Téléchargement sur votre Agenda de Poche avec Hepatoweb Edition Mobile

Mois de Septembre 2003

Archives depuis le 01/09/00

---

HEPATOLOGY

Table of Contents for September 2003 · Volume 38 · Number 3

Liver Failure and Liver Disease

Epidemiology of primary hepatic malignancies in U.S. children (*Human Study*)
Anil Darbari, Keith M. Sabin, Craig N. Shapiro, Kathleen B. Schwarz
The epidemiology of primary hepatic malignancies in U.S. children is poorly characterized. We analyzed the incidence, mortality, and characteristics of primary hepatic malignancies in U.S. residents less than 20 years of age. Fatal primary hepatic malignancies in persons less than 20 years of age, between 1979 and 1996, were identified using the multiple-cause-of-death database (National Center for Health Statistics). Histologically confirmed primary hepatic malignancies occurring between 1973 and 1997 were identified using the Surveillance, Epidemiology, and End Results (SEER) database. Between 1979 and 1996, 918 primary hepatic malignancy deaths (average, 0.7/1,000,000/year) were reported nationally among persons less than 20 years of age; rates were higher among Asians and among foreign-born children. Between 1973 and 1997, 271 primary hepatic malignancy cases were reported to SEER among persons less than 20 years of age, of which 184 (67%) and 83 (31%) were hepatoblastoma and hepatocellular carcinoma, respectively. Among children less than 5 years of age, hepatoblastoma accounted for 91% of primary hepatic malignancy cases, whereas among those 15 to 19 years of age, hepatocellular carcinoma accounted for 87% of cases. Five-year survival for hepatoblastoma was 52%, compared with 18% for hepatocellular carcinoma. In the SEER sites, between 1973 and 1977 and 1993 and 1997, hepatoblastoma rates increased (0.6 to 1.2/1,000,000, respectively), while hepatocellular carcinoma rates decreased (0.45 to 0.29/1,000,000, respectively). In conclusion, histologically confirmed hepatocellular carcinoma was reported in children less than 5 years of age, also, where hepatoblastoma is the predominant primary hepatic malignancy. Hepatocellular carcinoma has worse survival rates than hepatoblastoma, and its incidence has not increased. Better maintenance of databases may provide information about associated factors behind this unexpected occurrence. (HEPATOLOGY 2003;38:560-566.)

Altered expression of genes involved in hepatic morphogenesis and fibrogenesis are identified by cDNA microarray analysis in biliary atresia (*Human Study*)
Limin Chen, Andrew Goryachev, Jin Sun, Peter Kim, Hui Zhang, M. James Phillips, Pascale Macgregor, Sylvie Lebel, Aled M. Edwards, Qiongfang Cao, Katryn N. Furuya
Biliary atresia (BA) is characterized by a progressive, sclerosing, inflammatory process that leads to cirrhosis in infancy. Although it is the most common indication for liver transplantation in early childhood, little is known about its etiopathogenesis. To elucidate factors involved in this process, we performed comprehensive genome-wide gene expression analysis using complementary DNA (cDNA) microarrays. We compared messenger RNA expression levels of approximately 18,000 human genes from normal, diseased control, and end-stage BA livers. Reverse-transcription polymerase chain reaction (RT-PCR) and Northern blot analysis were performed to confirm changes in gene expression. Cluster and principal component analysis showed that all BA samples clustered together, forming a distinct group well separated from normal and diseased controls. We further identified 35 genes and ESTs whose expression differentiated BA from normal and diseased controls. Most of these genes are known to be associated with cell signaling, transcription regulation, hepatic development, morphogenesis, and fibrogenesis. In conclusion, this study serves to delineate processes that are involved in the pathogenesis of BA. (HEPATOLOGY 2003;38:567-576.)

Novel differential gene expression in human cirrhosis detected by suppression subtractive hybridization (*Human Study*)
Nicholas A. Shackel, Peter H. McGuinness, Catherine A. Abbott, Mark D. Gorrell, Geoffrey W. McCaughan
Pathogenic molecular pathways in cirrhotic liver diseases such as hepatitis C virus (HCV), autoimmune hepatitis (AIH) and primary biliary cirrhosis (PBC) are poorly characterized. Differentially expressed genes are often important in disease pathogenesis. Suppression subtractive hybridization (SSH) is a genome-wide approach that enriches for differentially expressed mRNA transcripts. We aimed to make novel observations of differential gene expression in cirrhosis using SSH combined with quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR). Liver transcriptomes in HCV cirrhosis, AIH cirrhosis, PBC, and nondiseased liver tissue were examined by SSH. Resulting complementary DNA (cDNA) clones were rescreened for differential expression by dot-blot hybridization and then sequenced. Selected gene expression was quantified by real-time RT-PCR. Following SSH, 694 clones were rescreened for differential gene expression, of which 145 were sequenced and found to derive from 89 different genes. Seven clones were homologous only with expressed sequence tag (EST) sequences encoding genes having no known function. Up-regulated expression of four genes was confirmed by real-time RT-PCR: transmembrane 4 superfamily member 3 (tetraspanin CO-029) in all forms of cirrhosis, hedgehog interacting protein (HIP) in AIH cirrhosis and chitinase 3-like-1 (HC gp-39 or ykl-40) and arginine-glutamic acid repeat (RERE) in HCV cirrhosis. RERE gene polymorphisms and splice variants were observed in all tissues examined. Tetraspanin CO-029 up-regulation was primarily localized to bile ductular cells. In conclusion, novel observations of differential gene expression in human cirrhosis were made using SSH as the primary discovery tool. In particular, further studies of the RERE gene and its products in HCV associated liver disease are warranted. (HEPATOLOGY 2003;38:577-588.)

Left ventricular hypertrophy in rats with biliary cirrhosis
Javier Inserte, Antonia Perelló, Luis Agulló, Marisol Ruiz-Meana, Klaus-Dieter Schlüter, Noelia Escalona, Mariona Graupera, Jaume Bosch, David Garcia-Dorado
Portal hypertension induces neuroendocrine activation and a hyperkinetic circulation state. This study investigated the consequences of portal hypertension on heart structure and function. Intrahepatic portal hypertension was induced in male Sprague-Dawley rats by chronic bile duct ligation (CBDL). Six weeks later, CBDL rats showed higher plasma angiotensin-II and endothelin-1 (P < .01), 56% reduction in peripheral resistance and 73% reduction in pulmonary resistance (P < .01), 87% increase in cardiac index and 30% increase in heart weight (P < .01), and increased myocardial nitric oxide (NO) synthesis. In CBDL rats, macroscopic analysis demonstrated a 30% (P < .01) increase in cross-sectional area of the left ventricular (LV) wall without changes in the LV cavity or in the right ventricle (RV). Histomorphometric analysis revealed increased cell width (12%, P < .01) of cardiomyocytes from the LV of CBDL rats, but no differences in myocardial collagen content. Myocytes isolated from the LV were wider (12%) and longer (8%) than right ventricular myocytes (P < .01) in CBDL rats but not in controls. CBDL rats showed an increased expression of ANF and CK-B genes (P < .01). Isolated perfused CBDL hearts showed pressure/end-diastolic pressure curves and response to isoproterenol identical to sham hearts, although generated wall tension was reduced because of the increased wall thickness. Coronary resistance was markedly reduced. This reduction was abolished by inhibition of NO synthesis with N-nitro-L-arginine. Expression of eNOS was increased in CBDL hearts. In conclusion, portal hypertension associated to biliary cirrhosis induces marked LV hypertrophy and increased myocardial NO synthesis without detectable fibrosis or functional impairment. This observation could be relevant to patients with cirrhosis. (HEPATOLOGY 2003;38:589-598.)

Upper digestive bleeding in cirrhosis. Post-therapeutic outcome and prognostic indicators (*Human Study*)
Gennaro D'Amico, Roberto De Franchis, Cooperative Study Group
Several treatments have been proven to be effective for variceal bleeding in patients with cirrhosis. The aim of this multicenter, prospective, cohort study was to assess how these treatments are used in clinical practice and what are the posttherapeutic prognosis and prognostic indicators of upper digestive bleeding in patients with cirrhosis. A training set of 291 and a test set of 174 bleeding cirrhotic patients were included. Treatment was according to the preferences of each center and the follow-up period was 6 weeks. Predictive rules for 5-day failure (uncontrolled bleeding, rebleeding, or death) and 6-week mortality were developed by the logistic model in the training set and validated in the test set. Initial treatment controlled bleeding in 90% of patients, including vasoactive drugs in 27%, endoscopic therapy in 10%, combined (endoscopic and vasoactive) in 45%, balloon tamponade alone in 1%, and none in 17%. The 5-day failure rate was 13%, 6-week rebleeding was 17%, and mortality was 20%. Corresponding findings for variceal versus nonvariceal bleeding were 15% versus 7% (P = .034), 19% versus 10% (P = .019), and 20% versus 15% (P = .22). Active bleeding on endoscopy, hematocrit levels, aminotransferase levels, Child-Pugh class, and portal vein thrombosis were significant predictors of 5-day failure; alcohol-induced etiology, bilirubin, albumin, encephalopathy, and hepatocarcinoma were predictors of 6-week mortality. Prognostic reassessment including blood transfusions improved the predictive accuracy. All the developed prognostic models were superior to the Child-Pugh score. In conclusion, prognosis of digestive bleeding in cirrhosis has much improved over the past 2 decades. Initial treatment stops bleeding in 90% of patients. Accurate predictive rules are provided for early recognition of high-risk patients. (HEPATOLOGY 2003;38:599-612.)

Viral Hepatitis
Viral and clinical factors associated with the fulminant course of hepatitis A infection (*Human Study*)
Guilhermo Rezende, Anne Marie Roque-Afonso, Didier Samuel, Michele Gigou, Elisabeth Nicand, Virginie Ferre, Elisabeth Dussaix, Henri Bismuth, Cyrille Féray
Fulminant hepatitis is a severe complication of hepatitis A virus infection. Its mechanism is unknown. Liver transplantation can be necessary, but spontaneous recovery is frequent. There are no data on the level of viral replication according to the clinical form of hepatitis A. We reviewed the files of 50 patients with acute hepatitis A. Nineteen patients had fulminant hepatitis (defined by encephalopathy and factor V <50%), and, from them, 10 patients underwent transplantation. Hepatitis A virus (HAV) RNA was quantified by real-time PCR on sera obtained at admission. The genotype was determined by phylogenetic analysis of HAV RNA. HAV RNA was detected in serum by RT-PCR in 39 out of 50 patients. Encephalopathy and low factor V level were significantly related to female gender, HAV PCR negativity (9/19 vs. 5/31, respectively; P = .03), a low serum HAV RNA level (log, 3.6 ± 0.6 vs. 4.4 ± 0.9, respectively; P = .02), genotypes other than IA, and acetaminophen intake. In multivariate analysis, low or undetectable HAV viral load and a high bilirubin level were independently associated with both low factor V levels and fulminant hepatitis and also with death or transplantation. In conclusion, HAV-related liver failure is due to an excessive host response associated with a marked reduction in viral load. Serum HAV RNA assay could be of help in the management of severe hepatitis A. (HEPATOLOGY 2003;38:613-618.)

Prevalence of HBV precore/core promoter variants in the United States (*Human Study*)
Chi-Jen Chu, Emmet B. Keeffe, Steven-Huy Han, Robert P. Perrillo, Albert D. Min, Consuelo Soldevila-Pico, William Carey, Robert S. Brown, Jr., Velimir A. Luketic, Norah Terrault, Anna S. F. Lok, The U.S. HBV Epidemiology Study Group
Variants in the precore (G1896A) and core promoter (A1762T, G1764A) regions of hepatitis B virus (HBV) may be related to serum HBV DNA levels and severity of liver disease. The aims of this nationwide study were to determine the prevalence of HBV precore/core promoter variants in the United States and the association between these variants and patient demographics, HBV genotypes, serum HBV DNA level, and severity of liver disease. A total of 694 consecutive chronic HBV-infected patients seen in 17 U.S. liver centers during a 1-year period were enrolled. Demographic, clinical, and laboratory data were collected. Sera were tested for HBV genotypes as well as precore and core promoter variants by line-probe assays. Quantitative HBV DNA levels were determined using Cobas Amplicor HBV Monitor kits. Precore and core promoter variants were found in 27% and 44% of patients with chronic HBV infection in the United States. Precore and core promoter variants were more common in hepatitis B e antigen (HBeAg)-negative than in HBeAg-positive patients (precore, 38% vs. 9%; core promoter, 51% vs. 36%; respectively, P < .001). The prevalence of these variants was related to ethnicity, place of birth, and HBV genotypes. Patients with core promoter variants were more likely to have hepatic decompensation. Precore and/or core promoter variants were associated with higher serum HBV DNA levels in HBeAg-negative but not in HBeAg-positive patients. In conclusion, HBV precore and core promoter variants are not rare in the United States. Physicians should be aware of the existence of HBV precore and core promoter variants and the clinical condition of "HBeAg-negative chronic hepatitis." (HEPATOLOGY 2003;38:619-628.)

Modulation of the outcome and severity of hepadnaviral hepatitis in woodchucks by antibodies to hepatic asialoglycoprotein receptor
Jingyu Diao, Darlene M. Slaney, Tomasz I. Michalak
Viral hepatitis is frequently accompanied by humoral autoimmune responses toward both organ-nonspecific and liver-specific antigens, but contribution of these reactivities to liver injury remains unrecognized. Infection with woodchuck hepatitis virus (WHV) has been identified as a potent inducer of autoantibodies against asialoglycoprotein receptor (anti-ASGPR), a molecule essentially unique to hepatocytes that mediates clearance of desialylated serum proteins. In this study, we applied the WHV-woodchuck model of hepatitis B to examine the effect of experimentally elicited anti-ASGPR on the progression and the severity of WHV hepatitis in initially healthy animals immunized with the receptor and then infected with WHV and in woodchucks with ongoing chronic WHV hepatitis. The results implied that the induction of anti-ASGPR prior to WHV infection tends to modulate acute viral hepatitis toward chronic outcome and, in animals with established chronic WHV infection, exacerbates histologic severity of liver lesions. The findings also suggest that the liver compromised by chronic hepadnavirus infection might be prone to anti-ASGPR-directed complement-mediated hepatocellular injury and that this is associated with formation of the ASGPR-anti-ASGPR immune complexes on hepatocyte surface. In conclusion, the host's immune response mounted against a hepatocyte-specific autoantigen may modulate both the outcome and the severity of liver injury in viral hepatitis. (HEPATOLOGY 2003;38:629-638.)

