bluuebunny/crossref_metadata_2025 huggingface dataset:
Emergence and takeover of YMDD motif mutant hepatitis B virus during long-term lamivudine therapy and re-takeover by wild type after cessation of therapy
Authors: Chayama Kazuaki , Suzuki Yoshiyuki , Kobayashi Masahiro , Kobayashi Mizuho , Tsubota Akihito , Hashimoto Michie , Miyano Yukiko , Koike Hiromi , Kobayashi Mariko , Koida Isao , Arase Yasuji , Saitoh Satoshi , Murashima Naoya , Ikeda Kenji , Kumada Hiromitsu
Published: July 2004

Treatment of hepatitis B virus (HBV) with lamivudine is effective in suppressing virus replication and results in reduced inflammatory activity. However, the emergence of lamivudine-resistant mutant virus, with amino acid substitution in the YMDD motif of DNA polymerase, has been reported. We report the emergence and takeover of YMDD mutant and re-takeover by wild type during and after long-term lamivudine therapy. YMDD mutants were detected in five patients who showed DNA breakthrough (HBV DNA becoming detectable after a period of DNA negativity), which occurred after 9 to 14 months of lamivudine therapy. Four of five mutants had amino acid sequence YIDD, and the remaining mutant had YVDD. Patients with high HBV-DNA titer and/or hepatitis B e antigen tended to develop breakthrough (P = .038). Using a sensitive and specific polymerase chain reaction (PCR)-based method developed in this study, the emergence of YMDD mutants was detected 1 to 4 months before DNA breakthrough, but not detected in any of the pretreatment sera. The mutants were predominant at breakthrough, but were replaced by wild-type virus 3 to 4 months after cessation of therapy in the two patients who discontinued therapy. One of these patients had a relapse of hepatitis. Mutant continued to replicate in the remaining three patients who continued to receive treatment, and relapse occurred in only one of these patients. Our results suggest that the replication of YMDD mutant viruses is less than wild type and is re-overtaken by wild type after cessation of therapy. Re-administration of lamivudine, possibly combined with other antiviral therapy, might be useful in some patients experiencing hepatitis with lamivudine-resistant variants.
Effect of alcohol consumption on serum hepatitis C virus RNA and histological lesions in chronic hepatitis C
Authors: Pessione Fabienne , Degos Françoise , Marcellin Patrick , Duchatelle Véronique , Njapoum Corinne , Martinot-Peignoux Michèle , Degott Claude , Valla Dominique , Erlinger Serge , Rueff Bernard
Published: July 2004

The role of alcohol intake in the occurrence of severe liver disease in chronic hepatitis C virus (HCV) carriers is still debated. A cross-sectional study has been conducted in 233 chronic hepatitis C virus carriers. Weekly self-reported alcohol consumption (SRAC) was evaluated, serum HCV RNA levels were measured by a branched DNA technique (Quantiplex 2.0) and HCV genotypes were determined. A liver biopsy was performed simultaneously and liver lesions were graded with the Knodell histological activity index. Data were examined by uni- and multivariate analyses. Alcohol consumption was relatively low (< 140 g/per week in 193/233 patients [80%]). We found a highly significant correlation between SRAC and serum HCV RNA levels (r = .26, P = .001). Fibrosis was significantly correlated with age and alcohol consumption. These results suggest that in HCV carriers, alcohol consumption, even with low alcohol intake, increases viremia and hepatic fibrosis. Chronic HCV carriers should be advised to avoid regular alcohol intake.
Role of alcohol in the progression of liver disease caused by hepatitis C virus infection
Authors: Ostapowicz George , Watson Katrina J. , Locarnini Stephen A. , Desmond Paul V.
Published: July 2004

In patients with chronic hepatitis C, alcohol consumption has been proposed as a risk factor for the progression of liver disease; however, evidence for this remains conflicting. Two hundred thirty-four anti-hepatitis C virus (HCV)-positive patients who had a liver biopsy performed within the past 24 months were studied. Demographic data and information on risk factors were recorded. A detailed lifetime alcohol consumption history was obtained. Viral studies included HCV viral titer and HCV genotype. Mean age (± SEM) of the group was 40.8 ± 0.7 years. One hundred sixty-six (71%) were male. A risk factor for HCV infection was found in 195 patients (86%). Genotype distribution was: 1b: 22%; 1a: 15%; 1(nonsubtypable): 15%; 3a: 34%; and 2: 7%. Fifty (21%) patients had cirrhosis. Patients with cirrhosis were older (51.6 ± 1.8 years) than those with chronic hepatitis (37.6 ± 0.6 years; P = .0001), were infected at an older age (25.9 ± 2.0 vs. 20.9 ± 0.6 years; P = .001), and had a longer duration of infection (20.5 ± 1.3 vs. 16.2 ± 0.5 years; P = .0008). Patients with cirrhosis had a greater total lifetime alcohol consumption (288,765 ± 58,115 g) than those with chronic hepatitis (189,941 ± 15,453 g; P = .018). Cirrhotic patients also had greater total alcohol consumption during the period of infection with HCV (240,962 ± 63,756 g vs. 146,510 ± 12,862 g; P = .02). On multivariate analysis, subject age and total alcohol consumption were independently associated with the presence of cirrhosis. Total lifetime alcohol consumption is a risk factor for the progression of liver disease caused by HCV.
Hepatic bile versus gallbladder bile: A comparison of protein and lipid concentration and composition in cholesterol gallstone patients
Authors: Keulemans Yolande C. , Mok Kam S. , de Wit Laurens Th , Gouma Dirk J. , Groen Albert K.
Published: July 2004

