bluuebunny/crossref_metadata_2025 huggingface dataset:
Absence of NO synthase type II expression in fetal liver from pregnant rats under septic shock conditions
Authors: Casado M , Velasco M , Bosca L , Martin-Sanz P
Published: July 2004

Treatment of rats at 21 days' gestation with lipopolysaccharide (LPS) induced type II NO synthase (iNOS) expression in maternal liver but completely failed to elicit this response in the corresponding fetal tissue. The impaired response of fetal liver cannot be attributed to alterations in the sensitivity of this organ to LPS or to proinflammatory cytokines involved in iNOS expression (tumor necrosis factor α [TNF-α], interferon gamma [IFN-γ]), because cultured fetal and adult hepatocytes similarly responded to these factors. Measurement of TNF-α and IFN-γ levels in maternal and fetal serum showed a restricted permeability of placenta to these cytokines, results that were in agreement with the absence of iNOS expression in fetal liver. Moreover, analysis of nuclear factor kappaB, which is required for iNOS expression, showed an activation in nuclear extracts from livers of mothers under septic shock conditions but not in the corresponding fetal livers. However, cultured fetal hepatocytes prepared from mothers in septic shock displayed an anomalous pattern of iNOS expression, including a decreased response to LPS as well as a shift in sensitivity to TNF-α. These hepatocytes from LPS-treated mothers showed significant expression of iNOS in the absence of added stimuli. These results suggest that there is a priming of the fetal hepatocytes from mothers with septic shock. The characterization of the factors causing this priming process might provide additional clues understanding the control of iNOS expression in liver.
Relaxing effect of interleukin-1 on rat cultured Ito cells
Authors: Sakamoto M , Ueno T , Sugawara H , Torimura T , Tsuji R , Sujaku K , Sata M , Tanikawa K
Published: July 2004

Interleukin-1β (IL-1β) is closely involved in liver disorders. IL-1β produces nitric oxide (NO) in vascular smooth muscle cells and relaxes vascular smooth muscle via cyclic guanosine 3′,5′-monophosphate (cGMP). In this study, we evaluated the relaxing effect of IL-1β on cultured Ito cells. Ito cells were isolated from the livers of male Wistar rats and cultured for 24 hours. Immunolocalization of inducible nitric oxide synthase (iNOS) and cGMP and intensity of fluorescence of cGMP were examined using a confocal laser microscope. Ito cells were treated with 0, 200, and 1,000 pmol/L IL-1β, and the intracellular cGMP concentration was measured after 12 hours. Moreover, Ito cells treated with 200 and 1,000 pmol/L IL-1β and not treated with IL-1β were observed over 12 hours, and the area of the same Ito cell was compared before and after the addition of IL-1β. Next, effects of NG-monomethyl-L-arginine (L-NMMA) and S-nitroso-N-acetyl-DL-penicillamine (SNAP) on Ito cell relaxation by IL-1β treatment were examined. In Ito cells, immunofluorescence of iNOS was observed, and fluorescent intensity of cGMP increased after addition of IL-1β. Intracellular cGMP concentration increased dose-dependently after addition of IL-1β. Cell area significantly increased in the IL-1β-treated group compared with the untreated group. Relaxation of Ito cells by IL-1β treatment was inhibited by L-NMMA in a dose-dependent manner, but was enhanced by SNAP. These results indicate that IL-1β produces NO in cultured Ito cells and relaxes the cells via cGMP.
Liver transplantation for alcoholic liver disease: Evaluation of a selection protocol
Authors: Anand A C , Ferraz-Neto B H , Nightingale P , Mirza D F , White A C , McMaster P , Neuberger J M
Published: July 2004

We have used a formal transplant protocol to select patients with alcoholic liver disease (ALD) for transplantation. We retrospectively analyzed all the patients with ALD who were referred specifically for transplantation to our Liver Unit between 1987 and 1994. Patients were selected for liver transplantation if they had end-stage liver disease and had remained abstinent from the time they were medically advised to stop alcohol intake. Of the 180 patients referred for transplantation, 43 (none of whom were transplanted) had case records insufficiently complete for full analysis; this may bias the analysis. Of the remaining 137 patients, 39 were transplanted and 4 were awaiting transplantation at the time of analysis. Of the patients who were not accepted for transplantation, 13 died during the assessment, 7 were considered to be unlikely to survive the procedure, 29 were found to be medically unsuitable, 16 psychologically unsuitable, 7 patients refused the offer of transplantation, and an additional 19 either showed clinical improvement or were considered too well for transplantation. Special investigations, such as brain computerized tomography (CT) scan and echocardiograph, changed the clinical decision to transplant in only a small number of cases (4% and 5%, respectively). Nine of the transplanted patients died and the remaining were followed up for a median of 25 (range, 7-63) months. One year actuarial survival for the transplanted patients was 79%, for those considered too sick was 0%, for medically unsuitable patients was 44%, for psychologically unsuitable patients was 65% and for those considered too well was 94%. Only 5 of the transplanted patients (13%) reverted to drinking. The observed actuarial survival of nontransplanted patients was compared with the expected survival calculated by ‘the Beclere model.’ The observed actuarial survival in the nontransplanted groups was much better than anticipated from the Beclere model, which therefore, is not applicable to our patients. The proportional hazards regression analysis of our nontransplanted patients identified serum bilirubin, serum albumin, blood urea, ascites, and spontaneous bacterial peritonitis as factors significantly predictive of their probability of survival. Using a model based on these parameters, the expected survival of our transplanted patients was calculated. Although we applied the model to a different population, the observed actuarial survival in the transplanted patients was found to be better than their expected survival (P < or = .001). Our protocol was useful in selecting suitable patients with ALD for liver transplantation, which resulted in significant survival advantage with low recidivism rate.
Survival after liver transplantation in cirrhotic patients with and without hepatocellular carcinoma: A comparative study
Authors: Figueras J , Jaurrieta E , Valls C , Benasco C , Rafecas A , Xiol X , Fabregat J , Casanovas T , Torras J , Baliellas C , Ibanez L , Moreno P , Casais L
Published: July 2004