High body mass index is an independent risk factor for nonresponse to antiviral treatment in chronic hepatitis C (*Human Study*)
Brian L. Bressler, Maha Guindi, George Tomlinson, Jenny Heathcote
The aim of this study was to determine if body mass index (BMI) was an independent predictor of response to antiviral treatment in patients with chronic hepatitis C. A retrospective review was performed of all patients at a single center with chronic hepatitis C treated with antiviral medication from 1989 to 2000. A sustained response was defined as either negative hepatitis C virus (HCV) RNA by polymerase chain reaction and/or normal alanine aminotransferase (ALT) level (only in those treated before availability of HCV RNA testing) 6 months following completion of therapy. All patients were classified into one of 3 groups according to BMI (normal, <25 kg/m2; overweight, 25-30 kg/m2; obese, >30 kg/m2). A total of 253 patients were treated with either interferon (IFN) monotherapy or IFN in combination with ribavirin. Patients were excluded if predetermined clinical characteristics were unavailable. Using logistic regression, and after adjusting for the examined variables (age, sex, history of alcohol consumption >50 g/d, cirrhosis on pretreatment biopsy, and BMI), likelihood ratio tests showed significant differences in response to treatment according to BMI group (P = .01), genotype (P < .01), and cirrhosis (P < .01). Those with genotypes 2 or 3 had an odds ratio (OR) for success of 11.7 compared with those with genotype 1, cirrhotic patients had an OR of 0.15 compared with noncirrhotic patients, and obese patients had an OR of 0.23 compared with normal and overweight patients. Hepatic steatosis was not an independent risk factor for response to antiviral treatment. In conclusion, obesity, only when defined as a BMI greater than 30 kg/m2, is an independent (of genotype and cirrhosis) negative predictor of response to hepatitis C treatment. (HEPATOLOGY 2003;38:639-644.)

Early virologic response to treatment with peginterferon alfa-2b plus ribavirin in patients with chronic hepatitis C (*Human Study*)
Gary L. Davis, John B. Wong, John G. McHutchison, Michael P. Manns, Joann Harvey, Janice Albrecht
Interferon-based regimens for the treatment of chronic hepatitis C have become increasingly effective and are able to eradicate virus in more than one half of cases. Early identification of patients who will not respond is desirable because treatment might be stopped, thereby avoiding the expense and inconvenience of unnecessary therapy. We examined the accuracy of different degrees of viral inhibition during the early weeks of treatment (early virologic response [EVR]) with pegylated interferon alfa-2b and ribavirin (PEG/R) in identifying patients who would not respond to therapy. The best definition of EVR was a reduction in hepatitis C virus (HCV) RNA by at least 2 logs after the first 12 weeks of treatment compared with baseline. Between 69% and 76% of patients achieved this threshold, depending on the treatment regimen, and sustained virologic response (SVR) occurred in 67% to 80% of these patients. Patients who did not reach EVR did not respond to further therapy. If treatment had been stopped in patients without EVR, drug costs would have been reduced by more than 20%. In conclusion, early confirmation of viral reduction following initiation of antiviral therapy for chronic hepatitis C is worthwhile. It provides a goal to motivate adherence during the first months of therapy and a milepost at which to reassess the need for continued treatment. Most patients who are able to complete the first 12 weeks of therapy achieve EVR and have a high probability of SVR. Patients who fail to achieve EVR will not clear virus even if an additional 9 months of therapy is received. Therapy can be confidently discontinued in those cases. (HEPATOLOGY 2003;38:645-652.)

Antibody-selected mimics of hepatitis C virus hypervariable region 1 activate both primary and memory Th lymphocytes (*Human Study*)
Loredana Frasca, Cristiano Scottà, Paola Del Porto, Alfredo Nicosia, Caterina Pasquazzi, Ilaria Versace, Anna Maria Masci, Luigi Racioppi, Enza Piccolella
An ideal strategy that leads to a vaccine aimed at controlling viral escape may be that of preventing the replication of escape mutants by eliciting a T- and B-cell repertoire directed against many viral variants. The hypervariable region 1 (HVR1) of the putative envelope 2 protein that presents B and T epitopes shown to induce protective immunity against hepatitis C virus (HCV), might be suitable for this purpose if its immunogenicity can be improved by generating mimics that induce broad, highly cross-reactive, anti-HVR1 responses. Recently we described a successful approach to select HVR1 mimics (mimotopes) incorporating the variability found in a great number of viral variants. In this report we explore whether these mimotopes, designed to mimic B-cell epitopes, also mimic helper T-cell epitopes. The first interesting observation is that mimotopes selected for their reactivity to HVR1-specific antibodies of infected patients also do express HVR1 T-cell epitopes, suggesting that similar constraints govern HVR1-specific humoral and cellular immune responses. Moreover, some HVR1 mimotopes stimulate a multispecific CD4+ T-cell repertoire that effectively cross-reacts with HVR1 native sequences. This may significantly limit effects as a T-cell receptor (TCR) antagonist frequently exerted by natural HVR1-variants on HVR1-specific T-cell responses. In conclusion, these data lend strong support to using HVR1 mimotopes in vaccines designed to prevent replication of escape mutants. (HEPATOLOGY 2003;38:653-663.)

Liver Biology and Pathobiology

Sympathetic nervous system inhibition increases hepatic progenitors and reduces liver injury
Jude A. Oben, Tania Roskams, Shiqi Yang, Huizhi Lin, Nicoletta Sinelli, Zhiping Li, Michael Torbenson, Jiawen Huang, Paul Guarino, Michel Kafrouni, Anna Mae Diehl
Recovery from liver damage might be enhanced by encouraging repopulation of the liver by endogenous hepatic progenitor cells. Oval cells are resident hepatic stem cells that promote liver regeneration and repair. Little is known about the mediators that regulate the accumulation of these cells in the liver. Parasympathetic nervous system inhibition reduces the number of oval cells in injured livers. The effect of sympathetic nervous system (SNS) inhibition on oval cell number is not known. Adrenergic inhibition mobilizes hematopoietic precursors into the circulation and has also been shown to promote liver regeneration. Thus, we hypothesized that SNS inhibition would promote hepatic accumulation of oval cells and reduce liver damage in mice fed antioxidant-depleted diets to induce liver injury. Our results confirm this hypothesis. Compared with control mice that were fed only the antioxidant-depleted diets, mice fed the same diets with prazosin (PRZ, an -1 adrenoceptor antagonist) or 6-hydroxydopamine (6-OHDA, an agent that induces chemical sympathectomy) had significantly increased numbers of oval cells. Increased oval cell accumulation was accompanied by less hepatic necrosis and steatosis, lower serum aminotransferases, and greater liver and whole body weights. Neither PRZ nor 6-OHDA affected the expression of cytokines, growth factors, or growth factor receptors that are known to regulate progenitor cells. In conclusion, stress-related sympathetic activity modulates progenitor cell accumulation in damaged livers and SNS blockade with -adrenoceptor antagonists enhances hepatic progenitor cell accumulation. (HEPATOLOGY 2003;38:664-673.)

Interleukin 6 is important for survival after partial hepatectomy in mice
Alex Blindenbacher, Xueya Wang, Igor Langer, Rocco Savino, Luigi Terracciano, Markus H. Heim
The response to partial hepatectomy (PH) is impaired in interleukin 6 (IL-6)-deficient mice. IL-6 is well known for its role in the induction of the acute phase (AP) response, and the impairment of this response after surgery and hepatectomy could explain the defective hepatocyte regeneration. In addition, it was proposed that IL-6 has an important role in stimulating the reentry of quiescent cells into the cell cycle within the first 2 to 4 hours after PH. To further analyze the role for IL-6, we performed two third hepatectomies in wild-type mice, in IL-6 knockout (KO) mice, and in IL-6 KO mice that were treated 30 minutes before surgery with intravenous (IV) (short acting) or subcutaneous (SC) (long acting) injections of recombinant IL-6. The high postoperative mortality of IL-6-deficient mice could be completely prevented by SC, but not by IV IL-6 treatment, showing the requirement of a sustained action of IL-6. However, there is a subset of IL-6 KO mice that survives a PH in good health even without IL-6 treatment. When we analyzed these mice, we found an intact liver regeneration and no indication of a block in cell cycle reentry. We conclude that the major role of IL-6 is the induction of an adaptive response to PH that ensures body homeostasis and survival. (HEPATOLOGY 2003;38:674-682.)

Timing and sequence of differentiation of embryonic rat hepatocytes along the biliary epithelial lineage
Robbert G. E. Notenboom, Marius A. van den Bergh Weerman, Koert P. Dingemans, Jacqueline L. M. Vermeulen, Stefan van den Eijnde, Chris P. Reutelingsperger, Hans Hut, Rob Willemsen, G. Johan A. Offerhaus, Wouter H. Lamers
To study the differentiation of hepatocytes along the biliary epithelial lineage in vivo, embryonic day 14 (E14) rat hepatocytes were isolated by differential centrifugation and transplanted as single-cell suspensions into the spleen of adult syngeneic rats. Hepatocytes and cholangiocytes were identified and their maturation characterized by the level of expression of -fetoprotein (AFP), glutamate dehydrogenase (GDH), and carbamoyl phosphate synthetase I (CPS); annexin IV, annexin V, cytokeratin 19 (CK-19), and cystic fibrosis transmembrane conductance regulator (CFTR); and electron microscopy. By correlating morphologic changes with the timing in the expression of these markers, we show that the organization of the transplanted E14 hepatocytes into lobular structures is accompanied by the formation and maturation of bile ducts around these developing lobules. Morphologic differentiation of the emerging bile ducts was accompanied by a gradual loss of hepatocyte markers and a gradual acquisition of cholangiocyte markers, with markers identifying a large-cholangiocyte phenotype appearing latest. Once fully differentiated, the intrasplenic liver lobules developed cholestatic features. The accompanying proliferation of bile ducts was due to cholangiocyte proliferation, but ductular transformation of hepatocytes was also observed. In conclusion, (1) bile duct formation at the interface between hepatocytes and connective tissue is an inherent component of liver development and (2) the susceptibility of developing hepatocytes to bile duct-inducing signals is highest in the fetal liver but that (3) this capacity is not irreversibly lost in otherwise mature hepatocytes. (HEPATOLOGY 2003;38:683-691.)

Sensitivity of the 2-oxoglutarate carrier to alcohol intake contributes to mitochondrial glutathione depletion
Olga Coll, Anna Colell, Carmen García-Ruiz, Neil Kaplowitz, J. C. Fernández-Checa
The mitochondrial pool of reduced glutathione (mGSH) is known to play a protective role against liver injury and cytokine-mediated cell death. However, the identification of the mitochondrial carriers involved in its transport in hepatocellular mitochondria remains unestablished. In this study, we show that the functional expression of the 2-oxoglutarate carrier from HepG2 cells in mitochondria from Xenopus laevis oocytes conferred a reduced glutathione (GSH) transport activity that was inhibited by phenylsuccinate, a specific inhibitor of the carrier. In addition, the mitochondrial transport of GSH and 2-oxoglutarate in isolated mitochondria from rat liver exhibited mutual competition and sensitivity to glutamate and phenylsuccinate. Interestingly, the kinetics of 2-oxoglutarate transport in rat liver mitochondria displayed a single Michaelis-Menten component with a Michaelis constant of 3.1 ± 0.3 mmol/L and maximum velocity of 1.9 ± 0.1 nmol/mg protein/25 seconds. Furthermore, the initial rate of 2-oxoglutarate was reduced in mitochondria from alcohol-fed rat livers, an effect that was not accompanied by an alcohol-induced decrease in the 2-oxoglutarate messenger RNA levels but rather by changes in mitochondrial membrane dynamics induced by alcohol. The fluidization of mitochondria by the fluidizing agent 2-(2-methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl) (A2C) restored the initial transport rate of both GSH and 2-oxoglutarate. Finally, these changes were reproduced in normal liver mitochondria enriched in cholesterol where the fluidization of cholesterol-enriched mitochondria with A2C restored the order membrane parameter and the mitochondrial 2-oxoglutarate uptake. In conclusion, these findings provide unequivocal evidence for 2-oxoglutarate as a GSH carrier and its sensitivity to membrane dynamics perturbation contributes in part to the alcohol-induced mGSH depletion. (HEPATOLOGY 2003;38:692-702.)

Potential role of PTEN phosphatase in ethanol-impaired survival signaling in the liver
Jong Eun Yeon, Sophia Califano, Julia Xu, Jack R. Wands, Suzanne M. De La Monte
Chronic ethanol consumption can cause sustained hepatocellular injury and inhibit the subsequent regenerative response. These effects of ethanol may be mediated by impaired hepatocyte survival mechanisms. The present study examines the effects of ethanol on survival signaling in the intact liver. Adult Long Evans rats were maintained on ethanol-containing or isocaloric control liquid diets for 8 weeks, after which the livers were harvested to measure mRNA levels, protein expression, and kinase or phosphatase activity related to survival or proapoptosis mechanisms. Chronic ethanol exposure resulted in increased hepatocellular labeling for activated caspase 3 and nuclear DNA damage as demonstrated using the TUNEL assay. These effects of ethanol were associated with reduced levels of tyrosyl phosphorylated (PY) IRS-1 and PI3 kinase, Akt kinase, and Erk MAPK activities and increased levels of phosphatase tensin homologue deleted on chromosome 10 (PTEN) mRNA, protein, and phosphatase activity in liver tissue. In vitro experiments demonstrated that ethanol increases PTEN expression and function in hepatocytes. However, analysis of signaling cascade pertinent to PTEN function revealed increased levels of nuclear p53 and Fas receptor mRNA but without corresponding increases in GSK-3 activity or activated BAD. Although fork-head transcription factor levels were increased in ethanol-exposed livers, virtually all of the fork-head protein detected by Western blot analysis was localized within the cytosolic fraction. In conclusion, chronic ethanol exposure impairs survival mechanisms in the liver because of inhibition of signaling through PI3 kinase and Akt and increased levels of PTEN. However, uncoupling of the signaling cascade downstream of PTEN that mediates apoptosis may account for the relatively modest degrees of ongoing cell loss observed in livers of chronic ethanol-fed rats. (HEPATOLOGY 2003;38:703-714.)

