bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of the Activin Receptor in Cultured Rat Hepatocytes
Authors: Zhang Y , Kanzaki M , Mashima H , Mine T , Kojima I
Published: November 2004

Activin A is an autocrine inhibitor of initiation of DNA synthesis in rat hepatocytes. The present study was conducted to characterize the cell–surface receptors for activin A in cultured rat hepatocytes by measuring 125I–activin A binding. Scatchard analysis of 125I–activin A binding indicated the existence of two classes of binding sites with apparent Kd values of 3 × 10-.10 mol/L and 3.5 × 10-.9 mol/L. Pretreatment of the cells with heparitinase reduced the number of low– affinity binding sites, whereas pretreatment with excess exogenous follistatin increased the number of low–affinity binding sites. Affinity cross–linking of 125I–activin A to hepatocytes revealed distinct protein complexes with molecular weights of approximately 48, 65, and 85 kd, which may represent cross–linked cell–bound follistatin, type I and type II activin receptors, respectively. Another band with a molecular weight of 180 kd was also found, which may represent the type III activin receptor. When hepatocytes were cultured with epidermal growth factor (EGF), both high– and low–affinity binding sites increased at 12 hours without altering their affinities. At 60 hours of the incubation with EGF, the high–affinity binding sites decreased while the number of low–affinity binding sites increased slightly. These results indicate that two classes of 125I–activin A binding sites exist in cultured hepatocytes: the high–affinity binding site may represent oligomeric complex of the type I and type II receptors, and at least part of the low–affinity binding site may represent cell–bound follistatin. The number of activin receptors in hepatocytes is increased after the stimulation with EGF.
Cirrhotic Cardiomyopathy: Getting to the Heart of the Matter
Authors: Ma Z , Lee S S
Published: November 2004

In cirrhosis, cardiac contractile function has been extensively documented to be abnormal. At baseline, cardiac output is increased, and this is one of the characteristics of hyperdynamic circulation. However, when cirrhotic patients are challenged by pharmacological or physiological stress, ventricular hyporesponsiveness is revealed. Similar patterns have been noted in cirrhotic animal models. This phenomenon has been termed “cirrhotic cardiomyopathy.” Although alcohol abuse may contribute to some cases of cirrhotic cardiomyopathy, it has been clearly documented to occur even in the absence of alcohol ingestion. Diminished myocardial β–adrenergic receptor signal transduction function, possibly caused by a persistent elevation in norepinephrine content, has been shown to play an important role. Alternation in cardiac plasma membrane properties due to impaired lipid metabolism is also crucial. Other possible pathogenic factors are reviewed, including accumulation of cardiodepressant substances caused by hepatocellular insufficiency, and ventricular overload secondary to increased blood volume and hyperdynamic circulation. Because the cardiac reserve function is borderline in patients with cirrhosis, cardiovascular status should be carefully monitored, especially when patients undergo stresses such as liver transplantation or portosystemic shunting procedures.
Detection of Common Hepatitis C Virus Subtypes With A Third–Generation Enzyme Immunoassa
Authors: Huber K R , Sebesta C , Bauer K
Published: November 2004

The causal agent of most posttransfusion non–A and non–B hepatitis infections was characterized in 1989 by molecular biological techniques as a positive–stranded, enveloped RNA virus, designated hepatitis C virus (HCV). Only since 1990 has it been possible to screen for an infection with antibody tests or direct amplification assays for the nucleic acid (i.e., reverse–transcription polymerase chain reaction [PCR]). However, these nucleic acid based tests are time consuming and rather expensive. Recently, third–generation enzyme immunoassays (EIAs) for HCV infection were introduced (i.e., Abbott Laboratories, North Chicago, IL; Ortho Diagnostics, Inc., Raritan, NJ; and Roche Diagnostics, Basel, Switzerland). The Roche Diagnostics EIA has been evaluated with our patient population. To this end, 1,090 samples were assayed by both EIA and PCR; 946 of all samples (87%) were negative, and 107 samples (9.8%) were positive by both tests. Thirty of the patients (2.7%) showed antibodies but no detectable virus, whereas 7 of all patients tested (0.6%) were PCR–positive but had not yet developed antibodies to the virus. Of these 7 patients, only one showed normal serum transaminases. Virus strain subtyping and quantification of viral load on the positive samples in parallel have been performed, and the fact that the EIA detects all the virus subtypes found in our hospital can be inferred. No correlation between subtypes, viral load, and immune response could be measured with this antibody test. Our results indicate that, in most circumstances (except in settings where immunocompromised patients are abundant), this EIA can replace the much more expensive PCR tests for the routine screening for HCV infection.
Receptor–Mediated Delivery of Hepatitis B Virus Dna and Antisense Oligodeoxynucleotides to Avian Liver Cells
Authors: Madon J , Blum H E
Published: November 2004

A highly efficient receptor–mediated delivery system for DNA and oligodeoxynucleotides (ODNs) to avian liver cells has been established, using complexes of nonmodified human adenovirus particles and a protein conjugate consisting of n–acetyl–glucosamine–modified bovine serum albumin, streptavidin, and Poly–l–lysine. The newly developed method of protein–conjugate preparation and purification yielded highly stable complexes with high DNA delivery efficiency for constructs expressing the

lacZ

gene, hepatitis B virus (HBV) DNA, and ODNs. Using this delivery system, an antisense ODN targeted to the encapsidation site of the HBV pregenome causes strong inhibition of HBV replication

in vitro

. The described, receptor–mediated DNA delivery method should prove useful for investigations of HBV replication, gene expression, and mode of action of antiviral agents in vitro, as well as for model studies of gene therapy for liver diseases from various etiologies, including viral hepatitides.
Hepatitis B Virus Carriers Without Precore Mutations in Hepatitis B E Antigen–Negative Stage Show More Severe Liver Damage
Authors: Lindh M , Horal P , Dhillon A P , Furuta Y , Norkrans G
Published: November 2004

