bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel chimeric fibroblast growth factor for liver parenchymal cells
Authors: Imamura T , Tanahashi T
Published: November 2004

Although fibroblast growth factor 1 (FGF-1) (formerly known as acidic FGF) but not FGF-2 (or basic FGF), has been suggested to play a pathophysiological role in liver regeneration, its clinical application has been restricted by its limited mitogenecity and heparin dependence. We report here that a chimeric human FGF protein, FGF-C(1211), is a heparin-independent potent mitogen for liver parenchymal cells both in vitro and in vivo. In the presence of serum and physiological concentration of insulin, FGF-C(1211) by itself induces as much as 55% of hepatocytes in culture to proliferate and up to 88% when used in combination with hepatocyte growth factor or epidermal growth factor. Furthermore, hepatocytes nuclear labeling in vivo with bromodeoxyuridine was markedly enhanced when FGF-C(1211) was injected intravenously into carbon tetrachloride-administered mice. Because FGF-C(1211) is heparin independent and nonantigenic, it has potential for clinical and preclinical applications.
Role of adhesion molecules in the development of massive hepatic necrosis in rats
Authors: Mochida S , Ohno A , Arai M , Tamatani T , Miyasaka M , Fujiwara K
Published: November 2004

Massive hepatic necrosis develops after endotoxin administration in rats pretreated with heat-killed Propionibacterium acnes as a result of microcirculatory disturbance caused by endothelial cell destruction by activated macrophages in the hepatic sinusoids. Immunohistochemical hepatic expression of intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen 1 α (LFA-1 α) and the effect of monoclonal antibodies against both adhesion molecules on liver necrosis provoked after endotoxin administration was studied in these rats. There were increased stains of ICAM-1 in endothelial cells and LFA-1 α in macrophages in the hepatic sinusoids in Propionibacterium acnes-pretreated rats compared with normal rats. Such stains were further increased soon after endotoxin administration, followed by development of hepatic necrosis. Monoclonal antibodies against both adhesion molecules significantly attenuated the extent of liver injury compared with controls, without affecting the infiltration and activation of hepatic macrophages. Polyclonal antibodies against polymorphonuclear leukocytes eradicated circulating neutrophils, but did not change such liver injury, although gum arabic, which suppressed macrophage activation, attenuated the extent of liver injury. Thus, adhesion between endothelial cells and activated macrophages in the hepatic sinusoids via ICAM-1 and LFA-1 α is essential for the initiation of massive hepatic necrosis of this type. Contribution of neutrophils seems less likely.
Adenosine does not mediate renal sodium retention and peripheral vasodilation elicited by partial portal vein ligation in rats
Authors: Murakami S , Bernardo J F , Branch R A , Sabra R
Published: November 2004

This study was conducted to assess the role of adenosine in the peripheral vasodilation and sodium retention that occurs after partial portal vein ligation (PVL) in the rat. The experiment was performed on day 1 after surgery when transient maximal sodium retention developed and day 7 when rats returned to sodium balance. Hemodynamic studies were conducted under anesthesia in portal hypertensive rats with sodium retention and in sham-operated controls. Measurements were obtained before and after administration of a nonselective A1 and A2 adenosine receptor antagonist 1,3-dipropyl-8-p-sulfophenylxanthine (DPSPX) (10 mg/kg intravenously followed by 150 μg/min). Under baseline conditions, portal hypertensive rats with sodium retention were hypotensive, with decreases in total peripheral resistance and filtration fraction on day 1 in comparison with the control group. Although hyperdynamic circulation was still maintained by day 7, there was a return to sodium balance. The adenosine receptor antagonist had a modest vasoconstrictor effect on systemic and renal vasculature in both groups, but less in portal hypertensive rats. However, no change in glomerular filtration rate was observed. DPSPX induced a natriuresis in control rats (from 0.40 ± 0.11 to 5.97 ± 0.61 mEq/min on day 1, from 0.48 ± 0.20 to 6.34 ± 0.45 mEq/min on day 7). This response was attenuated in portal hypertensive rats on day 1 (from 0.14 ± 0.04 to 1.67 ± 0.57 mEq/min), but not on day 7 (from 0.20 ± 0.06 to 5.11 ± 0.55 mEq/min). These results suggest that in portal hypertensive rats (1) adenosine is not responsible for vasodilation and sodium retention, (2) a sodium-retaining factor acting directly on the renal tubule is responsible for sodium retention.
Spontaneous bacterial empyema in cirrhotic patients: A prospective study
Authors: Xiol X , Castellví J M , Guardiola J , Sesé E , Castellote J , Perelló A , Cervantes X , Iborra M J
Published: November 2004

Spontaneous bacterial empyema (SBEM) is an infection of a preexisting hydrothorax in cirrhotic patients and has seldom been reported. To determine its incidence and primary characteristics, all cirrhotic patients with pleural effusion underwent thoracentesis at our hospital either on admission or when an infection was suspected. Pleural fluid (PF) study included biochemical analysis, polymorphonuclear (PMN) leukocyte count, and culture by two methods: conventional and modified (inoculation of 10 mL of PF into a blood culture bottle at the bedside). SBEM was defined according to previously reported criteria: PF culture positive or PMN count greater than 500 cells/μL, and exclusion of parapneumonic effusions. Sixteen of the 120 (13%) cirrhotic patients admitted with hydrothorax had 24 episodes of SBEM. In 10 of the 24 episodes (43%), SBEM was not associated with spontaneous bacterial peritonitis (SBP). PF culture was positive by the conventional method in 8 episodes (33%) and by the modified method (blood culture inoculation) in 18 (75%) (P = .004, McNemar). The microorganisms identified in PF were Escherichia coli in 8 episodes, Streptococcus species in 4, Enterococcus species in 3, Klebsiella pneumoniae in 2, and Pseudomonas stutzeri in 1. All episodes were treated with antibiotics without inserting a chest tube in any case. Mortality during treatment was 20%. We conclude that SBEM is a common complication of cirrhotic patients with hydrothorax. Almost half of the episodes were not associated with SBP; thus, thoracentesis should be performed in patients with cirrhosis, pleural effusion, and suspected infection. Culture of PF should be performed by inoculating 10 mL into a blood culture bottle at the bedside.
Etiology, evaluation, and outcome of jaundice in patients with acquired immunodeficiency syndrome
Authors: Chalasani N , Wilcox C M
Published: November 2004

