bluuebunny/crossref_metadata_2025 huggingface dataset:
Therapy of hepatitis C: Re-treatment with alpha interferon
Authors: Alberti A , Chemello L , Noventa F , Cavalletto L , De Salvo GianLuca
Published: July 2004

The long-term benefit of interferon therapy in chronic hepatitis C is limited. During therapy, serum alanine aminotransferase (ALT) levels decrease to normal and hepatitis C virus (HCV) RNA decreases in 40% to 60% of patients. However, most patients relapse after therapy withdrawal, so that no more than 15% to 25% achieve a sustained response. Re-treatment has been evaluated in studies using different regimens and forms of alpha interferon in different cohorts of patients at different times after initial therapy. Both end-of-treatment and sustained responses to re-treatment correlate with the type of response achieved during the initial course. Patients who do not respond or have only a partial response to the initial course of interferon have an extremely low rate of sustained response when re-treated, independently of the regimen used. Combining data from 13 studies, sustained responses occurred in no patients who were re-treated with 3 million units (MU) three times weekly for 6 months, and in only 2% to 3% of patients re-treated with higher doses and/or for longer periods. In contrast, a significant number of patients who responded during the initial course but subsequently relapsed have a sustained response when re-treated with interferon alone. Combining data from 11 published studies on patients who relapsed after an initial course, sustained responses occurred in 15% (95% confidence interval [CI], 10%-20%) of patients re-treated with 3 MU three times weekly for 6 months, in 29% (CI, 17%-40%) re-treated with a higher dose for 6 months, and in 43% (CI, 34%/50%) re-treated for at least 12 months. On the other hand, patients who relapsed after a 12-month course of interferon had only 4% rate (range, 0%-8%) of sustained response when re-treated. The best predictor of sustained response to re-treatment in patients who had relapsed was a negative serum HCV-RNA test by polymerase chain reaction at the end of the first course. These results, which have been confirmed in a recent prospective, randomized controlled trial, indicate that nonresponders to interferon should not be re-treated with interferon alone, whereas patients who relapse after a 6-month course of alpha interferon therapy have an indication to be re-treated for at least 12 months, especially if serum HCV RNA was negative at the end of the first course of treatment.
Therapy of hepatitis C: Other options
Authors: Bonkovsky H L
Published: July 2004

Because current standard therapy of chronic hepatitis C with α interferon is less than ideal, numerous other approaches have been studied. Iron in the liver, particularly that found in vascular endothelial cells of portal tracts, has been associated with decreased responsiveness to α interferon therapy. Iron reduction alone, generally achieved by therapeutic phlebotomy, regularly has been associated with biochemical improvement (decrease in serum alanine aminotransferase), but not with virological improvement. Iron reduction has been reported to increase the therapeutic response to α interferon. Most studies of this combination have been conducted in patients who had not responded to interferon alone; in these patients, improved responsiveness has been observed in some, but not all studies. In patients not previously treated, iron reduction was found in a recent trial to improve the sustained biochemical and virological response rate from 5% to 29%. Hepatic iron and chronic hepatitis C increase oxidative stress in the liver and are associated with decreases in hepatic glutathione levels. In one report, administration of N-acetyl cysteine, a sulfhydryl donor, led to improved response to interferon in chronic hepatitis C. Several cytokines and immunomodulators have undergone limited study; perhaps the most promising of these is thymosin α-1. In one small study, amantadine was found to produce some response in patients who previously had failed to respond to interferon. Ursodiol improves serum aminotransferase levels in chronic hepatitis C but has no antiviral effect, nor has it been found to improve histologic abnormalities. The future of therapy of chronic hepatitis C will likely include measures to decrease oxidative stress and injury and multidrug combinations, including inhibitors of the hepatitis C viral protease and RNA polymerase.
Enhanced cyclooxygenase-1 expression within the superior mesenteric artery of portal hypertensive rats: Role in the hyperdynamic circulation†
Authors: Hou Ming-Chih , Cahill Paul A. , Zhang Shuangmin , Wang Yi-Ning , Hendrickson Richard J. , Redmond Eileen M. , Sitzmann James V.
Published: July 2004

Portal hypertension (PHT) is characterized by splanchnic hyperemia due to enhanced production of vasodilator substances. Enhanced vasodilation and increased splanchnic blood flow contribute to the elevated portal pressure characteristic of PHT. The aim of this study was to determine whether cyclooxygenase (Cox) expression is altered in PHT vessels and whether chronic inhibition of this enzyme impacts on splanchnic blood flow in PHT. PHT was created in Sprague-Dawley rats by a partial portal vein ligation. Control animals were sham operated. Plasma 6-keto-PGF1α (prostaglandin F1α) levels were significantly elevated in PHT after 2 days as compared with sham and remained elevated up to day 15. Treatment with indomethacin (2 mg/kg ip daily for 15 days) resulted in a significant decrease in 6-keto-PGF1α levels, which was concomitant with a significant decrease in superior mesenteric artery blood flow (Qsma) after 15 days in PHT rats. Cox-I expression was differentially enhanced in the PHT superior mesenteric artery and thoracic aorta during the development and progression of PHT. In contrast, Cox-II messenger RNA (mRNA) and protein expression was not detected in either of these vessels throughout the development of PHT. These data suggest that PHT is associated with enhanced Cox-I expression within the splanchnic vasculature concomitant with elevated plasma prostacyclin levels and a significant pressor response to indomethacin in PHT animals. We conclude that enhanced Cox-I expression results in increased prostacyclin levels that partially contribute to the maintenance of the hyperemia typical of PHT.
Reversibility of hepatorenal syndrome by prolonged administration of ornipressin and plasma volume expansion
Authors: Guevara Mónica , Ginès Pere , Fernández-Esparrach Glòria , Sort Pau , Salmerón Joan Manuel , Jiménez Wladimiro , Arroyo Vicente , Rodés Juan
Published: July 2004

