bluuebunny/crossref_metadata_2025 huggingface dataset:
Nitric Oxide Does Not Reverse Pulmonary Hypertension Associated With End–Stage Liver Disease: A Preliminary Report
Authors: Ramsay M A , Schmidt A , Hein H A , Nguyen A T , Lynch K , East C A , Ramsay K J , Klintmalm G B
Published: August 2004

Pulmonary hypertension is a well known, though uncommon complication of end–stage liver disease (ESLD). Patients with severe pulmonary hypertension and ESLD undergoing orthotopic liver transplantation (OLT) may develop right ventricular failure and death. This study investigates the reversibility of pulmonary hypertension by the inhalation of nitric oxide in patients under evaluation for OLT. Ten patients with ESLD who were discovered to have moderate to severe pulmonary hypertension were administered nitric oxide via face mask in concentrations ranging from 0 to 80 ppm. Inhaled nitric oxide is a potent pulmonary vasodilator without apparent systemic effects. Nitric oxide had no demonstrable effect on mean pulmonary artery pressure (PAP) (37 vs. 37 mm Hg), transpulmonary gradient (TPG) (26 vs. 26 mm Hg), or pulmonary vascular resistance (PVR) (295 vs. 288 dynes.sec.cm -5). Two patients were discovered to have an elevated pulmonary artery occlusion pressure (PAOP) on baseline readings. The cause of pulmonary hypertension in these two patients was secondary to volume overload as a result of hepato–renal syndrome rather than primary pulmonary arteriolar pathology and was responsive to diuresis or dialysis but not to nitric oxide therapy. In conclusion nitric oxide does not reverse pulmonary hypertension associated with ESLD.
Continuous Versus Inpatient Prophylaxis of the First Episode of Spontaneous Bacterial Peritonitis With Norfloxacin
Authors: Novella M , Sola R , Soriano G , Andreu M , Gana J , Ortiz J , Coll S , Sabat M , Vila M C , Guarner C , Vilardell F
Published: August 2004

Cirrhotic patients with ascites and low ascitic fluid total protein and/or high serum bilirubin levels are at high risk to develop the first episode of spontaneous bacterial peritonitis during long–term follow–up. The aim of the present study was to determine the efficacy of continuous long–term selective intestinal decontamination with norfloxacin in the prevention of this complication. One hundred nine cirrhotic patients with ascites and ascitic fluid total protein levels of ≤ 1 g/dL or serum bilirubin levels of > 2.5 mg/dL without previous spontaneous bacterial peritonitis were prospectively randomized into two groups: group 1 (n = 56) received norfloxacin, 400 mg daily administered orally, and group 2 (n = 53) was the long–term control group, receiving norfloxacin only during hospitalization. During a mean follow–up of 43 +/– 3 weeks, there was one spontaneous bacterial peritonitis (1.8%) in group 1 and 9 (16.9%) in group 2 (P < .01). The incidence of community–acquired spontaneous bacterial peritonitis was lower in group 1 (1.8% vs. 13.2%, P < .05), whereas the incidence of nosocomial spontaneous bacterial peritonitis (0% vs. 3.7%) and the incidence of extraperitoneal infections (25% vs. 24.5%) were similar in both groups (P = NS). The actuarial probability of survival at 18 months was 75% in group 1 and 62% in group 2 (P = NS). Resistance to norfloxacin was observed in 9 of 10 (90%) Escherichia coli isolated in infections from group 1 and in 4 of 11 (36.3%) from group 2 (P < .05). The overall incidence of infections caused by norfloxacin–resistant bacteria was higher in group 1 (19.6% vs. 15%), but it did not reach statistical significance. Continuous long–term selective intestinal decontamination with norfloxacin is effective in preventing the first spontaneous bacterial peritonitis in cirrhotic patients at high risk. However, the emergence of infections caused by norfloxacin–resistant bacteria must be weighed carefully against the benefits of continuous long–term prophylaxis.
Autoimmune Hepatitis in Childhood: A 20–Year Experience
Authors: Gregorio G V , Portmann B , Reid F , Donaldson P T , Doherty D G , McCartney M , Mowat A P , Vergani D , Mieli-Vergani G
Published: August 2004

To determine the clinical, biochemical, and histological features, and outcome of childhood autoimmune hepatitis (AIH), we reviewed the medical records of 52 children with AIH, 32 (median age: 10 [2–15] years) anti–nuclear and/or smooth muscle antibody (ANA/SMA) positive, 20 (7 [0.8–14] years) liver/kidney microsomal antibody (LKM–1) positive, with median follow–up of 5 years (range 0.3–19). At presentation: 56% had symptoms of prolonged acute hepatitis; LKM–1 positive were younger (P = .011), with higher bilirubin (P = .007), and AST (P = .047); ANA/SMA positive had lower albumin (P = .023); 69% ANA/SMA positive, and 38% LKM–1 positive were cirrhotic (P = .080). ANA/SMA positive had increased frequency of HLA haplotype A1/B8/DR3/DR52a compared with controls (53% vs. 14%, P < .001). Of six (5 LKM–1 positive) with fulminant hepatitis, four were transplanted, one died, and one ANA/SMA positive improved with immunosuppression. Of 47 treated with immunosuppression, 2 (1 LKM–1 positive) died with no remission and 4 (2 LKM–1 positive) were transplanted 8 to 14 years after diagnosis. Immunosuppression was stopped successfully in 19% of ANA/SMA positive after a median of 3 years of treatment, but in none of LKM–1 positive. Baseline bilirubin and international normalized prothrombin ratio (INR) were independent variables predictive of outcome. In conclusion, ANA/SMA positive and LKM–1 positive AIH in childhood have clinical, biochemical, and histological differences, but similar severity and long–term outcome.
Daily Variation in Portal Blood Flow and the Effect of Propranolol Administration in A Randomized Study of Patients With Cirrhosis
Authors: Alvarez D , de las Heras M , Abecasis R , Terg R , Gerona S , Albornoz L , Galdame O , Torres J , Mastai R
Published: August 2004

