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Revue de Presse du Mois Mois de Décembre 2001

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HEPATOLOGY

Table of Contents for December 2001 · Volume 34 · Number 6

Biliary Tract

Carcinoembryonic antigen-related cell adhesion molecule 1 is the 85-kilodalton pronase-resistant biliary glycoprotein in the cholesterol crystallization promoting low density protein-lipid complex (*Human Study*)
Milan Jirsa, Lucie Muchová, Lubica Dráberová, Petr Dráber, Frantisek Smíd, Masahide Kuroki, Zdenk Mareek, Albert K. Groen
A pronase resistant 85-kd glycoprotein in the Concanavalin A­binding fraction (CABF) of biliary glycoproteins has been reported to act as a promotor of cholesterol crystallization. De Bruijn et al. (Gastroenterology 1996;110:1936-1944) found this protein in a low-density protein-lipid complex (LDP) with potent cholesterol crystallization promoting activity. This study identifies and characterizes this protein. An LDP was prepared from CABF by discontinuous gradient ultracentrifugation. Proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), blotting and immunochemical staining with anti-carcinoembryonic antigen, CEA-related adhesion molecule 1 (CEACAM1) cross-reacting antibodies. Biliary concentrations of CEA cross-reacting proteins were determined in patients with and without gallstones. Two isoforms of CEACAM1 (85- and 115-kd bands), CEA and 2 CEA cross-reacting protein bands of 40 and 50 kd were found in human bile. All bands were also present in CABF, but only a subfraction of the 85-kd band found in the LDP was resistant to digestion with pronase. CEACAM1-85 exhibited potent cholesterol crystallization promoting activity in vitro and accounted for most of the activity in CABF. Total CEA cross-reacting protein concentrations were the same in gallbladder biles from patients with cholesterol and pigment gallstones but only half of those in biles from nongallstone subjects. In conclusion, we have identified the protein component of the cholesterol crystallization promoting LDP to be CEACAM1-85. (HEPATOLOGY 2001;34:1075-1082.)

Chronic Liver Disease

The geographical distribution of primary biliary cirrhosis in a well-defined cohort (*Human Study*)
Martin I. Prince, Amanda Chetwynd, Peter Diggle, Mikala Jarner, Jane V. Metcalf, Oliver F. W. James
The incidence of primary biliary cirrhosis (PBC) varies widely between regions. However, little is known about variation within regions and the degree to which this may reflect environmental risk factors. The aim of this study was to describe the spatial distribution of cases of PBC in a defined region of Northeast England over a defined period, and to assess the magnitude of any departure from random spatial distribution. Seven hundred seventy patients with established PBC were identified in a previous comprehensive case finding study. A total of 3,044 control locations were randomly selected from postcode (zip code) data weighted for number of drop off points per postcode. Geographical analysis was performed by testing both for spatial variation in risk and local clustering by using previously described point process methods. Both tests used the same null hypothesis that risk of disease does not vary spatially and cases occur independently of each other. Statistically significant spatial variations in risk were found in the whole study region (P < .001) and in the major urban area within the region (P < .004). Risk was higher in the urban area of Tyneside than in the surrounding rural area. Within the rural area, spatial variation in risk was equivocal (P = .012), but there was significant (P = .001) clustering of cases (estimated average cluster effect approximately 10 excess cases within a 7-km radius). PBC occurred to a density of 10.7 cases/km2 in the highest risk areas. In conclusion, PBC is unevenly distributed in Northeast England. This may reflect one or more environmental risk factors in its etiology. (HEPATOLOGY 2001;34:1083-1088.)

Impact of gastroenterology consultation on the outcomes of patients admitted to the hospital with decompensated cirrhosis (*Human Study*)
Edmund J. Bini, Elizabeth H. Weinshel, Ramon Generoso, Loay Salman, Georges Dahr, Ivan Pena-Sing, Thomas Komorowski
Managed care has strongly discouraged generalists from referring patients to specialists in an effort to reduce the costs of health care. The aim of this study was to compare patient outcomes when generalists work together with gastroenterologists or alone in the management of patients admitted to the hospital with decompensated cirrhosis. Consecutive patients admitted to the hospital with decompensated cirrhosis over a 1-year period were identified. We compared the length of stay, cost of hospitalization, incidence of hospital readmission, and mortality for patients who did and those who did not have a gastroenterology (GI) consultation. A GI consultation was requested for 107 of the 197 patients (54.3%). Patients who had a GI consultation had a significantly shorter length of stay (5.6 ± 3.5 vs. 10.1 ± 5.8 days, P < .001) and a lower cost of hospitalization ($6,004 ± $4,994 vs. $10,006 ± $6,183, P < .001) than those patients who were managed by generalists alone. The 30-day incidence of readmission (13.3% vs. 27.8%, P = .01) and mortality (7.5% vs. 16.7%, P = .045) were significantly lower in the GI consultation group. During a median follow-up of 618 days (range, 2-970), patients who had a GI consultation during hospitalization had a significantly longer time to hospital readmission (P < .001) and improved survival (P = .02) compared with those who were managed by generalists alone. In conclusion, for patients admitted to the hospital with decompensated cirrhosis, individuals who were managed by generalists in conjunction with gastroenterologists had better outcomes than those who were managed by generalists alone. (HEPATOLOGY 2001;34:1089-1095.)

Propranolol for the prevention of first esophageal variceal hemorrhage: A lifetime commitment? (*Human Study*)
Diane R. Abraczinskas, Rie Ookubo, Norman D. Grace, Roberto J. Groszmann, Jaime Bosch, Guadalupe Garcia-Tsao, Caroline R. Richardson, Daniel S. Matloff, Juan Rodés, Harold O. Conn
Although blockers have had significant impact in the treatment of portal hypertension, the question of how long they should be continued for prevention of variceal hemorrhage remains unknown. Prospective studies on blockers to prevent variceal hemorrhage lack long-term follow-up, and indefinite administration of blockers for primary prevention of variceal bleeding has become standard practice. The aim of this study was to determine the outcomes of patients in whom blocker therapy was discontinued. Patients completing a prospective, randomized, double-blind, placebo-controlled trial of propranolol for the primary prevention of variceal hemorrhage were tapered off of propranolol and placebo and followed prospectively for subsequent events. Of the 49 patients in the follow-up study (25 former propranolol, 24 former placebo), 9 experienced variceal hemorrhage (6 former propranolol, 3 former placebo). Following withdrawal of propranolol, the freedom from variceal bleeding was not significantly different between these 2 groups of patients, suggesting that the protective effect of propranolol against variceal hemorrhage, noted previously, was no longer present. Seventeen patients died (12 former propranolol, 5 former placebo) during the follow-up study. Cumulative survival was longer in the placebo group. These trends for EVH and survival were opposite to those observed in the original study population while patients were taking medication. When propranolol is withdrawn, the risk of variceal hemorrhage returns to what would be expected in an untreated population. Patients who discontinue blockers experience increased mortality compared with an untreated population. These observations support the current practice of indefinite prophylactic therapy. (HEPATOLOGY 2001;34:1096-1102.)


Hepatic and intestinal cytochrome P450 3A activity in cirrhosis: Effects of transjugular intrahepatic portosystemic shunts (*Human Study*)
Naga Chalasani, J. Christopher Gorski, Nilesh H. Patel, Stephen D. Hall, Raymond E. Galinsky
Transjugular intrahepatic portosystemic shunt (TIPS) is performed to treat some complications of cirrhosis. This study investigated the effects of cirrhosis and TIPS on intestinal and hepatic cytochrome P450 3A (CYP3A) activity. Nine volunteers were cirrhotic patients with TIPS, 9 were cirrhotic controls (matched for sex, age, etiology, and Child-Pugh class), and 9 were sex- and age-matched healthy volunteers. Simultaneous doses of midazolam were given intravenously (0.05 mg/kg) and orally (3 mg of [15N3]midazolam). Peripheral and portal venous blood samples were assayed for midazolam and [15N3]midazolam. The systemic clearance of midazolam was significantly greater (P < .05) in healthy volunteers (0.42 ± 0.10 L · h­1 · kg­1) compared with cirrhotic controls (0.20 ± 0.05) and with cirrhotic patients with TIPS (0.21 ± 0.09). Hepatic availability followed the same trend. The bioavailability of midazolam was significantly higher (P < .05) in cirrhotic patients with TIPS (0.76 ± 0.20) compared with cirrhotic controls (0.27 ± 0.14) and with healthy volunteers (0.30 ± 0.10). The intestinal availability was significantly greater (P < .05) in cirrhotic patients with TIPS (0.83 ± 0.17) compared with cirrhotic controls (0.32 ± 0.16) and with healthy volunteers (0.42±0.15). As expected, hepatic CYP3A activity was reduced in cirrhosis. However, in cirrhotic patients with TIPS, there was a marked loss in first-pass metabolism of midazolam as a result of diminished intestinal CYP3A activity. (HEPATOLOGY 2001;34:1103-1108.)

Contrast-enhanced doppler ultrasonography in the diagnosis of hepatocellular carcinoma and premalignant lesions in patients with cirrhosis (*Human Study*)
Anna Ludovica Fracanzani, Larry Burdick, Mauro Borzio, Massimo Roncalli, Nicola Bonelli, Franco Borzio, Alessandra Maraschi, Gemino Fiorelli, Silvia Fargion
Hepatocellular carcinogenesis in cirrhosis is a multistage process that includes large regenerative nodules, dysplastic nodules, and hepatocarcinoma. The aim of this study was to establish whether contrast-enhanced Doppler ultrasonography (US) is able to distinguish between early hepatocellular carcinoma (HCC) and small nonmalignant nodules in cirrhosis. Between January 1998 and December 1999, 500 cirrhotic patients with no previous history of HCC or evidence of hepatic focal lesions were enrolled and prospectively followed-up with US every 6 months until December 2000. Sixty-one patients developed focal lesions, 12 multifocal, and 49 monofocal. Biopsy of focal lesions, contrast-enhanced Doppler US, and spiral computed tomography (CT) were performed in 41 consecutive patients with small (<3 cm) monofocal lesions. Twenty nodules were diagnosed as HCC and 21 as nonmalignant (14 large regenerative nodules, 3 low-grade, and 4 high-grade dysplastic nodules) by liver biopsy. Intratumoral arterial blood flow was detected in 19 of 20 (95%) HCC and 6 of 21 (28%) nonmalignant nodules by contrast-enhanced Doppler US (P<.0001). The mean peak resistance and pulsatility indices were 0.82 ± 0.09 and 1.56 ± 0.2 in HCC and 0.62 ± 0.08 and 0.82 ± 0.08 in dysplastic lesions (P = .002 and .0001), respectively. Spiral CT revealed arterial perfusion in 19 of 20 HCC and in 4 of 21 nonmalignant nodules (high-grade dysplastic nodules). Four of the apparently false-positive nodules at enhanced Doppler US were high-grade dysplastic nodules and 2 evolved to HCC during follow-up. In conclusion, contrast-enhanced Doppler US is a noninvasive, very sensitive technique in differentiating malignant and premalignant lesions from nonmalignant focal lesions in the liver. (HEPATOLOGY 2001;34:1109-1112.)

