bluuebunny/crossref_metadata_2025 huggingface dataset:
The Guanylate Cyclase-Coupled Natriuretic Peptide Receptor: A New Target for Prevention of Cold Ischemia-Reperfusion Damage of the Rat Liver
Authors: Gerbes Alexander L. , Vollmar Angelika M. , Kiemer Alexandra K. , Bilzer Manfred
Published: July 2004

The aim of our studies was to investigate hormonal prevention of hepatic preservation damage by the atrial natriuretic peptide (ANP) and the mechanisms involved. Isolated perfusion of rat livers was performed in a nonrecirculating fashion. Twenty minutes of preischemic perfusion was performed with or without different concentrations of ANP, followed by 24–hour storage in cold University of Wisconsin (UW) solution. Two hundred nanomoles of ANP prevented hepatocellular damage during a 2–hour reperfusion period as indicated by a marked attenuation of the sinusoidal efflux of lactate dehydrogenase (LDH) and purine nucleoside phosphorylase (PNP), and by reduced Trypan blue uptake. Furthermore, postischemic bile flow as an indicator of liver function was significantly improved by about 60% with 200 nmol/L ANP. No protection was conveyed by 20 nmol/L ANP nor by pretreatment with 200 nmol/L ANP for only 10 minutes. The effects of ANP seemed to be mediated by the guanylate cyclase-coupled A (GC–A) receptor and cyclic guanosine monophosphate (cGMP): whereas expression of both GC–A and GC–B receptors as well as of the GC–C receptor was found, cGMP did protect from ischemia–reperfusion damage, but selective ligands of the B and C receptor did not. To begin to determine the mechanisms of ANP–mediated protection, different parameters were investigated: ANP had no effect on portal pressure as an indicator of hepatic circulation, nor on intracellular energy depletion determined by adenosine nucleotide concentration. However, the marked augmentation of nuclear factor κB (NF–κB) binding activity during reperfusion was prevented in ANP–pretreated livers. In conclusion, pretreatment with ANP protects the rat liver from cold ischemia–reperfusion damage. This effect is mediated via the GC–A receptor and cGMP, and may be linked to an influence of ANP on NF–κB activation. Thus, ANP signaling via the GC–A receptor should be considered as a new pharmacological target to prevent preservation injury of the liver.
Acute and Chronic Ethanol Increases Reactive Oxygen Species Generation and Decreases Viability in Fresh, Isolated Rat Hepatocytes
Authors: Bailey Shannon M. , Cunningham Carol C.
Published: July 2004

Although reactive oxygen species (ROS) have been implicated in the etiology of alcohol–induced liver disease, neither their relative contribution to cell death nor the cellular mechanisms mediating their formation are known. The purpose of this study was to test the hypothesis that acute and chronic ethanol exposure enhances the mitochondrial generation of ROS in fresh, isolated hepatocytes. Acute ethanol exposure stimulated ROS production, increased the cellular NADH/NAD+ ratio, and decreased hepatocyte viability slightly, which was prevented by pretreatment with 4–methylpyrazole (4–MP), an inhibitor of alcohol dehydrogenase. Similarly, xylitol, an NADH–generating compound, enhanced hepatocyte ROS production and decreased viability. Incubation with pyruvate, an NADH–oxidizing compound, and cyanamide, an inhibitor of aldehyde dehydrogenase, significantly decreased ROS levels in acute ethanol–treated hepatocytes. Chronic ethanol consumption produced a sixfold increase in hepatocyte ROS production compared with levels measured in controls. Hepatocytes from ethanol–fed rats were less viable compared with controls, e.g., viability was 68% ± 2% (ethanol) versus 83% ± 1% (control) after 60 minutes of incubation. Antimycin A increased ROS production and decreased cell viability; however, the toxic effect of antimycin A was more pronounced in ethanol–fed hepatocytes. These results suggest that acute and chronic ethanol exposure exacerbates mitochondrial ROS production, contributing to cell death.
Interstitial Accumulation of Na+ and K+ During Flush–Out and Cold Storage of Rat Livers: Implications for Graft Survival
Authors: Tian Ying-hua , Fukuda Chifumi , Schilling Martin K.
Published: July 2004

Intracellular–type electrolyte solutions were introduced into organ preservation to prevent K+ efflux and Na+ and Cl- influx into cells and cell swelling during cold ischemia. We studied cation accumulation in the interstitial space by microdialysis, during rat liver cold storage and after flush–out with high–K+ and low–K+ solutions. The effect of Na+ and K+ on graft function and survival was studied in an isolated perfused liver model and an orthotopic transplantation model after rat liver storage in iso–osmolar high–K+ and low–K+ solutions. After 24 hours of cold ischemia [Na+]o dropped from 136 ± 2 mmol/L to 91.8 ± 1.1 mmol/L, and [K+]o increased from 5.9 ± 0.1 mmol/L to 12.2 ± 1.6 mmol/L (P < .001 vs. control). [Na+]o and [K+]o after flush–out did not equilibrate with [Na+]sol and [K+]sol after 24 hours of cold storage. Rat livers preserved in low–K+ solutions produced significantly more bile during isolated reperfusion and released less alanine transaminase, aspartate transaminase, and lactate dehydrogenase into the reperfusion medium than high–K+ solutions. Rat liver survival after 14 hours of preservation was higher in low–K+ solutions (13 of 13) than in high–K+ solutions (7 of 13). Those studies indicate that during cold storage of rat livers, transmembraneous Na+–K+ sodium–potassium exchange might not follow the 3:2 stochiometry of a sole sodium–potassium exchange via Na+–K+ sodium–potassium adenosine triphosphatase (ATPase), and that low–K+ solutions might improve graft function and survival after rat liver preservation.
Expression of the Apical Conjugate Export Pump, Mrp2, in the Polarized Hepatoma Cell Line, Wif–B
Authors: Nies Anne T. , Cantz Tobias , Brom Manuela , Leier Inka , Keppler Dietrich
Published: July 2004

