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Mois de Juin 2005

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HEPATOLOGY

Table of Contents for June 2005 • Volume 41, Issue 6

Liver Biology and Pathobiology

An intronic silencer element is responsible for specific zonal expression of glutamine synthetase in the rat liver (p 1225-1232)
Frank Gaunitz, Danilo Deichsel, Kerstin Heise, Max Werth, Ulf Anderegg, Rolf Gebhardt
The most striking phenomenon of glutamine synthetase (GS) expression in the liver is its unique restriction to cells surrounding the terminal hepatic venules. Expression is positively regulated by elements located in the 5-upstream region and in the first intron of the gene. It was long believed that transcription factors present in GS-positive cells and absent in GS-negative cells are responsible for the phenomenon of zonal expression. However, strong enhancers are equally active in both types of cells. Therefore, the existence of a silencer mechanism in GS-negative hepatocytes was postulated. In the present study, a GS silencer element was investigated that was previously identified within the first intron and was shown to be able to prevent glucocorticoid-induced expression in cells negative for a transacting factor designated GS silencer element-binding protein. Reporter gene assays with the silencer element in combination with the most potent 5-enhancer of the GS gene demonstrate that the silencer element is able to prevent enhancement mediated by the 5-enhancer in combination with a heterologous as well as with the homologous promoter. More importantly, the effect of the silencer is shown to be restricted to GS-negative hepatocytes. In conclusion, the phenomenon of zonal expression of GS in the liver is caused by a protein present in GS-negative cells and absent in GS-positive cells that interacts with the silencer element in the first intron and not by a differential expression of enhancer-binding proteins.

Effective angiostatic treatment in a murine metastatic and orthotopic hepatoma model (p 1233-1240)
Esther Raskopf, Christian Dzienisowicz, Tobias Hilbert, Christian Rabe, Ludger Leifeld, Nicolas Wernert, Tilman Sauerbruch, Jesús Prieto, Cheng Qian, Wolfgang H. Caselmann, Volker Schmitz
Vascular endothelial growth factor (VEGF) activity is correlated with a progressive tumor disease in patients with hepatocellular carcinoma (HCC). In spite of the well-recognized role of VEGF in HCC, there are few data available regarding therapeutic strategies to block VEGF activity. Therefore, we employed a recombinant adenoviral vector encoding a soluble dominant negative fragment of VEGF receptor 2 (Flk-1), AdsFlk-1, to control pre-established murine orthotopic and metastatic hepatomas. Vector function was confirmed via reverse-transcriptase polymerase chain reaction and ELISA, and angiostatic effects were analyzed in vitro and in vivo. Antitumoral effects of systemic AdsFlk-1 application were studied in a subcutaneous and orthotopic Hepa129 HCC model. Cell supernatant containing the truncated form of Flk-1 had no direct effect on cell proliferation of Hepa129 cells in vitro but reduced endothelial tube formation on matrigel matrix by approximately 80% in vitro. Endothelial-like cell infiltration into matrigel plugs in vivo was also decreased by 80%. Systemic treatment of tumor-bearing mice inhibited tumor growth by 84% compared with the corresponding control group within 16 days after vector application. Likewise, the survival rate was significantly improved in the AdsFlk-1 group compared with control. Orthotopic tumor growth was reduced by 82%, and development of malignant ascites was also retarded. In conclusion, systemic adenoviral-mediated gene transfer of an Flk-1 fragment significantly inhibited tumor growth in orthotopic and metastatic murine HCC. The data support the value of VEGF blockade as an effective target for HCC treatment.

Activation of CREB by tauroursodeoxycholic acid protects cholangiocytes from apoptosis induced by mTOR inhibition (p 1241-1251)
LiFu Wang, Anne-Christine Piguet, Karin Schmidt, Thierry Tordjmann, Jean-François Dufour
Tauroursodeoxycholic acid (TUDCA) is a cytoprotective bile acid frequently prescribed to patients with cholestatic diseases. Several mechanisms of action have been investigated, but the possibility that cyclic adenosine monophosphate responsive element binding protein (CREB), a transcription factor promoting cell survival, mediates TUDCA's protective effects has not been considered. We examined whether TUDCA activates CREB and whether this activation can protect biliary epithelial cells. Cholangiocytes were stressed by exposure to CCI-779, which inhibits signaling though the kinase mTOR (mammalian target of rapamycin), resulting in cell cycle arrest and apoptosis. Incubation of normal rat cholangiocytes (NRC) cells, with TUDCA resulted in phosphorylation of CREB (Western blotting analysis) and activation of CREB transcription activity (luciferase reporter assay). Inhibition of calcium signals and inhibition of protein kinase C prevented the TUDCA-induced activation of CREB. CCI-779 decreased the viability of rat cholangiocytes in a dose-dependent manner (MTT [3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay). TUDCA protected against CCI-779 cytotoxicity. A dominant negative form of CREB was stably transduced in NRC cells (NRC-M1). TUDCA protection was decreased in NRC-M1. While CCI-779 induced apoptosis in NRC cells as determined by caspase 3 activity, TUDCA attenuated CCI-779-induced apoptosis, an effect absent in NRC-M1. Finally, CCI-779 blocked proliferation of both NRC and NRC-M1 (thymidine incorporation) and this was unaffected by TUDCA. In conclusion, TUDCA activates CREB in cholangiocytes, reducing the apoptotic effect of CCI-779. These findings suggest a novel cytoprotective mechanism for this bile acid.

Dose- and time-dependent oval cell reaction in acetaminophen-induced murine liver injury (p 1252-1261)
Alexander V. Kofman, Glyn Morgan, Adam Kirschenbaum, Jon Osbeck, Mehboob Hussain, Scott Swenson, Neil D. Theise
We examined the response of murine oval cells, that is, the putative liver progenitor cells, to acetaminophen. Female C57BL/6J mice were injected intraperitoneally with varying doses of N-acetyl-paraaminophen (APAP) (250, 500, 750, and 1,000 mg/kg of weight) and sacrificed at 3, 6, 9, 24, and 48 hours. In preliminary studies, we showed that anticytokeratin antibodies detected A6-positive cells with a sensitivity and specificity of greater than 99%. The oval cell reaction was quantified, on immunostaining for biliary-type cytokeratins, as both number and density of oval cells per portal tract, analyzed by size of portal tract. Acetaminophen injury was followed by periportal oval cell accumulation displaying a moderate degree of morphological homogeneity. Oval cell response was biphasic, not temporally correlating with the single wave of injury seen histologically. Increases in oval cells were largely confined to the smallest portal tracts, in keeping with their primary derivation from the canals of Hering, and increased in a dose-dependent fashion. The timing of the two peaks of the oval cell reaction also changed with increasing dose, the first becoming earlier and the second later. In conclusion, our studies indicate a marked oval cell activation during the height of hepatic injury. Oval cells appear to be resistant to acetaminophen injury. The close fidelity of mechanism and histology of acetaminophen injury between mouse and human livers makes it a useful model for investigating liver regeneration and the participation of stem/progenitor cells in that process.

An inhibitor of cyclin-dependent kinase, stress-induced p21Waf-1/Cip-1, mediates hepatocyte mito-inhibition during the evolution of cirrhosis (p 1262-1271)
John G. Lunz III, Hirokazu Tsuji, Isao Nozaki, Noriko Murase, Anthony J. Demetris
During the evolution of cirrhosis, there is a relative decrease in volume percentage of hepatocytes and a relative increase in biliary epithelial cells and myofibroblasts. This is recognized histopathologically as a ductular reaction and leads to gradual distortion of the normal hepatic architecture. The final or decompensated stage of cirrhosis is characterized by a further decline in hepatocyte proliferation and loss of functional liver mass that manifests clinically as ascites, encephalopathy, and other signs of liver failure. In this report, we tested the hypothesis that p21-mediated hepatocyte mito-inhibition accelerates the evolution of cirrhosis using an established mouse model of decompensated biliary cirrhosis, p21-deficient mice, and liver tissue from humans awaiting liver replacement. Despite the same insult of long-term (12-week) bile duct ligation, mice prone to decompensation showed significantly more oxidative stress and hepatocyte nuclear p21 expression, which resulted in less hepatocyte proliferation, an exaggerated ductular reaction, and more advanced disease compared with compensation-prone controls. Mice deficient in p21 were better able than wild-type controls to compensate for long-term bile duct ligation because of significantly greater hepatocyte proliferation, which led to a larger liver mass and less architectural distortion. Mito-inhibitory hepatocyte nuclear p21 expression in humans awaiting liver replacement directly correlated with pathological disease stage and model of end-stage liver disease scoring. In conclusion, stress-induced upregulation of hepatocyte p21 inhibits hepatocyte proliferation during the evolution of cirrhosis. These findings have implications for understanding the evolution of cirrhosis and associated carcinogenesis.

NAD(P)H oxidase plays a crucial role in PDGF-induced proliferation of hepatic stellate cells (p 1272-1281)
Tohru Adachi, Hitoshi Togashi, Akihiko Suzuki, Shigenobu Kasai, Junitsu Ito, Kazuhiko Sugahara, Sumio Kawata
The proliferation of hepatic stellate cells (HSCs) is a critical step in hepatic fibrogenesis. Platelet-derived growth factor (PDGF) is the most potent mitogen for HSCs. We investigated the role of nonphagocytic NAD(P)H oxidase-derived reactive oxygen species (ROS) in PDGF-induced HSC proliferation. The human HSC line, LI-90 cells, murine primary-cultured HSCs, and PDGF-BB were used in this study. We examined the mechanism of PDGF-BB-induced HSC proliferation in relation to the role of a ROS scavenger and diphenylene iodonium, an inhibitor of NAD(P)H oxidase. We also measured ROS production with the aid of chemiluminescence. We showed that PDGF-BB induced proliferation of HSCs through the intracellular production of ROS. We also demonstrated that HSCs expressed key components of nonphagocytic NAD(P)H oxidase (p22phox, gp91phox, p47phox, and p67phox) at both the messenger RNA and protein levels. Diphenylene iodonium suppressed PDGF-BB-induced ROS production and HSC proliferation. Coincubation of H2O2 and PDGF-BB restored the proliferation of HSCs that was inhibited by diphenylene iodonium pretreatment. Phosphorylation of the mitogen-activated protein kinase (MAPK) family constitutes a signal transduction pathway of cell proliferation. Our data demonstrate that NAD(P)H oxidase-derived ROS induce HSC proliferation mainly through the phosphorylation of p38 MAPK. Moreover, an in vivo hepatic fibrosis model also supported the critical role of NAD(P)H oxidase in the activation and proliferation of HSCs. In conclusion, NAD(P)H oxidase is expressed in HSCs and produces ROS via activation of NAD(P)H oxidase in response to PDGF-BB. ROS further induce HSC proliferation through the phosphorylation of p38 MAPK.

Liver Failure and Liver Disease

MELD score and clinical type predict prognosis in hepatorenal syndrome: Relevance to liver transplantation (p 1282-1289)
Carlo Alessandria, Osman Ozdogan, Mónica Guevara, Tea Restuccia, Wladimiro Jiménez, Vicente Arroyo, Juan Rodés, Pere Ginès
Important progress has been made recently regarding the pathogenesis and treatment of hepatorenal syndrome (HRS). However, scant information exists about factors predicting outcome in patients with cirrhosis and HRS. Moreover, the prognostic value of the model of end-stage liver disease (MELD) score has not been validated in the setting of HRS. The current study was designed to assess the prognostic factors and outcome of patients with cirrhosis and HRS. The study included 105 consecutive patients with HRS. Forty-one patients had type 1 HRS, while 64 patients had type 2 HRS. Patients with type 1 HRS not only had more severe liver and renal failure than type 2 patients, they also had greater impairment of circulatory function, as indicated by lower arterial pressure and higher activation of vasoconstrictor factors. In the whole series, the median survival was 3.3 months. In a multivariate analysis of survival, only HRS type and MELD score were associated with an independent prognostic value. All patients with type 1 HRS had a high MELD score (20) and showed an extremely poor outcome (median survival: 1 mo). By contrast, the survival of patients with type 2 HRS was longer and dependent on MELD score (20, median survival 3 mo; <20, median survival 11 mo; P < .002). In conclusion, the outcome of patients with cirrhosis and HRS can be estimated by using two easily available variables, HRS type and MELD score. These data can be useful in the management of patients with HRS, particularly for patients who are candidates for liver transplantation.

Chronic liver injury during obstructive sleep apnea (p 1290-1296)
Florence Tanné, Frédéric Gagnadoux, Olivier Chazouillères, Bernard Fleury, Dominique Wendum, Elisabeth Lasnier, Bernard Lebeau, Raoul Poupon, Lawrence Serfaty
Patients with obstructive sleep apnea (OSA) are at risk for the development of fatty liver as a result of being overweight. Several data suggest that OSA per se could be a risk factor of liver injury; ischemic hepatitis during OSA has been reported, and OSA is an independent risk factor for insulin resistance. Therefore, we investigated liver damage and potential mechanisms in 163 consecutive nondrinking patients with nocturnal polysomnographic recording for clinical suspicion of OSA. Serum levels of liver enzymes were measured in all patients. Liver biopsy was offered to patients with elevated liver enzymes. Intrahepatic hypoxia was assessed by the expression of vascular endothelial growth factor (VEGF) on liver biopsy specimens. Severe OSA (apnea-hypopnea index [AHI] > 50/hr) was seen in 27% of patients; 52% had moderate OSA (AHI 10-50/hr), and 21% had no OSA. Overall, 20% had elevated liver enzymes. Independent parameters associated with elevated liver enzymes were body mass index (BMI) (OR: 1.13; CI: 1.03-1.2) and severe OSA (OR: 5.9; CI: 1.2-29). Liver biopsy was performed in 18 of 32 patients with elevated liver enzymes and showed steatohepatitis in 12 cases, associated with fibrosis in 7 cases. Patients with severe OSA were more insulin-resistant according to homeostasis model assessment, had higher percentage of steatosis as well as scores of necrosis and fibrosis, despite similar BMI. Hepatic immunostaining used as an indirect marker of hypoxia was not different between patients with or without severe OSA. In conclusion, severe OSA is a risk factor for elevated liver enzymes and steatohepatitis independent of body weight. Promotion of insulin resistance is probably involved. Further studies are needed to determine whether hypoxia contributes directly to liver injury.

A novel 3D hepatectomy simulation based on liver circulation: Application to liver resection and transplantation (p 1297-1304)
Shinichi Saito, Junichi Yamanaka, Koui Miura, Norio Nakao, Tomohiro Nagao, Takaaki Sugimoto, Tadamichi Hirano, Nobukazu Kuroda, Yuji Iimuro, Jiro Fujimoto
Fatigue is common in primary biliary cirrhosis (PBC). Altered central serotonergic neurotransmission may be involved in its pathogenesis. This multicenter, randomized, double-blind, placebo-controlled, crossover trial evaluated the efficacy of ondansetron, a selective 5-HT3 receptor subtype antagonist, for treating fatigue in PBC. A crossover design was chosen, allowing subjects to serve as their own controls - appropriate to evaluate fatigue, a subjective symptom. Sixty patients with clinically stable PBC, a Fatigue Severity Score (FSS) > 4, and no other identifiable cause for fatigue were enrolled. Subjects were randomized to receive ondansetron (4 mg) or placebo orally 3 times daily for 4 weeks (period 1). Subjects then crossed over, after a minimum 1-week washout period, for a further 4 weeks of ondansetron or placebo (period 2). Fatigue was measured at the beginning and end of each period by using the FSS and Fatigue Impact Scale (FIS). Six patients withdrew; the remaining 54 subjects had a mean baseline FSS of 5.55 (±0.1). Response to study medication in period 1 versus period 2 was not uniform; thus, it was necessary to analyze the trial periods separately. In period 1, there was no significant additional fatigue reduction on ondansetron over placebo. During period 2, FSS and FIS decreased significantly on ondansetron versus placebo (P = .001). However, period 2 results were invalidated because drug side effects unblinded subjects (constipation affected 63.0% of patients taking ondansetron, versus 13.3% on placebo). In conclusion, ondansetron administration did not confer clinically significant fatigue reduction when compared with placebo in our study population.

