bluuebunny/crossref_metadata_2025 huggingface dataset:
Hepatic jagged1 expression studies
Authors: Louis Anthonise A. , Van Eyken Peter , Haber Barbara A. , Hicks Carol , Weinmaster Gerry , Taub Rebecca , Rand Elizabeth B.
Published: July 2004

Mutations in Jagged1, a Notch ligand, have been shown to result in Alagille syndrome (AGS), however, the causal link between haploinsufficiency of Jagged1 and intrahepatic ductal paucity is unknown. This survey was performed to determine the expression pattern of Jagged1 in the fetal and postnatal liver. Reverse transcription polymerase chain reaction (RT-PCR) showed Jagged1 expression in all samples studied including rat liver embryonic days 16 to 21, 1-day-old, 1-week-old, and 2-month-old adult rats. RT-PCR detected Jagged1 in total liver RNA extracted from cadaver organ donor samples from reduced human grafts and explanted native livers from a variety of pediatric disorders including AGS, biliary atresia, congenital hepatic fibrosis, sclerosing cholangitis, cystic fibrosis, fulminant hepatic failure, tyrosinemia, and chronic rejection. Immunohistochemistry showed Jagged1 expression in human fetal samples localized to the ductal plate from 14-week gestation onward. Expression in the postnatal liver was seen in biliary epithelium and zone 3 hepatocytes. In conclusion, these studies show that Jagged1 is expressed in the fetal and postnatal liver in health and disease. We show localization of expression by immunohistochemistry to ductal plate epithelium in human fetal samples and to the biliary epithelium and zone 3 hepatocytes in human postnatal samples. Our results show the localization of Jagged1 in fetal liver and demonstration of Jagged1 expression in postnatal rat and human liver specimens. Further studies of Jagged1 and the Notch signaling pathway are expected to elucidate mechanisms of the regulation of biliary epithelial growth and development.
Inhibition of interleukin 6-mediated mitogen-activated protein kinase activation attenuates growth of a cholangiocarcinoma cell line
Authors: Park Joongwon , Tadlock Laura , Gores Gregory J. , Patel Tushar
Published: July 2004

Biliary tract malignancies represent challenges because of the lack of effective therapy and poor prognosis, in part because of the paucity of information regarding the mechanisms regulating their growth. We have recently identified a critical role for the p44/p42 mitogen-activated protein kinase (MAPK) pathway in interleukin 6 (IL-6)-stimulated growth of human cholangiocytes. Although IL-6 is a potential mitogen for cholangiocarcinoma, the role of this cytokine and its intracellular signaling pathways in cholangiocarcinoma growth is unknown. Thus, our aims were to determine the role of IL-6-mediated signaling mechanisms, and in particular the MAPK pathways, in the growth regulation of human cholangiocarcinoma. KMCH-1 cells (malignant cholangiocyte cells) secreted IL-6 constitutively, and increased IL-6 secretion in response to inflammatory cytokines such as tumor necrosis factor α (TNF-α) and IL-1β. Stimulation with IL-6 resulted in proliferation of malignant cholangiocytes. These cells also possessed the IL-6 receptor complex subunits as directly assessed by immunoblot analysis. Furthermore, proliferation was completely inhibited by preincubation with anti-IL-6 neutralizing antibodies, indicating that the proliferative response to IL-6 involved receptor-mediated signaling. Both p38 and p44/p42 MAPKs were constitutively present and active in malignant cholangiocytes, and increased activity of both was observed within 15 minutes of stimulation with IL-6. Selective inhibition of either the p44/p42 MAPK pathway, by PD098059, or of the p38 MAPK pathway, by SB203580, blocked proliferation in response to IL-6. Thus, IL-6 can contribute to the autocrine and/or paracrine growth stimulation of malignant cholangiocytes via activation of either p38 or p44/p42 MAPK signaling pathways.
Transjugular intrahepatic portosystemic shunt in the treatment of refractory bleeding from ruptured gastric varices
Authors: Barange Karl , Péron Jean-Marie , Imani Kamran , Otal Philippe , Payen Jean-Louis , Rousseau Herve , Pascal Jean-Pierre , Joffre Francis , Vinel Jean-Pierre
Published: July 2004

The optimal management of ruptured gastric varices in patients with cirrhosis has not been codified yet. The present study reports the use of transjugular intrahepatic portosystemic shunt (TIPS) in patients with refractory gastric variceal bleeding. Thirty-two consecutive patients were included. All had been unresponsive to vasoactive agents infusion, sclerotherapy, and/or tamponade and were considered poor surgical candidates. They were followed-up until death, transplantation, or at least 1 year (median: 509 days; range 4 to 2,230). Hemostasis was achieved in 18 out of 20 patients actively bleeding at the time of the procedure. In the whole sample of 32 patients, rebleeding rates were 14%, 26%, and 31%, respectively at 1 month, 6 months, and 1 year. De novo encephalopathy was observed in 5 (16%) patients. Seven patients experienced complications and consequently 4 of these patients died. TIPS primary patency rates were 84%, 74%, and 51%, respectively, at 1 month, 6 months, and 1 year. For the same periods of time, survival rates were 75%, 62%, and 59%. These results suggest that TIPS can be used in cirrhotic patients with refractory gastric variceal bleeding and are effective in achieving hemostasis as well as in preventing rebleeding.
Suppression of lipopolysaccharide-induced nitric oxide synthase expression by platelet-activating factor receptor antagonists in the rat liver and cultured rat kupffer cells
Authors: Mustafa Shamimunisa B. , Flickinger Brent D. , Olson Merle S.
Published: July 2004

