bluuebunny/crossref_metadata_2025 huggingface dataset:
Biodistribution, Stability, and Antiviral Efficacy of Liposome–Entrapped Phosphorothioate Antisense Oligodeoxynucleotides in Ducks for the Treatment of Chronic Duck Hepatitis B Virus Infection
Authors: Soni Paresh N. , Brown David , Saffie Roggieh , Savage Kay , Moore Duncan , Gregoriadis Gregory , Dusheiko Geoffrey M.
Published: July 2004

This study investigated the feasibility of using liposomes to increase the hepatic delivery and antiviral efficacy of phosphorothioate antisense oligodeoxynucleotides (PS–ODN) for thein vivotreatment of hepatitis B virus (HBV) infection. Ducks infected with duck hepatitis B virus (DHBV) were used as the model. We studied the stability of an antisense PS–ODN in duck plasma, its integrity during the process of liposome entrapment, itsin vivobiodistribution, plasma clearance, and excretion. In addition, the intrahepatic distribution of a labeled free and liposome–entrapped ODN was also investigated. The results of our studies show that: 1) phosphorothioate ODN remain stable during the process of liposome entrapment; 2) are stable in duck plasma for many hours; 3) are rapidly cleared from the plasma when injected intravenously; 4) intravenous injection of antisense ODNs entrapped within liposomes enhances delivery of the ODN to the liver; and 5) inhibit DHBV replication. Serum DHBV DNA levels fell rapidly, with a corresponding decrease in intrahepatic viral replicative intermediates at the end of the 5–day study period. Although inhibition of viral replication and a fall in the target protein was observed, a marked inhibition of viral replication was also observed with high doses of a random–sequence ODN. Thus, it is not certain that inhibition of viral replication was entirely through an antisense mechanism. Therefore, liposomes may be effective vehicles to improve the delivery of antisense oligonucleotides to the liver for the therapy of hepatotropic viruses.
Blunted Thrombopoietin Response to Interferon Alfa-Induced Thrombocytopenia During Treatment for Hepatitis C
Authors: Peck-Radosavljevic Markus , Wichlas Martina , Pidlich Johann , Sims Paul , Meng Gloria , Zacherl Johannes , Garg Shaily , Datz Christian , Gangl Alfred , Ferenci Peter
Published: July 2004

Thrombocytopenia is common in advanced–stage liver disease and is partly caused by inadequate thrombopoietin (TPO) production in the failing liver. Treatment of chronic hepatitis C with interferon alfa (IFN–α) often induces thrombocytopenia, sometimes even leading to discontinuation of treatment. TPO regulation in response to IFN–α-induced thrombocytopenia was studied in patients with chronic hepatitis C with and without cirrhosis (Child A). An in vitro culture system with HepG2 cells was used to demonstrate any direct effects of IFN–α on TPO mRNA expression, TPO synthesis, or TPO secretion from liver cells. Thrombocyte count was lower (U test: P < .05) in patients with hepatitis C cirrhosis compared with patients with chronic hepatitis C without cirrhosis before IFN therapy, and decreased in both patient groups (Wilcoxon matched–pairs test: P < .05) on IFN therapy, the median decrease in both groups being comparable (noncirrhotic patients, 35%; cirrhotic patients, 32%; U test: P = .57). TPO levels rose in noncirrhotic patients (Wilcoxon matched–pairs test: P < .05), but not in patients with cirrhosis (noncirrhotic patients' median increase: 43% vs. cirrhotic patients' median decrease: 5%; U test: P < .001). Even in patients without cirrhosis, the increase in TPO levels was relatively small for the decrease in platelet count. No effect of IFN–α could be demonstrated on TPO mRNA expression in vitro, but TPO secretion from liver cells was significantly reduced. Lower platelet counts but similar TPO levels in patients with chronic hepatitis C and cirrhosis compared with noncirrhotic patients and a moderate increase in TPO levels in noncirrhotic patients with a missing increase in cirrhotic patients during IFN–α-induced thrombocytopenia provide further evidence for an impairment of TPO production in patients with cirrhosis and during IFN therapy. Recombinant human TPO could be of value in patients developing severe thrombocytopenia under IFN–α therapy.
Quantitative Studies of Testicular Atrophy Following Portacaval Shunt in Rats
Authors: Zaitoun Abd M. , Apelqvist Gustav , Wikell Cecilia , Al-mardini Hanan , Bengtsson Finn , Record Christopher O.
Published: July 2004

To evaluate the differential effects of portacaval shunting (PCS) on the morphological changes that occur in humans with portal–systemic encephalopathy, male rats underwent either PCS (13) or sham operations (10). Normal adult rats (6) were used as controls. All animals were killed 5 to 7 weeks after the surgery. The wet weight of the testes was obtained. Hematoxylin–eosin (HE)-stained sections at 5–μm thickness were used for stereological analysis using an image analysis system. Apoptosis was assessed quantitatively in HE and in in situ end–labeling (ISEL)-stained slides, while mitotic activity and mast cell numbers were assessed in 20 high–power fields. There was a significant reduction in the testicular mass (664 mg) in PCS rats in comparison with sham (2,199 mg) and control (1,937 mg) rats (P < .00001). The thickness of germinal epithelium was significantly reduced in PCS rats (64 μm) compared with sham (126 μm) and control groups (108 μm). The number of tubules per square millimeter and the mean curvature were significantly increased in PCS rats (P < .00001). There was a 112–fold increase in apoptosis in PCS rats (112) in comparison with the control and sham–operation groups (1.2 and 0.7, respectively). Mitosis was significantly reduced in the PCS group (P = .0089), but mast cells were unchanged. The results suggest that PCS in the absence of liver dysfunction produces testicular atrophy by reduction in mitosis, maturation arrest, and increased apoptosis of the germinal epithelium. PCS may therefore be responsible for gonadal atrophy that occurs with advanced liver disease in humans.
Enhanced Release of Nitric Oxide in Response to Changes in Flow and Shear Stress in the Superior Mesenteric Arteries of Portal Hypertensive Rats
Authors: Hori Naoki , Wiest Reiner , Groszmann Roberto J.
Published: July 2004

