bluuebunny/crossref_metadata_2025 huggingface dataset:
Influence of Human Immunodeficiency Virus Infection on Chronic Hepatitis B in Homosexual Men
Authors: Colin Jean-François , Cazals-Hatem Dominique , Loriot Marie Anne , Martinot-Peignoux Michèle , Pham Bach Nga , Auperin Anne , Degott Claude , Benhamou Jean-Pierre , Erlinger Serge , Valla Dominique , Marcellin Patrick
Published: July 2004

The aim of this study was to assess the influence of human immunodeficiency virus (HIV) infection on chronic hepatitis B. In a series of 132 (65 anti–HIV positive) homosexual non-drug addicted men with chronic hepatitis B, the liver function was assessed with biochemical tests; the degree of hepatitis B virus (HBV) replication was assessed with serum HBV DNA level and with immunoperoxidase staining of hepatitis B core (HBc) antigen on liver specimens; and the severity of liver lesions was assessed with an histology activity index. Anti–HIV-positive and anti–HIV-negative patients were not different for serum aspartate transaminase activity, bilirubin, prothrombin, and histology activity index. Anti–HIV-positive patients had lower serum alanine transaminase activity levels (P = .0001), lower serum albumin levels (P = .0009), and higher serum HBV DNA levels (P = .01). There was a higher prevalence of cirrhosis in anti–HIV-positive patients (P = .04). In homosexual men with chronic hepatitis B, HIV infection is associated with a higher level of HBV replication and a higher risk for cirrhosis without increased liver necrotico–inflammatory process.
Expression of Hepatitis C Virus Ns5b Protein: Characterization of Its Rna Polymerase Activity and Rna Binding
Authors: Ishii Koji , Tanaka Yoshinobu , Yap Chan-Choo , Aizaki Hideki , Matsuura Yoshiharu , Miyamura Tatsuo
Published: July 2004

The nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) is considered to possess RNA–dependent RNA polymerase (RdRp) activity and to play an essential role for the viral replication. In this study, we expressed the NS5B protein of 65 kd by a recombinant baculovirus. With the highly purified NS5B protein, we established an in vitro system for RdRp activity by using poly(A) as a template and a 15–mer oligo(U) (oligo(U)15) as a primer. Optimal conditions of temperature and pH for primer–dependent polymerase activity of the NS5B were 32°C and pH 8.0. The addition of 10 mmol of Mg2+increased the activity. The importance of three motifs conserved in RdRp among other positive–strand RNA viruses was confirmed by introduction of an Ala residue to every amino acid of the motifs by site–directed mutagenesis. All mutants lost RdRp activity, but retained the RNA binding activity, except one mutant at Thr287/Asn291. Deletion mutant analysis indicated that the N–terminal region of NS5B protein was critical for the RNA binding. Inhibition of RdRp activity by (-)β–l–2′,3′–dideoxy–3′–thiacytidine 5′–triphosphate (3TC; lamivudine triphosphate) and phosphonoacetic acid (PAA) was observed after screening of nucleoside analogs and known polymerase inhibitors. These data provide us not only important clues for understanding the mechanism of HCV replication, but also a new target of antiviral therapy
Growth Inhibition by A Triple Ribozyme Targeted to Repetitive B2 Transcripts
Authors: Crone Tina M. , Schalles Shani L. , Benedict Catharine M. , Pan Weihua , Ren Ling , Loy Sarah E. , Isom Harriet , Clawson Gary A.
Published: July 2004

The B2 family represents a group of short repetitive sequences that are found throughout the rodent genome and are analogous to the human Alu sequences. Certain B2 subfamilies are transcribed by RNA polymerase III (pol III), and this transcription is in part controlled by the retinoblastoma protein. In addition to their putative role in retrotranspositional events, these actively transcribed B2 RNAs show a predicted highly stable secondary structure. Although B2 transcripts are normally confined to the nucleus, they demonstrate altered compartmentation after carcinogen treatment, in cancers, and in immortalized and/or transformed cell lines, the significance of which is unclear. Because modulation of B2 transcripts did not seem feasible with an antisense approach, we designed a triple ribozyme (TRz) construct to down–regulate B2 transcripts. The B2–targeted TRz undergoes efficient self–cleavage, resulting in liberation of the internal hammerhead Rz, which we targeted to a single–stranded region of the consensus B2 sequence. The liberated internal targeted Rz was 20 times more active than the corresponding double–G mutant construct that could not undergo self–cleavage, and 5 times more active than the same Rz flanked by nonspecific vector sequences. The B2–targeted TRz was used to develop stable transfectant clones from an SV40–immortalized hepatocyte cell line. These transfectant clones all showed variably reduced growth rates, accompanied by significant reductions in both cytoplasmic and nuclear B2 RNA levels: linear regression analyses showed that their growth rates were directly related to residual cytoplasmic B2 levels. Reverse–transcription polymerase chain reaction (RT–PCR) analyses documented efficient self–liberation of the internal targeted Rz in vivo, and showed that the relative cytoplasmic expression levels generally paralleled the magnitude of the decrease in B2 transcripts. The RT–PCR analyses further demonstrated that up to 20% of the Rz was located in the nucleus, which presumably reflects competition between autocatalytic processing and nucleocytoplasmic transport of the initial TRz transcript
Hepatic and Extrahepatic Malignancies in Primary Biliary Cirrhosis
Authors: Floreani Annarosa , Baragiotta Anna , Baldo Vincenzo , Menegon Tiziana , Farinati Fabio , Naccarato Remo
Published: July 2004

