This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The study will evaluate the causes of hyperacute lung rejection, based on different outcomes using different strains of pigs with well-defined genetic backgrounds and specific molecular alterations in pathways known or believed to be relevant to hyperacute lung rejection. In addition, various drugs or medications will be added to the blood (not given to blood donor) which may affect the rejection process and help us better understand it. Currently, the most effective therapy for end-stage organ failure is a transplant. As our surgical technique and patient management skills have been evolved to allow one-your survival rates approaching 90%, more patients are being considered for transplantation. This has worsened the organ shortage problem. Over 20% of those on the waiting list died waiting for an organ. Xenotransplantation (transplantation of organs from pig to man) offers a limitless supply of potential organs. It also offers the potential to limit the hardship on patients and families waiting for long intervals for organs. If a constant source of organs were available, donors could be selected which are specifically suited to individual recipients, and better reparation of the recipient and donor would be possible. Pigs are considered the most likely source of xenografts for man, because they are plentiful, commercially bred, and used by many in our society as a source of food and heart valves. Hyperacute rejection (immediate organ destruction) is the most important barrier to use of animal lungs as life-saving organ transplantation for man. The purpose of this study is to learn how best to overcome Hyperacute Lung Rejection, and specifically to define what are the components and steps of this reaction. Various different transgenic and knockout pig types are becoming available that provide a stepwise study approach. These transgenic and knockout pigs provide us the possibility to answer questions about individual pathways, critical to the understanding of mechanisms involved in xenograft lung hyperacute rejection. Additional drugs can be used to block injury pathways not controlled by the transgene. In a related component of this application, therapeutic strategies validated in the pig model will then be applied to a large animal model (e.g. pig lungs transplanted into baboons), final step before the eventual clinical application The general purpose of this study is to define and prevent rejection mechanisms occurring after transplantation of a pig lung (xenograft) into people. To achieve the goal of using xenotransplantation to treat human diseases, genetically engineered pigs are being created that are less sensitive to rejection. Our previous work using a model of ex-vivo perfusion of pig lungs that express human complement regulatory proteins (such as the decay- accelerating factor hDAF, or membrane cofactor protein MCP) by human blood identified several mechanisms by which transgenic lung xenografts were rejected, and that coagulation is one important mechanism driving this process. We have now confirmed that this mechanism is also involved when lungs lack the main carbohydrate antigen (Gal 1,3aGal) recognized by human anti-pig antibodies (using Gal-knock out or GalTKO pig lungs. In addition several other components of the innate immune system (non-Gal natural antibodies, complement, pulmonary intravascular macrophages or PIMs) affect both the tempo and character of lung hyperacute rejection. In this project we propose to use the model of pig lung perfused with human blood to dissect the cellular and molecular processes governing GalTKO HALR. To study the xenogenic effects during HAR, two units of fresh human blood are required (450 ml per donor, 900 ml per experiment). Fresh blood is necessary because the cellular components (white blood cells, plateletscritical to the hyperacute rejection response, and do not function physiologically in stored blood.
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