bluuebunny/crossref_metadata_2025 huggingface dataset:
The Roles of Intrahepatic Vα14+ Nk1.1+ T Cells for Liver Injury Induced By Salmonella Infection in Mice
Authors: Ishigami Masatoshi , Nishimura Hitoshi , Naiki Yoshikazu , Yoshioka Kentaro , Kawano Tetsu , Tanaka Yujiro , Taniguchi Masaru , Kakumu Shinichi , Yoshika Yasunobu
Published: July 2004

To investigate the roles of intrahepatic T cells in liver injury after Salmonella infection, we examined serum alanine transaminase (ALT), histopathology, and bacterial numbers in liver after infection with Salmonella choleraesuis strain 31N–1 in mice genetically lacking TCRαβ+, CD4+, CD8+, or NK1.1+T cells with C57BL/6 background. In control (+/+) mice, serum ALT reached a peak level by day 7 after an intraperitoneal inoculation of 2 × 106 CFU Salmonella choleraesuis 31N–1. In TCR–β-/- mice, liver injury, as assessed by serum ALT level and histological examination, was significantly suppressed on day 7 after Salmonella infection but the numbers of bacteria in liver did not differ from those in normal mice, suggesting that αβ T cells are responsible for liver injury induced by Salmonella infection. To further determine which subsets in αβ T cells are important for the liver injury, we compared serum ALT level in mice genetically lacking CD4, CD8, β2–microglobulin (β2m, IAβ, or Jα281 after Salmonella infection. In CD4-/- mice, serum ALT was significantly lower in comparison with control mice, but there was no difference in serum ALT levels in CD8-/- and IAβ-/- mice from that in control mice. Notably, serum ALT levels and pathological lesions in liver were significantly decreased in β2m-/- or Jα281-/- mice, which lacked in NK1.1+ T cells bearing TCR Vα14–Jα281 specific for β2m–associated CD1d, following Salmonella infection. Taken together, it is suggested that αβ T cells bearing NK1.1 and CD4 may be main effector cells for liver injury after Salmonella infection.
Small Hyperechoic Nodules in Chronic Liver Diseases Include Hepatocellular Carcinomas With Low Cyclin D1 and Ki–67 Expression
Authors: Yamagata Michiko , Masaki Tsutomu , Okudaira Takahito , Imai Yasuo , Shiina Shuichiro , Shiratori Yasushi , Omata Masao
Published: July 2004

In spite of the importance of periodic screening for hepatocellular carcinoma (HCC) by ultrasonography (US) in patients with underlying liver disease, the clinicopathological characteristics of hyperechoic nodules have not been clearly evaluated. The aim of this study was to characterize the pathological and proliferating features of small hyperechoic nodules. Tissue specimens of 55 hyperechoic and 107 hypoechoic nodules less than 20 mm in diameter in patients with chronic liver disease were obtained by echo–guided needle biopsy and examined histopathologically. Of these, 42 (76%) hyperechoic and 56 (52%) hypoechoic nodules were diagnosed as HCC, and 82% of hyperechoic HCCs contained fatty change and/or clear cell change. In addition, immunohistochemical staining using cyclin D1, p53, and Ki–67 was examined. A high–level expression of cyclin D1 was found in only 5% of hyperechoic HCCs, in contrast to 38% of hypoechoic HCCs (P < .02). The labeling index of Ki–67 in hyperechoic HCCs was lower than in hypoechoic HCCs (4.2% vs. 8.9%; P < .003). However, there was no difference on p53 staining between them. Retrospective follow–up study revealed that hyperechoic nodules showed slow growth (doubling time, median: 1,403 days) initially, and came to show rapid growth (doubling time, median: 56 days). From these results, small hyperechoic nodules in chronic liver diseases are worth notice as candidates for well–differentiated HCC with low cyclin D1 and Ki–67 expression.
The Relative Role of the Child–Pugh Classification and the Mayo Natural History Model in the Assessment of Survival in Patients With Primary Sclerosing Cholangitis
Authors: Kim Ray W. , Poterucha John J. , Wiesner Russell H. , LaRusso Nicholas F. , Lindor Keith D. , Petz Jan , Therneau Terry M. , Malinchoc Michael , Dickson Rolland E.
Published: July 2004

The Child–Pugh classification is a simple, convenient prognostic measure in patients with liver cirrhosis. We investigated the relative role of the Child–Pugh classification and the Mayo model in the assessment of survival in patients with primary sclerosing cholangitis (PSC). Of the 173 patients described in the original Mayo PSC natural history model, 147 patients had sufficient information in the medical record to allow computation of the Child–Pugh score. We used our most recent modification of the Mayo model to compute the risk score, based on patient's age, serum levels of bilirubin, albumin, and aspartate aminotransferase and history of variceal bleeding. Using the risk score (R), patients were divided into the low– (R < 0), intermediate– (0 ≤ R < 2), and high–risk (R ≥ 2) groups. Kaplan–Meier estimates and proportional hazards analysis were used to evaluate the two prognostic models. Although there was a statistically significant correlation between the Child–Pugh and Mayo risk scores, two–thirds of the patients had a Child–Pugh score of 5 or 6 and a relatively wide range of risk scores (-1.1–4.3). The probability of survival for 7 years in patients in the low–, intermediate–, and high–risk groups was 92%, 74%, and 40% for Child–Pugh class A (n = 96) and 100%, 62%, and 28% for Child–Pugh class B patients (n = 44), respectively. There were only a small number (n = 7) of Child–Pugh class C patients. In our age–adjusted multivariate analysis, each unit increase in the Mayo risk score was associated with a 2.5–fold increase in the risk of death (95% confidence interval: 1.8–3.4, P < .01), whereas Child–Pugh classification had no significant impact on survival (Child–Pugh B vs. A: risk ratio = 1.1 [95% confidence interval: 0.6–2.0]; Child–Pugh C versus A: risk ratio = 0.6 [95% confidence interval: 0.2–1.8]). In contrast to the Child–Pugh classification, which was developed for advanced liver cirrhosis, the Mayo model provides valid survival information, particularly in patients early in the course of PSC.
Formation of 4–Hydroxynonenal Adducts With Cytochrome C Oxidase in Rats Following Short–Term Ethanol Intake
Authors: Chen Juanjuan , Robinson Neal C. , Schenker Steven , Frosto Teri A. , Henderson George I.
Published: July 2004