Hepatocellular carcinoma (HCC) is rarely reported as a complication of primary biliary cirrhosis (PBC). However, data suggest that patients with PBC have an increased incidence of breast cancer when compared with the general population. Our aim was to analyze the incidence of malignancies in a large series of PBC patients from North–East Italy; to compare findings with those obtained in the general population of the same geographical area, as derived from the general cancer registry; and to study any possible adjunctive risk factor for malignancy. The overall sample included 175 patients (9 males, 166 females). The mean age at presentation was 50.8 years (range 23–77); 17 patients had histological stage I, 45 had stage II, 76 had stage III, and 37 had stage IV. The prevalence of gynecological diseases obtained from the past history of the females included 19.9% miscarriages, 12% hysterectomies, and 2.4% curettages. The follow–up period was 1,187 person/years (average 6.8 yrs per person as a mean). The comparison of the incidence of malignancies between the study group and the general population was obtained by the proportional incidence ratio (PIR), which is the ratio between the cases observed and the expected number of cases in the study group. Logistic regression analysis was performed utilizing the risk factors significantly associated with cancer development in the univariate analysis. Extrahepatic malignancies developed in eight cases (4.5%) and HCC in a further four cases (2.3%), all associated with cirrhosis. Two of the four patients with HCC had a superinfection with hepatitis C virus (HCV). Breast cancer developed only in two patients. The PIR for HCC was 26.27 (95% CI 6.8–46.5), whereas the PIR for breast cancer was 0.43. The logistic regression analysis indicated that a history of cigarette smoking and HCV–RNA positivity were independent variables for the development of HCC. HCC has a relatively high prevalence in PBC and HCV superinfection may play an important part in favoring HCC. The incidence of breast cancer is not significantly higher in PBC than in the general population of the same area
A Paucity of Unusual Trihydroxy Bile Acids in the Urine of Patients With Severe Liver Diseases
Authors: Nakashima Toshiaki , Sakamoto Yoshikuni , Inaba Koji , Mitsuyoshi Hironori , Ishikawa Hiroki , Nakajima Yoshiki , Sakai Motonari , Shima Toshihide , Kashima Kei
Published: July 2004

To clarify the relationship between the occurrence of unusual trihydroxy bile acids, namely hyocholic acid, ursocholic acid (UCA), and ω–muricholic acid (ω–MCA) in urine and liver disease severity, urinary bile acids were analyzed by gas–liquid chromatography in acute and late phases of acute hepatitis and before and after ursodeoxycholic acid (UDCA) loading in healthy adults and liver cirrhosis patients. In 11 patients with acute hepatitis, the occurrence rates and amounts of unusual trihydroxy bile acids were increased in the late (recovery) phase, as compared with those in the early phase. In 10 patients with severe acute hepatitis who had prothrombin times exceeding 16 seconds, these bile acids had completely disappeared from the urine in the early phase but reappeared in the late phase in those who had a good outcome, though never in a patient who died. After UDCA administration for a week, the amounts of unusual bile acids, especially UCA and ω–MCA, which are thought to be synthesized through 12 α– and 6 α–hydroxylations, respectively, from UDCA, were clearly increased in 10 healthy adults but only slightly changed in 10 patients with liver cirrhosis. In conclusion, hepatic hydroxylations of dihydroxy bile acids as a detoxification reaction were impaired in severe liver diseases, which may play a role in the intensification and perpetuation of hepatocellular injuries.
Pretransplant Prediction of Prognosis After Liver Transplantation in Primary Sclerosing Cholangitis Using A Cox Regression Model
Authors: Neuberger James , Gunson Bridget , Komolmit Piyawat , Davies Mervyn H. , Christensen Erik
Published: July 2004

Liver transplantation remains the only treatment for patients with end–stage primary sclerosing cholangitis (PSC); however, selection criteria for the procedure and its timing remains uncertain. The aim of this study was to identify pretransplant variables associated with survival after transplantation and to devise a Cox regression model for prediction of post–transplant survival. We studied 118 patients transplanted for PSC at the Queen Elizabeth Hospital, Birmingham, UK, being followed for up to 9¼ years after the procedure. The association between pretransplant data and the post–transplant survival up to 1 year was studied using the logrank test (univariate analyses) and Cox multiple regression analysis. Univariate analyses showed the following variables to be associated with a decreased post–transplant survival: high serum creatinine, high serum bilirubin, biliary tree malignancy, previous upper abdominal surgery, hepatic encephalopathy, ascites, and Crohn's disease, whereas ulcerative colitis was associated with increased post–transplant survival (allP≤ .05). The final multiple Cox regression model included the following significant variables: inflammatory bowel disease, ascites, previous upper abdominal surgery, serum creatinine, and biliary tree malignancy (allP< .03). Biliary tree malignancy could be omitted from the Cox model with only slight loss of information. The results were validated using the data of 30 independent PSC patients from another center. These results can improve selection of patients with PSC for liver transplantation. The developed prognostic model for transplantation can be used in parallel with previously published prognostic models for nontransplantation. The obtained prognostic estimates will provide additional information that is useful for optimal timing of liver transplantation in the individual patient
Multicenter Study of Lamivudine Therapy for Hepatitis B After Liver Transplantation
Authors: Perrillo Robert , Rakela Jorge , Dienstag Jules , Levy Gary , Martin Paul , Wright Teresa , Caldwell Stephen , Schiff Eugene , Gish Robert , Villeneuve Jean Pierre , Farr Gist , Anschuetz Gaya , Crowther Lynn , Brown Nathaniel
Published: July 2004

