bluuebunny/crossref_metadata_2025 huggingface dataset:
Tubulovesicular Transport of Horseradish Peroxidase in Isolated Rat Hepatocyte Couplets: Effects of Low Temperature, Cytochalasin B and Bile Acids
Authors: Sakisaka Shotaro , Harada Masaru , Gondo Kazuhisa , Yoshitake Masao , Tanikawa Kyuichi
Published: March 2007

The transcytotic vesicular pathway in isolated rat hepatocyte couplets was investigated using horseradish peroxidase. Ten to 20 min after horseradish peroxidase labeling, vesicles and tubules containing horseradish peroxidase were observed to be predominantly around the bile canaliculi. In hepatocytes incubated in a 4° C medium for 10 min after horseradish peroxidase labeling, few horseradish peroxidase–containing structures were observed around the bile canaliculi, and the fine reticular immunofluorescence of microtubules was reduced. Cells treated with cytochalasin B (a microfilament inhibitor) showed a fair number of horseradish peroxidase–containing structures around the markedly dilated bile canaliculi and the distribution of microtubules was preserved. Cells labeled by horseradish peroxidase and then incubated for 10 min in a horseradish peroxidase–free medium containing 50 μmol/L of taurocholic acid, ursodeoxycholic acid or tauroursodeoxycholic acid had more tubular structures containing horseradish peroxidase around the bile canaliculi than control cells, whereas 50 μmol/L of taurochenodeoxycholic acid, taurodeoxycholic acid, dehydrocholic acid and taurodehydrocholic acid each failed to increase the number of tubular structures. These findings show that horseradish peroxidase was transported in hepatocyte couplets from the cell periphery to the bile canalicular front through the tubulovesicular pathway, depending on cytoplasmic microtubules. Cytoplasmic microfilaments appeared to play a minor role in this transport. Several specific bile acids such as taurocholic acid, ursodeoxycholic acid and tauroursodeoxycholic acid each promoted the tubular transformation.(HEPATOLOGY 1994;20:1015–1023)
A Comparison of Copper Uptake by Liver Plasma Membrane Vesicles and Uptake by Isolated Cultured Rat Hepatocytes
Authors: Bingham Michelle J. , McArdle Harry J.
Published: March 2007

We studied copper uptake from copper dihistidine complexes by plasma membrane vesicles isolated from rat liver and compared the data with those for uptake under the same conditions by hepatocytes cultured from rat liver to determine whether membrane vesicles can be used to study copper uptake. Marker enzyme analysis showed a 28–fold increase in 5′–nucleotidase activity, a slight increase in endoplasmic reticulum and no contamination with mitochondrial membranes. Copper uptake by vesicles is temperature dependent, and solubilization with Triton X–100 results in a loss of accumulative capacity. Increasing osmotic pressure resulted in a decrease in copper levels in the vesicles at equilibrium, showing that uptake — as opposed to binding by the vesicles—occurred. Uptake by vesicles is concentration dependent, with evidence for cooperation in the uptake sites. The substrate concentration yielding 10% maximum uptake was 4.01 ± 0.5 μmol/L, maximum uptake was 10.8 ± 0.4 nmol/Cu/mg protein min and the n value was 1.5 ± 0.2. In contrast, uptake by cells showed no cooperation (n = 1.09 ± 0.06) and a significantly higher apparent Michaelis–Menten constant (17.4 ± 1.3 μmol/L). As expected, the maximum uptake was lower in the hepatocytes (1.82 ± 0.08 nmol/mg protein min). Albumin,

N

–ethylmaleimide and zinc all inhibited uptake in vesicles and in hepatocytes, and the degrees of inhibition were similar in both types of preparation. Vitamin C stimulated uptake in both vesicles and hepatocytes; again, there was a correlation between the increase in uptake at different concentrations. However, cadmium inhibited uptake and nickel stimulated uptake in vesicles and neither metal had any effect in hepatocytes. The data show that vesicles have value as a system in which to study copper uptake but that care must be taken in interpreting the information obtained.(HEPATOLOGY 1994;20:1024–1031)
Effects of Protein Kinase C and Cytosolic Ca2+ on Exocytosis in the Isolated Perfused Rat Liver
Authors: Bruck Rafael , Nathanson Michael H. , Roelofsen Han , Boyer James L.
Published: March 2007

Both protein kinase C and cytosolic Ca2+ are involved in the regulation of exocytosis in a number of cell types. However, the relative importance of each of these for apical exocytosis in the hepatocyte is unknown. To investigate this, we studied the effects of protein kinase C and Ca2+ agonists on horseradish peroxidase excretion in the isolated perfused rat liver. Vasopressin increased both horseradish peroxidase concentration and net horseradish peroxidase excretion in bile, and these effects were abolished by the protein kinase C inhibitor H–7. The protein kinase C activator phorbol dibutyrate also increased both net excretion and the concentration of biliary horseradish peroxidase. In contrast, the Ca2+ ionophore A23187 and the Ca2+ mobilizing agent 2′,5′–di (

tert

butyl)–1,4,–benzohydroquinone both had minimal effects on horseradish peroxidase concentration and inhibited the rate of horseradish peroxidase excretion. These results suggest that protein kinase C stimulates apical exocytosis in the hepatocyte, whereas increased Cai
2+ per se does not influence exocytosis and inhibits excretion only transiently by reducing bile flow. (Hepatology 1994;20:1032–1040).
G Proteins in Rat Liver Proliferation During Cholestasis
Authors: Rodriguez-Henche Nieves , Guijarro Luis G. , Couvineau Alain , Carrero Isabel , Arilla Eduardo , Laburthe Marc , Prieto Juan C.
Published: March 2007

