bluuebunny/crossref_metadata_2025 huggingface dataset:
Selective loss of nucleoside carrier expression in rat hepatocarcinomas
Authors: Dragan Yvonne , Valdés Raquel , Gomez-Angelats Mireia , Felipe Antonio , Casado Javier F. , Pitot Henry , Pastor-Anglada Marçal
Published: March 2004

Evidence that hepatoma cell lines show differential expression of concentrative nucleoside transporters (CNT1 and CNT2) prompted us to study the transporter proteins in 2 models of hepatocarcinogenesis, the chemically induced Solt and Farber model and the albumin-SV40 large T antigen (Alb-SV40) transgenic rat. CNT1 expression was lower in tumor biopsy specimens from Alb-SV40 rat livers than in normal tissue. Immunocytochemistry revealed that the CNT1 protein was indeed absent in the tumor lesions. CNT1 was also absent in a cell line, L25, derived from the Alb-SV40 transgenic rat liver tumors, whereas another cell line, L37, derived from the normal-appearing parenchyma, retained the expression of both carrier isoforms. The protein expression correlated with the nucleoside transport properties of these cell lines. Moreover, although CNT2 expression was highly dependent on the growth characteristics of the 2 cell lines, as was CNT1 (albeit to a lower extent) in L37 cells, it was not expressed in L25 cells at any stage of cell growth. In contrast to the transgenic model of hepatocarcinogenesis, in the chemically induced tumors the expression of CNT2 was lower, although still detectable. In summary, these data indicate that hepatocarcinogenesis leads to a selective loss or diminished expression of nucleoside carrier isoforms, a feature that may be relevant to our understanding of the molecular basis of the bioavailability of those drugs that are nucleoside derivatives and may be substrates of these carriers. The transport properties and isoform-expression profile of the L25 and L37 cell lines make them suitable hepatocyte culture models with which to study nucleoside transport processes and drug sensitivity.
Hepatocellular response to chemical stress in CD-1 mice: Induction of early genes and γ-glutamylcysteine synthetase
Authors: Kitteringham Neil R. , Powell Helen , Clement Yuri N. , Dodd Charlotte C. , Tettey Justice N. , Pirmohamed Munir , Smith Dennis A. , McLellan Lesley I. , Park Kevin B.
Published: March 2004

Exposure of cells to toxic chemical species can result in reduced glutathione (GSH) depletion, generation of free radicals, and/or binding to critical cell determinants. Chemical stress is usually followed by a concerted cellular response aimed at restoring homeostasis, although the precise initial stimulus for the response is unclear. We have focused on one component of this stress response, the up-regulation of γ-glutamylcysteine synthetase (γ-GCS) and the preceding molecular events involved in its regulation in an in vivo mouse model. Male CD-1 mice received buthionine sulphoximine (BSO; 7.2 mmol/kg), diethyl maleate (DEM; 4.2 mmol/kg), paracetamol (APAP; 3.5 and 1.0 mmol/kg), or carbon tetrachloride (CCl4; 1.0 and 0.2 mmol/kg). Biochemical (serum transaminase and hepatic GSH levels) and molecular (c-jun and c-fos messenger RNA [mRNA] levels and activator protein 1 [AP-1] DNA binding activity) parameters were measured, as well as the consequent effects on γ-GCS levels and activity. All compounds produced GSH depletion, but only the higher doses of APAP and CCl4 caused liver damage. DEM, APAP, and CCl4 increased c-jun and c-fos mRNA levels, together with an increase in AP-1 binding; BSO failed to induce AP-1 despite an increase in c-fos. Interestingly, the effects on γ-GCS varied markedly according to the compound: BSO and DEM increased γ-GCS enzyme activity, although only DEM, but not BSO, resulted in an increase in γ-GCSh mRNA and protein. In contrast, APAP and CCl4 both increased γ-GCSh mRNA and protein; however, there was a marked dose-dependent decrease in γ-GCS activity. These data indicate that the effect of chemical stress on the liver is compound specific and is not merely dependent on depletion of GSH.
Natural history of inoperable hepatocellular carcinoma: Estrogen receptors’ status in the tumor is the strongest prognostic factor for survival
Authors: Villa Erica , Moles Anna , Ferretti Ilva , Buttafoco Paola , Grottola Antonella , Del Buono Mariagrazia , De Santis Mario , Manenti Federico
Published: March 2004

Clinical course in hepatocellular carcinoma may be very different. We prospectively evaluated 96 patients with hepatocellular carcinoma unsuitable for radical therapy to investigate factors that could influence survival. Clinical, pathologic, and molecular data of patients were analyzed by univariate and multivariate analysis. The overall actuarial probability of survival at year 1, 2, 3, 4, 5, and 6 was 72%, 41%, 38%, 24%, 20%, and 9%. At univariate analysis, alpha-fetoprotein (AFP) (P= .0082); alkaline phosphatase (P= .0281); bilirubin (P= .0076); etiology (P= .0001); increment of tumor mass at month 3 (P= .0051); type of estrogen receptor (ER) in the tumor (P= .0000); prothrombin time (P= .0003); and portal vein thrombosis (P= .0000) had prognostic significance. At multivariate analysis, only type of ER (P= .0000) and bilirubin (P= .0030) showed independent predictive value for mortality. Survival was significantly longer in patients with wild-type estrogen receptors (P= .0000). Cumulative probability of survival at year 1, 2, 3, 4, 5, and 6 was 94%, 66%, 52%, 43%, 35%, and 18% for wild-type and 51%, 21%, 16%, and 9% for variant estrogen receptors (no patients alive after 4 years). Hepatitis B surface antigen (HBsAg)-positive patients with variant ERs had a median survival of 8 months versus 45 months in anti-hepatitis C virus-positive patients with wild-type ERs (P= .0001). In conclusion, (1) the presence of variant liver ER transcripts in the tumor was the strongest negative predictor of survival in inoperable hepatocellular carcinoma; (2) their presence was associated with spontaneous survival significantly worse than in patients with wild-type estrogen receptors; and (3) HBsAg-positive patients with variant receptors were characterized by the worst survival.
Alcoholic fatty liver differentially induces a neutrophil-chemokine and hepatic necrosis after ischemia-reperfusion in rat
Authors: Yamada Shinwa , Iida Takeshi , Tabata Takahiro , Nomoto Minoru , Kishikawa Hirofumi , Kohno Kimitoshi , Eto Sumiya
Published: March 2004

