bluuebunny/crossref_metadata_2025 huggingface dataset:
Acarbose Alone or in Combination With Ethanol Potentiates the Hepatotoxicity of Carbon Tetrachloride and Acetaminophen in Rats
Authors: Wang Pei-Yu , Kaneko Takashi , Wang Yuan , Sato Akio
Published: July 2004

Acarbose reduces the absorption of monosaccharides derived from dietary carbohydrates, which play an important role in the metabolism and toxicity of some chemical compounds. We studied the effects of acarbose on the hepatotoxicity of carbon tetrachloride (CCl4) and acetaminophen (AP) in rats, both of which exert their toxic effects through bioactivation associated with cytochrome P450 2E1 (CYP2E1). Male Sprague–Dawley rats were kept on a daily ration (20 g) of powdered chow diet containing 0, 20, 40, or 80 mg/100 g of acarbose, with drinking water containing 0% or 10% of ethanol (vol/vol). Three weeks later, the rats were either killed for an in vitro metabolism study or challenged with 0.50 g/kg CCl4 orally or 0.75 g/kg AP intraperitoneally. The ethanol increased the hepatic microsomal CYP2E1 level and the rate of dimethylnitrosamine (DMN) demethylation. The 40– or 80–mg/100 g acarbose diet, which alone increased the CYP2E1 level and the rate of DMN demethylation, augmented the enzyme induction by ethanol. The 40– or 80–mg/100 g acarbose diet alone potentiated CCl4 and AP hepatotoxicity, as evidenced by significantly increased levels of both alanine transaminase (ALT) and aspartate transaminase (AST) in the plasma of rats pretreated with acarbose. Ethanol alone also potentiated the toxicity of both chemicals. When the 40– or 80–mg/100 g acarbose diet was combined with ethanol, the ethanol–induced potentiation of CCl4 and AP hepatotoxicity was augmented. Our study demonstrated that high doses of acarbose, alone or in combination with ethanol, can potentiate CCl4 and AP hepatotoxicity in rats by inducing hepatic CYP2E1.
Acute Effect of Leptin on Hepatic Glycogenolysis and Gluconeogenesis in Perfused Rat Liver
Authors: Nemecz Martin , Preininger Kurt , Englisch Rainer , Fürnsinn Clemens , Schneider Barbara , Waldhäusl Wernes , Roden Michael
Published: July 2004

Leptin circulates in blood and is involved in body weight control primarily via hypothalamic receptors. To examine its direct metabolic action, effects of short–term portal leptin infusion: 1) on postprandial basal and epinephrine–stimulated glycogenolysis; and 2) on postabsorptive lactate–stimulated gluconeogenesis were studied in isolated perfused rat livers. Incremental epinephrine (150 pmol · min-1 · g-1 liver)–stimulated glucose release (in μmol/g liver within 30 minutes; control: 28.3 ± 2.8) was suppressed (P < .05) by 44% (15.8 ± 1.6), by 48% (14.6 ± 4.1), and by 53% (13.3 ± 2.1) during insulin (3 pmol · min-1 · g-1 liver), leptin (30 pmol · min-1 · g-1 liver), and simultaneous leptin + insulin infusion. Perfusate cyclic adenosine monophosphate increased approximately twofold during epinephrine stimulation in all groups. Neither leptin nor insulin affected hepatic lactate production, bile flow, or portal pressure in the fed state. In the postabsorptive state (20–hour fasting), rates of lactate (10 mmol/L)–dependent hepatic glucose release (in μmol · min-1 · g-1 liver; control: 0.12 ± 0.01) were increased (P < .01) to 0.35 ± 0.02 and to 0.24 ± 0.01 by glucagon (3 pmol · min-1 · g-1 liver) and by leptin (15 pmol · min-1 · g-1 liver), respectively. In parallel, lactate uptake rates (in μmol · min-1 · g-1 liver) were higher in the presence of both glucagon (0.90 ± 0.03) and leptin (0.84 ± 0.02) compared with control (0.68 ± 0.04). In conclusion, leptin modulates hepatic glucose fluxes and may contribute to direct humoral regulation of liver glycogen stores in the fasted as well as in the fed state.
Mice Deficient in the Urea–Cycle Enzyme, Carbamoyl Phosphate Synthetase I, Die During the Early Neonatal Period From Hyperammonemia
Authors: Schofield Paul J. , Schofield Paul J. , Cox Timothy M. , Caskey Thomas C. , Wakamiya Maki
Published: July 2004

Ammonia liberated during amino acid catabolism in mammals is highly neurotoxic and is detoxified by the five enzymes of the urea cycle that are expressed within the liver. Inborn errors of each of the urea cycle enzymes occur in humans. Carbamoyl phosphate synthetase I (CPSase I; EC 6.3.4.16) is located within the inner mitochondrial matrix and catalyzes the initial rate–limiting step of the urea cycle. Unless treated, complete deficiency of CPSase I, a rare autosomal recessive disease, causes death in newborn infants. Survivors are often mentally retarded and suffer frequent hyperammonemic crises during intercurrent illness or other catabolic stresses. Biochemically, CPSase I deficiency is characterized by high levels of blood ammonia, glutamine, and alanine, with low or absent citrulline and arginine levels. As a first step toward the development of gene therapy directed to the hepatocyte, we have generated a CPSase I-deficient mouse by gene targeting. Mice with homozygous disruption of CPSase I (CPSase [-/-] mice) die within 36 hours of birth with overwhelming hyperammonemia, and without significant liver pathology. This animal is a good model of human CPSase I deficiency.
Release of Osmolytes From Perfused Rat Liver on Perivascular Nerve Stimulation: α–Adrenergic Control of Osmolyte Efflux From Parenchymal and Nonparenchymal Liver Cells
Authors: Vom Dahl Stephan , Bode Johannes Georg , Reinehr Roland Michael , Mönnighoff Irmhild , Kubitz Ralf , Häussinger Dieter
Published: July 2004

