bluuebunny/crossref_metadata_2025 huggingface dataset:
Different Hepatitis B Virus Genotypes Are Associated With Different Mutations in the Core Promoter and Precore Regions During Hepatitis B E Antigen Seroconversion
Authors: Chan Henry L.Y. , Hussain Munira , Lok Anna S.F.
Published: July 2004

Mutations in the core promoter and precore regions are frequently found in hepatitis B e antigen (HBeAg)–negative patients, but precore stop codon mutation is restricted to hepatitis B virus (HBV) genotypes that have T at nucleotide 1858. The aims of this study were to determine the role of core promoter and/or precore mutations in HBeAg seroconversion and their impact on the subsequent course of liver disease, and to determine if core promoter mutations are more frequently selected in patients with HBV genotypes that preclude the development of precore stop codon mutation. Serial sera from 45 patients with chronic HBV infection were polymerase chain reaction (PCR)–amplified, and the HBV core promoter and precore regions were sequenced. Ninety–two percent of patients had core promoter or precore mutations after HBeAg seroconversion: 42% had core promoter changes only, 38% had precore stop codon mutations only, and 12% had changes in both regions. Seventy–three percent of the patients had persistently normal aminotransferases, and only 8% had multiple flares in aminotransferases after HBeAg seroconversion. Core promoter changes were significantly more common in patients infected with HBV who have C at nucleotide 1858 (91% vs. 27%; P < .01), while precore stop codon changes were exclusively found in patients infected with HBV who have T at nucleotide 1858 (87% vs. 0; P < .01). The vast majority of our patients had core promoter and/or precore mutations after HBeAg seroconversion. Nevertheless, most patients had sustained remission of liver disease. Our data suggest that core promoter changes are preferentially selected in patients infected with HBV genotypes that preclude the development of precore stop codon mutation.
Zidovudine (Azt) Causes An Oxidation of Mitochondrial Dna in Mouse Liver
Authors: de la Asunción José García , del Olmo María L. , Sastre Juan , Pallardó Federico V. , Viña José
Published: July 2004

Zidovudine (3′–azido–2′,3′–dideoxythymidine [AZT]) inhibits human immunodeficiency virus replication and delays progression of acquired immune deficiency syndrome. We have recently found that, in muscle, AZT causes oxidative damage to mitochondrial DNA (mtDNA) and other signs of mitochondrial oxidative damage. The aim of this work was to test if AZT causes oxidative damage to liver mtDNA. In our study, an experimental mouse model was used in which mice were administered AZT (10 mg/kg body weight/d) in drinking water. Liver mtDNA of mice treated with AZT had 40% more of the oxidized, mutagenic nucleoside, 8–oxo–7,8–dihydroxy–2′deoxyguanosine (8–oxo–dG) than untreated controls. This oxidative damage to mtDNA is caused by a significant increase (of over 240%) in peroxide production by liver mitochondria from AZT–treated mice, which was prevented by dietary administration with vitamins C and E.
Peroxynitrite Formation During Rat Hepatic Allograft Rejection
Authors: Yamaguchi Yasuo , Okabe Kazutoshi , Matsumura Fujio , Akizuki Eiji , Matsuda Teishi , Ohshiro Hajime , Liang Jian , Yamada Shinwa , Mori Katsutaka , Ogawa Michio
Published: July 2004

The role of nitric oxide (NO) on tissue injury of hepatic allografts during rejection remains controversial. We investigated inducible nitric oxide synthase (iNOS) expression and formation of peroxynitrite in ACI rat liver grafts implanted in recipients. Animals were divided into four experimental groups: group I, isografts; group II, untreated hepatic allografts; group III, allografts treated with FK506; and group IV, allografts pretreated with donor–specific blood transfusion (DST). Serum nitrite/nitrate, interferon–γ (IFN–γ), and tumor necrosis factor–α (TNF–α) concentrations increased significantly in group II rats after transplantation but were significantly lower in groups I, III, and IV. The numbers of macrophages that reacted with an antimacrophage iNOS monoclonal antibody as well as iNOS messenger RNA (mRNA) levels in liver specimens were also much lower in groups I, III, and IV as compared with group II. Immunostaining and Western blot analysis showed prominent tissue nitrotyrosine expression in untreated hepatic allografts, but not in allografts treated with FK506 or donor–specific blood. These results suggest that one of the mechanisms by which production of NO results in injury in rat hepatic allografts may be because of its reaction with superoxide to form peroxynitrite.
Expression of Cyclooxygenase–2 in Human Hepatocellular Carcinoma: Relevance to Tumor Dedifferentiation
Authors: Koga Hironori , Sakisaka Shotaro , Ohishi Masahito , Kawaguchi Takumi , Taniguchi Eitaro , Sasatomi Kurumi , Harada Masaru , Kusaba Taku , Tanaka Masatoshi , Kimura Rina , Nakashima Yutaka , Nakashima Osamu , Kojiro Masamichi , Kurohiji Toshihiko , Sata Michio
Published: July 2004