Patterns of regional sympathetic nerve traffic in preascitic and ascitic cirrhosis (*Human Study*)
Massimo Pozzi, Guido Grassi, Elena Redaelli, Raffaella Dell'Oro, Laura Ratti, Alessandro Redaelli, Gerardo Foglia, Alessandro Di Lelio, Giuseppe Mancia
Overactivity of the sympathetic nervous system and portal hypertension are key factors in the development of ascites in cirrhosis. The sympathoexcitation that characterizes the more advanced stages of liver diseases is less clearly defined in preascitic cirrhosis. We measured sympathetic nerve traffic to skeletal muscle (peroneal nerve) and to skin districts by microneurography in (1) 12 Child class A cirrhotic patients with clinically significant portal hypertension (portal pressure gradient > 10 mm Hg, 14.8 ± 1.2 mm Hg, mean ± SEM) but without actual or previous ascites, (2) 16 Child class C cirrhotic patients with tense ascites, and (3) 10 patients with mild congestive heart failure, a condition paradigmatic of a marked sympathetic activation. Muscle sympathetic nerve traffic was markedly increased in Child class C subjects as compared with controls (23.9 ± 1.6 bursts/min, P < .01) and superimposable to that recorded in heart failure patients (52.9 ± 4.7 vs. 60.3 ± 2 bursts/min, P = not significant). Muscle sympathetic nerve traffic was also increased in Child class A subjects (41.6 ± 2 bursts/min, P < .01 vs. controls) although to a lesser extent (P < .05 vs. Child class C patients). Skin sympathetic nerve traffic was within the normal range in all patients. Neurohormones were all markedly increased in Child class C subjects. Only norepinephrine was increased in Child class A patients. Our data show that sympathetic nerve traffic activation (1) is already detectable in Child class A cirrhosis when clinically significant portal hypertension is present but ascites never developed and (2) is not generalized because although muscle traffic is increased, skin traffic is within normal range. The role of drugs modulating sympathoactivation should be investigated in preascitic cirrhosis. (HEPATOLOGY 2001;34:1113-1118.)

Growth Regulation and Cancer

Activation of caspase-8 during N-(4-hydroxyphenyl)retinamide­induced apoptosis in FAS-defective hepatoma cells
Kyung-Ran You, Myung-Nym Shin, Rae-Kil Park, Seung-Ok Lee, Dae-Ghon Kim
We observed that N-(4-hydroxyphenyl)retinamide (4HPR), a chemopreventive and chemotherapeutic agent, effectively induced apoptosis in hepatoma cells. Interestingly, Fas-negative (Hep 3B and PLC/PRF/5) hepatoma cells were shown to be more susceptible to apoptosis induced by 4HPR than were Fas-positive (Hep G2 and SK-HEP-1) hepatoma cells. Thus, we explored the mechanisms underlying 4HPR-induced apoptosis in Fas-defective hepatoma cells. Hep 3B cells stably expressing the dominant-negative Fas-associated death domain (dnFADD) showed no alteration in 4HPR drug susceptibility, but when stably expressing E1B19K, Crm A, or dominant-negative FLICE (dnFLICE), Hep 3B cells were resistant, suggesting that 4HPR-induced apoptosis was mediated by caspase-8 activation. Furthermore, apoptosis could be completely blocked by Z-VAD-FMK (a general caspase inhibitor) or by IETD-CHO (a caspase-8 inhibitor), but was only partially blocked by Ac-DEVD-CMK (a caspase-3 inhibitor), by N-acetyl-L-cysteine (NAC) (an antioxidant), by N-acetyl-leucyl-leucyl-norleucinal (ALLN) (a calpain inhibitor I), or by Z-LEHD-FMK (a caspase-9 inhibitor). Time-sequence analysis of the induction of apoptosis by 4HPR revealed that an initial caspase-8 activation was followed by late mitochondrial cytochrome c release and minor caspase-9 activation, which suggested that caspase-8 activation is the primary upstream regulatory point. Activation of Bid or induction of proapoptotic Bax was not observed during apoptosis. In contrast, Bcl-xL expression was decreased during 4HPR-induced apoptosis. Taken together, these results indicate that 4HPR may be a potential chemotherapeutic drug, which is able to induce apoptosis in Fas-defective hepatoma cells through caspase-8 activation. (HEPATOLOGY 2001;34:1119-1127.)

Immunohistologic study on the expressions of -fetoprotein and protein induced by vitamin K absence or antagonist II in surgically resected small hepatocellular carcinoma (*Human Study*)
Miwako Fujioka, Yutaka Nakashima, Osamu Nakashima, Masamichi Kojiro
Sixty-eight cases of single hepatocellular carcinoma (HCC) with less than 3 cm of diameter were immunohistochemically examined for the expressions of -fetoprotein (AFP) and protein induced by vitamin K absence or antagonist II (PIVKA-II). In cancerous tissues, the expression rate was significantly higher for PIVKA-II (34 cases [50%]) than AFP (21 cases [31%]) (P < .05), suggesting a higher specificity of PIVKA-II to small HCC. Sixteen of the 68 cases (24%) were positive to both AFP and PIVKA-II, and in 8 of the 16 cases, AFP and PIVKA-II expressing areas within a nodule were clearly divided by a fibrous septum. According to histologic grades, PIVKA-II expression was confirmed in 2 of the 15 well-differentiated HCCs, and in the well-differentiated component of 6 of the 12 "nodule-in-nodule"­type well-differentiated HCCs. AFP expression was not found in well-differentiated HCCs, but found in 16 of the 40 moderately differentiated HCCs (40%) and in the moderately differentiated component of 3 of the 12 "nodule-in-nodule"­type well-differentiated HCCs. The positive rate in the tissues was correlated to the serum levels for both AFP and PIVKA-II. In addition, frequency of tissue­PIVKA-II expression was higher than tissue-AFP expression in the cases whose serum protein level was within the normal range. This indicates that AFP and PIVKA-II have different patterns of tissue expression and of secretion to the blood. In comparison with tissue-AFP­negative cases, tissue-AFP­positive HCCs had a larger tumor size, higher frequencies of portal vein invasion and intrahepatic metastasis, a high Ki-67 labeling index, and a lower rate of recurrence-free survival. Thus, tissue-AFP­positive HCCs are suggested to be biologically more malignant than those HCCs that are AFP-negative and PIVKA-II­positive. (HEPATOLOGY 2001;34:1128-1134.)

Spatiotemporal expression of angiogenesis growth factor receptors during the revascularization of regenerating rat liver
Mark A. Ross, Christina M. Sander, Talia B. Kleeb, Simon C. Watkins, Donna Beer Stolz
Regenerating liver was evaluated for the spatiotemporal expression of angiogenic growth factor receptors on endothelial cell (EC) membranes during revascularization resulting from 70% partial hepatectomy (PHx). Fractions enriched in EC membranes were examined by Western blot for angiogenic growth factor receptor expression from 1 to 14 days after PHx. Increases in vascular endothelial growth factor (VEGF) receptors Flt-1 and Flk-1/KDR, angiopoietin receptors Tie-1, Tie-2, and platelet-derived growth factor receptor beta (PDGF-R), modest increases in epidermal growth factor receptor (EGF-R), and no increase in hepatocyte growth factor receptor (c-Met) or fibroblast growth factor receptors (FGF-R) were observed in isolated membranes during EC proliferation. All receptors were tyrosine phosphorylated, and therefore activated, during peak expression. Immunofluorescence staining of regenerating liver identified populations with increased receptor expression, indicating cells receptive to ligand signaling. EGF-R was upregulated evenly throughout the sinusoidal membrane, whereas c-Met was observed on hepatocyte canaliculae, bile duct epithelium, and large vessel EC. Tie-2 and PDGF-R were increased on sinusoidal and large vessel EC, whereas Tie-1 was expressed in EC surrounding avascular hepatic islands. Flk-1/KDR was increased on large vessels with slight increases on sinusoidal EC, whereas Flt-1 was increased in arterioles, sinusoidal EC as well as in hepatocytes. Although Flt-1 was phosphorylated on isolated hepatocytes, vascular endothelial growth factor165 (VEGF165) did not induce a proliferative or motogenic response. Proliferation assays on isolated EC indicated responsiveness to VEGF165, but synergism among several growth factors including PDGF-BB was also observed. The data identify novel autocrine and paracrine interactions and indicate that each growth factor acts on a specific set of EC at specific times during revascularization of regenerating liver. (HEPATOLOGY 2001;34:1135-1148.)

Inflammation, Cytokines, and Fibrosis

Delivery of IB superrepressor gene with adenovirus reduces early alcohol-induced liver injury in rats
Takehiko Uesugi, Matthias Froh, Gavin E. Arteel, Blair U. Bradford, Erwin Gäbele, Michael D. Wheeler, Ronald G. Thurman
Chronic alcohol administration increases gut-derived endotoxin in the portal blood, which activates Kupffer cells through nuclear factor B (NF-B) to produce toxic mediators such as proinflammatory cytokines, leading to liver injury. Therefore, a long-term intragastric ethanol feeding protocol was used here to test the hypothesis that NF-B inhibition would prevent early alcohol-induced liver injury. Adenoviral vectors encoding either the transgene for IB superrepressor (AdIB-SR) or the bacterial -galactosidase reporter gene (AdlacZ) were administered intravenously to Wistar rats. Animals were fed a high-fat liquid diet with either ethanol or isocaloric maltose-dextrin (control) for 3 weeks. There was no significant difference in mean urine alcohol concentrations between the groups fed ethanol. IB-SR expression was increased for up to 2 weeks after injection, but was undetectable at 3 weeks. NF-B activation was increased by ethanol and associated with up-regulation of tumor necrosis factor (TNF-). These increases were blunted significantly up to 2 weeks by AdIB-SR. Dietary alcohol significantly increased liver to body weight ratios and serum alanine transaminase (ALT) levels in AdlacZ-treated animals, effects that were blunted significantly in AdIB-SR­treated rats. Ethanol caused severe steatosis, inflammation, and focal necrosis in AdlacZ-treated animals. These pathologic changes were significantly decreased by AdIB-SR. The protective effects of IB-SR were significant 2 weeks after injection, but were lost at 3 weeks when IB-SR was no longer expressed. Ethanol increased 4-hydroxynonenal as a maker of oxidative stress in both AdlacZ and AdIB groups. These data support the hypothesis that NF-B inhibition prevents early alcohol-induced liver injury even in the presence of oxidative stress. (HEPATOLOGY 2001;34:1149-1157.)

Gene expression of tumor necrosis factor and TNF-receptors, p55 and p75, in nonalcoholic steatohepatitis patients (*Human Study*)
Javier Crespo, Amalía Cayón, Pedro Fernández-Gil, Manuel Hernández-Guerra, Marta Mayorga, Agustín Domínguez-Díez, José Carlos Fernández-Escalante, Fernando Pons-Romero
The main objective of this study was to analyze the pathogenic role of the tumor necrosis factor (TNF-) system in the development of nonalcoholic steatohepatitis (NASH). Fifty-two obese patients were studied. We investigated: (1) the expression of mRNA of TNF- and their p55 and p75-receptors by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) in hepatic and adipose tissues; and (2) the relationship between TNF-, p55, and p75 and the severity of NASH. Obese patients without NASH were the control group. A remarkable increase in the expression of mRNA of TNF- was found in patients with NASH in hepatic tissue (0.65 ± 0.54) and in peripheral fat (0.43 ± 0.45); in the control samples, the mRNA expression was 0.28 ± 0.32, P < .007, and 0.26 ± 0.22, P < .018, respectively. Furthermore, we found a significant increase in the mRNA levels of p55 receptor (2.42 ± 1.81 vs. 1.56 ± 1.17; P < .05); however, the mRNA expression of the p75 receptor was similar in both patients. Those patients with NASH with significant fibrosis presented an increase in the expression of mRNA TNF- in comparison with those with a slight or nonexistent fibrosis. An overexpression of TNF- mRNA is found in the liver and in the adipose tissue of NASH patients. The levels of mRNA-p55 are increased in the liver tissue of NASH patients. This overexpression is more elevated in patients with more advanced NASH. These findings suggest that the TNF- system may be involved in the pathogenesis of NASH. (HEPATOLOGY 2001;34:1158-1163.)

