bluuebunny/crossref_metadata_2025 huggingface dataset:
Specific inhibition of hepatitis C viral gene expression by antisense phosphorothioate oligodeoxynucleotides
Authors: Alt Michael , Renz Renate , Hofschneider Peter H. , Paumgartner Gustav , Caselmann Wolfgang H.
Published: March 2007

The inhibitory effect of antisense phosphorothioate oligodeoxynucleotides (S-ODN) on hepatitis C viral gene expression was analyzed in an in vitro test system and in cell culture. S-ODN were directed against different stem loop structures in the 5′noncoding region (NCR) of the hepatitis C virus (HCV) RNA and against a nucleotide stretch, including the start codon of the polyprotein precursor. The inhibitory effect of these S-ODN was quantified employing a viral RNA consisting of the first 407 nucleotides of a HCV type 1b genome fused to the coding sequence of the firefly luciferase gene. For in vitro assays this RNA was generated by in vitro transcription and used as a template in a rabbit reticulocyte lysate in vitro translation system. The production of active luciferase in the absence or presence of S-ODN was monitored using an enzymatic assay. The best results were obtained with S-ODN 4 directed against nucleotides 326 to 348, comprising the start AUG of the poly-protein coding sequence. With this oligonucleotide, a specific and dose-dependent effect was observed with a maximal inhibition of 96 ± 1% at a S-ODN concentration of 4.14 μmol/L. For cell culture experiments, the hepatoblastoma cell line HepG2 was transfected with a plasmid expressing the HCV-luciferase fusion RNA. In this assay system S-ODN 2, complementary to nucleotides 264 to 282 of the HCV RNA, and S-ODN 4 were most efficient and reduced the viral translation by 96 ± 0.4% and 94 ± 0.7%, respectively, at a concentration of 0.3 μmol/L. The inhibition was specific (1) because the expression of the HCV-luciferase fusion RNA was not significantly impaired by the control S-ODN and (2) because the expression of an unrelated messenger RNA was not or only slightly downregulated. These data suggest that HCV gene expression can be inhibited effectively by antisense S-ODN. Therefore, this approach represents a promising perspective for the treatment of hepatitis C. (Hepatology 1995; 22:707-717.)
Cerebral blood flow autoregulation and transcranial doppler sonography in patients with cirrhosis
Authors: Larsen Fin Stolze , Olsen Karsten Skovgaard , Ejlersen Ellen , Hansen Bent Adel , Paulson Olaf B. , Knudsen Gitte Moos
Published: March 2007

Impairment of cerebral blood flow (CBF) autoregulation may have serious implications for patients with cirrhosis if arterial hypotension occurs during coma, anesthesia, bleeding, or sepsis. In this study, CBF autoregulation was investigated in patients with cirrhosis with no or mild encephalopathy. Ten patients (median age, 45 years; range, 30 to 61 years) and six healthy volunteers (median age, 30 years; range, 21 to 61 years) were included. Catheters were placed in a radial artery and in the internal jugular vein. Baseline CBF was measured using single-photon emission computed tomography (SPECT) with concomitant measurements of cerebral arteriovenous oxygen content differences (AVDO2). CBF autoregulation was evaluated using the AVDO2 method and changes in mean flow velocity in the middle cerebral artery (Vmean) as determined by transcranial Doppler (TCD). Mean arterial pressure (MAP) was increased by 30 mm Hg by intravenous norepinephrine, and subsequently decreased by a combination of lower body negative pressure and ganglion blockade, whereas AVDO2 and Vmean were measured at each 5 mm Hg change in MAP. CBF was 61 (range, 45 to 78) mL 100 g−1 min−1 in patients with cirrhosis and 65 (range, 53 to 88) mL 100 g−1 min−1 in volunteers (not significant [NS]). There were no regional differences in CBF between the two groups. Arterial carbon dioxide tension was 31 (23 to 35) mm Hg in patients with cirrhosis and lower, compared with 36 (range, 34 to 37) mm Hg in the volunteers (P ± .01). For evaluation of autoregulation, MAP was raised to 116 (range 100 to 145) and then decreased to 39 (range, 34 to 50) mm Hg. In 8 of 10 patients with cirrhosis, a lower limit of autoregulation of 74 (range 64 to 103) mm Hg was identified, similar to the value of 74 (range, 53 to 79) mm Hg in the controls. In the two remaining patients with cirrhosis, no lower limit could be established. A statistically significant correlation between Vmean and CBF below the lower limit of autoregulation was found in patients with cirrhosis (CBF = 22 + 0.75 ± Vmean; r
2 = .60; P ± .005). CBF autoregulatory capacity seems generally preserved in patients with cirrhosis but the lower limit may be impaired in some patients and can be reliably evaluated using TCD sonography. (Hepatology 1995; 22:730-736.)
Alterations of peroxisomes in steatosis of the human liver: A quantitative study
Authors: De Craemer Dirk , Pauwels Marina , Van den Branden Christiane
Published: March 2007

We investigated the hepatocellular peroxisomes in 27 patients with steatosis of the liver by means of catalase cytochemistry, light and electron microscopic study, and morphometry. Seven normal human livers were used as controls. In our patients, fatty liver was mainly associated with alcohol abuse or obesity. Indications for a slight decrease in catalase activity and for a proliferation were found by visual evaluation of the peroxisomes. Morphometric analysis showed a significant decrease in mean peroxisomal diameter (to 87%) and a simultaneous significant elevation in numerical density of the peroxisomes (to 188%); this resulted in a normal volume density and a significant increase (to 133%) in surface density. However, individual differences were found. No differences in peroxisomal characteristics were found between fatty livers of different causes. A significant inverse linear correlation between mean peroxisomal diameter and numerical density was found in patients with fatty livers. Because a similar correlation was also found when control data were added to the fatty liver data, we hypothesize that the peroxisomal compartment in human fatty livers is adapted in such a way to permit the same metabolic efficiency as in control livers. (Hepatology 1995; 22:744-752.)
Beneficial effects of intravenous albumin infusion on the hemodynamic and humoral changes after total paracentesis
Authors: Luca Angelo , García-Pagán Juan C. , Bosch Jaume , Feu Faust , Jiménez Wladimiro , Ginés Angels , Fernández Mercedes , Escorsell Angels , Arroyo Vicente , Rodés Joan
Published: March 2007