Hepatorenal syndrome is caused by a marked vasoconstriction of the renal circulation. It is suggested that the renal vasoconstriction is related to an overactivity of vasoconstrictor systems secondary to a vasodilation of the arterial circulation that causes a reduction in effective arterial blood volume. To test this hypothesis, 16 cirrhotic patients with hepatorenal syndrome were treated with a combination of ornipressin, a potent vasoconstrictor agent, and plasma volume expansion with albumin to improve effective arterial blood volume. The combined treatment was administered either for 3 or 15 days (8 patients each), and the effects on renal function, vasoactive systems, and systemic hemodynamics were assessed. The 3-day treatment with ornipressin and albumin was associated with a normalization of the overactivity of renin-angiotensin and sympathetic nervous systems, a marked increase in atrial-natriuretic peptide levels, and only a slight improvement in renal function. However, when ornipressin and albumin were administered for 15 days, a remarkable improvement in renal function was observed, with normalization of serum-creatinine concentration, a marked increase in renal plasma flow and glomerular filtration rate, and a persistent suppression in the activity of vasoconstrictor systems. However, 3 of 8 patients on 15-day therapy treatment had to be discontinued because of ischemic complications. In conclusion, the decrease in effective arterial blood volume and the activation of vasoconstrictor systems play a crucial role in the pathogenesis of hepatorenal syndrome. Although the prolonged administration of ornipressin combined with plasma volume expansion reverses hepatorenal syndrome, this treatment should be used with great caution in clinical practice because of the risk of ischemic complications.
Increased renal and vascular cytosolic phospholipase A2 activity in rats with cirrhosis and ascites
Authors: Niederberger Michel , Ginès Pere , Martin Pierre-Yves , St. John Judy , Woytaszek Paul , Xu Lieming , Tsai Phoebe , Nemenoff Raphael A. , Schrier Robert W.
Published: July 2004

Indirect evidence suggests that the renal and vascular production of prostaglandins is increased in cirrhosis with ascites. However, the activity of the enzymes regulating the prostaglandin pathway has not been investigated in cirrhosis. The aim of the current study was to determine the activity of phospholipase A2 (PLA2), the key enzyme in the regulation of prostaglandin synthesis, in kidney and vascular tissue obtained from rats with carbon tetrachloride-induced cirrhosis and ascites (n = 9) and control rats (n= 6). PLA2 activity was assayed in vitro using [14C]arachidonyl-phosphatidylcholine (PC) and [14C]arachidonyl-phosphatidylethanolamine (PE) as substrates in the presence of Ca2+. Kidneys from cirrhotic rats had significantly higher PLA2 activity compared with control rats, with both PC and PE (35 ± 5 and 40 ± 6 vs. 21 ± 2 and 26 ± 3 pmol/mg/min, respectively; P < .05 for both). PLA2 activity was increased in the renal cortex as well as in the renal medulla. Fractionation of the kidney extracts by Mono-Q anion-exchange chromatography showed that the elution position of PLA2 activity corresponded to the cytosolic PLA2 isoform (cPLA2). Increased amounts of cPLA2 protein were found in kidney extracts immunoblotted with an anti-cPLA2 antibody. However, reverse-transcriptase polymerase chain reaction (RT-PCR) analysis did not detect any difference in cPLA2 mRNA. PLA2 activity was also higher in aortic tissue from cirrhotic rats than in controls (PC 38 ± 5 vs. 26 ± 1 and PE 66 ± 8 vs. 41 ± 3 pmol/mg/min; P < .05 for both). Incubation of renal and aortic extracts from cirrhotic rats with anti-cPLA2 antibody reduced PLA2 activity by 64% and 88%, respectively. In conclusion, PLA2 activity is increased in kidneys and vascular tissue from cirrhotic rats with ascites. This can be accounted for by an induction of cPLA2, which would mediate, at least in part, the increased renal and vascular production of prostaglandins in cirrhosis.
Transjugular intrahepatic portosystemic shunt versus endoscopic sclerotherapy for the prevention of variceal bleeding in cirrhosis: A randomized multicenter trial
Authors: Merli Manuela , Salerno Francesco , Riggio Oliviero , de Franchis Roberto , Fiaccadori Franco , Meddi Patrizia , Primignani Massimo , Pedretti Giovanni , Maggi Alessandra , Capocaccia Livio , Lovaria Andrea , Ugolotti Ugo , Salvatori Filippo , Bezzi Mario , Rossi Plinio
Published: July 2004

Transjugular intrahepatic portosystemic shunt (TIPS), a new technique for the treatment of portal hypertension, has been successful in preliminary studies to treat acute variceal hemorrhage and to prevent variceal rebleeding. The purpose of this multicenter, randomized controlled trial is to compare the efficacy of TIPS with that of endoscopic sclerotherapy in the prevention of variceal rebleeding in cirrhosis. Eighty-one cirrhotic patients, with endoscopically proven variceal bleeding, were randomized to either TIPS (38 patients) or endoscopic sclerotherapy (43 patients). Randomization was stratified according to the following: if bleeding occurred < 1 week (stratum I); if bleeding occurred 1 to 6 weeks (stratum II); and if bleeding occurred 6 weeks to 6 months (stratum III) before enrollment. Follow-up included clinical, biochemical, Doppler Ultrasound, and endoscopic examinations every 6 months. During a mean follow-up of 17.7 months, 51% of the patients treated with sclerotherapy and 24% of those treated with TIPS rebled (P = .011). Mortality was 19% in sclerotherapy patients and 24% in TIPS patients (P)= .50). Hepatic encephalopathy (HE) developed in 26% and 55%, respectively (P= .006). A separate analysis of the three strata showed that TIPS was significantly more effective than sclerotherapy (P = .026) in preventing rebleeding only in stratum I patients. TIPS is significantly better than sclerotherapy in preventing rebleeding only when it is performed shortly after a variceal bleed; however, TIPS does not improve survival and is associated with a significantly higher incidence of HE. The overall performance of TIPS does not seem to justify the adoption of this technique as a first-choice treatment to prevent rebleeding from esophageal varices in cirrhotic patients.
Expression of apomucins in the intrahepatic biliary tree in hepatolithiasis differs from that in normal liver and extrahepatic biliary obstruction
Authors: Sasaki Motoko , Nakanuma Yasuni , Kim Young S.
Published: July 2004