This study addresses the role of the lipid peroxidation product, 4–hydroxynonenal (HNE), in ethanol–related damage of cytochrome c oxidase (COX) in vivo. It utilizes an animal model with acute ethanol exposure in which HNE levels in liver mitochondria are strikingly increased. Pregnant female Sprague–Dawley rats were administered 5 doses of ethanol (4 gm/kg, po at 12–hour intervals) beginning on day 17 of gestation and were sacrificed on day 19. Controls were pair–fed and received dextrose isocaloric to ethanol. Mitochondria were isolated from maternal and fetal livers and COX activities were measured spectrophotometrically. Compared with the pair–fed controls, COX activity was decreased with exposure to ethanol by 25% in maternal rats and 43% in fetal rats (P<.05). Western Blot with an HNE–Histidine antibody showed enhanced formation of HNE adducts with COX from ethanol–exposed rats, which was more pronounced in fetal than in adult livers. The HNE adducts were mainly with subunit IV of COX. The cause and effect relationship between HNE adduct formation and COX inhibition was examined in vitro by incubating purified COX with HNE. COX inhibition was accompanied by concentration–dependent HNE adduct formation that was consistent with those found in in vivo ethanol–exposed samples. These results suggest that the ethanol–related decreases in COX activity found in liver mitochondria could be attributable to HNE adduct formation with the enzyme complex. This could be an important mechanism by which modification of proteins occur in in vivo oxidative stress.
Dietary Iron Overload and Induced Lipid Peroxidation Are Associated With Impaired Plasma Lipid Transport and Hepatic Sterol Metabolism in Rats
Authors: Brunet Sylvain , Thibault Louise , Delvin Edgard , Yotov Wagner , Bendayan Moïse , Levy Emile
Published: July 2004

Although hemochromatosis is characterized by dramatic morphological and functional alterations in the liver, little is known about the effects of an excess of iron on lipid metabolism. Therefore, we determined the effect of chronic iron overload on plasma lipid profile and lipoprotein composition, as well as on hepatic cholesterol metabolism and biliary sterol output. Rats administered a diet enriched with 3% iron carbonyl for 12 weeks displayed a 30–fold increase in iron (P < .0001) and a 5–fold rise in malondialdehyde (P < .001) in the liver. When compared with pair–fed controls, iron–overload rats showed a significant increase in triglycerides (P < .005), free cholesterol (P < .006), cholesteryl ester (P < .007), and high–density lipoprotein (HDL)–cholesterol (P < .003). Triglyceride and cholesteryl ester enrichment, protein depletion, size increase, and apolipoprotein composition alterations characterized the very low density lipoprotein (VLDL) and HDL particles of iron–overload rats. Assessment of the activity of intracellular key enzymes for cholesterol homeostasis in these rats disclosed a reduction in 3–hydroxy–3–methylglutaryl coenzyme A (HMG–CoA) reductase (66%, P < .005) and cholesterol 7α–hydroxylase (58%, P < .0004) with an increment of acyl–CoA: cholesterol acyltransferase (62%, P < .002). The lack of optimal enzymatic activity may be a result of marked membrane lipid peroxidation that brings about fluidity drop (P < .029) in microsomes of iron–overload rats (5.00 ± 0.013) versus controls (8.20 ± 0.03), reflected by polarization. A decline of the pool size of cholesterol and bile acids was noted in iron–overload rats during a 6–hour bile drainage. Our results show that experimental iron overload causes marked perturbations in plasma lipid transport and hepatobiliary sterol metabolism. Given the positive correlation of malondialdehyde with most of the altered parameters, iron–catalyzed lipid peroxidation may well be one of the involved mechanisms.
Three Cases of Severe Subfulminant Hepatitis in Heart–Transplanted Patients After Nosocomial Transmission of A Mutant Hepatitis B Virus
Authors: Stuyver Lieven , De Gendt Sija , Cadranel Jean Fran , Van Geyt Caroline , Van Reybroeck Georges , Dorent Richard , Gandjbachkh Iranj , Rosenheim Michel , Charlotte Frederic , Opolon Pierre , Huraux Jean Marie , Lunel Françoise
Published: July 2004

Fulminant and severe viral hepatitis are frequently associated with mutant hepatitis B virus (HBV) strains. In this study, the genetic background of a viral strain causing severe subfulminant outcome in heart–transplanted patients was studied and compared with viral hepatitis B strains that were not linked to severe liver disease in the same setting. A total of 46 patients infected nosocomially with HBV genotype A were studied. Five different viral strains were detected, infecting 3, 9, 5, 24, and 5 patients, respectively. Only one viral strain was found to be associated with the subfulminant outcome and 3 patient deaths as a consequence of severe liver disease. The remaining 43 patients with posttransplantation HBV infection did not show this fatal outcome. Instead, symptoms of hepatitis were generally mild or clinically undiagnosed. Comparison of this virus genome with the four other strains showed an accumulation of mutations in the basic core promoter, a region that influences viral replication, but also in hepatitis B X protein (HBX) (7 mutant motifs), core (10 mutant motifs), the preS1 region (5 mutant motifs), and the HBpolymerase open reading frame (17 motifs). Some of these variations, such as those in the core region, were located on the tip of the protruding spike of the viral capsid (codons 60 to 90), also known in part as an important HLA class II-restricted epitope region. These mutations might therefore influence the immune–mediated response. The viral strain causing subfulminant hepatitis was, in addition, the only strain with a preCore stop codon mutation and, thus, hepatitis B e antigen (HBeAg) expression was never observed. The combination of these specific viral factors is thought to be responsible for the fatal outcome in these immune–suppressed heart–transplant recipients.
Characterization of A Reproducible Rat Model of Hepatic Veno–Occlusive Disease
Authors: DeLeve Laurie D. , McCuskey Robert S. , Wang Xiangdong , Hu Liping , McCuskey Margaret K. , Epstein Robert B. , Kanel Gary C.
Published: July 2004