Increased nitric oxide (NO) release has been implicated in the pathogenesis of the hyperdynamic circulation in portal hypertension. NOS 3 (eNOS) causes NO release from the endothelium in response to physical stimuli, such as increased blood flow and shear stress. We evaluated the functional activity of the endothelium in the superior mesenteric arterial bed of portal hypertensive rats through direct measurement of NO metabolites (NOx) during changes in flow and shear stress. The in vitro perfusion system (McGregor) was used in sham and portal vein-ligated (PVL) rats. Shear stress was applied gradually to superior mesenteric arterial beds by increasing the perfusion rate. Flow studies were performed serially before and after incubation with either Krebs solution alone or with the NO–inhibitor, NG
–monomethyl–l–arginine (l–NMMA). NOx concentrations in the perfusate were measured using chemiluminescence. The slope of NOx release versus flow–induced shear stress was calculated. Before l–NMMA administration, NOx concentrations and release of NO in PVL rats were significantly elevated in comparison with sham rats at flow rates of 32, 40, and 48 mL/min. The slope of NOx production versus shear stress index was significantly higher in PVL than in sham rats. After l–NMMA incubation, the decrease in slope was significantly larger in PVL rats. This study provides direct evidences of an increased NO synthesis by the superior mesenteric arterial vascular endothelium of PVL animals in response to shear stress. The increased NO output in response to shear stress suggests an adaptative mechanism developed by the vascular endothelial cells in response to a chronic increase in flow–mediated shear stress.
Kai1, A New Metastasis Suppressor Gene, Is Reduced in Metastatic Hepatocellular Carcinoma
Authors: Guo Xiao-Zhong , Friess Helmut , Di Mola Fabio F. , Heinicke Jean-Marc , Abou-Shady Mohamed , Graber Hans U. , Baer Hans U. , Zimmermann Arthur , Korc Murray , Büchler Markus W.
Published: July 2004

Down–regulation of KAI1 expression has been shown to be associated with formation of metastases or disease progression in prostate and pancreatic cancer. In the present study we analyzed the expression pattern of KAI1 in metastatic and nonmetastatic hepatocellular carcinomas (HCCs) in comparison with normal livers to evaluate whether alteration of KAI1 also facilitates the metastatic ability in this malignancy. Thirty–nine primary HCCs and 10 normal liver tissue samples were studied for KAI1 messenger RNA (mRNA) expression with use of Northern blot analysis and in situ hybridization. By Northern blot analysis, moderate to strong KAI1 mRNA expression was present in normal liver samples. In contrast, KAI1 mRNA expression in tissue samples of primary HCCs was markedly decreased compared with normal controls. The normal/tumor ratio of KAI1 mRNA expression was 2.6:1 (P< .01). Primary HCCs that gave rise to metastasis showed significantly lower KAI1 mRNA levels than nonmetastasized HCCs (P < .05). As seen by in situ hybridization, moderate to strong cytoplasmic KAI1 mRNA staining was present in almost all normal hepatocytes. Bile ducts, blood vessels, and connective tissue showed no or only faint KAI1 mRNA expression in the normal liver samples. In nonmetastatic HCCs, the cancer cells exhibited in situ hybridization signals that were similar to the normal controls. In contrast, most of the primary HCC cells in samples with metastases showed only faint or moderate KAI1 mRNA expression predominantly in the perinuclear regions. When KAI1 mRNA expression of primary hepatocellular cancer cells was compared with metastasized cancer cells in lymph nodes, with intrahepatic satellite metastasis, or with peritoneal metastasis in the same patients, significantly lower (P < .01) KAI1 mRNA levels were present in the metastasized HCC cells. Reduced KAI1 mRNA in HCC cells seems to influence their metastatic ability and thereby enhances the malignant potential of HCC
Growth–Related Expression of the Ectonucleotide Pyrophosphatase Pc–1 in Rat Liver
Authors: Stefan Cristiana , Stalmans Willy , Bollen Mathieu
Published: July 2004

Plasma cell differentiation antigen–1 (PC–1) is a 5′–ectonucleotide pyrophosphatase that has been implicated in various processes including insulin– and nucleotide–mediated signaling and cell growth. We show here that the expression of both PC–1 mRNA and protein in rat liver and in hepatoma cells is strictly growth–related. Thus, the level of PC–1 in FAO hepatoma cells increased with the cell density. PC–1 was not expressed in the neonatal rat liver, but gradually appeared in the first weeks of age, to reach adult levels around the weaning period. Furthermore, PC–1 protein and mRNA largely disappeared from the liver within 24 hours following a hepatectomy of 70%, but re–appeared in the later phases (3–15 days) of the ensuing regeneration period. An equally rapid loss of PC–1 protein and mRNA could also be provoked in normal livers by the administration of the translational inhibitor, cycloheximide, but the transcriptional inhibitors, actinomycin D and α–amanitin, did not show these effects. Nuclear run–on assays revealed that the loss of PC–1 mRNA after hepatectomy or after the administration of cycloheximide was not caused by a decreased transcription of the PC–1 gene, suggesting that the level of PC–1 is controlled by an mRNA–stabilizing protein that is lost after hepatectomy and has a high turnover.
Differential Expression and Regulation of Nucleoside Transport Systems in Rat Liver Parenchymal and Hepatoma Cells
Authors: del Santo Belén , Valdés Raquel , Mata João , Felipe Antonio , Casado Javier F. , Pastor-Anglada Marçal
Published: July 2004