The study investigated the hemodynamic and neuro-humoral effects of albumin infusion after total paracentesis in 18 patients with cirrhosis and tense ascites. Measurements of systemic and splanchnic hemodynamics, and vasoactive neurohumoral systems were performed before and immediately after total paracentesis. The patients were then randomized to receive albumin or not, and hemodynamic and humoral measurements were repeated at 24 hours. Hemodynamic and humoral changes just after paracentesis were similar in patients later randomized to receive albumin infusion or not. Twenty-four hours after total paracentesis, patients not receiving albumin had significant reductions in cardiac index (−13%; P = .005), femoral blood flow (−17%; P = .004), and pulmonary capillary pressure (−16%; P = .02), which were accompanied by significant increases in plasma renin activity (PRA) and plasma aldosterone (PA) and by significant decreases in atrial natriuretic factor (ANF) and plasma sodium. By contrast, there were no significant changes in patients receiving albumin, except for an increase in ANF and a further decrease in PA. In both groups, hepatic venous pressure gradient (HVPG) and azygos blood flow decreased just after paracentesis returning to baseline at 24 hours. This study shows that albumin infusion prevents the impairment in systemic hemodynamics, vasoactive neurohumoral systems, and plasma sodium after a large-volume paracentesis, without detrimental effects on portal pressure and portocollateral blood flow. (Hepatology 1995; 22:753-758.)s
A randomized, double-blind, placebo-controlled trial of ursodeoxycholic acid in primary biliary cirrhosis
Authors: Combes Burton , Carithers Robert L. , Maddrey Willis C. , Lin Danyu , McDonald Mary F. , Wheeler Donald E. , Eigenbrodt Edwin H. , Muñoz Santiago J. , Rubin Raphael , Garcia-Tsao Guadalupe , Bonner Gregory F. , West Alexander B. , Boyer James L. , Luketic Velimir A. , Shiffman Mitchell L. , Mills Scott A. , Peters Marion G. , White Heather M. , Zetterman Rowen K. , Rossi Stephen S. , Hofmann Alan F. , Markin Rodney S.
Published: March 2007

One hundred fifty-one patients with primary biliary cirrhosis (PBC) grouped into four strata based on entry serum bilirubin (<2 mg/dL vs. 2 mg/dL or greater) and liver histology (stages I, II vs. stages III, IV-Ludwig criteria) were randomized within each stratum to ursodiol or placebo given in a single dose of 10 to 12 mg/kg at bedtime for 2 years. Placebo- (n = 74) and ursodiol-treated (n = 77) patients were well matched at baseline for demographic and prognostic factors. Ursodiol induced major improvements in biochemical tests of the liver in strata 1 and 2 (entry bilirubin <2), but had less effect on laboratory tests in patients with entry serum bilirubin of ≥2 (strata 3 and 4). Histology was favorably affected by ursodiol in patients in strata 1 and 2 but not in strata 3 and 4. Ursodiol enrichment in fasting bile obtained at the conclusion of the trial was approximately 40% and comparable in all strata. Thus, differences in ursodiol enrichment of the bile acid pool do not explain better responses of laboratory tests and histology found in patients with less advanced PBC. Patients treated with ursodiol tended to develop a treatment failure less frequently than those who received placebo, particularly in strata 1 and 2 (ursodiol 42%, placebo 60%, P = .078). Development of severe symptoms (fatigue/pruritus) and doubling of serum bilirubin were reduced significantly in ursodiol-treated patients. Major complications of liver disease, progression to liver transplantation or death, occurred in 10.5% and 76.6%, respectively, in patients who had an entry serum bilirubin of <2 or ≥2 mg/dL. The incidence of these complications was comparable in ursodiol- and placebo-treated patients. Treatment failure occurred sooner in placebo than in ursodiol-treated patients in strata 1 and 2 but at the same rate in similarly treated patients in strata 3 and 4. Patients with advanced disease are unlikely to benefit from ursodiol. Trials longer than 2 years will likely be needed to determine whether ursodiol reduces major complications of liver disease in patients with milder disease. (Hepatology 1995;22:759-766.)
Acetaminophen (paracetamol) hepatotoxicity with regular intake of alcohol: Analysis of instances of therapeutic misadventure
Authors: Zimmerman Hyman J. , Maddrey Willis C.
Published: March 2007

