bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantitative Antibody Responses to Structural (Core) and Nonstructural (Ns3, Ns4, and Ns5) Hepatitis C Virus Proteins Among Seroconverting Injecting Drug Users: Impact of Epitope Variation and Relationship to Detection of Hcv Rna in Blood
Authors: Beld Marcel , Penning Maarten , van Putten Marieke , Lukashov Vladimir , van den Hoek Anneke , McMorrow Martin , Goudsmit Jaap
Published: July 2004

To gain insight into the natural history of hepatitis C virus (HCV), 13 human immunodeficiency virus (HIV)–seronegative injecting drug users were studied who seroconverted for HCV as determined by third–generation enzyme–linked immunosorbent assay, showed an ensuing antibody response to HCV, and were not treated with any antiviral drugs during follow–up. Subjects included 13 untreated HIV–negative individuals, of whom 5 (38.5%) apparently cleared HCV and were polymerase chain reaction (PCR)–negative in at least 3 consecutive samples, 3 (23.1%) showed transient viremia and were PCR–negative in 1 sample during the study period, and the other 5 (38.5%) showed persistent viremia. Viremia was determined longitudinally by reverse–transcription PCR (RT–PCR) and quantified by branched DNA (bDNA). HCV genotypes were determined on serial samples during follow–up. Quantitative antibody levels to core, NS3, NS4, and NS5 were determined using the Chiron RIBA HCV–titering Strip Immunoblot Assay, which is based on HCV genotype 1. The antibody responses to core, NS3, NS4, and NS5 were erratic. In individuals infected with HCV genotype 1, significantly higher median antibody responses to core (P = .02) and to NS4 (P = .04) were found as compared with those infected with other genotypes, showing a significant impact of HCV genotype specificity of the assay. In groups infected with HCV genotype 1, significantly higher median NS3 antibody titers (2.61 relative intensity [RI] vs. 0.38 RI; P = .003) were found in the individuals with persistent viremia than in those with apparent resolution of HCV RNA in blood. In groups infected with genotypes other than genotype 1, significantly higher median NS3 antibody titers (0.89 RI vs. 0.03 RI; P = .0004) and NS5 antibody titers (1.86 RI vs. 0.01 RI;P = .006) were found in the individuals with persistent viremia than in those with apparent resolution of HCV RNA in blood. Individuals with viral persistence had higher HCV–RNA loads with higher antibody responses as compared with individuals with apparent viral clearance from blood. Apparent viral clearance from blood was observed in an unexpectedly high percentage (38.5%), associated with a significant decrease of antibodies to NS3, independent of HCV genotype, as compared with individuals with persistent viremia (P < .005). Apparent viral clearance from blood with gradual loss of antibodies to various HCV proteins, independent of HCV genotype, was observed in 4 of the 5 individuals within approximately 1 year after HCV seroconversion, whereas 1 of these individuals apparently cleared the virus from blood, with complete seroreversion.
Gap Junctional Communication and Regulation of the Glycogenic Response to Insulin by Cell Density and Glucocorticoids in Cultured Fetal Rat Hepatocytes
Authors: Siddiqui Mashiat U. , Benatmane Samia , Zachayus Jean-Luc , Plas Christiane
Published: July 2004

Cell culture studies have revealed that metabolic functions of the adult hepatocyte are related to cell density. Development of the glycogenic response to insulin under glucocorticoid control was investigated in 15– and 18–day–old fetal rat hepatocytes plated at different cell densities. After culturing for 48 hours with glucocorticoids, the stimulatory effect of insulin on [14C]glucose incorporation into glycogen after 3 hours progressed from weak response (less than 1.4–fold) in sparse cultures to a maximal response in dense ones (3.0– to 4.5–fold), depending on the fetal stage. The response was always no more than 2.0–fold in the absence of glucocorticoids, even with dense cultures. Such a dual regulation pattern was not found for the glycogenolytic effect of glucagon similarly expressed regardless of culture conditions. When cells were clustered in limited circular regions of the dish, the insulin response was higher than for sparse cultures for a similar number of cells per culture. Using the scrape–loading technique with Lucifer Yellow CH, a positive dye transfer was obtained in clustered cultures providing that they were grown in the presence of glucocorticoids; insulin as well as glucagon stimulated twofold intercellular communication. Connexin32 (Cx32) and connexin26 (Cx26) protein levels were assayed by Western immunoblotting and developed according to age and exposure to glucocorticoids. Thus, glucocorticoids through development of gap junctions enabled establishment of intercellular communication that could be stimulated by insulin and glucagon in cultured fetal hepatocytes. Gap junction functioning and the biologic effect of insulin correlated closely
Histopathological Study of Primary Biliary Cirrhosis and the Effect of Ursodeoxycholic Acid Treatment on Histology Progression
Authors: Degott Claude , Zafrani Elie Serge , Callard Patrice , Balkau Beverley , Poupon Renée Eug , Poupon Raoul
Published: July 2004