Lack of a reproducible animal model has hampered progress in understanding hepatic veno–occlusive disease (HVOD). This article characterizes a reproducible model of HVOD. Rats gavaged with monocrotaline, 160 mg/kg, were killed between days 1 and 10. Sections were evaluated by light microscopy with a standardized scoring system, by immunoperoxidase staining with ED–1 (monocytes, macrophages) and ED–2 (Kupffer cells) antibodies, and by transmission (TEM) and scanning electron microscopy (SEM). On days 1 and 2, the earliest manifestations were progressive injury to the sinusoidal wall with loss of sinusoidal lining cells, sinusoidal hemorrhage, and mild damage to central vein (CV) endothelium. On days 3 through 5 (“early HVOD”), there was centrilobular coagulative necrosis, severe injury to sinusoids, severe sinusoidal hemorrhage, and severe CV endothelial damage; inflammation with ED–1-positive cells was most marked on these days. Days 6 and 7 (“late HVOD”) were characterized by subendothelial and advential fibrosis of CVs, damage of the CV endothelium with subendothelial hemorrhage, and some restoration of the sinusoidal wall. Between days 8 and 10, sections showed interindividual variation ranging from mild, residual fibrosis to severe, late HVOD. From days 1 through 10, ED–2-positive cells were decreased in number, and the number of ED–1-positive cells was increased. Sinusoidal damage is the earliest change in HVOD. Coagulative necrosis follows sinusoidal injury and resolves with improvement in sinusoidal endothelial cell (SEC) morphology. Moderate–to–severe CV fibrosis occurs after reappearance of sinusoidal lining cells and resolution of hepatocyte necrosis. The inflammatory response within the lobule and CVs is a result of recruitment of monocytes, whereas Kupffer cells are decreased in number.
Treatment of Hepatitis C Virus-Related Cirrhosis: A Randomized, Controlled Trial of Interferon Alfa–2B Versus No Treatment
Authors: Valla Dominique-Charles , Chevallier Michele , Marcellin Patrick , Payen Jean-Louis , Trepo Christian , Fonck Marianne , Bourliere Marc , Boucher Evelyne , Miguet Jean-Philippe , Parlier David , Lemonnier Catherine , Opolon Pierre
Published: July 2004

To examine the effects of interferon (IFN) therapy on clinical, biochemical, and histological features in patients with compensated hepatitis C virus (HCV)–related cirrhosis, we have conducted a randomized, controlled trial of IFN therapy versus observation. Eight centers included a total of 99 patients with biopsy–proven cirrhosis. IFN–α2b, 3 million units three times per week, or no antiviral therapy was given for 48 weeks. Twenty–three patients dropped out. End–of–treatment biochemical response was not observed in any of the 39 controls but was observed in 6 of the 47 treated patients (P < .02); sustained biochemical response was obtained in only 2 treated patients. Controls and treated patients did not significantly differ with regard to the changes in serum level of albumin, bilirubin, α–fetoprotein, in plasma prothrombin, in histological activity, or liver collagen content. During trial or follow–up (160 ± 57 weeks), hepatocellular carcinoma developed in 9 controls and 5 treated patients (NS); decompensation of cirrhosis occurred in 5 controls and 7 treated patients. Seven controls and 10 treated patients died. In conclusion, in patients with compensated HCV–related cirrhosis, a 48–week course of IFN therapy is safe and is able to induce end–of–treatment biochemical response in a significant proportion of patients. However, a 48–week course of IFN therapy usually fails to achieve sustained response and, within the limit of this study, did not significantly improve the 3–year outcome. Therefore, a longer course of IFN therapy or combination therapy with ribavirin should be evaluated in patients with HCV–related cirrhosis.
Bile Acids Modulate the Interferon Signalling Pathway
Authors: Podevin Philippe , Rosmorduc Olivier , Conti Filomena , Calmus Yvon , Meier Peter J. , Poupon Raoul
Published: July 2004

We have previously shown that cholestasis and bile acids inhibit 2′,5′ oligoadenylate synthetase (OAS) activity in the liver and in primary hepatocyte cultures. Here, we assessed the influence of bile acids on interferon (IFN) pathway activation in three hepatoma cell lines. In HepG2 cells, bile acids (100–200 μmol/L) inhibited IFN–induced 2′,5′ OAS activity to an extent depending on their surface activity index. In Western blot analysis, IFN–induced expression of two major antiviral proteins, MxA and OAS p100, was reduced by 54% ± 8% and 44% ± 12%, respectively, when cells were preincubated for 4 hours with 100 μmol/L chenodeoxycholic acid (CDCA). In the same conditions, CDCA did not modify the IFN–induced signal transducers and activators of transcription (STAT)s tyrosine phosphorylation. In contrast, it reduced IFN–induced MxA promoter activity by 60%. The inhibitory effect of CDCA was not mediated by a 4β–phorbol 12β–myristate 13α–acetate (PMA)–sensitive protein kinase C (PKC)–dependent pathway. Finally, using CHO cells stably expressing a functional human bile acid carrier (Na+–dependent taurocholate cotransporting polypeptide [NTCP]), we found that bile acid inhibition of the IFN pathway occurred in the range of more physiological concentrations (12–50 μmol/L). In summary, our results provide strong evidence that bile acids inhibit the induction of proteins involved in the antiviral activity of IFN. This might partly explain the lack of responsiveness to IFN therapy in some patients with advanced chronic viral liver diseases.
Enhancement of Mdr2 Gene Transcription Mediates the Biliary Transfer of Phosphatidylcholine Supplied by An Increased Biosynthesis in the Pravastatin–Treated Rat
Authors: Carrella Massimino , Feldman Douglas , Cogoi Susanna , Csillaghy Annalisa , Weinhold Paul A.
Published: July 2004

