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Revue de Presse du Mois Mois de Juin 2003

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HEPATOLOGY

Table of Contents for May 2003 · Volume 37 · Number 5

Liver Biology and Pathobiology

Differentiation of embryonic stem cells into hepatocytes: Biological functions and therapeutic application
Hanako Yamamoto, Gary Quinn, Akira Asari, Hiroko Yamanokuchi, Takumi Teratani, Masaaki Terada, Takahiro Ochiya
Embryonic stem (ES) cells provide a unique source for tissue regeneration. We examined whether mouse ES cells can efficiently differentiate into transplantable hepatocytes. ES cells were implanted into mouse livers 24 hours after carbon tetrachloride intoxication; ES-derived cells with several hepatocyte-cell-markers were generated. They were able to grow in vitro and showed morphology consistent with typical mature hepatocytes and expressed hepatocyte-specific genes. After transplantation into the carbon tetrachloride-injured mouse liver, ES-derived green fluorescent protein-positive cells were incorporated into liver tissue and rescued mice from hepatic injury. No teratoma formation was observed in the transplant recipients. In conclusion, ES cells can provide a valuable tool for studying the molecular basis for differentiation of hepatocytes and form the basis for cell therapies. (HEPATOLOGY 2003;37:983-993.)

Repopulation of rat liver by fetal hepatoblasts and adult hepatocytes transduced ex vivo with lentiviral vectors
Michael Oertel, Richard Rosencrantz, Yuan-Qing Chen, Prashanthi N. Thota, Jaswinderpal S. Sandhu, Mariana D. Dabeva, Annmarie L. Pacchia, Martin E. Adelson, Joseph P. Dougherty, David A. Shafritz
Recent studies have shown that nondividing primary cells, such as hepatocytes, can be efficiently transduced in vitro by human immunodeficiency virus-based lentivirus vectors. Other studies have reported that, under certain conditions, the liver can be repopulated with transplanted hepatocytes. In the present study, we combined these procedures to develop a model system for ex vivo gene therapy by repopulating rat livers with hepatocytes and hepatoblasts transduced with a lentivirus vector expressing a reporter gene, green fluorescent protein (GFP). Long-term GFP expression in vivo (up to 4 months) was achieved when the transgene was driven by the liver-specific albumin enhancer/promoter but was silenced when the cytomegalovirus (CMV) enhancer/promoter was used. Transplanted cells were massively amplified (~10 cell doublings) under the influence of retrorsine/partial hepatectomy, and both repopulation and continued transgene expression in individual cells were documented by dual expression of a cell transplantation marker, dipeptidyl peptidase IV (DPPIV), and GFP. In this system, maintenance or expansion of the transplanted cells did not depend on expression of the transgene, establishing that positive selection is not required to maintain transgene expression following multiple divisions of transplanted, lentivirus-transduced hepatic cells. In conclusion, fetal hepatoblasts (liver stem/progenitor cells) can serve as efficient vehicles for ex vivo gene therapy and suggest that liver-based genetic disorders that do not shorten hepatocyte longevity or cause liver damage, such as phenylketonuria, hyperbilirubinemias, familial hypercholesterolemia, primary oxalosis, and factor IX deficiency, among others, might be amenable to treatment by this approach. HEPATOLOGY 2003;37:994-1005.)

Keratin mutation in transgenic mice predisposes to Fas but not TNF-induced apoptosis and massive liver injury
Nam-On Ku, Roy M. Soetikno, M. Bishr Omary
Hepatocytes express keratins 8 and 18 (K8/18) as their only cytoskeletal intermediate filament (IF) proteins, and K8/18 mutations predispose their carriers to liver cirrhosis. Transgenic mice that overexpress mutant human K18 (Arg89Cys [R89C]) develop mild chronic hepatitis, hepatocyte fragility, keratin filament disruption, and increased susceptibility to drug-induced liver injury. K18 is a major caspase substrate during apoptosis, and K8- or K18-null mice are significantly predisposed to Fas- and possibly tumor necrosis factor (TNF)-mediated apoptosis in the liver. Here we tested the potential role of the K18 R89C mutation on Fas- or TNF-mediated apoptotic liver injury by injecting Fas antibody (Ab) or TNF- plus actinomycin D into mice that overexpress wild-type (WT) human K18 (with intact filament network, termed TG2 mice) or into K18 R89C mice (with disrupted filament network). K18 R89C mice are significantly more susceptible to Fas-mediated liver injury compared with nontransgenic and TG2 mice. This included differences in lethality, histology, apoptosis, and serum transaminase levels. In contrast, K18 WT and R89C mice manifest similar sensitivity to TNF-induced injury. Both Fas- and TNF-induced apoptosis in liver tissues are associated with caspase-mediated K18 degradation and increased keratin phosphorylation on several but not all sites. In conclusion, transgenic mouse K18 mutation and its consequent keratin filament disruption predispose hepatocytes to Fas- but not TNF-mediated apoptotic injury. This supports the association of keratin mutations with cirrhosis in patients with liver disease and suggests that keratins modulate apoptosis induced by Fas but not TNF. (HEPATOLOGY 2003;37:1006-1014.)

Overexpression of thioredoxin prevents acute hepatitis caused by thioacetamide or lipopolysaccharide in mice
Hiroaki Okuyama, Hajime Nakamura, Yasuyuki Shimahara, Shinichi Araya, Norifumi Kawada, Yoshio Yamaoka, Junji Yodoi
Thioredoxin (Trx) is a small redox-active protein with antioxidant and antiapoptotic effects. Trx transgenic (Tg) mice are more resistant to cerebral infarction and survive longer than wild-type (WT) C57BL/6 mice. The aim of the present study was to investigate the protective role of Trx in acute hepatitis models. The expression of endogenous Trx was decreased in thioacetamide (TAA)-induced acute hepatitis. TAA (100 µg/g) was injected intraperitoneally in WT and Tg mice. Survival rate after TAA injection was higher in Tg mice than in WT mice. The level of oxidative stress was significantly less in Tg mice than in WT mice, as shown by the protein carbonylation assay and lipid peroxidation assay. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)-positive cells were less in Tg mice than in WT mice, which was consistent with DNA laddering assay. Caspase-3 and caspase-9 activities and cytochrome c release were significantly inhibited in Tg mice compared with those in WT mice. In addition, lipopolysaccharide (LPS) plus D-galactosamine (GalN), or anti-Fas antibody (Jo2) were injected. Survival rate after LPS plus GalN injection was much higher in Tg mice than in WT mice. In contrast, there was no difference in survival rate after Jo2 injection between WT and Tg mice. In conclusion, transgene of Trx attenuated TAA- or LPS-induced acute lethal hepatitis. In addition to an antioxidant effect, Trx has the potential to protect acute liver injury via an antiapoptotic effect, which mainly inhibits mitochondria-mediated apoptosis signaling. (HEPATOLOGY 2003;37:1015-1025.)

Rat hepatocyte aquaporin-8 water channels are down-regulated in extrahepatic cholestasis
Flavia I. Carreras, Sergio A. Gradilone, Amelia Mazzone, Fabiana García, Bing Q. Huang, J. Elena Ochoa, Pamela S. Tietz, Nicholas F. LaRusso, Giuseppe Calamita, Raúl A. Marinelli
Hepatocytes express the water channel aquaporin-8 (AQP8), which is mainly localized in intracellular vesicles, and its adenosine 3´,5´-cyclic monophosphate (cAMP)-induced translocation to the plasma membrane facilitates osmotic water movement during canalicular bile secretion. Thus, defective expression of AQP8 may be associated with secretory dysfunction of hepatocytes caused by extrahepatic cholestasis. We studied the effect of 1, 3, and 7 days of bile duct ligation (BDL) on protein expression, subcellular localization, and messenger RNA (mRNA) levels of AQP8; this was determined in rat livers by immunoblotting in subcellular membranes, light immunohistochemistry, immunogold electron microscopy, and Northern blotting. One day of BDL did not affect expression or subcellular localization of AQP8. Three days of BDL reduced the amount of intracellular AQP8 (75%; P < .001) without affecting its plasma membrane expression. Seven days after BDL, AQP8 was markedly decreased in intracellular (67%; P < .05) and plasma (56%; P < .05) membranes. Dibutyryl cAMP failed to increase AQP8 in plasma membranes from liver slices, suggesting a defective translocation of AQP8 in 7-day BDL rats. Immunohistochemistry and immunoelectron microscopy in liver sections confirmed the BDL-induced decreased expression of hepatocyte AQP8 in intracellular vesicles and canalicular membranes. AQP8 mRNA expression was unaffected by 1-day BDL but was significantly increased by about 200% in 3- and 7-day BDL rats, indicating a posttranscriptional mechanism for protein level reduction. In conclusion, BDL-induced extrahepatic cholestasis caused posttranscriptional down-regulation of hepatocyte AQP8 protein expression. Defective expression of AQP8 water channels may contribute to bile secretory dysfunction of cholestatic hepatocytes. (HEPATOLOGY 2003;37:1026-1033.)

The normal liver harbors the vitamin D nuclear receptor in nonparenchymal and biliary epithelial cells
Marielle Gascon-Barré, Christian Demers, Ali Mirshahi, Stéphane Néron, Sylvia Zalzal, Antonio Nanci
The liver is generally considered negative for the vitamin D nuclear receptor (VDRn), even though several studies have shown significant effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on liver cell physiology. The low abundance of VDRn in the liver led us to propose that hepatocytes (the largest hepatic cell population) were most likely negative for the receptor, whereas the small hepatic sinusoidal and ductular cell populations that contain cell types known to express VDRn in other tissues should express the receptor. Using freshly isolated cells from normal livers as well as biliary and epithelial hepatic cell lines, our data show that the human, rat, and mouse hepatocytes express very low VDRn messenger RNA (mRNA) and protein levels. In contrast, sinusoidal endothelial, Kupffer, and stellate cells of normal rat livers as well as the mouse biliary cell line BDC and rat hepatic neonatal epithelial SD6 cells clearly expressed both VDRn mRNA and protein. In addition, specimens of human hepatocarcinoma as well as intrahepatic colon adenocarcinoma metastases were also found to express the VDRn gene transcript. Kupffer, stellate, and endothelial cells responded to 1,25(OH)2D3 by a significant increase in the CYP24, indicating that the VDRn is fully functional in these cells. In conclusion, selective hepatic cell populations are targets for the vitamin D endocrine/paracrine/intracrine system. (HEPATOLOGY 2003;37:1034-1042.)

Toll-like receptor 4 mediates inflammatory signaling by bacterial lipopolysaccharide in human hepatic stellate cells (*Human Study*)
Yong-Han Paik, Robert F. Schwabe, Ramón Bataller, Maria P. Russo, Christian Jobin, David A. Brenner
Bacterial lipopolysaccharide (LPS) stimulates Kupffer cells and participates in the pathogenesis of alcohol-induced liver injury. However, it is unknown whether LPS directly affects hepatic stellate cells (HSCs), the main fibrogenic cell type in the injured liver. This study characterizes LPS-induced signal transduction and proinflammatory gene expression in activated human HSCs. Culture-activated HSCs and HSCs isolated from patients with hepatitis C virus-induced cirrhosis express LPS-associated signaling molecules, including CD14, toll-like receptor (TLR) 4, and MD2. Stimulation of culture-activated HSCs with LPS results in a rapid and marked activation of NF-B, as assessed by in vitro kinase assays for IB kinase (IKK), IB steady-state levels, p65 nuclear translocation, NF-B-dependent luciferase reporter gene assays, and electrophoretic mobility shift assays. Lipid A induces NF-B activation in a similar manner. Both LPS- and lipid A-induced NF-B activation is blocked by preincubation with either anti-TLR4 blocking antibody (HTA125) or Polymyxin B. Lipid A induces NF-B activation in HSCs from TLR4-sufficient (C3H/OuJ) mice but not from TLR4-deficient (C3H/HeJ) mice. LPS also activates c-Jun N-terminal kinase (JNK), as assessed by in vitro kinase assays. LPS up-regulates IL-8 and MCP-1 gene expression and secretion. LPS-induced IL-8 secretion is completely inhibited by the IB super repressor (Ad5IB) and partially inhibited by a specific JNK inhibitor, SP600125. LPS also up-regulates cell surface expression of ICAM-1 and VCAM-1. In conclusion, human activated HSCs utilize components of TLR4 signal transduction cascade to stimulate NF-B and JNK and up-regulate chemokines and adhesion molecules. Thus, HSCs are a potential mediator of LPS-induced liver injury. (HEPATOLOGY 2003;37:1043-1055.)

ADAM12 in human liver cancers: TGF--Regulated expression in stellate cells is associated with matrix remodeling
Hélène Le Pabic, Dominique Bonnier, Ulla M. Wewer, Alexandre Coutand, Orlando Musso, Georges Baffet, Bruno Clément, Nathalie Théret
"A disintegrin and metalloproteinases" (ADAMs) form a family of cell-surface glycoproteins with potential protease and cell-adhesion activities. We have investigated ADAM expression in human liver cancers and their regulation by several cytokines involved in liver injury. Using degenerative RT-PCR, cDNA encoding sequences for ADAM9 and ADAM12 were identified in human activated hepatic stellate cells (HSCs). Northern blot analyses showed that HSCs, but not hepatocytes, expressed transcripts for ADAM9 messenger RNA (mRNA) and both the long and short forms of ADAM12. This expression was associated with the transition from quiescent to activated state of rat HSCs and markedly increased in human livers with cirrhosis. ADAM12 but not ADAM9 expression was up-regulated by transforming growth factor (TGF-) in human activated HSCs. The PI3K inhibitor LY294002 and the mitogen-activated protein kinase kinase (MEK) inhibitor UO126 prevented ADAM12 induction by TGF-, suggesting the involvement of PI3K and MEK activities. In vivo, the steady-state of both ADAM9 and ADAM12 mRNA levels was nearly undetectable in both normal livers and benign tumors and increased in hepatocellular carcinomas (up to 3- and 6-fold, respectively) and liver metastases from colonic carcinomas (up to 40- and 60-fold, respectively). The up-regulation of both ADAM9 and ADAM12 was correlated with an increase in matrix metalloproteinase 2 expression and activity. In conclusion, in liver cancers ADAM9 and ADAM12 expression is associated with tumor aggressiveness and progression. (HEPATOLOGY 2003;37:1056-1066.)

Conditional tetracycline-regulated expression of TGF-1 in liver of transgenic mice leads to reversible intermediary fibrosis
Elke Ueberham, Rainer Löw, Uwe Ueberham, Kai Schönig, Hermann Bujard, Rolf Gebhardt
Based on the tetracycline-regulated gene expression system, a double-transgenic mouse model for liver fibrosis was established in which the expression of transforming growth factor 1 (TGF-1) can be regulated deliberately by addition or removal of doxycycline hydrochloride to the drinking water. TGF-1 plasma levels in induced double-transgenic mice reached values ranging from 250 to 1,200 ng/mL, being 10 to 30 times above the normal plasma levels. By applying a cyclic induction-deinduction protocol, deleterious effects of the high plasma TGF-1 levels were overcome. By using this protocol, liver fibrosis occurred within a few cycles and progressed further to an intermediary fibrosis when cyclic induction was continued. On histochemical staining, a marked perisinusoidal deposition of extracellular matrix was detected accompanied by the activation of hepatic stellate cells as shown by alpha-smooth muscle actin (-SMA) expression. Apoptosis of hepatocytes was prominent in TGF-1 high producers, leading to a decreasing number of TGF-1-expressing cells with time. No compensatory proliferation of hepatocytes could be detected. In advanced stages, fibrogenesis could be stopped by switching off TGF-1 production and reversal of fibrosis could be shown by (immuno)histochemistry within 6 to 21 days. Determination of messenger RNA (mRNA) levels of procollagen I and III, laminin (B1), matrix metalloproteinase (MMP)-2, -9, and -13, and tissue inhibitor of matrix metalloproteinase (TIMP)-1 and -2 by real-time reverse-transcription polymerase chain reaction (RT-PCR) provided insight into some mechanistic details of the fibrogenic process and its reversal. In conclusion, this model will enable the analysis of fibrogenesis at progressive stages and help in elucidating the cellular changes during development and regression of liver fibrosis caused by elevated TGF-1 expression. (HEPATOLOGY 2003;37:1067-1078.)

