bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of Fetal Promoters of Insulinlike Growth Factors Ii Gene in Hepatitis C Virus-Related Chronic Hepatitis, Cirrhosis, and Hepatocellular Carcinoma
Authors: Nardone G , Romano M , Calabrò A , Pedone P V , de Sio I , Persico M , Budillon G , Bruni C B , Riccio A , Zarrilli R
Published: November 2004

Increased prevalence of hepatitis C virus (HCV) infection has been found in patients with hepatocellular carcinoma (HCC). The expression of insulinlike growth factor II (IGF–II) has been linked to hepatocarcinogenesis in the experimental animal and in humans. Since reactivation of fetal IGF–II transcripts has been observed in human HCC, we have analyzed the levels of adult P1 and fetal P3 and P4 IGF–II promoter–derived transcripts in the liver of patients with HCV–related chronic active hepatitis (CAH), cirrhosis, and HCC by means of a semiquantitative reverse–transcription polymerase chain reaction (RT–PCR) assay. Transcripts derived from adult P1 promoter were increasingly expressed from normals to patients with CAH and cirrhosis, but were undetectable in the tumorous area of 5 of 7 HCC patients and present at low levels in the nontumorous area of all HCC patients. Transcripts derived from fetal P3 promoter were not detectable in normal subjects, while they were expressed abundantly in most CAH and all cirrhotic patients. Transcripts from fetal P4 promoter were detected at high levels in 3 of 9 CAH patients and in the majority of cirrhotic patients. Increased expression of fetal promoter–derived transcripts was also found in the liver of HCC patients, although levels were lower than in cirrhosis. Also, the activity of fetal P3 and P4 promoters was higher in the nontumorous than in the tumorous area of the liver of HCC patients. The expression of IGF–II transcripts was correlated with the rate of cell mitotic activity by measuring the expression of the proliferating cell nuclear antigen (PCNA) gene. PCNA messenger RNA (mRNA) levels progressively increased from normals to CAH and to cirrhotic patients, and persisted at a high level in the tumorous and in the nontumorous area of HCC subjects, thus showing that the increase of IGF–II transcripts in CAH and cirrhosis is accompanied by an activation of cell mitosis in these samples. These data suggest that the activation of IGF–II gene expression from adult and fetal promoters may play a role in premalignant proliferation observed in HCV–related chronic liver disease.
Effect of Venovenous Bypass on Perioperative Renal Function in Liver Transplantation: Results of A Randomized, Controlled Trial
Authors: Grande L , Rimola A , Cugat E , Alvarez L , Garcia-Valdecasas J C , Taurá P , Beltran J , Fuster J , Lacy A M , González F J , Tabet J , Cifuentes A , Rull R , Ramos C , Visa J , Rodés J
Published: November 2004

Although venovenous bypass (VVBP) has been suggested to protect the kidneys during liver transplantation and its systematic use has therefore been recommended, this beneficial effect of VVBP has not been clearly demonstrated. In a prospective, randomized, controlled trial, 77 patients receiving liver transplants for chronic liver disease were allocated to be supported with VVBP (group 1, 38 patients) or not (group 2, 39 patients). Both groups were similar in relation to preoperative clinical and laboratory data and operative transfusion requirements. Inulin clearance and urinary β2–microglobulin and N–acetyl–β–D–glucosaminidase (NAG) excretion (to determine glomerular filtration rate and tubular damage, respectively) were measured at different perioperative periods (anesthesia induction, hepatectomy, anhepatic phase, biliary anastomosis, and 24 hours after surgery). A significant decrease in inulin clearance and increase in tubular damage markers were observed in the anhepatic phase, which only partly improved in the subsequent phases. No significant differences were observed between groups 1 and 2 at any perioperative phase, except during the anhepatic phase, in which a more marked renal function impairment occurred in group 2 patients. However, renal function on the 7th postoperative day and the need for hemodialysis/ hemofiltration during the 1st week were similar in both groups. Among 40 variables analyzed, only low mean arterial pressure at anesthesia induction was identified as an independent predictor for early postoperative severe renal failure (inulin clearance < 10 mL/min/1.73 m2 at the 24th postoperative hour), with no significant relationship between this complication and the use of venovenous bypass. Renal function markedly deteriorates during liver transplantation, and renal impairment persists during the early postoperative period. Because VVBP support is not associated with any clear benefit in renal function, its systematic use does not seem to be justified.
Expression of Epithelial Cadherin and α– and β–Catenins in Nontumoral Livers and Hepatocellular Carcinomas
Authors: Ihara A , Koizumi H , Hashizume R , Uchikoshi T
Published: November 2004

Expression of the cell adhesion molecule, epithelial cadherin (E–CD) and its binding proteins, α– and β–catenins, in normal liver, chronic liver diseases, and hepatocellular carcinomas (HCCs) was investigated immunohistologically. In normal liver, weak immunostaining of E–CD and catenins was observed at the lateral membranes of the hepatocytes, whereas at the interlobular bile duct epithelia, they stained strongly. No immunoreactions were seen in sinusoidal Kupffer cells. Similar results were observed in the majority of livers from chronic hepatitis and cirrhosis sufferers; however, hepatocytes undergoing regeneration and rosette formation, as well as Hering canals and proliferating ductules, showed markedly increased molecular expression. Analysis of 66 HCC lesions revealed that the majority (64.3–96.6%) of thin trabecular– and pseudoglandular–type tumors preserved or overexpressed E–CD and catenins, whereas thick trabecular–type HCCs frequently showed low E–CD and α–catenin expression (56.5–65.2% reduction), suggesting that the thick trabecular histology represented diffuse tumor cell growth. Likewise, the E–CD and catenin expression levels correlated with the HCC cell differentiation grades. These collective results indicate that intercellular adhesion mediated by the E–CD–catenin system plays a role in morphological changes in nonmalignant and malignant hepatic diseases.
Hepatocyte Proliferative Activity in Chronic Liver Damage As Assessed by the Monoclonal Antibody Mib1 Ki67 in Archival Material: the Role of Etiology, Disease Activity, Iron, and Lipid Peroxidation
Authors: Farinati F , Cardin R , D'Errico A , De Maria N , Naccarato R , Cecchetto A , Grigioni W
Published: November 2004

