- Email: [email protected]
Haemochromatosis associated mutations in heavy diinkers with decompensated liver disease
of human HPCwithout inducingterminal differentiation. HPCwas employedin in vitro regeneration caused by subcloning of blastospheres,suggesting the utility of HPC against liver tissue injury in vivo.
2761 Interleukin-16 Promoter Region PotymorphismsAnd Alcoholic Liver Disease Helen Newberry, Damitha Edirisinghe, Univ of Liverpool, Liverpool United Kingdom; Nell Fisher, Univ of Birmingham, Birmingham United Kingdom; lan T. Gilmore, ROYAL LIVERPOOLUniv Hosp, Liverpool United Kingdom; David Adams, Univ of Birmingham, Birmingham United Kingdom; Brian K. Park, Munir Pirmohamed, Univ of Liverpool, Liverpool United Kingdom
2759 First Clinical Application ot a Second Generation HepatAssist Bioadificial Liver Support System Without Charcoal Associated with Neurological Recovery in a Fulminant Hepatic Failure Patient Nikolaos Arkadopoulos, Univ of Athens S., Aretaieion, Athens Greece; Maria Alexandri, Aretaieion Hosp, Athens Greece; Nikitorita Poulakaki,Apostolos Papalois, Dimitris Papadimitriou, Andreas Prachalias, Aretaieion Hosp, Athens Greece; Chris Stevens, Circle Biomed, Lexington, MA; Lila Papadimitriou, Anesthisiology Unit, Arataieion Hosp, Athens Greece; John Papadimitriou
Background: Only 10-30% of individuals who misuse alcohol go on to develop alcoholic liver disease (ALD). There is a genetic component in the predisposition to ALD. Intarleukin-fO (IL10) is an anti-inflammatory,anti-proliferativeand anti-fibrotic cytokinethat may exert protective effects in ALD. The IL-lO gene has a polymorphic 5'-flanking region; it has recently been shown that the -627"A allele, which leads to low IL-lO expression, is associated with ALD (Groveet al, Gut, 2000, 46:540-5). We havetherefore investigatedwhether the IL-lO polymorphisms are associated with ALD in our cohort of patients. Methods: Patients with advanced ALD (n=290) who were Hepatitis C negative, diagnosed on either biopsy or unequivocal clinical evidence, were recruited from both Liverpool and Birmingham. The control groups comprised 269 healthy volunteers, and 59 alcohol misusers who did not have any clinical or biochemical evidenceof ALD. Subjects were genotypedfor the -627 (C,A) and -1117 (G,A) polymorphisms by PCR-RFLP analysis using Rsal and EcoNI, respectively, for restriction digestion. Results:The allele frequencies in the patients and controls are shown in the table. The -627"A allele frequency did not differ betweenthe patients with ALD and the two control groups (alcoholic patients with no liver diseaseand healthy volunteers) combined (odds ratio 0.89, 95% CI 0.68-1.19, p=O.4). Similarly, there was no difference in the -1117"A allele frequency between patients with ALD and the combined control group (odds ratio 0.99, 95% CI 0.79-1.24, p = 0.9). Conclusions: In contrast to the study by Grove at al (2000), we have not shown an association betweenthe -627 polymorphism in the IL-lO promoter region and ALD. The reason(s) for the discrepancy between the two studies are unknown, but may be the result of differences in linkage disequilibrium between the patient cohorts.
