bluuebunny/crossref_metadata_2025 huggingface dataset:
Propranolol plus isosorbide-5-mononitrate for portal hypertension in cirrhosis: Long-term hemodynamic and renal effects
Authors: Morillas Rosa Maria , Planas Ramon , Cabré Eduard , Galán Amparo , Quer Juan Carlos , Feu Faust , Pagán Joan Carles García , Bosch Jaume , Gassull Miquel Angel
Published: March 2007

The effect on kidney function, vasoactive systems and ascites outcome of long-term treatment with propranolol plus isosorbide-5-mononitrate, a combined therapy proven more effective than propranolol alone in decreasing portal pressure in the cirrhotic patient, is unknown. Thirty cirrhotic patients who survived acute variceal bleeding and were treated with propranolol plus isosorbide-5-mononitrate were studied. Portal and systemic hemodynamics (n = 15), inulin clearance, free water clearance, plasma renin activity, aldosterone concentration and prostaglandin E2 excretion (n = 20) were measured before and after 3 mo of treatment. In addition, data on ascites outcome in the entire series after a mean follow-up of 9.6 mo were compared with those of 30 patients undergoing elective sclerotherapy and with those of 30 patients treated with propranolol alone matched for age, sex, presence of ascites, Child-Pugh class and mean follow-up length included in other randomized controlled trials. Combined therapy significantly decreased the hepatic venous pressure gradient and azygos blood flow. In addition, no changes in inulin clearance, free water clearance, plasma renin activity, aldosterone concentration and prostaglandin E2 excretion occurred, despite a mild decrease in mean arterial pressure. Moreover, no differences among the three groups of patients studied in ascites outcome were found. These results suggest that long-term treatment with propranolol plus isosorbide-5-mononitrate does not impair kidney function, vasoactive systems or ascites outcome in cirrhotic patients. (Hepatology 1994;20:1502-1508).
Effects of obesity on bile formation and biliary lipid secretion in the genetically obese JCR:LA-corpulent rat
Authors: St. George Catherine M. , Russell James C. , Shaffer Eldon A.
Published: March 2007

Obesity, a known risk factor in cholesterol gallstone disease, is a favorable factor in the formation of bile supersaturated with cholesterol. Previous studies have been indirect and limited to human beings. To better define the hepatic secretory defect, we directly measured bile secretion and bile salt kinetics in a genetically obese rat. Common bile duct cannulations and 24-hr bile collections were performed on 11 obese (homozygous for the cp gene) and 11 lean (heterozygous cp) female rats of the JCR:LA-cp strain. In absolute terms, obese rats had normal bile salt secretion rates, 28% larger bile salt pools and 45% greater bile salt synthesis than lean controls. Obese rats had twice the total liver DNA content of lean controls, indicating twice the liver cell number. In terms relative to liver cell number, bile salt synthesis per milligram DNA was no different in the obese rats and controls. Biliary secretion under basal conditions, when expressed per total DNA content, was significantly decreased in the obese rats. Bile salt and phospholipid secretion per milligram DNA were decreased 54% and 49%, respectively, whereas a lesser 31% reduction in cholesterol secretion resulted in an increased cholesterol saturation index. The bile salt secretion rate and bile flow per milligram DNA were both decreased by half in obese rats, a cholestatic state likely reflecting the accumulation of intracellular microvesicular fat and mitochondrial damage. The secretory defect in this model of obesity is therefore less bile salt and phospholipid in relation to the cholesterol secreted per liver cell. (Hepatology 1994;20:1541-1547).
Potential role of bile duct collaterals in the recovery of the biliary obstruction: Experimental study in rats using microcholangiography, histology, serology and magnetic resonance imaging
Authors: Ni Yicheng , Lukito Gregorius , Marchal Guy , Cresens Erwin , Yu Jie , Petré Carine , Baert Albert L. , Fevery Johan
Published: March 2007

Obstructive cholestasis induced in animals at the level of the lobar and common bile ducts is known to be reversible with time. This study was conducted not only to test the hypothesis that formation of bile duct collaterals is responsible for the recovery of biliary obstruction but also to assess the potential of hepatobiliary agent-enhanced magnetic resonance imaging for visualizing cholestasis. A total of 52 rats were divided into three groups with selective biliary obstruction, total biliary obstruction and sham surgery. We studied the evolution of cholestasis by correlating microcholangiographic, histological findings with the results of liver tests and hepatobiliary agent-enhanced magnetic resonance imaging. Lobar cholestasis undetected by liver tests but seen on magnetic resonance imaging as a difference between ligated and unligated lobes, occurred in 15 out of 20 rats subjected to selective biliary obstruction within 48 hr after ligation, and recovered later on as a result of the development of bile duct collaterals. Five rats failed to show local cholestasis as a result of the existence of interlobar accessory bile channels. All 18 total biliary obstruction-treated rats were cholestatic soon after ligation, as confirmed by high serum bilirubin and alkaline phosphatase levels and as documented by poor liver enhancement on magnetic resonance imaging. Cholestasis recovered within 4 wk with normalization of liver enhancement on magnetic resonance imaging as a result of the formation of bile duct collaterals (as demonstrated by microcholangiographic and histological study). Bile duct collateral formation is responsible for the recovery from obstructive cholestasis in rats. A similar mechanism might be present in conditions of bile duct obstruction without cholestasis. Hepatobiliary agent-enhanced magnetic resonance imaging is more sensitive than blood tests in detecting local cholestasis and can be used to monitor noninvasively the evolution of biliary obstruction. (Hepatology 1994;20:1557-1566).
Fructose metabolism and cell survival in freshly isolated rat hepatocytes incubated under hypoxic conditions: Proposals for potential clinical use
Authors: Lefebvre Véronique , Goffin Isabelle , Buc-Calderon Pedro
Published: March 2007

