Volume 44, Issue 5, November 2006
Liver Failure and Liver Disease
Perspectives in Clinical Hepatology
Sclerosing cholangitis: A focus on secondary causes (p 1063-1074)
Rupert Abdalian, E. Jenny Heathcote
Secondary sclerosing cholangitis (SSC) is a disease that is morphologically similar to primary sclerosing cholangitis (PSC) but that originates from a known pathological process. Its clinical and cholangiographic features may mimic PSC, yet its natural history may be more favorable if recognition is prompt and appropriate therapy is introduced. Thus, the diagnosis of PSC requires the exclusion of secondary causes of sclerosing cholangitis and recognition of associated conditions that may potentially imitate its classic cholangiographic features. Well-described causes of SSC include intraductal stone disease, surgical or blunt abdominal trauma, intra-arterial chemotherapy, and recurrent pancreatitis. However, a wide variety of other associations have been reported recently, including autoimmune pancreatitis, portal biliopathy, eosinophillic and/or mast cell cholangitis, hepatic inflammatory pseudotumor, recurrent pyogenic cholangitis, primary immune deficiency, and AIDS-related cholangiopathy. This article offers a comprehensive review of SSC.
Beyond randomized controlled trials: A critical comparison of trials with nonrandomized studies (p 1075-1082)
Henrik Toft Sørensen, Timothy L. Lash, Kenneth J. Rothman
Observational analogs of randomized clinical trials (RCTs) are well accepted in the study of disease risk factors, diagnosis, and prognosis. There is controversy about observational studies when the focus is on the intended benefit due to lack of blinding and poor control for unmeasured confounding. Well-designed randomized clinical trials are costly both in time and money. Therefore, existing databases are used increasingly and are often the only feasible source with which to examine delayed health effects. We reviewed the reasons for possible discrepancies between RCTs and observational studies. There can be different patient populations, differences in therapeutic regimen, control of confounding, follow-up, measuring outcome, and differences arising from the intention-to-treat analysis. Observational studies cannot replace trials, nor do trials make observational studies unnecessary. Both designs are susceptible to particular bias, so neither provides perfect information.
Viral Hepatitis
A simple noninvasive index for predicting long-term outcome of chronic hepatitis C after interferon-based therapy (p 1086-1097)
Ming-Lung Yu, Shi-Ming Lin, Chuan-Mo Lee, Chia-Yen Dai, Wen-Yu Chang, Shinn-Cherng Chen, Li-Po Lee, Zu-Yau Lin, Ming-Yuh Hsieh, Liang-Yen Wang, Wan-Long Chuang, Yun-Fan Liaw
Changes in hepatic fibrosis after interferon-based therapy may be important in determining the long-term outcome of chronic hepatitis C (CHC). The use of liver biopsy for posttreatment assessment is not a viable option as a routine follow-up procedure. This study evaluated the predictive value of a simple noninvasive index, the aspartate aminotransferase (AST)-to-platelet ratio index assessed 6 months after end of treatment (APRI-M6). We evaluated APRI-M6, platelet-M6, AST-M6, and -fetoprotein-M6 of 776 CHC patients with interferon-based therapy as well as the parameters at baseline of 562 untreated patients who were evaluated to predict the risk of hepatocellular carcinoma (HCC) and mortality, during a mean follow-up period of 4.75 (1.0-12.2) and 5.15 (1.0-16) years, respectively. Based on analysis of receiver operating characteristics (ROC) and using optimized cutoff point, the APRI-M6 and platelet-M6 had superior prediction models for long-term outcome with area under the curve of 0.870-0.875 and 0.824-0.847, respectively, and accuracy of 78%-81% and 76%-78%, respectively, for interferon-based-treated patients. The predictive values of all 4 parameters were poor in untreated patients. In subgroup analysis, the APRI-M6 provided a more consistent prediction ratio than platelet-M6 for sustained responders and cirrhosis-free subgroups; both parameters had similar prediction power for nonresponders and were unsatisfactory in patients with cirrhosis. According to Cox proportional hazards analysis, cirrhosis and APRI-M6 were the 2 most important factors for predicting HCC. In conclusion, APRI-M6 can accurately predict the long-term outcome of patients subjected to interferon-based treatment. Nevertheless, the data needs further validation, particularly since the predictive accuracy for patients with cirrhosis is low.
Loss of IL-7 receptor alpha-chain (CD127) expression in acute HCV infection associated with viral persistence (p 1098-1109)
Lucy Golden-Mason, James R. Burton Jr, Nicole Castelblanco, Jared Klarquist, Salvador Benlloch, Chia Wang, Hugo R. Rosen
Interleukin-7 (IL-7) is required for the establishment and maintenance of memory CD4+ and CD8+ T lymphocytes, and cells lacking IL-7R (CD127) demonstrate impaired IL-2 secretion and have a short life-span. Chronic HCV is characterized by T cells that are functionally impaired and exhibit an immature phenotype. To investigate the potential role of IL-7/IL-7R in the outcome of HCV infection, we used multiparameter flow cytometry to characterize patients with acute infection (n = 24), long-term chronic infection (12) and normal subjects (13). HCV infection per se resulted in downregulation of CD127 on total CD4+ and CD8+ T lymphocytes as compared to normal controls. Total expression was lowest in those patients who subsequently developed persistence and intermediate in those patients with acute-resolving infection. This reduction affected both naïve and effector/memory T cells. CD127 correlated phenotypically with upregulation of chemokine receptors CCR7 and CXCR4, expression of the anti-apoptotic molecule B cell leukemia/lymphoma 2 (Bcl-2), and enhanced IL-2 production. In six HLA A2-positive patients, we longitudinally tracked tetramer responses to HCV and CMV epitopes; at baseline, reflecting the expression of CD127 on whole T cell populations, viral-specific CTLs in patients who became chronic demonstrated lower CD127. In conclusion, CD127 is a useful marker of functional CD4+ and CD8+ T cells and its expression correlates with virologic outcome of acute HCV. These data provide a mechanistic basis for the observation that CTLs generated in early infection rapidly decline as chronicity is established; CD127 expression should be considered in the design of novel immunotherapeutic approaches.
Randomized controlled study of tenofovir and adefovir in chronic hepatitis B virus and HIV infection: ACTG A5127 (p 1110-1116)
Marion G. Peters, Janet Andersen, Patrick Lynch, Tun Liu, Beverly Alston-Smith, Carol L. Brosgart, Jeffrey M. Jacobson, Victoria A. Johnson, Richard B. Pollard, James F. Rooney, Kenneth E. Sherman, Susan Swindells, Bruce Polsky, ACTG Protocol A5127 Team
Chronic hepatitis B virus (HBV) infection is an important cause of morbidity and mortality in subjects coinfected with HIV. Tenofovir disoproxil fumarate (TDF) and adefovir dipivoxil (ADV) are licensed for the treatment of HIV-1 and HBV infection, respectively, but both have in vivo and in vitro activity against HBV. This study evaluated the anti-HBV activity of TDF compared to ADV in HIV/HBV-coinfected subjects. ACTG A5127 was a prospective randomized, double-blind, placebo-controlled trial of daily 10 mg of ADV versus 300 mg of TDF in subjects with HBV and HIV coinfection on stable ART, with serum HBV DNA 100,000 copies/mL, and plasma HIV-1 RNA 10,000 copies/mL. This study closed early based on results of a prespecified interim review, as the primary noninferiority end point had been met without safety issues. Fifty-two subjects were randomized. At baseline, 73% of subjects had a plasma HIV-1 RNA < 50 copies/mL, 86% were HBeAg positive, 94% were 3TC resistant, median serum ALT was 52 IU/L, and 98% had compensated liver disease. The mean time-weighted average change in serum HBV DNA from baseline to week 48 (DAVG48) was -4.44 log10 copies/mL for TDF and -3.21 log10 copies/mL for ADV. There was no difference in toxicity between the 2 treatment arms, with 11 subjects (5 ADV and 6 TDF) experiencing elevations of serum ALT on treatment. In conclusion, over 48 weeks, treatment with either ADV or TDF resulted in clinically important suppression of serum HBV DNA. Both drugs are safe and efficacious for patients coinfected with HBV and HIV.