MHC class II-expressing hepatocytes function as antigen-presenting cells and activate specific CD4 T lymphocyutes
Johannes Herkel, Bettina Jagemann, Christiane Wiegard, Jose Francisco Garcia Lazaro, Stefan Lueth, Stephan Kanzler, Manfred Blessing, Edgar Schmitt, Ansgar W. Lohse
The ability to activate CD4 T cells is restricted to antigen-presenting cells that express major histocompatibility complex (MHC) class II molecules. Parenchymal cells normally do not express MHC class II molecules; however, in clinical hepatitis, viral or autoimmune, hepatocytes often exhibit aberrant MHC class II expression. It is not known whether MHC class II-expressing hepatocytes can function as antigen-presenting cells, but it has been suggested that aberrant MHC class II expression by parenchymal cells may cause autoimmune disease. Therefore, we generated transgenic mice that specifically overexpress class II transactivator molecules in hepatocytes. Hepatocytes from these mice exhibited stable MHC class II expression and were used to stimulate CD4 T cells from T-cell receptor transgenic mice and CD4 T-cell lines. MHC II-expressing hepatocytes featured costimulatory CD80 molecules and could serve as antigen-presenting cells that were able to process protein antigen and to activate specific CD4 T cells. Nevertheless, the transgenic mice with aberrant hepatocellular MHC class II expression did not exhibit any symptoms of autoimmune disease. In conclusion, MHC II-expressing hepatocytes, as found in clinical hepatitis, can present antigen and activate CD4 T cells. The ability of hepatocytes to present antigen on MHC II molecules does not seem to be a sufficient cause for inflammatory autoimmunity and hepatitis. However, we still need to explore whether such antigen presentation is occurring in vivo. The transgenic mice described in this study may serve as a model to study the immune interaction of hepatocytes and CD4 T cells in both in vitro and in vivo.(HEPATOLOGY 2003;37:1079-1085.)

Troglitazone induces p27Kip1-associated cell-cycle arrest through down-regulating Skp2 in human hepatoma cells (*Human Study*)
Hironori Koga, Masaru Harada, Motoaki Ohtsubo, Shoichiro Shishido, Hiroto Kumemura, Shinichiro Hanada, Eitaro Taniguchi, Katsumi Yamashita, Ryukichi Kumashiro, Takato Ueno, Michio Sata
Increasing evidence has confirmed that ligands for peroxisome proliferator-activated receptor (PPAR) exhibit antitumoral effects through inhibition of cell proliferation and induction of cell differentiation in several malignant neoplasms. Recently, we have documented the accumulation of a cyclin-dependent kinase inhibitor, p27Kip1, as well as an unexpected accumulation in cyclin E in G1-arrested human hepatoma cells treated with the PPAR ligand troglitazone. Simultaneous accumulations in both p27Kip1 and cyclin E are known to be characteristic phenotypes in cells derived from mice lacking Skp2, an F-box protein component of the SCF ubiquitin-ligase complex. Thus, the aim of the present study was to assess whether Skp2 might be involved in the down-regulation of p27Kip1 in troglitazone-treated human hepatoma cells. A striking decrease in Skp2 expression and a reciprocal increase in p27Kip1 expression were found in troglitazone-treated hepatoma cells but not in those cells treated with other PPAR ligands such as pioglitazone and ciglitazone. Quantitative real-time RT-PCR analysis showed that troglitazone down-regulated Skp2 at the mRNA levels. Consistently, ectopic overexpression in Skp2 brought resistance to troglitazone, resulting in a decreased population of arrested cells at the G1 phase compared with that in the mock-transfected cells. In surgically resected hepatocellular carcinoma (HCC) tissue, an increased expression in Skp2 was found in both the moderately differentiated HCCs and the poorly differentiated HCCs. In conclusion, troglitazone attenuated Skp2 expression, thereby promoting p27Kip1 accumulation in human hepatoma cells. This therapeutic potential of the ligand may lead to new cell-cycle-based antitumor strategies for advanced HCCs. (HEPATOLOGY 2003;37:1086-1096.)

Inhibition of cholangiocarcinoma growth by tannic acid
Carla Marienfeld, Laura Tadlock, Yoko Yamagiwa, Tushar Patel
Cholangiocarcinoma is an aggressive malignancy of the biliary tract for which effective treatment is lacking. Tannic acid (TA) is a naturally occurring polyphenolic compound with antioxidant and radical scavenging properties as well as anticarcinogenic effects. TA inhibited proliferation of malignant human cholangiocytes in vitro. Furthermore, the growth rate of Mz-ChA-1 cholangiocarcinoma xenografts in balb/c athymic mice was reduced from 10.9 ± 1.8 mm3/d in mice fed with normal water to 5.5 ± 1.2 mm3/d in mice fed with water containing 0.05% TA. Pretreatment with 50 µg/mL TA for 24 hours before xenograft implantation increased tumor latency by 2.5-fold compared with untreated controls, and decreased subsequent growth rates compared with controls in the absence of TA feeding. TA was not cytotoxic to Mz-ChA-1 cells in vitro, but enhanced sensitivity to camptothecin cytotoxicity. TA potently inhibited cell cycle progression, and increased expression of the cyclin-dependent kinase inhibitor p27KIP1. In addition, TA (0-50 µg/mL) inhibited proteasomal activity in cholangiocyte cell extracts in a concentration-dependent manner. In conclusion, the growth inhibitory effects of TA may result from dysregulation of cell cycle progression due to altered proteasomal degradation of these cell cycle regulatory proteins. TA warrants evaluation as a candidate for the treatment of human cholangiocarcinoma either by itself or in combination with other chemotherapeutic agents. (HEPATOLOGY 2003;37:1097-1104.)

Liver Failure and Liver Disease

Angiopoietins and tie-2 expression in angiogenesis and proliferation of human hepatocellular carcinoma (*Human Study*)
Noboru Mitsuhashi, Hiroaki Shimizu, Masayuki Ohtsuka, Yasuo Wakabayashi, Hiroshi Ito, Fumio Kimura, Hiroyuki Yoshidome, Atsushi Kato, Yuji Nukui, Masaru Miyazaki
Human hepatocellular carcinoma (HCC) is a hypervascular tumor but the mechanisms underlying the process of angiogenesis are not fully understood. Angiopoietins (Ang) have been recently identified as ligands for Tie-2 receptor and are thought to be important factors in vascular maturation and stability during angiogenesis. In this study, we investigated the expression of Ang-1, Ang-2, Tie-2, and vascular endothelial growth factor (VEGF) in surgically resected specimens from 46 patients with HCC to determine their potential role in tumor angiogenesis and its progression. VEGF messenger RNA (mRNA) was significantly up-regulated in HCC compared to normal liver tissue from patients with hepatic metastases. No differences were found between HCC and adjacent liver tissue. Meanwhile, Ang-2 mRNA expression in HCC was significantly increased when compared to adjacent liver tissue. On the other hand, Ang-1 and Tie-2 mRNA expression in HCC was not different from that in adjacent liver tissue. Immunohistochemical staining also showed increased Ang-2 protein in HCC. Furthermore, a high Ang-2/1 mRNA ratio in HCC was closely associated with tumor portal vein invasion, tumor diameter, and the microvessel density level as assessed by CD34 immunostaining. With regard to prognosis, the survival time for patients in the high Ang-2/1 mRNA ratio group was significantly poorer when compared with the low Ang-2/1 mRNA ratio group. In conclusion, an increased expression of Ang-2/1 in the presence of VEGF may play a critical role in promoting tumor angiogenesis and progression in human HCC. (HEPATOLOGY 2003;37:1105-1113.)

Des-gamma carboxyprothrombin can differentiate hepatocellular carcinoma from nonmalignant chronic liver disease in American patients (*Human Study*)
Jorge A. Marrero, Grace L. Su, Wei Wei, Dawn Emick, Hari S. Conjeevaram, Robert J. Fontana, Anna S. Lok
Mortality due to hepatocellular carcinoma (HCC) has not improved over the last 20 years. This is in part due to the poor performance of available tumor markers leading to delays in diagnosis. Des-gamma carboxy-prothrombin (DCP) has been reported to be more sensitive and specific for the diagnosis of HCC in Japanese patients compared with -fetoprotein (AFP). We conducted a cross-sectional case control study to evaluate whether DCP is more sensitive and specific than AFP for differentiating HCC from nonmalignant liver disease in a cohort of American patients from a single referral center. Four groups were studied: G1, normal healthy subjects; G2, patients with noncirrhotic chronic hepatitis; G3, patients with compensated cirrhosis; and G4, patients with histologically proven HCC. A total of 207 subjects were enrolled. Both DCP and AFP levels increased progressively from G1 to G4, but DCP values had less overlap among the groups than AFP. ROC curve indicated that a DCP value of 125 mAU/mL yielded the best sensitivity (89%; 95% CI, 77%-95%) and specificity (95%; 95% CI, 82%-96%) for differentiating patients with HCC from those with cirrhosis and chronic hepatitis. The optimal AFP cutoff value was 11 ng/mL and was inferior to the DCP value of 125 mAU/mL, the area under the ROC curves being 0.928 versus 0.810, respectively (P = .002). In conclusion, DCP was more sensitive and specific than AFP for differentiating HCC from nonmalignant chronic liver disease. Prospective studies to evaluate the role of DCP in early HCC are underway. (HEPATOLOGY 2003;37:1114-1121.)

Human WISP1v, a member of the CCN family, is associated with invasive cholangiocarcinoma (*Human Study*)
Shinji Tanaka, Keishi Sugimachi, Toshifumi Kameyama, Shin-ichiro Maehara, Ken Shirabe, Mitsuo Shimada, Jack R. Wands, Yoshihiko Maehara
Family members of the connective tissue growth factor, cysteine-rich 61, nephroblastoma over-expressed gene (CCN) encode cysteine-rich secreted proteins with roles in human fibrotic disorders and tumor progression. In this study, we identified a CCN family member, WISP1v, as over-expressed in human cholangiocarcinomas. Genetic analysis of WISP1v was performed on surgically resected specimens of cholangiocarcinoma. The WISP1v biological effects were analyzed using the HuCCT1 human cholangiocarcinoma cell line. The WISP1v gene was expressed in 19 of 39 cholangiocarcinoma tissues (49%) but not in normal livers. Expression of WISP1v was significantly associated with lymphatic and perineural invasion of tumor cells (P < .05), as well as a poor clinical prognosis (P < .01). In the intraductal papillary cholangiocarcinomas, WISP1v was detected only in the cases with duct wall invasion but not in the cases without duct wall invasion (P < .05). No mutation of WISP1v gene was detected in the examined samples. In vitro analysis revealed that WISP1v stimulated the invasive phenotype of cholangiocarcinoma cells with activation of both p38 and p42/p44 mitogen-activated protein kinases (MAPKs). Furthermore, WISP1v-induced cholangiocarcinoma invasion was significantly suppressed by the p38 MAPK inhibitor SB203580 but not by the p42/p44 MAPK kinase (MEK) inhibitor PD98059. Our findings suggest that WISP1v-mediated signaling is involved in the generation of invasive cellular properties and leads to progression of cholangiocarcinoma. (HEPATOLOGY 2003;37:1122-1129.)

Alterations in bronchoalveolar lavage fluid during ischemia-induced acute hepatic failure in the pig
Georgia Kostopanagiotou, Christina Routsi, Vassilios Smyrniotis, Marilena E. Lekka, Eirini Kitsiouli, Nikolaos Arkadopoulos, George Nakos
The objective of this controlled experimental animal study was to evaluate whether acute hepatic failure (AHF) can cause acute lung injury (ALI) and to investigate possible pathophysiologic mechanisms. Seventeen domestic pigs were randomly assigned to AHF and sham groups. AHF was induced by surgical devascularization of liver in 10 animals. Seven animals were sham operated. Hemodynamics, lung mechanics, extravascular lung water (EVLW), and intracranial pressure, blood gas, liver function tests, and serum endotoxin levels were measured. Cells count, total protein, and phospholipids and phospholipases A2 were determined in the bronchoalveolar lavage (BAL) fluid. Measurements were obtained after the insertion of central lines and 4 hours and 7 hours after the completion of the surgical procedure. Hemodynamic, biochemical, neuromonitoring, and histologic data confirmed the development of liver failure. Seven hours after devascularization, EVLW was higher in AHF (13.7 ± 1.8 mL/kg) compared with the sham group (5.9 ± 0.7 mL/kg) (P < .05); in AHF, increase of neutrophils (5% ± 8% to 25% ± 8%, P < .001), total protein (6.2 ± 3.7 to 11.2 ± 6.5 µg/mL, P < .048), and phospholipase A2 (1.43 ± 0.56 to 2.38 ± 1.38 nmoL/mL/h, P < .03) and decrease in PAF-acetylhydrolase (0.114 ± 0.128 to 0.039 ± 0.038 nmol/mL/h, P < .01) compared with baseline were observed; total phospholipids decreased in AHF and increased in the sham model. Histologic examination confirmed lesions compatible with acute lung injury. In conclusion, AHF due to hepatic devascularization induced acute lung injury, confirmed by the increase of inflammatory cells in the alveoli as well as by histologic findings. The decreased PAF-AcH and the increased phospholipase A2 may play a significant role in the perpetuation of inflammation accompanied by surfactant disorders. (HEPATOLOGY 2003;37:1130-1138.)

Sonographic criteria for the diagnosis of hepatic involvement in hereditary hemorrhagic telangiectasia (HHT) (*Human Study*)
Martin Caselitz, Matthias J. Bahr, Jörg S. Bleck, Ajay Chavan, Michael P. Manns, Siegfried Wagner, Michael Gebel
Hepatic involvement in hereditary hemorrhagic telangiectasia (HHT) is highly variable and may lead to severe clinical symptoms such as heart failure. This controlled, prospective study defined sonographic criteria for hepatic involvement in HHT. Color Doppler sonography and pulsed Doppler sonography were used to study 25 patients with HHT and liver involvement, 20 patients with HHT without liver involvement, 25 patients with cirrhosis, and 25 patients without liver disease. The diagnosis of hepatic manifestation was confirmed by computed tomography and/or angiography. Liver size, parenchymal changes of the liver, vessel diameters, and flow velocities of the portal vein and the hepatic artery were determined. Resistance index (RI) and pulsatility index (PI) were calculated. The diameter of the common hepatic artery was significantly dilated without overlap between HHT patients with liver involvement and the 3 control groups (mean 11.3 ± 2.8 mm [HHT with liver involvement], 4.6 ± 0.9 mm [HHT without liver involvement], 4.8 ± 1.0 mm [cirrhosis], and 4.4 ± 1.0 mm [healthy controls], P < .001). Doppler parameters of the proper hepatic artery differed significantly (all P < .001). In all patients with HHT and liver involvement, areas with intrahepatic hypervascularization caused by dilated intrahepatic arteries were observed in varying intensity. Cardiac output significantly correlated with the diameter of the common hepatic artery (r = 0.53, P = .007) and the portal vein (r = 0.42, P = .05). In conclusion, the diameter of the common hepatic artery (>7 mm) and intrahepatic hypervascularization are suitable sonographic diagnostic parameters of HHT with high sensitivity and specificity. Dilated diameters of the hepatic feeding vessels are indicators for systemic circulatory distress in these patients. (HEPATOLOGY 2003;37:1139-1146.)

