476
In vitro studies on vascular hyperacute xenograft rejection (HAR) are usually conducted on cultured endothelial cells (EC) exposed to human serum, or in complex whole organ perfusion models using heparinized blood. Here we describe a new model which allows for the use of non-heparinized human whole blood, and has the advantage of retaining the endothelial cells in their native environment. Method: A 2 cm segment of the porcine common iliac artery (PIA) is connected to a surface-heparinized, circular cardiovascular plastic tube and the vessel perfused by rocking of the device for 5, 15 or 60 minutes with 7-8 ml fresh non-heparinized human blood in an incubator at 37' C. A 2 cm section of the surface-heparinized cardiovascular tube is used to replace the vessel in control experiments. Thrombocytes (TRC). differential 1eukocyte count, C3a, soluble `Terminal Complement Complex (TCC) and Thrombin-Anti-Thrombin (TAT) was measured before and after perfusion. Snap-frozen sections of the porcine vessel were stained for human immunoglobulin (IgM, IgA, IgG), C3a, TCC. ant,-CD4la(TRC) and fibrin using IF and IH techniques. Results: The table below summarizes the findings at 60 minutes.
During perfusion of the porcine vessel there was a rapid consumption of TRC during the first 5-30 min and clotting could be observed at I5 minutes. At 60 minutes there was a depletion of neutrophils and monocytes but not of lymphocytes. IF and IH revealed extensive binding of human IgM, IgA, IgA, C3 and to some extent also TCC to the E.C. within 5 minutes. Fibrin and TRC were deposited within 5 minutes on the E.C. and thrombi were seen by 60 minutes. Conclusion Events characterizing HAR can be triggered in this model, and the findings are similar to those seen in whole organ xenograft perfusion models The present model offers a simple and reproducible way to study pig-to-human HAR. Also, the small blood volume needed permits testing inhibitors of HAR, which are not available in large quantities, or are very expensive.