BACKGROUND:Clinical experiencewith liver support devices utilizing biological components is limited. The HepatAssitTM Bioartiticial Liver Support System containing sevenbillion primary porcine hepatocytes and charcoal is currently being tested in a multicenter randomized controlled phase II/111clinical trial. We present herein the first clinical application of a secondgeneration bioartificial liver (BAL) which incorporates 28 billion primary porcine hepatocytes but no charcoal. METHODS:The novel plasma-perfusedBAL consists of a reservoir, a pump, a membrane oxygenatorand two parallel bioreactors made of polysulfone hollow-fibers each containing 14 billion primary porcine hepatocytes.A 29 year-old male patient with fulminant hepaticfailure (FHF)of unknown etiology, acute renal failure and stage IV hepaticencephalopathy with brain edema, underwent 6-hour long BAL sessions on two consecutive days. Prior to BAL application,all standard intensivecare measureshad beeninitiated including continuous veno-venoushemofiltration as well as full hemodynamic,metabolicand biochemical monitoring. The patient's cerebral perfusion status was monitored with transcranial doppler, cerebral oxymetry and serum S-lOg protein measurements. RESULTS: Both BAL treatments were associated with neurologic improvement demonstrated by increase of the mean velocity and lowering of the pulsatility index of the middle cerebralartery, improvementof cerebraloxymetry values and decreaseof the S-lOO levels. Biochemicalchangesincluded reduction of transaminase and ammonia values. Improvement of coagulation was evident by the stabilization of INR below 3.0 without the needfor fresh frozen plasma administration. After the second BAL treatment the patient underwent successful orthotopic liver transplantation and experienced complete neurologic recovery.CONCLUSION:A modified HepatAssistTM Liver Support System containing 28 billion primary porcine hepatocyteswithout charcoal was safely used to treat an FHF patient and associatedwith physiological, biochemical and clinical measurementsof neurologic improvement during treatment.
IL.10 polymorphisms Subjects Alcoholicliverdisease Alcoholics with no liver disease Healthyconb'ols
Vadantallele ft'equencies -627 polymorphism -11t7 polymorphism 22% 22.9% 24.3%
50% 54.6% 49.4%
2762 2760
Nonalcoholic Steatohepatitis Is Associated With Increased Histological Activity And Fibrosis In Patients With Chronic Hepatitis C Sanjay Ramrakhiani,Alex S. Befeler, Bruce R. Bacon, Saint Louis Univ, St. Louis, MO
A Common Mutation of the Long-Chain HydroxyacyI-Co-ADehydrogenaseEnzyme of Mitochondrial Fatty Acid Beta-Oxidation is not Associated with Non-Alcoholic SteatehepatiUs Raphael B. Merriman, Univ of CA, San Francisco, San Francisco, CA; Bruce R, Bacon, Elizabeth M. Brunt, Brent A. Tetri, St Louis Univ, St. Louis, MO; Marion G. Peters, Univ of CA, San Francisco, San Francisco, CA; Arnold W. Strauss, Vanderbilt Univ, Nashville, TN Introduction: Non-alcoholicsteatohepatitis(NASH)is characterizedby elevatedtransaminases, with steatosis and necroinftammatorychanges on liver biopsy in patients w~thout signilicant alcohol ingestion. The pathogenesisis not understood. Long-chain 3-hydroxyacyl coenzymeA dehydrogenase(LCHAD) catalyses the third step in the beta-oxidation of fatty acids in mitochondria.The most common LCHADmutation, G1528C,results in a changefrom glutamate to glutamine at amino-acid 474 in exon 15 (E4740). The heterozygote frequency of this mutation is 1 in 175 in the generalpopulation. We havepreviously shown that isolated LCHAD deficiency (homozygotemutation) is associatedwith acute fatty liver of pregnancyand HELLP syndrome. It is proposed that mutations in components of mitochondrial fatty acid oxidation or oxidative phosphorylation may predispose to the development of NASH in combination with other hepatic stressors. In a previous pilot study, we detected one G1528C mutation in a group of 12 patients with NASH (Gastroenterol 2000;118:2:A899) Aim: To determine the prevalence of this common LCHAD mutation (E474Q) in a new large group of patients (pts) with NASH. Methods: Fifty four pts with NASHwere evaluated,for whom liver biopsy material was availabe.DNAwas isolatedfrom formalin-fixed paraffin-embeddedliver biopsy specimens. PCR amplification of exon 15 of LCHADwas performed with [32p]deoxycytosine-triphosphate and previously described intronic primers and analysed for single-stranded conformational polymorphisms. Nucleotidesequenceswere then determinedby the dideoxy chain termination method using an automatedsequencerwith amplified exonicfragments. Results:The diagnosis of NASH was well establishedin all pts based upon biochemical data, exclusion of significant alcohol ingestion and compatible histological findings. Single stranded conformational polymorphism analysis indicated six pts with possible heterozygotemutations. Howeverdefinitive nucleotide sequence analysis did not indicate the presence of any common exon 15 LCHAD mutation. Conclusions: We could not demonstrate the presence of the common LCHAD mutation (E474Q) in a new large group of 54 with well-defined NASH. Though it appears unlikely that this particular mutation is relevant in NASH pts, the search should continue for other mitochondrial mutations that potentially predisposeto this disorder.
