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HEPATOLOGY

Table of Contents forDecember 2003 · Volume 38 · Number 6

Liver Failure and Liver Disease

Early change in bilirubin levels is an important prognostic factor in severe alcoholic hepatitis treated with prednisolone (*Human Study*)
Philippe Mathurin, Marcelle Abdelnour, Marie-José Ramond, Nicolas Carbonell, Laetitia Fartoux, Lawrence Serfaty, Dominique Valla, Raoul Poupon, Jean-Claude Chaput, Sylvie Naveau
Early identification of patients with severe (discriminant function 32) biopsy-proven alcoholic hepatitis (AH) who are not responding to corticosteroids would be clinically relevant. Our goal was to develop simple criteria that will help physicians to promptly identify nonresponders to corticosteroids. A total of 238 patients were included. We used 6 months survival as an end point because of the rule requiring 6 months for listing alcoholic patients for transplantation. Overall survival at 1 and 6 months was 85% ± 2.3% and 64.3% ± 3.3%, respectively. An early change in bilirubin levels (ECBL) at 7 days (defined as bilirubin level at 7 days lower than bilirubin level on the first day of treatment) was observed in 73% of patients. At 7 days, in patients with ECBL, bilirubin decreased (84 ± 75 µmol/L [4.94 ± 4.40 mg/dL]), whereas it increased in patients without ECBL (76.5 ± 77 µmol/L [4.50 ± 4.54 mg/dL], P < .0001). Ninety-five percent of patients with ECBL continued to have improved liver function during treatment. At 6 months, survival of patients with ECBL was significantly higher than that of patients without ECBL, 82.8% ± 3.3% versus 23% ± 5.8%, P < .0001. On multivariate analysis, ECBL, discriminant function and creatinine were independent prognostic variables, and ECBL had the most important prognostic value. In conclusion, ECBL is a very simple predictive factor for identifying nonresponders. A recommendation to discontinue corticosteroids after 7 days in patients without ECBL, suggested by our results, awaits additional confirmation. (HEPATOLOGY 2003;38:1363-1369.)

Humoral and cardiac effects of TIPS in cirrhotic patients with different "effective" blood volume (*Human Study*)
Francesco Salerno, Massimo Cazzaniga, Giovanni Pagnozzi, Ilaria Cirello, Antonio Nicolini, Daniele Meregaglia, Larry Burdick
The aim of this study was to evaluate the cardiac effects of transjugular intrahepatic portosystemic shunts (TIPS) in cirrhotic patients with different effective blood volume. Two-dimensional echocardiography was performed before and 7 and 28 days after TIPS insertion in 7 cirrhotic patients with PRA <4 ng/mL/h (group A, normal effective blood volume) and 15 with PRA >4 ng/mL/h (group B, reduced effective blood volume). Before TIPS, most cirrhotic patients showed diastolic dysfunction as indicated by reduced early maximal ventricular filling velocity (E)/late filling velocity (A) ratio. Patients of group B differed from patients of group A because of smaller left ventricular volumes and stroke volume, indicating central underfilling. After TIPS insertion, portal decompression was associated with a significant increase of cardiac output (CO) and a decrease of peripheral resistances. The most important changes were recorded in patients of group B, who showed a significant increase of both the end-diastolic left ventricular volumes and the E/A ratio and a significant decrease of PRA. In conclusion, these results show that the hemodynamic effects of TIPS differ according to the pre-TIPS effective blood volume. Furthermore, TIPS improves the diastolic cardiac function of cirrhotic patients with effective hypovolemia. This result is likely due to a TIPS-related improvement of the fullness of central blood volume. (HEPATOLOGY 2003;38:1370-1377.)

Effects of the V1a vasopressin agonist F-180 on portal hypertension-related bleeding in portal hypertensive rats
Josephine Morales, Eduardo Moitinho, Juan G. Abraldes, Mercedes Fernández, Jaime Bosch
F-180 is a new, long-acting analog of vasopressin with a selective agonist effect on the vascular V1a receptors, with the advantage of having no effect on renal V2 receptors. F-180 is approximately 20 times more powerful than terlipressin in reducing portal pressure and has less marked systemic effects. The present study investigated the effects of F-180 on the outcome of portal hypertension-related bleeding in hypovolemic rats. Partial portal vein-ligated rats were subjected to portal hypertension-related bleeding by sectioning a first-order branch of the ileocolic vein. After hemodynamic stabilization, a second sectioning of the first-order branch of the ileocolic vein section was performed in the already hypovolemic animals, and either F-180 or placebo was administered. Blood transfusion was adjusted to maintain mean arterial pressure (MAP) > 80 mm Hg. The first section of a first-order branch of the ileocolic vein induced a hemorrhage of similar severity in both groups of rats. After a 2nd sectioning of a first-order branch of the ileocolic vein section, F-180 was more effective than placebo in recovering shock (MAP, 21% ± 23% vs. 0% ± 13% in placebo; P < .05), preventing portal pressure (PP) increase during blood transfusion (PP: ­1% ± 19% vs. 47% ± 65% in placebo; P = .07), reducing transfusion requirements (2.9 ± 3.3 mL vs. 11.2 ± 6.0 mL in placebo; P < .01), diminishing the magnitude of collected blood losses (5.1 ± 2.2 g vs. 12.7 ±7.7 g in placebo; P < .05), and decreasing the mortality from the portal hypertension-related bleeding (10% vs. 60% in placebo; P < .05). In conclusion, in hypovolemic portal-hypertensive rats during a portal hypertension-related bleeding, F-180 rapidly recovers arterial pressure and decreases transfusion requirements, blood losses, and mortality. (HEPATOLOGY 2003;38:1378-1383.)

Impaired IRS-1/PI3-kinase signaling in patients with HCV: A mechanism for increased prevalence of type 2 diabetes (*Human Study*)
Serhat Aytug, David Reich, Lawrence E. Sapiro, David Bernstein, Najma Begum
Patients with hepatitis C virus (HCV) infection have a greater risk of developing type 2 diabetes mellitus. However, the mechanism of this association is unclear. In this study, we examined the potential defects in upstream insulin signaling pathways in liver specimens obtained from nonobese/nondiabetic subjects with HCV infection. Fasting liver biopsy specimens were obtained from 42 HCV-infected subjects and 10 non-HCV-infected subjects matched for age and body mass index. Liver tissues were exposed to insulin and examined for the contents and phosphorylation/activation status of the upstream insulin signaling molecules by immunoprecipitation and Western blot analysis. HCV infection resulted in a trend toward a 2-fold to 3-fold increase in insulin receptor (IR) and insulin receptor substrate (IRS)-1 contents when compared with non-HCV. In contrast, insulin-stimulated IRS-1 tyrosine phosphorylation was decreased by 2-fold in HCV-infected subjects compared with non-HCV-infected subjects (P < .05). The observed reductions in IRS-1 tyrosine phosphorylation were accompanied by a 3.4-fold decrease in IRS-1/p85 phosphatidylinositol 3-kinase (PI3-kinase) association and a 2.5-fold decrease in IRS-1-associated PI3-kinase enzymatic activity (P < .05 vs. non-HCV). This was accompanied by a marked reduction in insulin-stimulated Akt phosphorylation without any alterations in mitogen-activated protein kinase (MAPK) phosphorylation. Cellular contents of the hepatic p85 subunit of PI3-kinase were comparable between HCV-infected and non-HCV-infected subjects. In conclusion, we found that (1) HCV infection leads to a postreceptor defect in IRS-1 association with the IR and (2) insulin signaling defects in hepatic IRS-1 tyrosine phosphorylation and PI3-kinase association/activation may contribute to insulin resistance, which leads to the development of type 2 diabetes mellitus in patients with HCV infection. (HEPATOLOGY 2003;38:1384-1392.)

Role of reproductive factors in hepatocellular carcinoma: Impact on hepatitis B­ and C­related risk (*Human Study*)
Ming-Whei Yu, Hung-Chuen Chang, Shun-Chiao Chang, Yun-Fan Liaw, Shi-Ming Lin, Chun-Jen Liu, Shou-Dong Lee, Chih-Lin Lin, Pei-Jer Chen, Shee-Chan Lin, Chien-Jen Chen
Hepatocellular carcinoma (HCC) is more prevalent in men than in women. Estrogen may play some role in the development of HCC. We conducted a multicenter case-control study to evaluate the effects of reproductive factors on HCC risk, and to assess whether the association between each factor and HCC differs between hepatitis B surface antigen (HBsAg)-positive and -negative women, in which hepatitis C virus (HCV) is the major cause of HCC. The study included 218 women with HCC and 729 control women selected from nonbiological and first-degree female relatives of patients with HCC. The risk of HCC was inversely related to the number of full-term pregnancies (FTP) (Ptrend = .0216) and age at natural menopause (Ptrend = .0251 among women aged 45-55 without prior surgical menopause). Oophorectomy at age 50 during premenopausal years was also a risk factor (multivariate-adjusted OR, 2.57; 95% CI, 1.42-4.63). Use of hormone replacement therapy (HRT) (multivariate-adjusted OR, 0.46; 95% CI, 0.27-0.79) was associated with a lower risk of HCC, and there was a trend in the risk with increasing duration of HRT (Ptrend = 0.0013). All reproductive factors had a similar impact on HBsAg-positive and -negative women except for an early menarche (12 vs. 16 years), which increased HCC risk in HBsAg carriers (multivariate-adjusted OR, 6.96; 95% CI, 2.52-19.18) but posed no increased risk in noncarriers (Pinteraction = .0053). In conclusion, increased exposure to estrogen during adulthood may provide a protective effect against HCC. Nevertheless, an early menarche, which results in early estrogen exposure, does not confer protection for HBsAg carriers. (HEPATOLOGY 2003;38:1393-1400.)

Separate analysis of asialoglycoprotein receptors in the right and left hepatic lobes using 99mTc-GSA SPECT (*Human Study*)
Kazuhiko Sugahara, Hitoshi Togashi, Kazuei Takahashi, Yuya Onodera, Mai Sanjo, Keiko Misawa, Akihiko Suzuki, Tohru Adachi, Junitsu Ito, Kazuo Okumoto, Etsuko Hattori, Tadashi Takeda, Hisayoshi Watanabe, Koji Saito, Takafumi Saito, Yukio Sugai, Sumio Kawata
The asialoglycoprotein receptor (ASGPR) is abundantly expressed on the sinusoidal surfaces of hepatocytes. We aimed to clarify the clinical significance of the regional distribution of ASGPRs in the human liver, especially in chronic viral hepatitis. Eighteen volunteers, 34 patients with chronic hepatitis, and 33 patients with cirrhosis (11/Child-Pugh A, 11/Child-Pugh B, 11/Child-Pugh C) were studied using a newly developed, conventional technetium-99m-diethylenetriaminepentaacetic acid-galactosyl human serum albumin (99mTc-GSA), single photon emission computed tomography (SPECT) method. Using Cantlie's line as a guide, ASGPR dynamics were analyzed separately in the right and left lobes, as well as in the whole liver, using novel indices (the liver uptake ratio [LUR] and the liver uptake density [LUD], which reflect the amount and density of ASGPRs in the liver, respectively). Mean LUR and LUD values for the whole liver and the right and left lobes decreased with increasing progression of chronic viral hepatitis. The LUR for the whole liver correlated well with parameters measuring the hepatic functional reserve and the platelet count. The right LUR correlated particularly well with conventional liver function tests, and comparison of the right LUD with histologic findings showed that it was a good indicator of periportal and/or bridging necrosis and fibrosis. In conclusion, our 99mTc-GSA SPECT method was clinically useful in evaluating regional hepatic function and the progression of chronic viral hepatitis using dynamic changes in ASGPRs. (HEPATOLOGY 2003;38:1401-1409.)

Viral Hepatitis

Inhibitory effect of adefovir and lamivudine on the initiation of hepatitis B virus infection in primary tupaia hepatocytes
Josef Köck, Thomas F. Baumert, William E. Delaney, IV, Hubert E. Blum, Fritz von Weizsäcker
Adefovir dipivoxil and lamivudine are two safe and efficacious drugs licensed for the treatment of chronic hepatitis B virus (HBV) infection. Both drugs inhibit the viral polymerase, resulting in a profound suppression of virus production. Blocking the viral polymerase may also affect the initiation of HBV infection, because HBV virions harbor a partially double-stranded genome and productive infection requires completion of viral plus-strand DNA synthesis with subsequent formation of covalently closed circular DNA (cccDNA). To address this issue, we used primary hepatocytes from the tree shrew Tupaia belangeri that were recently shown to be susceptible to HBV infection. Treatment of cells with either drug partially inhibited initial HBV cccDNA formation. Adefovir was more effective than lamivudine, resulting in a 3-fold reduction of RNA synthesis and viral surface antigen production. However, prevention of initial cccDNA formation was incomplete even after combined treatment, whereas de novo synthesis of viral replicative intermediates was completely suppressed. A possible explanation for this observation is the genomic plus-strand gap of less than 200 bases in some virions, limiting the window for antiviral action. In conclusion, nucleos(t)ide analogues can target initial plus-strand DNA repair and reduce but not completely block HBV infection. (HEPATOLOGY 2003;38:1410-1418.)

Adefovir dipivoxil therapy for lamivudine-resistant hepatitis B in pre­ and post­liver transplantation patients (*Human Study*)
Eugene R. Schiff, Ching-Lung Lai, Stefanos Hadziyannis, Peter Neuhaus, Norah Terrault, Massimo Colombo, Hans L. Tillmann, Didier Samuel, Stefan Zeuzem, Leslie Lilly, Maria Rendina, Jean-Pierre Villeneuve, Nicole Lama, Craig James, Michael S. Wulfsohn, Hamid Namini, Christopher Westland, Shelly Xiong, Gavin S. Choy, Sally Van Doren, John Fry, Carol L. Brosgart, The Adefovir Dipivoxil Study 435 International Investigators Group
Three-hundred and twenty-four patients were enrolled in an open-label, multicenter, international study in which pre- and post-liver transplantation (LT) patients with recurrent chronic hepatitis B (CHB) and evidence of lamivudine-resistant HBV were treated with adefovir dipivoxil 10 mg once daily. In the pre- and post-LT cohorts, 128 and 196 patients were treated for a median duration of 18.7 and 56.1 weeks, respectively. In patients who received 48 weeks of treatment, 81% of the pre-LT and 34% of the post-LT cohort achieved undetectable serum hepatitis B virus (HBV) DNA (Roche Amplicor MonitorTM polymerase chain reaction [PCR] assay lower limit of quantification [LLQ] < 400 copies/mL) with a median change in serum HBV DNA from baseline of ­4.1 log10 and ­4.3 log10 copies/mL, respectively. Serum alanine aminotransferase (ALT), albumin, bilirubin, and prothrombin time normalized in 76%, 81%, 50%, and 83% of pre-LT patients and 49%, 76%, 75%, and 20% of post-LT patients. The Child-Pugh-Turcotte (CPT) score improved in over 90% of patients in both cohorts. Genotypic analysis of 122 HBV baseline samples revealed that 98% of these patients had lamivudine-resistant mutant HBV. No adefovir resistance mutations were identified in patients after 48 weeks of therapy. One-year survival was 84% for pre-LT and 93% for post-LT patients (Kaplan-Meier analysis). Treatment-related adverse effects associated with adefovir dipivoxil in this setting were primarily mild to moderate in severity. In conclusion, 48 weeks of adefovir dipivoxil resulted in significant improvements in virologic, biochemical, and clinical parameters in CHB patients pre- and post-LT with lamivudine-resistant HBV. (HEPATOLOGY 2003;38:1419-1427.)

Results of retransplantation for recurrent hepatitis C (*Human Study*)
Sasan Roayaie, Thomas D. Schiano, Swan N. Thung, Sukru H. Emre, Thomas M. Fishbein, Charles M. Miller, Myron E. Schwartz
Retransplantation for recurrent hepatitis C virus (HCV) has been evaluated in small series. In this study, patients undergoing transplantation for HCV-related cirrhosis with subsequent retransplantation more than 90 days for recurrent HCV (proven by pathologic examination of the explant and exclusion of other factors) were prospectively followed. This group was compared with a simultaneous cohort without HCV infection undergoing retransplantation more than 90 days after primary transplantation. Forty-two patients underwent retransplantation for recurrent HCV with a median survival of 12.9 ± 6.7 months after retransplantation. Twenty patients (48%) were dead at 6 months, and 13 (65%) of these deaths were due to sepsis. On univariate analysis, creatinine level greater than or equal to 3 mg/dL, platelet count less than 100,000/µL, prothrombin time (PT) greater than or equal to 16 seconds, alkaline phosphatase level less than or equal to 240 U/L, -glutamyltransferase level less than or equal to 130 U/L, and donor age of 60 years or greater all correlated significantly with shorter survival after retransplantation. PT and donor age were predictors of survival on multivariate analysis. Patients undergoing retransplantation for recurrent HCV had a significantly shorter median survival than the 55 patients undergoing retransplantation for other chronic reasons of graft loss (75.6 ± 17.7 months). In conclusion, median survival after liver retransplantation for recurrent HCV is significantly shorter than after retransplantation for other causes of late graft loss. Most deaths occur in the first 6 months and are due to sepsis. Candidates for retransplantation with a preoperative PT less than 16 seconds and those receiving grafts from donors younger than 60 years can expect a significantly longer median survival after retransplantation. (HEPATOLOGY 2003;38:1428-1436.)

