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Acellular materials of xenogenic origin are used worldwide as xenografts, and phase I trials of viable pig pancreatic islets are currently being performed. However, limited information is available on transmission of porcine endogenous retrovirus (PERV) after xenotransplantation and on the long-term immune response of recipients to xenoantigens. We analyzed the blood of burn patients who had received living pig-skin dressings for up to 8 wk for the presence of PERV as well as for the level and nature of their long term (maximum, 34 y) immune response against pig Ags. Although no evidence of PERV genomic material or anti-PERV Ab response was found, we observed a moderate increase in anti-αGal Abs and a high and sustained anti-non-αGal IgG response in those patients. Abs against the nonhuman sialic acid Neu5Gc constituted the anti-non-αGal response with the recognition pattern on a sialoglycan array differing from that of burn patients treated without pig skin. These data suggest that anti-Neu5Gc Abs represent a barrier for long-term acceptance of porcine xenografts. Because anti-Neu5Gc Abs can promote chronic inflammation, the long-term safety of living and acellular pig tissue implants in recipients warrants further evaluation.

BACKGROUND. Rabbit-generated antithymocyte globulins (ATGs), which target human T cells, are widely used as immunosuppressive agents during treatment of kidney allograft recipients. However, ATGs can induce immune complex diseases, including serum sickness disease (SSD). Rabbit and human IgGs have various antigenic differences, including expression of the sialic acid Neu5Gc and α-1-3-Gal (Gal), which are not synthesized by human beings. Moreover, anti-Neu5Gc antibodies have been shown to preexist and be elicited by immunization in human subjects. This study aimed to assess the effect of SSD on long-term kidney allograft outcome and to compare the immunization status of grafted patients presenting with SSD following ATG induction treatment. METHODS. We analyzed data from a cohort of 889 first kidney graft recipients with ATG induction (86 with SSD [[SSD.sup.+]] and 803 without SSD [[SSD.sup.-]]) from the Donnees Informatisees et Validees en Transplantation data bank. Two subgroups of [SSD.sup.+] and [SSD.sup.-] patients that had received ATG induction treatment were then assessed for total anti-ATG, anti-Neu5Gc, and anti-Gal antibodies using ELISA assays on sera before and after transplantation. RESULTS. SSD was significantly associated with long-term graft loss (>10 years, P = 0.02). Moreover, [SSD.sup.+] patients exhibited significantly elevated titers of anti-ATG (P = 0.043) and anti-Neu5Gc (P = 0.007) IgGs in late post-graft samples compared with [SSD.sup.-] recipients. CONCLUSION. In conclusion, our data indicate that SSD is a major contributing factor of late graft loss following ATG induction and that anti-Neu5Gc antibodies increase over time in [SSD.sup.+] patients. FUNDING. This study was funded by Societe d'Acceleration du Transfert de Technologies Ouest Valorisation, the European FP7 'Translink' research program, the French National Agency of Research, Labex Transplantex, the Natural Science and Engineering Research Council of Canada, and the Canadian Foundation for Innovation., Introduction Polyclonal anti-human T lymphocyte IgGs (antithymocyte globulins [ATGs]) from various animal sources have been used since the very beginning of allotransplantation (1, 2). ATGs are administered either immediately following [...]

Background: Fas ligand expression by cells of the vessel wall has been proposed to play a role in normal and pathologic conditions. Genetic engineering of vascularized organs for endothelial cell (EC) expression of FasL could protect the endothelium and underlying tissues from infiltrating Fas+ leukocytes. Nevertheless, the endogenous expression of FasL by ECs of different species and the potential deleterious effects of enforced FasL expression by ECs are largely unknown. In human ECs, levels of FLICE/caspase 8-inhibitory protein (FLIP) have been shown to control apoptosis mediated by Fas., Materials and Methods: Cell surface expression of FasL in rat, mouse, human, and pig ECs was obtained using recombinant adenoviruses or transient plasmid transfection assays. FasL expression was evaluated by FACS analysis and cytotoxicity assays. Apoptosis was evaluated using annexin V, TUNEL, and cytotoxicity assays. FLIP levels were evaluated by Western blot analysis and overexpression was obtained by transient transfection., Results: Analysis of ECs from different species showed that FasL was predominantly present in the cytoplasm, and depending on the species, little or no cell surface expression was detected. Enforced cell surface expression of FasL on rat or mouse ECs, either in culture or within the vessel wall resulted in massive apoptosis. In contrast, porcine or human ECs were completely resistant to apoptosis mediated by Fas-FasL interaction. Markedly reduced FLIP levels were observed in rat and mouse ECs compared to human and porcine ECs. Overexpression of FLIP in rat ECs conferred protection against cell surface expression of FasL., Conclusions: The consequences of FasL overexpression depend on the subcellular compartment and species in which FasL enforced expression is targeted and this is at least partially related to FLIP levels.

The disaccharide galactose(alpha)1,3 galactose (the alphaGal epitope) is the major xenoantigen responsible for the hyperacute vascular rejection occurring in pig-to-primates organ transplantation. The synthesis of the alphaGal epitope is catalyzed by the enzyme alpha1,3-galactosyltransferase (alpha1,3GalT). To be able to control porcine alpha1,3GalT gene expression specifically, we have analyzed the upstream portion of the alpha1,3GalT gene, and identified the regulatory sequences. Porcine alpha1,3GalT transcripts were detected by 5' RACE analysis, and the corresponding genomic sequences were isolated from a phage library. The porcine alpha1,3GalT gene consists of at least 10 different exons, four of which contain 5' untranslated sequence. Four distinct promoters, termed A-D, drive alpha1,3GalT gene transcription in porcine cells. A combination of alternative promoter usage and alternative splicing produces a series of transcripts that differ in their 5' portion, but encode the same protein. Promoters A-C have been isolated, and functionally characterized using luciferase reporter gene assays in transfected porcine endothelial cells (PEC-A). Promoter preference in porcine endothelial cells was estimated on the basis of relative transcript levels as determined by real-time quantitative PCR. More than 90% of the alpha1,3GalT transcripts in PEC-A cells originate from promoter B, which has characteristics of a housekeeping gene promoter. While promoter preference remains unchanged, alpha1,3GalT mRNA levels increase by 50% in 12 h upon tumour necrosis factor alpha-activation of PEC-A cells. However, the magnitude of this change induced by inflammatory conditions could be insufficient to affect cell surface alpha1,3-galactosylation.