Hepatitis B e antigen (HBeAg) is considered to be a major target for the immune response in chronic hepatitis B. The G → A mutation at nucleotide 1896 may mediate viral escape by creating a TAG stop codon in the precore region, thus preventing HBeAg production. This mutation frequently evolves during HBe seroconversion if thymine, but rarely if cytosine, is present in position 1858. Applying a combination of polymerase chain reaction (PCR) and restriction enzyme action, we have studied the relation of the TAG mutation and the nucleotide (nt) 1858 variants to liver damage assessed by histology activity index (HAI) scoring in 175 chronic hepatitis B virus (HBV) carriers. A TAG mutation was found in 68 of 71 (96%) of HBeAg–negative carriers infected with a T–1858 strain, but not in any of 33 carriers infected exclusively with a C–1858 strain. Four patients showed a mutation of the precore start codon, and 2 had a TAA stop mutation at codon 2. HBeAg–positive infection with a mixture of wild–type and TAG mutant virus indicated active liver damage, because 8 of 9 (89%) of such patients had a Knodell HAI greater than or equal to 8. In HBeAg–negative stage, both inflammation and fibrosis were more pronounced in carriers infected with wild–type HBV compared with precore mutants. C–1858 strains were associated with more inflammation and fibrosis compared with T–1858 strains. C–1858 strains were found in 71% of northern European, 17% of southern European, 31% of African, 2% of Middle Eastern, and 10% of Far Eastern carriers. Analysis of these variants and mutants may prove useful for clinical evaluation and choice of therapy, and may be facilitated by the methods described.
Osthole Increases Glycosylation of Hepatitis B Surface Antigen and Suppresses the Secretion of Hepatitis B Virus In Vitro
Authors: Huang R , Chen C , Huang Y , Hsieh D , Hu C , Chang C
Published: November 2004

During screening of Chinese herbal medicines for the activities against hepatitis B virus (HBV), a known pure compound, osthole, was found to inhibit the secretion of HBV surface antigens

in vitro

. The secretion of hepatitis B surface antigen (HBsAg) in culture medium of MS–G2 and HuH–7 cells transfected with HBV DNA decreased by 60% to 70% after osthole treatment, without any detectable cytotoxic effects. Both 42–nm Dane particles and 22–nm subviral HBsAg particles were significantly reduced as revealed by density gradient analysis of viral particles and the endogenous; HBV DNA polymerase assay. The production of naked HBcAg particles was not changed by osthole treatment. The steady–state level of HBV–specific messenger RNA (mRNA) was not altered by osthole treatment, as revealed by Northern blot analysis, Immunoprecipitation of culture medium showed that the ratio of glycosylated versus nonglycosylated HBsAg increased after osthole treatment. Kinetic studies revealed that osthole increased the glycosylation rate of HBsAg, thus altering the ratio of glycosylated to unglycosylated HBsAg. The increase of glycosylation of HBsAg by osthole was blocked by tunicamycin, an inhibitor of protein glycosylation. We conclude that osthole suppresses the secretion of HBV in cell culture probably because of its novel function in increasing the glycosylation of HBsAg.
Heparan Sulfate Proteoglycan Expression in Chronic Cholestatic Human Liver Diseases
Authors: Roskams T , Rosenbaum J , De Vos R , David G , Desmet V
Published: November 2004

The mechanism of bile ductular reaction and accompanying fibrogenesis depends on interactions of ductular cells with the matrix and growth factors. Heparan sulfate proteoglycans (HSPGs) are essential cofactors in cell–matrix adhesion processes, in cell–cell recognition systems, and in receptor–growth factor interactions. We used monoclonal antibodies specific for the cell surface HSPGs (syndecans, glypican), for matrix HSPG (perlecan), and for heparan sulfate carbohydrate (HS) to investigate their immunohistochemical expression in 20 specimens with chronic cholestatic liver disease and in five normal human liver specimens. Because activated hepatic stellate cells HSC are a major source of fibrosis in the liver, we also examined HSPG expression in primary cultures of human activated HSC using immunocytochemistry and Western blotting and for syndecan–1 also Northern blotting. In comparison with bile ductular cells of normal liver, reactive ductules in chronic cholestasis were marked by an elevated expression of syndecan–1, surrounded by an increased perlecan expression. In acinar zone 1, large stimulated macrophages and HSC, present in increased numbers, were strongly positive for syndecan–3. Cultured HSC showed a membranous staining pattern for syndecan–1, syndecan–3, and heparan sulfate, and in addition intracellular staining for syndecan–2, –3, and 4. Perlecan immunoreactivity was detected as intercellular strings. Western blotting revealed positive bands with all antibodies and Northern blotting for syndecan–1 was also positive. These results show that cultured human HSC can synthesize all four syndecans, glypican, and perlecan. These data reveal changes in the expression of syndecan–1, syndecan–3, and perlecan in human chronic cholestatic liver disease, that may be important in the deposition of matrix components and activation of growth factors that support ductular reaction and accompanying fibrogenesis.
The Diagnosis of Subclinical Hepatic Encephalopathy in Patients With Cirrhosis Using Neuropsychological Tests and Automated Electroencephalogram Analysis
Authors: Quero J C , Hartmann I J , Meulstee J , Hop W C , Schalm S W
Published: November 2004

Neuropsychological tests used for the assessment of subclinical hepatic encephalopathy (SHE) may overdiagnose SHE because scores are usually not corrected for age. The aim of this study was to estimate the prevalence of SHE using two easy administrable psychometric tests (Number Connection Test part A [NCT–A], Symbol Digit Test [SDT]) with age–related normal values. In addition, spectral electroencephalogram (EEG) was used, which is the in–house electrophysiological method for quantifying encephalopathy.