Cyclooxygenase (rlCOX) is a key enzyme in the synthesis of prostanoids. Two isoforms of this enzyme have been identified: COX–1 and COX–2. Recent studies have suggested that COX–2, but not COX–1, may play a role in colorectal tumorigenesis. In the present study, we investigated the expression of COX–2 as well as COX–1 in human hepatocellular carcinoma (HCC) tissues using immunohistochemistry and immunoblotting. Forty–four surgically resected HCC tissues with adjacent nontumorous livers (NTs), involving 17 cases of chronic viral hepatitis and 27 cases of cirrhosis, and 7 surgically resected, histologically normal liver tissues were used. The well–differentiated HCC expressed COX–2 more frequently and strongly than less–differentiated HCC or hepatocytes of NTs. Less–differentiated HCCs expressed less COX–2 than hepatocytes of NTs, which showed scattered, strong COX–2 expression. Histologically normal liver was weakly positive for COX–2. The expression of COX–1 was weaker than that of COX–2 in hepatic neoplastic and non–neoplastic parenchymal cells. An enhanced expression of COX–1 was not observed in well–differentiated HCCs. Immunoblotting also confirmed up–regulation of COX–2, but not COX–1, in well–differentiated HCCs. The present study is the first to demonstrate a high expression of COX–2 in well–differentiated HCC and a low expression in advanced HCC, in contrast to its continuous expression during colorectal carcinogenesis. These findings suggested that COX–2 may play a role in the early stages of hepatocarcinogenesis, but not in the advanced stages, and may consequently be related to HCC dedifferentiation.
Fibrosis in Chronic Hepatitis C Correlates Significantly With Body Mass Index and Steatosis
Authors: Hourigan Luke F. , Macdonald Graeme A. , Purdie David , Whitehall Vicki H. , Shorthouse Claudia , Clouston Andrew , Powell Elizabeth E.
Published: July 2004

Steatosis is a frequent histological finding in chronic hepatitis C infection; however, the pathophysiology of steatosis and its role in disease progression have not been established. We studied 148 consecutive patients with untreated chronic hepatitis C to assess the effect of body mass index, diabetes mellitus, alcohol consumption, hepatic iron content, and viral load on steatosis and hepatic fibrosis. Ninety–one patients (61%) had steatosis: grade 1 (<30% hepatocytes involved) in 61 (41%), grade 2 (30%–70% hepatocytes involved) in 17 (11%), and grade 3 (>70% hepatocytes involved) in 13 (9%). After adjusting for potential confounding variables, a highly significant relationship was found primarily between steatosis and body mass index (P < .0001). The mean (±SD) body mass index of patients with no steatosis was 23.9 ± 4.3 kg/m2, whereas for grade 1 steatosis it was 26.5 ± 5.1 kg/m2, and for grade 2 and 3 steatosis combined the body mass index was 28.4 ± 4.9 kg/m2. Hepatic fibrosis was significantly associated with age (P = .002). After adjusting for potential confounding variables, including age, hepatic fibrosis was also significantly associated with steatosis (P < .03). There was no significant association between hepatic iron content, alcohol intake, gender, and viral load and steatosis or fibrosis. These findings suggest that increasing body mass index has a role in the pathogenesis of steatosis in chronic hepatitis C and that steatosis may contribute to fibrosis. The association between body mass index and steatosis and fibrosis has important prognostic and therapeutic implications in the management of patients with chronic hepatitis C virus
Generation of Monoclonal Antibodies to Murine Bile Duct Epithelial Cells: Identification of Annexin V As A New Marker of Small Intrahepatic Bile Ducts
Authors: Katayanagi Kazuyoshi , Van de Water Judy , Kenny Thomas , Nakanuma Yasuni , Ansari Aftab A. , Coppel Ross , Gershwin Eric M.
Published: July 2004

Biliary epithelial cells (BECs) are distributed along the length of both the extrahepatic and intrahepatic biliary tree, but have distinctly different phenotypes and functions according to their anatomical location. It has been reasoned that the distinct appearance of pathogenic lesions in different biliary diseases may be associated with the expression of distinct proteins. These data prompted us to immunize rats with cultured murine BECs with the objective of determining if there are unique antigens on BECs. Of the 45 monoclonal antibodies (mAbs) produced, 12 mAbs (MBEC 1–12) were selected for detailed study based on their classification into three major groups. These groups included four antibodies (MBEC 1–4) that reacted in a staining pattern typical of mucin. A second group of mAbs, MBECs 5 to 8, reacted strongly along the biliary tract and by immunoblot analysis, reacted with several bands ranging from 44 kd to 64 kd. These antibodies were considered as markers of pan BECs and their staining pattern proved similar to that of a control polyclonal pan–cytokeratin. The final group of mAbs, MBECs 9 to 12, recognized a 36–kd antigen using lysates of murine BECs. These antibodies also predominantly stained small peripheral bile ducts. The reactive antigen was purified by immunoprecipitation and microsequenced; the peptides sequenced showed 100% homology with murine annexin V. The identification of annexin V with predominantly intrahepatic bile ducts, is of significant interest because of the multiple roles of annexin V, including that of membrane cytoskeletal interactions during transport and apoptosis
Characterization of the Overlap Syndrome of Primary Biliary Cirrhosis (Pbc) and Autoimmune Hepatitis: Evidence for It Being A Hepatitic Form of Pbc in Genetically Susceptible Individuals
Authors: Lohse Ansgar W. , zum Büschenfelde Karl-Hermann Meyer , Franz Beate , Kanzler Stephan , Gerken Guido , Dienes Hans-Peter
Published: July 2004

Some patients with autoimmune liver disease present with a clinical and/or histological picture showing characteristic findings of both autoimmune hepatitis (AIH) and primary biliary cirrhosis (PBC). Various names, mostly overlap syndrome, have been used to describe these cases, which have thus far not been more closely characterized. The aim of this study was the comparison of 20 patients with overlapping features to representative patients considered suffering from typical AIH or typical PBC (20 patients in each group). We found these patients to indeed show a very mixed picture of both conditions biochemically, serologically, and histologically. However, closer analysis suggested that all of these patients were primarily suffering from PBC as all of them had at least either bile duct destruction on histology or anti–M2 positive antimitochondrial antibodies (AMA). We suggest that these PBC patients because of their genetic susceptibility, evidenced by the AIH–characteristic histocompatibility leukocyte antigen (HLA) type B8, DR3, or DR4, developed a more hepatitic picture. Response to immunosuppressive therapy was excellent. We propose that the name “overlap syndrome” be abandoned for “PBC, hepatitic form.” These observations not only have pathophysiological implications, but also suggest that therapy of PBC should be guided by the degree of biochemical and histological hepatic involvement
Novel Recurrent Genetic Imbalances in Human Hepatocellular Carcinoma Cell Lines Identified by Comparative Genomic Hybridization
Authors: Zimonjic Drazen B. , Keck Catherine L. , Thorgeirsson Snorri S. , Popescu Nicholas C.
Published: July 2004