The half-life of hepatitis B virions (p 1117-1121)
John M. Murray, Robert H. Purcell, Stefan F. Wieland
The virion half-life of hepatitis B virus (HBV) is currently estimated at approximately 1 day. This estimate has been obtained from drug perturbation experiments with reverse transcriptase inhibitors. However, the analyses of those experiments have not considered the export of virions produced from preformed mature DNA-containing HBV capsids in infected cells. Data from 3 acutely infected chimpanzees indicates that there is approximately 10-fold more total intracellular HBV DNA than HBV DNA in blood, and therefore the half-life of virions for chimpanzees during acute infection is 10-fold shorter at 3.8 hours than the half-life associated with export of total intracellular HBV DNA. Mathematical model simulations duplicating the viral dynamics observed in drug perturbation experiments suggest a half-life of at most 4.4 hours for HBV virions in chronically infected humans, significantly shorter than current estimates, but consistent with the half-lives of virions for hepatitis C virus and HIV. This faster turnover of HBV in blood indicates a correspondingly higher replication rate and risk of mutation against hepatitis B antiviral therapy. In conclusion, we find the half-life of HBV virions is approximately 4 hours, significantly shorter than current estimates of 1 day. This new value is consistent with virion half-life estimates for HIV and hepatitis C virus.
Different signaling pathways in the livers of patients with chronic hepatitis B or chronic hepatitis C (p 1122-1138)
Masao Honda, Taro Yamashita, Teruyuki Ueda, Hajime Takatori, Ryuhei Nishino, Shuichi Kaneko
The clinical manifestations of chronic hepatitis B (CH-B) and chronic hepatitis C (CH-C) are different. We previously reported differences in the gene expression profiles of liver tissue infected with CH-B or CH-C; however, the signaling pathways underlying each condition have yet to be clarified. Using a newly constructed cDNA microarray consisting of 9614 clones selected from 256,550 tags of hepatic serial analysis of gene expression (SAGE) libraries, we compared the gene expression profiles of liver tissue from 24 CH-B patients with those of 23 CH-C patients. Laser capture microdissection was used to isolate hepatocytes from liver lobules and infiltrating lymphoid cells from the portal area, from 16 patients, for gene expression analysis. Furthermore, the comprehensive gene network was analyzed using SAGE libraries of CH-B and CH-C. Supervised and nonsupervised learning methods revealed that gene expression was correlated more with the infecting virus than any other clinical parameters such as histological stage and disease activity. Pro-apoptotic and DNA repair responses were predominant in CH-B with p53 and 14-3-3 interacting genes having an important role. In contrast, inflammatory and anti-apoptotic phenotypes were predominant in CH-C. These differences would evoke different oncogenic factors in CH-B and CH-C. In conclusion, we describe the different signaling pathways induced in the livers of patients with CH-B or CH-C. The results might be useful in guiding therapeutic strategies to prevent the development of hepatocellular carcinoma in cases of CH-B and CH-C.
Hepatitis C virus reinfection in injection drug users (p 1139-1145)
Jason Grebely, Brian Conway, Jesse D. Raffa, Calvin Lai, Mel Krajden, Mark W. Tyndall
Spontaneous clearance of hepatitis C (HCV) may provide protection against reinfection. In a large community-based cohort study of 3,553 inner-city residents (mainly injection drug users), we identified HCV-infected individuals in whom virological clearance had occurred and compared the rate of reinfection in this group with that observed in previously uninfected members of the same cohort. We identified 926 HCV-uninfected and 658 HCV-infected viremic subjects at baseline, with 152 of 658 (23.1%) spontaneously clearing viremia over a median follow-up of 5.2 years (IQR, 2.8-7.4). At baseline, individuals with HCV clearance were more likely to be HIV coinfected (P < .001) and to be engaged in frequent illicit drug use (P = .004) and injection drug use (P < .001). The occurrence of HCV infection was lower in individuals with previous infection (14/152, 9.2%) compared with that in those without previous infection (172/926, 18.6%), with incidence rates of 1.8 (95% CI, 0.9-3.0 cases/100 person-years) and 8.1 (95% CI, 6.9-9.4 cases/100 person-years) cases/100 person-years, respectively, after accounting for follow-up. In a logistic regression analysis, with previous HCV infection assessed as a covariate with other potential confounding variables (age, sex, ethnicity, HIV infection, housing status, and illicit and injection drug use), individuals with previous HCV infection and viral clearance were 4 times less likely to develop infection than those infected for the first time (adjusted odds ratio, 0.23; 95% CI, 0.10-0.51, P < .001). In conclusion, individuals with clearance of HCV infection may have a lower risk of acquiring HCV than individuals who have never been infected, despite ongoing exposure to HCV.
HCV kinetics, quasispecies, and clearance in treated HCV-infected and HCV/HIV-1-coinfected patients with hemophilia (p 1146-1157)
Norah J. Shire, Paul S. Horn, Susan D. Rouster, Sandra Stanford, M. Elaine Eyster, Kenneth E. Sherman, Multicenter Hemophilia Cohort HCV Study Group
Hepatitis C virus (HCV) treatment response rates remain low in HCV/HIV-1-coinfected individuals compared with those with HCV alone. Persons with inherited coagulation disorders have high rates of HCV and HIV-1 infection, but HCV treatment trials in this patient population are scarce. We hypothesized that differences by infection status in HCV viral kinetics would be associated with differences in HCV quasispecies complexity over time and with treatment response disparities. Coinfected and monoinfected patients were enrolled in a treatment trial for pegylated-interferon alpha-2a (peg-IFN) + ribavirin. Patients were treated for 48 weeks and followed for an additional 24. Quantitative HCV RNA was tested at multiple times during and after treatment. Viral kinetic parameters associated with response were estimated with a mathematical model. Quasispecies emergence was determined via heteroduplex complexity assay. Twenty-two patients were HCV RNA-positive at baseline, with no significant demographic or virological differences by infection status. Five of eleven (45%) of monoinfected and 3 of 11 (27%) of coinfected patients achieved sustained viral response (SVR). Peg-IFN efficacy () of 90% or greater was associated with probability of end-of-treatment response (ETR) (P = .001) and SVR (P = .06). Patients with SVR had lower baseline quasispecies complexity than those without SVR (P = .07). Those with of 90% or greater also had lower baseline complexity (P = .07). Coinfection status mediated changes in complexity over time (P = .04). In conclusion, low pretreatment quasispecies complexity may predict peg-IFN response; early peg-IFN response is critical for sustained HCV clearance and is altered in coinfection. Further studies are warranted.
Liver Biology and Pathobiology
Biology
Human UDP-glucuronosyltransferase (UGT)1A3 enzyme conjugates chenodeoxycholic acid in the liver (p 1158-1170)
Jocelyn Trottier, Mélanie Verreault, Susan Grepper, Didier Monté, Julie Bélanger, Jenny Kaeding, Patrick Caron, Ted T. Inaba, Olivier Barbier
Chenodeoxycholic acid (CDCA) is a liver-formed detergent and plays an important role in the control of cholesterol homeostasis. During cholestasis, toxic bile acids (BA) accumulate in hepatocytes causing damage and consequent impairment of their function. Glucuronidation, a conjugation reaction catalyzed by UDP-glucuronosyltransferase (UGT) enzymes, is considered an important metabolic pathway for hepatic BA. This study identifies the human UGT1A3 enzyme as the major enzyme responsible for the hepatic formation of the acyl CDCA-24glucuronide (CDCA-24G). Kinetic analyses revealed that human liver and UGT1A3 catalyze the formation of CDCA-24G with similar Km values of 10.6 to 18.6 mol/L, respectively. In addition, electrophoretic mobility shift assays and transient transfection experiments revealed that glucuronidation reduces the ability of CDCA to act as an activator of the nuclear farnesoid X-receptor (FXR). Finally, we observed that treatment of human hepatocytes with fibrates increases the expression and activity of UGT1A3, whereas CDCA has no effect. In conclusion, UGT1A3 is the main UGT enzyme for the hepatic formation of CDCA-24G and glucuronidation inhibits the ability of CDCA to act as an FXR activator. In vitro data also suggest that fibrates may favor the formation of bile acid glucuronides in cholestatic patients.
In vivo immune modulatory activity of hepatic stellate cells in mice (p 1171-1181)
Cheng-Hsu Chen, Liang-Mou Kuo, Yigang Chang, Wenhan Wu, Christina Goldbach, Mark A. Ross, Donna B. Stolz, Liepin Chen, John J. Fung, Lin Lu, Shiguang Qian
Accumulating data suggest that hepatic tolerance, initially demonstrated by spontaneous acceptance of liver allografts in many species, results from an immune regulatory activity occurring in the liver. However, the responsible cellular and molecular components have not been completely understood. We have recently described profound T cell inhibitory activity of hepatic stellate cells (HSCs) in vitro. In this study, we demonstrate in vivo evidence of immune modulatory activity of HSCs in mice using an islet transplantation model. Co-transplanted HSCs effectively protected islet allografts from rejection, forming a multi-layered capsule, which reduced allograft immunocyte infiltrates by enhancement of apoptotic death. The immune modulation by HSCs appeared to be a local effect, and regulated by inducible expression of B7-H1, an inhibitory molecule of B7 family. This may reflect an intrinsic mechanism of immune inhibition mediated by liver-derived tissue cells. In conclusion, these results may lead to better understanding of liver immunobiology and development of new strategies for treatment of liver diseases.