Cumulative recurrence after surgical resection for hepatocellular carcinoma (HCC) is very high. Several retrospective analyses have shown that liver transplantation was more effective than resection for patients with HCC at early tumor stages. Consequently, in January 1990, we decided to prospectively indicate orthotopic liver transplantation (OLT) as the first surgical treatment for small, localized HCC in cirrhotic patients without nodal involvement independently of the degree of liver function. The aim of this prospective cohort study was to analyze prognosis, recurrence rate, and survival after liver transplantation in patients in whom the main indication was HCC with cirrhosis. Thirty-eight patients in whom the main indication for liver transplantation was HCC and hepatic cirrhosis were compared with 136 transplantations because of cirrhosis without tumor, performed in our unit from January 1990 to December 1995. HCC arising in noncirrhotic livers and those incidently discovered after OLT were excluded from the study. Chemoembolization using doxorubicin, lipiodol, and Gelfoam was performed before OLT in 31 patients with good liver function. There were no differences in gender, but HCC patients were older (57 ± 7 vs. 50 ± 10 years [P < .001]). Liver function was better in HCC (Child-Pugh score: 6.9 ± 2 vs. 8.6 ± 1.8; P < .001), and hepatitis C virus antibody was positive in 31 (82%) vs. 51 (37%) (P < .007). Seven tumors had bilobar involvement (18%). Capsule was present in 22 (58%). The mean size of the tumor was 3.4 ± 2 cm. Seventeen tumors (45%) were larger than 3 cm, and 4 (11%) were larger than 5 cm. The average number of nodules was 2 ± 1. The tumor-node-metastasis stage of the tumors was pT1 in 6 patients (16%), 11 were pT2 (29%), 12 were pT3 (31%), and 9 were pT4 (24%). Seven patients were retransplanted in the HCC group (18%) and 19 (14%) in the nontumor group (not significant). Tumor recurrence was detected in three patients (8%). One, 3-, and 5-year survival rates were 82% vs. 79%, 75% vs. 71%, and 63% vs. 68%, respectively, for patients with and without HCC, and no differences were found between the two groups (P = .84). Survival was significantly reduced in patients with a macroscopic vascular invasion and tumors greater than 5 cm in diameter. Recurrence and mortality after liver transplantation in cirrhotic patients with carefully selected HCC are similar to the results in cirrhotic patients without tumor.
Hepatitis C in a French population-based survey, 1994: Seroprevalence, frequency of viremia, genotype distribution, and risk factors
Authors: Dubois F , Desenclos J , Mariotte N , Goudeau A
Published: July 2004

The aims of this study were the following: (1) to estimate the prevalence of hepatitis C virus (HCV) antibody (anti-HCV) in a population-based survey of French residents not selected for risk factors; (2) to investigate the association between anti-HCV seropositivity, viremia, the infecting HCV genotype, and the alanine transaminase (ALT) level; and (3) to identify risk factors for HCV infection by a nested case control study within this survey sample. The anti-HCV seroprevalence survey was performed in 6,283 volunteers (20- to 59-years-old) randomly selected from 45,377 consecutive individuals undergoing routine medical checkup in social security medical centers covering 4 of the 22 “regions” of France. Seventy-two volunteers were anti-HCV positive, a crude prevalence of 1.15%. Fifty percent of these positive volunteers also had an abnormal ALT level and 81% were HCV-RNA positive by polymerase chain reaction (PCR). The prevalence weighted for age, sex, and place of residence was 1.05% (95% CI: 0.75-1.34). The weighted prevalence was lower among men > 40-years-old (0.5%; 95% CI: 0.1-1.0) and was close to 1% in all other age and sex groups. Prevalence was inversely correlated with socioprofessional status with the highest rate being found among those with no paid employment (2.2%; 95% CI: 1.3-3.0). The HCV prevalence (1.7%; 95% CI: 1.0-2.3) was highest in southeastern France. Seventy-eight percent of positive intervenous (I.V.) drug abusers were infected with HCV genotypes 1a or 3, whereas 80% of the transfusion-associated cases were infected by HCV genotypes 1b or 2a. Only three variables were significantly associated with HCV seropositivity in multivariate analysis: I.V. drug abuse (21 cases, 14 men all < 40-years-old), previous transfusion (22 cases, 18 women), and not having paid employment. Although routes of transmission other than I.V. drug abuse and transfusion may not be formally excluded they were not found to be statistically significant. Hepatitis C appears to be a major public health concern in France. A more active screening policy may be required because only 17 of 72 cases (24%) were aware of their HCV seropositivity before enrollment in the study.
Clearance of hepatitis B surface antigen after bone marrow transplantation: Role of adoptive immunity transfer
Authors: Lau G K , Lok A S , Liang R H , Lai C L , Chiu E K , Lau Y L , Lam S K
Published: July 2004