Liver Cell Injury and Apoptosis

Adenosine monophosphate­activated protein kinase mediates the protective effects of ischemic preconditioning on hepatic ischemia-reperfusion injury in the rat
Carmen Peralta, Ramón Bartrons, Anna Serafin, Cristina Blázguez, Manuel Guzmán, Neus Prats, Carme Xaus, Blanca Cutillas, Emilio Gelpí, Joan Roselló-Catafau
Hepatic ischemia-reperfusion (I/R) injury associated with liver transplantation and hepatic resections are an unresolved problem in the clinical practice. Preconditioning is known to preserve energy metabolism in liver during sustained ischemia, but the molecular mechanisms underlying this effect are still unclear. Different metabolic signals, including adenosine monophosphate (AMP) and nitric oxide (NO), have been implicated in preconditioning. AMP-activated protein kinase (AMPK) protects cells by acting as a low-fuel warning system, becoming switched on by adenosine triphosphate (ATP) depletion. NO synthesis is induced by AMPK in the heart during ischemia. The aim of this study was to investigate: 1) whether preconditioning induces AMPK activation; and 2) if AMPK activation leads to ATP preservation and reduced lactate accumulation during prolonged ischemia and its relationship with NO. Preconditioning activated AMPK and concomitantly reduced ATP degradation, lactate accumulation, and hepatic injury. The administration of an AMPK activator, AICAR, before ischemia simulated the benefits of preconditioning on energy metabolism and hepatic injury. The inhibition of AMPK abolished the protective effects of preconditioning. The effect of AMPK on energy metabolism was independent of NO because the inhibition of NO synthesis in the preconditioned group and the administration of the NO donor before ischemia, or to the preconditioned group with previous inhibition of AMPK, had no effect on energy metabolism. Both preconditioning and AICAR pretreatment, through AMPK activation, may be useful surgical and pharmacologic strategies aimed at reducing hepatic I/R injury. (HEPATOLOGY 2001;34:1164-1173.)

Disturbances in hepatic cell-cycle regulation in mice with assembly-deficient keratins 8/18
Diana M. Toivola, Mikko I. Nieminen, Michael Hesse, Tao He, Hélène Baribault, Thomas M. Magin, M. Bishr Omary, John E. Eriksson
Simple epithelial tissues such as liver and pancreas express keratins 8 (K8) and 18 (K18) as their major intermediate filament proteins. K8 and K18 null mice and transgenic mice that express mutant K18 (K18C) manifest several hepatocyte abnormalities and demonstrate that K8/18 are important in maintaining liver tissue and cell integrity, although other potential functions remain uncharacterized. Here, we report an additional abnormal liver phenotype, which is similar in K8 null, K18 null, and K18C mouse models. Liver histologic examination showed large polynuclear areas that lacked cell membranes, desmosomal structures, and filamentous actin. Similar, but less prominent, areas were observed in the pancreas. The parenchyma outside the polynuclear areas displayed irregular sinusoidal structures and markedly enlarged nuclei. Most K8 null hepatocytes were positive for the proliferating cell nuclear antigen (PCNA) with a doubled DNA content in comparison with the predominantly PCNA-negative wild-type hepatocytes. The distribution of the 14-3-3 protein was also altered in K8 null mice. Taken together, our results indicate that absence of keratin filaments causes disturbances in cell-cycle regulation, driving cells into the S-G2 phase and causing aberrant cytokinesis. These effects could stem from disturbed functions of K8/18-dependent cell-cycle regulators, such as the signaling integrator, 14-3-3. (HEPATOLOGY 2001;34:1174-1183.)

Molecular Cell Biology

Notch receptor expression in adult human liver: A possible role in bile duct formation and hepatic neovascularization (*Human Study*)
Sarbjit S. Nijjar, Heather A. Crosby, Lorraine Wallace, Stefan G. Hubscher, Alastair J. Strain
Notch signaling is an evolutionarily conserved mechanism, used to regulate cell fate decisions. Four Notch receptors have been identified in man (Notch-1 to -4). In this study, semiquantitative reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry were used to examine the expression pattern of Notch receptor genes in whole adult human liver and isolated liver cell preparations. All 4 receptors were expressed in the adult liver, with no significant differences in the levels of Notch-1, -2, and -4 messenger RNA (mRNA) between normal and diseased liver. However, Notch-3 expression appeared to be increased in diseased tissue. The distribution of Notch-1 and -4 in normal tissue was similar, with Notch-1 also detectable at low levels in the sinusoidal endothelium. Notch-2 expression was more widely distributed, and detectable in hepatocytes, medium-sized bile ducts, and the sinusoidal endothelium. Notch-3 expression was seen on hepatocytes, with weaker expression detectable in portal veins, hepatic arteries, and the sinusoids. In normal liver tissue Notch-1, -2, and -3 were found to be coexpressed on bile duct epithelium; however, with the exception of Notch-3 in primary sclerosing cholangitis (PSC) livers, expression was absent on proliferating ductules in all disease states examined. Interestingly, the expression of Notch-2 and -3 was associated with numerous small vessels within the portal tract septa of diseased tissue. The absence of Notch receptor expression on proliferating bile ductules and its presence on neovessels suggests that Notch signaling may be important for normal bile duct formation and the aberrant neovascularization seen in diseased liver tissue. (HEPATOLOGY 2001;34:1184-1192.)

Viral Hepatitis

The influence of human immunodeficiency virus coinfection on chronic hepatitis C in injection drug users: A long-term retrospective cohort study (*Human Study*)
Vincent Di Martino, Pierre Rufat, Nathalie Boyer, Philippe Renard, Françoise Degos, Michèle Martinot-Peignoux, Sophie Matheron, Vincent Le Moing, François Vachon, Claude Degott, Dominique Valla, Patrick Marcellin
In this study we analyzed the influence of human immunodeficiency virus (HIV) infection on the course of chronic hepatitis C through multivariate analysis including age, alcohol consumption, immune status, and hepatitis C virus (HCV)-related virologic factors. Eighty HIV-positive and 80 HIV-negative injection drug users included between 1980 and 1995 were matched according to age, gender, and duration of HCV infection and followed-up during 52 months. The progression to cirrhosis was the primary outcome measure. The impact of HIV on HCV-RNA load, histologic activity index, response to interferon therapy, and liver-related death was also considered. In HIV-positive patients, chronic hepatitis C was characterized by higher serum HCV-RNA levels (P = .012), higher total Knodell score (P = .011), and poorer sustained response to interferon therapy (P = .009). High serum HCV-RNA level was associated with low CD4-lymphocyte count (P = .001). Necroinflamatory score was higher in HIV-positive patients (P = .023) independently of the CD4-lymphocyte count, whereas increased fibrosis was related to decreased CD4-lymphocyte count (P = .011). The progression to cirrhosis was accelerated in HIV-positive patients with low CD4 cell count (RR = 4.06, P = .024) and in interferon-untreated patients (RR = 4.76, P = .001), independently of age at HCV infection (P = .001). Cirrhosis caused death in 5 HIV-positive patients. The risk of death related to cirrhosis was increased in heavy drinkers (RR = 10.8, P = .001) and in HIV-positive patients with CD4 cell count less than 200/mm3 (RR = 11.9, P = .007). In this retrospective cohort study, HIV coinfection worsened the outcome of chronic hepatitis C, increasing both serum HCV-RNA level and liver damage and decreasing sustained response to interferon therapy. Age and alcohol were cofactors associated with cirrhosis and mortality. Interferon therapy had a protective effect against HCV-related cirrhosis no matter what the patient's HIV status was. (HEPATOLOGY 2001;34:1193-1199.)

Prevalence and risk factors for hepatitis C virus infection at an Urban Veterans administration medical center (*Human Study*)
Megan E. Briggs, Christiane Baker, Robert Hall, J. Michael Gaziano, David Gagnon, Natalie Bzowej, Teresa L. Wright
This study was designed to determine the seroprevalence and risk factors for hepatitis C virus (HCV) infection in veterans. Anti-HCV testing was performed in 1,032 patients and a questionnaire regarding sociodemographic characteristics and potential risk factors was administered. Adjusted prevalence of unique HCV-positive patients using outpatient services was 17.7% (95% confidence interval [CI] 17.2%, 18.2%). The following risk factors were associated with HCV infection: a history of injection drug use (IDU), receipt of blood transfusion prior to 1992, history of tattoo (odds ratio [OR], 2.93; 95% CI, 1.70-5.08), combat job as a medical worker (OR, 2.68; 95% CI, 1.25-5.60), history of incarceration over 48 hours (OR, 2.56; 95% CI, 1.52-4.32), greater than 15 lifetime sexual partners (OR, 1.61; 95% CI, 0.94-2.76) and sexual relations with a prostitute (OR, 0.46; 95% CI, 0.25-0.82). We concluded that HCV is common in veterans. Risk factors independently associated with infection are IDU, prior transfusion, prior tattoo, combat medical work, incarceration, and multiple opposite sex partners. Infection with HCV among veterans is strongly associated with traditional risk factors for infection and less strongly associated with combat-related risk. (HEPATOLOGY 2001;34:1200-1205.)

Different hepatitis C virus nonstructural protein 3 (Ns3)-DNA­expressing vaccines induce in HLA-A2.1 transgenic mice stable cytotoxic T lymphocytes that target one major epitope
Carine Brinster, Stéphanie Muguet, Yu-Chun Lone, Delphine Boucreux, Nathalie Renard, Anne Fournillier, François Lemonnier, Geneviève Inchauspé
The immunogenicity of the Hepatitis C virus (HCV) nonstructural protein 3 (NS3) was investigated using different DNA-based strategies and a preclinical mouse model transgenic for the HLA-A2.1 molecule. Plasmids expressing NS3 either as a wild-type protein, as a fusion with murine lysosome-associated-membrane protein-1 specific sequences, or under the control of the Semliki Forest virus replicase were evaluated in vitro and in vivo. All plasmids were shown to express the expected size protein. These 3 NS3-expressing vaccines induced overall comparable levels of CTLs when measured at different times postvaccination although mice injected with the NS3-LAMP expressing plasmid showed a particularly homogeneous and overall vigorous response (specific lysis ranged from 60% to 90 % for an E:T ratio of 33.3:1 with a mean CTL precursor frequency of 1:2.105 cells). Out of the four HLA-A2.1-restricted NS3 epitopes previously described in HCV infected patients (aa 1073-1081, aa 1406-1415; aa 1169-1177 and aa 1287-1296), the NS3-DNA generated CTLs were predominantly targeted at the aa 1073-1081 epitope. Peptide-based immunization showed that the mouse repertoire was intact for all epitopes tested except one (aa 1287-1296). In conclusion, the 3 NS3-DNA vaccines although based on different mode of action, shared a comparable efficacy at inducing CTL. Surprisingly, the breadth of such response was restricted to a single, major epitope. (HEPATOLOGY 2001;34:1206-1217.)

Hepatitis B virus X protein transactivates inducible nitric oxide synthase gene promoter through the proximal nuclear factor B-binding site: Evidence that cytoplasmic location of X protein is essential for gene transactivation (*Human Study*)
Pedro Majano, Enrique Lara-Pezzi, Manuel López-Cabrera, Arantxa Apolinario, Ricardo Moreno-Otero, Carmelo García-Monzón
Nitric oxide appears to play a central role in the pathogenesis of many inflammatory disorders. We have previously shown that there is an enhanced intrahepatic expression of the inducible nitric oxide synthase (iNOS) gene during chronic hepatitis B virus infection, and that viral X protein (HBx) transcriptionally activates this cellular gene, but the molecular basis for this activation remains to be defined. We aimed to explore the involvement of different cis-acting elements of the human iNOS promoter in the HBx-mediated up-regulation as well as the effect of the intracellular distribution of the HBx on its transacting function. Cotransfection of human hepatocyte-derived cells with wild-type or mutated iNOS promoter and with an HBx expression vector showed that functional inactivation of the proximal nuclear factor B (NF-B)­binding site of the iNOS promoter markedly reduced the HBx-mediated transcriptional activity. Mobility shift assays showed increased DNA-protein complexes comprising mainly the p50 and p65 members of NF-B family in the nuclear extracts from HBx-transfected cells. Transient transfection experiments with HBx-expressing plasmids containing distinct cellular localization sequences showed that cytoplasmic location of this viral protein activated the iNOS promoter but when HBx was targeted to the nucleus, the HBx-induced luciferase activity was almost completely abrogated. In conclusion, cytoplasmic location of HBx protein is essential for the transcriptional activation of the iNOS gene through the nuclear translocation of p50-p65 heterodimers. (HEPATOLOGY 2001;34:1218-1224.)


