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Mois d'Aout 2003

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HEPATOLOGY

Table of Contents for August 2003 · Volume 38 · Number 2

Liver Biology and Pathobiology

Visualization of early events in tumor formation of eGFP-transfected rat colon cancer cells in liver
Olaf R. F. Mook, Jan Van Marle, Heleen Vreeling-Sindelárová, Remmet Jonges, Wilma M. Frederiks, Cornelis J. F. Van Noorden
Colon cancer preferentially metastasizes to the liver. To determine cellular backgrounds of this preference, we generated an enhanced green fluorescent protein (eGFP)-expressing rat adenocarcinoma cell line (CC531s) that forms metastases in rat liver after administration to the portal vein. Intravital videomicroscopy (IVVM) was used to visualize early events in the development of tumors in livers of live animals from the time of injection of the cancer cells up to 4 days afterward. Based on information obtained with IVVM, tissue areas were selected for further analysis using confocal laser scanning microscopy (CLSM), electron microscopy (EM), and electron tomography. It was shown that initial arrest of colon cancer cells in sinusoids of the liver was due to size restriction. Adhesion of cancer cells to endothelial cells was never found. Instead, endothelial cells retracted rapidly and interactions were observed only between cancer cells and hepatocytes. Tumors developed exclusively intravascularly during the first 4 days. In conclusion, initial steps in the classic metastatic cascade such as adhesion to endothelium and extravasation are not essential for colon cancer metastasis in liver. (HEPATOLOGY 2003;38:295-304.)

Mechanism of 1-integrinmediated hepatoma cell growth involves p27 and S-phase kinaseassociated protein 2
Hao Zhang, Iwata Ozaki, Toshihiko Mizuta, Tohru Yoshimura, Sachiko Matsuhashi, Akitaka Hisatomi, Jutaro Tadano, Takahiro Sakai, Kyosuke Yamamoto
Although cooperative interactions between growth factors and integrins, cell surface receptors for extracellular matrices (ECM), have been reported, little is known about the interaction between hepatocyte growth factor (HGF) and integrin in hepatoma cells. We investigated the effects and mechanisms of integrin on the proliferation of hepatoma cells regulated by HGF. Human HepG2 hepatoma cells stably transfected with 1-integrin were treated with HGF and compared with parental and mock-transfected control cells. Cell proliferation and expression of cyclin-dependent kinase (Cdk) inhibitors and S-phase kinase-associated protein 2 (Skp2), were investigated. HGF dose-dependently suppressed the proliferation of parental and mock-transfected HepG2 cells. However, cells overexpressing 1-integrin exhibited increased proliferation in response to HGF. Although HGF increased p27 and decreased Skp2 expression in the parental and mock-transfected cells, the p27 and Skp2 levels in cells overexpressing 1-integrin were not altered by HGF. Interestingly, HepG2 cells overexpressing 1-integrin showed increased Skp2 expression. Furthermore, HGF did not reduce the proliferation of HepG2 cells transfected with antisense p27 or sense Skp2. Thus, HGF suppresses HepG2 cell proliferation by directly increasing p27 expression and indirectly decreasing Skp2 expression, and 1-integrin modulates the responsiveness of hepatoma cells to HGF via a p27-dependent manner by increasing Skp2. In conclusion, these results strongly suggest that integrin-mediated signals from the ECM can modulate growth factor-mediated signals in hepatoma cells, and may contribute to the growth of hepatocellular carcinomas. (HEPATOLOGY 2003;38:305-313.)

A common set of immediate-early response genes in liver regeneration and hyperplasia
Joseph Locker, Jianmin Tian, Robert Carver, Danilo Concas, Costanza Cossu, Giovanna M. Ledda-Columbano, Amedeo Columbano
Partial hepatectomy (PH) and some tumor-promoting agents stimulate hepatocyte cell proliferation, but each treatment acts through distinct transcription factors. We compared mouse immediate-early gene expression changes after PH with those induced by 1,4-bis[2-(3,5-dichoropyridyloxy)]benzene (TCPOBOP), a tumor-promoting liver mitogen. PH activates nuclear factor B (NF-B) and Stat3, whereas TCPOBOP is a ligand for the nuclear receptor, constitutive androstane receptor (CAR). RNA from 1 and 3 hours after each treatment was hybridized to a 9,000 complementary DNA (cDNA) microarray. Of about 6,000 messenger RNAs that had detectable expression, 127 showed reproducible up-regulation or down-regulation at a significant level. The TCPOBOP response was more discrete than the PH response; they amounted to 1% and 1.9% of positive hybridizations, respectively. Twenty-three genes were regulated only by TCPOBOP, 57 only by PH, and 59 by both treatments. More detailed analysis defined 16 clusters with common patterns of expression. These patterns and quantification of hybridization levels on the array were confirmed by Northern blots. TCPOBOP selectively activated expression of a number of detoxification enzymes. In conclusion, the genes that were regulated by both treatments suggest down-regulation of apoptosis, altered signal transduction, and early biogenesis of critical cell components. (HEPATOLOGY 2003;38:314-325.)

Massive liver growth in mice induced by systemic interleukin 6 administration
Teresa A. Zimmers, Iain H. McKillop, Robert H. Pierce, Joo-Yeon Yoo, Leonidas G. Koniaris
The multifunctional cytokine interleukin 6 (IL-6) is expressed in a wide variety of disease states and pathologic processes. Mice deficient in IL-6 display abnormal and delayed liver regeneration and repair. Currently, IL-6 is thought to influence liver growth indirectly by priming hepatocytes to respond to growth factors such as hepatocyte growth factor (HGF) by inducing expression of HGF and by inhibiting hepatocyte apoptosis, as distinct from the direct mitotic effects of IL-6 on myeloid and other cell types. Here, we show that systemic administration of IL-6 using CHO cell tumors in nude mice results in dramatic hepatomegaly and hepatocyte hyperplasia in the absence of liver injury. Liver mass and liver to body mass ratios increased to 2 to 3 times normal because of proliferation of hepatocytes. Liver growth was associated with high levels of serum IL-6 and with activation of the IL-6-signaling pathway, including increased expression of IL-6 receptor-/gp80, activation of the signal transducer and activator of transcription-3 (STAT-3), and mitogen-activated protein kinase (MAPK/ERK)-signaling pathways and induction of downstream target genes, including c-myc. HGF receptor and transforming growth factor (TGF-)/epidermal growth factor (EGF) receptor activation were decreased in hypertrophied livers, suggesting that IL-6-induced liver growth was independent of these known hepatocyte mitotic pathways. In conclusion, we suggest that IL-6 may function as a direct hepatic mitogen in vivo and, furthermore, that IL-6 warrants closer examination as a potent liver growth factor with potential clinical utility for increasing liver mass following injury. (HEPATOLOGY 2003;38:326-334.)

Nuclear factor B decoy oligodeoxynucleotides prevent endotoxin-induced fatal liver failure in a murine model
Ichiro Ogushi, Yuji Iimuro, Ekihiro Seki, Gakuhei Son, Tadamichi Hirano, Toshikazu Hada, Hiroko Tsutsui, Kenji Nakanishi, Ryuichi Morishita, Yasufumi Kaneda, Jiro Fujimoto
Endotoxin syndrome is a systemic inflammatory response mediated by inflammatory cytokines. Nuclear factor B (NF-B) is the dominant regulator of the production of these cytokines by inflammatory cells. The aim of this study was to assess the efficacy of in vivo transfer of synthetic double-stranded oligodeoxynucleotides (ODN) with high affinity against NF-B (NF-B/decoy/ODN) as a therapeutic strategy for treating endotoxin-induced fatal liver injury. Liver injury was induced by administration of lipopolysaccharide (LPS) to Propionibacterium acnes-primed BALB/C mice. NF-B/decoy/ODN was transferred into the portal vein using a fusigenic liposome with hemagglutinating virus of Japan. NF-B/decoy/ODN was preferentially transferred to Kupffer cells, and activation of NF-B after the LPS challenge was suppressed, leading to decreased inflammatory cytokine production. As a result, the massive necrosis and hepatocyte apoptosis observed in the control mice was dramatically attenuated and the survival rate improved. In conclusion, NF-B/decoy/ODN transfer in vivo effectively suppressed endotoxin-induced fatal liver injury in mice. (HEPATOLOGY 2003;38:335-344.)

Effects of proinflammatory cytokines on rat organic anion transporters during toxic liver injury and cholestasis
Andreas Geier, Christoph G. Dietrich, Sebastian Voigt, Suk-Kyum Kim, Thomas Gerloff, Gerd A. Kullak-Ublick, Johann Lorenzen, Siegfried Matern, Carsten Gartung
Hepatobiliary transporters are down-regulated in toxic and cholestatic liver injury. Cytokines such as tumor necrosis factor (TNF-) and interleukin 1 (IL-1) are attributed to mediate this regulation, but their particular contribution in vivo is still unknown. Thus, we studied the molecular mechanisms by which Ntcp, Oatp1, Oatp2, and Mrp2 are regulated by proinflammatory cytokines during liver injury. Rats were injected intraperitoneally with either carbon tetrachloride or endotoxin. Inactivation of TNF- and IL-1 was achieved by repetitive intraperitoneal injection of etanercept and anakinra, respectively. Messenger RNA (mRNA) levels of transporters and binding activities as well as nuclear protein levels of Ntcp, Oatp2, and Mrp2 transactivators were determined 20 to 24 hours later. In contrast to IL-1, TNF- inactivation alone fully prevented down-regulation of Ntcp, Oatp1, and Oatp2 mRNA as well as reduced binding activity of hepatocyte nuclear factor 1 (HNF-1) in CCl4-induced toxic injury. In endotoxemia, down-regulation of Mrp2, and partially in case of Ntcp, could be prevented by IL-1 but not TNF- blockade. However, inactivation of either cytokine led to preservation of HNF1 and partially of retinoid X receptor/retinoic acid receptor (RXR/RAR) binding activity. No effect of anticytokines was seen on pregnane X receptor (PXR) and constitutive androstane receptor (CAR) binding activity as well as nuclear protein mass. In conclusion, TNF- represents the master cytokine responsible for HNF1-dependent down-regulation of Ntcp, Oatp1, and Oatp2 in CCl4-induced toxic liver injury. IL-1 predominates in a complex signaling network of Ntcp and Mrp2 regulation in cholestatic liver injury. In contrast to in vitro studies, HNF1 and RXR/RAR-independent mechanisms appear to be more important in regulation of Mrp2 and Ntcp gene expression in endotoxemia. (HEPATOLOGY 2003;38:345-354.)

Neutrophils aggravate acute liver injury during obstructive cholestasis in bile ductligated mice
Jaspreet S. Gujral, Anwar Farhood, Mary Lynn Bajt, Hartmut Jaeschke
Obstruction of the common bile duct in a variety of clinical settings leads to cholestatic liver injury. An important aspect of this injury is hepatic inflammation, with neutrophils as the prominent cell type involved. However, the pathophysiologic role of the infiltrating neutrophils during cholestatic liver injury remains unclear. Therefore, we tested the hypothesis that neutrophils contribute to the overall pathophysiology by using bile duct-ligated (BDL) wild-type animals and mice deficient in the 2 integrin CD18. In wild-type animals, neutrophils were activated systemically as indicated by the increased expression of Mac-1 (CD11b/CD18) and L-selectin shedding 3 days after BDL. Histologic evaluation (48 ± 10% necrosis) and plasma transaminase levels showed severe liver injury. Compared with sham-operated controls (< 10 neutrophils per 20 high-power fields), large numbers of neutrophils were present in livers of BDL mice (425 ± 64). About 60% of these neutrophils had extravasated into the parenchyma. In addition, a substantial number of extravasated neutrophils were found in the portal tract. In contrast, Mac-1 was not up-regulated and plasma transaminase activities and the area of necrosis (21 ± 9%) were significantly reduced in CD18-deficient animals. These mice had overall 62% less neutrophils in the liver. In particular, extravasation from sinusoids and portal venules (PV) was reduced by 91% and 47%, respectively. Immunohistochemical staining for chlorotyrosine, a marker of neutrophil-derived oxidant stress, was observed in the parenchyma of BDL wild-type but not CD18-deficient mice. In conclusion, neutrophils aggravated acute cholestatic liver injury after BDL. This inflammatory injury involves CD18-dependent extravasation of neutrophils from sinusoids and reactive oxygen formation. (HEPATOLOGY 2003;38:355-363.)

Bilirubin rinse: A simple protectant against the rat liver graft injury mimicking heme oxygenase1 preconditioning
Yutaro Kato, Motohide Shimazu, Mieko Kondo, Koji Uchida, Yusuke Kumamoto, Go Wakabayashi, Masaki Kitajima, Makoto Suematsu
Heme oxygenase (HO)-1 preconditioning through genetic or pharmacologic interventions was shown experimentally to improve posttransplant outcome of liver grafts. However, its clinical application requires careful consideration because of the complexity and economic costs of the procedures. This study aimed to examine if graft preconditioning with HO-1 could be substituted by a simple treatment with heme-degrading products such as bilirubin. Rats were pretreated with or without hemin, an HO-1 inducer for preconditioning. Their livers were harvested as grafts in University of Wisconsin (UW) solution for 16 hours at 4°C and followed by reperfusion ex vivo or by transplantation in vivo. The control grafts were also treated with a rinse buffer containing varied concentrations of unconjugated bilirubin with different time intervals. The HO-1-preconditioned grafts ex vivo exhibited a marked improvement of bile output and cell injury that was cancelled by blocking HO with zinc protoporphyrin-IX. The aggravation of the graft viability by the inhibitor was repressed by supplementation of bilirubin but not by that of carbon monoxide. Furthermore, a short-term rinse treatment with micromolar levels of bilirubin attenuated biliary dysfunction and cell injury of the grafts both ex vivo and in vivo even without HO-1 preconditioning. The protective effects of HO-1 preconditioning or bilirubin rinse appeared to involve its inhibitory effects on lipid peroxidation in hepatocytes. In conclusion, these results suggest that bilirubin rinse serves as a simple strategy to ameliorate hyperacute oxidative stress and hepatobiliary dysfunction of the transplanted grafts, mimicking effects of HO-1-mediated preconditioning. (HEPATOLOGY 2003;38:364-373.)

Cotransport of reduced glutathione with bile salts by MRP4 (ABCC4) localized to the basolateral hepatocyte membrane
Maria Rius, Anne T. Nies, Johanna Hummel-Eisenbeiss, Gabriele Jedlitschky, Dietrich Keppler
The liver is the major source of reduced glutathione (GSH) in blood plasma. The transport protein mediating the efflux of GSH across the basolateral membrane of human hepatocytes has not been identified so far. In this study we have localized the multidrug resistance protein 4 (MRP4; ABCC4) to the basolateral membrane of human, rat, and mouse hepatocytes and human hepatoma HepG2 cells. Recombinant human MRP4, expressed in V79 hamster fibroblasts and studied in membrane vesicles, mediated ATP-dependent cotransport of GSH or S-methyl-glutathione together with cholyltaurine, cholylglycine, or cholate. Several monoanionic bile salts and the quinoline derivative MK571 were potent inhibitors of this unidirectional transport. The Km values were 2.7 mmol/L for GSH and 1.2 mmol/L for the nonreducing S-methyl-glutathione in the presence of 5 µmol/L cholyltaurine, and 3.8 µmol/L for cholyltaurine in the presence of 5 mmol/L S-methyl-glutathione. Transport of bile salts by MRP4 was negligible in the absence of ATP or without S-methyl-glutathione. These findings identify a novel pathway for the efflux of GSH across the basolateral hepatocyte membrane into blood where it may serve as an antioxidant and as a source of cysteine for other organs. Moreover, MRP4-mediated bile salt transport across the basolateral membrane may function as an overflow pathway during impaired bile salt secretion across the canalicular membrane into bile. In conclusion, MRP4 can mediate the efflux of GSH from hepatocytes into blood by cotransport with monoanionic bile salts. (HEPATOLOGY 2003;38:374-384.)