Randomized trial comparing albumin and saline in the prevention of paracentesis-induced circulatory dysfunction in cirrhotic patients with ascites (*Human Study*)
Javier Sola-Vera, Josep Miñana, Elena Ricart, Montserrat Planella, Begoña González, Xavier Torras, Jose Rodríguez, José Such, Sonia Pascual, Germán Soriano, Miguel Pérez-Mateo, Carlos Guarner
Paracentesis-induced circulatory dysfunction (PICD) is a recently described complication that can be prevented with the administration of plasma expanders. The aim of this study was to compare the efficacy of saline versus albumin in the prevention of PICD. Patients were randomized to receive albumin or saline after total paracentesis. Patients readmitted as a consequence of a second episode of tense ascites were treated with total paracentesis and the alternative plasma expander. After randomization, 35 patients received saline and 37 received albumin. Twenty-one patients were readmitted for tense ascites and treated with the alternative expander. Significant increases in plasma renin activity (PRA) were found 24 hours and 6 days after paracentesis when saline was used (baseline, 5.6 ± 5.7; 24 hours, 7.6 ± 6.9; 6 days, 8.5 ± 8.0 ng · mL­1 · hr­1; P < .05 and P < .01 vs. baseline, respectively), whereas no significant changes were observed with albumin. The incidence of PICD was significantly higher in the saline group versus the albumin group (33.3% vs. 11.4%, respectively; P = .03). However, no significant differences were found when less than 6 L of ascitic fluid was evacuated (6.7% vs. 5.6% in the saline and albumin groups, respectively; P = .9). Similar results were observed when analyzing patients who received 2 consecutive paracentesis (i.e., a significant increase in PRA after saline [P < .01] without significant variations after albumin). In conclusion, albumin is more effective than saline in the prevention of PICD. Saline is a valid alternative to albumin when less than 6 L of ascitic fluid is evacuated. (HEPATOLOGY 2003;37:1147-1153.)

Peripheral blood mononuclear cell expression of toll-like receptors and relation to cytokine levels in cirrhosis (*Human Study*)
Stephen M. Riordan, Narelle Skinner, Ammar Nagree, Helen McCallum, Christopher J. McIver, Jelica Kurtovic, John A. Hamilton, Stig Bengmark, Roger Williams, Kumar Visvanathan
Activation of macrophages by endotoxin is assumed responsible for increased circulating tumor necrosis factor (TNF-) and soluble TNF receptor (sTNFR) levels in cirrhosis. Relevant to this is expression of Toll-like receptor (TLR) 4 and TLR2, which is critically involved in production of TNF- in response to endotoxin and Gram-positive microbial stimuli, respectively. The first studies on this in cirrhosis are reported here. In 36 cirrhotic patients and 32 controls, we measured (1) circulating endotoxin, TNF-, and sTNFR levels; (2) peripheral blood mononuclear cell (PBMC) expression of TLR4 and TLR2, and (3) in vitro TNF- production by PBMCs stimulated with endotoxin or Staphylococcus aureus enterotoxin B (SEB). PBMC expression of TLR2, circulating TNF- levels, and in vitro TNF- production were reassessed after supplementation with a synbiotic regimen known to increase intestinal levels of Gram-positive bacteria. Endotoxin, TNF-, and sTNFR levels were significantly increased in cirrhosis. Endotoxin levels did not correlate significantly with other parameters. PBMC expression of TLR2 but not TLR4 was significantly up-regulated in cirrhosis and correlated significantly with serum TNF- and sTNFR levels. In vitro TNF- production by PBMCs stimulated by SEB was significantly blunted. Supplementation with the synbiotic regimen resulted in significant up-regulation of PBMC expression of TLR2. Serum TNF- levels were further increased and in vitro TNF- production further reduced in most patients. In conclusion, up-regulation of PBMC expression of TLR2 but not TLR4 occurs in cirrhosis, which implies, contrary to previous assumptions, an important stimulatory role for Gram-positive microbial components but not endotoxin. TLR2 likely contributes to increased circulating TNF- and sTNFR levels in cirrhosis. HEPATOLOGY 2003;37:1154-1164.)

Recombinant human interleukin-11 improves thrombocytopenia in patients with cirrhosis (*Human Study*)
Reem Ghalib, Cheryl Levine, Manal Hassan, Tricia McClelland, John Goss, Risë Stribling, Philip Seu, Yehuda Z. Patt
To elucidate the hematopoietic activity of recombinant human interleukin-11 (rhIL-11, [Neumega, Cambridge, MA]) in patients with cirrhosis and thrombocytopenia, we administered rhIL-11 at 50 µg/kg/d subcutaneously to 10 patients for 10 days with a 30-day follow-up period. All treated patients (n = 9) experienced a gradual, yet significant increase in their platelet count above the baseline value (P .01) reaching the peak value (median, 93,000/µL; range, 60,000-206,000/µL) at a median of 13 days (range, 6-23 days). Eight patients (89%) had a significant increase of 50% over the baseline value (P < .05). Moreover, further increases to 60,000/µL, 80,000/µL, and 100,000/µL were observed in 100%, 78%, and 33% of the patients, respectively. A subsequent decline in platelet count was observed at a median of 19 days (range, 7-26 days) after the occurrence of peak concentration. A significant increase in neutrophil count was also demonstrated starting on the third day of treatment (P .01). Concurrent with an increase in the serum level of fibrinogen, transaminase levels declined significantly during treatment period, while bilirubin levels continued to drop for up to 20 days after the initiation of treatment (P < .05). The most frequent effects were due to plasma volume expansion, including conjunctival redness and edema. In conclusion, rhIL-11 can improve platelet counts in patients with early cirrhosis and these patients could benefit from rhIL-11 treatment. However, given the high frequency of regimen-related toxicity, the use of rhIL-11 in patients with cirrhosis should be administered with caution. (HEPATOLOGY 2003;37:1165-1171.)

Viral Hepatitis

Long-term histologic and virologic outcomes of acute self-limited hepatitis B (*Human Study*)
Nobukazu Yuki, Takayuki Nagaoka, Masatoshi Yamashiro, Kiyoshi Mochizuki, Akira Kaneko, Keiji Yamamoto, Masao Omura, Kazumasa Hikiji, Michio Kato
The long-term impact of acute self-limited hepatitis B on the liver is unknown. Fourteen patients were recalled at a median of 4.2 years (range, 1.8-9.5 years) after the onset of acute hepatitis B. All showed clinical and serologic recovery with circulating hepatitis B surface antigen (HBsAg) clearance. Antibody to HBsAg (anti-HBs) had developed in 12 patients. Nine underwent liver biopsies at a median of 7.2 years, and histologic findings were evaluated using Ishak scores. Serum samples and frozen liver tissue were subjected to real-time detection polymerase chain reaction (PCR) to quantify the surface and X regions of the hepatitis B virus (HBV) genome and qualitative PCR to detect the covalently closed circular (ccc) HBV DNA replicative intermediate. Three patients had low levels of circulating HBV DNA up to 8.9 years after the onset, whereas both HBV DNA surface and X regions were found in the liver of all 9 patients examined, including 7 negative for serum HBV DNA. Liver viral loads assessed by the 2 regions showed a significant correlation (r = 0.946; P = .008), and all patients tested positive for ccc HBV DNA. Liver fibrosis and mild inflammation persisted in 8 patients. The fibrosis stage had relation to peak serum HBV DNA in the acute phase (P = .046) but not to liver viral loads in the late convalescent phase. In conclusion, occult HBV infection persists in the liver and is accompanied by abnormal liver histology for a decade after complete clinical recovery from acute self-limited hepatitis B. (HEPATOLOGY 2003;37:1172-1179.)

Gene expression associated with interferon alfa antiviral activity in an HCV replicon cell line
Haizhen Zhu, Hongshan Zhao, Christin D. Collins, Sarah E. Eckenrode, Qingguo Run, Richard A. McIndoe, James M. Crawford, David R. Nelson, Jin-Xiong She, Chen Liu
Interferon alfa (IFN-)-based treatment is the only therapeutic option for chronic hepatitis C viral infection. However, the molecular mechanisms of IFN- antiviral activity are not completely understood. The recent development of an HCV replicon cell culture system provides a feasible experimental model to investigate the molecular details of IFN-induced direct antiviral activity in hepatocytes. In this report, we show that IFN- can effectively inhibit HCV subgenomic RNA replication and suppress viral nonstructural protein synthesis. Using cDNA microarray analysis, we also show that the replicon cells have different gene expression profile compared with the parental hepatoma cells (Huh7). IFN- can induce a number of responsive genes in the replicon cells. One of the genes, 6-16 (G1P3), can enhance IFN- antiviral efficacy. In addition, we demonstrate that IFN- can significantly activate STAT3 in hepatoma cells, suggesting that this pathway plays a role in IFN- signaling. In conclusion, our results indicate that IFN- antiviral activity is associated with activation of STAT3-signaling pathway and intracellular gene activation. Our results also suggest that IFN--induced target genes may play an important role in IFN- anti-HCV activity. (HEPATOLOGY 2003;37:1180-1188.)

Detection of functionally altered hepatitis C virus-specific CD4+ T cells in acute and chronic hepatitis C (*Human Study*)
Axel Ulsenheimer, J. Tilman Gerlach, Norbert H. Gruener, Maria-Christina Jung, Carl-Albrecht Schirren, Winfried Schraut, Reinhart Zachoval, Gerd R. Pape, Helmut M. Diepolder
Chronic hepatitis C is characterized by a weak or absent hepatitis C virus (HCV)-specific CD4+ T-cell response in terms of antigen-specific proliferation or interferon gamma (IFN-) secretion. To clarify whether this is due to the absence or functional impairment of antigen-specific CD4+ T cells we developed an assay that relies on the induced expression of the T-cell activation marker CD25 and is therefore independent from cytokine secretion or proliferation. In 10 of 20 patients with chronic hepatitis C, a significant number of antigen-specific activated CD4+ T cells (mean 1.06%/patient; range, 0% to 5.2% of CD4+ T cells) could be shown, whereas antigen-specific proliferation was present in only 1 of 20 patients. IFN- secretion was absent in all 13 patients tested. However, significant antigen-specific interleukin 10 (IL-10) and transforming growth factor (TGF-) secretion was present in 6 of 10 and 3 of 10 patients, respectively. In 8 patients with acute hepatitis C, irrespective of disease outcome, HCV-specific CD4+ T cells were detected in all patients and at a significantly higher frequency (mean 3.7%/patient; range, 1.16% to 7.17%) in the first weeks of disease. A chronic course of disease was associated either with a loss of both IFN- secretion and proliferation, resembling an anergic state, or a loss of T-cell proliferation followed by a rapid decline in IFN--producing cells, corresponding to exhaustion of the specific immune response. In conclusion, functional changes of HCV-specific CD4+ T cells or failure to develop a long-lasting T-helper response may contribute to chronic hepatitis C viral persistence. (HEPATOLOGY 2003;37:1189-1198.)

Blood lactate as a prognostic marker in acetaminophen-induced acute liver failure
Lars E. Schmidt, Fin Stolze Larsen
Background: Although the King's College Hospital (KCH) selection criteria for emergency liver transplantation in paracetamol-induced acute liver failure are widely used, strategies to improve sensitivity and facilitate earlier transplantation are required. We investigated the use of arterial blood lactate measurement for the identification of transplantation candidates. Methods: In a single-centre study, we measured arterial blood lactate early (median 4 h) and after fluid resuscitation (median 12 h) in patients admitted to a tertiary-referral intensive-care unit. Threshold values that best identified individuals likely to die without liver transplantation were derived in a retrospective initial sample of 103 patients with paracetamol-induced acute liver failure and applied to a prospective validation sample of 107 patients. Predictive value and speed of identification were compared to those of the KCH criteria. Findings: In the initial sample, median lactate was significantly higher in non-surviving patients than in survivors both in the early samples (8.5 [range 1.7-21.0] vs. 1.4 [0.53-7.9] mmol/L, p<0.0001) and after fluid resuscitation (5.5 [1.3-18.6] vs. 1.3 [0.26-3.2], p<0.0001). Applied to the validation sample, a threshold value of 3.5 mmol/L early after admission had sensitivity 67%, specificity 95%, positive likelihood ratio 13, and negative likelihood ratio 0.35; the corresponding values for a threshold of 3.0 mmol/L after fluid resuscitation were 76%, 97%, 30, and 0.24. Combined early and postresuscitation lactate concentrations had similar predictive ability to KCH criteria but identified non-surviving patients earlier (4 [3-13] vs 10 [3.5-19.5] h, p=0.01). Addition of postresuscitation lactate concentration to KCH criteria increased sensitivity from 76% to 91% and lowered negative likelihood ratio from 0.25 to 0.10. Interpretation: Arterial blood lactate measurement rapidly and accurately identifies patients who will die from paracetamol-induced acute liver failure. Its use could improve the speed and accuracy of selection of appropriate candidates for transplantation.

Nonalcoholic steatohepatitis: Summary of an AASLD Single Topic Conference
Brent A. Neuschwander-Tetri, Stephen H. Caldwell
Fatty liver disease that develops in the absence of alcohol abuse is recognized increasingly as a major health burden. This report summarizes the presentations and discussions at a Single Topic Conference held September 20-22, 2002, and sponsored by the American Association for the Study of Liver Diseases. The conference focused on fatty liver disorders. Estimates based on imaging and autopsy studies suggest that about 20% to 30% of adults in the United States and other Western countries have excess fat accumulation in the liver. About 10% of these individuals, or fully 2% to 3% of adults, are estimated to meet current diagnostic criteria for nonalcoholic steatohepatitis (NASH). Sustained liver injury leads to progressive fibrosis and cirrhosis in a fraction, possibly up to one third, of those with NASH, and NASH may be a cause of cryptogenic cirrhosis. NASH is now a significant health issue for obese children as well, leading to cirrhosis in some. The diagnostic criteria for NASH continue to evolve and rely on the histologic findings of steatosis, hepatocellular injury (ballooning, Mallory bodies), and the pattern of fibrosis. Generally recognized indications for biopsy include establishing the diagnosis and staging of the injury, but strict guidelines do not exist. Liver enzymes are insensitive and cannot be used reliably to confirm the diagnosis or stage the extent of fibrosis. Older age, obesity, and diabetes are predictive of fibrosis. The pathogenesis of NASH is multifactorial. Insulin resistance may be an important factor in the accumulation of hepatocellular fat, whereas excess intracellular fatty acids, oxidant stress, adenosine triphosphate (ATP) depletion, and mitochondrial dysfunction may be important causes of hepatocellular injury in the steatotic liver. Efforts are underway to refine the role of insulin resistance in NASH and determine whether improving insulin sensitivity pharmacologically is an effective treatment. An altered lifestyle may be a more effective means of improving insulin sensitivity. The research agenda for the future includes establishing the role of insulin resistance and abnormal lipoprotein metabolism in NASH, determining the pathogenesis of cellular injury, defining predisposing genetic abnormalities, identifying better noninvasive predictors of disease, and defining effective therapy. (HEPATOLOGY 2003;37:1202-1219.)

Copyright © 2003 by the American Association for the Study of Liver Diseases. All rights reserved.

GASTROENTEROLOGY

Table of Contents for May 2003 · Volume 124 · Number 5

Rho kinase blockade prevents inflammation via nuclear factor B inhibition: Evidence in Crohn's disease and experimental colitis
Jean-Pierre Segain, Diane Raingeard de la Blétière, Vincent Sauzeau, Arnaud Bourreille, Gréory Hilaret, Chrystelle Cario-Toumaniantz, Pierre Pacaud, Jean-Paul Galmiche, Gervaise Loirand
Background & Aims: Rho proteins are involved in the regulation of several cellular functions. Data from in vitro studies suggest that RhoA could be involved in the inflammatory response. We investigated the role of RhoA and its downstream effector Rho kinase in intestinal inflammation. Methods: Activation of RhoA was assessed by pull-down assays. A specific inhibitor of Rho kinase, Y-27632, was used to examine the role of Rho kinase in inflammatory response in vivo and in vitro by molecular biology and by immunological and biochemical approaches. Results: Increased activation of RhoA was found in inflamed intestinal mucosa of patients with Crohn's disease and of rats with 2,4,6-trinitrobenzene sulfonic acid-induced colitis. Oral administration of Y-27632 in rats significantly reduced the colonic inflammation. In vitro, activation of RhoA alone was sufficient to induce tumor necrosis factor production. Y-27632 inhibited production of tumor necrosis factor- and interleukin-1 by lamina propria and peripheral blood mononuclear cells. Rho kinase inhibition prevented nuclear factor B activation and I-B phosphorylation and degradation. We showed that Rho kinase associates with and activates I-B kinase and that Y-27632 prevents I-B kinase activation. Conclusions: Our study provides the first evidence that Rho kinase activates I-B kinase and, thus, nuclear factor B, suggesting a key role of Rho kinase in inflammatory responses and intestinal inflammation. Specific inhibition of Rho kinase may be a promising approach for the treatment of patients with Crohn's disease.

