E. Rybak

Expression of human CD46 modulates inflammation associated with GalTKO lung xenograft injury

Evaluation of lungs from GalTKO.hCD46 pigs, genetically modified to lack the galactose‐α(1,3)‐galactose epitope (GalTKO) and to express human CD46, a complement regulatory protein, has not previously been described. Physiologic, hematologic and biochemical parameters during perfusion with heparinized fresh human blood were measured for 33 GalTKO.hCD46, GalTKO (n = 16), and WT pig lungs (n = 16), and 12 pig lungs perfused with autologous pig blood. Median GalTKO.hCD46 lung survival was 171 min compared to 120 for GalTKO (p = 0.27) and 10 for WT lungs (p < 0.001). Complement activation, platelet activation and histamine elaboration were significantly reduced during the first 2 h of perfusion in GalTKO.hCD46 lungs compared to GalTKO (ΔC3a at 120′ 812 ± 230 vs. 1412 ± 1047, p = 0.02; ΔCD62P at 120′ 9.8 ± 7.2 vs. 25.4 ± 18.2, p < 0.01; Δhistamine at 60′ 97 ± 62 vs. 189 ± 194, p = 0.03). We conclude that, in addition to significant down‐modulation of complement activation, hCD46 expression in GalTKO lungs diminished platelet and coagulation cascade activation, neutrophil sequestration and histamine release. Because GalTKO.hCD46 lung failure kinetics correlated directly with platelet and neutrophil sequestration, coagulation cascade activation and a rise in histamine levels within the first hour of perfusion, further progress will likely depend upon improved control of these pathways, by rationally targeted additional modifications to pigs and pharmacologic interventions.

Pig-to-baboon liver xenoperfusion utilizing GalTKO.hCD46 pigs and glycoprotein Ib blockade

Although transplantation of genetically modified porcine livers into baboons has yielded recipient survival for up to 7 days, survival is limited by profound thrombocytopenia, which becomes manifest almost immediately after revascularization, and by subsequent coagulopathy. Porcine von Willebrands factor (VWF), a glycoprotein that adheres to activated platelets to initiate thrombus formation, has been shown to constitutively activate human platelets via their glycoprotein Ib (GPIb) receptors. Here, we report our pig‐to‐primate liver xenoperfusion model and evaluate whether targeting the GPIb‐VWF axis prevents platelet sequestration.

Platelet sequestration and activation during GalTKO.hCD46 pig lung perfusion by human blood is primarily mediated by GPIb, GPIIb/IIIa, and von Willebrand Factor

Here, we ask whether platelet GPIb and GPIIb/IIIa receptors modulate platelet sequestration and activation during GalTKO.hCD46 pig lung xenograft perfusion.

PILOT EVALUATION OF ANTI-GPIB EFFECTS ON PLATELET SEQUESTRATION IN AN EX VIVO XENOGENEIC PIG LIVER PERFUSION MODEL: 3132

L. Burdorf1, R.N. Barth2, T. Zhang3, E. Rybak4, I.I. Salles5, K. Broos6, E. Welty4, C.J. Avon7, A. Laaris4, B. McCormick2, D. Ayares8, H. Deckmyn6, A.M. Azimzadeh2, R.N. Pierson III2 1Department Of Surgery, University of Maryland, Baltimore/MD/ UNITED STATES OF AMERICA, 2Surgery, University of Maryland, Baltimore/UNITED STATES OF AMERICA, 3Surgery, Univ. of Maryland & Baltimore VA, Baltimore/MD/UNITED STATES OF AMERICA, 4Department Of Surgery, University of Maryland, Baltimore/UNITED STATES OF AMERICA, 5Department Of Haematology, Imperial College London, London/UNITED KINGDOM, 6Laboratory For Thrombosis, KU Leuven Campus Kortrijk, Kortrijk/ BELGIUM, 7Surgery, University of Maryland and Baltimore VAMHCS, baltimore/MD/UNITED STATES OF AMERICA, 8, Revivicor Inc, Blacksburg/UNITED STATES OF AMERICA

Preemptive CD20+ B cell Depletion Attenuates Cardiac Allograft Vasculopathy in CD154-Treated Monkeys.

Background Anti-CD154 monotherapy is associated with antidonor allo-antibody (Ab) elaboration, cardiac allograft vasculopathy (CAV), and allograft failure in preclinical primate cell and organ transplant models. In the context of calcineurin inhibitors (CNI), these pathogenic phenomena are delayed by preemptive “induction” B cell depletion. Methods &agr;CD154 (IDEC-131)–treated cynomolgus monkey heart allograft recipients were given peritransplant rituximab (&agr;CD20) alone or with rabbit antihuman thymocyte globulin. Results Relative to previously reported reference groups, &agr;CD20 significantly prolonged survival, delayed Ab detection, and attenuated CAV within 3 months in &agr;CD154-treated recipients (&agr;CD154 + &agr;CD20 graft median survival time > 90 days, n = 7, vs 28 days for &agr;CD154 alone (IDEC-131), n = 21; P = 0.05). Addition of rabbit antihuman thymocyte globulin to &agr;CD154 (n = 6) or &agr;CD154 + &agr;CD20 (n = 10) improved graft protection from graft rejection and failure during treatment but was associated with significant morbidity in 8 of 16 recipients (6 infections, 2 drug-related complications). In &agr;CD20-treated animals, detection of antidonor Ab and relatively severe CAV were anticipated by appearance of CD20+ cells (>1% of lymphocytes) in peripheral blood and were associated with low &agr;CD154 trough levels (below 100 &mgr;g/mL). Conclusions These observations support the hypothesis that efficient preemptive “induction” CD20 B cell depletion consistently modulates pathogenic alloimmunity and attenuates CAV in this translational model, extending our prior findings with calcineurin inhibitors to the context of CD154 blockade.