A nocturnal increase in portal pressure and blood flow was demonstrated in patients with cirrhosis, suggesting that these hemodynamic changes may contribute to the triggering of the hemorrhagic episodes observed during the night in these patients. It is known that propranolol reduces portal flow, thus reducing the risk of variceal bleeding. In a double–blind, placebo–controlled study, we evaluated the effect of long– term propranolol administration on the daily fluctuation of systemic and splanchnic hemodynamic parameters in 14 patients with cirrhosis. Cardiac output and portal blood flow were measured by the Doppler technique. A daily fluctuation of both cardiac output and portal blood flow was observed, peaking at midnight. β–Adrenergic blockade was manifested by a significant reduction in heart rate (–21% ±4%, P < .01) and cardiac output (–12% ± 2%, P < .05). A significant decrease in portal blood flow (–20 % ± 4%, P < .01) was also observed in these patients. Propranolol administration blunted the time–related changes in cardiac output and portal blood flow. In contrast, patients receiving placebo had a nocturnal peak of both parameters similar to that observed under basal conditions. Our study shows that chronic propranolol administration abolishes the nocturnal peak of portal blood flow in patients with cirrhosis and indicates a preventive effect of propranolol in these patients.
Protective Effect of L–Carnitine in Ammonia–Precipitated Encephalopathy in the Portacaval Shunted Rat
Authors: Therrien G , Rose C , Butterworth J , Butterworth R F
Published: August 2004

l–carnitine administration prevents the neurological symptoms of acute ammonia toxicity. To further evaluate its efficacy in the prevention of hepatic encephalopathy in hyperammonemic conditions, l–carnitine (16 mmol/kg, intraperitoneally [i.p.) was administered 1 hour before ammonium acetate (NH4OAc) (8.5 mmol/kg, subcutaneously) to portacaval shunted (PCS) rats. Cerebrospinal fluid (CSF) ammonia, lactate, and amino acid levels were measured in relation to deteriorating neurological status in these animals. None of 35 l–carnitine–treated animals showed neurological deterioration after NH4OAC administration compared with saline–treated controls; the latter manifested severe encephalopathy progressing through loss of righting reflex to coma. Survival rate was 100% in the l–carnitine–treated group compared with 5% in saline–treated controls. Following NH4OAC administration to PCS rats, CSF ammonia increased to 0.93 ± 0.15 mmol/L and 1.24 ± 0.15 mmol/L at precoma and coma stages of encephalopathy (P < .01) respectively. Treatment with l–carnitine reduced CSF ammonia at both precoma and coma stages; the time–course of this protective effect paralleled blood and CSF l–carnitine accumulation. CSF alanine and lactate increases following NH4OAC administration to PCS rats were significantly attenuated following l–carnitine treatment. However, L– carnitine treatment did not lead to significant reductions in plasma ammonia nor CSF or brain glutamine in these animals. These findings show the therapeutic efficacy of l–carnitine in ammonia–precipitated coma in PCS rats and suggest that this protective effect is centrally mediated involving improved mitochondrial respiration. l–carnitine could be of therapeutic benefit in the prevention of hepatic encephalopathy precipitated by ammoniagenic conditions in humans with chronic liver disease.
Regulation of Cyclin–Dependent Kinase Inhibitor P21Waf1/Cip1/Sdi1 Gene Expression in Hepatic Regeneration
Authors: Albrecht J H , Meyer A H , Hu M Y
Published: August 2004

WAF1/Cip1/Sdi1 (p21) is the prototype of a family of proteins that inhibit cyclin–dependent kinases and regulate cell cycle progression in eukaryotic cells. In addition to normal cell cycle progression, p21 is involved in growth suppression mediated by p53 and transforming growth factor β (TGFβ), differentiation, and apoptosis. To gain insight into the possible involvement of p21 in liver cell growth, the expression and regulation of the p21 gene was evaluated in rodent models of liver regeneration and specimens of human liver diseases. Little p21 mRNA was detected in normal liver tissue. After growth stimulation in vivo by 70% partial hepatectomy (PH), the p21 transcript was upregulated in a biphasic manner, with enhanced expression during G1 phase and following S phase. The induction of p21 after PH was regulated primarily at the post–transcriptional level and was due to enhanced mRNA stability. Inhibition of protein synthesis with cycloheximide rapidly induced p21 expression, primarily by post–transcriptional stabilization of the transcript. Hepatic p21 mRNA was also induced by dietary protein deprivation in normal mice. Expression of the p21 gene after PH was similar in p53–deficient (p53 –/–) and wild–type mice, but was p53–dependent following protein deprivation. Primary hepatocytes in culture demonstrated increased p21 expression after treatment with hepatocyte growth factor, TGFβ, and activin A. p21 mRNA was upregulated in human liver diseases, suggesting a possible role in hepatic growth regulation in pathologic states. The present study demonstrates that p21 is regulated by p53–dependent and –independent pathways in the liver, and is influenced by both mitogenic and growth inhibitory stimuli.
Hematogenous Spreading of Hepatocellular Carcinoma Cells: Possible Participation in Recurrence in the Liver
Authors: Funaki N O , Tanaka J , Seto S I , Kasamatsu T , Kaido T , Imamura M
Published: August 2004