Hyperleptinemia prevents increased plasma ghrelin concentration during short-term moderate caloric restriction in rats
Rocco Barazzoni, Michela Zanetti, Marco Stebel, Gianni Biolo, Luigi Cattin, Gianfranco Guarnieri
Background & Aims: Ghrelin is an orexigenic hormone secreted by the stomach. Increased plasma ghrelin concentration was reported during diet-induced weight loss in obese humans, suggesting that ghrelin contributes to adaptive increment in appetite associated with caloric restriction. Leptin reduces spontaneous food intake and body weight in rodents. The current study tested the hypothesis that increased plasma leptin prevents the potential increase in plasma ghrelin concentration during moderate caloric restriction in lean rats. Methods: Six-month-old male rats (body weight, 367 ± 9 grams) were randomly assigned to one of the following treatments (8 rats each) for 1 week: (1) leptin subcutaneous infusion to induce moderate hyperleptinemia and moderate caloric restriction (­26% of ad libitum), (2) vehicle infusion and pair feeding, and (3) vehicle infusion and ad libitum feeding. Results: Leptin-treated (­19 ± 5 grams) and pair-fed (­19 ± 2) rats lost weight compared with ad libitum-fed rats (­3 ± 1, P < 0.05). Compared with control (6.8 ± 0.7 ng/mL), plasma leptin was higher in leptin-treated (18.6 ± 0.9 ng/mL, P < 0.01) rats and lower in pair-fed rats (4.3 ± 0.4 ng/mL, P < 0.05). Plasma ghrelin was substantially higher in calorie-restricted than control rats (2505 ± 132 pg/mL vs. 1790 ± 134 pg/mL, P < 0.01), and leptin treatment (1625 ± 117 pg/mL) completely prevented this change. Plasma ghrelin concentration was negatively correlated with body weight changes in calorie-restricted and control (r = ­0.75, P < 0.01) but not in leptin-treated rats (P > 0.8). Conclusions: Moderate hyperleptinemia prevents an increase of plasma ghrelin during moderate short-term caloric restriction. Satiety-inducing effects of leptin include suppression of gastric orexigenic signals and disruption of a potential feedback mechanism between body weight changes and plasma ghrelin in lean adult rats.

Clinical-alimentary Tract

Increased risk of noncardia gastric cancer associated with proinflammatory cytokine gene polymorphisms
Emad M. El-Omar, Charles S. Rabkin, Marilie D. Gammon, Thomas L. Vaughan, Harvey A. Risch, Janet B. Schoenberg, Janet L. Stanford, Susan T. Mayne, James Goedert, William J. Blot, Joseph F. Fraumeni, Jr, Wong-ho Chow
Background & Aims: Genetic variations in proinflammatory and anti-inflammatory cytokine genes influence individual response to carcinogenic exposures. Polymorphisms in interleukin (IL)-1 and its endogenous receptor antagonist are associated with risk of Helicobacter pylori-related gastric cancer. The aim of this study was to evaluate the role of proinflammatory cytokine gene polymorphisms in gastric and esophageal cancers defined by anatomic subsite. Methods: We assessed polymorphisms of the IL-1 gene cluster and 4 other cytokine genes in a population-based case-control study of upper gastrointestinal cancers, including gastric cardia (n = 126) and noncardia adenocarcinoma (n = 188), esophageal squamous cell carcinoma (n = 53), and adenocarcinoma (n = 108), and frequency-matched controls (n = 212). ORs for the different cancers were computed from logistic regression models adjusted for potential confounding factors. Results: Proinflammatory genotypes of tumor necrosis factor and IL-10 were each associated with more than doubling of the risk of noncardia gastric cancer. Carriage of multiple proinflammatory polymorphisms of IL-1Bo IL-1 receptor antagonist, tumor necrosis factor A, and IL-10 conferred greater risk, with ORs (and 95% confidence intervals) of 2.8 (1.6­5.1) for one, 5.4 (2.7­10.6) for 2, and 27.3 (7.4­99.8) for 3 or 4 high-risk genotypes. In contrast, these polymorphisms were not consistently related to the risks of esophageal or gastric cardia cancers. Polymorphisms in IL-4 and IL-6 were not associated with any of the cancers studied. Conclusions: A proinflammatory cytokine genetic profile increases the risk of noncardia gastric adenocarcinoma but not other upper gastrointestinal cancers, possibly by inducing a hypochlorhydric and atrophic response to gastric H. pylori infection.

Prophylaxis of pouchitis onset with probiotic therapy: A double-blind, placebo-controlled trial
Paolo Gionchetti, Fernando Rizzello, Ulf Helwig, Alessandro Venturi, Karen Manon Lammers, Patrizia Brigidi, Beatrice Vitali, Gilberto Poggioli, Mario Miglioli, Massimo Campieri
Background & Aims: We have recently documented the efficacy of a highly concentrated probiotic preparation (VSL#3) in the prevention of flare-up in patients with chronic pouchitis. The aim of this study was to compare probiotic therapy with VSL#3 versus placebo in the ability to prevent the onset of acute pouchitis during the first year after ileal pouch-anal anastomosis. Methods: Forty consecutive patients who underwent ileal pouch-anal anastomosis for ulcerative colitis were randomized to receive either VSL#3 (1 packet containing 900 billion bacteria/day) (n = 20) or an identical placebo (n = 20) immediately after ileostomy closure for 1 year. The patients were assessed clinically, endoscopically, and histologically after 1, 3, 6, 9, and 12 months. Health-related quality of life was assessed using the Inflammatory Bowel Disease Questionnaire. Results: Two of the 20 patients (10%) treated with VSL#3 had an episode of acute pouchitis compared with 8 of the 20 patients (40%) treated with placebo (log-rank test, z = 2.273; P < 0.05). Treatment with VSL#3 determined a significant improvement in Inflammatory Bowel Disease Questionnaire score, whereas this was not the case with placebo. Conclusions: Treatment with VSL#3 is effective in the prevention of the onset of acute pouchitis and improves quality of life of patients with ileal pouch-anal anastomosis.

Plasma citrulline: A marker of enterocyte mass in villous atrophy-associated small bowel disease
Pascal Crenn, Kouroche Vahedi, Anne Lavergne-Slove, Luc Cynober, Claude Matuchansky, Bernard Messing
Background & Aims: Plasma citrulline, a nonprotein amino acid produced by enterocytes, was suggested as a marker of remnant enterocyte mass in patients with short bowel. Our objective was to evaluate citrulline as a marker of severity and extent of villous atrophy in patients without intestinal resection. Methods: Forty-two patients with celiac disease and 10 patients with non-celiac villous atrophy disease were studied by plasma postabsorptive citrulline and biological dosages, biopsies of proximal (duodenojejunal) small bowel and distal ileum (n = 25), or measurement of vitamin B12 absorption (n = 4). Nine patients were reevaluated after following a gluten-free diet for 1 year. Controls were 51 healthy subjects and 10 severely malnourished patients with anorexia nervosa with no intestinal mucosal abnormalities. Results: Plasma citrulline concentration was lower (P < 0.001) in patients with villous atrophy (24 ± 13 µmol/L) than in healthy subjects (40 ± 10 µmol/L) and patients with anorexia nervosa (39 ± 9 µmol/L). Three thresholds were individualized: <10 µmol/L for patients with diffuse total villous atrophy (n = 10), 10­20 µmol/L for patients with proximal-only total villous atrophy (n = 12), and 20­30 µmol/L for patients with partial villous atrophy (n = 10). Plasma citrulline concentration was correlated to the severity and extent of villous atrophy (r = 0.81; P < 0.001) and to albuminemia (r = 0.47; P < 0.01). Receiver operating characteristic curves indicated that plasma citrulline concentration was the best biological variable to predict villous atrophy. Following a 1-year gluten-free diet, plasma citrulline concentration increased in histologically responsive (n = 6) but not in unresponsive (n = 3) patients. Conclusions: In patient villous atrophy diseases, plasma citrulline concentration may prove to be a simple and reliable marker of reduced enterocyte mass.

Antro-fundic dysfunctions in functional dyspepsia
Maria PÍa Caldarella, Fernando Azpiroz, Juan-R. Malagelada
Background & Aims: Symptoms in functional dyspepsia have been related to impaired accommodation and hypersensitivity of the proximal stomach. We hypothesized that identification of putative antral dysfunctions provides a more comprehensive pathophysiological interpretation. Methods: In 30 patients with functional dyspepsia and 22 healthy subjects, 2 consecutive studies were performed. In study I, with the subjects in the upright position, the proximal and distal stomach were selectively distended by bags containing air and water, respectively, while perception and fundic relaxation in response to antral distention were measured. In study II, by using air-filled bags connected to a tensostat, the proximal and the distal stomach were selectively distended by positioning the subjects on the right and left lateral decubitus, respectively, while perception, compliance, and the responses to intestinal nutrient infusion were measured. Results: Patients with dyspepsia showed hypersensitivity of both the proximal stomach (discomfort at 30 ± 3 g vs. 62 ± 2 g in controls; P < 0.05) and the antrum (discomfort at 31 ± 2 g vs. 53 ± 4 g in controls; P < 0.05). Fundic and antral fasting tone was normal, but reflex fundic relaxation induced either by antral distention (3 ± 16 mL at 80 mL of distention vs. 38 ± 10 mL in controls; P < 0.05) or by intestinal nutrients (35 ± 7 mL vs. 107 ± 8 mL in controls; P < 0.05) was markedly impaired. Conclusions: Antral and fundic dysfunctions interact to produce the symptoms of functional dyspepsia, and impaired control of fundic accommodation may lead to overload of a hypersensitive antrum.

Pain and biomechanical responses to distention of the duodenum in patients with systemic sclerosis
Jan Pedersen, Chunwen Gao, Henrik Egekvist, Peter Bjerring, Lars Arendt-nielsen, Hans Gregersen, Asbjørn Mohr Drewes
Background & Aims: Abnormalities of the small intestine have been indicated in systemic sclerosis. The aim was to use a new method to study the active-passive mechanical and sensory properties of the duodenum in these patients. Methods: A volume-controlled ramp-distention protocol was used in the duodenum in 9 patients and 8 healthy controls. The nonpainful/painful sensations, pressure, cross-sectional area, wall tension, and strain were evaluated. Using butylscopolamine for muscle relaxation, the active (contractile muscular component) and passive (other mechanical tissue components) were computed. Results: The contraction amplitude was smaller and the cross-sectional area higher in the patients (P < 0.05). Both the active and passive tension as function of strain was translated to the left in the patients, indicating a stiffer wall. The maximum active tension and the corresponding strain were 62% and 69% lower in the patients (P < 0.05). An association was found between the duration of the disease and the strain (P < 0.05). The perception score was higher as function of pressure, tension, and strain (P = 0.01, P = 0.03, and P < 0.01, respectively) in the patients than in the controls, with strain as the most sensitive variable to describe the sensory response. In 5 patients who complained of regular clinical symptoms, the referred pain area to distention was enlarged. Conclusions: Systemic sclerosis resulted in increased stiffness and impaired muscle function of the duodenum. The pain evoked by a controlled strain of the gut was increased and can explain many of the symptoms reported in the clinic.

Folate status, genomic DNA hypomethylation, and risk of colorectal adenoma and cancer: A case control study
Maria Pufulete, Reyad Al-Ghnaniem, Andrew J.M. Leather, Paul Appleby, Sally Gout, Catherine Terry, Peter W. Emery, Thomas A.B. Sanders
Background & Aims: Low folate intake may increase risk for colorectal cancer by inducing DNA hypomethylation. This study reports the influence of folate status, DNA methylation, and polymorphisms of methylenetetrahydrofolate reductase (MTHFR 677CT and 1298AC), methionine synthase (MS 2756AG), and cystathionine--synthase (CBS 844ins68) on risk for developing colorectal neoplasia. Methods: Thirty-five patients with adenoma, 28 patients with cancer, and 76 controls were recruited for a case control study. Recruitment consent rate was 98%. Blood samples were obtained for determination of blood folates, vitamin B12, homocysteine, DNA methylation, and genotypes. Tissue biopsy samples were obtained at colonoscopy for determination of DNA methylation in colonic mucosa. Folate status was assessed by constructing a score from estimates of dietary intake and serum and erythrocyte folate. Results: Cancer patients had 26% lower folate status (95% confidence interval [CI]: 6% to 44%, P = 0.01) and 21% lower serum vitamin B12 concentration (95% CI: ­38% to 1%, P = 0.06) compared with controls. [3H] methyl incorporation into colonic DNA was 26% higher in patients with adenoma (95% CI: 8% to 56%, P = 0.009) and 30% higher in patients with cancer (95% CI: ­3% to 48%, P = 0.08) compared with controls. High folate status was associated with decreased risk for cancer (P = 0.01 for trend). Colonic and leukocyte DNA hypomethylation were associated with increased risk for adenoma (P = 0.02 and P = 0.01 for trend, respectively) and a nonsignificantly increased risk for cancer (P = 0.09 and P = 0.08 for trend, respectively). Conclusions: Low folate status and DNA hypomethylation are associated with colorectal neoplasia.

Platelets trigger a CD40-dependent inflammatory response in the microvasculature of inflammatory bowel disease patients
Silvio Danese, Carol de la Motte, Andreas Sturm, Jon D. Vogel, Gail A. West, Scott A. Strong, Jeffry A. Katz, Claudio Fiocchi
Background & aims: Platelets circulate in an activated state in patients with inflammatory bowel disease (IBD), but their role in the pathogenesis of IBD is unclear. The recent demonstration that activated platelets express CD40 ligand (L) provides a mechanism of interaction with CD40-positive endothelial cells, inducing them to produce proinflammatory mediators. We investigated whether platelets from patients with IBD express enhanced levels of CD40L and induce human intestinal microvascular endothelial cells (HIMEC) to up-regulate cell adhesion molecule (CAM) expression and secrete chemokines. Methods: CD40L expression was assessed in resting and thrombin-activated platelets by flow cytometry and in mucosal microthrombi by confocal microscopy. Platelet-HIMEC cocultures were used to study CAM up-regulation, and interleukin (IL)-8 and RANTES production by HIMEC. Results: IBD platelets expressed significantly higher CD40L levels than those of healthy subjects, and CD40L-positive platelets were detected in IBD-involved mucosa. Activated platelets up-regulated expression of intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 as well as production of interleukin 8 by HIMEC in a CD40-dependent fashion. High levels of RANTES were present in platelet-HIMEC cocultures and platelets were identified as the source of this chemokine, which mediated T-cell adhesion to HIMEC. Conclusions: These results show that platelets can actively contribute to mucosal inflammation and represent a previously unrecognized component of IBD pathogenesis.