A randomized, controlled crossover trial of ondansetron in patients with primary biliary cirrhosis and fatigue (p 1305-1312)
Jeremy J. Theal, Mohssen N. Toosi, Larisa Girlan, Ronald J. Heslegrave, Pierre-Michel Huet, Kelly W. Burak, Mark Swain, George A. Tomlinson, E. Jenny Heathcote
Fatigue is common in primary biliary cirrhosis (PBC). Altered central serotonergic neurotransmission may be involved in its pathogenesis. This multicenter, randomized, double-blind, placebo-controlled, crossover trial evaluated the efficacy of ondansetron, a selective 5-HT3 receptor subtype antagonist, for treating fatigue in PBC. A crossover design was chosen, allowing subjects to serve as their own controls - appropriate to evaluate fatigue, a subjective symptom. Sixty patients with clinically stable PBC, a Fatigue Severity Score (FSS) > 4, and no other identifiable cause for fatigue were enrolled. Subjects were randomized to receive ondansetron (4 mg) or placebo orally 3 times daily for 4 weeks (period 1). Subjects then crossed over, after a minimum 1-week washout period, for a further 4 weeks of ondansetron or placebo (period 2). Fatigue was measured at the beginning and end of each period by using the FSS and Fatigue Impact Scale (FIS). Six patients withdrew; the remaining 54 subjects had a mean baseline FSS of 5.55 (±0.1). Response to study medication in period 1 versus period 2 was not uniform; thus, it was necessary to analyze the trial periods separately. In period 1, there was no significant additional fatigue reduction on ondansetron over placebo. During period 2, FSS and FIS decreased significantly on ondansetron versus placebo (P = .001). However, period 2 results were invalidated because drug side effects unblinded subjects (constipation affected 63.0% of patients taking ondansetron, versus 13.3% on placebo). In conclusion, ondansetron administration did not confer clinically significant fatigue reduction when compared with placebo in our study population.

Design and validation of a histological scoring system for nonalcoholic fatty liver disease (p 1313-1321)
David E. Kleiner, Elizabeth M. Brunt, Mark Van Natta, Cynthia Behling, Melissa J. Contos, Oscar W. Cummings, Linda D. Ferrell, Yao-Chang Liu, Michael S. Torbenson, Aynur Unalp-Arida, Matthew Yeh, Arthur J. McCullough, Arun J. Sanyal, Nonalcoholic Steatohepatitis Clinical Research Network
Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic steatosis in the absence of a history of significant alcohol use or other known liver disease. Nonalcoholic steatohepatitis (NASH) is the progressive form of NAFLD. The Pathology Committee of the NASH Clinical Research Network designed and validated a histological feature scoring system that addresses the full spectrum of lesions of NAFLD and proposed a NAFLD activity score (NAS) for use in clinical trials. The scoring system comprised 14 histological features, 4 of which were evaluated semi-quantitatively: steatosis (0-3), lobular inflammation (0-2), hepatocellular ballooning (0-2), and fibrosis (0-4). Another nine features were recorded as present or absent. An anonymized study set of 50 cases (32 from adult hepatology services, 18 from pediatric hepatology services) was assembled, coded, and circulated. For the validation study, agreement on scoring and a diagnostic categorization (NASH, borderline, or not NASH) were evaluated by using weighted kappa statistics. Inter-rater agreement on adult cases was: 0.84 for fibrosis, 0.79 for steatosis, 0.56 for injury, and 0.45 for lobular inflammation. Agreement on diagnostic category was 0.61. Using multiple logistic regression, five features were independently associated with the diagnosis of NASH in adult biopsies: steatosis (P = .009), hepatocellular ballooning (P = .0001), lobular inflammation (P = .0001), fibrosis (P = .0001), and the absence of lipogranulomas (P = .001). The proposed NAS is the unweighted sum of steatosis, lobular inflammation, and hepatocellular ballooning scores. In conclusion, we present a strong scoring system and NAS for NAFLD and NASH with reasonable inter-rater reproducibility that should be useful for studies of both adults and children with any degree of NAFLD. NAS of 5 correlated with a diagnosis of NASH, and biopsies with scores of less than 3 were diagnosed as not NASH.

Changes in gallbladder bile composition and crystal detection time in morbidly obese subjects after bariatric surgery (p 1322-1328)
Ulf Gustafsson, Lisbet Benthin, Lars Granström, Albert K. Groen, Staffan Sahlin, Curt Einarsson
The aim of the present study was to elucidate the mechanisms of development of cholesterol crystals and gallstones during weight reduction in obese subjects. Twenty-five morbidly obese, gallstone-free subjects underwent vertical-banded gastroplasty. Gallbladder bile was collected at the time of the operation via needle aspiration and 1.1-7.3 months after the operation via ultrasound-guided transhepatic puncture of the gallbladder. The mean weight loss was 17 kg. Two patients developed gallstones and 10 patients displayed cholesterol crystals in their bile. In patients with a follow-up time of less than 2 months (n = 13), cholesterol saturation increased from 90% to 114% but tended to decrease in the patients with a follow-up time of more than 2 months. The extraction of the concanavalin-A-binding fraction from gallbladder bile obtained after weight reduction in 7 patients prolonged crystallization detection time from 6 to 10 days. The hexosamine concentration, a marker for mucin, was increased by about 100% in bile obtained in 6 of 7 patients after weight reduction. In conclusion, the results indicate that crystallization-promoting compounds (mucin) are of great importance in the development of cholesterol crystals and gallstones in obese subjects during weight reduction, probably because of defective gallbladder emptying.

Th1 cytokine-induced downregulation of PPAR in human biliary cells relates to cholangitis in primary biliary cirrhosis (p 1329-1338)
Kenichi Harada, Kumiko Isse, Takashi Kamihira, Shinji Shimoda, Yasuni Nakanuma
Published Online: 2 May 2005
Peroxisome proliferator-activated receptor- (PPAR) is known to inhibit the production of proinflammatory cytokines. In Th1-predominant diseases, PPAR ligands can ameliorate clinical severity by downregulating the expression of proinflammatory cytokines. Primary biliary cirrhosis (PBC) is characterized by chronic destructive cholangitis with a Th1-predominant cytokine milieu. Unusual immune responses to infectious agents are suspected to underlie its etiopathogenesis. We examined the significance of PPAR in biliary inflammation in connection to PBC. To this end, we performed immunohistochemistry, quantitative polymerase chain reaction, and nuclear factor-kappaB (NF-B) DNA-binding assays to clarify the intrahepatic distribution of PPAR and the regulation of PPAR by inflammatory cytokines and PPAR ligand in five cultured biliary cell lines including one derived from PBC liver. In liver specimens from patients with PBC, PPAR protein was ubiquitously expressed in intrahepatic biliary epithelium, whereas the expression of PPAR protein and mRNA was reduced in damaged bile ducts. PPAR expression in cultured cells was upregulated by interleukin-4 (IL-4; Th2-type), but downregulated by IFN- (Th1-type). PPAR ligand negatively modulated lipopolysaccharide-induced NF-B activation. Moreover, this inhibitory effect of PPAR ligand was attenuated by pretreatment with IFN-. In conclusion, PPAR may be important to maintain homeostasis in the intrahepatic biliary epithelium, and its reduction in the bile ducts of PBC liver may be associated with the Th1-predominant milieu and with the development of chronic cholangitis in PBC. Immunosuppression using PPAR ligands may be of therapeutic benefit to attenuate biliary inflammation in PBC.

Genome-wide analysis of gene expression in human intrahepatic cholangiocarcinoma (p 1339-1348)
Kazutaka Obama, Katsuaki Ura, Meihua Li, Toyomasa Katagiri, Tatsuhiko Tsunoda, Akinari Nomura, Seiji Satoh, Yusuke Nakamura, Yoichi Furukawa
Intrahepatic cholangiocarcinoma is a neoplasm arising in the liver, and its incidence is increasing in Japan as well as in Western countries. Prognosis of patients with this type of tumor remains unsatisfactory because no effective chemotherapeutic drugs are available, we have no sensitive tumor markers to detect this tumor in its early stage, and it is difficult to identify a high-risk group for the disease. To clarify the molecular mechanism of tumorigenesis and identify molecular targets for diagnosis and treatment, we analyzed global gene-expression profiles of 25 intrahepatic cholangiocarcinomas using tumor cell populations purified by laser microbeam microdissection and a cDNA microarray containing 27,648 genes. We identified 52 genes that were commonly upregulated and 421 that were downregulated in intrahepatic cholangiocarcinomas compared with noncancerous biliary epithelial cells. From the 52 upregulated genes, we selected P-cadherin and survivin for further investigation and corroborated enhanced expression of their products in cancer tissues by immunohistochemical staining. Furthermore, comparison between tumors with lymph node metastasis and those without metastasis identified 30 genes that were associated with lymph node involvement. In conclusion, these data should be helpful for a better understanding of the tumorigenesis of intrahepatic cholangiocarcinoma and should contribute to the development of diagnostic and therapeutic strategies for this type of tumor.

Viral Hepatitis

Activity of stabilized short interfering RNA in a mouse model of hepatitis B virus replication (p 1349-1356)
David V. Morrissey, Karin Blanchard, Lucinda Shaw, Kristi Jensen, Jennifer A. Lockridge, Brent Dickinson, James A. McSwiggen, Chandra Vargeese, Keith Bowman, Chris S. Shaffer, Barry A. Polisky, Shawn Zinnen
To develop synthetic short interfering RNA (siRNA) molecules as therapeutic agents for systemic administration in vivo, chemical modifications were introduced into siRNAs targeted to conserved sites in hepatitis B virus (HBV) RNA. These modifications conferred significantly prolonged stability in human serum compared with unmodified siRNAs. Cell culture studies revealed a high degree of gene silencing after treatment with the chemically modified siRNAs. To assess activity of the stabilized siRNAs in vivo initially, an HBV vector-based model was used in which the siRNA and the HBV vector were codelivered via high-volume tail vein injection. More than a 3 log10 decrease in levels of serum HBV DNA and hepatitis B surface antigen, as well as liver HBV RNA, were observed in the siRNA-treated groups compared with the control siRNA-treated and saline groups. Furthermore, the observed decrease in serum HBV DNA was 1.5 log10 more with stabilized siRNA compared with unmodified siRNA, indicating the value of chemical modification in therapeutic applications of siRNA. In subsequent experiments, standard systemic intravenous dosing of stabilized siRNA 72 hours after injection of the HBV vector resulted a 0.9 log10 reduction of serum HBV DNA levels after 2 days of dosing. In conclusion, these experiments establish the strong impact that siRNAs can have on the extent of HBV infection and underscore the importance of stabilization of siRNA against nuclease degradation.

Long-term follow-up of peginterferon and lamivudine combination treatment in HBeAg-positive chronic hepatitis B (p 1357-1364)
Henry Lik-Yuen Chan, Alex Yui Hui, Vincent Wai-Sun Wong, Angel Mei-Ling Chim, May-Ling Wong, Joseph Jao-Yiu Sung
We have previously demonstrated that combination peginterferon and lamivudine treatment has superior antiviral efficacy to lamivudine monotherapy in chronic hepatitis B. In this study, we investigated the long-term posttreatment virological response to this combination treatment. Sustained virological response of patients who completed 32-week peginterferon and 52-week lamivudine combination treatment was compared to patients who completed 52-week lamivudine monotherapy. Sustained response was defined as sustained hepatitis B e antigen (HBeAg) loss and HBV DNA < 100,000 copies/mL from treatment cessation until the end of follow-up. Forty-eight patients receiving combination treatment and 47 patients receiving lamivudine monotherapy were studied. The posttreatment follow-up of patients who received combination treatment was 117 ± 34 weeks and that of patients receiving lamivudine monotherapy was 124 ± 29 weeks. At the end of treatment, HBeAg loss occurred in 63% of patients in the combination group and 28% of patients in the lamivudine group (P = .001). The probabilities of sustained response for combination treatment and lamivudine monotherapy were 33% and 13% at week 24, 31% and 11% at week 52, and 29% and 9% at week 76, respectively (log-rank test, P = .0015). No patients developed virological relapse after week 76 until the last visit in either treatment group. All sustained responders had no biochemical relapse (alanine aminotransferase [ALT] > 2 times upper limit of normal) during follow-up. Among the non-sustained responders, biochemical relapse occurred in 32 patients (94%) in the combination group and 38 patients (88%) in the lamivudine group, respectively. In conclusion, combination treatment of peginterferon and lamivudine has a higher sustained virological response than lamivudine monotherapy up to 3 years after treatment.

T-cell response relative to genotype and ethnicity during antiviral therapy for chronic hepatitis C (p 1365-1375)
David E. Kaplan, Kazushi Sugimoto, Fusao Ikeda, Jason Stadanlick, Mary Valiga, Kirti Shetty, K. Rajender Reddy, Kyong-Mi Chang
Viral genotype and host ethnicity are important predictors of viral clearance during antiviral therapy for chronic hepatitis C virus (HCV) infection. Based on the role of T cells in natural HCV clearance, we hypothesized that T cells may contribute to the genotypic and ethnic difference in treatment outcome. To test this hypothesis, T-cell response to HCV antigens (core, nonstructural NS3/4 and NS5) and control phytohemagglutinin (PHA) was monitored prospectively and was correlated with virological outcome in 41 patients chronically infected with HCV (27 genotype 1, 14 genotype 2 or 3; 19 black persons, 22 white persons) undergoing combined interferon alfa and ribavirin therapy. Interestingly, in patients with genotype 2 or 3 infection, enhanced virological response coincided with a greater T-cell response to HCV NS3/4 antigen at baseline (50% vs. 15%; P = .026) that augmented further during therapy (29% vs. 4%; P = .035) compared with genotype 1-infected patients. However, HCV-specific T-cell response remained weak in genotype 1-infected patients regardless of virological outcome or ethnicity. Furthermore, virological outcome was associated with a suppressed baseline proliferative response to phytohemagglutinin (P < .03) that increased during therapy (P < .003) independent of ethnicity or genotype. In conclusion, HCV-specific T-cell response was associated with HCV genotype but not with therapeutic clearance of HCV infection. The association between treatment outcome and phytohemagglutinin response suggests more global and antigen-nonspecific mechanisms for therapeutic HCV clearance.