To detect hepatocellular carcinoma (HCC) cells in the circulating peripheral blood, we previously designed a highly sensitive reverse–transcription polymerase chain reaction (RT–PCR) method targeting α–fetoprotein (AFP) messenger RNA (mRNA). Using this method, we analyzed peripheral blood of in– and out–patients bearing HCC for 2 months consecutively and examined the outcome thereafter. All 11 patients with recurrence either in the liver alone or in both the liver and the lung were positive for AFP mRNA. Among 10 recurrence–free patients, 2 patients were AFP mRNA–negative and remained recurrence–free during a period of 22 months observed. Four of 8 AFP mRNA–positive, recurrence–free patients developed a clinically evident recurrence in the liver after 2 to 16 months. Seven of 8 preoperative patients were already positive for AFP mRNA and the remaining negative patient became positive during surgery. Four of 6 preoperatively positive and still–alive patients had recurrence in the liver after 2 to 9 months. None of the HCC–negative patients were positive for AFP mRNA. We actually found an AFP protein–positive cell in peripheral blood obtained from one of the AFP mRNA–positive patients by immunostaining. The present results suggest that circulating HCC cells may have some relationship with the recurrence of HCC in the liver.
Localization of Thrombospondin in Hepatocellular Carcinoma
Authors: Hayashi K , Kurohiji T , Shirouzu K
Published: August 2004

The expression of thrombospondin (TSP) of hepatocellular carcinoma (HCC) was investigated by light and electron microscopic immunohistochemical studies, using an anti–human TSP antibody to HCC and liver specimens. Tissue specimens were obtained by surgical resection from 60 patients with HCC. In light microscopic immunohistochemistry, all specimens showed strong immunoreaction for TSP in fibrous tissues in HCC and surrounding nonmalignant areas. In the strong TSP–stained fibrous tissue, immunoreaction for TSP was observed in fibroblasts and vascular endothelial cells. In 31 of 60 cases, HCC cells revealed cytoplasmic staining for TSP. In electron microscopic immunohistochemistry, reaction products for TSP were revealed in the rough endoplasmic reticulum (RER) of HCC cells, and also in the RER of fibroblasts and endothelial cells. These findings proved that TSP was synthesized in HCC cells, in endothelial cells, and in fibroblasts surrounding the tumor, and further suggested that TSP in the fibrous tissues surrounding carcinoma was basically synthesized by nonparenchymal cells such as fibroblasts and endothelial cells.
Reduced P21Waf1/Cip1 Expression and P53 Mutation in Hepatocellular Carcinomas
Authors: Hui A M , Kanai Y , Sakamoto M , Tsuda H , Hirohashi S
Published: August 2004

p21WAF1/CIP1 is a universal cyclin–dependent kinase (cdk) inhibitor, the expression of which is regulated by p53–dependent and p53–independent pathways. We examined p21WAF1/CIP1 expression in and p53 status of 21 primary hepatocellular carcinomas (HCCs) by reverse–transcriptase polymerase chain reaction (RT–PCR) and by PCR single–strand conformation polymorphism (PCR–SSCP) analysis. p21WAF1/CIP1 messenger RNA expression was reduced markedly in 8 of 21 HCCs (38.1%) and 5 of these 8 HCCs bore p53 mutations. The relative p21WAF1/CIP1 messenger RNA expression value of HCCs with p53 mutations (.73 ± .13 U, n = 6) was significantly lower than that of HCCs with wild–type p53 (1.00 ± 0.21 U, n = 14; P < .01). The p21WAF1/CIP1 expression levels in cancerous tissues (.73 ± .13 U) were significantly reduced in comparison with those in noncancerous tissues (.97 ± .13 U) (P < .01) in the 6 cases with p53 mutations. These data indicate that p21WAF1/CIP1 expression in HCCs is predominantly regulated by dependence on p53 and that reduced p21WAF1/CIP1 expression may participate in hepatocarcinogenesis.
Difference in Gene Expression for Matrix Metalloproteinase–1 Between Early and Advanced Hepatocellular Carcinomas
Authors: Okazaki I , Wada N , Nakano M , Saito A , Takasaki K , Doi M , Kameyama K , Otani Y , Kubochi K , Niioka M , Watanabe T , Maruyama K
Published: August 2004

The histological observation that well–differentiated cancer cells in early hepatocellular carcinoma (HCC) invade portal tracts and/or fibrous bands and that these fibrous tissues then disappear suggests the participation of matrix metalloproteinase–1 (MMP–1) in the degradation of fibrous tissue. To confirm this hypothesis, the authors investigated the localization of both the MMP–1 protein and its messenger RNA (mRNA) in early HCC immunohistochemically and by in situ hybridization using complementary DNA (cDNA) and synthetic antisense probe of MMP–1; they then compared the results with those in advanced HCC. MMP–1 gene transcripts and protein were observed in well–differentiated cancer cells of early HCC but not in moderately or poorly differentiated cancer cells. Thus, cancer cells producing MMP–1 in early HCC may destroy the portal tract tissue adjacent to the cancer lesion and/or the fibrous band of cirrhosis. These results seem to have demonstrated a difference in the mechanism of cancer growth and invasion between early and advanced HCCs.
Epithelial–Mesenchymal Transition of Cultured Rat Neonatal Hepatocytes Is Differentially Regulated in Response to Epidermal Growth Factor and Dimethyl Sulfoxide
Authors: Pagan R , Martin I , Llobera M , Vilaro S
Published: August 2004