Impaired expression of peroxisome proliferator-activated receptor in ulcerative colitis
Laurent Dubuquoy, Emmelie Å. Jansson, Samir Deeb, Sabine Rakotobe, Mehdi Karoui, Jean-Frédéric Colombel, Johan Auwerx, Sven Pettersson, Pierre Desreumaux
Background & Aims: The peroxisome proliferator-activated receptor (PPAR) has been proposed as a key inhibitor of colitis through attenuation of nuclear factor B (NF-B) activity. In inflammatory bowel disease, activators of NF-B, including the bacterial receptor toll-like receptor (TLR)4, are elevated. We aimed to determine the role of bacteria and their signaling effects on PPAR regulation during inflammatory bowel disease (IBD). Methods: TLR4-transfected Caco-2 cells, germ-free mice, and mice devoid of functional TLR4 (Lpsd/Lpsd mice) were assessed for their expression of PPAR in colonic tissues in the presence or absence of bacteria. This nuclear receptor expression and the polymorphisms of gene also were assessed in patients with Crohn's disease (CD) and ulcerative colitis (UC), 2 inflammatory bowel diseases resulting from an abnormal immune response to bacterial antigens. Results: TLR4-transfected Caco-2 cells showed that the TLR4 signaling pathway elevated PPAR expression and a PPAR-dependent reporter in an I dependent fashion. Murine and human intestinal flora induced PPAR expression in colonic epithelial cells of control mice. PPAR expression was significantly higher in the colon of control compared with Lpsd/Lpsd mice. Although PPAR levels appeared normal in patients with CD and controls, UC patients displayed a reduced expression of PPAR confined to colonic epithelial cells, without any mutation in the PPAR gene. Conclusions: These data showed that the commensal intestinal flora affects the expression of PPAR and that PPAR expression is considerably impaired in patients with UC.

Clinical-liver, Pancreas, and Biliary Tract

Emergency sclerotherapy versus vasoactive drugs for variceal bleeding in cirrhosis: A cochrane meta-analysis
Gennaro D'Amico, Giada Pietrosi, Ilaria Tarantino, Luigi Pagliaro
Background & aims: Emergency sclerotherapy is used as a first-line therapy for variceal bleeding in cirrhosis, although pharmacologic treatment stops bleeding in most patients. We performed a meta-analysis comparing emergency sclerotherapy with pharmacologic treatment. Methods: MEDLINE (1968­2002), EMBASE (1986­2002), and the Cochrane Library (2002;4) were searched to retrieve randomized controlled trials comparing sclerotherapy with vasopressin (± nitroglycerin), terlipressin, somatostatin, or octreotide for variceal bleeding in cirrhosis. Outcome measures were failure to control bleeding, rebleeding, blood transfusions, adverse events, and mortality. Results: Fifteen trials were identified. Sclerotherapy was not superior to terlipressin, somatostatin, or octreotide for any outcome and to vasopressin for rebleeding, blood transfusions, death, and adverse events; it was superior to vasopressin for the control of bleeding in a single trial flawed by a potential detection bias. Sclerotherapy was associated with significantly more adverse events than somatostatin. In a predefined sensitivity analysis, combining all of the trials irrespective of the control treatment, risk differences (sclerotherapy minus control) and confidence intervals (CIs) were as follows: failure to control bleeding, ­0.03 (­0.06 to 0.01); mortality, ­0.035 (­0.07 to 0.008); adverse events, 0.08 (0.02 to 0.14). Mortality risk difference was ­0.01 (­0.07 to 0.04) in good-quality trials and ­0.08 (­0.14 to ­0.02) in poor-quality trials. Conclusions: Available evidence does not support emergency sclerotherapy as the first-line treatment of variceal bleeding in cirrhosis when compared with vasoactive drugs, which control bleeding in 83% of patients. Therefore, endoscopic therapy might be added only in pharmacologic treatment failures.

Risk factors for the development of pancreatic cancer in familial pancreatic cancer kindreds
Stephen J. Rulyak, Albert B. Lowenfels, Patrick Maisonneuve, Teresa A. Brentnall
Background & Aims: Approximately 10% of pancreatic cancers are inherited, but the factors that affect tumorigenesis in familial pancreatic cancer are unknown. We sought to determine whether smoking or other factors could predict cancer risk in familial pancreatic cancer kindreds. Methods: We conducted a nested case-control study including 251 members of 28 families. All families included 2 or more members with pancreatic cancer. We determined the effects of smoking, young age of onset within the family, diabetes mellitus, sex, and number/standing of affected relatives on the risk of pancreatic cancer. Results: Smoking was an independent risk factor for familial pancreatic cancer (odds ratio [OR], 3.7; 95% confidence interval [CI], 1.8­7.6), and the risk was greatest in males and subjects younger than 50 (OR, 5.2 and OR, 7.6, respectively). Smokers developed cancer 1 decade earlier than nonsmokers (59.6 vs. 69.1 years; P = 0.01), and the number of affected first-degree relatives also increased risk (OR, 1.4; 95% CI, 1.1­1.9 for each additional family member). Diabetes was not a risk factor for pancreatic cancer, although diabetes was associated with pancreatic dysplasia. One third of families demonstrated genetic anticipation, as the mean age of onset decreased by 2 decades between generations. Conclusions: Smoking is a strong risk factor in familial pancreatic cancer kindreds, particularly among males and those under age 50. Persons with multiple affected first-degree relatives are also at increased risk. These factors may be useful in selecting candidates for pancreatic cancer screening. Members of families with multiple pancreatic cancers should be counseled not to smoke.

Epigenetic and genetic alterations in duodenal carcinomas are distinct from biliary and ampullary carcinomas
Sang Geol Kim, Annie On-On Chan, Tsung-Teh Wu, Jean-Pierre J. Issa, Stanley R. Hamilton, Asif Rashid
Background & Aims: Carcinomas of the extrahepatic bile ducts, ampulla of Vater, and duodenum are uncommon, and their epigenetic and genetic alterations are not well characterized. Methods: We therefore compared the methylation profile and genetic alterations in 18 extrahepatic biliary, 9 ampullary, and 12 duodenal carcinomas. We evaluated methylation at p16, p14, and human Mut L homologue (hMLH1) by methylation- specific PCR (MSP), and at cyclooxygenase 2 (COX2), O6-methyl-guanine methyltransferase (MGMT), estrogen receptor (ER), retinoic acid receptor 2 (RAR), and T-type calcium channel (CACNA1G) genes, and methylated in tumor 1 (MINT1), MINT2, MINT25, MINT27, and MINT31 loci by combined bisulfite restriction analysis (COBRA); mutation of K-ras, p53, p16, and p14 genes by sequencing; loss of heterozygosity of chromosome 9p; and microsatellite instability (MSI). Results: Duodenal carcinomas were methylated more frequently or had increased methylation densities than biliary carcinomas at p14 (P = 0.04), hMLH1 (P = 0.04), MGMT (P = 0.01), MINT1 (P = 0.01), MINT25 (P = 0.01), MINT27 (P = 0.001), RAR (P = 0.03), and ER (P = 0.001), and than ampullary carcinomas at RAR (P = 0.02) and ER (P = 0.03). In contrast, the methylation profiles of biliary and ampullary carcinomas were not statistically different. Simultaneous methylation of 3 or more CpG islands (CpG island methylator phenotype-high) was more common in duodenal cancers (P = 0.004). MGMT methylation was associated with G-to-A mutation in K-ras (P = 0.006), and hMLH1 methylation was associated with MSI-high (P = 0.01). Conclusions: Our findings indicate that the methylation profile and genetic alterations of duodenal carcinomas are distinct from biliary and ampullary carcinomas, and that tumor-specific methylation is associated with gene mutation and MSI.

Frequent mutations of hepatocyte nuclear factor 1 in colorectal cancer with microsatellite instability
Pierre Laurent-puig, Olivier Plomteux, Olivier Bluteau, Franck Zinzindohoué, Emmanuelle Jeannot, Karin Dahan, Alex Kartheuser, Caroline Chapusot, Paul-henri Cugnenc, Jessica Zucman-rossi
Background & Aims: The TCF1 gene encoding hepatocyte nuclear factor 1 (HNF1), a transcription factor germline mutated in patients with maturity-onset diabetes of the young type 3, was recently found to be frequently inactivated by biallelic alterations in liver adenoma and in rare hepatocellular carcinomas. The impact of HNF1 in colorectal carcinogenesis has not been studied until now. Colorectal cancer is characterized by the existence of different molecular mechanisms known as microsatellite stable or unstable tumors. Methods: At first, a series of 10 adenomas and 29 colon cancers regardless of microsatellite instability status were screened for TCF1 mutations on the entire coding sequence. Results: Three mutations in microsatellite instability high (MSI-H) tumors were found in the exon 4 polymorphic poly-cytosin (C)8 or (C)9 tract and consisted of a cytosin deletion at position 291. To further characterize the prevalence of TCF1 mutations in the subgroup of MSI-H tumors, 52 additional MSI-H samples were screened for exon 4 alterations; 23% of MSI-H tumors (95% confidence interval, 14%­36%) were found to harbor frameshift at the poly-cytosin tract. The (C)9 allele was significantly more frequently mutated than the (C)8 allele (22% vs. 8%; P = 0.03), showing a higher instability of the longer repetition. Conclusions: These results show a role for HNF1 in MSI-H colorectal carcinogenesis.

Basic-alimentary Tract

A novel PPAR gene therapy to control inflammation associated with inflammatory bowel disease in a murine model
Kazufumi Katayama, Koichiro Wada, Atsushi Nakajima, Hiroyuki Mizuguchi, Takao Hayakawa, Shinsaku Nakagawa, Takashi Kadowaki, Ryozo Nagai, Yoshinori Kamisaki, Richard S. Blumberg, Tadanori Mayumi
Background & Aims: Peroxisome proliferator-activated receptor (PPAR) is one of the nuclear receptors that plays a central role in adipocyte differentiation and insulin sensitivity. PPAR has also recently been recognized as an endogenous regulator of intestinal inflammation. However, its levels are decreased during chronic inflammation in human and mice, thus limiting PPAR ligand therapy during established disease. We sought to determine whether this decrease in PPAR could be counteracted by a gene therapy approach. Methods: We characterized PPAR levels in experimental colitis associated with dextran sodium sulfate administration to mice. In this model, the therapeutic benefits of PPAR gene therapy using a replication-deficient adenovirus vector expressing PPAR (Ad-PPAR) was assessed. Results: PPAR protein levels were decreased in whole colonic tissue, lamina propria lymphocytes, and peritoneal exudate cells during the course of colitis. PPAR gene delivery using Ad-PPAR restored responsiveness to a PPAR ligand, resulting in marked amelioration of tissue inflammation associated with the colitis, which included attenuation of intercellular adhesion molecule-1, cyclooxygenase-2 and tumor necrosis factor- expression. Conclusions: Our results suggest that gene delivery of PPAR can be used to restore and/or enhance endogenous anti-inflammatory processes that are normally operative in mammalian tissues such as in the colon.

Characterization of the effects of pancreatic polypeptide in the regulation of energy balance
Akihiro Asakawa, Akio Inui, Hideki Yuzuriha, Naohiko Ueno, Goro Katsuura, Mineko Fujimiya, Masayuki A. Fujino, Akira Niijima¶, Michael M. Meguid, Masato Kasuga
Background & Aims: Pancreatic polypeptide (PP) belongs to a family of peptides including neuropeptide Y and peptide YY. We examined the role of PP in the regulation of body weight as well as the therapeutic potential of PP. Methods: We measured food intake, gastric emptying, oxygen consumption, and gene expression of hypothalamic neuropeptides, gastric ghrelin, and adipocytokines in mice after administering PP intraperitoneally. Peptide gene expression was also examined in PP-overexpressing mice. Vagal and sympathetic nerve activities were recorded after intravenous administration in rats. Effects of repeated administrations of PP on energy balance and on glucose and lipid metabolism were examined in both ob/ob obese mice and fatty liver Shionogi (FLS)-ob/ob obese mice. Results: Peripherally administered PP induced negative energy balance by decreasing food intake and gastric emptying while increasing energy expenditure. The mechanism involved modification of expression of feeding-regulatory peptides (decrease in orexigenic neuropeptide Y, orexin, and ghrelin along with an increase in anorexigenic urocortin) and activity of the vagovagal or vagosympathetic reflex arc. PP reduced leptin in white adipose tissue and corticotropinreleasing factor gene expression. The expression of gastric ghrelin and hypothalamic orexin was decreased in PP-overexpressing mice. Repeated administrations of PP decreased body weight gain and ameliorated insulin resistance and hyperlipidemia in both ob/ob obese mice and FLS-ob/ob obese mice. Liver enzyme abnormalities in FLS-ob/ob obese mice were also ameliorated by PP. Conclusions: These observations indicate that PP may influence food intake, energy metabolism, and the expression of hypothalamic peptides and gastric ghrelin.

Epidermal growth factor receptor-related protein: A potential therapeutic agent for colorectal cancer
Dorota J. Marciniak, Lathika Moragoda, Ramzi M. Mohammad, Yingjie Yu, Kiran K. Nagothu, Amro Aboukameel, Fazlul H. Sarkar, Volkan N. Adsay, Arun K. Rishi, Adhip P.N. Majumdar
Background & Aims: Epidermal growth factor receptor is frequently implicated in epithelial cancers and is, therefore, being considered as a potential target for therapy. Recently, we reported the isolation and characterization of epidermal growth factor receptor­related protein, a negative regulator of epidermal growth factor receptor. To discern whether epidermal growth factor receptor-related protein could be an effective therapeutic agent for colorectal cancer, we generated epidermal growth factor receptor­related protein fusion protein and studied its effect on the growth of colon cancer cells in vivo and in vitro. We also studied whether epidermal growth factor receptor-related protein expression is altered in colorectal cancer. Methods: A 55-kilodalton epidermal growth factor receptor­related protein fusion protein with V5 and His tags was generated in a drosophila expression system and subsequently purified by a His antibody affinity column. Rabbit polyclonal antibodies against epidermal growth factor receptor-related protein were used to examine the expression of epidermal growth factor receptor­related protein. Results: Epidermal growth factor receptor-related protein expression was found to be high in benign human colonic epithelium but low in adenocarcinoma. Exposure of the colon cancer cell lines HCT-116 and Caco-2 to purified recombinant epidermal growth factor receptor­related protein caused a marked inhibition of proliferation, as well as attenuation of basal and ligand-induced stimulation of epidermal growth factor receptor phosphorylation. Epidermal growth factor receptor-related protein-induced inhibition of proliferation of colon cancer cells was prevented by epidermal growth factor receptor­related protein antibodies. Reduced epidermal growth factor receptor phosphorylation was partly due to sequestration of epidermal growth factor receptor ligands by epidermal growth factor receptor-related protein, resulting in the formation of inactive heterodimers with epidermal growth factor receptor. Intratumoral or subcutaneous (away from the tumor site) injections of purified epidermal growth factor receptor­related protein caused regression of palpable colon cancer xenograft tumors in some severely compromised immunodeficient mice and arrested tumor growth in others. Conclusions: We propose that epidermal growth factor receptor-related protein inhibits cellular growth by attenuating epidermal growth factor receptor signaling processes and is an effective therapeutic agent for colorectal cancer.

Progastrin stimulates murine colonic epithelial mitosis after DNA damage
Penelope D. Ottewell, Alastair J.M. Watson, Timothy C. Wang, Andrea Varro, Graham J. Dockray, D. Mark Pritchard
Background & Aims: Transgenic mice that overexpress progastrin are more susceptible than either wild-type mice or mice that overexpress amidated gastrin to chemical carcinogen-induced colonic adenomas. We have investigated whether alterations in the regulation of apoptosis or mitosis after DNA damage contribute to the effects of progastrin on murine colonic epithelium. Methods: Apoptosis and mitosis were assessed on a cell positional basis in murine intestinal epithelium after -irradiation. Mice analyzed were progastrin overexpressing, gastrin overexpressing, gastrin knockout, and their wild-type counterparts. The expression of cell cycle regulators was analyzed by gene array and Western blotting. Results: Apoptosis was induced to similar levels in the small intestinal and colonic crypts of all mice 4.5 hours after 8 Gy -radiation. Colonic mitosis was inhibited to almost undetectable levels by 8Gy -radiation in wild-type, gastrin-knockout, and gastrin-overexpressing mice. However, significant colonic mitosis persisted in progastrin-overexpressing mice up to 24 hours after 8Gy -radiation. Increased levels of cdk4 and cyclin D1 proteins were found in the colonic epithelium of progastrin-overexpressing mice relative to wild-type animals after -radiation. Conclusions: After DNA damage by -radiation, mice with elevated progastrin exhibit significantly higher levels of colonic mitosis than wild-type or gastrin-overexpressing mice. Persistently elevated cdk4 and cyclin D1 in progastrin overexpressing mice accounts for the capacity of colon cells to continue with the cell cycle after DNA damage.