An increase of biliary lipid secretion is known to occur in the rat under sustained administration of statin–type 3–hydroxy–3–methylglutaryl (HMG) coenzyme A (CoA) reductase inhibitors. The present study has addressed critical mechanisms of hepatic lipid synthesis and phosphatidylcholine (PC) biliary transport in the rat fed with a 0.075% pravastatin diet for 3 weeks. After treatment, biliary secretion of PC and cholesterol increased to 233% and 249% of controls, while that of bile salts was unchanged. Activity of cytidylyltransferase (CT), a major regulatory enzyme in the CDP–choline pathway of PC synthesis, was raised in both microsomal and cytosolic fractions (226% and 150% of controls), and there was an increase to 187% in the mass of active enzyme as determined by Western blot of microsomal protein using an antibody specific to CT. Cytosolic activity of choline kinase, another enzyme of the CDP–choline pathway, also increased to 175% of controls. In addition, there was an over eightfold increase in the HMG CoA reductase activity and mRNA. Thus, an increased PC and cholesterol synthetic supply to hepatocytes appeared as a basic mechanism for the biliary hypersecretion of these lipids. Notwithstanding the increased synthesis, hepatic PC content was unchanged, suggesting an enhanced transfer of this lipid into bile. Indeed, there was a sevenfold increase of multidrug resistance gene 2 (mdr2) gene mRNA coding for a main PC canalicular translocase. Thus, hypersecretion of biliary PC in the model studied can be explained by an up–regulation of mdr2 gene transcription and its P–glycoprotein product mediating the biliary transfer of PC supplied by an increased biosynthesis.
Effects of Iron Loading on Pathogenicity in Hepatitis C Virus-Infected Chimpanzees
Authors: Bassett Suzanne E. , Di Bisceglie Adrian M. , Bacon Bruce R. , Sharp Mark R. , Govindarajan Sugantha , Hubbard Gene B. , Brasky Kathleen M. , Lanford Robert E.
Published: July 2004

Elevated iron levels have been associated with raised serum alanine transaminase (ALT) levels in hepatitis C virus (HCV)–infected humans. However, it is not clear if HCV infection causes increased iron accumulation by the liver or if the severity of HCV infection is actually worsened by higher iron levels in the host. To better understand the relationship between iron and persistent HCV infections, we examined the effect of excess dietary iron on disease severity in HCV–infected chimpanzees. Iron was supplemented in the diets of four HCV–infected and two uninfected chimpanzees for 29 weeks to achieve iron loading. Iron loading was confirmed by increases in serum iron levels, percentages of transferrin saturation, ferritin levels, elevations in hepatic iron concentration (HIC), and by histological examination. The majority of HCV–infected chimpanzees had higher iron levels before iron feeding than the uninfected animals. Although various degrees of iron loading occurred in all chimpanzees, HCV–infected animals exhibited increased loading in comparison with uninfected animals. The effects of iron loading on HCV disease expression was determined by comparing disease parameters during an extended baseline period before iron loading with the period during iron loading and immediately following iron loading. Iron loading did not influence the viral load, but did exacerbate liver injury in HCV–infected chimpanzees, as evidenced by elevated ALT and histological changes. Because all chimpanzees on high iron diets experienced iron loading, but pathological effects were only observed in HCV–infected chimpanzees, HCV infection appears to increase the susceptibility of the liver to injury following iron loading. These results confirm and extend previous observations made in human populations and serve to further validate the chimpanzee model of chronic hepatitis C.
Interferon Alfa Receptor Expression and Growth Inhibition by Interferon Alfa in Human Liver Cancer Cell Lines
Authors: Yano Hirohisa , Iemura Akihiro , Haramaki Makoto , Ogasawara Sachiko , Takayama Akiko , Akiba Jun , Kojiro Masamichi
Published: July 2004

Type I interferon (IFN) receptor consists of two chains (Hu–IFN–αR1 and Hu–IFN–αR2), and Hu–IFN–αR2 takes a soluble (Hu–IFN–αR2a), short (Hu–IFN–αR2b), or long (Hu–IFN–αR2c) form. We examined the expression of type I IFN receptor, the growth–suppression effect of IFN–α, and their relationship in 13 liver cancer cell lines. With reverse–transcription polymerase chain reaction (RT–PCR) analysis, the expressions of Hu–IFN–αR1, Hu–IFN–αR2a, and Hu–IFN–αR2c were confirmed in all cell lines, and that of Hu–IFN–αR2b in 12 cell lines. All cell lines expressed mRNAs of a transcriptional activator, interferon regulatory factor (IRF)–1, and its antagonistic repressor (IRF–2). Flow cytometry revealed weak expression of Hu–IFN–αR2 on the cell surface in 12 cell lines. The soluble–form protein of Hu–IFN–αR2 was detected at varying levels in culture supernatants of all cell lines with enzyme–linked immunosorbent assay (ELISA). Cell proliferation was suppressed in proportion to the dose of human natural IFN–α at 96 hours of culture, but it was not clearly related to the expression of Hu–IFN–αR2 protein on the cell surface. Investigations on the morphology, DNA, and cell cycle presented four growth suppression patterns as a result of IFN–α: 1) induction of apoptosis and blockage of cell cycle at the S phase (9 cell lines); 2) blockage at the S phase (2 cell lines); 3) induction of apoptosis and blockage at the G2 /M phase (1 cell line); and 4) blockage at the G1 phase (1 cell line). There was no evidence showing that changes in the expressions of Bcl–2, Bcl–xL, Bak, and Bax lead directly to IFN–α-mediated apoptosis. Our findings demonstrated that IFN–α would express growth–suppression effects at varying degrees by inducing inhibition of cell–cycle progression with or without apoptosis, regardless of the expression level of Hu–IFN–αR2 protein on the cell surface.
Interferon Alfa Subtypes and Levels of Type I Interferons in the Liver and Peripheral Mononuclear Cells in Patients With Chronic Hepatitis C and Controls
Authors: Castelruiz Yurdana , Larrea Esther , Boya Patricia , Civeira María-Pilar , Prieto Jesús
Published: July 2004