Primary graft nonfunction of steatotic liver allograft is one of the factors causing shortage of donor livers. Ischemia/reperfusion (I/R) injury is an important contributory factor to primary graft nonfunction. In this study, we investigated the complex chain of events from transcription factor activation to necrosis through cytokine induction and apoptosis in steatotic rat liver after warm I/R. Rats with alcoholic or nonalcoholic fatty liver were subjected to hepatic warm I/R and compared with control rats. Rats fed an ethanol diet for 6 to 8 weeks developed severe hepatic necrosis accompanied by increased neutrophil recruitment after I/R, compared with rats with nonalcoholic fatty liver or control. Hepatic apoptosis as assessed by DNA fragmentation at 4 hours after I/R, however, increased to a similar degree in each of the 2 fatty liver models compared with the control. Alcoholic fatty liver exposed to I/R showed a rapid increase in nuclear factor-κB (NF-κB) binding activity at 1 hour after I/R, which preceded an increased expression of tumor necrosis factor α (TNF-α) and cytokine-induced neutrophil chemoattractant-1 (CINC-1). In contrast, nonalcoholic fatty liver did not show such potentiation of either NF-κB activation or cytokine induction after I/R. Our results have indicated that alcoholic fatty liver may differentially induce CINC-1 production and hepatic necrosis after I/R. Furthermore, our results suggest that apoptosis per se does not always lead to necrosis in the liver following I/R.
Preclinical Evaluation of Two Human Anti-Hepatitis B Virus (HBV) Monoclonal Antibodies in the HBV-Trimera Mouse Model and in HBV Chronic Carrier Chimpanzees
Authors: Eren Rachel , Ilan Ehud , Nussbaum Ofer , Lubin Ido , Terkieltaub Dov , Arazi Yossi , Ben-Moshe Ofer , Kitchinzky Alberto , Berr Shoshana , Gopher Judy , Zauberman Arie , Galun Eithan , Shouval Danny , Daudi Nili , Eid Ahamed , Jurim Oded , Magnius Lars O. , Hammas Berit , Reisner Yair , Dagan Shlomo
Published: March 2004

Two human monoclonal antibodies (mAbs) against hepatitis B surface antigen (HBsAg) generated in the Trimera mouse system are described. Both mAbs 17.1.41 and 19.79.5 are of the IgG1 isotype and have high affinity constants for HBsAg binding in the range of 10−10 mol/L. Monoclonal antibody 17.1.41 recognizes a conformational epitope on the a determinant of HBsAg whereas mAb 19.79.5 recognizes a linear one. The 2 mAbs bind to a panel of hepatitis B virus (HBV) subtypes with distinct patterns. The neutralizing activity of these antibodies was tested in 2 different animal model systems. Administration of each mAb to HBV-Trimera mice, a system that provides a mouse model for human hepatitis B infection, reduced the viral load and the percentage of HBV-DNA-positive mice in a dose-dependent manner. These 2 mAbs were more effective than a polyclonal antibody preparation (Hepatect; Biotest Pharma, Dreieich, Germany) in both inhibition of HBV liver infection and reduction of viral load. A single administration of a mixture of these mAbs into HBV chronic carrier chimpanzees resulted in immediate reduction in HBsAg levels followed by recurrence to initial levels within few days. Thus, these mAbs may be potential candidates for preventive therapy or in combination with other antiviral agents against HBV. Further studies in humans are needed to assess these mAbs in various clinical indications.
Expression and Antitumor Effects of TRAIL in Human Cholangiocarcinoma
Authors: Tanaka Shinji , Sugimachi Keishi , Shirabe Ken , Shimada Mitsuo , Wands Jack R. , Sugimachi Keizo
Published: March 2004

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)/Apo2L has been recently identified as important in promoting programmed cell death in breast and colon adenocarcinomas. In this study, we investigated the expression and therapeutic potential of TRAIL in cholangiocarcinoma, one of the most devastating human hepatic malignancies. Expression of TRAIL receptors was determined in 13 patients with resectable intrahepatic cholangiocarcinoma. Cellular effects of TRAIL in promoting apoptosis of human cholangiocarcinoma cells were analyzed after exposure to recombinant protein, as well as following transfection with a cDNA expression construct. In vivo effects of TRAIL on tumor growth were investigated after subcutaneous injection of cholangiocarcinoma cells into nude mice. Analysis of 13 clinical and tissue samples revealed that TRAIL receptors containing the death domain were present in all cholangiocarcinomas as well as paired normal hepatic tissues derived from surgically resected margins. In contrast, 7 tumors did not express the TRAIL decoy receptors lacking the death domain; such receptors were detectable in all of the normal hepatic tissue counterparts. Recombinant TRAIL induced extensive programmed cell death in cholangiocarcinoma cell lines lacking decoy receptor expression. Transfection of the ectodomain of TRAIL also induced cellular apoptosis; this effect was abolished by introduction of the generalized lymphoproliferative disease-like mutation in the TRAIL protein. Finally, in vivo administration of recombinant TRAIL substantially inhibited subcutaneous tumor growth of human cholangiocarcinoma cells. Induction of apoptosis in tumor cells is possible with a biologically active TRAIL, and suggests that this cytokine is a promising antitumor agent against human cholangiocarcinoma.
Prednisolone Priming Enhances Th1 Response and Efficacy of Subsequent Lamivudine Therapy in Patients With Chronic Hepatitis B
Authors: Liaw Yun-Fan , Tsai Sun-Lung , Chien Rong-Nan , Yeh Chau-Ting , Chu Chia-Ming
Published: March 2004