Tacrine inhibits topoisomerases and DNA synthesis to cause mitochondrial DNA depletion and apoptosis in mouse liver
Abdellah Mansouri, Delphine Haouzi, Véronique Descatoire, Christine Demeilliers, Angela Sutton, Nathalie Vadrot, Bernard Fromenty, Gérard Feldmann, Dominique Pessayre, Alain Berson
After several weeks of treatment, levels of alanine aminotransferase (ALT) increase in 50% of patients treated with tacrine for Alzheimer's disease. We looked for progressive effects on DNA to explain delayed toxicity. We first studied the in vitro effects of tacrine on DNA replication and topoisomerase-mediated DNA relaxation. We then treated mice with doses of tacrine reproducing the human daily dose on a body area basis and studied the effects of tacrine administration for up to 28 days on hepatic DNA, mitochondrial function, and cell death. In vitro, tacrine impaired DNA polymerase -mediated DNA replication and also poisoned topoisomerases I and II to increase the relaxation of a supercoiled plasmid. In vivo, administration of tacrine markedly decreased incorporation of [3H]thymidine into mitochondrial DNA (mtDNA), progressively and severely depleted mtDNA, and partly unwound supercoiled mtDNA into circular mtDNA. Incorporation of [3H]thymidine into nuclear DNA (nDNA) was barely decreased, and nDNA levels were unchanged. After 12 to 28 days of treatment, administration of tacrine increased p53, Bax, mitochondrial permeability transition, cytosolic cytochrome c, and caspase-3 activity and triggered hepatocyte apoptosis and/or necrosis. In conclusion, the intercalating drug tacrine poisons topoisomerases and impairs DNA synthesis. Tacrine has been shown to accumulate within mitochondria, and it particularly targets mtDNA. After several weeks of treatment, the combination of severe mtDNA depletion and a genotoxic stress enhancing p53, Bax, and permeability transition trigger hepatocyte necrosis and/or apoptosis. (HEPATOLOGY 2003;38:715-725.)

Biliary lipid secretion, bile acid metabolism, and gallstone formation are not impaired in hepatic lipase-deficient mice
Ludwig Amigo, Pablo Mardones, Carla Ferrada, Silvana Zanlungo, Flavio Nervi, Juan Francisco Miquel, Attilio Rigotti
Whereas hepatic lipase (HL) has been implicated in lipoprotein metabolism and atherosclerosis, its role in controlling biliary lipid physiology has not been reported. This work characterizes plasma lipoprotein cholesterol, hepatic cholesterol content, bile acid metabolism, biliary cholesterol secretion, and gallstone formation in HL-deficient mice and C57BL/6 controls fed standard chow, a cholesterol-supplemented diet, or a lithogenic diet. Compared with C57BL/6 controls, HL knockout mice exhibited increased basal plasma high-density lipoprotein (HDL) cholesterol as well as reduced cholesterol levels transported in large lipoproteins in response to cholesterol-enriched diets. Hepatic cholesterol content and biliary cholesterol secretion of chow-fed HL knockout and wild-type mice were not different and increased similarly in both strains after feeding dietary cholesterol or a lithogenic diet. There were no differences in biliary bile acid secretion, bile acid pool size and composition, or fecal bile acid excretion between HL-deficient and control mice. HL knockout mice had a similar prevalence of gallstone formation as compared with control mice when both strains were fed with a lithogenic diet. In conclusion, the deficiency of HL has no major impact on the availability of lipoprotein-derived hepatic cholesterol for biliary secretion; HL expression is not essential for diet-induced gallstone formation in mice. (HEPATOLOGY 2003;38:726-734.)

A novel liver-specific zona pellucida domain containing protein that is expressed rarely in hepatocellular carcinoma
Zhi-Gang Xu, Jian-Jun Du, Xin Zhang, Zhi-Hong Cheng, Zhen-Zhong Ma, Hua-Sheng Xiao, Li Yu, Zhi-Qin Wang, Yu-Yang Li, Ke-Ke Huo, Ze-Guang Han
We currently identified a liver-specific gene that encodes a novel zona pellucida (ZP) domain-containing protein named liver-specific ZP domain-containing protein (LZP). The full-length complementary DNA (cDNA) of human LZP has 2,255 bp with a complete open reading frame (ORF) of 1,635 bp. The gene is localized on chromosome 10q21.3 and spans 40 kb with 9 encoding exons and 8 introns. The deduced protein sequence has 545 amino acid residues, with an N-terminal signal peptide followed by 3 epidermal growth factor (EGF)-like domains and a ZP domain in C-terminal section. Interestingly, human LZP is expressed specifically in liver out of 23 tissues examined, and its mouse counterpart was detected at very early stage during embryo development. Moreover, LZP can be secreted into blood, albeit the protein was localized mainly on the nuclear envelop of hepatocytes. Most importantly, LZP is down-regulated in hepatocellular carcinoma (HCC) and HCC cell lines; meanwhile, the decreased level of hLZP messenger RNA (mRNA) could, at least in some HCC samples, be related to the methylation status of the putative LZP promoter. However, overexpression of hLZP in HCC cell line SMMC-7721 and human liver cell line L02 by stable cell transfection did not inhibit cell growth, implying that the down-regulation of hLZP in HCC might be a consequence of the dedifferentiation involved in hepatocarcinogenesis. In conclusion, these data suggest that LZP is a liver-specific protein involved possibly in hepatocellular function and development, and the protein could be used as potential negative biomarker for HCC pathologic diagnosis. (HEPATOLOGY 2003;38:735-744.)

Transcriptional regulation of the human transferrin gene by GADD153 in hepatoma cells (*Human Study*)
Kyung-Ran You, Ming-Jie Liu, Xue-Ji Han, Zee-Won Lee, Dae-Ghon Kim
The transcription factor CHOP/GADD153 is reportedly induced by cellular stresses such as UV light, genotoxic agents, and protein misfolding in the endoplasmic reticulum. However, the mechanism whereby induction of the GADD153 gene is linked to a downstream pathway is still unclear. Previously, we observed that a synthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR) effectively impaired cell growth and survival (induction of growth arrest and apoptosis) in human hepatoma cells, which was accompanied by over expression of GADD153. Furthermore, GADD153-transfected Hep 3B cells were growth arrested and were sensitized to drug-induced apoptosis. Thus, in this study, we used suppression subtractive hybridization (SSH) to identify GADD153 target genes that were up-regulated or down-regulated in the GADD153 transfectants. We screened 614 sequence-verified clones by Northern blotting, of which 42 genes were scored as over expressed and 17 genes as under expressed in GADD153 transfectants compared with control vector transfectants. Of those genes, 49 corresponded to known genes in public databases. Among them, we further verified that the expression of transferrin (Tf), which is a negative acute-phase protein and is essential to cell survival as a growth factor, was highly modulated by drug-induced GADD153 over expression or by in vitro transfection. GADD153 significantly antagonized the C/EBP (C/EBP-, -, and -)-mediated transcriptional activation of the Tf gene. In conclusion, Tf and other target genes identified may play a functional role in the downstream pathway of GADD153. (HEPATOLOGY 2003;38:745-755.)

Cyclooxygenase-2 promotes hepatocellular carcinoma cell growth through AKT activation: Evidence for AKT inhibition in celecoxib-induced apoptosis
Jing Leng, Chang Han, A. Jake Demetris, George K. Michalopoulos, Tong Wu
Cyclooxygenase-2 (COX-2)-controlled prostaglandin (PG) metabolism recently has been implicated in the pathogenesis of hepatocellular carcinoma (HCC). However, the biologic role and molecular mechanism of COX-2-mediated PGs in the control of liver cancer growth have not been established. This study was designed to examine the direct effect of COX-2 and its inhibitor celecoxib on the growth control of liver cancer cells. Human HCC cell lines Hep3B and HepG2 transfected with COX-2 expression vector showed increased cell growth and enhanced phosphorylation of serine/threonine protein kinase B (Akt). The level of COX-2 expression and Akt phosphorylation is correlated positively in cultured HCC cells and human liver cancer tissues. Inhibition of Akt activation by phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY294002 significantly decreased the viability of Hep3B and HepG2 cells (P < .01). These results reveal a novel role of Akt activation in COX-2-induced HCC cell survival. Furthermore, HCC cells treated with the COX-2 inhibitor celecoxib showed significant reduction of Akt phosphorylation and marked morphologic and biochemical characteristics of apoptosis. Overexpression of COX-2 or addition of exogenous PGE2 partially prevented celecoxib-induced apoptosis (P < .01). In conclusion, our results suggest the involvement of COX-2-dependent and -independent mechanisms in celecoxib-mediated HCC cell apoptosis. (HEPATOLOGY 2003;38:756-768.)

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

GASTROENTEROLOGY

Table of Contents for September 2003 · Volume 125 · Number 3

Rapid Communication

Corticotropin-releasing factor receptor 1-deficient mice do not develop postoperative gastric ileus
Andrew Luckey, Lixin Wang, Pauline M. Jamieson, Nicole R. Basa, Mulugeta Million, Jozsef Czimmer, Wylie Vale, Yvette Taché
Background & Aims: Corticotropin-releasing factor (CRF) signaling pathways play a key role in the stress response through the activation of CRF1 and CRF2 receptors. We investigated the CRF receptor subtypes involved in gastric postoperative ileus. Methods: Adult male mice (C57BL/6, CRF1-deficient, and wild-type), fasted for 16­18 hours, were anesthetized for 10 minutes and had a midline celiotomy and cecal exteriorization and palpation for 30 or 60 seconds or no surgery (sham). Phenol red was given by gavage 100 minutes after anesthesia; 20 minutes later, gastric emptying and blood glucose level were measured. Results: In C57BL/6 mice, cecal palpation for 30 or 60 seconds significantly reduced gastric emptying to 30.3% ± 1.4% and 5.8% ± 3.4%, respectively, compared with 58.5% ± 4.4% in sham. The CRF1 antagonist CP-154,526 (20 mg/kg subcutaneously) completely prevented the 30-second cecal palpation-induced delayed gastric emptying (53.0% ± 7.9% vs. 28.0% ± 4.0% in vehicle + surgery), whereas the CRF2 antagonist astressin2-B injected subcutaneously had no effect. In CRF1-deficient mice, cecal palpation for 30 seconds did not delay gastric emptying (80.3% ± 4.5% compared with 84.7% ± 6.3% in sham); in wild-type mice, gastric emptying was decreased to 17.8% ± 16.1% (P < 0.05 vs. sham 72.0% ± 12.4%). Surgery increased glucose levels by 46% compared with sham in wild-type mice, while glycemia was not altered in CRF1-deficient mice. Basal emptying was similar in wild-type and CRF1-deficient mice and not influenced by CRF antagonists in C57BL/6 mice. Conclusions: These data show that CRF1 activation plays an important role in mediating the early phase of gastric ileus.

Gain-of-function mutations of platelet-derived growth factor receptor gene in gastrointestinal stromal tumors
Seiichi Hirota, Akiko Ohashi, Toshirou Nishida, Koji Isozaki, Kazuo Kinoshita, Yasuhisa Shinomura, Yukihiko Kitamura
Background & Aims: Most gastrointestinal stromal tumors (GISTs) have gain-of-function mutations of c-kit receptor tyrosine kinase (KIT) gene, but some GISTs do not. We investigated the cause of GISTs without KIT mutations. Because GISTs apparently expressed platelet-derived growth factor receptor (PDGFR) , we examined whether GISTs without KIT mutations had a mutation of PDGFR . Methods: Whole coding region of PDGFR complementary DNA (cDNA) was sequenced in GISTs with or without KIT mutations. Mutant PDGFR cDNA was transfected into 293T human embryonic kidney cells, and autophosphorylation of PDGFR was examined. Proliferation of Ba/F3 murine lymphoid cells stably transfected with mutant PDGFR cDNA was estimated by tritium thymidine incorporation. Wild-type KIT cDNA was cotransfected with mutant PDGFR cDNA, and immunoprecipitation by anti-KIT antibody was performed. Inhibitory effect of Imatinib mesylate on activated PDGFR was examined. Results: We found 2 types of constitutively activated mutations of PDGFR , Val-561 to Asp or Asp-842 to Val, in 5 of 8 GISTs without KIT mutations but not in 10 GISTs with KIT mutations. Stable transfection of each mutation induced autonomous proliferation of Ba/F3 cells. Constitutively activated mutant PDGFR bound and activated the cotransfected wild-type KIT. The constitutive activation of PDGFR with Val-561 to Asp was inhibited effectively by Imatinib mesylate but that of PDGFR with Asp-842 to Val was inhibited only weakly, even at the concentration of 10 µmol/L. Conclusions: The gain-of-function mutations of PDGFR appear to play an important role in development of GISTs without KIT mutations.