One hundred and thirty–seven consecutive patients (mean age 49 years, range 17–77) with cirrhosis without any clinical signs of encephalopathy, were screened for SHE. In addition, the Child–Pugh score and the arterial blood ammonia were determined. Patients with concurrent use of alcohol, benzodiazepines or anti–epileptics were excluded.

Fifty percent of the patients had an abnormal NCT according to the standard recommended procedure, in contrast only 7% of the patients had an abnormal NCT when scores corrected for age were used. Combining the results of the spectral EEG and the psychometric tests corrected for age yielded a higher prevalence of SHE (23%) than when each test method was used alone (17% vs. 10% abnormal, respectively). Severity of liver disease correlated with the presence of SHE, because the prevalence of abnormal tests increased from 14% in Child–Pugh grade A to 45% in Child–Pugh grade B or C. Age above 40 years and an elevated blood ammonia level were significant determinants related to an abnormal EEG. We conclude that the NCT uncorrected for age markedly overdiagnoses SHE and, therefore, should not be used as a test for the screening of SHE. Using a combination of spectral EEG and two psychometric tests with age–corrected normal values a low prevalence of SHE in patients with Child A liver cirrhosis is found. Older patients with an elevated arterial ammonia are more prone to develop SHE than younger patients with an equal arterial ammonia concentration.
Immunogenicity of Biliary Epithelium: Investigation of Antigen Presentation to Cd4+ T Cells
Authors: Leon M P , Bassendine M F , Wilson J L , Ali S , Thick M , Kirby J A
Published: November 2004

The intrahepatic biliary epithelium is susceptible to extensive T–cell– mediated damage during primary biliary cirrhosis, primary sclerosing cholangitis, and hepatic allograft rejection. During these processes, human intrahepatic biliary epithelial cells (HIBEC) become activated and express high levels of the lymphocyte adhesion molecules, intercellular adhesion molecule–1 (ICAM–1) and lymphocyte–associated antigen (LFA)–3, and of class II MHC antigens. It follows that activated HIBEC may also play a direct role in the activation of antigen–specific CD4+ T lymphocytes. The capacity of class II MHC antigen–expressing HIBEC to present antigen and induce specific proliferation of CD4+ T cells was examined in this study. Lines of purified HIBEC were activated by culture with the proinflammatory cytokines interferon γ (IFN–γ) and tumor necrosis factor a and were mixed in coculture with allogeneic CD4+ T cells. The result of interaction between these cells was assessed by measurement of lymphoproliferation and IL–2 production. Class II MHC antigen–expressing HIBEC failed to induce either lymphoproliferation or IL–2 production. However, both of these parameters of T–cell activation were positive in cocultures when a costimulation signal was delivered to T cells by adding bivalent anti–CD28 antibodies. The antigen–specific activation of these T cells was further enhanced by the addition of a cross–linking secondary antibody that caused CD28 receptor aggregation. The failure of cytokine–stimulated HIBEC to induce T–cell activation is consistent with the observation that HIBEC do not express the costimulatory CD28 ligands B7–1 or B7–2 at either mRNA or protein levels. It may be concluded that HIBEC are unlikely to play a direct role in activation of antigen–specific CD4+ T lymphocytes within the inflamed liver.
Apolipoprotein E4 Genotype and Gallbladder Motility Influence Speed of Gallstone Clearance and Risk of Recurrence After Extracorporeal Shock–Wave Lithotripsy
Authors: Portincasa P , van Erpecum K J , van de Meeberg P C , Dallinga-Thie G M , de Bruin T W A , van Berge-Henegouwen G P
Published: November 2004

Extracorporeal shock–wave lithotripsy (ESWL) is an effective treatment in selected gallstone patients, but stone recurrence is a major drawback. Factors potentially influencing gallstone clearance and recurrence were studied in 84 patients in whom stone dissolution was diagnosed after ESWL plus bile salt therapy for initial solitary (n = 55) or multiple (n = 29) radiolucent stones. Apolipoprotein E (apoE) genotyping and gallbladder motility (sonography) were studied in a representative subgroup of patients (n = 50). The median follow–up after ESWL was 36 months (range, 4.5–67 months). Gallstone clearance was achieved after 8.7 months (range, 0.2–30 months). Independent factors significantly enhancing gallstone clearance were the presence of E4 allele; small initial gallstone size and number; effectiveness of fragmentation; and good gallbladder emptying (

