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Expression of soluble and membrane-bound complement regulatory proteins by human hepatocytes
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171- EXPRESSION OF SOLUBLE AND MEMBRANEBOUND COMPLEMENT REGULATORY PROTEINS BY HUMAN HEPATOCYTES. Heiko Vogel, Jarkko Halme and
Michael Kirschtink, Institute of Immunology, University of Heidelberg, Germany Liver cell protein expression is traditionally investigated in hepatoma-derived cell lines, such as HepG2, which often have been claimed not to represent the physiologic behaviour of the nonmalignant hepatocyte. The current study was performed to analyze the potential regulatory effect of IL-6, ‘INF-a and IFN-y on the expression of complement regulatory proteins on isolated human primary hepatocytes. Synthesis of the soluble complement regulators was assessed by specific ELISA for the components C I inhibitor, factor 1, factor H and sCDS9. Expression of membrane regulatoy proteins CD59, CD46 and CD55 was tested by cytofluorometric analysis. IL-6 as well as IFN-y, but not TNF-a , dose-dependently stimulated the synthesis of Clinhibitor. Synthesis of factor I increased upon incubation with IL-6. TNF-a as well as IL-6 augmented sCD59 secretion. Factor H was never detected in supematents of isolated human hepatocytes, neither under basal conditions nor upon cytokine treatment. Freshly isolated non-stimulated human hepatocytes strongly expressed CD46 whereas only very low numbers of CD59 and no CD55 were detected. Expression of CD46, and more pronounced of CD59, was increased upon cytokine treatment. From these data we conclude that protection of hepatocytes by soluble and membrane complement regulators is modulated by proinflammatory cytokines.
17% TPIO (sCR1) INCREASES PROTECTION AGAINSTCOMPLEMENTMEDIATED DAMAGE FOLLOWING XENO-TRANSPLANTATION OF hDAF TRANSGENIC PIG KIDNEYS INTO CYNOMOLGUS MONKEYS. Richard Harrison, Conrad Mat, Bob Soin, Saqib Masmor, Gilda Chavez, John Bradley, Ken Smith, Sathia Thiru, David white, P&r Friend 8 Emanuele Cozzi. lmutran Ltd (A Novartts Pharma AG Company), PO Box 399, Cambridge CBZ 2YP. UK. Introduction: Organs frcm pigs transgenic for human DAF, transplanted into nonhuman primates, are protected against natural antibody mediated hyparacute rejectton (HAR). However, reperfusion exposes the xenograft to a complement insult far greater than that experienced by an allcgraft, and this study was performed to detemtine whether TPIO (sCR1) would give added protectton. !&!jg&: hDAF transgenic pig kidneys were transplanted after 6h and 12h cold ischemia (Cl) into cynomolgus monkeys. In addition to other immunosuppressive agents, hatf the animals received TPlO on days I, 0 and 1. C3a, sC5bQ and TPlO levels were monitored in recipients, and biopsies, taken 20 min after revasculartsation of the graft, were subjected to routtne andysts. Graft perfomrance was monitored using standard clinical parameters. &&g: While plasma C3a was elevated in all animals immediately post-reperfusion, the rise was significantly reduced in TPlMraated animals (6h Cl; TPlO= 2WSng/ml, controts= 496&5ngM, p=O.o02: 12h Cl; TPltI= 1&27ngM, controls= 46(k183ng/ml, p=O.O4). Plasma sC5b-9, present in contrd snimds (6h Cl; lOQ+Qnglml:12h Cl; QQ&n@ml) was absent in dl TPlO-treated animals. Histcdtemical examination of post-mperfuston biopsies showed lg, fibrin, C4, C3 and C9 depositton on capillary endothelid a& of control animds, but only lg and C4 deposition in TPllMeated animals. Discrete areas of hemorrhage, most evident in the inter&ha1 amas of the cortex, were seen in control but not TPlQtreaterJ animals. Initial ptssma crestinine was significantly lower in TPlC-treatsd vs. untreated animals. Mean grsfl survival in TPIC-treated animalswas 37.L12.2d vs. 24.L7.Qd (p=O.O5)in controls. Condusion: While the hDAF trsnsgsne prevents HAR, pstioparstive TPIO gives added pmtedion against complementinducad damage,and wntrtbutes to improvedgrsft performanceand survival.