T lymphocytes interact with hepatocytes through fenestrations in murine liver sinusoidal endothelial cells (p 1182-1190)
Alessandra Warren, David G. Le Couteur, Robin Fraser, David G. Bowen, Geoffrey W. McCaughan, Patrick Bertolino
The liver has an established ability to induce tolerance. Recent evidence indicates that this unique property might be related to its distinctive architecture allowing T cells to be activated in situ independently of lymphoid tissues. Unlike lymph node-activated T cells, liver-activated T cells are short-lived, a mechanism that might contribute to the liver tolerance effect. Although the potential role of hepatocytes as tolerogenic antigen-presenting cells has been demonstrated, the question as to whether these cells are able to interact with CD8+ T cells in physiological settings remains controversial. Contradicting the immunological dogma stating that naïve T lymphocytes are prevented from interacting with parenchymal cells within non-lymphoid organs by an impenetrable endothelial barrier, we show here that the unique morphology of the liver sinusoidal endothelial cell (LSEC) permits interactions between lymphocytes and hepatocytes. Using electron microscopy, we demonstrate that liver resident lymphocytes as well as circulating naïve CD8+ T cells make direct contact with hepatocytes through cytoplasmic extensions penetrating the endothelial fenestrations that perforate the LSECs. Furthermore, the expression of molecules required for primary T cell activation, MHC class I and ICAM-1, is polarized on hepatocytes to the perisinusoidal cell membrane, thus maximizing the opportunity for interactions with circulating lymphocytes. In conclusion, this study has identified, at the ultrastructural level, a unique type of interaction between naïve T lymphocytes and liver parenchymal cells in vivo. These results hold implications for the pathogenesis of viral hepatitis in which hepatocytes may represent the main antigen-presenting cell, and for the development of immune tolerance as lymphocytes pass through the liver.
Pathobiology
Protection against Western diet-induced obesity and hepatic steatosis in liver fatty acid-binding protein knockout mice (p 1191-1205)
Elizabeth P. Newberry, Yan Xie, Susan M. Kennedy, Jianyang Luo, Nicholas O. Davidson
Liver fatty acid-binding protein (L-Fabp) regulates murine hepatic fatty acid trafficking in response to fasting. In this study, we show that L-Fabp-/- mice fed a high-fat Western diet for up to 18 weeks are less obese and accumulate less hepatic triglyceride than C57BL/6J controls. Paradoxically, both control and L-Fabp-/- mice manifested comparable glucose intolerance and insulin resistance when fed a Western diet. Protection against obesity in Western diet-fed L-Fabp-/- mice was not due to discernable changes in food intake, fat malabsorption, or heat production, although intestinal lipid secretion kinetics were significantly slower in both chow-fed and Western diet-fed L-Fabp-/- mice. By contrast, there was a significant increase in the respiratory exchange ratio in L-Fabp-/- mice, suggesting a shift in energy substrate use from fat to carbohydrate, findings supported by an approximately threefold increase in serum lactate. Microarray analysis revealed increased expression of genes involved in lipid synthesis (fatty acid synthase, squalene epoxidase, hydroxy-methylglutaryl coenzyme A reductase), while genes involved in glycolysis (glucokinase and glycerol kinase) were decreased in L-Fabp-/- mice. Fatty acid synthase expression was also increased in the skeletal muscle of L-Fabp-/- mice. In conclusion, L-Fabp may function as a metabolic sensor in regulating lipid homeostasis. We suggest that L-Fabp-/- mice are protected against Western diet-induced obesity and hepatic steatosis through a series of adaptations in both hepatic and extrahepatic energy substrate use.
Bile acids inhibit interleukin-6 signaling via gp130 receptor-dependent and -independent pathways in rat liver (p 1206-1217)
Dirk Graf, Caroline Kohlmann, Katrin Haselow, Thor Gehrmann, Johannes G. Bode, Dieter Häussinger
Interleukin-6 (IL-6) is a major regulator of the acute phase reaction in the liver and is thought to mediate protective effects in response to hepatotoxins. In this study, the influence of bile acids on IL-6 signal transduction was analyzed. It was shown that hydrophobic bile acids such as glycochenodeoxycholate (GCDC) inhibited IL-6-induced tyrosine phosphorylation of signal transducer and activator of transcription (STAT) 3 in hepatocytes and in perfused rat liver. This inhibition was accompanied by GCDC-mediated downregulation of glycoprotein (gp) 130 expression, whereas gp130 and suppressor of cytokine signaling 3 messenger RNA and gp80 protein levels remained unaffected. The GCDC-induced downregulation of gp130 protein expression was insensitive to inhibition of proteasomal or lysosomal protein degradation but turned out to be sensitive to inhibition of caspase-3 or caspase-8 activity. Accordingly, treatment of cell extracts with active recombinant caspase-3 led to a decay of immunoreactive gp130. Moreover, activation of caspases by CD95 ligand or hyperosmotic stress also resulted in a downregulation of gp130 levels. This indicates that caspase activation antagonizes IL-6 signaling by decay of gp130 levels. However, caspase inhibition did not prevent GCDC-dependent inhibition of IL-6-induced STAT3 activation, which turned out to be at least partially sensitive to suppression of p38MAPK activation. In conclusion, hydrophobic bile acids compromise IL-6 signaling through both a caspase-mediated downregulation of gp130 and a p38MAPK-dependent inhibition of STAT3 phosphorylation. This may contribute to bile acid-induced hepatotoxicity in cholestasis through counteracting the known hepatoprotective effects of IL-6.
Inactivation of oxidized and S-nitrosylated mitochondrial proteins in alcoholic fatty liver of rats (p 1218-1230)
Kwan-Hoon Moon, Brian L. Hood, Bong-Jo Kim, James P. Hardwick, Thomas P. Conrads, Timothy D. Veenstra, Byoung J. Song
Increased oxidative/nitrosative stress is a major contributing factor to alcohol-mediated mitochondrial dysfunction. However, which mitochondrial proteins are oxidatively modified under alcohol-induced oxidative/nitrosative stress is poorly understood. The aim of this study was to systematically investigate oxidized and/or S-nitrosylated mitochondrial proteins and to use a biotin-N-maleimide probe to evaluate their inactivation in alcoholic fatty livers of rats. Binge or chronic alcohol exposure significantly elevated nitric oxide, inducible nitric oxide synthase, and ethanol-inducible CYP2E1. The biotin-N-maleimide-labeled oxidized and/or S-nitrosylated mitochondrial proteins from pair-fed controls or alcohol-fed rat livers were subsequently purified with streptavidin-agarose. The overall patterns of oxidized and/or S-nitrosylated proteins resolved by 2-dimensional polyacrylamide gel electrophoresis were very similar in the chronic and binge alcohol treatment groups. Seventy-nine proteins that displayed differential spot intensities from those of control rats were identified by mass spectrometry. These include mitochondrial aldehyde dehydrogenase 2 (ALDH2), ATP synthase, acyl-CoA dehydrogenase, 3-ketoacyl-CoA thiolase, and many proteins involved in chaperone activity, mitochondrial electron transfer, and ion transport. The activity of 3-ketoacyl-CoA thiolase involved in mitochondrial -oxidation of fatty acids was significantly inhibited in alcohol-exposed rat livers, consistent with hepatic fat accumulation, as determined by biochemical and histological analyses. Measurement of activity and immunoblot results showed that ALDH2 and ATP synthase were also inhibited through oxidative modification of their cysteine or tyrosine residues in alcoholic fatty livers of rats. In conclusion, our results help to explain the underlying mechanism for mitochondrial dysfunction and increased susceptibility to alcohol-mediated liver damage.
Cellular and humoral autoimmunity directed at bile duct epithelia in murine biliary atresia (p 1231-1239)
Cara L. Mack, Rebecca M. Tucker, Brandy R. Lu, Ronald J. Sokol, Andrew P. Fontenot, Yoshiyuki Ueno, Ronald G. Gill
Biliary atresia is an inflammatory fibrosclerosing lesion of the bile ducts that leads to biliary cirrhosis and is the most frequent indication for liver transplantation in children. The pathogenesis of biliary atresia is not known; one theory is that of a virus-induced, subsequent autoimmune-mediated injury of bile ducts. The aim of this study was to determine whether autoreactive T cells and autoantibodies specific to bile duct epithelia are present in the rotavirus (RRV)- induced murine model of biliary atresia and whether the T cells are sufficient to result in bile duct inflammation. In vitro analyses showed significant increases in IFN--producing T cells from RRV-diseased mice in response to bile duct epithelial autoantigen. Adoptive transfer of the T cells from RRV-diseased mice into naïve syngeneic SCID recipients resulted in bile duct-specific inflammation. This induction of bile duct pathology occurred in the absence of detectable virus, indicating a definite response to bile duct autoantigens. Furthermore, periductal immunoglobulin deposits and serum antibodies reactive to bile duct epithelial protein were detected in RRV-diseased mice. In conclusion, both cellular and humoral components of autoimmunity exist in murine biliary atresia, and the progressive bile duct injury is due in part to a bile duct epithelia-specific T cell-mediated immune response. The role of cellular and humoral autoimmunity in human biliary atresia and possible interventional strategies therefore should be the focus of future research.