Primary cultures of rat–liver parenchymal cells show carrier–mediated nucleoside uptake by a mechanism that mainly involves concentrative, Na+–dependent transport activity. In contrast, the hepatoma cell line FAO shows high nucleoside transport activity, although it is mostly accounted for by Na+–independent transport processes. This is associated with a low amount of sodium purine nucleoside transporter (SPNT) mRNA. SPNT encodes a purine–preferring transporter expressed in liver parenchymal cells. To analyze whether SPNT expression is modulated during cell proliferation, SPNT mRNA levels were determined in the early phase of liver growth after partial hepatectomy and in synchronized FAO cells that had been induced to proliferate. SPNT mRNA amounts increased as early as 2 hours after partial hepatectomy. FAO cells induced to proliferate after serum refeeding show an increase in SPNT mRNA levels, which is followed by an increase in Na+–dependent nucleoside uptake and occurs before the peak of 3H–thymidine incorporation into DNA. FAO cells also express significant equilibrative nucleoside transport activity, which may be accounted for by the expression of the nitrobenzylthioinosine (NBTI)–sensitive and –insensitive isoforms, rat equilibrative nucleoside transporter 1 (rENT1) and rENT2, respectively. Interestingly, rENT2 mRNA levels follow a similar pattern to that described for SPNT when FAO cells are induced to proliferate, whereas rENT1 appears to be constitutively expressed. Liver parenchymal cells show low and negligible mRNA levels for rENT1 and rENT2 transporters, respectively, although most of the equilibrative transport activity found in hepatocytes is NBTI–resistant. It is concluded that: 1) SPNT expression is regulated both in vivo and in vitro in a way that appears to be dependent on cell cycle progression; 2) SPNT expression may be a feature of differentiated hepatocytes; and 3) equilibrative transporters are differentially regulated, rENT2 expression being cell cycle-dependent. This is consistent with its putative role as a growth factor-induced delayed early response gene.
Primary Rat and Mouse Hepatic Stellate Cells Express the Macrophage Inhibitor Cytokine Interleukin–10 During the Course of Activation In Vitro
Authors: Thompson Kerry C. , Trowern Angus , Fowell Andrew , Marathe Mandar , Haycock Catherine , Arthur Michael J. , Sheron Nick
Published: July 2004

Activation of local tissue macrophages (Kupffer cells) and of quiescent hepatic stellate cells (HSCs) to a myofibroblast phenotype are two key events in liver inflammation and fibrosis. It is known that products of activated macrophages may activate stellate cells. We have hypothesized that the products of activated HSCs may also modulate the activity of Kupffer cells. The cytokine interleukin–10 (IL–10), produced by lymphocytes and macrophages, has profound inhibitory actions on macrophages. Normal rat and mouse HSCs that differentiate in vivo and in vitro to activated myofibroblasts were isolated by enzyme perfusion and density centrifugation with or without centrifugal elutriation, confirmed by vitamin A autofluorescence and positive immunostaining for the myofibroblast markers desmin and smooth muscle actin (SMA). Conditioned media and lysates from these cells were found to down–regulate lipopolysaccharide (LPS)–induced tumor necrosis factor–α (TNF–α) secretion by the mouse macrophage line RAW 267.4. In highly purified preparations of rat HSCs, messenger RNA (mRNA) for IL–10 was detected by reverse–transcription polymerase chain reaction (RT–PCR), from the time of isolation to up to 120 days of culture on plastic. Long–term cultures of unstimulated mouse HSCs secreted IL–10 protein as detected by immunoblotting and specific enzyme–linked immunosorbent assay (ELISA). IL–10 protein was undetectable by immunohistochemistry in mouse HSCs for the first 3 days in culture. After this, the percentage of IL–10-positive cells increased to 45% at day 7 and 100% by day 14, and expression of IL–10 continued in long–term cultures of up to 120 days. The expression of IL–10 by the stromal cells that govern the fibrotic process in the liver may have important implications for the regulation of inflammation and fibrosis in the liver.
The Significance of Microabscesses in Liver Transplant Biopsies: A Clinicopathological Study
Authors: Lamps Laura W. , Pinson Wright C. , Raiford David S. , Shyr Yu , Scott Margie A. , Washington Mary Kay
Published: July 2004

Parenchymal microabscesses (MA) in liver transplant biopsies are frequently associated with cytomegalovirus (CMV) infection. However, other potential causes of MA have not been fully investigated. We studied additional etiologies for MA via histological evaluation and clinicopathological correlation. Three hundred seventy–two liver transplant biopsies from 97 patients (from 1991 to 1997) were reviewed and stained immunohistochemically for CMV. Numerous histological features were evaluated including size and number of MA, lobular and portal inflammation, and cholestasis. Medical records were reviewed for radiographic, laboratory, and other clinical data from the time of biopsy. The χ2 or Fisher's Exact test and ANOVA with adjusted multiple comparisons were used to determine statistical significance. Sixty–two of 372 biopsies (17%) from 43 patients contained MA. Biopsies were obtained between 4 days and 2.3 years posttransplant (median, 14 days). Nineteen percent of biopsies had CMV infection at the time of biopsy; 27% were associated with other bacterial, viral, or fungal infections; 10% had graft ischemia; 15% had biliary obstruction/cholangitis; 3% had a combination of ischemia and sepsis; and no explanation was found in 26% of biopsies. Numerous MA within a biopsy (>9) correlated with CMV infection (P < .005); no other histological features, including size of MA, correlated with the etiology of MA. Overall, 43 of 97 (44%) liver transplantation patients at our institution had biopsies demonstrating MA at some point in their posttransplantation course. CMV infection appears responsible for only a minority of cases. MA, although nonspecific, are an important histological finding in numerous conditions that may have a significant impact on both graft survival and overall patient morbidity.
Expression of Adhesion Molecules on Circulating Polymorphonuclear Leukocytes During Orthotopic Liver Transplantation
Authors: Thiel Manfred , Imendörffer Swantje , Chouker Alexander , Groh Jochen , Briegel Josef , Anthuber Matthias , Krämling Hans J. , Arfors Karl E. , Peter Klaus , Messmer Konrad
Published: July 2004

