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HEPATOLOGY

Volume 39, Issue 1 - January 2004

Viral Hepatitis

Viral features of lamivudine resistant hepatitis B genotypes A and D (p 42-50)
Bernhard Zöllner, Jörg Petersen, Elisabeth Puchhammer-Stöckl, Josef Kletzmayr, Martina Sterneck, Lutz Fischer, Matthias Schröter, Rainer Laufs, Heinz-Hubert Feucht
Viral differences among lamivudine resistant hepatitis B (HBV) genotypes have not been yet investigated. Therefore, we analyzed the characteristics of these viral strains in vivo . Forty-one patients carrying lamivudine resistant HBV were enrolled. Twenty-six patients (63%) carried resistant HBV genotype A (group A) and 15 patients (37%) carried resistant HBV genotype D (group D). The rate of reverse transcriptase 204I mutants was significantly higher in group D (67%) compared with group A (19%), whereas rt204V mutants (81% in group A vs 33% in group D; P= .006) and rt180M mutants (81% in group A vs 40% in group D, P= .015) prevailed in group A. The median time of shift from rt204I to rt204V mutants was significantly shorter in group A (4 months in group A, >12 months in group D, P< .001). Additional resistance associated mutations were detected exclusively in group D ( P= .004). In a multivariate analysis, HBV genotype ( P= .039) and pretreatment serum HBV DNA ( P= .001) were independently associated with emerging rt204I or rt204V mutants, respectively. Serum HBV copy numbers after emergence of resistance were higher in group A (mean log 10 6.99 copies/ml; range 3-9) compared with group D (mean log 10 6.1 copies/ml; range 3.3-8; P= .04). There was no difference between both groups regarding core promoter/precore mutations, viral turnover, and number of flares or disease progression during follow-up. In conclusion, the mutational pattern during selection of lamivudine resistant HBV strains differs between genotypes A and D. This may have consequences for a salvage regimen initiated for treatment of lamivudine resistant HBV. (H EPATOLOGY 2004;39:42-50.)

High prevalence of occult hepatitis B in Baltimore injection drug users (p 51-57)
Michael Torbenson, Rajesh Kannangai, Jacquie Astemborski, Steffanie A. Strathdee, David Vlahov, David L. Thomas
Occult hepatitis B is defined by the presence of hepatitis B virus (HBV) DNA in a serum or liver in the absence of hepatitis B surface antigen (HBsAg). The prevalence and clinical correlates of occult hepatitis B remain incompletely defined. A cross-sectional study was performed to determine the prevalence of occult hepatitis B in a high-risk cohort composed of 188 injection drug users in Baltimore, Maryland. All individuals had chronic hepatitis C viral infections confirmed by RNA detection and liver biopsy. Serologic assays for HBsAg and core antibody (HBcAb) were performed. Serum HBV DNA was detected using the COBAS HBV AMPLICOR monitor assay (lower limit of detection, 200 HBV copies per milliliter) and a semi-nested polymerase chain reaction (PCR) assay (lower limit of detection, 15 HBV copies per milliliter). Although almost all individuals (96%) were anti-HBC positive, only 8 of 188 (4%) were HBsAg positive. Occult hepatitis B was not identified using the COBAS assay, but was found in 81 of 180 (45%) of individuals using semi-nested PCR. Of the 8 HBsAg positive individuals, HBV DNA was found in 1/8 using the COBAS assay and 6/8 using the nested PCR assay. Overall, liver disease was mild, with a median serum alanine aminotransferase (ALT) of 38 IU/L, median activity grade of 3/18, and median fibrosis stage of 1/6. No association was found between the serum AST (aspartate aminotransferase), activity grade, or stage of liver disease and the presence of occult hepatitis B. Serum ALT levels were slightly higher in patients without occult hepatitis B (46 vs. 35 IU/L), and the median years since first injection drug use was somewhat longer in those without occult hepatitis B (24 vs. 20 years). In conclusion, although further research is needed to assess its clinical significance, there is a high prevalence of occult HBV infection in this cohort of HCV-infected injection drug users. (H EPATOLOGY 2004;39:51-57.)

Pediatric fulminant hepatic failure in endemic areas of hepatitis B infection: 15 years after universal hepatitis B vaccination (p 58-63)
Huey-Ling Chen, Chee-Jen Chang, Man-Shan Kong, Fu-Chen Huang, Hung-Chang Lee, Chieh-Chung Lin, Ching-Chuan Liu, I-Hsien Lee, Tzee-Chung Wu, Shu-Fen Wu, Yen-Hsuan Ni, Hong-Yuan Hsu, Ding-Shinn Chen, Mei-Hwei Chang
To investigate the role of hepatitis B virus (HBV) infection in pediatric fulminant hepatic failure (FHF) after the launch of universal HBV vaccination, the authors analyzed the data from patients with FHF collected from a nationwide collaborative study group. Children aged 1 month to 15 years who were diagnosed with FHF (62 males and 33 females) between 1985-1999 were included. HBV infection (hepatitis B surface antigen [HBsAg] and/or immunoglobulin M hepatitis B core antibody [IgM anti-HBc] seropositive) accounted for 46% (43 of 95 cases) of all the cases of FHF. The average annual incidence of FHF in the time period 1985-1999 was 0.053/100,000 in the group of patients ages 1-15 years and 1.29/100,000 in those patients age < 1 year. Approximately 61% (58 of 95 cases) of all FHF cases were infants. The percentage of HBV infection was found to be higher in infants (57%) compared with children ages 1-15 years (27%) ( P= 0.004). The incidence rate ratio of those patients age < 1 year to those ages 1-15 years was 54.2 for HBV-positive FHF and 15.2 for HBV-negative FHF. Maternal HBsAg was found to be positive in 97% of the infants with HBV-positive FHF, and hepatitis B e antigen (HBeAg) was found to be negative in 84% of these infants. Approximately 74% of all HBV-positive FHF patients and 81% of the infantile HBV-positive patients had been vaccinated. In conclusion, within the first 15 years of universal vaccination, HBV was found to rarely cause FHF in children age > 1 year but remained a significant cause of FHF in infants. HBV-positive FHF was prone to develop in infants born to HBeAg-negative, HBsAg-carrier mothers; these infants had not received hepatitis B immunoglobulin according to the vaccination program in place. (H EPATOLOGY 2004;39:58-63.)

Identification of HBV DNA sequences that are predictive of response to lamivudine therapy (p 64-73)
Alessia Ciancio, Antonina Smedile, Mario Rizzetto, Marco Lagget, John Gerin, Brent Korba
Numerous studies have shown that resistance to long-term lamivudine therapy occurs in as many as of hepatitis B virus (HBV) chronic carriers. Additional studies have shown that reversion of HBV mutations in the precore/core promoter region conferring an HBeAg-negative phenotype/genotype can occur in up to 30% of lamivudine-treated patients. In this study, sequences of the HBV polymerase and precore/core coding regions in 26 HBV-infected patients (24 with HBeAg-negative virus infection, 25 genotype D, 1 genotype A) treated for 27 to 53 months with lamivudine were analyzed to determine the relationship between pretreatment HBV DNA sequence patterns and long-term treatment response, and the effect of therapy on the status of HBV precore mutations. Reversions of precore mutations A1762T/G1764A and G1896A were observed in 29% and 25% of patients, respectively, but none became HBeAg-positive. These data are consistent with previously published reversion frequencies for 2 other groups of lamivudine-treated patients. Two naturally-occurring DNA polymorphisms at aa91 and aa256 of the HBV polymerase were observed in the pretreatment serum samples, which correlated with extended treatment failure. In conclusion, reversion of mutations conferring an HBeAg-negative phenotype occur relatively frequently under lamivudine therapy. Furthermore, at least in HBeAg-negative patients infected predominately with HBV genotype D, specific viral DNA sequences which are present before therapy appear to be useful as predictors of long-term response to lamivudine treatment. (H EPATOLOGY 2004;39:64-73.)

Health-related quality of life in active injecting drug users with and without chronic hepatitis C virus infection (p 74-80)
Olav Dalgard, Atle Egeland, Kjell Skaug, Kostas Vilimas, Tore Steen
This study assessed the effect of chronic hepatitis C virus (HCV) infection on the health-related quality of life (HRQOL) of injecting drug users, comparing the HRQOL of injecting drug users with and without chronic HCV infection. The study included 199 injecting drug users of more than 18 years of age who participated in a needle exchange program. Blood samples were tested for the presence of HCV RNA in serum with a polymerase chain reaction method. HRQOL was measured using the questionnaire SF-36, measuring HRQOL over the last 4 wk. The HCV RNA test was positive in 102 (51%) and negative in 97 (49%) subjects. The HRQOL scores of actively injecting drug users were markedly reduced compared to the population norm. However, we did not find poorer HRQOL in injecting drug users with chronic HCV infection than in injecting drug users without HCV infection. HCV RNA positive injecting drug users who were aware of the infection had lower HRQOL scores than those unaware of the infection in 4 of the 8 SF-36 dimensions (general health, physical functioning, physical role, and vitality). HCV RNA negative subjects, who believed they were infected, scored worse in one dimension (general health) compared to those who did not believe they were infected. In conclusion, chronic HCV infection per se did not negatively affect the HRQOL of active injecting drug users. Those who thought they were infected had a lower HRQOL scores than those who believed they were not infected. (H EPATOLOGY 2004;39:74-80.)

Reactive oxygen species suppress hepatitis C virus RNA replication in human hepatoma cells (p 81-89)
Jinah Choi, Ki Jeong Lee, Yanyan Zheng, Ardath K. Yamaga, Michael M.C. Lai, Jing-hsiung Ou
Published Online: 5 Jan 2004
Hepatitis C virus (HCV) is a positive-stranded RNA virus that causes severe liver diseases, such as cirrhosis and hepatocellular carcinoma. HCV uses an RNA-dependent RNA polymerase to replicate its genome and an internal ribosomal entry site to translate its proteins. HCV infection is characterized by an increase in the concentrations of reactive oxygen species (ROS), the effect of which on HCV replication has yet to be determined. In this report, we investigated the effect of ROS on HCV replication, using a bicistronic subgenomic RNA replicon and a genomic RNA that can replicate in human hepatoma cells. The treatment with peroxide at concentrations that did not deplete intracellular glutathione or induce cell death resulted in significant decreases in the HCV RNA level in the cells. This response could be partially reversed by the antioxidant N- acetylcysteine. Further studies indicated that such a suppressive response to ROS was not due to the suppression of HCV protein synthesis or the destabilization of HCV RNA. Rather, it occurred rapidly at the level of RNA replication. ROS appeared to disrupt active HCV replication complexes, as they reduced the amount of NS3 and NS5A in the subcellular fraction where active HCV RNA replication complexes were found. In conclusion, our results show that ROS can rapidly inhibit HCV RNA replication in human hepatoma cells. The increased ROS levels in hepatitis C patients may therefore play an important role in the suppression of HCV replication. (H EPATOLOGY 2004;39:81-89.)

Long-term outcome (35 years) of hepatitis C after acquisition of infection through mini transfusions of blood given at birth (p 90-96)
Maria Antonietta Casiraghi, Massimo De Paschale, Luisa Romanò, Renato Biffi, Agnese Assi, Giorgio Binelli, Alessandro Remo Zanetti
Long-term follow up studies of hepatitis C virus (HCV) infection rarely exceed 20-25 yr. We studied the outcome of HCV infection in 35-yr-old adults infected at birth (1968) through mini transfusions of blood. A retrospective-prospective study was carried out. The cohort included 31 individuals who were given mini blood transfusions (21-30 ml) collected from a donor subsequently revealed to be HCV infected. At enrollment (1998), 18 of 31 (58.1%) recipients had anti-HCV antibody and 16 (88.9%) of them were HCV-RNA positive. All viremic recipients and the infectious donor had the same genotype 1b. Sequence analysis of E1/E2 and NS5b regions, coupled with phylogenetic analysis, indicated that HCV isolates from donor/recipients were linked. Eleven of the 16 viremic recipients gave consent to liver biopsy. Nine had no fibrosis or mild portal fibrosis and 2 had either discrete (Ishak's staging 3) or marked (Ishak's staging 4) fibrosis. During the prospective follow-up period (1998-2003), 2 patients were given therapy, one of whom achieved sustained clinical and virologic response. A second biopsy, performed in 5 patients at a 5 yr interval, revealed no substantial modifications in 4 cases and progression from absence of fibrosis to mild portal fibrosis in the fifth. In conclusion, taking into account the limited study sample, these findings suggest that HCV infection acquired early in life shows a slow progression and mild outcome during the first 35 yr of infection. (H EPATOLOGY 2004;39:90-96.)

Liver Biology & Pathobiology

Differential modulation of rat hepatic stellate phenotype by natural and synthetic retinoids (p 97-108)
Karine Hellemans, Peggy Verbuyst, Erik Quartier, Frans Schuit, Krista Rombouts, Ross A.S. Chandraratna, Detlef Schuppan, Albert Geerts
Activation of hepatic stellate cells (HSC) is a central event in the pathogenesis of liver fibrosis during chronic liver injury. We examined the expression of retinoic acid (RAR) and retinoid X receptors (RXR) during HSC activation and evaluated the influence of natural and synthetic retinoic acids (RA) on the phenotype of culture-activated HSC. The expression of the major RAR/RXR subtypes and isoforms was analyzed by Northern hybridization. Presence of functional receptor proteins was established by gel shift analysis. Retinoic acids, RAR, and RXR selective agonists and an RAR antagonist were used to evaluate the effects of retinoid signalling on matrix synthesis by Northern blotting and immunoprecipitation, and on cell proliferation by BrdU incorporation. The 9- cis RA and synthetic RXR agonists reduced HSC proliferation and synthesis of collagen I and fibronectin. All- trans RA and RAR agonists both reduced the synthesis of collagen I, collagen III, and fibronectin, but showed a different effect on cell proliferation. Synthetic RAR agonists did not affect HSC proliferation, indicating that ATRA inhibits cell growth independent of its interaction with RARs. In contrast, RAR specific antagonists enhance HSC proliferation and demonstrate that RARs control proliferation in a negative way. In conclusion, natural RAs and synthetic RAR or RXR specific ligands exert differential effects on activated HSC. Our observations may explain prior divergent results obtained following retinoid administration to cultured stellate cells or to animals subjected to fibrogenic stimuli. (H EPATOLOGY 2004;39:97-108.)

AQP4 transfected into mouse cholangiocytes promotes water transport in biliary epithelia (p 109-116)
Patrick L. Splinter, Anatoliy I. Masyuk, Raul A. Marinelli, Nicholas F. LaRusso
Rodent cholangiocytes express 6 of the 11 known channel proteins called aquaporins (AQPs) that are involved in transcellular water transport in mammals. However, clarifying the role of AQPs in mediating water transport in biliary epithelia has been limited in part because of the absence of physiologically relevant experimental models. In this study, we established a novel AQP4-transfected polarized mouse cholangiocyte cell line suitable for functional studies of transepithelial water transport, and, using this model, we define the importance of this AQP in water transport across biliary epithelia. Polarized normal mouse cholangiocytes (NMCs) lacking endogenous AQP4 were transfected stably with functional AQP4 or cotransfected with functional AQP4 and a transport-deficient AQP4 dominant negative mutant using a retroviral delivery system. In transfected NMCs, AQP4 is expressed on both the mRNA and protein levels and is localized at both the apical and basolateral membranes. In nontransfected NMCs, the transcellular water flow, Pf, value was relatively high ( i.e. , 16.4 ± 3.2 m/sec) and likely was a reflection of endogenous expression of AQP1 and AQP8. In NMCs transfected with AQP4, Pfincreased to 75.7 ± 1.4 m/sec, that is, by 4.6-fold, indicating the contribution of AQP4 in channel-mediated water transport across MNCs monolayer. In cotransfected NMCs, AQP4 dominant negative reduced Pftwofold; no changes in Pfwere observed in NMCs transfected with the empty vector. In conclusion, we developed a novel polarized mouse cholangiocyte monolayer model, allowing direct study of AQP4-mediated water transport by biliary epithelia and generated data providing additional support for the importance of AQP4 in cholangiocyte water transport. (H EPATOLOGY 2004;39:109-116.)