The effects of perivascular nerve stimulation and phenylephrine on osmolyte release were studied in the intact perfused rat liver and isolated liver parenchymal cells (PC) and nonparenchymal cells. In the perfused liver, electrical stimulation of perivascular nerves (20 Hz/2 ms/20 V) led to a phentolamine–sensitive increase of cell hydration by 6.5% ± 1.2% (n = 3) and a transient phentolamine–sensitive stimulation of taurine and inositol, but not betaine, release. These nerve effects were mimicked by phenylephrine, but not prostaglandin F2α, and were not affected by sodium nitroprusside (SNP) or ibuprofen. Nerve stimulation-induced taurine, but not inositol, release was inhibited by 4,4′–di–isothiocyanatostilbene–2,2′–disulphonic acid (DIDS) (50 μmol/L). Single–cell fluorescence studies with isolated liver PC, Kupffer cells (KC), sinusoidal endothelial cells (SEC), and hepatic stellate cells (HSC) revealed that phenylephrine induced an increase in cytosolic free Ca2+ only in PC and HSC, but not in KC and SEC, whereas extracellular uridine triphosphate (UTP) produced Ca2+ transients/oscillations in all liver cell types studied. Phenylephrine had no effect on osmolyte release from isolated KC and SEC, but increased taurine (but not inositol) release from PC and inositol (but not taurine) efflux from HSC. The data suggest that: 1) liver cell hydration and—consecutively—osmolyte content are modulated by hepatic nerves via an α–adrenergic mechanism, which does not involve eicosanoids or hemodynamic changes; 2) that PC and HSC are the primary targets for nerve–dependent α–adrenergic activation, whereas 3) KC and SEC probably do not express α–adrenoceptors coupled to Ca2+ mobilization or osmolyte efflux.
Different Lobular Distributions of Altered Hepatocyte Tight Junctions in Rat Models of Intrahepatic and Extrahepatic Cholestasis
Authors: Kawaguchi Takumi , Sakisaka Shotaro , Sata Michio , Mori Michio , Tanikawa Kyuichi
Published: July 2004

Hepatocyte tight junctions (TJs), the only intercellular barrier between the sinusoidal and the canalicular spaces, play a key role in bile formation. Although hepatocyte TJs are impaired in cholestasis, attempts to localize the precise site of hepatocyte TJ damage by freeze–fracture electron microscopy have produced limited information. Recently, several TJ–associated proteins like ZO–1 and 7H6 have been identified and characterized. Immunolocalization of 7H6 appears to closely correlate with paracellular permeability. We used rat models of intrahepatic cholestasis by ethinyl estradiol (EE) treatment and extrahepatic cholestasis by bile duct ligation (BDL) to precisely determine the site of TJ damage. Alterations in hepatocyte TJs were assessed by double–immunolabeling for 7H6 and ZO–1 using a confocal laser scanning microscope. In control rats, immunostaining for 7H6 and ZO–1 colocalized to outline bile canaliculi in a continuous fashion. In contrast, 7H6 and ZO–1 immunostaining was more discontinuous, outlining the bile canaliculi after BDL. Immunostaining for 7H6, not ZO–1, decreased and predominantly appeared as discrete signals in the submembranous cytoplasm of periportal hepatocytes after BDL. After EE treatment, changes in immunostaining for 7H6 and ZO–1 were similar to those seen in periportal hepatocytes after BDL, but distributed more diffusely throughout the lobule. This study is the first to demonstrate that impairment of hepatocyte TJs occurs heterogenously in the liver lobule after BDL and suggests that BDL and EE treatments produce different lobular distributions of increased paracellular permeability.
Detection of Hepatitis C Virus in Paraffin–Embedded Liver Biopsies of Patients Negative for Viral Rna in Serum
Authors: Dries Volker , von Both Ingo , Müller Marion , Gerken Guido , Schirmacher Peter , Odenthal Margarete , Bartenschlager Ralf , Drebber Uta , zum Büschenfelde Karl-Hermann Meyer , Dienes Hans Peter
Published: July 2004

The diagnosis of hepatitis C is based on serological testing for antibodies against various epitopes of the hepatitis C virus (HCV) and detection of HCV RNA in serum, because anti–HCV antibodies alone cannot discriminate patients who are infectious from those who have resolved the infection. If HCV RNA is not detected, which is the case in at least 20% of enzyme immunoassay (EIA)–positive patients, diagnosis remains unclear in a state of disease possibly well suited for therapeutic intervention. Therefore, we investigated if detection of HCV antigens or HCV RNA in routinely processed, formalin–fixed and paraffin–embedded (ffpe) liver biopsy specimens of patients positive for anti–HCV, but negative for HCV RNA in serum, could confirm diagnosis in this serological constellation. We detected HCV RNA by reverse–transcription polymerase chain reaction (RT–PCR) in 27 (61%) of 44 ffpe liver biopsies from EIA–positive, but HCV–RNA-seronegative, patients. Testing of 18 of these biopsies by a panel of polyclonal antibodies against structural and nonstructural HCV proteins revealed positive immunostaining in 6 cases (33%), which were also positive by RT–PCR. Most biopsies showed necroinflammation compatible with chronic hepatitis C, and the detection of tissue HCV RNA correlated significantly with a higher grade of inflammatory activity. Detectability of HCV RNA did not correlate with HCV subtype. In conclusion, the search for HCV RNA by RT–PCR within the liver biopsy specimen can establish rapid and unequivocal diagnosis of hepatitis C in at least 60% of anti–HCV antibody-positive patients who are seronegative for HCV RNA, and thus may help to avoid repeated testing and delayed therapy. Tissue RT–PCR may also be more efficient than serological testing for surveillance of interferon therapy response, because ongoing chronic active hepatitis C is clearly demonstrated in the absence of detectable serum HCV RNA.
Transient Selection of A Hepatitis B Virus Polymerase Gene Mutant Associated With A Decreased Replication Capacity and Famciclovir Resistance
Authors: Pichoud Christian , Seignères Béatrice , Wang Zhirui , Trépo Christian , Zoulim Fabien
Published: July 2004