Asian lamivudine trial has shown that hepatitis B e antigen (HBeAg) seroconversion rate during 1 year of lamivudine therapy was only 16% but was 64% in the subgroup of patients with a pretherapy serum alanine transaminase (ALT) level over 5 times the upper limit of normal (ULN). To test whether ALT rebound following corticosteroid priming enhances response to lamivudine therapy, a pilot study was conducted in 30 patients with ALT levels less than 5× ULN (43-169; N < 36 U/L). They received 30 mg of prednisolone daily for 3 weeks, 15 mg daily for 1 week, no treatment for 2 weeks, and then 150 mg of lamivudine daily for 9 months. Complete response (CR) was defined as ALT normalization with HBV-DNA seroclearance and HBeAg seroconversion. Peripheral blood mononuclear cell proliferation and cytokine secretion in response to recombinant HBV core antigen were serially assayed in 7 patients during priming and after withdrawal of prednisolone. Clinical rebound with an ALT over 5× ULN was observed in 20 patients (67%). Of these 20, 12 (60%) showed CR as compared with 1 (10%) of the 10 patients without significant ALT rebound (P < .002). The HBeAg seroconversion sustained in 70% of the patients 3 to 6 months after the end of lamivudine therapy. Immunological assays revealed that the responders showed Th1 dominant response and higher stimulation index to prednisolone priming. No serious side effect was encountered. These results suggest that corticosteroid priming induced immune/ALT rebound greatly enhances response to lamivudine therapy in chronic hepatitis B. Confirmation by randomized controlled trial is needed.
Unresponsiveness of Intrahepatic Lymphocytes to Bacterial Superantigen: Rapid Development of Suppressive Mac-1high Cells in the Mouse Liver
Authors: Terabe Masaki , Shimizu Masumi , Mabuchi Ayako , Matui So , Morikawa Hiroyasu , Kaneda Kenji , Kakiuchi Terutaka , Yokomuro Kozo
Published: March 2004

We previously found that a small dose (2 μg per mouse) of staphylococcal enterotoxin B (SEB) induced early emerging unresponsiveness in intrahepatic-lymphocyte populations (IHLs). The purpose of this study was to reveal the inducing role of accessory cells involved in IHLs in this phenomenon. IHLs prepared at 3 to 24 hours after SEB injection failed to proliferate in response not only to SEB but also to SEA, representing ligand-nonspecific unresponsiveness, whereas spleen cells (SPCs) and mesenteric lymph-node cells showed transient proliferation. Unresponsiveness in IHLs was related to a deficit of their accessory cell function as measured by coculture of irradiated IHLs and antigen-specific, type 1 T-helper (Th1) clone cells. High levels of nitrite were detected in the culture supernatant. Supplement of N
G-monomethyl-l -arginine lowered nitrite levels and concurrently restored the proliferative response of Th1 cells, indicating the involvement of nitric oxide in suppression. Adherent cells prepared from IHLs well reproduced these results. As shown by flow cytometry, Mac-1high Ia+ cells, which mainly included F4/80+ cells (macrophages) and a minor population of CD11c+ cells (dendritic cells), increased in proportion in IHLs but not in SPCs at 6 to 24 hours. Depletion of Mac-1high cells from IHLs with antibody-coated magnetic beads recovered the proliferative response. Depleted Mac-1high cells had a monocytoid appearance. In immunostained sections, Kupffer cells came to highly express both Mac-1 and Ia at 12 hours. These results indicate that Mac-1highIa+ adherent cells, largely Kupffer cells activated by SEB, nonspecifically suppress the proliferation of Th1 cells via nitric oxide production before manifestation of ligand-specific unresponsiveness.
Expression of GP73, a resident Golgi membrane protein, in viral and nonviral liver disease
Authors: Kladney Raleigh D. , Cui Xiaoyen , Bulla Gary A. , Brunt Elizabeth M. , Fimmel Claus J.
Published: March 2004

GP73 is a novel type II Golgi membrane protein of unknown function that is expressed in the hepatocytes of patients with adult giant-cell hepatitis (Gene 2000;249:53-65). Its expression pattern in human liver disease and the regulation of its expression in hepatocytes have not been systematically studied. The aims of the present study were to compare GP73 protein levels in viral and nonviral human liver disease and in normal livers, to identify its cellular sources, and to study the regulation of its expression in hepatoma cells in vitro. GP73 protein levels were quantitated in explant livers of patients with well-defined disease etiologies and compared with the levels in normal donor livers. GP73-expressing cells were identified immunohistochemically. GP73 expression in vitro was studied by Western blotting and immunofluorescence microscopy in HepG2 and SK-Hep-1 cells and in the HepG2-derived, hepatitis B virus (HBV)-transfected HepG2215 and HepG2T14.1 cell lines. Whole organ levels of GP73 were low in normal livers. Significant increases were found in liver disease due to viral causes (HBV, HCV) or nonviral causes (alcohol-induced liver disease, autoimmune hepatitis). In normal livers, GP73 was constitutively expressed by biliary epithelial cells but not by hepatocytes. Hepatocyte expression of GP73 was dramatically up-regulated in diseased livers, regardless of the etiology, whereas biliary epithelial cell expression did not change appreciably. GP73 was present at high levels in HepG2215 cells (a cell line that supports active HBV replication), but was absent in HepG2T14.1 cells (an HBV-transfected cell line that does not support HBV replication) and in HBV-free HepG2 cells. In SK-Hep-1 cells, GP73 expression was increased in response to interferon gamma (IFN-γ), and inhibited by tumor necrosis factor α (TNF-α). In conclusion, increased expression of GP73 in hepatocytes appears to be a general feature of advanced liver disease, and may be regulated via distinct pathways that involve hepatotropic viruses or cytokines.
Liver cell proliferation requires methionine adenosyltransferase 2A mRNA up-regulation
Authors: Pañeda Covadonga , Gorospe Itziar , Herrera Blanca , Nakamura Toshikazu , Fabregat Isabel , Varela-Nieto Isabel
Published: March 2004