To search for recurrent and specific genomic alterations in human hepatocellular carcinoma (HCC), we examined 18 cell lines by comparative genomic hybridization (CGH), a molecular cytogenetic approach that allows positional identification of gains and losses of DNA sequences of the entire tumor genome. We report here a distinct pattern of multiple recurrent DNA copy–number gains and losses that include alterations frequently seen in other neoplasias as well as changes potentially specific for HCC. The most frequent gains were localized on 1p34.3–35, 1p33–34.1, 1q21–23, 1q31–32, 6p11–12, 7p21, 7q11.2, 8q24.1–24.2, 11q11–13, 12q11–13, 12q23, 17q11.2–21, 17q23–24, and 20p11.1–q13.2. Recurrent losses were mapped on 3p12–14, 3q25, 4p12–14, 4q13–34, 5q21, 6q25–26, 8p11.2–23, 9p12–24, 11q23–24, 13q12–33, 14q12–13, 15q25–26, 18q11.2–22.2, and 21q21–22. Seventeen genomic imbalances are novel in HCC, thus extending significantly the map of genetic changes and providing a starting point for the isolation of new genes relevant in pathogenesis of liver neoplasia, as well as providing molecular probes for both diagnosis and monitoring treatment of the disease
Recurrent and New Hepatitis C Virus Infection After Liver Transplantation
Authors: Everhart James E. , Wei Yuling , Eng Heather , Charlton Michael R. , Persing David H. , Wiesner Russell H. , Germer Jeffrey J. , Lake John R. , Zetterman Rowen K. , Hoofnagle Jay H.
Published: July 2004

Chronic infection with the hepatitis C virus (HCV) is the most common reason for liver transplantation. We examined the results of laboratory tests for HCV on a cohort of patients who received a liver transplant between 1990 and 1994 at three large centers. Seven hundred twenty–two recipients and 604 donors were tested for antibody to HCV (anti–HCV) using a second–generation enzyme–linked immunoassay (EIA–2), followed by recombinant immunoblot (RIBA–2) and HCV RNA confirmation by reverse–transcription polymerase chain reaction (RT–PCR) (with genotyping and viral quantification). Diagnosis of posttransplantation infection required detection of serum HCV RNA that could be genotyped by sequencing or was repeatedly positive despite being unsequenceable. Twenty–five percent of transplantation candidates were seropositive for anti–HCV. Approximately 86% of anti–HCV-positive, 93% of RIBA–positive, and 97% of HCV RNA-positive candidates developed infection after transplantation. Pretransplantation HCV RNA was superior to RIBA–2 for predicting posttransplantation infection. Whereas HCV genotype was identified in nearly all candidates and changed little after transplantation, serum viral levels rose markedly after transplantation. Fifteen donors were either anti–HCV- or HCV RNA-positive. Recipients of grafts from donors with HCV RNA all developed infection, whereas infection was not detected in recipients of grafts from donors with anti–HCV but without detectable HCV RNA. The rate of new infection fell significantly (P = .02) after the introduction of EIA–2 screening of blood. Donor and candidate markers for HCV predict posttransplantation infection
Down–Regulation of Insulin–Like Growth Factor Binding Proteins and Growth Modulation in Hepatoma Cells by Retinoic Acid
Authors: Kim Dae-Ghon , Lee Dae-yeol , Cho Baik-Hwan , You Kyung-Ran , Kim Mi-Young , Ahn Deuk-Soo
Published: July 2004

We observed that all–trans–retinoic acid (RA) down–regulated insulin–like growth factor binding proteins (IGFBPs) in cultured human hepatoma cells (Hep 3B, PLC/PRF/5, and Hep G2); therefore, we characterized the role of this down–regulation in cell growth. Treatment with 10 μmol/L RA revealed a rapid decrease in IGFBP–3 within 2 days, and continued treatment with RA for 6 days resulted in a time–dependent stimulation of Hep 3B cell growth. However, RA treatment decreased IGFBP–1 in PLC/PRF/5 cells and in Hep G2 cells, and the growth–stimulatory activity of RA was transient and less prominent, and was finally obliterated in both cell lines. The addition of 5 ng/mL or 50 ng/mL insulin–like growth factors (IGFs) did not change the growth effects elicited by RA. The addition of IGFBP–3 (1,000 ng/mL) inhibited the growth of Hep 3B cells and counteracted the growth–stimulatory activity of RA, but not completely, suggesting that RA has direct growth–stimulatory activity and that this is enhanced by autocrine down–regulation of IGFBP–3. IGFBP–3 also inhibited the growth of PLC/PRF/5 cells and of Hep G2 cells. Treatment with phosphorylated IGFBP–1 (1,000 ng/mL) alone or with RA did not affect the growth of PLC/PRF/5 cells or Hep G2 cells. However, addition of dephosphorylated IGFBP–1, derived from in vivo dephosphorylation of the phosphorylated form, stimulated the growth of both cell lines, independent of interaction with IGF–I. From these observations, we propose that RA down–regulates IGFBPs, which in turn causes autocrine modulation of cell growth independent of IGF in hepatoma cells in vitro or in vivo. In addition, RA regulates IGFBPs at the posttranscriptional (Hep 3B cells and Hep G2 cells) or transcriptional level (PLC/PRF/5 cells) in a cell–specific manner
Mannose Binding Lectin Gene Mutations Are Associated With Progression of Liver Disease in Chronic Hepatitis B Infection
Authors: Yuen Man-Fung , Lau Chak-Sing , Lau Yu-Lung , Wong Wai-Man , Cheng Chi-Chung , Lai Ching-Lung
Published: July 2004