Although liver test abnormalities are frequently identified in patients with acquired immunodeficiency syndrome (AIDS), the causes, evaluation, and outcome of jaundice in these patients have not been systematically evaluated. From August 1, 1990 through September 1, 1994, all human immunodeficiency virus (HIV)-infected patients with liver test abnormalities seen by the gastroenterology service at a large, inner-city hospital were prospectively identified. Jaundice was defined as a serum bilirubin concentration ≥ 3 mg/dL. The etiology of jaundice was determined by the pattern of liver biochemistry test abnormalities, radiographic studies, liver biopsy, clinical follow-up, and autopsy. During the study period, 541 HIV-infected patients (511 with AIDS) were evaluated for liver disease by our service; 36 of these patients had jaundice (7%). The most common causes of jaundice were drug-induced hepatitis, occurring in 11 patients (31%), and alcoholic liver disease, occurring in 5 (13%). Opportunistic infections or neoplasms were identified as the cause of jaundice in 11 patients (30%), with 4 having intrahepatic disease and 7 having extrahepatic disease. Multiple potential causes were seen in 3 patients. Abdominal ultrasonography (US) and computed tomography (CT) were helpful in suggesting the underlying cause of disease. The short-term mortality was high, with 9 patients dying during the hospitalization (25%) and 7 patients dying within 6 months of evaluation. Liver disease was the cause of death in 7 of these patients. In conclusion, jaundice is uncommon in AIDS and may result from a variety of both opportunistic and non-opportunistic etiologies. Drug-induced hepatitis is the most common cause and may be fatal. Long-term survival was poor.
The prolyl 4-hydroxylase inhibitor HOE 077 prevents activation of Ito cells, reducing procollagen gene expression in rat liver fibrosis induced by choline-deficient l-amino acid-defined diet
Authors: Sakaida I , Matsumura Y , Kubota M , Kayano K , Takenaka K , Okita K
Published: November 2004

No effective therapy has yet developed for liver fibrosis by directory inhibiting the accumulation of extracellular matrix. The effect of a newly synthesized prolyl4-hydroxylase (PH) inhibitor, HOE 077 (pyridine- 2, 4-di-carboxylic-di(2-methoxyethyl)amide), was examined using the model of choline-deficient l-amino acid (CDAA) defined diet-induced liver fibrosis in 16-week-old male Wistar rats. HOE 077 at doses up to 200 ppm prevented fibrosis in a dose-dependent manner, as indicated by reduced hydroxyproline content in liver as well as inhibition of increased serum fibrotic markers (PIIIP, 7S, hyaluronic acid). HOE 077 at 200 ppm reduced expression of type III procollagen alpha1, messenger RNA (mRNA) in the liver, with a good correlation with serum PIIIP and hydroxyproline content of the liver. Histologically, HOE 077 at 200 ppm also reduced proliferation of myofibroblastlike cells (activated Ito cells). These results indicate that a PH inhibitor can prevent fibrosis by inhibiting not only the hydroxylation of proline but also the activation of Ito cells, which are considered the main collagen-producing cells, resulting in reduced expression of procollagen mRNA.
Assessment of microchimerism in rat liver transplantation by polymerase chain reaction
Authors: Tashiro H , Fukuda Y , Kimura A , Hoshino S , Ito H , Dohi K
Published: November 2004

Mixed chimerism has been demonstrated in organ transplant recipients surviving over a long period. However, little is known about the fate and movements of donor cells following liver transplantation. In this study, using rat male-to-female liver transplantation, we assessed microchimerism by semiquantitative polymerase chain reaction (PCR). A PCR specific for the Y-chromosome (sex-determining region Y [Sry]) allowed the distinction of small amounts of male cells in a large excess of female cells. Nonimmunosuppressed Lewis recipients of Lewis liver grafts survived > 60 days, and donor cells were detected in peripheral blood up to day 60 in four of six recipients. All immunosuppressed Lewis recipients survived for 60 days, and the donor cells were detected up to day 60. Semiquantitative PCR demonstrated that in immunosuppressed Lewis recipients surviving > 60 days with no acute rejection episode, the donor-to-recipient cell ratio remained > 1:1,000. In nonimmunosuppressed Lewis recipients of ACI liver allografts, donor cells in peripheral blood decreased rapidly by day 7. In immunosuppressed Lewis recipients of ACI liver allografts, donor cells were detected for longer periods, in proportion to the amount and duration of FK506 (Fujisawa Pharmaceutical Co., Osaka, Japan) administered. However, in recipients with deteriorating function due to ongoing rejection, small amounts (0.01 percent) of donor cells were detected. Our results indicate that the Sry-specific PCR is useful for assessing microchimerism following rat liver transplantation, and demonstrate correlation between microchimerism and graft outcome; high- level microchimerism (>0.l percent) correlated well with graft acceptance, whereas low-level microchimerism (<0.01 percent) did not.
n -Butyrate induces plasminogen activator inhibitor type 1 messenger RNA in cultured Hep G2 cells
Authors: Smith T J , Piscatelli J J , Andersen V , Wang H , Lance P
Published: November 2004