Mucin plays an important role in the development of hepatoliths, which are formed within the intrahepatic large bile ducts. To date, eight apomucins, components of mucin, have been identified. The purpose of this study is to characterize the expression of MUC1, MUC2, MUC3, MUC5, and MUC6 apomucins at both the protein and messenger RNA (mRNA) levels in the intrahepatic large bile ducts and peribiliary glands in 36 livers from patients with hepatolithiasis, in 25 livers from the patients with extrahepatic biliary obstruction, and in 23 histologically-normal livers. MUC3 and MUC6 were constantly and focally expressed, respectively, in the biliary epithelial cells in the intrahepatic large bile ducts in normal livers, extrahepatic biliary obstruction, and, also, hepatolithiasis. In hepatolithiasis, MUC1, MUC2, and MUC5 apomucins were also focally expressed in 33%, 64%, and 89%, respectively, of the large intrahepatic bile ducts examined, whereas such expression was infrequent in normal livers and in extrahepatic biliary obstruction. The markedly proliferated intramural and extramural peribiliary glands in hepatolithiasis frequently expressed MUC3 and MUC6 apomucins and focally expressed MUC2 and MUC5 apomucins. All of the apomucins mRNA expression, except that of MUC1, resembled each protein expression. In conclusion, the characteristics of apomucins in the intrahepatic biliary tree in hepatolithiasis differs from those in normal livers and extrahepatic biliary obstructrion. Increased expression of gel-forming mucins, such as MUC5 and MUC2, could be involved in the formation and development of hepatolithiasis.
Induction of DNA synthesis in primary cultures of rat hepatocytes by serotonin: Possible involvement of serotonin S2 receptor
Authors: Balasubramanian Sudha , Paulose Cheramadathikudyil S.
Published: July 2004

The involvement of serotonin and its receptor subtype in the induction of hepatocyte DNA synthesis was investigated in primary cultures of adult rat hepatocytes. Serotonin caused a dose-dependent increase in DNA synthesis in primary cultures of rat hepatocytes in the presence of epidermal growth factor (EGF) and insulin, as measured by [3H]thymidine incorporation. The serotonin S2receptor antagonists, ketanserin (10−6 mol/L) and spiperone (10−6 mol/L), blocked stimulation of DNA synthesis by serotonin. Displacement studies on [3H]5-hydroxytryptamine (5-HT) binding to crude membranes from control and regenerating liver tissue, using cold ketanserin and spiperone, showed an increased involvement of S2 receptors of serotonin in the regenerating liver during the DNA-synthetic phase. Serotonin enhanced the phosphorylation of a 40-kd substrate protein of protein kinase C (PKC) in the regenerating liver during the DNA synthetic phase of the hepatocyte cell cycle. This was blocked by ketanserin, indicating that serotonin S2receptor activates PKC, an important second messenger in cell growth and division, during rat liver regeneration. Our results show that serotonin can act as a potent hepatocyte comitogen and induce DNA synthesis in primary cultures of rat hepatocytes, which is suggested to be mediated through the serotonin S2 receptors of hepatocytes.
Prospective randomized controlled trial comparing percutaneous acetic acid injection and percutaneous ethanol injection for small hepatocellular carcinoma
Authors: Ohnishi Kunihiko , Yoshioka Hisashi , Ito Susumu , Fujiwara Kenji
Published: July 2004

To assess whether ultrasound-guided percutaneous acetic acid injection is superior to percutaneous ethanol injection in the treatment of small hepatocellular carcinoma (HCC), 60 patients with one to four HCCs smaller than 3 cm were entered onto a randomized controlled trial. Thirty-one and 29 patients, respectively, were treated by percutaneous acetic acid injection using 50% acetic acid or by percutaneous ethanol injection using absolute ethanol. There were no significant differences in age, sex ratio, Child-Pugh class, size of tumors, or number of tumors between the two groups. When there was no evidence of viable HCC from biopsy, plain and helical dynamic computed tomography, or angiography, the treatment was considered successful and was discontinued. All original tumors were treated successfully by either therapy. However, 8% of 38 tumors treated with percutaneous acetic acid injection and 37% of 35 tumors treated with percutaneous ethanol injection developed a local recurrence (P< .001) during the follow-up periods of 29 ± 8 months and 23 ± 10 months, respectively. The 1- and 2-year survival rates were 100% and 92% in percutaneous acetic acid injection and 83% and 63% in percutaneous ethanol injection (P= .0017). A multivariate analysis of prognostic factors revealed that treatment was an independent predictor of survival. The risk ratio of percutaneous acetic acid injection versus percutaneous ethanol injection was 0.120 (range, 0.027-0.528;P= .0050). In conclusion, percutaneous acetic acid injection is superior to percutaneous ethanol injection in the treatment of small HCC.
Control of mouse hepatocyte proliferation and ploidy by p53 and p53ser246 mutation in vivo
Authors: Yin Li , Ghebranious Nader , Chakraborty Subhendu , Sheehan Christine E. , Ilic Zoran , Sell Stewart
Published: July 2004

