bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of cytochrome P4502E1-dependent formation of hydroxyethyl free radical in the development of liver damage in rats intragastrically fed with ethanol
Authors: Albano E , Clot P , Morimoto M , Tomasi A , Ingelman-Sundberg M , French S W
Published: November 2004

We have previously shown that the treatment with diallyl sulfide (DAS) and phenylethyl isothiocyanate (PIC) of rats receiving ethanol in the alcohol tube-feeding model effectively suppressed the induction of cytochrome P4502E1 (CYP2E1) by ethanol. Here we report that rat treatment with DAS and PIC significantly decreased the trapping of hydroxyethyl free radicals in liver microsomes incubated in vitro with ethanol. Furthermore, these inhibitors also greatly reduced the production of hydroxyethyl radical-derived epitopes detectable in vivo in the liver of ethanol-fed rats. The action of DAS and PIC on the formation of hydroxyethyl radicals paralleled their inhibitory effect on lipid peroxidation as monitored using, respectively, liver malonildialdehyde (MDA) and plasma lipid hydroperoxide levels as well as by the titers of antibodies versus MDA adducts to proteins. Thus, these results indicated a link between the induction of CYP2E1 by ethanol, the formation of hydroxyethyl radicals and the stimulation of lipid peroxidation. The pathological scores in the livers of rats fed with ethanol plus or minus DAS and PIC also correlated with levels of hydroxyethyl radical-derived epitopes. Rats fed intragastrically with ethanol developed antibodies and the formation of these antibodies was greatly reduced by DAS and PIC. Taken together these results suggest that CYP2E1 plays an important role in the generation of hydroxyethyl radicals during chronic alcohol feeding and that ethanol-derived free radicals might play a role in the onset of liver injury in this model of alcohol administration.
A molecular analysis of viral persistence in surface antigen-negative chronic hepatitis B
Authors: Kato J , Hasegawa K , Torii N , Yamauchi K , Hayashi N
Published: November 2004

To identify the mechanisms of viral persistence in patients with chronic hepatitis B after the acquisition of anti-hepatitis B surface antigen antibodies (antiHBs), we serially analyzed the nucleotide sequence of the envelope region in a cohort of infected patients. Four patients with histological diagnoses of chronic hepatitis B who had at least 5 years of observance by our hospital staff were studied. All but one showed normalization of serum alanine aminotransferase (ALT) concentration after clearance of the hepatitis B surface of antigen (HBsAg) and the appearance of anti-HBs. Hepatitis B virus (HBV) DNA was still detectable by polymerase chain reaction (PCR) amplification assay in serum specimens from two patients, even in the presence of circulating anti-HBs. The envelope gene was amplified by PCR in serum samples obtained both before and after seroconversion, and direct cycle sequencing of the PCR products was performed. A mutation resulting in a premature stop codon was found in the pre-S1 region of one patient just prior to clearance of HBsAg. Two years later, the stop codon was converted to a leucine codon and three mutations developed in the “a” loop. In the other patient, 16 amino acids had been deleted between amino acids 8 and 23 in the pre-S2 region before clearance of HBsAg. After the appearance of circulating anti-HBs, the pre-S2 gene reverted to the wild type but three additional mutations appeared inside the “a” loop. These results suggest that HBV mutates when HBsAg is cleared, which may contribute to viral persistence due to an evasion of the host immune surveillance.
Hepatitis B virus nucleic acids associated with human peripheral blood mononuclear cells do not originate from replicating virus
Authors: Köck J , Theilmann L , Galle P , Schlicht H
Published: November 2004

There have been numerous reports suggesting that human peripheral blood mononuclear cells (PBMCs) can be productively infected with human hepatitis B virus (HBV). We therefore examined whether the PBMCs can be used to establish an in vitro infection system for HBV. Freshly purified PBMCs were incubated with HBV with or without mitogen stimulation. Successful infection was tested using a newly developed PCR method that can differentiate between the relaxed circular (RC) DNA of the virus inoculum and the covalently closed circular (CCC) DNA which is formed only after successful virus entry. This method enables virus uptake to be proven even if the infection is abortive because there is no gene expression because of the lack of liver specific gene expression factors. All attempts to detect CCC DNA after incubation of PBMCs with HBV failed. On the contrary, CCC DNA could easily be detected in infected liver or after in vitro infection of primary human hepatocytes. Because this result appeared to be contradictory to the published data, we analyzed PMBCs isolated from infected patients. We could confirm that HBV DNA and RNA are associated with these cells. However, even after restimulation with mitogens, we could only detect RC DNA. Moreover, we could also demonstrate that viral RNA is present in free virus. Apparently, a certain amount of defective particles do not reverse transcribe the packaged pregenomic RNA. In summary we found no evidence that PMBCs can be infected with HBV and conclude that all previous observations can be explained by adsorbed virus.
Purification of circulating liver plasma membrane fragments using a monoclonal antileucine aminopeptidase antibody
Authors: Deng J T , Hoylaerts M F , Nouwen E J , De Broe M E , Van Hoof V O
Published: November 2004

