Furthermore, in 2 baboons (B3907, “type”:”entrez-nucleotide”,”attrs”:”text”:”B18308″,”term_id”:”2316212″,”term_text”:”B18308″B18308) complement activity was eliminated by the administration of CVF (evidenced by the CH50 test, data not shown), and in 4 baboons the grafts were protected from complement activity by the over-expression of a human complement-regulatory protein (CD46), yet CC still developed. the onset of CC, often in the relative absence of features of acute humoral xenograft rejection. Prevention of recipient platelet activation may be crucial for successful pig-to-primate kidney Tx. study showed that, after contact with porcine aortic endothelial cells (PAECs), human platelets and monocytes are activated to express TF, and this is associated with increased TF activity (23). We hypothesize that, following pig organ Tx into primates, recipient platelets and leukocytes are activated to express functional TF and may be a causative factor in the development Rabbit Polyclonal to RAD17 of CC. We have explored this hypothesis in the present study. MATERIALS AND METHODS Animals Baboons (species, 6C20 kg, of known ABO blood type [n=9], Division of Animal Resources, Oklahoma University Health Sciences Center, Oklahoma City, OK) were recipients of pig kidney grafts. Wild-type (WT; n=2), 1,3-galactosyltransferase gene-knockout (GT-KO, n=1)(24), or GT-KO pigs transgenic for the complement-regulatory protein, CD46 (GT-KO/CD46, n=6) (all from Revivicor, Inc., Blacksburg, VA) were sources of kidneys; all were of blood group O (non-A). GT-KO was confirmed by negative expression of biotinylated GSIB4 lectin in major organs (24). High CD46 expression was confirmed on PAECs and peripheral blood mononuclear cells (PBMCs) by flow cytometry(25). All animal care was in accordance with the Principles of Laboratory Animal Care (NIH publication No. 86-23, revised 1985). Protocols were approved by the University of Pittsburgh Institutional Animal Care and Use Committee. Surgical procedures, supportive therapy, and monitoring Anesthesia, kidney harvesting in pigs, and renal Tx in baboons have been described previously (26-27). Both native baboon kidneys were excised. All recipient baboons received prophylactic intravenous (i.v.) antibiotics for the first 3 post-Tx days, and famotidine and ganciclovir throughout the post-operative period. Conventional heparin was begun on day 0 (day of Tx) and gradually increased in an effort to maintain the activated partial thromboplastin time (APTT) at approximately 150sec from day 3, as described previously(26), Mesaconine but was discontinued when the recipient developed features of CC, particularly if spontaneous bleeding occurred. Human albumin was administered i.v. from day 0 to prevent hypoalbuminemia that resulted from persistent proteinuria. Washed baboon red blood cells were administered when necessary to maintain the hematocrit 20%. Blood cell counts, chemistry, and coagulation parameters (prothrombin time, APTT, fibrinogen, D-dimer) were measured in the Central Laboratory of the University of Pittsburgh Medical Center. Kidney graft status was monitored by serum creatinine. CC was defined by the development of the following features:- (i) unexplained and persistent thrombocytopenia ( 30% fall in platelet count) and (ii) significant and persistent decrease in serum fibrinogen (and increase in serum D-dimer), followed by (iii) clinical lethargy, requiring euthanasia, and/or (iv) overt bleeding, requiring euthanasia. Immunosuppressive therapy Two baboons received grafts from WT (n=1) or GT-KO/CD46 (n=1) pigs without immunosuppressive therapy (Table 1). Immunosuppressive therapy was administered to 7 baboons that received grafts from WT (n=1), GT-KO (n=1), or GT-KO/CD46 (n=5) pigs with immunosuppressive therapy (Table 1). Immunosuppressive therapy varied, but was based on induction with anti-thymocyte globulin and maintenance with a human anti-CD154 monoclonal Ab (mAb; ABI193, a generous gift from Novartis Pharma, Basel, Switzerland), and mycophenolate mofetil (MMF, Roche, Nutley, NJ), with or without methylprednisolone . Cobra venom factor (CVF) was administered to two baboons (Table 1). Table 1 Graft type, immunosuppressive therapy, outcome, graft survival, and cause of death or euthanasia in pig-to-baboon kidney transplantation. porcine TF, since it was inhibited by an anti-human TF Ab. Because of a different avidity between the species, this Ab inhibits only human, but not pig, TF activity in the clotting assay (23) (Figure 2E). TF expression and activity on pig kidney grafts and native baboon organs At death or euthanasia of those baboons with CC (n=6), immunofluorescent staining of the pig Mesaconine kidney graft showed that TF was expressed on the platelet aggregates in the clot and on PBMCs (Figure 3A). Consistently, baboon TF activity was demonstrated by quantitative RT-PCR in the porcine grafts (Figure 3B). Open in a separate window Open in a separate window Open in a separate window Open in a separate window Figure 3 TF expression Mesaconine in the pig kidney grafts and native baboon organs(A) TF expression on platelets and PBMCs in the graft was examined by immunofluorescent staining. Grafts were stained with TF (green) and either CD42a (platelets, red, 400X) or CD45 (PBMCs, red, 400X), respectively. TF expression on platelet aggregates (upper two.