Liver proliferation appears to be dually regulated, in part by cyclic AMP levels. Here we studied the alterations in the stimulatory action of cholera toxin and other agents on the adenylyl cyclase system, as well as the status of Gs and Gi protein subunits during the liver proliferation that follows bile duct ligation in rats. The stimulatory effects of glucagon and vasoactive intestinal peptide (which act through membrane receptors) or guanosine 5′–[βγ–imido]triphosphate (which interacts with G proteins) and forskolin (which directly activates the adenylyl cyclase catalytic subunit) on liver adenylyl cyclase activity were blunted in cholestasis. The results indicated an impairment in the stimulatory interaction between the αs subunit of Gs protein and the adenylyl cyclase catalytic subunit. Indeed, we observed an important decrease in the stimulation of adenylyl cyclase activity by cholera toxin in cholestasis that was accompanied by a reduced extent of [32P]ADP ribosylation of αs protein catalyzed by cholera toxin, as revealed by the poor labeling of the 42,000 Da band in liver membranes from cholestatic rats. However, there was no change in the amount of αs or β proteins as measured with immunoblotting techniques. Experiments on [32P]ADP ribosylation of αi subunits of Gi proteins indicated an impairment in liver membranes from cholestatic rats, whereas Western blotting for the detection of αi subunits showed decreased αi3 and increased αi2 levels in this condition. Further efforts are needed to better understand the molecular mechanisms underlying the relationship between the observed divergent expression of Gs and Gi proteins and liver cell proliferation in the cholestatic liver. (Hepatology 1994;20:1041-1047).
Ursodeoxycholate–Induced Hypercholeresis in Cirrhotic Rats: Further Evidence for Cholehepatic Shunting
Authors: Elsing Christoph , Sägesser Hans , Reichen JÜRg
Published: March 2007

The aim of the investigation was to explore whether ursodeoxycholate, a tertiary bile acid with potential for treatment of chronic cholestasis in cirrhotic liver disease, has the same physiological effects in cirrhotic as in normal rats. Furthermore, we wanted to investigate whether ductular proliferation, as it occurred in this situation, increases the bicarbonate stimulatory effect of ursodeoxycholate. Rats (n = 16) were rendered cirrhotic by continuous exposure to phenobarbital—carbon tetrachloride; untreated animals (n = 13) served as controls. In cirrhotic rats

in vivo

, ursodeoxycholate (20 μmoles/min/kg) stimulated bile salt secretion and bile flow less than in controls. Nevertheless, the increment in ursodeoxycholate–induced biliary bicarbonate—the bicarbonate stimulatory potency—was increased by 29% in cirrhotic animals (0.55 ± 0.08 mmol vs. 0.71 ± 0.11 mmol; p < 0.05). This finding could be related to ductular proliferation because the volume fraction of bile ductules, determined stereologically, increased from 0.3% ± 0.1% to 2.7% ± 0.6% in cirrhotic rats (p < 0.005). To explore further the behavior of ductules during ursodeoxycholate stimulation, we carried out experiments in the

in situ

perfused rat liver. In the portally perfused organ, replacement of bicarbonate by tricine–acetate abolished ursodeoxycholate–induced hypercholeresis. In the dually perfused organ (perfusion of both portal vein and hepatic artery) perfusion of the hepatic artery with bicarbonate–containing buffer, ursodeoxycholate had a similar stimulatory effect as

in vivo

in both control and cirrhotic rats. When bicarbonate in the hepatic artery was replaced by tricine–acetate, a decrease in biliary bicarbonate and bile flow occurred in controls but not in cirrhotic rats. During ursodeoxycholate stimulation, a decrease in biliary bicarbonate occurred in both experimental groups. We conclude that ursodeoxycholate maintains its hypercholeretic properties in cirrhosis and that the ductular proliferation found in this model of cirrhosis conveys a larger bicarbonate–stimulating capacity to this tertiary bile acid. These results lend further support the cholehepatic shunt hypothesis to explain ursodeoxycholate–induced bicarbonate–rich hypercholeresis in rodents.(HEPATOLOGY 1994;20:1048–1054)
The Mallory Body: Morphological, Clinical and Experimental Studies (Part 1 of A Literature Survey)
Authors: Jensen Kenneth , Gluud Christian
Published: March 2007

To aid understanding of markers of disease and predictors of outcome in alcohol–exposed systems, we undertook a literature survey of more than 700 articles to view the morphological characteristics and the clinical and experimental epidemiology of the Mallory body. Mallory bodies are filaments of intermediate diameter that contain intermediate filament components (e.g., cytokeratins) observable by conventional light microscopy or immunohistochemical methods, identical in structure regardless of initiating factors or putative pathogenesis. Although three morphological types can be identified under electron microscopy (with fibrillar structure parallel, random or absent), they remain stereotypical manifestations of hepatocyte injury. A summary of the conditions associated with Mallory bodies in the literature and their validity and potential etiological relationships is presented and discussed, including estimates on the combined light microscopic and immunohistochemical prevalences and kinetics. Emphasis is placed on proper confounder control (in particular, alcohol history), which is highly essential but often inadequate. These conditions include (mean prevalence of Mallory bodies inparentheses): Indian childhood cirrhosis (73%), alcoholic hepatitis (65%), alcoholic cirrhosis (51%), Wilson's disease (25%), primary biliary cirrhosis (24%), nonalcoholic cirrhosis (24%), hepatocellular carcinoma (23%), morbid obesity (8%) and intestinal bypass surgery (6%). Studies in alcoholic hepatitis strongly suggest a hit–and–run effect of alcohol, whereas other chronic liver diseases show evidence of gradual increase in prevalence of Mallory bodies with severity of hepatic pathology. Mallory bodies in cirrhosis do not imply alcoholic pathogenesis. Obesity, however, is associated with alcoholism and diabetes, and Mallory bodies are only present in diabetic patients if alcoholism or obesity complicates the condition. In addition, case studies on diseases in which Mallory bodies have been identified, along with pharmacological side effects and experimental induction of Mallory bodies by various antimitotic and oncogenic chemicals, are presented. Mallory bodies occur only sporadically in abetalipoproteinemia, von Gierke's disease and focal nodular hyperplasia and during hepatitis due to calcium antagonists or perhexiline maleate. Other conditions and clinical drug side effects are still putative. Finally, a variety of experimental drugs have been developed that cause Mallory body formation, but markedly different cell dynamics and metabolic pathways may raise questions about the relevance of such animal models for human Mallory body formation. In conclusion, the Mallory body is indicative but not pathognomonic of alcohol involvement. A discussion on theories of development and pathological significance transcending the clinical frameworks will be presented in a future paper. (Hepatology 1994;20:1061-1077).
Induction of A Dose–Related Increase in Sulfobromophthalein Uptake Velocity in Freshly Isolated Rat Hepatocytes by Phenobarbital
Authors: Potter Barry J. , Ni Jia-Zhang , Wolfe Kathleen , Stump Decherd , Berk Paul D.
Published: March 2007