Ethanol metabolism results in a G2/M cell-cycle arrest in recombinant Hep G2 cells
Dahn L. Clemens, Lilian E. Calisto, Michael F. Sorrell, Dean J. Tuma
Previous studies using the Hep G2-based VA cells showed that ethanol metabolism resulted in both cytotoxicity and impaired DNA synthesis, causing reduced accumulation of cells in culture. To further characterize the ethanol oxidation-mediated impairment of DNA synthesis we analyzed the cell-cycle progression of VA cells. These studies showed approximately a 6-fold increase in the percentage of cells in the G2/M phase of the cell cycle after 4 days of ethanol exposure. The G2/M transition requires activity of the cyclin-dependent kinase, Cdc2. Cdc2 is positively regulated by association with cyclin B1, and negatively regulated by phosphorylation of amino acids Thr14 and Tyr15. Immunoblot analysis revealed that ethanol metabolism had little affect on total Cdc2 content in these cells, but resulted in the accumulation of up to 20 times the amount of cyclin B1, indicating that cyclin B1 was available for formation of Cdc2/cyclin B1 complexes. Co-immunoprecipitation revealed that 6 times more Cdc2/cyclin B1 complexes were present in the ethanol-treated cells compared with the controls. Investigation of the phosphorylation state of Cdc2 revealed that ethanol oxidation increased the amount of the phosphorylated inactive form of Cdc2 by approximately 3-fold. Thus, the impairment in cell-cycle progression could not be explained by a lack of cyclin B1, or the ability of Cdc2 and cyclin B1 to associate, but instead resulted, at least in part, from impaired Cdc2 activity. In conclusion, ethanol oxidation by VA cells results in a G2/M cell-cycle arrest, mediated by accumulation of the phosphorylated inactive form of Cdc2. (HEPATOLOGY 2003;38:385-393.)

Antimycin Ainduced defenestration in rat hepatic sinusoidal endothelial cells
Filip Braet, Michael Muller, Katrien Vekemans, Eddie Wisse, David G. Le Couteur
Liver sinusoidal endothelial cells (LSECs) possess fenestrae arranged in sieve plates. Hepatic endothelial fenestrae are open pores approximately 100 to 200 nm in diameter. Alterations in their number or diameter by hormones, xenobiotics, and diseases have important implications for hepatic microcirculation and function. Numerous reports of hepatotoxin-induced defenestration suggest that the cytoskeleton and the energy status of hepatic endothelial cells play a key role in the regulation of fenestrae. Therefore, we investigated the effect of antimycin A, an inhibitor of mitochondrial energy production, on the number of fenestrae in cultured LSECs using high-resolution microscopy and immunocytochemistry. Prolonged incubation (greater than 30 min) with antimycin A resulted in defenestrated cells and coincided with the appearance of F-actin dots, whereas the distribution of G-actin remained unchanged. Adenosine triphosphate (ATP) was depleted dramatically to less than 5% within 30 minutes within the LSECs. After treatment with antimycin A, unusual elevated fenestrated complexes were apparent, organized as a meshwork of anastomosing fenestrae at the center of and above the sieve plates. The position and appearance of these novel structures and their association with defenestration suggest that they are implicated in the process of defenestration. In conclusion, the results of experiments with antimycin A suggest that ATP is needed to maintain fenestrae and the underlying fenestrae-associated cytoskeleton rings that maintain fenestrae patency. Antimycin A-induced defenestration of LSECs is associated with the development of a structure in the sieve plate that appears to be intrinsically involved in defenestration. (HEPATOLOGY 2003;38:394-402.)

A liver tolerates a portal antigen by generating CD11c+ cells, which select Fas Ligand+ Th2 cells via apoptosis
Tomohiro Watanabe, Hiroaki Katsukura, Yasuhiko Shirai, Masashi Yamori, Toshiki Nishi, Tsutomu Chiba, Toru Kita, Yoshio Wakatsuki
Administration of an antigen (Ag) per oral route leads to apoptosis of Ag-specific CD4+ T cells and to development of Th2 cells expressing Fas ligand (FasL) in the liver. We determined whether presentation of an ingested Ag in the liver alone was enough to select these FasL+Th2 cells and explored how this selection was achieved in the liver. Ovalbumin (OVA) administered orally was colocalized with class II+ cells in the periportal and parenchymal area of the liver. On coculture with naive OVA-specific CD4+ T cells, hepatic CD11c+ cells from mice fed OVA generated Ag-specific Th2 cells. This was achieved by apoptosis of CD4+ T cells, decrease of interleukin 12 (IL-12) secretion, and increase of IL-18 secretion by the CD11c+ cells. Addition of IL-12 to this coculture prevented apoptosis of the CD4+ T cells, which was associated with up-modulation of IL-2 receptor chain expression. Administration of IL-12 to mice fed OVA prevented apoptosis of OVA-specific CD4+ T cells in the liver. Moreover, adoptive transfer of hepatic CD11c+ cells from mice fed OVA together with OVA-specific CD4+ T cells led to development of Th2 cells as well as apoptosis of the transferred CD4+ T cells in the lymph nodes of the recipient mice on immunization with OVA. In conclusion, presentation of an ingested Ag by hepatic CD11c+ cells selects Th2 cells resistant to apoptosis in the liver, which is mediated in part by down-regulation of IL-12 secretion by the former cells. (HEPATOLOGY 2003;38:403-412.)

Liver Failure and Liver Disease

Cytokines and NASH: A pilot study of the effects of lifestyle modification and vitamin E (*Human Study*)
Marcelo Kugelmas, Daniell B. Hill, Beverly Vivian, Luis Marsano, Craig J. McClain
There are few data evaluating plasma and/or peripheral blood monocyte cytokine concentrations/production or attempts to manipulate proinflammatory cytokines in nonalcoholic steatohepatitis (NASH). A pilot project in a general clinical research center evaluated the effects of a step 1 American Heart Association diet plus aerobic exercise with or without 800 IU of vitamin E daily on cytokine profiles and liver enzyme levels in 16 patients with biopsy-proven NASH. Biochemical assessment of liver function, lipid profiles, and body mass index significantly improved during the first 6 weeks of therapy and remained stable during the following 6 weeks. Plasma hyaluronic acid (HA) concentrations decreased in parallel with weight loss. Plasma tumor necrosis factor (TNF) concentrations were significantly elevated in patients with NASH and similar to patients with stable alcoholic cirrhosis but not as elevated as in patients with acute alcoholic steatohepatitis (AH). Although plasma TNF, interleukin 8 (IL-8), and IL-6 concentrations were all significantly elevated compared with control values, only plasma IL-6 levels significantly decreased with therapy. Peripheral blood monocyte TNF, IL-8, and IL-6 production was significantly elevated in patients with NASH but did not significantly decrease. Independent effects of vitamin E were not observed in this small sample. In conclusion, patients with NASH have dysregulated cytokine metabolism similar to, but less pronounced than abnormalities documented in AH. Cytokine values generally did not decrease significantly with weight loss with or without vitamin E over the duration of the study. Lifestyle modifications (low-fat diet and exercise) were associated with improvement in liver enzymes, cholesterol, and plasma HA levels in patients with NASH, whereas the level of vitamin E supplementation used in this short-term pilot study provided no apparent added benefit. (HEPATOLOGY 2003;38:413-419.)

Long-term outcomes of cirrhosis in nonalcoholic steatohepatitis compared with hepatitis C (*Human Study*)
Jason M. Hui, James G. Kench, Shivakumar Chitturi, Archana Sud, Geoffrey C. Farrell, Karen Byth, Pauline Hall, Mahbub Khan, Jacob George
Data on the long-term outcome of nonalcoholic steatohepatitis (NASH)-associated cirrhosis are few, and most reports describe cases of cryptogenic cirrhosis associated with risk factors for NASH but without histologic definition. In this prospective cohort study, we describe the long-term morbidity and mortality of 23 patients with NASH-associated cirrhosis defined by strict clinicopathologic criteria. Outcomes were compared with 46 age- and gender-matched patients with cirrhosis from chronic hepatitis C virus (HCV) infection: 23 untreated and 23 nonresponders to antiviral therapy. During follow-up (mean, 84 months; median, 60 months; range, 5-177 months), 9 of the 23 NASH-associated cirrhosis cases developed liver-related morbidity (8 ascites and/or encephalopathy, 1 variceal bleeding). The probability of complication-free survival was 83%, 77%, and 48% at 1, 3, and 10 years, respectively, and the cumulative probability of overall survival was 95%, 90%, and 84% at 1, 3, and 10 years, respectively. Five deaths were from liver failure, 1 from a non-liver-related cause. By multivariate analysis, bilirubin (P = .02) and platelet (P = .04) were independent predictors of complication-free survival; bilirubin (P = .05) was the only predictor for overall survival. After controlling for these factors, there was no difference in complication-free or overall survival between the NASH-cirrhosis cohort and either group of HCV-cirrhosis. However, 8 cases of liver cancer occurred in the HCV-cirrhosis groups compared with none among NASH cases. In conclusion, liver failure is the main cause of morbidity and mortality in NASH-associated cirrhosis. The prognosis is either similar or less severe than HCV-cirrhosis, except that HCC appears less common. (HEPATOLOGY 2003;38:420-427.)

CYP2E1 activity before and after weight loss in morbidly obese subjects with nonalcoholic fatty liver disease (*Human Study*)
Maurice G. Emery, Jeannine M. Fisher, Jenny Y. Chien, Evan D. Kharasch, E. Patchen Dellinger, Kris V. Kowdley, Kenneth E. Thummel
Previous studies suggest that hepatic cytochrome P450 2E1 (CYP2E1) activity is increased in individuals with chronic alcoholism, nonalcoholic steatohepatitis (NASH), and morbid obesity, and may contribute to liver disease. We studied 16 morbidly obese subjects with varying degrees of hepatic steatosis and 16 normal-weight controls. Obese subjects were evaluated at baseline, 6 weeks, and 1 year after gastroplasty, a procedure that leads to weight loss. Hepatic CYP2E1 activity was assessed by determination of the clearance of chlorzoxazone (CLZ), an in vivo CYP2E1-selective probe. Liver biopsy tissue was obtained during surgery for histopathology. Both the total and unbound oral CLZ clearance (Clu/F) was elevated approximately threefold in morbidly obese subjects compared with controls (P < .001). The Clu/F was significantly higher among subjects with steatosis involving >50% of hepatocytes, compared with those with steatosis in 50% of hepatocytes (P = .02). At postoperative week 6 and year 1, the median body mass index (BMI) of subjects who underwent gastroplasty decreased by 11% and 33%, total oral CLZ clearance declined by 16% (P < .01) and 46% (P < .05), and Clu/F decreased by 18% (P < .05) and 35% (P = .16), respectively. Moreover, those subjects with a year 1 BMI <30 kg/m2 exhibited a median Clu/F that was 63% lower (P = .02) than the respective clearance for all other subjects. In conclusion, hepatic CYP2E1 activity is up-regulated in morbidly obese subjects. A positive association between the degree of steatosis and CYP2E1 activity preoperatively and between the extent of obesity and CYP2E1 activity postoperatively, suggests that CYP2E1 induction is related to or caused by hepatic pathology that results from morbid obesity. (HEPATOLOGY 2003;38:428-435.)

Prevention of diet-induced fatty liver in experimental animals by the oral administration of a fatty acid bile acid conjugate (FABAC)
Tuvia Gilat, Alicia Leikin-Frenkel, Ilana Goldiner, Christine Juhel, Huguette Lafont, Diana Gobbi, Fred M. Konikoff
Fatty acid bile acid conjugates (FABACs) are a new family of synthetic molecules designed to solubilize biliary cholesterol. They were shown to prevent and dissolve cholesterol gallstones in inbred C57L/J mice fed a lithogenic, high-fat diet (HFD). In these mice, fatty liver was observed in the controls but not in the FABAC-treated ones. The present study was designed to study the effect of FABAC (arachidyl-amido-cholanoic acid) on diet-induced fatty liver in rats, hamsters, and mice. The fatty liver score (on a scale of 0-4 by light microscopy) was 4.0 in control hamsters and 0.3 in the FABAC-fed hamsters (P < .001). In mice it was 1.5 and 0.4, respectively (P < .01). The lipid/protein ratio in the liver was 1.3 ± 0.44 (mg lipid/mg protein) in control rats and 0.66 ± 0.04 in the FABAC group (P = .001) after 14 days. In hamsters it was 1.41 ± 0.27 and 1.11 ± 0.20, respectively (P = .03), after 21 days. In Imperial Charles River (ICR) mice the ratio was 0.34 ± 0.10 and 0.17 ± 0.07 (P = .03), respectively, after 24 days. Liver fat concentration, measured as mg lipid/g liver tissue, decreased similarly by FABAC feeding. The decrease in liver fat affected mainly the triglyceride levels. FABAC-fed animals gained weight similarly to the controls. Triglyceride absorption was unaffected by FABAC supplementation. In conclusion, oral FABAC therapy prevents/reduces the development of fatty liver in animals consuming a HFD. (HEPATOLOGY 2003;38:436-442.)

Fulminant hepatitis after grand mal seizures: Mechanisms and role of liver transplantation (*Human Study*)
Philippe Ichai, Emmanuel Huguet, Catherine Guettier, Daniel Azoulay, Maria Eugenia Gonzalez, Bernard Fromenty, Pascal Masnou, Faouzi Saliba, Bruno Roche, Fahed Zeitoun, Denis Castaing, Didier Samuel
Fulminant liver failure is a rare complication of grand mal seizures with a high mortality, the prognosis being largely determined by the combination of the hepatic and neurologic insults. The mechanisms of acute liver failure secondary to grand mal epilepsy and the place of liver transplantation in this context are poorly defined and are the subject of this report. A series of 6 such patients is presented. All had a history of chronic primary or post-traumatic epilepsy and presented with acute liver failure shortly after a grand mal fit. Detailed accounts of background, presentation, and management are given and integrated with blood, radiologic, and histologic investigations. Two of the 6 patients survived, 1 making a full recovery and the other with neurologic sequelae. Two patients underwent liver transplantation but died with severe neurologic sequelae despite improving liver function. The remaining 2 patients were considered too unwell to undergo liver transplantation and died in multiple organ failure. Liver histology from needle biopsy and/or native liver explants identified lesions compatible with a combination of steatosis and necrosis. Factor V and transaminase levels may allow early identification of patients in whom liver function is likely to improve spontaneously. In conclusion, the mechanisms of liver failure occurring after grand mal seizures appear multifactorial, including hypoxia, steatosis, and drug-induced components. The neurological prognosis and overall survival of these patients remains poor. (HEPATOLOGY 2003;38:443-451.)

Increased carbon monoxide production in patients with cirrhosis with and without spontaneous bacterial peritonitis (*Human Study*)
Dara De Las Heras, Javier Fernández, Pere Ginès, Andres Cárdenas, Rolando Ortega, Miguel Navasa, Joan Albert Barberá, Blas Calahorra, Mónica Guevara, Ramón Bataller, Wladimiro Jiménez, Vicente Arroyo, Juan Rodés
Carbon monoxide, a product of the heme-oxygenase (HO) pathway, is an important endogenous vasoactive substance. Production of CO has not been assessed in human cirrhosis. The aim of this study was to assess production of CO in patients with cirrhosis with and without spontaneous bacterial peritonitis (SBP). CO concentration in the exhaled air and blood carboxyhemoglobin (COHb) levels, as estimates of total HO activity, were determined in 16 healthy subjects, 32 noninfected cirrhotic patients (20 with ascites), and 19 patients with SBP, all nonsmokers. Noninfected cirrhotic patients had a CO concentration in the exhaled air and COHb levels significantly higher compared with values of healthy subjects (2.3 ± 0.2 ppm vs. 0.7 ± 0.1 ppm and 1.0% ± 0.1% vs. 0.6% ± 0.1%, respectively; P < .05 for both). Patients with ascites had the highest values. Both CO concentration in the exhaled air and COHb levels were very high in patients with SBP (5.6 ± 0.6 ppm and 1.9% ± 0.2%; P < .01 vs. the other 2 groups) and decreased slowly after resolution of the infection, reaching values similar to those of noninfected patients 1 month after SBP. In patients with SBP, there was a significantly direct correlation between CO and plasma renin activity (PRA) (r = 0.71, P < .001). In conclusion, these results support the existence of increased CO production in human cirrhosis, which further increases in the setting of SBP. Increased CO production may participate in the disturbance of circulatory function that occurs during severe bacterial infections in cirrhosis. (HEPATHOLOGY 2003;38:452-459.)

