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HEPATOLOGY

Table of Contents for October 2004 - Volume 40 - Issue 4 

Perspectives in Clinical Hepatology

Polycystic disease of the liver (p 774-782)
Gregory T. Everson, Matthew R. G. Taylor, R. Brian Doctor
Autosomal dominant polycystic disease is genetically heterogeneous with mutations in two distinct genes predisposing to the combination of renal and liver cysts (AD-PKD1 and AD-PKD2) and mutations in a third gene yielding isolated liver cysts (the polycystic liver disease gene). Transcription and translation of the PKD1 gene produces polycystin-1, an integral membrane protein that may serve as an extracellular receptor. Mutations occur throughout the PKD1 gene, but more severe disease is associated with N-terminal mutations. The PKD2 gene product, polycystin-2, is an integral membrane protein with molecular characteristics of a calcium-permeant cation channel. Mutations occur throughout the PKD2 gene, and severity of disease may vary with site of mutation in PKD2 and the functional consequence on the resultant polycystin-2 protein. Polycystic liver disease is genetically linked to protein kinase C substrate 80K-H (PRKCSH). The PRKCSH gene encodes hepatocystin, a protein that moderates glycosylation and fibroblast growth factor receptor signaling. More prominent in women, hepatic cysts emerge after the onset of puberty and dramatically increase in number and size through the child-bearing years of early and middle adult life. Although liver failure or complications of advanced liver disease are rare, some patients develop massive hepatic cystic disease and become clinically symptomatic. There is no effective medical therapy. Interventional and surgical options include cyst aspiration and sclerosis, open or laparoscopic cyst fenestration, hepatic resection, and liver transplantation. (HEPATOLOGY 2004;40:774-782.)

Liver Failure and Liver Disease

Influence of portal hypertension and its early decompression by TIPS placement on the outcome of variceal bleeding (p 793-801)
Alberto Monescillo, Francisco Martínez-Lagares, Luis Ruiz-del-Arbol, Angel Sierra, Clemencia Guevara, Elena Jiménez, José Miguel Marrero, Enrique Buceta, Juan Sánchez, Ana Castellot, Mónica Peñate, Ana Cruz, Elena Peña
Increased portal pressure during variceal bleeding may have an influence on the treatment failure rate, as well as on short- and long-term survival. However, the usefulness of hepatic hemodynamic measurement during the acute episode has not been prospectively validated, and no information exists about the outcome of hemodynamically defined high-risk patients treated with early portal decompression. Hepatic venous pressure gradient (HVPG) measurement was made within the first 24 hours after admission of 116 consecutive patients with cirrhosis with acute variceal bleeding treated with a single session of sclerotherapy injection during urgent endoscopy. Sixty-four patients had an HVPG less than 20 mm Hg (low-risk [LR] group), and 52 patients had an HVPG greater than or equal to 20 mm Hg (high-risk [HR] group). HR patients were randomly allocated into those receiving transjugular intrahepatic portosystemic shunt (TIPS; HR-TIPS group, n = 26) within the first 24 hours after admission and those not receiving TIPS (HR-non-TIPS group). The HR-non-TIPS group had more treatment failures (50% vs. 12%, P= .0001), transfusional requirements (3.7 ± 2.7 vs. 2.2 ± 2.3, P= .002), need for intensive care (16% vs. 3%, P< .05), and worse actuarial probability of survival than the LR group. Early TIPS placement reduced treatment failure (12%, P= .003), in-hospital and 1-year mortality (11% and 31%, respectively; P< .05). In conclusion , increased portal pressure estimated by early HVPG measurement is a main determinant of treatment failure and survival in variceal bleeding, and early TIPS placement reduces treatment failure and mortality in high risk patients defined by hemodynamic criteria. (HEPATOLOGY 2004;40:793-801.)

Persistent ascites and low serum sodium identify patients with cirrhosis and low MELD scores who are at high risk for early death (p 802-810)
Douglas M. Heuman, Souheil G. Abou-assi, Adil Habib, Leslie M. Williams, R. Todd Stravitz, Arun J. Sanyal, Robert A. Fisher, Anastasios A. Mihas
Despite the adoption of sickest first liver transplantation, pretransplant death remains common, and many early deaths occur despite initially low Model for End-stage Liver Disease (MELD) scores. From 1997-2003, we studied 507 cirrhotic United States veterans referred for consideration of liver transplantation to identify additional predictors of early mortality. Most of the patients were male (98%) with cirrhosis caused by hepatitis C and/or alcohol (88%). Data for 296 patients referred prior to February 27, 2002 (training group), were analyzed; findings were validated in 211 patients referred subsequently (validation group). In the training group, 61 patients (21%) died within 180 days without transplantation; their median initial MELD score was 21. MELD score, persistent ascites, and low serum sodium (<135 meq/L) were independent predictors of early mortality. In patients with a MELD score of less than 21, only low serum sodium and persistent ascites were independent predictors of mortality; for MELD scores above 21, only MELD was independently predictive. Prognostic significance of persistent ascites and low serum sodium for low MELD score patients was confirmed in the validation group. Risk varied continuously with worsening hyponatremia. Modifying MELD, by including points for persistent ascites and low serum sodium, improved prediction of early pretransplant mortality in low MELD score patients. In conclusion , persistent ascites and low serum sodium identify patients with cirrhosis with high mortality risk despite low MELD scores. Ascites, hyponatremia, and other findings indicative of hemodynamic decompensation merit further prospective study as prognostic indicators in patients awaiting liver transplantation, and should be considered in setting minimal listing criteria.

Effects of tilting on central hemodynamics and homeostatic mechanisms in cirrhosis (p 811-819)
Søren Møller, Annette Nørgaard, Jens H. Henriksen, Erik Frandsen, Flemming Bendtsen
Patients with cirrhosis have a hyperdynamic circulation and an abnormal blood volume distribution with central hypovolemia, an activated sympathetic nervous system (SNS) as well as the renin-angiotensin-aldosterone system (RAAS). As the hyperdynamic circulation in cirrhosis may be present only in the supine patient, we studied the humoral and central hemodynamic responses to changes with posture. Twenty-three patients with alcoholic cirrhosis (Child-Turcotte-Pugh classes A/B/C: 2/13/8) and 14 healthy controls were entered. Measurements of central hemodynamics and activation of SNS and RAAS were taken in the supine position, after 30° head-down tilting, and after 60° passive head-up tilting for a maximum of 20 minutes. After the head-up tilting, the central blood volume (CBV) decreased in both groups, but the decrease was significantly smaller in patients than in controls (-19% vs. -36%, P< .01). Central circulation time increased only in the patients (+30% vs. -1%, P< .01). The absolute increases in circulating norepinephrine and renin after head-up tilting were significantly higher in the patients than in the controls ( P< .05 and P< .01, respectively). In patients with cirrhosis, changes in SNS and RAAS were related to changes in arterial blood pressure, systemic vascular resistance, heart rate, non-CBV, plasma volume, and arterial compliance. In conclusion , cardiovascular and humoral responses to changes in posture are clearly abnormal in patients with cirrhosis. Head-up tilting decreases the CBV less in patients with cirrhosis, and the results suggest a differential regulation of central hemodynamics in patients with cirrhosis. (HEPATOLOGY 2004;40:811-819.)

Natural history of nonalcoholic steatohepatitis: A longitudinal study of repeat liver biopsies (p 820-826)
Eduardo Fassio, Estela Álvarez, Nora Domínguez, Graciela Landeira, Cristina Longo
Nonalcoholic steatohepatitis may cause severe fibrosis, cirrhosis, and hepatocellular carcinoma, but supporting evidence is based on indirect data. Few publications have examined the results of repeat liver biopsies to evaluate progression of fibrosis. The aims of this study were to assess rate of fibrosis progression in untreated patients with nonalcoholic steatohepatitis and to identify associated variables. Among 106 patients, a second liver biopsy was proposed to those who had undergone their first liver biopsy at least 3 years before. None of them had been given pharmacological therapy. Liver biopsy samples were evaluated blindly. Variables were compared between patients with (group P) and without (group NP) fibrosis progression, using a Wilcoxon rank-sum test for numerical variables and a difference of two binomial proportions for categorical ones. Twenty-two patients (median age, 45 years; age range, 20-69 years; 13 women; diabetes in 8 patients, obesity in 10 patients) underwent a second liver biopsy 4.3 years (range, 3.0-14.3 years) after the first. Fibrosis progression was found in 7 patients in group P (31.8%), no progression was found in 15 patients in group NP. There were no differences between both groups regarding age, gender, diabetes, hyperlipidemia, ALT levels, AST-to-ALT ratio levels, albumin levels, prothrombin activity, steatosis, or inflammation. Obesity was significantly more prevalent in group P (86%) than in group NP (27%; P= .01). Basal body mass index was higher in group P (median, 33.2; range, 29.1-38.2) than in group NP (median, 29.0; range, 24.0-38.1; P= .024). Time between biopsies was not different between groups. In conclusion , progression of liver fibrosis was found in a third of nonalcoholic steatohepatitis patients 4.3 years after the first liver biopsy, and obesity and body mass index were the only associated factors with such progression. (HEPATOLOGY 2004;40:820-826.)

Serum bilirubin levels in the U.S. population: Gender effect and inverse correlation with colorectal cancer (p 827-835)
Stephen D. Zucker, Paul S. Horn, Kenneth E. Sherman
Bilirubin, the primary end product of heme catabolism, is a key marker of liver and hematological disorders, and important cytoprotective properties have been ascribed to this bile pigment. The Third National Health and Nutrition Examination Survey, a comprehensive assessment of health and nutrition in the United States, was analyzed to determine the demographics and correlates of serum bilirubin levels in the general population. Men and women aged 17 and older were included in the weighted analysis, representing a total of 176,748,462 subjects. The mean serum total bilirubin in the adult population is 0.62 ± 0.003 mg/dL (SEM), with a 97.5% cut-off of 1.4 mg/dL. Serum bilirubin levels are significantly higher in men (0.72 ± 0.004) than in women (0.52 ± 0.003 mg/dL) and are lower in non-Hispanic blacks (0.55 ± 0.005 mg/dL) compared with non-Hispanic whites (0.63 ± 0.004 mg/dL) and Mexican Americans (0.61 ± 0.005 mg/dL). Bilirubin concentrations are unrelated to body weight but are reduced in active smokers. Individuals with a history of nondermatological malignancy exhibit significantly lower serum bilirubin concentrations compared with those who do not have a history of nondermatological cancer. In particular, each 1-mg/dL increase in serum bilirubin is associated with a markedly decreased prevalence of colorectal cancer (OR = 0.257; 95% CI 0.254-0.260). In conclusion , serum bilirubin levels vary significantly with gender, race, and smoking status. The observed inverse correlation between serum bilirubin concentrations and a history of nondermatological malignancy, particularly colorectal cancer, warrants further investigation of a potentially important chemopreventive function of bilirubin. (HEPATOLOGY 2004;40:827-835.)

Secretion of cytokines and growth factors into autosomal dominant polycystic kidney disease liver cyst fluid (p 836-846)
Matthew T. Nichols, Elsa Gidey, Tom Matzakos, Rolf Dahl, Greg Stiegmann, Raj J. Shah, Jared J. Grantham, J. Gregory Fitz, R. Brian Doctor
The principal extrarenal manifestation of autosomal dominant polycystic kidney disease (ADPKD) involves formation of liver cysts derived from intrahepatic bile ducts. Autocrine and paracrine factors secreted into the cyst would be positioned to modulate the rate of hepatic cyst growth. The aim of this study was to identify potential growth factors present in human ADPKD liver cyst fluid. Cytokine array and enzyme-linked immunosorbent assay analysis of human ADPKD liver cyst fluid detected epithelial neutrophil attractant 78, interleukin (IL)-6 (503 ± 121 pg/mL); and IL-8 (4,488 ± 355 pg/mL); and elevated levels of vascular endothelial growth factor compared with non-ADPKD bile (849 ± 144 pg/mL vs. 270 pg/mL maximum concentration). ADPKD liver cyst cell cultures also released IL-8 and vascular endothelial growth factor, suggesting that cystic epithelial cells themselves are capable of secreting these factors. Western blotting of cultured cyst cells and immunostaining of intact cysts demonstrate that cysteine-X-cysteine receptor 2, an epithelial neutrophil attractant 78 and IL-8 receptor, is expressed at the apical domain of cyst lining epithelial cells. Suggesting the cystic epithelial cells may exist in hypoxic conditions, electron microscopy of the ADPKD liver cyst epithelium revealed morphological features similar to those observed in ischemic bile ducts. These features include elongation, altered structure, and diminished abundance of apical microvilli. In conclusion , IL-8, epithelial neutrophil attractant 78, IL-6, and vascular endothelial growth factor may serve as autocrine and paracrine factors to direct errant growth of ADPKD liver cyst epithelia. Interruption of these signaling pathways may provide therapeutic targets for inhibiting liver cyst expansion. (HEPATOLOGY 2004;40:836-846.)

Viral Hepatitis

Allelic loss of chromosome 4q21 23 associates with hepatitis B virus-related hepatocarcinogenesis and elevated alpha-fetoprotein (p 847-854)
Shiou-Hwei Yeh, Ming-Wei Lin, Shu-Fen Lu, Dai-Chen Wu, Shih-Feng Tsai, Ching-Yi Tsai, Ming-Yang Lai, Hey-Chi Hsu, Ding-Shinn Chen, Pei-Jer Chen
Allelic loss of chromosome 4q is one of the most frequent genetic aberrations found in human hepatocellular carcinoma (HCC) and suggests the presence of putative tumor suppressor genes within this region. To precisely define the region containing these tumor suppressor genes for further positional cloning, we tried a detailed deletion mapping strategy in 149 HCCs by using 49 microsatellite markers covering 4q12 25. A common region with allelic loss has been identified based on the interstitial deletions occurring within it; this region is found between D4S1534 and D4S1572 (a 17.5-cM genetic interval). When we included all cases with limited aberration regions for comparison, 2 smaller regions were derived: 1 between D4S1534 and D4S2460 (3.52 cM) and 1 between D4S2433 and D4S1572 (8.44 cM). A few candidate genes were found to be down-regulated in HCCs, but without sequence mutations. In these HCCs, 4q alleleic loss was associated with hepatitis B virus infection status and the elevation of serum alpha-fetoprotein ( 400 ng/mL). In conclusion , the current study not only mapped a common allelic loss region on chromosome 4q, but it also revealed that its loss may be involved in hepatitis B virus-related hepatocarcinogenesis and the elevation of serum alpha-fetoprotein. ( HEPATOLOGY 2004;40:847-854.)