The polarized rat hepatoma/human fibroblast hybrid cell line, WIF–B, forms apical vacuoles into which cholephilic substances are secreted. We studied expression, localization, and function of the apical conjugate export pump, Mrp2, in WIF–B cells. Mrp2, the apical isoform of the multidrug resistance protein, alternatively termed canalicular Mrp (cMrp) or canalicular multispecific organic anion transporter (cMoat), is a 190–kd membrane glycoprotein mediating adenosine triphosphate (ATP)–dependent transport of glucuronides, glutathione S–conjugates, and other amphiphilic anions across the hepatocyte canalicular membrane into bile. Expression of the rat mrp2 gene in WIF–B cells was shown by reverse–transcription polymerase chain reaction (PCR), followed by sequencing of the amplified 789–bp fragment. Immunoblotting, using antibodies reacting with the amino–terminal or with the carboxyl–terminal sequence of rat Mrp2, detected the 190–kd glycoprotein in WIF–B cell homogenates. Immunofluorescence microscopy localized Mrp2 to the apical membrane domain. Preloading of WIF–B cells with a membrane–permeable ester of the calcium–dependent fluorescent indicator, Fluo–3, was followed by Mrp2–mediated secretion of the amphiphilic anion, Fluo–3, into the apical vacuoles. This transport was potently inhibited by cyclosporin A added to the culture medium. Direct measurements of ATP–dependent transport into Mrp2–containing plasma membrane vesicles in comparison with Mrp2–deficient vesicles established that Fluo–3 is transported by Mrp2 with a K
m value of 3.7 μmol/L. Our results indicate that the polarized WIF–B cells express the rat ortholog of the apical conjugate-transporting ATPase, Mrp2. The function of Mrp2 as well as the action of inhibitors can thus be analyzed by use of the fluorescent amphiphilic anion, Fluo–3.
Adenosine Triphosphate-Dependent Transport of Estradiol–17β(β– D –Glucuronide) in Membrane Vesicles by Mdr1 Expressed in Insect Cells
Authors: Huang Liyue , Hoffman Tim , Vore Mary
Published: July 2004

MDR1, an ABC transporter that confers multidrug resistance in tumor cells, is constitutively expressed in normal liver canalicular membrane. Human MDR1–expressing multidrug–resistant cells display increased resistance to estradiol–17β(β–d–glucuronide) (E217G). MDR1 substrates/modulators inhibit adenosine triphosphate (ATP)–dependent transport of E217G in the rat canalicular membrane and protect against E217G–mediated cholestasis in isolated perfused rat liver. The present studies were designed to determine if E2 17G is a substrate for MDR1 using a baculovirus expression system and if other estrogen glucuronides interact with MDR1. ATP–dependent transport of E217G (10 μmol/L) was linear for up to 2 minutes and yielded a rate of 45.6 pmol/min/mg protein in membrane vesicles from Sf9 cells infected with MDR1–baculovirus. This transport was saturable (K
m = 62 μmol/L) and occurred into an osmotically sensitive space. ATP–dependent transport of E2 17G (10 μmol/L) was inhibited 63% by 10 μmol/L daunomycin, but not by 100 μmol/L S–(2,4–dinitrophenyl)glutathione (GS–DNP) (a substrate for canalicular multispecific organic anion transporter [cMOAT]). Glucuronide conjugates of the estrogen D–ring (100 μmol/L), estriol–17β(β–d–glucuronide) (E317G) and estriol–16α(β–d–glucuronide) (E316G), inhibited MDR1–mediated E217G transport by 58% and 35%, respectively. In contrast, noncholestatic glucuronides, estradiol–3–(β–d–glucuronide) (E23G) or estradiol–3–sulfate–17β(β–d–glucuronide) (E23SO417G), had no effect. E217G neither stimulated MDR1 ATPase activity nor inhibited verapamil–stimulated ATPase activity. Infusion of 1.5 μmol/L doxorubicin or 1 μmol/L taxol protected against cholestasis induced by E316G and E317G in isolated perfused rat liver. These studies identify E217G, and probably E316G and E317G, as endogenous substrates for MDR1.
Search for Hepatitis C Virus Extrahepatic Replication Sites in Patients With Acquired Immunodeficiency Syndrome: Specific Detection of Negative–Strand Viral Rna in Various Tissues
Authors: Laskus Tomasz , Radkowski Marek , Wang Lian-Fu , Vargas Hugo , Rakela Jorge
Published: July 2004

The existence of extrahepatic reservoirs of hepatitis C virus (HCV) replication remains highly controversial. We searched for the presence of HCV–RNA negative strand in various tissues from eight HCV–infected patients who died of acquired immunodeficiency syndrome (AIDS)–related complications. Negative–strand RNA was detected by a Tth–based reverse–transcriptase polymerase chain reaction (RT–PCR), which was optimized for sensitivity and strand specificity on synthetic RNA templates. This assay was capable of detecting about 102 genomic Eq molecules of the correct strand while unspecifically detecting ≥108 genomic Eq molecules of the incorrect strand. Negative–strand viral RNA was detected in all but one liver, in lymph nodes (5 cases), in pancreas (5 cases), in adrenal gland (2 cases), in thyroid (2 cases), in bone marrow (1 case), and in spleen (1 case). These data suggest a possible presence of HCV replication sites outside the liver, at least in AIDS patients. Whether these findings relate to various extrahepatic manifestations of HCV infection remains to be determined.
Early Hepatitis C Virus-Rna Responses Predict Interferon Treatment Outcomes in Chronic Hepatitis C
Authors: Lee William M. , Reddy Rajender K. , Tong Myron J. , Black Martin , van Leeuwen Dirk J. , Hollinger Blaine F. , Mullen Kevin D. , Pimstone Neville , Albert Donald , Gardner Sheila
Published: July 2004

