bluuebunny/crossref_metadata_2025 huggingface dataset:
Reactivity of 13 in vitro expressed hepatitis B surface antigen variants in 7 commercial diagnostic assays
Authors: Ireland Jacqueline H. , O’Donnell Barbara , Basuni Ashraf A. , Kean Joy D. , Wallace Lesley A. , Lau George K. , Carman William F.
Published: March 2004

The primary marker of current hepatitis B infection is the surface antigen (HBsAg), however HBsAg negativity does not exclude hepatitis B viremia. HBsAg variants can be responsible for such diagnostic failures. Here 13 different HBsAg variants were cloned, variant protein produced in a mammalian expression system, and tested using 7 commercial HBsAg diagnostic assays. Of 12 variants analyzed, 6 samples displayed similar reactivity to the positive control (containing standard HBsAg sequence) in most of the assays, but 6 samples, containing various mutations throughout the entire major hydrophilic region (MHR), showed reduced reactivity. It was found that the loss of cysteine at amino acid (aa) 124 in 1 sample affected the secretion as well as the reactivity of HBsAg in the expression system. Thus, not all assays are equally able to detect HBsAg variants, implying that, to attain an acceptable level of sensitivity, the antibody repertoire of the current assays should be extended.
production and release of endothelin-1 from the gut and spleen in portal hypertension due to cirrhosis
Authors: Nagasue Naofumi , Dhar Dipok Kumar , Yamanoi Akira , Emi Yamaguchi , Udagawa Jun , Yamamoto Akitaka , Tachibana Mitsuo , Kubota Hirofumi , Kohno Hitoshi , Harada Takayuki
Published: March 2004

This study was aimed to evaluate the source of endothelin-1 (ET-1) in cirrhotic patients. ET-1 is implicated in the pathogenesis of portal hypertension. However, the mechanism and source for increased plasma ET-1 in cirrhotic patients are still obscure. Plasma ET-1 levels in systemic (SV), superior mesenteric (SMV), and splenic venous (SPV) blood were measured in 23 patients with cirrhosis and 8 controls with normal liver. Fourteen removed spleens were immunohistochemically studied for ET-1, CD34, CD68, and CD20. In situ hybridization was done to localize ET-1 messenger RNA (mRNA). In cirrhosis, ET-1 levels in both SMV and SPV were higher than in SV. ET-1 in SV and SPV were significantly higher in cirrhotic patients than in control patients. Three groups of cells in the spleen expressed both protein and mRNA of ET-1: endothelial cells in the sinus, which were also stained for CD34; cells in the germinal center; and cells in the marginal zone of lymphoid sheaths and follicles, which were also stained for CD20 but not for CD34 and CD68. The ET-1 concentration released from the spleen was in parallel with the grade of ET-1 expression in the spleen. The spleen is one of the major sites of ET-1 release in cirrhotic patients. Endothelial cells of the splenic sinus and possibly B lymphocytes in the germinal center and marginal zone of lymphoid sheaths and follicles seem to be the sites of ET-1 production in the spleen.
Predictive value of the variceal pressure response to continued pharmacological therapy in patients with cirrhosis and portal hypertension
Authors: Escorsell Àngels , Bordas Josep Maria , Castañeda Beatriz , Llach Josep , García-Pagán Joan Carles , Rodés Joan , Bosch Jaume
Published: March 2004

Noninvasive measurements of variceal pressure adequately reflect the hemodynamic effects of propranolol on portal hypertension. However, the prognostic value of variceal pressure responses during continued propranolol therapy has not been evaluated, and it is unclear whether this may substitute invasive measurements of portal pressure response. Fifty-five portal hypertensive patients with cirrhosis were studied before and at 4 months of continued propranolol therapy. Variceal pressure was measured using an endoscopic pressure gauge. Portal pressure was evaluated as the hepatic venous pressure gradient (HVPG). Over a 28 ± 11 month follow-up, 16 patients experienced variceal bleeding. Baseline characteristics were similar in bleeders and nonbleeders. At 4 months, reduction in variceal pressure was less marked in bleeders than in nonbleeders (5% ± 20% vs. −15% ± 24%; P = .03). A fall in variceal pressure 20% or greater of baseline was an independent predictor of absence of variceal bleeding; which occurred in 5% of patients with a 20% or greater fall in variceal pressure versus 42% of patients with less than a 20% reduction (P = .004). The HVPG response had similar independent prognostic value (decrease ≥20%: 6% bleeding; decrease <20%: 45% bleeding; P = .004) but identified different patients. Achieving a 20% decrease in either variceal pressure or HVPG was highly sensitive (85%) and specific (93%) identifying patients not bleeding on follow-up. Endoscopic measurements of variceal pressure response to continued pharmacotherapy provide useful prognostic information on the risk of variceal bleeding. As with HVPG response, a fall in variceal pressure of 20% or greater is associated with a very low risk of variceal bleeding. The combination of both parameters allows almost optimal prognostication.
Glutathione, lactobionate, and histidine: cryptic inhibitors of matrix metalloproteinases contained in university of wisconsin and histidine/tryptophan/ketoglutarate liver preservation solutions
Authors: Upadhya Gundumi Aravinda , Strasberg Steven M.
Published: March 2004