Spontaneous cholecysto- and hepatolithiasis in Mdr2 -/- mice: A model for low phospholipid-associated cholelithiasis (p 117-128)
Frank Lammert, David Q.-H. Wang, Sonja Hillebrandt, Andreas Geier, Peter Fickert, Michael Trauner, Siegfried Matern, Beverly Paigen, Martin C. Carey
Previously, we identified needle-like and filamentous, putatively anhydrous cholesterol crystallization in vitro at very low phospholipid concentrations in model and native biles. Our aim now was to address whether spontaneous gallstone formation occurs in Mdr2 (Abcb4 ) knockout mice that are characterized by phospholipid-deficient bile. Biliary phenotypes and cholesterol crystallization sequences in fresh gallbladder biles and non-fixed liver sections were determined by direct and polarizing light microscopy. The physical chemical nature and composition of crystals and stones were determined by sucrose density centrifugation and before mass and infrared spectroscopy. Gallbladder biles of Mdr2 -/- mice precipitate needle-like cholesterol crystals at 12 weeks of age on chow. After 15 weeks, more than 50% of Mdr2 -/- mice develop gallbladder stones, with female mice displaying a markedly higher gallstone-susceptibility. Although gallbladder biles of Mdr2 -/- mice contain only traces ( 1.1 mM) of phospholipid and cholesterol, they become supersaturated with cholesterol and plot in the left 2-phase zone of the ternary phase diagram, consistent with anhydrous cholesterol crystallization. Furthermore, more than 40% of adult female Mdr2 -/- mice show intra- and extrahepatic bile duct stones. In conclusion, spontaneous gallstone formation is a new consistent feature of the Mdr2 -/- phenotype. The Mdr2 -/- mouse is therefore a model for low phospholipid-associated cholelithiasis recently described in humans with a dysfunctional mutation in the orthologous ABCB4 gene. The mouse model supports the concept that this gene is a monogenic risk factor for cholesterol gallstones and a target for novel therapeutic strategies. (H EPATOLOGY 2004;39:117-128.)

Over-expression of the ribosomal protein L36a gene is associated with cellular proliferation in hepatocellular carcinoma (p 129-138)
Jong-Hyun Kim, Kyung-Ran You, In Hee Kim, Baik-Hwan Cho, Chan-Young Kim, Dae-Ghon Kim
Using messenger RNA (mRNA) differential display, we identified a single complementary DNA (cDNA) fragment (HG23T1) that was over-expressed in a hepatocellular carcinoma (HCC) specimen. We cloned the full-length HG23T1 gene by the rapid amplification of cDNA end (RACE) polymerase chain reaction (PCR) method. It perfectly matched the gene encoding human ribosomal protein L36a ( RPL36A also referred to as RPL44 ). RPL36A mRNA was preferentially over-expressed in 34 of 40 HCC cases (85%, P< .001) and in all of 8 HCC cell lines. Ectopically over-expressed L36a ribosomal protein localized in the nucleoli of cells, and this localization seemed to be controlled by the N-terminal or the internal tetrapeptide consensus with its adjacent N-terminal domain. Over-expression of L36a led to enhanced colony formation and cell proliferation, which may have resulted from rapid cell cycling, and an antisense cDNA effectively reversed these alterations. In conclusion, RPL36A plays a role in tumor cell proliferation and may be a potential target for anticancer therapy of HCC. (H EPATOLOGY 2004;39:129-138.)

Role of thromboxane derived from COX-1 and -2 in hepatic microcirculatory dysfunction during endotoxemia in mice (p 139-150)
Hiroyuki Katagiri, Yoshiya Ito, Ken-ichiro Ishii, Izumi Hayashi, Makoto Suematsu, Shohei Yamashina, Takahiko Murata, Shuh Narumiya, Akira Kakita, Masataka Majima
Although thromboxanes (TXs), whose synthesis is regulated by cyclooxygenase (COX), have been suggested to promote inflammation in the liver, little is known about the role of TXA 2in leukocyte endothelial interaction during endotoxemia. The present study was conducted to investigate the role of TXA 2as well as that of COX in lipopolysaccharide (LPS)-induced hepatic microcirculatory dysfunction in male C57Bl/6 mice. We observed during in vivo fluorescence microscopic study that LPS caused significant accumulation of leukocytes adhering to the hepatic microvessels and non-perfused sinusoids. Levels of serum alanine transaminase (ALT) and tumor necrosis factor alpha (TNF ) also increased. LPS raised the TXB 2level in the perfusate from isolated perfused liver. A TXA 2synthase inhibitor, OKY-046, and a TXA 2receptor antagonist, S-1452, reduced LPS-induced hepatic microcirculatory dysfunction by inhibiting TNF production. OKY-046 suppressed the expression of an intercellular adhesion molecule (ICAM)-1 in an LPS-treated liver. In thromboxane prostanoid receptor-knockout mice, hepatic responses to LPS were minimized in comparison with those in their wild-type counterparts. In addition, a selective COX-1 inhibitor, SC-560, a selective COX-2 inhibitor, NS-398, and indomethacin significantly attenuated hepatic responses to LPS including microcirculatory dysfunction and release of ALT and TNF . The effects of the COX inhibitors on hepatic responses to LPS exhibited results similar to those obtained with TXA 2synthase inhibitor, and TXA 2receptor antagonist. In conclusion, these results suggest that TXA 2is involved in LPS-induced hepatic microcirculatory dysfunction partly through the release of TNF , and that TXA 2derived from COX-1 and COX-2 could be responsible for the microcirculatory dysfunction during endotoxemia. (H EPATOLOGY 2004;39:139-150.)

Cloning the human betaretrovirus proviral genome from patients with primary biliary cirrhosis (p 151-156)
Lizhe Xu, Michael Sakalian, Zhiwei Shen, George Loss, James Neuberger, Andrew Mason
Patients with primary biliary cirrhosis (PBC) have both serologic and tissue evidence of infection. A recently identified human betaretrovirus was originally cloned from the biliary epithelium cDNA library of a patient with PBC. By conducting a BLASTN search, the initial partial pol gene fragment was found to have 95% to 97% nucleotide homology with mouse mammary tumor virus (MMTV) and with retrovirus sequences derived from human breast cancer samples. Using an anti-p27 CA MMTV antibody, viral proteins were detected in the perihepatic lymph nodes but not in liver tissue samples from patients with PBC, suggesting a higher viral burden in lymphoid tissue. Therefore, in the current study, we used lymph node DNA to clone the proviral genome of the human betaretrovirus from two patients with PBC using a polymerase chain reaction (PCR) walking methodology with conserved primers complementary to MMTV. The human betaretrovirus genome contains five potential open reading frames (ORF) for Gag, protease (Pro), polymerase (Pol), envelope (Env), and superantigen (Sag) proteins that are collinear with their counterparts in MMTV. Alignment studies performed with characterized MMTV and human breast cancer betaretrovirus amino acid sequences revealed a 93% to 99% identity with the p27 capsid proteins, a 93% to 97% identity with the betaretrovirus envelope proteins, and a 76% to 85% identity with the more variable superantigen proteins. Phylogenetic analysis of known betaretrovirus superantigen proteins showed that the human and murine sequences did not cluster as two distinct species. In conclusion, human betaretrovirus nucleic acid sequences have been cloned from patients with PBC. They share marked homology with MMTV and human breast cancer-derived retrovirus sequences. (H EPATOLOGY 2004;39:151-156.)

Reversal of liver fibrosis in aryl hydrocarbon receptor null mice by dietary vitamin A depletion (p 157-166)
Fausto Andreola, Diego F. Calvisi, Guillermo Elizondo, Sonia B. Jakowlew, Jennifer Mariano, Frank J. Gonzalez, Luigi M. De Luca
Aryl hydrocarbon receptor (AHR)- null mice display a liver fibrosis phenotype that is associated with a concomitant increase in liver retinoid concentration, tissue transglutaminase type II (TGaseII) activity, transforming growth factor (TGF ) overexpression, and accumulation of collagen. To test the hypothesis that this phenotype might be triggered by the observed increase in liver retinoid content, we induced the condition of retinoid depletion by feeding AHR- null mice a vitamin A- deficient diet with the purpose to reverse the phenotype. Liver retinoid content decreased sharply within the first few weeks on the retinoid-deficient diet. Analysis of TGF 1, TGF 2, and TGF 3 expression revealed a reduction to control levels in the AHR -/- mice accompanied by parallel changes in TGaseII protein levels. In addition, we observed an increase in the TGF receptors, TGF RI and TGF RII, as well as in Smad4, and their reduction to wild-type mouse liver levels in AHR -/- mice fed the retinoid-deficient diet. Reduction of peroxisomal proliferator-activated receptor (PPAR ) messenger RNA (mRNA) and protein levels in AHR -/- mice was consistent with the presence of hepatic stellate cell (HSC) activation and liver fibrosis. Vitamin A deficiency normalized PPAR expression in AHR -/- mice. In conclusion, livers from AHR -/- mice fed the vitamin A-deficient diet showed a decrease in collagen deposition, consistent with the absence of liver fibrosis. (H EPATOLOGY 2004;39:157-166.)

Genipin enhances Mrp2 (Abcc2)-mediated bile formation and organic anion transport in rat liver (p 167-178)
Junichi Shoda, Tetsuo Miura, Hirotoshi Utsunomiya, Koji Oda, Masahiro Yamamoto, Masahito Kano, Tadashi Ikegami, Naomi Tanaka, Hidetaka Akita, Kousei Ito, Hiroshi Suzuki, Yuichi Sugiyama
Inchin-ko-to (ICKT), an herbal medicine, and its ingredients exert potent choleretic effects by a bile acid-independent mechanism. The current study was designed to determine whether ICKT or its ingredients potentiate multidrug resistance-associated protein 2 (Mrp2; Abcc2)-mediated choleresis in vivo . Biliary secretion of Mrp2 substrates and the protein mass, subcellular localization, and messenger RNA (mRNA) level of Mrp2 were assessed in rat liver after infusion of genipin, an intestinal bacterial metabolite of geniposide, a major ingredient of ICKT. The function of Mrp2 was also assessed by the adenosine triphosphate (ATP)-dependent uptake of Mrp2-specific substrates using canalicular membrane vesicles (CMVs) from the liver. Infusion of genipin increased bile flow by 230%. It also increased biliary secretion of bilirubin conjugates and reduced glutathione (GSH) by 513% and 336%, respectively, but did not increase bile acid secretion. The ATP-dependent uptake of estradiol 17- -D-glucuronide (E 217 G; by 265%), leukotriene C4 (LTC 4; by 161%), taurolithocholate-3-sulfate (TLC-3S; by 266%), and methotrexate (MTX; by 234%) was significantly stimulated in the CMVs from the liver. These effects were not observed in Mrp2-deficient rats. Under these conditions, genipin treatment increased the protein mass of Mrp2 in the CMVs but not the mRNA level. In immunoelectron microscopic studies, a marked increase in Mrp2 density in the canalicular membrane (CM) and microvilli was observed in the genipin-treated liver tissue sections when compared with the vehicle-treated liver tissue sections. In conclusion, genipin may enhance the bile acid-independent secretory capacity of hepatocytes, mainly by stimulation of exocytosis and insertion of Mrp2 in the bile canaliculi. ICKT may be a potent therapeutic agent for a number of cholestatic liver diseases. (H EPATOLOGY 2004;39:167-178.)

Liver Failure & Liver Disease

Relative contribution of iron burden, HFE mutations, and insulin resistance to fibrosis in nonalcoholic fatty liver (p 179-187)
Elisabetta Bugianesi, Paola Manzini, Sergio D'Antico, Ester Vanni, Filomena Longo, Nicola Leone, Paola Massarenti, Antonio Piga, Giulio Marchesini, Mario Rizzetto
The mechanism(s) determining the progression from fatty liver to steatohepatitis is currently unknown. Our goal was to define the relative impact of iron overload, genetic mutations of HFE, and insulin resistance on the severity of liver fibrosis in a population of subjects with nonalcoholic fatty liver disease (NAFLD) who had low prevalence of obesity and no overt symptoms of diabetes. In a cohort of 263 prospectively enrolled patients with NAFLD, 7.4% of patients had signs of peripheral iron overload and 9% had signs of hepatic iron overload, but 21.1% had hyperferritinemia. The prevalence of C282Y and H63D HFE mutations was similar to the general population and mutations were not associated with iron overload. Although subjects were on average only moderately overweight, insulin sensitivity, measured both in the fasting state and in response to oral glucose, was lower. Univariate analysis demonstrated that the presence of severe fibrosis was independently associated with older age, female sex, overweight, aspartate/alanine aminotransferase ratio, serum ferritin level, fasting glucose and insulin levels, decreased insulin sensitivity, and with histologic features (degree of necroinflammation and steatosis). After adjustment for body mass index (BMI), age, sex, and degree of steatosis, ferritin levels (odds ratio [OR] = 1.77; 95% CI = 1.21- 2.58; P= .0032) and the oral glucose insulin sensitivity (OR = 0.53; CI = 0.33-0.87; P= .0113) were independent predictors of severe fibrosis. In conclusion, the current study indicates that insulin resistance is a major, independent risk factor for advanced fibrosis in patients with NAFLD. Increased ferritin levels are markers of severe histologic damage, but not of iron overload. Iron burden and HFE mutations do not contribute significantly to hepatic fibrosis in the majority of patients with NAFLD. (H EPATOLOGY 2004;39:179-187.)

A pilot study of pioglitazone treatment for nonalcoholic steatohepatitis (p 188-196)
Kittichai Promrat, Glen Lutchman, Gabriel I. Uwaifo, Renee J. Freedman, Alejandro Soza, Theo Heller, Edward Doo, Marc Ghany, Ahalya Premkumar, Yoon Park, T. Jake Liang, Jack A. Yanovski, David E. Kleiner, Jay H. Hoofnagle
Nonalcoholic steatohepatitis (NASH) is a common chronic liver disease for which there is no known effective therapy. A proportion of patients with NASH progress to advanced fibrosis and cirrhosis. NASH is considered one of the clinical features of the metabolic syndrome in which insulin resistance plays a central role. This prospective study evaluates the role of insulin-sensitizing agent in treatment of NASH. Eighteen nondiabetic patients with biopsy-proven NASH were treated with pioglitazone (30 mg daily) for 48 weeks. Tests of insulin sensitivity and body composition as well as liver biopsies were performed before and at the end of treatment. By 48 weeks, serum alanine aminotransferase values fell to normal in 72% of patients. Hepatic fat content and size as determined by magnetic resonance imaging decreased, and glucose and free fatty acid sensitivity to insulin were uniformly improved. Histological features of steatosis, cellular injury, parenchymal inflammation, Mallory bodies, and fibrosis were significantly improved from baseline (all P< 0.05). Using strict criteria, histological improvement occurred in two-thirds of patients. Pioglitazone was well tolerated; the main side effects were weight gain (averaging 4%) and an increase in total body adiposity. In conclusion, these results indicate that treatment with an insulin-sensitizing agent can lead to improvement in biochemical and histological features of NASH and support the role of insulin resistance in the pathogenesis of this disease. The long-term safety and benefits of pioglitazone require further study. (H EPATOLOGY 2004;39:188-196.)