The semiquantitative histopathological analysis of the liver biopsies obtained before and after 4 years of ursodeoxycholic acid (UDCA) therapy in a cohort of primary biliary cirrhosis (PBC) patients is reported. The relationships between elementary histological lesions before treatment and their progression under therapy were assessed. At baseline, two independent groups of lesions, each of which participate in the development of fibrosis, were individualized, i.e., florid bile duct lesions and ductopenia on one hand and lymphocytic piecemeal necrosis, ductular proliferation, and lobular necroinflammatory changes on the other hand. Four years of UDCA therapy were associated with a significant decrease in the prevalence of florid interlobular bile duct (ILBD) lesions, of epithelioid granuloma (P < .001) without any aggravation in the severity of bile duct paucity. Lobular inflammation and necrosis markedly improved (P < .001) whereas the degree of severity of the lymphocytic piecemeal necrosis and ductular proliferation at entry and at 4 years were similar. Worsening of fibrosis was observed in 14 patients (12 of them had a one grade progression) whereas stabilization was noted in 30 of the remaining patients. Severity of both the lymphocytic piecemeal necrosis and lobular inflammation and necrosis at entry was significantly associated with the progression of fibrosis. The results suggest that UDCA therapy influences the process leading to bile duct destruction. Patients with severe lymphocytic piecemeal necrosis and lobular inflammation may need additional therapeutic intervention because they have increased risk of fibrosis progression
Comparison of Immune Reactivity and Pharmacokinetics of Two Hepatitis B Immune Globulins in Patients After Liver Transplantation
Authors: Adler Ruth , Safadi Rifaat , Caraco Yoseph , Rowe Mina , Etzioni Amos , Ashur Yaffa , Shouval Daniel
Published: July 2004

Hepatitis B virus (HBV) immune globulin (HBIg) administration will prevent HBV graft reinfection in HBV patients after orthotopic liver transplantation (OLT). However, the expenditure for such prophylaxis is extremely high ranging between $2,000 to $10,000 per month in various countries for an undefined period and presumably for life. As a consequence, there is a need for introduction of additional and less expensive modes of treatment. In a preliminary clinical trial a new HBIg preparation has been shown to induce longer lasting levels of circulating antibodies to hepatitis B surface antigen (anti–HBs) in patients after OLT compared with previous experience with conventional HBIg preparations. In the present study the pharmacokinetics of this new HBIg, OMRI–Hep–B, were studied and compared with a conventional, licensed preparation, Hepatect. Fifteen post–OLT patients (2–8 years post–OLT, 18–62 years of age, 6 men, 9 women) were treated intravenously with 49 doses of OMRI–Hep–B or Hepatect given at least once, alternately, at 10,000 to 14,000 units per injection (≈130 IU/kg body weight). The new HBIg was well tolerated and no adverse effects were observed. Administration of OMRI–Hep–B was shown to induce high and long–lasting levels of circulating anti–HBs antibodies and greater areas under the curve (AUC) compared with the conventional preparation. Thus, anti–HBs half–life was 22 ± 1.3 days for OMRI–Hep–B recipients and 13 ± 1.3 days for Hepatect recipients (P< .001). Time to reach trough anti–HBs levels of 150 mIU/mL was significantly longer after administration of OMRI–Hep–B than after Hepatect (79 ± 4.5 and 52 ± 3.8 days, respectively;P< .001). In summary, the pharmacokinetic profile of the new HBIg, and in particular its prolonged elimination half–life, may reduce the cost of administration by approximately 30% and improve the quality of life of patients by extending the interval between repeated immune globulin injections.
After Portal Branch Ligation in Rat, Nuclear Factor κB, Interleukin–6, Signal Transducers and Activators of Transcription 3, C– Fos , C– Myc , and C– Jun Are Similarly Induced in the Ligated and Nonligated Lobes
Authors: Stärkel Peter , Horsmans Yves , Sempoux Christine , De Saeger Christine , Wary Johan , Lause Pascale , Maiter Dominique , Lambotte Luc
Published: July 2004

Several studies have emphasized the involvement of transcription factors, cytokines, and proto–oncogenes in initiating the regenerative process after partial hepatectomy. To assess whether these events do specifically occur in a cellular system undergoing regeneration, we studied the induction of nuclear factor κB (NFκB), interleukin–6 (IL–6), signal transducers and activators of transcription 3 (Stat3), c–fos, c–myc, c–jun, after portal branch ligation (PBL), which produces atrophy of the deprived lobes (70% of the liver parenchyma), whereas the perfused lobes undergo compensatory regeneration. Nuclear extracts and total RNA were prepared from control livers as well as from atrophying and regenerating lobes at 0.5, 1, 2, 5, and 8 after PBL. NFκB and Stat3 induction were studied by electrophoretic mobility shift assays and Western blotting. IL–6 and proto–oncogenes expressions were assessed by reverse transcription polymerase chain reaction and Northern blotting, respectively. Assays were also performed after a sham operation. NFκB and Stat3 protein expression and DNA binding were rapidly and similarly induced in nuclear extracts from the atrophying and regenerating lobes. IL–6 was elevated in both lobes from 1 to 8 hours after PBL as well as c–fos, c–myc, and c–jun during the first 2 hours. IL–6 and Stat3 but not NFκB were also elevated after a sham operation. These findings suggest that the cellular and molecular changes occurring early in a regenerating liver are nonspecific, possibly stress–induced, cellular responses. They do not indicate the future evolution towards atrophy or regeneration.
Differential Expression of Apolipoprotein E Messenger Rna Within the Rat Liver Lobule Determined By In Situ Hybridization
Authors: Massimi Mara , Lear Steven R. , Williams David L. , Jones Albert L. , Erickson Sandra K.
Published: July 2004