Suppression of HCV-specific T cells without differential hierarchy demonstrated ex vivo in persistent HCV infection (*Human Study*)
Kazushi Sugimoto, Fusao Ikeda, Jason Stadanlick, Frederick A. Nunes, Harvey J. Alter, Kyong-Mi Chang
Hepatitis C virus (HCV) has a high propensity for persistence. To better define the immunologic determinants of HCV clearance and persistence, we examined the circulating HCV-specific T-cell frequency, repertoire, and cytokine phenotype ex vivo in 24 HCV seropositive subjects (12 chronic, 12 recovered), using 361 overlapping peptides in 36 antigenic pools that span the entire HCV core, NS3-NS5. Consistent with T-cell-mediated control of HCV, the overall HCV-specific type-1 T-cell response was significantly greater in average frequency (0.24% vs. 0.04% circulating lymphocytes, P = .001) and scope (14/36 vs. 4/36 pools, P = .002) among the recovered than the chronic subjects, and the T-cell response correlated inversely with HCV titer among the chronic subjects (R = ­0.51, P = .049). Although highly antigenic regions were identified throughout the HCV genome, there was no apparent difference in the overall HCV-specific T-cell repertoire or type-1/type-2 cytokine profile relative to outcome. Notably, HCV persistence was associated with a reversible CD4-mediated suppression of HCV-specific CD8 T cells and with higher frequency of CD4+CD25+ regulatory T cells (7.3% chronic vs. 2.5% recovered, P = .002) that could directly suppress HCV-specific type-1 CD8 T cells ex vivo. In conclusion, we found that HCV persistence is associated with a global quantitative and functional suppression of HCV-specific T cells but not differential antigenic hierarchy or cytokine phenotype relative to HCV clearance. The high frequency of CD4+CD25+ regulatory T cells and their suppression of HCV-specific CD8 T cells ex vivo suggests a novel role for regulatory T cells in HCV persistence. (HEPATOLOGY 2003;38:1437-1448.)

Sampling variability of liver fibrosis in chronic hepatitis C
Pierre Bedossa, Delphine Dargère, Valerie Paradis
Fibrosis is a common endpoint of clinical trials in chronic hepatitis C, and liver biopsy remains the gold standard for fibrosis evaluation. However, variability in the distribution of fibrosis within the liver is a potential limitation. Our aim was to assess the heterogeneity of liver fibrosis and its influence on the accuracy of assessment of fibrosis with liver biopsy. Surgical samples of livers from patients with chronic hepatitis C were studied. Measurement of fibrosis was performed on the whole section by using both image analysis and METAVIR score (reference value). From the digitized image of the whole section, virtual biopsy specimens of increasing length were produced. Fibrosis was assessed independently on each individual virtual biopsy specimen. Results were compared with the reference value according to the length of the biopsy specimen. By using image analysis, the coefficient of variation of fibrosis measurement with 15-mm long biopsy specimens was 55%; and for biopsy specimens of 25-mm length it was 45%. By using the METAVIR scoring system, 65% of biopsies 15 mm in length were categorized correctly according to the reference value. This increased to 75% for a 25-mm liver biopsy specimen without any substantial benefit for longer biopsy specimens. Sampling variability of fibrosis is a significant limitation in the assessment of fibrosis with liver biopsy. In conclusion, this study suggests that a length of at least 25 mm is necessary to evaluate fibrosis accurately with a semiquantitative score. Sampling variability becomes a major limitation when using more accurate methods such as automated image analysis. (HEPATOLOGY 2003;38:1449-1457.)

Hepatitis C virus and liver disease: Global transcriptional profiling and identification of potential markers (*Human Study*)
Maria W. Smith, Zhaoxia N. Yue, Marcus J. Korth, Hao A. Do, Loreto Boix, Nelson Fausto, Jordi Bruix, Robert L. Carithers, Jr., Michael G. Katze
Microarray analysis of RNA from hepatitis C virus (HCV)-infected cirrhotic livers was performed to identify a gene expression signature of liver disease. The expression levels of approximately 13,600 genes were analyzed using surgical material and core biopsy specimens from HCV-infected cirrhotic liver explants in comparison with reference samples of normal nondiseased liver. In addition, normal liver samples were compared with each other to determine normal physiologic variation in gene expression. A set of genes, including some associated with stress, acute-phase immune response, and hepatic stellate cell activation, had variable expression levels in normal livers. These genes were subtracted from the sets of genes differentially expressed in cirrhotic livers. To exclude cancer-related genes from our marker sets, we subtracted genes that also were expressed differentially in hepatocellular carcinomas. The resultant HCV- and liver disease-associated gene set provided a molecular portrait of several processes occurring in the HCV-infected liver. It included (1) genes expressed in activated lymphocytes infiltrating the cirrhotic liver, and activated liver macrophages; (2) genes involved in remodeling of extracellular matrix-cell and cell-cell interactions associated with cytoskeleton rearrangements; (3) genes related to the anti-apoptotic pathway of Bcl-2 signaling; and (4) genes involved with the interferon response and virus-host interactions. In conclusion, our microarray analysis identified several potential gene markers of HCV-associated liver disease and contributed to our rapidly expanding database of experiments describing HCV pathogenesis. (HEPATOLOGY 2003;38:1458-1467.)

Association of genetic variants of the chemokine receptor CCR5 and its ligands, RANTES and MCP-2, with outcome of HCV infection (*Human Study*)
Simon Hellier, Angela J. Frodsham, Branwen J. W. Hennig, Paul Klenerman, Suzanne Knapp, Patricia Ramaley, Jack Satsangi, Mark Wright, Lyna Zhang, Howard C. Thomas, Mark Thursz, Adrian V. S. Hill
The effect of host genetic variation on the outcome of hepatitis C virus (HCV) infection and its treatment is poorly understood. The chemokine receptors CCR5, CCR2, and CCR3 and their ligands, RANTES, MCP-1, MCP-2, and MIP-1, are involved in the immune responses and the selective recruitment of lymphocytes to the liver in HCV infection. We studied 20 polymorphisms within these genes and investigated their association with persistent carriage of HCV, severity of liver disease, hepatic inflammation, and response to treatment in a large European cohort. Significant associations were found between CCR5-32 and reduced portal inflammation (P = .011, odds ratio [OR]: 2.3, 95% confidence interval [CI]: 1.09-4.84) and milder fibrosis (P = .015, OR: 1.97, 95% CI: 1.13-3.42). A promoter polymorphism at position ­403 in the RANTES gene was associated with less severe portal inflammation (P = .004). An amino acid change in MCP2, Q46K, was associated with severity of fibrosis (P = .018, OR: 2.29, 95% CI: 1.14-4.58). In conclusion, our study suggests a possible role of the polymorphisms CCR5-32, RANTES ­403, and MCP-2 Q46K in the outcome of HCV infection. (HEPATOLOGY 2003;38:1468-1476.)

Liver Biology and Pathobiology

Hepatic overexpression of caveolins increases bile salt secretion in mice
Mauricio Moreno, Hector Molina, Ludwig Amigo, Silvana Zanlungo, Marco Arrese, Attilio Rigotti, Juan Francisco Miquel
Caveolins are cholesterol-binding proteins involved in the regulation of several intracellular processes, including cholesterol transport. Because hepatocytes express caveolin-1 and caveolin-2, these proteins might modulate hepatic lipid metabolism and biliary lipid secretion. Our aim was to investigate the potential physiologic role of caveolins in hepatic cholesterol and bile salt (BS) metabolism and transport using adenoviral gene transfer. C57BL/6 mice were infected with recombinant human caveolin-1 and caveolin-2 adenoviruses. Mice infected with adenovirus lacking the transgene were used as controls. Hepatic caveolin expression was evaluated by immunochemical methods. Reverse-transcription polymerase chain reaction (RT-PCR) and immunoblotting were used to assess messenger RNA (mRNA) levels and protein mass of BS transporters (sodium taurocholate cotransporting polypeptide [Ntcp] and bile salt export pump [Bsep]). Serum, liver, biliary, and fecal biochemical determinations and BS maximal secretory rate (SRm) were performed by standard methods. Ad.Cav-1- and Ad.Cav-2-infected mice exhibited a 10- and 7-fold increase in hepatic caveolin-1 and caveolin-2 protein expression, respectively. Caveolin-1-overexpressing mice had a significant increase in plasma high-density lipoprotein (HDL) cholesterol and hepatic free cholesterol content, whereas total plasma cholesterol and triglyceride levels remained unchanged. Hepatic caveolin-1 and/or caveolin-2 overexpression significantly increased bile flow and secretion of all biliary lipids. Caveolin-1-overexpressing mice showed a 2.5-fold increase in taurocholate (TC) SRm, indicating increased canalicular BS transport capacity. BS pool size and fecal BS excretion remained within the normal range in mice with Cav-1 overexpression. No changes were seen in the protein mass of BS transporters Ntcp and Bsep. In conclusion, our findings indicate that caveolins may play an important role in regulating hepatic BS and cholesterol metabolism. (HEPATOLOGY 2003;38:1477-1488.)

Severe cholestasis induced by cholic acid feeding in knockout mice of sister of P-glycoprotein
Renxue Wang, Ping Lam, Lin Liu, Dana Forrest, Ibrahim M. Yousef, Diane Mignault, M. James Phillips, Victor Ling
Intrahepatic cholestasis is often associated with impairment of biliary bile acid secretion, a process mediated by the sister of P-glycoprotein (Spgp or Abcb11) also known as the bile salt export pump (Bsep). In humans, mutations in the Spgp gene are associated with a fatal childhood disease, type 2 progressive familial intrahepatic cholestasis (PFIC2). However in mice, the "knockout" of Spgp only results in mild cholestasis. In this study, we fed spgp­/­ knockout mice with a cholic acid (CA)-supplemented diet to determine whether a more pronounced PFIC2-like phenotype could be induced. Such mice developed severe cholestasis characterized by jaundice, weight loss, elevated plasma bile acid, elevated transaminase, cholangiopathy (proliferation of bile ductules and cholangitis), liver necrosis, high mortality, and wide-ranging changes in the mRNA expression of major liver genes (16/36 examined). A surprising observation was that the bile acid output and bile flow in CA-fed mutant mice was significantly higher than anticipated. This suggests that the spgp­/­ mice are able to utilize an alternative bile salt transport system. However, unlike Spgp, this system is insufficient to protect the knockout mice from cholestasis despite its high capacity. In conclusion, the spgp­/­ mice provide a unique model to investigate molecular pathways associated with cholestasis and related diseases. (HEPATOLOGY 2003;38:1489-1499.)

Thyrotropin-releasing hormone in the dorsal vagal complex stimulates hepatic blood flow in rats
Masashi Yoneda, Takashi Hashimoto, Kimihide Nakamura, Keisuke Tamori, Shiro Yokohama, Toru Kono, Hajime Watanobe, Akira Terano
Central administration of thyrotropin-releasing hormone (TRH) enhances hepatic blood flow in animal models. TRH nerve fibers and receptors are localized in the dorsal vagal complex (DVC), and retrograde tracing techniques have shown that hepatic vagal nerves arise mainly from the left DVC. However, nothing is known about the central sites of action for TRH to elicit the stimulation of hepatic blood flow. The effect of microinjection of a TRH analogue into the DVC on hepatic blood flow was investigated in urethane-anesthetized rats. After measuring basal flow, a stable TRH analogue (RX-77368) was microinjected into the DVC and hepatic blood flow response was observed for 120 minutes by laser Doppler flowmetry. Either left or right cervical vagotomy or hepatic branch vagotomy was performed 2 hours before the peptide. Microinjection of RX-77368 (0.5-5 ng) into the left DVC dose-dependently increased hepatic blood flow. The stimulation of hepatic blood flow by RX-77368 microinjection into the left DVC was eliminated by left cervical and hepatic branch vagotomy but not by right cervical vagotomy. By contrast, microinjection of RX-77368 into the right DVC did not significantly alter hepatic blood flow. These results suggest that TRH acts in the left DVC to stimulate hepatic blood flow through the left cervical and hepatic vagus, indicating that neuropeptides may act in the specific brain nuclei to regulate hepatic function. (HEPATOLOGY 2003;38:1500-1507.)

Bacterial translocation up-regulates GTP-cyclohydrolase I in mesenteric vasculature of cirrhotic rats
Reiner Wiest, Gregory Cadelina, Sheldon Milstien, Robert S. McCuskey, Guadalupe Garcia-Tsao, Roberto J. Groszmann
In cirrhosis, arterial vasodilation and the associated hemodynamic disturbances are most prominent in the mesenteric circulation, and its severity has been linked to bacterial translocation (BT) and endotoxemia. Synthesis of nitric oxide (NO), the main vasodilator implicated, is dependent on the essential cofactor tetrahydrobiopterin (BH4). The key enzyme involved in BH4 synthesis is GTP-cyclohydrolase I (GTPCH-I), which is stimulated by endotoxin. Therefore, we investigated GTPCH-I activity and BH4 biosynthesis in the mesenteric vasculature of cirrhotic rats with ascites, as well as their relationship with BT and endotoxemia, serum NO, and mean arterial pressure (MAP). GTPCH-I activity and BH4 content in mesenteric vasculature was determined by high-performance liquid chromatography. BT was assessed by standard bacteriologic culture of mesenteric lymph nodes (MLNs). Serum endotoxin was measured by a kinetic turbidimetric limulus amebocyte lysate assay, and serum NO metabolite (NOx) concentrations were assessed by chemiluminescence. BT was associated with local lymphatic and systemic appearance of endotoxin and was accompanied by increases in serum NOx levels. GTPCH-I activity and BH4 content in mesenteric vasculature were both increased in animals with BT and correlated significantly (r = 0.69, P < .01). Both GTPCH-I activity and BH4 levels significantly correlated with serum endotoxin and NOx levels (r = 0.69 and 0.54, 0.81 and 0.53, P < .05). MAP (a marker of systemic vasodilatation) correlated with endotoxemia (r = 0.58, P < .03) and with GTPCH-I activity (r = 0.69, P < .01). In conclusion, in cirrhotic animals BT appears to lead to endotoxemia, stimulation of GTPCH-I, increased BH4 synthesis, and further enhancement of vascular NO production that leads to aggravation of vasodilatation. (HEPATOLOGY 2003;38:1508-1515.)

DNA methylation controls the responsiveness of hepatoma cells to leukemia inhibitory factor
Frédéric Blanchard, Erin Tracy, Joseph Smith, Souvik Chattopadhyay, Yanping Wang, William A. Held, Heinz Baumann
The related members of the interleukin 6 (IL-6) family of cytokines, IL-6, leukemia inhibitory factor (LIF), and oncostatin M, act as major inflammatory mediators and induce the hepatic acute phase reaction. Normal parenchymal liver cells express the receptors for these cytokines, and these receptors activate, to a comparable level, the intracellular signaling through signal transducer and activator of transcription (STAT) proteins and extracellular-regulated kinase (ERK). In contrast, hepatoma cell lines show attenuated responsiveness to some of these cytokines that is correlated with lower expression of the corresponding ligand-binding receptor subunits. This study tests the hypothesis that the reduced expression of LIF receptor (LIFR) observed in hepatoma cells is mediated by altered DNA methylation. H-35 rat hepatoma cells that have a greatly reduced LIF responsiveness were treated with 5-aza-2´-deoxycytidine, an inhibitor of DNA methyltransferase. Surviving and proliferating cells showed reestablished expression of LIFR protein and function. Restriction landmark genomic scanning (RLGS) demonstrated genome-wide drug-induced alterations in DNA methylation status, with striking similarities in the demethylation pattern among independently derived clonal lines. Upon extended growth in the absence of 5-aza-2´-deoxycytidine, the cells exhibit partial reversion to pretreatment patterns. Demethylation and remethylation of the CpG island within the LIFR promoter that is active in normal liver cells correlate with increased and decreased usage of this promoter in H-35 cells. In conclusion, these results indicate that transformed liver cells frequently undergo epigenetic alterations that suppress LIFR gene expression and modify the responsiveness to this IL-6 type cytokine. (HEPATOLOGY 2003;38:1516-1528.)

Polyunsaturated fatty acids ameliorate hepatic steatosis in obese mice by SREBP-1 suppression
Motohiro Sekiya, Naoya Yahagi, Takashi Matsuzaka, Yuho Najima, Masanori Nakakuki, Ryozo Nagai, Shun Ishibashi, Jun-ichi Osuga, Nobuhiro Yamada, Hitoshi Shimano
Leptin-deficient ob/ob mice show many characteristics of obesity, including excess peripheral adiposity as well as severe hepatic steatosis, at least in part, due to increased hepatic lipogenesis. Polyunsaturated fatty acids (PUFAs) are not only ligands for peroxisome proliferator-activated receptor (PPAR) but are also negative regulators of hepatic lipogenesis, which is thought to be mediated by the repression of sterol regulatory element-binding protein (SREBP)-1. We have previously shown that the disruption of SREBP-1 in ob/ob mice decreased their liver triglyceride storage. To examine whether PUFAs could reduce hepatic triglyceride deposition, we challenged ob/ob mice with dietary PUFA. It is demonstrated that PUFA markedly decreased the mature form of SREBP-1 protein and thereby reduced the expression of lipogenic genes such as fatty acid synthase (FAS) and stearoyl-CoA desaturase 1 (SCD1) in the livers of ob/ob mice. Consequently, the liver triglyceride content and plasma alanine aminotransferase (ALT) levels were decreased. Furthermore, both hyperglycemia and hyperinsulinemia in ob/ob mice were improved by PUFA administration, similar to the effect of PPAR activators. In conclusion, PUFAs ameliorate obesity-associated symptoms, such as hepatic steatosis and insulin resistance, presumably through both down-regulation of SREBP-1 and activation of PPAR. (HEPATOLOGY 2003;38:1529-1539.)