A-543
Background: Liver biopsy is commonly used for evaluation of patients with hepatitis C and these patients often have other histologic lesions that are apparenton histologic examination. In particular, steatohepatltiscan coexist with chronic HCV. The role of steatohepatitison the course of chronic hepatitis C is poorly documented.Aim:We evaluatedthe impact of nonalcoholic steatohepatitis on histologic activity grade and stage in hepatitis C. Methods: Patients with HCV who had ~ndergo~ liver biopsy were studied, h retrospective chart review was performedto collect information about patient demographics,laboratorydata,and liver biopsy. Patients with a history of alcohol consumption >lOg/day were excludedfrom the study. The histologic activity gradeand stagewas determinedaccordingto the modified KnodellHistologic Activity Index. Results:201 patients (120 men and 81 women) with a mean age of 48 years were includedin the study. Of these, 35 (17%) had histologic featuresdiagnostic of steatohepatitis. The liver biopsies with steatohepatitis had significantly higher histologic activity grade (2.3 vs. 2.1; p =0.05) and stage (2.7 vs. 2.3; p= 0.03) comparedto those that did not show changes of steatohepatitis.The group with steatohepatitis was more likely to have elevated aminotransferases. There was no significant difference in two groups with regards to HCV genotypeand viral load. Conclusions:1)NASHoften coexistswith chronic HCVand is associated with greater histologic activity and fibrosis. 2) Developmentof NASH does not seem to be influenced by viral factors and hence host factors may be important.
2763 Haemochromatosis Associated Mutations in Heavy Drinkers With Decompensated Liver Disease. Keith L E Dear, Royal Hallamshire Hosp, Sheffield United Kingdom; K Palmer, V Chambers, Ann Dalton, M S, Tanner, Div of Child Health, Univ of Sheffield, Sheffield United Kingdom; Martin P. Bradley, Oermot C. Gleeson, Royal Hallamshire Hosp, Sheffield United Kingdom Background:Only 10% of heavy drinkers develop alcoholic liver disease (ALD). Candidate genes to explain the variable predisposition remain to be defined. 90% of UK patients with genetic haemochromatosis(GH) are homozygousfor the C282Y mutation. Heterozygosityfor C282Y has been associated with severity of liver disease in hep C, NASH and porphyria; studies in ALD are conflicting. Methods: We studied Caucasian heavy drinkers (>6OU/wk (M)or 40U/wk (F) for ~5yrs) (patients n = 156; 108 M, age 48, SD lOyrs)with decompensated liver disease (Childs B or C). Serum was -re for hep B & C, AMA & ANA antibodies in all. Controls were unselectedmale blood donors (n =3574).Genotyl~ingfor C262Y and HB3Dwas performed using a Bi-Pasa PCR method. Results: 142 of 156 patients did not have iron overload (-ve iron)based on (a) normal ferritin (n =59); (b) normal iron saturation n=t22);(c)liver histology consistent with ALD with -< grade 2 iron staining (n=49). 14 patients had possible iron overload, (+re iron) with persistently elevated ferritin & iron saturation. Distribution of The GH mutations of patients & controls is shown below. Summary: In ALD the frequency of GH associated mutations is not significantly greater than in the general population and is unlikely to contribute to the genetic predisposition of ALD. However the risk of iron overload in C282Y homozygoteswho drink heavily remainsto be determined.
