bluuebunny/crossref_metadata_2025 huggingface dataset:
Is Left Ventricular Diastolic Thickening Documented During Dobutamine and Pacing Stress Echocardiography Related to Myocardial Ischemia? An Animal Model Study
Authors: Schmidt André , De Almeida Filho Oswaldo César , Ayres‐Neto Elias Mello , Carneiro João José , Marin‐Neto José Antonio , Maciel Benedito Carlos
Published: July 2003

Transient increase in diastolic wall thickness (pseudohypertrophy) during pacing stress echocardiography has been reported in normal myocardium. To evaluate the occurrence of pseudohypertrophy and to investigate the contribution of myocardial ischemia on its production during pacing and dobutamine stress echocardiography, we produced a physiologically significant coronary stenosis in 14 open chest dogs. The stenosis in the circumflex artery was measured by quantitative coronary angiography (range: 50% to 89% reduction in luminal diameter), and no resting segmental wallmotion abnormalities were observed by epicardial echocardiography (short‐axis, papillary level). In each study, dobutamine (5–40 μg/kg/min) and pacing (up to 260 beats/min) were performed randomly. Positivity of stress echocardiography tests was quantitatively determined by a significant (P < 0.05) reduction or failure to increase in absolute and percent systolic wall thickening in the myocardial area supplied by the stenotic artery as compared to the left anterior descending (LAD) artery‐related areas. Diastolic wall thickness and left ventricular diastolic area were compared before and after each stress test in the circumflex and LAD artery‐related regions. Pseudohypertrophy was observed in 57% and 86% of dogs for pacing and dobutamine, respectively, in the circumflex region, and in 50% and 64% in the LAD region. Despite its increased incidence in the circumflex region, the augmented diastolic wall thickness did not correlate with coronary stenosis severity or stress test positivity, but correlated inversely with changes in left ventricular diastolic area. In addition, it correlated directly with changes in heart rate only for pacing. In conclusion, pseudohypertrophy was a frequent finding during pacing and dobutamine stress echocardiography tests but was not related to myocardial ischemia in this animal model.
Visualization of Early Events in Tumor Formation of Egfp–Transfected Rat Colon Cancer Cells in Liver
Authors: Mook Olaf R.F. , Van Marle Jan , Vreeling-Sindelárová Heleen , Jonges Remmet , Frederiks Wilma M. , Van Noorden Cornelius J.F.
Published: July 2003

Colon cancer preferentially metastasizes to the liver. To determine cellular backgrounds of this preference, we generated an enhanced green fluorescent protein (eGFP)–expressing rat adenocarcinoma cell line (CC531s) that forms metastases in rat liver after administration to the portal vein. Intravital videomicroscopy (IVVM) was used to visualize early events in the development of tumors in livers of live animals from the time of injection of the cancer cells up to 4 days afterward. Based on information obtained with IVVM, tissue areas were selected for further analysis using confocal laser scanning microscopy (CLSM), electron microscopy (EM), and electron tomography. It was shown that initial arrest of colon cancer cells in sinusoids of the liver was due to size restriction. Adhesion of cancer cells to endothelial cells was never found. Instead, endothelial cells retracted rapidly and interactions were observed only between cancer cells and hepatocytes. Tumors developed exclusively intravascularly during the first 4 days. In conclusion, initial steps in the classic metastatic cascade such as adhesion to endothelium and extravasation are not essential for colon cancer metastasis in liver.
Nuclear Factor κB Decoy Oligodeoxynucleotides Prevent Endotoxin–Induced Fatal Liver Failure in A Murine Model
Authors: Ogushi Ichiro , Iimuro Yuji , Seki Ekihiro , Son Gakuhei , Hirano Tadamichi , Hada Toshikazu , Tsutsui Hiroko , Nakanishi Kenji , Morishita Ryuichi , Kaneda Yasufumi , Fujimoto Jiro
Published: July 2003

Endotoxin syndrome is a systemic inflammatory response mediated by inflammatory cytokines. Nuclear factor κB (NF–κB) is the dominant regulator of the production of these cytokines by inflammatory cells. The aim of this study was to assess the efficacy of in vivo transfer of synthetic double–stranded oligodeoxynucleotides (ODN) with high affinity against NF–κB (NF–κB/decoy/ODN) as a therapeutic strategy for treating endotoxin–induced fatal liver injury. Liver injury was induced by administration of lipopolysaccharide (LPS) to Propionibacterium acnes–primed BALB/C mice. NF–κB/decoy/ODN was transferred into the portal vein using a fusigenic liposome with hemagglutinating virus of Japan. NF–κB/decoy/ODN was preferentially transferred to Kupffer cells, and activation of NF–κB after the LPS challenge was suppressed, leading to decreased inflammatory cytokine production. As a result, the massive necrosis and hepatocyte apoptosis observed in the control mice was dramatically attenuated and the survival rate improved. In conclusion, NF–κB/decoy/ODN transfer in vivo effectively suppressed endotoxin–induced fatal liver injury in mice.
A Common Set of Immediate–Early Response Genes in Liver Regeneration and Hyperplasia
Authors: Locker Joseph , Tian Jianmin , Carver Robert , Concas Danilo , Cossu Costanza , Ledda-Columbano Giovanna M. , Columbano Amedeo
Published: July 2003