Clinical-alimentary Tract

Improvement of gastroesophageal reflux symptoms after radiofrequency energy: A randomized, sham-controlled trial
Douglas A. Corley, Philip Katz, John M. Wo, Andreas Stefan, Marco Patti, Richard Rothstein, Steven Edmundowicz, Michael Kline, Rodney Mason, M.Michael Wolfe
Background & Aims: Gastroesophageal reflux disease is a prevalent disorder that often requires long-term medical therapy or surgery. The United States Food and Drug Administration recently cleared new endoluminal gastroesophageal reflux disease treatments; however, no controlled trials exist. Methods: We randomly assigned 64 gastroesophageal reflux disease patients to radiofrequency energy delivery to the gastroesophageal junction (35 patients) or to a sham procedure (29 patients). Principal outcomes were reflux symptoms and quality of life. Secondary outcomes were medication use and esophageal acid exposure. After 6 months, interested sham patients crossed over to active treatment. Results: At 6 months, active treatment significantly and substantially improved patients' heartburn symptoms and quality of life. More active vs. sham patients were without daily heartburn symptoms (n = 19 [61%] vs. n = 7 [33%]; P = 0.05), and more had a >50% improvement in their gastroesophageal reflux disease quality of life score (n = 19 [61%] vs. n = 6 [30%]; P = 0.03). Symptom improvements persisted at 12 months after treatment. At 6 months, there were no differences in daily medication use after a medication withdrawal protocol (n = 17 [55%] vs. n = 14 [61%]; P = 0.67) or in esophageal acid exposure times. There were no perforations or deaths. Conclusions: Radiofrequency energy delivery significantly improved gastroesophageal reflux disease symptoms and quality of life compared with a sham procedure, but it did not decrease esophageal acid exposure or medication use at 6 months. This procedure represents a new option for selected symptomatic gastroesophageal reflux disease patients who are intolerant of, or desire an alternative to, traditional medical therapies.

Possible endocannabinoid control of colorectal cancer growth
Alessia Ligresti, Tiziana Bisogno, Isabel Matias, Luciano De Petrocellis, Maria Grazia Cascio, Vittorio Cosenza, Giuseppe D'argenio, Giuseppe Scaglione, Maurizio Bifulco, Italo Sorrentini, Vincenzo Di Marzo
Background & Aims: The endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG) inhibit cancer cell proliferation by acting at cannabinoid receptors (CBRs). We studied (1) the levels of endocannabinoids, cannabinoid CB1 and CB2 receptors, and fatty acid amide hydrolase (FAAH, which catalyzes endocannabinoid hydrolysis) in colorectal carcinomas (CRC), adenomatous polyps, and neighboring healthy mucosa; and (2) the effects of endocannabinoids, and of inhibitors of their inactivation, on human CRC cell proliferation. Methods: Tissues were obtained from 21 patients by biopsy during colonoscopy. Endocannabinoids were measured by liquid chromatography-mass spectrometry (LC-MS). CB1, CB2, and FAAH expression were analyzed by RT-PCR and Western immunoblotting. CRC cell lines (CaCo-2 and DLD-1) were used to test antiproliferative effects. Results: All tissues and cells analyzed contain anandamide, 2-AG, CBRs, and FAAH. The levels of the endocannabinoids are 3- and 2-fold higher in adenomas and CRCs than normal mucosa. Anandamide, 2-AG, and the CBR agonist HU-210 potently inhibit CaCo-2 cell proliferation. This effect is blocked by the CB1 antagonist SR141716A, but not by the CB2 antagonist SR144528, and is mimicked by CB1-selective, but not CB2-selective, agonists. In DLD-1 cells, both CB1 and CB2 receptors mediate inhibition of proliferation. Inhibitors of endocannabinoid inactivation enhance CaCo-2 cell endocannabinoid levels and block cell proliferation, this effect being antagonized by SR141716A. CaCo-2 cell differentiation into noninvasive cells results in increased FAAH expression, lower endocannabinoid levels, and no responsiveness to cannabinoids. Conclusions: Endocannabinoid levels are enhanced in transformed colon mucosa cells possibly to counteract proliferation via CBRs. Inhibitors of endocannabinoid inactivation may prove useful anticancer agents.

Computerized tomographic colonography: Performance evaluation in a retrospective multicenter setting
C.Daniel Johnson, Alicia Y. Toledano, Benjamin A. Herman, Abraham H. Dachman, Elizabeth G. Mcfarland, Matthew A. Barish, James A. Brink, Randy D. Ernst, Joel G. Fletcher, Robert A. Halvorsen, Jr, Amy K. Hara, Kenneth D. Hopper, Robert E. Koehler, David S.K. Lu, Michael Macari, Robert L. Maccarty, Frank H. Miller, Martina Morrin, Erik K. Paulson, Judy Yee, Michael Zalis
Background & Aims: No multicenter study has been reported evaluating the performance and interobserver variability of computerized tomographic colonography. The aim of this study was to assess the accuracy of computerized tomographic colonography for detecting clinically important colorectal neoplasia (polyps 10 mm in diameter) in a multi-institutional study. Methods: A retrospective study was developed from 341 patients who had computerized tomographic colonography and colonoscopy among 8 medical centers. Colonoscopy and pathology reports provided the standard. A random sample of 117 patients, stratified by criterion standard, was requested. Ninety-three patients were included (47% with polyps 10 mm; mean age, 62 years; 56% men; 84% white; 40% reported colorectal symptoms; 74% at increased risk for colorectal cancer). Eighteen radiologists blinded to the criterion standard interpreted computerized tomography colonography examinations, each using 2 of 3 different software display platforms. Results: The average area under the receiver operating characteristic curve for identifying patients with at least 1 lesion 10 mm was 0.80 (95% lower confidence bound, 0.74). The average sensitivity and specificity were 75% (95% lower confidence bound, 68%) and 73% (95% lower confidence bound, 66%), respectively. Per-polyp sensitivity was 75%. A trend was observed for better performance with more observer experience. There was no difference in performance across software display platforms. Conclusions: Computerized tomographic colonography performance compared favorably with reported performance of fecal occult blood testing, flexible sigmoidoscopy, and barium enema. A prospective study evaluating the performance of computerized tomography colonography in a screening population is indicated.

Alterations of the intestinal transport and processing of gliadin peptides in celiac disease
Tamara Matysiak-Budnik, Celine Candalh, Christophe Dugave, Abdelkader Namane, Christophe Cellier, Nadine Cerf-Bensussan, Martine Heyman
Background & aims:The hypothesis of a defect in the intestinal transport and processing of toxic (31­49) or immunostimulant (57­68 and the 33-mer 56­89) gliadin peptides was tested in patients with active celiac disease (ACD), patients with treated celiac disease (TCD), and controls. Methods: Using duodenal biopsy specimens mounted in Ussing chambers, we measured electrical resistance, mucosal-to-serosal radiolabeled-peptide fluxes, and peptide processing during transport using radio-reverse-phase high-performance liquid chromatography. Results: Peptide 31­49 fluxes (24.7 µg · 3 h­1 · cm­2) were increased in patients with ACD compared with controls and patients with TCD (12.7 and 12.3 µg · 3 h­1 · cm­2; P < 0.01). In contrast, no increase was observed for peptide 57­68 or 56­89 (33-mer). Electrical resistance was decreased in patients with ACD versus controls (15.3 vs. 23.9 ohms · cm2; P < 0.001). Peptide 57­68 was partially degraded by brush-border peptidases in controls but not in patients with celiac disease. However, it was totally degraded after intestinal transport both in controls and patients with celiac disease. Peptides 31­49 and 56­89 were resistant to brush-border peptidases in all groups of patients but were totally degraded during intestinal transport in controls and patients with TCD. In patients with ACD, however, 50% of peptide 31­49 was delivered intact into the serosal compartment and only partial degradation of the 33-mer was observed. These abnormalities were not related to a nonspecific paracellular leakage. Conclusions: Our data indicate that gliadin peptides, although poorly or not digested by intraluminal enzymes, can be fully digested by enterocytes in controls and patients with TCD. In patients with ACD, incomplete degradation of the 33-mer and protected transport of the peptide 31­49 might favor their respective immunostimulatory and toxic effects.

Inactivating mutations of caspase-8 gene in colorectal carcinomas
Hong Sug Kim, Jong Woo Lee, Young Hwa Soung, Won Sang Park, Su Young Kim, Jong Heun Lee, Jik Young Park, Youg Gu Cho, Chang Jae Kim, Seong Whan Jeong, Suk Woo Nam, Sang Ho Kim, Jung Young Lee, Nam Jin Yoo, Sug Hyung Lee
Background & Aims: There has been evidence that dysregulation of apoptosis is involved in the pathogenesis of cancer development. Caspase-8 is an initiation caspase that activates the caspase cascade during apoptosis. The aim of this study was to explore the possibility that mutation of the caspase-8 gene might be involved in the development of colorectal cancer. Methods: We analyzed the entire coding region of the caspase-8 gene for the detection of somatic mutations in 180 colorectal tumors (98 invasive carcinomas and 82 adenomas) by polymerase chain reaction, single-strand conformation polymorphism, and DNA sequencing. Results: Overall, we detected a total of 5 somatic mutations in 98 invasive carcinomas (5.1%), but no mutations were detected in 82 adenomas (0%). The frequency of caspase-8 mutation in the carcinomas was significantly higher than that in adenomas (P < 0.05). The 5 mutations consisted of 1 frameshift, 1 nonsense mutation, and 3 missense mutations. We expressed the 5 tumor-derived caspase-8 mutants and found that 3 of the 5 mutations markedly decreased apoptosis activity of caspase-8. Furthermore, expression of the inactivating caspase-8 mutants interfered with apoptosis by death receptor overexpression, indicating that these mutants have dominant-negative inhibition of the death receptor-induced apoptosis. Conclusions: The presence of caspase-8 mutation in colon carcinomas suggests that caspase-8 gene mutation might lead to the loss of its apoptotic function and contribute to the pathogenesis of colorectal carcinomas, especially at the late stage of colorectal carcinogenesis.


The effect of the selective cyclooxygenase-2 inhibitor rofecoxib on human colorectal cancer liver metastases
Stephen W. Fenwick, Giles J. Toogood, J.Peter A. Lodge, Mark A. Hull
Background & Aims: Cyclooxygenase-2 (COX-2) is a potential target for chemotherapy of colorectal cancer (CRC). We tested the antineoplastic activity of the selective COX-2 inhibitor rofecoxib on human CRC liver metastases by measuring surrogate markers of tumor growth and angiogenesis in a randomized, double-blind, placebo-controlled trial. Methods: Patients undergoing liver resection surgery for metastatic disease were randomized to receive rofecoxib 25 mg daily or placebo before surgery (duration, >14 days). The apoptosis index (AI; neocytokeratin 18), proliferation index (PI; Ki-67), and microvessel density (MVD; CD31) were measured in metastases by immunohistochemistry. The effect of rofecoxib on COX-2-positive HCA-7 human CRC cell PGE2 synthesis, proliferation, and apoptosis in vitro was also investigated. Results: Patients who received rofecoxib (n = 23) and placebo (n = 21) were well matched regarding clinical and metastasis characteristics. The mean (range) duration of rofecoxib therapy was 26 (14­46) days. Rofecoxib-treated metastases had a 29% decrease in MVD (mean, 25.1 [SEM, 2.7] per hpf) compared with placebo-treated tissue (32.5 [SEM, 4.5] per hpf; P = 0.15). There was little difference in AI (rofecoxib mean, 2.03% [SEM, 0.43%] vs. placebo 1.39% [SEM, 0.39%]) or PI (rofecoxib 54.7% [SEM, 5.1%] vs. placebo 52.6% [SEM, 5.6%]). Rofecoxib-induced growth arrest and apoptosis of HCA-7 cells occurred only at concentrations (>10 µmol/L), which were significantly higher than the IC50 for COX-2 inhibition. Conclusions: Rofecoxib may negatively regulate angiogenesis in human CRC liver metastases. The absence of a significant, direct effect of rofecoxib on epithelial cells in liver metastases in vivo mirrors the lack of activity on human CRC cells at pharmacologically relevant concentrations in vitro.


Interleukin 15: A key to disrupted intraepithelial lymphocyte homeostasis and lymphomagenesis in celiac disease
Jean-Jacques Mention, Mélika Ben Ahmed, Bernadette Bègue, Ullah Barbe, Virginie Verkarre, Vahid Asnafi, Jean-frédéric Colombel, Paul-henri Cugnenc, Frank M. Ruemmele, Elisabeth Mcintyre, Nicole Brousse, Chistophe Cellier, Nadine Cerf-Bensussan
Background & Aims: The mechanism of intraepithelial lymphocyte hyperplasia, a hallmark of celiac disease, is unknown. We have investigated the role of epithelium-derived interleukin (IL)-15 in the alterations of epithelial homeostasis in refractory celiac sprue, a privileged situation to study the first step of lymphoid transformation and the contribution of intraepithelial lymphocytes to villous atrophy in celiac disease. Methods: IL-15 expression was assessed in biopsy specimens and isolated enterocytes by combining immunohistochemistry, flow cytometry, and real-time quantitative polymerase chain reaction. The ability of IL-15 to induce growth and survival of clonal intraepithelial lymphocytes lacking surface CD3 and to induce their cytotoxicity and secretion of interferon gamma was tested using soluble IL-15 and coculture in the presence of epithelial cell lines expressing membrane IL-15. Results: IL-15 was massively overexpressed not only in lamina propria but also in the intestinal epithelium of patients with active celiac disease and refractory celiac sprue. IL-15 was not secreted but delivered at the surface of enterocytes. IL-15 specifically induced the expansion and survival of the clonal abnormal intraepithelial lymphocytes that characterize refractory celiac sprue and triggered their secretion of interferon gamma and their cytotoxicity against intestinal epithelial cells. Comparable activating signals could be delivered by IL-15 expressed at the membrane of the T84 enterocyte cell line. Conclusions: These data provide strong evidence that uncontrolled overexpression of IL-15 in refractory celiac sprue perpetuates epithelial damage and promotes the emergence of T-cell clonal proliferations. Blocking IL-15 might prove useful to treat this severe complication of celiac disease.