IL-2 receptor -/- mice and the development of primary biliary cirrhosis (p 1240-1249)
Kanji Wakabayashi, Zhe-Xiong Lian, Yuki Moritoki, Ruth Y. Lan, Koichi Tsuneyama, Ya-Hui Chuang, Guo-Xiang Yang, William Ridgway, Yoshiyuki Ueno, Aftab A. Ansari, Ross L. Coppel, Ian R. Mackay, M. Eric Gershwin
Recently, we identified a child born with a genetic deficiency of IL-2 receptor (IL-2R, CD25) expression who had several clinical manifestations of primary biliary cirrhosis (PBC). In addition, there has been suggestive evidence in both patients with PBC and their first-degree relatives that a deficiency of regulatory T cells (Tregs) is an integral component for susceptibility to PBC. Based on these observations, we generated IL-2R/CD25 deficient (IL-2R-/-) mice and wild-type littermate controls and followed them longitudinally for the natural history of liver immunopathology and appearance of antimitochondrial antibodies (AMAs). The analyses included immunohistochemical staining of liver and portal tract infiltrates as well as FACS profiles of lymphoid subpopulations in liver and spleen. In addition, serum cytokine profiles were quantitated. Importantly, IL-2R-/-, but not littermate controls, develop portal inflammation and biliary ductular damage similar to human patients with PBC. CD4+ and CD8+ T cells predominate among portal cell infiltrates and sera reflect a Th1 cytokine bias with increased levels of IFN-, TNF-, IL-2 and IL-12p40. Of importance is the finding that the IL-2R-/- mice not only develop significantly increased serum levels of IgG and IgA, but they also develop AMAs with specificity for PDC-E2, which maps to the inner lipoyl domain of the autoantigen, all characteristics which are hallmarks of human PBC. In conclusion, the IL-2R-/- mice should facilitate studies of the early events in PBC and especially the tantalizing connection between Treg deficiency and autoimmunity specifically directed to mitochondrially located PDC-E2 and subsequent biliary ductular cell damage.
Noninvasive evaluation of liver repopulation by transplanted hepatocytes using 31P MRS imaging in mice (p 1250-1258)
Charles S. Landis, Kosho Yamanouchi, Hongchao Zhou, Sankar Mohan, Namita Roy-Chowdhury, David A. Shafritz, Alan Koretsky, Jayanta Roy-Chowdhury, Hoby P. Hepatocyte transplantation (HT) is being explored as a substitute for liver transplantation for the treatment of liver diseases. For the clinical application of HT, a preparative regimen that allows preferential proliferation of transplanted cells in the host liver and a noninvasive method to monitor donor cell engraftment, proliferation, and immune rejection would be useful. We describe an imaging method that employs the creatine kinase (CK) gene as a marker of donor hepatocytes. Creatine kinase is unique among marker genes, because it is normally expressed in brain and muscle tissues and is therefore not immunogenic. Preferential proliferation of transplanted CK-expressing hepatocytes was induced by preparative hepatic irradiation and expression of hepatocyte growth factor using a recombinant adenoviral vector. CK is normally not expressed in mouse liver and its expression by the donor cells led to the production of phosphocreatine in the host liver, permitting 31P magnetic resonance spectroscopic imaging of liver repopulation by engrafted hepatocytes. In conclusion, this study combined a noninvasive imaging technique to assess donor hepatocyte proliferation with a preparative regimen of partial liver irradiation that allowed regional repopulation of the host liver. Our results provide groundwork for future development of clinical protocols for HT.
Plasma cholesterol is hyperresponsive to statin in ABCG5/ABCG8 transgenic mice (p 1259-1266)
Weiqing Tang, Yinyan Ma, Liqing Yu
Interindividual variation exists in response to statin therapy. It has been hypothesized that subjects with higher baseline cholesterol synthesis rates are more sensitive to statins. To directly test this hypothesis, mice overexpressing the heterodimeric ATP-binding cassette (ABC) transporter G5/G8 (G5G8Tg mice) were treated with lovastatin because they have a compensatory increase in cholesterol biosynthesis as a result of increased cholesterol excretion into bile and feces. As expected, lovastatin treatment did not alter plasma and hepatic cholesterol levels in wild-type mice. Interestingly, this treatment significantly reduced plasma concentration and hepatic content of cholesterol by 42% and 17.3%, respectively, in the statin-treated versus untreated G5G8Tg mice despite a greater feedback upregulation of genes in the pathway of cholesterol biosynthesis in the lovastatin-treated G5G8Tg mice. The reduced plasma cholesterol concentration is unlikely to be attributed to LDL and HDL receptors because the protein levels of both receptors remained unchanged. Surprisingly, statin treatment resulted in an increase in biliary cholesterol concentration, which was associated with an upregulation in hepatic mRNA and protein levels of ABCG5 and ABCG8, and in hepatic mRNA levels of Niemann-Pick C1-Like 1 (NPC1L1), a gene that is required for intestinal cholesterol absorption. In conclusion, mice with higher endogenous cholesterol synthesis rates are more sensitive to statin. A synergistic hypocholesterolemic effect could be potentially achieved in humans by simultaneously inhibiting cholesterol biosynthesis and promoting ABCG5/ABCG8-mediated cholesterol excretion.
Sustained activation of Rac1 in hepatic stellate cells promotes liver injury and fibrosis in mice (p 1267-1277)
Steve S. Choi, Jason K. Sicklick, Qi Ma, Liu Yang, Jiawen Huang, Yi Qi, Wei Chen, Yin-Xiong Li, Pascal J. Goldschmidt-Clermont, Anna Mae Diehl
Rac, a small, GTP-binding protein in the Rho family, regulates several cellular functions, including the activation of NADPH oxidase, a major intracellular producer of reactive oxygen species (ROS). Hepatic stellate cells (HSCs) isolated from mice that are genetically deficient in NADPH oxidase produce less ROS, and their activation during chronic liver injury is abrogated, resulting in decreased liver fibrosis. Therefore, we hypothesized that HSC ROS production and activation would be enhanced, and fibrosis worsened, by increasing Rac expression in HSCs. To achieve this, we used transgenic mice that express constitutively active human Rac1 under the control of the -smooth muscle actin (-sma) promoter, because -sma expression is induced spontaneously during HSC activation. Transgene expression was upregulated progressively during culture of primary Rac-transgenic HSCs, and this increased HSC ROS production as well as expression of activation markers and collagen. Similarly, Rac mice treated with carbon tetrachloride (CCl4) accumulated greater numbers of activated HSCs and had more liver damage, hepatocyte apoptosis, and liver fibrosis - as well as higher mortality - than CCl4-treated wild-type mice. In conclusion, sustained activation of Rac in HSCs perpetuates their activation and exacerbates toxin-induced liver injury and fibrosis, prompting speculation that Rac may be a therapeutic target in patients with cirrhosis.
Sphingosine 1-phosphate protects rat liver sinusoidal endothelial cells from ethanol-induced apoptosis: Role of intracellular calcium and nitric oxide (p 1278-1287)
Dong-Mei Zheng, Tsuneo Kitamura, Kenichi Ikejima, Nobuyuki Enomoto, Shunhei Yamashina, Satoko Suzuki, Yoshiyuki Takei, Nobuhiro Sato
In alcoholic liver disease, ethanol-induced damage to sinusoidal endothelial cells (SECs) appears to be important in the progression of liver damage. However, little is known about the mechanisms responsible for protection of SECs against ethanol-induced injury. To elucidate the role of sphingosine 1-phosphate (S1P), which is stored in platelets and may be released from them on their activation, we investigated the effect of S1P on rat liver SECs in primary culture. Pretreatment of cells with 1 mol/L S1P attenuated ethanol-induced apoptosis. Electron microscopy confirmed this protective effect of S1P on damaged SECs in liver tissues after perfusion of ethanol. In the absence of ethanol, S1P increased DNA synthesis as determined via incorporation of bromodeoxyuridine. S1P also ameliorated the decreased DNA synthesis of cells induced by ethanol. Addition of S1P to cells induced an increase in intracellular calcium concentrations and NO production in cells. Western blotting revealed that S1P significantly induced the activation of endothelial NO synthase (eNOS), but not Akt, and that S1P-induced activation of eNOS was blocked by trifluoperazine, a calmodulin inhibitor. Furthermore, NG-nitro-L-arginine methyl ester, a NO synthase inhibitor, cancelled the effect of S1P on DNA synthesis, apoptosis, and NO production in vitro as well as the protective effect of S1P on cell damage in situ. In conclusion, the biological effect of S1P is at least partially mediated by Ca2+-sensitive eNOS activation and subsequent NO formation; extracellular S1P could contribute to sinusoidal protection and remodeling in alcoholic liver injury.