Mannose–binding lectin (MBL) plays an important role in immune defense. We examined the MBL gene mutations and MBL levels in Chinese hepatitis B and hepatitis C patients with and without symptomatic cirrhosis. We recruited 190 hepatitis B and C patients, and 117 normal Chinese as controls. Serum MBL levels were measured by enzyme–linked immunosorbent assay. MBL gene mutation at codons 52, 54, and 57 was detected by polymerase chain reaction (PCR) assay. In asymptomatic hepatitis B and C patients, there was no increase in codons 52, 54, and 57 mutation, but the MBL levels were significantly lower than those in the controls. Codon 54 mutation rate was increased to 44.4% (P = .007) in symptomatic hepatitis B cirrhosis and 64.3% (P = .0026) in patients with spontaneous bacterial peritonitis (SBP). There was no increase in codon 54 mutation rate in hepatitis B-related hepatocellular carcinoma (HCC). In chronic hepatitis B infection, the odds ratio for an individual with codon 54 mutation to develop cirrhosis was 1.84 (95% CI: 1.21–2.81) and to develop SBP was 4.58 (95% CI: 1.73–12.16). Chronic hepatitis B and hepatitis C infection lowered the MBL levels, probably by suppressing MBL production. Codon 54 mutation of MBL was associated with progression of disease in chronic hepatitis B infection
Mutations in the Basic Core Promotor and the Precore Region of Hepatitis B Virus and Their Selection in Children With Fulminant and Chronic Hepatitis B
Authors: Friedt Michael , Gerner Patrick , Lausch Ekkehart , Trübel Hubert , Zabel Bernhard , Wirth Stefan
Published: July 2004

The involvement of precore stop codon 1896–A and base exchanges in the AT–rich region at positions 1762 and 1764 of the hepatitis B core promotor has been controversely discussed in adults with fulminant hepatitis B. Because no data are currently available on children, we analyzed the basic core promotor (BCP) and precore region in children with chronic and fulminant hepatitis B. The BCP and precore region were sequenced directly and after cloning from mothers and infants. Thirteen children suffered from chronic liver disease, 6 of whom were treated with interferon alfa (IFN–α). All 13 patients seroconverted from hepatitis B e antigen (HBeAg) to hepatitis B e antigen antibodies (anti–HBe), and sera were analyzed before and after seroconversion. Nine vertically infected infants developed a fulminant course of hepatitis B. The occurrence of BCP (1762–T/1764–A, 7.7%) and precore (1896–A, 7.7%; 1899–A, 15%) mutations in chronic hepatitis B was rare. A genotype shift from D to A was observed in 3 patients after development of anti–HBe. A high number of base exchanges was detected in those infants with fulminant hepatitis B. Eight of nine showed a G–A exchange at positions 1896/97 (89%), 1899 (56%), and/or mutations at nucleotide (nt) positions 1762 (56%) and 1764 (78%). All virus strains belonged to genotype D, whereas in the only surviving infant, a D–to–A shift was detected. Hepatitis B virus (HBV) DNA clones were examined from 3 babies and 5 mothers. Our results showed a heterogeneous virus population in 4 of 5 mothers. In contrast, a homogeneous virus population emerged in the infants. According to our data, the analysis in children with fulminant and chronic hepatitis B revealed a striking presence of BCP and precore mutants in infants with fulminant hepatitis (FH) when compared with clinically inapparent anti–HBe-positive children (P < .002), which could be one factor in the pathogenesis of fulminant hepatitis B in children
The Role of Protein Phosphatases in the Expression of Inducible Nitric Oxide Synthase in the Rat Hepatocyte
Authors: Taylor Bradley S. , Liu Shubing , Villavicencio Raphael T. , Ganster Raymond W. , Geller David A.
Published: July 2004

Previously, we demonstrated that nuclear factor–κB (NF–κB) mediates cytokine–induced hepatic inducible nitric oxide synthase (iNOS) expression. NF–κB activation is regulated by kinases and phosphatases whose function is only beginning to be understood. Therefore, experiments were performed to determine the role of protein phosphatases (PPase) in cytokine–induced iNOS expression. Hepatocytes were stimulated with cytokines in the presence or absence of tyrosine phosphatase inhibitors (pervanadate [PV], phenylarsine oxide [PAO]) and a serine–threonine phosphatase inhibitor (okadaic acid [OA]). Cytokines induced hepatocyte iNOS mRNA, protein, and NO2
- production that was substantially decreased by the addition of the tyrosine phosphatase inhibitors (PAO and PV). The serine–threonine phosphatase inhibitor (OA) decreased NO release and protein levels in a concentration–dependent fashion; however, iNOS mRNA levels were not significantly reduced. Nuclear run–on experiments demonstrated that protein tyrosine phosphatases (PTPases) are required for iNOS transcription, while the serine–threonine phosphatase inhibitor (OA) had no effect on iNOS transcription. Electromobility shift assays (EMSAs) revealed that the tyrosine–phosphatase inhibitors blocked cytokine–induced NF–κB activation, while OA did not have a significant effect on NF–κB DNA binding activity. Therefore, tyrosine phosphatases are involved in the regulation of cytokine–induced activation of NF–κB, while serine–threonine phosphatases posttranscriptionally regulate iNOS translation. These results identify the regulatory role of specific protein phosphatases (PPases) in hepatic iNOS expression
High–Level Expression of Rat Class I Alcohol Dehydrogenase Is Sufficient for Ethanol–Induced Fat Accumulation in Transduced Hela Cells
Authors: Galli Andrea , Price Donna , Crabb David
Published: July 2004