Hepatitis B virus (HBV)– and hepatitis C virus (HCV)–related liver damage is linked to an increased risk of hepatocellular carcinoma, but the mechanisms underlying hepatitis C viral activity are not known. We therefore compared hepatocellular proliferative activity in chronic C virus–related hepatitis and in liver damage of other etiology. Hepatocyte proliferation rate was investigated in 56 patients with chronic hepatitis using the Ki67 MIB1 monoclonal antibody in archival material. According to etiology, the patients were subgrouped as follows: HCV (34), HBV (11), Alcohol (4), HCV + Alcohol (4), and Hemochromatosis (3). Proliferation rate was correlated with age, sex, etiology, disease activity, liver iron storage, free–radical production, and glutathione levels by regression and discriminant analysis. HCV–positive patients had significantly more MIB1–positive hepatocytes in the periportal area (

P

< .011) and in the low–proliferating perivenular area (zones 2 and 3) (

P

< .05). The number of MIB1–positive cells correlated directly with alanine transaminase (ALT) levels, Knodell index (KI), and, inversely, with iron saturation. By stepwise discriminant analysis, ALT levels and etiology were identified as single independent variables. These data suggest that HCV infection induces increased and abnormal hepatocyte proliferation, which might be related to the increased risk of hepatocellular carcinoma in patients with HCV–related liver damage.
Cloning of Variable Regions of An Antibody That Reacts With the Soluble Fraction of Human Liver Cells and Its Possible Value in Chronic Liver Disease
Authors: Saito H , Tada S , Ebinuma H , Atsukawa K , Masuda T , Inagaki Y , Tsuchimoto K , Morizane T , Ishii H
Published: November 2004

A gene encoding the variable regions of the heavy and light chains of a mouse monoclonal antibody designated H2, which specifically reacts with human liver cells, was cloned into a phagemid vector. The clone of the variable region was designed to be expressed as a separate protein, the structure of which is the same as that of the mouse antibody. The cloned phage protein specifically reacted with anti–idiotypic antibodies produced in rabbits against the original mouse antibody, and this reaction was specifically blocked by the original antibody. The soluble protein, expressed as a fusion protein, was detected as a single 30–kd band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and specifically bound to an anti–H2 idiotypic antibody as determined by Western blot analysis. Sera of patients with various diseases were assayed for antibodies to anti–H2 by sandwich enzyme–linked immunosorbent assay (ELISA). Only sera from patients with chronic liver disease reacted strongly. This binding was specifically blocked by the cloned soluble protein. The nucleotide sequences of the variable regions were determined by the dideoxy chain– termination method, and the sequences were approximately 95% identical to those of other mouse immunoglobulins. These findings suggest that a human antibody with the same idiotype as a mouse monoclonal antibody that reacts with human liver cells, can be detected in patients with chronic liver disease, suggesting that autoimmunity may be partly responsible for these diseases.
Transferrin Receptor–Independent Uptake of Differic Transferrin by Human Hepatoma Cells With Antisense Inhibition of Receptor Expression
Authors: Trinder D , Zak O , Aisen P
Published: November 2004

The hepatic uptake of transferrin–bound iron by a nontransferrin receptor (NTR)–mediated process was investigated using the human hepatoma cell line HuH7. Because HuH7 cells also acquire iron from transferrin by a receptor (TR)–mediated process, TR expression was inhibited by transfecting the cells with a plasmid containing human TR complementary DNA in antisense orientation relative to a human cytomegalovirus promoter/enhancer element. Cell clones were obtained that expressed a 50% to 60% reduction in cell surface TR, leading to a corresponding decrease in transferrin and iron uptake compared with wild–type cells. Uptake of transferrin by a second process was nonsaturable and not inhibited by a 100–fold excess of unlabeled transferrin. The amounts of transferrin taken up by the wild–type and antisense cells by this process were similar, showing that it did not involve TR. The proteolytic enzyme Pronase reduced the uptake of transferrin, suggesting that the NTR–mediated process entailed the nonsaturable binding of transferrin to plasma membrane proteins. This process, like the TR–mediated one, involved the internalization and recycling of transferrin, leading to accumulation of iron with time. Iron uptake mediated by NTR process was saturable and displaced by 100–fold excess unlabeled transferrin and reduced by weak bases and metabolic inhibitors. Therefore, the NTR–mediated process entailed transferrin adsorption to membrane–bound proteins, internalization, and release of iron from transferrin by a pH–dependent step followed by the intracellular transport of iron into ferritin and heme by a saturable carrier–mediated mechanism.
Expression of C– Fos And C– Jun During Hepatocellular Remodeling Following Ischemia/Reperfusion in Mouse Liver
Authors: Schlossberg H , Zhang Y , Dudus L , Engelhardt J F
Published: November 2004