To determine whether phenobarbital affects hepatocellular bilirubin/sulfobromophthalein uptake mechanism, we administered it to male Sprague–Dawley rats, body weight 175 ± 25 gm, at doses of 1 to 75 mg/kg body wt/day for 7 days. Control rats were given an equivalent volume of physiological saline solution. On day 8, hepatocytes were isolated by means of collagenase perfusion, suspended in Hanks' solution without albumin and incubated with high specific activity (3 Ci/mmol) [35S]sulfobromophthalein, which was synthesized in our laboratory and purified by means of a new reverse–phase high–pressure liquid chromatography procedure. The initial uptake rate of sulfobromophthalein was determined at sulfobromophthalein concentrations of 1 to 50 μmol/L with a rapid filtration technique. The maximum uptake velocity and Michaelis constant for sulfobromophthalein uptake at each phenobarbital dose were determined by means of a computer analysis. In control studies, maximum uptake and Michaelis constant were 48.0 ± 16.7 (mean ± S.D.) pmol/50,000 cells/min and 22 ± 4 μmol/L, respectively. Maximum uptake velocity increased linearly with the log of the phenobarbital dose (r = 0.98, p < 0.01), the increase achieving statistical significance at a dose of 3 mg/kg/day. Michaelis constant, however, was essentially unchanged at phenobarbital doses of 50 mg/kg/day or less. The maximal observed increase in maximum uptake velocity of sulfobromophthalein (to 619% of control values) was appreciably greater than the maximal increase in UDP–glucuronyltransferase activity (200% of control) or immunoreactive ligandin concentrations (260% of control) seen in earlier studies, suggesting a direct effect on the plasma membrane transport mechanism. (Hepatology 1994;20:1078-1085).
Expression of Three– and Four–Repeat Tau Isoforms in Mouse Liver
Authors: Kenner Lukas , El-Shabrawi Yosuf , Hutter Heinz , Forstner Michael , Zatloukal Kurt , Hoefler Gerald , Preisegger Karl-Heinz , Kurzbauer Robert , Denk Helmut
Published: March 2007

Tau protein is a member of the family of microtubule–associated proteins, which support microtubule polymerization and stability. Under pathological conditions, tau is a major constituent of neurofibrillary tangles in nerve cells of patients with Alzheimer's disease. Neurofibrillary tangles share some morphological, biochemical and immunological properties with cytoplasmic inclusions associated with other diseases, such as Mallory bodies in the livers of patients with alcoholic hepatitis and in corresponding mouse models. Recently a Mallory body component was identified that in molecular mass and isoelectric point resembles the abnormally phosphorylated tau of neurofibrillary tangles. There has been, however, so far no report describing the occurrence of tau in normal liver. We now demonstrate the expression of two tau isoforms containing three and four repeats, respectively, of the microtubule–binding domains in normal mouse liver and kidney. This finding provides evidence for a physiological role of tau in the liver and, consequently, the basis for the involvement of tau in pathological situations. (Hepatology 1994;20:1086-1089).
Expression of The C–Myc Protooncogene Product in Cells Infected With the Hepatitis Delta Virus
Authors: Tappero Gianfranco , Natoli Gioacchino , Anfossi Giovanni , Rosina Floriano , Negro Francesco , Smedile Antonina , Bonino Ferruccio , Angeli Alberto , Purcell Robert H. , Rizzetto Mario , Levrero Massimo
Published: March 2007

The intrahepatic accumulation of the c–

myc

protooncogene product was observed on immunoflourescence in each of six patients with chronic hepatitis delta virus infection who exhibited the hepatitis D antigen in their livers. The c–

myc

product was stained in the same nuclei that contained the hepatitis D antigen. C–

myc

was not observed in acute hepatitis D or in cases of chronic hepatitis delta virus infection without expression of the hepatitis D antigen. The protooncogene product was detected in only 1 of 32 viral and nonviral liver disorders unrelated to hepatitis delta virus. To confirm these observations, we transfected HBsAg–positive (PCL/PRF/5) and HBsAg–negative (HepG2) transformed liver cell lines with a plasmid containing a hepatitis delta virus cDNA trimer under the control of the SV40 early enhancer/promoter sequences. Whereas baseline c–

myc

expression was barely detectable in mock–transfected PLC/PRF/5 or HepG2 cells, strong c–

myc

nuclear fluorescence was observed when these same cells were transfected with the hepatitis D antigen expression vector. Similar results were obtained after infection of HeLa cells with a recombinant vaccinia virus expressing the hepatitis D antigen. Detection of c–

myc

mRNA sequences by means of

in situ

hybridization suggested that the c–

myc

product accumulation was not due to increased amounts of its mRNA. The c–

myc

protein accumulates selectively in the livers of patients with chronic hepatitis delta virus infection and in the same nuclei that contain the hepatitis D antigen. The expression of c–

myc

in hepatitis D antigencontaining cells does not require the presence of hepatitis B virus infection. (Hepatology 1994;20:1109-1114).
Increased Serum Hepatitis C Virus Rna Levels Among Alcoholic Patients With Chronic Hepatitis C
Authors: Oshita Masahide , Hayashi Norio , Kasahara Akinori , Hagiwara Hideki , Mita Eiji , Naito Masafumi , Katayama Kazuhiro , Fusamoto Hideyuki , Kamada Takenobu
Published: March 2007