Excessive nitric oxide (NO) generated by hepatic cells in response to lipopolysaccharide (LPS) and inflammatory substances (e.g., platelet-activating factor [PAF]) is a key contributor to the pathophysiological outcomes observed in the liver during sepsis. In rats subjected to liver-focused endotoxemia, inducible nitric oxide synthase (iNOS) levels in the intact liver were elevated by 6 hours; cell-specific expression of iNOS messenger RNA (mRNA) was Kupffer cells (KCs), endothelial cells, and hepatocytes. Elevated serum alanine transaminase (ALT) levels at 6 hours confirmed hepatic damage. Pretreatment of endotoxemic rats with PAF receptor antagonists BN 50739 or WEB 2170 reduced serum ALT and iNOS mRNA levels in the intact liver. Pretreatment of cultured KCs with BN 50739 or WEB 2170 inhibited both LPS and PAF-induced iNOS mRNA formation. In addition, LPS-induced iNOS protein levels in KCs pretreated with BN 50739 or WEB 2170 were decreased. Exposure of KCs to either LPS or PAF caused the translocation of the p65 subunit of nuclear factor kappa B (NF-κB) into the nucleus and this process was attenuated by BN 50739 and WEB 2170. There was concomitant inhibition of LPS-dependent degradation of the inhibitory protein IκBα and increase in intracellular Ca2+ in KC treated with BN 50739 or WEB 2170. Also, in KCs, LPS was able to induce iNOS mRNA expression independent of CD14. This response was inhibited by pretreatment of KCs with either BN 50739 or WEB 2170. Our findings indicate that PAF receptor antagonists convey protection against hepatocellular injury accompanied by a decrease in nitric oxide (NO) formation in the livers of endotoxemic rats.
Phospholipid metabolism of hypothermically stored rat hepatocytes
Authors: Kim Jae-Sung , Southard James H.
Published: July 2004

Isolated rat hepatocytes were suspended and stored in either Liebovitz-15 medium (37°C or 4°C) or University of Wisconsin (UW) solution (4°C) containing [3H] arachidonic acid (AA). At varying times, membrane phospholipids were separated by thin layer chromatography. AA labeled phospholipids similarly at both 4°C and 37°C. Analysis of the ratios of [3H] AA and [14C] glycerol incorporated into phosphatidic acid or other phospholipids in dual-labeled cells indicated that the deacylation/reacylation cycle was the major route of AA incorporation at hypothermia. This was supported by showing that blocking phospholipase A2 (PLA2) activity by trifluoperazine suppressed AA incorporation into phospholipids. PLA2 activity, measured by determining the release of AA, was slow during 48-hour cold storage, but increased significantly when ATP was depleted by inhibition of mitochondria and glycolysis. In the whole rat liver, there was no significant loss of phospholipids during 48-hour storage (total phospholipids [μmol phosphorus/L/mg] : 0.197 ± .001 at 0 hours) unless energy blockers were used (0.155 ± .005 at 48 hours) or glycogen depleted by fasting the rat (0.167 ± .001 at 48 hours). This study shows that a net PLA2 stimulated hydrolysis of phospholipids is seen only when ATP is depleted and its generation from anaerobic glycolysis inhibited. Thus, PLA2 hydrolysis of phospholipids is not a significant cause of liver cell injury during cold storage when livers are obtained in optimal condition. However, conditions affecting the generation of ATP during cold storage could alter PLA2 leading to membrane damage.
Kupffer cell-independent acute hepatocellular oxidative stress and decreased bile formation in post-cold-ischemic rat liver
Authors: Kumamoto Yusuke , Suematsu Makoto , Shimazu Motohide , Kato Yutaro , Sano Tsuyoshi , Makino Nobuya , Hirano Ken-ichiro , Naito Makoto , Wakabayashi Go , Ishimura Yuzuru , Kitajima Masaki
Published: July 2004

The purpose of this study was to examine distribution and time history of oxidative stress during the hyperacute period of reperfusion in the liver grafts undergoing cold ischemia and to investigate roles of Kupffer cells as a potential oxidant source. Rat livers were harvested at 4°C in University of Wisconsin solution and followed by reperfusion with Krebs-Henseleit buffer under monitoring bile excretion. To investigate oxidative changes, laser-confocal microfluorography was performed in reperfused livers preloaded with dichlorodihydrofluorescein diacetate succinimidyl ester, a fluorescence precursor sensing intracellular hydroperoxide generation. Livers undergoing the 16-hour cold storage displayed an impaired recovery of bile acid-dependent bile output concurrent with a marked increase in hydroperoxide generation in hepatocytes, which occurred as early as 5 minutes after the onset of reperfusion, whereas the status of lobular perfusion was well maintained. Pretreatment with liposome-encapsulated dichloromethylene diphosphonate, a Kupffer cell-depleting reagent, did neither alter the reperfusion-induced periportal oxidative changes nor improve the recovery of bile output in the graft. On the other hand, EPCK, a hepatotropic antioxidant composed of vitamin E phosphate ester bound to vitamin C, not only diminished the oxidative changes but also improved the reduction of bile acid-dependent bile output. Furthermore, the reagent was capable of inhibiting H2 O2-induced oxidative stress in cultured hepatocytes. These results suggest that hepatocytes constitute a major site of the oxidative insult triggered through Kupffer cell-independent mechanisms and serve as an important cellular component to be protected by antioxidant therapeutics.
Ursodeoxycholic acid as adjunctive therapy for problematic type 1 autoimmune hepatitis: A randomized placebo-controlled treatment trial
Authors: Czaja Albert J. , Carpenter Herschel A. , Lindor Keith D.
Published: July 2004

