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Revue de Presse du Mois Mois de Fevrier 2003

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HEPATOLOGY

Table of Contents for February 2003 · Volume 37 · Number 2

Liver Biology and Pathobiology

Selective Na+/H+ exchange inhibition by cariporide reduces liver fibrosis in the rat
Antonio Di Sario, Emanuele Bendia, Silvia Taffetani, Marco Marzioni, Cinzia Candelaresi, Paola Pigini, Ursula Schindler, Heinz-Werner Kleemann, Luciano Trozzi, Giampiero Macarri, Antonio Benedetti
The aim of this study was to evaluate the effect of cariporide, a selective Na+/H+ exchange inhibitor, on isolated and cultured hepatic stellate cells (HSCs) and in 2 in vivo models of rat liver fibrosis. Platelet-derived growth factor (PDGF)-induced HSC proliferation, evaluated by measuring the percentage of bromodeoxyuridine-positive cells, was significantly inhibited by cariporide, with a maximal effect at 10 µmol/L. Incubation with cariporide did not inhibit PDGF-induced extracellular-regulated kinase 1/2 (ERK1/2), Akt (a downstream component of the phosphatidylinositol [PI]-3 kinase pathway), and protein kinase C (PKC) activation but reduced PDGF-induced activation of the Na+/H+ exchanger, with a maximal effect at 10 µmol/L. Rats treated with dimethylnitrosamine (DMN; 10 mg/kg) for 1 and 5 weeks received a diet with or without 6 ppm cariporide. Treatment with cariporide reduced the degree of liver injury, as determined by alanine aminotransferase (ALT) values, also when administered after the induction of hepatic damage. This was associated with reduced HSC activation and proliferation and reduced collagen deposition, as determined by morphometric evaluation of ­smooth muscle actin (SMA)/proliferating cell nuclear antigen­positive cells and percentage of Sirius red­positive parenchyma, respectively. Moreover, cariporide was also able to reduce (1)I procollagen messenger RNA (mRNA) expression. Similar effects were observed in bile duct­ligated (BDL) rats. In conclusion, selective inhibition of the Na+/H+ exchanger by cariporide may represent an effective therapeutic strategy in the treatment of hepatic fibrosis. (HEPATOLOGY 2003;37:256-266.)

DNase I­hypersensitive sites enhance 1(I) collagen gene expression in hepatic stellate cells
Yutaka Yata, Andrew Scanga, Andrea Gillan, Liu Yang, Shimon Reif, Michael Breindl, David A. Brenner, Richard A. Rippe
Liver fibrosis is characterized by a dramatic increase in the expression of type I collagen. Several deoxyribonuclease (DNase) I­hypersensitive sites (HS) have been located in the distal 5'-flanking region of the 1(I) collagen gene that are specific to collagen-producing cells. To assess the role of the DNase I­HS in regulating 1(I) collagen gene expression in hepatic stellate cells (HSCs), 3 transgenic mouse lines expressing collagen-1(I) reporter genes were used (Krempen et al. Gene Expr 1999;8:151-163). The pCol9GFP transgene contains the collagen gene promoter (­3122 to +111) linked to the green fluorescent protein (GFP) reporter gene. The pCol9GFP-HS4,5 transgene contains HS4,5 and pColGFP-HS8,9 contains HS8,9 positioned upstream of the collagen promoter in pCol9GFP. HSCs isolated from transgenic mice containing pCol9GFPHS4,5 and pColGFP-HS8,9 showed earlier and higher GFP expression patterns than HSCs isolated from pCol9GFP mice. HSCs from pCol9GFP-HS4,5 showed the highest levels of GFP expression and culture-induced expression correlated with induction of the endogenous 1(I) collagen gene. After CCl4 administration, pCol9GFP-HS4,5 mice showed increased GFP expression compared with pCol9GFP mice in both whole liver extracts and isolated HSCs. Several sites for DNA-protein interactions in both HS4 and HS5 were identified that included a binding site for activator protein 1. In conclusion, DNase I­HS4,5 enhance expression of the 1(I) collagen gene promoter in HSCs both in vitro and in vivo after a fibrogenic stimulus. The collagen-GFP transgenic mice provide a convenient and reliable model system to investigate the molecular mechanisms controlling increased collagen expression during fibrosis. (HEPATOLOGY 2003;37:267-276.)

Mechanisms of hepatocyte protection against hypoxic injury by atrial natriuretic peptide
Rita Carini, Maria Grazia De Cesaris, Roberta Splendore, Cinzia Domenicotti, Maria Paola Nitti, Maria Adelaide Pronzato, Emanuele Albano
Atrial natriuretic peptide (ANP) reduces ischemia and/or reperfusion damage in several organs, but the mechanisms involved are largely unknown. We used freshly isolated rat hepatocytes to investigate the mechanisms by which ANP enhances hepatocyte resistance to hypoxia. The addition of ANP (1 µmol/L) reduced the killing of hypoxic hepatocytes by interfering with intracellular Na+ accumulation without ameliorating adenosine triphosphate (ATP) depletion and pH decrease caused by hypoxia. The effects of ANP were mimicked by 8-bromo-guanosine 3', 5'-cyclic monophosphate (cGMP) and were associated with the activation of cGMP-dependent kinase (cGK), suggesting the involvement of guanylate cyclase­coupled natriuretic peptide receptor (NPR)-A/B ANP receptors. However, stimulating NPR-C receptor with des-(Gln18, Ser19,Gly20,Leu21,Gly22)-ANP fragment 4-23 amide (C-ANP) also increased hepatocyte tolerance to hypoxia. C-ANP protection did not involve cGK activation but was instead linked to the stimulation of protein kinase C (PKC)- through Gi protein­ and phospholipase C­mediated signals. PKC- activation was also observed in hepatocytes receiving ANP. The inhibition of phospholipase C or PKC by U73122 and chelerythrine, respectively, significantly reduced ANP cytoprotection, indicating that ANP interaction with NPR-C receptors also contributed to cytoprotection. In ANP-treated hepatocytes, the stimulation of both cGK and PKC- was coupled with dual phosphorylation of p38 mitogen-activated protein kinase (MAPK). The p38 MAPK inhibitor SB203580 abolished ANP protection by reverting p38 MAPK-mediated regulation of Na+ influx by the Na+/H+ exchanger. In conclusion, ANP recruits 2 independent signal pathways, one mediated by cGMP and cGK and the other associated with Gi proteins, phospholipase C, and PKC-. Both cGK and PKC- further transduce ANP signals to p38 MAPK that, by maintaining Na+ homeostasis, are responsible for ANP protection against hypoxic injury. (HEPATOLOGY 2003;37:277-285.)

Induction of cellular resistance against Kupffer cell­derived oxidant stress: A novel concept of hepatoprotection by ischemic preconditioning
Rolf J. Schauer, Alexander L. Gerbes, Daniel Vonier, Mark op den Winkel, Peter Fraunberger, Manfred Bilzer
Ischemic preconditioning (IP) triggers protection of the liver from prolonged subsequent ischemia. However, the underlying protective mechanisms are largely unknown. We investigated whether and how IP protects the liver against reperfusion injury caused by Kupffer cell (KC)-derived oxidants. IP before 90 minutes of warm ischemia of rat livers in vivo significantly reduced serum alanine aminotransferase (AST) levels and leukocyte adherence to sinusoids and postsinusoidal venules during reperfusion. This protective effect was mimicked by postischemic intravenous infusion of glutathione (GSH), an antioxidative strategy against KC-derived H2O2. Interestingly, no additional protection was achieved by infusion of GSH to preconditioned animals. These findings and several additional experiments strongly suggest IP mediated antioxidative effects: IP prevented oxidant cell injury in isolated perfused rat livers after selective KC activation by zymosan. Moreover, IP prevented cell injury and pertubations of the intracellular GSH/GSSG redox system caused by direct infusion of H2O2 (0.5 mmol/L). IP-mediated resistance against H2O2 could neither be blocked by the adenosine A2a antagonist DMPX nor mimicked by A2a agonist CGS21680. In contrast, H2O2 resistance was abolished by the p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB203580, but induced when p38 MAPK was directly activated by anisomycin. In conclusion, we propose a novel concept of hepatoprotection by IP: protection of liver cells by enhancing their resistance against KC-derived H2O2. Activation of p38 MAPK and preservation of the intracellular GSH/oxidized glutathione (GSSG) redox system, but not adenosine A2a receptor stimulation, seems to be pivotal for the development of H2O2 resistance in preconditioned livers. (HEPATOLOGY 2003;37:286-295.)

Stat4 and Stat6 signaling in hepatic ischemia/reperfusion injury in mice: HO-1 dependence of Stat4 disruption-mediated cytoprotection
Xiu-Da Shen, Bibo Ke, Yuan Zhai, Feng Gao, Dean Anselmo, Charles R. Lassman, Ronald W. Busuttil, Jerzy W. Kupiec-Weglinski
Ischemia/reperfusion (I/R) injury remains an important problem in clinical organ transplantation. There is growing evidence that T lymphocytes, and activated CD4+ T cells in particular, play a key role in hepatic I/R injury. This study analyzes the role of signal transducer and activator of transcription 4 (Stat4) and Stat6 signaling in liver I/R injury. Using a partial lobar warm ischemia model, groups of wild-type (WT), T cell­deficient, Stat4-/Stat6-deficient knockout (KO) mice were assessed for the extent/severity of I/R injury. Ninety minutes of warm ischemia followed by 6 hours of reperfusion induced a fulminant liver failure in WT and Stat6 KO mice, as assessed by hepatocellular damage (serum alanine aminotransferase [sALT] levels), neutrophil accumulation (myeloperoxidase [MPO] activity) and histology (Suzuki scores). In contrast, T cell deficiency (nu/nu mice) or disruption of Stat4 signaling (Stat4 KO mice) reduced I/R insult. Unlike adoptive transfer of WT or Stat6-deficient T cells, infusion of Stat4-deficient T cells failed to restore hepatic I/R injury and prevented tumor necrosis factor (TNF-) production in nu/nu mice. Diminished TNF-/Th1-type cytokine messenger RNA (mRNA)/protein elaborations patterns, along with overexpression of heme oxygenase-1 (HO-1)­accompanied hepatic cytoprotection in Stat4 KO recipients. In contrast, HO-1 depression restored hepatic injury in otherwise I/R resistant Stat4 KOs. In conclusion, Stat4 signaling is required for, whereas Stat4 disruption protects against, warm hepatic I/R injury in mice. The cytoprotection rendered by Stat4 disruption remains HO-1­dependent. (HEPATOLOGY 2003;37:296-303.)

Endogenous IL-13 protects hepatocytes and vascular endothelial cells during ischemia/reperfusion injury
Atsushi Kato, Tomohisa Okaya, Alex B. Lentsch
Hepatic ischemia/reperfusion injury involves a complex inflammatory cascade resulting in neutrophil-mediated injury of hepatocytes. Previous studies from our laboratory have established that exogenous administration of the anti-inflammatory cytokines interleukin 10 (IL-10) and IL-13 can ameliorate the inflammatory response and significantly reduce hepatocellular injury. The purpose of the present study was to determine if IL-10 and IL-13 function as endogenous regulators of the hepatic inflammatory response to ischemia/reperfusion. Wild-type, IL-10­, and IL-13­deficient (IL-10­/­, IL-13­/­) mice were exposed to 90 minutes of partial hepatic ischemia and up to 24 hours of reperfusion. In wild-type mice, expression of IL-10 and IL-13 shared similar expression profiles with maximal production after 8 hours of reperfusion. There were no significant differences between wild-type and IL-10­/­ mice in response to hepatic ischemia and reperfusion. IL-13­/­ mice had much greater liver injury, as assessed biochemically and histologically, than wild-type mice. There were no differences between wild-type and IL-13­/­ mice in their production of inflammatory cytokines, but IL-13­/­ mice displayed disrupted neutrophil accumulation, with less neutrophils present in the hepatic parenchyma and far more neutrophils adherent to the endothelium of large hepatic venules than wild-type mice. These observations were associated with increased liver endothelial cell injury in IL-13­/­ mice, as measured by serum levels of hyaluronic acid. In vitro, IL-13 protected hepatocytes from H2O2-induced cytotoxicity. In conclusion, IL-10 is not an important endogenous regulator of the inflammatory response to hepatic ischemia/reperfusion. In contrast, endogenous IL-13 appears to be critical for the control of this response, with prominent protective effects on hepatocytes and hepatic endothelial cells. (HEPATOLOGY 2003;37:304-312.)

Effect of cold preservation on intracellular calcium concentration and calpain activity in rat sinusoidal endothelial cells
G. Aravinda Upadhya, Stefan A. Topp, Richard S. Hotchkiss, John Anagli, Steven M. Strasberg
This study was performed to determine the role of intracellular calcium concentration and calpain activity on the cellular events that occur in rat sinusoidal endothelial cells (SEC) in the cold. Intracellular calcium concentrations were measured in isolated cold preserved rat SEC. Dantrolene or 1,2-bis(o-Aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM) was added in some studies. In other studies, calpain activity and m-calpain and µ-calpain expression were measured during cold preservation in the presence or absence of calpain inhibitors. The effect of addition of dantrolene to preservation solutions on function of whole livers after preservation was determined. Cold preservation caused an increase in intracellular calcium concentration first detected at 1 hour of preservation. This was associated with cell rounding and actin disassembly. Dantrolene and BAPTA-AM prevented the increase in intracellular calcium concentration and reduced cell rounding and actin disassembly. Cold preservation also resulted in increased calpain activity and expression on SEC. Calpain expression was reduced by dantrolene. Calpain inhibitors N-acetyl-leu-leu-norleucinal (ALLN) and N-acetyl-leu-leu-methioninal (ALLM) reduced calpain activity and expression and restored SEC cell shape and actin morphology. Dantrolene improved function of livers preserved in Eurocollins (EC) solution when tested on the isolated perfused rat liver (IPRL). In conclusion, exposure of SEC to cold results sequentially in elevated intracellular calcium concentration, increased calpain activity, and actin disassembly. (HEPATOLOGY 2003;37:313-323.)