Inhibition of CK2 activity by TGF-1 promotes IB- protein stabilization and apoptosis of immortalized hepatocytes
Lakita G. Cavin, Raphaelle Romieu-Mourez, Ganesh R. Panta, Jiyuan Sun, Valentina M. Factor, Snorri S. Thorgeirsson, Gail E. Sonenshein, Marcello Arsura
Nuclear factor B (NF-B) is an antiapoptotic factor involved in development, regeneration, and neoplastic progression of the liver. Previously, we have shown that stabilization of inhibitor B (IB)- protein following treatment of hepatocytes with transforming growth factor (TGF)-1 promoted NF-B repression, which then permitted induction of AP-1/SMAD-mediated liver cell death. Because basal IB- protein turnover is regulated by protein kinase CK2, here we have elucidated the regulation of CK2 kinase activity and its role in control of NF-B levels following treatment with TGF-1. We show that both messenger RNA (mRNA) and protein levels of the CK2 catalytic subunit are down-regulated following TGF-1 stimulation in murine hepatocyte cells. The ensuing inhibition of CK2 kinase activity promotes stabilization of IB protein, which is followed by the shutoff of constitutive NF-B activity and induction of apoptosis. Ectopic expression of CK2 inhibits TGF-1-induced apoptosis through sustained activation of NF-B. Conversely, expression of a kinase-dead mutant of CK2 potentiates TGF-1 cell killing. Importantly, we show that hepatocellular carcinomas (HCCs) derived from TGF-1 transgenic mice and human HCC cell lines display enhanced CK2 IB kinase activity that contributes in part to an elevated NF-B activity in vivo. In conclusion, inhibition of CK2 expression levels by TGF-1 is crucial for the induction of apoptosis of hepatocytes. Circumvention of this process by up-regulation of CK2 activity in transformed cells may contribute to the promotion of TGF-1-induced liver carcinogenesis. (HEPATOLOGY 2003;38:1540-1551.)

Growth hormone stimulates proliferation of old-aged regenerating liver through forkhead box m1b
Katherine Krupczak-Hollis, Xinhe Wang, Margaret B. Dennewitz, Robert H. Costa
The Forkhead Box (Fox) proteins are an extensive family of transcription factors that shares homology in the winged helix DNA-binding domain and the members of which play essential roles in cellular proliferation, differentiation, and longevity. Reduced cellular proliferation during aging is associated with a progressive decline in both growth hormone (GH) secretion and Foxm1b expression. Liver regeneration studies with 12-month-old (old-aged) transgenic mice indicated that increased hepatocyte expression of Foxm1b alone is sufficient to restore hepatocyte proliferation to levels found in 2-month-old (young) regenerating liver. GH therapy in older people has been shown to cause an increase in cellular proliferation, but the transcription factors that mediated this stimulation in proliferation remain uncharacterized. In this study, we showed that human GH administration to old-aged Balb/c mice dramatically increased both expression of Foxm1b and regenerating hepatocyte proliferation. This increase in old-aged regenerating hepatocyte proliferation was associated with elevated protein expression of Cdc25A, Cdc25B, and cyclin B1, with reduced protein levels of cyclin-dependent kinase inhibitor p27Kip1 (p27). GH treatment also was found to stimulate hepatocyte proliferation and expression of Foxm1b protein without partial hepatectomy (PHx). Furthermore, GH treatment of young Foxm1b ­/­ mice failed to restore regenerating hepatocyte DNA replication and mitosis caused by Foxm1b deficiency. These genetic studies provided strong evidence that the presence of Foxm1b is essential for GH to stimulate regenerating hepatocyte proliferation. In conclusion, our old-aged liver regeneration studies show that increased Foxm1b levels are essential for GH to stimulate hepatocyte proliferation, thus providing a mechanism for GH action in the elderly. (HEPATOLOGY 2003;38:1552-1562.)

Dexamethasone inhibits early regenerative response of rat liver after cold preservation and transplantation
Fotini Debonera, Alyssa M. Krasinkas, Andrew E. Gelman, Xavier Aldeguer, Xingye Que, Abraham Shaked, Kim M. Olthoff
Regeneration is crucial for the recovery of hepatic mass following liver transplantation. Glucocorticoids, immunosuppressive and antiinflammatory agents commonly used in transplantation, are known to inhibit the expression of specific cytokines and growth factors. Some of these proteins, namely tumor necrosis factor (TNF-) and interleukin 6 (IL-6), play a critical role in the initiation of liver regeneration. Following cold preservation and reperfusion of the transplanted liver, the normal recovery process is marked by increased expression of TNF- and IL-6, followed by activation of cytokine-responsive transcription factors and progression of the cell cycle resulting in hepatocyte proliferation. We hypothesized that glucocorticoids may influence the repair mechanisms initiated after extended cold preservation and transplantation. Using a rat orthotopic liver transplant model, recipient animals were treated with dexamethasone at the time of transplantation of liver grafts with prolonged cold storage (16 hours). Treatment with dexamethasone suppressed and delayed the expression of TNF- and IL-6 compared with animals receiving no treatment and attenuated downstream nuclear factor B (NF-B), signal transduction and activator of transcription 3 (STAT3), and activation protein 1 (AP-1) activation. This suppression was accompanied by poor cell-cycle progression, delayed cyclin D1 nuclear transposition, and impaired hepatocyte proliferation by BrdU uptake. Histologically, the liver grafts in treated animals demonstrated more injury than controls, which appeared to be necrosis, rather than apoptosis. In conclusion, these data provide evidence that the administration of glucocorticoids at the time of transplantation inhibits the initiation of the regenerative process and may have a deleterious effect on the recovery of liver grafts requiring significant regeneration. This may be particularly relevant for transplantation of partial liver grafts in the living donor setting. (HEPATOLOGY 2003;38:1563-1572.)

A key pathogenic role for the STAT1/T-bet signaling pathway in T-cell­mediated liver inflammation
Jürgen Siebler, Stefan Wirtz, Sonja Klein, Martina Protschka, Manfred Blessing, Peter R. Galle, Markus F. Neurath
TH1 cytokines have been suggested to contribute to the pathogenesis of T-cell-mediated liver injury and inflammation. However, the molecular signaling pathways involved in such injury are still poorly understood. In the present study, we investigated the role of the STAT1/T-bet signaling pathway in a murine model of T-cell-mediated liver inflammation induced by the application of concanavalin A (Con A) using newly created STAT1 transgenic mice as well as STAT1- and T-bet-deficient mice. Liver injury induced by Con A was associated with an increase of both pSTAT1 and T-bet levels in the liver. Furthermore, functional studies suggested a pathogenic role for STAT1 in Con A-induced liver injury, because transgenic mice overexpressing STAT1 under the control of the CD2 promoter/enhancer construct showed elevated interferon gamma (IFN-) and IRF-1 levels as well as significantly augmented liver injury following administration of Con A. Consistently, we observed that both STAT1-deficient and T-bet-deficient mice were protected from such T-cell-dependent liver injury. In conclusion, these findings suggest a key pathogenic role for the STAT1/T-bet signaling pathway for T-cell activation in the Con A model of T-cell-mediated liver pathology. (HEPATOLOGY 2003;38:1573-1580.)

Therapy for HDV!
John M. Taylor
Hepatitis delta virus (HDV) can dramatically worsen liver disease in patients coinfected with hepatitis B virus (HBV). No effective medical therapy exists for HDV. The HDV envelope requires HBV surface antigen proteins provided by HBV. Once inside a cell, however, HDV can replicate its genome in the absence of any HBV gene products. In vitro, HDV virion assembly is critically dependent on prenyl lipid modification, or prenylation, of its nucleocapsid-like protein large delta antigen. To overcome limitations of current animal models and to test the hypothesis that pharmacologic prenylation inhibition can prevent the production of HDV virions in vivo, we established a convenient mouse-based model of HDV infection capable of yielding viremia. Such mice were then treated with the prenylation inhibitors FTI-277 and FTI-2153. Both agents were highly effective at clearing HDV viremia. As expected, HDV inhibition exhibited duration-of-treatment dependence. These results provide the first preclinical data supporting the in vivo efficacy of prenylation inhibition as a novel antiviral therapy with potential application to HDV and a wide variety of other viruses.

A new survival trick of hepatitis C virus: Blocking the activation of interferon regulatory factor-3
Markus H. Heim
Persistent infections with hepatitis C virus (HCV) are likely to depend on viral inhibition of host defenses. We show that the HCV NS3/4A serine protease blocks the phosphorylation and effector action of interferon regulatory factor-3 (IRF-3), a key cellular antiviral signaling molecule. Disruption of NS3/4A protease function by mutation or a ketoamide peptidomimetic inhibitor relieved this blockade and restored IRF-3 phosphorylation after cellular challenge with an unrelated virus. Furthermore, dominant-negative or constitutively active IRF-3 mutants, respectively, enhanced or suppressed HCV RNA replication in hepatoma cells. Thus, the NS3/4A protease represents a dual therapeutic target, the inhibition of which may both block viral replication and restore IRF-3 control of HCV infection.

Resistance of HBV to adefovir dipivoxil: A case for combination antiviral therapy?
Costica Aloman, Jack R. Wands
Background & Aims: Adefovir dipivoxil effectively inhibits both hepatitis B virus (HBV) replication and disease activity in patients with chronic hepatitis B. Resistance to treatment was not observed in 2 recent large placebo-controlled 48-week studies with this drug. The aim of this study was to characterize adefovir resistance in a patient who developed clinical and virologic evidence of breakthrough during a 96-week course of treatment. Methods: HBV DNA was PCR amplified and sequenced. Phenotypic studies used patient-derived HBV as well as specific mutations created by site-directed mutagenesis of a HBV/baculovirus recombinant. Results: Following the commencement of treatment with adefovir dipivoxil, the patient initially responded with a 2.4 log10 decrease in serum HBV DNA and normalization of alanine aminotransaminase levels by week 16. During the second year of treatment, however, serum HBV DNA rose progressively, eventually returning to near-pretreatment levels. This increase in viral replication was associated with a marked increase in alanine aminotransferase and mild changes in bilirubin, albumin, and prothrombin time. Comparison of pretreatment and posttreatment HBV DNA by polymerase chain reaction sequencing identified a novel asparagine to threonine mutation at residue rt236 in domain D of the HBV polymerase. In vitro testing of a laboratory strain encoding the rtN236T mutation and testing of patient-derived virus confirmed that the rtN236T substitution caused a marked reduction in susceptibility to adefovir. Conclusions: The development of this novel mutation in the HBV polymerase confers resistance to adefovir dipivoxil. The patient responded to subsequent lamivudine therapy, achieving normalization of alanine aminotransferase and a significant decrease in serum HBV DNA.

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

GASTROENTEROLOGY

Table of Contents for Volume 139, Number 6, December 2003

Clinlcal-alimentary Tract

Survival and cause-specific mortality in ulcerative colitis: Follow-up of a population-based cohort in Copenhagen County
Karen Vanessa Winther, Tine Jess, Ebbe Langholz, Pia Munkholm, Vibeke Binder
Background & Aims: A population-based cohort from Copenhagen County comprising 1160 patients diagnosed with ulcerative colitis between 1962 and 1987 was followed-up until 1997 to describe survival and cause-specific mortality. Methods: Observed vs. expected deaths were presented as standardized mortality ratio (SMR) with exact 95% confidence intervals (CI) calculated by using individually registered person-years at risk and Danish 1995 mortality rates. Cumulative survival curves were calculated. Results: A total of 261 deaths occurred, not significantly different from the expected number of 249 (SMR, 1.05; 95% CI, 0.92­1.19). The median age at death among men was 70 years (range, 6­96 years) and among women 74 years (range, 25­96 years). Twenty-five deaths (9.6%) were caused by complications to ulcerative colitis, mostly infectious and cardiovascular postoperative complications. Patients older than 50 years of age at diagnosis and with extensive colitis showed an increased mortality within the first 2 years because of ulcerative colitis-associated causes. The mortality from colorectal cancer was not increased and that of cancer in general was significantly lower than expected: 50 vs. 71 (SMR, 0.70; 95% CI, 0.52­0.93). A significantly increased mortality from pulmonary embolism and pneumonia was found. Among women only, death from genitourinary tract diseases and suicide was significantly increased. Conclusions: Despite an overall normal life expectancy for patients with ulcerative colitis, patients >50 years of age and with extensive colitis at diagnosis had increased mortality within the first 2 years after diagnosis, owing to colitis-associated postoperative complications and comorbidity.

Mortality in inflammatory bowel disease: A population-based cohort study
Tim Card, Richard Hubbard, Richard F.A. Logan
Background & Aims: There is no consensus regarding any increase in mortality with inflammatory bowel disease (IBD). In general, previous studies were not contemporary and were unable to correct for likely confounders. We have performed a large cohort study to examine contemporary IBD related mortality in the United Kingdom. Methods: We selected subjects within the General Practice Research Database with a coded diagnosis of inflammatory bowel disease and up to 5 matched controls for each. We derived the date of recorded deaths and information on smoking and a variety of medical conditions. We calculated both the absolute risk of death and the relative risk as a hazard ratio corrected for available confounders by Cox regression. Results: We included 16,550 IBD cases with 1047 deaths and 82,917 controls with 3758 deaths. The mortality rate was 17.1 per 1000 person-years overall for IBD cases and 12.3 for controls; this difference was greatest in the elderly. Conversion of these figures to hazard ratios by Cox regression gave hazard ratios of 1.54 (1.44­1.65) for all IBD, 1.44 (1.31­1.58) for ulcerative colitis (UC), and 1.73 (1.54­1.96) for Crohn's disease. The greatest hazard ratio for UC was among the 40­59-year age group (1.79 [1.42­2.27]) and for Crohn's disease among 20­39-year-olds (3.82 [2.17­6.75]). Conclusions: IBD is associated with an overall small increase in mortality rate greatest in relative terms in younger subjects but in absolute terms in the elderly.

Inflammatory bowel disease and the risk of fracture
Tjeerd-Pieter Van staa, Cyrus Cooper, Listy Samuels Brusse, Hubert Leufkens, Muhammad K. Javaid, Nigel K. Arden
Background & Aims: Although patients with inflammatory bowel disease (IBD) have reduced bone mass, there is controversy whether there is an increased risk of fracture. This study examines the risk of fracture and its predictors in patients with IBD.Methods: In a primary care- based nested case-control study, 231,778 fracture cases and 231,778 age- and sex-matched controls were recruited. A history of IBD was assessed from medical records. Results: The prevalence of IBD was 156 and 282 per 100,000 for Crohn's disease (CD) and ulcerative colitis (UC), respectively. Patients with IBD had an increased risk of vertebral fracture (odds ratio [OR], 1.72; 95% confidence interval [CI], 1.13­2.61) and hip fracture (OR, 1.59; 95% CI, 1.14­2.23). The risk of hip fracture was greater in patients with CD (OR, 1.86; 95% CI, 1.08­3.21) compared with UC (OR, 1.40; 95% CI, 0.92­2.13). Disease severity, assessed by the number of symptoms, predicted fracture even after adjusting for corticosteroid use (OR, 1.46; 95% CI, 1.04­2.04). Only 13% of patients with IBD who had already sustained a fracture were on any form of antifracture treatment. Conclusions: Patients with IBD have a higher risk of fracture due to both disease activity and use of oral corticosteroids. However, few of these patients are receiving optimal bone-sparing therapy, highlighting the importance of increasing awareness of osteoporosis in those managing these patients.

Subclinical intestinal inflammation and sacroiliac changes in relatives of patients with ankylosing spondylitis
Ingvar Bjarnason, Kristjan O. Helgason, Árni J. Geirsson, Gudmundur Sigthorsson, Inga Reynisdottir, Daniel Gudbjartsson, Anna S. Einarsdottir, Roy Sherwood, Kristleifur Kristjansson, Ólafur Kjartansson, Bjarni Thjodleifsson
Background & Aims: It has been suggested that subclinical intestinal inflammation plays a pathogenic role in the spondylarthropathy of ankylosing spondylitis (AS). We assessed the possible presence and inheritance pattern of subclinical intestinal inflammation in first-degree relatives of patients with AS. The relationship between this inflammation and the subjects' HLA-B27 genotype as well as computerized tomographic sacroiliac abnormalities was also assessed. Methods: A total of 124 of 213 (58%) available first-degree relatives of 47 patients with AS in Iceland underwent investigation for intestinal inflammation (fecal calprotectin concentration), HLA-B27 genotyping, and computerized tomography of the sacroiliac joints. Results: A total of 41% of the first-degree relatives had subclinical intestinal inflammation, whereas 15 of 17 spouses were normal. Variance components analyses suggest that the inheritance pattern of this inflammation is affected by a major additive gene. Some sacroiliac changes, suggestive of early AS, differed significantly between subjects with and without subclinical intestinal inflammation (mean diameter of subchondral cysts [2.9 vs. 1.2 mm; P = 0.026] and blurring of joint margins [9 of 44 (20%) vs. 1 of 41 (2%); P = 0.02]). Intestinal inflammation and sacroiliac changes did not relate to the subjects' HLA-B27 status. Conclusions: Many first-degree relatives of patients with AS appear to have an inherited abnormality that leads to subclinical intestinal inflammation. The association between the presence of this inflammation and the sacroiliac changes suggests that it may play a pathogenic role in the spondylarthropathy of AS.