Prolonged therapy for chronic hepatitis B (HBV) with nucleoside analogs may result in the emergence of HBV mutants resistant to antivirals. Here, we describe the transient selection of an HBV polymerase gene mutant that was associated with viral persistence in an immune competent patient treated with famciclovir. Viral polymerase gene sequence was analyzed directly on polymerase chain reaction (PCR) products and also after cloning. The results showed the transient selection of a V542I mutant in the C domain of the viral polymerase. This mutation was associated with a stop codon at amino acid position 199 in the overlapping S gene. The mutated sequence was subcloned in a vector expressing the entire HBV pregenome to study its replication capacity after transient transfection in cultured hepatoma cells. The results showed that the V542I mutant has a decreased replication capacity compared with wild type virus and does not produce HBsAg. The sensitivity of the V542I mutant to penciclovir, the active metabolite of famciclovir, was further studied in tissue culture. This mutant was shown to be resistant to penciclovir, but remained sensitive to lamivudine, as was subsequently observed in vivo. These findings indicate that a prolonged administration of famciclovir may allow for the selection of HBV polymerase gene mutants in immune competent patients. The impaired replication capacity of this V542I mutant may have contributed to the absence of outgrowth of this viral strain in vivo. The study of the in vitro sensitivity of HBV polymerase mutants to nucleoside analogs will be important to design new anti–HBV strategies.(Hepatology 1999;29:230–237.)
Kinetics of Hepatitis B Surface Antigen-Specific Immune Responses in Acute and Chronic Hepatitis B or After Hbs Vaccination: Stimulation of The In Vitro Antibody Response by Interferon Gamma
Authors: Böcher Wulf Otto , Herzog-Hauff Sabine , Schlaak Jörg , zum Büschenfelde Karl-Hermann Meyer , Löhr Hanns Friedrich
Published: July 2004

Because cellular and humoral immune responses against the hepatitis B virus (HBV) surface antigen (HBs) might be crucial to overcome HBV infection, HBs–specific B– and T–cell responses of HBV patients and HBs vaccine recipients were analyzed quantitatively and functionally. In patients with acute hepatitis B (AHB), transient high anti–HBs-secreting B–cell frequencies were observed early after clinical onset, whereas 1 patient who probably developed chronic infection and chronic HBV carriers had absent or weak B– and T–cell responses. In HBs vaccine recipients, maximal HBs–specific B– and T–cell responses were detected after the first injection that decreased gradually before anti–HBs antibodies appeared in serum. Years after vaccination, anti–HBs-secreting B cells were enriched in the bone marrow. After in vitro stimulation with HBsAg, peripheral blood mononuclear cells (PBMC) of only 1 of 5 acute and 1 of 6 chronic HBV patients, but of all 6 vaccine recipients, secreted varying amounts of interferon gamma (IFN–γ), but no interleukin–4 (IL–4) or IL–5. Furthermore, the addition of IFN–γ, but not of IL–2, –4, –12, or IFN–α, resulted in strong increases of anti–HBs-secreting B cells in vaccine recipients and chronic carriers. In conclusion, circulating anti–HBs-secreting B cells were significantly higher in early acute hepatitis B or early after HBs vaccination than in chronic hepatitis B and decreased in the follow–up as a result of compartmentalization to lymphoid tissues. Release of IFN–γ by antigen–stimulated T cells might be critical for anti–HBs formation.
High Incidence of Allograft Cirrhosis in Hepatitis C Virus Genotype 1B Infection Following Transplantation: Relationship With Rejection Episodes
Authors: Prieto Martín , Berenguer Marina , Rayón José Miguel , Córdoba Juan , Argüello Lidia , Carrasco Domingo , García-Herola Antonio , Olaso Vicente , De Juan Manuel , Gobernado Miguel , Mir José , Berenguer Joaquín
Published: July 2004

The natural history of hepatitis C virus (HCV) infection following liver transplantation and predictors of disease severity remain controversial. The aims of the study were to assess in a homogeneous population of 81 cyclosporine–based HCV–infected liver transplant recipients mostly infected with genotype 1b and undergoing strict protocol annual biopsies: 1) the histological progression of posttransplantation HCV disease and, in particular, the incidence of HCV–related graft cirrhosis within the first 5 years after surgery; and 2) the relationship between progression to cirrhosis and i) rejection episodes and ii) first–year liver biopsy findings. We studied 81 consecutive HCV–RNA-positive patients (96% genotype 1b) undergoing liver transplantation between 1991 and 1996 with a minimum histological follow–up of 1 year. All patients received cyclosporine–based immunosuppression and underwent protocol yearly liver biopsies for the first 5 years. The mean histological follow–up was 32 months (range, 12–60 months). Biopsies were scored according to the histological activity index (HAI), with separate evaluation of grade (activity) and stage (fibrosis). Histological hepatitis, present in 97% of patients in the most recent biopsy, was moderate or severe in 64%. Twelve patients developed HCV–related cirrhosis at a median time of 24 months (range, 12–48 months), with an actuarial rate of HCV–cirrhosis of 3.7%, 8.5%, 16%, 28%, and 28% at 1, 2, 3, 4, and 5 years, respectively. Rejection was significantly more common among patients with cirrhosis versus those without (83% vs. 48%; P = .02), with an association between the incidence of cirrhosis and the number of rejection episodes: 5%, 15%, and 50% in patients without rejection, one and two episodes, respectively (P = .001). The degree of activity and fibrosis score in the first–year biopsy were higher in patients who developed cirrhosis than in those who did not (P = .008 and .18, respectively). In conclusion, HCV genotype 1b-infected liver recipients are at a high risk of developing graft cirrhosis in the first 4 to 5 years following transplantation, especially those with previous rejection episodes. First–year liver biopsies may help to sooner identify patients at the highest risk, improving further patient management.
Impact of Hepatitis B and C Virus on Kidney Transplantation Outcome
Authors: Mathurin Philippe , Mouquet Catherine , Poynard Thierry , Sylla Cheickna , Benalia Hadjira , Fretz Catherine , Thibault Vincent , Cadranel Jean-François , Bernard Brigitte , Opolon Pierre , Coriat Pierre , Bitker Marc Olivier
Published: July 2004