Comparison and validation of simple noninvasive tests for prediction of fibrosis in chronic hepatitis C (p 1376-1382)
Carolin Lackner, Gerd Struber, Bernadette Liegl, Sebastian Leibl, Petra Ofner, Csilla Bankuti, Bernd Bauer, Rudolf E. Stauber
Liver biopsy is recommended before antiviral treatment, particularly for patients with hepatitis C virus (HCV) genotype 1 infection, but it may cause complications and is limited by sampling error. Several non-invasive tests comprising routine laboratory parameters (simple fibrosis tests) have been proposed to predict fibrosis in chronic HCV. The aim of the current study was to validate and compare the diagnostic accuracies of the simple fibrosis tests, aspartate aminotransferase (AST)/alanine aminotransferase (ALT) ratio (AAR), cirrhosis discriminant score (CDS), age-platelet (AP) index, Pohl score, AST-to-platelet ratio index (APRI), and platelet count per se. Staging was performed in liver biopsy specimens of 194 treatment-naïve patients with chronic HCV according to Ishak et al. by two independent pathologists. Receiver operating characteristic curve analysis showed comparable diagnostic accuracies of CDS, AP index, APRI, and platelet count for prediction of significant fibrosis (F3-F6) (area under the ROC curve [AUROC], 0.71, 0.74, 0.80, and 0.71, respectively; pathologist A) and for prediction of cirrhosis (F5-F6) (AUROC, 0.91, 0.91, 0.90, and 0.89, respectively; pathologist A). Diagnostic accuracy of APRI for prediction of significant fibrosis was superior to that of AAR (P < .05). Significant fibrosis was reliably predicted by APRI 1.5 and platelet count <150 ?109/L in 24% and 22% of the patients, respectively, whereas cirrhosis was reliably excluded by APRI <2.0 and platelet count 150 ?109/L in 85% and 78% of the patients, respectively. In conclusion, simple fibrosis tests may render liver biopsy unnecessary only in a minority of patients with chronic HCV. Improved serum fibrosis markers with greater sensitivity for severe fibrosis or cirrhosis are needed.

Molecular analysis of HLA class II associations with hepatitis B virus clearance and vaccine nonresponsiveness (p 1383-1390)
Andrew Godkin, Miles Davenport, Adrian V.S. Hill
Clearance of acute hepatitis B virus (HBV) infection is associated with a vigorous CD4+ T-cell response focusing on the core protein. HLA class II glycoproteins present viral peptides to CD4+ T cells and influence the immune responses. HLA-DRB1*1301/2 have been associated with viral clearance, and HLA-DRB1*0301 is associated with nonresponse to vaccination with envelope proteins. Binding affinities of overlapping peptides covering the core and envelope proteins of HBV were measured to HLA glycoproteins encoded by HLA-DRA1*0101,-DRB1*0101 (HLA-DR1), HLA-DRA1*0101,-DRB1*0301 (HLA-DR3), HLA-DRA1*0101,-DRB1*0701 (HLA-DR7) and HLA-DRA1*0101,-DRB1*1301 (HLA-DR13) molecules and compared with published peptide-specific CD4+ T-cell responses. There are more high-affinity ligands (IC50 < 1 mol/L) derived from the core protein than the surface antigen (P < .04 for HLA-DR1/7/13), but there was no increase in the number or the affinity of ligands for HLA-DR13. Clusters of particular core peptides bound to multiple HLA types, explaining the immunodominance of these regions for T-cell responses. Within the envelope protein, the low-affinity ligands (IC50 < 10 mol/L) are found mainly in the surface antigen, with a marked paucity of ligands for HLA-DR3 (HLA-DR3 vs. non-DR3; P < .05) consistent with the lower vaccination responses for this HLA type. Of all peptides tested, 8 to 10 bound mainly to one HLA type, allowing a substantially greater breadth of response in heterozygotes. In conclusion, these data offer a mechanistic explanation for the dominant response to the HBV core protein during infection and support the direct involvement of the HLA-DRB1 gene in vaccine nonresponsiveness but not altered susceptibility to viral persistence.

Susceptibility to antivirals of a human HBV strain with mutations conferring resistance to both lamivudine and adefovir (p 1391-1398)
Marie-Noëlle Brunelle, Anne-Carole Jacquard, Christian Pichoud, David Durantel, Sandra Carrouée-Durantel, Jean-Pierre Villeneuve, Christian Trépo, Fabien Zoulim
Mutations within the hepatitis B virus (HBV) polymerase gene conferring drug-resistance are selected during prolonged lamivudine (3TC) or adefovir dipivoxil (ADV) treatment. Because there is no other approved drug against HBV, treatments with 3TC or ADV are used either sequentially or in addition, depending on treatment response or failure. Considering the use of de novo or add-on 3TC+ADV bitherapy, we investigated the possibility of the emergence of an HBV strain harboring polymerase mutations conferring resistance to both 3TC (rtL180M+M204V) and ADV (rtN236T). We constructed the L180M+M204V+N236T mutant and determined its replication capacity and its susceptibility to different nucleos(t)ide analogs in transiently transfected hepatoma cell lines. The triple mutant replicates its genome in vitro, but less efficiently than either the wild-type (wt) HBV or L180M+M204V and N236T mutants. Phenotypic assays indicated that the L180M+M204V+N236T mutant is resistant to pyrimidine analogs (3TC, -FTC, -L-FD4C, L-FMAU). Compared with wt HBV, this mutant displays a 6-fold decreased susceptibility to ADV and entecavir and a 4-fold decreased susceptibility to tenofovir. Interferon alfa inhibited equally the replication of wt and L180M+M204V+N236T HBV. In conclusion, the combination of rtL180M+M204V and rtN236T mutations impairs HBV replication and confers resistance to both 3TC and ADV in vitro. These results suggest that the emergence of the triple mutant may be delayed and associated with viral resistance in patients treated with 3TC+ADV. However, other nucleos(t)ide analogs in development showed an antiviral activity against this multiresistant strain in vitro. This provides a rationale for the clinical evaluation of de novo combination therapies.

Copyright © 2005 by the American Association for the Study of Liver Diseases. All rights reserved.

GASTROENTEROLOGY

Rapid Communication

Differentiation of In Vitro–Modified Human Peripheral Blood Monocytes Into Hepatocyte–like and Pancreatic Islet-like Cells
Maren Ruhnke, Hendrik Ungefroren, Andreas Nussler, Franz Martin, Marc Brulport, Wiebke Schormann, Jan G. Hengstler, Wolfram Klapper, Karin Ulrichs, James A. Hutchinson, Bernat Soria, Reza M. Parwaresch, Peter Heeckt, Bernd Kremer, Fred Fändrich
Background & Aims: Adult stem cells provide a promising alternative for the treatment of diabetes mellitus and end-stage liver diseases. We evaluated the differentiation potential of human peripheral blood monocytes into hepatocyte-like and pancreatic islet-like cells. Methods: Monocytes were treated with macrophage colony-stimulating factor and interleukin 3 for 6 days, followed by incubation with hepatocyte and pancreatic islet-specific differentiation media. Cells were characterized by flow cytometry, gene-expression analysis, metabolic assays, and transplantation for their state of differentiation and tissue-specific functions. Results: In response to macrophage colony-stimulating factor and interleukin 3, monocytes resumed cell division in a CD115-dependent fashion, which was associated with a down-regulation of the PRDM1 and ICSBP genes. These programmable cells of monocytic origin were capable of differentiating into neohepatocytes, which closely resemble primary human hepatocytes with respect to morphology, expression of hepatocyte markers, and specific metabolic functions. After transplantation into the liver of severe combined immunodeficiency disease/nonobese diabetic mice, neohepatocytes integrated well into the liver tissue and showed a morphology and albumin expression similar to that of primary human hepatocytes transplanted under identical conditions. Programmable cells of monocytic origin-derived pancreatic neoislets expressed ? cell-specific transcription factors, secreted insulin and C peptide in a glucose-dependent manner, and normalized blood glucose levels when xenotransplanted into immunocompetent, streptozotocin-treated diabetic mice. Programmable cells of monocytic origin retained monocytic characteristics, notably CD14 expression, a monocyte-specific methylation pattern of the CD115 gene, and expression of the transcription factor PU.1. Conclusions: The ability to reprogram, expand, and differentiate peripheral blood monocytes in large quantities opens the real possibility of the clinical application of programmable cells of monocytic origin in tissue repair and organ regeneration.

Positron Emission Tomography Imaging of Adenoviral-Mediated Transgene Expression in Liver Cancer Patients
Iván Peñuelas, Guillermo Mazzolini, José F. Boán, Bruno Sangro, Josep Martí-Climent, María Ruiz, Juan Ruiz, Nagichettiar Satyamurthy, Cheng Qian, Jorge R. Barrio, Michael E. Phelps, José A. Richter, Sanjiv S. Gambhir, Jesús Prieto
Background & Aims: In gene-therapy protocols, imaging of gene expression is needed to evaluate the transduction efficiency of the vector, its tissue distribution, and the duration of transgene expression and to assess the feasibility of repeated vector administration. Methods: We have used positron emission tomography with a fluorine-18-labeled penciclovir analogue to monitor thymidine kinase gene expression after intratumoral injection of a first-generation recombinant adenovirus in patients with hepatocellular carcinoma. Patients were enrolled in a pilot clinical trial and treated with escalating doses of the vector. Two days after adenovirus inoculation, transgene expression was evaluated during the first hours after administration of the radiotracer both on the treated lesion and on a whole-body basis. Results: Transgene expression in the tumor was dependent on the injected dose of the adenovirus and was detectable in all patients who received ≥1012 viral particles. However, when the study was repeated 9 days after vector injection, no expression could be observed. It is interesting to note that no specific expression of the transgene could be detected in distant organs or in the surrounding cirrhotic tissue in any of the cases studied. Conclusions: Our findings show the real possibility of imaging transgene expression in humans by using viral vectors. We show that hepatocarcinoma is a permissive tumor for adenoviral infection and that the nontumoral cirrhotic liver is spared from transduction when the vector is administered by intratumoral injection. These results show that positron emission tomography imaging may help in the design of gene-therapy strategies and in the clinical assessment of new-generation vectors.
 
Human Colorectal Cancer Cells Induce T-Cell Death Through Release of Proapoptotic Microvesicles: Role in Immune Escape
Veronica Huber, Stefano Fais, Manuela Iero, Luana Lugini, Paola Canese, Paola Squarcina, Annamaria Zaccheddu, Marisa Colone, Giuseppe Arancia, Massimo Gentile, Ettore Seregni, Roberta Valenti, Giuseppina Ballabio, Filiberto Belli, Ermanno Leo, Giorgio Parmiani, Licia Rivoltini
Background & Aims: Normal and neoplastic cells release microvesicles, whose effects on the immune system still need to be elucidated. Because human colorectal cancer cells are hypothesized to escape immune recognition by expressing proapoptotic molecules, we investigated whether microvesicles bearing Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand and inducing apoptosis of activated T cells are secreted by colorectal cancer cells both in vitro and in affected patients. Methods: Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand expression were analyzed in colorectal cancer cells and purified microvesicles by flow cytometry, Western blotting, and immunoelectron microscopy. Microvesicle tumor origin was assessed through simultaneous detection of lysosomal (CD63) and adenocarcinoma (carcinoembryonic antigen) markers. Proapoptotic activity of microvesicles was evaluated by annexin V/propidium iodide staining and caspase activation in T cells, including CD8+ T lymphocytes from colorectal cancer patients. Results: Colorectal cancer cells showed a granular pattern of tumor necrosis factor-related apoptosis-inducing ligand and Fas ligand expression, suggesting a secretory behavior. These proapoptotic molecules were detected on isolated microvesicles, together with class I HLA, CD63, and carcinoembryonic antigen. Microvesicles induced Fas ligand-mediated and tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis of activated CD8+ T cells generated from colorectal cancer patients. Microvesicles with comparable phenotypes and functions were found in plasma from patients with advanced disease, whereas vesicular structures expressing Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand were also detected in colorectal cancer specimens. Conclusions: These data show that colorectal cancer induces T-cell apoptosis through the release of Fas ligand-bearing and tumor necrosis factor-related apoptosis-inducing ligand-bearing microvesicles both in vitro and in vivo. This mechanism of immune escape has potential implications as a prognostic factor and could be targeted for the development of new antitumor therapies in colorectal cancer patients.
 
Clinical-alimentary Tract
 
Infliximab as Rescue Therapy in Severe to Moderately Severe Ulcerative Colitis: A Randomized, Placebo-Controlled Study
Gunnar Järnerot, Erik Hertervig, Ingalill Friis-Liby, Lars Blomquist, Per Karlén, Christer Grännö, Mogens Vilien, Magnus Ström, Åke Danielsson, Hans Verbaan, Per M. Hellström, Anders Magnuson, Bengt Curman
Background & Aims: Despite treatment with corticosteroids, severe to moderately severe attacks of ulcerative colitis have a high colectomy rate. We intended to find a rescue therapy other than cyclosporin A, which imposes a high risk of side effects and cyclosporine-related mortality. Methods: This was a randomized double-blind trial of infliximab or placebo in severe to moderately severe ulcerative colitis not responding to conventional treatment. Patients were randomized to infliximab/placebo either on day 4 after the initiation of corticosteroid treatment if they fulfilled the index criteria for fulminant ulcerative colitis on day 3 or on day 6–8 if they fulfilled index criteria on day 5–7 for a severe or moderately severe acute attack of ulcerative colitis. Results were analyzed according to the intention-to-treat principle. The primary end point was colectomy or death 3 months after randomization. Secondary end points were clinical and endoscopic remission at that time in patients who did not undergo operation. Results: Forty-five patients were included (24 infliximab and 21 placebo). No patient died. Seven patients in the infliximab group and 14 in the placebo group had a colectomy (P = .017; odds ratio, 4.9; 95% confidence interval, 1.4–17) within 3 months after randomization. No serious side effects occurred. Three patients in the placebo group required operation for septic complications. Conclusions: Infliximab 4–5 mg/kg is an effective and safe rescue therapy in patients experiencing an acute severe or moderately severe attack of ulcerative colitis not responding to conventional treatment.

A Randomized, Double-Blind, Controlled Withdrawal Trial in Crohn’s Disease Patients in Long-term Remission on Azathioprine
Marc Lémann, Jean-Yves Mary, Jean-Frédéric Colombel, Bernard Duclos, Jean-Claude Soule, Eric Lerebours, Robert Modigliani, Yoram Bouhnik
Background & Aims: An open study reported that patients with Crohn’s disease in remission who have taken azathioprine for longer than 3.5 years are at low risk of relapse when azathioprine is discontinued. To confirm this observation, we performed a multicenter, double-blind, noninferiority withdrawal study. Methods: Patients who were in clinical remission on azathioprine for ≥42 months were randomized to continue azathioprine or to receive an equivalent placebo for 18 months. The primary end point was clinical relapse at 18 months. Results: Forty patients were randomly assigned to receive azathioprine and 43 to receive placebo. Characteristics of patients at entry were similar in the 2 study groups. At 18 months, 3 patients had a relapse in the azathioprine group, and 9 had a relapse in the placebo group. Kaplan-Meier estimates of the relapse rate at 18 months were 8% ± 4% and 21% ± 6%, respectively. The hypothesis that placebo was inferior to azathioprine was not rejected (P = .195). Among the baseline variables, C-reactive protein level >20 mg/L, time without steroids <50 months, and hemoglobin level <12 g/dL were found to be predictive of relapse in the multivariate analysis. Conclusions: This study shows that azathioprine withdrawal is not equivalent to continued therapy with azathioprine for maintenance of remission in patients with Crohn’s disease who have been in remission on azathioprine for ≥3.5 years. Thus, azathioprine maintenance therapy should be continued beyond 3.5 years.