Apolipoprotein (Apo) E plays a key role in the metabolism of lipoproteins. It also modulates immunoregulation, cell growth and differentiation and the response to nerve injury. The liver is a major site of ApoE synthesis. Most of the circulating ApoE is thought to be of hepatic origin with most synthesized in hepatocytes. We showed that total liver ApoE messenger RNA (mRNA) levels were greater in normal adult female rats than in male and that gender–specific patterns of liver ApoE mRNA expression were present by in situ hybridization. In the male liver, the signal was strongest in the portal area, decreasing toward the central vein with the weakest signal in pericentral hepatocytes, resulting in a hepatic lobular gradient of expression. In female liver, a strong periportal signal also was observed that decreased in Zone 2, similar to that in males, but which then increased in pericentral hepatocytes resulting in a bowl–like distribution in marked contrast with that of the male. The results suggest that ApoE mRNA level is regulated differentially in hepatocytes within the liver plate and that the regulation is gender–dependent. Further, the results suggest that in males, hepatocytes in the portal area are the major contributors of ApoE to the plasma and/or sinusoidal pool, whereas in females, both portal and central area hepatocytes play an equal role.
P–Selectin Mediates Reperfusion Injury Through Neutrophil and Platelet Sequestration in the Warm Ischemic Mouse Liver
Authors: Yadav Surinder S. , Howell David N. , Steeber Douglas A. , Harland Robert C. , Tedder Thomas F. , Clavien Pierre-Alain
Published: July 2004

Hepatic damage following ischemia–reperfusion injury involves polymorphonuclear leukocytes (PMN) and platelet sequestration, however the mechanisms of adhesion remain elusive. In this study, using gene–targeted deficient mice, we evaluated P–selectin and its contribution to PMN and platelet adhesion in hepatic damage. In an in vivo warm ischemia model, hepatic injury was assessed by serum transaminase levels, survival, PMN adhesion by histological analysis, and platelet sequestration by immunostaining. Serum transaminase levels were strikingly reduced (by up to threefold) in the P–selectin deficient mice, particularly at 90 minutes of ischemia, when compared with wild–type controls. PMN adhesion and platelet sequestration was also significantly decreased in P–selectin deficient mice following 90 minutes of partial ischemia. Animal survival was significantly improved after 75 minutes of total hepatic ischemia in P–selectin deficient mice when compared with wild–type mice. Survival was also achieved after 90 minutes of ischemia in the mutant mice whereas none of the wild–type animals survived. These data show that P–selectin plays a critical role in PMN and platelet adhesion following ischemia–reperfusion injury to the liver.
Effect of Chronic Coadministration of Endotoxin and Ethanol on Rat Liver Pathology and Proinflammatory and Anti–Inflammatory Cytokines
Authors: Järveläinen Harri A. , Fang Che , Ingelman-Sundberg Magnus , Lindros Kai O.
Published: July 2004

To better understand how gut–derived endotoxins influence alcohol–induced liver injury and the expression of inflammatory cytokines a new animal model was developed. After 2 weeks on a modified ethanol–containing liquid diet, some rats also were infused with endotoxin via osmotic minipumps for 4 additional weeks. Ethanol diet alone increased plasma endotoxin threefold to 9.3 pg/mL. Endotoxin infusion increased the levels to 388 and 513 pg/mL in controls and ethanol–fed animals, respectively. Panlobular macrovesicular and microvesicular steatosis and inflammatory foci were observed in livers from both ethanol– and ethanol–endotoxin-treated animals, but there was no significant potentiation by endotoxin. Only minor changes, mainly polymorphonuclear infiltration, were seen in animals treated with endotoxin alone although the messenger RNA (mRNA) expression of both proinflammatory cytokines tumor necrosis factor α (TNF–α), interleukin 1β (IL–1β) and anti–inflammatory cytokines IL–4 and IL–10 were markedly increased, as shown by competitive polymerase chain reaction (PCR) analysis using cyclophilin as standard. The effect of endotoxin infusion on cytokine mRNA expression in ethanol–fed animals was not significantly different. Expression of transforming growth factor β1 (TGF–β1 ) mRNA was increased twofold by ethanol, eightfold by endotoxin, but only threefold by ethanol–endotoxin treatment. The mRNA expression of lipopolysaccharide binding protein (LBP) and CD14 endotoxin receptor was not significantly increased by chronic endotoxin treatment, contrasting with the marked elevation observed after acute endotoxin challenge. These results suggest that the tolerance observed despite sustained hepatic expression of proinflammatory cytokines is counteracted by the anti–inflammatory cytokines and by down–regulation of CD14 and LBP. Furthermore, a similar adaptation may occur in alcoholics with continuous endotoxemia.
Regulation of Rat Hepatocyte Protein Kinase C β Isoenzymes by the Lipid Peroxidation Product 4–Hydroxy–2,3–Nonenal: A Signaling Pathway to Modulate Vesicular Transport of Glycoproteins
Authors: Chiarpotto Elena , Domenicotti Cinzia , Paola Dimitri , Vitali Antonella , Nitti Mariapaola , Pronzato Maria A. , Biasi Fiorella , Cottalasso Damiano , Marinari Umberto M. , Dragonetti Antonella , Cesaro Patrizia , Isidoro Ciro , Poli Giuseppe
Published: July 2004

A major aldehydic end product of the peroxidation of arachidonic acid, 4–hydroxy–2,3–nonenal (HNE), has recently been considered for its potential involvement in a variety of cell functions. Here we report on the differential regulation of rat hepatocyte protein kinase C (PKC) isoforms by concentrations of HNE actually detectable in specific biological fluids or tissues. PKC βI and, to a much greater extent, PKC βII activities were markedly increased by 0.1 μmol/L HNE (final concentration in cell medium) whereas they were unaffected or even inhibited by 1 to 10 μmol/L HNE. On the contrary, the calcium independent PKC δ activity was inhibited by 0.1 μmol/L and increased by 1 and 10 μmol/L. Further, we show here that HNE–induced stimulation of PKC βI and βII activities, both in cytosolic and in membrane fractions, is paralleled by a marked stimulation of the anterograde transport of a lysosomal enzyme within the central vacuolar system. In fact, the treatment with 0.1 μmol/L HNE accelerated the PKC–dependent transport of lysosomal procathepsin D from the trans–Golgi network to the endosomal–lysosomal compartment and, in addition, increased the exocytosis of mature cathepsin D (CD) from these compartments. On the other hand, hepatocyte cotreatment with a selective inhibitor of classic PKCs prevented the aldehyde–induced activation of CD transport. These results support the possible involvement of HNE in the PKC–dependent regulation of the traffic of secretory glycoproteins, and point to remarkable implications of this aldehyde in the pathophysiology of various exocytic processes including hepatocyte lipoprotein secretion.
Incidence of Cancer in Primary Biliary Cirrhosis: the Mayo Experience
Authors: Nijhawan Pardeep K. , Therneau Terry M. , Dickson Rolland E. , Boynton Jessica , Lindor Keith D.
Published: July 2004

