Table of Contents for Volume 41, Issue 1 (January 2005)
Liver Failure and Liver Disease
Alpha-fetoprotein is a predictor of outcome in acetaminophen-induced liver injury (p 26-31)
Lars E. Schmidt, Kim Dalhoff
An increase in alpha-fetoprotein (AFP) following hepatic necrosis is considered indicative of hepatic regeneration. This study evaluated the prognostic value of serial AFP measurements in patients with severe acetaminophen-induced liver injury. Prospectively, serial measurements of AFP were performed in 239 patients with acetaminophen intoxication and a peak alanine aminotransferase (ALT) level above 1,000 U/L. AFP was measured using an enzyme-linked immunoassay (EIA) with a detection limit below 0.4 g/L. The optimum threshold of AFP to discriminate nonsurvivors was identified. An increase in AFP above 4 g/L occurred in 158 (79%) of 201 survivors compared with 11 of 33 nonsurvivors (33%; P < .00001). The increase in AFP occurred a mean of 1.0 days (range, -2 to +6 days) after peak ALT in survivors compared with 4.1 days (range, +2 to +7 days) in nonsurvivors (P < .00001). Starting on the day of peak ALT, AFP values were significantly higher in survivors than in nonsurvivors. A threshold AFP of 3.9 g/L on day +1 after peak ALT to identify nonsurvivors had a sensitivity of 100%, a specificity of 74%, a positive predictive value of 45%, and a negative predictive value of 100%. In conclusion, an increase in AFP was strongly associated with a favorable outcome in patients with acetaminophen-induced liver injury. AFP may be useful as a supplement to existing prognostic criteria. We suggest that the introduction of highly sensitive EIAs for the detection of AFP will require a reevaluation of AFP as a prognostic marker in acute nonneoplastic liver disease. (HEPATOLOGY 2005;41:26-31.)
Serum sodium predicts mortality in patients listed for liver transplantation (p 32-39)
Scott W. Biggins, Harry J. Rodriguez, Peter Bacchetti, Nathan M. Bass, John P. Roberts, Norah A. Terrault
With the implementation of the model for end-stage liver disease (MELD), refractory ascites, a known predictor of mortality in cirrhosis, was removed as a criterion for liver allocation. Because ascites is associated with low serum sodium, we evaluated serum sodium as an independent predictor of mortality in patients with cirrhosis who were listed for liver transplantation and whether the addition of serum sodium to MELD was superior to MELD alone. This is a single-center retrospective cohort of all adult patients with cirrhosis listed for transplantation from February 27, 2002, to December 26, 2003. Listing laboratories were those nearest the listing date ±2 months. Of the 513 patients meeting inclusion criteria, 341 were still listed, while 172 were removed from the list (105 for transplantation, 56 for death, 11 for other reasons). The median serum sodium and MELD scores were 137 mEq/L (range, 110-155) and 15 (range, 6-51), respectively, at listing. Median follow-up was 201 (range, 1-662) days. The risk of death with serum sodium < 126 mEq/L at listing or while listed was increased, with hazard ratios of 7.8 (P < .001) and 6.3 (P < .001), respectively, and the association was independent of MELD. The c-statistics of receiver operating characteristic curves for predicting mortality at 3 months based upon listing MELD with and without listing serum sodium were 0.883 and 0.897, respectively, and at 6 months were 0.871 and 0.905, respectively. In conclusion, serum sodium < 126 mEq/L at listing or while listed for transplantation is a strong independent predictor of mortality. Addition of serum sodium to MELD increases the ability to predict 3- and 6-month mortality in patients with cirrhosis. (HEPATOLOGY 2005;41:32-39.)
Identification of a new marker of hepatocellular carcinoma by serum protein profiling of patients with chronic liver diseases (p 40-47)
Valérie Paradis, Francoise Degos, Delphine Dargère, Nanou Pham, Jacques Belghiti, Claude Degott, Jean-Louis Janeau, Annie Bezeaud, Dominique Delforge, Myriam Cubizolles, Ingrid Laurendeau, Pierre Bedossa
Surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF MS) is a proteomic technique that enables the profiling of proteins present in any biological material studied. We used this approach to identify new biomarkers of hepatocellular carcinoma (HCC) in the sera of patients with cirrhosis. Sera from 82 patients with cirrhosis, either without (n = 38) or with (n = 44) HCC, were analyzed by SELDI-TOF MS, and the results of the two groups were compared. The most efficient protein peaks leading to discrimination of patients with HCC were selected (receiver operative characteristic curves). The highest-scoring peak combination was established in a first group of serum samples (multinomial regression) and was tested in an independent group. The protein corresponding to the highest discrimination was purified and characterized further. The intensity of 30 protein peaks significantly differed between cirrhotic patients with and without HCC. An algorithm including the six highest-scoring peaks allowed correct classification (presence or absence of HCC) of 92.5% of patients in the test sample set and 90% in the validation sample set. The highest discriminating peak (8,900 Da) was purified further and was characterized as the C-terminal part of the V10 fragment of vitronectin. An in vitro study suggested that the increase of the 8,900-Da fragment in the serum of patients with HCC may proceed from the cleavage of native vitronectin with metalloproteases, a family of enzymes whose activity is enhanced in HCC. In conclusion, global protein profiling is an efficient approach that enabled us to identify a catalytic fragment of vitronectin as a new serum marker of HCC in patients with chronic liver diseases. (HEPATOLOGY 2005;41:40-47.)
Noninvasive assessment of liver fibrosis by measurement of stiffness in patients with chronic hepatitis C (p 48-54)
Marianne Ziol, Adriana Handra-Luca, Adrien Kettaneh, Christos Christidis, Frédéric Mal, Farhad Kazemi, Victor de Lédinghen, Patrick Marcellin, Daniel Dhumeaux, Jean-Claude Trinchet, Michel Beaugrand
Liver fibrosis is the main predictor of the progression of chronic hepatitis C, and its assessment by liver biopsy (LB) can help determine therapy. However, biopsy is an invasive procedure with several limitations. A new, noninvasive medical device based on transient elastography has been designed to measure liver stiffness. The aim of this study was to investigate the use of liver stiffness measurement (LSM) in the evaluation of liver fibrosis in patients with chronic hepatitis C. We prospectively enrolled 327 patients with chronic hepatitis C in a multicenter study. Patients underwent LB and LSM. METAVIR liver fibrosis stages were assessed on biopsy specimens by 2 pathologists. LSM was performed by transient elastography. Efficiency of LSM and optimal cutoff values for fibrosis stage assessment were determined by a receiver-operating characteristics (ROC) curve analysis and cross-validated by the jack-knife method. LSM was well correlated with fibrosis stage (Kendall correlation coefficient: 0.55; P < .0001). The areas under ROC curves were 0.79 (95% CI, 0.73-0.84) for F 2, 0.91 (0.87-0.96) for F 3, and 0.97 (0.93-1) for F = 4; for larger biopsies, these values were, respectively, 0.81, 0.95, and 0.99. Optimal stiffness cutoff values of 8.7 and 14.5 kPa showed F 2 and F = 4, respectively. In conclusion, noninvasive assessment of liver stiffness with transient elastography appears as a reliable tool to detect significant fibrosis or cirrhosis in patients with chronic hepatitis C. (HEPATOLOGY 2005;41:48-54.)
Genetic polymorphisms influencing xenobiotic metabolism and transport in patients with primary biliary cirrhosis (p 55-63)
Yasuhiko Kimura, Carlo Selmi, Patrick S. C. Leung, Tin K. Mao, Joseph Schauer, Mitchell Watnik, Shigeki Kuriyama, Mikio Nishioka, Aftab A. Ansari, Ross L. Coppel, Pietro Invernizzi, Mauro Podda, M. Eric Gershwin
Epidemiological data suggest that environmental factors may trigger autoimmunity in genetically susceptible individuals. In primary biliary cirrhosis (PBC), it has been postulated that halogenated xenobiotics can modify self-molecules, facilitating the breakdown of tolerance to mitochondrial antigens. The transport and metabolism of xenobiotics is highly dependent on key genetic polymorphisms that alter enzymatic phenotype. We analyzed genomic DNA from 169 patients with PBC and 225 geographically and sex-matched healthy subjects for polymorphisms of genes coding for cytochromes P450 (CYPs) 2D6 (CYP2D6*4, CYP2D6*3, CYP2D6*5, and CYP2D6*6) and 2E1 (c1/c2), multidrug resistance 1 (MDR1 C3435T) P-glycoprotein, and pregnane X receptor (PXR C-25385T, C8055T, and A7635G). We compared the genotype frequencies in patients and controls and also correlated polymorphisms with PBC severity. The distributions of the studied genotypes did not significantly differ between patients and controls. However, when clinical characteristics of patients with PBC were compared according to genotype, the CYP2E1 c2 allele was associated with signs of more severe disease. In conclusion, genetic polymorphisms of CYP 2D6 and 2E1, PXR, and MDR1 do not appear to play a role in the onset of PBC. (HEPATOLOGY 2005;41:55-63.)
Chronological development of elevated aminotransferases in a nonalcoholic population (p 64-71)
Ayako Suzuki, Paul Angulo, James Lymp, Jennifer St. Sauver, Ayako Muto, Toshihide Okada, Keith Lindor
The incidence and risk factors of nonalcoholic fatty liver disease (NAFLD) have never been prospectively determined. To determine the frequency and risk factors of NAFLD and chronological ordering between NAFLD, weight gain, and features of insulin resistance, a historical cohort study was conducted in a Japanese workplace. A cohort free of previous liver injury, alcohol consumption of more than 140 g/wk, and hepatitis B or C infection (529 of 1,537 subjects), and a subcohort of 287 subjects free of insulin resistance-related features were identified. Elevated aminotransferases in nonalcoholics were used as a surrogate for NAFLD. High aminotransferases together with weight gain of more than 2 kg and insulin resistance-related features in the subcohort were sought for up to 5 years. The incidence of high aminotransferases was 31 per 1,000 person-years (71 events). A significant interaction occurred between age and sex in the development of high aminotransferases. In subjects younger than age 40 years, male sex (hazard ratio [HR]: 4.6), elevated body mass index (HR: 2.1), hypertension (HR: 2.6), and low high-density lipoprotein cholesterol (HR: 2.8) increased the risk of high aminotransferases, whereas age (HR: 0.6 for each 5 years) decreased the risk. In subjects older than age 40 years, glucose intolerance (HR: 5.3) was the only significant risk factor. In the subcohort, weight gain preceded high aminotransferases and other insulin resistance-related features, which appeared sequentially in order of low high-density lipoprotein cholesterol, hypertriglyceridemia/hypertransaminasemia/hypertension, and glucose intolerance. In conclusion, this cohort study clearly showed chronological ordering and an association between development of elevated aminotransferases and risk factors of NAFLD. (HEPATOLOGY 2005;41:64-71.)