After ischemia–reperfusion, polymorphonuclear leukocytes (PMNLs) become activated by inflammatory mediators, adhere to the vascular endothelium via the interaction of specific adhesion molecules, and cause tissue injury by the release of cytotoxic oxygen radicals and enzymes. Results obtained in animal experiments suggest a key role for PMNLs in ischemia–reperfusion injury of transplanted livers; therefore, we studied the expression of adhesion molecules on circulating PMNLs (β2 –integrins [CD18] and l–selectin [CD62L]) in 20 patients undergoing orthotopic liver transplantation (study group). To determine the effects of surgical trauma to the liver in the absence of ischemia and reperfusion, the expression of PMNL adhesion molecules was measured in 10 patients scheduled for elective partial liver resection without hepatic vascular exclusion (control group). Patients were classified as responders or nonresponders based on changes in the expression of adhesion molecules elicited by reperfusion. In the control group, all patients remained nonresponders, showing that surgical trauma of the liver alone does not cause activation of circulating PMNLs. In contrast, 8 of 20 patients in the study group were classified as responders. In responders, postoperative serum liver enzyme activities were significantly higher than in nonresponders, indicating that activation of PMNL was associated with damage to hepatocellular integrity. Because expression of adhesion molecules was already changed during surgery, monitoring of the expression of β2 –integrins and l–selectin on circulating PMNLs during orthotopic liver transplantation might be useful in prediction of early graft dysfunction.
Augmented Interleukin–1β-Induced Depression of Locomotor Activity in Cholestatic Rats
Authors: Swain Mark G. , Beck Paul , Rioux Kevin , Le Tai
Published: July 2004

“Sickness behaviors” such as lethargy, fatigue, and malaise occur commonly in patients with cholestatic liver diseases and contribute significantly to the morbidity associated with these diseases. However, the cause of these symptoms is unknown. Interleukin–1β (IL–1β) released within the brain has been implicated in the genesis of a number of “sickness behaviors,” including malaise and lethargy. Therefore, we investigated whether experimental cholestatic liver disease caused by bile duct resection (BDR) in rats is associated with enhanced central sensitivity to IL–1β-induced “sickness behaviors.” The central infusion of IL–1β at a dose that produced an insignificant decrease in locomotor activity in control rats produced a striking reduction in locomotor activity in cholestatic rats. The anorectic response to central IL–1β infusion was similar in cholestatic and noncholestatic animals and did not parallel our locomotor activity findings. Therefore, cholestatic liver injury is characterized by augmented central responsiveness to IL–1β with respect to a decrease in locomotor activity. These findings may explain, at least in part, the high incidence of symptoms such as fatigue, malaise, and lethargy that occur in cholestatic patients and may open novel future avenues for their treatment.
Activation of Rat Hepatic Stellate Cells in Culture Is Associated With Increased Sensitivity to Endothelin 1
Authors: Reinehr Roland M. , Kubitz Ralf , Peters-Regehr Thorsten , Bode Johannes G. , Häussinger Dieter
Published: July 2004

The effect of endothelin (ET) 1 on intracellular Ca2+transients in cultured rat hepatic stellate cells (HSCs) during transformation was studied by use of single–cell fluorescence. Regardless of the duration of HSC culture, ET–1 caused a BQ–123-sensitive but IRL–1038-insensitive elevation of [Ca2+]i, indicating the involvement of ETAbut not ETBreceptors. HSCs in early culture (“quiescent HSCs”) were mildly responsive to ET–1: the ET–1 concentration required to obtain a [Ca2+]itransient in 50% of the cells (RC50) was 7 nmol/L, and all cells responded to ET–1 concentrations above 40 nmol/L. With culture time, α-smooth muscle actin (α–SMA) expression increased, as did the ET–1 sensitivity of cells, resulting in a shift of the RC50value from 7 nmol/L to 13 pmol/L within 8 days. Independent of the duration of culture, ET–1 sensitivity was higher in α–SMA-expressing cells. On the other hand, sensitivity of HSCs to produce a [Ca2+]iresponse to extracellular uridin 5′–triphosphate (UTP) or phenylephrine did not change during the activation process. There was no difference between quiescent and activated HSCs with respect to the sharing of intracellular Ca2+stores, which could be mobilized by ET–1, UTP, and phenylephrine, respectively. The data suggest three conclusions. (1) A marked increase in ET–1 sensitivity of HSCs during the activation process suggests a potentiation of autocrine/paracrine stimulation. (2) HSCs are susceptible to α–adrenergic and purinergic stimulation, but sensitivity to phenylephrine and UTP is not affected during the transformation process. (3) The ET–1-mobilizable Ca2+store is contained in and is smaller than the Ca2+pool, which is mobilized by phenylephrine or UTP.
Xanthine Oxidase Inhibition Attenuates Kupffer Cell Production of Neutrophil Chemoattractant Following Ischemia–Reperfusion in Rat Liver
Authors: Matsumura Fujio , Yamaguchi Yasuo , Goto Mataro , Ichiguchi Osamu , Akizuki Eiji , Matsuda Teishi , Okabe Kazutoshi , Liang Jian , Ohshiro Hajime , Iwamoto Takeshi , Yamada Shinwa , Mori Katsutaka , Ogawa Michio
Published: July 2004