In previous studies employing interferons (IFNs) in the treatment of chronic hepatitis C, there have been few reliable predictors of sustained responses. We retrospectively evaluated the predictive value of hepatitis C virus (HCV)–RNA measurements in the first few months during consensus interferon (CIFN) treatment using a sensitive reverse–transcriptase polymerase chain reaction assay to determine sustained responses. Data from two large treatment trials, one of IFN–naive patients and one of retreated relapsers and nonresponders, were used, including serum samples at 2–week intervals in the naive study and 8–week intervals in the retreatment study. Patients received initial CIFN (9 μg) treatment for 6 months and were assessed 6 months after treatment. There were 28 sustained viral responders of 232 CIFN–treated patients. Of the sustained responders, 48% had already cleared HCV RNA from serum (<100 copies/mL) by week 2, 78% by week 4, 81% by week 6, and 96% by week 12. Patients with early HCV–RNA clearance were more likely to have sustained responses than those who responded later. Early clearance of HCV from serum was also associated with greater likelihood of a sustained response to 48 weeks of retreatment with 15 μg CIFN. Ninety–five percent of the sustained responders were HCV–RNA-negative by week 8 of retreatment. Early assessment of HCV RNA may help in the prediction of sustained responses to IFN and allow the value of continued treatment to be determined early in the course of IFN therapy.
Histopathology of the Liver in Children With Chronic Hepatitis C Viral Infection
Authors: Badizadegan Kamran , Jonas Maureen M. , Ott Mary Jane , Nelson Suzanne P. , Perez-Atayde Antonio R.
Published: July 2004

Although the epidemiology, natural history, and pathological aspects of chronic hepatitis C are well–defined in the adult population, little is known about the characteristics of chronic hepatitis C infection in children. Reports on the histological features and progression of hepatitis C in children are scarce, and consist primarily of multicenter studies in Japanese and European children. Given the geographic variations in viral genotype and the association of pathology with genotype, whether the Japanese and European studies can be extended to the North American populations is unclear. We report the histopathology of the liver in 40 children with chronic hepatitis C infection treated in a single North American institution. The children included 19 males and 21 females ranging in age from 2.0 to 18.6 years at the time of liver biopsy (mean ± SD: 11.4 ± 4.3 years). Our findings indicate that the characteristic histopathological lesions of chronic hepatitis C infection, including sinusoidal lymphocytosis, steatosis, portal lymphoid aggregates/follicles, and bile duct epithelial damage, occur with approximately the same frequencies in children as have been reported in adults. Necroinflammatory activity was generally mild. Portal fibrosis was present in 78% of the specimens, including fibrous portal expansion (26%), bridging fibrosis (22%), bridging fibrosis with architectural distortion (22%), and cirrhosis (8%). Centrilobular pericellular fibrosis, which has not been previously reported in the context of chronic hepatitis C infection in adults or children, was also a prominent feature in our series, occurring with a similar frequency as steatosis or portal lymphoid aggregates/follicles. Our data suggest that in spite of mild histological necroinflammatory activity in general, the stage of fibrosis in children can be severe in spite of relatively short duration of infection.
Effect of Intraduodenal Bile Salt on Pancreaticobiliary Responses to Bombesin and to Cholecystokinin in Humans
Authors: Thimister Paul W. , Hopman Wim P. , Tangerman Albert , Rosenbusch Gerd , Willems Hans L. , Jansen Jan B.
Published: July 2004

Bile salts modulate postprandial gallbladder emptying and pancreatic enzyme secretion, possibly by interfering with plasma cholecystokinin (CCK) responses. The regulatory role of bile salts in the absence of nutrients from the gut is poorly understood. Therefore, we studied the effect of intraduodenal sodium chenodeoxycholate on bombesin (BBS)– or CCK–stimulated plasma CCK levels, plasma pancreatic polypeptide levels, gallbladder motility, and pancreatic enzyme secretion. In a crossover design, saline without or with chenodeoxycholate was perfused intraduodenally for 3 hours in healthy volunteers. During the last hour, either BBS (n = 9) or CCK (n = 10) was infused intravenously. Chenodeoxycholate inhibited BBS–stimulated gallbladder emptying from 59% ± 4% to 34% ± 6% (P < .05) and intraduodenal bilirubin output from 41 ± 9 to 21 ± 5 μmol/h (P < .05), but it increased integrated plasma CCK levels from 157 ± 19 to 184 ± 19 pmol/L · 60 min (P = .01). Similarly, chenodeoxycholate administration inhibited gallbladder emptying and bilirubin output in response to intravenous CCK. Chenodeoxycholate also tended to reduce pancreatic polypeptide release and intraduodenal amylase output in response to intravenous BBS or CCK. It is concluded that intraduodenal chenodeoxycholate administration inhibits BBS– or CCK–stimulated gallbladder emptying, probably by diminishing target organ sensitivity to circulating CCK.
Macrophage–Derived Nitric Oxide Induces Apoptosis of Rat Hepatoma Cells In Vivo
Authors: Nishikawa Manabu , Sato Eisuke F. , Kuroki Tetsuo , Utsumi Kozo , Inoue Masayasu
Published: July 2004