173- SOLUBLE COMPLEMENT RECEPTOR (sCR1) 1 RESCUES RATS FROM LETHAL SHOCK INDUCED BY PRIMINGOFSUBLETHALDOSEOF LIPOPOLYSACCHARIDE (LPS) AND ANTI-CRRY ANTIBODIES. Masashi Mizuno@, Kazuhiro Nishikawaa, Noriko Okada*, and Hidechika Okada*, and Seiichi Matsuo@ Q3rd Dept of Int Med, Nagoya UnivSchl of Med, Nagoya, JAPAN; *Dept. of Molecular Biology, Nagoya City Univ Schl of Med, Nagoya, JAPAN. Mortalthy of endotoxin shock is still high in spite of recent improvements of supportive therapies, and no conclusive therapy is available in ctinicat situation. To develop new therapeutic strategies, appropriate animal models will be useful. All the rats pretreated with sublethal amount of LPS (0.01 mg/kg)developed severe decreaseof blood pressure and died after functional suppression of amembrane complement (C) regulator, rat Crry. Severity of decrease of blood pressure was not directly correlated with serum concentration of LPS or TNF-a. The lethal reaction was inhibited by depletion of leukocytes. Using this model, we investigated the possibility of the therapy of sCR1 in patients with endotoxin shock. SCRl (AVANT lmmunotherapeutics & Yamanouchi Pharm. Co.) prevented lethal shock when injected 30 min before the challenge of LPS-primed rats. Moreover, sCR1 prevented the lethal reaction even when administered 1 min after injection of mAb 512, although a transient decrease in blood pressure was observed before recovery. sCR1, regulator of C activation, protected LPSprimed ratsfromlethal shock, even if the lethal reaction had already been initiated by mAb 512. Regulation of C activation by sCR1 may be auseful strategy for treatment of lethal shock involving significant Cactivation.
174- DEVELOPMENT OF A MEMBRANE-TARGETED :OMPLEMENT INHIBITOR FOR CLINICAL USE I.Dodd, ?.G.Oldroyd, SPowell, L.J.Affleck, L.Bamber, S.Gattagher, ).J.E.Rowling, C.Ragnauth, G.P.Smith, J.R.Pratt’, Q.H.Sacks’, S.M.Linton*, B.P.Morgan’and R.A.G. Smith IdProTech plc. Unit 3.2 Orchard Rd, Royston, Herts SG5 jHD. UK ;‘Dept. Nephrology and Transplantation, UMDS, &y’s Hospital, London SE19 9RT UK; Dept. Med. 3iochem., IWCM, Heath Park, Cardiff CF4 4XN, UK We have developed a potent inhibitor of complement activation that differs from existing agents by its ability to be ncorporated exogenously into cell membranes. APT070 is jerived from the first three short consensus repeats (SCRI-3) >f CRl(CD35) modified at the C-terminus with a myriStOytslectrostatic switch peptide. The latter conferred a large ncrease in potency in vitro in an anti-hemolytic assay. Using FACS, optical biosensot and other techniques, we found that 4PT070 bound to a variety of cell types in a dose-dependent nanner. In viva, APT070 was active in a rat vascular shock model of acute basement membrane disease. Biodistribution studies ndicsted that such activity is probably due to localization in the microvasculature. Specific cell binding was also found upon local administration of the drug to perfused rat kidneys sx-viva and within rat knee joints upon intra-articular administration. Localization at these sites has enabled evaluation of APT070 in models of renal transplantation and arthritis. Further aspects of preclinicel development of APT070 will be presented including its large-scale production.
