bluuebunny/crossref_metadata_2025 huggingface dataset:
Zinc Mesoporphyrin Represses Induced Hepatic 5–Aminolevulinic Acid Synthase and Reduces Heme Oxygenase Activity in A Mouse Model of Acute Hepatic Porphyria
Authors: Schuurmans Macé M. , Hoffmann Francine , Lindberg Raija L. , Meyer Urs A.
Published: August 2002

Zinc mesoporphyrin (ZnMP) is a potent inhibitor of heme oxygenase (HO) and represses 5–aminolevulinic acid synthase (ALAS). These properties make it a potential candidate for treatment of inducible acute hepatic porphyrias, diseases characterized by neurovisceral symptoms, and massive ALAS induction. Effects of intraperitoneal ZnMP (2.5–10 μmol/kg/d) and heme arginate (3–6 mg/kg/d) on plasma levels of 5–aminolevulinic acid (ALA), on messenger RNA (mRNA), and activity of hepatic ALAS and HO were studied in porphobilinogen deaminase-deficient mice treated with phenobarbital (100 mg/kg/d) to induce ALAS. ZnMP (5 μmol/kg/d) led to a significant reduction of plasma ALA levels to 31% of controls (P < .01) by lowering the activity of hepatic mitochondrial and cytosolic ALAS to 29% and 25% of controls, respectively (P < .03). ZnMP decreased the mRNA levels of hepatic ALAS to 53% (P < .03) of controls and this repression was more pronounced than that achieved with heme arginate. In contrast to heme arginate, ZnMP led to a significant reduction of HO activity. We conclude that the combined effect of ZnMP on highly induced ALAS and on HO may be of potential benefit for human acute hepatic porphyrias and therefore merits further in vivo investigations addressing questions raised by this study. (Hepatology 2001;33:1217–1222.)
Characterization of the Mouse Bile Salt Export Pump Overexpressed in the Baculovirus System
Authors: Noe Johannes , Hagenbuch Bruno , Meier Peter J. , St-Pierre Marie V.
Published: August 2002

The bile salt export pump (Bsep), a member of the ATP–binding cassette superfamily of transporters, mediates the ATP–dependent canalicular secretion of bile salts. We have cloned and expressed the mouse Bsep (mBsep) protein in Sf9 insect cells, and characterized its transport and ATPase properties. Because its deduced amino acid sequence predicts multiple phosphorylation sites for protein kinase A, protein kinase C (PKC) and Ca2+–calmodulin dependent kinase II, we have also tested whether mBsep undergoes phosphorylation. MBsep transports both glycine and taurine conjugated bile salts. Sf9 cell membranes that express mBsep exhibit higher basal ATPase activity than control membranes, and this is further stimulated by bile salts and inhibited by vanadate. Taurochenodeoxycholate is transported with the highest affinity and is the most potent inducer of ATPase activity. Cyclosporin A, glibenclamide and rifamycin SV, all competitive inhibitors of Bsep transport, also reduced the bile salt–stimulated ATPase activity. MBsep exists as a phospho–protein when expressed in Sf9 cells and the immunoprecipitated mBsep complex is a substrate for the catalytic subunit of PKC. When mBsep and the α–isoform of mouse PKC are co–expressed in Sf9 cells, a ninefold stimulation of phosphorylation occurs. This is further increased to 18–fold after activation by phorbol ester. Given that bile salts activate selected PKC isoforms in hepatocytes, including the α isoform, the phosphorylation of mBsep by PKCα may represent a point of regulation for this transporter that is mediated by its own substrate. (Hepatology 2001;33:1223–1231.)
Malaria Sporozoites Actively Enter and Pass Through Rat Kupffer Cells Prior to Hepatocyte Invasion
Authors: Pradel Gabriele , Frevert Ute
Published: August 2002

Malaria sporozoites have to cross the layer of sinusoidal liver cells to reach their initial site of multiplication in the mammalian host, the hepatocytes. To determine the sinusoidal cell type sporozoites use for extravasation, endothelia or Kupffer cells, we quantified sporozoite adhesion to and invasion of sinusoidal cells isolated from rat liver. In vitro invasion assays reveal that Plasmodium berghei and P. yoelii sporozoites attach to and enter Kupffer cells, but not sinusoidal endothelia. Unlike hepatocytes and other nonphagocytic cells, which are invaded in vitro only within the first hour of parasite exposure, the number of intracellular sporozoites in Kupffer cells increases for up to 12 hours. By confocal and electron microscopy, sporozoites are enclosed in a vacuole that does not colocalize with lysosomal markers. Inhibition of phagocytosis with gadolinium chloride has no effect on Kupffer cell invasion, but abolishes phagocytosis of inactivated sporozoites. Furthermore, sporozoites traverse in vitro from Kupffer cells to hepatocytes where they eventually develop into exoerythrocytic schizonts. Thus, malaria sporozoites selectively recognize and actively invade Kupffer cells, avoid phagosomal acidification, and safely passage through these phagocytes.
Sen Virus Infection and Its Relationship to Transfusion–Associated Hepatitis
Authors: Umemura Takeji , Yeo Anthony E.T. , Sottini Alessandra , Moratto Daniele , Tanaka Yasuhito , Wang Richard Y.-H. , Shih Wai-Kuo J. , Donahue Peter , Primi Daniele , Alter Harvey J.
Published: August 2002

