HEPATOLOGY
Volume 45, Issue 1, January 2007
Original Article
Long-acting octreotide versus placebo for treatment of advanced HCC: A randomized controlled double-blind study (p 9-15)
Gerhild Becker, Hans-Peter Allgaier, Manfred Olschewski, Andreas Zähringer, Hubert Erich Blum, HECTOR Study Group
Although numerous treatment modalities have been explored in patients with advanced HCC, the therapeutic options are still limited. Somatostatin has been shown to have antimitotic activity in endocrine as well as in a variety of nonendocrine tumors. Expression of somatostatin receptors is found in HCCs, but the efficacy of the somatostatin analogue octreotide remains controversial. Therefore, a randomized double-blind placebo-controlled multicenter trial was performed to assess the efficacy of long-acting octreotide for the treatment of advanced HCC. One hundred twenty untreated patients with histologically confirmed HCC were randomized to receive either long-acting octreotide (Sandostation LAR 30 mg) intramuscularly every 4 weeks or placebo. The study groups were comparable with respect to clinical characteristics. There was no difference in the cumulative survival. The median survival time was 4.7 months in the octreotide group compared with 5.3 months in the control group. Six-month survival rates were 41% for octreotide patients and 42% for control patients, respectively. The unadjusted relative risk for mortality in the octreotide group compared with patients in the control group was 1.11 (95% CI 0.76-1.63; P = 0.59). When adjusted for Okuda, CTP, and Cancer of the Liver Italian Program (CLIP) scores, the relative risk for octreotide did not change markedly and was 1.05 (95% CI 0.71-1.55; P = 0.83). The CLIP score seems to predict survival better than both Okuda and CTP score. Conclusion: The randomized controlled double-blind HECTOR trial showed no survival benefit for HCC patients treated with long-acting octreotide compared with placebo.
Hepatitis B virus promotes hepatocarcinogenesis in transgenic mice (p 16-21)
Yanyan Zheng, Wen-ling Chen, Stan G. Louie, T. S. Benedict Yen, Jing-hsiung James Ou
HBV is a major risk factor for hepatocellular carcinoma (HCC). However, whether HBV can directly cause HCC or only indirectly via the induction of chronic liver inflammation has been controversial. By using transgenic mice carrying the entire HBV genome as a model, we now demonstrate that HBV by itself is an inefficient carcinogen. However, it can efficiently promote hepatocarcinogenesis initiated by the carcinogen diethylnitrosamine (DEN). This effect of HBV does not involve chronic liver inflammation, is apparently due to enhanced hepatocellular apoptosis and compensatory regeneration following DEN treatment, and does not require the HBV X protein. Conclusion: Our results demonstrate a direct role of HBV in a hepatocarcinogenesis pathway that involves the interaction between this virus and a dietary carcinogen.
Intrahepatic delivery of -galactosylceramide-pulsed dendritic cells suppresses liver tumor (p 22-30)
Tomohide Tatsumi, Tetsuo Takehara, Shinjiro Yamaguchi, Akira Sasakawa, Ryotaro Sakamori, Kazuyoshi Ohkawa, Keisuke Kohga, Akio Uemura, Norio Hayashi
Alpha-galactosylceramide, a glycosphingolipid, mediates interaction of dendritic cells (DCs) and NKT cells, leading to activation of both innate and acquired immunity. For cancer treatment, conventional DC-based vaccine has been tried, but its clinical efficacy is limited against liver cancer. Intrahepatic injection of -Galactosylceramide-pulsed DCs (GCDC) has not yet been tested in the liver that contains abundant immune cells such as NK, NKT, and T cells. In the present study, we examined the efficacy of GCDC administration in comparison with p53 peptide-pulsed DCs using a well-established murine CMS4 tumor model. Injection of GCDC into CMS4 liver tumors resulted in complete tumor rejection and established long-term survival of the animals, while injection of p53232-240 peptide-pulsed DCs (pepDC) only partially suppressed tumor growth in the liver. The levels of IFN- in sera of GCDC-treated mice were significantly higher than those of pepDC-treated mice. Hepatic NK cells were efficiently activated by GCDC injection and played a critical role in liver tumor rejection as evidenced by an in vivo antibody-mediated NK cell depletion study. Injection of GCDC into liver tumor led to higher p53232-240 peptide-specific CD8+ T cell response than that of pepDC. The mice that had been protected from CMS4 liver tumor by GCDC injection became resistant to subcutaneous CMS4 rechallenge, but not to Colon26 rechallenge. Conclusion: These results demonstrate that GCDC injection into the liver can efficiently activate NK cells that in turn reject liver tumors to establish potent acquired immunity against the original tumor.
Transforming growth factor-beta differentially regulates oval cell and hepatocyte proliferation (p 31-41)
Lananh N. Nguyen, Momoko H. Furuya, Lawrence A. Wolfraim, Anthony P. Nguyen, Matthew S. Holdren, Jean S. Campbell, Belinda Knight, George C. T. Yeoh, Nelson Fausto, W. Tony Parks
Oval cells are hepatocytic precursors that proliferate in late-stage cirrhosis and that give rise to a subset of human hepatocellular carcinomas. Although liver regeneration typically occurs through replication of existing hepatocytes, oval cells proliferate only when hepatocyte proliferation is inhibited. Transforming growth factor- (TGF-) is a key inhibitory cytokine for hepatocytes, both in vitro and in vivo. Because TGF- levels are elevated in chronic liver injury when oval cells arise, we hypothesized that oval cells may be less responsive to the growth inhibitory effects of this cytokine. To examine TGF- signaling in vivo in oval cells, we analyzed livers of rats fed a choline-deficient, ethionine-supplemented (CDE) diet for phospho-Smad2. Phospho-Smad2 was detected in more than 80% of hepatocytes, but staining was substantially reduced in oval cells. Ki67 staining, in contrast, was significantly more common in oval cells than hepatocytes. To understand the inverse relationship between TGF- signaling and proliferation in oval cells and hepatocytes, we examined TGF- signaling in vitro. TGF- caused marked growth inhibition in primary hepatocytes and the AML12 hepatocyte cell line. Two oval cell lines, LE/2 and LE/6, were less responsive. The greater sensitivity of the hepatocytes to TGF--induced growth inhibition may result from the absence of Smad6 in these cells. Conclusion: Our results indicate that oval cells, both in vivo and in vitro, are less sensitive to TGF--induced growth inhibition than hepatocytes. These findings further suggest an underlying mechanism for the proliferation of oval cells in an environment inhibitory to hepatocytic proliferation.
Transcriptome classification of HCC is related to gene alterations and to new therapeutic targets (p 42-52)
Sandrine Boyault, David S. Rickman, Aurélien de Reyniès, Charles Balabaud, Sandra Rebouissou, Emmanuelle Jeannot, Aurélie Hérault, Jean Saric, Jacques Belghiti, Dominique Franco, Paulette Bioulac-Sage, Pierre Laurent-Puig, Jessica Zucman-Rossi
Hepatocellular carcinomas (HCCs) are a heterogeneous group of tumors that differ in risk factors and genetic alterations. We further investigated transcriptome-genotype-phenotype correlations in HCC. Global transcriptome analyses were performed on 57 HCCs and 3 hepatocellular adenomas and validated by quantitative RT-PCR using 63 additional HCCs. We determined loss of heterozygosity, gene mutations, promoter methylation of CDH1 and CDKN2A, and HBV DNA copy number for each tumor. Unsupervised transcriptome analysis identified 6 robust subgroups of HCC (G1-G6) associated with clinical and genetic characteristics. G1 tumors were associated with low copy number of HBV and overexpression of genes expressed in fetal liver and controlled by parental imprinting. G2 included HCCs infected with a high copy number of HBV and mutations in PIK3CA and TP53. In these first groups, we detected specific activation of the AKT pathway. G3 tumors were typified by mutation of TP53 and overexpression of genes controlling the cell cycle. G4 was a heterogeneous subgroup of tumors including TCF1-mutated hepatocellular adenomas and carcinomas. G5 and G6 were strongly related to -catenin mutations that lead to Wnt pathway activation; in particular, G6 tumors were characterized by satellite nodules, higher activation of the Wnt pathway, and E-cadherin underexpression. Conclusion: These results have furthered our understanding of the genetic diversity of human HCC and have provided specific identifiers for classifying tumors. In addition, our classification has potential therapeutic implications because 50% of the tumors were related to WNT or AKT pathway activation, which potentially could be targeted by specific inhibiting therapies.
Ethanol-induced oxidative stress suppresses generation of peptides for antigen presentation by hepatoma cells (p 53-61)
Natalia A. Osna, Ronda L. White, Sandra Todero, Benita L. Mc Vicker, Geoffrey M. Thiele, Dahn L. Clemens, Dean J. Tuma, Terrence M. Donohue Jr.
