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Revue de Presse du Mois Mois d'Avril 2003

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HEPATOLOGY

Table of Contents for April 2003 · Volume 37 · Number 4

Viral Hepatitis

Durability of serologic response after lamivudine treatment of chronic hepatitis B (*Human Study*)
Jules L. Dienstag, Janusz Cianciara, Selim Karayalcin, Kris V. Kowdley, Bernard Willems, Stanilav Plisek, Mary Woessner, Stephen Gardner, Eugene Schiff
Forty subjects with chronic hepatitis B and hepatitis B e antigen (HBeAg) seroconversion following lamivudine therapy in previous trials were monitored after treatment to assess the durability of serologic responses. Patient follow-up began a median of 4.3 months after completion of therapy in previous trials. At months 2, 4, 6, 9, and 12 of year 1, and every 6 months thereafter, we tested for HBeAg and hepatitis B surface antigen (HBsAg), hepatitis B virus (HBV) DNA, and alanine aminotransferase (ALT). After a median (range) of 36.6 (4.8-45.6) months of follow-up monitoring, HBeAg seroconversion was demonstrated at the last visit by 77% (30 of 39) of patients. In a post hoc analysis of a slightly different population of all 65 patients with HBeAg seroconversion in previous trials, the 3-year durability of HBeAg seroconversion measured from the time immediately after discontinuing lamivudine therapy was 64%. Nine (9 of 40, 23%) patients were HBsAg negative at the last assessment. Seventy-four percent (17 of 23) of patients with baseline undetectable HBV DNA and normal ALT maintained these responses at the last visit. Eight patients (8 of 40, 20%) initiated retreatment for reappearance of HBV markers, and 7 showed biochemical and/or virologic improvement (including regained HBeAg seroconversion in 2). No safety issues of concern emerged. In conclusion, most HBeAg responses achieved during lamivudine therapy were durable, and most responders experienced prolonged clinical benefit after HBeAg seroconversion and subsequent discontinuation of lamivudine. Lamivudine retreatment for reappearance of hepatitis B markers can achieve resumption of viral suppression. (HEPATOLOGY 2003;37:748-755.)

Long-term suppression of hepatitis B e antigen-negative chronic hepatitis B by 24-month interferon therapy (*Human Study*)
Pietro Lampertico, Ersilio Del Ninno, Mauro Viganò, Raffaella Romeo, Maria Francesca Donato, Erwin Sablon, Alberto Morabito, Massimo Colombo
To assess whether extended treatment with interferon improves the outcome of hepatitis B e antigen (HBeAg)-negative chronic hepatitis B, 101 consecutive patients were treated with 6 MU of interferon alfa 2b 3 times weekly for 24 months. During the 68-month study, 30 patients (30%) had a sustained response (i.e., normal serum transaminase levels and undetectable hepatitis B virus DNA by non-polymerase chain reaction [PCR] assays), and 15 cleared serum surface antigen. Twenty-five nonresponders, 16 relapsers, and 30 who discontinued treatment were considered treatment failures. Multivariate analysis predicted a sustained response for young age (odds ratio, 0.94; 95% confidence interval, 0.89-0.99; P = .041) and high pretreatment serum levels of immunoglobulin M (IgM) anti-hepatitis B core antigen (HBc) (odds ratio, 4.52; 95% confidence interval, 1.63-12.5; P = .004). Liver disease progressed in none of the sustained responders but in 16 with treatment failure (0% vs. 22%, P = .002); hepatocellular carcinoma (HCC) developed with similar frequency in both groups (7%). Overall, estimated 8-year complication-free survival was longer for the 30 sustained responders than the 71 patients with treatment failure (90% vs. 60%, P < .001), but 8-year patient survival was similar in the 2 groups (100% and 90%). Short complication-free survival was predicted by failure to respond to interferon (hazard ratio, 7.8; 95% confidence interval, 1.8-34.0; P = .006) and high scores for liver fibrosis (hazard ratio, 1.71; 95% confidence interval, 1.17-2.50; P = .005). In conclusion, 24 months of treatment with interferon alfa 2b led to sustained disease suppression in a significant proportion of patients with HBeAg-negative chronic hepatitis B. (HEPATOLOGY 2003;37:756-763.)

Inhibition of hepatitis B virus expression and replication by RNA interference
Amir Shlomai, Yosef Shaul
RNA interference (RNAi) is the process of sequence-specific gene silencing, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the target gene. Because it has been shown that RNAi can be accomplished in cultured mammalian cells by introducing small interfering RNAs (siRNAs), much effort has been invested in exploiting this phenomenon for experimental and therapeutic means. In this study, we present a series of experiments showing a significant reduction in hepatitis B virus (HBV) transcripts and proteins in cell culture, as well as in the viral replicative forms, induced by siRNA-producing vectors. The antiviral effect is sequence-specific and does not depend on active viral replication. In conclusion, our data suggest that RNAi may provide a powerful therapeutic tool, acting both on replication-competent and on replication-incompetent HBV. (HEPATOLOGY 2003;37:764-770.)

Role of type 1 versus type 2 immune responses in liver during the onset of chronic woodchuck hepatitis virus infection
Yun Wang, Stephan Menne, James R. Jacob, Bud C. Tennant, John L. Gerin, Paul J. Cote
Immune response messenger RNAs (mRNA) were compared in liver during self-limited (resolved) and chronic neonatal woodchuck hepatitis virus (WHV) infection. At week 14 postinfection (mid-acute phase), mRNAs for leukocyte markers (CD3, CD4, CD8), type 1 cytokines and related transcription factors (IFN-, TNF-, STAT4, T-bet), and IL-10 were increased in livers from resolving infections, but mRNAs of other type 1 (IL-2) and type 2 (IL-4, STAT6, and GATA3 markers remained at baseline levels. Increased coexpression of IFN- and TNF- mRNAs correlated in most cases with lower levels of intrahepatic WHV covalently closed circular DNA (cccDNA). At the same time point postinfection, livers from woodchucks that eventually progressed to chronic infection had baseline or slightly elevated levels of CD and type 1 mRNAs, which were significantly lower (or elevated less frequently) compared with resolving woodchucks. Earlier, at week 8, there were no differences between the two outcome settings. During these early time points and at a later stage in chronic infection (15 months), type 2 mRNAs in carrier liver remained at baseline levels or, when elevated, were never in excess of those in resolving woodchucks. In conclusion, the onset and maintenance of neonatal chronic WHV infection are not associated with antagonistic type 2 immunoregulation of type 1 responses in liver. Accordingly, chronicity develops in association with a primary deficiency in the intrahepatic CD responses, especially involving CD8+ T lymphocytes, and in both extracellular (cytokine) and intracellular (transcriptional) type 1 response mediators. This has relevant implications for future treatment of chronic hepatitis B virus (HBV) infection in humans. (HEPATOLOGY 2003;37:771-780.)

Primary lymphoma of the liver: Clinical-pathological features and relationship with HCV infection in French patients (*Human Study*)
Jean-Pierre Bronowicki, Catherine Bineau, Pierre Feugier, Olivier Hermine, Nicole Brousse, Frédéric Oberti, Marie-Christine Rousselet, Sébastien Dharancy, Philippe Gaulard, Jean-François Flejou, Dominique Cazals-Hatem, Eric Labouyrie
Primary lymphoma of the liver (PLL) is rare. In some cases, the hepatic lymphoma has been diagnosed in patients who were infected by the hepatitis C virus (HCV). It has been suggested that HCV plays a role in the pathogenesis of lymphoma. The aim of our multicentric retrospective study was to assess the characteristics of PLL and to determine the prevalence of HCV infection in PLL. Thirty-one immunocompetent patients (anti-human immunodeficiency virus, anti-human T-cell leukemia/lymphoma virus negative, no history of allograft) with PLL fulfilled the entire selection criteria. The liver biopsy specimens were reassessed by the same pathologist. The non-Hodgkin's lymphomas were classified according to the World Health Organization classification. Blood samples were tested in 28 patients for antibodies to HCV, and HCV RNA was detected by reverse transcription polymerase chain reaction. In the majority of cases, the clinical, biologic, and radiologic data were nonspecific. Twenty-seven of 31 patients presented a B-cell lymphoma corresponding to the centroblastic morphologic variant of a diffuse, large B-cell lymphoma (22 cases), a Burkitt's lymphoma (1 case), an extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue type (3 cases), and unclassified, small B-cell lymphoma (1 case). The 4 other cases were T-cell lymphomas. The prevalence of HCV infection was 21% (6 of 28 cases). All of these patients were positive for HCV RNA by polymerase chain reaction in blood. Most of the HCV-infected patients presented a high-grade, B-cell type lymphoma. In conclusion, our study confirms the rarity of PLL and demonstrates an increased prevalence of HCV infection. (HEPATOLOGY 2003;37:781-787).

The relationship of in vivo 31P MR spectroscopy to histology in chronic hepatitis C (*Human Study*)
Adrian K. P. Lim, Nayna Patel, Gavin Hamilton, Joseph V. Hajnal, Robert D. Goldin, Simon D. Taylor-Robinson
Liver biopsy remains the gold standard for characterizing diffuse liver disease and is associated with significant morbidity and, rarely, mortality. Our aim was to investigate whether a noninvasive technique, in vivo phosphorus 31 (31P)-magnetic resonance spectroscopy (MRS), could be used to assess the severity of hepatitis C virus (HCV)-related liver disease. Fifteen healthy controls and 48 patients with biopsy-proven HCV-related liver disease were studied prospectively. Based on their histologic fibrosis (F) and necroinflammatory (NI) scores, patients were divided into mild hepatitis (F 2/6, NI 3/18), moderate/severe hepatitis (3 F < 6 or NI 4/18), and cirrhosis (F = 6/6). Hepatic 31P MR spectra were obtained using a 1.5-T spectroscopy system. Quantitation of the 31P signals was performed in the time domain using the Advanced MAgnetic RESonance algorithm. There was a monotonic increase in the mean ± 1 standard error phosphomonoester (PME) to phosphodiester (PDE) ratios for the control, mild disease, moderate disease, and cirrhosis groups: 0.15 ± 0.01, 0.18 ± 0.02, 0.25 ± 0.02, 0.38 ± 0.04, respectively (ANOVA, P < .001). An 80% sensitivity and specificity was achieved when using a PME/PDE ratio less than or equal to 0.2 to denote mild hepatitis and a corresponding ratio greater than or equal to 0.3 to denote cirrhosis. No other significant spectral changes were observed. In conclusion, 31P MRS can separate mild from moderate disease and these 2 groups from cirrhosis. The ability to differentiate these populations of patients has therapeutic implications and 31P MRS, in some situations, would not only complement a liver biopsy but could replace it and be of particular value in assessing disease progression. (HEPATOLOGY 2003;37:788-794.)

Racial differences in the relationship between hepatitis C infection and iron stores (*Human Study*)
George N. Ioannou, Jason A. Dominitz, Noel S. Weiss, Patrick J. Heagerty, Kris V. Kowdley
Black race and increased hepatic iron stores predict poor response to interferon treatment for chronic hepatitis C virus (HCV) infection. We tested the hypothesis that these 2 observations are linked by investigating whether HCV-infected African-Americans have increased iron stores relative to uninfected persons. Using data from the third National Health and Nutrition Examination Survey (NHANES III), we determined the risk of having increased iron stores, defined as elevation of both serum ferritin and transferrin-iron saturation (TS), in HCV-RNA-positive blacks (n = 100) and nonblacks (n = 126) relative to HCV-RNA-negative blacks (n = 4,002) and nonblacks (n = 10,943). HCV-positive blacks were 5.4 times (95% CI, 1.2 to 24) more likely to have increased iron stores than HCV-positive nonblacks. The proportion of HCV-positive blacks who had increased iron stores was 16.4% among those with abnormal liver enzymes and 2.8% among those with normal liver enzymes, compared with only 0.6% among HCV-negative blacks. After adjustment for age, alcohol intake, gender, menopausal status, education, body mass index, and poverty index, HCV-positive blacks with abnormal liver enzymes had an elevated risk of having increased iron stores (odds ratio, 17.8; 95% CI, 5.1 to 63). In contrast, among persons of other races, there was a much smaller difference in the proportion of persons with increased iron stores between HCV-positive persons with (3.4%) or without (1.4%) abnormal liver enzymes and HCV-negative persons (0.9%). In conclusion, a greater proportion of blacks than persons of other races respond to HCV infection with an increase in iron stores. This finding may partly explain the reduced response of HCV-positive African-Americans to antiviral treatment. (HEPATOLOGY 2003;37:795-801.)

Hepatitis C virus infection in the general population: A community-based study in West Bengal, India (*Human Study*)
Abhijit Chowdhury, Amal Santra, Susmita Chaudhuri, Gopal Krishna Dhali, Sujit Chaudhuri, Satya Gopal Maity, Trailokya Nath Naik, Sujit Kumar Bhattacharya, Debendra Nath Guha Mazumder
Limited information is available about the prevalence and genotype distribution of hepatitis C virus (HCV) in the general population of India. A community-based epidemiologic study was carried out in a district in West Bengal, India. By a 1:3 sampling method, 3,579 individuals were preselected from 10,737 inhabitants of 9 villages of the district, of whom 2,973 (83.1%) agreed to participate. Twenty-six subjects (0.87%) were HCV antibody positive. The prevalence increased from 0.31% in subjects <10 years of age to 1.85% in those 60 years. No difference in prevalence between men and women was observed. Serum alanine aminotransferase (ALT) levels were elevated in 30.8% (8 of 26) of anti-HCV-positive subjects compared with 3.2% (94 of 2,947) anti-HCV-negative subjects (P < .001). HCV RNA was detectable in 80.8% (95% CI, 65.6%-95.91%) of the anti-HCV-positive subjects by reverse transcription-primed polymerase chain reaction (RT-PCR). The participants were HCV types 1b in 2 (9.5%), 3a in 8 (38.1%), 3b in 6 (28.6%), and unclassified in 5 (23.8%). Nucleotide sequencing and phylogenetic analysis assigned the unclassified type to genotype 3e. In conclusion, this study provides general population-based estimates of HCV prevalence, including genotypes, from a South Asian country. Although the prevalence of HCV infection in this population was lower than that reported from industrialized countries of the west, the total reservoir of infection is significant and calls for public health measures, including health education to limit the magnitude of the problem.(HEPATOLOGY 2003;37:802-809.)

