bluuebunny/crossref_metadata_2025 huggingface dataset:
Cholestasis with altered structure and function of hepatocyte tight junction and decreased expression of canalicular multispecific organic anion transporter in a rat model of colitis
Authors: Kawaguchi Takumi , Sakisaka Shotaro , Mitsuyama Keiichi , Harada Masaru , Koga Hironori , Taniguchi Eitaro , Sasatomi Kurumi , Kimura Rina , Ueno Takato , Sawada Norimasa , Mori Michio , Sata Michio
Published: March 2004

Cholestasis is frequently associated with inflammatory bowel disease. Because some cholestasis is resulted from altered hepatocyte tight junctions (TJs) or the canalicular multispecific organic anion transporter, we have investigated the following topics in a rat model of inflammatory bowel disease: (1) alterations in hepatocyte TJs and in the canalicular multispecific organic anion transporter, (2) etiologic factors for cholestasis, and (3) effects of antibiotics on cholestasis. Rats with trinitrobenzene sulfonic acid-induced colitis were studied 24 hours after treatment. Hepatocyte TJs and the canalicular multispecific organic anion transporter were evaluated by immunostaining for TJ-associated proteins, 7H6 and ZO-1, and multidrug resistance protein 2 (mrp2). To investigate etiologic factors causing cholestasis, portal endotoxin and proinflammatory cytokines were examined. The effects of polymyxin B, penicillin G, or metronidazole on immunostaining for 7H6, ZO-1, mrp2, and cholestasis were investigated. (1) Immunostaining for 7H6 and ZO-1 colocalized outlining the bile canaliculi and immunostaining for mrp2 localized on the canalicular membrane in controls. Treatment with trinitrobenzene sulfonic acid induced significant cholestasis and caused translocation of immunostaining for 7H6, but not that for ZO-1, to the cytoplasm and diminished immunostaining for mrp2 on the canaliculus membrane. (2) The levels of portal endotoxin, but not proinflammatory cytokines, was increased. (3) Polymyxin B, but not the other antibiotics, prevented alterations in immunostaining for both 7H6 and mrp2, and cholestasis. We described that both hepatocyte TJs and the canalicular multispecific organic anion transporter were altered and that gut-derived endotoxin levels in the portal blood were increased in this rat colitis model.
Viral clearance in hepatitis C (1b) infection: Relationship with human leukocyte antigen class II in a homogeneous population
Authors: Fanning Liam J. , Levis John , Kenny-Walsh Elizabeth , Wynne Freda , Whelton Michael , Shanahan Fergus
Published: March 2004

The aim of this study was to investigate the possibility of a significant relationship between human leukocyte antigen (HLA) class II and the clearance of hepatitis C virus (HCV). The study group consisted of 156 Irish women who iatrogenically received HCV 1b-contaminated Anti-D immunoglobulin between May 1977 and November 1978. Thus, the study population was homogeneous in terms of gender, source of infection, and ethnicity. On Screening in 1994, all individuals were anti-HCV antibody positive by recombinant immunoblot assay, while 46% (n = 72) of the group were HCV-positive by reverse transcriptase-polymerase chain reaction (RT-PCR). HLA DRB1 and DQB1 status was molecularly defined by high resolution reverse line probe hybridization methodology. Clearance of HCV 1b was found to be associated with DRB1*01. However, this association was lost after Bonferroni correction for multiple comparisons. Extended haplotype analysis between specific DRB1 and DQB1 allelic combinations identified a significant reduction in the frequency of DQB1*0501 in the presence of DRB1*0701 in the persistently infected individuals in the study group (P < .05). No associations with either viral clearance or persistence were found at the DQB1 locus. Our results suggest that HLA DRB1*01 appears to contribute to the spontaneous resolution of a primary HCV infection in the Irish population. The presence of DRB1*0701 in the absence of DQB1*0501 possibly reflects an influence of this allele in persistence of HCV infection. Defined and homogeneous patient populations offer the best opportunity to illuminate previously disguised immunogenetic factors important in the clearance of HCV 1b.
Differential expression of lymphoid and myeloid markers on differentiating hematopoietic stem cells in normal and tumor-bearing adult human liver
Authors: Golden-Mason Lucy , Curry Michael P. , Nolan Niamh , Traynor Oscar , McEntee Gerry , Kelly Jacinta , Hegarty John E. , O’Farrelly Cliona
Published: March 2004

The presence and phenotype of lineage-committed hematopoietic progenitors in the normal adult human liver (AHL) were investigated and compared with the profiles of differentiating hematopoietic precursor populations detected in liver bearing metastases of colonic origin. Levels of hematopoietic stem cells (HSCs) (CD34+CD45+) detected in hepatic mononuclear cell (HMNC) populations were increased 6-fold when compared with matched peripheral blood samples. In normal liver, less than 5% of HSCs expressed the myeloid-associated antigen, CD33, whereas considerable proportions expressed lymphoid-associated markers (T cell, 33.39%; B cell, 17.39%; and natural killer [NK] cell, 37.17%). Significant increases were observed in the relative proportions of hepatic HSCs coexpressing CD33 (20.53%; P = .001), and the T-cell marker (CD7, 58.13%; P = .02) in tumor-bearing liver compared with normal liver. HSCs with B-cell progenitor phenotype (CD19+) were significantly decreased in tumor-bearing liver (0.06%; P = .02). Despite these differences, the activation status of hematopoiesis, as measured by the coexpression of the differentiation and activation markers, CD38 and CD45RA, did not differ significantly between normal and tumor-bearing liver. These results indicate that the normal AHL harbors lineage-committed hematopoietic progenitors, and the vast majority of these progenitors express lymphoid-associated antigens with changes occurring in both the myeloid and lymphoid compartments of the hepatic hematopoietic pathway on tumor challenge. While tumor-bearing livers are enriched for intrahepatic myeloid precursors and T-cell progenitor cells, further studies are required to establish the origin and in situ development potential of hepatic HSCs in the adult human and their role in tumor immunity.
Bile acid synthesis in cultured human hepatocytes: support for an alternative biosynthetic pathway to cholic acid
Authors: Axelson Magnus , Ellis Ewa , Mörk Birgitta , Garmark Kristina , Abrahamsson Anna , Björkhem Ingemar , Ericzon Bo-Göran , Einarsson Curt
Published: March 2004

