bluuebunny/crossref_metadata_2025 huggingface dataset:
Vasopressin reverses mesenteric hyperemia and vasoconstrictor hyporesponsiveness in anesthetized portal hypertensive rats
Authors: Heinemann Akos , Wachter Christof H. , Fickert Peter , Trauner Michael , Stauber Rudolf E.
Published: July 2004

We recently reported that vasopressin analogues correct the in vitro vascular hyporeactivity to adrenergic vasoconstrictors in portal hypertensive rats. The aim of the present study was to determine whether vasopressin reduces splanchnic blood flow in portal vein-ligated (PVL) rats by restoring vasoconstrictor responsiveness in vivo. The ultrasonic transit time-shift technique was used for blood flow measurements. At basal conditions, blood flow through the superior mesenteric artery was elevated 1.6-fold in PVL rats as compared with sham-operated (SHAM) control rats. PVL rats also exhibited blunted mesenteric constrictor responses to the adrenoceptor agonist, phenylephrine (0.03-1 μmol · min−1 · kg−1). Terlipressin (2-20 μg · kg−1) and arginine vasopressin (3-300 pmol · min−1 · kg−1) dose-dependently reduced, and at the highest doses, even abolished, the difference in mesenteric blood flow (MBF) between PVL and SHAM rats. When expressed as percent changes relative to baseline, mesenteric arterial responses to terlipressin and arginine vasopressin were found to be enhanced in PVL rats as compared with SHAM rats. Moreover, pretreatment with terlipressin (20 μg · kg−1) reversed the mesenteric hyporesponsiveness to phenylephrine of PVL rats. These vasopressin effects were independent of the nitric oxide (NO) pathway, because they were not mimicked by inhibition of NO synthesis with N
G-nitro-l -arginine methyl ester (l -NAME) (0.1-10 mg · kg−1). These data indicate that pharmacological doses of vasopressin reverse the splanchnic hyperemia by restoring the responsiveness to adrenergic vasoconstrictors in portal hypertensive rats.
Beneficial hemodynamic effects of bosentan, a mixed ETA and ETB receptor antagonist, in portal hypertensive rats
Authors: Sogni Philippe , Moreau Richard , Gomola Alexandra , Gadano Adrian , Cailmail Stephane , Calmus Yvon , Clozel Martine , Lebrec Didier
Published: July 2004

In patients with cirrhosis, the plasma level of endothelin, a potent vasoconstrictor peptide, is elevated, and endothelin plays a role in increased intrahepatic vascular resistance. Thus, the aim of this study was to evaluate the hemodynamic effects of bosentan, a mixed ETA and ETB endothelin receptor antagonist in three models of portal hypertension. In all groups of rats, endothelin (2 μg/kg intravenously) administration significantly increased intrahepatic vascular resistance. In rats with secondary biliary cirrhosis, bosentan (30 mg/kg) significantly reduced portal pressure from 14.6 ± 1.2 to 12.1 ± 0.6 mm Hg, while portal blood flow and cardiac output increased by 45% and 57%, respectively. Thus, hepatocollateral vascular resistance decreased significantly from 177 ± 19 to 101 ± 9 dyn · s · cm−5 × 10−3. Similar results were observed in rats with CCl4-induced cirrhosis. In isolated perfused cirrhotic rat livers, bosentan (1 to 100 μmol/L) had no significant effect on hepatic vascular resistance. In portal vein-stenosed rats, bosentan administration significantly decreased portal pressure from 13.1 ± 0.6 to 11.4 ± 0.5 mm Hg by reducing portosystemic vascular resistance, because bosentan had no effect on vascular resistance of normal rat liver. In conclusion, bosentan administration decreased portal pressure in vivo by reducing hepatocollateral vascular resistance in rats with cirrhosis. Thus, mixed endothelin receptor antagonists might be a new approach in the pharmacological treatment of portal hypertension.
Effects of protein kinase C modulators on Na+/K+ adenosine triphosphatase activity and phosphorylation in aortae from rats with cirrhosis
Authors: Lahaye Philippe , Tazi Khalid A. , Rona Jean-Pierre , Dellis Olivier , Lebrec Didier , Moreau Richard
Published: July 2004

Protein kinase C (PKC) modulates the activity and phosphorylation of the catalytic α-subunit of sodium-potassium-adenosine triphosphatase (Na+/K+ ATPase) in normal arteries. Because PKC is altered in cirrhotic aortae, Na+/K+ ATPase may also be altered in these arteries. The aim of the present study was to investigate α-subunit activity and phosphorylation in aortae from normal and cirrhotic rats, under baseline conditions and during exposure to PKC modulators. α-Subunit activity was assessed by measuring the amount of 32P released by hydrolysis of [γ-32P]ATP in freshly isolated cell membranes (in the absence of PKC modulators only) and membrane depolarization caused by ouabain-induced α-subunit inhibition in isolated aortae (in the absence and presence of PKC modulators). α-Subunit phosphorylation was assessed by incorporation of 32P into α-subunits. Staurosporine, a PKC inhibitor, and phorbol 12,13-dibutyrate (PDBU), a PKC activator, were used. In addition, α-subunit expression was studied by Western blot analysis. In the absence of PKC modulators, the amount of 32P released by hydrolysis of [γ-32P]ATP and ouabain-induced membrane depolarization were significantly lower in cirrhotic than in normal aortae. Staurosporine suppressed ouabain-induced membrane depolarization in cirrhotic and normal arteries. Ouabain-induced membrane depolarization was similar in cirrhotic aortae exposed to PDBU and in normal arteries studied under baseline conditions. α-Subunit phosphorylation was significantly lower in cirrhotic than in normal aortae, in aortae under baseline conditions, and in arteries exposed to staurosporine. Phosphorylation of the α-subunit was similar in cirrhotic aortae exposed to PDBU and in normal arteries under baseline conditions. Western blot analysis showed that the amount of α-subunit did not significantly differ between cirrhotic and normal aortae. In conclusion, a decrease in baseline Na+/K+ ATPase α-subunit activity occurs in aortae from cirrhotic rats as a result of reduced basal PKC activity. This PKC-dependent decreased α-subunit activity may be caused by a reduction in PKC-induced α-subunit phosphorylation.
Renal effects of transjugular intrahepatic portosystemic shunt in cirrhosis: Comparison of patients with ascites, with refractory ascites, or without ascites
Authors: Gerbes Alexander L. , Gülberg Veit , Waggershauser Tobias , Holl Josef , Reiser Maximilian
Published: July 2004

