Genetically engineered pig red blood cells for clinical transfusion: initial in vitro studies

Long, Cassandra; Hara, Hidetaka; Pawlikowski, Zachary; Koike, Naoko; D'Arville, Thomas; Yeh, Peter C.; Ezzelarab, Mohamed; Ayares, David; Yazer, Mark H.; Cooper, David K. C.

doi:10.1111/j.1537-2995.2009.02306.x

Cited by 31 publications

(50 citation statements)

References 40 publications

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“…As our group has previously reported [23, 24], both Gal and Neu5Gc were expressed on wild-type pig RBCs and AECs, but hCD46 was not (Fig 1). RBCs and AECs from GTKO/CD46 pigs expressed Neu5Gc, but not Gal; the AECs expressed hCD46, but RBCs did not (as the transgene is not expressed in RBCs as they have no nuclei).…”

Section: Resultssupporting

confidence: 71%

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Anti-Neu5Gc and anti-non-Neu5Gc antibodies in healthy humans

et al. 2017

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Our group previously investigated the levels of anti-Gal and anti-nonGal IgM and IgG in a cohort of 75 healthy humans of various backgrounds, and found some significant differences related to factors such as age, gender, ABO blood group, diet, vaccination history, and geographic location during childhood. We have now expanded our cohort (n = 84) to investigate the levels of anti-Neu5Gc and anti-nonGal/nonNeu5Gc antibodies in healthy humans. Anti-nonGal and anti-nonGal/nonNeu5Gc human IgM and IgG binding to pRBCs and pAECs from GTKO/CD46 and GTKO/CD46/Neu5GcKO pigs were measured by flow cytometry. Anti-Gal and anti-Neu5Gc IgM and IgG levels were measured by ELISA. In summary, (i) the great majority (almost 100%) of humans had anti-Neu5Gc IgM and IgG antibodies that bound to pAECs and approximately 50% had anti-Neu5Gc antibodies that bound to pRBCs, (ii) there was significantly less human antibody binding to pig cells that did not express either Gal or Neu5Gc compared with those that did not express Gal alone, (iii) the levels of both IgM and IgG binding to GTKO/CD46/Neu5GcKO pRBCs and pAECs were low, (iv) the level of anti-Neu5Gc IgG was higher in men than women, (v) the level did not change with age or diet, and there was some variability associated with (vi) previous vaccination history and (vii) the geographic region in which the individual spent his or her childhood. Our study confirms that human antibody binding to RBCs and AECs from GTKO/CD46/Neu5GcKO pigs is greatly reduced compared to binding to GTKO/CD46 cells. However, all humans appear to have a low level of antibody that binds to pAECs that is not directed to either Gal or Neu5Gc. Our findings require consideration in planning clinical trials of xenotransplantation.

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Section: Resultssupporting

confidence: 71%

“…RBCs (which express carbohydrate antigens but do not express swine leukocyte antigens [SLA]) were isolated as previously described [24]. Briefly, RBCs were separated from whole blood, washed x3 with phosphate-buffered saline (PBS, Invitrogen, Carlsbad, CA), and centrifuged at 700 g for 5min at 4°C.…”

Section: Methodsmentioning

confidence: 99%

Anti-Neu5Gc and anti-non-Neu5Gc antibodies in healthy humans

et al. 2017

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“…Most early studies have been focused on antibody-mediated destruction/rejection of xenogeneic RBCs, which involve complement-mediated cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC). A recent study showed that RBCs from α1,3GalT KO pigs are significantly less susceptible than those from wild-type pigs to antibody-dependent complement-mediated cytotoxicity and ADCC by human macrophages [38], indicating that α1,3Gal is an important antigen causing destruction of porcine RBCs. However, depletion of α1,3Gal is not enough to overcome rejection, and α1,3GalT KO porcine RBCs were still rapidly and vigorously rejected after infusion into baboons [39].…”

Section: Macrophages In the Rejection Of Xenogeneic Red Blood Cellsmentioning

confidence: 99%

Innate cellular immunity and xenotransplantation

Wang¹,

Yang²

2012

Current Opinion in Organ Transplantation

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Purpose of review This review assesses the recent progress in xenograft rejection by innate immune responses, with a focus on innate cellular xenoreactivity. Recent findings Current literature was reviewed for new insights into the role of innate cellular immunity in xenograft rejection. Increasing evidence confirms that vigorous innate immune cell activation is accounted for by a combination of xenoantigen recognition by activating receptors, and incompatibility in inhibitory receptor-ligand interactions. Although both innate humoral and cellular xenoimmune responses are predominantly elicited by preformed and induced xenoreactive antibodies in nonhuman primates following porcine xenotransplantation, innate immune cells can also be activated by xenografts in the absence of antibodies. The latter antibody-independent response will likely persist in recipients even when adaptive xenoimmune responses are suppressed. In addition to xenograft rejection by recipient innate immune cells, phagocytic cells within liver xenografts are also deleterious to recipients by causing thrombocytopenia. Summary Strategies of overcoming innate immune responses are required for successful clinical xenotransplantation. In addition to developing better immunosuppressive and tolerance induction protocols, endeavors towards further genetic modifications of porcine source animals are ultimately important for successful clinical xenotransplantation.

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“…The number of migrated monocytes was assessed using a 96-well monocyte cell migration assay (EMD Millipore, San Diego, CA) based on the Boyen-chamber principle. Human monocytes were isolated from PBMCs, using monocyte isolation kit II (Miltenyl Biotec, Auburn, CA) as previously described 41 . Supernatants obtained from pCECs culture with/without Y27632 after hTNF-α stimulation were collected, and used in the monocytes migration assay.…”

Section: Methodsmentioning

confidence: 99%

Effect of Rho-kinase Inhibitor, Y27632, on Porcine Corneal Endothelial Cell Culture, Inflammation and Immune Regulation

Lee

Miyagawa

Long

et al. 2015

Ocular Immunology and Inflammation

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Purpose To investigate the effect of the Rho-kinase inhibitor, Y27632, on pig corneal endothelial cell (pCEC) culture, and on inflammation and immune regulation of the responses of human cells to pCECs. Methods pCECs were cultured with/without Y27632 to assess cell proliferation and in vitro wound healing assay. The level of MCP-1 and VEGF in pCECs stimulated with human TNF-α were measured. Proliferation of human PBMCs stimulated with pCECs, and cytokine production in human T cells, and monocyte migration after stimulation were investigated. Results Y27632 promoted pCEC proliferation, prevented pCEC death, and enhanced in vitro wound healing. After stimulation, there were significantly lower levels of MCP-1 and VEGF measured in pCECs cultured with Y27632, and significantly reduced human PBMC proliferation, cytokine production, and monocyte migration. Conclusions The application of the Rho-kinase inhibitor will be beneficial when culturing pCECs, and may provide a novel therapy to reduce inflammation after corneal xenotransplantation.

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Genetically engineered pig red blood cells for clinical transfusion: initial in vitro studies

Cited by 31 publications

References 40 publications

Anti-Neu5Gc and anti-non-Neu5Gc antibodies in healthy humans

Anti-Neu5Gc and anti-non-Neu5Gc antibodies in healthy humans

Innate cellular immunity and xenotransplantation

Effect of Rho-kinase Inhibitor, Y27632, on Porcine Corneal Endothelial Cell Culture, Inflammation and Immune Regulation

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