The biosynthesis of bile acids by primary cultures of normal human hepatocytes has been investigated. A general and sensitive method for the isolation and analysis of sterols and bile acids was used, based on anion exchange chromatography and gas chromatography-mass spectrometry (GC/MS). Following incubation for 5 days, 8 oxysterols and 8 C27- or C24-bile acids were identified in media and cells. Cholic and chenodeoxycholic acids conjugated with glycine or taurine were by far the major steroids found, accounting for 70% and 24% of the total, respectively, being consistent with bile acid synthesis in human liver. Small amounts of sulfated 3β-hydroxy-5-cholenoic acid and 3β,7α-dihydroxy-5β-cholanoic acid were also detected. Nine steroids were potential bile acid precursors (2% of total), the major precursors being 7α,12α-dihydroxy-3-oxo-4-cholenoic acid and its 5β-reduced form. These 2 and 5 other intermediates formed a complete metabolic sequence from cholesterol to cholic acid (CA). This starts with 7α-hydroxylation of cholesterol, followed by oxidation to 7α-hydroxy-4-cholesten-3-one and 12α-hydroxylation. Notably, 27-hydroxylation of the product 7α,12α-dihydroxy-4-cholesten-3-one and further oxidation and cleavage of the side chain precede A-ring reduction. A-Ring reduction may also occur before side-chain cleavage, but after 27-hydroxylation, yielding 3α,7α,12α-trihydroxy-5β-cholestanoic acid as an intermediate. The amounts of the intermediates increased in parallel to those of CA during 4 days of incubation. Suppressing 27-hydroxylation with cyclosporin A (CsA) resulted in a 10-fold accumulation of 7α,12α-dihydroxy-4-cholesten-3-one and a decrease of the production of CA and its acidic precursors. These results suggest that the observed intermediates reflect an alternative biosynthetic pathway to CA, which may be quantitatively significant in the cells.
Autoimmune cholangitis within the spectrum of autoimmune liver disease
Authors: Czaja Albert J. , Carpenter Herschel A. , Santrach Paula J. , Moore Breanndan S.
Published: March 2004

Autoimmune cholangitis is an idiopathic disorder with mixed hepatocellular and cholestatic findings. Our goal was to characterize the disease prospectively by application of uniform diagnostic criteria. Twenty patients were identified and compared with 242 patients with conventional forms of autoimmune liver disease. Patients with autoimmune cholangitis were distinguished from type 1 autoimmune hepatitis (AIH) by lower serum levels of aspartate transaminase (AST), γ-globulin, and immunoglobulin G; higher serum levels of alkaline phosphatase; and lower frequencies of autoantibodies. They were distinguished from primary biliary cirrhosis (PBC) by higher serum levels of AST and bilirubin, lower serum concentrations of immunoglobulin M, and greater occurrence of autoantibodies. Their female predominance, lower serum alkaline phosphatase levels, higher frequency of autoantibodies, and absence of inflammatory bowel disease differentiated them from primary sclerosing cholangitis (PSC). Laboratory findings ranged widely and did not characterize individual patients. HLA risk factors were similar to those of type 1 AIH and PBC, and different from those of PSC. Treatment responses to corticosteroids or ursodeoxycholic acid were poor. Composite histological patterns resembled mainly PBC or PSC. We conclude that autoimmune cholangitis diagnosed by prospective analysis cannot be assimilated into a single, conventional, diagnostic category. It may represent variant forms of diverse conditions, a transition stage, or a separate entity with varying manifestations.
Cold liver ischemia-reperfusion injury critically depends on liver T cells and is improved by donor pretreatment with interleukin 10 in mice
Authors: Le Moine Olivier , Louis Hubert , Demols Anne , Desalle Fabrice , Demoor François , Quertinmont Eric , Goldman Michel , Devière Jacques
Published: March 2004

Kupffer cells are thought to mediate most of the deleterious effects of liver ischemia-reperfusion injury. The role of liver T cells and the impact of resident cell deactivation by interleukin 10 (IL-10) have never been addressed. Using a model of ex vivo liver cold ischemia and reperfusion, we assessed liver injury, tumor necrosis factor (TNF) and interferon gamma (IFN-γ) release from livers of balb/c mice, nude mice, nude mice reconstituted with T cells, and gadolinium balb/c pretreated mice. The anti-inflammatory cytokine IL-10 was then used to define the best strategy of administration potentially able to modulate ischemia-reperfusion injury. For this purpose IL-10 was administered to the donor before liver harvesting, in the preservation medium during cold ischemia or during reperfusion. TNF and IFN-γ were released time dependently and paralleled liver injury after reperfusion of cold preserved livers. Reperfused livers from nude or gadolinium pretreated mice disclosed a dramatic decrease in TNF and IFN-γ release. Tissue injury was reduced by 51% in the absence of T cells and by 88% when Kupffer cells were deactivated. This effect was reverted by T-cell transfer to nude mice. Only donor pretreatment with IL-10 or IL-10 infusion during reperfusion led to a significant decrease in liver injury, TNF, and IFN-γ release (−66% or −41%, −95% or −94%, and −70% or −70%, respectively). In conclusion, liver resident T cells are critically involved in cold ischemia-reperfusion injury and pretreatment of the donor with IL-10 decreases liver injury and the release of T-cell- and macrophage-dependent cytokines.
Combination of ribavirin and interferon-alfa surpasses high doses of interferon-alfa alone in patients with genotype-1b-related chronic hepatitis C
Authors: Pol Stanislas , Nalpas Bertrand , Bourlière Marc , Couzigou Patrice , Tran Albert , Abergel Armand , Zarski Jean-Pierre , Berthelot Pierre , Bréchot Christian
Published: March 2004