Partial hepatectomy (PH) and some tumor–promoting agents stimulate hepatocyte cell proliferation, but each treatment acts through distinct transcription factors. We compared mouse immediate–early gene expression changes after PH with those induced by 1,4–bis[2–(3,5–dichoropyridyloxy)]benzene (TCPOBOP), a tumor–promoting liver mitogen. PH activates nuclear factor κB (NF–κB) and Stat3, whereas TCPOBOP is a ligand for the nuclear receptor, constitutive androstane receptor (CAR). RNA from 1 and 3 hours after each treatment was hybridized to a 9,000 complementary DNA (cDNA) microarray. Of about 6,000 messenger RNAs that had detectable expression, 127 showed reproducible up–regulation or down–regulation at a significant level. The TCPOBOP response was more discrete than the PH response; they amounted to 1% and 1.9% of positive hybridizations, respectively. Twenty–three genes were regulated only by TCPOBOP, 57 only by PH, and 59 by both treatments. More detailed analysis defined 16 clusters with common patterns of expression. These patterns and quantification of hybridization levels on the array were confirmed by Northern blots. TCPOBOP selectively activated expression of a number of detoxification enzymes. In conclusion, the genes that were regulated by both treatments suggest down–regulation of apoptosis, altered signal transduction, and early biogenesis of critical cell components.
Bilirubin Rinse: A Simple Protectant Against the Rat Liver Graft Injury Mimicking Heme Oxygenase-1 Preconditioning
Authors: Kato Yutaro , Shimazu Motohide , Kondo Mieko , Uchida Koji , Kumamoto Yusuke , Wakabayashi Go , Kitajima Masaki , Suematsu Makoto
Published: July 2003

Heme oxygenase (HO)–1 preconditioning through genetic or pharmacologic interventions was shown experimentally to improve posttransplant outcome of liver grafts. However, its clinical application requires careful consideration because of the complexity and economic costs of the procedures. This study aimed to examine if graft preconditioning with HO–1 could be substituted by a simple treatment with heme–degrading products such as bilirubin. Rats were pretreated with or without hemin, an HO–1 inducer for preconditioning. Their livers were harvested as grafts in University of Wisconsin (UW) solution for 16 hours at 4°C and followed by reperfusion ex vivo or by transplantation in vivo. The control grafts were also treated with a rinse buffer containing varied concentrations of unconjugated bilirubin with different time intervals. The HO–1–preconditioned grafts ex vivo exhibited a marked improvement of bile output and cell injury that was cancelled by blocking HO with zinc protoporphyrin–IX. The aggravation of the graft viability by the inhibitor was repressed by supplementation of bilirubin but not by that of carbon monoxide. Furthermore, a short–term rinse treatment with micromolar levels of bilirubin attenuated biliary dysfunction and cell injury of the grafts both ex vivo and in vivo even without HO–1 preconditioning. The protective effects of HO–1 preconditioning or bilirubin rinse appeared to involve its inhibitory effects on lipid peroxidation in hepatocytes. In conclusion, these results suggest that bilirubin rinse serves as a simple strategy to ameliorate hyperacute oxidative stress and hepatobiliary dysfunction of the transplanted grafts, mimicking effects of HO–1–mediated preconditioning.
Increased Carbon Monoxide Production in Patients With Cirrhosis With and Without Spontaneous Bacterial Peritonitis
Authors: De Las Heras Dara , Fernández Javier , Ginès Pere , Cárdenas Andres , Ortega Rolando , Navasa Miguel , Barberá Joan Albert , Calahorra Blas , Guevara Mónica , Bataller Ramón , Jiménez Wladimiro , Arroyo Vicente , Rodés Juan
Published: July 2003

Carbon monoxide, a product of the heme–oxygenase (HO) pathway, is an important endogenous vasoactive substance. Production of CO has not been assessed in human cirrhosis. The aim of this study was to assess production of CO in patients with cirrhosis with and without spontaneous bacterial peritonitis (SBP). CO concentration in the exhaled air and blood carboxyhemoglobin (COHb) levels, as estimates of total HO activity, were determined in 16 healthy subjects, 32 noninfected cirrhotic patients (20 with ascites), and 19 patients with SBP, all nonsmokers. Noninfected cirrhotic patients had a CO concentration in the exhaled air and COHb levels significantly higher compared with values of healthy subjects (2.3 ± 0.2 ppm vs. 0.7 ± 0.1 ppm and 1.0% ± 0.1% vs. 0.6% ± 0.1%, respectively; P < .05 for both). Patients with ascites had the highest values. Both CO concentration in the exhaled air and COHb levels were very high in patients with SBP (5.6 ± 0.6 ppm and 1.9% ± 0.2%; P < .01 vs. the other 2 groups) and decreased slowly after resolution of the infection, reaching values similar to those of noninfected patients 1 month after SBP. In patients with SBP, there was a significantly direct correlation between CO and plasma renin activity (PRA) (r = 0.71, P < .001). In conclusion, these results support the existence of increased CO production in human cirrhosis, which further increases in the setting of SBP. Increased CO production may participate in the disturbance of circulatory function that occurs during severe bacterial infections in cirrhosis.
Cytokines and Nash: A Pilot Study of the Effects of Lifestyle Modification and Vitamin E
Authors: Kugelmas Marcelo , Hill Daniell B. , Vivian Beverly , Marsano Luis , McClain Craig J.
Published: July 2003

