bluuebunny/crossref_metadata_2025 huggingface dataset:
Variceal pressure is a factor predicting the risk of a first variceal bleeding: A prospective cohort study in cirrhotic patients
Authors: Nevens Frederik , Bustami Rami , Scheys Ilse , Lesaffre Emmanuel , Fevery Johan
Published: July 2004

Predictive criteria for a first variceal hemorrhage lack substantial accuracy. Cross-sectional studies suggest a close relationship between variceal pressure (VP) and the occurrence of variceal bleeding. In the present prospective cohort study, the significance of VP measurement for prediction of a first variceal bleed was assessed. Eighty-seven patients with cirrhosis and large esophageal varices who had never developed variceal bleeding were followed for 12 months. The endpoint of the study was the presentation or not of a variceal hemorrhage. Thirty-four patients (39%) were in Child’s class A, 37 in class B (43%), and 16 in class C (18%). The median interval between endoscopic diagnosis of varices and the beginning of the study was 15 months. Twenty-eight patients (32%) developed a variceal hemorrhage with a bleeding-related mortality of 18% (n=5). The 1-year mortality overall was 16% (n=14). Variables predictive of a first bleed identified by Cox proportional hazards regression model were: the level of VP, the North Italian Endoscopic Club (NIEC) score, and the interval between the diagnosis of varices and the start of the study. By adding VP to NIEC, a significant gain in prognostic accuracy was obtained (P = .003). In conclusion, the present study provides evidence that the level of VP is a major predictive factor for variceal hemorrhage, and that it provides further prognostic information in addition to the NIEC index.
Q-T interval prolongation in cirrhosis: Prevalence, relationship with severity, and etiology of the disease and possible pathogenetic factors
Authors: Bernardi Mauro , Calandra Salvatore , Colantoni Alessandra , Trevisani Franco , Raimondo Maria L. , Sica Giuseppe , Schepis Filippo , Mandini Marina , Simoni Patrizia , Contin Manuela , Raimondo Giovanni
Published: July 2004

Prolonged Q-T interval predicts severe arrhythmias and sudden death, and has been shown to occur in alcoholic liver disease and cirrhotic patients who are candidates for liver transplantation. This study first evaluated the prevalence of prolonged Q-T interval in a large population of unselected patients with cirrhosis, and assessed the relationship between abnormal Q-T, etiology, and severity of liver disease and mortality of patients. Possible causes of Q-T abnormality were also explored. Ninety-four patients with cirrhosis without overt heart disease and 37 control subjects with mild chronic active hepatitis were enrolled. Rate-corrected Q-T interval (Q-Tc) was assessed along with routine liver tests, Child-Pugh score, serum bile salts, electrolytes and creatinine, plasma renin activity, aldosterone, norepinephrine, atrial natriuretic factor and, gonadal hormones. Q-Tc was longer in patients with cirrhosis than in controls (440.3 ± 3.2 vs. 393.6 ± 3.7 ms; P < .001) and prolonged (>440 ms) in 44 patients (46.8%) and 2 controls (5.4%; P < .001).Q-Tc length was not influenced by the etiology of cirrhosis and correlated with Child-Pugh score (r = .53; P < .001), liver tests such as prothrombin activity, and serum concentrations of albumin and bilirubin, plasma bile salts, and plasma norepinephrine. Multivariate analysis showed that only Child-Pugh score and plasma norepinephrine were independently correlated with Q-Tc duration. Over a median follow-up period of 19 months (range, 2-33 months), patients with Q-Tc longer than 440 ms had a significantly lower survival rate than those with normal Q-Tc. Q-T interval is frequently prolonged in patients with cirrhosis, regardless the etiology of the disease, worsens in parallel with the severity of the disease, and may have an important prognostic meaning. In addition to other undefined factors related to the severity of cirrhosis, sympathoadrenergic hyperactivity may play a pathogenetic role.
Right-sided pedunculated hepatocellular carcinoma: A form of adrenal metastasis
Authors: Okuda Kunio , Arakawa Masahiro , Kubo Yasuhiko , Sakata Kenji , Kage Masayoshi , Iwamoto Shozo , Takeda Shigeaki , Sonoda Koshi , Sanefuji Hayato
Published: July 2004

Pedunculated hepatocellular carcinoma (HCC) or extrahepatic growth of HCC is an uncommon but not rare pathological form, but its genesis is unknown. Right-sided adrenal metastases of HCC that were abutting on or about to fuse with the right hepatic lobe were resected in three patients. The masses seemed to have originated in the para-adrenal tissue, leaving the adrenal gland intact. They were partially supplied by the hepatic artery as well as by the suprarenal artery. Four cases of autopsied pedunculated HCC of the right lobe were also studied. The mass was protruding caudad from the noncancerous parenchyma of the right lobe. Postmortem angiography carried out on one liver showed that only a small portion of the mass toward the liver was supplied from the hepatic artery. These observations suggest that some, perhaps most, of the right-sided pedunculated HCCs represent fusion of the right lobe and para-adrenal or adrenal metastatic HCC. This phenomenon may be explained by possible transport of cancer cells toward the right adrenal gland through the so-called adrenohepatic fusion, a relatively common anatomical change in advanced age.
Nitric oxide suppression reversibly attenuates mitochondrial dysfunction and cholestasis in endotoxemic rat liver†
Authors: Shiomi Masaya , Wakabayashi Yoshiyuki , Sano Tsuyoshi , Shinoda Yuichi , Nimura Yuji , Ishimura Yuzuru , Suematsu Makoto
Published: July 2004