Liver Biology and Pathobiology

Nitric oxide-mediated cytoprotection of hepatocytes from glucose deprivation-induced cytotoxicity: Involvement of heme oxygenase-1
Byung-Min Choi, Hyun-Ock Pae, Young-Myeong Kim, Hun-Taeg Chung
Heme oxygenase-1 (HO-1) is the rate-limiting enzyme in heme catabolism, which leads to the generation of carbon monoxide (CO), biliverdin, and free iron. One of 3 mammalian HO isoforms, HO-1, is a stress-responsive protein and known to modulate such cellular functions as cytokine production, cell proliferation, and apoptosis to protect organs and tissues from acute injury. Although nitric oxide (NO)-mediated cytoprotective effects against cytotoxicity induced by glucose deprivation have been well recognized, the underlying mechanisms remain to be elucidated. Thus, we investigate the involvement of HO-1 in the cytoprotective effects of NO. Deprivation of glucose markedly reduced the viability of BNL CL.2 cells and primary rat hepatocytes. Pretreatment with NO donor, sodium nitroprusside (SNP), protected hepatocytes from glucose deprivation-induced cytotoxicity; zinc protoporphyrin (ZnPP) IX, an inhibitor of HO, was found to block the SNP-induced cytoprotection. SNP increased the induction of HO-1 protein as well as its activity in hepatocytes. A cytoprotective effect comparable to SNP was observed when the cells were transfected with HO-1 gene or preincubated with another HO-1 inducer, hemin. Additional experiments revealed the involvement of CO in the cytoprotective effect of SNP/HO-1 in BNL CL.2 cells. CO mediated cytoprotective effect through suppression of ERK MAPK activation. In conclusion, our results show that SNP protects hepatocytes from glucose deprivation-induced cytotoxicity through up-regulation of HO-1. Thus, HO-1 might be an important cellular target of NO donor with clinical implications for the prevention of acute liver injury in several pathological conditions. (HEPATOLOGY 2003;37:810-823.)

c-Jun-N-terminal kinase drives cyclin D1 expression and proliferation during liver regeneration
Robert F. Schwabe, Cynthia A. Bradham, Tetsuya Uehara, Etsuro Hatano, Brydon L. Bennett, Robert Schoonhoven, David A. Brenner
The c-Jun-N-terminal kinase (JNK) pathway is strongly activated after partial hepatectomy (PH), but its role in hepatocyte proliferation is not known. In this study, JNK activity was blocked with the small molecule inhibitor JNK SP600125 in vivo and in vitro as shown by a reduction of c-Jun phosphorylation, AP-1 DNA binding activity, and c-jun messenger RNA (mRNA) expression. SP600125 inhibited proliferating cell nuclear antigen (PCNA) expression, cyclin D1 mRNA and protein expression and reduced mitotic figures after PH. Survival was reduced significantly 3 days after PH in SP600125-treated versus vehicle-treated rats (3 of 11 vs. 8 of 9, P < .01). In epidermal growth factor (EGF)-treated primary cultures of rat hepatocytes, SP600125 decreased 3H-thymidine uptake, cyclin D1 mRNA and protein expression, and inhibited the EGF-induced transcription of a cyclin D1 promoter-driven reporter gene. The defective regeneration and the decreased survival in SP600125-treated rats did not result from a major increase in apoptosis as shown by normal levels of caspase 3 activity and only slight increases in apoptotic figures. In conclusion, our data show that JNK drives G0 to G1 transition in hepatocytes and that cyclin D1 is a downstream target of the JNK pathway during liver regeneration. (HEPATOLOGY 2003;37:824-832.)

p18(INK4c) collaborates with other CDK-inhibitory proteins in the regenerating liver
Tom Luedde, Maria E. Rodriguez, Frank Tacke, Yue Xiong, David A. Brenner, Christian Trautwein
p18(INK4c) belongs to the family of cyclin-dependent kinase inhibitory proteins that target the cyclin-dependent kinases and inhibit their catalytic activity. The role of p18(INK4c) for cell cycle progression in vivo is characterized poorly. Therefore, we studied the expression and physiologic relevance of p18 in quiescent and proliferating hepatocytes during liver regeneration. For our analysis we used single- (p18[INK4c], p27[KIP1], p21[CIP1/WAF1]), and double-mutant (p18/p21, p18/p27) mice. p18 expression was found in quiescent hepatocytes and a slight up-regulation was evident after partial hepatectomy (PH). p18 knockout animals showed normal cell cycle progression after PH. However, when p18/p21 and p18/p27 double-mutant mice were used, differences in cell cycle progression were evident compared with wild-type (wt) and single knockout animals. In p18/p21 knockout animals, the G1 phase was shortened as evidenced by an earlier onset of cyclin D and proliferating cell nuclear antigen (PCNA) expression and cyclin-dependent kinase (CDK) activation after PH. In contrast, in p18/p27 knockout animals, the G1 phase was unchanged, but the amount of proliferating hepatocytes (5-bromo-2´-deoxyuridine [BrdU] and PCNA positive) 48 hours after PH was elevated. In conclusion, our results suggest that p18 is involved in cell cycle progression after PH. Additionally we provide evidence that timing and strength of DNA synthesis in hepatocytes after PH is regulated tightly through the collaboration of different cell cycle inhibitors. (HEPATOLOGY 2003;37:833-841.)

Changes in mitochondrial adenine nucleotides and in permeability transition in two models of rat liver regeneration
Rolando Hernández-Muñoz, Lourdes Sánchez-Sevilla, Alejandro Martínez-Gómez, Myrna A. R. Dent
Although enhanced phosphorylative activity can be a requisite for later DNA synthesis during liver regeneration (LR), mitochondrial generation of reactive oxygen species could lead to altered mitochondrial membrane permeability during the prereplicative phase of LR. Therefore, the role of mitochondrial permeability transition (MPT) was evaluated during rat LR, induced by either partial hepatectomy (PH) or after CCl4 administration. Parameters indicative of mitochondrial function and membrane potentials, those of oxidative stress, and in vivo changes of the intramitochondrial pool of adenine nucleotides were determined. Twelve hours after PH, mitochondrial oxidative and phosphorylative activities and adenosine diphosphate (ADP) content were increased, reaching a maximal peak at 24 hours after surgery (maximal DNA synthesis). Parameters suggestive of oxidant stress were enhanced, but mitochondrial volume and membrane electrical potential remained unaltered. Interestingly, moderate mitochondrial swelling and depolarization were found at later post-PH times (72 hours). In CCl4-treated animals, it was found that an active liver cell necrosis delayed mitotic activity and mitochondrial uncoupled respiration. Starting 12 hours after CCl4 intoxication, a drastic increase of inorganic phosphate occurred within swollen and strongly depolarized mitochondria, suggesting changes in the MPT. Despite expression of messenger RNA (mRNA) for mitochondrial transcription, factor A showed a similar time course in both experimental models. The so-called augmenter liver regeneration was found significantly elevated only in PH rats. In conclusion, onset of MPT could be associated with cell necrosis and inflammation after CCl4 treatment, whereas this mitochondrial event could constitute a putative effector mechanism, through which growth or inflammatory factors inhibiting cell proliferation could initiate LR termination. (HEPATOLOGY 2003;37:842-851.)

Homozygous deletion scanning in hepatobiliary tumor cell lines reveals alternative pathways for liver carcinogenesis
Pascal Pineau, Agnès Marchio, Seishi Nagamori, Shuichi Seki, Pierre Tiollais, Anne Dejean
Despite high rates of loss of heterozygosity affecting various chromosomes, the number of tumor suppressor genes (TSGs) found to be consistently involved in primary liver cancer is low. In the past decade, characterization of homozygous deletions (HDs) in tumors has become instrumental to identify new TSGs or to reveal the influence of a particular TSG on the development of a specific tumor type. We performed a detailed HD profiling at 238 critical loci on a collection of 57 hepatobiliary tumor cell lines (hepatocellular, cholangiocellular, and bile duct carcinomas, hepatoblastomas, and immortalized hepatocytes). We identified HDs at 9 independent loci, the analysis of which was extended to 17 additional hepatobiliary tumor cell lines. In total, 34 homozygous losses involving 9 distinct genes were detected in the 74 cell lines analyzed. Besides expected deletions at the p16-INK4A/p14-ARF, FHIT, AXIN1, and p53 genes, we detected HDs at the PTEN, NF2, STK11, BAX, and LRPDIT genes that were formerly not known to be implicated in human liver tumorigenesis. In conclusion, our data suggest that these genes may represent novel liver tumor suppressive targets. Additional tumorigenic pathways should be carefully considered in hepatocarcinogenesis. (HEPATOLOGY 2003;37:852-861.)

Reduced hepatic tumor incidence in cyclin G1-deficient mice
Michael Rugaard Jensen, Valentina M. Factor, Anna Fantozzi, Kristian Helin, Chang-Goo Huh, Snorri S. Thorgeirsson
Cyclin G1 is a transcriptional target of the tumor suppressor p53, and its expression is increased after DNA damage. Recent data show that cyclin G1 can regulate the levels of p53 by a mechanism that involves dephosphorylation of Mdm2 by protein phosphatase 2A. To understand the biologic role of cyclin G1, we have generated cyclin G1-deficient mice. In agreement with previous results, we showed that these mice develop normally, and that proliferation and induction of cellular senescence in cyclin G1-deficient mouse embryo fibroblasts are indistinguishable from wild-type fibroblasts. However, we found that the p53 levels in the cyclin G1-deficient mice are 2-fold higher that in wild-type mice. Moreover, we showed that treatment of mice with the alkylating agent 1,4-bis[N,N´-di(ethylene)-phosphamide]piperazine (Dipin), followed by partial hepatectomy, decreased G1-S transition in cyclin G1-null hepatocytes as compared with wild type. Finally, we found a significant decrease in tumor incidence, mass, and malignancy in both male and female cyclin G1-null mice after treatment with the potent hepatocarcinogen N-diethylnitrosamine. Taken with recent published data, our results suggest that cyclin G1, together with Mdm2, constitute a part of a negative feedback system that attenuates the activity of p53. In conclusion, our data suggest that the decreased tumor susceptibility after loss of cyclin G1 function is caused by the increased tumor suppressor action of p53. (HEPATOLOGY 2003;37:862-870.)

Microdissection-based allelotyping discriminates de novo tumor from intrahepatic spread in hepatocellular carcinoma
Sydney D. Finkelstein, Wallis Marsh, Anthony J. Demetris, Patricia A. Swalsky, Eizaburo Sasatomi, Andrew Bonham, Michael Subotin, Igor Dvorchik
A total of 103 cases of hepatocellular carcinoma (HCC) arising in native livers discovered at the time of transplantation underwent allelic loss analysis. HCC mutational allelotyping targeted 10 genomic loci (1p, 3p, 5q, 7q, 8q, 9p, 10q, 17p, 17q, 18q) using 18 polymorphic microsatellite markers situated in proximity to known tumor suppressor genes associated with human carcinogenesis. Gene analysis was performed on microdissected tissue samples removed from 4-µm thick histologic sections at specific topographic sites selected on the basis of representative cellular characteristics. Microdissection targets included largest tumor nodule at 2 locations as well as up to 3 additional tumor nodules in each case. HCC genotyping characteristics including mutational profile and cumulative fractional allelic loss (FAL) were correlated with clinical and pathologic features. Individual nodules of HCC showed 2 patterns of mutational change: (1) essentially concordant mutational profiles consistent with intrahepatic spread of tumor, or (2) discordant mutational profiles consistent with independent primary cancer formation. In 15 of 56 cases (27%) in which the HCC was in a multinodular, bilobar form (T4), sufficient discordance in the allelic loss profile enabled a more accurate T-stage classification with better prediction of recurrence-free survival. In conclusion, microdissection genotyping of HCC is an effective and objective means to (1) distinguish between de novo HCC tumor formation versus intrahepatic spread of cancer and to (2) improve on current methods for prediction of tumor aggressiveness and recurrence-free survival after liver transplantation. (HEPATOLOGY 2003;37:871-879.)

Influence of ursodeoxycholate-enriched diet on liver tumor growth in HBV transgenic mice
Michele Barone, Eugenio Maiorano, Roberta Ladisa, Rosario Cuomo, Antonia Pece, Pasquale Berloco, Maria Lucia Caruso, Anna Maria Valentini, Achille Iolascon, Antonio Francavilla, Alfredo Di Leo, Enzo Ierardi
Hepatitis B virus (HBV) transgenic mice (official designation, Tg [Alb-1 HBV] Bri 44) invariably develop macroscopically evident tumors within the 20th month of life. Sustained proliferative activity seems to play an important role in the development of these lesions. We previously showed that ursodeoxycholate (UDC) stimulates hepatocyte proliferation in various experimental settings. Herein, we tested the assumption that biological factors able to further increase liver cell proliferation, such as UDC, could accelerate tumor development in this animal model. For this study, 22 eight-week-old male transgenic mice were divided into 2 groups; 11 animals received a standard diet, and 11 received a UDC-enriched diet. The 2 groups were further divided into 2 subgroups of 5 and 6 animals each and were sacrificed at 3 and 15 months of age, respectively. These different times were chosen to exclude diet-related toxicity (in 3-month-old mice) and evaluate tumor growth (in 15-month-old mice). In addition, hepatocyte proliferation was assessed in all animals. In 3-month-old mice receiving UDC, cholestatic and cytolytic indices as well as liver histology were comparable to those in controls. At 15 months, all UDC-treated mice showed large multinodular tumors whereas only 33% of controls developed smaller uninodular neoplasms. Hepatocyte proliferation was increased in all animals receiving UDC compared with controls. In conclusion, the increase in serum UDC (undetectable in mice fed a standard diet), in the absence of any toxic effect on the liver, suggests the involvement of this bile salt in the stimulation of hepatocyte proliferation and tumor growth. (HEPATOLOGY 2003;37:880-886.)