Many studies have demonstrated that gallbladder bile (but not hepatic bile) of animals or patients with cholesterol gallstones contains higher protein concentrations than does gallbladder bile of control patients without stones or with pigment stones. The underlying defect has not been elucidated. To establish whether there is net production or net absorption/degradation of protein by gallbladder epithelium for different classes of protein, paired samples of hepatic and gallbladder bile were obtained from fourteen patients with cholesterol gallstones during elective cholecystectomy. In these paired samples, lipid and protein composition were determined. To obtain the concentration ratio (CR) of protein and lipid, its concentration in the gallbladder was divided by the concentration determined in the paired hepatic bile sample. The CR of bile salts was used as a parameter for water absorption in the gallbladder. Of the biliary proteins that were determined only mucin, albumin, immunoglobulin (Ig) G, and aminopeptidase N appeared to increase in the gallbladder from another cause than water absorption. A strong correlation was found between mucin, albumin, and IgG. Haptoglobin, α1-acid glycoprotein, IgM, and IgA appeared to be absorbed by gallbladder epithelium in the majority of patients. In cholesterol gallstone patients, total protein concentration in gallbladder bile of cholesterol gallstone patients is increased when compared with hepatic bile. The increase in protein concentration cannot be explained for all bile samples solely by water absorption. In this study we show that the defect is largely caused by a selective increase in albumin, mucin, and IgG. All other proteins which were investigated are taken up by the gallbladder.
Optimal timing of liver transplantation for primary biliary cirrhosis
Authors: Kim Ray W. , Wiesner Russell H. , Therneau Terry M. , Poterucha John J. , Porayko Michael K. , Evans Roger W. , Klintmalm Goran B. , Crippin Jeffrey S. , Krom Ruud A. , Dickson Rolland E.
Published: July 2004

In 1989, we reported on the efficacy of liver transplantation in primary biliary cirrhosis (PBC) by demonstrating that the actual patient survival following transplantation was significantly better than without transplantation as predicted by a mathematical survival model (“Mayo natural history model”). Our aim in this investigation was to determine an optimal time to perform liver transplantation in PBC. One hundred forty-three patients with PBC undergoing liver transplantation were followed prospectively. Disease severity was measured immediately before transplantation by a summary score (“risk score”) used in the Mayo natural history model, namely age, bilirubin, albumin, prothrombin time, and the presence or absence of edema. Proportional hazards analyses were performed assessing patient survival following transplantation. The influence of disease severity immediately pretransplantation on resource utilization for liver transplantation was assessed. Compared with our report in 1989, liver transplantation was performed at an earlier stage of disease (e.g., median risk score: 7.5 vs. 8.3; P < .01). Following transplantation, patient survival probabilities at 1, 2, and 5 years were 93%, 90%, and 88%, respectively. In the proportional hazards analysis, the risk of death following transplantation remained low until reaching a risk score of 7.8. In contrast, risk scores greater than 7.8 were associated with a progressively increased mortality. Resource utilization measured by the days in the intensive care unit (ICU) and hospital and the requirement for intraoperative blood transfusions was significantly greater in recipients who had higher risk scores before transplantation. Our data suggest that an optimal timing for liver transplantation, as determined by patient survival and resource utilization, appears to be at a risk score around 7.8 in patients with PBC.
Increased cerebrovascular resistance in cirrhotic patients with ascites
Authors: Guevara Mónica , Bru Concepción , Ginès Pere , Fernández-Esparrach Gloria , Sort Pau , Bataller Ramón , Jiménez Wladimiro , Arroyo Vicente , Rodés Juan
Published: July 2004

Studies assessing regional hemodynamics in patients with cirrhosis and ascites have shown vasodilation in the splanchnic circulation and vasoconstriction in the renal circulation and in the brachial and femoral artery vascular territories.The aim of this study was to assess the cerebral vascular resistance in cirrhotic patients with ascites.The resistive index in the middle cerebral artery (an index of the cerebral vascular resistance)and in a renal interlobar artery were measured by Doppler ultrasonography in 7 healthy subjects: 13 patients with compensated cirrhosis and 24 patients with ascites (13 with renal failure).The arterial blood pressure and the activity of the renin-angiotensin and sympathetic nervous systems, as estimated by plasma renin activity and plasma norepinephrine concentration, respectively, were also measured. The resistive index in the middle cerebral artery was significantly increased in patients with ascites (0.68 ± 0.05, mean ± SD)as compared with patients without ascites (0.60 ± 0.01, P<.05) and with healthy patients (0.52 ± 0.01, P<.01).Renal resistive index was also increased in patients with ascites (0.77 ± 0.01) compared with the other two groups (0.68 ± 0.02 and 0.62 ± 0.00, respectively; P < .001). The resistive index in the middle cerebral artery showed a direct correlation with renal resistive index (r = .73, P< .01), plasma renin activity (r= .61, P < .01), and norepinephrine (r= .53, P< .01). The resistive index in the middle cerebral artery showed an inverse correlation with mean arterial pressure (r=−.45, P < .01). These results indicate that in patients with cirrhosis and ascites there is a cerebral vasoconstriction which is probably related with the arterial hypotension and the overactivity of vasoconstrictor systems.
A novel human tumor necrosis factor alfa mutein, F4614, inhibits in vitro and in vivo growth of murine and human hepatoma: Implication for immunotherapy of human hepatocellular carcinoma
Authors: Atarashi Yoshinari , Yasumura Satoshi , Nambu Shuji , Yoshio Yukimatsu , Murakami Jun , Takahara Terumi , Higuchi Kiyohiro , Watanabe Akiharu , Miyata Keizo , Kato Masanari
Published: July 2004

