bluuebunny/crossref_metadata_2025 huggingface dataset:
The role of iNOS in alcohol-dependent hepatotoxicity and mitochondrial dysfunction in mice
Authors: Venkatraman Aparna , Shiva Sruti , Wigley Amanda , Ulasova Elena , Chhieng David , Bailey Shannon M. , Darley-Usmar Victor M.
Published: August 2004

Nitric oxide (NO) is now known to control both mitochondrial respiration and organelle biogenesis. Under conditions of ethanol-dependent hepatic dysfunction, steatosis is increased, and this is associated with increased expression of inducible nitric oxide synthase (iNOS). We have previously shown that after chronic exposure to ethanol, the sensitivity of mitochondrial respiration to inhibition by NO is enhanced, and we have proposed that this contributes to ethanol-dependent hypoxia. This study examines the role of iNOS in controlling the NO-dependent modification of mitochondrial function. Mitochondria were isolated from the livers of both wild-type (WT) and iNOS knockout (iNOS−/−) mice that were fed an isocaloric ethanol-containing diet for a period of 5 weeks. All animals that consumed ethanol showed some evidence of fatty liver; however, this was to a lesser extent in the iNOS−/− mice compared to controls. At this early stage in ethanol-dependent hepatic dysfunction, infiltration of inflammatory cells and the formation of nitrated proteins was also decreased in response to ethanol feeding in the iNOS−/− animals. Mitochondria isolated from wild-type ethanol-fed mice showed a significant decrease in respiratory control ratio and an increased sensitivity to NO-dependent inhibition of respiration relative to their pair-fed controls. In contrast, liver mitochondria isolated from iNOS−/− mice fed ethanol showed no change in the sensitivity to NO-dependent inhibition of respiration. In conclusion, the hepatic response to chronic alcohol-dependent cytotoxicity involves a change in mitochondrial function dependent on the induction of iNOS. (Hepatology 2004;40:565-573.)
Reduced expression of cell cycle regulator p18INK4C in human hepatocellular carcinoma
Authors: Morishita Asahiro , Masaki Tsutomu , Yoshiji Hitoshi , Nakai Seiji , Ogi Tomohiro , Miyauchi Yoshiaki , Yoshida Shuhei , Funaki Toshiharu , Uchida Naohito , Kita Yuko , Funakoshi Fumi , Usuki Hisashi , Okada Setsuo , Izuishi Kunihiko , Watanabe Seishiro , Kurokohchi Kazutaka , Kuriyama Shigeki
Published: August 2004

Cyclins, cyclin-dependent kinases (Cdks), and Cdk inhibitors (CdkIs) are frequently altered in human cancer. p18INK4C, a member of the INK4 family of CdkIs, is a potential tumor-suppressor gene product. However, the expression of p18INK4C in hepatocellular carcinoma (HCC) remains unknown. The aim of this study was to examine the expression of p18INK4C in various liver diseases including HCC and to assess its clinical significance in HCC. To that end, we examined the expression of p18INK4C by immunohistochemistry in various liver diseases, including 51 HCCs, and also studied the relationship between p18INK4C expression, the phosphorylation of retinoblastoma protein (pRb), and the activity level of Cdk4 and Cdk6. Immunohistochemical analysis revealed the frequent loss of p18INK4C expression in HCC, especially in poorly differentiated HCC. The loss of p18INK4C expression was shown to be associated with a poor prognosis compared with that associated with p18INK4C- positivity. Further, the kinase activity of Cdk4 was found to be higher in p18INK4C-negative HCCs than in p18INK4C- positive HCCs. However, the level of Cdk6 activity was similar in the 2 groups of HCCs. In p18INK4C- positive HCCs, p18INK4C dominantly interacted with Cdk4 rather than with Cdk6. pRb phosphorylated at serine(Ser) 780 was detected more frequently in p18INK4C - negative than in p18INK4C - positive HCCs. In conclusion, the loss of p18INK4C expression may play a role in the differentiation and development of HCC through the up-regulation of Cdk4 activity. (Hepatology 2004;40:677-686.)
Rapamycin-sensitive induction of eukaryotic initiation factor 4F in regenerating mouse liver
Authors: Goggin Melissa M. , Nelsen Christopher J. , Kimball Scot R. , Jefferson Leonard S. , Morley Simon J. , Albrecht Jeffrey H.
Published: August 2004

Following acute injuries that diminish functional liver mass, the remaining hepatocytes substantially increase overall protein synthesis to meet increased metabolic demands and to allow for compensatory liver growth. Previous studies have not clearly defined the mechanisms that promote protein synthesis in the regenerating liver. In the current study, we examined the regulation of key proteins involved in translation initiation following 70% partial hepatectomy (PH) in mice. PH promoted the assembly of eukaryotic initiation factor (eIF) 4F complexes consisting of eIF4E, eIF4G, eIF4A1, and poly-A binding protein. eIF4F complex formation after PH occurred without detectable changes in eIF4E-binding protein 1 (4E-BP1) phosphorylation or its binding eIF4E. The amount of serine 1108-phosphorylated eIF4G (but not Ser209-phosphorylated eIF4E) was induced following PH. These effects were antagonized by treatment with rapamycin, indicating that target of rapamycin (TOR) activity is required for eIF4F assembly in the regenerating liver. Rapamycin inhibited the induction of cyclin D1, a known eIF4F-sensitive gene, at the level of protein expression but not messenger RNA (mRNA) expression. In conclusion, increased translation initiation mediated by the mRNA cap-binding complex eIF4F contributes to the induction of protein synthesis during compensatory liver growth. Further study of factors that regulate translation initiation may provide insight into mechanisms that govern metabolic homeostasis and regeneration in response to liver injury. (Hepatology 2004;40:537-544.)
Improved survival after variceal bleeding in patients with cirrhosis over the past two decades
Authors: Carbonell Nicolas , Pauwels Arnaud , Serfaty Lawrence , Fourdan Olivier , Lévy Victor George , Poupon Raoul
Published: August 2004