Regulation of liver cell proliferation is a key event to control organ size during development and liver regeneration. Methionine adenosyltransferase (MAT) 2A is expressed in proliferating liver, whereas MAT1A is the form expressed in adult quiescent hepatocytes. Here we show that, in H35 hepatoma cells, growth factors such as hepatocyte growth factor (HGF) and insulin up-regulated MAT2A expression. HGF actions were time- and dose-response dependent and required transcriptional activity. Mitogen-activated protein (MAP) kinase and phosphatidylinositol 3-phosphate kinase (PI 3-K) pathways were required for both HGF-induced cell proliferation and MAT2A up-regulation. Furthermore, in H35 cells treated with HGF, the inhibition of these pathways was associated with the switch from the expression of fetal liver MAT2A to the adult liver MAT1A isoform. Fetal liver hepatocytes exhibited an identical response pattern. Treatment of H35 hepatoma cells with MAT2A antisense oligonucleotides decreased cell proliferation induced by HGF; this decrease correlated with the decay in MAT2A messenger RNA (mRNA) levels. Finally, growth inhibitors such as transforming growth factor (TGF) β blocked HGF-induced MAT2A up-regulation while increasing MAT1A mRNA levels in H35 cells. In conclusion, our results show that MAT2A expression not only correlates with liver cell proliferation but is required for this process.
Small-duct primary sclerosing cholangitis: A long-term follow-up study
Authors: Angulo Paul , Maor-Kendler Yaakov , Lindor Keith D.
Published: March 2004

Some patients with inflammatory bowel disease (IBD) have chronic cholestasis and hepatic histology compatible with primary sclerosing cholangitis (PSC) but normal findings on cholangiography. These patients with small-duct PSC have remained largely unstudied. Our aim was to determine the prevalence and long-term outcomes of patients with small-duct PSC. Eighteen patients with small-duct PSC (7 female and 11 male patients; mean age, 39.9 ± 15.3 years [range, 13-68 years]) seen over a 4-year period were matched blindly by age and sex to 36 patients with classic PSC and followed up for 32.5 years. Small-duct PSC represented 5.8% of patients (18 of 309) with sclerosing cholangitis. Subsequent endoscopic retrograde cholangiography (ERC) performed in 5 patients with small-duct PSC showed progression to typical PSC in 3 patients at 4, 5.5, and 21 years of follow-up. None of the patients with small-duct PSC but 4 of the patients with classic PSC developed hepatobiliary malignancy. There were 3 deaths (17%) or liver transplantations in patients with small-duct PSC (2 after progressing to classic PSC) and 15 (42%) in the classic PSC group. Survival free of liver transplantation was significantly greater in the small-duct than in the classic PSC group (P = .04). Compared with the general U.S. population, survival in patients with small-duct PSC was similar (P = .4) but significantly lower in patients with classic PSC (P < .001). In conclusion, small-duct PSC may represent an earlier stage of PSC associated with a significantly better long-term prognosis. Some patients, however, progress to classic PSC and/or end-stage liver disease with the consequent necessity of liver transplantation.
Role of MAP kinases and their cross-talk in TGF-β1-induced apoptosis in FaO rat hepatoma cell line
Authors: Park Hyun-Jin , Kim Byung-Chul , Kim Seong-Jin , Choi Kyeong Sook
Published: March 2004

Transforming growth factor (TGF) β1 is a potent inducer of apoptosis in the liver. During TGF-β1-induced apoptosis, 3 mitogen-activated protein (MAP) kinases (extracellular signal-regulated kinase [ERK], c-Jun N-terminal kinase [JNK], and p38 kinase) showed simultaneously sustained activation in FaO rat hepatoma cells. TGF-β1-induced apoptosis was markedly enhanced when ERK activation was selectively inhibited by the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor PD98059. In contrast, both interfering with p38 activity by overexpression of the dominant negative (DN) MKK6 mutant and inhibition of the JNK pathway by overexpression of the DN SEK1 mutant resulted in suppression of mitochondrial cytochrome c release, abrogating TGF-β1-induced apoptosis. In addition, antiapoptotic Bcl-2 blocked mitochondrial cytochrome c release, suppressing TGF-β1-induced activation of JNK and p38. Inhibition of ERK activity enhanced TGF-β1-induced p38 and JNK activation. However, inhibition of the JNK pathway suppressed p38 but induced transient ERK activation. Similarly, interfering with the p38 pathway also attenuated JNK activation but generated transient ERK activation in response to TGF-β1. These results indicate that disrupting one MAP kinase pathway affects the TGF-β1-induced activation of other MAP kinases, suggesting cross-talk among MAP kinase pathways. In conclusion, we propose that the balance and integration of MAP kinase signaling may regulate commitment to TGF-β1-induced apoptosis modulating the release of cytochrome c from mitochondria.
Altered localization and activity of canalicular Mrp2 in estradiol-17β-D-glucuronide-induced cholestasis
Authors: Mottino Aldo D. , Cao Jingsong , Veggi Luis M. , Crocenzi Fernando , Roma Marcelo G. , Vore Mary
Published: March 2004