The protective effect of fructose with regard to hypoxia-induced cell injury was investigated. The addition of fructose (2 to 20 mmol/L) protected hepatocytes against hypoxia-mediated cell lysis in a concentration-dependent way. The intracellular ATP content was initially decreased as a result of fructose-1-phosphate formation, but it remained constant during the hypoxic incubation. Conversely, high initial ATP values observed at low fructose concentrations progressively declined. Cellular protection was observed only when fructose was added before (and not after) the start of hypoxia. In addition, a sufficient amount of fructose-1-phosphate rapidly accumulated before the induction of hypoxia, and the linear production of lactate, during hypoxic incubation, indicated that cells synthesized ATP continuously. The lack of cell protection by fructose added after the onset of the hypoxia may be explained by a lesser fructose-1-phosphate formation and a subsequently low accumulation leading to insufficient glycolytic ATP production. Under aerobic conditions, both glycolysis (lactate formation) and gluconeogenesis (glucose formation) were carried out in fructose-1-phosphate-loaded cells with the same initial rates, whereas under hypoxic conditions glycolysis was the main metabolic event. The fact that protein synthesis activity recovered faster during reoxygenation of previously hypoxic fructose-treated cells than in glucose-treated cells led us to hypothesize that in situ perfusion of liver with fructose, before its removal, would improve its metabolic capacity during the hypoxic cold preservation and subsequent transplantation. (Hepatology 1994;20:1567-1576).
Ursodeoxycholate protects oxidative mitochondrial metabolism from bile acid toxicity: Dose-response study in isolated rat liver mitochondria
Authors: Krähenbühl Stephan , Fischer Sven , Talos Christine , Reichen Jürg
Published: March 2007

The effect of ursodeoxycholate and tauroursodeoxycholate on the toxicity of lipophilic bile acids (chenodeoxycholate and lithocholate) on the function of the electron transport chain was investigated in isolated rat liver mitochondria. At a concentration of 30 μmol/L, both chenodeoxycholate and lithocholate reduced state 3 oxidation rates and respiratory control ratios of L-glutamate, succinate and duroquinol. In contrast, ADP/O ratios of these substrates and oxidative metabolism of ascorbate were not significantly affected. Ursodeoxycholate did not impair mitochondrial oxidative metabolism up to concentrations of 100 μmol/L; at 300 μmol/L, however, it decreased state 3 oxidation rates and respiratory control ratios of L-glutamate, succinate and duroquinol. Tauroursodeoxycholate had no significant inhibitory effect on state 3 oxidation rates of L-glutamate and succinate at concentrations up to 300 μmol/L. When ursodeoxycholate (final concentration, 30 μmol/L or 100 μmol/L) was added to mitochondrial incubations containing chenodeoxycholate or lithocholate, the toxic effects of lipophilic bile acids on mitochondrial oxidative metabolism were partially reversed. However, 300 μmol/L ursodeoxycholate, in combination with chenodeoxycholate or lithocholate, exhibited greater toxicity compared with incubations containing only the individual bile acids. In contrast to ursodeoxycholate, tauroursodeoxycholate did not reduce the toxic effects of chenodeoxycholate or lithocholate on mitochondrial metabolism. Ursodeoxycholate (100 μmol/L) significantly decreased the incorporation of chenodeoxycholate into mitochondrial membranes, whereas the decrease in lithocholate incorporation was not statistically significant. These studies demonstrate that ursodeoxycholate, but not tauroursodeoxycholate, decreases the toxicity of lipophilic bile acids on the function of the electron transport chain up to a concentration of 100 μmol/L but increases bile acid-induced mitochondrial toxicity at higher concentrations. The protective effect of ursodeoxycholate may partially be explained by decreased incorporation of bile acids into mitochondrial membranes. (Hepatology 1994;20:1595-1601).
Nonenzymatic glycosylation of poly-l-lysine: A new tool for targeted gene delivery
Authors: Martinez-Fong Daniel , Mullersman Jerald E. , Purchio Anthony F. , Armendariz-Borunda Juan , Martinez-Hernandez Antonio
Published: March 2007

The basic approach in targeted gene delivery relies on the formation of a complex between a vector and a molecule that will be selectively internalized by the target cells. In the case of hepatocytes, asialoglycoproteins are convenient targeting molecules because of the high affinity and avidity of the hepatocyte galactose receptor. In this system, poly-l-lysine is crosslinked to an asialoglycoprotein, and the resulting conjugate is complexed with the expression vector (DNA). The electrostatic binding between DNA and poly-l-lysine—asialoglycoprotein ensures delivery of the intravenously injected complex to the liver, where it is subjected to endocytosis by hepatocytes. However, the poly-l-lysine—asialoglycoprotein complexes tend to be unstable, of limited solubility and of fixed carbohydrate content. For these reasons we searched for a simpler alternative. We exploited the known capacity of reducing sugars to be reductively coupled to the -amino groups in proteins and used lactose to obtain poly-l-lysine with „exposed” galactose. Glycosylation with sodium cyanoborohydride at high pH in borate buffer is a simple, reproducible procedure. The „lactosylated” poly-l-lysine has proved very stable, highly soluble and easily bound to plasmids. In a set of experiments we compared the asialofetuin—poly-l-lysine vector complexes with lactosylated poly-l-lysine vector complexes by transfecting hepatoma cells (HepG2) in culture. For these experiments we used a pRc/cytomegalovirus eukaryotic expression vector containing a mutant TGF-β1 complementary DNA. On Northern-blot analysis, cells transfected with lactosylated poly-l-lysine expressed 10 to 20 times more TGF-β1 messenger RNA than did cells transfected with the same plasmid coupled to asialofetuin—poly-l-lysine. Therefore glycosylated poly-l-lysine is a simple, highly effective alternative to poly-l-lysine—asialoprotein complexes. Furthermore the use of different disaccharides may permit targeting of the complexes to different cell types. (Hepatology 1994;20:1602-1608).
Increased nitric oxide—dependent vasorelaxation in aortic rings of cirrhotic rats with ascites
Authors: Clària Joan , Jiménez Wladimiro , Ros Josefa , Rigol Montserrat , Angeli Paolo , Arroyo Vicente , Rivera Francisca , Rodés Joan
Published: March 2007