To evaluate the efficacy of ursodeoxycholic acid as adjunctive therapy in type 1 autoimmune hepatitis, 37 patients who had experienced treatment failure, repeated relapse, or incomplete response were randomized to ursodeoxycholic acid (13-15 mg/kg daily) or placebo for 6 months in addition to their usual corticosteroid schedule. Serum aspartate transaminase (70% vs.31%,P= .04) and alkaline phosphatase (47% vs. 7%,P= .02) levels improved more commonly in the 21 patients randomized to ursodeoxycholic acid. Mean serum levels, however, were similar before and after the treatment period. The frequency of dose reduction or corticosteroid withdrawal was comparable in both groups (29% versus 31%,P>.9), and clinical improvement (48% vs. 44%,P>.9)orits absence (52% vs. 56%,P> .9) occurred as commonly in patients receiving ursodeoxycholic acid or placebo.The modifed histological activity score (3.5 ± 0.8 vs. 3.5 ± 0.9)and the modified fibrosis score (2.4 ± 0.4 vs. 2.4 ± 0.4)were similar before and after treatment with ursodeoxycholic acid and no different than after placebo therapy.We conclude that ursodeoxycholic acid can improve certain laboratory tests in problematic patients with type 1 autoimmune hepatitis when administered adjunctively for 6 months. Short-term therapy, however, does not facilitate reduction in the dose of corticosteroids or its withdrawal, affect clinical outcome, or reduce histological activity.
Regional cerebral blood flow during mechanical hyperventilation in patients with fulminant hepatic failure
Authors: Strauss Gitte Irene , Høgh Peter , Møller Kirsten , Knudsen Gitte Moos , Hansen Bent Adel , Larsen Fin Stolze
Published: July 2004

Hyperventilation is frequently used to prevent or postpone the development of cerebral edema and intracranial hypertension in patients with fulminant hepatic failure (FHF). The influence of such therapy on regional cerebral blood flow (rCBF) remains, however, unknown. In this study the CBF-distribution pattern was determined within the first 12 hours after development of hepatic encephalopathy (HE) stage 4 before and during hyperventilation. Ten consecutive patients (median age 48 [range 33-57] years) with FHF and 9 healthy controls (median age 54 [24-58] years) had rCBF determined by single photon emission computed tomography (SPECT) using intravenous injection of 133Xenon. For determination of high resolution CBF pattern, the patients were also studied with 99mTc-hexa-methylpropyleneamine oxime (HMPAO) in the hyperventilation condition. There was no significant difference in the rCBF distribution pattern during normoventilation as compared with hyperventilation. The anterior to posterior (AP) ratio was significantly lower in patients as compared with healthy controls. After hepatic recovery and disappearance of HE, 3 patients had restored normal rCBF distribution pattern as compared with healthy controls. We conclude that in sedated patients with FHF, a relatively lower rCBF is found in the frontal regions and in the basal ganglia as compared with posterior regions. This rCBF-distribution pattern was not aggravated during hyperventilation. It is speculated that this change of rCBF in patients with FHF may render the frontal brain regions more susceptible to hypoxia. The relative frontal rCBF decrease was shown to be reversible with hepatic recovery and alleviation of HE.
Evolution of hepatitis C virus quasispecies in patients with severe cholestatic hepatitis after liver transplantation
Authors: Pessoa Mario G. , Bzowej Natalie , Berenguer Marina , Phung Yume , Kim Michael , Ferrell Linda , Hassoba Howayda , Wright Teresa L.
Published: July 2004

Evolution of hepatitis C quasispecies may be one mechanism by which fibrosing cholestatic hepatitis develops after liver transplantation. In this study, we compared changes in quasispecies complexity and/or divergence in (1) hepatitis C-infected immunosuppressed transplant recipients and in immunocompetent controls; (2) transplant recipients with mild recurrence, and in those with the most severe form of posttransplantation recurrence. Quasispecies were measured in 12 hepatitis C-infected patients pretransplantation and posttransplantation (6 with mild and 6 with severe recurrence), and in 5 immunocompetent patients with similar follow-up, and characterized by heteroduplex mobility and sequence analysis of the hypervariable region. Although the number of variants (complexity) did not change with time in either group, there was a qualitative change in the variants with time (divergence) in immunocompromised, but not in immunocompetent patients. These changes were most marked with severe recurrence, and preceded the development of severe disease. Phylogenetic analysis confirmed that most posttransplantation variants were unrelated to those detected pretransplantation. These observations suggest that in the absence of immune suppression, there is minor evolution of quasispecies. With immune suppression, divergence of quasispecies is enhanced, resulting in selection/emergence of many new variants, particularly in those with fibrosing cholestatic hepatitis. Thus, quasispecies may influence disease progression in immune suppressed populations.
Role for tumor necrosis factor α receptor 1 and interleukin-1 receptor in the suppression of mouse hepatocyte apoptosis by the peroxisome proliferator nafenopin
Authors: West Douglas A. , James Neil H. , Cosulich Sabina C. , Holden Peter R. , Brindle Richard , Rolfe Mark , Roberts Ruth A.
Published: July 2004

Peroxisome proliferators (PPs) cause rodent liver enlargement and tumors. In vitro, PPs induce rat and mouse hepatocyte DNA synthesis and suppress apoptosis, a response mimicked by exogenous tumor necrosis factor α (TNFα). Here, we determine the role of TNF receptor 1 (TNFR1), TNF receptor 2 (TNFR2), and nuclear factor κ beta (NFκB) in the response of mouse hepatocytes to the PP, nafenopin. Nafenopin (50 μmol/L) induced DNA synthesis as measured by bromodeoxyuridine (BrdU) incorporation, suppressed cell death as measured by Hoechst 33258 staining, induced peroxisomal β-oxidation as measured by cyanide insensitive palmitoyl CoA oxidation (PCO) and caused activation of nuclear factor kappa β (NFκB) as determined by electrophoretic mobility gel shift assay (EMSA). The induction of DNA synthesis and the suppression of apoptosis in response to nafenopin was abrogated completely by blocking antibodies to TNFR1 but not to TNFR2. In contrast, the induction of peroxisomal β-oxidation by nafenopin was not blocked by the anti-TNFR1 antibody. Next, we evaluated the response of hepatocytes to interleukin-1 (IL-1), another proinflammatory cytokine. IL-1α (2.5 ng/mL) and, to a lesser extent, IL-1β (5 ng/mL), shared the ability of TNFα to induce DNA synthesis and suppress apoptosis. In addition, anti-IL-1 receptor, type 1/p80 (IL-1R) antibodies were able to abrogate the response to nafenopin. IL-1α was still able to perturb hepatocyte growth in the presence of the anti-TNFR1 antibody suggesting that IL-1α acts independently rather than by elaborating TNFα. In summary, these data provide additional evidence for a role for hepatic cytokines in the perturbation of hepatocyte growth by PPs such as nafenopin.
The canals of hering and hepatic stem cells in humans
Authors: Theise Neil D. , Saxena Romil , Portmann Bernard C. , Thung Swan N. , Yee Herman , Chiriboga Luis , Kumar Ashok , Crawford James M.
Published: July 2004