Hepatic injury in alcoholics due to intake of acetaminophen (APAP or acetylparaaminophenol) with therapeutic intent has been reported, but the extent of the phenomenon is not clear, pertinent details of the association remain insufficiently clarified, and the importance of the phenomenon is not widely appreciated. The present report describes 67 patients who developed hepatic injury after ingestion of APAP with therapeutic intent. All were regular users of alcohol. Sixty-four percent of the patients were considered to be “alcoholic” or reported intakes greater than 80 g/d, 35% took 60 g/d or less, and the remainder were vague in their reporting. Doses of APAP were in the “nontoxic” range (<6 g/d) in 60% of the group, within the recommended range (<4 g/d) in 40%, and at 4.1 to 6 g/d in 20%. Characteristic feature was the towering level reached by aspartate transaminase (AST) with figures ranging from 3,000 to 48,000 IU in more than 90% of cases. Almost 20% of the patients died. The data on these patients were similar to 94 cases of injury from APAP taken with therapeutic intent reported in the literature. This study provides further evidence of hepatic injury in regular users of alcohol, especially chronic alcoholics, who take APAP with therapeutic intent. Susceptibility is presumably caused by induction of cytochrome P-4502EI by ethanol and by depletion of glutathione (GSH) because of the effects of alcohol, the malnutrition often associated with alcoholism, and the depletion associated with chronic use of APAP and impaired glucuronidation caused by fasting perhaps as well. The syndrome of liver injury is distinctive, marked by uniquely elevated levels of AST, and poses a significant threat. A greater awareness of the phenomenon by the medical and lay community is essential. (Hepatology 1995;22:767-773.)
p53 protein immunoreactivity in extrahepatic bile duct and gallbladder cancer: Correlation with tumor grade and survival
Authors: Diamantis Ioannis , Karamitopoulou Eva , Perentes Elias , Zimmermann Arthur
Published: March 2007

Forty-five extrahepatic bile duct carcinomas (i.e., tumors of the region proximal to the duct junction, including Klatskin tumors, tumors of the lower mid-region, and tumors of the ampulla of Vater) and 11 gallbladder carcinomas were immunohistochemically examined for p53 protein expression, using the DO-7 monoclonal (mAb) and the CM-1 polyvalent (pAb) antibodies and an antigen retrieval method. Because the DO-7 mAb was found to be significantly more reliable than the CM-1 pAb in detecting p53 immunoreactivity, the immunohistochemical results obtained with the former antibody were used for comparing p53 protein immunoreactivity with tumor site, tumor grade, and survival of patients. Approximately one third (3 of 10) of the proximal tumors were found to express weak p53 immunopositivity, whereas moderate immunopositivity and higher rate (18 of 29) was observed in tumors of the lower mid-region. Finally, moderate and marked p53 immunopositivity was observed in tumors of the ampulla (5 of 6) and gallbladder (8 of 11). In tumors of the lower mid-region of the ampulla and the gallbladder, a significantly higher p53 positivity was noted in high-grade compared with lowgrade neoplasms. For the cases in which complete follow-up was available (11 tumors of the low mid-region), median survival of patients with p53-negative tumors was 25.7 months, whereas survival of those with p53-positive tumors was 5.2 months. In conclusion, our findings suggest that (1) p53 protein is differentially expressed in extrahepatic bile duct carcinomas, thus stressing the clinical and histological distinction between proximal and lower duct tumors, (2) in a subset of the above carcinomas, p53 protein expression appears to be related to higher grades of malignancy, and (3) the presence of p53 immunopositivity might be associated with a shorter survival of the patients. (Hepatology 1995; 22:774-779.)
Thrombin stimulates proliferation of liver fat-storing cells and expression of monocyte chemotactic protein-1: Potential role in liver injury
Authors: Marra Fabio , Grandaliano Giuseppe , Valente Anthony J. , Abboud Hanna E.
Published: March 2007

Liver fat-storing cells (FSC) proliferate and secrete extracellular matrix in experimental models of liver injury. In this study, we determined if thrombin, a serine protease produced during acute and chronic tissue injury, modulates the functions of FSC. Thrombin stimulated DNA synthesis and proliferation of FSC, as assessed by [3H]-thymidine incorporation assay and measurement of cell number, respectively. Thrombin also increased the secretion of monocyte chemotactic protein-1 (MCP-1) in a time-and dose-dependent fashion. The effect of thrombin on both DNA synthesis and MCP-1 secretion was neutralized by pretreatment of thrombin with hirudin. The increased MCP-1 secretion was associated with increased steady-state levels of MCP-1 messenger RNA. Pretreatment of FSC with 5 μmol/L retinol for 48 hours inhibited the mitogenic effects of thrombin but not the induction of MCP-1 secretion. FSC express specific transcripts encoding for the human thrombin receptor, as shown by Northern blot analysis of poly (A)+ RNA. Proteolytic activation of the thrombin receptor results in the formation of a new N-terminus that functions as a tethered ligand. We studied the effects of a thrombin receptor activating peptide (TRAP) corresponding to the newly formed N-terminus, on FSC. TRAP mimicked the effects of thrombin on [3H]-thymidine incorporation, MCP-1 secretion, and MCP-1 gene expression. This study suggests that thrombin may be involved in modulating FSC proliferation and monocyte chemotaxis during human liver disease, through proteolytic activation of its receptor. (Hepatology 1995; 22:780-787.)
Human myofibroblastlike cells obtained by outgrowth are representative of the fibrogenic cells in the liver
Authors: Blazejewski Sylvie , Preaux Anne-Marie , Mallat Ariane , Brocheriou Isabelle , Mavier Philippe , Dhumeaux Daniel , Hartmann Daniel , Schuppan Detlef , Rosenbaum Jean
Published: March 2007