Liver Failure and Liver Disease

A randomized controlled trial of ursodeoxycholic acid in patients with alcohol-induced cirrhosis and jaundice (*Human Study*)
Gilles Pelletier, Dominique Roulot, Thierry Davion, Claude Masliah, Xavier Causse, Frédéric Oberti, Jean-Jacques Raabe, Claire Van Lemmens, Hélène Labadie, Lawrence Serfaty, URSOMAF Group
The aim of our multicenter study was to assess the efficacy of ursodeoxycholic acid (UDCA) on the survival of patients with alcohol-induced cirrhosis and jaundice. We included patients with histologically proven alcohol-induced cirrhosis and serum bilirubin >50 µmol/L. After randomization, patients received either UDCA (13-15 mg/kg/d) or a placebo for 6 months. Two hundred twenty-six patients (113 in each group) were included in 24 centers. There were 139 men and 87 women, mean age of 50.3 years. Seventy-four percent had associated alcohol-induced hepatitis, and 24% received a corticosteroid therapy. At inclusion, the 2 groups were comparable for the main clinical and biologic parameters, but serum bilirubin was higher in the UDCA group than in the placebo group (163 µmol/L vs. 145 µmol/L, P < .03). The percentage of patients lost at follow-up or who resumed their alcoholism during the study was comparable in the 2 groups. During the study, 55 patients died, 35 in the UDCA group and 20 in the placebo group. In the intention to treat analysis, the probability of survival at 6 months (Kaplan-Meier method) was lower in the UDCA than in the P group (69% vs. 82%, respectively; P = .04, log-rank test). After adjustment on the bilirubin level at entry (Cox model), the independent predictive value of the treatment group did not reach the statistical level (RR = 1.64, CI 0.85-2.85; P = .077). In conclusion, UDCA administered at the dose recommended in primary biliary cirrhosis has no beneficial effect on the 6-month survival of patients with severe alcohol-induced cirrhosis. An inappropriate dosage of UDCA cannot be excluded as an explanation for the lack of therapeutic benefit.(HEPATOLOGY 2003;37:887-892.)

Rapid diagnosis of spontaneous bacterial peritonitis by use of reagent strips (*Human Study*)
José Castellote, Carmen López, Joan Gornals, Gemma Tremosa, Eva Rodríguez Fariña, Carmen Baliellas, Alicia Domingo, Xavier Xiol
We studied the use of reagent strips for diagnosis of spontaneous bacterial peritonitis (SBP) in cirrhotic patients with ascites. A reagent strip for leukocyte esterase designed for the testing of urine with a colorimetric 5-grade scale (0 to 4) was used to evaluate ascitic fluid in 228 nonselected paracentesis performed in 128 cirrhotic patients. We diagnosed 52 SBP and 5 secondary bacterial peritonitis by means of polymorphonuclear cell count and classical criteria. When we considered positive a reagent strip result of 3 or 4, sensitivity was 89% (51 of 57), specificity was 99% (170 of 171), and positive predictive value was 98%. When we considered positive a reagent strip result of 2 or more, sensitivity was 96% (55 of 57), specificity was 89% (152 of 171), and negative predictive value was 99%. In conclusion, the use of reagent strips is a rapid, easy to use, and inexpensive tool for diagnosis of ascitic fluid infection. A positive result should be an indication for empirical antibiotic therapy, and a negative result may be useful as a screening test to exclude SBP. (HEPATOLOGY 2003;37:893-896.)

Spontaneous bacterial peritonitis in asymptomatic outpatients with cirrhotic ascites (*Human Study*)
Luke T. Evans, W. Ray Kim, John J. Poterucha, Patrick S. Kamath
The prevalence and natural history of spontaneous bacterial peritonitis in asymptomatic patients with ascites secondary to cirrhosis is unknown. From a prospectively recorded database, we reviewed the clinical and laboratory features of all outpatients with cirrhotic ascites undergoing paracentesis between July 1994 and December 2000. The prevalence of spontaneous bacterial peritonitis in the population of 427 cirrhotic outpatients as defined by neutrocytic ascites (absolute neutrophil count 250 cells/mm3) was 3.5%. Of the 15 patients with neutrocytic ascites, 6 were culture positive (1.4%) and 9 culture negative (2.1%). Eight other patients (1.9%) had bacterascites. The organisms cultured from ascitic fluid in these asymptomatic patients with culture positive neutrocytic ascites and bacterascites were predominantly gram positive. No patient developed hepatorenal syndrome, and 1-year survival of 67% was better than historical data from hospitalized patients with spontaneous bacterial peritonitis. Moreover, patients who did not receive antibiotics for neutrocytic ascites fared no worse than patients who did receive antibiotics. In conclusion, spontaneous bacterial peritonitis in outpatients with cirrhotic ascites is less frequent, occurs in patients with less advanced liver disease, and may have a better outcome than its counterpart in hospitalized patients. In addition, the organisms cultured from ascitic fluid in outpatients are predominantly gram positive. A reassessment of diagnostic criteria for spontaneous bacterial peritonitis in outpatients may be required. (HEPATOLOGY 2003;37:897-901.)

Hemodynamic response to pharmacological treatment of portal hypertension and long-term prognosis of cirrhosis (*Human Study*)
Juan G. Abraldes, Ilaria Tarantino, Juan Turnes, Juan Carlos Garcia-Pagan, Juan Rodés, Jaime Bosch
In cirrhotic patients under pharmacologic treatment for portal hypertension, a reduction in hepatic venous pressure gradient (HVPG) of 20% of baseline or to 12 mm Hg markedly reduces the risk of variceal rebleeding. This study was aimed at evaluating whether these hemodynamic targets also prevent other complications of portal hypertension and improve long-term survival. One hundred five cirrhotic patients included in prospective trials for the prevention of variceal rebleeding were studied. Seventy-three of the patients had 2 separate HVPG measurements, at baseline and under pharmacologic therapy with propranolol ± isosorbide mononitrate. Patients were followed for up to 8 years. Survival and risk of developing portal hypertension-related complications were compared between responders and nonresponders. Twenty-eight patients showed a reduction of HVPG 20% of baseline or to 12 mm Hg (responders), and 45 patients were nonresponders. Nonresponders had a significantly greater risk of developing variceal rebleeding (P = .013), ascites (P = .025), spontaneous bacterial peritonitis (P = .003), hepatorenal syndrome (P = .026), and hepatic encephalopathy (P = .024) than responders. Eight-year cumulative probability of survival was significantly lower in nonresponders than in responders (52% vs. 95%, respectively, P = .003). At multivariate analysis, being a nonresponder was independently associated with the risk of developing rebleeding, ascites, spontaneous bacterial peritonitis, and lower survival. In conclusion, in cirrhotic patients receiving pharmacologic treatment for prevention of variceal rebleeding, a decrease in HVPG 20% or to 12 mm Hg is associated with a marked reduction in the long-term risk of developing complications of portal hypertension and with improved survival. (HEPATOLOGY 2003;37:902-908.)

Dietary habits and their relations to insulin resistance and postprandial lipemia in nonalcoholic steatohepatitis (*Human Study*)
Giovanni Musso, Roberto Gambino, Franco De Michieli, Maurizio Cassader, Mario Rizzetto, Marilena Durazzo, Emanuela Fagà, Barbara Silli, Gianfranco Pagano
The relations of dietary habits to insulin sensitivity and postprandial triglyceride metabolism were evaluated in 25 patients with nonalcoholic steatohepatitis (NASH) and 25 age-, body mass index (BMI)-, and gender-matched healthy controls. After a 7-day alimentary record, they underwent a standard oral glucose tolerance test (OGTT), and the insulin sensitivity index (ISI) was calculated from the OGTT; an oral fat load test was also performed in 15 patients and 15 controls. The dietary intake of NASH patients was richer in saturated fat (13.7% ± 3.1% vs. 10.0% ± 2.1% total kcal, respectively, P = .0001) and in cholesterol (506 ± 108 vs. 405 ± 111 mg/d, respectively, P = .002) and was poorer in polyunsaturated fat (10.0% ± 3.5% vs. 14.5% ± 4.0% total fat, respectively, P = .0001), fiber (12.9 ± 4.1 vs. 23.2 ± 7.8 g/d, respectively, P = .000), and antioxidant vitamins C (84.3 ± 43.1 vs. 144.2 ± 63.1 mg/d, respectively, P = .0001) and E (5.4 ± 1.9 vs. 8.7 ± 2.9 mg/d, respectively, P = .0001). The ISI was significantly lower in NASH patients than in controls. Postprandial total and very low density lipoproteins triglyceride at +4 hours and +6 hours, triglyceride area under the curve, and incremental triglyceride area under the curve were higher in NASH compared with controls. Saturated fat intake correlated with ISI, with the different features of the metabolic syndrome, and with the postprandial rise of triglyceride. Postprandial apolipoprotein (Apo) B48 and ApoB100 responses in NASH were flat and strikingly dissociated from the triglyceride response, suggesting a defect in ApoB secretion. In conclusion, dietary habits may promote steatohepatitis directly by modulating hepatic triglyceride accumulation and antioxidant activity as well as indirectly by affecting insulin sensitivity and postprandial triglyceride metabolism. Our findings provide further rationale for more specific alimentary interventions, particularly in nonobese, nondiabetic normolipidemic NASH patients. (HEPATOLOGY 2003;37:909-916.)

Nonalcoholic fatty liver, steatohepatitis, and the metabolic syndrome
Giulio Marchesini, Elisabetta Bugianesi, Gabriele Forlani, Fernanda Cerrelli, Marco Lenzi, Rita Manini, Stefania Natale, Ester Vanni, Nicola Villanova, Nazario Melchionda, Mario Rizzetto
Nonalcoholic fatty liver disease (NAFLD) has been associated with the insulin-resistance syndrome, at present defined as the metabolic syndrome, whose limits were recently set. We assessed the prevalence of the metabolic syndrome in 304 consecutive NAFLD patients without overt diabetes, on the basis of 3 or more criteria out of 5 defined by the U.S. National Institutes of Health (waist circumference, glucose, high-density lipoprotein [HDL]-cholesterol, triglycerides, and arterial pressure). The prevalence of the metabolic syndrome increased with increasing body mass index, from 18% in normal-weight subjects to 67% in obesity. Insulin resistance (Homeostasis Model Assessment method) was significantly associated with the metabolic syndrome (odds ratio [OR], 2.5; 95% CI, 1.5-4.2; P < .001). Liver biopsy was available in 163 cases (54%). A total of 120 patients (73.6%) were classified as having nonalcoholic steatohepatitis (NASH); 88% of them had a metabolic syndrome (vs. 53% of patients with pure fatty liver; P < .0001). Logistic regression analysis confirmed that the presence of metabolic syndrome carried a high risk of NASH among NAFLD subjects (OR, 3.2; 95% CI, 1.2-8.9; P = .026) after correction for sex, age, and body mass. In particular, the syndrome was associated with a high risk of severe fibrosis (OR, 3.5; 95% CI, 1.1-11.2; P = .032). In conclusion, the presence of multiple metabolic disorders is associated with a potentially progressive, severe liver disease. The increasing prevalence of obesity, coupled with diabetes, dyslipidemia, hypertension, and ultimately the metabolic syndrome puts a very large population at risk of forthcoming liver failure in the next decades. (HEPATOLOGY 2003;37:917-923.)

Cytochrome P450 2E1 genotype and the susceptibility to antituberculosis drug-induced hepatitis (*Human Study*)
Yi-Shin Huang, Herng-Der Chern, Wei-Juin Su, Jaw-Ching Wu, Shi-Chuan Chang, Chi-Huei Chiang, Full-Young Chang, Shou-Dong Lee
Most cases with antituberculosis drug-induced hepatitis have been attributed to isoniazid. Isoniazid is metabolized by hepatic N-acetyltransferase (NAT) and cytochrome P450 2E1 (CYP2E1) to form hepatotoxins. However, the role of CYP2E1 in this hepatotoxicity has not yet been reported. The aim of this study was to evaluate whether the polymorphism of the CYP2E1 gene is associated with antituberculosis drug-induced hepatitis. A total of 318 tuberculosis patients who received antituberculosis treatment were followed prospectively. Their CYP2E1 and NAT2 genotypes were determined using a polymerase chain reaction with restriction fragment length polymorphism method. Twenty-one healthy volunteers were recruited for CYP2E1 phenotype study using a chlorzoxazone test. Forty-nine (15.4%) patients were diagnosed to have drug-induced hepatotoxicity. Patients with homozygous wild genotype CYP2E1 c1/c1 had a higher risk of hepatotoxicity (20.0%; odds ratio [OR], 2.52) than those with mutant allele c2 (CYP2E1 c1/c2 or c2/c2, 9.0%, P = .009). If CYP2E1 c1/c2 or c2/c2 genotype combined with rapid acetylator status was regarded as the reference group, the risk of hepatotoxicity increased from 3.94 for CYP2E1 c1/c1 with rapid acetylator status to 7.43 for CYP2E1 c1/c1 with slow acetylator status. After adjustment for acetylator status and age, the CYP2E1 c1/c1 genotype remained an independent risk factor for hepatotoxicity (OR, 2.38; P = .017). Furthermore, under the administration of isoniazid, the volunteers with CYP2E1 c1/c1 genotype had higher CYP2E1 activity than those with other genotypes had and, hence, might produce more hepatotoxins. In conclusion, CYP 2E1 genetic polymorphism may be associated with susceptibility to antituberculosis drug-induced hepatitis. (HEPATOLOGY 2003;37:924-930.)