SEN virus (SEN–V) is a recently identified single–stranded, circular DNA virus. Two SEN–V variants (SENV–D and SENV–H) were assayed by polymerase chain reaction (PCR) to investigate their role in the causation of transfusion–associated non-A to E hepatitis. The incidence of SEN–V infection after transfusion was 30% (86 of 286) compared with 3% (3 of 97) among nontransfused controls (P < .001). Transfusion risk increased with the number of units transfused (P < .0001) and donor–recipient linkage for SEN–V was shown by sequence homology. The prevalence of SEN–V in 436 volunteer donors was 1.8%. Among patients with transfusion–associated non-A to E hepatitis, 11 of 12 (92%) were infected with SEN–V at the time of transfusion compared with 55 of 225 (24%) identically followed recipients who did not develop hepatitis (P < .001). No effect of SEN–V on the severity or persistence of coexistent hepatitis C virus (HCV) infection was observed. In 31 infected recipients, SEN–V persisted for greater than 1 year in 45% and for up to 12 years in 13%. SEN–V-specific RNA (a possible replicative intermediate) was recovered from liver tissue. In summary, SENV–D and –H were present in nearly 2% of US donors, and were unequivocally transmitted by transfusion and frequently persisted. The strong association of SEN–V with transfusion–associated non-A to E hepatitis compared with controls raises the possibility, but does not establish that SEN–V might be a causative agent of posttransfusion hepatitis. The vast majority of SEN–V-infected recipients did not develop hepatitis. (Hepatology 2001;33:1303–1311.)
Protective Immune Response to Hepatitis C Virus in Chimpanzees Rechallenged Following Clearance of Primary Infection
Authors: Bassett Suzanne E. , Guerra Bernadette , Brasky Kathleen , Miskovsky Emil , Houghton Michael , Klimpel Gary R. , Lanford Robert E.
Published: August 2002

Hepatitis C virus (HCV) infections were evaluated in chimpanzees that had previously cleared HCV and were rechallenged. Animals that had previously cleared HCV infection rapidly cleared homologous and heterologous virus upon rechallenge, indicative of a strong protective immunity. In one animal, sterilizing immunity was observed with regard to viremia, although viral RNA was transiently detected in the liver. Accelerated viral clearance following rechallenge with HCV was observed in animals that had not been exposed to HCV for over 16 years, suggesting that long–lasting protective immunity may be possible. The ability of peripheral blood mononuclear cells (PBMC) to recognize HCV proteins was evaluated during the course of the rechallenge experiments. A very early and strong in vitro recall response to HCV nonstructural proteins appeared to be associated with viral clearance. In contrast, proliferative responses to HCV proteins were not observed in 4 persistently infected chimpanzees, and a weak proliferative response was observed in 1 of 2 animals during acute resolving infection. The results suggest that a strong T–cell proliferative response is induced upon rechallenge of chimpanzees with HCV and that this response is associated with rapid viral clearance. The antibody response to HCV proteins increased by over 1,000–fold in all animals following rechallenge as well. A more complete understanding of the role of the cellular immune response in the clearance of HCV and the nature of the protective immune response following viral clearance may aid in the generation of therapies and vaccines.
Earlier Expression of the Transcription Factor Hfh–11B Diminishes Induction of P21Cip1/Waf1 Levels and Accelerates Mouse Hepatocyte Entry Into S–Phase Following Carbon Tetrachloride Liver Injury
Authors: Wang Xinhe , Hung Nai-Jung , Costa Robert H.
Published: August 2002

Partial hepatectomy (PH) or toxic liver injury induces the proliferation of terminally differentiated hepatic cells to regenerate the original size of the adult liver. Previous PH liver regeneration studies showed that premature transgenic expression of the Forkhead Box M1b (FoxM1b, HFH–11B) transcription factor accelerated hepatocyte entry into DNA replication (S–phase). In this study, we used carbon tetrachloride (CCl4) liver injury to induce a different type of mouse liver regeneration and show that premature hepatic HFH–11B levels also accelerate the onset of hepatocyte S–phase in this injury model. Unlike PH liver regeneration, earlier hepatocyte proliferation after CCl4 liver injury is correlated with diminished transgenic hepatic levels of p21CIP1/WAF1 at the G1/S transition of the cell cycle. Differential hybridization of cDNA arrays and RNase protection studies determined that CCl4 regenerating liver of transgenic mice displayed early stimulated expression of the S–phase promoting cyclin D1 and cyclin E and sustained levels of Cdc25a phosphatase genes. Compared with previous PH liver regeneration studies, our data suggest that premature expression of HFH–11B activates distinct S–phase promotion pathways in the CCl4 liver injury model. Although proliferating transgenic hepatocytes induced by either PH or CCl4 liver injury displayed early expression of identical M–phase cyclin genes (cyclin B1, B2, A2, and F), only CCl4 regenerating transgenic liver exhibited earlier expression of the M–phase promoting Cdc25b. These studies suggest that CCl4 injury of transgenic liver not only uses the same mechanisms as PH to mediate accelerated hepatocyte entry into mitosis, but also promotes M–phase entry by stimulating Cdc25b expression.
Interferon Alfa Down–Regulates Cd81 in Patients With Chronic Hepatitis C
Authors: Kronenberger Bernd , Rüster Brigitte , Elez Robert , Weber Stephan , Piiper Albrecht , Lee Jung-Hun , Roth Kurt W. , Zeuzem Stefan
Published: August 2002

CD81 protein has been shown to bind hepatitis C virus (HCV) envelope 2 (E2) glycoprotein in vitro and may act as a (co)receptor for HCV. Regulation of CD81 expression by interferon alfa (IFN–α) and ribavirin could thereby affect the response to antiviral therapy. In the present study, the effects of IFN–α and ribavirin on CD81 protein and CD81 mRNA were assessed in peripheral blood lymphocytes (PBL) and isolated human hepatocytes by fluorescence–activated cell sorter (FACS) analysis and real–time polymerase chain reaction (PCR), respectively. In addition, regulation of CD81 in PBL was investigated in 10 patients treated with combination therapy. Incubation with IFN–α (50 U/mL) down–regulated total CD81 in PBL (81.7 ± 11.6% of control; P = .003) and in isolated human hepatocytes (91.6 ± 8.1% of control; P = .034). Incubation with IFN–α with and without ribavirin (2.2 μg/mL) significantly reduced cell surface-associated CD81 protein (83.9 ± 10.3% of control; P = .003). PBL of untreated patients chronically infected with HCV had significantly higher levels of total CD81 protein compared with PBL obtained from healthy donors (631.1 ± 93.3 vs. 538.9 ± 95.2 relative fluorescence units [RFU]; P = .030). Pretreatment cell surface-associated CD81 protein was lower in patients infected with genotype HCV–3 than those infected with HCV–1 (111.8 ± 15.0 vs. 162.0 ± 41.3 RFU; P = .019). Furthermore, cell surface-associated CD81 protein was lower 4 weeks after initiation of therapy in patients with an initial virologic response compared with initial virologic nonresponders (110.5 ± 8.5 vs. 139.8 ± 27.5 RFU; P = .057). In conclusion, IFN–α and ribavirin regulate CD81 expression in vitro and in vivo. CD81 expression correlates with HCV genotype and initial virologic response in patients with chronic hepatitis C.
Increasing Incidence and Mortality of Primary Intrahepatic Cholangiocarcinoma in the United States
Authors: Patel Tushar
Published: August 2002