Although treatment for hepatocellular carcinoma (HCC) has recently improved, most patients still relapse and die from this disease. The development of new therapeutic and preventive strategies for HCC is, therefore, required. A novel mutant protein (mutein) of human tumor necrosis factor alfa (TNF-α mutein F4614,1SSSRGDSD··· 29V ··· 155L) was developed to decrease several adverse effects of TNF-α. F4614 is known to lack hypotensive effects of human TNF-α without losing its anti-tumor effect in mice transplanted with Meth-A sarcoma. Our study investigated the anti-tumor effects of F4614 against hepatoma cells in vitro and in vivo. F4614 significantly inhibited growth of all four tumor cells in vitro. A murine hepatoma cell line, MH134, when incubated in the presence of F4614, exhibited upregulation of surface major histocompatibility complex (MHC) class-I, intercellular adhesion molecule-1 (ICAM-1) and B7-1 molecules, and a decreased proportion of cells in the G2/M phase of the cell cycle. In addition, F4614 induced apoptosis in a significant number of MH134 cells. TNF-α and F4614 (5 μg/mouse daily for 5 days) showed similar anti-tumor activities in syngeneic MH134-bearing mice and heterogeneic PLC/PRF/5-bearing athymic nude mice. Intratumoral injection of F4614 or TNF-α was more effective than intravenous injection. Immunohistochemical analysis of the tumors treated by F4614 revealed that tumors were surrounded with a large number of Mac-1+ cells and a small number of CD4+ and CD8+ T cells; that suggests that intratumoral injection of F4614 elicited host immunoreactions. Thus, F4614 may be a new strategy for immunotherapy of HCC.
Expression of vascular endothelial growth factor in human hepatocellular carcinoma
Authors: Yamaguchi Rin , Yano Hirohisa , Iemura Akihiro , Ogasawara Sachiko , Haramaki Makoto , Kojiro Masamichi
Published: July 2004

Vascular endothelial growth factor (VEGF) is thought to take an important role in tumor angiogenesis. The present study examined VEGF expression immunohistochemically in hepatocellular carcinomas (HCCs) in various histological grades and sizes. In HCCs that were composed of cancerous tissues of single histological grade, VEGF expression was the highest in well-differentiated HCCs, followed by moderately differentiated HCCs, and then poorly differentiated HCCs. VEGF positivity gradually decreased with the increase in tumor size. In the nodules larger than 3.0 cm, 36.8% were VEGF-negative. In HCCs consisting of cancerous tissues of two different histological grades, the expression was less intensive in the higher-grade HCC component. VEGF was not expressed in sarcomatous areas, while VEGF was expressed in the surrounding HCC tissues. The expression was also remarkable in the noncancerous tissues in which inflammatory cell infiltration was apparent. VEGF expression was also examined in six HCC cell lines. In reverse-transcription polymerase chain reaction (RT-PCR) analysis, expressions of the two secretion types (VEGF121 and VEGF165)were the highest. Thus, VEGF protein in culture supernatant was measured by using enzyme-linked immunosorbent assay (ELISA) with or without inflammatory cytokines, i.e., interleukin (IL)-1β, interferon (IFN)-α, IFN-γ, and tumor necrosis factor (TNF)-α; and growth factors, i.e., epidermal growth factor (EGF), platelet-derived growth factor (PDGF)-BB, basic fibroblast growth factor (bFGF), and transforming growth factor (TGF)-α. As a result, secretion of VEGF from the cell lines was up-regulated at various degrees. Based on these findings, VEGF expression in HCC tissues was thought to be related to the histological grade. The findings also indicate that various cytokines and growth factors could cooperatively act to enhance VEGF expressions in HCC.
Genomic fluidity is a necessary event preceding the acquisition of tumorigenicity during spontaneous neoplastic transformation of WB-F344 rat liver epithelial cells
Authors: Hooth Michelle J. , Vincent Jack L. , Coleman William B. , Presnell Sharon C. , Grisham Joe W. , Smith Gary J.
Published: July 2004

The genomic evolution of a cohort of WB-F344 rat liver epithelial cell lineages undergoing spontaneous neoplastic transformation was followed to define the mechanistic relationship between genomic instability and progression to the neoplastic phenotype. Eighteen independent populations of WB-F344 cells (initiated from a single diploid-founding population) were subjected to 12 cycles of selective growth at confluent cell density, and cellular DNA contents were measured after each selection cycle. Flow cytometry demonstrated significant gains in the amount of G1 DNA after selection cycles 3, 6, and 7 in 44% (8 of 18), 89% (16 of 18), and 39% (7 of 18) of the cell populations, respectively. All populations subsequently lost DNA and returned to a diploid or pseudo-diploid DNA content within 1 to 2 selection cycles after the appearance of an increased DNA content. Additionally, appearance and subsequent disappearance of aneuploid or tetraploid subpopulations was observed in 11% (2 of 18) and 83% (15 of 18) of the experimental lineages, respectively. Although perturbations of G1 DNA content were apparent as early as selection cycle 3, at least 8 cycles of selective growth were required for the acquisition of tumorigenicity. While the independent lineages demonstrated significant fluctuations in G1 DNA content between selection cycles 3 and 8, the majority (11 of 13) of the populations contained a diploid or pseudo-diploid DNA content at the time tumorigenicity was expressed. Genomic instability preceded the acquisition of tumorigenic potential in rat liver epithelial cells subjected to selective growth conditions of maintenance at confluence, and may be required for its expression.
Allelic imbalance of insulin-like growth factor II gene expression in cancerous and precancerous lesions of the liver
Authors: Aihara Tomohiko , Noguchi Shinzaburo , Miyoshi Yasuo , Nakano Hiroshi , Sasaki Yo , Nakamura Yusuke , Monden Morito , Imaoka Shingi
Published: July 2004