The nitric oxide donor, V-PYRRO/NO, protects against acetaminophen-induced hepatotoxicity in mice
Jie Liu, Chengxiu Li, Michael P. Waalkes, James Clark, Page Myers, Joseph E. Saavedra, Larry K. Keefer
The liver-selective nitric oxide (NO) donor, O2-vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (V-PYRRO/NO), is metabolized by P-450 enzymes to release NO in the liver, and is shown to protect the liver from tumor necrosis factor (TNF-)-induced apoptosis and D-glactosamine/endotoxin­induced hepatotoxicity. This study was undertaken to examine the effects of V-PYRRO/NO on acetaminophen-induced hepatotoxicity in mice. Mice were given V-PYRRO/NO via osmotic pumps (1.8-5.4 mg/mL, 8 µL/h) 4 to 16 hours before a hepatotoxic dose of acetaminophen (600 mg/kg, intraperitoneally [ip]). V-PYRRO/NO administration dramatically reduced acetaminophen-induced hepatotoxicity in a dose- and time-dependent manner, as evidenced by reduced serum alanine aminotransferase (ALT) activity, reduced hepatic congestion, apoptosis, and improved hepatocellular pathology. The protection afforded by V-PYRRO/NO does not appear to be caused by a decrease in the formation of toxic acetaminophen metabolites, which consumes glutathione (GSH), because V-PYRRO/NO did not alter acetaminophen-induced hepatic GSH depletion. Acetaminophen-induced lipid peroxidation, as determined by the concentrations of 4-hydroxyalkenals (4-HNE) and malondialdehyde (MDA), was reduced significantly by V-PYRRO/NO treatment. Although pretreatment was most effective, administration of V-PYRRO/NO simultaneously with acetaminophen also was able to reduce acetaminophen hepatotoxicity. Genomic analysis of the liver samples 10 hours after acetaminophen intoxication showed the enhanced expression of genes associated with stress/oxidative stress, apoptosis/cell death, and DNA damage/repair. Acetaminophen-induced alterations in gene expression were attenuated significantly by V-PYRRO/NO. Real-time reverse-transcription polymerase chain reaction (RT-PCR) and Western-blot analysis confirmed microarray results. In conclusion, V-PYRRO/NO is effective in blocking acetaminophen-induced hepatotoxicity in mice. This protection may involve the reduction of oxidative stress, the inhibition of apoptosis, and possibly the maintenance of hepatic vasculature to prevent congestion. (HEPATOLOGY 2003;37:324-333.)

Benzodiazepine-induced protein tyrosine nitration in rat astrocytes
Boris Görg, Natalie Foster, Roland Reinehr, Hans J. Bidmon, Andrea Höngen, Dieter Häussinger, Freimut Schliess
See Editorial on Page 245
Recent studies indicate that ammonia and hypoosmotic astrocyte swelling can induce protein tyrosine nitration (PTN) in astrocytes with potential pathogenetic relevance for hepatic encephalopathy (HE). Because HE episodes are known to be precipitated also by sedatives, the effects of benzodiazepines on PTN in cultured rat astrocytes and rat brain in vivo were studied. In cultured rat astrocytes, diazepam, PK11195, Ro5-4864, and the benzodiazepine binding inhibitor (DBI), which acts on peripheral-type benzodiazepine receptors, induced PTN. Clonazepam, a specific ligand of the central benzodiazepine receptor, failed to induce PTN. Nanomolar concentrations of DBI and PK11195 were sufficient to increase PTN, and diazepam effects were already observed at concentrations of 1 µmol/L. Diazepam-induced PTN was insensitive to NOS inhibition and uric acid but was blunted by MK-801, BAPTA-AM, W13, and catalase, suggesting an involvement of NMDA-receptor activation, elevation of the cytosolic Ca2+ concentration [Ca2+]i, and hydrogen peroxide. Diazepam induced a plateau-like increase in [Ca2+]i and the generation of reactive oxygen intermediates (ROIs), which are both blunted by MK-801 and BAPTA-AM. The expression of functional N-methyl-D-aspartate (NMDA) receptors on cultured rat astrocytes was confirmed by reverse transcriptase polymerase chain reaction, Western blot analysis, immunhistochemistry, and receptor autoradiography. Astroglial PTN is also found in brains from rats challenged with diazepam, indicating the in vivo relevance of the present findings. In conclusion, production of ROIs and increased PTN by benzodiazepines may alter astrocyte function and thereby contribute to the precipitation of HE episodes. (HEPATOLOGY 2003;37:334-342.)

Probiotics and antibodies to TNF inhibit inflammatory activity and improve nonalcoholic fatty liver disease
Zhiping Li, Shiqi Yang, Huizhi Lin, Jiawen Huang, Paul A. Watkins, Ann B. Moser, Claudio DeSimone, Xiao-yu Song, Anna Mae Diehl
Ob/ob mice, a model for nonalcoholic fatty liver disease (NAFLD), develop intestinal bacterial overgrowth and overexpress tumor necrosis factor (TNF-). In animal models for alcoholic fatty liver disease (AFLD), decontaminating the intestine or inhibiting TNF- improves AFLD. Because AFLD and NAFLD may have a similar pathogenesis, treatment with a probiotic (to modify the intestinal flora) or anti-TNF antibodies (to inhibit TNF- activity) may improve NAFLD in ob/ob mice. To evaluate this hypothesis, 48 ob/ob mice were given either a high-fat diet alone (ob/ob controls) or the same diet + VSL#3 probiotic or anti-TNF antibodies for 4 weeks. Twelve lean littermates fed a high-fat diet served as controls. Treatment with VSL#3 or anti-TNF antibodies improved liver histology, reduced hepatic total fatty acid content, and decreased serum alanine aminotransferase (ALT) levels. These benefits were associated with decreased hepatic expression of TNF- messenger RNA (mRNA) in mice treated with anti-TNF antibodies but not in mice treated with VSL#3. Nevertheless, both treatments reduced activity of Jun N-terminal kinase (JNK), a TNF-regulated kinase that promotes insulin resistance, and decreased the DNA binding activity of nuclear factor B (NF-B), the target of IKK, another TNF-regulated enzyme that causes insulin resistance. Consistent with treatment-related improvements in hepatic insulin resistance, fatty acid -oxidation and uncoupling protein (UCP)-2 expression decreased after treatment with VSL#3 or anti-TNF antibodies. In conclusion, these results support the concept that intestinal bacteria induce endogenous signals that play a pathogenic role in hepatic insulin resistance and NAFLD and suggest novel therapies for these common conditions. (HEPATOLOGY 2003;37:343-350.)

Uroporphyria caused by ethanol in Hfe(­/­) mice as a model for porphyria cutanea tarda
Peter R. Sinclair, Nadia Gorman, Heidi W. Trask, William J. Bement, Juliana G. Szakacs, George H. Elder, Dominic Balestra, Jacqueline F. Sinclair, Glenn S. Gerhard
Two major risk factors for the development of porphyria cutanea tarda (PCT) are alcohol consumption and homozygosity for the C282Y mutation in the hereditary hemochromatosis gene (HFE). To develop an animal model, Hfe knockout mice were treated continuously with 10% ethanol in drinking water. By 4 months, uroporphyrin (URO) was detected in the urine. At 6 to 7 months, hepatic URO was increased and hepatic uroporphyrinogen decarboxylase (UROD) activity was decreased. Untreated Hfe(­/­) mice or wild-type mice treated with or without ethanol did not show any of these biochemical changes. Treatment with ethanol increased hepatic nonheme iron and hepatic 5-aminolevulinate synthase activity in Hfe(­/­) but not wild-type mice. The increases in nonheme iron in Hfe(­/­) mice were associated with diffuse increases in iron staining of parenchymal cells but without evidence of significant liver injury. In conclusion, the results of this study suggest that the uroporphyrinogenic effect of ethanol is mediated by its effects on hepatic iron metabolism. Ethanol-treated Hfe(­/­) mice seem to be an excellent model for studies of alcohol-mediated PCT. (HEPATOLOGY 2003;37:351-358.)

Liver Failure and Liver Disease

Nadolol plus spironolactone in the prophylaxis of first variceal bleed in nonascitic cirrhotic patients: A preliminary study (*Human Study*)
Raquel Abecasis, David Kravetz, Eduardo Fassio, Beatriz Ameigeiras, Daniel Garcia, Rogelio Isla, Graciela Landeira, Nora Dominguez, Gustavo Romero, Julio Argonz, Ruben Terg
Treatment with -blockers fails to decrease portal pressure in nearly 40% of cirrhotic patients. Recent studies have suggested that treatment with spironolactone reduces pressure and flow in the portal and variceal systems. This trial was designed to assess if nadolol plus spironolactone is more effective than nadolol alone to prevent the first variceal bleeding. One hundred patients with medium and large varices who had never bled and were without ascites were included in a prospective, randomized, multicenter, double-blind, placebo-controlled trial. The patients were randomized into 2 groups: 51 received nadolol plus placebo (N + P) and 49 received nadolol plus spironolactone 100 mg/d (N + S). Hepatic venous pressure gradient (HVPG) and activity of the renin-aldosterone system (plasma renin activity/plasma aldosterone levels) were measured in 24 patients. There were no significant differences in the appearance of variceal bleeding and ascites between groups at a mean follow-up of 22 ± 16 months. However, analyzing both complications together, the incidence was significantly higher in the N + P group than in the N + S group (39% vs. 20%; P < .04). Clinical ascites was also higher in patients in the N + P group than in the N + S group (21% vs. 6%; P < .04). Significant increases in plasma renin activity and plasma aldosterone levels were only observed in patients in the N + S group (P < .01). The cumulative probabilities of remaining free of bleeding and ascites were similar in both groups after 70 months of follow-up. In conclusion, these results suggest that nadolol plus spironolactone does not increase the efficacy of nadolol alone in the prophylaxis of the first variceal bleeding. However, when bleeding and ascites were considered together, the combined therapy effectively reduced the incidence of both portal-hypertensive complications. (HEPATOLOGY 2003;37:359-365.)

Endoscopic screening for esophageal varices in cirrhosis: Is it ever cost effective?
Brennan M. R. Spiegel, Laura Targownik, Gareth S. Dulai, Hetal A. Karsan, Ian M. Gralnek
Current guidelines for the management of patients with compensated cirrhosis recommend universal screening endoscopy followed by prophylactic -blocker therapy to prevent initial hemorrhage in those found to have esophageal varices. However, the cost-effectiveness of this recommendation has not been established. Our objective was to determine whether screening endoscopy is cost-effective compared with empiric medical management in patients with compensated cirrhosis. Decision analysis with Markov modeling was used to calculate the cost-effectiveness of 6 competing strategies: (1) universal screening endoscopy (EGD) followed by -blocker (BB) therapy (EGDBB) if varices are present, (2) EGD followed by endoscopic band ligation (EBL) (EGDEBL) if varices are present, (3) selective screening endoscopy (sEGD) in high risk patients followed by BB therapy if varices are present (sEGDBB), (4) selective screening endoscopy followed by EBL (sEGDEBL) if varices are present, (5) empiric -blocker therapy in all patients, and (6) no prophylactic therapy ("Do Nothing"). Cost estimates were from a third-party payer perspective. The main outcome measure was the cost per initial variceal hemorrhage prevented. The "Do Nothing" strategy was the least expensive yet least effective approach. Compared with the "Do Nothing" strategy, the empiric -blocker strategy cost an incremental $12,408 per additional variceal bleed prevented. Compared with the empiric -blocker strategy, in turn, both the EGDBB and the EGDEBL strategies cost over $175,000 more per additional bleed prevented. The sEGDBB and sEGDEBL strategies were more expensive and less effective than the empiric -blocker strategy. In conclusion, empiric -blocker therapy for the primary prophylaxis of variceal hemorrhage is a cost-effective measure, as the use of screening endoscopy to guide therapy adds significant cost with only marginal increase in effectiveness. (HEPATOLOGY 2003;37:366-377.)

Low doses of isosorbide mononitrate attenuate the postprandial increase in portal pressure in patients with cirrhosis
Lia Bellis, Annalisa Berzigotti, Juan G. Abraldes, Eduardo Moitinho, Juan C. García-Pagán, Jaime Bosch, Juan Rodés
Postprandial hyperemia is associated with a significant increase in portal pressure in cirrhosis, which may contribute to progressive dilation and rupture of gastroesophageal varices. In cirrhosis, an insufficient hepatic production of nitric oxide (NO) may impair the expected hepatic vasodilatory response to increased blood flow, further exaggerating the postprandial increase in portal pressure. This study was aimed at investigating whether low doses of an oral NO donor might counteract the postprandial peak in portal pressure. Twenty-three portal hypertensive cirrhotics, 8 of them under propranolol therapy, were randomized to receive orally 5-isosorbide mononitrate (ISMN; 10 mg; n = 11) or placebo (n = 12) and a standard liquid meal 15 minutes later. Hepatic venous pressure gradient (HVPG), mean arterial pressure (MAP), and hepatic blood flow (HBF) were measured at baseline and 15, 30, and 45 minutes after a meal. ISMN significantly attenuated the postprandial increase in portal pressure as compared with placebo (peak HVPG increase: 2.4 ± 1.4 mm Hg vs. 5.2 ± 2.1 mm Hg, P = .002). Percentual increases in HBF were similar in both groups. MAP decreased slightly in ISMN group (­7.5% ± .5%; P < .01 vs. baseline). These effects were also observed in patients on chronic propranolol therapy. In conclusion, hepatic NO supplementation by low doses of ISMN effectively reduces the postprandial increase of portal pressure in cirrhosis, with only a mild effect on arterial pressure. The same was observed in patients receiving propranolol. Our results suggest that therapeutic strategies based on selective hepatic NO delivery may improve the treatment of portal hypertension. (HEPATOLOGY 2003;37:378-384.)

Functional significance of hepatic arterial flow reserve in patients with cirrhosis (*Human Study*)
Alexander Zipprich, Norbert Steudel, Curd Behrmann, Frank Meiss, Ursula Sziegoleit, Wolfgang E. Fleig, Gerhard Kleber
In cirrhosis, hepatic arterial vasodilatation occurs in response to reduced portal venous blood flow. However, although the hepatic arterial flow reserve is high in patients with cirrhosis, its impact on hepatic function is unknown. This study investigated the effect of adenosine-induced hepatic arterial vasodilatation on different markers of liver function. In 20 patients with cirrhosis (Child-Pugh class A/B/C: n = 2/7/11) adenosine (2-30 µg · min­1 · kg body wt­1) was infused into the hepatic artery and hepatic arterial average peak flow velocities (APV), pulsatility indices (PI), and blood flow volumes (HABF) were measured using digital angiography and intravascular Doppler sonography. Indocyanine green (ICG), lidocaine, and galactose were administered intravenously in doses of 0.5, 1.0, and 500 mg/kg body weight in the presence of adenosine-induced hepatic arterial vasodilatation and, on a separate study day, without adenosine. ICG disappearance, galactose elimination capacity (GEC), and formation of the lidocaine metabolite monoethylglycinxylidide (MEGX) were assessed. Adenosine markedly increased APV and HABF and markedly decreased PI. Serum MEGX concentrations were 63.7 ± 18.2 (median, 62; range, 36-107) and 99.0 ± 46.3 (82.5; 49-198) ng/mL in the absence and presence of adenosine infusion, respectively (P = .001). Adenosine-induced changes in MEGX concentrations were correlated inversely to changes in APV (r = ­0.5, P = .02) and PI (r = ­0.55, P = .01) and were more marked in Child-Pugh class C compared with Child-Pugh class A patients (57.4 ± 49.9 [44; ­14 to 140] vs. 8.4 ± 16.5 [13; ­11 to 35] ng/mL, P < .01). In conclusion, hepatic arterial vasodilatation provides substantial functional benefit in patients with cirrhosis. The effect does not depend directly on hepatic arterial macroperfusion and is observed preferentially in patients with decompensated disease. (HEPATOLOGY 2003;37:385-392.)

