Browsing by Subject "Xenotransplantation"

Now showing 1 - 10 of 11
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    Blocking porcine sialoadhesin improves extracorporeal porcine liver xenoperfusion with human blood
    (Wiley, 2013-07) Waldman, Joshua P.; Vogel, Thomas; Burlak, Christopher; Coussios, Constantin; Dominguez, Javier; Friend, Peter; Rees, Michael A.; Surgery, School of Medicine
    BACKGROUND: Patients in fulminant hepatic failure currently do not have a temporary means of support while awaiting liver transplantation. A potential therapeutic approach for such patients is the use of extracorporeal perfusion with porcine livers as a form of "liver dialysis". During a 72-h extracorporeal perfusion of porcine livers with human blood, porcine Kupffer cells bind to and phagocytose human red blood cells (hRBC) causing the hematocrit to decrease to 2.5% of the original value. Our laboratory has identified porcine sialoadhesin expressed on Kupffer cells as the lectin responsible for binding N-acetylneuraminic acid on the surface of the hRBC. We evaluated whether blocking porcine sialoadhesin prevents the recognition and subsequent destruction of hRBCs seen during extracorporeal porcine liver xenoperfusion. METHODS: Ex vivo studies were performed using wild type pig livers perfused with isolated hRBCs for 72-h in the presence of an anti-porcine sialoadhesin antibody or isotype control. RESULTS: The addition of an anti-porcine sialoadhesin antibody to an extracorporeal porcine liver xenoperfusion model reduces the loss of hRBC over a 72-h period. Sustained liver function was demonstrated throughout the perfusion. CONCLUSIONS: This study illustrates the role of sialoadhesin in mediating the destruction of hRBCs in an extracorporeal porcine liver xenoperfusion model.
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    Co-expression of HLA-E and HLA-G on genetically modified porcine endothelial cells attenuates human NK cell-mediated degranulation
    (Frontiers Media, 2023-07-17) Cross-Najafi, Arthur A.; Farag, Kristine; Isidan, Abdulkadir; Li, Wei; Zhang, Wenjun; Lin, Zhansong; Walsh, Julia R.; Lopez, Kevin; Park, Yujin; Higgins, Nancy G.; Cooper, David K. C.; Ekser, Burcin; Li, Ping; Surgery, School of Medicine
    Natural killer (NK) cells play an important role in immune rejection in solid organ transplantation. To mitigate human NK cell activation in xenotransplantation, introducing inhibitory ligands on xenografts via genetic engineering of pigs may protect the graft from human NK cell-mediated cytotoxicity and ultimately improve xenograft survival. In this study, non-classical HLA class I molecules HLA-E and HLA-G were introduced in an immortalized porcine liver endothelial cell line with disruption of five genes (GGTA1, CMAH, β4galNT2, SLA-I α chain, and β-2 microglobulin) encoding three major carbohydrate xenoantigens (αGal, Neu5Gc, and Sda) and swine leukocyte antigen class I (SLA-I) molecules. Expression of HLA-E and/or HLA-G on pig cells were confirmed by flow cytometry. Endogenous HLA-G molecules as well as exogenous HLA-G VL9 peptide could dramatically enhance HLA-E expression on transfected pig cells. We found that co-expression of HLA-E and HLA-G on porcine cells led to a significant reduction in human NK cell activation compared to the cells expressing HLA-E or HLA-G alone and the parental cell line. NK cell activation was assessed by analysis of CD107a expression in CD3-CD56+ population gated from human peripheral blood mononuclear cells. CD107a is a sensitive marker of NK cell activation and correlates with NK cell degranulation and cytotoxicity. HLA-E and/or HLA-G on pig cells did not show reactivity to human sera IgG and IgM antibodies. This in vitro study demonstrated that co-expression of HLA-E and HLA-G on genetically modified porcine endothelial cells provided a superior inhibition in human xenoreactive NK cells, which may guide further genetic engineering of pigs to prevent human NK cell mediated rejection.