P

= .002). Gallstone recurrence was seen in 30 patients after 18.6 months (range, 1.0–50 months). Cumulative gallstone recurrence rate (life–table analysis) was 15% within 1 year, increasing to 60% within 5.5 years. Although the probability of gallstone recurrence tended to be smaller in patients with initial solitary stones than in those with multiple stones during early follow–up, differences disappeared after long–term follow–up. Effective gallbladder emptying (residual volume < or = 6 mL) and apolipoprotein E4 (apoE4) independently influenced gallstone recurrence. Recurrence rate was higher (log rank test, P = .037) in those patients who were homozygous and heterozygous for the E4 allele compared with the individuals who were not expressing the apoE4 allele. Accordingly, there was an overrepresentation of the allele frequency for E4 in the group with gallstone recurrence (

P

= .03). Patients with small postprandial residual gallbladder volumes (≤ 6 mL) had a lower probability of stone recurrence than those with large residual gallbladder volumes (log rank test,

P

= .0215). Biliary pain was more frequent with recurrence (55% vs. 13%,

P

= .001). The present study indicates that apoE4 genotype is associated with increased speed of gallstone clearance as well as a high risk of recurrence after ESWL. This finding points to a possible role for genetic factors in the pathogenesis of cholesterol gallstones. Effective gallbladder emptying is important for speed of clearance and prevention of recurrence. Patients with initial solitary stones have a decreased early– but not long–term gallstone recurrence rate.
Hepatocyte Growth Factor and C– Met Expression in Long–Evans Cinnamon Rats With Spontaneous Hepatitis and Hepatoma
Authors: Nakayama N , Kashiwazaki H , Kobayashi N , Hamada J , Ogiso Y , Itakura Y , Matsumoto K , Nakamura T , Koike T , Kuzumaki N , Takeichi N
Published: November 2004

The Long–Evans Cinnamon (LEC) rat is characterized by the spontaneous onset of acute and chronic hepatitis, followed by occurrence of liver cancer, and is thus able to provide a unique experimental model for human genetical liver disease, Wilson's disease. Hepatocyte growth factor (HGF) is a potent hepatotrophic factor in liver regeneration, and its expression is up–regulated in response to liver injuries. We found that the plasma HGF level in LEC rats rose markedly during the fulminant hepatitis phase, fell during the phase of chronic/cholangiofibrosis, and fluctuated during the hepatoma phase. Immunohistological staining of the liver revealed that the number of HGF–positive cells increased remarkably during the fulminant hepatitis phase, and that many of these cells were localized at the portal triads. Fewer HGF–positive cells were observed during the phase of chronic hepatitis. The surface of the hepatocellular carcinoma (HCC) cells and the cytoplasm of the nonepithelial cells in cancerous liver tissues were HGF–positive. The HGF–messenger RNA (mRNA) level in the liver rose in the fulminant hepatitis phase, fell in the chronic hepatitis phase, and was intermediate or high during the hepatoma phase. The expression of c–

met

mRNA was strong in the tissues of LEC rats with fulminant hepatitis and, especially, in the cholangiofibrosis tissues. c–

met

mRNA was also detected in HCCs. These results suggest that the HGF–c–

met

system may play an important role in the regeneration of hepatocytes as well as in the development of HCC in paracrine or autocrine mechanisms.
Differential Induction of Carcinogen Metabolizing Enzymes in A Transgenic Mouse Model of Fulminant Hepatitis
Authors: Chemin I , Takahashi S , Belloc C , Lang M A , Ando K , Guidotti L G , Chisari F V , Wild C P
Published: November 2004

The objective of this work is to examine the possible modulation of carcinogen metabolism (activation by cytochrome P450s and detoxification by conjugation via glutathione S–transferases [GST]) in relation to hepatitis B virus (HBV)–associated liver injury. In HBV transgenic mouse lineage 107.5, the hepatitis B surface antigen (HBsAg) is expressed at noncytopathic concentrations but after injection of an HBsAg–specific, major histocompatibility complex (MHC) class I restricted cytotoxic T–lymphocyte (CTL) clone, the mice develop a severe acute necroinflammatory liver disease that reaches maximum severity within 3 days and gradually subsides during the next 2 to 3 weeks. In this model, using immunohistochemical analysis, we observed an increase of P450s (CYP1A and 2A5), both involved in aflatoxin B1, metabolism, but minor changes or no changes for others (2B, 2C, 2E, 3A). There was a fivefold decrease in the total liver P450 microsomal content 3 days' post–CTL injection with the result that the relative proportion of CYP2A5 and 1A compared with other P450s is increased. Individual microsomal P450 enzyme contents estimated by Western blotting; Northern blot analysis of liver CYP messenger RNA (mRNA) levels as well as

in vitro

metabolism of specific substrates for different P450 isoenzymes were consistent with the immunohistochemical data. Immunohistochemical staining with antibodies to cytosolic pi class GST was increased 1 and 3 days postinjection followed by a progressive decrease at later time points (the same phenomenon was observed to a lesser extent for GST α). The activity of hepatic cytosols toward substrates specific for different subclasses of GST (mu, pi, α) showed that while GST mu was not changed in the CTL–injected HBV transgenic mice, GST pi and, to a lesser extent, α were increased as compared with controls. These results suggest that liver cell injury induced by a process of acute fulminant–like hepatitis can lead to the induction of some carcinogen metabolizing enzymes notably, Cyp 1A, 2A5 and GST pi in the mouse.
Selective Enhancement of Lipid Peroxidation in Plasma Membrane in Two Experimental Models of Liver Regeneration: Partial Hepatectomy and Acute Ccl4 Administration
Authors: Aguilar-Delfin I , López-Barrera F , Hernández-Munoz R
Published: November 2004