Cardiac hepatopathy: Clinical, hemodynamic, and histologic characteristics and correlations (*Human Study*)
Robert P. Myers, Raimondo Cerini, Raymond Sayegh, Richard Moreau, Claude Degott, Didier Lebrec, Samuel S. Lee
Cardiac hepatopathy, hepatic injury caused by cardiac dysfunction, is a common entity but has been characterized incompletely, particularly the relationship between hemodynamics and histology. We aimed to describe the clinical, biochemical, hemodynamic, and histologic characteristics of this disorder. Eighty-three patients from 2 tertiary referral centers were studied. Patients were divided into 3 groups based on the duration of cardiac dysfunction: (1) acute (n = 12); (2) chronic (n = 53); and (3) acute on chronic (n = 18). Results showed that serum aminotransferase levels were increased typically, particularly in the acute group (median aspartate aminotransferase level was 30.2 times the upper limit of normal [range, 1-100]; P < .0001 vs. the chronic group). The most salient hemodynamic features were elevated right atrial (14 mm Hg [range, 1-29]), and hepatic venous pressures (wedged: 18 mm Hg [range, 5-35]; free: 15 mm Hg [range, 2-30]). The hepatic venous pressure gradient was normal in most (81%), correlated moderately with the aminotransferase levels (aspartate aminotransferase level: r = .59; P < .0001), and associated with the presence of centrilobular necrosis and inflammation, periportal necrosis, and stainable hepatic iron (P < .05 for all comparisons), but not fibrosis. Sinusoidal dilatation was associated with higher right atrial (P = .047) and free hepatic venous pressures (P = .06). Although cirrhosis was rare (n = 1), centrilobular fibrosis was common (74%) and not associated with any hemodynamic measurement. In conclusion, cardiac hepatopathy has diverse clinical, hemodynamic, and histologic manifestations that vary with the temporal course of cardiac dysfunction. Hepatic fibrosis is common, but does not correlate with systemic or hepatic hemodynamics. (HEPATOLOGY 2003;37:393-400.)

Diagnosis of portopulmonary hypertension in candidates for liver transplantation: A prospective study (*Human Study*)
Isabelle O. Colle, Richard Moreau, Erica Godinho, Jacques Belghiti, Florence Ettori, Alain Cohen-Solal, Hervé Mal, Jacques Bernuau, Jean Marty, Didier Lebrec, Dominique Valla, François Durand
Portopulmonary hypertension represents a major risk factor for transplantation; therefore, preoperative detection is crucial. The aims of this study were to determine (1) whether Doppler echocardiography performed at evaluation is a reliable tool for detecting portopulmonary hypertension and (2) the incidence of acquired portopulmonary hypertension profile after evaluation. One hundred sixty-five patients had Doppler echocardiography and right heart catheterization at evaluation over a 9-year period. All patients had a prospective follow-up, and the results of catheterization at evaluation were compared with those obtained at the time of transplantation. Seventeen of 165 patients met the criteria for portopulmonary hypertension on Doppler echocardiography. Portopulmonary hypertension was confirmed by catheterization in 10 patients and ruled out in 7. There were no false negatives for echocardiography. Mean pulmonary artery pressure was significantly higher during the initial phase of transplantation than at evaluation (17.8 ± 4.3 vs. 20.3 ± 5.5 mm Hg, respectively, P < .0001), and there was no significant correlation between values obtained at these 2 time points. Three patients showed to have acquired portopulmonary hypertension profile while waiting for a graft within time intervals ranging from 2.5 to 5 months. In conclusion, Doppler echocardiography is a highly sensitive tool for detecting portopulmonary hypertension. However, because this technique has a poor positive predictive value, right heart catheterization is recommended for confirming portopulmonary hypertension. In addition, the absence of portopulmonary hypertension at evaluation does not exclude the occasional occurrence of acquired portopulmonary hypertension profile after listing. (HEPATOLOGY 2003;37:401-409.)

Genetic and epigenetic factors in autoimmune reactions toward cytochrome P4502E1 in alcoholic liver disease (*Human Study*)
Matteo Vidali, Stephen F. Stewart, Roberta Rolla, Ann K. Daly, Yuanneng Chen, Elisa Mottaran, David E. J. Jones, Julian B. Leathart, Christopher P. Day, Emanuele Albano
Autoimmune reactions are often associated with alcoholic liver disease; however, the mechanisms responsible are largely unknown. This study investigates the potential role of the immune response against hydroxyethyl free radical (HER)-derived antigens and of polymorphisms in immunoregulatory genes in the development of anti-cytochrome P4502E1 (CYP2E1) autoantibodies in alcohol abusers. Immunoglobulin G (IgG) recognizing human CYP2E1 and HER-derived epitopes were measured by microplate immunosorbent assay in the sera of 90 patients with alcoholic fibrosis/cirrhosis (ALD), 37 heavy drinkers without liver disease or steatosis only (HD), and 59 healthy subjects. Single nucleotide polymorphisms in the interleukin 10 (IL-10) promoter and in exon 1 of the cytotoxic T-lymphocyte antigen-4 (CTLA-4) gene were genotyped by polymerase chain reaction-restriction fragment length polymorphism analysis. The titers and frequency of anti-CYP2E1 autoantibodies were significantly higher in ALD than in HD subjects or controls. ALD patients with anti-HER IgG had higher titers and a 4-fold increased risk (OR: 4.4 [1.8-10.9]) of developing anti-CYP2E1 autoantibodies than subjects without anti-HER antibodies. The mutant CTLA-4 G allele, but not the IL-10 polymorphism, was associated with an enhanced risk of developing anti-CYP2E1 IgG (OR: 3.8 [1.4-10.3]). CTLA-4 polymorphism did not influence antibody formation toward HER-antigens. ALD patients with concomitant anti-HER IgG and the CTLA-4 G allele had a 22-fold higher (OR: 22.9 [4.2-125.6]) risk of developing anti-CYP2E1 autoreactivity than subjects negative for these factors. In conclusion, antigenic stimulation by HER-modified CYP2E1 combined with an impaired control of T-cell proliferation by CTLA-4 mutation promotes the development of anti-CYP2E1 autoantibodies that might contribute to alcohol-induced liver injury. (HEPATOLOGY 2003;37:410-419.)

Selective increase of brain lactate synthesis in experimental acute liver failure: Results of a [1H-13C] nuclear magnetic resonance study
Claudia Zwingmann, Nicolas Chatauret, Dieter Leibfritz, Roger F. Butterworth
Acute liver failure (ALF) results in alterations of energy metabolites and of glucose-derived amino acid neurotransmitters in brain. However, the dynamics of changes in glucose metabolism remain unclear. The present study was undertaken using 1H and 13C nuclear magnetic resonance (NMR) spectroscopy to determine the rates of incorporation of glucose into amino acids and lactate via cell-specific pathways in relation to the severity of encephalopathy and brain edema in rats with ALF because of hepatic devascularization. Early (precoma) stages of encephalopathy were accompanied by significant 2- to 4.5-fold (P < .001) increases of total brain glutamine and lactate concentrations. More severe (coma) stages of encephalopathy and brain edema led to a further significant increase in brain lactate but no such increase in glutamine. Furthermore, 13C isotopomer analysis showed a selective increase of de novo synthesis of lactate from [1-13C]glucose resulting in 2.5-fold increased fractional 13C enrichments in lactate at coma stages. [2-13C]glutamine, synthesized through the astrocytic enzyme pyruvate carboxylase, increased 10-fold at precoma stages but showed no further increase at coma stages of encephalopathy. 13C-label incorporation into [4-13C]glutamate, synthesized mainly through neuronal pyruvate dehydrogenase, was selectively reduced at coma stages, whereas brain GABA synthesis was unchanged at all time points. In conclusion, increased brain lactate synthesis and impaired glucose oxidative pathways rather than intracellular glutamine accumulation are the major cause of brain edema in ALF. Future NMR spectroscopic studies using stable isotopes and real-time measurements of metabolic rates could be valuable in the elucidation of the cerebral metabolic consequences of ALF in humans. (HEPATOLOGY 2003;37:420-428.)

Systematic review of randomized trials for unresectable hepatocellular carcinoma: Chemoembolization improves survival
Josep M. Llovet, Jordi Bruix, for the Barcelona-Clínic Liver Cancer Group
There is no standard treatment for patients with unresectable hepatocellular carcinoma (HCC). Survival benefits derived from medical interventions are controversial. The aim of this systematic review was to assess the evidence of the impact of medical treatments on survival. Randomized controlled trials (RCTs) that were published as full papers assessing survival for primary treatments of HCC were included. MEDLINE, the Cochrane Library, CANCERLIT, and a manual search from 1978 to May 2002 were used. The primary end point was survival, and the secondary end point was response to treatment. Estimates of effect were calculated according to the random effects model. Sensitivity analysis included methodological quality. We identified 61 randomized trials, but only 14 met the criteria to perform a meta-analysis assessing arterial embolization (7 trials, 545 patients) or tamoxifen (7 trials, 898 patients). Arterial embolization improved 2-year survival compared with control (odds ratio [OR], 0.53; 95% confidence interval [CI], 0.32-0.89; P = .017). Sensitivity analysis showed a significant benefit of chemoembolization with cisplatin or doxorubicin (OR, 0.42; 95% CI, 0.20-0.88) but none with embolization alone (OR, 0.59; 95% CI, 0.29-1.20). Overall, treatment induced objective responses in 35% of patients (range, 16%-61%). Tamoxifen showed no antitumoral effect and no survival benefits (OR, 0.64; 95% CI, 0.36-1.13; P = .13), and only low-quality scale trials suggested 1-year improvement in survival. In conclusion, chemoembolization improves survival of patients with unresectable HCC and may become the standard treatment. Treatment with tamoxifen does not modify the survival of patients with advanced disease. (HEPATOLOGY 2003;37:429-442.)

Viral Hepatitis

Adherence and mental side effects during hepatitis C treatment with interferon alfa and ribavirin in psychiatric risk groups (*Human Study*)
Martin Schaefer, Folkhard Schmidt, Christian Folwaczny, Reinhard Lorenz, Gaby Martin, Norbert Schindlbeck, Walter Heldwein, Michael Soyka, Heinz Grunze, August Koenig, Klaus Loeschke
Psychiatric disorders or drug addiction are often regarded as contraindications against the use of interferon alfa (IFN-) in patients with chronic hepatitis C. Our aim was to obtain prospective data on adherence to as well as efficacy and mental side effects of treatment with IFN- in different psychiatric risk groups compared with controls. In a prospective trial, 81 patients with chronic hepatitis C (positive hepatitis C virus[HCV] RNA and elevated alanine aminotransferase[ALT] level) and psychiatric disorders (n = 16), methadone substitution (n = 21), former drug addiction (n = 21), or controls without a psychiatric history or drug addiction (n = 23) were treated with a combination of IFN--2a 3 MU 3 times weekly and ribavirin (1,000-1,200 mg/d). Sustained virologic response (overall, 37%) did not differ significantly between subgroups. No significant differences between groups were detected with respect to IFN-­related development of depressions during treatment. However, in the psychiatric group, significantly more patients received antidepressants before and during treatment with IFN- (P < .001). Most of those who dropped out of the study were patients with former drug addiction (43%; P = .04) compared with 14% in the methadone group, 13% in the control group, and 18% in the psychiatric group. No patient in the psychiatric group had to discontinue treatment because of psychiatric deterioration. In conclusion, our data do not confirm the supposed increased risk for IFN-­induced mental side effects and dropouts in psychiatric patients if interdisciplinary care and antidepressant treatment are available. Preexisting psychiatric disorders or present methadone substitution should no longer be regarded as contraindications to treatment of chronic hepatitis C with IFN- and ribavirin in an interdisciplinary setting. (HEPATOLOGY 2003;37:443-451.)

Modulation of cellular immune response against hepatitis C virus nonstructural protein 3 by cationic liposome encapsulated DNA immunization
Xuanmao Jiao, Richard Y.-H. Wang, Zhiming Feng, Harvey J. Alter, James Wai-Kuo Shih
A vaccine strategy directed to increase Th1 cellular immune responses, particularly to hepatitis C virus (HCV) nonstructural protein 3 (NS3), has considerable potential to overcome the infection with HCV. DNA vaccination can induce both humoral and cellular immune responses, but it became apparent that the cellular uptake of naked DNA injected into muscle was not very efficient, as much of the DNA is degraded by interstitial nucleases before it reaches the nucleus for transcription. In this paper, cationic liposomes composed of different cationic lipids, such as dimethyl-dioctadecylammonium bromide (DDAB), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), or 1,2-dioleoyl-sn-glycerol-3-ethylphosphocholine (DOEPC), were used to improve DNA immunization in mice, and their efficiencies were compared. It was found that cationic liposome-mediated DNA immunization induced stronger HCV NS3-specific immune responses than immunization with naked DNA alone. Cationic liposomes composed of DDAB and equimolar of a neutral lipid, egg yolk phosphatidylcholine (EPC), induced the strongest antigen-specific Th1 type immune responses among the cationic liposome investigated, whereas the liposomes composed of 2 cationic lipids, DDAB and DOEPC, induced an antigen-specific Th2 type immune response. All cationic liposomes used in this study triggered high-level, nonspecific IL-12 production in mice, a feature important for the development of maximum Th1 immune responses. In conclusion, the cationic liposome-mediated gene delivery is a viable HCV vaccine strategy that should be further tested in the chimpanzee model. (HEPATOLOGY 2003;37:452-460.)

A recombinant adenovirus encoding hepatitis C virus core and E1 proteins protects mice against cytokine-induced liver damage
Juan José Lasarte, Pablo Sarobe, Patricia Boya, Noelia Casares, Laura Arribillaga, Ascensión López-Díaz de Cerio, Marta Gorraiz, Francisco Borrás-Cuesta, Jesús Prieto
Hepatitis C virus (HCV) infection has a strong tendency to evolve to chronicity despite up-regulation of proapoptotic cytokines in the inflamed liver. The mechanisms responsible for persistent viral replication in this inflammatory environment are obscure. It is conceivable that viral replication would be facilitated if the infected hepatocytes are rendered resistant to cytokine-induced cytotoxicity. In this study, we investigated if an adenovirus encoding HCV core and E1 (RAdCE1) could reduce liver cell injury in different in vivo models of cytokine-mediated hepatotoxicity in mice. We show that RAdCE1 markedly attenuates hepatocellular apoptosis and the increase in serum transaminase levels after concanavalin A (con A) challenge. This protective effect is accompanied by an inhibition of nuclear translocation of nuclear factor B (NF-B); reduced expression of inducible nitric oxide synthase (iNOS); decreased hepatic messenger RNA levels of chemokines macrophage inflammatory protein 2 (MIP-2), monocyte chemoattractant protein 1 (MCP-1), and interferon-inducible protein 10 (IP-10); and abrogation of liver leukocyte infiltration. RAdCE1 also causes a reduction in serum transaminase levels and inhibits hepatocellular apoptosis in mice given tumor necrosis factor (TNF)- plus D-galactosamine. In conclusion, HCV structural antigens can protect liver cells against the proapoptotic effects of proinflammatory cytokines. The antiapoptotic status of infected liver cells may represent a mechanism favoring viral persistence. Our findings also suggest that, in chronic hepatitis C, the burden of hepatocellular damage mainly affects noninfected liver cells. (HEPATOLOGY 2003;37:461-470.)