Adoptive immunity transfer has been reported to be effective in clearing chronic hepatitis B virus (HBV) infection. Two hundred twenty-six patients who received allogeneic bone marrow transplantation (BMT) between May 1990 and September 1995 were screened for hepatitis B markers. Twenty-one patients were hepatitis B surface antigen (HBsAg) positive before BMT. The median follow-up period was 20 months (range, 2-59 months). Two of these patients had sustained clearance of HBV infection after transplantation. Both patients were hepatitis B e antigen (HBeAg)-negative, hepatitis B e antibody (anti-HBe)-positive, and serum HBV DNA-negative (by dot-blot hybridization) before BMT. Both had a flare in the serum alanine transaminase (ALT) level around the time of HBsAg clearance. Sustained clearance of HBsAg was observed in 2 of the 5 patients who received hepatitis B surface antibody (anti-HBs)-positive marrow but in none of the 16 patients who received anti-HBs-negative marrow (P < .05). One additional patient who received anti-HBs-positive marrow had transient HBsAg seroconversion. Among the 18 patients who remained persistently HBsAg-positive after BMT, 3 had HBeAg seroconversion and 3 had reversion to HBeAg positivity. In this study, we found a significant association between clearance of HBV infection and anti-HBs-positive bone marrow donors. Adoptive immunity transfer is effective in clearing HBV from patients with chronic HBV infection.
Interferon α induces disorder of lipid metabolism by lowering postheparin lipases and cholesteryl ester transfer protein activities in patients with chronic hepatitis C
Authors: Shinohara E , Yamashita S , Kihara S , Hirano K , Ishigami M , Arai T , Nozaki S , Kameda-Takemura K , Kawata S , Matsuzawa Y
Published: July 2004

The effect of recombinant interferon α 2a (rIFN-α2a) on serum lipoprotein metabolism was assessed in 39 patients with chronic viral hepatitis C. rIFN-α2a was administered intramuscularly at a dose of 9 x 106 U/d for 2 weeks and then for 3 times a week over 6 months. The serum cholesterol concentration significantly decreased one week after rIFN-α;2a administration. Approximately 67% of this decrease was attributed to the reduction of high-density lipoprotein (HDL)-cholesterol; a decrease in HDL2-cholesterol was more evident. By contrast, serum triglyceride levels, largely derived from very-low density lipoprotein (VLDL), significantly increased following rIFN-α2a treatment. Lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) activities in the postheparin plasma were reduced by 75.7% and by 79.4%, respectively, and decreases in plasma cholesteryl ester transfer protein (CETP) activity and its protein mass were also observed. However, prothrombin time was ameliorated by rIFN-α2a, suggesting that the decrease in LPL, HTGL, and CETP activities may not be due to a reduction in protein synthesis by the liver. Simple correlation analysis demonstrated that the changes in LPL activity before and after 2 weeks of treatment with rIFN-α2a showed a significant negative correlation with changes in serum triglyceride and VLDL-triglyceride and a positive correlation with changes in HDL-cholesterol and HDL2-cholesterol. These results suggest a major contribution of reduced LPL activity with regard to the lipoprotein disorders. In conclusion, rIFN-α2a treatment on patients with chronic hepatitis C causes marked changes in serum lipoprotein metabolism associated with decreases in LPL, HTGL, and CETP activities.
A mutation in the human canalicular multispecific organic anion transporter gene causes the Dubin-Johnson syndrome
Authors: Paulusma C C , Kool M , Bosma P J , Scheffer G L , ter Borg F , Scheper R J , Tytgat G N , Borst P , Baas F , Elferink R O
Published: July 2004

The human Dubin-Johnson syndrome (DJS) is a rare autosomal recessive liver disorder characterized by chronic conjugated hyperbilirubinemia. Patients have impaired hepatobiliary transport of non-bile salt organic anions. A highly similar phenotype has been described for a mutant Wistar rat strain, the transport-deficient (TR-) rat, which is defective in the canalicular multispecific organic anion transporter (cmoat). This protein mediates adenosine triphosphate-dependent transport of a broad range of endogenous and xenobiotic compounds across the (apical) canalicular membrane of the hepatocyte. The complementary DNA (cDNA) encoding rat cmoat has recently been cloned, and the mutation underlying the defect in TR- rats has been identified. In the present study, we have isolated the human homologue of rat cmoat, human cMOAT, and analyzed the corresponding cDNA from fibroblasts of a DJS patient for mutations. Our results show that a mutation in this gene is the cause of DJS.
Cytotoxicity of bile salts against biliary epithelium: A study in isolated bile ductule fragments and isolated perfused rat liver
Authors: Benedetti A , Alvaro D , Bassotti C , Gigliozzi A , Ferretti G , La Rosa T , Di Sario A , Baiocchi L , Jezequel A M
Published: July 2004