UW solution and HTK solution are both used for cold preservation of liver allografts. Although they are about equally effective, their compositions are very different, and they were formulated using different rationales. The authors recently showed an important role for MMPs in liver preservation injury and consequently postulated that these preservation solutions contain cryptic inhibitors of MMP activity. To determine this possibility, the ability of these solutions to inhibit MMP activity was studied. The source of MMP2 and MMP9 was human liver effluents obtained at the time of liver transplantation or commercially available human recombinant MMP2 and MMP9. MMP2 and MMP9 showed gelatinolytic activity at 37°C and also at 4°C, although activity at 4°C was reduced. Activity was inhibited by University of Wisconsin (UW) and Histidine/Tryptophan/ Ketoglutarate (HTK) solutions. Examination of individual ingredients disclosed that reduced glutathione (GSH) and lactobionate in UW solution and histidine in HTK solution were the cryptic inhibitors. HTK solution was a more effective inhibitor than UW solution. GSH inhibited the activity of both enzymes, but was a much more effective inhibitor of MMP9 than MMP2. Oxidized glutathione(GSSG) was a much less effective inhibitor of the enzymes. The inhibitor constants (Ki) of GSH for MMP2 and MMP9 were 34 μmol/L and 3 μmol/L, respectively. The authors conclude that MMP inhibition is a cryptic property of both commonly used liver preservation solutions and contributes importantly to their action. Furthermore, GSH appears to be an effective inhibitor of gelatinases at concentrations at which it is normally present in extracellular fluid.
An exploratory population-based case-control study of primary biliary cirrhosis
Authors: Howel Denise , Fischbacher Colin M. , Bhopal Raj S. , Gray Jackie , Metcalf Jane V. , James Oliver F.
Published: March 2004

Primary biliary cirrhosis (PBC) is a disease of unknown etiology, with unexplained geographical variation. Various exposures have been suggested as triggers for disease development—possibly in susceptible individuals, but the evidence was not always well founded. We therefore conducted a population-based case-control study in Northeast England to investigate these and other exposures. All cases incident during 1993 to 1995 in a defined area of Northeast England were identified, and age- and sex-matched population controls were identified from primary care population registers. Cases and controls were sent postal self-completion questionnaires covering medical history and lifestyle. Information was received from 100 cases and 223 controls. The familial tendency of PBC was found to be less marked than has been claimed: Only weak associations were found with other autoimmune diseases. Among factors considered previously, no significant associations were found with surgical procedures, events in pregnancy, past infections, vaccinations, and medications. No significant associations were found for previously unconsidered lifestyle factors (drinking alcohol, previous pets, or stressful events), but there was an unexpected association with past smoking (ever smoked: 76% in cases vs. 57% in controls, odds ratio 2.4; smoked for 20 years or more: 64% vs. 35%, odds ratio 3.5). There were also unexpected significant associations with psoriasis (13% in cases vs. 3% in controls, odds ratio 4.6) and eczema (3% in cases vs. 11% in controls, odds ratio 0.13). These findings merit further investigation.
Critical Role of Reactive Oxygen Species and Mitochondrial Permeability Transition in Microcystin-Induced Rapid Apoptosis in Rat Hepatocytes
Authors: Ding Wen-Xing , Shen Han-Ming , Ong Choon-Nam
Published: March 2004

Microcystin-LR (M-LR) is a specific hepatotoxin. At present, the exact toxic mechanism of its action remains unclear though apoptosis is believed to be involved. This study was designed to investigate the role of reactive oxygen species (ROS) and mitochondrial permeability transition (MPT) in the M-LR-induced apoptotic process. Morphologic changes such as cell shrinkage, externalization of cell membrane phosphatidylserine, DNA fragmentation, and nuclear condensation suggest that M-LR causes rapid apoptosis in hepatocytes. Confocal microscopy revealed that M-LR exposure led to the onset of MPT and mitochondrial depolarization, evidenced by (1) redistribution of calcein fluorescence from cytosol to mitochondria, and (2) loss of mitochondrial tetramethyrhodamine methyl ester (TMRM) fluorescence; both occurred before apoptosis. Moreover, there was a significant and rapid increase of ROS level before the onset of MPT and loss of MMP, indicating a critical role of ROS in M-LR-induced apoptosis. Deferoxamine (DFO), an iron chelator, prevented the increase of ROS production, delayed the onset of MPT, and, subsequently, cell death. In addition, a specific MPT inhibitor, cyclosporin A (CsA), blocked the M-LR-induced ROS formation, onset of MPT, and mitochondrial depolarization as well as cell death. Thus, we conclude that the M-LR-induced ROS formation leads to the onset of MPT and apoptosis.
Triple Antiviral Therapy as a New Option for Patients With Interferon Nonresponsive Chronic Hepatitis C
Authors: Brillanti Stefano , Levantesi Fabio , Masi Livia , Foli Mauro , Bolondi Luigi
Published: March 2004

The aim of the study was to evaluate the efficacy of triple antiviral therapy with interferon, ribavirin, and amantadine in comparison with interferon and ribavirin combination treatment in patients with interferon-nonresponsive chronic hepatitis C. We performed an open-label, prospective randomized controlled trial at a secondary referral center. We used a 2:1 ratio, patients received interferon, ribavirin, and amantadine, or interferon and ribavirin for 12 months, and were followed up for an additional 6 months. Ninety-four consecutive adult interferon nonresponders with chronic hepatitis C were screened. Sixty consecutive elected patients entered the study. No patients withdrew because of adverse effects. Forty patients received interferon alfa (5 megaunits on alternate days), ribavirin (800-1,000 mg daily), and amantadine (200 mg daily) for 12 months, and 20 patients received the same treatment without amantadine. At the end of follow-up, alanine transaminase (ALT) level normalization was maintained in 23 of 40 patients (57%) after triple therapy, but in 2 of 20 patients (10%) after double therapy (P < .001, RR = 2.11, 95% CI, 1.43-3.12), whereas disappearance of serum HCV RNA persisted in 19 of 40 patients (48%) and in 1 of 20 patients (5%), respectively (P < .001, RR = 1.81, 95% CI, 1.32-2.47). The safety profile was similar in the 2 groups. In conclusion, in patients with interferon-nonresponsive chronic hepatitis C, triple antiviral therapy for 1 year results in a high rate of sustained biochemical and virologic responses.
Endoscopic Variceal Ligation Plus Nadolol and Sucralfate Compared With Ligation Alone for the Prevention of Variceal Rebleeding: A Prospective, Randomized Trial
Authors: Lo Gin-Ho , Lai Kwok-Hung , Cheng Jin-Shiung , Chen Mei-Hsiu , Huang Hui-Chun , Hsu Ping-I , Lin Chiun-Ku
Published: March 2004