Validation and refinement of survival models for liver retransplantation (*Human Study*)
Hugo R. Rosen, Martin Prieto, Teresa Casanovas-Taltavull, Valentin Cuervas-Mons, Olaf Guckelberger, Paolo Muiesan, Russell W. Strong, Wolf O. Bechstein, John O'Grady, Atif Zaman, Benjamin Chan, Joaquin Berenguer, Roger Williams, Nigel Heaton, Peter Neuhaus
Orthotopic liver retransplantation (re-OLT) is highly controversial. The objectives of this study were to determine the validity of a recently developed United Network for Organ Sharing (UNOS) multivariate model using an independent cohort of patients undergoing re-OLT outside the United States, to determine whether incorporation of other variables that were incomplete in the UNOS registry would provide additional prognostic information, to develop new models combining data sets from both cohorts, and to evaluate the validity of the model for end-stage liver disease (MELD) in patients undergoing re-OLT. Two hundred eighty-one adult patients undergoing re-OLT (between 1986 and 1999) at 6 foreign transplant centers comprised the validation cohort. We found good agreement between actual survival and predicted survival in the validation cohort; 1-year patient survival rates in the low-, intermediate-, and high-risk groups (as assigned by the original UNOS model) were 72%, 68%, and 36%, respectively (P < .0001). In the patients for whom the international normalized ratio (INR) of prothrombin time was available, MELD correlated with outcome following re-OLT; the median MELD scores for patients surviving at least 90 days compared with those dying within 90 days were 20.75 versus 25.9, respectively (P = .004). Utilizing both patient cohorts (n = 979), a new model, based on recipient age, total serum bilirubin, creatinine, and interval to re-OLT, was constructed (whole model 2 = 105, P < .0001). Using the c-statistic with 30-day, 90-day, 1-year, and 3-year mortality as the end points, the area under the receiver operating characteristic (ROC) curves for 4 different models were compared. In conclusion, prospective validation and use of these models as adjuncts to clinical decision making in the management of patients being considered for re-OLT are warranted. (HEPATOLOGY 2003;38:460-469.)

Cellular retinol-binding protein-1 in hepatocellular carcinoma correlates with -catenin, Ki-67 index, and patient survival (*Human Study*)
Annette Schmitt-Gräff, Viktoria Ertelt, Hans-P. Allgaier, Konrad Koelble, Manfred Olschewski, Roland Nitschke, Marie-L. Bochaton-Piallat, Giulio Gabbiani, Hubert E. Blum
The cellular retinol-binding protein-1 (CRBP-1) plays a key role in the esterification and intercellular transfer of retinol. By in situ hybridization, immunohistochemistry, and confocal laser scanning microscopy (CLSM), we show that, in normal liver, CRBP-1 is strongly expressed in the cytoplasm of hepatic stellate cells (HSCs) and myofibroblasts (MFs) with only low CRBP-1 levels in hepatocytes. By contrast, in 196 hepatocellular carcinoma (HCC) specimens CRBP-1 expression in MFs was down-regulated in 83%. Patients with high CRBP-1 expression in MFs had a significantly higher 2-year survival as compared with patients with low CRBP-1 expression (52% vs. 29%, respectively; P = .034). An aberrant nuclear CRBP-1 accumulation resulting from cytoplasmic invagination was found in 29% of HCCs. Nuclear CRBP-1 staining correlated positively with a favorable tumor stage (Okuda stage I; P = .01) and negatively with the Ki-67+ proliferation fraction (PF). A Ki-67+ PF of 10% was associated with a lower 2-year survival probability as compared with patients with a Ki-67+ PF of <10% (12% vs. 40%, respectively; P = .015). Prognosis did not correlate with the nuclear -catenin expression. There was, however, a close correlation between nuclear CRBP-1 inclusions and nuclear -catenin staining in HCCs (P = .008), suggesting a cross talk between CRBP-1 and the Wnt/wingless signal transduction pathway. In conclusion, our findings demonstrate that CRBP-1 detection may be useful for the discrimination between nonneoplastic and neoplastic liver cells and suggest that modulation of CRBP-1 expression in HCCs contributes to tumor growth and progression via retinoid-mediated signaling and disruption of cellular vitamin A homeostasis. (HEPATOLOGY 2003;38:470-480.)

Viral Hepatitis

Biochemical surrogate markers of liver fibrosis and activity in a randomized trial of peginterferon alfa-2b and ribavirin (*Human Study*)
Thierry Poynard, John McHutchison, Michael Manns, Rob P. Myers, Janice Albrecht
Liver fibrosis and activity indexes were validated in patients infected by hepatitis C virus (HCV) nontreated and treated by interferon. The aim was to validate their usefulness as surrogate markers of histologic features using the data of a randomized trial of combination peginterferon alfa-2b and ribavirin. Three hundred fifty-two patients who had had 2 interpretable liver biopsies and stored serum sample before and after treatment were selected. Two hundred eight patients received peginterferon alfa-2b 1.5 mcg per kg and ribavirin and 144 patients interferon alfa-2b 3 MU three times a week and ribavirin for 48 weeks. A fibrosis and an activity index combining 5 and 6 biochemical markers were assessed at baseline and at end of follow-up (24 weeks after treatment). The biochemical markers have significant predictive values both for the diagnosis of fibrosis and for activity. For the diagnosis of bridging fibrosis and/or moderate necroinflammatory activity, the area under the receiver operating characteristics curve of the activity index was 0.76 ± 0.03 at baseline and 0.82 ± 0.02 at end of follow-up. A cutoff of activity index at 0.30 (range, 0.00-1.00) had 90% sensitivity and 88% positive predictive value for the diagnosis of bridging fibrosis or moderate necroinflammatory activity. Sensitivity analyses with biopsy specimens of size greater than 15 mm suggest that a part of discordances between biochemical markers and histology were due to biopsy specimen sampling error. In conclusion, these biochemical markers of fibrosis and activity could be used as surrogate markers for liver biopsy in patients with chronic hepatitis C, both for the initial evaluation and for follow-up. (HEPATOLOGY 2003;38:481-492.)

Favorable prognosis of chronic hepatitis C after interferon therapy by long-term cohort study (*Human Study*)
Fumio Imazeki, Osamu Yokosuka, Kenichi Fukai, Hiromitsu Saisho
The prognosis of patients with chronic hepatitis C after interferon (IFN) therapy is still poorly defined. The present study evaluated the effect of IFN therapy on survival in a cohort of such patients. The study included 459 patients with biopsy-proven C-viral chronic liver disease who were followed for 8.2 ± 2.9 years (range, 7-183 months). Survival status was examined by medical records or direct questionnaires. Fifteen (14%) of 104 IFN-untreated patients and 33 (9%) of 355 patients treated with IFN died during follow-up. Among the treated patients, 4 (3%) of 116 with sustained virologic response and 29 (12%) of 239 without sustained virologic response died. Liver-related death was shown in 32 (67%) patients, and hepatocellular carcinoma (HCC) caused 25 (52%) of the 48 deaths. Multivariate Cox proportional regression analysis revealed that IFN treatment decreased the risk ratio for overall death to 0.521 (confidence interval [CI]: 0.263-1.034) and for liver-related death to 0.208 (CI: 0.088-0.495) compared with untreated patients, and sustained virologic response showed a decrease in the risk ratio for overall death to 0.219 (CI: 0.068-0.710) and for liver-related death to 0.030 (CI: 0.003-0.267). IFN treatment showed no association with liver-unrelated death. Furthermore, the standardized mortality ratios for all causes of death and liver-related death were reduced in IFN-treated patients compared with untreated patients (1.4 vs. 2.0 for total death and 7.9 vs. 19.7 for liver-related death). In conclusion, the present data suggest that IFN therapy has a long-term clinical benefit for patients with chronic hepatitis C patients by reducing liver-related death, especially in patients with sustained virologic response. (HEPATOLOGY 2003;38:493-502.)

A potent and specific morpholino antisense inhibitor of hepatitis C translation in mice
Anton P. McCaffrey, Leonard Meuse, Mobin Karimi, Christopher H. Contag, Mark A. Kay
Hepatitis C virus (HCV) is an RNA virus infecting one in every 40 people worldwide. Current treatments are ineffective and HCV is the leading cause of liver failure leading to transplantation in the United States and Europe. Translational control of HCV is a prime therapeutic target. We assessed the inhibitory potential of morpholino phosphoramidate antisense oligonucleotides (morpholinos) on HCV translation by codelivering them with reporter plasmids expressing firefly luciferase under the translational control of the HCV internal ribosome entry site (IRES) into the livers of mice. Real-time imaging of HCV IRES luciferase reporter messenger RNA (mRNA) translation in living mice showed that a 20-mer complementary to nucleotides 345-365 of the IRES inhibited translation by greater than 95% for at least 6 days and showed mismatch specificity. No significant nonspecific inhibition of a cap-dependent luciferase or encephalomyocarditis virus (EMCV) IRES luciferase reporter translation was observed. Inhibition by the 20-mer morpholino was dose dependent, with 1 nmol/mouse giving the highest inhibition. In conclusion, morpholino antisense oligonucleotides are potent inhibitors of HCV IRES translation in a preclinical mouse model; morpholinos have potential as molecular therapeutics for treating HCV and other viral infections. The in vivo model described is a broadly applicable, straightforward, and rapid readout for inhibitor efficacy. As such, it will greatly facilitate the development of novel therapeutic strategies for viral hepatitis. Notably, the level of antisense inhibition observed in this in vivo model is similar to the maximal inhibition we have obtained previously with RNA interference in mice. (HEPATOLOGY 2003;38:503-508.)

Dynamics of alanine aminotransferase during hepatitis C virus treatment (*Human Study*)
Ruy M. Ribeiro, Jennifer Layden-Almer, Kimberly A. Powers, Thomas J. Layden, Alan S. Perelson
Studies of the kinetics of hepatitis C virus (HCV) decline during interferon (IFN)-based therapy have led to insights into treatment efficacy. However, the kinetics of serum alanine aminotransferase (ALT), an enzyme used as a surrogate of liver damage, have not been closely monitored, and it is not known if they correlate with those of HCV RNA. Here we describe the associations between ALT and HCV dynamics. We analyzed 35 patients treated daily with 10 mIU IFN-2b with or without ribavarin for 28 days followed by standard IFN/ribavirin therapy. Patients exhibited 4 patterns of ALT change: (1) exponential decay of ALT, (2) transient increase in ALT followed by a decrease to pretreatment or normal levels, (3) increase in ALT to a new level, and (4) no significant change. By simultaneously modeling HCV and ALT dynamics, we successfully fit the observed changes. We found ALT decays with t1/2 = 12.7 hours. The transient increase in ALT observed in some patients suggested a mild hepatotoxic effect of IFN. However, patients with a smaller initial ALT increase achieved higher rates of viral negativity by week 72 (P = .02). The week-4 ALT decline correlated with the HCV log drop (P = .006) and the efficacy of therapy (P = .025). In conclusion, our results suggest the use of ALT as a surrogate marker for treatment effect in patients with elevated ALT. (HEPATOLOGY 2003;38:509-517.)

A simple noninvasive index can predict both significant fibrosis and cirrhosis in patients with chronic hepatitis C (*Human Study*)
Chun-Tao Wai, Joel K. Greenson, Robert J. Fontana, John D. Kalbfleisch, Jorge A. Marrero, Hari S. Conjeevaram, Anna S.-F. Lok
Information on the stage of liver fibrosis is essential in managing chronic hepatitis C (CHC) patients. However, most models for predicting liver fibrosis are complicated and separate formulas are needed to predict significant fibrosis and cirrhosis. The aim of our study was to construct one simple model consisting of routine laboratory data to predict both significant fibrosis and cirrhosis among patients with CHC. Consecutive treatment-naive CHC patients who underwent liver biopsy over a 25-month period were divided into 2 sequential cohorts: training set (n = 192) and validation set (n = 78). The best model for predicting both significant fibrosis (Ishak score 3) and cirrhosis in the training set included platelets, aspartate aminotransferase (AST), and alkaline phosphatase with an area under ROC curves (AUC) of 0.82 and 0.92, respectively. A novel index, AST to platelet ratio index (APRI), was developed to amplify the opposing effects of liver fibrosis on AST and platelet count. The AUC of APRI for predicting significant fibrosis and cirrhosis were 0.80 and 0.89, respectively, in the training set. Using optimized cut-off values, significant fibrosis could be predicted accurately in 51% and cirrhosis in 81% of patients. The AUC of APRI for predicting significant fibrosis and cirrhosis in the validation set were 0.88 and 0.94, respectively. In conclusion, our study showed that a simple index using readily available laboratory results can identify CHC patients with significant fibrosis and cirrhosis with a high degree of accuracy. Application of this index may decrease the need for staging liver biopsy specimens among CHC patients. (HEPATOLOGY 2003;38:518-526.)

GASTROENTEROLOGY

Table of Contents for August 2003 · Volume 125 · Number 2

Rapid Communications

Resistance to adefovir dipivoxil therapy associated with the selection of a novel mutation in the HBV polymerase
Peter Angus, Rhys Vaughan, Shelly Xiong, Huiling Yang, William Delaney, Craig Gibbs, Carol Brosgart, Danielle Colledge, Rosalind Edwards, Anna Ayres, Angeline Bartholomeusz, Stephen Locarnini
Background & aims: Adefovir dipivoxil effectively inhibits both hepatitis B virus (HBV) replication and disease activity in patients with chronic hepatitis B. Resistance to treatment was not observed in 2 recent large placebo-controlled 48-week studies with this drug. The aim of this study was to characterize adefovir resistance in a patient who developed clinical and virologic evidence of breakthrough during a 96-week course of treatment.

Methods: HBV DNA was PCR amplified and sequenced. Phenotypic studies used patient-derived HBV as well as specific mutations created by site-directed mutagenesis of a HBV/baculovirus recombinant.

Results: Following the commencement of treatment with adefovir dipivoxil, the patient initially responded with a 2.4 log10 decrease in serum HBV DNA and normalization of alanine aminotransaminase levels by week 16. During the second year of treatment, however, serum HBV DNA rose progressively, eventually returning to near-pretreatment levels. This increase in viral replication was associated with a marked increase in alanine aminotransferase and mild changes in bilirubin, albumin, and prothrombin time. Comparison of pretreatment and posttreatment HBV DNA by polymerase chain reaction sequencing identified a novel asparagine to threonine mutation at residue rt236 in domain D of the HBV polymerase. In vitro testing of a laboratory strain encoding the rtN236T mutation and testing of patient-derived virus confirmed that the rtN236T substitution caused a marked reduction in susceptibility to adefovir.

Conclusions: The development of this novel mutation in the HBV polymerase confers resistance to adefovir dipivoxil. The patient responded to subsequent lamivudine therapy, achieving normalization of alanine aminotransferase and a significant decrease in serum HBV DNA.

6-thioguanine can cause serious liver injury in inflammatory bowel disease patients
Marla C. Dubinsky, Eric A. Vasiliauskas, Hardeep Singh, Maria T. Abreu, Kostas A. Papadakis, Tram Tran, Paul Martin, John M. Vierling, Stephen A. Geller, Stephan R. Targan, Fred F. Poordad
Background & aims: Thioguanine (6-TG) has been studied as an alternative thiopurine in inflammatory bowel disease (IBD). Short-term safety and efficacy data were favorable. Experience with 6-TG in patients with acute lymphoblastic leukemia raised long-term safety concerns when implicated in nodular regenerative hyperplasia (NRH) of the liver and portal hypertension. The aim of this study was to describe the association between 6-TG and NRH in IBD.

Methods: Liver chemistries and complete blood counts were monitored, and patients were encouraged to undergo liver biopsy. Clinical data were collected by chart review, and associations were tested by univariate and multivariable analyses. Patients were classified based on the presence (group 1) or absence (group 2) of laboratory abnormalities.

Results: Laboratory abnormalities occurred in 29 of 111 patients (26%). Elevations of liver enzymes and a decrease in platelet counts (<200,000) were most commonly observed. Male gender (odds ratio, 2.9; 95% CI, 1.17.3; P < 0.03) and preferential 6-methylmercaptopurine production on 6-mercaptopurine/azathioprine (odds ratio, 3.0; 95% CI, 1.27.4; P < 0.04) were independently associated with laboratory abnormalities. No association was seen with duration of 6-TG treatment, cumulative dose, or 6-TG nucleotide levels. The median increase in alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase levels was 39, 30, and 75 U/L, respectively, in group 1, and the median decrease in platelet count was 115,000 in group 1 versus 7000 in group 2 (P < 0.001). NRH occurred in 76% of patients undergoing biopsy in group 1 and 33% in group 2.

Conclusions: NRH is a common finding in 6-TG-treated patients with IBD. The progression or reversibility of NRH remains unknown. Our findings suggest that 6-TG should not be considered as therapy for patients with IBD.