Renal effects of the transjugular intrahepatic portosystemic shunt (TIPS) were compared in 6 patients without ascites (group 1), 11 patients with ascites responding to diuretic treatment (group 2), and 6 patients with refractory ascites (group 3). Seven days after insertion of TIPS, 24-hour urinary sodium excretion had increased in patients with ascites: 113 ± 16 mmol to 170 ± 30 mmol (P = .012) in group 2, and 22 ± 8 mmol to 77 ± 27 mmol (P = .050) in group 3. In group 3, fractional sodium excretion tended to increase from 0.26% ± 0.14% to 0.62% ± 18% (P = .081). The relative increase of urinary sodium excretion (to 444% ± 122%) and fractional sodium excretion (to 413% ± 127%) in group 3 was significantly (P < .05) higher than in group 1 and group 2, respectively. Creatinine clearance and 24-hour urinary volume were not significantly changed in either group. Patients with Child-Pugh class C had a more pronounced effect of TIPS on urinary sodium excretion (increase to 396% ± 115% vs. 139% ± 15%; P= .066) and on fractional sodium excretion (increase to 415% ± 103% vs. 94% ± 15%; P = .020) than patients with less-severe liver disease. Fractional sodium excretion of less than 0.35% before TIPS was found to be an indicator of renal response to TIPS. The effect of TIPS on urinary sodium excretion and on fractional sodium excretion was related to the patients’ Child-Pugh score (r = .55; P = .007 and r = .68; P= .001, respectively) and inversely to their fractional sodium excretion (r = −.44; P = .047 and r= −.54; P = .012, respectively) before TIPS. These data demonstrate that TIPS affects renal sodium handling in patients with ascites, particularly in patients with refractory ascites. Severity of liver disease and fractional sodium excretion before TIPS are parameters to predict the extent of the renal response.
Bone mineral density, serum insulin-like growth factor I, and bone turnover markers in viral cirrhosis
Authors: Gallego-Rojo Francisco J. , Gonzalez-Calvin Jorge L. , Muñoz-Torres Manuel , Mundi Jose L. , Fernandez-Perez Ramon , Rodrigo-Moreno Dolores
Published: July 2004

Previous studies suggest that low bone mass is a complication of alcoholic liver disease. Nevertheless, little is known about bone mass and bone metabolism in viral cirrhosis. To evaluate the prevalence and magnitude of hepatic osteopenia in these patients, bone remodeling status, and its relationship with the severity of liver disease and serum levels of insulin-like growth factor I (IGF-I), we studied 32 consecutive patients with viral cirrhosis and no history of alcohol intake. Bone mineral density (BMD) was measured by dual x-ray absorptiometry in the lumbar spine (LS) and femoral neck (FN), and the values were expressed as the z score. Bone metabolism markers and hormone profiles were measured. Patients with viral cirrhosis showed reduced BMD in all sites (LS: −1.27 ± 1.06, P < .001; FN: −0.48 ± 0.96; P < .01). Of the 32 patients, 53% met the diagnostic criteria for osteoporosis. In patients, urine deoxypyridinoline (D-Pyr) as a marker of bone resorption and serum bone alkaline phosphatase (b-AP) as a marker of bone formation were significantly higher than in control subjects (P < .001 and P < .01, respectively). Serum IGF-I was lower than in control subjects (P < .001), and significant differences were also found between patients with and without osteoporosis (P < .05). BMD in LS correlated with severity of the disease, with serum levels of IGF-I, and with urine D-Pyr. Our findings show that viral cirrhosis is a major cause of osteoporosis in men, and that low serum IGF-I levels seem to play a role in the bone mass loss in these patients. The biochemical markers of bone remodeling suggest high-turnover osteoporosis in patients with viral cirrhosis.
Hepatocyte growth factor induces hepatocyte proliferation in vivo and allows for efficient retroviral-mediated gene transfer in mice
Authors: Patijn Gijsbert A. , Lieber Andre , Schowalter David B. , Schwall Ralph , Kay Mark A.
Published: July 2004

Recombinant retroviral vectors are an attractive means of transferring genes into the liver because they integrate into the host cell genome and result in permanent gene expression. However, efficientin vivogene transfer is limited by the requirement of active cell division for integration. Traditional approaches to induce liver proliferation have the disadvantage of inducing hepatocellular injury by delivery of toxins or by surgical partial hepatectomy. As a nontraumatic alternative, we show that exogenous hepatocyte growth factor (HGF) is a powerful and safe mitogen for the mature intact murine liver when delivered continuously into the portal vein. A 5-day infusion of human HGF (5 mg/kg/d) resulted in >140% increase in relative liver mass, which returned to normal in 4 to 5 weeks. This clearly shows that an exogenous growth factor can induce robust liver proliferationin vivo.In addition, we show that the HGF-induced proliferation was independent of interleukin-6, an essential cytokine involved in liver regeneration after partial hepatectomy. When recombinant retroviral vectors were infused in combination with HGF, 30% of hepatocytes were stably transduced with no indication of hepatic injury or histopathology. These results show the ability to obtain a clinically relevant transduction efficiency with retroviral vectorsin vivowithout the prior induction of liver injury. The level of hepatic gene transfer achieved has the potential to be curative for a large number of genetic liver diseases.
Levels of transforming growth factor β and transforming growth factor β receptors in rat liver during growth, regression by apoptosis and neoplasia
Authors: Grasl-Kraupp Bettina , Rossmanith Walter , Ruttkay-Nedecky Branislav , Müllauer Leonhard , Kammerer Bettina , Bursch Wilfried , Schulte-Hermann Rolf
Published: July 2004