Clinical-liver, Pancreas, and Biliary Tract

Duodenal cytochrome B and hephaestin expression in patients with iron deficiency and hemochromatosis
Heinz Zoller, Igor Theurl, Robert O. Koch, Andrew T. Mckie, Wolfgang Vogel, Gü Weiss
Background & Aims: An increased duodenal expression of the iron transporters, divalent-metal-transporter-1, and ferroportin is observed in patients with iron deficiency or hereditary hemochromatosis. Two oxidoreductases, termed duodenal cytochrome b and hephaestin, are proposed to co-operate with divalent-metal-transporter-1 and FPN1, respectively, to transfer iron from the duodenal lumen to the circulation. Methods: In the present study, we investigated the mRNA and protein expression of Dcytb and hephaestin in duodenal biopsies from patients with iron deficiency, HFE, and non-HFE-associated hemochromatosis and in control subjects by means of real-time polymerase chain reaction, Western blot, and immunofluorescence. Results: In iron deficiency a coordinated upregulation of the iron transporters divalent-metal-transporter-1 and ferroportin and of duodenal-cytochrome b and hephaestin was found, whereas in patients with HFE and non-HFE-associated hemochromatosis duodenal-cytochrome b and hephaestin protein and mRNA expression were not significantly different from control subjects. However, HFE but not non-HFE hemochromatosis patients presented with an increased duodenal ferric reductase activity. Spearman rank correlations showed that Dcytb, hephaestin, FPN1, and DMT1 mRNA expression are positively related to each other independently of the underlying disease, which ensures an efficient transepithelial transport of absorbed iron. Conclusions: Our data show that duodenal-cytochrome b activity in iron deficiency is stimulated via enhanced protein expression, whereas in HFE hemochromatosis it is up-regulated post-translationally. This points to different kinetics of intestinal iron uptake between iron deficiency and HFE hemochromatosis and also indicates that duodenal iron accumulation in HFE and non-HFE hemochromatosis is pathophysiologically different.

Infection and the progression of hepatic encephalopathy in acute liver failure
Javier Vaquero, Julie Polson, Chuhan Chung, Irene Helenowski, Frank V. Schiodt, Joan Reisch, William M. Lee, Andres T. Blei
Background & Aims:Progression of hepatic encephalopathy (HE) is a major determinant of outcome in acute liver failure (ALF). Our aim was to identify predictive factors of worsening HE, including the relation of encephalopathy with the systemic inflammatory response (SIRS) and infection. Methods:We included 227 consecutive patients with stage I-II HE prospectively enrolled in the U.S. Acute Liver Failure Study. Univariate and multivariate analysis of 27 variables at admission were performed separately for acetaminophen (n = 96) and nonacetaminophen (n = 131) etiologies. Results:On multivariate analysis, acquisition of infection during stage I-II HE (P < 0.01), increased leukocyte levels at admission (P < 0.01), and decreased platelet count (P < 0.05) were predictive factors of worsening HE in the acetaminophen group. By contrast, only increased pulse rate (P < 0.05) and AST levels (P < 0.05) at admission were predictors in nonacetaminophen patients. In patients who progressed to deep HE, the first confirmed infection preceded progression in 15 of 19 acetaminophen patients compared with 12 of 23 nonacetaminophen patients. In patients who did not demonstrate positive microbiologic cultures, a higher number of components of SIRS at admission was associated with more frequent worsening of HE (25% vs. 35% vs. 50% for 0, 1, and 2 components of SIRS, P < 0.05). Conclusions:This prospective evaluation points to infection and/or the resulting systemic inflammatory response as important factors contributing to worsening HE in ALF, mainly in patients with acetaminophen- induced ALF. The use of prophylactic antibiotics in these patients and the mechanisms by which infection triggers hepatic encephalopathy require further investigation.

Basic-Alimentary Tract

An endogenous cannabinoid tone attenuates cholera toxin-induced fluid accumulation in mice
Angelo A. Izzo, Francesco Capasso, Anna Costagliola, Tiziana Bisogno, Giovanni Marsicano, Alessia Ligresti, Isabel Matias, Raffaele Capasso, Luisa Pinto, Francesca Borrelli, Aldo Cecio, Beat Lutz, Nicola Mascolo, Vincenzo Di Marzo
Background & Aims: Cholera toxin (CT) is the most recognizable enterotoxin causing secretory diarrhea, a major cause of infant morbidity and mortality throughout the world. In this study, we investigated the role of the endogenous cannabinoid system (i.e., the cannabinoid receptors and their endogenous ligands) in CT-induced fluid accumulation in the mouse small intestine. Methods: Fluid accumulation was evaluated by enteropooling; endocannabinoid levels were measured by isotope-dilution gas chromatography mass spectrometry; CB1 receptors were localized by immunohistochemistry and their messenger RNA (mRNA) levels were quantified by reverse-transcription polymerase chain reaction (PCR). Results: Oral administration of CT to mice resulted in an increase in fluid accumulation in the small intestine and in increased levels of the endogenous cannabinoid, anandamide, and increased expression of the cannabinoid CB1 receptor mRNA. The cannabinoid receptor agonist CP55,940 and the selective cannabinoid CB1 receptor agonist arachidonoyl-chloro-ethanolamide inhibited CT-induced fluid accumulation, and this effect was counteracted by the CB1 receptor antagonist SR141716A, but not by the CB2 receptor antagonist SR144528. SR141716A, per se, but not the vanilloid VR1 receptor antagonist capsazepine, enhanced fluid accumulation induced by CT, whereas the selective inhibitor of anandamide cellular uptake, VDM11, prevented CT-induced fluid accumulation. Conclusions: These results indicate that CT, along with enhanced intestinal secretion, causes overstimulation of endocannabinoid signaling with an antisecretory role in the small intestine.

Dietary glycine prevents chemical-induced experimental colitis in the rat
Isao Tsune, Kenichi Ikejima, Miyoko Hirose, Mutsuko Yoshikawa, Nobuyuki Enomoto, Yoshiyuki Takei, Nobuhiro Sato
Background & Aims: In this study, the effect of dietary glycine on experimental colitis induced by 2,4,6-trinitrobenzene sulphonic acid (TNBS) and dextran sulfate sodium (DSS) in the rat was evaluated. Methods: Male Wistar rats were fed a diet containing 5% glycine or casein as controls starting 3 days before experiments, and were given a single intracolonic injection of TNBS (50 mg/rat, dissolved in 50% ethanol). Similarly, some rats were given 3% DSS orally in drinking water for 5 days to induce colitis as a second model. The severity of colitis was evaluated pathologically, and tissue myeloperoxidase (MPO) activity was measured. Further, mRNA and protein levels for interleukin (IL)-1, tumor necrosis factor (TNF)-, cytokine-induced neutrophil chemoattractant (CINC), and macrophage inflammatory protein (MIP)-2 were detected by reverse-transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Results: A diet containing glycine ameliorated diarrhea and body weight loss caused by TNBS, and improved both macroscopic and histologic scores of colitis significantly. TNBS-induced increases in MPO activities in the colonic tissue were blunted significantly in glycine-fed animals. Further, dietary glycine largely prevented increases in IL-1 and TNF- in the colon 2 days after TNBS, and TNBS induction of CINC and MIP-2 in the colonic tissue also was abrogated by glycine. Importantly, the protective effect of glycine was significant even when TNBS colitis was once established. Moreover, dietary glycine also was preventive in a second, DSS-induced colitis model. Conclusions: Dietary glycine prevents chemical-induced colitis by inhibiting induction of inflammatory cytokines and chemokines. It is postulated that glycine may be useful for the treatment of inflammatory bowel diseases as an immunomodulating nutrient.

Rectal intraganglionic laminar endings are transduction sites of extrinsic mechanoreceptors in the guinea pig rectum
Penny A. Lynn, Catharina Olsson, Vladimir Zagorodnyuk, Marcello Costa, Simon J.H. Brookes
Background & aims: Vagal afferent mechanoreceptors in the upper gut have recently been identified morphologically as intraganglionic laminar endings (IGLEs), but little is known about the structure of mechanoreceptive endings elsewhere in the gastrointestinal tract. We have morphologically characterized the nerve endings of specialized mechanoreceptors in the rectum. Methods: Extracellular recordings from guinea pig rectal and colonic nerves were made, in vitro, in combination with rapid anterograde transport of biotinamide, to reveal the morphology of recorded fibers. Controlled distentions were used to activate mechanoreceptive afferent units, and von Frey hairs were used to identify their transduction sites. Results: Rectal mechanoreceptors were present in high density, had low thresholds, and adapted slowly to maintained distention. Each afferent unit had multiple small (<200-µm diameter) transduction sites ("hot spots") at which they could be activated locally by application of a light von Frey hair (0.08­7 mN). Anterograde dye filling revealed characteristic rectal intraganglionic laminar endings (rIGLEs) in myenteric ganglia, significantly associated with hot spots, comparable to the IGLEs of vagal tension receptors, but smaller and less complex. Afferent fibers with these morphologic and physiologic features could not be recorded from colonic nerves innervating the large bowel proximal to the rectum. Conclusions: The rectum receives a dense afferent innervation by a distinct population of low-threshold, slowly adapting mechanoreceptors with specialized intraganglionic laminar endings (rIGLEs), which are not found more proximally in the colon.

Stress-induced disruption of colonic epithelial barrier: Role of interferon- and myosin light chain kinase in mice
Laurent Ferrier, Ludmilla Mazelin, Nicolas Cenac, Pierre Desreumaux, Anne Janin, Dominique Emilie, Jean-Frederic Colombel, Rafael Garcia-Villar, Jean Fioramonti, Lionel Bueno
Background & Aims: Stressful life events are supposed to be involved in various diseases such as inflammatory bowel diseases and irritable bowel syndrome. Impairment of the intestinal epithelial barrier function is a suspected consequence of stress, but the underlying mechanisms remain unclear. This study aimed to determine the mechanisms through which stress modulates the colonic epithelial barrier. Methods: Cytokine messenger RNA (mRNA) expression was evaluated in murine colon, liver, and spleen by competitive reverse-transcription polymerase chain reaction after 1­4 days of daily 2-hour stress sessions. Colonic paracellular permeability was measured as the in vivo lumen-to-blood ratio of 51Cr-ethylenediaminetetraacetic acid. The effect of a myosin light chain (MLC) kinase inhibitor (ML-7) was assessed on stress-induced interferon (IFN)- mRNA expression and colonic epithelial barrier impairment, and MLC phosphorylation was determined by immunoblot. Finally, the incidence of repeated stress sessions on bacterial translocation was determined. Results: Repeated stress induced an overexpression of colonic IFN-. In the liver, higher levels of IFN-, interleukin (IL)-4, and IL-10 mRNAs were detected and were associated with bacterial translocation, inflammation, and apoptosis. Stress increased colonic permeability of control mice, but not of SCID and IFN--deficient mice. ML-7 inhibited the stress-induced increased permeability, bacterial translocation, and cytokine overexpression in the liver and restored a normal histology. Larger amounts of phosphorylated MLC were detected in stressed animals. Conclusions: Repeated stress sessions drive organ-specific cytokine expression patterns and alter colonic mucosal barrier functions associated with bacterial translocation. This effect depends on the presence of CD4+ T cells and requires IFN- production and MLC phosphorylation.

Effect of 5-hydroxytryptamine on duodenal mucosal bicarbonate secretion in mice
Bi-guang Tuo, Jon I. Isenberg
Background & Aims: 5-hydroxytryptamine (5-HT) is an important neurotransmitter and intercellular messenger that modulates many gastrointestinal functions. Because little is known about the role of 5-HT in the regulation of duodenal bicarbonate secretion, we examined the role of 5-HT on duodenal bicarbonate secretion and define neural pathways involved in the actions of 5-HT. Methods: Duodenal mucosa from National Institutes of Health Swiss mice was stripped of seromuscular layers and mounted in Ussing chambers. The effect of 5-HT on duodenal bicarbonate secretion was determined by the pH stat technique. Acetylcholine (ACh) release from duodenal mucosa was assessed by preincubating the tissue with [3H] choline and measuring 5-HT-evoked release of tritium. Results: 5-HT added to the serosal bath markedly stimulated duodenal bicarbonate secretion and short circuit current (Isc) in a dose-dependent manner (10­7 mol/L to 10­3 mol/L; P < 0.0001), whereas mucosally added 5-HT was without effect. 5-HT-stimulated bicarbonate secretion was independent of luminal Cl­. Pretreatment with tetrodotoxin (TTX) (10­6 mol/L) or atropine (10­5 mol/L) markedly reduced 5-HT-stimulated duodenal bicarbonate secretion (by 60% and 65%, respectively; P < 0.001) and Isc (by 45% and 27%, respectively; P < 0.001 and P < 0.05). Pretreatment with N-nitro-L-arginine methyl ester (L-NAME) (10­3 mol/L), propranolol (10­5 mol/L), or phentolamine (10­5 mol/L) did not significantly alter 5-HT-stimulated duodenal mucosal bicarbonate secretion or Isc. 5-HT concentration-dependently evoked ACh release from duodenal mucosal preparations (P < 0.0001). TTX markedly inhibited 5-HT-evoked ACh release (P < 0.001). Conclusions: 5-HT is a potent activator of duodenal mucosal bicarbonate secretion in mice. Duodenal bicarbonate secretion induced by 5-HT in vitro occurs principally via a cholinergic neural pathway.