Hepatitis viruses and alcohol are major causes of liver disease. This study was aimed at investigating the effect of alcohol intake on the replication of hepatitis C virus and the efficacy of interferon therapy. Fiftythree patients who were histologically proved to have chronic hepatitis C were tested. Of these, 16 were diagnosed as habitual drinkers whose cumulative total consumption of alcohol was more than 100 kg or who had consumed at least 60 gm of ethanol daily for at least 5 yr. The quantities of hepatitis C virus RNA in serum were measured with a competitive assay that combined reverse transcription and polymerase chain reaction. The subjects received a 26–wk course of interferon–α therapy. There were no significant differences in age and ALT levels between habitual drinkers and nonhabitual drinkers. The titer of viral RNA (logarithmic transformed copy numbers per milliliter of serum) of habitual drinkers (8.5 ± 0.5) was higher than that of nonhabitual drinkers (7.7 ± 0.8) (p < 0.01). Neopterin levels in serum, a marker for the activation of cellmediated immunity, were lower for habitual drinkers (5.7 ± 1.5 pmol/ml) than for nonhabitual drinkers (8.1 ± 5.0 pmol/ml) (p < 0.01). Eleven of the nonhabitual drinkers (30%) were long–term responders whose alanine aminotransferase levels remained within normal range during the 24 wk after interferon therapy, whereas only one (6%) of the habitual drinkers was a long–term responder (p = 0.06). These findings suggest that alcohol intake increases hepatitis C virus RNA levels in serum—at least in part—impairment of cellular immunity, and modulates the efficacy of interferon therapy. (Hepatology 1994;20:1115-1120).
Influence of Viral Quasispecies on Effectiveness of Interferon Therapy in Chronic Hepatitis C Patients
Authors: Kanazawa Yoshiyuki , Hayashi Norio , Mita Eiji , Li Tiancheng , Hagiwara Hideki , Kasahara Akinori , Fusamoto Hideyuki , Kamada Takenobu
Published: March 2007

The quasispecies nature of hepatitis C virus genome distribution is most evident in hypervariable regions of the putative envelope 2 domain. Eight patients with chronic hepatitis C treated with interferon–α were studied as to heterogeneity of the hypervariable regions to clarify the implications of quasispecies. More than 10 recombinant clones generated from polymerase chain reaction–amplified products of the hypervariable regions were sequenced. The sets of clones derived from long–term responders before interferon therapy showed a significantly lower (p < 0.05) degree of sequence complexity of the hypervariable region 1 quasispecies than those from short–term ones or nonresponders. The values of nucleotide diversity (the average number of nucleotide differences per site between two randomly chosen sequences) in hypervariable region 1 before interferon therapy were also significantly lower (p < 0.05) for long–term responders (mean, 2.31 × 10) than for short–term ones or nonresponders (13.02 × 10-2). In some cases, nucleotide diversity decreased remarkably during interferon therapy, whereas the values remained unchanged in other cases. In one interesting case, a short–term response was first noted with the nucleotide diversity decreasing from 13.98 × 10-2 to 0.21 × 10-2; namely, the diversity of the quasispecies was significantly reduced, and then a long–term response was observed after an additional course of interferon therapy. Thus, the degree of quasispecies' complexity and diversity of hypervariable region 1 was closely correlated with the responsiveness to interferon therapy in chronic hepatitis C patients, and thus may have some influence on interferon efficacy. (Hepatology 1994;20:1121-1130).
Superinfection of Chimpanzees Carrying Hepatitis C Virus of Genotype Ii/1B With That of Genotype Iii/2A or I/1A
Authors: Okamoto Hiroaki , Mishiro Shunji , Tokita Hajime , Tsuda Fumio , Miyakawa Yuzo , Mayumi Makoto
Published: March 2007

Three chimpanzees persistently infected with hepatitis C virus of genotype II/1b were challenged with hepatitis C virus of genotype III/2a and 6 wk later with hepatitis C virus of genotype I/1a. They were tested for titers of total and genotype–specific hepatitis C virus RNA, as well as for serum transaminase levels, until 52 wk after the first challenge. One chimpanzee (CH489) with intermittent low hepatitis C virus RNA titers of genotype II/1b in serum was superinfected with hepatitis C virus of genotype III/2a between wk 1 and 7 after the challenge; superinfection was accompanied by fluctuating high transaminase levels. Later, the animal was superinfected with hepatitis C virus of genotype I/1a. Superinfection was accompanied by persistently high transaminase levels immediately after the challenge. Hepatitis C virus of genotype I/1a persisted, whereas hepatitis C virus of genotype II/1b was undetectable 22 wk after the challenge and thereafter. In another chimpanzee (CH353) with intermittent low hepatitis C virus RNA titers of genotype II/1b, hepatitis C virus of genotype III/2a induced fluctuating high levels of serum transaminases without revealing itself in serum. Then, HCV of genotype I/1a superinfected her, induced persistently high transaminase levels and took over HCV of genotype II/1b at 22 wk after the challenge and thereafter. The third chimpanzee (CH451) with persistently high HCV RNA titers of genotype II/1b did not reveal HCV RNA of genotype III/2a in serum after the challenge, although transaminases sharply increased. Low–titered HCV RNA of genotype I/1a was detected at 18 wk after the challenge. He continued to have elevated transaminase levels and high–titered HCV RNA of genotype II/1b throughout the observation period. These results indicate superinfection with HCV of different genotypes in chimpanzees, newly introduced HCV replacing or being expelled by the predecessor HCV with each infection inducing an episode of liver injury. (Hepatology 1994;20:1131-1136).
The Course of Hepatitis C Virus Infection After Liver Transplantation
Authors: Féray Cyrille , Gigou Michelle , Samuel Didier , Paradis Valérie , Wilber Judith , David Marie France , Urdea Mickey , Reynes Michel , Bréchot Christian , Bismuth Henri
Published: March 2007