Both β-blockers and endoscopic variceal ligation (EVL) have proven to be valuable alternatives to sclerotherapy in the prevention of variceal rebleeding. Sucralfate is a mucosal protector. The effects of combinations of β-blocker, band ligation, and sucralfate (triple therapy) remain unknown. A total of 122 patients with a history of esophageal variceal bleeding were randomized to receive EVL only (group A, 62 patients) or triple therapy (group B, 60 patients). The procedure for the triple therapy included ligation with the addition of sucralfate granules until variceal obliteration. In addition, nadolol was administered during the course of the study or until death. After a median follow-up of 21 months, recurrent upper gastrointestinal bleeding developed in 29 patients (47%) in group A and 14 patients (23%) in group B (P = .005). Recurrent bleeding from esophagogastric varices occurred in 18 patients in group A and 7 patients in group B (P = .001). Twenty-one patients in group A (50%) and 12 patients (26%) in group B experienced variceal recurrence after variceal obliteration (P < .05). Treatment failure occurred in 11 patients (18%) in group A and in 4 patients (7%) in group B (P = .05). Twenty patients from group A and 10 patients from group B died (P = .08); 9 and 4 of these deaths, respectively, were attributed to variceal hemorrhage (P = .26). The combination of ligation, nadolol, and sucralfate (triple therapy) proved more effective than banding ligation alone in terms of prevention of variceal recurrence and upper gastrointestinal rebleeding as well as variceal rebleeding.
Radial artery wall alterations in genetic hemochromatosis before and after iron depletion therapy
Authors: Failla Monica , Giannattasio Cristina , Piperno Alberto , Vergani Anna , Grappiolo Alessandra , Gentile Gaetano , Meles Ester , Mancia Giuseppe
Published: March 2004

Iron overload is believed to have an adverse influence on the cardiovascular system and animal studies have shown that iron may be involved in the events that lead to atherosclerosis via an enhancement of smooth muscle cell proliferation, lipid oxidation, and free radical production. There are no data on the effect of iron overload on arterial structural and mechanical properties in humans. We measured wall thickness and distensibility (D) by ultrasonography of the radial artery in 12 patients with uncomplicated genetic hemochromatosis (GH) who were normotensive and without atherosclerotic plaques. Twelve age- and sex-matched patients were taken as controls. Nine patients were evaluated also after iron depletion. Wall thickness was greater in patients with GH than in controls (+50%, P< .01) whereas D was slightly reduced in the former group compared with the latter group, though the difference was not statistically significant. After iron depletion, a significant reduction of wall thickness and a significant increase in D were observed (−24% and +33%, P < .05 for both). Thus, in patients with hemochromatosis, arterial wall thickness is increased before the onset of cardiovascular complications. This alteration is reverted by iron depletion, which also can improve the initial and modest radial artery wall stiffening associated with this condition. Thus, functional and structural alterations in midsize muscle arteries may be an early abnormality of hemochromatosis.
Pharmacokinetics and Tissue Distribution of a Ribozyme Directed Against Hepatitis C Virus RNA Following Subcutaneous or Intravenous Administration in Mice
Authors: Lee Patrice A. , Blatt Lawrence M. , Blanchard Karin S. , Bouhana Karyn S. , Pavco Pamela A. , Bellon Laurent , Sandberg Jennifer A.
Published: March 2004

A nuclease resistant ribozyme targeting the 5′ untranslated region (5′ UTR) of hepatitis C virus (HCV) at site 195 has been identified. To investigate the therapeutic utility of this ribozyme, we evaluated the pharmacokinetics and tissue distribution with two labeled forms of this ribozyme. [32P]-labeled ribozyme was administered as a single subcutaneous (SC) or intravenous (IV) bolus at a dose of 10 mg/kg or 30 mg/kg in C57Bl/6 mice. Regardless of route of administration, peak liver concentrations achieved were greater than the concentration necessary to inhibit HCV-IRES-luciferase expression in cell culture. The ribozyme was well absorbed after SC administration (89%) and had an elimination half-life of 23 minutes. To show intracellular localization of the ribozyme in target tissue, a tetramethyl rhodamine (TMR)-labeled ribozyme was administered as a single SC or IV bolus at a dose of 30 mg/kg in C57Bl/6 mice. Mice treated SC or IV with TMR-labeled ribozyme had positive fluorescence in the liver from 15 minutes to 48 hours after dosing. Definite positive fluorescence was still present at 72 hours in the mice dosed via the IV route. At early time points (15 and 30 minutes postinjection), nuclear and possibly cytoplasmic fluorescence was present in the hepatocytes, and sinusoidal fluorescence was intense. At the later time points, fluorescence became more punctate. Abundant staining was often present in Kupffer cells. This study confirms the retention of ribozyme in liver cells and supports the potential of an anti-HCV ribozyme as a therapeutic agent for treatment of chronic hepatitis C.