Patients with primary biliary cirrhosis (PBC) may be at increased risk for malignancies. Several studies have addressed the risk of specific malignancies; however, there is little information about overall incidences of malignancies in these patients. We hypothesize that these patients may be at an increased risk for cancer. We performed a retrospective chart review evaluating patients with the diagnosis of PBC and malignancies. We reviewed records of patients with PBC presenting to the Mayo Clinic between 1976 and 1985. The diagnosis of PBC was made using evidence of cholestasis, positive antimitochondrial antibody titers and liver biopsy findings consistent with PBC. The incidence of malignancies were then compared with published data by the Surveillance, Epidemiology and End Results (SEER) program of the National Cancer Institute. Of the 1,692 patients with PBC in the Mayo Clinic data base, 114 patients were identified with primary cancer. The number of malignancies was higher than would be anticipated by chance alone; with 93 observed versus 62.4 expected events (P < .001). Hepatobiliary malignancies had a relative risk of 46 (P < .0001) for women and 55 (P < .0001) in men. There was a dramatic increased risk for development of hepatobiliary malignancies. PBC patients might benefit from more aggressive surveillance for hepatobiliary malignancies during their lifetime
Lipopolysaccharide From Escherichia Coli Stimulates Mucin Secretion by Cultured Dog Gallbladder Epithelial Cells
Authors: Choi JaeWoon , Klinkspoor Henri J. , Yoshida Tadashi , Lee Sum P.
Published: July 2004

Biliary infection is associated with mucin hypersecretion by the biliary epithelium. Mucins have been identified as potent pronucleators of cholesterol in bile. The aim of the present study was to determine whether lipopolysaccharides (LPS) from different bacteria are capable of stimulating mucin secretion by cultured dog gallbladder epithelial (DGBE) cells, and to investigate the mechanism by which LPS stimulate mucin secretion. Mucin secretion by confluent monolayers of DGBE cells was quantified by measuring the secretion of [3H]–N–acetyl–d–glucosamine-labeled glycoproteins. Cell viability was evaluated by measuring the leakage of the enzyme, lactate dehydrogenase (LDH), into the culture medium. LPS, derived from Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa (200 μg/mL), all caused an increase in mucin secretion by the DGBE cells, without causing concomitant cell lysis. LPS from E. coli was found to be the most potent stimulator of mucin secretion, and increased mucin secretion by the DGBE cells to 252% ± 14% of control. LPS from E. coli had no effect on intracellular cyclic adenosine monophosphate (cAMP) levels in the DGBE cells. Addition of the nitric oxide (NO)–releasing compound, NOR–4 (0.125–1 mmol/L), to the cells did not result in increased mucin secretion, and the NO synthase inhibitor, Nω
–nitro–l–arginine methyl ester (l–NAME) (4 or 10 mmol/L), did not inhibit the LPS–stimulated mucin secretion. Exogenous tumor necrosis factor α (TNF–α) (1–10 ng/mL) did cause a minor increase in mucin secretion by the DGBE cells, but the effect of LPS from E. coli on mucin secretion could not be inhibited by preincubation with a TNF–α antibody (10 μg/mL). We conclude that LPS stimulates mucin secretion by the gallbladder epithelium. Whether this stimulation is mediated by TNF–α remains to be determined.(Hepatology 1999;29:1352–1357.)
Inhibition of Rat Hepatocyte Proliferation by Transforming Growth Factor β and Glucagon Is Associated With Inhibition of Erk2 and P70 S6 Kinase
Authors: Dixon Mark , Agius Loranne , Yeaman Stephen J. , Day Christopher P.
Published: July 2004

Stimulation of hepatocyte proliferation by epidermal growth factor (EGF) and insulin is inhibited by transforming growth factor β (TGF–β) and by glucagon. It is also suppressed by inhibitors of various protein kinases, including rapamycin, which blocks activation of p70 S6 kinase (p70S6k), PD98059, which inhibits the activation of extracellular–regulated kinase (ERK), and SB 203580, an inhibitor of the p38 mitogen–activated protein kinase (p38 MAPK). In this study, we investigated whether the inhibition of proliferation by TGF–β involves these protein kinase cascades. Culture of hepatocytes with TGF–β for 16 hours decreased the stimulation by EGF of ERK2 and p70S6k (by 50% and 35%, respectively), but did not affect the stimulation of either p38 MAPK, c–jun NH2 –terminal kinase (JNK), or protein kinase B (PKB). Culture of hepatocytes with glucagon for 16 hours also inhibited the stimulation by EGF of activation of ERK2 and p70S6k (by ≈50%). The inhibitory effects of glucagon were observed when the hormone was added either 10 minutes or 60 minutes before EGF addition, whereas no effects of TGF–β were observed after 10–minute or 60–minute incubation. These results suggest that the inhibition of hepatocyte proliferation by TGF–β may be in part mediated by inhibition of ERK2 and p70S6k, but does not involve PKB, JNK, or p38 MAPK. Unlike glucagon, the effects of TGF–β are not elicited in response to short–term treatment
Contribution of Increased Mitochondrial Free Ca2+ to the Mitochondrial Permeability Transition Induced By Tert –Butylhydroperoxide in Rat Hepatocytes
Authors: Byrne Aaron M. , Lemasters John J. , Nieminen Anna-Liisa
Published: July 2004