Hepatitis B after liver transplantation is often fatal, and no proven medical therapy exists for this condition. We chose to study the potential efficacy of lamivudine therapy for patients with chronic hepatitis B after liver transplantation. Fifty–two patients with chronic hepatitis B after liver transplantation were treated in an open label, multicenter study. Each had detectable hepatitis B virus (HBV) DNA in serum and 45 (87%) had detectable serum hepatitis B e antigen before treatment. Patients were treated for 52 weeks with lamivudine (100 mg daily). The primary endpoint was undetectability of HBV DNA; secondary endpoints included normalization of serum alanine transaminase (ALT) levels, disappearance of hepatitis B e antigen, and improvement in liver histology. After treatment, 60% of patients had undetectable HBV DNA by solution hybridization assay, 14 (31%) of the initially positive patients lost hepatitis B e antigen; hepatitis B surface antigen was undetectable in 3 (6%); and serum ALT levels normalized in 71%. Blinded histological assessments showed improvement in the histological activity index (P= .007 for periportal necrosis, .001 for lobular necrosis, and .013 for portal inflammation). YMDD variants of HBV, potentially associated with drug resistance, were detected in 14 (27%) of the patients. Repeat liver biopsies in 7 patients with the mutated virus were unchanged in 2, improved in 2, and worse in 3. We conclude that lamivudine is a potentially effective therapy for hepatitis B after liver transplantation.
Inherited Hfe–Unrelated Hemochromatosis in Italian Families
Authors: Camaschella Clara , Fargion Silvia , Sampietro Maurizio , Roetto Antonella , Bosio Sandra , Garozzo Giovanni , Arosio Cristina , Piperno Alberto
Published: July 2004

Hemochromatosis (HH) is usually caused by the homozygous state for C282Y mutation in the HFE gene. A minority of iron loaded patients have no mutations in this gene. An infrequent subset shows an early–onset aggressive disorder, denoted juvenile hemochromatosis (JH), which has no linkage to 6p. In this report we describe six patients from three unrelated Italian families, four men and two women, aged 21 to 44 with the typical hemochromatosis phenotype, who are homozygous for the wild type allele at the HFE gene. In two families the disorder is unlinked to 6p; in one family some features of the juvenile form are seen, but linkage to 6p is not excluded. Our results point to genetic forms of hemochromatosis not associated with HFE and raise the problem of whether non–HFE hemochromatosis in Italy is related to the “juvenile” form. They also emphasize the importance of phenotypic as well as genetic diagnosis of HH.
Multiple Genetic Alterations, 4Q28, A New Suppressor Region, and Potential Gender Differences in Human Hepatocellular Carcinoma
Authors: Hammond Christine , Jeffers Lennox , Carr Brian I. , Simon Daniela
Published: July 2004

Primary hepatocellular carcinomas (HCCs) of different etiologies were studied to determine the rate of alteration of several genetic regions previously associated with the HCC phenotype. The focus of our study was to identify the frequency of genetic alterations within individual HCCs and their distribution among male and female cases. Genetic differences were evaluated between DNA isolated from tumor (T) and corresponding non–tumor (N) tissue using short tandem repeat (STR)–microsatellites and restriction fragment length polymorphism (RFLP) analyses. Twenty–eight HCC cases were studied with polymorphic markers from different parts of the genome. Three or more loci were identified with genetic alterations from 28 loci tested in 63% of HCC cases. The highest frequency of alteration occurred in the chromosome regions 1p36, 13q14, 17p13, and the 4q28 region identified in HCCs for the first time. High loss of heterozygosity (LOH) in 1p36, 4q28, 13q14, and 17p13 regions indicates important HCC suppressors within the regions. The study documents no association between cause and specific genetic change or their frequency in HCCs analyzed. The data document a significant gender distortion for genetic alteration in chromosome 13q14 and 17p13 regions and a concordant gender alteration rate in the 1p36 and 4q28 regions. An overall higher frequency of genetic alterations was identified in male cases. Future study with an extended number of HCC cases should substantiate the frequency of alteration between genders and identify tentative suppressors in the 1p36 and 4q28 regions.
Reorganization of Cholangiocyte Membrane Domains Represents An Early Event in Rat Liver Ischemia
Authors: Doctor Brian R. , Dahl Rolf H. , Salter Kelli D. , Fitz Gregory J.
Published: July 2004

Cholangiocytes contribute significantly to bile formation through the vectorial secretion of water and electrolytes and are a focal site of injury in a number of diseases including liver ischemia and post–transplantation liver failure. Using ischemia in intact liver and adenosine triphosphate (ATP) depletion in cultured cells to model cholangiocyte injury, these studies examined the effects of metabolic inhibition on cholangiocyte viability and structure. During 120 minutes of ischemia or ATP depletion, cell viability and tight junctional integrity in cholangiocytes were maintained. However, both the in vivo and in vitro models displayed striking alterations in the secondary structure of the plasma membrane. After 120 minutes, the basolateral (BL) interdigitations were diminished and the apical (Ap) microvilli were significantly decreased in number. The BL and Ap membrane surface areas decreased by 42 ± 8% and 63 ± 2%, respectively. Despite these changes, F–actin remained predominantly localized to the membrane domains. In contrast, in a time course that paralleled the loss of microvilli, the actin–membrane linking protein ezrin progressively dissociated from the cytoskeleton. These studies indicate that cholangiocyte ATP depletion induces characteristic, domain–specific changes in the plasma membrane and implicate alterations in the membrane–cytoskeletal interactions in the initiation of the changes. Pending the re–establishment of the differentiated domains, the loss of specific secondary structures may contribute to impaired vectorial bile duct secretion and postischemic cholestasis.
Chronic Ethanol Consumption Impairs Receptor–Mediated Endocytosis of Formaldehyde–Treated Albumin by Isolated Rat Liver Endothelial Cells
Authors: Thiele Geoffrey M. , Miller Jacqueline A. , Klassen Lynell W. , Tuma Dean J.
Published: July 2004