(Hepatology 2000;32:640-646.)
JAGGED1 Gene Expression During Human Embryogenesis Elucidates the Wide Phenotypic Spectrum of Alagille Syndrome
Authors: Crosnier Cécile , Attié-Bitach Tania , Encha-Razavi Férechté , Audollent Sophie , Soudy Fardouss , Hadchouel Michelle , Meunier-Rotival Michèle , Vekemans Michel
Published: March 2004

Mutations of the JAGGED1 gene, encoding a NOTCH receptor ligand, cause Alagille syndrome (AGS), a complex malformative disorder affecting mainly the liver, heart, vertebrae, eye, and face. Minor and occasional features involving kidney, pharynx, systemic arteries, skeleton, and ear are in some cases associated with the syndrome. To describe the expression of JAGGED1 during human embryogenesis and to study its relationship with all the features of AGS, we performed in situ hybridization studies on human embryos and fetal tissue sections. JAGGED1 was mainly expressed in the cardiovascular system. In the liver, JAGGED1 transcripts were only detected in blood vessels. JAGGED1 was also expressed in other structures of mesenchymal origin (distal mesenchyme of limb buds; mesonephric and metanephric tubules of the kidney) and in epithelial structures including the ciliary margin of the retina and the posterior part of the lens, the ventral epithelium of the otic vesicle, the neurosensory epithelium of the ear vestibule, the epithelium of pharyngeal arches, and the developing central nervous system. The strong JAGGED1 expression during human embryo- and feto-genesis both in the vascular system and in other mesenchymal and epithelial tissues implicates abnormal angiogenesis in the pathogenesis of Alagille syndrome and particularly the paucity of interlobular bile ducts. However, it is probably not the only mechanism of the disease. Except for the central nervous system, there is a strong correlation between JAGGED1 expression and all the features of AGS. This implies that the features occasionally associated with the syndrome are not coincidental.
Practices of Liver Biopsy in France: Results of a Prospective Nationwide Survey
Authors: Cadranel Jean-François , Rufat Pierre , Degos Françoise
Published: March 2004

A nationwide prospective study was conducted in France in 89 university and primary referral hospitals’ liver units to evaluate practices of liver biopsy and the occurrence of complications. A total of 2,084 biopsies were analyzed, recording the indication, hemostasis parameters, experience of operator, route of biopsy, use of ultrasonography (US), type of hospitalization, side effects, and complications. Pain, anxiety, and discomfort were evaluated by patients by visual analogue scale (VAS). Biopsies were performed by experienced physicians (>150 procedures performed) in 72%, and hepato-gastroenterologists in 89% of the cases. Hepatitis C was the indication in 54%. Sedation or premedication (atropine) was given in 46%. US-guidance was used in 56% of the cases. A day-care procedure was used in 27%. No deaths occurred, but severe complications were observed in 0.57% and increased with the number of passes and decreased with experience of operator, use of atropine, and US-guidance. Pain was independently related to general anesthesia, experience of the operator, female sex, and hepatitis C. Anxiety was increased in women. Discomfort was increased by venous access and decreased with an experienced operator. Acceptance of additional biopsies was related to a day-care procedure and independently related to general anesthesia and multiples passes. This study showed that (1) liver biopsy procedures vary greatly in France, (2) hepatitis C is the main indication for liver biopsy at present, (3) US-guidance should be developed to reduce severe complications, and (4) day-care procedures increase acceptance of a future biopsy and should also be used more often.
Standardization of Hepatitis C Virus RNA Quantification
Authors: Pawlotsky Jean-Michel , Bouvier-Alias Magali , Hezode Christophe , Darthuy Francoise , Remire Jocelyne , Dhumeaux Daniel
Published: March 2004

It was recently recommended that hepatitis C virus (HCV) RNA quantification be used to tailor the duration of combined interferon alfa (IFN-α)/ribavirin therapy in patients infected by HCV genotypes 1, 4, and 5. This recommendation has been difficult to implement in the absence of standardized quantitative units for HCV RNA. The aim of this work was to define clinically relevant HCV RNA loads in standardized international units (IU), for use in routine clinical and research applications based on standardized quantitative assays. Two hepatitis C virus RNA quantitative assays were used: (1) the Superquant assay (National Genetics Institute, Los Angeles, CA), for which possibly relevant thresholds were established; and (2) the semi-automated Cobas Amplicor HCV Monitor assay version 2.0 (Cobas v2.0, Roche Molecular Systems, Pleasanton, CA) that measures HCV RNA loads in IU/mL. Quantification in the Cobas v2.0 assay was linear over the entire range of values tested, including viral loads higher than 850,000 IU/mL after 100-fold dilution. The accuracy and precision of the measures in IU/mL were satisfactory with Cobas v2.0. The results obtained with Superquant and Cobas v2.0 correlated (r = .932; P < .0001). A value of 2,000,000 copies/mL (6.3 log10 copies/mL) with Superquant was converted to nearly 800,000 IU/mL (5.9 log10 IU/mL). In conclusion, all HCV RNA quantitative assays should give HCV RNA loads in international units and be validated with appropriate calibrated panels; 800,000 IU/mL in any of these assays should be used as the decision threshold to tailor the IFN-α/ribavirin treatment duration in patients infected by HCV genotypes 1, 4, and 5.
Hyperstimulation With Interleukin 6 Inhibits Cell Cycle Progression After Hepatectomy in Mice
Authors: Wüstefeld Torsten , Rakemann Tim , Kubicka Stefan , Manns Michael P. , Trautwein Christian
Published: March 2004

Interleukin 6 (IL-6) is an important mediator of hepatocyte proliferation after hepatectomy. However, elevated IL-6 levels are found in patients with chronic liver disease. Therefore, it is unclear if hyperstimulation with IL-6 may have an influence on liver regeneration. We investigated whether a strong activation of IL-6-dependent pathways may change the course of hepatocyte proliferation after hepatectomy. Transgenic mice overexpressing the human soluble IL-6 receptor/gp80 (hsgp80) in hepatocytes were stimulated with or without hepatectomy with human IL-6 (hIL-6). Nuclear extracts were prepared and activation of gp130-dependent pathways was studied by Western blot and gel shift experiments. Cell cycle progression of hepatocytes after hepatectomy was investigated by monitoring cell cycle-specific factors. hIL-6 strongly activates Stat3 for more than 48 hours in human soluble hsgp80 transgenic mice. In contrast, no major differences were evident in the regulation of the Ras/MAP kinase pathway compared with wild-type (wt) mice. Also when hsgp80 mice were stimulated with hIL-6 3 hours before hepatectomy Stat3 is activated for more than 72 hours, whereas in unstimulated mice this event is restricted to the early hours. Strong activation of Stat3 resulted in a delay and inhibition of hepatocyte proliferation as measured by 5-bromo-2′-deoxyuridine (BrdU) staining and Cyclin A and E expression. This observation directly correlates with the induction of the cell cycle inhibitor p21. In summary, strong IL-6-dependent activation of Stat3 before hepatectomy results in delay and inhibition of cell cycle progression after hepatectomy. Therefore our results suggest that hyperstimulation with IL-6 can inhibit liver regeneration.
Glycine Prevents Apoptosis of Rat Sinusoidal Endothelial Cells Caused by Deprivation of Vascular Endothelial Growth Factor
Authors: Zhang Yan-jun , Ikejima Kenichi , Honda Hajime , Kitamura Tsuneo , Takei Yoshiyuki , Sato Nobuhiro
Published: March 2004