Processing of peptides for antigen presentation is catalyzed by antigen-trimming enzymes, including the proteasome and leucine aminopeptidase. Oxidative stress suppresses proteasome function. We hypothesized that in liver cells, processing of antigenic peptides is altered by ethanol metabolism. To address this issue, soluble extracts of ethanol-metabolizing VL-17A cells treated with 100 mM ethanol or left untreated were incubated with C-extended or N-extended 18-27 HBV core peptides. Peptide cleavage was measured by recovery after HPLC. Ethanol exposure to VL-17A cells increased CYP2E1 and decreased proteasome peptidase activities. The latter effect was prevented by treatment of cells with inhibitors, 4-methylpyrazole and diallyl sulfide. Ethanol treatment of VL-17A cells also reduced the activity of leucine aminopeptidase (LAP). Consequently, cleavage of both C-extended and N-extended peptides by cytosolic extracts was suppressed by pretreatment of cells with ethanol. Treatment of cells with interferon gamma, which enhances proteasome activity, did not reverse the effects of ethanol. Ethanol exerted similar effects on WIFB cells, indicating that its effects are not unique to one cell type. Conclusion: Ethanol metabolism suppresses activities of antigen-trimming enzymes, thereby decreasing the cleavage of C-extended and N-extended peptides. This defect may potentially result in decreased MHC class I-restricted antigen presentation on virally infected liver cells.
Increasing dimethylarginine levels are associated with adverse clinical outcome in severe alcoholic hepatitis (p 62-71)
Rajeshwar P. Mookerjee, Mohammed Malaki, Nathan A. Davies, Stephen J. Hodges, R. Neil Dalton, Charles Turner, Sambit Sen, Roger Williams, James Leiper, Patrick Vallance, Rajiv Jalan
Previous studies suggest reduced hepatic endothelial nitric oxide synthase activity contributes to increased intrahepatic resistance. Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor, undergoes hepatic metabolism via dimethylarginine-dimethylamino-hydrolase, and is derived by the action of protein-arginine-methyltransferases. Our study assessed whether ADMA, and its stereo-isomer symmetric dimethylarginine (SDMA), are increased in alcoholic hepatitis patients, and determined any relationship with severity of portal hypertension (hepatic venous pressure gradient measurement) and outcome. Fifty-two patients with decompensated alcoholic cirrhosis were studied, 27 with acute alcoholic hepatitis and cirrhosis, in whom hepatic venous pressure gradient was higher (P = 0.001) than cirrhosis alone, and correlated with ADMA measurement. Plasma ADMA and SDMA were significantly higher in alcoholic hepatitis patients and in nonsurvivors. Dimethylarginine-dimethylamino-hydrolase protein expression was reduced and protein-arginine-methyltransferase-1 increased in alcoholic hepatitis livers. ADMA, SDMA and their combined sum, which we termed a dimethylarginine score, were better predictors of outcome compared with Pugh score, MELD and Maddrey's discriminant-function. Conclusion: Alcoholic hepatitis patients have higher portal pressures associated with increased ADMA, which may result from both decreased breakdown (decreased hepatic dimethylarginine-dimethylamino-hydrolase) and/or increased production. Elevated dimethylarginines may serve as important biological markers of deleterious outcome in alcoholic hepatitis.
Uridine supplementation antagonizes zalcitabine-induced microvesicular steatohepatitis in mice (p 72-79)
Dirk Lebrecht, Yetlanezi A. Vargas-Infante, Bernhard Setzer, Janbernd Kirschner, Ulrich A. Walker
Zalcitabine is an antiretroviral nucleoside analogue that exhibits long-term toxicity to hepatocytes by interfering with the replication of mitochondrial DNA (mtDNA). Uridine antagonizes this effect in vitro. In the present study we investigate the mechanisms of zalcitabine-induced hepatotoxicity in mice and explore therapeutic outcomes with oral uridine supplementation. BalbC mice (7 weeks of age, 9 mice in each group) were fed 0.36 mg/kg/d of zalcitabine (corresponding to human dosing adapted for body surface), or 13 mg/kg/d of zalcitabine. Both zalcitabine groups were treated with or without Mitocnol (0.34 g/kg/d), a dietary supplement with high bioavailability of uridine. Liver histology and mitochondrial functions were assessed after 15 weeks. One mouse exposed to high dose zalcitabine died at 19 weeks of age. Zalcitabine induced a dose dependent microvesicular steatohepatitis with abundant mitochondria. The organelles were enlarged and contained disrupted cristae. Terminal transferase dUTP nick end labeling (TUNEL) assays showed frequent hepatocyte apoptosis. mtDNA was depleted in liver tissue, cytochrome c-oxidase but not succinate dehydrogenase activities were decreased, superoxide and malondialdehyde were elevated. The expression of COX I, an mtDNA-encoded respiratory chain subunit was reduced, whereas COX IV, a nucleus-encoded subunit was preserved. Uridine supplementation normalized or attenuated all toxic abnormalities in both zalcitabine groups, but had no effects when given without zalcitabine. Uridine supplementation was without apparent side effects. Conclusion: Zalcitabine induces mtDNA-depletion in murine liver with consequent respiratory chain dysfunction, up-regulated synthesis of reactive oxygen species and microvesicular steatohepatitis. Uridine supplementation attenuates this mitochondrial hepatotoxicity without apparent intrinsic effects.
Race, insulin resistance and hepatic steatosis in chronic hepatitis C (p 80-87)
Hari S. Conjeevaram, David E. Kleiner, Jay E. Everhart, Jay H. Hoofnagle, Steven Zacks, Nezam H. Afdhal, Abdus S. Wahed, Virahep-C Study Group
Hepatic steatosis is common in chronic hepatitis C and has been linked to concurrent obesity, insulin resistance, diabetes, disease severity, and poor response to therapy. Racial differences in rates of obesity and diabetes may contribute to racial differences in hepatic steatosis and treatment response. The aim of the present study was to compare hepatic steatosis and its associations between African American (AA) and Caucasian American (CA) patients with chronic hepatitis C, genotype 1, participating in a prospective study of peginterferon and ribavirin therapy. Liver biopsy results were available from 194 AA patients and 205 CA patients. The 2 groups were compared for anthropometric, clinical, and biochemical features and insulin resistance estimated by the homeostasis model assessment index (HOMA-IR). Sixty-one percent of the AA patients and 65% of the CA patients had hepatic steatosis (P = 0.38). In univariable analysis, steatosis was associated with HOMA-IR, body mass index, waist circumference, serum triglycerides, aminotransferase level, and histological scores for inflammation and fibrosis. After adjusting for these features, AA patients had a lower risk of steatosis than did CA patients (OR 0.54, 95% CI 0.32-0.91, P = 0.02). Insulin resistance but not steatosis was associated with a lower rate of sustained virological response when adjusted for known factors that predict response (relative risk 0.87, 95% CI 0.77-0.99, P = 0.028). Conclusion: After adjusting for the higher prevalence of features associated with hepatic steatosis, AA patients had a lower prevalence of hepatic steatosis than did CA patients with chronic hepatitis C, genotype 1. Insulin resistance but not steatosis was independently associated with lower sustained virological response.
Keratin 18 overexpression but not phosphorylation or filament organization blocks mouse Mallory body formation (p 88-96)
Masaru Harada, Pavel Strnad, Evelyn Z. Resurreccion, Nam-On Ku, M. Bishr Omary
Several human liver diseases are associated with formation of Mallory body (MB) inclusions. These hepatocyte cytoplasmic deposits are composed primarily of hyperphosphorylated keratins 8 and 18 (K8/K18). Feeding a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-containing diet is a well-established mouse model of MBs. K8 overexpression, and K8-null or K18-null mouse models, indicate that a K8-greater-than-K18 expression ratio is critical for MB formation. We used established transgenic mouse models to study the effect of K18 overexpression and phosphorylation, or keratin filament disorganization, on MB formation. Five mouse lines were used: nontransgenic, those that overexpress wild-type K18 or the K18 phosphorylation mutants Ser33-to-Ala (S33A) or Ser52-to-Ala (S52A), and mice that overexpress K18 Arg89-to-Cys, which causes collapse of the keratin filament network into dots. DDC feeding induced MBs in nontransgenic livers, but MBs were rarely seen in any of the K18 transgenic mice. Wild-type K18 overexpression protected mice from DDC-induced liver injury. Conclusion: K18 overexpression protects mice from MB formation and from DDC-induced liver injury, which supports the importance of the K8-to-K18 ratio in MB formation. The effect of K18 on MB formation is independent of hepatocyte keratin filament organization or K18 Ser33/Ser52 phosphorylation. Keratin filament collapse, which is a major risk for acute liver injury, is well tolerated in the context of chronic DDC-mediated liver injury.
