“Blastosphere”: A new culture method for human fetal hepatic progenitor cells

2754 Management of Fulminant Hepatic Failure in the USA: Results from a Survey of 14 Liver Transplant Programs J E. Ray, Mayo Clin, Rochester, MN; 6 McGuire, Univ of Alabama, Birmingham, AL; G Ostapowicz, W M. Lee, Univ of Texas Southwestern Medical Ctr, Dallas, 1)( Acute liver failure (ALF) is an uncommon condition of high mortality requiring urgent diagnosis and facilities for intensive care and liver transplantation (OLT). With few therapeutic trials in the USA, little is known about standard managementof ALF. Aims: To assess the therapeutic approach to ALF in USA and to identify acceptedand controversial aspects of care. Methods: A questionnaireto assessthe managementof ALF was completedby the physician representative of each of 14 liver transplant centers participating in the muiticenter Acute Liver Failure Study Group;these centers perform 30% of all liver transplants in USA. Results:The following care is instituted in most centers (# of centers): peptic ulcer prophylaxis, mainly H2 blockers (13); enteral nutrition if tolerated (13); hemostatic monitoring by PT/INR and platelet count (14); FFP (14) and platelets (13) prior to invasive procedure; use of N-acetylcysteine in acetaminophenoverdose (13); surveillancecultures of blood and urine (12); elevationof head of bed (11); liver biopsy only with clinical suspicion of unusual diagnosis (11). With stages Ill/IV encephalopathyand/or needfor mechanicalventilation, therapy includes hemodynamic monitoring by arterial (14), central venous (14) and pulmonaryartery catheters(13); intracranial pressure monitoring (ICP) with epidural or subdural catheters in OLT candidates (13); head scanning by CT (14); hyperventilationas first line therapy to treat Cerebraledema (11); mannitol therapy to treat cerebral edema (14); renal dialysis by CWHD (13); and use of King's College criteria for assessmentfor OLT (13). More controversial areas include use of medical agents for PSE (10), barbiturate coma to treat cerebral edema (10), prophylactic therapy of cerebral edema (6), vitamin K therapy (10), surveillance cultures, use and type of prophylactic antibiotics, blood products to prevent bleeding (4), and nutritional support with TPN (6) or BCAA (4). Conclusions: The patient with ALF requires specializedcare in a liver transplant center where aggressivemeasures - intensive hemodynamic, ICP monitoring, use of CWHD - are almost universal. Also universal are some less invasive measures (e.g. surveillance cultures, peptic ulcer prophylaxis) which can be instituted early in the referring center prior to transfer. Many areas of supportive care remain controversial and optimal therapy for ALF requires further investigation.

received standard med=caltreatment (SMT) for 3-5 days 24 patients who did not respond to SMT and did not meet exclusion criteria (lack of consent, hepatorenal syndrome, major bleedings etc.) where raodomised to either SMT alone (control group) or SMT plus daily albumin dialysis (treatment group) for 6 hours as long as serum bilirubin was higher than 15 mg/dl up to a maximum of 10 treatments. Impact on bilirubin, albumin, prothrombin time value, hepatic encephalopathyand ascites as major liver function parameterscumulating in the Child Turquotte Pugh (CTP) Index, mean arterial blood pressure (MAP) and renal function where monitored, After 30-days a survival analysis was done. Results: Out of 12 patients randomised to SMT alone only 6 patients survived whereas 11 out of 12 patients receiving SMT plus albumin dialysis where still alive after 30 days. There was a significant reduction of serum bilirubin and an improvement of serum albumin in the treatment group that was not seen in the control group. There was a significant progression of hepatic encephalopathy and Glasgow Coma Score in the control group that was not seen in the treatment group. With all this, there was a significant improvementof ChildTurquotte Pugh Index in the treatment group comparedto the control group. Interpretation: In chronic liver diseasecomplicated by cholestasis extracorporealliver assist devices providing excretory liver function can improve sundval by supporting the patients liver and enabling its partial recovery.