n-Butyrate, a short-chain aliphatic carboxylic acid with pleiotropic actions, is present at high concentrations in the portal circulation and thus may play an important role in the regulation of specific gene expression in the mammalian liver. We report here that n-butyrate can increase substantially the level of plasminogen activator inhibitor type 1 (PAI-1) messenger RNA (mRNA) in Hep G2 cells, up to eightfold above control cultures. Maximal effects occurred at a concentration of 3 mmol/L n-butyrate and with a treatment period of 8 to 12 hours. Increases in PAI-1 mRNA were accompanied by modest increases (twofold) in the encoded protein as assessed by specific enzyme-linked immunosorbent assay and by [35S]methionine incorporation into immunoprecipitable PAI-1 in the culture medium. Nuclear run-on studies showed that the rate of transcription of the PAI-1 gene did not appear altered by treatment with 3 mmol/L n-butyrate for 6 hours. The increases in steady-state PAI-1 mRNA caused by exposure to n-butyrate can be blocked by cycloheximide. Enhanced stability of mature PAI-1 transcript could not be demonstrated in Hep G2 cells treated with the carboxylic acid. We have reported previously that n-butyrate can reduce the level of beta-galactoside alpha 2,6-sialyltransferase expression in Hep G2 cells. That effect was attenuated with inhibitors of protein and RNA synthesis and was mediated at the post-transcriptional level. Thus, n-butyrate can influence the expression of multiple genes in this hepatoblastoma cell through its actions on events that appear to be posttranscriptional. These observations may be relevant to the normal physiology of the mammalian liver because of the high concentrations of n-butyrate and related compounds to which the organ is ordinarily exposed.
Acetaldehyde and malondialdehyde react together to generate distinct protein adducts in the liver during long-term ethanol administration
Authors: Tuma D J , Thiele G M , Xu D , Klassen L W , Sorrell M F
Published: November 2004

Acetaldehyde and the lipid peroxidation-derived aldehyde malondialdehyde (MDA), are reactive compounds that are generated during ethanol metabolism in the liver, and both aldehydes have been shown to be capable of binding to proteins and forming stable adducts. Because similar concentrations of MDA and acetaldehyde can coexist in the liver during ethanol oxidation, protein adduct formation in the presence of both of these aldehydes was studied under both in vitro and in vivo conditions. When proteins were incubated in the presence of both MDA and acetaldehyde, MDA caused a marked and concentration-dependent increase in the stable binding of acetaldehyde to proteins. Maximum stimulation of binding occurred at approximately a fourfold molar excess of MDA relative to acetaldehyde when concentrations of 1.0 mmol/L and 0.1 mmol/L were tested. The formation of highly fluorescent product or products was associated with the MDA stimulation of acetaldehyde binding, indicating that new and distinct products were being generated. These hybrid adducts of MDA and acetaldehyde have been designated as MAA adducts. An affinity-purified polyclonal antibody was produced that specifically recognized MAA epitopes on proteins and did not cross-react with carrier proteins or proteins modified with either acetaldehyde or MDA alone. A quantitative competitive enzyme-linked immunosorbent assay (ELISA) was developed and detected the presence of MAA-modified proteins in liver cytosol from ethanol-fed rats but not in pair-fed controls. Quantification of the data from the competitive ELISA indicated the presence of approximately 75 pmoles protein-bound MAA per milligram liver cytosol proteins of the ethanol-fed animals. These results indicate that acetaldehyde and MDA can react together in a synergistic manner and generate hybrid adducts (MAA-adducts) and further suggest that MAA adducts may represent a major species of adducts formed in the liver during ethanol metabolism in vivo.
Hepatic Expression of Hepatitis C Virus Rna in Chronic Hepatitis C: A Study By In Situ Reverse–Transcription Polymerase Chain Reaction
Authors: Lau G K , Davis G L , Wu S P , Gish R G , Balart L A , Lau J Y
Published: November 2004

Hepatitis C virus (HCV) replicates at a low rate and this makes its detection and intrahepatic localization difficult. To evaluate the clinical implications and effect of interferon alfa (IFN–α) therapy on hepatic expression of HCV RNA, HCV RNA was detected by

in situ

reverse–transcription polymerase chain reaction (IS–RT–PCR) in formalin–fixed paraffin–embedded liver sections from 26 patients with chronic hepatitis C. Results were compared with RT–PCR of HCV RNA extracted from liver sections/tissue. Twenty–four paired post–IFN–α treatment biopsy specimens were also assessed. Using RT–PCR of the extracted RNA as a positive standard and non–HCV liver sections as the negative standard, the sensitivity and specificity of IS–RT–PCR were 69% and 100%, respectively. HCV RNA was detected in the cytoplasm of hepatocytes (median, 5% hepatocytes positive; range, 0 to 35%) and very occasionally in infiltrating mononuclear cells. There was no correlation between hepatic expression of HCV RNA and the clinical, biochemical parameters, total and activity scores of histology activity index. Presence of HCV RNA in liver as detected by IS–RT–PCR was associated with higher serum HCV RNA levels (4.9 × 106 vs. 0.4 × 106 genome Eq/mL,