Brain Response to Visceral Aversive Conditioning: A Functional Magnetic Resonance Imaging Study
Lidia Yágüez, Steven Coen, Lloyd J. Gregory, Edson Amaro, Christian Altman, Michael J. Brammer, Edward T. Bullmore, Steven C.R. Williams, Qasim Aziz
ackground & Aims: Brain-imaging studies to date have confounded visceral pain perception with anticipation. We used functional magnetic resonance imaging of the human brain to study the neuroanatomic network involved in aversive conditioning of visceral pain and, thus, anticipation. Methods: Eight healthy volunteers (5 male) participated in the study. We used a classic conditioning paradigm in which 3 neutral stimuli (differently colored circles) that acted as conditioned stimuli were paired with painful esophageal distention, air puff to the wrist, or nothing, which acted as unconditioned stimuli. Neural activity was measured during learning, anticipation (pairing only 50% of conditioned stimuli with their unconditioned stimuli), and extinction (unpaired conditioned stimuli) phases. For magnetic resonance imaging, axial slices depicting blood oxygen level-dependent contrast were acquired with a 1.5-T system. Results: Neural responses during the learning phase included areas commonly associated with visceral pain (anterior cingulate cortex, insula, and primary and secondary somatosensory cortices) and innocuous somatosensory perception (primary and secondary somatosensory cortices and insula). During the anticipation and extinction phases of aversive stimulation, brain activity resembled that seen during actual painful esophageal stimulation. In contrast, anticipation and extinction of the innocuous somatic stimulus failed to show that effect. Conclusions: We have shown that actual and anticipated visceral pain elicit similar cortical responses. These results have implications for the design and interpretation of brain-imaging studies of visceral pain. They not only contribute to our understanding of the processing of visceral pain, but also have clinical implications for the management of chronic pain states.

Vitamin B6 Intake, Alcohol Consumption, and Colorectal Cancer: A Longitudinal Population-Based Cohort of Women
Susanna C. Larsson, Edward Giovannucci, Alicja Wolk
Background & Aims: Vitamin B6 has a crucial role in 1-carbon metabolism, which involves DNA synthesis and DNA methylation. Aberrations in these processes have been implicated in colorectal carcinogenesis. We examined the association between long-term dietary vitamin B6 intake and risk of colorectal cancer and whether this association is modified by consumption of alcohol, which may disrupt 1-carbon metabolism. Methods: Our study population comprised 61,433 women in the population-based Swedish Mammography Cohort. The women were aged 40 to 76 years, had no history of cancer, and completed a food-frequency questionnaire at baseline in 1987–1990. Dietary information was updated in 1997. During a mean follow-up of 14.8 years, 805 incident colorectal cancer cases were diagnosed. Results: After controlling for age and other potential confounders, long-term intake of dietary vitamin B6 was significantly inversely associated with risk of colorectal cancer (P value for trend = .002). Compared with women in the lowest quintile of vitamin B6 intake, those in the highest quintile had a 34% lower risk (multivariate rate ratio, 0.66; 95% confidence interval, 0.50–0.86). The association was most pronounced among women with moderate to high alcohol consumption. The multivariate rate ratio of colorectal cancer comparing extreme quintiles of vitamin B6 intake was 0.28 (95% confidence interval, 0.13–0.59) among women who consumed ≥30 g/wk of alcohol (approximately equivalent to 2 drinks per week). Conclusions: Findings of this study suggest that vitamin B6 may play a role in the prevention of colorectal cancer, particularly among women who drink alcohol.

Helicobacter pylori “Test and Treat” or Endoscopy for Managing Dyspepsia: An Individual Patient Data Meta-analysis
Alexander C. Ford, Michelle Qume, Paul Moayyedi, Nicolaas L.A. Arents, Annmarie T. Lassen, Richard F.A. Logan, Kenneth E.L. McColl, Paul Myres, Brendan C. Delaney
Background & Aims: Helicobacter pylori “test and treat” has been recommended for the management of young dyspeptic patients without alarm symptoms, and trials have suggested that it is as effective as endoscopy. However, none of these trials have had sufficient sample size to confirm that “test and treat” costs less or to detect small differences in effect. A collaborative group has prospectively registered trials comparing prompt endoscopy with a “test and treat” approach, with the aim of performing an individual patient data meta-analysis of both effect and resource utilization data. Methods: Researchers provided data for meta-analysis, pooling effects of interventions on individual dyspepsia symptoms. Standardized unit costs were applied to resource utilization, and net benefit was calculated at patient level. Effects, costs, and net benefit were then pooled at study level. Results: Five trials were identified, containing 1924 patients (946 endoscopy [mean age, 40 years], 978 “test and treat” [mean age, 41 years]). The relative risk (RR) of remaining symptomatic after 1 year was reduced with endoscopy compared with “test and treat” (RR = 0.95; 95% confidence interval [CI]: 0.92–0.99). “Test and treat” cost $389 less per patient (95% CI: $275–$502). Using the net benefit approach, at no realistic level of willingness to pay per patient symptom-free did prompt endoscopy become cost-effective. Conclusions: Prompt endoscopy confers a small benefit in terms of cure of dyspepsia but costs more than “test and treat” and is not a cost-effective strategy for the initial management of dyspepsia.

Long-term Outcome of Helicobacter pylori–Negative Idiopathic Bleeding Ulcers: A Prospective Cohort Study
Lawrence C.T. Hung, Jessica Y.L. Ching, Joseph J.Y. Sung, Ka-Fai To, Aric J. Hui, Vincent W.S. Wong, Rupert W.L. Leong, Henry L.Y. Chan, Justin C.Y. Wu, Wai-Keung Leung, Yuk-Tong Lee, S.C. Sydney Chung, Francis K.L. Chan
Background & Aims: Helicobacter pylori-negative idiopathic ulcers are increasingly recognized. The secular trend and long-term outcome of this condition are unknown. Methods: We prospectively studied consecutive patients with bleeding gastroduodenal ulcers from January to December 2000. The incidence and etiology of ulcers during this period were compared with that between September 1997 and August 1998. H pylori–negative idiopathic ulcers were defined as negative tests for H pylori, no exposure to analgesics within 4 weeks, and absence of other risk factors for ulcers. After the ulcers had healed, patients with H pylori–negative idiopathic ulcers and patients with H pylori ulcers who received eradication therapy were followed up for 12 months without anti-ulcer drugs. Results: Six hundred thirty-eight patients had bleeding ulcers: 213 (33.4%) were H pylori ulcers, and 120 (18.8%) were H pylori–negative idiopathic ulcers (vs 480 [50.3%] H pylori ulcers and 40 [4.2%] H pylori–negative idiopathic ulcers in 1997–1998; P < .001). H pylori–negative idiopathic ulcers accounted for 16.1% of patients who were admitted for bleeding and 42.4% of patients who bled while in the hospital (P < .0001); 28.3% of patients with H pylori–negative idiopathic ulcers had histologic evidence of past H pylori infection. The probability of recurrent ulcer complications in 12 months was 13.4% (95% CI: 7.3%–19.5%) in patients with H pylori–negative idiopathic ulcers and 2.5% (95% CI: 0.4%–4.6%) in patients with H pylori ulcers who received eradication therapy (P = .0002). Conclusions: The incidence of H pylori–negative idiopathic bleeding ulcers is rising. These ulcers are prone to recurrent complications.

Increase of Bone Marrow–Derived Secretory Lineage Epithelial Cells During Regeneration in the Human Intestine
Tomoko Matsumoto, Ryuichi Okamoto, Tomoharu Yajima, Takehiko Mori, Shinichiro Okamoto, Yasuo Ikeda, Makio Mukai, Motomi Yamazaki, Shigeru Oshima, Kiichiro Tsuchiya, Tetsuya Nakamura, Takanori Kanai, Hideyuki Okano, Johji Inazawa, Toshifumi Hibi, Mamoru Watanabe
Background & Aims: We have previously reported that bone marrow (BM)-derived cells contribute to the regeneration of the human intestinal epithelium. To analyze further how these cells arise, proliferate, and differentiate as epithelial cells, histologic analysis was conducted using endoscopic specimens. Methods: Thirty biopsy specimens from 14 female, sex-mismatched BM-transplantation recipients were examined. BM-derived cells were identified by fluorescent in situ hybridization (FISH) for the Y chromosome and immunohistochemistry. Multicolor FISH was used to exclude cell fusion. These cells were further analyzed for various differentiation or proliferation markers. Results: No evidence of cell fusion was detected. BM-derived cells did not distribute within the crypt as stem cells and rarely expressed Musashi-1. However, BM-derived epithelial cells frequently expressed Ki-67, and some of these cells appeared as pairs of adjacent cells. These cells also expressed markers of all 4 lineages of terminally differentiated cells. During regeneration following graft-vs-host disease, the number of BM-derived cells was substantially increased within Ki-67–positive cells. Interestingly, the number of cells expressing markers for secretory lineage cells was significantly increased within BM-derived cells. This change was unique for BM-derived cells, resulting in a significantly increased proportion of BM-derived cells among secretory lineage cells. Conclusions: BM-derived epithelial cells arise via a mechanism other than cell fusion and rarely give rise to stem cells. However, a small proportion of these cells express proliferation markers, and a majority reside as terminally differentiated cells. During regeneration BM-derived cells increase as secretory lineage cells, thereby contributing to restore epithelial functions.

Peripheral and Intestinal Regulatory CD4+CD25high T Cells in Inflammatory Bowel Disease
Jochen Maul, Christoph Loddenkemper, Pamela Mundt, Erika Berg, Thomas Giese, Andreas Stallmach, Martin Zeitz, Rainer Duchmann
Background & Aims: Regulatory CD25+ T cells (Treg) are effective in the prevention and down-regulation of inflammatory bowel disease (IBD) in animal models. Functional Treg cells are characterized by the expression of the transcription factor FOXP3 and show a CD4+CD25high phenotype in humans. The aim of this study was to determine whether disease activity in IBD correlates with changes in frequency of Treg cells and their distribution in the intestinal mucosa. Methods: Treg cells were analyzed from peripheral blood and from biopsy specimens of IBD patients, inflammatory controls, and healthy volunteers by flow cytometry (CD4+CD25high), immunochemistry (FOXP3), and real-time PCR (FOXP3). Regulatory properties of purified peripheral CD4+CD25high Treg cells were determined by their suppressive effect on the proliferation of CD4+CD25? T cells. Results: In peripheral blood, CD4+CD25high T cells from IBD patients retain their suppressive activity. CD4+CD25high and FOXP3+ Treg cells are increased during remission but decreased during active disease. This contrasts with their strong increase in peripheral blood of patients with acute diverticulitis. Different than peripheral blood, inflamed IBD mucosa contains an increased number of CD4+CD25high T cells, FOXP3+ T cells, and transcripts for FOXP3 compared with noninflamed mucosa. However, the increase of FOXP3+ T cells in IBD lesions is significantly lower compared with inflammatory controls. Conclusions: The frequency of CD4+CD25+ Treg cells varies with IBD activity. Active IBD is not associated with a functional defect but with a contraction of the peripheral blood Treg pool and an only moderate expansion in intestinal lesions. Thus, compensatory mechanisms, numerically, are not successfully achieved in these diseases.

Human Intestinal IgA Response Is Generated in the Organized Gut-Associated Lymphoid Tissue but Not in the Lamina Propria
Laurent Boursier, John N. Gordon, Sivashankari Thiagamoorthy, Jonathan D. Edgeworth, Jo Spencer
Background & Aims: The intestinal lamina propria has traditionally been viewed as the effector site of mucosal immune responses. However, this view has been challenged with the identification, in the murine lamina propria, of an in situ class switch DNA recombination pathway to IgA. In this study, we tested the hypothesis that in situ class switching occurs in the human lamina propria. Methods: Immunohistochemistry was used to analyze tissue microenvironments and RT-PCR to look for molecular evidence of Ig class switching and to track clonally related cells of B lineage. Results: We found no evidence of proliferation of either lamina propria CD20+ or CD19+ cells or evidence of activation-induced cytidine deaminase mRNA expression outside the organized gut-associated lymphoid tissue, although I?-C? immunoglobulin germ-line gene transcript expression could be identified in the lamina propria. We identified clonally related cells, including IgA and IgM isotype-switched variants, in multiple samples known to be free of activation-induced cytidine deaminase, organized lymphoid tissue, or cellular proliferation. For 4 groups of cells, the patterns of somatic mutations on the rearranged IgVH5 gene segment were more similar between cells from distant sites than from their immediate neighbors, implying dissemination of cells from a common set of precursors. Conclusions: Our data are inconsistent with a model in which precursors of human IgA-secreting plasma cells are induced or expanded in the lamina propria. The human lamina propria is therefore likely to solely be an effector site of intestinal secretory IgA responses that originate from the organized gut-associated lymphoid tissues.

Clinical-liver, Pancreas, and Biliary Tract
 
Intrahepatic Hepatitis B Virus Covalently Closed Circular DNA Can Be a Predictor of Sustained Response to Therapy
Joseph J.Y. Sung, May-Ling Wong, Scott Bowden, Choong-Tsek Liew, Alex Y. Hui, Vincent W.S. Wong, Nancy W.Y. Leung, Stephen Locarnini, Henry L.Y. Chan
Background & Aims: This study aimed to determine whether intrahepatic hepatitis B virus (HBV) covalently closed circular (ccc) DNA and total HBV DNA levels at the end of therapy would predict sustained response to therapy. Methods: Hepatitis B e antigen (HBeAg)-positive chronic hepatitis B patients receiving either lamivudine monotherapy or combination of peginterferon and lamivudine had liver biopsy at the end of 1 year therapy and were followed for 52 more weeks after cessation of therapy. Serum HBV DNA, intrahepatic HBV ccc DNA, and total HBV DNA levels were determined. Results: Forty-seven patients, including 34 males and 13 females, were studied. Twenty-seven patients received combination therapy, and 20 patients received lamivudine monotherapy. Twenty-nine patients had end-of-treatment virologic response, and 15 patients had sustained response 52 weeks after therapy. At the end of treatment, log serum HBV DNA levels correlated well with log intrahepatic HBV cccDNA and log intrahepatic total HBV DNA levels. Log intrahepatic cccDNA and log intrahepatic total DNA levels were significantly lower among patients with sustained virologic response. The adjusted odds ratio for log cccDNA was 5.3 (95% CI: 1.5–18.2, P = .009) and, for log intrahepatic HBV DNA, was 4.4 (95% CI: 1.3–14.7, P = .015) to predict sustained virologic response. Using log cccDNA at ?0.80 copies/genome equivalent as cutoff, the sensitivity, specificity, and positive and negative predictive values and accuracy of predicting sustained virologic response were 73%, 78%, 56%, 86%, and 77% respectively. Conclusions: Intrahepatic HBV cccDNA and intrahepatic total HBV DNA levels at the end of therapy are superior to serum HBV DNA as surrogates of sustained virologic response.

