479 Unraveling the Mechanistic Pathways of the Instant Blood Mediated Inflammatory Reaction
Tuesday November 17, 2015 from 11:00 to 12:30
Room 109

David Liuwantara, Australia

Postdoctoral Scientist

Center for Transplantation and Renal Research

Westmead Millennium Institute


Unraveling the Mechanistic Pathways of the Instant Blood Mediated Inflammatory Reaction

David Liuwantara1, Yi Vee Chew1, Joanne Hawkes1, Heather Burns1, Sussan Davies1, Lindy Williams1, Emmanuele J Favaloro3, Philip J O'Connell1,2, Wayne J Hawthorne1,2.

1Center for Transplantation and Renal Research, Westmead Millennium Institute, Sydney, Australia; 2Medicine, University of Sydney, Sydney, Australia; 3Haematology, Institute of Clinical Pathology and Medical Research, Sydney, Australia

xenotransplantation. islet transplantation.

The Instant Blood Mediated Inflammatory Reaction (IBMIR) is the initial barrier inhibiting survival of porcine neonatal islet cell clusters (NICC) following xenotransplantation.  Whilst IBMIR is a significant cause of islet loss, little is known about the various mechanisms involved and the resulting function and viability of the islets surviving this initial insult.
Aim: As such we investigated the molecular pathways contributing to IBMIR and assessed islet viability following IBMIR.
Methods: NICC from outbred pigs were exposed to either human blood plasma and/ or neutrophils to trigger IBMIR.  Continuous thrombin generation kinetics were measured in plasma for 2-hours following exposure to NICC.  Complement levels were also measured in plasma following exposure to islets for 30 or 60 minutes.  Neutrophil activation was assessed using FACS following interaction with NICC ± plasma or purified rabbit complement.  Extracellular flux analyses were performed using an XF24 analyzer to measure NICC viability following xenogeneic IBMIR.
Results: We found that xenogeneic NICC-induced IBMIR activates thrombin generation even in the absence of platelets. However, the presence of platelets accelerates thrombin generation and produces up to 73% stronger thrombin response compared to when platelets are absent. Xenogeneic complement activation in response to NICC was not platelet dependent and we observed formation of anaphylatoxins C3a, C4a and C5a as early as 30-min (4.4-fold, 1.52-fold and 2.08-fold respectively, compared to plasma alone, p < 0.05) following exposure of NICC to human blood plasma. Formation of membrane attack complex C5-b9 however, was not observed in NICC sections until after 60-min. Neutrophil activation was measured by detecting the expression of surface markers CD66b and CD11b following exposure of NICC to purified human neutrophils. Interestingly, we found that NICC alone could not trigger activation of neutrophils in vitro. We found however, that complement activation was necessary to induce NICC-mediated neutrophil activation (CD66b: 1.48-fold, CD11b: 1.18-fold, compared to expression of surface markers on control neutrophils, p< 0.0005). Extracellular Flux analysis of NICC following exposure to human blood plasma showed that up to 40% of islet function was lost during IBMIR (153.00 vs. 98.25 pMoles/min/ μg DNA, p < 0.05). We also observed reduced cellular maximum capacity (173.8 vs. 107.50 pMoles/min/ μg DNA, p < 0.05), which could be indicative of mitrochondrial damage.
Conclusion: In the xenograft setting IBMIR causes significantly detrimental effects on the graft both in regards to survival and function. Our data shows complement activation plays a pivotal role in regulation of xenogeneic IBMIR. Therefore, significant gains can be achieved by using genetic modification of donor pigs targeting complement regulators to dampen the xenogeneic IBMIR response.

Lectures by David Liuwantara

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