To assess whether aortic vessels of rats with cirrhosis and ascites possess an enhanced vascular response to endothelium-derived, nitric oxide—dependent vasodilators, we performed relaxation studies in isolated aortic rings of 21 control rats and 24 rats with carbon tetrachloride—induced cirrhosis and ascites. We carried out studies after contracting the vessels with norepinephrine. We measured endothelium-dependent vasodilator response by administering increasing concentrations of acetylcholine (10−6 to 10−2 mol/L) and ADP (10−7 to 10−4 mol/L). We evaluated endothelium-independent response by giving increased concentration of sodium nitrite (10−5 to 10−2 mol/L). The maximal absolute tension developed in response to norepinephrine was significantly decreased in cirrhotic rings (816 ± 72 mg, p < 0.025) compared with control (1,425 ± 75 mg) rings. Dose-response curves for endothelium-dependent vasodilators were shifted to the left in aortic rings of cirrhotic rats, and EC50 for acetylcholine and ADP were significantly decreased in cirrhotic (0.8 ± 0.15 mmol/L and 0.42 ± 0.16 μmol/L, p < 0.025 and p < 0.01, respectively) than in control rings (1.91 ± 0.33 mmol/L and 3.09 ± 0.82 μmol/L). In both acetylcholine- and ADP-stimulated vessels, differences between cirrhotic and control rings disappeared after nitric oxide synthesis inhibition with Nω-nitro-l-arginine (10−4 mol/L). No difference in the relaxing effect of sodium nitrite was observed between cirrhotic and control rings. These results therefore demonstrate for the first time enhanced in vitro vascular responsiveness to nitric oxide—dependent vasodilators in rats with cirrhosis and ascites, giving further support to the concept that nitric oxide activity is increased in cirrhosis. (Hepatology 1994;20:1615-1621).
Identifying the hepatic organic anion transporter: One of many?
Authors: Weinman Steven A.
Published: March 2007

Using expression cloning in Xenopus laevis oocytes, we have isolated a cDNA encoding a rat liver organic anion-transporting polypeptide (oatp). The cloned oatp mediated Na+-independent uptake of sulfobromophthalein (BSP) which was Cl−-dependent in the presence of bovine serum albumin (BSA) at low BSP concentrations (e.g., 2 μM). Addition of increasing amounts of BSA had no effects on the maximal velocity of initial BSP uptake, but it increased the Km value from 1.5 μM (no BSA) to 24 μM (BSA/BSP molar ratio, 3.7) and 35 μM (BSA/BSP ratio, 18.4). In addition to BSP, the cloned oatp also mediated Na+-independent uptake of conjugated (taurocholate) and unconjugated (cholate) bile acids. Sequence analysis of the cDNA revealed an open reading frame of 2010 nucleotides coding for a protein of 670 amino acids (calculated molecular mass, 74 kDa) with four possible N-linked glycosylation sites and 10 putative transmembrane domains. Translation experiments in vitro indicated that the transporter was indeed glycosylated and that its polypeptide backbone had an apparent molecular mass of 59 kDa. Northern blot analysis with the cloned probe revealed crossreactivity with several mRNA species from rat liver, kidney, brain, lung, skeletal muscle, and proximal colon as well as from liver tissues of mouse and rabbit, but not of skate (Raja erinacea) and human.
Gene array analysis and the liver
Authors: Shackel Nicholas A. , Gorrell Mark D. , McCaughan Geoffrey W.
Published: March 2007

Functional genomics methods promise a previously unparalleled high-throughput examination of intrahepatic gene expression. Profiling transcriptomes as well as examining the coordinate expression of many genes in diverse pathobiologic pathways is now pssible with techniques such as gene array analysis. However, the nature of the hepatic transcriptome, limitations of the functional genomics methokologies used, and analysis of the data generated are often poorly understood. Further, the use of replicates during analysis and the downstream confirmation of results using a supplemental methokology are important but frequently neglected aspects of experimenta design. This review presents an overview of functional genomics methodoogies in the organ-specific context of studying intrahepatic gene expression. The focus of the review is the most widely used functional genomic methodology, gene array analysis. The nature of the hepatic transcriptome is discussed, together with a summary of gene array analysis methodologies. Analysis of data generated by gene array analysis, determination of intrahepatic cellular origin of transcript expression, and studies of intrahepatic gene expression using gene array analysis are also reviewed. Finally, the importance of a coordinate experimental approach examining both liver protein and mRNA expression is presented. (Hepatology 2002;36: 1313-1325).
Cerebral microdialysis in patients with fulminant hepatic failure
Authors: Tofteng Flemming , Jorgensen Linda , Hansen Bent Adel , Ott Peter , Kondrup Jens , Larsen Fin Stolze
Published: March 2007

