title: Rhesus monkeys and baboons develop clotting factor VIII inhibitors in response to porcine endothelial cells or islets.
abstract: BACKGROUND
  Xenotransplantation of porcine organs holds promise of solving the human organ donor shortage. The use of α-1,3-galactosyltransferase knockout (GTKO) pig donors mitigates hyperacute rejection, while delayed rejection is currently precipitated by potent immune and hemostatic complications. Previous analysis by our laboratory suggests that clotting factor VIII (FVIII) inhibitors might be elicited by the structurally restricted xenoantibody response which occurs after transplantation of either pig GTKO/hCD55/hCD59/hHT transgenic neonatal islet cell clusters or GTKO endothelial cells.
  
  
  METHODS
  A recombinant xenoantibody was generated using sequences from baboons demonstrating an active xenoantibody response at day 28 after GTKO/hCD55/hCD59/hHT transgenic pig neonatal islet cell cluster transplantation. Rhesus monkeys were immunized with GTKO pig endothelial cells to stimulate an anti-non-Gal xenoantibody response. Serum was collected at days 0 and 7 after immunization. A two-stage chromogenic assay was used to measure FVIII cofactor activity and identify antibodies which inhibit FVIII function. Molecular modeling and molecular dynamics simulations were used to predict antibody structure and the residues which contribute to antibody-FVIII interactions. Competition ELISA was used to verify predictions at the domain structural level.
  
  
  RESULTS
  Antibodies that inhibit recombinant human FVIII function are elicited after non-human primates are transplanted with either GTKO pig neonatal islet cell clusters or endothelial cells. There is an apparent increase in inhibitor titer by 15 Bethesda units (Bu) after transplant, where an increase greater than 5 Bu can indicate pathology in humans. Furthermore, competition ELISA verifies the computer modeled prediction that the recombinant xenoantibody, H66K12, binds the C1 domain of FVIII.
  
  
  CONCLUSIONS
  The development of FVIII inhibitors is a novel illustration of the potential impact the humoral immune response can have on coagulative dysfunction in xenotransplantation. However, the contribution of these antibodies to rejection pathology requires further evaluation because "normal" coagulation parameters after successful xenotransplantation are not fully understood.
authors.affiliation: Division of Human Anatomy, School of Medicine, Loma Linda University, Loma Linda, CA, USA.
authors.forename: John M
authors.initials: JM
authors.lastname: Stewart
authors.forename: Alice F
authors.initials: AF
authors.lastname: Tarantal
authors.forename: Wayne J
authors.initials: WJ
authors.lastname: Hawthorne
authors.forename: Evelyn J
authors.initials: EJ
authors.lastname: Salvaris
authors.forename: Philip J
authors.initials: PJ
authors.lastname: O'Connell
authors.forename: Mark B
authors.initials: MB
authors.lastname: Nottle
authors.forename: Anthony J F
authors.initials: AJ
authors.lastname: d'Apice
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Cowan
authors.forename: Mary
authors.initials: M
authors.lastname: Kearns-Jonker
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D000910:Antibodies, Heterophile / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D003198:Computer Simulation; D042783:Endothelial Cells / Q000276:immunology / Q000637:transplantation; D005169:Factor VIII / Q000037:antagonists & inhibitors* / Q000737:chemistry; D005700:Galactosyltransferases / Q000235:genetics / Q000276:immunology; D055786:Gene Knockout Techniques; D006801:Humans; D016381:Islets of Langerhans Transplantation / Q000009:adverse effects* / Q000276:immunology; D008253:Macaca mulatta / Q000276:immunology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010215:Papio / Q000276:immunology*; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D034421:Sus scrofa; D014183:Transplantation, Heterologous / Q000009:adverse effects*
chemical_list: D000910:Antibodies, Heterophile; D011994:Recombinant Proteins; D005169:Factor VIII; D005700:Galactosyltransferases; C530267:alpha-1,3-galactosyltransferase 1, porcine
keywords: clotting factor VIII; clotting factor VIII inhibitor; non-human primate; porcine; xenoantibody