Sampling Variability of Liver Biopsy in Nonalcoholic Fatty Liver Disease
Vlad Ratziu, Frédéric Charlotte, Agnès Heurtier, Sophie Gombert, Philippe Giral, Eric Bruckert, André Grimaldi, Frédérique Capron, Thierry Poynard
Background & Aims: In nonalcoholic fatty liver disease (NAFLD), the distinction between steatosis and steatohepatitis (NASH) and the assessment of the severity of the disease rely on liver histology alone. The aim of this study was to assess the sampling error of liver biopsy and its impact on the diagnosis and staging of NASH. Methods: Fifty-one patients with NAFLD underwent percutaneous liver biopsy with 2 samples collected. The agreement between paired biopsy specimens was assessed by the percentage of discordant results and by the ? reliability test. Results: No features displayed high agreement; substantial agreement was only seen for steatosis grade; moderate agreement for hepatocyte ballooning and perisinusoidal fibrosis; fair agreement for Mallory bodies; acidophilic bodies and lobular inflammation displayed only slight agreement. Overall, the discordance rate for the presence of hepatocyte ballooning was 18%, and ballooning would have been missed in 24% of patients had only 1 biopsy been performed. The negative predictive value of a single biopsy for the diagnosis of NASH was at best 0.74. Discordance of 1 stage or more was 41%. Six of 17 patients with bridging fibrosis (35%) on 1 sample had only mild or no fibrosis on the other and therefore could have been under staged with only 1 biopsy. Intraobserver variability was systematically lower than sampling variability and therefore could not account for most of the sampling error. Conclusions: Histologic lesions of NASH are unevenly distributed throughout the liver parenchyma; therefore, sampling error of liver biopsy can result in substantial misdiagnosis and staging inaccuracies.

Basic-alimentary Tract
 
Oncogenic K-ras Stimulates Wnt Signaling in Colon Cancer Through Inhibition of GSK-3?
Jingnan Li, Yusuke Mizukami, Xiaobo Zhang, Won-Seok Jo, Daniel C. Chung
Background & Aims: Two key genetic events underlying the development of colon cancer are activation of the K-ras and Wnt signaling pathways. We have previously shown that these 2 pathways can cooperate to regulate vascular endothelial growth factor (VEGF) gene expression. The goal of this study was to define the molecular basis for this interaction. Methods: The effects of K-rasVal12 on VEGF and T-cell factor 4 (TCF-4) promoter activity, nuclear levels of ?-catenin and ?-catenin/TCF-4 complexes, glycogen synthase kinase 3? (GSK-3?) phosphorylation, and GSK-3? kinase activity were measured. LY294002 and PD98059 were used to define the role of specific ras effector pathways. Results: Oncogenic K-ras up-regulated the activity of the VEGF promoter, and selective mutagenesis of TCF-4 binding sites significantly blocked this induction. K-rasVal12 also induced the activity of a heterologous TCF-4 reporter construct in Caco-2 and HeLa cells. LY294002 and dominant negative phosphatidylinositol 3-kinase nearly completely blocked this induction. K-rasVal12 increased the stability of ?-catenin, the levels of nuclear ?-catenin, and the formation of nuclear ?-catenin/TCF-4 complexes, and these effects were also blocked by LY294002. Finally, K-rasVal12 inhibited the kinase activity of total cellular GSK-3? and GSK-3? complexed with Axin. This effect was not mediated through phosphorylation at serine 9 but did depend on phosphatidylinositol 3-kinase. Conclusions: Our results suggest a unique cooperative interaction between 2 critical oncogenic pathways in colorectal tumorigenesis and highlight the pivotal role of GSK-3?.

Poly(ADP-Ribose) Polymerase-1 Is a Component of the Oncogenic T-Cell Factor-4/?-Catenin Complex
Masashi Idogawa, Tesshi Yamada, Kazufumi Honda, Satoshi Sato, Kohzoh Imai, Setsuo Hirohashi
Background & Aims: T-cell factor (TCF)-4 regulates a certain set of genes related to growth and differentiation of intestinal epithelial cells. Aberrant transactivation of these TCF-4-regulated genes by ?-catenin protein plays a crucial role in early intestinal carcinogenesis, and the transcriptional machinery of the TCF-4/?-catenin complex is likely to contain targets for molecular therapy. We explored the molecular composition of the TCF-4/?-catenin transcriptional complex by means of proteomics. Methods & Results: A protein of approximately 112 kilodaltons was consistently coimmunoprecipitated with FLAG-tagged TCF-4 transiently expressed in HEK293 cells, and the protein was identified by mass spectrometry as poly(ADP-ribose) polymerase-1 (PARP-1). PARP-1 physically interacted with TCF-4 and augmented the transcriptional activity of the ?-catenin/TCF-4 complex. Knockdown of PARP-1 by RNA interference significantly suppressed both transcriptional activity and proliferation by colorectal cancer cells. Auto-polyADP-ribosylation of the PARP-1 protein induced by DNA damage inhibited the functional interaction of PARP-1 with TCF-4. PARP-1 was overexpressed in the intestinal adenomas of patients with familial adenomatous polyposis and multiple intestinal polyposis mice. The expression of PARP-1 was closely associated with the accumulation of ?-catenin and with the undifferentiated status of intestinal epithelial cells. Conclusions: In this study, we identified PARP-1 as a novel coactivator of the ?-catenin/TCF-4 complex. Although PARP-1 has been believed to play a protective role against carcinogenesis, these expression patterns and functional properties of PARP-1 were highly suggestive of its participation in early colorectal carcinogenesis.

Helicobacter felis Eradication Restores Normal Architecture and Inhibits Gastric Cancer Progression in C57BL/6 Mice
Xun Cai, Jane Carlson, Calin Stoicov, Hanchen Li, Timothy C. Wang, JeanMarie Houghton
Background & Aims: The impact of Helicobacter eradication therapy on the progression or regression of gastric lesions is poorly defined. This study examined the effects of eradication therapy on inflammation, atrophy, metaplasia, dysplasia, and cancer progression. Methods: C57BL/6 mice were infected with Helicobacter felis and received bacterial eradication therapy after 2, 6, or 12 months of infection. The gastric mucosa was examined at early, mid, and late intervals after eradication and graded for histology, expression pattern of ?-catenin and ?-catenin, and IQGAP1. Results: Eradication of Helicobacter infection after 2 or 6 months of infection led to a regression of inflammation, restoration of parietal cell mass, and reestablishment of normal architecture. Progression to adenocarcinoma was prevented. Bacterial eradication at 1 year was associated with the reappearance of parietal cells, partial regression of inflammation, and restoration of architecture. Hyperplasia scores significantly improved, and dysplasia did not progress. Infected mice developed antral adenocarcinoma and gastric outlet obstruction by 24 months. Only 30% of the mice receiving bacterial eradication therapy at 12 months developed antral carcinoma. Bacterial eradication at any time during the first year of infection prevented death due to gastric outlet obstruction. The expression pattern of ?-catenin, ?-catenin, and IQGAP1 varied with cell type and paralleled histologic changes. Conclusions: Inflammation, metaplasia, and dysplasia are reversible with early eradication therapy; progression of dysplasia was arrested with eradication therapy given as late as 1 year and prevented gastric cancer-related deaths.

A Model of Neural Cross-Talk and Irritation in the Pelvis: Implications for the Overlap of Chronic Pelvic Pain Disorders
Michael A. Pezzone, Ruomei Liang, Matthew O. Fraser
Background & Aims: Irritable bowel syndrome, interstitial cystitis, and other chronic pelvic pain (CPP) disorders often occur concomitantly. Neural cross-talk may play a role in the overlap of CPP disorders via the convergence of pelvic afferents. We investigated the hypothesis that afferent irritation of one pelvic organ may adversely influence and sensitize another via neural interactions. Methods: We measured pelvic organ smooth muscle and striated muscle reflexes during micturition and colorectal distention (CRD) in urethane-anesthetized rats. The effects of acute cystitis on distal colonic sensory thresholds to CRD and the effects of acute colonic irritation on micturition parameters were assessed. Results: External urethral sphincter (EUS) electromyography (EMG) was typical for the rat, with phasic firing during micturition. External anal sphincter EMG also showed phasic firing during micturition in synchrony with EUS activity but, in addition, showed both tonic bursts and phasic firing independent of EUS activity. Before bladder irritation, graded CRDs to 40 cm H2O produced no notable changes in abdominal wall EMG activity. Following acute bladder irritation, dramatic increases in abdominal wall EMG activity in response to CRD were observed at much lower distention pressures, indicating colonic afferent sensitization. Analogously, following acute colonic irritation, bladder contraction frequency increased 66%, suggesting sensitization of lower urinary tract afferents. Conclusions: We report compelling evidence of bidirectional cross-sensitization of the colon and lower urinary tract in a novel experimental model. This cross-sensitization may account for the substantial overlap of CPP disorders; however, further studies are needed to fully characterize these pathways.
 
Synergistic Inhibitory Effects of Gastrin and Histamine Receptor Antagonists on Helicobacter-Induced Gastric Cancer
Shigeo Takaishi, Guanglin Cui, Dana M. Frederick, Jane E. Carlson, JeanMarie Houghton, Andrea Varro, Graham J. Dockray, Zhongming Ge, Mark T. Whary, Arlin B. Rogers, James G. Fox, Timothy C. Wang
Background & Aims: Apart from its importance as an acid secretogogue, the role of histamine as a downstream target of gastrin has not been fully explored. Previous studies have shown that the combination of hypergastrinemia and Helicobacter infection resulted in accelerated gastric cancer in mice. We used this model to examine the role of cholecystokinin 2 (CCK2)/gastrin receptor and histamine H2-receptor signaling in the development of gastric atrophy and cancer. Methods: Male hypergastrinemic mice (INS-GAS mice) were infected with Helicobacter felis and given the CCK2/gastrin receptor antagonist YF476 and/or the histamine H2-receptor antagonist loxtidine for 3 or 6 months. In addition, mice were treated with omeprazole alone or in combination with either YF476 or loxtidine for 3 months. Results: Mice treated with YF476 or loxtidine alone showed partial suppression of both gastric acid secretion and progression to neoplasia. The combination of YF476 plus loxtidine treatment resulted in nearly complete inhibition of both parameters. YF476 and/or loxtidine treatment did not alter the overall level of H felis colonization but did result in significant down-regulation of the growth factors regenerating gene I and amphiregulin. Loxtidine treatment, with or without YF476, induced a mild shift in T-helper cell polarization. In contrast, omeprazole treatment resulted in mild progression of gastric hyperplasia/dysplasia, which was ameliorated by the addition of YF476 or loxtidine. Conclusions: The combination of CCK2/gastrin- and histamine H2-receptor antagonists has synergistic inhibitory effects on development of gastric atrophy and cancer in H felis/INS-GAS mice, while the proton pump inhibitor showed no such effects. These results support an important role for the gastrin-histamine axis in Helicobacter-induced gastric carcinogenesis.

A Regenerative Role for Bone Marrow Following Experimental Colitis: Contribution to Neovasculogenesis and Myofibroblasts
Mairi Brittan, Victoria Chance, George Elia, Richard Poulsom, Malcolm R. Alison, Thomas T. MacDonald, Nicholas A. Wright
Background & Aims: Bone marrow (BM) cells form differentiated adult lineages within nonhematopoietic tissues, with a heightened propensity with increasing regenerative pressure dictated by disease. We have previously shown that BM cells engraft into the gut and contribute substantially to the subepithelial intestinal myofibroblast population in the lamina propria. To investigate the reparative capacity of BM in inflammatory bowel disease (IBD), a well-established model of experimental colitis was used. Methods: Lethally irradiated female mice were rescued by a BM transplant from male donors. Colitis was induced 6 weeks posttransplantation by injection of trinitrobenzene sulfonic acid (TNBS), and tissues were analyzed 1–14 days later. Donor-derived cells were detected by in situ hybridization using a Y chromosome-specific probe, and their phenotype was determined by immunohistochemistry. Results: TNBS-induced colitis was manifest as patchy lesions that increased in severity between days 1 and 8, and the mucosa gradually regenerated between days 8 and 14. The contribution of BM to intestinal myofibroblasts was significantly increased in regions of colitis compared with noninflamed regions. Furthermore, BM-derived endothelial cells, pericytes, and vascular smooth muscle cells were frequently interspersed throughout blood vessels, suggesting that these cells facilitate angiogenesis in tissue repair, substantiated by a significant increase in the incidence of BM-derived vascular smooth muscle cells in colitic compared with noninflamed regions. Blood vessels formed entirely from BM-derived cells were also seen, suggesting a role for BM in neovasculogenesis. Conclusions: Our data show that BM contributes to multiple intestinal cell lineages in colitis, with an important function in tissue regeneration and vasculogenesis after injury.

Rectal Instillation of Butyrate Provides a Novel Clinically Relevant Model of Noninflammatory Colonic Hypersensitivity in Rats
Sophie Bourdu, Michel Dapoigny, Eric Chapuy, Fabrice Artigue, Marie-Paule Vasson, Pierre Dechelotte, Gilles Bommelaer, Alain Eschalier, Denis Ardid
Background & Aims: The treatment of irritable bowel syndrome (IBS), characterized by abdominal pain and bloating, is empirical and often poorly efficient. Research lacks suitable models for studying the pathophysiologic mechanisms of the colonic hypersensitivity and new pharmacologic targets. The present study aimed to develop a novel model of colonic hypersensitivity possessing several of the characteristics encountered in patients with IBS. Methods: Rats received enemas of a butyrate solution (8–1000 mmol/L) twice daily for 3 days. A time course was determined for colonic hypersensitivity (colorectal distention test) and referred cutaneous lumbar hyperalgesia (von Frey hairs). Macroscopic and histologic analyses were performed on colonic mucosa. The efficacy of morphine, U50488H (a ? opioid agonist), and trimebutine on the 2 pain parameters was determined. Finally, the involvement of peptidergic C-fibers was evaluated using capsaicin-pretreated animals and treatments with calcitonin gene-related peptide (CGRP) and neurokinin 1 receptor antagonists. Results: Butyrate enemas induced a sustained, concentration-dependent colonic hypersensitivity and, to a lesser extent, a referred cutaneous mechanical hyperalgesia, particularly in female rats, but no macroscopic and histologic modifications of the colonic mucosa, as observed in patients with IBS. Both pain parameters were sensitive to morphine, U50488H, trimebutine, neonatal capsaicin treatment, and the CGRP receptor antagonist but not to the neurokinin 1 receptor antagonist. Conclusions: These results present our noninflammatory model of chronic colonic hypersensitivity as a useful novel tool for studying IBS. The CGRP receptor antagonist-induced reduction of colonic hypersensitivity suggests that CGRP receptors may provide a promising target for treatment of IBS.