A randomized controlled trial of lamivudine to treat acute hepatitis B (p 97-101)
M. Kumar, S. Satapathy, R. Monga, K. Das, S. Hissar, C. Pande, B. C. Sharma, S. K. Sarin
The role of antivirals in patients with acute viral hepatitis B (AVH-B) has not been evaluated in controlled trials. The aim of this study was to evaluate the efficacy of lamivudine in patients with AVH-B. AVH-B patients with serum bilirubin of more than 5 mg/dL were randomized to receive either 100 mg of lamivudine daily for 3 months (group 1, n = 31) or placebo (group 2, n = 40). Patients were considered to have severe AVH-B if they fulfilled 2 of 3 criteria: (1) hepatic encephalopathy; (2) serum bilirubin 10.0 mg/dL; and (3) international normalized ratio (INR) 1.6. At week 4, HBV DNA levels were significantly lower (P = 0.037) in group 1 (median: 3.6721 log copies/mL) than group 2 (median: 4.2721 log copies/mL). Thereafter, HBV DNA levels were comparable in the 2 groups. The improvement in serum bilirubin, ALT, and INR values was similar in the 2 groups. Twenty-two patients (71%) in group 1 and 25 patients (62.5%) in group 2 had severe AVH-B. Results were similar when patients with severe AVH-B were analyzed separately. After 12 and 18 months, 93.5% and 92.5%, respectively, of patients in the lamivudine group and 96.7% and 97.5%, respectively, of patients in the placebo group lost HBsAg. There were no deaths in either group. After 1 year, 21 patients (67.7%) in group 1 and 34 patients (85%) in group 2 developed protective anti-HBs titers (P = 0.096). All HBeAg-positive patients in both groups lost e antigen and anti-HBe developed in 71% and 87.5% of patients in groups 1 and 2, respectively (P = 0.132). Conclusion: Though lamivudine causes a greater decrease in levels of HBV DNA, it does not cause significantly greater biochemical and clinical improvement as compared to placebo in patients with acute hepatitis B.
Regulation of Toll-like receptor-2 expression in chronic hepatitis B by the precore protein (p 102-110)
Kumar Visvanathan, Narelle A. Skinner, Alex J.V. Thompson, Stephen M. Riordan, Vitini Sozzi, Roslyn Edwards, Sally Rodgers, Jelica Kurtovic, Judy Chang, Sharon Lewin, Paul Desmond, Stephen Locarnini
Toll-like receptors (TLRs) play a key role in the innate immune response. The aim of this study was to examine the expression of TLR2 and TLR4 in chronic hepatitis B (CHB). The TLR2 and TLR4 expression on hepatocytes and Kupffer cells from fresh liver biopsies was measured from 21 patients with untreated hepatitis B e antigen (HBeAg)-positive and HBeAg-negative CHB. Parallel studies were also undertaken on monocytes from their peripheral blood. Expression of TLR2 on hepatocytes, Kupffer cells, and peripheral monocytes was significantly reduced in patients with HBeAg-positive CHB in comparison with HBeAg-negative CHB and controls, whereas it was significantly increased in HBeAg-negative CHB compared with controls. The level of TLR4 expression did not differ significantly between the groups. These results were confirmed in vitro using hepatic cell lines transduced with recombinant HBV baculovirus expressing wild-type HBV (HBeAg-positive), precore stop codon (G1896A) mutant HBV (HBeAg-negative). The functional relevance of these findings was established by the demonstration of significantly reduced cytokine production (TNF-) and phospho-p38 kinase expression in the presence of the HBeAg. In the absence of HBeAg, HBV replication was associated with up-regulation of the TLR2 pathway leading to increased TNF- production. Conclusion: This study demonstrates a potentially important interaction between HBeAg, HBV, and the innate immune response.
Hepatitis C virus eradication in intravenous drug users maintained with subcutaneous naltrexone implants (p 111-117)
Gary P. Jeffrey, Gerry MacQuillan, Fern Chua, Sam Galhenage, Judith Bull, Emma Young, Gary Hulse, George O'Neil
The effectiveness of HCV antiviral therapy in patients who have undergone recent drug dependency treatment and continue to inject drugs sporadically is presently not clear. Patients attending a community-based drug rehabilitation and naltrexone implant clinic from October 2002 until March 2005 were screened for HCV infection and if positive offered further assessment and treatment with interferon and ribavirin therapy. The first 50 patients to commence HCV therapy and complete at least 6 months follow-up were prospectively studied. ETR response (HCV PCR negative) was 34/50 (68%) and SVR 6 months post-treatment was 31/50 (62%). Viral eradication was maintained in those 22 patients that have had 12 months or more post-treatment follow-up. Eleven (22%) patients stopped therapy early due to side effects or poor compliance. Only two patients with an ETR likely reinfected due to unsafe injection practices. One was re-treated and achieved an SVR. Of the patients achieving a 6-month SVR, 17 of 31 patients reported no further IDU and 13 of 31 patients occasional IDU during treatment and this was maintained after HCV treatment cessation. 46% of patients received antidepressant and/or antipsychotic medication during treatment. Conclusion: This study of HCV treatment in a community-based subcutaneous naltrexone implant clinic found antiviral therapy resulted in a 62% SVR. This result is comparable to that reported in hospital-based clinics in non-IDU patients. The side effect profile and compliance was also similar. HCV antiviral therapy should be offered to this large and currently under treated group.
Anti-gp210 and anti-centromere antibodies are different risk factors for the progression of primary biliary cirrhosis (p 118-127)
Minoru Nakamura, Hisayoshi Kondo, Tsuyoshi Mori, Atsumasa Komori, Mutsumi Matsuyama, Masahiro Ito, Yasushi Takii, Makiko Koyabu, Terufumi Yokoyama, Kiyoshi Migita, Manabu Daikoku, Seigo Abiru, Hiroshi Yatsuhashi, Eiichi Takezaki, Naohiko Masaki, Kazuhiro Sugi, Koichi Honda, Hiroshi Adachi, Hidehiro Nishi, Yukio Watanabe, Yoko Nakamura, Masaaki Shimada, Tatsuji Komatsu, Akira Saito, Takeo Saoshiro, Hideharu Harada, Takeshi Sodeyama, Shigeki Hayashi, Akihide Masumoto, Takehiro Sando, Tetsuo Yamamoto, Hironori Sakai, Masakazu Kobayashi, Toyokichi Muro, Michiaki Koga, Zakera Shums, Gary L. Norman, Hiromi Ishibashi
The predictive role of antinuclear antibodies (ANAs) remains elusive in the long-term outcome of primary biliary cirrhosis (PBC). The progression of PBC was evaluated in association with ANAs using stepwise Cox proportional hazard regression and an unconditional stepwise logistic regression model based on the data of 276 biopsy-proven, definite PBC patients who have been registered to the National Hospital Organization Study Group for Liver Disease in Japan (NHOSLJ). When death of hepatic failure/liver transplantation (LT) was defined as an end-point, positive anti-gp210 antibodies (Hazard ratio (HR) = 6.742, 95% confidence interval (CI): 2.408, 18.877), the late stage (Scheuer's stage 3, 4) (HR = 4.285, 95% CI:1.682,10.913) and male sex (HR = 3.266, 95% CI: 1.321,8.075) were significant risk factors at the time of initial liver biopsy. When clinical progression to death of hepatic failure/LT (i.e., hepatic failure type progression) or to the development of esophageal varices or hepatocellular carcinoma without developing jaundice (Total bilirubin < 1.5 mg/dL) (i.e., portal hypertension type progression) was defined as an end-point in the early stage (Scheuer's stage 1, 2) PBC patients, positive anti-gp210 antibodies was a significant risk factor for hepatic failure type progression [odds ratio (OR) = 33.777, 95% CI: 5.930, 636.745], whereas positive anti-centromere antibodies was a significant risk factor for portal hypertension type progression (OR = 4.202, 95% CI: 1.307, 14.763). Histologically, positive anti-gp210 antibodies was most significantly associated with more severe interface hepatitis and lobular inflammation, whereas positive anticentromere antibodies was most significantly associated with more severe ductular reaction. Conclusion: These results indicate 2 different progression types in PBC, hepatic failure type and portal hypertension type progression, which may be represented by positive-anti-gp210 and positive-anticentromere antibodies, respectively.
Regulation of the UGT1A1 bilirubin-conjugating pathway: Role of a new splicing event at the UGT1A locus (p 128-138)
Eric Lévesque, Hugo Girard, Kim Journault, Johanie Lépine, Chantal Guillemette
UDP-glucuronosyltransferase 1A1 (UGT1A1) is involved in a wide range of biological and pharmacological processes because of its critical role in the conjugation of a diverse array of endogenous and exogenous compounds. We now describe a new UGT1A1 isoform, referred to as isoform 2 (UGT1A1_i2), encoded by a 1495-bp complementary DNA isolated from human liver and generated by an alternative splicing event involving an additional exon found at the 3 end of the UGT1A locus. The N-terminal portion of the 45-kd UGT1A1_i2 protein is identical to UGT1A1 (55 kd, UGT1A1_i1); however, UGT1A1_i2 contains a unique 10-residue sequence instead of the 99-amino acid C-terminal domain of UGT1A1_i1. RT-PCR and Western blot analyses with a specific antibody against UGT1A1 indicate that isoform 2 is differentially expressed in liver, kidney, colon, and small intestine at levels that reach or exceed, for some tissues, those of isoform 1. Western blots of different cell fractions and immunofluorescence experiments indicate that UGT1A1_i1 and UGT1A1_i2 colocalize in microsomes. Functional enzymatic data indicate that UGT1A1_i2, which lacks transferase activity when stably expressed alone in HEK293 cells, acts as a negative modulator of UGT1A1_i1, decreasing its activity by up to 78%. Coimmunoprecipitation of UGT1A1_i1 and UGT1A1_i2 suggests that this repression may occur via direct protein-protein interactions. Conclusion: Our results indicate that this newly discovered alternative splicing mechanism at the UGT1A locus amplifies the structural diversity of human UGT proteins and describes the identification of an additional posttranscriptional regulatory mechanism of the glucuronidation pathway.