There are few data evaluating plasma and/or peripheral blood monocyte cytokine concentrations/production or attempts to manipulate proinflammatory cytokines in nonalcoholic steatohepatitis (NASH). A pilot project in a general clinical research center evaluated the effects of a step 1 American Heart Association diet plus aerobic exercise with or without 800 IU of vitamin E daily on cytokine profiles and liver enzyme levels in 16 patients with biopsy–proven NASH. Biochemical assessment of liver function, lipid profiles, and body mass index significantly improved during the first 6 weeks of therapy and remained stable during the following 6 weeks. Plasma hyaluronic acid (HA) concentrations decreased in parallel with weight loss. Plasma tumor necrosis factor (TNF) concentrations were significantly elevated in patients with NASH and similar to patients with stable alcoholic cirrhosis but not as elevated as in patients with acute alcoholic steatohepatitis (AH). Although plasma TNF, interleukin 8 (IL–8), and IL–6 concentrations were all significantly elevated compared with control values, only plasma IL–6 levels significantly decreased with therapy. Peripheral blood monocyte TNF, IL–8, and IL–6 production was significantly elevated in patients with NASH but did not significantly decrease. Independent effects of vitamin E were not observed in this small sample. In conclusion, patients with NASH have dysregulated cytokine metabolism similar to, but less pronounced than abnormalities documented in AH. Cytokine values generally did not decrease significantly with weight loss with or without vitamin E over the duration of the study. Lifestyle modifications (low–fat diet and exercise) were associated with improvement in liver enzymes, cholesterol, and plasma HA levels in patients with NASH, whereas the level of vitamin E supplementation used in this short–term pilot study provided no apparent added benefit.
Effects of Proinflammatory Cytokines on Rat Organic Anion Transporters During Toxic Liver Injury and Cholestasis
Authors: Geier Andreas , Dietrich Christoph G. , Voigt Sebastian , Kim Suk-Kyum , Gerloff Thomas , Kullak-Ublick Gerd A. , Lorenzen Johann , Matern Siegfried , Gartung Carsten
Published: July 2003

Hepatobiliary transporters are down–regulated in toxic and cholestatic liver injury. Cytokines such as tumor necrosis factor α (TNF–α) and interleukin 1β (IL–1β) are attributed to mediate this regulation, but their particular contribution in vivo is still unknown. Thus, we studied the molecular mechanisms by which Ntcp, Oatp1, Oatp2, and Mrp2 are regulated by proinflammatory cytokines during liver injury. Rats were injected intraperitoneally with either carbon tetrachloride or endotoxin. Inactivation of TNF–α and IL–1β was achieved by repetitive intraperitoneal injection of etanercept and anakinra, respectively. Messenger RNA (mRNA) levels of transporters and binding activities as well as nuclear protein levels of Ntcp, Oatp2, and Mrp2 transactivators were determined 20 to 24 hours later. In contrast to IL–1β, TNF–α inactivation alone fully prevented down–regulation of Ntcp, Oatp1, and Oatp2 mRNA as well as reduced binding activity of hepatocyte nuclear factor 1 (HNF–1) in CCl4–induced toxic injury. In endotoxemia, down–regulation of Mrp2, and partially in case of Ntcp, could be prevented by IL–1β but not TNF–α blockade. However, inactivation of either cytokine led to preservation of HNF1 and partially of retinoid X receptor/retinoic acid receptor (RXR/RAR) binding activity. No effect of anticytokines was seen on pregnane X receptor (PXR) and constitutive androstane receptor (CAR) binding activity as well as nuclear protein mass. In conclusion, TNF–α represents the master cytokine responsible for HNF1–dependent down–regulation of Ntcp, Oatp1, and Oatp2 in CCl4–induced toxic liver injury. IL–1β predominates in a complex signaling network of Ntcp and Mrp2 regulation in cholestatic liver injury. In contrast to in vitro studies, HNF1 and RXR/RAR–independent mechanisms appear to be more important in regulation of Mrp2 and Ntcp gene expression in endotoxemia.
Massive Liver Growth in Mice Induced by Systemic Interleukin 6 Administration
Authors: Zimmers Teresa A. , McKillop Iain H. , Pierce Robert H. , Yoo Joo-Yeon , Koniaris Leonidas G.
Published: July 2003

The multifunctional cytokine interleukin 6 (IL–6) is expressed in a wide variety of disease states and pathologic processes. Mice deficient in IL–6 display abnormal and delayed liver regeneration and repair. Currently, IL–6 is thought to influence liver growth indirectly by priming hepatocytes to respond to growth factors such as hepatocyte growth factor (HGF) by inducing expression of HGF and by inhibiting hepatocyte apoptosis, as distinct from the direct mitotic effects of IL–6 on myeloid and other cell types. Here, we show that systemic administration of IL–6 using CHO cell tumors in nude mice results in dramatic hepatomegaly and hepatocyte hyperplasia in the absence of liver injury. Liver mass and liver to body mass ratios increased to 2 to 3 times normal because of proliferation of hepatocytes. Liver growth was associated with high levels of serum IL–6 and with activation of the IL–6–signaling pathway, including increased expression of IL–6 receptor–α/gp80, activation of the signal transducer and activator of transcription–3 (STAT–3), and mitogen–activated protein kinase (MAPK/ERK)–signaling pathways and induction of downstream target genes, including c–myc. HGF receptor and transforming growth factor α (TGF–α)/epidermal growth factor (EGF) receptor activation were decreased in hypertrophied livers, suggesting that IL–6–induced liver growth was independent of these known hepatocyte mitotic pathways. In conclusion, we suggest that IL–6 may function as a direct hepatic mitogen in vivo and, furthermore, that IL–6 warrants closer examination as a potent liver growth factor with potential clinical utility for increasing liver mass following injury.
Biochemical Surrogate Markers of Liver Fibrosis and Activity in A Randomized Trial of Peginterferon Alfa–2B and Ribavirin
Authors: Poynard Thierry , McHutchison John , Manns Michael , Myers Rob P. , Albrecht Janice
Published: July 2003