Fulminant hepatic failure (FHF) is often complicated by high intracranial pressure (ICP) and fatal brain damage. In this study, we determined if a rise in [glutamate] ec and [lactate] ec preceded surges of high ICP in patients with FHF (median age, 42; range, 20-55 years; 7 women; 3 men) by inserting a microdialysis catheter into the brain-cortex together with an ICP catheter. The microdialysis catheter was perfused with artificial cerebrospinal-fluid at a rate of 0.3 μL/min. Dialysate was collected approximately every 30 minutes or when ICP increased. A total of 352 microdialysis samples were collected during a median of 3 days and allowed for ˜ 1,760 bedside analyses of the collected dialysate. In 5 patients that later developed surges of high ICP, the initial values of [glutamate]ec and [lactate]ec were 2 to 5 times higher compared with patients with normal ICP. [Glutamate] ec then tended to vanish with time in both groups of patients. An increase in [glutamate] ec did not precede high ICP in any of the cases. In contrast, [lactate] ec was high throughout the study in the high ICP group and increased further before surges of high ICP. We conclude that in patients with FHF, cerebral [glutamate] ec and [lactate] ec are elevated. However, the elevated [glutamate] ec is not correlated to high ICP. In contrast, elevations in [lactate] ec preceded surges of high ICP. In conclusion, accelerated glycolysis with lactate accumulation is implicated in vasodilatation and high ICP in patients with FHF. The data suggest that bedside cerebral microdialysis is a valuable tool in monitoring patients with FHF and severe hyperammonemia. (Hepatology2002;36:1333-1340).
NAFLD may be a common underlying liver disease in patients with hepatocellular Carcinoma in the United States
Authors: Marrero Jorge A. , Fontana Robert J. , Su Grace L. , Conjeevaram Hari S. , Emick Dawn M. , Lok Anna S.
Published: March 2007

The incidence of hepatocellular carcinoma (HCC) in the United States is increasing, but the clinical characteristics of American patients with HCC have not been well described. The aims of this study were to determine the etiology of liver disease and short-term outcome among HCC patients presenting to a single center in the United States. One hundred five consecutive patients with HCC were studied; mean age was 59 years, 67% were men, and 76% were non-Hispanic white. The most common etiology of liver disease was hepatitis C (51%) and cryptogenic cirrhosis (29%). Half of the patients with cryptogenic cirrhosis had histologic or clinical features associated with nonalcoholic fatty liver disease (NAFLD). Fifty-three (50%) patients had HCC detected during surveillance (group I), whereas the remaining patients had symptomatic tumors (group II). Group I patients had smaller tumors (P = 0.01), were more likely to be eligible for surgical treatment (P = 0.005), and had a better medican survival compared with patients in group II (P = 0.001). Patients with cryptogenic cirrhosis were less likely to have undergone HCC surveillance and had larger tumors at diagnosis. In conclusion, hepatitis C and cryptogenic liver disease are the most common etiologies of diseases in our patients with HCC. NAFLD accounted for at least 13% of the cases. Patients who underwent surveillance had smaller tumors and were more likely to be candidates for surgical or local ablative therapies. Because of the increasing incidence of NAFLD, further studies are needed to determine the risk of HCC in patients with NAFLD. (Hepatology2002;36:1349-1354).
Valine-alanine manganese superoxide dismutase polymorphism is not associated with alcohol-induced oxidative stress or liver fibrosis
Authors: Stewart Stephen F. , Leathart Julian B. , Chen Yuanneng , Daly Ann K. , Rolla Roberta , Vay Daria , Mottaran Elisa , Vidali Matteo , Albano Emanuele , Day Chris P.
Published: March 2007

The role of genetic factors in the pathogenesis of alcohol-induced liver disease (ALD) is receiving increasing attention. Recently, it has been reported that homozygosity for a valine to alanine substitution in the mitochondrial targeting sequence of manganese superoxide dismutase (Mn-SOD) represents a risk factor for severe ALD. Because this mutation is postulated to modify enzyme transport into mitochondria, we have sought confirmatory evidence of this association in a larger group of patients and investigated whether this polymorphism might influence alcohol-induced oxidative stress. Genotyping for the valinealanine (Val-Ala) polymorphism of the Mn-SOD gene in 281 patients with advanced ALD (cirrhosis/fibrosis) and 218 drinkers without liver disease showed no differences in either the heterozygote (55% vs. 50%) or the homozygote (19% vs. 23%) frequency for the alanine allele. By measuring the titers of circulating antibodies against oxidized cardiolipin (OX-CL) and malondialdehyde (MDA) or hydroxy-ethyl radical (HER) adducts as markers of oxidative stress, we found a significant increase in ALD patients compared with healthy controls. However, the carriers of the alanine Mn-SOD allele had titers of anti-MDA, anti-HER, and anti-OX-CL IgG comparable with heterozygotes and patients homozygous for the valine allele. Similarly, the frequency of subjects with antibody titers above the 95th percentile of controls was not increased among homozygotes for the alanine Mn-SOD allele. In conclusion, in our population Val-Ala polymorphism in Mn-SOD influences neither susceptibility to alcohol-induced liver fibrosis nor alcohol-induced oxidative stress. (Hepatology2002;36:1355-1360).
Characterization of recombinant monoclonal IgA anti—PDC-E2 autoantibodies derived from patients with PBC
Authors: Fukushima Nobuyoshi , Nalbandian Greg , Van De Water Judy , White Kandra , Ansari Aftab A. , Leung Patrick , Kenny Thomas , Kamita Shizuo G. , Hammock Bruce D. , Coppel Ross L. , Stevenson Freida , Ishibashi Hiromi , Gershwin Eric M.
Published: March 2007