Viral infections stimulate the transcription of interferon type I, which includes IFN–alfa (IFN–α) (13 subtypes) and IFN–β (a single substance). Hepatitis C virus (HCV) infection is remarkable by its ability to evade host antiviral defenses; however, there is little information as to whether endogenous IFN is activated or not in this disease. Additionally, despite the fact that the various IFN–α subtypes may differ in biological activity, there are no data concerning the IFN–α subtypes specifically expressed in normal and diseased liver tissue. Thus, we have analyzed the IFN–α subtypes and the mRNA levels of type I IFNs in samples of normal liver tissue and in liver from patients with chronic hepatitis C. Similar studies were performed in peripheral blood mononuclear cells (PBMC) from patients and controls. After amplification and cloning of IFN–α cDNA, we observed that 98 of the 100 clones from normal liver tissue corresponded to the IFN–α5 subtype. However, in livers with chronic hepatitis C and in PBMC from controls and patients, a variety of subtypes, in addition to IFN–α5, were detected, suggesting a participation of infiltrating leukocytes in the production of IFN–α in livers with chronic hepatitis C. As compared with controls, patients with chronic hepatitis C showed a significant increase in IFN–β mRNA in both the liver and PBMC, while IFN–α mRNA was significantly increased in PBMC but markedly reduced in liver tissue. In conclusion, IFN–α5 is the sole IFN–α subtype expressed in normal liver tissue. The hepatic levels of IFN–α are reduced in chronic hepatitis C, an event that may favor viral persistence.
Hepatic Thrombopoietin Mrna Levels in Acute and Chronic Liver Failure of Childhood
Authors: Wolber Eva-Maria , Ganschow Rainer , Burdelski Martin , Jelkmann Wolfgang
Published: July 2004

The liver is the main production site of the hormone thrombopoietin (TPO), the major regulator of megakaryopoiesis. To investigate the role of an impaired TPO gene expression in the pathogenesis of thrombocytopenia in pediatric patients suffering from liver failure, we measured hepatic TPO mRNA in children with acute or chronic end–stage liver disease undergoing orthotopic liver transplantation. Tissue samples for RNA extraction were obtained from 12 children with compensated cirrhosis (CC), 22 children with decompensated cirrhosis (DC), and 9 children with acute liver failure (ALF). TPO mRNA was quantitated by competitive polymerase chain reaction (PCR), following reverse transcription (RT). Furthermore, in 9 children with ALF, serum TPO levels were measured by enzyme–linked immunosorbent assay before and 10 to 14 days after liver transplantation. The hepatic TPO mRNA concentration was highest in children with CC (median, 50.9 amol/μg RNA). This value was significantly reduced in children with DC (30.2 amol/μg RNA) or ALF (13.8 amol/μg RNA). Children with ALF (139 cells/nL) or DC (200 cells/nL) had lower platelet counts than children with CC (368 cells/nL). The serum TPO concentration increased from a median of 156 pg/mL in patients with ALF to 547 pg/mL after liver transplantation. These results show that the thrombocytopenia in children with liver failure is associated with reduced hepatic TPO mRNA levels. It remains to be investigated whether the serum TPO level and platelet counts are markers for the severity of liver damage that may serve as a prognostic indicator.
Development of A New, Simple Rat Model of Early Alcohol–Induced Liver Injury Based on Sensitization of Kupffer Cells
Authors: Enomoto Nobuyuki , Yamashina Shunhei , Kono Hiroshi , Schemmer Peter , Rivera Chantal A. , Enomoto Ayako , Nishiura Teruhiro , Nishimura Tetsuro , Brenner David A. , Thurman Ronald G.
Published: July 2004

The continuous intragastric in vivo enteral feeding model in the rat developed by Tsukamoto and French has been very useful; however, it requires surgical expertise. Recently, we found that Kupffer cells isolated from rats treated only once with ethanol were sensitized to endotoxin 24 hours later. Accordingly, these experiments were designed to determine if a new, simple animal model of ethanol hepatotoxicity could be developed based on Kupffer cell sensitization. Female Wistar rats were given ethanol (5 g/kg body weight) once every 24 hours intragastrically. Livers were stained with hematoxylin–eosin to assess steatosis, inflammation, and necrosis, and tissue triglycerides, serum transaminases, and plasma endotoxin were measured. Kupffer cells were isolated 0 to 24 hours after one intragastric dose of ethanol daily, and intracellular Ca2+ ([Ca2+]i) was measured using fura–2, while tumor necrosis factor α (TNF–α) was measured by enzyme–linked immunosorbent assay. CD14 was evaluated by Western and Northern analysis. Ethanol caused steatosis, necrosis, and inflammation in only a few weeks, and after 8 weeks, serum aspartate transaminase (AST) levels were doubled. Values were similar to levels achieved in the enteral feeding model. Triglycerides were also increased significantly by ethanol as expected, and endotoxin levels were increased to 70 to 80 pg/mL. This latter increase was prevented (<20 pg/mL) by antibiotics implicating endotoxin. In isolated Kupffer cells from untreated control rats, [Ca2+]i increased to 82 ± 7 nmol/L after addition of lipopolysaccharide (LPS) (100 ng/mL), and levels were elevated about twofold by ethanol given 24 hours earlier (174 ± 15 nmol/L). In addition, TNF–α production by Kupffer cells was increased fourfold in cells isolated from rats treated with ethanol 24 hours earlier. Sterilization of the gut with antibiotics blocked all effects of ethanol on [Ca2+]i and TNF–α release completely. Moreover, 4 weeks after ethanol, CD14 in Kupffer cells was elevated about twofold. A new, simple chronic model of ethanol hepatotoxicity has been developed here based on sensitization of Kupffer cells to endotoxin
Expression of Delta F508 Cystic Fibrosis Transmembrane Conductance Regulator Protein and Related Chloride Transport Properties in the Gallbladder Epithelium From Cystic Fibrosis Patients
Authors: Dray-Charier Nathalie , Paul Annick , Scoazec Jean-Yves , Veissière Danielle , Mergey Martine , Capeau Jacqueline , Soubrane Olivier , Housset Chantal
Published: July 2004