The expression of the immediate early genes (IEGs) c–

fos

and e–

jun

have been hypothesized to potentially play key roles in mediating cellular responses following injury to the liver. In this study, we sought to evaluate the potential involvement of c–

jun

and c–

fos

as determinants either of cellular regeneration or programmed cell death following ischemia/reperfusion (I/R) in mouse liver. To this end, we have analyzed the

in situ

messenger RNA (mRNA) expression patterns of c–

jun

and c–

fos

following lobar I/R in mouse liver. The expression patterns of c–

jun

and c–

fos

were correlated with four criteria for tissue repair and injury, including: 1) morphological determinations of regeneration using immunocytochemical detection of proliferating cell nuclear antigen (PCNA), 2) programmed cell death (apoptosis) using the

in situ

terminal deoxynucleotidyl transferase–mediated dUTP–biotin nick end–labeling (TUNEL) method, 3) histopathologic assessment of hepatocellular necrosis, and 4) serum glutamic pyruvic transaminase (GPT) levels. Increasing lengths of lobar ischemia for 3, 60, and 90 minutes followed by reperfusion directly correlated with the extent of liver injury as determined by serum transaminases and hepatocellular necrosis. PCNA expression in the liver was elevated at 1 to 6 hours following liver reperfusion and returned to baseline levels by 20 hours in both ischemic and nonischemic lobes. In contrast, apoptotic responses peaked only in ischemic lobes at 6 hours′ postreperfusion and remained elevated out to 20 hours. Two distinct patterns of c–

jun

and c–

fos

expression were observed during the acute (1–3 hours) and subacute (6–20 hours) phases of liver responses to I/R including: 1) coexpression of c–

jun

and c–

fos

mRNA within damaged regions of the liver at 1 to 3 hours′ postreperfusion, and 2) a decline in c–

fos

expression with sustained high levels of c–

jun

expression within a subset of cells bordering necrotic/apoptotic regions of the liver at 6 to 20 hours′ postreperfusion. These findings suggest that coexpression of both c–

jun

and c–

fos

may be involved in mediating early tissue repair processes in liver remodeling following I/R. In contrast, the onset of hepatocellular apoptosis correlated with sustained c–

jun

expression, in the absence of c–

fos

, and suggests that these changes in the molecular profile of immediate early gene expression may regulate cellular responses that signal hepatocytes for programmed cell death.
Possible Roles of Nonparenchymal Cells in Hepatocyte Proliferation Induced by Lead Nitrate and by Tumor Necrosis Factor α
Authors: Shinozuka H , Ohmura T , Katyal S L , Zedda A I , Ledda-Columbano G M , Columbano A
Published: November 2004

A single intravenous injection of lead nitrate (LN) to rats induces liver cell proliferation without causing cell necrosis (direct hyperplasia). We suggested that liver cell proliferation in this model may be triggered by the induction of liver tumor necrosis factor α (TNF–α). Because administration of TNF–α

in vivo

has been shown to induce proliferation of both parenchymal and nonparenchymal cells of the liver, we analyzed the temporal sequences of DNA synthesis in both cell populations following LN and recombinant TNF–α treatment by 5– bromo–2–deoxyuridine (BrdU) immunohistochemistry. The patterns of cell proliferation induced by these agents were further compared with those induced by a single dose of nafenopin (NAF), a direct mitogen which does not induce liver TNF–α messenger RNA (mRNA). In male Wistar rats given a single dose of LN (100 μmol/kg), BrdU incorporation of hepatocytes and nonparenchymal cells (Kupffer cells, endothelial cells and periportal nondescript cells) became evident 12 hours after the treatment. The labeling of all cell types reached a peak after 36 hours and declined thereafter. Rats given a single intravenous injection of human recombinant TNF–α (46 μg/rat) showed an increase of BrdU labeling in nonparenchymal cells after 24 hours, whereas the labeling of hepatocytes became evident at 36 hours. A single intragastric administration of NAF resulted in a rapid increase in the number of labeled hepatocytes with no substantial labeling of nonparenchymal cells. These results add further support to the notion that LN–induced liver cell proliferation is mediated by TNF–α, and suggest that different cell populations are involved in the initial proliferative response of the liver to mitogens, depending on the capacity of the mitogens to stimulate TNF–α production.
The Translational Inhibitor Cycloheximide Represses Growth Factor Depletion–Induced Apoptosis in An Alb–Sv40t Transgenic Rat Liver Cell Line
Authors: Bulera S J , Sattler C A , Pitot H C
Published: November 2004

A transgenic rat line carrying the alb–SV40A transgene has been described by this laboratory. Several cell lines have been established from the livers of two of these rats. One of these cell lines, L37, exhibits a large nuclear/cytoplasmic ratio and a well–differentiated cytoplasm containing numerous organelles. When L37 cells are placed into culture medium lacking necessary growth factors, cellular proliferation continues for 48 hours after medium change. Subsequent to the initial 48 hours, cells begin to shrink and lose contact with adjacent cells, eventually sloughing off the culture plate surface, with most cell deaths occurring between 48 and 96 hours after medium change. Microscopic examination of sloughing cells indicates they possess highly convoluted and blebbed plasma membranes, a morphological characteristic of apoptosis. Ultrastructural studies demonstrate the ubiquitous presence of apoptotic bodies. When DNA isolated from growth factor–depleted cells is resolved on agarose gels, DNA fragmentation ladders are observed at times of maximum apoptotic change. Quantitative analysis of L37 cells between 48 and 96 hours after the removal of the culture medium shows that 59% ± 2% of the cells undergo apoptosis. When cycloheximide, puromycin, or actinomycin D is added to the L37 cultures, only cycloheximide is able to repress apoptosis, indicating that the mechanism of apoptosis in the L37 liver–derived cell line requires a cycloheximide–sensitive translational event. The extremely high rate of apoptosis, together with the maintenance of hepatocellular characteristics, indicates the usefulness of this cell line as a model in which to study the mechanisms of hepatocellular apoptosis.
Slow Intestinal Transit: A Motor Disorder Contributing to Cholesterol Gallstone Formation in the Ground Squirrel
Authors: Xu Q , Scott R B , Tan D T , Shaffer E A
Published: November 2004