Viral Hepatitis
Reconstitution of hepatitis C virus-specific T-cell-mediated immunity after liver transplantation (p 72-81)
Scott J. Weston, Rachel L. Leistikow, K. Rajender Reddy, Maria Torres, Anne M. Wertheimer, David M. Lewinsohn, Sunwen Chou, Michael P. Davey, Christopher Corless, Cliona O'Farrelly, David R. Nelson, Hugo R. Rosen
Hepatitis C virus (HCV)-related liver failure is the leading indication for liver transplantation worldwide. After transplantation, virological recurrence is the rule, but the spectrum of histological injury is wide, ranging from the development of allograft cirrhosis within a few years to minimal hepatitis despite long-term follow-up. The immunological correlates of this variable natural history are poorly understood. Here, we studied the kinetics of the cellular immune responses, viral replication, and allograft histology in 24 patients who had undergone liver transplantation for HCV-related liver failure. Using direct ex vivo methodologies (i.e., interferon-gamma ELISPOT and major histocompatibility complex class I-peptide tetrameric complexes), we found that patients who experienced viral eradication after antiviral therapy showed restoration of HCV-specific T-cell responses, whereas patients with progressive HCV recurrence that failed to respond to therapy showed declining frequencies of these viral-specific effector cells. The cytotoxic T lymphocytes that peripherally reconstituted after transplantation were clonotypically identical to those present within the recipient explant liver, defined at the level of the T-cell receptor beta chain (one epitope/one clone). Moreover, the subset of patients who spontaneously demonstrated minimal histologic recurrence had more vigorous CD4+ T-cell responses in the first 3 months, particularly targeting nonstructural proteins. We provide evidence that T-cell responses emerge after liver transplantation, and their presence correlates with improved histological and clinical outcomes. In conclusion, these results may help identify patients more likely to develop severe HCV recurrence and therefore benefit from current antiviral therapy, as well as provide a rationale for the future use of novel immunotherapeutic approaches.
Impact of steatosis on progression of fibrosis in patients with mild hepatitis C (p 82-87)
Laetitia Fartoux, Olivier Chazouillères, Dominique Wendum, Raoul Poupon, Lawrence Serfaty
In patients with mild hepatitis C, the usefulness of antiviral therapy is subject of debate, as a low risk for progression of fibrosis is assumed. Several studies have shown that steatosis is a strong and independent predictor of the severity as well as the progression of fibrosis in chronic hepatitis C. Therefore, this study assessed the impact of steatosis on the progression of fibrosis between paired liver biopsies in untreated patients with mild hepatitis on index biopsy. One hundred thirty-five untreated patients (mean age, 38 years; M/F sex ratio, 1.43) with one known risk factor of infection (68 transfusions, 67 injecting drug use) had 2 liver biopsies after a median interval of 61 months (18-158). All had METAVIR score of A1F1 or lower at first liver biopsy. Unequivocal progression of fibrosis was considered if patients had a fibrosis score of 3 or 4 at the second liver biopsy. The probability of progression of fibrosis was estimated by using the Kaplan-Meier method. During follow-up, progression of fibrosis occurred in 21 patients (16%) after a median delay of 65 months. Cumulative probabilities of the progression of fibrosis at 4 and 6 years were 5.2% and 19.8%, respectively. In multivariate analysis, steatosis was the only independent factor predictive of progression of fibrosis (RR, 4.8; CI, 1.3-18.3). Probability of progression of fibrosis was significantly related to the percentage of hepatocytes with steatosis. In conclusion, steatosis is a major determinant of the progression of fibrosis in mild hepatitis C, regardless of the genotype. Our results argue for antiviral treatment in the subgroup of patients with mild hepatitis and steatosis. (HEPATOLOGY 2005;41:82-87.)
Elevated prevalence of hepatitis C infection in users of United States veterans medical centers (p 88-96)
Jason A. Dominitz, Edward J. Boyko, Thomas D. Koepsell, Patrick J. Heagerty, Charles Maynard, Jennifer L. Sporleder, VA Cooperative Study Group 488
Several studies suggest veterans have a higher prevalence of hepatitis C virus infection than nonveterans, possibly because of military exposures. The purpose of this study was to estimate the prevalence of anti-hepatitis C antibody and evaluate factors associated with infection among users of Department of Veterans Affairs medical centers. Using a two-staged cluster sample, 1,288 of 3,863 randomly selected veterans completed a survey and underwent home-based phlebotomy for serological testing. Administrative and clinical data were used to correct the prevalence estimate for nonparticipation. The prevalence of anti-hepatitis C antibody among serology participants was 4.0% (95% CI, 2.6%-5.5%). The estimated prevalence in the population of Veterans Affairs medical center users was 5.4% (95% CI, 3.3%-7.5%) after correction for sociodemographic and clinical differences between participants and nonparticipants. Significant predictors of seropositivity included demographic factors, period of military service (e.g., Vietnam era), prior diagnoses, health care use, and lifestyle factors. At least one traditional risk factor (transfusion or intravenous drug use) was reported by 30.2% of all subjects. Among those testing positive for hepatitis C antibody, 78% either had a transfusion or had used injection drugs. Adjusting for injection drug use and nonparticipation, seropositivity was associated with tattoos and incarceration. Military-related exposures were not found to be associated with infection in the adjusted analysis. In conclusion, the prevalence of hepatitis C in these subjects exceeds the estimate from the general US population by more than 2-fold, likely reflecting more exposure to traditional risk factors among these veterans. (HEPATOLOGY 2005;41:88-96.)
Fas and TNFR1, but not cytolytic granule-dependent mechanisms, mediate clearance of murine liver adenoviral infection (p 97-105)
Marwan S. Abougergi, Sarah J. Gidner, David K. Spady, Bonnie C. Miller, Dwain L. Thiele
After intravenous injection of replication-deficient adenovirus, hepatocytes are transduced and express high levels of adenovirus-encoded genes. However, adenovirally encoded gene expression is ablated rapidly by CD8+ T-cell-dependent mechanisms. Thus, this model is suitable for examining intrahepatic cytotoxic T lymphocyte (CTL) effector mechanisms. In the present studies, recombinant adenoviruses encoding secreted (human apolipoprotein A-I) or intracellular (-galactosidase) gene products were infused into mice with genetic deficiencies affecting the granule exocytosis-, Fas-, or tumor necrosis factor receptor 1 (TNFR1)-mediated pathways of CTL and natural killer cell effector function; the rates of clearance of adenovirus-encoded gene products were assessed. Clearance of secreted or intracellular adenoviral gene products was not delayed in perforin-deficient mice or dipeptidyl peptidase I-deficient mice, which fail to process and activate granzyme A or granzyme B. TNFR1-deficient mice also exhibited no delay in clearance of adenoviral gene products. However, adenoviral clearance from Fas-deficient mice was delayed, and such delays were much greater in mice deficient in both TNFR1 and Fas. In contrast, chimeric mice lacking both hepatic Fas and lymphocyte perforin function exhibited no greater delay in adenoviral clearance than chimeras deficient only in hepatic Fas expression. In conclusion, Fas-dependent mechanisms are required for efficient clearance of virally infected hepatocytes and, in Fas-deficient animals, TNFR1-dependent mechanisms provide an alternative mechanism for hepatic adenovirus clearance. In contrast, perforin- and granule protease-dependent cytotoxicity mechanisms play no apparent role in clearance of adenovirus from the liver. (HEPATOLOGY 2005;41:97-105.)
Persistence of hepatitis C virus in patients successfully treated for chronic hepatitis C (p 106-114)
Marek Radkowski, Juan F. Gallegos-Orozco, Joanna Jablonska, Thomas V. Colby, Bozena Walewska-Zielecka, Joanna Kubicka, Jeffrey Wilkinson, Debra Adair, Jorge Rakela, Tomasz Laskus
It is unclear whether the current antiviral treatment for chronic hepatitis C virus (HCV) infection results in complete elimination of the virus, or whether small quantities of virus persist. Our study group comprised 17 patients with chronic HCV who had sustained virological response (SVR) after interferon/ribavirin treatment. Serum and peripheral blood mononuclear cells were collected 2 to 3 times at 3- to 6-month intervals starting 40 to 109 months (mean, 64.2 ± 18.5 months) after the end of therapy. In addition, lymphocyte and macrophage cultures were established at each point. In 11 patients, frozen liver tissue samples were available from follow-up biopsies performed 41 to 98 months (mean, 63.6 ± 16.7 months) after therapy. Presence of HCV RNA was determined by sensitive reverse-transcriptase polymerase chain reaction, and concentration of positive and negative strands was determined by a novel quantitative real-time reverse transcriptase polymerase chain reaction. Only 2 of 17 patients remained consistently HCV RNA negative in all analyzed compartments. HCV RNA was detected in macrophages from 11 patients (65%) and in lymphocytes from 7 patients (41%). Viral sequences were also detected in 3 of 11 livers and in sera from 4 patients. Viral replicative forms were found in lymphocytes from 2 and in macrophages from 4 patients. In conclusion, our results suggest that in patients with SVR after therapy, small quantities of HCV RNA may persist in liver or macrophages and lymphocytes for up to 9 years. This continuous viral presence could result in persistence of humoral and cellular immunity for many years after therapy and could present a potential risk for infection reactivation. (HEPATOLOGY 2005;41:106-114.)
Nosocomial transmission of HCV in the liver unit of a tertiary care center (p 115-122)
Xavier Forns, Eva Martínez-Bauer, Anna Feliu, Montserrat García-Retortillo, Marta Martín, Eugeni Gay, Miquel Navasa, Jose María Sánchez-Tapias, Miquel Bruguera, Juan Rodés
Despite its medical and legal implications, there are no prospective studies analyzing the incidence and mechanisms involved in the nosocomial transmission of hepatitis C virus (HCV) in liver units. This study prospectively investigates the nosocomial transmission of HCV in the liver unit of a tertiary care center from August 2000 to October 2002. The median prevalence of HCV infection among hospitalized patients was 50%. Anti-HCV-negative patients admitted to the liver unit during the study period were prospectively followed, and serum markers of HCV infection were repeated 6 months after discharge. All known risk factors for HCV transmission (including the physical allocation of HCV-infected and noninfected patients during hospitalization) were recorded. Complete follow-up data were available in 1,301 (84.5%) of 1,540 patients. Six patients (0.46%) acquired HCV infection (annual incidence: 0.27/100 admissions). Phylogenetic analyses of recovered HCV sequences identified the source of infection as an HCV-infected roommate (3 cases) and a patient receiving care by the same nurse team (1 case). The most relevant risk factors associated with HCV acquisition were duration of hospitalization (>10 days; OR, 35; 95% CI, 1.96-622) and hospitalization with an HCV-infected roommate (>5 days; OR, 12; 95% CI, 1.39-103). In fact, HCV infection occurred in 1.7% of the 357 patients hospitalized longer than 10 days. In conclusion, HCV nosocomial infection appears to occur via patient-to-patient transmission in liver units, particularly in individuals who require long hospitalizations. Continuous reinforcement of universal prevention measures and, when possible, isolation of patients at higher risk might further reduce nosocomial HCV transmission. (HEPATOLOGY 2005;41:115-122.)