Liver fibrosis and activity indexes were validated in patients infected by hepatitis C virus (HCV) nontreated and treated by interferon. The aim was to validate their usefulness as surrogate markers of histologic features using the data of a randomized trial of combination peginterferon alfa–2b and ribavirin. Three hundred fifty–two patients who had had 2 interpretable liver biopsies and stored serum sample before and after treatment were selected. Two hundred eight patients received peginterferon alfa–2b 1.5 mcg per kg and ribavirin and 144 patients interferon alfa–2b 3 MU three times a week and ribavirin for 48 weeks. A fibrosis and an activity index combining 5 and 6 biochemical markers were assessed at baseline and at end of follow–up (24 weeks after treatment). The biochemical markers have significant predictive values both for the diagnosis of fibrosis and for activity. For the diagnosis of bridging fibrosis and/or moderate necroinflammatory activity, the area under the receiver operating characteristics curve of the activity index was 0.76 ± 0.03 at baseline and 0.82 ± 0.02 at end of follow–up. A cutoff of activity index at 0.30 (range, 0.00–1.00) had 90% sensitivity and 88% positive predictive value for the diagnosis of bridging fibrosis or moderate necroinflammatory activity. Sensitivity analyses with biopsy specimens of size greater than 15 mm suggest that a part of discordances between biochemical markers and histology were due to biopsy specimen sampling error. In conclusion, these biochemical markers of fibrosis and activity could be used as surrogate markers for liver biopsy in patients with chronic hepatitis C, both for the initial evaluation and for follow–up.
Long–Term Outcomes of Cirrhosis in Nonalcoholic Steatohepatitis Compared With Hepatitis C
Authors: Hui Jason M. , Kench James G. , Chitturi Shivakumar , Sud Archana , Farrell Geoffrey C. , Byth Karen , Hall Pauline , Khan Mahbub , George Jacob
Published: July 2003

Data on the long–term outcome of nonalcoholic steatohepatitis (NASH)–associated cirrhosis are few, and most reports describe cases of cryptogenic cirrhosis associated with risk factors for NASH but without histologic definition. In this prospective cohort study, we describe the long–term morbidity and mortality of 23 patients with NASH–associated cirrhosis defined by strict clinicopathologic criteria. Outcomes were compared with 46 age– and gender–matched patients with cirrhosis from chronic hepatitis C virus (HCV) infection: 23 untreated and 23 nonresponders to antiviral therapy. During follow–up (mean, 84 months; median, 60 months; range, 5–177 months), 9 of the 23 NASH–associated cirrhosis cases developed liver–related morbidity (8 ascites and/or encephalopathy, 1 variceal bleeding). The probability of complication–free survival was 83%, 77%, and 48% at 1, 3, and 10 years, respectively, and the cumulative probability of overall survival was 95%, 90%, and 84% at 1, 3, and 10 years, respectively. Five deaths were from liver failure, 1 from a non–liver–related cause. By multivariate analysis, bilirubin (P = .02) and platelet (P = .04) were independent predictors of complication–free survival; bilirubin (P = .05) was the only predictor for overall survival. After controlling for these factors, there was no difference in complication–free or overall survival between the NASH–cirrhosis cohort and either group of HCV–cirrhosis. However, 8 cases of liver cancer occurred in the HCV–cirrhosis groups compared with none among NASH cases. In conclusion, liver failure is the main cause of morbidity and mortality in NASH–associated cirrhosis. The prognosis is either similar or less severe than HCV–cirrhosis, except that HCC appears less common.
Cellular Retinol–Binding Protein–1 in Hepatocellular Carcinoma Correlates With β–Catenin, Ki–67 Index, and Patient Survival
Authors: Schmitt-Gräff Annette , Ertelt Viktoria , Allgaier Hans P. , Koelble Konrad , Olschewski Manfred , Nitschke Roland , Bochaton-Piallat Marie L. , Gabbiani Giulio , Blum Hubert E.
Published: July 2003

The cellular retinol–binding protein–1 (CRBP–1) plays a key role in the esterification and intercellular transfer of retinol. Byin situhybridization, immunohistochemistry, and confocal laser scanning microscopy (CLSM), we show that, in normal liver, CRBP–1 is strongly expressed in the cytoplasm of hepatic stellate cells (HSCs) and myofibroblasts (MFs) with only low CRBP–1 levels in hepatocytes. By contrast, in 196 hepatocellular carcinoma (HCC) specimens CRBP–1 expression in MFs was down–regulated in 83%. Patients with high CRBP–1 expression in MFs had a significantly higher 2–year survival as compared with patients with low CRBP–1 expression (52% vs. 29%, respectively;P= .034). An aberrant nuclear CRBP–1 accumulation resulting from cytoplasmic invagination was found in 29% of HCCs. Nuclear CRBP–1 staining correlated positively with a favorable tumor stage (Okuda stage I;P= .01) and negatively with the Ki–67+proliferation fraction (PF). A Ki–67+PF of ≥10% was associated with a lower 2–year survival probability as compared with patients with a Ki–67+PF of <10% (12% vs. 40%, respectively;P= .015). Prognosis did not correlate with the nuclear β–catenin expression. There was, however, a close correlation between nuclear CRBP–1 inclusions and nuclear β–catenin staining in HCCs (P= .008), suggesting a cross talk between CRBP–1 and the Wnt/wingless signal transduction pathway. In conclusion, our findings demonstrate that CRBP–1 detection may be useful for the discrimination between nonneoplastic and neoplastic liver cells and suggest that modulation of CRBP–1 expression in HCCs contributes to tumor growth and progressionviaretinoid–mediated signaling and disruption of cellular vitamin A homeostasis.
Fulminant Hepatitis After Grand Mal Seizures: Mechanisms and Role of Liver Transplantation
Authors: Ichai Philippe , Huguet Emmanuel , Guettier Catherine , Azoulay Daniel , Gonzalez Maria Eugenia , Fromenty Bernard , Masnou Pascal , Saliba Faouzi , Roche Bruno , Zeitoun Fahed , Castaing Denis , Samuel Didier
Published: July 2003