Basic-Liver, Pancreas, and Biliary Tract

Effects of hypothermia on brain glucose metabolism in acute liver failure: A 1H/13C-nuclear magnetic resonance study
Nicolas Chatauret, Claudia Zwingmann, Christopher Rose, Dieter Leibfritz, Roger F. Butterworth
Background & Aims: Mild hypothermia has a protective effect on brain edema and encephalopathy in both experimental and human acute liver failure. The goals of the present study were to examine the effects of mild hypothermia (35°C) on brain metabolic pathways using combined 1H and 13C-Nuclear Magnetic Resonance (NMR) spectroscopy, a technique which allows the study not only of metabolite concentrations but also their de novo synthesis via cell-specific pathways in the brain. Methods: 1H and 13C NMR spectroscopy using [1-13C] glucose was performed on extracts of frontal cortex obtained from groups of rats with acute liver failure induced by hepatic devascularization whose body temperature was maintained either at 37°C (normothermic) or 35°C (hypothermic), and appropriate sham-operated controls. Results: At coma stages of encephalopathy in the normothermic acute liver failure animals, glutamine concentrations in frontal cortex increased 3.5-fold compared to sham-operated controls (P < 0.001). Comparable increases of brain glutamine were observed in hypothermic animals despite the absence of severe encephalopathy (coma). Brain glutamate and aspartate concentrations were respectively decreased to 60.9% ± 7.7% and 42.2% ± 5.9% (P < 0.01) in normothermic animals with acute liver failure compared to control and were restored to normal values by mild hypothermia. Concentrations of lactate and alanine in frontal cortex were increased to 169.2% ± 15.6% and 267.3% ± 34.0% (P < 0.01) respectively in normothermic rats compared to controls. Furthermore, de novo synthesis of lactate and alanine increased to 446.5% ± 48.7% and 707.9% ± 65.7% (P < 0.001), of control respectively, resulting in increased fractional 13C-enrichments in these cytosolic metabolites. Again, these changes of lactate and alanine concentrations were prevented by mild hypothermia. Conclusions: Mild hypothermia (35°C) prevents the encephalopathy and brain edema resulting from hepatic devascularization, selectively normalizes lactate and alanine synthesis from glucose, and prevents the impairment of oxidative metabolism associated with this model of ALF, but has no significant effect on brain glutamine. These findings suggest that a deficit in brain glucose metabolism rather than glutamine accumulation is the major cause of the cerebral complications of acute liver failure.

Role of farnesoid X receptor in determining hepatic ABC transporter expression and liver injury in bile duct-ligated mice
Martin Wagner, Peter Fickert, Gernot Zollner, Andrea Fuchsbichler, Dagmar Silbert, Oleksiy Tsybrovskyy, Kurt Zatloukal, Grace L. Guo, John D. Schuetz, Frank J. Gonzalez, Hanns-Ulrich Marschall, Helmut Denk, Michael Trauner
Background & Aims: Cholestasis induces changes in hepatic adenosine triphosphate-binding cassette (ABC) transporter expression. We aimed to investigate the role of the nuclear bile acid receptor (farnesoid X receptor [FXR]) in mediating changes in ABC transporter expression and in determining liver injury. Methods: Hepatic ABC transporter (multidrug resistance-associated proteins [Mrp] 2­4 and bile salt export pump [Bsep]) expression and localization were studied in common bile duct-ligated (CBDL) FXR knockout (FXR­/­), wild-type (FXR+/+), and sham-operated mice. Serum alanine aminotransferase, alkaline phosphatase, bilirubin and bile acid levels, hepatic bile acid composition, and liver histology were investigated. Cholangiomanometry and bile duct morphometry were performed. Results: CBDL induced expression of Mrp 3 and Mrp 4 in FXR+/+ and even more in FXR­/­, whereas Mrp 2 expression remained unchanged. Bsep expression was maintained in CBDL FXR+/+ but remained undetectable in CBDL FXR­/­. Alanine aminotransferase levels and mortality rates did not differ between CBDL FXR+/+ and FXR­/­. CBDL increased biliary pressure and induced bile ductular proliferation and bile infarcts in FXR+/+, whereas FXR­/­ had lower biliary pressures, less ductular proliferation, and developed disseminated liver cell necroses. Conclusions: Overexpression of Mrp 3 and Mrp 4 in CBDL mice is FXR independent and could play an important role in the adaptive hepatic ABC transporter response to cholestasis. Maintenance of Bsep expression strictly depends on FXR and is a critical determinant of the cholestatic phenotype. Lack of bile infarcts in CBDL FXR­/­ suggests that development of bile infarcts is related to bile acid-dependent bile flow and biliary pressure. This information is relevant for the potential use of FXR modulators in the treatment of cholestatic liver diseases.

Bile salt-induced hepatocyte apoptosis involves epidermal growth factor receptor-dependent CD95 tyrosine phosphorylation
Roland Reinehr, Dirk Graf, Dieter Häussinger
Background & aims: Hydrophobic bile acids induce CD95-dependent hepatocyte apoptosis. Methods: The mechanisms of bile acid-induced CD95 activation were studied in 24-hour cultured rat hepatocytes, in situ-perfused rat livers, and livers from bile duct-ligated rats. Results: Within 1 minute, the proapoptotic bile salts taurolithocholate-3-sulfate and glycochenodeoxycholate induced oxidative stress and EGF receptor (EGF-R) tyrosine phosphorylation followed by rapid c-Jun-N-terminal kinase (JNK) activation. Thereafter, EGF-R associated with CD95 with subsequent CD95 tyrosine phosphorylation, CD95 membrane targeting, and death-inducing signal complex (DISC) formation. All of these responses were also triggered by taurochenodeoxycholate except that DISC formation only occurred in the presence of phosphatidylinositol 3-kinase inhibitors. No activation of EGF-R or CD95 was observed with tauroursodeoxycholate or taurocholate. Taurolithocholate-3-sulfate-induced EGF-R phosphorylation was sensitive to N-acetylcysteine (NAC) and genistein, whereas CD95/EGF-R association was inhibited by NAC, JNK, or protein kinase C inhibition but not by AG1478. However, the latter compound as well as NAC, genistein, inhibition of JNK, or protein kinase C inhibited CD95 tyrosine phosphorylation, membrane trafficking, and DISC formation. Conclusions: Induction of apoptosis by hydrophobic bile salts involves EGF-R activation and EGF-R-dependent CD95 tyrosine phosphorylation, which triggers CD95 membrane targeting and Fas-associated death domain/caspase-8 recruitment. The latter step is apparently also controlled by phosphatidylinositol 3-kinase.

Bid activates multiple mitochondrial apoptotic mechanisms in primary hepatocytes after death receptor engagement
Yongge Zhao, Wen-xing Ding, Ting Qian, Simon Watkins, John J. Lemasters, Xiao-ming Yin
Background & Aims: Activation of Fas or tumor necrosis factor receptor 1 (TNF-R1) on hepatocytes leads to apoptosis, which requires mitochondria activation. The pro-death Bcl-2 family protein, Bid, mediates this pathway by inducing mitochondrial releases of cytochrome c and other apoptotic factors. How Bid activates mitochondria has been studied in vitro with isolated mitochondria. We intended to study the mechanisms in intact hepatocytes so that findings could be made in a proper cellular context and would be more physiologically relevant. Methods: Hepatocytes were isolated from wild-type and bid-deficient mice and treated with anti-Fas or TNF-. Mechanisms of mitochondria activation were dissected with genetic, biochemical, and morphologic approaches. Results:bid-deficient hepatocytes were much more resistant to apoptosis. Bid was required for permeability transition and mitochondria depolarization in addition to the previously defined release of cytochrome c. Permeability transition inhibitors cyclosporin A and aristolochic acid could inhibit mitochondria activation effectively, but not as much as the deletion of the bid gene, and they could not inhibit Bak oligomerization. In addition, mitochondria depolarization also could be induced by caspases, whose activation was mainly dependent on Bid. Conclusions: Bid may activate mitochondria by 2 mechanisms, one is related to permeability transition and the other is related to Bak oligomerization. Bid can further affect mitochondria potentials by indirectly regulating caspase activity. This in vivo study provides novel findings not previously disclosed by in vitro studies, and indicates the importance of several mechanisms in contributing Bid-mediated mitochondria dysfunction that could be potential cellular targets of intervention.

FXR and ABCG5/ABCG8 as determinants of cholesterol gallstone formation from quantitative trait locus mapping in mice
Henning Wittenburg, Malcolm A. Lyons, Renhua Li, Gary A. Churchill, Martin C. Carey, Beverly Paigen
Background & Aims: Cholesterol gallstone formation is a complex genetic trait. To identify additional cholesterol gallstone susceptibility loci, we performed a quantitative trait locus analysis using an intercross of PERA/Ei and I/LnJ inbred strains of mice. Methods: Mice of both sexes were examined for gallstone weight and evaluated according to a scoring system for the physical chemistry of cholelithiasis during feeding of a lithogenic diet. Intercross offspring were genotyped, and linkage analysis was performed by interval mapping. Differences in messenger RNA expression of positional candidate genes were determined using reverse-transcription and real-time polymerase chain reaction. Results: We identified significant loci associated with gallstone weight on chromosomes 10 and 4, named Lith7 and Lith8, respectively (both susceptibility alleles conferred by strain I/LnJ). Positional candidate genes with higher expression in I/LnJ mice are Fxr (official symbol, Nr1h4), encoding the nuclear bile salt receptor, on chromosome 10 and Shp1 (official symbol, Nr0b2), encoding the small heterodimer partner 1, on chromosome 4. A significant locus associated with gallstone score on chromosome 17, named Lith9 (susceptibility allele conferred by strain PERA/Ei), colocalizes with the genes Abcg5 and Abcg8 that encode the canalicular cholesterol transporter. Higher hepatic messenger RNA expression of Abcg5 and Abcg8 in strain PERA/Ei correlates positively with higher biliary cholesterol levels. Conclusions: Our findings suggest a primary role of the nuclear bile salt receptor FXR and the canalicular cholesterol transporter ABCG5/ABCG8 in the genetic susceptibility and pathogenesis of cholesterol cholelithiasis in these strains of inbred mice.

Sinusoidal obstruction syndrome (veno-occlusive disease) in the rat is prevented by matrix metalloproteinase inhibition
Laurie D. DeLeve, Xiangdong Wang, Jeffrey Tsai, Gary Kanel, Steven Strasberg, Zoltan A. Tokes
Background & Aims: The mechanical origins of the obstruction in sinusoidal obstruction syndrome are initiated by dehiscence of sinusoidal endothelial cells from the space of Disse. The biochemical changes that permit the dehiscence of the sinusoidal endothelial cells were investigated. Methods: In vitro and in vivo studies examined changes induced by monocrotaline, a pyrrolizidine alkaloid that induces sinusoidal obstruction syndrome in both humans and experimental animals. Results: In the monocrotaline-induced rat model of sinusoidal obstruction syndrome, there was an early increase of matrix metalloproteinase-9 and a later, lower-magnitude increase of matrix metalloproteinase-2 in the liver. In vitro studies of sinusoidal endothelial cells, hepatocytes, stellate cells, and Kupffer cells showed that sinusoidal endothelial cells are the major source of both basal and monocrotaline-induced matrix metalloproteinase-9/matrix metalloproteinase-2 activity. Monocrotaline caused depolymerization of F-actin in sinusoidal endothelial cells, and blocking of F-actin depolymerization prevented the increase in matrix metalloproteinase activity. Administration of matrix metalloproteinase inhibitors prevented the signs and histological changes associated with sinusoidal obstruction syndrome. Conclusions: Monocrotaline causes depolymerization of F-actin in sinusoidal endothelial cells, which leads to increased expression of metalloproteinase-9 and matrix metalloproteinase-2 by sinusoidal endothelial cells. Inhibition of matrix metalloproteinase-9 and matrix metalloproteinase-2 prevents the development of sinusoidal obstruction syndrome, establishing that matrix metalloproteinase inhibitors may be a therapeutically viable strategy for prevention.

Activated signal transducer and activator of transcription 3 (STAT3) supports the malignant phenotype of human pancreatic cancer
Arne Scholz, Sandra Heinze, Katharina M. Detjen, Michael Peters, Martina Welzel, Peter Hauff, Michael Schirner, Bertram Wiedenmann, Stefan Rosewicz
Background & aims: Constitutive activation of signal transducer and activator of transcription 3 (STAT3) has been implicated in regulation of growth and malignant transformation. We therefore analyzed the expression and biologic significance of STAT3 in human pancreatic cancer cells. Methods: Expression and activation of STAT3 were investigated by immunohistochemistry and immunoblotting. Functional inactivation of STAT3 was achieved by stable transfection of dominant-negative STAT3 constructs in 2 pancreatic cancer cell lines and confirmed by electrophoretic mobility shift assay and immunoblotting. Cell proliferation and tumorigenicity were evaluated by cell counting, colony formation in soft agar, and xenotransplantation in nude mice. STAT3-dependent cell cycle distribution was monitored by flow cytometry, immunoprecipitation, immunoblotting, and histone H1 and GST-Rb kinase assays. Results: Compared with nontransformed human pancreas, activated STAT3 is overexpressed in ductal carcinoma cells but not in ducts from chronic pancreatitis. Constitutive activation was also observed in all human pancreatic cancer cell lines examined. Functional inactivation of STAT3 resulted in significant inhibition of anchorage-dependent and -independent proliferation in vitro and reduced tumor growth in vivo. Cell cycle analysis showed a delay of G1/S-phase progression due to inhibition of cyclin-dependent kinase 2 activity based on increased expression of p21WAF1 in vitro and in vivo. Blocking of the STAT3 upstream activator Janus kinase 2 by tyrphostin also resulted in growth arrest because of delayed G1/S-phase progression and increased expression of p21WAF1. Conclusions: On malignant transformation, activated STAT3 promotes cellular proliferation by acceleration of G1/S-phase progression and thereby contributes to the malignant phenotype of human pancreatic cancer.


