Introduction: Progresses in the field of xenotransplantation (XTx) with the development of transgenic pigs has allowed the recent pig to human XTx.
The main initiating factor in the human hyperacute rejection to pig organs is the presence of preformed human natural antibodies; therefore, the characterization of human anti-pig xenoantibodies (XAb) is required to elucidate the mechanisms involved in XTx rejection.
Aim: Analyze in patients exposed to pig hepatocytes following Bioartificial Liver (BAL) treatment: 1) the long-term XAb response to pig endothelial cells (PEC)  from wild-type (wt) and transgenic pigs lacking the alfa-Gal epitope (GalT-KO), 2) the cytotoxicity of patients' plasma to PEC from wt and GalT-KO. 
Material & Methods: The BAL was used as a support treatment in patients with acute liver failure (ALF) awaiting liver transplantation, and consists of patients’ plasma perfused through a hollow-fiber cartridge seeded with porcine hepatocytes attached to beads. 
Six long-term follow-up ALF patients treated with BAL were included in this study. Each patient received 1 to 6 BAL treatments; 3 patients subsequently received a liver transplant, the other patients recovered to avoid the need of transplant. 
Patients' plasma collected before (day 0) and at different time-points post-BAL treatment (up to 1 year) was analyzed by ELISA for IgG and IgM XAb binding to PEC from wt and GalT-KO pigs, and to bovine thyroglobulin (contains alfa-Gal). The cytotoxicity of patients' plasma was analyzed by flow cytometric toxicity assay using PEC from wt and GalT-KO as target cells. 
Results: Anti-pig IgG and IgM XAb to wt, to GalT-KO PEC (non-alfa-Gal) and to thyroglobulin (alfa-Gal epitopes), were present in patients' plasma before exposure to pig antigens (day 0), with high variability in the XAb levels among patients. Repetitive BAL treatments were associated with an immediate decreased of the IgG/IgM XAb titers (up to 50%), indicating that XAb were absorbed during BAL treatment. A marked increase of XAb was observed by 1-2 weeks post-BAL treatments followed by a gradual decrease over time; the increase being more pronounced for alfa-Gal XAb. The cytotoxicity assay showed that patient's plasma prior to porcine exposure has a degree of cytotoxicity to PEC from wt (0-20%) and minimal cytotoxicity to GalT-KO pigs. By 1-2 weeks post-BAL treatment a sharp increase of cytotoxicity to wt PEC was observed (22-80%), however, patients' plasma was minimally cytotoxic to GalT-KO-PEC (cytotoxicity <5% in 5 patients). 
Conclusions: -Patients' plasma contains both IgG and IgM XAb that recognize and bind to alfa-Gal and non-alfa-Gal epitopes on PEC from wt and GalT-KO pigs. 
-An immediate XAb absorption was observed during repetitive BAL treatments, followed by an increase in IgG and IgM XAb titers by 1-2 weeks post-BAL. 
-Human XAb produced in response to pig hepatocytes exposure are cytotoxic to PEC from wt pigs; however very low cytotoxicity was observed toward PEC from GalT-KO.