintenance caused a further decrease in serum alanine aminotransferase levels, but hepatitis C virus (HCV) RNA levels increased. Only 2 of the 38 patients (5%) had a sustained virologic response. Several patients required treatment with erythropoietin for anemia. In conclusion, IFN- and ribavirin are effective in a small proportion of liver allograft recipients with recurrent hepatitis C. Adverse effects occur commonly, requiring dose reductions and treatment withdrawal. (HEPATOLOGY 2002;36:1253-1258.)

Southeast Asian patients with chronic hepatitis C: The impact of novel genotypes and race on treatment outcome (*Human Study*)
Anouk T. Dev, Rhonda McCaw, Vijaya Sundararajan, Scott Bowden, William Sievert
Hepatitis C virus (HCV) genotype and other host and viral factors influence treatment outcome in chronic HCV infection. We evaluated the effect of race and genotype on interferon and ribavirin treatment outcome in 70 Southeast Asian (SEA) and 50 white patients. Genotype was based on the 5' untranslated region (5'UTR) with a commonly used line probe assay (INNO-LiPA HCV II) that may mistype genotype 7, 8, or 9 as 1b. HCV core region sequencing resulted in reclassification of 8 genotype 1 and 25 genotype 1b SEA subjects as genotype 7, 8, or 9. Twenty-six SEA genotype 7, 8, and 9 (79%) and 10 SEA true genotype 1b (59%) patients achieved a sustained virologic response (SVR) compared with 15 (34%) white genotype 1b patients. Logistic regression analysis showed that SEA patients with genotype 7, 8, or 9 were more likely to achieve a SVR than white genotype 1b patients (OR 16.56; 95%CI 4.16, 65.91) as were SEA true genotype 1b patients compared with white genotype 1b patients (OR 4.63; 95%CI 1.19, 18.04). In conclusion, a proportion of SEA patients classified by INNO-LiPA as genotype 1b were in reality genotype 7, 8, or 9. In comparison with white genotype 1b patients, both SEA genotype 1b and SEA genotype 7, 8, and 9 patients showed a significantly greater SVR. HCV core sequencing was necessary to determine genotype accurately in persons potentially exposed to HCV genotypes 7, 8, or 9. This study also supports the concept that race and ethnicity are important determinants of treatment outcome in HCV infected patients.(HEPATOLOGY 2002;36:1259-1265.)

Hepatitis C virus genotype 3 is cytopathic to hepatocytes: Reversal of hepatic steatosis after sustained therapeutic response (*Human Study*)
Dinesh Kumar, Geoffrey C. Farrell, Caroline Fung, Jacob George
On the basis of cross-sectional studies, it has been proposed that hepatic steatosis is a cytopathic effect of hepatitis C virus (HCV) genotype 3 but not genotype 1 infections. We tested this hypothesis by examining whether antiviral treatment altered hepatic steatosis in chronic hepatitis C. In 28 patients with genotype 1 and 34 with genotype 3 HCV, we determined the severity of steatosis in pre- and posttreatment liver biopsies using computer-assisted morphometric image analysis as well as conventional semiquantitative scoring. Before treatment, hepatic steatosis was present in 16 (57%) patients infected with HCV genotype 1 and 21 (62%) of those with genotype 3. Sustained viral response (SVR) was achieved in 9 (32%) patients with genotype 1 and 22 (65%) with genotype 3. In neither group were there significant changes in body weight or alcohol consumption between pre- and posttreatment biopsies. In patients with HCV genotype 1, there was no change in hepatic steatosis after treatment, irrespective of the treatment response. Among those infected with genotype 3, SVR significantly reduced steatosis (P < .001), but there was no change in steatosis among those without a SVR. By logistic regression analysis, SVR was the only variable predictive of improvement in hepatic steatosis (OR = 36, 95% CI = 2.7-481, P = .007). In conclusion, these data provide strong support for a direct causal association between HCV genotype 3 infection and hepatic steatosis. (HEPATOLOGY 2002;36:1266-1272.)

Neutropenia during combination therapy of interferon alfa and ribavirin for chronic hepatitis C (*Human Study*)
Alejandro Soza, James E. Everhart, Marc G. Ghany, Edward Doo, Theo Heller, Kittichai Promrat, Yoon Park, T. Jake Liang, Jay H. Hoofnagle
Interferon therapy of hepatitis C causes a decrease in neutrophil counts, and neutropenia is a common reason for dose adjustment or early discontinuation. However, it is unclear whether neutropenia caused by interferon is associated with an increased rate of infection. In this study, we assessed factors associated and clinical consequences of neutropenia before and during interferon therapy of chronic hepatitis C. A total of 119 patients with chronic hepatitis C treated with the combination of interferon alfa and ribavirin were analyzed. In these studies, neutropenia was not used as an exclusion or dose modification criterion. In multivariate analysis, only black race was associated with baseline neutropenia. During treatment, neutrophil counts decreased by an average of 34%. Among 3 blacks with baseline neutropenia without cirrhosis or splenomegaly, there was little or no decrease in neutrophil counts (despite typical decreases in platelet and lymphocyte counts). Documented or suspected bacterial infections developed in 22 patients (18%), but in no patient with neutropenia. United States population estimates suggest that 76,000 blacks with hepatitis C have neutrophil counts below 1,500 cells/µL and might be denied therapy if this exclusion criterion was generally applied. In conclusion, neutropenia is frequent during treatment of hepatitis C with interferon and ribavirin, but it is not usually associated with infection. Constitutional neutropenia, which is common among blacks, should not exclude patients from therapy with interferon as these patients usually have minimal further decreases in neutrophil counts on therapy and are not excessively prone to bacterial infections. (HEPATOLOGY 2002;36:1273-1279.)

Recombinant alfa-interferon plus ribavirin therapy in children and adolescents with chronic hepatitis C (*Human Study*)
Stefan Wirth, Thomas Lang, Stephan Gehring, Patrick Gerner
Treatment with alfa-interferon alone yielded poor results in children with chronic hepatitis C and was not generally recommended. Owing to limited experience with combination therapy in children, the aim of the study was to evaluate the efficacy and tolerability of alfa-interferon 2b in combination with ribavirin in these patients with different routes of viral transmission. In an uncontrolled pilot study, 41 children and adolescents ranging from 3 to 16 years were treated with alfa-interferon at a dose of 3 or 5 MU/m2 3 times weekly in combination with oral ribavirin (15 mg/kg/d) for 12 months. The mode of infection was unknown in 4, parenterally transmitted in 16, and vertically transmitted in 21 children. Forty patients completed the study. Eleven children, who remained hepatitis C virus (HCV)-RNA positive 6 months after the beginning, discontinued therapy. One boy stopped treatment because of side effects. At the end of treatment 25 patients were HCV-RNA negative (61%). All individuals remained HCV-RNA negative during the 6-month follow-up period. Nine of 15 children with parenteral (56.3%), 14 of 21 with vertical (66.6%), and 2 of 4 with unknown route of infection responded. Side effects included minor clinical signs such as fever, flu-like symptoms, anorexia, and more severe signs (21.4%) such as the development of thyroid autoantibodies and impairment of thyroid function. In conclusion, combination of alfa-interferon with ribavirin seems to be an important advance in the treatment of chronic hepatitis C in children and adolescents. This also is true for both vertically infected patients and for individuals with normal transaminase levels before therapy. (HEPATOLOGY 2002;36:1280-1284.)

HBV superinfection in hepatitis C virus chronic carriers, viral interaction, and clinical course (*Human Study*)
Evangelista Sagnelli, Nicola Coppola, Vincenzo Messina, Domenico di Caprio, Cecilia Marrocco, Anna Marotta, Mirella Onofrio, Carlo Scolastico, Pietro Filippini
We enrolled 44 patients with hepatitis B virus (HBV) acute infection, 21 anti-hepatitis C virus (HCV)­positive for at least 1 year (case BC group), 20 anti-HCV­negative (control B group), and 3 with HBV/HCV acute concurrent infection. For each case BC, a subject with chronic HCV infection alone was selected (control C group). At the first observation, 85.7% of patients in case BC group and 85% of those in control B group were HBV-DNA­positive (polymerase chain reaction [PCR]), with a similar trend towards a decrease and negativization in about 20 days; in the case BC group, seroconversion to antibody to hepatitis B e antigen (anti-HBe) was more rapid. HCV-RNA (PCR) was undetectable in all case BC patients but 1, who shortly became negative, whereas 85.7% of subjects in control C group were positive (P < .001). Severe acute hepatitis was more frequent in the case BC group than in the control B group (28.6% vs. 0%, P < .05). Of the 14 patients in the case BC group and of the 16 in the control B group followed up for more than 6 months, 1 in the first and 1 in the second group became hepatitis B surface angiten (HBsAg) chronic carriers. Of the 13 patients in case BC group who recovered, 1 cleared both anti-HCV and HCV-RNA, 6 became HCV-RNA­positive, and 6 remained HCV-RNA­negative. In patients with HBV/HCV acute concurrent infection, HBV-DNA became undetectable in 15 days, and HCV-RNA and anti-HCV became positive at days 30 and 45, respectively; these patients developed HCV-RNA­positive chronic hepatitis. In conclusion, HBV superinfection in chronic HCV carriers has a severe clinical course and strongly and persistently depresses HCV. (HEPATOLOGY 2002;36:1285-1291.)

Hepatorenal syndrome: Is it truly the end of the road?

Florence Wong, M.D., F.R.C.P
Background & Aims: Type 1 hepatorenal syndrome (HRS) is a severe complication of cirrhosis associated with a short median survival time (<2 weeks). Although the administration of terlipressin improves renal function, its effect on survival is unknown. This study investigated predictive factors of survival in patients with type 1 HRS treated with terlipressin.
Methods: Ninety-nine patients with type 1 HRS treated with terlipressin in 24 centers were retrospectively studied. Terlipressin-induced improved renal function was defined as a decrease in serum creatinine value to <130 micromol/L or a decrease of at least 20% at the end of treatment.
Results: At inclusion, the Child-Pugh score was 11.8±1.6 (mean±SD). Terlipressin (3.2±1.3 mg/day) was administered for 11±12 days. Renal function improved in 58 (64%) patients (serum creatinine decreased by 46%±17% from 272±114 micromol/L). Median survival time was 21 days. Survival rate was 40% at 1 month. Multivariate analysis showed that improved renal function and Child-Pugh score 11 at inclusion were independent predictive factors of survival (P<0.0001 and 0.02, respectively). Thirteen patients underwent liver transplantation (92±95 days after HRS onset), 10 of whom had received terlipressin and had had improved renal function.
Conclusions: This retrospective uncontrolled study shows that in patients with type 1 HRS, terlipressin-induced improved renal function is associated with an increase in survival. Thus, a randomized trial investigating the effect of terlipressin on survival in patients with type 1 HRS should be performed.