P

< .01). There was no difference in the pretreatment proportion of HCV RNA–positive hepatocytes among patients with different biochemical responses to IFN–α therapy. In the posttreatment samples, HCV RNA was undetectable by IS–RT–PCR in 16 of 24 patients (

P

< .01), including all 4 patients who had complete and sustained response (SR). We conclude that HCV RNA was detected by IS–RT–PCR in 0 to 35% of hepatocytes in patients with chronic HCV infection, detection of HCV RNA in liver by IS–RT–PCR was associated with higher viremia levels and IFN–α therapy reduced hepatocytic expression of HCV RNA.
Serum Levels of Hepatitis C Virus Core Protein in Patients With Chronic Hepatitis C Treated With Interferon Alfa
Authors: Tanaka E , Kiyosawa K , Matsumoto A , Kashiwakuma T , Hasegawa A , Mori H , Yanagihara O , Ohta Y
Published: November 2004

The quantitation of hepatitis C virus (HCV) viremia can be helpful in the diagnosis, therapy, and monitoring of patients with chronic hepatitis C. A sensitive and quantitative fluorescence enzyme immunoassay (FEIA) has recently been developed for assaying HCV core protein in serum. To assess the utility of measurements of serum HCV core protein during the course of treatment of chronic hepatitis C, we studied 27 patients who were treated with a single schedule of interferon alfa (IFN–α) (9 million units per dose for 24 weeks; total dose, 720 million units). Eleven of the 27 patients responded with clearance of HCV RNA and fall of aminotransferase to normal; 16 patients did not respond to treatment. Before therapy, HCV core antigen was detectable in 25 of the 27 patients (93%). The initial serum concentration of HCV core protein was significantly (

P

< .01) higher in the nonresponders versus the responders. Two weeks after initiating IFN–α therapy, HCV core protein was not detectable in any of the 11 responders, but was detected in 8 of 16 nonresponders (

P

< .01). All responders, but none of the nonresponders, remained negative for core protein after IFN–α therapy. The measurement of HCV core protein by FEIA may be useful for predicting the response to IFN–α and for monitoring its therapeutic efficacy.
The Long–Term Pathological Evolution of Chronic Hepatitis C
Authors: Yano M , Kumada H , Kage M , Ikeda K , Shimamatsu K , Inoue O , Hashimoto E , Lefkowitch J H , Ludwig J , Okuda K
Published: November 2004

Most patients infected with hepatitis C virus (HCV) develop chronic hepatitis. Unfortunately, the pathological evolution of this disease over time is not completely understood. We studied 70 HCV–positive patients, from whom 2 to 10 liver biopsy specimens (mean, 3.9) had been obtained during an interval of 1 to 26 years (mean, 8.8 years). Each biopsy specimen was evaluated independently by four pathologists who each provided a numerical score for the grade of portal/periportal necroinflammation (0–4), grade of lobular necroinflammation (0–4), their sum (final grade), and the stage of fibrosis (1–4). The scores were correlated with progression of disease, if any, and transition to cirrhosis. During follow–up, 35 patients (50%) developed cirrhosis. Cirrhosis developed in all patients with a high final grade (≥ 5) of necroinflammation on initial biopsy who were followed for 10 years and in 96% of patients with an intermediate final grade (3.5–4.9) who were followed for 17 years. Only 30.4% of patients with low final grade (≤ 3.4) on initial biopsy developed cirrhosis after 13 years. All patients with evidence of septal fibrosis with incomplete nodularity (stage 3.0–3.4) in the initial biopsy progressed to unequivocal cirrhosis by 10 years. The rate of progression to cirrhosis was accelerated in patients whose initial biopsies showed high–grade and –stage lesions. This study demonstrates the importance of grading and staging liver biopsy lesions in chronic hepatitis C, particularly for patients with high–grade necroinflammation, septal fibrosis, and regions of modularity on initial biopsy who are at high risk of developing advanced cirrhosis in the ensuing decade.
Chemical Mediators Released by Primary–Cultured Human Hepatic Macrophages in Patients With and Without Cirrhosis: A Study in Tumor–Bearing Patients
Authors: Funaki N , Arii S , Monden K , Higashitsuji H , Furutani M , Mise M , Tanaka J , Imamura M
Published: November 2004

To elucidate the possible role of chemical mediators in modulating the host–defense activity of patients with cirrhosis, primary–cultured human hepatic macrophages (HHMΦ) were obtained from cirrhotic and noncirrhotic patients who received liver resections because of the presence of malignant liver tumors. The cirrhotic and noncirrhotic groups consisted of patients with similar malignancies: noncirrhotic patients had normal liver function and normal liver histology for nontumorous portions. The cultured HHMΦ were analyzed for their ability to release chemical mediators with specific activities in the host defense system. Dose–dependent increases in superoxide release, interleukin–1 (IL–1) release, and, within a relatively narrow range, prostaglandin–E2 (PGE2) release were observed in opsonized zymosan (oz)–stimulated HHMΦ derived from both cirrhotic and noncirrhotic patients. The release of O2– and PGE2 from HHMΦ derived from cirrhotic patients was significantly less than HHMΦ derived from noncirrhotic patients, whereas the release of IL–1 was significantly greater. Although, because of the limited sample availability, only tumor–bearing patients were studied, the mediator–releasing ability of HHMΦ derived from cirrhotic patients was significantly different from the ability of HHMΦ derived from noncirrhotic patients with similar malignancies. This phenomenon may be related to altered host defenses in patients with cirrhosis.
Quantitation of α–Fetoprotein and Albumin Messenger Rna in Human Hepatocellular Carcinoma
Authors: Niwa Y , Matsumura M , Shiratori Y , Imamura M , Kato N , Shiina S , Okudaira T , Ikeda Y , Inoue T , Omata M
Published: November 2004

