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HEPATOLOGY Vol. 22, No. 4, Pt. 2, 1995 AASLD ABSTRACTS 487A 1521 A NEW APPROACH TO STUDY THE METABOLIC FATE OF APOLIPOPROTEIN A-I. HH-J Schmidt, C Strassburg, R Haas, J Genschel, and MP Manns. Abt. Gastroenterologie und Hepatologie, Medizinische Hochschule Harmover, Hannover, Germany. Analysis of patients with reduced plasma concentration of HDL has revealed that accelerated catabolism of apolipoprotein A-I (apoA-I) is the most cormnon cause of low HDL levels. The mechanism, however, for the increased clearance of apoA-I has not been delineated. Although the aminoterminal domain of the mature form of apoA-I can undergo limited proteolysis, the carboxytenninal domain has been shown to be particularly sensitive to proteolysis. In vitro stgdies suggest that proteolysis of apoA-I may possibly occur in vivo. Preliminary data of a 26 kDa proteolyzed fragment of apoA-I with an intact aminoterminal domain showed rapid in vivo catabolism. Generating various carboxyterminal truncation mutants we demonstrated that the carboxytemainal region (residue 227-243) of apoA-I is critical in modulating the association of apoA-I with lipoproteins and in vivo metabolism of apoA-I (Schmidt HH-J et al, JBC 1995, 270:5469-5475). To investigate the suggested carboxyterminal proteolysis we expressed ApoA-I which is modified by a carboxyterminal octapeptide (FLAG) in Baculovirus. The FLAG peptide consists of eight amino acids, is hydrophilic, and due to its antigenic property there is a specific antibody commercially available. 11.5% of total protein was the amount of expressed recombinant apoA-I/FLAG. DNA sequence analysis, the estimated molecular weight and the isoelectric point after protein purification were confirmed. Currently we analyze this modified apoA-I in vitro and in vivo to study the proteolysis of apoA-I. In conclusion, we have generated a modified apoA-I in the Baculovirus expression system, which should give us more insights about proteolysis and the metabolic fate of apoA-I. 1522 LIPID- AND LIPOPROTEINCHANGESIN MILD CHRONIC HEPATITIS B AND C. H. Seb.ncll-KretsehmCr, A. Kurz, G. Ambrosch*,C. Lulev*. H.U. Kl6___~r and the PBC-Studv-Gronv.*Clinical Cbemistry ! ~ , University of Magdeburg and 3rd Medical Department, Universityof Gielk~, Germany~ The liver plays an importantrole in liver and lipoproteinmetabolism. Severefiver disease is therefore associated with marked abnormalities of lipid metabolism. However, it is nnknown,weathermild m ~ r a t e liver disease such as chronic viral hepatitis showssimilarchanges. We studied 30 patients with established chronic viral hepatitis B or C with no ~ t abnormality of liver funotion except mild to moderate elevation of transaimonses (>2x normal) for abnormalities in lipids, lipoprotein fraction.s, - particles and atmlipoproteins. Lipids and lipoproteinswere analyzed using standard techniques and scqecntiel uitraccntrifugntion. Apolipoproteins and lipopreteinparticles were determined by electroimmanmssay. Plasma total cholesterol(rc) as well as VLDL..Cand LDL..C were normal while HDL-C was slightly reduced: Plasma triglyzerides (TG) were normal, while LDL-TG and HDL-TG was markedly elevated. The TC/TG-ratio was highly abnormal in both fractians. Plasma pbespholipids (PL) were slightly reduced with significant decrease in I-IDL-PL and a relative increase in LDL-PL. Plasma Apo AI was slightlylower than normal. The lipoprotein-parti¢le subfractionLP- All was normal, while LP-AI:AII was significantly reduced. Correspondingly, plasma APO All was also doercas~, Plasma APO B was moderately reduced, mainly in LDL. Plasma Apo CIII was in the low normal range. The Apo CIII- ~ of VLDL was extremiyhiw, while it was mlafivlyincreased in LDL and HDL. Plasma Apo E was normal with marked reduetioa in VLDL-Apo E and concomitant inereaso in LDL and, particularly, in HDL, where abnormal LP- B:E-Partieles could be demonstrated. We were able to demmstrme a variety of subtle lipid-, fipoprotein- and apolipoprotein-changes in patients with chreqnic hepalitis without clinical sympu~s of hepatic insufficiency. We therefore propose t o m e modem techniques of lipid- and lipopmtein-analysis for the evaluation of incipkat hepatic dysfunctionin chronic fiverdisease. 