Fulminant liver failure is a rare complication of grand mal seizures with a high mortality, the prognosis being largely determined by the combination of the hepatic and neurologic insults. The mechanisms of acute liver failure secondary to grand mal epilepsy and the place of liver transplantation in this context are poorly defined and are the subject of this report. A series of 6 such patients is presented. All had a history of chronic primary or post–traumatic epilepsy and presented with acute liver failure shortly after a grand mal fit. Detailed accounts of background, presentation, and management are given and integrated with blood, radiologic, and histologic investigations. Two of the 6 patients survived, 1 making a full recovery and the other with neurologic sequelae. Two patients underwent liver transplantation but died with severe neurologic sequelae despite improving liver function. The remaining 2 patients were considered too unwell to undergo liver transplantation and died in multiple organ failure. Liver histology from needle biopsy and/or native liver explants identified lesions compatible with a combination of steatosis and necrosis. Factor V and transaminase levels may allow early identification of patients in whom liver function is likely to improve spontaneously. In conclusion, the mechanisms of liver failure occurring after grand mal seizures appear multifactorial, including hypoxia, steatosis, and drug–induced components. The neurological prognosis and overall survival of these patients remains poor.
Validation and Refinement of Survival Models for Liver Retransplantation
Authors: Rosen Hugo R. , Prieto Martin , Casanovas-Taltavull Teresa , Cuervas-Mons Valentin , Guckelberger Olaf , Muiesan Paolo , Strong Russell W. , Bechstein Wolf O. , O'Grady John , Zaman Atif , Chan Benjamin , Berenguer Joaquin , Williams Roger , Heaton Nigel , Neuhaus Peter
Published: July 2003

Orthotopic liver retransplantation (re–OLT) is highly controversial. The objectives of this study were to determine the validity of a recently developed United Network for Organ Sharing (UNOS) multivariate model using an independent cohort of patients undergoing re–OLT outside the United States, to determine whether incorporation of other variables that were incomplete in the UNOS registry would provide additional prognostic information, to develop new models combining data sets from both cohorts, and to evaluate the validity of the model for end–stage liver disease (MELD) in patients undergoing re–OLT. Two hundred eighty–one adult patients undergoing re–OLT (between 1986 and 1999) at 6 foreign transplant centers comprised the validation cohort. We found good agreement between actual survival and predicted survival in the validation cohort; 1–year patient survival rates in the low–, intermediate–, and high–risk groups (as assigned by the original UNOS model) were 72%, 68%, and 36%, respectively (P< .0001). In the patients for whom the international normalized ratio (INR) of prothrombin time was available, MELD correlated with outcome following re–OLT; the median MELD scores for patients surviving at least 90 days compared with those dying within 90 days were 20.75 versus 25.9, respectively (P= .004). Utilizing both patient cohorts (n = 979), a new model, based on recipient age, total serum bilirubin, creatinine, and interval to re–OLT, was constructed (whole model χ2= 105,P< .0001). Using the c–statistic with 30–day, 90–day, 1–year, and 3–year mortality as the end points, the area under the receiver operating characteristic (ROC) curves for 4 different models were compared. In conclusion, prospective validation and use of these models as adjuncts to clinical decision making in the management of patients being considered for re–OLT are warranted.
Favorable Prognosis of Chronic Hepatitis C After Interferon Therapy by Long–Term Cohort Study
Authors: Imazeki Fumio , Yokosuka Osamu , Fukai Kenichi , Saisho Hiromitsu
Published: July 2003

The prognosis of patients with chronic hepatitis C after interferon (IFN) therapy is still poorly defined. The present study evaluated the effect of IFN therapy on survival in a cohort of such patients. The study included 459 patients with biopsy–proven C–viral chronic liver disease who were followed for 8.2 ± 2.9 years (range, 7–183 months). Survival status was examined by medical records or direct questionnaires. Fifteen (14%) of 104 IFN–untreated patients and 33 (9%) of 355 patients treated with IFN died during follow–up. Among the treated patients, 4 (3%) of 116 with sustained virologic response and 29 (12%) of 239 without sustained virologic response died. Liver–related death was shown in 32 (67%) patients, and hepatocellular carcinoma (HCC) caused 25 (52%) of the 48 deaths. Multivariate Cox proportional regression analysis revealed that IFN treatment decreased the risk ratio for overall death to 0.521 (confidence interval [CI]: 0.263–1.034) and for liver–related death to 0.208 (CI: 0.088–0.495) compared with untreated patients, and sustained virologic response showed a decrease in the risk ratio for overall death to 0.219 (CI: 0.068–0.710) and for liver–related death to 0.030 (CI: 0.003–0.267). IFN treatment showed no association with liver–unrelated death. Furthermore, the standardized mortality ratios for all causes of death and liver–related death were reduced in IFN–treated patients compared with untreated patients (1.4 vs. 2.0 for total death and 7.9 vs. 19.7 for liver–related death). In conclusion, the present data suggest that IFN therapy has a long–term clinical benefit for patients with chronic hepatitis C patients by reducing liver–related death, especially in patients with sustained virologic response.
A Potent and Specific Morpholino Antisense Inhibitor of Hepatitis C Translation in Mice
Authors: McCaffrey Anton P. , Meuse Leonard , Karimi Mobin , Contag Christopher H. , Kay Mark A.
Published: July 2003