Transforming growth factor β1 (TGF-β1) has been implicated as inhibitor of cell proliferation and a potent inducer of apoptosis in vitro and in vivo after the administration of high doses. To assess the role of endogenous TGF-β1, we quantitated the cytokine and its receptors in rat liver during regenerative and hyperplastic growth, regression by apoptosis, and in hepatocellular carcinoma (HCC). This was accomplished by Northern blot analysis and by RNase protection assay of the messenger RNA (mRNA) of TGF-β1 and TGF-β receptors (TβR) types I to III and by an activity bioassay of the TGF-β proteins. Untreated rat livers were found to contain 15.6 ± 4.8 ng TGF-β1 protein/g tissue; TGF-β2 protein was not detected. To induce toxic cell death and subsequent regenerative DNA synthesis in the liver, rats were treated with a necrogenic dose of carbon tetrachloride (CCl4). After 24 and 48 hours, there was an upregulation of TGF-β1 (mRNA, up to tenfold; protein, about twofold) and of TβRs (mRNA: two- to fourfold); that indicates an overall enhanced production of and sensitivity to TGF-β1, which may serve to confine the regenerative response. Hyperplastic liver growth and regression of the hyperplasia were induced by treatment with cyproterone acetate (CPA) or nafenopin (NAF) followed by withdrawal; neither mRNAs of TGF-β1 and TβR types I to III nor TGF-β1 protein exhibited significant changes during the growth phase or during regression by apoptosis. We also studied neoplastic growth. HCC, obtained after long-term treatment with NAF, exhibited high rates of cell replication and apoptosis. The majority of lesions contained mRNA and protein of TGF-β1 and mRNA of TβR types I to III at concentrations similar to those of the surrounding tissue. In conclusion, during liver regeneration there is a pronounced upregulation of expression of both TGF-β1 and TβRs I to III, but not during mitogen-induced liver growth or regression. It appears that apoptosis is induced via altered local concentration of TGF-β1 in a paracrine and/or autocrine way. By this mechanism the lethal effects of TGF-β1 may be locally confined, and overshoots of apoptosis in the liver may be prevented.
Expression of cyclin-dependent kinase inhibitor p21 in human liver
Authors: Crary Gretchen S. , Albrecht Jeffrey H.
Published: July 2004

The p21 protein is a universal inhibitor of cyclin-dependent kinases and of cell-cycle progression and is involved in numerous growth-inhibitory pathways in cell culture systems. Recent studies suggest that p21 regulates hepatocyte cell cycle progression in models of liver regeneration. The present study was designed to investigate the possible involvement of p21 in the control of hepatocyte proliferation in human liver diseases. To examine that, the expression of p21 in clinical liver biopsy specimens was determined by immunohistochemistry. This was correlated with hepatocyte Ki-67 immunostaining (a marker of hepatocyte proliferation in vivo) as well as histologic features. Little p21 or Ki-67 expression was detected in normal human liver or in specimens of nonalcoholic steatohepatitis. In patients with alcoholic hepatitis, increased expression of p21, but not of Ki-67, was observed. In specimens with chronic hepatitis C, hepatocyte p21 expression was significantly correlated with Ki-67 immunostaining, as well as with the degree of inflammation and fibrosis. These results indicate that hepatocyte p21 expression is upregulated in response to hepatic injury and correlates with histologic markers of proliferation and disease activity. This study provides evidence that p21 plays a role in the regulation of hepatocyte proliferation in human liver diseases.
Chronic alcohol intake reduces retinoic acid concentration and enhances AP-1 (c-Jun and c-Fos) expression in rat liver†
Authors: Wang Xiang-Dong , Liu Chun , Chung Jayong , Stickel Felix , Seitz Helmut K. , Russell Robert M.
Published: July 2004

Chronic ethanol intake may interfere with retinoid signal transduction by inhibiting retinoic acid synthesis and by enhancing activator protein-1 (AP-1) (c-Jun and c-Fos) expression, thereby contributing to malignant transformation. To determine the effect of ethanol on hepatic retinoid levels, retinoic acid receptors (RARs) and AP-1 (c-Jun and c-Fos) gene expression, chronic ethanol (36% of total calorie intake) pair-feeding was conducted on rats for a 1-month period. Retinoic acid, retinol, and retinyl ester concentrations in both liver and plasma were examined by using high-performance liquid chromatography (HPLC). Both retinoic acid receptor (α, β, γ) and AP-1 (c-Jun and c-Fos) expression in the rat liver were examined by using Western blot analysis. Treatment with high-dose ethanol led to a significant reduction of retinoic acid concentration in both the liver and the plasma (11- and 8.5-fold reduction, respectively), as compared with animals pair-fed an isocaloric control diet containing the same amount of vitamin A. Similar to the retinoic acid reductions, both retinol and retinyl palmitate levels in the livers of the alcohol-fed group decreased significantly, but in smaller fold reduction (6.5- and 2.6-fold reduction, respectively). Ethanol did not modulate the expression of RARα, -β, and -γ genes in the liver. However, chronic alcohol feeding enhanced AP-1 (c-Jun and c-Fos) expression by 7- to 8-fold, as compared with the control group. These data suggest that functional down-regulation of RARs by inhibiting biosynthesis of retinoic acid and up-regulation of AP-1 gene expression may be important mechanisms for causing malignant transformation by ethanol.
Portal branch ligation with a continuous hepatocyte growth factor supply makes extensive hepatectomy possible in cirrhotic rats
Authors: Kaido Toshimi , Yoshikawa Akira , Seto Shin-ichi , Yamaoka Shoji , Sato Maki , Ishii Takehisa , Imamura Masayuki
Published: July 2004