Cystic fibrosis transmembrane conductance regulator (CFTR), the cystic fibrosis (CF) gene product, functions as an adenosine 3′,5′–cyclic monophosphate (cAMP)–regulated chloride channel in the apical membrane of biliary epithelial cells, including gallbladder epithelial cells. It has been shown that △F508, the most common CF mutation, impedes CFTR trafficking to the apical surface of epithelial cells. To elucidate the mechanisms of CF biliary disease, we examined structural features, CFTR expression, and chloride transport properties in gallbladder epithelial cells from nine △F508 homozygous liver transplant recipients. Three CF patients had microgallbladders, characterized by severe histological abnormalities. Microgallbladder epithelial cells displayed aberrant immunolocalization of CFTR and of other normally apical proteins in the lateral domain of their plasma membrane and in their cytoplasm. This pattern was mimicked by chronic cholecystitis in non–CF patients. In the 6 remaining CF patients, CFTR was predominantly apical in the gallbladder epithelium, consistent with the detection of a fully glycosylated form by Western blot. In CF as compared with non–CF gallbladder epithelial cells in primary culture, chloride efflux was lower in response to cAMP and tended to be higher in response to exogenous adenosine 5′–triphosphate (ATP). The CF cells exhibited a residual cAMP–dependent chloride secretion that was inversely correlated with ATP–induced chloride secretion, and almost completely blunted in the cells derived from microgallbladders. Our results suggest that epithelial structural alterations aggravate △F508 CFTR mislocalization in the gallbladder epithelium. The associated decrease in residual cAMP–dependent chloride secretion may contribute to biliary damage despite the up–regulation of alternative chloride transport pathways.
Failure to Detect Genetic Alteration of the Mannose–6–Phosphate/Insulin–Like Growth Factor 2 Receptor (M6p/Igf2r) Gene in Hepatocellular Carcinomas in Japan
Authors: Wada Ikuo , Kanada Hiroaki , Nomura Kimie , Kato Yo , Machinami Rikuo , Kitagawa Tomoyuki
Published: July 2004

The mannose–6–phosphate/insulin–like growth factor 2 receptor (M6P/IGF2R) suppresses cell growth through binding to the insulin–like growth factor 2 (IGF2) and latent complex of the transforming growth factor–β (TGF–β). Recently, it was reported in the United States that loss of heterozygosity (LOH) and mutations in exons 27, 28, and 31 of the M6P/IGF2R gene are frequent in hepatocellular carcinomas (HCCs) and adenomas. In view of the possible importance of this finding, especially for differential diagnosis of small hepatic lesions, we analyzed 43 primary HCCs, 2 adenomatous hyperplasias (AHs), and 3 regenerative nodules (RNs) developing in 42 Japanese patients in Japan for LOH using the polymorphic locus and for mutations by both single strand conformation polymorphism (SSCP) and direct sequencing methods. In the LOH study, 21 out of 22 informative HCCs and all of the informative AHs and RNs showed no allelic loss. In mutational studies of exons 27, 28, and 31, no mutations were detected either by SSCP or direct sequencing analysis in any of the 48 lesions. Thus inactivation of the M6P/IGF2R gene because of genetic alteration does not appear to be essential for hepatocarcinogenesis in Japan.
Association of Clonally Expanded T Cells With the Syndrome of Primary Biliary Cirrhosis and Limited Scleroderma
Authors: Mayo Marlyn J. , Jenkins Robert N. , Combes Burton , Lipsky Peter E.
Published: July 2004

Clinical features of the CREST (calcinosis cutis, Raynaud's syndrome, esophageal dysmotility, sclerodactyly, and telangiectasias) syndrome are sometimes exhibited in patients with primary biliary cirrhosis (PBC), but the postulated autoimmune mechanisms behind these conditions are poorly understood. Clonally expanded T cells may play an important role in disease pathogenesis. In this study, overrepresentation of one T–cell receptor beta chain variable region, TCRBV3, was documented in patients with PBC and/or CREST. Overrepresentation of the TCRBV3 gene mRNA was demonstrated by semiquantitative reverse–transcriptase polymerase chain reaction (RT–PCR). T cells expressing TCRBV3 were analyzed by flow cytometry, were primarily CD8+, and contained activated cells as assessed by expression of CD69. Clonally expanded T cells within this population were documented by both complementarity determining region 3 (CDR3) length polymorphism analysis and sequencing of T–cell receptor CDR3 cDNA. TCRBV3+ clonal expansions were stable when followed for up to 5 years. The results of this study demonstrate that the T–cell repertoire of patients with PBC and CREST is characterized by expanded clonal populations of CD8+ TCRBV3+ T cells. These clonal expansions provide evidence that stimulation of clonal populations of CD8+ T cells is associated with the clinical syndrome of PBC with CREST.
Pretreatment of mice with macrophage inactivators decreases acetaminophen hepatotoxicity and the formation of reactive oxygen and nitrogen species
Authors: Michael Sherryll L. , Pumford Neil R. , Mayeux Philip R. , Niesman Michael R. , Hinson Jack A.
Published: July 2004