Clinical observations suggest a recent increase in intrahepatic biliary tract malignancies. Thus, our aim was to determine recent trends in the epidemiology of intrahepatic cholangiocarcinoma in the United States. Reported data from the Surveillance, Epidemiology, and End Results (SEER) program and the United States Vital Statistics databases were analyzed to determine the incidence, mortality, and survival rates of primary intrahepatic cholangiocarcinoma. Between 1973 and 1997, the incidence and mortality rates from intrahepatic cholangiocarcinoma markedly increased, with an estimated annual percent change (EAPC) of 9.11% (95% CI, 7.46 to 10.78) and 9.44% (95%, CI 8.46 to 10.41), respectively. The age–adjusted mortality rate per 100,000 persons for whites increased from 0.14 for the period 1975–1979 to 0.65 for the period 1993–1997, and that for blacks increased from 0.15 to 0.58 over the same period. The increase in mortality was similar across all age groups above age 45. The relative 1– and 2–year survival rates following diagnosis from 1989 to 1996 were 24.5% and 12.8%, respectively. In conclusion, there has been a marked increase in the incidence and mortality from intrahepatic cholangiocarcinoma in the United States in recent years. This tumor continues to be associated with a poor prognosis.
Initial Amplification of Duck Hepatitis B Virus Covalently Closed Circular Dna After In Vitro Infection of Embryonic Duck Hepatocytes Is Increased by Cell Cycle Progression
Authors: Borel Christelle , Schorr Olivier , Durand Isabelle , Zoulim Fabien , Kay Alan , Trepo Christian , Hantz Olivier
Published: August 2002

The relationship between the cell cycle and early amplification of duck hepatitis B virus covalently closed circular (CCC) DNA was studied after in vitro infection of fetal hepatocytes. We first showed that embryonic hepatocytes proliferated for at least 6 days after plating and that complete viral replication including CCC DNA amplification occurred in these proliferating cells. Addition of sodium butyrate or aphidicolin reversibly blocked cells in the G1 phase and diminished CCC DNA synthesis, which was restored after drug withdrawal, concomitantly with the entry of cells into S phase. Cell cycle progression of fetal hepatocytes can be triggered by stimulation with epidermal growth factor (EGF), hepatocyte growth factor (HGF), and tumor growth factor α (TGF–α). CCC DNA synthesis increased with progression to the S phase induced by EGF, HGF, and TGF–α alone or in combination. By contrast, tumor growth factor β (TGF–β) alone or in combination with EGF inhibited cell proliferation and viral DNA synthesis. By double labeling, viral nucleocapsids were found predominantly in bromodeoxyuridine–positive hepatocytes, indicating that high viral replication occurs preferentially in proliferating hepatocytes. CCC DNA was also detected mainly in cells in the S and G2/M phases separated from cells in the G1 phase by cell sorting. Taken together, these results show that hepatocyte proliferation may positively regulate the initial amplification of CCC DNA of avian hepadnaviruses, and may explain why mitosis is not necessarily associated with loss of CCC DNA.
Suppression of Bile Acid Synthesis, But Not of Hepatic Cholesterol 7α–Hydroxylase Expression, by Obstructive Cholestasis in Humans
Authors: Bertolotti Marco , Carulli Lucia , Concari Mauro , Martella Pietro , Loria Paola , Tagliafico Enrico , Ferrari Sergio , Del Puppo Marina , Amati Barbara , De Fabiani Emma , Crestani Maurizio , Amorotti Claudio , Manenti Antonio , Carubbi Francesca , Pinetti Adriano , Carulli Nicola
Published: August 2002

Regulation of bile acid synthesis, a key determinant of cholesterol homeostasis, is still incompletely understood. To elucidate the feedback control exerted on bile acid biosynthesis in humans with obstructive cholestasis, 16 patients with bile duct obstruction were studied. In vivo 7α–hydroxylation, reflecting bile acid synthesis, was assayed in 13 of them by tritium release analysis. Serum 27–hydroxycholesterol was determined by gas chromatography–mass spectrometry. In a subgroup, hepatic cholesterol 7α–hydroxylase mRNA was assayed by real–time polymerase chain reaction (PCR), enzyme activity was determined by isotope incorporation, and microsomal cholesterol content was assayed by gas chromatography-mass spectrometry. Age–matched control subjects were studied in parallel. Hydroxylation rates were lower in cholestatic patients (108 ± 33 mg of cholesterol per day, mean ± SEM; controls: 297 ± 40 mg/d; P < .01). The reduction was proportional to the severity of cholestasis, and synthetic rates were normalized in 4 subjects restudied after resolution of biliary obstruction. Consistent findings were obtained by analysis of serum 7α–hydroxycholesterol levels. On the other hand, hepatic cholesterol 7α–hydroxylase mRNA, microsomal enzyme activity, and cholesterol content tended to be increased in cholestasis. Finally, serum 27–hydroxycholesterol levels were slightly reduced in cholestatic subjects and were not related with the severity of the disease. Suppression of in vivo bile acid synthesis with no corresponding reduction in tissue 7α–hydroxylase expression and activity is consistent with nontranscriptional, posttranslational levels of regulation; these may play a role in the feedback control of bile acid synthesis in particular conditions. Alteration of the alternate biosynthetic pathway seems unlikely according to the present data.
A Potent Antiviral Effect on Hepatitis C Viral Dynamics in Serum and Peripheral Blood Mononuclear Cells During Combination Therapy With High–Dose Daily Interferon Alfa Plus Ribavirin and Intravenous Twice–Daily Treatment With Interferon Beta
Authors: Asahina Yasuhiro , Izumi Namiki , Uchihara Masakatsu , Noguchi Osamu , Tsuchiya Kaoru , Hamano Kosei , Kanazawa Nobuhiko , Itakura Jun , Miyake Shozo , Sakai Takahiro
Published: August 2002