In a cirrhotic liver, the regenerative ability and specific functions are so impaired that excessive resection easily complicates postoperative liver dysfunction, which frequently leads to life-threatening multiple-organ failure. Hepatocyte growth factor (HGF), first identified as the most potent stimulator of DNA synthesis in primary hepatocytes, not only stimulates liver regeneration, but also accelerates hepatic function, improves fibrosis, and protects liver cells against injury. Therefore, we investigated the efficacy of preoperative portal branch ligation (PBL) (which can induce compensatory hypertrophy of the unaffected lobes) combined with a continuous HGF supply in the performance of extensive hepatectomy in cirrhotic rats. Cirrhosis was induced by intraperitoneal injections of dimethylnitrosamine (DMN) three times per week for 3 weeks. Five days after the last injection, when 70% hepatectomy is lethal, the rats underwent portal ligation of the left lateral and median branches (corresponding to approximately 70% of the total volume of the liver). Simultaneously, they were continuously treated with either recombinant human HGF (rhHGF) or vehicle from an intraperitoneally implanted osmotic pump. Four days after the portal ligation, the occluded lobes were resected. The HGF treatment rapidly increased both the wet weight of the unoccluded lobes and the hepatocellular DNA synthesis. The blood chemical analysis indicated that HGF significantly suppressed the posthepatectomy liver dysfunction. Most importantly, the HGF treatment markedly improved the survival rate of the rats at 48 hours after the major hepatectomy. In conclusion, PBL combined with a continuous HGF supply makes extensive hepatectomy possible in cirrhotic rats, mainly by promoting the hypertrophy of the unaffected lobes.
Parenchymal cell apoptosis as a signal for sinusoidal sequestration and transendothelial migration of neutrophils in murine models of endotoxin and fas-antibody-induced liver injury
Authors: Lawson Judy A. , Fisher Michael A. , Simmons Carol A. , Farhood Anwar , Jaeschke Hartmut
Published: July 2004

Endotoxin (ET) induces neutrophil sequestration in hepatic sinusoids, the activation of proinflammatory transcription factors (nuclear factor κB [NF-κB]) with up-regulation of adhesion molecules on sinusoidal endothelial cells and hepatocytes. However, if galactosamine (Gal) is co-administered with ET, neutrophils transmigrate and attack parenchymal cells. This suggests that a signal from parenchymal cells triggers neutrophil transmigration. In this study, we tested the hypothesis that parenchymal cell apoptosis may induce neutrophil transendothelial migration in the Gal/ET model. Treatment of C3Heb/FeJ mice with 700 mg/kg Gal and 100 μg/kg ET induced tumor necrosis factor α (TNF-α) formation (13.25 ± 0.75 ng/mL) and hepatic NF-κB activation at 90 minutes; the generation of the C-X-C chemokine KC (2.86 ± 0.30 ng/mL at 5 hours); sinusoidal neutrophil sequestration (380 ± 21 polymorphonuclear leukocytes/50 high-power fields) and apoptosis (925% ± 29% increase of DNA fragmentation; and a 45-fold increase of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells) at 6 hours, followed by transmigration of neutrophils and development of substantial necrosis (38% ± 3% of hepatocytes; alanine transaminase [ALT]: 1,500 ± 300 U/L) at 7 hours. Administration of uridine (1,000 mg/kg) did not reduce plasma levels of TNF-α and KC, NF-κB activation, or polymorphonuclear leukocyte sequestration, but attenuated apoptosis by 90% to 94%. In these livers, neutrophils did not transmigrate and liver injury was prevented (necrosis: <5%; ALT: 40 ± 3 U/L). However, massive apoptosis and liver injury initiated by the anti-Fas antibody, Jo2, did not recruit neutrophils into the liver. We conclude that excessive parenchymal cell apoptosis represents an important signal for transmigration of primed neutrophils sequestered in sinusoids during endotoxemia in vivo. However, apoptosis per se does not cause neutrophil sequestration in the liver vasculature.
Hepatic preconditioning in rats is defined by a balance of adenosine and xanthine
Authors: Peralta Carmen , Closa Daniel , Xaus Carme , Gelpí Emilio , Roselló-catafau Joan , Hotter Georgina
Published: July 2004