New challenge of hepatorenal syndrome: Prevention and treatment
Florence Wong, Laurence Blendis
Hepatorenal syndrome (HRS) remains one of the major therapeutic challenges in hepatology today. The pathogenesis is complex, but the final common pathway seems to be that sinusoidal portal hypertension, in the presence of severe hepatic decompensation, leads to splanchnic and systemic vasodilatation and decreased effective arterial blood volume. Renal vasoconstriction increases concomitantly, renal hemodynamics worsens, and renal failure occurs. Renal failure was shown 15 years ago to be potentially reversible after liver transplantation. This potential reversibility together with increased understanding of the pathogenesis has led to successful preliminary attempts to reverse HRS nonsurgically with combinations of splanchnic vasoconstrictors and colloid volume expansion, insertion of transjugular intrahepatic portovenous shunt radiologically, and improved forms of dialysis. Recent classification of HRS into the acute onset or severe type 1 with virtually 100% mortality and the more insiduous less severe type II promises to shed more light on the pathogenesis of HRS, especially on the currently unrecognized precipitating factors. It is hoped that this classification will be included in the necessary and carefully performed clinical trials, which should lead to clearer indications for the available therapies. The challenge now is to use all this information to improve our management of cirrhotic patients to prevent occurrence of HRS in the future. (HEPATOLOGY 2001;34:1242-1251.)

GASTROENTEROLOGY

Table of Contents for December 2001 · Volume 121 · Number 6

WNT1 Inducible Signaling Pathway Protein 3, WISP-3, a Novel Target Gene in Colorectal Carcinomas With Microsatellite Instability
LIN THORSTENSEN, CHIEU B. DIEP, GUNN I. MELING, TRUDE H. AAGESEN, CHRISTIAN H. AHRENS, TORLEIV O. ROGNUM, and RAGNHILD A. LOTHE
Gastroenterology 2001 121: 1275-1280. Published online Nov 14 2001.Background & Aims: Microsatellite instability (MSI) is the phenotype of colorectal carcinomas with defect mismatch repair. Genes with repetitive sequences within their coding regions are targets for mutations in these tumors. We have evaluated 2 novel candidate genes for potential involvement in development of MSI colorectal carcinomas and compared them with alterations in known target genes. Methods: The MSI status was determined by multiplex polymerase chain reactions (PCRs) of 5-17 markers in a Norwegian series of 275 colorectal carcinomas. All MSI tumors were analyzed for gene mutations using fluorescence PCR followed by capillary electrophoresis. Two novel candidate genes, WNT1-inducible signaling pathway protein 3 (WISP-3) and caspase-1, and 9 known target genes were analyzed. Results: Thirteen percent of the tumors were MSI-high (H) and 12% were MSI-low (L). Thirty-three of 37 MSI-H vs. 1 of 34 MSI-L tumors showed mutations in the target genes (P < 0.001). WISP-3 was mutated in 31% of the MSI-H tumors. The frequencies of frameshift mutations in the known target genes were comparable with other studies. Conclusions: The relative high frequency of mutation, higher than those seen for other known target genes, the predicted truncation of the protein product, and the homology with WISP-1 and WISP-2, 2 proteins induced downstream of WNT1 signaling, strongly suggest WISP-3 as a novel target in development of MSI-H colorectal carcinomas.

CLINICAL SCIENCE:

Gastric Surgery Is Not a Risk for Barrett's Esophagus or Esophageal Adenocarcinoma
BENJAMIN AVIDAN, AMNON SONNENBERG, THOMAS G. SCHNELL, and STEPHEN J. SONTAG
Gastroenterology 2001 121: 1281-1285. Published online Nov 29 2001.Background & Aims: The contribution of duodeno-gastroesophageal reflux to the development of Barrett's esophagus has remained an interesting but controversial topic. The present study assessed the risk for Barrett's esophagus after partial gastrectomy. Methods: The data of outpatients from a medicine and gastroenterology clinic who underwent upper gastrointestinal endoscopy for any reason were analyzed in a case-control study. A case population of 650 patients with short- segment and 366 patients with long-segment Barrett's esophagus was compared in a multivariate logistic regression to a control population of 3047 subjects without Barrett's esophagus or other types of gastroesophageal reflux disease. Results: In the case population, 25 (4%) patients with short-segment and 15 (4%) patients with long-segment Barrett's esophagus presented with a history of gastric surgery compared with 162 (5%) patients in the control population, yielding an adjusted odds ratio of 0.89 with a 95% confidence interval of 0.54-1.46 for short-segment and an adjusted odds ratio of 0.71 (0.30-1.72) for long-segment Barrett's esophagus. Similar results were obtained in separate analyses of 64 patients with Billroth-1 gastrectomy, 105 patients with Billroth-2 gastrectomy, and 33 patients with vagotomy and pyloroplasty for both short- and long-segment Barrett's esophagus. Caucasian ethnicity, the presence of hiatus hernia, and alcohol consumption were all associated with elevated risks for Barrett's esophagus. Conclusions: Gastric surgery for benign peptic ulcer disease is not a risk factor for either short- or long-segment Barrett's esophagus. This lack of association between gastric surgery and Barrett's esophagus suggests that reflux of bile without acid is not sufficient to damage the esophageal mucosa.

Risk of Adenocarcinomas of the Esophagus and Gastric Cardia in Patients With Gastroesophageal Reflux Diseases and After Antireflux Surgery
WEIMIN YE, WONG-HO CHOW, JESPER LAGERGREN, LI YIN, and OLOF NYRÉN
Gastroenterology 2001 121: 1286-1293. Published online Nov 29 2001. Background & Aims: Gastroesophageal reflux has been proposed as an important risk factor for esophageal and gastric cardia adenocarcinoma, but prospective data are lacking. Furthermore, the effect of antireflux surgery has not yet been studied. We conducted a population-based retrospective cohort study to fill these gaps. Methods: A cohort of 35,274 male and 31,691 female patients with a discharge diagnosis of gastroesophageal reflux diseases, and another cohort of 6406 male and 4671 female patients who underwent antireflux surgery, were identified in the Swedish Inpatient Register. Follow-up was attained through record linkage with several nationwide registers. Standardized incidence ratio (SIR) was used to estimate relative risk of upper gastrointestinal cancers, using the general Swedish population as reference. Results: After exclusion of the first year follow-up, 37 esophageal and 36 gastric cardia adenocarcinomas were observed among male patients who did not have surgery (SIR, 6.3, 95% confidence interval [CI], 4.5-8.7; SIR, 2.4, 95% CI, 1.7-3.3, respectively). SIR for esophageal adenocarcinoma increased with follow-up time (P = 0.03 for trend). Among male patients who had undergone antireflux surgeries, risks were also elevated (16 esophageal adenocarcinoma, SIR, 14.1, 95% CI, 8.0-22.8; 15 gastric cardia adenocarcinomas, SIR, 5.3, 95% CI, 3.0-8.7) and remained elevated with time after surgery. The cancer risk pattern in women was similar to that for men, but the number of cases were much smaller. Conclusions: Gastroesophageal reflux is strongly associated with the risk of esophageal adenocarcinoma, and to a lesser extent, with gastric cardia adenocarcinoma. The risk of developing adenocarcinomas of the esophagus and gastric cardia remains increased after antireflux surgery.

Extraesophageal Associations of Gastroesophageal Reflux Disease in Children Without Neurologic Defects
HASHEM B. EL-SERAG, MARK GILGER, MARK KUEBELER, and LINDA RABENECK
Gastroenterology 2001 121: 1294-1299. Published online Nov 29 2001. Background & Aims: The potential association between gastroesophageal reflux disease (GERD) and extraesophageal manifestations remains unknown in children without neurological defects. We conducted a large case-control study to examine the association between GERD and several upper and lower respiratory disorders in these children. Methods: We identified all patients between 2 and 18 years of age with GERD who were seen at Texas Children's Hospital between 1996 and 2000. Patients seen during the same time period without GERD were randomly selected as controls (4:1 ratio). Patients with mental retardation, cerebral palsy, or congenital esophageal anomalies were excluded. We compared the presence of several predefined upper and lower respiratory disorders in cases and controls. Results: We identified 1980 patients with GERD and 7920 controls without GERD. Cases and controls were without neurological deficits or congenital esophageal anomalies. Cases were older than controls (9.2 years ± 4.6 vs. 8.6 ± 4.9, P < 0.0001), and were more likely to be female (51.2% vs. 47.2%, P = 0.0028) and white (60.2% vs. 41.2%, P < 0.0001). Compared with controls in univariate analyses, cases with GERD had more sinusitis (4.2% vs. 1.4%, P < 0.0001), laryngitis (0.7% vs. 0.2%), asthma (13.2% vs. 6.8%, P < 0.0001), pneumonia (6.3% vs. 2.3%, P < 0.0001), and bronchiectasis (1.0% vs. 0.1%, P < 0.0001). However, otitis media was less common in cases than controls (2.1% vs. 4.6%, P < 0.0001). After adjusting for differences in age, gender, and ethnicity in the regression analyses, GERD remained a significant risk factor for sinusitis (adjusted odds ratio [OR], 2.3; 95% confidence intervals [CI], 1.7-3.2; P < 0.0001), laryngitis (OR, 2.6; CI, 1.2-5.6; P = 0.0228), asthma (OR, 1.9; CI, 1.6-2.3; P < 0.0001), pneumonia (OR, 2.3; CI, 1.8-2.9; P < 0.0001), and bronchiectasis (OR, 2.3; CI, 1.1-4.6; P = 0.0193). Conclusions: GERD in children without neurological defects is associated with a several-fold increase in the risk of sinusitis, laryngitis, asthma, pneumonia, and bronchiectasis. Further studies are needed to examine whether a cause-effect relationship exists between GERD and these disorders in children.

Extensive Methylation of hMLH1 Promoter Region Predominates in Proximal Colon Cancer With Microsatellite Instability
YASUYUKI MIYAKURA, KOKICHI SUGANO, FUMIO KONISHI, AKIRA ICHIKAWA, MASATO MAEKAWA, KAZUHISA SHITOH, SEIJI IGARASHI, KENJIRO KOTAKE, YASUO KOYAMA, and HIDEO NAGAI
Gastroenterology 2001 121: 1300-1309. Published online Nov 29 2001. Background & Aims: Methylation of the hMLH1 promoter region has been suggested to cause microsatellite instability (MSI) in sporadic colorectal carcinoma (CRC). We studied the methylation profile in a wide region of the hMLH1 promoter and compared with the hMLH1 protein expression and MSI status in 88 cases of sporadic CRC. Methods: Na-bisulfite treatment and polymerase chain reaction single-strand conformation polymorphism analysis was performed using 5 sets of polymerase chain reaction primers spanning the promoter region of the hMLH1 to examine methylation status. Results were compared with immunostaining using anti-hMLH1 monoclonal antibody and MSI status of the tumor samples. Results: Methylation status was classified as full or partial methylation. Full methylation indicates the methylation of all CpG sites in the examined regions. Methylation of the hMLH1 promoter was observed in 88.9% (16 of 18) of CRCs showing high frequency MSI (MSI-H), among which 89% (14 of 16) had full methylation with reduced hMLH1 protein expression. All cases showing full methylation were proximal colon tumors with MSI-H. In cases with partial methylation, only the upstream region of the hMLH1 promoter was methylated. Partial methylation was also shown in 33.3% (6 of 18) of the normal mucosa of MSI-H cases. Frequencies of methylation were significantly correlated with female gender (P = 0.0009) and aging (P = 0.007). Conclusions: Full methylation of the hMLH1 promoter region and subsequent gene inactivation may play a crucial role in the carcinogenesis of MSI-H CRCs in the proximal colon. Methylation upstream of the hMLH1 promoter appears to be an early event in the carcinogenesis of MSI-H tumors.

