Shuji Miyagawa

The mechanism of discordant xenograft rejection.

The mechanism of discordant xenograft rejection using the guinea pig-to-rat heart graft model was studied. In this model, we found that (A) Rejection occurred rapidly, in 17.5 +/- 8.3 min (mean +/- SD) (n = 8). (B) The graft survived longer when the recipient rat was pretreated with cobra venom facter (CVF)

Complete Loss of Ndel1 Results in Neuronal Migration Defects and Early Embryonic Lethality

ABSTRACT Regulation of cytoplasmic dynein and microtubule dynamics is crucial for both mitotic cell division and neuronal migration. NDEL1 was identified as a protein interacting with LIS1, the protein product of a gene mutated in the lissencephaly. To elucidate NDEL1 function in vivo, we generated null and hypomorphic alleles of Ndel1 in mice by targeted gene disruption. Ndel1 −/− mice were embryonic lethal at the peri-implantation stage like null mutants of Lis1 and cytoplasmic dynein heavy chain. In addition, Ndel1 −/− blastocysts failed to grow in culture and exhibited a cell proliferation defect in inner cell mass. Although Ndel1 +/− mice displayed no obvious phenotypes, further reduction of NDEL1 by making null/hypomorph compound heterozygotes (Ndel1 cko/− ) resulted in histological defects consistent with mild neuronal migration defects. Double Lis1 cko/+ -Ndel1 +/− mice or Lis1 +/− -Ndel1 +/− mice displayed more severe neuronal migration defects than Lis1 cko/+ -Ndel1 +/ + mice or Lis1 +/− -Ndel1 +/+ mice, respectively. We demonstrated distinct abnormalities in microtubule organization and similar defects in the distribution of β-COP-positive vesicles (to assess dynein function) between Ndel1 or Lis1-null MEFs, as well as similar neuronal migration defects in Ndel1- or Lis1-null granule cells. Rescue of these defects in mouse embryonic fibroblasts and granule cells by overexpressing LIS1, NDEL1, or NDE1 suggest that NDEL1, LIS1, and NDE1 act in a common pathway to regulate dynein but each has distinct roles in the regulation of microtubule organization and neuronal migration.

EFFECTS OF TRANSFECTED COMPLEMENT REGULATORY PROTEINS, MCP, DAF, AND MCP/DAF HYBRID, ON COMPLEMENT-MEDIATED SWINE ENDOTHELIAL CELL LYSIS

We established several swine endothelial cell (SEC) lines, expressing human MCP (CD46), DAF (CD55), and MCP/DAF hybrid by transfection of cDNA, and assessed the function of these transfectant molecules on complement-mediated cell lysis as an in vitro hyper-acute rejection model of swine to human discordant xenograft. Discordant organ xenografts are hyper-acutely rejected by complement activation. Amelioration of complement-mediated lysis by these transfectant molecules was tested in each SEC line by lactate dehydrogenase assay. Naive swine endothelial cells were markedly damaged by human complement mainly via the classical pathway, activating only minimally the alternative pathway of human complement. Both MCP and DAF protected SEC from human complement attack in parallel with the expression density, with DAF being more effective than MCP. The MCP/DAF hybrid was more effective than MCP alone, and as effective as DAF in this system. The results suggest that the transfection of DAF or the MCP/DAF hybrid cDNA into organs to be transplanted could protect against hyperacute rejection.

Remodeling of the Major Pig Xenoantigen by N-Acetylglucosaminyltransferase III in Transgenic Pig

We have been successful in generating several lines of transgenic mice and pigs that contain the human β-d-mannoside β-1,4-N-acetylglucosaminyltransferase III (GnT-III) gene. The overexpression of the GnT-III gene in mice and pigs reduced their antigenicity to human natural antibodies, especially the Galα1–3Galβ1–4GlcNAc-R, as evidenced by immunohistochemical analysis. Endothelial cell studies from the GnT-III transgenic pigs also revealed a significant down-regulation in antigenicity, including Hanganutziu-Deicher antigen, and dramatic reductions in both the complement- and natural killer cell-mediated pig cell lyses. Changes in the enzymatic activities of other glycosyltransferases, such as α1,3-galactosyltransferase, GnT-IV, and GnT-V, did not support cross-talk between GnT-III and these enzymes in the transgenic animals. In addition, we demonstrated the effect of GnT-III in down-regulating the xenoantigen of pig heart grafts, using a pig to cynomolgus monkey transplantation model, suggesting that this approach may be useful in clinical xenotransplantation in the future.

A study of the xenoantigenicity of adult pig islets cells.