Small, extraportal, hepatic parenchymal cells, positive for biliary-type cytokeratins, may represent hepatic stem cells, canals of Hering (CoH), and/or ductal plate remnants. We evaluated these cells 3 dimensionally in normal human liver and massive necrosis. Tissues from normal human livers and from 1 liver with acetaminophen-induced massive necrosis were serially sectioned, immunostained for cytokeratin 19 (CK19), and sequentially photographed. Images were examined to determine 3-dimensional relationships among CK19-positive cells. Immunostains for other hepatocyte and progenitor cell markers were examined. In normal livers, intraparenchymal CK19-positive cells lined up as linear arrays in sequential levels. One hundred of 106 (94.3%) defined, complete arrays within levels examined, most having 1 terminus at a bile duct, the other in the lobule, beyond the limiting plate. In massive necrosis, there were 767 individual CK19-positive cells or clusters around a single portal tract, 747 (97.4%) of which were spatially related forming arborizing networks connected to the interlobular bile duct by single tributaries. C-kit was positive in normal CoH. CK19 co-expressed with HepPar1, c-kit, and α-fetoprotein (AFP) in parenchymal cells in massive necrosis. Small, extraportal, biliary-type parenchymal cells represent cross-sections of the CoH that radiate from the portal tract, usually extending past the limiting plate into the proximate third of the hepatic lobule. The 3-dimensional structure of ductular reactions in massive necrosis suggests that these reactions are proliferations of the cells lining the CoH. Therefore, the CoH consist of, or harbor, facultative hepatic stem cells in humans.
Interleukin-11 reduces T-cell-dependent experimental liver injury in mice
Authors: Bozza Mary , Bliss Judith L. , Maylor Richard , Erickson Jamie , Donnelly Lori , Bouchard Page , Dorner Andrew J. , Trepicchio William L.
Published: July 2004

Recombinant human interleukin-11 (rhIL-11) is a multifunctional cytokine that can reduce inflammation through the downregulation of multiple pro-inflammatory mediators from activated macrophages. rhIL-11 also inhibits production of several immunostimulatory cytokines such as IL-12 and interferon γ (IFN-γ) and has shown biological activity in multiple animal models of inflammatory disease consistent with immunomodulatory effects on macrophages and T cells. To further elucidate the anti-inflammatory activity of rhIL-11 in vivo, the effect of rhIL-11 in a model of Concanavalin A (Con-A)-induced T-cell-mediated hepatotoxicity was examined. Administration of a single dose of rhIL-11 before Con-A administration reduced centrilobular liver necrosis and enhanced survival. A dose-dependent reduction in serum levels of liver enzymes, tumor necrosis factor α (TNF-α), and IFN-γ corresponded with this amelioration of liver damage. No significant change in infiltrating lymphocyte populations in the liver was observed following rhIL-11 treatment. Taken together, these results indicate that rhIL-11 ameliorates T-cell-mediated hepatic injury and suggests its therapeutic potential to treat inflammatory liver disease.
The human multidrug resistance protein 2 gene: Functional characterization of the 5′-flanking region and expression in hepatic cells
Authors: Tanaka Toshiya , Uchiumi Takeshi , Hinoshita Eiji , Inokuchi Akihiko , Toh Satoshi , Wada Morimasa , Takano Hiroshi , Kohno Kimitoshi , Kuwano Michihiko
Published: July 2004

The human multidrug resistance protein 2 (MRP2), also termed as the canalicular multispecific organic anion transporter (cMOAT), is a member of the adenosine triphosphate-binding cassette transporter superfamily. In the liver, MRP2 mediates the multispecific efflux of various types of organic anions, including glucuronate, sulfate, and glutathione conjugates, across the canalicular hepatocyte membrane to the bile. To investigate how the MRP2 gene is expressed in liver cells, the 5′-flanking region of the human MRP2 gene was isolated from a human placental genomic library. Sequence analysis of the MRP2 promoter showed a number of consensus binding sites for both ubiquitous and liver-enriched transcription factors. Transfection of human hepatic HepG2 cells with a series of 5′-deleted promoter luciferase constructs identified a putative silencer element localized in the −1,659/−491 region and a liver-specific positive regulatory element localized in the −491/−258 region. This latter region contained the liver-abundant transcription factor CCAAT-enhancer binding protein β (C/EBPβ). The transcriptional activity of the promoter construct containing a mutation in the C/EBPβ binding site was significantly decreased in HepG2 cells. This study suggests that C/EBPβ (−356 to −343) may regulate the liver expression of the MRP2 gene.
Cholangiocarcinomas express fas ligand and disable the fas receptor
Authors: Que Florencia G. , Phan Vy A. , Phan Van H. , Celli Adrianne , Batts Kenneth , LaRusso Nicholas F. , Gores Gregory J.
Published: July 2004