Hepatitis C virus—induced liver disease is becoming a main indication for liver transplantation. Recurrence of hepatitis after transplantation has been reported, but its long–term consequences are unknown. Seventynine patients positive for hepatitis C virus (group 1) and 106 subjects negative for hepatitis C virus antibody (group 2) with a mean follow–up of 4 yr were retrospectively studied by means of serology, nested polymerase chain reaction and branched–DNA technology before and after liver transplantation. The actuarial rates of hepatitis C virus—related acute hepatitis were 72% and 20% at 4 yr in groups 1 and 2, respectively. Progression to chronic active hepatitis occurred in 61% and 36% of the subjects within 3 yr of the onset of recurrent and acquired hepatitis, respectively. No case of acute graft failure and two cases of cirrhosis were related to recurrent or acquired hepatitis C virus liver disease. Hepatitis C virus RNA levels were significantly increased in cases of hepatitis after transplantation. In contrast, the pretransplant hepatitis C virus RNA level was not predictive of recurrence. Our results establish the general persistence of hepatitis C virus infection after liver transplantation, the frequency and the severe course of recurrent liver disease. However, liver transplantation in hepatitis C virus antibody—positive patients still has a good medium–term prognosis. (Hepatology 1994;20:1137-1143).
Changes in Cytokine Production During Therapy With Granulocyte–Macrophage Colony–Stimulating Factor in Patients With Chronic Hepatitis B
Authors: Martín Julio , Quiroga Juan A. , Bosch Orencio , Carreño Vicente
Published: March 2007

Recombinant human granulocyte–macrophage colony–stimulating factor therapy significantly reduces serum hepatitis B virus DNA levels, associated with increased 2′, 5′–oligoadenylate synthetase activity in cultured mononuclear cells of patients with chronic hepatitis B. To assess changes in immune function during therapy of chronic hepatitis B patients, spontaneous and mitogen–induced production of tumor necrosis factor–α, interleukin–1β, interleukin–6, interferon–α and interferon–γ were measured–along with serum levels of soluble CD4, soluble CD8, soluble interleukin–2 receptor and β2–microglobulin–before, during and after a 6–wk course of granulocyte–macrophage colony–stimulating factor in nine patients with chronic hepatitis B. Treatment statistically enhanced spontaneous production of tumor necrosis factor–α (p < 0.05) and interleukin–1β (p < 0.02). Furthermore, spontaneous interleukin–6 production correlated negatively with hepatitis B virus DNA levels (p < 0.03), and spontaneous interleukin–1β production correlated positively with 2′, 5′–oligoadenylate synthetase activity (p < 0.0005). In addition, statistically significant increases were found during therapy in serum levels of soluble interleukin–2 receptor (p < 0.01), soluble CD4 (p < 0.01) and β2–microglobulin (p < 0.05). Levels of soluble interleukin–2 receptor and soluble CD4 correlated negatively with levels of hepatitis B virus DNA (p < 0.05), and levels of soluble interleukin–2 receptor and β2–microglobulin correlated positively with 2′, 5′–oligoadenylate synthetase activity (p < 0.003 and p < 0.02, respectively). Thus recombinant human granulocyte–macrophage colony–stimulating factor administration may induce reductions in hepatitis B virus DNA levels, perhaps by altering the immune status and increasing cytokine production. (Hepatology 1994;20:1156-1161).
Cerebral Phosphorus–31 Magnetic Resonance Spectroscopy in Patients With Chronic Hepatic Encephalopathy
Authors: Taylor-Robinson Simon D. , Sargentoni Janet , Mallalieu Rosemary J. , Bell Jimmy D. , Bryant David J. , Coutts Glyn A. , Morgan Marsha Y.
Published: March 2007

Cerebral phosphorus–31 magnetic resonance spectroscopy was undertaken in 33 patients with biopsyproven cirrhosis: 6 had no evidence of neuropsychiatric impairment on standard clinical, psychometric and electrophysiological testing; 8 had evidence of subclinical hepatic encephalopathy; and 19 were classified as having overt hepatic encephalopathy. The reference population comprised 15 healthy volunteers. Unlocalized spectra were acquired from the entire head with a 45–degree pulse angle and repetition times of 1 and 5 sec. Spectra localized to the basal ganglia were acquired with a 45–degree pulse angle and a repetition time of 1 sec. Peak area ratios of phosphomonoesters, inorganic phosphate, phosphodiesters and phosphocreatine relative to β–ATP were measured in the spectra acquired. We noted no consistent change in the ratios of inorganic phosphate to ATP and phosphocreatine to ATP. Mean values of the ratios of phosphomonoesters to ATP and phosphodiesters to ATP were significantly lower in the total patient population than in the reference population, and they correlated with the patients' neuropsychiatric status. Thus we found no significant reductions in the mean ratios of phosphomonoesters to ATP and phosphodiesters to ATP in patients who were neuropsychiatrically unimpaired, but significant reductions were observed in the mean ratios of phosphomonoesters to ATP and phosphodiesters to ATP in patients with both subclinical and overt hepatic encephalopathy. The most marked reductions in these metabolite ratios were observed in patients with overt encephalopathy. (Hepatology 1994;20:1173-1178).
Relationship of Ornithine Decarboxylase Activity and Histological Findings in Human Hepatocellular Carcinoma
Authors: Tamori Akihiro , Nishiguchi Shuhel , Kuroki Tetsuo , Seki Shuichi , Kobayashi Kenzo , Kinoshita Hiroaki , Otani Shuzo
Published: March 2007