Case Reports

 Multiple ulcers with perforation of the small intestine in buerger's disease: A case report
Atsushi Kurata, Takahiro Nonaka, Yasuo Arimura, Masao Nunokawa, Yuichi Terado, Kenichi Sudo, Yasunori Fujioka
We present the case of a young man with Buerger's disease that involved the gastrointestinal tract. The patient experienced sudden onset of abdominal pain during the period of treatment for peripheral manifestations. Radiographic workup revealed free air in the abdomen. A laparotomy was performed, and the resected specimen revealed regularly distanced and multiple ulcers in proximity of the perforation, which was accompanied by occluded intramural arteries of the small intestine. Although visceral localization of Buerger's disease is unusual, several cases have been previously reported. Nevertheless, the manifestation of regularly distanced skip intestinal ulcers in Buerger's disease is apparently unique. In addition, detailed pathological findings delineated this rare condition.

Special Reports and Reviews

Protective strategies against ischemic injury of the liver
Nazia Selzner, Hannes Rudiger, Rolf Graf, Pierre-Alain Clavien
This article summarizes strategies to protect the liver from injuries caused by ischemia and reperfusion. Three different sections (i.e., surgical and pharmacologic strategies and gene therapy) present approaches to enhance the survival and viability of the liver in various surgical procedures including liver transplantation. The first section reviews approaches using surgical interventions such as ischemic preconditioning and intermittent clamping. Their protective effects are discussed with respect to the mechanism of injury. In the second section, pharmacologic agents targeting microcirculation, oxidative stress, proteases, and inflammation are described. Mechanisms of injury and their suppression by a wide variety of drugs are discussed. The third section focuses on gene therapy. Potential target genes have been identified (e.g., superoxide dismutase or heme oxygenase). Animal experiments in which the liver injury is reduced successfully may pave the way to novel strategies applied to different liver diseases in humans.

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

---

JOURNAL OF HEPATOLOGY

Table of Contents for Journal of Hepatology Volume 39, Issue 3, September 2003

Chronic Liver Diseases

Structure and liver cell expression pattern of the HFE gene in the rat
Petra Holmström et al.
Background/Aims: Very little is known about the HFE gene in the rat. The aim of the present study was to determine: (1) the structure of the rat HFE gene; and (2) the tissue expression of the HFE mRNA in the rat, with special emphasis on the liver. Methods: Cloning of the rat HFE gene was performed using library screening and PCR. Exon-intron borders were assigned by DNA sequencing. Parenchymal and non-parenchymal liver cells were isolated by fractionation of normal rat liver. HFE mRNA levels were determined by Northern blot (tissues) and real-time PCR (isolated liver cells). Results: The rat HFE gene contained six exons and five introns. The HFE gene is expressed in multiple tissues in the rat, including bone marrow, with the highest expression in the liver. We observed HFE transcripts in several categories of isolated rat liver cells. Unexpectedly, expression also occurred in rat hepatocytes. Conclusions: The exon-intron pattern of the HFE gene is strongly conserved between rat and mouse. The pattern of tissue expression of the HFE gene is rather similar in humans and rodents. The finding of HFE gene expression in rat hepatocytes raises interesting questions regarding its role in the hepatocyte iron metabolism. Keywords: HFE; Gene structure; Gene expression; Liver; Rat; Hepatocytes; Iron metabolism

Cirrhosis and its Complications

Cardiac dysfunction in portal hypertension among patients with cirrhosis and non-cirrhotic portal fibrosis
Binay K. De et al.
Background/Aims: In cirrhosis, diastolic dysfunction of heart is well documented. Contribution of portal hypertension towards cardiac changes in cirrhosis is difficult to assess. We examined the patients of non-cirrhotic portal fibrosis who have portal hypertension without liver insufficiency to understand the contribution of portal hypertension in causing cardiac changes.

Methods: Cardiac function was studied in four groups of patients: normal controls, patients with non-cirrhotic portal fibrosis (having portal hypertension without liver dysfunction) and cirrhotics with and without ascites. Cardiac function was evaluated by echocardiography. Additional measurements of plasma renin activity and aldosterone levels were performed.

Results: Diastolic function as assessed by the ratio between E wave and A wave (E/A ratio), was significantly lower in patients with non-cirrhotic portal fibrosis (median 1.3) compared to normal controls (median 1.52). However, even lower values were observed in cirrhotics without ascites (median 1.05) and with ascites (median 0.94). There was a significant correlation (r=0.75) between plasma aldosterone levels and the E/A ratio in cirrhotics.

Conclusions: Diastolic dysfunction is not only present in cirrhosis but also in non-cirrhotic portal fibrosis. It indicates that portal hypertension is an important factor in the genesis of cardiac dysfunction.
Keywords: Non-cirrhotic portal fibrosis; Portal hypertension; Diastolic dysfunction

Memory function in early hepatic encephalopathy
Karin Weissenborn, Susanne Heidenreich, Kathrin Giewekemeyer, Norbert Rückert and Hartmut Hecker

Background: Early hepatic encephalopathy (HE) is characterized by deficits in motor performance, visual perception, visuo-constructive abilities and attention. Whether defective memory is a feature of early HE is controversial.

Aims: To analyze memory function in patients with early HE.

Methods: Memory tests were applied to cirrhotic patients with grade 0 HE, minimal HE and grade I HE (n=45) and controls (n=52). The battery included short and long term memory tests requiring free recall or recognition. Minimal HE was diagnosed by assessing the psychometric hepatic encephalopathy score using the PSE-Syndrom-Test and by carrying out a neurological examination. Group differences of the test results were analyzed using analysis of covariance.

Results: HE 0 patients achieved test results similar to the controls in all but two tests. Patients with early HE (minimal and grade I HE) scored lower than the controls in all tests applied. A detailed analysis of test performance showed that the patients' deficits were in attention and visual perception, rather than memory.

Conclusions: Patients with early HE score lower than controls in memory tasks predominantly because of deficits in attention and visual perception.
Keywords: Memory; Hepatic encephalopathy; Test

Quantitative liver-spleen scan using single photon emission computerized tomography (SPECT) for assessment of hepatic function in cirrhotic patients
Eli Zuckerman et al.
Background/Aims: Accurate quantitative determination of liver function is critical in cirrhotic patients in order to predict outcome, particularly in patients who undergo hepatic resection or non-hepatic surgery. As colloid uptake by perfused Kupffer cells is proportional to perfused hepatocyte mass, quantitative liver spleen scan may be used as an index of perfused hepatocyte mass. Thus, this study was conducted to evaluate quantitative single photon emission computerized tomography (SPECT) of Tc-99mm-phytate colloid uptake by the liver as a test for hepatic function in cirrhotic patients.

Methods: Quantitative SPECT was used to measure liver volume, quantitative colloid uptake by the liver and percentage of injected dose/ml of liver tissue in cirrhotic patients (n=75), non-cirrhotic patients with chronic liver disease (n=52) and patients without liver disease (n=36).

Results: Although liver volume was similar among the three groups, the cirrhotic patients had significantly lower total quantitative uptake and quantitative uptake/ml compared to groups 2 and 3 (P<0.001). Quantitative liver uptake in the cirrhotic patients was highly correlated with Child-Pugh score (r=0.64, P<0.0001) and with indocyanine green retention at 15 min (r=0.84, P<0.0001).

Conclusions: Quantitative SPECT of the liver may be an additional, useful, non-invasive quantitative test for assessment of hepatic function and severity of liver disease in cirrhotic patients.
Keywords: Liver; Single photon emission computerized tomography; Cirrhotic patients

Liver Cell Injury and Liver Failure

Silibinin protects mice from T cell-dependent liver injury
Jens Schümann et al.
Background/Aims: Silibinin is the major pharmacologically active compound of the Silybum marianum fruit extract silymarin. Its well-known hepatoprotective activities are mostly explained by antioxidative properties, inhibition of phosphatidylcholine synthesis or stimulation of hepatic RNA and protein synthesis. Here, we characterized the hepatoprotective potential of silibinin as an immune-response modifier in T cell-dependent hepatitis in vivo.

Methods: Silibinin was tested in the mouse model of concanavalin A (ConA)-induced, T cell-dependent hepatitis. Liver injury was assessed by quantification of plasma transaminase activities and intrahepatic DNA fragmentation. Plasma cytokine concentrations were determined by enzyme-linked immunosorbent assay (ELISA), intrahepatic cytokine and inducible NO synthase (iNOS) mRNA levels by reverse transcriptase polymerase chain reaction, intrahepatic iNOS expression by immunofluorescent staining, and intrahepatic nuclear factor kappa B (NF-B) activation by electrophoretic mobility shift assay.

Results: Silibinin significantly inhibited ConA-induced liver disease. Silibinin proved to be an immune-response modifier in vivo, inhibiting intrahepatic expression of tumor necrosis factor, interferon-, interleukin (IL)-4, IL-2, and iNOS, and augmenting synthesis of IL-10. In addition, silibinin inhibited intrahepatic activation of NF-B.

Conclusions: Silibinin, suppressing T cell-dependent liver injury as an immune-response modifier, might be a valuable drug in therapeutic situations in which intrahepatic immunosuppression is required.
Keywords: Silibinin; T cells; Liver injury

Atrial natriuretic peptide preconditioning protects against hepatic preservation injury by attenuating necrotic and apoptotic cell death
Tobias Gerwig et al.
Background/Aims: Preconditioning of livers with the atrial natriuretic peptide (ANP) markedly reduces hepatic ischemia-reperfusion injury. Aim of this study was to characterize the influence of ANP preconditioning on necrotic and apoptotic cell death and on proliferation.

Methods: Rat livers were perfused with Krebs-Henseleit buffer with or without ANP or its second messenger analogue 8-Bromo cyclic guanosine monophosphate (8-Br cGMP) for 20 min, stored in cold University of Wisconsin solution (24 h), and reperfused for up to 120 min. Apoptosis and necrosis were determined using biochemical and morphological criteria, proliferation was assessed by Ki67 histochemistry.

Results: Apoptosis peaked after 24 h of cold ischemia. Preconditioning with both ANP and 8-Br-cGMP significantly reduced caspase-3-like activity and the number of triphosphate nick-end labelling-positive cells. Reduction of apoptosis was significant for hepatocytes, but not for endothelial cells. After ischemia, degenerative cell changes were clearly reduced in ANP pretreated livers. After reperfusion, ANP preconditioning led to a significant reduction of necrotic hepatocytes and endothelial cells in periportal zones. Cell proliferation was not affected by preconditioning.

Conclusions: ANP reduces necrotic and apoptotic cell death without affecting the proliferation status. The protection takes place mainly in the periportal area and seems to be most prominent against necrosis of hepatocytes and endothelial cells during reperfusion.
Keywords: Hormonal preconditioning; Liver transplantation; cGMP; Ischemia-reperfusion

Resistance to indomethacin-induced down-regulation of hepatic cytochrome P450 enzymes in the mice with non-functional Toll-like receptor 4
Yasuhiro Masubuchi and Toshiharu Horie
Background/Aims: Repetitive indomethacin administration induces down-regulation of hepatic cytochrome P450 (CYP) enzymes. We tested the hypothesis that an increase in intestinal permeability by indomethacin-induced intestinal injury leads to entry of bacterial endotoxin and reaching into liver via the portal vein, resulting in down-regulations of CYPs.

Methods: C3H/HeJ mice, which are resistant to endotoxin, have a mutation in Toll-like receptor 4 gene. The sensitivity to indomethacin-induced impairment of hepatic CYPs in the lipopolysaccharide (LPS)-resistant mice was examined along with LPS-sensitive (C3H/He) mice.

Results: Treatment of the LPS-sensitive mice with intraperitoneal indomethacin (5 mg/kg per day, 3 days) significantly decreased enzyme activities for CYP3A11, CYP2D9 and CYP1A2 but not CYP2E1. The LPS-resistant mice were resistant to the indomethacin-induced impairment of CYP2D9. The mice were also less sensitive to the effects on CYP3A11 and CYP1A2, but the activities for these isozymes in the indomethacin-treated mice were still lower than in untreated mice. Immunoblot analysis with anti-CYP3A2 and anti-CYP2D2 sera indicated that indomethacin-induced decreases in expression of the proteins recognized by the antibodies were attenuated in the LPS-resistant mice.

Conclusions: We conclude that Toll-like receptor 4 is involved in the indomethacin-induced down-regulation of hepatic CYP enzymes, indicating the pivotal role of gut-derived endotoxin in the hepatic effects.
Keywords: Non-steroidal anti-inflammatory drug; Indomethacin; Cytochrome P450; Enteropathy; Endotoxin; Toll-like receptor 4; Peptidoglycan; C3H/HeJ mouse

Liver Growth and Cancer

Regeneration of hepatocyte `buds' in cirrhosis from intrabiliary stem cells
Olga Falkowski et al.
Background/Aims: In massive hepatic necrosis, hepatic stem cells constitute a canal of Hering derived, cytokeratin 19 (CK19) positive `ductular reaction' (DR). Whether DRs in cirrhosis are activated stem cells (so called `buds') or biliary metaplasia of cholestatic, injured hepatocytes is still debated. We investigate derivation of intraseptal hepatocytes (ISHs) from DRs and from the biliary tree in cirrhosis.

Methods: Explants of hepatitis B and C, alcohol, primary biliary cirrhosis and primary sclerosing cholangitis-related cirrhosis were examined. ISHs were quantified and their associations with DRs and cholestasis recorded. 3D-reconstruction of ISHs and nearby bile ducts was performed in blocks from hepatitis C and primary sclerosing cholangitis cirrhosis.

Results: Seven hundred seventy five/830 (94%) ISHs were associated with CK19 positive DRs. ISHs without ductular reactions were more likely to show cholestatic features (P<0.0001). In 3D, ISHs were seen to bud directly from the biliary tree. In summary: ISHs: (1) are usually associated with stem cell-like DRs; (2) are rarely cholestatic, leaving the associated DRs unexplained; and (3) are linked to the biliary tree in 3D. Dynamic proliferation rates in hepatitis C over time suggest that hepatocyte replication diminishes in late stages, with an associated activation of the biliary stem cell compartment.