To analyze gene expression of α–fetoprotein (AFP) and albumin in hepatocellular carcinoma (HCC), messenger RNAs (mRNAs) of these proteins in six human hepatoma cell lines and in 30 cases of HCC were quantitatively analyzed by competitive reverse transcription (RT) followed by polymerase chain reaction (PCR). The transcriptional levels of both AFP and albumin genes in HepG2 and Huh 7 cell lines were 1010 copies/μg RNA, in contrast to approximately 105 copies/μg RNA in HLE and HLF cell lines. AFP and albumin mRNA levels in three normal livers were 105 and 1010 transcripts/μg RNA, respectively. In 30 cases with HCC AFP mRNA level in neoplasm was 10 to 105–fold enhanced as compared with that of nonneoplastic portion, and correlated with serum AFP level and tumor size (

P

< .01). In contrast, albumin mRNA level was not reduced in the neoplasms presenting enhanced AFP mRNA levels, indicating that AFP and albumin gene expression in situ is not necessarily mutually exclusive. Prospective analysis revealed that an increased serum AFP was shown at the time of recurrence among patients with enhanced AFP mRNA levels in neoplasm only, indicating that AFP mRNA levels in neoplasm could be a clinically predictable tool.
Ursodeoxycholic Acid for Liver Disease Associated With Cystic Fibrosis: A Double–Blind Multicenter Trial
Authors: Colombo C , Battezzati P M , Podda M , Bettinardi N , Giunta A
Published: November 2004

Liver disease is increasingly recognized as a major cause of morbidity in cystic fibrosis (CF). Preliminary data suggest that ursodeoxycholic acid (UDCA) may be beneficial for treatment of this manifestation. We performed a double–blind, multicenter trial in these patients to establish efficacy and safety of UDCA in terms of the improvement of clinical and nutritional indicators besides standard liver function tests. We also intended to establish whether taurine supplementation has a beneficial effect in patients receiving UDCA. From June to December 1990, we enrolled in 12 centers 55 CF patients with liver disease (39 male subjects; median age, 13.8 years). They were randomly assigned to receive for 1 year one of the following treatments: UDCA (15 mg/kg body weight daily) plus taurine (30 mg/kg body weight daily), UDCA plus placebo, placebo plus taurine, or double placebo. Clinical and laboratory evaluations were performed every 3 months. After 1 year, deterioration of overall clinical conditions, as indicated by the Shwachman–Kulczycki score (SKS), occurred in patients who received placebo but not in those who received UDCA (

P

= .025). Patients treated with UDCA also showed an improvement in γ–glutamyl transpeptidase (GGT) (

P

= .004) and 5′–nucleotidase (

P

= .006) levels. Treatment with taurine was followed by a significant increase in serum prealbumin levels (

P

= .053), a trend toward a reduction in fat malabsorption, and no effect on the biochemical profile. No severe side effects occurred with any treatment. Thus, we concluded that UDCA administration improves clinical and biochemical parameters in CF patients with liver disease. Taurine supplementation may be indicated in patients with severe pancreatic insufficiency and poor nutritional status.
Insulin–Like Growth Factor Ii/Mannose 6–Phosphate–Receptor Expression in Liver and Serum During Acute Ccl4 Intoxication in the Rat
Authors: De Bleser P J , Scott C D , Niki T , Xu G , Wisse E , Geerts A
Published: November 2004

The liver is reported to be the main source of soluble insulin–like growth factor–II/mannose 6–phosphate (IGF–II/M6P) receptor in adults. In view of the role of this receptor in the activation of transforming growth factor β (TGF–β) during hepatic fibrogenesis, we have investigated the correlation between serum levels and tissue expression of the receptor during acute CCl4 intoxication of the rat. Sixteen hours after CCl4, injection, the level of the soluble receptor in serum, as measured by radioimmunoassay (RIA), increased threefold. At 24 hours, values almost returned to normal, but increased again by twofold at 48 hours. By 96 hours, nearly normal values were obtained. Northern blot analysis showed peaks in tissue IGF–II/M6P receptor messenger RNA (mRNA) levels at 24 hours and at 48 hours. In normal liver, immunostaining for IGF–II/M6P receptor showed weak positivity in parenchymal cells. CCl4–induced hydropic changes appeared in centrilobular parenchymal cells (PCs) at 8 hours. These changes extended to the midzonal region at 16 hours. Hydropic cells were devoid of receptor staining. The hydropic wave became extinct at 32 hours. At 48 hours, we observed a collapse of PCs in the centrilobular zone, coinciding with strongly positive staining for IGF–II/M6P receptor in fat–storing cells (FSCs), identified by dual IGF–II/M6P receptor and desmin immunostaining. Between 48 and 72 hours, the liver gradually regained its normal appearance. As shown by Western blotting,

in vitro

differentiated FSCs released soluble receptor in the medium. Northern blot analysis showed this release to be preceded by an increased receptor–mRNA expression, whereas immunostaining showed an increase of intracellular receptor. In conclusion, we have shown that acute CCl4 intoxication induces two peaks in serum levels of soluble receptor. While the first peak at 16 hours coincides with the loss of receptor– staining in hydropically damaged PCs, the second peak at 48 hours is paralleled by an increase in positive staining in FSCs and tissue mRNA level. Differentiated FSCs shed soluble receptor

in vitro

. As a consequence, these cells might contribute to the serum levels of soluble receptor

in vivo

. These results indicate that measuring serum soluble IGF–II/M6P receptor might useful in the diagnosis of early acute liver damage.
Effects of Ethanol Administration on Components of the Ubiquitin Proteolytic Pathway in Rat Liver
Authors: Born L J , Kharbanda K K , McVicker D L , Zetterman R K , Donohue T M
Published: November 2004