We evaluated cytotoxic effects of different unconjugated and glycine- and taurine-conjugated bile salts (BS) against bile duct epithelial cells in isolated bile ductule fragments and isolated perfused rat liver. Ultrastructural morphometric studies were performed in polarized rat bile ductule fragments exposed in vitro to increasing concentrations (10-100 μmol/L) of lithocholate (LCA), deoxycholate (DCA), chenodeoxycholate (CDCA), cholate (CA), ursodeoxycholate (UDCA), their taurine-conjugates, and glycoconjugates of cholic (GCA) or chenodeoxycholic acid (GCDCA) for 20, 30, or 75 minutes. To evaluate the cytotoxicity of unconjugated hydrophobic bile salts against biliary epithelium (BDE) in the whole liver, livers were isolated from rats with impaired taurine-conjugation capacity (β-alanine treatment) and perfused for 70 minutes with 2 μmol/min LCA (n = 6), CDCA (n = 6), CA (n = 6), or 0.5 μmol/min tauro-LCA (n = 4). In isolated bile ductule fragments, hydrophobic unconjugated bile salts (LCA, CDCA, DCA) induced a marked damage of intracellular organelles, mainly mitochondria. The damage started at a concentration of 10 μmol/L and became prominent at concentrations higher than 50 μmol/L. No damage of the apical and basolateral membrane was seen and tight junctions appeared intact. UDCA, taurine and glycoconjugated bile salts failed to induce any evident ultrastructural alteration. In taurine- depleted isolated livers, perfused with LCA, CDCA, or CA, bile duct epithelial cells showed no evidence of intracellular damage, despite the increased biliary excretion of unconjugated BS. Marked alterations of the apical cell membrane were seen only in livers perfused with LCA and in isolated segments of the biliary epithelium. In contrast with biliary epithelium, hepatocytes showed prominent subcellular damage with CA and CDCA, and profound alterations of the canalicular membrane with LCA and tauro-LCA. We have shown that, in vitro, BDE cells are not damaged by taurine- or glycine-conjugated BS, but they are very sensitive to cytotoxicity of hydrophobic unconjugated BS. Such sensitivity is not present in the whole liver, probably because of the specificity of BS transport processes, the microvascular architecture of the bile ductal system, and the presence in bile of a physiological surfactant, such as phospholipids.
Does antimitochondrial antibody status affect response to treatment in patients with primary biliary cirrhosis? Outcomes of ursodeoxycholic acid therapy and liver transplantation
Authors: Kim W R , Poterucha J J , Jorgensen R A , Batts K P , Homburger H A , Dickson E R , Krom R A , Wiesner R H , Lindor K D
Published: July 2004

Approximately 5% to 10% of patients with features otherwise consistent with primary biliary cirrhosis (PBC) lack antimitochondrial antibodies (AMA). Most of these patients have other autoantibodies, a syndrome recently named “autoimmune cholangitis.” We report our experience in patients with AMA-negative PBC treated with ursodeoxycholic acid (UDCA) and/or liver transplantation (OLT). The study of response to UDCA was performed as follows. While recruiting patients for a previously reported multicenter trial, we identified 8 patients with AMA-negative PBC. The patients were given UDCA and followed up at regular intervals. The characteristics of AMA-negative patients at presentation were similar to those of AMA-positive patients with PBC. The clinical outcomes and sequential liver biochemistries of UDCA treatment were also comparable with those of AMA-positive patients. The study of outcome of OLT was performed as follows. We identified OLT recipients at the Mayo Clinic who had clinical, radiological, and histological features compatible with PBC. Their pretransplant AMA status was determined, and each AMA-negative patient was paired with 2 AMA-positive patients. Of 85 OLT recipients with a diagnosis of PBC, 6 (7.1%) were AMA negative, including 1 who had undergone UDCA therapy. After a median of 36 months of follow-up, graft and patient survival rates and subsequent histological changes (disease recurrence and steroid-resistant or late rejections) were comparable in AMA-negative and -positive PBC patients. In summary, in our experience of 13 AMA-negative PBC patients (including 9 who met the criteria for a diagnosis of autoimmune cholangitis), treatment with UDCA or OLT resulted in similar outcomes to those found in AMA-positive patients. We conclude that AMA status does not affect the response in PBC patients to treatment with UDCA or OLT.
Enhanced G-protein-induced relaxation in portal hypertensive rats: Role of nitric oxide
Authors: Hou M , Cahill P A , Zhang S , Redmond E M , Sitzmann J V
Published: July 2004

Portal hypertension (PHT) is characterized by splanchnic hyperemia due to a reduction in mesenteric vascular resistance. The reasons for the decreased resistance include an increased responsiveness to a vasodilator substance. Because the activation of an inhibitory guanine nucleotide regulatory (Gi) protein can result in endothelium-dependent relaxation, we tested the hypothesis that exaggerated Gi-protein induced relaxation via a nitric oxide (NO)-dependent pathway partly reflects the enhanced Gi-protein expression in PHT vessels. PHT was created in Sprague-Dawley rats by a partial portal-vein ligation. Control animals were sham operated. Using isolated vascular rings in the absence or presence of an intact endothelium, N
G-nitro-L-arginine methyl ester (L-NAME), and pertussis toxin, dose response relationships for sodium fluoride (NaF; range, 0.1-4 mmol/L), a Gi protein activator, were determined in a cumulative manner. Gi-protein expression was determined by Western blotting. NaF caused a dose-dependent relaxation in both sham and portal hypertensive pre-contracted vessels, an effect that was significantly inhibited by pertussis toxin, endothelial denudation, and L-NAME. Concentrations of NaF greater than 4 mmol/L caused contractions, an effect that was unaffected by L-NAME. The NaF-induced relaxation response was significantly greater in PHT vessels as compared with sham concomitant with increased Gi-protein expression in PHT vessels. These data suggest that the enhanced endothelial Gi-protein-induced relaxation in PHT vessels may partly reflect enhanced expression of Gi-proteins in PHT vessels and may, thus, represent an important mechanism for exaggerated NO-dependent relaxation in the PHT vasculature.
Hemodynamic evaluation of the addition of isosorbide-5-mononitrate to nadolol in cirrhotic patients with insufficient response to the β- blocker alone
Authors: Merkel C , Sacerdoti D , Bolognesi M , Enzo E , Marin R , Bombonato G , Angeli P , Gatta A
Published: July 2004