This study aimed to examine whether nitric oxide (NO) plays a causal role in endotoxin-induced dysfunction of biliary transport. Rats were treated with intraperitoneal injection of endotoxin (O111B4, 4 mg/kg). At 2 hours, the liver was excised and perfused ex vivowith taurocholate (TC)-containing Krebs-Ringer solution under monitoring bile output and NO2in the perfusate and tissue cyclic guanosine monophosphate (cGMP) levels as indices of NO production. The endotoxin treatment evoked a marked decrease in the bile acid-dependent bile formation concurrent with the increasing NO2output, cGMP elevation, and a reduction of hepatic adenosine triphosphate (ATP) contents and oxygen consumption. Perfusion with 1 mmol/L aminoguanidine (AG), an inhibitor of inducible NO synthase, but not with l -nitroarginine methyl ester, an inhibitor of the constitutive form of the enzyme, significantly reversed the endotoxin-induced increment of the bile formation in concert with the recovery of oxygen consumption and ATP levels. Laser confocal microfluorography of the liver lobules using rhodamine 123 (Rh), a fluoroprobe sensitive to mitochondrial membrane potential, revealed that endotoxin elicited a significant mitochondrial dysfunction panlobularly. The AG administration reversed the endotoxin-induced decrease in mitochondrial membrane potential. Collectively, up-regulation of NO by inducible NO synthase accounts for a mechanism through which endotoxin impairs the bile formation, and its suppression serves as a therapeutic strategy for improvement of hepatobiliary function.
Hepatic cytochrome P450 2E1 is increased in patients with nonalcoholic steatohepatitis
Authors: Weltman Martin D. , Farrell Geoffrey C. , Hall Pauline , Ingelman-Sundberg Magnus , Liddle Christopher
Published: July 2004

Nonalcoholic steatohepatitis (NASH) has multiple etiologic associations, but the pathogenesis is poorly understood. Cytochrome P450 (CYP) 2E1 is induced in the liver of patients who drink alcohol to excess and is important in the pathogenesis of alcoholic liver disease (ALD). We have previously shown that hepatic CYP2E1 is also increased in a rat dietary model of steatohepatitis. The aim of the present study was to test the hypothesis that hepatic CYP2E1 is induced in the liver of patients with NASH, defined on the basis of compatible liver histology and the exclusion of excessive alcohol intake. Sections of paraffin-embedded liver biopsy material from 31 subjects with NASH were evaluated and compared with sections from 10 histologically normal livers and 6 patients with ALD. Hepatic CYP2E1 and CYP3A were detected in liver sections by immunohistochemistry using specific anti-human CYP2E1 and CYP3A antibodies. As expected, normal livers showed CYP2E1 immunostaining confined to a rim, two to three cells thick, around terminal hepatic venule, while livers from alcoholic hepatitis patients showed increased CYP2E1 staining. CYP2E1 immunostaining was also increased in livers from patients with NASH, irrespective of the etiologic association. Further, the pattern of CYP2E1 distribution was similar to ALD, with increased perivenular intensity and more extensive acinar distribution of staining. As in the rat model, the hepatic distribution of CYP2E1 corresponded to that of steatosis. In contrast to CYP2E1, CYP3A immunostaining was decreased in patients with NASH. We conclude that hepatic CYP2E1 is increased in patients with NASH compared with normal livers. Thus, despite many possible etiologic factors for NASH, the pathogenetic mechanisms may be similar and, like alcoholic steatohepatitis, may involve induction of CYP2E1.
Management of adult patients with ascites caused by cirrhosis†
Authors: Runyon Bruce A.
Published: July 2004

Ascites is the most common of the major complications of cirrhosis. The development of ascites is an important landmark in the natural history of cirrhosis and has been proposed as an indication for liver transplantation. The initial evaluation of a patient with ascites should include a history, physical evaluation, and abdominal paracentesis with ascitic fluid analysis. Treatment should consist of abstinence from alcohol, sodium restricted diet, and diuretics. This regimen is effective in ≈90% of patients. The treatment options for the diuretic-resistant patients include serial therapeutic paracenteses, liver transplantation, and peritoneovenous shunting.
Evidence for altered central noradrenergic function in experimental acute liver failure in the rat
Authors: Michalak Adrianna , Rose Christopher , Buu Paul N. , Butterworth Roger F.
Published: July 2004

These is increasing evidence to suggest that central noradrenergic mechanisms may contribute to the central nervous system manifestations of acute liver failure. To further elucidate this possibility, extracellular brain concentrations of the monoamines, noradrenaline (NA), dopamine (DA), and serotonin, were measured by high-performance liquid chromatography with electrochemical detection in microdialysates from the extracellular compartment of frontal cortex in rats with acute (ischemic) liver failure at various times during the progression of encephalopathy and brain edema, as well as in obligate control groups of animals. In addition, binding sites for the noradrenergic receptor subtype ligands, [3H]-prazosin (α1 sites), [3H]-RX821002 (α2 sites), and [125]I-iodopindolol (β sites), were assessed using quantitative receptor autoradiography in regions of the brains of rats at coma stage of acute liver failure and of control groups of animals. Coma stages of encephalopathy in acute liver failure were associated with selectively increased noradrenaline concentrations (P < .05) and a concomitant selective loss of α1 and β1 sites in frontal cortex and thalamus. These findings add to a growing body of evidence that central noradrenergic function is modified in acute liver failure and suggest that α1/β1 receptor-mediated noradrenergic mechanisms may play a role in the pathogenesis of brain edema and encephalopathy in this condition.
Cerebral blood flow and metabolism in patients with chronic liver disease undergoing orthotopic liver transplantation
Authors: Philips Barbara J. , Armstrong Ian R. , Pollock Anthony , Lee Alistair
Published: July 2004