Apoptosis of sinusoidal endothelial cells (SECs) is one of the initial events in the development of ischemia-reperfusion injury of the liver. Glycine has been shown to diminish ischemia-reperfusion injury in the liver and improve graft survival in the rat liver transplantation model. Here, we investigated the effect of glycine on apoptosis of primary cultured rat SECs induced by vascular endothelial growth factor (VEGF) deprivation. Isolated rat SECs were cultured in EBM-2 medium supplemented with 10% fetal bovine serum (FBS) and growth factors including 20 ng/mL VEGF for 3 days. SECs at 3 days of culture showed spindle-like shapes; however, cells started shrinking and detaching from dishes by VEGF deprivation. Apoptosis was detected by terminal deoxynucleotidyl transferase (TdT)-mediated d-uridine triphosphate (dUTP)-biotin nick end labeling (TUNEL) staining in these conditions. Control SECs contained only a few percent of TUNEL-positive cells; however, they started increasing 4 hours after VEGF deprivation, and the percentage of TUNEL-positive cells reached about 50% at 8 hours and almost 100% at 16 hours after VEGF deprivation. Interestingly, this increase in TUNEL-positive cells after VEGF deprivation was prevented significantly when glycine (1-10 mmol/L) was added to the medium, the levels being around 60% of VEGF deprivation without glycine. Furthermore, strychnine (1 μmol/L), a glycine receptor antagonist, inhibited this effect of glycine, suggesting the possible involvement of the glycine receptor/chloride channel in the mechanism. Moreover, Bcl-2 protein levels in SECs were decreased 8 hours after VEGF deprivation, which was prevented almost completely by glycine. It is concluded that glycine prevents apoptosis of primary cultured SECs under VEGF deprivation.
A Dose-Ranging Study of Pegylated Interferon Alfa-2b and Ribavirin in Chronic Hepatitis C
Authors: Glue Paul , Rouzier-Panis Regine , Raffanel Claude , Sabo Ron , Gupta Samir K. , Salfi Margaret , Jacobs Shiela , Clement Robert P.
Published: March 2004

The objectives of this study were to assess the safety, pharmacokinetics, and efficacy of pegylated interferon alfa-2b (PEG-Intron) plus ribavirin in patients with chronic hepatitis C. A total of 72 patients (35 men/37 women, age range 20-68 years) with clinically compensated chronic hepatitis C virus (HCV) were enrolled into this open-label, randomized, active controlled study. Patients received either PEG-Intron 0.35, 0.7, or 1.4 μg/kg subcutaneously weekly for 24 weeks alone, or in combination with ribavirin 600, 800, or 1,000 to 1,200 mg orally daily. Patients were evaluated during treatment and after a 24-week follow-up period for safety and efficacy. Detailed pharmacokinetic assessments were performed at weeks 1 and 4. PEG-Intron alone produced expected dose-related reductions in white cells, neutrophils and platelets. Addition of ribavirin reduced hemoglobin levels in a dose-related manner, did not further reduce PEG-Intron-induced decreases in neutrophil or white cell count, and increased platelet counts. Neutrophil function tests (C5a and FMLP migration, killing curves) were unaltered. Reported adverse events (flu-like symptoms, asthenia) were qualitatively similar in all dose groups. Anti-HCV activity, as measured by loss of detectable serum HCV RNA (i.e. <100 copies/mL) at the end of treatment (week 24) and after 24 weeks of follow-up (week 48) showed dose-response trends for PEG-Intron. At each PEG-Intron dose level, anti-HCV activity was higher in patients coadministered ribavirin than in patients treated with PEG-Intron monotherapy. There was no evidence of pharmacokinetic interactions with either drug. We conclude that the safety and tolerability of combined PEG-Intron/ribavirin and PEG-Intron alone were comparable. Combined PEG-Intron/ribavirin showed dose-related synergistic anti-HCV effects, which were numerically superior to those obtained with PEG-Intron monotherapy.

(Hepatology 2000;32:647-653.)
Priming of hepatitis C virus-specific cytotoxic t lymphocytes in mice following portal vein injection of a liver-specific plasmid DNA
Authors: Lee Alexander Y. , Manning William C. , Arian Christopher L. , Polakos Noelle K. , Barajas Jose L. , Ulmer Jeffrey B. , Houghton Michael , Paliard Xavier
Published: March 2004

The immunology of hepatitis C virus (HCV) infection should be studied in the context of HCV antigen expression in the liver, because HCV primarily infects this organ. Indeed, the nature, function, and fate of T cells primed after antigen expression in the liver might differ from those primed when antigens are expressed systemically or in other organs, because the nature of the antigen-presenting cells (APCs) involved may be different. In addition, the normal liver contains a resident population of lymphocytes that differ from those present at other sites. Thus, we investigated whether HCV-specific CD8+ cytotoxic T cells (CTLs) could be elicited following portal vein (PV) injection of plasmid DNA in mice whose hepatic veins were transiently occluded. We show that PV injection of mice with “naked” DNA expressing the HCV-NS5a protein, under the control of a liver-specific enhancer/promoter, resulted in NS5a expression in the liver and the priming of HCV-specific CTLs. These results suggested that such a model might be relevant to the study of HCV-specific immune responses primed during natural infection.
Interferon tau-induced hepatocyte apoptosis in sheep
Authors: Kim Hun-Taek , Stoica George , Bazer Fuller W. , Ott Troy L.
Published: March 2004