Receptor–mediated endocytosis (RME) by a scavenger receptor on sinusoidal liver endothelial cells (LECs) for formaldehyde–treated bovine serum albumin (f–Alb) has previously been shown to be impaired following chronic ethanol consumption. These studies were initially performed by in situ perfusion, making it difficult to determine the point in the process at which RME is affected. Therefore, it was the purpose of this study to use isolated LECs to begin elucidating at what point in the process chronic ethanol consumption affects RME. Initial studies showed that degradation at the single–cell level were similarly decreased at levels that had been observed for in situ studies, suggesting that the ethanol effects can be repeated using isolated LECs, making them useful for in vitro studies. Binding studies with 125I–formaldehyde-treated bovine serum albumin (125I-f–Alb) demonstrated there was a slight, but significantly different, decrease in binding by LECs from ethanol–fed rats when compared with pair–fed or chow–fed rats. However, the affinity of these receptors was not different between these groups. In contrast, a defect in the initial stages of receptor–ligand internalization was indicated as less surface–bound ligand was internalized and subsequently degraded in cells from the ethanol–treated animals as compared with controls. Additionally, once the data were adjusted for the amount of ligand internalized, the degradation of the internalized ligand was only slightly impaired. These results indicate that chronic ethanol feeding impairs the process of RME by the liver; the major cause of this impairment appears to be caused by a decreased ability of these cells to internalize all of the surface–bound ligand, with a minimal defect in postinternalization events.
Delineation of A Novel Hepatic Route for the Selective Transfer of Unesterified Sterols From High–Density Lipoproteins to Bile: Studies Using the Perfused Rat Liver
Authors: Robins Sander J. , Fasulo Joan M.
Published: July 2004

Cholesterol is principally excreted from the body in bile as unesterified cholesterol (UC). Using the unesterified plant sterol, sitostanol (SIT), as a nonexchangeable analog for UC, we have found that high–density lipoproteins (HDL), but not low–density lipoproteins, provide a vehicle for the direct delivery of cholesterol to bile. To determine the mechanism for preferential cholesterol transport from HDL to bile, isolated rat livers were perfused with a reconstituted HDL, made with radiolabeled unesterified SIT, UC, and cholesteryl esters (CE). Total biliary sterol secretion was independent of the concentration of HDL added to perfusions, but with increasing HDL–SIT perfused, the proportion of SIT to cholesterol in bile was linearly increased. Biliary SIT secretion was rapid (detected within 2 to 4 minutes after reconstituted HDL was added to perfusions) and was dependent on the immediate presence of SIT in the perfusate, but independent of the amount of SIT that had accumulated in the liver. The ratio of SIT to UC was seven– to ninefold greater in bile than in the liver, consistent with preferential mobilization of membrane sterols delivered from HDL. Although radiolabeled UC as well as SIT was taken up from HDL by the liver and secreted in bile, net UC uptake could not be quantitated because of both UC exchange and a sizable enrichment of HDL with UC mass that approximated the SIT removed during the passage of HDL through the liver. These results are consistent with sterol transport to bile from HDL by a direct plasma membrane pathway and by a mechanism that appears to involve substitution of unesterified (exogenous) sterol from HDL for plasma membrane UC during transport. By this process, HDL can promote reverse cholesterol transport from peripheral tissues to bile, even without an increase in biliary cholesterol secretion.
Lipoic Acid Prevents Development of the Hyperdynamic Circulation in Anesthetized Rats With Biliary Cirrhosis
Authors: Marley Richard , Holt Steve , Fernando Bimbi , Harry David , Anand Radhi , Goodier David , Davies Susan , Moore Kevin
Published: July 2004

Chronic bile duct ligation is associated with the development of oxidant injury, biliary cirrhosis, portal hypertension, and a hyperdynamic circulation. We have previously demonstrated that the hyperdynamic circulation in the partial portal vein-ligated rat can be prevented by the administration of N–acetylcysteine. To extend these findings, we have examined the effect of lipoic acid, a thiol–containing antioxidant, on hemodynamics, oxidative stress, and nitric oxide (NO) production in bile duct-ligated (BDL) cirrhotic rats. Lipoic acid was given continuously in drinking water to normal and BDL rats; control rats received ordinary drinking water, and animals were studied at 24 days following surgery. Lipoic acid prevented the development of the hyperdynamic circulation (cardiac index [CI]: 15.7 ± 2.0 vs. 29.5 ± 2.1 mL · min-1 · 100 g-1; P < .05) and significantly attenuated the rise in portal pressure (PP) (12.7 ± 0.8 vs. 15.2 ± 0.5 mm Hg; P < .05). Hepatic nitric oxide synthase (NOS) activity and plasma nitrite/nitrate concentration increased significantly following bile duct ligation, and both of these were prevented by lipoic acid. Lipoic acid had no effect on the biochemical or histological parameters of liver function in the cirrhotic group. We conclude that lipoic acid prevents the development of the hyperdynamic circulation in the rat model of biliary cirrhosis, and that this is associated with decreased synthesis of NO.(Hepatology 1999;29:1358–1363.)
Possible Contribution to Hepatocarcinogenesis of X Transcript of Hepatitis B Virus in Japanese Patients With Hepatitis C Virus
Authors: Tamori Akihiro , Nishiguchi Shuhei , Kubo Shoji , Koh Noritoshi , Moriyama Yoshinori , Fujimoto Shunsuke , Takeda Tadashi , Shiomi Susumu , Hirohashi Kazuhiro , Kinoshita Hiroaki , Otani Shuzo , Kuroki Tetsuo
Published: July 2004