1523 IMMUNOLOGICALLY NEGATIVE HEPATITIS B VIRUS MUTANT IN A KIDNEY TRANSPLANT RECIPIENT. M. Schories H.-J. Schlayer, T.Peters, B. Hiller. H.E. Blum and J. Rasenack. Department of internal Medicine, Albert-Ludwigs-University, Freiburg, Germany Low-titred HBV infections without apparent immunological markers can be found in patients with acute or chronic hepatitis, in patients with cirrhosis, and with hepatoceUular carcinoma and in patients without liver disease. The aim of this study was the sequencing of the ttBV genomes from the serum of a patient with such an atypical infection. Prior to sequencing the viral genome was amplified. Two overlapping fragments were cloned and 22 different clones of each fragment sequenced. At least three different HBV variants of the subtype adw2, genotype A (Norder) were present. In comparison with 21 published I-IBV genomic sequences 97 point mutations were found. 78 of these exchanges were observed only in one or two clones, 12 point mutations in 3-7 clones and 4 point mutations were found in the majority of the clones. Three point mutations were found in aU clones sequenced in parallel. 55 point mutations were localized in the preS/S ORF, leading to 51 amino acid exchanges, 12 point mutations were in the X ORF, leading to 11 amino acid exchanges, 85 point mutations were found in the P-ORF, four of them lead to a stop eodon and 64 to an amino acid exchange. The preC-region was unaffected, the c-ORF had 14 pm. In addition to the point mutations a 4 bp deletion was observed in ten clones and a 1 bp deletion in another 7 clones. Both deletions lead to a truncated C-protein. Our results demonstrate a mixed infection with at least three different HBV variants. The mutations we observed could be responsible for the diminished rate of replication and the subsequent low-titred, immunologically negative I-IBV infection. 1524 SOLUBLE COLLAGENS MODULATE THE PROLIFERATION OF HEPATOCELLULAR CARCINOMA CELLS. D Sehannan. C Fisseau. J Atkinson. *P Schirmacher M Ruehl. EO Riecken. Dept. of Gastroenterology and Hepatology, Klinikum Benjamin Franklin, Free University of Berlin, and *Dept. of Pathology, Universityof Mainz, Germany. BACKGROUND: Most hepatocelinlar carcinomas (HCCs) harbor a colla- genous extraeellular matrix (ECM)which is primarily derived from stromal cells recruited into the growing tumor. Since molecules of the ECM are known to modulate cellular phenotype, we studied the influence of several collagens on the growth of human HEP-1 and rat Morris-AHCC cells. METHODS: Serum-starved hepatomacells cultured on polystyrenemicrotiter wells were exposed to soluble, native, human placental collagens type I, III, IV and VI (CI, CIIII, CIV, CVI) at 0.3-3 ttg/well (5-100 nM/1) for 20 h followed by measurementof cell proliferation (BrdU-incorporationover 4 h) and cell number. Inhibition studies were performed with the denatured collagen chains and blocking anti-ECM-integrin antibodies. RESULTS: All collagens enhanced cell proliferation in Morris-A but not HEP-I cells. However, cell number was only increased by soluble CVI in both cell types and by CIV in HEP-1 cells. Growth factor contamination was rigorously excluded and soluble collagens did not affect adhesion. The effect could be blocked by denatured CVI, but not by antibodies to ECM-intcgrins. CI CIII CIV CVI (Prol/C-No) (ProYC-No) (ProYC-No) (ProYC-No) HEP-1 80/88% 110/96% 90/139% 110/148% Morris A 219/93 % 212/93 % 2!0/102 % 592/271% Proliferation (Prol) and cell number (C-No) of 3 independent experiments in triplicate (SD <15%) are shown as ~ of controls (no addition of collagen). CONCLUSIONS: 1. Collagens, especially CVI, may serve as stimulators for HCC cell growth. 2. The efficacy of CVI in molar terms is comparableto that of the most potent growth factors. 3. The effect can not be blocked by a spectrum of antibodies to ECM-integrins. 4: Since the growth response to CVI can be inhibited by the denatured molecule, identification of the peptide sequences involved may lead to the design of novel anti-tumorous agents.