The mechanisms by which ethanol causes fatty liver are complex. Reducing equivalents generated during ethanol oxidation inhibit tricarboxylic acid cycle activity and fatty acid oxidation. In addition, ethanol inhibits lipoprotein export and increases fatty acid uptake and lipid peroxidation. To test the role that alcohol metabolism by alcohol dehydrogenase (ADH) has on cellular lipid metabolism, a cell line expressing rat ADH was generated by transducing HeLa cells with an ADH–expressing retrovirus. The cells expressed high levels of ADH protein and had ADH activity similar to that of liver. Exposure of the cells to 20 mmol/L ethanol for 24 hours led to substantial accumulation of free fatty acids and triacylglycerol in the transduced, but not wild–type, HeLa cells. The rate of synthesis of saponifiable lipid was increased significantly by ethanol under these conditions. Ethanol exposure also promoted triacylglycerol accumulation when the cells were incubated with linoleic acid. This was associated with a decrease in the rate at which the cells oxidized 1–[14–C]–linoleic acid. Fat accumulation was not prevented by including α–tocopherol in the medium, arguing against a role for lipid peroxidation. However, the presence of methylene blue completely prevented the fat accumulation. This was associated with a return of the elevated lactate/pyruvate ratio toward normal. These data suggest that generation of reducing equivalents by ADH was sufficient to cause fat accumulation in this cell model
The Long–Term Outcomes of Patients With Compensated Hepatitis C Virus-Related Cirrhosis and History of Parenteral Exposure in the United States
Authors: Hu Ke-Qin , Tong Myron J.
Published: July 2004

It is well known that hepatitis C virus (HCV) infection may progress to cirrhosis and is linked to the development of hepatocellular carcinoma (HCC). Previous studies have shown that compensated HCV–cirrhosis is related to a certain morbidity and mortality in European patients, but little is known in regard to the clinical outcomes of a similar group of patients in the United States. This study investigated this category of patients in terms of the incidence of decompensation, development of HCC, mortality, and the predictive risk factors for morbidity and mortality. The potential effects of interferon (IFN) therapy on outcomes of the disease also were assessed. A total of 112 patients with compensated HCV–cirrhosis and a documented history of either intravenous drug abuse (IVDA) or transfusion were consecutively enrolled. The mean follow–up interval was 4.5 (2–7.7) years. The cumulative probabilities for decompensation and development of HCC were 22.2% and 10.1% in 5 years, with an estimated yearly incidence of 4.4% and 2.0%, respectively. The cumulative survival probability was 82.8% from entry and 51.1% from decompensation in 5 years, with estimated yearly events of mortality and liver transplantation of 3.4% and 9.8%, respectively. It was found that age at entry and initial exposure, initial levels of albumin, platelet count, and prothrombin time (PT) were predictive risk factors for developing decompensation, whereas age at entry and initial exposure, history of transfusion, lower initial levels of albumin, platelet count, and viral load were predictive risk factors for events of mortality and liver transplantation. The incidence of decompensation was significantly lower in patients treated with IFN, but age may have played a contributory role. In contrast, neither HCC development nor mortality was significantly altered by IFN therapy. In conclusion, our study indicated that patients with compensated HCV–cirrhosis in the United States progressed slowly and experienced eventual morbidity and mortality. Once decompensation develops, the disease will be more progressive and result in even higher mortality. Further studies will be required to determine the efficacy of IFN on clinical outcomes in this group of patients.

(Hepatology 1999;29:1311–1316.)
Two Control Elements in the Hepatitis B Virus S–Promoter Are Important for Full Promoter Activity Mediated by Ccaat–Binding Factor
Authors: Bock Claus-Thomas , Kubicka Stefan , Manns Michael Peter , Trautwein Christian
Published: July 2004

Natural occurring mutations in the preS–region are frequently found during chronic hepatitis B virus (HBV) infection. Here we used the mutated preS–region from a patient to study the transcriptional regulation of the S–promoter. The mutations were a CCAAT–box (MUT1) point mutation, a 6–base pair (bp) deletion (MUT2) 3′ of the CCAAT–box, and a 153 bp deletion (MUT3) in the preS2 genome. Transfection experiments revealed for MUT1 and 2 30% to 40% and MUT3 75% of the wildtype (wt) S–promoter activity. In electro–mobility shift assays experiments, binding of a nuclear protein was impaired with MUT1. Ultraviolet cross–linking, South–Western, and gel shift experiments revealed a 30– to 40–kd protein interacting with the wt CCAAT–motif. Computer–assisted analysis and supershift experiments showed that CCAAT–binding factor (CBF) is the CCAAT–box binding protein. Cotransfection experiments with expression vectors for dominant–negative CBF or wt CBF showed that the wt S–promoter but not MUT1 could be regulated through CBF. Additionally, the CBF constructs did not modulate the basal activity of MUT2 but changes the activity of MUT3 like wt HBV. Artificial mutations were introduced in the MUT2 reporter constructs. Transfection experiments revealed that wt promoter activity could not be reconstituted. Therefore these experiments indicated the sterical position of CBF being essential for full S–promoter activity. Our study shows that the CCAAT–box and a second region is essential to mediate full S–promoter activity dependent on CBF. As these mutations also lead to retention of S–protein in the endoplasmic reticulum our results indicate that mutational changes in the preS–region might be linked to the progression of HBV–related liver disease
Recurrence of Primary Sclerosing Cholangitis Following Liver Transplantation
Authors: Graziadei Ivo W. , Wiesner Russell H. , Batts Kenneth P. , Marotta Paul J. , LaRusso Nicholas F. , Porayko Michael K. , Hay Eileen J. , Gores Gregory J. , Charlton Michael R. , Ludwig Jurgen , Poterucha John J. , Steers Jeffery L. , Krom Ruud A.
Published: July 2004