Cholangiocarcinoma is a highly-malignant adenocarcinoma originating from cholangiocytes. Current concepts support escape from immune surveillance using aberrant expression of Fas ligand (FasL) and dysregulation of receptor (FasR) signaling as a potential mechanism for tumor progression. Our aims were to determine if altered expression of FasR and FasL or changes in expression of FLICE inhibitor (I-FLICE) allow cholangiocarcinoma cells to escape immune surveillance. Human cholangiocarcinoma cell lines were evaluated for the functional expression of FasR and FasL by (1) quantitating apoptosis after incubation of cells with agonistic antibodies and (2) an in vitro cell death assay involving coculture of cholangiocarcinoma cells with Fas-sensitive thymocytes. I-FLICE antisense treatment was performed by stable transfection with complementary DNA (cDNA) for I-FLICE in the reverse orientation. We found that normal cholangiocytes in vivo express FasL. Human cholangiocarcinoma cell lines express both FasL and FasR and I-FLICE. FasL expressed by cholangiocarcinomas in vitro induced lymphocyte cell death (70% after 24 hours). Despite the expression of FasR, exposure of the cells to agonistic antibodies (500 ng/mL) induced only minimal apoptosis in the Jurkat cells. Antisense treatment of cholangiocarcinomas in vitro with I-FLICE reduced protein expression of I-FLICE by 90% to 95% and increased Fas-mediated apoptosis 2-fold. We concluded that cholangiocarcinomas escape immune surveillance either by disabling FasR signaling through the expression of I-FLICE and/or increased FasL expression to induce apoptosis of invading T cells. Reduction of I-FLICE expression in cholangiocarcinoma cells restored Fas-mediated apoptosis. Therapeutic maneuvers to inhibit expression of I-FLICE may aid in the treatment of cholangiocarcinoma.
Phase I and Phase II drug-metabolizing enzymes are expressed and heterogeneously distributed in the biliary epithelium
Authors: Lakehal Fatima , Wendum Dominique , Barbu Véronique , Becquemont Laurent , Poupon Raoul , Balladur Pierre , Hannoun Laurent , Ballet François , Beaune Philippe H. , Housset Chantal
Published: July 2004

Tissue expression of drug-metabolizing enzymes influences susceptibility to drugs and carcinogens. Because the biliary epithelium, exposed to bile-borne chemicals, may give rise to drug-induced cholangiopathies and to cholangiocarcinomas, we determined the pattern of expression of drug-metabolizing enzymes in this epithelium. We first demonstrated by blot analyses that biliary epithelial cells (BEC) isolated from human gallbladders display cytochrome P450 (CYP) 1A, 2E1, and 3A, microsomal epoxide hydrolase (mEH), α, μ, and π glutathione S-transferase (GST), transcripts and proteins. We also identified CYP-associated steroid 6β-hydroxylase activity in BEC. CYP and mEH expression was 5- to 20-fold lower in BEC than in autologous hepatocytes, and further differed by a higher ratio of CYP3A5/CYP3A4, and by CYP1A1 predominance over CYP1A2. αGST was highly expressed in both hepatocytes and BEC, while πGST was restricted to BEC. In approximately 50% of individuals, μGST was expressed in hepatocytes and at lower levels in BEC. By using the same antibodies as those used in immunoblots, we could show by immunohistochemistry that CYP2E1, CYP3A, mEH, α, μ, and πGST immunoreactivities are expressed and display a heterogeneous distribution in the epithelium lining the entire biliary tract except for small intrahepatic bile ducts that were devoid of CYP3A and αGST immunoreactivities. In conclusion, BEC contribute to phase II, and although to a lesser extent than hepatocytes, to phase I biotransformation. The distribution of drug-metabolizing enzymes in BEC suggest that they are heterogeneous in their ability to generate and detoxicate reactive metabolites, which may contribute to specific distributions of cholangiopathies.
The transforming growth factor β1-inducible transcription factor, TIEG1, mediates apoptosis through oxidative stress
Authors: Ribeiro Afonso , Bronk Steven F. , Roberts Patricia J. , Urrutia Raul , Gores Gregory J.
Published: July 2004

Transforming growth factor β1(TGF-β1)-inducible transcription factors have recently elicited interest because of their critical role in the regulation of cell proliferation, differentiation, and apoptosis. We have previously reported that the TGF-β1-inducible transcription factor, TIEG1, induces apoptosis in a pancreas-derived cell line. However, the mechanisms underlying the apoptotic effects of this transcription factor remain to be defined. In this study, using the TGF-β1-sensitive Hep 3B cell line, we have defined the mechanistic sequence of events that characterize TIEG1-mediated apoptosis and compared these events with the changes observed during TGF-β1-induced apoptosis. Both TGF-β1- and TIEG1-induced cell death were accompanied by an increase in the generation of reactive oxygen species and a loss of the mitochondrial membrane potential preceding the morphological changes of apoptosis. In contrast, increases in caspase 3-like activity and glutathione (GSH) depletion occurred later in the apoptotic process, concurrent with the morphological features of apoptosis. The antioxidant, trolox, decreased the formation of reactive oxygen species and apoptosis. These results demonstrate that similar to TGF-β1, TIEG1 induces apoptosis by a mechanism involving the formation of reactive oxygen species.
Differential role of ethanol and acetaldehyde in the induction of oxidative stress in HEP G2 cells: Effect on transcription factors AP-1 and NF-κB
Authors: Román Juan , Colell Anna , Blasco Carmen , Caballeria Joan , Parés Albert , Rodés Joan , Fernández-Checa José C.
Published: July 2004