The purpose of this study was to compare interferon-alfa alone (12-month course with high initial doses) with a combination of interferon-alfa and ribavirin in patients infected with genotype 1b. Three hundred and seven patients were randomized into 3 groups to receive 6 mega units (MU) of interferon-alfa-2b subcutaneously 3 times weekly for 6 months followed by 3 MU for 6 months (n = 95, group A); 10 MU for 3 months followed by 6 MU for 3 months, followed by 3 MU for 6 months (n = 83, group B); or the group-A schedule in combination with ribavirin (n = 129, group C) for 4 (n = 46), 6 (n = 44), or 12 months (n = 39). Negative polymerase chain reaction (PCR) was more frequent in group C than in groups A or B after 3 months of treatment (P< .006), at the end of treatment (P= .017), and at the end of follow-up (32.8%, 16.9%, and 14.1%, respectively, P< .003). A complete response (negative PCR and normal alanine transaminase) was higher in group C than in the other groups and when comparing 12- to 4- and 6-month combination therapy at the end of treatment (P = .05) and of follow-up (45.2% vs. 25.4%, respectively, P = .05). The greater efficacy of the combination was related to the higher rate of primary virological response and also to a decrease in the percentage of breakthrough and of relapse. In 1b-infected patients, the combination of high doses of interferon-alfa (6 MU) and ribavirin for 12 months appears to be the best therapy, with a high rate of sustained response.
Decreased carnitine biosynthesis in rats with secondary biliary cirrhosis
Authors: Krähenbühl Stephan , Brass Eric P. , Hoppel Charles L.
Published: March 2004

Carnitine biosynthesis was investigated in rats with secondary biliary cirrhosis induced by bile duct ligation (BDL) for 4 weeks (n = 5) and in pair-fed, sham-operated control rats (n = 4). Control rats were pair-fed to BDL rats, and all rats were fed an artificial diet with negligible contents of carnitine, butyrobetaine, or trimethyllysine. Biosynthesis of carnitine and its precursors was determined by measuring their excretion in urine and accumulation in the body of the animals. Four weeks after BDL, total carnitine content was increased by 33% in livers from BDL rats when compared with control rats, but was unchanged in skeletal muscle and whole carcass. The plasma total carnitine concentration averaged 29.0 ± 4.1 vs. 46.4 ± 7.3 μmol/L in BDL rats and control rats, respectively. Urinary total carnitine excretion was reduced by 56% in BDL rats as compared with control rats. Carnitine biosynthesis was significantly decreased in BDL rats (0.45 ± 0.19 vs. 0.93 ± 0.08 μmol/100 g body weight/d in BDL and control rats, respectively). The tissue content of free and protein-linked trimethyllysine, a carnitine precursor, and trimethyllysine plasma concentrations were not different between BDL and control rats. However, urinary trimethyllysine excretion was increased 5-fold in BDL rats and approximated glomerular filtration. In contrast, urinary excretion of butyrobetaine, the direct carnitine precursor, was decreased by 40% in BDL rats as compared with control rats. Trimethyllysine biosynthesis was not different, but butyrobetaine biosynthesis was decreased by 51% in BDL as compared with control rats. In conclusion, carnitine biosynthesis is decreased in BDL rats as a result of a defect in the conversion of trimethyllysine to butyrobetaine.
Isosorbide mononitrate and propranolol compared with propranolol alone for the prevention of variceal rebleeding
Authors: Gournay Jérôme , Masliah Claude , Martin Thierry , Perrin Daniel , Galmiche Jean-Paul
Published: March 2004

The aim of this study was to test the effectiveness of isosorbide-5-mononitrate (IM) as an adjunct to propranolol (PR) in the prevention of variceal rebleeding. Ninety-five cirrhotic patients with variceal bleeding were randomly assigned to treatment with PR + IM (46 patients) or PR alone (49 patients). Eighteen patients in the PR + IM group and 28 in the PR group had rebleeding during the 2 years after randomization. The actuarial probability of rebleeding 2 years after randomization was lower in the PR + IM group (40.4% vs. 57.4%) but the difference was not significant (P = .09). However, the decrease in the risk of rebleeding reached statistical significance after stratification according to age, i.e. less than 50 versus ≥50 years old, (P = .03) or by adding an additional year of follow-up (P = .05). No significant difference was found in rebleeding index and survival. The multivariate Cox analysis indicated first, that both treatment (P = .03) and age (P = .001) were factors predictive of rebleeding and second, that PR + MI reduced the risk of rebleeding by half (relative risk: 0.51, 95% confidence interval: 0.28-0.95). Seven patients in the PR + MI group and 1 patient in the PR group had to discontinue one of the drugs because of adverse events (P = .03). These results suggest that the addition of IM improves the efficacy of PR alone in the prevention of variceal rebleeding in cirrhotic patients. However no beneficial effects were observed on other parameters reflecting the efficacy of treatment.
Unique epithelial cell production of hepatocyte growth factor/scatter factor by putative precancerous intestinal metaplasias and associated “intestinal-type” biliary cancer chemically induced in rat liver
Authors: Lai Guan-Hua , Radaeva Svetlana , Nakamura Toshikazu , Sirica Alphonse E.
Published: March 2004