Previously, we showed that the oxidant chemical, tert–butylhydroperoxide (t–BuOOH), induces a mitochondrial permeability transition (MPT) in intact hepatocytes, causing lethal cell injury. Here, we investigated the role of mitochondrial free Ca2+ in t–BuOOH cytotoxicity to 1–day–cultured rat hepatocytes using confocal microscopy of autofluorescence and parameter–indicating fluorophores. t–BuOOH (100 μmol/L) caused an early increase of mitochondrial free Ca2+, as assessed by confocal microscopy of Rhod–2 fluorescence. Increased mitochondrial Ca2+ was followed by onset of the MPT, as evidenced by permeation of cytosolic calcein into mitochondria and loss of the mitochondrial membrane potential-indicating dye, tetramethylrhodamine methylester. Preincubation with an intracellular Ca2+ chelator (BAPTA–AM and its derivatives) partially blocked the late phase of mitochondrial NAD(P)H oxidation after t–BuOOH, but failed to prevent the early oxidation of mitochondrial NAD(P)H. Ca2+ chelation also prevented the increase of mitochondrial Ca2+, generation of mitochondrial reactive oxygen species (ROS), onset of the MPT, and subsequent cell death. Confocal images showed that protection occurred when loading of the Ca2+ chelator was predominantly mitochondrial. The antioxidant, desferal, also diminished increased mitochondrial Ca2+ after t–BuOOH and prevented cell death. We conclude that oxidative stress induced by t–BuOOH enhances mitochondrial Ca2+ uptake, leading to increased matrix Ca2+, increased ROS formation, onset of the MPT, and cell death.
Determination of the Clonal Origin of Multiple Human Hepatocellular Carcinomas by Cloning and Polymerase Chain Reaction of the Integrated Hepatitis B Virus Dna
Authors: Yamamoto Toshiki , Kajino Kazunori , Kudo Masatoshi , Sasaki Yo , Arakawa Yasuyuki , Hino Okio
Published: July 2004

The poor prognosis of hepatocellular carcinoma (HCC) is partly the result of the high rate of recurrence that is caused either by intrahepatic metastasis (IM) or independent multicentric occurrence (MO). For convenience, discrimination of IM and MO is based on pathological findings, but reliable parameters are not sufficiently established. In the case of hepatitis B virus (HBV)–associated HCC, molecular discrimination of IM from MO can be achieved by comparison of integrated HBV DNAs. However, Southern blotting cannot be used for this purpose when one tumor is saved in frozen form and the other is in paraffin–embedded form. To solve this problem, we employed polymerase chain reaction (PCR) assays to confirm the clonality of primary and recurrent tumors. From the frozen tissue, we determined the junction between the integrated HBV and flanking genomic DNA by molecular cloning, and checked the existence of an identical junction in the DNA of paraffin–embedded tissue by PCR. Using this method, as well as Southern blotting, we proved in 6 of 8 patients that two nodular HCC lesions resected metachronously or simultaneously were caused by MO, while the remaining 2 cases were caused by IM. In 1 IM case, band patterns between two HCCs detected by Southern blotting were not identical
Overexpression of C–Neu and C–Met During Rat Liver Cholangiocarcinogenesis: A Link Between Biliary Intestinal Metaplasia and Mucin–Producing Cholangiocarcinoma
Authors: Radaeva Svetlana , Ferreira-Gonzalez Andrea , Sirica Alphonse E.
Published: July 2004

Based on limited but compelling immunohistochemical data demonstrating individual overexpression of the tyrosine kinase growth factor receptors, c–erbB–2 and c–met, in significant percentages of human cholangiocarcinoma (ChC), we investigated if combined overexpression of both c–neu, the rat homologue of c–erbB–2, and c–met, the receptor for hepatocyte growth factor/scatter factor (HGF/SF), might represent a characteristic, early event associated with furan–induced cholangiocarcinogenesis in rat liver. Specifically, through the use of immunohistochemistry,in situhybridization (ISH), and Western and Northern blotting, we found that both c–neu and c–met are prominently overexpressed in intestinal metaplastic lesions in early putative precancerous cholangiofibrotic tissue formed in the livers of rats after 6 weeks of furan treatment when compared with normal and hyperplastic intrahepatic biliary epithelia. We further demonstrated that c–neu and c–met are concordantly overexpressed in neoplastic glandular epithelia in later–developed primary “intestinal–type” of ChC formed in the livers of furan–treated rats, as well as in subsequently derived transplantable mucin–producing tumors. Overexpression of c–neu and c–met correlated with increased proliferating cell nuclear antigen (PCNA)–labeling indices, which were determined to be three to four times higher in intestinal metaplastic glands in precancerous cholangiofibrotic tissue and in neoplastic glands in the primary “intestinal type” of ChC than in hyperplastic bile ductular structures within either cholangiofibrotic or bile duct-ligated (BDL) livers. The c–neu and c–met receptor proteins overexpressed in differentin vivopassages of a transplantable ChC each contained immunoreactive phosphotyrosines, indicating an activated state. However, we did not detect evidence of either gene amplification of c–neu or c–met or of a common transmembrane–activating mutation in c–neu expressed in transplantable ChC. Our findings indicate that altered expression of c–neu and c–met occurs relatively early in the process of furan–induced cholangiocarcinogenesis in rat liver and may play a potentially important role in its pathogenesis. They further indicate a common alteration in tyrosine kinase growth factor receptor expression linking early putative precancerous intestinal metaplastic lesions in liver to later–developed mucin–producing biliary cancer.
Permissiveness of Human Biliary Epithelial Cells to Infection by Hepatitis C Virus
Authors: Loriot Marie-Anne , Bronowicki Jean-Pierre , Lagorce David , Lakehal Fatima , Persico Tiziana , Barba Giovanna , Mergey Martine , Vons Corinne , Franco Dominique , Belghiti Jacques , Giacca Mauro , Housset Chantal , Bréchot Christian
Published: July 2004