Although nitric oxide (NO) has been postulated to play important roles in host defense mechanisms against tumor cells, a direct evidence supporting this hypothesis is lacking. To obtain molecular insights into the antitumor action of NO, its metabolism and effect on ascites hepatoma (AH–130) cells were investigated in tumor–bearing rats. Kinetic analysis revealed that substantial amounts of nitrite and nitrate, metabolites of NO, appeared in plasma and ascites of AH–130-inoculated rats. Western blot analysis revealed that a large number of macrophages that expressed inducible type of NO synthase (iNOS) appeared in cancerous ascites, particularly during 1 to 2 weeks after inoculation of AH–130 cells. When NO generation by peritoneal macrophages increased, a significant fraction of AH–130 in ascites fluid underwent apoptosis as judged from the fragmentation of their nuclear DNA. Kinetic analysis revealed that NO strongly inhibited mitochondrial electron transport and changed calcium status in AH–130 cells, particularly under low oxygen tensions such as in cancerous ascites. Intraperitoneal injection of NO donor strongly enhanced DNA fragmentation of AH–130 cells. Antimycin A, a specific inhibitor for mitochondrial electron transport, also induced DNA fragmentation of AH–130 cells by a mechanism that was inhibited by adding ascorbate and tetramethyl–p–phenylene diamine (TMPD) as electron donors. These results indicate that NO derived from peritoneal macrophages inhibits mitochondrial electron transport and disturbs calcium homeostasis in ascites hepatoma AH–130 cells, thereby inducing their apoptosis in vivo.
Vascular Endothelial Growth Factor Tightly Regulates In Vivo Development of Murine Hepatocellular Carcinoma Cells
Authors: Yoshiji Hitoshi , Kuriyama Shigeki , Yoshii Junichi , Yamazaki Masaharu , Kikukawa Masaji , Tsujinoue Hirohisa , Nakatani Toshiya , Fukui Hiroshi
Published: July 2004

Angiogenesis is essential for the development of a solid tumor, including hepatocellular carcinoma (HCC). HCC is a well–known hypervascular tumor. Vascular endothelial growth factor (VEGF) is one of the most potent angiogenic factors. Its role has not been clarified in vivo in HCC development. We used a self–contained, tetracycline–regulated retroviral vector system to elucidate the effect of VEGF on murine HCC development in a xenograft experimental model. By delivering the VEGF gene within the retroviral vector and under the control of a tetracycline–regulated promoter, we were able to manipulate VEGF expression in vivo tumor by providing tetracycline in the drinking water. Overexpression of VEGF showed a marked increase in tumor development accompanied by augmentation of neovascularization. The degree of tumor enlargement corresponded to the level of VEGF gene expression. Suppression of VEGF led to a decrease in tumor growth at the established tumor size, whether relatively small or large. The level of VEGF expression did not alter the proliferation of HCC cells in vitro. In a double–chamber chemoinvasion assay, the in vitro invasion activity of VEGF–transduced cells was not changed. In the presence of endothelial cells (EC), however, VEGF–transduced cells showed a marked increase in their in vitro invasion activity. These results suggested that VEGF plays a critical role in the development of HCC in cooperation with EC
Enhanced Expression of Thrombospondin–1 and Hypovascularity in Human Cholangiocarcinoma
Authors: Kawahara Naoyuki , Ono Mayumi , Taguchi Ken-ichi , Okamoto Masahiro , Shimada Mitsuo , Takenaka Kenji , Hayashi Kenshi , Mosher Dean F. , Sugimachi Keizo , Tsuneyoshi Masazumi , Kuwano Michihiko
Published: July 2004

Cholangiocarcinoma (CCC) is relatively hypovascular, in contrast to hepatocellular carcinoma (HCC), which is often highly vascular. We investigated if the diminished vascularity of CCC is related to altered expression of thrombospondin–1 (TSP–1), an antiangiogenic factor, and/or vascular endothelial growth factor (VEGF), a potent angiogenic factor, comparing the relationships with those of high– and low–vascular HCC. We also investigated the relationship between the mutation of the p53 gene and TSP–1 expression or VEGF expression. Northern blot analysis and immunohistochemical staining were performed on surgically resected human CCC and HCC. The ratios of TSP–1 mRNA level in cancer cells versus adjacent noncancerous cells (T/N ratios) were significantly higher in CCC (n = 11) than in HCC with high vascularity (n = 15). In contrast, T/N ratios of VEGF mRNA level in CCC (n = 11) were comparable with those in HCC with low vascularity (n = 5). In CCC, the cancer cells and fibroblasts were positively stained with anti-TSP–1 antibody. We observed that T/N ratios of VEGF mRNA level, but not those of the TSP–1 mRNA level, were significantly correlated with vascularity in HCC. The relative increase in TSP–1 and the relative decrease in VEGF in tumors compared with normal tissue may underlie the limited angiogenesis of CCC. The p53 gene did not affect the expression of TSP–1 in CCC or VEGF in HCC.
Antifibrotic and Hemodynamic Effects of the Early and Chronic Administration of Octreotide in Two Models of Liver Fibrosis in Rats
Authors: Fort Joël , Oberti Frédéric , Pilette Christophe , Veal Nary , Gallois Yves , Douay Olivier , Rousselet Marie Christine , Rosenbaum Jean , Calès Paul
Published: July 2004

The aim of this study was to assess the effect of the early and chronic administration of octreotide in the prevention of hepatic fibrosis and portal hypertension (PHT). Two experimental models of liver fibrosis caused by bile duct ligation (BDL) or CCl4 were divided into 4 rat groups: sham, placebo, and octreotide (10 and 100 μg/kg twice daily, subcutaneously). Liver fibrosis was assessed by the area of fibrosis (image analysis), liver hydroxyproline and fibronectin mRNA contents, and serum hyaluronate. Systemic and splanchnic hemodynamic changes were also evaluated, including the splenorenal shunt blood flow by the transit–time ultrasound (TTU) technique. In both models, splenorenal shunt blood flow was significantly lower in the octreotide groups than in the placebo group (P < .05), while portal pressure was not significantly decreased. There was a significant decrease in fibrosis by octreotide in the CCl4 model only: area of fibrosis: 13.9% ± 3.7% vs. 9.8% ± 2.5% (P < .01), hydroxyproline: 1.8 ± 0.6 vs. 1.3 ± 0.4 mg/g wet liver (P < .05), respectively, placebo vs. octreotide 10 μg/kg. There was a significant correlation between the area of fibrosis and hydroxyproline liver content (r = .87 in the biliary model and r = .91 in the CCl4 model;P < .0001). The early and chronic administration of octreotide prevents the development of portocollateral blood flow without reducing portal pressure in two models of liver fibrosis and the development of liver fibrosis in the CCl4 model.
Identification of Glutathione S–Transferase Isozymes and γ–Glutamylcysteine Synthetase As Negative Acute–Phase Proteins in Rat Liver
Authors: Buetler Timo M.
Published: July 2004