Recently, we observed that Met, the receptor for hepatocyte growth factor/scatter factor (HGF/SF), is overexpressed in epithelial cells of both early-appearing intestinal metaplastic glands in precancerous hepatic cholangiofibrotic tissue and neoplastic glands in later developed intestinal-type of cholangiocarcinoma originated from the furan rat model of cholangiocarcinogenesis when compared with normal and hyperplastic intrahepatic biliary epithelia. We now show that HGF/SF is also aberrantly expressed in a manner closely paralleling that of its receptor in the neoplastic epithelial cells of furan-induced rat cholangiocarcinomas and in a majority of metaplastic epithelial cells within earlier formed precancerous hepatic cholangiofibrotic tissue. Usingin situhybridization and reverse transcription-polymerase chain reaction (RT-PCR), we further showed specific expression of HGF/SF messenger RNA (mRNA) in a novel rat cholangiocarcinoma epithelial cell line overexpressing Met. This cholangiocarcinoma cell line, termed C611B, was established from tumorigenic cells isolated from a furan-induced transplantable tumor. Moreover, we detected byin situhybridization strong expression of HGF/SF mRNA transcripts in the cancerous epithelial glands of cholangiocarcinoma developed in recipient rats afterin vivocell transplantation of C611B cells. In contrast, mRNA transcripts and protein immunoreactivity for this cytokine were not detected in hepatocytes and biliary epithelial cells in adult normal rat liver nor in rat hyperplastic intrahepatic biliary epithelium. Our results clearly show that HGF/SF becomes aberrantly expressed in cholangiocarcinoma epithelium and in putative precancerous intestinal metaplastic epithelium induced in the liver of furan-treated rats.
Low frequency of cirrhosis in a hepatitis C (genotype 1b) single-source outbreak in germany: A 20-year multicenter study
Authors: Wiese Manfred , Berr Frieder , Lafrenz Michael , Porst Heiner , Oesen Ute
Published: March 2004

From August 1978 until March 1979, 14 batches of anti-D immune globulin contaminated with hepatitis C virus (HCV) genotype 1b (20,000-480,000 copies/dose) from a single erythrocyte donor had been administered for prophylaxis of rhesus isoimmunization throughout East Germany. All 2,867 women involved had been recalled after January 12, 1979 for repeated screening of alanine transaminase (ALT). They were prospectively followed in regional centers. We have reexamined a cohort of 1,018 women (median age 24, range 16-38 years at infection) on follow-up for 20 years in 9 representative centers. Within 6 months after anti-D administration, 10% of these women had no evidence of disease and 90% had acute hepatitis C (n = 917) including 49% with symptomatic and 22% with icteric course. After 20 years, 85% of the 917 affected women still tested positive for HCV antibodies (among them 3% responded to interferon treatment) and 55% were positive for HCV RNA (among them 7% were nonresponders to interferon and 3% were apparent HCV carriers). Only 4 (0.4%) had overt cirrhosis. Two (0.2%) died of superinfected fulminant hepatitis B or alcoholism and cirrhosis, respectively. Histology obtained in 44% of the viremic women showed hepatitis of minimal to moderate grade in 96%, portal fibrosis in 47%, and septal fibrosis in 3% of the cases. In conclusion, formerly healthy young women, without hepatic comorbidity, may clear HCV (1b) infection in half of the cases or develop mild chronic hepatitis C with low risk of progression to cirrhosis within 20 years.
Human placenta sphingomyelinase, an exogenous acidic pH-optimum sphingomyelinase, induces oxidative stress, glutathione depletion, and apoptosis in rat hepatocytes
Authors: García-Ruiz Carmen , Marí Montse , Morales Albert , Colell Anna , Ardite Esther , Fernández-Checa José C.
Published: March 2004

Ceramide has been identified as a putative lipid messenger that mediates diverse cellular processes including cell death. Since glutathione (GSH) depletion is known to sensitize cells to many cytotoxic agents and as a result of the reported regulation of neutral sphyngomyelinase (NSMase) by GSH, the present study compared the role of individual SMases in the induction of oxidative stress, regulation of cellular GSH, and apoptosis of rat hepatocytes. Exposure of cultured rat hepatocytes to exogenous Bacillus cereus sphingomyelinase (bSMase), a neutral SMase, or human placenta sphingomyelinase (hSMase), an acidic SMase (ASMase), generated similar ceramide levels in a dose-dependent manner. However, whereas bSMase increased hepatocellular GSH levels, hSMase depleted GSH stores, an effect that was prevented by monensin and mannose 6-phosphate (M-6-P), suggesting that exogenous hSMase enters hepatocytes by endocytosis and is delivered to an endosomal/lysosomal acidic compartment. Interestingly, despite the differential effect of either SMases on cell GSH levels, both bSMase and hSMase increased γ-glutamylcysteine synthetase heavy-subunit chain (γ-GCS-HS) mRNA levels. Consistent with these findings on GSH regulation, hSMase, but not bSMase, generated reactive oxygen species (ROS), being accompanied by mitochondrial depolarization, suggesting that hSMase targeted mitochondria, leading to oxidative stress. Accordingly, hepatocytes displayed a selective sensitivity to hSMase in contrast to bSMase exposure, and depletion of GSH stores enhanced susceptibility to hSMase as a result of potentiation of ROS formation and caspase 3 activation. Thus, these findings reveal the ability of ASMase to induce oxidative stress as a result of the targeting of mitochondria, and that GSH depletion sensitizes hepatocytes to the ASMase-induced apoptosis.
Long-term follow-up study of core gene deletion mutants in children with chronic hepatitis B virus infection
Authors: Ni Yen-Hsuan , Chang Mei-Hwei , Hsu Hong-Yuan , Chen Huey-Ling
Published: March 2004