171- EXPRESSION OF SOLUBLE AND MEMBRANEBOUND COMPLEMENT REGULATORY PROTEINS BY HUMAN HEPATOCYTES. Heiko Vogel, Jarkko Halme and
Michael Kirschtink, Institute of Immunology, University of Heidelberg, Germany Liver cell protein expression is traditionally investigated in hepatoma-derived cell lines, such as HepG2, which often have been claimed not to represent the physiologic behaviour of the nonmalignant hepatocyte. The current study was performed to analyze the potential regulatory effect of IL-6, ‘INF-a and IFN-y on the expression of complement regulatory proteins on isolated human primary hepatocytes. Synthesis of the soluble complement regulators was assessed by specific ELISA for the components C I inhibitor, factor 1, factor H and sCDS9. Expression of membrane regulatoy proteins CD59, CD46 and CD55 was tested by cytofluorometric analysis. IL-6 as well as IFN-y, but not TNF-a , dose-dependently stimulated the synthesis of Clinhibitor. Synthesis of factor I increased upon incubation with IL-6. TNF-a as well as IL-6 augmented sCD59 secretion. Factor H was never detected in supematents of isolated human hepatocytes, neither under basal conditions nor upon cytokine treatment. Freshly isolated non-stimulated human hepatocytes strongly expressed CD46 whereas only very low numbers of CD59 and no CD55 were detected. Expression of CD46, and more pronounced of CD59, was increased upon cytokine treatment. From these data we conclude that protection of hepatocytes by soluble and membrane complement regulators is modulated by proinflammatory cytokines.
17% TPIO (sCR1) INCREASES PROTECTION AGAINSTCOMPLEMENTMEDIATED DAMAGE FOLLOWING XENO-TRANSPLANTATION OF hDAF TRANSGENIC PIG KIDNEYS INTO CYNOMOLGUS MONKEYS. Richard Harrison, Conrad Mat, Bob Soin, Saqib Masmor, Gilda Chavez, John Bradley, Ken Smith, Sathia Thiru, David white, P&r Friend 8 Emanuele Cozzi. lmutran Ltd (A Novartts Pharma AG Company), PO Box 399, Cambridge CBZ 2YP. UK. Introduction: Organs frcm pigs transgenic for human DAF, transplanted into nonhuman primates, are protected against natural antibody mediated hyparacute rejectton (HAR). However, reperfusion exposes the xenograft to a complement insult far greater than that experienced by an allcgraft, and this study was performed to detemtine whether TPIO (sCR1) would give added protectton. !&!jg&: hDAF transgenic pig kidneys were transplanted after 6h and 12h cold ischemia (Cl) into cynomolgus monkeys. In addition to other immunosuppressive agents, hatf the animals received TPlO on days I, 0 and 1. C3a, sC5bQ and TPlO levels were monitored in recipients, and biopsies, taken 20 min after revasculartsation of the graft, were subjected to routtne andysts. Graft perfomrance was monitored using standard clinical parameters. &&g: While plasma C3a was elevated in all animals immediately post-reperfusion, the rise was significantly reduced in TPlMraated animals (6h Cl; TPlO= 2WSng/ml, controts= 496&5ngM, p=O.o02: 12h Cl; TPltI= 1&27ngM, controls= 46(k183ng/ml, p=O.O4). Plasma sC5b-9, present in contrd snimds (6h Cl; lOQ+Qnglml:12h Cl; QQ&n@ml) was absent in dl TPlO-treated animals. Histcdtemical examination of post-mperfuston biopsies showed lg, fibrin, C4, C3 and C9 depositton on capillary endothelid a& of control animds, but only lg and C4 deposition in TPllMeated animals. Discrete areas of hemorrhage, most evident in the inter&ha1 amas of the cortex, were seen in control but not TPlQtreaterJ animals. Initial ptssma crestinine was significantly lower in TPlC-treatsd vs. untreated animals. Mean grsfl survival in TPIC-treated animalswas 37.L12.2d vs. 24.L7.Qd (p=O.O5)in controls. Condusion: While the hDAF trsnsgsne prevents HAR, pstioparstive TPIO gives added pmtedion against complementinducad damage,and wntrtbutes to improvedgrsft performanceand survival.