Primary biliary cirrhosis (PBC) is an autoimmune liver disease characterized by the presence of autoantibodies to mitochondria (AMA). Recent evidence suggests that PBC develops after a locally driven response in the mucosa, where immunoglobulin A (IgA) is the dominant antibody isotype. In this study, we produced recombinant pyruvate dehydrogenase complex (PDC-E2)—specific dimeric human IgA monoclonal antibodies (mAbs) in a baculovirus expression system. By using 2 anti—PDC-E2 IgG mAbs derived from patients with PBC, we constructed 2 recombinant baculoviruses, each containing heavy chains with the Cα constant region. These were simultaneously co-infected into Sf9 insect cells with recombinant baculovirus containing the J chain. A sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) immunoblotting profile of the IgA using a 6% nonreducing gel verified the dimeric nature of the autoantibodies. Both recombinants retained their original specificity for PDC-E2. In addition, the antibody showed a mitochondrial staining pattern in HEp2 cells and apically stained the biliary epithelial cells (BECs) in the liver of a patient with PBC but not a normal patient. Transcytosis experiments performed using human polymeric immunoglobulin receptor (pIgR) expressing Madine-Darby canine kidney (MDCK) cells showed that one of the recombinants showed a high degree of colocalization with PDC-E2. In conclusion, these data provide further support of the hypothesis that PDC-E2-specific IgA may enter biliary epithelial cells of PBC patients via the pIgR and complex with PDC-E2, thereby potentially contributing to the pathology of BECs. Moreover, this recombinant PDC-E2-specific mAb provides a tool for further determination of the role of anti-PDC-E2 IgA in the pathogenesis of PBC. (Hepatology2002;36:1383-1392).
Cellular vacuolization and apoptosis induced by hepatitis B virus large surface protein
Authors: Foo Ngee-Chih , Ahn Byung Y. , Ma Xiaohong , Hyun William , Benedict Yen T. S.
Published: March 2007

Fibrosing cholestatic hepatitis (FCH) is a rapidly progressive form of viral hepatitis B that occurs in severely immunosuppressed patients. Pathologically, the liver in FCH is characterized by widespread hepatocyte vacuolization and apoptosis, which, in contrast to more common forms of hepatitis B, is only rarely associated with significant inflammation. Therefore, it has been proposed that, in FCH, hepatocytes may be injured by a direct cytopathic effect of the virus rather than by the host immune response. In support of this hypothesis, we present evidence that cultured hepatoma cells that had been transfected with a plasmid selectively expressing the viral large surface protein form numerous large vacuoles and undergo apoptosis. The similarity of the cytopathology in FCH in vivo and in these transfected cells in vitro strongly implicates the large surface protein as the direct cause of this acute liver disease. This conclusion is further supported by the published demonstration that hepatocytes tend to accumulate large surface protein in FCH, which may reflect its overexpression by the virus. In conclusion, our data implicate the large surface protein as a major cause of hepatocyte injury in fibrosing cholestatic hepatitis. (Hepatology2002;36:1400-1407).
Quantitative serum HBV DNA levels during different stages of chronic hepatitis B infection
Authors: Chu Chi-Jen , Hussain Munira , Lok Anna S. F.
Published: March 2007

The goals of this retrospective study were to determine whether there is a threshold hepatitis B virus (HBV) DNA value associated with spontaneous or antiviral therapy—related hepatitis B e antigen (HBeAg) clearance. We also investigated whether there is an HBV DNA value that can be used for differentiating inactive carriers from patients with HBeAg-negative chronic hepatitis B. HBV DNA levels in sequential serum samples of 165 Chinese patients with different stages of chronic HBV infection were quantified by a polymerase chain reaction (PCR)—based assay. Our results showed that almost all of the patients (89%) who remained HBeAg-positive had HBV DNA levels that were persistently above 105 copies/mL. Serum HBV DNA levels decreased by a mean of 3 log10 in patients with HBeAg loss, but 51% had levels above 105 copies/mL at the time HBeAg first became undetectable. Mean serum HBV DNA levels were significantly lower in HBeAg-negative patients. HBV DNA value above 105 copies/mL would exclude all inactive carriers, but 45% of patients with HBeAg-negative chronic hepatitis would also be excluded if testing were only performed at presentation and 30% would be excluded if testing were performed on 3 occasions. In conclusion, serum HBV DNA levels decreased significantly in patients with HBeAg loss, but there was no threshold HBV DNA level associated with HBeAg clearance. Given the fluctuating course of HBeAg-negative chronic hepatitis, it is not possible to define a single cutoff HBV DNA value for differentiating inactive carriers from patients with HBeAg-negative chronic hepatitis. (Hepatology2002;36:1408-1415).
Genetic polymorphisms in interferon pathway and response to interferon treatment in hepatitis B patients: A pilot study
Authors: King Jennifer K. , Yeh Shiou-Hwei , Lin Ming-Wei , Liu Chun-Jen , Lai Ming-Yang , Kao Jia-Horng , Chen Ding-Shinn , Chen Pei-Jer
Published: March 2007