Because acute infection and inflammation affect drug metabolism and drug–metabolizing enzymes, the effect of the acute–phase response on the expression of glutathione S–transferase (GST) isoenzymes, glutathione synthesis, and several antioxidant enzymes was investigated. Hepatic expression of GST isozymes, positive and negative acute–phase reactants, and antioxidant enzymes were determined by Northern blotting and hybridization with gene–specific oligonucleotide probes after lipopolysaccharide treatment of rats. Lipopolysaccharide caused the expected acute–phase response as judged by the increased expression of positive and decreased expression of negative acute–phase proteins. The messenger RNA (mRNA) expression of the major hepatic rat GST isozymes A1, A2, A3, M1, and M2 was decreased 50% to 90%. Total hepatic GST activity toward 1–chloro–2,4–dinitrobenzene was also significantly decreased. mRNA expression of γ–glutamylcysteine synthetase (GCS) large subunit and catalase was reduced by approximately 60%. GCS enzyme activity was also decreased, resulting in a 35% decrease in the hepatic content of reduced glutathione 4 days after lipopolysaccharide challenge. Mn–Superoxide dismutase expression was increased 13–fold, and thioredoxin level was elevated 3–fold after lipopolysaccharide challenge. The expression of all parameters determined returned to near control levels 7 days after treatment. Together, these data show that GSTs and GCS are negative acute–phase proteins and that decreased GCS activity results in a decrease in hepatic glutathione content. Thus, in addition to the phase I drug–metabolizing enzymes known to be decreased during the acute–phase response, some phase II enzymes involved in the elimination of xenobiotics and carcinogens are also decreased.
Subcellular Localization of (Latent) Transforming Growth Factor β and the Latent Tgf–β Binding Protein in Rat Hepatocytes and Hepatic Stellate Cells
Authors: Roth-Eichhorn Sylke , Kühl Kitty , Gressner Axel M.
Published: July 2004

Recently, the existence of the large latent transforming growth factor β (TGF–β) complex, consisting of TGF–β, the N–terminal part of its precursor (latency–associated peptide [LAP]), and the latent TGF–β binding protein (LTBP), was demonstrated in rat liver parenchymal cells (PC) and stellate cells (HSC). However, in contrast to HSC, in freshly isolated PC, no message of these proteins is detectable. This study was performed to investigate the subcellular distribution of the proteins forming the latent TGF–β complex in PC and HSC from rat liver to obtain more information about their origin and potential intracellular functions. PC and HSC were isolated from rat liver by protease reperfusion and investigated for TGF–β1,-2,-3, β1–LAP, and LTBP–1 after cultivation using double–immunofluorescent staining, followed by high–resolution confocal microscopic analysis. Subcellular fractions obtained by standard differential centrifugation of rat liver homogenate were analyzed using a TGF–β1 enzyme–linked immunosorbent assay (ELISA) and Western blotting for β1–LAP and LTBP–1. By confocal microscopy, a diffuse distribution of TGF–β and LAP in the cytoplasm of PC is noticed, whereas the LTBP immunostaining predominates at plasma membranes. In PC, distinct intracellular granules were superimposed with TGF–β, LAP, and LTBP stainings identified as lysosomal compartments and mitochondria by ELISA and immunoblotting of subcellular fractions. In HSC, stainings of colocalized TGF–β, LAP, and LTBP are strongest in the perinuclear area, indicating synthesis and secretion via endoplasmic reticulum and Golgi, respectively. Partially, the proteins were also found in HSC nuclei. During the transformation of HSC to myofibroblasts, LAP and LTBP become strongly colocalized with other components of the cytoskeletal network like smooth muscle–α–actin, desmin, and talin. The results confirm biochemical data about the existence and expression of the large latent TGF–β complex in PC and HSC, respectively. Baseline information is provided from which new hypotheses regarding intracellular functions of TGF–β, LAP, and LTBP in liver parenchymal and stellate cells can be concluded.
Interleukin–10 Expression and Function in Experimental Murine Liver Inflammation and Fibrosis
Authors: Thompson Kerry , Maltby Julia , Fallowfield Jon , McAulay Martin , Millward-Sadler Harry , Sheron Nick
Published: July 2004