Liver Failure and Liver Disease
Liver Failure
Adrenal insufficiency in patients with cirrhosis and septic shock: Effect of treatment with hydrocortisone on survival (p 1288-1295)
Javier Fernández, Angels Escorsell, Michel Zabalza, Vanessa Felipe, Miguel Navasa, Antoni Mas, Antonio M. Lacy, Pere Ginès, Vicente Arroyo
Relative adrenal insufficiency is frequent in patients with severe sepsis and is associated with hemodynamic instability, renal failure, and increased mortality. This study prospectively evaluated the effects of steroids on shock resolution and hospital survival in a series of 25 consecutive patients with cirrhosis and septic shock (group 1). Adrenal function was evaluated by the short corticotropin test within the first 24 hours of admission. Patients with adrenal insufficiency were treated with stress doses of intravenous hydrocortisone (50 mg/6 h). Data were compared to those obtained from the last 50 consecutive patients with cirrhosis and septic shock admitted to the same intensive care unit in whom adrenal function was not investigated and who did not receive treatment with steroids (group 2). Incidence of adrenal insufficiency in group 1 was 68% (17 patients). Adrenal dysfunction was frequent in patients with advanced cirrhosis (Child C: 76% vs. Child B: 25%, P = .08). Resolution of septic shock (96% vs. 58%, P = .001), survival in the intensive care unit (68% vs. 38%, P = .03), and hospital survival (64% vs. 32%, P = .003) were significantly higher in group 1. The main causes of death in group 1 were hepatorenal syndrome or liver failure (7 of 9 patients). In contrast, refractory shock caused most of the deaths in group 2 (20 of 34 patients). In conclusion, relative adrenal insufficiency is very frequent in patients with advanced cirrhosis and septic shock. Hydrocortisone administration in these patients is associated with a high frequency of shock resolution and high survival rate.
Upregulation of the tumor suppressor gene menin in hepatocellular carcinomas and its significance in fibrogenesis (p 1296-1307)
Pierre J. Zindy, Annie L'Helgoualc'h, Dominique Bonnier, Antony Le Béchec, Katia Bourd-Boitin, Chang Xian Zhang, Orlando Musso, Denise Glaise, Marie Bérangère Troadec, Olivier Loréal, Bruno Turlin, Jean Léger, Bruno Clément, Nathalie Théret
The molecular mechanisms underlying the progression of cirrhosis toward hepatocellular carcinoma were investigated by a combination of DNA microarray analysis and literature data mining. By using a microarray screening of suppression subtractive hybridization cDNA libraries, we first analyzed genes differentially expressed in tumor and nontumor livers with cirrhosis from 15 patients with hepatocellular carcinomas. Seventy-four genes were similarly recovered in tumor (57.8% of differentially expressed genes) and adjacent nontumor tissues (64% of differentially expressed genes) compared with histologically normal livers. Gene ontology analyses revealed that downregulated genes (n = 35) were mostly associated with hepatic functions. Upregulated genes (n = 39) included both known genes associated with extracellular matrix remodeling, cell communication, metabolism, and post-transcriptional regulation gene (e.g., ZFP36L1), as well as the tumor suppressor gene menin (multiple endocrine neoplasia type 1; MEN1). MEN1 was further identified as an important node of a regulatory network graph that integrated array data with array-independent literature mining. Upregulation of MEN1 in tumor was confirmed in an independent set of samples and associated with tumor size (P = .016). In the underlying liver with cirrhosis, increased steady-state MEN1 mRNA levels were correlated with those of collagen 2(I) mRNA (P < .01). In addition, MEN1 expression was associated with hepatic stellate cell activation during fibrogenesis and involved in transforming growth factor beta (TGF-)-dependent collagen 2(I) regulation. In conclusion, menin is a key regulator of gene networks that are activated in fibrogenesis associated with hepatocellular carcinoma through the modulation of TGF- response.
Liver Disease
Aiming at minimal invasiveness as a therapeutic strategy for Budd-Chiari syndrome (p 1308-1316)
Aurélie Plessier, Annie Sibert, Yann Consigny, Antoine Hakime, Magaly Zappa, Marie-Hélène Denninger, Bertrand Condat, Olivier Farges, Carine Chagneau, Victor de Ledinghen, Claire Francoz, Alain Sauvanet, Valérie Vilgrain, Jacques Belghiti, François Durand, Dominique Valla
The 1-year spontaneous mortality rate in patients with Budd-Chiari syndrome (BCS) approaches 70%. No prospective assessment of indications and impact on survival of current therapeutic procedures has been performed. We evaluated a therapeutic strategy uniformly applied during the last 8 years in a single referral center. Fifty-one consecutive patients first received anticoagulation and were treated for associated diseases. Symptomatic patients were considered for hepatic vein recanalization; then for transjugular intrahepatic portosystemic shunt (TIPS), and finally for liver transplantation. The absence of a complete response led to the next procedure. Assessment was according to the strategy, whether procedures were technically applicable and successful. At entry, median (range) Child-Pugh score and Clichy prognostic index were 8 (5-12), and 5.4 (3.1-7.7), respectively. A complete response was achieved on medical therapy alone in 9 patients; after recanalization in 6, TIPS in 20, liver transplantation in 9, and retransplantation in 1. Of the 41 patients considered for recanalization, the procedure was not feasible in 27 and technically unsuccessful in 3. Of the 34 patients considered for TIPS, the procedure was considered not feasible in 9 and technically unsuccessful in 4. At 1 year of follow-up, a complete response to TIPS was achieved in 84%. One- and 5-year survival from starting anticoagulation were 96% (95% CI, 90-100) and 89% (95% CI, 79-100), respectively. In conclusion, excellent survival can be achieved in BCS patients when therapeutic procedures are introduced by order of increasing invasiveness, based on the response to previous therapy rather than on the severity of the patient's condition.
Gabapentin in patients with the pruritus of cholestasis: A double-blind, randomized, placebo-controlled trial (p 1317-1323)
Nora V. Bergasa, Monnie McGee, Iona H. Ginsburg, Danielle Engler
Pruritus is defined as the second order of nociception, the first being pain; thus, there is a rationale to study gabapentin, a drug that increases the threshold to experience nociception. The aim of this double-blind, randomized, placebo-controlled trial was to study the effect of gabapentin on the perception of pruritus and its behavioral manifestation, scratching, in cholestasis. The participants were 16 women with chronic liver disease and chronic pruritus. Hourly scratching activity (HSA) was continuously recorded for up to 48 hours at baseline and on treatment for at least 4 weeks in an inpatient setting. The perception of pruritus was assessed by interviews and by a visual analog score (VAS) of pruritus recorded every hour while patients were awake. Patients were randomized to the study drug (gabapentin or placebo) at a starting dose of 300 mg orally per day in divided doses to a maximum of 2,400 mg or until relief from pruritus. Gabapentin was associated with an increase in mean HSA, in contrast to the placebo, which was associated with a decrease. The mean VAS decreased significantly among those taking the placebo and in some patients on gabapentin. In conclusion, gabapentin did not provide a significant therapeutic advantage over the placebo; in fact, it was associated with an increase in the perception of pruritus and in HSA in some patients.
Gilbert's disease and atazanavir: From phenotype to UDP-glucuronosyltransferase haplotype (p 1324-1332)
Tim O. Lankisch, Ulrike Moebius, Michael Wehmeier, Georg Behrens, Michael P. Manns, Reinhold E. Schmidt, Christian P. Strassburg
Gilbert's disease leads to intermittent non-hemolytic hyperbilirubinemia by a reduction of hepatic bilirubin glucuronidation associated with the presence of the UDP-glucuronosyltransferase (UGT) 1A1*28 polymorphism. It is considered benign because it does not result in hepatocellular damage. However, pharmacogenetic analyses have linked UGT1A1*28 to drug toxicity and cancer predisposition. The protease inhibitor atazanavir (ATV) is an inhibitor of hepatic UGT activity leading to hyperbilirubinemia in individual patients. Whether this is linked specifically to UGT1A1*28 or to more complex variants influencing glucuronidation is unclear. One hundred and six ATV-treated patients were characterized and genotyped for UGT1A1*28, the UGT1A3 (-66C) and UGT1A7 (-57G) promoter variants, and UGT1A7129K/131K. ATV treatment increased median bilirubin levels from 10 to 41 mol/L (P = .001) with hyperbilirubinemia exceeding 43 mol/L in 37%. Hyperbilirubinemia over 43 mol/L was significantly associated not only with UGT1A1*28 but also with UGT1A3-66C, UGT1A7-57G, and UGT1A7129K/131K, although these variants do not naturally occur in linkage dysequilibrium in blood donors. Homozygous combinations of UGT1A1*28 with the other variants increased from 7.4% (normal bilirubin to 42 mol/L) to 41% to 46.1% (43 to >85 mol/L), and 100% (>85 mol/L). All six patients with hyperbilirubinemia greater than 85 mol/L were homozygous for all four variants identifying a haplotype inherited on a single allele. In conclusion, the genetic variant associated with Gilbert's disease is identified as part of a haplotype of four UGT1A variants spanning three genes at the UGT1A gene locus. This haplotype predisposes to hyperbilirubinemia in ATV treatment and may have an additional role as a pharmacogenomic risk factor for drug therapy.