Core gene deletion mutants of hepatitis B virus (HBV) have been identified in adults. Because the acquisition of HBV occurs mainly in infancy and childhood in hyperendemic areas, this study aimed to learn the temporal profile of such mutants in children with chronic HBV infection. We have followed up 365 HBV-infected children for more than 10 years and screened out HBV core gene deletion from their sera. Serial serum samples of positive cases were subjected to HBV-DNA nucleotide sequence analyses and quantification. Deletion mutants were found in 18 of the 365 patients (4.9%). Most cases (15 of 18) with deletion mutants heralded hepatitis B e antigen (HBeAg) seroconversion phase, while the other cases (3 of 18) remained in HBeAg-seropositive phase. Deletion mutants disappeared after HBeAg seroconversion except in 1 child. Decreased HBV-DNA levels accompanied deletion mutants for those who finally underwent HBeAg seroconversion, but the HBV-DNA level did not decline if there was no seroconversion. Deletion mutants were not associated with a particularly high peak liver enzyme. Core gene deletion mutants could appear as early as the age of 5. The duration of their appearance was 0.5 to 5 years. Horizontal rather than perinatal transmission of HBV was a favorable factor for these mutants to develop. Deletion fragments were located in the middle part of core gene. The emergence of the mutants was likely the result of host-viral interaction and mostly signified HBeAg seroconversion within 1 year. Core gene deletion mutants appeared preferably in children acquiring HBV by horizontal transmission.
Rifamycin SV and rifampicin exhibit differential inhibition of the hepatic rat organic anion transporting polypeptides, Oatp1 and Oatp2
Authors: Fattinger Karin , Cattori Valentino , Hagenbuch Bruno , Meier Peter J. , Stieger Bruno
Published: March 2004

The antibiotics, rifamycin SV and rifampicin, are known to interfere with hepatic bile salt and organic anion uptake. The aim of this study was to explore which transport systems are affected. In short-term-cultured rat hepatocytes, low concentrations (10 μmol/L) of both compounds inhibited mainly sodium-independent taurocholate uptake, whereas higher concentrations (100 μmol/L) also inhibited sodium-dependent taurocholate uptake. In Xenopus laevis oocytes expressing the Na+/taurocholate cotransporting polypeptide (Ntcp), high rifamycin SV and rifampicin concentrations were required for inhibition of taurocholate uptake. In contrast, sodium-independent taurocholate uptake mediated by the organic anion transporting polypeptides, Oatp1 and Oatp2, was already substantially inhibited by 10 μmol/L rifamycin SV. Rifampicin potently inhibited Oatp2-mediated taurocholate uptake, but did not interfere with Oatp1-mediated taurocholate uptake. Similar effects of rifamycin SV and rifampicin were found for Oatp1- and Oatp2-mediated estradiol-17β-glucuronide transport. Dixon plot analysis yielded a pattern compatible with competitive inhibition of estradiol-17β-glucuronide transport with Ki estimates of 6.6 μmol/L and 7.3 μmol/L for rifamycin SV-induced inhibition of Oatp1 and Oatp2, respectively, and of 1.4 μmol/L for rifampicin-induced inhibition of Oatp2. These results demonstrate that rifamycin SV and rifampicin exhibit differential inhibition on Oatp1 and Oatp2, and identify rifampicin as a selective Oatp2 inhibitor. The data indicate that these inhibitors can be used to determine the in vivo relevance of Oatp1 and Oatp2 for the overall bioavailability and disposition of drugs and other Oatp1/2 substrates.
Adefovir dipivoxil for the treatment of lamivudine-resistant hepatitis B mutants
Authors: Perrillo Robert , Schiff Eugene , Yoshida Eric , Statler Ann , Hirsch Kenneth , Wright Teresa , Gutfreund Klaus , Lamy Patrick , Murray Alison
Published: March 2004

Lamivudine has been shown to be an effective therapy for chronic hepatitis B, but resistance to this nucleoside agent is common after prolonged use. Five patients with chronic hepatitis B virus (HBV) infection developed resistance to lamivudine after 9 to 19 months of treatment. In 4 patients this occurred after liver transplantation and the remaining individual had stable cirrhosis. In each case, resistance was confirmed to be caused by one or more mutations in the HBV-DNA polymerase gene and was associated with active underlying liver disease. The patients were treated with adefovir dipivoxil in a dose of 5 to 30 mg daily. Two to 4 log10 reductions in HBV-DNA levels were observed in 4 cases, and the fifth patient became negative by quantitative polymerase chain reaction (PCR) after retransplantation in conjunction with hepatitis B immunoglobulin (HBIg). Virologic improvement was associated with stable or declining serum alanine transaminase levels in 4 patients. HBV-DNA suppression has been sustained during a mean treatment period of 13 months (range 11 to 15 months), including 1 patient in whom lamivudine has been discontinued. Mild changes in renal function were observed during treatment in most cases but did not require early discontinuation of the drug. This study provides evidence that adefovir dipivoxil can be an effective treatment for lamivudine-resistant HBV mutants as well as wild-type HBV.
Iron reduction as an adjuvant to interferon therapy in patients with chronic hepatitis C who have previously not responded to interferon: A multicenter, prospective, randomized, controlled trial
Authors: Di Bisceglie Adrian M. , Bonkovsky Herbert L. , Chopra Sanjiv , Flamm Stephen , Reddy Rajender K. , Grace Norman , Killenberg Paul , Hunt Christine , Tamburro Carlo , Tavill Anthony S. , Ferguson Roy , Krawitt Edward , Banner Barbara , Bacon Bruce R.
Published: March 2004