A new strategy for studying in vitro the drug susceptibility of clinical isolates of human hepatitis B virus (p 855-864)
David Durantel, Sandra Carrouée-Durantel, Bettina Werle-Lapostolle, Marie-Noëlle Brunelle, Christian Pichoud, Christian Trépo, Fabien Zoulim
Resistance of hepatitis B virus (HBV) to antivirals has become a major clinical problem. Our objective was to develop a new method for the cloning of naturally occurring HBV genomes and a phenotypic assay capable of assessing HBV drug susceptibility and DNA synthesis capacity in vitro . Viral DNA was extracted from sera and was amplified by polymerase chain reaction, and amplicons were cloned into vectors that enable, after cell transfection, the initiation of the intracellular HBV replication cycle. Single or multiple clones were used to transfect Huh7 cells. The viral DNA synthesis capacity and drug susceptibility were determined by measuring the level of intracellular DNA intermediate, synthesized in absence or presence of antiviral, using Southern blot analysis. We have developed, calibrated, then used this phenotypic assay to determine the drug susceptibility of HBV quasispecies isolated throughout the course of therapy from patients selected according to their mutation profile. A multiclonal and longitudinal analysis enabled us to measure the variation of drug susceptibility of different viral quasispecies by comparison of IC 50 /IC 90 s with standards. The presence of famciclovir- or lamivudine-induced mutations in the viral population caused a change in viral DNA synthesis capacity and drug susceptibility in vitro , demonstrating the clinical relevance of the assay. In conclusion , our phenotypic assay enables the in vitro characterization of DNA synthesis capacity and drug susceptibility of HBV quasispecies isolated from patients. This assay should allow a better monitoring of patients undergoing antiviral therapy, as well as the screening of novel drugs on emerging resistant strains. (HEPATOLOGY 2004;40:855-864.)

Factors associated with fulminant liver failure during an outbreak among injection drug users with acute hepatitis B (p 865-873)
Richard S. Garfein, William A. Bower, Cherry M. Loney, Yvan J. F. Hutin, Guo-liang Xia, Jaspaul Jawanda, Amy V. Groom, Omana V. Nainan, James S. Murphy, Beth P. Bell
Death related to acute hepatitis B occurs in approximately 1% of patients. We investigated an outbreak of hepatitis B virus (HBV) infections among injection drug users (IDUs) resulting in several deaths. We conducted a case-control study of fulminant (case patients) and nonfulminant (control patients) HBV infections. We directly sequenced the entire HBV genome from fulminant and nonfulminant cases. From October 1998 to July 2000, 21 acute HBV infections, including 10 fulminant hepatitis B cases, were identified. The median age was 30 (range, 18-49) years, 12 (57%) were female, 20 (95%) were American Indians, and 20 (95%) reported injecting illicit drugs. All patients with fulminant hepatitis B died (case-fatality rate = 47.6%). Case patients (n = 5) and control patients (n = 9) were similar with respect to age, sex, race, and hepatitis C virus serostatus. All case patients used acetaminophen during their illness compared with 44% of control patients ( P= .08). Compared with control patients, case patients lost more weight in the 6 months before illness ( P= .04); during their illness, they used more alcohol ( P= .03) and methamphetamine ( P= .04). All 9 isolates sequenced were genotype D, shared 99.7% homology, and included mutations previously described in association with fulminant hepatitis B. In conclusion , a high prevalence of exposure to factors potentiating hepatic damage with acute hepatitis B contributed to the outbreak's high mortality rate; mutations present in the outbreak strain might also have been a factor. Improved vaccination coverage among IDUs has the potential to prevent similar outbreaks in the future. (HEPATOLOGY 2004;40:865-873.)

Induction or expansion of T-cell responses by a hepatitis B DNA vaccine administered to chronic HBV carriers (p 874-882)
Maryline Mancini-Bourgine, Hélène Fontaine, Daniel Scott-Algara, Stanislas Pol, Christian Bréchot, Marie-Louise Michel
Despite the availability of effective hepatitis B vaccines for many years, over 370 million people remain persistently infected with hepatitis B virus (HBV). Viral persistence is thought to be related to poor HBV-specific T-cell responses. A phase I clinical trial was performed in chronic HBV carriers to investigate whether HBV DNA vaccination could restore T-cell responsiveness. Ten patients with chronic active hepatitis B nonresponder to approved treatments for HBV infection were given 4 intramuscular injections of 1 mg of a DNA vaccine encoding HBV envelope proteins. HBV-specific T-cell responses were assessed by proliferation, ELISpot assays, and tetramer staining. Secondary end points included safety and the monitoring of HBV viraemia and serological markers. Proliferative responses to hepatitis B surface antigen were detected in two patients after DNA injections. Few HBV-specific interferon -secreting T cells were detectable before immunization, but the frequency of such responses was significantly increased by 3 DNA injections. Immunization was well tolerated. Serum HBV DNA levels decreased in 5 patients after 3 vaccine injections, and complete clearance was observed in 1 patient. In conclusion , this study provides evidence that HBV DNA vaccination is safe and immunologically effective. We demonstrate that DNA vaccination can specifically but transiently activate T-cell responses in some chronic HBV carriers who do not respond to current antiviral therapies.

Clinical outcome of HBeAg-negative chronic hepatitis B in relation to virological response to lamivudine (p 883-891)
Vito Di Marco, Alfredo Marzano, Pietro Lampertico, Pietro Andreone, Teresa Santantonio, Piero Luigi Almasio, Mario Rizzetto, Antonio Craxì, fot the Italian Association for the Study of the Liver (AISF) Lamivudine Study Group, Italy
The effect of lamivudine treatment on the outcome of patients with hepatitis B e antigen (HBeAg)-negative chronic hepatitis is unclear. In a retrospective multicenter study, we have analyzed the virological events observed during lamivudine therapy in patients with HBeAg-negative chronic hepatitis and evaluated the correlation between virological response and clinical outcomes. Among 656 patients (mean age 49.1 years) included in the database, 54% had chronic hepatitis, 30% had Child-Turcotte-Pugh (CTP) A cirrhosis, and 16% had CTP B/C cirrhosis. On therapy (median 22 months, range 1-66), a virological response was obtained in 616 patients (93.9%). The rate of maintained virological response was 39% after 4 years. During follow-up, 47 (7.2%) patients underwent liver transplantation, liver disease worsened in 31 (4.7%), hepatocellular carcinoma (HCC) developed in 31 (4.7%), and 24 patients (3.6%) died of liver-related causes. Patients who had cirrhosis and who maintained virological response were less likely than those with viral breakthrough to develop HCC ( P< .001) and disease worsening ( P< .001). Survival was better in CTP A patients with cirrhosis and maintained virological response ( P= .01 by rank test). Multivariate analysis revealed that presence of cirrhosis and viral breakthrough were independently related to mortality and development of HCC. In conclusion , lamivudine is highly effective in reducing viral load in HBeAg-negative patients. After 4 years of therapy, 39% of patients maintain a virological and biochemical response. Loss of virological response may lead to clinical deterioration in patients with cirrhosis. (HEPATOLOGY 2004;40:883-891.)

Influence of alcohol use, race, and viral coinfections on spontaneous HCV clearance in a US veteran population (p 892-899)
Barbara A. Piasecki, James D. Lewis, K. Rajender Reddy, Scarlett L. Bellamy, Steven B. Porter, Robert M. Weinrieb, Donald D. Stieritz, Kyong-Mi Chang
The hepatitis C virus (HCV) F protein is a recently described, frameshift product of HCV core encoding sequence of genotype 1a. Its function and antigenic properties are unknown. Using enzyme-linked immunosorbent assay, we assessed the prevalence of anti-F antibodies in 154 patients chronically infected with HCV, 65 patients with other liver diseases, and 121 healthy controls. For this purpose, we expressed a highly purified HCV F recombinant protein from HCV genotype 1a in Escherichia coli . Because the F protein shares the 10 first amino acids with the core protein, the anti-HCV F response was also assessed by a F recombinant protein deleted of its 10 first amino acids [ (1-10)-F]. Ninety-six (62%) of the 154 HCV serum samples reacted with the complete F recombinant protein, whereas 39 (25%) showed a weaker anti- (1-10)F reactivity and 150 (97%) had anti-core antibodies. No reactivity against F, (1-10)F, or core was detected in any of the controls. To exclude a potential cross-reaction of anti-F antibodies with anti-core antibodies, a specific enzyme-linked immunosorbent assay was performed for anti-core antibodies. The specificity of anti-F antibodies was confirmed using an F synthetic peptide. The prevalence of anti-F antibodies did not correlate with HCV RNA serum level, genotype, or stage of liver disease. Sequence analysis from 8 anti-F-positive and 5 anti-F-negative serum samples did not reveal any particular difference potentially accounting for their respective anti-F responses. In conclusion , the F protein elicits specific antibodies in 62% of individuals chronically infected with HCV; such anti-F response does not seem to be affected by the F sequence heterogeneity. (HEPATOLOGY 2004;40:900-909.)

Antigenic relevance of F protein in chronic hepatitis C virus infection (p 900-909)
Florence Komurian-Pradel, Alain Rajoharison, Jean-Luc Berland, Valérie Khouri, Magali Perret, Mark van Roosmalen, Stanislas Pol, Francesco Negro, Glaucia Paranhos-Baccalà
The hepatitis C virus (HCV) F protein is a recently described, frameshift product of HCV core encoding sequence of genotype 1a. Its function and antigenic properties are unknown. Using enzyme-linked immunosorbent assay, we assessed the prevalence of anti-F antibodies in 154 patients chronically infected with HCV, 65 patients with other liver diseases, and 121 healthy controls. For this purpose, we expressed a highly purified HCV F recombinant protein from HCV genotype 1a in Escherichia coli . Because the F protein shares the 10 first amino acids with the core protein, the anti-HCV F response was also assessed by a F recombinant protein deleted of its 10 first amino acids [ (1-10)-F]. Ninety-six (62%) of the 154 HCV serum samples reacted with the complete F recombinant protein, whereas 39 (25%) showed a weaker anti- (1-10)F reactivity and 150 (97%) had anti-core antibodies. No reactivity against F, (1-10)F, or core was detected in any of the controls. To exclude a potential cross-reaction of anti-F antibodies with anti-core antibodies, a specific enzyme-linked immunosorbent assay was performed for anti-core antibodies. The specificity of anti-F antibodies was confirmed using an F synthetic peptide. The prevalence of anti-F antibodies did not correlate with HCV RNA serum level, genotype, or stage of liver disease. Sequence analysis from 8 anti-F-positive and 5 anti-F-negative serum samples did not reveal any particular difference potentially accounting for their respective anti-F responses. In conclusion , the F protein elicits specific antibodies in 62% of individuals chronically infected with HCV; such anti-F response does not seem to be affected by the F sequence heterogeneity. (HEPATOLOGY 2004;40:900-909.)

Liver Biology and Pathobiology

Overexpression of cholesterol transporter StAR increases in vivo rates of bile acid synthesis in the rat and mouse (p 910-917)
Shunlin Ren, Phillip B. Hylemon, Dalila Marques, Emily Gurley, Patricia Bodhan, Elizabeth Hall, Kaye Redford, Gregorio Gil, William M. Pandak
Bile acid synthesis (BAS) occurs mainly via two pathways: the neutral pathway, which is initiated by highly regulated microsomal CYP7A1, and an acidic pathway, which is initiated by mitochondrial CYP27A1. Previously, we have shown that overexpression of the steroidogenic acute regulatory protein (StAR), a mitochondrial cholesterol transport protein, increases bile acid biosynthesis more than 5-fold via the acidic pathway in primary rat hepatocytes. This observation suggests that mitochondrial cholesterol transport is the rate-limiting step of BAS via this pathway. The objective of this study was to determine the effect of increased StAR on rates of BAS in vivo . Overexpression of StAR and CYP7A1 were mediated via infection with recombinant adenoviruses. BAS rates were determined in chronic biliary-diverted rats and mice, and in mice with an intact enterohepatic circulation. The protein/messenger RNA levels of StAR and CYP7A1 increased dramatically following overexpression. Overexpression of StAR or CYP7A1 led to a similar 2-fold ( P< .01) increase in BAS over up-regulated (approximately 2-fold) 3-day chronic biliary-diverted control rats. Additionally, overexpression of StAR led to more than 3- and 6-fold increases over controls in the rates of BAS in biliary-diverted and intact mice, respectively ( P< .01). In conclusion , in both rats and mice in vivo , overexpression of StAR led to a marked increase in the rates of BAS initiated by delivery of cholesterol to mitochondria containing CYP27A1.

Ex vivo transduced liver progenitor cells as a platform for gene therapy in mice (p 918-924)
Sihong Song, Rafal P. Witek, Yuanqing Lu, Young-Kook Choi, Donghang Zheng, Marda Jorgensen, Chengwen Li, Terence R. Flotte, Byron E. Petersen
Allogeneic stem cell-based transplants may be limited by allograft rejection, as is seen with conventional organ transplantation. One way to avert such a response is to use autologous stem cells, but that may carry the risk of recurrence of the original disease, particularly in the context of a genetic defect. We investigated the potential for gene modification of autologous stem cells to avoid both problems, using recombinant adenoassociated virus vector expressing human 1-antitrypsin in murine liver progenitor cells. We showed that recombinant adenoassociated virus 1 was the most efficient vector for liver progenitor cell transduction among five different serotypes of recombinant adenoassociated virus vectors. Ex vivo infected green fluorescent protein-positive liver progenitor cells from C57BL/6 mice with recombinant adenoassociated virus 1-vector-expressing human 1 antitrypsin were transplanted into the liver of monocrotaline-treated and partial-hepatectomized C57BL/6 recipients. Using green fluorescent protein as a donor marker, we were able to determine that at 18 weeks after transplantation, approximately 40% to 50% of the regenerated liver was green fluorescent protein positive. In addition, transgene expression (serum human 1-antitrypsin) was sustained for the length of the study (18 weeks after transplantation). Immunostaining revealed approximately 5% to 10% of repopulating liver cells expressing human 1-antitrypsin. In conclusion , this study demonstrated the feasibility of long-term engraftment and stability of transgene expression from genetically modified liver progenitor cells with a recombinant adenoassociated virus vector and implies a novel approach to gene therapy for treatment of liver diseases, such as 1-antitrypsin deficiency. (HEPATOLOGY 2004;40:918-924.)