The effect of expression of the p53 gene, in the presence or absence of the p53ser246 mutation (p53*), on ploidization (image cytometry), proliferation (expression of proliferating cell nuclear antigen and radioactive thymidine histoautoradiography), and apoptosis (in situ detection of DNA fragments) is determined in hepatocytes of p53-null and p53-transgenic mice. The mouse p53ser246 mutation is equivalent to the p53ser249 mutation found in human hepatomas associated with hepatitis B virus infection and aflatoxin exposure. The hepatocytes of heterozygous or homozygous p53-knockout mice (p53+/−; p53−/−), as well as knockout mice expressing one allele of p53ser246 (p53+/−, p53*; p53−/−, p53*), do not undergo normal polyploidization with aging and show an increase in the number of cycling (G1-, S-, and M-phase) cells. In addition, p53ser246-transgenic mice (p53+/+, p53*; p53+/−, p53*; and p53−/−, p53*) have a greatly increased number of hepatocytes in the G1 phase. No differences in rates of apoptotic hepatocytes are found among any of the mouse groups studied, so the increased proliferation results in a hyperplasia manifested by a increased number of small periportal cells. We conclude that loss of p53 removes blocks in the cell cycle, leading to increased proliferation, whereas expression of the p53ser246 mutation stimulates G0to G1and/or M to G1transition of hepatocytes. Increased proliferation of hepatocytes, combined with no concomitant increase in apoptosis, may in part explain the enhanced development of hepatocellular carcinomas in p53-knockout and p53-transgenic mice exposed to aflatoxin.
Stimulation of inducible nitric oxide synthase in rat liver by hyaluronan fragments
Authors: Rockey Don C. , Chung John J. , McKee Charlotte M. , Noble Paul W.
Published: July 2004

Hepatic injury and chronic wounding are characterized by increased synthesis of extracellular matrix proteins including hyaluronan (HA). Recently, it has been recognized that low-molecular-weight fragments of HA, but not native HA (e.g., high-molecular-weight HA), induce inflammatory gene expression, and activate the transcriptional regulator, nuclear factor κB (NF-κB). The inducible isoform of nitric oxide synthase (iNOS) is induced by cytokines and/or lipopolysaccharide (LPS) through the NF-κB signal transduction pathway. Because of this association, we hypothesized that HA fragments might also stimulate iNOS gene transcription. The aims of this study were therefore to determine whether HA or HA fragments induced iNOS in hepatic cells, and to characterize the signaling pathway. HA fragments (100 μg/mL) markedly stimulated iNOS messenger RNA (mRNA) in endothelial and Kupffer cells, but minimally induced this mRNA in hepatocytes and stellate cells. High-molecular-weight HA (200 μg/mL) had no effect on iNOS mRNA in any cell type. The addition of interferon gamma (IFN-γ) to HA fragments resulted in stimulation of iNOS mRNA 2-, 3-, 4-, and 10-fold above that for HA fragments alone in hepatocytes, endothelial, Kupffer, and stellate cells, respectively. The combination of HA fragments and LPS did not result in an incremental increase in iNOS mRNA induction. iNOS protein and nitrite levels (used as a measure of NO production and NOS enzymatic activity) paralleled closely iNOS mRNA expression and increased proportionally to HA fragment concentration in a dose-dependent fashion. At 1 hour following stimulation, NF-κB DNA binding activity was detected in extracts from Kupffer cells stimulated with HA fragments, but not in those exposed to media alone or to high-molecular-weight HA. Finally, inhibitors of NF-κB blocked HA fragment-dependent iNOS mRNA induction in Kupffer and sinusoidal endothelial cells. The data indicate that HA fragments, but not high-molecular-weight HA, induce iNOS in liver, having the greatest effects on endothelial and Kupffer cells. We speculate that HA fragments may be an important stimulus for NO production in various forms of liver disease, particularly as a cofactor with inflammatory cytokines.
Interleukin-10 expression is autoregulated at the transcriptional level in human and murine kupffer cells
Authors: Knolle Percy A. , Uhrig Anja , Protzer Ulrike , Trippler Martin , Duchmann Rainer , zum Büschenfelde Karl-Hermann Meyer , Gerken Guido
Published: July 2004

Interleukin 10 (IL-10) is known to downregulate immune responses. The regulation of IL-10 gene expression therefore determines the outcome of local immune reactions. We investigated time course and downregulation of IL-10 production in primary Kupffer’s cells (KC), which are known to secrete IL-10 in response to endotoxin challenge. Human and murine KC were isolated by centrifugal elutriation and investigated for IL-10 gene expression by a two-step amplification procedure (reverse transcriptase-polymerase chain reaction [PCR] followed by T7-polymerase chain reaction). We show that IL-10 messenger ribonucleic acid (mRNA) showed a >450 fold increase in KC 2 hours after endotoxin challenge. IL-10 protein release from KC strictly depended on de novo protein synthesis. Endotoxin mediated increase in IL-10 gene expression was downregulated by exogenous (>350-fold reduction of IL-10 mRNA level), as well as endogenous IL-10 protein, showing a negative autoregulatory feedback loop. IL-10 receptor expression was found to be constitutive and functional in KC. Early expression of IL-10 in KC may be of functional relevance to the outcome of immune and inflammatory reactions in the liver sinusoid. The negative autoregulation of IL-10 expression may represent a mechanism to regain a state of functional responsiveness in the microenvironment towards new proinflammatory stimuli. In conclusion, autoregulatory downregulation of IL-10 expression in KC may account for important regulatory steps of local immune response in the liver sinusoid.
Cytotoxic effect of 7α-hydroxy-4-cholesten-3-one on HepG2 cells: Hypothetical role of acetaldehyde-modified Δ4-3-ketosteroid-5β-reductase (the 37-kd-liver protein) in the pathogenesis of alcoholic liver injury in the rat
Authors: Lin Renee C. , Fillenwarth Michael J. , Du Xiangnan
Published: July 2004