GASTROENTEROLOGY

Table of Contents for February 2003 · Volume 124 · Number 2

Clinical­Alimentary Tract

Serious lower gastrointestinal clinical events with nonselective NSAID or coxib use
L. Laine, L. G. Connors, A. Reicin, C. J. Hawkey, R. Burgos­Vargas, T. J. Schnitzer, Q. Yu, C. Bombardier
Background & Aims: Epidemiologic studies suggest nonsteroidal anti-inflammatory drugs (NSAIDs) increase the risk for lower gastrointestinal (GI) clinical events, but data from prospective trials are lacking. Cyclooxygenase (COX)-2­selective inhibitors decrease upper GI clinical events but the effect on lower GI events has not been determined. We performed a post hoc analysis of serious lower GI clinical events with a nonselective NSAID and a COX-2­selective agent in a prospective, double-blind, randomized GI outcomes trial.
Methods: A total of 8076 rheumatoid arthritis patients 50 years or older (or 40 years or older on corticosteroid therapy) expected to require NSAIDs for 1 year or greater were randomly assigned to naproxen 500 mg twice daily or rofecoxib 50 mg daily. The rate of serious lower GI clinical events, defined as bleeding with a 2 g/dL drop in hemoglobin or hospitalization, or hospitalization for perforation, obstruction, ulceration, or diverticulitis, was determined.
Results: The rate of serious lower GI events per 100 patient-years was 0.41 for rofecoxib and 0.89 for naproxen (relative risk, 0.46; 95% confidence interval [CI], 0.22­0.93; P = 0.032). Serious lower GI events accounted for 39.4% of all serious GI events (complicated upper GI event or lower GI event) among patients taking naproxen and 42.7% among those taking rofecoxib.
Conclusions: Serious lower GI events occurred at a rate of 0.9% per year in rheumatoid arthritis patients taking the nonselective NSAID naproxen, accounting for nearly 40% of the serious GI events that developed in these patients. Serious lower GI events were 54% lower with the use of the selective COX-2 inhibitor rofecoxib.

Low-dose growth hormone in adult home parenteral nutrition­dependent short bowel syndrome patients: A positive study
D. Seguy, K. Vahedi, N. Kapel, J.­C. Souberbielle, B. Messing
Background & Aims: Controversy surrounds a 3-week treatment with a high-dose (0.14 mg · kg­1 · day­1) growth hormone (GH), glutamine, and high carbohydrate diet in home parenteral nutrition (HPN)-dependent patients with short-bowel syndrome (SBS). This study assessed treatment with low-dose GH in these patients.
Methods: Twelve adult HPN-dependent (duration, 7 ± 1 years; mean ± SEM) patients with SBS (small-bowel remnant length, 48 ± 11 cm) who were on an unrestricted hyperphagic diet were randomized in a double-blind, placebo-controlled, crossover study. Patients received daily low-dose GH (0.05 mg · kg­1 · day­1) and placebo for two 3-week periods separated by a 1-week washout period. Net intestinal absorption of macronutrients was assessed using a duplicate diet; nutritional assessment and blood tests were performed. Data from each group were compared using Wilcoxon rank sum test.
Results: Treatment with GH increased intestinal absorption of energy (15% ± 5%, P < 0.002), nitrogen (14% ± 6%, P < 0.04), carbohydrates (10% ± 4%, P < 0.04), and fat (12% ± 8%, NS). The increased food absorption represented 37% ± 16% of total parenteral energy delivery. Body weight (P < 0.003), lean body mass (P < 0.006), D-xylose absorption (P < 0.02), insulin-like growth factor 1 (P < 0.002), and insulin-like growth factor binding protein 3 (P < 0.002) increased, whereas uptake of GH binding protein decreased (P < 0.01), without any major adverse effect.
Conclusions: Three weeks of low-dose GH significantly improved intestinal absorption in HPN-dependent SBS patients who were on a hyperphagic western diet.

The cost-effectiveness of psychotherapy and paroxetine for severe irritable bowel syndrome
F. Creed, L. Fernandes, E. Guthrie, S. Palmer, J. Ratcliffe, N. Read, C. Rigby, D. Thompson, B. Tomenson, on Behalf of The North of England IBS Research Group
Background & Aims: Psychotherapy and antidepressants are effective in patients with severe irritable bowel syndrome (IBS), but the cost-effectiveness of either treatment in routine practice has not been established.
Methods: Patients with severe IBS were randomly allocated to receive 8 sessions of individual psychotherapy, 20 mg daily of the specific serotonin reuptake inhibitor (SSRI) antidepressant, paroxetine, or routine care by a gastroenterologist and general practitioner. Primary outcome measures of abdominal pain, health-related quality of life, and health care costs were determined after 3 months of treatment and 1 year later.
Results: A total of 257 subjects (81% response rate) from 7 hospitals were recruited; 59 of 85 patients (69%) randomized to psychotherapy and 43 of 86 (50%) of the paroxetine group completed the full course of treatment. Both psychotherapy and paroxetine were superior to treatment as usual in improving the physical aspects of health-related quality of life (SF-36 physical component score improvement, 5.2 [SEM, 1.26], 5.8 [SEM, 1.0], and ­0.3 [SEM, 1.17]; P < 0.001), but there was no difference in the psychological component. During the follow-up year, psychotherapy but not paroxetine was associated with a significant reduction in health care costs compared with treatment as usual (psychotherapy, $976 [SD, $984]; paroxetine, $1252 [SD, $1616]; and treatment as usual, $1663 [SD, $3177]).
Conclusions: For patients with severe IBS, both psychotherapy and paroxetine improve health-related quality of life at no additional cost.

Clinical­Liver, Pancreas, and Biliary Tract

Hepatitis C, iron status, and disease severity: Relationship with HFE mutations
B. Y. Tung, M. J. Emond, M. P. Bronner, S. D. Raaka, S. J. Cotler, K. V. Kowdley
Background & Aims: Mild to moderate hepatic iron loading is common in patients with chronic hepatitis C. We sought to determine whether mutations in the hemochromatosis gene, HFE, are associated with iron overload and acceleration of disease progression in hepatitis C patients.
Methods: A total of 316 patients with chronic hepatitis C were studied: 198 consecutive patients undergoing liver biopsy for compensated liver disease and 118 who underwent liver transplantation for end-stage liver disease. Serum iron studies, quantitative hepatic iron concentration, histologic activity index, and HFE genotype were determined.
Results: Among patients with compensated liver disease, the presence of HFE mutations was independently associated with elevations in serum iron level, serum transferrin-iron saturation, serum ferritin level, and hepatic iron index (P < 0.05). After adjustment for duration of infection with hepatitis C virus, HFE mutations were also independently associated with the presence of bridging fibrosis or cirrhosis (odds ratio, 18; 95% confidence interval, 1.7­193). HFE mutations were not independently associated with iron loading in patients with end-stage liver disease. There was no significant difference in the prevalence of HFE mutations between patients with compensated and end-stage liver disease (42% vs. 33%, respectively; P = 0.67).
Conclusions: The presence of HFE mutations is independently associated with iron loading and advanced fibrosis in patients with compensated liver disease from chronic hepatitis C, especially after controlling for duration of disease. These results suggest that HFE mutations accelerate hepatic fibrosis in hepatitis C but may not be responsible for progression to end-stage liver disease.

Basal core promoter mutations of hepatitis B virus increase the risk of hepatocellular carcinoma in hepatitis B carriers
J.­H. Kao, P.­J. Chen, M.­Y. Lai, D.­S. Chen
Background & Aims: Hepatitis B viral (HBV) genotype C is associated with the development of hepatocellular carcinoma (HCC) compared with genotype B; however, the virologic factors contributing to the pathogenic differences remain unknown. We investigated the prevalence of T1762/A1764 basal core promoter mutant in a cohort of 250 genotype B- or C-infected HBV carriers with different stages of liver disease to clarify a possible role for this mutant in hepatocarcinogenesis.
Methods: The sequences of basal core promoter of HBV genome were determined in 60 inactive HBV carriers and 190 patients with histologically verified chronic liver disease and HCC.
Results: Genotype C has a higher prevalence of T1762/A1764 mutation than genotype B (odds ratio, 5.18; 95% confidence interval [CI], 2.59­10.37; P < 0.001). The likelihood of T1762/A1764 mutation parallels the progression of liver disease, from 3% in inactive carriers to 64% in HCC patients (odds ratio, 20.04; 95% CI, 7.25­55.41; P < 0.001). By multiple logistic regression analysis, patients with T1762/A1764 mutation were significantly associated with the development of HCC than those without (odds ratio, 10.60; 95% CI, 4.92­22.86; P < 0.001), and the risk was observed for both genotypes B and C. In addition, the prevalence of T1762/A1764 mutation in younger HCC patients was comparable with older HCC patients but was significantly higher than that in age-matched inactive carriers, irrespective of genotypes.
Conclusions: Our data suggest that HBV carriers with T1762/A1764 basal core promoter mutant are at increased risk for HCC and that this mutant may contribute to the pathogenesis of HBV infection.

Defective cellular localization of mutant ATP7B in Wilson's disease patients and hepatoma cell lines
D. Huster, M. Hoppert, S. Lutsenko, J. Zinke, C. Lehmann, J. Mössner, F. Berr, K. Caca
Background & Aims: Wilson's disease, a hereditary disorder caused by mutations in the Wilson's disease gene (ATP7B), leads to hepatic and/or neurological pathology resulting from cellular copper overload. In vitro studies showed that ATP7B, located in the trans-Golgi network, traffics to a cytoplasmic vesicular compartment in response to increased copper concentration. Mislocalization and failed intracellular trafficking of ATP7B mutants are suggested to be among disease-causing mechanisms; however, the effect of mutations on ATP7B localization in human tissues has not been directly shown. Therefore, we characterized the subcellular localization of normal and mutant ATP7B in human livers and in hepatoma cell lines.
Methods: Subcellular distribution of ATP7B in liver tissue from 3 control individuals and 3 Wilson's disease patients harboring a homozygous H1069Q-ATP7B mutation was analyzed by using immunogold electron microscopy. In addition, 14 ATP7B mutants tagged to green fluorescent protein were generated and expressed in HuH-7 and HepG2 cells; intracellular localization of these mutants was characterized by confocal microscopy.
Results: In hepatocytes, ATP7B was localized in trans-Golgi vesicles, whereas H1069Q-ATP7B was trapped in the endoplasmic reticulum. Similar results were observed for wild-type ATP7B and H1069Q-ATP7B expressed in hepatoma cells. Most ATP7B proteins harboring missense mutations were distributed similarly to wild-type ATP7B. In contrast, truncated ATP7B mutants showed a diffuse, clustered, cytoplasmic pattern, distinct from the trans-Golgi network or endoplasmic reticulum.
Conclusions: These results provide a detailed demonstration of the ATP7B distribution in control and diseased human livers and indicate that several Wilson's disease mutations lead to incorrect localization of ATP7B to distinct cell compartments.

Liver transplantation improves hepatic myelopathy: Evidence by three cases
K. Weissenborn, U. J. F. Tietge, M. Bokemeyer, B. Mohammadi, U. Bode, M. P. Manns, M. Caselitz
Background & Aims: Hepatic myelopathy is a rare complication of chronic liver disease, causing progressive spastic paraparesis. Today, no therapy of this disorder has been established. Commonly used therapeutic strategies for hepatic encephalopathy aiming at the reduction of plasma ammonia levels such as protein restriction, oral neomycin, lactulose, or ornithine aspartate fail to improve the symptoms of hepatic myelopathy. The aim of this study was to find out whether orthotopic liver transplantation (OLT) may improve hepatic myelopathy.
Methods: Follow-up examinations of 3 patients with severe hepatic myelopathy before and after OLT.
Results: In all 3 patients, the neurologic status improved significantly after liver transplantation. The grade of improvement was related to the time interval between onset of the first symptoms of hepatic myelopathy and liver transplantation.
Conclusions: Early recognition of hepatic myelopathy is important because timely liver transplantation as an established therapy for end-stage liver disease offers the chance of complete recovery from hepatic paraparesis.

Associations of chemokine system polymorphisms with clinical outcomes and treatment responses of chronic hepatitis C
K. Promrat, D. H. McDermott, C. M. Gonzalez, D. E. Kleiner, D. E. Koziol, M. Lessie, M. Merrell, A. Soza, T. Heller, M. Ghany, Y. Park, H. J. Alter, J. H. Hoofnagle, P. M. Murphy, T. J. Liang
Background & Aims: CCR532, a 32­base pair deletion of the CC chemokine receptor (CCR) 5 gene, is associated with slowed human immunodeficiency virus disease progression in heterozygotes and protection against infection in homozygotes. A recent study found a higher than expected frequency of CCR532/32 in patients with hepatitis C virus infection. The roles of other disease-associated chemokine system polymorphisms have not been evaluated in hepatitis C virus infection.
Methods: Six chemokine system polymorphisms (CCR532, CCR5 promoter 59029­G/A, CCR2 -64I, RANTES [regulated upon activation, normal T cells expressed and secreted] -403 -G/A, and -28 -C/G and stromal derived factor 1 -3'A) were studied in 417 patients with liver diseases (339 with hepatitis C) and 2380 blood donors. The clinical parameters of hepatitis C virus infection were compared between carriers and noncarriers of each genetic variant.
Results: The frequency of CCR532 homozygosity was 0.8% in whites with hepatitis C virus and 1.1% in controls (P = 0.75). The CCR532 allele was not associated with any of the clinical parameters of hepatitis C virus infection. Hepatitis C virus­seropositive whites with the RANTES -403-A allele were less likely to have severe hepatic inflammation compared with those without (odds ratio, 0.34; P = 0.03). In multivariate analysis, the CCR5 promoter 59029 -A allele was marginally associated with a sustained response to interferon therapy (odds ratio, 3.07; P = 0.048).
Conclusions: In this cohort, the frequency of CCR532 homozygosity in patients with hepatitis C was similar to controls. The high prevalence of CCR532 homozygosity in the hepatitis C virus patients of the earlier study likely reflects resistance to human immunodeficiency virus infection in hemophiliacs rather than a susceptibility to hepatitis C virus infection. Expression of CCR5 and RANTES may be important in the modulation of hepatic inflammation and response to interferon therapy in chronic hepatitis C.