N-Methyl-d-Aspartate Receptors Mediate Endogenous Opioid Release in Enteric Neurons After Abdominal Surgery
Simona Patierno, Wubanche Zellalem, Anthony Ho, Chris G. Parsons, K.C. Kent Lloyd, Marcello Tonini, Catia Sternini
Background & Aims: We tested the hypothesis that N-methyl-D-aspartate (NMDA) receptors mediate surgery-induced opioid release in enteric neurons. Methods: We used ? opioid receptor (?OR) internalization as a measure of opioid release with immunohistochemistry and confocal microscopy. ?OR internalization was quantified in enteric neurons from nondenervated and denervated ileal segments of guinea pig after abdominal laparotomy with and without pretreatment with NMDA-receptor antagonists acting at different recognition sites (+)-5-methyl-10, 11-dihydro-5H-dibenzo [a, b] cyclohepten-5, 10-imine (MK-801) or (D) 2-amino-5-phosphopenoic acid (AP-5) at .5, 1 mg/kg; 8-chloro-4-hydroxy-1-oxo-1, 2-dihydropyridazinol [4,5-]quinoline-5-oxide choline (MRZ 2/576) or 8-chloro-1, 4-dioxo-1,2,3,4-tetrahydropyridazinol [4,5-]quinoline choline salt (MRZ 2/596) at .3, 1 mg/kg, or with an antagonist for the ?-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, 6-cyano-7-nitroquinoxaline-2, 3-dione (1, 3 mg/kg). To determine whether NMDA stimulation induces opioid release, (1) ilea were exposed to NMDA (100 ?mol/L) and D-serine (10 ?mol/L) with or without the antagonist MK-801 or AP-5 (50 ?mol/L); and (2) neuromuscular preparations of the ileum were stimulated electrically (20 Hz, 20 min) with or without MK-801 or AP-5 (50 ?mol/L). Results: ?OR endocytosis induced by abdominal laparotomy was inhibited significantly by NMDA-receptor antagonists in nondenervated and denervated ileal segments, but not by the AMPA-receptor antagonist. ?OR endocytosis in neurons exposed to NMDA or electrical stimulation was prevented by NMDA-R antagonists. Conclusions: Abdominal laparotomy evokes local release of glutamate that results in endogenous opioid release through the activation of peripheral NMDA receptors. This suggests an interaction between the glutamatergic and opioid systems in response to the noxious and perhaps mechanosensory stimulation of surgery.

Antibodies to CBir1 Flagellin Define a Unique Response That Is Associated Independently With Complicated Crohn’s Disease
Stephan R. Targan, Carol J. Landers, Huiying Yang, Michael J. Lodes, Yingzi Cong, Konstantinos A. Papadakis, Eric Vasiliauskas, Charles O. Elson, Robert M. Hershberg
Background & Aims: Antibody responses to certain microbial antigens define heterogeneous groups of Crohn’s patients; multiple and high-level responses to these antigens are associated with aggressive clinical phenotypes. The flagellin, CBir1, identified by investigations in the C3H/HeJBir mouse model, has been identified as a dominant antigen capable of inducing colitis in mice and eliciting antibody responses in a subpopulation of patients with Crohn’s disease (CD). The aim of this study was to evaluate serum response to CBir1 flagellin in CD patients and to compare this response to responses defined previously to oligomannan (anti-Saccharomyces cerevisiae antibody), I2, OmpC, and neutrophil nuclear autoantigens (pANCA), and to determine anti-CBir1-associated phenotypes. Methods: A total of 484 sera from the Cedars Sinai Medical Center repository, previously typed for anti-Saccharomyces cerevisiae antibody, anti-I2, anti-OmpC, and pANCA were tested for anti-CBir1 by enzyme-linked immunosorbent assay, and results were assessed for clinical phenotype associations. Results: The presence and level of immunoglobulin G anti-CBir1 were associated with CD independently. Anti-CBir1 was present in all antibody subgroups and expression increased in parallel with increases in the number of antibody responses. pANCA+ CD patients were more reactive to CBir1 than were pANCA+ ulcerative colitis patients. Anti-CBir1 expression is associated independently with small-bowel, internal-penetrating, and fibrostenosing disease features. Conclusions: Serum responses to CBir1 independently identify a unique subset of patients with complicated CD. This bacterial antigen was identified in a murine model and has a similar pattern of aberrant reactivity in a subset of CD patients.

Basic-liver, Pancreas, and Biliary Tract

Use of Adenovirus-Delivered siRNA to Target Oncoprotein p28GANK in Hepatocellular Carcinoma
Honghai Li, Xiaoyong Fu, Yao Chen, Yi Hong, Yexiong Tan, Huifang Cao, Mengchao Wu, Hongyang Wang
Background & Aims: RNA interference (RNAi) is a powerful tool to silence gene expression. The adenoviral vector expressing small interfering RNA (siRNA) is highly effective in mammalian cells. However, its potential use as a therapeutic tool to target an oncogene specifically remains to be seen. We applied the adenovirus-delivered siRNA (AdSiRNA) to inhibit a hepatocellular carcinoma (HCC) oncogene, p28GANK, in HCC cell lines and investigated its antitumor effects. Methods: The T7-RNA polymerase system was used to screen the specific target site. Double-strand oligonucleotide for transcription of short hairpin RNA was constructed into the adenoviral vector. Four HCC cell lines were infected with the RNAi-containing adenovirus. The RNAi effects on HCC were studied in cultured cells as well as in animal models. Results: p28GANK expression was suppressed by up to 80% in HCC cells. Depletion of p28GANK inhibited HCC cell growth and tumorigenesis, enhanced dephosphorylation of RB1, and decreased transcription activity of E2F-1 in HuH-7 cells. Furthermore, depletion of p28GANK induced caspase-8- and caspase-9-mediated apoptosis of HCC cells. Finally, targeting p28GANK by adenovirus injection inhibited the growth of established tumors in nude mice. Conclusions: This study shows that the T7-system screening-based AdSiRNA can be used successfully to silence an oncogene. We proved the therapeutic potential of AdSiRNA on the treatment of HCC by targeting p28GANK. Our results indicate that p28GANK may serve as a novel therapeutic target for treating HCC.

Interferon-Alpha Inhibits Hepatitis B Virus–Induced NF-?B Activation Through Nuclear Localization of NF-kB–Inducing Kinase
Sung Gyoo Park, Hyun Mi Ryu, Seong-Oe Lim, Yong-Il Kim, Soon B. Hwang, Guhung Jung
Background & Aims: Nuclear factor-?B (NF-?B) signaling pathway is an important regulating pathway in liver diseases, including hepatocellular carcinoma. In our study, immunohistochemical analysis showed that NF-?B-inducing kinase (NIK), an upstream kinase of I?B kinases, nuclear localization occurs only in liver tissues obtained from hepatitis B surface antigen (HBsAg)(+) patients but not in tissues from HBsAg(?) patients. The aim of the present study was to identify the inducer of NIK nuclear localization and determine whether the NIK nuclear localization affects the hepatitis B virus (HBV)-mediated NF-?B activation. Methods: The experiments were performed on HepG2.2.15 cells and on HepG2 cells transfected with pHBV1.2?, a plasmid encoding all HBV messages, using NF-?B-dependent luciferase reporter gene assay, electrophoretic mobility shift assay, immunoblot analysis, and fluorescent microscopy analysis. Results: HBV induced NIK-dependent NF-?B activation. However, interferon (IFN)-? induced NIK nuclear localization and inhibited NF-?B activation in HepG2.2.15 cells and in HepG2 cells transfected with pHBV1.2?. When NIK nuclear localization was inhibited by deletion of nuclear localization signal on NIK, IFN-? did not induce the NIK nuclear localization and did not inhibit NF-?B activation. Conclusions: IFN-? selectively inhibits HBV-mediated NF-?B activation. This inhibition is accomplished by NIK nuclear localization, which is a novel mechanism of NF-?B inhibition.

Interleukin-6 Contributes to Mcl-1 Up-regulation and TRAIL Resistance via an Akt-Signaling Pathway in Cholangiocarcinoma Cells
Shogo Kobayashi, Nathan W. Werneburg, Steven F. Bronk, Scott H. Kaufmann, Gregory J. Gores
Background & Aims: Cholangiocarcinomas often arise within a background of chronic inflammation suggesting that inflammation imparts survival signals to this cancer. Previous studies have also shown that the inflammatory cytokine interleukin (interleukin [IL]-6) contributes to survival signals in an autocrine fashion and that myeloid cell leukemia-1 (Mcl-1), an antiapoptotic member of the B-cell leukemia-2 family, is an important participant in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance in this neoplasm. The present study evaluated the possibility that IL-6 signaling contributes to Mcl-1 up-regulation in cholangiocarcinoma. Methods: Protein kinase B (Akt) and Mcl-1 expression in human tissue was assessed by immunohistochemistry. The relationship between IL-6 signaling, Akt activity, and Mcl-1 expression was examined in cell lines. Results: Immunohistochemistry showed that the serine/threonine kinase Akt and Mcl-1 are strongly expressed in the preneoplastic bile duct inflammatory disease primary sclerosing cholangitis and in human cholangiocarcinoma specimens. Immunoblotting showed that Akt is expressed and constitutively phosphorylated in 3 human cholangiocarcinoma lines. Further analysis showed that treatment with anti-IL-6-neutralizing antiserum led to reduced Akt phosphorylation, diminished Mcl-1 expression, and enhanced TRAIL sensitivity. Likewise, the Akt inhibitor A443654.3 led to diminished signaling through the Akt pathway, decreased Mcl-1 expression, and enhanced TRAIL-mediated apoptosis. Conclusions: These findings not only show that an autocrine IL-6/Akt signaling pathway enhances Mcl-1 expression in cholangiocarcinoma but also suggest a strategy for overcoming the resulting apoptosis resistance.

Early Growth Response-1 Transcription Factor Is Essential for Ethanol-Induced Fatty Liver Injury in Mice
Megan R. McMullen, Michele T. Pritchard, Qifang Wang, Carrie A. Millward, Colleen M. Croniger, Laura E. Nagy
Background & Aims: Early growth response-1 (Egr-1), an immediate early gene/zinc-finger transcription factor, is required for maximal stimulation of tumor necrosis factor ? (TNF-?) transcription in response to lipopolysaccharide (LPS). Because chronic ethanol exposure sensitizes macrophages to LPS-stimulated TNF-? expression, we have investigated the role of Egr-1 in mediating increased LPS-stimulated TNF-? expression after chronic ethanol feeding. Furthermore, because TNF-? contributes to alcoholic liver injury, we tested the hypothesis that Egr-1 is required for the development of ethanol-induced fatty liver injury in wild type and egr-1 ?/? mice. Methods: Wild-type and egr-1 ?/? mice were fed ethanol-containing diets or pair-fed control diets for 6 weeks. Results: Wild-type mice fed the ethanol diet developed hepatic steatosis characterized by micro- and macrovesicular lipid accumulation. However, egr-1 ?/? mice did not develop steatosis after ethanol feeding. Alanine transferase and TNF-? concentrations in serum were increased after ethanol feeding in wild-type but not egr-1 ?/? mice. In wild-type mice, challenge with LPS increased Egr-1 messenger RNA (mRNA) and DNA binding activity in liver; this response to LPS was enhanced after chronic ethanol feeding. LPS challenge also increased hepatic TNF-? mRNA and serum TNF-? to a greater extent after ethanol feeding compared with pair-fed wild-type mice. However, chronic ethanol feeding did not enhance LPS-stimulated TNF-? mRNA or serum TNF-? in egr-1 ?/? mice. Conclusions: These data show that Egr-1 contributes to increased LPS-mediated TNF-? expression after chronic ethanol and that the absence of Egr-1 prevents chronic ethanol-induced fatty liver, as well as increased sensitivity to LPS.

Role of Connective Tissue Growth Factor in Oval Cell Response During Liver Regeneration After 2-AAF/PHx in Rats
Liya Pi, Seh-Hoon Oh, Thomas Shupe, Bryon E. Petersen
Background & Aims: Recruitment and proliferation of Thy-1+ oval cells is a hallmark of liver regeneration after 2-acetylaminofluorene (2-AAF)/partial hepatectomy (PHx) in rats. To understand the molecular mechanism underlying this process, we investigated the role of connective tissue growth factor (CTGF), one of the candidate genes differentially expressed in Thy-1+ oval cells, in this liver injury model. Methods: Northern and Western analyses were performed to examine the induction of CTGF in total liver homogenate. Quantitative real-time polymerase chain reaction (PCR), immunofluorescent staining, and in situ hybridization were performed to confirm the expression and localization of CTGF in Thy-1+ oval cells. Finally, a known inhibitor of CTGF synthesis, Iloprost, was administered to 2-AAF/PHx treated rats to investigate the effect of Iloprost on oval cell response. Results: CTGF was found to be up-regulated at both the RNA and protein levels and occurred concurrently with an up-regulation of transforming growth factor ?1 (TGF-?1). Sorted Thy-1+ oval cells expressed a high level of CTGF gene in a quantitative PCR assay. Colocalization of Thy-1 antigen and ctgf signals by in situ hybridization further confirmed that Thy-1+ oval cells were a source of CTGF. Iloprost administration blocked CTGF induction in treated animals but did not affect TGF-?1 expression. The inhibition of CTGF induction by Iloprost was associated with a significant decrease in oval cell proliferation and a lower level of ?-fetoprotein expression as compared with control animals. Conclusions: These results show that CTGF induction is important for robust oval cell response after 2-AAF/PHx treatment in rats.


Long-term Extensive Expansion of Mouse Hepatic Stem/Progenitor Cells in a Novel Serum-Free Culture System
Atsunori Tsuchiya, Toshio Heike, Hisanori Fujino, Mitsutaka Shiota, Katsutsugu Umeda, Momoko Yoshimoto, Yasunobu Matsuda, Takafumi Ichida, Yutaka Aoyagi, Tatsutoshi Nakahata
Background & Aims: The liver has high regenerative potential. We attempted to establish a novel culture system for extensive expansion of fetal mouse hepatic stem/progenitor cells and to characterize cultured cells. Methods: Hepatic spheroids collected from 6-day floating cultures were cultured on collagen-coated dishes in serum-free conditions in medium containing growth factors. Cultured cells were mainly characterized by immunocytochemistry and flow cytometry or transplanted into adult mice. Results: Approximately 400 expanding hepatic spheroids were generated from every 1 ? 106 fetal liver cells. Subsequently, highly replicative colonies were subcultured with maintaining colony formation on collagen-coated dishes. These colonies consisted of small immature ?-fetoprotein-positive cells and hepatocytic and cholangiocytic lineage-committed cells. The immature ?-fetoprotein-positive cells could be expanded in a reproducible manner at least 5 ? 105-fold (which involved at least 30 passages over >6 months) without losing differentiation potential. Flow cytometric analysis showed that all cultured cells expressed CD49f, but not CD34, Thy-1, c-kit, or CD45. Nearly 15% of the cells expressed Sca-1, and approximately 5%–20% of the cells were side population cells. Both sorted side population cells and Sca-1-positive cells (especially side population cells) produced a large number of ?-fetoprotein-positive cells and lineage-committed cells. Expanded cells had bidirectional differentiation potential and improved serum albumin levels in mice with severe liver damage. Conclusions: Long-term extensive expansion of transplantable hepatic stem/progenitor cells was reproducibly achieved in a novel serum-free culture system. Moreover, this culture system yielded side population and Sca-1-positive cell populations that included hepatic stem/progenitor cells with differentiation and proliferation properties.