Estradiol-17β-D-glucuronide (E217G), an endogenous metabolite of estradiol, induces a potent dose-dependent and reversible inhibition of bile flow in the rat. We analyzed the effect of a single dose of E217G (15 μmol/kg, intravenously) to female rats on bile flow and the endocytic retrieval and function of the canalicular multidrug resistance-associated protein 2 (Mrp2) and the effect of pretreatment with dibutyryl-cyclic AMP (DBcAMP; 20 μmol/kg) on these measures. Bile flow was maximally inhibited by 85% within 10 minutes of E217G and returned to 50% and 100% of control levels within 75 and 120 minutes, respectively. Western analysis of total homogenates and mixed plasma and intracellular membranes suggested partial internalization of Mrp2 during the acute phase of cholestasis at 20 minutes and during the period of recovery from cholestasis at 75 minutes, which returned to control levels by 180 minutes after E217G. Confocal analysis confirmed Western studies and demonstrated endocytic retrieval of Mrp2 from the canalicular membrane into pericanalicular and intracellular domains. The biliary concentration and excretion of the model Mrp2 substrate, dinitrophenyl-S-glutathione (DNP-SG), was impaired in parallel with the extent of Mrp2 retrieval. Pretreatment with DBcAMP partially protected against maximal E217G cholestasis and the endocytic retrieval and decreased function of Mrp2 at 20 minutes and significantly accelerated the exocytic insertion of Mrp2 into the canalicular membrane and the recovery of bile flow and biliary excretion of DNP-SG. In conclusion, these data indicate that E217G induces endocytic internalization of Mrp2, which occurs in parallel with decreased bile flow and Mrp2 transport activity.
Maturation of fetal hepatocytes in vitro by extracellular matrices and oncostatin M: Induction of tryptophan oxygenase
Authors: Kamiya Akihide , Kojima Nobuhiko , Kinoshita Taisei , Sakai Yasuyuki , Miyaijma Atsushi
Published: March 2004

Previously, we described that embryonic day 14.5 (E14.5) mouse fetal hepatocytes differentiate to express tyrosine amino transferase (TAT) and glucose-6-phosphatase, which are expressed in the perinatal liver, in response to oncostatin M (OSM) or in high-cell-density culture. However, under such conditions, fetal hepatic cells failed to express genes for adult liver-specific enzymes, such as tryptophan oxygenase (TO). Although phenobarbital (PB) and dimethylsulfoxide (DMSO) have been known to maintain the functions of adult hepatocytes in vitro, they failed to induce TO expression in fetal hepatic cells. Thus far, no system has been developed that reproduces terminal differentiation of fetal hepatocytes in vitro. Here, we describe that extracellular matrices derived from Engelbreth-Holm-Swarm sarcoma (EHS) in combination with OSM or high-cell-density culture induced expression of TO as well as cytochrome P450 genes that are involved in detoxification. However, EHS alone was insufficient to induce expression of TO, although it induced TAT expression in fetal hepatocytes. In addition, high-density culture further augmented differentiation. In conclusion, the combination of signals by cytokines, cell-cell contact, and cell-matrix interaction is required for induction of adult liver functions in fetal hepatocytes in vitro. This primary culture system will be useful for studying the mechanism of liver development.
Partial external biliary diversion for intractable pruritus and xanthomas in Alagille syndrome
Authors: Emerick Karan M. , Whitington Peter F.
Published: March 2004

Alagille syndrome (AGS) causes intractable pruritus and disfiguring xanthomas because of retained bile acids and cholesterol. This study was performed to determine whether partial external biliary diversion (PEBD) is effective for relief of pruritus and xanthomas in AGS patients who fail conventional medical therapy. Between the years 1985 and 2001, 9 AGS patients underwent PEBD. Complete follow-up data were available for all patients. The average age at PEBD was 4.8 (range 1.4-10) years. The average duration of follow-up was 7.5 (range 0.5-16.0) years. All 9 patients had severe, mutilating pruritus (grade 4) prior to diversion. At 1 year post-PEBD, the average pruritus score was 1.1; 8 patients had only mild scratching when undistracted. Three patients with extensive xanthomas prior to PEBD had complete resolution within 1 year. Mean serum bile salt levels (n = 5) decreased from 136.5 to 37.1 μmol/L and mean cholesterol (n = 7) from 724 to 367 mg/dL 1 year after PEBD. A single 21-year-old patient with PEBD for 14 years experienced an increase in pruritus from grade 1 to grade 4 within 2 months of elective reversal of PEBD. In conclusion, PEBD is effective for treating severe pruritus and hypercholesterolemia in AGS patients without cirrhosis who did not respond to medical therapy. PEBD should be considered as a therapeutic option for these patients before referral for liver transplantation because of morbid complications
Cardiovascular effects of canrenone in patients with preascitic cirrhosis
Authors: La Villa Giorgio , Barletta Giuseppe , Romanelli Roberto Giulio , Laffi Giacomo , Del Bene Riccarda , Vizzutti Francesco , Pantaleo Pietro , Mazzocchi Valeria , Gentilini Paolo
Published: March 2004