Hepatic iron concentration has consistently been observed as being directly correlated with the response to interferon therapy in chronic hepatitis C virus (HCV). We therefore conducted a randomized, controlled trial comparing iron reduction by phlebotomy with iron reduction followed by retreatment with interferon in 96 patients with chronic hepatitis C who had previously not responded to a course of interferon. During the initial phase when all patients were undergoing phlebotomy, we found that serum alanine transaminase (ALT) activities decreased but by less than 50% from baseline in 67 patients (89%), decreased by more than 50% in 12 patients (13%) and became normal in 9 patients (9%) with no overall change in HCV-RNA levels. Subsequently no patient in either treatment group achieved a sustained virologic response. Improvements in necroinflammatory changes were noted in liver biopsy specimens in those patients receiving phlebotomy plus interferon (mean index 8.59 vs. 7.37, P < .05). A slight but not statistically significant decrease in histologic activity index was noted in those subjects treated by phlebotomy alone (mean index 8.4 vs. 7.75, P not significant). We conclude that, although prior phlebotomy therapy does not improve the rate of sustained response to interferon retreatment, it does result in less liver injury manifested by a decrease in serum transaminase activity and a slight improvement in liver histopathology.
Cold-preservation-induced sensitivity of rat hepatocyte function to rewarming injury and its prevention by short-term reperfusion
Authors: Vajdová Katarína , Smreková Renáta , Mišlanová Csilla , Kukan Marián , Lutterová Martina
Published: March 2004

With increasing time of cold preservation, levels of high-energy nucleotides in the liver are reducing. The authors hypothesized that cold preservation sensitizes hepatocyte function to ischemic injury occurring during graft rewarming and that the injury can be prevented by short-term reperfusion. Rat livers were cold-preserved in University of Wisconsin solution for 0 to 18 hours and ischemically rewarmed for 0 to 45 minutes to simulate the implantation stage of transplantation. Hepatobiliary function was assessed using a blood-free perfusion model. In comparison with controls, neither 18-hour preservation nor 45-minute ischemic rewarming significantly influenced hepatocyte function. Compared with livers subjected to 45-minute ischemic rewarming, livers subjected to 9-hour preservation and 45-minute rewarming, and livers subjected to 18-hour preservation and 45-minute rewarming exhibited, respectively: 3.8 and 24 times reduced bile production, 4.3- and 116-fold decreased taurocholate excretion, and 3.1 and 42 times depressed bromosulfophthalein excretion. Thirty-minute oxygenated warm reperfusion after 9- and 18-hour preservation nearly completely blunted sensitization of hepatocyte function to rewarming ischemia. The authors found that short-term oxygenated reperfusion restored adenine nucleotides in liver tissue to the values found before organ preservation and that reperfusion with energy substrate containing solutions increased tissue adenosine triphosphate concentration to a higher level than that found before preservation. In conclusion, sensitization of hepatocyte function to rewarming ischemia increases disproportionally with storage time, suggesting that this phenomenon may play a role in graft dysfunctions with increasing liver preservation time. Short-term oxygenated reperfusion of the liver may protect hepatocyte functions against warm ischemic insult, even after extended preservation.
The function of alkaline phosphatase in the liver: Regulation of intrahepatic biliary epithelium secretory activities in the rat
Authors: Alvaro Domenico , Benedetti Antonio , Marucci Luca , Delle Monache Marco , Monterubbianesi Rita , Di Cosimo Emanuele , Perego Lucia , Macarri Giampiero , Glaser Shannon , Le Sage Gene , Alpini Gianfranco
Published: March 2004

We studied the effects of alkaline phosphatase (AP) on the secretory processes of the rat intrahepatic biliary epithelium as well as the role of the intrahepatic biliary epithelium in the uptake and biliary secretion of exogenous AP. The effects of acute and chronic administration of AP on bile secretory parameters were investigatedin vivoin normal and bile duct ligated (BDL) rats andin vitroin isolated rat bile duct units (IBDU).In vivo,acute AP administration decreased bile flow and biliary bicarbonate excretion and abolished secretin choleresis in BDL rats but not in normal rats. On the contrary, the AP inhibitor, levamisole, increased in BDL rat bile flow and biliary bicarbonate excretion.In vitro, basal and secretin-stimulated Cl−/HCO3−exchanger activity in IBDU was immediately inhibited by AP intraluminal microinjection (apical exposure) but only after a prolonged exposure to the basolateral pole. Levamisole increased the Cl−/HCO3−exchanger activity of IBDU. A significant basolateral uptake of AP occurs in IBDU with a progressive transport to the apical domain. AP chronic treatment increased AP and γ-glutamyltranspeptidase (γ-GT) activities in the intrahepatic bile ducts and hepatocyte canalicular pole, promoted enlargement of bile canaliculi, and decreased bile flow and biliary bicarbonate excretion. In conclusion, the intrahepatic biliary epithelium plays a role in the uptake and biliary secretion of serum AP. AP inhibits the secretory processes of the intrahepatic biliary epithelium and induces features of intrahepatic cholestasis after chronic administration. These findings indicate that AP plays an active role in down-regulating the secretory activities of the intrahepatic biliary epithelium.
Pretransplantation hepatitis C virus quasispecies may be predictive of outcome after liver transplantation
Authors: Pelletier Shawn J. , Raymond Daniel P. , Crabtree Traves D. , Iezzoni Julia C. , Sawyer Robert G. , Hahn Young S. , Pruett Timothy L.
Published: March 2004