Oxidative stress as a trigger for cellular immune responses in patients with alcoholic liver disease (p 197-203)
Stephen F. Stewart, Matteo Vidali, Christopher P. Day, Emanuele Albano, David E.J. Jones
Serum antibodies reactive with neo-antigens generated during ethanol metabolism have been identified in patients with alcoholic liver disease (ALD), although their role in the pathogenesis of disease remains unclear. In this study, we characterized peripheral blood mononuclear cell (PBMC) T-cell and antibody responses to human serum albumin (HAS) adducted with acetaldehyde under reducing conditions (AcA-HSA) or with malondialdehyde (MDA-HSA) in patients with advanced ALD (AALD, n= 28), heavy drinkers with no liver disease (NALD, n= 14), and mild/moderate drinking controls ( n= 22). Peak proliferative responses of PBMC were assessed in vitro by tritiated thymidine incorporation after the addition of optimized concentrations of antigen or OKT3. Antibody titers were determined by enzyme-linked immunosorbent assay (ELISA). MDA-HSA induced PBMC T-cell proliferation was significantly higher in ALD than in NALD or control patients. Moreover, 10 of 28 (36%) of ALD patients had significant T-cell proliferative responses to MDA-HSA compared to 0 of 14 (0%, P= .02) of the NALD group and 2 of 22 (9%, P< .05) of controls. No significant difference in PBMC T-cell response to Aca-HSA was seen between subject groups. Patients with positive cellular responses to MDA had higher serum anti-MDA antibody titers than those not exhibiting a positive cellular response ( P< .005). In conclusion, the pattern of cellular and humoral responses to MDA adducts suggests that the development of these responses may be a susceptibility factor for the development of advanced alcoholic liver disease. The apparent importance of T-cell responses to MDA adducts suggests that oxidative stress may represent an important stimulus for the development of cellular immune responses associated with advanced ALD. (H EPATOLOGY 2004;39:197-203.)

Prevalence and predictors of esophageal varices in patients with primary sclerosing cholangitis (p 204-210)
Claudia O. Zein, Keith D. Lindor, Paul Angulo
Patients with primary sclerosing cholangitis (PSC) may develop and bleed from esophageal varices. However, the exact prevalence of esophageal varices in patients with PSC remains unknown and potential predictors of esophageal varices in this population have not been identified. Our aim was to determine the prevalence of esophageal varices in patients with PSC and the variables that predict their presence. Data were collected on 283 patients with PSC treated for the first time at the Mayo Clinic (Rochester, MN) during 8 consecutive years. Thirty-six percent (102 of 283) of patients had esophageal varices including 56% (57 of 102) with moderate/large varices. After excluding 28 patients with a history of variceal bleeding, data on 183 patients were analyzed to identify independent predictors of esophageal varices and of moderate/large size varices. Platelet count, albumin level, and advanced histologic disease were independent predictors of esophageal varices (area under the receiver operator characteristic [ROC] curve = 0.88). After controlling for the presence of advanced histologic stage and albumin levels, the odds ratios (OR) of platelet count less than 150 ? 10 3/dL for the presence of esophageal varices was 6.3 (95% CI: 2.6-15.8). The diagnostic accuracy of these results was corroborated by cross-validation of the data in an independent set of 72 patients with PSC (area under the ROC = 0.90). In conclusion, in patients with PSC, noninvasive markers of portal hypertension and of advanced liver disease predict the presence of esophageal varices. Our results suggest a clinically applicable and useful approach to identify patients with PSC who are more likely to benefit from endoscopic screening for esophageal varices. (H EPATOLOGY 2004;39:204-210.)

The Gambia Liver Cancer Study: Infection with hepatitis B and C and the risk of hepatocellular carcinoma in West Africa (p 211-219)
Gregory D. Kirk, Olufunmilayo A. Lesi, Maimuna Mendy, Aliu O. Akano, Omar Sam, James J. Goedert, Pierre Hainaut, Andrew J. Hall, Hilton Whittle, Ruggero Montesano
Hepatocellular carcinoma (HCC) is the most common cancer in The Gambia. Hepatitis B virus (HBV) infection is endemic, with 15% to 20% of the population being chronic carriers, whereas hepatitis C virus (HCV) prevalence is low. We recruited 216 incident cases of HCC and 408 controls from three sites. HBV carriage was present in 61% (129/211) of HCC patients and 16% (64/402) of controls, whereas 19% (36/191) of HCC patients were HCV seropositive compared with 3% (11/382) of controls. HCC patients with HCV were notably older and were more likely to be female than those with HBV. Increased HCC risk was strongly associated with chronic HBV (odds ratio, 16.7; 95% CI, 9.7-28.7), HCV (16.7; 6.9-40.1), and dual infection (35.3; 3.9-323). We interpret the additive nature of risk with coinfection as representative of HBV and HCV acting primarily through shared steps in the multistage process of hepatocarcinogenesis. HCV infection was not observed among younger participants, suggesting a possible cohort effect. Reasons for the striking age and gender differences in HCC associated with HBV compared with HCV are unclear, but transmission patterns and age at exposure may be factors. In conclusion, in a standardized evaluation of well-characterized study participants from The Gambia, most cases of HCC are attributable to HBV (57%), but HCV adds a significant fraction (20%), especially among older patients and females. If HCV transmission is not perpetuated in future cohorts, focusing available resources on HB vaccination efforts could greatly ameliorate a major cause of cancer death in sub-Saharan Africa. (H EPATOLOGY 2004;39:211-219.).

MUC4 is a novel prognostic factor of intrahepatic cholangiocarcinoma-mass forming type (p 220-229)
Hiroaki Shibahara, Shugo Tamada, Michiyo Higashi, Masamichi Goto, Surinder K. Batra, Michael A. Hollingsworth, Kohzoh Imai, Suguru Yonezawa
Complete surgical resection of the tumor is the sole approach to improve the cure rate of patients with intrahepatic cholangiocarcinoma-mass forming type (ICC-MF). Although patients are treated by curative resection, many of them show poor outcome. Mucin (MUC)4 expression has been implicated as a marker for diagnosis and progression of pancreatic adenocarcinomas, but there is no study of the relationship between MUC4 expression and patient's prognosis in ICC-MF. In the present study, we examined the expression profile of MUC4 in ICC-MF tissue from 27 patients using immunohistochemistry. MUC4 was expressed in the carcinoma tissues of 10 (37%) of the 27 ICC-MF tumors, whereas it was not expressed in normal liver tissue. Because MUC4 is an intramembrane ligand for receptor tyrosine kinase ErbB2 and is related with regulation of p27, we also compared the MUC4 expression with ErbB2 and p27 expressions in ICC-MFs. The patients with MUC4 and ErbB2 double positive expression showed a short survival period compared to non-expressing patients. MUC4 and p27 showed no relationship. The univariate analysis showed that tumor size, intrahepatic metastasis, lymph node metastasis, MUC4 expression, and MUC1 expression were statistically significant risk factors affecting the outcome of the patients with ICC-MF. The multivariate analysis demonstrated that MUC4 expression, as well as surgical margin, were statistically significant independent risk factors. In conclusion, the results suggest that expression of MUC4 in ICC-MF is a new independent factor for poor prognosis and is a useful marker to predict the outcome of the patients with ICC-MF. (H EPATOLOGY 2004;39:220-229.)

Prediction of survival after liver retransplantation for late graft failure based on preoperative prognostic scores (p 230-238)
Francis Y. Yao, Sammy Saab, Nathan M. Bass, Ryutaro Hirose, David Ly, Norah Terrault, Ann A. Lazar, Peter Bacchetti, Nancy L. Ascher, John P. Roberts
The current policy for determining priority for organ allocation is based on the model for end stage liver disease (MELD). We hypothesize that severity of graft dysfunction assessed by either the MELD score or the Child-Turcotte-Pugh (CTP) score correlates with mortality after liver retransplantation (re-OLT). To test this hypothesis, we analyzed the outcome of 40 consecutive patients who received re-OLT more than 90 days after primary orthotopic liver transplantation (OLT). The Kaplan-Meier 1-year and 5-year survival rates after re-OLT were 69% and 62%, respectively. The area under the curve (AUC) values generated by the receiver operating characteristics (ROC) curves were 0.82 (CI 0.70-0.94) and 0.68 (CI 0.49-0.86), respectively ( P= .11), for the CTP and MELD models in predicting 1-year mortality after re-OLT. The 1-year and 5-year survival rates for patients with CTP scores less than 10 were 100% versus 50% and 40%, respectively, for CTP scores of at least 10 ( P= .0006). Patients with MELD scores less than or equal to 25 had 1-year and 5-year survival rates of 89% and 79%, respectively, versus 53% and 47%, respectively, for MELD scores greater than 25 ( P= .038). Other mortality predictors include hepatic encephalopathy, intensive care unit (ICU) stay, recurrent hepatitis C virus (HCV) infection, and creatinine level of 2 mg/dL or higher. Analysis of an independent cohort of 49 patients showed a trend for a correlation between CTP and MELD scores with 1-year mortality, with AUC of 0.59 and 0.57, in respective ROC curves. In conclusion, our results suggest that severity of graft failure based on CTP and MELD scores may be associated with worse outcome after re-OLT and provide a cautionary note for the sickest first policy of organ allocation. (H EPATOLOGY 2004;39:230-238.)

Vascular involvement of the liver in Turner's syndrome (p 239-247)
Dominique Roulot, Claude Degott, Olivier Chazouillères, Frédéric Oberti, Paul Calès, Nicolas Carbonell, Said Benferhat, Solange Bresson-Hadni, Dominique Valla
Unexplained liver test abnormalities are frequent in patients with Turner's syndrome. This cohort study was performed to clarify the histopathologic features, causes, and long-term outcome of liver involvement in these patients. Thirty patients with persistently abnormal liver test results were followed-up for 8.8 ± 5.2 years. Liver specimens were available in 27 patients. Marked architectural changes were present in 10 patients, including nodular regenerative hyperplasia in six, multiple focal nodular hyperplasia in two, and cirrhosis in two patients. These changes frequently were associated with obliterative portal venopathy lesions and with aortic malformations. There was mild to moderate portal fibrosis in 15 of the 17 other patients, inflammatory infiltrates in nine patients, and nonalcoholic fatty liver disease in 11 patients. Bile duct alterations resembling small duct sclerosing cholangitis were observed in 21 patients (with or without architectural changes). There was no viral, alcoholic, autoimmune, or drug-induced liver damage. Portal hypertension was observed in four patients with marked architectural changes, including three in whom refractory ascites or recurrent variceal bleeding developed, one of whom underwent transplantation. None of the patients without marked architectural changes experienced progressive or decompensated liver disease. There was no evidence of liver toxicity from estrogen replacement therapy. In conclusion, the main causes of liver involvement in Turner's syndrome are vascular disorders, probably of a congenital origin, and nonalcoholic fatty liver disease. In patients with vascular disorders, severe liver disease requiring liver transplantation may develop. Estrogen therapy does not appear to be pathogenically implicated. (H EPATOLOGY 2004;39:239-247.)

Save Article Copyright © 2004 by the American Association for the Study of Liver Diseases. All rights reserved.

GASTROENTEROLOGY

The F508 mutation results in loss of CFTR function and mature protein in native human colon
Marcus Mall, Silvia M. Kreda, April Mengos, Timothy J. Jensen, Stephanie Hirtz, Hans H. Seydewitz, James Yankaskas, Karl Kunzelmann, John R. Riordan, Richard C. Boucher
Background & Aims :Deletion of the codon for phenylalanine at position 508 ( F508) is the most frequent disease-causing mutation in the cystic fibrosis transmembrane conductance regulator ( CFTR ) gene. In heterologous cells, defective processing of the F508 protein results in endoplasmic reticulum retention, proteolytic degradation, and absence of adenosine 3´,5´-cyclic monophosphate (cAMP)-dependent plasma membrane Cl –conductance. However, data with respect to the processing block of F508 protein in native epithelia are limited and conflicting. Methods :To characterize both the fate and function of F508 protein in a native epithelium, we measured CFTR-mediated Cl –secretion, localization of the CFTR protein, and CFTR maturation in rectal biopsy specimens from normal individuals and F508 homozygous patients with cystic fibrosis (CF). Results :Ussing chamber studies showed that cAMP-dependent and cholinergic Cl –secretion was absent from rectal tissues freshly excised from F508 homozygous patients with CF. By immunohistochemistry, we detected wild-type but not F508 CFTR at the luminal membrane of crypt colonocytes. By sequential immunoprecipitation and immunoblotting analyses, mature CFTR protein was detected in normal but not in F508 homozygous tissues. Conclusions :Collectively, these data show that there is insufficient maturation and transport of F508 CFTR from the endoplasmic reticulum to the apical membrane to support CFTR-mediated Cl –secretion in the CF colon.

The role of mismatch repair gene defects in the development of adenomas in patients with HNPCC
Andrea E. De Jong, Hans Morreau, Marjo Van Puijenbroek, Paul H.C. Eilers, Juul Wijnen, Fokko M. Nagengast, Gerrit Griffioen, Annemieke Cats, Fred H. Menko, Jan H. Kleibeuker, Hans F.A. Vasen
Background & Aims :The adenoma-carcinoma sequence in hereditary nonpolyposis colorectal cancer (HNPCC) is accelerated. It remains unknown whether the mismatch repair (MMR) defect also promotes the development of adenomas. The aim of this study was to compare the risk of developing colorectal adenoma and carcinoma in HNPCC carriers and noncarriers (controls) and to compare the features of adenomas in both groups. Methods :Eighty-six families with a known MMR gene mutation from the Dutch HNPCC Registry were analyzed. Subjects with known mutation status with colonoscopies performed for the purpose of surveillance were selected for this study. Information on the surveillance examinations was obtained from medical reports. The histology of all adenomas was confirmed. Immunohistochemistry was performed in a subgroup of adenomas. Results :We identified 249 carriers and 247 controls. The proportion of subjects free of an adenoma at the age of 60 years was 29.7% for carriers and 70.8% for controls ( P< 0.05). The adenomas in carriers were larger, and a higher proportion had villous components and/or high-grade dysplasia ( P< 0.05, all analyses). The adenomas and carcinomas of the carriers were located predominantly in the proximal colon. Most adenomas showed absent staining of the MMR proteins. Conclusions :This study indicates that the MMR defect is involved in the early stages of development of adenomas. We recommend immunohistochemical staining of large adenomas with high-grade dysplasia in young patients (younger than 50 years) to identify patients with suspected HNPCC.