GASTROENTEROLOGY

Table of Contents for November 2002 · Volume 123 · Number 5

Clinical­Alimentary Tract

Cancer incidence in a population-based cohort of individuals hospitalized with celiac disease or dermatitis herpetiformis
J. Askling, M. Linet, G. Gridley, T. S. Halstensen, K. Ekström, A. Ekbom
Background & Aims: Studies of cancer risk in celiac disease (CD) or dermatitis herpetiformis (DH) indicate increased risks for malignant lymphoma and occasionally other neoplasms, but are characterized by small numbers, lack of systematic cancer assessment, and subjects identified from referral institutions.
Methods: By using Swedish population­based inpatient and cancer registry data, we followed-up 12,000 subjects with CD or DH, and evaluated cancer incidence by using standardized incidence ratios (SIR).
Results: Adults (but not children and adolescents) with CD had an elevated overall risk for cancer (SIR = 1.3) that declined with time and eventually reached unity. Elevated risks were found for malignant lymphomas, small-intestinal, oropharyngeal, esophageal, large intestinal, hepatobiliary, and pancreatic carcinomas. The excess occurrence of malignant lymphomas was confined to adults, decreased with time of follow-up evaluation, and decreased over successive calendar periods. Decreased risks were found for breast cancer. Subjects with DH had a slightly increased overall cancer risk (SIR = 1.2) owing to excesses of malignant lymphoma and leukemia, but no increases of gastrointestinal carcinomas.
Conclusions: Albeit increased, the relative risks for lymphomas and gastrointestinal cancers in this study are lower (and declining) than in most previous reports. The overall cancer risk is only moderately increased, and nonelevated during childhood and adolescence.

Primary chronic interstitial nephritis in Crohn's disease
H. Izzedine, J. Simon, A.-M. Piette, M. Lucsko, A. Baumelou, D. Charitanski, E. Kernaonet, A.-C. Baglin, G. Deray, H. Beaufils
Background & Aims: In Crohn's disease, cases of interstitial nephritis with renal failure have been reported in connection with the use of mesalamine.
Methods: We observed 4 patients with severe interstitial nephritis proven by examination of kidney biopsy specimens. Renal failure was discovered before or simultaneously with the diagnosis of Crohn's disease, and patients were not treated with mesalamine. Impairment of renal function progressed to end-stage renal failure in 3 of the 4 patients.
Results: Our results show that the kidney can be an extraintestinal target of Crohn's disease.
Conclusions: Several unanswered questions remain concerning the frequency of interstitial nephritis in patients with Crohn's disease, as well as the exact role of mesalamine in the development of chronic interstitial nephritis.

Significance of pelvic floor muscles in anal incontinence
X. Fernández-Fraga, F. Azpiroz, J.-R. Malagelada
Background & Aims: The pathophysiology of anal incontinence may be elusive using current parameters. Our aim was to establish the role of the levator ani in anal continence.
Methods: In 53 patients with anal incontinence, 30 with constipation as disease controls, and 15 healthy controls, we evaluated incontinence severity by a 0­12 scale, anorectal function by standard manometric tests, and levator ani contraction by a perineal dynamometer.
Results: Patients with incontinence exhibited various physiologic abnormalities (3.2 ± 0.3 per patient), but multiple regression analysis showed that levator ani contraction was the independent variable with strongest relation to the severity of incontinence (R = ­0.84; P < 0.0001), as well as a predictive factor of the response to treatment (R = 0.53; P < 0.01). Furthermore, in contrast to other physiologic parameters, clinical improvement in response to treatment (4.4 ± 0.5 score vs. 7.9 ± 0.5 score pre; P < 0.001) was associated with a marked and significant strengthening of levator ani contraction (448 ± 47 g vs. 351 ± 35 g pre; P < 0.05).
Conclusions: We have shown the importance of levator ani failure in understanding the etiology of anal incontinence and in predicting response to treatment.

Do changes in visceral sensory function determine the development of dyspepsia during treatment with aspirin?
G. Holtmann, J. Gschossmann, L. Buenger, G. Gerken, N. J. Talley
Background & Aims: We hypothesized that the development of dyspeptic symptoms during treatment with nonsteroidal anti-inflammatory drugs (NSAIDs) would be linked to alterations in gastric mechanosensory function and gastric emptying.
Methods: In the first study, gastric mechanosensory thresholds (barostat technique) and gastric emptying (13C-octanoic breath test) were measured and endoscopy was performed at entry and after 5 days of treatment with aspirin (500 mg 3 times daily) in 8 patients with functional dyspepsia (initially without symptoms) and 8 healthy controls. In a second, double-blind, placebo-controlled, cross-over study, 6 new patients with functional dyspepsia and 6 controls were started with either placebo or aspirin for 5 days. Sensory thresholds were tested after the fifth day of aspirin or placebo treatment. Abdominal symptoms were assessed daily.
Results: In the first study, 6 of 8 patients and 3 of 8 controls, and in the second trial 6 of 6 patients and 1 of 6 healthy subjects, developed dyspepsia on aspirin (P < 0.005 patients vs. healthy subjects). No symptoms occurred during placebo treatment. Lanza scores were not associated with symptoms. After aspirin, sensory thresholds increased in both studies in subjects without development of symptoms (by 25.9% ± 7.9%, and 31.0% ± 4.1%, respectively, all P < 0.05), whereas there was no significant increase in subjects who developed symptoms (­11.2% ± 5.3% and ­3.4% ± 13.4%, all P > 0.4). Neither thresholds nor symptoms were linked with the severity of mucosal damage, baseline gastric emptying (t1/2), or changes of gastric emptying (all P > 0.4).
Conclusions: Failure to increase sensory thresholds during treatment with aspirin is associated with the development of dyspepsia.

Enteric nerves and interstitial cells of Cajal are altered in patients with slow-transit constipation and megacolon
T. Wedel, J. Spiegler, S. Soellner, U. J. Roblick, T. H. K. Schiedeck, H.-P. Bruch, H.-J. Krammer
Background & Aims: A variety of gastrointestinal motility disorders have been attributed to alterations of interstitial cells of Cajal and malformations of the enteric nervous system. This study evaluates both the distribution of interstitial cells of Cajal and the pathohistology of the enteric nervous system in 2 severe human colorectal motility disorders.
Methods: Colonic specimens obtained from patients with slow-transit constipation (n = 11), patients with megacolon (n = 6), and a control group (n = 13, nonobstructing neoplasia) were stained with antibodies against c-kit (marker for interstitial cells of Cajal) and protein gene product 9.5 (neuronal marker). The morphometric analysis of interstitial cells of Cajal included the separate registration of the number and process length within the different regions of the muscularis propria. The structural architecture of the enteric nervous system was assessed on microdissected whole-mount preparations.
Results: In patients with slow-transit constipation, the number of interstitial cells of Cajal was significantly decreased in all layers except the outer longitudinal muscle layer. The myenteric plexus showed a reduced ganglionic density and size (moderate hypoganglionosis) compared with the control group. Patients with megacolon were characterized by a substantial decrease in both the number and the process length of interstitial cells of Cajal. The myenteric plexus exhibited either complete aganglionosis or severe hypoganglionosis.
Conclusions: The enteric nervous system and interstitial cells of Cajal are altered concomitantly in slow-transit constipation and megacolon and may play a crucial role in the pathophysiology of colorectal motility disorders.

Dysregulated peripheral and mucosal Th1/Th2 response in Whipple's disease
T. Marth, N. Kleen, A. Stallmach, S. Ring, S. Aziz, C. Schmidt, W. Strober, M. Zeitz, T. Schneider
Background & Aims: An impaired monocyte function and impaired interferon (IFN)- production has been suggested as a possible pathogenetic factor in Whipple's disease (WD) and as a cause for the delayed elimination of Tropheryma whipplei in some patients.
Methods: We studied, in a series of 20 WD patients with various degrees of disease activity, cellular immune functions.
Results: We found an increased in vitro production of interleukin (IL)-4 by peripheral mononuclear blood cells as determined by enzyme-linked immunosorbent assay, but reduced secretion of IFN- and IL-2 as compared with age- and sex-matched controls. In addition, we observed a significantly reduced monocyte IL-12 production in response to various stimuli in WD patients whereas other cytokines were comparable with controls; these immunologic alterations were not significantly different in patients with various disease activities. At the mucosal level, we found decreased CD4 T-cell percentage and a significantly impaired IFN- secretion.
Conclusions: Our data define a defective cellular immune response in a large series of WD patients and point to an important pathogenetic role of impaired Th1 responses. The decreased monocyte IL-12 levels may result in reduced peripheral and mucosal IFN- production and lead to an increased susceptibility to T. whipplei infection in certain hosts.

Prognostic significance of calcium-binding protein S100A4 in colorectal cancer
S. Gongoll, G. Peters, M. Mengel, P. Piso, J. Klempnauer, H. Kreipe, R. von Wasielewski
Background & Aims: Prognostication in colon cancer almost exclusively still rests on the tumor stage. Furthermore, tumor-derived markers to improve discrimination of low- and high-risk subtypes generally are not in use. S100A4 has been reported to be associated with invasion and metastasis; however, no data are available on its prognostic value in colorectal carcinoma. Therefore, we investigated the prognostic significance of immunohistochemical S100A4 expression in colorectal carcinoma compared with clinicopathologic parameters and expression of cell-cycle markers p16, p21, p27, p53, Ki-67, and RB.
Methods: Archival tissue from 709 patients with colorectal cancer were retrieved, applied in tissue array technology, and investigated immunohistochemically. Univariate and multivariate survival analyses were carried out on all investigated parameters.
Results: Sixteen percent of cases showed high; 31%, low; and 53%, no S100A4 expression. In Kaplan­Meier analysis, S100A4 positively stained cases showed a significantly decreased survival time compared with negatively stained cases (P < 0.0001). In multivariate regression analysis, S100A4 expression emerged as a highly significant independent parameter (P < 0.001) with the highest relative-risk factor among other covariates. Nodal status (pN) lost its prognostic value if S100A4 was added to the model. High S100A4 expression was associated with tumor stage pT3/4, secondary metastasis, women, p16, and RB expression.
Conclusions: S100A4 expression represents a highly significant prognostic marker in colorectal carcinoma, which is able to identify a subset of patients at high risk. In this respect, it is superior to established prognostic markers such as nodal status, pT stage, and p53 expression.