Basic­Alimentary Tract

Peroxisome proliferator-activated receptor ligands suppress colon carcinogenesis induced by azoxymethane in mice
E. Osawa, A. Nakajima, K. Wada, S. Ishimine, N. Fujisawa, T. Kawamori, N. Matsuhashi, T. Kadowaki, M. Ochiai, H. Sekihara, H. Nakagama
Background & Aims: Peroxisome proliferator-activated receptor (PPAR) is known to regulate growth arrest and terminal differentiation of adipocytes and is used clinically as a new class of antidiabetic drugs. Recently, several studies have reported that treatment of cancer cells with PPAR ligands induces cell differentiation and apoptosis, suggesting a potential application as chemopreventive agents against carcinogenesis. However, contradictory results have been reported with regards to the biologic role of PPAR in carcinogenesis. Tanaka et al.24 have recently reported the suppressive effect of a PPAR ligand, troglitazone (Tro), on the formation of aberrant crypt foci (ACF) in rats. In the present study, 3 different kinds of PPAR ligands were subjected to the experiments to confirm their suppressive effects on colon carcinogenesis.
Methods: Three PPAR ligands, pioglitazone (Pio) (200 ppm), rosiglitazone (Rosi) (200 ppm), and Tro (1000 ppm) were investigated on the induction of ACF, a putative precancerous lesion of the colon, and colon tumor formation using an azoxymethane (AOM)-induced colon cancer model in BALB/c mice, and dose dependency of a PPAR ligand was also examined.
Results: PPAR ligands reduced the ACF formation by AOM (10 mg/kg body weight) and induction of colon tumors were also markedly suppressed by a continuous feeding of Pio at 200 ppm.
Conclusions: Our findings indicate that PPAR ligands are indeed potential chemopreventive agents for colon carcinogenesis.

Effects of HIV-1 Tat protein on ion secretion and on cell proliferation in human intestinal epithelial cells
R. Berni Canani, P. Cirillo, G. Mallardo, V. Buccigrossi, A. Secondo, L. Annunziato, E. Bruzzese, F. Albano, F. Selvaggi, A. Guarino
Background & Aims: Severe diarrhea and enteropathy of unknown origin are frequent in patients infected with human immunodeficiency type 1 virus (HIV-1). The HIV-1 transactivating factor protein (Tat) is a key factor in the pathogenesis of acquired immunodeficiency syndrome. We investigated whether Tat could directly induce ion secretion and cell damage in enterocytes.
Methods: Electrical parameters (ion transport studies) were measured in Caco-2 cell monolayers and in human colonic mucosa specimens mounted in Ussing chambers. The effect of Tat on intestinal mucosa integrity was determined by monitoring the transepithelial electrical resistance of Caco-2 cell monolayers. 3H-thymidine incorporation and cell count were used to evaluate the effect of Tat on cell growth. Intracellular calcium concentrations were measured at the single-cell level using microfluorometry technique.
Results: Tat protein induced ion secretion in Caco-2 cells and in human colonic mucosa similar to that induced by bacterial enterotoxins. It also significantly prevented enterocyte proliferation. In both instances, the effect of Tat was maximum at concentrations within the range detected in the sera of HIV-1­infected patients. Anti-Tat antibodies inhibited both effects. Ion secretion and the antiproliferative effects were mediated by L-type Ca2+ channels. An increase in intracellular calcium concentration in Caco-2 cells was found after addition of Tat.
Conclusions: These results indicate that Tat may be involved in HIV-1­related intestinal disease through direct interaction with enterocytes.

Inhaled carbon monoxide suppresses the development of postoperative ileus in the murine small intestine
B. A. Moore, L. E. Otterbein, A. Türler, A. M. K. Choi, A. J. Bauer
Background & Aims: The induction of heme oxygenase (HO-1), the rate-limiting enzyme in heme metabolism, is protective against injury in acute and chronic inflammation. Inhalation of low levels of carbon monoxide (CO), a byproduct of heme metabolism, has anti-inflammatory effects equal to HO-1 induction. This study examined whether inhaled CO was protective against the development of postoperative ileus.
Methods: Ileus was induced by surgical anesthesia and gentle manipulation of the mouse small intestine. Animals were exposed to CO (250 ppm) in air 1 hour before and continuously for 24 hours after surgery.
Results: CO inhalation prevented the manipulation-induced suppression of circular muscle contractility in vitro, and significantly improved gastrointestinal transit in vivo. Proinflammatory messenger RNA (mRNA) expression (interleukin [IL]-6, IL-1, cyclooxygenase 2 [COX-2], inducible nitric oxide [iNOS]) and anti-inflammatory mediator expression (IL-10 and HO-1) were elevated 3 to 6 hours after surgery relative to controls. CO treatment reduced IL-1 and iNOS peak expression by 75%, but not IL-6 or COX-2. In manipulated mice treated with CO, HO-1 expression peaked earlier (3 hours after surgery) and at levels 300% higher than in mice not exposed to CO. IL-10 expression at 3 hours also was 300% higher after CO treatment.
Conclusions: These findings suggest that CO attenuates postoperative ileus by inhibiting selective elements within the inflammatory cascade and by enhanced induction of the anti-inflammatory cytokine IL-10. In addition, the early and enhanced induction of HO-1 potentially amplifies the anti-inflammatory effects of the HO-1 pathway by protection from free radical stress and by increasing the tissue availability of CO directly at the sites of inflammation.

Bacteroides fragilis enterotoxin induces c-Myc expression and cellular proliferation
S. Wu, P. J. Morin, D. Maouyo, C. L. Sears
Background & Aims: Enterotoxigenic Bacteroides fragilis that secrete a zinc-dependent metalloprotease toxin termed the B. fragilis toxin (BFT) have been associated with acute diarrheal disease. BFT rapidly cleaves the extracellular domain of E-cadherin, leading to the complete degradation of the E-cadherin protein. E-cadherin is the primary intercellular adhesion protein of the zonula adherens, and its cytoplasmic domain associates with the nuclear signaling protein -catenin. The goal of this study was to examine if BFT triggers -catenin nuclear signaling in intestinal epithelial cells.
Methods: Cell biologic and biochemical techniques were combined to address -catenin nuclear signaling stimulated by BFT.
Results: Loss of membrane-associated E-cadherin after BFT treatment of human colonic epithelial cells (HT29/C1 clone) triggers -catenin nuclear localization within 3 hours. Subsequently, c-myc transcription and translation are induced and persistent cellular proliferation ensues, mediated in part by -catenin/T-cell factor­dependent transcriptional activation. Cellular proliferation is stimulated by as little as 5 ¥ 10­10 mol/L BFT.
Conclusions: To our knowledge, BFT is the first bacterial toxin reported to activate T-cell factor­dependent -catenin nuclear signaling in intestinal epithelial cells. These results suggest that genetic evolution of this common colonic commensal has rendered an organism with the potential to contribute to oncogenic transformation in the colon.

Gastric electrical stimulation with short pulses reduces vomiting but not dysrhythmias in dogs
J. D. Z. Chen, L. Qian, H. Ouyang, J. Yin, Enteric Neuromuscular Disorders and Pain Group
Background & Aims: The aim of this study was to investigate the acute effects of 3 different methods of electrical stimulation in the prevention of vasopressin-induced emetic response and gastric dysrhythmias.
Methods: Seven female hound dogs chronically implanted with 4 pairs of electrodes on gastric serosa were used in a 5-session study. Saline and vasopressin were infused in sessions 1 and 2, respectively. In the other 3 sessions with vasopressin infusion, 3 different methods of electrical stimulation (short-pulse stimulation, long-pulse stimulation, and electroacupuncture) were applied. Gastric slow waves and vomiting and behaviors suggestive of nausea were recorded in each session. In a separate study, additional experiments were performed in 5 vagotomized dogs to investigate vagally mediated mechanisms.
Results: Vasopressin induced gastric dysrhythmias, uncoupling of slow waves, and vomiting and behaviors suggestive of nausea (P < 0.02, analysis of variance). Long-pulse stimulation, but not short-pulse stimulation or electroacupuncture, was capable of preventing vasopressin-induced gastric dysrhythmias and gastric slow wave uncoupling. Short-pulse stimulation and electroacupuncture, but not long-pulse stimulation, prevented vomiting and significantly reduced the symptom scores, which was not noted in the dogs with truncal vagotomy.
Conclusions: Long-pulse stimulation normalizes vasopressin-induced slow wave abnormalities with no improvement in vomiting and behaviors suggestive of nausea. Short-pulse stimulation and electroacupuncture prevent vomiting and behaviors suggestive of nausea induced by vasopressin but have no effects on slow waves, and their effects are vagally mediated.

Ameliorating effect of anti-inducible costimulator monoclonal antibody in a murine model of chronic colitis
T. Totsuka, T. Kanai, R. Iiyama, K. Uraushihara, M. Yamazaki, R. Okamoto, T. Hibi, K. Tezuka, M. Azuma, H. Akiba, H. Yagita, K. Okumura, M. Watanabe
Background & Aims: Inducible costimulator (ICOS)/B7RP-1 represents a newly described receptor/ligand pair involved in costimulation of T cells by antigen-presenting cells. We investigated the involvement of the ICOS/B7RP-1 interaction in the pathogenesis of colitis and the therapeutic potential of anti-ICOS monoclonal antibody (mAb) in experimental colitis
Methods: We administered anti-ICOS or anti­B7RP-1 mAb to mice with experimental colitis induced by transfer of CD4+CD45RBhigh T cells from normal mice into SCID mice. The ability of CD4+CD45RBhigh cells derived from ICOS­/­ mice to induce colitis was assessed. Th2 cytokine production and apoptosis in infiltrating T cells was examined after administration of anti-ICOS mAb.
Results: ICOS was strongly induced on CD4+ T cells, and B7RP-1 was expressed by macrophages in the inflamed mucosa of colitic mice. Anti-ICOS mAb, but not anti­B7RP-1, ameliorated chronic colitis when administered in prevention or therapeutic protocols. Transfer of CD4+CD45RBhigh T cells from ICOS­/­ mice induced colitis. Treatment with anti-ICOS mAb did not enhance the production of Th2 cytokines, but a single dose of anti-ICOS mAb induced massive apoptosis of infiltrating ICOS-expressing T cells.
Conclusions: ICOS/B7RP-1 interactions are not required for the development of colitis. However, treatment with anti-ICOS mAb can prevent and reverse intestinal inflammation by inducing apoptosis of ICOS-expressing T lymphocytes.

Basic­Liver, Pancreas, and Biliary Tract

Treatment of cirrhosis and liver failure in rats by hepatocyte xenotransplantation
H. Nagata, M. Ito, J. Cai, A. S. Edge, J. L. Platt, I. J. Fox
Background & Aims: Hepatocyte transplantation has been proposed as an alternative to liver transplantation for the treatment of hepatic failure. A major limitation to this form of therapy is the availability of human livers as a source of hepatocytes. The use of porcine hepatocytes might address this problem; however, xenogeneic hepatocytes are thought to be functionally incompatible across species and susceptible to irreversible rejection.
Methods: Liver cirrhosis was induced with phenobarbital and carbon tetrachloride. Only rats with decompensated liver failure that did not correct 4 weeks after the discontinuation of carbon tetrachloride were subjected to intrasplenic rat or porcine hepatocyte transplantation. The immunologic integrity of cirrhotic rats was assessed by allogeneic skin grafting, and the immune response to transplanted porcine hepatocytes was assessed by enzyme-linked immunosorbent assay.
Results: Porcine hepatocytes restored metabolic function and prolonged the survival of cirrhotic rats, as well as rat hepatocytes. Cirrhotic rats retained the ability to reject allogeneic skin grafts and showed an immune response to the engrafted hepatocytes. Despite this, survival of transplanted porcine hepatocytes was accepted in cirrhotic rats for a period of weeks without immunosuppression. Conventional immunosuppression with FK506 allowed successful retransplantation with hepatocytes from a second porcine donor.
Conclusions: Hepatocytes transplanted between widely divergent species can function to correct liver failure in cirrhotic rats and prolong their survival. Conventional immunosuppression allows long-term functioning of xenogeneic hepatocyte retransplants and suggests that hepatocyte xenotransplantation might be useful as a bridge to liver transplantation and could potentially provide long-term hepatic support.

Telomerase reconstitution immortalizes human fetal hepatocytes without disrupting their differentiation potential
H. Wege, H. T. Le, M. S. Chui, L. Liu, J. Wu, R. Giri, H. Malhi, B. S. Sappal, V. Kumaran, S. Gupta, M. A. Zern
Background & Aims: The availability of in vitro expandable human hepatocytes would greatly advance liver-directed cell therapies. Therefore, we examined whether human fetal hepatocytes are amenable to telomerase-mediated immortalization without inducing a transformed phenotype and disrupting their differentiation potential. Telomerase is a ribonucleoprotein that plays a pivotal role in maintaining telomere length and chromosome stability. Human somatic cells, including hepatocytes, exhibit no telomerase activity. Consequently, their telomeres progressively shorten with each cell cycle until critically short telomeres trigger replicative senescence.
Methods: The catalytic subunit, telomerase reverse transcriptase, was expressed in human fetal hepatocytes. Transduced cells were characterized for telomerase activity, telomere length, proliferative capacity, hepatocellular functions, oncogenicity, and their in vivo maturation potential.
Results: The expression of human telomerase reverse transcriptase restored telomerase activity in human fetal hepatocytes. Telomerase-reconstituted cells were capable of preserving elongated telomeres, propagated in culture beyond replicative senescence for more than 300 cell doublings (to date), and maintained their liver-specific nature, as analyzed by a panel of hepatic growth factors, growth factor receptors, and transcription factors as well as albumin, glucose-6-phosphatase, glycogen synthesis, cytochrome P450 (CYP) expression profiles, and urea production. Moreover, the immortalized cells exhibited no oncogenicity, and no up-regulation of c-Myc was detected. The cells engrafted and survived in the liver of immunodeficient mice with hepatocellular gene expression.
Conclusions: Reconstitution of telomerase activity induces indefinite replication in human fetal hepatocytes and offers unique opportunities for examining basic biologic mechanisms and for considering development of stable cell lines for liver-directed therapies.