The cellular tropism of hepatitis C virus (HCV) is an important but much debated issue. Permissivity to HCV of biliary cells has never been demonstrated. In this context, we used gallbladder epithelial cells (GBEC) as a model of the more proximal biliary epithelium. These cells were isolated from HCV–positive and –negative individuals and cultured for up to 40 days. Biliary cells from HCV–negative subjects were infected in vitro with various inocula. The retention of GBEC functional characteristics was assessed by the expression of cystic fibrosis transmembrane conductance regulator (CFTR). All 12 GBEC tested from HCV–negative patients were successfully infected by HCV. This was assessed by: 1) the detection of HCV–RNA positive and negative strands; 2) the detection of the viral capsid by immunofluorescence; and 3) the combination of single–strand conformation polymorphism (SSCP) and HVR1 sequence analysis demonstrating the distinct majoritary HCV genomes in serum and in GBEC. The level of HCV RNA in cell extracts and supernatants was low, but HCV infection was highly reproducible. Our results expand those showing the cellular tropism of HCV, and demonstrate the sensitivity of biliary cells to HCV infection. This might have an important impact in terms of pathogenesis and pathological features of HCV infection. In addition, given the easy access to these cells and the high reproducibility of in vitro infection, they should constitute an important tool for studies aimed at analyzing the issue of HCV penetration and neutralizing antibodies.
Cloning and Functional Expression of A Mouse Liver Organic Cation Transporter
Authors: Green Richard M. , Lo Kathy , Sterritt Coreen , Beier David R.
Published: July 2004

Hepatic uptake of organic cations is essential for the metabolism and secretion of numerous endobiotics and drugs. Several hepatic organic cation transporters have been kinetically defined, yet have not been isolated or cloned. We have isolated a complementary DNA (cDNA) from both murine liver and kidney cDNA libraries (mOct1/Slc22a1), and have functionally expressed it in Xenopus laevis oocytes. Although mOct1/Slc22a1 is homologous to previously cloned rat and human organic cation transporters, organic cation transport kinetics differed markedly. mOct1/Slc22a1–RNA injection of oocytes resulted in the saturable, time– and temperature–dependent uptake of the quaternary organic cation [14C]–tetraethylammonium ([14C]–TEA), with a Km of 38 μmol/L. TEA uptake was inhibited by several other organic cation drugs, but was not inhibited by the organic cation n–methyl–nicotinamide (NMN), being instead stimulated by it (fourfold). [14C]–TEA uptake was also stimulated by an inside–outside proton gradient. mOct1/Slc22a1–injected oocytes transported the organic cations [3H]–1–methyl–4–phenylpyridium and [3H]–choline chloride, but did not transport other classes of organic compounds. mOct1/Slc22a1 encodes for a hepatic and renal organic cation transporter which may be important for the uptake and secretion of cationic drugs and endobiotics.
Macronutrient Preference, Dietary Intake, and Substrate Oxidation Among Stable Cirrhotic Patients
Authors: Davidson Isobel H. , Richardson Rosemary , Sutherland Donald , Garden James O.
Published: July 2004

Anorexia in liver disease is common; however, its association with aberrant metabolism and the type of cirrhosis has not been considered. Dietary intake, nutritional status, fasting substrate oxidation, and macronutrient preference were examined in controls (n = 18) and 65 patients with hepatocellular (n = 31) or biliary cirrhosis (n = 34). Energy intakes were lowest in hepatocellular patients (controls: 9.0 ± 0.48 megajoules/day compared with biliary: 7.0 ± 0.40 MJ/day, P < .05; controls compared with hepatocellular 6.5 ± 0.39 megajoules/day, P < .01). Triceps skinfold was lower only in hepatocellular patients (controls: 109 ± 9.2% compared with hepatocellular 79 ± 5.6%, P < .05). The fasting rate of lipid oxidation was elevated in hepatocellular patients when compared with controls and biliary patients (controls: 40.9 ± 15.1 mg/min compared with hepatocellular 62.8 ± 16.8 mg/min, P < .001, and biliary: 45.5 ± 17.0 mg/min compared with hepatocellular, P < .001). Control subjects exhibited a greater preference for the high fat, moderate carbohydrate food (controls: median 7.0 IQR 2.0 compared with biliary: median 5.0 interquartile range [IQR] 4.7, P < .01) (controls compared with hepatocellular: median 6.0 IQR 4.0, P < .01). Cirrhotic patients' spontaneous dietary intake is lower than that of controls and recommended intakes. Although macronutrient preference ratings were different within cirrhotic patient groups it remains unclear whether associated nutrient deficits are metabolically driven and dictated by primary cause
Ten–Year Survival in Ursodeoxycholic Acid-Treated Patients With Primary Biliary Cirrhosis
Authors: Poupon Renée Eugénie , Bonnand Anne-Marie , Chrétien Yves , Poupon Raoul
Published: July 2004