The effect of antiretroviral therapy on liver disease among adults with HIV and hepatitis C coinfection (p 123-131)
Shruti H. Mehta, David L. Thomas, Michael Torbenson, Sherilyn Brinkley, Lisa Mirel, Richard E. Chaisson, Richard D. Moore, Mark S. Sulkowski
In the era of antiretroviral therapy (ART), liver disease has emerged as an important cause of death among persons with human immunodeficiency virus (HIV)/hepatitis C virus (HCV) coinfection. The objective of this study was to estimate the burden of liver disease and evaluate determinants of liver fibrosis and necroinflammatory activity among HIV/HCV coinfected patients receiving ART. We studied 112 randomly selected and 98 referred HCV-infected patients undergoing care in the Johns Hopkins University HIV clinic. Liver disease was characterized clinically and histologically. Of the 210 individuals studied - 64% of whom had received ART within 2 years of liver disease assessment - 33% had no fibrosis (F0), and 26% had bridging fibrosis or cirrhosis (F3). The median necroinflammatory activity score was 3 (range, 0-9 of 18). ART was not associated with fibrosis; however, significantly less hepatic necroinflammatory activity was observed among persons who had received highly active antiretroviral therapy longer (P = .02) and more effectively (defined by HIV RNA suppression; P < .01). Twelve percent of individuals had previous ART-associated liver enzyme elevations (grades 3-4), but liver fibrosis was not more severe if the liver enzyme elevation resolved. On the other hand, liver fibrosis was more severe in persons with persistent liver enzyme elevations (grades 1-4). In conclusion, despite widespread exposure to ART and documented instances of ART-related hepatitis, we found no evidence that ART caused serious histological liver disease. Recognition of bridging fibrosis and cirrhosis in some but not most patients underscores the importance of identifying and treating liver disease in HIV/HCV coinfected persons. (HEPATOLOGY 2005;41:123-131.)
Liver Biology and Pathobiology
Liver tissue engineering at extrahepatic sites in mice as a potential new therapy for genetic liver diseases (p 132-140)
Kazuo Ohashi, Jacob M. Waugh, Michael D. Dake, Takashi Yokoyama, Hiroyuki Kuge, Yoshiyuki Nakajima, Masaki Yamanouchi, Hiroyuki Naka, Akira Yoshioka, Mark A. Kay
Liver tissue engineering using hepatocyte transplantation has been proposed as an alternative to whole-organ transplantation or liver-directed gene therapy to correct various types of hepatic insufficiency. Hepatocytes are not sustained when transplanted under the kidney capsule of syngeneic mice. However, when we transplanted hepatocytes with the extracellular matrix components extracted from Engelbreth-Holm-Swarm cells, hepatocytes survived for at least 140 days and formed small liver tissues. Liver engineering in hemophilia A mice reconstituted 5% to 10% of normal clotting activity, enough to reduce the bleeding time and have a therapeutic benefit. Conversely, the subcutaneous space did not support the persistent survival of hepatocytes with Engelbreth-Holm-Swarm gel matrix. We hypothesized that establishing a local vascular network at the transplantation site would reduce graft loss. To test this idea, we provided a potent angiogenic agent before hepatocyte transplantation into the subcutaneous space. With this procedure, persistent survival was achieved for the length of the experiment (120 days). To establish that these engineered liver tissues also retained their native regeneration potential in vivo, we induced two different modes of proliferative stimulus to the naïve liver and confirmed that hepatocytes within the extrahepatic tissues regenerated with activity similar to that of naïve liver. In conclusion, our studies indicate that liver tissues can be engineered and maintained at extrahepatic sites, retain their capacity for regeneration in vivo, and used to successfully treat genetic disorders. (HEPATOLOGY 2005;41:132-140.)
Diosgenin-induced biliary cholesterol secretion in mice requires Abcg8 (p 141-150)
Astrid Kosters, Raoul J. J. M. Frijters, Cindy Kunne, Edwin Vink, Marit S. Schneiders, Frank G. Schaap, Catherina P. Nibbering, Shailendra B. Patel, Albert K. Groen
The plant sterol diosgenin has been shown to stimulate biliary cholesterol secretion in mice without affecting the expression of the adenosine triphosphate-binding cassette transporter heterodimer Abcg5/g8. The aim of this study was to investigate the mechanism of diosgenin-induced cholesterol hypersecretion and to identify the genes involved. Surprisingly, despite its lack of effect on Abcg5/g8 expression in wild-type mice, diosgenin did not stimulate biliary cholesterol secretion in mice deficient for Abcg8. Analysis of the kinetics of cholesterol secretion suggested that diosgenin probably activates a step before Abcg5/g8. To identify potential diosgenin targets, gene expression profiling was performed in mice fed a diosgenin-supplemented diet. Diosgenin feeding increased hepatic expression of genes involved in cholesterol synthesis as well as genes encoding for several cytochrome P450s. No significant change in expression of known cholesterol transporters was found. Comparison with published expression-profiling data for Srebp2-overexpressing mice, another mouse model in which biliary cholesterol secretion is elevated, revealed a number of genes with unknown function that were upregulated in both diosgenin-fed mice and mice overexpressing Srebp2. In conclusion, we found that although Abcg8 is essential for most diosgenin-induced biliary cholesterol hypersecretion, diosgenin probably does not interact directly with Abcg5/Abcg8, but rather increases cholesterol delivery to the heterodimer.
Biliary epithelial cells regulate autoreactive T cells: Implications for biliary-specific diseases (p 151-159)
Takashi Kamihira, Shinji Shimoda, Minoru Nakamura, Teruhumi Yokoyama, Yasushi Takii, Akira Kawano, Mizuki Handa, Hiromi Ishibashi, M. Eric Gershwin, Mine Harada
The biliary epithelial cell (BEC) is the target for several human immune mediated liver diseases, including primary biliary cirrhosis, but it is not always clear whether the BEC functions as an accessory cell or an antigen presenting cell, although it is well documented that BECs express high levels of human leukocyte antigen Class II, intercellular adhesion molecule-1, and lymphocyte function-associated antigen-3. To examine this issue, we established autoreactive T-cell clones from human leukocyte antigen-DR53 patients with primary biliary cirrhosis and characterized BEC function as a function of the ability of BECs to regulate T-cell activation. We report herein that BEC-mediated T-cell activation occurs partially via programmed death 1 ligands in a cell-contact-dependent manner. Further, such activation occurs via prostaglandin E2 production in a cell-contact-independent fashion. Moreover, the production of prostaglandin E2 was partially controlled by interleukin-1 and tumor necrosis factor . In conclusion, the regulatory activities of BECs are important for the maintenance of peripheral immune tolerance. Further, modulation of BEC function may be used for therapeutic modulation. (HEPATOLOGY 2005;41:151-159.)
Quantitative isolation of 1AT mutant Z protein polymers from human and mouse livers and the effect of heat (p 160-167)
Jae-Koo An, Keith Blomenkamp, Douglas Lindblad, Jeffrey H. Teckman
Alpha-1-antitrypsin (1AT) deficiency in its most common form is caused by homozygosity for the 1AT mutant Z gene. This gene encodes a mutant Z secretory protein, primarily synthesized in the liver, that assumes an abnormal conformation and accumulates within hepatocytes causing liver cell injury. Studies have shown that mutant 1ATZ protein molecules form unique protein polymers. These Z protein polymers have been hypothesized to play a critical role in the pathophysiology of liver injury in this disease, although a lack of quantitative methods to isolate the polymers from whole liver has hampered further analysis. In this study, we demonstrate a quantitative 1ATZ polymer isolation technique from whole liver and show that the hepatocellular periodic acid-Schiff-positive globular inclusions that are the histopathological hallmark of this disease are composed almost entirely of the polymerized 1ATZ protein. Furthermore, we examine the previously proposed but untested hypothesis that induction of 1ATZ polymerization by the heat of physiological fever is part of the mechanism of hepatic 1ATZ protein accumulation. The results, however, show that fever-range temperature elevations have no detectable effect on steady-state levels of intrahepatic Z protein polymer in a model in vivo system. In conclusion, methods to separate insoluble protein aggregates from liver can be used for quantitative isolation of 1ATZ protein polymers, and the effect of heat from physiological fever may be different in vivo compared with in vitro systems. (HEPATOLOGY 2005;41:160-167.)
Combined loss of orphan receptors PXR and CAR heightens sensitivity to toxic bile acids in mice (p 168-176)
Hirdesh Uppal, David Toma, Simrat P.S. Saini, Songrong Ren, Thomas J. Jones, Wen Xie
Efficient detoxification of bile acids is necessary to avoid pathological conditions such as cholestatic liver damage and colon cancer. The orphan nuclear receptors PXR and CAR have been proposed to play an important role in the detoxification of xeno- and endo-biotics by regulating the expression of detoxifying enzymes and transporters. In this report, we showed that the combined loss of PXR and CAR resulted in a significantly heightened sensitivity to bile acid toxicity in a sex-sensitive manner. A regimen of lithocholic acid treatment, which was tolerated by wild-type and PXR null mice, caused a marked accumulation of serum bile acids and histological liver damage as well as an increased hepatic lipid deposition in double knockout males. The increased sensitivity in males was associated with genotype-specific suppression of bile acid transporters and loss of bile acid-mediated downregulation of small heterodimer partner, whereas the transporter suppression was modest or absent in females. The double knockout mice also exhibited gene- and tissue-specific dysregulation of PXR and CAR target genes in response to PXR and CAR agonists. In conclusion, although the cross-regulation of target genes by PXR and CAR has been proposed, the current study represents in vivo evidence of the combined loss of both receptors causing a unique pattern of gene regulation that can be translated into physiological events such as sensitivity to toxic bile acids. (HEPATOLOGY 2005;41:168-176.)
Microarray analysis in human hepatocytes suggests a mechanism for hepatotoxicity induced by trovafloxacin (p 177-186)
Michael J. Liguori, Mark G. Anderson, Stanley Bukofzer, James McKim, Jeffrey F. Pregenzer, Jacques Retief, Brian B. Spear, Jeffrey F. Waring
Idiosyncratic drug toxicity, defined as toxicity that is dose independent, host dependent, and usually cannot be predicted during preclinical or early phases of clinical trials, is a particularly confounding complication of drug development. An understanding of the mechanisms that lead to idiosyncratic liver toxicity would be extremely beneficial for the development of new compounds. We used microarray analysis on isolated human hepatocytes to understand the mechanisms underlying the idiosyncratic toxicity induced by trovafloxacin. Our results clearly distinguish trovafloxacin from other marketed quinolone agents and identify unique gene changes induced by trovafloxacin that are involved in mitochondrial damage, RNA processing, transcription, and inflammation that may suggest a mechanism for the hepatotoxicity induced by this agent. In conclusion, this work establishes the basis for future microarray analysis of new compounds to determine the presence of these expression changes and their usefulness in predicting idiosyncratic hepatotoxicity.