Peptide antibiotic human beta-defensin-1 and -2 contribute to antimicrobial defense of the intrahepatic biliary tree (p 925-932)
Kenichi Harada, Kazuo Ohba, Satoru Ozaki, Kumiko Isse, Toshiya Hirayama, Akihiro Wada, Yasuni Nakanuma
Human beta-defensins (hBDs) are important antimicrobial peptides that contribute to innate immunity at mucosal surfaces. This study was undertaken to investigate the expression of hBD-1 and hBD-2 in intrahepatic biliary epithelial cells in specimens of human liver, and 4 cultured cell lines (2 consisting of biliary epithelial cells and 2 cholangiocarcinoma cells). In addition, hBD-1 and hBD-2 were assayed in specimens of bile. hBD-1 was nonspecifically expressed immunohistochemically in intrahepatic biliary epithelium and hepatocytes in all patients studied, but expression of hBD-2 was restricted to large intrahepatic bile ducts in 8 of 10 patients with extrahepatic biliary obstruction (EBO), 7 of 11 with hepatolithiasis, 1 of 6 with primary biliary cirrhosis (PBC), 1 of 5 with primary sclerosing cholangitis (PSC), 0 of 6 with chronic hepatitis C (CH-C), and 0 of 11 with normal hepatic histology. hBD-2 expression was evident in bile ducts exhibiting active inflammation. Serum C reactive protein levels correlated with biliary epithelial expression of hBD-2. Real-time PCR revealed that in all of 28 specimens of fresh liver, including specimens from patients with hepatolithiasis, PBC, PSC, CH-C and normal hepatic histology, hBD-1 messenger RNA was consistently expressed, whereas hBD-2 messenger RNA was selectively expressed in biliary epithelium of patients with hepatolithiasis. Immunobloting analysis revealed hBD-2 protein in bile in 1 of 3 patients with PSC, 1 of 3 with PBC, and each of 6 with hepatolithiasis; in contrast, hBD-1 was detectable in all bile samples examined. Four cultured biliary epithelial cell lines consistently expressed hBD-1; in contrast these cell lines did not express hBD-2 spontaneously but were induced to express hBD-2 by treatment with Eschericia coli , lipopolysaccharide, interleukin-1 or tumor necrosis factor- .In conclusion , these findings suggest that in the intrahepatic biliary tree, hBD-2 is expressed in response to local infection and/or active inflammation, whereas hBD-1 may constitute a preexisting component of the biliary antimicrobial defense system.

Interleukin 6 alleviates hepatic steatosis and ischemia/reperfusion injury in mice with fatty liver disease (p 933-941)
Feng Hong, Svetlana Radaeva, Hong-na Pan, Zhigang Tian, Richard Veech, Bin Gao
Fatty liver, formerly associated predominantly with excessive alcohol intake, is now also recognized as a complication of obesity and an important precursor state to more severe forms of liver pathology including ischemia/reperfusion injury. No standard protocol for treating fatty liver exists at this time. We therefore examined the effects of 10 days of interleukin 6 (IL-6) injection in 3 murine models of fatty liver: leptin deficient ob/ob mice, ethanol-fed mice, and mice fed a high-fat diet. In all 3 models, IL-6 injection decreased steatosis and normalized serum aminotransferase. The beneficial effects of IL-6 treatment in vivo resulted in part from an increase in mitochondrial oxidation of fatty acid and an increase in hepatic export of triglyceride and cholesterol. However, administration of IL-6 to isolated cultured steatotic hepatocytes failed to decrease lipid contents, suggesting that the beneficial effects of IL-6 in vivo do not result from its effects on hepatocytes alone. IL-6 treatment increased hepatic peroxisome proliferator-activated receptor (PPAR) and decreased liver and serum tumor necrosis factor (TNF) . Finally, 10 days of treatment with IL-6 prevented the susceptibility of fatty livers to warm ischemia/reperfusion injury. In conclusion , long-term IL-6 administration ameliorates fatty livers and protects against warm ischemia/reperfusion fatty liver injury, suggesting the therapeutic potential of IL-6 in treating human fatty liver disease.

Genetic factors influence ethanol-induced uroporphyria in Hfe (-/-) mice (p 942-950)
Nadia Gorman, Heidi W. Trask, William J. Bement, Juliana G. Szakacs, George H. Elder, Dominic Balestra, Nicholas J. Jacobs, Judith M. Jacobs, Jacqueline F. Sinclair, Glenn S. Gerhard, Peter R. Sinclair
Two major risk factors for porphyria cutanea tarda (PCT) are alcohol consumption and homozygosity for the C282Y mutation in the hereditary hemochromatosis gene ( HFE ). We recently described an animal model for alcohol-induced uroporphyria, using Hfe (-/-) mice. In the present study we show that this effect is dependent on genetic background and ethanol dose. In the 129S6/SvEvTac (129) strain, treatment with 15% ethanol in the drinking water for 6.5 months produced an accumulation of hepatic uroporphyrin (URO) 4-fold higher than that observed with 10% ethanol, a 90% decrease in uroporphyrinogen decarboxylase activity (UROD), and further increased the activities of hepatic 5-aminolevulinate synthase (ALAS) and CYP1A2. Hepatic nonheme iron (NHFe) and hepatocyte iron staining were not further increased by 15% compared to 10% ethanol. Treatment of C57BL/6 Hfe (-/-) mice with 15% ethanol for 6.5 months did not increase hepatic URO. Although NHFe was increased by ethanol, the resulting level was only half that of ethanol-treated 129 Hfe (-/-) mice. ALAS induction was similar in both Hfe (-/-) strains. In wild-type 129 mice treated with ethanol for 6 to 7 months, administration of iron dextran increased hepatic URO accumulation and decreased UROD activity. In conclusion , this study demonstrates a strong effect of genetic background on ethanol-induced uroporphyria, which is probably due to a greater effect of ethanol on iron metabolism in the susceptible strain. (HEPATOLOGY 2004;40:942-950.)

Interleukin 1 inhibits CAR-induced expression of hepatic genes involved in drug and bilirubin clearance (p 951-960)
Eric Assenat, Sabine Gerbal-Chaloin, Dominique Larrey, Jean Saric, Jean-Michel Fabre, Patrick Maurel, Marie-José Vilarem, Jean Marc Pascussi
During the inflammatory response, intrahepatic cholestasis and decreased drug metabolism are frequently observed. At the hepatic level, the orphan nuclear constitutive androstane receptor (CAR) (NR1I3) controls phase I (cytochrome P450 [CYP] 2B and CYP3A), phase II (UGT1A1), and transporter (SLC21A6, MRP2) genes involved in drug metabolism and bilirubin clearance in response to xenobiotics such as phenobarbital or endobiotics such as bilirubin. We investigated the negative regulation of CAR, a glucocorticoid-responsive gene, via proinflammatory cytokine interleukin 1 (IL-1 ) and lipopolysaccharides (LPSs) in human hepatocytes. We show that IL-1 decreases CAR expression and decreases phenobarbital- or bilirubin-mediated induction of CYP2B6, CYP2C9, CYP3A4, UGT1A1, GSTA1, GSTA2, and SLC21A6 messenger RNA. This occurs via nuclear factor B (NF- B) p65 activation, which interferes with the enhancer function of the distal glucocorticoid response element that we have identified recently in the CAR promoter. We demonstrate that: (1) LPSs, IL-1 , or overexpression of p65RelA inhibit glucocorticoid receptor (GR)-mediated CAR transactivation; (2) these suppressive effects can be blocked both by pyrrolidine dithiocarbamate, an inhibitor of NF- B activation, or by overexpression of SRIkB , a NF- B repressor; and (3) the GR agonist dexamethasone induces histone H4 acetylation at the proximal CAR promoter region, whereas LPSs and IL-1 inhibit this acetylation as assessed via chromatin immunoprecipitation assay. In conclusion , GR/NF- B interaction affects CAR gene transcription through chromatin remodeling and provide a mechanistic explanation for the long-standing observation that inflammation and sepsis inhibit drug metabolism while inducing intrahepatic cholestasis or hyperbilirubinemia.

Bile acids induce mitochondrial ROS, which promote activation of receptor tyrosine kinases and signaling pathways in rat hepatocytes (p 961-971)
Youwen Fang, Song Iy Han, Clint Mitchell, Seema Gupta, Elaine Studer, Steven Grant, Phillip B. Hylemon, Paul Dent
Previous studies have demonstrated in hepatocytes that deoxycholic acid (DCA) promotes inactivation of protein tyrosine phosphatases (PTPases) and activation of ERBB1 and the extracellular-regulated kinase (ERK) 1/2 pathway. The present studies have determined the biochemical mechanism(s) through which these events occur. DCA and taurodeoxycholic acid (TDCA) (100 mol/L) caused activation of ERBB1, insulin receptor, and the ERK1/2 and AKT pathways in primary rodent hepatocytes. DCA- and TDCA-induced receptor and signaling pathway activations were blocked by the reactive oxygen species (ROS) scavengers N-acetyl cysteine (NAC) and Trolox (TX), as well as by cyclosporin A (CsA) and bongkrekic acid (BKA). DCA activated the ERK1/2 pathway in HuH7 human hepatoma cells that was blocked by the incubation of cells with an ERBB1 inhibitor, NAC, TX, CsA, or BKA. DCA did not activate the ERK1/2 pathway in mitochondria-defective HuH7 Rho 0 cells. In HuH7 cells and primary hepatocytes, DCA enhanced the production of ROS, an effect that was abolished in Rho 0 cells and by prior incubation of cells with CsA or BKA. In hepatocytes and HuH7 cells, DCA inhibited PTPase activity. Incubation of hepatocytes with either CsA or BKA prevented DCA-induced inhibition of PTPase activity. Loss of mitochondrial function in Rho 0 cells also abolished the inhibitory effects of DCA on PTPase activity. In conclusion , DCA and TDCA cause ROS generation in hepatocytes that is dependent on metabolically active mitochondria. The generation of ROS is essential for PTPase inactivation, receptor tyrosine kinase activation, and enhanced signaling down the ERK1/2 and AKT pathways.

Peroxisome proliferator-activated receptor protects against alcohol-induced liver damage (p 972-980)
Tamie Nakajima, Yuji Kamijo, Naoki Tanaka, Eiko Sugiyama, Eiji Tanaka, Kendo Kiyosawa, Yoshimitsu Fukushima, Jeffrey M. Peters, Frank J. Gonzalez, Toshifumi Aoyama
The mechanisms underlying alcoholic liver disease are not completely understood, but lipid accumulation seems to be central to the cause of this disease. The peroxisome proliferator-activated receptor (PPAR ) plays an important role in the control of lipid homeostasis, metabolism of bioactive molecules, and modulation of inflammatory responses. To investigate the roles of PPAR in alcoholic liver injury, wild-type and PPAR -null mice were continuously fed a diet containing 4% ethanol, and liver injury was analyzed. PPAR -null mice fed ethanol exhibited marked hepatomegaly, hepatic inflammation, cell toxicity, fibrosis, apoptosis, and mitochondrial swelling. Some of these hepatic abnormalities were consistent with those of patients with alcoholic liver injury and were not found in wild-type mice. Next, the molecular mechanisms of ethanol-induced liver injury in PPAR -null mice were investigated, and changes related to ethanol and acetaldehyde metabolism, oxidative stress, inflammation, hepatocyte proliferation, fibrosis, and mitochondrial permeability transition activation occurred specifically in PPAR -null mice as compared with wild-type mice. In conclusion , these studies suggest a protective role for PPAR in alcoholic liver disease. Humans may be more susceptible to liver toxicity induced by ethanol as PPAR expression in human liver is considerably lower compared to that of rodents. (HEPATOLOGY 2004;40:972-980.)

Aging does not reduce the hepatocyte proliferative response of mice to the primary mitogen TCPOBOP (p 981-988)
Giovanna M. Ledda-Columbano, Monica Pibiri, Costanza Cossu, Francesca Molotzu, Joseph Locker, Amedeo Columbano
It has been shown that the magnitude of DNA synthesis and the time at which maximal DNA synthesis occurs after two-thirds partial hepatectomy (PH) is greatly reduced in the liver of aged rodents compared to young animals. This reduction could represent an intrinsic defect in proliferation or a more specialized change in the response to PH. We therefore evaluated the proliferative capacity of hepatocytes in aged animals, following treatment with primary liver mitogens. We show that treatment of 12-month-old CD-1 mice with the hepatomitogen 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) caused an increase in hepatocyte proliferation similar to that seen in young (8-week-old) mice. The labeling index was 82% in the livers of aged mice versus 76% in young animals. Histological observation demonstrated that the number of hepatocytes entering mitoses was similar in both groups; the mitotic indices were 2.5 per thousand and 2.7 per thousand, respectively. Additional experiments showed that the timing of DNA synthesis and M phase were nearly identical in both aged and young mice. Stimulation of hepatocyte DNA synthesis was associated with increased expression of several cell cycle-associated proteins (cyclin D1, cyclin A, cyclin B1, E2F, pRb, and p107); all were comparable in aged mice and young mice. TCPOBOP treatment also increased expression of the Forkhead Box transcription factor m1b (Foxm1b) to a similar degree in both groups. In conclusion , hepatocytes retain their proliferative capacity in old age despite impaired liver regeneration. These findings suggest that therapeutic use of mitogens would alleviate the reduction in hepatocyte proliferation observed in the elderly. (HEPATOLOGY 2004;40:981-988.)

S-adenosylhomocysteine sensitizes to TNF- hepatotoxicity in mice and liver cells: A possible etiological factor in alcoholic liver disease (p 989-997)
Zhenyuan Song, Zhanxiang Zhou, Silvia Uriarte, Lipeng Wang, Y. James Kang, Theresa Chen, Shirish Barve, Craig J. McClain
In alcoholic liver disease, tumor necrosis factor- (TNF ) is a critical effector molecule, and abnormal methionine metabolism is a fundamental acquired metabolic abnormality. Although hepatocytes are resistant to TNF -induced killing under normal circumstances, previous studies have shown that primary hepatocytes from rats chronically fed alcohol have increased TNF cytotoxicity. Therefore, there must be mechanisms by which chronic alcohol exposure sensitizes to TNF hepatotoxicity. S-adenosylhomocysteine (SAH) is product of methionine in transsulfuration pathway and a potent competitive inhibitor of most methyltransferases. In this study, we investigated the effects of increased SAH levels on TNF hepatotoxicity. Our results demonstrated that chronic alcohol consumption in mice not only decreased hepatic S-adenosylmethionine levels but also increased hepatic SAH levels, which resulted in a significantly decreased S-adenosylmethionine-to-SAH ratio. This was associated with significant increases in hepatic TNF levels, caspase-8 activity, and cell death. In vitro studies demonstrated that SAH-enhancing agents sensitized hepatocytes to TNF killing, and the death was associated with increased caspase-8 activity, which was blocked by a caspase-8 inhibitor. In addition, increased intracellular SAH levels had no effect on nuclear factor B activity induced by TNF .In conclusion , these results provide a new link between abnormal methionine metabolism and abnormal TNF metabolism in alcoholic liver disease. Increased SAH is a potent and clinically relevant sensitizer to TNF hepatotoxicity. These data further support improving the S-adenosylmethionine-to-SAH ratio and removal of intracellular SAH as potential therapeutic options in alcoholic liver disease.