Unsedated ultrathin EGD is well accepted when compared with conventional sedated EGD: A multicenter randomized trial
Ruel T. Garcia, John P. Cello, Mindie H. Nguyen, Stanley J. Rogers, Alex Rodas, Huy N. Trinh, Neil H. Stollman, Gail Schlueck, Kenneth R. McQuaid
Background & Aims: In the United States, upper gastrointestinal endoscopy is usually performed using intravenous sedation. Sedation increases the rate of both complications and costs of endoscopy. Unsedated esophagogastroduodenoscopy (EGD) using conventional 8­11-mm endoscopes is an alternative to sedated endoscopy but is generally perceived as unacceptable to many American patients. Unsedated EGD using ultrathin 5­6-mm endoscopes is better tolerated. A randomized trial comparing unsedated ultrathin EGD (UT-EGD) with sedated conventional EGD (C-EGD) in a diverse American population is needed. Methods: In this multicenter, randomized, controlled trial, 80 patients scheduled to undergo elective outpatient EGD were randomized to unsedated UT-EGD or sedated C-EGD. The study was carried out at San Francisco General Hospital, San Francisco Veterans Affairs Medical Center, and the Liver and Digestive Health Medical Clinic, San Jose. Results: Baseline characteristics of patients randomized to unsedated UT-EGD and sedated C-EGD were similar. Moreover, there were no significant differences in overall patient satisfaction and willingness to repeat endoscopy in the same manner among the 2 study groups. There was, however, a significant difference in median total procedure time between the 2 study groups of 1.5 hours (P < 0.0001). The mean (± SD) total procedure cost was $512.4 (± $100.8) for sedated C-EGD and $328.6 (± $70.3) for unsedated UT-EGD (P < 0.0001). Conclusions: Patients undergoing unsedated UT-EGD are as satisfied as patients undergoing sedated C-EGD and are just as willing to repeat an unsedated UT-EGD. Unsedated UT-EGD was also faster, less costly, and may allow greater accessibility to this procedure.

Expression of LL-37 by human gastric epithelial cells as a potential host defense mechanism against Helicobacter pylori
Koji Hase, Masamoto Murakami, Mitsutoshi Iimura, Sheri P. Cole, Yoshimune Horibe, Takaaki Ohtake, Marygorret Obonyo, Richard L. Gallo, Lars Eckmann, Martin F. Kagnoff
Background & Aims : LL-37/human cationic antimicrobial peptide 18 (hCAP18) is a human cathelicidin with broad-spectrum antimicrobial, lipopolysaccharide binding, and chemotactic activities. This study examined the role of LL-37/hCAP18 in gastric innate immune defense by characterizing its constitutive and regulated expression by human gastric mucosa and its bactericidal activity against the gastric pathogen Helicobacter pylori . Methods : LL-37/hCAP18 messenger RNA expression in normal and H. pylori -infected gastric mucosa and gastric epithelial cells was determined by in situ hybridization, real-time polymerase chain reaction, immunostaining, and immunoblot analysis. Bactericidal activity was measured by using a colony-forming unit assay. Results : LL-37/hCAP18 messenger RNA and protein were expressed in a distinct distribution by surface epithelial cells as well as chief and parietal cells in the fundic glands of normal gastric mucosa. LL-37/hCAP18 was significantly increased in the epithelium and gastric secretions of H. pylori -infected patients, but not in individuals with non-H. pylori -induced gastric inflammation. Infection of cultured gastric epithelial cells with a wild-type but not an isogenic cagE mutant strain of H. pylori increased LL-37/hCAP18 expression, indicating that H. pylori -induced regulation of LL-37/hCAP18 production required an intact type IV secretion system. LL-37, the C-terminal peptide of LL-37/hCAP18, alone or in synergy with human -defensin 1, was bactericidal for several H. pylori strains. Conclusions : These data indicate that H. pylori up-regulates production of LL-37/hCAP18 by gastric epithelium and suggest this cathelicidin contributes to determining the balance between host mucosal defense and H. pylori survival mechanisms that govern chronic infection with this gastric pathogen.

Impact of lymph node staging on therapy of esophageal carcinoma
Enrique Vazquez-Sequeiros, Maurits J. Wiersema, Jonathan E. Clain, Ian D. Norton, Michael J. Levy, Yvonne Romero, Diva Salomao, Ross Dierkhising, Alan R. Zinsmeister
Background & Aims: Therapy of esophageal carcinoma is stage dependent. The role of EUS-guided fine-needle aspiration (EUS FNA) in this setting is unclear. The aims of this study were to compare the performance characteristics of CT, EUS, and EUS FNA for preoperative nodal staging of esophageal carcinoma and to measure the impact of each staging test on treatment decisions. Methods: From December 1999 to March 2001, all patients with esophageal carcinoma seen at the Mayo Clinic Rochester were prospectively evaluated with CT, EUS, and EUS FNA. The impact of tumor stage on final therapy was assessed. Results: A total of 125 patients with esophageal carcinoma were enrolled. EUS FNA was more sensitive (83% vs. 29%; P < 0.001) than CT and more accurate than CT (87% vs. 51%; P < 0.001) or EUS (87% vs. 74%; P = 0.012) for nodal staging. Direct surgical resection was contraindicated in 77% of patients evaluated due to advanced locoregional/metastatic disease. Tumor location, patient age, comorbidities, and tumor stage determined by CT, EUS, and EUS FNA were associated with treatment decisions (P < 0.05). EUS FNA resulting in a higher/worse stage than CT (41 patients) was associated with a greater rate of treatments that were not direct surgeries compared with cases in which the stage was the same or better. Conclusions: EUS FNA is more accurate for nodal staging and impacts on therapy of patients with esophageal carcinoma. EUS FNA should be included in the preoperative staging algorithm of these patients.

Meta-analysis of the relationship between cagA seropositivity and gastric cancer
Jia Qing Huang, Ge Fan Zheng, Katica Sumanac, E.Jan Irvine, Richard H. Hunt
Background & Aims : Reports in the literature regarding the relationship of infection with cagA -positive strains of Helicobacter pylori to gastric cancer over and above H. pylori infection alone are conflicting. The aim of this study was to estimate the magnitude of the risk for gastric cancer associated with cagA seropositivity and to identify any sources of heterogeneity between studies. Methods : A meta-analysis of case-control studies with age- and sex-matched controls, which provided raw data on the infection rates with H. pylori and cagA strains of H. pylori as detected by serology or polymerase chain reaction DNA, was performed. Results : A comprehensive literature search identified 16 qualified studies with 2284 cases and 2770 controls. H. pylori and cagA seropositivity significantly increased the risk for gastric cancer by 2.28- and 2.87-fold, respectively. Among H. pylori -infected populations, infection with cagA -positive strains further increased the risk for gastric cancer by 1.64-fold (95% confidence interval [CI], 1.21­2.24) overall and by 2.01-fold (95% CI, 1.21­3.32) for noncardia gastric cancer. Gastric cancer at the cardia is not associated with H. pylori infection or cagA -positive strains of H. pylori . Patient age and site of gastric cancer contributed to the heterogeneity between studies. Conclusions : Infection with cagA -positive strains of H. pylori increases the risk for gastric cancer over the risk associated with H. pylori infection alone. Searching for cagA status over H. pylori infection may confer additional benefit in identifying populations at greater risk for gastric cancer.

Cancer-attributable costs of diagnosis and care for persons with screen-detected versus symptom-detected colorectal cancer
Scott D. Ramsey, Margaret T. Mandelson, Kristin Berry, Ruth Etzioni, Robert Harrison
Background & aims: Colorectal cancer screening is effective and cost-effective, but little data from health plan settings are available inform decision-makers regarding direct economic implications of colorectal cancer screening programs. The purpose of this study was to compare the prediagnosis evaluation and first-year treatment costs of persons diagnosed with colorectal cancer, stratified by whether the cancer was detected by screening using fecal occult blood testing or evaluation of symptoms. Methods: This retrospective study analyzed personsdiagnosed with colorectal cancer from 1993 to 1999 in Group Health Cooperative, a large health maintenance organization in Washington state. Total health care costs during 3 months before and 12 months following diagnosis were compared for screen-detected versus symptom-detected individuals. Results: During thistime, 206 cancers were detected by screening and 717 by symptoms. In the 3 months before diagnosis, total costs were $7346 for persons with screen-detected versus $10,042 for those with symptom-detected cancer (P < 0.01). Stratified by stage, diagnosis costs were significantly lower for persons with stage B cancer ($7282 vs. $11,682; P < 0.01) and nonsignificantly lower for other stages. A total of 53% of screen-detected cases were Dukes' stage A or in situ at diagnosis versus 30% of symptom-detected cases (P < 0.01). Overall costs were lower for the screen-detected group in the 12 months following diagnosis ($22,369 vs. $29,471; P < 0.01). Conclusions: Colorectal cancer screening can substantially reduce prediagnosisevaluation costs. These savings are of interest to health plans and should be factored into cost-effectiveness evaluations of screening programs.

Relative importance of enterochromaffin cell hyperplasia, anxiety, and depression in postinfectious IBS
Simon P. Dunlop, David Jenkins, Keith R. Neal, Robin C. Spiller
Background & Aims: Both psychological and mucosal changes (increased enterochromaffin [EC] cells and T lymphocytes) have been associated with postinfectious irritable bowel syndrome (PI-IBS). However, previous studies have been underpowered to determine the relative importance of these changes in predicting the development of PI-IBS. Our aim was to prospectively determine the relative importance of both psychological and histologic factors in the development of PI-IBS after Campylobacter infection. Methods: Questionnaires detailing psychological and bowel symptoms were sent to 1977 patients 3 months after infection. Twenty-eight patients with new-onset PI-IBS, 28 age- and sex-matched patient controls who were asymptomatic after infection, and 34 healthy volunteers underwent rectal biopsy, which was assessed for serotonin-containing EC cells, mast cells, and lamina propria T lymphocytes. Results: PI-IBS, predominantly of the diarrhea-predominant subtype, occurred in 103 of 747 (13.8%) of those infected. EC cell counts per high-power field (hpf) were higher in patients with PI-IBS (35.8 ± 1.2) compared with patient controls (30.6 ± 1.9; P = 0.022) and volunteers (29.1 ± 1.8; P = 0.006). Lamina propria T lymphocytes per hpf were higher in patients with PI-IBS (127.1 ± 8.7) and patient controls (113.4 ± 6.2) in contrast to healthy volunteers (97.1 ± 5.7) (P = 0.006 and P = 0.058, respectively). Anxiety, depression, and fatigue were significantly increased in patients with PI-IBS compared with patient controls. Multivariate analysis indicated that increased EC cell counts and depression were equally important predictors of developing PI-IBS (relative risk, 3.8 and 3.2 for each standard deviation increase in respective values). Conclusions: Both increased EC cells and depression are important independent predictors of developing PI-IBS.

Natural history of primary eosinophilic esophagitis: A follow-up of 30 adult patients for up to 11.5 years
Alex Straumann, Hans-peter Spichtin, Leticia Grize, Kathleen A. Bucher, Christoph Beglinger, Hans-uwe Simon
Background & Aims: Primary eosinophilic esophagitis is a chronic, increasingly recognized, interleukin 5-driven inflammatory disorder of the esophagus. The leading symptom in adults is uniform attacks of dysphagia, and the established histologic sign is a dense eosinophilic infiltration of the esophageal epithelium. Before this study, the natural course of eosinophilic esophagitis had not been defined and information regarding potential long-term risks was lacking. Methods: This prospective case series included 30 adult patients with eosinophilic esophagitis (22 men and 8 women; mean age, 40.6 years) whose diagnosis had been made >1 year before study debut based on typical history, consistent endoscopic abnormalities, and infiltration of the esophageal epithelium with >24 eosinophils/high-power field. After a mean of 7.2 years, patients underwent a comprehensive follow-up examination. Results: All patients survived the study period in good health and stable nutritional state. Dysphagia persisted in 29 patients, exerting a major negative effect on socioprofessional activities on 1 patient and a minor impact on 15. Attacks of dysphagia were more frequent in patients with blood eosinophilia or pronounced endoscopic alterations. The esophageal eosinophilic infiltration persisted in all symptomatic patients, but cell numbers spontaneously decreased significantly (78.7 vs. 40.3 cells/high-power field). The inflammatory process evoked fibrosis of the esophageal lamina propria but did not spread to the stomach or duodenum. No case evolved to a hypereosinophilic syndrome. Conclusions: Eosinophilic esophagitis, a primary and chronic disease restricted to the esophagus, leads to persistent dysphagia and structural esophageal alterations but does not impact the nutritional state. To date, no malignant potential has been associated with this disease.

Screening for Barrett's esophagus in colonoscopy patients with and without heartburn
Douglas K. Rex, Oscar W. Cummings, Michael Shaw, Mark D. Cumings, Roy K.H. Wong, Raj S. Vasudeva, Donal Dunne, Emad Y. Rahmani, Debra J. Helper
Background & Aims : The population prevalence of Barrett's esophagus (BE) is uncertain. Our aim was to describe the prevalence of BE in a volunteer population. Methods : Upper endoscopy (EGD) was performed in 961 persons with no prior history of EGD who were scheduled for colonoscopy. Symptom questionnaires were completed prior to endoscopy. Biopsy specimens were taken from the gastric cardia and any columnar mucosa extending 5 mm into the tubular esophagus and from the stomach for H. pylori infection in the last 812 patients. Results : The study sample was biased toward persons undergoing colonoscopy, males, and persons with upper GI symptoms. The prevalence of BE was 65 of 961 (6.8%) patients, including 12 (1.2%) with long-segment BE (LSBE). Among 556 subjects who had never had heartburn, the prevalences of BE and LSBE were 5.6% and 0.36%, respectively. Among 384 subjects with a history of any heartburn, the prevalences of BE and LSBE were 8.3% and 2.6%, respectively. In a univariate analysis, LSBE was more common in those with any heartburn vs. those with no heartburn (P = 0.01), but the sample size was insufficient to allow multivariate analysis of predictors of LSBE. In a multivariate analysis, BE was associated with increasing age (P = 0.02), white race (P = 0.03), and negative H. pylori status (P = 0.04). Overall, BE was not associated with heartburn, although heartburn was more common in persons with LSBE or circumferential short segments. Conclusions : LSBE is very uncommon in patients who have no history of heartburn. SSBE is relatively common in persons age 40 years with no prior endoscopy, irrespective of heartburn history.

Congenital sucrase-isomaltase deficiency because of an accumulation of the mutant enzyme in the endoplasmic reticulum
Valentina Ritz, Marwan Alfalah, Klaus-peter Zimmer, Jacques Schmitz, Ralf Jacob, Hassan Y. Naim
Background & Aims: Congenital sucrase-isomaltase deficiency (CSID) is an autosomal recessive human disorder characterized by reduced activities of the brush border enzyme sucrase-isomaltase (SI). Here, we elucidate the pathogenesis of a new variant of CSID at the cellular and molecular level. Methods: Assessment of the CSID phenotype was achieved by enzymatic activity measurements, biosynthetic labeling of intestinal biopsy specimens, immunoprecipitation of SI, and immunoelectronmicroscopy. The putative mutation was identified by sequencing of the SI cDNA isolated by RT-PCR from intestinal biopsy samples. The function of the mutation was verified by immunoprecipitation and confocal microscopy of transiently transfected cells. Results: Biosynthetic labeling and immunoelectron microscopy reveal a predominant localization of SI in the endoplasmic reticulum (ER) similar to phenotype I of CSID. Unlike phenotype I, however, a partial conversion of SI to a complex glycosylated mature form takes place. The SI cDNA in this phenotype revealed 3 mutations, 2 of which, Val to Phe at residue 15 and Ala to Thr at residue 231, had no effect on the structure or function of SI. By contrast, the third mutation resulted in an exchange of leucine by proline at position 620 (L620P) and revealed in transfected COS cells structural features and subcellular localization similar to the phenotype identified in the patient's enterocytes. Conclusions: This is the first identification at the molecular and subcellular levels of a novel variant of CSID in which SI accumulates predominantly in the ER, and a minor proportion is further processed and transported to the apical membrane of enterocytes.