The impact of hepatitis B (HBV) and C (HCV) on patient survival after kidney transplantation is controversial. The aims of this study were (1) to assess the independent prognostic values of HBsAg and anti–HCV in a large renal transplant population, (2) to compare infected patients with noninfected patients matched for factors possibly associated with graft and patient survival, and (3) to assess the prognostic value of biopsy–proven cirrhosis. Eight hundred thirty–four transplanted patients were included: 128 with positive HBsAg (group I), 216 with positive anti–HCV (group II), and 490 without serological markers of HBV and HCV (group III). Fifteen percent and 29% of patients were HBsAg–positive and anti–HCV-positive, respectively. Ten–year survivals of group I (55 ± 6%) and group II (65 ± 5%) were significantly lower than survival of group III (80 ± 3%,P< .001). At 10 years, among overall patients with HCV screening (n = 834), four variables had independent prognostic values in patient survival: age at transplantation (P< .0001), year of transplantation (P= .02), biopsy–proven cirrhosis (P= .03), and presence of HCV antibodies (P= .02). In the case control study, comparison of infected patients with their matched control patients showed that age at transplantation (P< .05), HBsAg (P= .005), and anti–HCV (P= .005) were independent prognostic factors. HCV, biopsy–proven cirrhosis, and age are independent prognostic factors of 10–year survival in patients with kidney grafts. The case–control study showed that anti–HCV and HBsAg were independently associated with patient and graft survivals. In infected patients, a routine liver histological analysis would improve selection of patients for renal transplantation.
Reduction of Health–Related Quality of Life in Chronic Hepatitis C and Improvement With Interferon Therapy
Authors: Bonkovsky Herbert L. , Woolley Michael J.
Published: July 2004

The natural history, prognosis, and clinical significance of chronic hepatitis C are highly variable and somewhat controversial. The purpose of this study was to evaluate the effect of chronic hepatitis C infection on patients' perceptions of health–related quality of life (HRQOL) and to evaluate whether treatment with interferon improves HRQOL. A total of 642 patients with compensated liver disease who were enrolled in a multicenter trial of interferon therapy for chronic hepatitis C had evaluation of HRQOL using the SF–36 and other instruments derived from the Medical Outcomes Study (MOS). These instruments were self–administered by patients at baseline and at the end of a 24–week post–treatment observation period after 24 weeks of interferon treatment. Patients with chronic hepatitis C were compared with healthy controls (n = 750) selected from a representative sample of adults in the United States. Unadjusted and age/gender–adjusted results were similar, as were analyses using parametric or nonparametric methods. Compared with healthy controls, patients with chronic hepatitis C at baseline had lower HRQOL on all eight scales of the SF–36 (P < .001 for all). Patients without cirrhosis (n = 284 ) showed similar although slightly smaller differences. The differences were highly significant, clinically and socially relevant, and greatest for those scales that were more reflective of physical than mental or emotional disease. Patients who had a sustained viral response to interferon therapy (n = 41) exhibited marked improvements in HRQOL, and these improvements exceeded those of nonresponders on 13 of 14 HRQOL scales (8 were statistically significant). Similar improvements were noted in patients with sustained biochemical responses. The authors concluded that patients with chronic hepatitis C with or without cirrhosis have markedly reduced HRQOL. Patients who had a sustained response (virological or biochemical) to interferon therapy experienced significant improvements in perceived wellness and functional status. Successful interferon therapy provides meaningful improvements in HRQOL in patients with chronic hepatitis C.
Clinical Significance of Serum Bilirubin Levels Under Ursodeoxycholic Acid Therapy in Patients With Primary Biliary Cirrhosis
Authors: Bonnand Anne-Marie , Heathcote Jenny E. , Lindor Keith D. , Poupon Renée Eugénie
Published: July 2004

We determined whether the normalization of serum bilirubin level (SBL) induced by ursodeoxycholic acid (UDCA) therapy was associated with an improved clinical outcome in patients with primary biliary cirrhosis (PBC). We estimated the prognostic values of SBL measured after 6 months of UDCA treatment for survival free of orthotopic liver transplantation (OLT). We used a database of 548 patients with PBC followed in three trials of UDCA. Among UDCA–treated patients, we compared survival free of OLT in patients with normalized SBL (≤17 μmol/L) with those who had persistently elevated SBL. Difference in survival was tested between UDCA–treated patients whose SBL normalized with treatment and placebo patients who had normal baseline SBL. We evaluated, in each treatment group, the prognostic value of 6–month SBL. Survival was estimated using the Kaplan–Meier method and compared by the Cox model. Survival free of OLT was significantly longer in patients who had normalized SBL (P < .0001; relative risk [RR]: 3.7, UDCA group). Survival free of OLT was not significantly different between UDCA patients with normalized SBL and placebo patients with a normal baseline SBL (P = .69). For several cutoffs of 6–month SBL, RRs of OLT or death were similar in UDCA–treated and placebo patients: the RR of OLT or death associated with a 6–month SBL more than 30 μmol/L was 6.0 for UDCA and 5.7 for placebo groups. In conclusion, normalization of SBL during therapy is associated with improved clinical outcome. SBL under UDCA therapy is a prognostic factor in PBC. SBL under UDCA therapy should be interpreted as in untreated patients.
The Economic Impact of Esophageal Variceal Hemorrhage: Cost–Effectiveness Implications of Endoscopic Therapy
Authors: Gralnek Ian M. , Jensen Dennis M. , Kovacs Thomas O. , Jutabha Rome , Machicado Gustavo A. , Gornbein Jeffrey , King Joy , Cheng Susie , Jensen Mary Ellen
Published: July 2004