Lipopolysaccharide-binding protein modulates acetaminophen-induced liver injury in mice (p 187-195)
Grace L. Su, Ke Qin Gong, Ming Hui Fan, William M. Kelley, Jason Hsieh, Jian Min Sun, Mark R. Hemmila, Saman Arbabi, Daniel G. Remick, Stewart C. Wang
Acetaminophen toxicity is the most common cause of acute liver failure in the United States and Europe. Although much is known about the metabolism of acetaminophen, many questions remain regarding the pathogenesis of liver injury. In this study, we examined the role of lipopolysaccharide-binding protein (LBP), a protein important in mediating cellular response to lipopolysaccharides, by using LBP wild-type and knockout (KO) mice. We found that LBP KO mice were protected from acetaminophen-induced hepatotoxicity. At 350 mg/kg of acetaminophen, LBP KO mice had significantly less liver injury and necrosis than wild-type mice. Repletion studies in LBP KO mice using an LBP-adenoviral construct resulted in significantly more hepatic injury and necrosis after acetaminophen exposure compared with mice receiving the control adenoviral construct. In conclusion, LBP KO mice are protected from toxicity with a decrease in hepatic necrosis following acetaminophen challenge. This suggests a novel role for LBP in modulating acetaminophen-induced liver injury.
Eradication of advanced hepatocellular carcinoma in rats via repeated hepatic arterial infusions of recombinant VSV (p 196-203)
Katsunori Shinozaki, Oliver Ebert, Savio L.C. Woo
Viruses that replicate selectively in cancer cells hold considerable promise as novel therapeutic agents for the treatment of malignancy. Vesicular stomatitis virus (VSV) is a nonpathogenic RNA virus with intrinsic oncolytic specificity due to attenuated antiviral responses in many tumors. We report that repeated hepatic arterial infusion of recombinant syncytia-forming VSV vector in advanced multifocal hepatocellular carcinoma (HCC)-bearing rats at a 10-fold reduced vector dose resulted in sustained tumor-selective virus replication until the onset of high-titer neutralizing antibodies in blood. No significant elevations in serum transaminases and liver pathology were noted, indicating a lack of hepatotoxicity. Substantially improved tumor response was achieved with completely necrotic tumor nodules surrounded by mononuclear phagocytic cells, followed by fibrosis and calcification of the lesions, angiogenesis, and regeneration of normal hepatic parenchyma. Survival of tumor-bearing rats treated with repeated vector infusions was not only significantly improved over that of animals after a single injection at 10 times the vector dose (P = .001), but 18% of animals in the former treatment group also achieved long-term and tumor-free survival compared with 0% of animals in the latter treatment group. In conclusion, this treatment regimen will be very useful in the future development of VSV-mediated virotherapy as a novel therapeutic modality for patients with advanced HCC. (HEPATOLOGY 2005;41:196-203.)
Special Article
Treatment challenges and investigational opportunities in autoimmune hepatitis (p 207-215)
Albert J. Czaja, Francesco B. Bianchi, Herschel A. Carpenter, Edward L. Krawitt, Ansgar W. Lohse, Michael P. Manns, Ian G. McFarlane, Giorgina Mieli-Vergani, Gotaro Toda, Diego Vergani, John Vierling, Mikio Zeniya
New drugs and advances in molecular biology afford opportunities to upgrade the treatment of autoimmune hepatitis. The aims of this study were to define treatment problems, identify possible solutions, and stimulate investigations to improve patient care. A clinical subcommittee of the International Autoimmune Hepatitis Group reviewed current management difficulties and proposed corrective actions. The assessment of new front-line and salvage therapies for adults and children were given top priority. Cyclosporine and mycophenolate mofetil were endorsed as drugs worthy of rigorous study in severe disease, and budesonide was endorsed for study as front-line therapy in mild disease. Diagnostic criteria and treatment regimens for children required codification, and pharmacokinetic studies were encouraged to develop optimal dosing schedules based on therapeutic ranges. Collaborative efforts were proposed to help understand racial, geographical, and genetic factors affecting outcome and to establish definitions and therapies for variant syndromes and graft dysfunction after transplantation. The development of experimental animal models was deemed essential for the study of site-specific molecular interventions, and gene therapy was endorsed as a means of bolstering reparative processes. In conclusion, evolving pharmacological and technical advances promise to improve the treatment of autoimmune hepatitis, and investigations of these advances are timely, feasible, and necessary. (HEPATOLOGY 2005;41:207-215.)
Copyright © 2005 by the American Association for the Study of Liver Diseases. All rights reserved.
Table of Contents for January 2005 • Volume 128 • Number 1
Clinical-alimentary Tract
Connective tissue growth factor inhibits metastasis and acts as an independent prognostic marker in colorectal cancer
Been-Ren Lin, Cheng-Chi Chang, Ting-Fang Che, Szu-Ta Chen, Robert Jeen-Chen Chen, Ching-Yao Yang, Yung-Ming Jeng, Jin-Tung Liang, Po-Huang Lee, King-Jen Chang, Yat-Pang Chau, Min-Liang Kuo
Background & Aims: Connective tissue growth factor (CTGF) has been shown to be implicated in tumor development and progression. The aim of this study was to investigate the role of CTGF in progression of colorectal cancer (CRC). Methods: Immunohistochemical staining of specimens from 119 patients with CRC was performed. Liposome-mediated transfection was used to introduce a CTGF expression vector into CRC cell lines. Transfectants were tested in invasive ability and experimental hepatic metastasis in BALB/c mice. Furthermore, a FOPflash/TOPflash reporter assay was performed to investigate CTGF on the ?-catenin/T-cell factor signaling pathway. Results: Patients with stage II and stage III CRC whose tumors displayed high CTGF expression had a significantly higher overall survival and a disease-free advantage over patients with CRC with low CTGF expression. Alterations in the CTGF level in CRC cell lines modulated their invasive ability with an inverse correlation. In addition, a reduction in the CTGF level of CT26 cells after stable transfection with antisense CTGF resulted in increased liver metastasis in BALB/c mice. The activity of the ?-catenin/T-cell factor signaling pathway and its downstream effector gene matrix metalloproteinase 7 in these CTGF-transfected cells was strongly attenuated. Blockage of matrix metalloproteinase 7 with its neutralizing antibodies inhibited increased invasiveness in antisense CTGF-transfected CT26 cells. Conclusions: Our results implicate CTGF as a key regulator of CRC invasion and metastasis, and it appears to be a useful and better prognosis factor for patients with stage II and stage III CRC.
Clinical-liver, Pancreas, and Biliary Tract
Coffee and caffeine consumption reduce the risk of elevated serum alanine aminotransferase activity in the United States
Constance E. Ruhl, James E. Everhart
Background & Aims: Based on experimental and epidemiologic studies, we investigated whether coffee and caffeine consumption reduced the risk of elevated alanine aminotransferase (ALT) activity in persons at high risk for liver injury in a national, population-based study. Methods: Participants were 5944 adults in the Third US National Health and Nutrition Examination Survey, 1988–1994, with excessive alcohol consumption, viral hepatitis, iron overload, overweight, or impaired glucose metabolism. Liver injury was indicated by abnormal serum ALT activity (>43 U/L). Results: Elevated ALT activity was found in 8.7% of this high-risk population. In unadjusted analysis, lower ALT activity was associated with increasing consumption of coffee (P = .001) and caffeine (P = .001). Multivariate logistic regression analyses showed that the risk of elevated ALT activity declined with increasing intake of coffee (P for trend = .034) and caffeine (P < .001). Comparing persons who drank more than 2 cups per day with noncoffee drinkers, the odds ratio was .56 (95% confidence interval, .31–1.0). Comparing persons in the highest caffeine quintile with the lowest, the odds ratio was .31 (95% confidence interval, .16–.61). These relationships were consistent across subgroups at risk for liver injury and were relatively unchanged when analyses included the entire population or when limited to persons without impaired liver function or right upper quadrant pain. Fasting insulin concentrations did not mediate the effects. Conclusions: In this large, national, population-based study, among persons at high risk for liver injury, consumption of coffee and especially caffeine was associated with lower risk of elevated ALT activity.
Relation between hepatocyte G1 arrest, impaired hepatic regeneration, and fibrosis in chronic hepatitis C virus infection
Aileen Marshall, Simon Rushbrook, Susan E. Davies, Lesley S. Morris, Ian S. Scott, Sarah L. Vowler, Nicholas Coleman, Graeme Alexander
Backgrounds & Aims: An increased risk of hepatitis C virus (HCV)-related cirrhosis is associated with hepatic steatosis, older age, and high alcohol consumption, which could be explained by synergistic effects on cell proliferation. We aimed to investigate hepatocyte cell cycle state and phase distribution in chronic HCV infection. Methods: Liver biopsy specimens diagnostic for chronic HCV (70), liver regeneration following transplant-related ischemic-reperfusion injury (15), and “normal” liver adjacent to colorectal cancer metastasis (10) were studied. Immunohistochemistry was used to detect cell cycle phase markers cyclin D1 (maximal in G1), cyclin A (S), cyclin B1 (cytoplasmic during G2) and phosphorylated histone 3 protein (mitosis), mini-chromosome maintenance protein 2 (Mcm-2; present throughout the cell cycle), and cyclin-dependent kinase inhibitor p21, which inhibits G1/S progression. Results: Hepatocyte Mcm-2 expression was elevated in chronic HCV and liver regeneration (13% vs 26.4%) but negligible in “normal” liver. In proportion to Mcm-2, there was no difference in cyclin D1 between chronic HCV infection and liver regeneration (51.6% of Mcm-2-positive hepatocytes vs 52.6%). In contrast, there was a striking reduction in cyclin A (3% vs 16.3%), cyclin B1 (.4% vs 2.3%), and phosphorylated histone 3 protein (0% vs 3.8%) in chronic HCV infection compared with liver regeneration. In chronic HCV infection, Mcm-2 and p21 expression were associated with fibrosis stage and positive serum HCV RNA. Conclusions: The data are consistent with hepatocyte G1 arrest in chronic HCV infection. This could impair hepatocellular function and limit hepatic regeneration.
Basic-alimentary Tract
Pyloric electrical stimulation reduces food intake by inhibiting gastric motility in dogs
Xiaohong Xu, Hongbing Zhu, Jiande D.Z. Chen
Background & Aims: The pylorus plays an important role in regulating gastric emptying. The aim of this study was to investigate the therapeutic potential of pyloric electrical stimulation (PES) for obesity in dogs. Methods: The study was composed of 3 separate experiments. The first experiment was designed to study the effects of PES with various parameters on gastric emptying and gastric slow waves in 5 sessions. The second experiment was used to test the effects of PES on antral contractions. The acute effect of PES on food intake was studied in the third experiment. Results: (1) Pyloric myoelectrical recording showed dual frequencies. The lower frequency was identical to the frequency of the gastric slow waves, and the higher frequency was similar to that of the intestinal slow waves. (2) Gastric emptying was significantly delayed with PES, and the delay in gastric emptying was significantly and negatively correlated with stimulation energy (r = ?.673; P < .001). (3) PES significantly impaired the regularity and coupling of the intrinsic gastric myoelectrical activity in an energy-dependent manner. The delayed gastric emptying was significantly correlated with the impairment of the coupling of gastric myoelectrical activity (r = .441; P < .02). (4) Antral contractions on the fed state were significantly and substantially inhibited with PES. (5) Acute PES significantly reduced food intake. Conclusions: PES reduces food intake that may be attributed to its inhibitory effects on intrinsic gastric myoelectrical activity, antral contractions, and gastric emptying.