Novel hepatic progenitor cell surface markers in the adult rat liver (p 139-149)
Mladen I. Yovchev, Petar N. Grozdanov, Brigid Joseph, Sanjeev Gupta, Mariana D. Dabeva
Hepatic progenitor/oval cells appear in injured livers when hepatocyte proliferation is impaired. These cells can differentiate into hepatocytes and cholangiocytes and could be useful for cell and gene therapy applications. In this work, we studied progenitor/oval cell surface markers in the liver of rats subjected to 2-acetylaminofluorene treatment followed by partial hepatectomy (2-AAF/PH) by using rat genome 230 2.0 Array chips and subsequent RT-PCR, immunofluorescent (IF), immunohistochemical (IHC) and in situ hybridization (ISH) analyses. We also studied expression of the identified novel cell surface markers in fetal rat liver progenitor cells and FAO-1 hepatoma cells. Novel cell surface markers in adult progenitor cells included tight junction proteins, integrins, cadherins, cell adhesion molecules, receptors, membrane channels and other transmembrane proteins. From the panel of 21 cell surface markers, 9 were overexpressed in fetal progenitor cells, 6 in FAO-1 cells and 6 are unique for the adult progenitors (CD133, claudin-7, cadherin 22, mucin-1, ros-1, Gabrp). The specificity of progenitor/oval cell surface markers was confirmed by ISH and double IF analyses. Moreover, study of progenitor cells purified with Ep-CAM antibodies from D-galactosamine injured rat liver, a noncarcinogenic model of progenitor cell activation, verified that progenitor cells expressed these markers. Conclusion: We identified novel cell surface markers specific for hepatic progenitor/oval cells, which offers powerful tool for their identification, isolation and studies of their physiology and pathophysiology. Our studies also reveal the mesenchymal/epithelial phenotype of these cells and the existence of species diversity in the hepatic progenitor cell identity.
Linkage between a new splicing site mutation in the MDR3 alias ABCB4 gene and intrahepatic cholestasis of pregnancy (p 150-158)
Gudrun Schneider, Teresa C. Paus, Gerd A. Kullak-Ublick, Peter J. Meier, Thomas F. Wienker, Thomas Lang, Patricia van de Vondel, Tilman Sauerbruch, Christoph Reichel
Intrahepatic cholestasis of pregnancy (ICP) is defined as pruritus and elevated bile acid serum concentrations in late pregnancy. Splicing mutations have been described in the multidrug resistance p-glycoprotein 3 (MDR3, ABCB4) gene in up to 20% of ICP women. Pedigrees studied were not large enough for linkage analysis. Ninety-seven family members of a woman with proven ICP were asked about pruritus in earlier pregnancies, birth complications and symptomatic gallstone disease. The familial cholestasis type 1 (FIC1, ATP8B1) gene, bile salt export pump (BSEP, ABCB11) and MDR3 gene were analyzed in 55 relatives. We identified a dominant mode of inheritance with female restricted expression and a new intronic MDR3 mutation c.3486+5G>A resulting in a 54 bp (3465-3518) inframe deletion via cryptic splicing site activation. Linkage analysis of the ICP trait versus this intragenic MDR3 variant yielded a LOD score of 2.48. A Bayesian analysis involving MDR3, BSEP, FIC1 and an unknown locus gave a posterior probability of >0.9966 in favor of MDR3 as causative ICP locus. During the episode of ICP the median -glutamyl transpeptidase (-GT) activity was 10 U/l (95% CI, 6.9 to 14.7 U/l) in the index woman. Four stillbirths were reported in seven heterozygous women (22 pregnancies) and none in five women (14 pregnancies) without MDR3 mutation. Symptomatic gallstone disease was more prevalent in heterozygous relatives (7/21) than in relatives without the mutation (1/34), (P = 0.00341). Conclusion: This study demonstrates that splicing mutations in the MDR3 gene can cause ICP with normal -GT and may be associated with stillbirths and gallstone disease.
Prostaglandin I2 and E2 mediate the protective effects of cyclooxygenase-2 in a mouse model of immune-mediated liver injury (p 159-169)
Hao Yin, Linling Cheng, Robert Langenbach, Cynthia Ju
Studies of the molecular and cellular mechanisms of concanavalin A (ConA)-induced liver injury have provided important knowledge on the pathogenesis of many liver diseases involving hepatic inflammation. However, studies identifying hepato-protective factors based on the mechanistic understanding of this model are lacking. Evidence suggests that certain prostaglandin (PG) products of cyclooxygenase (COX)-1 and COX-2 provide important anti-inflammatory and cytoprotective functions in some pathophysiological states. In the present study, we demonstrate a protective role of COX-2 derived PGs in ConA-induced liver injury. COX-2-/- mice developed much more severe liver damage upon ConA treatment compared with wild-type and COX-1-/- mice. Treatment of COX-2-/- mice with misoprostol (a PGE1/2 analog) or beraprost (a PGI2 analog) significantly decreased ConA-induced liver injury. Data from both in vivo and in vitro experiments demonstrated that misoprostol and beraprost acted directly on hepatic leukocytes, including natural killer (NK)T and T cells, and down-regulated their production of interferon (IFN)-, which are critical in mediating ConA-induced tissue damage. Collectively, the results provide strong evidence that the protective effects of COX-2 within the liver are mediated through the production of PGE2 and PGI2, which exert anti-inflammatory functions. These findings suggest that COX-2-derived PGs may have great therapeutic potentials in treating patients with inflammatory liver diseases.
Thyroid hormone preconditioning: Protection against ischemia-reperfusion liver injury in the rat (p 170-177)
Virginia Fernández, Iván Castillo, Gladys Tapia, Pamela Romanque, Sebastián Uribe-Echevarría, Mario Uribe, Denise Cartier-Ugarte, Gonzalo Santander, María T. Vial, Luis A. Videla
Recently, we reported that oxidative stress due to 3,3,5-triiodothyronine (T3)-induced calorigenesis up-regulates the hepatic expression of mediators promoting cell protection. In this study, T3 administration in rats (single dose of 0.1 mg/kg intraperitoneally) induced significant depletion of reduced liver glutathione (GSH), with higher protein oxidation, O2 consumption, and Kupffer cell function (carbon phagocytosis and carbon-induced O2 uptake). These changes occurred within a period of 36 hours of T3 treatment in animals showing normal liver histology and lack of alteration in serum AST and ALT levels. Partial hepatic ischemia-reperfusion (IR) (1 h of ischemia via vascular clamping and 20 h reperfusion) led to 11-fold and 42-fold increases in serum AST and ALT levels, respectively, and significant changes in liver histology, with a 36% decrease in liver GSH content and a 133% increase in that of protein carbonyls. T3 administration in a time window of 48 hours was substantially protective against hepatic IR injury, with a net 60% and 90% reduction in liver GSH depletion and protein oxidation induced by IR, respectively. Liver IR led to decreased DNA binding of nuclear factor-B (NF-B) (54%) and signal transducer and activator of transcription 3 (STAT3) (53%) (electromobility shift assay), with 50% diminution in the protein expression of haptoglobin (Western blot), changes that were normalized by T3 preconditioning. Conclusion: T3 administration involving transient oxidative stress in the liver exerts significant protection against IR injury, a novel preconditioning maneuver that is associated with NF-B and STAT3 activation and acute-phase response.
Immune role of hepatic TLR-4 revealed by orthotopic mouse liver transplantation (p 178-186)
Beena John, Ingo Klein, I. Nicholas Crispe
Activated CD8+ T cells migrate to the liver at the end of an immune response and go through apoptosis there, but this mechanism is impaired in mice lacking Toll-like receptor-4. This allowed us to test the importance of liver trapping in an ongoing immune response. In the absence of Toll-like receptor-4, reduced liver accumulation was associated with an increase in the circulating CD8+ T cell pool, more long-lived memory T cells and increased CD8+ T cell memory responses. Using experimental orthotopic liver transplantation, we showed that the effect of Toll-like receptor-4 on the formation of the CD8+ T cell memory resides in the liver. Conclusion: These studies reveal a new function for the liver, which is to regulate the magnitude of T cell memory responses through a Toll-like receptor-4-dependent mechanism.