Clinical­Liver, Pancreas, and Biliary Tract

Evolution of hepatitis C viral quasispecies after liver transplantation
A. C. Lyra, X. Fan, D. M. Lang, K. Yusim, S. Ramrakhiani, E. M. Brunt, B. Korber, A. S. Perelson, A. M. Di Bisceglie
Background & Aims: To determine whether HCV quasispecies diversity correlated positively with liver disease progression after orthotopic liver transplantation (OLT).
Methods: We studied 11 patients undergoing OLT for HCV-related cirrhosis with recurrent hepatitis C in 2 groups according to the stage of hepatic fibrosis on follow-up. The mild group had stage 1 or 2 fibrosis; the severe group, stage 3 or 4 fibrosis. HCV quasispecies diversity was assessed by cloning and sequencing in pretransplantation and posttransplantation serum samples.
Results: In the mild fibrosis group, intrasample hypervariable region 1 (HVR1) genetic distance and nonsynonymous substitutions increased after OLT, whereas in the severe fibrosis group, these parameters decreased in follow-up. In contrast, intrasample diversity progressed similarly in both groups in the adjacent sequences flanking HVR1. There was an inverse correlation between the stage of hepatic fibrosis and amino acid complexity after OLT. Among all patients, the estimated rate of amino acid change was greater initially and became more constant after 36 months.
Conclusions: After OLT, a more complex HCV HVR1 quasispecies population was associated with mild disease recurrence. Among those patients with severe recurrent hepatitis C, HCV appeared to be under greater immune pressure. The greatest change in viral amino acid sequences occurred in the first 36 months after OLT.

Long-term impact of renal transplantation on liver fibrosis during hepatitis C virus infection
L. Alric, V. Di-Martino, J. Selves, P. Cacoub, F. Charlotte, D. Reynaud, J.-C. Piette, J.-M. Péron, J.-P. Vinel, D. Durand, J. Izopet, T. Poynard, M. Duffaut, L. Rostaing
Background & Aims: During hepatitis C virus (HCV) infection, liver fibrosis progression after renal transplantation remains controversial. The aim of this cohort study with controls was to compare liver histopathologic features during HCV infection between renal transplant recipients and matched groups of hemodialyzed patients or controls without renal disease and untreated for HCV.
Methods: Each renal transplant recipient (group 1, n = 30) was matched at first liver biopsy (LB) using the main factors known to influence progression of fibrosis with one HCV hemodialyzed patient (group 2, n = 30) and one HCV-infected patient (nonhemodialyzed, nontransplanted; group 3, n = 30). Patients from group 1 were also matched with those of group 3 on the time between 2 consecutive LBs performed 37 months apart. LBs were evaluated according to the Knodell index, METAVIR score, and rate of fibrosis progression per year (fibrosis unit).
Results: The rate of fibrosis progression per year between the first and second LBs was significantly lower (P = 0.03) in group 1 (0.067; 95% confidence interval: ­0.05, 0.18) than group 3 (0.20; 95% confidence interval: 0.13, 0.26). At the second LB, the Knodell index and activity or fibrosis in METAVIR were lower in group 1 than group 3 (4.2 ± 0.4 vs. 7.5 ± 0.6, 0.5 ± 0.1 vs. 1.3 ± 0.2, and 1.4 ± 0.2 vs. 2.3 ± 0.2 respectively, P < 0.01).
Conclusions: Our study suggests that liver fibrosis progression is low in most HCV-infected renal transplant recipients with moderate liver disease at baseline.

Study of recurrence after surgical resection of intraductal papillary mucinous neoplasm of the pancreas
S. T. Chari, D. Yadav, T. C. Smyrk, E. P. DiMagno, L. J. Miller, M. Raimondo, J. E. Clain, I. A. Norton, R. K. Pearson, B. T. Petersen, M. J. Wiersema, M. B. Farnell, M. G. Sarr
Background & Aims: The aim of this study was to determine recurrence and long-term survival after resection of pancreatic intraductal papillary mucinous neoplasm and to correlate recurrence and survival with histology, extent of resection, and duration of follow-up.
Methods: A single pathologist, without knowledge of previous interpretations of histology or clinical data, retrospectively reviewed and classified 113 resected intraductal papillary mucinous neoplasms as invasive carcinoma (n = 40) or as noninvasive neoplasms (adenoma, borderline, or carcinoma in situ; n = 73). Data on recurrence (locoregional or metastatic), follow-up, and cause of death were obtained from patient records and/or by contacting patients and their physicians.
Results: In invasive intraductal papillary mucinous neoplasm, recurrence was similar after partial pancreatectomy (18/27; 67%) and total pancreatectomy (8/13; 62%) and occurred within 3 years of resection in 91%. Among noninvasive neoplasms, 5 of 60 (8%) recurred after partial pancreatectomy (median follow-up, 37 months); none recurred after total pancreatectomy (n = 13; median follow-up, 32 months). Recurrence after resection in noninvasive neoplasms was diagnosed after a median of 40 months (range, 23­75 months); recurrence was noninvasive in 3 and invasive cancer in 2. Five-year survival was better for noninvasive compared with invasive intraductal papillary mucinous neoplasm (84.5% vs. 36%; P < 0.001).
Conclusions: Invasive intraductal papillary mucinous neoplasm recurs frequently even after a complete "curative" resection and portends poor survival. In contrast, noninvasive intraductal papillary mucinous neoplasm recurs infrequently after resection, and survival is excellent regardless of the degree of epithelial dysplasia in the tumor.

Basic­Alimentary Trac

3 adrenergic stimulation inhibits the opossum lower esophageal sphincter
M. Dimarino, K. Banwait, S. Rattan, S. Cohen, A. J. Dimarino
Background & Aims: Previous studies have identified adrenergic receptor sites in the lower esophageal sphincter (LES) of animals and humans. A 3 adrenoceptor has been identified and cloned. The binding site for this receptor has been found in the rat LES in vitro. The aim of the study was to assess the role of a specific 3 agonist (CL316243) on LES pressure (LESP) in vivo.
Methods: Anesthetized adult opossums were given CL316243 and isoproterenol intravenously as boluses before and after continuous infusion of L748337 (a specific 3 antagonist), propranolol, and bethanechol. Blood pressure, heart rate, and LESP were continuously recorded.
Results: CL316243 caused a dose-dependent maximal inhibition of LESP of 88.5% ± 4.8%. The mean duration of inhibition was 62.2 ± 9.2 minutes with minimal change in cardiovascular parameters. Isoproterenol caused dose-dependent maximal inhibition of 89.4% ± 4.7% with mean duration of action of 5.1 ± 0.9 minutes but was associated with significant hypotension and tachycardia. L748337 and propranolol significantly blocked the effects of CL316243 and isoproterenol, respectively. CL316243 and isoproterenol inhibited the bethanechol-mediated hypertensive LES.
Conclusions: (1) A selective 3 agonist, CL316243, caused significant, prolonged, and dose-dependent inhibition of LESP and, unlike isoproterenol, had minimal effect on heart rate and mean arterial pressure. (2) The 3 antagonist, L748337, selectively inhibited CL316243 without altering the isoproterenol response. (3) CL316243 and isoproterenol both caused inhibition of cholinergic-mediated hypertensive LESP.

Expansion of CD8+ T cells with regulatory function after interaction with intestinal epithelial cells
M. Allez, J. Brimnes, I. Dotan, L. Mayer
Background & Aims: Regulatory T cells play a role in the control of immune responses in the intestinal mucosa and their absence may predispose to inflammatory bowel disease (IBD). We have previously shown that T cells activated by intestinal epithelial cells (IECs) are suppressive in function. Our goal was to characterize the phenotype and function of T cells proliferating after interaction with IECs.
Methods: Irradiated human IECs, isolated from normal resection specimens, were cultured with carboxy fluorescein succinimidyl ester (CFSE) labeled T cells. Flow cytometric analysis of T cells was performed at days 5­10. CD8+ T cells proliferating in culture with IECs were sorted and added to suppressive assays.
Results: The precursor frequency of T cells proliferating in response to IECs ranged from 0.3%­0.9%. Several subpopulations were shown to proliferate (CD8+CD28­/CD8+CD28+/CD4+CD25+), but one population (CD8+CD28­CD101+CD103+) appeared to be dependent on contact with the CD8 ligand gp180. After sorting, culture in the presence of interleukin (IL)-7 and IL-15 allowed for the generation of cell lines. IEC-activated CD8+ T cells, but not nonactivated CD8+ T cells, were suppressive in function. Suppression belonged to the CD101+CD103+ subset of IEC-activated CD8+ T cells and appeared to require cell contact. CD8+ lamina propria T cells also showed suppressive function, suggesting the presence of CD8+ regulatory T cells in the mucosa.
Conclusions: IECs are able to induce the proliferation of a small fraction of CD8+ peripheral T cells. The CD8+CD28­ subset of IEC-activated CD8+ T cells, which express CD101 and CD103, interacts with IECs through gp180 and has regulatory function.

Rapid development of colitis in NSAID-treated IL-10­deficient mice
D. J. Berg, J. Zhang, J. V. Weinstock, H. F. Ismail, K. A. Earle, H. Alila, R. Pamukcu, S. Moore, R. G. Lynch
Background & Aims: Interleukin (IL)-10 is an anti-inflammatory and immune regulatory cytokine. IL-10­deficient mice (IL-10­/­) develop chronic inflammatory bowel disease (IBD), indicating that endogenous IL-10 is a central regulator of the mucosal immune response. Prostaglandins are lipid mediators that may be important mediators of intestinal inflammation. In this study we assessed the role of prostaglandins in the regulation of mucosal inflammation in the IL-10­/­ mouse model of IBD.
Methods: Prostaglandin (PG) synthesis was inhibited with nonselective or cyclooxygenase (COX)-isoform selective inhibitors. Severity of inflammation was assessed histologically. Cytokine production was assessed by ribonuclease protection analysis and enzyme-linked immunosorbent assay. PGE2 levels were assessed by enzyme immunoassay. COX-1 and COX-2 expression was assessed by Western blot analysis.
Results: Nonsteroidal anti-inflammatory drug (NSAID) treatment of wild-type mice had minimal effect on the colon. In contrast, NSAID treatment of 4-week-old IL-10­/­ mice resulted in rapid development of colitis characterized by infiltration of the lamina propria with macrophages and interferon gamma­producing CD4+ T cells. Colitis persisted after withdrawal of the NSAID. NSAID treatment decreased colonic PGE2 levels by 75%. Treatment of IL-10­/­ mice with sulindac sulfone (which does not inhibit PG production) did not induce colitis whereas the NSAID sulindac induced severe colitis. COX-1­ or COX-2­selective inhibitors used alone did not induce IBD in IL-10­/­ mice. However, the combination of COX-1­ and COX-2­selective inhibitors did induce colitis.
Conclusions: NSAID treatment of IL-10­/­ mice results in the rapid development of severe, chronic IBD. Endogenous PGs are important inhibitors of the development of intestinal inflammation in IL-10­/­ mice.