During human fibrogenesis, myofibroblastlike cells proliferate and are the main source of fibrosis components. We have used cultured myofibroblastlike cells obtained by outgrowth from explants of human liver to study the expression of extracellular matrix (ECM) components and matrix-metalloproteinase-2 (MMP-2). These cells contained types I, III, IV, and V procollagen messenger RNAs (mRNAs). They also expressed mRNAs for laminin B1 chain and for cellular and plasma fibronectin. The corresponding proteins were detected by immunocytochemistry. MMP-2 expression was shown by Northern blot and gelatin zymography. Because transforming growth factor β1 (TGFβ1) is considered an important mediator in liver fibrogenesis, we examined its effect on expression of ECM components by cultured human myofibroblastlike cells. TGFβ1 increased collagen mRNAs steady-state levels and total collagen secretion in the culture medium. It also increased fibronectin mRNA levels but had no effect on laminin mRNA or MMP-2 expression. In summary, cultured human myofibroblastlike cells express those ECM components that accumulate during hepatic fibrogenesis, indicating the usefulness of this model to study mechanisms of human liver fibrogenesis. In addition to the mitogenic effect of TGFβ1 on human myofibroblastlike cells, we now demonstrate its stimulation of ECM accumulation in these cells, thus emphasizing the central role of TGFβ1 and myofibroblastlike cells in the pathophysiology of human hepatic fibrosis. (Hepatology 1995; 22:788-797.)
A clinical study of lectin-reactive alpha-fetoprotein as an early indicator of hepatocellular carcinoma in the follow-up of cirrhotic patients
Authors: Shiraki Katsuya , Takase Koujirou , Tameda Yukihiko , Hamada Minoru , Kosaka Yoshitane , Nakano Takeshi
Published: March 2007

Levels of two types of lectin-reactive alpha-fetoprotein (AFP), designated AFP-L3 and AFP-P4+P5, were analyzed with Lens culinaris agglutinin A and AFP-P4+P5 with erythroagglutinating phytohemagglutinin, respectively, in an attempt to determine the utility and significance of these macromolecules as early indicators of hepatocellular carcinoma during the periodic follow-up of cirrhotic patients. The subjects were 51 of 190 consecutive cirrhotic patients in whom hepatocellular carcinoma developed during a 6-year follow-up period and 21 cirrhotic patients without hepatocellular carcinoma. Serum AFP levels were of limited value to diagnose and predict hepatocellular carcinoma. The relative levels of AFP-L3 and AFP-P4+P5 in patients with hepatocellular carcinoma at the time of tumor detection were significantly higher than those in patients with cirrhosis. The sensitivity was 61%, and the specificity was 90%. Fourteen patients (48%) of 29 patients with small hepatocellular carcinomas less than 2 cm in diameter showed elevated percentage of lectin-reactive AFP. Retrospective examination of 21 patients who were positive for lectin-reactive AFP at diagnosis of hepatocellular carcinoma showed that 41% of them had already expressed lectin-reactive AFP 12 months before the direct detection of hepatocellular carcinoma by diagnostic imaging. These results lead us to conclude that the level of lectin-reactive AFP is a suitable predictive marker for the early recognition of hepatocellular carcinoma in the follow-up of patients with cirrhosis, and that measurements of the level of lectin-reactive AFP should be added to the screening methods that are now in use. (Hepatology 1995; 22:802-807.)
Effect of anti-ulcer drugs on DNA synthesis in adult normal human hepatocytes in culture
Authors: Blanc Pierre , Liautard Jacques , Greuet Jölle , Daures Jean Pierre , Fabre Jean-Michel , Larrey Dominique , Michel Henri , Maurel Patrick
Published: March 2007

The aim of this work was to investigate the effect of four H2 receptor antagonists, cimetidine, ranitidine, famotidine, nizatidine, and of two proton pump inhibitors, omeprazole and lansoprazole, on the mitotic response of human hepatocytes in primary culture. After plating at subconfluent density, cells were exposed to 0.2 to 20 μmol/L of these drugs for 48 hours, either in the absence or in the presence of epidermal growth factor (EGF). The rate of DNA synthesis was evaluated by [3H]-thymidine incorporation into genomic DNA. Both the basal rate of DNA synthesis and the extent of stimulation by EGF exhibited a wide interindividual variability, and were not correlated with the viability of freshly prepared cells. In contrast, the effects of anti-ulcer drugs on the rate of DNA synthesis were clearly reproducible from one culture to another. H2 receptor antagonists had no significant effect (P > .3) over the entire range of concentration tested, whereas omeprazole and lansoprazole significantly inhibited the rate of DNA synthesis by 60% to 90% at 20 μmol/L (P = .016). This effect was concentration dependent between 2 and 20 μmol/L. Neither of the drugs tested was cytotoxic under the conditions used in this work, as assessed by measurements of the de novo protein synthesis. We conclude that, in contrast to H2 receptor antagonists, omeprazole and lansoprazole are able to interfere with the replicative synthesis of DNA in human hepatocytes in culture, at suprapharmacological concentrations. Whether or not this effect is clinically significant remains to be established. (Hepatology 1995; 22:814-819.)
Iron-responsive element-binding protein in hemochromatosis liver and intestine
Authors: Flanagan Peter R. , Hajdu Attila , Adams Paul C.
Published: March 2007