Biliary papillary tumors share pathological features with intraductal papillary mucinous neoplasm of the pancreas (p 1333-1343)
Yoh Zen, Takahiko Fujii, Keita Itatsu, Koichi Nakamura, Hiroshi Minato, Satomi Kasashima, Hiroshi Kurumaya, Kazuyoshi Katayanagi, Atsuhiro Kawashima, Shinji Masuda, Hideki Niwa, Takeshi Mitsui, Yasuyuki Asada, Shouji Miura, Tetsuo Ohta, Yasuni Nakanuma
Recently, attention has been drawn to papillary neoplasm of the pancreatobiliary systems. In the pancreas, the disease entity of intraductal papillary mucinous neoplasm (IPMN-P) is widely recognized. In contrast, the pathological characteristics of biliary papillary tumors, such as biliary papilloma(tosis) and papillary cholangiocarcinoma, have not yet been well documented. In this study, we compared the pathological features and post-operative prognosis among biliary papillary tumors (10 cases of biliary papilloma(tosis) and 22 cases of papillary cholangiocarcinoma), conventional non-papillary cholangiocarcinoma (15 cases), and IPMN-P (31 cases). Macroscopically, all biliary papillary tumors were characterized by the prominent intraductal papillary proliferation, and macroscopic mucin-hypersecretion was seen in 9 of 32 cases (28%). Histologically, biliary papillary tumors consisted of three types of tumor cells (pancreaticobiliary, intestinal and gastric types), whereas only the pancreaticobiliary type was observed in non-papillary cholangiocarcinoma. Immunohistochemically, biliary papillary tumors were characterized by the common expression of MUC2, CDX2 and cytokeratin 20. In addition, biliary papillary tumors could be associated with two types of invasive lesions: tubular adenocarcinoma (9 cases) and mucinous carcinoma (5 cases). Patients with tubular adenocarcinoma had a poor prognosis compared to non-invasive papillary tumor or papillary tumor with mucinous carcinoma. These pathological characteristics and the survival status of biliary papillary tumors were different from those of non-papillary cholangiocarcinoma, and rather closely resembled those of IPMN-P. In conclusion, biliary papillary tumors may be the biliary counterpart (intraductal papillary neoplasm of the bile duct) of IPMN-P.
Copyright © 2006 by the American Association for the Study of Liver Diseases. All rights reserved.
Novembre 2006 en attente de Parution
© 2006 American Gastroenterological Association (AGA) Institute. Published by Elsevier Inc. All rights reserved.
Volume 45, Issue 5, November 2006
"Seuls les abstracts des articles Originaux sont mis sur cette page. Les autres articles sont en ligne en FREE sur le site du Journal"
Original Articles
Viral Hepatitis
Stable HepG2- and Huh7-based human hepatoma cell lines for efficient regulated expression of infectious hepatitis B virus, 7 July 2006
Sun D, Nassal M
Background/Aims
Hepatitis B virus (HBV) cannot be propagated in cultured cells but two human hepatoma cell lines, HepG2 and Huh7, support virus replication when transfected with HBV DNA. If standardization is required stably transfected cell lines provide distinct advantages. One such line, HepG2.2.15, is widely used in antiviral research but HBV production is limited and difficult to control. Our aim was to establish stable, inducibly HBV producing HepG2 and Huh7 cell lines that overcome these limitations.
Methods
Based on the tetracycline (Tet)-regulated TetOFF system, a Tet-responsive promoter-controlled HBV genome was introduced into separately established, well-regulatable HepG2 and Huh7 lines expressing Tet-responsive trans-activators (tTAs). Stable clones were analyzed for regulatability and levels of HBV expression, quality of the virus produced, and responsiveness towards antivirals.
Results
HepG2- and Huh7-based cell lines were established which, Tet-controllably, produce more HBV than HepG2.2.15 cells. The secreted virions were infectious for primary tupaia hepatocytes, and the cell lines responded as well as HepG2.215 cells to different antivirals.
Conclusions
The new HBV cell lines should be valuable tools for academic and pharmaceutical HBV research. The parental tTA-cells will facilitate the generation of additional lines, producing HBV variants, or other genes, in an identical host cell background.
Specific mutations in enhancer II/core promoter of hepatitis B virus subgenotypes C1/C2 increase the risk of hepatocellular carcinoma, 10 August 2006
Tanaka Y, Mukaide M, Orito E, Yuen MF, Ito K, Kurbanov F, Sugauchi F, Asahina Y, Izumi N, Kato M, Lai CL, Ueda R, Mizokami M
Background/Aims
Hepatitis B virus genotype C (HBV/C) has been classified into two geographically distinct subgenotypes; HBV/C1/Cs (Southeast Asia) and HBV/C2/Ce (East Asia).
Methods
Viral differences in enhancer II/core promoter and precore regions between the subgenotypes and their association with hepatocellular carcinoma (HCC) were assessed in a matched cross-sectional control study of 118 carriers (from Hong Kong) with HBV/C1/Cs (48.0 years, 81% male, 40% HBeAg+, 44% HCC) and 210 HBV/C2/Ce (172 from Japan, 38 from Hong Kong) (50.2 years, 78% male, 30% HBeAg+, 46% HCC).
Results
Univariate analyses showed that mutation V1753 was predictive for HCC among HBeAg-positive-C1/Cs-carriers (P=0.0055), and T1653 among HBeAg-positive-C2/Ce-carriers (P=0.018), and T1653 or V1753 or T1762/A1764 among HBeAg-negative-C2/Ce-carriers (P<0.05). In the multivariate analysis on all HBV/C subjects, independent predictive factors for HCC were subgenotype C2/Ce (odds ratio, 4.21; 95% confidence interval, 1.07–16.23), T1653 (3.64; 1.93–6.86), V1753 (3.07; 1.66–5.65) and T1762/A1764 (2.58; 1.21–5.49) mutations, age (?50 years), gender (male) and HBeAg (positive).
Conclusions
Our data indicate that T1653 and/or V1753 mutations in addition to T1762/A1764 are differently associated with HCC in context of HBeAg status among HBV/C1/Cs and C2/Ce-carriers. HBV/C subgenotypes have specific mutation patterns, which is probably responsible for increased carcinogenesis of HBV/C2/Ce.
Cirrhosis and its Complications
Hypolocomotion in rats with chronic liver failure is due to increased glutamate and activation of metabotropic glutamate receptors in substantia nigra, 10 August 2006
Cauli O, Llansola M, Erceg S, Felipo V
Background/Aims
Patients with hepatic encephalopathy show altered motor function, psychomotor slowing and hypokinesia. The underlying mechanisms remain unclear. This work’s aims were: (1) to analyse in rats with chronic liver failure due to portacaval shunt (PCS) the neurochemical alterations in the basal ganglia–thalamus–cortex circuits; (2) to correlate these alterations with those in motor function and (3) to normalize motor activity of PCS rats by pharmacological means.
Methods
Extracellular neurotransmitters levels were analysed by in vivo brain microdialysys. Motor activity was determined by counting crossings in open field.
Results
Extracellular glutamate is increased in substantia nigra pars reticulata (SNr) of PCS rats. Blocking metabotropic receptor 1 (mGluR1) in SNr normalizes motor activity in PCS rats. In ventro-medial thalamus of PCS rats GABA is increased and it is normalized by blocking mGluR1 in SNr. Blocking mGluR1 in SNr increases and mGluR1 activation reduces glutamate in motor cortex and motor activity.
Conclusions
Increased extracellular glutamate and activation of mGluR1 in SNr are responsible for reduced motor activity in rats with chronic liver failure. Blocking mGluR1 in SNr normalizes motor activity in PCS rats, suggesting that, under appropriate conditions, similar treatments could be useful to treat the psychomotor slowing and hypokinesia in patients with hepatic encephalopathy.