Hepatitis C virus (HCV) is known to infect and replicate within peripheral blood mononuclear cells (PBMC), thereby enabling the direct evaluation of antiviral mechanisms by analyzing HCV dynamics in PBMC. To address potential molecular differences associated with distinct antiviral regimens, we studied HCV dynamics in both serum and PBMC in 44 patients with HCV genotype 1b and high viral load who were randomly assigned to the following 4 different treatment groups: 1) combination therapy with 6 MU daily of interferon alfa 2b (IFN–α2b) plus 800 mg of ribavirin; 2) monotherapy with 6 MU daily of IFN–α2b; 3) monotherapy with twice–daily intravenous administration with 3MU of IFN–β; and 4) monotherapy with daily intravenous administration with 6 MU of IFN–β. HCV–RNA levels were measured serially using highly sensitive real–time detection polymerase chain reaction (PCR). HCV dynamics in both the serum and PBMC showed a “biphasic” pattern. The exponential decay slopes of the second phase were significantly higher in the combination or twice–daily dosing regimen groups compared with groups 2 or 4 (0.10 ± 0.08 vs. 0.02 ± 0.09 or 0.16 ± 0.09 vs. 0.02 ± 0.04 day-1; P < .05 or P < .0005, respectively). Moreover, the viral half–lives in the second phase were significantly shorter in these groups (73.2 ± 42.5 vs. 240.1 ± 120.7 or 56.0 ± 44.6 vs. 361.6 ± 293.5 hours; P < .05 or P < .05, respectively). Additionally, the slope of HCV decline in PBMC tended to be higher in the combination regimens, as compared with monotherapy. Taken together, our data on HCV dynamics provide molecular insight into utilization of combination or twice–daily dosing regimens to increase rates of sustained viral eradication of HCV.
Factors Affecting Liver Fibrosis in Human Immunodeficiency Virus-And Hepatitis C Virus-Coinfected Patients: Impact of Protease Inhibitor Therapy
Authors: Benhamou Yves , Di Martino Vincent , Bochet Marie , Colombet Geneviève , Thibault Vincent , Liou Amélie , Katlama Christine , Poynard Thierry
Published: August 2002

Hepatitis C virus (HCV)–related liver fibrosis progression is accelerated in human immunodeficiency virus (HIV)–infected patients. The effect of protease inhibitor (PI) therapy on liver fibrosis is unknown. The aim of this work was to analyze the impact of PI therapy on HCV–related liver fibrosis in HIV/HCV coinfected patients. We evaluated in a long–term follow–up retrospective cohort study the influence of antiretroviral therapy containing PI on liver fibrosis in 182 consecutive HIV/HCV coinfected patients. At liver biopsy, 63 patients had received PI and 119 patients had never been treated with PI. Relationships between liver histologic features, age, alcohol consumption, CD4 cell count, HIV–RNA load, and antiretroviral regimens were analyzed. Liver fibrosis stage was lower in patients receiving PIs by comparison with patients who had never received PIs (P = .03). The 5–, 15–, and 25–year cirrhosis rates were 2% versus 5%, 5% versus 18%, and 9% versus 27%, respectively, in patients who had received PIs compared with PI–untreated patients (P = .0006). Multivariate analysis identified 4 independent predictors of progression to cirrhosis: absence of protease inhibitor therapy (relative risk [RR] = 4.74, 95% confidence interval [CI], 1.34–16.67), heavy alcohol consumption (≥ 50 g daily) (RR = 4.71, 95% CI, 1.92–11.57), low CD4 cell count (<200/μL) (RR = 2.74, 95% CI, 1.17–6.41), and age at HCV contamination (≥20 years) (RR = 2.37, 95% CI, 1.04–5.38). In conclusion, protease inhibitor therapy might not accelerate progression to HCV–related cirrhosis. Furthermore, chronic use of antiretroviral therapy containing PI together with reduction of alcohol consumption and maintenance of high CD4 count could have a beneficial impact on liver fibrosis progression in HIV/HCV coinfected patients.
Short–Term Prognosis in Critically Ill Patients With Cirrhosis Assessed by Prognostic Scoring Systems
Authors: Wehler Markus , Kokoska Judith , Reulbach Udo , Hahn Eckhart Georg , Strauss Richard
Published: August 2002

The short–term prognosis of acutely ill patients with cirrhosis is influenced by the degree of hepatic insufficiency and by dysfunction of extrahepatic organ systems. The purpose of this study was to assess and compare the prognostic accuracy of the Child–Pugh classification, the Acute Physiology and Chronic Health Evaluation (APACHE) II system and the Sequential Organ Failure Assessment (SOFA) for predicting hospital mortality in patients with cirrhosis when used 24 hours after admission to a medical intensive care unit (ICU). Prospective data were recorded on 143 patients. Cumulative mortality rates were 36% in the ICU, 46% in the hospital, and 56% at 6–month follow–up. By using the area under receiver operating characteristic (AUROC) curves, the SOFA showed an excellent discriminative power (AUROC 0.94), which was clearly superior to the APACHE II (AUROC 0.79) and the Child–Pugh system (AUROC 0.74). Hospital mortality rates below and above a cutoff of 8 SOFA points were 4% and 88%, respectively (P < .0005). The SOFA score also reflected resource use during the ICU treatment as measured by daily workload and length of stay. The SOFA is an easily applied tool with excellent prognostic abilities and can be used to enhance clinical judgment of prognosis as well as providing patients and families with objective information.
A Bioinformatical Approach Suggests the Function of the Autoimmune Hepatitis Target Antigen Soluble Liver Antigen/Liver Pancreas
Authors: Kernebeck Thomas , Lohse Ansgar W. , Grötzinger Joachim
Published: August 2002