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    Corrigendum to "The Role of Costimulation Blockade in Solid Organ and Islet Xenotransplantation"
    (Hindawi, 2018-03-22) Samy, Kannan P.; Butler, James R.; Li, Ping; Cooper, David K. C.; Ekser, Burcin; Surgery, School of Medicine
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    Differences in platelet aggregometers to study platelet function and coagulation dysregulation in xenotransplantation
    (Wiley, 2021-01) Isidan, Abdulkadir; Chen, Angela M.; Saglam, Kutay; Yilmaz, Sezai; Zhang, Wenjun; Li, Ping; Ekser, Burcin; Surgery, School of Medicine
    Xenotransplantation (ie, cross-species transplantation) using genetically engineered pig organs could be a limitless source to solve the shortage of organs and tissues worldwide. However, despite prolonged survival in preclinical pig-to-nonhuman primate xenotransplantation trials, interspecies coagulation dysregulation remains to be overcome in order to achieve continuous long-term success. Different platelet aggregometry methods have been previously used to study the coagulation dysregulation with wild-type and genetically engineered pig cells, including the impact of possible treatment options. Among these methods, while thromboelastography and rotational thromboelastometry measure the change in viscoelasticity, optical aggregometry measures the change in opacity. Recently, impedance aggregometry has been used to measure changes in platelet aggregation in electrical conductance, providing more information to our understanding of coagulation dysregulation in xenotransplantation compared to previous methods. The present study reviews the merits and differences of the above-mentioned platelet aggregometers in xenotransplantation research.
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    Expression of NeuGc on Pig Corneas and Its Potential Significance in Pig Corneal Xenotransplantation
    (Wolters Kluwer, 2016-01) Lee, Whayoung; Miyagawa, Yuko; Long, Cassandra; Ekser, Burcin; Walters, Eric; Ramsoondar, Jagdeece; Ayares, David; Tector, A. Joseph; Cooper, David K. C.; Hara, Hidetaka; Department of Surgery, IU School of Medicine
    PURPOSE: Pigs expressing neither galactose-α1,3-galactose (Gal) nor N-glycolylneuraminic acid (NeuGc) take xenotransplantation one step closer to the clinic. Our aims were (1) to document the lack of NeuGc expression on corneas and aortas and cultured endothelial cells [aortic endothelial cells (AECs); corneal (CECs)] of GTKO/NeuGcKO pigs, and (2) to investigate whether the absence of NeuGc reduced human antibody binding to the tissues and cells. METHODS: Wild-type (WT), GTKO, and GTKO/NeuGcKO pigs were used for the study. Human tissues and cultured cells were negative controls. Immunofluorescence staining was performed using anti-Gal and anti-NeuGc antibodies, and human IgM and IgG binding to tissues was determined. Flow cytometric analysis was used to determine Gal and NeuGc expression on cultured CECs and AECs and to measure human IgM/IgG binding to these cells. RESULTS: Both Gal and NeuGc were detected on WT pig corneas and aortas. Although GTKO pigs expressed NeuGc, neither humans nor GTKO/NeuGcKO pigs expressed Gal or NeuGc. Human IgM/IgG binding to corneas and aortas from GTKO and GTKO/NeuGcKO pigs was reduced compared with binding to WT pigs. Human antibody binding to GTKO/NeuGcKO AECs was significantly less than that to GTKO AECs, but there was no significant difference in binding between GTKO and GTKO/NeuGcKO CECs. CONCLUSIONS: The absence of NeuGc on GTKO aortic tissue and AECs is associated with reduced human antibody binding, and possibly will provide a better outcome in clinical xenotransplantation using vascularized organs. For clinical corneal xenotransplantation, the absence of NeuGc expression on GTKO/NeuGcKO pig corneas may not prove an advantage over GTKO corneas.
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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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    Nuclease-based editing in the porcine genome : a strategy to facilitate porcine-to human xenotransplantation
    (2017-04-18) Butler, James R.; Tector, A. Joseph; White, Kenneth E.; Schmidt, C. Max; Radovich, Milan
    Solid organ transplantation is severely limited by a shortage of available donor allografts. Pig-to-human xenotransplantation offers a potential solution to this growing problem. For xenotransplantation to achieve clinical relevance, both immunologic and physiologic barriers must be understood. Genetic modification of pigs has proven to be a valuable means of both studying and eliminating these barriers. The present body of work describes a method for greatly increasing the efficiency and precision of genome editing within the porcine genome. By combining non-integrating selection and homologous recombination of exogenous oligonucleotides, a method for rapidly creating genetic modification without reliance on phenotypic sorting was achieved. Furthermore this work employs the technique of CRISPR/Cas9-directed mutagenesis to create and analyze several new animal models of porcine-to-human xenotransplantation with respect to both immunologic and physiologic parameters. First, Isoglobotrihexosylceramide -a controversial glycan to the field of xenotransplantation- was studied in a knockout model and found not to affect human-anti-porcine humoral reactions. Second, a new combination of glycan modifications is described that significantly lowers the human anti-porcine humoral immune response. This model animal suggests that glycan-deletion alone will be sufficient to promote clinical application, and that conventional immunosuppression will be successful in mediating the human cellular response. Finally, two potential physiologic barriers to xenotransplantation are studied in genetically modified model animals. Xenogenic consumption of human platelets was studied across hepatic and renal organ systems; xenogenic platelet consumption was reduced by glycan modifications to the porcine liver while human platelet sequestration was not identified in the study of renal endothelium. Porcine FcRN –an essential receptor expressed in kidneys to maintain serum proteostasis- was studied as a final potential barrier to pig-to human renal transplantation. Because albumin is the primary driver of serum oncotic pressure, the protein-protein interaction of endogenous porcine FcRN and human albumin was studied. Porcine FcRN was found capable of binding human albumin under physiologic parameters. In summary, the results of the present work suggest that the salient barriers to clinical xenotransplantation have been removed and that porcine-to human renal transplantation may soon offer an answer to the current organ shortage.