Mechanoreceptors of the proximal stomach: Role in triggering transient lower esophageal sphincter relaxation
Roberto Penagini, Stefania Carmagnola, Paolo Cantù, Mariangela Allocca, Paolo A. Bianchi
Background & Aims :The role of fundic tension and stretch mechanoreceptors in triggering transient lower esophageal sphincter (LES) relaxation is still unknown. This information would be useful for the development of effective pharmacologic strategies. To elucidate this topic, gastric contractile activity was modified during isovolumetric gastric distention at 2 different volumes. Methods :LES (Dentsleeve) and gastric (barostat) motility were recorded in 21 healthy subjects during studies comprising two 30-minute isovolumetric gastric distentions (placebo and glucagon or erythromycin). Glucagon (bolus of 4.8 µg/kg plus infusion of 9.6 µg · kg –1 · h –1 ) was administered at high intragastric volume (i.e., 75% of the threshold volume for discomfort; n = 7) and erythromycin (3 mg/kg) at high (n = 7) and low intragastric volume (i.e., at perception threshold; n = 7). Results :Glucagon decreased ( P< 0.05) baseline intragastric pressure and abolished gastric contractions (0 vs. 16.7 ± 2.3), whereas erythromycin increased ( P< 0.05) baseline pressure and doubled ( P< 0.05) the rate of gastric contractions at both volumes. Neither drug affected the rate of transient LES relaxations. Low intragastric volume induced a lower rate of transient LES relaxations (1.7 ± 0.3 vs. 5.7 ± 1.1; P< 0.01) and gastric contractions (11.8 ± 2.5 vs. 20.5 ± 3.1; P< 0.05) compared with high volume but similar baseline intragastric pressure (10.6 ± 0.6 vs. 11.9 ± 0.9 mm Hg). Conclusions :Stretch receptors (gastric volume) seem to be more relevant than tension receptors in triggering transient LES relaxation.

Predictive value of the balloon expulsion test for excluding the diagnosis of pelvic floor dyssynergia in constipation
Miguel Minguez, Belen Herreros, Vicente Sanchiz, Vicent Hernandez, Pedro Almela, Ramon Añon, Francisco Mora, Adolfo Benages
Background & Aims :The aim of this study was to establish a simple method to exclude the possibility of pelvic floor dyssynergia (PFD) in constipated patients and thus avoid unnecessary expensive physiologic studies. Methods :Patients with suspicion of functional constipation (FC) were studied prospectively between 1994 and 2002, excluding those with severe systemic, psychological, or symptomatic anorectal/colonic disorders or taking medications that might modify symptoms or results of studies. Diagnosis of PFD was established retrospectively by manometric plus defecographic findings according to Rome II criteria. Two groups of patients were identified: FC without PFD (FC group) and PFD group. A 30-day symptom diary and balloon expulsion test results were evaluated in all patients. Clinical differences and results of the expulsion test were statistically compared between groups. Results :Of 359 patients evaluated, 130 were included (FC group, 106; PFD group, 24). According to data from the diary, only anal pain was more frequent in the PFD group compared with the FC group (anal pain in >25% of defecations, 70.8% vs. 40.6%; P< 0.05, 2test). The expulsion test was pathologic in 21 of 24 patients with PFD and 12 of 106 without PFD. The specificity and negative predictive value of the test for excluding PFD were 89% and 97%, respectively. Conclusions :The balloon expulsion test is a simple and useful screening procedure to identify constipated patients who do not have PFD. Symptoms are not enough to differentiate between subtypes of constipation.

CD40-mediated immune-nonimmune cell interactions induce mucosal fibroblast chemokines leading to T-Cell transmigration
Jon D. Vogel, Gail A. West, Silvio Danese, Carol de la Motte, Manijeh H. Phillips, Scott A. Strong, Joseph Willis, Claudio Fiocchi
Background & Aims :The CD40 pathway is a key mediator of inflammation and autoimmunity. We investigated cell adhesion molecule (CAM) up-regulation and chemokine production by CD40-positive human intestinal fibroblasts (HIF) and microvascular endothelial cells (HIMEC) induced by CD40 ligand (CD40L)-positive T cells and soluble CD40L and their effect on T-cell adhesion and transmigration. Methods :Expression of CD40, CD40L, and CAM was assessed by immunohistochemistry, confocal microscopy and flow cytometric analysis, and chemokine production using enzyme-linked immunosorbent assay. Calcein-labeled T cells were used to assay HIF adhesion and Transwell HIMEC transmigration. Results :Ligation of CD40-positive HIF and HIMEC by CD40L-positive T cells or soluble CD40L induced up-regulation of CAM expression as well as interleukin-8 and RANTES production. The specificity of these responses was shown by inhibition with a CD40L blocking antibody and by CD40 signaling-dependent p38 mitogen-activated protein kinase phosphorylation. On CD40 ligation, HIF increased their T-cell binding capacity and generated chemoattractants able to induce T-cell migration through HIMEC monolayers. Conclusions :Activation of the CD40/CD40L system in the gut mucosa may trigger a self-sustaining loop of immune-nonimmune cell interactions leading to an antigen-independent influx of T cells that contributes to chronic inflammation.


Clinical-liver, Pancreas, and Biliary Tract

Adefovir dipivoxil added to ongoing lamivudine in chronic hepatitis B with YMDD mutant hepatitis B virus
Robert Perrillo, Hie-won Hann, David Mutimer, Bernard Willems, Nancy Leung, William M. Lee, Alison Moorat, Stephen Gardner, Mary Woessner, Eric Bourne, Carol L. Brosgart, Eugene Schiff
Background & Aims :Prolonged lamivudine therapy is associated with treatment-resistant YMDD mutant hepatitis B virus (HBV). We evaluated the efficacy and safety of adding adefovir dipivoxil to lamivudine in 135 patients with chronic hepatitis B (CHB) and YMDD mutant HBV. Methods :Ninety-five patients with compensated CHB (group A) were randomized to adefovir 10 mg daily (n = 46) or placebo (n = 49) for 52 weeks while continuing treatment with lamivudine. Forty patients with decompensated hepatitis B or post-liver transplantation (group B) received adefovir and lamivudine. The primary end point was a decline in serum HBV DNA level to 10 5copies/mL or a >2 log 10 reduction from baseline at weeks 48 and 52. Results :HBV DNA response occurred in 85% of patients (39 of 46) in group A given combined therapy versus 11% (5 of 46) receiving lamivudine alone ( P< 0.001), with a significant change in HBV DNA level from baseline ( P< 0.001) between treatment groups (median, –4.6 vs. +0.3 log 10 copies/mL, respectively). Normalization of alanine aminotransferase levels occurred in 31% of patients (14 of 45) receiving combined therapy versus 6% (3 of 48) receiving lamivudine alone ( P= 0.002). Ninety-two percent of patients (36 of 39) in group B had an HBV DNA response (median change of –4.6 log 10 copies/mL) and improved liver chemistries ( P0.001). Both treatment regimens were well tolerated, and renal function abnormalities were not observed in either group. Conclusions :The addition of adefovir dipivoxil to lamivudine in patients with CHB with compensated or decompensated liver disease due to YMDD mutant HBV is associated with virologic and biochemical improvement during 52 weeks of treatment and is well tolerated.


Adefovir dipivoxil alone or in combination with lamivudine in patients with lamivudine-resistant chronic hepatitis B
Marion G. Peters, H.W. Hann, Paul Martin, E. Jenny Heathcote, P. Buggisch, R. Rubin, M. Bourliere, K. Kowdley, C. Trepo, D.F. Gray, M. Sullivan, K. Kleber, R. Ebrahimi, S. Xiong, Carol L. Brosgart
Background & Aims :Adefovir dipivoxil possesses potent in vitro and in vivo antiviral activity in wild-type hepatitis B. This study assessed the safety and efficacy of adefovir dipivoxil alone and in combination with lamivudine compared with ongoing lamivudine therapy in patients with chronic hepatitis B with compensated liver disease and lamivudine-resistant hepatitis B virus (HBV). Methods :Fifty-nine hepatitis B e antigen (HBeAg)-positive patients with genotypic evidence of lamivudine-resistant HBV, serum alanine aminotransferase (ALT) level 1.2 times the upper limit of normal, and serum HBV DNA level 6 log 10 copies/mL despite ongoing treatment with lamivudine were randomized to adefovir dipivoxil 10 mg, lamivudine 100 mg, or addition of adefovir dipivoxil to ongoing lamivudine daily. The primary end point was the time-weighted average change from baseline in serum HBV DNA level (DAVG) up to week 16. Results :Rapid reductions in serum HBV DNA level were seen by 4 weeks in all recipients of adefovir dipivoxil; DAVG 16 was –0.07 in the lamivudine group compared with –2.45 and –2.46 log 10 copies/mL in the adefovir dipivoxil/lamivudine and adefovir dipivoxil monotherapy groups, respectively ( P< 0.001). Median change from baseline in serum HBV DNA level at week 48 was 0.0, –3.59, and –4.04 log 10 copies/mL in the lamivudine, adefovir dipivoxil/lamivudine, and adefovir dipivoxil groups, respectively. ALT level normalized in 10 of 19 (53%) and 9 of 18 (47%) recipients of adefovir dipivoxil/lamivudine and adefovir dipivoxil, respectively, compared with 1 of 19 (5%) recipients of lamivudine. Three patients receiving adefovir dipivoxil or adefovir dipivoxil/lamivudine and none receiving lamivudine monotherapy were HBeAg negative at week 48 and one became hepatitis B surface antigen negative. Conclusions :These data, limited to patients with compensated liver disease, indicate that adefovir dipivoxil alone or in combination with ongoing lamivudine therapy provides effective antiviral therapy in patients with lamivudine-resistant HBV.

Hepatitis B virus maintains its pro-oncogenic properties in the case of occult HBV infection
Teresa Pollicino, Giovanni Squadrito, Giovanni Cerenzia, Irene Cacciola, Giuseppina Raffa, Antonio Crax, Fabio Farinati, Gabriele Missale, Antonina Smedile, Claudio Tiribelli, Erica Villa, Giovanni Raimondo
Background & Aims :Occult hepatitis B virus (HBV) infection is characterized by persistence of HBV DNA into the tissue of hepatitis B surface antigen-negative individuals. The clinical relevance of this peculiar infection is still under debate. In particular, the impact of occult HBV infection in cases of hepatocellular carcinoma (HCC) is uncertain. We investigated the prevalence and molecular status of occult HBV in patients with HCC. Methods :We tested tumor tissues from 107 patients with HCC and the corresponding nontumor liver tissue from 72 of these patients for HBV DNA. We also examined liver specimens from 192 patients with chronic hepatitis. All cases were hepatitis B surface antigen negative. Covalently closed circular HBV genomes, HBV transcripts, and viral integrated forms were investigated in cases of HCC found positive for occult HBV. Results :Viral DNA was detected in 68 of 107 cases of HCC (63.5%) and in 63 of 192 cases of chronic hepatitis (32.8%) ( P< 0.0001; odds ratio, 3.6; 95% confidence interval, 2.2–5.9). The significant association of occult HBV with HCC was irrespective of age, sex, and contemporary hepatitis C virus infection. Both integrated viral DNA and covalently closed circular HBV genomes were detected in patients with occult HBV. Moreover, the presence of free HBV genomes was associated with persistence of viral transcription and replication. Conclusions :Our findings provide clear evidence that occult HBV is a risk factor for development of HCC and show that the potential mechanisms whereby overt HBV might induce tumor formation are mostly maintained in cases of occult infection.


Basic-alimentary Tract
 
Bone morphogenetic protein 2 is expressed by, and acts upon, mature epithelial cells in the colon
James C.H. Hardwick, Gijs R. Van Den Brink, Sylvia A. Bleuming, Isabel Ballester, Jan. M.H. Van Den Brande, Josbert J. Keller, G. Johan A. Offerhaus, Sander J.H. Van Deventer, Maikel P. Peppelenbosch
Background & Aims :The recent findings of bone morphogenetic protein (BMP) receptor Ia mutations in juvenile polyposis and frequent Smad4 mutations in colon cancer suggest a role for BMPs in the colonic epithelium and colon cancer. We investigated the role of BMP2 in the colon. Methods :We assessed BMP receptor expression in cell lines using the reverse-transcribed polymerase chain reaction and immunoblotting. We investigated the effect of BMP2 on cell lines using the MTT assay and by immunoblotting for markers of differentiation, proliferation, and apoptosis. We assessed the expression of BMP2, its receptors, and signal transduction elements in mouse and human colon tissue using immunohistochemistry. We also investigated the effect of the BMP antagonist noggin in vivo in mice by assessing colon tissue with immunohistochemistry and immunoblotting. Finally, we investigated the expression of BMP2 in microadenomas from familial adenomatous polyposis patients. Results :BMP receptors (BMPR) Ia, BMPR Ib, and BMPR II are all expressed in colonic epithelial cell lines. BMP2 inhibits colonic epithelial cell growth in vitro, promoting apoptosis and differentiation and inhibiting proliferation. BMP2, BMPRIa, BMPRIb, BMPRII, phosphorylated Smad1, and Smad4 are expressed predominantly in mature colonocytes at the epithelial surface in normal adult human and mouse colon. Noggin inhibits apoptosis and proliferation in mouse colonic epithelium in vivo. BMP2 expression is lost in the microadenomas of familial adenomatous polyposis patients. Conclusions :These data suggest that BMP2 acts as a tumor suppressor promoting apoptosis in mature colonic epithelial cells.

Akt2, phosphatidylinositol 3-kinase, and PTEN are in lipid rafts of intestinal cells: Role in absorption and differentiation
Xshang Li, Sharon Leu, Alice Cheong, Huiping Zhang, Boris Baibakov, Chris Shih, Morris J. Birnbaum, Mark Donowitz
Background & Aims :In intestinal Na absorptive cells, phosphatidylinositol 3-kinase (PI 3-K) is involved in rapid epidermal growth factor (EGF) stimulation of Na absorption by the brush border membrane (BBM) Na +/H +exchanger NHE3. However, how NHE3 is regulated by the PI 3-K pathway and the role of Akt2 are poorly defined. Methods :The localization of Akt, PI 3-K, and NHE3 was determined by either immunocytochemistry and/or membrane fractionation using OptiPrep density gradient centrifugation. Results :In ileum, active total Akt was present most in the villi and basal layer of the crypts, and Akt2 was mostly in villi. In villus cells, PI 3-K and Akt2 were mostly at the apical surface at which they were present partially in lipid rafts (LR). EGF increased PI 3-K and active Akt2 in ileal BBM at the same time that it increased PI 3-K-dependent trafficking of NHE3 to BBM and stimulation of Na absorption. However, Akt2 was only active in the detergent soluble (DS) pool and not LR of ileal BBM, which correlated with the presence of PTEN in LR. In Caco-2 cells, while EGF stimulated BB NHE3, Akt2 was active in both LR and DS pools. This correlated with the lack of PTEN in the LR of Caco-2 membranes. Akt2 also correlated with epithelial cell differentiation. Akt2 amount and activity were greater in differentiated than undifferentiated Caco-2 cells. Conclusions :These results suggest that LR may play an important role in determining the function of PI 3-K/Akt2 signaling, including stimulation of intestinal Na absorption. These results also suggest that LR-associated Akt2 may be involved in enterocyte differentiation.

Cyclooxygenase-2 inhibitor (SC-236) suppresses activator protein-1 through c-Jun NH2-terminal kinase
Benjamin Chun-yu Wong, Xiao Hua Jiang, Marie C.M. Lin, Shui Ping Tu, Jian Tao Cui, Shi Hu Jiang, Wai Man Wong, Man Fung Yuen, Shiu Kum Lam, Hsiang Fu Kung
Background & Aims :Aspirin exerts antitumor effect partly through blocking tumor promoter-induced activator protein-1 (AP-1) activation. The aim of this study is to determine how specific COX-2 inhibitor SC-236 mediates antitumor effect by modulation of AP-1-signaling pathway. Methods :AP-1 transcriptional activity and DNA-binding activity were detected by luciferase reporter assay and gel shift assay, separately. Mitogen-activated protein kinase (MAPK) activation was determined by Western blot and in vitro kinase assay. Antisense oligonucleotide against c-Jun-N-terminal kinase (JNK) was used to suppress JNK expression. Results :We showed that SC-236 inhibited 12-O-tetradecanoylphorbol-13-acetate (PMA)-induced cell transformation in a dose-dependent manner in JB6 cells. At a dose range (12.5–50 µmol/L) that inhibited cell transformation, SC-236 also inhibited anchorage-independent cell growth and AP-1-activation in 3 gastric cancer cells, independent of COX-prostaglandin synthesis. SC-236 down-regulated c-Jun-NH2-terminal kinase phosphorylation and activity. Suppression of JNK activity reversed the inhibitory effect on AP-1 activity by SC-236 and suppressed gastric cancer cell growth, indicating that the inhibitory effect of SC-236 on AP-1 activation and cell growth was through interaction with JNK. Conclusions :The inhibitory effect on JNK-c-Jun/AP-1 activation contributes to the antitumor effect of COX-2-specific inhibitor, and inhibition of JNK activation may have a therapeutic benefit against gastric cancer.