Cystic Fibrosis Gene Mutations and Pancreatitis Risk: Relation to Epithelial Ion Transport and Trypsin Inhibitor Gene Mutations
PEADAR G. NOONE, ZHAOQING ZHOU, LAWRENCE M. SILVERMAN, PAUL S. JOWELL, MICHAEL R. KNOWLES, and JONATHAN A. COHN
Gastroenterology 2001 121: 1310-1319. Published online Nov 29 2001. Background & Aims: Nonalcoholic chronic pancreatitis is usually idiopathic and often associated with cystic fibrosis gene (CFTR) mutations. It is unknown whether pancreatitis risk correlates with having 1 or 2 CFTR mutations, abnormal epithelial ion transport, or mutations of other genes. Methods: We tested 39 patients with idiopathic chronic pancreatitis (mean age at diagnosis, 33 years) for common mutations of CFTR and of genes encoding a trypsin inhibitor (PSTI) and trypsinogen (PRSS1). To exclude hereditary pancreatitis, we initially relied on family history and subsequently tested for PRSS1 mutations. Twenty subjects were tested for rare CFTR mutations (DNA sequencing) and 11 were tested for extrapancreatic CFTR function (clinical and physiologic evaluation). Results: Mutations were identified in 24 of 39 subjects. Nine patients had cystic fibrosis-causing mutations, 8 of whom also had mild-variable mutations. Eight others had only mild-variable mutations. Nine subjects had the N34S PSTI mutation and 1 had hereditary pancreatitis (R122H, PRSS1). Pancreatitis risk was increased approximately 40-fold by having 2 CFTR mutations (P < 0.0001), 20-fold by having N34S (P < 0.0001), and 900-fold by having both (P < 0.0001). Subjects with 2 CFTR mutations had abnormal nasal epithelial ion transport and clinical findings suggesting residual CFTR function between that in cystic fibrosis and in carriers. By contrast, subjects with only PSTI mutations had normal CFTR function. Conclusions: CFTR-related pancreatitis risk correlates with having 2 CFTR mutations and reduced extrapancreatic CFTR function. The N34S PSTI mutation increased risk separately. Testing for pancreatitis-associated CFTR and PSTI genotypes may be useful in nonalcoholic pancreatitis.

Epithelial Barrier Defects in Ulcerative Colitis: Characterization and Quantification by Electrophysiological Imaging
ALFRED H. GITTER, FRIEDERIKE WULLSTEIN, MICHAEL FROMM, and JÖRG DIETER SCHULZKE
Gastroenterology 2001 121: 1320-1328. Published online Nov 29 2001.Background & Aims: In ulcerative colitis (UC), the epithelial barrier is impaired by erosion/ulcer-type lesions and epithelial apoptosis causing local leaks, and generalized tight junction alterations increasing the basal permeability. We quantified the contribution of these mechanisms to the increased colonic ion permeability. Methods: Sigmoid colon was stripped, and the spatial distribution of current clamped across the viable epithelium was recorded by a microelectrode probe, using the conductance scanning method. Local leaks (circumscribed conductive peaks) were marked, and structural changes were studied in H&E-stained series sections. Results: Overall conductivity increased from 8.4 ± 0.7 mS/cm2 (mean ± SEM) in controls to 11.7 ± 0.6 in specimens with mild inflammation (i.e., with intact epithelium) and 34.4 ± 6.2 mS/cm2 in moderate-to-severe inflammation (i.e., with visible epithelial lesions). Only in part this was caused by a generalized increase in basal conductivity (12.2 ± 1.5 mS/cm2 in moderate-to-severe UC vs. 8.3 ± 0.7 in controls). More importantly, the spatial distribution of conductivity, which was even in controls, showed dramatic leaks in UC. Leaks found in mild inflammation without epithelial lesion turned out to be foci of epithelial apoptosis. In moderate-to-severe inflammation, leaks correlated with epithelial erosion/ulcer-type lesions or crypt abscesses. Conclusions: In early UC, but not in controls, seemingly intact epithelium comprises leaks at apoptotic foci. With more intensive inflammation, erosion/ulcer-type lesions are highly conductive, even if covered with fibrin. Local leaks contribute 19% to the overall epithelial conductivity in mild and 65% in moderate-to-severe inflammation.

Celiac Disease-like Abnormalities in a Subgroup of Patients With Irritable Bowel Syndrome
ULRICH WAHNSCHAFFE, R. ULLRICH, E. O. RIECKEN, and J. D. SCHULZKE
Gastroenterology 2001 121: 1329-1338. Published online Nov 29 2001. Background & Aims: Abdominal symptoms in the absence of mucosal abnormalities are features of both the irritable bowel syndrome (IBS) and latent/potential celiac disease (cd). To identify a possible subgroup of IBS patients with latent/potential cd, surrogate markers of cd were investigated in IBS patients. Methods: IBS patients suffering from diarrhea (n = 102), and patients with active cd (n = 10), treated cd (n = 26), and latent cd (n = 5) were included in the study. We measured serum immunoglobulin (Ig) A against gliadin and tissue-transglutaminase, and IgA and IgM against gliadin, tissue-transglutaminase (intestinal cd-associated antibodies), and the dietary proteins -lactoglobulin and ovalbumin in duodenal aspirate by enzyme-linked immunosorbent assay. Intraepithelial lymphocytes (IELs) were counted in histology sections, and the expression of HLA-DQ2 (A1*0501/B1*0201) was investigated by polymerase chain reaction. In 26 IBS patients, the effect of 6 months of gluten withdrawal was examined. Results: Most cd patients expressed HLA-DQ2 and had increased intestinal cd-associated antibodies, whereas cd-associated serum IgA and IEL counts were increased in active cd in contrast to treated or latent cd. In IBS patients, 35% were HLA-DQ2-positive, 23% had increased IEL counts, and 0% and 30% had increased cd-associated antibodies in serum and duodenal aspirate, respectively. Furthermore, stool frequency and intestinal IgA decreased significantly under a gluten-free diet in the subgroups of HLA-DQ2-positive and intestinal antibody-positive IBS patients when compared with IBS patients without these markers. Conclusions: HLA-DQ2 expression and increased intestinal cd-associated antibodies are markers that can identify latent/potential cd in a subgroup of IBS patients who consequently appear to profit from a gluten-free diet.

-Up-regulation of Cyclooxygenase 2 Gene Expression Correlates With Tumor Angiogenesis in Human Colorectal Cancer
FABIO CIANCHI, CAMILLO CORTESINI, PAOLO BECHI, ORNELLA FANTAPPIÈ, LUCA MESSERINI, ALFREDO VANNACCI, IACOPO SARDI, GIANNA BARONI, VIERI BODDI, ROBERTO MAZZANTI, and EMANUELA MASINI
Gastroenterology 2001 121: 1339-1347. Published online Nov 29 2001. Background & Aims: Recent studies have shown that cyclooxygenase (COX)-2 and its products, prostaglandins (PGs), may be involved in colorectal carcinogenesis. The aim of this study was to determine whether COX-2 expression and PGE2 production correlate with microvessel density, vascular endothelial growth factor (VEGF) expression, and tumor metastasis in human colorectal cancer. Methods: Tumor samples and adjacent normal mucosa were obtained from 31 surgical specimens. Immunohistochemical expression of COX-2, VEGF, and CD31 was analyzed on paraffin-embedded tissue sections. COX-2 and COX-1 proteins were determined by Western blot analysis. COX-2 and VEGF messenger RNA expressions were evaluated using Northern blot analysis. PGE2 production was determined by specific radioimmunoassay. Results: The immunohistochemical expressions of both COX-2 and VEGF were significantly correlated with microvessel density (P = 0.02 and P = 0.002, respectively). A significant correlation was found between COX-2 and VEGF expression (P = 0.004). Western analysis confirmed the up-regulation of COX-2 protein expression. COX-2 and VEGF genes were overexpressed in tumor specimens as compared with normal mucosa. PGE2 levels were significantly higher in metastatic tumors than in nonmetastatic ones (P = 0.03). Conclusions: COX-2 is related to tumor angiogenesis in colorectal cancer. It is likely that VEGF is one of the most important mediators of the COX-2 angiogenic pathway.

Incidence of Gastric Cancer and Breast Cancer in CDH1 (E-Cadherin) Mutation Carriers From Hereditary Diffuse Gastric Cancer Families
PAUL D. P. PHAROAH, PARRY GUILFORD, CARLOS CALDAS, and THE INTERNATIONAL GASTRIC CANCER LINKAGE CONSORTIUM
Gastroenterology 2001 121: 1348-1353. Published online Nov 29 2001. Background & Aims: Germline mutations in CDH1 are known to cause hereditary diffuse gastric cancer (HDGC). Breast and colorectal cancer have also been reported in CDH1-associated HDGC. The purpose of this study was to estimate the cumulative risk of gastric and breast cancer in CDH1 mutation carriers. Methods: Family data were collected by member groups of the International Gastric Cancer Linkage Consortium. Eligible families had at least 3 cases of diffuse gastric cancer, and at least 1 affected member had tested positive for a mutation in CDH1. Eleven families met these criteria. We used the pedigree information to estimate penetrance using the MENDEL program. The conditional likelihood of the pedigree was maximized given the phenotype of the pedigree and genotype of the index case at ascertainment. We parameterized the model in terms of log relative risks for mutation carriers compared with risks in the general population of the United Kingdom. Noncarriers of the gene were assumed to develop the disease at population incidence rates. Results: The estimated cumulative risk of gastric cancer by age 80 years was 67% for men (95% confidence interval [95% CI], 39-99) and 83% for women (95% CI, 58-99). For women, the cumulative risk of breast cancer was 39% (95% CI, 12-84). The combined risk of gastric cancer and breast cancer in women was 90% by age 80 years. Conclusions: These penetrance estimates should be useful for genetic counseling in multiple-case families. However, they may not apply to individuals with a minimal family history, in whom the risks may be lower.

BASIC SCIENCE:

Prostanoid Production Via COX-2 as a Causative Mechanism of Rodent Postoperative Ileus
NICOLAS T. SCHWARZ, JÖRG C. KALFF, ANDREAS TÜRLER, BRITTA M. ENGEL, SIMON C. WATKINS, TIMOTHY R. BILLIAR, and ANTHONY J. BAUER
Gastroenterology 2001 121: 1354-1371. Published online Nov 29 2001. Background & Aims: This study demonstrates a significant role for cyclooxygenase (COX)-2 and prostanoid production as mechanisms for surgically induced postoperative ileus. Methods: Rats, COX-2+/+, and COX-2/ mice underwent simple intestinal manipulation. Reverse-transcription polymerase chain reaction and immunohistochemistry were used to detect and localize COX-2 expression. Prostaglandin levels were measured from serum, peritoneal lavage fluid, and muscularis culture media. Jejunal circular muscle contractions were measured in an organ bath, and gastrointestinal transit was measured in vivo. Results: The data show that intestinal manipulation induces COX-2 messenger RNA and protein within resident muscularis macrophages, a discrete subpopulation of myenteric neurons and recruited monocytes. The manipulation-induced increase in COX-2 expression resulted in significantly elevated prostaglandin levels within the circulation and peritoneal cavity. The source of these prostanoids could be directly attributed to their release from the inflamed muscularis externa. As a consequence of the molecular up-regulation of COX-2, we observed a decrease in in vitro jejunal circular muscle contractility and gastrointestinal transit, both of which could be alleviated pharmacologically with selective COX-2 inhibition. These studies were corroborated with the use of COX-2/ mice. Conclusions: Prostaglandins, through the induction of COX-2, are major participants in rodent postoperative ileus induced by intestinal manipulation.