Allelic imbalance of the insulin-like growth factor II (IGF II) gene expression is often seen in hepatocellular carcinoma (HCC). To investigate the role of allelic imbalance in hepatocarcinogenesis, we have studied allelic expression status of the IGF II gene in dysplastic nodules, which are precancerous lesions of HCC, as well as in HCCs of different histological grade, and the influence of the allelic imbalance on IGF II gene expression has also been examined. Allelic imbalance was observed in 3 of 7 dysplastic nodules, in 7 of 9 well-differentiated HCCs, and in 8 of 9 moderately differentiated HCCs. IGF II gene expression level, which was studied by a semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR), was significantly higher (3.6-fold) in the dysplastic nodules than the control livers, but a significant increase in the IGF II gene expression was not observed in well- and moderately differentiated HCCs as compared with the control livers. These results demonstrate that the allelic imbalance of the IGF II gene expression is seen in the early stage (precancerous lesions) of hepatocarcinogenesis. Association of the allelic imbalance with an increased expression of the IGF II gene in the precancerous lesions might suggest a possible involvement of an IGF II autocrine loop in the pathogenesis of these lesions.
Microsatellite instability and loss of heterozygosity at DNA mismatch repair gene loci occurs during hepatic carcinogenesis
Authors: Macdonald Graeme A. , Greenson Joel K. , Saito Koji , Cherian Sajeev P. , Appelman Henry D. , Boland Richard C.
Published: July 2004

DNA mismatch repair is an important mechanism involved in maintaining the fidelity of genomic DNA. Defective DNA mismatch repair is implicated in a variety of gastrointestinal and other tumors; however, its role in hepatocellular carcinoma (HCC) has not been assessed. Formalin-fixed, paraffin-embedded archival pathology tissues from 46 primary liver tumors were studied by microdissection and microsatellite analysis of extracted DNA to assess the degree of microsatellite instability, a marker of defective mismatch repair, and to determine the extent and timing of allelic loss of two DNA mismatch repair genes, human Mut S homologue-2 (hMSH2) and human Mut L homologue-1 (hMLH1), and the tumor suppressor genes adenomatous polyposis coli gene (APC), p53, and DPC4. Microsatellite instability was detected in 16 of the tumors (34.8%). Loss of heterozygosity at microsatellites linked to the DNA mismatch repair genes, hMSH2 and/or hMLH1, was found in 9 cases (19.6%), usually in association with microsatellite instability. Importantly, the pattern of allelic loss was uniform in 8 of these 9 tumors, suggesting that clonal loss had occurred. Moreover, loss at these loci also occurred in nonmalignant tissue adjacent to 4 of these tumors, where it was associated with marked allelic heterogeneity. There was relatively infrequent loss of APC, p53, or DPC4 loci that appeared unrelated to loss of hMSH2 or hMLH1 gene loci. Loss of heterozygosity at hMSH2 and/or hMLH1 gene loci, and the associated microsatellite instability in premalignant hepatic tissues suggests a possible causal role in hepatic carcinogenesis in a subset of hepatomas.
Collagen XVIII is localized in sinusoids and basement membrane zones and expressed by hepatocytes and activated stellate cells in fibrotic human liver
Authors: Musso Orlando , Rehn Marko , Saarela Janna , Théret Nathalie , Liétard Jocelyne , Hintikka Elina , Lotrian Dominique , Campion Jean-Pierre , Pihlajaniemi Taina , Clément Bruno
Published: July 2004

Type XVIII collagen is a recently discovered nonfibrillar collagen associated with basement membranes in mice and expressed at high levels in human liver. We studied the origin, distribution, and RNA levels of type XVIII collagen in normal and fibrotic human livers by in situ hybridization, immunohistochemistry, and Northern and dot blots and compared procollagen α1(XVIII) RNA levels with those of procollagen α1(IV) and laminin γ1, the two major components of liver basement membranes. In normal liver, type XVIII collagen was heavily deposited in perisinusoidal spaces and basement membrane zones. The major source of type XVIII collagen was hepatocytes and, to a lesser extent, endothelial, biliary epithelial, and vascular smooth muscle cells and peripheral nerves. In cirrhosis, type XVIII collagen formed a thick deposit along capillarized sinusoids. Grain counts after in situ hybridization showed myofibroblasts to increase their expression 13-fold in active and twofold in quiescent fibrosis, whereas hepatocytes increased their expression only twofold in both active and quiescent fibrosis. Activated stellate cells in vitro expressed type XVIII collagen at high levels. These data indicate that type XVIII collagen is a component of the perisinusoidal space and is associated with basement membrane remodeling. Hepatocytes and activated stellate cells are important sources of type XVIII collagen in normal and fibrotic liver respectively, which suggests tissue-specific regulation of its expression.
Initial heme uptake from albumin by short-term cultured rat hepatocytes is mediated by a transport mechanism differing from that of other organic anions
Authors: Noyer Charles M. , Immenschuh Stephan , Liem Heng H. , Muller-Eberhard Ursula , Wolkoff Allan W.
Published: July 2004