Antibodies to a soluble liver antigen/liver pancreas (SLA/LP) appear to be highly specific for the diagnosis of autoimmune hepatitis. The SLA/LP target antigen was recently identified as a hitherto unknown gene encoding 474 amino acid residues. The function of this antigen remains unclear, because it does not share sequence homology with proteins of known function stored in any of the publicly accessible databases. Therefore we used a new theoretical method called fold recognition and could show that the SLA/LP sequence is compatible with the architecture of the superfamily of pyridoxal phosphate (PLP; vitamin B6)–dependent transferases. Its function is likely to be that of a serine hydroxymethyltransferase and may be an important enzyme in the thus far poorly understood selenocysteine pathway.
Mechanisms of cell death induced by suicide genes encoding purine nucleoside phosphorylase and thymidine kinase in human hepatocellular carcinoma cells in vitro
Authors: Krohne Tim U. , Shankara Srinivas , Geissler Michael , Roberts Bruce L. , Wands Jack R. , Blum Hubert E. , Mohr Leonhard
Published: August 2002

For gene therapy of hepatocellular carcinoma (HCC), the Escherichia coli purine nucleoside phosphorylase (PNP)/fludarabine suicide gene system may be more useful than the herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) system as a result of a stronger bystander effect. To analyze the molecular mechanisms involved in PNP/fludarabine-mediated cell death in human HCC cells in comparison with HSV-tk/GCV, we transduced human HCC cells of the cell lines, HepG2 and Hep3B, with PNP or HSV-tk using adenoviral vectors, followed by prodrug incubation. Both systems predominantly induced apoptosis in HepG2 and Hep3B cells. PNP/fludarabine induced strong p53 accumulation and a more rapid onset of apoptosis in p53-positive HepG2 cells as compared with p53-negative Hep3B cells, but efficiency of tumor cell killing was similar in both cell lines. In contrast, HSV-tk/GCV-induced apoptosis was reduced in p53-negative Hep3B cells as compared with p53-positive HepG2 cells. HSV-tk/GCV, but not PNP/fludarabine, caused up-regulation of Fas in p53-positive HepG2 cells and of Fas ligand (FasL) in both HCC cell lines. These results demonstrate cell line-specific differences in response to treatment with PNP/fludarabine and HSV-tk/GCV, respectively, and indicate that PNP/fludarabine may be superior to HSV-tk/GCV for the treatment of human HCC because of its independence from p53 and the Fas/FasL system. (Hepatology 2001;34:511-518.)
Graft dysfunction mimicking autoimmune hepatitis following liver transplantation in adults
Authors: Heneghan Michael A. , Portmann Bernard C. , Norris Suzanne M. , Williams Roger , Muiesan Paolo , Rela Mohamed , Heaton Nigel D. , O'Grady John G.
Published: August 2002

In children, a type of graft dysfunction associated with autoimmune features has been described. We have identified 7 adult liver-transplant (LT) recipients from a series of over 1,000 consecutive transplant recipients who presented between 0.3 years and 7.2 years following transplantation with characteristic symptoms, autoantibody profiles, and histologic findings of autoimmune disease. The indications for transplantation were Ecstasy overdose, alcohol-related cirrhosis, primary sclerosing cholangitis (PSC) (2), primary biliary cirrhosis (PBC), hepatitis C cirrhosis, and cryptogenic cirrhosis. Two patterns of de novo autoantibody development were noted; anti-liver-kidney-microsome (LKM) antibody development at high titer in association with an aspartate transaminase (AST) > 500 and antinuclear (ANA) and antismooth muscle (AMA) antibody development at titers >1/80 with lower AST levels. All cases had elevated IgG. Liver biopsies showed changes of an autoimmune-type hepatitis with portal and periportal hepatitis in association with a marked infiltrate of plasma cells, lymphocytes, and bridging collapse. Two patients lost their grafts because of the disease. Patients were treated with reintroduction of steroids and azathioprine in cases in which it had been withdrawn. Major histocompatibility class I and II mismatching did not incur risk. Eight of 12 liver allografts were acquired from either DRB*0301- or DRB*0401-positive donors, and 4 recipients were DRB*0301-positive. This series illustrates that both symptoms and histologic findings of graft dysfunction compatible with autoimmune hepatitis (AIH) exist in adult LT recipients. Graft loss may be a consequence. This entity may represent a specific type of rejection that should currently be classified as “graft dysfunction mimicking autoimmune hepatitis.” (Hepatology 2001;34:464-470.)
Smoking and Hypoxemia Caused by Hepatopulmonary Syndrome Before and After Liver Transplantation
Authors: Rolla Giovanni , Brussino Luisa , Dutto Luca , Ottobrelli Antonio , Bucca Caterina
Published: August 2002

