Up to 9-day survival and control of thrombocytopenia following alpha1,3-galactosyl transferase knockout swine liver xenotransplantation in baboons

Abstract

Background: With standard miniature swine donors, survivals of only 3 days have been achieved in primate liver-transplant recipients. The recent production of alpha1,3-galactosyl transferase knockout (GalT-KO) miniature swine has made it possible to evaluate xenotransplantation of pig organs in clinically relevant pig-to-non-human primate models in the absence of the effects of natural anti-Gal antibodies. We are reporting our results using GalT-KO liver grafts.

Methods: We performed GalT-KO liver transplants in baboons using an immunosuppressive regimen previously used by our group in xeno heart and kidney transplantation. Post-operative liver function was assessed by laboratory function tests, coagulation parameters and histology.

Results: In two hepatectomized recipients of GalT-KO grafts, post-transplant liver function returned rapidly to normal. Over the first few days, the synthetic products of the donor swine graft appeared to replace those of the baboon. The first recipient survived for 6 days and showed no histopathological evidence of rejection at the time of death from uncontrolled bleeding, probably caused by transfusion-refractory thrombocytopenia. Amicar treatment of the second and third recipients led to maintenance of platelet counts of over 40 000 per μl throughout their 9- and 8-day survivals, which represents the longest reported survival of pig-to-primate liver transplants to date. Both of the last two animals nevertheless succumbed to bleeding and enterococcal infection, without evidence of rejection.

Conclusions: These observations suggest that thrombocytopenia after liver xenotransplantation may be overcome by Amicar therapy. The coagulopathy and sepsis that nevertheless occurred suggest that additional causes of coagulation disturbance must be addressed, along with better prevention of infection, to achieve long-term survival.

Fig 1

The hematocrit fell in all animals to critical levels, requiring the transfusion of substantial amounts of packed red blood cells (PRBC). The volume of transfused blood per day is represented by bar graphs.

Fig 2

Thrombocytes were lost immediately after reperfusion. However, the levels remained slightly above the critical value of 20 000 per μl after Amicar treatment was started. The red and green dotted horizontal lines indicate Amicar infusion in B291 and B317, respectively. The interruption of the green dotted line indicates the accidental discontinuation of Amicar in B291 overnight. The blue arrow marks the time point of transfusion of allogenic platelets in B274 (details see text). B274 did not receive Amicar, while B317 received Amicar throughout its post-operative course.

Fig 3

(A, B) Coagulation factors in naïve swine and baboon and in the recipient of a porcine liver (B274). Coagulation factors approached swine baseline levels after transplantation. The Y-axis depicts per cent, with 100% being the normal value in humans. The half-lives of the clotting factors ranged from 6 to 12 h, making a transition from baboon to pig highly likely (A). (B) Shows the stable PTT/INR in all three animals with the exception of B 274, which experienced post-operative ischaemia and hypoxia.

Fig 4

Liver function tests (A) Alanine aminotransferase (ALT) levels confirmed stable hepatic function in animal B291 and B317, while B274 exhibited a progressive release of ALT in line with declining liver function, likely due to hypoxia (B) aspartate aminotransferase levels peaked twice after surgery in recipient Baboon 274, and release could be from hepatocytes or haemolyzed red blood cells. AST release appeared less in animals treated with Amicar. (C) Total bilirubin levels rose progressively in all animals. This was likely caused by the continued haemorrhage and haemolysis as the differential analysis showed that it was mostly indirect bilirubin. (D) Albumin levels remained in normal range throughout the animals’ life, but frequent exogenous administration makes interpretation difficult.

Fig 5

Normal liver tissue without any evidence of rejection, haematoxylin and eosin. The sample was taken on POD 7, B 291. It was representative for all liver samples of all animals in that none of the biopsies ever showed histologic evidence of rejection.