Although it is known that circulating heme accumulates in liver cells, the process by which heme enters hepatocytes is only partly understood. Hemopexin and a putative hemopexin receptor on hepatocyte membranes may mediate the uptake process. However, whether there are sufficient hemopexin receptors on rat hepatocytes to account for the bulk of heme entering cells is unknown. It is likely that heme may be transferred directly from albumin with the help of a plasma membrane heme transporter. To clarify the transport mechanism of heme into liver cells, we studied the uptake by short-term cultured rat hepatocytes of 55Fe-heme incubated with rat serum albumin. In these cells, the initial uptake of 55Fe-heme at 37°C was five- to eightfold higher than that at 4°C, linear for at least 5 minutes, and saturable. The K
m of heme uptake was 0.95 ± 0.27 μmol/L, and the V
max was 0.12 ± 0.01 pmol/min/mg protein (n = 3). Neither isosmotic substitution of sucrose for NaCl in the medium nor adenosine triphosphate (ATP) depletion, perturbations that are known to reduce uptake of bilirubin, sulfobromophthalein (BSP), and taurocholate, had any influence on 55Fe-heme uptake. In addition, heme uptake was not reduced in the presence of a greater than 500-fold molar excess of BSP. These results indicate that hepatocytes take up heme by a process that is distinct from that of these other organic anions.
Expression of cytosolic β-glucosidase in guinea pig liver cells
Authors: Hays William S. , Wheeler Donald E. , Eghtesad Bijan , Glew Robert H. , Johnston David E.
Published: July 2004

The cytosolic β-glucosidase of mammalian liver has been implicated in the metabolic transformation of plant glycosides, such as vicine and amygdalin, which are associated with the development of toxic syndromes. We investigated which cell types express cytosolic β-glucosidase in guinea pig liver, and characterized the contribution of this enzyme to the hydrolysis of aromatic glucosides in cultured cells and in tissue slices. Cytosolic β-glucosidase was expressed in hepatocytes and not in Kupffer or endothelial cells as determined by enzyme-specific activity and Western blots of liver cell extracts. Intracellular β-glucosidase activity was visualized using the fluorescent β-glucosidase substrate, resorufin β-d-glucoside, and shown to be caused by the cytosolic β-glucosidase using the inhibitors, conduritol β-epoxide and dinitrophenol-2-deoxy-2-fluoro-β-d-glucopyranoside (DNP2FGlc). Staining of fresh liver slices with resorufin β-glucoside revealed that cytosolic β-glucosidase is expressed in all hepatocytes, with no significant portal-central gradient. These data indicate that cytosolic β-glucosidase is a hepatocyte-specific enzyme, and support the hypothesis that cytosolic β-glucosidase in the liver functions to hydrolyze small glucosides absorbed by the intestine. Furthermore, toxic injury to cultured hepatocytes by CCl4 resulted in release of cytosolic β-glucosidase in parallel with the hepatocyte marker enzymes alanine transaminase and lactate dehydrogenase. This suggests that acute increases in serum levels of cytosolic β-glucosidase in animal models of liver injury may reflect direct injury of hepatocytes.
Lack of coordinate control of ferritin and transferrin receptor expression during rat liver regeneration
Authors: Cairo Gaetano , Tacchini Lorenza , Pietrangelo Antonello
Published: July 2004

Transferrin receptor (TfR) and ferritin, key proteins of cellular iron metabolism, are coordinately and divergently controlled by cytoplasmic proteins (iron regulatory proteins, IRP-1 and IRP-2) that bind to conserved mRNA motifs called iron-responsive elements (IRE). IRP, in response to specific stimuli (low iron levels, growth and stress signals) are activated and prevent TfR mRNA degradation and ferritin mRNA translation by hindering ferritin mRNA binding to polysomes. We previously found that, in regenerating liver, IRP activation was accompanied by increased TfR mRNA levels, but not by reduced ferritin expression. The basis for this unexpected behavior was investigated in the present study. Liver regeneration triggered by carbon tetrachloride (CCl4) stimulated by four- to fivefold the synthesis of both L and H ferritin chains. This increase was accompanied with a transcriptionally regulated twofold rise in the amount of ferritin mRNAs. Moreover, polysome-associated ferritin transcripts were fourfold higher in CCl4-treated animals than in control animals. Because RNA bandshift assays showed a fourfold increase in IRP-2 binding activity after CCl4administration, activated IRP in regenerating liver seemed unable to prevent ferritin mRNAs binding to polysomes. This was confirmed by direct demonstration in the wheat germ translation system that the efficiency of IRP as a translational repressor of a mRNA bearing an IRE motif in front of a reporter transcript is impaired in CCl4-treated rats in spite of an enhanced IRE-binding capacity. In conclusion, we show for the first time that the paradigm of coordinate and opposite control of ferritin and TfR by IRP is contradicted in liver regeneration. Under these circumstances, growth-dependent signals may activate ferritin gene transcription and at the same time hamper the ability of activated IRP-2 to repress translation of ferritin mRNAs, thus preserving for growing liver cells an essential iron-storage compartment.
Continuous human cell lines inducibly expressing hepatitis C virus structural and nonstructural proteins
Authors: Moradpour Darius , Kary Petra , Rice Charles M. , Blum Hubert E.
Published: July 2004