Changes in cerebral hemodynamics and metabolism associated with anesthesia and liver transplantation may present particular hazards for patients with cirrhosis. Fifteen patients undergoing liver transplantation were studied, 7 of whom had encephalopathy. Cerebral blood flow (CBF) was measured at the start of surgery, during veno-venous bypass and post reperfusion, using a method based on the Kety-Schmidt method. Cerebral metabolism was assessed by measuring the cerebral metabolic rate for oxygen (CMRO2) and the lactate oxygen index (LOI). The cerebral vascular reactivity to carbon dioxide (CO2) was studied during the preanhepatic and post reperfusion phases. During the preanhepatic period, the median CBF was 44 mL/100 g/min at an arterial carbon dioxide tension (PaCO2) of 3.8 kPa. After reperfusion the CBF increased (P < .02) to 102 mL/100 g/min, the arterial hydrogen ion concentration increased from 39 nmol/L to 53 nmol/L (P < .02) and the jugular venous oxygen saturation from 74% to 89% (P < .02). CBF was similar in patients with and without encephalopathy. The cerebral vascular reactivity to CO2 remained intact, although after reperfusion, the CBF for a given PaCO2 was greater, and the slope of the CBF/CO2 response curve diminished. The CMRO2was normal in patients without encephalopathy. In the encephalopathic patients, the CMRO2 was low during all stages of transplantation (0.54, 0.86, 1.24 mL/100 g/min, respectively). Patients with encephalopathy may be at increased risk of hypoxemic brain injury during transplantation. To minimize this possibility, more detailed neurological monitoring may be useful.
Quantitation of telomerase activity in hepatocellular carcinoma: A possible aid for a prediction of recurrent diseases in the remnant liver
Authors: Suda Takeshi , Isokawa Osamu , Aoyagi Yutaka , Nomoto Minoru , Tsukada Kazuhiro , Shimizu Takeaki , Suzuki Yasufumi , Naito Akira , Igarashi Hirotaka , Yanagi Masahiko , Takahashi Toru , Asakura Hitoshi
Published: July 2004

Because telomerase activity is necessary for cell immortality and probably associated with tumor progression, we have evaluated a possible aid for quantitation of the activity to predict intrahepatic recurrences after surgery in patients with hepatocellular carcinoma (HCC). HCC tissues obtained by surgical resection from 20 patients were studied. Telomerase activity was expressed as peaks with a periodicity through a fluorescence-based telomeric repeat amplification protocol using an autosequencer, and the quantity of activity was calculated from peak areas. A ratio of fluorescence intensity depending on telomerase to that of an internal standard was used as a value of relative telomerase activity (RTA). RTA in serially diluted S100 extracts from HepG2 cells was well correlated with the amount of the extracts. The mean RTA value of 36.4 ± 27.8 (mean ± SD, 3.21 to 105) in 9 patients suffering from early recurrences after surgery was significantly higher than that (9.84 ± 7.65; mean ± SD, 3.00 to 29.0) in 11 patients without intrahepatic recurrences during the early period (P = .004). These results indicate that RTA value can be a useful predictor for intrahepatic recurrences during the early period after surgical resection of HCC.
Role of glutathione in nitric oxide-dependent regulation of energy metabolism in rat hepatoma cells
Authors: Nishikawa Manabu , Sato Eisuke F. , Kashiba Misato , Kuroki Tetsuo , Utsumi Kozo , Inoue Masayasu
Published: July 2004

Previous studies in this laboratory revealed that nitric oxide (NO) reversibly inhibits the respiration of isolated mitochondria and ascites hepatoma (AH-130) cells by an oxygen concentration-dependent mechanism. The inhibitory effect of NO on the respiration of AH-130 cells was enhanced by treating with digitonin that selectively permeabilized plasma membranes and released cytosolic low-molecular-weight compounds. Reduced glutathione (GSH) is the most abundant cytosolic thiol that easily reacts with NO. To elucidate the mechanism by which digitonin enhanced the inhibitory action of NO, the effect of GSH and related thiols was studied with AH-130 cells and their mitochondria. The inhibitory effect of NO on the respiration of digitonin-treated cells was suppressed by either GSH, l -cysteine, or N-acetylcysteine, but not by oxidized glutathione. The inhibitory effect of NO on the respiration of their mitochondria was also decreased by GSH. In contrast, the inhibitory effect of NO was markedly enhanced with AH-130 cells obtained from animals that were pretreated with l -buthionine sulfoximine (BSO), a specific inhibitor for GSH synthesis. Kinetic analysis revealed that NO dose-dependently decreased GSH levels in AH-130 cells with concomitant generation of S-nitrosothiols. Although S-nitrosoglutathione (GSNO), a slow releaser of NO, also inhibited the respiration of tumor cell mitochondria, its effect was significantly lower than that of NO. These results suggest that cellular GSH might play pivotal roles in the regulation of energy metabolism in hepatoma cells by modulating free forms of NO.
Hepatic oval cells express the hematopoietic stem cell marker thy-1 in the rat
Authors: Petersen Bryon E. , Goff Julie P. , Greenberger Joel S. , Michalopoulos George K.
Published: July 2004