The role of dietary microparticles and calcium in apoptosis and interleukin-1 release of intestinal macrophages
S. M. Evans, P. Ashwood, A. Warley, F. Berisha, R. P. H. Thompson, J. J. Powell
Background & Aims: The intestinal mucosa is exposed to micron-sized, man-made exogenous particles (e.g., titanium dioxide) and freshly formed endogenous particles (calcium phosphate). A role for such microparticles in inflammation has been proposed, and here we examined their effects on lamina propria mononuclear cells.
Methods: Lamina propria mononuclear cells were isolated from patients with and without inflammatory bowel disease and incubated with lipopolysaccharide, titanium dioxide, and calcium ± citrate, as well as a conjugate of lipopolysaccharide, calcium, and titanium dioxide. Interleukin-1 and interleukin-1 receptor antagonist were measured by enzyme-linked immunosorbent assay in culture supernatants and macrophage apoptosis by flow cytometry. Mechanistic studies were undertaken in normal peripheral blood mononuclear cells.
Results: Baseline levels of interleukin-1 and macrophage apoptosis were greater in inflammatory bowel disease than in normal lamina propria mononuclear cells. Lipopolysaccharide and titanium dioxide had no additional effect, but calcium, and more so the conjugate, induced interleukin-1 release in proportion to the degree of inflammation. Citrate, used to prevent in situ calcium phosphate formation, negated lamina propria mononuclear cell stimulation. Macrophage apoptosis was also increased by calcium and the conjugate. In peripheral blood mononuclear cells, inhibition of caspase 1 reduced interleukin-1 secretion, whereas blockade of phagocytosis inhibited calcium-induced apoptosis and interleukin-1 release.
Conclusions: The endogenous luminal microparticle calcium phosphate Promotes apoptosis of intestinal macrophages. Concomitantly, interleukin-1 is released, which is enhanced in the presence of inflamed cells and/or exogenous dietary microparticles. Endogenous or exogenous microparticles could aggravate the ongoing inflammation of inflammatory bowel disease.

Serine proteases excite myenteric neurons through protease-activated receptors in guinea pig small intestine
C. Gao, S. Liu, H.-Z. Hu, N. Gao, G. Y. Kim, Y. Xia, J. D. Wood
Background & Aims: Serine proteases are postulated to influence gastrointestinal function by stimulating protease-activated receptors (PARs). This study identified the effects on myenteric neurons of activating PARs and investigated underlying mechanisms of action.
Methods: Intracellular electrophysiologic methods were used to study the effects of proteases on electrical and synaptic behavior of morphologically identified neurons in the guinea pig enteric nervous system. Fluorescent immunohistochemistry was used to study the chemical coding of neurons that responded to PARs stimulation.
Results: Application of thrombin, trypsin, or mast cell tryptase evoked slowly activating excitatory responses reminiscent of slow synaptic excitation in enteric neurons. Synthetic activating peptides for PAR-1, -2, and -4 receptors mimicked the actions of the proteases. The depolarizing responses evoked by PARs were insensitive to cyclooxygenase inhibitors and were suppressed by agents that inhibit phospholipase C (PLC) or block intraneuronal receptors for inositol triphosphate. A majority of PAR-sensitive uniaxonal neurons expressed immunoreactivity for nitric oxide synthase. Most of the PAR-sensitive AH Dogiel morphologic type II neurons were immunoreactive for calbindin.
Conclusions: Excitatory responses to the serine proteases are mediated by PAR-1, -2, and -4 receptors. The mechanism of signal transduction involves stimulation of PLC and intraneuronal calcium mobilization and is independent of prostanoid formation.

CDX2 regulates liver intestine­cadherin expression in normal and malignant colon epithelium and intestinal metaplasia
T. Hinoi, P. C. Lucas, R. Kuick, S. Hanash, K. R. Cho, E. R. Fearon
Background & Aims: The intestine-specific caudal-related homeobox transcription factor CDX2 seems to play a key role in intestinal development and differentiation. Inactivation of one Cdx2 allele predisposes mice to develop colon polyps, and loss of CDX2 expression is a feature of some poorly differentiated colon carcinomas in humans. Conversely, aberrant CDX2 expression is often seen in intestinal metaplasias in the stomach and esophagus and in some gastric carcinomas. To better understand CDX2 function, we sought to define CDX2-regulated genes.
Methods: HT-29 colon cancer cells with minimal endogenous CDX2 expression were engineered to express exogenous CDX2, and gene expression changes relative to control cells were assessed using high-density oligonucleotide arrays.
Results: The gene for liver intestine (LI)-cadherin (cadherin 17) was strongly induced by CDX2 in HT-29. In other colorectal cancer lines, endogenous CDX2 and LI-cadherin expression were well correlated. Activation of a ligand-regulated form of CDX2 rapidly induced LI-cadherin gene expression, even in the presence of protein synthesis inhibitor. Analysis of the 5'-flanking region of the LI-cadherin gene defined 2 CDX2 responsive elements, and chromatin immunoprecipitation assays indicate CDX2 binds to the elements. In primary colorectal cancers and intestinal metaplasias in the stomach, CDX2 and LI-cadherin expression were tightly correlated.
Conclusions: CDX2 regulates LI-cadherin gene expression in normal, metaplastic, and neoplastic tissues of the gastrointestinal tract via binding to elements in the 5'-flanking region of the gene. Given the well-established roles of cadherins in morphogenesis and differentiation, LI-cadherin may be a key factor mediating CDX2 function in intestinal cell fate determination.

Motilin regulates interdigestive gastric blood flow in dogs
C. Jin, S. Naruse, M. Kitagawa, H. Ishiguro, W. Muxin, M. Nakajima, K. Yokohata, O. Ito, T. Hayakawa
Background & Aims: Gastric blood flow exhibits cyclical increases in phase with the interdigestive contractions and secretion of the stomach in dogs. The aim of this study is to clarify the regulatory role of motilin in interdigestive gastric blood flow in dogs.
Methods: Blood flow of the left gastric (LGA) and superior mesenteric (SMA) arteries were measured by ultrasound transit-time blood-flow meters in 5 conscious dogs. Motilin was infused intravenously with or without Phe-cyclo[Lys-Tyr(3-tBu)-Ala-] · trifluoroacetate (GM-109; motilin antagonist), granisetron (5-HT3 antagonist), atropine, hexamethonium (C6), phenoxybenzamine, propranolol, or cimetidine.
Results: Motilin (12.5, 25, 50, and 100 pmol · kg­1 · h­1) induced LGA blood-flow responses, consisting of a sustained increase and a rapid phasic change coupled with a contraction, without affecting the blood pressure, heart rate, and SMA blood flow. GM-109 completely abolished the LGA, motility, and secretory responses to motilin (100 pmol · kg­1 · h­1). Atropine abolished motilin-induced gastric contractions, secretion, and phasic changes of LGA blood flow but failed to affect the sustained flow increase. However, atropine partially inhibited the LGA responses to lower doses of motilin. The LGA flow responses to motilin were not inhibited by granisetron, C6, -adrenergic, -adrenergic, or H2 blockers. Motilin induced significantly larger gastric vasodilatation than the equivalent doses of VIP.
Conclusions: Motilin has a potent and selective gastric vasodilator effect, which appears to be mediated by both cholinergic and noncholinergic mechanisms. Motilin plays an important role in the regulation of interdigestive gastric blood flow in dogs.

Restoration of acid secretion following treatment with proton pump inhibitors
J. M. Shin, G. Sachs
Background & Aims: Proton pump inhibitors (PPIs) are covalent inhibitors of the gastric H+,K+-adenosine triphosphatase (ATPase) forming disulfide bonds. Recovery of acid secretion after PPI inhibition may be due to de novo synthesis of pump protein and/or disulfide reduction and reactivation of inhibited pump. The half-time of recovery of acid secretion in rats following omeprazole treatment is ~15 hours, whereas pump protein half-life is 54 hours. In humans, the half-life of the inhibitory effect on acid secretion is ~28 hours for omeprazole and ~46 hours for pantoprazole. Whereas all PPIs bind to cysteine 813, pantoprazole additionally binds to cysteine 822, deeper in the membrane domain of TM6. Their different durations of action may reflect different rates of pump reactivation due to differing accessibility of the disulfides to glutathione.
Methods: Rats were stimulated and treated with 30 mg/kg of each PPI. Gastric ATPase was prepared and reversal of inhibition of the H+,K+-ATPase was measured as the time-dependent restoration of activity by incubation with dithiothreitol or glutathione.
Results: One hundred percent reactivation of ATPase following inhibition in vivo by omeprazole or its enantiomers was seen with dithiothreitol and 89% with glutathione. Similar data were found for lansoprazole or rabeprazole. No reactivation by either reducing agent was seen following inhibition by pantoprazole.
Conclusions: Recovery of acid secretion following inhibition by all PPIs, other than pantoprazole, may depend on both protein turnover and reversal of the inhibitory disulfide bond. In contrast, recovery of acid secretion after pantoprazole may depend entirely on new protein synthesis.

Cyclooxygenase-2­derived lipoxin A4 increases gastric resistance to aspirin-induced damage
S. Fiorucci, O. Menezes de Lima, Jr., A. Mencarelli, B. Palazzetti, E. Distrutti, W. McKnight, M. Dicay, L. Ma, M. Romano, A. Morelli, J. L. Wallace
Background & Aims: Cyclooxygenase-2 (COX-2) has been implicated as contributing to mucosal defense. Acetylation of COX-2 by aspirin can result in production of an antiinflammatory substance, 15(R)-epi-LXA4. We determined whether aspirin-triggered lipoxin (LX) production via COX-2 diminishes aspirin-induced damage in the rat stomach.
Methods: Rats were treated with aspirin plus or minus celecoxib or rofecoxib. Gastric generation of LXA4 was measured. Effect of exogenous LXA4 or an LXA4 receptor antagonist on gastric resistance to aspirin-induced damage was examined. Aspirin-induced leukocyte adherence in mesenteric venules, and the effects of LXA4, were examined by intravital microscopy.
Results: Celecoxib and rofecoxib significantly increased the severity of aspirin-induced gastric damage. Aspirin rapidly up-regulated COX-2 expression in the stomach and caused a significant increase in gastric 15(R)-epi-LXA4 production, which was abolished by celecoxib. LXA4 dose dependently (0.25­2.5 µg/kg, intraperitoneally) reduced the severity of aspirin-induced gastric damage and suppressed aspirin-induced leukocyte adherence, whereas an LXA4 antagonist had the opposite effects.
Conclusions: Aspirin administration results in elevated production of 15(R)-epi-LXA4 via COX-2. LXA4 exerts very potent protective actions on the gastric mucosa. Co-administration of aspirin and a selective COX-2 inhibitor results in substantially more severe gastric injury than is produced with either agent alone.