Impaired gallbladder motility is an established factor in cholesterol gallstone formation. We assessed whether altered small intestinal smooth muscle contractility with slow transit might potentiate gallstone formation by further impeding enterohepatic cycling of bile acids. Ground squirrels were fed a 1% or a trace (controls) cholesterol diet. Small intestinal transit was evaluated from 51Cr distribution in conscious, fasted animals 20 minutes after infusion into the proximal jejunum. Small intestinal and gallbladder smooth muscle contractility was determined

in vitro

. Biliary lipid secretion was measured from the cannulated common duct and the bile salt pool size calculated by isotope dilution. Gas–liquid chromatography (GLC) assessed bile salt profile. In animals on the 1% cholesterol diet, aboral transit was significantly delayed, the maximal contractile response to bethanechol was markedly increased (

P

<.05) with no change in median effective concentration in either circular or longitudinal muscle strips from both the jejunum and ileum, and the gallbladder contractile responses to bethanechol and cholecystokinin (CCK) were decreased. Cholesterol saturation index and the fraction of deoxycholic acid in the pool doubled, whereas the total bile salt pool size remained unchanged in cholesterol–fed animals. In this model, a high–cholesterol diet is associated with altered small intestinal smooth muscle contractility and prolonged small intestinal transit, in addition to diminished gallbladder contractility. The resulting sluggish enterohepatic cycling of bile salts, associated with expanded deoxycholate pool, contributes to cholesterol gallstone formation.
Hepatitis B Virus Genomes of Patients With Fulminant Hepatitis Do Not Share A Specific Mutation
Authors: Sterneck M , Gunther S , Santantonio T , Fischer L , Broelsch C E , Greten H , Will H
Published: November 2004

The pathogenesis of fulminant hepatitis B virus (HBV) infection is not well understood. The aim of this study was to investigate whether there is an association between specific viral variants and a fulminant disease course. The entire HBV genomes from the serum of eight patients with fulminant HBV infection and one patient with fulminant hepatitis during reinfection after liver transplantation were investigated. After isolation and amplification of viral DNA by polymerase chain reaction (PCR), plus and minus strands were directly sequenced. Sequence data were analyzed by comparative sequence alignments with 35 and 2 complete HBV genome sequences from patients without and with fulminant hepatitis, respectively. Several point mutations were present in all regions of the genomes. Many nucleotide changes had never or rarely been found in the reported HBV isolates from patients without fulminant hepatitis. A distinct mutation present in all genomes was not identified. Clusters of rare and unique mutations were observed in the enhancer II core promoter region. Mutations previously suggested to be associated with fulminant HBV infection were not consistently found. A precore stop codon mutation at nucleotide position 1896 or an A–to–T mutation at nucleotide position 1762 and a G–to–A mutation at nucleotide position 1764 in the core promoter region were present in four and three cases, respectively. Fulminant HBV infection does not appear to be caused by a specific genomic mutation. However, various mutations clustering in the enhancer II core promoter region may contribute to a fulminant disease course.
Comparison of the Characteristics of Hepatocellular Carcinoma Between Hepatitis B and C Viral Infection: Tumor Multicentricity in Cirrhotic Liver With Hepatitis C
Authors: Miyagawa S , Kawasaki S , Makuuchi M
Published: November 2004

Clinicopathological and prognostic features in patients who had undergone hepatectomy for hepatocellular carcinoma (HCC) were examined in relation to viral infection. Among 175 patients, cirrhosis was diagnosed histologically in 134, while 41 had noncirrhotic livers. One hundred twenty–four patients were positive for antibody to hepatitis C virus (anti–HCV) (HC group), 32 for hepatitis B virus surface antigen (HBsAg) (HB group), and 19 negative for both anti–HCV and HBsAg (non–B, non–C group). In the HB group, the mean patient age was significantly younger, and liver function in terms of the plasma retention rate of indocyanine green at 15 minutes and the serum total bilirubin level was significantly better than in either the HC or the non–B, non–C group. Seventeen patients had synchronous multicentric HCCs: the HC group showing a significantly higher incidence than the HB group (

P

< .05). In the HC group, the proportion of cirrhotic liver in patients with multicentric HCCs was significantly larger than in patients with unicentric HCC (

P

< .05). No significant differences in disease–free survival rate after hepatectomy were observed between the three groups. The present retrospective study of surgically treated patients showed that anti–HCV–positive HCCs tended to occur in older individuals who showed worse liver function and a higher incidence of cancer multicentricity compared with HBsAg–positive HCCs. The prognosis of anti–HCV–positive HCCs, which had the disadvantageous characteristic of multicentricity, did not differ from that of HBsAg–positive HCCs.
Expression of Hepatocyte Growth Factor, Transforming Growth Factor α, and Transforming Growth Factor β 1 Messenger Rna in Various Human Liver Diseases and Correlation With Hepatocyte Proliferation
Authors: Masuhara M , Yasunaga M , Tanigawa K , Tamura F , Yamashita S , Sakaida I , Okita K
Published: November 2004