Effect of iron and ascorbate on uroporphyria in ascorbate-requiring mice as a model for porphyria cutanea tarda (p 187-194)
Nadia Gorman, Adrian Zaharia, Heidi S. Trask, Juliana G. Szakacs, Nicholas J. Jacobs, Judith M. Jacobs, Dominic Balestra, Jacqueline F. Sinclair, Peter R. Sinclair
Excess hepatic iron is known to enhance both porphyria cutanea tarda (PCT) and experimental uroporphyria. Since previous studies have suggested a role for ascorbate (AA) in suppressing uroporphyria in AA-requiring rats (in the absence of excess iron), the present study investigated whether AA could suppress uroporphyria produced by excess hepatic iron. Hepatic URO accumulation was produced in AA-requiring Gulo(-/-) mice by treatment with 3,3,4,4,5-pentachlorbiphenyl, an inducer of CYP1A2, and 5-aminolevulinic acid. Mice were administered either sufficient AA (1000 ppm) in the drinking water to maintain near normal hepatic AA levels or a lower intake (75 ppm) that resulted in 70 % lower hepatic AA levels. The higher AA intake suppressed hepatic URO accumulation in the absence of administered iron, but not when iron dextran (300-500 mg Fe/kg) was administered. This effect of iron was not due to hepatic AA depletion since hepatic AA content was not decreased. The effect of iron to prevent AA suppression of hepatic URO accumulation was not observed until a high hepatic iron threshold was exceeded. At both low and high AA intakes, hepatic malondialdehyde (MDA), an indicator of oxidative stress, was increased three-fold by high doses of iron dextran. MDA was considerably increased even at low iron dextran doses, but without any increase in URO accumulation. The level of hepatic CYP1A2 was unaffected by either AA intake. Conclusion: In this mouse model of PCT, AA suppresses hepatic URO accumulation at low, but not high hepatic iron levels. These results may have implications for the management of PCT.
Wnt'er in liver: Expression of Wnt and frizzled genes in mouse (p 195-204)
Gang Zeng, Farrukh Awan, Wade Otruba, Peggy Muller, Udayan Apte, Xinping Tan, Chandrashekhar Gandhi, Anthony J. Demetris, Satdarshan P. S. Monga
The Wnt signaling pathway is essential for a wide array of developmental and physiological processes. Wnts are extracellular ligands that bind to frizzled (Fz) receptors at the membrane, canonically inducing -catenin nuclear translocation and activation. Although -catenin has been shown to be critical in liver biology, the expression of the 19 Wnt and 10 Fz genes in liver remains undetermined. We report comprehensive analysis of Wnt and Fz expression in whole liver as well as individual cell types: freshly isolated and plated hepatocytes, biliary epithelial cells, normal and activated stellate and Kupffer cells, and sinusoidal endothelial cells (SECs). Oligonucleotides for the 19 Wnt, 10 frizzled receptors genes, and secreted Frizzled-related protein-1 (sFRP or Fzb) were synthesized based on the available sequences. A total of 11 Wnts and 8 Fz genes and Fzb were expressed in normal liver. Although only 6 Wnt and 5 Fz genes were expressed in freshly isolated hepatocytes, 8 Wnt genes, 7 Fz genes, and Fzb were expressed in plated hepatocytes. Although 12 Wnt and 7 Fz genes were expressed in biliary tree, additional Fz9 and Fzb were only expressed in cultured biliary epithelial cells. The same 14 Wnt and 7 Fz genes were expressed in both activated and normal stellate and Kupffer cells; only Fzb was expressed in their activated state. Also, 11 Wnt, seven Fz, and Fzb genes were expressed in SECs. Conclusion: These data indicate that most Wnt and frizzled genes are expressed in the liver and might be playing important roles in liver pathobiology via canonical and noncanonical pathways.
Arsenic stimulates sinusoidal endothelial cell capillarization and vessel remodeling in mouse liver (p 205-212)
Adam C. Straub, Donna B. Stolz, Mark A. Ross, Araceli Hernández-Zavala, Nicole V. Soucy, Linda R. Klei, Aaron Barchowsky
Trivalent arsenic [As(III)] is a well-known environmental toxicant that causes a wide range of organ-specific diseases and cancers. In the human liver, As(III) promotes vascular remodeling, portal fibrosis, and hypertension, but the pathogenesis of these As(III)-induced vascular changes is unknown. To investigate the hypothesis that As(III) targets the hepatic endothelium to initiate pathogenic change, mice were exposed to 0 or 250 parts per billion (ppb) of As(III) in their drinking water for 5 weeks. Arsenic(III) exposure did not affect the overall health of the animals, the general structure of the liver, or hepatocyte morphology. There was no change in the total tissue arsenic levels, indicating that arsenic does not accumulate in the liver at this level of exposure. However, there was significant vascular remodeling with increased sinusoidal endothelial cell (SEC) capillarization, vascularization of the peribiliary vascular plexus (PBVP), and constriction of hepatic arterioles in As(III)-exposed mice. In addition to ultrastructural demonstration of SEC defenestration and capillarization, quantitative immunofluorescence analysis revealed increased sinusoidal PECAM-1 and laminin-1 protein expression, suggesting gain of adherens junctions and a basement membrane. Conversion of SECs to a capillarized, dedifferentiated endothelium was confirmed at the cellular level with demonstration of increased caveolin-1 expression and SEC caveolae, as well as increased membrane-bound Rac1-GTPase. Conclusion: These data demonstrate that exposure to As(III) causes functional changes in SEC signaling for sinusoidal capillarization that may be initial events in pathogenic changes in the liver.
Bone marrow-derived cells express matrix metalloproteinases and contribute to regression of liver fibrosis in mice (p 213-222)
Reiichi Higashiyama, Yutaka Inagaki, Yun Yu Hong, Miwa Kushida, Sachie Nakao, Maki Niioka, Tetsu Watanabe, Hideyuki Okano, Yumi Matsuzaki, Goshi Shiota, Isao Okazaki
Liver fibrosis is usually progressive, but it can occasionally be reversible if the causative agents are adequately removed or if patients are treated effectively. However, molecular mechanisms responsible for this reversibility of liver fibrosis have been poorly understood. To reveal the contribution of bone marrow (BM)-derived cells to the spontaneous regression of liver fibrosis, mice were treated with repeated carbon tetrachloride injections after hematopoietic reconstitution with enhanced green fluorescent protein (EGFP)-expressing BM cells. The distribution and characteristics of EGFP-positive (EGFP+) cells present in fibrotic liver tissue were examined at different time points after cessation of carbon tetrachloride intoxication. A large number of EGFP+ cells were observed in liver tissue at peak fibrosis, which decreased during the recovery from liver fibrosis. Some of them, as well as EGFP-negative (EGFP-) liver resident cells, expressed matrix metalloproteinase (MMP)-13 and MMP-9. Whereas MMP-13 was transiently expressed mainly in the cells clustering in the periportal areas, MMP-9 expression and enzymatic activity were detected over the resolution process in several different kinds of cells located in the portal areas and along the fibrous septa. Therapeutic recruitment of BM cells by granulocyte colony-stimulating factor (G-CSF) treatment significantly enhanced migration of BM-derived cells into fibrotic liver and accelerated the regression of liver fibrosis. Experiments using transgenic mice overexpressing hepatocyte growth factor (HGF) indicated that G-CSF and HGF synergistically increased MMP-9 expression along the fibrous septa. Conclusion: Autologous BM cells contribute to the spontaneous regression of liver fibrosis, and their therapeutic derivation could be a new treatment strategy for intractable liver fibrosis.
Renal failure and bacterial infections in patients with cirrhosis: Epidemiology and clinical features (p 223-229)
Silvano Fasolato, Paolo Angeli, Lucia Dallagnese, Giulio Maresio, Erika Zola, Elena Mazza, Freddy Salinas, Silvio Donà, Stefano Fagiuoli, Antonietta Sticca, Giacomo Zanus, Umberto Cillo, Ilaria Frasson, Carla Destro, Angelo Gatta
The aim of the study was to investigate the prevalence and clinical course of renal failure that was induced by the various types of bacterial infections in patients with cirrhosis and ascites. Three hundred and nine patients, who were consecutively admitted to the 3 major hospitals of Padova, Italy, during the first 6 months of 2005, were studied prospectively. Of these, 233 patients (75.4%) had evidence of ascites. In 104 patients with cirrhosis and ascites (44.6%) a bacterial infection was diagnosed. A bacterial infection-induced renal failure was observed in 35 of 104 patients (33.6%). The prevalence of renal failure was higher in biliary or gastrointestinal tract infections and in spontaneous bacterial peritonitis (SBP) and in than in other types of infections. In addition, the progressive form of renal failure was only precipitated by biliary or gastrointestinal tract infections, SBP, and urinary tract infections (UTI). In a multivariate analysis only MELD score (P = 0.001), the peak count of neutrophil leukocyte in blood (P = 0.04), and the lack of resolution of infection (P = 0.03) had an independent predictive value on the occurrence of renal failure. Conclusion: The results of the study show that the development of bacterial-induced renal failure in patients with cirrhosis and ascites is related to the MELD score, and to both the severity and the lack of resolution of the infection. A progressive form of renal failure occurs only as a consequence of biliary or gastrointestinal tract infections, SBP, and UTI.
Copyright © 2007 by the American Association for the Study of Liver Diseases. All rights reserved.