2761 Interleukin-16 Promoter Region PotymorphismsAnd Alcoholic Liver Disease Helen Newberry, Damitha Edirisinghe, Univ of Liverpool, Liverpool United Kingdom; Nell Fisher, Univ of Birmingham, Birmingham United Kingdom; lan T. Gilmore, ROYAL LIVERPOOLUniv Hosp, Liverpool United Kingdom; David Adams, Univ of Birmingham, Birmingham United Kingdom; Brian K. Park, Munir Pirmohamed, Univ of Liverpool, Liverpool United Kingdom
2759 First Clinical Application ot a Second Generation HepatAssist Bioadificial Liver Support System Without Charcoal Associated with Neurological Recovery in a Fulminant Hepatic Failure Patient Nikolaos Arkadopoulos, Univ of Athens S., Aretaieion, Athens Greece; Maria Alexandri, Aretaieion Hosp, Athens Greece; Nikitorita Poulakaki,Apostolos Papalois, Dimitris Papadimitriou, Andreas Prachalias, Aretaieion Hosp, Athens Greece; Chris Stevens, Circle Biomed, Lexington, MA; Lila Papadimitriou, Anesthisiology Unit, Arataieion Hosp, Athens Greece; John Papadimitriou
Background: Only 10-30% of individuals who misuse alcohol go on to develop alcoholic liver disease (ALD). There is a genetic component in the predisposition to ALD. Intarleukin-fO (IL10) is an anti-inflammatory,anti-proliferativeand anti-fibrotic cytokinethat may exert protective effects in ALD. The IL-lO gene has a polymorphic 5'-flanking region; it has recently been shown that the -627"A allele, which leads to low IL-lO expression, is associated with ALD (Groveet al, Gut, 2000, 46:540-5). We havetherefore investigatedwhether the IL-lO polymorphisms are associated with ALD in our cohort of patients. Methods: Patients with advanced ALD (n=290) who were Hepatitis C negative, diagnosed on either biopsy or unequivocal clinical evidence, were recruited from both Liverpool and Birmingham. The control groups comprised 269 healthy volunteers, and 59 alcohol misusers who did not have any clinical or biochemical evidenceof ALD. Subjects were genotypedfor the -627 (C,A) and -1117 (G,A) polymorphisms by PCR-RFLP analysis using Rsal and EcoNI, respectively, for restriction digestion. Results:The allele frequencies in the patients and controls are shown in the table. The -627"A allele frequency did not differ betweenthe patients with ALD and the two control groups (alcoholic patients with no liver diseaseand healthy volunteers) combined (odds ratio 0.89, 95% CI 0.68-1.19, p=O.4). Similarly, there was no difference in the -1117"A allele frequency between patients with ALD and the combined control group (odds ratio 0.99, 95% CI 0.79-1.24, p = 0.9). Conclusions: In contrast to the study by Grove at al (2000), we have not shown an association betweenthe -627 polymorphism in the IL-lO promoter region and ALD. The reason(s) for the discrepancy between the two studies are unknown, but may be the result of differences in linkage disequilibrium between the patient cohorts.