The association β-blockers plus isosorbide-5-mononitrate (I5M) has been proposed for the treatment of portal hypertension in patients with insufficient response to β-blockers alone, according to hemodynamic criteria. The mechanism of action in these patients is not clearly defined. Fifteen patients with cirrhosis and esophageal varices were evaluated by hepatic venous pressure gradient (HVPG) measurement and duplex-Doppler ultrasonography before and after 1 month of treatment with nadolol. Nine patients who did not exhibit a decrease in HVPG to 12 mm Hg or a percent decrease greater than 20% were classified as poor responders, and were studied again with the same methodology after 3 months of chronic administration of nadolol + I5M 20 mg twice per day. In poor responders, mean HVPG decrease after nadolol was 8.9% ± 2.8%, and after the combination, it was 25.7% ± 1.7% (P = .004). All patients except one became good responders to the association. Portal blood flow (PBF) decreased significantly after nadolol (P = .004), and remained unchanged after the addition of nitrates. Resistance to portal blood flow (RPBF) increased after nadolol (P = .02) and returned to baseline values during combined treatment (P = .03). In good responders, an adequate decrease in HVPG was associated with a decrease in PBF (P = .06) but no change in RPBF. A wide spectrum of combined changes in PBF and in RPBF after nadolol was observed in poor responders, ranging from no change in either parameter to a marked decrease in PBF counterbalanced by a marked increase in RPBF. The addition of I5M was followed in most cases by larger effects on resistance than on flow. Doppler parameters were not significantly correlated with the HVPG response to nadolol alone or associated with I5M. It is concluded that good hemodynamic responders to nadolol differ from poor responders in the lack of increase in RPBF after the drug. The addition of nitrates to nadolol is effective in decreasing portal pressure in most poor responders to nadolol alone. A decrease in outflow resistance is the main mechanism involved.
Taste perception in cirrhosis: Its relationship to circulating micronutrients and food preferences
Authors: Madden A M , Bradbury W , Morgan M Y
Published: July 2004

Impairment of gustatory acuity may influence nutrient intake and hence nutritional status. The aim of this study was to evaluate gustatory acuity in patients with cirrhosis and its relationship to circulating concentrations of micronutrients, and food preferences. Gustatory evaluation was undertaken, using a rinsing technique, in 75 cirrhotic patients and 75 comparable healthy volunteers. Circulating concentrations of magnesium, zinc, vitamin A, and α- and β-carotene were measured, and food preferences were assessed by questionnaire. The cirrhotic patients showed impaired gustatory function with significantly higher (less sensitive) median thresholds for detection of salt, sweet, and sour and for recognition of bitter, salt, sweet, and sour, together with a higher overall median gustatory score (P < .0001). Mean circulating concentrations of magnesium, zinc, vitamin A, and α- and β-carotene were significantly lower in the patient population. Serum magnesium was significantly negatively associated with detection of salt (P = .02) and gustatory score (P = .02). Patients' subjective assessment of taste acuity did not correspond with objective measurements. Overall, no differences were observed in food preferences between the two groups, nor was any association found between food preferences and gustatory acuity. Patients with cirrhosis have impaired gustatory acuity that is associated with hypomagnesemia but apparently does not affect food selection.
Co-expression and regulation of Met and Ron proto-oncogenes in human hepatocellular carcinoma tissues and cell lines
Authors: Chen Q , Seol D , Carr B , Zarnegar R
Published: July 2004

Met and ron proto-oncogenes encode the cell surface receptors for hepatocyte growth factor (HGF) and hepatocyte growth factor-like (HLP) protein, respectively, and induce mitogenesis, motogenesis, morphogenesis, and metastatic activity in various cell types. Overexpression of met in human carcinoma has been reported by several groups including ours; however, the mechanisms that control met gene expression are thus far unclear. The present study focuses on the expression and regulation of the Met and Ron receptors in human hepatocellular carcinoma (HCC). We report here that abnormal expression of met and ron proteins occurs in some cases of human HCC. Using several HCC cell lines as a model system, we show that HGF, as well as other cytokines, such as epidermal growth factor (EGF), interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor α (TNF-α), induce met and ron expression. Using several chimeric constructs consisting of various lengths of the met promoter region fused to the reporter gene of chloramphenicol acetyl transferase (CAT), and by performing transient transfection of these constructs into HepG2 cells, we show that induction of met gene expression by HGF and other cytokines is, at least in part, through up-regulation of met gene promoter activity. The DNA region conferring responsiveness to cytokine induction was located within 0.2 kb of the met core promoter. Interestingly, EGF did not stimulate met promoter activity in any of the met-CAT chimeric constructs. These results provide evidence that met and ron are modulated in the liver by a similar cytokine network. In the case of met expression, the 0.2-kb region in the met gene promoter may play an important role in mediating its gene induction in response to HGF and other cytokines. Our results also suggest that unregulated expression of met and ron may be associated with pathological conditions, such as HCC, in the liver.
Mechanism of the impairment of the glucagon-stimulated phosphoenolpyruvate carboxykinase gene expression by interleukin-6 in rat hepatocytes: Inhibition of the increase in cyclic 3′,5′ adenosine monophosphate and the downstream cyclic 3′,5′ adenosine monophosphate action
Authors: Christ B , Nath A , Jungermann K
Published: July 2004