Over the past two decades, new treatment modalities have been introduced for the management of variceal bleeding. The aim of this retrospective study in a single center was to assess whether these treatments have improved the prognosis for cirrhotic patients with variceal bleeding. We reviewed the clinical records of all patients with cirrhosis admitted to our Liver Intensive Care Unit due to variceal bleeding during the years 1980, 1985, 1990, 1995, and 2000. Whereas balloon tamponade was still the first-line treatment in 1980, patients treated in 2000 received a vasoactive agent, an endoscopic treatment, and an antibiotic prophylaxis in, respectively, 90%, 100%, and 94% of cases. The in-hospital mortality rate steadily decreased over the study period: 42.6%, 29.9%, 25%, 16.2%, and 14.5% in 1980, 1985, 1990, 1995, and 2000, respectively (P < .05). Mortality decreased from 9% in 1980 to 0% in 2000 in Child-Turcotte-Pugh class A patients, from 46% to 0% in class B patients, and from 70% to 32% in class C patients. This improved survival was associated with a decrease of rebleeding (from 47% in 1980 to 13% in 2000) and bacterial infection rates (from 38% to 14%). On multivariable analysis, endoscopic therapy and antibiotic prophylaxis were independent predictors of survival. In conclusion, in-hospital mortality of patients with cirrhosis and variceal bleeding decreased threefold over the past two decades, in concurrence with an early and combined use of pharmacological and endoscopic therapies and short-term antibiotic prophylaxis. (Hepatology 2004;40:652-659.)
In vivo regulation of murine CYP7A1 by HNF-6: A novel mechanism for diminished CYP7A1 expression in biliary obstruction
Authors: Wang Minhua , Tan Yongjun , Costa Robert H. , Holterman Ai-Xuan L.
Published: August 2004

Disruption of the enterohepatic bile acid circulation during biliary tract obstruction leads to profound perturbation of the cholesterol and bile acid metabolic pathways. Several families of nuclear receptor proteins have been shown to modulate this critical process by regulating hepatic cholesterol catabolism and bile acid synthesis through the transcriptional control of cholesterol 7-α hydroxylase (CYP7A1). Hepatocyte nuclear factor (HNF) 6 (also known as OC-1) is a member of the ONECUT family of transcription factors that activate numerous hepatic target genes essential to liver function. We have previously shown that hepatic expression of mouse HNF-6 messenger RNA (mRNA) and protein significantly decrease following bile duct ligation. Because CYP7A1 contains potential HNF-6 binding sites in its promoter region, we tested the hypothesis that HNF-6 transcriptionally regulates CYP7A1. Following bile duct ligation, we demonstrated that diminished HNF-6 mRNA levels correlate with a reduction in CYP7A1 mRNA expression. Increasing hepatic levels of HNF-6 either by infection with recombinant adenovirus vector expressing HNF-6 cDNA by growth hormone treatment leads to an induction of CYP7A1 mRNA. To directly evaluate if HNF-6 is a transcriptional activator for CYP7A1, we used deletional and mutational analyses of CYP7A1 promoter sequences and defined sequences −206/−194 to be critical for CYP7A1 transcriptional stimulation by HNF-6 in cotransfection assays. In conclusion, the HNF-6 protein is a component of the complex network of hepatic transcription factors that regulates the expression of hepatic genes essential for bile acid homeostasis and cholesterol/lipid metabolism in normal and pathological conditions. (Hepatology 2004;40:600-608.)
Selective priming to Toll-like receptor 4 (TLR4), not TLR2, ligands by P. acnes involves up-regulation of MD-2 in mice
Authors: Romics Laszlo , Dolganiuc Angela , Kodys Karen , Drechsler Yvonne , Oak Shilpa , Velayudham Arumugam , Mandrekar Pranoti , Szabo Gyongyi
Published: August 2004

Lipopolysaccharide (LPS) triggers cytokine production through Toll-like receptor 4 (TLR4), which shares downstream signaling pathways with TLR2. We investigated the roles of TLR2 and TLR4 in Propionibacterium acnes (P. acnes)-primed, LPS-induced liver damage using selective TLR ligands. Stock LPS induced interleukin 8 in both TLR4- and TLR2-expressing human embryonic kidney (HEK) 293 cells. Purified LPS (TLR4 ligand) activated HEK/TLR4 cells, while peptidoglycan and lipoteichoic acid (TLR2 ligands) activated HEK/TLR2 cells, respectively. In mice, P. acnes priming resulted in increased liver messenger RNA (mRNA) and serum levels of tumor necrosis factor α, interleukin 12, and interferon γ (IFN-γ) by both stock LPS and purified LPS challenges compared with nonprimed controls. In contrast, P. acnes failed to sensitize to TLR2 ligands (peptidoglycan + lipoteichoic acid). In the liver, P. acnes-priming was associated with up-regulation of TLR4 and MD-2 proteins, and subsequent LPS challenge further increased MD-2 and CD14 mRNA levels. The lack of sensitization to TLR2 ligands by P. acnes correlated with no increase in hepatic TLR1 or TLR6 mRNA. In vitro, P. acnes pretreatment desensitized RAW macrophages to a secondary stimulation via both TLR2 and TLR4. However, IFN-γ could selectively prevent desensitization to TLR4 but not to TLR2 ligands. Furthermore, P. acnes induced production of IFN-γ in vivo as well as in isolated splenocytes. In vitro, P. acnes-primed Hepa 1-6 hepatocytes but not RAW macrophages produced increased MD-2 and CD14 mRNA levels after an LPS challenge. In conclusion, P. acnes priming to selective TLR4-mediated liver injury is associated with up-regulation of TLR4 and MD-2 and is likely to involve IFN-γ and prevent TLR4 desensitization by P. acnes. (Hepatology 2004;40:555-564.)
Exploring interactions between rat hepatocytes and nonparenchymal cells using gene expression profiling
Authors: Khetani Salman R. , Szulgit Greg , Del Rio Jo A. , Barlow Carrolee , Bhatia Sangeeta N.
Published: August 2004