Membrane-bound liver alkaline phosphatase (Mem-LiALP, EC 3.1.3.1) is a high-molecular-mass liver alkaline phosphatase (ALP) present in metastatic, infiltrative and cholestatic liver disease. Shedding of hepatocyte plasma membrane fragments (LiPMF) is thought to be responsible for the appearance of Mem-LiALP in the circulation. Several other membrane-bound enzymes, such as γ-glutamyltransferase (γ-GT), leucine aminopeptidase (LAP), and 5′-nucleotidase (5′-Nu) are present in the membrane of the shedded LiPMF. By means of immunohistochemical and immunoassay procedures, we presently show that AD-1, a specific monoclonal antibody originally produced against Mem-LiALP, reacts with LAP, a constituent of the human liver plasma membrane. Using AD-1 as an immunosorbant, we isolated circulating LiPMF from cholestatic sera to a high level of purity and separated it from other high-molecular-mass material, such as liver ALP ˜ lipoprotein-X complexes. These purified membrane fragments retained their biochemical characteristics. Glycosyl-phosphatidylinositol anchor bearing liver ALP (Anch-LiALP) could be released from the LiPMF by Triton X-100. Whereas ALP was released upon treatment of AD-1 purified LiPMF with phospholipase C, phospholipase D only cleaved the glycosyl-phosphatidylinositol anchor following detergent solubilization of the enzyme. Serum LiPMF from patients with different kinds of cholestatic liver disease were bound onto AD-1 coated nitrocellulose disks and the activity of four membrane-bound enzymes (LAP, ALP, 5′Nu, γ-GT) was analyzed. A considerable interindividual variation of enzyme activities was observed, suggesting some heterogeneity in the membrane composition of these fragments.
Presence of autonomic neuropathy is a poor prognostic indicator in patients with advanced liver disease
Authors: Fleckenstein J F , Frank S M , Thuluvath P J
Published: November 2004

Autonomic neuropathy (AN) is seen in both alcohol-induced and non-alcohol-induced liver disease, and when present is an independent predictor of mortality. We postulated that patients who were awaiting liver transplantation are likely to have a high prevalence of autonomic neuropathy with an associated increase in mortality. To test our hypothesis, we evaluated the presence of autonomic neuropathy using a battery of tests in 33 patients awaiting liver transplantation and prospectively followed them to determine their prognosis. Twenty-two of 33 (67%) patients with liver disease had evidence of autonomic neuropathy; of these, 12 (36%) had evidence of definite and 10 (31%) had early autonomic neuropathy. The prevalence of AN was similar in alcohol-induced and non-alcohol-induced liver disease. Using Child-Pugh classification, 14.3% Child A, 31.3% Child B, and 60% Child C had definite autonomic neuropathy. Six patients died during a median observation period of 10 months, and all had AN. Kaplan-Meier survival analysis showed a significantly higher mortality (P=.05) in patients with AN. On the basis of this observation, we suggest that consideration should be given for early liver transplantation in patients with advanced liver disease and autonomic neuropathy.
Identification of bipotential progenitor cells in human liver development
Authors: Haruna Y , Saito K , Spaulding S , Nalesnik M A , Gerber M A
Published: November 2004

Intermediate filament proteins have been reported to be expressed in a cell lineage-specific manner during morphogenesis. We studied the expression of cytokeratin (CK)14, CK19, and vimentin and of the hepatocyte-specific HepPar1 antigen during the development of human liver. Nineteen fetal livers (gestational ages 4 to 40 weeks), 3 normal infant livers, and 3 normal adult livers were studied by immunoperoxidase staining of paraffin sections with monoclonal anti-CK19, anti-vimentin, and HepPar1 antibodies and polyclonal anti-CK14 antibodies. Double-immunostaining for CK14 and CK19 as well as bile duct cytokeratin and HepPar1 antigen was also done. CK19 and HepPar1 antigen were the first markers detected in immature progenitor cells of the liver primordium at 4 weeks’ gestation. During subsequent liver development, the progenitor cells expressed HepPar1 antigen, CK14, and CK19, from 8 to 14 weeks’ gestation. As hepatocyte differentiation progressed, expression of HepPar1 antigen increased, and CK14 and CK19 were abrogated from hepatoblasts at 14 to 16 weeks’ gestation. In contrast, as progenitor cells transformed into ductal plate cells, CK19 expression increased and persisted in differentiated bile ducts, whereas CK14 and HepPar1 antigen were lost. Vimentin was detected in ductal plate and biliary epithelial cells from 9 to 36 weeks’ gestation, but not in hepatoblasts or hepatocytes. Double-immunostaining confirmed coexpression of CK14 and CK19 in the progenitor cells for a short time (8 to 14 weeks’ gestation) during early development. Double immunostaining for bile duct CK and HepPar1 antigen clearly demonstrated the divergence of the hepatocyte and bile duct epithelial cell lineages. Our findings suggest that hepatic progenitor cells differentiate in steps marked by the acquisition or loss of specific phenotypic characteristics. Commitment of the HepPar1+CK19+ progenitor cells to either hepatocyte or bile duct epithelial cell lineages results in increased expression of one marker and loss of the other marker. These characteristics clearly identify bipotential hepatic progenitor cells in the developing human liver.
Studies of liver repopulation using the dipeptidyl peptidase IV-deficient rat and other rodent recipients: Cell size and structure relationships regulate capacity for increased transplanted hepatocyte mass in the liver lobule
Authors: Rajvanshi P , Kerr A , Bhargava K K , Burk R D , Gupta S
Published: November 2004