Interferon alfa (IEN-α) therapy remains a mainstay of treatment in active hepatitis B. However, sustained remission rates remain relatively low, and the search for factors important for response to therapy continues. Our study aimed to identify the host single nucleotide polymorphisms (SNPs) that predict IFN response in hepatitis B patients. We selected genes in the IFN pathway involved in antiviral and signaling activities and sequenced 22 SNPs for each of our 82 patients. Our results identified 2 SNPs in the antiviral pathway that may influence IFN response. One SNP in the regulatory region of the eIF-2α gene revealed A/G alleles. The rate of A/G heterozygotes is 22% in nonresponders (NR) and 2% in sustained responders (R), with an odds ratio (OR) of 12.82 (95% CI: 1.52-107.85, P = 0.009). After adjustment for age, sex, and HBV DNA level, the OR reaches 14.94 (95% CI: 1.45-153.71, P = 0.023). This marker revealed greater significance than HBV DNA levels (OR 5, 95% CI: 1.01-2.43, P = 0.033) as a marker for IFN response, suggesting its potential advantage over conventional predictors. In addition, borderline significance for the SNP in MxA gene promoter at nt -88 revealed G/T alleles, with the G/T heterozygote rate being 19% in nonresponders and 43% in sustained R (P = 0.061), concurring with a previous study involving hepatitis C patients. In conclusion, this pilot identified SNPs as potential markers that could predict hepatitis B patient response. These observations may help guide future large-scale studies in examining host SNPs for their clinical utility in predicting IFN response. (Hepatology2002;36:1416-1424).
HBV genotype B is associated with better response to interferon therapy in HBeAg( + ) chronic hepatitis than genotype C
Authors: Wai Chun Tao , Chu Chi-Jen , Hussain Munira , Lok Anna S. F.
Published: March 2007

Hepatitis B virus (HBV) genotype and precore/core promoter mutations have been implicated in spontaneous and interferon alfa (IFN-α)—related hepatitis B e antigen (HBeAg) seroconversion. We performed a retrospective analysis of a previously reported randomized controlled trial to determine the effects of HBV genotype and precore/core promoter mutations on IFN-α response in patients with HBeAg-positive chronic hepatitis. Clinical data and stored sera from 109 (95%) patients in the original trial were analyzed. Seventy-three patients received IFN-α and 34 received no treatment (controls). Almost all patients had HBV genotypes B (38%) and C (60%). Antiviral response was achieved in 39% and 17% of IFN-α—treated patients (P = 0.03) and in 10% and 8% of untreated controls (P = 0.88) with HBV genotype B and C, respectively. Multivariate analysis identified HBV genotype B, elevated pretreatment alanine aminotransferase (ALT) levels, and low pretreatment HBV-DNA levels but not IFN-α treatment as independent factors associated with antiviral response. Among the 66 patients with elevated pretreatment ALT level, antiviral response was achieved in 57% and 21% of IFN-α—treated patients (P = 0.019), and in 25% and 8% of untreated controls (P = 0.45) with HBV genotype B and C, respectively. Multivariate analysis showed that genotype B and low pretreatment HBV-DNA levels were independent predictors of antiviral response. In conclusion, our data showed that HBV genotype B was associated with a higher rate of IFN-induced HBeAg clearance compared with genotype C. Stratification for HBV genotypes should be considered in future clinical trials of antiviral therapy of chronic hepatitis B. (Hepatology2002;36:1425-1430).
Expression profiling of liver cell lines expressing entire or parts of hepatitis C virus open reading frame
Authors: Aizaki Hideki , Harada Takashi , Otsuka Motoyuki , Seki Naohiko , Matsuda Mami , Li Yue Wei , Kawakami Hayato , Matsuura Yoshiharu , Miyamura Tatsuo , Suzuki Tetsuro
Published: March 2007

Although hepatitis C virus (HCV) is a causative agent of liver diseases, its mechanism of pathogenesis is still unclear, mainly because of the lack of adequate cell culture systems to support HCV infection and replication. In this report, we describe development and characterization of human hepatoma cell lines constitutively expressing entire (Hep394) or parts (Hep352, Hep3294) of the HCV open reading frame (ORF). The viral and cellular proteolytic machinery involved in the viral precursor processing was consistently functional, and processed HCV proteins were synthesized in these established cell lines. By using a cDNA microarray analysis coupled with semiquantitative reverse-transcription polymerase chain reaction (RT-PCR), we identified 12 genes up-regulated and 4 genes down-regulated in Hep394 cells. With regard to genes related to cell growth regulation, we found up-regulation of forkhead transcription factor FREAC-1, poly (A) binding protein PABP2, and Ras suppressor Rsu-1. Another category of changes in gene expression includes MHC antigens, which play an important role in the T-cell-mediated immune reaction in the liver. In conclusion, functional genomic approaches comparing expression among the different cell lines expressing parts of the HCV genome may promote our understanding of the molecular basis of pathogenicity of HCV infection. (Hepatology2002;36:1431-1438).
Endothelin-1 and heme oxygenase-1 as modulators of sinusoidal tone in the stress-exposed rat liver
Authors: Rensing Hauke , Bauer Inge , Zhang Jian X. , Paxian Markus , Pannen Benedikt H. J. , Yokoyama Yukihiro , Clemens Mark G. , Bauer Michael
Published: March 2007