Iron-responsive element-binding protein (IRE-BP) activity was studied in liver and intestinal samples of hemochromatosis and control patients using a short 32P-IRE-RNA probe on “retardation” nondenaturing polyacrylamide gels. IRE-BP activity was assessed in liver biopsy specimens in 36 patients—16 hemochromatosis homozygotes, 4 hemochromatosis heterozygotes, 6 patients with secondary iron overload, and 10 control patients with normal hepatic iron concentrations. Intestinal IRE-BP activity was assessed in 14 hemochromatosis homozygotes and 16 normal subjects. Endogenous IRE-BP activity was determined from 32P retarded on the gel, and total IRE-BP activity was assessed after reducing tissue samples with 2-mercaptoethanol. Hepatic endogenous IRE-BP activity was inversely related to hepatic iron concentration (r = −.59, P < .0002). Mean hepatic endogenous IRE-BP activity in the hemochromatosis homozygotes, 0.25 ± 0.04 pmol/mg protein, was also significantly decreased compared with values in the normal controls, 0.45 ± 0.06 pmol/mg protein, P < .05. Hepatic total IRE-BP was also significantly decreased in the hemochromatosis patients by gel retardation assay and Western blotting with anti-IRE-BP antibody. Intestinal endogenous IRE-BP activity, total IRE-BP activity, and iron concentration did not significantly differ between hemochromatosis patients and normal control subjects. This suggests that both endogenous IRE-BP activity and the total amount of the protein are downregulated in the liver by tissue iron. Intestinal IRE-BP activity that regulates intestinal transferrin receptor expression is normal in hemochromatosis and appropriate for the intracellular iron concentration. (Hepatology 1995; 22:828-832.)
Prevalence of hypertransaminasemia in adult celiac patients and effect of gluten-free diet
Authors: Bardella Maria Teresa , Fraquelli Mirella , Quatrini Maurizio , Molteni Nicoletta , Bianchi Paolo , Conte Dario
Published: March 2007

The prevalence of hypertransaminasemia and the effect of gluten-free diet (GFD) were evaluated in 158 consecutive adult celiac patients, 127 women and 31 men, aged 18 to 68 years (mean, 32). At diagnosis, 67 patients (42%) had raised aspartate and/or alanine transaminase levels (AST and ALT; mean, 47 IU/L, range, 30 to 190; and 61 IU/L, range, 25 to 470, respectively), whereas 91 patients had normal liver function tests (LFT). Patients with and without hypertransaminasemia were comparable for epidemiological data, body mass index (18.5 vs. 19.6), and severity of intestinal histological involvement. All patients were given a strict GFD and were followed for 1 to 10 years (median, 4). At 1 year, a highly significant improvement in intestinal histology was observed in both groups (P < .0001). In the 67 patients with raised transaminase levels body mass index (BMI) also increased significantly (from 18.5 to 21.0, P < .001), and transaminase levels normalized in 60 (95%). In the other seven cases liver biopsy showed fatty infiltration in two and chronic active hepatitis (CAH) in the other five, related to chronic infection with hepatitis B virus in three and hepatitis C virus in one, and to autoimmune type in the fifth. We conclude that in adult celiac patients elevated serum transaminases are a frequent finding and normalize in most cases after GFD. When they persist, liver biopsy is mandatory to further investigate hepatic involvement, which in our series was mainly attributable to CAH. (Hepatology 1995; 22:833-836.)
Organellar clusters formed by mitochondrial-rough endoplasmic reticulum associations: An ordered arrangement of mitochondria in hepatocytes
Authors: Cascarano Joseph , Chambers Patricia A. , Schwartz Eileen , Poorkaj Parvoneh , Gondo Roy E.
Published: March 2007

Our objective was to determine if mitochondrial-rough endoplasmic reticulum (mt-RER) associations provide for an ordered arrangement of mitochondria in the cell. If such an ordered arrangement exists, it might be manifested by grouping of mitochondria according to size and biochemical properties. Liver homogenate was subjected to rate zonal centrifugation for fractionating mitochondrial clusters. These clusters were then examined for morphological and biochemical characteristics. Scanning electron microscopy (SEM) showed that (1) mitochondria were held together in clusters by rough endoplasmic reticulum, (2) clusters consisted of mitochondria of comparable size, and (3) a 45-fold difference in average mitochondrial volume existed between the organelles of the fastest and slowest sedimenting clusters. Transmission electron microscopy (TEM) affirmed that all of the organellar clusters examined were mitochondria associated with rough endoplasmic reticulum. Cytochrome oxidase and mitochondrial DNA were found to be proportional to mitochondrial volume, indicating that these components were synthesized in proportion to increases in volume. Conversely, succinic dehydrogenase and ornithine carbamoyl transferase were increased disproportionately (2.9-fold and six-fold, respectively) with increase in mitochondrial volume. It is evident from this biochemical heterogeneity that clusters composed of larger mitochondria differ functionally from clusters of smaller mitochondria. The size-ordered arrangement suggests that this organization is in some way related to the biogenesis of hepatocyte mitochondria. It is also conjectured that the biochemical heterogeneity is a consequence of addition of selected proteins (e.g., succinic dehydrogenase and carbamoyl transferase) to mitochondria in a developmental process as they mature into larger organelles. (Hepatology 1995; 22:837-846.)
In vivo transfection of hepatitis C virus complementary DNA into rodent liver by asialoglycoprotein receptor mediated gene delivery
Authors: Yamamoto Masato , Hayashi Norio , Miyamoto Yasuhide , Takehara Tetsuo , Mita Eiji , Seki Makoto , Fusamoto Hideyuki , Kamada Takenobu
Published: March 2007

An in vivo model of hepatitis C virus (HCV) infection is needed to enable investigation of the mechanism of the liver injury that it causes. In this study, we used asialoglycoprotein receptor mediated gene delivery to obtain expression of the complementary DNA (cDNA) coding the core and part of the envelope 1 protein of HCV because selective delivery to the hepatocytes has been reported to be attained with this method. The optimum carrier-DNA ratio was examined using in vitro transfection and found to be important for the efficiency of this method. In transfection in vivo, microautoradio-graphical examination showed that the transfected plasmids were delivered selectively to the liver parenchymal cells. To obtain an immunohistochemically detectable level of protein expression in rodent liver, some modifications for increasing the in vivo transfection efficiency were performed; a lysosomal enzyme inhibitor, chloroquine, was used and the administration route of the carrier-DNA complex was changed from the tail vein to the portal vein. On the bases of these results, in vivo transfection with expression vector of HCV core/E1 region was performed. In rat liver transfected by intraportal injection with chloroquine, the transcript RNA and the core protein were detected. These results indicated that the HCV core/E1 expression vector was not merely delivered but also successfully expressed in the liver using asialoglycoprotein receptor mediated gene delivery. The number of the HCV core expressing cells in the transfected liver was similar to that in patients with hepatitis C. These in vivo transfected animals should be useful for investigating the role of this region in the liver injury caused by HCV. (Hepatology 1995; 22:847-855.)
Mucin-vesicle interactions in model bile: Evidence for vesicle aggregation and fusion before cholesterol crystal formation
Authors: Afdhal Nezam H. , Niu Niu , Nunes David P. , Bansil Rama , Cao Xing Xiang , Gantz Donald , Small Donald M. , Offner Gwynneth D.
Published: March 2007