The evolution of hepatitis C virus (HCV) envelope variation was studied using a liver-transplant model to evaluate the role of HCV quasispecies for hepatocyte infection. Twelve HCV polymerase chain reaction (PCR)-positive liver-transplant recipients (6 with posttransplantation biochemical hepatitis and 6 without hepatitis [controls]) were prospectively evaluated and underwent detailed quasispecies analysis of pre- and postoperative serum samples. HCV amino acid sequence diversity and complexity at the first hypervariable region (HVR1) of the second envelope protein (E2) was correlated with outcome. Recurrence of HCV-induced allograft injury was defined by persistently elevated serum alanine transaminase (ALT) levels. The major variant (sequences >10% of all clones) of recipients with hepatitis accounted for a significantly smaller percent of all preoperative clones compared with controls (41% ± 6% vs. 69% ± 8%; P < .015). Recipients with hepatitis had an increased number of pretransplantation major variants (2.5 ± 0.3 vs. 1.1 ± 0.2; P < .006). Eighty-three percent of controls had a predominant variant (accounting for >50% of clones) compared with 17% of those with recurrence (P < .05). These differences did not persist postoperatively. In addition, recipients without a pretransplantation predominant variant demonstrated an increased allograft fibrosis score (2.3 ± 0.3 vs. 0.5 ± 0.3; P < .015) at 181 to 360 days posttransplantation compared with those in whom a predominant variant was present. Increased HCV envelope complexity may act as a stronger antigenic stimulus or improve hepatocyte receptor binding and lead to allograft hepatitis and fibrosis. Although pretransplantation differences in HCV quasispecies did not persist postoperatively, pretransplantation quasispecies may be a predictor of HCV-induced hepatitis and graft fibrosis after liver transplantation.
Human homologue of maid: A dominant inhibitory helix-loop-helix protein associated with liver-specific gene expression
Authors: Terai Shuji , Aoki Hiroshi , Ashida Kozo , Thorgeirsson Snorri S.
Published: March 2004

The helix-loop-helix (HLH) family of transcriptional regulatory proteins are key regulators in numerous developmental processes. The class I HLH proteins, such as E12 are ubiquitously expressed. Class II HLH proteins, such as MyoD, are expressed in a tissue-specific manner. Class I and II heterodimers can bind to E-boxes (CANNTG) and regulate lineage commitments of embryonic cells. In an attempt to identify partners for the E12 protein that may exert control during liver development, we performed the yeast 2-hybrid screen using an expression complementary DNA library from human fetal liver. A novel dominant inhibitory HLH factor, designated HHM (human homologue of maid), was isolated and characterized. HHM is structurally related to the Id family and was highly expressed in brain, pituitary gland, lung, heart, placenta, fetal liver, and bone marrow. HHM physically interacted with E12 in vitro and in mammalian cells. Comparison of the dominant inhibitory effects of HHM and Id2 on the binding of E12/MyoD dimer to an E-box element revealed a weaker inhibition by HHM. However, HHM but not Id2 specifically inhibited the luciferase gene activation induced by hepatic nuclear factor 4 (HNF4) promoter. The HHM was transiently expressed during stem-cell-driven regeneration of the liver at the stage in which the early basophilic foci of hepatocytes started to appear. These results suggest that HHM is a novel type of dominant inhibitory HLH protein that might modulate liver-specific gene expression.
Specific detection of minus-strand hepatitis C virus RNA by reverse-transcription polymerase chain reaction on polyA+-purified RNA
Authors: Takyar Seyed Taghi , Li Dong-sheng , Wang Yong-hong , Trowbridge Rachel , Gowans Eric J.
Published: March 2004

A full-length complementary DNA (cDNA) clone of the hepatitis C virus (HCV) genome was used to prepare full-length plus- and minus-strand RNA. The minus-strand RNA, which contains a polyA+ tract complementary to the polyU tract found in the plus strand (genomic) RNA, but not the plus strand RNA, was captured with a commercial polyA+-tract isolation system. After elution, the minus strand was amplified by reverse-transcription polymerase chain reaction (RT-PCR). The combination of this procedure and RT-PCR using rTth resulted in an unprecedented level of discrimination of 10 logs10. HCV minus-strand RNA isolation was unaffected by the addition of an excess of 104 of plus strands or by the addition of cellular RNA, and although the polyA+ isolation step removed 99.99% of plus strands, there was no loss of minus-strand signal. Minus-strand RNA was detected in RNA extracted from 4/4 liver samples and 4/8 peripheral blood mononuclear cells (PBMC) samples examined. Because the titer of plus-strand HCV RNA in any sample makes a significant contribution to false, random, and self-priming, removal of the plus strand in this manner results in the most accurate method yet devised to confirm the replication of HCV in a population of cells.
Regulatory mechanisms for transforming growth factor β as an autocrine inhibitor in human hepatocellular carcinoma: Implications for roles of Smads in its growth
Authors: Matsuzaki Koichi , Date Masataka , Furukawa Fukiko , Tahashi Yoshiya , Matsushita Masanori , Sugano Yasushi , Yamashiki Noriyo , Nakagawa Taichi , Seki Toshihito , Nishizawa Mikio , Fujisawa Junichi , Inoue Kyoichi
Published: March 2004

Transforming growth factor β (TGF-β) initiates signaling through heteromeric complexes of transmembrane type I and type II serine/threonine kinase receptors. Activated TGF-β type I receptor phosphorylates receptor-regulated Smads (2 and 3). Antagonistic Smad 7 forms stable association with the activated TGF-β type I receptor, blocking phosphorylation of receptor-regulated Smads. On the other hand, elevated serum concentration of TGF-β along with resistance to its growth-inhibitory effect is commonly observed in human hepatocellular carcinoma (HCC) patients. In this study, we investigated the mechanisms of resistance to tumor-derived TGF-β in human HCC and hepatoblastoma-derived cell lines, focusing on the roles of receptor-regulated Smads and antagonistic Smad 7. HuH-7 and HepG2 cells showed poor response to TGF-β-mediated growth inhibition. Because neutralization of TGF-β in the medium or blockage of signal transduction pathway by inductions of dominant negative Smad 2/3 resulted in a stimulation of cell growth, tumor-derived TGF-β signal acts on cell growth negatively. However, Smad 7 induced by TGF-β negatively regulated Smad 2 action and rendered most Smad 2 proteins in the cytoplasm. Taken together, these results indicate that endogenous TGF-β-mediated induction of Smad 7 results in a higher “threshold” for the antiproliferative signals mediated by receptor-regulated Smads, and can be involved in reduced responsiveness to the cytokine in some human HCC cells.
Activation of intracellular signaling by hepatitis B and C viruses: C-viral core is the most potent signal inducer
Authors: Kato Naoya , Yoshida Hideo , Ono-nita Suzane Kioko , Kato Jun , Goto Tadashi , Otsuka Motoyuki , Lan Keng-hsin , Matsushima Kouji , Shiratori Yasushi , Omata Masao
Published: March 2004