To evaluate the correlation between the polyamine metabolism and the degree of malignancy in hepatocellular carcinoma, we measured ornithine decarboxylase activity and polyamine concentrations in neoplastic tissue and adjacent noncancerous tissue from resected specimens of liver from 30 patients. Ornithine decarboxylase activity, polyamines (putrescine, spermidine and spermine) and ornithine decarboxylase mRNA levels were significantly higher in hepatoma tissue than in noncancerous tissue. The activity of this enzyme in the tumor tissue had a negative correlation with the histological degree of differentiation judged according to a modification of the Edmondson and Steiner classification. Resected hepatoma tissue was stained immunohistochemically with antibodies for proliferating cell nuclear antigen (also called cyclin), a marker of cell proliferation. We noted correlation between ornithine decarboxylase activity and the number of cells stained for this antigen (r = 0.882, p < 0.001). These results indicate that ornithine decarboxylase activity is high in human hepatocellular carcinoma, leading to increased intracellular concentrations of polyamines. Ornithine decarboxylase activity also reflected the rate of tumor proliferation and was correlated with the histological findings. (Hepatology 1994;20:1179-1186).
Diurnal Fluctuations of Portal and Systemic Hemodynamic Parameters in Patients With Cirrhosis
Authors: Alvarez Daniel , Golombek Diego , Lopez Patricia , De Las Heras Marcelo , Viola Luis , Sanchez Susana , Kolker Miguel , Mastai Ricardo
Published: March 2007

A close temporal relationship between higher levels of portal pressure during the night and the peak incidence of acute variceal bleeding has recently been demonstrated in patients with cirrhosis. Because hemodynamic changes may have a role in triggering this hemorrhagic episode, we measured systemic and portal hemodynamic parameters at 4–hr intervals for 24 hr in 12 cirrhotic patients. These results were compared with those obtained in eight healthy subjects. Cardiac output, femoral and portal blood flows were measured by Doppler technique. In cirrhotic patients, heart rate and mean arterial pressure remained constant throughout the whole study period. A marked and significant increase in portal blood flow (917 ± 248 ml/min) (mean ± S.D.) as compared with mesor values (649 ± 114 ml/min, p < 0.001) was observed at midnight. This effect was accompanied by a mild but significant rise of cardiac output (from 6.5 ± 0.7 to 6.8 ± 0.7 l/min, p < 0.01) at 2400 hr. A significant correlation between both hemodynamic parameters was found (r = 0.78, p < 0.01). Cosinor analysis showed a significant (p < 0.05) circadian rhythm for both portal blood flow and cardiac output with an acrophase at 0050 hr. In the healthy subjects group, a significant decrease of mean arterial pressure and heart rate was observed at 2400 hr. Cosinor analysis confirmed the presence of significant rhythm for both hemodynamic parameters. In contrast with cirrhotic patients, no significant changes were observed in portal blood flow and cardiac output in healthy subjects. Our results show that in patients with cirrhosis, maximal increases in portal blood flow occur at night. The circadian variation of portal blood flow could have important implications in the management of these patients. (Hepatology 1994;20:1198-1203).
Enzyme Inhibitory Autoantibodies to Pyruvate Dehydrogenase Complex in Primary Biliary Cirrhosis: Applications of A Semiautomated Assay
Authors: Teoh Khay-Lin , Rowley Merrill J. , Zafirakis Helen , Dickson Rolland E. , Wiesner Russell H. , Gershwin Eric M. , Mackay Ian R.
Published: March 2007

Sera from patients with primary biliary cirrhosis inhibit the activity of the mitochondrial pyruvate dehydrogenase complex. We utilized this effect to develop a simple, miniaturized, semiautomated spectrophotometric assay as a diagnostic aid. The sera studied were from 71 patients with primary biliary cirrhosis and 62 other subjects. The assays included enzyme inhibition, immunofluorescence on HEp–2 cells, enzyme–linked immunosorbent assay using recombinant pyruvate dehydrogenase complex–E2 and immunoblotting on bovine heart mitochondria. With the 71 primary biliary cirrhosis sera, on which M2 antibody was detected by immunofluorescence in 64 (90%), antibodies against pyruvate dehydrogenase complex were detected in 53 (83%) by means of enzyme inhibition, in 57 (89%) by means of enzyme–linked immunosorbent assay and in 60 (94%) by means of immunoblotting. Of the 64 sera positive by immunofluorescence, 60 reacted with pyruvate dehydrogenase complex–E2 on immunoblotting, and the miniaturized enzyme inhibition assay was positive in 53 of these. The enzyme inhibition assay and enzyme–linked immunosorbent assay were calibrated to give a specificity of 100%. At this level, the sensitivities for detection of pyruvate dehydrogenase complex antibody were 83% and 87%, respectively. We found no significant changes in levels of reactivity with the enzyme inhibition assay or enzyme–linked immunosorbent assay according to disease stage. Treatment with cyclosporine was accompanied by a significant decrease in levels of antibody to pyruvate dehydrogenase complex–E2 that matched improved indexes of biochemical liver function. The semiautomated enzyme inhibition assay as described provides an additional diagnostic procedure in pyruvate dehydrogenase complex; this type of assay involving specific enzyme inhibition could also be generically applied to any autoantibody antigen system. (Hepatology 1994;20:1220-1224).
Expression of the C– Met Protooncogene in Human Hepatocellular Carcinoma
Authors: Suzuki Kunio , Hayashi Norio , Yamada Yukinori , Yoshihara Harumasa , Miyamoto Yasuhide , Ito Yoshiki , Ito Toshihumi , Katayama Kazuhiro , Sasaki Yutaka , Ito Akihiko , Kisida Yutaka , Kashiwagi Toru , Fusamoto Hideyuki , Kamada Takenobu
Published: March 2007