Clinical applications of Type I interferon (IFN) are limited by adverse side effects mediated largely by unknown mechanisms. This study examined the mechanisms of acute hepatic injury in lambs treated with systemic administration of IFN-τ, a Type I IFN. Liver tissues were collected at 24, 48, or 96 hours after treatment with either IFN-τ or saline. Histopathology revealed acute hepatopathy including cellular swelling, cytoplasmic aggregates, and apoptosis in all IFN-τ-treated lambs, which were accompanied by elevation of aspartate transaminase (AST) (P < .01). The number of apoptotic hepatocytes in IFN-τ-treated lambs was higher than for control lambs (P < .001). Immunohistochemistry for proliferating cell nuclear antigen (PCNA) revealed that IFN-τ induced hepatocyte growth arrest at the G0/G1 phase of the cell cycle and that the majority of hepatocytes in S or G2 phase were eliminated by apoptosis. We investigated expression of bax-α and bcl-2, acting as pro- and antiapoptotic molecules, in IFN-τ-induced apoptosis. Northern blot analysis revealed increased expression of bax-α messenger RNA (mRNA) in IFN-τ-treated lambs (P < .01) compared with control lambs, consistent with the expression pattern for bax-α protein. However, there was no detectable difference in expression of bcl-2 proteins between control and IFN-τ-treated lambs. The levels of bax-α associated with the mitochondria also increased during IFN-τ treatment. Bax-α immunostaining showed scattered immunoreactive hepatocytes with morphological hallmarks of apoptosis. These results suggest that IFN-τ induces growth arrest as well as apoptosis by regulating bax-α expression. These pathological effects of IFN-τ on sheep liver indicate potential mechanisms of Type 1 IFN-induced hepatotoxicity in animals and humans.
Cloning and functional characterization of the bile acid-sensitive methotrexate carrier from rat liver cells
Authors: Honscha Walther , Dötsch Kerstin U. , Thomsen Nadine , Petzinger Ernst
Published: March 2004

We have cloned two complementary DNAs (cDNAs), RL-Mtx-1 and RL-Mtx-2, corresponding to the bile acid- sensitive methotrexate carrier from rat liver by direct full-length rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR) using degenerated primers that were deduced from published sequences of tumor cell methotrexate transporters. When expressed in Xenopus laevis oocytes and cosM6 cells, both clones mediate methotrexate and bumetanide transport. RL-Mtx-1 consists of 2,445 bp with an open reading frame of 1,536 bp. The corresponding protein with 512 amino acids has a molecular weight of 58 kd. RL-Mtx-2 (2,654 bp) differs by an additional insert of 203 bp. This insert is located in frame at position 1,196 of the RL-Mtx-1 and contains the typical splice junction sites at the 5′ and 3′ end, indicating that the RL-Mtx-2 messenger RNA (mRNA) is generated by alternative splicing. The insert contains a stop codon that shortens the RL-Mtx-2 protein to 330 amino acids (38 kd). Both cDNAs contain the binding site sequence for the dioxin/nuclear translocator responsive element (Ah/Arnt-receptor) in conjunction with a barbiturate recognition sequence (Barbie box). Preliminary results show that the Barbie box acts as a negative regulatory element. The two liver cDNA clones show homologies to the published sequences of folate and the reduced folate carriers, but no homology is found to the transport systems for organic anions like the Ntcp1, oatp1, OAT-K1, and OAT1. Expression of the mRNA for the methotrexate carrier is found in liver, kidney, heart, brain, spleen, lung, and skeletal muscle, but not in the testis as revealed by Northern blot analysis. The highest abundance of the mRNA is found in the kidney.
Increased natriuretic efficiency of furosemide in rats with carbon tetrachloride-induced cirrhosis
Authors: Jonassen Thomas E.N. , Sørensen Anne-Mette , Petersen Jørgen S , Andreasen Frederik , Christensen Sten
Published: March 2004

The authors examined the natriuretic efficiency of furosemide in rats with cirrhosis induced by carbon tetrachloride (CCl4). Rats were treated for 17 weeks with intraperitoneal injections of CCl4 in groundnut oil twice a week throughout the study. Control rats were treated with vehicle (groundnut oil). Studies in metabolic cages showed that sodium retention was present from week 14. Renal clearance experiments were performed in chronically, instrumented conscious rats at the end of week 14 and at the termination of the study (end week 16) when ascites and hyponatremia were present. After 14 weeks, cirrhotic rats had sodium retention along with increased renal plasma flow, normal GFR, normal renal lithium handling, and a significantly increased diuretic (+41% vs. control) and natriuretic (+56% vs. control) response to a test dose furosemide (7.5 mg/kg b.w., intravenously). The natriuretic efficiency of furosemide, i.e., the natriuresis expressed relative to the furosemide excretion rate (△UNaV/UFURV) was increased by 51% versus control. After 17 weeks, ascites and hyponatremia had developed, and significant decreases in renal plasma flow (−33%), GFR (−30%), and fractional lithium excretion (−44%) were observed. At this stage urinary recovery of furosemide was significantly decreased and the diuretic (−27% vs. Control) and natriuretic (−38% vs. control) responses to furosemide were significantly impaired. However, the increased natriuretic efficiency of furosemide was still present (+34% vs. control). Together these results suggest that increased sodium reabsoprtion in the thick ascending limb of Henle’s loop is involved in the renal sodium retention in cirrhosis in rats that eventually results in decompensation with the formation of ascites.
Influence of immunogenetic background on the outcome of recurrent hepatitis C after liver transplantation
Authors: Belli Luca Saverio , Zavaglia Claudio , Alberti Alberto Battista , Poli Francesca , Rondinara Gianfranco , Silini Enrico , Taioli Emanuela , de Carlis Luciano , Scalamogna Mario , Forti Domenico , Pinzello Giovambattista , Idèo Gaetano
Published: March 2004