Clinical-alimentary Tract

Virtual colonoscopy using oral contrast compared with colonoscopy for the detection of patients with colorectal polyps
Benoit C. Pineau, Electra D. Paskett, G. John Chen, Mark A. Espeland, Kim Phillips, James P. Han, Claudia Mikulaninec, David J. Vining
Background & Aims: Virtual colonoscopy using abdominal spiral computed tomography scanning allows total colonic evaluation with minimal invasiveness. Two-dimensional images and selective 3-dimensional images of the colon are used to detect colorectal lesions. This trial used conventional colonoscopy with segmental unblinding to determine the ability of virtual colonoscopy to identify patients with colorectal lesions who need conventional colonoscopy.

Methods: We studied 205 patients with virtual colonoscopy using oral iodinated contrast preceding conventional colonoscopy. Colonic lavage was achieved with an oral sodium phosphosoda preparation and colonic distention with a carbon dioxide electronic insufflator.

Results: The overall sensitivity and specificity of virtual colonoscopy in identifying patients with colorectal lesions was 61.8% and 70.7%, respectively. Virtual colonoscopy was more accurate in identifying patients with lesions 6 mm (sensitivity 84.4% and specificity 83.1%) and those with lesions 10 mm (sensitivity 90% and specificity 94.6%). The negative predictive value of virtual colonoscopy was 95% for a 6-mm cutoff size and 98.9% for a 10-mm cutoff. Using a 10-mm cutoff, virtual colonoscopy precludes the need for conventional colonoscopy in 86% of patients with a 1% false-negative rate (68% with a 3.4% false-negative rate when using a 6-mm cutoff).

Conclusions: Virtual colonoscopy has a high sensitivity and specificity for detecting patients with significant colorectal lesions. Its high negative predictive value may help reduce the number of negative screening colonoscopies. Further studies are needed to determine what lesion cutoff size is clinically acceptable and the appropriate interval time for repeat virtual colonoscopy when it detects lesions below this cutoff size.

Prospective blinded evaluation of computed tomographic colonography for screen detection of colorectal polyps
C. Daniel Johnson, William S. Harmsen, Lynn A. Wilson, Robert L. Maccarty, Timothy J. Welch, Duane M. Ilstrup, David A. Ahlquist
Background & Aims: This study used a low lesion prevalence population reflective of the screening setting to estimate the sensitivity and specificity of computerized tomographic (CT) colonography for detection of colorectal polyps.

Methods: This prospective, blinded study comprised 703 asymptomatic persons at higher-than-average risk for colorectal cancer who underwent CT colonography followed by same-day colonoscopy. Two of 3 experienced readers interpreted each CT colonography examination.

Results: Overall lesion prevalence for adenomas 1 cm in diameter was 5%. Seventy percent of all lesions were proximal to the descending colon. With colonoscopy serving as the gold standard, CT colonography detected 34%, 32%, 73%, and 63% of the 59 polyps 1 cm for readers 1, 2, 3, and double-reading, respectively; and 35%, 29%, 57%, and 54% of the 94 polyps 59 mm for readers 1, 2, 3, and double-reading, respectively. Specificity for CT colonography ranged from 95% to 98% and 86% to 95% for >1 cm and 59-mm polyps, respectively. Interobserver variability was high for CT colonography with statistic values ranging from 0.67 to 0.89.

Conclusions: In a low prevalence setting, polyp detection rates at CT colonography are well below those at colonoscopy. These rates are less than previous reports based largely on high lesion prevalence cohorts. High interobserver variability warrants further investigation but may be due to the low prevalence of polyps in this cohort and the high impact on total sensitivity of each missed polyp. Specificity, based on large numbers, is high and exhibits excellent agreement among observers.

Corticosteroids and immunomodulators: Postoperative infectious complication risk in inflammatory bowel disease patients
Faten N. Aberra, James D. Lewis, David Hass, John L. Rombeau, Benjamin Osborne, Gary R. Lichtenstein
Background & Aims: Many patients with inflammatory bowel disease receive corticosteroids and 6-mercaptopurine/azathioprine during elective bowel surgery. We investigated the postoperative infection risk for patients undergoing elective bowel surgery who were receiving corticosteroids and/or 6-mercaptopurine/azathioprine before surgery compared with patients not receiving these medications.

Methods: A retrospective cohort study was conducted on 159 patients with inflammatory bowel disease who underwent elective bowel surgery. There were 56 patients receiving corticosteroids alone, 52 patients receiving 6-mercaptopurine/azathioprine alone or with corticosteroids, and 51 patients receiving neither corticosteroids nor 6-mercaptopurine/azathioprine. Postoperative infectious complications to time of discharge were categorized into major and minor complications.

Results: Patients receiving corticosteroids had an adjusted odds ratio for any and major infectious complications of 3.69 (95% confidence interval [CI], 1.2410.97) and 5.54 (95% CI, 1.1227.26), respectively. The adjusted odds ratio for patients receiving 6-mercaptopurine/azathioprine for any and major infectious complications was 1.68 (95% CI, 0.654.27) and 1.20 (95% CI, 0.373.94), respectively.

Conclusions: Preoperative use of corticosteroids in patients with inflammatory bowel disease who are undergoing elective bowel surgery is associated with an increased risk of postoperative infectious complications. 6-Mercaptopurine/azathioprine alone and the addition of 6-mercaptopurine/azathioprine for patients receiving corticosteroids was not found to significantly increase the risk of postoperative infectious complications.

Kaposi of kaposi's sarcoma
William S. Haubrich, M.D.
Background & Aims: Epidemiologic and experimental studies have suggested that aspirin intake reduces the risk for colorectal carcinogenesis. However, the available data are not sufficient to serve as the basis for firm recommendations.

Methods: We randomly assigned 272 patients with a history of colorectal adenomas (at least one more than 5 mm in diameter, or more than 3) to daily lysine acetylsalicylate (160 or 300 mg/day) or placebo for 4 years. The primary end points were adenoma recurrence after 1 and 4 years. These results are those of the year 1 colonoscopy.

Results: Among the 238 patients who completed the year 1 colonoscopy, at least one adenoma was observed in 38 patients of the 126 (30%) in the aspirin group and in 46 of the 112 (41%) in the placebo group; relative risk was 0.73 (95% confidence interval [CI]: 0.521.04; P = 0.08). At least one adenoma of more than 5 mm diameter was observed in 13 patients (10%) in the aspirin group and 26 (23%) in the placebo group (P = 0.01). The corresponding numbers for adenomas more than 10 mm in diameter were one (1%) and 7 (6%) (P = 0.05). Stepwise regression showed that independent factors associated with lower adenoma recurrence are aspirin treatment (adenoma >5 mm, P = 0.01), absence of personal history of adenoma before the entry colonoscopy (P = 0.01), and initial adenomatous polyp burden less than 10 mm (P = 0.001).

Conclusions: Daily soluble aspirin is associated with a reduction in the risk for recurrent adenomas found at colonoscopy 1 year after starting treatment.

Daily soluble aspirin and prevention of colorectal adenoma recurrence: One-year results of the APACC trial
Robert Benamouzig, Jacques Deyra, Antoine Martin, Bernard Girard, Eric Jullian, Benoit Piednoir, Daniel Couturier, Thierry Coste, Julian Little, Stanislas Chaussade
Background & Aims: Epidemiologic and experimental studies have suggested that aspirin intake reduces the risk for colorectal carcinogenesis. However, the available data are not sufficient to serve as the basis for firm recommendations.

Conclusions: Daily soluble aspirin is associated with a reduction in the risk for recurrent adenomas found at colonoscopy 1 year after starting treatment.

Intestinal T-cell responses to high-molecular-weight glutenins in celiac disease
ØYvind Molberg, Nina Solheim flÆte, Tore Jensen, Knut E. A. Lundin, Helene Arentz-Hansen, Olin D. Anderson, Anne Kjersti Uhlen, Ludvig M. Sollid
Background & Aims: The chronic, small intestinal inflammation that defines celiac disease is initiated by a HLA-DQ2 restricted T-cell response to ingested gluten peptides after their in vivo deamidation by tissue transglutaminase (TG2). To date, celiac disease can only be treated by a lifelong abstinence from foods that contain wheat, rye, or barley; better therapeutic options are hence needed. An attractive target would be to identify nontoxic wheat cultivars or components thereof with intact baking qualities. Because these qualities are mainly determined by the high molecular weight (HMW) glutenin proteins of gluten, it is critical to know if these proteins are toxic or, more specifically, if they will trigger the activation of T cells in the celiac lesion.

Methods: Different, highly purified HMW glutenins were isolated from wheat cultivars or expressed as recombinant proteins. The proteins were first tested for recognition by a large panel of gluten-specific T-cell lines established from celiac lesions and then applied during ex vivo challenges of celiac biopsies to allow for a direct identification of HMW specific T cells.

Results: Intestinal T-cell responses to TG2-deamidated HMW glutenins but not the corresponding native proteins were detectable in 9 of the 22 adult and childhood celiac disease patients tested.

Conclusions: T cells within celiac lesions frequently recognize deamidated HMW glutenin proteins. This finding questions the possibility of implementing these proteins in novel food items destined to be nontoxic for celiac disease patients.

Human small-intestinal epithelium contains functional natural killer lymphocytes
Francisco León, Ernesto Roldán, Laura Sanchez, Cristina Camarero, Alfredo Bootello, Garbiñe Roy
Background & Aims: CD3 non-T lymphocytes constitute the second most abundant lymphoid subset in the human small-bowel epithelium, and these CD3 intraepithelial lymphocytes are virtually absent in active celiac disease. Phenotypically, they resemble natural killer cells and have been termed natural killer-like intraepithelial lymphocytes. Because of the limited availability of appropriate human samples, functional studies have not yet been reported, and it is not yet clear whether these are true natural killer cells.

Methods: We used magnetic bead-based purification and flow cytometry to study several aspects of normal human small-bowel natural killer-like intraepithelial lymphocytes: intracellular cytokine content (basally and after activation); ability to lyse natural killer-sensitive K562 target cells; and expression of perforins, Fas ligand, and other functional markers.

Results: CD3 intraepithelial lymphocytes cultured in interleukin-2 showed a higher lymphokine-activated killer activity than CD3+ intraepithelial lymphocytes (48%83% lysis exerted by CD3 intraepithelial lymphocytes at an effector-target cell ratio of 2:1 vs. 8%18% by CD3+ intraepithelial lymphocytes). Perforin content correlated with this lytic potential (75% ± 4% in CD3 vs. 5% ± 4% in CD3+ intraepithelial lymphocytes). Both CD3 and CD3+ cells displayed a type I cytokine profile (interferon- > tumor necrosis factor- > interleukin-2; undetectable interleukin-4 and interleukin-10). In addition to their activated phenotype, subsets of natural killer-like intraepithelial lymphocytes expressed CD8 and intracellular CD3 chain, showing the existence of heterogeneity within this cell lineage.

Conclusions: This is the first demonstration of functional natural killer cells within the human gut epithelium. These cells might play an important role in innate mucosal immunity (host defense and tumor surveillance) and tolerance.

Childhood constipation: Longitudinal follow-up beyond puberty
Rijk van Ginkel, Johannes B. Reitsma, Hans A. Büller, Michiel P. van wijk, Jan A.J.M. Taminiau, Marc A. Benninga
Background & Aims: Sparse data exist about the prognosis of childhood constipation and its possible persistence into adulthood.

Methods: A total of 418 constipated patients older than 5 years at intake (279 boys; median age, 8.0 yr) participated in studies evaluating therapeutic modalities for constipation. All children subsequently were enrolled in this follow-up study with prospective data collection after an initial 6-week intensive treatment protocol, at 6 months, and thereafter annually, using a standardized questionnaire.

Results: Follow-up was obtained in more than 95% of the children. The median duration of the follow-up period was 5 years (range, 18 yr). The cumulative percentage of children who were treated successfully during follow-up was 60% at 1 year, increasing to 80% at 8 years. Successful treatment was more frequent in children without encopresis and in children with an age of onset of defecation difficulty older than 4 years. In the group of children treated successfully, 50% experienced at least one period of relapse. Relapses occurred more frequently in boys than in girls (relative risk 1.73; 95% confidence interval, 1.152.62). In the subset of children aged 16 years and older, constipation still was present in 30%.

Conclusions: After intensive initial medical and behavioral treatment, 60% of all children referred to a tertiary medical center for chronic constipation were treated successfully at 1 year of follow-up. One third of the children followed-up beyond puberty continued to have severe complaints of constipation. This finding contradicts the general belief that childhood constipation gradually disappears before or during puberty.

A proinflammatory genetic profile increases the risk for chronic atrophic gastritis and gastric carcinoma
José Carlos Machado, Céu Figueiredo, Paulo Canedo, Paul Pharoah, Ralph Carvalho, Sérgio Nabais, Catarina Castro Alves, Maria Luisa Campos, Leen-jan Van Doorn, Carlos Caldas, Raquel Seruca, Fátima Carneiro, Manuel Sobrinho-Simões
Background & Aims: Pro-inflammatory polymorphisms within the genes interleukin (IL)-1B and IL-1RN are associated with risk for gastric carcinoma (GC) in Helicobacter pylori-infected individuals. We aimed to determine the association between variation of the tumor necrosis factor (TNF)- gene and the risk for chronic atrophic gastritis (CAG) and GC. We also investigated the extent to which the combined effect of proinflammatory genetic polymorphisms (IL-1B, IL-1RN, and TNF-), and the combined effect of TNF- and bacterial genotypes each influence such a risk.

Methods: In a case-control study including 306 controls, 221 individuals with chronic gastritis, and 287 GC patients, the TNF--308 and IL-1B-511 bi-allelic polymorphisms, the IL-1RN variable number of tandem repeats (VNTR), and the H. pylori genes vacA (s and m regions) and cagA were genotyped.

Results: We found that carriers of the TNF--308*A allele are at increased risk for GC development with an odds ratio (OR) of 1.9 (95% confidence interval [CI], 1.32.7). For both CAG and GC, the odds of developing disease increased with the number of high-risk genotypes. Individuals carrying high-risk genotypes at the 3 loci are at increased risk for CAG and GC with an OR of 5.8 (95% CI, 1.131.0) and 9.7 (95% CI, 2.636.0), respectively. The risk for GC was not affected significantly by the combination of bacterial and TNF--308 genotypes.

Conclusions: These findings show that a proinflammatory polymorphism in the TNF- gene is associated with increased risk for GC, and that it is possible to define a specific genetic profile associated with highest risk for CAG and GC.

Rates of dyspepsia one year after Helicobacter pylori screening and eradication in a Danish population
Mette Wildner-Christensen, Jane MØller Hansen, Ove B. Schaffalitzky De Muckadell
Background & Aims: Helicobacter pylori (Hp) is strongly correlated with peptic ulcer and is a risk factor for gastric cancer. The aim of this study was to assess whether screening and eradication of Hp in a general population would reduce the prevalence of dyspepsia and the incidence of peptic ulcer and thus save health care resources and improve quality of life.

Methods: Twenty thousand individuals aged 40 to 65 years were randomized to screening and eradication for Hp or to the control group. Hp status was assessed by a whole blood Hp test, a positive result confirmed by a 13C-urea breath test. Hp-positive individuals were offered Hp eradication therapy. The prevalence of dyspepsia and the quality of life were assessed through a mailed questionnaire. Information on the use of endoscopies and the use of prescription medication was obtained from registers.

Results: The response rate was 62.6%. The prevalence of Hp was 17.5%. The Hp eradication rate was 95%. In the intervention group, the prevalence of dyspepsia decreased from 24.3% at inclusion to 20.5% at 1-year follow-up. The reduction was similar in Hp-negative and Hp-positive persons. In the control group, dyspepsia increased from 20.3% to 21.5%. Gastroesophageal reflux symptoms improved slightly in Hp-eradicated participants. Except for a decreased consultation rate for dyspepsia, there were no visible savings in health care.

Conclusions: Dyspepsia was modestly reduced after the screening and treatment procedure, and the result was not sufficiently extensive to have an effect on the use of health care or to improve quality of life.

Tacrolimus for the treatment of fistulas in patients with crohn's disease: A randomized, placebo-controlled trial
William J. Sandborn, Daniel H. Present, Kim L. Isaacs, Douglas C. Wolf, Eugene Greenberg, Stephen B. Hanauer, Brian G. Feagan, Lloyd Mayer, Therese Johnson, Joseph Galanko, Christopher Martin, Robert S. Sandler
Background & Aims: This study determined the effectiveness of tacrolimus for the treatment of Crohn's disease fistulas.