Recurrence of primary sclerosing cholangitis (PSC) following liver transplantation has been suggested; however, it has not been fully defined because of numerous complicating factors and the lack of diagnostic criteria. In the present study, we investigated the recurrence of PSC by developing strict criteria and applying them to a large cohort of PSC patients who underwent liver transplantation. Between March 1985 and June 1996, 150 PSC patients underwent liver transplantation at the Mayo Clinic; mean follow up was 55 months. The incidence of nonanastomotic biliary strictures and hepatic histologic findings suggestive of PSC were compared between patients transplanted for PSC and a non–PSC transplant control group. Our definition of recurrent PSC was based on characteristic cholangiographic and histologic findings that occur in nontransplant PSC patients. By using strict criteria, 30 patients with other known causes of posttransplant nonanastomotic biliary strictures were excluded leaving 120 patients for analysis of recurrence of PSC. We found evidence of PSC recurrence after liver transplantation in 24 patients (20%). Of these, 22 out of 24 patients showed characteristic features of PSC on cholangiography and 11 out of 24 had compatible hepatic histologic abnormalities with a mean time to diagnosis of 360 and 1,350 days, respectively. Both cholangiographic and hepatic histologic findings suggestive of PSC recurrence were seen in nine patients. The higher incidence and later onset of nonanastomotic biliary strictures in patients with PSC compared with a non–PSC control group is supportive of the fact that PSC does recur following liver transplantation. We were unable to identify specific clinical risk factors for recurrent PSC, and the overall patient and graft survival in patients with recurrent PSC was similar to those without evidence of recurrence. Our observations provide convincing evidence that PSC frequently recurs in the hepatic allograft using strict inclusion and exclusion criteria
Mitochondrial Proteins That Regulate Apoptosis and Necrosis Are Induced in Mouse Fatty Liver
Authors: Rashid Asif , Wu Tzyy-Choou , Huang Chao-Cheng , Chen Chien-Hung , Lin Hui Zhi , Yang Shi Qi , Lee Fung Ye , Diehl Anna Mae
Published: July 2004

Fatty liver is common in nonalcoholic, obese individuals and in lean people who consume alcohol chronically. Although fatty liver is typically benign, a subset of individuals with steatosis develop steatohepatitis and eventually cirrhosis. The disparate outcomes of fatty liver suggest that it reflects a generally beneficial, adaptive response to obesity or alcohol–related stress, but may also increase hepatocyte vulnerability to other challenges. Thus, both protective factors (e.g., Bcl–2 and Bcl–xL) and factors that promote hepatocyte death by apoptosis (e.g., Bax) or necrosis (e.g., UCP2) may be increased in fatty livers. To evaluate this possibility, hepatocyte apoptosis, necrosis, and the expression of factors that regulate cellular viability were assessed in two models of fatty liver (i.e., genetically obese [ob/ob] mice and ethanol [EtOH]–fed lean mice). Findings in mice with fatty livers were compared with lean, control mice that did not have hepatic steatosis. Immunohistochemistry showed striking induction of hepatocyte proteins that promote (e.g., Bax) and inhibit (e.g., Bcl–2 and Bcl–xL) apoptosis in both groups with fatty liver. Both models of fatty liver also increased hepatic transcripts for UCP2, a mitochondrial uncoupling protein, and the protein itself was induced in ob/ob hepatocytes. Despite the up–regulation of factors that threaten cell viability, hepatocyte death was not increased in either ob/ob or EtOH–fed mice, confirming that the liver's protective responses were sufficient under the conditions studied. However, if UCP2 induction reduces the efficiency of adenosine triphosphate (ATP) synthesis, this initially harmless response might enhance the vulnerability of hepatocytes to necrosis
A Phase I/Ii Study of Recombinant Human Interleukin–12 in Patients With Chronic Hepatitis C
Authors: Zeuzem Stefan , Hopf Uwe , Carreno Vicente , Diago Moisés , Shiffman Mitchell , Grüne Stefan , Dudley Francis J. , Rakhit Ashok , Rittweger Karen , Yap Sing Hiem , Koff Raymond S. , Thomas Howard C.
Published: July 2004

Interleukin–12 (IL–12) plays a central role in mounting an effective cellular immune response directed towards elimination of intracellular pathogens. The present open–label, multicenter, dose–escalation phase I/II study was designed to assess tolerability, pharmacokinetics, pharmacodynamics, and efficacy of subcutaneously administered recombinant human interleukin–12 (rHuIL–12) in the treatment of chronic hepatitis C. Sixty patients (42 men, 18 women, aged 24–60) were treated with 0.03 μg/kg (n = 16), 0.1 μg/kg (n = 14), 0.25 μg/kg (n = 15), or 0.5 μg/kg rHuIL–12 (n = 15) for 10 consecutive weeks. rHuIL–12 was generally well tolerated, with 2 patients (3.3%) being withdrawn from treatment for adverse events. Treatment was associated with temporary decreases in neutrophils and lymphocyte counts and with elevations in serum transaminases and bilirubin. Serum IL–12 levels observed were higher at 0.5 μg/kg compared with 0.25 μg/kg doses, suggesting a dose–related increase in systemic exposure of IL–12. Measurable levels of interferon gamma (IFN–γ) were also observed at the highest dose of 0.5 μg/kg. At the end of treatment hepatitis C virus (HCV) RNA was detectable in all patients. A more than 50% decrease in pretreatment HCV RNA levels was observed in 3 of 16 patients of the 0.03–μg/kg dose group, in 3 of 14 of the 0.10–μg/kg dose group, in 6 of 15 of the 0.25–μg/kg dose group, and in 8 of 15 patients of the 0.5–μg/kg dose group. Although in several cases serum alanine transaminase (ALT) levels decreased either during or after treatment, ALT normalization was observed in only 4 patients at the end of treatment and in 5 patients at the end of follow–up. Significant anti-rHuIL–12 antibody titers were not detectable in any patient. In conclusion, antiviral activity of rHuIL–12 in patients with chronic hepatitis C does not appear advantageous in comparison with other currently available treatments.
Zonal Regulation of Gene Expression During Liver Regeneration of Urokinase Transgenic Mice
Authors: Locaputo Stephanie , Carrick Terri L. , Bezerra Jorge A.
Published: July 2004