-Blockade of Endogenous IL-18 Ameliorates TNBS-Induced Colitis by Decreasing Local TNF- Production in Mice
TESSA TEN HOVE, ANNE CORBAZ, HAGIT AMITAI, SHUKI ALONI, ILANA BELZER, PIERRE GRABER, PAUL DRILLENBURG, SANDER J. H. VAN DEVENTER, YOLANDE CHVATCHKO, and ANJE A. TE VELDE
Gastroenterology 2001 121: 1372-1379. Published online Nov 29 2001. Background & Aims: Interleukin (IL) 18 has proinflammatory effects. IL-18 plays a pivotal role in Th1 responses, but its proinflammatory activities extend beyond Th1 cells, including macrophages and production of tumor necrosis factor (TNF)  and IL-1. IL-18 is up-regulated in colonic specimens of patients with Crohn's disease. The goal of this study was to evaluate the role of IL-18. Methods: Activity of IL-18 was neutralized using recombinant human IL-18 binding protein isoform a (rhIL-18BPa) in trinitrobenzene sulfonic acid (TNBS)-induced colitis. Results: Mice treated daily with rhIL-18BPa (8 mg/kg) had significant reductions in clinical score, body weight loss, and colon weight increase compared with saline-treated mice. Histologic analysis showed that rhIL-18BPa-treated mice developed only mild colitis without signs of ulceration, with a mean total score of9.8 ± 1.3 points compared with 15.9 ± 1.1 points observed in saline-treated mice with colitis. Analysis of cytokine levels in colon homogenates showed a significant decrease in TNF-, IL-6, and IL-1 after rhIL-18BPa treatment but no effect on interferon . The therapeutic potential of rhIL-18BPa treatment was confirmed in TNBS mice that were treated only on days 8 and 9 after the start of the experiment. In these mice, significant reductions in total colitis score and colon weight were also observed. Conclusions: These findings show that inhibition of rhIL-18BPa bioactivity, via rhIL-18BPa, may be beneficial for the treatment of IBD.

Human Pancreatic Acinar Cells Lack Functional Responses to Cholecystokinin and Gastrin
BAOAN JI, YAN BI, DIANE SIMEONE, RICHARD M. MORTENSEN, and CRAIG D. LOGSDON
Gastroenterology 2001 121: 1380-1390. Published online Nov 29 2001. Background & Aims: Pancreatic acinar cells from various species express cholecystokinin (CCK) A, CCK-B, or a combination of these CCK receptor subtypes. The presence and functional roles of CCK receptors on human acinar cells remain unclear. Methods: Acini isolated from human pancreas were treated with CCK receptor agonists, CCK-8 and gastrin, and an agonist for m3 muscarinic acetylcholine receptors (m3 AchR), carbachol. Functional parameters measured included intracellular [Ca2+], amylase secretion, and ERK phosphorylation. Binding studies were performed using 125I-CCK-8. Expression of messenger RNAs (mRNAs) was determined using real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR) and localized by in situ hybridization. Results: Human acini did not respond to CCK agonists. In contrast, they responded to carbachol with robust increases in each of the functional parameters. Moreover, the cells responded to CCK agonists after adenoviral-mediated gene transfer of CCK-A or CCK-B receptors. A low level of specific and a high level of nonspecific binding of 125I-CCK-8 were observed. Quantitative RT-PCR indicated that the message levels for CCK-A receptors were ~30-fold lower than those of CCK-B receptors, which were ~10-fold lower than those of m3 Ach receptors. In situ hybridization indicated the presence of m3 Ach receptor and insulin mRNA but not CCK-A or CCK-B receptor mRNAs in adult human pancreas. Conclusions: These data indicate that human pancreatic acinar cells do not respond to CCK receptor agonists in terms of expected functional parameters and show that this is due to an insufficient level of receptor expression.

Duodenal Reflux Induces Cyclooxygenase-2 in the Esophageal Mucosa of Rats: Evidence for Involvement of Bile Acids
FAN ZHANG, NASSER K. ALTORKI, YU-CHUNG WU, ROBERT A. SOSLOW, KOTHA SUBBARAMAIAH, and ANDREW J. DANNENBERG
Gastroenterology 2001 121: 1391-1399. Published online Nov 29 2001. Background & Aims: Reflux of duodenal contents including bile acids is believed to contribute to esophageal injury and Barrett's esophagus. Cyclooxygenase (COX)-2, an inducible form of COX, has been implicated in both inflammation and carcinogenesis. In this study, we investigated the effects of bile acids and duodenal reflux on COX-2 expression in cultured esophageal cells and tissue, respectively. Methods: Immunoblotting and Northern blotting were used to assess the effects of bile acids on COX-2 expression in esophageal cell lines. Immunoblotting and immunohistochemistry were performed to evaluate the effects of duodenal reflux on COX-2 expression and cell proliferation in esophageal tissue. Results: Unconjugated bile acids were about fivefold more potent inducers of COX-2 messenger RNA, COX-2 protein, and prostaglandin synthesis than conjugated bile acids. Acidifying the culture medium sensitized esophageal cells to bile acid-mediated induction of COX-2. The induction of COX-2 by bile acids was mediated by phosphatidylinositol-3 kinase and extracellular signal-regulated kinase 1/2 mitogen-activated protein kinases. In experimental animals, duodenoesophageal reflux led to esophagitis, marked thickening of the esophageal mucosa, and enhanced expression of COX-2. Increased immunoreactivity for Ki-67 and cyclin D1 indicated that enhanced cell proliferation contributed to mucosal thickening. Conclusions: Reflux of duodenal contents into the esophagus led to increased COX-2 expression and mucosal thickening. Bile acids are likely to contribute to these effects.

D-Glucose Releases 5-Hydroxytryptamine from Human BON Cells as a Model of Enterochromaffin Cells
MINSOO KIM, HELEN J. COOKE, NAJMA H. JAVED, HANNAH V. CAREY, FIEVOS CHRISTOFI, and HELEN E. RAYBOULD
Gastroenterology 2001 121: 1400-1406. Published online Nov 29 2001. Background & Aims: 5-Hydroxytryptamine (5-HT) is released from enterochromaffin cells and activates neural reflex programs regulating motility and secretion. Although sugars are reported to release 5-HT in vivo, it is unclear whether they act directly on enterochromaffin cells or indirectly through an intermediary messenger. The aim was to determine if D-glucose is a stimulus for 5-HT release. Methods: Human BON cells, derived from enterochromaffin cells, were treated with D-glucose, galactose, and the nonmetabolizable methyl -D-glucopyranoside, or with fructose. Results: Reverse-transcription polymerase chain reaction together with Western blot analysis revealed an SGLT-like protein. D-Glucose caused a concentration-dependent increase in 5-HT release, which was mimicked by methyl -D-glucopyranoside and galactose but not fructose. D-Glucose-stimulated 5-HT release was significantly reduced by phloridzin. Concentrations of mannitol below 75 mmol/L were ineffective in releasing 5-HT. Brefeldin A abolished forskolin-stimulated 5-HT release without affecting basal or constitutive release. Conclusions: The results show that high concentrations of metabolizable and nonmetabolizable hexoses activate signal transduction pathways, leading to release of 5-HT. These findings imply a role for enterochromaffin cells as "glucose sensors" during ingestion of a meal.

The Role of Mutant Apc in the Development of Dysplasia and Cancer in the Mouse Model of Dextran Sulfate Sodium-Induced Colitis
HARRY S. COOPER, LYNETTE EVERLEY, WEN-CHI CHANG, GORDON PFEIFFER, BRYAN LEE, SREEKANT MURTHY, and MARGIE L. CLAPPER
Gastroenterology 2001 121: 1407-1416. Published online Nov 29 2001. Background & Aims: Differences in genetic background may play a role in the development of ulcerative colitis (UC)-related neoplasia. Loss of heterozygosity (LOH) of APC has been reported in human UC-associated neoplasia. To investigate the role of genetic differences in UC-associated neoplasia, we compared differences in dextran sodium sulfate (DSS) colitis-associated neoplasia between wild-type C57BL/6J mice (WT-DSS) and C57BL/6J mice with a germline mutation in Apc (Min-DSS). Methods: DSS colitis was induced in female wild-type and Min mice. Age- and sex-matched non-DSS-treated Mins were also studied. Animals were sacrificed after 1 and 2 cycles of DSS. The cecums and large intestines were studied for numbers of dysplasias/cancers. Dysplasias were studied for LOH of Apc. Results: No WT-DSS, 100% of Min-DSS, and 50% of non-DSS-treated Mins had dysplasia. The mean numbers of lesions per mouse were 0 (WT-DSS), 15.6 and 29.3 (1 and 2 cycles Min-DSS, respectively), 1.2 and 1.9 (age-matched control Min, 1 and 2 cycle equivalents, respectively; P < 0.0002, Min-DSS vs. WT-DSS and non-DSS-treated Min; P = 0.03, Min-DSS 2 cycle vs. Min-DSS 1 cycle). Cancers were seen in 0%, 22%, and 40% of non-DSS Min, Min-DSS-1 cycle, and Min-DSS-2 cycle animals, respectively. LOH of Apc was observed in 90.6% of dysplasias and 6% of nondysplastic mucosa. Conclusions: A germline mutation in Apc contributes significantly to the development of colitis-associated neoplasia. Colitis markedly accelerates the development of dysplasia and cancer in the Min mouse. Dysplasia in Min-DSS occurs through LOH of Apc.

Antral Mucosa Expresses Functional Leptin Receptors Coupled to STAT-3 Signaling, Which Is Involved in the Control of Gastric Secretions in the Rat
HÉLÈNE GOÏOT, SAMIR ATTOUB, STÉPHANIE KERMORGANT, JEAN-PIERRE LAIGNEAU, BERNARD LARDEUX, THÉRÈSE LEHY, MIGUEL J. M. LEWIN, and ANDRÉ BADO
Gastroenterology 2001 121: 1417-1427. Published online Nov 29 2001. Background & Aims: Leptin is a circulating hormone that communicates the peripheral nutritional status to the hypothalamus, which controls food intake, energy expenditure, and body weight. This study characterizes leptin receptors and leptin-sensitive STAT proteins in the antrum and investigates the effects of leptin on gastric secretions. Methods: The effects of leptin on gastrin messenger RNA (mRNA), plasma gastrin, gastric acid in vivo in the rat, and on somatostatin and gastrin secretions by isolated antral cells were determined in vitro. Leptin receptors were investigated in isolated rat antral cells by reverse transcription-polymerase chain reaction and binding of [125I]-leptin studies. The effects of in vivo and in vitro leptin on transduction signal STAT proteins were investigated by immunoblotting antral extracts. Results: Peripheral injection of leptin inhibited in a dose-dependent manner, basal gastric secretion, gastrinemia, and mucosal gastrin mRNA in vivo. mRNAs encoding the long (Ob-Rb) and short (Ob-Ra) receptor forms were detected in rat antral mucosa, as were STAT-1, -3, and -5b immunoreactive proteins. Isolated antral cells specifically bound [125I]-leptin, and addition of leptin to these cells inhibited the release of somatostatin and increased the release of gastrin. These effects were associated with an increase in nuclear STAT-3 proteins in vitro and in vivo. Conclusions: This study provides the first molecular evidence for the coexpression of leptin receptors and STAT-3 in antral mucosa. It provides further evidence for the involvement of leptin in the control of gastric secretions.