Delivery of matrix metalloproteinase-1 attenuates established liver fibrosis in the rat
Y. Iimuro, T. Nishio, T. Morimoto, T. Nitta, B. Stefanovic, S. K. Choi, D. A. Brenner, Y. Yamaoka
Background & Aims: During hepatic fibrogenesis, the hepatic extracellular matrix changes to fibrillar collagens types I and III, and cirrhosis is believed to produce an irreversible scar. In this study, we investigated whether gene delivery of human matrix metalloproteinase-1, which degrades collagens types I and type III, would attenuate established hepatic fibrosis in the rat, induced by either thioacetamide or bile duct ligation.
Methods: Hepatic fibrosis induced by thioacetamide for 7 weeks was persistent for at least 2 months, even after discontinuation of the treatment. The rats were infected once with a recombinant adenovirus, Ad5MMP-1, into which human pro­human matrix metalloproteinase-1 complementary DNA was packaged, or with a control adenovirus, Ad5LacZ.
Results: In Ad5MMP-1­infected, but not in Ad5LacZ-infected, rats, the fibrosis was dramatically attenuated at 2 weeks after the infection. It is interesting to note that the number of activated hepatic stellate cells was also decreased in Ad5MMP-1­infected rats. Moreover, disorganization of the hepatic trabecula, heterogeneity in the size of hepatocytes, and increased dried liver weight were observed only in Ad5MMP-1­treated rats, suggesting that human matrix metalloproteinase-1 stimulated hepatocyte proliferation, which was confirmed by bromodeoxyuridine staining. After 4 weeks, the proliferative effect of human matrix metalloproteinase-1 almost disappeared, but the hepatic fibrosis remained attenuated, whereas the fibrosis in Ad5LacZ-treated rats persisted. Furthermore, the administration of Ad5MMP-1, but not Ad5LacZ, decreased type I collagen and generated a small collagen fragment in hepatic fibrosis induced by bile duct ligation.
Conclusions: Our findings show that transient human matrix metalloproteinase-1 overexpression in the liver effectively attenuates established fibrosis and induces hepatocyte proliferation.

Antiproliferative property of sphingosine 1-phosphate in rat hepatocytes involves activation of Rho via Edg-5
H. Ikeda, H. Satoh, M. Yanase, Y. Inoue, T. Tomiya, M. Arai, K. Tejima, K. Nagashima, H. Maekawa, N. Yahagi, Y. Yatomi, S. Sakurada, Y. Takuwa, I. Ogata, S. Kimura, K. Fujiwara
Background & Aims: Sphingosine 1-phosphate (S1P), a ligand for G protein-coupled endothelial differentiation gene-1 (Edg-1), Edg-3, Edg-5, Edg-6, and Edg-8, elicits a variety of responses by cells. Prominent among these is cell proliferation. S1P is abundantly stored in platelets and released upon their activation, suggesting that S1P plays a pathophysiologic role in vivo. Because the major part of injected S1P was distributed into the liver in mice, we wondered whether the liver would be one of its targets. The effects of S1P on hepatocytes, the major constituent cells in the liver, were examined.
Methods & Results: Northern blot analysis revealed the expression of Edg-1 and Edg-5 messenger RNA (mRNA) in cultured rat hepatocytes, in which S1P decreased DNA synthesis induced by hepatocyte growth factor (HGF) or epidermal growth factor (EGF) without affecting total protein synthesis. This inhibitory effect was attenuated by inactivation of small GTPase Rho with C3 exotoxin but not by inactivation of Gi with pertussis toxin. Moreover, in the presence of JTE-013, a newly developed and specific binding antagonist for Edg-5, the inhibitory effect was also cancelled. Finally, the administration of S1P after 70% partial hepatectomy in rats reduced the peak of DNA synthesis in hepatocytes with increased Rho activity. Furthermore, Edg-5 but not Edg-1 mRNA expression was enhanced in hepatocytes 24­72 hours after partial hepatectomy, which coincides with decreasing hepatocyte proliferation.
Conclusions: S1P has an antiproliferative property in rat hepatocytes by activating Rho via Edg-5. Our results raise the possibility that S1P is a negative regulator in liver regeneration.

Circulating bile salt­dependent lipase originates from the pancreas via intestinal transcytosis
N. Bruneau, M. Bendayan, D. Gingras, L. Ghitescu, E. Levy, D. Lombardo
Background & Aims: Bile salt­dependent lipase (BSDL) has been detected in human blood, where it is assumed to play a substantial role in atherosclerosis. The origin of this circulating enzyme is unknown. The aim of this study was to show that blood BSDL originates from pancreatic exocrine secretions via a transcytotic motion across the intestinal epithelium.
Methods: Fluorescein isothiocyanate­ or [125I]-labeled human pancreatic BSDL was instilled into the lumen of intestinal loops of the rat, and combined biochemical and immunocytochemical investigations were performed in intestinal cells and in the blood of these animals.
Results: In vivo pancreatic BSDL is internalized by duodenal enterocytes. The pancreatic enzyme was associated with microvilli and present in endocytic vesicles and Golgi apparatus as well as along the basolateral membrane of enterocytes. It was also detected in intestinal interstitial spaces. Radiolabeled pancreatic BSDL internalized by the duodenal epithelium is the one further detected in circulation. The radiolabeled protein was immunoprecipitated from plasma and had a 100-kilodalton molecular mass compatible with native pancreatic BSDL. In blood, BSDL was mainly associated with low-density lipoproteins.
Conclusions: These in vivo data show that BSDL, normally present in blood, originates from exocrine pancreatic secretion and support the pathophysiologic relevance of BSDL transcytosis through the intestinal mucosa cell lining. Based on this, the implication of circulating BSDL in atherosclerosis merits careful attention.

Severe acute pancreatitis and reduced acinar cell apoptosis in the exocrine pancreas of mice deficient for the Cx32 gene
J.­L. Frossard, L. Rubbia­Brandt, M. A. Wallig, M. Benathan, T. Ott, P. Morel, A. Hadengue, S. Suter, K. Willecke, M. Chanson
Background & Aims: The early events leading to acinar cell injury during acute pancreatitis are poorly characterized. Signaling through gap junction channels contributes to the homeostasis of the exocrine pancreas by coordinating acinar cell activity within an acinus. To explore the role of gap junctional communication in acinar cell response to injury, we analyzed the course of acute pancreatitis induced by injection of cerulein in mice deficient for Cx32, the major gap junction protein expressed in the exocrine pancreas.
Methods: The severity of pancreatitis was evidenced by measuring serum amylase activity, pancreatic edema, acinar cell necrosis, pancreatic tumor necrosis factor concentration, and myeloperoxidase activity. Acinar cell apoptosis was detected by terminal deoxynucleotidyl transferase­mediated deoxyuridine triphosphate nick-end labeling (TUNEL), caspase-3 activity, and Bax/Bcl-2 expression. Expression and function of connexin were evaluated by immunofluorescence and dye coupling.
Results: Cx32-deficient mice exhibited a deleterious course of acute pancreatitis with increased necrosis, edema, and inflammation of the exocrine pancreas. In addition, the exocrine pancreas of Cx32-deficient mice showed a decreased number of TUNEL-positive acinar cells and decreased caspase-3 activity but no change in Bax or Bcl-2 pancreatic expression. Interestingly, chemicals known to induce apoptosis in vivo had no effect on Cx32-deficient pancreatic acinar cells.
Conclusions: Deficiency of a pancreatic connexin converts a mild reversible form of acute pancreatitis into a severe disease and decreases the sensitivity of acinar cells to apoptotic stimuli. The results show that acinar cell-to-cell communication plays a key role in the modulation of severity of acute pancreatitis.

Mitochondrial permeability transition in the switch from necrotic to apoptotic cell death in ischemic rat hepatocytes
J.­S. Kim, T. Qian, J. J. Lemasters
Background & Aims: Ischemia/reperfusion can initiate both necrotic and apoptotic death of hepatocytes. Previous work showed that onset of the mitochondrial permeability transition (MPT) can initiate necrotic cell death after reperfusion, but the MPT is also implicated in apoptosis. Here, we investigated factors regulating how cell death switches from necrosis to apoptosis after ischemia/reperfusion injury.
Methods: Overnight cultured rat hepatocytes were incubated in anoxia at pH 6.2 for 4 hours and reoxygenated at pH 7.4 to simulate ischemia/reperfusion. Some cells were incubated with fructose plus glycine just before and then continuously after reperfusion. Development of apoptosis was evaluated by examining chromatin condensation, nuclear DNA fragmentation, and caspase 3 activity.
Results: Reperfusion with the glycolytic substrate fructose plus the cytoprotective amino acid glycine prevented necrotic cell killing. Instead, apoptosis developed within 12 hours as shown by nuclear chromatin changes, TUNEL staining, and caspase 3 activation. This apoptotic cell killing was prevented by cyclosporin A, an MPT blocker, and by pancaspase and caspase 3 inhibition, but not by caspase 8 inhibition. Cyclosporin A also blocked caspase-3 activation. Reperfusion with glycine alone prevented necrotic cell death but did not induce apoptosis and only poorly promoted recovery of ATP, whereas fructose alone during reperfusion promoted both ATP recovery and apoptosis.
Conclusions: Glycolytic ATP generation after reperfusion prevents necrotic killing of hepatocytes after simulated ischemia/reperfusion despite onset of the MPT. Instead, the MPT promotes caspase-and ATP-dependent apoptosis. Thus, the MPT is a common mechanism responsible for both necrosis and apoptosis after ischemia/reperfusion.

Antifibrogenic effects of canrenone, an antialdosteronic drug, on human hepatic stellate cells
A. Caligiuri, R. M. S. De Franco, R. G. Romanelli, A. Gentilini, M. Meucci, P. Failli, L. Mazzetti, K. Rombouts, A. Geerts, M. Vanasia, P. Gentilini, F. Marra, M. Pinzani
Background & Aims: Several lines of evidence indicate that aldosterone antagonists may exert direct antifibrogenic effects. The aim of this study was to evaluate the possible direct antifibrogenic effects of canrenone, the active metabolite of spironolactone, in activated human hepatic stellate cells.
Methods: The effects of canrenone were assessed on platelet-derived growth factor­induced mitogenic and chemotactic effects and the increased de novo synthesis of different extracellular matrix components induced by transforming growth factor-1.
Results: Canrenone dose-dependently reduced platelet-derived growth factor­induced cell proliferation and motility. This effect was not associated with either changes in the phosphorylation of platelet-derived growth factor receptor and phospholipase C or in the activation of the Ras/extracellular signal-regulated kinase pathway, whereas it was accompanied by a dose-dependent inhibition of platelet-derived growth factor­induced phosphatidylinositol 3-kinase activity. In addition, canrenone inhibited the activity of the Na+/H+ exchanger 1 induced by platelet-derived growth factor. The effect of canrenone on Na+/H+ exchanger 1 activity was reproduced by phosphatidylinositol 3-kinase inhibitors, thus supporting an inhibitory action of canrenone on phosphatidylinositol 3-kinase activity. To further address this possibility, the action of canrenone was compared with that of 2 established Na+/H+ exchanger 1 inhibitors: ethylisopropylamiloride and cariporide. Whereas ethylisopropylamiloride was able to inhibit platelet-derived growth factor­induced phosphatidylinositol 3-kinase activity, cariporide was without any effect. Both compounds reproduced the effects of canrenone on platelet-derived growth factor­induced mitogenesis and chemotaxis. Finally, canrenone was able to reduce transforming growth factor-1­induced de novo synthesis of procollagen type I/IV and fibronectin and thrombin-induced hepatic stellate cell contraction.
Conclusions: These results indicate that canrenone may be active as an antifibrogenic drug.

Special Reports and Reviews

The genetics of inflammatory bowel disease
D. K. Bonen, J. H. Cho
The inflammatory bowel diseases (IBD) comprise complex genetic disorders, with multiple contributing genes. Linkage studies have implicated several genomic regions as likely containing IBD susceptibility genes, with some observed uniquely in Crohn's disease (CD) or ulcerative colitis (UC), and others common to both disorders. The best replicated linkage region, IBD1, on chromosome 16q contains the CD susceptibility gene, NOD2/CARD15. NOD2/CARD15 is expressed in peripheral blood monocytes and is structurally related to the plant R proteins, which mediate host resistance to microbial pathogens. Three major coding region polymorphisms within NOD2/CARD15 have been highly associated with CD among patients of European descent. Having one copy of the risk alleles confers a 2­4-fold risk for developing CD, whereas double-dose carriage increases the risk 20­40-fold. All 3 major CD variants exhibit a deficit in NF-B activation in response to bacterial components. Carriage of NOD2/CARD15 risk alleles is associated with ileal location, earlier disease onset, and stricturing phenotype. Other IBD genomic regions include IBD2 on chromosome 12q (observed more in UC), and IBD3, containing the major histocompatibility complex region. A short genomic region has been associated with CD on chromosome 5q, but the precise contributing gene is as yet unidentified. The characterization of additional IBD susceptibility genes could potentially lead to the identification of novel therapeutic agents for IBD, make possible a molecular reclassification of disease, and increase understanding of the contribution of environmental factors (notably, tobacco and the intestinal microbial milieu) to intestinal inflammation.

Case Reports

Consecutive regression of concurrent laryngeal and gastric MALT lymphoma after anti­Helicobacter pylori therapy
G. Caletti, T. Togliani, P. Fusaroli, E. Sabattini, E. Khodadadian, B. Gamberi, M. Gobbi, S. Pileri
The most common primary lymphoma of the gastrointestinal tract is B-cell lymphoma arising from mucosa-associated lymphoid tissue known as MALT lymphoma. Although the majority of these lesions affect the stomach and are associated with Helicobacter pylori organisms, sites other than the gastrointestinal tract may be affected. This case report describes a patient with concomitant laryngeal MALT lymphoma and Helicobacter pylori­related gastric MALT lymphoma derived from the same clone as confirmed by PCR. Treatment of Helicobacter pylori infection in this patient using antibiotics led to regression of both lesions. This patient remains in remission at 46-month follow-up. This is the first case report on the regression of a laryngeal MALT lymphoma after Helicobacter pylori eradication. We suggest that all patients presenting with extragastric MALT lymphoma should undergo upper gastrointestinal endoscopy with gastric biopsies for the determination of Helicobacter pylori status and presence of concomitant gastric MALT lymphoma, followed by a course of anti­Helicobacter pylori antibiotic therapy. Nonresponders may subsequently be considered for surgery and/or chemo/radiation therapy.

American Gastroenterological Association

Colorectal cancer screening and surveillance: Clinical guidelines and rationale-Update based on new evidence
S. Winawer, R. Fletcher, D. Rex, J. Bond, R. Burt, J. Ferrucci, T. Ganiats, T. Levin, S. Woolf, D. Johnson, L. Kirk, S. Litin, C. Simmang, for the U.S. Multisociety Task Force on Colorectal Cancer
We have updated guidelines for screening for colorectal cancer. The original guidelines were prepared by a panel convened by the U.S. Agency for Health Care Policy and Research and published in 1997 under the sponsorship of a consortium of gastroenterology societies. Since then, much has changed, both in the research rature and in the clinical context. The present report summarizes new developments in this field and suggests how they should change practice. As with the previous version, these guidelines offer screening options and encourage the physician and patient to decide together which is the best approach for them. The guidelines also take into account not only the effectiveness of screening but also the risks, inconvenience, and cost of the various approaches. These guidelines differ from those published in 1997 in several ways: we recommend against rehydrating fecal occult blood tests; the screening interval for double contrast barium enema has been shortened to 5 years; colonoscopy is the preferred test for the diagnostic investigation of patients with findings on screening and for screening patients with a family history of hereditary nonpolyposis colorectal cancer; recommendations for people with a family history of colorectal cancer make greater use of risk stratification; and guidelines for genetic testing are included. Guidelines for surveillance are also included. Follow-up of postpolypectomy patients relies now on colonoscopy, and the first follow-up examination has been lengthened from 3 to 5 years for low-risk patients. If this were adopted nationally, surveillance resources could be shifted to screening and diagnosis. Promising new screening tests (virtual colonoscopy and tests for altered DNA in stool) are in development but are not yet ready for use outside of research studies. Despite a consensus among expert groups on the effectiveness of screening for colorectal cancer, screening rates remain low. Improvement depends on changes in patients' attitudes, physicians' behaviors, insurance coverage, and the surveillance and reminder systems necessary to support screening programs.