The present work investigates the relationship between adenosine, nitric oxide (NO), and free radicals during ischemic preconditioning in rat liver. For this purpose, we evaluated: 1) the efficacy of different periods of preconditioning; 2) the changes in the concentration of adenine nucleotides during preconditioning; 3) the importance of adenosine and xanthine concentrations in the induction of preconditioning; and 4) the possible effect of xanthine oxidase-derived superoxide anion on NO during preconditioning. Results show that just a 10- to 15-minute period of ischemia followed by 10-minute reperfusion prevents the ischemic damage that would be induced by a subsequent 90 minutes of ischemia followed by 90 minutes of reperfusion. Administration of xanthine or metabolization of endogenous adenosine abolishes the protective effect of preconditioning. When rats have been subjected to a period of preconditioning not within the effective time window (10-15 minutes), and thus offering no protection, the administration of a NO donor was found to restore the protection. The dose needed to restore protection appears to be proportional to the endogenous xanthine concentration. In addition, when xanthine oxidase was inhibited, preconditioning effectively offered protection in front of ischemia and reperfusion, independently of the xanthine concentration. Altogether, this indicates that the time window of ischemia capable to induce preconditioning in liver is defined by the relative tissue concentrations of adenosine and xanthine. The lower limit of this window (10 minutes) is defined by the amount of adenosine able to induce NO generation. Its upper limit (15 minutes) is defined by the concentration of xanthine able to remove the generated NO.
Modulation of endothelin and nitric oxide: A rational approach to improve canine hepatic microcirculation
Authors: Dhar Dipok Kumar , Yamanoi Akira , Ohmori Haruyuki , Nakashima Yuichi , Yamamoto Akitaka , Osama El-Assal N. , Kubota Hirofumi , Kohno Hitoshi , Nagasue Naofumi
Published: July 2004

ET receptor blocker (TAK-044) and NO donor (FK409) were used to improve the hepatic microcirculation following ischemia-reperfusion injury. In the first experiment (60 minutes of ischemia), 15 dogs were divided into three groups: group A (control), saline; group B, TAK 5 mg/kg; and group C, FK 0.4 mg/kg. In the second experiment (90 minutes of ischemia), 38 dogs were divided into six groups that underwent 90 minutes of hepatic ischemia followed by reperfusion: group I (control), saline only; group II, TAK 5 mg/kg and FK 3.2 mg/kg; group III, TAK 5 mg/kg and FK 0.4 mg/kg; group IV, TAK 5 mg/kg; group V, FK 0.4 mg/kg; and group VI, FK3.2 mg/kg. All drugs were administered through the portal vein. Following 60 minutes of ischemia, both FK and TAK produced significant improvement in hepatic microcirculation and enzymatic status when compared with the control group. After 90 minutes of ischemia, low doses of FK and TAK significantly improved hepatic microcirculation and reduced portal pressure following reperfusion in group III compared with group I. Leakage of hepatic enzymes was prevented and tissue injury score was significantly lower in group III. In group VI, early protection was obtained to some extent; however, blood pressure was reduced significantly following reperfusion compared with group I. In contrast, hepatocellular function deteriorated and the tissue injury score was higher in group II animals. TAK pretreatment with low doses of FK provided the best protection for the hepatic microcirculation in ischemia-reperfusion injury of the liver.
Maternal cholestasis does not affect the ontogenic pattern of expression of the Na+/Taurocholate cotransporting polypeptide (ntcp) in the fetal and neonatal rat liver
Authors: Arrese Marco , Trauner Michael , Ananthanarayanan Meenakshisundaram , Boyer James L. , Suchy Frederick J.
Published: July 2004

To assess the effects of cholestasis during pregnancy on fetal and neonatal mRNA expression, protein mass, and function of the Na+/taurocholate cotransporting polypeptide (Ntcp), common bile duct ligation (BDL) was performed in pregnant rats on day 14 of pregnancy (maternal cholestasis [MC] group), and livers were harvested at days 20 and 21 of fetal life, as well as at days 4, 7, 14, 21, and 28 after birth. Sham-operated rats and their litters were used as controls.Ntcpsteady-state mRNA levels, protein mass, and function were determined by Northern blotting, immunoblotting, and taurocholate (TC) transport studies in isolated short-term cultured hepatocytes, respectively. In addition, protein mass and function of the organic anion transporting polypeptide (Oatp1), another sinusoidal bile acid transporter, were studied at 4 weeks of age. The majority of pregnant cholestatic rats (94%) were able to carry pregnancy to term. Body and liver weights of the offspring from the MC group were lower than those from sham-operated animals at all postnatal time points.Ntcpsteady-state mRNA levels, protein mass, and function were unaffected by MC. The ontogenic pattern of expression was identical in offspring from MC and controls with detection of theNtcpmRNA at day 21 of fetal life. There was a significant increase in mRNA postnatally, reaching adult levels by 7 days of age. Protein mass and function of Ntcp as well as of Oatp1 were similar in offspring from MC and control groups at 4 weeks of age. In conclusion, maternal obstructive cholestasis during the last third of pregnancy does not affect the fetal/neonatal expression of the basolateral bile acid transporters,NtcpandOatp1. This suggests that the impaired bile acid excretion described in this experimental model is not related to altered uptake of bile acids in the affected neonate.
Dissection of human humoral immune response against hepatitis C virus E2 glycoprotein by repertoire cloning and generation of recombinant fab fragments
Authors: Burioni Roberto , Plaisant Paola , Manzin Aldo , Rosa Domenico , Carri Valeria Delli , Bugli Francesca , Solforosi Laura , Abrignani Sergio , Varaldo Pietro E. , Fadda Giovanni , Clementi Massimo
Published: July 2004

Demonstration of antibodies inhibiting key viral functions is the basis for the design of an effective vaccine. Dissection of the human antibody response by repertoire cloning may be a powerful means to address this issue. In this study, a panel of human monoclonal recombinant Fab fragments specific for hepatitis C virus (HCV) E2 envelope protein was generated. The selection procedure was designed to select for cross-genotype reactive antibodies. Sequences coding five different human recombinant Fabs specific for the HCV/E2 protein were cloned and characterized. The ability of the cloned antibody fragments to inhibit adhesion of recombinant envelope E2 protein to target cells was assayed. While affinity of the different antibody fragments appeared similar, activity in inhibiting E2 binding to target cells varied considerably from one Fab fragment to another. Two Fabs were not able to inhibit E2 binding at high concentration (40 μg/mL), while three other Fab clones were active in neutralizing 50% of the E2 binding at concentrations ranging from 3 to 0.35 μg/mL.
Cellular immunity to hepatitis C virus core protein and the response to interferon in patients with chronic hepatitis C
Authors: Lasarte Juan-José , García-Granero Marta , López Ascensión , Casares Noelia , García Nicolás , Civeira María-Pilar , Borrás-Cuesta Francisco , Prieto Jesús
Published: July 2004