Esophageal variceal hemorrhage (EVH) is a serious and expensive sequela of chronic liver disease, leading to increased utilization of resources. Today, endoscopic sclerotherapy (ES) and endoscopic ligation (EL) are the accepted, community standards of endoscopic treatment of patients with EVH. However, there are no published studies comparing the economic costs of treating EVH using these interventions. As part of a prospective, randomized trial comparing ES and EL for the treatment of EVH, we estimated the direct costs of health care utilization and cost–effectiveness for the prevention of variceal rebleeding and patient survival at 1–year follow–up. Treatment groups were similar in incidence of variceal rebleeding (41.9% vs. 42.9%), variceal obliteration (41.9% vs. 40.0%), hospital days, blood transfusions, shunt requirements, and survival (71.0% vs. 60.0%). There were significantly more treatment failures for active bleeding using EL (42% vs. 0%; P = .027) and esophageal stricture formation in the ES–treated patients (19.4% vs. 2.9%; P = 0.03). Median total direct cost outcomes were similar between groups (EL = $9,696 and ES = $13,197; P = .46). EL and ES had similar cost/variceal rebleeding prevented ($28,678 vs. $29,093) and cost/survival ($27,313 vs. $23,804). In the subgroup of active bleeders, ES had a substantially lower cost/survival ($28,523 vs. $51,696). We conclude that resource utilization was similar between treatment groups and that the choice of endoscopic therapy for EVH must still rely on clinical grounds. Further studies comparing costs and resource utilization in this patient population are needed.
Regulation of Electrogenic Anion Secretion in Normal and Cystic Fibrosis Gallbladder Mucosa
Authors: Chinet Thierry , Fouassier Laura , Dray-Charier Nathalie , Imam-Ghali Mama , Morel Hugues , Mergey Martine , Dousset Bertrand , Parc Rolland , Paul Annick , Housset Chantal
Published: July 2004

Fluid and ion transport across biliary epithelium contributes to bile flow. Alterations of this function may explain hepatobiliary complications in cystic fibrosis (CF). We investigated electrogenic anion transport across intact non–CF and CF human gallbladder mucosa in Ussing–type chambers. In non–CF tissues, baseline transmural potential difference (PD), short–circuit current (Isc), and resistance (R) were -2.2 ± 0.3 mV (lumen negative), 40.7 ± 7.8 μA/cm2, and 66.5 ± 9.6 Ω · cm2, respectively (n = 14). The addition of forskolin (10-5mol/L) to the apical and basolateral baths and that of adenosine 5′–triphosphate (ATP) (10-4mol/L) to the apical bath induced significant increases in Isc by 8.0 ± 1.4 and 10.3 ± 1.8 μA/cm2, respectively. Depletion of bathing solutions in Cl-and HCO3-significantly reduced baseline Isc and the forskolin– and ATP–induced increases in Isc. Anion secretion was stimulated by extracellular ATP via P2Y2purinoceptors, as indicated by the effects of different nucleotides on Isc and on36Cl efflux in cultured gallbladder epithelial cells. This effect was mediated by cytosolic calcium increase and Ca2+/calmodulin–dependent protein kinase II, as ascertained by using inhibitors. In CF preparations, basal PD and Isc were lower than in non–CF, and the response to forskolin was abolished, whereas the response to ATP was enhanced (P< .05 for all). We conclude that electrogenic anion secretion occurs in human gallbladder mucosa under basal state and is stimulated by an adenosine 3′,5′–cyclic monophosphate (cAMP)–dependent pathway mediated by cystic fibrosis transmembrane conductance regulator (CFTR), and by exogenous ATP via a CFTR–independent pathway that is up–regulated in CF and involves P2Y2purinoceptors and a calcium–dependent pathway.
Role of β1 Integrins in Adhesion and Invasion of Hepatocellular Carcinoma Cells
Authors: Masumoto Akihide , Arao Shuichi , Otsuki Makoto
Published: July 2004

To investigate the role of integrins in hepatocellular carcinoma (HCC) invasion, we analyzed the relationship between the expression and activity of β1 integrins and the invasive ability of multiple HCC cell lines. Human HCC cell lines, PLC/PRF/5, Hep3B, HepG2, HLE, HuH7, and C3A cells, had high expression of β1 and α6 subunits, and various levels of α1, α2, α3, and α5 expression as determined by cell surface flow cytometry. Activity of β1 integrins was evaluated by cell adhesion to collagen, fibronectin, and laminin in the presence or absence of the stimulatory anti–β1 monoclonal antibody (mAb) TS2/16. Different types of HCC cells showed various levels of constitutive activity of β1 integrins as assessed by the TS2/16 requirement in cell adhesion. TS2/16 rapidly stimulated constitutively inactive or partially active β1 integrins to fully active states, and as the result, the levels of cell adhesion to each ligand correlated with the expression levels of corresponding β1 integrins. Thus, in the presence of TS2/16 stimulation, the levels of cell adhesion to collagen, fibronectin, and laminin correlated predominantly with the expression levels of α2, α5, and α6, respectively. Remarkably, as a result of in vitro chemoinvasion assay, the levels of constitutive activity of β1 integrins correlated with the invasive ability of HCC cells. The inhibitory anti–β1 mAb 13 almost completely blocked the invasion of PLC/PRF/5 and Hep3B cells that are the most invasive HCC cell lines. Alternatively, the stimulatory anti–β1 mAb TS2/16 strongly inhibited the invasion. These results not only show an essential role of β1 integrins in invasion of HCC cells but also suggest subtle regulatory mechanisms of cell invasion.
Targeted Gene Transfer to Hepatocellular Carcinoma Cells In Vitro Using A Novel Monoclonal Antibody-Based Gene Delivery System
Authors: Mohr Leonhard , Schauer Julia I. , Boutin Raymond H. , Moradpour Darius , Wands Jack R.
Published: July 2004