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JOURNAL OF HEPATOLOGY

Table of Contents for Volume 38, Issue 2, February 2003

Biliary Tract and Cholestasis

Taurohyodeoxycholate- and tauroursodeoxycholate-induced hypercholeresis is augmented in bile duct ligated rats
Leonardo Baiocchi et al.
Background/Aims: Taurohyodeoxycholate (THDCA) and tauroursodeoxycholate (TUDCA) induce more bile flow per molecule excreted compared to endogenous bile acids. The aim of this study is to determine if the hypercholeretic effect of tauroursodeoxycholate or taurohyodeoxycholate in normal and bile duct ligated (BDL) rats is due to increased ductal secretion. Methods: Normal or BDL rats were infused with tauroursodeoxycholate or taurohyodeoxycholate and bile flow, bicarbonate, bile salt, cholesterol, and phospholipid secretion were measured. Cholangiocytes were stimulated with taurohyodeoxycholate or tauroursodeoxycholate, and secretin-stimulated secretion was measured. Results: Taurohyodeoxycholate and tauroursodeoxycholate increased bile flow more in BDL than normal rats. Tauroursodeoxycholate increased bicarbonate secretion more in BDL compared to normal rats. Taurohyodeoxycholate when infused with taurocholate increased bile flow (but not phospholipid excretion) to a greater degree in BDL compared to normal rats. Taurohyodeoxycholate and tauroursodeoxycholate decreased secretin-stimulated cholangiocyte secretion. Conclusions: Consistent with a ductal origin for bile acid-induced hypercholeresis, taurohyodeoxycholate and tauroursodeoxycholate produced a greater hypercholeresis in BDL than normal rats. Tauroursodeoxycholate- (but not taurohyodeoxycholate-) stimulated hypercholeresis is associated with increased HCO3 secretion. Tauroursodeoxycholate increases biliary HCO3 secretion by a mechanism unrelated to secretin-stimulated cholangiocyte secretion. Taurohyodeoxycholate-induced hypercholeresis in BDL rats is unrelated to enhanced phospholipid excretion.

Down-regulation of the Na+/taurocholate cotransporting polypeptide during pregnancy in the rat
Marco Arrese et al.
Background: Experimental studies have shown decreased bile acid (BA) uptake and reduced excretion of cholephilic compounds in pregnant rodents. Aim: To assess the expression and function of the main BA importer, the Na+/taurocholate cotransporting polypeptide (Ntcp) in pregnant rats. Methods: BA uptake and Ntcp expression were studied in control and timed-pregnant rats in late gestation. Ntcp protein, messenger RNA (mRNA) expression, and Ntcp tissue localization were determined by Northern blotting, Western analysis, and tissue immunofluorescence. The activity of three transactivators of the Ntcp promoter: hepatocyte nuclear factor 1- (HNF1-), nuclear receptor heterodimer retinoid X receptor:retinoid acid receptor (RXR:RAR) and signal transducer and activator of transcription 5 (Stat5) was assessed using gel electrophoretic mobility shift assays. Results: A significantly reduced BA uptake and decreased Ntcp mRNA levels (40%) and protein mass (60%) was observed in pregnant rats. Nuclear extracts from pregnant rats showed a marked decrease of HNF1- and RXR:RAR binding activities by 80 and 40% of basal activity, respectively. In contrast, binding activity of Stat-5 was increased by 50% in nuclear extracts from pregnant rats. Conclusions: Pregnancy is associated with reduced Ntcp expression and function in the rat. Our findings suggest that Ntcp down-regulation during pregnancy occurs primarily at the transcriptional level.

Cell Biology, Metabolism and Transport

The hepatocyte is a direct target for transforming-growth factor activation via the insulin-like growth factor II/mannose 6-phosphate receptor
Laurence Villevalois-Cam et al.
Background/Aims: The cation-independent mannose 6-phosphate receptor (CIMPR) is overexpressed in hepatocytes during liver regeneration and has been implicated in the maturation of latent pro-transforming growth factor (TGF). In this study, we have: (1) kinetically characterized the changes in CIMPR expression in regenerating liver and cultured proliferating hepatocytes; and (2) assessed the contribution of hepatocyte via the CIMPR to latent pro-TGF activation. Methods: The expression of CIMPR protein and mRNA in livers collected after partial hepatectomy and hepatocyte primary cultures was analyzed by Western and Northern blotting. Activity of latent pro-TGF was assessed by inhibition of [3H] methylthymidine incorporation into DNA. Results: The expression of the CIMPR protein and/or mRNA progressively increased after 8 h in regenerating liver and 42-46 h in cultured hepatocytes, prior to the onset of DNA replication. Both mature TGF and latent pro-TGF inhibited epidermal growth factor-stimulated DNA synthesis in hepatocytes in a dose-dependent manner. The effect of latent pro-TGF was reversed by two ligands of the CIMPR: -galactosidase, a mannose 6-phosphate containing protein, and a CIMPR antibody. Conclusions: (1) The induction of the CIMPR gene during liver regeneration and hepatocyte culture occurs in mid G1 phase; and (2) the CIMPR mediates latent proTGF activation and thus may act, by targeting TGF to hepatocytes, as a negative regulator of hepatocyte growth.

The canine copper toxicosis gene MURR1 does not cause non-Wilsonian hepatic copper toxicosis
Thomas Müller et al.
Background: Non-Wilsonian hepatic copper toxicosis includes Indian childhood cirrhosis (ICC), endemic Tyrolean infantile cirrhosis (ETIC) and the non-Indian disease known as idiopathic copper toxicosis (ICT). These entities resemble the hepatic copper overload observed in livers of Bedlington terriers with respect to their clinical presentation and biochemical and histological findings. We recently cloned the gene causing copper toxicosis in Bedlington terriers, MURR1, as well as the orthologous human gene on chromosome 2p13-p16. Aim: To study the human orthologue of the canine copper toxicosis gene as a candidate gene for ICC, ETIC, and ICT. Methods: We sequenced the exons and the intron-exon boundaries of the human MURR1 gene in 12 patients with classical ICC, one patient with ETIC, and 10 patients with ICT to see whether these patients display any mutations in the human orthologue of the canine copper toxicosis gene. Results: No mutations in the MURR1 gene, including the intron-exon boundaries, were identified in a total of 23 patients with non-Wilsonian hepatic copper toxicosis. Conclusions: Our results demonstrate that copper toxicosis in Bedlington terriers is not an animal model for the non-Wilsonian hepatic copper toxicosis described in this study.

Inflammation and Fibrosis

Adenoviral-mediated transfer of p53 or retinoblastoma protein blocks cell proliferation and induces apoptosis in culture-activated hepatic stellate cells
Bärbel Abriss, Günter Hollweg, Axel M. Gressner and Ralf Weiskirchen
Background/Aims: The principal cellular effectors of fibrosis in liver are hepatic stellate cells (HSC). In response to liver injury these quiescent cells undergo a phenotypic change to a myofibroblastic cell type, proliferate and secrete matrix components. Thus, removal of activated HSC should be beneficial for the prognosis of hepatic fibrogenesis and preserve organ function. Methods: The purpose of this study was to investigate whether administration of adenoviruses constitutively expressing the p53 tumor suppressor or the retinoblastoma protein (pRb) is sufficient to induce cell arrest or apoptosis in culture-activated HSC. The expression of the transgenes was confirmed by Western blot analysis and immunohistochemistry. Results: Both proteins were expressed mainly in the nucleus and their expression was associated with a marked inhibition of cell proliferation and induction of apoptosis as determined by measurement of phosphatidylserine exposed at the surface, proliferation assay, induction of the p21 cyclin-dependent kinase inhibitor, and an increase of caspase-3 activity. Additionally, electron microscopic analysis confirmed that activation of the p53-mediated pathway in HSC results in chromatin and cytoplasmic condensation, typical features of ongoing apoptosis. Conclusions: Our results indicate that transduction of p53 or pRb offers a feasible approach to induce apoptosis in activated HSC. Thus, targeted transfer of these proteins into HSC may be potentially useful for the treatment of hepatic fibrosis.

Liver Cell Injury and Liver Failure

Selective blockade of mGlu5 metabotropic glutamate receptors is protective against acetaminophen hepatotoxicity in mice
Marianna Storto et al
Background/Aims: mGlu5 metabotropic glutamate receptor antagonists protect rat hepatocytes against hypoxic death. Here, we have examined whether mGlu5 receptor antagonists are protective against liver damage induced by oxidative stress. Methods: Toxicity of isolated hepatocytes was induced by tert-butylhydroperoxide (t-BuOOH) after pretreatment with the mGlu5 receptor antagonists, MPEP, SIB-1757 and SIB-1893. The effect of these drugs was also examined in mice challenged with toxic doses of acetaminophen. Results: Addition of tBuOOH (0.5 mM) to isolated hepatocytes induced cell death (70±5% at 3 h). Addition of MPEP or SIB-1893 to hepatocytes reduced both the production of reactive oxygen species (ROS) and cell toxicity induced by t-BuOOH (tBuOOH=70±5%; tBuOOH+MPEP=57±6%; tBuOOH+SIB-1893=40±4%). In mice, a single injection of acetaminophen (300 mg/kg, i.p.) induced centrilobular liver necrosis, which was detectable after 24 h. MPEP (20 mg/kg, i.p.) substantially reduced liver necrosis and the production of ROS, although it did not affect the conversion of acetaminophen into the toxic metabolite, N-acetylbenzoquinoneimine. MPEP, SIB-1893 and SIB-1757 (all at 20 mg/kg, i.p.) also reduced the increased expression and activity of liver iNOS induced by acetaminophen. Conclusions: We conclude that pharmacological blockade of mGlu5 receptors might represent a novel target for the treatment of drug-induced liver damage.

Cerebral oxygenation determined by near-infrared spectrophotometry in patients with fulminant hepatic failure
Henning Bay Nielsen, Flemming Tofteng, Lars Peter Wang and Fin Stolze Larsen
Background/Aims: In severe cases of acute liver failure (ALF), cerebral hyperperfusion may result in high intracranial pressure and brain damage. The aim of this study was to determine if near-infrared spectrophotometry (NIRS) could detect a raise in cerebral blood flow and oxygenation induced by noradrenaline (NA) infusion. Methods: In seven ALF patients (five females and two males; median age 49 years (range 20-70)) changes in cerebral concentration of oxy-(HbO2) and total-haemoglobin (HbT) were compared to the jugular bulb saturation (SvjO2) and cerebral blood flow velocity (Vmean) during NA infusion. Results: Mean arterial pressure increased from 68 (64-86) to 103 (87-118) mmHg and the cerebral perfusion pressure from 61 (53-79) to 95 (74-110) mmHg (P<0.05), while the intracranial pressure (7 (6-15) mmHg) was not significantly changed. In six patients cerebral HbO2 and HbT increased 2.7 (0.3-9.6) and 2.0 (0.3-14.8) µmol l1, respectively, but cerebral oxygenation decreased in one patient. SvjO2 increased from 68 (55-76) to 74 (64-78) % (P<0.05) concomitant with an increase in Vmean from 47 (34-65) to 68 (50-86) cm s1 (P<0.05). HbO2 covariated with changes in SvjO2 during NA in all but one patient. Conclusions: In ALF patients, a change in cerebral perfusion was detected by NIRS. The combination of NIRS and transcranial Doppler sonography may be valuable non-invasive techniques to detect cerebral hyperperfusion before intracranial hypertension becomes manifest.

Kupffer cells modulate splenic interleukin-10 production in endotoxin-induced liver injury after partial hepatectomy
Kiyotaka Kurachi et al.
Background/Aims: This study was conducted to investigate the implication of Kupffer cells and the spleen in interleukin (IL)-10 production in endotoxin-induced liver injury after hepatectomy. Methods: Rats were divided into five groups: the S group, sham-operation; the SG group, sham-operation followed by intravenous gadolinium chloride (GdCl3: 7 mg/kg) administration to inhibit Kupffer cell function; the H group, two-thirds hepatectomy; the HG group, hepatectomy and subsequent GdCl3 administration; the HGS group, hepatectomy and splenectomy with GdCl3 administration. Lipopolysaccharide (1.5 mg/kg) was intravenously administered for each group 48 h after surgery. Results: GdCl3 treatment significantly suppressed the elevation of plasma tumor necrosis factor (TNF)- levels by lipopolysaccharide administration with completely inhibited induction of hepatic TNF- and IL-10 mRNAs. In the HG group, marked increase in plasma IL-10 levels associated with enhanced splenic IL-10 mRNA was observed 1 h after lipopolysaccharide administration when compared to those in the H and HGS groups. Plasma TNF-/IL-10 ratio 1 h after lipopolysaccharide administration was higher in the order of H, HGS and HG groups. Hepatic parenchymal damage and the 24-h mortality were lowest in group HG, followed by groups HGS and H. Conclusions: Kupffer cells after hepatectomy may aggravate endotoxin-induced liver injury via down-regulation of IL-10 production in the spleen.
Keywords: Kupffer cell; Spleen; Gadolinium chloride; Tumor necrosis factor-; Interleukin-10; Endotoxin; Endotoxemia; Hepatectomy; Cytokine; Lipopolysaccharide

Liver Growth and Cancer

Risk factors contributing to early and late phase intrahepatic recurrence of hepatocellular carcinoma after hepatectomy
Hiroshi Imamura et al.
Background/Aims: We conducted a retrospective cohort study to investigate factors to early and late phase recurrence of hepatocellular carcinoma (HCC). Methods: The study population consisted of 249 patients including 157 with cirrhosis who underwent hepatectomy for HCC. The endpoint was time-to-recurrence. Using a Cox regression model, factors to early and late phase recurrences were investigated censoring recurrence-free patients at the 2-year time point and in patients without recurrence at 2 years. Results: Actuarial probability of overall recurrence at 1, 3, and 5 years were 0.301, 0.623, and 0.790, respectively, with a median follow-up of 624 days. Early recurrence was observed in 123 out of 249 patients; while late recurrence was found in 61 out of 113 patients. Factors to early recurrence were as follows: non-anatomical resection, presence of microscopic vascular invasion, and serum alpha-fetoprotein level 32ng/ml. Those contributing to late phase recurrence were higher grade of hepatitis activity, multiple tumors, and gross tumor classification. Conclusions: Variables associated with metastatic recurrence were factors to early phase recurrence; whereas those related with elevated carcinogenesis contributed to late phase recurrence, thus providing an epidemiological evidence that different mechanisms, i.e. metastasis and de novo, are involved in intrahepatic recurrence after hepatectomy for HCC.