Liver Failure, Growth and Cancer
Interferon-gamma produced by interleukin-12-activated tumor infiltrating CD8+T cells directly induces apoptosis of mouse hepatocellular carcinoma, 6 July 2006
Komita H, Homma S, Saotome H, Zeniya M, Ohno T, Toda G
Background/Aims
Interleukin-12 (IL-12), a cytokine with antitumor activity, was examined for the suppressive effect on hepatocellular carcinoma (HCC) in mouse model, and its mechanism of antitumor activity was analyzed.
Methods
Mice implanted with MIH-2 HCC cells were treated with recombinant mouse IL-12 (500ng/mouse). Involvement of CD4+, CD8+, NK cells and interferon (IFN)-gamma on tumor suppression by IL-12 was examined by treatment of mice with each antibody. Interferon-gamma (IFN-gamma) production by tumor infiltrating cells was analyzed by immunofluorescence microscopy and flow cytometric analysis. Signal transduction for apoptosis induction was examined by immunoblot analysis.
Results
The growth of implanted MIH-2 tumors was significantly suppressed by IL-12 and the suppression was inhibited by depletion of CD8+T cells. IL-12 treatment caused numerous IFN-gamma-producing CD8+T cells to infiltrate into MIH-2 tumors. Antitumor activity of IL-12 was blocked by treating mice with anti-IFN-gamma mAb. CD8+T cells from IL-12-treated mice attached to MIH-2 cells and produced IFN-gamma in vitro. Cell attachment might be associated with intercellular adhesion molecule-1 induced by IFN-gamma. In vitro treatment with IFN-gamma induced apoptosis of MIH-2 cells via a mitochondria-dependent pathway.
Conclusions
IL-12 suppressed HCC growth in mouse model. IFN-gamma produced by IL-12-activated tumor-infiltrating CD8+T cells directly induced apoptosis of HCC cells.
Deregulation of the activin/follistatin system in hepatocarcinogenesis, 27 July 2006
Grusch M, Drucker C, Peter-Vörösmarty B, Erlach N, Lackner A, Losert A, Macheiner D, Schneider WJ, Hermann M, Groome NP, Parzefall W, Berger W, Grasl-Kraupp B, Schulte-Hermann R
Background/Aims
Activins A and E negatively regulate hepatic cell number by inhibiting cell replication and inducing apoptosis. Follistatin and follistatin-like 3 bind activins and antagonise their biological activities. Aim of our study was to investigate, whether activins and follistatins may play a role in hepatocarcinogenesis.
Methods
Expression levels of follistatin, follistatin-like 3, and activin subunits ?A as well as ?E were investigated in chemically induced rat and human liver tumours by real-time PCR and immunohistochemistry. In addition, the effects of follistatin and activin A on DNA synthesis of normal as well as preneoplastic hepatocytes and hepatoma cells were analysed.
Results
Follistatin was overexpressed while both activin subunits were downregulated in the majority of rat and human liver tumours. Follistatin-like 3 expression was low in normal but enhanced in malignant rat liver. In human normal liver, in contrast, it was abundantly expressed but downregulated in liver cancer. Administration of follistatin to normal and preneoplastic hepatocytes stimulated DNA synthesis preferentially in preneoplastic rat hepatocytes, whereas activin A repressed it.
Conclusions
The balanced expression of follistatins and activins becomes deregulated during hepatocarcinogenesis. The sensitivity of preneoplastic hepatocytes to activin signals suggests the activin/follistatin system as promising target for therapeutic intervention.
Molecular and Cell Biology
In vivo selection of transduced hematopoietic stem cells and little evidence of their conversion into hepatocytes in vivo, 30 May 2006
Yamaguchi K, Itoh K, Masuda T, Umemura A, Baum C, Itoh Y, Okanoue T, Fujita J
Background/Aims
FMEV-type retroviral vector provides high transgene expression in hepatocytes and hematopoietic stem cells (HSCs). Here, we examined whether these vectors could provide a sufficient drug-resistance gene expression in HSCs and whether transduced HSCs could differentiate into hepatocytes in vivo.
Methods
The CD45+/Lin? cells were transduced in vitro by FMEV-type vectors containing human O6-methylguanine–DNA methyltransferase (MGMT)/reporter genes and transferred into recipient mice. After the treatment with temozolomide and O6-benzylguanine (TMZ/BG) in vivo, we analyzed the transgene expression in peripheral blood cells by flow-cytometry. Immunohistochemistry was performed on the liver slices in partial hepatectomized recipient mice.
Results
After TMZ/BG treatment, transduced host cells were enriched in recipient mice. In the liver, we observed the efficient transgene expression in many small cells along sinusoids. However, only few large cells in hepatic lobules expressed albumin. They also expressed both a transgene and a recipient marker gene, suggesting the fusion of donor HSCs with recipient hepatocytes.
Conclusions
This vector expressed a drug-resistance gene in HSCs highly enough to protect them from the drugs. But, the conversion of HSCs into hepatocytes in vivo might be a rare event in this model.
Blockage of HGF/c-Met system by gene therapy (adenovirus-mediated NK4 gene) suppresses hepatocellular carcinoma in mice, 30 May 2006
Son G, Hirano T, Seki E, Iimuro Y, Nukiwa T, Matsumoto K, Nakamura T, Fujimoto J
Background/Aims
Hepatocyte growth factor promotes cancer development through cell motility-promoting and angiogenic effects. NK4, a fragment of hepatocyte growth factor, acts as its receptor antagonist. We assessed effects of NK4 gene therapy against human hepatocellular carcinoma cells (HUH7) transplanted into mice.
Methods
NK4 gene transduction was mediated by adenovirus (AdCMV.NK4). LacZ expression adenovirus (AdCMV.LacZ) was used as a control. NK4 effects on HUH7 cells first were studied in vitro. Subcutaneous HUH7 tumors established in athymic nude mice were injected with AdCMV.NK4 (n=6) or AdCMV.Lacz (n=6). Finally, after HUH7 cells were injected into the portal vein in mice with severe combined immunodeficiency to establish hepatic tumors, mice systemically were injected with AdCMV.NK4 (n=6) or AdCMV.LacZ (n=6).
Results
NK4 inhibited hepatocyte growth factor-induced phosphorylation of c-Met in HUH7 cells. Invasion and migration of HUH7 cells were inhibited by NK4 transfection, which also suppressed growth of transplanted subcutaneous and liver tumors (p<0.001, p<0.01 respectively), and improved mouse survival (p<0.05). Angiogenesis assessed by small vessel density was significantly decreased in the NK4-treated group.
Conclusions
NK4 inhibited tumor cell motility and angiogenesis, greatly suppressing growth of HUH7 tumors transplanted into mouse liver. NK4 gene therapy thus showed apparent promise for treatment of hepatocellular carcinoma.
Precision-cut fibrotic rat liver slices as a new model to test the effects of anti-fibrotic drugs in vitro, 25 May 2006
van de Bovenkamp M, Groothuis GMM, Meijer DKF, Olinga P
Background/Aims
Current cell culture models contributed significantly to the study of liver fibrosis and the testing of anti-fibrotic drugs but mimic the complex in vivo milieu poorly. Therefore, we evaluated fibrotic rat liver slices as a new, more physiologic in vitro model to test anti-fibrotic compounds.
Methods
Precision-cut slices (8mm diameter, 250?m thickness) were prepared from fibrotic rat livers three weeks after bile-duct ligation and incubated for 0–48h, during which viability and progression of the fibrotic process was evaluated. In addition, the effects of pentoxifylline, gleevec, and dexamethasone on mRNA expression of markers for fibrosis were determined.
Results
Fibrotic liver slices remained viable during 48h of incubation, with increasing ?SMA and pro-collagen 1a1 mRNA expression, and ?SMA and collagen protein content after prolonged incubation. Addition of pentoxifylline, gleevec, or dexamethasone during incubation dose-dependently inhibited pro-collagen-1a1 and ?SMA mRNA expression after 24h of incubation without influencing slice viability.
Conclusions Fibrotic liver slices are a promising tool to test anti-fibrotic drugs in vitro in a multicellular, fibrotic milieu, which cannot be achieved in vitro using other models. Importantly, this method may provide the opportunity to study anti-fibrotic compounds not only in animal but also in fibrotic human liver tissue.
Cholestasis and Autoimmune Liver Disease
Analysis of MAdCAM-1 and ICAM-1 polymorphisms in 365 Scandinavian patients with primary sclerosing cholangitis, 25 April 2006
Bowlus CL, Karlsen TH, Broomé U, Thorsby E, Vatn M, the IBSEN study group , Schrumpf E, Lie BA, Boberg KM
Background/Aims
Mucosal addressin cellular adhesion molecule-1 (MAdCAM-1) has been implicated in the aberrant homing of intestinal lymphocytes to the liver in primary sclerosing cholangitis (PSC). Intercellular adhesion molecule-1 (ICAM-1) has also been implicated in the pathogenesis of PSC and the E/E genotype of the K469E polymorphism has been reported to be associated with PSC susceptibility. The aims of this study were to determine if MAdCAM-1 polymorphisms or the K469E polymorphism of ICAM-1 are associated with PSC in Scandinavia.