Neonatal rat hepatocytes cultured in the absence of added growth factors dedifferentiate by epithelial–mesenchymal transition (EMT). This involves the loss of their typical differentiation markers, the acquisition of a migrating morphology, and a change in the expression of the intermediate filament (IF) proteins. We attempted to determine whether the EMT of cultured neonatal rat hepatocytes could be modulated by factors that maintain and promote the differentiation state of adult and fetal hepatocytes such as epidermal growth factor (EGF) and dimethyl sulfoxide (DMSO). By (3H)–thymidine incorporation, Western blotting analysis, flow cytometry, and double–immunofluorescence studies, we found that both factors have marked but opposite effects on the EMT and on proliferation of neonatal liver cells. In DMSO treatment, albumin levels were higher than in the nontreated cells at all days studied. Moreover, DMSO reduced cytokeratin levels and inhibited cell proliferation, acquisition of the fibroblast–like morphology, and vimentin expression typical of the EMT. The increase in vimentin–positive cells in serum–free medium was not observed in DMSO cultures. EGF also increased albumin levels at all days studied. In contrast, EGF treatment induced hepatocyte proliferation and enhanced vimentin and cytokeratin expression. However, the increase in vimentin levels did not correlate with a significant increase in the number of vimentin–positive cells. Moreover, vimentin–positive cells in EGF treatment were also cytokeratin–positive and albumin–positive, and they maintained epithelioid morphology in spite of the vimentin network. These results indicate that EMT of cultured rat neonatal hepatocytes is differentially regulated in response to EGF and DMSO.
Adenovirus–Mediated Expression of Cytokine–Induced Neutrophil Chemoattractant in Rat Liver Induces A Neutrophilic Hepatitis
Authors: Maher J J , Scott M K , Saito J M , Burton M C
Published: August 2004

C–X–C chemokines are potent chemoattractants that are believed to mediate neutrophilic inflammation in several organs. Recent studies suggest a role for C–X–C chemokines in the pathogenesis of neutrophilic hepatitis but do not prove causation. We investigated the biological consequences of hepatic chemokine production in vivo by transiently overexpressing cytokine–induced neutrophil chemoattractant (CINC), a member of the C–X–C chemokine family, in intact rats. Rats were injected intraportally with a replication–defective recombinant adenovirus containing the CINC complementary DNA (cDNA). Within 4 days, treated animals had high levels of CINC in both liver tissue and plasma Rats overexpressing CINC exhibited an eightfold increase in circulating neutrophils; they also developed severe hepatic injury, characterized by a 6– to 25–fold increase in plasma transaminases and marked hepatic inflammation on biopsy. Liver disease in CINC–producing rats correlated positively with the number of neutrophils sequestered in the hepatic parenchyma. Tissue injury was attributed directly to chemokine overproduction, because control rats infected with adenoviruses lacking the CINC cDNA did not produce CINC and developed only minor hepatic abnormalities. These experiments provide direct evidence that C–X–C chemokines, when expressed in sufficient quantity in the liver in vivo, induce neutrophil recruitment and tissue invasion and provoke severe liver injury. The data suggest that C–X–C chemokines have important pathogenic potential in both clinical and experimental liver disease.
Selective Loss of Binding Sites for the Glutamate Receptor Ligands [3H]Kainate and (S)–[3H]5–Fluorowillardiine in the Brains of Rats With Acute Liver Failure
Authors: Michalak A , Butterworth R F
Published: August 2004

There is increasing evidence that alterations of glutamatergic function are implicated in the pathogenesis of central nervous system consequences of acute liver failure. The aim of the study was to assess the integrity of glutamate receptors in the brain in experimental ischemic liver failure using quantitative receptor autoradiography and the selective ligands [3H]MK801 (for N–methyl–D–aspartate [NMDA] sites), [3H]5–fluorowillardiine (for non–NMDA, α–amino–3–hydroxy–5–methyl–4–isoxazole propionic acid [AMPA] subclass sites), and [3H]kainate (for non–NMDA, kainate subclass sites). At coma stages of encephalopathy, a selective loss of up to 60% of binding sites for the kainate– and AMPA–receptor ligands was observed in cerebral cortical and hippocampal structures as well as in the hypothalamus and cerebellum. The finding of a selective loss of AMPA sites at coma stages of encephalopathy in this model of acute liver failure is consistent with previous electrophysiological reports of inhibition of AMPA–mediated neuronal depolarization resulting from exposure of hippocampal neurons to millimolar concentrations of ammonia. On the other hand, the present study showed that binding sites for the NMDA–receptor ligand [3H]MK801 at coma stages of encephalopathy in acute liver failure were within normal limits in all brain structures examined. NMDA sites are uniquely neuronal, whereas kainate and AMPA sites are localized on both neurons and astrocytes. Therefore, the selective loss of non–NMDA sites in acute liver failure may also reflect astrocytic changes in this condition. Because astrocytic glutamate receptors are implicated in K+ and neurotransmitter reuptake, alterations in their density could result in altered neuronal excitability and thus be responsible for the neurological dysfunction characteristic of hepatic encephalopathy in acute liver failure.
Neutrophil–Mediated Sinusoidal Endothelial Cell Injury After Extensive Hepatectomy in Cholestatic Rats
Authors: Ohtsuka M , Miyazaki M , Kondo Y , Nakajima N
Published: August 2004