An upstream polymorphism associated with lactase persistence has increased enhancer activity
Jesper T. Troelsen, Jørgen Olsen, Jette Møller, Hans SjÖstrÖm
Background & Aims: Intestinal lactase activity declines during childhood in some humans. This phenotypic polymorphism of lactase persistence or nonpersistence into adult life has been shown in a recent study to be 100% associated with a T/C nucleotide polymorphism at position ­13910 and approximately 97% with an A/G nucleotide polymorphism at position ­22018. The aim of this study was to investigate the role of these nucleotide polymorphisms for lactase-phlorizin hydrolase (LPH) gene expression. Methods: The ­13910 and ­22018 regions were cloned from lactase-persistent and -nonpersistent individuals, and the regions were analyzed for gene regulatory activity of a luciferase reporter gene by transfection experiments using the intestinal cell line Caco-2. Electrophoretic mobility shift assays (EMSAs) were used to investigate protein/DNA interactions with the ­13910 sequence. Results: We show that the ­13910 region contains a strong enhancer. The ­13910 regions from both lactase persistent (­13910T variant) and lactase nonpersistent (­13910C variant) have enhancer activity. However, the ­13910T variant enhances the LPH promoter approximately 4 times more than the ­13910C variant when analyzed in differentiated Caco-2 cells. A nuclear factor from both an intestinal and a nonintestinal extract binds strongly to the ­13910T variant whereas the binding to the ­13910C variant is much weaker. Conclusions: The discovery of a functional difference between the 2 alleles at position ­13910 supports the notion that the molecular difference between lactase persistence and nonpersistence is caused by the mutation at position ­13910.

Insulin resistance is associated with chronic hepatitis C and virus infection fibrosis progression
Jason M. Hui, Archana Sud, Geoffrey C. Farrell, Priyanka Bandara, Karen Byth, James G. Kench, Geoffrey W. McCaughan, Jacob George
Background & Aims: Chronic hepatitis C virus infection is associated with an increased prevalence of type 2 diabetes. We hypothesized that virus-induced insulin resistance may be a mechanism for fibrogenesis in chronic hepatitis C virus infection. Methods: In 260 hepatitis C virus-infected subjects, we examined the relationship between histological findings and anthropometric and biochemical data, including insulin resistance determined by the homeostasis model assessment (HOMA-IR). We also compared fasting serum insulin, C peptide, and HOMA-IR levels between the subset of 121 hepatitis C virus patients with stage 0 or 1 hepatic fibrosis and 137 healthy volunteers matched by sex, body mass index, and waist-hip ratio. Results: Hepatitis C virus-infected subjects with stage 0 or 1 hepatic fibrosis had higher levels of insulin, C peptide, and HOMA-IR (all P 0.01) compared with matched healthy controls. In the 250 hepatitis C virus patients (fibrosis stage 0 to 4), viral genotype and portal, but not lobular, inflammation were univariate predictors of HOMA-IR. By multiple linear regression analysis, independent predictors of HOMA-IR included body mass index (P < 0.001), previous failed antiviral treatment (P < 0.001), portal inflammatory grade (P < 0.001), and genotype 3 status (P = 0.01). Genotype 3 had significantly lower HOMA-IR than other genotypes (which were comparable when adjusted for effects of the remaining independent predictors). HOMA-IR was an independent predictor for the degree of fibrosis (P < 0.001) and the rate of fibrosis progression (P = 0.03). Conclusions: Hepatitis C virus may induce insulin resistance irrespective of the severity of liver disease, and this effect seems to be genotype specific. Further, our findings support the hypothesis that insulin resistance may contribute to fibrotic progression in chronic hepatitis C virus infection.

Autoreactivity to lipoate and a conjugated form of lipoate in primary biliary cirrhosis
Sylvaine F.A. Bruggraber, Patrick S.C. Leung, Katsushi Amano, Chao Quan, Mark J. Kurth, Michael H. Nantz, Gordon D. Benson, Judy Van de Water, Velimer Luketic, Thomas E. Roche, Aftab A. Ansari, Ross L. Coppel, M.Eric Gershwin
Background & Aims: Although considerable effort has been directed toward the mapping of peptide epitopes by autoantibodies, the role of nonprotein molecules has been less well studied. The immunodominant autoantigen in primary biliary cirrhosis (PBC), E2 components of pyruvate dehydrogenase complexes (PDC-E2), has a lipoate molecule bonded to the domain to which autoantibodies are directed. Methods: We examined sera from patients with PBC (n = 105), primary sclerosing cholangitis (n = 70), and rheumatoid arthritis (n = 28) as well as healthy volunteers (n = 43) for reactivity against lipoic acid. The lipoic acid hapten specificity of the reactive antibodies in PBC sera was determined following incubation of aliquots of the sera with human serum albumin (HSA), lipoylated HSA (HSA-LA), PDC-E2, lipoylated PDC-E2, polyethylene glycol (PEG), lipoylated PEG, free lipoic acid, and synthetic molecular mimics of lipoic acid. Results: Anti-lipoic acid specific antibodies were detected in 81% (79 of 97) of antimitochondrial antibody (AMA)-positive patients with PBC but not in controls. Two previously unreported specificities in AMA-positive sera that recognize free lipoic acid and a carrier-conjugated form of lipoic acid were also identified. Conclusions: We hypothesize that conjugated form(s) of native or xenobiotic lipoic acid mimics contribute to the initiation and perpetuation of autoimmunity by at first breaking self-tolerance and participating in subsequent determinant spreading. The variability in the immunoreactive carrier/lipoate conjugates provides an experimental framework on which potential mechanisms for the breakdown of self-tolerance following exposure to xenobiotics can be investigated. The data have implications for patients taking lipoic acid as a dietary supplement.

Long-term safety of lamivudine treatment in patients with chronic hepatitis B
Anna S.F. Lok, Ching-Lung Lai, Nancy Leung, Guang-Bi Yao, Zhen-Yu Cui, Eugene R. Schiff, Jules L. Dienstag, E. Jenny Heathcote, Nancy R. Little, Dorothea A. Griffiths, Stephen D. Gardner, Mary Castiglia
Background & aims: Data on thelong-term safety of lamivudine are limited. The aim of this analysis was to determine the incidence of hepatitis flares, hepatic decompensation, and liver-disease-related (LDR) serious adverse events (SAE) during long-term lamivudine treatment. Methods: We reviewed data on 998 patients with HBeAg-positive compensated chronic hepatitis B who received lamivudine for up to 6 years (median, 4 years) and 200 patients who received placebo for 1 year.Results: Hepatitis flares occurred in 10% of thelamivudine-treated patients in year 1 and in 18%­21% in years 2­5. A temporal association between hepatitis flares and lamivudine-resistant mutations increased from 43% in year 1 to >80% in year 3. Ten hepatic decompensation events occurred in 8 (<1%) lamivudine-treated patients. Fifty-three (5%) lamivudine-treated patients experienced a total of 60 LDR SAEs. Four patients died, 2 from liver-related causes. The proportion of patients with a documented lamivudine-resistant mutation increased from 23% in year 1 to 65% in year 5. During each year of the study, patients with lamivudine-resistant mutations experienced significantly more hepatitis flares than patients without lamivudine-resistant mutations (P < 0.005). The occurrence of hepatic decompensation (0%­2%) and LDR SAEs (1%­10%) among patients with lamivudine resistance remained stable during the first 4 years with mutations and increased afterward to 6% (P = 0.03) and 20% (P = 0.009), respectively. Conclusions: This study demonstrated that lamivudine treatment for up to 6 years has an excellent safety profile in patients with HBeAg-positive compensated liver disease, but patients with long-standing lamivudine-resistant mutations may experience worsening liver disease.

Prevalence of hepatitis C virus infection in B-cell non-Hodgkin's lymphoma: Systematic review and meta-analysis
Javier P. Gisbert, Luisa Garca-Buey, José Mara Pajares, Ricardo Moreno-Otero
Background & Aims: The aim of our study was to conduct a systematic review of studies evaluating prevalence of hepatitis C virus (HCV) infection in B-cell non-Hodgkin's lymphoma (B-NHL) and to perform a meta-analysis of case-control studies comparing this prevalence with that of a reference group.Methods: Data sources: Electronic databases and the Cochrane Controlled Trials Register. Study selection: Studies evaluating prevalence of HCV infection in patients with B-NHL. Studies comparing HCV prevalence in B-NHL (cases) and in a reference group (controls) were included in the meta-analysis. Data extraction: Author/country, diagnostic method (serology/PCR), control type, matching/design, and VHC prevalence. Data synthesis: Prevalence of HCV infection and meta-analysis combining the odds ratios (OR).Results: Forty-eight studies (5542 patients) were identified. Mean HCV infection prevalence was 13% (95% CI: 12%­14%), which was higher in Italy (20%) and Japan (14%). Ten studies compared HCV prevalence in B-NHL (17%) and healthy controls (1.5%) (OR: 10.8; 95% CI: 7.4­16), results being homogeneous; OR increased up to 14.1 when only Italian studies were considered. Sixteen studies compared HCV prevalence in B-NHL (13%) and in other hematologic malignancies (2.9%) (OR: 4.2; 95% CI: 2.5­7), also with homogeneous results; OR increased up to 7.8 when subanalysis included only Italian studies.Conclusions: HCV prevalence in patients with B-NHL is approximately 15%, higher than that reported not only in general population (1.5%) but also in patients with other hematologic malignancies (2.9%), suggesting a role of HCV in the etiology of B-NHL. The striking geographic variation in this association suggests that genetic and/or environmental factors are also involved in the pathogenesis of this disorder.

Cancer risk in patients with hereditary hemochromatosis and in their first-degree relatives
Maria Elmberg, Rolf Hultcrantz, Anders Ekbom, Lena Brandt, Sigvard Olsson, Rolf Olsson, Stefan Lindgren, Lars Lööf, Per Stål, Sven Wallerstedt, Sven Almer, Hanna Sandberg-Gertzén, Johan Askling
Background & Aims: Iron overload may be carcinogenic. Patients with hereditary hemochromatosis (HH) are reportedly at a 20­200-fold risk of intrahepatic cancer, but the reported risks for nonhepatobiliary cancers are conflicting. The risk of cancer in heterozygous individuals (estimated allele frequency, 1/10 to 1/20) is unknown. This study aimed to better assess these risks. Methods: We performed a population-based cohort study of 1847 Swedish patients with HH and 5973 of their first-degree relatives using nationwide, population-based health and census registers. We used standardized incidence ratios (SIRs) as relative risk. Results: With 62 liver cancers and 128 nonhepatobiliary cancers, patients with HH were at a 20-fold risk of liver cancer (SIR, 21; 95% confidence interval [CI], 16­22) but an almost unaltered risk of all other cancers (SIR, 1.2; 95% CI, 1.0­1.4), including nonelevated risks for several gastrointestinal tract cancers. At 10 years of follow-up, the absolute risk of liver cancer was 6% among men and 1.5% among women. With 21 liver cancers and 508 nonhepatobiliary cancers, first-degree relatives were at an unaltered risk of extrahepatic cancer (SIR, 1.0; 95% CI, 0.9­1.1, including unelevated risks for gastrointestinal cancers) but at a modest and historic increased risk of hepatobiliary cancer (SIR, 1.5; 95% CI, 1.0­2.4), the histopathologic spectrum of which differed from the patients. Conclusions: Patients (particularly men) with HH are at increased risk for hepatocellular cancer, although the magnitude of the risk is lower than previous estimates. Overall cancer risk in first-degree relatives does not seem to be increased.

Early is superior to deferred preemptive lamivudine therapy for hepatitis B patients undergoing chemotherapy
George K.K. Lau, Harry H.Y. Yiu, Daniel Y.T. Fong, Hoi-Ching Cheng, Wing-Yan Au, Lydia S.F. Lai, Micheal Cheung, Hai-Ying Zhang, Albert Lie, Roger Ngan, Raymond Liang
Background & Aims: Hepatitis B virus reactivation is a serious cause of morbidity and mortality in hepatitis B surface antigen-positive patients treated with chemotherapy. We compared the efficacy of early and deferred preemptive lamivudine therapy in reducing the incidence of hepatitis due to hepatitis B virus reactivation in hepatitis B surface antigen-positive lymphoma patients treated with chemotherapy.Methods: Thirty consecutive hepatitis B surface antigen-positive lymphoma patients undergoing intensive chemotherapy were randomized (1:1) to receive lamivudine 100 mg daily 1 week before chemotherapy (group 1) or to have this treatment deferred until there was serological evidence of hepatitis B virus reactivation on the basis of serial 2-week-interval serum hepatitis B virus DNA monitoring by a Digene Hybrid Capture II assay (group 2).Results: Eight (53%) patients in group 2 and none in group 1 had hepatitis B virus virological reactivation after chemotherapy (P = 0.002). Seven patients in group 2 still had hepatitis (5 anicteric hepatitis, 1 icteric hepatitis, and 1 hepatic failure). Survival free from hepatitis due to hepatitis B virus reactivation in group 1 patients was significantly longer than that in group 2 (P = 0.002 on the log-rank test). The median onset of hepatitis B virus reactivation in these patients was 16 weeks (range, 4­36 weeks) after the initiation of chemotherapy. Three (13%) of the 23 patients treated with lamivudine had hepatitis B virus-related hepatitis after lamivudine withdrawal.Conclusions: Lamivudine should be considered preemptively before or at the initiation of chemotherapy for all hepatitis B surface antigen-positive lymphoma patients undergoing intense chemotherapy.

Basic-alimentary Tract

A murine model of chronic inflammation-induced intestinal fibrosis down-regulated by antisense NF-B
Ian C. Lawrance, Feng Wu, AndrÉ Z.A. Leite, Joseph Willis, Gail A. West, Claudio Fiocchi, Shukti Chakravarti
Background & Aims: To elucidate extracellular matrix (ECM) changes underlying intestinal fibrosis, a frequent complication of inflammatory bowel disease, we developed a murine model of chronic colitis associated with intestinal fibrosis. Methods: Chronic inflammation was established by weekly intrarectal administration of trinitrobenzene sulfonic acid (TNBS). In 2 variations of the model an antisense oligonucleotide for nuclear factor B (NF-B) p65 was given prophylactically or therapeutically to block chronic inflammation-associated fibrosis. Colonic inflammation and fibrosis were determined by histology. Total collagen level was estimated by hydroxyproline quantification. Colonic expression of collagens (Col1a2, Col3a2), ECM remodeling genes (matrix metalloproteinase [MMP]-1, -3, and tissue inhibitor of matrix metalloproteinase [TIMP]-1), and inflammation-modulating cytokines (tumor necrosis factor [TNF-], interferon [IFN-], transforming growth factor 1 [TGF-1], and insulin-like growth factor 1 [IGF-1]) were assessed by semiquantitative reverse-transcription polymerase chain reaction. Control and TNBS-treated colonic mesenchymal cells were characterized by morphology, phenotype, and functional response to TNF- and IFN-. Results: Colons of TNBS-treated mice contained acute and chronic inflammatory infiltrates, increased collagen, fibrogenic tissue architecture, and increased expression of TNF-, TGF-1, IGF-1, Col1a2, MMP-1, and TIMP-1. Colonic mesenchymal cells from TNBS-treated mice were also morphologically distinct from those of the control mice, with increased TIMP-1 expression in response to IFN- treatment. Fibrosis persisted for 2­4 weeks after cessation of the TNBS treatment. In mice given NF-B antisense prophylactically, 67% were fibrosis-free, whereas of those treated after establishing chronic inflammation, 43% were free of fibrosis. Conclusions: Extended TNBS treatment of mice yielded chronic intestinal inflammation-associated fibrosis with extensive fibrogenic ECM changes that could be counteracted by specific blockade of NF-B.

Inhibition of indoleamine 2,3-dioxygenase augments trinitrobenzene sulfonic acid colitis in mice
Gregory J. Gurtner, Rodney D. Newberry, Suzanne R. Schloemann, Keely G. McDonald, William F. Stenson
Background & Aims: Indoleamine 2,3-dioxygenase (IDO), an interferon -induced intracellular enzyme, inhibits lymphocyte proliferation through tryptophan degradation. IDO is highly expressed in the mammalian intestine. We sought to determine whether IDO played a regulatory role in the T-cell helper 1 (Th1)-mediated trinitrobenzene sulfonic acid (TNBS) model of colitis. Methods: Intrarectal TNBS was given to SJL/J mice along with either placebo or a specific IDO inhibitor. IDO protein and mRNA expression were assessed by Western blotting and real-time PCR. Colonic lamina propria mononuclear cells (LPMNCs) were isolated, fractionated, and cultured, in the presence and absence of IFN-, to determine the cell type(s) expressing IDO.Results: IDO is expressed by professional antigen-presenting cells in the lamina propria. Induction of TNBS colitis resulted in a significant increase in IDO mRNA (P = 0.005) and protein expression. IDO inhibition during TNBS colitis resulted in an 80% mortality compared with 10% for placebo-treated animals (P = 0.0089). IDO inhibition resulted in a more severe colitis both histologically and morphologically (P < 0.05) and significantly increased colonic proinflammatory cytokine expression compared with placebo-treated animals.Conclusions: IDO is expressed in the normal colon and is up-regulated in the setting of TNBS colitis. Inhibition of IDO during TNBS colitis resulted in increased mortality and an augmentation of the normal inflammatory response. These findings suggest that IDO plays an important role in the down-regulation of Th1 responses within the gastrointestinal tract.