Actinin-4 increases cell motility and promotes lymph node metastasis of colorectal cancer
Kazufumi Honda, Tesshi Yamada, Yasuharu Hayashida, Masashi Idogawa, Satoshi Sato, Fumio Hasegawa, Yoshinori Ino, Masaya Ono, Setsuo Hirohashi
Background & Aims: Enhanced motility of cancer cells by remodeling of the actin cytoskeleton seems crucial in the process of cancer invasion and metastasis. We previously identified an actin-binding protein, actinin-4, as a new biomarker of cancer invasion and an indicator of prognosis for patients with breast cancer. However, its involvement in the mechanisms of cancer invasion and metastasis remains undetermined. The current study tested the role of actinin-4 in the motility and metastatic potential of colorectal cancer cells. Methods & Results: Quantitative immunofluorescence histochemistry showed that the expression level of the actinin-4 protein was increased in 73.1% (19/26) of the cases of colorectal cancer over the corresponding normal intestinal epithelium. The increased expression of actinin-4 was most significant in dedifferentiated cancer cells at the invasive front. A colorectal cancer cell clone capable of inducing actinin-4 using the tetracycline-regulatory system (designated DLD1 Tet-off ACTN-4) was established. Upon the induction of actinin-4, DLD1 Tet-off ACTN-4 cells spread filopodia and significantly increased their motility (P = .00027); actinin-4 protein was concentrated at the leading edges of these actin-rich podia. When injected into the mesocecum of severe combined immunodeficient mice, DLD1 Tet-off ACTN4 cells, but not the control cells, metastasized into regional mesenteric lymph nodes, resembling the behavior of clinical cancers. The expression of actinin-4 in focally dedifferentiated cancer cells at the invasive front was significantly correlated with the frequency of lymph node metastasis of colorectal cancer (P = .038). Conclusions: Actinin-4 actively increases cell motility and promotes lymph node metastasis of colorectal cancer.
Survivin: A novel target for indomethacin-induced gastric injury
Shiun-Kwei Chiou, Tetsuya Tanigawa, Tomohiko Akahoshi, Basim Abdelkarim, Michael K. Jones, Andrzej S. Tarnawski
Background & Aims: Nonsteroidal anti-inflammatory drugs (NSAIDs) cause gastrointestinal erosions and ulcers. Apoptosis is one of the mechanisms. The role of survivin, an antiapoptosis protein, in NSAID-induced gastric injury is unknown. We examined the role of survivin in NSAID-induced gastric mucosal and gastric cell injury. Methods: We examined: (1) the effects of indomethacin (nonselective NSAID), celecoxib and NS-398 (cyclooxygenase [COX]-2-selective NSAIDs), SC-560 (a COX-1-selective NSAID), and SC-560 plus celecoxib on survivin expression and extent of injury in rat gastric mucosa; (2) the effects of indomethacin, NS-398, SC-560, and SC-560 plus NS-398 on survivin expression and injury in gastric epithelial (RGM-1) cells; and (3) the effects of survivin suppression with small interfering RNA (siRNA) on RGM-1 cell integrity at baseline and following indomethacin injury. Results: Indomethacin treatment dose-dependently reduced survivin protein levels and caused severe injury of gastric mucosa and RGM-1 cells. Suppression of survivin expression with siRNA in RGM-1 cells caused cell damage and increased susceptibility to injury by indomethacin. Celecoxib treatment caused exfoliation of the mucosal surface epithelium, but neither caused deep erosions or altered survivin expression. Neither NS-398 nor SC-560 treatment altered survivin levels or produced injury in vivo or in vitro. COX-1 and COX-2 inhibitor combination caused injury in vivo and in vitro but did not decrease survivin expression. Conclusions: (1) Indomethacin, but not selective COX-1 or COX-2 inhibitors alone or in combination, reduces survivin expression in gastric mucosal cells and (2) significant reduction of survivin precedes greater severity of gastric injury.
A major quantitative trait locus on mouse chromosome 3 is involved in disease susceptibility in different colitis models
Michelle E.A. Borm, Jianping He, Brian Kelsall, A. Salvador Peña, Warren Strober, Gerd Bouma
Background & Aims: Mice with a disrupted gene for the G-protein ? inhibitory 2 chain (Gnai2?/?) develop a spontaneous colitis resembling human inflammatory bowel disease. Disease expression differs markedly between inbred strains of mice, indicating genetic control of disease susceptibility. We performed a genome-wide screen to localize the chromosomal regions regulating disease expression. Methods: A total of 284 F2 mice derived from resistant C57BL/6J Gnai2?/? mice and susceptible C3H/HeN Gnai2?/? mice were analyzed in a genome-wide screen for colitis susceptibility and severity. Results: A highly significant locus on chromosome 3 (Gpdc1) contributed to colitis susceptibility and severity (likelihood ratio statistics [LRS] = 32.4; LOD score = 7; P < 1.0 ? 10?5). The peak linkage of this locus at 62 cM colocalizes exactly with a previously identified locus controlling colitis susceptibility in interleukin-10–deficient mice. In addition, evidence for linkage with a locus on chromosome 1 (Gpdc2; LRS = 19.7; LOD = 4.3) was found, and the 2 loci interacted epistatically (combined LRS = 68.2). A third locus (Gpdc3) was found on chromosome 9 and this locus interacted epistatically with a locus on chromosome 7, which by itself did not have an effect on the trait. Conclusions: The identification of a major locus on chromosome 3 that controls susceptibility to spontaneous colitis in 2 different gene-knockout models indicates that this locus harbors a gene(s) that plays a key role in maintaining mucosal homeostasis. Identification of this gene(s) may contribute to further understanding of the mechanisms underlying human inflammatory bowel disease.
Basic-liver, Pancreas, and Biliary Tract
Acyclic retinoid inhibits human hepatoma cell growth by suppressing fibroblast growth factor-mediated signaling pathways
Run-Xuan Shao, Motoyuki Otsuka, Naoya Kato, Hiroyoshi Taniguchi, Yujin Hoshida, Masaru Moriyama, Takao Kawabe, Masao Omata
Background & Aims: Hepatocellular carcinoma (HCC) is one of the most common human malignancies. Its high mortality rate is mainly a result of high intrahepatic recurrence. The novel synthetic retinoid acyclic retinoid (ACR) has been reported to prevent the recurrence of human HCC after surgical resection of primary tumors, but the molecular mechanisms underlying its effects remain to be elucidated. In this study, we clarified the molecular targets of ACR. Methods: The inhibitory effects by ACR on growth were examined. Intracellular signaling induced by ACR was comprehensively studied by a reporter assay. Gene expression changes by ACR were examined using a microarray. From these results, a candidate signaling pathway modulated by ACR was determined and whether antagonizing this pathway reverses the effect was examined. Results: We show that ACR inhibits the growth of HCC cells through the down-regulation of fibroblast growth factor (FGF) receptor 3 expression and FGF-mediated signaling, which in turn suppresses the activity of Rho and serum response factor-mediated transcription. Conversely, overexpression of the active form of FGF receptor 3 or the addition of FGF reverses the ACR-mediated inhibition of growth. In addition, silencing the FGF receptor 3 gene by RNA interference inhibits cell growth. Conclusions: These studies show that ACR is a potent inhibitor of FGF signaling and that selective blocking of the FGFmediated pathway could be a promising therapeutic approach for the management of patients with HCC.
Hepatitis C virus core protein, cytochrome P450 2E1, and alcohol produce combined mitochondrial injury and cytotoxicity in hepatoma cells
Kazuhiro Otani, Masaaki Korenaga, Michael R. Beard, Kui Li, Ting Qian, Lori A. Showalter, Aman K. Singh, Ting Wang, Steven A. Weinman
Background & Aims: Alcohol consumption exacerbates liver injury in chronic hepatitis C, and enhanced mitochondrial oxidative stress is one possible mechanism. The aim of this study was to determine whether hepatitis C virus core protein and alcohol-inducible cytochrome P450 2E1 contribute to reactive oxygen species production and cytotoxicity in human hepatoma cells. Methods: Huh-7 cells expressing core protein, cytochrome P450 2E1, or both were exposed to 0.1 mmol/L tertiary butyl hydroperoxide, tumor necrosis factor ?, and/or 25 mmol/L ethanol. Cytotoxicity, reactive oxygen species production, glutathione content, and mitochondrial membrane potential were measured. Results: Expression of core/cytochrome P450 2E1 synergistically enhanced cell death induced by either tertiary butyl hydroperoxide or tumor necrosis factor ?. After tertiary butyl hydroperoxide treatment, total reactive oxygen species production was increased more than 3-fold compared with cells that did not express core and cytochrome P450 2E1. Mitochondrial depolarization and reduced glutathione depletion occurred as well, and cell death was prevented by inhibition of mitochondrial permeability transition or caspase activity. Confocal microscopy showed that the mitochondria themselves were the origin of the reactive oxygen species. In the absence of core/cytochrome P450 2E1 expression, mitochondrial changes and cell death did not occur. Ethanol treatment further decreased mitochondrial reduced glutathione content and exacerbated mitochondrial reactive oxygen species production, depolarization, and cell death. All these effects were prevented by the antioxidant N-acetylcysteine. Conclusions: Mitochondrial reactive oxygen species production is induced by hepatitis C virus core and cytochrome P450 2E1, resulting in a reduction of mitochondrial antioxidant capacity and sensitivity to oxidants and tumor necrosis factor ?. Alcohol further depletes mitochondrial reduced glutathione, which exacerbates depolarization and cell death. Sensitization of mitochondria to oxidative insults is thus a potential mechanism for alcohol-related exacerbation of liver injury in chronic hepatitis C.
