Xenotransplantation (Xtr) is of interest because of the insufficient supply of human organs. The pig is considered a likely donor species for humans, but there are major barriers to successful Xtr. The first two barriers, hyperacute rejection (HAR) and acute vascular rejection (AVR), are mediated primarily by humoral immunity. HAR can be abrogated, but then AVR will occur. Strategies to interfere with AVR include reducing humoral immunity or inducing tolerance in the recipient, and decreasing immunogenicity or inducing protection in the graft. Our long-term goal is to investigate induction of protection in the donor organ against AVR. HYPOTHESIS: The cytokine IL-4 is capable of inducing broad protection against xenogeneic injury of porcine endothelial cells (ECs) and can be used to prepare an organ for Xtr, such that the organ is protected from HAR and AVR.
SPECIFIC AIMS. I. Investigate protection induced by IL-4 in porcine ECs against xenogeneic activation. The activation response of ECs deficient in the main antigen responsible for graft rejection will be studied for IL-1 production and other mediators of EC activation, and the requirements and mechanisms for IL-4 protection will be defined. II. Characterize the mechanisms that participate in protection against complement killing. The participation of the signaling protein Akt is required for IL-4-induced protection. Akt is known to activate several substrates that may result in protection;therefore the identification and characterization of these pathways will be carried out. III. Investigate the role of anti-apoptotic proteins in IL-4-induced protection against apoptosis. The role of Bcl-xL and other protective molecules will be studied in protection from apoptosis elicited with TNF-alpha and with human natural killer cells. In these first three specific aims the protective pathways will be studied with kinase inhibitors and the functional role of candidate proteins will be defined with siRNA. IV. Investigate the protective effect of IL-4 overexpression against HAR and AVR in the mouse-to-rat model of Xtr. The AVR process in this animal model is similar to that in pig-to-primate models and will allow us to define the mechanisms of protection. Mouse hearts will be infected with an adenovirus carrying the IL-4 gene, or an irrelevant gene as control, and then transplanted into rats for studies of graft survival, histology, and gene expression and activation. Studies will be performed at different periods after grafting to establish the efficacy of IL-4 in preventing the manifestations of activation and the pathological course of rejection. It is expected that there will be an attenuation of the intensity and a delay in the manifestation of such processes, and prolongation of graft survival. Accomplishing these goals will result in understanding the mechanisms of protection of IL-4 in donor organs from a different species and to what extent they contribute to successful Xtr, which is essential for those patients with end-stage organ failure for whom no human organs are available.

Public Health Relevance

The supply of human organs for transplantation is insufficient, and a potential solution to overcome this shortage is xenotransplantation (using organs from donor animals such as the pig). Accomplishing the goals of this proposal will define a novel approach to modify donor organs for clinical xenotransplantation. This will also provide knowledge for new treatments for vascular diseases, including ischemia and atherosclerosis.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Transplantation, Tolerance, and Tumor Immunology (TTT)
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Schwartz, Lisa
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University of Minnesota Twin Cities
Schools of Medicine
United States
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Benson, Barbara A; Vercellotti, Gregory M; Dalmasso, Agustin P (2015) IL-4 and IL-13 induce protection from complement and melittin in endothelial cells despite initial loss of cytoplasmic proteins: membrane resealing impairs quantifying cytotoxicity with the lactate dehydrogenase permeability assay. Xenotransplantation 22:295-301
Dalmasso, Agustin P; Goldish, Daniel; Benson, Barbara A et al. (2014) Interleukin-4 induces up-regulation of endothelial cell claudin-5 through activation of FoxO1: role in protection from complement-mediated injury. J Biol Chem 289:838-47
Dalmasso, Agustin P (2012) On the intersections of basic and applied research in xenotransplantation. Xenotransplantation 19:137-43
Black, Sylvester M; Benson, Barbara A; Idossa, Damé et al. (2011) Protection of porcine endothelial cells against apoptosis with interleukin-4. Xenotransplantation 18:343-54
Black, Sylvester M; Schott, Megan E; Batdorf, Bjorn H et al. (2010) IL-4 induces protection of vascular endothelial cells against killing by complement and melittin through lipid biosynthesis. Eur J Immunol 40:803-12
Black, S M; Schott, M E; Benson, B A et al. (2008) Interleukin-4 induces lipogenesis in porcine endothelial cells, which in turn is critical for induction of protection against complement-mediated injury. Transplant Proc 40:638-40
Black, Sylvester M; Grehan, John F; Rivard, Andrew L et al. (2006) Porcine endothelial cells and iliac arteries transduced with AdenoIL-4 are intrinsically protected, through Akt activation, against immediate injury caused by human complement. J Immunol 177:7355-63
Grehan, John F; Levay-Young, Brett K; Fogelson, Jeremy L et al. (2005) IL-4 and IL-13 induce protection of porcine endothelial cells from killing by human complement and from apoptosis through activation of a phosphatidylinositide 3-kinase/Akt pathway. J Immunol 175:1903-10
Grehan, John F; Levay-Young, Brett K; Benson, Barbara A et al. (2005) Alpha Gal ligation of pig endothelial cells induces protection from complement and apoptosis independently of NF-kappa B and inflammatory changes. Am J Transplant 5:712-9
Grubbs, Brian C; Benson, Barbara A; Dalmasso, Agustin P (2003) Characteristics of CD59 up-regulation induced in porcine endothelial cells by alphaGal ligation and its association with protection from complement. Xenotransplantation 10:387-97

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