Investigation of the hepatitis C virus (HCV) life cycle and the evaluation of novel antiviral strategies are limited by the lack of an efficient cell culture system. Therefore, continuous human cell lines inducibly expressing the entire HCV open reading frame were generated with use of a tetracycline-regulated gene expression system. HCV transgenes were found to be chromosomally integrated in a head-to-tail configuration. Northern blot analyses revealed a tightly regulated unspliced transcript of approximately 9 kilobases (kb). HCV structural and nonstructural proteins were faithfully processed, indicating that the cellular and viral proteolytic machineries and posttranslational modification pathways are fully functional in these cell lines. Steady state expression levels could be regulated over a broad range by the concentration of tetracycline present in the culture medium. Kinetic analyses revealed a half-life of less than 1 hour for the HCV RNA whereas a half-life of approximately 9.5, 12, 11, and 10 hours was found for core, NS3, NS4A, and NS5A proteins, respectively. Viral proteins were found to colocalize in the cytoplasm in a pattern characteristic of the endoplasmic reticulum. High-level expression of HCV proteins in the fully induced state was toxic to the cells. These cell lines provide a unique in vitro system to analyze structural and functional properties of HCV proteins, their interactions with cellular proteins and pathways, and the requirements for HCV morphogenesis. In addition, they should prove useful for the evaluation of novel antiviral strategies against hepatitis C in a well-defined and reproducible cellular context.
Human and murine antibody recognition is focused on the ATPase/Helicase, but not the protease domain of the hepatitis C virus nonstructural 3 protein
Authors: Chen Margaret , Sällberg Matti , Sönnerborg Anders , Jin Ling , Birkett Ashley , Peterson Darrell , Weiland Ola , Milich David R.
Published: July 2004

The hepatitis C virus (HCV) nonstructural (NS) 3 protein has been shown to possess at least two enzymatic domains. The amino terminal third contains a serine-protease domain, whereas the carboxy terminal two thirds is comprised of an adenosine triphosphatase (ATPase)/helicase domain. These domains are essential for the maturation of the carboxy-terminal portion of the HCV polyprotein and catalyze the cap synthesis of the RNA genome. In this report, human and murine antibody responses induced by NS3 were characterized using a recombinant full-length NS3 (NS3-FL) protein, or the isolated protease or ATPase/helicase domains, expressed and purified from Escherichia coli. Sera from 40 patients with chronic HCV infection were assayed in enzyme-linked immunoassays (EIAs) for antibody binding to the panel of NS3 proteins. Virtually all patient sera contained antibodies specific for NS3-FL and the ATPase/helicase domain, whereas only 10% of sera reacted with the protease domain of NS3. Human antibodies reactive with NS3-FL were highly restricted to the immunoglobulin G1 (IgG1) isotype and were inhibited by soluble ATPase/helicase, but not by the protease domain. The anti-NS3 (ATPase/helicase) reactivity decreased on denaturation by sodium dodecyl sulfate (SDS) and β -mercaptoethanol (2ME), suggesting the recognition of nonlinear or conformational B-cell determinants. Similar to infected humans, mice immunized with NS3-FL developed high-titered primary antibody responses to the NS3 ATPase/helicase domain, whereas an anti-NS3 protease response was not observed after primary or secondary immunizations. Thus, the human and murine humoral immune responses to the HCV NS3 protein are focused on the ATPase/helicase domain, are restricted to the IgG1 isotype in humans, and are conformationally dependent. Unexpectedly, in both species, the NS3 protease domain, present in the context of the full-length NS3, appears to possess low intrinsic immunogenicity in terms of antibody production.
Intrahepatic hepatitis C virus-specific cytotoxic T lymphocyte activity and response to interferon alfa therapy in chronic hepatitis C
Authors: Nelson David R. , Marousis Constantine G. , Ohno Tomoyoshi , Davis Gary L. , Lau Johnson Y.N.
Published: July 2004

Hepatitis C virus (HCV)-specific cytotoxic T lymphocytes (CTL) have been shown to play a role in host defense and pathogenesis of chronic HCV infection. Our aim was to test the hypothesis that intrahepatic HCV-specific CTL activity may impact subsequent response to interferon alfa (IFN-α) therapy. Of the 37 patients that we have prospectively evaluated for HCV-specific CTL activity in liver, 21 received IFN therapy, and 19 completed a 6-month course and attended 6 to 18 months of follow-up. Intrahepatic CD8+ cells were isolated from liver biopsy tissue and tested against target cells expressing HCV antigens to determine intrahepatic CTL activity. The relationship between treatment response and HCV-specific CTL activity and other factors known to associate with response (genotype, viremia, histology) was analyzed. HCV-specific CTL activity was detected in 9 of 21 patients (and 9 of 19 who completed therapy). After 6 months of IFN therapy, 8 of 19 (42%) patients had normal serum alanine transaminase (ALT) (complete responders). After 18 months of follow-up, only 3 patients (16%) had a sustained biochemical response. Of the 9 patients with detectable HCV-specific CTL activity in their liver before treatment, 7 (78%) developed a complete response. In contrast, only 1 of the 10 patients with no detectable HCV-specific CTL activity developed a complete response to IFN (P < .01). In 6 of 8 patients with a complete response, including the 3 sustained responders, the CTL response appeared to be directed predominately to the HCV core region. These data suggest that the host immune response, particularly that mediated by CD8+ CTL, may be important in determining the outcome of IFN therapy for chronic HCV infection. Further understanding of the mechanism of action of IFN should impact the design of better therapeutic strategies against chronic HCV infection.
Sero-clearance of hepatitis B surface antigen in chronic carriers does not necessarily imply a good prognosis
Authors: Huo Teh-ia , Wu Jaw-ching , Lee Pui-ching , Chau Gar-yang , Lui Wing-yu , Tsay Shyh-haw , Ting Ling-tan , Chang Full-young , Lee Shou-dong
Published: July 2004