Conclusions: We therefore suggest that the biliary tree, from at least its smaller branches up to the canals of Hering, are composed of or at least harbor facultative hepatic stem cells, and that ISH largely represent `buds' of newly formed hepatocytes.
Keywords: Progenitor cells; Liver

Liver regeneration in a retrorsine/CCl4 - induced acute liver failure model: do bone marrow-derived cells contribute?
Marc H. Dahlke et al.
Background/Aims: Adult bone marrow contains progenitors capable of generating hepatocytes. Here a new liver failure model is introduced to assess whether bone marrow-derived progeny contribute to liver regeneration after acute hepatotoxic liver failure.

Methods: Retrorsine was used to inhibit endogenous hepatocyte proliferation, before inducing acute liver failure by carbon tetrachloride. Bone marrow chimeras were generated before inducing liver failure to trace bone marrow-derived cells. Therefore, CD45 and major histocompatibility complex (MHC) class I dimorphic rat models were applied.

Results: Early after acute liver failure a multilineage inflammatory infiltrate was observed, mainly consisting of granulocytes. In long-term experiments small numbers of CD90+/CD45 cells of donor origin occurred in clusters associated with portal triads. Bone marrow cell infusion was not able to enhance liver regeneration. Cellular hypertrophy was the predominant way of liver mass regeneration in models applying retrorsine.

Conclusions: Retrorsine pretreatment did not affect sensitivity for carbon tetrachloride. A multilineage inflammatory infiltrate was observed in rats whether pretreated with retrorsine or not. Few donor cells co-expressing CD90 (THY 1) were present in recipient livers, which may resemble donor-derived hematopoietic progenitors or oval cells. No other donor cells within liver parenchyma were detected. This is in contrast to other cell infusion models of acute cell death.
Keywords: Transdifferentiation; Retrorsine; Acute liver failure; Adult stem cells; Oval cells; Small hepatocyte like progenitor cell; Liver regeneration

Trichostatin A induces differential cell cycle arrests but does not induce apoptosis in primary cultures of mitogen-stimulated rat hepatocytes
Peggy Papeleu et al.

Background/Aims: The effects of Trichostatin A (TSA), a drug candidate for cancer therapy, on proliferation and survival of primary hepatocytes, the major site of xenobiotic biotransformation and primary target of drug-induced toxicity, were investigated.

Methods: DNA replication was measured using [methyl-3H]-thymidine incorporation. Cell cycle markers were analyzed by Western and Northern blottings. Necrosis and apoptosis were monitored by LDH release, caspase-3-activation, respectively.

Results: We identified two distinct cell cycle arrests, prior DNA replication, in two experimental conditions. First, perfusion of the liver in presence of TSA, prevented c-jun and cyclin D1 induction, characteristic for G1 entry and progression through late G1, respectively. Secondly, TSA treatment of isolated hepatocytes, located in early G1, led to an early S-phase arrest evidenced by the absence of the S/G2/M marker, CDK1. TSA upregulated the expression of the anti-apoptotic protein BclxL and did not increase caspase-3-activity and LDH release.

Conclusions: TSA inhibits hepatocyte proliferation at different steps of the cell cycle. Our data suggest that this inhibition may involve downregulation of distinct subsets of genes. TSA does not induce apoptosis in primary hepatocytes, in contrast to what has been observed in hepatoma cells. This finding supports its use in the treatment of proliferative disorders.
Keywords: Histone deacetylase inhibitor; Growth arrest; c-jun; Immediate-early oncogenes; Cyclin D1; Apoptosis; Caspase 3

The lack of a specific association between arsenic in drinking water and hepatocellular carcinoma
How-Ran Guo
Background/Aims: Liver cancers, particularly angiosarcomas, among patients with arsenic intoxication have been reported for more than half a century. Studies on cancers of urinary system and skin showed the carcinogenic effect of arsenic was cell-type specific. To evaluate whether this is also true for liver cancers, a study was conducted in 243 townships in Taiwan.

Methods: Cases of liver cancer were identified through the National Cancer Registry Program. The distribution of major cell-types was compared between an endemic area of arsenic intoxication, including 5 of the 243 townships, and the rest of the townships.

Results: A total of 40,832 patients with liver cancer, including 32,034 men and 8798 women, were diagnosed between January 1, 1980 and December 31, 1999 in the study townships. Distributions of the two major cell-types, hepatocellular carcinoma and cholangiocarcinoma, did not appear to be different between the arsenic intoxication endemic area and the other areas. The rest of the cell-types did not have enough cases to provide stable estimates.

Conclusions: Convincing evidence in the literature has suggested a specific association between angiosarcoma of liver and arsenic ingestion, but the current study did not find such an association for hepatocellular carcinoma, although it is the major cell-type.
Keywords: Hepatocelluar carcinoma; Cholangiocarcinoma; Hepatic angiosarcoma; Blackfoot disease; Cell-type specificity

Transplantation

Antiviral therapy of patients with decompensated cirrhosis to prevent recurrence of hepatitis C after liver transplantation
Xavier Forns et al.
Background/Aims: After liver transplantation (LT) infection of the graft with the hepatitis C virus (HCV) is almost universal and chronic hepatitis and cirrhosis develop in a significant proportion of patients. One of the possible strategies to prevent HCV infection recurrence is to eradicate HCV before LT.

Methods: We evaluated the efficacy and safety of antiviral therapy to prevent HCV recurrence in 30 HCV-cirrhotic patients awaiting LT. At the time of inclusion 15 patients were Child-Pugh A and 15 Child-Pugh B/C. The infecting genotype was 1b in 25 patients. Treatment with interferon -2b 3 MU/day and ribavirin 800 mg/day was initiated when the expected time for LT was less than 4 months and continued until LT. The median duration of treatment was 12 weeks.

Results: Nine patients (30%) achieved a virological response and 21 did not respond to therapy. In nine (43%) of the 21 non-responders viral load decreased \ge 2 log10 during treatment. A viral load decrease \ge 2 log10 at week 4 of treatment was the strongest predictor of virological response. All nine virological responders have already undergone LT; six patients remain free of infection after a median follow-up of 46 weeks and HCV infection recurred in three patients after LT. In one of these patients HCV-RNA was still detectable in the explanted liver. Side effects were frequent and dose reduction was necessary in 19 (63%) of the 30 patients; no patient died while on therapy.

Conclusions: Our data support the utilization of antiviral therapy in HCV-infected patients awaiting LT as one of the strategies to prevent hepatitis C recurrence after transplantation.
Keywords: Interferon; Ribavirin; Liver graft; Virological response; Viral load

Antiviral treatment of recurrent hepatitis C virus (HCV) infection after liver transplantation: association of a strong, multispecific, and long-lasting CD4+ T cell response with HCV-elimination
Carl Albrecht Schirren et al.
Background/Aims: Patients with recurrent hepatitis C virus (HCV)-infection after liver transplantation (OLTx) could develop an early, multispecific, preferentially intrahepatic CD4+ T cell response. We asked now whether there is a correlation between the HCV-specific CD4+ T cell response and treatment outcome in patients who receive interferon (IFN)-/ribavirin.

Methods: Liver- and blood-derived T cell lines of 20 patients were studied in parallel before, under, at the end and after antiviral treatment. Virus-specific IFN- production at a single cell level to HCV-proteins (core, non-structural protein (NS)3/4, NS5) was determined by enzyme-linked immunospot assay.

Results: In 6/7 non-responders a weak HCV-specific CD4+ T cell response was detectable. All six sustained responders developed a strong, at NS3/4 and NS5 directed and long-lasting CD4+ T cell response which was mainly detected in peripheral blood mononuclear cells. This reaction was significantly stronger: (1) in the responders than in the non-responders; and (2) within the responders at the end of treatment than before (P<0.03). Seven transient-responders showed a weak and/or transient HCV-specific CD4+ T cell response.

Conclusions: In patients with recurrent HCV-infection after OLTx, who receive antiviral treatment, a strong, at NS3/4 and NS5 directed and long-lasting CD4+ T cell response is associated with HCV-elimination whereas no or a weak/transient response is associated with treatment failure.
Keywords: Hepatitis C virus-specific T cell response; Recurrent hepatitis C virus-infection; Liver transplantation; Enzyme-linked immunospot; Antiviral therapy

Abbreviations: HCV, hepatitis C virus; OLTx, orthotopic liver transplantation; IFN, interferon; HCV-RNA, HCV-ribonucleotid acid; IL-2, interleukin-2; NS, non-structural protein; C, core; PBMC, peripheral blood mononuclear cells; ELISPOT, enzyme-linked immunospot; HCC, hepatocellular carcinoma; CTL, cytotoxic T lymphocytes; aa, amino acid; HAI, histological activity index; RAI, rejection activity index; PBS, phosphate-buffered saline

Viral Hepatitis

Induction of antibodies to the PreS region of surface antigens of woodchuck hepatitis virus (WHV) in chronic carrier woodchucks by immunizations with WHV surface antigens
Mengji Lu et al.
Background/Aims: One goal of therapeutic vaccinations against chronic hepatitis B virus infection is to stimulate the B-cell responses to viral surface antigens in chronic carriers. Here we investigated the induction of antibody responses to hepadnaviral surface antigens in the woodchuck model, with emphasis on the vaccination of woodchucks chronically infected with woodchuck hepatitis virus (WHV).

Methods: Naive and chronically WHV-infected woodchucks were immunized with plasma-derived WHV surface antigens (p-WHsAg) containing the S and PreS sequences. Antibody responses to WHsAg and the WHV PreS region and viral load in immunized woodchucks were monitored.

Results: After repeated immunizations with WHsAg, 17 of 18 chronic WHV carriers developed a persistent antibody response to WHsAg. These antibodies were mainly directed to epitopes within the PreS region and detectable by Western blotting. However, neither WHV DNA nor WHsAg concentrations in these woodchucks changed significantly by immunizations and during the follow up. Sequence analysis of WHV genomes showed that no WHV mutants emerged after the induction of anti-WHs/anti-WHpreS antibodies. No immunopathological changes in livers of immunized animals were recognized thus far.

Conclusions: Our study demonstrated that the immunological unresponsiveness of chronically WHV-infected woodchucks to WHsAg can be partially overcome by repeated immunizations with WHsAg.
Keywords: Woodchuck model; Therapeutic immunization; Anti-WHpreS response; Hepatitis B virus

Hepatitis B virus enhances tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) cytotoxicity by increasing TRAIL-R1/death receptor 4 expression
Harry L.A. Janssen, Hajime Higuchi, Ahmad Abdulkarim and Gregory J. Gores
Background/Aims: Apoptosis by death receptors, such as Fas and tumor necrosis factor (TNF)-alpha receptor-1, play a significant role in the pathogenesis of hepatitis B virus (HBV)-infections. Although liver also expresses death receptors for TNF-related apoptosis-inducing ligand (TRAIL), information is lacking regarding the effects of HBV on apoptosis by TRAIL. Thus, the aims of this study were to examine the effects of HBV replication on TRAIL cytotoxicity.

Methods: Hep G2 and Hep G2.215 cells, the latter which is stably transfected with HBV, were employed for these studies.

Results: TRAIL-mediated cell killing was concentration-dependent and greater in Hep G2.2.15 cells at all doses as compared to the parent cell line, Hep G2 cells. Cell death by apoptosis was confirmed by demonstrating caspase activation and inhibition of cell killing by a caspase inhibitor, zVAD-fmk. TRAIL-R1/DR4 protein expression was enhanced in Hep G2.2.15 cells as compared to Hep G2 cells. Lamivudine treatment reduced TRAIL-mediated apoptosis and TRAIL-R1/DR4 expression in Hep G2.2.15 cells. In Hep G2 cells transfected with the HBV-encoded X antigen (HBxAg), sensitivity to TRAIL-mediated apoptosis and TRAIL-R1/DR4 expression were both increased.

Conclusions: TRAIL-induced apoptosis is enhanced by the level of HBV replication in human hepatocytes, in part, by HBxAg-dependent upregulation of TRAIL-R1/DR4.
Keywords: Apoptosis; Lamivudine

Interferon-stimulated gene expression and hepatitis C viral dynamics during different interferon regimens
Yasuhiro Asahina et al.
Background/Aims: To address the molecular mechanism for enhanced antiviral efficacy associated with a frequent dosing of interferon (IFN)-beta.

Methods: Serum hepatitis C viral (HCV) dynamics, double-stranded RNA-activated protein kinase (PKR) mRNA and MxA mRNA levels in peripheral blood mononuclear cells (PBMC) were analyzed serially in 140 patients who were randomly assigned to a twice daily (3MU bid) or once daily (6MU qd) administration group.

Results: In twice daily group, the rate of HCV decline during the second phase was 2-fold greater than in the once daily group (P=0.04). Peak PKR and MxA gene expression levels in the first phase (observed 4 h after a single administration) were 2-fold higher in the once daily group. However, the expression in the second phase was maintained at a significantly higher level in the twice daily group. Initial and peak expression levels were related to initial viral load. Basal expressions in PBMC were significantly correlated with those in the liver tissue (PKR, r=0.81; MxA, r=0.75, respectively, P<0.0001).

Conclusions: Our data suggest that elimination of HCV-infected cells is enhanced by twice daily dosing of IFN-beta, and that this enhanced effect is associated with a higher intracellular expression of PKR and MxA during the second phase.
Keywords: Double-stranded RNA-activated protein kinase (PKR); MxA; Viral kinetics; Peripheral blood mononuclear cell; Real-time detection PCR; Interferon-beta

Abbreviations: IFN, interferon; ISG, interferon-stimulated gene; PXR, double-stranded RNA-activated protein kinase; HCV, hepatitis C virus; ALT, alanine aminotransferase; PBMC, peripheral blood mononuclear cell; G3PDH, glyceraldehyde-3-phosphate dehydrogenase

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

BRITISH MEDICAL JOURNAL

© 2003 BMJ Publishing Group Ltd

NEW ENGLAND JOURNAL

The New England Journal of Medicine is owned, published, and copyrighted © 2003 Massachusetts Medical Society. All rights reserved.

LANCET

The Lancet, published, and copyrighted © 2003. All rights reserved.