Abstract:  Background:  The pig pancreas is considered to be the most suitable source of islets for xenotransplantation into patients with type I diabetes. The purpose of this study was to assess the antigenicity of pig islets, including the Galα1‐3Galβ1‐4GlcNAc‐R (the α‐Gal) and Hanganutziu‐Deicher (H‐D) antigens, and the pathway involved in human complement activation.

Prolonging discordant xenograft survival with anticomplement reagents K76COOH and FUT175.

The guinea pig heart, when transplanted into the rat heterotopically, is rejected within 30 min via activation of the alternative complement pathway. Natural antibody does not contribute to rejection. This xenotransplantation model was used to assess the effect of anti-complement reagents on discordant xenograft survival. In vivo administration of K76COOH (K76) to rats induced only slight suppression of factors B and D and a marked decrease of C3, leading to the depression of ACH50 (reflecting the potency of the alternative pathway). On the other hand, FUT175 (FUT) reduced C3 activity by about 80% and inhibited factor B activity nearly 100% < 1 hr after the administration, but inhibited factor D activity only marginally. FUT abrogated ACH50 for > 6 hr. Of note, the xenograft beating time was prolonged approximately 3 times by FUT but not by K76, suggesting that direct inhibition of plasma serine protease factor B results in the complete suppression of ACH50 and graft survival. The administration of both K76 and FUT resulted in the longest graft survival, but the effects of these reagents were abolished by additional antigraft antibody. Anticomplement reagents that block factor B and C3 are therefore effective for prolongation of discordant xenograft survival when the graft rejection is associated with the complement alternative pathway.

Survival of adult islet grafts from transgenic pigs with N-acetylglucosaminyltransferase-III (GnT-III) in cynomolgus monkeys.

Abstract:  Background:  Because of a severe shortage of human donor pancreases, pig islets are considered to be an attractive donor source. Our previous in vitro study revealed that adult pig islets have strong non‐Galα1‐3Galβ1‐4GlcNAc‐R (α‐Gal) antigenicity, including the Hanganutziu‐Deicher (H‐D) antigen, especially in N‐linked sugars. In this study, the issue of whether islets from N‐acetylglucosaminyltransferase‐III (GnT‐III) transgenic pigs can prolong their survival in cynomolgus monkeys was examined.

Production of alpha 1,3‐Galactosyltransferase gene‐deficient pigs by somatic cell nuclear transfer: A novel selection method for gal alpha 1,3‐Gal antigen‐deficient cells

The objective of the present study was to isolate alpha 1,3‐galactosyltransferase (GalGT)‐gene double knockout (DKO) cells using a novel simple method of cell selection method. To obtain GalGT‐DKO cells, GalGT‐gene single knockout (SKO) fetal fibroblast cells were cultured for three to nine passages and GalGT‐null cells were separated using a biotin‐labeled IB4 lectin attached to streptavidin‐coated magnetic beads. After 15–17 days of additional cultivation, seven GalGT‐DKO cell colonies were obtained from a total of 2.5 × 107 GalGT‐SKO cells. A total of 926 somatic nuclear transferred embryos reconstructed with the DKO cells were transferred into eight recipient pigs, producing four farrowed, three liveborns, and six stillborns. Absence of GalGT gene in the cloned pigs was confirmed by PCR and Southern blotting. Flow cytometric analysis revealed that αGal antigens were not present in the cells of the cloned DKO pigs. Mol. Reprod. Dev. 75: 1372–1378, 2008.

Evidence That Graft Coronary Arteriosclerosis Begins In The Early Phase After Transplantation And Progresses Without Chronic Immunoreaction: Histopathological Analysis Using a Retransplantation Model

The histopathological features of chronic rejection and its initiation were assessed using rat heterotopic heart transplantation and retransplantation models. Fully allogeneic or minor, non-MHC antigen-mismatch heart grafts transplanted into recipient rats treated with a short course of FK506 showed long-term survival but developed graft atherosclerosis after 40 days posttransplantation. Retransplantation of allografts back into the original donor strain did not prevent graft atherosclerosis if the grafts had resided in the primary recipient for up to 5 days; residence in the primary allogeneic recipient for less than 4 days did not result in graft atherosclerosis in the secondary recipient. Short-course administration of FK506 did not affect the production of these changes. Graft coronary arteriosclerosis begins between 3 and 5 days posttransplantation and progresses without continuous allogeneic immunological drive. The present findings will provide a new means by which to approach the analysis of development of chronic allograft rejection.

Complement regulation in the GalT KO era

Miyagawa S, Yamamoto A, Matsunami K, Wang D, Takama Y, Ueno T, Okabe M, Nagashima H, Fukuzawa M. Complement regulation in the GalT KO era.
Xenotransplantation 2010; 17: 11–25.