The c–

met

protooncogene is a growth factor receptor with tyrosine kinase activity. Recently the hepatocyte growth factor was identified as the ligand for this receptor. Because the hepatocyte growth factor is a most potent mitogen in hepatocytes, possible involvement of c–

met

expression in hepatocarcin genesis is suspected. In this study, we examined c–

met

expression in 23 hepatocellular carcinoma cases by means of Northern–blot analysis and an immunohistochemical study. Northern–blot analysis revealed c–

met

mRNA expression in the tumors of 6 of 19 patients (31.6%); in the immunohistochemical study, c–

met

protein was detected in 16 of 23 patients (69.6%). With both methods, c–

met

was found to be overexpressed in hepatocellular carcinoma compared with the surrounding normal liver. Comprehensive analysis showed that c–

met

protein expression was correlated with poor–to–moderate differentiation of cancer cells (p < 0.05). Tumor proliferative activity of hepatocellular carcinoma was evaluated by means of Ki–67 labeling index. All cases with increased tumor proliferative activity showed c–

met

protein expression, although the elevation of proliferative activity in the c–

met

-positive group was not statistically significant. These data suggest that the overexpression of c–

met

plays an important role in the development of hepatocellular carcinoma. (Hepatology 1994;20:1231-1236).
Hepatocyte Proliferation and Gene Expression Induced by Triiodothyronine In Vivo And In Vitro
Authors: Francavilla Antonio , Carr Brian I. , Azzarone Alessandro , Polimeno Lorenzo , Wang Ziqiu , Van Thiel David H. , Subbotin Vladimir , Prelich John G. , Starzl Thomas E.
Published: March 2007

Subcutaneous injections of hormone triiodothyronine in rats resulted in peak blood levels at 24 hr with return to baseline by 96 hr. The injections stimulated a liver regeneration response that resembled in timing and in magnitude of DNA synthesis (peak, 24 hr) that induced by 40% hepatic resection. The principal proliferation was of hepatocytes. Although there were some temporal differences from the gene expression of transforming growth factor–α, transforming growth factor–β, and c–Ha–

ras

that are known to follow partial hepatectomy, the overall profile of these changes was similar to those after partial resection. The effect was liver specific and could be reproduced three times with no diminution in response in the same animal with injections at 10–day intervals. No response was detected in kidney or intestine. This effect in intact animals contrasted with the minimal ability of triiodothyronine to stimulate hepatocytes in culture. However, when the culture medium was enriched with epidermal growth factor, there was a dose–related response to triiodothyronine. The totality of these experiments provides a preliminary basis for the creation with pharmacological techniques of an

in vivo

hyperplastic hepatic condition permissive of transfection of new genes, as an alternative to partial hepatectomy. Although triiodothyronine was the test agent used, other hepatic growth factors singly or in combination could be candidates for this purpose. (Hepatology 1994;20:1237-1241).
Human Hepatocytes Are More Resistant Than Rat Hepatocytes to Anoxia–Reoxygenation Injury
Authors: Caraceni Paolo , Gasbarrini Antonio , Nussler Andreas , Silvio Mauricio Di , Bartoli Fabio , Borle Andre B. , Van Thiel David H.
Published: March 2007

We performed this study to determine whether perfused isolated human and rat hepatocytes have different sensitivities to anoxia–reoxygenation injury. Oxygen free radicals were detected by lucigenin–enhanced chemiluminescence. Lipid peroxidation was assessed by measuring malondialdehyde release. Cell injury was evaluated by measuring lactate dehydrogenase release and trypan blue uptake. During the control period, lucigenin–enhanced chemiluminescence, malondialdehyde and lactate dehydrogenase release and trypan blue uptake were similar in rat and human hepatocytes. During 3.5 hr of anoxia, lucigenin–enhanced chemiluminescence decreased to background levels and malondialdehyde release remained constant in both groups. In contrast, lactate dehydrogenase release increased eightfold in rat hepatocytes but only threefold in human hepatocytes. With reoxygenation after 2.5 hr of anoxia, in rat hepatocytes lucigenin–enhanced chemiluminescence increased 13–fold within 15 min and then declined toward control levels. Malondialdehyde release doubled after 1 hr of reoxygenation. The rate of lactate dehydrogenase release increased to a level almost twice that observed in cells kept continuously anoxic. In contrast, with human hepatocytes lucigenin–enhanced chemiluminescence increased only fourfold, whereas malondialdehyde and lactate dehydrogenase releases did not differ significantly from those levels measured in cells perfused continuously under anoxic conditions. At the end of the experiment, the increase in trypan blue uptake was significantly greater with rat hepatocytes than with human hepatocytes. These results demonstrate that (a) during reoxygenation following 2.5 hr of anoxia, isolated human hepatocytes generate fewer oxygen free radical, and lipoperoxides than do rat hepatocytes, and (b) human hepatocytes are more resistant to cell injury during anoxia–reoxygenation than are rat hepatocytes. (Hepatology 1994;20:1247-1254).
Gastric Microcirculatory Changes of Portal–Hypertensive Rats Can Be Attenuated by Long–Term Estrogen–Progestagen Treatment
Authors: Panés Julián , Casadevall Maria , Fernández Mercedes , Piqué Josep M. , Bosch Jaume , Casamitjana Roser , Cirera Isabel , Bombí Josep A. , Terés Josep , Rodés Juan
Published: March 2007