Hepatic oval cells (HOC) are a small subpopulation of cells found in the liver when hepatocyte proliferation is inhibited and followed by some type of hepatic injury. HOC can be induced to proliferate using a 2-acetylaminofluorene (2-AAF)/hepatic injury (i.e., CCl4, partial hepatectomy [PHx]) protocol. These cells are believed to be bipotential, i.e., able to differentiate into hepatocytes or bile ductular cells. In the past, isolation of highly enriched populations of these cells has been difficult. Thy-1 is a cell surface marker used in conjunction with CD34 and lineage-specific markers to identify hematopoietic stem cells. Thy-1 antigen is not normally expressed in adult liver, but is expressed in fetal liver, presumably on the hematopoietic cells. We report herein that HOC express high levels of Thy-1. Immunohistochemistry revealed that the cells expressing Thy-1 were indeed oval cells, because they also expressed α-fetoprotein (AFP), γ-glutamyl transpeptidase (GGT), cytokeratin 19 (CK-19), OC.2, and OV-6, all known markers for oval cell identification. In addition, the Thy-1+ cells were negative for desmin, a marker specific for Ito cells. Using Thy-1 antibody as a new marker for the identification of oval cells, a highly enriched population was obtained. Using flow cytometric methods, we isolated a 95% to 97% pure Thy-1+ oval cell population. Our results indicate that cell sorting using Thy-1 could be an attractive tool for future studies, which would facilitate both in vivo and in vitro studies of HOC.
Pathology of the liver in budd-chiari syndrome: Portal vein thrombosis and the histogenesis of veno-centric cirrhosis, veno-portal cirrhosis, and large regenerative nodules
Authors: Tanaka Masaya , Wanless Ian R.
Published: July 2004

Hepatic vein (HV) thrombosis causes ascites, hepatomegaly, and severe congestion of the liver (Budd-Chiari syndrome [BCS]). Severe hepatic fibrosis develops in this syndrome with a variety of histological patterns. Some livers have a pattern of cirrhosis in which there is fibrous bridging between HVs and portal tracts (veno-portal cirrhosis). Other livers have a pattern of “reversed-lobulation cirrhosis” (veno-centric cirrhosis), in which fibrous bridging between HVs and portal tracts is minimal. The prevalence and pathogenesis of these forms of cirrhosis and the effect of portal vein (PV) thrombosis in this disease have not been studied. We examined 15 resected livers from patients with BCS to determine the distribution of vascular obstruction and the character of the parenchymal response. Six livers had veno-portal cirrhosis, and all of these had severe PV obliteration caused by thrombosis. Three livers had veno-centric cirrhosis and had normal medium and large PVs. The remaining six livers had mixed veno-centric/veno-portal cirrhosis and had moderate PV obliteration. The nodules in veno-centric cirrhosis had evidence of an unusual circulation with small arteries supplying a midzonal venous plexus that appeared to drain retrogradely into patent small PVs. Nine livers had large regenerative nodules histologically similar to focal nodular hyperplasia. PV thrombosis is a frequent occurrence in BCS. The correlation between PV thrombosis and the pattern of cirrhosis suggests a role for PV obliteration in the genesis of veno-portal bridging fibrosis in this disease and possibly in other diseases leading to cirrhosis.
Identification of B10, an alkaline phosphodiesterase of the apical plasma membrane of hepatocytes and biliary cells, in rat serum: Increased levels following bile duct ligation and during the development of cholangiocarcinoma
Authors: Meerson Nirina Rajho , Delautier Danièle , Durand-Schneider Anne-Marie , Moreau Alain , Schilsky Michael L. , Sternlieb Irmin , Feldmann Gérard , Maurice Michèle
Published: July 2004

Alkaline phosphodiesterase (APDE) is associated with the cellular plasma membrane of many organs. Several isoforms are also detected in normal human serum and their respective amounts vary in liver diseases but their significance is unknown. The aims of this study were: 1) to identify a serum form of B10, an APDE exclusively localized at the apical pole of the plasma membrane of rat hepatocytes and biliary cells; 2) to gain insight into its origin; and 3) to investigate its behavior, in two liver diseases in which an abnormal membrane expression of B10 has been reported, namely cholestasis and cholangiocarcinoma. A soluble form of B10 was immunoprecipitated from normal rat serum, which amounted to 13% of total serum APDE activity. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the size of the serum enzyme was 125 kd, which is slightly lower than that found in the plasma membrane (130 kd). In bile, a 120-kd and a 130-kd form was found. A sixfold and fivefold increase of B10 APDE activity was observed in the serum of bile duct-ligated rats and in the Long-Evans Cinnamon (LEC) rats which spontaneously develop cholangiocarcinoma. The molecular size of the form present in serum was unchanged. A threefold increase was also observed in LEC rats which had not yet developed a cholangiocarcinoma. In conclusion, we identified a soluble form of B10 in normal rat serum. The increase in serum B10 in the experimental and pathological conditions investigated does not seem to result from passage of the biliary form to the serum but seems to be caused by increased cleavage of the membrane form. Its rise early during the onset of cholangiocarcinoma suggests that B10 in the serum might be a marker of carcinogenesis and/or be involved in the development of cholangiocarcinoma.
Efficient In Vitro vectorial transport of a fluorescent conjugated bile acid analogue by polarized hepatic hybrid WIF-B and WIF-B9 cells
Authors: Bravo Pilar , Bender Virginie , Cassio Doris
Published: July 2004