Hepatic protein accumulation during ethanol administration may result partly from an ethanol–elicited decline in hepatic protein degradation, which we have previously shown. We conducted the current studies to examine the effects of ethanol administration on the levels of hepatic ubiquitin, an 8.5–kd protein which is an important mediator of extralysosomal protein catabolism. Rats were pair–fed liquid diets containing either ethanol (36% of calories) or isocaloric maltose–dextrin for 1 to 5 weeks. Ubiquitin was immunochemically quantified by competitive enzyme–linked immunosorbent assay (ELISA) in crude cytosol fractions from whole liver and in 12,000g supernatants of hepatocyte lysates. Ubiquitin levels in hepatic cytosol fractions of ethanol–fed rats exceeded those of controls by about 30%. Isolated hepatocytes from ethanol–fed animals also showed a 40% to 75% elevation of ubiquitin above that in cells of pair–fed controls and this difference exceeded the relative rise in hepatocellular protein. In hepatocyte lysates subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and immunoblotting, we detected monomeric ubiquitin and higher molecular mass ubiquitin–protein conjugates. However, the immunoblot analyses revealed no quantitative changes in the level of either free or conjugated ubiquitin. The ubiquitin conjugating activity of crude and diethyl aminoethyl–fractionated liver cytosols of ethanol–fed rats had equal capacities to those from controls in catalyzing the formation of ubiquitin–protein conjugates. Our findings indicate that chronic ethanol consumption increased the level of immunoreactive ubiquitin in rat liver. This may have resulted from enhanced ubiquitin production because of an ethanol–elicited stress response and/or decreased catabolism of ubiquitin and its conjugates. Our findings also provide no indication that the ethanol–elicited reduction in hepatic proteolysis is because of a ubiquitin–mediated mechanisms.
Effect of Tumor Necrosis Factor α on Intrahepatic Bile Duct Epithelial Cell of Rat Liver
Authors: Mano Y , Ishii M , Okamoto H , Igarashi T , Kobayashi K , Toyota T
Published: November 2004

Tumor necrosis factor α (TNF–α), which is primarily produced by macrophages, is a cytokine with various biological activities. Macrophage infiltration often accompanies experimental cholangitis in rats, and chronic cholangitis in humans. The pathophysiologic significance of TNF–α in cholangitis is not known. We used cultured, polarized intrahepatic bile duct epithelial cells (IBDECs) from rat liver to determine whether TNF–α directly affects the organization of IBDEC monolayers. The addition of recombinant TNF–α (rTNF–α) to culture media at concentrations from 10 to 200 U/mL lacked cytotoxicity to the IBDECs as judged by trypan blue exclusion and lactate dehydrogenase (LDH) release. rTNF–α transiently reduced transepithelial electrical resistance in a dose–dependent manner. During this decrease in resistance, the cellular tight junctions became leaky, allowing horseradish peroxidase (HRP) penetration. rTNF–α, at concentrations up to 200 U/mL, did not detach IBDECs from Matrigel, an artificial basement membrane. Electron microscopy and immunohistochemistry for F–actin showed a well–preserved cell structure and organization of IBDECs. Results suggest that TNF–α is nontoxic to IBDECs, and that it increases the permeability of tight junctions. TNF–α may thus disturb the barrier function of the bile duct.
Platelet–Activating Factor Augments Lipopolysaccharide–Induced Nitric Oxide Formation by Rat Kupffer Cells
Authors: Mustafa S B , Howard K M , Olson M S
Published: November 2004

Acute endotoxic shock is accompanied by an increase in the production of nitric oxide (NO) by several different hepatic cell types. Platelet–activating factor (PAF) is a potent proinflammatory mediator with many pathophysiological actions and, in fact, elevated plasma and tissue levels of PAF are observed in animal models of endotoxic shock. The current study demonstrates that PAF induced nitrite formation, the end product of nitric oxide synthesis, by Kupffer cells in a dose– and time– dependent manner. Moreover, PAF was seen to initiate NO synthase gene expression and protein synthesis. PAF augmented lipopolysaccharide (LPS)–induced expression of inducible nitric oxide synthase messenger RNA (mRNA), protein, nitrite and cyclic guanosine monophosphate (cGMP) levels in Kupffer cells. Treatment of Kupffer cells with actinomycin D or cycloheximide inhibited PAF– and LPS–stimulated nitrite and nitric oxide synthase protein formation confirming that de novo synthesis of the enzyme occurred. In Kupffer cells, the presence of an arginine analog, NG–methyl–L–arginine, attenuated nitrite formation induced by PAF and LPS alone or in combination. L–arginine is the principal substrate for nitric oxide synthase. PAF and LPS individually and in combination induced a time–dependent uptake of L–[3H]–arginine into the Kupffer cell, and this response was sensitive to cycloheximide. The current study indicates that exogenous PAF contributes to the induction of nitric oxide synthase by LPS in cultured rat Kupffer cells.
Dexamethasone Alters Messenger Rna Levels But Not Synthesis of Collagens, Fibronectin, or Laminin by Cultured Rat Fat–Storing Cells
Authors: Niki T , Schuppan D , de Bleser P J , Vrijsen R , Pipeleers-Marichal M , Beyaert R , Wisse E , Geerts A
Published: November 2004