Impaired gastric secretion and lack of trophic responses to hypergastrinemia in M3 muscarinic receptor knockout mice
Takeshi Aihara, Teruaki Fujishita, Keiko Kanatani, Kazuharu Furutani, Eiji Nakamura, Makoto M. Taketo, Minoru Matsui, Duan Chen, Susumu Okabe
Background & Aims: The physiologic significance of the M3 muscarinic receptor is unclear due to an absence of specific ligand. In the present study, M3 receptor knockout (KO) mice were used to elucidate the role of M3 receptors in gastric acid secretion and gastric mucosal integrity. Methods: M3 KO versus wild-type mice aged 1 month to 2 years were included. Gastric acid secretion was assessed by both direct intragastric pH measurement and pylorus ligation. Serum gastrin and gastric mucosal histamine levels were determined by radioimmunoassay and enzyme-linked immunosorbent assay, respectively. Morphologic analysis was performed by both immunohistochemistry and transmission electron microscopy. Results: Fasted M3 KO mice exhibited higher intragastric pH, lower acid output after pylorus ligation, a lower proportion of active parietal cells, and higher serum gastrin levels than fasted wild-type mice. Acid secretion in response to carbachol, histamine, gastrin 17, and 2-deoxy-D-glucose was impaired in the mutant mice. Although carbachol was still able to cause ~30% acid output in M3 KO mice, the acid secretion was inhibited by pirenzepine or famotidine. Despite remarkable hypergastrinemia in M3 KO mice, there were no trophic responses in the oxyntic mucosa with respect to the mucosal thickness, proliferation rate, and numbers of parietal and enterochromaffin-like cells. Cholecystokinin type 2 receptor antagonist YM022 was without the effect in M3 KO mice. Conclusions:The present study shows that M3 receptors are essential for basal acid secretion, a fully acid secretory response to histamine and gastrin, and the trophic responses of oxyntic mucosa to gastrin.

Colonic bacterial superantigens evoke an inflammatory response and exaggerate disease in mice recovering from colitis
Jun Lu, Arthur Wang, Sara Ansari, Robert M. Hershberg, Derek M. Mckay
Background & Aims: There is renewed interest in commensal bacteria as triggers of idiopathic disease, a concept that is prominent in inflammatory bowel disease (IBD). Here the effect of intracolonic instillation of Staphylococcus aureus enterotoxin B (SEB), a model superantigen (SAgs: potent T-cell stimuli), into mice was examined.Methods: Mice (Balb/c, severe combined immunodeficient [SCID], V8+ ovalbumin transgenic [OVA-Tg], interleukin 10 [IL-10] knockout [KO]) received a single intrarectal (IR) dose of SAg and colonic form (histology, myeloperoxidase [MPO] activity) and function (ion transport) were assessed 12­72 hours later. In subsequent studies the potential for SEB to reactivate disease in mice recovering from dextran sodium sulfate (DSS)-induced colitis (5 days at 4% [wt/vol] followed by 14 days normal water) was examined.Results: SEB-treated Balb/c mice displayed a time- and dose-dependent colonic inflammation (increased MPO, histologic damage score, and macrophage number). Similar events occurred in response to other SAgs, namely S. aureus enterotoxin A (SEA) and Yersinia pseudotuberculosis mitogen. Ion transport, the driving force for water movement, was unaffected by SEB treatment. SCID mice developed no inflammation after IR SEB delivery, whereas OVA Tg mice displayed enhanced responsiveness. Although SEB treatment of IL-10 KO mice did elicit a response, the inflammation was transitory and did not hasten the spontaneous colitis seen in these mice. Finally, mice recovering from DSS-induced colitis showed a worsening of the disease when challenged with SEB; IR SEB evoked significant increases in MPO, macrophage infiltration, T-cell activation (i.e., CD25 expression), and perturbed epithelial ion transport. Conclusions: Lumen-derived bacterial SAgs can elicit a local inflammation and aggravate enteric inflammatory disorders in which they were not the causative agent.

Basic-liver, Pancreas, and Biliary Tract

Enhanced carbon tetrachloride-induced liver fibrosis in mice lacking adiponectin
Yoshihiro Kamada, Shinji Tamura, Shinichi Kiso, Hitoshi Matsumoto, Yukiko Saji, Yuichi Yoshida, Koji Fukui, Norikazu Maeda, Hitoshi Nishizawa, Hiroyuki Nagaretani, Yoshihisa Okamoto, Shinji Kihara, Jun-ichiro Miyagawa, Yasuhisa Shinomura, Tohru Funahashi, Yuji Matsuzawa
Background & Aims: Obesity is one of the risk factors for liver fibrosis, in which plasma adiponectin, an adipocytokine, levels are decreased. Hepatic stellate cells play central roles in liver fibrosis. When they are activated, they undergo transformation to myofibroblast-like cells. Adiponectin suppresses the proliferation and migration of vascular smooth muscle cells, whose characteristics are similar to those of hepatic stellate cells. Adiponectin could have biological significances in liver fibrosis. Methods: The role of adiponectin on liver fibrosis induced by the administration of carbon tetrachloride twice a week for 12 weeks was tested by using adiponectin-knockout mice and an adenovirus-mediated adiponectin-expression system. We also investigated the effect of adiponectin in activated hepatic stellate cells. Results: When mice were administered carbon tetrachloride (300 µL/kg body weight) twice a week for 12 weeks, knockout mice showed extensive liver fibrosis with an enhanced expression of transforming growth factor-1 and connective tissue growth factor compared with wild-type mice (P < 0.05). Injection of adenovirus producing adiponectin (AdADN) before carbon tetrachloride (1000 µL/kg body weight) treatment prevented liver fibrosis in wild-type mice (P < 0.001). Injection of AdADN at 6 weeks attenuated liver fibrosis even though carbon tetrachloride was given for an additional 6 weeks (total of 12 weeks). In cultured hepatic stellate cells, adiponectin suppressed platelet-derived growth factor-induced proliferation and migration and attenuated the effect of transforming growth factor-1 on the gene expression of transforming growth factor-1 and connective tissue growth factor and on nuclear translocation of Smad2. Conclusions: The findings indicate that adiponectin attenuates liver fibrosis and could be a novel approach in its prevention.

Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon
Takanobu Kato, Tomoko Date, Michiko Miyamoto, Akihiro Furusaka, Katsutoshi Tokushige, Masashi Mizokami, Takaji Wakita
Background & Aims: Although the hepatitis C virus (HCV) subgenomic replicon system has been widely used in the study of HCV, this system is available only for a few related genotypes. To develop a new replicon system, the genotype 2a clone JFH-1 was isolated from a patient with fulminant hepatitis. Methods: A genotype 2a replicon was constructed by isolating the consensus sequence of JFH-1, transfecting G418-selectable subgenomic transcripts into Huh7 cells, and estimating the replication efficiency. Results: The colony formation efficiency of the JFH-1 replicon was 53,200 colonies/µg RNA, significantly higher than that of the genotype 1b cell-adapted replicon, at 909 colonies/µg RNA (P < 0.05). The JFH-1 replicon RNA was transmissible to naive Huh7 cells by transfection of cellular RNA from cells containing the replicon. Sequencing of cloned replicon RNAs revealed that all but 1 had at least 1 nonsynonymous mutation. One of these mutations was shown to enhance the colony formation efficiency of the JFH-1 replicon. Furthermore, the JFH-1 replicon RNA replicated efficiently without G418 selection in a transient replication assay. Conclusions: The genotype 2a subgenomic replicon was established in Huh7 cells and replicated efficiently with or without G418 selection. This subgenomic replicon could replicate without common amino acid mutations; however, some of the mutations found in the clones might be important in conferring higher replication phenotypes. This system provides a powerful new tool for researching HCV.

Binge ethanol exposure increases liver injury in obese rats
Michal Carmiel-Haggai, Arthur I. Cederbaum, Natalia Nieto
Background & Aims: The objective of this study was to address the hepatic effects of acute alcohol consumption in obesity by simulating an alcohol binge in genetically obese fa/fa rats compared with lean Fa/? rats. Methods: Ethanol 4 g/kg or saline was administered by gavage every 12 hours for 3 days. Results: Plasma alcohol levels were similar in both groups. Binge ethanol exposure caused liver injury in obese fa/fa but not in lean Fa/? rats, as assessed by alanine aminotransferase and H&E staining. Obesity impaired the antioxidant defense because basal levels of glutathione, glutamate cysteine ligase modulatory subunit, catalase, glutathione reductase, and superoxide dismutase were lower in fa/fa compared with Fa/? rats; the ethanol binge further decreased these antioxidants in fa/fa rats and also decreased glutathione peroxidase activity. Nonesterified fatty acids and lipid peroxidation were increased after ethanol treatment in fa/fa rats. Cytochrome P450 2E1 was down-regulated in fa/fa compared with Fa/? rats; however, the ethanol binge increased cytochrome P450 2E1 in both genotypes. Adenosine triphosphate decreased and uncoupling protein 2 increased in fa/fa rats treated with ethanol. 3-Nitrotyrosine protein adducts were detected only in fa/fa rats treated with ethanol, and this was accompanied by an induction of inducible nitric oxide synthase. Ethanol binge increased caspase-3 and caspase-8 activity, the expression of Fas ligand, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling in fa/fa rats. Conclusions: These data indicate that binge drinking increases apoptosis and liver injury in obese rats more than in lean controls and suggest that the injury may involve oxidative and nitrosative damage.

Inducible nitric oxide synthase is required in alcohol-induced liver injury: Studies with knockout mice
Stephen E. McKim, Erwin Gäbele, Fuyumi Isayama, Jason C. Lambert, Lindsay M. Tucker, Michael D. Wheeler, Henry D. Connor, Ronald P. Mason, Mark A. Doll, David W. Hein, Gavin E. Arteel
Background & Aims : Oxidative stress contributes to early alcohol-induced liver injury, and superoxide (O2·­) production from NADPH oxidase plays a key role. However, the production of the free radical nitric oxide (NO·) by inducible nitric oxide synthase (iNOS) could also be involved. Methods : To test this hypothesis, iNOS knockout (B6.129P2-Nos2 tm1 Lau ) and wild-type mice were fed high-fat control or ethanol-containing diets for 4 weeks. Results : Mean body weight gains were not significantly different between treatment groups, and average urine ethanol concentrations were similar in wild-type and iNOS knockout mice. After 4 weeks, serum alanine aminotransferase (ALT) levels were increased significantly about 4-fold over control values (29 ± IU/L) by enteral ethanol (113 ± 20) in wild-type mice; this effect of ethanol was significantly blunted in iNOS knockout mice (50 ± 9). Similar protective effects against liver damage were observed if wild-type mice were treated with the iNOS inhibitor N -(3-aminomethyl)benzyl-acetamindine (1400W). Enteral ethanol also caused severe fatty accumulation, mild inflammation, and necrosis in the liver in wild-type mice but had no effect in iNOS knockout mice. The accumulation of 4-hydroxynonenal (lipid peroxidation) and 3-nitrotyrosine (reactive nitrogen species formation) protein adducts caused by alcohol was completely blocked in iNOS knockout mice. Conclusions : These data strongly support the hypothesis that iNOS is required for the pathogenesis of early alcohol-induced hepatitis by production of nitric oxide-derived pro-oxidants (e.g., peroxynitrite).

Nonoxidative ethanol metabolites alter extracellular matrix protein content in rat pancreas
Aurelia Lugea, Ilya Gukovsky, Anna S. Gukovskaya, Stephen J. Pandol
Background & aims: The mechanisms involved in ethanol-induced pancreas fibrosis are poorly understood. Here we show that fatty acid ethyl esters (FAEEs), nonoxidative ethanol metabolites, increase extracellular matrix (ECM) protein levels in pancreas. Methods: Rat pancreatic acini were incubated for 1­4 hours with FAEEs or acetaldehyde. In another set of experiments, rats received an intravenous infusion of FAEEs for 6 hours. Collagens were assessed by a hydroxyproline assay. Laminin and fibronectin were analyzed by Western blotting. Gene expression of ECM proteins was measured by conventional and real-time reverse-transcription polymerase chain reaction (RT-PCR). Matrix metalloproteinase (MMP), plasmin, and urokinase-type plasminogen activator (uPA) activities were determined by zymography and fluorogenic assays. Results: FAEEs increased collagen, laminin, and fibronectin levels in pancreatic acini without affecting messenger RNA (mRNA) expression for these proteins. Actinomycin D, a transcriptional inhibitor, did not block the increase in ECM proteins induced by FAEEs. FAEEs reduced the activity of the serine protease, plasmin, and that of the uPA. Consistent with these results, the serine protease inhibitor aprotinin reproduced the effects of FAEEs and prevented the further increase in ECM proteins induced by FAEEs. In vivo administration of FAEEs reduced plasmin and uPA activities and increased ECM protein levels in pancreas. Acetaldehyde had minor effects on ECM protein levels and did not affect plasmin activity. Conclusions: FAEEs increase ECM protein levels in pancreas. The results suggest that this effect is caused primarily by an inhibition in ECM degradation via serine proteases including the plasminogen system.

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

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JOURNAL OF HEPATOLOGY

Table of Contents for Volume 39, Issue 6, December 2003

Cell Biology, Metabolism and Transport

Extensive changes in liver gene expression induced by Hereditary Tyrosinemia type I are not normalized by treatment with 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione
Marjanka C. Luijerink et al.
Background: Hereditary Tyrosinemia type I, caused by deficiency of fumarylacetoacetate hydrolase (FAH), is characterized by liver and kidney damage. Administration of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) corrects the tyrosinemia phenotype, but does not prevent development of hepatocellular carcinoma. Aim: To gain insight into the pathophysiological changes associated with liver damage induced by tyrosinemia and the preventive action of NTBC on these changes. Methods: Differential gene expression patterns in livers of tyrosinemia-affected and healthy mice, and of tyrosinemia-affected and NTBC-treated Fah/ mice were investigated by suppression subtractive hybridization. Results: Transcripts encoding proteins playing a role in protein turnover, growth and proliferation, RNA processing, and signal transduction were primarily induced in tyrosinemia-affected livers. Transcripts mainly contributing to the profile of suppressed genes encode proteins that are secreted by the liver, or are necessary for intermediate metabolism. NTBC treatment fails to normalize the tyrosinemia-induced alterations in expression of transcripts encoding proteins involved in protein turnover, signal transduction, and cell growth and proliferation. Conclusions: The failure of NTBC to normalize liver gene expression of Fah/ mice may play a role in rendering the tyrosinemia-affected liver susceptible to development of hepatocellular carcinoma under NTBC treatment.
Keywords: Fumarylacetoacetate hydrolase; 2-(2-Nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC); Hepatocellular carcinoma; Liver damage; Suppression subtractive hybridization; Liver gene profiling

Specific, functional effector/memory CD8+ T cells are found in the liver post-vaccination
Nektarios Dikopoulos, Ieva Jomantaite, Reinhold Schirmbeck and Jörg Reimann
Background: The liver efficiently eliminates activated CD8+ T blasts. It is unknown if vaccine-primed CD8+ T blasts migrate to and establish functional CD8+ T cell immunity in the liver post-immunization. Aims: We tested, if functional CD8+ T cell populations can be detected in the liver post-vaccination. Methods: Murine CD8+ T cells with different epitope/restriction specificities were primed by intramuscular injection of protein- or DNA-based vaccines. The kinetics of appearance in the liver, as well as the surface phenotype and functional competence of intrahepatic, specific CD8+ T cell populations was tested. Results: High numbers of specific CD8+ T cells appear in the liver after vaccination that are activated (CD69+ CD44+), express effector functions (CD27lo/CD28lo phenotype, interferon secretion, specific cytolytic reactivity), but show no evidence of apoptosis (annexin V, B220lo, similar numbers/kinetics in primed, congenic lpr/lpr mice). Specific CD8+ T cells from the liver adoptively transferred into a naïve, syngeneic host successfully reconstitute specific CD8+ T cell immunity. Conclusions: Specific, functionally competent CD8+ effector/memory T cell populations are established in the liver for months post-vaccination.
Keywords: Hepatic T cells; Hepatic tolerance; Hepatic CD8+ T cell immunity

Preferential accumulation of CD103+ T cells in human livers; its association with extrathymic T cells
Yukihiro Shimizu et al.
Background/Aims: CD103, a mucosal integrin E7, binds to E-cadherin expressed on hepatocytes and bile duct epithelium in the liver. Although CD103+ T cells are enriched in intestinal intraepithelial lymphocytes, the localization of those cells in the liver is unknown Methods: We investigated whether CD103+ cells are present in human livers, and how they are associated with the intrahepatic development of T cells by flow cytometry and immunohistochemistry. Results: Human livers contain significantly (P<0.001) higher percentages of CD103+ cells in CD4+ and CD8+ T cells (25.7±13.5 and 27.1±19.3%, respectively) than peripheral blood lymphocytes. Moreover, CD103+ cells in the liver, but not in peripheral blood, contained T cells with intermediate expression level of T cell receptor . Those cells consist of mostly CD4+ and CD4CD8 cells, and expressed low level of CD56 and interleukin-2 receptor beta chain in most of the population. These characteristics are distinct from natural killer T cells, which have been thought to be extrathymic T cells in human livers. Moreover, intrahepatic CD103+ cells expressed mRNA for recombination-activating gene-1, -2 and pre T cell receptor-alpha detected by reverse transcription-polymerase chain reaction Conclusions: CD103+ T cells are preferentially accumulated in human livers, and those T cells show characteristics of extrathymic T cells.
Keywords: Extrathymic T cells; CD103; Human liver