Kupffer cells (KC) play a central role in the initiation and perpetuation of hepatic inflammation, which, if uncontrolled, can result in tissue damage, fibrosis, and cirrhosis. Interleukin–10 (IL–10) can inhibit a range of macrophage functions. We hypothesized that the transcription, synthesis, and release of IL–10 may influence the development of liver injury. Rat KC were activated in vitro with lipopolysaccharide (LPS), and expression of IL–10 mRNA compared with IL–13 and IL–1β by reverse–transcription polymerase chain reaction (RT–PCR). The effects of pretreatment with recombinant IL–10 (rIL–10) on KC phagocytosis, production of superoxide (SO), and tumor necrosis factor α (TNF–α) were examined by fluorescent activated cell sorter (FACS), reduction of ferricytochrome C, and bioassay, respectively. Rats were administered intraperitoneal carbon tetrachloride (CCl4), and expression of IL–10 mRNA and protein in vivo compared with IL–13 and IL–1β by RT–PCR and immunoblotting. Results were correlated with histological inflammatory changes. Finally, IL–10 gene–deleted (IL–10-/-) mice and wild–type (WT) controls were administered intraperitoneal CCl4 biweekly for up to 70 days, and the development of inflammation and fibrosis compared by scoring histological changes. IL–10 mRNA was up–regulated early, both in KC in vitro and in whole liver in vivo, concurrent with that of IL–1β. IL–10 was able to inhibit KC production of both SO and TNF–α in vitro, and this was achieved more effectively than IL–4 or IL–13; no such effects were seen on KC phagocytosis. After 70 days of treatment with CCl4, IL–10-/- mice showed significantly more severe fibrosis and exhibited higher hepatic TNF–α levels than WT controls. These results suggest that IL–10 synthesized during the course of liver inflammation and fibrosis may modulate KC actions, and influence subsequent progression of fibrosis.
Interleukin–10 Controls Neutrophilic Infiltration, Hepatocyte Proliferation, and Liver Fibrosis Induced by Carbon Tetrachloride in Mice
Authors: Louis Hubert , Van Laethem Jean-Luc , Wu Wei , Quertinmont Eric , Degraef Chantal , Van den Berg Kit , Demols Anne , Goldman Michel , Le Moine Olivier , Geerts Albert , Devière Jacques
Published: July 2004

The role of the anti–inflammatory cytokine interleukin–10 (IL–10) was investigated in the mouse model of liver injury induced by carbon tetrachloride (CCl4). To address the role of endogenous IL–10 production, acute hepatitis was induced by CCl4 in C57Bl/6 IL–10 gene knock out (KO) and wild–type (WT) mice. After CCl4 challenge, serum and liver levels of tumor necrosis factor–alpha (TNF–α) and serum levels of transforming growth factor–beta 1 (TGF–β1) increased and were significantly higher in IL–10 KO mice, whereas IL–6 serum levels were only slightly increased compared with WT mice. At histological examination, the livers disclosed a significantly more prominent neutrophilic infiltration in IL–10 KO mice 12 and 24 hours after CCl4 injection. In contrast, hepatocyte necrosis, evaluated by histological examination and serum alanine aminotransferase levels, was only marginally affected. The proliferative response of hepatocytes, assessed by the proliferating cell nuclear–antigen labeling index, was significantly increased in IL–10 KO mice, compared with WT mice 48 hours after CCl4 injection. Finally, repeated CCl4 injections led to more liver fibrosis in IL–10 KO mice after 7 weeks. In conclusion, endogenous IL–10 marginally affects the hepatocyte necrosis although it controls the acute inflammatory burst induced by CCl4. During liver repair, it limits the proliferative response of hepatocytes and the development of fibrosis.
Connexin 32 Gap Junctions Enhance Stimulation of Glucose Output by Glucagon and Noradrenaline in Mouse Liver
Authors: Stümpel Frank , Ott Thomas , Willecke Klaus , Jungermann Kurt
Published: July 2004

Gap junctions connect neighboring cells via intercellular channels composed of connexins (Cx). Connexin 32 (Cx32) is the main connexin in hepatocytes. Gap junctions propagate a signal from periportal to perivenous hepatocytes generated by electrical stimulation of sympathetic liver nerves. Therefore, it was the aim of this study to examine the involvement of hepatocellular gap junctions in hormonal regulation. In perfused livers from wild–type mice and Cx32–deficient mice, the stimulation of glucose release by varying noradrenaline and glucagon concentrations was investigated. At saturating hormone concentrations, glucose release was the same in wild–type and Cx32–deficient livers. However, glucose output was significantly smaller in Cx32–deficient than wild–type livers at half–maximally effective hormone concentrations. Because the two hormones circulate at less than half–saturating concentrations and because they are degraded during passage of blood through the liver, they lose efficiency from the periportal to the perivenous zone. In wild–type livers, this decrease in efficiency can be partially compensated by intercellular signal propagation through gap junctions, resulting in higher hormone actions than in Cx32–deficient livers. It is concluded that gap junctions are not only involved in intercellular propagation of nervous, but also of hormonal signals from periportal to perivenous hepatocytes.
Nitric Oxide and Guanosine 3′,5′–Cyclic Monophosphate Stimulate Bile Secretion in Isolated Rat Hepatocyte Couplets, But Not in Isolated Bile Duct Units
Authors: Trauner Michael , Mennone Albert , Gigliozzi Alessandro , Fraioli Flavia , Boyer James L.
Published: July 2004

Nitric oxide (NO) and guanosine 3′,5′–cyclic monophosphate (cGMP) have recently been shown to stimulate bile acid-independent bile flow in the isolated perfused rat liver (IPRL). However, the cellular origin and mechanisms of this choleresis have not yet been determined. To address these questions, we examined the effects of NO and cGMP on bile secretion in isolated rat hepatocyte couplets (IRHC) and in isolated bile duct units (IBDU), both of which are isolated cell systems in which cell polarity is maintained and secretion can be measured directly. Changes in the area of the canalicular and ductular lumens were determined in IRHC and IBDU, respectively, as indicators of the rate of fluid secretion using video microscopy. In addition, Cl-/HCO3
- exchanger activity in IBDU was evaluated by measuring changes in intracellular pH (pHi ) after Cl- removal/readmission by microfluorometric methods. In the presence of HCO3
-, both the NO donor, S–nitroso–acetyl–penicillamine (SNAP), and the cell–permeant cGMP analogue, dibutyryl cGMP (DBcGMP), stimulated canalicular bile secretion (P < .05), as did the cell–permeant cAMP analogue, dibutyryl cAMP (DBcAMP) (P < .05). Removal of HCO3
- from the buffer completely abolished the choleretic effects of DBcGMP, but had no effect on NO–induced choleresis. In contrast, secretion in IBDU was not stimulated following incubations with SNAP or DBcGMP over 30 minutes, whereas DBcAMP and secretin, a cholangiocyte secretagogue and cAMP agonist, both had a marked effect on ductular secretion over this same time interval (P < .05). SNAP also had no effect on Cl-/HCO3
- exchanger activity in IBDU, and inhibition of endogenous NO synthesis by N
G–monomethyl–l–arginine (l–NMMA) did not alter secretin–induced stimulation of ductular bile secretion and Cl-/HCO3
- exchanger activity. In summary, NO and cGMP stimulate bile secretion exclusively at the the level of hepatocytes, whereas cAMP mediates choleresis at both hepatocyte and bile duct levels. These findings may have important implications for the regulation of ductular bile secretion by hormones and neuropeptides, as well as under pathological conditions with increased hepatic NO synthesis.
Endotoxin Suppresses the Oltipraz–Mediated Induction of Major Hepatic Glutathione Transferases and Cytochromes P450 in the Rat
Authors: Mahéo Karine , Morel Fabrice , Antras-Ferry Jocelyne , Langouët Sophie , Desmots Fabienne , Corcos Laurent , Guillouzo André
Published: July 2004