BACKGROUND:Clinical experiencewith liver support devices utilizing biological components is limited. The HepatAssitTM Bioartiticial Liver Support System containing sevenbillion primary porcine hepatocytes and charcoal is currently being tested in a multicenter randomized controlled phase II/111clinical trial. We present herein the first clinical application of a secondgeneration bioartificial liver (BAL) which incorporates 28 billion primary porcine hepatocytes but no charcoal. METHODS:The novel plasma-perfusedBAL consists of a reservoir, a pump, a membrane oxygenatorand two parallel bioreactors made of polysulfone hollow-fibers each containing 14 billion primary porcine hepatocytes.A 29 year-old male patient with fulminant hepaticfailure (FHF)of unknown etiology, acute renal failure and stage IV hepaticencephalopathy with brain edema, underwent 6-hour long BAL sessions on two consecutive days. Prior to BAL application,all standard intensivecare measureshad beeninitiated including continuous veno-venoushemofiltration as well as full hemodynamic,metabolicand biochemical monitoring. The patient's cerebral perfusion status was monitored with transcranial doppler, cerebral oxymetry and serum S-lOg protein measurements. RESULTS: Both BAL treatments were associated with neurologic improvement demonstrated by increase of the mean velocity and lowering of the pulsatility index of the middle cerebralartery, improvementof cerebraloxymetry values and decreaseof the S-lOO levels. Biochemicalchangesincluded reduction of transaminase and ammonia values. Improvement of coagulation was evident by the stabilization of INR below 3.0 without the needfor fresh frozen plasma administration. After the second BAL treatment the patient underwent successful orthotopic liver transplantation and experienced complete neurologic recovery.CONCLUSION:A modified HepatAssistTM Liver Support System containing 28 billion primary porcine hepatocyteswithout charcoal was safely used to treat an FHF patient and associatedwith physiological, biochemical and clinical measurementsof neurologic improvement during treatment.
IL.10 polymorphisms Subjects Alcoholicliverdisease Alcoholics with no liver disease Healthyconb'ols
Vadantallele ft'equencies -627 polymorphism -11t7 polymorphism 22% 22.9% 24.3%
50% 54.6% 49.4%
2762 2760
Nonalcoholic Steatohepatitis Is Associated With Increased Histological Activity And Fibrosis In Patients With Chronic Hepatitis C Sanjay Ramrakhiani,Alex S. Befeler, Bruce R. Bacon, Saint Louis Univ, St. Louis, MO
A Common Mutation of the Long-Chain HydroxyacyI-Co-ADehydrogenaseEnzyme of Mitochondrial Fatty Acid Beta-Oxidation is not Associated with Non-Alcoholic SteatehepatiUs Raphael B. Merriman, Univ of CA, San Francisco, San Francisco, CA; Bruce R, Bacon, Elizabeth M. Brunt, Brent A. Tetri, St Louis Univ, St. Louis, MO; Marion G. Peters, Univ of CA, San Francisco, San Francisco, CA; Arnold W. Strauss, Vanderbilt Univ, Nashville, TN Introduction: Non-alcoholicsteatohepatitis(NASH)is characterizedby elevatedtransaminases, with steatosis and necroinftammatorychanges on liver biopsy in patients w~thout signilicant alcohol ingestion. The pathogenesisis not understood. Long-chain 3-hydroxyacyl coenzymeA dehydrogenase(LCHAD) catalyses the third step in the beta-oxidation of fatty acids in mitochondria.The most common LCHADmutation, G1528C,results in a changefrom glutamate to glutamine at amino-acid 474 in exon 15 (E4740). The heterozygote frequency of this mutation is 1 in 175 in the generalpopulation. We havepreviously shown that isolated LCHAD deficiency (homozygotemutation) is associatedwith acute fatty liver of pregnancyand HELLP syndrome. It is proposed that mutations in components of mitochondrial fatty acid oxidation or oxidative phosphorylation may predispose to the development of NASH in combination with other hepatic stressors. In a previous pilot study, we detected one G1528C mutation in a group of 12 patients with NASH (Gastroenterol 2000;118:2:A899) Aim: To determine the prevalence of this common LCHAD mutation (E474Q) in a new large group of patients (pts) with NASH. Methods: Fifty four pts with NASHwere evaluated,for whom liver biopsy material was availabe.DNAwas isolatedfrom formalin-fixed paraffin-embeddedliver biopsy specimens. PCR amplification of exon 15 of LCHADwas performed with [32p]deoxycytosine-triphosphate and previously described intronic primers and analysed for single-stranded conformational polymorphisms. Nucleotidesequenceswere then determinedby the dideoxy chain termination method using an automatedsequencerwith amplified exonicfragments. Results:The diagnosis of NASH was well establishedin all pts based upon biochemical data, exclusion of significant alcohol ingestion and compatible histological findings. Single stranded conformational polymorphism analysis indicated six pts with possible heterozygotemutations. Howeverdefinitive nucleotide sequence analysis did not indicate the presence of any common exon 15 LCHAD mutation. Conclusions: We could not demonstrate the presence of the common LCHAD mutation (E474Q) in a new large group of 54 with well-defined NASH. Though it appears unlikely that this particular mutation is relevant in NASH pts, the search should continue for other mitochondrial mutations that potentially predisposeto this disorder.