Methods
Seven single nucleotide polymorphisms (SNPs) in MAdCAM-1 and the G421R and K469E ICAM-1 SNPs were genotyped in 365 PSC patients from Norway and Sweden. 327 Norwegian ulcerative colitis (UC) patients and 368 Norwegian bone marrow donors served as controls.
Results
No significant association with PSC was found for any of the MAdCAM-1 or ICAM-1 SNPs. Allele frequencies for these polymorphisms were not significantly different between PSC patients with UC, UC patients and healthy controls.
Conclusions
Polymorphisms in MAdCAM-1 are not likely to significantly affect PSC susceptibility. In addition, the E/E genotype of the K469E in ICAM-1 does not influence PSC susceptibility in Scandinavia.
Biliary candida infections in primary sclerosing cholangitis, 10 August 2006
Kulaksiz H, Rudolph G, Kloeters-Plachky P, Sauer P, Geiss H, Stiehl A
Background/Aims
Patients with Primary Sclerosing Cholangitis (PSC) frequently develop dominant stenoses of the bile ducts and bacterial infections represent a major problem in such patients. In the present study, the role of fungal infections of the bile ducts has been evaluated.
Methods
In a prospective non-randomized trial, in 67 consecutive patients with PSC, 148 bile samples, each taken at one endoscopic examination, were microbiologically analysed.
Results
Candida species were found in 8/67 patients whereas Aspergillus was not detected. Seven patients with biliary Candida had a dominant stenosis and one had a wide papillotomy with chronic ascending cholangitis. Altogether 7/49 of patients with dominant stenosis and 1/18 of patients without dominant stenosis had Candida in their bile. All patients with biliary Candida intermittently had received antibiotics and had advanced disease with cholestasis. Candida disappeared spontaneously in 2/7 patients, cleared after antifungal treatment in 2, and persisted in 3 patients. Patients with biliary Candida had more severe cholangitis with higher CRP and serum bilirubin compared to those without Candida infection.
Conclusions
This is the first report on the identification of Candida species in the bile of patients with PSC. Apart from bacterial also fungal infection of the bile ducts should be considered in the treatment of such patients.
Genetic and Metabolic Liver Disease
Predominant role of sterol response element binding proteins (SREBP) lipogenic pathways in hepatic steatosis in the murine intragastric ethanol feeding model, 20 June 2006
Ji C, Chan C, Kaplowitz N
Background/Aims
Alcohol-induced fatty liver is associated with induction of sterol response element binding proteins (SREBPs), transcription factors which regulate expression of genes of lipid synthesis. The contribution of SREBP-1c to alcohol-induced fatty liver and injury was studied.
Methods
Wild type and SREBP1c null mice were fed alcohol or control diet by intragastric infusion for 4 weeks. H&E and TUNEL staining, real-time PCR, RT-PCR, and immunoblotting were applied to analyze alcohol-induced liver injury.
Results
ALT, plasma homocysteine, liver cholesterol, and TUNEL positive hepatocytes were increased in alcohol-fed mice as compared to control in both genotypes. Liver triglycerides were increased 4-fold in alcohol-fed wild type mice (87.2±7.5 vs. control 22.3±3.1mg/g liver) but 1.8-fold in alcohol-fed null mice (27.9±4 vs. control 14.5±3.8mg/g liver). SREBP-2 and HMG CoA reductase were higher in the null than in wild type. Betaine feeding prevented partially the alcohol-induced changes of hepatic lipids and injury in both genotypes. mRNA of Insig-1 was reduced in both genotypes fed alcohol. No change was detected for the SREBP cleavage-activating protein (Scap) or S1P in either genotype fed alcohol.
Conclusions
The predominant mechanism of hepatic triglyceride accumulation in the intragastric alcohol fed mouse requires the participation of SREBP-1c. SREBP-2 regulated cholesterol accumulation still occurs.
Hepatic gene expression profile associated with non-alcoholic steatohepatitis protection by S-nitroso-N-acetylcysteine in ob/ob mice, 5 July 2006
Souza de Oliveira CPM, Stefano JT, de Lima VMR, de Sá SV, Simplicio FI, de Mello ES, Corrêa-Giannella ML, Ferreira Alves VA, Martins Laurindo FR, de Oliveira MG, Giannella-Neto D, Carrilho FJ
Background/Aims
To understand the molecular mechanisms underlying non-alcoholic steatohepatitis (NASH) prevention by S-nitroso-N-acetylcysteine (SNAC), an NO donor that inhibits lipid peroxidation, we examined hepatic differentially expressed genes between ob/ob mice receiving or not SNAC treatment concomitantly with a methionine–choline deficient (MCD) diet.
Methods
Ob/ob mice were assigned to receive oral SNAC fed solution (MCD+SNAC group) or vehicle (MCD group) by gavage. After four weeks, histopathological analysis and microarray hybridizations were conducted in liver tissues from groups. GeneSifter® system was used to identify differentially expressed genes and pathways according to Gene Ontology.
Results
NASH was absent in the MCD+SNAC group and no significant changes in food intake or body weight were observed in comparison to MCD group. After SNAC treatment, several genes belonging to oxidative phosphorylation, fatty acid biosynthesis, fatty acid metabolism and glutathione metabolism pathways were down-regulated in comparison to the MCD group.
Conclusions
SNAC treatment promotes down regulation of several genes from fatty acid (FA) metabolism related pathways, possibly through abrogation of the cytotoxic effects of reactive oxygen species and lipid peroxides with consequent prevention of mitochondrial overload. Further studies are required to investigate the clinical implications of these findings, in attempt to develop novel therapeutic strategies for NAFLD treatment.
Copyright © 2001-2006 European Association for the Study of the Liver. All rights reserved.
Copyright © 2006 BMJ Publishing Group Ltd
Volume 355:1863-1872 November 2, 2006
Colonoscopy in Colorectal-Cancer Screening for Detection of Advanced Neoplasia
Jaroslaw Regula, M.D., Maciej Rupinski, M.D., Ewa Kraszewska, M.Sc., Marcin Polkowski, M.D., Jacek Pachlewski, M.D., Janina Orlowska, M.D., Marek P. Nowacki, M.D., and Eugeniusz Butruk, M.D.
Background Recommendations for colorectal-cancer screening are based solely on age and family history of cancer, not sex.
Methods We performed a cross-sectional analysis of the data from a large colonoscopy-based screening program that included 50,148 participants who were 40 to 66 years of age. People 40 to 49 years of age were eligible only if they had a family history of cancer of any type. Of the 43,042 participants 50 to 66 years of age, 13.3% reported a family history of colorectal cancer, as did 66.3% of the 7106 participants who were 40 to 49 years of age. We defined advanced neoplasia as cancer or adenoma that was at least 10 mm in diameter, had high-grade dysplasia, or had villous or tubulovillous histologic characteristics, or any combination thereof. We used multivariate logistic regression to identify associations between participants' characteristics and advanced neoplasia in a primary (or derivation) data set, and we confirmed the associations in a secondary (or validation) data set.
Results Advanced neoplasia was detected in 2553 (5.9%) participants 50 to 66 years of age and in 243 (3.4%) participants 40 to 49 years of age. The rate of complications during colonoscopy was 0.1%, and no participants died. In the validation set, a logistic-regression model showed that male sex was independently associated with advanced neoplasia (adjusted odds ratio, 1.73; 95% confidence interval, 1.52 to 1.98; P<0.001). In each age group (40 to 49 years, 50 to 54 years, 55 to 59 years, and 60 to 66 years), the number of persons who would have to undergo colorectal-cancer screening in order to detect one advanced neoplasia was significantly lower in men than in women (23 vs. 36, 17 vs. 28, 12 vs. 22, and 10 vs. 18, respectively).
Conclusions We detected advanced neoplasia at a significantly higher rate in men than in women, which may warrant refinement of the screening recommendations for colorectal cancer.
Source Information
From the Department of Gastroenterology, Medical Center for Postgraduate Education, and the Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology (J.R., M.R., M.P., J.P., J.O., E.B.); and the Departments of Biostatistics (E.K.) and Colorectal Cancer (M.P.N.), Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology — all in Warsaw, Poland.
Address reprint requests to Dr. Regula at the Department of Gastroenterology, Maria Sklodowska-Curie Memorial Cancer Center, Roentgen St. 5, 02-784 Warsaw, Poland, or at jregula@coi.waw.pl..
The New England Journal of Medicine is owned, published, and copyrighted © 2006 Massachusetts Medical Society. All rights reserved.
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