Interleukin-11-induced heat shock protein 25 confers intestinal epithelial-specific cytoprotection from oxidant stress
Mark J. Ropeleski, Jun Tang, Margaret M. Walsh-Reitz, Mark W. Musch, Eugene B. Chang
Background & Aims: The mechanisms of interleukin-11 (IL-11) cytoprotection in intestinal epithelial injury are largely unknown. IL-11 protects barrier integrity during oxidant stress, a common endpoint of numerous types of intestinal injury including ischemia and immune-mediated inflammation. Because heat shock proteins (hsp) are cytoprotective in intestinal epithelia, we hypothesized that IL-11-conferred cytoprotection is mediated by inducible hsps. Methods: IL-11 receptor (IL-11R) activation was determined using phospho-specific antibodies to STAT3. IL-11 induction of hsp72 and hsp25 was determined by immunoblot in IEC-18 crypt and young adult mouse colon colonic epithelial cells. Epithelial resistance to oxidant injury by monochloramine was determined by 51Cr release. Stable hsp anti-sense IEC-18 cell clones were obtained by electroporation and hygromycin B selection. The IL-11 effect on hsp25 distribution was characterized by analysis of Triton ¥-100 insoluble fractions, 2-D isoelectric focusing gels, and confocal microscopy. Results: IL-11R signaling was detected in all cells under study. IL-11 induces hsp25 in an intestinal epithelial-specific manner that significantly preserves cellular viability in the presence of monochloramine. This effect was significantly reversed in intestinal epithelia stably expressing anti-sense to hsp25. IL-11 induced a shift of hsp25 to Triton ¥-100 insoluble fractions containing cytoskeletal elements, which was not associated with altered hsp25 phosphorylation. The shift was not paralleled by increased hsp25 co-localization with F-actin by confocal microscopy. Conclusions: The induction of hsp25 by IL-11 confers epithelial-specific cytoprotection that is independent of phosphorylation-dependent co-localization of hsp25 to F-actin, thereby contributing to the protective effects of IL-11 in models of intestinal epithelial injury.

NF-B activation by oxidative stress and inflammation suppresses contractility in colonic circular smooth muscle cells
Xuan-Zheng Shi, Paul F. Lindholm, Sushil K. Sarna
Background & Aims: Transcription factor nuclear factor B (NF-B) plays a critical role in transcriptional changes in several diseases, including inflammation. The aim of this study was to investigate whether NF-B is activated by inflammation and oxidative stress in colonic circular smooth muscle cells and whether that leads to suppression of their contractility. Methods: The experiments were performed on freshly dissociated single cells using electrophoretic mobility shift assay, Western immunoblotting, and immunofluorescence imaging. Results: The NF-B DNA binding was ~6-fold greater in cells from the inflamed colon vs. those from the normal colon. Supershift assay indicated that the antibodies to p65, p50, and c-Rel, but not that to p52, shifted the NF-B band. Western immunoblotting and immunofluorescence imaging also demonstrated the presence of p65, p50, and c-Rel proteins in the cytoplasm and their translocation to the nucleus by H2O2-induced oxidative stress. H2O2 treatment degraded IB, but not IB, to translocate NF-B to the nucleus. Hydrogen peroxide concentration and time dependently activated NF-B DNA binding and suppressed cell contraction to acetylcholine. NF-B inhibitors significantly inhibited these effects. Inhibition of NF-B prior to and during inflammation in intact dogs also reversed the suppression of contractility. Conclusions: Transcription factor NF-B is activated in colonic circular muscle cells by inflammation and oxidative stress. This activation of NF-B mediates the suppression of cell contractility.

Galectin-1 suppresses experimental colitis in mice
Luca Santucci, Stefano Fiorucci, Natalia Rubinstein, Andrea Mencarelli, Barbara Palazzetti, Barbara Federici, Gabriel A. Rabinovich, Antonio Morelli
Background & Aims: Uncontrolled T-cell activation plays a critical role in the pathogenesis of inflammatory bowel diseases. Therefore, pharmacologic strategies directed to restore the normal responsiveness of the immune system by deleting inappropriately activated T cells could be efficacious in the treatment of these pathologic conditions. Galectin-1 is an endogenous lectin expressed in lymphoid organs that plays a role in the maintenance of central and peripheral tolerance. The aim of the present study was to evaluate the therapeutic effects of galectin-1 on T-helper cell type 1-mediated experimental colitis induced by intrarectal administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS) in mice. Methods: Cells and tissues from mice with TNBS colitis receiving treatment with several doses of human recombinant galectin-1 (hrGAL-1) were analyzed for morphology, cytokine production, and apoptosis. Results: Prophylactic and therapeutic administration of rhGAL-1 resulted in a striking improvement in the clinical and histopathologic aspects of the disease. hrGAL-1 reduced the number of hapten-activated spleen T cells, decreased inflammatory cytokine production, and profoundly reduced the ability of lamina propria T cells to produce IFN in vitro. Moreover, hrGAL-1 led to the appearance of apoptotic mononuclear cells in colon tissue when administered in vivo and induced selective apoptosis of TNBS-activated lamina propria T cells in vitro. Conclusion: Collectively, these data show that hrGAL-1 exerts protective and immunomodulatory activity in TNBS-induced colitis and it might be effective in the treatment of inflammatory bowel diseases.

Enteric flora and lymphocyte-derived cytokines determine expression of heat shock proteins in mouse colonic epithelial cells
Keishi Kojima, Mark W. Musch, Hongyu Ren, David L. Boone, Barbara A. Hendrickson, Averil Ma, Eugene B. Chang
Background & Aims: Inducible heat shock proteins (hsps), particularly hsp25 and hsp72, are expressed by surface colonocytes and may have a role in protecting intestinal epithelial cells against injury. This study is aimed at determining if enteric bacteria and/or immune signals regulate their physiologic expression. Methods: Intestinal hsp25, hsp72, and constitutive hsc73 expression were studied in immunodeficient RAG-1­/­ mice and in normal mice. Mucosal permeability was measured by mannitol flux and transepithelial resistance. Hsp expression in intestinal YAMC cells was assessed after incubation with recombinant cytokines, activated lamina propria lymphocytes (LPLs), or Bacteroides fragilis. Results: Chronic metronidazole treatment decreases colonic mucosal hsp25 and hsp72 expression, an effect associated with increased susceptibility of mucosal barrier function to C. difficile toxin A. Hsp expression also was increased in YAMC cells incubated with B. fragilis, an effect mediated by lipopolysaccharide and other bacteria-derived factors. Colonic hsp72, but not hsp25 or hsc73, expression is decreased in RAG-1­/­ mice. Recombinant IL-2 and other cytokines enhance YAMC hsp25 and/or hsp72 expression. Activated LPLs induce YAMC hsp expression, an effect blocked by IL-2 neutralizing antibody. Conclusions: Enteric flora and mucosal lymphocytes play a role in maintaining physiologic expression of colonocyte hsp25 and hsp72.

Chronic Helicobacter pylori infections induce gastric mutations in mice
Eliette Touati, Valérie Michel, Jean-Michel Thiberge, Nicole Wuscher, Michel Huerre, Agnès Labigne
Background & Aims: Helicobacter pylori is an important etiologic factor in the development of gastric cancer. The aim of this study was to analyze the role of H. pylori infections in the induction of mutagenic events in gastric epithelial cells. The effect of a high-salt diet as a genotoxic risk factor was also investigated. Methods: Big Blue transgenic male mice (C57Bl/6) were inoculated with H. pylori (strain SS1) or Helicobacter felis (strain CS1) for 6 and 12 months. The frequency and spectrum of mutations at the stomach level were assessed. Inflammatory host response and inducible nitric oxide synthase (iNOS) expression by reverse-transcription polymerase chain reaction and immunohistochemistry analysis were also performed. Results: After 6 months, the gastric mutant frequency was 4-fold and 1.7-fold higher in mice infected with H. pylori and H. felis, respectively, than in uninfected mice. It was associated with a high frequency of transversions (AT CG and GC TA) known to result from oxidative damages. The Helicobacter-infected mice exhibited severe gastritis and a high level of iNOS messenger RNA expression. Hyperplasia developed 12 months after inoculation, and both the mutagenic effects and iNOS expression decreased in H. pylori- and H. felis-infected mice. No synergistic effects of a high-salt diet and Helicobacter infection were observed regarding the frequency of gastric mutation. Conclusions: A direct gastric mutagenic effect due to H. pylori infection in the Big Blue transgenic mouse model has been shown 6 months after inoculation. This genotoxicity can be attributable to oxidative DNA damage involving the inflammatory host response.

Delineation of a CD1d-restricted antigen presentation pathway associated with human and mouse intestinal epithelial cells
Yvonne van de Wal, Nadia Corazza, Matthieu Allez, Lloyd F. Mayer, Hideki Iijima, Mark Ryan, Steven Cornwall, Dominique Kaiserlian, Robert Hershberg, Yasuhiko Koezuka, Sean P. Colgan, Richard S. Blumberg
Background & Aims: CD1d, a major histocompatibility complex (MHC) class I-related molecule that is responsible for the presentation of glycolipid antigens to subsets of natural killer T (NK-T) cells, is expressed by intestinal epithelial cells (IECs). However, CD1d-restricted antigen presentation has not yet been examined on IECs. Methods: A mouse intestinal epithelial cell line (MODE-K), a human epithelial cell line (T84), T84 cells transfected with CD1d and/or MHC class II, and freshly isolated human IECs were examined for their ability to present model glycolipid antigens to NK-T cells as defined by interleukin (IL)-2 or IL-4 secretion. Results: MODE-K and freshly isolated human IECs exhibited dose-dependent, CD1d-restricted presentation of the functional glycolipid antigen, -galactosylceramide (GalCer), to the mouse NK-T cell hybridoma, DN32.D3. The human IEC line, T84, mainly presented GalCer when transfected with human CD1d. Presentation of GalCer by CD1d-transfected T84 cells (T84d) to DN32.D3 cells was greater along the basal surface in comparison with the apical surface. Induction of the MHC class II antigen presentation machinery by cotransfecting T84d with the MHC class I transactivator (CIITA) did not alter this polarity of presentation. Neither MODE-K nor T84 cells transfected with CD1d, CD1d plus CIITA, or CD1d plus HLA-DR were able to present glycolipid antigens requiring intracellular processing. The MODE-K cell line could also present GalCer to primary mouse NK-T cells. Conclusions: CD1d is expressed functionally on IECs with a polarity of presentation (basal > apical) predicting a role in presentation of mucosal glycolipid antigens to local CD1d-restricted T cells.

Basic-liver, Pancreas, and Biliary Tract

Localization of the ammonium transporters, Rh B glycoprotein and Rh C glycoprotein, in the mouse liver
I. David Weiner, R. Tyler Miller, Jill W. Verlander
Background & aims: Hepatic ammonium metabolism is critical for maintenance of normal health. Three mammalian members of an ammonium transporter family have recently been identified: Rh A glycoprotein (RhAG), Rh B glycoprotein (RhBG), and Rh C glycoprotein (RhCG). This study examined which of these are expressed in the mouse liver and in which cells they are expressed. Methods: Normal Balb/c mice were used. Messenger RNA (mRNA) expression was detected using either conventional or real-time reverse-transcription polymerase chain reaction (RT-PCR). Protein expression was examined using immunoblot analysis and either immunohistochemical or immunofluorescent microscopy. Results: We confirmed hepatic RhBG mRNA expression using real-time RT-PCR. Immunoblot analysis identified expression of a ~45-kilodalton protein. Immunohistochemical and immunofluorescent microscopy identified basolateral RhBG immunoreactivity in 1­2 cell layers of hepatocytes surrounding central veins. No immunoreactivity was identified in periportal or midzonal hepatocytes. Perivenous hepatocyte-specific expression was confirmed by colocalization with glutamine synthetase. A second ammonium transporter, RhCG, was expressed but at substantially lower levels. Real-time RT-PCR quantified hepatic RhCG mRNA expression at ~0.4% of RhBG mRNA expression. Immunoblot analysis confirmed RhCG protein expression, and immunofluorescence microscopy identified RhCG expression in bile duct epithelia. In contrast to RhBG and RhCG, RhAG mRNA was not identified by RT-PCR. Conclusions: RhBG and RhCG are expressed by the mouse liver. Basolateral RhBG is expressed by perivenous hepatocytes, where it may mediate ammonium uptake, and RhCG immunoreactivity is present in bile duct epithelial cells, where it may contribute to ammonium secretion into bile fluid.

IFN-2a protects mice against a helminth infection of the liver and modulates immune responses
Véronique Godot, Saïd. Harraga, Guennady Podoprigora, Martine Liance, Karine Bardonnet, Dominique A. Vuitton
Background & aims: Hepatic alveolar echinococcosis (AE), caused by the larval growth of Echinococcus multilocularis, is one of the most lethal helminthic diseases with no satisfactory treatment. Advances in the understanding of the host's immune response (Th2 responses associated with a progressive form of AE), have driven the research towards immune stimulation as an alternative possibility to treat patients. We previously reported clinical stabilization associated with a shift from a Th2 to a Th1 cytokine profile in an AE patient treated with interferon (IFN). Methods: The effects of recombinant IFN-2a were analyzed in the susceptible C57BL/6J E. multilocularis infected mice. Parasitic burden, macrophage functions, and specific T-cell responses were studied 15, 45, and 90 days postinfection. Results: After 90 days postinfection, 75% of infected IFN-2a-treated mice had no hepatic lesions and half were fully protected. IFN-2a treatment markedly decreased the abnormally elevated production of IL-10 in both spleen cell cultures and peritoneal macrophage cultures from infected mice and restored phagocytosis and oxidative metabolism of macrophages. It also inhibited IL-6 and IL-13 antigen-induced secretions in spleen cell cultures. Conclusions: Through its immunoregulatory properties, IFN-2a may be effective in a helminthic liver infection and is a promising candidate for clinical application in AE.

Leptin-specific mechanisms for impaired liver regeneration in ob/ob mice after toxic injury
Isabelle A. Leclercq, Jacqueline Field, Geoffrey C. Farrell
Background & aims: Profound impairment of liver regeneration in rodents with dysfunctional leptin signaling has been attributed to non-alcohol-induced fatty liver disorders (NAFLD). Our aim was to establish whether defective liver regeneration in ob/ob mice is a direct consequence of leptin-dependent, intracellular signaling mechanisms controlling cell-cycle regulation in hepatocytes. Methods: After exposure to a single hepatotoxic dose of (CCl4), the regenerative response to hepatic injury was studied in leptin-deficient ob/ob and control mice. The effects of leptin supplementation (100 µg · kg­1 · day­1) were examined. We assessed entry into and progression through the cell cycle and activation of key signaling intermediates and transcriptional regulators. Results: CCl4-induced liver injury was equally severe in ob/ob and control mice. In leptin-deficient mice, it was associated with exaggerated activation of NF-B and STAT3 during the priming phase, abrogation of tumor necrosis factor (TNF) and interleukin (IL)-6 release at the time of G1/S transition, and failure of hepatocyte induction of cyclin D1 and cell cycle entry. Leptin replacement corrected these defects in ob/ob mice by restoring TNF and IL-6 release and inducing cyclin D1. Hepatocytes entered S phase and progressed, as in wild-type mice, to vigorous mitosis and normal hepatic regenerative response. In ob/ob mice, low doses of TNF before CCl4 also were associated with restitution of TNF release and proliferative capabilities. Conclusions: Impaired liver regeneration in ob/ob mice is caused by leptin deficiency. We propose that altered cytokine production in ob/ob mice is part of the mechanisms responsible for impaired proliferation in response to hepatic injury.