To investigate the involvement of T-cell response against hepatitis C virus (HCV) antigens in viral clearance after interferon therapy, we measured interleukin-2 (IL-2) production by peripheral mononuclear cells in response to HCV core in patients with chronic hepatitis C. In a cohort of 43 patients, we investigated the frequency of circulating core-specific T-helper (Th) cell precursors by the limiting-dilution assay, and in a second cohort of 60 patients, we analyzed the response to specific core epitopes using 52 synthetic 15-mer overlapping peptides. We observed that the frequency of core-specific Th cell precursors was significantly higher in patients with sustained biochemical and virological response (SR) after interferon (IFN) therapy (median, 1/55,736) than in untreated patients (1/274,023) or that in patients who remained viremic after completion of the treatment—nonresponders (NR) plus transient responders (TR) (1/1,909,972). Patients who failed to respond to IFN (NR) and those who relapsed after IFN discontinuation (TR) had a similarly low number of precursors. The number of core peptides recognized by SR, TR, NR, UT, and healthy controls was 8.2 ± 1.5, 6.5 ± 1.2, 2.0 ± 0.5, 2.7 ± 0.9, and 0.3 ± 0.2, respectively. In SR, the intensity of the proliferative response to core peptides as estimated by the summation of stimulation indexes (ΣSI) was significantly higher than in NR and than in UT, but not different from that of TR. Our results indicate that both expansion of HCV-specific Th cell precursors and Th cell recognition of multiple core epitopes seem to be important in the elimination of HCV after IFN therapy.
Predictors of patient and graft survival following liver transplantation for hepatitis C
Authors: Charlton Michael , Seaberg Eric , Wiesner Russell , Everhart James , Zetterman Rowen , Lake John , Detre Katherine , Hoofnagle Jay
Published: July 2004

End-stage liver disease secondary to hepatitis C virus (HCV) infection is the leading indication for liver transplantation in the United States. Recurrence of HCV infection is nearly universal. We studied the patients enrolled in the National Institute of Diabetes and Digestive and Kidney Diseases Liver Transplantation Database to determine whether pretransplantation patient or donor variables could identify a subset of HCV-infected recipients with poor patient survival. Between April 15, 1990, and June 30, 1994, 166 HCV-infected and 509 HCV-negative patients underwent liver transplantation at the participating institutions. Median follow-up was 5.0 years for HCV-infected and 5.2 years for HCV-negative recipients. Pretransplantation donor and recipient characteristics, and patient and graft survival, were prospectively collected and compared. Cumulative patient survival for HCV-infected recipients was similar to that of recipients transplanted for chronic non-B-C hepatitis, or alcoholic and metabolic liver disease, better than that of patients transplanted for malignancy or hepatitis B (P= .02 andP= .003, respectively), and significantly worse than that of patients transplanted for cholestatic liver disease (P= .001). Recipients who had a pretransplantation HCV-RNA titer of ≥1 × 106vEq/mL had a cumulative 5-year survival of 57% versus 84% for those with HCV-RNA titers of <1 × 106vEq/mL (P= .0001). Patient and graft survival did not vary with recipient gender, HCV genotype, or induction immunosuppression regimen among the HCV-infected recipients. While long-term patient and graft survival following liver transplantation for end-stage liver disease secondary to HCV are generally comparable with that of most other indications, higher pretransplantation HCV-RNA titers are strongly associated with poor survival among HCV-infected recipients.
TT-virus infection in north american blood donors, patients with fulminant hepatic failure, and cryptogenic cirrhosis
Authors: Charlton Michael , Adjei Philip , Poterucha John , Zein Nizar , Moore Breanndan , Therneau Terry , Krom Ruud , Wiesner Russell
Published: July 2004

A novel DNA virus, TT-virus (TTV), has been reported in patients with non-A-G posttransfusion hepatitis in Japan. We sought to determine whether TTV infection occurs in North American blood donors and to further determine the prevalence of TTV infection in several groups of patients with liver disease, including patients with cryptogenic cirrhosis and idiopathic fulminant hepatic failure. TTV infection was sought by detection of TTV DNA in serum by polymerase chain reaction (PCR) using primers generated from a conserved region of the TTV genome. Blood donors, patients with cryptogenic cirrhosis, idiopathic fulminant hepatic failure, and patients with other forms of advanced liver disease with and without a history of parenteral exposures were studied. TTV infection was present in 1% (1 of 100) of blood donors, 15% (5 of 33) of patients with cryptogenic cirrhosis, 27% (3 of 11) of patients with idiopathic fulminant hepatic failure, 18% (2 of 11) of patients with a history of exposure to blood products, and 4% (1 of 25) of patients without parenteral risk factors. For all patients tested, a history of prior exposure to blood products was associated with an increased risk of TTV infection (relative risk, 4.5; 90% confidence intervals, 0.6-43.9). We conclude that TTV infection is present among North American blood donors and is common in patients with liver disease, including cryptogenic cirrhosis and fulminant hepatic failure. Further studies are required to determine the role of TTV in the pathogenicity of acute and/or chronic liver disease.
Purinergic regulation of acid/base transport in human and rat biliary epithelial cell lines
Authors: Zsembery Ákos , Spirlì Carlo , Granato Anna , LaRusso Nicholas F. , Okolicsanyi Lajos , Crepaldi Gaetano , Strazzabosco Mario
Published: July 2004