Methods: The study was a randomized, double-blind, placebo-controlled, multicenter clinical trial. Forty-eight patients with Crohn's disease and draining perianal or enterocutaneous fistulas were randomized to treatment with oral tacrolimus 0.2 mg · kg1 · day1 or placebo for 10 weeks. The primary outcome measure was fistula improvement as defined by closure of 50% of particular fistulas that were draining at baseline and maintenance of that closure for at least 4 weeks. A secondary outcome measure was fistula remission as defined by closure of all fistulas and maintenance of that closure for at least 4 weeks.

Results: Forty-three percent of tacrolimus-treated patients had fistula improvement compared with 8% of placebo-treated patients (P = 0.004). Ten percent of tacrolimus-treated patients had fistula remission compared with 8% of placebo-treated patients (P = 0.86). Adverse events significantly associated with tacrolimus, including headache, increased serum creatinine level, insomnia, leg cramps, paresthesias, and tremor, were managed with dose reduction.

Conclusions: Oral tacrolimus 0.2 mg · kg1 · day1 is effective for fistula improvement, but not fistula remission, in patients with perianal Crohn's disease. Adverse events associated with tacrolimus can be managed by dose reduction. Lower doses of tacrolimus should be evaluated.

Gastrointestinal health care resource utilization with chronic use of COX-2-specific inhibitors versus traditional NSAIDs
Loren Laine, Jenifer Wogen, Holly Yu
Background & aims: Cyclooxygenase 2 (COX-2)-specific inhibitors (coxibs) decrease gastrointestinal (GI) events in controlled trials, but results in clinical practice are unknown. We assessed GI-related resource use and costs in patients switching from chronic nonsteroidal anti-inflammatory drug (NSAID) therapy to chronic coxib therapy and in patients starting chronic NSAID therapy vs. chronic coxib therapy in a U.S. administrative claims database of >8 million lives.

Methods: "Switchers" (n = 2246) were assessed in the 12-month periods before and after switching from chronic NSAID therapy to coxib therapy. "New NSAID" (n = 25,989) and "new coxib" (n = 2125) groups were assessed for the 12-month periods before and after the initial prescription. Proportions of patients with GI resource use (odds ratio [OR] adjusted for relevant covariates) and costs were compared.

Results: The adjusted OR for any GI resource use (coxib vs. NSAID period) among switchers was 0.86 (0.740.99). The decrease was due to less GI cotherapy (OR = 0.82 [0.690.97]). Costs were not significantly lower after switching to coxibs (mean difference, $19; 95% CI: $139, $55), although after adding NSAID/coxib costs, the total cost in the coxib period was significantly higher (mean increase, $377; $271, $488). Adjusted OR for GI resource use for new-coxib vs. new-NSAID was 1.04 (0.921.16), but GI costs were significantly lower in new-NSAID patients.

Conclusions: Patients switching from chronic NSAID therapy to chronic coxib therapy had a slight decrease in the proportion using GI-related resources but not in GI costs. When NSAID/coxib drug costs were included, costs were significantly less with NSAIDs than with coxibs. The potential GI-related cost savings suggested in coxib clinical trials may not be fully realized in "real-world" settings.

A randomized trial comparing heater probe plus thrombin with heater probe plus placebo for bleeding peptic ulcer
N.I. Church, H.J. Dallal, J. Masson, N.A.G. Mowat, D.A. Johnston, E. Radin, M. Turner, G. Fullarton, R.J. Prescott, K.R. Palmer
Background & Aims: This multicenter, double-blind, controlled trial compared the efficacy of combined endoscopic hemostatic treatment using the heater probe plus thrombin injection with that of the heater probe plus placebo injection as treatment for peptic ulcers with active bleeding or nonbleeding visible vessels. Efficacy was defined in terms of primary hemostasis, prevention of rebleeding, and need for urgent surgery.

Methods: Two hundred forty-seven patients presenting with major peptic ulcer bleeding were randomized to heater probe plus thrombin or to heater probe plus placebo. The groups were well matched for all risk categories including age, endoscopic stigmata, shock, and severity of comorbid diseases. Endoscopic therapy was applied using the heater probe followed by injection of thrombin or placebo.

Results: Successful primary hemostasis was achieved in 97% of patients. Rebleeding developed in 19 (15%) of thrombin plus heater probe patients and 17 (15%) of placebo plus heater probe patients. Emergency surgery was necessary in 16 and 13 patients, respectively. Eight patients in the thrombin group had adverse events compared with 4 in the placebo group. Eight (6%) of thrombin plus heater probe patients and 14 (12%) of placebo plus heater probe patients died (P = 0.21).

Conclusions: The combination of thrombin and the heater probe does not confer an additional benefit over heater probe and placebo as endoscopic treatment for bleeding peptic ulcer. Our trial does not support the use of this combination of hemostatic therapy.

The cyclooxygenase-2-selective inhibitors rofecoxib and celecoxib prevent colorectal neoplasia occurrence and recurrence
Elham Rahme, Alan N. Barkun, Youssef Toubouti, Marc Bardou
Background & Aims: Colorectal cancer is one of the leading causes of cancer death. Most colorectal cancers are believed to develop from colorectal adenomas. We examined the effect of the selective cyclooxygenase-2 inhibitors rofecoxib and celecoxib, nonselective nonsteroidal anti-inflammatory drugs, aspirin, and acetaminophen on colorectal neoplasia (colorectal cancer, colorectal adenoma, or both).

Methods: This was a nested case-control study, which used data from a government insurance database on patients 65 years and older who underwent a diagnostic test or procedure for colorectal neoplasia between January and June 2001. Logistic regression models were used to determine the effect of exposure to the drugs of interest for at least 3 months on the occurrence or recurrence of colorectal neoplasia.

Results: The control group included 2568 patients found to be free of colorectal neoplasia; 730 patients were diagnosed with colorectal adenoma, and 179 were diagnosed with colorectal cancer. Patients more likely to have colorectal adenoma (odds ratio, 95% confidence interval) were those diagnosed with colorectal adenoma (4.12, 3.275.18) or colorectal cancer (3.74, 2.326.03) in the previous 13 years and those with hemorrhage of the rectum or unspecified anemia in the prior month (3.19, 2.464.12). Exposures to rofecoxib (0.67, 0.460.98) and nonselective nonsteroidal anti-inflammatory drugs (0.41, 0.210.83) reduced the risk of colorectal adenoma. Rofecoxib, celecoxib, and nonselective nonsteroidal anti-inflammatory drugs were all protective against both neoplasias (0.64, 0.450.91; 0.73, 0.540.99; and 0.47, 0.260.86, respectively).

Conclusions: Rofecoxib, celecoxib, and nonselective nonsteroidal anti-inflammatory drugs seem to protect against the development of colorectal neoplasia.

Clostridium difficile toxin B is an inflammatory enterotoxin in human intestine
Tor C. Savidge, Wei-hua Pan, Paul Newman, Michael O'Brien, Pauline M. Anton, Charalabos Pothoulakis
Background & aims: Clostridium difficile causes antibiotic-associated diarrhea and pseudomembranous colitis, diseases afflicting millions of people each year. Although C. difficile releases 2 structurally similar exotoxins, toxin A and toxin B, animal experiments suggest that only toxin A mediates diarrhea and enterocolitis. However, toxin A-negative/toxin B-positive strains of C. difficile recently were isolated from patients with antibiotic-associated diarrhea and colitis, indicating that toxin B also may be pathogenic in humans.

Methods: Here we used subcutaneously transplanted human intestinal xenografts in immunodeficient mice to generate a chimeric animal model for C. difficile toxin-induced pathology of human intestine.

Results: We found that intraluminal toxin B, like equivalent concentrations of toxin A, induced intestinal epithelial cell damage, increased mucosal permeability, stimulated interleukin (IL)-8 synthesis, and caused an acute inflammatory response characterized by neutrophil recruitment and tissue damage. Laser capture microdissection and real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR) showed that intestinal epithelial cell-specific IL-8 gene expression also was increased significantly after luminal exposure to C. difficile toxins in vivo.

Conclusions: We conclude that C. difficile toxin B, like toxin A, is a potent inflammatory enterotoxin for human intestine. Future therapeutic or vaccine strategies for C. difficile infection therefore need to target both toxins.

Gastric electrical stimulation for medically refractory gastroparesis
Thomas Abell, Richard McCallum, Michael Hocking, Kenneth Koch, Hasse Abrahamsson, Isabelle LeBlanc, Greger Lindberg, Jan Konturek, Thomas Nowak, Eammon M.M. Quigley, Gervais Tougas, Warren Starkebaum
Background & aims: This study investigated the efficacy of gastric electrical stimulation for the treatment of symptomatic gastroparesis unresponsive to standard medical therapy.

Methods: Thirty-three patients with chronic gastroparesis (17 diabetic and 16 idiopathic) received continuous high-frequency/low-energy gastric electrical stimulation via electrodes in the muscle wall of the antrum connected to a neurostimulator in an abdominal wall pocket. After implantation, patients were randomized in a double-blind crossover design to stimulation ON or OFF for 1-month periods. The blind was then broken, and all patients were programmed to stimulation ON and evaluated at 6 and 12 months. Outcome measures were vomiting frequency, preference for ON or OFF, upper gastrointestinal tract symptoms, quality of life, gastric emptying, and adverse events.

Results: In the double-blind portion of the study, self-reported vomiting frequency was significantly reduced in the ON vs. OFF period (P < 0.05) and this symptomatic improvement was consistent with the significant patient preference (P < 0.05) for the ON vs. OFF period determined before breaking the blind. In the unblinded portion of the study, vomiting frequency decreased significantly (P < 0.05) at 6 and 12 months. Scores for symptom severity and quality of life significantly improved (P < 0.05) at 6 and 12 months, whereas gastric emptying was only modestly accelerated. Five patients had their gastric electrical stimulation system explanted or revised because of infection or other complications.

Conclusions: High-frequency/low-energy gastric electrical stimulation significantly decreased vomiting frequency and gastrointestinal symptoms and improved quality of life in patients with severe gastroparesis.

Fracture risk in people with celiac disease: A population-based cohort study
Joe West, Richard F.A. Logan, Tim R. Card, Chris Smith, Richard Hubbard
Background & Aims: People with celiac disease are at risk of developing osteoporosis, but the extent of any increased fracture risk is unclear. We performed a population-based cohort study by using the General Practice Research Database to quantify the fracture risk in people with celiac disease compared with the general population.

Methods: We identified 4732 people with celiac disease, of whom 1589 were "incident" cases, and 23,620 age- and sex-matched control subjects. We used Cox regression to estimate the hazard ratios for any fracture, hip fracture, and ulna or radius fracture in the celiac disease cohort compared with the general population.

Results: In the incident subjects with celiac disease, the mean age at diagnosis was 44 years, and 67% were women. The overall hazard ratio for any fracture was 1.30 (95% confidence interval, 1.161.46), for hip fracture was 1.90 (95% confidence interval, 1.203.02), and for ulna or radius fracture was 1.77 (95% confidence interval, 1.352.34). The absolute difference in the overall fracture rate was 3.20 per 1000 person-years and for hip fracture it was 0.97 per 1000 person-years in those older than 45 years. In 1589 incident subjects, the excess fracture risk was slightly lower compared with the "prevalent" subjects (hazard ratio for any fracture, 1.19 vs. 1.40, respectively).

Conclusions: There were small increases in both the absolute and relative fracture risks in people with celiac disease; the excess risks were slightly lower in those with a more recent diagnosis. Our data indicate that concerns regarding a markedly increased fracture risk in celiac disease are unwarranted.

Clinical-liver, Pancreas, and Biliary Tract

Hepatocyte apoptosis and fas expression are prominent features of human nonalcoholic steatohepatitis
Ariel E. Feldstein, Ali Canbay, Paul Angulo, Makiko Taniai, Lawrence J. Burgart, Keith D. Lindor, Gregory J. Gores
Background & Aims: The pathogenesis of nonalcoholic steatohepatitis (NASH) remains poorly understood. Although apoptosis is a common mechanism of liver injury, the extent and clinical significance of apoptosis in NASH has not been examined. Thus, the aims of this study were to quantify hepatocyte apoptosis in NASH, correlate it with disease severity, and identify possible mechanisms of apoptosis induction.

Methods: Hepatocyte apoptosis was assessed in NASH, simple steatosis, alcoholic hepatitis, and controls without liver disease using the TUNEL assay and immunohistochemistry for activated caspases 3 and 7. Liver specimens were also graded according to the magnitude of inflammation and fibrosis.

Results: TUNEL-positive cells were significantly increased in liver biopsy specimens from patients with NASH compared with simple steatosis and controls. Unexpectedly, TUNEL-positive cells were also greater in NASH vs. alcoholic hepatitis. Immunohistochemistry demonstrated active caspases 3 and 7 in NASH specimens, confirming the occurrence of apoptosis in this disease. A positive correlation was observed between hepatocyte apoptosis and hepatic fibrosis and inflammatory activity, respectively. The Fas receptor was strongly expressed in hepatocytes in liver specimens from NASH patients as compared with controls.

Conclusions: Hepatocyte apoptosis is significantly increased in patients with NASH and correlates with disease severity, suggesting that antiapoptotic therapy may be useful in this syndrome.

Hepatitis B virus genotypes in the United States: Results of a nationwide study
Chi-Jen Chu, Emmet B. Keeffe, Steven-Huy Han, Robert P. Perrillo, Albert D. Min, Consuelo Soldevila-Pico, William Carey, Robert S. Brown, Jr, Velimir A. Luketic, Norah Terrault, Anna S.F. Lok
Background & Aims: Hepatitis B virus (HBV) genotypes may be related to severity of liver disease and treatment response. The aims of this nationwide study were to determine the prevalence of HBV genotypes in the United States and the association between HBV genotypes and patient demographics, mode of infection, and clinical status.

Methods: A total of 694 consecutive chronic HBV-infected patients seen in 17 U.S. liver centers during a 1-year period were enrolled. Demographic, clinical, and laboratory data were collected. Sera were tested for HBV genotyping, precore, and core promoter variants by line-probe assays.

Results: All 7 HBV genotypes (AG) were found, with genotypes A and C the most common. The prevalence of HBV genotypes was different in different regions of the United States. A strong correlation was found between HBV genotypes and ethnicity. HBV genotype A was prevalent among white and black patients, whereas genotypes B and C were most common among Asian patients. The predominant genotype among patients born in the United States, Europe, the Far East, and Southeast Asia were A, D, C, and B, respectively. Genotypes A and C were associated with a higher prevalence of hepatitis B e antigen. Precore variant was detected in 27% of patients and core promoter variant in 44% of patients.

Conclusions: Our study suggests that the epidemiology of HBV infection in the United States may have changed over time as a result of immigration from countries with a high prevalence of HBV infection. HBV genotypes may account for the heterogeneity in disease manifestations among patients with chronic HBV infection.

ABCB4 gene mutation-associated cholelithiasis in adults
Olivier Rosmorduc, Brigitte Hermelin, Pierre-Yves Boelle, Rolland Parc, Jacques Taboury, Raoul Poupon
Background & Aims: We recently put forward arguments in favor of ABCB4 gene (adenosine triphosphate-binding cassette, subfamily B, member 4) defects as a risk factor for symptomatic cholelithiasis in adults. In this study, we characterized ABCB4 gene mutations in a series of patients with symptomatic cholelithiasis to determine the genetic basis and the clinical phenotype of ABCB4 gene mutation-associated cholelithiasis.

Methods: We analyzed the entire ABCB4 gene coding sequences in a first group of 32 patients who had a clinical history compatible with the syndrome previously described, in a second group of 28 patients who presented with a classic gallstone disease that justified a cholecystectomy, and in a third group of 33 patients without a history of cholelithiasis.

Results: We identified both heterozygous and homozygous ABCB4 gene point mutations in 18 of 32 (56%) patients who presented with clinical criteria of the syndrome, whereas no mutation was detected in the 2 other groups of patients (P < 0.001). Three independent clinical features were strongly associated with point mutations: recurrence of symptoms after cholecystectomy (odds ratio, 8.5); intrahepatic hyperechoic foci, intrahepatic sludge, or microlithiasis (odds ratio, 6.1); and age <40 years at the onset of symptoms (odds ratio, 3.0). ABCB4 gene point mutations were detected exclusively in the patients who showed 2 or 3 of these clinical features.