Albumin Olaltrsis as a New Tool for Extracorporaal Liver Support-Clinical Data from 19 Centers Reporting 103 Patients and Two Prospective Randomised Controlled Tda|s. Jan Stange, UCSD Medical Ctr, San Diego, CA; Steffen R. Mitzner, Univ of Rostock, Rostock Germany;,Tsrek I. Hassanein,Ravindra Mehta, UCSD Medical Ctr, San Diego, CA; Robert Bartlett, Univ of Michigan, Ann Arbor, MI Background: Extracorporeal liver support aims to prolong survival time in liver failure by providing liver function. Hemodlalysisusing albumin as a Molecular Adsorbent in the dialysis solution can be used to track even tightly albumin bound toxins from blood into the dialysate if the albumin's binding sites are on line purified by a sorbentJdialysisbasedRecyclingSystem (MARS). The present work summarizesdata reporting clinical effects of this new technology that provides excretory liver function to support a tailing liver. Method: 19 centers reporting 103 patients and two prospective randomised controlled trials including 37 patients were recorded.Clinical results were analyzedfor chronic liver disease(CLD) complicatedby hepatorenal syndrome (HRS) and cholostesis (CS) as well as for CLD complicated by CS alone. Furthermore, results for acute liver failure (ALF) and primary liver graft dysfunction (PLGD) were analysed,Results:All canters reportedthat the MARS-technologywas capableto remove water soluble andalbumin bound toxins and to provide renal support in case of renal failure. 14 centers have reported that MARS-treatment improved mental status in liver failure if hepatic encephalopathywas present. In liver failure and cholestasis, MARS was associated by hemodynamicstabilization,improvementof hepatic and kidneyfunction and disappearance of pruritus. The use of MARSin another26 patientssuffering from ALF or PLGDwas associated with spontaneousrecoveryin 10/26 and bridge to re- or transplantationin 13/26. A prospective randomizedcontrolledtrial studyingthe effect of MARSin CLDcomplicatedby CS(bilirubin >20 rag/all) and HRS including 13 patients has shown that MARS-treatmentwas able to prolong survival time significantly. Another prospectiverandomizedcontrolled trial studying the effect of MARS in CLD complicated by CS (bilirubin>20 mg/dl) alone including 24 patients has shown that MARS-treatment was associated with improvement of hepatic encephalopatby, Child Turcotte Pugh Index, Glasgow Coma Score and Mean Arterial Pressure, ending into a significant improvementof survival. Conclusion: 19 centers reporting 103 patientshaveshown, that MARS-treatment is safe, easy to handle, feasible and effective. For chronic liver disease complicated by cholestasis with or without hepatorenal syndrome there is some evidence, that MARS can prolong survival time.

2755 The Use of the King's College Hospital Criteria (KCHC) on Admission to Predict Mortality or the Need for Orthotopic Liver Transplantation (OLT) in Patients Referred to Emory University Hospital (EUH) for Fulminant Hepatic Failure (FHF). Peter C. Fenton, Christine J. Bruno, Paolo Ricci, Emory Univ, Atlanta, GA BACKGROUND: FHF as defined by O'Grady is hepatic encephalopathywithin 12 weeks of jaundice.The aims of this retrospectivestudy was to determine: (1) If the KCHCfor acetaminophen (acetamin.) and non-acetamincphen (ncn-acetamin.) causes (~,2) on admission have similar prognostic value for mortality or need for OLT in our patients Comparedto the prior Pittsburgh study by Shakil et a13.;(2) If KCHCcan predict outcome in patients with FHF due to viral hepatitis (VH). METHODS:KCHCon admission were reviewedin 76 patients with FHF admitted to EUI-Ibetween 9/30/1990 and 9/30/2000. Chi-Squaredtest was used for statistical analysis. RESULTS:Definitions used in the table below are: ~- Criteria present for:. (a) 3 of 5 variables for non-acetamin. (age40; cause - non-A non-B hepatitis, halothane hepatitis or idiosyncratic clrug reaction; encephalopathy>7days after jaundice; total bilirubin>17.5 or INR>3.6) (b) Acetamin. - INR>6.6, creatinine>3.4 and grade Ill-IV encephalnpathy. 2_Criteria presentfor INR>6.5 for non-acetamin,and pH<7.3 for acetamin. More patients with VH and KCHC present were alive without OLT compared to patients with non-Viral hepatitis (P-O.OO2)CONCLUSION:KCHCwhen used on admission provides similar prognostic information in our population comparedto the Pittsburgh study3. The criteria had its greatest specificity and PPV in patients with acefamin, overdose. In patients with VH the KCHCwere far less useful. The KCHC should be used with caution in predicting mortality or need for OLT in Ft-IFdue to VH. Prospectivestudies are neededto identify better criteria for mortality or need for OLT in patients with FHF caused by VH. Patientswith FHF