Glucocorticoids have been shown to suppress collagen synthesis and gene expression by fibroblasts. However, little is known about their effects on fat–storing cells, the major matrix–producing cells in liver fibrosis. In this study we investigated the effect of dexamethasone on the extracellular matrix expression by cultured rat fat–storing cells. Fat–storing cells were isolated from male Wistar rats by collagenase/pronase digestion and purified by density gradient centrifugation. Fat–storing cells in early primary culture (3–day–old, representing a relatively quiescent phenotype) and in subculture (one passage, about 2–week–old, representing an activated phenotype) were treated with 10–6 mol/L dexamethasone for messenger RNA (mRNA) study or with 10–8 to 10–6 mol/L dexamethasone for protein study. Expression of collagen type I, III, IV, fibronectin, and laminin was analyzed at the mRNA level by Northern hybridization, and at the protein level by metabolic labeling and immunoprecipitation.

Dexamethasone had a variable effect on the expression of collagen α1(I) mRNA level. While a tendency for modest suppression was observed (5%–50%) in primary cells, the difference was not statistically significant. Variable response was observed in subcultured cells. Collagen α1(III) mRNA level showed a tendency for stimulation. Dexamethasone stimulated the expression of collagen α1 (IV), fibronectin, and laminin B1 mRNA levels by 1.4–, 2.4–, and 1.6–fold respectively, in primary fat–storing cells. Subcultured cells showed a similar response, but the magnitude of stimulation was more variable than that of primary cells. Unexpectedly, at the protein level dexamethasone had no effect on the expression of these proteins. Our results indicate that glucocorticoids do not possess a net suppressive effect on extracellular matrix synthesis by fat–storing cells. Beneficial effects of glucocorticoids may be attributable to other mechanisms of action, such as their anti–inflammatory effect.
Inhibition of Viral Replication by Genetically Engineered Mutants of the Duck Hepatitis B Virus Core Protein
Authors: von Weizsäcker F , Wieland S , Blum H E
Published: November 2004

The hepatitis B virus (HBV) nucleocapsid consists of 240 viral core proteins that are arranged in a highly symmetrical structure, HBV replication can only take place inside intact nucleocapsids. In the present study, we investigated whether genetically engineered core mutants can inhibit viral replication by interfering with the formation of intact nucleocapsids. Using the duck hepatitis B virus (DHBV) model, a series of core protein mutants was generated. Polymerase chain reaction–amplified fragments from the bacterial lacZ gene expressing up to 282 amino acids were added either to the amino– or carboxy–terminus of the DHBV core protein. In addition, carboxy–terminal extensions were generated by fusing the DHBV core protein with the DHBV small surface protein or various fragments of the viral polymerase. Finally, the green fluorescent protein (GFP) was fused in–frame to the carboxy–terminus of the DHBV core protein. In this chimeric protein, GFP is still functional and can act as a reporter molecule. The various core protein mutants were tested for their potential antiviral activity by cotransfection with a replication–competent DHBV construct into the avian hepatoma cell line LMH. Carboxy–terminal, but not amino–terminal, DHBV core mutants inhibited DHBV replication by up to 90% at an effector–to–target ratio of 1:10, thus displaying a dominant negative phenotype. Antiviral activity was species–specific and caused by posttranslational interference with viral replication. The DHBV core–GFP fusion protein should be an ideal tool to assess the antiviral potential of dominant negative core proteins

in vivo

.
Current Therapy for Crigler–Najjar Syndrome Type 1: Report of A World Registry
Authors: van der Veere C N , Sinaasappel M , McDonagh A F , Rosenthal P , Labrune P , Odiévre M , Fevery J , Otte J , McClean P , Bürk G , Masakowski V , Sperl W , Mowat A P , Vergani G M , Heller K , Wilson J P , Shepherd R , Jansen P L
Published: November 2004

This study represents a multicenter survey on the management of patients with Crigler–Najjar syndrome (CNS) type 1. The aim of the survey was to find guiding principles for physicians in the care of these patients. Fifty–seven patients were included. At the time of inclusion, 21 patients had received a liver transplant (37%). The average age at transplantation was 9.1 ± 6.9 years (range, 1-23 years); the age of the patients who had not been transplanted at the time of inclusion was 6.9 ± 6.0 years (range, 0-23 years). Brain damage had developed in 15 patients (26%). Five patients died, and 10 are alive with some degree of mental or physical handicap. In 2 patients, ages 22 and 23 years, early signs of bilirubin encephalopathy could be reversed, in 1 by prompt medical intervention followed by liver transplantation and in the other by prompt liver transplantation. Seven patients underwent transplantation with some degree of brain damage at the time of the surgery; 1 of these died after retransplantation, 2 improved neurologically, and 4 remained neurologically impaired. The age of 8 patients with and 13 without brain damage at or before transplantation was 14.3 ± 5.9 and 5.9 ± 5.4 years (

P

< .01), respectively. Therapy of CNS type 1 consists of phototherapy (12 h/d), followed by liver transplantation. Phototherapy, although initially very effective, is socially inconvenient and becomes less efficient in the older age group, thus also decreasing compliance. Currently, liver transplantation is the only effective therapy. This survey shows that, in a significant number of patients, liver transplantation is performed after some form of brain damage has already occurred. From this, one must conclude that liver transplantation should be performed at a young age, particularly in situations in which reliable administration of phototherapy cannot be guaranteed.
Interferon γ Down–Regulates Cytochrome P450 3A Genes in Primary Cultures of Well–Differentiated Rat Hepatocytes
Authors: Tapner M , Liddle C , Goodwin B , George J , Farrell G C
Published: November 2004