The feasibility of liver repopulation with hepatocytes has been shown, although clinical applications demand significant hepatic replacement. To show whether portal vascular bed in large animals could accomodate a greater cell number, we analyzed liver repopulation in syngeneic Fischer 344 rats deficient in dipeptidyl peptidase IV. This system allowed localization of transplanted normal hepatocytes in liver or various ectopic sites, as well as dual studies for analysis of gene expression. Interestingly, the product of a dipeptidyl peptidase IV substrate inactivated bile canalicular adenosine triphosphatase (ATPase) activity in normal but not in dipeptidyl peptidase IV- deficient rats, which allowed localization of dipeptidyl peptidase IV- deficient hepatocytes in normal rat liver for additional reversed transplantation systems. Further studies with genetically marked cells showed that because of the size difference between hepatocytes and portal vein radicles, intrasplenically transplanted cells were distributed in periportal areas (zone 1) in mice, whereas in larger animals (rats or rabbits) cells were also distributed downstream to midlobular (zone 2) or perivenous (zone 3) areas. Transplantation of an escalating number of hepatocytes showed that adult rats tolerated intrasplenic injection of a large cell number in single sessions (up to 1 × 108, ˜ 10% to 15% of the host hepatocyte mass). Morphometric analysis of recipient livers showed survival of a significantly greater cell number with incorporation in host liver plates. At 4 weeks, transplantation of 2 × 107 hepatocytes into adult rats led to a survival of 1.4 ± 1.0 × 106 transplanted cells/cm3 liver, whereas after transplantation of 5 × 107 cells or 7.5 × 107 cells, the number of surviving transplanted cells in the liver significantly increased to 4.1 ± 1.4 × 106 transplanted cells/cm3 liver (mean, 2.9-fold; P<.003) and 5.5 ± 1.3 × 106 transplanted cells/cm3 liver (mean, 3.9-fold; P<.003), respectively. When cells were injected in greater numbers, transplanted hepatocytes retained normal function and produced more serum albumin or hepatitis B surface antigen in deficient hosts. These data indicate the feasibility in larger animals of significant liver repopulation with hepatocyte transplantation. Use of dipeptidyl peptidase IV-deficient rats should help further analysis of mechanisms in liver repopulation.
Exaggeration of acute liver damage by hepatic sympathetic nerves and circulating catecholamines in perfused liver of rats treated with d-galactosamine
Authors: Iwai M , Shimazu T
Published: November 2004

Effects of electrical stimulation of the hepatic nerves on acute liver damage were examined using isolated rat liver perfused in situ, 24 hours after intraperitoneal injection with d-galactosamine (800 mg/kg). The leakage of lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) from the liver was used as markers of acute liver damage. In perfused livers after treatment with galactosamine, nerve stimulation (20 V, 20 Hz, 2 ms) increased the leakage of LDH and AST about 3-fold over the basal level accompanied by the decrease in flow rate, whereas with control livers the leakage of LDH and AST into the effluent was almost undetectable throughout the perfusion. The rapid increase in the leakage of LDH and AST was observed during nerve stimulation even under conditions where perfusion flow was maintained constant. Such effects of hepatic nerve stimulation on galactosamine-treated livers were mimicked well by infusion of noradrenaline or phenylephrine, and inhibited by the α1-antagonist bunazosin. Artificial reduction of perfusion flow alone did not induce the rapid leakage of LDH and AST into the effluent. On the other hand, low concentration (10 nmol/L) of noradrenaline only minimally decreased the flow rate but apparently augmented liver cell damage. The acute liver damage augmented by noradrenaline was dependent on extracellular Ca2+. These results indicate that in the liver, already having been injured slightly, the activation of hepatic sympathetic nerves and circulating catecholamines exaggerates acute liver damage through an action on liver cells, which depends on the influx of extracellular Ca2+.
Inhibition of 5-lipoxygenase promotes the regeneration of the liver after partial hepatectomy in normal and icteric rats
Authors: Urade M , Izumi R , Kitagawa H
Published: November 2004

The role of leukotriene (LT) on liver regeneration after hepatectomy is still unknown. LTB4 stagnates in the liver with obstructive jaundice, because LTB4 is excreted in the bile; therefore, LTB4 may have an effect on liver regeneration after hepatectomy with obstructive jaundice. Release of obstructive jaundice and simultaneous 70% hepatectomy was performed in rats to study the effect of 5-lipoxygenase inhibitor (AA-861) on liver regeneration. Group 1 underwent hepatectomy with administration of 0.1 mL dimethyl sulfoxide (DMSO), group 2 underwent hepatectomy with administration of AA-861 (20 mg/kg/d) dissolved in 0.1 mL DMSO, group 3 underwent hepatectomy with administration of AA-861 (40 mg/kg/d) dissolved in 0.1 mL DMSO, group 4 underwent release of obstructive jaundice and hepatectomy with administration of 0.1 mL DMSO, and group 5 underwent relief of obstructive jaundice and hepatectomy with administration of AA-861 (20 mg/kg/d). DMSO or AA-861 was administered 24 hours before, during, and 24 hours after hepatectomy in each group. Whole blood LTB4 and serum alanine aminotransferase (ALT), total bilirubin, and bromodeoxyuridine labeling index (LI) were measured before and after hepatectomy. The LTB4 level increased during obstructive jaundice and after hepatectomy. LTB4 and serum ALT levels were significantly lower after hepatectomy in the rats that were administered AA-861, and a significantly higher LI was observed at 24 hours after hepatectomy in rats receiving AA-861. Inhibition of 5-lipoxygenase promotes liver regeneration and decreases hepatocyte injury after hepatectomy associated with obstructive jaundice.
Induction of rat liver parenchymal cell apoptosis by hepatic myofibroblasts via transforming growth factor β
Authors: Gressner A M , Polzar B , Lahme B , Mannherz H
Published: November 2004