Nucleation of cholesterol monohydrate crystals from bile is a critical step in the formation of cholesterol gallstones. Measurement of nucleation in model bile systems and the characteristics of the initial nucleus have proven elusive. In this study we have used three separate physical chemical techniques to examine vesicle aggregation and fusion, including dynamic light scattering (DLS), transmission electron microscopy (TEM), and fluorescent biochemical assays. These assays enabled us to quantify the effect of biliary proteins, such as gallbladder mucin, on vesicle fusion and aggregation. In the absence of mucin, fusion is a relatively slow process occurring over 24 hours, whereas physiological concentrations of mucin are able to accelerate almost complete fusion of vesicles within 6 hours. Vesicle fusion and aggregation as characterized by TEM result in the formation of aggregates of multilamellar vesicles and giant fusion bodies associated with a background of mucin. These mucin-vesicle aggregate bodies may represent true nuclei and precede cholesterol monohydrate crystal nucleation. In future studies, these vesicle fusion assays can be used to quantitatively examine the effect of putative pro- and anti-nucleating proteins on the earliest steps of cholesterol crystal nucleation. (Hepatology 1995; 22:856-865.)
Differential expression and release of CD54 induced by cytokines
Authors: Mickelson Judith K. , Kukielka Gilbert , Bravenec Stanley J. , Mainolfi Elizabeth , Rothlein Robert , Hawkins Hal K. , Kelly James H. , Smith Wayne C.
Published: March 2007

Intercellular adhesion molecule-1 (ICAM-1, CD54) is upregulated in many cell types stimulated by cytokines. A human hepatoblastoma cell line (C3A, a subclone of HepG2/C3 that is currently being used as a surrogate liver) and human lung adenocarcinoma cells (A549) were stimulated with interleukin-β (IL-β), tumor necrosis factor-α (TNFα), interferon-γ (EFNγ), or IL-6 to determine any differences in cell type responsiveness to individual cytokines for ICAM-1 upregulation. Time courses were performed with each cytokine evaluating ICAM-1 mRNA, surface expression, and cICAM-1 in the cell culture media. Between 3 and 6 hours, IL-β (30 U/mL) stimulated the greatest increase in hepatocyte ICAM-1 mRNA, followed by IFNγ (100 U/mL), TNFα (30 U/mL), and IL-6 (100 U/mL) in order of potency. Except for EL-6, cytokine-induced hepatocyte surface levels of ICAM-1 (immunofluorescence flow cytometry, mAb R6.5) were dose dependent, with inhibition at higher concentration. Highest levels followed stimulation with IFNγ (P < .05). Significantly less was found after both EL-1β and TNFα; none was detected after IL-6 (P < .05). In contrast, IL-1β stimulated significantly more cICAM-1 release from hepatocytes than the other cytokines (P < .001), and IL-6 stimulated modest cICAM-1. Between 3 and 6 hours in the A549 cells, IL-1β stimulated the greatest increase in ICAM-1 mRNA, followed by TNFα. Both responses were greater than that observed in the hepatocytes. IFNγ-and IL-6-induced ICAM-1 mRNA synthesis was not different from unstimulated A549 cells. Cytokine-induced A549 surface levels of ICAM-1 (immunofluorescence flow cytometry, mAb R6.5) was highest for IL-1β (peak levels similar to hepatocyte response), modest with TNFα (peak levels less than hepatocytes), detectable with IFNγ (much less than hepatocytes), and nondetectable after IL-6. No cICAM-1 release from A549 cells was induced under any condition. In hepatocytes the amount of ICAM-1 mRNA was best accounted for by considering both cell surface levels of ICAM-1 and cICAM-1 levels. In human lung adenocarcinoma cells, the cytokine induction of ICAM-1 mRNA could potentially be accounted for by observing cell surface levels of ICAM-1 because no cICAM-1 was produced. These results suggest that surface ICAM-1 and cICAM-1 may be differentially controlled by each cytokine and by each parenchymal cell type. (Hepatology 1995; 22:866-875.)
The growth inhibitory effects of vitamins K and their actions on gene expression
Authors: Wang Ziqiu , Wang Meifang , Finn Frances , Carr Brian I.
Published: March 2007