GASTROENTEROLOGY
Décembre 2006 en attente (Mise en ligne vers le 15/01/07)
© 2007 American Gastroenterological Association (AGA) Institute. Published by Elsevier Inc. All rights reserved.
JOURNAL OF HEPATOLOGY
Volume 46, Issue 1, January 2007
"Seuls les abstracts des articles Originaux sont mis sur cette page. Les autres articles sont en ligne en FREE sur le site du Journal"
Original Articles
Viral Hepatitis
Outbreak of hepatitis C virus infection during sclerotherapy of varicose veins: Long-term follow-up of 196 patients (4535 patient-years), 21 September 2006
de Lédinghen V, Trimoulet P, Mannant PR, Dumas F, Champbenoît P, Baldit C, Foucher J, Faure M, Vergniol J, Castéra L, Bertet J, Fleury H, Couzigou P, Bernard PH
Background/Aims
The aim of this study was to describe the natural history of a HCV infection outbreak in 196 patients who had sclerotherapy by a same physician and to confirm patient-to-patient transmission using phylogenetic analysis in a large series of patients.
Methods
Demographic information included clinical and biological parameters. Fibrosis evaluation was performed using liver biopsy or transient elastography. Follow-up was maintained until death, or the end of the observation period. In order to determine if the virus had been transmitted between the HCV genotype 2 patients, sequence analysis was undertaken of a part of the NS5b region of the genomes in samples of patients.
Results
The mean duration of follow-up was 23.1±6.7 years (4535 patient-years). In patients with fibrosis evaluation, 55.7% had no or mild fibrosis and 44.3% had significant fibrosis. No patient died from HCV-related disease. Nucleotide sequence analysis of a part of the NS5b region revealed that patients were all infected with the same HCV subtype (genotype 2d). The most evident feature of the tree is the clustering of all patients involved in the outbreak without any unrelated isolates.
Conclusion
This study emphasizes the risk for nosocomial spread of HCV during intravenous therapy.
Serum-derived hepatitis C virus infectivity in interferon regulatory factor-7-suppressed human primary hepatocytes, 30 October 2006
Aly HH, Watashi K, Hijikata M, Kaneko H, Takada Y, Egawa H, Uemoto S, Shimotohno K
Background/Aims
The development of an efficient in vitro infection system for HCV is important in order to develop new anti-HCV strategy. Only Huh7 hepatocyte cell lines were shown to be infected with JFH-1 fulminant HCV-2a strain and its chimeras. Here we aimed to establish a primary hepatocyte cell line that could be infected by HCV particles from patients’ sera.
Methods
We transduced primary human hepatocytes with human telomerase reverse transcriptase together with human papilloma virus 18/E6E7 (HPV18/E6E7) genes or simian virus large T gene (SV40 T) to immortalize cells. We also established the HPV18/E6E7-immortalized hepatocytes in which interferon regulatory factor-7 was inactivated. Finally we analyzed HCV infectivity in these cells.
Results
Even after prolonged culture HPV18/E6E7-immortalized hepatocytes exhibited hepatocyte functions and marker expression and were more prone to HCV infection than SV40 T-immortalized hepatocytes. The susceptibility of HPV18/E6E7-immortalized hepatocytes to HCV infection was further improved, in particular, by impairing signaling through interferon regulatory factor-7.
Conclusions
HPV18/E6E7-immortalized hepatocytes are useful for the analysis of HCV infection, anti-HCV innate immune response, and screening of antiviral agents with a variety of HCV strains.
Clinical evaluation (Phase I) of a human monoclonal antibody against hepatitis C virus: Safety and antiviral activity, 23 October 2006
Galun E, Terrault NA, Eren R, Zauberman A, Nussbaum O, Terkieltaub D, Zohar M, Buchnik R, Ackerman Z, Safadi R, Ashur Y, Misrachi S, Liberman Y, Rivkin L, Dagan S
Background/Aims:
HCV-AB68, a human monoclonal antibody against the envelope protein of hepatitis C virus (HCV), neutralizes HCV in cell-culture and in the HCV-Trimera mouse model. A Phase 1 clinical trial was designed to test safety, tolerability, and antiviral activity of HCV-AB68 in patients with chronic HCV-infection.
Methods/Results:
Single doses of HCV-AB68, 0.25–40mg, administered to 15 patients were well tolerated with no moderate or serious adverse events (SAEs) reported. In six patients, HCV-RNA levels transiently decreased by 2- to 100-fold immediately following infusion and rebound to baseline in 24–48h. Multiple doses of HCV-AB68, 10–120mg, were administered to 25 patients. Doses were given weekly for 3 weeks, then 3? a week during the fourth week, after which patients were followed for 3 months. No drug-related SAEs were reported and no specific pattern of adverse events was evident. Eight out of 25 patients had at least a 1-log reduction and 17 had at least a 0.75-log reduction in HCV-RNA levels from baseline at one or more time points following HCV-AB68 infusion.
Conclusions:
These data support the investigation of HCV-AB68 in the prevention of recurrent HCV-infection in patients who had received hepatic allografts for end-stage liver disease.
Interferon therapy in HBeAg positive chronic hepatitis reduces progression to cirrhosis and hepatocellular carcinoma, 23 October 2006
Lin SM, Yu ML, Lee CM, Chien RN, Sheen IS, Chu CM, Liaw YF
Background/Aims
The long-term outcomes of interferon-alpha (IFN-?) therapy in hepatitis B e antigen (HBeAg) seropositive patients remain controversial. This study was conducted to address this issue.
Methods
The long-term outcomes were compared in 233 IFN-treated patients and 233 well-matched untreated controls.
Results
The cumulative incidence at the end of 15 years of follow-up (median 6.8 years, range 1.1–16.5 years) in the IFN-treated patients and controls was: HBeAg seroconversion 74.6% vs. 51.7% (P=0.031); hepatitis B surface antigen (HBsAg) seroclearance 3% vs. 0.4% (P=0.03); cirrhosis 17.8% vs. 33.7% (P=0.041); and hepatocellular carcinoma (HCC) 2.7% vs. 12.5% (P=0.011). Significant reduction of HCC was only observed in patients with pre-existing cirrhosis (P<0.01). Compared with untreated controls with persistent HBeAg, HBeAg seroconverters in untreated and IFN-treated group showed significantly lower incidence of cirrhosis and HCC (P=0.003–0.031), while non-seroconverters of IFN-treated group had marginally significant lower incidence of cirrhosis (P=0.065). Multivariate analysis showed that IFN therapy, HBeAg seroconversion and genotype B HBV infection are independent factors for better long-term outcomes.
Conclusions
IFN therapy reduces cirrhosis and HCC development.
Intracellular accumulation of hepatitis C virus proteins in a human hepatoma cell line, 26 October 2006
Brazzoli M, Crotta S, Bianchi A, Bagnoli F, Monaghan P, Wileman T, Abrignani S, Merola M
Background/Aims
The establishment of HCV replicon systems strongly improved the research on the replication processes but poorly advanced our knowledge on the subcellular localization of the structural glycoproteins, mainly due to their low expression. We sought to verify whether reinforcing E1E2 expression in the context of both HCV genomic and subgenomic replicon from either homologous or heterologous strains leads to formation of supramolecular structures including structural and nonstructural proteins.
Methods
Robust expression of HCV glycoproteins was achieved by stable expression of E1E2p7 from genotype 1a and 1b.
Results
In these cells, E1 and E2 triggered the formation of dot-like structures in which they co-localized with core and the nonstructural proteins NS3 and NS5A. Confocal microscopy analyses suggested that accumulation of HCV proteins occurs in an ER-derived subcompartment. Moreover, by labeling de novo-synthesized HCV RNA, we showed that these structures constitute a site of viral RNA synthesis.
Conclusions
Expression in trans of HCV glycoproteins in the context of replicative viral genome or subgenome generates accumulation of structural and nonstructural viral proteins in peculiar cytoplasmic structures. The simultaneous presence of viral RNA, structural and nonstructural protein suggests that these complexes represent not only sites of HCV replication but also potential places of viral pre-budding.
Cirrhosis and its Complications
Long-term portosystemic shunt patency as a determinant of outcome in Budd–Chiari syndrome, 20 October 2006
Bachet JB, Condat B, Hagège H, Plessier A, Consigny Y, Belghiti J, Valla D
ackground/Aims
Portosystemic shunting, whether surgical or transjugular intrahepatic, has been a cornerstone of therapy for Budd–Chiari syndrome. However, the long-term impact of shunt dysfunction remains unknown. We have assessed this long-term impact in patients with surgical shunting.
Methods
Thirty-nine consecutive patients operated on between 1978 and 2000 were analyzed using time-dependent multivariate Cox model.
Results
Median follow-up was 110 months. Prosthetic shunts and high preshunt portal venous pressure were predictors of subsequent shunt dysfunction. Among 19 patients with persistently patent shunt, as compared to 20 patients with shunt dysfunction, 1 versus 18 developed refractory ascites; 1 versus 7 had variceal bleeding; 7 versus 2 had encephalopathy; 3 versus 11 (55%) died or underwent liver transplantation; and 0 versus 10 died from end-stage liver disease. Shunt dysfunction was associated with a shorter survival (p=0.001). Out of 20 patients with shunt dysfunction, seven had successful revision of the shunt. None of these seven patients had refractory ascites after revision or died from end-stage liver disease.