"Blastasphere": A New CuHura Method for Human Fetal Hepatic Progenitor Cells. Tetsuro Hirose, Inst Frontier Medical Science, Kyoto Univ, Kyoto Japan; Kentaro Yasuchika, Takahisa Fujikawa, Hideaki Fujii, Shoshiro Oe, HisayaAzuma, Iwao Ikai, Yoshio Yamaoka, Dept Gastroenterol Surg Grad Sch Med Kyoto Univ, Kyoto Japan

King's College Hospital Criteda (KCHC)

EUH patients Sensitivity Viral Hepatitis4 23/76 69% Acetamin.4 19/76 54% Non. 711-64% acetamin?

Specificity 28% 100% 71~-932%

Acetamin?

1001-502% 1001-752% 55~-62:~o

571-672%

PPV 69% 100% 961-992%

Background: Donor-shortage is one of main problems in transplantation today. Growing evidences have indicated that hepatocytetransplantation is efficient in some cases of liver failure. However, in order to introduce this strategy into practical medicine, development of cell source other than donatedorgan is necessary.Consideringinfection from different species, human-derived cells are desirable. For these reasons, we focus on utilization of human fetal hepatic progenitor cells (HPC) as a substitute for hepatocytes.To enable expansion of HPC according to demand, in vitro culture system which do not induce terminal differentiation and maintain growth potential of HPC is indispensable.This time we developeda new isolating and culture method for human RPC.Materials and Methods: Humanfetal liver was dissociated by enzymedigestion. HPC were characterizedby immunodetectionof alpha-fetoprotein(AFP), cytokeratin 19 (CK19), and BrdU. E-cadherin was detected either by Westemblotting or by immunocytochemistry, and its binding was blocked using neutralizing antibody HECD-1. Nucleus of HPC was stained by Hoechst33342. Results: Fetal liver cells lost E-cadherin expression during isolation. When cultured in suspension according to our method, they formed spheroids, concomitantly with E-cadberinre-expression.This spheroid formation was blocked by HECD-I. This E-cadherin dependent spheroids consisted from AFP and CK19 positive HPC, although a majority of cells migrating and surrounding spheroids expressed CK19 but not AFP. Hencethese spheroids was denominatedas "blastosphere". Blastospheres could he subcloned. HPC reproducibly proliferated and migrated from them. 8rdU uptakewas detected inside the blastospheres early after subcloning. In contrast, after migrating cells surrounded blastospheres,BrdUwas uptakenmainly by surrounding cells, nonethelessnucleus inside blastosphere remained intact. Using this blastosphere culturing system, human HPC was cultured for 4 months without inducing terminal differentiation and maintaining HPC character. Conclusion: We developeda new isolation and culture method for HPC depending on E-cadherinexpressionand denominatedas "blastosphere". This enabledlong-term culture

NPV 28% 61% 211-22~/o

2756 A New Extracorporeal Liver Support System for Chronic Liver Disease Complicated by Cholestasis and Liver Failure-Results of a Prospective Controlled Randomised Trial. Jan Stange, Steffen R. Mitzner, Sebastian Klammt, Reinhardt Schmkit, Univ of Rostock, Rostock Germany; UIIrich Treichel, Jan Loock, Guido Gerken, Uwe Heemann, Univ of Essen, ~ssen Germany Background: Cholestasis complicating end stage liver disease may result in life threatening liver failure. Extracorporealblood treatment providing assistance in excretory liver function by removing toxic molecules offers the chance to support the liver until the patient recovers or a transplant becomesavailable.The presentpaperdescribesthe clinical effects of hemodialysis using a specific membraneand albumin as a Molecular Adsorbent in the dialysis solution that is on-line purified in a Recycling System (MARS) as a new method to remove albumin bound toxins which are generally considered to be important in liver failure. Methods: 45 Patients having chronic liver diseaseand serum bilirubin>2O mo/dl representingcholestasis

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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.

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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.