In cultured rat hepatocytes, the gluconeogenic key enzyme, phosphoenolpyruvate carboxykinase (PCK), is induced by glucagon via elevation of cyclic 3′,5′ adenosine monophosphate (cAMP). The proinflammatory cytokine, interleukin-6 (IL-6), which in the liver together with IL-1β and tumor necrosis factor α triggers the acute-phase response, had been shown to attenuate the glucagon-induced increase in PCK gene transcription, messenger (mRNA) levels, and enzyme activity. The molecular mechanism of this inhibition was investigated in the present study. Glucagon increased cyclic cAMP and PCK mRNA levels to a transient maximum twofold and fivefold, respectively. The increases were attenuated by IL-6. Forskolin, which stimulates adenylate cyclase activity, increased cAMP and PCK mRNA levels 1.6-fold and fivefold, respectively. However, IL-6 attenuated the forskolin- stimulated increase in PCK mRNA but not the increase in cAMP. This showed that IL-6 inhibited PCK mRNA increase in part by the attenuation of cAMP increase, but also beyond cAMP formation. This was confirmed in experiments in which PCK mRNA levels were increased by the nonhydrolyzable cAMP-analogue, chlorophenylthio (CPT)-cAMP. The increase in PCK mRNA was again attenuated by IL-6. In pertussis toxin- and in isobutylmethylxanthine-treated hepatocytes, IL-6 still inhibited the glucagon-stimulated increase in cAMP, indicating that IL-6 did not activate an inhibitory G-protein or phosphodiesterase, which could cause the impairment of cAMP increase. To demonstrate whether the inhibition of PCK gene expression by IL-6 beyond cAMP might be caused by the inhibition of the activation of the PCK gene promoter by cAMP, cultured rat hepatocytes were transfected with a luciferase reporter gene construct under the control of a PCK gene promoter fragment (base −979 to base +32). Luciferase activity was determined after stimulation of the cells with CPT-cAMP in the absence or presence of IL-6. CPT-cAMP increased luciferase activity by 1.7-fold, which was inhibited in the presence of IL-6. It is concluded that IL-6 had a dual inhibitory effect on the stimulation of PCK gene expression by glucagon. It inhibited the increase in cAMP at a site before cAMP formation by adenylate cyclase and at a site after cAMP formation, the activation of the PCK gene promoter by cAMP.
In situ detection of lipid peroxidation by-products in chronic liver diseases
Authors: Paradis V , Kollinger M , Fabre M , Holstege A , Poynard T , Bedossa P
Published: July 2004

Lipid peroxidation is an autocatalytic mechanism leading to oxidative destruction of cellular membranes. The deleterious consequences of this mechanism are related in part to the formation of reactive aldehydic products that bind to intra- or extracellular molecules to form adducts. Specific antibodies directed against malondialdehyde (MDA) and 4-hydroxynonenal (HNE) adducts, major aldehydic metabolites of lipid peroxidation, allowed us to investigate in situ, with an immunohistochemical procedure, the occurrence of lipid peroxidation in a panel of different chronic liver diseases. Intracellular HNE and MDA adducts were detected respectively in 24 of 39 cases (62%) and in 12 of 34 cases investigated (35%). They were localized mainly in the cytoplasm of hepatocytes, with the strongest staining observed in hemochromatosis, Wilson's disease, and in areas of acute alcoholic hepatitis in cases of alcoholic liver diseases. A peculiar pattern of immunostaining was observed in primary biliary cirrhosis where biliary cells of destroyed but also intact bile ducts strongly expressed HNE adducts. The liver extracellular matrix also displayed MDA adducts (30 of 34 cases, 88%) and HNE adducts (23 of 39 cases, 59%). While HNE adducts were specifically localized on large bundles of collagen fibers, MDA adducts were detected in a thin reticular network and in sinusoidal cells around portal tracts or fibrous septa. In conclusion, lipid peroxidation by-products are detectable in chronic liver diseases. Immunohistochemical results suggest that this mechanism is implicated very early in the pathogenesis of some of these diseases.
Differential activation of heat shock and nuclear factor κB transcription factors in postischemic reperfused rat liver
Authors: Tacchini L , Radice L , Pogliaghi G , Bernelli-Zazzera A
Published: July 2004

The aim of this study was to investigate the behavior of the transcription factors, heat-shock factor (HSF) and nuclear factor κB (NF-κB), in postischemic reperfused liver, with particular attention paid to possible differences in the time-course and mechanism of activation, which may help in defining their role in the response of the liver to reperfusion. Ischemia was induced by clamping the hilar pedicle of the left lateral and median liver lobes; the clamp was removed after 1 hour. Some rats were treated intraperitoneally with IL-1 receptor antagonist (IL-1RA) 30 minutes before ischemia and at the time of reperfusion. Binding of NF-κB to the corresponding consensus sequence is activated after 30 minutes of reperfusion, and is still increased 1 hour after reperfusion. Activation is suppressed in rats treated with IL-1RA; NF-κB persists in the cytosol associated with the inhibitor, IκB, and can be artifactually activated in vitro. Super-gel shift experiments revealed that the two subunits, p50 and p65, are involved in the activation of binding. In contrast, binding of HSF to the corresponding consensus sequence, heat shock element (HSE), is already activated at the end of ischemia, shows a further increase after 30 minutes of reperfusion, but declines 1 hour after reperfusion; more importantly, it is not inhibited by pretreatment of the rat with IL-1RA. In conclusion, although both HSF and NF-κB are activated by ischemia-reperfusion, there are clear differences in time-course and mechanism of activation of the two transcription factors. Activation of HSF depends directly on some events occurring during ischemia; NF-κB is activated only after reperfusion and the concurrent oxidative stress, by an indirect mechanism that can be suppressed by IL-1RA. The possibility of dissociating the activation of these two transcription factors in postischemic reperfusion can have a prospective clinical relevance.
Dose-dependent acute clearance of hepatitis C genotype 1 virus with interferon alfa
Authors: Lam N P , Neumann A U , Gretch D R , Wiley T E , Perelson A S , Layden T J
Published: July 2004