Antibody Blockade of ICAM-1 and VCAM-1 Ameliorates Inflammation in the SAMP-1/Yit Adoptive Transfer Model of Crohn's Disease in Mice
R. CARTLAND BURNS, JESUS RIVERA-NIEVES, CHRISTOPHER A. MOSKALUK, SATOSHI MATSUMOTO, FABIO COMINELLI, and KLAUS LEY
Gastroenterology 2001 121: 1428-1436. Published online Nov 29 2001. Background & Aims: Integrins (4 and 2) and their endothelial ligands vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) play key roles in leukocyte recruitment to areas of inflammation. ICAM-1 and VCAM-1 are expressed in inflamed intestinal tissues. This study investigates a possible causative role of adhesion molecules ICAM-1, VCAM-1, and 4 integrins in mediating the inflammatory response in a murine model of Crohn's disease (CD). Methods: CD4+ mesenteric lymph node cells from SAMP-1/Yit donor mice were adoptively transferred into major histocompatibility complex-matched severe combined immunodeficiency disease mice. Six weeks later, these mice were left untreated or treated for 3 days with monoclonal antibodies (mAbs) to ICAM-1, VCAM-1, or both, and 4, or both ICAM-1 and 4, dexamethasone, or nonblocking isotype control antibodies. On day 4 after treatment, tissues were investigated for expression of ICAM-1, VCAM-1, and for severity of inflammation using a semiquantitative inflammatory score. Dexamethasone treatment resolved all measures of intestinal inflammation. Results: Blocking either ICAM-1, VCAM-1, or 4 integrins had no significant beneficial effect. However, blocking ICAM-1 and 4, or blocking ICAM-1 and VCAM-1, showed a 70% resolution of the active inflammation, but not chronic inflammation. Conclusions: These findings suggest that blocking ICAM-1 and VCAM-1 may have therapeutic benefit for the acute inflammatory component of Crohn's disease.

Phosphorylation of the Serine 60 Residue Within the Cdx2 Activation Domain Mediates Its Transactivation Capacity
EDMOND H. H. M. RINGS, FRANÇOIS BOUDREAU, JENNIFER K. TAYLOR, JENNIFER MOFFETT, EUN RAN SUH, and PETER G. TRABER
Gastroenterology 2001 121: 1437-1450. Published online Nov 29 2001. Background & Aims: Cdx2 is critical in intestinal proliferation and differentiation. Modulation of Cdx2 function in response to cellular signaling is to be elucidated. We hypothesize that phosphorylation of the Cdx2 activation domain can modulate its function. Methods: The Cdx2 activation domain was delineated in transient transfections using different portions of Cdx2 fused to the Gal4-DNA binding domain. In vivo phosphorylation was studied by metabolic labeling with 32P-orthophosphate. To study a potential phosphorylation site, polyclonal antibodies were generated: CNL was raised against amino acids 54-66 of Cdx2 and P-Cdx2-S60 against the same epitope in which serine 60 was phosphorylated. Results: A critical region for transactivation resides within amino acids 60-70. Substitution of serine 60 with alanine reduces incorporation of 32P-orthophosphate substantially. S60-phosphorylation decreases Cdx2 transactivation. Phosphorylation of serine 60 can be inhibited with the mitogen-activated protein kinase inhibitors PD98059 or UO126. P-Cdx2-S60 recognizes phosphorylated serine 60 mainly in proliferative compartment of the intestinal epithelial layer. In contrast, CNL recognizes Cdx2 predominantly in the differentiated compartment. Conclusions: The Cdx2 activation domain is phosphorylated at serine 60 via the mitogen-activated protein kinase pathway. S60-phosphorylated and S60-nonphosphorylated Cdx2 have different transcriptional activity, as well as different spatial expression patterns in the intestinal epithelium.

The Superoxide Dismutase Mimetic MnTBAP Prevents Fas-Induced Acute Liver Failure in the Mouse
BENOÎT MALASSAGNE, PIERRE-JACQUES FERRET, RENAUD HAMMOUD, MICHELINE TULLIEZ, SASSIA BEDDA, HÉLÈNE TRÉBÉDEN, PATRICK JAFFRAY, YVON CALMUS, BERNARD WEILL, and FRÉDÉRIC BATTEUX
Gastroenterology 2001 121: 1451-1459. Published online Nov 29 2001. Background & Aims: Acute liver failure (ALF) of viral origin results from massive hepatocyte apoptosis induced by the interaction between Fas expressed on hepatocytes and Fas ligand on activated T lymphocytes. Because Fas-induced apoptosis of hepatocytes involves mitochondrial damages and potential reactive oxygen species (ROS) overproduction, we investigated whether manganese III tetrakis (5,10,15,20 benzoic acid) (MnTBAP), a nonpeptidyl mimic of superoxide dismutase (SOD), can inhibit Fas-induced ALF. Methods: An agonist anti-Fas monoclonal antibody was used to induce hepatocyte apoptosis in vitro and ALF in vivo. Results: Preventive and curative treatments by MnTBAP significantly increased survival rates and significantly reduced aspartate aminotransferase levels and parenchymal lesions. ROS generation was suggested by those beneficial effects and significant increases in SOD and Gpx activities after anti-Fas injection. Flow cytometry of isolated hepatocytes incubated with anti-Fas monoclonal antibody showed that ROS production was associated with the collapse of transmembrane potential and loss of cardiolipin content. After injection of anti-Fas monoclonal antibody, mitochondrial Bcl-2 was decreased, cytochrome c released, and caspase-3 activated. Mitochondrial alterations and their consequences were abrogated by MnTBAP. Conclusions: ROS are key executioners in Fas-induced hepatocyte apoptosis. This finding explains why a nonpeptidyl mimic of SOD can cure ALF in a model of viral hepatitis, pointing out the potential interest of this molecule in humans.

Kupffer Cell-Mediated Recruitment of Rat Dendritic Cells to the Liver: Roles of N-Acetylgalactosamine-Specific Sugar Receptors
RYOSUKE UWATOKU, MAKOTO SUEMATSU, TAICHI EZAKI, TAKAHITO SAIKI, MAKOTO TSUIJI, TATSURO IRIMURA, NORIFUMI KAWADA, TATSUO SUGANUMA, MAKOTO NAITO, MASAYASU ANDO, and KENJIRO MATSUNO
Gastroenterology 2001 121: 1460-1472. Published online Nov 29 2001. Background & Aims: Tissue recruitment of dendritic cells (DCs) is essential for antigen presentation. This study aimed to examine cellular and molecular mechanisms for DC recruitment to the liver. Methods: Purified rat DCs were injected into circulation and their traffics were analyzed in normal and Kupffer cell-depleted rats by intravital confocal microscopy and immunohistology. Affinities of DCs to sinusoidal cells were examined by a cell-binding assay. DC precursor recruitment was induced by particulate injection. Results: Both DC precursors and DCs at the antigen-transporting stage could be recruited to the liver, and their majority initially showed a selective binding to Kupffer cells. In the Kupffer cell-depleted rats, DCs could neither be recruited to the liver nor adhere to sinusoidal walls. Pretreatment with varied monosaccharides showed that sugar residues consisting of N-acetylgalactosamine were necessary for this binding. The binding was calcium-dependent, implying the C-type lectin involvement. Furthermore, DCs could endocytose N-acetylgalactosamine polymers in a receptor-specific manner. Conclusions: The DC-Kupffer cell binding through N-acetylgalactosamine-specific C-type lectin-like receptors is crucial for DC recruitment to the liver. Rat DCs at least partly possess receptors for endocytosis of galactosylated antigens. These DC receptors as well as Kupffer cell lectins are presumably responsible for this binding.

Adaptive Regulation of Bile Salt Transporters in Kidney and Liver in Obstructive Cholestasis in the Rat
JOHN LEE, FRANCESCO AZZAROLI, LIN WANG, CAROL J. SOROKA, ALESSANDRO GIGLIOZZI, KENNETH D. R. SETCHELL, WERNER KRAMER, and JAMES L. BOYER
Gastroenterology 2001 121: 1473-1484. Published online Nov 29 2001. Background & Aims: Cholestasis results in adaptive regulation of bile salt transport proteins in hepatocytes that may limit liver injury. However, it is not known if changes also occur in the expression of bile salt transporters that reside in extrahepatic tissues, particularly the kidney, which might facilitate bile salt excretion during obstructive cholestasis. Methods: RNA and protein were isolated from liver and kidney 14 days after common bile duct ligation in rats and assessed by RNA protection assays, Western analysis, and tissue immunofluorescence. Sodium-dependent bile salt transport was also measured in brush border membrane vesicles from the kidney. Results: After common bile duct ligation, serum bile salts initially rose and then declined to lower levels after 3 days. In contrast, urinary bile salt excretion rose progressively over the 2-week period. By that time, the ileal sodium-dependent bile salt transporter messenger RNA and protein expression in total liver had increased to 300% and 200% of controls, respectively, while falling to 46% and 37% of controls, respectively, in the kidney. Sodium-dependent uptake of 3H-taurocholate in renal brush border membrane vesicles was decreased. In contrast, the multidrug resistance-associated protein 2 expression in the kidney was increased 2-fold, even 1 day after ligation. Immunofluorescent studies confirmed the changes in the expression of these transporters in liver and kidney. Conclusions: These studies show that the molecular expression of bile salt transporters in the kidney and cholangiocytes undergo adaptive regulation after common bile duct obstruction in the rat. These responses may facilitate extrahepatic pathways for bile salt excretion during cholestasis.

CASE REPORTS:

Treatment of Vitamin D Deficiency Due to Crohn's Disease With Tanning Bed Ultraviolet B Radiation
POLYXENI KOUTKIA, ZHIREN LU, TAI C. CHEN, and MICHAEL F. HOLICK
Gastroenterology 2001 121: 1485-1488. Published online Nov 29 2001.POLYXENI KOUTKIA, ZHIREN LU, TAI C. CHEN, and MICHAEL F. HOLICK

Vitamin D, Skin, and Bone Research Laboratory, Department of Medicine, Endocrinology, Nutrition, and Diabetes Section, Boston University School of Medicine, Boston, Massachusetts

In Crohn's disease, severe skeletal demineralization, secondary hyperparathyroidism, and muscle weakness can occur. This may be caused by impaired vitamin D absorption, resulting from extensive intestinal disease and resection of duodenum and jejunum, where vitamin D is absorbed. We report a 57-year-old woman with a long history of Crohn's disease and short-bowel syndrome who had only 2 feet of small intestine remaining after 3 bowel resections. She was taking a daily multivitamin containing 400 IU of vitamin D3 and was dependent on total parenteral nutrition that contained 200 IU of vitamin D and calcium (18 mEq in a 1-L bag infused over 8 hours daily) for a period of 36 months. Despite the above replacement, she complained of bone pain and muscle weakness, and she continued to be vitamin D-deficient with a 25(OH)D level <20 ng/mL. She was then exposed to ultraviolet B (UVB) radiation in a tanning bed wearing a 1-piece bathing suit for 10 minutes, 3 times a week for 6 months at the General Clinical Research Center, Boston University Medical Center. She tolerated the irradiation well without evidence of erythema. After 4 weeks, her serum 25(OH)D level increased by 357% from 7 to 32 ng/mL, parathyroid hormone level decreased by 52% from 92 to 44 pg/mL, and the serum calcium level increased from 7.8 to 8.5 mg/dL. After 6 months of UVB treatment, her serum 25(OH)D level was maintained in the normal range and was free of muscle weakness, and bone and muscle pain.

SPECIAL REPORTS AND REVIEWS:

Molecular Aspects of Iron Absorption and HFE Expression
SEPPO PARKKILA, ONNI NIEMELÄ, ROBERT S. BRITTON, ROBERT E. FLEMING, ABDUL WAHEED, BRUCE R. BACON, and WILLIAM S. SLY
Gastroenterology 2001 121: 1489-1496. Published online Nov 29 2001. Hereditary hemochromatosis, a disease of iron overload, occurs in about 1 in 200-400 Caucasians. The gene mutated in this disorder is termed HFE. The product of this gene, HFE protein, is homologous to major histocompatibility complex class I proteins, but HFE does not present peptides to T cells. Based on recent structural, biochemical, and cell biological studies, transferrin receptor (TfR) is a ligand for HFE. This association directly links HFE protein to the TfR-mediated regulation of iron homeostasis. Although evidence is accumulating that binding of HFE to TfR is critical for the effects of HFE, the final pieces in the HFE puzzle have not been established. This review focuses on recent advances in HFE research and presents a hypothetical model of HFE function.