Conclusions: Our results show that ABCB4 gene mutations represent a major genetic risk factor in a symptomatic and recurring form of cholelithiasis in young adults.

Heme oxygenase-1 is an antifibrogenic protein in human hepatic myofibroblasts
Liying Li, Pascale Grenard, Jeanne Tran Van Nhieu, Boris Julien, Ariane Mallat, Ada Habib, Sophie Lotersztajn
Background & Aims: Hepatic myofibroblasts play a key role in the development of liver fibrosis associated with chronic liver diseases. We have shown that oxidative stress is a messenger of 15-deoxy-delta-12,14-prostaglandin J2 (15-d-PGJ2) in human hepatic myofibroblasts. The aim of the present study was to investigate the role of a stress-inducible protein, heme oxygenase-1 (HO-1), in the action of 15-d-PGJ2.

Methods: Expression of HO-1 was characterized in biopsy specimens of normal human liver and active cirrhosis by immunohistochemistry, and in cultured human hepatic myofibroblasts by Northern and Western blot analysis. Functional studies also were performed in cultured human hepatic myofibroblasts.

Results: Immunohistochemistry showed that in biopsy specimens from normal livers, HO-1 protein expression was restricted to Kupffer cells. Biopsy specimens from cirrhotic patients displayed HO-1 protein both in macrophages and in myofibroblasts within fibrotic septa. HO-1 messenger RNA (mRNA) and protein also were detected in cultured human hepatic myofibroblasts and increased in response to 15-d-PGJ2 in a time- and dose-dependent manner. Induction of HO-1 in human hepatic myofibroblasts mediated 2 major antifibrogenic properties of 15-d-PGJ2, namely, inhibition of proliferation and of procollagen I mRNA expression. These effects were ascribed to bilirubin, one of the products of HO-1-mediated heme degradation.

Conclusions: This study shows that HO-1 is expressed in human hepatic myofibroblasts and induced during chronic liver injury. Moreover, these data unravel HO-1 as a major antifibrogenic pathway.

Basic-alimentary Tract

Diarrhea caused by enterotoxigenic Escherichia coli infection of humans is inhibited by dietary calcium
Ingeborg M.J. Bovee-Oudenhoven, Mischa L.G. Lettink-Wissink, Wim Van Doesburg, Ben J.M. Witteman, Roelof Van Der Meer
Background & aims: In several rat infection experiments, we have shown that dietary calcium inhibits intestinal colonization and translocation of invasive salmonella. The aim of the present study was to find out whether calcium is also protective against enterotoxigenic Escherichia coli (ETEC) infection. This was first tested in our rat model and subsequently verified in a human infection study.

Methods: Rats were fed a purified diet with either a low or a high amount of calcium phosphate and orally infected with ETEC. In addition, a parallel, double-blind, placebo-controlled intervention study of 3 weeks was performed with 32 healthy men. Subjects largely maintained their habitual diet and consumed either regular milk products (calcium supply, 1100 mg/day) or placebo milk products (calcium supply, 60 mg/day). On day 10, subjects ingested a live but attenuated ETEC strain (strain E1392/75-2A), able to induce mild although short-lived symptoms. Primary outcomes studied were infection-induced diarrhea (total fecal output and relative fecal dry weight) and fecal mucin excretion.

Results: In humans, ETEC induced diarrhea in both groups, in that total fecal output doubled and mean relative fecal dry weight dropped from 25% to 20%. Additionally, fecal mucin excretion was increased in both groups. All these fecal parameters were completely normalized in the calcium group on the second infection day, in contrast to the placebo group, which recovered on the third infection day. Likewise, supplemental calcium inhibited ETEC colonization and diarrhea in rats.

Conclusions: Calcium in milk products improves human resistance to ETEC infection as it inhibits infectious diarrhea.

Identification of a quantitative trait locus for ileitis in a spontaneous mouse model of Crohn's disease: SAMP1/YitFc
Kosuke Kozaiwa, Kazuhiko Sugawara, Michael F. Smith, Jr, Virginia Carl, Vladimir Yamschikov, Brian Belyea, Sherri B. Mcewen, Christopher A. Moskaluk, Theresa T. Pizarro, Fabio Cominelli, Marcia Mcduffie
Background & aims: The SAMP1/Yit mouse strain develops spontaneous ileitis with histologic features of Crohn's disease. Disease expression in the SAMP1/YitFc subline (SAMP1/Fc) is partially inhibited by outcross to C57BL/6J (B6) mice, suggesting complex genetic control of disease susceptibility with both dominant and recessive determinants. We performed a genetic analysis of a (B6 Å~ SAMP1/Fc)F2 cross to localize the genes regulating intestinal inflammation in this model.

Methods: A genome-wide scan was performed using a panel of microsatellite loci determined to be informative for this cross. Quantitative trait loci were identified with Map Manager QT using a serial regression approach. Positional candidate genes were selectively sequenced at the genomic level to identify potential susceptibility genes for functional screening.

Results: A genome-wide scan of (B6 Å~ SAMP1/Fc)F2 mice identified a SAMP-derived quantitative trait loci with additive effects on chromosome 9 in a region likely to have been inherited from the AKR mouse strain. The candidate interval contains several genes of interest because of their potential role in either immune system function, intestinal epithelial function, or both. Suggestive evidence for additional loci was also observed on chromosomes 6 and X.

Conclusions: The SAMP1/Fc allele for a locus, designated Ibdq1, promotes inflammation-associated epithelial damage in these mice. Consistent with persistent mild ileitis in (B6 Å~ SAMP1/Fc)F1 mice, this locus appears to function in an additive fashion. Two genes in this interval, encoding the interleukin 10 receptor chain and interleukin 18, are excellent candidates for Ibdq1.

CCR5 mediates specific migration of Toxoplasma gondii-primed CD8+ lymphocytes to inflammatory intestinal epithelial cells
Souphalone Luangsay, Lloyd H. Kasper, Nicolas Rachinel, Laurie A. Minns, Franck J.D. Mennechet, Alain Vandewalle, Dominique Buzoni-Gatel
Background & Aims: Toxoplasma gondii, an obligate intracellular parasite, can invade intestinal epithelial cells and elicit a robust Th1 immune response. In this model of intestinal inflammation, CD8+ intraepithelial lymphocytes (IELs) secrete transforming growth factor (TGF)-, which appears necessary for the maintenance of homeostasis in the intestine. However, the mechanism responsible for the IEL migration to the inflamed intestine is still unclear.

Methods: An in vitro coculture cell system was used to quantify the IEL attraction by an infected intestinal epithelial cell line (m-ICcl2). We used CCR5-deficient mice to determine which chemokine receptor-chemokine interaction could be responsible for the recruitment of antigen-specific CD8+ IELs to the small intestine for the promotion of parasite clearance and host recovery.

Results: We observed increased expression of several chemokine receptors (CCR1, CCR2, CCR5, CXCR3) in the infected ileum. In particular, CCR5 expression was markedly increased in antigen-primed CD8+ IELs. Experiments using recombinant chemokines as well as blocking antibodies showed that macrophage inflammatory protein (MIP)-1 and MIP-1 were critical for their homing. CD8+ IELs isolated from CCR5-deficient mice (CCR5/), despite their high production of TGF- and overexpression of activation markers, were impaired in their ability to migrate in vitro to the m-ICcl2 monolayer or in vivo to the inflamed intestine after adoptive transfer.

Conclusions: Our data emphasize the biologic role of CCR5 as an important component in the migration of intraepithelial CD8+ T cells and the regulation of the inflammatory response following parasite infection.

Pathogenicity of the hereditary colorectal cancer mutation hMLH1 del616 linked to shortage of the functional protein
Tiina E. Raevaara, Carlos Vaccaro, Wael M. Abdel-Rahman, Esteban Mocetti, Shashi Bala, Karin E. Lönnqvist, Reetta Kariola, Henry T. Lynch, Päivi Peltomäki, Minna Nyström-Lahti
Background & Aims: Hereditary nonpolyposis colorectal cancer is associated with mismatch repair deficiency. Most predisposing mutations prevent the production of functional mismatch repair protein. Thus, when the wild-type copy is also inactivated, the cell becomes mismatch repair deficient, and this leads to a high degree of microsatellite instability in tumors. However, tumors linked to nontruncating mutations may display positive or partly positive immunohistochemical staining of the mutated protein and low or atypical microsatellite instability status, which suggests impaired functional activity but not a total lack of mismatch repair. We found human mutL homology (hMLH) 1 del616, one of the most widespread recurring mutations in hereditary nonpolyposis colorectal cancer, segregating in a large hereditary nonpolyposis colorectal cancer family. Because the predicted coding change is a deletion of only 1 amino acid, the pathogenicity of the mutation was evaluated.

Methods: Many analyses were performed to assess the pathogenicity of hMLH1 del616 and to study the expression and function of the mutated messenger RNA and protein.

Results: Genetic and immunohistochemical evidence supported hMLH1-linked cancer predisposition in this family. Microsatellite instability varied from low to high, and the hMLH1 protein was lost in 2 tumors but was partly detectable in 1 tumor. Whereas similar optimal amounts of mutated hMLH1 del616 and wild-type hMLH1 proteins were equally functional in an in vitro mismatch repair assay, the amount of in vivo-expressed hMLH1 del616 was much lower than the amount of wild-type protein; this suggests that the deletion imparts instability to the mutant protein.

Conclusions: Our results suggest that the pathogenicity of hMLH1 del616 is not linked to nonfunctionality, but to shortage of the functional protein.

Transcriptional regulation of the human trefoil factor, TFF1, by gastrin
Zara E. Khan, Timothy C. Wang, Guanglin Cui, Alfred L. Chi, Rod Dimaline
Background & Aims: This study aimed to identify gastrin-sensitive genes that may mediate the effects of this hormone on gastric epithelial architecture.

Methods: Gastrin-sensitive genes were identified by messenger RNA (mRNA) differential display of the gastric fundus from gastrin-deficient (GAS-KO) or wild-type mice. Gastrin-stimulated expression of the trefoil peptide TFF1 in mouse fundus and in the gastric cancer cell line AGS-GR was determined by Northern blot and real-time polymerase chain reaction. Transcriptional regulation of TFF1 in AGS-GR cells was studied using promoter-reporter assays and electrophoretic mobility shift assay. Expression of TFF1 and the cholecystokininB receptor in response to gastric mucosal injury was determined by immunohistochemistry.

Results: mRNA differential display identified TFF1 as a gastrin-regulated gene. TFF1 mRNA was reversibly reduced in GAS-KO mice and increased in a hypergastrinemic transgenic strain versus respective background strains. TFF1 mRNA expression was rapidly and potently induced by gastrin in a gastric cancer cell line that expresses the gastrin/cholecystokininB receptor. Gastrin responsiveness of the human TFF1 promoter mapped to a G-C rich region 300 base pairs upstream of the transcriptional start site. This region bound the transcription factors SP3 and MAZ. Gastrin activated transcription through a Raf-, Mek- and Erk-dependent but Ras-independent pathway. TFF1 expression was induced both directly and by transactivation between neighboring cells. Neither direct nor indirect gastrin-induced TFF1 expression required activation of the epidermal growth factor receptor.

Conclusions: Gastrin exerts tonic control of TFF1 expression but also has the potential for rapid up-regulation of this trefoil factor. TFF1 is a potential candidate to counterbalance the proliferative effects of gastrin.

Basic-liver, Pancreas, and Biliary Tract

Transduction of the liver with activated Akt normalizes portal pressure in cirrhotic rats
Manuel Morales-ruiz, Pilar Cejudo-martn, Guillermo Fernández-varo, Sònia Tugues, Josefa Ros, Paolo Angeli, Francisca Rivera, Vicente Arroyo, Juan Rodés, William C. Sessa, Wladimiro Jiménez
Background & Aims: Portal hypertension in cirrhosis is secondary to an increase in hepatic resistance that occurs mainly through collagen deposition. However, recent evidence points to a major contribution by other factors, such as an intrahepatic reduction in nitric oxide production. Akt is a major activator of the endothelial nitric oxide synthase (eNOS) enzyme, but its potential role in intrahepatic resistance in cirrhosis is unknown. For this reason the aims of the present study were to determine whether there is an impaired Akt activation in cirrhotic livers and how this phenomenon relates to the decrease in NO production associated with portal hypertension.

Methods: Cirrhosis was induced in rats by carbon tetrachloride inhalation. Protein abundance and phosphorylation status of Akt and eNOS were examined by Western blotting. The role of Akt in the liver of cirrhotic rats was investigated through infection with adenoviruses encoding either -galactosidase (-gal) or constitutively active Akt (myr-Akt).

Results: The liver of cirrhotic animals showed a significant reduction in Akt and eNOS phosphorylation. Adenoviral delivery of myr-Akt restored eNOS phosphorylation and increased the intrahepatic concentration of guanosine 3´,5´-cyclic monophosphate. These events were associated with normalization in portal pressure and a significant increase in mean arterial pressure after 3 days of adenoviral infection. In contrast, transduction of livers with -gal did not produce any change in these hemodynamic parameters.

Conclusions: myr-Akt gene therapy restored Akt activation and NO production in the cirrhotic liver, suggesting that this therapy may be useful for the treatment of portal hypertension.

Lack of gp130 expression in hepatocytes promotes liver injury
Konrad L. StreetZ, Torsten Wüstefeld, Christian Klein, Karl-Josef Kallen, Francois Tronche, Ullrich A.K. Betz, Günther Schütz, Michael P. Manns, Werner Müller, Christian Trautwein
Background & aims: Interleukin 6 (IL-6) contributes via its signal transducer gp130 to the acute phase response (APR) in hepatocytes. Recent studies indicated that IL-6 is involved in the regulation of different pathophysiologic conditions of the liver. To define the IL-6-dependent intracellular pathways more specifically, we generated a hepatocyte-specific gp130 knockout mouse.

Methods: Hepatocyte-specific gp130-deficient mice were generated using the Cre-loxP system. Expression of the Cre recombinase was under the control of a hepatocyte-specific control element. Adult mice were challenged with IL-6, oncostatin M (OSM), and LPS.

Results: Cre expression started at day 10.5 postconception, and a complete deletion of gp130 in hepatocytes was found at day 14 during liver development. The adult liver of these mice showed no abnormalities; however, after IL-6 and OSM stimulation, gp130-dependent pathways (STAT3, APR gene expression) were completely blocked in the liver of these animals. Additionally, challenging hepatocyte-specific gp130 knockout animals with lipopolysaccharides (LPS) lead to an onset of acute liver injury with an increase of hepatocyte apoptosis associated with elevated tumor necrosis factor (TNF-) serum levels and reduced nuclear factor B (NF-B) activation in hepatocytes.

Conclusions: Our findings demonstrate that gp130 is of minor relevance for embryonal development of hepatocytes. However, the molecule has an essential role in controlling acute phase gene expression and provides hepatocellular protection after LPS challenge.

Farnesoid X receptor agonists suppress hepatic apolipoprotein CIII expression
Thierry Claudel, Yusuke Inoue, Olivier Barbier, Daniel Duran-Sandoval, Vladimir Kosykh, Jamila Fruchart, Jean-Charles Fruchart, Frank J. Gonzalez, Bart Staels
Background & aims: Increased serum triglyceride levels constitute a risk factor for coronary heart disease. Apolipoprotein CIII (Apo CIII) is a determinant of serum triglyceride metabolism. In this study, we investigated whether activators of the nuclear farnesoid X receptor (FXR) modulate Apo CIII gene expression.

Methods: The influence of bile acids and synthetic FXR activators on Apo CIII and triglyceride metabolism was studied in vivo by using FXR wild-type and FXR-deficient mice and in vitro by using human primary hepatocytes and HepG2 cells.

Results: In mice, treatment with the FXR agonist taurocholic acid strongly decreased serum triglyceride levels, an effect associated with reduced Apo CIII serum and liver messenger RNA levels. By contrast, no change was observed in FXR-deficient mice. Incubation of human primary hepatocytes and HepG2 cells with bile acids or the nonsteroidal synthetic FXR agonist GW4064 resulted in a dose-dependent down-regulation of Apo CIII gene expression. Promoter transfection experiments and mutation analysis showed that bile acid-activated FXR decrease human Apo CIII promoter activity via a negative FXR response element located in the I4 footprint between nucleotides 739 and 704. Chromatin immunoprecipitation experiments showed that bile acid treatment led to binding of FXR/retinoid X receptor heterodimers to and displacement of HNF4 from this site. Bile acid treatment still repressed liver Apo CIII gene expression in hepatic HNF4-deficient mice, suggesting an active rather than a competitive mechanism of Apo CIII repression by the FXR.