Induced hyperammonemia alters neuropsychology, brain MR spectroscopy and magnetization transfer in cirrhosis (*Human Study*)
Sherzad Balata, Steven W. M. Olde Damink, Karen Ferguson, Ian Marshall, Peter C. Hayes, Nicolaas E. P. Deutz, Roger Williams, Joanna Wardlaw, Rajiv Jalan
Hyperammonemia is a universal finding after gastrointestinal hemorrhage in cirrhosis. We administered an oral amino acid solution mimicking the hemoglobin molecule to examine neuropsychological changes, brain glutamine levels, and brain magnetization transfer ratio (MTR). Forty-eight metabolically stable patients with cirrhosis and no evidence of "overt" hepatic encephalopathy (HE) were randomized to receive 75 g of amino acid solution or placebo; measurements were performed before and 4 hours after administration. Neuropsychological tests included the Trails B Test, Digit Symbol Substitution Test, memory subtest of the Randt battery, and reaction time. Plasma was collected for ammonia and amino acid measurements, and brain metabolism was studied using proton magnetic resonance (MR) spectroscopy in the first 16 randomized patients. In 7 other patients, MTR was measured. A significant increase in ammonia levels was observed in the amino acid group (amino acid group, 76 ± 7.3 to 121 ± 6.4 µmol/L; placebo, 83 ± 3.3 to 78 ± 2.9 µmol/L; P < .001). Neuropsychological function improved significantly in the placebo group, but no significant change in neuropsychological function was observed in the amino acid group. Brain glutamate/glutamine (Glx)/creatine (Cr) ratio increased significantly in the amino acid group. MTR decreased significantly from 30 ±2.9 to 23 ± 4 (P < .01) after administration of the amino acid solution. In conclusion, an improvement in neuropsychological test results followed placebo, which was not observed in patients administered the amino acid solution. Induced hyperammonemia resulted in an increase in brain Glx/Cr ratio and a decrease in MTR, which may indicate an increase in brain water as the operative mechanism. (HEPATOLOGY 2003;37:931-939.)


The ABCs of biliary cholesterol secretion and their implication for gallstone disease
Silvana Zanlungo, Juan Francisco Miquel, Attilio Rigotti, Flavio Nervi
Two ATP-binding cassette (ABC) transporters, ABCG5 and ABCG8, have been proposed to limit sterol absorption and to promote biliary sterol excretion in humans. To test this hypothesis, a P1 clone containing the human ABCG5 and ABCG8 genes was used to generate transgenic mice. The transgenes were expressed primarily in the liver and small intestine, mirroring the expression pattern of the endogenous genes. Transgene expression only modestly affected plasma and liver cholesterol levels but profoundly altered cholesterol transport. The fractional absorption of dietary cholesterol was reduced by about 50%, and biliary cholesterol levels were increased more than fivefold. Fecal neutral sterol excretion was increased three- to six-fold and hepatic cholesterol synthesis increased two- to four-fold in the transgenic mice. No significant changes in the pool size, composition, and fecal excretion of bile acids were observed in the transgenic mice. Transgene expression attenuated the increase in hepatic cholesterol content induced by consumption of a high cholesterol diet. These results demonstrate that increased expression of ABCG5 and ABCG8 selectively drives biliary neutral sterol secretion and reduces intestinal cholesterol absorption, leading to a selective increase in neutral sterol excretion and a compensatory increase in cholesterol synthesis.

Regulation of apoptotic signaling pathways in hepatocytes in vivo
Hartmut Jaeschke, Mary Lynn Bajt
The mitochondrial pathway is critical for the efficient execution of death receptor-initiated apoptosis in certain cell types. Questions remain as to why the mitochondria are required in that scenario. We investigated the molecular events that determined the need for the mitochondria by using an in vivo model of anti-Fas-induced hepatocyte apoptosis. In wild-type mice, Fas stimulation resulted in normal activation of caspase-3, with the generation of the active p19-p12 complex. In bid-deficient mice, caspase-3 activation was arrested after the initial cleavage at Asp175. This allowed the generation of the p12 small subunit, but the p20 large subunit could not be further processed to the p19 subunit. The p20-p12 complex generated by Fas stimulation in bid-deficient hepatocytes was inactive, arresting the death program. Failure of p20/p12 caspase-3 to mature and to exhibit activity was because of the inhibition by the inhibitor-of-apoptosis proteins (IAPs), such as XIAP, and also to a low caspase-8 activity. This block could be overcome in wild-type mice by two mechanisms. Smac was released from mitochondria early following Fas activation and was competitively bound to the IAPs to reverse their effects. XIAP could also be cleaved, and this occurred later and was likely mediated by enhanced caspase activities. Both mechanisms were dependent on Bid and thus were not operative in bid-deficient hepatocytes. In conclusion, mitochondrial activation by Bid is required for reversing the IAP inhibition through Smac release. It is also required for the alternative activation of caspases through cytochrome c release, as demonstrated previously. Together, these events ensure a successful progression of the death program initiated by the death receptor activation in the hepatocyte.

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

GASTROENTEROLOGY

Table of Contents for April 2003 · Volume 124 · Number 4

Rapid Communications

Methylene blue-aided chromoendoscopy for the detection of intraepithelial neoplasia and colon cancer in ulcerative colitis
R. Kiesslich, J. Fritsch, M. Holtmann, H. H. Koehler, M. Stolte, S. Kanzler, B. Nafe, M. Jung, P. R. Galle, M. F. Neurath
Background & Aims: Timely diagnosis of intraepithelial neoplasias (IN) and colitis-associated colon carcinomas (CRC) is crucially important for the treatment of ulcerative colitis (UC). We performed a randomized, controlled trial to test whether chromoendoscopy (CE) might facilitate early detection of IN and CRC in UC. Methods: A total of 263 patients with long-standing UC (8 years) were screened for potential inclusion in the study, 165 of whom were randomized at a 1:1 ratio to undergo conventional colonoscopy or colonoscopy with CE using 0.1% methylene blue. Five mucosal biopsy specimens were taken every 10 cm between the rectum and cecum. Circumscript lesions in the colon were evaluated according to a modified pit pattern classification. Results: In the CE group, there was a significantly better correlation between the endoscopic assessment of degree (P = 0.0002) and extent (89% vs. 52%; P < 0.0001) of colonic inflammation and the histopathologic findings compared with the conventional colonoscopy group. More targeted biopsies were possible, and significantly more IN were detected in the CE group (32 vs. 10; P = 0.003). Using the modified pit pattern classification, both the sensitivity and specificity for differentiation between non-neoplastic and neoplastic lesions were 93%. Conclusions: Based on our prospective randomized trial, CE permits more accurate diagnosis of the extent and severity of the inflammatory activity in UC compared with conventional colonoscopy. In addition, CE with methylene blue is a novel tool for the early detection of IN and CRC in patients with UC. These findings have important implications for medical and surgical interventions.

Clinical-Alimentary Tract

Ursodeoxycholic acid as a chemopreventive agent in patients with ulcerative colitis and primary sclerosing cholangitis
D. S. Pardi, E. V. Loftus, Jr, W. K. Kremers, J. Keach, K. D. Lindor
Background & Aims: Ursodeoxycholic acid (UDCA) has shown effectiveness as a colon cancer chemopreventive agent in preclinical studies. In addition, a recent report suggests that it also may decrease the risk for developing colorectal dysplasia in patients with ulcerative colitis (UC) and primary sclerosing cholangitis (PSC). We sought to evaluate the effect of UDCA on colorectal neoplasia in a group of patients with UC and PSC enrolled in a randomized, placebo-controlled trial. Methods: From a prior, randomized, placebo-controlled trial of UDCA therapy in PSC at our center, we followed-up patients with concomitant UC to assess the effect of UDCA on the development of colorectal dysplasia and cancer as compared with placebo. Results: Fifty-two subjects were followed-up for a total of 355 person-years. Those originally assigned to receive UDCA had a relative risk of 0.26 for developing colorectal dysplasia or cancer (95% confidence interval, 0.06-0.92; P = 0.034). Many of the patients originally assigned to the placebo group eventually received open-label UDCA. Assigning these patients to the UDCA group from the time they began active therapy did not change the magnitude of the protective effect (relative risk, 0.26; 95% confidence interval, 0.07-0.99; P = 0.049). Conclusions: UDCA significantly decreases the risk for developing colorectal dysplasia or cancer in patients with UC and PSC.

Colonic fermentation influences lower esophageal sphincter function in gastroesophageal reflux disease
T. Piche, S. B. des Varannes, S. Sacher-Huvelin, J. J. Holst, J. C. Cuber, J. P. Galmiche
Background & Aims: Colonic fermentation of carbohydrates is known to influence gastric and esophageal motility in healthy subjects. This study investigated the effects of colonic fermentation induced by oral administration of fructooligosaccharides (FOS) in patients with gastroesophageal reflux disease (GERD). Methods: In the cross-over design used in the study, 9 patients with symptomatic GERD were administered a low-residue diet (i.e., 10 g fiber/day) during 2, 7-day periods, receiving either 6.6 g of FOS or placebo 3 times daily after meals. Each period was separated by a wash out of at least 3 weeks. On day 7, esophageal motility and pH were recorded in fasting conditions and after a test meal containing 6.6 g of FOS or placebo. Breath hydrogen concentrations (reflecting colonic fermentation) and plasma concentrations of glucagon-like peptide 1 (GLP-1), peptide YY, and cholecystokinin were monitored. Results: Compared with placebo, FOS led to a significant increase in the number of transient lower esophageal sphincter relaxations (TLESRs) and reflux episodes, esophageal acid exposure, and the symptom score for GERD. The integrated plasma response of GLP-1 was significantly higher after FOS than placebo. Conclusions: Colonic fermentation of indigestible carbohydrates increases the rate of TLESRs, the number of acid reflux episodes, and the symptoms of GERD. Although different mechanisms are likely to be involved, excess release of GLP-1 may account, at least in part, for these effects.

Heterogeneity of symptom pattern, psychosocial factors, and pathophysiological mechanisms in severe functional dyspepsia
B. Fischler, J. Tack, V. De Gucht, Z. Shkedy, P. Persoons, D. Broekaert, G. Molenberghs, J. Janssens
Background & Aims: Categorization of functional dyspepsia into subgroups is based on expert opinion according to (dominant) symptoms or on underlying pathophysiological mechanisms. We used an evidence-based approach to the determination of subtypes of functional dyspepsia. Methods: Consecutive functional dyspepsia patients were recruited from a tertiary referral center. The following were performed: (1) exploratory (EFA) and confirmatory factor analysis (CFA) of symptom patterns in a large group of patients with functional dyspepsia; (2) external validation of these factors by the determination of their association pattern with physio- and psychopathological mechanisms, and with health-related quality of life and sickness behavior; and (3) cluster analysis of their distribution in this population. Results: Both EFA and CFA do not support the existence of functional dyspepsia as a homogeneous (unidimensional) condition. A 4-factor model is found to be valid, with differential distribution within the patient population according to cluster analysis. Factor 1 is characterized by nausea, vomiting, early satiety, and weight loss and factor 2 by postprandial fullness and bloating. Both factor 1 and 2 are associated with delayed emptying, but only factor 1 is associated with younger age, female sex, and sickness behavior. Factor 3 is characterized by pain symptoms and associated with gastric hypersensitivity and several psychosocial dimensions including medically unexplained symptoms and health-related quality of life dimensions. Factor 4, characterized by belching, is also associated with hypersensitivity, but is unrelated to psychosocial dimensions. Conclusions: In a tertiary care population, functional dyspepsia is a heterogeneous condition characterized by 4 major dimensions differentially associated with psychopathological and physiopathological mechanisms.

Extracolonic findings at CT colonography: Evaluation of prevalence and cost in a screening population
T. M. Gluecker, C. D. Johnson, L. A. Wilson, R. L. MacCarty, T. J. Welch, D. J. Vanness, D. A. Ahlquist
Background & Aims: To assess the prevalence and spectrum of extracolonic findings in a screening population undergoing computed tomography colonography (CTC), and to evaluate the short-term direct medical costs incurred from subsequent radiologic follow-up evaluation. Methods: Six hundred and eighty-one asymptomatic patients undergoing colonoscopy screening consented to a CTC examination. Extracolonic CT findings were classified into high, medium, and low importance. Clinical and radiologic follow-up, missed lesions, and outcomes were assessed by chart review (time interval, 410-1513 days; median, 913 days). Short-term direct medical costs of radiologic follow-up were determined based on Medicare 2002 reimbursement rates. Results: Extracolonic findings were found commonly. These were categorized as high clinical importance in 71 (10%) individuals, as medium importance in 183 individuals (27%), and as low importance in 341 individuals (50%). Subsequent medical or surgical interventions resulted from these findings in 9 of the 681 patients (1.3%). Costs of subsequent radiologic follow-up studies were calculated as $23,380.59 (average added costs per CTC examination $34.33). Conclusions: CTC commonly detects extracolonic findings that can be considered clinically important when applied to an asymptomatic screening population. Although such incidental findings add benefit to the screening intervention, moderate incremental costs are incurred based on additional radiologic procedures generated during short-term follow-up.