We investigated the effects of the xanthine oxidase inhibitor, BOF–4272, on the production of cytokine–induced neutrophil chemoattractant (CINC) following reperfusion injury in rat liver. Ischemia was induced for 30 minutes by portal vein occlusion. Animals were pretreated with intravenous injection of BOF–4272 (1 mg/kg) or heparin (50 U/kg) 5 minutes before vascular clamp. Both BOF–4272 and heparin limited increases in the chemoattractant compared with nonpretreated rats. Pretreatment with BOF–4272 plus heparin resulted in an additive effect. Most cells immunostained for chemoattractant were macrophages in sinusoids.In vitrochemoattractant production by Kupffer cells isolated from animals pretreated with heparin or BOF–4272 was significantly lower than by Kupffer cells from nonpretreated animals. Expression of transcripts in liver for chemoattractant peaked 3 hours after reperfusion in nonpretreated animals, while pretreatment with heparin or BOF–4272 significantly decreased chemoattractant mRNA levels.In vitrochemoattractant transcription and production could be induced in naive Kupffer cells by hypoxanthine and xanthine oxidase, but BOF–4272 prevented these increases. We conclude that Kupffer cells release chemoattractant in response to oxygen radicals reducible by xanthine oxidase inhibition.
Sodium Taurocholate Cotransporting Polypeptide Is A Serine, Threonine Phosphoprotein and Is Dephosphorylated by Cyclic Adenosine Monophosphate
Authors: Mukhopadhyay Sunil , Ananthanarayanan M. , Stieger Bruno , Meier Peter J. , Suchy Frederick J. , Anwer Sawkat M.
Published: July 2004

Na+/taurocholate (Na+/TC) cotransport in hepatocytes is mediated primarily by Na+/TC cotransporting polypeptide (Ntcp), and cyclic adenosine monophosphate (cAMP) stimulates Na+/TC cotransport by inducing translocation of Ntcp to the plasma membrane. The aim of the present study was to determine if Ntcp is a phosphoprotein and if cAMP alters Ntcp phosphorylation. Freshly prepared hepatocytes from rat livers were incubated with carrier–free 32PO4 for 2 hours, followed by incubation with 10 μmol/L 8–chlorophenylthio adenosin 3′:5′–cyclic monophosphate (CPT–cAMP) for 15 minutes. Subcellular fractions isolated from 32P–labeled hepatocytes were subjected to immunoprecipitation using Ntcp antibody, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and autoradiography to determine if Ntcp is phosphorylated. Ntcp immunoprecipitated from plasma membranes isolated from nonlabeled hepatocytes was subjected to immunoblot analysis using anti–phosphoserine, anti–phosphothreonine, or anti–phosphotyrosine antibody to determine whether Ntcp is a serine, threonine, or tyrosine phosphoprotein. Hepatocytes were loaded with bis–(2–amino–5–methylphenoxy)–ethane–N,N,N′,N′–tetraacetic acid (MAPTA), a Ca2+ buffering agent, and the effect of CPT–cAMP on TC uptake, cytosolic [Ca2+], and ntcp phosphorylation and translocation was determined. In addition, the effect of cAMP on protein phosphatases 1 and 2A (PP1/2A) was determined in homogenates and plasma membranes obtained from CPT–cAMP-treated hepatocytes. Phosphorylation study showed that phosphorylated Ntcp is detectable in plasma membranes, and cAMP treatment resulted in dephosphorylation of Ntcp. Immunoblot analysis with phosphoamino antibodies revealed that Ntcp is a serine/threonine, and not a tyrosine, phosphoprotein, and cAMP inhibited both serine and threonine phosphorylation. In MAPTA–loaded hepatocytes, CPT–cAMP failed to stimulate TC uptake, failed to increase cytosolic [Ca2+], and failed to induce translocation and dephosphorylation of Ntcp. cAMP did not alter the activity of PP1/2A in either homogenates or in plasma membranes. Taken together, these results suggest that Ntcp is a serine/threonine phosphoprotein and is dephosphorylated by cAMP treatment. Activation of PP1/2A is not involved in cAMP–mediated dephosphorylation of Ntcp. Both translocation and dephosphorylation of Ntcp may be involved in the regulation of hepatic Na+/TC cotransport.
Up–Regulation of the Multidrug Resistance Genes, Mrp1 And Mdr1b , and Down–Regulation of the Organic Anion Transporter, Mrp2, and the Bile Salt Transporter, Spgp, in Endotoxemic Rat Liver
Authors: Vos Thera A. , Hooiveld Guido J. , Koning Hans , Childs Sarah , Meijer Dirk K. , Moshage Han , Jansen Peter L. , Müller Michael
Published: July 2004

Endotoxin–induced cholestasis is mainly caused by an impaired canalicular secretion. Mrp2, the canalicular multispecific organic anion transporter, is strongly down–regulated in this situation, and canalicular bile salt secretion is also reduced. We hypothesized that other adenosine triphosphate-binding cassette (ABC) transporters may compensate for the decreased transport activity to protect the cell from cytokine–induced oxidative damage. Therefore, we examined the expression of ABC–transport proteins in membrane fractions of whole liver and of isolated hepatocytes of endotoxin–treated rats and performed reverse–transcriptase polymerase chain reaction (RT–PCR) on mRNA isolated from these livers. In addition, the localization of these transporters was examined using confocal scanning laser microscopy. By 6 hours after endotoxin administration, we found a clear increase of mrp1 mRNA and protein, whereas mrp2 mRNA and protein were decreased. This was confirmed in isolated hepatocytes. In addition,mdr1bmRNA was strongly increased, whereasmdr1aandmdr2mRNA did not change significantly. Both the mRNA and protein levels of the sister of P–glycoprotein (spgp), the recently cloned bile salt transporter, decreased. After endotoxin treatment, the normally sharply delineated canalicular staining of mrp2 and spgp had changed to a fuzzy pattern, suggesting localization in a subapical compartment. We conclude that endotoxin–induced cholestasis is caused by decreased mrp2 and spgp levels, as well as an abnormal localization of these proteins. The simultaneous up–regulation of mrp1 and mdr1b may confer resistance to hepatocytes against cytokine–induced metabolic stress.
Hematopoietic Support and Cytokine Expression of Murine–Stable Hepatocyte Cell Lines (Mmh)
Authors: Aiuti Alessandro , Cicchini Carla , Bernardini Silvia , Fedele Giorgio , Amicone Laura , Fantoni Antonio , Tripodi Marco
Published: July 2004