Cocultivation of primary hepatocytes with a plethora of nonparenchymal cells (from within and outside the liver) has been shown to support hepatic functions in vitro. Despite significant investigation into this phenomenon, the molecular mechanism underlying epithelial-nonparenchymal interactions in hepatocyte cocultures remains poorly understood. In this study, we present a functional genomic approach utilizing gene expression profiling to isolate molecular mediators potentially involved in induction of liver-specific functions by nonparenchymal cells. Specifically, primary rat hepatocytes were cocultivated with closely related murine fibroblast cell types (3T3-J2, NIH-3T3, mouse embryonic fibroblasts) to allow their classification as “high,” “medium,” or “low” inducers of hepatic functions. These functional responses were correlated with fibroblast gene expression profiles obtained using Affymetrix GeneChips. Microarray data analysis provided us with 17 functionally characterized candidate genes in the cell communication category (cell surface, extracellular matrix, secreted factors) that may be involved in induction of hepatic functions. Further analysis using various databases (i.e., PubMed, GenBank) facilitated prioritization of the candidates for functional characterization. We experimentally validated the potential role of two candidates in our coculture model. The cell surface protein, neural cadherin (N-cadherin), was localized to hepatocyte-fibroblast junctions, while adsorbed decorin up-regulated hepatic functions in pure cultures as well as cocultures with low-inducing fibroblasts. In the future, identifying mediators of hepatocyte differentiation may have implications for both fundamental hepatology and cell-based therapies (e.g., bioartificial liver devices). In conclusion, the functional genomic approach presented in this study may be utilized to investigate mechanisms of cell-cell interaction in a variety of tissues and disease states. (Hepatology 2004;40:545-554.)
Peroxynitrite alters the catalytic activity of rodent liver proteasome in vitro and in vivo
Authors: Osna Natalia A. , Haorah James , Krutik Viatcheslav M. , Donohue Terrence M.
Published: August 2004

The proteasome is an important multicatalytic enzyme complex that degrades misfolded and oxidized proteins, signal transduction factors, and antigenic peptides for presentation. We investigated the in vitro effects of peroxynitrite (PN) on the peptidase activity of both crude 20S and 26S and purified 20S proteasome preparations from rat liver as well as proteasome activity in Hep G2 cells and in mouse liver. Crude and purified proteasome preparations were exposed to PN or to the PN donor, 3-morpholinosydnonimine hydrochloride (SIN-1), and then assayed for chymotrypsin-like activity. For in vivo experiments, mice were treated with molsidomine, which is metabolized to SIN-1 in liver. PN and SIN-1 dose-dependently modulated the chymotrypsin-like activity of the 20S proteasome: lower concentrations enhanced proteasome activity, and higher concentrations caused its decline. The NO donor S-nitroso-N-acetylpenicillamine (SNAP), at all concentrations, suppressed 20S proteasome activity. We observed similar results when liver soluble fractions (S-100) were treated with PN, SIN-1, or SNAP, except that enzyme activity in S-100 fractions was less sensitive than the purified enzymes to these agents. Treatment of Hep G2 cells with 0.01 or 0.1 mmol/L SIN-1 stimulated in situ proteasome activity in these cells, while 1 mmol/L SIN-1 suppressed it. SNAP treatment did not affect proteasome activity in Hep G2 cells. Mice treated with molsidomine had enhanced liver proteasome activity 6 hours after treatment, but after 24 hours enzyme activity declined below control levels. In conclusion, PN dose-dependently modulated proteasome activity, regulating protein degradation by the proteasome in liver cells. (Hepatology 2004;40:574-582.)
Quantitation of covalently closed circular hepatitis B virus DNA in chronic hepatitis B patients
Authors: Wong Danny Ka-Ho , Yuen Man-Fung , Yuan HeJun , Sum Simon Siu-Man , Hui Chee-Kin , Hall Jeff , Lai Ching-Lung
Published: August 2004

This study examined a signal amplification assay, the Invader assay, for the quantitation of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) in liver biopsies and sera. DNA was extracted from liver biopsy and serum samples were collected from 16 hepatitis B e antigen (HBeAg)-positive and 36 antibody-to-HBeAg-positive (anti-HBe-positive) chronic hepatitis B patients. The amount of total HBV DNA and cccDNA was measured using the Invader assay. Anti-HBe-positive patients had lower median total intrahepatic HBV DNA (P < .001) and intrahepatic cccDNA levels (P = .001) than HBeAg-positive patients. Intrahepatic cccDNA correlated positively with the total intrahepatic HBV DNA (r = 0.950, P < .001). However, the proportion of intrahepatic HBV DNA in the form of cccDNA was inversely related to the amount of total intrahepatic HBV DNA (r = −0.822, P < .001). A small amount of cccDNA was detected in 39 of 52 (75%) serum samples. Anti-HBe-positive patients had lower median serum cccDNA levels than HBeAg-positive patients (P = .002). Serum HBV DNA correlated positively with intrahepatic total HBV DNA (r = 0.778, P < .001) and intrahepatic cccDNA (r = 0.481, P = .002). In conclusion, the Invader assay is a reliable assay for the quantitation of cccDNA. Serum and intrahepatic total HBV DNA and cccDNA levels become lower as the disease progresses from HBeAg-positive to anti-HBe-positive phase, with cccDNA becoming the predominant form of intrahepatic HBV DNA. (Hepatology 2004;40:727-737.)
Hepatitis C recurrence is more severe after living donor compared to cadaveric liver transplantation
Authors: Garcia-Retortillo Montserrat , Forns Xavier , Llovet Josep M. , Navasa Miquel , Feliu Anna , Massaguer Anna , Bruguera Miquel , Fuster Josep , Garcia-Valdecasas Juan Carlos , Rimola Antoni
Published: August 2004