It has been proposed that lipid peroxidation (LP) might be a modulator of cell division, influencing initiation and cessation of mitosis in regenerating liver. However, the understanding of the participating role of this event in the onset of liver proliferation has been hampered by the fact that both higher or lower LP have been reported after two–thirds partial hepatectomy (PH). Therefore, the present study deals with the extent of LP in the main subcellular fractions from rat liver at early stages of regeneration, induced by either PH of 70% or acute CCl4 administration. Our results, using several methods to monitor LP, indicate a differential effect in the peroxidative pattern of specific subcellular fractions from regenerating liver after 24 hours of PH: a decrease in microsomes and an increase confined to plasma membrane and cytosolic fractions, peaking after 24 hours of PH. In CCl4–treated rats, higher LP was also noted in plasma membrane and cytosol, being maximal at the replicative stage in this experimental model (48 hours). In addition, increased LP was found in microsomal and nuclear fractions, declining before the 48 hours. In hepatectomized rats, changes in LP seem to be an organ–specific event and related to only PHs capable of triggering a synchronized proliferative response, namely above 40%. These results show that LP, promoted by PH and CCl4 administration, is qualitatively distinct among subcellular fractions and may indeed be a normal cell event of physiological importance in the regenerating liver.
Expression of Intracellular Adhesion Molecule 1 by Activated Hepatic Stellate Cells
Authors: Hellerbrand C , Wang S C , Tsukamoto H , Brenner D A , Rippe R A
Published: November 2004

The hepatic stellate cell (HSC), following a fibrogenic stimulus, is transformed from a quiescent to an activated cell. HSC activation results in numerous changes in cellular morphology, cellular metabolism, and in the pattern of gene expression. Many of the changes that are observed in activated HSCs in animal models of hepatic fibrosis are also seen when these cells are activated by culturing on plastic. These changes include morphological changes to a myofibroblast– like cell with the appearance of smooth muscle α–actin, a loss of the retinol stores, an increase in the rough endoplasmic reticulum, and increases in extracellular matrix production, including a dramatic increase in type I collagen. To identify additional genes that are induced or suppressed during HSC activation, we used the differential polymerase chain reaction (PCR) display technique. Using this technique, we isolated a complementary DNA (cDNA) fragment for the intercellular adhesion molecule 1 (ICAM–1). Northern blotting confirmed that the ICAM–1 messenger RNA (mRNA) was expressed in HSCs activated by culture, but not in quiescent, freshly isolated HSCs. The presence of ICAM–1 protein was demonstrated in culture–activated HSCs, but not in quiescent cells by Western blot analysis and immunohistochemical staining. A functional assay was performed, demonstrating that lymphocytes will adhere to activated HSCs and that treatment of these cells with tumor necrosis factor α (TNF–α) increases lymphocyte adherence. Furthermore, ICAM–1 mRNA levels were increased in HSCs activated in rats in vivo after 1 week of bile duct ligation (BDL). Together, these data indicate that ICAM–1 expression is induced following HSC activation and that the HSC may have a direct role in the transmigration of leukocytes from the hepatic sinusoid to sites of tissue damage during the inflammatory response in the liver.
Inhibition of Anaphylatoxin C3a– and C5a– But Not Nerve Stimulation– or Noradrenaline–Dependent Increase in Glucose Output and Reduction of Flow in Kupffer Cell–Depleted Perfused Rat Livers
Authors: Puschel G P , Nolte A , Schieferdecker H L , Rothermel E , Gotze O , Jungermann K
Published: November 2004

In isolated in situ perfused rat livers, infusion of anaphylatoxins C3a and C5a, activation peptides of the complement system, as well as stimulation of sympathetic hepatic nerves have been shown to increase hepatic glucose output and to reduce hepatic flow. These effects were mediated via an at least partially prostanoid–dependent intercellular signalling chain between nonparenchymal cells and hepatocytes. Kupffer cells have been implicated as the source of prostanoids in the anaphylatoxin–dependent signalling chain and Ito cells in the nerve stimulation–dependent signalling chain, because anaphylatoxins and noradrenaline increased prostanoid synthesis in isolated Kupffer and Ito cells, respectively. To further corroborate this hypothesis, anaphylatoxins were infused and hepatic nerves were stimulated in perfused rat livers in which Kupffer cells had been largely depleted by treatment of the animals with gadolinium chloride. Native human anaphylatoxin C3a (nhC3a) and recombinant rat anaphylatoxin C5a (rrC5a) increased prostanoid formation as well as glucose output and reduced flow in perfused rat liver. In Kupffer cell–depleted rat livers, the nhC3a– and rrC5a–mediated prostanoid formation was nearly abolished, and the increase in glucose output and the reduction of flow were reduced to between 30% and 50% (area under the curve [AUC]) of control livers. In contrast, stimulation of hepatic nerves increased glucose output and reduced flow to a similar extent in Kupffer cell–depleted livers as in control livers. These results indicate that Kupffer cells were not involved in the prostanoid–mediated nerve stimulation–dependent increase in glucose output and reduction of flow. Kupffer cells seemed, however, to be at least one major source of the anaphylatoxin–mediated prostanoid formation and, consequently, stimulation of glucose release and flow reduction in perfused liver. Because the metabolic and hemodynamic anaphylatoxin effects were not completely blocked in livers of gadolinium–treated animals, either Kupffer cells may not have been entirely eliminated, or yet another nonparenchymal cell type and mediator might be involved in the anaphylatoxin–elicited intercellular communication between nonparenchymal cells and hepatocytes.
Pk 11195 Aggravates 3,5–Diethoxycarbonyl–1,4–Dihydrocollidine–Induced Hepatic Porphyria in Rats
Authors: Fonia O , Weizman R , Coleman R , Kaganovskaya E , Gavish M
Published: November 2004