It was recently reported that transgenic expression in the liver of truncated human Met renders hepatocytes constitutively resistant to apoptosis and reproducibly permits their immortalization. The derived stable cell lines (MMH from Met murine hepatocyte) are highly differentiated and nontransformed. In this report, the capacity of MMHs to support in vitro hematopoiesis is characterized. By reverse–transcription polymerase chain reaction, the expression by MMHs of cytokines involved in the survival and self–renewal of early progenitor cells (stem cell factor and FLT3 ligand) as well as those acting at different stages of progenitor differentiation (interleukin [IL] 1β, IL–3, leukemia inhibitory factor, IL–6, granulocyte–macrophage colony-stimulating factor, granulocyte colony-stimulating factor, macrophage colony-stimulating factor, and thrombopoietin) was shown. A ribonuclease protection assay further substantiated the presence of at least six cytokine transcripts in MMH lines. Cocultures between MMH layers and progenitor–enriched fetal liver hematopoietic cells resulted in a 40–fold to 80–fold expansion of total hematopoietic cells and in a 2.5–fold expansion of clonogenic progenitors after 1 to 2 weeks. Hematopoiesis was maintained for up to 6 weeks with formation of typical cobblestone cell areas and continuous differentiation of precursor into cells at various degrees of maturation. At 5 weeks of coculture, clonogenic progenitors were maintained at 20% of the input level in coculture with embryonic–derived hepatocytes, showing the ability of hepatocyte feeder layer to support survival and possibly self–renewal of clonogenic progenitors. Therefore, the data emphasize a direct role of the hepatocyte in sustaining hematopoietic cell proliferation and differentiation.
Better Efficacy of A 12–Month Interferon Alfa–2B Retreatment in Patients With Chronic Hepatitis C Relapsing After A 6–Month Treatment: A Multicenter, Controlled, Randomized Trial
Authors: Payen Jean-Louis , Izopet Jacques , Galindo-Migeot Virginie , Lauwers-Cances Valérie , Zarski Jean-Pierre , Seigneurin Jean-Marie , Dussaix Elizabeth , Voigt Jean-Jacques , Selves Janick , Barange Karl , Puel Jacqueline , Pascal Jean-Pierre
Published: July 2004

We studied the efficacy of three interferon alfa–2b (IFN–α2b) regimens for the retreatment of patients with chronic hepatitis C (CHC) with prior complete response followed by relapse. Consecutive patients with CHC who had a complete biochemical response but relapse after a first course of 6 months of IFN with 3 million units (MU) given subcutaneously three times per week were enrolled in the study. Six to 24 months after the end of the first treatment, the patients were randomly assigned to receive IFN with either the same regimen (group 1), a regimen of 12 months with 3 MU (group 2), or a regimen of 6 months with 10 MU (group 3). Sustained biochemical response was defined as normal serum alanine transaminase (ALT) values during the follow–up and sustained virological response as a clearance of hepatitis C virus (HCV) RNA from the serum at the end of follow–up (6 months' posttreatment). Histological improvement was defined as a decrease of 1 point in Metavir score between the first liver biopsy and a biopsy performed at 6 months' postretreatment. Two hundred forty–seven patients were randomized: 75 to group 1, 91 to group 2, and 81 to group 3. In an intent–to–treat analysis, 12%, 36.3%, and 18.5% of patients had a sustained biochemical response after retreatment in groups 1, 2, and 3, respectively (P < .001); 13.8%, 32.4%, and 17.2% of patients had a sustained virological response after retreatment in groups 1, 2, and 3, respectively (P < .05). A low viral load and patients in group 2 were independently associated with a sustained biochemical response. A low Knodell score index before treatment, patients with a high level of ALT before retreatment, genotype 3, low viral load, and patients in group 2 were independently associated with sustained virological response. Younger age, a high level of ALT, a low level of γ–glutamyl transferase before retreatment, low viral load, and patients in group 2 were independently associated with sustained biochemical and virological response. Among the 80 patients with repeated liver biopsies, 47.6% had improved histological activity scores; this improvement was associated with a sustained biochemical and virological response. In patients with CHC initially treated with 3 MU of IFN given subcutaneously three times per week over a 6–month period, and who subsequently developed a relapse after a biochemical response, retreatment with a regimen of 3 MU of IFN given three times per week for 12 months produced better biochemical and virological sustained response rates than regimens involving a higher dose or a shorter duration of retreatment. The biochemical and virological sustained response was associated with histological improvement.
Chronic Hepatitis C in Children: A Clinical and Immunohistochemical Comparative Study With Adult Patients
Authors: García-Monzón Carmelo , Jara Paloma , Fernández-Bermejo Miguel , Hierro Loreto , Frauca Esteban , Camarena Carmen , Díaz Carmen , De la Vega Angela , Larrauri Javier , García-Iglesias Consuelo , Borque María J. , Sanz Paloma , García-Buey Luisa , Moreno-Monteagudo José A. , Moreno-Otero Ricardo
Published: July 2004