Conclusions
In patients with Budd–Chiari syndrome treated with portosystemic shunting, shunt dysfunction has a major impact on morbidity and mortality.
Liver Failure, Growth and Cancer
Pravastatin reduces lung metastasis of rat hepatocellular carcinoma via a coordinated decrease of MMP expression and activity, 27 July 2006
Taras D, Blanc JF, Rullier A, Dugot-Senant N, Laurendeau I, Vidaud M, Rosenbaum J
Background/Aims
Statins have beneficial effects in early pre-clinical models of hepatocellular carcinoma (HCC). Our aim was to test the efficacy of pravastatin on the progression of established HCC in rat, and to study its mechanisms.
Methods
HCC was induced with diethylnitrosamine and N-nitrosomorpholine. After 14 weeks, all rats developed HCC and then received pravastatin or its solvent for 10 weeks (10 rats/group).
Results
Liver tumor mass was lower in pravastatin group (PG) than control group (CG), as estimated from the number of liver tumors (p<0.004) and the liver weight/body weight ratio (p<0.04). Every CG rat surviving at 24 weeks (4/4) had lung metastasis, against only 5/8 in PG. Moreover, the percentage of lung surface occupied by metastasis was 10-fold smaller in PG than CG (p<0.016). Pravastatin decreased liver matrix metalloproteinase (MMP)-9 activity and mostly suppressed MMP-2 activation (p<0.004), likely because it decreased expression of MMP-14 and tissue inhibitor of matrix metalloproteinases-2 (p<0.01), required for MMP-2 activation.
Conclusions
Pravastatin reduces progression and limits metastatic diffusion of established HCC. This could be linked to the decreased MMP activity. These results, obtained in a very aggressive HCC model, further suggest the potential benefit of statins in human HCC.
Role of the Fas/FasL pathway in combination therapy with interferon-? and fluorouracil against hepatocellular carcinoma in vitro, 25 September 2006
Nakamura M, Nagano H, Sakon M, Yamamoto T, Ota H, Wada H, Damdinsuren B, Noda T, Marubashi S, Miyamoto A, Takeda Y, Umeshita K, Nakamori S, Dono K, Monden M
Background/Aims
Several studies have reported the efficacy of combination therapy of interferon (IFN) ? and 5-fluorouracil (5-FU) for hepatocellular carcinoma (HCC). However, the mechanism underlying the clinical anti-tumor effects of this treatment is not well understood. The aim of this study was to determine the role of Fas/FasL signaling in the anti-tumor effect of this combination therapy.
Methods and Results
We used six human hepatoma cell lines, three of which are known Fas-expressing cells. Growth of Fas-positive hepatoma cell lines was inhibited by an agonistic anti-Fas antibody in a dose-dependent manner, and these effects were enhanced by IFN? or 5-FU alone, but even more so by combination therapy using both agents. Annexin-V assay implicated apoptosis as the main mechanism underlying these growth inhibitory effects, although changes in Fas expression regulated by IFN? and/or 5-FU did not correlate with increased apoptosis. Caspase-3 activation was exclusively increased by IFN?/5-FU combination treatment, which was compatible with enhancement of the synergistic apoptotic effect, and other caspases and apoptotic factors (FLIP, BCL-xl, and Bax) were also regulated by IFN?/5-FU. 51Cr-release assay revealed that pretreatment with IFN activated cytotoxicity of peripheral blood mononuclear cells (PBMCs) against HCC cells. The largest interaction was observed when both PBMC and HCC cells were pretreated with the combination of IFN?/5-FU. These cytotoxicities were markedly inhibited by a neutralizing anti-Fas antibody.
Conclusions
Our results indicated that IFN?/5-FU combination treatment enhances the induction of apoptosis and the cytotoxic effect of PBMCs via the Fas/FasL pathway. The Fas/FasL pathway seems, at least in part, to contribute to the anti-tumor effects of IFN?/5-FU against HCCs.
Prospero-related homeobox 1 (PROX1) is frequently inactivated by genomic deletions and epigenetic silencing in carcinomas of the bilary system, 27 September 2006
Laerm A, Helmbold P, Goldberg M, Dammann R, Holzhausen HJ, Ballhausen WG
Background/Aims
Functional deletion of the transcription factor Prospero-related homeobox 1 (PROX1) causes abnormal cellular proliferation via down-regulated expression of the cell cycle inhibitors p27kip1 and p57kip2. Hence, we examined whether inactivation of the PROX1 gene can be demonstrated in malignant tumors of the bilary system.
Methods
Seventeen paraffin-embedded specimens of carcinomas of the bilary system were subjected to loss-of-heterozygosity (LOH) and microsatellite instability analyses, methylation-specific polymerase-chain reaction (MSP) and immunohistochemical detection of PROX1 protein in tumor sections.
Results
The marker D1S213 located close to PROX1 at 1q41 indicated LOH events in 50% of informative tumor samples analyzed. In contrast to intense cytoplasmic and nuclear staining of normal bile duct epithelia, PROX1 protein was absent or drastically reduced in 10 of 16 (63%) carcinomas. MSP revealed significant PROX1 promoter hypermethylation in 8 out of 17 clinical cases (47%). A correlation between clinicopathological characteristics and reduced PROX1 expression was not observed.
Conclusions
We demonstrate that mechanisms like genomic deletions and hypermethylation, which are prototypic for the inactivation of tumor suppressor genes, inactivate PROX1 in carcinomas of the bilary system. Our findings prompt the elucidation of molecular pathways involved in PROX1 dependent misregulation of differentiation and proliferation processes in bilary tract carcinomas.
Complete eradication of hepatocellular carcinomas by combined vasostatin gene therapy and B7H3-mediated immunotherapy, 25 September 2006
Ma L, Luo L, Qiao H, Dong X, Pan S, Jiang H, Krissansen GW, Sun X
Background/Aims
B7H3 immunogene therapy is able to completely eradicate tumors when combined with an anti-vascular agent. The aim of this study was to determine whether vasostatin, a potent anti-angiogenic agent, could synergize with B7H3-mediated immunotherapy to combat hepatocellular carcinoma (HCC).
Methods
Vasostatin and B7H3 expression plasmids were constructed, and the in vitro and in vivo expression and anti-angiogenic activity of recombinant vasostatin were measured. The anti-tumor activities of B7H3 and vasostatin alone and in combination were assessed using single and multiple H22 tumor models.
Results
Gene transfer of vasostatin inhibited the proliferation of aortic endothelial cells, and angiogenesis in the chorioallantoic membrane assay. Subcutaneous H22 tumors established in BALB/c mice were completely eradicated in response to intratumoral injection of B7H3-expressing plasmids followed 24h later by vasostatin-expressing plasmids. In contrast, neither vasostatin nor B7H3 monotherapy was effective. Gene transfer of vasostatin inhibited tumor angiogenesis and enhanced infiltration of NK cells, whereas B7H3 therapy activated CD8+ and NK cells and increased their infiltration into tumors, and enhanced the levels of circulating IFN-?. B7H3 and vasostatin combination gene therapy was effective in combating a systemic challenge of parental H22 cells, and caused the complete regression of multiple distant tumor nodules.
Conclusions
Combining vasostatin anti-angiogenic therapy with B7H3-mediated immunotherapy warrants investigation as a therapeutic strategy to combat HCC, and other malignancies.
Antibody detection using tumor-associated antigen mini-array in immunodiagnosing human hepatocellular carcinoma, 25 September 2006
Zhang JY, Megliorino R, Peng XX, Tan EM, Chen Y, Chan EKL
Background/Aims
Previous studies have demonstrated that during transition from chronic liver disease to hepatocellular carcinoma (HCC), autoantibodies can appear which are not detected in the prior pre-malignant conditions. These antibody responses may be stimulated by cellular proteins involved in carcinogenesis. This study determines the prevalence of antibodies to a selected panel of eight tumor-associated antigens (TAAs) in sera from patients with chronic hepatitis, liver cirrhosis and HCC, and considers the possibility and usefulness of antibodies to such a panel of TAAs in differentiating between these conditions. The panel of eight TAAs includes Imp1, p62, Koc, p53, c-myc, cyclin B1, survivin and p16 full-length recombinant proteins.
Methods
Enzyme-linked immunosorbent assay (ELISA) was used to detect antibodies against eight selected TAAs in 30 sera from chronic hepatitis, 30 from liver cirrhosis, and 142 from HCC. Positive results were also confirmed by slot blot, Western blotting and immunoprecipitation assay.
Results
Antibody frequency to any individual TAA in HCC varied from 9.9% to 21.8%. With the successive addition of TAAs to a final total of eight antigens, there was a stepwise increase of positive antibody reactions reaching a frequency of 59.8% with whole cohort of HCC patients. This was significantly higher than the frequency of antibodies in chronic hepatitis (20%), liver cirrhosis (30%) and normal individuals (12.2%).