Hepatocyte growth factor (HGF) and transforming growth factor α (TGF–α) stimulate liver regeneration, whereas transforming growth factor β 1 (TGF–β 1) inhibits it in rats. However their significance in human liver diseases, especially in severe acute liver injury, remains unclear. We studied HGF, TGF–α, and TGF–β 1 messenger RNA (mRNA) expression in the livers of patients with live diseases using a competitive reverse transcriptase polymerase chain reaction. As little as a twofold difference in mRNA expression could be detected from minute liver biopsy samples. We then examined cell proliferation using proliferating cell nuclear antigen (PCNA) staining. HGF mRNA levels were significantly higher (approximately threefold) in acute hepatitis (AH) than in exacerbation of chronic liver disease (EX) (

P

< .05). TGF–α mRNA levels were significantly greater in AH (approximately twofold) than EX (

P

< .05), and the levels were significantly higher (approximately threefold) in chronic hepatitis (CH) than in EX (

P

< .05). The TGF–β 1 mRNA levels in all the groups were not significantly different. In acute liver injury (AH and EX), there was a significant correlation between HGF mRNA expression and the PCNA labeling index (LI) in the liver (r = .87,

P

< .0005). TGF–α mRNA expression also correlated with the PCNA LI (r = .92,

P

< .0001). There was no significant correlation between the serum HGF and the PCNA LI in the liver. In conclusion, HGF and TGF–α produced in the liver stimulate hepatocyte proliferation in response to acute liver injury in humans.
Transplantation of Conditionally Immortalized Hepatocytes to Treat Hepatic Encephalopathy
Authors: Schumacher I K , Okamoto T , Kim B , Chowdhury N R , Chowdhury J R , Fox I J
Published: November 2004

Transplantation of hepatocytes has been shown to provide metabolic support during liver failure in experimental models. The potential clinical application of hepatocyte transplantation, however, is limited by the need for readily available, well–characterized cells, and a worldwide shortage of donor organs. A clonal hepatocyte cell line that could be grown economically

in vitro

and would exhibit a differentiated, nontransformed phenotype following transplantation would be an attractive solution to this problem. To test this alternative, primary Lewis rat hepatocytes were conditionally immortalized by retroviral transduction with a thermolabile mutant Simian virus 40 (SV40) large T antigen. The cloned immortalized cells proliferate in culture at 33 degrees C and stop growing at 37 degrees C to 39 degrees C. Transplanted into normal livers, these hepatocytes integrate normally into liver cords. When transplanted into the spleens of portacaval–shunted rats, they protect recipients from hyperammonemia–induced hepatic encephalopathy. The cells engrafted in the spleen exhibit normal morphology, secrete bile, and express albumin messenger RNA. The protection from hyperammonemia is reversed by splenectomy. These studies show that hepatocytes can be conditionally immortalized, expanded in culture, and are capable of providing metabolic support in chronic liver insufficiency. Safeguards that could make these cells clinically useful can be accomplished using currently available technology.
The Peptide–Based Thrombin Inhibitor Crc 220 Is A New Substrate of the Basolateral Rat Liver Organic Anion–Transporting Polypeptide
Authors: Eckhardt U , Horz J A , Petzinger E , Stüber W , Reers M , Dickneite G , Daniel H , Wagener M , Hagenbuch B , Stieger B , Meier P J
Published: November 2004

The peptidomimetic thrombin inhibitor CRC 220, 4–methoxy–2,3,6–trimethylphenylsulfonyl–l–aspartyl–d–4–amidinop henylalanyl– piperidide, is taken up into isolated rat hepatocytes through active, carrier–mediated transport. This uptake is inhibited by bile acids. Functional expression in Xenopus laevis oocytes was performed to identify the transport system responsible for the hepatocellular CRC 220 uptake. Injection of poly(A)+RNA in

X. laevis

oocytes resulted in a two– to three–times higher uptake of CRC 220, compared with uninjected or water–injected control oocytes. Taurocholate (200 mumol/L) inhibited this uptake completely. No uptake of the peptidomimetic thrombin inhibitor was observed, when

X. laevis

oocytes were injected with complementary RNA (cRNA) encoding either the cloned rat liver Na+– dependent taurocholate transporter Ntcp, the renal oligopeptide carrier rhaPT or the intestinal oligopeptide transporter PepT1. However, after injection of cRNA of the cloned rat liver Na+–independent organic anion transporting polypeptide oatp, a specific and saturable CRC 220 uptake was observed (Michaelis–Menten constant 29.5 mumol/L). Cis–inhibition with known oatp–substrates, e.g., 20 mumol/L Bromsulphalein (BSP), 2007 mumol/L taurocholate and 2007 mumol/L cholate, occurred in oatp–expressing

X. laevis

oocytes, whereas substrates of the two peptide carriers as well as dipeptide– and single–amino acid constituents of the thrombin inhibitor itself lacked any significant inhibitory effects. These data show that the modified dipeptide CRC 220 is a highly selective substrate of the organic anion transporting polypeptide oatp in the basolateral plasma membrane of rat hepatocytes.
Nimodipine, A Dihydropyridine–Type Calcium Channel Blocker, Prevents Alcoholic Hepatitis Caused by Chronic Intragastric Ethanol Exposure in the Rat
Authors: Iimuro Y , Ikejima K , Rose M L , Bradford B U , Thurman R G
Published: November 2004