Extracellular polyamines regulate fluid secretion in rat colonic crypts via the extracellular calcium-sensing receptor
Sam X. Cheng, John P. Geibel, Steven C. Hebert
Background & aims :Polyamines are essential for the normal postnatal development, maintenance, and function of gastrointestinal epithelia. The extracellular Ca 2+ (Ca 2+ o/nutrient)-sensing receptor is expressed on both luminal and basolateral membranes of colonocytes, and, in other cell systems, this receptor has been shown to respond to polyamines. Thus, the Ca 2+ -sensing receptor could provide a mechanism for modulation of colonocyte function by dietary and systemic extracellular polyamines. In the present study, we investigated the interaction of polyamines, particularly spermine, and extracellular Ca 2+ on second messenger generation by, and on function of, rat distal colonic crypts. Methods :Calcium-sensing receptor activation was assessed in colonic epithelial cells and intact crypts freshly isolated from distal colon by monitoring intracellular IP 3and Ca 2+ accumulation using radioimmunoassay and Fluo-3 fluorometry, respectively. Interactions of extracellular Ca 2+ and spermine on regulation of both basal and forskolin-stimulated fluid transport were measured in crypts microperfused in vitro. Results :Polyamine (spermine > spermidine > putrescine)-mediated enhancement of intracellular D-myo-inositol 1,4,5-trisphosphate (IP 3) and Ca 2+ accumulation required extracellular Ca 2+ , and the EC 50 for extracellular Ca 2+ -mediated activation of the calcium-sensing receptor was reduced by polyamines. Extracellular spermine modulated both basal and forskolin-stimulated fluid secretion in perfused colonic crypts, and the EC 50 for spermine-induced reduction in forskolin-stimulated fluid secretion was inversely dependent on extracellular Ca 2+ (Ca 2+ o). Conclusions :The interactions of extracellular Ca 2+ and polyamines on second messenger accumulation and fluid secretion support a role for the luminal and basolateral calcium-sensing receptors in mediating some of the effects of polyamines on distal colonic epithelial cells.

Selective jejunal manipulation causes postoperative pan-enteric inflammation and dysmotility
Nicolas T. Schwarz, Jörg C. Kalff, Andreas Türler, Nicola Speidel, Jennifer R. Grandis, Timothy R. Billiar, Anthony J. Bauer
Background & aims :Small bowel manipulation initiates an intense molecular and cellular inflammatory response within the jejunal muscularis, which causes ileus. The current objective was to investigate pan-enteric inflammatory molecular and functional motility alterations of the muscularis from the unmanipulated stomach and colon initiated by selective jejunal manipulation. Methods :Rat jejunum was manipulated, and animals sacrificed between 0–24 hours. In vivo gastric emptying, gastrointestinal transit, and in vitro colonic circular muscle recordings were measured. Reverse-transcriptase polymerase chain reaction (RT-PCR) and electromobility shift assay (EMSA) of gastric, jejunal, and colonic muscularis extracts were performed. Whole mounts were histochemically stained for myeloperoxidase leukocytes. Results :Surgical manipulation suppressed jejunal contractions that were significantly prevented by dexamethasone pretreatment. Selective jejunal manipulation also suppressed in vivo gastric emptying, gastrointestinal transit, and in vitro colonic circular muscle contractility. Nuclear factor interleukin-6 (NF-IL-6) was activated within the gastric and colonic muscularis. RT-PCR showed a 14.9-, 8.1-, and 11.4-fold up-regulation of IL-6 messenger RNA within the jejunal, gastric, and colonic muscularis, respectively. EMSA showed a 30.6-, 14.2-, and 20.8-fold increased activation of signal transducer and activator of transcription (STAT) proteins in jejunal, gastric, and colonic muscularis extracts, respectively. Tumor necrosis factor- , cyclooxygenase-2, and inducible nitric oxide synthase showed a significant up-regulation in the manipulated jejunum, as well as the unmanipulated gastric and colonic muscularis. Neutrophils were significantly recruited into all gastrointestinal regions. Conclusion :Selective small bowel manipulation leads to a molecular, cellular, and functional pan-enteric “field effect” phenomenon in the unmanipulated gastric and colonic muscularis.

Exacerbated colitis associated with elevated levels of activated CD4+ T cells in TCR chain transgenic mice
Immo Prinz, Uwe Klemm, Stefan H.E. Kaufmann, Ulrich Steinhoff
Background & Aims :An unconventional CD4+ TCR –+cell population mediates the development of colitis resembling ulcerative colitis in T-cell receptor mutant (TCR –/– ) mice. However, the significance of such T cells in individuals with an intact TCR locus remains unclear. Because a substantial proportion of naturally rearranged TCR chains fails to pair with TCR chains, the aim of this study was to analyze the development of CD4+ TCR –+cells and the course of colitis in the presence of such a TCR chain. Methods :TCR chain transgenic TCR –/– mice were generated and compared with wild-type and TCR –/– mice by flow cytometric analysis of T lymphocytes with respect to their TCR expression and activation status and by histological analysis of colon tissue. The colitogenic potential of the unconventional CD4+ TCR –+cells was assessed by adoptive transfer experiments. Furthermore, the half-life of TCR chains was determined by pulse-chase labeling and immunoprecipitation. Results :Transgenic expression of a TCR V 7.2 chain led to increased frequencies of CD4+ TCR –+cells that caused rapid onset of colitis, reminiscent of, but even more severe than, that in TCR –/– mice. This unconventional T-cell population displayed a constitutively activated phenotype in normal and transgenic TCR –/– mice. An extended half-life of newly synthesized TCR chains suggests a chaperone function of the TCR V 7.2 chain in TCR –/– mice. Conclusions :Physiological TCR rearrangement can promote the formation of chronically activated CD4+ TCR –+T cells and may play a role in the etiology of UC.

Divalent cations regulate acidity within the lumen and tubulovesicle compartment of gastric parietal cells
Andrea Gerbino, Aldebaran M. Hofer, Breda McKay, Bonnie W. Lau, David I. Soybel
Background & Aims :Until recently, it has not been possible to evaluate factors that regulate the acidity of the microenvironment within the tubulovesicles and luminal (TV/L) spaces of the gastric gland. The goal of this study was to develop a method for monitoring the mechanisms that regulate acidity in the TV/L compartment. Methods :Isolated rabbit gastric glands (intact or permeabilized with S. aureus -toxin) were loaded with a recently characterized fluorescent dye, LysoSensor Yellow-Blue DND 160 (Molecular Probes, Eugene, OR), which localizes to highly acidic compartments and can be used to monitor acidity ratiometrically. Results :In resting glands, the pH of the TV/L compartment was ~3.4. Moderate alkalizations (~0.5 to 1.0 pH unit alkalization) were observed during exposure to inhibitors of the apical H +/K +ATPase (omeprazole and SCH28080), thereby unmasking a stable, low-level leak of H +ions from the TV/L compartment. Similar changes were observed in -toxin permeabilized glands following depletion of ATP in the cytoplasm. In intact and permeabilized glands, we used the cell-permeant, divalent cation chelator, tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) to probe the effects of lowering divalent cation content of the TV/L compartment. Exposure to relatively low concentrations (20 µmol/L, 50 µmol/L) of TPEN reversibly promoted H +leakage. At these concentrations, simultaneous inhibition using SCH28080 led to marked enhancement of the rate of alkalization. Conclusions :The effects of low-dose TPEN suggests that acidity within the TV/L compartment of the gastric gland may be regulated, at least in part, by its content of divalent cations such as Zn 2+ , for which TPEN has high affinity.

Gastric cancer development in mice lacking the SHP2 binding site on the IL-6 family co-receptor gp130
Louise M. Judd, Barbara M. Alderman, Meegan Howlett, Arthur Shulkes, Chris Dow, Jill Moverley, Diane Grail, Brendan J. Jenkins, Matthias Ernst, Andrew S. Giraud
Background & Aims :We have developed a mouse model of gastric cancer that resembles human intestinal-type adenocarcinoma. The aim of this study was to determine the identity and temporal changes in mediators of IL-6 signaling regulating tumor development. Methods :gp130 757F/F Mice that lack the SHP2-binding site on the IL-6 family receptor gp130 and have increased STAT 3 activity and wild-type littermates were used. Cohorts were assessed by quantitative histology and immunohistochemistry for gastric cell phenotype and proliferation markers from 4 to 40 weeks of tumor development. Northern blotting and in situ hybridization were used to quantify expression of the tumor suppressor TFF1 and the mitogens gastrin and Reg I. Expression of epidermal growth factor receptor (EGFr) and its ligands was measured by RT-PCR analysis. Age-matched differences in gene expression profiles were tested by ANOVA. Results :Hyperplastic antral tumors with inflammation and ulceration were evident in gp130 757F/F mice at 4 weeks of age and reached maximum size by 20 weeks. Tumor progression was marked by gastritis, atrophy, intestinal metaplasia, dysplasia, and submucosal invasion after 30 weeks. Both TFF1 and gastrin expression were progressively inhibited during tumorigenesis, whereas Reg I was stimulated. The EGFr and its ligands transforming growth factor (TGF)- and heparin-binding EGF had increased expression corresponding to maximal tumor growth. Conclusions :gp130 757F/F Mice rapidly develop distal stomach tumors, with loss of SHP2/Erk/AP-1 transcriptional regulation exemplified by decreased TFF1 expression and increased STAT1/3 regulated genes such as Reg I. Tumor development occurs in a hypogastrinemic environment. Balanced IL-6 signaling is required for maintaining gastric homeostasis.

A protective role of protease-activated receptor 1 in rat gastric mucosa
Atsufumi Kawabata, Hiroyuki Nishikawa, Hitomi Saitoh, Yumiko Nakaya, Kaori Hiramatsu, Satoko Kubo, Minoru Nishida, Naoyuki Kawao, Ryotaro Kuroda, Fumiko Sekiguchi, Mitsuhiro Kinoshita, Kazuaki Kakehi, Naoki Arizono, Hisakazu Yamagishi, Kenzo Kawai
Background & Aims :On activation, protease-activated receptor (PAR)-2 modulates multiple gastric functions and exerts mucosal protection via activation of sensory neurons. The role of PAR-1, a thrombin receptor, in the stomach remains unknown. We thus examined if the PAR-1 agonist could protect against gastric mucosal injury in rats. Methods :Gastric mucosal injury was created by oral administration of ethanol/HCl or absolute ethanol in conscious rats. Gastric mucosal blood flow and acid secretion were determined in anesthetized rats. Immunohistochemical analyses of PAR-1 and cyclooxygenase (COX)-1 were also performed in rat and human stomach. Results :The PAR-1 agonist TFLLR-NH 2, administered intravenously in combination with amastatin, protected against the gastric mucosal injury induced by ethanol/HCl or absolute ethanol. The protective effect of TFLLR-NH 2was abolished by indomethacin or a COX-1 inhibitor but not by ablation of sensory neurons with capsaicin. TFLLR-NH 2produced an NO-independent increase in gastric mucosal blood flow that was partially inhibited by blockade of the endothelium-derived hyperpolarizing factor pathway. This inhibitory effect was promoted by indomethacin. TFLLR-NH 2suppressed carbachol-evoked acid secretion in an indomethacin-reversible manner. Immunoreactive PAR-1 and COX-1 were expressed abundantly in rat gastric muscularis mucosae and smooth muscle, and the former protein was also detectable in blood vessels. Similar staining was observed in human gastric muscularis mucosae. Conclusions :The PAR-1 agonist, given systemically, protects against gastric mucosal injury via COX-1-dependent formation of prostanoids, modulating multiple gastric functions. Our data identify a novel protective role for PAR-1 in gastric mucosa, and the underlying mechanism is entirely different from that for PAR-2.

Bax is required for resection-induced changes in apoptosis, proliferation, and members of the extrinsic cell death pathways
Yuzhu Tang, Deborah A. Swartz-Basile, Elzbieta A. Swietlicki, Lu Yi, Deborah C. Rubin, Marc S. Levin
Background & Aims :To define better the homeostatic mechanisms contributing to small intestinal adaptation following partial resection, the relative contributions of apoptosis, cell proliferation, and enterocyte migration and the comparative roles of the intrinsic (mitochondrial) and extrinsic (death receptor-mediated) apoptotic pathways were assessed. Methods :After 50% jejunoileal resections or transections, adaptation was analyzed in duodenal-jejunal and ileal segments from C57BL/6 Bax +/+ (16, 48, and 168 hours postoperative) and Bax –/– mice (168 hours). Results :Basal apoptotic rates were equivalent in all mice. By 1-week postresection, villus heights and crypt depths were increased in the duodenal-jejunal and ileal remnants of both genotypes. In Bax +/+ mice, adaptation occurred in concert with increased crypt proliferative and apoptotic indices. Bax –/– mice did not show increases in proliferation or apoptosis, yet adaptive increases in villus height were enhanced relative to Bax +/+ mice. Enterocyte migration increased in both genotypes. Postresection, the expression of caspases and genes involved in death receptor-mediated apoptosis was decreased in Bax –/– compared with Bax +/+ mice. Conclusions :Postresection adaptation involves parallel changes in crypt proliferation and apoptosis, but, as observed in Bax –/– mice, it can occur without increased proliferation. These studies demonstrate that spontaneous gut apoptosis is Bax independent, whereas adaptation-related apoptosis is Bax-dependent. Differences between resected Bax +/+ and Bax –/– mice suggest that apoptosis in the adapting gut utilizes the extrinsic pathway, but this requires linkage to the mitochondrial pathway via Bax. The increased adaptive response in Bax –/– mice indicates that modulation of apoptosis may be useful for enhancing adaptation.


Basic-liver, Pancreas, and Biliary Tract
 TOP 

hSulf1 sulfatase promotes apoptosis of hepatocellular cancer cells by decreasing heparin-binding growth factor signaling
Jin-ping Lai, Jeremy R. Chien, David R. Moser, Julie K. Staub, Ileana Aderca, Damian P. Montoya, Tori A. Matthews, David M. Nagorney, Julie M. Cunningham, David I. Smith, Eddie L. Greene, Viji Shridhar, Lewis R. Roberts
Background & aims :The heparin-binding growth factors fibroblast growth factor (FGF) and hepatocyte growth factor (HGF) are potent mitogens for hepatocellular carcinomas (HCCs). Heparin-binding growth factor signaling is regulated by sulfation of cell-surface heparan sulfate proteoglycans (HSPGs). We hypothesized that hSulf1, a recently described sulfatase, regulates growth signaling in HCCs. Methods :Expression of hSulf1 in human HCC tumors was determined by real-time PCR. Down-regulation of hSulf1 expression was investigated by analyzing loss of heterozygosity (LOH) at the hSulf1 locus and the effect of the DNA methylation inhibitor 5-aza-deoxycytidine on hSulf1 expression. The subcellular location of hSulf1 and sulfation state of cell-surface HSPGs were assessed by immunocytochemistry. FGF and HGF signaling was examined by phospho-specific immunoblot analysis. Cell growth was measured by trypan blue exclusion, and the MTT assay and apoptosis were quantitated by fluorescence microscopy. Results :hSulf1 expression was decreased in 29% of HCCs and 82% of HCC cell lines. There was LOH at the hSulf1 locus in 42% of HCCs. Treatment with 5-aza-deoxycytidine reactivated hSulf1 expression in hSulf1-negative cell lines. Low hSulf1-expressing cells showed increased sulfation of cell-surface HSPGs, enhanced FGF and HGF-mediated signaling, and increased HCC cell growth. Conversely, forced expression of hSulf1 decreased sulfation of cell-surface HSPGs and abrogated growth signaling. HCC cells with high-level hSulf1 expression were sensitive to staurosporine- or cisplatin-induced apoptosis, whereas low expressing cells were resistant. Transfection of hSulf1 into hSulf1-negative cells restored staurosporine and cisplatin sensitivity. Conclusions :Down-regulation of hSulf1 contributes to hepatocarcinogenesis by enhancing heparin-binding growth factor signaling and resistance to apoptosis.