Preliminary reports suggested that hepatitis C virus (HCV) infection has a more aggressive course following living donor liver transplantation (LDLT) compared to cadaveric liver transplantation (CLT). The aim of this prospective study was to establish if HCV disease recurrence differs between LDLT and CLT. A cohort of 116 consecutive HCV-infected patients undergoing 117 LTs in a single center from March 2000 to August 2003 were followed-up, including systematic liver biopsies. Severe recurrence (SR) was defined as biopsy-proven cirrhosis and/or the occurrence of clinical decompensation. After a median follow-up of 22 months (2.6-44 months), 26 (22%) patients developed SR (decompensation in 12), involving 17 (18%) of 95 patients undergoing CLT and 9 (41%) of 22 undergoing LDLT. The 2-year probability of presenting SR was significantly higher in LDLT compared to CLT (45% vs. 22%, P = .019). By univariate analysis LDLT (P = .019) and an ALT higher than 80 IU/L 3 months after LT (P = .022) were predictors of SR. In 93 patients from whom a liver biopsy was available 3 months after LT, a lobular necroinflammatory score >1 (P < .01), LDLT (P < .01), and biliary complications (P = .046) were associated with SR. However, the only variables independently associated with SR were LDLT (odds ratio [OR], = 2.8; 95% CI,1.19-6.6; P = .024) and a lobular necroinflammatory score >1 (OR, 3.1; 95% CI, 1.2-8; P = .013). In conclusion, HCV recurrence is more severe in LDLT compared to CLT. Although our results were based on a single-center experience, they should be considered in the decision-making process of transplant programs, since severe HCV recurrence may ultimately compromise graft and patient survival. (Hepatology 2004; 40:699-707.)
Gas exchange mechanism of orthodeoxia in hepatopulmonary syndrome
Authors: Gómez Federico P. , Martínez-Pallí Graciela , Barberà Joan A. , Roca Josep , Navasa Miquel , Rodríguez-Roisin Robert
Published: August 2004

The mechanism of orthodeoxia (OD), or decreased partial pressure of arterial oxygen (PaO2) from supine to upright, a characteristic feature of hepatopulmonary syndrome (HPS), has never been comprehensively elucidated. We therefore investigated the intrapulmonary (shunt and ventilation-perfusion [V̇A/𝑄̇] mismatching) and extrapulmonary factors governing PaO2 in 20 patients with mild to severe HPS (14 males, 6 females; 50 ± 3 years old SE) at upright and supine, in random order. We set out a cutoff value for OD, namely a PaO2 decrease ≥5% or ≥4 mm Hg (area under the receiver operating characteristic curve, 0.96 each). Compared to supine, 5 patients showed OD (PaO2 change, −11% ± 2%, −7 ± 1 mm Hg, P < .05) with further V̇A/𝑄̇ worsening (shunt + low V̇A/𝑄̇ mode increased from 19% ± 7% to 21% ± 7% of cardiac output [𝑄̇T], P < .05), as opposed to 15 patients who did not (+2% ± 2%, +1± 1 mm Hg) with V̇A/𝑄̇ improvement (from 20% ± 4% to 16% ± 4% of 𝑄̇T, P < .01). Cardiac output was significantly lower in OD patients in both positions. Changes in extrapulmonary factors at upright, such as increased minute ventilation and decreased 𝑄̇T, were of similar magnitude in both subsets of patients. In conclusion, our data suggest that gas exchange response to OD in HPS points to a more altered pulmonary vascular tone inducing heterogeneous blood flow redistribution to lung zones with prominent intrapulmonary vascular dilatations. (Hepatology 2004;40:660-666.)
Serum from patients with fulminant hepatic failure causes hepatocyte detachment and apoptosis by a β1-integrin pathway
Authors: Newsome Philip N. , Tsiaoussis John , Masson Steven , Buttery Robert , Livingston Cameron , Ansell Ian , Ross James A. , Sethi Tariq , Hayes Peter C. , Plevris John N.
Published: August 2004

Hepatocyte transplantation is restricted by the impaired ability of hepatocytes to engraft and survive in the damaged liver. Understanding the mechanisms that control this process will permit the development of strategies to improve engraftment. We studied changes in liver matrix during acute injury and delineated the mechanisms that perturb the successful adhesion and engraftment of hepatocytes. Collagen IV expression was increased in sinusoidal endothelium and portal tracts of fulminant hepatic failure explants, whereas there were minimal changes in the expression of fibronectin, tenascin, and laminin. Using an in vitro model of cellular adhesion, hepatocytes were cultured on collagen-coated plates and exposed to serum from patients with liver injury to ascertain their subsequent adhesion and survival. There was a rapid, temporally progressive decrease in the adhesive properties of hepatocytes exposed to such serum that occurred within 4 hours of exposure. Loss of activity of the β1-integrin receptor, which controls adhesion to collagen, was seen to precede this loss of adhesive ability. Addition of the β1-integrin activating antibody (TS2/16) to cells cultured with liver injury serum significantly increased their adhesion to collagen, and prevented significant apoptosis. In conclusion, we have identified an important mechanism that underpins the failure of infused hepatocytes to engraft and survive in liver injury. Pretreating cells with an activating antibody can improve their engraftment and survival, indicating that serum from patients with liver injury exerts a defined nontoxic biological effect. This finding has important implications in the future of cellular transplantation for liver and other organ diseases. (Hepatology 2004;40:636-645.)
Negative regulation of hepatocellular carcinoma cell growth by signal regulatory protein α1
Authors: Yan He-Xin , Wang Hong-Yang , Zhang Rui , Chen Lei , Li Bao-An , Liu Shu-Qin , Cao Hui-Fang , Qiu Xiu-Hua , Shan Yun-Feng , Yan Zhong-Hua , Wu Hong-Ping , Tan Ye-Xiong , Wu Meng-Chao
Published: August 2004