Severe hypoxemia may occur in patients with liver disease as a result of abnormal intrapulmonary vasodilatations (hepatopulmonary syndrome, HPS). Liver transplantation (LT) is the only effective treatment of HPS, with a quite variable delay of improvement of oxygenation. Smoking, by decreasing respiratory nitric oxide (NO), apparently contributed to improved oxygenation in a 44–year–old man with alcohol–induced cirrhosis, complicated by HPS, who underwent LT. The patient quit smoking just before LT, when his PaO2 was 29 mm Hg and exhaled NO (eNO) 28 ppb, a value far above the normal limits (9.6 ± 3.2 ppb). After LT, oxygenation remained poor and eNO remained high for more than 4 months, when the patient started to smoke again (blood HbCO going up to 5%). At that time eNO decreased to 6 ppb and PaO2 increased to 67 mm Hg. The strict relationship between eNO and oxygenation observed in this case reinforces the hypothesis that NO is the most important vasodilating mediator in HPS. Smoking may have hastened the resolution of HPS after LT by inhibiting respiratory NO and/or through a generalized impairment of endothelium–dependent vasodilation.
Sequential treatment with lamivudine and interferon monotherapies in patients with chronic hepatitis B not responding to interferon alone: Results of a pilot study
Authors: Serfaty Lawrence , Thabut Dominique , Zoulim Fabien , Andreani Tony , Chazouillères Olivier , Carbonell Nicolas , Loria Alain , Poupon Raoul
Published: August 2002

Sustained viral suppression using monotherapy with interferon alfa (IFN-α) or lamivudine can only be achieved in a small percentage of patients with chronic hepatitis B. The concomitant administration of lamivudine and IFN-α does not enhance efficacy. We postulated that the optimal timing of therapy might be sequential treatment with lamivudine and IFN-α. The aim of this study was therefore to assess the efficacy of sequential treatment in patients resistant to IFN-α alone. Fourteen male patients, with a median age of 40 years, nonresponders to IFN-α with hepatitis B virus (HBV) DNA > 100 pg/mL (branched DNA [bDNA] Chiron) and positive hepatitis B e antigen (HBeAg) in 11 of 14 patients, were treated with lamivudine 100 mg/d alone for 20 weeks, then with both IFN-α2b 5 MU 3 times per week and lamivudine for 4 weeks, and lastly with IFN-α alone for 24 weeks. At the end of lamivudine therapy, all patients had undetectable serum HBV DNA, and none exhibited an emergence of HBV polymerase mutant or breakthrough. Sustained serum HBV-DNA clearance 6 months after the end of sequential treatment was achieved in 8 of 14 patients, HBeAg-to-anti-HBe seroconversion in 5 of 11 patients, and HBeAg and hepatitis B surface antigen (HBsAg) seroconversions in 3 of 14 patients (anti-HBs > 100 IU/mL). All sustained responders had normalized their alanine transaminase (ALT) values and exhibited histologic improvements. In conclusion, the results of this pilot study suggest that sequential treatment with lamivudine and IFN-α can induce a sustained virologic response, including HBs seroconversion, in patients with chronic hepatitis B not responding to IFN-α alone, without the selection of drug-resistant mutants. This therapeutic schedule warrants further evaluation in clinical trials. (Hepatology 2001;34:573-577.)
Distinct epitopes on formiminotransferase cyclodeaminase induce autoimmune liver cytosol antibody type 1
Authors: Muratori Luigi , Sztul Elizabeth , Muratori Paolo , Gao Ya-sheng , Ripalti Alessandro , Ponti Cristina , Lenzi Marco , Landini Maria Paola , Bianchi Francesco B.
Published: August 2002

Liver cytosol antibody type 1 (LC1) is regarded as a serologic marker of type 2 autoimmune hepatitis, in addition to liver kidney microsomal antibody type 1. Among 38 patients with type 2 autoimmune hepatitis, 23 were positive for LC1 antibodies. The antigen recognized by LC1 has been identified as a liver-specific 58-kd metabolic enzyme named formiminotransferase cyclodeaminase (FTCD). All 23 LC1-positive sera immunoprecipitated rat FTCD, and 22 gave an identity reaction with rat FTCD by immunodiffusion. No reaction was observed with sera from 10 patients with type 1 autoimmune hepatitis, 10 with primary biliary cirrhosis, 10 with chronic hepatitis C, and 10 healthy controls. By Western immunoblotting all 23 LC1-positive sera and all the controls tested negative, suggesting that all the antigenic epitopes were destroyed by denaturation. FTCD is a bifunctional protein composed of distinct globular FT and CD domains connected by a short linker. To identify epitopes that trigger the LC1 autoimmune response, we tested LC1 antibodies against FTCD constructs encoding the N-terminal FT domain (amino acids 1-339), or the C-terminal CD domain (amino acids 332-541). Of 20 sera positive against full-length FTCD, 8 (40%) recognized the FT domain and the CD domain, 7 (35%) recognized only the FT domain, and 5 (25%) did not recognize either construct. No sera reacted with only the CD domain. These data indicate that multiple regions of FTCD trigger the LC1 autoimmune response, and that LC1 reactivity is mainly directed to conformation-sensitive epitopes located in the FT region of FTCD. (Hepatology 2001;34:494-501.)
Hepatitis B virus variants in patients receiving lamivudine treatment with breakthrough hepatitis evaluated by serial viral loads and full-length viral sequences
Authors: Liu Chun-Jen , Chen Pei-Jer , Lai Ming-Yang , Kao Jia-Horng , Chen Ding-Shinn
Published: August 2002