Heme oxygenase (HO)-1 is up-regulated after ischemia/reperfusion and contributes to maintenance of hepatic perfusion and integrity. Blockade of HO-1 leads to an increased portal pressor response in the stress-exposed liver. We tested whether the increase in portal pressure reflects unmasking of a concomitant up-regulation of the vasoconstrictor endothelin (ET)-1. Hemorrhagic shock induced messenger RNAs encoding HO-1 (16-fold) and ET-1 (9-fold) with a similar time course in the liver. At maximum induction of both mediators, rats received either vehicle or the endothelin ETA/B antagonist bosentan (10 mg/kg intravenously). Subsequently, the HO pathway was blocked in all animals by tin-protoporphyrin (SnPP)-IX (50 μmol/kg intravenously). Portal and sinusoidal hemodynamics were measured using microflow probes and intravital microscopy, respectively. Blockade of the HO pathway led to a significant increase in portal resistance (sham/SnPP-IX, 0.17 ± 0.046 mm Hg · min · mL−1; shock/vehicle/SnPP-IX, 0.57 ± 0.148 mm Hg · min · mL−1; P < 0.05) and a decrease in sinusoids conducting flow (shock/vehicle/SnPP-IX: baseline, 28.3 ± 0.85 sinusoids/mm; 10 minutes after SnPP-IX, 23.1 ± 1.09 sinusoids/mm; P < 0.05). Intravital microscopy showed narrowing of failing sinusoids colocalizing with stellate cells after blockade of the HO pathway. Blockade of ETA/B receptors attenuated the increase in portal resistance (shock/bosentan/SnPP-IX, 0.29 ± 0.051 mm Hg · min · mL−1) and prevented sinusoidal perfusion failure (shock/bosentan/SnPP-IX: baseline, 28.2 ± 0.97 sinusoids/mm; 10 minutes after SnPP-IX, 28.8 ± 1.18 sinusoids/mm) as well as sinusoidal narrowing. In conclusion, a functional interaction of the up-regulated vasodilatory HO system and the vasoconstrictor ET-1 on the sinusoidal level exists under stress conditions. Both mediator systems affect sinusoidal diameter via direct action on hepatic stellate cells in vivo. (Hepatology2002;36:1453-1465).
Epidermal growth factor—induced activation of the insulin-like growth factor I receptor in rat hepatocytes
Authors: Hallak Hazem , Moehren Giesla , Tang Jei , Kaou Mohamad , Addas Mouhamad , Hoek Jan B. , Rubin Raphael
Published: March 2007

Insulin-like growth factor I (IGF-I) plays a critical role in the induction of cell cycle progression and survival in many cell types. However, there is minimal IGF-I binding to hepatocytes, and a role for IGF-I in hepatocyte signaling has not been elucidated. The dynamics of IGF-I receptor (IGF-IR) activation were examined in freshly isolated rat hepatocytes. IGF-I did not activate the IGF-IR. However, des(1-3)IGF-I, which weakly binds IGF binding protein-3 (IGFBP-3), induced IGF-IR phosphorylation. IGFBP-3 surface coating was identified by confocal immunofluorescence microscopy. In contrast with the inactivity of IGF-I, epidermal growth factor (EGF) induced the tyrosine phosphorylation of the IGF-IR in parallel with EGF receptor phosphorylation. Transactivation of the IGF-IR by EGF was inhibited by tyrphostin I-Ome-AG538, a tyrosine kinase inhibitor with high specificity for the IGF-IR. Src kinase inhibitors pyrazolopyrimidine PP-1 and PP-2 inhibited transactivation of the IGF-IR by EGF. EGF stimulated the tyrosine phosphorylation of Src, and induced its association with the IGF-IR. EGF-induced phosphorylations of insulinrelated substrate (IRS)-1, IRS-2, Akt, and p42/44 mitogen-activated protein kinases (MAPKs) were inhibited variably by I-Ome-AG538. In conclusion, the data show an EGF- and Src-mediated transactivation pathway for IGF-IR activation in hepatocytes, and indicate a role for the IGF-IR in hepatocyte intracellular signaling. The findings also show a role for IGFBP-3 in the inhibition of IGF-I signaling in hepatocytes. (Hepatology2002;36:1509-1518).
Dysregulation of glycogen synthase kinase-3β signaling in hepatocellular carcinoma cells
Authors: Desbois-Mouthon Christèle , Eggelpoë Marie-José Blivet-Van , Beurel Eléonore , Boissan Mathieu , Delélo Roland , Cadoret Axelle , Capeau Jacqueline
Published: March 2007

It has been reported that upstream components of the insulin-like growth factor (IGF) signaling axis could be overexpressed during hepatocarcinogenesis in humans and rodents. However, the signal transduction pathways activated downstream have been poorly studied. Here, we examined whether glycogen synthase kinase-3β (GSK-3β) could be a target in human hepatoma cell lines and transgenic ASV mice with hepatic expression of the SV40 large T antigen. In HuH7, Mahlavu, and Hep3B cells, basal levels of GSK-3βSer9 phosphorylation were strongly elevated, indicating that GSK-3β was inhibited. GSK-3β phosphorylation was insensitive to exogenous IGFs and was blocked with an IGF-1 receptorneutralizing antibody in Mahlavu and Hep3B cells. By using LY294002 and ML-9, which act as phosphatidylinositol 3-kinase (PI3-K) and Akt inhibitors, respectively, we showed that GSK-3β phosphorylation required P13-K activation in both cell lines whereas downstream Akt activation was required only in Mahlavu cells. However, in the 2 cell lines, GSK-3βSer9 phosphorylation was controlled by protein kinase C (PKC)ξ because it was blocked by an inhibitory PKCξ peptide. The blockage of GSK-3β phosphorylation markedly inhibited glycogen synthesis and decreased β-catenin expression. In addition, the overexpression of a constitutively active GSK-3β reduced AP-1-mediated gene transcription in Hep3B cells. Finally, we observed that reexpression of IGF-2 in tumoral livers from ASV mice was associated with a marked phosphorylation of GSK-3β. In conclusion, our results identify GSK-3β as a molecular target of the constitutive activation of the IGF axis in in vitro and in vivo models of hepatocarcinogenesis. Persistent phosphorylation of GSK-3β could be critical for regulation of glycogen metabolism and cell growth in hepatoma cells. (Hepatology2002;36:1528-1536).
Hepatitis C Virus Core Protein Activates Erk and P38 Mapk in Cooperation With Ethanol in Transgenic Mice
Authors: Tsutsumi Takeya , Suzuki Tetsuro , Moriya Kyoji , Shintani Yoshizumi , Fujie Hajime , Miyoshi Hideyuki , Matsuura Yoshiharu , Koike Kazuhiko , Miyamura Tatsuo
Published: March 2007