Liver gene transcription plays a fundamental role in the hepatic reparative response to injury. However, little is known about the functional relationship of gene expression between diseased and regenerative compartments following a liver injury. To address the hypothesis that the control of gene expression and the cellular proliferative response are specific to diseased and regenerative liver compartments independently, we assessed the expression of liver growth modulators, hepatocyte proliferation, and apoptosis in transgenic livers overexpressing the urokinase–type plasminogen activator (uPA). uPA livers have regenerative nodules that are visually distinct from the surrounding diseased compartments. Northern analyses using RNA from microdissected regenerative and diseased compartments showed that, among the known liver growth factors studied, there was a selective increase in the expression of hepatocyte growth factor (HGF) in diseased compartments above the levels seen in regenerative compartments and in livers of nontransgenic littermates. Despite the high level of HGF mRNA in diseased compartments, hepatocyte proliferation was low. In contrast, in regenerative compartments, where HGF mRNA was low, hepatocyte proliferation was abundant. For growth inhibitors, mRNA expression for transforming growth factor β1(TGF–β1), p53, and activin A was increased in diseased compartments, where hepatocytes displayed apoptosis. These findings define a zone–specific regulation of gene expression in injured livers and point to an important role of the diseased microenvironment in the fate of hepatocytes during the regenerative process
Increased Angiogenesis in Portal Hypertensive Rats: Role of Nitric Oxide
Authors: Sumanovski Lazar T. , Battegay Edouard , Stumm Michael , van der Kooij Maaike , Sieber Cornel C.
Published: July 2004

Systemic and especially splanchnic arterial vasodilation accompany chronic portal hypertension. Different soluble mediators causing this vasodilation have been proposed, the strongest evidence being for nitric oxide (NO). No data exist if structural vascular changes may partly account for this vasodilatory state. Here, we developed a new in vivo quantitative angiogenesis assay in the abdominal cavity and determined if: 1) portal hypertensive rats show increased angiogenesis; and 2) angiogenesis is altered by inhibiting NO formation. Portal hypertension was induced by partial portal vein ligation (PVL). Sham–operated rats served as controls (CON). During the index operation (day 0), a teflon ring filled with collagen I (Vitrogen 100) was sutured in the mesenteric cavity. After 16 days, rings were explanted, embedded in paraffin, and ingrown vessels counted using a morphometry system. The role of NO was tested by adding an antagonist of NO formation (N
ω–nitro–l–arginine [NNA], 3.3 mg/kg/d) into the drinking water. The mean number of ingrown vessels per implant was significantly higher in PVL rats compared with CON rats, i.e., 1,453 ± 187 versus 888 ± 116, respectively (P < .05; N = 5 per group). NNA significantly (P < .01) inhibited angiogenesis in PVL (202 ± 124; N = 5) and in CON (174 ± 25; N = 6) rats, respectively. In contrast, the β–adrenergic blocker, propranolol, did not prevent angiogenesis either in PVL or CON rats in a separate set of experiments (data not shown). The conclusions drawn from this study are that: 1) rats with portal hypertension show increased angiogenesis; and 2) inhibition of NO formation significantly prevents angiogenesis in both PVL and CON rats. Therefore, splanchnic vasodilation in chronic portal hypertension may also be a result of structural changes.

(Hepatology 1999;29:1044–1049.)
Infection and Hemostasis in Decompensated Cirrhosis: A Prospective Study Using Thrombelastography
Authors: Papatheodoridis George V. , Patch David , Webster George J. , Brooker Jim , Barnes Ellie , Burroughs Andrew K.
Published: July 2004

Bacterial infections are common complications in decompensated cirrhosis, but their relationship with hemostasis has not been studied. We prospectively assessed whether infection affects hemostasis in cirrhosis using routine hemostasis tests and thrombelastography (TEG), a global test of hemostatic function. Eighty–four cirrhotic patients (Child–Pugh B: 26; C: 58) without overt bleeding or blood–product transfusion were prospectively evaluated with routine hemostasis tests and TEG on admission and/or the first day with signs of infection and 5 days later. There were 30 patients with infection; 15 had infection on admission, and 15 developed infection in hospital. In the patients who developed infection in hospital, there was a significant deterioration in all routine hemostasis tests except platelet count (PLT) and in all TEG parameters, on the first day of infection compared with 7 ± 3 days previously. The same parameters significantly improved from the first day of infection to day 5 and after (P < .02) only in the 22 patients whose infection resolved, while the r, k, and α TEG parameters significantly worsened in the 8 patients with persistent infection. In those who developed infection in hospital and were cured (n = 11), the 5–day parameters did not differ from their preinfection values. In conclusion, bacterial infections frequently impair hemostasis in decompensated cirrhotic patients. Successful treatment of infection usually restores hemostasis parameters to preinfection levels in 5 days. Thus, infection may have a role in the bleeding diathesis of cirrhosis
Effect of Interferon Therapy on Hepatocellular Carcinogenesis in Patients With Chronic Hepatitis Type C: A Long–Term Observation Study of 1,643 Patients Using Statistical Bias Correction With Proportional Hazard Analysis
Authors: Ikeda Kenji , Saitoh Satoshi , Arase Yasuji , Chayama Kazuaki , Suzuki Yoshiyuki , Kobayashi Masahiro , Tsubota Akihito , Kobayashi Mizuho , Nakamura Ikuo , Murashima Naoya , Kumada Hiromitsu , Kawanishi Masahiro
Published: July 2004