Conclusions: We identified bile acid and synthetic activators of the nuclear FXR as negative regulators of Apo CIII expression, an effect that may contribute to the triglyceride-decreasing action of FXR agonists.

JTE-522, a cyclooxygenase-2 inhibitor, is an effective chemopreventive agent against rat experimental liver fibrosis
Hirofumi Yamamoto, Motoi Kondo, Shoji Nakamori, Hiroaki Nagano, Ken-ichi Wakasa, Yurika Sugita, Jin Chang-de, Shogo Kobayashi, Bazarragchaa Damdinsuren, Keizo Dono, Koji Umeshita, Mitsugu Sekimoto, Masato Sakon, Nariaki Matsuura, Morito Monden
Background & Aims: The aim of this study was to assess the effects of cyclooxygenase (COX)-2 inhibition on rat experimental liver fibrogenesis.

Methods: We investigated the inhibitory effects of a selective COX-2 inhibitor, JTE-522, on liver fibrosis induced by a choline-deficient, L-amino acid-defined diet (CDAA). Inhibitory effect was also tested in a second model of thioacetamide (TAA)-induced liver fibrosis.

Results: CDAA induced liver fibrosis and preneoplastic foci at 12 weeks and cirrhosis at 36 weeks. Hepatocellular carcinoma was noted in 13 of 15 rats (87%). JTE-522 significantly inhibited fibrosis and development of preneoplastic lesions in a dose-dependent manner and completely inhibited generation of cirrhosis and hepatocellular carcinoma at both low and high doses (10 and 30 mg/kg body wt/day, respectively). JTE-522 administrated only from 12 weeks to 36 weeks also prevented cirrhosis and formation of hepatocellular carcinoma. JTE-522 itself did not cause local or systemic gross or histopathologic changes at 36 weeks. Mechanistic studies indicated that the CDAA model displayed up-regulation of several biomarkers, including COX-2, arachidonate metabolite (prostaglandin E2), serum aspartate aminotransferase, and c-myc expression. The model also showed an increased proportion of activated hepatic stellate cells, proliferating cell nuclear antigen index, and CD45-positive inflammatory cells in the liver. JTE-522 effectively diminished these changes. JTE-522 exhibited similar antifibrosis effects in the TAA model.

Conclusions: Our results suggest that COX-2 is involved in CDAA- and TAA-induced liver fibrosis. Our data also indicate that JTE-522 is a potent chemopreventive agent of rat liver fibrosis with low toxicity.

Insulin signaling through insulin receptor substrate 1 and 2 in normal liver development
Leila Khamzina, Philip A. Gruppuso, Jack R. Wands
Background & Aims: The insulin growth factor signal transduction pathway is an important regulator of adult hepatocyte proliferation. The purpose of this study was to determine the roles of the insulin receptor substrate (IRS-1 and IRS-2)-mediated growth cascades in rapidly growing fetal rat liver.

Methods: We determined the expression and tyrosyl phosphorylation of the insulin receptor subunit (IR), IRS-1 and IRS-2, the binding of phosphatidylinositol 3-kinase (PI3K), and activation of the mitogen-activated protein kinase (MAPK) pathway in the presence or absence of insulin stimulation in vivo during development and in the adult liver. In addition, activation of other downstream components including PI3K, Akt, GSK3, Bad, and p70S6 kinase was studied.

Results: We observed reduced expression and tyrosyl phosphorylation of IRS-1 in the fetal liver compared with the adult liver. These developmental changes resulted in a lack of sensitivity to insulin stimulation and subsequent downstream activation of the PI3K and MAPK cascades until the postneonatal period. In contrast, there was a high level of IRS-2 expression and insulin-stimulated tyrosyl phosphorylation as early as embryonic day 15 with robust PI3K binding and activation, which may enhance hepatocyte survival during the rapid growth phase of the liver.

Conclusions: The IRS-1 signal transduction pathway does not play a major role in fetal liver growth because IRS-2 functions as the major insulin responsive molecule in early development. However, insulin-mediated IRS-1/MAPK cascade activation contributes to growth in the adult.

Case Report

Pain as a complication of use of opiate antagonists for symptom control in cholestasis
Christine A. Mcrae, Martin I. Prince, Mark Hudson, Christopher P. Day, Oliver F.W. James, David E.J. Jones
Controlled trials have suggested that opiate antagonist therapy may be effective for the treatment of the symptoms of cholestasis. The oral opiate antagonist naltrexone in particular has started to enter into routine clinical use for amelioration of cholestatic itch. Attention regarding the side effects of opiate antagonist therapy has, to date, largely focused on an opiate withdrawal-type reaction (which can be controlled effectively by titrated therapy introduction regimens). Here we describe 3 cases of a further clinically important side effect, loss of control of pain resulting from other pathologies, which in each case necessitated the withdrawal of hitherto clinically effective opiate antagonist therapy. Of the 14 patients treated by our unit with opiate antagonist agents for the control of cholestatic symptoms, 13 (93%) showed resolution of, or significant improvement in, symptoms. Of the 13 patients showing a clinical response, 7 (54%) subsequently had to discontinue therapy because of side effects (including the 3 patients with uncontrolled pain). It is our experience that in the routine clinical setting, opiate antagonists are highly effective for the treatment of cholestatic symptoms. In practice, however, their usefulness is limited by their side-effect profile.

JOURNAL OF HEPATOLOGY

Table of Contents for Journal of Hepatology Volume 39, Issue 2, August 2003

Biliary Tract and Cholestasis

Resistance of rat hepatocytes against bile acid-induced apoptosis in cholestatic liver injury is due to nuclear factor-kappa B activation
Marieke H. Schoemaker et al.
Background/Aims: To examine the extent and mechanisms of apoptosis in cholestatic liver injury and to explore the role of the transcription factor nuclear factor-kappa B in protection against bile acid-induced apoptosis.

Methods: Cholestatic liver injury was induced by bile duct ligation in Wistar rats. Furthermore, primary cultures of rat hepatocytes were exposed to glycochenodeoxycholic acid (GCDCA), tauroursodeoxycholic acid (TUDCA), taurochenodeoxycholic acid (TCDCA) and to cytokines. Apoptosis was determined by TUNEL-staining, active caspase-3 staining, activation of caspase-8, -9 and -3.

Results: Limited hepatocyte apoptosis and an increased expression of NF-B-regulated anti-apoptotic genes A1 and cIAP2 were detected in cholestatic rat livers. Bcl-2 expression was restricted to bile duct epithelium. In contrast to TCDCA and TUDCA, GCDCA induced apoptosis in a Fas-associated protein with death domain (FADD)-independent pathway in hepatocytes. Although bile acids do not activate NF-B, NF-B activation by cytokines (induced during cholestasis) protected against GCDCA-induced apoptosis in vitro by upregulating A1 and cIAP2.

Conclusions: GCDCA induces apoptosis in a mitochondria-controlled pathway in which caspase-8 is activated in a FADD-independent manner. However, bile acid-induced apoptosis in cholestasis is limited. This could be explained by cytokine-induced activation of NF-B-regulated anti-apoptotic genes like A1 and cIAP2.
Keywords: Cholestasis; Bile acids; Hepatocytes; Apoptosis; Caspases; Inflammation; Nuclear factor-kappa B; cIAP2

Abbreviations: TNF-, tumor necrosis factor ; TNFR-1, TNF- type I receptor; FADD, Fas-associated protein with death domain; GCDCA, glycochenodeoxycholic acid; TCDCA, taurochenodeoxycholic acid; TUDCA, tauroursodeoxycholic acid; IAP, inhibitor of apoptosis protein; HIAP, human IAP; iNOS, inducible NO synthase; CM, cytokine mixture; IL-1, interleukin-1; IFN-, interferon-; Ad5IBAA, adenoviral dominant-negative IB-; Ad5LacZ, adenoviral -galactosidase; Ad5dnFADD, adenoviral dominant-negative FADD; AdHIAP1, adenoviral human IAP1; EMSA, electrophoretic mobility shift assay; ALAT, alanine aminotransferase; ASAT, aspartate aminotransferase; GGT, gamma glutamyltransferase

Cell Biology, Metabolism and Transport

Purification of adult hepatic progenitor cells using green fluorescent protein (GFP)-transgenic mice and fluorescence-activated cell sorting
Takahisa Fujikawa et al.
Background/Aims: Recent advances in stem cell research have revealed that hepatic stem/progenitor cells may play an important role in liver development and regeneration. However, a lack of detectable definitive markers in viable cells has hindered their primary culture from adult livers.

Methods: Enzymatically dissociated liver cells from green fluorescent protein (GFP)-transgenic mice, which express GFP highly in liver endodermal cells, were sorted by GFP expression using a fluorescence-activated cell sorter. Sorted cells were characterized, and also low-density cultured for extended periods to determine their proliferation and clonal differentiation capacities.

Results: When CD45TER119 side-scatterlow GFPhigh cells were sorted, -fetoprotein-positive immature endoderm-characterized cells, having high growth potential, were present in this population. Clonal analysis and electron microscopic evaluation revealed that each single cell of this population could differentiate not only into hepatocytes, but also into biliary epithelial cells, showing their bilineage differentiation activity. When surface markers were analyzed, they were positive for Integrin-6 and -1, but negative for c-Kit and Thy1.1.

Conclusions: Combination of GFP-transgenic mice and fluorescence-activated cell sorting enabled purification of hepatic progenitor cells from adult mouse liver. Further analysis of this population may lead to purification of their human correspondence that would be an ideal cell-source candidate for regenerative medicine.
Keywords: Adult hepatic progenitor cell; Green fluorescent protein; Transgenic mouse; Fluorescence-activated cell sorting; Hepatocyte; Biliary epithelial cell; Regenerative medicine

Downregulation of cytochromes P450 in growth-stimulated rat hepatocytes: role of c-Myc induction and impaired C/EBP binding to DNA
Marina Tinel et al.
Background/Aims: Several cytochromes P450 (CYPs) are expressed in differentiated hepatocytes, but downregulated in growth-stimulated cells. We determined the signals involved in CYP downregulation by epidermal growth factor (EGF).

Methods: Rat hepatocytes were cultured with or without diverse substances for 72 h and EGF for the last 48 h.

Results: EGF increased c-myc mRNA and protein, and decreased CYP mRNAs and proteins; both effects were prevented by two agents blocking c-myc transcription (retinoic acid and DMSO) and two antisense c-myc oligomers. Despite unchanged CCAAT-enhancer binding protein (C/EBP) and increased C/EBP levels, nuclear proteins of EGF-treated cells did not bind to a C/EBP DNA probe in a gel mobility shift assay. This binding was restored when cells were co-treated with both EGF and c-myc antisense oligomers (preventing c-Myc induction). The N-terminal c-Myc domain added to control nuclear extracts prevented C/EBP DNA binding. A monoclonal anti-c-Myc antibody co-immunoprecipitated c-Myc, C/EBP and C/EBP from nuclear extracts. In cells not treated with EGF, an antisense C/EBP oligomer decreased CYP expression.

Conclusions: EGF overexpresses c-Myc, decreases C/EBP binding to DNA and downregulates CYPs. We suggest that c-Myc may form inactive complexes with C/EBPs, thus decreasing C/EBP-mediated CYP transactivation.
Keywords: Epidermal growth factor; c-Myc; CCAAT-enhancer binding protein; Cytochrome P450; CYP; Cell differentiation; Cell growth

Chronic Liver Diseases

Serum levels of YKL-40 and PIIINP as prognostic markers in patients with alcoholic liver disease
Camilla Nøjgaard et al.
Background/Aims: YKL-40 (growth factor) and PIIINP (N-terminal propeptide of Type III procollagen) are potential markers of liver fibrosis. The aim was to evaluate the prognostic value of serum YKL-40 and PIIINP levels in patients with alcoholic liver disease.

Methods: Three hundred and seventy patients with alcoholic liver disease were studied in a trial of malotilate with a median follow-up period of 470 days; 75 patients died; 336 patients had a liver biopsy on entry. Serum levels of YKL-40 and PIIINP were determined by radioimmunoassay (RIA).

Results: Serum YKL-40 and PIIINP were elevated in the patients compared to controls. Patients with steatosis or no fibrosis had the lowest serum levels of YKL-40 and PIIINP, whereas patients with alcoholic hepatitis and/or cirrhosis had the highest levels. Serum YKL-40 was associated with the presence of fibrosis, and serum PIIINP was also associated with the different grades of fibrosis. Patients with elevated serum YKL-40 or PIIINP had shorter survival than patients with normal serum levels of YKL-40 (P<0.0001) or PIIINP (P=0.044). High degree of fibrosis predicted shorter survival (P=0.004).

Conclusions: Serum levels of YKL-40 and PIIINP are elevated in alcoholic patients, related to the presence of liver fibrosis and may provide prognostic information.
Keywords: YKL-40; Human cartilage glycoprotein; PIIINP; N-terminal propeptide of Type III procollagen; Alcoholic liver disease; Liver fibrosis; Prognostic marker

Cirrhosis and its Complications

Spironolactone alone or in combination with furosemide in the treatment of moderate ascites in nonazotemic cirrhosis. A randomized comparative study of efficacy and safety
Justiniano Santos et al.
Background/Aims: The most rational treatment of moderate ascites is spironolactone alone or in combination with furosemide. However, it is unknown which of these two treatment schedules is preferable.

Methods: One hundred nonazotemic cirrhotic patients with moderate ascites were randomly assigned to be treated with spironolactone and furosemide (Group 1: 50 patients) or with spironolactone alone (Group 2: 50 patients). If no response was obtained, the doses of diuretics were increased up to 400 mg/day of spironolactone and 160 mg/day of furosemide. In patients of group 2 not responding to 400 mg/day of spironolactone, furosemide was added. In cases with an excessive response, the dosage of diuretics was reduced.

Results: The response rate (98% in Group 1 vs. 94% in Group 2), the rapidity of ascites mobilization and the incidence of complications induced by diuretic therapy was similar in both groups. The need to reduce the diuretic dosage was significantly higher in Group 1 than Group 2 (68% vs. 34%; P=0.002).

Conclusions: In the treatment of moderate ascites, spironolactone alone seems to be as safe and effective as spironolactone associated with furosemide. Since spironolactone alone requires less dose adjustment, it would be more suitable for treating ascites on an outpatient basis.
Keywords: Spironolactone; Furosemide; Ascites; Cirrhosis

Vasopressin accelerates experimental ammonia-induced brain edema in rats after portacaval anastomosis
Chuhan Chung, Javier Vaquero, Jeanne Gottstein and Andres T. Blei
Background/Aims: Cerebral hyperemia is an important contributor to the development of brain edema in fulminant hepatic failure. Rats receiving an ammonia infusion after portacaval anastomosis (PCA) demonstrate a rise in cerebral blood flow (CBF) with brain edema at 180 min. Vasopressin (VP), a systemic vasoconstrictor which in the rat dilates cerebral vessels through V2 receptors, was used to ascertain the effects of increasing CBF.

Methods: Changes in CBF were measured with Laser Doppler flowmetry (LDF). Absolute CBF was measured with radioactive microspheres to calculate oxygen and ammonia uptake.

Results: Compared to the NH3+Vehicle group, VP+NH3 infusion accelerated the rise in CBF (117±21 vs. 6±12%, P<0.01), and the development of brain edema (81.09±0.17 vs. 80.29±0.06%, P<0.01). Radioactive microspheres confirmed these results (254±44 vs. 106±9.5 ml/min/100 g, P<0.01). Oxygen uptake was similar. Ammonia uptake was more than twofold higher in the VP+NH3 group. A V1 antagonist negated the higher mean arterial pressure (MAP) that occurs with VP but cerebral hyperemia still occurred. A V2 antagonist resulted in similar systemic pressures, CBF and brain water compared to the VP+NH3 group.

Conclusions: In this model, an increase in CBF with VP hastens the development of brain edema while increasing ammonia delivery to the brain.
Keywords: Cerebral blood flow; Portacaval anastomosis; Vasopressin

Kupffer cells are a major source of increased platelet activating factor in the CCl4-induced cirrhotic rat liver
Yongping Yang, Stephen A.K. Harvey and Chandrashekhar R. Gandhi
Background/Aims: Endothelin-1 (ET-1) stimulates the synthesis of platelet-activating factor (PAF) by Kupffer cells in vitro. Hepatic concentrations of both ET-1 (a potent vasoconstrictor) and PAF (a mediator of hepatic vasoconstriction and the cirrhotic hyperdynamic state) increase in cirrhosis. The aim of this study was to determine if the responsiveness of Kupffer cells to produce PAF upon ET-1 challenge is modified by cirrhosis.