We recently identified △4-3-ketosteroid-5β-reductase as the 37 kd liver protein which is highly susceptible to acetaldehyde modification in rats continuously fed alcohol. The 5β-reductase is a key enzyme involved in bile acid synthesis. We report here that the ability to degrade 7α-hydroxy-4-cholesten-3-one (HCO) was lower in the liver cytosol of alcohol-fed rats than in control animals, suggesting an inhibition of the 5β-reductase enzyme activity by acetaldehyde modification. We also showed that HCO exhibited a time- and concentration-dependent cytotoxicity to HepG2 cells. HCO cytotoxicity was noticeable at a concentration of 2.5 μg/mL. When 10 μg/mL of HCO was added to confluent cell monolayers, 57% and 37% of cells remained viable after 24 and 48 hours of treatment. The decrease in cell viability was accompanied by an increased lactic dehydrogenase activity in the culture medium. DNA extracted from HCO-treated cells showed no evidence of DNA fragmentation when analyzed by agarose gel electrophoresis. Staining with propidium iodide showed no nuclear condensation in cells. Thus, cell death by HCO treatment was caused by necrosis and not by apoptosis. Various agents, including, serum proteins, hormones, bile acids, antioxidants, Ca++-chelators, Fe++-chelator, CYP450 inhibitor, adenylate cyclase inhibitor, protease inhibitors, and nitric oxide synthase inhibitor, did not protect against HCO cytotoxicity. We speculate that HCO concentrations may be elevated around the pericentral area in the liver after chronic alcohol ingestion, causing local cell necrosis. The release of cellular contents and protein-acetaldehyde adducts (PAAs) may activate nonparenchymal cells and provoke autoimmune reaction. Thus, the formation of the 37 kd-PAA may play an important role in the initiation of alcoholic liver injury.
Mallory body formation by ethanol feeding in drug-primed mice
Authors: Zhang-Gouillon Zhi-Qi , Yuan Qi-X. , Hu Bing , Marceau Norman , French Barbara A. , Gaal Karl , Nagao Yasuki , Wan Y.-J. Yvonne , French Samuel W.
Published: July 2004

Drug-primed mice, created by a 5-month feeding of diethyl-1,4-dihydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate (DDC), followed by a 1-month withdrawal, were refed ethanol or isocaloric dextrose (control) diets intragastrically for 7 days. The formation of Mallory bodies (MBs) was monitored by immunofluorescence and immunoperoxidase microscopy using antibodies to cytokeratin and ubiquitin, and also by electron microscopy. The changes in cytokeratin 55 (CK55), ubiquitin conjugate, nuclear factor κB (NFκB) p65, NFκB p50, inhibitor κBα, c-myc, tumor necrosis factor α, and cytochrome P450 2E1 (CYP2E1) contents were determined by Western blotting using appropriate antibodies. The messenger RNA (mRNA) for CYP2E1, cytokeratin, ubiquitin, hepatocyte growth factor activator, and tissue transglutaminase was quantitated. MBs were present at 5 to 7 days’ postfeeding with ethanol, but not with dextrose. They developed in clusters of “empty hepatocytes,” where the cytokeratin antibody failed to recognize the typical filament structures seen in normal hepatocytes. MBs were larger and more numerous in the subcapsular region. Northern blots showed that CK55 mRNA was decreased by the ethanol treatment, but protein levels were increased, suggesting a decreased turnover of the cytokeratin. Likewise, the increase in CYP2E1 protein in the face of a lack of an increase in mRNA for CYP2E1 could be explained by a decreased turnover of this cytochrome. This is the first report of MB formation induced by ethanol ingestion in an experimental model.
Selective bowel decontamination of recipients for prevention against liver injury following orthotopic liver transplantation: Evaluation with rat models†
Authors: Arai Masahiro , Mochida Satoshi , Ohno Akihiko , Arai Shin , Fujiwara Kenji
Published: July 2004

Gut-derived substances can activate Kupffer cells to provoke hepatic necrosis after partial hepatectomy in rats. A similar situation may occur during orthotopic liver transplantation (OLT), as congestion in the intestinal wall, caused by portal vein occlusion, is inevitable during the operation. The contribution of such substances to liver injury following OLT was investigated in rats. Oral administration of polymyxin B sulfate for 7 days significantly altered intestinal bacterial flora in rats; Enterobacteriaceae diminished and anaerobes such as Bifidobacterium, Lactobacillus,Bacteroides, and Eubacterium increased in number, compared with the control rats. Also, this treatment significantly reduced endotoxin concentration in the portal blood 30 minutes after blood reflow following portal vein occlusion. When OLT was performed in rats using the liver preserved in cold University of Wisconsin solution for 18 hours, tissue factor activity in Kupffer cells (KC) isolated from the transplanted liver 1 hour after the operation was significantly higher than in that of normal rats. This increase was significantly reduced by pretreatment of the recipients with polymyxin B sulfate. In these recipients, serum alanine aminotransferase activity, tumor necrosis factor α (TNFα) concentration, and histological extent of liver necrosis were significantly attenuated at 24 hours after the operation compared with those of control rats. We conclude that the substances derived from bacilli sensitive to polymyxin B sulfate in the gut may be a contributing factor to liver injury following OLT in rats; we feel that this probably occurs by entering of the substances into the portal blood during the ahepatic phase of the operation to activate KC. Selective bowel decontamination of recipients with polymyxin B sulfate would be a candidate for protection against early graft failure following OLT.
Carrier-mediated entry of 4-methylumbelliferyl sulfate: Characterization by the multiple-indicator dilution technique in perfused rat liver
Authors: Chiba Masato , Schwab Andreas J. , Goresky Carl A. , Pang Sandy K.
Published: July 2004