Signal regulatory protein (SIRP) α1 is a member of the SIRP family that undergoes tyrosine phosphorylation and binds SHP-2 tyrosine phosphatase in response to various mitogens. The expression levels of SIRPα1 were decreased in HCC tissues, compared with the matched normal tissues. Exogenous expression of wild type SIRPα1, but not of a mutant SIRPα1 lacking the tyrosine phosphorylation sites, in SIRPα1-negative Huh7 human HCC cells resulted in suppression of tumor cell growth both in vitro and in vivo. Treatment of Huh7 transfectants with EGF or HGF induced tyrosine phosphorylation of SIRPα1 and its association with SHP-2, which were accompanied by reduced ERK1 activation. Expression of SIRPα1 significantly suppressed activation of NF-κB and also sensitized Huh7 cells to TNFα or cisplatin-induced cell death. In addition, SIRPα1-transfected Huh7 cells displayed reduced cell migration and cell spreading in a fashion that was dependent on SIRPα1/SHP-2 complex formation. In conclusion, a negative regulatory effect of SIRPα1 on hepatocarcinogenesis is exerted, at least in part, through inhibition of ERK and NF-κB pathways. (Hepatology 2004;40:618-628.)
TNF-α regulates mouse fetal hepatic maturation induced by oncostatin M and extracellular matrices
Authors: Kamiya Akihide , Gonzalez Frank J.
Published: August 2004

Fetal hepatic maturation consists of multisteps and is regulated by several cytokines and cell-cell or cell-matrices interactions. In the mid-to-late fetal stage, hepatocytes have few metabolic functions associated with adult liver homeostasis. Cultured fetal hepatocytes acquire the expression of several mature liver-specific genes through stimulation with hepatic maturation factor oncostatin M (OSM) and matrigel. Tumor necrosis factor-α (TNFα) regulates fetal hepatic maturation stimulated by OSM and matrigel. TNFα suppressed expression of mature liver-specific genes such as tyrosine aminotransferase and apolipoproteins. In addition, the expression of hematopoietic cytokines and cyclin A2, repressed by OSM and matrigel, is induced by TNFα in the fetal hepatic cultures coincident with cell division. TNFα inhibited the induction of hepatocyte nuclear factor 4α induced by OSM and matrigel, suggesting that down-regulation of hepatocyte nuclear factor 4α expression is involved in the mechanism of suppression of hepatic maturation by TNFα. Interestingly, TNFα is expressed in the prenatal and postnatal liver but not in adult liver, whereas TNFR1, a TNFα receptor, is expressed in both fetal and adult livers. In conclusion, TNFα is a suppressive factor of hepatic maturation. The balance between hepatic maturation factor (OSM and extracellular matrices) and TNFα is important for liver development. (Hepatology 2004;40:527-536.)
In vitro differentiation of rat liver derived stem cells results in sensitization to TNFα-mediated apoptosis
Authors: Sánchez Aránzazu , Factor Valentina M. , Espinoza Luis A. , Schroeder Insa S. , Thorgeirsson Snorri S.
Published: August 2004

Hepatic stem cells are activated after liver damage and have a critical role in tissue homeostasis and repair. Characterization of molecular and cellular events accompanying the expansion and differentiation of liver stem cells is essential for understanding the basic biology of stem cells and for facilitating clinical application of the stem cells. We assessed whether in vitro differentiation of putative hepatic progenitor (rat liver epithelial [RLE]) cells toward hepatocytic lineage affects the response to TNFα-mediated cytotoxicity, a common determinant of liver injury. The data show that 50% of differentiated cells underwent apoptosis after 6 hours of TNFα treatment whereas control RLE cells were resistant. Both cell types displayed mitochondrial depolarization and release of cytochrome c but the TNFα treatment resulted in activation of caspases 9 and 3 and the execution of apoptosis only in differentiated RLE cells. Apoptotic death was associated with increased ROS production and depletion of glutathione. Antioxidants completely prevented both glutathione depletion and apoptosis induced by TNFα in differentiated RLE cells. Conversely, glutathione-depleting agents sensitized control RLE cells to TNFα induced apoptosis. In conclusion, efficient antioxidant defense system involving glutathione renders hepatic progenitor cells resistant to TNFα-mediated apoptosis and acquisition of sensitivity to death stimuli is an implicit feature of the differentiation process. Supplementary material for this article can be found on the Hepatology website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html). (Hepatology 2004;40:590-599.)
Randomized controlled study of TIPS versus paracentesis plus albumin in cirrhosis with severe ascites
Authors: Salerno Francesco , Merli Manuela , Riggio Oliviero , Cazzaniga Massimo , Valeriano Valentina , Pozzi Massimo , Nicolini Antonio , Salvatori Filippo
Published: August 2004