Serological research suggests that hepatitis B virus (HBV) and hepatitis C virus (HCV) are associated with the development of hepatocellular carcinoma (HCC). It is unclear how genes of hepatitis viruses participate in hepatocarcinogenesis in patients infected with HCV. We investigated the expression of hepatitis virus-related RNAs in resected liver from 51 patients with HCV antibodies (Ab) and without hepatitis B surface antigen (HBsAg). mRNA transcripts of the genes HBx, HBc, HBs, nonstructural (NS) region 3 of HCV, the 5′–untranslated region (UTR) of HCV, and the 5′–UTR of hepatitis G virus (HGV) were amplified by reverse–transcription polymerase chain reaction (RT–PCR) with specific primers for each gene. The HBx transcript was detected in 19 (37%) tumors and in 8 (16%) specimens of noncancerous tissues (P = .014). The NS3 gene of HCV was detected in 35 (69%) tumors and 41 (80%) noncancerous tissues. HGV RNA was detected in 3 tumors (6%). Patients with HBx transcripts were younger than patients without HBx transcripts (P = .012). HBx transcripts were detected in 3 (33%) of 9 well–differentiated HCCs, in 8 (31%) of 26 moderately differentiated HCCs, and in 8 (50%) of 16 poorly differentiated HCCs. Codon 130 (AAG) and codon 131 (GTC) of HBx were changed to ATG and ATC, respectively, in all HCCs with HBx transcripts. In conclusion, we found that the HBx gene was expressed in many HCCs; the gene might promote hepatocarcinogenesis in patients with HCVAb and without HBsAg, but HGV is not closely related to hepatocarcinogenesis in such patients
A Fresh Look At Augmenter of Liver Regeneration in Rats
Authors: Gandhi Chandrashekhar R. , Kuddus Ruhul , Subbotin Vladimir M. , Prelich John , Murase Noriko , Rao Abdul S. , Nalesnik Michael A. , Watkins Simon C. , DeLeo Albert , Trucco Massimo , Starzl Thomas E.
Published: July 2004

Augmenter of liver regeneration (ALR) is a hepatotrophic protein originally identified by bioassay in regenerating rat and canine livers following partial hepatectomy and in the hyperplastic livers of weanling rats, but not in resting adult livers. The ALR gene and gene product were subsequently described, but little is known about the cellular/subcellular sites of ALR synthesis in the liver, or about the release and dissemination of the peptide. To obtain this information in rats, we raised antibodies in rabbits against rat ALR for development of an enzyme–linked immunosorbent assay (ELISA). ALR concentrations were then determined in intact livers of unaltered weanling and adult rats; in regenerating residual liver after partial hepatectomy; in cultured hepatocytes and nonparenchymal cells (NPCs); and in culture medium and serum. ALR in the various liver cells was localized with immunohistochemistry. In addition, hepatic ALR and ALR mRNA were assayed with Western blotting and reverse–transcriptase polymerase chain reaction (RT–PCR), respectively. The hepatocyte was the predominant liver cell in which ALR was synthesized and stored; the cultured hepatocytes secreted ALR into the medium in a time–dependent fashion. Contrary to previous belief, the ALR peptide and ALR mRNA were present in comparable concentrations in the hepatocytes of both weanling and resting adult livers, as well as in cultured hepatocytes. A further unexpected finding was that hepatic ALR levels decreased for 12 hours after 70% hepatectomy in adult rats and then rose with no corresponding change in mRNA transcripts. In the meantime, circulating (serum) ALR levels increased up to 12 hours and declined thereafter. Thus, ALR appears to be constitutively expressed in hepatocytes in an inactive form, and released from the cells in an active form by unknown means in response to partial hepatectomy and under other circumstances of liver maturation (as in weanling rats) or regeneration
Differential Effect of Thioacetamide on Hepatic Methionine Adenosyltransferase Expression in the Rat
Authors: Huang Zong-Zhi , Mato José M. , Kanel Gary , Lu Shelly C.
Published: July 2004

Liver–specific and non-liver–specific methionine adenosyltransferase (MAT) are products of two genes, MAT1A and MAT2A, respectively, that catalyze the formation of S–adenosylmethionine (SAM), the principal methyl donor. Mature liver expresses mainly MAT1A. We showed a switch from MAT1A to MAT2A gene expression in human liver cancer cells that may offer a growth advantage. To gain a better understanding of the chronology and significance of the change in MAT expression, we examined changes in hepatic MAT expression after acute treatment of rats with a hepatocarcinogen, thioacetamide (TAA). TAA treatment for 3 weeks did not change the MAT1A mRNA level but reduced the liver–specific MAT protein level to below 30% of control. TAA also acutely reduced the activity of liver–specific MAT when added to normal liver homogenates. In contrast, both the mRNA and protein levels of non-liver–specific MAT were induced. Because liver–specific MAT exhibits a much higher K
m for methionine (mmol/L) than non-liver–specific MAT (≈10 μmol/L), MAT activity was decreased at 5 mmol/L but increased at 20 μmol/L methionine concentration. The SAM level, SAM–to-S–adenosylhomocysteine (SAH) ratio, and DNA methylation all fell during treatment. In summary, TAA treatment induced differential changes in hepatic MAT expression. The reduction in liver–specific MAT protein level represents a novel mechanism of inactivation of liver–specific MAT. This along with induction in MAT2A contributed to a fall in the SAM–to–SAH ratio. The resulting DNA hypomethylation may be important in the process of hepatocarcinogenesis.
Albumin Modified With Mannose 6–Phosphate: A Potential Carrier for Selective Delivery of Antifibrotic Drugs to Rat and Human Hepatic Stellate Cells
Authors: Beljaars Leonie , Molema Grietje , Weert Betty , Bonnema Harmke , Olinga Peter , Groothuis Geny M. , Meijer Dirk K. , Poelstra Klaas
Published: July 2004