The aim of this study was to assess the hypothesis that hepatic failure after extensive hepatectomy in patients with obstructive jaundice (OJ) may be mediated by polymorphonuclear neutrophils (PMN). In the OJ group, rats underwent a partial hepatectomy of 78% after 2 weeks of cholestasis and subsequent external biliary drainage for 5 days. In the sham–operated control group, rats were partially hepatectomized 19 days after the sham surgery. The concentration of the serum cytokine–induced neutrophil chemoattractant (CINC), which is homologous with the growth–related oncogene (gro) product, a member of the human interleukin (IL)–8 family, and a major neutrophil chemotactic factor in rats, increased concomitantly with accumulation of PMNs in the hepatic sinusoids during cholestasis and subsequent external drainage. However, changes in the serum purine nucleoside phosphorylase (PNP)/alanine transaminase (ALT) ratio as a marker of sinusoidal endothelial cell (SEC) injury showed no significant differences between the two groups. Intercellular adhesion molecule–1 (ICAM–1) expression on SECs was not affected by cholestasis and external drainage. After partial hepatectomy, the serum CINC concentration immediately elevated more prominently in the OJ group than in the sham–operated control group, and accumulation of PMNs in the sinusoids was more obvious and prolonged in the former. ICAM–1 expression was enhanced in both groups with a peak between 24 and 48 hours after partial hepatectomy. At this peak period, a significantly higher PNP/ALT ratio was observed in the OJ group. These results suggest that accumulation of PMNs in the sinusoidal space and ICAM–1 expression on SECs might be closely associated with the development of SEC injury after extensive hepatectomy in cholestasis.
Angiotensin–Converting Enzyme Inhibition by Enalapril: A Novel Approach to Reduce Ischemia/Reperfusion Damage After Experimental Liver Transplantation
Authors: Anthuber M , Farkas S , Rihl M , Menger M D , Schildberg F W , Jauch K W , Messmer K
Published: August 2004

Angiotensin–converting enzyme (ACE) inhibitors have proven to be effective in the reduction of ischemia/reperfusion damage after myocardial ischemia. Whether this favorable effect can be related to other models of ischemia and reperfusion has not yet been investigated. Therefore, we studied in a model of syngeneic liver transplantation in the rat the effect of recipient enalapril treatment on postischemic liver injury. Untreated animals served as the control group. Treatment with enalapril was started 5 minutes before reperfusion by intravenous infusion of enalapril at a dosage of 5 mg/kg/h. By means of in vivo microscopy, the sinusoidal perfusion rate and leukocyte adherence in sinusoids and postsinusoidal venules were analyzed during 45 to 60 minutes of reperfusion. Liver function was monitored by measuring bile output over a period of 60 minutes. Analysis of coagulation factors (prothrombin time, factor V, fibrinogen) and liver enzymes (alanine transaminase [ALT], aspartate transaminase [AST]) served for the evaluation of organ dysfunction and damage secondary to ischemia/reperfusion injury. The sinusoidal perfusion rate was significantly improved by enalapril treatment (94.7% [1.0] vs. 75.3% [3.8]; mean [SEM]; P = .005). In addition, leukocyte–sticking in both liver sinusoids and postsinusoidal venules was remarkably reduced in enalapril–treated animals as compared with controls (stickers/lobule: 21.0 [3.3] vs. 59.2 [2.1]; P = .0004; stickers/mm2 venular surface: 20.5 [4.7] vs. 110.3 [18.1]; P = .0004). Moreover, bile output was increased (1.13 [0.35] vs. 0.43 [0.18] g bile/60 min x 100 g liver; P = .06). Values for PT (22.5% [2.1] vs. 9.7% [1.8]; P = .005), factor V 99.4% [9.5] vs. 49.5% [8.5]; P = .007), and fibrinogen (64.1% [7.7] vs. 12.8% [3.2]; P = .001) were significantly improved, paralleled by a remarkable reduction in serum ALT (1,428 U/L [190] vs. 2,315 [248]; P = .02). Our data show for the first time that ACE inhibition in the liver recipient by enalapril attenuates hepatic ischemia/reperfusion damage after experimental liver transplantation. Our results may offer a novel approach to reduce ischemia/reperfusion injury in clinical liver transplantation.
Banff Schema for Grading Liver Allograft Rejection: An International Consensus Document
Authors: Demetris Anthony J. , Batts Kenneth P. , Dhillon Amar P. , Ferrell Linda , Fung John , Geller Stephen A. , Hart John , Hayry Pekka , Hofmann Walter J. , Hubscher Stephan , Kemnitz Josef , Koukoulis George , Lee Randall G. , Lewin Klaus J. , Ludwig Jurgen , Markin Rod S. , Petrovic Lidija M. , Phillips James M. , Portmann Bernard , Rakela Jorge , Randhawa Parmjeet , Reinholt Finn P. , ReynÈS Michel , Robert Marie , Schlitt Hans , Solez Kim , Snover Dale , Taskinen Eero , Thung Swan N. , Tillery Weldon G. , Wiesner Russell H. , Wight Derek D.G. , Williams James W. , Yamabe Hirohiko
Published: August 2004

A panel of recognized experts in liver transplantation pathology, hepatology, and surgery was convened for the purpose of developing a consensus document for the grading of acute liver allograft rejection that is scientifically correct, simple, and reproducible and clinically useful. Over a period of 6 months pertinent issues were discussed via electronic communication media and a consensus conference was held in Banff, Canada in the summer of 1995. Based on previously published data and the combined experience of the group, the panel agreed on a common nomenclature and a set of histopathological criteria for the grading of acute liver allograft rejection, and a preferred method of reporting. Adoption of this internationally accepted, common grading system by scientific journals will minimize the problems associated with the use of multiple different local systems. Modifications of this working document to incorporate chronic rejection are expected in the future.
Effects of Extended Cold Preservation and Transplantation on the Rat Liver Microcirculation
Authors: Imamura H , Brault A , Huet P M
Published: August 2004