BRITISH MEDICAL JOURNAL

8 December 2001 (Volume 323, Issue 7325)

Prescriptions for antiulcer drugs in Australia: volume, trends, and costs
Johanna I Westbrook, Anne E Duggan, and Jean H McIntosh
BMJ 2001; 323: 1338-1339. [Full text]

H2 receptor antagonists and proton pump inhibitors have markedly changed the management of peptic ulcer and gastro-oesophageal reflux disease; they have also changed the profile of national drug budgets. Antiulcer drugs have retained the leading position in drug sales worldwide: sales of antiulcer drugs were valued at $US12.9 billion (£8.6bn) in 1998 and were increasing at 3% a year.1

Since 1992 the Australian government's pharmaceutical benefits scheme has required prescribers of proton pump inhibitors to certify the presence of peptic ulcer disease or ulcerating oesophagitis (confirmed by endoscopy, radiography, or surgery) and refractory to treatment with other drugs, scleroderma oesophagus, or Zollinger-Ellison syndrome. The aim of this study was to assess how these restrictions have affected prescribing of antiulcer drugs....

1 December 2001 (Volume 323, Issue 7324)

ABC of the upper gastrointestinal tract: Indigestion: When is it functional?
Nicholas J Talley, Nghi Phung, and Jamshid S Kalantar
BMJ 2001; 323: 1294-1297. [Full text]  

Patients often complain of indigestion, but what do they mean? Indigestion is an old English word that means lack of adequate digestion, but patients and doctors interpret this in different ways. Many patients mean heartburn or acid regurgitation, the classic symptoms of gastro-oesophageal reflux disease. Some describe belching, abdominal rumblings, or even bad breath as indigestion. Others mean pain localised to the epigastrium or a non-painful discomfort in the upper abdomen which may be described as fullness, bloating, or an inability to finish a normal meal (early satiety). Dyspepsia is best restricted to mean pain or discomfort centred in the upper abdomen......

NEW ENGLAND JOURNAL

Volume 345:1734-1739 December 13, 2001 Number 24

Naltrexone in the Treatment of Alcohol Dependence
John H. Krystal, M.D., Joyce A. Cramer, B.S., William F. Krol, Ph.D., Gail F. Kirk, M.S., Robert A. Rosenheck, M.D., for the Veterans Affairs Naltrexone Cooperative Study 425 Group

Background Although naltrexone, an opiate-receptor antagonist, has been approved by the Food and Drug Administration for the treatment of alcohol dependence, its efficacy is uncertain. Methods We conducted a multicenter, double-blind, placebo-controlled evaluation of naltrexone as an adjunct to standardized psychosocial treatment. We randomly assigned 627 veterans (almost all men) with chronic, severe alcohol dependence to 12 months of naltrexone (50 mg once daily), 3 months of naltrexone followed by 9 months of placebo, or 12 months of placebo. All patients were offered individual counseling and programs to improve their compliance with study medication and were encouraged to attend Alcoholics Anonymous meetings. Results There were 209 patients in each group; all had been sober for at least five days before randomization. At 13 weeks, we found no significant difference in the number of days to relapse between patients in the two naltrexone groups (mean, 72.3 days) and the placebo group (mean, 62.4 days; 95 percent confidence interval for the difference between groups, ­3.0 to 22.8). At 52 weeks, there were no significant differences among the three groups in the percentage of days on which drinking occurred and the number of drinks per drinking day. Conclusions Our findings do not support the use of naltrexone for the treatment of men with chronic, severe alcohol dependence.

EDITORIAL (texte)

 Treatment for alcohol dependence has been limited almost entirely to various types of counseling. An exception has been the use of the medication disulfiram, which acts indirectly by making a person feel ill if he or she drinks alcohol. The efficacy of disulfiram is limited, however, because compliance is often poor, and it is not widely used.

Counseling patients with alcoholism leads to rates of remission similar to those achieved with treatment of other chronic medical conditions, such as asthma.1 Nonetheless, the large number of people dependent on alcohol in the United States (over 8.1 million2) and the substantial costs of alcoholism to society - an estimated $185 billion and 100,000 deaths each year - make it imperative that treatment be improved. Thus, it is not surprising that reports in 19923,4 that the opiate antagonist naltrexone reduced the relapse rate in people dependent on alcohol generated substantial interest. The reports suggested that treatment could be improved through the use of medication in conjunction with counseling.

Since the initial report by Volpicelli et al.3 and the confirmation by O'Malley et al.,4 there have been additional randomized, placebo-controlled clinical trials of the effectiveness of naltrexone in treating alcoholism.5,6,7,8,9,10,11 Six5,6,7,8,9,10 found that naltrexone was effective in preventing relapse in compliant patients. The clinical end points varied. Four studies3,6,7,8 defined relapse as having five or more drinks on one occasion, having five or more drinking occasions in one week, or arriving at a clinic visit intoxicated (blood alcohol concentration above 100 mg per deciliter). Two studies4,5 used the time to the first day of heavy drinking as a measure of the time to relapse; one of these4 also used the time to any drinking. One study9 found a greater reduction in the amount of alcohol consumed and in the levels of -glutamyltransferase in patients who were compliant with the naltrexone regimen, whereas another10 found significantly fewer heavy drinking days and fewer drinks on the days patients drank.

A double-blind, placebo-controlled trial of another opioid antagonist, nalmefene, found that it also prevented relapse of heavy drinking.12 A controlled study of naltrexone, nefazodone, and placebo in 183 patients found no benefit from naltrexone; naltrexone was associated with more adverse events than placebo and more attrition from the study than nefazodone and placebo.11 Finally, a recent meta-analysis found that naltrexone was moderately effective in reducing alcohol consumption in alcoholics.13

In this issue of the Journal, Krystal and his colleagues from the Veterans Affairs Naltrexone Cooperative Study 425 Group report the results of a multicenter, double-blind, placebo-controlled evaluation of naltrexone as an adjunct to standardized counseling for alcohol dependence.14 In 627 veterans (almost all men), they found no benefit from naltrexone, as measured by the time to relapse, the percentage of days on which drinking occurred, or the number of drinks per drinking day. Although the study by Krystal et al. is larger than the previous studies, additional factors may explain the discordant findings.

The typical patient in the study by Krystal et al. was about 10 years older than the patients in the previous studies (except for one study, in which the objective was to study naltrexone in older patients7). The typical patient had also been drinking for a longer period of time. Alcoholics who have families and are employed have a better prognosis than those who live alone or are unemployed. Only one third of the veterans in the study by Krystal et al. were married or living with a partner; these are smaller percentages than in most of the previous studies. Employment data are not given, but one third of the veterans were receiving disability pensions, a fact that may have affected their motivation to stop drinking. As Krystal et al. acknowledge, their results may not be generalizable to people who are not veterans, to women, or to patients whose dependence on alcohol is of shorter duration.

One of the early studies of naltrexone also involved patients treated at a Veterans Affairs hospital.1 The study was conducted at a single site, and for three months patients returned to the hospital daily for six to eight hours of rehabilitation activities. In the study by Krystal et al.,14 which involved multiple sites, patients received outpatient counseling for one hour per week for 16 weeks and then at a decreasing frequency for the remainder of a year. It is possible that in order to benefit from naltrexone, patients with more severe alcohol dependence require more intensive counseling.

The studies involving naltrexone also vary in the types of counseling used in conjunction with the medication. Four of the studies that showed a benefit4,5,6,8 used coping-skills therapy or relapse-prevention training. These types of counseling are commonly used in the treatment of alcoholism. In both, patients are taught to identify situations or mood states that place them at high risk for a relapse and are helped to develop coping skills to reduce the probability of a relapse. Although a negative study11 also used a relapse-prevention approach, the preponderance of the evidence suggests that this approach should be used in combination with naltrexone. Coping-skills therapy is different from the 12-step facilitation counseling used in the study by Krystal et al., which encourages involvement in Alcoholics Anonymous.

As the value of any medication is being established, randomized clinical trials are not always consistent in their findings. Our institute is currently supporting a multicenter, placebo-controlled clinical trial of naltrexone and acamprosate, another medication for the treatment of alcoholism, alone or in combination in a younger group of patients (30 percent of whom were women) than those in the study by Krystal et al. The efficacy of acamprosate appears to be similar to that of naltrexone. It is not an opiate antagonist, although the precise mechanism of its action is not known. Acamprosate affects the activity of N-methyl-d-aspartate receptors, which are components of the glutamate system; the effects of alcohol are mediated to some extent by this system. At least 12 of 14 controlled clinical trials conducted in Europe support its effectiveness.15 Diarrhea may be a side effect of acamprosate but is usually not severe enough to require cessation of treatment. Acamprosate is approved for use in most European countries, several Latin American countries, and Australia. In the United States, acamprosate is under review by the Food and Drug Administration.

If additional studies find that naltrexone works, it will be important to identify which patients are likely to benefit and which are not. If no benefit is found, such results, coupled with the results of the study by Krystal et al., would weigh against the use of naltrexone as an adjunctive treatment for alcoholism. Until we have more information, we recommend that physicians continue to prescribe naltrexone for patients they think might benefit. Such patients appear to be those who have been drinking heavily for 20 years or less and have stable social support and living situations.

Advances in our understanding of the neurobiology of alcohol dependence provide the foundation for developing new medications to treat the disorder. In addition to the opioid system, preclinical studies have shown that alcohol affects neurotransmitters in the brain, including dopamine, serotonin, glutamate, -aminobutyric acid, and norepinephrine. Second-messenger systems, such as the isoform of protein kinase C and the hypothalamic­pituitary­adrenal axis, are involved in problem drinking. The hypothalamic­pituitary­adrenal axis also has a pivotal role in stress. A number of compounds affecting these systems have shown efficacy in reducing alcohol consumption in studies in animals. They include an N-methyl-d-aspartate agonist, a compound that is both a serotonin 1A­receptor agonist and a serotonin 2­receptor antagonist, and a corticotropin-releasing­factor antagonist. At the clinical level, a recent trial indicated that ondansetron, a selective serotonin 3­receptor antagonist,16 shows promise for the treatment of people who become dependent on alcohol before the age of 25 years. Because so many new approaches seem promising, we are optimistic that more effective medications will be found.

Richard K. Fuller, M.D.
Enoch Gordis, M.D.
National Institute on Alcohol Abuse and Alcoholism
Bethesda, MD 20892

LANCET

Volume 358, Number 9297 08 December 2001

Effect on treatment outcome of coinfection with SEN viruses in patients with hepatitis C [Full Text]
Basil Rigas, Izhar Hasan, Raja Rehman, Peter Donahue, Knut M Wittkowski, Edward Lebovics
The newly discovered SEN D and SEN H viruses are transmitted parenterally and can cause post-transfusion hepatitis. We assessed whether coinfection of patients with chronic hepatitis C and SEN D or SEN H correlates with the outcome of treatment with interferon and ribavirin. Of 31 patients with hepatitis C studied, six were positive for SEN D and seven for SEN H (one was positive for both). All of those positive for SEN D and five of those positive for SEN H failed to respond to therapy. Overall response (RNA titre and alanine aminotransferase concentration after treatment) was lower in SEN-infected patients than uninfected patients (p=0·025). We conclude that coinfection with SEN viruses is frequent in chronic hepatitis C patients and might adversely affect the outcome of treatment with interferon and ribavirin.

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