The activity of interferon (IFN) is not elucidated from the viewpoint of cancer prevention in chronic hepatitis C patientsen masse. The hepatocellular carcinogenesis rate was analyzed statistically in 1,643 patients with chronic hepatitis C: 1,191 patients with IFN therapy and 452 without IFN therapy. Hepatocellular carcinogenesis rates in the treated and untreated groups were 2.1% and 4.8% at the end of the 5th year, and 7.6% and 12.4% at the 10th year, respectively (P= .0036). Multivariate analysis showed that IFN slightly decreased the risk of carcinogenesis by 33%, compared with that of untreated patients (P= .14), adjusting for the confounding effects of age, fibrotic stage, gender, and γ–glutamyl transpeptidase (GGTP) value. Among 1,191 patients with IFN, 461 patients attained persistent loss of hepatitis C virus (HCV) RNA, and the other 145 patients retained normal alanine transaminase (ALT) values without loss of HCV RNA. The hazard of carcinogenesis in these 606 patients with persistent normal ALT with or without HCV–RNA clearance was significantly lower than that of untreated patients (hazard ratio: 0.32;P= .012) and that of the abnormal aminotransferase group. Among patients with chronic hepatitis C, IFN significantly decreased the hepatocellular carcinogenesis rate in those patients with normal or persistent low ALT values
Secretory Low-Molecular–Weight Phospholipases A2 and Their Specific Receptor in Bile Ducts of Patients With Intrahepatic Calculi: Factors of Chronic Proliferative Cholangitis
Authors: Shoda Junichi , Kano Masahito , Asano Toru , Irimura Tatsuro , Ueda Tetsuya , Iwasaki Ryu , Furukawa Masato , Kamiya Junichi , Nimura Yuji , Todoroki Takeshi , Matsuzaki Yasushi , Tanaka Naomi
Published: July 2004

Intrahepatic calculi is characterized by an intractable course and frequent recurrences, requiring multiple operative interventions. Chronic proliferative cholangitis, an active and long–standing inflammation of the stone–containing bile ducts with the hyperplasia of epithelia and the proliferation of the duct–associated mucus glands, may underlie the complex nature of the disease. In terms of the pathophysiology, interest has been focused on the role of secretory low-molecular–weight phospholipases A2 (sPLA2 s) as inflammatory mediators or factors modulating cell functions via their specific sPLA2 –receptor, and also on the production and secretion of altered mucin molecules from the inflamed bile ducts. In search of factors involving chronic proliferative cholangitis, the sPLA2 isoforms in the bile such as the pancreatic–type sPLA2 (group IB sPLA2 ) and the arthritic–type sPLA2 (group IIA sPLA2 ), were assayed to correlate protein masses of the sPLA2 s with alterations in biliary composition. Furthermore, the steady–state messenger RNA (mRNA) levels of the sPLA2 s, the membrane–bound sPLA2 –receptor, cystic fibrosis transmembrane conductance regulator (CFTR), and mucin core polypeptide (MUC) genes in the bile ducts were assayed by reverse– transcriptase polymerase chain reaction (RT–PCR). Immunoreactive sPLA2 –IB and sPLA2 –IIA levels were significantly higher in the bile from the stone–containing hepatic ducts (2315 ± 677 for sPLA2 –IB; 281 ± 42 for sPLA2 –IIA ng/dL, mean ± SEM; n = 20) than in the ductal bile from gallbladder stone patients (609 ± 92, P < .01; 22 ± 2, P < .01; n = 24). The increased sPLA2 levels were associated with a concomitant increase in lysophosphatidylcholine, prostaglandin E2 (PGE2 ), and total mucin concentrations. The affected bile ducts showed an increased mRNA level of sPLA2 –IB and sPLA2 –IIA compared with the ducts from control subjects, in whom the mRNAs of the sPLA2 –receptor and other sPLA2 isoforms, such as groups V and X sPLA2 s, were coincidently expressed. Reflecting the increased amounts of total biliary mucins, the affected ducts showed an increase in mRNA levels of CFTR as well as MUC2, MUC3, MUC5AC, MUC5B, and MUC6 compared with the ducts from control subjects. In intrahepatic calculi, an enhanced expression of the sPLA2 s and their possible cross–talk via sPLA2 –receptor may be of pathophysiological significance for the chronic proliferative cholangitis, in association with the enhanced CFTR expression and the alterations in mucin gene expression in the bile ducts, probably through potentiating arachidonate metabolism with associated biliary alterations favoring growth of preexisting stones and even further progressions.

(Hepatology 1999;29:1026–1036.)
Enhanced Duck Hepatitis B Virus Gene Expression Following Aflatoxin B Exposure
Authors: Barraud Luc , Guerret Sylviane , Chevallier Michele , Borel Christelle , Jamard Catherine , Trepo Christian , Wild Christopher P. , Cova Lucyna
Published: July 2004

Epidemiological studies have suggested synergistic interactions between chronic hepatitis B virus (HBV) infection and aflatoxin B1 (AFB1) exposure in the etiology of hepatocellular carcinoma (HCC), although the molecular mechanisms of their interactions are still not understood. The aim of this study was to use the Pekin duck model to investigate the impact of AFB1 exposure on duck hepatitis B virus (DHBV) replication during the early stages of virus–carcinogen interactions. Six–week–old chronic DHBV–carrier or uninfected ducks were exposed to AFB1 for 5 weeks or treated with dimethylsulfoxide (DMSO) as a control. Animals were observed for 6 to 13 weeks after AFB1 treatment to study the influence of AFB1 exposure on DHBV replication and liver pathologies. Histological analysis showed more marked changes in the livers of AFB1–treated ducks, and these were enhanced by DHBV infection. A significant increase in serum and liver DHBV DNA level was observed in AFB1–treated ducks as compared with DMSO–treated controls. In addition, viral RNAs, in particular the pregenomic RNA that is the template of viral replication, and intrahepatic DHBV DNA replicative intermediates, were significantly increased by AFB1 treatment. Moreover, an overexpression and accumulation of DHBV large envelope (L) protein was observed in the hepatocytes of AFB1–exposed animals. The in vitrostudy has further confirmed an increase in intracellular viral DNA and in virus release in AFB1–treated primary duck hepatocytes. Taken together, our results indicate that AFB1 exposure leads to an increase in virus gene expression associated with intrahepatic accumulation of DHBV L protein and enhanced liver pathology.