The induction of apoptosis of rat liver parenchymal cells (PC) by transforming growth factor β (TGF-β)-expressing transforming fat-storing cells (FSC), i.e., myofibroblasts (MFB), was studied under culture conditions and compared with the apoptotic effect of human recombinant TGF-β1. MFB were obtained by subculture of FSC. The TGF-β concentration in the conditioned medium of myofibroblast (MFBcM) determined with the Mink cell proliferation inhibition assay was <0.25 ng/mL/24 in the native medium, but 1.9 ng/mL24 h after transient acidification. MFBcM added in various dilutions and for different times to PC monolayers induced progressive cell detachment from the plastic support and increase of lactate dehydrogenase (LDH) activity in the medium. The reduction of mitochondrial dehydrogenase activity in PC (XTT or WST-1 test) was an early sign of MFBcM-induced functional impairment of PC. Short term exposure of PC with MFBcM for 3 hours was sufficient to induce the deleterious effects on PC, but neither native (nonactivated) MFBcM nor conditioned medium of untransformed FSC (FSCcM), in which TGF-β was not detectable, were able to impair function and viability of PC. Activated MFBcM increased strongly (up to 21-fold) the concentration of oligonucleosomal DNA fragments both in the adherent and detached fraction of PC. Internucleosomal DNA fragments (DNA ladder) were demonstrated by electrophoresis of extracted DNA on agarose gels and by in situ end-labeling of DNA breaks (TUNEL reaction) only in MFBcM-exposed PC. MFBcM-treated PC exhibited intense fluorescence after staining with DNA-binding dye Hoechst 33342 and an increased number of cells with fragmented nuclei. All these criteria point to MFBcM-generated apoptosis of cultured PC, which were found to be very similar to those induced by human recombinant TGF-β1. The exclusive role of active TGF-β in MFBcM as mediator of the apoptotic effects of MFB was proven by preincubation of the conditioned medium with human recombinant latency-associated peptide, which reversed completely MFBcM induced reduction of the XTT-test and the MFBcM-generated increase of oligonucleosomal DNA fragments. Partial reversibility was reached by preincubation of the medium with recombinant soluble type II TGF-β receptor. The data let us conclude that transformed FSC, i.e., MFB in damaged liver, could participate in the mechanisms of PC apoptosis by paracrine loops involving TGF-β.
Biliary excretion of estradiol-17β-glucuronide in the rat
Authors: Takikawa H , Yamazaki R , Sano N , Yamanaka M
Published: November 2004

Estradiol-17β-glucuronide (E217G) is a cholestatic agent and is considered to be related to the pathogenesis of intrahepatic cholestasis of pregnancy. In the current study, we examined the mechanism of the biliary excretion of E217G and estradiol metabolites in rats. Biliary excretion of tracer doses of [3H]estradiol-17β-glucuronide and [14C]estradiol or [3H]taurocholate and ]14C]vinblastine, a P-glycoprotein (P-GP) substrate, intravenously administered as a bolus to bile-drained control rats or EHBR was studied. Biliary excretion of E217G and estradiol metabolites EHBR was markedly delayed. Analyses of biliary metabolites after estradiol injection showed less polar conjugates in EHBR. In contrast, the excretion of taurocholate and vinblastine (VLB) was only slightly delayed in EHBR. Although phenothiazine treatment to induce the expression of P-GP increased biliary vinblastine excretion, it did not affect biliary excretion of a tracer dose of [3H]estradiol-17β-glucuronide. However, phenothiazine treatment inhibited the cholestasis induced by E217G infused at the rate of 0.075 μmol/min/100g for 20 minutes and increased biliary E217G excretion. Sulfobromophthalein infusion (0.2 μmol/min/100 g body 0 weight) markedly inhibited the biliary excretion of E217G and estradiol metabolites, whereas dibromosulfophthalein (DBSP) at the same infusion rate had no effect. These findings indicate that EG17G is excreted into bile by a canalicular organic anion carrier for sulfobromopthalein (BSP), not for DBSP, under physiological conditions, and that P-GP influences E217G excretion only at a high dose. under physiological conditions, and that P-GP influence s E217G excretion only at a high dose.
Quantitative analysis of antibody to hepatitis C virus envelope 2 glycoprotein in patients with chronic hepatitis C virus infection
Authors: Yuki N , Hayashi N , Kasahara A , Hagiwara H , Mita E , Ohkawa K , Katayama K , Fusamoto H , Kamada T
Published: November 2004

The significance of circulating antibody to hepatitis C virus (HCV) envelope glycoprotein 2 (E2)/nonstructural protein 1 (NS1) glycoprotein was studied in 83 patients with chronic HCV infection diagnosed by polymerase chain reaction (PCR). E2/NS1 antibody was quantitatively examined by a passive hemagglutination test using recombinant E2/NS1 glycoprotein encompassing amino acids 388 to 664 of the HCV-H strain. The results were correlated with clinical and virological features such as genotypes and viremic levels assessed by a competitive reverse-transcription PCR assay. E2/NS1 antibody was found in 73 patients (88%), and its occurrence was related to viremic levels. E2/NS1 antibody titers were low in asymptomatic HCV carriers with low levels of viral replication; 9 of 17 such patients tested positive for E2/NS1 antibody (53%), compared with 64 of 66 chronic hepatitis C patients (97%) (P < .01). A significant direct relationship was observed between viremic levels and E2/NS1 antibody titers (r = .52, P < .01). Of the 13 patients with low viremic levels of < 106 copies/mL, only 5 tested positive for E2/NS1 antibody (38%), whereas 68 of the 70 patients with viremic levels of > or = 106 copies/mL had it (97%) (P < .01). As for the relation to HCV genotypes, no difference was seen in E2/NS1 antibody titers among genotypes examined (1b, 2a, and 2b). These findings suggest that the E2/NS1 antibody tested exhibits no neutralizing activity in chronic HCV infection but may serve as a serological indicator of active virus replication.
The effect of long-term treatment with spironolactone on variceal pressure in patients with portal hypertension without ascites
Authors: Nevens F , Lijnen P , VanBilloen H , Fevery J
Published: November 2004