Both viral loads and genome variations have been implicated in the pathogenesis of acute exacerbation of chronic hepatitis B. Hepatitis B exacerbation in patients receiving lamivudine treatment represented a unique setting to clarify their importance. Three organ recipients with posttransplantation hepatitis B exacerbation and 3 patients with chronic hepatitis B were studied. All received lamivudine treatment and their alanine aminotransferase (ALT) levels and hepatitis B virus (HBV) loads were regularly followed. Full-length genomic sequences before and during lamivudine treatment were determined in patients who had breakthrough of serum HBV DNA or elevation of serum ALT. Breakthrough of serum HBV DNA occurred after 6 to 15 months of lamivudine treatment in all. A rapid increase of viral load accompanying the emergence of tyrosine-methionine-aspartate-aspartate (YMDD) variant was followed by hepatitis B exacerbation in each patient. The mean number of nucleotide and amino acid substitutions per genome pair was equivalent in immunosuppressed or immunocompetent patients (6.3 vs. 6.3 for nucleotide, P > .05; 6.0 vs. 6.7 for amino acid, P > .05). Changes of nucleotide and amino acid beyond the YMDD motif were distributed along the whole HBV genome but none occurred within the known B-cell epitopes and human leukocyte antigen class I- or II-restricted T-cell epitopes. Our results suggest that a resurgence of viral load rather than changes of the known immunogenic viral epitopes is more closely associated with the development of hepatitis B exacerbation after the emergence of YMDD variants in patients receiving lamivudine treatment. (Hepatology 2001;34:583-589.)
Increase in de novo HBV DNA integrations in response to oxidative DNA damage or inhibition of poly(ADP-ribosyl)ation
Authors: Dandri Maura , Burda Martin R. , Bürkle Alexander , Zuckerman David M. , Will Hans , Rogler Charles E. , Greten Heimer , Petersen Joerg
Published: August 2002

Chronic infection with hepatitis B virus (HBV) is associated with an increased risk for the development of cirrhosis and hepatocellular carcinoma (HCC). Although clonal HBV DNA integrations are detected in nearly all HCCs the role of these integrations in hepatocarcinogenesis is poorly understood. We have used a cloning protocol that allows studying the frequency and the natural history of HBV DNA integrations in cell culture. Southern blot analysis of the genomic DNA of HepG2 2.2.15 subclones, which replicate HBV, enabled us to detect new HBV DNA integrations in approximately 10% of the HepG 2.2.15 subclones over 4 rounds of sequential subcloning, whereas no loss of any preexisting HBV DNA integrations was observed. Treatments of HepG2 cells with H2O2, designed to increase DNA damage, increased the frequency of HBV integrations to approximately 50% of the subclones and treatments designed to inhibit DNA repair, by inhibiting Poly(ADP-ribosyl)ation, also increased the frequency of HBV integration to 50%. These findings suggest that DNA strand breaks induced by oxidative stress during persistent HBV infection in humans may increase HBV DNA integration events, whereas PARP-1 activity may function to limit the occurrence of de novo HBV DNA integrations.
Insulin signaling in the transcriptional and posttranscriptional regulation of CYP2E1 expression
Authors: Woodcroft Kimberley J. , Hafner Mikehl S. , Novak Raymond F.
Published: August 2002

Diabetes has been reported to increase the expression of cytochrome P450 (CYP) 2E1 messenger RNA (mRNA) and protein several-fold, and enhanced expression has been associated with elevated ketone bodies. Primary cultured rat hepatocytes were used to explore ketone body and insulin regulation of CYP2E1 expression. Hydroxybutyrate and acetoacetate (AC), alone or in combination, either failed to affect or decreased CYP2E1 mRNA levels by up to 90% relative to untreated hepatocytes. Insulin produced a concentration-dependent decrease in CYP2E1 mRNA levels, and insulin receptor immunoprecipitation showed a correspondence between receptor phosphorylation and the decrease in CYP2E1 mRNA levels at physiologic levels of insulin. Phosphatase inhibitors decreased CYP2E1 mRNA levels by greater than 95%. The phosphatidylinositol 3-kinase (PI3-kinase) inhibitors wortmannin or LY294002 and rapamycin, an inhibitor of p70 S6 kinase phosphorylation, ameliorated the insulin-mediated decrease in CYP2E1 mRNA levels. Geldanamycin, which inhibits Src kinase, also abrogated the insulin-mediated decrease in CYP2E1 mRNA levels. In contrast, the protein kinase C (PKC) inhibitor bisindolylmaleimide, the mitogen-activated protein kinase kinase (MEK) inhibitor PD98059, and the p38 mitogen-activated protein (MAP) kinase inhibitor SB202190 did not affect the insulin-mediated decrease in CYP2E1. CYP2E1 mRNA half-life decreased from ˜48 hours in the absence of insulin to ˜15 hours at 10 nmol/L insulin, and this decrease was prevented by wortmannin. The half-life of CYP2B mRNA was increased by insulin, whereas that of CYP3A was unaffected. Analysis of CYP2E1 gene transcription using heterogeneous nuclear RNA (hnRNA) showed that insulin suppressed CYP2E1 transcription. In conclusion, these data show involvement of transcriptional and posttranscriptional mechanisms in the insulin-mediated regulation of CYP2E1 and implicate PI3-kinase, p70 S6 kinase, and Src kinase in mediating these effects.)
Complex HBV populations with mutations in core promoter, C gene, and pre-S region are associated with development of cirrhosis in long-term renal transplant recipients
Authors: Preikschat Petra , Günther Stephan , Reinhold Simone , Will Hans , Budde Klemenz , Neumayer Hans H. , Krüger Detlev H. , Meisel Helga
Published: August 2002

Long-term immunosuppressed renal transplant recipients with chronic hepatitis B virus (HBV) infection often develop liver cirrhosis (LC) and end-stage liver disease (ESLD). This study investigated accumulation and persistence of specific HBV mutants in relation to the clinical course in these patients (n = 38; mean follow-up, 3.5 years). HBV was analyzed longitudinally via length polymorphism of polymerase chain reaction (PCR) fragments (median, 6.5 serum samples per patient) as well as by cloning and partial sequencing of 346 full-length HBV genomes. Fourteen patients (group 1) developed LC or died from ESLD, whereas 24 patients (group 2) showed no evidence of LC during follow-up. Development of LC and ESLD was associated with persistence of HBV mutant populations characterized by deletions/insertions in core promoter plus deletions in the C gene and/or deletions in the pre-S region (86% of group 1 vs. 17% of group 2; P < .0001). HBV without these mutations or with core promoter mutations alone were predominantly found in group 2 (14% of group 1 vs. 75% of group 2). In patients infected with core promoter mutants, the additional appearance and persistence of deletions in the C gene and/or the pre-S region were accompanied or followed by development of LC and ESLD. The mutations were distributed on individual genomes in various combinations, leading to a high complexity of the virus population. In conclusion, these data suggest that accumulation and persistence of specific HBV populations characterized by mutations in 3 subgenomic regions play a role in pathogenesis of LC and ESLD in long-term renal transplant recipients.
Varied assembly and RNA editing efficiencies between genotypes I and II hepatitis D virus and their implications
Authors: Hsu Sheng-Chieh , Syu Wan-Jr , Sheen I-Jane , Liu Hui-Ting , Jeng King-Song , Wu Jaw-Ching
Published: August 2002