Gene therapy approaches for the treatment of malignant tumors will require high–level expression of therapeutic genes in tumors compared with normal tissues. This may be achieved either by targeted gene delivery to tumor cells or by the use of tumor–specific promoters. Here, we describe the use of a novel conjugate consisting of a tumor–reactive monoclonal antibody (mAb), designated AF–20, coupled to a DNA–binding cationic amphiphile, cholesteryl–spermine, for gene delivery to hepatocellular carcinoma (HCC) cells. The high–affinity mAb, AF–20, recognizes a rapidly internalized 180–kd cell–surface glycoprotein that is abundantly expressed on HCC and other human tumors. The AF–20 mAb and an isotype–matched control antibody (C7–57) were covalently coupled to cholesteryl–spermine. Binding and internalization of AF–20-cholesteryl–spermine was confirmed by fluorescence microscopy using fluorescein isothiocyanate (FITC)–labeled anti–mouse IgG antibody. Following transfection of FITC–labeled oligonucleotides and ethidium monoazide-labeled plasmid DNA, cellular uptake and intracellular localization of nucleic acids were examined by laser scanning confocal microscopy. Transfection of luciferase or β–galactosidase reporter genes complexed to AF–20-cholesteryl–spermine resulted in high levels of gene expression in AF–20 antigen–positive tumor cells. Very low levels of gene expression were observed using the control compound (C7–57–cholesteryl–spermine), which does not recognize the AF–20 tumor antigen or when AF–20 antigen–negative NIH 3T3 cells were transfected with AF–20-cholesteryl–spermine. Thus, covalent coupling of the AF–20 mAb to cholesteryl–spermine generated a highly specific and efficient nonviral vector system for targeted gene delivery to AF–20 antigen–positive HCC cells.
Morphogenetic Events in Mixed Cultures of Rat Hepatocytes and Nonparenchymal Cells Maintained in Biological Matrices in the Presence of Hepatocyte Growth Factor and Epidermal Growth Factor
Authors: Michalopoulos George K. , Bowen William C. , Zajac Valerie F. , Beer-Stolz Donna , Watkins Simon , Kostrubsky Vsevolod , Strom Steven C.
Published: July 2004

Hepatocytes were grown in chemically defined hepatocyte growth medium (HGM) containing hepatocyte growth factor (HGF) and epidermal growth factor (EGF) on collagen–coated polystyrene beads in roller bottle cultures, forming clusters of beads, and proliferating hepatocytes and nonparenchymal cells, including fenestrated endothelium–forming vascular structures. Desmin–positive cells surrounded hepatocytes. Collagen types I and III were deposited in a diffuse manner whereas collagen type IV surrounded the clusters of the epithelial cells, forming a basement membrane. When the mixed cell clusters were implanted in Matrigel (Collaborative Research, Bedford, MA), hepatocytes grew in three dimensions, forming plates and ducts. Many single, long plates of hepatocytes were seen, suggesting progressive linear assembly guided by hepatocyte specific structural parameters. HGF, EGF, and transforming growth factor–α (TGF–α) enhance these phenomena. HGF plus EGF elicited maximal response. TGF–β1 suppressed formation of the ducts and plates. Within three months in Matrigel, the cultures established monolayers composed of plates, ducts, and a well–delineated canalicular network. The mixed cultures expressed albumin, A1AT, AFP, transferrin, and CYPIIB1. Following implantation of the cell clusters in Matrigel, there was decreased expression of c–met, urokinase, urokinase receptor, and TGF–β1. Electron microscopy showed differentiated hepatocytes with nearly normal ultrastructure. The proliferating cell nuclear antigen (PCNA) labeling index was high (more than 80%) whereas the Bromo–deoxyaridine labeling index of ongoing DNA synthesis varied from 10% to 15%. These results show that the mixed cultures of proliferating hepatocytes and nonparenchymal cells can reproduce the hallmark structures of hepatic histological architecture while maintaining differentiation and the capacity to proliferate.
Hypoxic Hepatitis Caused by Acute Exacerbation of Chronic Respiratory Failure: A Case–Controlled, Hemodynamic Study of 17 Consecutive Cases
Authors: Henrion Jean , Minette Philippe , Colin Lucien , Schapira Michael , Delannoy Andre , Heller Francis R.
Published: July 2004