Inhibition of inhibitor of ?B kinases stimulates hepatic stellate cell apoptosis and accelerated recovery from rat liver fibrosis
Fiona Oakley, Muriel Meso, John P. Iredale, Karen Green, Carylyn J. Marek, Xiaoying Zhou, Michael J. May, Harry Millward-Sadler, Matthew C. Wright, Derek A. Mann
Background & Aims: Resolution of liver fibrosis is associated with clearance of hepatic myofibroblasts by apoptosis; development of strategies that promote this process in a selective way is therefore important. The aim of this study was to determine whether the inhibitor of ?B kinase suppresser sulfasalazine stimulates hepatic myofibroblast apoptosis and recovery from fibrosis. Methods: Hepatic myofibroblasts were generated by culture activation of rat and human hepatic stellate cells. Fibrosis was established in rat livers by chronic injury with carbon tetrachloride followed by recovery with or without sulfasalazine (150 mg/kg) treatment. Results: Treatment of hepatic stellate cells with sulfasalazine (0.5–2.0 mmol/L) induced apoptosis of activated rat and human hepatic stellate cells. A single in vivo administration of sulfasalazine promoted accelerated recovery from fibrosis as assessed by improved fibrosis score, selective clearance of smooth muscle ?-actin-positive myofibroblasts, reduced hepatic procollagen I and tissue inhibitor of metalloproteinase 1 messenger RNA expression, and increased matrix metalloproteinase 2 activity. Mechanistic studies showed that sulfasalazine selectively blocks nuclear factor-?B-dependent gene transcription, inhibits hepatic stellate cell expression of Gadd45?, stimulates phosphorylation of Jun N-terminal kinase 2, and promotes apoptosis by a mechanism that is prevented by the Jun N-terminal kinase inhibitor SP600125. As further evidence for a survival role for the inhibitor of ?B kinase/nuclear factor-?B pathway in activated hepatic stellate cells, a highly selective cell-permeable peptide inhibitor of ?B kinase activation also stimulated hepatic stellate cell apoptosis via a Jun N-terminal kinase-dependent mechanism. Conclusions: Inhibition of the inhibitor of ?B kinase/nuclear factor-?B pathway is sufficient to increase the rate at which activated hepatic stellate cells undergo apoptosis both in vitro and in vivo, and drugs that selectively target inhibitor of ?B kinase have potential as antifibrotics.
Autocrine/paracrine regulation of the growth of the biliary tree by the neuroendocrine hormone serotonin
Marco Marzioni, Shannon Glaser, Heather Francis, Luca Marucci, Antonio Benedetti, Domenico Alvaro, Silvia Taffetani, Yoshiyuki Ueno, Tania Roskams, Jo Lynne Phinizy, Juliet Venter, Giammarco Fava, Gene D. LeSage, Gianfranco Alpini
Background & Aims: The biliary tree is the target of cholangiopathies that are chronic cholestatic liver diseases characterized by loss of proliferative response and enhanced apoptosis of cholangiocytes, the epithelial cells lining the biliary tree. The endogenous factors that regulate cholangiocyte proliferation are poorly understood. Therefore, we studied the role of the neuroendocrine hormone serotonin as a modulator of cholangiocyte proliferation. Methods: The presence of the serotonin 1A and 1B receptors on cholangiocytes was evaluated. We then tested whether the activation of such receptors by the administration of the selective agonists modifies cholangiocyte proliferation and functional activity both in vivo and in vitro. In addition, the intracellular signal mediating the serotonin receptor action in cholangiocytes was characterized. We studied the expression and secretion of serotonin by cholangiocytes and the effects of the neutralization of the secreted hormone on the growth of the biliary tree. Results: Cholangiocytes express the serotonin 1A and 1B receptors. Their activation markedly inhibits the growth and choleretic activity of the biliary tree in the bile duct-ligated rat, a model of chronic cholestasis. Such changes are mediated by enhanced D-myo-inositol 1,4,5-triphosphate/Ca2+/protein kinase C signaling and the consequent inhibition of the adenosine 3?,5?-cyclic monophosphate/protein kinase A/Src/extracellular signal-regulated kinase 1/2 cascade. Cholangiocytes secrete serotonin, the blockage of which enhances cholangiocyte proliferation in the course of cholestasis. Conclusions: We observed the existence of an autocrine loop based on serotonin that limits the growth of the biliary tree in the course of chronic cholestasis. Our novel findings might open new approaches for the management of cholangiopathies.
Suppression of macrophage infiltration inhibits activation of hepatic stellate cells and liver fibrogenesis in rats
Michio Imamura, Tadashi Ogawa, Yasuyuki Sasaguri, Kazuaki Chayama, Hikaru Ueno
Background & Aims: Monocytes/macrophages infiltrate into injured livers. We tried to clarify their roles in inflammation and subsequent fibrogenesis by inhibiting their infiltration with a mutated form (7ND; 7 amino acids at the N-terminal were deleted) of monocyte chemoattractant protein 1, which may function as a dominant-negative mutant. Methods: Rats were injected via the tail vein with an adenovirus expressing either human 7ND (Ad7ND), a truncated type II transforming growth factor ? receptor (AdT?-TR), which works as a dominant-negative receptor, bacterial ?-galactosidase (AdLacZ), or saline. Seven days later, the rats were treated with dimethylnitrosamine for 1–21 days. Results: Within 24 hours after a single dimethylnitrosamine injection, macrophages were observed in livers. With a 3-day dimethylnitrosamine treatment, activated hepatic stellate cells were detectable in livers in AdLacZ-, AdT?-TR–, and saline-injected rats. In contrast, in the Ad7ND-treated rats, infiltration of macrophages was markedly reduced, and activated hepatic stellate cells were not detectable. After a 3-week dimethylnitrosamine treatment, fibrogenesis was almost completely inhibited, and activated hepatic stellate cells were hardly seen in livers in both Ad7ND- and AdT?-TR–treated rats. Conclusions: Our results show that blockade of macrophage infiltration inhibits activation of hepatic stellate cells and leads to suppression of liver fibrogenesis. The presence of activated hepatic stellate cells in the initial phase after injury and its absence at a later phase in the AdT?-TR–treated livers indicate that transforming growth factor ? is not an activating factor for hepatic stellate cells, and this suggests that transforming growth factor ? is required for the survival of activated hepatic stellate cells. Our study suggests that infiltrated macrophages may themselves produce an activating factor for hepatic stellate cells.
Increased fecal neutral sterol loss upon liver X receptor activation is independent of biliary sterol secretion in mice
Janine K. Kruit, Torsten Plösch, Rick Havinga, Renze Boverhof, Pieter H.E. Groot, Albert K. Groen, Folkert Kuipers
Background & Aims: Reverse cholesterol transport (RCT) is defined as high-density lipoprotein (HDL)-mediated flux of excess cholesterol from peripheral cells to liver, followed by secretion into bile and disposal via the feces. Various steps of this pathway are controlled by the liver X receptor (LXR). We addressed the role of the intestine in LXR-dependent stimulation of fecal cholesterol excretion. Methods: To segregate biliary from intestine-derived cholesterol, wild-type and Mdr2 P-glycoprotein-deficient mice (Mdr2?/?), which are unable to secrete cholesterol into bile, were treated with the LXR agonist GW3965. Results: Treatment with GW3965 increased biliary cholesterol secretion by 74% in wild-type mice but had no effect in Mdr2?/? mice. LXR activation increased fecal neutral sterol excretion 2.1-fold in wild-type mice. Surprisingly, an identical increase was observed in Mdr2?/? mice. Fractional cholesterol absorption was reduced on LXR activation in both strains but was more pronounced in Mdr2?/? mice, coinciding with reduced Npc111 expression. Intestinal gene expression of ATP-binding cassette transporters (Abc) Abca1, Abcg1, Abcg5, and Abcg8 was strongly induced upon LXR activation in both strains, whereas expression of HMGCoA reductase, controlling cholesterol synthesis, remained unaffected. Additionally, LXR activation stimulated the excretion of plasma-derived [3H]cholesterol into the fecal neutral sterol fraction in Mdr2?/? mice. Conclusions: Increased fecal cholesterol loss upon LXR activation is independent of biliary cholesterol secretion in mice. An important part of excess cholesterol is excreted directly via the intestine, supporting the existence of an alternative, quantitatively important route for cholesterol disposal.
Case Reports
Use of a mixed endothelin receptor antagonist in portopulmonary hypertension: A safe and effective therapy?
Christian Kuntzen, Veit Gülberg, Alexander L. Gerbes
Background & Aims: Portopulmonary hypertension (PPHTN), a severe complication of portal hypertension is observed in 3%–6% of patients evaluated for liver transplantation. Endothelin-1, a potent vasoconstrictor, is likely to play a role in the pathogenesis of primary pulmonary hypertension, and, in 2 recent trials, the dual endothelin receptor antagonist bosentan has shown beneficial effects in this disease. A role for endothelins in the development of both pulmonary hypertension and cirrhosis has been suggested. We therefore hypothesized that endothelin receptor blockade may be beneficial in the treatment of PPHTN. Methods: We report a case of a 42-year-old patient with PPHTN and alcoholic cirrhosis treated with the mixed endothelin receptor antagonist bosentan. Results: The patient rapidly improved from NYHA IV to stage II, experienced a remarkable improvement of 6-minute walking distance from 0 to 590 m within 6 months, and resumed working full-time as a locksmith after 7 months of treatment. Improvement of cardiovascular parameters included a reduction of pulmonary vascular resistance by 60%, a decrease of mean pulmonary artery pressure (mPAP) from 55 to 44 mm Hg at 9 months, and a decline of plasma B-type natriuretic peptide (BNP) from 339 pg/mL to 19 pg/mL after 1 year. There were no adverse events except for a transient decrease in systemic blood pressure. Conclusions: To our knowledge, this is the first report of a patient with PPHTN treated with an endothelin receptor antagonist. The marked and sustained improvement supports the undertaking of controlled studies of the safety and efficacy of bosentan in PPHTN.
Endosonographic diagnosis of recurrent gastrointestinal stromal tumors associated with Carney’s syndrome
Michael T. Lipcan III, David E. Loren, Juan P. Palazzo, Marluce Bibbo, Anthony J. Dimarino, Sidney Cohen
Assay of molecular markers in stool represents a promising noninvasive approach to screen colorectal cancer. Given that neoplasms exfoliate abundantly into the lumen and that DNA recovered from stool can be assayed with sensitive techniques, there is a strong biologic rationale to pursue this emerging technology. A challenge with DNA-based testing relates to the selection of markers. Because of the molecular heterogeneity of cancer, no single marker has yielded perfect sensitivity. Several combinations of markers in early stool assays have produced high detection rates of both colorectal cancer and advanced adenomas in selected patient groups, but observations from large representative populations are lacking at present. Potential expanded applications of stool DNA testing include detection of supracolonic aerodigestive cancers and of dysplasia in inflammatory bowel disease. Further marker discovery and technologic refinements should translate into improved test performance and fuel a continued evolution with this screening approach.
Copyright © 2001-2005 by the American Gastroenterological Association. All rights reserved.
Table of Contents for January 2005 (Vol. 42, Issue 1)
Viral Hepatitis
Insulin resistance is associated with liver fibrosis in non-diabetic chronic hepatitis C patients, 22 October 2004
Muzzi A, Leandro G, Rubbia-Brandt L, James R, Keiser O, Malinverni R, Dufour JF, Helbling B, Hadengue A, Gonvers JJ, Müllhaupt B, Cerny A, Mondelli MU, Negro F, For the Swiss Hepatitis C Cohort Study (SCCS)
pages 41-46
Background/Aims
Liver steatosis is a frequent finding in chronic hepatitis C. An association has been suggested between steatosis and fibrosis progression rate, but the pathogenetic mechanisms linking fatty infiltration and collagen deposition are unknown.