The hepatocellular entry of 4-methylumbelliferyl sulfate (4MUS) a highly ionized and highly bound anion capable of futile cycling, was examined in the single-pass albumin-free perfused rat liver preparation. Desulfation of 4MUS to 4-methylumbelliferone (4MU) was verified in vitro to be a low-affinity, high-capacity process (K
m= 731 μmol/L;V
max= 414 nmol min−1g−1 liver). With 4MUS given to the perfused rat liver, sulfation of 4MU, the formed metabolite, was attenuated in the presence of 2,6-dichloro-4-nitrophenol (DCNP), a sulfation inhibitor, and when sulfate ion was substituted by chloride ion. 4MU sulfation, being a high-affinity system, was reduced most effectively at the lowest 4MUS concentration (15 μmol/L) used, evidenced by the increased (24%) net hepatic extraction ratio of 4MUS and reduced utilization (72%) of infused tracer 35SO4
2− by 4MU for 4MU35S formation. Single-pass multiple indicator dilution (MID) studies were thus conducted under identical conditions (DCNP and absence of inorganic sulfate), with injection of [3H]4MUS and a set of noneliminated vascular and cellular reference indicators into the portal vein (prograde) or hepatic vein (retrograde), against varying background bulk concentrations of 4MUS (5 to 900 μmol/L). The steady-state removal rate of 4MUS and formation rates of 4MU and its glucuronide conjugate (4MUG) were not altered with perfusion flow direction, suggesting the presence of even or parallel distributions of 4MUS desulfation and 4MU glucuronidation activities. When the outflow dilution profile of [3H]4MUS was evaluated with the barrier-limited model of Goresky, a slight red cell carriage effect was found for 4MUS. The permeability surface area product for cellular entry for prograde showed a dramatic concentration-dependent decrease (from 0.13 to 0.01 mL sec−1 g−1, or 7.4 to 0.56 times the blood perfusate flow rate) and was resolved as saturable and nonsaturable components, while data for retrograde were more scattered, varying from 2.8 to 1 times the blood perfusate flow rate. Efflux (coefficient = 0.0096 ± 0.0024 and 0.0088 ± 0.0062 mL sec−1 g−1, respectively) was relatively insensitive to concentration and flow direction. The same was observed for the removal capacity for metabolism and excretion (sequestration coefficient: for prograde, 0.0056 ± 0.0017 mL sec−1g−1; for retrograde, 0.0056 ± 0.003 mL sec−1 g−1). The decrease in the apparent partition coefficient (ratio of 4MUS concentration estimated in tissue to unbound plasma concentration) and the increase in relative throughput component with concentration further substantiate the claim on the presence of concentrative processes at the sinusoidal membrane.
Changes in glutathione homeostasis during liver regeneration in the rat
Authors: Huang Zong-Zhi , Li Hongyan , Cai Jiaxin , Kuhlenkamp John , Kaplowitz Neil , Lu Shelly C.
Published: July 2004

We have shown previously that plating primary cultures of rat hepatocytes under low density, which stimulates hepatocytes to shift from the G0 to the G1phase of the cell cycle, resulted in increased levels of glutathione (GSH) and cysteine, and increased activity of γ-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in GSH synthesis (Lu et al., Am. J. Physiol. 1992;263:C1181-C1189). In the current work we examined changes in GSH homeostasis after two-thirds partial hepatectomy (PH). Male Sprague-Dawley rats underwent two-thirds PH or sham operation. GSH, oxidized glutathione (GSSG), cysteine, GSH efflux, DNA synthesis, changes in GCS subunit messenger RNA (mRNA), and protein levels were measured 12 and 24 hours after PH. Both liver GSH and cysteine levels were doubled at 12 hours and remained elevated at 24 hours after PH. GSSG levels also increased, but the ratio of GSH to GSSG levels remained unchanged. The increase in GSH and cysteine levels preceded the increase in DNA synthesis. Sinusoidal GSH efflux was unchanged after two-thirds PH, but biliary GSH efflux decreased. However, total GSH efflux was minimally altered after two-thirds PH. The increase in GSH can be largely accounted for by the increase in both cysteine availability and the activity of GCS. The steady-state mRNA and protein levels of the GCS heavy subunit were increased at 12 hours after PH. The mRNA level of the GCS light subunit was unchanged. In summary, early in the course of liver regeneration the steady-state hepatic GSH levels double because of an increase in the biosynthesis of GSH.
Regulation of early cholesterol biosynthesis in rat liver: Effects of sterols, bile acids, lovastatin, and BM 15.766 on 3-hydroxy-3-methylglutaryl coenzyme A synthase and acetoacetyl coenzyme A thiolase activities
Authors: Honda Akira , Salen Gerald , Nguyen Lien B. , Xu Guorong , Tint Stephen G. , Batta Ashok K. , Shefer Sarah
Published: July 2004

Cytosolic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase catalyzes the formation of HMG-CoA, the substrate for the rate-controlling enzyme in the cholesterol biosynthetic pathway. To explore the regulation in liver, we developed a new, accurate, and reliable reversed-phase ion-pair chromatographic assay that uses nonradioactive substrates and n-propionyl coenzyme A as an internal recovery standard. The hepatic activities were measured in rats treated with cholesterol, sitosterol, cholic acid, deoxycholic acid, ursodeoxycholic acid, cholestyramine, bile fistula, lovastatin, and BM 15.766, an inhibitor of 7-dehydrocholesterol Δ7-reductase, and were compared with microsomal HMG-CoA reductase and cytosolic acetoacetyl coenzyme A (AcAc-CoA) thiolase activities. HMG-CoA synthase activity was effectively suppressed in synchrony with HMG-CoA reductase activity by treatments with cholesterol (−41%, P< .05), cholic acid (−72%, P< .005), and deoxycholic acid (−62%, P< .05). However, ursodeoxycholic acid increased activity 84% (P< .05) and intravenous sitosterol did not change activity. AcAc-CoA thiolase activities also paralleled HMG-CoA reductase and HMG-CoA synthase activities, but differences were not statistically significant. In contrast to inhibition, up-regulation of hepatic HMG-CoA synthase activities by cholestyramine, bile fistula, and lovastatin was much less than HMG-CoA reductase activities. In addition, BM 15.766 did not stimulate synthase activity, whereas lovastatin increased activity 2.4-fold. Thus, hepatic HMG-CoA synthase activity was regulated coordinately with HMG-CoA reductase, and responded more forcefully to regulatory stimuli than acetoacetyl-CoA thiolase activity but usually less than HMG-CoA reductase.
Pharmacological expression in rat hepatocytes of a gene transferred by an adenovirus vector enabled by a chimeric promoter containing multiple cyclic adenosine monophosphate response elements
Authors: Suzuki Motoyoshi , Singh Ravi N. , Crystal Ronald G.
Published: July 2004