Chronic Liver Diseases

Haemochromatosis-associated HFE genotypes in English blood donors: age-related frequency and biochemical expression
Vanessa Chambers et al.
Background/Aims: There are limited data on the frequency and biochemical expression of the haemochromatosis-associated mutations C282Y and H63D in healthy people. Methods: We genotyped (bi-directional PCR amplification of specific alleles method) and performed serum iron studies in randomly selected English male blood donors (<4 previous units donated) in four age bands <30, 30-40, 40-50 and >50 years. Results: In 6261 subjects, frequency of C282Y homozygosity (+/+) was 0.3%, C282Y/H63D compound heterozygosity (+/) 2.0%, and H63D and C282Y heterozygosity +/, 21.7 and 10.4%, respectively. Genotype distribution was within Hardy-Weinberg equilibrium in each age band. C282Y +/ frequency fell from 11.7% in subjects <30 years to 8.2% in subjects >50 (Chi2 7.19; P<0.005). No such trend was seen for C282Y +/+. In C282Y +/+ subjects, median serum ferritin was 247 (range 60-2449) µg/l and exceeded >500 µg/l in only two of 18 subjects. Compared to wild/wild (/) subjects, C282Y and H63D +/ subjects had slightly higher serum iron and lower unsaturated iron binding concentrations, similar overall serum ferritin values but higher serum ferritin values in subjects who had previously donated blood. Conclusions: C282Y +/+ shows limited biochemical expression and no trend towards age-related attrition. C282Y and H63D +/ may protect against iron deficiency.
Keywords: Haemochromatosis; Genetics; Blood donors; Iron indices

Cirrhosis and its Complications

NCX-1000, a nitric oxide-releasing derivative of ursodeoxycholic acid, ameliorates portal hypertension and lowers norepinephrine-induced intrahepatic resistance in the isolated and perfused rat liver
Stefano Fiorucci et al.
Background/Aims: We studied whether acute administration of NCX-1000, a nitric oxide (NO)-releasing derivative of ursodeoxycholic acid (UDCA), to animals with established liver cirrhosis decreases intrahepatic resistance and modulates hepatic vascular hypereactivity to norepinephrine (NE). Methods: Four-week bile duct ligated (BDL) cirrhotic and control, sham-operated, rats were treated orally with 28 mg/kg per day NCX-1000 or 15 mg/kg per day UDCA for 5 days. Isolated normal and cirrhotic livers were perfused with NE, from 10 nM to 30 µM, in a recirculating system. Results: NCX-1000 administration to BDL cirrhotic rats decreased portal pressure (P<0.01) without affecting mean arterial pressure and heart rate. In the isolated perfused liver system, administration of NE resulted in a dose-dependent increase of intrahepatic resistance. Vasoconstriction caused by 30 µM NE was reduced by 60% in animals treated with NCX-1000 (P<0.001), while UDCA was uneffective. The same portal pressure lowering effect was documented in cirrhotic and sham operated rats. Administration of NCX-1000 to BDL and sham operated rats resulted in a similar increase of nitrite/nitrate and cGMP concentrations in the liver. Conclusions: By selectively delivering NO to the liver, NCX-1000 increases cGMP concentrations and effectively counteracts the effect of endogenous vasoconstrictors on the hepatic vascular tone.
Keywords: Portal hypertension; Nitric oxide; Ursodeoxycholic acid; cGMP; Norepinephrine

A liver-specific nitric oxide donor improves the intra-hepatic vascular response to both portal blood flow increase and methoxamine in cirrhotic rats
Mauricio R. Loureiro-Silva, Gregory W. Cadelina, Yasuko Iwakiri and Roberto J. Groszmann
Background/Aims: A decreased intra-hepatic nitric oxide (NO) production participates on the pathogenesis of portal hypertension in cirrhosis. We tested the hemodynamic effects of a liver-specific NO donor (NCX-1000) derived from ursodeoxycholic acid in portal hypertensive cirrhotic rats. Methods: After a 14-day treatment with ursodeoxycholic acid or NCX-1000 by gavage, ascitic cirrhotic rats (CCl4-induced) were used in two studies: (1) in vivo mean arterial pressure (MAP), portal pressure (PP) and superior mesenteric artery (SMA) blood flow measurements before and during progressive blood volume expansion (blood infusion); and (2) in situ liver perfusion to obtain dose/response curves to methoxamine (alpha1-adrenergic agonist) and flow/pressure curves. Results: Basal heart rate, MAP, and PP were similar in both groups. During blood infusion, similar MAP and SMA flow increases were observed in both groups; however, PP increase observed in control rats was blunted in NCX-1000 treated rats (P=0.015). In liver perfusions, flow/pressure curves were similar in both groups; however, NCX-1000-treated livers showed a lower response to methoxamine (P=0.016). cGMP concentration in NCX-1000-treated livers was higher (P=0.015) than in controls. Conclusions: Treatment with a liver-specific NO donor improves the portal system adaptability to portal blood flow increase and ameliorates the intra-hepatic response to methoxamine in cirrhotic rats.
Keywords: Rat liver perfusion; Portal hypertension; Portal pressure; Superior mesenteric artery flow; Intra-hepatic microcirculation

Improvement of prognostic power of the Child-Pugh classification of liver cirrhosis by hyaluronan
Thomas Körner et al.
Background/Aims: Modifications of the Child-Pugh classification of liver cirrhosis by incorporation of hyaluronan were tested to improve the prognostic power for long term evaluation of liver cirrhosis in 126 patients observed over a period of 10 years. Methods: Serum concentrations of HA were determined at study entry. Statistical analysis included Kaplan-Meier life tables and stepwise multivariant Cox-regression analysis for each parameter of Child-Pugh classification and hyaluronan. Prognostic models were developed by exchanging prothrombin time, albumin and encephalopathy by HA in different combinations. Results: Based on a good single correlation between hyaluronan (0.62) and clinical course (P<0.01) we conclude that models with hyaluronan instead of albumin or encephalopathy and with or without shifted threshold values of bilirubin and albumin are superior for the prediction of the long term prognosis. In Cox-regression analysis, apart from hyaluronan and bilirubin, no other parameters contributed to an improvement. Conclusions: We conclude that a modification of the Child-Pugh classification of liver cirrhosis by inclusion of HA significantly improves the predictive power of CP, especially in alcoholic etiology. A prospective validation of the newly defined scores needs to be done in the future.
Keywords: Liver cirrhosis; Hyaluronan; Prognosis; Child's classification

Hemostatic effect of activated recombinant factor VII (rFVIIa) in liver disease: studies in an in vitro model
Raúl Tonda et al.
Background/Aims: There is clinical evidence for the efficacy of activated recombinant factor VII (rFVIIa) in patients with cirrhosis. The exact mechanism of action of rFVIIa in this clinical condition is unknown. We have explored effects of rFVIIa on hemostasis in cirrhotic patients using an in vitro perfusion technique. Methods: Blood samples were drawn from control donors or from 11 patients previously diagnosed with cirrhosis (seven Child-Pugh B and four Child-Pugh C) and anticoagulated with low molecular weight heparin. rFVIIa was added to blood samples at therapeutic concentrations (0.5 or 1 µg/ml of plasma) and blood was recirculated through annular chambers containing damaged vascular segments. Presence of platelets and fibrin on the subendothelium were morphometrically quantified. Results: Cirrhotic patients showed a diminished platelet interaction with the subendothelium compared to healthy donors (17.3% (9.28-28.88%) vs. 26.16% (19.96-54.5%), P<0.05). After addition of rFVIIa to cirrhotic samples, no differences in platelet covered surface were observed. However, fibrin formation was significantly improved after the addition of rFVIIa (from 51.81% (3.02-86.68%) to 86.94% (30.03-93.18%) and 89.05% (45.65-93.84%), respectively, P<0.05). Conclusions: Our data confirm a defective interaction of platelets with the subendothelium in cirrhotic patients. rFVIIa improved local fibrin formation at damaged sites and this mechanism could explain the beneficial action of rFVIIa in cirrhotic patients.
Keywords: Activated recombinant factor VII (rFVIIa); Cirrhosis; Hemostasis; Procoagulant action

Inflammation and Fibrosis

Apoptosis of hepatic stellate cells in carbon tetrachloride induced acute liver injury of the rat: analysis of isolated hepatic stellate cells
Jung Il Lee et al.
Background/Aims: Analysis of isolated hepatic stellate cells (HSCs) from the injured liver may provide direct information on HSC apoptosis. However, it has not been established whether apoptotic HSCs would be isolated using the usual density gradient centrifugation method. The aim of this study was to observe the serial pattern of proliferation and apoptosis in isolated HSCs in comparison with that of liver tissue sections in CCl4 induced acute liver injury. Methods: Male Sprague-Dawley rats were treated with a single intraperitoneal injection of carbon tetrachloride (CCl4) and were killed at various time points after the treatment. Results: HSC proliferation showed a maximal increase at 32 h after CCl4 injection. Apoptosis of HSC, examined by quantitative analysis of annexin-V-fluorescein isothiocyanate (FITC)staining, showed the maximal increase at 64 h. Apoptosis of HSC in liver tissue sections examined by counting desmin and Tdt-mediated-dUTP biotin nick end labeling (TUNEL) double staining cells, peaked at 64 h. The number of TUNEL positive HSCs in liver tissue sections correlated significantly with annexin-V-FITC binding of isolated HSC. Conclusions: Studying apoptosis using apoptotic HSCs isolated by a usual density gradient centrifugation method from injured tissue sections would be feasible since it correlated with in vivo apoptosis of HSC.
Keywords: Hepatic stellate cell; In vivo; Ex vivo

Pro-fibrotic polymorphisms predictive of advanced liver fibrosis in the severely obese
John B. Dixon et al.
Background/Aims: Insulin resistance and systemic hypertension are predictors of advanced fibrosis in obese patients with non-alcoholic fatty liver disease (NAFLD). Genetic factors may also be important. We hypothesize that high angiotensinogen (AT) and transforming growth factor-1 (TGF-1) producing genotypes increase the risk of liver fibrosis in obese subjects with NAFLD. Methods: One hundred and five of 130 consecutive severely obese patients having a liver biopsy at the time of laparoscopic obesity surgery agreed to have genotype analysis. Influence of specific genotype or combination of genotypes on the stage of hepatic fibrosis was assessed after controlling for known risk factors. Results: There was no fibrosis in 70 (67%), stages 1-2 in 21 (20%) and stages 3-4 fibrosis in 14 (13%) of subjects. There was no relationship between either high AT or TGF-1 producing genotypes alone and hepatic fibrosis after controlling for confounding factors. However, advanced hepatic fibrosis occurred in five of 13 subjects (odds ratio 5.7, 95% confidence interval 1.5-21.2, P=0.005) who inherited both high AT and TGF-1 producing polymorphisms. Conclusions: The combination of high AT and TGF-1 producing polymorphisms is associated with advanced hepatic fibrosis in obese patients with NAFLD. These findings support the hypothesis that angiotensin II stimulated TGF-1 production may promote hepatic fibrosis.
Keywords: Fibrosis; Obese patients; Non-alcoholic fatty liver disease

Liver Cell Injury and Liver Failure

Sub-lethal oxidative stress triggers the protective effects of ischemic preconditioning in the mouse liver
Hannes A. Rüdiger, Rolf Graf and Pierre-Alain Clavien
Background/Aims: While ischemic preconditioning confers significant protection against subsequent prolonged periods of ischemia, the mechanisms triggering protection remain speculative. We hypothesize that a sub-lethal oxidative stress during ischemic preconditioning induces defense mechanisms preventing subsequent lethal injury. Methods: We used mouse models of partial and total hepatic ischemia for 75 min. Ischemic preconditioning consisted of 10-min ischemia and 15-min reperfusion prior to the prolonged ischemic insult. Results: Tissue levels of peroxides increased about three times after 10 min of ischemia and normalized within 15 min of reperfusion. This limited oxidative stress during ischemic preconditioning prevented the negative effects of subsequent prolonged ischemia as assessed by AST-levels, TUNEL-staining of hepatocytes and animal survival. N-Acetylcysteine inhibited the mild oxidative burst of ischemic preconditioning, and fully reversed the protective effects of preconditioning. The protective role of a sub-lethal oxidative stress was supported by the benefit of delivery of an H2O2-analog through the portal vein prior to a long ischemic insult. This challenge conferred similar protection as ischemic preconditioning. Conclusions: We conclude that the mild burst of oxidative stress generated during ischemic preconditioning triggers protective mechanisms against subsequent, otherwise lethal, ischemic injury. The pathway possibly includes enhancement of natural anti-oxidative stress mechanisms.
Keywords: Liver; Ischemia-reperfusion injury; Ischemic preconditioning; Apoptosis; Oxidative stress

Diet associated hepatic steatosis sensitizes to Fas mediated liver injury in mice
Ariel E. Feldstein et al.
Background/Aims: Hepatic steatosis sensitizes the liver to injury and inflammation by unclear mechanisms. Because Fas has been linked to liver injury and inflammation, Fas expression and sensitization to Fas signaling was examined in models of hepatic steatosis. Methods: Mice were fed a carbohydrate diet while control animals received standard chow. Sensitization to Fas was examined following administration of Jo2 antibody. For the in vitro experiments, HepG2 cells were incubated with or without a mixture of long chain fatty acids (2:1 oleate:palmitate). Sensitization of the cells to Fas was examined using the CH11 antibody. Results: Mice fed a high caloric diet developed hepatic steatosis, hyperlipidemia, insulin resistance, and hyperleptinemia, all features of the human syndrome. Fas expression in hepatocytes was increased as compared to lean animals and was coupled to cytotoxic signaling. Indeed, hepatocyte apoptosis, liver injury and chemokine generation were all accentuated in obese animals following administration of Jo-2, a Fas agonist. Hep G2 cells cultured in the presence of free fatty acids also developed `cellular steatosis', upregulated Fas expression and were more sensitive to apoptosis by a Fas agonist. Conclusions: Collectively, these data implicate Fas as a link between obesity associated fatty liver and increased susceptibility to liver damage.
Keywords: Obesity; Non-alcoholic fatty liver disease; Fatty acids; Fas (CD95); Apoptosis

Prometheus® - a new extracorporeal system for the treatment of liver failure
Kinan Rifai et al.
Background/Aims: Extracorporeal detoxification systems for supportive therapy of liver failure have recently gained much interest. We herein report results from the first clinical application of Prometheus®, a new liver support system in which albumin-bound substances are directly removed from blood by special adsorber. In a simultaneous step, high-flux hemodialysis is performed. We assessed safety, adsorber efficiency and clinical efficacy of the Prometheus® system. Methods: Eleven patients with acute-on-chronic liver failure and accompanying renal failure were treated with Prometheus® on 2 consecutive days for >4 h. Results: Prometheus® treatment significantly improved serum levels of conjugated bilirubin, bile acids, ammonia, cholinesterase, creatinine, urea and blood pH. There were no significant changes in hemoglobin and platelet levels, whereas leucocytes increased without signs of systemic infection. No treatment-related complications except a blood pressure drop in two patients with systemic infection were noted. In one patient (Child-Pugh score: 15) Prometheus® treatment could not be completed due to onset of uncontrolled bleeding 16 h after dialysis. Conclusions: Prometheus® is a safe supportive therapy for patients with liver failure. A significant improvement of the biochemical milieu was observed already after two treatments. Prospective controlled studies with the Prometheus® system are necessary to evaluate hard clinical end-points.
Keywords: Albumin dialysis; Liver failure; Hepatorenal syndrome; Hepatic encephalopathy; Clinical study; Humans; Prometheus

Liver Growth and Cancer

Expression of ephrin-B1 in hepatocellular carcinoma: possible involvement in neovascularization
Yoshiyuki Sawai et al.
Background/Aims: Hepatocellular carcinoma (HCC) is, in general, a hypervascular tumor. Ephrin/Eph molecules have recently been reported as possible regulators of angiogenesis. We aimed to clarify the role of ephrin-Bs (B1-B3) in the progression of HCC. Methods: Ephrin-Bs transcripts in 26 HCC and their corresponding non-tumor liver tissues were analyzed by the quantitative reverse transcription-polymerase chain reaction. We established ephirn-B1 overexpressing cell in a human HCC cell line, PLC/PRF/5 cell, and their in vivo growth monitored after subcutaneous injection into nude mice. Neovascularization in the inoculated tumors was evaluated by the immunohistochemical staining of CD31. The migration and proliferation of human umbilical vein endothelial cells (HUVECs) in response to soluble ephrin-B1-Fc was examined. Results: The expression of the ephrin-B1 transcript but not -B2 and -B3 transcripts was significantly higher in HCC tissues than in non-tumor tissues (P<0.05). The ephrin-B1 overexpressing cells developed tumors more rapidly than controls in vivo (P<0.05), although in vitro cell growth was not affected. The tumor vessel number significantly increased in the ephrin-B1 overexpressing tumors (P<0.0001). In addition, in vitro studies revealed that ephrin-B1 induced migration and proliferation of HUVECs. Conclusions: Ephrin-B1 may be involved in in vivo tumor progression by promoting neovascularization in HCC.
Keywords: Ephrin-B1; Hepatocellular carcinoma; Neovascularization