Methods
We measured the levels of insulin resistance (as HOMA score) and leptin in 221 non-diabetic chronic hepatitis C patients, to assess their impact on liver steatosis and fibrosis, relative to other factors, using a multivariable logistic regression.
Results
When all 221 patients were considered, steatosis was associated with excessive alcohol intake, genotype 3, and serum HCV RNA level, whereas fibrosis was associated with HOMA score and age. In 152 patients infected with genotype non-3, steatosis was associated with alcohol abuse and HCV RNA level, and fibrosis with HOMA score and age. In the 69 patients with genotype 3, steatosis and fibrosis were associated with each other. The association between fibrosis and HOMA score held also when 22 obese patients were excluded from the analysis. Levels of insulin resistance were not correlated with the presence of steatosis.
Conclusions
Thus, insulin resistance (but not leptin) may play a role in fibrogenesis in chronic hepatitis C patients infected with genotype non-3.
Molecular evolutionary analyses implicate injection treatment for schistosomiasis in the initial hepatitis C epidemics in Japan, 25 October 2004
Tanaka Y, Hanada K, Orito E, Akahane Y, Chayama K, Yoshizawa H, Sata M, Ohta N, Miyakawa Y, Gojobori T, Mizokami M
pages 47-53
Background/Aims
The mortality due to hepatocellular carcinoma (HCC) has ranged widely in various areas of Japan since 30 years ago and the incidence was particularly high in once Schistosoma japonicum (Sj)-endemic areas. Our aim was to estimate the spread time of hepatitis C virus (HCV) infection in the past with possible relevance to a higher incidence of HCC in once Sj-endemic than Sj-nonendemic areas.
Methods
During 2001, 131 strains of HCV-1b were obtained from patients in three previously Sj-endemic areas, as well as Sj-nonendemic areas in Japan and a cross-sectional study was conducted on them with molecular evolutionary analyses.
Results
A phylogenetic tree reconstructed on HCV-1b sequences in the NS5B region disclosed 2 independent clusters for Sj-positive and -negative groups with a high bootstrap value. The estimated effective number of HCV-infections indicated a transition from quiescence to rapid exponential growth in the 1920s among patients with schistosomiasis, which is 20 years earlier than that among patients without schistosomiasis.
Conclusions
The estimated spread time in previously Sj-endemic areas in Japan coincides with injection treatment for Sj since 1921. A high incidence of HCC there would be attributed to a long duration of HCV infection since 1920s.
Chronic infection with hepatitis B viruses and antiviral drug evaluation in uPA mice after liver repopulation with tupaia hepatocytes, 3 November 2004
Dandri M, Burda MR, Zuckerman DM, Wursthorn K, Matschl U, Pollok JM, Rogiers X, Gocht A, Köck J, Blum HE, Weizsäcker Fv, Petersen J
pages 54-60
Background/Aims
Transplantation of primary human hepatocytes and establishment of hepatitis B virus (HBV) infection in immunodeficient urokinase plasminogen activator (uPA) transgenic mice was shown. However, the availability of usable primary human hepatocytes is very limited. Therefore, alternative and more accessible sources of hepatocytes permissive for HBV infection are highly desirable. Here we investigated the potential of primary hepatocytes from the tree shrew Tupaia belangeri that were shown to be susceptible to HBV infection.
Methods
Freshly isolated or cryopreserved primary tupaia hepatocytes were transplantated via intrasplenic injection into immunodeficient uPA/RAG-2 mice. Engrafted mice were then infected with HBV and woolly monkey (WM)-HBV positive sera.
Results
Extensive proliferation of xenografted cells was demonstrated by the stable production of tupaia alpha1-antitrypsin in serum and liver of transplanted mice. Quantitative PCR assays demonstrated the presence of circulating viral particles as well as intracellular viral DNA, including covalently closed circular (ccc) DNA, in transplanted mice. Viral infection could be serially passaged in mice. Furthermore, viral replication was strongly inhibited by treating mice with adefovir dipivoxil.
Conclusions
uPA mice repopulated with tupaia hepatocytes represent a useful and more accessible model for HBV infection studies, including the evaluation of antiviral therapy and cccDNA.
Comparable functions of plasmacytoid and monocyte-derived dendritic cells in chronic hepatitis C patients and healthy donors, 12 October 2004
Piccioli D, Tavarini S, Nuti S, Colombatto P, Brunetto M, Bonino F, Ciccorossi P, Zorat F, Pozzato G, Comar C, Abrignani S, Wack A
pages 61-67
Background/Aims
Dendritic cells (DCs) play a key role in immune responses through antigen presentation and cytokine secretion. Hepatitis C virus (HCV) is able to escape elimination by the immune system and often establishes a chronic infection. To investigate whether DC dysfunction is involved in this process, we have studied monoycte-derived DCs (Mo-DCs) and plasmacytoid DCs (pDCs), which produce large amounts of IFN-?, from chronic HCV patients and healthy donors.
Methods
We have assessed TNF-? and IFN-? production by pDCs using intracellular staining after total PBMCs stimulation with unmethylated CG dinucleotides (CpGs). The induction of allogeneic T cell proliferation by immature Mo-DCs was measured using the MLR assay. The up-regulation of maturation markers and the production of TNF-? in response to LPS were analyzed using flow cytometry and ELISA, respectively.
Results
We have detected comparable frequencies of pDCs producing TNF-? and IFN-? in both chronic HCV patients and healthy donors and we have found that immature Mo-DCs from both patients and donors similarly induce allogeneic T cell proliferation and mature and secrete TNF-? in response to LPS.
Conclusions
Our results demonstrate that both pDC and Mo-DCs are not impaired in HCV infected patients.
Cirrhosis and its Complications
Cardiac alterations in cirrhosis: reversibility after liver transplantation, 11 October 2004
Torregrosa M, Aguadé S, Dos L, Segura R, Gónzalez A, Evangelista A, Castell J, Margarit C, Esteban R, Guardia J, Genescà J
pages 68-74
Background/Aims
Liver cirrhosis induces cardiac alterations. We aimed to define these alterations and assess their reversibility after transplantation.
Methods
Cirrhotic patients (n=40) and controls (n=15) underwent echocardiography and stress ventriculography. Fifteen cirrhotics were reevaluated 6–12 months after transplantation.
Results
Cirrhotics had higher left ventricular wall thickness (9.6±1.2 vs. 8.8±1.2mm; P<0.05) and ejection fraction (73±6 vs. 65±4%, P<0.001) than controls. Basal diastolic function was similar. During stress, cirrhotics presented lower increases of heart rate, left ventricular ejection fraction, stroke volume and cardiac index (P<0.05 for all), and diastolic dysfunction with lower ventricular peak filling rate (P=0.001). Exercise capacity was reduced (48±21 vs. 76±24W; P<0.001). Ascitic patients exhibited more diastolic dysfunction at rest and during stress compared to non-ascitic patients. Liver transplantation caused regression of ventricular wall thickness (10.2±1.3 vs. 9.5±1.2mm; P<0.05), improvement of diastolic function, and normalization of systolic response and exercise capacity during stress (significant increases in heart rate, ventricular ejection fraction, stroke volume and cardiac index; P<0.05 for all).
Conclusions
Cardiac alterations in cirrhosis present with mild increases in ventricular wall thickness, diastolic dysfunction that worsens with ascites and physical stress, and abnormal systolic response to stress limiting exercise capacity. Liver transplantation reverses these alterations.
The selective cyclooxygenase-2 inhibitor celecoxib modulates the formation of vasoconstrictor eicosanoids and activates PPAR?. Influence of albumin, 11 October 2004
López-Parra M, Clària J, Titos E, Planagumà A, Párrizas M, Masferrer JL, Jiménez W, Arroyo V, Rivera F, Rodés J
pages 75-81
Background/Aims
Selective cyclooxygenase (COX)-2 inhibitors do not adversely affect renal function in experimental cirrhosis. In the current study, we investigated the molecular mechanisms underlying the effects of the selective COX-2 inhibitor, celecoxib, and assessed the influence of albumin on its actions.
Methods
Rat mesangial cells (RMC) were incubated with celecoxib in the absence or presence of albumin, and levels of selected vasoconstrictor eicosanoids, renin release and ?-smooth muscle actin (?-SMA) expression were determined. The effects of celecoxib on PPAR? were assessed in RMC co-transfected with PPAR? and luciferase reporter constructs.
Results
Under resting conditions, RMC expressed COX-1, COX-2 and 12/15-lipoxygenase and mainly generated prostaglandin (PG)E2, thromboxane (TX)B2, 12-hydroxyeicosatetraenoic acid (12-HETE) and 8-epi-PGF2?. Celecoxib, in addition to reducing PGE2, significantly decreased 8-epi-PGF2? formation. In the presence of albumin, celecoxib also reduced TXB2 and 12-HETE. Albumin per se inhibited PGE2 as well as renin release. In trans-activation assays, celecoxib acted as a PPAR? agonist whereas albumin inhibited PPAR? as well as 15d-PGJ2-induced PPAR? activation. Finally, celecoxib and albumin potentiated the inhibitory effect of 15d-PGJ2 on ?-SMA expression.
Conclusions
These data provide novel molecular mechanisms of celecoxib and their modulation by albumin, that may be relevant to prevent renal dysfunction in conditions of unbalanced effective blood volume.
Transplantation
The predictive value of admission and follow up factor V and VII levels in patients with acute hepatitis and coagulopathy, 11 October 2004
Elinav E, Ben-Dov I, Hai-Am E, Ackerman Z, Ofran Y
pages 82-86
Background/Aims
Low factor V and VII levels are bad prognostic indicators in fulminant hepatic failure (FHF). The prognostic importance of admission versus follow up levels of these factors in patients with acute hepatitis and coagulopathy without encephalopathy has not been evaluated.
Methods
Clinical and laboratory data from 68 consecutive patients with acute hepatitis and coagulopathy but without encephalopathy, during a 6-year period, was retrospectively evaluated.
Results
Sixty patients (88%) demonstrated improvement in liver function and coagulation (‘survivors’), while 8 patients (12%) died or underwent OLT (‘non-survivors’). Survivors had higher admission (P<0.005) and follow up factor VII levels (P<0.005) than non-survivors. Follow up factor V levels were higher in survivors (P<0.02), while admission factor V level was not different between groups (P=NS). Multivariate logistic regression analysis demonstrated that admission factor VII levels predicted outcome (P<0.006). Area under the ROC curve of factor VII was larger than that of factor V (0.885 and 0.715, respectively, P<0.02). After 3 days of hospitalization, factor V levels, but not factor VII, independently predicted outcome (P<0.04).
Conclusions
In patients with hepatitis and coagulopathy without encephalopathy at presentation, admission factor VII level may serve as a reliable prognostic marker. Subsequently, during hospitalization, changes in factor V are better outcome indicators.