To determine if the clearance of hepatitis C genotype 1 virus (HCV) is dependent on the dose of interferon alfa-2b (IFN-α2b), the acute clearance of HCV after a single dose of either 3, 5, or 10 mIU of IFN-α was compared in patients with chronic hepatitis C. HCV-RNA levels following IFN-α administration were measured. At 24 hours, mean percentage serum viral reduction was 41.4%, 63.7%, and 85.5% for 3, 5, and 10 mIU, respectively (P < .001). At 48 hours, the mean viral reduction was consistently less than the reduction at 24 hours, averaging 22.9%, 61.9%, and 74.3%, respectively (P < .001), indicating that the drug effect diminishes before 48 hours. Regression analysis showed a positive correlation between dose and percent reduction of HCV- RNA levels (r = .6; P < .001). A mathematical model showed that such dose dependence is expected if IFN-α partially blocks viral production. Minimum clearance and production rates of HCV were estimated from measurements of HCV-RNA levels after the 10-mIU dose. HCV decay followed an exponential decline with a minimum estimate of the viral clearance rate constant of 2.8 per day, corresponding to a virion half-life of 0.3 days or less. A minimal estimate of the daily HCV production and clearance is 3.7 × 1011 virions per day, indicating a high rate of replication and turnover. These results indicate that there is a dose-dependent effect of IFN-α in clearance of HCV genotype 1. Because the virion production rate is very rapid and because the current recommended dose of IFN-α (3 mIU) is often ineffective, larger doses should be considered to treat genotype 1-infected patients.
Circulatory effects of graded diversion of portal blood flow to the systemic circulation in rats: Role of nitric oxide
Authors: Bernadich C , Bandi J C , Piera C , Bosch J , Rodes J
Published: July 2004

Total portal-systemic shunting in normal animals is associated with splanchnic and systemic vasodilation, suggesting that vasodilation in cirrhosis may be facilitated by spontaneous shunts promoted by portal hypertension. However, the mechanism of this vasodilation is unknown. The aim is to study the acute effects of the graded diversion of portal blood flow to the systemic circulation in normal and portal hypertensive rats. Portal and systemic hemodynamics were measured in normal and portal hypertensive rats before and during graded portacaval diversion of portal blood flow, in basal conditions, and after nitric oxide inhibition. In portal hypertensive rats, graded portal flow diversion caused a rate-related decrease in portal pressure (from 15.3 ± 0.6 to 11.2 ± 0.7 mm hg at 6 mL × min(-1), P < .001) and a redistribution of portal-collateral blood flow from the spontaneous portal-systemic collaterals to the portacaval circuit, without changing total portal-systemic shunting. Graded portal diversion caused an immediate systemic vasodilation, with reduced peripheral resistance. This vasodilatory response was more pronounced in normal than in portal hypertensive rats (increase in cardiac index 23.6% ± 2.8% vs. 8.5% ± 4.9%, P < .02, fall in peripheral resistance -24.5% ± 3.4% vs. -16.5% ± 2.6%, P = .08), and was totally prevented by nitric oxide inhibition. The graded diversion of portal blood flow caused a flow- rate-related reduction in portal pressure and blood flow through spontaneous portal-systemic collaterals in portal hypertensive rats, and caused a nitric-oxide dependent systemic vasodilatory response, which was greater in normal than in portal hypertensive rats. These results suggest that portal-systemic shunting per se may contribute to the vasodilatation in portal hypertension.
Cerebral benzodiazepine receptor binding in vivo in patients with recurrent hepatic encephalopathy
Authors: Macdonald G A , Frey K A , Agranoff B W , Minoshima S , Koeppe R A , Kuhl D E , Shulkin B L , Lucey M R
Published: July 2004

Increased activation of the central benzodiazepine receptor (BZR) appears to play an important role in hepatic encephalopathy (HE). However, there is controversy regarding whether the density or affinity of BZRs is altered. A previous positron emission tomography (PET) study using the BZR antagonist [11C]flumazenil (FMZ) found two- to threefold greater cerebral cortical tracer uptake in recurrent HE, but did not account for impaired FMZ metabolism due to liver disease or assess the relative contributions of tracer delivery versus BZR binding. We hypothesized that correcting for these factors would affect estimations of BZR binding in HE. Nine patients with recurrent HE and 13 age-comparable controls were studied with [11C]FMZ PET. After intravenous administration of [11C]FMZ, arterial blood samples were collected, and PET images were acquired over 60 minutes. FMZ transport and binding maps were calculated for each subject by using a physiological tracer kinetic model. In agreement with the previous report, we found that FMZ reached a much higher level and was retained longer in the HE cerebral cortex despite similar total blood radioactivity levels in the two groups. However, the patients showed impaired hepatic metabolism of FMZ. After physiological modeling incorporating these data, significant increases in BZR binding were found in the thalamus (13%), cerebellum (20%), and pons (23%). There were minor, statistically insignificant increases in cerebral cortical (10%), putamen (12%), and whole brain (12%) BZR binding in patients with recurrent HE. These findings are in general agreement with results of autopsy studies, confirming a lack of major increases in cortical or basal ganglial BZR binding in HE. They emphasize that physiological tracer modeling should be used and altered peripheral radioligand metabolism considered in future PET studies of HE.
Cell growth and differentiation of a novel mouse Ito (fat-storing) cell line transformed by a temperature-sensitive mutant of simian virus 40
Authors: Kitamura Y , Tanigawa T , Katsumoto T , Tomita K , Wang H , Hirai K , Ichihara K , Terada T
Published: July 2004