There is evidence to suggest that peripheral–type benzodiazepine receptors (PBR) are involved in porphyrin transport during erythroid differentiation, and it is possible that these receptors have an important role in heme biosynthesis. We examined the biochemical and ultrastructural alterations in rat liver following experimentally induced acute hepatic porphyria, as well as the effects of the administration of a selective PBR ligand, PK 11195. The most severe pathological conditions were found in rats that received a combined treatment of the porphyrinogenic agent 3,5–diethoxycarbonyl–1,4–dihydrocollidine (DDC) and PK 11195. Transmission electron microscopy showed a correlation between the ultrastructural pathology of the liver, the total porphyrin levels in urine and liver, and the porphobilinogen levels in urine. Hepatocytes in this acute porphyria showed the development of large secondary lysosomes containing crystalline aggregates of protoporphyrin. Bile canaliculi were grossly enlarged, contained aggregates of protoporphyrin crystals, and showed the presence of bile thrombi. In addition, prominent bundles of collagen fibers (fibrosis) were commonly found in livers of rats that had been treated with DDC or DDC and PK 11195. We conclude that the administration of PK 11195 to porphyric rats aggravates porphyrin accumulation and cellular damage in the liver. Perhaps this evidence suggests that PK 11195 blocks the binding of protoporphyrin IX to PBR, thus elevating the content of protoporphyrin IX in liver.
T– and B–Cell Responses to Different Hepatitis C Virus Antigens in Patients With Chronic Hepatitis C Infection and in Healthy Anti– Hepatitis C Virus—Positive Blood Donors Without Viremi
Authors: Lechmann M , Ihlenfeldt H G , Braunschweiger I , Giers G , Jung G , Matz B , Kaiser R , Sauerbruch T , Spengler U
Published: November 2004

As the host's immune response may determine the course of hepatitis C virus (HCV) infection, we studied the humoral and cellular immune responses to HCV–related antigens in subjects with different outcomes of HCV infection. Lymphoproliferative responses and circulating antibodies to a panel of HCV core– and E1–related 25–mer peptides were examined in 10 healthy anti–HCV–seropositive blood donors (group A) and in 29 patients with chronic hepatitis C (group B). In addition, cellular recognition of recombinant HCV proteins (core, NS3, NS4A, NS5A, NS5B) were investigated. In group A, stronger T–cell responses were detected against both HCV proteins (core,

P

= .03; NS4,

P

= .005; NS5B,

P

= .03) and peptides. Proliferation was induced by the same peptides in each group, defining at least five distinctive epitopes within core (amino acids [aa] of 20–44, aa 39–63, aa 79–103, aa 118–152 and aa 148–172) and three regions within E1(aa 198–252, aa 308–372, and aa 368–392). Subjects with strong T–cell responses had low or no detectable levels of peptide–specific antibodies, and vice versa. In particular, T–cell responses were more common in group A; B–cell responses were more common in group B. From our data, we conclude that a benign course of HCV infection may be the consequence of the effective activation of T–helper lymphocytes.
Differential Induction of Prehepatic and Hepatic Metabolism of Verapamil by Rifampin
Authors: Fromm M F , Busse D , Kroemer H K , Eichelbaum M
Published: November 2004

Cytochrome P450 (CYP) enzymes, which metabolize numerous drugs, are expressed both in liver and in extrahepatic tissues. CYP3A4 for example is present and inducible by rifampin in epithelial cells of the gastrointestinal tract. It has been shown that such prehepatic metabolism contributes substantially to total clearance of CYP3A4 substrates (e.g., cyclosporine) before and even more pronounced during enzyme induction. We examined the effect of enzyme induction on prehepatic and hepatic metabolism of the model compound R/S–verapamil after simultaneous oral and intravenous administration using a stable isotope technology. This approach allows us to exclude intraindividual day–to–day variability and is therefore suitable to quantitatively assess prehepatic extraction of high–clearance drugs. Moreover, because verapamil is administered as a race–mate with the S–enantiomer being preferentially metabolized, we investigated the influence of induction on stereoselectivity of prehepatic and hepatic metabolism. Eight male volunteers received 120 mg of racemic verapamil bid for 24 days. Rifampin (600 mg daily) was given from day 5 to day 16. Systemic clearance and bioavailability of the verapamil enantiomers were determined by coadministering deuterated verapamil intravenously on day 4, on day 16, and on day 24. Effects of verapamil on atrioventricular conduction after oral and intravenous (iv) administration were assessed by measuring the maximum PR–interval prolongation Rifampin increased the systemic clearance of the active S–verapamil 1.3–fold (

P

< .001). In contrast, rifampin increased the apparent oral clearance of S– verapamil 32–fold (

P

< .001) and decreased its bioavailability 25–fold (

P

< .001), with partial recovery after rifampin withdrawal (

P

< .01). With rifampin, the effect of oral verapamil on atrioventricular conduction was nearly abolished (

P

< .01), whereas no significant changes were observed after intravenous administration. Induction caused a considerable reduction of stereoselectivity after both intravenous and oral administration (