Viral Hepatitis

Hepatic immunohistochemical staining with a monoclonal antibody against HCV-E2 to evaluate antiviral therapy and reinfection of liver grafts in hepatitis C viral infection
Chris Verslype et al.
Background/Aims: A simple and reproducible hepatic immunohistochemical staining (IHS) for hepatitis C virus (HCV) is not available. We aimed to validate hepatic IHS with monoclonal antibody (Mab) IG222, directed against the HCV-envelope 2 (E2) protein. Methods: A three-step indirect immunoperoxidase method was used for frozen sections and a two-step indirect EnVision technique was used for paraffin-embedded sections. Results: Naturally or in vitro HCV infected primary human hepatocytes were immunoreactive to HCV-E2. In the patient study (n=253), IHS had a sensitivity of 96% and a specificity of 91%. Six patients who showed positivity in the liver with Mab IG222, but remained anti-HCV and HCV-RNA negative, had hepatitis C-like changes in their liver biopsy. In one patient HCV-RNA could be detected in the liver biopsy. We confirmed early graft reinfection in patients transplanted for HCV-related disease (34 patients with serial biopsies). Treatment for acute cellular rejection with steroids was associated with an increase in staining intensity. In nine patients with clearance of HCV-RNA during antiviral therapy, seven achieved negativation of immunoreactivity and two a marked reduction. Conclusions: The IHS with Mab IG222 is an accurate tool for diagnosis and clinical management of chronic hepatitis C.

Assessment of cost-effectiveness of universal hepatitis B immunization in a low-income country with intermediate endemicity using a Markov model
Rakesh Aggarwal, Uday C. Ghoshal and Subhash R. Naik
Background/Aims: Most countries with high hepatitis B (HB) virus endemicity and most high-income countries have introduced immunization programmes against this infection. However, several low-income countries with intermediate HB endemicity have not done so. We performed a cost-effectiveness analysis of universal childhood HB immunization in such countries using India as an example, since available data on this aspect are limited. Methods: Marginal cost of every life-year and quality-adjusted life-year (QALY) gained with universal HB vaccination was calculated using a Markov model. Two types of analyses (including and excluding expenditure on treatment of long-term complications of HB infection) were done. Several sensitivity analyses and Monte-Carlo simulation were performed. Results: Universal immunization reduced the HB carrier rate by 71%, and increased the number of years and QALY lived by a birth-cohort by 0.173 years (61.072 vs. 60.899 years) and 0.213 years (61.056 vs. 60.843 years), respectively. Marginal costs were US$16.27 per life-year gained and US$13.22 per QALY gained, much lower than annual per capita income. One-way sensitivity analysis and Monte-Carlo simulation confirmed the robustness of the conclusions. Conclusions: Universal HB immunization is highly cost-effective in low-income countries with intermediate endemicity rates.

Histopathological study of chronic hepatitis B and C: a comparison of two scoring systems
Rekha Rozario and Banumathi Ramakrishna
Background/Aims: Several histological scoring systems are used to evaluate chronic viral hepatitis. This study was undertaken to determine the correlation between the Ishak system (modified histological activity index, HAI) and the METAVIR system, in Indian patients with chronic viral hepatitis. Methods: Liver biopsies from 127 patients with chronic viral hepatitis B or C were examined, and scored using the Ishak and METAVIR systems, and weighted kappa analysis of correlation was done. Correlation of necroinflammatory activity with serum transaminase levels was analyzed, and prevalence of specific histological features compared in hepatitis B virus (HBV) and HCV biopsies. Results: HBV infection accounted for 64.6% of cases, and HCV for 35.4%; 91.3% of patients had minimal or mild hepatitis. The necroinflammatory scores of the Ishak and METAVIR systems correlated moderately well (weighted kappa 0.627), while there was excellent correlation with regard to fibrosis (weighted kappa 0.998). Similar concordance was found when HBV and HCV cases were analyzed separately. HAI showed poor correlation with serum transaminases (weighted kappa 0.21). Micronodular cirrhosis, lymphoid aggregates, bile duct damage, bile ductular proliferation and steatosis were significantly more common in HCV biopsies compared to HBV. Conclusions: Concordance between Ishak and METAVIR scoring systems is good for necroinflammatory change, and excellent for fibrotic change.

Enhanced monocyte Th1 cytokine production in HCV-infected cryoglobulinemic patients
Stefano Loffreda et al.
Background/Aims: The etiologic link between chronic hepatitis C virus (HCV) infection and mixed cryoglobulinemia is well established, while its prognostic significance within the context of HCV-related hepatitis is not as clear. Patients with an HCV-related cryoglobulinemic syndrome oft have mild liver disease, an aspect that can be influenced by an individual's Th1/Th2 orientation. Our goal was to document stigmata of differentiate cytokine production in this subgroup of patients. Methods: Fifteen patients with chronic HCV-related liver disease (CLD) and a cryoglobulinemic syndrome (CRYO) were compared to age/sex matched CLD controls with negative cryocrit. Cultured monocytes were stimulated with either Staphylococcus aureus (SAC) or lipopolysaccharide (LPS). Results: The protein concentrations of TNF- and of the Th1-type cytokines interleukin (IL)-12 and IL-18 were significantly greater in the CRYO group, while IL-10 (a Th2 cytokine) levels were greater in the control group. Conclusions: The clinical distinctiveness of the two groups was reflected at the cytokine level. The cryoglobulinemic patients studied showed a greater Th1 polarization than their cryoglobulin-negative counterparts. This enhanced production of Th1-type cytokines is seemingly not able to rid the host of infection but may account for a milder course of liver disease.

BRITISH MEDICAL JOURNAL

NEW ENGLAND JOURNAL

Volume 348:791-799 February 27, 2003 Number 9

Multiple Colorectal Adenomas, Classic Adenomatous Polyposis, and Germ-Line Mutations in MYH
Oliver M. Sieber, B.Sc., Lara Lipton, M.B., B.S., Michael Crabtree, M.B., B.S., Karl Heinimann, Ph.D., Paulo Fidalgo, M.D., Robin K.S. Phillips, M.D., Marie-Luise Bisgaard, M.D., Torben F. Orntoft, M.D., Lauri A. Aaltonen, Ph.D., Shirley V. Hodgson, D.M., Huw J.W. Thomas, Ph.D., and Ian P.M. Tomlinson, Ph.D.

Background Germ-line mutations in the base-excision­repair gene MYH have been associated with recessive inheritance of multiple colorectal adenomas. Tumors from affected persons displayed excess somatic transversions of a guanine­cytosine pair to a thymine­adenine pair (G:CT:A) in the APC gene. Methods We screened for germ-line MYH mutations in 152 patients with multiple (3 to 100) colorectal adenomas and 107 APC-mutation­negative probands with classic familial adenomatous polyposis (>100 adenomas). Subgroups were analyzed for changes in the related genes MTH1 and OGG1. Adenomas were tested for somatic APC mutations. Results Six patients with multiple adenomas and eight patients with polyposis had biallelic germline MYH variants. Missense and protein-truncating mutations were found, and the spectrums of mutations were very similar in the two groups of patients. In the tumors of carriers of biallelic mutations, all somatic APC mutations were G:CT:A transversions. In the group with multiple adenomas, about one third of patients with more than 15 adenomas had biallelic MYH mutations. In the polyposis group, no patient with biallelic MYH mutations had severe disease (>1000 adenomas), but three had extracolonic disease. No clearly pathogenic MTH1 or OGG1 mutations were identified. Conclusions Germ-line MYH mutations predispose persons to a recessive phenotype, multiple adenomas, or polyposis coli. For patients with about 15 or more colorectal adenomas - especially if no germ-line APC mutation has been identified and the family history is compatible with recessive inheritance - genetic testing of MYH is indicated for diagnosis and calculation of the level of risk in relatives. Clinical care of patients with biallelic MYH mutations should be similar to that of patients with classic or attenuated familial adenomatous polyposis.

Adefovir Dipivoxil for the Treatment of Hepatitis B e Antigen­Negative Chronic Hepatitis B
Stephanos J. Hadziyannis, M.D., Nicolaos C. Tassopoulos, M.D., E. Jenny Heathcote, M.D., Ting-Tsung Chang, M.D., George Kitis, M.D., Mario Rizzetto, M.D., Patrick Marcellin, M.D., Seng Gee Lim, M.D., Zachary Goodman, M.D., Michael S. Wulfsohn, M.D., Ph.D., Shelly Xiong, Ph.D., John Fry, B.Sc., Carol L. Brosgart, M.D., for the Adefovir Dipivoxil 438 Study Group

Background Adefovir dipivoxil, a nucleotide analogue, demonstrated clinically significant antiviral activity in patients with chronic hepatitis B in phase 1 and 2 clinical trials. Methods We randomly assigned 185 patients with chronic hepatitis B who were negative for hepatitis B e antigen (HBeAg) to receive either 10 mg of adefovir dipivoxil or placebo once daily for 48 weeks in a 2:1 ratio and a double-blind manner. The primary end point was histologic improvement. Results At week 48, 64 percent of patients who had base-line liver-biopsy specimens available in the adefovir dipivoxil group had improvement in histologic liver abnormalities (77 of 121), as compared with 33 percent of patients in the placebo group (19 of 57, P<0.001). Serum hepatitis B virus (HBV) DNA levels were reduced to fewer than 400 copies per milliliter in 51 percent of patients in the adefovir dipivoxil group (63 of 123) and in 0 percent of those in the placebo group (0 of 61, P<0.001). The median decrease in log-transformed HBV DNA levels was greater with adefovir dipivoxil treatment than with placebo (3.91 vs. 1.35 log copies per milliliter, P<0.001). Alanine aminotransferase levels had normalized at week 48 in 72 percent of patients receiving adefovir dipivoxil (84 of 116), as compared with 29 percent of those receiving placebo (17 of 59, P<0.001). No HBV polymerase mutations associated with resistance to adefovir were identified. The safety profile of adefovir dipivoxil was similar to that of placebo. Conclusions In patients with HBeAg-negative chronic hepatitis B, 48 weeks of adefovir dipivoxil treatment resulted in significant histologic, virologic, and biochemical improvement, with an adverse-event profile similar to that of placebo. There was no evidence of the emergence of adefovir-resistant HBV polymerase mutations.

Adefovir Dipivoxil for the Treatment of Hepatitis B e Antigen­Positive Chronic Hepatitis B
Patrick Marcellin, M.D., Ting-Tsung Chang, M.D., Seng Gee Lim, M.D., Myron J. Tong, Ph.D., M.D., William Sievert, M.D., Mitchell L. Shiffman, M.D., Lennox Jeffers, M.D., Zachary Goodman, M.D., Ph.D., Michael S. Wulfsohn, M.D., Ph.D., Shelly Xiong, Ph.D., John Fry, B.Sc., Carol L. Brosgart, M.D., for the Adefovir Dipivoxil 437 Study Group

Background In preclinical and phase 2 studies, adefovir dipivoxil demonstrated potent activity against hepatitis B virus (HBV), including lamivudine-resistant strains. Methods We randomly assigned 515 patients with chronic hepatitis B who were positive for hepatitis B e antigen (HBeAg) to receive 10 mg of adefovir dipivoxil (172 patients), 30 mg of adefovir dipivoxil (173), or placebo (170) daily for 48 weeks. The primary end point was histologic improvement in the 10-mg group as compared with the placebo group. Results After 48 weeks of treatment, significantly more patients who received 10 mg or 30 mg of adefovir dipivoxil per day than who received placebo had histologic improvement (53 percent [P<0.001], 59 percent [P<0.001], and 25 percent, respectively), a reduction in serum HBV DNA levels (by a median of 3.52 [P<0.001], 4.76 [P<0.001], and 0.55 log copies per milliliter, respectively), undetectable levels (fewer than 400 copies per milliliter) of serum HBV DNA (21 percent [P<0.001], 39 percent [P<0.001], and 0 percent, respectively), normalization of alanine aminotransferase levels (48 percent [P<0.001], 55 percent [P<0.001], and 16 percent, respectively), and HBeAg seroconversion (12 percent [P=0.049], 14 percent [P=0.01], and 6 percent, respectively). No adefovir-associated resistance mutations were identified in the HBV DNA polymerase gene. The safety profile of the 10-mg dose of adefovir dipivoxil was similar to that of placebo; however, there was a higher frequency of adverse events and renal laboratory abnormalities in the group given 30 mg of adefovir dipivoxil per day. Conclusions In patients with HBeAg-positive chronic hepatitis B, 48 weeks of 10 mg or 30 mg of adefovir dipivoxil per day resulted in histologic liver improvement, reduced serum HBV DNA and alanine aminotransferase levels, and increased the rates of HBeAg seroconversion. The 10-mg dose has a favorable risk­benefit profile for long-term treatment. No adefovir-associated resistance mutations were identified in the HBV DNA polymerase gene.

LANCET

Volume 361, Number 9355   01 February 2003

Intrahepatic arterial versus intravenous fluorouracil and folinic acid for colorectal cancer liver metastases: a multicentre randomised trial
David J Kerr, Colin S McArdle, Jonathan Ledermann, Irving Taylor, David J Sherlock, Peter M Schlag, John Buckels, David Mayer, Dionne Cain, Richard J Stephens, on behalf of the MRC and EORTC colorectal cancer study groups

Background The liver is the most frequent site for metastases of colorectal cancer, which is the second largest contributor to cancer deaths in Europe. We did a randomised trial to compare an intrahepatic arterial (IHA) fluorouracil and folinic acid regimen with the standard intravenous de Gramont fluorouracil and folinic acid regimen for patients with adenocarcinoma of the colon or rectum, with metastases confined to the liver. Methods We randomly allocated 290 patients from 16 centres to receive either intravenous chemotherapy (folinic acid 200 mg/m2, fluorouracil bolus 400 mg2 and 22-h infusion 600 mg/m2, day 1 and 2, repeated every 14 days), or IHA chemotherapy designed to be equitoxic (folinic acid 200 mg/m2, fluorouracil 400 mg/m2 over 15 mins and 22-h infusion 1600 mg/m2, day 1 and 2, repeated every 14 days). The primary endpoint was overall survival, and analysis was by intention to treat. Findings 50 (37%) patients allocated to IHA did not start their treatment, and another 39 (29%) had to stop before receiving six cycles of treatment because of catheter failure. The IHA group received a median of two cycles (0-6), compared with 8·5 (6-12) for the intravenous group. 45 (51%) IHA patients who did not start or did not receive six cycles switched to intravenous treatment. In both groups, grade 3 or 4 toxicity was uncommon. Median overall survival was 14·7 months for the IHA group and 14·8 months for the intravenous group (hazard ratio 1·04 [95% CI 0·80-1·33], log-rank test p=0·79). Similarly, there was no significant difference in progression-free survival. Interpretation Our results showed no evidence of an advantage in progression-free survival or overall survival for the IHA group; thus continued use of this regimen cannot be recommended outside of a clinical trial.

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