Using the adenovirus vector AdCF126(CRE8).Luc to deliver an expression cassette containing multiple cyclic adenosine monophosphate (cAMP) response elements driving the luciferase reporter gene, this study is directed toward evaluating the possibility of controlling genes transferred to the liver using pharmacological agents that raise hepatocyte cAMP levels. Infection of primary rat hepatocytes with AdCF126(CRE8).Luc yielded a low level of luciferase activity that was enhanced 16-fold by the addition of forskolin. Direct intrahepatic administration of the Ad vector in C57Bl/6 mice resulted in low-level luciferase activity that was increased 76-fold by the administration of theophylline and 8-bromo-cAMP to increase cAMP levels. In contrast, animals receiving intrahepatic administration of a control vector containing a constitutively active Rous sarcoma virus (RSV) viral promoter driving the luciferase gene had no response to elevated cAMP. Strikingly, delivery of the vector to the liver by the intravenous route permitted a 258-fold enhancement of liver luciferase activity following administration of the same cAMP-elevating agents. In comparison, a control Ad vector with the RSV promoter was not activated by the elevation in cAMP. The maximum luciferase levels achieved by the combination of AdCF126(CRE8).Luc and pharmacological cAMP elevation was 45-fold greater than that with the RSV promoter. These results show the feasibility of using a chimeric promoter to permit pharmacological induction of high-level expression from an expression cassette transferred to the liver with an adenovirus vector, an approach that may be useful in a variety of liver-related gene-transfer strategies.
A two-year prospective study of the effect of ursodeoxycholic acid on urinary bile acid excretion and liver morphology in cystic fibrosis-associated liver disease
Authors: Lindblad Anders , Glaumann Hans , Strandvik Birgitta
Published: July 2004

The efficacy of 2 years of treatment with ursodeoxycholic acid (UDCA) in cystic fibrosis (CF)-associated liver disease was evaluated by liver biopsies and liver function tests in 10 patients aged 8 to 28 years. The metabolism of UDCA was investigated by analysis of urinary bile acids with fast atom bombardment mass spectrometry (FABMS) and gas-liquid chromatography-mass spectrometry. Eight patients responded with normalization of liver function tests (LFT) and all with decreased serum levels of immunoglobulin G (IgG). Blind evaluation of liver biopsies indicated improved liver morphology with less inflammation and/or bile duct proliferation than before treatment with UDCA in 7 patients. Only 1 patient had signs of progression of clinical liver disease. The proportion of UDCA and isoUDCA in urine varied, but increased during treatment from a mean (median) of approximately 4% (3%) to 40% (40%) of total bile acids. The increase was not related to LFT. The secondary bile acids, such as lithocholic acid (LCA) and deoxycholic acid (DCA), did not increase significantly. The excretion pattern of glycosidic conjugates of UDCA and its metabolites was similar to that found in healthy individuals, UDCA and isoUDCA being mainly excreted in conjugation withN-acetylglucosamine. This study shows that UDCA modulates inflammation in CF-associated liver disease and indicates improvement of liver morphology during 2 years of treatment.
Lipoprotein changes in children after liver transplantation: Mild hypertriglyceridemia and a decrease in HDL/HDL ratio
Authors: Granot Esther
Published: July 2004

Hyperlipidemia is frequently observed in patients who undergo renal, cardiac, bone marrow, or liver transplantation, and its contribution to the long-term morbidity and survival of patients with organ transplants may be substantial. In the few studies that have focused on the pediatric age group, findings have been inconsistent. The lipoprotein profile of 10 children after liver transplantation was characterized and compared with those in normal population controls and 10 healthy siblings. Plasma triglyceride and cholesterol concentrations were determined, lipoprotein fractions (very-low-density lipoprotein [VLDL], low-density lipoprotein [LDL], and high-density lipoproteins [HDL2and HDL3]) were isolated, their chemical compositions were analyzed (protein, phospholipids, triglycerides, free cholesterol, and cholesteryl ester), and the percent relative weight composition of the particles was calculated. Plasma triglyceride and VLDL cholesterol levels were higher post-liver transplantation (P < .05): triglycerides (mean ± SD), 115.1 ± 58.7 mg% versus 76.6 ± 20.9 mg% in siblings and 60.0 ± 25.0 mg% in normal population controls; very-low-density lipoprotein cholesterol (VLDL-C), 23.0 ± 11.7 mg% versus 15.3 ± 4.7 mg% and 13.0 ± 8.0 mg%, respectively. Plasma triglyceride levels did not correlate with the length of the period after liver transplantation. Levels of LDL-C and total HDL-C and the relative weight composition of VLDL, LDL, HDL2, and HDL3 particles did not differ between post-liver transplantation children and controls. Posttransplantation, levels of HDL3, the normally predominant HDL subfraction, were decreased relative to HDL2levels (HDL3, 1.3; HDL2, 2.3). Because this observed relative increase in larger cholesteryl ester-rich HDL particles (HDL2) may result from inhibition of cholesteryl ester-triglyceride transfer processes, cholesteryl ester transfer protein activity was assayed. Cholesteryl ester transfer protein activity did not differ between patients and controls. Thus, the lipoprotein changes observed in children post-liver transplantation are mild hypertriglyceridemia and a significant increase in HDL2relative to HDL3. Because HDL2 is regarded as protective against atherosclerosis, this may be of clinical relevance.
High prevalence of the His63Asp HFE mutation in italian patients with porphyria cutanea tarda
Authors: Sampietro Maurizio , Piperno Alberto , Lupica Loredana , Arosio Cristina , Vergani Anna , Corbetta Noemi , Malosio Ida , Mattioli Michela , Fracanzani Anna Ludovica , Cappellini Maria Domenica , Fiorelli Gemino , Fargion Silvia
Published: July 2004