The oxidative metabolism of ethanol by the cytochrome P450 2E1 (CYP2E1) has been recognized to contribute to the ethanol-induced deleterious effects through the induction of oxidative stress. This study compared the effect of ethanol and acetaldehyde in the induction of oxidative stress and activation of transcription factors nuclear factor-κB (NF-κB) and activating protein 1 (AP-1) in HepG2 cells, which do not express CYP2E1, and HepG2 cells transfected with CYP2E1 (E47 cells). Neither ethanol (80 mmol/L) nor acetaldehyde (25-200 μmol/L) caused oxidative stress in HepG2 cells, an effect that was independent of blocking reduced glutathione (GSH) synthesis with buthionine-l -sulfoximine (BSO). However, BSO preincubation caused an overproduction of peroxides and activation of NF-κB and AP-1 in E47 cells even in the absence of ethanol. Furthermore, the incubation of E47 cells with ethanol (80 mmol/L for up to 5 days) depleted cellular GSH stores in both cytosol and mitochondria, reflecting the induction of oxidative stress. Ethanol activated NF-κB and AP-1 in E47 cells, an effect that was prevented by 4-methylpyrazole, potentiated by cyanamide, and attenuated by trolox C. Interestingly, however, despite the inability of acetaldehyde to induce oxidative stress in HepG2, acetaldehyde activated NF-κB and AP-1; in contrast, ethanol failed to activate these transcription factors in HepG2. Thus, our findings indicate that activation of NF-κB and AP-1 by ethanol and acetaldehyde occurs through distinct mechanisms. CYP2E1 is indispensable in the induction of oxidative stress from ethanol, whereas the activation of NF-κB and AP-1 by acetaldehyde is independent of oxidative stress.
Effect of propranolol on the factors promoting bacterial translocation in cirrhotic rats with ascites
Authors: Pérez-Paramo María , Muñoz Javier , Albillos Agustín , Freile Isabel , Portero Francisca , Santos Martín , Ortiz-Berrocal José
Published: July 2004

Bacterial translocation appears to be an important mechanism in the pathogenesis of spontaneous infections in cirrhosis. Cirrhotic patients are commonly treated with β-adrenoceptor blockers, but the impact of this treatment in the factors promoting bacterial translocation has not been investigated. This study was aimed at investigating in cirrhotic rats with ascites the effect of propranolol on intestinal bacterial load, transit, and permeability of the bowel and on the rate of bacterial translocation. Bacterial translocation to mesenteric lymph nodes and intestinal bacterial overgrowth, permeability (urinary excretion of 99mTc-diethylenetriaminepentaacetic acid [99mTc-DTPA]), and transit (geometric center ratio of 51Cr) were assessed in 29 rats with carbon tetrachloride (CCl4) cirrhosis and 20 controls. These variables were then measured in 12 placebo- and in 13 propranolol-treated ascitic cirrhotic rats. Bacterial translocation was present in 48% of the cirrhotic rats and in none of the controls. Cirrhotic rats with intestinal bacterial overgrowth had a significantly higher rate of translocation and slower intestinal transit than those without it. Among the 15 rats with overgrowth and a 99mTc-DTPA excretion greater than 10%, 15 had translocation and 2 had bacterial peritonitis. Only 1 of the 14 rats with either intestinal overgrowth or a 99mTc-DTPA excretion greater than 10% presented translocation. Compared with the placebo group, propranolol-treated animals had significantly lower portal pressure, faster intestinal transit, and lower rates of bacterial overgrowth and translocation. In ascitic cirrhotic rats, bacterial translocation results from intestinal overgrowth and severe damage to gut permeability. In this setting, intestinal overgrowth is associated with intestinal hypomotility. Propranolol accelerates the intestinal transit, decreasing the rates of bacterial overgrowth and translocation.
Cytotoxic T lymphocyte antigen-4 (CTLA-4) gene polymorphisms and susceptibility to type 1 autoimmune hepatitis
Authors: Agarwal Kosh , Czaja Albert J. , Jones David E. , Donaldson Peter T.
Published: July 2004

Genetic susceptibility to type 1 autoimmune hepatitis is indicated by a preponderance of female subjects and strong associations with human leukocyte antigens (HLA) DRB1*0301 and DRB1*0401. The gene encoding cytotoxic T-lymphocyte antigen-4 (CTLA-4) on chromosome 2q33 may also influence autoimmunity. To determine the frequency and significance of the exon 1 adenine (A)-guanine (G) base-exchange polymorphism for CTLA-4 in patients with type 1 autoimmune hepatitis, 155 northern European Caucasoid patients and 102 ethnically-matched control subjects were tested by polymerase chain reaction. The genotype distribution was significantly different in patients compared to controls (AA = 50/155 patients vs. 51/102 controls; AG = 84/155 patients vs. 38/102 controls; GG = 21/155 patients vs. 13/102 controls, χ2 = 8.94, P = .011). This difference was caused by a significant over-representation of the G allele in patients compared to controls (105/155 patients vs. 51/102 controls, χ2 = 8.34, P = .004, odds ratio = 2.12). The GG genotype was associated with a significantly higher mean serum aspartate transaminase level (P = .03), greater frequency of antibodies to thyroid microsomal antigens (P = .004) and was found more commonly in patients with HLA
DRB1*0301 (P = .02). Treatment outcomes, however, were not affected by the genotype. The CTLA-4 G allele is more common in patients with type 1 autoimmune hepatitis and may represent a second susceptibility allele. Furthermore, there may be synergy between the HLA-DRB1*0301 and the GG genotype in terms of disease risk.
Randomized controlled trial of interferon treatment for advanced hepatocellular carcinoma
Authors: Llovet Josep M. , Sala Margarita , Castells Lluís , Suarez Yanette , Vilana Ramon , Bianchi Lluís , Ayuso Carmen , Vargas Víctor , Rodés Joan , Bruix Jordi
Published: July 2004