The effect of spironolactone on esophageal variceal pressure (VP) in patients without ascites was investigated. VP was assessed using a noninvasive endoscopic gauge. Spironolactone was administered during a 6-week period at a dosage of 100 mg/d. This treatment decreased VP from 16.8 ± 1.9 (SD) to 14.1 ± 2.7 mm Hg (P < .001) in a group of 12 patients and from 18.6 ± 2.1 to 13.7 ± 4.1 mm Hg (P < .01) in another group of 8 patients who still had high VP despite chronic intake of propranolol. In both groups, placebo administration to 12 and 8 comparable patients did not significantly alter VP. Spironolactone induced a significant reduction of plasma volume (42.1 ± 5.5 to 36.1 ± 6.6 mL/kg body weight, P < .01) and of the concentration of α-atrial natriuretic peptide (α-ANP) (39.8 ± 22 to 27.7 ± 20 pg/mL, P < .01); in addition, a pronounced increase in plasma renin activity (PRA) (1.1 ± 0.9 to 7.5 ± 3.4 ng/mL/h, P < .001) was induced by the treatment. No significant changes in systemic hemodynamics were observed during the studies. Severe side effects were not observed except for a high incidence (55%) of painful gynecomasty in the male patients. In conclusion, chronic spironolactone administration effectively lowers VP, even in patients under chronic propranolol therapy. The combination of propranolol and spironolactone deserves further study as a prophylactic therapy of variceal hemorrhage, but development of gynecomasty might be a problem. Finally, we confirmed the reproducibility of VP measurements with the noninvasive gauge in chronic conditions.
The role of ultrasonography and automatic-needle biopsy in outpatient percutaneous liver biopsy
Authors: Lindor K D , Bru C , Jorgensen R A , Rakela J , Bordas J M , Gross J B , Rodes J , McGill D B , Reading C C , James E M , Charboneau J W , Ludwig J , Batts K P , Zinsmeister A R
Published: November 2004

The risk of complications from percutaneous liver biopsy is low, but discomfort is common and complications require hospitalization in approximately 4% of patients. The optimal method of performing these biopsies is unknown. The goal of our study was to determine whether the use of ultrasonography in the biopsy room immediately prior to or during the procedure would lessen the risk of complications and to compare the safety and efficacy in obtaining tissue by use of a Trucut needle versus an automatic biopsy needle. Between 1992 and 1994, 836 patients were entered into a randomized study (489 in Rochester, MN; 347 in Barcelona, Spain). Patients were randomized immediately prior to liver biopsy into four groups: Trucut needle, or automatic biopsy needle, and with or without ultrasonography. Fisher’s Exact Test and a logistic regression model were also used to assess the effect of needle and ultrasonography on the odds for complications. The four biopsy groups were well-matched at entry with respect to age, sex, underlying liver disease, hemoglobin, prothrombin time, and platelet count. The use of ultrasound was associated with a decreased rate of hospitalization for pain, hypotension, or bleeding (2 vs. 9, P < .05). No difference in safety was found between the two types of needles. The number of passes needed to obtain specimens was similar for all four groups. The average length of the specimen was slightly greater with ultrasonographic-guided biopsies (1.7 mm vs. 1.6 mm, P < .05) and with biopsies obtained using the automatic biopsy needle when compared with the Trucut needle (1.7 mm vs. 1.5 mm, P < .05), but this did not seem to be clinically important. The addition of ultrasonography reduces complications in patients undergoing percutaneous liver biopsy. The type of needle appears to offer little difference in safety or yield of diagnostic tissue. The use of ultrasonography for guidance of percutaneous liver biopsy will lead to a lower rate of complications. The value of this benefit must be weighed against the added cost of ultrasonographic guidance.
Zinc supplementation and amino acid-nitrogen metabolism in patients with advanced cirrhosis
Authors: Marchesini G , Fabbri A , Bianchi G , Brizi M , Zoli M
Published: November 2004

Zinc deficiency is common in cirrhosis and has been involved in the altered nitrogen metabolism. In this study, we measured the effects of zinc supplementation on the dynamics of amino acid-derived urea synthesis in cirrhosis with mild or latent encephalopathy. The hepatic conversion of amino acids into urea was studied in eight patients with advanced cirrhosis under controlled conditions of substrate availability (continuous alanine infusion), before and after 3-month oral zinc sulfate supplementation (600 mg/d). Eight more patients, matched for hepatocellular failure and encephalopathy, served as controls. Plasma zinc levels were reduced in all patients and returned to normal after oral zinc. The alanine-stimulated urea nitrogen synthesis rate in relation to α-amino-N concentration-the functional hepatic nitrogen clearance-increased by 25% after zinc supplementation, i.e., more urea was produced at any α-amino-N concentration. Basal and alanine-induced glucagon decreased by 50%, and the ammonia response to alanine decreased by 30%. Psychometric tests improved, as did routine and dynamic liver function tests and the Child- Pugh score. Also, the plasma concentration of lipid peroxides was reduced by zinc. No significant changes were observed in the control group. Our data indicate that long-term oral zinc speeds up the kinetics of urea formation from amino acids and ammonia. Changes in the hormonal drive and/or the antioxidant activity of zinc might be involved in the general improvement in liver function, whereas the beneficial effects on encephalopathy might stem from decreased ammonia.
Relationship between serum and hepatic 7S fragments of type IV collagen in chronic liver disease
Authors: Suou T , Yamada S , Hosho K , Yoshikawa N , Kawasaki H
Published: November 2004