Hepatotoxic doses of acetaminophen to mice produce not only acetaminophen-protein adducts in the centrilobular cells of the liver, but nitrotyrosine-protein adducts in the same cells, the site of the necrosis. Nitration of tyrosine occurs with peroxynitrite, a species formed by reaction of nitric oxide (NO·) with superoxide (O2
·−). Because NO· and O2
·− may be produced by activated Kupffer cells and/or infiltrated macrophages, we pretreated mice with the macrophage inactivators/depeleters gadolinium chloride (7 mg/kg, intravenously [iv]) or dextran sulfate (10 mg/kg, iv) 24 hours before administration of acetaminophen (300 mg/kg). Mice treated with acetaminophen plus gadolinium chloride, or acetaminophen plus dextran sulfate, had significantly less evidence of hepatotoxicity as evidenced by lower serum alanine transaminase (ALT) levels (28 ± 1 IU/L and 770 ± 240 IU/L, respectively) at 8 hours compared with acetaminophen (6,380 ± 408 IU/L). Analysis of hepatic homogenates for acetaminophen-protein adducts at 2 hours, a time of maximal covalent binding and before hepatocyte lysis, indicated that these pretreatments did not decrease covalent binding. Western blot analysis for the macrophage marker protein F4/80 in homogenates revealed not only the expected decrease by the macrophage inactivators/depleters, but also an apparent increase in acetaminophen-only-treated mice. At 8 hours nitrotyrosine-protein adducts were present in the acetaminophen-only-treated mice, but not in the acetaminophen plus gadolinium chloride-treated mice, or acetaminophen plus dextran sulfate-treated mice. High levels of heme-protein adducts, a measure of oxidative stress, were detected in livers of the 8 hour acetaminophen-only-treated mice. These data suggest that acetaminophen hepatotoxicity is mediated by an initial metabolic activation and covalent binding, and subsequent activation of macrophages to form O2
·−, NO·, and peroxynitrite. Nitration of tyrosine correlates with toxicity.
Long-term ursodeoxycholic acid therapy is associated with reduced risk of biliary pain and acute cholecystitis in patients with gallbladder stones: A cohort analysis
Authors: Tomida Shinji , Abei Masato , Yamaguchi Takashi , Matsuzaki Yasushi , Shoda Junichi , Tanaka Naomi , Osuga Toshiaki
Published: July 2004

Whether ursodeoxycholic acid (UDCA) therapy alters the long-term clinical course of gallstones (GS) without stone dissolution remains unknown. We aimed to clarify the relationship between long-term UDCA therapy and risks of biliary pain or acute cholecystitis in GS patients. We also aimed to identify factors affecting the natural course, and to explore a simple patient selection criteria for UDCA therapy. A cohort of 527 uncomplicated GS patients with or without UDCA (600 mg/d) followed for up to 18 years was analyzed. Patients who had frequent attacks or were complicated with cholecystitis were converted to cholecystectomy. History and UDCA therapy were identified on Cox analysis as 2 factors affecting the long-term clinical course. In patients without therapy, history was the only predictor of biliary pain among various patient or stone characteristics; biliary pain was rare in asymptomatic patients, while frequent in symptomatic patients (P< .001). UDCA therapy was associated with reduced risk for biliary pain in both symptomatic (62% vs. 92% in untreated patients at 10 years;P< .001; relative risk, 0.19; 95% CI, 0.10-0.34) and asymptomatic patients (6% vs. 12% in untreated patients at 10 years;P= .037; relative risk, 0.19; 95% CI, 0.04-0.91). Risk for the conversion was also reduced in UDCA-treated symptomatic patients (26% vs. 88% in untreated patients at 10 years,P< .001; relative risk, 0.08; 95% CI, 0.03-0.22). These effects were independent of stone dissolution. Three factors were identified on Cox analysis as affecting GS dissolution: radiolucency, small size (<10 mm) of stones, and visualized gallbladder (GB) on cholecystogram. A selection criteria based on these appears to exhibit high sensitivity (74%) and specificity (95%) for dissolution. UDCA therapy might be considered in symptomatic patients fulfilling these criteria, and also in patients who have significant surgical risk, because the longterm therapy is clearly associated with reduced risk of biliary pain and acute cholecystitis
The long-term course of chronic hepatitis B
Authors: Di Marco Vito , Iacono Oreste Lo , Cammà Calogero , Vaccaro Alessandra , Giunta Marco , Martorana Giuseppe , Fuschi Patrizia , Almasio Piero L. , Craxì Antonio
Published: July 2004

The aim of this study was to assess the long-term outcome in hepatitis B virus (HBV)-infected patients according to HBV, hepatitis C virus (HCV), and hepatitis D virus (HDV) replication, focusing on survival, liver failure, and hepatocellular carcinoma (HCC). A cohort of 302 hepatitis B surface antigen (HBsAg)-positive subjects (mean age, 34 ± 15.3 years; male/female 214/88; 39 subjects under 14 years) with biopsy-proven chronic hepatitis (86 with cirrhosis) was prospectively assessed, with a median follow-up of 94 ± 37.6 months. One hundred nine patients received interferon alfa (IFN). At baseline, 86 subjects (28.5%) were hepatitis B e antigen (HBeAg)-positive (wild-type HBV), 80 (26.5%) were HBeAg-negative, HBV-DNA-positive, 76 (25.2%) had HDV infection, 43 (14.2%) had dual HBV/HCV infection, and 17 (5.6%) were negative for HBV-DNA, anti-HCV, and anti-HDV. During follow-up, decompensation of disease occurred in 46 subjects: 8 developed HCC, 36 developed ascites, and 2 developed jaundice. Five patients underwent transplantation. Thirty-five subjects died: 33 of hepatic and 2 of nonhepatic causes. Overall mortality was 5.2-fold that of the general population (95% CI: 3.6-7.3; 35 deaths observed, 6.7 expected;P< .0001). By Cox regression analysis, survival was independently predicted by young age, absence of cirrhosis at baseline, and sustained normalization of aminotransferases during follow-up. Survival without decompensation was independently predicted by the same factors and by IFN treatment. Chronic hepatitis B infection increases mortality in comparison with the general population in our area regardless of specific virological profiles at presentation. Presence of cirrhosis and persistent necroinflammation markedly increase the risk of death.
Carbon monoxide-mediated alterations in paracellular permeability and vesicular transport in acetaminophen-treated perfused rat liver
Authors: Mori Mikiji , Suematsu Makoto , Kyokane Takanori , Sano Tsuyoshi , Suzuki Hidekazu , Yamaguchi Tokio , Ishimura Yuzuru , Ishii Hiromasa
Published: July 2004