The effect of Escherichia coli lipopolysaccharide (LPS), a classic inducer of the acute–phase response, has been analyzed on both constitutive and oltipraz (a chemoprotective agent)–inducible messenger RNAs (mRNAs) and enzyme activities of major cytochromes P450 (CYPs) and glutathione transferases (rGSTs) in rat liver. At the dose administered (1 mg/kg) and the time studied (6 and 24 hours), endotoxin had no effect on the expression of either CYPs and GSTs with the exception of CYP1A2, which was reduced at both mRNA and activity levels. A strong increase of rGSTA1/2, rGSTM1, rGSTP1, CYP1A2, CYP2B1/2, and CYP2E1 was observed after 3 days of treatment with oltipraz (0.075%, wt/wt). Oltipraz induction of these enzymes (with the exception of CYP2E1) was found to be suppressed at both mRNA, protein, and activity levels during the acute–phase response to endotoxin. Moreover, it is shown that oltipraz induction of CYP1A2 and CYP2B1/2 and its suppression by E. coli LPS occurred at a transcriptional level. These data support the idea that the chemoprotective effect of oltipraz is altered in the course of inflammation and that adaptation in chemoprotective strategies should be considered in certain physiopathologic situations.
Perioperative Change in Albumin Messenger Rna Levels in Patients With Hepatocellular Carcinoma
Authors: Gion Tomonobu , Taketomi Akinobu , Shimada Mitsuo , Shirabe Ken , Hasegawa Hirofumi , Takenaka Kenji , Sugimachi Keizo
Published: July 2004

A quantitative assay of albumin messenger RNA (mRNA) in blood samples was designed using the competitive reverse–transcription polymerase chain reaction, and the significance of measuring albumin mRNA levels in the blood of patients with hepatocellular carcinoma (HCC) in hepatic resection was evaluated. Albumin mRNA levels were measured in the following: (1) peripheral blood in 11 patients with HCC and 20 control subjects without liver disease, (2) blood in the portal and hepatic veins in five patients with HCC immediately after laparotomy, and (3) a perioperative series of peripheral blood in eight patients with HCC. Two patients with HCC whose albumin mRNA level in peripheral blood was markedly high were both at stage IVa. On the other hand, 20 control subjects showed negative or <5 × 103 transcripts/μg RNA of albumin mRNA expression. Immediately after laparotomy, the albumin mRNA levels in the tumor–draining hepatic vein were greater than in the portal and non-tumor–draining hepatic veins in four of five patients with HCC. Albumin mRNA levels in peripheral blood showed a marked increase after mobilization and/or resection of the liver and, thereafter, gradually decreased at postoperative day 7 in all eight patients with HCC. A new method to measure the albumin mRNA levels in blood samples was developed, and high albumin mRNA levels in the peripheral blood of patients with advanced–stage HCC suggest the presence of HCC cells in the circulation. Increased levels in the tumor–draining hepatic vein could indicate the spontaneous release of tumor cells or nontumorous hepatocytes or an increased albumin transcription in activated blood mononuclear cells. An increase in the levels in peripheral blood during an operation is intermittent. Therefore, an increased albumin mRNA level in the tumor–draining vein suggests, but does not prove, that the increased albumin mRNA level reflects tumor cells entering the systemic circulation. This alone does not prove that the prognosis is worsened.
Mutations in Hepatitis B Dna Polymerase Associated With Resistance to Lamivudine Do Not Confer Resistance to Adefovir In Vitro
Authors: Xiong Xiaofeng , Flores Carmina , Yang Huiling , Toole John J. , Gibbs Craig S.
Published: July 2004

To determine whether adefovir is active against lamivudine–resistant hepatitis B virus (HBV), the inhibition constants of adefovir diphosphate and lamivudine triphosphate for wild–type and mutant human HBV DNA polymerases, which contain amino acid substitutions associated with lamivudine resistance, were compared. Recombinant wild–type and mutant human HBV DNA polymerases were expressed and substantially purified using a baculovirus expression system and immunoaffinity chromatography. HBV DNA polymerase mutants M552I, M552V, and L528M/M552V showed resistance to lamivudine triphosphate with inhibition constants (Ki
) increased by 8.0–fold, 19.6–fold, and 25.2–fold compared with that of wild–type HBV DNA polymerase. However, these mutants remained sensitive to adefovir diphosphate with the inhibition constants increasing by 1.3–fold and 2.2–fold or decreasing by 0.79–fold. The L528M single mutation, identified in patients with increasing HBV DNA levels during therapy with famciclovir, also remained sensitive to adefovir diphosphate with the inhibition constant increased by only 2.3–fold.
Mutations in Ns5a Region of Hepatitis C Virus Genome Correlate With Presence of Ns5a Antibodies and Response to Interferon Therapy for Most Common European Hepatitis C Virus Genotypes
Authors: Frangeul Lionel , Cresta Pascale , Perrin Michele , Lunel Françoise , Opolon Pierre , Agut Henri , Huraux Jean-Marie
Published: July 2004