It has been suggested that estrogen–progestagen therapy may be useful in preventing bleeding from gastric angiodysplasia, a vascular lesion similar to that described in portal–hypertensive gastropathy. In this study we assessed the effects of estrogen–progestagen therapy on gastric microcirculation and systemic and splanchnic hemodynamics in portal–hypertensive and sham–operated rats. One week after the surgical procedure (partial portal vein ligation or sham surgery), animals were given an intramuscular injection of a slow–release preparation of estrogen–progestagen or its vehicle. Two weeks later, gastric mucosal blood flow was measured by means of hydrogen gas clearance, a morphometrical analysis of gastric mucosal blood vessels was performed and systemic and splanchnic hemodynamics were evaluated with a radiolabeled–microspheres technique. In portal–hypertensive rats, estrogen–progestagen therapy induced a significant reduction in gastric mucosal blood flow, number of blood vessels and relative area of vessels. Similar changes, although of lesser magnitude, were achieved with estrogen or progestagen given separately and with the low dose of combined estrogen–progestagen. Estrogen–progestagen treatment also induced significant reductions in portal pressure and portocollateral resistance without changing systemic or splanchnic hemodynamics. In contrast, estrogen–progestagen treatment did not induce changes in any of the parameters studied in sham–operated rats. We conclude that long–term estrogen–progestagen therapy reduces the gastric hyperemia, increased mucosal vessel density and portal pressure in portal–hypertensive rats. (Hepatology 1994;20:1261-1270).
Relationship Between Sigma–Like Site and Progesterone–Binding Site of Adult Male Rat Liver Microsomes
Authors: Yamada Morio , Nishigami Takashi , Nakasho Keiji , Nishimoto Yukiyasu , Miyaji Hideki
Published: March 2007

An increasing amount of evidence suggests that the sigma (σ) sites, putative targets for a variety of psychotomimetic and antipsychotic drugs, exist not only in the brain but also in various peripheral organs. However, there are many ambiguities as to their biological roles, subcellular distributions, endogenous ligands and so on. We therefore performed our study for clarification of some of these ambiguities. As a result, we demonstrated that adult male rat liver microsomes, especially smooth endoplasmic reticulum, possessed a saturable haloperidol–binding site closely resembling the σ site, with a high affinity (Kd 1.0 ± 0.3 nmol/L) and high capacity (Bmax 9.3 ± 1.5 pmol/mg protein) and with the rank order of affinity of the ligands: haloperidol > reduced haloperidol > clorgyline > ifenprodil > 1,3–di(2–tolyl)guanidine, (—)–butaclamol > GBR–12909 > SKF–525A > progesterone > 5α–dihydrotestosterone > R(+)–3– (hydroxyphenyl)–

N

–propylpiperidine > testosterone ± corticosteroids, estradiol–17β, cholesterol and neuroactive compounds displaying high affinities for other neurotransmitter receptors such as dopamine D2, serotonin (5–HT1A and 5–HT2) and α1–adrenergic and GABAA receptors. This rank order showed a high correlation (r = 0.908) with that of a large portion (˜85%) of specific progesterone–binding site (Kd 31.0 ± 3.5 nmol/L, Bmax 5.7 ± 0.2 pmol/mg protein) of the same source. Therefore, these two sites were suggested to be the same or closely related. Furthermore, we provide a strong suggestion that these sites neither are identical with some subforms of the microsomal cytochromes P–450 or other steroid/drug–metabolizing enzymes nor participate universally and directly in the progesterone–metabolizing processes. (Hepatology 1994;20:1271-1280).
Identification of the Thromboxane A2 Receptor in Hepatic Sinusoidal Endothelial Cells and Its Role in Endotoxin–Induced Liver Injury in Rats
Authors: Ishiguro Satoshi , Arii Shigeki , Monden Kazunobu , Adachi Yukito , Funaki Naomi , Higashitsuji Hiroaki , Fujita Shin-Ichi , Furutani Masaharu , Mise Masahiro , Kitao Tadahiro , Nakamura Toshio , Ushikubi Fumitaka , Nakamura Ken-Ichi , Narumiya Shuh , Enomoto Katsuhiko , Ohmura Tousei , Mori Michio , Imamura Masayuki
Published: March 2007

The presence of the thromboxane A2 receptor in sinusoidal endothelial cells was investigated and its pathogenic role in endotoxin–induced liver injury examined. The receptor was measured with a binding assay using a specific thromboxane A2 receptor antagonist, [3H]S–145. Scatchard analysis of the binding indicated the presence of a single class of high–affinity binding sites with a dissociation constant of 5.00 ± 0.96 nmol/L, a maximal binding of 22.85 ± 2.71 fmol/106 cells and 13.80 ± 1.60 × 103 binding sites per cell. The addition of a cyclooxygenase inhibitor, indomethacin, during the cell preparation increased the maximal binding value and the number of binding sites of 37.34 ± 3.01 and 22.50 ± 1.80 × 103 sites/cell, respectively. The binding was displaced by various thromboxane A2 analogs such as ONO–3708 and STA2 but was not effectively competed for by other prostaglandins. Endotoxin injection reduced dissociation constant, maximal binding and the number of binding sites in sinusoidal endothelial cells to 3.49 ± 0.87 nmol/L, 6.03 ± 0.64 fmol/106 cells and 3.65 ± 0.39 × 103 sites/cell, respectively. A cyclooxygenase inhibitor and a Kupffer cell inhibitor added before endotoxin treatment significantly prevented the reduction in the number of thromboxane A2 receptors. It is possible that these effects were due to a reduction in the agonist–induced internalization of the thromboxane A2 receptor brought about by the prevention of thromboxane A2 production. Preadministration of both a cyclooxygenase inhibitor and a thromboxane A2 receptor antagonist attenuated the degree of endotoxininduced liver injury. These findings indicated that the thromboxane A2—thromboxane A2 receptor system in the hepatic sinusoids plays a significant role in the pathogenesis of endotoxin–dependent liver damage. (Hepatology 1994;20:1281-1286).