The aim of this randomized controlled trial was to assess the efficacy of interferon alfa-2b (IFN) for the treatment of advanced hepatocellular carcinoma (HCC). Fifty-eight patients with HCC who were not suitable for resection, transplantation, ethanol injection, or arterial embolization were stratified according to their Okuda stage and randomized to receive IFN (3 × 106, 3 times a week, for 1 year) (n = 30) or symptomatic treatment (n = 28). Both groups were identical in terms of age, sex, performance status, presence of constitutional syndrome, Child-Pugh class, Okuda stage, multinodularity, portal thrombosis, and extrahepatic spread. Adhesion to IFN treatment was adequate in 27 patients, with a mean duration of treatment of 8 ± 3 months. However, IFN treatment was associated with side effects in 23 patients, leading to treatment discontinuation in 13 patients. Two of the 30 patients (6.6%) presented a partial response with greater than 50% size reduction and normalization of α-fetoprotein levels. The survival at 1 and 2 years according to intention to treat was not different between the 2 groups (58% and 38% vs. 36% and 12%, respectively, Breslow P = .19, log rank P = .14) and the absence of difference was maintained when dividing patients according to their Okuda stage. The probability of presenting tumor progression (P = .17), or deterioration of Child-Pugh class (P= .37), performance status (P = .07), or Okuda stage (P = .44) was not modified by IFN treatment. These results indicate that IFN is not properly tolerated in patients with cirrhosis and advanced HCC and that its administration prompts no benefit in terms of tumor progression rate and survival.
Heterogeneity of growth potential of adult rat hepatocytes in vitro
Authors: Tateno Chise , Takai-Kajihara Kaori , Yamasaki Chihiro , Sato Hajime , Yoshizato Katsutoshi
Published: July 2004

Nearly pure populations of small hepatocytes (SHs), parenchymal hepatocytes (PHs), and nonparenchymal cells (NPCs) were prepared from the adult rat, and cocultures of hepatocytes and NPCs were reconstituted from them first to obtain the direct evidence that NPCs promote the growth of hepatocytes and second to compare the growth potential between SHs and PHs. SHs and PHs underwent multiple divisions when cocultured with NPCs, whereas neither SHs nor PHs formed colonies at 10 days when cultured alone. Stellate cells in the NPCs were shown to be responsible for this growth promotion. SHs showed a higher growth capacity than PHs. To clearly show the relationship between the growth potential and the size of hepatocytes, SHs and PHs were further fractionated by a fluorescence-activated cell sorter, because the size distribution of SHs and PHs was half overlapped. SHs produced 2 cell populations, SH-R2 and SH-R3. The former showed a greater extent of granularity and autofluorescence than the latter. In contrast, PHs produced only 1 population (PH-R2), which corresponded to the SH-R2. The size of hepatocytes of SH-R3 was smaller (17.1 ± 0.2 μm) than those of SH-R2 (22.6 ± 0.5 μm) and PH-R2 (24.1 ± 0.1 μm) and there was not a significant overlap in the size distribution between the 2 groups. The hepatocytes of SH-R3 were highly replicative and 4 or 5 times higher in their growth potential than those of SH-R2 and PH-R2. We concluded that the growth potential of hepatocytes is heterogeneous and is correlated with their size and the extent of their granularity and autofluorescence.
Expression and activation of pro-MMP-2 and pro-MMP-9 during rat liver regeneration
Authors: Kim Tae-Hyoung , Mars Wendy M. , Stolz Donna B. , Michalopoulos George K.
Published: July 2004

Partial hepatectomy triggers a variety of biological phenomena, which culminate in regeneration of the liver mass. Hepatocyte proliferation is a major feature of the regenerating liver after partial hepatectomy. Previous studies in our laboratory suggested that hepatic matrix remodeling might be a prerequisite process for hepatocyte proliferation in the regenerating liver. In the present study we use immunohistochemical staining, Western blot analysis, and gelatin zymography to show that the inactive matrix metalloproteinases (MMPs), pro-MMP-2 and pro-MMP-9, are elevated at 30 minutes and activated at 6 to 12 hours and at 3 to 6 hours, respectively, after hepatectomy. Sham-operated livers did not show an increase in inactive pro-MMP-2 and pro-MMP-9 and did not contain active MMP-2 or MMP-9. To examine whether tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) is changed to regulate the activities of MMPs after partial hepatectomy, the level of TIMP-1 protein was analyzed by both immunohistochemistry and Western blot analysis. The level of TIMP-1 protein appears to increase by 6 to 18 hours, implying that an increase in TIMP-1 regulates activities of active MMP-2 and MMP-9. Taken together, these results suggest that hepatic matrix remodeling is mediated by activated MMPs, which contribute to modulation of the environment surrounding hepatocytes during rat liver regeneration.
Peroxisome proliferator-activated receptor γ transcriptional regulation is involved in platelet-derived growth factor-induced proliferation of human hepatic stellate cells
Authors: Galli Andrea , Crabb David , Price Donna , Ceni Elisabetta , Salzano Renata , Surrenti Calogero , Casini Alessandro
Published: July 2004

During liver injury, hepatic stellate cells (HSC) acquire a myofibroblast-like phenotype associated with reduction of lipid droplets, increased collagen synthesis, and proliferation. Peroxisome proliferator-activated receptor γ (PPARγ) regulates adipocyte differentiation and controls gene transcription in response to various activators including prostanoids and antidiabetic thiazolidinediones. We explored whether the presence of PPARγ and its transcriptional activity were involved in control of HSC proliferation in vitro. PPARγ ligands, 15-deoxy-△12
14 prostaglandin J2 (15d-PGJ2) and ciglitizone, significantly decrease platelet-derived growth factor (PDGF)-induced proliferation in activated human HSC and inhibit α smooth muscle actin (α-SMA) expression during HSC transdifferentiation. Treatment with 9-cis retinoic acid (9-cisRA) and LG268, ligands of the heterodimerization partner retinoic X receptor (RXR), had a negligible effect in PDGF-treated cells but caused a further reduction of proliferation when used in combination with ciglitizone. Transfection experiments with a reporter gene consisting of 3 copies of a PPAR response element (peroxisome proliferator response element [PPRE]3-tk-luciferase) showed a progressive reduction of PPAR transcriptional activity during plastic-induced HSC transdifferentiation. Cotransfection with human PPARγ expression vector restored the PPRE3-tk-luciferase reporter expression and the increased level of the receptor in activated HSC-inhibited cell proliferation in a dose-dependent manner. Incubation of human PPARγ-cotransfected HSC with PDGF strongly inhibited luciferase activity and this effect was blocked by the inhibition of the mitogen-activated protein (MAP) kinase signal cascade. Our results indicate that depression of PPARγ expression and activity is involved in HSC proliferation and that the PPARγ ligand-mediated activation exerts a previously unrecognized inhibition of PDGF-induced mitogenesis in activated human HSC.
Tyrosine phosphorylation of focal adhesion kinase by PDGF is dependent on Ras in human hepatic stellate cells
Authors: Carloni Vinicio , Pinzani Massimo , Giusti Sabrina , Romanelli Roberto G. , Parola Maurizio , Bellomo Giorgio , Failli Paola , Hamilton Andrew D. , Sebti Said M. , Laffi Giacomo , Gentilini Paolo
Published: July 2004