P

< .001). Rifampin altered the pharmacokinetics and the pharmacological effects of verapamil to a much greater extent after oral administration compared with intravenous administration. These data clearly indicate that prehepatic metabolism of verapamil (presumably in the gut wall) is preferentially induced compared with hepatic metabolism and that stereoselectivity of verapamil metabolism is affected by induction.
Systemic Antibiotic Prophylaxis After Gastrointestinal Hemorrhage in Cirrhotic Patients With A High Risk of Infection
Authors: Pauwels A , Mostefa-Kara N , Debenes B , Degoutte E , Levy V
Published: November 2004

In cirrhotic patients with gastrointestinal hemorrhage, bacterial infections are frequent and play a significant role in mortality. We have previously found that patients with a Child–Pugh's class C or a rebleeding are a subgroup of cirrhotic patients with a high risk of infection. The aims of the study were (1) to validate these indicators and (2) to assess the effectiveness of a systemic antibiotic treatment in preventing bacterial infections in bleeding cirrhotics with a high risk of infection. One hundred and nineteen bleeding cirrhotic patients were divided into 3 groups. Patients with a Child–Pugh's class A–B and no rebleeding (i.e., with a low risk of infection) constituted group 1 (n = 55). Patients with a high risk of infection were randomly allocated to serve as controls (group 2, n = 34) or to receive the ciprofloxacin and a combination of amoxicillin and clavulanic acid for 3 days after hemorrhage (group 3, n = 30). This antibiotic prophylaxis was administered first intravenously and then orally when the bleeding was controlled. The study period was defined as 10 days after hemorrhage. Incidence of bacterial infections was significantly higher in patients from group 2 than in patients from group 1 (52.9% vs. 18.2%;

P

< .001). Moreover, infections were more severe in group 2: a sepsis syndrome or a septic shock developed in 66.7% of infected patients from this group, but in only 20% of infected patients from group 1. Incidence of bacterial infections was much lower in patients from group 3 than in those from group 2 (13.3% vs. 52.9%;

P

< .001). Eight patients from group 2 (23.5%) and 4 patients from group 3 (13.3%) died during the first four weeks (P–not significant). Septic shock was the cause of death in 3 patients from group 2 and in only 1 patient from group 3. The cost of antibiotic therapy, including antibiotic prophylaxis in group 3, was $208 ± $63 per patient in group 2 and $167 ± $42 per patient in group 3 (

P

< .05). We conclude that (1) patients with a Child–Pugh's class C and/or a rebleeding are a subgroup of cirrhotic patients with a high risk of infection after gastrointestinal hemorrhage and that (2) in these patients, a prophylactic treatment with systemic antibiotics is very effective in preventing bacterial infections.
Pathomorphological Study of Kupffer Cells in Hepatocellular Carcinoma and Hyperplastic Nodular Lesions in the Liver
Authors: Tanaka M , Nakashima O , Wada Y , Kage M , Kojiro M
Published: November 2004

A pathomorphological study was conducted to clarify the localization of Kupffer cells in hepatocellular carcinoma (HCC) tissues and such hyperplastic nodular lesions as adenomatous hyperplasia (AH) and focal nodular hyperplasia (FNH). Materials were surgical specimens of 50 HCCs, 7 AHs, and 13 FNHs. These tissues were immunohistochemically stained with an anti–human macrophage antibody (anti–CD68 antibody). Among CD68–positive cells, short spindle–shaped cells were considered as Kupffer cells, and Kupffer cell numbers in tumor lesions and surrounding liver tissues of each specimen were comparatively examined. As a result, the number of Kupffer cells in well–differentiated HCC tissues less than 1 cm in diameter was 27.8 ± 3.3 (mean ± SE/0.25 mm2); in noncancerous tissues, it was 30.2 ± 3.2, showing no statistically significant differences. The number of Kupffer cells in cancerous tissues decreased in comparison with the number in noncancerous tissues, as the tumor size increased and histological grade decreased. In hyperplastic nodular lesions, the number was higher in nodular lesions than in the surrounding liver tissues in 4 of 7 AHs (57.1%) and 6 of 13 FNHs (46.2%). This could explain the reason why enhanced MRI, which utilizes the selective taken–up mechanism of chondroitin sulfate iron colloid and superparamagnetic iron oxide into the reticuloendothelial system of the liver and spleen, depicts well– differentiated HCC and AH at the same signal intensity as in the surrounding liver tissues. Our findings also indicate that there is a limitation in differentiating or diagnosing small HCC and hyperplastic nodular lesions by using enhanced MRI, which utilizes Kupffer cell functions.
Clinical Hepatology: Profile of An Urban, Hospital–Based Practic
Authors: Byron D , Minuk G Y
Published: November 2004

To dispel the common notion that the practice of hepatology in North America largely consists of the care of middle–aged male patients with alcohol–induced liver disease, and, in the process, provide undergraduate and postgraduate students with a clearer picture of what patient profiles might resemble in an urban, hospital–based hepatology practice, 1,226 charts derived from referrals between July 1, 1987, and January 1, 1994, were retrospectively reviewed for the following information: year of referral, age and sex of the patient, practice of the referring physician, status of ongoing care, and principal diagnosis. The results of the study revealed the following: 1 referrals for assessment and care of patients with liver disease are increasing at a rapid rate, 2 the majority of referred patients are between the ages of 25 and 45 years, 3 there is an equal distribution of men and women, 4 referrals are most often from general practitioners (57%) and internists (27%), 5 turnover is common, with 56% of referred patients no longer being followed, and 6 a variety of liver disorders are seen, including acute and chronic viral hepatitis (47%), nonalcoholic steatohepatitis (11%), drug–induced liver disease (6%), alcoholic liver disease (5%), primary biliary cirrhosis (4%), primary sclerosing cholangitis (3%), autoimmune chronic hepatitis (3%), “cryptogenic” cirrhosis (3%), and miscellaneous or undiagnosed causes (20%). Thus, contrary to popular belief, in this urban, hospital–based practice, the majority of liver disease patients are not middle–aged alcoholics, but rather young adults with a variety of hepatobiliary disorders.
Effects of Brain Death and Glucose Infusion on Hepatic Glycogen and Blood Hormones in the Pig
Authors: Roelsgaard K , Bøtker H E , Stødkilde-Jørgensen H , Andreasen F , Jensen S L , Keiding S
Published: November 2004