Biliary epithelial cells (cholangiocytes) are responsible for rapid regulation of bile volume and alkalinity. Secretin and other hormones raising intracellular cyclic adenosine monophosphate (cAMP) concentrations promote biliary HCO3 secretion by stimulating apical Cl− channels and Cl−/HCO3
− exchange (AE2). Cholangiocyte ion transport may also be stimulated by locally acting mediators; for example, adenosine 5′-triphosphate (ATP), a secretagogue that can be released into the bile by hepatocytes and cholangiocytes, activates Cl− conductances and Na+/H+ exchange (NHE) in cholangiocyte cell lines. To further explore the role of extracellular ATP in the paracrine regulation of carrier mechanisms regulating cholangiocyte H+/HCO3
− secretion, we investigated the effects of nucleotides on intracellular pH regulation (measured by microfluorimetry with 2′7′-bis(2-carboxyethyl)-5,6,carboxyfluorescein [BCECF]) in human (MZ-ChA-1) and rat (NRC-1) cholangiocyte cell lines. In MZ-ChA-1 cells, 10 mol/L ATP, uridine 5′-triphosphate (UTP), and ATPγs significantly increased NHE activity. The pharmacological profile of agonists was consistent with that anticipated for receptors of the P2Y2 class. ATP did not increase AE2 activity, but, when given to cells pretreated with agents raising intracellular cAMP, had a synergistic stimulatory effect that was inhibited by amiloride. To assess the polarity of purinergic receptors, monolayers of NRC-1 cells were exposed to apical or basolateral nucleotides. Apical administration of purinergic agonists, but not adenosine, increased basolateral NHE activity (ATPγS > UTP > ATP). Basolateral administration of purinergic agonists induced a weaker activation of NHE, which was instead strongly stimulated by adenosine and by adenosine receptor agonists (NECA = R-PIA = S-PIA). In conclusion, this study demonstrates that, consistent with the proposed role for biliary ATP in paracrine and autocrine control of cholangiocyte ion secretion, extracellular ATP stimulates cholangiocyte basolateral NHE activity through P2Y2 receptors that are predominantly expressed at the apical cell membrane.
Clonal analysis of macronodules in cirrhosis
Authors: Paradis Valerie , Laurendeau Ingrid , Vidaud Michel , Bedossa Pierre
Published: July 2004

Several arguments suggest that most hepatocellular carcinomas (HCCs) occurring in human cirrhotic livers arise from large hepatocellular nodules or macronodules. Except for nodules with obvious features of HCC, there exist no consistent criteria enabling the differentiation between benign regenerative and neoplastic, potentially malignant macronodules. Surrogate markers able to accurately discriminate those lesions that will evolve toward a HCC are required. In this study, we investigated the clonality of 26 macronodules isolated from eight cases of explanted cirrhotic livers in women by analyzing X-chromosome inactivation, as indicated by the methylation status of the human androgen receptor gene (HUMARA). For each macronodule, a large set of pathological features was evaluated and used to classify the macronodules into four groups: entirely benign-looking nodule (type 1), low-grade dysplastic nodule (type 2), high-grade dysplastic nodule (type 3), and HCC (type 4). Clonal analysis showed that 14 macronodules (54%) were monoclonal and 12 (46%) were polyclonal. Monoclonality was detected in 5 of 11 (45%) nodules from groups of entirely benign-looking and low-grade dysplastic nodules (types 1 and 2) and in 9 of 15 (60%) nodules from the group of high-grade dysplastic nodule and HCC (types 3 and 4). Neither the etiology of cirrhosis nor the size or histological classification of macronodules was correlated with the clonal status. In conclusion, clonal analysis of macronodules enables the differentiation between mono- and polyclonal macronodules in cirrhosis. Because monoclonal macronodules are prone to evolve to HCC, the determination of the clonal status of a macronodule could provide additional information for evaluating the prognosis of these lesions.
Analysis of liver regeneration in mice lacking type 1 or type 2 tumor necrosis factor receptor: Requirement for type 1 but not type 2 receptor
Authors: Yamada Yasuhiro , Webber Eric M. , Kirillova Irina , Peschon Jacques J. , Fausto Nelson
Published: July 2004

We used KO mice lacking either TNF receptor 1 (TNFR-1) or receptor 2 (TNFR-2) to determine whether signaling at the start of liver regeneration after partial hepatectomy (PH) involves only one or both TNF receptors and to analyze in more detail the abnormalities caused by lack of TNFR-1 receptor, which is required for the initiation of liver regeneration. Lack of TNFR-2 had little effect on NF-κB and STAT3 binding, and no effect in interleukin-6 production after PH, but caused a delay in AP-1 and C/EBP binding and in the expression of c-jun and c-myc messenger RNA (mRNA). In contrast to mice lacking TNFR-1, which had deficient hepatocyte DNA synthesis and massive lipid accumulation in hepatocytes, TNFR-2 KO mice had normal liver structure and similar levels of hepatocyte DNA replication as those of wild type mice. We conclude that TNFR-1, but not TNFR-2, is necessary for liver regeneration, and that NF-κB and STAT3 binding are activated by signals transduced by TNFR-1. Inhibition of AP-1 and C/EBP binding and in the expression of c-jun and c-myc mRNA in the first 4 hours after PH, as well as the apparent lack of Fos in AP-1 complexes, had no effect on the timing or extent of DNA replication.
Clinical relevance of transforming growth factor α, epidermal growth factor receptor, p53, and Ki67 in colorectal liver metastases and corresponding primary tumors
Authors: De Jong Koert P. , Stellema Rudi , Karrenbeld Arend , Koudstaal Jan , Gouw Annette S. , Sluiter Wim J. , Peeters Paul M. J. G. , Slooff Maarten J. H. , De Vries Elisabeth G. E.
Published: July 2004