Reduced oncotic necrosis in Fas receptor-deficient C57BL/6J- lpr mice after bile duct ligation (p 998-1007)
Jaspreet S. Gujral, Jie Liu, Anwar Farhood, Hartmut Jaeschke
Neutrophils aggravate cholestatic liver injury after bile duct ligation (BDL). Recently, it was suggested that hepatocellular apoptosis might be critical for liver injury in this model. To test the hypothesis that apoptosis could be a signal for neutrophil extravasation and injury, we assessed parameters of apoptosis and inflammation after BDL using 2 different approaches: (1) wild-type and Fas receptor-deficient lpr mice of the C57BL/6J or C3H/HeJ strains, and (2) treatment with the pancaspase inhibitor z-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk)in C3HeB/FeJ mice. After BDL for 3 days, total cell death was estimated to be between 10% and 50% of all cells evaluated. However, less than 0.1% of hepatocytes showed apoptotic morphology in all 3 strains. Processing of procaspase-3, caspase-3 enzyme activities, and immunohistochemical staining for cytokeratin 18 cleavage products indicated no activation of caspases. Real-time reverse-transcriptase polymerase chain reaction analysis revealed increased expression of many inflammatory mediators but no effect on proapoptotic genes. More than 50% of all accumulated neutrophils were extravasated and colocalized with foci of oncotic hepatocytes and chlorotyrosine adducts. z-VAD-fmk treatment had no effect on apoptosis or liver injury after BDL but eliminated apoptosis after galactosamine/endotoxin in C3HeB/FeJ mice. In Fas receptor-deficient lpr mice (C57BL/6J), expression of inflammatory mediators, neutrophil accumulation and extravasation, chlorotyrosine adduct formation, and liver injury were reduced. This protection was not observed in lpr mice of the endotoxin-resistant C3H/HeJ strain. In conclusion , liver injury (oncotic necrosis) after BDL correlated with the severity of the inflammatory response. The minimal amount of apoptosis had no effect on inflammation or on the overall injury. (HEPATOLOGY 2004;40:998-1007.)

Hepatology Elsewhere

STAT-3 and the liver: A new STATion on our way to understand diabetes? (p 1008-1010)
Marcin T. Kortylewski, Andreas Barthel
The transcription factor, signal transducer and activator of transcription-3 (STAT-3) contributes to various physiological processes. Here we show that mice with liver-specific deficiency in STAT-3, achieved using the Cre-loxP system, show insulin resistance associated with increased hepatic expression of gluconeogenic genes. Restoration of hepatic STAT-3 expression in these mice, using adenovirus-mediated gene transfer, corrected the metabolic abnormalities and the alterations in hepatic expression of gluconeogenic genes. Overexpression of STAT-3 in cultured hepatocytes inhibited gluconeogenic gene expression independently of peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1 alpha), an upstream regulator of gluconeogenic genes. Liver-specific expression of a constitutively active form of STAT-3, achieved by infection with an adenovirus vector, markedly reduced blood glucose, plasma insulin concentrations and hepatic gluconeogenic gene expression in diabetic mice. Hepatic STAT-3 signaling is thus essential for normal glucose homeostasis and may provide new therapeutic targets for diabetes mellitus.

Outfoxing liver cancer with p19 ARF tumor suppressor? (p 1010-1012)
Snorri S. Thorgeirsson
Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide. Here, we provide evidence that the Forkhead Box (Fox) m1b (Foxm1b or Foxm1) transcription factor is essential for the development of HCC. Conditionally deleted Foxm1b mouse hepatocytes fail to proliferate and are highly resistant to developing HCC in response to a Diethylnitrosamine (DEN)/Phenobarbital (PB) liver tumor-induction protocol. The mechanism of resistance to HCC development is associated with nuclear accumulation of the cell cycle inhibitor p27 Kip1 protein and reduced expression of the Cdk1-activator Cdc25B phosphatase. We showed that the Foxm1b transcription factor is a novel inhibitory target of the p19 ARF tumor suppressor. Furthermore, we demonstrated that conditional overexpression of Foxm1b protein in osteosarcoma U2OS cells greatly enhances anchorage-independent growth of cell colonies on soft agar. A p19 ARF 26-44 peptide containing nine D-Arg to enhance cellular uptake of the peptide was sufficient to significantly reduce both Foxm1b transcriptional activity and Foxm1b-induced growth of U2OS cell colonies on soft agar. These results suggest that this (D-Arg) 9-p19 ARF 26-44 peptide is a potential therapeutic inhibitor of Foxm1b function during cellular transformation. Our studies demonstrate that the Foxm1b transcription factor is required for proliferative expansion during tumor progression and constitutes a potential new target for therapy of human HCC tumors

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

GASTROENTEROLOGY

Rapid Communication

Suppression of Peutz—Jeghers polyposis by inhibition of cyclooxygenase-2
Lina Udd, Pekka Katajisto, Derrick J. Rossi, Anna Lepistö, Anna—Maria Lahesmaa, Antti Ylikorkala, Heikki J. Järvinen, Ari P. Ristimäki, Tomi P. Mäkelä
Background & Aims: Peutz—Jeghers syndrome (PJS) is typically manifested as severe gastrointestinal polyposis. Polyps in PJS patients and in Lkb1 +/? mice that model PJS polyposis are frequently characterized by elevated cyclooxygenase-2 (COX-2). This study was designed to determine whether COX-2 inhibition would reduce tumor burden in Lkb1 +/? mice or Peutz–Jeghers patients. Methods: Genetic interactions between Cox-2 and Lkb1 in polyp formation were analyzed in mice with combined deficiencies in these genes. Pharmacologic inhibition of COX-2 was achieved by supplementing the diet of Lkb1 +/? mice with 1500 ppm celecoxib between 3.5–10 and 6.5–10 months. In PJS patients, COX-2 was inhibited with a daily dose of 2 ? 200 mg celecoxib for 6 months. Results: Total polyp burden in Lkb1 +/? mice was significantly reduced in a Cox-2 +/? (53%) and in a Cox-2 ?/? (54%) background. Celecoxib treatment initiating before polyposis (3.5–10 months) led to a dramatic reduction in tumor burden (86%) and was associated with decreased vascularity of the polyps. Late treatment (6.5–10 months) also led to a significant reduction in large polyps. In a pilot clinical study, a subset of PJS patients (2/6) responded favorably to celecoxib with reduced gastric polyposis. Conclusions: These data establish a role for COX-2 in promoting Peutz–Jeghers polyposis and suggest that COX-2 chemoprevention may prove beneficial in the treatment of PJS.

Clinical-alimentary Tract

Celecoxib versus diclofenac plus omeprazole in high-risk arthritis patients: Results of a randomized double-blind trial
Francis K.L. Chan, Lawrence C.T. Hung, Bing Y. Suen, Vincent W.S. Wong, Aric J. Hui, Justin C.Y. Wu, Wai K. Leung, Yuk T. Lee, Ka F. To, S.C. Sydney Chung, Joseph J.Y. Sung
Background & Aims: The gastric safety of cyclooxgenase-2 inhibitors and prophylactic antisecretory therapy in high-risk arthritis patients is unclear. We studied the ulcer incidence and factors predicting ulcer recurrence in a prospective, double-blinded trial. Methods: We studied patients who presented with nonsteroidal anti-inflammatory drug-associated ulcer bleeding. After ulcer healing, patients who were negative for Helicobacter pylori were randomly assigned to celecoxib 200 mg twice a day plus omeprazole placebo once daily or diclofenac 75 mg twice daily plus omeprazole 20 mg once daily for 6 months. Patients underwent endoscopy if they developed recurrent bleeding. Those without recurrent events underwent endoscopy at their last follow-up visit. Results: Two hundred eighty-seven patients were enrolled; 24 had recurrent gastrointestinal complications. Among 259 patients without events, 222 underwent endoscopy (116 received celecoxib and 106 received diclofenac plus omeprazole). The probability of recurrent ulcers in 6 months was 18.7% in the celecoxib group and 25.6% in the diclofenac plus omeprazole group (difference, ?6.7%; 95% CI: ?17.8% to 3.9%) ( P= 0.21). Combining bleeding and endoscopic ulcers, 24.1% in the celecoxib group and 32.3% in the diclofenac plus omeprazole group had recurrent ulcers in 6 months (difference, ?8.2%; 95% CI: ?19.5% to 2.9%) ( P= 0.15). Treatment-induced significant dyspepsia (hazard ratio, 5.3; 95% CI: 2.6–10.8), age ≥75 (hazard ratio, 2.0; 95% CI: 1.1–3.5), and comorbidity (hazard ratio, 2.1; 95% CI: 1.2–3.7) independently predicted ulcer recurrence. Conclusions: Among patients with previous ulcer bleeding, neither celecoxib nor diclofenac plus omeprazole adequately prevents ulcer recurrence. Treatment-induced significant dyspepsia is an indication for endoscopic evaluation.

Insulin therapy and colorectal cancer risk among type 2 diabetes mellitus patients
Yu–Xiao Yang, Sean Hennessy, James D. Lewis
Background & Aims: Endogenous hyperinsulinemia in the context of type 2 diabetes mellitus is potentially associated with an increased risk of colorectal cancer. We aimed to determine whether insulin therapy might increase the risk of colorectal cancer among type 2 diabetes mellitus patients. Methods: We conducted a retrospective cohort study among all patients with a diagnosis of type 2 diabetes mellitus in the General Practice Research Database from the United Kingdom. We excluded patients with <3 years of colorectal cancer-free database follow-up after the diabetes diagnosis as well as those insulin users who developed colorectal cancer after <1 year of insulin therapy. The remaining insulin users and the noninsulin-using type 2 diabetic patients were followed for the occurrence of colorectal cancer. Hazard ratios (HR) were determined in Cox proportional hazard analysis. A nested case-control study was conducted to perform multivariable analysis and to determine a duration-response effect. Results: The incidence of colorectal cancer in insulin users (n = 3160) was 197 per 100,000 person-years, compared with 124 per 100,000 person-years in type 2 diabetes mellitus patients not receiving insulin (n = 21,758). The age- and sex-adjusted HR of colorectal cancer associated with ≥1 year of insulin use was 2.1 (95% CI: 1.2–3.4, P= 0.005). The positive association strengthened after adjusting for potential confounders. The multivariable odds ratio associated with each incremental year of insulin therapy was 1.21 (95% CI: 1.03–1.42, P= 0.02). Conclusions: Chronic insulin therapy significantly increases the risk of colorectal cancer among type 2 diabetes mellitus patients.

Incidence of juvenile-onset Crohn’s disease in Scotland: Association with northern latitude and affluence
Emma L. Armitage, Marian C. Aldhous, Niall Anderson, Hazel E. Drummond, Rudolph A. Riemersma, Subrata Ghosh, Jack Satsangi
Background & Aims :The incidence of Crohn’s disease in Scottish children has increased steadily over 30 years. Many studies have investigated genetic influence or possible links with childhood events. We aimed to study sociodemographic and/or geographic distribution of juvenile=onset Crohn’s disease in Scotland. Methods :Using a previously established and validated database covering the entire Scottish population, 580 Scottish children (<16 years of age at symptom onset) with inflammatory bowel disease incident between 1981 and 1995 were identified. Postcodes of incident cases were classed for geographic location and material deprivation. Incidence rates (/100,000/year) were sex standardized to the 1991 census population. The effects of sex, geographic location, time, and deprivation category were estimated from a multifactorial Poisson regression model. Results :The incidence of juvenile-onset Crohn’s disease was 2.3 (95% CI: 2.0–2.5) for the time period 1981 to 1995 and was significantly higher in northern (3.1, 95% CI: 2.6–3.8) than in southern Scotland (2.1, 95% CI: 1.9–2.4, P< 0.001). The incidence of juvenile-onset ulcerative colitis did not show north/south variation ( P= 0.677). The relative risks of developing CD were significantly lower in postcode areas with deprivation categories 2–7 as compared with deprivation score 1 (most affluent, P= 0.033). This pattern was not seen for UC. Conclusions :There was an increased incidence of juvenile-onset Crohn’s disease in northern compared with southern Scotland. Children from more affluent areas had a higher relative risk of developing Crohn’s disease. Juvenile onset ulcerative colitis did not show north/south variation in incidence or association with affluence.

Role of tension receptors in dyspeptic patients with hypersensitivity to gastric distention
Jan Tack, Philip Caenepeel, Maura Corsetti, Jozef Janssens
Background & Aims: Studies in health have shown that tension-sensitive mechanoreceptors mediate sensitivity to gastric distention. A role for these mechanoreceptors in perception or symptoms in hypersensitive functional dyspepsia (FD) has not been established. Tension-sensitive mechanoreceptors are activated during phasic contractions and inactivated during gastric relaxation. The aim of the present study was to investigate whether hypersensitive FD patients perceive spontaneous changes in fundic wall tension and whether fundus-relaxing drugs decrease sensitivity to gastric distention and meal-related symptoms. Methods: Fifty patients were selected after a barostat study established gastric hypersensitivity. In 12 patients, an intragastric balloon was inflated with a fixed volume just below perception thresholds and patients were asked to indicate changes in perception on a keypad, and the relationship between perception and contractions was analyzed. In 20 patients, we studied the influence of the fundus-relaxing drug sumatriptan on sensitivity to gastric distention. In, respectively, 10 and 8 patients, we studied the influence of the fundus-relaxing drugs sumatriptan and clonidine on meal-related symptoms. Results: The majority of patients had a statistically significant association between perception and phasic isovolumetric contractions. Pretreatment with sumatriptan increased both pressures and volumes needed to induce first perception and discomfort. Pretreatment with sumatriptan and clonidine both significantly decreased meal-induced symptoms. Conclusions: Patients with hypersensitivity to gastric distention perceive isovolumetric phasic contractions of the proximal stomach. Fundus-relaxing drugs decrease sensitivity to gastric distention and decrease meal-induced symptoms in these patients. The findings are compatible with involvement of tension mechanoreceptors in symptom generation in hypersensitive FD.

Endoscopic evaluation of patients with dyspepsia: Results from the national endoscopic data repository
David Lieberman, M. Brian Fennerty, Cynthia D. Morris, Jennifer Holub, Glenn Eisen, Amnon Sonnenberg
Background & Aims: Endoscopy is commonly performed to evaluate symptoms of dyspepsia. The aim of this study was to characterize patients who receive endoscopy for dyspepsia and measure predictors of primary endoscopic outcomes, utilizing a large national endoscopic database. Methods: The Clinical Outcomes Research Initiative (CORI) receives endoscopy reports from a network of 74 sites in the United States. Sixty-one percent of reports come from private practice settings. Patients with reflux dyspepsia and nonreflux dyspepsia were identified from January 2000 to June 2002. Patients with dysphagia and known Barrett’s esophagus were excluded. Primary endoscopic outcomes included esophageal inflammation and stricture, gastric ulcer, duodenal ulcer, suspected Barrett’s esophagus (≥2cm), and suspected esophageal and gastric malignancy. The presence or absence of alarm symptoms (vomiting, weight loss, and evidence of GI blood loss) was determined. Adjusted relative risk (RR) for predicting serious outcomes was calculated in a multivariate model. Results: We received 117,497 endoscopic reports, representing 99,558 unique patients. Dyspepsia, with and without reflux symptoms, accounted for 43% of upper endoscopies. Among dyspeptic patients, 36.5% were younger than 50 years of age without alarm symptoms. Esophageal or gastric malignancy in patients with dyspepsia was associated with increasing age, male sex, Asian race, Native American race, and symptoms of weight loss and vomiting. Suspected Barrett’s esophagus (≥2cm) was associated with reflux symptoms, male sex, age, and white race. Ulcers were associated with evidence of bleeding, vomiting, male sex, black race, and Hispanic ethnicity. Conclusions These practice-based data reveal important practice behaviors and outcomes.