The hallmark of liver fibrosis is an increased extracellular matrix deposition, caused by an activation of hepatic stellate cells (HSC). Therefore, this cell type is an important target for pharmacotherapeutic intervention. Antifibrotic drugs are not efficiently taken up by HSC or may produce unwanted side–effects outside the liver. Cell–specific delivery can provide a solution to these problems, but a specific drug carrier for HSC has not been described until now. The mannose 6–phosphate/insulin–like growth factor II (M6P/IGF–II) receptor, which is expressed in particular upon HSC during fibrosis, may serve as a target–receptor for a potential carrier. The aim of the present study was to examine if human serum albumin (HSA) modified with mannose 6–phosphate (M6P) is taken up by HSC in fibrotic livers. A series of M6Px –modified albumins were synthetized: x = 2, 4, 10, and 28. Organ distribution studies were performed to determine total liver uptake. The hepatic uptake of M6Px–HSA increased with increasing M6P density. M6Px–HSA with a low degree of sugar loading (x = 2–10) remained in the plasma and accumulated for 9% ± 0.5% or less in fibrotic rat livers. An increase in the molar ratio of M6P:HSA to 28:1 caused an increased liver accumulation to 59% ± 9% of the administered dose. Furthermore, we determined quantitatively the in vivo intrahepatic distribution of M6Px–HSA using double–immunostaining techniques. An increased substitution of M6P was associated with an increased accumulation in HSC; 70% ± 11% of the intrahepatic staining for M6P28–HSA was found in HSC. We also demonstrate that M6P–modified bovine serum albumin (BSA) accumulates in slices of normal and cirrhotic human livers. After incubation of this neoglycoprotein with human tissue, the protein is found in nonparenchymal liver cells. Because M6P–modified albumins are taken up by HSC in fibrotic livers, this neoglycoprotein can be applied as a selective drug carrier for HSC. This technology may create new opportunities for the pharmacological intervention of liver fibrosis
Modeling the Hepatitis C Virus Epidemic in France
Authors: Deuffic Sylvie , Buffat Laurent , Poynard Thierry , Valleron Alain-Jacques
Published: July 2004

A backcalculation approach allows a reconstruction of the history of hepatitis C virus (HCV) infection in France and predictions of mortality from hepatocellular carcinoma (HCC) related to the virus. The model uses information from the literature about the natural history of the disease, epidemiological data about infected subjects in three French cohorts, and mortality data from national statistics. It seeks to determine the annual transition probabilities from chronic hepatitis to cirrhosis and the HCV incidence per year in the past. These unknowns are found by fitting the observed deaths from HCC that are attributable to HCV. Optimal values for these unknowns then allow to project the number of HCC deaths attributable to HCV for each year through 2025 (for patients infected before 1996). The model traces the HCV epidemic in France back to around the 1940s. It predicts that HCC mortality related to HCV will continue to increase through 2020 in the absence of treatment, with a 150% increase in the yearly incidence among men and 200% among women. The model also confirms that progression to cirrhosis depends strongly on sex and age. At any age, the annual probability of progression is 10 times greater for men than for women. Moreover, for men aged between 61 and 70 years, this probability is 300 times greater than that for men aged between 21 and 40 years.
Prospective Study of Interferon Therapy for Compensated Cirrhotic Patients With Chronic Hepatitis C by Monitoring Serum Hepatitis C Rna
Authors: Shiratori Yasushi , Yokosuka Osamu , Nakata Ryo , Ihori Masashi , Hirota Katsutaro , Katamoto Tetsuro , Unuma Tadao , Okano Ken'ichi , Ikeda Yusei , Hirano Masanori , Kawase Tateo , Takano Susumu , Matsumoto Kazunori , Ohashi Yasuo , Omata Masao
Published: July 2004

Because interferon therapy exhibits low efficacy for cirrhotic patients infected with hepatitis C virus, this prospective study was conducted to determine effective interferon regimens tailored to treatment response by monitoring HCV RNA status. A total of 157 cirrhotic patients were enrolled to receive 9 million units (MU) of interferon three times a week. The HCV RNA values were drawn 8 weeks apart and the patients were randomized to a further 16 or 32 weeks of treatment after two sequential findings of negativity for HCV RNA. A total of 73 out of 157 patients (46%) proceeded to randomization to different durations of treatment, 37 short–course and 36 long–course (duration: 38 ± 8 and 49 ± 13 weeks; total amount of interferon: 940 ± 240 and 1130 ± 390 MU, respectively). The remaining 84 patients without two sequential negative serum HCV RNA determinations received 44.8 ± 27.4 weeks of interferon (IFN) therapy with total amount of 993 ± 633 MU. Of these 157 patients, sustained virological and biochemical response was shown in 32 (20%) and 37 patients (24%), respectively. Sustained virological and biochemical response rate in the randomized patients was significantly higher than in nonrandomized patients (41% vs. 2%, and 38% vs. 11%; each P < .01). Of the 73 randomized patients, the rate of sustained virological response in patients with long–course treatment (50%) was significantly higher than that of patients with short–course treatment (32%) (P = .026: log–rank test), and in patients with early disappearance of HCV RNA especially within 8 weeks, in patients with low virus load (≤106.3 copies/mL) and with HCV 2a. Multivariate analysis revealed that HCV RNA level and subtypes were the most important factors contributing to sustained virological response. Interferon is effective even in cirrhotic patients with low viral load and HCV 2a, but requires a longer course of administration.
Effects of Chronic Octreotide Treatment on Renal Changes During Cirrhosis in Rats
Authors: Jonassen Thomas E.N , Christensen Sten , Sørensen Anne-Mette , Marcussen Niels , Flyvbjerg Allan , Andreasen Frederik , Petersen Jørgen S
Published: July 2004