Lipid- and lipoproteinchanges in mild chronic hepatitis B and C . *Clinical Chemistry Department, University of Magdeburg and 3rd Medical Department, University of Gie?en, Germany

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Page 1: Lipid- and lipoproteinchanges in mild chronic hepatitis B and C . *Clinical Chemistry Department, University of Magdeburg and 3rd Medical Department, University of Gie?en, Germany

HEPATOLOGY Vol. 22, No. 4, Pt. 2, 1995 AASLD A B S T R A C T S 487A

1521 A NEW APPROACH TO STUDY THE METABOLIC FATE OF APOLIPOPROTEIN A-I. HH-J Schmidt, C Strassburg, R Haas, J Genschel, and MP Manns. Abt. Gastroenterologie und Hepatologie, Medizinische Hochschule Harmover, Hannover, Germany.

Analysis of patients with reduced plasma concentration of HDL has revealed that accelerated catabolism of apolipoprotein A-I (apoA-I) is the most cormnon cause of low HDL levels. The mechanism, however, for the increased clearance of apoA-I has not been delineated. Although the aminoterminal domain of the mature form of apoA-I can undergo limited proteolysis, the carboxytenninal domain has been shown to be particularly sensitive to proteolysis. In vitro s tgdies suggest that proteolysis of apoA-I may possibly occur in vivo. Preliminary data of a 26 kDa proteolyzed fragment of apoA-I with an intact aminoterminal domain showed rapid in vivo catabolism. Generating various carboxyterminal truncation mutants we demonstrated that the carboxytemainal region (residue 227-243 ) of apoA-I is critical in modulating the association of apoA-I with lipoproteins and in vivo metabolism of apoA-I (Schmidt HH-J et al, JBC 1995, 270:5469-5475). To investigate the suggested carboxyterminal proteolysis we expressed ApoA-I which is modified by a carboxyterminal octapeptide (FLAG) in Baculovirus. The FLAG peptide consists of eight amino acids, is hydrophilic, and due to its antigenic property there is a specific antibody commercially available. 11.5% of total protein was the amount of expressed recombinant apoA-I/FLAG. DNA sequence analysis, the estimated molecular weight and the isoelectric point after protein purification were confirmed. Currently we analyze this modified apoA-I in vitro and in vivo to study the proteolysis of apoA-I.

In conclusion, we have generated a modified apoA-I in the Baculovirus expression system, which should give us more insights about proteolysis and the metabolic fate of apoA-I.

1522 LIPID- AND LIPOPROTEINCHANGES IN MILD CHRONIC HEPATITIS B AND C. H. Seb.ncll-KretsehmCr, A. Kurz, G. Ambrosch*, C. Lulev*. H.U. Kl6___~r and the PBC-Studv-Gronv.*Clinical Cbemistry ! ~ , University of Magdeburg and 3rd Medical Department, University of Gielk~, Germany~

The liver plays an important role in liver and lipoprotein metabolism. Severe fiver disease is therefore associated with marked abnormalities of lipid metabolism. However, it is nnknown, weather mild m ~ r a t e liver disease such as chronic viral hepatitis shows similar changes. We studied 30 patients with established chronic viral hepatitis B or C with no ~ t abnormality of liver funotion except mild to moderate elevation of transaimonses (>2x normal) for abnormalities in lipids, lipoprotein fraction.s, - particles and atmlipoproteins. Lipids and lipoproteins were analyzed using standard techniques and scqecntiel uitraccntrifugntion. Apolipoproteins and lipopretein particles were determined by electroimmanmssay. Plasma total cholesterol (rc) as well as VLDL..C and LDL..C were normal while HDL-C was slightly reduced: Plasma triglyzerides (TG) were normal, while LDL-TG and HDL-TG was markedly elevated. The TC/TG-ratio was highly abnormal in both fractians. Plasma pbespholipids (PL) were slightly reduced with significant decrease in I-IDL-PL and a relative increase in LDL-PL. Plasma Apo AI was slightly lower than normal. The lipoprotein-parti¢le subfraction LP- All was normal, while LP-AI:AII was significantly reduced. Correspondingly, plasma APO All was also doercas~, Plasma APO B was moderately reduced, mainly in LDL. Plasma Apo CIII was in the low normal range. The Apo CIII- ~ of VLDL was extremiy hiw, while it was mlafivly increased in LDL and HDL. Plasma Apo E was normal with marked reduetioa in VLDL-Apo E and concomitant inereaso in LDL and, particularly, in HDL, where abnormal LP- B:E-Partieles could be demonstrated. We were able to demmstrme a variety of subtle lipid-, fipoprotein- and apolipoprotein-changes in patients with chreqnic hepalitis without clinical sympu~s of hepatic insufficiency. We therefore propose t o m e modem techniques of lipid- and lipopmtein-analysis for the evaluation of incipkat hepatic dysfunction in chronic fiver disease.