Ursodeoxycholic acid (UDCA) treatment has been shown to increase survival without orthotopic liver transplantation (OLT) in patients with primary biliary cirrhosis (PBC) at 4 years. Whether this beneficial effect was maintained over the long term remained to be established. In a large cohort of UDCA–treated patients with PBC, we aimed to determine the 10–year outcome of these patients using two endpoints: (1) survival without OLT, and (2) survival. The cohort was comprised of 225 patients with PBC treated with UDCA (13–15 mg/kg/d) monitored from the beginning of treatment until time of last follow–up, OLT, or death. Because of the absence of a control group, survival without OLT was compared with survival predicted by the Mayo model (first 7 years), and observed 10–year survival with an estimation of survival of a standardized control cohort of the French population. Observed survival without OLT of UDCA–treated patients was significantly higher (P < .04) than survival predicted by the Mayo model. Observed survival was significantly lower (P < .01) than survival predicted from the French population. Observed survival of noncirrhotic patients was not different (P > .9) from that of the French control population but survival of cirrhotic patients was significantly lower (P < .0001). Twenty–two patients died; 13 patients died of hepatic causes and 4 patients died after OLT. In conclusion, survival without OLT among patients treated with UDCA for PBC is higher than that of untreated patients, as predicted by the Mayo model. Ten–year survival among UDCA–treated patients is slightly lower than that of an age– and sex–matched general population, the difference mainly being explained by mortality among cirrhotic patients
Antibiotic Prophylaxis for the Prevention of Bacterial Infections in Cirrhotic Patients With Gastrointestinal Bleeding: A Meta–Analysis
Authors: Bernard Brigitte , Grangé Jean-Didier , Khac Eric Nguyen , Amiot Xavier , Opolon Pierre , Poynard Thierry
Published: July 2004

In cirrhotic patients with gastrointestinal bleeding, antibiotic prophylaxis decreases the incidence of infections but most randomized trials have not shown an increase in survival. The aim of this meta–analysis was to assess the efficacy of antibiotic prophylaxis in the prevention of infections and its effect on survival rate in cirrhotic patients with gastrointestinal bleeding. Four end points were assessed: infection, bacteremia and/or spontaneous bacterial peritonitis (SBP), incidence of SBP, and death. For each end point, heterogeneity and treatment efficacy were assessed by Der Simonian and Peto methods. Five trials including 534 patients, 264 treated with antibiotic prophylaxis for 4 to 10 days and 270 without, were identified. Mean follow–up was 12 days. Antibiotic prophylaxis significantly increased the mean percentage of patients free of infection (32% mean improvement rate, 95% confidence interval [CI]: 22–42, P< .001), bacteremia and/or SBP (19% mean improvement rate, 95% CI: 11–26, P< .001), and SBP (7% mean improvement rate, 95% CI: 2.1–12.6, P = .006). Antibiotic prophylaxis also significantly increased the mean survival rate (9.1% mean improvement rate, 95 % CI: 2.9–15.3, P = .004), without significant heterogeneity. In cirrhotic patients with gastrointestinal bleeding, short–term antibiotic prophylaxis significantly increases the mean percentage of patients free of infection and significantly increases short–term survival rate
Hepatitis C Virus Infection and Bone Marrow Transplantation: A Cohort Study With 10–Year Follow–Up
Authors: Strasser Simone I. , Myerson David , Spurgeon Catherine L. , Sullivan Keith M. , Storer Barry , Schoch Gary H. , Kim Sang , Flowers Mary E. , McDonald George B.
Published: July 2004

Before the introduction of routine blood donor screening in 1991, marrow transplant recipients were at significant transfusion–associated risk for infection with hepatitis C virus (HCV). We followed a cohort of 355 patients undergoing transplant in Seattle during 1987 to 1988 to determine (1) the impact of pretransplant HCV infection on the occurrence and severity of venocclusive disease (VOD); (2) the impact of HCV infection on liver dysfunction, other than VOD, occurring between 21 and 60 days after transplantation; and (3) the natural history of post–transplant HCV liver disease with a 10–year follow–up. HCV–RNA status was determined on serum stored before transplant and at day 100 post–transplant. Sixty–two (17%) patients were HCV–RNA positive before transplant, and 113 (32%) were HCV–RNA positive by day 100 post–transplant (or before death). Severe VOD developed in 22 of 46 (48%) evaluable patients with pretransplant HCV infection and in 150 of 229 (14%) evaluable patients without HCV (P < .0001). In multivariable analysis of risk factors for developing VOD, pretransplant HCV infection associated with elevated serum aspartate transaminase (AST) levels predicted the development of severe VOD (relative risk, 9.6; P = .0001). The presence of HCV with normal AST levels before transplant was not a risk factor for severe VOD. Between 21 and 60 days after transplant, HCV–RNA positive-patients had higher AST levels (median 101 U/L), but similar alkaline phosphatase and total bilirubin levels compared with HCV–negative patients, suggesting that cholestatic liver disease (particularly graft–versus–host disease [GVHD]) was not related to HCV infection. An acute flare of hepatitis (AST >10 times the upper limit of normal) developed at a mean of 136 ± 58 days in 31% of HCV–positive patients; no patients developed fulminant hepatitis. Between 5 and 10 years after transplant, 57% of HCV–positive and 6% of HCV–negative patients had mild to moderate elevations of AST (P < .0001), but HCV infection was not associated with excess mortality between 3 and 10 years after bone marrow transplantation. In summary, HCV infection with elevated AST levels is a significant risk factor for severe VOD after marrow transplant. However, the decision to proceed to transplantation in HCV–positive patients must balance the absolute risk of death from VOD against the risks of the underlying disease. In long–term survivors, HCV infection is not associated with excess mortality over 10 years of follow–up.
Prevention of Lethal Acute Hepatic Failure by Antimacrophage Migration Inhibitory Factor Antibody in Mice Treated With Bacille Calmette–Guerin and Lipopolysaccharide
Authors: Kobayashi Seiji , Nishihira Jun , Watanabe Satoshi , Todo Satoru
Published: July 2004