We examined the effect of a new long–acting release formula (LAR) of the somatostatin analogue, octreotide, on development of sodium retention and functional and structural changes in the thick ascending limb of Henle's loop (TAL) in rats with cirrhosis induced by common bile duct ligation (CBL). CBL and sham–operated control rats were treated withoctreotide–LAR (10 mg/kg bodyweight subcutaneously, as a singledose) or vehicle at the time of CBL or sham–CBL. The rats were instrumented with chronic catheters, and sodium balance and renal function were examined 4 weeks after CBL or sham operation. Octreotide–LAR treatment significantly inhibited sodium retention in CBL rats and prevented renalvasodilatation without changes in glomerular filtration rate (GFR). The natriuretic response to a test dose of furosemide (7.5 mg/kg body weight intravenously) was significantly increased in CBL rats, and when expressed in terms of natriuretic efficiency (mmol Na/mg furosemide in urine), thenatriuretic response was increased by 57% relative to sham–operatedcontrols. Stereological examination of kidneys demonstrated a 53% increase in the volume of the inner stripe of the outer medulla and a 108% increase in the volume of TAL epithelium in cirrhotic rats relative to controls. The increased natriuretic efficiency of furosemide as well as the hypertrophy of the inner stripe and the TAL in this renal zone were absent in CBL rats treated with octreotide–LAR. These results suggest that octreotide–LAR treatment inhibits sodium retention in cirrhotic rats, partly by inhibition of increased furosemide–sensitive sodium reabsorption in the TAL
Deranged Blood Coagulation Equilibrium As A Factor of Massive Liver Necrosis Following Endotoxin Administration in Partially Hepatectomized Rats
Authors: Mochida Satoshi , Arai Masahiro , Ohno Akihiko , Yamanobe Fumio , Ishikawa Keiko , Matsui Atsushi , Maruyama Ikuo , Kato Hisao , Fujiwara Kenji
Published: July 2004

Activated Kupffer cells provoke massive liver necrosis after endotoxin stimulation through microcirculatory disturbance caused by sinusoidal fibrin deposition in rats undergoing 70% hepatectomy. In these rats, serum activities of purine nucleoside phosphorylase (PNP) and alanine transaminase (ALT) were increased at 1 and 5 hours, respectively, following endotoxin administration. When 70% resected liver was perfused with Dulbecco's modified Eagle medium (DMEM) containing heat–inactivated fetal calf serum, the increase in both enzyme activities was not affected by addition of endotoxin during perfusion, suggesting that activated Kupffer cells injured neither sinusoidal endothelial cells nor hepatocytes. The activity of tissue factor, an initiator of blood coagulation cascade, was much higher in Kupffer cells isolated from partially hepatectomized rats than in those from normal rats. In contrast, mRNA expressions of tissue factor pathway inhibitor (TFPI) as well as thrombomodulin were almost undetectable in normal and partially resected livers. When recombinant human TFPI was injected intravenously in 70% hepatectomized rats, TFPI was markedly stained on the surfaces of sinusoidal endothelial cells and microvilli of hepatocytes on immunohistochemistry. In these rats, endotoxin–induced liver injury was significantly attenuated compared with rats given no TFPI. Similar attenuation was also found in rats receiving recombinant human thrombomodulin. These results suggest that fibrin deposition developing in 70% hepatectomized rats after endotoxin administration may be caused by deranged blood coagulation in the hepatic sinusoids through increasing tissue factor activity in Kupffer cells and minimal TFPI and thrombomodulin in endothelial cells. The destruction of sinusoidal endothelial cells as well as hepatocytes may occur as a result of microcirculatory disturbance caused by such sinusoidal fibrin deposition.
Hepatocellular Carcinoma Cell Lines From Diethylnitrosamine Phenobarbital–Treated Rats. Characterization and Sensitivity to Endothall, A Protein Serine/Threonine Phosphatase–2A Inhibitor
Authors: Thièry Jean-Paul , Blazsek Istvàn , Legras Stéphane , Marion Sylvie , Reynes Michel , Anjo Aurora , Adam René , Misset Jean Louis
Published: July 2004

Primary hepatocellular carcinoma (HCC) is probably one of the most common fatal forms of liver cancer. We have established permanent cell lines from diethylnitrosamine/phenobarbital induced primary rat liver carcinomas to study new anticancer therapies. The rat hepatocellular carcinoma cell lines (HR–2, HR–3, and HR–4) have been maintained in culture for over 3 years. They form tumors when transplanted sc or im into young syngeneic rats. Immunocytology (α–fetoprotein, albumin), biochemical (γ–glutamyl transferase), and histochemical (glycogen) marker studies and electron microscopy (biliary canaliculi) showed unique, stable differentiation patterns in these tumor lines. They overproduced the c–met protooncogene product and formed colonies spontaneously in semisolid culture with high cloning efficiency (HR–2: 50%–80%, HR–3: 35%–50% and HR–4: 50%–65%). The sensitivity of these cell lines to inhibitors of protein ser/thr phosphatase–2A (PP2A), a key enzyme in the control of G1/S and G2/M cell cycle phase transitions in eukaryotes, was studied in vitro. The specific, weak inhibitor of PP2A, endothall, caused dose– and time–dependent cytostasis specifically in G2/M. The cells died later by apoptosis, which was confirmed by cytology (annexin V–FITC labeling, propidium iodide painting of apoptotic bodies) and by fluorescent activated cell sorter (FACS) DNA measurements. The HR–2, HR–3, HR–4, and Zajdela hepatocellular carcinomas were most sensitive to endothall (IC50 of 1.7, 1.2, 0.9, and 1.7 μg/mL), whereas newborn rat hepatocytes growing exponentially in primary culture (IC50 = 6.2 μg/mL), rat DHD/K12 colon carcinoma cells (IC50 = 3.6 μg/mL), or human HT–29 colon carcinoma cells (IC50 = 4.9 μg/mL) were less sensitive. Thus, endothall inhibits preferentially HCC growth and these new rat hepatocellular carcinoma lines may be useful for further biochemical and pharmacological studies on PP2A inhibitors, and for testing new forms of treatment of hepatic cell carcinomas
Increased Circulating Leptin in Alcoholic Cirrhosis: Relation to Release and Disposal
Authors: Henriksen Jens H. , Holst Jens Juul , Møller Søren , Brinch Kim , Bendtsen Flemming
Published: July 2004