It has been shown recently that inactivation of Kupffer cells prevents free radical formation and early alcohol–induced liver injury, and that hypoxia subsequent to a hypermetabolic state caused by activated Kupffer cells is likely involved in the mechanism. Calcium is essential for the activation of Kupffer cells, which contain L–type voltage–dependent Ca2+ channels. Therefore, the purpose of this study was to determine whether a Ca2+ channel blocker, nimodipine, prevents early alcohol–induced liver injury

in vivo

and to evaluate its effect on intracellular calcium ([Ca2+]i) in Kupffer cells

in vitro

. Male Wistar rats were exposed to ethanol (10–12 g/kg/d) continuously for up to 4 weeks via intragastric feeding using an enteral model developed by Tsukamoto and French. In this model, ethanol causes steatosis, necrosis, and inflammation in only a few weeks. In the experimental group, nimodipine (10 mg/kg/d) was added to the diet and was shielded from direct light. Nimodipine had no effect on body weight over a 4–week treatment period, nor were mean ethanol concentrations or their cyclic pattern in urine affected. The mean urine ethanol values were 154 ± 11 mg/dL in ethanol–fed and 144 ± 38 mg/dL in ethanol + nimodipine–fed rats. After 4 weeks, serum aspartate transaminase (AST) levels were elevated in ethanol–treated rats to 183 ± 78 U/L. In contrast, values only reached 101 ± 9 U/L in rats given nimodipine + ethanol–values which were significantly lower. Steatosis and necrosis assessed histologically were also reduced significantly by nimodipine. Nimodipine (10 micrograms/kg) also blocked the swift increase in alcohol metabolism and elevated oxygen consumption in perfused livers from rats treated with alcohol in vivo. Further, in cultured Kupffer cells, nimodipine (1 mumol/L) largely prevented the elevation in [Ca2+]i caused by lipopolysaccharide (LPS) (LPS, 200 ± 11 nmol/L; LPS + nimodipine, 94 ± 31 nmol/L;

P

< .05). These results indicate that nimodipine prevents alcoholic hepatitis, possibly by inhibition of endotoxin–mediated Kupffer cell activation.
Expression of E–Selectin and E–Selectin Ligands in Human Liver Inflammation
Authors: Adams D H , Hubscher S G , Fisher N C , Williams A , Robinson M
Published: November 2004

Lymphocyte recruitment to tissue is regulated by homing molecules expressed on subsets of leukocytes that bind to endothelial adhesion molecules termed addressins in target tissues. The addressins and homing molecules involved in recruiting lymphocytes to inflamed human liver are not known. E–selectin, which acts as an addressin for a subset of skin–infiltrating T cells that express a distinct E–selectin ligand called the cutaneous lymphocyte antigen (CLA), can be detected on endothelium in inflammatory liver diseases suggesting it might also act as an addressin for liver–infiltrating lymphocytes. To determine whether E–selectin does play such a role we looked for concomitant expression of E–selectin on hepatic endothelium and E–selectin ligands on infiltrating lymphocytes in liver sections from patients with primary biliary cirrhosis (PBC), acute allograft rejection, alcoholic liver disease, and normal liver. E–selectin was detected on vascular endothelium in association with lymphocytic infiltrates in all three liver diseases but not on normal endothelium. However, less that 10% of infiltrating lymphocytes expressed the only known lymphocyte E–selectin ligand, CLA. We used E–selectin chimeric fusion proteins to probe for novel E–selectin ligands on liver–infiltrating lymphocytes; however, less than 10% of lymphocytes had t he ability to bind the fusion proteins. Thus, E–selectin is unlikely to act as an addressin for liver–infiltrating lymphocytes, suggesting that other molecules are involved in lymphocyte recruitment to the liver. Despite the lack of binding to lymphocytes other structures in the liver expressed CLA and bound the E–selectin fusion proteins. Inflamed, but not normal, intrahepatic bile ducts expressed CLA and bound E–selectin, suggesting that they express activation dependent carbohydrate binding receptors. Furthermore, Kupffer cells did not express CLA but bound to both E–selectin fusion proteins, suggesting that they express potentially novel E–selectin ligands.
Comparison of High–Resolution Endoluminal Sonography to Video Endoscopy in the Detection and Evaluation of Esophageal Varices
Authors: Miller L S , Schiano T D , Adrain A , Cassidy M , Liu J , Ter H , Bellary S V , Dabezies M A , Black M
Published: November 2004

High–resolution endoluminal sonography (HRES) was used to image and measure esophageal varices in control subjects and patients with portal hypertension and compared with endoscopic findings. Nine control patients and 68 patients with known cirrhosis or noncirrhotic portal hypertension underwent videotaped HRES and videotaped esophagoscopy (EGD). Two blinded investigators reviewed the videotapes to determine the presence and size of the largest esophageal varix in each patient. The largest varix by HRES was measured with the esophagus at rest at a point where the varix appeared most circular. The largest varix seen on EGD was graded on a 5–point scale. All nine of the control patients were correctly identified by both EGD and HRES as grade I (no varices). Eight of the 10 patients with no varices seen on EGD had varices identified by HRES. The interobserver correlation for HRES was r = .88 and for EGD was r = .79. The correlation between EGD and HRES was r = .50. High resolution endoluminal sonography allows quantitative measurement of variceal size, is a more sensitive and reproducible imaging modality than esophagoscopy for the detection and sizing of esophageal varices. There is poor correlation between monographic measurement and endoscopic grading of esophageal varices. Assessment of esophageal variceal presence and the measurement of variceal size by high resolution endoluminal sonography is an accurate, reproducible method of determining the size of esophageal varices.
Microfilament–Disrupting Agent Latrunculin A Induces and Increased Number of Fenestrae in Rat Liver Sinusoidal Endothelial Cells: Comparison With Cytochalasin B
Authors: Braet F , De Zanger R , Jans D , Spector I , Wisse E
Published: November 2004