A characteristic defect occurs in rat and human hepatocellular carcinoma (HCC) resulting in a loss of function of the vitamin K-dependent enzyme gamma-glutamyl-carboxylase in the tumor but not in the underlying liver. This causes the secretion of elevated levels of the immature or des-gamma-carboxylated form of prothrombin, which is used as a marker of HCC. We investigated whether, using the defined conditions of growing HCC cell lines in tissue culture, addition of the naturally occurring vitamins K1 or K2 or the synthetic vitamin K3 could influence the secretion of immature prothrombin. We found that vitamins K1, K2, and K3 all suppressed the secretion of immature prothrombin into the tissue culture medium. Vitamins K2 and K3 were also found to inhibit growth of the HCC cell line, in an apparently nontoxic and reversible manner. The influence of the vitamins K on the expression of some genes related to vitamin K action was examined and compared with that of another growth inhibitor, TGFβ1 protein. The vitamins K were found to increase the expression of prothrombin and carboxylase messenger RNA and c-myc messenger RNA, but had no effects on the expression of TGFβ1 messenger RNA. By contrast, TGFβ1 increased TGFβ1 messenger RNA levels, but had no effects on the other genes, suggesting a different pathway. The previously studied vitamin K3-mediated inhibition of growth was antagonized by the addition of catalase to the culture medium, but the inhibitory effects of vitamin K2 were not antagonized. These experiments show that the vitamins K are a class of growth inhibitors that have a novel mechanism, possibly involving carboxylation. (Hepatology 1995; 22:876-882.)
The differential effects of three forms of interferon alfa on hepatic regeneration after partial hepatectomy in the rat
Authors: Wong Stephen , Gauthier Tony , Kaita Kelly D. E. , Minuk Gerald Y.
Published: March 2007

The purpose of this study was to determine whether all commercially available forms of interferon alfa (IFN α) have the same inhibitory effect on hepatic regeneration and whether this inhibitory effect can be prevented by putrescine, a hepatic growth promotor. Adult male Sprague-Dawley rats (n = 92) received either IFN α-2a, 2b, n1, or saline, 0 and 8 hours after partial hepatectomy (PHx) or 16 hours before PHx. A subgroup of 29 rats being treated with IFN α-2a or saline also received putrescine (5, 50, or 500 mg/kg) 16 hours before PHx. Hepatic regeneration was documented by determining [3H]-thymidine incorporation into hepatic DNA (DNA synthesis), hepatic putrescine levels, and, in selected cases, ornithine decarboxylase (ODC) activity at 24 hours after PHx. The results of the study showed that hepatic regeneration was unaffected when IFN α was administered 0 and 8 hours after PHx. When administered at −16 hours, only IFN α-2a significantly inhibited DNA synthesis and was associated with decreased hepatic putrescine levels. Inhibition was dose-dependent in that a 10-fold increase in IFN α-2a caused a further decrease in both DNA synthesis and hepatic putrescine levels. At the higher dose, IFN α-2b and n1 also inhibited DNA synthesis and lowered hepatic putrescine levels and ODC activity. Exogenous putrescine (5 and 50 mg/kg) restored hepatic regenerative activity to normal but was toxic at high concentrations (500 mg/kg). These data indicate that not all commercially available forms of IFN α inhibit hepatic regeneration in the rat to the same extent. (Hepatology 1995; 21:883-886.)
Endothelin association with the cultured rat Kupffer cell: Characterization and regulation
Authors: Stephenson Katherine , Harvey Stephen A. K. , Mustafa Shamimunisa B. , Eakes Ann T. , Olson Merle S.
Published: March 2007

Circulating endothelin (ET) levels are elevated in conditions such as endotoxemia, hepatic ischemia-reperfusion injury, or orthotopic liver transplantation, and this potent peptide may contribute to hepatic pathophysiology. We measured the surface binding of [125I]ET-1 to rat Kupffer cells in primary culture at 4°C; the apparent dissociation constant (Kd) was 270 pmol/L, and the apparent Bmax was 3,000 receptors/cell. At 37°C, total association (surface binding plus internalization) was much greater than at 4°C, indicating that internalization of the receptor-ligand complex is rapid; the apparent Kd was 30 pmol/L, comparable with other reports for hepaticderived cells. Studies using [125I]ET-1, [125I]ET-3, and specific ET (ant)agonists showed that Kupffer cells possess predominantly ETB type receptors. Prior treatment with 500 pmol/L unlabeled endothelin rapidly (<15 minutes) occluded 60% of subsequent [125I]ET association; using 5 nmol/L unlabeled ET, this occlusion occurred within 1 minute. [125I]ET association with Kupffer cells was unaffected by short-term (<1 hour) treatment with cyclic adenosine monophosphate (cAMP), but long-term (20 hour) treatment resulted in a twofold increase in [125I]ET association with no change in the apparent Kd. Stimulation of protein kinase C in Kupffer cells by phorbol 12-myristate acetate had a dual regulatory effect on [125I]ET association. Short-term (<1 hour) treatment with phorbol 12-myristate acetate decreased [125I]ET-3 association by 50%, whereas prolonged treatment (20 hour) increased association twofold. In both cases, the apparent Kd for [125I]-endothelin was unaltered. (Hepatology 1995; 22:896-905.)
Role of nitric oxide and prostacyclin in the control of renal perfusion in experimental cirrhosis
Authors: Ros Josefa , Clària Joan , Jiménez Wladimiro , Bosch-Marcé Marta , Angeli Paolo , Arroyo Vicente , Rivera Francisca , Rodés Joan
Published: March 2007