The incidence of delayed hepatitis B surface antigen (HBsAg) clearance in the natural history of chronic hepatitis B virus (HBV)-infected patients was low. Previous studies regarding the prognosis in such patients were controversial. Among 1,355 chronic carriers from 1985 to 1997, spontaneous HBsAg clearance was observed in 55 patients. During a mean follow-up period of 23 months, 18 (32.7%; all were male subjects) developed serious complications, including 11 with hepatocellular carcinoma (HCC) (9 of them underwent surgical resection), 6 with cirrhosis, and 1 with subfulminant liver failure. The overall cumulative probability of complications was 29.8% at 4 years, and it was higher in males (P= .044) and patients aged 45 years or more (P= .006); the latter carried an 8.6-fold increased risk (95% CI: 1.2-64.6; P= .037) of adverse events. Histories of acute or chronic infection by hepatitis A virus, C virus (HCV), or D virus (HDV) were present in 42% of patients. Patients seropositive for antibodies against HCV (anti-HCV) or HDV (anti-HDV) had higher alanine transaminase (ALT) levels (>40 U/L;P= .008) after sero-clearance. HBV DNA was detectable in 31% of 51 subjects, in 20% of 20 with antibodies against HBsAg, in 40% of 20 with anti-HCV or anti-HDV, and also in an HCC patient’s serum and tumor. Staining of liver HBsAg was positive in 30% of 10 HCC patients. In conclusion, our results demonstrated that hepatitis B viremia may persist, and adverse complications were not rare in HBsAg-clearance patients. All such patients should be closely monitored, which may allow for earlier detection of HCC.
Modulation of immune responses to hepatitis C virus envelope E2 protein following injection of plasmid DNA using single or combined delivery routes
Authors: Fournillier Anne , Nakano Isao , Vitvitski Ludmila , Depla Erik , Vidalin Olivier , Maertens Geert , Trépo Christian , Inchauspé Genevieve
Published: July 2004

Different delivery routes of plasmid DNA may result in the induction of differential humoral and cellular immunity. We have studied the influence of two main routes of plasmid injection, performed intramuscularly and intraepidermally using a gene gun, for the induction of immune responses specific to hepatitis C virus (HCV) envelope protein E2. Three plasmids expressing different immunogenic domains of E2 (amino acids [aa] 384-443, aa 504-555, and aa 384-746) were injected into BALB/c mice according to five different protocols using various combinations of intramuscular (i.m.) or intraepidermal (i.e.) primary and booster injections. Seroconversion rates, antibody titers and isotypes, epitope recognition, and T-helper (Th) release cytokine profiles were analyzed. Antibody titers and epitope recognition were linked to either or both the nature of the immunogen expressed and the delivery route chosen. In all cases, the lowest antibody titers were obtained using single i.m.-based protocols. Independently of the antibody titers generated, only some specific i.e.-combined delivery routes induced antibodies able to recognize determinants located in the N-terminal of E2 (aa 384-411 and aa 411-437) and mimicked by synthetic peptides. By contrast, the antibody isotypes and the splenic cytokine production identified were independent of the plasmids used and the delivery route implemented. All conditions resulted in Th-1 like responses suggested by the exclusive detection of IgG2a and 2b antibodies and the production of interferon gamma (INF-γ) but no interleukin-4 (IL-4). Overall, our results suggest that the combination of i.m. and i.e. delivery routes provides the most efficient way to induce a broad immune response against HCV-E2.
Hepatitis C virus dynamics in vivo : Effect of ribavirin and interferon alfa on viral turnover
Authors: Zeuzem Stefan , Schmidt Jürgen M. , Lee Jung-hun , von Wagner Michael , Teuber Gerlinde , Roth Kurt W.
Published: July 2004

Treatment of patients with chronic hepatitis C with recombinant interferon alfa (rIFN-α) can cause a decrease of serum transaminases and hepatitis C virus (HCV) RNA. Recent trials evaluating combination therapy of IFN-α and ribavirin suggested a potential synergistic effect. From serial measurements of serum HCV RNA concentrations following treatment-induced perturbation of the balance between virus production and clearance, we compared the antiviral efficacy of both IFN-α alone and IFN-α in combination with ribavirin. Chronically HCV-infected patients were treated with either 3 × 3 MU or 3 × 6 MU rIFN-α per week or 3 × 6 MU rIFN-α plus 14 mg/kg of body weight ribavirin per day. The time-dependent HCV RNA concentrations during antiviral treatment were analyzed by iterative least-squares regression. After initiation of antiviral therapy, HCV RNA declined exponentially below the detection limit of the reverse-transcription polymerase chain reaction assay (1,000 HCV RNA molecules per milliliter) in 10 of 26 (39%), 10 of 19 (53%), and 10 of 18 patients (56%) treated with 3 × 3 MU, 3 × 6 MU rIFN-α without and with ribavirin, respectively. Viral clearance from serum was faster in patients treated with 3 × 6 MU rIFN-α (t1/2= 0.23 ± 0.15) compared with patients treated with 3 × 3 MU rIFN-α per week (0.67 ± 0.36 days) (P< .004). However, half-lives of viral clearance were similar in patients treated with rIFN-α or rIFN-α plus ribavirin. For virus release from infected hepatocytes, absence and presence of ribavirin yielded half-lives oft1/2= 2.54 ± 2.10 andt1/2= 1.99 ± 1.70, respectively, indicating that ribavirin does not significantly inhibit HCV production. In conclusion, the data of the present study indicate that higher rIFN-α doses accelerate viral clearance from serum. Ribavirin (14 mg/kg/d), however, lacks synergistic antiviral effects in the treatment of chronic hepatitis C with 3 × 6 MU rIFN-α per week.
Carbon monoxide as a regulator of bile canalicular contractility in cultured rat hepatocytes
Authors: Shinoda Yuichi , Suematsu Makoto , Wakabayashi Yoshiyuki , Suzuki Tsuneharu , Goda Nobuhito , Saito Shuji , Yamaguchi Tokio , Ishimura Yuzuru
Published: July 2004