We wished to study the effects of intravenous glucose/ insulin infusion to brain–dead pigs on the hepatic glycogen content. Four groups of 40– kg pigs were studied: brain–dead and control pigs given isotonic saline or glucose/insulin (7.5 mg glucose/kg/min, 1.25 mU insulin/kg/ min) (n = 5 to 10 in each group). Brain death was induced by inflating a balloon placed in the epidural space. In brain–dead pigs given saline, liver glycogen decreased from 45 ± 11 mmol/g DNA (mean ± SEM) to 7 ± 3 mmol/ g DNA after 6 hours. Thereafter, it increased to 28 ± 9 mmol/g DNA after 9 hours (

P

= .05 compared with the 6–hour measurement). These changes were accompanied by transient increases in plasma adrenaline, glucose, free fatty acids (FFA), and glucagon. Following glucose/ insulin infusion, hepatic glycogen increased steadily and was approximately double after 12 hours (

P

< .01) in both brain–dead and in non–brain–dead pigs. In brain–dead pigs, the increases in the aforementioned blood measurements were smaller following glucose/insulin infusion than following saline infusion. However, studies of longer duration will be needed to examine these effects on a time scale that is relevant to human organ donors. In conclusion, the decrease in hepatic glycogen content after brain death could be prevented by intravenous glucose/insulin infusion probably because of a reduction of the adrenaline response to the induction of brain death.
Differential Expression Pattern Of S –Adenosylmethionine Synthetase Isoenzymes During Rat Liver Development
Authors: Gil B , Casado M , Pajares M A , Bosca L , Mato J M , Martin-Sanz P , Alvarez L
Published: November 2004

The pattern of expression of liver–specific and extrahepatic

S

–adenosylmethionine (SAM) synthetase in developing rat liver was established by determining steady–state levels of the respective messenger RNAs (mRNAs) and protein content. Levels of liver–specific SAM synthetase mRNA increased progressively from day 20 of gestation, increased 10–fold immediately after birth, and reached a peak at 10 days of age, decreasing slightly by adulthood. Conversely, mRNA levels of extrahepatic isoenzyme decreased toward birth, increased threefold in the newborn, and decreased further in the postnatal life, reaching a minimum in the adult. Similar expression profiles were observed in isolated hepatocytes, indicating that both mRNAs are differentially regulated in the same cell type. Western blot analysis showed that levels of immunoreactive liver–specific isoenzyme followed a trend similar to the mRNA, indicating that developmental regulation of this enzyme is mediated at the mRNA level. Developmental patterns of expression of albumin and α–fetoprotein (AFP) mRNAs were closely related to those for liver–specific and extrahepatic isoenzymes, respectively. Therefore, it is suggested that liver–specific SAM synthetase may be a marker for hepatocyte differentiation. Incubation of primary cultures of hepatocytes from 21–day–old fetuses with permeant cyclic adenosine monophosphate (cAMP) analogues elicited an up–regulation of the mRNA for the liver–specific isoenzyme with a concomitant down–regulation of the extrahepatic message, suggesting a physiological role for the increased postnatal glucagonemia in the control of this isoenzyme switching. In contrast with the isoenzyme expression profiles, the levels of SAM, the product of SAM synthetase reaction, were determined to be greater during gestation than in immediate postnatal periods. These results indicate that synthesis and utilization of SAM may be regulated differentially in fetal and adult hepatocytes.
Impaired Stress and Interleukin–1β–Induced Hypothalamic Expression of the Neuronal Activation Marker Fos in Cholestatic Rats
Authors: Swain M G , Maric M
Published: November 2004

Both interleukin (IL)–1β and psychological stress activate the hypothalamic–pituitary–adrenal (HPA) axis by stimulating neuronal activity within the paraventricular nucleus of the hypothalamus via different pathways. Because it has been shown previously that cholestatic rats show defective cytokine–and stress–induced HPA axis activation, neuronal activation in the paraventricular nucleus of rats with cholestasis caused by bile duct resection and in noncholestatic sham–resected (sham) controls was examined. To do this, the expression of the neuronal activation marker FOS in the hypothalamic paraventricular nucleus of bile duct–resected (BDR) and sham rats exposed to restraint stress and intracerebroventricularly infused IL–1β, two stimuli known to activate the HPA axis by stimulating neurons in the paraventricular nucleus, was quantitated. BDR rats showed decreased FOS expression in the paraventricular nucleus after both stimuli compared with similarly treated sham controls. These data show that BDR rats show an impaired ability to activate hypothalamic neurons within the paraventricular nucleus and suggest that hypofunctioning of the HPA axis in response to immune (i.e., IL–1β) and psychological stressors, which have been documented previously for BDR rats, may be caused by a specific defect in the intracellular machinery involved in the activation of neurons within the hypothalamic paraventricular nucleus.