In human chronic hepatitis C, alcohol intake is a synergistic factor for the acceleration of hepatocarcinogenesis. Recently, we showed a significant increase of reactive oxygen species (ROS) in hepatitis C virus (HCV) core–transgenic mice fed ethanol–containing diets. Because previous studies indicated that ROS is closely associated with mitogen–activated protein kinases (MAPK), we examined activities of c–Jun N–terminal kinase, p38 MAPK, and extracellular signal–regulated kinase (ERK) in the liver of core–transgenic and nontransgenic mice with short–term ethanol feeding. Activity of ERK and p38 MAPK was increased in core–transgenic mice compared with nontransgenic mice, whereas neither ERK nor p38 MAPK was activated in core–transgenic mice with normal diets. In addition, activity of cyclic–AMP and serum responsive element, downstream pathways of p38 MAPK and ERK, was also increased. Comparison of gene expression profiles by cDNA microarray and real–time PCR revealed that galectin–1, which is associated with cell transformation, was significantly increased in ethanol–fed core–transgenic mice. On the other hand, glutathione S–transferase (GST), which plays a key role in protecting cells from oxidative stress, was decreased. In conclusion, these results suggest that HCV core protein cooperates with ethanol for the activation of some MAPK pathways, and leads to the modulation of several genes, contributing to the pathogenesis of liver disease of HCV– infected patients with high ethanol consumption. (Hepatology 2003;38:820-828).
Phenotypic and Functional Characterization of Intrahepatic T Lymphocytes During Chronic Hepatitis C
Authors: Leroy Vincent , Vigan Ines , Mosnier Jean-Francois , Dufeu-Duchesne Tania , Pernollet Martine , Zarski Jean-Pierre , Marche Patrice N. , Jouvin-Marche Evelyne
Published: March 2007

The pathogenesis of liver cell injury during chronic hepatitis C (CHC) is poorly understood. The cellular immune response is thought to play a key role in both inhibition of viral replication and liver pathology. However, little is currently known about which lymphocyte populations and which immune effectors contribute to or control liver damage. We investigated a panel of 15 phenotypic and functional markers of intrahepatic T–lymphocyte subsets irrespective of their antigen specificity in 48 hepatitis C virus (HCV)–infected patients and 8 healthy control subjects. Lymphocyte characteristics were evaluated from liver biopsy specimens both at gene expression level by real–time quantitative reverse–transcription polymerase chain reaction (RT–PCR) and by immunochemistry, in relation with the degree of liver injury and with intrahepatic HCV–RNA levels. As compared with controls, we found major changes in T–lymphocyte subsets in HCV–infected patients, with a significant decrease of T–cell antigen receptor (TCR) δ and CD56 gene expression, associated with a concomitant increase of TCRα and CD8β that were correlated with cytotoxic factors, proinflammatory chemokines, and chemokine receptors including peforin, interferon gamma (IFN–γ), tumor necrosis factor α (TNF–α), RANTES, and CXCR3. The gene expression of CD8β, a specific marker for conventional TCRα+CD8+ lymphocytes, was correlated by multivariate analysis with both alanine aminotransferase (ALT) serum levels and histologic activity index. Furthermore, CD8 staining was observed by immunochemistry in the areas of lobular and piecemeal necrosis. In contrast, no lymphocyte marker was correlated with viral load, measured both in serum and in liver. In conclusion, these results strongly suggest key roles for CD8+ T cells as effectors of liver damage during chronic HCV infection and for their inability to control viral replication. (Hepatology 2003;38:829-841).
Identification of A Ribavirin–Resistant Ns5b Mutation of Hepatitis C Virus During Ribavirin Monotherapy
Authors: Young Kung-Chia , Lindsay Karen L. , Lee Ki-Jeong , Liu Wen-Chun , He Jian-Wen , Milstein Susan L. , Lai Michael M.C.
Published: March 2007

Ribavirin (RBV), a guanosine analogue, has been suggested to exert an antiviral action against hepatitis C virus (HCV) by causing lethal mutations and suppressing RNA polymerase in vitro, but the mechanism of its clinical therapeutic effects is currently unknown. To test the hypothesis that RBV could act both as an RNA mutagen and inhibit viral RNA synthesis in vivo, we studied the evolution of the nucleotide sequences of HCV RNA at the nonstructural (NS) 5B region in patients receiving RBV, placebo, or interferon alfa (IFN–α) monotherapy. The RBV group showed a slightly more accelerated evolution rate of HCV RNA quasispecies than either the IFN–α or placebo group. RBV caused preferentially A–to–G and U–to–A mutations. Interestingly, an NS5B amino acid 415 Phe–to–Tyr (F415Y) mutation emerged in all (5 of 5) patients infected with HCV genotype 1a during the RBV treatment. Subsequently, the parental 415F strain reemerged in some patients after the treatment was discontinued. The effect of the amino acid substitution at NS5B415 on HCV RNA replication was then investigated using an HCV subgenomic replicon in Huh7 cells. We showed that treatment of replicon cells with RBV reduced the HCV RNA level of NS5B415F replicon, but not NS5B415Y, in a dose–dependent manner. Thus, NS5B F415Y mutation represents an RBV–resistant variant. The 3–dimensional modeling and structure analysis of NS5B protein revealed that the 415th amino acid is located at the P helix region of the thumb subdomain, which may interact with the minor groove of the template–primer duplex in the putative RNA–binding cleft. In conclusion, RBV could work as a weak mutagen for HCV RNA in HCV–infected patients. Furthermore, the selection of an RBV–resistant variant with a single amino acid substitution in NS5B suggested that RBV may directly interact with HCV RNA polymerase, thus interfering with its enzymatic activity. (Hepatology 2003;38:869-878).