Inhibition of bile salt-induced apoptosis by cyclic AMP involves serine/threonine phosphorylation of CD95
Roland Reinehr, Dieter Häussinger
Background & Aims :Cyclic AMP (cAMP) inhibits bile salt-induced hepatocyte apoptosis; the underlying mechanisms are unclear. Methods :The effects of cAMP on taurolithocholate-3-sulfate-(TLCS)- or glycochenodesoxycholate (GCDC)-induced CD95 (Fas/APO-1) activation and apoptosis were studied in 24-hour cultured rat hepatocytes and in perfused rat liver. Results :TLCS induced a rapid oxidative stress response, c-Jun-N-terminal kinase (JNK) and epidermal growth factor (EGF) receptor (EGF-R) activation, subsequent EGF-R/CD95 association and CD95 tyrosine phosphorylation, CD95 membrane targeting, death-inducing signal complex (DISC) formation and hepatocyte apoptosis. None of these responses was triggered by cAMP; however, cAMP induced H89-sensitive serine/threonine phosphorylation of CD95. Similar data were obtained with GCDC, another proapoptotic bile acid. cAMP did not prevent the TLCS-induced oxidative stress response, JNK activation and EGF-R/CD95 association, however abolished EGF-R activation and subsequent CD95 tyrosine phosphorylation, CD95 membrane trafficking, and DISC formation in a H89-sensitive way. Also in presence of TLCS, cAMP induced rapid Ser/Thr phosphorylation of CD95 within 10 min. The effects of cAMP on the various steps of CD95 activation were also found in the intact perfused rat liver. Evidence is given that a cAMP-induced Ser/Thr phosphorylation favors internalization of CD95. Conclusions :Inhibition of bile salt-induced apoptosis by cAMP involves both PKA-dependent Ser/Thr phosphorylation of the CD95 and inhibition of EGF-R activation, which results in an inhibition of CD95 tyrosine phosphorylation, CD95 membrane targeting, and DISC formation. CD95 regulation involves complex phosphorylations with CD95-tyrosine phosphorylation favoring CD95 membrane trafficking and DISC formation, whereas CD95 Ser/Thr phosphorylation inhibits these processes.

Hepatitis C virus inhibits interferon signaling through up-regulation of protein phosphatase 2A
Francois H.T. Duong, Magdalena Filipowicz, Marco Tripodi, Nicola La Monica, Markus H. Heim
Background & Aims :To establish a chronic infection, hepatitis C virus (HCV) has to evade the host defense. Expression of HCV proteins in cultured cells and in hepatocytes of transgenic mice inhibits interferon- -induced intracellular signaling through the Jak-STAT (signal transducer and activator of transcription) pathway. It is not known if interferon- signaling is inhibited in patients with chronic hepatitis C as well, and the molecular mechanisms are not well defined. Methods :Interferon- -induced signaling was investigated in liver biopsies from patients with chronic hepatitis C. The molecular mechanisms of HCV interference with Jak-STAT signaling were analyzed in cultured cells, HCV transgenic mice, and liver biopsies. Results :Interferon- -induced DNA binding of STAT1 was significantly impaired in liver biopsies from patients with chronic hepatitis C compared with controls. Tyrosine and serine phosphorylation of STAT1 were intact, but methylation of STAT1 on arginine 31 was reduced. Hypomethylated STAT1 associated with PIAS1, an inhibitor of STAT DNA binding. Increased expression levels of Protein Phosphatase 2A were found in liver extracts from HCV transgenic mice and in liver biopsies of patients with chronic hepatitis C. Overexpression of PP2Ac in Huh7 cells resulted in hypomethylation of STAT1, increased binding to PIAS1, and reduced interferon- -induced DNA binding of STAT1. Conclusions :We conclude that HCV interferes with interferon- signaling via up-regulation of PP2Ac, hypomethylation of STAT1, and increased STAT1-PIAS1 association, resulting in reduced transcriptional activation of interferon- -stimulated genes.

Prolonged and inducible transgene expression in the liver using gutless adenovirus: A potential therapy for liver cancer
Lin Wang, RubÉN. HernÁndez-Alcoceba, Vijay Shankar, Maider Zabala, Stefan Kochanek, Bruno Sangro, M. Gabriela Kramer, Jesus Prieto, Cheng Qian
Background & Aims :Gene therapy of liver diseases would benefit from systems allowing prolonged, regulable, and tissue-specific transgene expression. We attempted to produce a vector fulfilling these requirements. Methods :We generated gutless adenoviral vectors containing a mifepristone (RU486)-inducible system for controlled and liver-specific expression of human interleukin-12 (hIL-12) (GL-Ad/RUhIL-12) and mouse IL-12 (mIL-12) (GL-Ad/RUmIL-12). The properties of these vectors were tested both in vitro and in vivo. Results :Infection of cells with GL-Ad/RUhIL-12 resulted in high level of hIL-12 expression in the presence of RU486 only in hepatocytic cells. In animals injected with GL-Ad/RUhIL-12, the administration of RU486 induced a transient rise of serum hIL-12 that peaked at 10 hours and completely disappeared by 72 hours. The peak value of hIL-12 was dependent on the doses of the vector and the inducer. High and sustained serum levels of hIL-12 could be attained by continuing administration of RU486 every 12 or 24 hours. Repetitive induction of hIL-12 could be obtained over, at least, a period of 48 weeks after a single injection of GL-Ad/RUhIL-12. Although the vector was detected in many tissues after systemic injection, transcription of the transgene was only found in the liver. Treatment of liver metastases with 5 ? 10 8infectious units of GL-Ad/RUmIL-12 plus RU846 resulted in complete tumor regression in all animals. Conclusion :Gutless adenoviral vectors allow liver-specific and regulable transgene expression for prolonged periods of time. These vectors are promising tools for gene therapy of liver cancer and could also be useful for other forms of hepatic disease.

Sitosterolemia in ABC-Transporter G5-deficient mice is aggravated on activation of the liver-X receptor
Torsten Plösch, Vincent W. Bloks, Yuko Terasawa, Sara Berdy, Karen Siegler, Fjodor van der Sluijs, Ido P. Kema, Albert K. Groen, Bei Shan, Folkert Kuipers, Margrit Schwartz
Background & aims :Mutations in either adenosine triphosphate- binding cassette (ABC) half-transporter G5 or G8 cause sitosterolemia. It has been proposed that ABCG5/ABCG8 heterodimers mediate secretion of plant sterols and cholesterol by hepatocytes into bile and their efflux from enterocytes into the intestinal lumen. Methods :To test whether deficiency of ABCG5 alone is sufficient to induce sitosterolemia, Abcg5-null mice were generated and characterized with respect to sterol metabolism. Results :Abcg5 deficiency was associated with strongly elevated plasma levels of -sitosterol (37-fold) and campesterol (7.7-fold) as well as reduced plasma cholesterol concentrations (–40%). Retention of orally administered [ 3H] -sitosterol in the intestinal wall (+550%) and plasma (+640%) was higher in Abcg5-null mice than in wild-type controls. Surprisingly, high plasma -sitosterol and campesterol concentrations were even further elevated in Abcg5-null mice on treatment with the synthetic LXR agonist T0901317 (0.015% dietary supplementation, 10 days), whereas these concentrations were reduced by ~75% in wild-type mice. Both cholesterol and phospholipid concentrations in gallbladder bile were decreased, but, unexpectedly, cholesterol/phospholipid ratios were unchanged in the absence of Abcg5 and increased in both genotypes on LXR activation. Hepatic expression of Abcg8 was reduced by about 35% in Abcg5-deficient mice when compared with controls. No compensatory overexpression of other ABC transporters potentially involved in hepatic cholesterol trafficking was observed on messenger RNA level. Conclusions :Our data show that disruption of the Abcg5 gene alone is sufficient to cause hyperabsorption of dietary plant sterols and sitosterolemia in mice, whereas the ability to secrete cholesterol into bile is maintained.


Case Report

Hereditary persistence of -fetoprotein is due to both proximal and distal hepatocyte nuclear factor-1 site mutations
Youssef Al j, Maria Georgiakaki, Jean-François Savouret, FrÉdÉric Mal, Pierre Attali, Gilles Pelletier, Catherine Fourré, Edwin Milgrom, Catherine Buffet, Anne Guiochon-Mantel, Gabriel Perlemuter
Background & Aims :The molecular mechanism of hereditary persistence of -fetoprotein (HPAFP) has been previously described in a large Scottish family, consisting of a –119G>A substitution in the distal hepatocyte nuclear factor 1 (HNF-1) binding site of the -fetoprotein (AFP) gene promoter. We report here the molecular mechanisms of HPAFP in 2 new unrelated families. Methods :Family 1 was of Bengali origin, and family 2 was Italian. Four of 5 subjects (family 1) and 3 of 9 (family 2) showed HPAFP. The AFP gene promoter was studied in all available family members. Results :All subjects with high AFP levels had mutated promoter sequences. Family 1 showed the reported –119G>A substitution. Family 2 showed –55C>A and –65C>T substitutions in the proximal putative HNF-1 binding region of the promoter. The –55C>A mutation increased the similarity of the proximal HNF-1 binding region to a consensus binding region. Gel shift assays confirmed its increased affinity toward HNF-1, and transfection experiments revealed an increased level of gene transcription. The –65C>T substitution theoretically created a CCAAT box. However, gel shift and transfection experiments failed to show any biological effect of this substitution that is associated with the –55C>A mutation. Conclusions :Two different mutations localized in either HNF-1 binding sites of the AFP gene promoter may result in HPAFP. This highlights the importance of HNF-1 in AFP gene expression. Unexplained persistent AFP should lead to family study and/or AFP gene promoter sequencing to avoid inappropriate explorations and treatment decisions.

PDGFRA germline mutation in a family with multiple cases of gastrointestinal stromal tumor
Agnès Chompret, Caroline Kannengiesser, Michel Barrois, Philippe Terrier, Philippe Dahan, Thomas Tursz, Gilbert M. Lenoir, Brigitte Bressac-De Paillerets
Familial gastrointestinal stromal tumor (GIST) is a rare autosomal dominant genetic disorder associated with KIT germline mutations. In sporadic forms of the disease, somatic mutations target either KIT or PDGFRA genes. In a kindred in which 5 individuals had GIST, no germline mutation in KIT coding sequence has been detected. We hypothesized that the PDGFRA gene could be a predisposing gene in familial GIST. We sequenced PDGFRA exons 12 and 18 because several somatic mutations were identified within this region. We detected a germline PDGFRA missense mutation, 2675G > T, resulting in a tyrosine substitution for the highly conserved aspartic acid at codon 846. This mutation showed perfect cosegregation with the GIST phenotype among the 7 family members tested. Interestingly, PDGFRA Asp846 is homologous to codon 820, which is located in the KIT tyrosine kinase II domain. In a previous study, a KIT germline Asp820Tyr mutation was detected in a Japanese kindred in which 6 individuals had GIST. Transfection of a KIT820Tyr complementary DNA in nude mice was found to be tumorigenic confirming the oncogenic potential of this mutation. The present study shows that PDGFRA is a second familial GIST predisposing gene. These results indicate a further example of involvement of structurally related genes in familial cancer syndromes.

Copyright © 2001-2004  © 2003 by the American Gastroenterological Association. All rights reserved.

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JOURNAL OF HEPATOLOGY

Table of Contents for Volume 40, Issue 1, january 2004

Biliary Tract and Cholestasis

Anna Strömsten et al.
Studies on the mechanism of accumulation of cholesterol in the gallbladder mucosa. Evidence that sterol 27-hydroxylase is not a pathogenetic factor
Background/Aims : Cholesterolosis is characterized by accumulation of esterified cholesterol in human gallbladder mucosa. The present study aimed at investigating possible pathogenetic factors for cholesterolosis. The hypothesis was tested that a reduced sterol 27-hydroxylase or an increased amount of ACAT-1 enzyme may be of importance.
Methods : Gall bladder mucosa and bile were obtained from patients with cholesterol gallstones undergoing cholecystectomy (30 with and 43 without cholesterolosis).
Results : In cholesterolosis, the gall bladder mucosa was characterized by a several-fold increase in esterified cholesterol and normal content of free cholesterol. The amount of ACAT-1 protein, measured by immunoblotting, was similar in patients with and without cholesterolosis. The level of 27-hydroxycholesterol in gallbladder mucosa was elevated sevenfold as compared with cholesterol in patients with cholesterolosis. Most (87%) of this oxysterol was esterified and the accumulation is most probably secondary to the higher total amount of cholesterol in the cells. Patients with cholesterolosis had normal levels of both sterol 27-hydroxylase mRNA (real time polymerase chain reaction) and protein (immunoblotting). The enzymatic activity of the sterol 27-hydroxylase in gallbladder mucosa was normal or increased in cholesterolosis.
Conclusions : The pathogenesis of cholesterolosis may be multifactorial, but is not caused by reduced efflux of cholesterol due to a defect sterol 27-hydroxylase mechanism.
Keywords: Acyl-coenzyme A: Cholesterol 0-acyltransferase (ACAT-1) ;Biliary lipids ;Cholesterolosis

Kathleen M. Campbell, Gregg E. Sabla and Jorge A. Bezerra
Transcriptional reprogramming in murine liver defines the physiologic consequences of biliary obstruction
Abstract
Background/Aims : While the metabolic and histological responses to cholestasis are recognized, the consequences of impaired biliary flow on liver gene expression are largely undefined. We hypothesized that biliary obstruction results in transcriptional reprogramming that dictates the physiologic response.
Methods : We determined global gene expression in murine livers 1-21 days following bile duct ligation. Total hepatic cRNA from experimental and sham mice was hybridized to Affymetrix gene chips. Gene expression data was analyzed by GeneSpring ®software and validated by Northern analysis.
Results : We found 92 genes over-expressed 2-fold at one or more time points following bile duct ligation. Functional classification of these genes revealed the activation of three main biological processes in a sequential and time-restricted fashion. At day 1, genes involved in sterol metabolism were uniquely over-expressed, including HMG-CoA reductase, the rate-limiting enzyme of cholesterol biosynthesis. This was followed by an increased expression of growth-promoting genes at day 7, the time point coinciding with peak cholangiocyte proliferation. In later phases (days 14-21), the liver over-expressed genes encoding structural proteins and proteases.
Conclusions : Transcriptional reprogramming in the liver following biliary obstruction favors the activation of genes regulating metabolism, cell proliferation, and matrix remodeling in a time-restricted and sequential fashion.
Keywords: Cholestasis ;Bile duct ligation ;Microarray ;Cholangiocyte proliferation ;Cholesterol metabolism
Corresponding author. Division of Gastroenterology, Hepatology and Nutrition, Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA. Tel.: +1-513-636-3008; fax: +1-513-636-5581