Host-dependent zonulin secretion causes the impairment of the small intestine barrier function after bacterial exposure
R. El Asmar, P. Panigrahi, P. Bamford, I. Berti, T. Not, G. V. Coppa, C. Catassi, A. Fasano
Background & Aims: Enteric infections have been implicated in the pathogenesis of both food intolerance and autoimmune diseases secondary to the impairment of the intestinal barrier. On the basis of our recent discovery of zonulin, a modulator of small-intestinal tight junctions, we asked whether microorganisms might induce zonulin secretion and increased small-intestinal permeability.
Methods: Both ex vivo mammalian small intestines and intestinal cell monolayers were exposed to either pathogenic or nonpathogenic enterobacteria. Zonulin production and changes in paracellular permeability were monitored in Ussing chambers and micro-snapwells. Zonula occludens 1 protein redistribution after bacteria colonization was evaluated on cell monolayers.
Results: Small intestines exposed to enteric bacteria secreted zonulin. This secretion was independent of either the species of the small intestines or the virulence of the microorganisms tested, occurred only on the luminal aspect of the bacteria-exposed small-intestinal mucosa, and was followed by a decrease in small-intestinal tissue resistance (transepithelial electrical resistance). The transepithelial electrical resistance decrement was secondary to the zonulin-induced tight junction disassembly, as also shown by the disengagement of the protein zonula occludens 1 protein from the tight junctional complex.
Conclusions: This zonulin-driven opening of the paracellular pathway may represent a defensive mechanism, which flushes out microorganisms and contributes to the host response against bacterial colonization of the small intestine.

Xenin-immunoreactive cells and extractable xenin in neuroendocrine tumors of duodenal origin
G. E. Feurle, M. Anlauf, G. Hamscher, R. Arnold, G. Klöppel, E. Weihe
Background & Aims: Xenin is a 25­amino acid peptide produced by specific endocrine cells of the duodenal mucosa. We investigated whether xenin is expressed in neuroendocrine tumors.
Methods: Seventy-two foregut and midgut neuroendocrine tumors were examined by means of immunohistochemistry, confocal laser microscopy with an antibody against the C-terminus of xenin, and high-pressure liquid chromatography after acidic extraction, assessed by radioimmunoassay.
Results: We found xenin-immunoreactive cells in 23 of 26 duodenal neuroendocrine tumors, including gastrinomas, somatostatinomas, and nonfunctioning and enterochromaffin cell tumors. In these tumors, up to 20% of the endocrine cells were xenin immunoreactive, and xenin immunoreactivity was concentrated in secretory granules. Xenin was coexpressed with chromogranin A. We found no xenin expression in gastrin-, somatostatin-, and serotonin-immunoreactive cells. High-pressure liquid chromatography after acidic extraction revealed 497 ± 285 pmol of xenin per gram of tissue in 5 duodenal gastrinomas. The other neuroendocrine tumors, such as bronchial carcinoids, gastric enterochromaffin-like cell carcinoids, gastric and ileal enterochromaffin cell carcinoids, insulinomas, and gastrinomas of pancreatic origin, did not contain immunoreactive xenin.
Conclusions: Xenin is a peptide marker specific to neuroendocrine tumors of the duodenum. This finding may be useful in tumor classification and in the differential diagnosis of neuroendocrine tumors of the upper gut.

High-conductance chloride channels generate pacemaker currents in interstitial cells of Cajal
J. D. Huizinga, Y. Zhu, J. Ye, A. Molleman
Background & Aims: Interstitial cells of Cajal (ICCs) are responsible for slow, wave-driven, rhythmic, peristaltic motor patterns in the gastrointestinal tract. The aim was to identify and characterize the ion channels that generate the underlying pacemaker activity.
Methods: Single ion channel recordings were obtained from nonenzymatically isolated ICCs and studied by using the cell attached and inside-out configurations of the patch clamp technique.
Results: A high-conductance chloride channel was observed in ICCs that was spontaneously and rhythmically active at the same frequency as the rhythmic inward currents defining ICC pacemaker activity, 20­30 cycles/min at room temperature. Main conductance levels occurred between 122­144 pS and between 185­216 pS. Periodicity in the channel opening coincided with periodicity in membrane potential change, hence, at the single channel level, chloride channels were seen to be associated with the generation of rhythmic changes in membrane potential.
Conclusions: ICCs harbor high-conductance chloride channels that participate in the generation of pacemaker activity and may become a target for pharmacologic treatment of gut motor disorders.

Global analysis of Helicobacter pylori gene expression in human gastric mucosa
J. E. Graham, R. M. Peek, Jr., U. Krishna, T. L. Cover
Background & Aims: Helicobacter pylori inhabits a highly restricted ecological niche in the human gastric mucosa. Microbial gene expression in the context of persistent infection remains largely uncharacterized.
Methods: An RNA analysis method, selective capture of transcribed sequences, was used in conjunction with genomic array hybridization to characterize H. pylori complementary DNAs (cDNAs) obtained from both human and experimentally infected gerbil gastric tissue specimens.
Results: Bacterial cDNAs obtained by selective capture of transcribed sequences from tissues hybridized to arrayed DNA fragments representing approximately 70% of open reading frames in the H. pylori genome. RNAs for most of these open reading frames were also detected by array hybridization analyses of total RNA prepared from the isolated H. pylori strains cultured in vitro. However, a subset of H. pylori RNAs detected in gastric tissue specimens was consistently undetectable in bacteria grown in vitro. The majority of these RNAs encode factors unique to H. pylori that are potentially produced in response to interactions with mammalian gastric mucosa.
Conclusions: The combination of selective capture of transcribed sequences with array hybridization has allowed a global analysis of bacterial gene expression occurring in human tissues during a natural infection.

Basic­Liver, Pancreas, and Biliary Tract

The human bile salt export pump: Characterization of substrate specificity and identification of inhibitors
J. A. Byrne, S. S. Strautnieks, G. Mieli-Vergani, C. F. Higgins, K. J. Linton, R. J. Thompson
Background & Aims: The bile salt export pump (BSEP) is the major bile salt transporter in the liver canalicular membrane. Our aim was to determine the affinity of the human BSEP for bile salts and identify inhibitors.
Methods: Human BSEP was expressed in insect cells. Adenosine triphosphatase (ATPase) assays were performed, and bile salt transport studies were undertaken.
Results: The BSEP gene, ABCB11, was cloned and a recombinant baculovirus was generated. Infected insect cells expressed a 140-kilodalton protein that was absent in uninfected and in mock-infected cells. An ATPase assay showed BSEP to have a high basal ATPase activity. Transport assays were used to determine the Michaelis constant for taurocholate as 4.25 µmol/L, with a maximum velocity of 200 pmol · min­1 · mg­1 protein. Inhibition constant values for other bile salts were 11 µmol/L for glycocholate, 7 µmol/L for glycochenodeoxycholate, and 28 µmol/L for taurochenodeoxycholate. Cyclosporin A, rifampicin, and glibenclamide were proved to be competitive inhibitors of BSEP taurocholate transport, with inhibition constant values of 9.5 µmol/L, 31 µmol/L, and 27.5 µmol/L, respectively. Progesterone and tamoxifen did not inhibit BSEP.
Conclusions: The human BSEP is a high-affinity bile salt transporter. The relative affinities for the major bile salts differ from those seen in rodents and reflect the different bile salt pools. BSEP is competitively inhibited by therapeutic drugs. This is a potentially significant mechanism for drug-induced cholestasis.

Functional expression of the canalicular bile salt export pump of human liver
J. Noé, B. Stieger, P. J. Meier
Background & Aims: Hepatic bile salt secretion is an essential function of vertebrate liver. Rat and mouse bile salt export pump (Bsep) are adenosine triphosphate (ATP)-dependent bile salt transporters. Mutations in human BSEP were identified as the cause of progressive familial intrahepatic cholestasis type 2. BSEP protein is highly identical with its rat and mouse orthologs and has not yet been functionally characterized; the effect of BSEP mutations on its function has also not been studied. Therefore, the aim of this study was to functionally characterize human BSEP.
Methods: Complementary DNA for BSEP was isolated from human liver and expressed with the baculovirus system in Sf9 cells. ATP-dependent bile salt transport assays were performed with Sf9 cell vesicles expressing BSEP and a rapid filtration assay.
Results: Cloning of human BSEP required the inactivation of a bacterial cryptic promoter motif within its coding region. BSEP expressed in Sf9 cells transports different bile salts in an ATP-dependent manner with Michaelis constant values as follows: taurocholate, 7.9 ± 2.1 µmol/L; glycocholate, 11.1 ± 3.3 µmol/L; taurochenodeoxycholate, 4.8 ± 1.7 µmol/L; tauroursodeoxycholate, 11.9 ± 1.8 µmol/L. The rank order of the intrinsic clearance of bile salts was taurochenodeoxycholate > taurocholate > tauroursodeoxycholate > glycocholate.
Conclusions: This study characterizes human BSEP as an ATP-dependent bile salt export pump with transport properties similar to its rat and mouse orthologs. Expression of BSEP in Sf9 cells will enable functional characterization of the consequences of mutations in the human BSEP gene.

Up-regulation of components of the renin-angiotensin system in the bile duct­ligated rat liver
G. Paizis, M. E. Cooper, J. M. Schembri, C. Tikellis, L. M. Burrell, P. W. Angus
Background & Aims: Angiotensin II (ANG II) has profibrotic actions in the heart and kidney, whereas blockade of the renin angiotensin system (RAS) attenuates injury. This study examines whether the RAS is present in the liver and examines its regulation in the bile duct­ligation model of hepatic fibrogenesis.
Methods: Sham-operated and bile duct­ligated (BDL) Sprague-Dawley rats were studied. Gene and protein expression of hepatic renin, angiotensinogen, angiotensin-converting enzyme (ACE), and the angiotensin receptors AT1 and AT2 were assessed using real-time reverse-transcription polymerase chain reaction, in vitro autoradiography, and immunohistochemistry.
Results: Angiotensinogen and renin messenger RNA were detected in sham liver but were not increased following BDL. Angiotensinogen protein was widely distributed in hepatocytes in both normal and injured livers, but in BDL livers, it was also expressed within areas of active fibrogenesis. Both ACE and AT1 receptor genes were up-regulated following BDL. The low level of ACE activity in sham animals was significantly increased in areas of active fibrogenesis in BDL livers. The AT1 receptor was present in both normal and diseased liver parenchyma, with increased AT1 receptor binding seen in fibrotic areas in the diseased liver. The AT2 receptor gene was not detected in normal or diseased liver.
Conclusions: Key elements of the RAS are present in normal liver tissue, and there is major up-regulation of the system in the bile duct-ligated liver. These findings are in keeping with recent experimental studies that have demonstrated antifibrotic effects of RAS blockade in the bile duct­ligated liver.