Out of a prospective series of 142 consecutive episodes of hypoxic (ischemic) hepatitis (HH), we identified 17 episodes associated with an acute exacerbation of chronic respiratory failure (CRF) without left cardiac failure. In the aim to evaluate the role of arterial hypoxemia in the pathogenesis of HH associated with respiratory failure, these 17 episodes of HH (study group) were hemodynamically compared with a control group of 17 episodes of HH associated with congestive heart failure (CHF) (control group 1) and a group of 16 episodes of acute respiratory failure (ARF) not complicated by HH (control group 2). Arterial hypoxemia was significantly more severe in the study group (arterial blood tension in O2 [Pao2], 34 mm Hg) than in control group 1 (Pao2, 70 mm Hg; P < .0001) and control group 2 (Pao2, 45.5 mm Hg; P = .002). The role of arterial hypoxemia, however, appeared weakened by comparable degrees of systemic hypotension and liver passive congestion in episodes of HH associated with CRF and episodes of HH associated with CHF. Finally, the causative role of arterial hypoxemia emerged from hemodynamic measurements of cardiac index (CI), systemic vascular resistances (SVR), and oxygen transport: systemic hypotension in HH associated with CHF (control group 1) was the result of a fall in CI (median, 2.33 L/min · m2; range, 1.21–3.14 L/min · m2) associated with high SVR (median, 2,492 dyn · s/cm5 · m2; range, 1,382–4,053 dyn · s/cm5 · m2), whereas in HH associated with respiratory failure (study group), systemic hypotension was the result of a fall in SVR (median, 1,053 dyn · s/cm5 · m2; range, 646–3,148 dyn · s/cm5 · m2), resulting in high CI (median, 4.23 L/min · m2; range, 1.9–5.32 L/min · m2) (P = .0087 and .0038 for cardiac index and SVR, respectively). Moreover, measurements of oxygen transport in patients with HH associated with respiratory failure showed low values of O2 delivery (Do2) (median, 376 mL/min · m2; range, 253–427 mL/min · m2) as a result of extreme arterial hypoxemia despite high CI. In conclusion, these hemodynamic results and additional measurements of hepatic blood flow (HBF) by the method of galactose clearance at a low concentration suggest that in the setting of HH associated with respiratory failure, the liver is not “ischemic,” despite hypotension, but rather “hypoxic” as a result of the combination of severe arterial hypoxemia and elevated central venous pressure (CVP)
Brain Histamine Levels and Neocortical Slow–Wave Activity in Rats With Portacaval Anastomosis
Authors: Lozeva Violina , Valjakka Antti , Anttila Eeva , MacDonald Ewen , Hippeläinen Mikko , Tuomisto Leena
Published: July 2004

To determine whether the increased histamine levels in the brain of rats with portacaval anastomosis (PCA) are associated with the development of sleep disturbances during the light phase, the neocortical slow–wave activity of PCA–operated rats was examined with electroencephalography (EEG) 1 month and 6 months after the surgery. The tissue levels of histamine, tele–methylhistamine, 5–hydroxytryptamine (5–HT) (serotonin), and 5–hydroxyindole–3–acetic acid (5–HIAA) in frontal cortex were assayed by high–performance liquid chromatography 6 months after the surgery. PCA surgery led to changes in the synchronized, low–frequency, high–amplitude frontal cortex EEG activity recorded during the light phase. Delta–wave amplitude but not delta time was significantly decreased, whereas both spindle amplitude and spindling time were significantly decreased. There werealso significant age–related changes, presented as increases in the duration of spindles and the amplitude of both delta waves and spindles. PCA–operated rats showed a change in the pattern of EEG activity with increasing age similar to sham–operated rats. This suggests that once established, the resetting of the systems regulating the sleep–waking behavior is being maintained with time. The tissue levels of both histamine and metabolite in the frontal cortex were increased, whereas the serotonin system showed only an increase in the level of the metabolite. There was a significant negative correlation between the spindling time and the tissue histamine levels. We suggest that histamine, which participates in the control of vigilance, sleep, and wakefulness, as well as in the modulation of circadian rhythmicity, may play a role in the development of sleep disturbances in rats with PCA.
Hepatic Blood Flow and Splanchnic Oxygen Consumption in Patients With Liver Failure. Effect of High–Volume Plasmapheresis
Authors: Clemmesen Jens Otto , Gerbes Alexander L. , Gülberg Veit , Hansen Bent Adel , Larsen Fin Stolze , Skak Claus , Tygstrup Niels , Ott Peter
Published: July 2004

Liver failure represents a major therapeutic challenge, and yet basic pathophysiological questions about hepatic perfusion and oxygenation in this condition have been poorly investigated. In this study, hepatic blood flow (HBF) and splanchnic oxygen delivery (DO2,sp ) and oxygen consumption (VO2,sp) were assessed in patients with liver failure defined as hepatic encephalopathy grade II or more. Measurements were repeated after high–volume plasmapheresis (HVP) with exchange of 8 to 10 L of plasma. HBF was estimated by use of constant infusion of D–sorbitol and calculated according to Fick's principle from peripheral artery and hepatic vein concentrations. In 14 patients with acute liver failure (ALF), HBF (1.78 ± 0.78 L/min) and VO2,sp (3.9 ± 0.9 mmol/min) were higher than in 11 patients without liver disease (1.07 ± 0.19 L/min, P < .01) and (2.3 ± 0.7 mmol/min, P< .001). In 9 patients with acute on chronic liver disease (AOCLD), HBF (1.96 ± 1.19 L/min) and VO2,sp(3.9 ± 2.3 mmol/min) were higher than in 18 patients with stable cirrhosis (1.00 ± 0.36 L/min, P< .005; and 2.0 ± 0.6 mmol/min, P< .005). During HVP, HBF increased from 1.67 ± 0.72 to 2.07 ± 1.11 L/min (n=11) in ALF, and from 1.89 ± 1.32 to 2.34 ± 1.54 L/min (n=7) in AOCLD, P < .05 in both cases. In patients with ALF, cardiac output (thermodilution) was unchanged (6.7 ± 2.5 vs. 6.6 ± 2.2 L/min, NS) during HVP. Blood flow was redirected to the liver as the systemic vascular resistance index increased (1,587 ± 650 vs. 2,020 ± 806 Dyne · s · cm-5 · m2, P< .01) whereas splanchnic vascular resistance was unchanged. In AOCLD, neither systemic nor splanchnic vascular resistance was affected by HVP, but as cardiac output increased from 9.1 ± 2.8 to 10.1 ± 2.9 L/min (P< .01) more blood was directed to the splanchnic region. In all liver failure patients treated with HVP (n=18), DO2,spincreased by 15% (P< .05) whereas VO2,sp was unchanged. Endothelin–1 (ET–1) and ET–3 were determined before and after HVP. Changes of ET–1 were positively correlated with changes in HBF (P< .005) and VO2,sp (P< .05), indicating a role for ET–1 in splanchnic circulation and oxygenation. ET–3 was negatively correlated with systemic vascular resistance index before HVP (P< .05) but changes during HVP did not correlate. Our data suggest that liver failure is associated with increased HBF and VO2,sp. HVP further increased HBF and DO2,sp but VO2,spwas unchanged, indicating that splanchnic hypoxia was not present.
Entry and Integration of Transplanted Hepatocytes in Rat Liver Plates Occur by Disruption of Hepatic Sinusoidal Endothelium
Authors: Gupta Sanjeev , Rajvanshi Pankaj , Sokhi Rana , Slehria Sanjeev , Yam Ana , Kerr Andrew , Novikoff Phyllis M.
Published: July 2004