Efficient transport of bile acids, a typical characteristic of hepatocytes, is partially lost in most hepatoma cell lines and in normal hepatocytes after some days in culture. We have tested whether the polarized rat hepatoma-human fibroblast hybrid WIF (hybrids between W138 and Fao cells) cells previously obtained by our group were able to perform vectorial transport of the fluorescent bile acid derivative cholylglycylamidofluorescein (CGamF) towards the bile canaliculi (BC). Four different WIF clones were analyzed. All were well polarized, as shown by the formation of spherical and even tubular BC-like structures and by the restricted localization at the BC, visualized by immunofluorescence, of the apical membrane marker HA4, a possible bile acid carrier. WIF-B and its subclone WIF-B9 were found to accumulate CGamF in 65% to 75% of their BC. This transport was time, temperature, and partly sodium dependent and was inhibited by coincubation with the parental natural bile salt cholylglycine. Dinitrophenyl glutathione, a substrate of the canalicular multispecific organic anion transporter, did not inhibit CGamF canalicular secretion, whereas it greatly impaired the canalicular secretion of a non-bile acid organic anion, fluorescein, generated intracellularly from fluorescein diacetate. Confocal microscopy confirmed the presence of CGamF in the cytoplasm, supporting a transcellular route from medium to BC. In contrast, two other polarized clones exhibited a poor ability (WIF 12-6) or no ability (WIF12-1 TGδ) to vectorially transport CGamF. In conclusion, WIF-B and WIF-B9 exhibit not only structural but also functional polarity, at least as far as vectorial organic anion transport is concerned.
Purification of rat hepatic stellate cells by side scatter-activated cell sorting
Authors: Geerts Albert , Niki Toshiro , Hellemans Karine , De Craemer Dirk , Van Den Berg Kit , Lazou Jean-Marc , Stange Geert , Van De Winkel Marnix , De Bleser Pieter
Published: July 2004

In this study, we present a new method to obtain pure, viable, freshly isolated hepatic stellate cells. Stellate cells were purified by cell sorting using their high side scatter (SSC) of incident light. Purity of the cells was established by light and transmission electron microscopy (TEM). Starting from stellate cells that were 50% to 70% enriched by centrifugation in 11% Nycodenz, the cell purity after sorting was found to be 96.6% ± 2.9%. Viability of the sorted cells was 90.8% ± 2.2% as measured by the Trypan blue exclusion test and was confirmed by cell culturing. Per hour of sorting, 1.4 ± 0.4 million stellate cells were obtained. Sorting runs of up to 4 hours were practically feasible, resulting in yields of 5 to 6 million cells per rat liver. Cells attached to plastic substratum within 24 hours. Subsequently, they spread and underwent spontaneous transition into myofibroblast-like cells. The purity of sorted cells was documented by reverse-transcriptase polymerase chain reaction (RT-PCR) experiments using specific primer pairs for messenger RNA (mRNA) species that were only present in parenchymal (preproalbumin), endothelial (endothelial cell nitric oxide synthase [eNOS]), stellate (desmin), or Kupffer cells (77- to 88-kd fucose receptor). Contaminating mRNA species were absent in sorted stellate cells. Next, we examined freshly sorted stellate cells by Western blotting to confirm the presence of relevant cytoskeletal proteins. Cells were positive for vimentin, desmin, and glial fibrillary acidic protein (GFAP), but negative for α-smooth muscle actin (α-SMA). Sorted and cultured cells were immunophenotyped for the presence of collagen types I, III, and IV, laminin, and the cytoskeletal proteins, α-SMA, desmin, vimentin, and GFAP. At 90 hours in culture, cells expressed all the investigated extracellular matrix proteins. Desmin was present in 82%± 1%, vimentin in 96%± 2.5%, and GFAP in 91% ± 4.5% of cells. α-SMA was present in 91% ± 2% of cultured cells. We conclude that cell sorting based on SSC of incident light is a convenient method to obtain virtually pure stellate cells that can be used for direct analysis or for culturing. Although the yields obtained with this method are lower than with standard methods, and additional equipment is required, SSC-activated sorting offers the possibility of very pure cells when essential for analyses based on sensitive detection methods such as RT-PCR.
Accurate separation of biliary lipid aggregates requires the correct intermixed micellar/intervesicular bile salt concentration
Authors: Donovan Joanne M. , Jackson Audrey A.
Published: July 2004

The intermixed micellar/intervesicular bile salt (BS) concentration (IMC), composed of BS monomers and simple micelles, is in dynamic equilibrium with mixed micelles and vesicles. Accurate separation of biliary lipid aggregates is believed to depend on accurately measuring the IMC. Using centrifugal ultrafiltration, we measured the IMC of cholesterol-supersaturated model biles that were physiologically composed. Gel chromatography was performed using eluants containing the following: 1) the IMC; 2) the same BS composition as the IMC but higher or lower BS concentrations; 3) the same BS concentration as the IMC, but with more hydrophilic or hydrophobic BS; and 4) 10 mmol/L cholate. Compared with an eluant containing the same BS composition as the IMC, an eluant containing the same relative BS composition but 75% of the IMC increased the proportion of cholesterol in vesicles and decreased the vesicular cholesterol/egg yolk phosphatidylcholine (EYPC) ratio. In contrast, an eluant containing 150% of the IMC entirely transformed vesicles to micelles. Eluants containing slightly more hydrophobic or more hydrophilic BS eliminated or increased vesicular cholesterol content, respectively. An eluant of 10 mmol/L cholate overestimated vesicular cholesterol and in concentrated biles reproducibly produced an incompletely separated intermediate peak, possibly because of re-equilibration between mixed micelles and vesicles. Further, in concentrated biles, fractions eluting at volumes corresponding to mixed micelles were visibly turbid, irrespective of the eluant used. The correct IMC allows accurate separation of biliary lipid aggregates, but differences in BS concentration or composition substantially alter the vesicular percentage of cholesterol as well as the cholesterol/EYPC ratio. Elution with 10 mmol/L cholate may introduce artifactual gel-filtration peaks and inadequate separation of particles with widely differing molecular weights, both of which have confused previous analyses of biliary lipid aggregates.
Characterization of antimitochondrial antibodies in healthy adults
Authors: Mattalia Alberto , Quaranta Stefano , Leung Patrick S.C. , Bauducci Marzia , Van de Water Judy , Calvo Pier Luigi , Danielle Franca , Rizzetto Mario , Ansari Aftab , Coppel Ross L. , Rosina Floriano , Gershwin Eric M.
Published: July 2004