A novel mouse Ito (fat-storing) cell line (A640-IS) was established by transformation with a temperature-sensitive mutant of simian virus 40 (SV40) and the relationships between the expression of SV40 large T antigen and the growth, differentiation, and functions of A640-IS cells were investigated. A640-IS cells expressed large T antigen when cultured at 33 degrees C. At this temperature, the cells grew actively, assumed a fibroblastic shape, and showed few Ito cell characteristics. In contrast, when large T-antigen expression was inhibited by culture at 39 degrees C, the cells did not grow but differentiated into Ito cells as assessed by both morphological and functional characteristics. Expression of the transcription factor SCL (stem cell leukemia), which plays a role in the development and differentiation of blood cells, was observed at both 33 degrees C and 39 degrees C, although expression was greater at 33 degrees C. Therefore, opposite patterns of cell growth and the functions of differentiated cells occurred at 33 degrees C and 39 degrees C in this novel Ito cell line. Transforming growth factor β1 stimulated A640-IS cells to produce fibronectin, collagen type III, and laminin. This unique Ito cell line provides a useful model to address important questions regarding the nature of these cells.
S-adenosyl-L-methionine protects the liver against the cholestatic, cytotoxic, and vasoactive effects of leukotriene D4: A study with isolated and perfused rat liver
Authors: Cincu R N , Rodriguez-Ortigosa C M , Vesperinas I , Quiroga J , Prieto J
Published: July 2004

Cysteinyl-leukotrienes can cause cholestasis and liver damage when administered at nanomolar concentrations. Using the isolated and perfused rat liver we analyzed whether S-adenosyl-L-methionine (SAMe) may protect this organ against the noxious effects of leukotriene-D4 (LTD4). We observed that a 2 nmol bolus of this compound decreased bile flow (-12.6% ± 1.6%, P < .02), and bile salt excretion (-23.5% ± 2.2%, P < .02; both compared with baseline values), caused the release of glutamic-oxaloacetic transaminase (GOT) and lactic dehydrogenase (LDH) to the hepatic effluent, and increased significantly the perfusion pressure as compared with a control group not receiving LTD4 (6.0 ± 1.1 vs. 0.2 ± 0.02 mm hg, respectively; P < .001). The cholestatic effect of LTD4 was attenuated by infusion of SAMe which, at rates of 67 and 100 microg/min, totally prevented the decrease in bile salt excretion. Likewise, in SAMe infused livers, the release to the effluent of GOT and LDH was lower than in the group receiving LTD4 only, and was even lower than in the control group. We also found that the increase in perfusion pressure induced by LTD4 was prevented by SAMe in a dose-dependent manner. Of interest, SAMe increased the biliary excretion of the eicosanoid in a dose-related fashion. We conclude that SAMe reverts the cholestatic, cytotoxic, and hemodynamic effects of LTD4 on the liver, and that these protective effects might be partly because of a stimulation of the biliary excretion of the leukotriene.
Role of ischemia in causing apoptosis, atrophy, and nodular hyperplasia in human liver
Authors: Shimamatsu K , Wanless I R
Published: July 2004

Ischemia is known to be a cause of hepatocellular apoptosis and atrophy in experimental animals, but the effect of vascular obstruction on such lesions in the normal or cirrhotic human liver has not been studied. The purpose of this study was to investigate the role of ischemia in the development of apoptosis, atrophy, and nodular hyperplasia in cirrhotic and noncirrhotic human livers. Apoptosis, focal atrophy, and nodular hyperplasia were semiquantitated in 203 liver specimens obtained at transplantation, segmental resection, or autopsy. These parameters were correlated with etiology, stage, activity, and acute and healed portal vein thrombosis (PVT). Large numbers of apoptotic cells were found in livers with acute PVT (17.2/medium-power field [MPF]) and in infarcts of Zahn caused by obstruction of portal veins (PVs) by tumor (16.4/MPF). Smaller numbers of apoptotic cells were found in cirrhosis of various etiologies (3.8-10.0/MPF) and rarely in normal livers (0.16/MPF). Evidence of healed PVT was found in 47% of cirrhotic livers and was associated with nodular hyperplasia (58% vs. 32%, P < .01) and focal atrophy (79% vs. 49%, P < .002). Apoptotic cells were found equally in those with and without healed PVT (40% vs. 38%, not significant). These observations suggest that apoptosis is a transient response to acute ischemia and that atrophy and nodular hyperplasia are chronic responses to ischemia. Vascular obstruction may be an important cause of the apoptosis and atrophy, which are found in nodular regenerative hyperplasia (NRH), infarct of Zahn, chronic hepatitis, and cirrhosis.
Oxygen-free radical-mediated injury to cultured rat hepatocytes during cold incubation in preservation solutions
Authors: Rauen U , Reuters I , Fuchs A , de Groot H
Published: July 2004

We have previously shown that the injury to cultured liver endothelial cells during cold incubation in University of Wisconsin (UW) solution is energy-dependent and is mediated by reactive oxygen species. Here we demonstrate that this reactive oxygen-mediated injury is specific neither to endothelial cells nor to UW solution: cultured hepatocytes incubated in cold (4 degrees C) UW solution or histidine-tryptophan-ketoglutarate (HTK) solution were injured under normoxic conditions (loss of viability, 63% ± 10% after 48 hours of cold incubation in UW solution and 82% ± 11% after 24 hours of cold incubation in HTK solution), whereas hypoxia was protective (loss of viability, 29% ± 12% [UW] and 13% ± 3% [HTK] after the same cold incubation times). The injury under normoxic conditions was also largely decreased by adding either the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) or the flavonoid silibinin to the solutions, or by preincubating the cells with the iron chelator deferoxamine before the hypothermic incubation. Marked lipid peroxidation was observed during cold incubation in both preservation solutions. These results suggest that the injury to cultured hepatocytes during cold incubation in UW and HTK solutions is mediated by reactive oxygen species as is the injury to cultured liver endothelial cells.