Limited information is available regarding the characteristics of the hepatitis C virus (HCV) infection in children. We compared the epidemiological background along with the virological and histological features as well as the intrahepatic immunologic phenotype of both children and adults with chronic hepatitis C (CHC). Serum samples of 24 pediatric and 32 adult patients were drawn for alanine transaminase (ALT) levels, HCV–typing, and viral load. The histological diagnosis and a semiquantitative immunohistochemical assessment were performed in all patients. The majority of children (62%) had been transfused and the mean duration of viral infection in these cases was 11 ± 4 years, being similar in adults (11 ± 9 years, not significant). Although genotype distribution was similar, viral load was lower in children than in adults. The mildest histological forms of chronic hepatitis along with a weak intrahepatic immunological phenotype were significantly more frequent among children than adult patients. In conclusion, in children with CHC, perinatal blood transfusion was the most frequent source of viral infection and the liver disease was characterized by both low ALT level and viral load, as well as the mildest histological and immunohistochemical forms of chronic hepatitis.
Apoptosis in Human Hepatoma Cell Lines by Chemotherapeutic Drugs Via Fas–Dependent and Fas–Independent Pathways
Authors: Jiang Shunai , Song Moon Jung , Shin Eui-Cheol , Lee Mi-Ock , Kim Se Jong , Park Jeon Han
Published: July 2004

Many chemotherapeutic drugs have been found to exert their mode of action via induction of apoptosis in cancer cells. The mechanisms involved in this process are not clear. Recent studies have shown that the Fas/Fas ligand (FasL) system is a key factor controlling apoptotic cell death. In the present study, the involvement of Fas in chemotherapeutic drug–induced apoptosis in hepatoma cell lines was investigated. Five different human hepatoma cell lines, Hep G2, Hep G2.2.15, Hep 3B, SK–Hep–1, and PLC/PRF/5, were used. It was found that they expressed different levels of Fas. However, all five cell lines were susceptible to apoptosis when treated with chemotherapeutic drugs such as 5–fluorouracil (5–FU) or cisplatin. In Hep G2 that constitutively expressed Fas, 5–FU or cisplatin treatment caused an increase in the expression of Fas before the formation of oligonucleosomal DNA fragments, a typical feature of apoptosis. However, in Hep 3B, where Fas is undetectable, apoptosis could also be induced by 5–FU or cisplatin without induction of Fas. The agonistic anti–Fas antibody (CH–11) was capable of inducing apoptosis by itself and promoted drug–induced apoptosis in Hep G2 but not in Hep 3B. The antagonistic anti–Fas antibody (ZB4) inhibited drug–induced apoptosis in Hep G2. Our results suggest that apoptosis can be induced in hepatoma cell lines via both Fas–dependent and Fas–independent pathways.
Reconstruction of Hepatic Organoid by Rat Small Hepatocytes and Hepatic Nonparenchymal Cells
Authors: Mitaka Toshihiro , Sato Fumihiko , Mizuguchi Toru , Yokono Takeshi , Mochizuki Yohichi
Published: July 2004

Hepatic cells isolated from an adult rat liver, consisting of small hepatocytes (SHs), mature hepatocytes (MHs), liver epithelial cells (LECs), Kupffer cells, sinusoidal endothelial cells, and stellate cells, were cultured in a medium supplemented with 10% fetal bovine serum, 10 mmol/L nicotinamide, 1 mmol/L ascorbic acid 2–phosphate, 10 ng/mL epidermal growth factor, and 1% dimethyl sulfoxide. The SHs rapidly proliferated and formed a colony. About 10% of cytokeratin 8 (CK8)–positive cells formed SH colonies. All SHs at day 10 immunocytochemically showed positivity for albumin, transferrin, CK8, and CK18, which are markers for hepatocytes. In contrast, α–fetoprotein (AFP)–, CK14–, OC2–, and glutathione S–transferase placental type (GST–P)–positive cells, which are thought to be markers for hepatic immature cells, were rarely observed. At day 20 some cells in the colonies were positive for AFP, CK7, CK19, and GST–P. LECs and stellate cells proliferated and surrounded the colonies. About 2 weeks after plating, piled up cells were often observed on the SH colonies. In those colonies LECs and stellate cells invaded under the colonies. The invasion of the cells and gradual deposits of extracellular matrix (ECM) such as type I collagen, type IV collagen, and laminin induced alteration of the shape of the SHs from relatively flat to cuboidal or rectangular. With the cellular structural changes, the expression of albumin, connexin 32 (Cx32), and tryptophan 2,3–dioxygenase (TO) messenger RNAs increased. In addition, overlapping nonparenchymal cells (NPCs) on the piled up cells induced the formation of duct– or cyst–like structures consisting of MHs. In the present experiment we showed that SHs could differentiate to MHs by interacting with NPCs and ECM. Thus, SHs may be “committed progenitor cells” that can further differentiate into MHs.
The Protective Role of Adenosine in Inducing Nitric Oxide Synthesis in Rat Liver Ischemia Preconditioning Is Mediated by Activation of Adenosine A2 Receptors
Authors: Peralta Carmen , Hotter Georgina , Closa Daniel , Prats Neus , Xaus Carme , Gelpí Emilio , Roselló-Catafau Joan
Published: July 2004

This study aims to determine if the protective role of adenosine in liver ischemic preconditioning is mediated by the activation of adenosine receptors and to ascertain which of these receptors is implicated in the process. Administration of adenosine A1 and A2 receptor antagonists to preconditioned animals indicates that hepatic preconditioning is mediated by the activation of adenosine A2 receptors. Propentofylline (an inhibitor of adenosine transport into cells) in the preconditioned group, subjected to previous administration of an adenosine A2 receptor antagonist, prevented the negative effect of the latter on the protection offered by preconditioning. An increase of NO production was detected just immediately after hepatic preconditioning, and the administration of an adenosine A2 receptor antagonist to the preconditioning group prevented this increase, thus abolishing the protective effect of preconditioning. However, the administration of a NO donor to the preconditioned group subjected to previous administration of the adenosine A2 receptor antagonist was able to maintain the preconditioning effects. In conclusion, these results indicate that, in preconditioning, the protective effect of adenosine could be a result of an increase in extracellular adenosine. This in turn would induce the activation of adenosine A2 receptors, which, by eliciting an increase in NO generation, would protect against the injury associated with hepatic ischemia–reperfusion.
Expression of the C–Kit Proto–Oncogene in Rat Hepatic Allografts During Acute Rejection
Authors: Yamaguchi Yasuo , Okabe Kazutoshi , Matsumura Fujio , Akizuki Eiji , Matsuda Teishi , Ohshiro Hajime , Liang Jian , Ishihara Kojiroh , Mori Katsutaka , Ogawa Michio
Published: July 2004