The transjugular intrahepatic portosystemic shunt (TIPS) has been shown to be effective in the control of refractory or recidivant ascites. However, the effect of TIPS on survival as compared with that of large-volume paracentesis plus albumin is uncertain. A multicenter, prospective, clinical trial was performed in 66 patients with cirrhosis and refractory or recidivant ascites (16 Child-Turcotte-Pugh class B and 50 Child-Turcotte-Pugh class C) randomly assigned to treatment with TIPS (n = 33) or with large-volume paracentesis plus human albumin (n = 33). The primary endpoint was survival without liver transplantation. Secondary endpoints were treatment failure, rehospitalization, and occurrence of complications. Thirteen patients treated with TIPS and 20 patients treated with paracentesis died during the study period, 4 patients in each group underwent liver transplantation. The probability of survival without transplantation was 77% at 1 year and 59% at 2 years in the TIPS group as compared with 52% and 29% in the paracentesis group (P = .021). In a multivariate analysis, treatment with paracentesis and higher MELD score showed to independently predict death. Treatment failure was more frequent in patients assigned to paracentesis, whereas severe episodes of hepatic encephalopathy occurred more frequently in patients assigned to TIPS. The number and duration of rehospitalizations were similar in the two groups. In conclusion, compared to large-volume paracentesis plus albumin, TIPS improves survival without liver transplantation in patients with refractory or recidivant ascites. (Hepatology 2004;40:629-635.)
A case-control study for differences among hepatitis B virus infections of genotypes A (subtypes Aa and Ae) and D †
Authors: Tanaka Yasuhito , Hasegawa Izumi , Kato Takanobu , Orito Etsuro , Hirashima Noboru , Acharya Subrat K. , Gish Robert G. , Kramvis Anna , Kew Michael C. , Yoshihara Namiko , Shrestha Santosh Man , Khan Mobin , Miyakawa Yuzo , Mizokami Masashi
Published: August 2004

There are two subtypes of hepatitis B virus genotype A (HBV/A) and they are provisionally designated Aa (“a” standing for Africa/Asia) and Ae (“e” for Europe). In a case-control study, 78 HBV/Aa, 78HBV/Ae, and 78HBV/D carriers from several countries were compared. The prevalence of HBe antigen (HBeAg) in serum was significantly lower in carriers of HBV/Aa than in carriers of HBV/Ae (31% vs. 49%; P = .033), with a difference more obvious in the carriers aged 30 years or younger (34% vs. 67%; P = .029). HBV DNA levels in the carriers of HBV/Aa (median, 3.46 log copies/mL; 95% CI, 2.93-3.95) were significantly lower than those of carriers of HBV/Ae (6.09 log copies/mL; 95% CI, 4.24-7.64) or of carriers of HBV/D (5.48 log copies/mL; 95% CI, 4.06-7.02), regardless of the HBeAg status (P < .001). The most specific and frequent substitutions in 54 HBV/Aa isolates were double substitutions for T1809 (100%) and T1812 (96%) immediately upstream of the precore initiation codon, which would interfere with the translation of HBeAg in HBV/Aa infections. They were not detected in 57 HBV/Ae or 61 HBV/D isolates examined. The double mutation in the core promoter (T1762/A1764) was more frequent in both HBV/Aa (50%) and HBV/Ae (44%) than in HBV/D isolates (25%; P < .01), whereas the precore mutation (A1896) occurred in HBV/D isolates only (48%; P < .0001). In conclusion, the clearance of HBeAg from serum may occur by different mechanisms in HBV/Aa, HBV/Ae, and HBV/D infections, which may influence clinical manifestations in the Western countries where both genotypes A and D are prevalent. (Hepatology 2004;40:747-755.)
Functional impairment of myeloid and plasmacytoid dendritic cells of patients with chronic hepatitis B
Authors: van der Molen Renate G. , Sprengers Dave , Binda Rekha S. , de Jong Esther C. , Niesters Hubert G. M. , Kusters Johannes G. , Kwekkeboom Jaap , Janssen Harry L. A.
Published: August 2004

Dendritic cells (DC) play an important role in the induction of T-cell responses. We hypothesize that the hampered antiviral T-cell response in chronic hepatitis B patients is a result of impaired dendritic cell function. In this study, we compared the number, phenotype and functionality of two important blood precursor DC, myeloid DC (mDC) and plasmacytoid DC (pDC), of chronic hepatitis B patients with healthy volunteers. No differences in percentages of mDC and pDC in peripheral blood mononuclear cells were observed between chronic hepatitis B patients and healthy controls. The allostimulatory capacity of isolated and in vitro matured mDC, but not of pDC, was significantly decreased in patients compared to controls. Accordingly, a decreased percentage of mDC expressing CD80 and CD86 was observed after maturation, compared to controls. In addition, mDC of patients showed a reduced capacity to produce tumor necrosis factor α after a stimulus with synthetic double-stranded RNA and interferon γ. Purified pDC from patients produced less interferon α, an important antiviral cytokine, in response to stimulation with Staphylococcus aureus Cowan strain I than pDC isolated from controls. In conclusion, mDC and pDC are functionally impaired in patients with chronic hepatitis B. This might be an important way by which hepatitis B virus evades an adequate immune response, leading to viral persistence and disease chronicity. (Hepatology 2004;40:738-746.)
Hepatitis C infection and injection drug use: The role of hepatologists in evolving treatment efforts
Authors: Kresina Thomas F. , Seeff Leonard B. , Francis Henry
Published: August 2004

Treatment regimens for both substance abuse and hepatitis C infection are complex and evolving. New pharmacotherapy for opioid addiction allows for office-based treatment and, thus, an opportunity for expanded treatment in the context of hepatitis C infection. The current article addresses the newly evolving, complex issues in the medical management of hepatitis C and injection drug use. (Hepatology 2004;40:516-519.)
Role of the renin-angiotensin system in hepatic ischemia reperfusion injury in rats
Authors: Guo Luping , Richardson Katharine S. , Tucker Lindsay M. , Doll Mark A. , Hein David W. , Arteel Gavin E.
Published: August 2004