A-543
Background: Liver biopsy is commonly used for evaluation of patients with hepatitis C and these patients often have other histologic lesions that are apparenton histologic examination. In particular, steatohepatltiscan coexist with chronic HCV. The role of steatohepatitison the course of chronic hepatitis C is poorly documented.Aim:We evaluatedthe impact of nonalcoholic steatohepatitis on histologic activity grade and stage in hepatitis C. Methods: Patients with HCV who had ~ndergo~ liver biopsy were studied, h retrospective chart review was performedto collect information about patient demographics,laboratorydata,and liver biopsy. Patients with a history of alcohol consumption >lOg/day were excludedfrom the study. The histologic activity gradeand stagewas determinedaccordingto the modified KnodellHistologic Activity Index. Results:201 patients (120 men and 81 women) with a mean age of 48 years were includedin the study. Of these, 35 (17%) had histologic featuresdiagnostic of steatohepatitis. The liver biopsies with steatohepatitis had significantly higher histologic activity grade (2.3 vs. 2.1; p =0.05) and stage (2.7 vs. 2.3; p= 0.03) comparedto those that did not show changes of steatohepatitis.The group with steatohepatitis was more likely to have elevated aminotransferases. There was no significant difference in two groups with regards to HCV genotypeand viral load. Conclusions:1)NASHoften coexistswith chronic HCVand is associated with greater histologic activity and fibrosis. 2) Developmentof NASH does not seem to be influenced by viral factors and hence host factors may be important.
2763 Haemochromatosis Associated Mutations in Heavy Drinkers With Decompensated Liver Disease. Keith L E Dear, Royal Hallamshire Hosp, Sheffield United Kingdom; K Palmer, V Chambers, Ann Dalton, M S, Tanner, Div of Child Health, Univ of Sheffield, Sheffield United Kingdom; Martin P. Bradley, Oermot C. Gleeson, Royal Hallamshire Hosp, Sheffield United Kingdom Background:Only 10% of heavy drinkers develop alcoholic liver disease (ALD). Candidate genes to explain the variable predisposition remain to be defined. 90% of UK patients with genetic haemochromatosis(GH) are homozygousfor the C282Y mutation. Heterozygosityfor C282Y has been associated with severity of liver disease in hep C, NASH and porphyria; studies in ALD are conflicting. Methods: We studied Caucasian heavy drinkers (>6OU/wk (M)or 40U/wk (F) for ~5yrs) (patients n = 156; 108 M, age 48, SD lOyrs)with decompensated liver disease (Childs B or C). Serum was -re for hep B & C, AMA & ANA antibodies in all. Controls were unselectedmale blood donors (n =3574).Genotyl~ingfor C262Y and HB3Dwas performed using a Bi-Pasa PCR method. Results: 142 of 156 patients did not have iron overload (-ve iron)based on (a) normal ferritin (n =59); (b) normal iron saturation n=t22);(c)liver histology consistent with ALD with -< grade 2 iron staining (n=49). 14 patients had possible iron overload, (+re iron) with persistently elevated ferritin & iron saturation. Distribution of The GH mutations of patients & controls is shown below. Summary: In ALD the frequency of GH associated mutations is not significantly greater than in the general population and is unlikely to contribute to the genetic predisposition of ALD. However the risk of iron overload in C282Y homozygoteswho drink heavily remainsto be determined.