The detection of antimitochondrial antibodies (AMAs) is an important criterion for the diagnosis of primary biliary cirrhosis (PBC). During the last decade, the mitochondrial autoantigens have been cloned, sequenced, and identified as members of the 2-oxo-acid dehydrogenase pathway, including the E2 subunits of pyruvate dehydrogenase (PDC-E2), branched-chain 2-oxo-acid dehydrogenase (BCOADC-E2), and 2-oxo-glutarate dehydrogenase (OGDC-E2). We have developed a rapid and sensitive diagnostic test for use in PBC based on a triple hybrid recombinant molecule (r-MIT3) that contains the autoepitopes of PDC-E2, BCOADC-E2, and OGDC-E2. To help understand the frequency and antigen specificity of AMAs in an asymptomatic population and to identify patients with early disease, we investigated the prevalence of AMA, by enzyme-linked immunosorbent assay (ELISA), in a cohort of 1,530 people from northern Italy. Positive sera were further analyzed for immunoglobulin (Ig) isotypes, subclasses, and epitopes of AMA by a combination of ELISA and immunoblotting. In this cohort of 1,530 people, 9 (0.5%) reacted to r-MIT3 by ELISA. Of the 9 reactive sera, 2 recognized PDC-E2, 2 of 9 recognized BCOADC-E2, 1 of 9 recognized OGDC-E2, 2 of 9 recognized both PDC-E2 and BCOADC-E2, and 1 of 9 recognized PDC-E2 and OGDC-E2. AMA reactivity was primarily IgM and IgA. Epitope mapping revealed an AMA pattern of reactivity to PDC-E2 that differed from that found in patients with histologically proven PBC in most of the sera. However, 1 sera of a 72-year-old female with a normal alkaline phosphatase had an AMA profile identical to typical PBC. After a variable follow-up period (8-14 months), sera from 8 of 9 of these people were re-obtained for AMA and relative epitope mapping. Interestingly, the reactivity had a wider AMA pattern than before.
Risk of colorectal neoplasia in patients with primary sclerosing cholangitis and ulcerative colitis following orthotopic liver transplantation
Authors: Loftus Edward V. , Aguilar Humberto I. , Sandborn William J. , Tremaine William J. , Krom Ruud A. , Zinsmeister Alan R. , Graziadei Ivo W. , Wiesner Russell H.
Published: July 2004

Several centers have identified colorectal cancer in patients with primary sclerosing cholangitis (PSC) and ulcerative colitis (UC) who have undergone orthotopic liver transplantation. Increased frequency of surveillance colonoscopy and prophylactic colectomy has been recommended. To address these concerns, we reviewed the posttransplantation experience with colorectal neoplasia in patients with PSC and UC at our institution. All patients with PSC and UC who underwent liver transplantation at Mayo Clinic between April 1985 and December 1993 were followed up through their complete history for colorectal dysplasia, cancer, or death. Eighty-one of 108 patients with PSC (75%) had concomitant inflammatory bowel disease (all but 1 had UC). Proctocolectomy had been performed before transplantation in 24 (30%). Median follow-up after transplantation was 4.2 years. Among the 57 patients with intact colons, 3 developed colorectal cancer, an incidence of approximately 1% per person per year. The cumulative incidence of dysplasia was 15% at 5 years and 21% at 8 years. Overall actuarial survival stratified by presence or absence of an intact colon at transplantation was similar (86% and 86%, respectively, at 5 years). The risk of carcinoma after transplantation compared with that expected for patients during a comparable (pretransplantation) period was increased fourfold but was not statistically significant. The risk of colorectal neoplasia (dysplasia and cancer) after liver transplantation in patients with PSC and UC is clinically important. However, this risk had no impact on patient survival. Prophylactic proctocolectomy does not appear necessary, but annual surveillance colonoscopy is recommended.
Evaluation of assays for antibody to hepatitis E virus by a serum panel
Authors: Mast Eric E. , Alter Miriam J. , Holland Paul V. , Purcell Robert H.
Published: July 2004

Few data are available to evaluate the performance of existing assays for antibody to the hepatitis E virus (anti-HEV). A panel of 164 randomized and coded sera was tested for anti-HEV by 12 different assays. The panel included a dilution series of an early convalescent human serum, known-positive sera (undiluted human sera obtained 2 months to 13 years after acute hepatitis E, and postinoculation chimpanzee sera), known-negative sera (preinoculation chimpanzee sera; sera from chimpanzees with hepatitis A virus, hepatitis B virus, or hepatitis C virus infection; and normal human sera), and sera obtained from previously tested U.S. blood donors without a history of hepatitis. Six tests detected anti-HEV in ≥90% of undiluted known-positive sera. The sensitivity of all of the assays with known-positive sera ranged from 17% to 100%, and the limit of detection by endpoint dilution ranged from 1:5 to 1:160. Ten tests were nonreactive for all of the 22 known-negative sera, one test was reactive for one serum, and one test was reactive for 5 sera. In pairwise comparisons of different tests in blood donor sera, the overall concordance ranged from 49% to 94% (median, 69%) and the concordance among reactive sera ranged from 0% to 89% (median, 32%). Several of these tests performed well in detecting anti-HEV in known positive sera. However, highly discrepant results among U.S. blood donor sera indicate that anti-HEV seroprevalence data in non-HEV-endemic countries may be unreliable and should be interpreted with caution.
Role of cyclic guanosine monophosphate and K+ channels as mediators of the mesenteric vascular hyporesponsiveness in portal hypertensive rats
Authors: Atucha Noemí M. , Ortíz Clara M. , Fortepiani Lourdes A. , Ruiz Francisco M. , Martínez Concepción , García-Estañ Joaquín
Published: July 2004