Genetic variants of the mannan-binding lectin are associated with immune reactivity to mannans in Crohn’s disease
Frank Seibold, Astrid Konrad, Beatrice Flogerzi, Beatrice Seibold-Schmid, Stephan Arni, Simone Jüliger, Jürgen F.J. Kun
Background & Aims : Some patients with Crohn’s disease (CD) develop antibodies against mannan, a component of the yeast Saccharomyces cerevisiae cell wall. Mannan-binding lectin (MBL), a component of the innate immune system, can bind to S. cerevisiae . MBL concentration depends on genetic polymorphisms. The aim of this study was to evaluate whether low MBL contributes to anti- S. cerevisiae antibody (ASCA) production. Methods : ASCA and MBL concentrations in sera from patients with CD (n = 74), ulcerative colitis (UC) (n = 22), and healthy controls (n = 32) were measured by an enzyme-linked immunosorbent assay (ELISA). Genetic MBL variants were determined from 58 CD patients, 18 UC patients, and 47 controls by DNA sequencing. Lymphocytes were tested for proliferative response after stimulation with mannan. Results : ASCA were found in 47% of the patients with CD and in 0% of the controls. More ASCA-positive patients (52%) had low serum MBL concentrations compared with ASCA-negative patients (4%) ( P< 0.0001). T-cell proliferation in response to mannan stimulation was observed in ASCA-positive patients and could be inhibited by the addition of MBL. These patients had significantly lower MBL serum concentrations than patients whose lymphocytes did not proliferate on mannan stimulation ( P< 0.0001). Homozygous or compound heterozygous MBL mutations in the exon 1 and promoter occurred in 12 patients with cellular or humoral immune reactivity to mannan as compared with only 1 nonreactive patient ( P< 0.0001). Conclusions : A subgroup of CD patients is characterized by ASCA positivity, T-cell proliferation on mannan stimulation, and mutations in the MBL gene that result in MBL deficiency. Thus, we propose that enhanced mannan exposure stimulates specific immune responses in a subgroup of CD patients with genetically determined low MBL concentrations. This enhanced exposure contributes to the generation of ASCA.

CFTR Cl? channel function in native human colon correlates with the genotype and phenotype in cystic fibrosis
Stephanie Hirtz, Tanja Gonska, Hans H. Seydewitz, Jörg Thomas, Peter Greiner, Joachim Kuehr, Matthias Brandis, Irmgard Eichler, Herculano Rocha, Ana—Isabel Lopes, Celeste Barreto, Anabela Ramalho, Margarida D. Amaral, Karl Kunzelmann, Marcus Mall
Background & Aims: Cystic fibrosis (CF) is caused by over 1000 mutations in the cystic fibrosis transmembrane conductance regulator ( CFTR ) gene and presents with a widely variable phenotype. Genotype-phenotype studies identified CFTR mutations that were associated with pancreatic sufficiency (PS). Residual Cl ?channel function was shown for selected PS mutations in heterologous cells. However, the functional consequences of most CFTR mutations in native epithelia are not well established. Methods: To elucidate the relationships between epithelial CFTR function, CFTR genotype, and patient phenotype, we measured cyclic adenosine monophosphate (cAMP)-mediated Cl ?secretion in rectal biopsy specimens from 45 CF patients who had at least 1 non-?F508 mutation carrying a wide spectrum of CFTR mutations. We compared CFTR genotypes and clinical manifestations of CF patients who expressed residual CFTR-mediated Cl ?secretion with patients in whom Cl ?secretion was absent. Results: Residual anion secretion was detected in 40% of CF patients, and was associated with later disease onset ( P< 0.0001), higher frequency of PS ( P< 0.0001), and less severe lung disease ( P< 0.05). Clinical outcomes correlated with the magnitude of residual CFTR activity, which was in the range of ?12%–54% of controls. Conclusions: Specific CFTR mutations confer residual CFTR function to rectal epithelia, which is related closely to a mild disease phenotype. Quantification of rectal CFTR-mediated Cl ?secretion may be a sensitive test to predict the prognosis of CF disease and identify CF patients who would benefit from therapeutic strategies that would increase residual CFTR activity.

TNF? suppresses human colonic circular smooth muscle cell contractility by SP1- and NF-?B-mediated induction of ICAM-1
Konrad Pazdrak, Xuan-Zheng Shi, Sushil K. Sarna
Background & Aims: Intercellular adhesion molecule 1 (ICAM-1) receptors are expressed at low levels on human colonic circular smooth muscle cells (HCCSMCs) and their expression is increased in patients with Crohn’s disease. We investigated the roles of transcription factors Sp1 and nuclear factor ? B (NF-?B) in the regulation of ICAM-1 expression on HCCSMCs and examined whether ICAM-1 expression mediates the suppression of contractility in response to TNF?. Methods: Experiments were performed on primary cultures of HCCSMCs and fresh human colonic circular muscle strips. Results: TNF? treatment of HCCSMCs induced rapid and prolonged accumulation of ICAM-1 messenger RNA (mRNA) and protein. NF-?B inhibition before, but not after, 1 hour of TNF?-stimulation blocked the expression of ICAM-1. TNF? significantly enhanced Sp1/DNA binding. Sp1 bound to the 3? flanking region of a variant ?B site in the ?192/?172 region of ICAM-1 promoter. Mutation of this region abolished the response to TNF?. The treatment of HCCSMCs with Sp1 antisense oligonucleotides (ODNs) blocked the expression of ICAM-1, but sense ODNs had no effect. Protein kinase C ? (PKC?) inhibition before or 3 hours after stimulation with TNF? also blocked the expression of ICAM-1. TNF? treatment of circular muscle strips pretreated with ICAM-1 sense ODNs or control medium significantly reduced their response to acetylcholine, whereas pretreatment with antisense ODNs blocked this effect. Conclusions: The expression of ICAM-1 on HCCSMCs in response to TNF? is regulated by transcription factors Sp1 and NF-?B binding independently to the ?192/?172 region of the ICAM-1 promoter. The expression of ICAM-1 plays a critical role in the suppression of cell contractility in response to TNF?.

Clinical-liver, Pancreas, and Biliary Tract

Functional consequences of frizzled-7 receptor overexpression in human hepatocellular carcinoma
Philippe Merle, Suzanne De La Monte, Miran Kim, Marc Herrmann, Shinji Tanaka, Annette Von Dem Bussche, Michael C. Kew, Christian Trepo, Jack R. Wands
Background & Aims: The molecular pathogenesis of human hepatocellular carcinoma (HCC) is understood poorly. In some tumors, activation of the Wnt/?-catenin pathway as a result of ?-catenin gene mutations has been found. However, in many other HCCs, activation of the Wnt/?-catenin pathway has been shown in the absence of such mutations. Methods: We previously have identified the upstream human Frizzled-7 receptor ( FZD7 ) gene of this pathway. In the present study, a quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR) assay for FZD7 was developed and overexpression of FZD7 was detected in 90% of tumors, most of which were related to chronic hepatitis B virus infection. FZD7 also was overexpressed in the 6 HCC cell lines tested and functional analysis showed that FZD7 messenger RNA (mRNA) levels correlated with enhanced cellular motility. Results: Transfection of HCC cells with dominant-negative mutant constructs encoding a C-terminally truncated FZD7 protein decreased wild-type ?-catenin protein accumulation and reduced cell motility. More importantly, we observed ?-catenin accumulation in human HCC tumors containing the wild-type ?-catenin gene in the context of high-level FZD7 expression. Conclusions: These observations suggest that the Wnt/?-catenin signal transduction pathway is involved much more commonly in the molecular pathogenesis of HCC than previously recognized because FZD7 overexpression occurred early in the disease process, stabilized wild-type ?-catenin levels, and contributed to enhanced tumor cell migration.

Recombinant factor VIIa for upper gastrointestinal bleeding in patients with cirrhosis: A randomized, double-blind trial
Jaime Bosch, Dominique Thabut, Flemming Bendtsen, Gennaro D’amico, Agustín Albillos, Juan González Abraldes, Soeren Fabricius, Elisabeth Erhardtsen, Roberto De Franchis
Background & Aims: Upper gastrointestinal bleeding (UGIB) is a severe and frequent complication of cirrhosis. Recombinant coagulation factor VIIa (rFVIIa) has been shown to correct the prolonged prothrombin time in patients with cirrhosis and UGIB. This trial aimed to determine efficacy and safety of rFVIIa in cirrhotic patients with variceal and nonvariceal UGIB. Methods: A total of 245 cirrhotic patients (Child-Pugh < 13; Child-Pugh A = 20%, B = 52%, C = 28%) with UGIB (variceal = 66%, nonvariceal = 29%, bleeding source unknown = 5%) were randomized equally to receive 8 doses of 100 ?g/kg rFVIIa or placebo in addition to pharmacologic and endoscopic treatment. The primary end point was a composite including: (1) failure to control UGIB within 24 hours after first dose, or (2) failure to prevent rebleeding between 24 hours and day 5, or (3) death within 5 days. Results: Baseline characteristics were similar between rFVIIa and placebo groups. rFVIIa showed no advantage over standard treatment in the whole trial population. Exploratory analyses, however, showed that rFVIIa significantly decreased the number of failures on the composite end point ( P= 0.03) and the 24-hour bleeding control end point ( P= 0.01) in the subgroup of Child-Pugh B and C variceal bleeders. There were no significant differences between rFVIIa and placebo groups in mortality (5- or 42-day) or incidence of adverse events including thromboembolic events. Conclusions: Although no overall effect of rFVIIa was observed, exploratory analyses in Child-Pugh B and C cirrhotic patients indicated that administration of rFVIIa significantly decreased the proportion of patients who failed to control variceal bleeding. Dosing with rFVIIa appeared safe. Further studies are needed to verify these findings.

Evolution of hepatitis B virus during primary infection in humans: Transient generation of cytotoxic T-cell mutants
Simon A. Whalley, David Brown, George J.M. Webster, Ruth Jacobs, Stephanie Reignat, Antonio Bertoletti, Chong–Gee Teo, Vincent Emery, Geoffrey M. Dusheiko
Background & Aims: Acute hepatitis B is a highly dynamic human viral infection during which the hepatitis B virus can generate many genetic variants. Methods: We analyzed the evolution of the hepatitis B virus genome in sequential serum samples from a unique cohort of patients with acute infection acquired from a single source. Results: We showed that most mutations were nonsynonymous, that genetic diversity was greatest at the peak of viremia, and that patients who resolved their infection (“resolvers”) showed a significantly higher level of diversity in the core, surface, and polymerase genes compared with those who progressed to chronic infection. Overall, the core gene showed the greatest genetic diversity. In resolvers who possessed an HLA-A*0201 haplotype, the emergence of mutants in the immunodominant HLA-A*0201–restricted core 18–27 epitope was observed. Functional studies showed that these mutants were less able to stimulate interferon-? release from core 18–27 specific CD8 +T-cell lines. However, they appeared only as a transient low-abundance species and were rapidly displaced by wild-type sequences before resolution of infection, and their overall significance is uncertain. Conclusions: Overall, genetic evolution of the hepatitis B virus differs at early time points between patients who experience acute resolving hepatitis B and those who progress to chronicity. These observations suggest that the rapid development of broadly reactive host immune responses leads to clearance of hepatitis B virus, even in the presence of possible CD8 +T-cell immune escape variants.

Basic-alimentary Tract

Apobec-1 protects intestine from radiation injury through posttranscriptional regulation of cyclooxygenase-2 expression
Shrikant Anant, Nabendu Murmu, Courtney W. Houchen, Debnath Mukhopadhyay, Terrence E. Riehl, Stephen G. Young, Aubrey R. Morrison, William F. Stenson, Nicholas O. Davidson
Background & Aims: This study aimed to determine the role of the RNA binding protein apobec-1 in radioprotection of the intestine. Methods: Apobec-1-deleted mice ( APOBEC -1 ?/? ) and wild-type controls were treated with 12 Gy of whole-body ?-irradiation in a cesium irradiator. The number of surviving intestinal crypts was assessed 3.5 days after irradiation by using a clonogenic assay. Cyclooxygenase-2 messenger RNA and protein expression were determined by real-time polymerase chain reaction and Western blot, respectively. RNA stability was studied by examining the turnover of a chimeric transcript containing the cyclooxygenase-2 3? untranslated region cloned downstream of luciferase complementary DNA. Apobec-1 binding to the cyclooxygenase-2 3? untranslated region was studied by electrophoretic mobility shift and UV crosslinking assays. Results: After ?-irradiation, the survival of intestinal stem cells decreased significantly in APOBEC -1 ?/? mice. In wild-type mice treated with lipopolysaccharide before ?-irradiation, intestinal stem cells were protected by marked increases in prostaglandin E 2mediated by cyclooxygenase-2. No such effect was observed in the APOBEC -1 ?/? mice. The mechanism of this radioprotective effect involves the binding of apobec-1 to AU-rich sequences in the first 60 nucleotides of the 3? untranslated region of cyclooxygenase-2. Upon binding to the AU-rich sequences, apobec-1 stabilizes cyclooxygenase-2 messenger RNA. This stabilization process does not seem to be mediated by p38 mitogen-activated protein kinase pathways. Conclusions: Lipopolysaccharide increases intestinal stem cell survival through apobec-1-mediated regulation of cyclooxygenase-2 messenger RNA stability.

The Akt and MAPK signal-transduction pathways regulate growth factor actions in isolated gastric parietal cells
Vinzenz Stepan, Nonthalee Pausawasdi, Saravanan Ramamoorthy, Andrea Todisco
Background & Aims: Incubation of purified (>95%) canine parietal cells in primary culture with epidermal growth factor for 7–16 hours stimulates H +K+-adenosine triphosphatase gene expression. In this study, we examined the effect of prolonged stimulation (72 hours) of the parietal cells with epidermal growth factor. Methods: H+K+-adenosine triphosphatase protein and gene expression were assessed by immunohistochemistry and Northern blots. Mitogen-activated protein kinase and Akt activation were quantitated by kinase assays and Western blots with specific antiphospho antibodies. Akt overexpression was achieved by adenovirus-mediated gene transfer of a constitutively active Akt gene. Results: Epidermal growth factor changed the morphology of the cultured cells, which acquired the appearance of fusiform cells, and it inhibited H +K+-adenosine triphosphatase gene expression. Staining of the cells both with anti–H +K+-adenosine triphosphatase antibodies and with Texas Red–labeled Dolichos biflorus lectin confirmed that the fusiform cells expressed markers of parietal cell differentiation. Epidermal growth factor stimulated mitogen-activated protein kinase with 2 peaks of activation, observed after 5 minutes and 72 hours, whereas it activated Akt after 5 minutes but not 72 hours of incubation. Overexpression of Akt blocked both epidermal growth factor–induced morphological transformation and inhibition of H +K+-adenosine triphosphatase gene expression. Identical results were observed in the presence of the mitogen-activated protein kinase inhibitor PD98059. Conclusions: Activation of the Akt signal-transduction pathway seems to be a crucial event for the induction of parietal cell maturation and differentiation.