Kupffer cells participate in early clearance of syngeneic hepatocytes transplanted in the rat liver
B. Joseph, H. Malhi, K. K. Bhargava, C. J. Palestro, R. S. McCuskey, S. Gupta
Background & Aims: Kupffer cells are activated shortly after deposition of hepatocytes in liver sinusoids, with clearance of a significant fraction of transplanted cells, especially when cells are entrapped in portal spaces. We determined whether perturbation of Kupffer cells would improve transplanted cell engraftment.
Methods: Dipeptidyl peptidase IV­deficient rats were used as recipients of syngeneic Fischer 344 rat hepatocytes. Kupffer cell function was analyzed by measuring phagocytic activity with carbon particle or 99mTc-sulfur colloid incorporation. Transplanted cell survival and integration in the liver parenchyma was determined by histochemical analysis of tissues. Transplanted cell proliferation was analyzed in rats conditioned with retrorsine and partial hepatectomy.
Results: Gadolinium chloride significantly impaired Kupffer cell function, especially in periportal areas, where transplanted cells were localized. Transplanted cell survival increased by approximately 2-fold in animals treated with gadolinium chloride 24 hours before cell transplantation. In gadolinium-treated rats, more transplanted cells were observed in portal vein radicles, as well as in liver sinusoids, albeit integration of cells in the liver parenchyma was slower in gadolinium-treated rats and cells separated from other hepatocytes in portal vein radicles that failed to exhibit bile canalicular reconstitution. Finally, hepatocyte transplantation in rats primed with retrorsine and partial hepatectomy showed accelerated kinetics of liver repopulation in animals pretreated with gadolinium chloride.
Conclusions: Perturbation of Kupffer cell activity will benefit liver repopulation with cells and further analysis of clinically suitable approaches to exploit this mechanism will be appropriate.

---

JOURNAL OF HEPATOLOGY

Volume 37, Issue 5, November 2002

Biliary Tract and Cholestasis

Background/Aims: As compared to other chronic liver diseases, cholestatic disorders are associated with a better outcome of variceal bleeding and less blood loss at transplantation, suggesting the presence of a hypercoagulable state. We have assessed plasmatic coagulation and platelet function in patients with cholestatic and non-cholestatic liver disease. Methods: Thirty-seven patients with chronic cholestatic liver disease (primary biliary cirrhosis (PBC)/primary sclerosing cholangitis (PSC)), 53 patients with chronic hepatitis C (HCV) or alcoholic cirrhosis (C2), and 62 healthy controls were studied. Results: Thrombelastography revealed a hypercoagulable state in non-cirrhotic patients with PBC/PSC, but not in those with HCV (ma-value: 6.54[6.25-6.92, 95%CI] vs. 5.39[5.11-5.58], P<0.05) possibly due to higher fibrinogen levels in PBC/PSC patients (369[329-418]mg/dl vs. 263[250-275]mg/dl, P<0.05). PFA-100 closure time was prolonged in HCV/C2 patients with advanced cirrhosis, but not in cirrhotic patients with PBC/PSC (Child B; epinephrine stimulation: 192[161-229]s vs. 132[105-158]s, P<0.05). Flow cytometric studies of platelet receptors and granules revealed a higher surface expression of CD42b (112[105-119]% vs. 100[95-104]%, P<0.05) and LIBS-1 (261[184-348]% vs. 121[92-145]%, P<0.05) in patients with PBC/PSC than in those with HCV/C2. Conclusions: These results indicate that platelet function differs between patients with cholestatic and non-cholestatic liver disease and is stable or even hyperactive in patients with PBC and PSC.

Henk Wolters et al.
Effects of bile salt flux variations on the expression of hepatic bile salt transporters in vivo in mice
Background/Aims: Expression of hepatic bile salt transporters is partly regulated by bile salts via activation of nuclear farnesoid X-activated receptor (Fxr). We investigated the physiological relevance of this regulation by evaluating transporter expression in mice experiencing different transhepatic bile salt fluxes. Methods: Bile salt flux was manipulated by dietary supplementation with taurocholate (0.5% w/w) or cholestyramine (2% w/w) or by disruption of the cholesterol 7-hydroxylase-gene (Cyp7A/ mice) leading to reduced bile salt pool size. Expression of hepatic transporters was assessed (polymerase chain reaction (PCR), immunoblotting, and immunohistochemistry). Results: Biliary bile salt secretion was increased (+350%) or decreased (50%) after taurocholate or cholestyramine feeding, respectively, but plasma bile salt concentrations and hepatic Fxr expression were not affected. The bile salt uptake system Na+-taurocholate co-transporting polypeptide (Ntcp) and organic anion transporting polypeptide-1 (Oatp1) were down-regulated by taurocholate and not affected by cholestyramine feeding. Cyp7A/ mice did not show altered Ntcp or Oatp1 expression. Canalicular bile salt export pump (Bsep) was up-regulated by 65% in taurocholate-fed mice, and slightly down-regulated in Cyp7A/ mice. Conclusions: Large variations in hepatic bile salt flux have minor effects on expression of murine Ntcp and Bsep in vivo, suggesting that these transporters are abundantly expressed and able to accommodate a wide range of `physiological' bile salt fluxes.

Corinne Lang et al.
Impaired ketogenesis is a major mechanism for disturbed hepatic fatty acid metabolism in rats with long-term cholestasis and after relief of biliary obstruction
Background/Aims: Rats with long-term cholestasis have reduced ketosis of unknown origin. Methods: Fatty acid metabolism was studied in starved rats with biliary obstruction for 4 weeks (bile duct ligated rats= BDL rats), and 3, 7, 14, 28 and 84 days after reversal of biliary obstruction by Roux-en-Y anastomosis (RY rats), and in sham-operated control rats. Results: BDL rats had reduced -hydroxybutyrate concentrations in plasma (0.25±0.10 vs. 0.75±0.20mmol/l) and liver (2.57±0.20 vs. 4.63±0.61µmol/g) which increased after restoring bile flow. Hepatic expression and activity of carnitine palmitoyltransferase I (CPT I) or CPT II were unaffected or decreased in BDL rats, respectively, and increased after restoring bile flow. Oxidative metabolism of different substrates by isolated liver mitochondria and activation of palmitate were reduced in BDL rats and recovered 7-14 days after restoring bile flow. Ketogenesis was decreased in mitochondria from BDL rats and recovered 3 months after restoring bile flow. Both mRNA and protein expression of hydroxymethylglutaryl-coenzyme A synthase (HMG-CoA synthase), the rate-limiting enzyme of ketogenesis, was reduced in livers of BDL rats and increased after reversing biliary obstruction. Conclusions: In BDL rats, impairment of hepatic fatty acid metabolism is multifactorial. After reversing biliary obstruction, reduced activity of HMG-CoA synthase is the major factor.

Chronic Liver Diseases

Edoardo Giannini et al.
Serum thrombopoietin levels are linked to liver function in untreated patients with hepatitis C virus-related chronic hepatitis
Background: Thrombocytopenia can be found in patients with chronic hepatitis related to hepatitis C virus (HCV). Both hypersplenism and decreased liver production of thrombopoietin (TPO) have been hypothesized as mechanisms responsible for thrombocytopenia. Aims: To assess the presence of relationships among platelet count, spleen size, TPO serum levels, liver histology, and liver function in a group of patients with HCV-related chronic hepatitis. Methods: Platelet count, TPO serum levels, and spleen size were assessed in 25 untreated HCV positive chronic hepatitis patients undergoing liver biopsy. These parameters were correlated to liver histology and liver function as evaluated by means of [13C]aminopyrine breath test (ABT). Results: Both platelet counts (146±48 vs. 202±56¥109/1, P<0.03) and TPO serum levels (103±24 vs. 158±7 1 pg/ml, P<0.02) were lower among patients with high fibrosis scores as compared to patients with low fibrosis scores. Patients with thrombocytopenia as well as patients with high fibrosis scores had lower ABT results as compared to patients with normal platelet counts and patients with no or mild fibrosis, respectively. TPO serum levels were correlated to platelet count (rs=0.493, P=0.016), and negatively correlated to fibrosis stage (rs=0.545, P=0.008). Lastly, low TPO serum levels were associated to a decrease in liver function. Conclusions: Our study showed that in patients with chronic hepatitis related to HCV infection serum TPO levels are correlated to liver functional impairment and to the degree of liver fibrosis.

Cirrhosis and its Complications

Hiroyuki Sugimoto, Tetsuya Kaneko, Masashi Hirota, Ekmel Tezel and Akimasa Nakao
Earlier hepatic vein transit-time measured by contrast ultrasonography reflects intrahepatic hemodynamic changes accompanying cirrhosis
Background/Aims: Non-invasive diagnosis of cirrhosis by transit-time analysis of an ultrasound contrast agent has been reported, even though the mechanism by which contrast arrives to the hepatic vein earlier in cirrhosis than in normal controls is unknown. The aim of this study is to assess whether the earlier appearance of contrast in the hepatic vein depends on intrahepatic or extrahepatic causes. Methods: There were 15 participants: six volunteers, three patients with hepatitis, and six with cirrhosis. The contrast agent was given intravenously, and transit-time analysis of the hepatic artery, portal vein and hepatic vein was performed. The time-acoustic intensity curves in the three vessels were analyzed by an image and cineloop display and quantification software package. Results: The hepatic artery and portal vein arrival times were not significantly different among the three groups. On the other hand, hepatic vein arrival times were significantly earlier in cirrhosis (median 18 seconds) compared with arrival times in hepatitis patients (median 30 seconds, P<0.001) and in healthy volunters (median 31 seconds, P<0.001). These results give support to a previous pilot study and indicate that most of the time delay in hepatic vein arrival time between cirrhosis and the other groups originated from intrahepatic circulation abnormalities. Conclusions: This study confirms that the earlier appearance of contrast in the hepatic vein observed in cirrhosis is due to intrahepatic, and not extrahepatic, hemodynamic changes.