1523 IMMUNOLOGICALLY NEGATIVE HEPATITIS B VIRUS MUTANT IN A KIDNEY TRANSPLANT RECIPIENT. M. Schories H.-J. Schlayer, T.Peters, B. Hiller. H.E. Blum and J. Rasenack. Department of internal Medicine, Albert-Ludwigs-University, Freiburg, Germany

Low-titred HBV infections without apparent immunological markers can be found in patients with acute or chronic hepatitis, in patients with cirrhosis, and with hepatoceUular carcinoma and in patients without liver disease. The aim of this study was the sequencing of the ttBV genomes from the serum of a patient with such an atypical infection. Prior to sequencing the viral genome was amplified. Two overlapping fragments were cloned and 22 different clones of each fragment sequenced. At least three different HBV variants of the subtype adw2, genotype A (Norder) were present. In comparison with 21 published I-IBV genomic sequences 97 point mutations were found. 78 of these exchanges were observed only in one or two clones, 12 point mutations in 3-7 clones and 4 point mutations were found in the majority of the clones. Three point mutations were found in aU clones sequenced in parallel. 55 point mutations were localized in the preS/S ORF, leading to 51 amino acid exchanges, 12 point mutations were in the X ORF, leading to 11 amino acid exchanges, 85 point mutations were found in the P-ORF, four of them lead to a stop eodon and 64 to an amino acid exchange. The preC-region was unaffected, the c-ORF had 14 pm. In addition to the point mutations a 4 bp deletion was observed in ten clones and a 1 bp deletion in another 7 clones. Both deletions lead to a truncated C-protein. Our results demonstrate a mixed infection with at least three different HBV variants. The mutations we observed could be responsible for the diminished rate of replication and the subsequent low-titred, immunologically negative I-IBV infection.

1524 SOLUBLE COLLAGENS MODULATE THE PROLIFERATION OF HEPATOCELLULAR CARCINOMA CELLS. D Sehannan. C Fisseau. J Atkinson. *P Schirmacher M Ruehl. EO Riecken. Dept. of Gastroenterology and Hepatology, Klinikum Benjamin Franklin, Free University of Berlin, and *Dept. of Pathology, University of Mainz, Germany.

BACKGROUND: Most hepatocelinlar carcinomas (HCCs) harbor a colla- genous extraeellular matrix (ECM)which is primarily derived from stromal cells recruited into the growing tumor. Since molecules of the ECM are known to modulate cellular phenotype, we studied the influence of several collagens on the growth of human HEP-1 and rat Morris-A HCC cells. METHODS: Serum-starved hepatoma cells cultured on polystyrene microtiter wells were exposed to soluble, native, human placental collagens type I, III, IV and VI (CI, CIIII, CIV, CVI) at 0.3-3 ttg/well (5-100 nM/1) for 20 h followed by measurement of cell proliferation (BrdU-incorporation over 4 h) and cell number. Inhibition studies were performed with the denatured collagen chains and blocking anti-ECM-integrin antibodies. RESULTS: All collagens enhanced cell proliferation in Morris-A but not HEP-I cells. However, cell number was only increased by soluble CVI in both cell types and by CIV in HEP-1 cells. Growth factor contamination was rigorously excluded and soluble collagens did not affect adhesion. The effect could be blocked by denatured CVI, but not by antibodies to ECM-intcgrins.

CI CIII CIV CVI (Prol/C-No) (ProYC-No) (ProYC-No) (ProYC-No)

HEP-1 80/88% 110 /96% 90/139% 110/148% Morris A 219/93 % 212/93 % 2!0/102 % 592/271%

Proliferation (Prol) and cell number (C-No) of 3 independent experiments in triplicate (SD <15%) are shown as ~ of controls (no addition of collagen).

CONCLUSIONS: 1. Collagens, especially CVI, may serve as stimulators for HCC cell growth. 2. The efficacy of CVI in molar terms is comparable to that of the most potent growth factors. 3. The effect can not be blocked by a spectrum of antibodies to ECM-integrins. 4: Since the growth response to CVI can be inhibited by the denatured molecule, identification of the peptide sequences involved may lead to the design of novel anti-tumorous agents.