We evaluated the mechanism of increased serum concentrations of the 7S fragment of the N-terminal domain of type IV collagen (7S collagen) in chronic liver disease. We measured the concentrations of hepatic-free and deposited 7S collagens after extraction with Tris-HCl buffer and bacterial collagenase, then compared them with the serum levels in 8 normal controls and 48 patients with chronic liver disease. The hepatic 7S collagen levels extracted with Tris-HCl buffer and collagenase accounted for 7% and 93%, respectively, of the total 7S collagen levels in normal controls. Both hepatic 7S collagen levels as well as serum levels increased in accordance with the progress of liver disease. Serum levels of 7S collagen showed a closer correlation with the hepatic 7S collagen levels extracted with Tris-HCl buffer (r = .822), compared with those extracted with collagenase (r = .382). On the other hand, the histological degrees of liver fibrosis were highly correlated with the hepatic collagenase-extracted 7S collagen levels (r = .822), compared with serum and the hepatic Tris-HCl buffer-extracted levels (r = .478 and r = .537, respectively). Although there was no difference in serum and hepatic 7S collagen levels between B and C viral patients, the serum and hepatic Tris-HCl buffer-extracted 7S collagen levels were higher in patients with alcoholic cirrhosis than patients with viral cirrhosis. However, the hepatic collagenase-extracted levels were similar in both groups. Gel filtration demonstrated that the serum and hepatic Tris-HCl buffer-extracted 7S collagens were mainly eluted in the macromolecular 7S collagen-reactive fraction in cirrhosis, whereas the hepatic collagenase-extracted 7S collagen was eluted in the authentic 7S collagen-reactive fraction. The results suggest that serum 7S collagen levels are not a particularly reliable measure of hepatic fibrosis but reflect the enhanced metabolism, especially synthesis of type IV collagen in the liver.
Vasoactive intestinal peptide in cirrhotic rats: Hemodynamic effects and mesenteric arterial receptor characteristics
Authors: Lee S S , Huang M , Ma Z , Rorstad O
Published: November 2004

Vasoactive intestinal peptide (VIP) blood levels in cirrhosis are elevated, but its hemodynamic and receptor characteristics remain unclarified. We aimed to quantify VIP receptor characteristics in mesenteric arteries, plasma VIP concentration by radioimmunoassay (RIA), and the hemodynamic effects of VIP infusion in bile duct-ligated (BDL) cirrhotic and sham-operated control rats. Mesenteric arterial membranes were prepared by ultracentrifugation, and receptor characteristics were studied using 125I-labeled VIP as a radioligand. For the hemodynamic study, there were four groups: cirrhotic and sham-operated rats were infused with either VIP (50 ng/kg/min for 15 minutes) or equivolumic isotonic saline. Regional blood flows were measured in conscious rats with radioactive microspheres. Receptor studies showed high-and low-affinity binding sites for VIP, which had similar equilibrium dissociation constants (binding affinities) and receptor densities for both the cirrhotic and control rats. Plasma VIP concentrations were significantly elevated in the cirrhotic rats. In both cirrhotic and sham-operated rats, VIP infusion produced plasma levels approximately twofold to threefold increased over the basal levels observed in cirrhotic rats. In cirrhotic rats, VIP infusion did not affect any hemodynamic parameter, whereas in the sham-operated rats VIP infusion significantly increased the mesenteric visceral blood flow. These results show that the hyporesponsiveness to VIP in cirrhotic rats is not attributable to receptor downregulation, implying postreceptor alterations. This suggests that VIP may not play a major role in the maintenance of splanchnic hyperemia in cirrhosis.
Expression of HLA class I molecules and the transporter associated with antigen processing in hepatocellular carcinoma
Authors: Kurokohchi K , Carrington M , Mann D L , Simonis T B , Alexander-Miller M A , Feinstone S M , Akatsuka T , Berzofsky J A
Published: November 2004

The expression of the HLA class I molecules on the cell surface was investigated in hepatocellular carcinoma (HCC) cell lines using complement-mediated cytotoxicity (CMC) and flow cytometric analysis. Although HLA-A antigens were detected by CMC in all cell lines tested, HLA-B and -C antigens were not detectable in six of seven HCC cell lines. These results were also confirmed by flow cytometric analysis focusing on HLA-Bw4 and Bw6 public antigens. Furthermore, complementary DNA (cDNA) from each cell line was tested for the expression of HLA-A, - B, -C and the transporter associated with antigen processing genes (TAP1 and TAP2). Two cell lines showed a reduced level of one or both of the TAP messenger RNAs (mRNAs), and one of these showed a reduction of HLA-B and -C gene expression as well, but the others had detectable mRNA levels. These results demonstrate that hepatocellular carcinoma cell lines tested in the current study lose or decrease the expression of HLA-B and -C alleles on the cell surface, even though mRNA encoding these alleles is present, suggesting that the loss of the HLA molecules might be caused by posttranscriptional events or failure to transport and load peptides necessary for HLA expression. The selective loss of HLA-B and -C, but not -A, molecules (which also excludes a β2-microglobulin defect) is intriguing, and may be attributable to the ability of some of the HLA-A molecules to load signal peptides not requiring TAP transport, or to natural selection of HLA-B or -C locus- specific immune surveillance.
Kupffer cell depletion by liposome-delivered drugs: Comparative activity of intracellular clodronate, propamidine, and ethylenediaminetetraacetic acid
Authors: Van Rooijen N , Sanders A
Published: November 2004

Macrophages such as Kupffer cells in the liver are multifunctional cells. They are involved in host defense mechanisms and have a regulatory role in many biomedical processes. Their selective depletion, using liposome-encapsulated drugs, forms a widely accepted approach to studying their functional aspects in vivo. We have compared the Kupffer cell-depleting activities of liposome-encapsulated clodronate, propamidine, and ethylenediaminetetraacetic acid (EDTA) for this purpose. These molecules represent the drug families of bisphosphonates, diamidines (or aromatic polyamidines), and polyaminopolycarboxylic acid-chelating agents, respectively. The Kupffer cell-depleting activity of the liposome-encapsulated antimicrobial drug propamidine exceeded that of clodronate by about a factor of 10. EDTA appeared to be inefficacious for depletion of Kupffer cells in the rat.
Mitogen-activated protein kinase activation in hepatocyte growth factor-stimulated rat hepatocytes: Involvement of protein tyrosine kinase and protein kinase C
Authors: Adachi T , Nakashima S , Saji S , Nakamura T , Nozawa Y
Published: November 2004