Inflammation and Fibrosis

Antonio Di Sario et al.
Effect of pirfenidone on rat hepatic stellate cell proliferation and collagen production
Background/Aims: Pirfenidone has been recently shown to reduce dimethynitrosamine-induced liver fibrosis in the rat, but no information are available on the effect of this drug on cultured hepatic stellate cells (HSC). Methods: HSC proliferation was evaluated by measuring bromodeoxyuridine incorporation; PDGF-receptor autophosphorylation, extracellular signal-regulated kinase (ERK1/2) and pp70S6K activation were evaluated by western blot; protein kinase C activation was evaluated by western blot and by ELISA; type I collagen accumulation and 1(I) procollagen mRNA expression were evaluated by ELISA and northern blot, respectively. Results: Pirfenidone significantly inhibited PDGF-induced HSC proliferation, starting at a concentration of 1 µM, with a maximal effect at 1000 µM, without affecting HSC viability and without inducing apoptosis. The inhibition of PDGF-induced HSC proliferation was associated neither with variations in PDGF-receptor autophosphorylation, or with ERK1/2 and pp70S6K activation. On the other hand, pirfenidone was able to inhibit PDGF-induced activation of the Na+/H+ exchanger, which is involved in PDGF-induced HSC proliferation in HSC, with a maximal effect at 1000 µM and inhibited PDGF-induced protein kinase C activation. Pirfenidone 100 and 1000 µM inhibited type I collagen accumulation in the culture medium induced by transforming growth factor1 by 54% and 92%, respectively, as well as TGF1-induced 1(I) procollagen mRNA expression. Results: Pirfenidone could be a new candidate for antifibrotic therapy in chronic liver diseases.

Cell Biology, Metabolism and Transport

Luisa Ibáñez, Eulàlia Pérez, Xavier Vidal, Joan-Ramon Laporte and the Grup d'Estudi Multicèntric d'Hepatotoxicitat Aguda de Barcelona (GEMHAB)
Prospective surveillance of acute serious liver disease unrelated to infectious, obstructive, or metabolic diseases: epidemiological and clinical features, and exposure to drugs
Background/Aims: Acute serious liver disease which is unrelated to infectious, obstructive, or metabolic disease is uncommon. Many drugs have been implicated. Data on its epidemiology are scarce. We performed a population-based prospective study of acute serious liver disease in Catalonia (Spain). Methods: A collaborating hospital network was set up. All patients with acute serious liver disease and negative viral hepatitis serological markers, without an obvious cause of liver disease, were included. Results: The incidence of acute serious liver disease was 7.4 per 106 inhabitants per year (95% CI; 6.0-8.8), which increased with age. The incidence of hepatocellular acute serious liver disease (3.84 per 106 per year) was greater than that of cholestatic and mixed patterns. The case-fatality ratio was 11.9% and mortality 0.8 per million person-years. The risk of death was similar among patients with hepatocellular and cholestatic patterns. Non-steroidal antiinflammatory drugs, analgesics, and antibacterials were the most frequently used drugs. Conclusions: Acute serious liver disease which is unrelated to infectious, obstructive, or metabolic disease is rare. Its incidence increases with age. The prognosis of cholestatic acute serious liver disease does not significantly differ from that of the hepatocellular pattern. Non-steroidal antiinflammatory drugs, analgesics, and antibacterials were the most common drugs likely to be responsible for acute liver disease.

Liver Cell Injury and Liver Failure

Gianluigi Vendemiale, Ignazio Grattagliano, Luigi Lupo, Vincenzo Memeo and Emanuele Altomare
Hepatic oxidative alterations in patients with extra-hepatic cholestasis. Effect of surgical drainage
Background/Aims: The mechanisms of liver injury in conditions of biliary obstruction are poorly understood. Hepatic oxidative injury has been observed in experimental models of cholestasis. Little is known in humans. This study aimed to gain more insights into the hepatic redox status in human cholestasis. Methods: Liver concentrations of total glutathione, protein sulfhydryls and malondialdehyde (end-product of lipid peroxidation) were measured in hepatic specimens of 12 patients with obstructive jaundice before and after the application of an external biliary drainage and in six control subjects. Results: Compared to control subjects, biliary obstructed patients showed significantly (P<0.001) lower concentrations of hepatic glutathione and protein sulfhydryls, and higher (P<0.001) levels of malondialdehyde, in the presence of comparable protein concentrations. Two-weeks after the application of external biliary drainage, cholestatic indices were significantly improved and the observed changes in glutathione, protein sulfhydryls and malondialdehyde levels, significantly decreased. Conclusions: This study shows that cholestasis is associated with a decreased protein and non-protein sulfhydryl content in the liver and with an increased lipid peroxidation. These alterations reversed almost completely after biliary drainage, indicating the cholestasis itself as the determining factor for the redox status impairment observed in the liver of patients with extra-hepatic biliary obstruction.

Lars Mueller et al.
The induction of the immediate-early-genes Egr-1, PAI-1 and PRL-1 during liver regeneration in surgical models is related to increased portal flow
Background: The environmental triggers which control liver regeneration following partial hepatectomy (PH) are not clear. With respect to haemodynamic changes, the model of rat portal branch ligation (PBL) provides the unique opportunity to discriminate transcriptional events, which selectively result from increased portal flow. Aims: The potential role of portal over-flow on early expression of early growth response gene-1 (Egr-1), type-1 plasminogen activator inhibitor (PAI-1) and phosphatase of regenerating liver-1 (PRL-1) was analysed by a comparative experimental study using PBL and PH. Methods: Operative procedures were carried out in male Wistar rats. Growth kinetics were measured by liver weight indices. S-phase-specific mRNA-levels of H2B-histone protein (H2B), as well as expression analysis of Egr-1, PAI-1 and PRL-1 were examined by Northern blot experiments. Results: Growth patterns did not differ significantly between PBL and PH, whereas peak H2B expression occurred earlier after PH. Egr-1 and PAI-1 were specifically induced during the first few hours in the hyper-perfused lobes following PBL and PH. PRL-1-expression selectively peaked 3h after PH and PBL in the hyper-perfused lobes. Conclusions: Increased portal flow after PBL and PH was associated with induction of Egr-1, PAI-1 and PRL-1. Thus, haemodynamic changes affect the molecular immediate-early response during liver regeneration.

Ludger Leifeld et al.
Inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) expression in fulminant hepatic failure
Background/Aims: Inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) have important functions in inflammation and vasoregulation but their role in fulminant hepatic failure (FHF) is not well understood. Methods: Intrahepatic in situ staining and semi-quantification of iNOS and eNOS by immunohistochemistry in 25 patients with FHF, in 40 patients with chronic liver diseases (CLD) and in ten normal controls (NC). Results: Expression patterns of iNOS and eNOS differed. While in NC only faint iNOS expression was found in some Kupffer cells/macrophages and hepatocytes, eNOS was expressed constitutively in sinusoidal and vascular endothelial cells. In CLD, iNOS expression was induced in Kupffer cells/macrophages and hepatocytes, representing the main iNOS expressing cell types. Additionally, bile ducts, vascular endothelial cells and lymphocytes also expressed iNOS (P=0.001). In contrast, no differences were found between eNOS expression in CLD and NC (P=0.64). The same cell types expressed eNOS and iNOS in FHF but numbers of both were significantly enhanced, exceeding the levels seen in CLD (P<0.001, P=0.017). Conclusions: Our data demonstrate that iNOS and eNOS are differently regulated in physiologic conditions and in liver disease. While eNOS seems to be involved in the physiological regulation of hepatic perfusion, strong upregulation of iNOS might contribute to inflammatory processes in FHF.

Liver Growth and Cancer

Bruno Turlin et al.
High vascular endothelial growth factor (VEGF) expression in chemically-induced hepatic microcancers in mice
Background/Aims: In experimental carcinogenesis microcancers are defined as foci of altered hepatocytes showing vascular invasion. Vascular endothelial growth factor (VEGF) could be involved in such vascular extension. The aim of our study was to evaluate the in situ VEGF expression in chemically-induced microcancers. Methods: Fourteen C3H male mice were submitted to a diethyl nitrosamine-induced carcinogenesis. Iron-dextran overload was performed in parallel in order to localize all iron-negative lesions. Animals were sacrificed at 24, 39 and 52 weeks of age. Liver sections were histologically (haematoxylin eosin safron (HES), Orcein and Perls' stains) and immunohistochemically (VEGF) studied. Results: Microcancers represented 8% of 424 lesions that we found as compared to foci (35%), adenomas (51%), and overt cancers (6%). VEGF hepatocyte positivity was found in 74% of lesions, with a more frequent, intense, and homogenous expression in microcancers than in other lesions, especially at 24 and 39 weeks. Conclusions: In our model, we found a high VEGF expression in microcancer exhibiting progression in vessels. Early overexpression of VEGF, formerly named vascular permeating factor, could act as a permeability factor and favors the development of vascular breakdown in microcancers.

Paul J. Gaglio et al.
Liver regeneration investigated in a non-human primate model (Macaca mulatta)
Background/Aims: An adequate model to study liver regeneration in humans is presently unavailable. We explored the feasibility of studying liver regeneration in a genetically similar species to man, the non-human primate Rhesus macaque. Methods: Five animals were studied; two underwent 60% hepatectomy, one underwent 30% hepatectomy, and cholecystectomy alone was performed on two animals. Laparoscopic-guided or open liver biopsies were performed on days 1, 2, 7, 14, 21, 30 and 60 following all surgeries. Liver regeneration was evaluated by measuring Ki-67, proliferating cell nuclear antigen expression and mitotic index, calculating changes in the surface area of the liver remnant and assessing intrahepatic production of cytokines. Results: Significant liver regeneration was induced in the animals that underwent 60% hepatectomy, peaking between days 21-30 postoperatively. Regeneration was minimal in all other animals studied. Cytokine production followed a similar pattern. Maximal liver regeneration correlated with restoration of surface area in the liver remnant. Conclusions: Sixty percent hepatectomy in a non-human primate model induced significant liver regeneration, maximizing 21-30 days following partial hepatectomy, suggesting a significant interspecies difference when compared to a rodent hepatectomy model. A partial hepatectomy model in Rhesus macaques may allow further characterization of liver regeneration in a species closer to humans.