Liver sinusoidal endothelial cell impairment and/or microcirculatory disturbances are thought to induce storage–related graft failure; however, the respective roles of changes induced by extended cold preservation and transplantation, as well as their interactions, are still unknown. To this end, the alterations of the liver microcirculation and of hepatocyte and sinusoidal endothelial cell functions induced by extended cold preservation and/or transplantation were assessed using an isolated perfused rat liver model combined to an orthotopic rat liver transplantation model. Liver microcirculation remained minimally altered following extended cold ischemia alone, despite a marked deterioration of sinusoidal endothelial cell function, while liver microcirculation alterations were mainly characterized by areas of no–reflow following transplantation alone. It was only when both procedures were associated that hepatocyte function became markedly compromised, without further deterioration in liver microcirculation. It is concluded that extended cold preservation and transplantation as such are not associated with lethal liver injury. However, the sinusoidal cell impairment and the liver microcirculatory disturbances, induced by both conditions combined, are important factors leading to secondary hepatic nonfunction, which might be triggered by extrahepatic events.
Deletion of Hydroxyethylstarch From University of Wisconsin Solution Induces Cell Shrinkage and Proteolysis During and After Cold Storage of Rat Liver
Authors: Neveux N , De Bandt J , Charrueau C , Savier E , Chaumeil J C , Hannoun L , Giboudeau J , Cynober L A
Published: August 2004

Among the numerous components of the University of Wisconsin (UW) solution used for organ preservation, the usefulness of hydroxyethylstarch (HES), the colloido–osmotic support of this solution, is controversial. The aim of our study was to determine the influence of HES on hepatic metabolism and intracellular hydration state during hypothermic preservation and after reperfusion in a model of isolated perfused rat liver. Three groups of eight livers were perfused either immediately or after 18 hours of cold storage in a UW– based preservation solution with or without HES. Omission of HES results in (1) a stimulation of protein degradation shown by the marked increase in branched–chain amino acid (BCAA) release (211 ± 55 vs. 87 ± 28 nmol/min/g; P < .05, modified UW group vs. UW group), (2) an increase in oxygen consumption (81.7 ± 4.8 vs. 61.5 ± 5.0 μmol/h/g; P < .05), (3) a decrease in glucose production (2.3 ± 0.6 vs. 5.0 ± 0.6 μmol/min/g; P < .05), and (4) a reduction in intracellular volume (414 ± 36 vs. 557 ± 41 μL/g; P < .05). We conclude that HES plays an important role in liver preservation by limiting proteolysis, possibly through the observed preservation of cell volume.
Hepatitis C Viral Quasispecies and Liver Damage in Patients With Chronic Hepatitis C Virus Infection
Authors: Hayashi J , Kishihara Y , Yamaji K , Furusyo N , Yamamoto T , Pae Y , Etoh Y , Ikematsu H , Kashiwagi S
Published: August 2004

To clarify the virological differences in patients with chronic hepatitis C virus (HCV) infection with and without liver damage, we assessed HCV markers in 306 patients from a rural area of Japan. Genotypes of HCV RNA were determined by polymerase chain reaction, and levels of RNA were determined by branched DNA signal–amplification assay. All patients had undergone annual tests for alanine aminotransferase (ALT) levels from 1986 to 1995. Patients were categorized into three groups: group A, 121 patients (39.5%) with normal ALT levels on all occasions for 10 years; group B, 127 patients (41.5%) with intermittently abnormal ALT levels; and group C, 58 patients (19.0%) with consistently abnormal ALT levels. There were no significant differences in serum RNA levels or distribution of genotypes among the three groups. We selected 10 patients from group A with normal ALT levels and 10 from group C with abnormal levels for sequence analysis of the HCV core region (nt 169–378) of five clones from each patient. More mutations were found in the 50 clones from the 10 patients from group C than in the 10 patients from group A. In group A, all mutations were synonymous so that the deduced amino acid sequences were identical among clones from each patient, whereas in group C 16 of 57 mutations were nonsynonymous so that the deduced amino acid sequences showed differences in the five clones of eight of 10 patients. In conclusion, the HCV core region was highly conserved in patients with normal liver biochemical test results but not in those with abnormal results. Our results suggest that abnormal liver biochemical test results in patients with chronic HCV infection may be associated with high degrees of virus quasispecies diversity.
Cytotoxic T Lymphocyte Response and Viral Load in Hepatitis C Virus Infection
Authors: Hiroishi K , Kita H , Kojima M , Okamoto H , Moriyama T , Kaneko T , Ishikawa T , Ohnishi S , Aikawa T , Tanaka N , Yazaki Y , Mitamura K , Imawari M
Published: August 2004

A cytotoxic T lymphocyte (CTL) response to the hepatitis C virus (HCV) nucleoprotein residues 88–96 that are the minimal and optimal epitope for human leukocyte antigen (HLA) B44–restricted CTLs was assessed in 27 HLA B44–positive patients with chronic HCV infection. Serum HCV RNA concentration and the amino acid sequence of the residues 81–100 were also determined. Three patients were infected with HCV with uncommon amino acid substitutions within the epitope. One was infected with HCV with an amino acid substitution in the flanking residues of the epitope. To stimulate CTLs in the peripheral blood, 9–mer peptides that corresponded to the residues 88–96 of the individual patients were synthesized and used. Seven of the 27 patients demonstrated a CTL response to the residues 88–96 with specific cytotoxic activities higher than 20%. The CTL activities were significantly higher in patients with a low titer of serum HCV RNA than in those with a high titer of serum HCV RNA (P = .0006). Some of the patients that demonstrated a CTL response to the residues 88–96 also demonstrated a CTL response to a newly identified HLA B44–restricted CTL epitope or a known HLA A11–restricted CTL epitope or both. No apparent association was observed between the CTL response and the stage of disease, or between the CTL response and the grade of necroinflammatory activity. The results suggest that the HLA B44–restricted CTLs together with other HCV–specific CTLs may inhibit the outgrowth of HCV and that high– titer infection with HCV may suppress the CTL responses.
The Predominance of Hepatitis Delta Virus Genotype I Among Chronically Infected Italian Patients
Authors: Niro G A , Smedile A , Andriulli A , Rizzetto M , Gerin J L , Casey J L
Published: August 2004