It has been shown that the renin-angiotensin system (RAS) plays key roles in the development of fibrosis in numerous organs, including the liver. Other studies have suggested that the RAS also may play roles in diseases of chronic inflammation. However, whether the RAS also can mediate acute inflammation in liver is unclear. The purpose of this study therefore was to determine the effect of the RAS inhibitors captopril and losartan on acute liver damage and inflammation caused by hepatic ischemia and subsequent reperfusion. Accordingly, male rats were subjected to 1 hour of hepatic ischemia (70%) followed by reperfusion; animals were killed 3, 8, or 24 hours after reperfusion. The effect of captopril or losartan (100 or 5 mg/kg intragastrically, respectively) was compared with that of vehicle (saline). The expression of angiotensinogen in liver increased fivefold 3 hours after reperfusion. Indices of liver damage and inflammation (e.g., alanine aminotransferase levels, pathological features, tumor necrosis factor-α levels, and intercellular adhesion molecule-1 expression) all were significantly elevated in vehicle-treated animals after hepatic ischemia and subsequent reperfusion. Ischemia and reperfusion also caused an increase in the accumulation of protein adducts of 4-hydroxynonenal, an index of oxidative stress. Captopril or losartan treatment showed profound protective effects under these conditions, significantly blunting the increase in all these parameters caused by ischemia and reperfusion. In conclusion, RAS inhibitors prevent acute liver injury in a model of inflammation caused by ischemia and reperfusion. These data further suggest that the RAS may play a key role in mediating such responses in the liver and suggest a novel role for this system. (Hepatology 2004;40:583-589.)
Effect of high-dose ursodeoxycholic acid on its biliary enrichment in primary sclerosing cholangitis
Authors: Rost Daniel , Rudolph Gerda , Kloeters-Plachky Petra , Stiehl Adolf
Published: August 2004

Ursodeoxycholic acid (UDCA) has beneficial effects in cholestatic liver diseases. In primary sclerosing cholangitis (PSC), there is evidence that high doses (±20 mg/kg) of UDCA may be more effective than average doses. Biliary enrichment of UDCA at such high doses may represent the decisive factor for its beneficial effect. Up to now it is not clear how high-dose UDCA correlates with its biliary enrichment and whether bacterial degradation of large amounts of UDCA may lead to an increased bacterial formation of more toxic hydrophobic bile acids. We determined the biliary bile acid composition in 56 patients with PSC including 30 patients with repeat bile samples treated with various doses of UDCA. At a UDCA dose of 10-13 mg/kg/d (n = 18) biliary UDCA represented 43.1% + 0.3% (mean + SD) of total bile acids; at a UDCA dose of 14-17 mg/kg (n = 14), its biliary content increased to 46.9% + 0.3%, at 18-21 mg/kg (n = 34) to 55.9% + 0.2%, at 22-25 mg/kg (n = 12) to 58.6% + 2.3%, and at 26-32 mg/kg (n = 8) to 57.7% + 0.4%. During UDCA treatment, the biliary content of all other bile acids was unchanged or decreased. In conclusion, biliary enrichment of UDCA increases with increasing dose and reaches a plateau at 22-25 mg/kg. There was no increase of toxic hydrophobic bile acids. If biliary enrichment of UDCA represents the decisive factor for its clinical effect, it seems likely that UDCA doses of up to 22-25 mg/kg may be more effective than lower doses. (Hepatology 2004;40:693-698.)
Hepatitis C virus NS5A-regulated gene expression and signaling revealed via microarray and comparative promoter analyses
Authors: Girard Sophie , Vossman Erik , Misek David E. , Podevin Philippe , Hanash Samir , Bréchot Christian , Beretta Laura
Published: August 2004

Most individuals exposed to hepatitis C virus (HCV) become chronically infected and are predisposed to liver disease. The mechanisms underlying viral persistence and disease progression are unknown. A role for the HCV NS5A protein in viral replication and interferon resistance has been demonstrated. To identify mechanisms affected by NS5A, we analyzed the gene expression of Huh7 cells expressing NS5A and control cells using oligonucleotide microarrays. A set of 103 genes (43 up-regulated, 60 down-regulated) whose expression was modified by at least twofold was selected. These included genes involved in cell adhesion and motility, calcium homeostasis, lipid transport and metabolism, and genes regulating immune responses. The finding of modulated expression of genes related to the TGF-β superfamily and liver fibrosis was observed. Interestingly, both the tumor necrosis factor and lymphotoxin beta receptors were down-regulated by NS5A. Similar data were obtained following expression of four NS5A mutants obtained from patients who were not responsive or were sensitive to interferon therapy. Through computational analysis, we determined that 39 of the 43 genes up-regulated by NS5A contained one or more nuclear factor κB (NF-κB) binding sites within their promoter region. Using the Gibbs sampling method, we also detected enrichment of NF-κB consensus binding sites in the upstream regions of the 43 coexpressed genes. Activation of NF-κB by NS5A was subsequently demonstrated in luciferase reporter assays. Adenovirus-mediated expression of IκBα reverted NS5A mediated up-regulation of gene expression. In conclusion, this study suggests a role of NS5A and NF-κB in HCV pathogenesis and related liver disease. Supplementary material for this article can be found on the Hepatology website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html). (Hepatology 2004;40:708-718.)
Proteomic signature corresponding to alpha fetoprotein expression in liver cancer cells
Authors: Yokoo Hideki , Kondo Tadashi , Fujii Kazuyasu , Yamada Tesshi , Todo Satoru , Hirohashi Setsuo
Published: August 2004