Nathalie Wong et al.
Frequent loss of chromosome 3p and hypermethylation of RASSF1A in cholangiocarcinoma
Background/Aims: Cholangiocarcinoma comprises 5-20% of all primary malignant tumors of the liver. However, the lack of information about the genetic basis of cholangiocarcinoma has impeded characterization and understanding of its biological behavior. Methods: In this study, genome-wide aberrations in 13 cases of cholangiocarcinoma were examined by the molecular cytogenetic technique, comparative genomic hybridization. Results: Frequent gains of 1q, 3q, 8q, 15q and 17q, and common losses on 3p, 4q, 6q, 9p, 17p and 18q were found. The finding of common chromosome 3p loss (~40%) with a minimal deleted region of 3p13-p21 has prompted our further investigation on the tumor suppressor gene RASSF1A, located at 3p21.3. Using bisulphite modification followed by methyl-specific PCR, a high incidence of methylated RASSF1A promoter region was detected in our current series (9/13 cases; 69%). Further expression analysis on the nine cases with promoter hypermethylation indicated much reduced RASSF1A expression compared to normal livers. Conclusions: Our current molecular cytogenetic investigation on primary cholangiocarcinoma provided overall karyotypic information and represents the first report on the methylation status of RASSF1A in cholangiocarcinoma. The high incidence of 3p loss and RASSF1A promoter hypermethylation detected may have implications for this tumor suppressor gene in the malignant progression of cholangiocarcinoma.

Transplantation and Surgery

Catherine M. Pastor et al.
Effect of hyperthermic preconditioning on cold preserved rat portal veins
Background/Aims: Little information is available regarding the effect of cold storage and hyperthermic preconditioning on the contractile responses of hepatic vessels. We then studied, after cold preservation, the in vitro contractile responses of rat portal veins (RPV) isolated from normal rats or from rats previously subjected to hyperthermia. Methods: Rats were or were not subjected to hyperthermia 24h before the RPV isolation. Then, RPV were stored at 4°C in Krebs-Henseleit bicarbonate (KHB) or University of Wisconsin solution or conserved at 20°C in KHB solution. Control RPV were tested after rat sacrifice. Results: The contractile responses were importantly decreased in RPV conserved at room temperature. The morphology of the vessels was altered and the heat shock protein 70 (Hsp70) protein expression disappeared. These abnormalities were prevented by cold preservation. The type of preservation solution did not interfere with the beneficial effect. Hyperthermic preconditioning increased the expression of Hsp70 protein in freshly isolated and cold preserved RPV but decreased the contractile responses. In RPV conserved at room temperature, hyperthermic preconditioning further worsened the decreased contractile response. Conclusions: Thus, hyperthermic preconditioning, which is beneficial in protecting hepatic injury following cold preservation, alters the contractile responses of RPV.

Irmeli Lautenschlager et al.
Human herpesvirus-6 infection is associated with adhesion molecule induction and lymphocyte infiltration in liver allografts
Background/Aims: Human herpesvirus-6 (HHV-6) infection has been recently described in liver transplants. HHV-6 may infect the transplant and cause graft dysfunction. Some association between HHV-6 and rejection has also been recorded. We have now investigated the possible involvement of HHV-6 in the intragraft immunological processes, adhesion molecules induction and lymphocyte activation. Methods: HHV-6 was detected in liver biopsies of 19 patients transplanted in the period from 1996 to 2000. Patients with other infections or rejection were excluded from the study. Finally, 19 biopsies of eight allografts with pure HHV-6 infection were available. Adhesion molecules (ICAM-1, VCAM-1, ELAM-1) and their ligands (LFA-1, VLA-4, sLeX) and lymphoid activation markers (MHC class II, IL-2R) were demonstrated in liver biopsies by immunohistochemistry. Five biopsies from patients with normal graft function and without rejection or infection were used as controls for immune staining, and ten biopsies with acute rejection but without infection were used as positive controls. Results: Biopsy histology demonstrated mild to moderate lymphocyte infiltration associated with HHV-6 infection. HHV-6 significantly (P0.05) increased the vascular expression of ICAM-1 and VCAM-1, and the number of graft infiltrating lymphocytes positive for LFA-1, VLA-4 and class II antigens. A total of 3/8 grafts developed chronic rejection. Conclusions: HHV-6 infection increased adhesion molecule expression and lymphocyte infiltration in liver allografts.

Viral Hepatitis

Raquel Muñoz et al.
A pilot study of -interferon for treatment of patients with chronic hepatitis B who failed to respond to -interferon
Background/Aims: Alpha-interferon achieves persistent loss of hepatitis B virus (HBV) in about 30-40% of patients with chronic hepatitis B. In non-responder patients, the disease may progress leading to complications such as cirrhosis and hepatocellular carcinoma. The aim of the current study was to evaluate the efficacy of beta-interferon in patients with chronic hepatitis B who did not respond to one course of alpha-interferon. Methods: Twenty nine alpha-interferon-non-responder patients with chronic hepatitis B (11 hepatitis B e antigen, HBeAg-positive; 18 HBeAg-negative) were treated with 6 million units beta-interferon five times a week for 24 weeks. The post-treatment follow-up lasted for 48 weeks. Results: At the end of treatment, 38% of patients (18% HBeAg-positive; 50% HBeAg-negative) had normal serum aminotransferase levels and negative serum HBV DNA. At the end of follow-up, HBV DNA was no longer detectable in serum in 21% of patients (18% HBeAg-positive; 22% HBeAg-negative). Beta-interferon was well tolerated and safe. Conclusions: This pilot study suggests that beta-interferon therapy is effective and safe in the retreatment of patients with chronic hepatitis B who had not responded to a previous alpha-interferon cycle.

Deborah E. Sullivan et al.
Construction and characterization of an intracellular single-chain human antibody to hepatitis C virus non-structural 3 protein
Background/Aims: We developed a single-chain antibody fragment (scFv) to the non-structural 3 protein (NS3) of hepatitis C virus (HCV) and tested its ability to interfere with the HCV replication cycle in infected hepatocytes. Methods: The variable regions of the human monoclonal antibody CM3.B6 that recognizes a conformational epitope within the helicase domain of NS3 were introduced into adenoviral vectors for expression in mammalian hepatocytes. Expression and binding properties of the scFv were analyzed by immunological assays. Effects of intracellular expression of the scFv on HCV replication were assessed in primary hepatocytes isolated from explanted livers of patients with chronic HCV infection by reverse transcription-polymerase chain reaction. Results: Transduction of HepG2 cells by the recombinant adenoviruses resulted in stable, efficient expression of scFv in the cytoplasm that was non-toxic to the cells. The scFv specifically bound to its cognate antigen. Significantly, intracellular expression of scFv resulted in a decrease in HCV genomic RNA in HCV infected hepatocytes. Conclusions: These results indicate that specific binding of a scFv to NS3 may inhibit one or more functions of this essential viral protein thus interfering with the HCV replication cycle.

Chuan-Mo Lee et al.
Durability of lamivudine-induced HBeAg seroconversion for chronic hepatitis B patients with acute exacerbation
Background/Aims: Lamivudine-induced hepatitis B e antigen (HBeAg) seroconversion in patients with chronic hepatitis B was reported to be durable by several studies but controversy still exists. The aim of this study was to evaluate the durability of the responses of lamivudine treatment. Methods: Among 53 chronic hepatitis B patients who had acute exacerbation and had finished lamivudine therapy after at least 6 months of treatment, 31 patients achieved full HBeAg seroconversion twice at least 1 month apart, and subsequently stopped lamivudine therapy. Post-treatment monitoring was continued for up to 87 weeks. Alanine transaminase (ALT), HBeAg and hepatitis B virus (HBV) DNA were used as indicators for relapse. Results: The cumulative relapse rates at 48 and 72 weeks post-treatment were 45.4% and 56.3%, respectively. During follow up, normal ALT levels precluded relapse while ALT levels over two times the upper limit of normal indicated relapse, which correlated well with HBeAg or HBV DNA reappearance. Patients older than 25 years were more likely to experience post-treatment relapse. Conclusions: Lamivudine-induced full HBeAg seroconversion was not durable in the Taiwanese population. ALT levels were useful for relapse detection. Age was the only independent predictive factor for relapse.

BRITISH MEDICAL JOURNAL

9 November 2002 (Volume 325, Issue 7372)

Effect of zinc supplementation started during diarrhoea on morbidity and mortality in Bangladeshi children: community randomised trial
Abdullah H Baqui, Robert E Black, Shams El Arifeen, Mohammad Yunus, Joysnamoy Chakraborty, Saifuddin Ahmed, and J Patrick Vaughan
BMJ 2002; 325: 1059.

Objective: To evaluate the effect on morbidity and mortality of providing daily zinc for 14 days to children with diarrhoea.
Design: Cluster randomised comparison.
Setting: Matlab field site of International Center for Diarrhoeal Disease Research, Bangladesh.
Participants: 8070 children aged 3-59 months contributed 11 881 child years of observation during a two year period.
Intervention: Children with diarrhoea in the intervention clusters were treated with zinc (20 mg per day for 14 days); all children with diarrhoea were treated with oral rehydration therapy.
Main outcome measures: Duration of episode of diarrhoea, incidence of diarrhoea and acute lower respiratory infections, admission to hospital for diarrhoea or acute lower respiratory infections, and child mortality.
Results: About 40% (399/1007) of diarrhoeal episodes were treated with zinc in the first four months of the trial; the rate rose to 67% (350/526) in month 5 and to >80% (364/434) in month 7 and was sustained at that level. Children from the intervention cluster received zinc for about seven days on average during each episode of diarrhoea. They had a shorter duration (hazard ratio 0.76, 95% confidence interval 0.65 to 0.90) and lower incidence of diarrhoea (rate ratio 0.85, 0.76 to 0.96) than children in the comparison group. Incidence of acute lower respiratory infection was reduced in the intervention group but not in the comparison group. Admission to hospital of children with diarrhoea was lower in the intervention group than in the comparison group (0.76, 0.59 to 0.98). Admission for acute lower respiratory infection was lower in the intervention group, but this was not statistically significant (0.81, 0.53 to 1.23). The rate of non-injury deaths in the intervention clusters was considerably lower (0.49, 0.25 to 0.94).
Conclusions: The lower rates of child morbidity and mortality with zinc treatment represent substantial benefits from a simple and inexpensive intervention that can be incorporated in existing efforts to control diarrhoeal disease.

NEW ENGLAND JOURNAL

LANCET

Role of peroxisome proliferator-activated receptor and retinoid X receptor heterodimer in hepatogastroenterological diseases
Laurent Dubuquoy, Sébastien Dharancy, Sophie Nutten, Sven Pettersson, Johan Auwerx, Pierre Desreumaux

The peroxisome proliferator-activated receptor (PPAR) and its partner the retinoid X receptor (RXR) are two nuclear receptors that are expressed mainly in adipose tissue and which have a role in lipid metabolism and insulin sensitisation. New sites of PPAR/RXR expression have been described, especially in the intestinal tract, pancreas, and liver. Concomitantly, new functions have been attributed to this heterodimer in regulation of inflammation, by its inhibition of nuclear factor (NF)-B and via stress-kinase pathways. These new sites and functions of PPAR/RXR have led to novel ideas about pathophysiology of different inflammatory digestive diseases and to development of innovative treatment strategies with PPAR activators. (Online)

---