Hepatitis C virus (HCV) is an RNA virus infecting one in every 40 people worldwide. Current treatments are ineffective and HCV is the leading cause of liver failure leading to transplantation in the United States and Europe. Translational control of HCV is a prime therapeutic target. We assessed the inhibitory potential of morpholino phosphoramidate antisense oligonucleotides (morpholinos) on HCV translation by codelivering them with reporter plasmids expressing firefly luciferase under the translational control of the HCV internal ribosome entry site (IRES) into the livers of mice. Real–time imaging of HCV IRES luciferase reporter messenger RNA (mRNA) translation in living mice showed that a 20–mer complementary to nucleotides 345–365 of the IRES inhibited translation by greater than 95% for at least 6 days and showed mismatch specificity. No significant nonspecific inhibition of a cap–dependent luciferase or encephalomyocarditis virus (EMCV) IRES luciferase reporter translation was observed. Inhibition by the 20–mer morpholino was dose dependent, with 1 nmol/mouse giving the highest inhibition. In conclusion, morpholino antisense oligonucleotides are potent inhibitors of HCV IRES translation in a preclinical mouse model; morpholinos have potential as molecular therapeutics for treating HCV and other viral infections. The in vivo model described is a broadly applicable, straightforward, and rapid readout for inhibitor efficacy. As such, it will greatly facilitate the development of novel therapeutic strategies for viral hepatitis. Notably, the level of antisense inhibition observed in this in vivo model is similar to the maximal inhibition we have obtained previously with RNA interference in mice.
Cotransport of Reduced Glutathione With Bile Salts by Mrp4 (Abcc4) Localized to the Basolateral Hepatocyte Membrane
Authors: Rius Maria , Nies Anne T. , Hummel-Eisenbeiss Johanna , Jedlitschky Gabriele , Keppler Dietrich
Published: July 2003

The liver is the major source of reduced glutathione (GSH) in blood plasma. The transport protein mediating the efflux of GSH across the basolateral membrane of human hepatocytes has not been identified so far. In this study we have localized the multidrug resistance protein 4 (MRP4; ABCC4) to the basolateral membrane of human, rat, and mouse hepatocytes and human hepatoma HepG2 cells. Recombinant human MRP4, expressed in V79 hamster fibroblasts and studied in membrane vesicles, mediated ATP–dependent cotransport of GSH or S–methyl–glutathione together with cholyltaurine, cholylglycine, or cholate. Several monoanionic bile salts and the quinoline derivative MK571 were potent inhibitors of this unidirectional transport. The K
m values were 2.7 mmol/L for GSH and 1.2 mmol/L for the nonreducing S–methyl–glutathione in the presence of 5 μmol/L cholyltaurine, and 3.8 μmol/L for cholyltaurine in the presence of 5 mmol/L S–methyl–glutathione. Transport of bile salts by MRP4 was negligible in the absence of ATP or without S–methyl–glutathione. These findings identify a novel pathway for the efflux of GSH across the basolateral hepatocyte membrane into blood where it may serve as an antioxidant and as a source of cysteine for other organs. Moreover, MRP4–mediated bile salt transport across the basolateral membrane may function as an overflow pathway during impaired bile salt secretion across the canalicular membrane into bile. In conclusion, MRP4 can mediate the efflux of GSH from hepatocytes into blood by cotransport with monoanionic bile salts.
Ethanol Metabolism Results in A G2/M Cell–Cycle Arrest in Recombinant Hep G2 Cells
Authors: Clemens Dahn L. , Calisto Lilian E. , Sorrell Michael F. , Tuma Dean J.
Published: July 2003

Previous studies using the Hep G2–based VA cells showed that ethanol metabolism resulted in both cytotoxicity and impaired DNA synthesis, causing reduced accumulation of cells in culture. To further characterize the ethanol oxidation–mediated impairment of DNA synthesis we analyzed the cell–cycle progression of VA cells. These studies showed approximately a 6–fold increase in the percentage of cells in the G2/M phase of the cell cycle after 4 days of ethanol exposure. The G2/M transition requires activity of the cyclin–dependent kinase, Cdc2. Cdc2 is positively regulated by association with cyclin B1, and negatively regulated by phosphorylation of amino acids Thr14 and Tyr15. Immunoblot analysis revealed that ethanol metabolism had little affect on total Cdc2 content in these cells, but resulted in the accumulation of up to 20 times the amount of cyclin B1, indicating that cyclin B1 was available for formation of Cdc2/cyclin B1 complexes. Co–immunoprecipitation revealed that 6 times more Cdc2/cyclin B1 complexes were present in the ethanol–treated cells compared with the controls. Investigation of the phosphorylation state of Cdc2 revealed that ethanol oxidation increased the amount of the phosphorylated inactive form of Cdc2 by approximately 3–fold. Thus, the impairment in cell–cycle progression could not be explained by a lack of cyclin B1, or the ability of Cdc2 and cyclin B1 to associate, but instead resulted, at least in part, from impaired Cdc2 activity. In conclusion, ethanol oxidation by VA cells results in a G2/M cell–cycle arrest, mediated by accumulation of the phosphorylated inactive form of Cdc2.
Neutrophils Aggravate Acute Liver Injury During Obstructive Cholestasis in Bile Duct-Ligated Mice
Authors: Gujral Jaspreet S. , Farhood Anwar , Bajt Mary Lynn , Jaeschke Hartmut
Published: July 2003

Obstruction of the common bile duct in a variety of clinical settings leads to cholestatic liver injury. An important aspect of this injury is hepatic inflammation, with neutrophils as the prominent cell type involved. However, the pathophysiologic role of the infiltrating neutrophils during cholestatic liver injury remains unclear. Therefore, we tested the hypothesis that neutrophils contribute to the overall pathophysiology by using bile duct–ligated (BDL) wild–type animals and mice deficient in the β2 integrin CD18. In wild–type animals, neutrophils were activated systemically as indicated by the increased expression of Mac–1 (CD11b/CD18) and L–selectin shedding 3 days after BDL. Histologic evaluation (48 ± 10% necrosis) and plasma transaminase levels showed severe liver injury. Compared with sham–operated controls (< 10 neutrophils per 20 high–power fields), large numbers of neutrophils were present in livers of BDL mice (425 ± 64). About 60% of these neutrophils had extravasated into the parenchyma. In addition, a substantial number of extravasated neutrophils were found in the portal tract. In contrast, Mac–1 was not up–regulated and plasma transaminase activities and the area of necrosis (21 ± 9%) were significantly reduced in CD18–deficient animals. These mice had overall 62% less neutrophils in the liver. In particular, extravasation from sinusoids and portal venules (PV) was reduced by 91% and 47%, respectively. Immunohistochemical staining for chlorotyrosine, a marker of neutrophil–derived oxidant stress, was observed in the parenchyma of BDL wild–type but not CD18–deficient mice. In conclusion, neutrophils aggravated acute cholestatic liver injury after BDL. This inflammatory injury involves CD18–dependent extravasation of neutrophils from sinusoids and reactive oxygen formation.
Cyp2e1 Activity Before and After Weight Loss in Morbidly Obese Subjects With Nonalcoholic Fatty Liver Disease
Authors: Emery Maurice G. , Fisher Jeannine M. , Chien Jenny Y. , Kharasch Evan D. , Dellinger Patchen E. , Kowdley Kris V. , Thummel Kenneth E.
Published: July 2003