In patients with cirrhosis and portal hypertension, standing induces a reduction in cardiac index (CI) and an increase in systemic vascular resistance index. Our previous studies indicate that this abnormal hemodynamic response to standing is due to an altered myocardial function, because cirrhotic patients are unable to compensate for the reduced preload with an increase in left ventricular (LV) ejection fraction (EF) and stroke volume. To evaluate whether the cardiac dysfunction in cirrhosis is influenced by canrenone, an aldosterone antagonist, 8 patients with preascitic, nonalcoholic cirrhosis, and portal hypertension underwent echocardiographic assessment of LV function and systemic hemodynamics and determinations of plasma volume, urinary sodium excretion, and plasma renin activity (PRA), aldosterone (PAC), and norepinephrine (PNE) when on a 150-mmol/d-sodium diet (baseline), after 1 month on canrenone (100 mg/d) plus a 40-mmol/d-sodium diet and after 1 month on canrenone plus a 150-mmol/d-sodium diet. Echocardiographic evaluation was performed with the patient in the supine position and during active standing. At baseline, patients had high plasma volume and normal renal function, PRA, PAC, and PNE. CI, LVEF, and stroke volume index were also normal. Standing caused a significant reduction in CI and LVEF. After canrenone and either sodium diet, CI significantly decreased, and PRA and PNE increased in the supine position. On standing, LVEF and CI did not decrease further. Plasma volume significantly decreased only after low-sodium diet plus canrenone. In conclusion, canrenone normalizes the cardiac response to the postural challenge in patients with preascitic cirrhosis.
Defective regulation of cholangiocyte Cl−/HCO− 3 and Na+/H+ exchanger activities in primary biliary cirrhosis
Authors: Melero Saida , Spirlì Carlo , Zsembery Ákos , Medina Juan F. , Joplin Ruth E. , Duner Elena , Zuin Massimo , Neuberger James M. , Prieto Jesús , Strazzabosco Mario
Published: March 2004

Primary biliary cirrhosis (PBC) is a disorder of unknown origin with autoimmune features. Recently, impaired biliary secretion of bicarbonate has been shown in patients with PBC. Here we have investigated whether bile duct epithelial cells isolated from PBC patients exhibit defects in transepithelial bicarbonate transport by analyzing the activities of 2 ion exchangers, Cl−/HCO−
3 anion exchanger 2 (AE2) and Na+/H+ exchanger (NHE) in isolated cholangiocytes. AE2 and NHE activities were studied in basal conditions and after stimulation with cyclic adenosine monophosphate (cAMP) and extracellular adenosine triphosphate (ATP), respectively. Cholangiocytes were grown from needle liver biopsies from 12 PBC patients, 8 normal controls, and 9 patients with other liver diseases. Also, intrahepatic cholangiocytes were cultured after immunomagnetic isolation from normal liver tissue (n = 6), and from recipients undergoing liver transplantation for end-stage PBC (n = 9) and other forms of liver disease (n = 8). In needle-biopsy cholangiocytes, basal AE2 activity was significantly decreased in PBC as compared with normal livers and disease controls. In addition, we observed that though cAMP increased AE2 activity in cholangiocytes from both normal and non-PBC livers, this effect was absent in PBC cholangiocytes. Similarly, though in cholangiocytes from normal and disease control livers extracellular ATP induced a marked enhancement of NHE activity, cholangiocytes from PBC patients failed to respond to purinergic stimulation. In conclusion, our findings provide functional evidence that PBC cholangiocytes exhibit a widespread failure in the regulation of carriers involved in transepithelial H+/HCO−
3 transport, thus, providing a molecular basis for the impaired bicarbonate secretion in this cholestatic syndrome.
Cyclosporine a protects against arachidonic acid toxicity in rat hepatocytes: Role of CYP2E1 and mitochondria
Authors: Wu Defeng , Cederbaum Arthur I.
Published: March 2004

Diets high in polyunsaturated fatty acids (PUFA) are important for the development of alcoholic liver injury. The goal of this report was to characterize toxicity by arachidonic acid (AA), its enhancement by salicylate, and the role of mitochondrial injury in the pathway leading to toxicity in hepatocytes from pyrazole-treated rats. AA caused toxicity that was increased by sodium salicylate. This synergistic toxicity was reduced by diallyl sulfide (DAS), an inhibitor of CYP2E1; Trolox ([±] 6-hydroxy, 2, 5, 7, 8-tetramethylchroman-2-carboxylic acid), an inhibitor of lipid peroxidation; Z-Val-Ala-Asp(OMe)-fluoromethylketone (ZVAD-FMK), a pan caspase inhibitor; and by cyclosporine A (CsA), an inhibitor of the mitochondrial permeability transition. Mitochondrial membrane potential also was reduced, and this was prevented by cyclosporine, diallyl sulfide, and Trolox. There was release of mitochondrial cytochrome c into the cytosol and activation of caspase 3, which were prevented by cyclosporine, diallylsulfide, and Trolox. Toxicity was prevented by expression of catalase either in the cytosolic or the mitochondrial compartment. Levels of CYP2E1 rapidly declined, and this was partially prevented by salicylate. These results are consistent with a model in which CYP2E1-dependent production of reactive oxygen species enhances lipid peroxidation when AA is added to hepatocytes. This results in damage to the mitochondria, with initiation of a membrane permeability transition and a decline in membrane potential, followed by release of cytochrome c, caspase 3 activation, and cellular toxicity. In conclusion, damage to mitochondria appears to play an important role in the CYP2E1 plus AA toxicity.
Cell cycle deregulation in liver lesions of rats with and without genetic predisposition to hepatocarcinogenesis
Authors: Pascale Rosa M. , Simile Maria M. , De Miglio Maria R. , Muroni Maria R. , Calvisi Diego F. , Asara Giuseppina , Casabona Daniela , Frau Maddalena , Seddaiu Maria A. , Feo Francesco
Published: March 2004