Focal adhesion kinase (FAK) is a widely expressed nonreceptor tyrosine kinase found in focal adhesions. FAK has been indicated as a point of convergence of other signaling pathways including platelet-derived growth factor (PDGF) receptors, and recently, FAK tyrosine phosphorylation has been shown to be stimulated by PDGF. In the present study we assessed the role of Ras as a possible intermediate protein regulating PDGF-induced FAK tyrosine phosphorylation in human hepatic stellate cells (HSCs), liver-specific pericytes primarily involved in the pathogenesis of liver fibrosis. For this purpose, cells were first subjected to retroviral-mediated gene transfer with a dominant-negative mutant of Ras (N17Ras). This resulted in a marked inhibition of PDGF-induced FAK tyrosine phosphorylation together with the expected reduction of PDGF-induced extracellular signal-regulated kinase activity (ERK). Afterward, the effects of pharmacological agents potentially affecting Ras isoprenylation were evaluated. PDGF-induced FAK tyrosine phosphorylation, ERK activity and intracellular calcium increase, as well as the biological effects of this growth factor, (i.e., mitogenesis and cell migration) were effectively blocked by GGTI-298, an inhibitor of geranylgeranyltransferase I. Inhibition of Ras processing obtained with FTI-277, an inhibitor of farnesyltransferase, resulted in detectable effects only at high doses. Taken together, these results establish that Ras operates as a protein-linking PDGF-β receptor to FAK in human HSCs, and that signaling molecules requiring geranylgeranylation may also be involved in this process.
Antituberculosis drug-related liver dysfunction in chronic hepatitis B infection
Authors: Wong Wai-Man , Wu Pui-Chee , Yuen Man-Fung , Cheng Chi-Chung , Yew Wing-Wai , Wong Poon-Chuen , Tam Cheuk-Ming , Leung Chi-Chiu , Lai Ching-Lung
Published: July 2004

Liver toxicity is a common side effect of antituberculosis (anti-TB) drugs. We studied the differences in liver dysfunction observed during anti-TB treatment between hepatitis B virus carriers (HBV) and noncarriers. Three hundred twenty-four patients on anti-TB drugs were recruited and followed up for 1 year. Forty-three patients with HBV and 276 non-HBV patients were included for analysis. Liver function tests and viral markers were monitored monthly. Liver biopsy was requested whenever the alanine transaminase (ALT) was persistently abnormal. Eighty-six HBV carriers who were not given anti-TB drugs were chosen as a second control and evaluated prospectively. The incidence of liver dysfunction was significantly higher in HBV carriers given anti-TB drugs (34.9%) when compared to noncarriers (9.4%, P < .001) and with HBV carriers not given anti-TB drugs (8.1%, P < .001). For patients given anti-TB drugs, HBV carriers who developed liver dysfunction were younger (P = .011) and had more severe liver injury compared with noncarriers (P = .008). By multiple logistic regression analysis, age (P = .002) and hepatitis B infection (P < .001) were the only 2 significant risk factors for hepatotoxicity related to anti-TB therapy.
Lamivudine treatment for decompensated cirrhosis resulting from chronic hepatitis B
Authors: Villeneuve Jean-Pierre , Condreay Lynn D. , Willems Bernard , Pomier-Layrargues Gilles , Fenyves Daphna , Bilodeau Marc , Leduc Raymond , Peltekian Kevork , Wong Florence , Margulies Mariana , Heathcote Jenny E.
Published: July 2004

The prognosis of decompensated cirrhosis resulting from chronic hepatitis B is poor, and the benefits of treatment with interferon are outweighed by serious side effects and by the risk of fatal exacerbation of disease activity. Lamivudine rapidly reduces hepatitis B virus (HBV)-DNA in serum to undetectable levels. We have treated 35 patients with chronic hepatitis B and decompensated cirrhosis with lamivudine 100 mg or 150 mg orally once daily. Pretreatment, all were positive for HBV-DNA in serum. Ten had Child-Pugh class B and 25 had Child-Pugh class C liver disease. Seven patients underwent liver transplantation within 6 months of treatment initiation, 5 patients died within 6 months, and 23 patients were treated for at least 6 months (mean = 19 months). In a majority of these 23 cases, there was a slow but marked improvement in liver function, which was most apparent after 9 months of treatment, with a decrease in serum bilirubin from 67 ± 13 to 30 ± 4 μmol/L (P< .05, baseline vs. 9 months), an increase in serum albumin from 27 ± 1 to 34 ± 1g/L (P< .05), and a decrease in Child-Pugh score from 10.3 ± 0.4 to 7.5 ± 0.5(P< .05). Three patients developed resistance to lamivudine because of a mutation in the YMDD motif, but liver function did not deteriorate. We conclude that inhibition of viral replication with lamivudine results in a significant improvement of liver function in patients with decompensated HBV cirrhosis, but the long-term benefits remain uncertain.