173- SOLUBLE COMPLEMENT RECEPTOR (sCR1) 1 RESCUES RATS FROM LETHAL SHOCK INDUCED BY PRIMINGOFSUBLETHALDOSEOF LIPOPOLYSACCHARIDE (LPS) AND ANTI-CRRY ANTIBODIES. Masashi Mizuno@, Kazuhiro Nishikawaa, Noriko Okada*, and Hidechika Okada*, and Seiichi Matsuo@ Q3rd Dept of Int Med, Nagoya UnivSchl of Med, Nagoya, JAPAN; *Dept. of Molecular Biology, Nagoya City Univ Schl of Med, Nagoya, JAPAN. Mortalthy of endotoxin shock is still high in spite of recent improvements of supportive therapies, and no conclusive therapy is available in ctinicat situation. To develop new therapeutic strategies, appropriate animal models will be useful. All the rats pretreated with sublethal amount of LPS (0.01 mg/kg)developed severe decreaseof blood pressure and died after functional suppression of amembrane complement (C) regulator, rat Crry. Severity of decrease of blood pressure was not directly correlated with serum concentration of LPS or TNF-a. The lethal reaction was inhibited by depletion of leukocytes. Using this model, we investigated the possibility of the therapy of sCR1 in patients with endotoxin shock. SCRl (AVANT lmmunotherapeutics & Yamanouchi Pharm. Co.) prevented lethal shock when injected 30 min before the challenge of LPS-primed rats. Moreover, sCR1 prevented the lethal reaction even when administered 1 min after injection of mAb 512, although a transient decrease in blood pressure was observed before recovery. sCR1, regulator of C activation, protected LPSprimed ratsfromlethal shock, even if the lethal reaction had already been initiated by mAb 512. Regulation of C activation by sCR1 may be auseful strategy for treatment of lethal shock involving significant Cactivation.
174- DEVELOPMENT OF A MEMBRANE-TARGETED :OMPLEMENT INHIBITOR FOR CLINICAL USE I.Dodd, ?.G.Oldroyd, SPowell, L.J.Affleck, L.Bamber, S.Gattagher, ).J.E.Rowling, C.Ragnauth, G.P.Smith, J.R.Pratt’, Q.H.Sacks’, S.M.Linton*, B.P.Morgan’and R.A.G. Smith IdProTech plc. Unit 3.2 Orchard Rd, Royston, Herts SG5 jHD. UK ;‘Dept. Nephrology and Transplantation, UMDS, &y’s Hospital, London SE19 9RT UK; Dept. Med. 3iochem., IWCM, Heath Park, Cardiff CF4 4XN, UK We have developed a potent inhibitor of complement activation that differs from existing agents by its ability to be ncorporated exogenously into cell membranes. APT070 is jerived from the first three short consensus repeats (SCRI-3) >f CRl(CD35) modified at the C-terminus with a myriStOytslectrostatic switch peptide. The latter conferred a large ncrease in potency in vitro in an anti-hemolytic assay. Using FACS, optical biosensot and other techniques, we found that 4PT070 bound to a variety of cell types in a dose-dependent nanner. In viva, APT070 was active in a rat vascular shock model of acute basement membrane disease. Biodistribution studies ndicsted that such activity is probably due to localization in the microvasculature. Specific cell binding was also found upon local administration of the drug to perfused rat kidneys sx-viva and within rat knee joints upon intra-articular administration. Localization at these sites has enabled evaluation of APT070 in models of renal transplantation and arthritis. Further aspects of preclinicel development of APT070 will be presented including its large-scale production.