Alpha fetoprotein (AFP) has been implicated in the development of hepatocellular carcinoma and is considered to be a diagnostic and prognostic tumor marker. Because elevated expression of AFP is associated with many characteristics of hepatocellular carcinoma tissues, we hypothesized that multiple proteins may function in a coordinated manner with AFP. To identify such proteins, we performed global protein expression analysis, namely a proteomic study. The protein expression profiles of 9 AFP-producing liver cancer cell lines (JHH-5, HuH-1, PLC/PRL/5, Hep3B, HT-17, JHH-7, HuH-7, HepG2, Li-7) and 7 nonproducing liver cancer cell lines (HLE, JHH-6, Sk-Hep-1, JHH-4, HLF, RBE, SSP-25) were generated by fluorescence 2-dimensional difference gel electrophoresis. In fluorescence 2-dimensional difference gel electrophoresis, proteins are labeled with fluorescent dyes before electrophoresis for more accurate quantitative expression analysis. We identified 11 protein spots that distinguished AFP-producing cell lines from nonproducing cell lines by multivariate studies. The spots showed consistent alterations in amount in AFP-producing cell lines (6 up-regulated and 5 down-regulated). An additional 5 liver cancer cell lines (KIM-1, KYN-2, KYN-3, PH5-CH, PH5-T) also were correctly grouped with respect to their AFP production on the basis of the intensity of the 11 protein spots. The proteins corresponding to the 11 selected spots were identified by mass spectrometry and were categorized into 4 groups based on their known role in apoptosis, glucose metabolism, cytoskeletal organization, or translation. In conclusion, we found a novel association of AFP with other proteins. Their interaction should provide insight into the biology of AFP-producing hepatocellular carcinoma cells. (Hepatology 2004;40:609-617.)
Effects of contrast media on renal function in patients with cirrhosis: A prospective study
Authors: Guevara Mònica , Fernández-Esparrach Glòria , Alessandria Carlo , Torre Aldo , Terra Carlos , Montañà Xavier , Piera Carlos , Alvarez Maria Luisa , Jiménez Wladimiro , Ginès Pere , Arroyo Vicente
Published: August 2004

Patients with cirrhosis are frequently submitted to radiological procedures that require the administration of contrast media. Contrast media is a well-known cause of renal failure, particularly in the presence of some predisposing conditions. However, it is not known whether cirrhosis constitutes a risk factor for contrast media-induced renal failure. The aim of this study was to assess the possible nephrotoxicity of contrast media in patients with cirrhosis. In a first protocol, renal function was evaluated with sensitive methods (glomerular filtration rate using iothalamate I 125 clearance and renal plasma flow using iodohippurate I 131 clearance) before and 48 hours after the administration of contrast media in 31 patients with cirrhosis (20 with ascites, 5 with renal failure). Solute-free water clearance, urine sodium, prostaglandins, and markers of tubular damage were also measured. The administration of contrast media was not associated with significant changes in renal function tests, neither in the whole group of patients nor in patients with ascites or renal failure. Urinary prostaglandin E2 and N-acetyl-β-d-glucosaminidase increased significantly, but sodium and solute-free water excretion remained unchanged. In a second protocol, a different series of 60 patients with cirrhosis and renal failure were examined prospectively. No patient had renal failure due to contrast media. Only in 1 patient with septic shock was contrast media a possible contributing factor. In conclusion, the administration of contrast media is not associated with adverse effects on renal function in patients with cirrhosis. Cirrhosis does not appear to be a risk factor for the development of contrast media-induced nephrotoxicity. (Hepatology 2004;40:646-651.)
A dose-finding study of once-daily oral telbivudine in HBeAg-positive patients with chronic hepatitis B virus infection
Authors: Lai Ching-Lung , Lim Seng Gee , Brown Nathaniel A. , Zhou Xiao-Jian , Lloyd Deborah M. , Lee Yin-Mei , Yuen Man-Fung , Chao George C. , Myers Maureen W.
Published: August 2004

Current therapy for chronic hepatitis B is suboptimal as a result of limited durable response rates, cumulative viral resistance, and/or poor tolerability. Telbivudine has potent antiviral activity against hepatitis B virus (HBV) in vitro and in the woodchuck model and has a promising preclinical safety profile. In this first clinical study of telbivudine, safety, antiviral activity, and pharmacokinetics were assessed in 43 adults with hepatitis B e antigen-positive chronic hepatitis B. This placebo-controlled dose-escalation trial investigated 6 telbivudine daily dosing levels (25, 50, 100, 200, 400, and 800 mg/d); treatment was given for 4 weeks, with 12 weeks’ follow-up. Serum HBV DNA levels were monitored via quantitative polymerase chain reaction. The results indicate that telbivudine was well tolerated at all dosing levels, with no dose-related or treatment-related clinical or laboratory adverse events. telbivudine plasma pharmacokinetics were dose-proportional within the studied dose range. Marked dose-related antiviral activity was evident, with a maximum at telbivudine doses of 400 mg/d or more. In the 800mg/d cohort, the mean HBV DNA reduction was 3.75 log10 copies/mL at week 4, comprising a 99.98% reduction in serum viral load. Correspondingly, posttreatment return of viral load was slowest in the high-dose groups. Viral dynamic analyses suggested a high degree of efficiency of inhibition of HBV replication by telbivudine and helped refine selection of the optimal dose. In conclusion, these results support expanded clinical studies of this new agent for the treatment of hepatitis B. (Hepatology 2004;40:719-726.)
Classification and prediction of survival in hepatocellular carcinoma by gene expression profiling
Authors: Lee Ju-Seog , Chu In-Sun , Heo Jeonghoon , Calvisi Diego F. , Sun Zongtang , Roskams Tania , Durnez Anne , Demetris Anthony J. , Thorgeirsson Snorri S.
Published: August 2004

We analyzed global gene expression patterns of 91 human hepatocellular carcinomas (HCCs) to define the molecular characteristics of the tumors and to test the prognostic value of the expression profiles. Unsupervised classification methods revealed two distinctive subclasses of HCC that are highly associated with patient survival. This association was validated via 5 independent supervised learning methods. We also identified the genes most strongly associated with survival by using the Cox proportional hazards survival analysis. This approach identified a limited number of genes that accurately predicted the length of survival and provides new molecular insight into the pathogenesis of HCC. Tumors from the low survival subclass have strong cell proliferation and antiapoptosis gene expression signatures. In addition, the low survival subclass displayed higher expression of genes involved in ubiquitination and histone modification, suggesting an etiological involvement of these processes in accelerating the progression of HCC. In conclusion, the biological differences identified in the HCC subclasses should provide an attractive source for the development of therapeutic targets (e.g., HIF1a) for selective treatment of HCC patients. Supplementary material for this article can be found on the Hepatology Web site (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html) (Hepatology 2004;40:667-676.)