Cystic fibrosis gene mutation reduces epithelial cell acidification and injury in acid-perfused mouse duodenum
Masahiko Hirokawa, Tetsu Takeuchi, Sahaoyou Chu, Yasutada Akiba, Vincent Wu, Paul H. Guth, Eli Engel, Marshall H. Montrose, Jonathan D. Kaunitz
Background & Aims :Dysfunction of the cystic fibrosis transmembrane regulator (CFTR) is associated with diminished duodenal HCO 3?secretion, despite a reported lack of clinical duodenal ulceration in affected subjects. We hypothesized that duodenal epithelial cells expressing a mutant CFTR have enhanced resistance to acid-induced injury. To test this hypothesis, we measured duodenal epithelial cell intracellular pH (pH i), injury, and acid back-diffusion in response to a luminal acid challenge in transgenic mice. Methods :A murine colony was established for the CFTR ?F508 (?F) mutation. Epithelial cell pH iwas measured by microscopy with a trapped, fluorescent pH-sensitive dye in living C57BL/6 and ?F/?F, +/?F, and +/+ mice. In vivo confocal microscopy confirmed the localization of the dye in the cytoplasm of the epithelial cells. Epithelial injury was measured fluorometrically using propidium iodide. Duodenal epithelial bicarbonate secretion and proton permeability were measured by back-titration. Bicarbonate secretion and acid back-diffusion were measured in a perfused duodenal loop. Results :Basal and post-acid exposure bicarbonate secretion were reduced in ?F/?F mice, although acid back-diffusion was similar to controls. Epithelial pH iof CFTR ?F/?F mice during luminal acid exposure was significantly higher than pH iin +/?F, +/+, or C57BL/6 mice. Acid-related epithelial injury was markedly less in ?F/?F mice in comparison with the other groups. Conclusions :Increased cellular buffering power of the epithelial cells of ?F/?F mice likely protects against acidification and injury during acid exposure. We speculate that this protective mechanism partially underlies the perceived relative lack of peptic ulceration in patients affected by cystic fibrosis.

Basic-liver, Pancreas, and Biliary Tract

Regulation of hepatic stellate cell activation and growth by transcription factor myocyte enhancer factor 2
Xuemin Wang, Xiaoli Tang, Xiaoming Gong, Efsevia Albanis, Scott L. Friedman, Zixu Mao
Background & Aims: Hepatic stellate cells (HSCs) undergo activation during the development of liver fibrosis. Transcriptional regulation plays a key role in this process. We studied the role of transcription factor myocyte enhancer factor 2 (MEF2) during HSC activation. Methods: Culture of HSCs isolated from rat liver on plastic dishes and HSC-T6 on a basement membrane–like matrix were used as models of HSC activation and deactivation, respectively. The expression and activity of MEF2 were correlated with HSC activation. The roles of MEF2 during HSC activation were assessed in vitro and in vivo by animal models of fibrosis. Results: Early induction of MEF2 messenger RNA and protein accompanied culture-induced HSC activation. This was associated with enhanced MEF2 DNA binding and transactivation activity. p38 mitogen-activated protein kinase but not extracellular signal–regulated kinase pathway was required for increased MEF2 activity during HSC activation. Increased MEF2 protein also correlated with fibrosis in vivo. Reversal of HSC activation was paralleled by a marked decrease in MEF2 protein and activity. Functionally, enhancing MEF2 significantly increased the expression of ?–smooth muscle actin (?-SMA), activated collagen I promoter activity, and stimulated HSC proliferation. MEF2 interference RNA significantly inhibited expression of ?-SMA, collagen ?1(I), and proliferating cell nuclear antigen. Conclusions: The studies provide the first evidence for the presence of MEF2 in the liver and show that MEF2 regulates multiple aspects of HSC activation. These studies show a novel role of MEF2 as a key nuclear mediator that may participate in the pathologic process of liver fibrogenesis in vivo.

Hepatocyte-specific disruption of Bcl-xL leads to continuous hepatocyte apoptosis and liver fibrotic responses
Tetsuo Takehara, Tomohide Tatsumi, Takahiro Suzuki, Edmund B. Rucker, Lothar Hennighausen, Masahisa Jinushi, Takuya Miyagi, Yoshiyuki Kanazawa, Norio Hayashi
Background & Aims: Recent research has suggested that apoptosis could be involved in the development of fibrosis, although it is generally considered to be a mechanism of cell removal without consequences to the tissue. Bcl-x L, an antiapoptotic member of the Bcl-2 family, is expressed in hepatocytes and up-modulated during various pathologic conditions. The aim of this study was to explore the function of Bcl-x Lin hepatocytes using the Cre- lox P system and to analyze the consequences of long-term apoptosis in hepatocytes. Methods: Hepatocytes isolated from mice homozygous for a floxed bcl-x allele ( bcl-x fl/fl ) were infected with recombinant adenovirus expressing the Cre recombinase gene (AdexCre). Bcl-x fl/fl mice were crossed with Alb-Cre transgenic mice, which express Cre under regulation of the albumin gene promoter to generate hepatocyte-specific Bcl-x L-deficient mice. Results: On AdexCre infection, primary cultured bcl-x fl/fl hepatocytes reduced their expression of Bcl-x Land rapidly underwent apoptosis associated with mitochondrial damage. In vivo hepatocyte-specific disruption of Bcl-x Lresulted in spontaneous apoptosis of hepatocytes for more than 6 months. The Bcl-x L-deficient mice showed liver fibrosis with advanced age that was preceded by an increase in hepatic transforming growth factor ? production. In vitro, macrophages and hepatocytes produced transforming growth factor ? on exposure to apoptotic hepatocytes. Conclusions: The present study identified Bcl-x Las a critical apoptosis antagonist in hepatocytes. Furthermore, it offers proof that persistent apoptosis of parenchymal cells is sufficient to induce fibrotic responses and suggests a mechanistic link between apoptosis and fibrosis.

Nerve growth factor modulates the proliferative capacity of the intrahepatic biliary epithelium in experimental cholestasis
Alessandro Gigliozzi, Gianfranco Alpini, Gianluca Svegliati Baroni, Luca Marucci, Veronica Drudi Metalli, Shannon S. Glaser, Heather Francis, Maria Grazia Mancino, Yoshiyuki Ueno, Barbara Barbaro, Antonio Benedetti, Adolfo F. Attili, Domenico Alvaro
Background & Aims :We evaluated the expression of neurotrophins in rat cholangiocytes and the role and mechanisms by which nerve growth factor (NGF) modulates cholangiocyte proliferation. Methods :The expression of neurotrophins and their receptors was investigated by immunohistochemistry in liver sections and reverse-transcription polymerase chain reaction and immunoblots in isolated cholangiocytes. In vitro, the effect of NGF on cholangiocyte proliferation and signal transduction was investigated by immunoblotting for proliferating cell nuclear antigen, phosphorylated AKT (p-AKT), phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2), phosphorylated c-jun-N-terminal kinase, and phosphorylated p38. In vivo, rats that had undergone bile duct ligation (BDL) were treated with an anti-NGF antibody to immunoneutralize NGF and bile duct mass, proliferation, apoptosis, and inflammation were investigated by immunohistochemistry. Results :NGF and its TrkA receptor were expressed by normal rat cholangiocytes and up-regulated following BDL. Cholangiocytes secrete NGF, and secretion is increased in proliferating BDL cholangiocytes. In vitro, NGF stimulated cholangiocyte proliferation, which was associated with enhanced p-AKT and p-ERK1/2 expression. NGF proliferation in vitro was partially blocked by the MEK inhibitor (UO126) and completely ablated by the phosphatidylinositol 3-kinase inhibitor (wortmannin). In vitro, NGF and estrogens have an additive effect on cholangiocyte proliferation by acting on phosphorylated TrkA and p-ERK1/2. In vivo, immunoneutralization of NGF decreased bile duct mass in BDL rats, which was associated with depressed proliferation and enhanced apoptosis and with increased portal inflammation. Conclusions :Cholangiocytes secrete NGF and express NGF receptors. NGF induces cholangiocyte proliferation by activating the ERK and, predominantly, the phosphatidylinositol 3-kinase pathway and exerts an additive effect in combination with estrogens on proliferation.

Hepatic response to right ventricular pressure overload
Roben G. Gieling, Jan M. Ruijter, Adri A.W. Maas, Marius A. Van Den Bergh Weerman, Koert P. Dingemans, Fibo J.W. Ten Kate, Ronald H. Lekanne Dit Deprez, Antoon F.M. Moorman, Wouter H. Lamers
Background & Aims : Modifying the afferent blood supply to the liver does not change the zonal expression pattern of hepatic enzymes in the rat. Methods : We used pulmonary trunk banding (PTB) to study the effect of an efferent hindrance of blood flow on hepatic architecture and zonation of gene expression. Results : Most PTB rats developed right ventricular hypertrophy and congestive heart failure. The hepatic response to PTB developed concomitantly with the decline in heart function. Enzyme expression in the periportal region was not affected, but the pericentral rim of hepatocytes expressing glutamine synthetase, ornithine aminotransferase, and NADPH cytochrome P-450 reductase (CYPred) first declined in diameter, then became discontinuous, and finally disappeared. Meanwhile, ornithine aminotransferase and especially CYPred, became re-expressed in the periportal zone. These changes occurred without appreciable cell death or fibrotic changes; the expression of fibronectin and ?-smooth muscle actin increased perisinusoidally, but that of collagen did not. Electron microscopic analysis revealed normal fenestration of the sinusoidal endothelial cells without detectable deposition of basement membrane material, but both the width of the space of Disse and the length and number of hepatic microvilli were significantly reduced, implying a decreased flow of fluid in the space of Disse. Conclusions : The reprogramming of gene expression in livers with a postsinusoidal hindrance of blood flow results from declining access of the hepatocytes to intrasinusoidal signal-transduction molecules and suggest that the impaired biotransformation that accompanies right ventricular failure is caused by a central-to-portal shift in expression of the corresponding enzymes.

Gene therapy for human ?1-antitrypsin deficiency in an animal model using SV40-Derived vectors
Yu–You Duan, Jian Wu, Jian–Liang Zhu, Shu–Ling Liu, Iwata Ozaki, David S. Strayer, Mark A. Zern
Background & Aims: In most genetic diseases, the goal of gene therapy is to deliver a particular transgene; however, sometimes a deleterious gene product must be eliminated. Because of the promise of recombinant simian virus 40 (rSV40) vectors, we tested their ability to deliver a transgene and to target a transcript for destruction by direct administration of the vectors to the liver of an animal model for human ? 1-antitrypsin (? 1-AT) deficiency. Methods: Therapy of human ? 1-AT deficiency requires stable transduction of resting hepatocytes, both to deliver wild-type ? 1-AT and to inhibit production of mutant ? 1-AT. Transgenic mice carrying the mutant human ? 1-AT PiZ allele were treated through an indwelling portal vein catheter with a simian virus 40 (SV40)-derived vector carrying a ribozyme designed to target the human transcript. Results: Treated transgenic mice showed marked decreases of human ? 1-AT messenger RNA and the protein in the liver, and serum levels of human ? 1-AT were decreased to 50% ± 5% of pretreatment values 3–16 weeks after transduction. Moreover, when normal mice were treated with an SV40-derived vector containing a modified human ? 1-AT complementary DNA engineered to be resistant to cleavage by the ? 1-AT ribozyme, they expressed human ? 1-AT messenger RNA and protein in their livers and serum levels of human ? 1-AT remained >1 ?g/mL for 1 year. Conclusions: These results represent the initial steps toward a novel approach to the gene therapy of ? 1-AT deficiency.

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

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JOURNAL OF HEPATOLOGY

October 2004 (Vol. 41 , Issue 4)

Biliary Tract and Cholestasis

cAMP stimulates the secretory and proliferative capacity of the rat intrahepatic biliary epithelium through changes in the PKA/Src/MEK/ERK1/2 pathway
Francis H, Glaser S, Ueno Y, LeSage G, Marucci L, Benedetti A, Taffetani S, Marzioni M, Alvaro D, Venter J, Reichenbach R, Fava G, Lynne Phinizy J, Alpini G pages 528-537
Background/Aims
To evaluate if increased cholangiocyte cAMP levels alone are sufficient to enhance cholangiocyte proliferation and secretion.
Methods
Normal rats were treated in vivo with forskolin for two weeks. Cholangiocyte apoptosis, proliferation and secretion were evaluated. Purified cholangiocytes from normal rats were treated in vitro with forskolin in the absence or presence of Rp-cAMPs (a PKA inhibitor), PP2 (an Src inhibitor) or PD98059 (a MEK inhibitor). Subsequently, we evaluated cholangiocyte proliferation by determination of proliferating cellular nuclear antigen (PCNA) protein expression by immunoblots. We evaluated if the effects of forskolin on cholangiocyte functions are associated with changes in the cAMP/PKA/Src/MEK/ERK1/2 pathway.
Results
Chronic administration of forskolin to normal rats increased the number of ducts, cAMP levels, and secretin-induced choleresis compared to controls. Forskolin-induced increases in cholangiocyte proliferation and secretion were devoid of cholangiocyte necrosis, inflammation and apoptosis. In vitro, in pure isolated cholangiocytes, forskolin increased cholangiocyte proliferation, which was ablated by Rp-cAMPs, PP2 and PD98059. The effects of forskolin on cholangiocyte proliferation were associated with increased activity of PKA, Src Tyrosine 139 (Tyr 139) and ERK1/2.
Conclusions
Modulation of the PKA/Src/MEK/ERK1/2 pathway may be important in the regulation of cholangiocyte growth and secretion observed in cholestatic liver diseases.

Cell Biology, Metabolism and Transport

Up-regulation of the high-affinity pyrimidine-preferring nucleoside transporter concentrative nucleoside transporter 1 by tumor necrosis factor-alpha and interleukin-6 in liver parenchymal cells , 19 August 2004
Fernández-Veledo S, Valdés R, Wallenius V, Casado F J, Pastor-Anglada M pages 538-544
Background/Aims
Concentrative nucleoside transporter 1 (CNT1), a high affinity transporter for pyrimidine nucleosides, is responsible for their Na +-dependent concentrative uptake into hepatocytes. Though CNT1 protein amounts increase in rat liver soon after partial hepatectomy, the physiological regulators of CNT1 expression have not yet been identified.
Methods
Rat hepatoma cell lines and hepatocytes isolated from fetuses and adult rats were used to identify single agents able to up-regulate CNT1 expression and activity in liver. TNF-? receptor-I (TNFRI) and IL-6 knock-out mice were also used to study CNT1 regulation in vivo.
Results
TNF-? and IL-6 independently induced CNT1 protein expression in cultured liver parenchymal and FAO hepatoma cells by PI-3 kinase- and ERK-dependent mechanisms, respectively. In vivo data showed that transporter protein levels were low in livers from TNFRI knock-out mice, but not in those from IL-6 deficient animals. However, IL-6 administration only partially restored CNT1 expression in the former model.
Conclusions
This study identifies TNF-? as a major in vivo modulator of the nucleoside transporter CNT1 and suggests a secondary role for IL-6 in mediating CNT1 up-regulation by TNF-? in vivo. Evidence is provided that two independent pathways are involved in the up-regulation of CNT1 by TNF-? and IL-6.