Preneoplastic and neoplastic hepatocytes undergo c-Myc up-regulation and overgrowth in rats genetically susceptible to hepatocarcinogenesis, but not in resistant rats. Because c-Myc regulates the pRb-E2F pathway, we evaluated cell cycle gene expression in neoplastic nodules and hepatocellular carcinomas (HCCs), induced by initiation/selection (IS) protocols 40 and 70 weeks after diethylnitrosamine treatment, in susceptible Fisher 344 (F344) rats, and resistant Wistar and Brown Norway (BN) rats. No interstrain differences in gene expression occurred in normal liver. Overexpression of c-myc, Cyclins D1, E, and A, and E2F1 genes, at messenger RNA (mRNA) and protein levels, rise in Cyclin D1-CDK4, Cyclin E-CDK2, and E2F1-DP1 complexes, and pRb hyperphosphorylation occurred in nodules and HCCs of F344 rats. Expression of Cdk4, Cdk2, p16INK4A, and p27KIP1
did not change. In nodules and/or HCCs of Wistar and BN rats, low or no increases in c-myc, Cyclins D1, E, and A, and E2F1 expression, and Cyclin-CDKs complex formation were associated with p16INK4A
overexpression and pRb hypophosphorylation. In conclusion, these results suggest deregulation of G1 and S phases in liver lesions of susceptible rats and block of G1-S transition in lesions of resistant strains, which explains their low progression capacity.
TFDP1, CUL4A, and CDC16 identified as targets for amplification at 13q34 in hepatocellular carcinomas
Authors: Yasui Kohichiroh , Arii Shigeki , Zhao Chen , Imoto Issei , Ueda Masakazu , Nagai Hisaki , Emi Mitsuru , Inazawa Johji
Published: March 2004

We carried out molecular cytogenetic characterization of 11 cell lines derived from hepatocellular carcinomas (HCCs) and 51 primary HCCs. Comparative genomic hybridization (CGH) revealed frequent amplification at 13q34, where we had detected amplification in several other types of tumor, including esophageal squamous cell carcinomas (ESC). Previously, we suggested possible involvement of TFDP1, encoding a transcription factor DP-1, in the 13q34 amplification observed in a primary ESC. Therefore, we investigated amplifications and expression levels of 5 genes mapped on the amplified region, including TFDP1, for exploring amplification targets at 13q34 in HCCs. 3 of those genes, TFDP1, CUL4A (cullin 4A), and CDC16 (cell division cycle 16), showed distinct amplification and consequent over-expression in some cell lines. Moreover, each was amplified in 3 or 4 of the 51 primary HCCs, and all 3 were amplified in 2 tumors, in which their expression patterns correlated with amplification patterns. To elucidate the functional role of TFDP1 in HCC, we examined expression levels of genes downstream of TFDP1 with real-time quantitative polymerase chain reaction (PCR). Expression of cyclin E gene (CCNE1) correlated closely with that of TFDP1 in not only cell lines, but also primary tumors. Treatment of HCC cells with the antisense oligonucleotide targeting TFDP1 resulted in down-regulation of CCNE1, suggesting that TFDP1 overexpression led to up-regulation of CCNE1 that encoded a positive regulator for cell cycle G1/S transition. In conclusion, our findings suggest that TFDP1, CUL4A, and CDC16 are probable targets of an amplification mechanism and therefore may be involved, together or separately, in development and/or progression of some HCCs.
Pretransplantation tumor necrosis factor-α production predicts acute rejection after liver transplantation
Authors: Bathgate Andrew J. , Lee Patricia , Hayes Peter C. , Simpson Kenneth J.
Published: March 2004

Immunosuppressive therapy has many adverse effects in both the short and longer term. Tailoring immunosuppression might be possible if pretransplantation parameters predicted rejection. We investigated production of the proinflammatory cytokine, tumor necrosis factor-α (TNF-α), and the anti-inflammatory cytokine, interleukin-10 (IL-10), pretransplantation to determine whether there is a relation with acute rejection. Peripheral-blood mononuclear cells were obtained from patients with chronic liver disease on the waiting list for orthotopic liver transplantation and healthy controls. Cells (0.5 × 106) were stimulated with 200 ng of lipopolysaccharide. Preincubation for 30 minutes with tacrolimus, cyclosporine, and dexamethasone at concentrations of 10 and 100 ng was also performed. TNF-α and IL-10 levels were measured by enzyme-linked immunosorbent assay. Acute rejection was defined on clinical and histological grounds. Pretransplantation in vitro production of TNF-α significantly (P < .05) increased in the group of patients with acute rejection (n = 9) compared with those who did not develop rejection (n = 12). Preincubation with dexamethasone significantly (P < .001) reduced TNF-α and IL-10 production in both patients and controls (n = 8). IL-10 production pretransplantation was not different in those who developed acute rejection (n = 9) compared with those who did not (n = 9). Preincubation with tacrolimus augmented (P < .05) the production of IL-10 in patients (n = 18), but not controls (n = 6). Pretransplantation TNF-α production is increased in patients who go on to develop acute rejection posttransplantion.
Predicting clinical and economic outcomes after liver transplantation using the mayo primary sclerosing cholangitis model and child-pugh score
Authors: Talwalkar Jayant A. , Seaberg Eric , Kim Ray W. , Wiesner Russell H.
Published: March 2004

