Tamotsu Shigehisa

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.

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.

In Vitro and in Vivo Anti-platelet Effects of Enzymatic Hydrolysates of Collagen and Collagen-related Peptides

Collagen-related peptides, Gly-Pro-Arg and its analogues, were examined for their inhibitory effects on platelet aggregation induced by the addition of ADP. Human platelet aggregation was suppressed by more than 50% with each of Gly-Pro-Arg and such Gly-Pro-Arg-containing peptides as Gly-Pro-Arg-Gly, Gly-Pro-Arg-Gly-Pro, Gly-Pro-Arg-Pro-Pro, and Gly-Pro-Arg-Pro-Pro-Pro at a concentration of 0.3 mm. The inhibitory effects of these peptides were about 10 times higher in human PRP than in rat PRP. Other Gly-Pro-Arg analogues such as Sar-Pro-Arg, Gly-Pro-Lys, Gly-Ala-Arg, and Ala-Gly-Pro-Arg had no inhibitory effect at a concentration from 0.1 to 0.8 mm even in human PRP. Intravenous and oral administrations of Gly-Pro-Arg and enzymatic hydrolysates of collagen suppressed the decrease in platelet count for endotoxin-induced DIC in rats. Collagen itself has been regarded as a potent inducer of platelet aggregation, but these findings suggest that collagen-related peptides and enzymatic hydrolysates of collagen prevent platelet aggregation.