This report describes the effect of the actin–disrupting marine toxin latrunculin A on the cytoskeleton and fenestrae of liver endothelial cells (LECs). Fluorescence microscopy and whole mount–transmission electron microscopic preparations of isolated, purified, and cultured LECs showed that latrunculin A, which sequesters actin monomers and depolymerizes actin filaments, caused profound changes in microfilament organization in LECs. Scanning electron microscopic preparations showed that latrunculin A almost doubles the number of fenestrae within 10 minutes, whereas the diameter is only slightly reduced. All new fenestrae possess the earlier described fenestrae–associated cytoskeleton ring. Cytochalasin B, which disrupts the network of actin filaments, principally by capping the fast growing end of actin filaments, produced comparable effects with regard to actin organization and the number and size of fenestrae. After 1 hour of treatment, an equal maximum number of fenestrae was observed for both agents. The effect of latrunculin A was obtained at concentrations about 100 times lower than cytochalasin B. Thus, two agents that alter the state of actin organization in LECs, albeit by different mechanisms, cause the doubling of the number of fenestrae within 10 to 30 minutes. This indicates that the state of assembly of the actin cytoskeleton is important in the numerical dynamics of LEC fenestrae and that the actin cytoskeleton of LECs is probably the main mechanical regulator for sieving between the sinusoidal blood and the parenchymal cells. Latrunculin A represents a new agent in the study of the de novo formation of fenestrae.
Long–Term Ethanol Administration Alters the Degradation of Acetaldehyde Adducts by Liver Endothelial Cells
Authors: Thiele G M , Miller J A , Klassen L W , Tuma D J
Published: November 2004

Previous reports have shown that long–term ethanol administration alters receptor–mediated endocytosis (RME) of a variety of macromolecules by liver endothelial cells (LEC). Acetaldehyde is the major metabolic product of ethanol metabolism and has been shown to bind to proteins to form adducts. In this study, the level of protein modification by acetaldehyde necessary for the uptake and degradation of acetaldehyde–modified proteins by LEC was investigated. Bovine serum albumin (BSA) acetaldehyde adducts were prepared by incubation of albumin with acetaldehyde at 100 mmol/L for 1 hour at 37 degrees C, and 1 mmol/L or 0.2 mmol/L for 5 days at 37 degrees C.

In situ

liver perfusion in the presence of these adducts resulted in the degradation of 107 +/– 10.02 microg, 69.82 +/– 5 microg, and 2.5 +/– 0.42 microg of acetaldehyde–adducted albumin, respectively, during a 4–hour period. These values were decreased by 53%, 67%, and nearly 100%, respectively, in livers from ethanol–fed rats. Additionally, modification of protein with 1 mmol/L of acetaldehyde for different periods of time and/or pH altered the amount of 14C–acetaldehyde binding, but no significant changes in degradation were observed. Finally, an excess of formaldehyde–modified albumin totally inhibited the degradation of acetaldehyde adducts, suggesting that they use the same receptor. These data show that acetaldehyde–modified proteins may be taken up and degraded by the scavenger receptor on LEC. This uptake and degradation are dependent on the extent modification of the protein by acetaldehyde, and long–term ethanol consumption decreases the degradation of acetaldehyde–protein adducts.
Induction of Endotoxin Tolerance in Transgenic Mouse Liver Expressing Creatine Kinase
Authors: Hatano E , Tanaka A , Iwata S , Satoh S , Kitai T , Tsunekawa S , Inomoto T , Shinohara H , Chance B , Yamaoka Y
Published: November 2004

The liver plays an important role in maintaining homeostasis during endotoxin–triggered systemic inflammatory response. To study the effects of phosphocreatine on hepatic energy metabolism after endotoxin administration, we used transgenic mice whose livers express creatine kinase (CK). CK catalyzes a phosphocreatine/creatine reaction, that is, an adenosine triphosphate (ATP) reservoir system. Because dietary supplementation with creatine leads to an accumulation of creatine and phosphocreatine in transgenic livers, we compared the CK transgenic mice fed with creatine with the normally fed CK transgenic mice. In the creatine–fed mice, hepatic ATP, energy charge ([ATP + 0.5 adenosine diphosphate (ADP)]/[ATP + ADP + adenosine monophosphate (AMP)]), and mitochondrial oxidative phosphorylation activities remained at high levels after injection of 10 mg/kg of lipopolysaccharide (LPS) as compared with those in normally fed mice. Furthermore, there were beneficial effects on the functional reserve for ATP synthesis and work–cost performance, as calculated by free cytoplasmic ADP and the Michaelis constant (Km). Interestingly, a reduction of tissue necrosis factor α and interleukin–lα (IL–lα), and suppression of the decrease in glucose levels after LPS injection were observed in the creatine fed mice. Survival rates at 72 hours after injection of 10 mg/kg of LPS significantly increased in the creatine fed mice compared with the normally fed mice (80% vs. 24%,

P

< .001). Therefore, we concluded that the presence of phosphocreatine in the liver maintains energy metabolism and attenuates cytokine response, resulting in endotoxin tolerance.
Concanavalin A Induces Perforin–Mediated But Not Fas–Mediated Hepatic Injury
Authors: Watanabe Y , Morita M , Akaike T
Published: November 2004