Jacqueline R.M. Plass et al.
A progressive familial intrahepatic cholestasis type 2 mutation causes an unstable, temperature-sensitive bile salt export pump
Abstract
Background/Aims : Progressive familial intrahepatic cholestasis type 2 (PFIC-2) patients have a defect in the hepatocanalicular bile salt secretion. The disease is caused by mutations in the bile salt export pump (BSEP). Ten different missense mutations have been described. In this study, we analysed the effect of the D482G PFIC-2 mutation on BSEP function.
Methods : Adenosine triphosphatase (ATPase) and taurocholate transport assays were performed with full-length mouse Bsep (mBsep) with and without the D482G mutation. The effect on expression and subcellular sorting was studied in HepG2 cells, stably expressing enhanced green fluorescent protein (EGFP)-tagged mBsep proteins.
Results : The D482G mutation did not significantly affect the taurocholate transport activity of mBsep, even though the bile salt-inducible ATPase activity of the mutant protein was slightly reduced. Protein expression and canalicular sorting were strongly affected by the D482G mutation. Mutant EGFP-mBsep protein was only partly glycosylated and detected in both the canalicular membrane and the cytoplasm. At 30°C, the mutant mRNA and protein levels were strongly increased, and the protein was predominantly glycosylated and efficiently targeted to the canalicular membrane.
Conclusions : These data suggest that PFIC-2 patients with the D482G mutation express a functional, but highly unstable, temperature-sensitive bile salt export pump.
Keywords: ATPase activity ;Transport ;ATP-binding cassette transporters ;Genetic cholestatic disease ;Missense mutation ;Temperature sensitivity
Abbreviations: ABC, ATP-binding cassette ;ATPase, adenosine triphosphatase ;BCV, bile canalicular-like vacuole ;BSEP, human bile salt export pump ;mBsep, mouse bile salt export pump ;D482G, aspartate-to-glycine mutation at position 482 in BSEP/Bsep ;EGFP, enhanced green fluorescent protein ;NBD, nucleotide binding domain ;PCR, polymerase chain reaction ;PFIC, progressive familial intrahepatic cholestasis ;PNGase F, peptide N-glycosidase F ;SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis ;TCA, taurocholate

Dimitrios-Petrou Bogdanos et al.
Microbial mimics are major targets of crossreactivity with human pyruvate dehydrogenase in primary biliary cirrhosis
Abstract
Background/Aims : Previous studies on patients with primary biliary cirrhosis (PBC) have shown extensive cross-reactivity between the dominant B- and T-cell epitopes of human pyruvate dehydrogenase complex-E2 (PDC-E2), and microbial mimics. Such observations have suggested microbial infection as having a role in the induction of anti-mitochondrial antibodies, through a mechanism of molecular mimicry. However the biological significance of these cross-reactivities is questionable, because PDC-E2 is so highly conserved among various species.
Methods : Interrogating protein databases, ten non-PDC-E2 microbial sequences with high degree of similarity to PDC-E2 212-226 were found in Escherichia coli (6), Helicobacter pylori ,Pseudomonas aeruginosa , Cytomegalovirus, and Haemophilus influenzae . We report on a study testing reactivity and competitive cross-reactivity against these respective peptides, and in some cases the parent protein, using sera from 55 patients with PBC, compared to reactivity of 190 pathological and 28 healthy controls.
Results : Cross-reactivity to E. coli mimics was commonly seen in PBC, and in a subset of pathological controls except where there was no evidence of urinary tract infection and correlated with anti-mitochondrial reactivity.
Conclusions :E. coli /PDC-E2 cross-reactive immunity characterizes primary biliary cirrhosis; the large number of E. coli immunogenic mimics may account for the dominance of the major PDC-E2 autoepitope.
Keywords: Primary biliary cirrhosis ;Microbial mimics ;Escherichia coli

Chronic Liver Diseases

Esteban Mezey, James J. Potter, Lynda Rennie-Tankersley, Juan Caballeria and Albert Pares
A randomized placebo controlled trial of vitamin E for alcoholic hepatitis
Abstract
Background/Aims : The effect of vitamin E administration on clinical and laboratory parameters of liver function and on markers of fibrogenesis was assessed in patients with mild to moderate alcoholic hepatitis in a double blind placebo controlled randomized trial.
Methods : Twenty-five patients received 1000 I.U. of vitamin E per day, while 26 patients received placebo for 3 months. The patients were followed for 1 year after entry into the trial.
Results : Vitamin E did not result in significant greater decreases in serum aminotransferases and serum bilirubin or in greater increases in serum albumin as compared with placebo. Prothrombin time did not change, while serum creatinine remained in the normal range. Monocyte nuclear nuclear factor- B binding activity decreased in patients who remained abstinent, regardless of whether they received vitamin E. As regards markers of hepatic fibrogenesis, vitamin E treatment decreased serum hyaluronic acid ( P<0.05) while serum aminoterminal peptide of type III procollagen did not change in either group. Four patients in the treatment group and five in the placebo group died during the 1-year study.
Conclusions : Vitamin E treatment improves serum hyaluronic acid but has no beneficial effects on tests of liver function in patients with mild to moderate alcoholic hepatitis.
Keywords: -Tocopherol ;Hyaluronic acid ;Aminoterminal peptide of type III procollagen ;Nuclear factor- B

Mary E. Rinella and Richard M. Green
The methionine-choline deficient dietary model of steatohepatitis does not exhibit insulin resistance
Abstract
Background/Aims : Non-alcoholic steatohepatitis is an important disease whose pathophysiology remains incompletely understood, although in humans a strong association with insulin resistance exists. Mice fed a methionine-choline deficient (MCD) diet develop steatohepatitis, however the influence of insulin in this model is unknown.
Methods : Male FVB/NJ mice were fed the MCD, MCD control or chow diet for 10 or 28 days. Fasting glucose, ALT, triglyceride and insulin was measured. Glucose tolerance tests (GTT) and insulin tolerance tests (ITT) were performed followed by quantitative insulin sensitivity check index (QUICKI) determination.
Results : ALT levels were significantly higher in the MCD group. Fasting glucose was 81±5 mg/dl in MCD diet fed mice, compared to MCD controls (196±46 mg/dl) and chow (199±15 mg/dl) ( P<0.0001). During GTT and ITT, the effect of glucose administration on blood glucose was dampened, and the insulin effect more pronounced in the MCD group ( P=0.026 and P<0.001). QUICKI in MCD fed mice was significantly higher than in the chow fed mice.
Conclusions : GTT, ITT and QUICKI confirmed the absence of insulin resistance in the MCD fed mice. This model causes fibrosing steatohepatitis and may help delineate the non-insulin resistant mechanisms involved in human steatohepatitis.
Keywords: Alanine transaminase ;Steatohepatitis ;Non-alcoholic steatohepatitis ;Glucose ;Insulin resistance ;Quantitative insulin sensitivity check index ;Liver

Inflammation and Fibrosis

Ryuichiro Sakata et al.
Green tea polyphenol epigallocatechin-3-gallate inhibits platelet-derived growth factor-induced proliferation of human hepatic stellate cell line LI90
Abstract
Background/Aims : 4-Hydroxynonenal (HNE) is a putative pro-fibrogenic product of oxidative stress able to elicit apoptosis and cytotoxicity in several cell types. This study has been performed to evaluate its `in vivo' levels in injured liver and whether HNE may induce apoptosis and/or affect selected phenotypic responses in activated human hepatic stellate cells (HSC/MF).
Methods/Results : During the development of acute liver injury induced by CCl 4, liver tissue HNE levels were in the range 0.5-10 µM, as shown by high performance liquid chromatography analysis. Cultured human HSC/MF, developed cytotoxicity only if exposed to very high HNE concentrations (25-50 µM) without any sign of induction of classic, caspase-dependent apoptosis, as assessed by evaluating morphology and biochemical parameters of cell death. HNE, at non-cytotoxic doses, up-regulated procollagen type I and tissue inhibitor of metalloproteinases-1 gene expression and/or protein synthesis without significantly affecting chemotaxis (wound healing and haptotaxis assay), matrix metalloproteinases 1 and 2 mRNA expression and activity as well as basal DNA synthesis.
Conclusions : HNE, at concentrations compatible with those detected in vivo, does not elicit HSC/MF classic apoptosis but, rather, may act as a potent pro-fibrogenic stimulus for the expression of genes involved in excess extracellular matrix deposition and proposed as survival signals for HSC/MF.
Keywords: 4-Hydroxynonenal ;Oxidative stress ;Liver fibrosis ;Apoptosis ;Hepatic stellate cells ;Liver myofibroblasts ;Tissue inhibitor of metalloproteinases-1 ;Matrix metalloproteinases ;Chemotaxis

Inflammation and Fibrosis

Elena Zamara et al.
4-Hydroxynonenal as a selective pro-fibrogenic stimulus for activated human hepatic stellate cells
Background/Aims : 4-Hydroxynonenal (HNE) is a putative pro-fibrogenic product of oxidative stress able to elicit apoptosis and cytotoxicity in several cell types. This study has been performed to evaluate its `in vivo' levels in injured liver and whether HNE may induce apoptosis and/or affect selected phenotypic responses in activated human hepatic stellate cells (HSC/MF).
Methods/Results : During the development of acute liver injury induced by CCl 4, liver tissue HNE levels were in the range 0.5-10 µM, as shown by high performance liquid chromatography analysis. Cultured human HSC/MF, developed cytotoxicity only if exposed to very high HNE concentrations (25-50 µM) without any sign of induction of classic, caspase-dependent apoptosis, as assessed by evaluating morphology and biochemical parameters of cell death. HNE, at non-cytotoxic doses, up-regulated procollagen type I and tissue inhibitor of metalloproteinases-1 gene expression and/or protein synthesis without significantly affecting chemotaxis (wound healing and haptotaxis assay), matrix metalloproteinases 1 and 2 mRNA expression and activity as well as basal DNA synthesis.
Conclusions : HNE, at concentrations compatible with those detected in vivo, does not elicit HSC/MF classic apoptosis but, rather, may act as a potent pro-fibrogenic stimulus for the expression of genes involved in excess extracellular matrix deposition and proposed as survival signals for HSC/MF.
Keywords: 4-Hydroxynonenal ;Oxidative stress ;Liver fibrosis ;Apoptosis ;Hepatic stellate cells ;Liver myofibroblasts ;Tissue inhibitor of metalloproteinases-1 ;Matrix metalloproteinases ;Chemotaxis

Lucia Wickert, Muna Abiaka, Ursula Bolkenius and Axel M. Gressner
Corticosteroids stimulate selectively transforming growth factor (TGF)- receptor type III expression in transdifferentiating hepatic stellate cells
Abstract
Background/Aims : Transforming growth factor (TGF)- receptors mediate TGF- signaling in activated hepatic stellate cells (HSC). This leads to pleiotropic cellular effects, e.g. to the production of extracellular matrix which is a hallmark for the development of liver fibrosis. Glucocorticoids and their receptors interact with the TGF- signaling pathway on the transcriptional and translational level.
Methods : To characterize TGF- receptor expression during HSC transdifferentiation and to study the influence of corticosteroids on receptor transcription in several liver cells, we established a real-time polymerase chain reaction procedure for mRNA quantification with gene-specific standards.
Results : All three TGF- receptor mRNAs are present in HSC and myofibroblasts. Whereas TGF receptor type I (T RI) shows a comparable mRNA expression during HSC transdifferentiation, T RII and T RIII mRNA concentration decreases in the course of time. In comparison with activated HSC T RIII mRNA is very low expressed in freshly isolated Kupffer cells and hepatocytes. Eight hours after corticosteroid treatment T RIII mRNA increased significantly in a time-and dose-dependent manner while the mRNA expression of T RI and T RII is not altered. The degree of induction of T RIII mRNA levels is also dependent upon the nature of the stimulating hormone: dexamethasone, hydrocortisone and aldosterone show different effects.
Conclusions : The increase of T RIII by corticosteroids indicates that these hormones are important regulators of this receptor and thereby they can modulate TGF- signaling.

Liver Cell Injury and Liver Failure

Stefano Realdon et al.
Proapoptotic effect of hepatitis C virus CORE protein in transiently transfected cells is enhanced by nuclear localization and is dependent on PKR activation
Abstract
Background/Aims : HCV-CORE protein has been implicated in the regulation of apoptosis of infected cells acting as full-length or C-terminus deleted forms and resulting in both proapoptotic and antiapoptotic effects in different experimental conditions.
Methods : We have fused full-length and C-terminus deleted CORE with GFP to assess intracellular localization in transiently transfected cell lines and primary hepatocytes. Apoptosis of cells expressing different levels of chimeric proteins was quantified by cytometry.
Results : Full-length CORE localized mainly in the cytoplasm, but nuclear staining was also observed, being more evident in primary human hepatocytes. Nuclear staining only was observed in cells expressing truncated CORE. Full-length CORE induced apoptosis in ~15-20% of transfected cells with low expression and in ~40-50% of those with high expression of viral protein. Interestingly, 40-50% of cells transfected with truncated CORE underwent apoptosis, independently of protein expression levels. CORE-induced apoptosis was significantly reduced in the presence of a protein kinase R (PKR) inhibiting peptide and truncated CORE was able to enhance translocation of PKR into nucleoli where CORE/PKR colocalization was observed.
Conclusions : These results suggest that nuclear forms of HCV-CORE are generated in vivo in primary hepatocytes and induce PKR-dependent apoptosis, a mechanism that might have a relevant role during natural infection.
Keywords: Hepatitis C virus ;CORE ;Apoptosis ;Protein kinase R ;Primary hepatocytes ;Nuclear localization

Rafael Bruck et al.
Melatonin inhibits nuclear factor kappa B activation and oxidative stress and protects against thioacetamide induced liver damage in rats
Abstract
Background/Aims : Free radical-mediated oxidative stress has been implicated in the pathogenesis of acute liver injury. The aim of our study was to investigate whether melatonin, a potent free radical scavenger could prevent fulminant hepatic failure in rats.
Methods : Liver damage was induced by two consecutive injections of thioacetamide (TAA, 300 mg/kg/i.p.) at 24 h intervals. Treatment with melatonin (3 mg/kg/daily, i.p) was initiated 24 h prior to TAA.
Results : Twenty-four h after the second TAA injection, serum liver enzymes and blood ammonia were lower in rats treated with TAA+melatonin compared to TAA ( P<0.001). Liver histology was significantly improved and the mortality in the melatonin-treated rats was decreased ( P<0.001). The increased nuclear binding of nuclear factor B in the livers of the TAA-treated rats, was inhibited by melatonin. The hepatic levels of thiobarbituric acid reactive substances, protein carbonyls and inducible nitric oxide synthase were lower in the TAA+melatonin-treated group ( P<0.01), indicating decreased oxidative stress and inflammation.
Conclusions : In a rat model of TAA-induced fulminant hepatic failure, melatonin improves survival and reduces liver damage and oxidative stress. The results suggest a causative role of oxidative stress in TAA-induced hepatic damage and suggest that melatonin may be utilized to reduce liver injury associated with oxidative stress.
Keywords: Fulminant hepatitis ;Melatonin ;Thioacetamide ;Oxidative stress