This study aimed to examine the mechanism(s) by which carbon monoxide (CO), a product of heme oxygenase reaction, controls the contractility of bile canaliculus (BC) in hepatocytes. When BCs associated with the couplet cells in cultured rat hepatocyte suspension were observed using time-lapse video microscopy, they exhibited periodical contractions with a most-probable interval of 6 minutes under our experimental conditions. The addition of 1 μmol/L zinc protoporphyrin IX (ZnPP), a potent inhibitor of heme oxygenase, to the culture medium elicited a 40% shortening of the interval time together with an increase in intracellular calcium concentrations, while the same concentration of iron protoporphyrin IX did not induce such changes. The production of CO, which was 0.5 nmol/h/108 cells in the absence of ZnPP, diminished to less than 0.1 nmol/h/108 cells upon application of ZnPP. The ZnPP-elicited increases in both contractile frequency and intracellular calcium concentrations were attenuated by the addition of 1 μmol/L CO or 50 μmol/L 1,2-bis(2-aminophenoxy) ethane-tetraacetate, a calcium chelator. Clotrimazole or metyrapone, inhibitors of cytochrome P450-dependent monooxygenase activities, also attenuated the ZnPP-induced elevation of the contractile frequency. On the other hand, intracellular cyclic guanosine monophosphate (cGMP) contents were not altered significantly by the application of ZnPP or by CO. These results indicate that CO generated by heme oxygenase controls the BC function by changing intracellular calcium concentrations presumably through a mechanism involving the cytochrome P450 reaction.
Primary biliary cirrhosis-autoimmune hepatitis overlap syndrome: Clinical features and response to therapy
Authors: Chazouillères Olivier , Wendum Dominique , Serfaty Lawrence , Montembault Sarah , Rosmorduc Olivier , Poupon Raoul
Published: July 2004

The association of primary biliary cirrhosis (PBC) and autoimmune hepatitis (AIH) is thought to be rare, and its optimal treatment is unknown. Of 130 consecutive patients with a diagnosis of PBC, we identified 12 cases (9.2%) of overlap syndrome (10 females, 2 males; median age, 50 years) strictly defined by the presence of at least two of the three recognized biochemical, serological, and histological criteria of each disease. One patient had initially pure PBC and developed AIH characterized by a flare of alanine transaminase (ALT) (1,330 IU/L; N < 35), elevated immunoglobulin G (IgG) (42 g/L; N < 14.0), and presence of anti-smooth muscle antibodies (ASMA) after 20 months of ursodeoxycholic acid (UDCA) therapy. A complete clinical and biochemical remission was achieved under combination of corticosteroids and UDCA. Eleven patients had features of both diseases at presentation: high serum levels of alkaline phosphatase (AP) (median: 280 IU/L; N < 100), ALT (140 IU/L), and IgG (30.8 g/L), presence of mitochondrial antibodies (n = 9) or ASMA (n = 9), florid bile duct lesions (n = 8), and moderate or severe periportal or periseptal lymphocytic piecemeal necrosis (n = 11). UDCA (13-15 mg/kg/d) given alone in 5 patients induced a significant decrease in biochemical cholestasis but not in ALT levels, and liver fibrosis progressed in 3 patients. Corticosteroids given alone in 6 patients induced a significant decrease in ALT, IgG, and AP levels, but none had a biochemical normalization. The patients with persistently abnormal liver tests under either UDCA or corticosteroids received both UDCA and corticosteroids. A further marked biochemical improvement was observed, and all patients became asymptomatic. We conclude that, in patients with PBC: 1) overlap syndrome with AIH is not rare; 2) flares of AIH may occur either spontaneously or under UDCA; and 3) combination of UDCA and corticosteroids is required in most patients to obtain a complete biochemical response. Overlap syndrome may represent an important and unrecognized cause of resistance to UDCA in patients with PBC.
Frequency and nature of the variant syndromes of autoimmune liver disease
Authors: Czaja Albert J.
Published: July 2004

To determine the frequency and nature of variant syndromes in autoimmune liver disease, 162 patients with type 1 autoimmune hepatitis, 37 patients with primary biliary cirrhosis, and 26 patients with primary sclerosing cholangitis were assessed in a uniform fashion, and the strength of the original diagnosis was evaluated by use of a scoring system. Variant forms, including syndromes with autoimmune hepatitis and primary biliary cirrhosis (7%) or primary sclerosing cholangitis (6%) and autoimmune cholangitis (11%), were common in the 225 patients (18%). Individuals with autoimmune hepatitis and primary biliary cirrhosis entered remission during corticosteroid therapy as commonly as individuals with definite autoimmune hepatitis (75% vs. 64%, P = .5), and they responded better than patients with autoimmune hepatitis and primary sclerosing cholangitis (75% vs. 22%, P = .03) or autoimmune cholangitis (75% vs. 0%, P = .009). Patients with autoimmune hepatitis and primary sclerosing cholangitis had a higher frequency of a poor result than other variants (78% vs. 17%, P = .04), and they died of liver failure or required liver transplantation more often than patients with definite autoimmune hepatitis (33% vs. 8%, P = .05). None of the patients entering remission had serum alkaline phosphatase levels greater than twofold the reference value. In conclusion, variants of autoimmune liver disease are common. Corticosteroid therapy can be effective in patients with features of autoimmune hepatitis and primary biliary cirrhosis. A serum alkaline phosphatase level of less than twofold the reference value characterizes corticosteroid-responsive individuals.