The mechanisms mediating the hyporesponsiveness to vasoconstrictors in portal hypertension are not completely established. In the present study, we evaluated the role of cyclic guanosine monophosphate (cGMP) and potassium channels as contributors to the pressor hyporesponsiveness to methoxamine (MTX) of the mesenteric vascular bed of portal vein-ligated (PVL) hypertensive rats. In basal conditions, and compared with sham-operated control rat (SHAM) vessels, PVL preparations showed a blunted pressor response (maximum: 39.3 ± 6.1 vs. 94.5 ± 8.9 mm Hg), which increased by pretreatment with methylene blue (MB), a guanylate cyclase inhibitor (118.7 ± 8.9 vs. 152.0 ± 10.0, respectively), and even more with the nitric oxide (NO) synthesis inhibitor N
ω-nitro-l-arginine (NNA) (159.9 ± 7.4 vs. 194.1 ± 5.7, respectively), suggesting that NO acts through cGMP-dependent and independent mechanisms. In all cases, however, the pressor responses of PVL vessels were lower than those of SHAM. Pretreatment of the vessels with the potassium channel inhibitors, tetraethylammonium (TEA), glibenclamide (GLB), or charybdotoxin (CHX), did not improve the reduced pressor responses of the PVL rats. However, when the preparations were simultaneously pretreated with MB and TEA or with NNA and TEA, the pressor responses were potentiated with respect to groups treated with MB or NNA alone, and the differences between PVL and SHAM vessels were completely corrected. These data suggest that both NO and potassium channels mediate the vascular hyporesponsiveness to methoxamine of the PVL mesenteric vasculature. Our results also disclose that NO blunts the pressor response of the PVL vessels by a dual mechanism of action, through activation of potassium channels and through the formation of cGMP. Finally, the NO-independent component mediated by potassium channels can be only seen when the main cGMP-NO component is inactivated. In conclusion, both cGMP and potassium channels mediate the vascular hyporesponsiveness to MTX of the mesenteric bed of portal hypertensive rats.
Tumor necrosis factor α regulates nitric oxide synthase expression in portal hypertensive gastric mucosa of rats
Authors: Ohta Masayuki , Tarnawski Andrzej S. , Itani Rabiha , Pai Rama , Tomikawa Morimasa , Sugimachi Keizo , Sarfeh James I.
Published: July 2004

Anti-tumor necrosis factor α (TNF-α) treatment decreases nitric oxide (NO) synthesis and ameliorates the hyperdynamic circulation in portal hypertensive rats. We have recently demonstrated that nitric oxide synthase isoform 3 (NOS3) is overexpressed in portal hypertensive gastric mucosa and that resultant NO overproduction probably is responsible for the increased susceptibility of the mucosa to damage. In the present study, we examined whether TNF-α is overexpressed in portal hypertensive gastric mucosa and whether anti-TNF-α treatment affects gastric NOS3 messenger RNA (mRNA) and protein expression. We examined plasma concentrations of TNF-α and its protein expression in gastric specimens from portal hypertensive and sham-operated rats using Western blotting and immunohistochemistry. We also measured gastric mucosal blood flow, gastric expression of NOS3 mRNA and protein, and NOS3 enzyme activity in rats with and without TNF-α- neutralizing antibody treatment. The TNF-α protein levels in portal hypertensive stomachs were significantly increased by 57% compared with levels in sham-operated controls. TNF-α antibody treatment normalized gastric mucosal blood flow in portal hypertensive stomachs and significantly reversed overexpression of gastric NOS3 mRNA, protein, and its enzyme activity in portal hypertensive rats by 48%, 45%, and 33%, respectively. These results suggest that TNF-α may regulate NOS3 expression in the portal hypertensive stomach and that anti-TNF-α treatment may ameliorate the pathophysiological abnormalities of portal hypertensive gastric mucosa.
Expression of e-cadherin, α-catenin, β-catenin, and CD44 (standard and variant isoforms) in human cholangiocarcinoma: An immunohistochemical study
Authors: Ashida Keigo , Terada Tadashi , Kitamura Yukisato , Kaibara Nobuaki
Published: July 2004

Immunolocalization of E-cadherin (E-cad), α-catenin, β-catenin, and CD44 has rarely been investigated in human cholangiocarcinoma (CC). We, therefore, immunohistochemically examined the expression of E-cad, α-catenin, β-catenin, CD44 standard (CD44s), and CD44 variants (CD44v) including CD44v5, CD44v6, CD44v7-8, and CD44v10 in normal adult livers and in 47 cases of CC; and the results were then correlated with tumor grade, vascular invasion, metastasis, p53 expression, proliferative fraction (Ki-67 labeling), and c-erbB2 expression. In normal livers, E-cad, α-catenin and β-catenin, but not CD44s, CD44v5, CD44v6, CD44v7-8, and CD44v10, were expressed at the cell membrane of normal intrahepatic bile ducts. In CC, membranous expression of E-cad, α-catenin, and β-catenin was the same or reduced when compared with non-cancerous bile ducts in the majority of CC. We found that the down-regulation of E-cad, α-catenin, and β-catenin expression significantly correlated with tumor high grade, but not with vascular invasion, metastasis, p53 expression, Ki-67 labeling, or c-erbB2 expression, except for β-catenin, the down-regulation of which was associated with c-erbB2 down-regulation. CD44s, CD44v5, CD44v6, CD44v7-8 and CD44v10 were frequently expressed at the membrane of CC cells. There were, however, no significant correlations between these aberrant CD44 expression and tumor grade, metastasis, vascular invasion, p53 expression, Ki-67 labeling, or c-erbB2 expression, with a few exceptions of CD44s and CD44v5. We found that CD44s aberrant expression significantly correlated with absence of metastasis and vascular invasion, and that CD44v5 aberrant expression significantly correlated with p53 under-expression. These results suggest that membranous expression of E-cad, α-catenin, and β-catenin is reduced in a majority of CC and this down-regulation correlates with CC high grade, and that β-catenin down-regulation is associated with c-erbB2 down-regulation. The data also suggested that CD44s, CD44v5, CD44v6, CD44v7-8, and CD44v10 may be neoexpressed during carcinogenesis of CC but this neoexpression does not correlate with tumor progression in CC, with the exception of CD44s and CD44v5.
Acetaminophen hepatotoxicity in tumor necrosis factor/lymphotoxin-α gene knockout mice
Authors: Boess Franziska , Bopst Martin , Althaus Roland , Polsky Stacey , Cohen Steven D. , Eugster Hans-Pietro , Boelsterli Urs A.
Published: July 2004

