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Effect of intravenous immunoglobulin (IVIg) on primate complement-dependent cytotoxicity of genetically engineered pig cells: relevance to clinical xenotransplantation.

ABSTRACT: Triple-knockout (TKO) pigs may be ideal sources of organs for clinical xenotransplantation because many humans have no preformed antibody to TKO pig cells. Intravenous immunoglobulin (IVIg) is widely used for severe infection or the treatment/prevention of antibody-mediated rejection in allotransplantation. Anti-pig antibodies in IVIg could be harmful in clinical xenotransplantation. It is unknown whether anti-TKO pig antibodies are present in IVIg. The main aim of this study was to investigate in vitro whether IVIg contains anti-TKO pig antibodies with cytotoxic effect to pig cells. Undiluted pooled human serum (HS) and five different commercial preparations of IVIg were tested for IgM and IgG binding to red blood cells (RBCs) from wild-type (WT), ?1,3-galactosyltransferase gene-knockout (GTKO), and TKO pigs by flow cytometry. Complement-dependent lysis of IVIg against these pig pRBCs was measured by hemolytic assay. Pooled HS and 4 of 5 IVIg commercial preparations contained anti-pig IgG that bound to WT and GTKO pRBCs, but not to TKO pRBCs. One preparation of IVIg contained antibodies that bound to TKO pRBCs, but there was no cytotoxicity of IVIg to TKO pRBCs. The results suggest that IVIg administration to human recipients of TKO pig grafts would be safe. However, the specific preparation of IVIg would need to be screened before its administration.

SUBMITTER: Yamamoto T

PROVIDER: S-EPMC7367287 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Effect of intravenous immunoglobulin (IVIg) on primate complement-dependent cytotoxicity of genetically engineered pig cells: relevance to clinical xenotransplantation.

Yamamoto Takayuki T Cui Yehua Y Patel Diyan D Jagdale Abhijit A Iwase Hayato H Ayares David D Cooper David K C DKC Hara Hidetaka H

Scientific reports 20200716 1

Triple-knockout (TKO) pigs may be ideal sources of organs for clinical xenotransplantation because many humans have no preformed antibody to TKO pig cells. Intravenous immunoglobulin (IVIg) is widely used for severe infection or the treatment/prevention of antibody-mediated rejection in allotransplantation. Anti-pig antibodies in IVIg could be harmful in clinical xenotransplantation. It is unknown whether anti-TKO pig antibodies are present in IVIg. The main aim of this study was to investigate ...[more]

PMID: 32678137

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Project description:Kidney xenotransplantation is expected to contribute to resolving the shortage of kidneys from deceased human donors. Although progress in experimental life-supporting pig renal xenotransplantation has been encouraging, there are still issues to be considered before a clinical trial can be initiated. We attempted to clarify some of these by an in vitro study. Blood was drawn from healthy volunteers (Volunteers, n=20), patients with end-stage renal disease (ESRD, n=20) pre-operation (Pre), and on Day 1 (POD 1) and Day 14 (POD 14) after renal allotransplantation, brain-dead organ donors (DBD, n=20), and renal allotransplant recipients who were currently experiencing T cell-mediated rejection (Allo-TCMR, n=20). Serum IgM/IgG binding to, and complement-dependent cytotoxicity (CDC) of, PBMCs and RBCs from (a) wild-type (WT), (b) α1,3-galactosyltransferase gene-knockout (GTKO), (c) GTKO/beta-1,4-N-acety1 galactosaminyltransferase 2-knockout (GTKO/β4GalNT2KO), (d) GTKO/cytidine monophosphate-N-acetylneuraminic acid hydroxylase-knockout (GTKO/CMAHKO), and (e) GTKO/β4GalNT2KO/CMAHKO/hCD55 (TKO/hCD55) pigs were measured by flow cytometry. We obtained the following results: (i) Serum IgM/IgG binding and CDC in Volunteers were significantly greater to WT, GTKO, and GTKO/β4GalNT2KO PBMCs or RBCs than to GTKO/CMAHKO and TKO/hCD55 cells; (ii) ESRD, DBD, and Allo-TCMR serum antibody binding and CDC to WT pig PBMCs were significantly greater than to GTKO, GTKO/β4GalNT2KO, GTKO/CMAHKO, and TKO/hCD55 cells; (iii) antibody binding to GTKO/CMAHKO pig cells was significantly lower in hemodialysis than peritoneal dialysis patients. (iv) Two of twenty allotransplantation recipients' serum IgG binding to GTKO pig PBMCs increased on POD14 compared with Pre, but IgG binding to GTKO pig RBCs did not; (v) In all sera, the lowest antibody binding and CDC were to GTKO/CMAHKO and TKO/CD55 pig cells. We conclude (i) CMAHKO in the pig may be critical to the success of clinical pig kidney xenotransplantation, and may be the most important after GTKO, at least in Chinese patients; (ii) subjects with ESRD, or who are immunosuppressed after kidney allotransplantation, and DBD, have lower levels of antibody binding and CDC to genetically-engineered pig cells than do volunteers; (iii) TKO pigs with selected human 'protective' transgenes, e.g., CD55, are likely to prove to be the optimal sources of kidneys for clinical xenotransplantation.

Project description:BackgroundIschemic stroke causes a high rate of deaths and permanent neurological damage in survivors. Ischemic stroke triggers the release of damage-associated molecular patterns (DAMPs) such as high-mobility group box 1 (HMGB1), which activate toll-like receptors (TLRs) and receptor for advanced glycation endproducts (RAGE) in the affected area, leading to an exaggerated inflammatory response and cell death. Both TLRs and RAGE are transmembrane pattern recognition receptors (PRRs) that have been shown to contribute to ischemic stroke-induced brain injury. Intravenous immunoglobulin (IVIg) preparations obtained by fractionating human blood plasma are increasingly being used as an effective therapeutic agent in the treatment of several inflammatory diseases. Its use as a potential therapeutic agent for treatment of stroke has been proposed, but little is known about the direct neuroprotective mechanisms of IVIg. We therefore investigate whether IVIg exerts its beneficial effects on the outcome of neuronal injury by modulating HMGB1-induced TLR and RAGE expressions and activations.MethodsPrimary cortical neurons were subjected to glucose deprivation or oxygen and glucose deprivation conditions and treated with IVIg and recombinant HMGB1. C57/BL6J mice were subjected to middle cerebral artery occlusion, followed by reperfusion, and IVIg was administered intravenously 3 h after the start of reperfusion. Expression of TLRs, RAGE and downstream signalling proteins in neurons and brain tissues were evaluated by immunoblot.ResultsTreatment of cultured neurons with IVIg reduced simulated ischemia-induced TLR2, TLR4, TLR8 and RAGE expressions, pro-apoptotic caspase-3 cleavage and phosphorylation of the cell death-associated kinases such as c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK) as well as the p65 subunit of nuclear factor kappa B (NF-κB). These results were recapitulated in an in vivo model of stroke. IVIg treatment also upregulated the anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) in cortical neurons under ischemic conditions. Finally, IVIg protected neurons against HMGB1-induced neuronal cell death by modulating TLR and RAGE expressions and signalling pathways.ConclusionsTaken together, these results provide a rationale for the potential use of IVIg to target inappropriately activated components of the innate immune system following ischemic stroke.

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Effect of intravenous immunoglobulin (IVIg) on primate complement-dependent cytotoxicity of genetically engineered pig cells: relevance to clinical xenotransplantation.

Publications

Effect of intravenous immunoglobulin (IVIg) on primate complement-dependent cytotoxicity of genetically engineered pig cells: relevance to clinical xenotransplantation.

Similar Datasets

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