A part of the hepatitis C virus (HCV) nonstructural protein 5A (NS5A) amino acid sequence, designated as an interferon (IFN)–sensitive determining region (ISDR), has been shown to be correlated with a response to IFN in Japanese patients. We have shown previously that the presence of NS5A antibodies (Abs) detected by the INNOLIA test (IL–NS5A Ab) is also correlated with a response to IFN. The aim of this study was to investigate, in a wide range of patients, the possible relationship within the NS5A protein between the sequence of ISDR and that used in the INNOLIA test designated as IL3R. Serum samples from 52 patients infected by HCV genotypes 1, 2, and 3 were analyzed before and after treatment. The patients were classified as nonresponders (NRs), responder–relapsers (RRs), or long–term responders (LTRs). We amplified the NS5A region for 42 patients using polymerase chain reaction (PCR), and these amplicons were sequenced directly. The 10 remaining patients were analyzed using PCR with mutation–specific primers. No correlation was found between the IL3R sequence of the HCV strains and the presence of the IL–NS5A Ab for all genotypes. However, for the subtype 1b, only 2 of 11 NR patients tested had an arginin in position 2218 within the ISDR versus 3 of 3 LTR and 10 of 13 RR patients. All patients with R–2218 had IL–NS5A Ab. For the genotype 1a, 2 of 2 LTR and 1 of 3 RR were mutated in position 2216–2218 in comparison to three NR sequences. For the genotype 3, no mutations were found in the region homologous to 1b–ISDR, but 4 of 5 LTR and RR patients had a mutation T–2161 to A or V versus 0 of 3 NR patients. A close correlation was found between arginin in position 2218 in ISDR, the presence of IL–NS5A Ab, and the response to IFN therapy for genotype 1b, but this association did not predict a long–term response. For genotype 3, a potential ISD mutation could be located at the codon 2161.
Prognosis of Chronic Hepatitis C: Results of A Large, Prospective Cohort Study
Authors: Niederau Claus , Lange Stefan , Heintges Tobias , Erhardt Andreas , Buschkamp Marlies , Hürter Dietmar , Nawrocki Marek , Kruska Lothar , Hensel Frank , Petry Wolfgang , Häussinger Dieter
Published: July 2004

The prognosis of chronic hepatitis C virus (HCV) infection is still ill–defined. The present study prospectively evaluated mortality and complications in a large cohort of patients with chronic hepatitis C. The study included 838 anti–HCV and HCV–RNA-positive patients who were followed for 50.2 ± 26.9 months (mean ± SD; range, 6–122 months) in a prospective protocol. During follow–up, 62 patients died (31 from liver disease and 31 from other causes), and 12 patients needed liver transplantation. When compared with a matched general population, hepatitis C increased mortality mainly when cirrhosis was present and in patients who were less than 50 years old at study entry. During follow–up, a further 30 patients developed nonlethal complications of cirrhosis. By multivariate regression, survival was decreased by cirrhosis, long disease duration, history of intravenous drug abuse, and excessive alcohol consumption, whereas interferon therapy improved survival. Alanine transaminase (ALT), bilirubin, sex, and genotype had no effect on survival. The risk of hepatocellular carcinoma (HCC) (n = 17) was increased by cirrhosis and to a lesser degree by long disease duration and high bilirubin, whereas interferon therapy, genotype, and other factors had no effect. Chronic hepatitis C is a disease with considerable mortality and morbidity when cirrhosis is present at diagnosis. Patients who acquire the infection early in life have a markedly increased mortality even when cirrhosis is absent at diagnosis. The age at diagnosis therefore should play a major role in therapeutic considerations. The present data also suggest that interferon therapy has a long–term clinical benefit, although it did not reduce the risk of liver cancer.
Sequential Serum Hepatitis C Viral Rna Levels Longitudinally Assessed by Branched Dna Signal Amplification
Authors: Gordon Stuart C. , Dailey Peter J. , Silverman Ann L. , Khan Bilal A. , Kodali Vallistram P. , Wilber Judith C.
Published: July 2004

The aim of this study was to determine the stability of viral load over an extended period in patients with chronic hepatitis C virus (HCV). Sequential serum specimens collected from fourteen non–alcoholic adult patients with chronic HCV between 1990 and 1997 were tested retrospectively for HCV RNA levels by branched DNA assay (Quantiplex HCV RNA 2.0 [Chiron Diagnostics, Emeryville, CA]). A minimum of three serum samples was obtained at various intervals from each patient. None of the patients received antiviral therapy. Liver biopsies, available for 10 of 14 patients, showed mild or moderate hepatitis in seven and cirrhosis in three (one developed cirrhosis during follow–up). RIBA strip immunoassay showed that 7, 3, and 4 patients had viral genotypes 1, 2, and 3, respectively. The follow–up time averaged 5.3 years (range, 3.7 to 6.6 years). Eight patients (57.2%) showed increased viral levels from baseline to follow–up, the remaining six patients (42.8%) showed decreased viral levels. The three cirrhotic patients had the highest viral levels over time. The mean change was a 0.29–fold decrease (median, -5.08; range, -17.49 to +7.32). A less than twofold change in either direction was demonstrated for six patients (42.8%), and a less than threefold change was demonstrated for 10 patients (71.4%). Variation from baseline to last follow–up as calculated by log determination showed that the viremic load varied less than one log10 in all but one individual. These results show that viral load remains relatively stable over prolonged periods in most untreated patients with chronic hepatitis C.