Issues in the selection and timing of liver transplantation for primary sclerosing cholangitis (PSC) remain controversial. Although the Child-Pugh classification (CP) score and Mayo PSC model have similar abilities to estimate pretransplantation survival, a comparison of these 2 scores in predicting survival after liver transplantation has not been conducted. The aim of this study is to compare the Mayo PSC model and CP score in predicting patient survival and related economic outcomes after liver transplantation. Data from 128 patients with PSC, identified from the NIDDK database, were used to calculate patient-specific Mayo PSC and CP scores before transplantation. Levels reflecting a poor outcome were defined a priori. Receiver operating characteristic (ROC) curves and regression methods (Cox proportional hazards and linear regression models) were used to assess the relationship between these 2 scores and 5 post liver transplantation outcome measures. CP score was found to be a significantly (P < .05) better predictor of death 4 months or less after liver transplantation than: (a) length of hospital stay >21 days (or death before discharge) and (b) resource utilization >200,000 units (measured by area under the ROC curve). The Cox model identified statistically significant (P < .05) associations between CP score and each outcome after adjusting for the Mayo PSC risk score. Similar results were not observed for the Mayo PSC model when adjusted for CP score. Among patients with PSC undergoing liver transplantation, CP score was a better overall predictor of both survival and economic resource utilization compared with the Mayo PSC model.
Auxiliary heterotopic liver transplantation with portal vein arterialization for fulminant hepatic failure
Authors: Margarit Carlos , Bilbao Itxarone , Charco Ramón , Lázaro José Luis , Hidalgo Ernest , Allende Elena , Murio Enrique
Published: March 2004

Auxiliary liver transplantation for patients with fulminant hepatic failure supports the patient’s failing liver for a period of time until the native liver (NL) has recovered and immunosuppression can be withdrawn. Auxiliary heterotopic liver transplantation (AHLT) with portal vein arterialization (PVA) has several advantages over auxiliary orthotopic liver transplantation: NL resection is not required, and the hepatic hilum is left untouched; thus, the chances of liver regeneration are optimal. The successful application of emergency AHLT with PVA in a young patient who developed toxic fulminant hepatic failure caused by tuberculostatic drugs is described. Two and one-half months after the procedure, the NL had completely regenerated; the graft was removed, and immunosuppression was suspended.
Interpreting the significance of drinking by alcohol-dependent liver transplant patients: Fostering candor is the key to recovery
Authors: Weinrieb Robert M. , Van Horn Deborah H.A. , McLellan Thomas A. , Lucey Michael R.
Published: March 2004

Few studies have examined the value of treating alcohol addiction either before or after liver transplantation. Nevertheless, most liver transplant programs and many insurance companies require 6 months to 1 year of abstinence from alcohol as a condition of eligibility for liver transplantation (the 6-month rule). We believe there are potentially harsh clinical consequences to the implementation of this rule. For example, the natural history of alcohol use disorders often involves brief fallbacks to drinking (“slips”), but when alcoholic liver transplant candidates slip, most are removed from consideration for transplantation or are required to accrue another 6 months of sobriety. Because there is no alternative treatment to liver transplantation for most patients with end-stage liver disease, the 6-month rule could be lethal in some circumstances. In this review, we survey the literature concerning the ability of the 6-month rule to predict drinking by alcoholic patients who undergo liver transplantation and examine its impact on the health consequences of drinking before and after liver transplantation. We believe that fostering candor between the alcoholic patient and the transplant team is the key to recovery from alcoholism. We conclude that it is unethical to force alcoholic liver patients who have resumed alcohol use while waiting for or after transplantation to choose between hiding their drinking to remain suitable candidates for transplantation or risk death by asking for treatment of alcoholism. Consequently, we advocate a flexible approach to clinical decision making for the transplant professional caring for an alcoholic patient who has resumed drinking and provide specific guidelines for patient management.
Impact of liver transplantation on health-related quality of life
Authors: Younossi Zobair M. , McCormick Marilyn , Price Lori Lyn , Boparai Navdeep , Farquhar Lou , Henderson Michael J. , Guyatt Gordon
Published: March 2004

This study is designed to measure the impact of orthotopic liver transplantation (OLT) on patients’ health-related quality of life. Two types of health-related quality-of-life questionnaires were administered at baseline and after OLT: generic (Medical Outcomes Study Short Form 36) and liver specific (Chronic Liver Disease Questionnaire). We also recorded clinical, demographic, and laboratory data. Pre-OLT scores of liver transplant candidates were compared with those of the general population and patients with congestive heart failure (CHF) and chronic obstructive pulmonary disease (COPD). Thirty-seven liver transplant candidates were evaluated: 25 men and 12 women; age, 50.2 ± 12 years; Child’s class A, 3 patients; class B, 30 patients; class C, 4 patients; and galactose elimination capacity, 277 ± 81. Health-related quality-of-life scores for patients awaiting liver transplants were significantly lower than those for patients with COPD and CHF and those in the general population. Sex and cause of liver disease did not affect the scores. There was a weak but significant inverse correlation between some aspects of health-related quality of life and both age (r = -0.31 to -0.34) and worsening of the Child-Pugh score (r = -0.32 to -0.43). All measured aspects of health-related quality of life significantly improved after OLT, and mental health scores were indistinguishable from the population norms. Similar improvements were evident in physical and disease-specific aspects of health-related quality of life, but some residual dysfunction persisted.
Split-cava technique: Liver splitting for two adult recipients
Authors: Gundlach Matthias , Broering Dieter , Topp Stefan , Sterneck Martina , Rogiers Xavier
Published: March 2004

Split-liver transplantation for 2 adult recipients is a challenging procedure because of the need to split through the midplane of the donor liver. In applied techniques, usually the middle hepatic vein is retained with the left split and the vena cava retained with the right split graft, particularly to avoid serious venous congestion of the right graft after reperfusion. The indispensable division of the caudate lobe veins lead to uncertain viability of liver segment I, and resection might be necessary. To provide optimal venous drainage of both hemiliver grafts, we developed the split-cava technique. This article describes our new technique of liver splitting, which has been successfully used in 2 in situ harvesting procedures.