Leptin is a cytokine peptide that decreases appetite and thereby food intake and increases energy expenditure. It is produced in fat cells, but recent animal experiments have shown expression of leptin in modified stellate hepatic cells. Because a change in circulating leptin in cirrhosis could be caused by an altered production rate, altered disposal rate, or both, the present study was undertaken to identify regions of leptin overflow into the blood stream and regions of leptin extraction. Patients with alcoholic cirrhosis (n = 16) and control patients without liver disease (n = 12) were studied during catheterization with elective blood sampling from different vascular beds. Blood samples for leptin determination (radioimmunoassay) were taken simultaneously from artery/hepatic vein, artery/renal vein, artery/iliac vein, and artery/cubital vein. Patients with cirrhosis had significantly increased circulating leptin (7.3 vs. control 2.6 ng/mL, P < .002) that correlated directly to ascitic–free body mass index (r = 0.71, P < .005). A significant renal extraction ratio of leptin was observed in control patients (0.16) and in patients with cirrhosis (0.07), but the latter value was significantly lower than in the control patients (-44%, P < .05) and inversely correlated to serum creatinine (r = -0.60, P < .05). A significant, but equal, hepatosplanchnic extraction of leptin was observed in cirrhotic patients and control patients (0.08 vs. 0.07). In patients with cirrhosis a significant cubital venous–arterial difference in leptin was observed, but not in control patients. The iliac venous/arterial leptin ratio was significantly above 1.0 in both groups and of similar size (1.16 vs. 1.15), but a higher difference in concentration was found in the cirrhotic patients (+33%, P < .05). The spillover rates of leptin in cirrhotic patients may be even higher than estimated from the increased systemic veno–arterial gradients. In conclusion, the elevated circulating leptin in patients with cirrhosis is most likely caused by a combination of decreased renal extraction and increased release from subcutaneous abdominal, femoral, gluteal, retroperitoneal pelvic, and upper limb fat tissue areas. The hepatosplanchnic bed drained through hepatic veins could not be identified as a source of increased circulating leptin in cirrhosis, but a contribution by the portosystemic collateral flow cannot be excluded.
The “Early–Sorting” Endocytic Compartment of Rat Hepatocytes Is Involved in the Intracellular Pathway of Caveolin–1 (Vip–21)
Authors: Pol Albert , Calvo Maria , Lu Albert , Enrich Carlos
Published: July 2004

The sinusoidal plasma membrane of the hepatocyte is organized into functional and structural microdomains whose origin, maintenance, and functioning are closely related with the endocytic compartment. Three different subcellular fractions, from rat liver, containing caveolin–1, the structural protein of caveolae, were morphologically and biochemically characterized. A caveolae–enriched plasma membrane fraction (CEF), contains large membrane structures surrounding attached internal plasmalemmal vesicles; the receptor–recycling compartment (RRC), contains tubules and vesicles with similar morphology to the internal vesicles observed by electron microscopy in CEF; and finally, caveolin–1 was also detected in early–sorting endosomes (CURL, compartment of uncoupling receptors and ligands). In this study, we show that following an intravenous administration of retinol–binding protein (RBP), there was a redistribution of caveolin–1 from the plasma membrane (CEF) to intracellular endocytic compartments (RRC and early–sorting endosomes). Thus, these results indicate that, in the hepatocyte, caveolae are dynamic structures actively interacting with the endocytic compartment.
High Prevalence of Multinodular Hepatocellular Carcinoma in Patients With Cirrhosis Attributable to Multiple Risk Factors
Authors: Fasani Pierangelo , Sangiovanni Angelo , De Fazio Cristina , Borzio Mauro , Bruno Savino , Ronchi G. , Del Ninno Ersilio , Colombo Massimo
Published: July 2004

To see whether or not there is an association between the cause of cirrhosis and the number of hepatocellular carcinoma (HCC) nodules, we analyzed 178 consecutive patients in whom HCC was detected during a prospective screening by abdominal ultrasound (US). The relevant information was obtained from the database of the screening programs operating at four hospitals in the Milan area. One hundred twenty–nine (72%) patients had a single tumor nodule detected by US and 49 (28%) patients had multinodular disease. Ninety–eight (55%) patients had normal serum values of α–fetoprotein (AFP). Tumor staging with biphasic computed tomography (CT) scan or hepatic arteriography with lipiodol revealed that 101 (57%) patients had single tumor nodules and 77 (43%) patients had more than one HCC nodule. After staging, multinodular HCC was more common in patients with multiple risk factors than in the hepatitis C virus (HCV) carriers (56% vs. 38%, P = .05). Interestingly, single tumors were as common in the 126 patients undergoing 6–month interval screening as in the 52 patients who were studied at yearly intervals. The former patients, however, had more small tumors than the latter ones (91% vs. 74%, P = .04). The 22 patients who were alcohol abusers had normal levels of serum AFP more often than the hepatitis B virus (HBV) or HCV carriers or those with multiple risk factors (86% vs. 57%, P < .04; vs. 47%, P < .002; vs. 52%, P < .006, respectively). We concluded that multinodular HCC was underdetected by real time US; it prevailed among patients with multiple risk factors. In these patients, screening with US exams every 6 months may be inadequate for early detection of liver cancer.