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Browsing by Author "Martens, Gregory R."
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Item Porcine iGb3s gene silencing provides minimal benefit for clinical xenotransplantation(Wiley, 2016-03) Butler, James R.; Skill, Nicholas J.; Priestman, David; Platt, Frances; Li, Ping; Estrada, Jose L.; Martens, Gregory R.; Ladowski, Joseph M.; Tector, Matthew; Tector, A. Joseph; Department of Surgery, IU School of MedicineBackground The Galα(1,3)Gal epitope (α-GAL), created by α-1,3-glycosyltransferase-1 (GGTA1), is a major xenoantigen causing hyperacute rejection in pig-to-primate and pig-to-human xenotransplantation. In response, GGTA1 gene-deleted pigs have been generated. However, it is unclear whether there is a residual small amount of α-Gal epitope expressed in GGTA1−/− pigs. Isoglobotrihexosylceramide synthase (iGb3s), another member of the glycosyltransferase family, catalyzes the synthesis of isoglobo-series glycosphingolipids with an α-GAL-terminal disaccharide (iGb3), creating the possibility that iGb3s may be a source of α-GAL epitopes in GGTA1−/− animals. The objective of this study was to examine the impact of silencing the iGb3s gene (A3GalT2) on pig-to-primate and pig-to-human immune cross-reactivity by creating and comparing GGTA1−/− pigs to GGTA1−/−- and A3GalT2−/−-double-knockout pigs. Methods We used the CRISPR/Cas 9 system to target the GGTA1 and A3GalT2 genes in pigs. Both GGTA1 and A3GalT2 genes are functionally inactive in humans and baboons. CRISPR-treated cells used directly for somatic cell nuclear transfer produced single- and double-gene-knockout piglets in a single pregnancy. Once grown to maturity, the glycosphingolipid profile (including iGb3) was assayed in renal tissue by normal-phase liquid chromatography. In addition, peripheral blood mononuclear cells (PBMCs) were subjected to (i) comparative cross-match cytotoxicity analysis against human and baboon serum and (ii) IB4 staining for α-GAL/iGb3. Results Silencing of the iGb3s gene significantly modulated the renal glycosphingolipid profile and iGb3 was not detected. Moreover, the human and baboon serum PBMC cytotoxicity and α-GAL/iGb3 staining were unchanged by iGb3s silencing. Conclusions Our data suggest that iGb3s is not a contributor to antibody-mediated rejection in pig-to-primate or pig-to-human xenotransplantation. Although iGb3s gene silencing significantly changed the renal glycosphingolipid profile, the effect on Galα3Gal levels, antibody binding, and cytotoxic profiles of baboon and human sera on porcine PBMCs was neutral.Item Silencing porcine CMAH and GGTA1 genes significantly reduces xenogeneic consumption of human platelets by porcine livers(Wolters Kluwer, 2016-03) Butler, James R.; Paris, Leela L.; Blankenship, Ross L.; Sidner, Richard A.; Martens, Gregory R.; Ladowski, Joeseph M.; Li, Ping; Estrada, Jose L.; Tector, Matthew; Tector, A. Joseph; Department of Surgery, IU School of MedicineBACKGROUND: A profound thrombocytopenia limits hepatic xenotransplantation in the pig-to-primate model. Porcine livers also have shown the ability to phagocytose human platelets in the absence of immune-mediated injury. Recently, inactivation of the porcine ASGR1 gene has been shown to decrease this phenomenon. Inactivating GGTA1 and CMAH genes has reduced the antibody-mediated barrier to xenotransplantation; herein, we describe the effect that these modifications have on xenogeneic consumption of human platelets in the absence of immune-mediated graft injury. METHODS: Wild type (WT), ASGR1, GGTA1, and GGTA1CMAH knockout pigs were compared for their xenogeneic hepatic consumption of human platelets. An in vitro assay was established to measure the association of human platelets with liver sinusoidal endothelial cells (LSECs) by immunohistochemistry. Perfusion models were used to measure human platelet uptake in livers from WT, ASGR1, GGTA1, and GGTA1 CMAH pigs. RESULTS: GGTA1, CMAH LSECs exhibited reduced levels of human platelet binding in vitro when compared with GGTA1 and WT LSECs. In a continuous perfusion model, GGTA1 CMAH livers consumed fewer human platelets than GGTA1 and WT livers. GGTA1 CMAH livers also consumed fewer human platelets than ASGR1 livers in a single-pass model. CONCLUSIONS: Silencing the porcine carbohydrate genes necessary to avoid antibody-mediated rejection in a pig-to-human model also reduces the xenogeneic consumption of human platelets by the porcine liver. The combination of these genetic modifications may be an effective strategy to limit the thrombocytopenia associated with pig-to-human hepatic xenotransplantation.Item Swine Leukocyte Antigen Class II Is a Xenoantigen(Wolters Kluwer, 2018-02) Ladowski, Joseph M.; Reyes, Luz M.; Martens, Gregory R.; Butler, James R.; Wang, Zheng-Yu; Eckhoff, Devin E.; Tector, Matt; Tector, A. Joseph; Surgery, School of MedicineBACKGROUND: Over 130 000 patients in the United States alone need a lifesaving organ transplant. Genetically modified porcine organs could resolve the donor organ shortage, but human xenoreactive antibodies destroy pig cells and are the major barrier to clinical application of xenotransplantation. The objective of this study was to determine whether waitlisted patients possess preformed antibodies to swine leukocyte antigen (SLA) class II, homologs of the class II HLA. METHODS: Sera from people currently awaiting solid organ transplant were tested for IgG binding to class II SLA proteins when expressed on mammalian cells. Pig fibroblasts were made positive by transfection with the class II transactivator. As a second expression system, transgenes encoding the alpha and beta chains of class II SLA were transfected into human embryonic kidney cells. RESULTS: Human sera containing IgG specific for class II HLA molecules exhibited greater binding to class II SLA positive cells than to SLA negative cells. Sera lacking antibodies against class II HLA showed no change in binding regardless of the presence of class II SLA. These antibodies could recognize either SLA-DR or SLA-DQ complexes. CONCLUSIONS: Class II SLA proteins may behave as xenoantigens for people with humoral immunity toward class II HLA molecules.