Nitric oxide (NO) and prostacyclin (PGI2) are two important modulators of renal function under normal conditions; however, little is known on their contributory role in cirrhosis with ascites. In this study, mean arterial pressure, renal hemodynamics, and sodium excretion were measured in 15 rats with cirrhosis and ascites and 16 control rats. Animals were studied in normal conditions, after inhibiting the synthesis of NO (N
ω-nitro-l-arginine, 50 μg ± kg−1 ± min−1) or prostaglandins (lysine acetylsalicylate, 15 mg·kg−1·min−1) and following the concomitant inhibition of both systems. Cirrhotic rats showed increased systemic pressure sensitivity and blunted renal vasoconstrictor response to nitric oxide inhibition as compared with control rats. As a consequence, the glomerular filtration rate increased in cirrhotic rats but not in control rats. In both groups of animals, NO inhibition was associated with significant increased urinary sodium and fractional sodium excretion. The only significant effect observed after prostaglandin biosynthesis inhibition was a decrease in renal plasma flow in cirrhotic rats. The concomitant inhibition of both systems reduced renal plasma flow and did not change glomerular filtration rate, with no differences between control and cirrhotic rats. Prostaglandin inhibition did not prevent the natriuretic effect of the NO inhibitor in both groups of animals. These results indicate that in experimental cirrhosis both NO and PGI2 play an important role in the maintenance of renal perfusion within normal limits. (Hepatology 1995; 22:915-920.)
Flow cytometric DNA-ploidy and proliferative activity of diethylnitrosamine-induced hepatocellular carcinoma and pulmonary metastases in monkeys
Authors: Lapis Károly , Bocsi József , Lapis Péter , Thorgeirsson Unnur P.
Published: March 2007

Flow cytometric DNA analysis was carried out on diethylnitrosamine (DEN) induced primary hepatocellular carcinomas (HCC) and lung metastases in monkeys. In analyzing one sample from each of 113 HCC cases, 76 (67.2%) were diploid and 37 (32.7%) aneuploid. When more samples were analyzed from the same tumorous liver, all of the 76 diploid cases maintained their pattern, whereas 5 (13.5%) of the aneuploid cases displayed both diploid and aneuploid DNA. Studies of lung metastases from 44 (28 diploid, 16 aneuploid) HCC cases showed that the DNA-ploidy pattern characterizing the primary HCC was preserved in the metastases in 78.6% of the diploid and 93.7% of the aneuploid cases. The average synthetic phase fraction (SPF) value for the diploid tumors was 7.7% and the aneuploid tumors 14.9%. The difference is highly significant (P < .01). Highly significant correlation was found between the DNA ploidy and the SPF values, both in the primary HCC (P = .0001) and the metastases (P = .0266). Of different tumor and host features examined, statistically significant correlation was only found between DNA-ploidy/SPF and the cytological tumor grade. This study represents the first DNA-ploidy analysis of HCC in monkeys. The data showed that diploid and aneuploid tumors displayed comparable metastatic potential. The DNA-ploidy pattern was preserved in the metastases in the majority of the cases. (Hepatology 1995; 22:952-961.)
Excretion of F2-isoprostanes in bile: A novel index of hepatic lipid peroxidation
Authors: Awad Joseph A. , Morrow Jason D.
Published: March 2007

Lipid peroxidation is believed to be an important mechanism of liver injury caused by some xenobiotics. However, it has been difficult to demonstrate and quantify this process in vivo. Moreover, little is known about the disposition of lipids oxidized in the liver. F2-isoprostanes are prostanoids produced by nonenzymatic free radical-catalyzed peroxidation of arachidonic acid esterified to phospholipids. Hydrolysis of F2-isoprostanes from phospholipids by phospholipases yields free F2-isoprostanes. Excretion of F2-isoprostanes, both free and esterified to phospholipids, was measured in bile after administration of CCI4. The concentration of lipid-esterified F2-isoprostanes in bile exceeded that of free F2-isoprostanes. CCI4, caused a dose-dependent increase in biliary F2-isoprostane excretion that correlated better with the increase in liver F2-isoprostaes than it did with the increase in plasma F2-isoprostanes. Pretreatment with colchicine ameliorated CCI4-liver injury but did not affect baseline or CCI4-induced biliary F2-isoprostane excretion. Administration of diquat to selenium-deficient rats, which causes hepatic and renal necrosis, was associated with a 13-fold elevation of plasma F2-isoprostanes. However, both hepatic F2-isoprostane concentrations and biliary F2-isoprostane excretion were increased only threefold. These data suggest that quantification of F2-isoprostane excretion in bile may provide a sensitive and quantitative index of hepatic lipid peroxidation. (Hepatology 1995; 22:962-968.)
Effects of extracellular matrices on tube formation of cultured rat hepatic sinusoidal endothelial cells
Authors: Shakado Satoshi , Sakisaka Shotaro , Noguchi Kazunori , Yoshitake Masao , Harada Masaru , Mimura Yoshihiro , Sata Michio , Tanikawa Kyuichi
Published: March 2007

To determine the effects of extracellular matrices on the function and morphology of hepatic sinusoidal endothelial cells, isolated rat hepatic sinusoidal endothelial cells were cultured in three-dimensional fashion on collagen gel containing various extracellular matrix components. Cells cultured on type I collagen gel with or without type IV collagen formed a cobblestone appearance on the surface of the gel. Cells cultured on laminincontaining type I collagen gel invaded the gel and exhibited three-dimensional tube formation with a decreased number of characteristic endothelial pores. Morphometrically, there was a significant relationship between the length of the tube formed and the concentration of laminin in the type I collagen gel. Cells cultured on Matrigel, which contains high concentrations of laminin, type IV collagen, fibroblast growth factor, tissue plasminogen activator, and other growth factors, formed a great number of tubes into a network on the surface of the gel, as is observed in in situ hepatic sinusoidal endothelial cells. Ultrastructurally, tube-forming endothelial cells cultured on Matrigel had many endothelial pores on the cell surface, with tubes (approximately 10 μm in diameter) formed by two or three hepatic sinusoidal endothelial cells. These results indicated that extracellular matrix components, especially laminin, induced the formation of tubes in cultured rat hepatic sinusoidal endothelial cells. Tube-forming sinusoidal endothelial cells cultured on Matrigel could provide more advantages than the two-dimensional culture model for investigating the function and morphology of these cells in vitro. (Hepatology 1995; 22:969-973.)