Expression of hepatitis c virus proteins inhibits interferon signaling in the liver of transgenic mice
Alex Blindenbacher, Francois H.T. Duong, Lukas Hunziker, Simone T.D. Stutvoet, Xueya Wang, Luigi Terracciano, Darius Moradpour, Hubert E. Blum, Tonino Alonzi, Marco Tripodi, Nicola La Monica, Markus H. Heim
Background & aims Hepatitis C virus (HCV) is a major cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma worldwide. The majority of patients treated with interferon alpha do not have a sustained response with clearance of the virus. The molecular mechanisms underlying interferon resistance are poorly understood. Interferon-induced activation of the Jak-STAT (signal transducer and activator of transcription) signal transduction pathway is essential for the induction of an antiviral state. Interference of viral proteins with the Jak-STAT pathway could be responsible for interferon resistance in patients with chronic HCV. Methods We have analyzed interferon-induced signal transduction through the Jak-STAT pathway in transgenic mice that express HCV proteins in their liver cells. STAT activation was investigated with Western blots, immunofluorescence, and electrophoretic mobility shift assays. Virus challenge experiments with lymphocytic choriomeningitis virus were used to demonstrate the functional importance of Jak-STAT inhibition. Results STAT signaling was found to be strongly inhibited in liver cells of HCV transgenic mice. The inhibition occurred in the nucleus and blocked binding of STAT transcription factors to the promoters of interferon-stimulated genes. Tyrosine phosphorylation of STAT proteins by Janus kinases at the interferon receptor was not inhibited. This lack in interferon response resulted in an enhanced susceptibility of the transgenic mice to infection with a hepatotropic strain of lymphocytic choriomeningitis virus. Conclusions Interferon-induced intracellular signaling is impaired in HCV transgenic mice. Interference of HCV proteins with interferon-induced intracellular signaling could be an important mechanism of viral persistence and treatment resistance.

Involvement of integrins and Src in tauroursodeoxycholate-induced and swelling-induced choleresis
Dieter Häussinger, Anna Kordelia Kurz, Matthias Wettstein, Dirk Graf, Stephan Vom Dahl, Freimut Schliess
Background & Aims: Stimulation of canalicular secretion by tauroursodeoxycholate (TUDC) involves dual activation of p38 mitogen-activated protein kinase (p38MAPK) and extracellular signal-regulated kinase (ERK). This study investigates the sensing and upstream signaling events of TUDC-induced choleresis. Methods: TUDC and hypo-osmolarity effects on protein kinase activities and taurocholate excretion were studied in perfused rat liver. Results: TUDC induced a rapid activation of focal adhesion kinase (FAK) and Src, as shown by an increase in Y418 phosphorylation and a decrease in Y529 phosphorylation of Src. Inhibition of Src by PP-2 abolished the TUDC-induced activation of p38MAPK but not of FAK and ERKs. An integrin-inhibitory peptide with an RGD motif blocked TUDC-induced FAK, Src, ERK, and p38MAPK activation, suggesting that integrin signaling toward FAK/Src is required for TUDC-induced MAPK activation. The RGD peptide and PP-2 also abolished the stimulation of taurocholate excretion in perfused rat liver in response to TUDC. Integrin-dependent Src activation was also identified as an upstream event in hypo-osmotic signaling toward MAPKs and choleresis. Conclusions: TUDC-induced stimulation of canalicular taurocholate excretion involves integrin sensing, FAK, and Src activation as upstream events for dual MAPK activation. Integrins may also represent one long-searched sensor for cell hydration changes in response to hypo-osmolarity.

Betaine decreases hyperhomocysteinemia, endoplasmic reticulum stress, and liver injury in alcohol-fed mice
Cheng Ji, Neil Kaplowitz
Background & Aims: Alcohol-induced hyperhomocysteinemia has been reported in rats and humans. Hyperhomocysteinemia has been associated with endoplasmic reticulum (ER) stress leading to the activation of ER-dependent apoptosis or up-regulation of lipid synthesis. This novel ER stress mechanism of alcoholic liver injury was studied in the model of intragastric alcohol-fed mice. Methods: Effects of alcohol on gene expression were analyzed using cDNA microarrays, RT-PCR, and Western blots over a period of 6 weeks. Liver injury was examined by histologic staining and TUNEL. Results: We observed fatty liver, increased hepatic necroinflammation and apoptosis, and hyperhomocysteinemia. Of 1176 toxicology-related genes, glucose-regulated proteins (GRP-78 and -94), growth arrest/DNAdamage-inducible protein 153 (CHOP/GADD153), and caspase-12 indicative of an ER stress response were among the alcohol-responsive genes. Sterol regulatory element binding protein (SREBP-1) and HMG-CoA reductase also were enhanced with alcohol administration. RT-PCR and selective Western blots confirmed the alcohol-induced expression of ER stress-related apoptosis and lipid synthesis genes. Addition of 0.5% and maximal 1.5% betaine to the alcohol diet reduced the elevated level of plasma homocysteine by 54% and more than 80% accompanied by a decrease in hepatic lipids and ER stress response. Betaine did not attenuate the ethanol-induced increase in tumor necrosis factor or CD14 mRNA. Conclusions: The results strongly suggest that alcohol may modulate both apoptotic and fat synthetic gene expression through homocysteine-induced ER stress in chronic alcoholic mouse liver and that correction of hyperhomocysteinemia by betaine or other approaches may be useful to prevent alcoholic liver disease.

The role of nitric oxide synthase isoforms in extrahepatic portal hypertension: Studies in gene-knockout mice
Nicholas G. Theodorakis, Yi-ning Wang, Nicholas J. Skill, Matthew A. Metz, Paul A. Cahill, Eileen M. Redmond, James V. Sitzmann
Background & Aims: Considerable debate exists concerning which isoform of nitric oxide synthase (NOS) is responsible for the increased production of NO in PHT. We used the portal vein ligation model of PHT in wild-type and eNOS- or iNOS-knockout mice to definitively determine the contribution of these isoforms in the development of PHT. Methods: The portal vein of wild-type mice, or those with targeted mutations in the nos2 gene (iNOS) or the nos3 gene (eNOS), was ligated and portal venous pressure (Ppv), abdominal aortic blood flow (Qao), and portosystemic shunt determined 2 weeks later. Results: In wild-type mice, as compared with sham-operated controls, portal vein ligation (PVL) resulted in a time-dependent increase in Ppv (7.72 ± 0.37 vs 17.57 ± 0.51 cmH2O, at 14 days) concomitant with a significant increase in Qao (0.12 ± 0.003 vs 0.227 ± 0.005 mL/min/g) and portosystemic shunt (0.47% ± 0.01% vs 84.13% ± 0.09% shunt). Likewise, PVL in iNOS-deficient mice resulted in similar increases in Ppv, Qao, and shunt development. In contrast, after PVL in eNOS-deficient animals, there was no significant change in Ppv (7.52 ± 0.22 vs 8.07 ± 0.4 cmH20) or Qao (0.111 ± 0.01 vs 0.14 ± .023 mL/min/g). However, eNOS (­/­) mice did develop a substantial portosystemic shunt (0.33% ± 0.005% vs 84.53% ± 0.19% shunt), comparable to that seen in wild-type animals after PVL. Conclusions: These data support a key role for eNOS, rather than iNOS, in the pathogenesis of PHT.

Copyright 2003 Elsevier Science (USA). All rights reserved.

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JOURNAL OF HEPATOLOGY

Table of Contents for Journal of Hepatology Volume 38, Issue 5, May 2003

Cirrhosis and its Complications

Increased pulmonary vascular endothelin B receptor expression and responsiveness to endothelin-1 in cirrhotic and portal hypertensive rats: a potential mechanism in experimental hepatopulmonary syndrome
Bao Luo et al.

Background/Aims: In experimental hepatopulmonary syndrome (HPS), hepatic endothelin-1 (ET-1) release during common bile duct ligation (CBDL) and ET-1 infusion in pre-hepatic portal hypertension after portal vein ligation (PVL) initiate vasodilatation through an endothelin B receptor mediated increase in pulmonary endothelial nitric oxide synthase (eNOS). We evaluated if pulmonary ET receptor expression changes in experimental cirrhosis and portal hypertension and confers susceptibility to HPS. Methods: In normal, PVL and CBDL animals, lung ET receptor expression and localization were assessed and ET receptor levels and functional analysis of ET-1 effects on eNOS levels were evaluated in intralobar pulmonary artery (PA) and aortic (AO) segments. Normal rats underwent evaluation for HPS after ET-1 infusion. Results: There was a selective increase in ETB receptor expression in the pulmonary vasculature from PVL and CBDL animals. ET-1 stimulated NO production and an ETB receptor mediated increase in eNOS levels in PA segments from PVL and CBDL animals, but not normal animals. ET-1 did not alter lung eNOS levels or cause HPS in normal rats. Conclusions: ETB receptor expression and ET-1 mediated eNOS and NO production are enhanced in the lung vasculature in cirrhotic and portal hypertensive animals and correlate with in vivo susceptibility to ET-1 mediated HPS.

Inflammation and Fibrosis

Effect of HMG-CoA reductase inhibitors on proliferation and protein synthesis by rat hepatic stellate cells
Krista Rombouts et al.

Background/Aims: 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors called statins, have besides their cholesterol-lowering function, therapeutic value in conditions such as neo-angiogenesis and atherosclerosis. We investigated the effect of two statins on the proliferation rate and protein steady state levels of hepatic stellate cells (HSC). Methods: Cellular DNA synthesis under the influence of statins and/or platelet derived growth factor (PDGF) and mevalonate was evaluated by measuring BrdU incorporation. Synthesis of collagens type I, III, IV and fibronectin was quantified by ELISA. Additionally, we examined the influence of simvastatin on isoprenylation of Ras and RhoA proteins. Results: Lovastatin and simvastatin induced a dose-dependent inhibition of the proliferation rate of HSC. Subsequent addition of PDGF and/or mevalonate, after long-term exposure of simvastatin to HSC, did not reverse simvastatins' antiproliferative effect. Lovastatin and simvastatin reduced the protein steady state level of collagens type I (40%), III (45%) and IV (27%). Membrane bound Ras steady state levels decreased under the influence of simvastatin. Membrane bound RhoA remained unaltered, whereas, cytosolic RhoA protein level was strongly reduced. Conclusions: Our data showed that lovastatin and simvastatin inhibited HSC proliferation and collagen steady state levels by mechanisms independent of their lipid reducing activities.

Liver Cell Injury and Liver Failure

Detection and analysis of intracytoplasmic cytokines in peripheral blood mononuclear cells in patients with drug-induced liver injury
Hiroyuki Murata, Yukihiro Shimizu, Kazuhiko Okada, Kiyohiro Higuchi and Akiharu Watanabe

Background/Aims: Idiosyncratic immune response to drugs causes two types of liver injury, cholestasis or hepatitis. However, the underlying immune mechanisms of drug-induced liver injury are presently unclear. Methods: We examined the cytokine production of peripheral blood mononuclear cells (PBMCs) from 17 patients with drug-induced liver injury and healthy controls during their incubation with and without the drug by flow cytometry. We also analyzed the cytokine production in PBMCs from eight patients after stimulation with the drug-pulsed HepG2 lysates to examine the possibility that the drug or its metabolites conjugated with a putative molecule derived from HepG2 cells might be more immunogenic. Results: Among several cytokines produced by the drug or the drug-pulsed HepG2 lysates, interferon- production from CD8+ cells was associated with hepatocellular injury, and tumor necrosis factor- production from CD14+ cells was with cholestasis. Especially, the latter was apparent when the drug-pulsed HepG2 lysates were used as stimulants, suggesting that a complex consist of the drug, or its metabolite, and a putative molecule derived from HepG2 cells might be more immunogenic than the drug itself. Conclusions: The analysis of intracytoplasmic cytokine in PBMCs after stimulation with the drug or the drug-pulsed HepG2 lysates is useful to analyze the immune mechanism underlying drug-induced liver injury.

The effects of early and late administration of inhibitors of inducible nitric oxide synthase in a thioacetamide-induced model of acute hepatic failure in the rat
Tony Manibur Rahman and Humphrey Julian Francis Hodgson

Background/Aims: Nitric oxide (NO) is a pivotal mediator of inflammation. Its role in acute hepatic failure (AHF) is controversial. We investigated the role of NO, and the hypothesis that inhibition of inducible NO synthase (iNOS) activity would improve outcome in liver failure in rats, using the iNOS inhibitors L-NAME and aminoguanidine (AMG). Methods: AHF was induced by two intraperitoneal injections of thioacetamide (TAA). Seven groups (n=10) were studied. Group I: TAA alone. Groups II, III and IV were additionally pre-treated with the NO precursor L-arginine (300 mg/kg i.p.), or iNOS inhibitors AMG (100 mg/kg s.c.), or NG-nitro-L-arginine methyl ester (L-NAME) (100 mg/kg s.c.) for 5 days, respectively. Groups V, VI and VII received L-arginine, AMG or L-NAME commencing immediately after TAA administration. Clinical and biochemical parameters were assessed serially, and mortality investigated in further similar cohorts for each regime. Results: AMG, pre-treatment but not post-treatment, significantly improved outcome including mortality (10 vs. 70%, P<0.005). The less selective iNOS inhibitor L-NAME was not beneficial. Arginine pre-and post-treatment, and iNOS inhibition post-treatment, worsened clinical parameters of TAA-induced liver failure. Conclusions: Administration of the iNOS inhibitor AMG prior to insult reduces the severity of damage and improves mortality.

Liver Growth and Cancer

p27Kip1 is an independent predictor of recurrence after surgical resection in patients with small hepatocellular carcinoma
Carolina Armengol et al.

Background/Aims: Alterations in p27Kip1 (p27) and cyclin E (cycE) expression are found in tumors and are related to poor prognosis. This study assesses the role of these cell cycle regulators in the development of recurrence after surgical resection in 46 cirrhotic patients (age: 61.3±7 years, 30 males, 44 Child-Pugh's A, 30 HCV-positive) with small hepatocellular carcinoma (HCC, size: 3.1±1.5cm, 40 solitary at pathological examination). Methods: p27 and cycE expression in tumoral and non-tumoral liver were analyzed by Western blot (WB). p27 was also assessed by immunohistochemistry (IHC). Results: Tumor p27 underexpression (50% decreased vs. non-tumoral liver) occurred in 12 cases. Throughout follow-up, 26 patients developed recurrence, which was significantly higher in patients with p27 underexpression than in those without (3-year recurrence: 80 vs. 44%, respectively, P=0.026). IHC showed concordant inverse findings: 13 tumors showed high p27 staining that was related to lower recurrence rate (P=0.019). Multivariate analysis identified p27 measured by WB as an improved predictor of recurrence (OR: 3.09, 95% CI: 1.26-7.08, P=0.016). By contrast, cycE, increased in 66% of the tumors, had no impact on recurrence but was associated to poor differentiation (P=0.015) and microvascular invasion (P=0.016). Conclusions: p27 underexpression is frequent in relatively early stages of HCC and constitutes an independent predictor of recurrence after surgical resection.

The mitogenic activity of the liver growth factor is mediated by tumor necrosis factor alpha in rat liver
Juan J. Díaz-Gil et al.

Background/Aims: Liver growth factor (LGF) is a hepatic mitogen, however, the hepatic stimulation pathway remains to be characterized. The aim of this study was to determine whether tumor necrosis factor alpha (TNF-) stimulation constitutes a step in the mitogenic pathway of LGF. Methods: Rats were injected with 4.5 µg LGF/rat, and LGF activity was measured both by liver DNA synthesis stimulation and `proliferating cell nuclear antigen (PCNA)-positive' hepatocytes in rats injected with LGF or +anti-TNF-. TNF- expression was evaluated by reverse-transcription polymerase chain reaction. TNF--producing cells were immunodetected. Human endothelial cells (HUVEC) were stimulated by LGF. TNF- was detected in the supernatant, and the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular endothelial adhesion molecule-1 (VCAM-1) by flow cytometry analysis. Results: LGF-injected rats showed higher intrahepatic TNF- expression. DNA synthesis and PCNA-positive hepatocytes induced by LGF were inhibited by anti-TNF-, PCNA-positive hepatocytes being especially abundant around the central vein when LGF was injected alone, but TNF- exhibited increased signal intensity in endothelial cells of the portal vein. LGF stimulated TNF- secretion in HUVEC, but did not stimulate ICAM-1 or VCAM-1 up-regulation. Conclusions: The mitogenic cascade initiated by LGF in rat liver in vivo depends, at least in part, on TNF- stimulation. Portal vein endothelial cells seem to be a source of TNF-.A

Activation of the ATF6, XBP1 and grp78 genes in human hepatocellular carcinoma: a possible involvement of the ER stress pathway in hepatocarcinogenesis
Masahiro Shuda et al.