Sporadic porphyria cutanea tarda (PCT) is caused by a reduced activity of uroporphyrinogen decarboxylase (URO-D) in the liver. Mild to moderate iron overload is common in PCT, as iron is one of the factors which trigger the clinical manifestations of the disease through the inactivation of URO-D. A role for genetic hemochromatosis in the development of iron overload in sporadic PCT has been hypothesized in the past. The aim of this work was to investigate whether mutations of HFE, which is a candidate gene for hemochromatosis, play the role of genetic susceptibility factors for PCT in Italian patients, who have a high prevalence of acquired triggering factors, such as hepatitis C virus (HCV) chronic infection and alcohol. We determined HFE genotypes of 68 male patients with PCT. Our data do not confirm an association of PCT with the Cys282Tyr HFE mutation, strongly associated with hemochromatosis in Northern European countries. A second mutation of HFE, His63Asp, however, had a significantly increased frequency as it was present in half of the patients. Surprisingly, the presence of the His63Asp mutation was not related to the iron status of patients, suggesting that a subtle abnormality of iron metabolism induced by this mutation could escape detection by the standard parameters of iron status. In PCT patients with liver disease, the presence of the mutation could contribute to the inactivation of URO-D, either directly or through a synergistic action with other factors that cause liver damage.
Effect of dihydrotestosterone on turnover of alcohol dehydrogenase in rat hepatocyte culture
Authors: Mezey Esteban , Rennie-Tankersley Lynda , Potter James J.
Published: July 2004

Dihydrotestosterone decreased alcohol dehydrogenase (ADH) activity and enzyme-protein in rat hepatocytes in culture. This effect was observed after the hepatocytes had been exposed to dihydrotestosterone for 3 days at concentrations of 0.5 μmol/L or higher. Dihydrotestosterone did not decrease alcohol dehydrogenase messenger RNA (mRNA) but, rather, resulted in small increases in ADH mRNA after 3 days of exposure. To further determine the mechanism for the effects of dihydrotestosterone in decreasing the enzyme, the turnover of ADH was determined after incorporation of [3H]-leucine into the enzyme protein. Dihydrotestosterone did not alter the initial 2-hour incorporation of [3H]-leucine into the enzyme protein. Dihydrotestosterone, however, resulted in an increase in the fractional rate of degradation (Kd) of the enzyme from 0.12 ± 0.013 to 0.23 ± 0.004 per hour (P < .001) accompanied by a much smaller increase in the fractional rate of synthesis (K s) from 0.12 ± 0.028 to 0.17 ± 0.031 per hour (P > .05). Hence, the mechanism for the fall in ADH in the presence of dihydrotestosterone is an increase in enzyme degradation which is not accompanied by a sufficient increase in enzyme synthesis.
Enhanced Na+-dependent bile salt uptake by WIF-B cells, a rat hepatoma hybrid cell line, following growth in the presence of a physiological bile salt
Authors: Konieczko Elisa M. , Ralston Amy K. , Crawford Aleta R. , Karpen Saul J. , Crawford James M.
Published: July 2004

Although bile salts are toxic to the liver at high plasma concentrations, the effects of physiological concentrations of bile salts on normal hepatic function are poorly understood. We examined the effect of taurocholate (TC) on the basolateral uptake of [3H]TC in WIF-B cells, a hybrid cell line stably exhibiting in vitro the structural and functional polarity of hepatocytes. Cells were grown in the absence or presence of TC (50 μmol/L) over 12 days, and then incubated with [3H]TC concentrations ranging from 1 to 250 μmol/L. For both control and TC-grown cells, uptake of [3H]TC was linear over 2 minutes. In control cells, the K
mfor [3H]TC Na+-dependent uptake over 1 minute was 6 ± 5 μmol/L, and the Vmax was 45 ± 6 pmol TC/mg protein/min (± SEM). TC-grown cells exhibited no significant change in K
m but showed a doubling of V
max to 87 ± 6 pmol TC/mg protein/min (P< .005). In both control and TC-grown cells, maximal uptake of [3H]TC occurred following 10 to 12 days in culture, with TC-grown cells consistently showing greater rates of [3H]TC uptake from 4 to 14 days in culture. Western blots immunostained for the basolateral Na+-dependent plasma membrane protein, ntcp, revealed the appropriate ≈50-kd band in control and TC-grown cells, and confocal immunofluorescence microscopy demonstrated staining along the basolateral plasma membrane. Northern blots hybridized with a cDNA probe directed against ntcp indicated a modest TC-induced increase in mRNA levels. Reverse-transcriptase polymerase chain reaction (RT-PCR) using RNA isolated from WIF-B cells and oligonucleotide primers specific for rat ntcp or human NTCP transcripts revealed only the presence of the rat ntcp transcript. We conclude that bile salts, at concentrations normally found in mammalian portal blood, may be capable of promoting enhanced hepatocellular bile salt uptake via an increase in basolateral Na+-dependent plasma membrane transport capacity.