Hepatocyte growth factor (HGF) stimulated mitogen-activated protein (MAP) kinases and MAP kinase kinase in primary cultured rat hepatocytes. Inhibitors for protein kinase C (PKC), Ro31-8425, H-7, and calphostin C, reduced HGF-induced MAP kinase activity. A PKC activator, phorbol myristate acetate (PMA), induced MAP kinase activation in a concentration-dependent manner. Protein tyrosine kinase (PTK) inhibitors, genistein, and ST638 also inhibited HGF-induced MAP kinase activation. Furthermore, HGF increased formation of Ras guanosine triphosphate (GTP) complex, indicating Ras activation. Genistein inhibited HGF-induced Ras activation, but Ro31-8425 was without effect. On the other hand, Ro31-8425 decreased HGF-induced [3H]arachidonic acid (AA) release and [3H]thymidine incorporation. Genistein also prevented [3H]AA release and [3H]-thymidine incorporation. Moreover, a commonly used phospholipase A2 (PLA2) inhibitor, quinacrine, decreased HGF- induced [3H]AA release and [3H]thymidine incorporation. The inhibitory profile of [3H]AA release was well correlated with that of [3H]thymidine incorporation in Ro31-8425-, genistein-, and quinacrine- treated cells. A cyclooxygenase inhibitor, indomethacin, which suppressed HGF-induced DNA synthesis, had minimal effect on MAP kinase activation. In contrast, prostaglandin (PG) E1, E2, or F2α, which stimulate [3H]thymidine incorporation to the same level as that caused by HGF in hepatocytes, caused very weak activation of MAP kinases. These results suggest that PTK, Ras, and PKC play roles in MAP kinase activation induced by HGF and that MAP kinase activation resulting in AA release is involved in DNA synthesis in rat hepatocytes.
Immunoregulatory Cytokines in Chronic Hepatitis C Virus Infection: Pre– and Posttreatment With Interferon Alfa
Authors: Cacciarelli T V , Martinez O M , Gish R G , Villanueva J C , Krams S M
Published: November 2004

T lymphocytes and immunoregulatory cytokines may be important in the host response to hepatitis C virus (HCV) infection. T–helper type 1 (Th1) cytokines (interleukin [IL]–2, interferon gamma [IFN–gamma]) are required for host antiviral immune responses, including cytotoxic T–cell generation and natural killer cell activation, while T–helper type 2 (Th2) cytokines (IL–4,IL–10) can inhibit the development of these effector mechanisms. In this study, the serum levels of Th1 and Th2 cytokines in patients (n = 23) infected with HCV were measured and compared with biochemical (alanine transaminase [ALT]) and viral (HCV RNA) indicators of infection. Serial cytokine levels were measured in a subset of 11 patients at 1 and 12 weeks during and at 1 week after interferon alfa (IFN–α) therapy (n = 33 samples). Levels of circulating IL–2, IL–4, IL–10, and IFN–γ were significantly elevated in HCV patients versus normal controls (128 vs. 25 pg/mL, 3,045 vs. 29 pg/mL, 2,949 vs. 18 pg/mL, and 307 vs. 24 pg/mL respectively;

P

< .01). Treatment with IFN–α decreased the levels of IL–4 (321 ± 224 pg/mL), and IL–10 (1,011 ± 344 pg/mL), which paralleled a decrease in HCV RNA (114 ± 27 vs. 25 ± 20 Eq/ml X 105, pre– vs. post–IFN–α [12 weeks];

P

<.05). These findings indicate that an activated T–cell response, as manifest by increased circulating immunoregulatory cytokines, is present in patients with HCV liver disease. Furthermore, treatment with HCV liver disease. Furthermore, treatment with IFN–α diminishes the Th2 cytokine response. Thus, modulation of T–cell function and cytokine production may be one mechanism whereby IFN–α therapy results in reduced viral burden.
Improved Sustained Response Following Treatment of Chronic Hepatitis C by Gradual Reduction in the Interferon Dose
Authors: Shiffman M L , Hofmann C M , Luketic V A , Sanyal A J , Contos M J , Mills A S
Published: November 2004

Interferon (IFN) treatment of chronic hepatitis C virus (HCV) is associated with a high rate of relapse. IFN is thought to exert its effect against HCV via direct viral inhibition and immune stimulation. We have hypothesized that relapse following termination of therapy results from the sudden withdrawal of this immune modulatory effect and that gradual reduction in the IFN dose may decrease the incidence of relapse. One hundred six patients with chronic HCV were enrolled into this 24–month controlled, randomized prospective trial. All were treated with 5 mU of interferon–alpha–2b three times a week for 6 months. Patients who achieved biochemical response were randomized to either stop or taper IFN gradually at monthly intervals as follows; 3 mU, 2 mU, 1 mU, and 0.5 mU (all three times a week). 0.5 mU twice weekly and then once weekly. Liver histology was assessed by Knodell index and HCV RNA was measured by a quantitative polymerase chain reaction (PCR) assay. Of the 92 patients who completed the initial 6 months of IFN treatment, 47 (51%) achieved biochemical response. Twenty–one of these patients were randomized to stop IFN treatment and 25 to taper (1 drop–out). At randomization patients were well matched with respect to age, sex, race, serum alanine transaminase (ALT), and liver histology. Biochemical relapse was observed in 19 of 21 (91%) patients who stopped IFN treatment compared with only 60% who tapered IFN (