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    Porcine UL-16 Binding Protein 1 Is Not a Functional Ligand for the Human Natural Killer Cell Activating Receptor NKG2D
    (MDPI, 2023-11-07) Lopez, Kevin J.; Spence, John Paul; Li, Wei; Zhang, Wenjun; Wei, Barry; Cross-Najafi, Arthur A.; Butler, James R.; Cooper, David K. C.; Ekser, Burcin; Li, Ping; Surgery, School of Medicine
    Natural killer (NK) cells play a vital role in xenotransplantation rejection. One approach to induce NK cell immune tolerance is to prevent the NK cell-mediated direct killing of porcine cells by targeting the interaction of the activating receptor NKG2D and its ligands. However, the identity of porcine ligands for the human NKG2D receptor has remained elusive. Previous studies on porcine UL-16 binding protein 1 (pULBP-1) as a ligand for human NKG2D have yielded contradictory results. The goal of the present study was to clarify the role of pULBP-1 in the immune response and its interaction with human NKG2D receptor. To accomplish this, the CRISPR/Cas9 gene editing tool was employed to disrupt the porcine ULBP-1 gene in a 5-gene knockout porcine endothelial cell line (GGTA1, CMAH, β4galNT2, SLA-I α chain, and β-2 microglobulin, 5GKO). A colony with two allele mutations in pULBP-1 was established as a 6-gene knockout pig cell line (6GKO). We found that pULBP-1-deficient pig cells exhibited a reduced binding capacity to human NKG2D-Fc, a recombinant chimera protein. However, the removal of ULBP-1 from porcine endothelial cells did not significantly impact human NK cell degranulation or cytotoxicity upon stimulation with the pig cells. These findings conclusively demonstrate that pULBP-1 is not a crucial ligand for initiating xenogeneic human NK cell activation.
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    Study of Physiologic and Immunologic Incompatibilities of Pig to Human Transplantation
    (2014) Chihara, Ray K.; Burlak, Christopher; Tector, A. Joseph; Basile, David P.; Tune, Johnathan D.
    Solid organ transplantation is limited by available donor allografts. Pig to human transplantation, xenotransplantation, could potentially solve this problem if physiologic and immunologic incompatibilities are overcome. Genetic modifications of pigs have proven valuable in the study of xenotransplantation by improving pig to human compatibility. More genetic targets must be identified for clinical success. First, this study examines platelet homeostasis incompatibilities leading to acute thrombocytopenia in liver xenotransplantation. Mechanisms for xenogeneic thrombocytopenia were evaluated using liver macrophages, Kupffer cells, leading to identification of CD18, beta-2 integrin, as a potential target for modification. When disruption of CD18 was accomplished, human platelet binding and clearance by pig Kupffer cells was inhibited. Further, human and pig platelet surface carbohydrates were examined demonstrating significant differences in carbohydrates known to be involved with platelet homeostasis. Carbohydrate recognition domains of receptors responsible for platelet clearance Macrophage antigen complex-1 (CD11b/CD18) and Asialoglycoprotein receptor 1 in pigs were found to be different from those in humans, further supporting the involvement of platelet surface carbohydrate differences in xenogeneic thrombocytopenia. Second, immunologic incompatibilities due to antibody recognition of antigens resulting in antibody-mediated rejection were studied. Identification of relevant targets was systematically approached through evaluation of a known xenoantigenic protein fibronectin from genetically modified pigs. N-Glycolylneuraminic acid, a sialic acid not found in humans, was expressed on pig fibronectin and was identified as an antigenic epitope recognized by human IgG. These studies have provided further insight into xenogeneic thrombocytopenia and antibody-mediated rejection, and have identified potential targets to improve pig to human transplant compatibility.