To clarify the effects of hepatitis C virus (HCV) infection on hepatocytes, we analyzed and compared the induction of intracellular signals by HCV and hepatitis B virus (HBV) proteins. We examined the influence of 7 HCV (core, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) and 4 HBV (precore, core, polymerase, and X) proteins on 5 well-defined intracellular signaling pathways associated with cell proliferation, differentiation, and apoptosis by use of a reporter assay. Viral protein-expression vectors were cotransfected into mammalian cells with reporter vectors having a luciferase gene driven by the following inducible cis-enhancer elements: the cyclic adenosine monophosphate response element, the serum response element (SRE), and the binding sites for nuclear factor κB (NF-κB), activator protein 1 (AP-1), and serum response factor (SRF). In addition, the activation of signals by HCV proteins was examined in a reporter plasmid having a natural interleukin-8 (IL-8) promoter upstream of a luciferase gene. Of 11 HCV and HBV proteins, HCV core had the strongest influence on intracellular signals, especially NF-κB-, AP-1-, and SRE-associated pathways. HCV core’s activation level exceeded that of HBV X protein, a well-characterized transactivator of these signals. Moreover, HCV core activated the IL-8 promoter through NF-κB and AP-1. For the other proteins, HCV NS4B showed signal activation, but signals were activated at a lesser extent. The luciferase reporter assay, a recently introduced technique, helped in the elucidation of molecular events underlying the inflammatory and proliferation process in the liver induced by HCV.
Proteome analysis of rat hepatic stellate cells
Authors: Kristensen Dan Bach , Kawada Norifumi , Imamura Kunihiko , Miyamoto Yuka , Tateno Chise , Seki Shuichi , Kuroki Tetsuo , Yoshizato Katsutoshi
Published: March 2004

Proteome analysis was performed on cellular and secreted proteins of normal (quiescent) and activated rat hepatic stellate cells. The stellate cells were activated either in vitro by cultivating quiescent stellate cells for 9 days or in vivo by injecting rats with carbon tetrachloride for 8 weeks. A total of 43 proteins/polypeptides were identified, which altered their expression levels when the cells were activated in vivo and/or in vitro. Twenty-seven of them showed similar changes in vivo and in vitro, including up-regulated proteins such as calcyclin, calgizzarin, and galectin-1 as well as down-regulated proteins such as liver carboxylesterase 10 and serine protease inhibitor 3. Sixteen of them showed different expression levels between in vivo and in vitro activated stellate cells. These results were reproducibly obtained in 3 independent experiments. The up-regulation of calcyclin, calgizzarin, and galectin-1, as well as the down-regulation of liver carboxylesterase 10 were directly confirmed in fibrotic liver tissues. Northern blots confirmed up-regulation of the messenger RNAs (mRNAs) of calcyclin, calgizzarin, and galectin-1 in activated stellate cells, indicating that these changes were controlled at the mRNA level. In addition a list compiling over 150 stellate cell proteins is presented. The data presented here thus provide a significant new protein-level insight into the activation of hepatic stellate cells, a key event in liver fibrogenesis.
A randomized, placebo-controlled study to evaluate the efficacy of 12-month famciclovir treatment in patients with chronic hepatitis B e antigen-positive hepatitis B
Authors: de Man Robert A. , Marcellin Patrick , Habal Flavio , Desmond Paul , Wright Teresa , Rose Toby , Jurewicz Regina , Young Clarence
Published: March 2004

We conducted a randomized, placebo-controlled clinical study evaluating famciclovir (500 mg 3 times daily and 1.5 g once daily) for 1 year (6 months post-treatment follow-up) in patients with chronic hepatitis B e antigen (HBeAg)-positive hepatitis B virus (HBV) infection. The study was conducted in 80 centers in North America, Europe, and Australia/New Zealand. A total of 417 patients with histologically documented chronic hepatitis B (histologic activity index [HAI] 9.5-11.0) received famciclovir (500 mg 3 times daily or 1.5 g once daily) or placebo. Famciclovir 500 mg 3 times daily significantly reduced HBV DNA and median HAI scores versus placebo. By week 8, median HBV DNA decreased from 1,645 to 283 MEq/mL (famciclovir 500 mg 3 times daily) and from 1,147 to 304 MEq/mL (famciclovir 1.5 g once daily), while increasing for placebo (1,617 to 1,685 MEq/mL). Median change in HBV DNA at the end of therapy was −76% (famciclovir 500 mg 3 times daily;P< .01) and −60% (famciclovir 1.5 g once daily;P= .25) versus −37% for placebo. Median change in HAI was −1.5 points (famciclovir 500 mg 3 times daily;P= .02) and −1.0 point (famciclovir 1.5 g once daily;P= .35) and zero for placebo. Fifty percent of patients receiving famciclovir 500 mg 3 times daily (P= .07) and 43% receiving 1.5 g once daily (P= .41) experienced ≥2 points improvement in HAI versus 37% for placebo. Nine percent of patients treated with famciclovir 500 mg 3 times daily underwent anti-HBeAg seroconversion with undetectable HBV DNA at end of follow-up versus 3% in the placebo group (P= .05). Famciclovir was well tolerated; the incidence of post-treatment alanine transaminase (ALT) elevations was comparable with placebo. In conclusion, famciclovir 500 mg 3 times daily gave modest suppression of viral replication, but translated into significant histologic improvement in median HAI score at 1 year.