In immunocompetent patients, specific human leukocyte antigen (HLA) class II alleles have been associated with the severity of hepatitis C virus (HCV)-related disease, in particular, HLA-DRB1*11 has been found to exert a protective effect. The authors have analyzed the role of HLA class I and II alleles in determining the frequency, timing, and progression of histologically proven recurrent hepatitis C in 89 patients who underwent a liver transplant for HCV-related cirrhosis. In addition, the influence of HLA mismatch between donor and recipient, HCV genotype, and use of steroid pulses was also evaluated. Median patient follow up was 35 months (range 4-119). HLA-DRB1 typing was performed by genomic analysis in all cases. Liver biopsies were obtained routinely and at least at yearly intervals. Histologically proven recurrent hepatitis was observed in 46 patients (52%), 10 patients progressing to stage 5-6 fibrosis in most cases within 2 years after transplant. By univariate analysis, 3 variables, HLA-B14, HLA-DRB1*04, and HLA-DRB1 donor/recipient mismatch, showed a significant effect on time to recurrent hepatitis C disease. These parameters were included in a multivariate regression model along with HCV genotype, treatment with steroid pulses and DRB1*11. HLA-B14, HLA-DRB1*04, and HLA-DRB1 donor/recipient mismatch were confirmed to provide a significant and independent contribution to the risk of hepatitic disease recurrence. As for the severity of the disease, none of the 10 patients with stage 5-6 hepatitis carried the HLA-DRB1*11 allele, in line with what was observed in nontransplant subjects. Our results suggest that in posttransplant recurrent hepatitis C, immunogenetic factors are relevant in determining HCV infection outcome.
Expression of aquaporin-4 water channels in rat cholangiocytes
Authors: Marinelli Raúl A. , Pham Linh D. , Tietz Pamela S. , LaRusso Nicholas F.
Published: March 2004

We recently reported that secretin induces the exocytic insertion of functional aquaporin-1 water channels (AQP1) into the apical membrane of cholangiocytes and proposed that this was a key process in ductal bile secretion. Because AQP1 is present on the basolateral cholangiocyte membrane in low amounts, we hypothesized that another AQP must be expressed at this domain to facilitate transbasolateral water movement. Thus, we investigated the expression, subcellular localization, possible regulation by secretin, and functional activity of AQP4, a mercury-insensitive water channel expressed in other fluid transporting epithelia. Using reverse transcription-polymerase chain reaction (RT-PCR) on RNA prepared from purified rat cholangiocytes, we amplified a product of 311 bp that was 100% homologous to the reported AQP4 sequence. RNase protection assay confirmed the presence of an appropriate size transcript for AQP4 in cholangiocytes. Immunoblotting detected a band of approximately 31 kd corresponding to AQP4 in basolateral but not apical membranes of cholangiocytes. Secretin did not alter the amount of plasma membrane AQP4 but, as expected, induced AQP1 redistribution from intracellular to apical plasma membranes. Functional studies showed that AQP4 accounts for about 15% of total cholangiocyte membrane water permeability. Our results indicate that: (1) cholangiocytes express AQP4 messenger RNA (mRNA) and protein and (2) in contrast to AQP1, which is targeted to the apical cholangiocyte membrane by secretin, AQP4 is constitutively expressed on the basolateral cholangiocyte membrane and is secretin unresponsive. The data suggest that AQP4 facilitates the basolateral transport of water in cholangiocytes, a process that could be relevant to ductal bile formation.
Mutations within the E2 and NS5A protein in patients infected with hepatitis C virus type 3a and correlation with treatment response
Authors: Sarrazin Christoph , Kornetzky Ilka , Rüster Brigitte , Lee Jung-Hun , Kronenberger Bernd , Bruch Katharina , Roth Kurt W. , Zeuzem Stefan
Published: March 2004

Defined regions of hepatitis C virus (HCV) envelope 2 (E2), PePHD, and nonstructural 5A (NS5A) protein (PKR-binding domain) have been shown to interact with interferon alfa (IFN-α)-inducible double-stranded RNA-activated protein kinase (PKR) in vitro, suggesting a possible mechanism of HCV to evade antiviral effects of IFN-α. The clinical correlation between amino acid mutations within the E2 PePHD or the NS5A PKR-binding domain and response to antiviral treatment in HCV-3a-infected patients is unknown. Thirty-three patients infected with HCV-3a isolates were treated with IFN-α with or without ribavirin. The carboxyterminal half of E2 and of the NS5A gene were sequenced. Sixteen patients achieved a sustained virological response (SR), 6 patients an end-of-treatment response with relapse thereafter (ETR), and 11 patients were nonresponders (NR). Within the PePHD of the E2 protein 0.5 (range, 0-2) mutations were observed in SR patients, whereas the number of mutations in ETR or NR patients was 0.2 (0-1). Quasispecies analyses showed almost no heterogeneity. The mean number of mutations within the PKR-binding domain of the NS5A protein was 1.6 (range, 0-4) in SR patients, 1 (0-2) in ETR patients, and 1.6 (0-3) in NR patients. Patients with higher numbers of mutations within the E2 or NS5A region showed a trend towards lower pretreatment viremia. Phylogenetic and conformational analyses of E2 or NS5A sequences allowed no differentiation between sensitive and resistant isolates. However, mutations within the E2 PePHD in SR patients were frequent, and hydrophobic mutations within the hydrophilic area of PePHD at codon 668 and 669 were exclusively observed in sustained virological responders.
Association between hepatitis B virus infection and HLA-DR type in Korea
Authors: Ahn Sang Hoon , Han Kwang-Hyub , Park Jeong Youp , Lee Chun Kyon , Kang Shin Wook , Chon Chae Yoon , Kim Yu Seun , Park Kiil , Kim Dong Kee , Moon Young Myoung
Published: March 2004