Basic fibroblast growth factor promotes the trans-differentiation of mouse bone marrow cells into hepatic lineage cells via multiple liver-enriched transcription factors , 19 August 2004
Saji Y, Tamura S, Yoshida Y, Kiso S, Iizuka AS, Matsumoto H, Kawasaki T, Kamada Y, Matsuzawa Y, Shinomura Y pages 545-550
Background/Aims
Evidence that bone marrow cells have trans-differentiating potential to hepatocytes has been described in recent reports. However, the molecular mechanism underlying this phenomenon is unclear. To address this issue, we investigated the parameters involved in the trans-differentiation of bone marrow cells into a hepatic lineage.
Methods
Mouse BM cells were cultured in a collagen gel without or with growth factors including basic fibroblast growth factor. The expression of hepatocyte-specific markers, cholangiocyte-specific marker and liver-enriched transcription factors was identified by RT-PCR and immunohistochemistry.
Results
Basic fibroblast growth factor was found to be the most effective for inducing albumin in cultured BM cells. Furthermore, on stimulation of basic fibroblast growth factor, BM cells were found to express other hepatocyte-specific markers and a cholangiocyte-specific marker. This conversion was found to be associated with the induction of transcription factors including hepatocyte nuclear factors and GATA family proteins.
Conclusions
We established an in vitro culture system in which mouse bone marrow cells could trans-differentiate to hepatic lineage cells in response to growth factors, without cell fusion. In particular, basic fibroblast growth factor has the ability to induce the trans-differentiation into hepatic lineage cells from BM cells.

Chronic Liver Diseases

Immunosurveillance of alveolar echinococcosis by specific humoral and cellular immune tests: long-term analysis of the Swiss chemotherapy trial (1976–2001) , 6 September 2004
Ammann RW, Renner EC, Gottstein B, Grimm F, Eckert J, Renner EL, Swiss Echinococcosis Study Group  pages 551-559
Background/Aims
Long-term chemotherapy with benzimidazoles is beneficial in non-resectable alveolar echinococcosis (AE). Criteria to track early therapeutic efficacy are lacking and the clinical impact of immunosurveillance is unsettled. We aimed to analyze this issue particularly for assessing the putative parasitocidal efficacy of chemotherapy.
Methods
The present study is part of our prospective Swiss trial outlined previously and comprises 57 patients with a median follow-up of 18.5 (3–30) years and with repeated tests of humoral and cell-mediated immunity. The series was subdivided into group A ( n=23; curative surgery) and group B ( n=34: non-resectable AE).
Results
Long-term survival was 87% (group A) and 76% (group B). The profiles of specific antibodies against EmII/3-10 antigen normalized within 3 years in most group A-patients, but remained above the cut-off value in 40% of group B-patients. This lack of normalization was associated with lower bioavailability of mebendazole. AE-recurrence after ‘radical’ surgery (up to 13 years) was associated with high anti-EmII/3-10 concentrations in 7 of 8 cases. Following abrogation of longterm chemotherapy in group B, no AE-recurrence occurred in 9/18 patients, suggestive of parasitocidal efficacy and documented by a normal EmII/3-10 profile.
Conclusions
The EmII/3-10 profile is of value in monitoring AE after surgery and/or chemotherapy.

Cirrhosis and its Complications

Bleeding ectopic varices—treatment with transjugular intrahepatic porto-systemic shunt (TIPS) and embolisation , 13 September 2004
Vangeli M, Patch D, Terreni N, Tibballs J, Watkinson A, Davies N, Burroughs AK pages 560-566
Background/Aims
Bleeding ectopic varices due to cirrhosis can be difficult to manage. We report our experience of uncontrolled bleeding from ectopic varices treated with transjugular intrahepatic porto-systemic shunt (TIPS).
Methods
We selected the 21 cirrhotics who underwent TIPS for bleeding ectopic varices from our database: Child-Pugh grade A (2), B (11) and C (8). Site of bleeding was rectal (11), colonic (2), ileal 1, jejunal 1, duodenal 1, and stomal (5).
Results
TIPS was performed successfully in 19/21 (90%) patients. All except 1 had either a reduction in portosystemic pressure gradient ≤ 12mmHg ( n=12) or reduction by 25–50% of baseline ( n=6). TIPS alone was used in 12/19: 7 of these 12 had no further bleeding; 5 (42%) rebled within 48h, and had embolisation, 4 without further bleeding. In 7 of 19, TIPS and embolisation were performed together: 2 patients (28%) rebled; further embolisation stopped the bleeding.
Conclusions
Ectopic varices do rebleed despite a reduction of porto-systemic pressure gradient ≤ 12mmHg or by 25–50% of baseline, following TIPS. Embolisation stopped bleeding in all but 1 patient. We recommend performing embolisation at the time of the initial TIPS to control bleeding from ectopic varices.

Inflammation and Fibrosis

Superoxide-induced apoptosis of activated rat hepatic stellate cells , 19 August 2004
Thirunavukkarasu C, Watkins S, Harvey SAK, Gandhi CR pages 567-575
Background/Aims
During liver injury, reactive oxygen species (ROS) are produced by the resident macrophages (Kupffer cells) and infiltrating blood cells such as neutrophils. ROS cause transformation of desmin-positive quiescent hepatic stellate cells (HSCs) into the proliferating activated phenotype that expresses ?-smooth muscle actin (?-SMA). The highly fibrogenic and contractile activated HSCs (aHSCs) produce various cytokines and growth factors, and play important role in the pathophysiology of chronic liver disease. However, apoptotic aHSCs are also observed during active fibrogenesis in the injured liver. Therefore, we investigated the mechanisms of apoptosis of aHSCs in relation to ROS.
Methods
HSCs, isolated from normal rat liver, were activated in culture and effects of superoxide were determined between subcultures 3 and 5.
Results
Treatment with superoxide caused apoptosis of aHSCs as determined by flow cytometry, TUNEL assay and DNA laddering analysis. The mechanisms of superoxide-induced apoptosis involved release of cytochrome c, increased Bax expression, increased caspase-3 activity, and hydrolysis of polyADP-ribose polymerase. Superoxide also increased the expression of antiapoptotic Bcl-xL and nuclear translocation of NF?B. Caspase-3 inhibitor (DEVD-fmk) and antioxidants ( N-acetylcysteine, vitamin E and superoxide dismutase) inhibited superoxide-induced apoptosis.
Conclusions
Superoxide-induced apoptosis of aHSCs may be a novel mechanism of limiting chronic fibrotic liver injury
Inflammation and Fibrosis

Serum markers of hepatic fibrogenesis in cystic fibrosis liver disease , 19 August 2004
Pereira TN, Lewindon PJ, Smith JL, Murphy TL, Lincoln DJ, Shepherd RW, Ramm GA pages 576-583
Background/Aims
Hepatic fibrosis contributes to adverse outcome in cystic fibrosis (CF). Early detection of CF liver disease (CFLD) may identify patients at risk of significant complications. To evaluate the utility of serum markers to detect hepatic fibrosis in children with CFLD vs. CF patients without liver disease (CFnoLD) and controls.
Methods
Sera from 36 CFLD, 30 CFnoLD and 39 controls were assessed for tissue inhibitor of matrix metalloproteinase (MMP) (TIMP)-1, collagen (CL)-IV, MMP-2, hyaluronic acid (HA) and prolyl hydroxylase (PH) by enzyme immunoassay and were correlated with hepatic fibrosis score in CFLD.
Results
TIMP-1, PH and CL-IV were increased in CFLD vs. CFnoLD and controls. Fibrosis score was negatively correlated with TIMP-1 ( r=?0.34, P=0.06) and PH ( r=?0.48, P=0.008). Receiver-operating characteristics analysis showed CL-IV (AUC 0.785, P<0.0001) and TIMP-1 (AUC 0.765, P<0.0001) differentiated CFLD from CFnoLD and controls, while PH (AUC 0.814, P<0.0001) predicted early fibrogenesis. Diagnostic accuracy improved using logistic regression combining (i) CL-IV, TIMP-1, PH to identify CFLD (AUC 0.831, P<0.0001) and (ii) TIMP-1, PH to identify CFLD patients with no fibrosis (AUC 0.852, P<0.02).
Conclusions
Elevated TIMP-1, CL-IV, PH may be indicators of hepatic fibrogenesis in CF. Increased TIMP-1, PH may be early markers of CFLD.

Inflammation and Fibrosis

Japanese herbal medicine Inchin-ko-to as a therapeutic drug for liver fibrosis , 26 August 2004
Inao M, Mochida S, Matsui A, Eguchi Y, Yulutuz Y, Wang Y, Naiki K, Kakinuma T, Fujimori K, Nagoshi S, Fujiwara K pages 584-591
Background/Aims
Inchin-ko-to (TJ-135) is an herbal medicine used in Japan for treatment of icteric patients with cirrhosis. Its efficacy as an anti-fibrogenic drug was evaluated in relation to stellate cell activation.
Methods/Results
Liver fibrosis was induced in rats by repeated injections of carbon tetrachloride (CCl 4) or pig-serum. Oral administration of TJ-135 improved the mortality of rats given CCl 4with reduced extents of liver necrosis and fibrosis. Similar improvement of liver fibrosis was found in rats given pig-serum showing no liver necrosis.
DNA synthesis of stellate cells activated in vitro after isolation from normal rat liver was decreased by culture with TJ-135 in a dose-related manner, accompanied by decreased smooth muscle ? actin expression and contractility. Such attenuation was not found in the cells cultured with geniposide, an iridoid compound of TJ-135, but genipin, an aglycone of geniposide formed in the gut by action of bacterial flora, markedly decreased stellate cell activation without affecting synthesis of proteins other than collagen.
Conclusions
TJ-135 may be useful for treatment of liver fibrosis and portal hypertension through suppression of activated hepatic stellate cell function by genipin, an absorbed form of its component.

Pentoxifylline attenuates steatohepatitis induced by the methionine choline deficient diet , 19 August 2004
Koppe SWP, Sahai A, Malladi P, Whitington PF, Green RM
pages 592-598
Background/Aims
Feeding mice a methionine choline deficient (MCD) diet serves as a nutritional model of non-alcoholic steatohepatitis (NASH). NASH and alcohol-induced steatohepatitis are histologically similar, suggesting a similar pathogenesis. Pentoxifylline (PTX) attenuates TNF-alpha production, acts as an antioxidant and decreases mortality in alcoholic steatohepatitis. The aim of our study is to determine if PTX attenuates MCD diet induced steatohepatitis and determine the mechanism of this effect.
Methods
Mice were placed on an MCD or control diet for 2 weeks and were treated with or without PTX. Serum ALT, liver histology, and inflammatory mechanisms were evaluated.
Results
PTX attenuates MCD diet induced steatohepatitis, decreasing both serum ALT levels and hepatic inflammation. Serum ALT levels were reduced approximately 50% in the MCD+PTX group compared to the MCD group. Hepatic glutathione levels were significantly higher in the MCD+PTX group compared to the MCD group. There was also a reduction in TNF-alpha mRNA in female mice treated with PTX. MCD+PTX mice had increased hepatic triglyceride content compared to the MCD mice, but less histologic evidence of inflammation despite the increased steatosis. Serum lipid and bile salt levels also were similar in PTX and vehicle control treated mice.
Conclusions
PTX decreases serum ALT levels and hepatic inflammation in the MCD model of steatohepatitis, likely via increasing glutathione levels or reducing TNF-alpha expression.

Liver Cell Injury and Liver Failure

Improvement of C3A cell metabolism for usage in bioartificial liver support systems , 19 August 2004
Filippi C, Keatch SA, Rangar D, Nelson LJ, Hayes PC, Plevris JN
pages 599-605
Background/Aims
The use of cell lines in bioartificial liver support systems (BALSS) to treat fulminant hepatic failure (FHF) is hindered by their reduced metabolic functions, which could be further decreased by the patient's serum/plasma. Hence, the aim of this study was to (i) test the effect of the FHF serum on C3A cell metabolism; (ii) precondition the cells to improve their metabolic capacity.
Methods
C3A cells were preconditioned in a medium developed at the University of Edinburgh (UoE) or a 10% FHF serum medium. Metabolism capacity was assessed on days 3, 7 and 10 and compared with primary porcine hepatocytes. Preconditioned-cell metabolism was reassessed after (i) passage and (ii) incubation with 10% FHF serum.
Results
UoE-preconditioned cells showed time-dependent increase in gluconeogenesis (500%), ureogenesis (200%), galactose elimination (240%) albumin synthesis (250%). These results were in the same order of magnitude as the ones obtained with primary porcine hepatocytes and were further enhanced by cell passage. UoE-preconditioning prevented the decrease of metabolism induced by acute incubation with FHF serum on control C3A cells. Preconditioning with FHF serum did not improve cell metabolism.
Conclusions
Cell preconditioning with UoE-medium increases metabolic capacity and would greatly improve BALSS efficacy
Thalidomide salvages lethal hepatic necroinflammation and accelerates recovery from cirrhosis in rats , 19 August 2004
Yeh TS, Ho YP, Huang SF, Yeh JN, Jan YY, Chen MF
pages 606-612
Background/Aims
The authors investigated the feasibility of thalidomide employed to treat liver fibrosis.
Methods
A cirrhotic model was established using Sprague–Dawley rats fed thioacetamide. Thalidomide-treated group was given thalidomide (10mg/kg/day) intraperitoneally for 10 consecutive days. Mortality, histopathological changes, TNF?, TGF?1, TIMP-1 and TIMP-2 were determined. Expression of TNF? and TGF?1 mRNA of Kupffer's cells derived from the experimental rats were determined.
Results
The mortality rates of thalidomide-treated group and vehicle-treated group were 8 and 32%, respectively. The total Knodell score of thalidomide-treated rats was lower than those of vehicle-treated rats. Micro-nodular cirrhosis resolved grossly in thalidomide-treated rats on day 28; while vehicle-treated rats continued to display uneven liver surface on day 28. Expression of TNF?, TGF?1, TIMP-1, and TIMP-2 was decreased in thalidomide-treated rats compared to those treated with vehicles. Finally, the expression of TNF? and TGF?1 mRNA of Kupffer's cells derived from rats treated with thalidomide were much lower than those treated with vehicle.
Conclusions
Thalidomide salvages lethal hepatic necroinflammation, accelerates recovery from cirrhosis in rats, and works by suppressing of TNF? and TGF?1 production of Kupffer's cells.