The role of the c–kit proto–oncogene in organ allograft rejection is not known. We investigated the level of c–kit expression following allogeneic transplantation of ACI rat liver grafts into LEW recipients. We studied c–kit mRNA and protein expression in groups of transplant recipients receiving hepatic isografts, hepatic allografts, or hepatic allografts after donor–specific blood transfusion (DST). Pretransplantation DST significantly prolonged survival of hepatic allografts. Infiltrates expressing c–kit were observed in allografts to untreated rats but not in groups receiving isografts or allografts following DST. Northern analysis also demonstrated abundant c–kit mRNA transcripts in the untreated allograft group in contrast to the isograft and the DST–treated groups. In addition, significantly more transcripts for interleukin–12 (IL–12), which is synergistic with c–kit, were present in untreated than in DST–treated allograft groups. In contrast, transforming growth factor β (TGF–β), which inhibits c–kit synthesis, was expressed abundantly in hepatic allografts to DST–treated rats but not in allografts to untreated animals. Transcripts for IL–10, which inhibits IL–12 production, were significantly more plentiful in hepatic allografts following DST than in those without DST. The results suggest that c–kit proto–oncogene expression in infiltrating cells is associated with rat hepatic allograft rejection.
Effects of Chronic Ethanol Administration on Rat Liver Proteasome Activities: Relationship With Oxidative Stress
Authors: Fataccioli Virginie , Andraud Evelyne , Gentil Monique , French Samuel W. , Rouach Helene
Published: July 2004

We previously reported that ethanol elicits an increased protein oxidation in the liver of rats receiving chronic ethanol by continuous intragastric infusion (Tsukamoto–French method). This accumulation of oxidized proteins could result from a decrease in the cytosolic proteolysis, related specifically to alkaline protease and its major components, the proteasomes. Because several studies suggest that intracellular proteolysis depends on the severity of oxidative stress, we investigated the cytosolic proteolytic activity under two chronic ethanol treatment paradigms associated with varying degrees of oxidative stress. For 4 weeks, male rats received chronic ethanol by continuous intragastric infusion or by oral administration (10% ethanol ad libitum as sole drinking fluid). A significant decrease was evident for alkaline protease activity as well as for sodium dodecyl sulfate (SDS)–activated latent 20S proteasome (chymotrypsine–like [ChT–L] and peptidylglutamyl peptide hydrolase [PGPH] activities) in the liver of rats receiving ethanol by continuous intragastric infusion. Free radical production and related processes appeared to be contributing events in proteolysis inhibition, because phenethyl isothiocyanate (PIC), an inhibitor of cytochrome P4502E1 (CYP2E1), reduced the inhibition of the ethanol–related ChT–L activity. Moreover, the lipid peroxidation level was inversely correlated with ChT–L activity. In contrast, no such changes were observed in ChT–L and PGPH activities or in cellular free radical targets following the oral ad libitum consumption of 10% ethanol. It appears, thus, that only the alcohol treatment paradigm associated with an overt oxidative stress produced a significant inhibition of the proteasome activity. The mechanisms of proteasome inhibition could involve the formation of an endogenous inhibitor such as protein aggregates or aldehyde–derivative peptides. Whatever the mechanism, the inhibition of cytosolic proteolysis and the subsequent accumulation of damaged proteins may be involved in the oxidatively challenged alcoholic livers and play a pathogenic role in experimental alcoholic liver disease.
Monocyte Chemotactic Protein–1 As A Chemoattractant for Human Hepatic Stellate Cells
Authors: Marra Fabio , Romanelli Roberto G. , Giannini Carlo , Failli Paola , Pastacaldi Sabrina , Arrighi Maria Cristina , Pinzani Massimo , Laffi Giacomo , Montalto Paolo , Gentilini Paolo
Published: July 2004

Following liver injury, hepatic stellate cells (HSC) undergo proliferation and migrate into damaged areas in response to chemotactic factors. HSC have been shown to regulate leukocyte trafficking by secreting monocyte chemotactic protein–1 (MCP–1), a chemokine that recruits monocytes and lymphocytes. In this study, we explored whether MCP–1 exerts biological actions on HSC. HSC were isolated from normal human livers, cultured on plastic, and studied in their myofibroblast–like phenotype, and three different cells lines were used. Chemotaxis was measured in modified Boyden chambers. Phosphatidylinositol 3–kinase (PI 3–K) was assayed on phosphotyrosine immunoprecipitates. Exposure of HSC to MCP–1 stimulated migration of HSC in a dose–dependent fashion. Maximal stimulation was obtained with 250 ng/mL MCP–1, which resulted in a 3– to 4–fold stimulation of cell migration. Checkerboard analysis showed that the increase in cell migration was almost completely a result of chemotaxis rather than chemokinesis. In contrast, in quiescent HSC, MCP–1 did not exert any effect on cell migration. In leukocytes, MCP–1 activates the pertussis toxin–sensitive CCR2 receptor. However, transcripts for CCR2 could not be shown in HSC, and pertussis toxin only modestly inhibited MCP–1–induced migration. Exposure of HSC to MCP–1 was associated with an increase in cytosolic calcium concentration, PI 3–K activity, protein tyrosine phosphorylation. Blocking calcium influx or pretreatment of HSC with the PI 3–K inhibitor wortmannin markedly reduced cell migration. This study shows, for the first time, a potential direct profibrogenic action of MCP–1 via HSC chemotaxis. MCP–1-dependent signals in these cells are not transduced by CCR2 and may be mediated by alternative chemokine receptors.