Intravenous hydrocortisone premedication reduces antibodies to infliximab in Crohn's disease: A randomized controlled trial
R. J. Farrell, M. Alsahli, Y.-T. Jeen, K. R. Falchuk, M. A. Peppercorn, P. Michetti
Background & Aims: We assessed the relationship between antibodies to infliximab (ATI) and the loss of response postinfliximab, infusion reactions and, in a randomized trial, investigated whether intravenous hydrocortisone premedication can reduce ATI. Methods: Initially, we prospectively evaluated clinical response, adverse events, and ATI levels in 53 consecutive patients with Crohn's disease who received 199 infliximab (5 mg/kg) infusions. Subsequently, 80 patients with Crohn's disease were randomized to intravenous hydrocortisone 200 mg or placebo immediately before their first and subsequent infliximab infusions. The primary endpoint was reduction in median ATI levels at week 16. Analysis was by intention to treat. Results: Nineteen of our initial 53 patients (36%) developed ATI, including all 7 patients with serious infusion reactions (median ATI level, 19.6 µg/mL). Eleven of 15 patients (73%) who lost their initial response were ATI positive compared with none of 21 continuous responders, (8.9 vs. 0.7 µg/mL, P < 0.0001). Administering a second infusion within 8 weeks of the first (OR, 0.13; 95% CI, 0.03-0.5; P = 0.0007) or concurrent immunosuppressants (OR, 0.19; 95% CI, 0.04-1.03; P = 0.007) significantly reduced ATI formation. In the placebo-controlled trial, ATI levels were lower at week 16 among hydrocortisone-treated patients (1.6 vs. 3.4 µg/mL, P = 0.02), and 26% of hydrocortisone-treated patients developed ATI compared with 42% of placebo-treated patients, P = 0.06. Conclusions: Loss of initial response and infusion reactions post-infliximab is strongly related to ATI formation and level. Administering a second infusion within 8 weeks of the first and concurrent immunosuppressant therapy significantly reduce ATI formation. Intravenous hydrocortisone premedication significantly reduces ATI levels but does not eliminate ATI formation or infusion reactions.

Clinical-Liver, Pancreas, and Biliary Tract

Epidemiologic and virologic characteristics of hepatitis B virus genotype B having the recombination with genotype C
F. Sugauchi, E. Orito, T. Ichida, H. Kato, H. Sakugawa, S. Kakumu, T. Ishida, A. Chutaputti, C.-L. Lai, R. G. Gish, R. Ueda, Y. Miyakawa, M. Mizokami
Background & Aims: Hepatitis B virus (HBV) isolates of genotype B (HBV/B) with or without the recombination with genotype C over the precore region plus core gene have been reported. Methods: All the 41 HBV/B isolates having the recombination with genotype C (HBV/Ba) possessed the nucleotide 1838 of A in contrast to that of G in all 29 of those without the recombination (HBV/Bj). Taking advantage of this single nucleotide polymorphism, a restriction fragment length polymorphism method was developed that distinguished HBV/Ba from HBV/Bj. Results: HBV/Bj was detected in 90 of the 97 (93%) carriers of HBV/B from Japan, whereas HBV/Ba occurred in all 177 carriers of HBV/B from other countries (China, 20; Hong Kong, 45; Taiwan, 32; Thailand, 30; Vietnam, 30; and the United States, 20 [all of an Asian ethnicity]). In a case-control study, hepatitis B e antigen (HBeAg) and the double mutation in the core promoter (T1762/A1764) were significantly more frequent in 80 carriers each of HBV/Ba than HBV/Bj (35% vs. 18%, P < 0.05 and 33% vs. 15%, P < 0.05, respectively). Differences in the prevalence of HBeAg were more conspicuous between the carriers of HBV/Bj and HBV/Ba older than 30 years (5 of 66 or 8% vs. 16 of 62 or 26%, P < 0.01). Conclusions: HBV/B having the recombination with genotype C is frequent in Asia, except in Japan, and HBeAg is more prevalent in carriers of HBV/Ba than HBV/Bj.

Therapy of hyponatremia in cirrhosis with a vasopressin receptor antagonist: A randomized double-blind multicenter trial
A. L. Gerbes, V. Gülberg, P. Ginès, G. Decaux, P. Gross, H. Gandjini, J. Djian, VPA Study Group
Background & Aims: Dilutional hyponatremia is a frequent complication of cirrhosis partly because of nonosmotic vasopressin release. No effective therapy exists for this complication. Therefore, we investigated the effects of VPA-985, an orally active vasopressin V2 receptor antagonist, in patients with cirrhosis and dilutional hyponatremia. Primary endpoint was normalization of serum sodium (serum sodium 136 mmol/L). Methods: Sixty patients with cirrhosis and dilutional hyponatremia were randomly assigned to 100 or 200 mg/day of VPA-985 or placebo in a double-blind study. Treatment was given with fluid restriction (1000 mL/day) until normalization of serum sodium or for 7 days. Results: Normalization of serum sodium concentration was achieved in 27% and 50% of patients in the VPA-985 100 mg/day and 200 mg/day groups, respectively, but in none of the patients in the placebo group (P < 0.05 and P < 0.001, respectively). Treatment with VPA-985 was associated with a significant reduction in urine osmolality and body weight. Thirst sensation increased significantly in the VPA 200 mg group but not in the VPA 100 mg or placebo group. Serious adverse events were similar among the 3 groups. Conclusions: An orally active vasopressin receptor antagonist can correct hyponatremia in patients with cirrhosis and ascites. This represents a novel therapy of water retention in cirrhosis.

Methionine adenosyltransferase II subunit gene expression provides a proliferative advantage in human hepatoma
M. L. Martínez-Chantar, E. R. García-Trevijano, M. Ujue Latasa, A. Martín-Duce, P. Fortes, J. Caballería, M. A. Avila, J. M. Mato
Background & Aims: Of the 2 genes (MAT1A, MAT2A) encoding methionine adenosyltransferase, the enzyme that synthesizes S-adenosylmethionine, MAT1A, is expressed in liver, whereas MAT2A is expressed in extrahepatic tissues. In liver, MAT2A expression associates with growth, dedifferentiation, and cancer. Here, we identified the subunit as a regulator of proliferation in human hepatoma cell lines. The subunit has been cloned and shown to lower the Km of methionine adenosyltransferase II 2 (the MAT2A product) for methionine and to render the enzyme more susceptible to S-adenosylmethionine inhibition. Methods: Methionine adenosyltransferase II 2 and subunit expression was analyzed in human and rat liver and hepatoma cell lines and their interaction studied in HuH7 cells. Subunit expression was up- and down-regulated in human hepatoma cell lines and the effect on DNA synthesis determined. Results: We found that subunit is expressed in rat extrahepatic tissues but not in normal liver. In human liver, subunit expression associates with cirrhosis and hepatoma. Subunit is expressed in most (HepG2, PLC, and Hep3B) but not all (HuH7) hepatoma cell lines. Transfection of subunit reduced S-adenosylmethionine content and stimulated DNA synthesis in HuH7 cells, whereas down-regulation of subunit expression diminished DNA synthesis in HepG2. The interaction between methionine adenosyltransferase II 2 and subunit was demonstrated in HuH7 cells. Conclusions: Our findings indicate that subunit associates with cirrhosis and cancer providing a proliferative advantage in hepatoma cells through its interaction with methionine adenosyltransferase II 2 and down-regulation of S-adenosylmethionine levels.

Up-regulation, nuclear import, and tumor growth stimulation of the adhesion protein p120ctn in pancreatic cancer
J. Mayerle, H. Friess, M. W. Büchler, J. Schnekenburger, F. U. Weiss, K.-P. Zimmer, W. Domschke, M. M. Lerch
Background & Aims: Cell adhesion proteins have been implicated as tumor suppressors because they prevent malignant cells from dissociating their cell contacts. We have studied the role of p120ctn, a recently discovered member of the cadherin/catenin family, in human pancreatic cancer. Methods: In 32 resection specimens of pancreatic adenocarcinoma and 10 control samples the expression of p120ctn was studied by Northern blot, immunocytochemistry, and immunogold electron microscopy. Patient survival data, tumor grading, and staging were correlated to the experimental results. In PaTu 8889 T pancreatic cancer cells, p120ctn expression was suppressed with 21-nucleotide small interfering RNA (siRNA) duplexes and proliferation was determined by bromodeoxyuridine (BrdU) incorporation. Results: In pancreatic cancer p120ctn messenger RNA (mRNA) was increased 3- to 4-fold. Although p120ctn was localized exclusively at cell contacts in controls it was found in the cytosol and nucleus of pancreatic cancer cells. This redistribution correlated to the degree of tumor dedifferentiation but was independent of tumor stage. The mean survival of patients with predominant membrane localization of p120ctn was 24 ± 7 (SEM) months vs. 9 ± 2 months for patients with predominant cytoplasmic p120ctn expression (P < 0.05). Silencing of p120ctn with siRNA duplexes reduced pancreatic cancer cell growth by 40%. Conclusions: Up-regulation, cytoplasmic redistribution, and nuclear import of p120ctn are associated with a more malignant phenotype of pancreatic cancer. This study further represents conclusive evidence for a direct involvement of p120ctn in malignant tumor cell proliferation. Both p120ctn-defective tumor cell contacts and p120ctn-mediated growth signals appear to contribute to the aggressive spread of pancreatic cancer.

Basic-Alimentary Tract

Therapeutic effects of vasoactive intestinal peptide in the trinitrobenzene sulfonic acid mice model of Crohn's disease
C. Abad, C. Martinez, M. G. Juarranz, A. Arranz, J. Leceta, M. Delgado, R. P. Gomariz
Background & Aims: Crohn's disease (CD) is a chronic debilitating disease of unknown etiology that is characterized by severe inflammation of the colon. Vasoactive intestinal peptide (VIP) has recently emerged as a promising candidate for treatment of inflammatory Th1-driven diseases. We studied the effect of VIP in trinitrobenzene sulfonic acid (TNBS)-induced colitis, which has clinical and molecular features in common with CD. Methods: A 3-mg enema of TNBS was given to BALB/c mice, and VIP (1 nmol) was given either as a single dose at 12 hours or every other day. Weight loss, histopathology, and chemokine and cytokine levels in serum and colon extracts were assessed. VIP was also tested given 5 days after the onset of TNBS-induced colitis, and its effect was analyzed given a second dose of TNBS. Results: Treatment with VIP reduced the clinical and histopathologic severity of TNBS-induced colitis, abrogating body weight loss, diarrhea, and macroscopic and microscopic intestinal inflammation. The therapeutic effects of VIP were associated with down-regulation of both inflammatory and Th1-driven autoimmune responses, including tumor necrosis factor , interleukin 1, and interleukin 6 in colon extracts and serum as well as interferon gamma by splenic and lamina propria CD4+ T cells. VIP reduced disease severity when given after disease onset and dramatically reduced disease recurrence given a second dose of TNBS. Conclusions: Our data suggest that VIP has beneficial prophylactic and therapeutic effects in TNBS-induced colitis and is a promising candidate to test for potential benefits in CD.

Emergence of perianal fistulizing disease in the SAMP1/YitFc mouse, a spontaneous model of chronic ileitis
J. Rivera-Nieves, G. Bamias, A. Vidrich, M. Marini, T. T. Pizarro, M. J. McDuffie, C. A. Moskaluk, S. M. Cohn, F. Cominelli
Background & Aims: SAMP1/Yit mice spontaneously develop chronic terminal ileitis, reminiscent of the human disease described by Crohn et al. in 1932. Several new phenotypic features have appeared in our colony after more than 20 generations of brother-sister mating. In this report, we describe the distinguishing features of the SAMP1/YitFc substrain at the University of Virginia, compared with the Japanese SAMP1/Yit parental strain. Methods: A colony of SAMP1/Yit mice was established at the University of Virginia in 1996, from 2 breeding pairs obtained from Japan. A systematic characterization of their phenotypic and immunologic characteristics was performed at 4, 10, 40, and more than 60 weeks of age. Results: The following differences were observed: (1) SAMP1/YitFc mice displayed established ileitis as early as 10 weeks of age, (2) the incidence of skin lesions inversely correlated with the occurrence of intestinal inflammation, (3) mice develop chronic ileitis with prominent muscular hypertrophy and focal collagen deposition in inflamed segments, (4) mesenteric lymph node lymphocytes acquired an activated phenotype coincident with disease progression, (5) high interferon- production was detected by 4 weeks of age and preceded the onset of ileitis, and (6) a subgroup of mice (~5%) developed perianal disease with ulceration and fistulae. Conclusions: The SAMP1/YitFc substrain exhibits unique characteristics when compared with the original Japanese strain. Of particular interest is the emergence of perianal fistulizing disease, to our knowledge the first report of such occurrence in an animal model of inflammatory bowel disease.

Helicobacter pylori induces matrix metalloproteinase-9 through activation of nuclear factor B
N. Mori, H. Sato, T. Hayashibara, M. Senba, R. Geleziunas, A. Wada, T. Hirayama, N. Yamamoto
Background & Aims: Matrix metalloproteinases (MMPs), enzymes capable of degrading extracellular matrix components, are believed to be active in connective tissue remodeling associated with various physiologic processes and in pathologic conditions. The aim of this study was to analyze the molecular mechanism responsible for Helicobacter pylori-mediated MMP expression. Methods: Expression of MMP messenger RNA and MMP activity were assessed by reverse-transcription polymerase chain reaction and zymography, respectively. Chloramphenicol acetyltransferase assay was used to monitor activation of the MMP-9 gene promoter, and electrophoretic mobility shift assay was used to explore the binding of transcription factors to this promoter. Gastric tissue samples were immunohistochemically stained for MMP-9. Results: H. pylori induced MMP-9 expression in 2 gastric epithelial cell lines but had no effect on MMP-2. Induction of MMP-9 was dependent on an intact cag pathogenicity island. Activation of the MMP-9 promoter by H. pylori occurred through the action of nuclear factor B. Transfection of kinase-deficient mutants of IB kinase and nuclear factor B-inducing kinase inhibited H. pylori-mediated activation of MMP-9. MMP-9 expression was higher in epithelial cells of H. pylori-positive tissue compared with those of H. pylori-negative tissue. Conclusions: H. pylori induced activation of nuclear factor B through an intracellular signaling pathway that involved IB kinase and nuclear factor B-inducing kinase, leading to MMP-9 gene transcription. MMP-9 induction by H. pylori may play an important role in gastric inflammation, ulcer formation, and carcinogenesis.