Previous studies suggest that hepatic cytochrome P450 2E1 (CYP2E1) activity is increased in individuals with chronic alcoholism, nonalcoholic steatohepatitis (NASH), and morbid obesity, and may contribute to liver disease. We studied 16 morbidly obese subjects with varying degrees of hepatic steatosis and 16 normal–weight controls. Obese subjects were evaluated at baseline, 6 weeks, and 1 year after gastroplasty, a procedure that leads to weight loss. Hepatic CYP2E1 activity was assessed by determination of the clearance of chlorzoxazone (CLZ), an in vivo CYP2E1–selective probe. Liver biopsy tissue was obtained during surgery for histopathology. Both the total and unbound oral CLZ clearance (Clu/F) was elevated approximately threefold in morbidly obese subjects compared with controls (P < .001). The Clu/F was significantly higher among subjects with steatosis involving >50% of hepatocytes, compared with those with steatosis in ≤50% of hepatocytes (P = .02). At postoperative week 6 and year 1, the median body mass index (BMI) of subjects who underwent gastroplasty decreased by 11% and 33%, total oral CLZ clearance declined by 16% (P < .01) and 46% (P < .05), and Clu/F decreased by 18% (P < .05) and 35% (P = .16), respectively. Moreover, those subjects with a year 1 BMI <30 kg/m2 exhibited a median Clu/F that was 63% lower (P = .02) than the respective clearance for all other subjects. In conclusion, hepatic CYP2E1 activity is up–regulated in morbidly obese subjects. A positive association between the degree of steatosis and CYP2E1 activity preoperatively and between the extent of obesity and CYP2E1 activity postoperatively, suggests that CYP2E1 induction is related to or caused by hepatic pathology that results from morbid obesity.
A Liver Tolerates A Portal Antigen by Generating Cd11c+ Cells, Which Select Fas Ligand+ Th2 Cells Via Apoptosis
Authors: Watanabe Tomohiro , Katsukura Hiroaki , Shirai Yasuhiko , Yamori Masashi , Nishi Toshiki , Chiba Tsutomu , Kita Toru , Wakatsuki Yoshio
Published: July 2003

Administration of an antigen (Ag) per oral route leads to apoptosis of Ag–specific CD4+ T cells and to development of Th2 cells expressing Fas ligand (FasL) in the liver. We determined whether presentation of an ingested Ag in the liver alone was enough to select these FasL+Th2 cells and explored how this selection was achieved in the liver. Ovalbumin (OVA) administered orally was colocalized with class II+ cells in the periportal and parenchymal area of the liver. On coculture with naive OVA–specific CD4+ T cells, hepatic CD11c+ cells from mice fed OVA generated Ag–specific Th2 cells. This was achieved by apoptosis of CD4+ T cells, decrease of interleukin 12 (IL–12) secretion, and increase of IL–18 secretion by the CD11c+ cells. Addition of IL–12 to this coculture prevented apoptosis of the CD4+ T cells, which was associated with up–modulation of IL–2 receptor β chain expression. Administration of IL–12 to mice fed OVA prevented apoptosis of OVA–specific CD4+ T cells in the liver. Moreover, adoptive transfer of hepatic CD11c+ cells from mice fed OVA together with OVA–specific CD4+ T cells led to development of Th2 cells as well as apoptosis of the transferred CD4+ T cells in the lymph nodes of the recipient mice on immunization with OVA. In conclusion, presentation of an ingested Ag by hepatic CD11c+ cells selects Th2 cells resistant to apoptosis in the liver, which is mediated in part by down–regulation of IL–12 secretion by the former cells.
Dynamics of Alanine Aminotransferase During Hepatitis C Virus Treatment
Authors: Ribeiro Ruy M. , Layden-Almer Jennifer , Powers Kimberly A. , Layden Thomas J. , Perelson Alan S.
Published: July 2003

Studies of the kinetics of hepatitis C virus (HCV) decline during interferon (IFN)–based therapy have led to insights into treatment efficacy. However, the kinetics of serum alanine aminotransferase (ALT), an enzyme used as a surrogate of liver damage, have not been closely monitored, and it is not known if they correlate with those of HCV RNA. Here we describe the associations between ALT and HCV dynamics. We analyzed 35 patients treated daily with 10 mIU IFN–α2b with or without ribavarin for 28 days followed by standard IFN/ribavirin therapy. Patients exhibited 4 patterns of ALT change: (1) exponential decay of ALT, (2) transient increase in ALT followed by a decrease to pretreatment or normal levels, (3) increase in ALT to a new level, and (4) no significant change. By simultaneously modeling HCV and ALT dynamics, we successfully fit the observed changes. We found ALT decays with t1/2= 12.7 hours. The transient increase in ALT observed in some patients suggested a mild hepatotoxic effect of IFN. However, patients with a smaller initial ALT increase achieved higher rates of viral negativity by week 72 (P= .02). The week–4 ALT decline correlated with the HCV log drop (P= .006) and the efficacy of therapy (P= .025). In conclusion, our results suggest the use of ALT as a surrogate marker for treatment effect in patients with elevated ALT.
Mechanism of β1–Integrin-Mediated Hepatoma Cell Growth Involves P27 and S–Phase Kinase-Associated Protein 2
Authors: Zhang Hao , Ozaki Iwata , Mizuta Toshihiko , Yoshimura Tohru , Matsuhashi Sachiko , Hisatomi Akitaka , Tadano Jutaro , Sakai Takahiro , Yamamoto Kyosuke
Published: July 2003