The sera of 46 Italian patients with chronic hepatitis delta virus (HDV) infection were analyzed for HDV RNA by polymerase chain reaction (PCR) amplification. Genetic analysis of sequences amplified from two regions of the HDV genome indicated that all HDV RNA–positive patients (98%) were infected with HDV genotype I. In Italy, infection with this genotype appeared to be associated with a broad spectrum of chronic disease. No subtypes of HDV genotype I were identified, nor were genetic variations clearly associated with different disease patterns; however, clustering of some sequences suggested correlations with geography and transmission route. Italian HDV genotype I sequences were more diverse than those from east Asia and North America, suggesting that HDV genotype I was likely introduced to Italy earlier and/or from multiple sources as compared to those areas. All sequences analyzed were predicted to fold into the unbranched rod structure typical of HDV RNA. Within this structure, three conserved features were identified, including sequences around the RNA editing site and the polyadenylation signal site. We conclude that in Italy, where HDV infection has been endemic, the overwhelmingly predominant genotype of HDV is genotype I.
Mutations in the Nonstructural 5A Gene of European Hepatitis C Virus Isolates and Response to Interferon Alfa
Authors: Zeuzem S , Lee J H , Roth W K
Published: August 2004

The response rate to interferon alfa (IFN–α) in patients infected with hepatitis C virus (HCV) genotype 1 isolates is poor. A region associated with sensitivity to IFN has been identified in subtype HCV– 1b isolates from Japanese patients in the carboxyterminal half of the nonstructural protein NS5A (between codon 2209 and 2248). HCV–1b isolates with at least four amino acid changes in this region compared with the HCV–1b prototype sequence were sensitive, whereas isolates identical to the prototype sequence were resistant to IFN–α. Patients infected with HCV–1b isolates carrying 1 to 3 mutations in NS5A2209–2248 showed an intermediate response pattern. Because of the large geographical differences observed for HCV it is unknown whether this putative IFN–α sensitivity determining region is also predictive for European isolates. We analyzed 32 patients chronically infected with HCV–1a or HCV–1b isolates who were treated with 3 million units of recombinant IFN–α three times per week for 1 year. Before initiation, during, and after treatment serum HCV–RNA levels were assessed by a quantitative reverse–transcription polymerase chain reaction (RT–PCR) assay. The amino acid sequence of NS5A2209–2248was determined by direct sequencing of the PCR–amplified HCV genome and was compared with the reference sequence HCV–J. In patients chronically infected with subtype HCV–1a or HCV–1b the initial or sustained response to IFN–α was not related to the number of amino acid substitutions in the NS5A2209–2248 region. In addition, the number of amino acid changes in NS5A2209–2248 was not related to pretreatment HCV–RNA serum levels. In three patients with a pronounced initial decline of HCV–RNA levels (> 3 log) sequence analyses of NS5A2209–2248 were performed before and after therapy. Compared with the pretreatment amino acid sequence the HCV isolates of these patients revealed more mutations in the NS5A2209–2248 region after therapy. These findings from European patients indicate that the NS5A2209–2248 region of HCV does not represent a common interferon sensitivity determining region.
Pretreatment Virus Load and Multiple Amino Acid Substitutions in the Interferon Sensitivity-Determining Region Predict the Outcome of Interferon Treatment in Patients With Chronic Genotype 1B Hepatitis C Virus Infection
Authors: Chayama K , Tsubota A , Kobayashi M , Okamoto K , Hashimoto M , Miyano Y , Koike H , Koida I , Arase Y , Saitoh S , Suzuki Y , Murashima N , Ikeda K , Kumada H
Published: August 2004

Hepatitis C virus (HCV) genotype 1b and high pretreatment virus load are predictive factors of poor response to interferon therapy in patients with chronic hepatitis C. To further examine the factors predicting the response to interferon in patients with genotype 1b infection, we analyzed 110 consecutive patients with HCV who were treated with a total of 624 million units of lymphoblastoid interferon alfa. Thirty–six patients (33%) were responders, while the remaining 74 patients (67%) were nonresponders. Multivariate analysis showed that a high virus titer (assessed by serum core protein level, P = .0021) and the presence of more than two amino acid substitutions in the interferon sensitivity–determining region (ISDR) (P = .0036) correlated significantly with the response to interferon therapy. Because mutations analyzed by direct sequencing of polymerase chain reaction (PCR) products may reflect artifacts of direct sequencing, we further analyzed quasispecies of HCV in this region by cloning and sequencing. Although PCR–based analysis of responders with multiple amino acid substitutions in the ISDR showed the presence of a small amount of wild–type strain in their serum, the results obtained by direct sequencing and cloning were essentially the same. A longitudinal study of quasispecies in 2 patients who showed a dramatic change in the virus titer showed no conversion from wild type to mutant or vice versa. Our results indicate that amino acid substitutions and virus load are independent predictors of the response to interferon therapy. The ability of some patients with no mutation in the ISDR or high virus load to eliminate the virus suggests the presence of other unidentified factors, host or viral, that influence the response to interferon therapy.
Hepatitis C Virus Genotypes and Risk of Hepatocellular Carcinoma in Cirrhosis: A Prospective Study
Authors: Bruno S , Silini E , Crosignani A , Borzio F , Leandro G , Bono F , Asti M , Rossi S , Larghi A , Cerino A , Podda M , Mondelli M U
Published: August 2004