Conclusions
This study demonstrates that malignant transition to HCC is associated with increased autoantibody responses to certain cellular proteins which might have a role in tumorigenesis, and shows that a mini-array of eight TAAs enhanced antibody detection for diagnosis of HCC. More studies in patients with HCC and precursor conditions such as chronic hepatitis, alcoholic hepatitis and liver cirrhosis using enlarged TAA mini-array panels might further improve the sensitivity and specificity of this mode of cancer immunodiagnosis. Its additional usefulness might be in the early detection of cancer in some patients with predisposing conditions.
Rosiglitazone attenuates suppression of RXR?-dependent gene expression in inflamed liver, 23 October 2006
Ghose R, Mulder J, von Furstenberg RJ, Thevananther S, Kuipers F, Karpen SJ
Background/Aims
A recently determined target of lipopolysaccharide (LPS) and cytokine signaling in liver is the central Type II nuclear receptor (NR) heterodimer partner, retinoid X receptor ? (RXR?). We sought to determine if Rosiglitazone (Rosi), a peroxisome proliferator activated receptor ? (PPAR?) agonist with anti-inflammatory properties, can attenuate LPS and cytokine-induced molecular suppression of RXR?-regulated genes.
Methods
In vivo, mice were gavage-fed Rosi for 3 days, prior to intraperitoneal injection of LPS, followed by harvest of liver and serum. In vitro, HepG2 cells were treated with IL-1?, ± short-term Rosi pretreatment. RNA was analyzed by quantitative RT-PCR, while nuclear and cytoplasmic proteins were analyzed by immunoblotting and gel shifts.
Results
Rosi attenuated LPS-mediated suppression of RNA levels of several Type II NR-regulated genes, including bile acid transporters and the major drug metabolizing enzyme, Cyp3a11, without affecting cytokine expression, suggesting a novel, direct anti-inflammatory effect in hepatocytes. Rosi suppressed the inflammation-induced nuclear export of RXR?, in both LPS-injected mice and IL-1?-treated HepG2 cells, leading to maintenance of nuclear RXR? levels and heterodimer binding activity.
Conclusions
Rosi directly attenuates the suppressive effects of inflammation-induced cell signaling on nuclear RXR? levels in liver.
Molecular and Cell Biology
Leptin represses matrix metalloproteinase-1 gene expression in LX2 human hepatic stellate cells, 21 September 2006
Cao Q, Mak KM, Lieber CS
Background/Aims
Collagen accumulation in liver fibrosis is due in part to decreased expression of matrix metalloproteinase (MMP)-1 relative to TIMP-1. LX-2 hepatic stellate cells produce increased amounts of collagen and tissue inhibitor of metalloproteinase (TIMP)-1 in response to leptin. The effect of leptin on MMP-1 production has not been reported.
Methods
LX-2 cells were treated with leptin with or without inhibitors. We determined: phosphorylation of Janus kinase (JAK) 1 and -2, signal transducer and activator of transcription (STAT)3 and -5, extracellular signal-regulated kinase (ERK)1/2 and p38 by Western blot; H2O2 concentration by a colorimetric method; MMP-1 mRNA levels and stability by Northern blot; MMP-1 promoter activity as well as pro-MMP-1 by ELISA; and active MMP-1 by fluorescence.
Results
LX-2 cells constitutively expressed the MMP-1 gene and leptin repressed the basal level of MMP-1 mRNA and its promoter activity. The repression was mediated by JAK/STAT pathway in synergism with JAK-mediated H2O2-dependent ERK1/2 and p38 pathways. ERK1/2 inhibited MMP-1 promoter activity, whereas p38 decreased the message stability, contributing to mRNA down-regulation. Inhibition of MMP-1 gene diminished secreted pro-MMP-1 and active MMP-1.
Conclusions
Leptin represses MMP-1 gene expression via the synergistic actions of the JAK/STAT pathway and JAK-mediated H2O2-dependent ERK1/2 and p38 pathways.
Attenuated liver progenitor (oval) cell and fibrogenic responses to the choline deficient, ethionine supplemented diet in the BALB/c inbred strain of mice, 9 October 2006
Knight B, Akhurst B, Matthews VB, Ruddell RG, Ramm GA, Abraham LJ, Olynyk JK, Yeoh GC
Background/Aims
Liver regeneration following chronic injury is associated with inflammation, the proliferation of liver progenitor (oval) cells and fibrosis. Previous studies identified interferon-gamma as a key mediator of oval cell proliferation. Interferon-? is known to regulate Th1 cell activities during immune challenge. Therefore, we hypothesised that progenitor cell-mediated regeneration is associated with a Th1 immune response.
Methods
C57Bl/6 (normal Th1 response) and BALB/c mice (deficient in Th1 signalling) were placed on a carcinogenic diet to induce liver injury, progenitor cell proliferation and fibrosis.
Results
Serum transaminases and mortality were elevated in BALB/c mice fed the diet. Proliferation of liver progenitor cells was significantly attenuated in BALB/c animals. The pattern of cytokine expression and inflammation differed between strains. Liver fibrosis and hepatic stellate cell activation were significantly inhibited in BALB/c mice compared to C57Bl/6. In addition, interferon-? knockout mice also showed reduced fibrosis compared to wild type. These findings are in contrast to published results, in which interferon-gamma is shown to be anti-fibrogenic.
Conclusions
Our data demonstrate that the hepatic progenitor cell response to a CDE diet is inhibited in mice lacking Th1 immune signalling and further show that this inhibition is associated with reduced liver fibrosis.
Fibrogenic cell fate during fibrotic tissue remodelling observed in rat and human cultured liver slices, 4 October 2006
Guyot C, Combe C, Balabaud C, Bioulac-Sage P, Desmoulière A
Background/Aims
Fibrotic liver remodelling was studied in culture of precision-cut liver slices (PCLS) derived from fibrotic liver.
Methods
Fibrosis was induced in rats by carbon tetrachloride (CCl4) treatment or bile duct ligation. Human fibrotic livers were also used. PCLS were cultured for 6, 24, 48, or 72h, and the expression of ?-smooth muscle (SM) actin, platelet-derived growth factor (PDGF) receptor-?, and active caspase 3 was studied by immunohistochemistry.
Results
Before culture, in CCl4-treated or bile duct ligated animals, fibrosis was observed around centrolobular veins, or in portal zones, respectively. In PCLS derived from CCl4-treated animals, ?-SM actin expression disappeared after 24h in culture while PDGF receptor-? expression decreased progressively after 48h. These changes were observed in absence of massive apoptosis. In PCLS derived from bile duct ligated animals, both ?-SM actin and PDGF receptor-? expression decreased after 48h in culture with a massive apoptosis. In PCLS derived from human fibrotic livers, ?-SM actin expression was dramatically reduced after 48h in culture.
Conclusions
After CCl4 treatment, a proportion of myofibroblasts derived from hepatic stellate cells seems to dedifferentiate while in bile duct ligation model, myofibroblasts derived from portal fibroblasts disappear by apoptosis, underlining the relevance of this model to evaluate the mechanisms involved in fibrotic liver remodelling.
Genetic and Metabolic Liver Disease
Hyperhomocysteinemia due to cystathionine beta synthase deficiency induces dysregulation of genes involved in hepatic lipid homeostasis in mice, 21 September 2006
Hamelet J, Demuth K, Paul JL, Delabar JM, Janel N
Background/Aims
Cystathionine beta synthase (CBS) deficiency leads to severe hyperhomocysteinemia, which confers diverse clinical manifestations, notably fatty liver. Recently, abnormal lipid metabolism has been demonstrated in CBS-deficient mice, a murine model of severe hyperhomocysteinemia. To gain further insights into effects of CBS deficiency on hepatic cholesterol metabolism, the expression of hepatic genes involved in biosynthesis, uptake and efflux was determined in CBS-deficient mice.
Methods
Gene expression analysis was performed on liver of CBS-deficient mice using quantitative real-time PCR.
Results
We found that CBS-deficiency in liver mice significantly increases expression of genes induced by endoplasmic reticulum stress and genes that regulate the expression of enzymes required for cholesterol and fatty acid biosynthesis and uptake, notably the scavenger receptor class B type I (SR-BI), concomitant with overexpression of SR-BI at the protein level. Moreover, we also found increased mRNA levels of ABCG5, ABCG8, ABCG1 and ABCA1, which play important roles in reverse cholesterol transport, associated with an upregulation of liver X receptors and a downregulation of the peroxisome proliferators-activated receptor ?.
Conclusions
We found that several ATP-binding cassette transporters and nuclear hormone receptors involved in liver lipid homeostasis are differentially expressed in liver of CBS-deficient mice.
Copyright © 2007 European Association for the Study of the Liver. All rights reserved.
BRITISH MEDICAL JOURNAL
Copyright © 2007 BMJ Publishing Group Ltd
NEW ENGLAND JOURNAL
The New England Journal of Medicine is owned, published, and copyrighted © 2007 Massachusetts Medical Society. All rights reserved.
LANCET
The Lancet, published, and copyrighted © 2007. All rights reserved.