Cytokines and Peroxisome Proliferator-Activated Receptor ? Ligand Regulate Phagocytosis by Pancreatic Stellate Cells
Kyoko Shimizu, Makio Kobayashi, Junko Tahara, Keiko Shiratori
Background & Aims: Pancreatic stellate cells have been characterized as the major source of extracellular matrix and cytokine production in the pancreas. This study showed that pancreatic stellate cells have a phagocytic function. Methods: The morphological features of periacinar phagocytic cells were investigated by immunohistochemically staining serial sections of the pancreas from male WBN/Kob rats and an animal model of acute pancreatitis for glial fibrillary acidic protein and ?-smooth muscle actin. Pancreatic stellate cells were assayed for phagocytic activity by incubating them with senescent polymorphonuclear neutrophils or fluorescence-labeled latex beads in the presence or absence of cytokines, growth factors, and peroxisome proliferator-activated receptor ? ligand. The role of CD36 and peroxisome proliferator-activated receptor ? in phagocytosis was investigated by blocking endogenous CD36 and peroxisome proliferator-activated receptor ? activity with anti-CD36 antibody and peroxisome proliferator-activated receptor ? small interfering RNAs, respectively. Results: Phagocytic cells were observed in areas of inflammation, and they were identical to the glial fibrillary acidic protein-positive and ?-smooth muscle actin-positive cells, thus suggesting that they were pancreatic stellate cells. Aged polymorphonuclear neutrophils were ingested into the cytoplasm of the pancreatic stellate cells. Transforming growth factor ?, tumor necrosis factor ?, and interleukin 1? decreased the phagocytic activity of pancreatic stellate cells, whereas troglitazone induced a dose-dependent increase in both phagocytic activity and expression of CD36. Blockade of CD36 reduced troglitazone-induced phagocytosis. Silencing of the peroxisome proliferator-activated receptor ? gene decreased phagocytosis and expression of CD36. Conclusions: Pancreatic stellate cells act as resident phagocytic cells, and CD36 promotes troglitazone-induced phagocytic activity via peroxisome proliferator-activated receptor ? transactivation. Because phagocytosis is essential to limit the extent of inflammation, enhancement of phagocytic activity may provide an important approach to the treatment of pancreatic diseases.

Case Report

Diagnosing Helicobacter pylori In Vivo by Confocal Laser Endoscopy
Ralf Kiesslich, Martin Goetz, Juergen Burg, Manfred Stolte, Ekkehard Siegel, Markus J. Maeurer, Steven Thomas, Dennis Strand, Peter R. Galle, Markus F. Neurath
Background & Aims: Confocal laser endomicroscopy enables subsurface microscopic imaging of living tissue during ongoing endoscopy. This case report describes the in vivo detection of Helicobacter pylori by endomicroscopy.Methods: Endomicroscopy (Pentax, Tokyo, EC-3870CIFK) was performed by using two different contrast stains: Topical Acriflavine in addition to intravenously applied fluorescein netted the surface and allowed identification of focal accumulation of Helicobacter pylori at the surface and in deeper layer of the gastric epithelium. Biopsies were performed at the antrum and corpus for urease testing and histology. In addition, biopsies were cultured for Helicobacter pylori. Cultured bacteria were re-assessed ex vivo using confocal microscopy with and without acriflavine staining.Results: Helicobacter pylori infection could be detected in a 70-year-old male by endomicroscopy. Accumulated, as well as single bacteria, could be observed and the distinct shape and flagella of Helicobacter pylori could be identified. Helicobacter pylori infection was proved by histology. Furthermore, ex vivo examination of cultures proved the presence of Helicobacter pylori and the active uptake of acriflavine into the bacteria.Conclusions: Endomicroscopy is a new diagnostic approach, which enables the immediate diagnosis of Helicobacter pylori in vivo during standard video endoscopy.

Microarrays and Other New Technologies
 
Molecular Analysis to Detect Pancreatic Ductal Adenocarcinoma in High-Risk Groups
Li Yan, Christopher McFaul, Nathan Howes, Jane Leslie, Gillian Lancaster, Theresa Wong, Jane Threadgold, Jonathan Evans, Ian Gilmore, Howard Smart, Martin Lombard, John Neoptolemos, William Greenhalf
Background & Aims: Screening of high-risk groups for pancreatic cancer has not been adopted because of concerns regarding specificity and sensitivity. Suitability of a combination of 3 novel molecular screening techniques was investigated. Methods: Pancreatic juice was extracted from 146 patients with pancreatic ductal adenocarcinoma, chronic pancreatitis, or biliary tract stones. p53 mutations were analyzed by using a modified yeast functional assay, K-ras status was analyzed using mutation-specific real-time PCR and the proportion of p16INK4a promoter methylation was estimated using comparative methylation-specific real-time PCR. Results: p53 mutations were detected in 20 of 48 (42%) cancer cases, none of 49 controls, and 2 of 49 (4%) patients with pancreatitis. K-ras mutations were detected in 31 of 57 (54%) cancer patients, 13 of 61 (21%) controls, and 23 of 67 (34%) patients with pancreatitis. Twenty-six of 42 (62%) cancer patients had promoter methylation levels > 12%, compared with 3 of 24 (13%) controls, and 2 of 26 (8%) with pancreatitis. Mutations in p53 or high-level p16INK4a promoter methylation occurred in 29 of 36 (80%) patients with cancer, 3 of 24 (13%) controls, and 3 of 22 (13%) with pancreatitis. Three patients (8%) of 36 with cancer; 14 of 24 (58%) controls, and 13 of 22 (59%) patients with pancreatitis had no marker. The gallstone disease patients had a high rate of positive K-ras mutations, possibly reflecting the fact that they were not disease free. Conclusions: Combination molecular analysis increased the discrimination between patients with malignant and benign disease. This level of discrimination would allow patients in high-risk groups to be stratified from negligible risk to over 50% probability of an early cancer.

Copyright © 2001-2005 by the American Gastroenterological Association. All rights reserved.

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JOURNAL OF HEPATOLOGY

Table of Contents for June 2005 (Vol. 42, Issue 6)

Viral Hepatitis

Prevention of interferon-alpha associated depression in psychiatric risk patients with chronic hepatitis C, 4 April 2005
Schaefer M, Schwaiger M, Garkisch AS, Pich M, Hinzpeter A, Uebelhack R, Heinz A, van Boemmel F, Berg T
Background/Aims: Interferon-alpha (IFN-?) induced depression is a major limitation for the treatment of chronic hepatitis C, especially for patients with psychiatric disorders. We prospectively studied the efficacy of a pre-emptive treatment with the antidepressant citalopram to prevent depression during hepatitis C treatment with pegylated IFN-?-2b plus ribavirin.
Methods: 14 HCV infected patients with psychiatric disorders received a prophylactic medication with citalopram (20mg/day) before and during therapy with IFN-?. The incidence of major depression was compared with 22 HCV infected patients with psychiatric disorders (group B; n=11) and without psychiatric risk factors (group C; n=11), who underwent IFN-? treatment without a pre-emptive antidepressant therapy. Depression was diagnosed by DSM-IV criteria.
Results: Pre-treatment of psychiatric patients with citalopram significantly reduced the incidence of major depression during the first 6 months of antiviral treatment as compared to the two control groups (group A 14% vs. 64% and 55% in group B and C; log-rank 6.89; df=2; P=0.032). Patients who developed symptoms of major depression during IFN therapy could also be improved by antidepressive treatment.
Conclusions: Our open label pilot study, though small, clearly indicates that IFN alpha induced depression in psychiatric risk patients can be ameliorated by both the use of antidepressants as well as by intensive psychiatric care. However, larger, double blind placebo controlled trials in other patient populations are required to confirm these preliminary findings.

Liver disease as a major cause of death among HIV infected patients: role of hepatitis C and B viruses and alcohol, 4 April 2005
Salmon-Ceron D, Lewden C, Morlat P, Bévilacqua S, Jougla E, Bonnet F, Héripret L, Costagliola D, May T, Chêne G, The ‘Mortality 2000’ study group Background/Aims
We analyzed the characteristics of HIV infected patients who died from liver disease, focusing on hepatitis virus co-infection.
Methods One-hundred and eighty-five French hospital departments involved in HIV/AIDS management prospectively notified all deaths occurring in 2000. Patients whose hepatitis C (HCV) and hepatitis B (HBV) serostatus was known were classified as being infected by HCV alone, HBV alone (HBsAg positive), both HCV and HBV, or neither HCV nor HBV.
Results Among 822 HIV infected patients, 29% were infected by HCV alone, 8% by HBV alone, and 4% by both HCV and HBV. The most frequent causes of death were liver disease (31% of cases) and AIDS (29%) among HIV–HCV co-infected patients, and AIDS (38%) and liver disease (22%) among HIV-HBV co-infected patients. Liver disease was a more frequent cause of death among patients co-infected by both HCV and HBV (44% of cases). Hepatocellular carcinoma was present in 15% of patients who died from liver disease, and was associated with HBV co-infection. Nearly half the patients who died from liver disease had more than 200 CD4/mm3. Conclusions
Liver disease is now a leading cause of death among HIV-HCV co-infected patients and is becoming an important cause of death among HIV-HBV co-infected patients. The risk of death from liver disease is highest in patients co-infected by both HCV and HBV.

Hepatitis B virus infection in lymphatic tissues in inactive hepatitis B carriers, 1 April 2005
Umeda M, Marusawa H, Seno H, Katsurada A, Nabeshima M, Egawa H, Uemoto S, Inomata Y, Tanaka K, Chiba T
Background/Aims Hepatitis B virus (HBV) infection in extrahepatic tissues is controversial. To clarify whether episomal HBV can infect nonhepatic tissues, we investigated the molecular forms of HBV in the lymphatic cells of inactive HBV carriers who lacked viremia, thus avoiding contamination with HBV genomes originating from the viral particles present in the serum.
Methods We assessed HBV genome, replicative forms, and viral integrants in the liver, serum, peripheral blood mononuclear cells (PBMC), and lymph nodes of 21 inactive HBV carriers who tested positive for antibodies against the HBV core antigen (anti-HBc).
Results Of the 21 anti-HBc positive individuals, HBV-DNA was detected in liver samples of 15 (71.4%), in the lymph nodes of 11 (52.4%), and in PBMC of three (14.3%). However, none of the detected HBV genomes from lymphatic tissues included the replicative forms of HBV. In one case, integrated HBV was present in the lymphatic tissues and the host–viral junction was present in the intronic sequences of chromosome 17.
Conclusions These data suggest that human lymphatic tissues cannot support viral replication in anti-HBc positive inactive HBV carriers, while retaining the viral genome as an integrated form.

Occult hepatitis B virus infection in hematopoietic stem cell donors in a hepatitis B virus endemic area, 31 March 2005
Hui Ck, Sun J, Au Wy, Lie AKW, Yueng Yh, Zhang Hy, Lee NP, Hou Jl, Liang R, Lau GKK
Background/Aims The acquisition of hepatitis B virus (HBV) infection following organ transplantation from donors with occult HBV infection is an important cause of morbidity and mortality. The aim of this study is to determine the prevalence of occult HBV in allogeneic hematopoietic stem cell (HSC) transplantation donors.
Methods We performed a retrospective study on 124 consecutive hepatitis B surface antigen negative HSC donors. Their serum samples were analyzed by PCR for the pre-S/S, pre-core/core and X regions of the virus. Samples reactive by at least two PCR assays were considered HBV-DNA positive.
Results Nineteen of the 124 HSC donors (15.3%) had occult HBV infection. Sixteen of these 19 donors with occult HBV infection (84.2%) tested positive for hepatitis B core antibody while 78 of 105 subjects (74.3%) without occult HBV infection were also positive (P=0.56). Fourteen of the 19 donors (73.7%) with occult HBV infection tested positive for hepatitis B surface antibody while 67 of the 105 subjects without occult HBV infection were also positive (P=0.45).
Conclusions The prevalence of occult HBV infection among HSC donors in Hong Kong is high. Anti-HBc and anti-HBs status had no significant correlation with the presence of occult HBV infection.

Cirrhosis and its Complications

MELD score and hepatocellular carcinoma identify patients at different risk of short-term mortality among cirrhotics bleeding from esophageal varices, 4 April 2005
Amitrano L, Guardascione MA, Bennato R, Manguso F, Balzano A
Background/Aims The role of model for end stage liver disease (MELD) and the presence of hepatocellular carcinoma (HCC) as risk factors of short-term mortality in patients bleeding from esophageal varices were evaluated.
Methods From February 2002 to August 2003, 172 cirrhotic patients admitted for the first episode of bleeding from esophageal varices received vasoactive and endoscopic therapy. Patients' survival was evaluated at 6 weeks and 3 months. The role of MELD and HCC as independent risk factors of mortality was evaluated.
Results In the 172 patients, the overall mortality was 21.5% at 6 weeks and 30.2% at 3 months. MELD score resulted a good predictor of mortality either at 6 weeks or 3 months. Fifty-four patients (31.3%) had HCC. The presence of advanced-HCC was an independent risk factor of mortality at 3 months. Patients with MELD score >15 and advanced-HCC had a significantly worse survival than patients with MELD ≤15 and without HCC or with early-HCC either at 6 weeks or 3 months
Conclusions MELD score and the presence of HCC allow to identify patients at different risk of short-term mortality among cirrhotic patients at first episode of bleeding from esophageal varices.

Evaluation of the increase in model for end-stage liver disease (?MELD) score over time as a prognostic predictor in patients with advanced cirrhosis: risk factor analysis and comparison with initial MELD and Child–Turcotte–Pugh score, 31 March 2005
Huo TI, Wu JC, Lin HC, Lee FY, Hou MC, Lee PC, Chang FY, Lee SD
Background/Aims The model for end-stage liver disease (MELD) has been used to prioritize cirrhotic patients awaiting liver transplantation. The change in MELD score over time (?MELD) may have additional prognostic value. We investigated the ability of ?MELD to predict the outcome of advanced cirrhosis and prospectively assessed the factors associated with increasing ?MELD.
Methods Risk factors were determined in 58 prospectively followed-up patients. The predictive power of ?MELD, initial MELD and Child–Turcotte–Pugh (CTP) score was compared by using c-statistic in 351 patients.
Results Ascites (P=0.020) and hepatic encephalopathy (P=0.023) were significantly associated with increasing MELD score at 3 months. The area under receiver operating characteristic (ROC) curve for ?MELD/month was 0.779 compared with 0.718 for MELD (P=0.130) and 0.528 for CTP score (P<0.001) at 6 months; the area was 0.822, 0.744 and 0.528, respectively (P=0.018 and <0.001, respectively) at 12 months. ?MELD/month >2.5 was the only significant prognostic predictor at 6 (odds ratio: 9.8, P<0.001) and 12 months (odds ratio: 16.3, P<0.001) in multivariate logistic analysis.
Conclusions Increasing MELD score is associated with the onset of ascites and encephalopathy. ?MELD is superior to initial MELD and CTP scores to predict intermediate term outcome in patients with advanced cirrhosis.

Liver Failure, Growth and Cancer

Cloning and characterization of hepaCAM, a novel Ig-like cell adhesion molecule suppressed in human hepatocellular carcinoma, 12 April 2005
Chung Moh M, Hoon Lee L, Shen S
Background/Aims Previously, we reported on gene HEPN1 that was silenced in hepatocellular carcinoma (HCC) and capable of arresting cell growth. In this study, we identified another novel gene hepaCAM from the liver, which contains the full-length HEPN1 on its antisense strand in the 3?-noncoding region, and assessed its expression, characteristics and functions in HCC.
Methods Full-length hepaCAM cDNA was isolated by rapid amplification of cDNA ends. The gene expression was examined by semi-quantitative RT-PCR in 23-paired HCC liver specimens and 5 HCC cell lines. Transfection studies, coupled with immunocytochemistry, cellular interaction analyses, colony formation and microtetrazolium assay, were employed to elucidate the localization and functions of hepaCAM.
Results The expression of hepaCAM was decreased in 20/23 of HCC samples and undetectable in 5 HCC cell lines tested. The gene product consisting of 416 amino acids displayed the typical structure of Ig-like cell adhesion molecules. The protein was glycosylated and predominantly localized on the cytoplasmic membrane. When re-expressed in HepG2, hepaCAM accelerated cell spreading (P<0.001), increased cell motility (P=0.0011), reduced colony formation (P=0.0022), and inhibited cell growth (P<0.001).
Conclusions Gene hepaCAM, frequently silenced in HCC, encodes an Ig-like transmembrane glycoprotein and is involved in cell adhesion and growth control.