CARD15/NOD2 functions as an antibacterial factor in human intestinal epithelial cells
T. Hisamatsu, M. Suzuki, H.-C. Reinecker, W. J. Nadeau, B. A. McCormick, D. K. Podolsky
Background & Aims: Mutations in the CARD15/NOD2 gene, a putative intracellular pattern recognition receptor, have been linked to the risk for Crohn's disease. Because intestinal epithelial cells play a role as the barrier to luminal microorganisms, we investigated the expression and function of CARD15/NOD2 in intestinal epithelial cells. Methods: Expression of CARD15/NOD2 messenger RNA (mRNA) in intestinal epithelial cell lines and primary intestinal epithelial cells was assessed by reverse-transcription polymerase chain reaction (RT-PCR). Regulation of expression of CARD15/NOD2 by cytokines was determined by Northern blot using the SW480 cell line. Active CARD15/NOD2 protein in SW480 cells was assessed by the combination of immunoprecipitation and immunoblotting using anti-CARD15/NOD2 antisera. To identify the functional role of CARD15/NOD2 in intestinal epithelial cells, gentamicin protection assays of Salmonella typhimurium were performed using Caco2 cells stably transfected with either wild-type CARD15/NOD2 or the 3020insC mutant associated with Crohn's disease. Results: CARD15/NOD2 mRNA was expressed in both intestinal epithelial cell lines and primary intestinal epithelial cells. CARD15/NOD2 mRNA and protein were up-regulated by tumor necrosis factor (TNF) in SW480 cells. The number of viable internalized S. typhimurium in Caco2 cells stably transfected with CARD15/NOD2 expression plasmid was lower than untransfected Caco2 cells or MOCK transfectant. In contrast, expression of a variant associated with Crohn's disease was unable to constrain bacterial survival. Conclusions: CARD15/NOD2 is expressed in intestinal epithelial cells and may serve as a key component of innate mucosal responses to luminal bacteria as an antibacterial factor. Failure in this activity may contribute to the development of Crohn's disease.

TNF- and IFN- regulate the expression of the NOD2 (CARD15) gene in human intestinal epithelial cells
P. Rosenstiel, M. Fantini, K. Bräutigam, T. Kühbacher, G. H. Waetzig, D. Seegert, S. Schreiber
Background & Aims: NOD2, a member of the NOD1/Apaf-1 family, was recently identified as the first susceptibility gene for Crohn's disease. The aim of this report was to describe the regulation and functional significance of NOD2 expression in intestinal epithelial cells. Methods: Expression of NOD2 messenger RNA was determined by reverse-transcription polymerase chain reaction (RT-PCR); NOD2 protein was detected by Western blot. Promoter activity was assessed by reporter gene assays and DNA-binding of NF-B by electrophoretic mobility shift assays. IL-8 production was investigated by RT-PCR and enzyme-linked immunosorbent assay. Results: TNF- induced an up-regulation of NOD2 in epithelial cell lines (HT-29, SW620, SW948, HeLa S3) and in primary colonic epithelial cells. A synergism was seen by cotreatment with IFN-. Two NF-B binding sites were identified in the promoter. Deletion of either site or overexpression of dominant negative IB led to reduced levels of TNF-/IFN--stimulated reporter gene activity. The identified B3 site was bound by NF-B as determined by gelshift assays. Elevated amounts of NOD2 protein were also found in colonic epithelial cells from patients with IBD. LPS induced high levels of IL-8 production in SW620 cells overexpressing NOD2. Conclusions: TNF-(/IFN-) treatment up-regulates the expression of the NOD2 gene in intestinal epithelial cells and subsequently increases their LPS susceptibility. Together with the mutation-derived truncation and functional change of the NOD2 protein, this could be part of the complex pathophysiology of barrier disruption as it is observed in inflammatory bowel diseases.

Basic-Liver, Pancreas, and Biliary Tract

Angiotensin-converting enzyme inhibitor attenuates pancreatic inflammation and fibrosis in male Wistar Bonn/Kobori rats
A. Kuno, T. Yamada, K. Masuda, K. Ogawa, M. Sogawa, S. Nakamura, T. Nakazawa, H. Ohara, T. Nomura, T. Joh, T. Shirai, M. Itoh
Background & Aims: Pancreatic stellate cells have some similarities to hepatic stellate cells and an intrinsic renin-angiotensin system is present in the pancreas and is enhanced in acute pancreatitis and chronic pancreatic hypoxia. We assessed the effects of lisinopril, an angiotensin-converting enzyme (ACE) inhibitor, on spontaneously occurring chronic pancreatitis. Methods: Lisinopril in drinking water (20, 50, or 200 mg/L) was administered to 10-week-old male Wistar Bonn/Kobori (WBN/Kob) rats for 10 weeks and then the inflammatory parameters, fibrosis, serum and pancreatic ACE activity, and expression of transforming growth factor-1 (TGF-1) messenger RNA (mRNA) as well as positive immunostaining for -smooth muscle actin (-SMA) were assessed. Results: Lisinopril attenuated gross alterations in the pancreas. This protective effect was confirmed quantitatively by significant increases in pancreatic weights and decreases in pancreatic myeloperoxidase (MPO) activity (an index of granulocyte infiltration), pancreatic hydroxyproline content (an index of collagen deposition), ratio of fibrous tissue, and histologic scores. Lisinopril significantly reduced serum ACE activity but it did not affect pancreatic activity. High doses of lisinopril suppressed the overexpression of TGF-1 mRNA measured by reverse-transcription polymerase chain reaction (RT-PCR) and decreased the number of -SMA-positive cells (activated pancreatic stellate cells) in the pancreas. Conclusions: Lisinopril alleviated chronic pancreatitis and fibrosis in male WBN/Kob rats. It suppressed the expression of TGF-1 mRNA, resulting in the prevention of pancreatic stellate cell activation, which may be involved in the observed protection. We propose that an ACE inhibitor may be useful for treating chronic pancreatitis.

Overexpression of heparin-binding EGF-like growth factor in mouse pancreas results in fibrosis and epithelial metaplasia
A. L. Means, K. C. Ray, A. B. Singh, M. K. Washington, R. H. Whitehead, R. C. Harris, Jr., C. V. E. Wright, R. J. Coffey, Jr., S. D. Leach
Background & Aims: Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is expressed in both normal pancreatic islets and in pancreatic cancers, but its role in pancreatic physiology and disease is not known. This report examines the effects of HB-EGF overexpression in mouse pancreas. Methods: Transgenic mice were established using a tissue-specific promoter to express an HB-EGF complementary DNA in pancreatic cells, effectively elevating HB-EGF protein 3-fold over endogenous levels. Results: Mice overexpressing HB-EGF in pancreatic islets showed both endocrine and exocrine pancreatic defects. Initially, islets from transgenic mice failed to segregate , , , and PP cells appropriately within islets, and had impaired separation from ducts and acini. Increased stroma was detected within transgenic islets, expanding with age to cause fibrosis of both endocrine and exocrine compartments. In addition to these structural abnormalities, subsets of transgenic mice developed profound hyperglycemia and/or proliferation of metaplastic ductal epithelium. Both conditions were associated with severe stromal expansion, suggesting a role for islet/stromal interaction in the onset of the pancreatic disease initiated by HB-EGF. Supporting this conclusion, primary mouse fibroblasts adhered to transgenic islets when the 2 tissues were cocultured in vitro, but did not interact with nontransgenic islets. Conclusions: An elevation in HB-EGF protein in pancreatic islets led to altered interactions among islet cells and among islets, stromal tissues, and ductal epithelium. Many of the observed phenotypes appeared to involve altered cell adhesion. These data support a role for islet factors in the development of both endocrine and exocrine disease.

Case Reports

A multidrug resistance 3 gene mutation causing cholelithiasis, cholestasis of pregnancy, and adulthood biliary cirrhosis
J.-F. Lucena, J. I. Herrero, J. Quiroga, B. Sangro, J. Garcia-Foncillas, N. Zabalegui, J. Sola, M. Herraiz, J. F. Medina, J. Prieto
We describe a 47-year-old patient who developed cholelithiasis in adolescence, followed by recurrent intrahepatic cholestasis of pregnancy, and finally biliary cirrhosis in adulthood. In our patient, the consecutive presentation of the 3 mentioned disorders raised the suspicion of a defect of MDR3, the canalicular protein involved in the transport of phospatidylcholine to bile. Mutational analysis in our patient showed a heterozygous missense mutation of the MDR3 gene that has not been described previously, which occurs in exon 14 at codon 535, and results in the substitution of glycine for aspartic acid. Further analysis of 7 members of the family showed the same mutation in her daughter who, on follow-up, developed cholestasis of pregnancy and persisting high serum levels of -glutamyl transpeptidase and alkaline phosphatase after delivery. Although biliary cirrhosis associated with MDR3 deficiency typically appears before the age of 25 years, in our case, the relatively mild MDR3 dysfunction allowed for a slower progression of the disease with established, well-advanced cirrhosis in the fifth decade of life. The present case, which accumulates the 3 clinical disorders assocaited with MDR3 deficiency, shows that this condition should be suspected not only in children or young people with high -glutamyl transpeptidase cholestasis but also in middle-aged or older patients with chronic idiopathic cholestasis, especially when there is a previous history of cholestasis of pregnancy or juvenile cholelithiasis.

Special Reports and Reviews

Lipoxins: Pro-resolution lipid mediators in intestinal inflammation
J. Goh, C. Godson, H. R. Brady, P. MacMathuna
Many inflammatory processes are self-limiting, suggesting the existence of endogenous anti-inflammatory mechanisms. Among the lipid mediators generated during cell-cell interactions are the lipoxins (LX, including LXA4 and B4), a distinct class of lipoxygenase-derived eicosanoids. Aspirin acetylation of cyclooxygenase 2 also promotes the generation of a series of 15-epimers of LXA4, known as aspirin-triggered lipoxins (ATL), that may account for some of the bioactivity profile of aspirin and possibly of nonsteroidal anti-inflammatory drugs. Native LX are rapidly inactivated in vivo, and stable analogs of LXA4, LXB4, and ATL have been synthesized that possess enhanced bioavailability and potency as anti-inflammatory eicosanoids. Here, we review current in vitro, ex vivo, and in vivo evidence supporting cytoprotective and proresolution roles for LX in intestinal inflammation. LXA4, LXA4 analogs, and ATL analogs inhibit neutrophil chemotaxis, adhesion to epithelium, and epithelial cell chemokine release. In addition, LX blunt TNF--stimulated inflammatory responses, cyclooxygenase product generation, and epithelial cell apoptosis and are cytoprotective for cytokine-activated colonic mucosa ex vivo. LX, ATL, and synthetic LX analogs have already been demonstrated to possess impressive antiinflammatory and proresolution efficacy in a range of experimental models of inflammation in vivo. Further elucidation of the role of LX in intestinal epithelial cell biology and immune function may yield novel therapeutic approaches in inflammatory bowel disease and possibly gastrointestinal cancer.

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

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JOURNAL OF HEPATOLOGY

Table of Contents for Journal of Hepatology Volume 38, Issue 4, April 2003

Biliary Tract and Cholestasis

Peter Fickert et al.
Mallory body formation in primary biliary cirrhosis is associated with increased amounts and abnormal phosphorylation and ubiquitination of cytokeratins
Background/Aims: Animal studies revealed a key role of toxic bile acids in the regulation of hepatocytic cytokeratin (CK) expression and Mallory body (MB) formation. In this study, we compared CK expression, phosphorylation, and ubiquitination in primary biliary cirrhosis (PBC), chronic hepatitis C (CHC) and control livers to determine whether bile acid-induced CK alterations are associated with cytoskeletal alterations and MB formation in a prototypic chronic cholestatic liver disease. Methods: CK 8 and CK 18 mRNA and protein levels were investigated by reverse transcriptase-polymerase chain reaction and Western blotting. Intermediate filament (IF) cytoskeletal alterations were assessed by immunofluorescence microscopy using antibodies against CKs, CK phosphoepitopes, MBs, and ubiquitin. Results: Despite unchanged mRNA levels, CK 8 and CK 18 protein levels were significantly elevated in PBC suggesting stabilization of CKs, possibly due to decreased degradation. CK-IF alterations in PBC comprised increased density with abnormal phosphorylation of the IF network of hepatocytes in acinar zone 1 and in the periphery of cirrhotic nodules. In addition, in these areas hepatocytes with diminished IF network containing MBs consisting of abnormally phosphorylated and ubiquitinated CK were observed. Conclusions: These findings support our concept that IF cytoskeletal alterations and MB formation in cholestatic liver diseases are related to bile acid-induced cell stress.

Ferenc Szalay et al.
High serum osteoprotegerin and low RANKL in primary biliary cirrhosis
Background/Aims: Osteoprotegerin is decoy receptor for osteoclast activating factor, RANKL, and impairs osteoclast funtion. Since osteoporosis is common in primary biliary cirrhosis (PBC), we investigated osteoprotegerin, RANKL and markers of bone turnover in PBC. Methods: Serum osteoprotegerin, RANKL, osteocalcin (OC) and C-terminal cross-linking telopeptide of type I collagen (CTX-I) were measured by ELISA in 41 patients with PBC, 16 women with chronic hepatitis C (CHC), and as controls in 44 age-matched healthy and 74 post-menopausal osteopenic otherwise healthy women. Results: Serum osteoprotegerin levels were higher in PBC patients (7.8±3.0pmol/l) than in healthy controls (4.4±2.3pmol/l) and osteopenic women (4.0±1.0pmol/l, P<0.0001 for both). RANKL levels were lower in PBC (0.9±1.8pmol/l, P<0.0001) than in healthy controls (1.3±0.5pmol/l). In CHC both osteoprotegerin (9.7±4.2pmol/l) and RANKL (3.2±4.7pmol/l) were elevated compared to the control groups (P<0.0001, for both). There was a positive correlation between serum osteoprotegerin and OC, CTX-I and AST but not with bone mineral density in PBC. Conclusions: The mechanisms and role of elevated osteoprotegerin and low RANKL in PBC are unclear, but it might partly represent a compensatory mechanism to negative balance of bone remodeling. High OPG and RANKL levels found in CHC might suggest that inflammatory process in the liver could also contribute to the elevation of osteoprotegerin.