Background/Aims: We identified the glucose-regulated protein (grp) 78 as a transformation-associated gene in hepatocellular carcinoma (HCC). Grp78 is a molecular chaperone involved in the unfolded protein response, the expression of which can be regulated by the transcription factors ATF6 and XBP1. Thus, we investigated the regulatory mechanisms of the grp78 gene in liver malignancy. Methods: Expression of grp78, ATF6 and XBP1 was examined by Northern blot, RT-PCR, immunoblot and immunohistochemical analyses. A reporter assay of the grp78 promoter was also performed. Results: Elevation of grp78 and ATF6 mRNAs and the splicing of XBP1 mRNA, resulting in the activation of XBP1 product, occurred in HCC tissues with increased histological grading. Higher accumulation of the grp78 product in the cytoplasm, concomitantly with marked nuclear localization of the activated ATF6 product (p50ATF6), was observed in moderately to poorly differentiated HCC tissues. Cooperation between the distal DNA segment and the proximal endoplasmic reticulum stress response elements was essential for maximum transcription of the grp78 promoter in HCC cells. Conclusions: The endoplasmic reticulum stress pathway mediated by ATF6 and by IRE1-XBP1 systems seems essential for the transformation-associated expression of the grp78 gene in HCCs.

Antisense oligodeoxynucleotides directed against aspartyl (asparaginyl) -hydroxylase suppress migration of cholangiocarcinoma cells
Takashi Maeda et al.

Background: Aspartyl (asparaginyl) -hydroxylase (AAH) is an -ketoglutarate-dependent dioxygenase that hydroxylates aspartate and asparagine residues in EGF-like domains of proteins. The consensus sequence for AAH -hydroxylation occurs in signaling molecules such as Notch and Notch homologs, which have roles in cell migration. Aim: This study evaluated the potential role of AAH in cell migration using cholangiocarcinoma cell lines as models due to their tendency to widely infiltrate the liver. Methods: Five human cholangiocarcinoma cell lines established from human tumors were examined for AAH expression and motility. The effect of antisense oligodeoxynucleotide inhibition of AAH on cholangiocarcinoma cell migration was investigated. Results: Western blot analysis detected the ~86 kDa AAH protein in all five cholangiocarcinoma cell lines, and higher levels of AAH in cell lines derived from moderately or poorly differentiated compared with well-differentiated tumors. Immunocytochemical staining and fluorescence activated cell sorting analysis revealed both surface and intracellular AAH immunoreactivity. Using the phagokinetic non-directional migration assay and a novel ATPLite luminescence-based directional migration assay, we correlated AAH expression with motility. Correspondingly, antisense and not sense or mutated antisense AAH oligodeoxynucleotides significantly inhibited AAH expression and motility in cholangiocarcinoma cells. Conclusions: AAH over-expression may contribute to the infiltrative growth pattern of cholangiocarcinoma cells by promoting motility.

A possible role of cholesterol-sphingomyelin/phosphatidylcholine in nuclear matrix during rat liver regeneration
Elisabetta Albi, Samuela Cataldi, Graziella Rossi and Mariapia Viola Magni

Background/Aims: Phospholipids and cholesterol in chromatin have been previously demonstrated. The lipid fraction changes during cell proliferation in relation to activation of enzymes of phospholipid metabolism. The aim of the present work is to clarify if chromatin lipids may derive or not from nuclear matrix and if they have different roles. Methods: The subnuclear fractions were isolated from rat hepatocyte nuclei and the lipid fraction was extracted and analysed by chromatography in normal and regenerating liver. The phosphatidylcholine-sphingomyelin metabolism enzymes activity was assayed, by using radioactive substrates. Results: In nuclear matrix, cholesterol and sphingomyelin are respectively five and three times higher than those present in chromatin; the amount of phosphatidylcholine, which it is enriched in saturated fatty acids, is lower, thus indicating a less fluid structure. The lower content in phosphatidylcholine may be justified by the phosphatidylcholine-dependent phospholipase C activity, which increases during liver regeneration, reaching a peak at the beginning of S-phase, when also cholesterol and sphingomyelin increase. Conclusions: The nuclear matrix lipids are independent from chromatin lipids; the ratio cholesterol-sphingomyelin/phosphatidylcholine is higher and, as a consequence, nuclear matrix is less fluid in relation to DNA synthesis, suggesting a specific role of nuclear matrix as a structure involved in DNA duplication.

Transplantation and Surgery

Alcohol relapse after liver transplantation for alcoholic liver disease: does it matter?
Georges-Philippe Pageaux et al.

Background/Aims: The aim of this study was to distinguish the types of alcohol consumption after liver transplantation (LT) for alcoholic cirrhosis and to assess the consequences of heavy drinking. Methods: Patients transplanted for alcoholic cirrhosis were studied. Alcoholic relapse diagnosis was based upon patient's and family members' reports, liver enzyme tests, graft biopsy, and use of urine alcohol test. Results: One hundred twenty-eight patients were studied, with a mean follow-up of 53.8 months. After LT, 69% of patients were abstinent, 10% were occasional drinkers, and 21% were heavy drinkers. Actuarial survival rates were not different, but three of the seven deaths observed among heavy drinkers were directly related to alcohol relapse. Although there was no difference between the three groups concerning the rejection rates, all rejection episodes observed in the group of heavy drinkers were related to poor compliance with immunosuppressive drugs. One heavy drinker developed alcoholic cirrhosis. Conclusions: The present study indicates that patients can resume heavy alcohol consumption after LT for alcoholic liver disease (ALD) and their grafts can be injured because of poor compliance with immunosuppressive drugs and alcohol-related liver injury. Although patient survival was not influenced by alcohol relapse, heavy alcohol consumption can be responsible for patients' death.

Viral Hepatitis

The effects of hepatitis B virus core promoter mutations on hepatitis B core antigen distribution in hepatocytes as detected by laser-assisted microdissection
Keiko Kawai, Norio Horiike, Kojiro Michitaka and Morikazu Onji

Background/Aims: The severity of liver damage in patients with chronic hepatitis B is dependent on several factors such as subcellular localization of hepatitis B core antigen (HBcAg) and mutation of hepatitis B virus (HBV) DNA. Here we studied the interrelationship between these two factors both in situ and in vitro. Methods: Hepatocytes from liver biopsies showing expression of HBcAg only in the cytoplasm (n=6), only in the nucleus (n=4) and in both cytoplasms or nucleus of different hepatocytes (n=5) were picked up by laser-assisted microdissection and were checked for nucleotide sequences of core promoter region of HBV DNA. HepG2 and Huh7 cell lines transfected with wild and mutant HBV DNA were checked for localization of HBcAg. Results: The frequencies of core promoter mutations at nucleotide (nt) 1762 and nt 1764 was significantly higher in hepatocytes with cytoplasmic expression of HBcAg compared to that of nuclear expression of HBcAg (P<0.05). Cytoplasmic expression of HBcAg was observed more frequently in HepG2 and Huh7 cells transfected with HBV mutant type (nt 1762 and 1764) than HBV wild type (P<0.05). Conclusions: Cytoplasmic localization of HBcAg was associated with HBV DNA mutations at nt 1762 and 1764.

Expansion of innate CD5pos B cells expressing high levels of CD81 in hepatitis C virus infected liver
Michael P. Curry et al.

Background/Aims: Association of hepatitis C virus (HCV) with increased autoantibodies, mixed cryoglobulinaemia, non-Hodgkin's B-cell lymphoma and increased peripheral innate (CD5pos) B cells suggests a role for B-lymphocytes in the pathogenesis of HCV-infection. Methods: Flow cytometry was used to estimate CD5pos B cell levels and CD81 co-expression in chronic HCV infection. Viral load was assessed using PCR. Results: We demonstrate expansion of innate B cells in HCV-infected liver from patients with fibrosis score less than stage II (39%, % of total B cells, P=0.002) and end stage HCV cirrhosis (20%, P<0.05) compared with normal liver (8%). Expression of CD81, a signal transducing molecule and putative HCV receptor, was significantly increased on peripheral blood CD5pos B cells compared with conventional B cells (P=0.0001). Higher levels of CD81 on CD5pos B cells were more dramatic in the liver of HCV-infected individuals. However, no significant difference was observed in the viral load of CD5posCD81High B cells and CD5negCD81Low B cells. Conclusions: Increased expression of CD81 on innate B cells, a population that is expanded in the livers and peripheral blood of chronic HCV-infected patients, suggests a role in viral specific activation and clonal proliferation in chronic HCV infection.

T lymphocytes infiltrating the liver during chronic hepatitis C infection express a broad range of T-cell receptor beta chain diversity
Ines Vigan et al.

Background/Aims: During viral chronic hepatitis C (CHC), the intra-hepatic lymphocyte infiltrate is mainly composed of T lymphocytes expressing T-cell receptors (TCR). Since little is known about the TCR diversity of intra-hepatic T lymphocytes (IHL), we evaluated the IHL repertoire from CHC patients (n=8) as compared to healthy subjects (n=4), total peripheral blood mononuclear cells, and purified peripheral and intra-hepatic CD8+ cells (n=2). Methods: The diversity of TCR receptors was evaluated by determining the size and the sequence of the TCR chain complementarity determining region 3 (CDR3). The number of total T lymphocytes in liver was estimated by real-time quantitative reverse transcription-polymerase chain reaction of TCR and CD3 transcripts. Results: Our results show that transcripts encoding all TCR V beta (BV) families and all TCR J beta (BJ) segments were present in healthy and CHC livers. No biased TCR repertoire, in terms of preferential BV or BJ gene use or restricted CDR3 sequence, was observed in infected livers. When corrected for equivalent numbers of T lymphocytes, BJ segments utilization and CDR3 length diversity were similar in IHL and PBMC, indicating that the TCR chain diversity is comparable in both cases. In addition, TCR diversity was similar in both peripheral and intra-hepatic CD8+ T cells. Conclusions: This study shows limited expansions of intra-hepatic T lymphocytes in CHC patients. The increase of T lymphocytes in infected livers correlates with diversification of TCR, arguing for the establishment of a multi-specific immune response.

Hepatocyte growth factor activates endothelial proangiogenic mechanisms relevant in chronic hepatitis C-associated neoangiogenesis
Jesús Medina et al.

Background: Angiogenesis occurs in inflamed portal tracts of chronic hepatitis C (CHC) patients. Aims: To characterize this phenomenon, by investigating the molecular mechanisms involved in neovessel formation in the livers of CHC patients and the angiogenic effects of hepatocyte growth factor (HGF) on human endothelial cells. Methods: Vascular endothelial growth factor (VEGF), VE-cadherin and v3 integrin were determined in CHC biopsies by Western blot and immunohistochemistry. Effects of HGF on VEGF and cell adhesion molecules expression by cultured human microvascular endothelial cells were evaluated by Western blot, Northern blot or immunofluorescence. HGF effects on cell proliferation were assessed by [3H]thymidine incorporation. Results: VEGF, VE-cadherin and v3 integrin were increased in CHC liver samples. In cultured endothelial cells, HGF transcriptionally increased VEGF expression, an effect which was blocked by an anti-VEGF receptor antibody. HGF transiently decreased VE-cadherin expression and its associated cytoskeleton-linking molecule -catenin, thus weakening intercellular contacts. HGF increased v3 integrin at focal contacts, and cell proliferation, an effect which was inhibited by an anti-VEGF receptor antibody. Conclusions: Our results show that HGF and VEGF modulate the expression of cell adhesion and migration molecules and induce proliferation in endothelial cells, mechanisms through which these factors may contribute to CHC-associated liver angiogenesis.

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

BRITISH MEDICAL JOURNAL

NEW ENGLAND JOURNAL

LANCET

Volume 361, Number 9368 03 May 2003

Dietary fibre and colorectal adenoma in a colorectal cancer early detection programme
Ulrike Peters, Rashmi Sinha, Nilanjan Chatterjee, Amy F Subar, Regina G Ziegler, Martin Kulldorff, Robert Bresalier, Joel L Weissfeld, Andrew Flood, Arthur Schatzkin, Richard B Hayes, for the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial Project Team

Background Although dietary fibre has been reported to have no association with colorectal adenoma and cancer, in some studies this topic remains controversial.

Methods We used a 137-item food frequency questionnaire to assess the relation of fibre intake and frequency of colorectal adenoma. The study was done within the Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial, a randomised controlled trial designed to investigate methods for early detection of cancer. In our analysis, we compared fibre intake of 33 971 participants who were sigmoidoscopy-negative for polyps, with 3591 cases with at least one histologically verified adenoma in the distal large bowel (ie, descending colon, sigmoid colon, or rectum). Odds ratios were estimated by logistic regression analysis.

Findings High intakes of dietary fibre were associated with a lower risk of colorectal adenoma, after adjustment for potential dietary and non-dietary risk factors. Participants in the highest quintile of dietary fibre intake had a 27% (95% CI 14-38, ptrend=0·002) lower risk of adenoma than those in the lowest quintile. The inverse association was strongest for fibre from grains and cereals and from fruits. Risks were similar for advanced and non-advanced adenoma. Risk of rectal adenoma was not significantly associated with fibre intake.

Interpretation Dietary fibre, particularly from grains, cereals, and fruits, was associated with decreased risk of distal colon adenoma.

Lancet 2003; 361: 1491-95

Dietary fibre in food and protection against colorectal cancer in the European Prospective Investigation into Cancer and Nutrition (EPIC): an observational study  
Sheila A Bingham, Nicholas E Day, Robert Luben, Pietro Ferrari, Nadia Slimani, Teresa Norat, Françoise Clavel-Chapelon, Emmanuelle Kesse, Alexandra Nieters, Heiner Boeing, Anne Tjønneland, Kim Overvad, Carmen Martinez, Miren Dorronsoro, Carlos A Gonzalez, Timothy J Key, Antonia Trichopoulou, Androniki Naska, Paolo Vineis, Rosario Tumino, Vittorio Krogh, H Bas Bueno-de-Mesquita, Petra HM Peeters, Göran Berglund, Göran Hallmans, Eiliv Lund, Guri Skeie, Rudolf Kaaks, Elio Riboli

Background Dietary fibre is thought to protect against colorectal cancer but this view has been challenged by recent prospective and intervention studies that showed no protective effect.

Methods We prospectively examined the association between dietary fibre intake and incidence of colorectal cancer in 519 978 individuals aged 25-70 years taking part in the EPIC study, recruited from ten European countries. Participants completed a dietary questionnaire in 1992-98 and were followed up for cancer incidence. Relative risk estimates were obtained from fibre intake, categorised by sex-specific, cohort-wide quintiles, and from linear models relating the hazard ratio to fibre intake expressed as a continuous variable.

Findings Follow-up consisted of 1939011 person-years, and data for 1065 reported cases of colorectal cancer were included in the analysis. Dietary fibre in foods was inversely related to incidence of large bowel cancer (adjusted relative risk 0·75 [95% CI 0·59-0·95] for the highest versus lowest quintile of intake), the protective effect being greatest for the left side of the colon, and least for the rectum. After calibration with more detailed dietary data, the adjusted relative risk for the highest versus lowest quintile of fibre from food intake was 0·58 (0·41-0·85). No food source of fibre was significantly more protective than others, and non-food supplement sources of fibre were not investigated.

Interpretation In populations with low average intake of dietary fibre, an approximate doubling of total fibre intake from foods could reduce the risk of colorectal cancer by 40%.

Lancet 2003; 361: 1496-501

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