P

= .04). Virological relapse occurred in 90% of patients who stopped and only 48% of persons who tapered IFN therapy. At completion of the 24–month study patients who achieved long–term sustained biochemical response had a significantly lower mean Knodell score (3.5 vs. 6.5) and a significantly greater number were HCV RNA negative in serum (85% vs. 18%) compared with relapsers. We conclude that gradual reduction in IFN dose is associated with a significant higher rate of sustained response and clearance of HCV RNA from serum compared with abruptly stopping treatment. This in turn is associated with a significant improvement in hepatic histology supporting the premise that response to IFN therapy can prevent progression to cirrhosis.
Elevated Aflatoxin Exposure and Increased Risk of Hepatocellular Carcinoma
Authors: Chen Chien-Jen , Wang Li-Yu , Lu Sheng-Nan , Wu Mei-Huei , You San-Lin , Zhang Yu-Jing , Wang Lian-Wen , Santella Regina M
Published: November 2004

To elucidate the importance of aflatoxin in the etiology of hepatocellular carcinoma (HCC), a community–based cohort study combined with molecular dosimetry of aflatoxin exposure was performed in the Penghu Islets where the HCC mortality rate is highest in Taiwan. A total of 6,487 residents aged 30 to 65 years were recruited in the two– stage screening survey and underwent regular follow–up examination. Among 33 newly diagnosed HCC cases, 31 (94%) were chronic hepatitis B surface antigen (HBsAg) carriers and 3 (9%) were positive for antibodies against hepatitis C virus (HCV). Among 20 HCC patients and 86 matched healthy controls whose serum samples were tested for aflatoxin B1 (AFB1)–albumin adducts by competitive enzyme–linked immunosorbant assay (ELISA), 13 (65%) HCC patients and 32 (37%) matched controls were seropositive, showing a statistically significant multivariate–adjusted odds ratio of 5.5 with a 95% confidence interval of 1.2 to 24.5. The results imply the elevated risk of HCC among Penghu residents may be attributable to their heavy exposure to aflatoxins and high HBsAg carrier rate.
The Ancestral Hemochromatosis Haplotype Is Associated With A Severe Phenotype Expression in Italian Patients
Authors: Piperno A , Arosio C , Fargion S , Roetto A , Nicoli C , Girelli D , Sbaiz L , Gasparini P , Boari G , Sampietro M , Camaschella C
Published: November 2004

We evaluate the relation between genotype and phenotype in 47 Italian male patients with homozygous genetic hemochromatosis (GH). Phenotype evaluation was based on the ratio of amount of iron removed (IR) by phlebotomy and age (IR/age). Patients were divided in two classes of phenotype expression: class I included 26 patients with less severe iron overload (IR/age ≤0.33) and class II included 21 patients with a more marked one (IR/age >0.33). Genetic variability was assessed by haplotype analysis combining alleles at HLA–B, D6S265, HLA–A, and D6S105 loci. A common ancestral haplotype carrying D6S265–1, HLA–A3, and D6S105–8 alleles was present in 13 of 52 (25%) chromosomes in class I and in 24 of 42 (57%) chromosomes in class II (

P

= .0027). Homozygotes and heterozygotes for the ancestral haplotype had higher iron indices than patients carrying two haplotypes other than the ancestral one. Seven of the eight patients homozygous for the ancestral haplotype were in class II, heterozygotes were equally distributed between the two classes, whereas 14 of 18 carriers of other haplotype combinations were in class I. Our results suggests that the gene defect linked to the ancestral haplotype is the result of a single, severe mutation. The high variability of phenotype expression in heterozygotes for the ancestral haplotype could be accounted for the contribution of the mutation carried by the second haplotype. Combination of different mutations could be responsible for the variable degrees of iron overload found in patients with GH.
Enhanced Expression of the Protein Kinase Substrate Annexin I in Human Hepatocellular Carcinoma
Authors: Masaki T , Tokuda M , Ohnishi M , Watanabe S , Fujimura T , Miyamoto K , Itano T , Matsui H , Arima K , Shirai M , Maeba T , Sogawa K , Konishi R , Taniguchi K , Hatanaka Y , Hatase O , Nishioka M
Published: November 2004

Annexin (AX) constitutes a new family of Ca2+–dependent membrane–binding proteins; 13 of them have been described. Among these, annexin–1 (AX–I) has displayed many biological functions

in vitro

. Its actual role

in vivo

, however, remains unknown. We already reported that AX–I was expressed in proliferating (regenerating) hepatocytes at both protein and messenger RNA (mRNA) levels. The role of AX–I in human hepatocellular carcinoma (HCC) remains obscure. In this study, the amounts of AX–I at protein and mRNA levels, as well as its localization, have been determined in the normal human liver, chronic hepatitis liver, and nontumorous and tumorous portions of HCC. AX–I was rarely found in normal and chronic liver tissues, whereas it is overexpressed at both the transcriptional and translational levels in tumorous and nontumorous regions of HCC. In addition, more AX–I was expressed in the tumorous portion than the nontumorous portion of HCC. AX–I was present in the hepatocytes and HCC cells, localized mainly in the cytoplasm. AX–I was expressed in poorly differentiated cancer cells. Furthermore, AX–I was tyrosine–phosphorylated in HCC. We also found that some of the AX–I–positive hepatocytes in the nontumorous tissues were derived from a particular subset of parenchymal cells (stem or oval cells). These results indicate that AX–I plays an important role in the malignant transformation process leading to HCC and that it is closely related to the histological grade of HCC. HCC would offer a novel tool with which to study the function of AX–I in malignant transformation.