Pietro Invernizzi et al.
Peculiar HLA polymorphisms in Italian patients with primary biliary cirrhosis
Background/Aims: Primary biliary cirrhosis (PBC) is an autoimmune cholestatic liver disease of unknown etiology with a highly variable progression rate and prevalence among different geographical areas. Data concerning human leukocyte antigen (HLA) polymorphisms in PBC come from a limited number of geographical areas, from which the association with the HLA-DRB1*08 allele has been consistently reported. Methods: To investigate whether HLA polymorphisms contribute toward disease susceptibility, we compared 186 well-defined Italian PBC patients with 558 healthy subjects matched by age, gender and geographical area (Northern, Central and Southern Italy). Patients and controls were HLA typed at low resolution by PCR-sequence specific oligonucleotides for the loci A and B; HLA-DRB1 alleles were typed by reverse line blot assay of PCR-amplified DNA. Results: HLA-DRB1*11 was associated with a markedly reduced risk of developing PBC (OR: 0.3; 95% CI: 0.2-0.5). No association was found with HLA-DRB1*08. The B*15 (2.5; 1.3-4.6), B*41 (12.0; 2.7-72.1), B*55 (2.9; 1.1-7.5) and B*58 alleles (6.8; 1.1-46.3) were more frequent in PBC. The frequency of HLA polymorphisms was similar in PBC patients with progressive or non-progressive disease, and in those with or without anti-mitochondrial antibodies. Conclusions: Our data on a large series of Italian patients suggest that PBC may have a peculiar genetic background in the Mediterranean area.

Cell Biology, Metabolism and Transport

Masaki Kaibori et al.
Hepatocyte growth factor inhibits insulin-stimulated glycogen synthesis in primary cultured hepatocytes
Background/Aims: Hepatocyte growth factor (HGF) plays an important role as a mitogen in liver regeneration. However, little is known about the metabolic effects of HGF in the liver. Studies were performed to examine whether HGF influences carbohydrate metabolism, which is drastically changed in the early course of the regeneration. Methods: Primary cultured rat hepatocytes were treated with glucoregulatory hormones such as insulin, glucagon and adrenaline in the presence or absence of HGF. Cellular glycogen deposition and activities of its metabolic enzymes were compared. Results: HGF inhibited insulin-stimulated glycogen deposition, but had no effect on glycogen degradation stimulated by glucagon and adrenaline. HGF decreased glycogen synthase activity and increased glycogen phosphorylase activity in insulin-stimulated hepatocytes, resulting in the inhibition of glycogen synthesis. Experiments with immunoprecipitation revealed that HGF had no effect on the upstream of insulin signaling including an activation of its receptor and association of insulin receptor substrate with phosphatidylinositol 3-kinase, indicating that HGF presumably affects further downstream of these events. Conclusions: These results demonstrate that HGF interacts with insulin on glucose metabolism in hepatocytes. HGF may be involved in glucose regulation, and contribute to cell growth and maturation in addition to its mitogenic action during liver regeneration.

Chronic Liver Diseases

Shahin Merat et al.
Probucol in the treatment of non-alcoholic steatohepatitis: a double-blind randomized controlled study
Background/Aims: A final step in the pathology of non-alcoholic steatohepatitis (NASH) is oxidative damage to hepatocytes. Probucol is a lipid-lowering agent with strong antioxidant properties. We designed a double-blind randomized controlled study to evaluate the effects of probucol in NASH. Methods: Thirty cases of biopsy-proven NASH were included. Subjects were randomly allocated to either the treatment group or to the control group by a 2:1 ratio. The treatment group was given 500mg of probucol daily for 6 months, and the control group, an identically appearing placebo. Results: Twenty-seven cases completed the study. The mean aspartate transaminase (AST) and alanine transaminase (ALT) levels changed from 81.9 to 36.2 and 102.2 to 44.7 in the treatment group and from 57.6 to 49.6 and 96.8 to 96.2 in the control group, respectively. The decrease in ALT level in the treatment group as compared to the control group was significant at the P<0.005 level (95% confidence interval: 20.2-93.7IU). Both AST and ALT levels dropped to normal in nine cases of the treatment group (50%) but none of the control group (P=0.01). Conclusions: Probucol appears to be significantly effective in decreasing the ALT levels in patients with NASH.

Herbert Tilg et al.
Anti-tumor necrosis factor-alpha monoclonal antibody therapy in severe alcoholic hepatitis
Background/Aims: Severe alcoholic hepatitis (AH) is associated with high mortality. Tumor necrosis factor-alpha (TNF) has been demonstrated to play an important role in its pathophysiology. Methods: Twelve patients with biopsy-confirmed AH and a Maddrey discriminant factor >32 were treated with a single infusion of the anti-TNF monoclonal antibody Infliximab at a dose of 5mg/kg body weight. Serial measurements were made for various cytokines using specific enzyme-linked immunoassays (ELISA). In four patients, liver biopsy samples were available pretreatment and on day+28 of therapy. Results: Ten of the 12 patients are alive at a median of 15 (12-20) months. Two patients died within 30 days from septicemia. Serum bilirubin levels, Maddrey score, neutrophil count and C-reactive protein fell significantly within the first month. There was an early, though not significant, decrease in plasma levels of proinflammatory cytokines (interleukins (IL)-1, IL-6, IL-8, interferon-gamma), whereas plasma levels of TNF remained near the sensitivity limit of the assay throughout the treatment course. While TNF mRNA expression in the liver did not change, expression of IL-8, a cytokine regulated mainly by TNF, was almost absent on day+28. Conclusions: Our data suggest that randomized controlled trials of anti-TNF antibody in severe AH are warranted.

Kim R. Bridle et al.
Evidence for a sub-morphological inflammatory process in the liver in haemochromatosis
Background/Aims: The role of cytokines in hepatic injury has been examined for many liver diseases however little is known of the cytokine involvement in haemochromatosis. The aim of the current study was to examine the hepatic gene expression of potential proinflammatory and profibrogenic cytokines in haemochromatosis. Methods: Interferon-, interleukin-10, transforming growth factor-1 and tumor necrosis factor- mRNA expression was assessed in liver tissue from 20 haemochromatosis patients, eight controls and eight chronic hepatitis C patients. To assess the immunophenotype of the inflammatory infiltrate in haemochromatosis, liver sections were subjected to immunohistochemistry using markers for macrophages (CD68, HAM56, MAC387) or T cells (CD3 and CD45RO). Results: Interferon- mRNA was increased in both haemochromatosis (0.29±0.08%, P=0.01) and hepatitis C patients (1.02±0.32%, P=0.03) compared to controls (0.04±0.01%). Interleukin-10 mRNA was significantly decreased in both haemochromatosis and hepatitis C patients (0.01±0.003%, P=0.008 and 0.03±0.015%, P=0.02, respectively) compared to controls (0.12±0.01%). CD3 positive T-cell number was significantly correlated with increasing hepatic iron concentration (r=0.56, P=0.03). Conclusions: This study has demonstrated a distinct pattern of cytokine gene expression in haemochromatosis, which resembles that of inflammatory conditions such as chronic hepatitis C. These factors may play a role in the development of iron-induced hepatic fibrosis in haemochromatosis.

Brent A. Neuschwander-Tetri et al.
Interim results of a pilot study demonstrating the early effects of the PPAR- ligand rosiglitazone on insulin sensitivity, aminotransferases, hepatic steatosis and body weight in patients with non-alcoholic steatohepatitis
Background/Aims: Hyperinsulinemia may cause hepatic steatosis and non-alcoholic steatohepatitis (NASH). The aims of this pilot study were to examine the safety of using the insulin-sensitizing peroxisomal proliferator activated receptor (PPAR) ligand rosiglitazone in patients with NASH and determine whether improved insulin sensitivity correlates with improved fatty liver. Methods: Thirty subjects with NASH and elevated alanine aminotransferase (ALT) levels received rosiglitazone, 4mg twice daily for 48 weeks; the preliminary results presented here were obtained at 24 weeks. Insulin sensitivity was measured using fasting insulin and glucose levels and liver fat content was estimated by CT imaging. Results: By 24 weeks, rosiglitazone improved insulin sensitivity and reduced liver fat content. The mean ALT decreased from 86 to 37U/l (P<0.01). Four subjects (13%) withdrew, one because of a rise in ALT from 59 to 277U/l that coincided with concomitant prednisone use. Subjects experienced a mean weight gain of 3.5% and hemoglobin drop of 1.1g/dl. Conclusions: Treatment of NASH with rosiglitazone for 24 weeks improved insulin sensitivity, reduced liver fat content and improved biochemical evidence of hepatocellular injury. These preliminary data provide evidence that hyperinsulinemia may be a cause of NASH. Strategies to improve insulin sensitivity as a treatment of NASH deserve further investigation.

Cirrhosis and its Complications

Francesca Nicolao et al.
Role of determination of partial pressure of ammonia in cirrhotic patients with and without hepatic encephalopathy
Background/Aims: To compare venous, arterial and partial pressure of ammonia (pNH3) in 27 consecutive cirrhotics with hepatic encephalopathy, 15 cirrhotics without hepatic encephalopathy and nine controls; to reevaluate all parameters after the improvement of encephalopathy. Methods: Patients were studied by clinical examination and psychometric testing. pNH3 was calculated from arterial ammonia and pH. Results: In patients with encephalopathy, each form of ammonia was higher than in both controls and patients without encephalopathy. The correlation with the severity of hepatic encephalopathy was similar for venous (r=0.72), arterial ammonia (r=0.76) and pNH3 (r=0.75). The sensitivity and specificity of each variable in correctly classifying the patients as having or not having hepatic encephalopathy was also similar. Each form of ammonia decreased after the resolution or amelioration of symptoms. However, even in the 17 patients with complete resolution of hepatic encephalopathy, all three ammonia determinations resulted unchanged or increased in some patients. Conclusions: Despite the significant correlation between pNH3 and hepatic encephalopathy, our study suggests that neither pNH3 nor arterial ammonia are, from a clinical point of view, more useful than venous ammonia: all three determinations being limited both for the diagnosis of hepatic encephalopathy and for the clinical management of the patients.

Frank Tacke et al.
Ghrelin in chronic liver disease
Background/Aims: Ghrelin is a novel endogenous ligand for the growth hormone (GH) secretagogue receptor involved in energy metabolism, glucose homeostasis and food intake. We investigated the role of ghrelin and insulin-like growth factor-1 (IGF-1), the mediator of the GH axis, in patients with chronic liver diseases (CLD). Methods: Ghrelin and IGF-1 serum levels were determined in 105 CLD patients and 97 healthy controls and correlated with clinical and biochemical parameters. Results: Ghrelin was significantly elevated and IGF-1 reduced in CLD patients compared with healthy controls. IGF-1 serum levels inversely correlated with Child's classification. Ghrelin levels were significantly elevated in Child C cirrhosis patients independent of the aetiology of liver disease. Ghrelin levels did not correlate with liver function. In contrast, there was a correlation of ghrelin with clinical (gastrointestinal bleeding, ascites, encephalopathy) and biochemical (anaemia, inflammatory markers, hypoglycaemia, renal dysfunction) parameters. In a subgroup of patients with CLD and hepatocellular carcinoma (HCC), we observed a strong inverse correlation between alpha-fetoprotein (AFP) and ghrelin levels. Conclusions: Unlike IGF-1, ghrelin is not correlated with liver function, but increases in Child C cirrhosis and with complications of CLD. The inverse correlation with AFP in HCC patients requires further studies on the potential impact of ghrelin on the pathogenesis of anorexia-cachexia syndrome.

Wilma Debernardi Venon et al.
Effects of long-term Irbesartan in reducing portal pressure in cirrhotic patients: comparison with propranolol in a randomised controlled study
Background/Aims: The role of angiotensin II (AT-II) type I receptor antagonists in the treatment of portal hypertension remains controversial. We tested the efficacy of Irbesartan (Irb) vs. Propranolol (Pro) in reducing portal pressure and evaluated its systemic haemodynamic effects. Methods: Thirty-four patients were randomly assigned to receive either Irb 300mg/day (19 patients) or Pro 40-120mg/day (15 patients) for 2 months. Results: Irb was discontinued in five patients (26%). No major side effect occurred in the Pro group. On an average, the portal pressure gradient decreased significantly more in the Pro than in the Irb group (median 19.5%, range 11/31% vs. 4.8%, +2.5/10%, P<0.001). A clinically significant decrease was seen in one (7%) of the patients given Irb vs. five (33%) given Pro (P<0.02). The fall in mean arterial pressure was significantly higher with Irb than with Pro (median 29%, range 15/45% vs. 4.9%, +8/19%, P<0.02). Irb significantly modified the blood creatinine clearance (median 29ml/m, range +9/61ml/m, 30, 24/35% P<0.0001 vs. basal). Conclusions: Irb offers no advantage over Pro in the control of portal hypertension. Moreover, its therapeutic profile is limited by important side effects.