A prospective study was performed to establish whether infection with specific hepatitis C virus (HCV) genotypes was associated with an increased risk of development of hepatocellular carcinoma (HCC) in cirrhosis. A cohort of 163 consecutive hepatitis C virus antibody (anti– HCV)–positive cirrhotic patients was prospectively evaluated for the development of HCC at 6–month intervals by ultrasound (US) scan and α–fetoprotein (AFP) concentration. HCV genotypes were determined according to Okamoto. Risk factors associated with cancer development were analyzed by univariate and multivariate statistics. At enrollment, 101 patients (62%) were infected with type 1b, 48 (29.5%) were infected with type 2a/c, 2 (1.2%) were infected with type 3a, 1 (0.6%) was infected with type 1a, 3 (1.8%) had a mixed–type infection, and, in 8 patients (4.9%), genotype could not be assigned. After a 5– to 7–year follow–up (median, 68 months), HCC developed in 22 of the patients, 19 infected with type 1b and 3 with type 2a/c (P < .005). Moreover, HCC developed more frequently in males (P < .01), patients with excessive alcohol intake (P < .01), those over 60 years of age (P < .02), and in patients who did not receive interferon treatment (P < .02). Multivariate analysis showed that type 1b was the most important risk factor associated with tumor development (odds ratio 6.14, 1.77–21.37 95% confidence interval). Other independent risk factors were older age and male sex. Cirrhotic patients infected with HCV type 1b carry a significantly higher risk of developing HCC than patients infected by other HCV types. The latter may require a less intensive clinical surveillance for the early detection of neoplasia.
Intercellular Gap Junctional Communication Is Required for An Optimal Metabolic Response of the Functional Units of Liver
Authors: Saez J C
Published: August 2004

The gap junction protein connexin32 is expressed in hepatocytes, exocrine pancreatic cells, Schwann's cells, and other cell types. We have inactivated the connexin32 gene by homologous recombination in the mouse genome and have generated homozygous connexin32–deficient mice that were viable and fertile but weighed on the average approximately ≈17% less than wild–type controls. Electrical stimulation of sympathetic nerve in connexin32–deficient liver triggered a 78% lower amount of glucose mobilization from glucose stores, when compared with wild–type liver. Thus, connexin32–containing gap junctions are essential in mouse liver for maximal intercellular propagation of the noradrenaline signal from sympathetic nerve endings, to perivenous (downstream) hepatocytes. In connexin32–defective liver, the amount of connexin26 protein expressed was found to be lower than in wild–type liver and the total area of gap junction plaques was approximately ≈1,000–fold smaller than in wild–type liver. In contrast to patients with connexin32 defects suffering from X chromosome–linked Charcot–Marie–Tooth disease (CMTX) due to demyelination in Schwann cells of peripheral nerves, connexin32–deficient mice did not show neurological abnormalities when analyzed at 3 months of age. It is possible, however, that they may develop neurodegenerative symptoms at an older age.
Adenosine Triphosphate Mediates Intercellular Communication in Liver: Talk Ain't Exactly Cheap
Authors: Lidofsky S
Published: August 2004

Intercellular communication among certain cell types can occur via ATP secretion, which leads to stimulation of nucleotide receptors on target cells. In epithelial cells, however, intercellular communication is thought to occur instead via gap junctions. Here we examined whether one epithelial cell type, hepatocytes, can also communicate via nucleotide secretion. The effects on cytosolic Ca2+ ([Ca2+]i) of mechanical stimulation, including microinjection, were examined in isolated rat hepatocytes and in isolated bile duct units using confocal fluorescence video microscopy. Mechanical stimulation of a single hepatocyte evoked an increase in [Ca2+]i in the stimulated cell plus an unexpected [Ca2+]i rise in neighboring noncontacting hepatocytes. Perifusion with ATP before mechanical stimulation suppressed the [Ca2+]i increase, but pretreatment with phenylephrine did not. The P2 receptor antagonist suramin inhibited these intercellular [Ca2+]i signals. The ATP/ADPase apyrase reversibly inhibited the [Ca2+]i rise induced by mechanical stimulation, and did not block vasopressin– induced [Ca2+]i signals. Mechanical stimulation of hepatocytes also induced a [Ca2+]i increase in cocultured isolated bile duct units, and this [Ca2+]i increase was inhibited by apyrase as well. Finally, this form of [Ca2+]i signaling could be elicited in the presence of propidium iodide without nuclear labeling by that dye, indicating that this phenomenon does not depend on disruption of the stimulated cell. Thus, mechanical stimulation of isolated hepatocytes, including by microinjection, can evoke [Ca2+]i signals in the stimulated cell as well as in the neighboring noncontacting hepatocytes and bile duct epithelia. This signaling is mediated by release of ATP or other nucleotides into the extracellular space. This is an important technical consideration given the widespread use of microinjection techniques for examining mechanisms of signal transduction. Moreover, the evidence provided suggests a novel paracrine signaling pathway for epithelia, which previously were thought to communicate exclusively via gap junctions.