Browsing by Author "Estrada, Jose L."

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    Erythrocytes from GGTA1/CMAH knockout pigs: implications for xenotransfusion and testing in non-human primates
    (Wiley, 2014-07) Wang, Zheng-Yu; Burlak, Christopher; Estrada, Jose L.; Li, Ping; Tector, Matthew F.; Tector, A. Joseph; Department of Surgery, IU School of Medicine
    Background Pig erythrocytes are potentially useful to solve the worldwide shortage of human blood for transfusion. Domestic pig erythrocytes, however, express antigens that are bound by human preformed antibodies. Advances in genetic engineering have made it possible to rapidly knock out the genes of multiple xenoantigens, namely galactose α1,3 galactose (aGal) and N-glycolylneuraminic acid (Neu5Gc). We have recently targeted the GGTA1 and CMAH genes with zinc finger endonucleases resulting in double knockout pigs that no longer express aGal or Neu5Gc and attract significantly fewer human antibodies. In this study, we characterized erythrocytes from domestic and genetically modified pigs, baboons, chimpanzees, and humans for binding of human and baboon natural antibody, and complement mediated lysis. Methods Distribution of anti Neu5Gc IgG and IgM in pooled human AB serum was analyzed by ELISA. Erythrocytes from domestic pigs (Dom), aGal knockout pigs (GGTA1 KO), aGal and Neu5Gc double knockout pigs (GGTA1/CMAH KO), baboons, chimpanzees, and humans were analyzed by flow cytometry for aGal and Neu5Gc expression. In vitro comparative analysis of erythrocytes was conducted with pooled human AB serum and baboon serum. Total antibody binding was accessed by hemagglutination; complement-dependent lysis was measured by hemolytic assay; IgG or IgM binding to erythrocytes was characterized by flow cytometry. Results The pooled human AB serum contained 0.38 μg/ml anti Neu5Gc IgG and 0.085 μg/ml anti Neu5Gc IgM. Both Gal and Neu5Gc were not detectable on GGTA1/CMAH KO erythrocytes. Hemagglutinaion of GGTA1/CMAH KO erythrocytes with human serum was 3.5-fold lower compared to GGTA1 KO erythrocytes, but 1.6-fold greater when agglutinated with baboon serum. Hemolysis of GGTA1/CMAH KO erythrocytes by human serum (25%) was reduced 9-fold compared to GGTA1 KO erythrocytes, but increased 1.64-fold by baboon serum. Human IgG binding was reduced 27-fold on GGTA1/CMAH KO erythrocytes compared to GGTA1 KO erythrocytes, but markedly increased 3-fold by baboon serum IgG. Human IgM binding was decreased 227-fold on GGTA1/CMAH KO erythrocytes compared to GGTA1 KO erythrocytes, but enhanced 5-fold by baboon serum IgM. Conclusions Removal of aGal and Neu5Gc antigens from pig erythrocytes significantly reduced human preformed antibody-mediated cytotoxicity but may have complicated future in vivo analysis by enhancing reactivity from baboons. The creation of the GGTA1/CMAH KO pig has provided the xenotransplantion researcher with organs and cells that attract fewer human antibodies than baboon and our closest primate relative, chimpanzee. These finding suggest that while GGTA1/CMAH KO erythrocytes may be useful for human transfusions, in vivo testing in the baboon may not provide a direct transplation to the clinic.
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    N-linked glycan profiling of GGTA1/CMAH knockout pigs identifies new potential carbohydrate xenoantigens
    (Wiley Online Library, 2015-10) Burlak, Christopher; Bern, Marshall; Brito, Alejandro E.; Isailovic, Dragan; Wang, Zheng-Yu; Estrada, Jose L.; Li, Ping; Tector, A. Joseph; Department of Surgery, IU School of Medicine
    BACKGROUND: The temporary or long-term xenotransplantation of pig organs into people would save thousands of lives each year if not for the robust human antibody response to pig carbohydrates. Genetically engineered pigs deficient in galactose α1,3 galactose (gene modified: GGTA1) and N-glycolylneuraminic acid (gene modified: CMAH) have significantly improved cell survival when challenged by human antibody and complement in vitro. There remains, however, a significant portion of human antibody binding. METHODS: To uncover additional xenoantigens, we compared the asparagine-linked (N-linked) glycome from serum proteins of humans, domestic pigs, GGTA1 knockout pigs, and GGTA1/CMAH knockout pigs using mass spectrometry. Carbohydrate structures were determined with assistance from GlycoWorkbench, Cartoonist, and SimGlycan software by comparison to existing database entries and collision-induced dissociation fragmentation data. RESULTS: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of reduced and solid-phase permethylated glycans resulted in the detection of high-mannose, hybrid, and complex type N-linked glycans in the 1000-4500 m/z ion range. GGTA1/CMAH knockout pig samples had increased relative amounts of high-mannose, incomplete, and xylosylated N-linked glycans. All pig samples had significantly higher amounts of core and possibly antennae fucosylation. CONCLUSIONS: We provide for the first time a comparison of the serum protein glycomes of the human, domestic pig, and genetically modified pigs important to xenotransplantation.
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    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 Medicine
    Background 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.
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    Reduced human platelet uptake by pig livers deficient in the asialoglycoprotein receptor 1 protein
    (Wiley, 2015-05) Paris, Leela L.; Estrada, Jose L.; Li, Ping; Blankenship, Ross L.; Sidner, Richard A.; Reyes, Luz M.; Montgomery, Jessica B.; Burlak, Christopher; Butler, James R.; Downey, Susan M.; Wang, Zheng-Yu; Tector, Matthew; Tector, A. Joseph; Surgery, School of Medicine
    BACKGROUND: The lethal thrombocytopenia that accompanies liver xenotransplantation is a barrier to clinical application. Human platelets are bound by the asialoglycoprotein receptor (ASGR) on pig sinusoidal endothelial cells and phagocytosed. Inactivation of the ASGR1 gene in donor pigs may prevent xenotransplantation-induced thrombocytopenia. METHODS: Transcription activator-like effector nucleases (TALENs) were targeted to the ASGR1 gene in pig liver-derived cells. ASGR1 deficient pig cells were used for somatic cell nuclear transfer (SCNT). ASGR1 knock out (ASGR1-/-) fetal fibroblasts were used to produce healthy ASGR1 knock out piglets. Human platelet uptake was measured in ASGR1+/+ and ASGR1-/- livers. RESULTS: Targeted disruption of the ASGR1 gene with TALENs eliminated expression of the receptor. ASGR1-/- livers phagocytosed fewer human platelets than domestic porcine livers during perfusion. CONCLUSIONS: The use of TALENs in liver-derived cells followed by SCNT enabled the production of healthy homozygous ASGR1 knock out pigs. Livers from ASGR1-/- pigs exhibit decreased human platelet uptake. Deletion of the ASGR1 gene is a viable strategy to diminish platelet destruction in pig-to-human xenotransplantation.
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    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 Medicine
    BACKGROUND: 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.