In human hepatocellular carcinoma in cirrhosis proliferating cell nuclear antigen (PCNA) is involved in cell proliferation and cooperates with P21 in DNA repair
Laura Gramantieri et al.
Background: Proliferating cell nuclear antigen (PCNA) is a nuclear protein involved in DNA-synthesis and repair. During DNA-synthesis and repair the only active PCNA fraction is tightly bound to DNA. Similarly, during DNA-repair, a fraction of p21 colocalizes with PCNA in a detergent-insoluble form. Aim: The aim of the study was to analyze to what extent the presence of DNA-bound PCNA and p21 correlates with cell proliferation and DNA-repair in hepatocellular carcinoma (HCC). Methods: Twenty-six HCCs and surrounding cirrhosis were studied. The DNA-bound and detergent-soluble fractions of PCNA and p21 were analyzed by immunoblotting. P53 and Ki67-Labeling Index (Ki67-LI) were evaluated by immunocytochemistry. Results: Soluble fractions of PCNA and p21 were found in all samples. One out of 26 cirrhotic samples displayed a DNA-bound fraction of PCNA while no case expressed DNA-bound p21. Fourteen HCCs showed a DNA-bound PCNA fraction. A highly significant correlation was found between Ki67-LI and DNA-bound PCNA but not with detergent-soluble PCNA. DNA-bound p21 and PCNA, indicating ongoing DNA repair activity, were present in 6 of these 14 HCCs and correlated with a high histological grade and high Ki67-LI. Conclusions: Our results suggest that in HCC PCNA participates both in DNA synthesis and repair and that highly proliferating HCCs may display a sustained DNA-repair.
Keywords: Hepatocellular carcinoma; Proliferating cell nuclear antigen; p21/waf1/cip1/sdi1; DNA repair; Cell proliferation

Viral Hepatitis

Different hepatitis C virus dynamics of free-virions and immune-complexes after initiation of interferon- in patients with chronic hepatitis C
Naoki Fujita et al.
Background/Aims: Hepatocellular carcinoma (HCC) usually develops following chronic liver inflammation caused by hepatitis C or B virus. Through expression profiling in a rare type of HCC, for which the causes are unknown, we sought to find key genes responsible for each step of hepatocarcinogenesis in the absence of viral influence. Methods: We used 68 non-B, non-C liver tissues (20 HCC, 17 non-tumor, 31 normal liver) for expression profiling with PCR-array carrying 3072 genes known to be expressed in liver tissues. To select the differentially expressed genes, we performed random permutation testing. A weighted voting classification algorithm was used to confirm the reliability of gene selection. We then compared these genes with the results of previous expression profiling studies. Results: A total of 220 differentially expressed genes were selected by random permutation tests. The classification accuracies using these genes were 91.8, 92.0 and 100.0% by a leave-one-out cross-validation, an additional PCR-array dataset and a Stanford DNA microarray dataset, respectively. By comparing our results with previous reports on virus-infected HCC, four genes (ALB, A2M, ECHS1 and IGFBP3) were commonly selected in some studies. Conclusions: The 220 differentially expressed genes selected by PCR-array are potentially responsible for hepatocarcinogenesis in the absence of viral influence.
Keywords: Hepatocellular carcinoma; DNA microarray; Expression profile; Cryptogenic cirrhosis; IGFBP3

Alcohol and hepatitis C virus core protein additively increase lipid peroxidation and synergistically trigger hepatic cytokine expression in a transgenic mouse model
Gabriel Perlemuter et al.
Background/Aims: To elucidate the mechanisms of action of interferon (IFN) against hepatitis C virus (HCV), we studied the serum HCV dynamics of free-virions (FV) and immune-complexes (IC) in patients treated with IFN. Methods: FV and IC were separated by immunoprecipitation using anti-human immunoglobulin and quantified serially using real-time detection-polymerase chain reaction. Results: Initially [1st phase (0-24 h)], the FV decreased more rapidly compared to IC [exponential decay slope (EDS)=1.78±0.42 vs. 0.99±0.31 log10/day, P<0.001; half-life=5.65±2.02 vs. 12.5±2.83 h, P<0.0001], but at the 2nd phase (1-14 days), half-life of FV was significantly longer than that of IC (101±117 vs. 14.2±1.08 h, P<0.005). Regarding response to IFN, the decline slope was not significantly different at the 1st phase, but at the 2nd phase, the FV-HCV RNA decreased more slowly in non-responders than in sustained responders to IFN (EDS=0.05±0.02 vs. 0.34±0.19 log10/day, P<0.005; half-life=186±112 vs. 15.3±1.85 h, P<0.005). Conclusions: The presence of escape mutants from the neutralizing antibodies may be involved in resistance to IFN. Analyzes of FV- and IC-HCV dynamics are useful for predicting the IFN efficacy and understanding the mechanism of IFN action in chronic hepatitis patients.
Keywords: Chronic hepatitis C; Interferon; Viral kinetics; Immunoprecipitation; Real-time detection-polymerase chain reaction; Hepatitis C virus genotype; Humoral immunity

Dynamics of plasma hepatitis B virus levels after highly active antiretroviral therapy in patients with HIV infection
Chi-Tai Fang et al.
Background/Aims: Alcohol consumption accelerates the appearance of liver fibrosis and hepatocellular carcinoma in patients with chronic hepatitis C virus (HCV) infection, but the mechanisms of these interactions are unknown. We therefore investigated the effects of chronic ethanol consumption in HCV core protein-expressing transgenic mice. Methods: Ethanol was progressively added (up to 20%) to the drinking water that was given ad libidum. Results: In vivo fatty acid oxidation was not inhibited by ethanol consumption and/or HCV core expression. Both chronic ethanol consumption and HCV core expression decreased hepatic lipoprotein secretion and caused steatosis, but had no additive effects on lipoprotein secretion or steatosis. However, chronic ethanol consumption and HCV core protein additively increased lipid peroxidation and acted synergistically to increase the hepatic expression of transforming growth factor- (TGF-) and, to a less extent, tumor necrosis factor- (TNF-). Conclusions: HCV core protein expression and chronic alcohol consumption have no effects on in vivo fatty acid oxidation and do not additively impair hepatic lipoprotein secretion, but additively increase hepatic lipid peroxidation and synergistically increase hepatic TNF- and TGF- expression. These effects may be involved in the activation of fibrogenesis and the development of hepatocellular carcinoma in patients cumulating alcohol abuse and HCV infection.
Keywords: Hepatitis C virus (HCV); HCV core; Ethanol; Alcohol; Peroxidation; Tumor necrosis factor-; Transforming growth factor-

Epidemiological and clinical burden of chronic hepatitis B virus/hepatitis C virus infection. A multicenter Italian study
Giovanni B. Gaeta et al.
Background/Aims: The optimal strategy to prescribe highly active antiretroviral therapy (HAART) in patients infected with both hepatitis B virus (HBV) and human immunodeficiency virus (HIV) remains unsettled. This study aimed to compare the HBV dynamics between HBeAg-positive and HBeAg-negative coinfected patients treated with lamivudine-containing HAART. Methods: We retrospectively analyzed the serial changes of plasma HBV DNA levels in 24 HBsAg-positive HIV-infected patients who entered the HAART program. A polymerase chain reaction-based assay, capable of quantifying as few as 400 HBV copies/ml, was used. The median follow-up time was 18 months. Results: HAART containing lamivudine 300 mg/day effectively suppressed plasma HBV-DNA to 103-105-fold of the baseline levels, but a multi-phasic decay of HBV DNA was observed. The later phases became flat, as a persistent residual HBV viremia, in eight of the studied 10 HBeAg-positive patients; in contrast, residual HBV viremia was not observed in the 10 HBeAg-negative patients studied (8/10 vs. 0/10, P=0.0007, Fisher's exact test). HAART without lamivudine did not suppress plasma HBV DNA levels in the remaining four patients. Conclusions: HAART containing lamivudine 300 mg/day effectively suppress HBV replication in HBeAg-negative HIV/HBV-coinfected patients. Nevertheless, residual HBV replication persisted in most HBeAg-positive coinfected patients.
Keywords: Hepatitis B; Human immunodeficiency virus infection; Lamivudine; Highly active antiretroviral therapy

In overweight patients with chronic hepatitis C, circulating insulin is associated with hepatic fibrosis: implications for therapy
Ingrid J. Hickman et al.
Background/Aims: This study assess prevalence, risk factors, and clinical and virological features of dual hepatitis B virus (HBV)/hepatitis C virus (HCV) infection. Methods: We evaluated 837 hepatitis B surface antigen positive patients, prospectively enrolled in 14 Italian units. Results: Anti-HCV was present in 59 cases (7%); age specific prevalences were 4.5% (0-30 years), 4.4% (>30-50) and 14% (>50). Independent predictors of dual infection were age >42 years, history of I.V. drug use (IDU), blood transfusion and residence in the South of the country. The strength of the association with IDU was high, but this exposure accounted for five coinfection cases only. Cirrhosis was present in 107 of the 709 patients with HBV alone (15.1%), in 30 of 69 with hepatitis D virus coinfection (43%) and in 17 of 59 with HCV coinfection (28.8%); a light alcohol use was marginally associated with cirrhosis. Of 36 B/C coinfected patients, 16 (44.4%) had only HBV-DNA in serum, (median age=47.5 years) five (13.9%) had both HBV-DNA and HCV-RNA (age=53), nine (25%) had HCV-RNA alone (age=59) and six (16.7%) tested negative for both. Conclusions: This study depicts the epidemiological and clinical burden of dual HBV/HCV infection in Italy.
Keywords: Hepatitis; Cirrhosis; Hepatitis B virus-DNA; Hepatitis C virus-RNA

Fibrogenic impact of high serum glucose in chro nic hepatitis C
Vlad Ratziu et al.
Background/Aims: Host factors such as increased body mass index (BMI) and genotype-specific viral factors contribute to the development of steatosis in patients with chronic hepatitis C (HCV). We hypothesized that host metabolic factors associated with increased BMI may play a role in disease progression. Methods: Fasting serum was collected from 160 patients with chronic HCV at the time of liver biopsy and 45 age, gender and BMI matched controls, and assessed for levels of insulin, c-peptide and leptin. Results: Patients with viral genotype 3 had more severe steatosis (P=0.0001) and developed stages 1 and 2 fibrosis at a younger age (P<0.05) than patients with genotype 1. For both genotypes, overweight patients had significantly more steatosis and increased insulin and leptin levels. In contrast to lean patients, there was a statistically significant increase in circulating insulin levels with increasing fibrosis in overweight patients with chronic HCV (P=0.03). Following multivariate analysis, insulin was independently associated with fibrosis (P=0.046) but not inflammation (P=0.83). There was no association between serum leptin levels and stage of fibrosis. Conclusions: Increasing circulating insulin levels may be a factor responsible for the association between BMI and fibrosis in patients with HCV, irrespective of viral genotype.
Keywords: Hepatitis C (HCV); Body mass index; Obesity; Insulin; Fibrosis; Steatosis; Inflammation; Leptin

When and how to treat acute hepatitis C?
Anna Licata et al.
Background: Appropriate treatment of acute hepatitis C is still a matter of controversy due to the lack of large controlled trials. Aim: To assess the effectiveness of interferon as treatment for acute hepatitis C by meta-analysis. Methods: MEDLINE search (1985-2002) was supplemented with manual searches of reference lists. Studies were included if they were controlled trials comparing interferon to no treatment and if they included patients with either post-transfusion or sporadic acute hepatitis C. Twelve trials were analyzed (414 patients). The outcome assessed was the sustained virological response (SVR) rate (undetectable hepatitis C virus RNA in serum at least 6 months after cessation of therapy). Results: Interferon significantly increased the SVR (risk difference 49%; 95% confidence interval 32.9-65%) in comparison to no treatment. The risk difference of SVR increased from 5 to 90% when trials were ordered by increasing interferon weekly dose. Delaying therapy by 8-12 weeks after the onset of disease does not compromise the SVR rate. Conclusions: Current evidence is sufficient to recommend interferon treatment of patients with acute hepatitis C. A later initiation of therapy yields the same likelihood of response as early treatment. A daily induction dose during the 1st month is the best option of treatment.
Keywords: Acute hepatitis C; Interferon; Sustained virological response Abbreviations: RCTs, randomized controlled trials; NRCTs, non-randomized controlled trials; IFN, interferon

Core promoter/pre-core mutations are associated with lamivudine-induced HBeAg loss in chronic hepatitis B with genotype C
Yasuhiro Asahina et al.
Background/Aims: To clarify the factors associated with the efficacy of lamivudine. Methods: Variables including basic core promoter (BCP) and pre-core (PreC) mutations were evaluated in 60 chronic hepatitis B e antigen (HBeAg)-positive patients with genotype C. Thirty patients were treated with lamivudine and the remaining 30 patients were age- and sex-matched controls. Results: Severe fibrosis was significantly more frequent in patients with the BCP-mutant/PreC-wild (MW) and BCP-mutant/PreC-mutant (MM) patterns compared to BCP-wild/PreC-wild (WW) pattern (P=0.02). The cumulative rates of HBeAg loss at 6, 12 and 18 months were significantly higher in the lamivudine group (14.2, 36.3, and 60.9%) compared with the control group (17.6, 17.6, and 24.5%, P=0.03), and was especially pronounced in patients with the MW pattern (P=0.04). The rate of lamivudine-related HBeAg loss was significantly lower in patients with the WW pattern (P=0.03). Factors correlating with HBeAg loss were histological fibrosis and activity, hepatitis B virus-DNA levels, BCP/PreC mutation and lamivudine therapy. Multivariate analysis revealed BCP/PreC mutations and fibrosis were independent factors for HBeAg loss. Conclusions: With specific reference to the genotype C, we found earlier HBeAg loss was expected in patients carrying MM and MW patterns, while the efficacy of lamivudine was limited in patients with the WW pattern.
Keywords: Hepatitis B virus; Hepatitis B e antigen; Therapy; Viral mutation; Case-control study

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

BRITISH MEDICAL JOURNAL

Volume 362, Number 9399, 06 December 2003

Screening for hereditary haemochromatosis within families and beyond
C Anne McCune, David Ravine, Mark Worwood, Helen A Jackson, H Martyn Evans, David Hutton

Screening programmes for haemochromatosis that include follow-up identification of relatives are claimed to be cost effective. We assessed uptake of screening by first-degree relatives of two groups of index cases: people homozygous for the C282Y mutation ascertained by genetic screening of blood donors; and patients presenting clinically with haemochromatosis. Only 40 (24%) of 165 relatives of blood donors had been tested. By contrast, testing uptake in 121 relatives of patients diagnosed clinically was more than double that (53%), despite unstructured provision of genetic information. A substantial number of untested relatives had undiagnosed iron overload. Overall efficacy of population screening for haemochromatosis is undermined by these observations.

© 2003 BMJ Publishing Group Ltd

NEW ENGLAND JOURNAL

Volume 349:2191-2200 December 4, 2003, Number 23

Computed Tomographic Virtual Colonoscopy to Screen for Colorectal Neoplasia in Asymptomatic Adults
Perry J. Pickhardt, M.D., J. Richard Choi, Sc.D., M.D., Inku Hwang, M.D., James A. Butler, M.D., Michael L. Puckett, M.D., Hans A. Hildebrandt, M.D., Roy K. Wong, M.D., Pamela A. Nugent, M.D., Pauline A. Mysliwiec, M.D., M.P.H., and William R. Schindler, D.O.

Background We evaluated the performance characteristics of computed tomographic (CT) virtual colonoscopy for the detection of colorectal neoplasia in an average-risk screening population. Methods A total of 1233 asymptomatic adults (mean age, 57.8 years) underwent same-day virtual and optical colonoscopy. Radiologists used the three-dimensional endoluminal display for the initial detection of polyps on CT virtual colonoscopy. For the initial examination of each colonic segment, the colonoscopists were unaware of the findings on virtual colonoscopy, which were revealed to them before any subsequent reexamination. The sensitivity and specificity of virtual colonoscopy and the sensitivity of optical colonoscopy were calculated with the use of the findings of the final, unblinded optical colonoscopy as the reference standard. Results The sensitivity of virtual colonoscopy for adenomatous polyps was 93.8 percent for polyps at least 10 mm in diameter, 93.9 percent for polyps at least 8 mm in diameter, and 88.7 percent for polyps at least 6 mm in diameter. The sensitivity of optical colonoscopy for adenomatous polyps was 87.5 percent, 91.5 percent, and 92.3 percent for the three sizes of polyps, respectively. The specificity of virtual colonoscopy for adenomatous polyps was 96.0 percent for polyps at least 10 mm in diameter, 92.2 percent for polyps at least 8 mm in diameter, and 79.6 percent for polyps at least 6 mm in diameter. Two polyps were malignant; both were detected on virtual colonoscopy, and one of them was missed on optical colonoscopy before the results on virtual colonoscopy were revealed. Conclusions CT virtual colonoscopy with the use of a three-dimensional approach is an accurate screening method for the detection of colorectal neoplasia in asymptomatic average-risk adults and compares favorably with optical colonoscopy in terms of the detection of clinically relevant lesions.

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

LANCET

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