Hideto Tsuchiya et al.
Pirfenidone prevents endotoxin-induced liver injury after partial hepatectomy in rats
Abstract
Background/Aims : Massive liver resection causes a variety of complications including endotoxemia. Pirfenidone (PFD) is a new experimental drug used as antifibrotic agent. Studies were performed to investigate whether PFD influences the survival rate of animals with endotoxin-induced liver injury after partial hepatectomy, and the mechanisms involved.
Methods : Rats were treated with lipopolysaccharide (LPS) 48 h after 70% hepatectomy. PFD was administered orally before LPS injection.
Results : PFD improved the survival rate of LPS-treated rats after hepatectomy. PFD prevented increases in serum enzymes and total bilirubin related to liver injury. Histopathological analysis revealed that PFD inhibited the enhancement in hepatic necrosis and apoptosis. Further, PFD inhibited increases of inflammatory cytokines and cytokine-induced neutrophil chemoattractant (CINC) in serum and liver, followed by decreases of number of infiltrating neutrophils into liver. Electrophoretic mobility shift assay revealed that PFD inhibited the activation of transcription factor nuclear factor- B (NF- B) induced by LPS. PFD also reduced the induction of inducible nitric oxide synthase (iNOS) in the liver of LPS-treated rats.
Conclusions : These results indicate that PFD inhibits the productions of inflammatory cytokines, CINC and iNOS in part through the inhibition of NF- B activation, resulting in the prevention of endotoxin-induced liver injury after hepatectomy.
Keywords: Pirfenidone ;Partial hepatectomy ;Endotoxin-induced liver injury ;Inflammatory cytokines ;Cytokine-induced neutrophil chemoattractant ;Nuclear factor- B;Inducible nitric oxide synthase

Liver Growth and Cancer

Alessandro Zerbini et al.
Ex vivo characterization of tumor-derived melanoma antigen encoding gene-specific CD8+cells in patients with hepatocellular carcinoma
Abstract
Background/Aims : Members of the melanoma antigen encoding gene family are expressed in tumors of different histological types but not in normal tissue. For this reason, they are attractive targets for cancer immunotherapy.
Methods : In the present study, we analyzed the expression of MAGE-1 and -3 genes in the hepatocellular carcinoma (HCC) tissue as well as frequency, phenotype and function of circulating and tumor infiltrating CD8+ cells specific for HLA-A1 and -A2 restricted epitopes of MAGE-1 and -3.
Results : Our study shows for the first time the presence of MAGE/tetramer+ CD8 cells in the tumor tissue of patients with HCC. These cells are able to recognize the MAGE-1 sequence 161-169 and the MAGE-3 sequence 271-279. In a patient with a particularly high frequency of MAGE-1 sequence 161-169-specific T cells, phenotypic and functional analysis was performed showing a phenotype of recently-primed CD8 cells (CD28+CD27+CD45RA CCR7).
Conclusions : The observation of a spontaneous in vivo priming of a MAGE-specific T cell response in patients with HCC and the high frequency of MAGE antigens expression in this tumor, makes this antigen a potential candidate for a MAGE-specific immunotherapy in hepatocellular carcinoma.
Keywords: Hepatocellular carcinoma ;Melanoma antigen encoding gene-1 ;MAGE-3 ;Immunotherapy ;Tumor immunity

Kiyotaka Okada, Shigeru Ueshima, Motohiro Imano, Kazuo Kataoka and Osamu Matsuo
The regulation of liver regeneration by the plasmin/ 2-antiplasmin system
Abstract
Background/Aims : The regeneration after liver injury is regulated by the release and activation of several growth factors. The role of the plasmin/ 2-antiplasmin ( 2-AP) system in liver regeneration was investigated.
Methods : CCl 4was injected intraperitoneally into the mice deficient ( /) in fibrinolytic factors: 2-AP /, plasminogen (Plg) /, and Plg /·2-AP /, and wild-type (WT) mice. The liver tissue was examined for its microscopic appearance, fibrinolytic activity, and fibronectin levels.
Results : In the gene deficient and WT mice, the livers exhibited the same extent of necrosis 2 days after the CCl 4injection. The livers of the WT mice normalized after 7 days, and the 2-AP /mice normalized after 5 days. In contrast, the livers of the Plg /and Plg /·2-AP /mice remained in the damaged state until 14 days after the liver injury. The injection of anti- 2-AP antibody in the WT mice improved the regeneration after the liver injury, and the injection of tranexamic acid in the 2-AP /mice reduced.
Conclusions : These results suggest that the plasmin/ 2-AP system played an important role in hepatic repair via clearance from the injury area.
Keywords: Liver ;Regeneration ;Plasminogen ;Plasmin ;2-Antiplasmin

Seiichi Yasuda et al.
Hexokinase II and VEGF expression in liver tumors: correlation with hypoxia-inducible factor-1 and its significance
Abstract
Background/Aims : We analyzed the expressions of hexokinase II (HK II), a key enzyme in glycolysis, and VEGF in hepatocellular carcinoma (HCC) and metastatic liver cancer in relation to tumor vascularity, and the participation of hypoxia-inducible factor-1 (HIF-1) was studied.
Methods : A real-time quantitative reverse transcription-polymerase chain reaction was performed to examine the HK II and VEGF mRNA expression. Expression of HIF-1 and HK II protein, and microvessel density (MVD) were examined immunohistochemically.
Results : MVD was significantly higher in HCCs than in metastatic liver cancers, and VEGF mRNA expression was positively correlated only with MVD of HCCs. HK II mRNA expression was significantly higher in metastatic liver cancers, however, some cases of HCC pretreated with transcatheter arterial embolization (TAE) showed marked HK II mRNA expression. Both HIF-1 and HK II protein expressions were co-localized in the cancer cells near necrosis, and the intensity of HIF-1 protein expression was significantly correlated with HK II mRNA expression in both tumors.
Conclusions: These results suggest that, in metastatic liver cancers, glycolysis induced by HIF-1 is the predominant energy source under the hypoxic environment and, at least in some TAE-pretreated HCC cases, cancer cells obtain energy for growth by switching the metabolic profile to glycolysis through HIF-1.
Keywords: Hexokinase II ;Hypoxia-inducible factor-1 ;Liver tumors

Umberto Cillo et al.
The critical issue of hepatocellular carcinoma prognostic classification: which is the best tool available?
Abstract
Background/Aims : Prognosis assessment in patients with hepatocellular carcinoma (HCC) remains controversial. The most widely used HCC prognostic tool is the Okuda classification, but new staging systems (Cancer of the Liver Italian Program score, Chinese University Prognostic Index, French classification and Barcelona Clinic Liver Cancer, BCLC, staging) have been recently described. We investigated the value of known prognostic systems in the particular setting of a surgically oriented Liver Unit where 187 HCC Italian patients were mainly treated with radical therapies (resection and percutaneous ablation).
Methods : A retrospective analysis of 187 HCCs observed at a single Institution from 1990 and 1999 was performed. By using survival time as the only outcome measure (Kaplan-Meier method and Cox regression), the performance of any prognostic system was assessed according the criteria of discriminatory and stratification abilities between different stages, homogeneity of survival within each stage and additional explanatory power respect to the other classifications.
Results : In the particular cohort studied, BCLC proved the best HCC prognostic system. This was true for the whole study group and for the 2 subgroups of surgical and non-surgical patients.
Conclusions : BCLC staging showed the best interpretation of the survival distribution in an HCC population comprising a large proportion of tumors treated with potentially radical therapies.
Keywords: Hepatocellular carcinoma ;Prognosis ;Staging ;Therapeutic decision ;Tumor stage ;Liver function

Manuel A. Fernández, Silvia Turró, Mercedes Ingelmo-Torres, Carlos Enrich and Albert Pol
Intracellular trafficking during liver regeneration - Alterations in late endocytic and transcytotic pathways
Abstract
Background/Aims : Liver growth, induced by partial hepatectomy of the organ is a precisely regulated process during which a radical reorganisation of metabolism occurs as the hepatocytes become committed to enter the cell cycle. Recent studies have shown the importance of the endocytic compartment in the control of lipid and protein intracellular trafficking but also in the control of the signal transduction events, which eventually will trigger the initiation of DNA synthesis and the subsequent cell division.
Methods : We isolated endosomes at different times after partial hepatectomy in male rats and compared with endosomes isolated from sham-operated animals. Also, bile was collected and analysed by 2D-gel electrophoresis.
Results : The amount of late endosomes isolated from regenerating livers decreased, concomitant with decreased cathepsin D specific enzyme activity. Furthermore, secretion of horseradish peroxidase, pIgA and transferrin increased in the pre-replicative phase of liver regeneration.
Conclusions : At the early stages of liver regeneration, the hepatocellular transport pathway towards degradation (late endosomes and lysosomal pathway) decreases, but the transcytosis and the bile secretion of several major proteins increases.
Keywords: Liver regeneration ;Endosomes ;Transcytosis ;Degradation ;Bile proteins

Transplantation

Tea Restuccia et al.
Effects of treatment of hepatorenal syndrome before transplantation on posttransplantation outcome. A case-control study
Abstract
Background : Pretransplant renal function is the major determinant of survival after liver transplantation (LTx). Patients with hepatorenal syndrome (HRS) have a poor outcome after LTx compared with patients transplanted without HRS.
Aim : To analyze the impact of treatment of HRS before LTx on outcome after transplantation.
Methods : The outcome of patients with HRS ( n=9) treated with vasopressin analogues before LTx was compared with that of a contemporary control group of patients without HRS ( n=27) matched by age, severity of liver failure, and type of immunosuppression.
Results : Cases and controls were similar with respect to pretransplantation characteristics. Three-year survival probability was similar between the two groups (HRS-treated: 100% vs control: 83%, P=0.15). No significant differences were found between the two groups with respect to the incidence of impairment of renal function after LTx (HRS-treated: 22% vs control: 30%), severe infections (22 vs 33%), acute rejection (33 vs 41%), days in Intensive Care Unit (6±1 vs 8±1), days in hospital (27±4 vs 31±4), and transfusion requirements (11±3 vs 10±2 units).
Conclusions : Patients with HRS treated with vasopressin analogues before LTx have a posttransplantion outcome similar to that of patients transplanted with normal renal function. These results suggest that HRS should be treated before LTx.
Keywords: Hepatorenal syndrome ;Liver transplantation ;Posttransplantation

Viral Hepatitis

Ke-Qin Hu et al.
Overweight and obesity, hepatic steatosis, and progression of chronic hepatitis C: a retrospective study on a large cohort of patients in the United States
Abstract
Background : Hepatic steatosis has been associated with chronic hepatitis C (CHC), but its prevalence, risk factors, and clinical significance remain to be determined.
Aims : The present study determined the frequency of, and risk factors for hepatic steatosis and its association with activity and progression of CHC in a large cohort of U.S. patients.
Methods : This is a retrospective study that utilized systematic chart review and statistical analyzes to investigate 324 U.S. patients with CHC from a university medical center and a regional VA medical center.
Results : The frequency of hepatic steatosis was 66.0%. We demonstrated that not only being obese, but also overweight (i.e. body mass index 25 kg/m 2) was independently associated with hepatic steatosis. In our cohort of patients with CHC, hepatic steatosis, especially grade II/III steatosis, was significantly associated with elevated aspartate aminotransferase at entry, persistently elevated alanine aminotransferase, and stage III/IV fibrosis. Grade II/III steatosis, was significantly associated with a higher histology activity index as well. Multivariate analysis indicated that steatosis, especially grade II/III steatosis, was independently associated with stage III/IV fibrosis.
Conclusions : Being overweight/obese serves as an independent risk factor for hepatic steatosis in U.S. patients with CHC. Steatosis accelerates activity and progression of CHC, and is independently associated with stage III/IV hepatic fibrosis in these patients.
Keywords: Hepatitis C virus ;Chronic hepatitis C ;Hepatic steatosis ;Obesity ;Body mass index ;Hepatic fibrosis

Case Report

S. Sharma et al.
Pharmacological thrombolysis in Budd Chiari syndrome: a single centre experience and review of the literature
Abstract
Background/Aims
To review our experience of thrombolytic therapy in patients with acute Budd Chiari syndrome (BCS).
Methods
Records of 10 patients with BCS, treated by thrombolysis over a 12-year period were retrospectively analysed for demographics, clinical presentation/duration, primary disease, thrombolytic regimen, and follow-up. The same characteristics were also studied in previously reported patients. The agent used was recombinant tissue plasminogen activator (tPA) in all patients.
Results
Thrombolysis was used 12 times in 10 patients. Infusion was made systemically in three patients, into the hepatic artery in one patient, locally into a hepatic vein and/or IVC in four patients and locally within TIPS/portal vein in two patients. Only one infusion made systemically was partially successful. Adjunctive balloon angioplasty and/or stent insertion was undertaken for all eight procedures (in six patients) where local infusion was into the hepatic vein or TIPS. Six of these were ultimately successful (in five patients) and two were unsuccessful. Thrombolysis was more likely to be successful in the presence of a short history of thrombosis, when the thrombolytic agent was locally infused and when it was combined with a successful radiological procedure. Mean follow-up was 4.5 years (range 1-10 years). No serious bleeding complication occurred.
Conclusions
We observed no benefit from thrombolysis when delivered systemically or arterially except in one case. Thrombolysis was useful in adjunctive management of BCS when the drug was infused locally into recently thrombosed veins that had appreciable flow following partial recanalisation. Thrombolysis was clearly of benefit in the repermeation of occluded/partially occluded hepatic veins/TIPS when early detection of new thrombus followed interventional procedures such as balloon angioplasty or stenting of hepatic veins.
Keywords: Thrombolysis ;Budd Chiari syndrome ;TIPS ;Hepatic venous outflow obstruction

Copyright © 2001-2004  European Association for the Study of the Liver. All rights reserved.

BRITISH MEDICAL JOURNAL

10 January 2004 (Vol 328, No 7431)

Pancreatic cancer is associated with long term use of aspirin
David Spurgeon
Women who regularly use aspirin over a long period may be increasing their risk of having pancreatic cancer, says an 18 year study of almost 90 000 women in the United States taking part in the nurses’ health study.
The authors, led by Dr Eva Schernhammer, of Brigham and Women’s Hospital and Harvard Medical School, conclude that their findings contradict other research indicating that analgesics reduce the risk of pancreatic cancer and instead point to an increasing risk that rises with dose ( Journal of the National Cancer Institute 2003;96:22-8). During the 18 years of follow up 161 new cases of pancreatic cancer developed among the 88 378 women studied. Overall, there was no significant difference in risk between women who did take aspirin and those who did not. But when the investigators examined data on the duration of use, they found that the risk of pancreatic cancer among women who took more than two tablets a week over 20 years was 58% higher than the risk among women who did not regularly take aspirin (relative risk 1.58 (95% confidence interval 1.03 to 2.43)). This difference rose to 86% among women who took 14 or more tablets a week (1.86 (1.03 to 3.35)).
The relative risk was 1.11 (95% confidence interval 0.70 to 1.76) for women taking one to three tablets a week, 1.29 (0.70 to 2.40) for those taking four to six tablets, and 1.41 (0.76 to 2.61) for those taking seven to 13 tablets (P for trend 0.02).
In an accompanying editorial Dr John Baron, of Dartmouth Medical School, wrote: "There are no easy answers to the question of what aspirin and other NSAIDs [non-steroidal anti-inflammatory drugs] do to pancreatic carcinogenesis. The findings by Schernhammer et al are provocative and force us to think carefully about the actions of aspirin and other NSAIDs and the mechanisms underlying pancreatic cancer."

© 2004 BMJ Publishing Group Ltd

NEW ENGLAND JOURNAL

The New England Journal of Medicine is owned, published, and copyrighted © 2004 Massachusetts Medical Society. All rights reserved.

LANCET

The Lancet, published, and copyrighted © 2004. All rights reserved.