To establish the process by which transplanted cells integrate into the liver parenchyma, we used dipeptidyl peptidase IV–deficient F344 rats as hosts. On intrasplenic injection, transplanted hepatocytes immediately entered liver sinusoids, along with attenuation of portal vein radicles on angiography. However, a large fraction of transplanted cells (>70%) was rapidly cleared from portal spaces by phagocyte/macrophage responses. On the other hand, transplanted hepatocytes entering the hepatic sinusoids showed superior survival. These cells translocated from sinusoids into liver plates between 16 and 20 hours after transplantation, during which electron microscopy showed disruption of the sinusoidal endothelium. Interestingly, production of vascular endothelial growth factor was observed in hepatocytes before endothelial disruptions. Portal hypertension and angiographic changes resulting from cell transplantation resolved promptly. Integration of transplanted hepatocytes in the liver parenchyma required cell membrane regenesis, with hybrid gap junctions and bile canaliculi forming over 3 to 7 days after cell transplantation. We propose that strategies to deposit cells into distal hepatic sinusoids, to disrupt sinusoidal endothelium for facilitating cell entry into liver plates, and to accelerate cell integrations into liver parenchyma will advance applications of hepatocyte transplantation
Hepatic Sequestration and Modulation of the Canalicular Transport of the Organic Cation, Daunorubicin, in the Rat
Authors: Hayes Julia H. , Soroka Carol J. , Rios-Velez Laura , Boyer James L.
Published: July 2004

In contrast to organic anions, substrates for the canalicular mdr1a and b are usually organic cations and are often sequestered in high concentrations in intracellular acidic compartments. Because many of these compounds are therapeutic agents, we investigated if their sequestration could be regulated. We used isolated perfused rat liver (IPRL), isolated rat hepatocyte couplets (IRHC), and WIF–B cells to study the cellular localization and biliary excretion of the fluorescent cation, daunorubicin (DNR). Despite rapid (within 15 minutes) and efficient (>90%) cellular uptake in the IPRL, only ≈10% of the dose administered (0.2–20 μmol) was excreted in bile after 85 minutes. Confocal microscopy revealed fluorescence predominantly in vesicles in the pericanalicular region in IPRL, IRHC, and WIF–B cells. Treatment of these cells with chloroquine and bafilomycin A, agents that disrupt the pH gradient across the vesicular membrane, resulted in a loss of vesicular fluorescence, reversible in the case of bafilomycin A. Taurocholate (TC) and dibutyryl cAMP (DBcAMP), stimulators of transcytotic vesicular transport, increased the biliary recovery of DNR significantly above controls, by 70% and 35%, respectively. The microtubule destabilizer, nocodazole, decreased biliary excretion of DNR. No effect on secretion was noted in TR- mutant rats deficient in mrp2. Coadministration of verapamil, an inhibitor of mdr1, also decreased DNR excretion. While TC and DBcAMP did not affect the fluorescent intensity or pattern of distribution in IRHC, nocodazole resulted in redistribution of DNR to peripheral punctuate structures. These findings suggest that the organic cation, DNR, is largely sequestered in cells such as hepatocytes, yet its excretion can still be modulated
Effects of Fibrillar C–Terminal Fragment of Cleaved α1–Antitrypsin on Cholesterol Homeostasis in Hepg2 Cells
Authors: Janciauskiene Sabina , Lindgren Stefan
Published: July 2004

Amyloid fibrils of diverse origin are known to disturb vital cellular functions and induce cell death. In this study, the effects of amyloid fibrils from the C–terminal fragment (C–36) of cleaved α1 –antitrypsin (AAT) on low–density lipoprotein (LDL) metabolism were investigated in HepG2 cells. Treatment of the cells with C–36 fibrils (10 μmol/L) enhanced 125I–LDL binding and uptake 10 to 15 times, and highly up–regulated levels of LDL receptor mRNA, as compared with control cells. Competition experiments using excess of unlabeled LDL and blockage experiments with a monoclonal LDL receptor antibody diminished or completely abolished the stimulatory effects of fibrils on LDL binding and LDL receptor mRNA levels, suggesting that fibrils act via the LDL receptor pathway. However, C–36 fibrils had no significant effect on [2–14C]acetate incorporation into cholesterol biosynthesis and cholesterol ester formation, but inhibited125I–LDL degradation by 20% and reduced bile acid biosynthesis up to 48% in a dose–dependent manner. Preincubation of the cells with fibrils before the addition of LDL totally abolished the LDL inhibitory effect on unesterified cholesterol synthesis, further confirming the LDL receptors to be the target for C–36 fibrils. Moreover, the expression of sterol regulatory element binding protein–1 (SREBP–1) was found to increase twofold and more after 24 hours of incubation of the cells with several concentrations of C–36 fibrils. Our study suggests that the cytotoxicity of C–36 fibrils on HepG2 cells is associated with perturbed intracellular cholesterol homeostasis, induced through fibril–stimulated expression of the LDL receptors via the sterol–responsive element