Concanavalin A (Con A) induces T–cell–mediated hepatic injury

in vivo

, although Con A–stimulated lymphocytes are not cytotoxic to normal hepatocytes in vitro. This contradiction makes the mechanism of Con A–induced hepatitis elusive. In this study, we demonstrate that Con A but not tumor necrosis factor α (TNF–α), interferon γ (IFN–γ), or actinomycin D (ActD) induced the susceptibility of hepatocytes to activated autologous lymphocyte cytotoxicity. Con A sensitized hepatocytes within 30 minutes after the stimulation in a dose–dependent fashion. The cytotoxicity was dose–dependently inhibited by either a Con A ligand, α–methyl mannoside, or a perforin inhibitor, concanamycin A (CMA), but not by anti–Fas ligand antiserum. In addition, Con A–treated hepatocytes were not sensitive to autologous activated lymphocytes from a perforin–deficient mouse, while hepatocytes from lpr mice were sensitized by Con A. In fact, Con A did not induce liver injury in perforin–deficient mice within the concentration employed in this study. Therefore, we conclude that the cytotoxicity was mediated through perforin/granzymes but not through the Fas/Fas ligand pathway. The cytotoxicity was inhibited by anti– intercellular adhesion molecule–1 (ICAM–1)/LFA–1 antibodies, but not by anti–VCAM–1/VLA–4 antibodies, both

in vitro

and

in vivo

. The cytotoxicity appears to be caused by CD8+ T cells; however, the cytokines from activated CD4+ T cells play a critical role in the pathogenesis of the hepatitis in vivo, because administration of anti– IFN–γ antibodies inhibited the occurrence of the hepatitis. In conclusion, Con A–induced hepatitis is thought to be dominantly mediated by a perforin–dependent pathway through ICAM–1/LFA–1 interaction.
Long–Term Ganciclovir Chemotherapy for Congenital Duck Hepatitis B Virus Infection In Vivo : Effect on Intrahepatic–Viral Dna, Rna, and Protein Expression
Authors: Luscombe C , Pedersen J , Uren E , Locarnini S
Published: November 2004

Long–term antiviral chemotherapy using the nucleoside analogue ganciclovir was undertaken with the aim of eliminating hepadnaviral covalently closed circular (CCC) DNA from the livers of ducks that were congenitally infected with the duck hepatitis B virus (DHBV). Twenty– four weeks of ganciclovir therapy caused a substantial reduction in viremia, intrahepatic viral DNA replicative intermediates, and viral core proteins. Unfortunately, ganciclovir therapy did not substantially affect CCC DNA or viral RNA levels, and the treatment resulted in an increase in the intrahepatic expression of the viral envelope proteins, pre–S and S. By the completion of therapy, the viral envelope proteins had assembled into large aggregates within the cytoplasm of most hepatocytes. Viral replication in the bile duct epithelial cells and in the extrahepatic sites was likewise not affected by long–term ganciclovir therapy. In conclusion, 24 weeks of ganciclovir therapy decreased most viral replication markers within the liver, except for those of viral CCC DNA, RNA, and envelope proteins. Long–term therapeutic strategies using nucleoside analogs such as ganciclovir should be used with caution in chronic hepatitis B virus (HBV) infection. The careful monitoring of serum and hepatic markers of viral replication may therefore be important to avoid possible toxic consequences, such as the selective accumulation of viral proteins.
A Randomized Controlled Trial of Thymosin–α1 Versus Interferon Alfa Treatment in Patients With Hepatitis B E Antigen Antibody—And Hepatitis B Virus Dna—Positive Chronic Hepatitis B
Authors: Andreone P , Cursaro C , Gramenzi A , Zavaglia C , Rezakovic I , Altomare E , Severini R , Franzone J S , Albano O , Ideo G , Bernardi M , Gasbarrini G
Published: November 2004

It has recently been shown that thymosin–α1(T–α1), a synthetic polypeptide of thymic origin, is able to promote disease remission and inhibition of hepatitis B virus (HBV) replication in patients affected by hepatitis B e antigen (HBeAg)–positive chronic active hepatitis. We evaluated the efficacy and safety of T–α1 treatment in patients with hepatitis B e antibody (anti–HBe) and HBV–DNA–positive chronic hepatitis. Thirty–three patients were randomly assigned to receive either T–α1 900 microg/m2 body surface area twice weekly (17 patients) or 5 MU of interferon alfa (IFN–α) three times weekly (16 patients) for 6 months. At baseline, both groups were comparable concerning age, sex, liver histology, and alanine transaminase (ALT) levels. At the end of treatment, complete response (defined as ALT normalization and HBV–DNA loss) occurred in 5 of 17 (29.4%) in the T–α1 group and in 7 of 16 (43.8%) in the IFN–α group (

P

= not significant). After a follow–up period of 6 months, a complete response was observed in 7 of 17 (41.2%) in the T–α1 group and in 4 of 16 (25%) in the IFN–α group (

P

= n.s.). Compared with the results observed in a group of 15 patients never treated with IFN–α and followed for 12 months, the rate of complete response was significantly higher in the IFN–α group at the end of therapy (1 of 15 vs. 7 of 16, respectively;

P

< .05) and in the T–α1 group at the end of follow–up (1 of 15 vs. 7 of 17, respectively; P < .05). Unlike IFN– α, T–α1 was well tolerated by all patients. The only side effect, reported by some, was local discomfort at injection sites. The results of this trial suggest that T–α1 is able to reduce HBV replication in patients affected by anti–HBe–positive chronic hepatitis. Furthermore, compared with IFN–α, T–α1 is better tolerated and seems to induce a gradual and more sustained ALT normalization and HBV–DNA loss. In conclusion, T–α1 appears to be a safe and effective alternative treatment for anti–HBe–positive chronic hepatitis. The benefit of this agent in producing long–term inhibition of HBV replication must be confirmed by future trials.