During the past few years, the biological functions of macrophage migration inhibitory factor (MIF) have been extensively re–evaluated. This has been found to be protein involved in broad–spectrum pathophysiological states as an inflammatory cytokine, pituitary–derived hormone, and glucocorticoid–induced immunomodulator. In this study, we investigated the involvement of this cytokine in the pathogenesis of lethal liver injury. Injecting a small dose of lipopolysaccharide (LPS) into bacille Calmette–Guerin (BCG)–primed Jcl:ICR mice caused a lethal hepatic injury mimicking fulminant hepatitis, in which 8 of 11 mice died within 48 hours (27% survival rate). Massive necrosis of parenchymal hepatocytes with marked mononuclear cell infiltration was observed by histological examination. Immunohistochemical analysis showed that most of the infiltrating mononuclear cells were Kupffer cells, macrophages, and, to a lesser extent, T cells. In parallel, serum aminotransferase and tumor necrosis factor–α (TNF–α) levels were increased. When an anti–MIF polyclonal antibody (0.3 mg IgG fraction/mouse) was intraperitoneally injected into mice primed with BCG, it protected them from acute hepatic failure (90% survival rate) with concomitant improvement of histological features. Injection of the antibody also suppressed the up–regulation of TNF–α and T–cell infiltration induced by LPS. Taken together, these results suggested that treatment with the anti–MIF antibody suppresses the endotoxin–induced fatal hepatic failure by regulating production of inflammatory cytokines and T–cell infiltration.
The Role of Tt Virus Infection in Acute Viral Hepatitis
Authors: Kanda Tatsuo , Yokosuka Osamu , Ikeuchi Tetsu , Seta Toshikatsu , Kawai Shigenobu , Imazeki Fumio , Saisho Hiromitsu
Published: July 2004

Recently, transfusion–transmitted virus (TTV) was discovered to be a potential causative agent for non-A–E hepatitis. Little is known about the relation between TTV and the clinical courses of various types of acute viral hepatitis. One hundred twenty–five patients with acute viral hepatitis who were admitted to the Chiba University Hospital between 1984 and 1998 and 100 persons with normal liver function tests were tested for the presence of TTV in their sera. Serum samples were tested for TTV DNA and genotype by polymerase chain reaction (PCR). TTV DNA was detected in 15 of 35 patients (43%) with non-A–E hepatitis, 14 of 33 patients (42%) with hepatitis C, 8 of 28 patients (29%) with hepatitis A, 7 of 29 patients (24%) with hepatitis B, and 37 of 100 subjects with normal liver function tests (37%). The detection rate did not differ statistically between non-A–E hepatitis and hepatitis A, B, C, or controls. The distribution of TTV genotypes was similar in non-A–E, A, B, C types, and controls. The clinical characteristics of the acute illnesses were similar for patients with or without TTV in hepatitis non-A–E, A, B, or C. Although TTV was detected frequently in non-A–E acute hepatitis, no etiologic role for TTV could be established.
Biliary Bile Acids in Primary Biliary Cirrhosis: Effect of Ursodeoxycholic Acid
Authors: Combes Burton , Carithers Robert L. , Maddrey Willis C. , Munoz Santiago , Garcia-Tsao Guadalupe , Bonner Gregory F. , Boyer James L. , Luketic Velimir A. , Shiffman Mitchell L. , Peters Marion G. , White Heather , Zetterman Rowen K. , Risser Richard , Rossi Stephen S. , Hofmann Alan F.
Published: July 2004

Bile acid composition in fasting duodenal bile was assessed at entry and at 2 years in patients with primary biliary cirrhosis (PBC) enrolled in a randomized, double–blind, placebo–controlled trial of ursodeoxycholic acid (UDCA) (10–12 mg/kg/d) taken as a single bedtime dose. Specimens were analyzed by a high–pressure liquid chromatography method that had been validated against gas chromatography. Percent composition in bile (mean ± SD) for 98 patients at entry for cholic (CA), chenodeoxycholic (CDCA), deoxycholic (DCA), lithocholic (LCA), and ursodeoxycholic (UDCA) acids, respectively, were 57.4 ± 18.6, 31.5 ± 15.5, 8.0 ± 9.3, 0.3 ± 1.0, and 0.6 ± 0.9. Values for CA were increased, whereas those for CDCA, DCA, LCA, and UDCA were decreased when compared with values in normal persons. Bile acid composition of the major bile acids did not change after 2 years on placebo medication. By contrast, in patients receiving UDCA for 2 years, bile became enriched with UDCA on average to 40.1%, and significant decreases were noted for CA (to 32.2%) and CDCA (to 19.5%). No change in percent composition was observed for DCA and LCA. Percent composition at entry and changes in composition after 2 years on UDCA were similar in patients with varying severity of PBC. In patients whose bile was not enriched in UDCA (entry and placebo–treated specimens), CA, CDCA, DCA, and the small amount of UDCA found in some of these specimens were conjugated to a greater extent with glycine (52%–64%) than with taurine (36%–48%). Treatment with UDCA caused the proportion of all endogenous bile acids conjugated with glycine to increase to 69% to 78%, while the proportion conjugated with taurine (22%–31%) fell (P < .05). Administered UDCA was also conjugated predominantly with glycine (87%)