Background: Inception of GalT knock out (GalT-KO) miniature swine has helped to ameliorate rapid xenograft rejection, now allowing for investigations into the utility of miniature swine xenografts as longer-term replacement therapy. However, before xenotransplantation of organs with complex metabolic functions such as the liver can be considered for clinical use, precise interrogation of graft liver function in situ is essential. We report for the first time the assessment of GalT-KO liver functionality after orthotopic transplantation in baboons by comparative ELISA-based measurement and quantification of multiple swine liver proteins. Methods: We performed orthotopic GalT-KO liver transplants in baboons using a standard immunosuppressive regimen. Post-operative liver xenograft function was assessed by means of ELISA-based quantification of swine liver proteins using healthy swine and primate serum as controls for optical density (OD) comparison. After optimal dilutions were established for each hepatic protein measured, OD comparisons were made between subjects and the relevant control, reflecting the outcome as a percentage of normal expression levels. Finally, reference values were used to convert these percentages to absolute values. Results: Four non-human-primate recipients of orthotopically transplanted GalT-KO liver grafts were observed to be cinically well immediately following surgery with no evidence of post-operative complications or signs of infection. Production of liver proteins by the swine xenografts was robust, with levels of Plasminogen, Fibrinogen, Albumin and Haptoglobulin reaching by day 3 post-transplant as much as 100% of the amount found in serum of naÏve swine. Concurrently, the levels of native liver protein decreased significantly by day 3, as expected following total native hepatectomy. Conclusion: These observations illustrate a novel, precise method of assessing in situ functionality of swine liver xenografts orthotopically transplanted in non-human-primates. Going forward, novel approaches to graft assessment such as this will allow us to more precisely determine areas of liver functionality that may contribute to graft loss or xeno-hepatic insufficiency and aid in prolongation of survival in pig-to-primate models of liver transplantation.

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