Although the mechanism of susceptibility to chronic persistent hepatitis B virus (HBV) infection is not well clarified, immunogenetic factors of the host may have a role. Recently, a strong association between HLA-DR13 and the self-limited course of HBV infection has been reported. To determine whether the elimination of HBV is related to a particular HLA allele, we studied the HBV markers and HLA-DR phenotypes of 1,272 Koreans who had visited Yonsei University Medical Center for renal transplantation. They included 330 renal transplant donors. Subjects were categorized into 3 different groups: the “Unexposed Group” (UE; n = 946) with negative HBV markers, the “Chronic Carrier Group” (CC; n = 83), who were hepatitis B surface antigen (HBsAg)-positive, and the “Spontaneously Cleared Group” (SC; n = 243), who were HBsAg-negative with antibodies to HBsAg (anti-HBs) and hepatitis B core antigen (anti-HBc). HLA-DR4 was the most common type in all groups. HLA-DR6 was significantly more frequent in 69 of 243 subjects with SC (28.4%) than in 8 of 83 subjects with CC (9.6%) (P < .001; relative risk [RR] = 3.72). HLA-DR9 was significantly more frequent in CC than in SC (P < .001; RR = 0.33). HLA-DR13 showed a stronger association with the clearance of HBV than the other HLA-DR6 subgroup. The distribution of HLA-DR phenotypes was similar regardless of renal disease. Our data indicate that HLA-DR6, especially HLA-DR13, is one of the host factors, which influences the immune response to HBV, and may be associated with self-elimination of HBV in Koreans.
Mimicry peptides of human PDC-E2 163-176 peptide, the immunodominant T-cell epitope of primary biliary cirrhosis
Authors: Shimoda Shinji , Nakamura Minoru , Shigematsu Hirohisa , Tanimoto Hironori , Gushima Toshihumi , Gershwin Eric M. , Ishibashi Hiromi
Published: March 2004

The human PDC-E2 163-176 peptide (GDLLAEIETDKATI) is an immunodominant autoreactive T-cell epitope in patients with primary biliary cirrhosis (PBC), restricted by HLA DRB4*0101. We have previously reported that the ExDK sequence is essential for recognition of this epitope and identified 1 mimicry peptide, Escherichia coli PDC-E2 peptide (EQSLITVEGDKASM), which can activate human PDC-E2 163-176 peptide-reactive T-cell clones. In the present study, to further investigate mimicry peptides possibly involved in PBC, we generated 13 different T-cell clones reactive to the human PDC-E2 163-176 peptide following repeated in vitro stimulation of peripheral T lymphocytes with the human PDC-E2 163-176 peptide (native peptide) and tested for the reactivity of these T-cell clones to 30 different mimicry peptides derived from various self- and nonself proteins that have an ExDK-sequence. We found 7 mimicry peptides derived from microbial proteins that can activate at least 1 of these T-cell clones; 7 of 7 T-cell clones from patients with PBC and 2 of 6 T-cell clones from healthy subjects were activated by at least 1 to 6 different mimicry peptides. Two of 6 T-cell clones from healthy subjects were activated by specific mimicry peptides more strongly than by the native peptide, and 2 of 6 T-cell clones from healthy subjects were not activated by any mimicry peptides tested. Thus, the pattern and degree of activation by mimicry peptides differed in each T-cell clone, indicating the presence of a diverse spectrum of autoreactive T cells that are reactive to a single minimal epitope of the human PDC-E2 163-176 peptide.
Mrp2 is essential for estradiol-17β(β-d -glucuronide)-induced cholestasis in rats
Authors: Huang Liyue , Smit Johan W. , Meijer Dirk K. , Vore Mary
Published: March 2004

The present study evaluates the roles of the multidrug resistance-1 P-glycoprotein, Mdr1a/1b, the bile salt export pump (Bsep), and the multidrug resistance-associated protein-2 (Mrp2) in mediating cholestasis induced by estradiol-17β(β-d -glucuronide) (E2 17G). Administration of [3H]E2 17G (18 nmol/g body weight) gave a similar degree of cholestasis and biliary excretion of E2 17G-equivalents in wild-type and Mdr1a-/-/1b-/- mice. When expressed in Sf9 cells, Bsep-mediated adenosine triphosphate (ATP)-dependent transport of taurocholate (TC, 1 μmol/L) in membrane vesicles was 110% ± 12.5% and 108% ± 17.3% of control in the presence of 10 and 50 μmol/L E2 17G, respectively, whereas in rat canalicular membrane, both E2 17G and the choleretic estradiol-3-β-d -glucuronide (E2 3G) inhibited ATP-dependent transport of TC to the same extent. Infusion of [3H]E2 17G (24 μmol) did not induce cholestasis in Mrp2-deficient TR− rats whereas 2 μmol of [3H]E2 17G inhibited bile flow by 51% in control Wistar rats. The maximal biliary concentration of E2 17G was 3.5 and 2.5 mmol/L in control and TR− rats, respectively. However, 2.2 mmol/L of E2 17G in bile is associated with inhibition of bile flow in control rats. These data show that (1) Mdr1a/1b are not essential for E2 17G-mediated cholestasis, (2) direct inhibition of Bsep-mediated bile acid transport is not the mechanism for E2 17G cholestasis, and (3) accumulation of E2 17G in bile alone is not sufficient to induce cholestasis. These data indicate that the process of Mrp2-mediated transport of high concentrations of E2 17G is essential for its induction of cholestasis.