To determine whether the expression of transforming growth factor α (TGF-α), its receptor (epidermal growth factor receptor [EGFr]), p53 nuclear protein, and proliferation influences prognosis of patients with liver metastases, a study was performed in 45 liver metastases and 33 corresponding primary colorectal carcinomas in patients referred for liver surgery. The expression of TGF-α, EGFr, p53 nuclear protein, and proliferation rate was correlated with clinicopathological characteristics and survival after partial liver resection. In liver metastases, TGF-α expression was low in 42%, intermediate in 35%, and high in 23%. TGF-α expression was higher in liver metastases derived from lymph node-positive primary carcinomas, in synchronous and in irresectable liver metastases compared with those derived from lymph node-negative primary carcinomas, metachronous, and resectable liver metastases. Nuclear p53 expression was found in 83% of primary tumors and 71% of liver metastases. p53 expression did not correlate with the various clinicopathological characteristics. Ki67 expression was not associated with clinicopathological characteristics in primary and metastatic tumors. In the 38 patients in whom a partial liver resection was performed, median survival was 25 months in patients with a higher TGF-α expression in the metastasis than in the primary tumor and 60 months in patients with comparable or lower TGF-α expression in the metastasis than in the primary tumor (P = .036). Median survival after liver resection was 21 months in patients with p53-negative liver metastases and 58 months in patients with p53-positive metastases (P = .043). By multivariate analysis, p53 and EGFr expression on liver metastases were the best predictors of disease-free survival after partial liver resection, with relative risks of 2.38 and 3.33, respectively. In patients with colorectal liver metastases, referred for liver surgery, a higher TGF-α expression is associated with unfavorable tumor characteristics, whereas p53 and absence of EGFr expression is associated with a better survival after partial liver resection.
Immunolocalization of OV-6, a putative progenitor cell marker in human fetal and diseased pediatric liver
Authors: Crosby Heather A. , Hubscher Stefan G. , Joplin Ruth E. , Kelly Deirdre A. , Strain Alastair J.
Published: July 2004

The existence of progenitor (stem) cells in the human liver remains a matter of debate. In rodent models of hepatocarcinogenesis and injury, oval cells proliferate in the periportal regions of the portal tracts and are suggested to derive from a stem cell compartment, because they are capable of differentiating into hepatocytes or biliary epithelial cells. In this study, the rat oval cell marker, OV-6 has been used to investigate the hypothesis that there are stem cells present in fetal and pediatric human liver. The pattern of OV-6 expression was compared with the established adult biliary cell markers human epithelial antigen-125 (HEA-125) and cytokeratin-19 (CK-19). In normal pediatric liver (n = 7), bile ducts and ductules were immunostained with CK-19 and HEA-125, whereas OV-6 staining was consistently negative. In fetal tissue (n = 10), ductal plate cells, primitive bile ducts, and hepatoblasts were stained with CK-19 and HEA-125 although only some of the ductal plate cells and hepatoblasts were OV-6 positive. In biliary atresia (n = 6) and α1, anti-trypsin deficiency (α1,AT) (n = 4), CK-19 and HEA-125 immunostained ductular proliferative cells that tended to form finely anastomosing ductules, whereas OV-6 staining was found more on discrete cells confined to portal tract margins. Additionally, in diseased liver, OV-6 was strongly positive in hepatocyte lobules with greatest intensity in the periseptal regions. This widespread hepatocyte OV-6 positivity suggests that the antibody may identify cells of a less differentiated phenotype (transitional hepatocytes) that have replaced the mature cells. Therefore, it is proposed that in human liver, OV-6 is recognizing cells with a progenitor stem cell-like phenotype with the capacity to differentiate into OV-6 positive ductular cells or lobular hepatocytes.
Expression of human macrophage metalloelastase gene in hepatocellular carcinoma: Correlation with angiostatin generation and its clinical significance
Authors: Rivas Manuel J. , Arii Shigeki , Furutani Masaharu , Harada Tomika , Mizumoto Masaki , Nishiyama Hiroyuki , Fujita Jun , Imamura Masayuki
Published: July 2004

Macrophage metalloelastase, a member of the human matrix metalloproteinase family, is believed to play an important role in angiostatin generation, which, in experimental studies, has an antiangiogenic function and is a key molecule in tumor dormancy. However, no clinical studies have been reported regarding the correlation between human macrophage metalloelastase (HME) gene expression and angiostatin production. Therefore, the present study was designed to evaluate the HME messenger RNA (mRNA) expression and angiostatin generation in hepatocellular carcinoma (HCC). Tumorous and contiguous nontumorous tissues were obtained from 40 HCC patients who underwent curative partial hepatectomy. By using Northern blot hybridization, HME mRNA was detected in 25 of the 40 HCC samples and, in all of these cases, the expression in tumorous tissues was stronger than in the nontumorous tissues.In situhybridization identified the HCC cells as mainly responsible for the signals shown in Northern blot. Angiostatin was detected by Western blot mainly in tumors and showed significant association with HME mRNA expression in tumorous tissues (P= .0008). The patients whose tumors did not express HME mRNA and, thus, did not produce angiostatin, demonstrated poorer survival than those whose tumors showed high expression of HME mRNA and angiostatin generation (P= .002). The univariate and multivariate analyses revealed that HME mRNA expression is a new and independent variable affecting overall survival (P= .001 andP= .03, respectively). These findings show that the HME gene is expressed in HCC being significantly associated with angiostatin generation by such tumors and that HME mRNA expression may serve as a new molecular prognostic marker in HCC patients after partial hepatectomy.