Pathogenesis of intracranial hypertension in acute liver failure: inflammation, ammonia and cerebral blood flow , 19 August 2004
Jalan R, Olde Damink SWM, Hayes PC, Deutz NEP, Lee A pages 613-620
Background/Aims
The study aims were to determine the role of inflammation in the pathogenesis of increased intracranial pressure (ICP) in patients with acute liver failure (ALF) and its interplay with cerebral blood flow (CBF) and ammonia.
Methods
Twenty-one patients with ALF were studied from the time they were ventilated for grade 4 encephalopathy until receiving specific treatment for increased ICP. Depending upon the ICP, the patients were divided into two groups; those that required specific treatment (ICP>20 mmHg, group 1: n=8, ICP: 32 (28–54) mmHg); and those that did not (ICP≤20 mmHg, group 2: n=13, ICP: 15 (10–20) mmHg).
Results
Inflammatory markers, arterial ammonia and CBF were significantly higher in the group 1 patients. TNF? levels correlated with CBF ( r=0.80). Four patients from group 2 developed surges of increased ICP (32 (15–112) hours from enrolment). These were associated increases in markers of inflammation and TNF?, and an increase in CBF. There was no change in these inflammatory markers, CBF or ICP in the other 9 group 2 patients.
Conclusions
The results of this study suggest that inflammation plays an important synergistic role in the pathogenesis of increased ICP possibly through its effects on CBF.

Increased expression of toll-like receptor 4 enhances endotoxin-induced hepatic failure in partially hepatectomized mice , 19 August 2004
Takayashiki T, Yoshidome H, Kimura F, Ohtsuka M, Shimizu Y, Kato A, Ito H, Shimizu H, Ambiru S, Togawa A, Miyazaki M pages 621-628
Background/Aims
Liver failure associated with infections after hepatectomy remains a cause of mortality. It has recently been reported that toll-like receptor 4 (TLR4) is involved in recognizing lipopolysaccharides (LPS). The aim of this study was to investigate the role of TLR4 in endotoxin-induced liver injury after hepatectomy.
Methods
C3H/HeN and C3H/HeJ mice underwent 70% hepatectomy or sham surgery, and LPS was administered 48h after surgery. Expression of TLR4 mRNA, nuclear factor-?B (NF-?B) activation, tumor necrosis factor-? (TNF-?) and serum ALT levels, histological findings, and myeloperoxidase content were examined. Survival after LPS administration was also determined.
Results
Hepatic expression of TLR4 was significantly increased 6–72h after hepatectomy. In mice with endotoxemia after hepatectomy, hepatic NF-?B activation was greatly increased. Hepatic mRNA and serum levels of TNF-?, and ALT levels were significantly elevated compared with sham operated controls. Focal necrosis with neutrophil infiltration was apparent, which is consistent with increased myeloperoxidase contents in endotoxemia after hepatectomy in C3H/HeN mice. These were completely absent in C3H/HeJ mice. Survival of C3H/HeN mice with endotoxemia after hepatectomy was significantly lower than that of C3H/HeJ mice.
Conclusions
Upregulated TLR4 expression and function after hepatectomy plays a pivotal role in endotoxin-induced liver injury after hepatectomy.

Liver Growth and Cancer

Overexpression of cortactin is involved in motility and metastasis of hepatocellular carcinoma , 19 August 2004
Chuma M, Sakamoto M, Yasuda J, Fujii G, Nakanishi K, Tsuchiya A, Ohta T, Asaka M, Hirohashi S pages 629-636
Background/Aims
The molecular basis of the metastasis of hepatocellular carcinoma (HCC) is not fully understood. The aim of this study was to elucidate the crucial genes involved in metastasis of HCC.
Methods
We compared expression profiles among highly metastatic HCC cell lines and non-metastatic HCC cell lines by using oligonucleotide array to identify genes associated with metastasis. We further investigated the effect of identified gene on cell motility and metastasis in vitro and in vivo. Finally, we examined immunohistochemistry in human tissue samples.
Results
We identified 39 genes whose expression levels were significantly correlated with metastatic ability ( P<0.05). Of these genes, we further investigated cortactin, because this cortical actin-associated protein is a substrate of Src, whose activation has been shown to be involved in HCC cell migration and metastasis. Overexpression of cortactin in a non-metastatic HCC cell line increased cell motility, and resulted in metastasis in an orthotopic model. Furthermore, immunohistochemical expression of cortactin revealed its significant overexpression in HCC with intrahepatic metastasis compared with HCC without intrahepatic metastasis ( P<0.005).
Conclusions
Overexpression of cortactin may play a role in the metastasis of HCC by influencing cell motility, and cortactin could be a sensitive marker for HCC with intrahepatic metastasis.

Liver Growth and Cancer

Ninjurin1 increases p21 expression and induces cellular senescence in human hepatoma cells , 19 August 2004
Toyama T, Sasaki Y, Horimoto M, Iyoda K, Yakushijin T, Ohkawa K, Takehara T, Kasahara A, Araki T, Hori M, Hayashi N pages 637-643
Background/Aims
Ninjurin1 is a novel adhesion molecule that has a role in promoting nerve regeneration. Although ninjurin1 is ubiquitously expressed in various human tissues, including the liver, the biologic functions of ninjurin1 in tissues other than the nervous system remain unknown. The aim of this study was to investigate the function of ninjurin1 in hepatocytes.
Methods
The effect of ninjurin1 overexpression was examined in Huh-7 hepatoma cells. Ninjurin1 expression was examined by Western blot in human hepatocellular carcinoma tissues as well as their adjacent liver tissues.
Results
Ninjurin1-overexpressing clones exhibited strong growth inhibition due to G1 cell cycle arrest, which is associated with a posttranscriptional increase in p21 WAF1/Cip1 , a decrease of cyclin-dependent kinase 2 activity and the hypophosphorylation of Rb. The ninjurin1-overexpressing clones had increased senescence-associated ?-galactosidase activity and autofluorescent pigment, characteristic features of cellular senescence. The levels of ninjurin1 expression were higher in hepatocellular carcinoma tissues than those in adjacent liver tissues.
Conclusions
The present study provides the first evidence that ninjurin1 is able to induce the senescence program. Ninjurin1 may be involved in the regulation of cellular senescence in the liver during carcinogenesis.

Viral Hepatitis

Alcohol is an important co-factor for both steatosis and fibrosis in Northern Italian patients with chronic hepatitis C , 19 August 2004
Fabris P, Floreani A, Carlotto A, Giordani MT, Baldo V, Stecca C, Marchioro L, Tramarin A, Bertin T, Negro F, de Lalla F pages 644-651
Background/Aims
Steatosis in patients with chronic hepatitis C (CHC) may be the result of both viral and host factors. To evaluate: (1) the relationship between steatosis and either host or viral factors; (2) the correlation between steatosis and fibrosis in patients with CHC.
Methods
A consecutive series of 349 patients were evaluated for steatosis. At liver biopsy, patients were tested for virological, and laboratory analysis and questioned for alcohol consumption.
Results
Logistic regression analysis demonstrated that steatosis was independently associated with genotype 3a (odds ratio, OR 3.5), alcohol intake at the time of biopsy (OR 2.6) and age >35 years (OR 2.7). In multivariate analysis the presence of fibrosis was associated with past alcohol abuse (OR 3.7), and age older than 44 years (OR 2.2). Overall, a weak correlation was found between grade of steatosis and fibrosis score ( r=0.861, P=0.05), which disappeared excluding patients without past or current alcohol intake. A direct correlation emerged between grade of steatosis and both ‘grading’ and ‘staging’ only in patients with genotypes other than 3a.
Conclusions
Genotype 3a is the main risk factor for steatosis in patients with CHC. The grade of steatosis correlated with both grading and staging only in patients with genotypes other than 3a and this relationship is strictly linked to alcohol consumption.

Influence of interleukin 12B (IL12B) polymorphisms on spontaneous and treatment-induced recovery from hepatitis C virus infection , 19 August 2004
Mueller T, Mas-Marques A, Sarrazin C, Wiese M, Halangk J, Witt H, Ahlenstiel G, Spengler U, Goebel U, Wiedenmann B, Schreier E, Berg T pages 652-658
Background/Aims
Interleukin-12 (IL-12) governs the Th1-type immune response, affecting the spontaneous and treatment-induced recovery from HCV-infection. We investigated whether the IL12B polymorphisms within the promoter region (4bp insertion/deletion) and the 3?-UTR (1188-A/C), which have been reported to influence IL-12 synthesis, are associated with the outcome of HCV infection.
Methods
We analyzed 186 individuals with spontaneous HCV clearance, 501 chronically HCV infected patients, and 217 healthy controls. IL12B 3?-UTR and promoter genotyping was performed by Taqman-based assays with allele-specific oligonucleotide probes and PCR-based allele-specific DNA-amplification, respectively.
Results
The proportion of IL12B promoter and 3?-UTR genotypes did not differ significantly between the different cohorts. However, HCV genotype 1-infected patients with high baseline viremia carrying the IL12B 3?-UTR 1188-C-allele showed significantly higher sustained virologic response (SVR) rates (25.3% vs. 46% vs. 54.5% for A/A, A/C and C/C) due to reduced relapse rates (24.2% vs. 12% vs. zero % for A/A, A/C and C/C).
Conclusions
IL12B 3?-UTR 1188-C-allele carriers appear to be capable of responding more efficiently to antiviral combination therapy as a consequence of a reduced relapse rate. No association of IL12B polymorphisms and self-limited HCV infection could be demonstrated.

Quantification and genotyping of hepatitis B virus in a single reaction by real-time PCR and melting curve analysis , 19 August 2004
Yeh SH, Tsai CY, Kao JH, Liu CJ, Kuo TJ, Lin MW, Huang WL, Lu SF, Jih J, Chen DS, Chen PJ pages 659-666
Background/Aims
Both viral titer and genotype of hepatitis B virus (HBV) play critical roles in determining clinical outcome and response to antiviral treatment in hepatitis B patients. In this study, a method was developed to determine both parameters in a single-tube reaction.
Methods
The method contains two consecutive steps, the first step used real-time PCR for quantification and second step used melting curve analysis for genotyping. For accurate quantification, the PCR primers and hybridization probes were selected from highly conserved regions to ensure the equivalent amplification and hybridization of all genotypes of HBVs. Within the sensor probe there exists signature single nucleotide polymorphisms (SNPs), which could effectively differentiate different HBV genotypes by showing different melting temperatures.
Results
The quantification results showed great consistency with the commercial assays in linear range from 10 2to 10 11 copies/ml. By comparison with the traditional restriction fragment length polymorphism (RFLP) methods, 99% of samples were accurately genotyped by current assay, and with a higher detection rate. In addition, this method can detect mixed HBV infections.
Conclusions
Currently, this methodology can be applied to areas prevalent with HBV genotypes B and C, providing an efficient alternative for clinical diagnosis and large-scaled longitudinal studies.

Interferon-? stimulation of liver cells enhances hepatitis delta virus RNA editing in early infection , 19 August 2004
Hartwig D, Schoeneich L, Greeve J, Schütte C, Dorn I, Kirchner H, Hennig H pages 667-672
Background/Aims
RNA editing controls the formation of hepatitis-delta-antigen-S and -L and therefore plays a central role in the hepatitis-delta-virus (HDV) life-cycle. Editing is catalyzed by the enzyme Adenosine-deaminase-acting-on-RNA1 (ADAR1) of which two different forms, ADAR1-L and ADAR1-S, exist. As ADAR1-L is induced by interferon (IFN)-?, we examined the influence of IFN-?-stimulation of host cells on HDV-RNA editing.
Methods
Editing was studied in Huh-7-cells transfected with HDV-RNA on days 7, 14, 21 and 28 after transfection. ADAR1-L mRNA was measured by RT-PCR.
Results
IFN-?-treatment led to a 5-fold higher expression of ADAR1-L and to an increase in editing from 14±2% (SD) in unstimulated controls to 27±4% (SD) on day 7 after transfection. Editing further increases over time to the same maximum level of 35% in IFN-?-treated as well as untreated cells.
Conclusions
By IFN-?-stimulation both ADAR1-L expression and editing are increased in Huh-7-cells at day 7, and the maximum level of edited antigenomes is reached earlier with IFN-?-treatment as compared to untreated cells. Thus, ADAR1-L appears to be able to increase editing, but the HDV genome apparently has an intrinsic negative feed-back regulation mechanism that limits editing to roughly a third of the genomes.

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

BRITISH MEDICAL JOURNAL

Copyright © 2004 BMJ Publishing Group Ltd

NEW ENGLAND JOURNAL

Volume 351:1521-1531 October 7, 2004 Number 15

Lamivudine for Patients with Chronic Hepatitis B and Advanced Liver Disease
Yun-Fan Liaw, M.D., Joseph J.Y. Sung, M.D., Wan Cheng Chow, M.D., Geoffrey Farrell, M.D., Cha-Ze Lee, M.D., Hon Yuen, M.D., Tawesak Tanwandee, M.D., Qi-Min Tao, M.D., Kelly Shue, M.R.Pharm.S., Oliver N. Keene, M.Sc., Jonathan S. Dixon, Ph.D., D. Fraser Gray, Ph.D., Jan Sabbat, M.B., B.S., for the Cirrhosis Asian Lamivudine Multicentre Study Group

Background The effectiveness of antiviral therapy in preventing disease progression in patients with chronic hepatitis B and advanced fibrosis or cirrhosis is unknown.

Methods Patients with chronic hepatitis B who had histologically confirmed cirrhosis or advanced fibrosis were randomly assigned in a 2:1 ratio to receive lamivudine (100 mg per day) or placebo for a maximum of five years. Of 651 patients, 436 were assigned to receive lamivudine and 215 to receive placebo. The primary end point was time to disease progression, defined by hepatic decompensation, hepatocellular carcinoma, spontaneous bacterial peritonitis, bleeding gastroesophageal varices, or death related to liver disease. An independent data and safety monitoring board monitored the progress of the study and performed interim analyses of the data.

Results We randomly assigned 651 patients (98 percent Asian and 85 percent male) to receive lamivudine or placebo. The study was terminated after a median duration of treatment of 32.4 months (range, 0 to 42) owing to a significant difference between treatment groups in the number of end points reached. End points were reached by 7.8 percent of the patients receiving lamivudine and 17.7 percent of those receiving placebo (hazard ratio for disease progression, 0.45; P=0.001). The Child–Pugh score increased in 3.4 percent of the patients receiving lamivudine and 8.8 percent of those receiving placebo (hazard ratio, 0.45; P=0.02), whereas hepatocellular carcinoma occurred in 3.9 percent of those in the lamivudine group and 7.4 percent of those in the placebo group (hazard ratio, 0.49; P=0.047). Genotypic resistance YMDD mutations developed in 49 percent of the patients treated with lamivudine, and the Child–Pugh score was more likely to increase in patients with these mutations than in the other patients treated with lamivudine (7 percent vs. <1 percent). Overall, 12 percent of the patients in the lamivudine group and 18 percent of the patients in the placebo group reported serious adverse events.

Conclusions Continuous treatment with lamivudine delays clinical progression in patients with chronic hepatitis B and advanced fibrosis or cirrhosis by significantly reducing the incidence of hepatic decompensation and the risk of hepatocellular carcinoma.

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

LANCET

Volume 364, Number 9441 02 October 2004

Vitamins to prevent cancer: supplementary problems (abstract)
David Forman, Douglas Altman

Calcium supplementation for preventing colorectal cancer: where do we stand?
Robert Benamouzig, Stanislas Chaussade (abstract)

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