Administration of interferons of both the γ and alfa/β classes down–regulates hepatic cytochrome P450 (CYP) genes when administered to humans or rats. In male rats, interferons decrease expression of CYP3A2 at a pretranslational level, but because interferons also release other cytokines

in vivo

, it is unclear whether this is a direct effect on hepatocytes. We therefore examined the effects of rat recombinant interferon γ (IFN–γ) on CYP3A2, other 3A genes, and 2C11 in stable primary cultures of male rat hepatocytes. Hepatocytes were cultured on matrigel in Williams' E, and messenger RNAs (mRNAs) for 3A2, 3A1–like CYPs, and 2C11 mRNA were determined by RNase protection assays. CYP3A and 2C11 proteins were immunoquantified, and their catalytic activities were estimated by testosterone hydroxylation pathways. In control cells, 3A2 mRNA decreased initially but then recovered, and stable levels (15% of freshly isolated cells) were attained between days 3 and 7. Phenobarbital increased 3A2 mRNA to 60-120% values of freshly isolated cells, and mRNA for 3A1–like CYPs were increased 20–fold. In both control and phenobarbital–treated hepatocytes, rat recombinant IFN–γ (33 U/mL) reduced mRNA for 3A2 and 3A1–like CYPs, as well as 3A protein and testosterone 6 β–hydroxylase activity. Interferon had no effect on CYP2C11 at mRNA or protein levels in untreated cells, although a reduction in 2C11 protein was evident in phenobarbital–treated cultures. It is concluded that interferon directly alters expression of constitutive and inducible CYP3A genes in well–differentiated male rat hepatocytes in culture, but has no effect on constitutive expression of CYP2C11.
Oxidative Metabolism in A Rat Hepatoma (N13) and Isolated Rat Hepatocytes: A Flow Cytometric Comparative Study
Authors: Juan G , Gil-Benso R , O'Connor J E , Callaghan R C
Published: November 2004

Recently, we have developed a new and fast kinetic method for assessing mitochondrial membrane potential by flow cytometry, based on the quantitation of the initial rate of rhodamine 123 (Rh123) uptake by living cells. This test has proved suitable to detect metabolic and toxic effects on mitochondria. To characterize energy metabolism in a rat hepatoma cell line (N13), we applied this method to assess several metabolic pathways that eventually generate mitochondrial membrane potential. Using this approach, we found that N13 hepatoma cells retain an oxidative capacity comparable with that observed in isolated hepatocytes under the same conditions. These results show that this cell line may represent an adequate biological model to perform metabolic and toxicological studies

in vitro

.
Functional Differences Between Hepatocytes and Biliary Epithelial Cells in Handling Polymeric Immunoglobulin A2 in Humans, Rats, and Guinea Pigs
Authors: Sakisaka S , Gondo K , Yoshitake M , Harada M , Sata M , Kobayashi K , Tanikawa K
Published: November 2004

Immunoglobulin A (IgA) in bile plays an important role in preventing the biliary tract from infection. In the present study, to clarify the functional differences between hepatocytes and biliary epithelial cells (BEC) in handling polymeric IgA2 (pIgA2), the major and important IgA in bile, we have determined in different species the binding characteristics of 125I–labeled pIgA2 to tissue sections of human, rat, and guinea pig livers. We have also examined the binding and transport features of 125I–labeled and gold–labeled pIgA2 in cultured hepatocytes and cultured BEC of rat and guinea pig. Asialofetuin, an asialoglycoprotein, or an antisecretory component antibody was used for determining the binding characteristics of pIgA2 to the cells. Grains of 125I–pIgA2 were morphometrically analyzed. In tissue sections, 125I–pIgA2 was predominantly bound to rat hepatocytes as well as to human and guinea pig BEC. The binding of 125I–pIgA2 to the cells was significantly inhibited by pretreatment with an antisecretory component (SC) antibody (

P

< .001), but not by preloaded asialofetuin. In cultured cells, labeled pIgA2 was observed binding predominantly to rat hepatocytes and guinea pig BEC as compared with rat BEC and guinea pig hepatocytes (both

P

< .001), respectively, and gold particles of gold–labeled pIgA2 were localized in the tubulovesicular structures and biliary luminal spaces of those cells. These results suggested that pIgA2 was bound selectively to hepatocytes in the rat liver, and to BEC in the human and guinea pig livers, through the SC but not through an asialoglycoprotein receptor, and was transported transcellularly and secreted into bile.
Development of the Ornithine Cycle in Rat Liver: Zonation of A Metabolic Pathway
Authors: Dingemanse M A , De Jonge W J , De Boer P A J , Mori M , Lamers W H , Moorman A F
Published: November 2004

Ammonia–fixation in mammalian livers is, via the ornithine cycle and glutamine synthetase, strictly compartmentalized, occurring in a wide upstream periportal compartment and in the very last downstream pericentral hepatocytes, respectively. This conclusion is based on the well–known distribution patterns of carbamoyl phosphate synthetase I (CPS) and glutamine synthetase in the developing and adult liver. To determine to what extent the expression patterns of the ornithine cycle enzymes are coordinated with that of CPS, we investigated the patterns of expression of the individual messenger RNAs (mRNAs) of the ornithine cycle pathway in developing and adult rat liver. Our results show that, within the liver, all mRNAs of the ornithine cycle pathway are zonated. The patterns of expression of the different mRNAs match almost perfectly, with variations only in the steepness of the gradients of the mRNAs, suggesting that, in the rat, common regulatory factors are involved in the establishment of the zonation pattern.