RGD peptides confer survival to hepatocytes via the ?1-integrin-ILK-pAkt pathway, 11 October 2004
Pinkse GGM, Jiawan-Lalai R, Bruijn JA, Heer Ed
pages 87-93
Background/Aims
Allogeneic cell transplantation is characterized by a lack of sustained survival of the transplanted cells in the recipient. Activation of the appropriate integrin-linked signaling pathways in cells can promote cell survival. The purpose of this study was to determine how presence or absence of anti-?1 integrin chain antibodies or RGD peptides affects the survival of hepatocytes.
Methods
Hepatocytes of BN rats were isolated. Hepatocyte survival was tested after the hepatocytes had been cultured in the presence of anti-?1 integrin antibodies or RGD peptides. Hepatocytes that had been given a different treatment were stained for caspase 3 (apoptosis marker) and phospho-Akt Ser 473 (survival marker) and were measured for their integrin-linked kinase (ILK) activity.
Results
Ligation of integrins using antibodies against the ?1 integrin chain or RGD peptides protected isolated hepatocytes from apoptosis and resulted in an increased ILK activity and persistent phosphorylation of protein kinase B/Akt at serine 473.
Conclusions
Integrin activation in isolated hepatocytes contributes to the activation of ILK, phosphorylation of Akt on serine residue 473, and inhibition of apoptosis. Integrin signaling through the ILK-phospho Akt pathway protects isolated hepatocytes from apoptosis. This notion may potentially be applied to render the transplantation of hepatocytes more effective.
Liver Failure, Growth and Cancer
Free radical scavenger (edaravone) prevents endotoxin-induced liver injury after partial hepatectomy in rats, 14 October 2004
Tsuji K, Kwon AH, Yoshida H, Qiu Z, Kaibori M, Okumura T, Kamiyama Y
pages 94-101
Background/Aims
Infection after major surgery, such as massive hepatectomy, induces liver dysfunction, occasionally leading to multiple organ failure and death. We demonstrated the anti-inflammatory effects and functional mechanisms of 3-methyl-1-phenyl-2-pyrazolin-5-one (edaravone), a newly synthesized free radical scavenger, on an experimental model of endotoxemia after partial hepatectomy in rats.
Methods
Rats were treated with lipopolysaccharide (LPS) 48h after 70% hepatectomy. Edaravone was administered intravenously before LPS-treatment.
Results
Edaravone markedly improved the survival rate of LPS-treated rats after hepatectomy and inhibited increases in serum levels of AST and LDH. Histopathological analysis demonstrated that edaravone prevented inflammatory changes in the liver, kidney and spleen. Edaravone inhibited the formation of one of the markers of oxidative damage, malondialdehyde. Increases in inflammatory cytokines and cytokine-induced neutrophil chemoattractant (CINC) in serum and liver tissue were inhibited in the edaravone-treated group. An electrophoretic mobility shift assay revealed that edaravone inhibited the activation of the transcription factor, nuclear factor-kappa B (NF-?B). Edaravone also reduced the induction of inducible nitric oxide synthase (iNOS).
Conclusions
Edaravone prevents endotoxin-induced liver injury after partial hepatectomy not only by attenuating oxidative damage, but also by reducing the production of inflammatory cytokines, CINC and iNOS, in part through the inhibition of NF-?B activation.
Molecular and Cell Biology
Kupffer cell depletion with liposomal clodronate prevents suppression of Ntcp expression in endotoxin-treated rats, 18 October 2004
Sturm E, Havinga R, Baller JFW, Wolters H, van Rooijen N, Kamps JAAM, Verkade HJ, Karpen SJ, Kuipers F
pages 102-109
Background/Aims
In sepsis-associated cholestasis, expression of many genes involved in bile acid transport, including Ntcp, is suppressed by cytokines. Kupffer cells (KC) are an important source of cytokines in sepsis. To assess the consequences of KC depletion on hepatic Ntcp expression in endotoxemic rats.
Methods
Sprague–Dawley rats received liposomal clodronate (CLO) or vehicle (PBS) to deplete KC prior to lipopolysaccharide (LPS) exposure. Plasma and liver samples were taken 1 and 16h after LPS exposure.
Results
Complete CLO-depletion of KC by was demonstrated by immunohistochemistry. Hepatic gene expression of IL-1? and TNF? as well as TNF? plasma levels in CLO/LPS-injected animals were significantly reduced to a mean of 41, 36 and 23% of controls injected with LPS only. Ntcp RNA- and protein expression was significantly higher whereas plasma bile salt concentration was lower in CLO/LPS animals vs. animals injected with LPS only. Binding activity of transcription factors RXR:RAR and HNF1? was decreased in LPS only controls but preserved in CLO/LPS treated animals.
Conclusions
Clodronate-depletion of KC blocks cytokine-mediated Ntcp suppression upon endotoxin exposure. KC may represent pharmacological targets for treatment of sepsis-associated cholestasis.
Involvement of mitochondrial permeability transition in acetaminophen-induced liver injury in mice, 12 October 2004
Masubuchi Y, Suda C, Horie T
pages 110-116
Although mitochondria have been demonstrated as primary targets in acetaminophen hepatotoxicity, the mechanism for mitochondria-mediated toxicity has not been defined. We examined the role of mitochondrial permeability transition (MPT) in the acetaminophen-induced liver injury.
Methods
Male CD-1 mice were given intraperitoneally acetaminophen (350mg/kg) without or with cyclosporin A (50mg/kg), a specific inhibitor of MPT. Serum alanine aminotransferase (ALT), a marker of liver injury, and other biochemical parameters were determined.
Results
Acetaminophen-induced ALT leakage was attenuated by co-administration of cyclosporin A. Cyclosporin A did not affect acetaminophen-induced early decrease in hepatic reduced glutathione (GSH) contents, indicating lack of the effect on the metabolic activation. Acetaminophen-induced decrease in mitochondrial GSH and ATP contents, and cytosolic leakage of cytochrome c were attenuated by cyclosporin A, suggesting that mitochondrial oxidative stress and ATP depletion resulting from MPT are principle mechanisms involved in acetaminophen-induced liver injury. Mitochondrial swelling by calcium was exacerbated in the mitochondria isolated from the acetaminophen-treated mice. In vitro exposure of intact mitochondria to N-acetyl-p-benzoquinone imine (NAPQI) with calcium caused mitochondrial swelling.
Conclusions
The present data indicate that the MPT is the principal mechanism in the acetaminophen-induced liver injury and NAPQI is a candidate to open the transition pore.
Overexpression of thioredoxin prevents thioacetamide-induced hepatic fibrosis in mice, 18 October 2004
Okuyama H, Nakamura H, Shimahara Y, Uyama N, Kwon YW, Kawada N, Yamaoka Y, Yodoi J
pages 117-123
Background/Aims
Thioredoxin is a small redox-active protein with anti-oxidant and anti-apoptotic effects. We have previously reported that thioacetamide-induced acute hepatitis was attenuated in thioredoxin transgenic mice. The aim of the present study was to investigate the protective effect of thioredoxin for hepatic fibrosis.
Methods
We subjected thioredoxin transgenic mice to thioacetamide-induced hepatic fibrosis. We also studied the effect of thioredoxin on the activation process of primary-cultured hepatic stellate cell.
Results
The expression of endogenous thioredoxin was induced in hepatocytes of thioacetamide-induced murine and rat fibrotic livers. Overexpression of thioredoxin inhibited tumor necrosis factor-?-induced apoptosis of HepG2 cells. Thioacetamide-induced fibrosis and accumulation of malondialdehyde were suppressed in transgenic mice as compared with wild type mice. Hepatic stellate cells isolated from transgenic mice were less proliferative than those isolated from wild type mice. Recombinant thioredoxin significantly inhibited DNA synthesis of primary-cultured stellate cells under serum or platelet-derived growth factor stimulation.
Conclusions
Thioredoxin has a potential to attenuate hepatic fibrosis via suppressing oxidative stress and inhibiting proliferation of stellate cells.
Cholestasis and Autoimmune Liver Disease
Evidence of angiogenesis in primary biliary cirrhosis: an immunohistochemical descriptive study, 26 October 2004
Medina J, Sanz-Cameno P, García-Buey L, Martín-Vílchez S, López-Cabrera M, Moreno-Otero R
pages 124-131
Background/Aims
The intrahepatic inflammatory process occurring during primary biliary cirrhosis contributes to bile duct destruction, but the cellular and molecular pathways involved are largely unknown. Furthermore, additional pathogenetic mechanisms may exist. We aimed at evaluating the cellular infiltrate phenotype; the expression of lymphocyte activation, antigen recognition and cell-adhesion molecules; the occurrence of hepatic angiogenesis and the molecules involved.
Methods
Immunohistochemical investigations were performed in frozen liver biopsy sections from primary biliary cirrhosis patients.
Results
CD8+ and CD69+ T cells were predominant in inflammatory infiltrates around damaged cholangiocytes; ?2-microglobulin conformational epitope and intercellular adhesion molecule-1 expression were enhanced in bile ducts and hepatocytes. Inflamed portal areas showed vascular cell adhesion molecule-1 up-regulation; formation of tubule-like structures (neovessels) by endothelial cells expressing vascular endothelial-cadherin and CD-31; vascular endothelial growth factor expression in surrounding sinusoidal endothelial cells; and enhanced expression of angiopoietins 1 and 2, their receptor Tie-2 and endoglin, suggesting their involvement in new vascular structure formation.
Conclusions
The inflammatory infiltrate in primary biliary cirrhosis shows an increased reactivity for lymphocyte activation, antigen recognition and cell- and vascular-adhesion molecules. Additionally, intrahepatic angiogenesis occurs, involving vascular endothelial growth factor, angiopoietins 1 and 2, Tie-2 and endoglin in neovessel formation.
Genetic and Metabolic Liver Disease
The histological course of nonalcoholic fatty liver disease: a longitudinal study of 103 patients with sequential liver biopsies, 12 October 2004
Adams LA, Sanderson S, Lindor KD, Angulo P
pages 132-138
Background/Aims
The histological course of nonalcoholic fatty liver disease (NAFLD) remains undescribed. Therefore, we examined the liver histology of NAFLD patients who had undergone sequential liver biopsies.
Methods
Data on 103 patients who underwent serial liver biopsies in the absence of effective treatment were reviewed, and biopsies scored in a blind fashion.
Results
Mean interval between biopsies was 3.2±3.0 years (range 0.7–21.3). Fibrosis stage apparently progressed in 37%, remained stable in 34% and regressed in 29%. Severity of steatosis, inflammation, hepatocyte ballooning and Mallory's hyaline improved significantly. Aminotransferases decreased significantly between biopsies, paralleling improvement in steatosis and inflammatory features but not fibrosis stage. The rate of fibrosis change ranged from ?2.05 to 1.7 stages/year. By multivariate analysis, diabetes (P=0.007) and low initial fibrosis stage (P<0.001) were associated with higher rate of fibrosis progression, as was higher body mass index (P=0.008) when cirrhotics were excluded.
Conclusions
Fibrosis in NAFLD progresses slowly over time with considerable variability in the rate of changes among patients. Changes of aminotransferases do not parallel changes in fibrosis stage. Diabetic patients with elevated BMI and low fibrosis stage are at risk for higher rates of fibrosis progression.
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