This study aimed to examine whether acetaminophen (AAP), an anti-inflammatory agent producing hepatocellular damages with its overdose, evokes hepatocellular dysfunction through mechanisms involving carbon monoxide (CO) generated by heme oxygenase (HO). In perfused rat livers, CO and bilirubin were determined in venous perfusate and bile samples as indices of heme degradation. Biliary excretion of transportally injected horseradish peroxidase was also determined to assess paracellular junctional permeability and vesicular transport across hepatocytes. AAP at 20 mmol/L induced a transient choleresis, followed by a reduction of bile output. Under these circumstances, the release of CO and bilirubin IXα, terminal products of the HO-mediated heme degradation, became 2.5-fold greater than the control. The rate of CO production appeared stoichiometric to the degradation rate of microsomal cytochrome P-450. Mechanisms for the AAP-induced cholestasis involved an increase in the junctional permeability that coincided with a reduction of vesicular transport across hepatocytes. Clotrimazole, a cytochrome P-450 inhibitor, or zinc protoporphyrin IX, an HO inhibitor, but not copper protoporphyrin IX, which did not inhibit HO, attenuated these AAP-induced changes. Furthermore, administration of CO at concentrations comparable with those induced by AAP elicited a marked elevation of the paracellular junctional permeability concurrent with a reduction of transcellular vesicular transport, mimicking effects of the AAP administration. Thus, CO serves as a putative regulator of hepatocellular function that is overproduced through acute heme degradation during xenobiotic transformation
CD95-mediated murine hepatic apoptosis requires an intact glutathione status
Authors: Hentze Hannes , Künstle Gerald , Volbracht Christiane , Ertel Wolfgang , Wendel Albrecht
Published: July 2004

Agonistic engagement of the cytokine receptor CD95 in mice leads to activation of hepatic caspases, followed by massive hepatocyte apoptosis, acute liver failure, and death. This mechanism of cell death is thought to be associated with several human liver disorders. Because hepatic glutathione represents the major defense against toxic liver injury, we investigated its role in CD95-mediated liver failure, which represents a model for hyperinflammatory organ destruction. As a tool for modulating the liver glutathione status of mice in vivo, we used the GSH transferase substrate, phorone, which rapidly depleted hepatic glutathione in a dose-dependent manner. When GSH was depleted, CD95-initiated hepatic caspase-3-like activity and DNA fragmentation were completely blocked, and animals were protected from liver injury dose-dependently as assessed by histological examination and determination of liver enzymes in plasma. Conversely, repletion of hepatic glutathione by treatment with the permeable glutathione monoethylester restored susceptibility of GSH-depleted mice toward CD95-mediated liver injury. In contrast, the antioxidants, GSH, N-acetyl cysteine, α-tocopherol, butyl-hydroxytoluene, and catalase failed to do so. Animals treated once with phorone survived for more than 3 months after an otherwise lethal injection of the activating anti-CD95 antibody. We investigated the thiol sensitivity of recombinant caspase-3 in vitro and observed that its activity was dependent on the presence of a reducing agent such as GSH, while GSSG attenuated proteolytic activity. Based on our finding that CD95-mediated hepatocyte apoptosis requires an intact intracellular glutathione status, we propose that the activation of apoptosis-executing caspases is controlled by reduced glutathione.
Interferon alfa for chronic hepatitis B infection: Increased efficacy of prolonged treatment
Authors: Janssen Harry L. , Gerken Guido , Carreño Vicente , Marcellin Patrick , Naoumov Nikolai V. , Craxi Antonio , Ring-Larsen Helmer , Kitis George , van Hattum Jan , de Vries Richard A. , Michielsen Peter P. , ten Kate Fiebo J. , Hop Wim C. , Heijtink Rudolf A. , Honkoop Pieter , Schalm Solko W.
Published: July 2004

Interferon alfa (IFN-α) is the primary treatment for chronic hepatitis B. The standard duration of IFN-α therapy is considered 16 weeks; however, the optimal treatment length is still poorly defined. We evaluated the efficacy and acceptability of prolonged IFN-α treatment in patients with chronic hepatitis B. To investigate whether treatment prolongation could enhance the rate of hepatitis B e antigen (HBeAg) seroconversion, we conducted a prospective, controlled, multicenter trial in which all patients were treated with a standard regimen of 10 million units IFN-α 3 times per week over 16 weeks. Patients who were still HBeAg-positive after 16 weeks of therapy were randomized to prolongation of the identical regimen up to 32 weeks (prolonged therapy) or discontinuation of treatment (standard therapy). Among the 162 patients who entered the study, 27 (17%) were HBeAg-negative after the first 16 weeks of treatment, and 118 were randomized to standard or prolonged therapy. After randomization, a response (HBeAg seroconversion and sustained hepatitis B virus [HBV]-DNA negativity) was observed in 7 of the 57 (12%) patients assigned to standard therapy versus 17 of the 61 (28%) patients assigned to prolonged therapy (P = .04). A low level of viral replication after 16 weeks of treatment, as indicated by serum HBV-DNA values under 10 pg/mL, was found to be the only independent predictor of response (52% vs. 0%; P < .001) during prolonged therapy. The prolonged IFN-α schedule was well tolerated in the large majority of patients. In chronic hepatitis B, prolongation of IFN-α therapy up to 32 weeks is superior to a standard course of 16 weeks. Those patients who exhibit a low level of viral replication at the end of the standard regimen benefit most from prolonged treatment.