Recent evidence suggests that macrophages and/or other nonparenchymal cells may release important mediators contributing to the hepatic necrosis induced by high doses of acetaminophen (APAP). The nature and causative role of these mediators has remained elusive, however. To investigate the role of the proinflammatory cytokine, tumor necrosis factor (TNF) in the initiation and early propagation of APAP-induced liver injury, we have used mice deficient in both TNF and the closely related lymphotoxin-α (LT-α). Male TNF/LT-α knockout mice and C57BL/6 wild-type mice were treated with a hepatotoxic dose of APAP (400 mg/kg, intraperitoneally), and the development of liver injury was monitored over 8 hours. Both genotypes exhibited similar basal activities of hepatic cytochrome P450 2E1 and 1A2. After APAP administration, both the rate of glutathione consumption and the extent of subsequent selective protein binding did not differ significantly in the knockout and wild-type mice. The TNF/LT-α-deficient mice developed severe centrilobular necrosis and exhibited highly increased levels of serum alanine aminotransferase and aspartate aminotransferase, the extent of which was not significantly different from that in wild-type mice. In C57BL/6 mice exposed to APAP, no increases in hepatic transcripts of TNF or LT-α were found by reverse transcription-polymerase chain reaction, nor was immunoreactive serum TNF detected by enzyme-linked immunosorbent assay over 8 hours posttreatment. These data indicate that, in the absence of the genes encoding for TNF and LT-α, APAP bioactivation was not altered and mice still developed severe hepatic necrosis. Thus, TNF is unlikely to be a key mediator in the early pathogenesis of APAP-induced hepatotoxicity.
Ethanol up-regulates fatty acid uptake and plasma membrane expression and export of mitochondrial aspartate aminotransferase in HepG2 cells
Authors: Zhou Sheng-Li , Gordon Ronald E. , Bradbury Michael , Stump Decherd , Kiang Chih-Li , Berk Paul D.
Published: July 2004

To explain the increased plasma mitochondrial aspartate aminotransferase (mAspAT) observed in alcoholics, we cultured HepG2 hepatoma cells in ethanol. Acute (24 hour) exposure to 0, 20, 40, or 80 mmol/L ethanol produced a dose-dependent (r = .98) increase in mAspAT messenger RNA (mRNA) of ≤ thirteen-fold, with no significant change in the cellular content of mAspAT or of several other enzymes. The recovery of mAspAT in the medium over 24 hours of ethanol exposure correlated with both ethanol concentration and with mAspAT mRNA (r = .90), reaching 808% of cellular enzyme content/24 hours at 80 mmol/L. Recovery of all other enzymes studied was ≤ 20% of cellular content and unaffected by ethanol. Plasma membrane mAspAT content also correlated with mAspAT mRNA (r = .96) and mitochondrial levels were unchanged. No mitochondrial morphologic abnormalities were observed at any ethanol concentration studied. In cells cultured chronically at 0 to 80 mmol/L ethanol, fatty acid uptake Vmax increased in parallel with plasma membrane expression of mAspAT (r = .98). Cellular triglyceride content was highly correlated with Vmax. Thus, the data suggest that: 1) the increased plasma mAspAT observed in alcoholics may reflect pharmacologic upregulation of mAspAT mRNA and of mAspAT synthesis by ethanol; and 2) increased mAspAT-mediated fatty acid uptake may contribute to alcoholic fatty liver.
The surface of rat hepatocytes can transfer iron from stable chelates to external acceptors
Authors: Scheiber Barbara , Goldenberg Hans
Published: July 2004

The chelator diethylenetriaminepentaacetate (DTPA) forms a stable complex with iron that does not donate iron to transferrin under physiological conditions, i.e., pH above 7 and isotonic milieu. It does, however, deliver iron to hepatocytes. This uptake is initiated by a mobilization of the metal from the complex by the cell surface. When an external chelator is added simultaneously, it can bind the iron and inhibit its accumulation by the cells. This is shown here with the impermeant siderophore conjugate hydroxyethyl-starch coupled desferrioxamine, as well as with apo-transferrin. We also demonstrate exchange of iron between DTPA and holo-transferrin, or at least movement from the chelator to the protein, which may have lost its iron to the cell in advance, providing new binding sites for mobilized iron. The efficient hepatocyte iron donor lactoferrin greatly stimulates iron uptake from DTPA, apparently by binding iron and transferring it into the cells by endocytosis. Ferritin is unable to do this; therefore, the mobilization of iron is not caused by a reducing activity at the cell surface, because iron is readily transferred from DTPA to ferritin by the reductant ascorbic acid. The transfer process is dependent on the temperature, the time, and the amount of cells present, and is partly inhibited by sulfhydryl reagents. We conclude that this activity represents a hitherto unidentified first step in the movement of iron through the cell membrane and may be relevant for transferrin-bound, as well as for non-transferrin-bound, iron uptake by hepatocytes.