The mechanisms that link genotypes of hepatitis D virus (HDV) with clinical outcomes have not yet been elucidated. Genotypic variations are unevenly distributed along the sequences of hepatitis delta antigens (HDAgs). Of these variations, the packaging signal at the C-terminus has a divergence of 74% between genotypes I and II. In this report, we address the issue of whether these high variations between genotypes affect assembly efficiency of HDV particles and editing efficiency of RNA. Viral package systems of transfection with expression plasmids of hepatitis B surface antigen and HDAgs or whole genomes of HDV consistently indicate that the package efficiency of genotype I HDV is higher than that of genotype II. Segment swapping of large-form HDAg indicates that the C-terminal 19-residue region plays a key role for the varied assembly efficiencies. Also, the editing efficiency of genotype I HDV is higher than that of genotype II. The nucleotide and structural changes surrounding the editing site may explain why genotype II HDV has a low RNA editing efficiency. The findings of in vitro assembly systems were further supported by the observations that patients infected with genotype II had significantly lower alanine transaminase (ALT) levels, more favorable outcomes (P < .05), and a trend to have lower serum HDV RNA levels as compared with those infected with genotype I HDV (P = .094). In conclusion, genotype II HDV secretes fewer viral particles than genotype I HDV does, which in turn may reduce the extent of infection of hepatocytes and result in less severe hepatic inflammation.
Antibodies to conformational epitopes of soluble liver antigen define a severe form of autoimmune liver disease
Authors: Ma Yun , Okamoto Manabu , Thomas Mark G. , Bogdanos Dimitrios P. , Lopes Agnel R. , Portmann Bernard , Underhill James , Dürr Ralf , Mieli-Vergani Giorgina , Vergani Diego
Published: August 2002

Prevalence and clinical relevance of antibodies to soluble liver antigen (tRNP(Ser)Sec/SLA) in autoimmune hepatitis (AIH) have been investigated using partially purified or prokaryotically expressed antigen. The aim of this study was to improve the detection of anti-tRNP(Ser)Sec/SLA by establishing an immunoassay that was able to identify antibodies directed to conformational epitopes and to investigate the clinical implication of this autoantibody in autoimmune liver disease. By using eukaryotically expressed tRNP(Ser)Sec/SLA as target in a radioligand assay (RLA), 81 patients with autoimmune liver disease (AILD) (33 type 1 AIH, 31 type 2 AIH, and 17 autoimmune sclerosing cholantitis [ASC]), 147 pathologic, and 56 healthy controls were investigated. RLA results were compared with those obtained using a commercial enzyme-linked immunosorbent assay (ELISA) and immunoblot. Reactivity to tRNP(Ser)Sec/SLA was present in 58% of patients with type 1 and type 2 AIH, 41% with ASC, but in only 3 pathologic controls. RLA was similarly disease-specific but remarkably more sensitive than ELISA and immunoblot. A prospective study showed that anti-tRNP(Ser)Sec/SLA-positive patients run a severe clinical course, having worse histology, needing longer to achieve remission, relapsing and requiring liver transplantation or dying more frequently than anti-tRNP(Ser)Sec/SLA negative patients. Anti-tRNP(Ser)Sec/SLA production was favored by the possession of DR3 and A1-B8-DR3 in AIH type 1 and ASC, and prevented by the possession of A2 in all 3 types of AILD, particularly in type 2 AIH. In conclusion, anticonformational tRNP(Ser)Sec/SLA reactivity is frequent in type 1 and type 2 AIH and ASC, defining patients with a worse prognosis.
Hematopoietic Stem Cell Transplantation: A Glimpse of the past and a View of the Future
Authors: Secola Rita
Published: August 2002

Hematopoietic stem cell transplantation (HSCT) is still the only definitive treatment for some malignant and nonmalignant diseases. The amount of knowledge that has been gained regarding this treatment is enormous. Research and development has been focused on minimizing transplant-related toxicity and disease recurrence. Early experiences of bone marrow administration to patients include marrow given by mouth, by intramuscular injection, intramedullary, and by intravenous route. Radiation injury in humans was a major stimulus for research in bone marrow transplantation. Early research was done on animals and then logically applied to humans. Over the last four decades, there was key progress made in the development of conditioning regimens, the selection of patients, the selection of donors, and prevention and treatment of complications associated with the HSCT procedure. It is believed that the recent and future advances of HSCT will make the transplant process less complex and more successful. In the 21st century, there will be marked improvements scientifically, technologically, and clinically in the HSCT procedure. If the current rate of research, clinical development, and dedication continues, HSCT should be even more rewarding and exciting for all clinicians who care for these patients and families.
Rural Families' Perspectives on having a Child with Cancer
Authors: Scott-Findlay Shannon , Chalmers Karen
Published: August 2002

The goal of this study was to explore and describe the experience of being or having a child with cancer in a rural geographic area. An exploratory, descriptive study was conducted that used semistructured, in-depth, tape-recorded interviews as the primary data collection method. Ten rural families who each had a child with cancer formed the sample. Data were analyzed with qualitative analysis procedures. Families' descriptions revealed that their experiences were fraught with challenges that were due to living a great distance from the cancer treatment center. Descriptions of their experiences are presented in this report. Recommendations for health care professionals, particularly pediatric oncology nurses, to assist rural families in coping with their challenges are also offered.