Methods: Kupffer cells, isolated from the livers of control and CCl4-induced cirrhotic rats, were placed in serum-free medium after overnight culture. PAF and ET-1 receptors, ET-1-induced PAF synthesis, and PAF- and ET-1-induced prostaglandin E2 (PGE2) synthesis were determined 24 h later.

Results: Both basal and ET-1-stimulated PAF synthesis was increased in cirrhotic Kupffer cells as indicated by increased cell-associated and released PAF. Cirrhotic Kupffer cells also had elevated densities of functional receptors for both PAF and ET-1 (exclusively ETB), as measured by ligand binding, mRNA expression of the respective receptors, and ligand-stimulated PGE2 synthesis.

Conclusions: Cirrhosis sensitizes Kupffer cells to both ET-1 and PAF by elevating their respective receptor levels. Since both mediators individually cause portal hypertension, an increase in ET-1-stimulated PAF synthesis in Kupffer cells will exacerbate the hepatic and extrahepatic complications of cirrhosis.
Keywords: Cirrhosis; Kupffer cells; Platelet-activating factor; Endothelin-1; Receptor

Liver Growth and Cancer

Impact of large regenerative, low grade and high grade dysplastic nodules in hepatocellular carcinoma development
Mauro Borzio et al.
Background/Aims: The natural outcome of ultrasound-detected macronodules in cirrhosis is still poorly understood. In this study we assessed the incidence and predictors of malignant transformation in a prospective study of 90 consecutive ultrasound-detected macronodules in cirrhosis.

Methods: Macronodules classification was based on recently proposed histological criteria. Extranodular large (LCC) and small cell changes were also evaluated. The follow-up included ultrasound and serum alfa-fetoprotein determination every 3 months. Independent predictors of hepatocellular carcinoma were evaluated by Cox proportional hazards regression analysis.

Results: During a mean follow-up of 33 months, 28 (31%) nodules transformed into hepatocellular carcinoma. The incidence of hepatocellular carcinoma per 100 person-years of follow-up was 11.3%, with a malignant transformation rate of 3.5, 15.5, 31 and 48.5% at 1, 2, 3, and 5 years respectively. High-grade dysplastic nodules (HGDN) (hazard risk=2.4; CI 95%=1.1-5.0) and LCC (hazard risk=3.1; CI 95%=1.2-7.8) were independent predictors of malignant transformation. Eight additional hepatocellular carcinomas developed outside the original lesions raising the overall malignant transformation rate to 40% while 15 macronodules (17%) became undetectable at ultrasound (US).

Conclusions: Macronodules characterize a cirrhotic subpopulation with high risk of hepatocellular carcinoma. HGDN and LCC are strong predictors of malignant transformation; subjects with simultaneous presence of both these two conditions are at highest risk of cancer development. The management of cirrhotics with macronodules should be based on morphologic features detected on liver microsamples.
Keywords: Large regenerative nodule; Low-grade dysplastic nodule; High-grade dysplastic nodule hepatocellular carcinoma; Cirrhosis

Diagnosis of intrahepatic metastasis and multicentric carcinogenesis by microsatellite loss of heterozygosity in patients with multiple and recurrent hepatocellular carcinomas
Osakuni Morimoto et al.
Background/Aims: The prognosis of hepatocellular carcinoma (HCC) is poor because of frequent intrahepatic metastasis (IM) or multicentric carcinogenesis (MC). We compared the effectiveness of loss of heterozygosity (LOH) analysis in the diagnosis of these two forms with that of histopathological diagnosis.

Methods: Using LOH analysis of 15 specific DNA microsatellite loci, tumor clonality was assessed in 37 cases.

Results: LOH was observed in 30% of seven solitary tumors. According to these results, the selected threshold to diagnose MC was a difference in the LOH status at more than 30% of the analyzed loci, when comparing two samples in the same liver. In nine multiple HCCs, identical genetic and histopathological diagnoses were found in four (IM: 2, MC: 2). Of 21 recurrent tumors, 19 showed LOH for at least one marker. IM and MC were genetically diagnosed in five and ten patients, respectively. Genetic and histopathological diagnoses were identical in ten of 19 patients (IM: 5. MC: 5). Five genetic MC were histopathologically diagnosed as IM (3) and `undetermined' (2).

Conclusions: Genetic diagnosis by LOH analysis may be more strict and specific than histopathological diagnosis in the differential diagnosis of IM and MC.
Keywords: Hepatocellular carcinoma; Loss of heterozygosity; Intrahepatic metastasis; Multicentric carcinogenesis

Viral Hepatitis

Prediction of liver histological lesions with biochemical markers in patients with chronic hepatitis B
Robert P. Myers et al.
Background Aims: Liver biopsy is the gold standard for assessing hepatitis B virus (HBV)-related histology. The aim was to determine the diagnostic utility of noninvasive serum markers in patients with chronic hepatitis B.

Methods: The aminotransferases and indices including 2-macroglobulin, apolipoprotein A1, haptoglobin, -glutamyl-transpeptidase (GGT), and total bilirubin (Fibrotest), and ALT (Actitest) were compared with liver histology. The primary outcomes were A2-A3 activity and F2-F4 fibrosis (METAVIR).

Results: Two hundred and nine patients were included. Forty-one patients (20%) had A2-A3 activity and 61 (29%) had F2-F4 fibrosis. AST and GGT (P<0.001) were independently associated with A2-A3 activity. AST, ALT, and Actitest accurately predicted activity ((areas under receiver operating characteristic (ROC) curves (AUROC), 0.81-0.82±0.04)); an AST or ALT30IU/l excluded significant activity with 96% certainty. Fibrotest accurately predicted F2-F4 fibrosis (AUROC, 0.78±0.04). Fibrotest scores (range, 0-1.0) 0.20 and >0.80 had negative and positive predictive values of 92%, respectively. Restricting biopsy to patients with intermediate scores (>0.20 and 0.80) may prevent liver biopsies in 46% of patients while maintaining 92% accuracy.

Conclusions: The aminotransferases and an index including five biochemical markers are accurate noninvasive markers of HBV-related activity and fibrosis, respectively.
Keywords: -Macroglobulins; Apolipoprotein A1; Biopsy; Diagnosis; Fibrosis; Haptoglobins; Histology; Hepatitis B; Receiver operating characteristic curve; Transaminases

Quality of life and cognitive function in hepatitis C at different stages of liver disease
Juan Córdoba et al.
Background/Aims: Hepatitis C has been associated with a decrease in quality of life and with neurological abnormalities. The aim of our study was to investigate the relationship between quality of life and cognitive function.

Methods: Quality of life, clinical variables and neuropsychological function were evaluated in 120 patients with hepatitis C (mild chronic hepatitis, compensated cirrhosis and decompensated cirrhosis) and in healthy controls (n=40, in each group).

Results: Patients with chronic hepatitis or compensated cirrhosis showed a decrease in quality of life, in spite of unimpaired neuropsychological tests. Patients with decompensated cirrhosis exhibited a further decrease in quality of life and neuropsychological abnormalities. The decrease in quality of life was associated with the severity of liver failure, neuropsychological abnormalities and treatment with beta-blockers or diuretics. However, in the multivariable analysis, only treatment with beta-blockers or diuretics (which was limited to decompensated cirrhosis) was independently associated with quality of life.

Conclusions: Hepatitis C causes a decrease in quality of life even in the absence of major cognitive impairment. The mechanisms that worsen quality of life are unknown. However, in cirrhotic outpatients with prior decompensations, treatment with beta-blockers or diuretics appears to have an important effect on quality of life.
Keywords: Quality of life; Hepatitis C; Liver disease; Mild cognitive disorder; Hepatic encephalopathy

Impact of liver biopsy size on histological evaluation of chronic viral hepatitis: the smaller the sample, the milder the disease
Guido Colloredo, Maria Guido, Aurelio Sonzogni and Gioacchino Leandro
Background/Aims: In chronic viral hepatitis, liver biopsy is performed for assessing disease activity and fibrosis. In this study, we evaluated the impact of the size of liver biopsy on the grading and staging.

Methods: We selected 161 liver biopsies from patients with chronic types B and C hepatitis on the basis of their length (3 cm) and width (1.4 mm). Ishak scoring system was used for grading and staging. The score was blindly repeated reducing the length of the specimen from 3 to 1.5 cm and to 1 cm long and width from 1.4 to 1 mm.

Results: Reducing the length of the biopsy led to an increase of cases with mild grades: 49.7% in 3 cm, 60.2% in 1.5 cm and 86.6% in 1 cm long specimens (P<0.001). Similarly, cases staged as having mild fibrosis significantly increased in the shorter specimens: 59% in 3 cm, 68.3% in 1.5 cm and 80.1% in 1 cm long specimens (P<0.001). As for the width, both grade and stage were significantly underscored in the 1 mm samples, regardless of their length.

Conclusions: Liver biopsy size strongly influences the grading and staging of chronic viral hepatitis. The use of fine needles should be discouraged in this setting.
Keywords: Liver biopsy; Chronic hepatitis; Grading; Staging; Sample size

Effects of the CCR5-32 mutation on antiviral treatment in chronic hepatitis C
Golo Ahlenstiel et al.
Background/Aims: The CC-chemokine receptor (CCR) 5-32 mutation may predispose to chronic liver disease and high level viremia in hepatitis C. However, it is unclear whether CCR5-32 also affects the response to antiviral treatment.

Methods: We determined CCR5 genotypes in patients with hepatitis C treated with either interferon- (N=78) or interferon and ribavirin (N=78). In each group, rates of end of treatment responses (ETRs) and sustained virological responses (SVRs) were compared between CCR5-32 carriers and homozygous CCR5 wildtype patients.

Results: ETR and SVR were achieved in 25 and 12 patients with interferon- and in 52 and 45 patients with interferon/ribavirin treatment, respectively. CCR5-32 carriers had significantly lower ETR rates than homozygous CCR5 wildtype patients (10.5 vs. 39.0%; P=0.02), whereas SVR rates only showed a non-significant trend (5.3 vs. 18.6%). Multivariate analysis confirmed CCR5-32 carriage as an independent negative predictor for ETR in interferon- monotherapy (odds ratio: 0.16; 95% confidence limits: 0.032-0.82; P=0.03). In interferon/ribavirin treated patients CCR-32 carriers and CCR5 wildtype patients had similar ETR rates [19.2% vs. 23.1%] and SVR rates [20.0% vs. 21.2%].

Conclusions: Response rates to interferon- monotherapy are reduced in hepatitis C virus (HCV)-infected patients carrying the CCR5-32 mutation. However, interferon/ribavirin combination treatment may overcome this negative effect of CCR5-32.

Characterisation of hepatitis B virus X protein mutants in tumour and non-tumour liver cells using laser capture microdissection
Massimo Iavarone et al.
Background/Aims: The analysis of hepatitis B virus (HBV) X protein genetic variability and is correlation with liver disease severity have only been addressed, so far, on whole liver extracts. We have studied, therefore, the HBV X protein (HBx) gene sequence in morphologically well-characterised tumour and non-tumour liver cells from patients with HBV-related hepatocellular carcinoma.

Methods: Using laser capture microdissection (LCM), we picked up six to eight groups of tumour and non-tumour hepatocytes in serial frozen sections from six patients. After global DNA preamplification followed by HBx-specific polymerase chain reaction, the HBx gene was sequenced in each group of microdissected cells. We also validated the quantification of HBV-DNA in microdissected hepatocytes using HBV Amplicor®.

Results: Heterogeneous mutations in HBx gene were found in distinct cirrhotic nodules and tumour areas from the same patient. Mutations at aa 127, 130 and 131 were frequently detected but there was no distinct point mutation profile between tumour and non-tumour samples. In contrast, deletions in HBx gene, which were found in five/six patients, were more frequent in tumour-derived sequences (6/18) than in non-tumour-derived sequences (1/20).

Conclusions: We have shown that LCM provides a direct insight of intrahepatic HBV infection. Using this technique, we demonstrated the persistence of distinct HBx encoding sequences in clonally expanding cells, thus supporting the hypothesis that HBx deletions may be implicated in liver carcinogenesis.
Keywords: Hepatitis B; X protein; Hepatocellular carcinoma; Laser capture microdissection

The absence of up-regulation of telomerase activity during regeneration after partial hepatectomy in hepatitis B virus X gene transgenic mice
Hiroshige Kojima et al.
Background/Aims: Transgenic mice that express HBV X protein (HBx) have increased sensitivity to hepatocarcinogens. In the present study, we hypothesized that HBx interferes with the DNA protective increases in telomerase activity that occur in proliferating hepatocytes.

Methods: Male CD-1 mice (4-6/grp) were killed and hepatic telomerase activity measured at 0, 6, 12, 24, 36, 48 h post partial hepatectomy (PHx). Four HBx transgenic mice were killed at 12 h post-PHx when maximum telomerase activity was observed in CD-1 non-transgenic mice. mRNA of the telomerase catalytic subunit; murine telomerase reverse transcriptase (mTERT), was measured by reverse transcription-polymerase chain reaction. Telomerase activity and human TERT (hTERT) were also measured in Chang and PLC/PRF/5 cells following transient transfection with HBx cDNA.

Results: Telomerase activity peaked at 12 h post-PHx in normal mice, however, in HBx transgenic mice, telomerase activity was significantly lower, both at baseline (P<0.05) and 12 h post-PHx (P<0.01). Following PHx, mTERT mRNA expression remained constant in normal mice but decreased significantly (P<0.01) in HBx transgenic mice. Transfection of HBx in Chang and PLC/PRF/5 cells had no effect on telomerase activity or hTERT mRNA expression.

Conclusions: The results of this study suggest that HBx expression may play a role in hepatocellular carcinogenesis by interfering with telomerase activity during hepatocyte proliferation.
Keywords: Telomerase; Murine telomerase reverse transcriptase; Telomere; HBV X protein; Transgenic mice; Hepatocellular carcinoma; Hepatitis B

Abbreviations: HCC, Hepatocellular carcinoma; HBV, hepatitis B; HBx, HBV X protein; PHx, partial hepatectomy; TERT, telomerase reverse transcriptase; PCR, polymerase chain reaction; ELISA, enzyme-linked immunosorbent assay

Copyright © 2001-2003 European Association for the Study of the Liver. All rights reserved.

BRITISH MEDICAL JOURNAL

NEW ENGLAND JOURNAL

LANCET

Volume 362, Number 9385 30 August 2003

Operative morbidity of living liver donors in Japan
Koji Umeshita, Kenji Fujiwara, Kendo Kiyosawa, Masatoshi Makuuchi, Susumu Satomi, Keizo Sugimachi, Koichi Tanaka, Morito Monden, for the Japanese Liver Transplantation Society

Background Deaths of living liver donors have been reported in western countries, whereas the morbidity and mortality of such donors in Japan, one of the leading countries for living liver transplantation, have not been reported in detail. We aimed to review the operative morbidity and mortality of such donors in Japan.

Methods 1853 donors of 1852 living liver transplants done in 46 liver transplant centres, and registered in the database of the Japanese Liver Transplantation Society, were assessed for eight donor-related factors of morbidity and mortality. Data for 1841 donors were analysed.

Findings No perioperative mortality was recorded since inception of the liver transplantation programme in Japan from Nov 13, 1989, to April 11, 2002. 244 postoperative complications were reported in 228 (12%) donors. The frequency of complications was significantly higher in donors of the right liver lobe than in those involving the lateral segment, and left lobe graft (p<0·0001, and p<0·0001, respectively). Postoperative hospital stay was significantly longer in donors of the right lobe (mean 19·7 [SD 13·0]) than in those of the lateral segment (14·2 [7·6]), left lobe (14·0 [6·5]), and left lobe and caudate lobe (16·3 [12·1]). Re-operation related to donor hepatectomy was done in 23 donors.

Interpretation By contrast with western countries, no perioperative mortality was recorded in living liver donors in Japan. However, a proportion of these donors developed serious complications. This morbidity should be reduced to maintain zero mortality in living liver donors.

Lancet 2003; 362: 687-90. Published online August 19, 2003