Activation of the canonical Wnt/?-catenin pathway confers growth advantages in c-Myc/E2F1 transgenic mouse model of liver cancer, 12 April 2005
Calvisi DF, Conner EA, Ladu S, Lemmer ER, Factor VM, Thorgeirsson SS
Background/Aims Previously, we showed that activation of the ?-catenin/Wnt pathway is a dominant event during c-Myc/E2F1 hepatocarcinogenesis. Majority of c-Myc/E2F1 HCCs displayed nuclear accumulation of ?-catenin in the absence of ?-catenin mutations, suggesting that alterations in other members of the Wnt pathway might be responsible for nuclear localization of ?-catenin. Here, we investigated the mechanisms responsible for nuclear translocation of wild-type ?-catenin and addressed the potential contribution of the Wnt pathway in c-Myc/E2F1 hepatocarcinogenesis.
Methods Status of the members of the Wnt pathway was determined through microsatellite and Western blot analysis.
Results Majority of c-Myc/E2F1 HCCs exhibited multiple abnormalities in the Wnt pathway regardless of the presence of ?-catenin mutations. The observed abnormalities included overexpression of Wnt-1, Frizzled 1 and 2 receptors, Dishevelled-1, downregulation of Secreted frizzled-related protein-1, GSK-3Ō inactivation, microsatellite instability at the Axin locus as well as induction of ?-catenin target genes, such as glutamine synthetase, glutamate transporter-1, and Wisp-1. HCCs with Ō-catenin activation displayed significantly higher proliferation rate and larger tumor size when compared with Ō-catenin negative tumors.
Conclusions The data demonstrate that multiple abnormalities in the members of the Wnt pathway lead to nuclear accumulation of ?-catenin and suggest that activation of Wnt pathway provides proliferative advantages in c-Myc/E2F1-driven hepatocarcinogenesis.

JNK mediates hepatic ischemia reperfusion injury, 12 April 2005
Uehara T, Bennett B, Sakata ST, Satoh Y, Bilter GK, Westwick JK, Brenner DA
Background/Aims Hepatic ischemia followed by reperfusion (I/R) is a major clinical problem during transplantation, liver resection for tumor, and circulatory shock, producing apoptosis and necrosis. Although several intracellular signal molecules are induced following I/R including NF-?B and c-Jun N terminal kinase (JNK), their roles in I/R injury are largely unknown. The aim of this study is to assess the role of JNK during warm I/R injury using novel selective JNK inhibitors.
Methods Male Wistar rats (200±25g) are pretreated with vehicle or with one of three compounds (CC0209766, CC0223105, and CC-401), which are reversible, highly selective, ATP-competitive inhibitors of JNK. In the first study, rats are assessed for survival using a model of ischemia to 70% of the liver for 90min followed by 30% hepatectomy of the non-ischemic lobes and then reperfusion. In the second study, rats are assessed for liver injury resulting from 60 or 90min of ischemia followed by reperfusion with analysis over time of hepatic histology, serum ALT, hepatic caspase-3 activation, cytochrome c release, and lipid peroxidation.
Results In the I/R survival model, vehicle-treated rats have a 7-day survival of 20–40%, while rats treated with the three different JNK inhibitors have survival rates of 60–100% (P<0.05). The decrease in mortality correlates with improved hepatic histology and serum ALT levels. Vehicle treated rats have pericentral necrosis, neutrophil infiltration, and some apoptosis in both hepatocytes and sinusoidal endothelial cells, while JNK inhibitors significantly decrease both types of cell death. JNK inhibitors decrease caspase-3 activation, cytochrome c release from mitochondria, and lipid peroxidation. JNK inhibition transiently blocks phosphorylation of c-Jun at an early time point after reperfusion, and AP-1 activation is also substantially blocked. JNK inhibition blocks the upregulation of the pro-apoptotic Bak protein and the degradation of Bid.
Conclusions Thus, JNK inhibitors decrease both necrosis and apoptosis, suggesting that JNK activity induces cell death by both pathways.

Global gene repression in hepatocellular carcinoma and fetal liver, and suppression of dudulin-2 mRNA as a possible marker for the cirrhosis-to-tumor transition, 11 April 2005
Coulouarn C, Derambure C, Lefebvre G, Daveau R, Hiron M, Scotte M, François A, Daveau M, Salier JP
Background/Aims It has been suspected but not proven that the transcriptional reprogramming induced by hepatocellular carcinoma could recapitulate that of the developing liver.
Methods With a complete coverage of the liver transcriptome by microarray using adult livers as controls, we searched for similarities and differences in mRNA abundances between hepatocellular carcinoma nodules and fetal livers taken before (early) or after (late) the 22–24th week of gestation. Changes in some mRNA levels were studied in further liver samples by quantitative RT-PCR.
Results Altered gene expression in hepatocellular carcinoma mostly results in down-regulated mRNAs which largely overlap with those repressed in the late fetal liver. Different frequencies of transcription factor binding sites in the down-regulated genes vs control genes as well as changes in abundance of mRNAs for relevant transcription factors point to a transcriptional repression. The down-regulated mRNAs code for proteins involved in (i) transcription and translation, (ii) specific functions of the differentiated hepatocyte or (iii) activation of proliferation and apoptosis.
Conclusions Apoptosis limitation is likely to predominate over active proliferation during liver development and hepatocellular carcinoma. Repression of the apoptosis-associated dudulin-2 mRNA points to a potential marker for the transition from a carcinoma-free to carcinoma-associated cirrhosis.

Molecular and Cell Biology

Insulin prevents liver damage and preserves liver function in lipopolysaccharide-induced endotoxemic rats, 23 February 2005
Jeschke MG, Rensing H, Klein D, Schubert T, Mautes AE, Bolder U, Croner RS
Background/Aims Liver integrity and function are crucial for survival of patients suffering from trauma, operations or infections. Insulin decreased mortality and prevented the incidence of multi organ failure and infection in critically ill patients. The aim of the present study was to determine whether insulin exerts positive effects on hepatic homeostasis and function during endotoxemia.
Methods Endotoxemic rats received either saline or insulin. Hepatic morphology and function was determined by measuring the effect of insulin on liver proteins, enzymes, hepatocyte apoptosis and proliferation including caspases-3 and -9 and Bcl-2. Intrahepatic ATP, glucose and lactate concentration were determined by bioluminescence. To determine possible molecular changes the effect of insulin on hepatic cytokine mRNA and gene profile analysis were assessed.
Results Insulin significantly improved hepatic protein synthesis by increasing albumin and decreasing c-reactive protein, P<0.05. Insulin attenuated hepatic damage by decreasing AST and ALT, P<0.05. Improved liver morphology was due to decreased hepatocyte apoptosis along with decreased caspase-3 concentration and increased hepatocyte proliferation along with Bcl-2 concentration, P<0.05. Insulin decreased hepatic IL-1?, IL-6 and MIF mRNA and improved hepatic glucose metabolism and glycolysis, P<0.05. GeneChip analysis revealed an anti-inflammatory effect of insulin.
Conclusions Insulin improves hepatic integrity, hepatic glucose metabolism and hepatic function by increasing cell survival and attenuating the hepatic inflammatory response in endotoxemic rats.

Interferon-alpha-induced modulation of glucocorticoid and serotonin receptors as a mechanism of depression, 4 April 2005
Cai W, Khaoustov VI, Xie Q, Pan T, Le W, Yoffe B
Background/Aims The mechanism of interferon (IFN)-?-induced depression remains poorly understood. Recently, modulation of glucocorticoid receptor (GR) and serotonin receptor 1A (5-HTR1A) were implicated in mechanism(s) leading to depression. To gain insight into this mechanism, we assessed the effect of IFN-? on the modulation of GR and 5-HTR1A expression.
Methods Hepatoblastoma, myelocyte-derived and T cell leukemia-derived cell lines were treated with titrated doses of IFN-? for different incubation times and analyzed by Western blot, RT-PCR, and microarrays. Dose- and time-dependent decreases of proteins and mRNA levels of GR and 5-HTR1A were observed.
Results The expression of GR and 5-HTR1A in cells treated for 6 days decreased by 74 and 72%, respectively. Recovery was observed following IFN-? withdrawal. Co-incubation with tricyclic antidepressants (desipramine) or serotonin reuptake inhibitors (fluoxetine) attenuated the effect of IFN-? on GR or 5-HTR1A. GR and 5-HTR1A were unaffected by treatment with either IFN-? or TUDCA. However, the effect of IFN-? on GR was abolished when used in combination with TUDCA.
Conclusions In conclusion, IFN-? downregulated GR and 5-HTR1A levels in cell lines. These levels of GR and 5-HTR1A, following IFN-?-induced downregulation, recovered after withdrawal of IFN-? or addition of desipramine or fluoxetine. These data provide insights regarding pathogenesis of IFN-?-induced depression.

Generation of a monoclonal human single chain antibody fragment to hepatic stellate cells – a potential mechanism for targeting liver anti-fibrotic therapeutics, 12 April 2005
Elrick LJ, Leel V, Blaylock MG, Duncan L, Drever MR, Strachan G, Charlton KA, Koruth M, Porter AJ, Wright MC
Background/Aims Hepatic stellate cells are pivotal to fibrogenesis in the liver and many potential anti-fibrotic therapeutics are required to act on targets within hepatic stellate cells. The aim of this study was to generate a human antibody fragment to hepatic stellate cells.
Methods Phage display was used to generate a human monoclonal antibody fragment to a peptide sequence present on an extracellular domain of synaptophysin, a protein expressed on the surface of hepatic stellate cells.
Results An antibody fragment was isolated (termed C1-3), expressed in bacteria and purified. Fluorescently-labelled C1-3 antibody associated with human hepatic stellate cells but not hepatocytes in culture. Binding of fluorescently labelled C1-3 to hepatic stellate cells was blocked by the extracellular synaptophysin peptide sequence and uptake of the antibody intracellularly was inhibited by monensin. The toxin tributyl tin—when conjugated to C1-3—retained the ability to kill hepatic stellate cells confirming that C1-3 is sequestered intracellularly.
Conclusions This antibody fragment may be an effective means to target therapeutics to human hepatic stellate cells.

A distinct microarray gene expression profile in primary rat hepatocytes incubated with ursodeoxycholic acid, 12 April 2005
Castro RE, Solá S, Ma X, Ramalho RM, Kren BT, Steer CJ, Rodrigues CMP
Background/Aims Ursodeoxycholic acid (UDCA) and its taurine-conjugated derivative, TUDCA, modulate cell death and cell cycle regulators, such as E2F-1 and p53. However, precise pathways underlying UDCA's effects are not fully understood. The aim of this study was to identify specific cellular targets of UDCA.
Methods The expression profile of primary rat hepatocytes incubated with UDCA was determined using Affymetrix GeneChip Rat 230A arrays. Hybridization data were processed to identify genes with significant expression changes. RT-PCR and immunoblot analyses of a selected target confirmed microarray data.
Results The results showed that >440 genes were modulated with UDCA by >1.5-fold; ?25% were significantly different from controls. Genes affected by UDCA included new regulatory molecules, such as Apaf-1. RT-PCR and immunoblotting confirmed a decrease in Apaf-1. Other altered genes were directly involved in cell cycle (cyclin D1, cadherin 1, HMG-box containing protein 1) and apoptosis (prothymosin-?) events. The E2F-1/p53/Apaf-1 pathway appears to be targeted by UDCA. Finally, transcripts for proteins with kinase activity and transcription factors were specifically modulated by TUDCA.
Conclusions This study expands our knowledge of the biological effects of UDCA in hepatocytes. Most of the identified genes represent novel potential targets of UDCA, which may ultimately explain its therapeutic properties.

Effects of betaine supplementation on hepatic metabolism of sulfur-containing amino acids in mice, 1 April 2005
Kim SK, Kim YC
Background/Aims We previously reported that acute betaine treatment induced significant changes in the hepatic glutathione and cysteine levels in mice and rats. The present study was aimed to determine the effects of dietary betaine on the metabolism of sulfur-containing amino acids. Methods/Results Male mice were supplemented with betaine (1%) in drinking water for up to 3 weeks. Changes in hepatic levels of major sulfur amino acid metabolites and products were stabilized after 2 weeks of betaine supplementation. Betaine intake increased methionine, S-adenosylmethionine, and S-adenosylhomocysteine levels significantly, but homocysteine and cystathionine were reduced. Methionine adenosyltransferase activity was elevated to three-fold of control. Cysteine catabolism to taurine was inhibited as evidenced by a decrease in cysteine dioxygenase activity and taurine levels in liver and plasma. Despite the significant changes in the transsulfuration reactions, neither hepatic cysteine nor glutathione was altered. Betaine supplementation decreased the hepatotoxicity induced by chloroform (0.5ml/kg, ip) significantly.
Conclusions Betaine supplementation enhances recycling of homocysteine for the generation of methionine and S-adenosylmethionine while reducing its utilization for the synthesis of cystathionine and cysteine. However, the hepatic levels of cysteine or glutathione are not affected, most probably due to the depression of taurine generation from cysteine.

Genetic and Metabolic Liver Disease

Association of myeloperoxidase promotor polymorphism with cirrhosis in patients with hereditary hemochromatosis, 4 April 2005
Österreicher CH, Datz C, Stickel F, Hellerbrand C, Penz M, Hofer H, Wrba F, Penner E, Schuppan D, Ferenci P
Background/Aims Hereditary hemochromatosis (HHC) is a disorder of iron metabolism with variable penetrance. Oxidative stress plays a central role in the progression to cirrhosis. Several enzymes involved in the production or degradation of reactive oxidants, like myeloperoxidase (MPO) and heme oxygenase (HO)-1 are influenced by promotor polymorphisms. This study assessed the impact of polymorphisms of the MPO (?463G/A) and the HO-1 promotors of Vienna (GT)n on the evolution of cirrhosis in patients with HHC.
Methods One-hundred and fifty-eight C282Y homozygotes without cofactors for fibrosis progression (119 males; mean age: 51.0±13.3) were studied. All patients underwent liver biopsy. Hepatic iron content was measured by atom absorption spectrophotometry. MPO polymorphism was assessed by RFLP analysis; HO-1 microsatellite polymorphism by a laser-based semi-automated DNA sequencer.
Results The MPO genotypes GG, GA, and AA were found in 102 (64.6%), 45 and 11 patients, respectively. The GG-genotype was more common in patients with cirrhosis than in those without (78.7 vs. 55.7%, P=0.003). The distribution of HO-1 genotypes was not different. Logistic regression analysis revealed MPO genotype-GG, serum ferritin, age and male sex as independent predictors for cirrhosis.
Conclusions MPO genotype GG is associated with cirrhosis in patients with hereditary hemochromatosis.

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