Hypodermin A, a new inhibitor of human complement for the prevention of xenogeneic hyperacute rejection.

BACKGROUND: Hyperacute rejection (HAR) of discordant xenografts in the pig-to-human combination can be prevented using tranplants expressing transgenic molecules that inhibit human complement. Hypodermin A (HA), a serine esterase that degrades C3, was tested in the guinea-pig-to-rat and in the pig-to-human combinations. METHODS: Hypodermin A was tested in vitro, ex vivo, and in vivo models of HAR in the guinea-pig-to-rat combination. Hamster ovary cells (CHO) and a line of porcine aortic endothelial cells (PAEC11) were transfected with HA complementary DNA (cDNA). RESULTS: The pattern of degradation of rat and human C3 by HA was different (multiple bands lower than 40 kDa) from the physiologic pattern observed after spontaneous degradation of rat C3 or physiologic activation of human C3. The CH50 activity in serum was significantly lower in rats treated with 3.2 mg HA/kg than in untreated rats (45 +/- 16 U/ml vs. 700 +/- 63 U/ml, P < 0.05). Sera from rats injected with 3.2 mg/kg of HA were less effective in lysing guinea-pig endothelial cells (12 +/- 7%) than normal rat sera (79 +/- 3%; P < 0.001). Ex vivo, guinea-pig hearts perfused by rat serum supplemented with HA survived longer than those perfused by non-treated serum (210 +/- 34 and 154 +/- 71 min, respectively; P < 0.05). In vivo, guinea-pig hearts transplanted into HA treated rats survived longer than in non-treated rats (27 +/- 5 min vs. 13 +/- 4 min; P < 0.001). In the presence of human serum, smaller amounts of C6 and C5b-9 were deposited onto HA-transfected CHO cells than onto control cells. The mHA-PAEC11 cells were significantly more resistant to lysis by human C than control PAEC11 cells. CONCLUSIONS: These data suggest that transgenic HA could be used to prevent hyperacute xenogeneic rejection.

[A new model of isolated perfused heart. Study of hypodermin A in hyperacute xenograft rejection]. / Mise au point d'un modèle de coeur isolé perfusé. Application à l'étude de l'hypodermine A dans la prévention du rejet suraigu xénogénique.

AIMS OF THE STUDY: Isolated perfused heart (IPH) system and heart transplantation in the guinea-pig/rat combination represent a good model for the study of hyperacute xenograft rejection (HAR) in which the component plays a central role. Hypodermin A (HA), a protease cleaving the component, could be used to delay the HAR. METHODS: Creation of an original IPH working with rat serum (30 mL) and ex vivo study of HAR and I'HA. RESULTS: Study of HAR is possible with this IPH system. The mean guinea-pig heart survival after perfusion by normal rat serum was 38 +/- 7 min and was lower than survival observed after perfusion by guinea-pig serum (210 +/- 34 min) (p < 0.001), by decomplemented rat serum (177 +/- 45 min) (p < 0.001), and by rat serum with 20 micrograms/mL of HA (154 +/- 71 min) (p < 0.001). CONCLUSION: We developed an original system of isolated perfused heart allowing ex vivo study of HAR. HA delayed the occurrence of the HAR and confirmed the central role of the component in the HAR.

A newly established porcine aortic endothelial cell line: characterization and application to the study of human-to-swine graft rejection.

The establishment of cell lines allows reproductible in vitro studies that would be far more difficult to perform using primary cells that rapidly undergo phenotypical alterations in culture. The purpose of this work was to establish an endothelial cell line appropriate for in vitro study of endothelial cell activation during xenograft rejection. Porcine aortic endothelial cells were transfected with the early region of SV40 and selected on the basis of morphological, phenotypical, and functional features. By light and electron microscopy, the porcine aortic endothelial cell line (PAEC11) and primary cells were similar except that PAEC11 was slightly smaller. PAEC11 displayed endothelial cell characteristics since it endocytosed acetylated low density lipoproteins, produced von Willebrand factor, and expressed E-selectin. Human natural antibodies bound to the same xenoantigens on PAEC11 and primary cells. That binding was followed by human complement activation and cell lysis. In addition, PAEC11 was found appropriate for genetic engineering since it could be transfected with a plasmid encoding a foreign gene. Therefore, this cell line should be a useful model for in vitro study of endothelial cell function in general and human-to-swine xenograft rejection in particular.

The RU5 ('R') region from human leukaemia viruses (HTLV-1) contains an internal ribosome entry site (IRES)-like sequence.

RNA fragments containing the complete R region and the beginning of the U5 region ('R') from the human T cell leukaemia virus 1 (HTLV-1) stimulated the translation of the second cistrons in bicistronic mRNAs. The 5' untranslated region from SV40 early genes (SU) which was unable to stimulate translation of second cistrons amplified markedly the internal ribosome entry site (IRES) effect of the HTLV-1 'R' fragments. The 'R' regions from HTLV-1 have therefore properties similar to internal ribosome entry sites (IRES) originally found in picornavirus. The beginning of the U5 region from HTLV-1 contains a polypyrimidine sequence which is known to play an essential role in the IRES activity in picornavirus. The same experiments carried out using the 'R' region from bovine leukaemia virus (BLV) showed that this sequence has at most a weak IRES effect. One retroviruses, HTLV-1 and perhaps others contain therefore an IRES activity. Interestingly, the combined SU 'R' sequence worked efficiently with different cistrons, different promoters and in all tested cell lines, whereas the poliovirus IRES was active in CHO cells but not in the mouse mammary cell line HC11. The SU 'R' sequence may therefore preferably be used to generate active bicistronic mRNAs.