Although cooperative interactions between growth factors and integrins, cell surface receptors for extracellular matrices (ECM), have been reported, little is known about the interaction between hepatocyte growth factor (HGF) and integrin in hepatoma cells. We investigated the effects and mechanisms of integrin on the proliferation of hepatoma cells regulated by HGF. Human HepG2 hepatoma cells stably transfected with β1–integrin were treated with HGF and compared with parental and mock–transfected control cells. Cell proliferation and expression of cyclin–dependent kinase (Cdk) inhibitors and S–phase kinase–associated protein 2 (Skp2), were investigated. HGF dose–dependently suppressed the proliferation of parental and mock–transfected HepG2 cells. However, cells overexpressing β1–integrin exhibited increased proliferation in response to HGF. Although HGF increased p27 and decreased Skp2 expression in the parental and mock–transfected cells, the p27 and Skp2 levels in cells overexpressing β1–integrin were not altered by HGF. Interestingly, HepG2 cells overexpressing β1–integrin showed increased Skp2 expression. Furthermore, HGF did not reduce the proliferation of HepG2 cells transfected with antisense p27 or sense Skp2. Thus, HGF suppresses HepG2 cell proliferation by directly increasing p27 expression and indirectly decreasing Skp2 expression, and β1–integrin modulates the responsiveness of hepatoma cells to HGF via a p27–dependent manner by increasing Skp2. In conclusion, these results strongly suggest that integrin–mediated signals from the ECM can modulate growth factor–mediated signals in hepatoma cells, and may contribute to the growth of hepatocellular carcinomas.
A Simple Noninvasive Index Can Predict Both Significant Fibrosis and Cirrhosis in Patients With Chronic Hepatitis C
Authors: Wai Chun-Tao , Greenson Joel K. , Fontana Robert J. , Kalbfleisch John D. , Marrero Jorge A. , Conjeevaram Hari S. , Lok Anna S.-F.
Published: July 2003

Information on the stage of liver fibrosis is essential in managing chronic hepatitis C (CHC) patients. However, most models for predicting liver fibrosis are complicated and separate formulas are needed to predict significant fibrosis and cirrhosis. The aim of our study was to construct one simple model consisting of routine laboratory data to predict both significant fibrosis and cirrhosis among patients with CHC. Consecutive treatment–naive CHC patients who underwent liver biopsy over a 25–month period were divided into 2 sequential cohorts: training set (n = 192) and validation set (n = 78). The best model for predicting both significant fibrosis (Ishak score ≥ 3) and cirrhosis in the training set included platelets, aspartate aminotransferase (AST), and alkaline phosphatase with an area under ROC curves (AUC) of 0.82 and 0.92, respectively. A novel index, AST to platelet ratio index (APRI), was developed to amplify the opposing effects of liver fibrosis on AST and platelet count. The AUC of APRI for predicting significant fibrosis and cirrhosis were 0.80 and 0.89, respectively, in the training set. Using optimized cut–off values, significant fibrosis could be predicted accurately in 51% and cirrhosis in 81% of patients. The AUC of APRI for predicting significant fibrosis and cirrhosis in the validation set were 0.88 and 0.94, respectively. In conclusion, our study showed that a simple index using readily available laboratory results can identify CHC patients with significant fibrosis and cirrhosis with a high degree of accuracy. Application of this index may decrease the need for staging liver biopsy specimens among CHC patients.
Antimycin A-Induced Defenestration in Rat Hepatic Sinusoidal Endothelial Cells
Authors: Braet Filip , Muller Michael , Vekemans Katrien , Wisse Eddie , Le Couteur David G.
Published: July 2003

Liver sinusoidal endothelial cells (LSECs) possess fenestrae arranged in sieve plates. Hepatic endothelial fenestrae are open pores approximately 100 to 200 nm in diameter. Alterations in their number or diameter by hormones, xenobiotics, and diseases have important implications for hepatic microcirculation and function. Numerous reports of hepatotoxin–induced defenestration suggest that the cytoskeleton and the energy status of hepatic endothelial cells play a key role in the regulation of fenestrae. Therefore, we investigated the effect of antimycin A, an inhibitor of mitochondrial energy production, on the number of fenestrae in cultured LSECs using high–resolution microscopy and immunocytochemistry. Prolonged incubation (greater than 30 min) with antimycin A resulted in defenestrated cells and coincided with the appearance of F–actin dots, whereas the distribution of G–actin remained unchanged. Adenosine triphosphate (ATP) was depleted dramatically to less than 5% within 30 minutes within the LSECs. After treatment with antimycin A, unusual elevated fenestrated complexes were apparent, organized as a meshwork of anastomosing fenestrae at the center of and above the sieve plates. The position and appearance of these novel structures and their association with defenestration suggest that they are implicated in the process of defenestration. In conclusion, the results of experiments with antimycin A suggest that ATP is needed to maintain fenestrae and the underlying fenestrae–associated cytoskeleton rings that maintain fenestrae patency. Antimycin A–induced defenestration of LSECs is associated with the development of a structure in the sieve plate that appears to be intrinsically involved in defenestration.