Franco Trevisani et al.
QT interval in patients with non-cirrhotic portal hypertension and in cirrhotic patients treated with transjugular intrahepatic porto-systemic shunt
Background/Aims: A prolonged QT interval is frequent in chronic liver disease and its aetiology remains unsettled. The study's aim was to assess the role of portal hypertension in the pathogenesis of QT prolongation. Methods: We measured the QT interval in: (1) 10 patients with non-cirrhotic portal hypertension (NCPH) and preserved liver function; (2) 19 cirrhotic patients before, 1-3 and 6-9 months after transjugular intrahepatic porto-systemic shunt (TIPS) insertion. Results: Baseline corrected maximum QT interval (QTcmax) was prolonged (>440ms) in eight NCPH and 16 cirrhotic patients, and its value did not differ between the two groups (453±8 vs. 465±6ms, P=NS). No patients showed an abnormal baseline QT dispersion. In cirrhotic individuals, QTcmax further increased 1-3 months after TIPS (P=0.042), thereafter remaining steadily elevated. QT dispersion only increased at the second post-TIPS determination (P=0.030). Such changes occurred despite no deterioration of liver function, plasma electrolytes and haemoglobin. Conclusions: QT interval is frequently prolonged in patient with both non-cirrhotic and cirrhotic portal hypertension and portal decompression by TIPS worsens this abnormality. These results suggest that the porto-systemic shunting is responsible for the altered ventricular repolarisation possibly through a dumping into the systemic circulation of splanchnic-derived cardioactive substances.

Inflammation and Fibrosis

Toshihiro Nishio et al.
Increased expression of collagenase in the liver induces hepatocyte proliferation with cytoplasmic accumulation of -catenin in the rat
Background/Aims: Since the hepatic extracellular matrix is remodeled in liver regeneration, we investigated whether increased collagenase activity in the liver can induce hepatocyte proliferation in vivo. Methods: To increase hepatic collagenase activity, human matrix metalloproteinase-1 was delivered to the rat liver by the recombinant adenoviral vector Ad5MMP-1. Results: Hepatic delivery of Ad5MMP-1 increased the 5-bromo-2-deoxyuridine labeling index and mitotic index in hepatocytes, causing an increase in the dry liver weight; control adenovirus, Ad5LacZ, had minimal effect. Hepatocyte proliferation started approximately 48 h after infection with Ad5MMP-1 and ended after about 2 weeks. The increase in the dry liver weight also returned to baseline after 2 weeks. Transient liver injury by Ad5MMP-1, reflected by increased aspartate and alanine aminotransferase levels, peaked around 1 week, and was associated with hepatocyte apoptosis. Collagenase-induced hepatocyte proliferation was accompanied by cytoplasmic accumulation of -catenin and a transient decrease in E-cadherin expression. Conclusions: Modification of the hepatic extracellular matrix by collagenase induces transient hepatocyte proliferation in vivo, suggesting that the condition of the hepatic extracellular matrix per se plays a pivotal role in regulating hepatocyte proliferation.

Liver Cell Injury and Liver Failure

Thomas S. Weiss, Sascha Pahernik, Irmgard Scheruebl, Karl-Walter Jauch and Wolfgang E. Thasler
Cellular damage to human hepatocytes through repeated application of 5-aminolevulinic acid
Background/Aims: 5-Aminolevulinic acid (ALA), a precursor of porphyrins is used for photodynamic diagnosis and therapy within topical or systemic applications. A potential toxic effect on the human liver is of major interest and therefore we investigated the impact of a repeated application of ALA without illumination on cultures of human hepatocytes. Methods: After ALA treatment of hepatocytes in vitro the porphyrin synthesis, albumin secretion, liver-specific enzyme release, and malondialdehyde levels were determined. In order to reduce levels of reactive oxygen substances, mannitol and the antioxidant enzymes superoxide dismutase and catalase were supplemented. Results: Porphyrin biosynthesis by human hepatocytes in vitro was repeatedly stimulated by ALA (0.001-1.0 mM), which was indicated by an accumulation of protoporphyrin IX. A repetitive treatment (up to four times) of hepatocytes with ALA resulted in an impairment of the hepatic function and viability, depending on the ALA concentration (0.1-1.0 mM) and frequency of application (2-3 times). This was also accompanied by increased malondialdehyde levels indicating enhanced lipid peroxidation. Only superoxide dismutase was able to reduce cellular damage and prevent specific function. Conclusions: Repeated, not single, ALA treatment without illumination may cause deleterious effects to the liver, which are mediated by oxygen radicals and inhibited by an antioxidant.

Fuminori Hirano, Keiji Komura, Etsushi Fukawa and Isao Makino
Tumor necrosis factor (TNF-)-induced RANTES chemokine expression via activation of NF-B and p38 MAP kinase: roles of TNF- in alcoholic liver diseases
Background/Aims: Increased concentration of plasma tumor necrosis factor (TNF-) correlates with the clinical course of alcoholic liver diseases. In addition, hepatic RANTES which migrates CD4 T lymphocytes to liver is increased in patients with alcoholic hepatitis. We investigated that roles of TNF- on RANTES expression in hepatocytes. Methods: HLE cells were treated with TNF- in the presence, or absence of several inhibitors. Enzyme-linked immunoassay and reverse transcriptase-polymerase chain reaction were performed for the measurement of protein production and mRNA of RANTES, respectively. Moreover, DNA-binding activity of NF-B was investigated using electrophoretic mobility shift assay. To examine effects of TNF- on RANTES gene expression, luciferase assay was performed. Results: TNF- clearly up-regulated RANTES expression in a time-dependent fashion and induced DNA-binding activity of NF-B. Moreover, TNF--induced RANTES expression was completely inhibited by SB203580, but not calphostin C and wortmannin. Luciferase assay showed that TNF- increased RANTES gene expression and mutation of NF-B binding sites in the RANTES promoter ablated TNF- inducibility. Conclusions: We showed that RANTES was transcriptionally induced in human hepatoma cells by treatment with TNF- via activation of NF-B and p38 MAP kinase, presumably suggesting that TNF--induced expression of RANTES plays important roles in cell-mediated liver injury in alcoholic liver diseases.

Cell Injury and Liver Failure

Alexandra K. Kiemer et al.
Kupffer-cell specific induction of heme oxygenase 1 (hsp32) by the atrial natriuretic peptide role of cGMP
Background/Aims: Pretreatment with atrial natriuretic peptide (ANP) attenuates ischemia-reperfusion injury of livers via cGMP. Heme oxygenase-1 (HO-1) is known as a protective mediator in ischemia-reperfusion injury. The aim of this study was to investigate whether ANP affects the expression of HO-1. Methods: Rat livers were perfused with KH-buffer with/without ANP or 8-Br-cGMP, kept in UW solution (4,°C, 24 h), and reperfused. HO-1 mRNA and protein was determined by Northern and Western blot, in situ hybridization, and immunohistochemistry in livers or isolated liver cells. Results: ANP significantly elevated HO-1 mRNA expression at the end of the preconditioning period and was without effects at the end of ischemia and during reperfusion. 8-Br-cGMP did not affect HO-1 mRNA expression. In situ hybridization as well as immunohistological double-staining revealed that Kupffer cells but not hepatocytes showed HO-1 mRNA and protein expression. Hepatocytes revealed no changes in HO-1 protein whereas Kupffer cells showed a marked increase in HO-1 protein after ANP treatment. Inhibition of HO-1 did not abrogate hepatoprotection conveyed by ANP. Conclusion: Our data show the potency of ANP to specifically induce HO-1 in Kupffer cells independently of cGMP. This increased expression of HO-1 is not involved in hepatoprotection conferred by ANP being in line with the knowledge that ANP mediates hepatoprotection via cGMP.

Viral Hepatitis
Silvia Fargion et al.
Sustained response to combination therapy in patients with chronic hepatitis C who failed to respond to interferon
Background/Aims: The best treatment for chronic hepatitis C patients who do not respond to interferon is still unknown. Reported rates of response to treatment vary as the result of heterogeneous definitions of non-responders and small study size. Methods: One hundred nineteen hepatitis C virus (HCV) RNA-positive non-responders to high-dose interferon monotherapy received alpha-interferon, 5 MU tiw plus oral ribavirin, 1000-1200 mg/day for 48 weeks (Group A, n=74) or alpha-interferon, 5 MU daily for 4 weeks, followed by 5 MU tiw plus oral ribavirin, 1000-1200 mg/day for 44 weeks (Group B, n=45) according to the Institution where they were followed. Persistently normal alanine aminotransferase and negative HCV RNA up to 72 weeks from treatment onset defined a sustained response. Results: Eighteen patients discontinued treatment (13 developed anemia, two mucositis, one granulocytopenia; two were dropouts), none for serious adverse events. There were 24 (20%) sustained responders, with similar final response rates in Groups A and B. Sustained response was more frequent in patients aged 40 years (36% vs. 13%; P=0.006) and in those with non-1 genotype (44% vs. 14%; P=0.002). Among genotype 1 patients, the younger ones showed higher response rates (32% vs. 7%; P=0.005). Compared with patients harboring non-1 genotypes, the odds ratio of being a non-responder was 1.68 (confidence interval (CI): 0.53-5.37; P=0.381) in younger genotype 1 patients and 9.53 (CI: 2.84-32; P<0.001) in older genotype 1 patients. Conclusions: Chronic hepatitis C patients who are non-responders to interferon monotherapy and infected by non-1 genotypes should undergo re-treatment with combination therapy. Treatment should be extended to younger genotype 1 patients who are more susceptible to liver disease worsening because of longer life expectancy and have a higher probability of being long lasting responders than their older counterparts.

Fatih Besisik et al.
Occult HBV infection and YMDD variants in hemodialysis patients with chronic HCV infection
Background/Aims: End-stage renal disease patients on chronic hemodialysis are at risk for both hepatitis B virus (HBV) and hepatitis C virus (HCV) infection. Although the prevalence is unknown in hemodialysis patients, occult HBV infection is frequent in subjects with chronic HCV infection. We aimed to investigate (1) the prevalence and clinical impact of occult HBV infection in hemodialysis patients with chronic HCV infection, and (2) the frequency of YMDD variants (tyrosine-methionine-aspartate-aspartate amino acid motif of HBV polymerase) in this setting. Methods: Thirty-three anti-HCV and HCV-RNA-positive, HBsAg-negative hemodialysis patients (mean age 36.9±10.4 years, 22 male) were admitted to this study. HBV-DNA (Innogenetics kit) and HCV-RNA (Cobas Amplicor HCV kit) were investigated by polymerase chain reaction technique (PCR). YMDD mutation was studied in all HBV-DNA-positive patients by the BOOM method. Results: HBV-DNA was detected in 12 of 33 patients (36.4%) by PCR. Their mean age was 33.0±9.0 years. Age, dialysis period (years) and biochemical parameters were not significantly different in patients with and without occult HBV infection. YMDD variants were identified in six of 12 (50%) patients with occult HBV infection. Conclusions: Occult HBV infection is frequent in hemodialysis patients with chronic HCV infection. YMDD variants are common in this setting.

Chee-Kin Hui, Tigist Belaye, Kevin Montegrande and Teresa Lyn Wright
A comparison in the progression of liver fibrosis in chronic hepatitis C between persistently normal and elevated transaminase
Background: Detectable serum hepatitis C virus (HCV) RNA in HCV patients with persistently normal alanine transaminase (PNALT) has been found to be associated with significant liver damage. Aims: The primary outcome of this study was to compare the histological progression of fibrosis in patients with PNALT and elevated alanine transaminase (ALT). Methods: Forty patients with PNALT (Group 1) and 41 patients with elevated ALT (Group 2) were recruited into this study. Only patients with fibrosis of F0 to F2 were recruited into this study. Results: The median time to second liver biopsies was 6.3 (range 2.0-11.1) years. Nine patients (22.5%) in Group 1 and 17 patients (41.5%) from Group 2 had progression of fibrosis. There was a trend towards a significantly higher cumulative probability of fibrosis progression in Group 2 (P=0.06). In patients with an initial F0 to F1 fibrosis, there was a significant difference in cumulative probability of fibrosis progression between Groups 1 and 2 (22.6% (7/31) vs. 43.3% (13/31), respectively, P=0.02). Conclusions: Anti-HCV patients with PNALT with an initial fibrosis of F0 or F1 were less likely to develop progression of fibrosis than those with elevated ALT, although patients with PNALT may have histologically and clinically progressive disease.

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

BRITISH MEDICAL JOURNAL

5 April 2003 (Volume 326, Issue 7392)

Prevalence of Helicobacter pylori in patients with gastro-oesophageal reflux disease: systematic review
Anan Raghunath, A Pali S Hungin, David Wooff, and Susan Childs
BMJ 2003; 326: 737. Prevalence of Helicobacter pylori in patients with gastro-oesophageal reflux disease: systematic review

Objectives: To ascertain the prevalence of Helicobacter pylori in patients with gastro-oesophageal reflux disease and its association with the disease.
Design: Systematic review of studies reporting the prevalence of H pylori in patients with and without gastro-oesophageal reflux disease.
Data sources: Four electronic databases, searched to November 2001, experts, pharmaceutical companies, and journals.
Main outcome measure: Odds ratio for prevalence of H pylori in patients with gastro-oesophageal reflux disease.
Results: 20 studies were included. The pooled estimate of the odds ratio for prevalence of H pylori was 0.60 (95% confidence interval 0.47 to 0.78), indicating a lower prevalence in patients with gastro-oesophageal reflux disease. Substantial heterogeneity was observed between studies. Location seemed to be an important factor, with a much lower prevalence of H pylori in patients with gastro-oesophageal reflux disease in studies from the Far East, despite a higher overall prevalence of infection than western Europe and North America. Year of study was not a source of heterogeneity.
Conclusion: The prevalence of H pylori infection was significantly lower in patients with than without gastro-oesophageal reflux, with geographical location being a strong contributor to the heterogeneity between studies. Patients from the Far East with reflux disease had a lower prevalence of H pylori infection than patients from western Europe and North America, despite a higher prevalence in the general population.

NEW ENGLAND JOURNAL

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