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.
: 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.
Methods:
The expression of α‐Gal on islets from adult pigs was investigated by immunohistochemical staining and flowcytometric analysis. The α1,3 galactosyltransferase (α1,3GT) activity of islets was determined by high‐performance liquid chromatography. Antigenicity to human natural antibodies, including the H‐D antigen of pig islets was next examined by treatment of pig islets with tunicamycin, D‐threo‐1‐phenyl‐2‐decanoylamino‐3‐morpholino‐1‐propanol (PDMP) and/or neuraminidase. In addition, complement‐mediated islets lysis was examined using factor D‐deficient and C1‐deficient sera.
Results: Adult pig islets expressed negligible amounts of α‐Gal epitope, and α1,3GT activity was also undetectable. However, human natural antibodies, immunoglobulin G and M, and the anti H‐D antibody react to the adult islet. Treatment of pig islets with tunicamycin, but not PDMP, led to a drastic reduction in antigenicity to human serum, indicating the importance of N‐linked sugars on the islets. Neuraminidase treatment indicated the presence of, not only the H‐D antigen, but also other sialic acid antigens that reacted with the human natural antibody. The complement deposition of C4, C3 and factor B on islets was demonstrated. The alternative pathway‐mediated pig islet killing accounted for approximately 30% of that by the total complement pathway.
Conclusion: The origin of antigenicity of pig islets is mainly N‐linked sugars including sialic acid antigens, but not the α‐Gal, and pig islets can be injured by both the classical and the alternative complement pathway in human serum.
: Background: The pig pancreas is considered to be the most suitable source of islets for xenotransplantation in patients with type I diabetes. The objective of this study was to assess the ...antigenicity of neonatal porcine islet‐like cell clusters (NPCC), including the Galα1–3Galβ1–4GlcNAc‐R (α‐Gal) and Hanganutziu–Deicher (H–D) antigens, and the pathway involved in human complement activation. The efficiency of expression of human decay‐accelerating factor (DAF: CD55) on NPCC by adenoviral transduction was also examined, and the functional capacity of DAF was also estimated.
Methods: The deposition of human natural antibodies, immunoglobulin (Ig)G and IgM, and the expression of α‐Gal and H–D antigens on NPCC were investigated by FACS analysis. The downregulation in the antigenicity to human natural antibodies, including the α‐Gal and H–D antigens on NPCC by treatment with tunicamycin, PDMP and neuraminidase were also examined. In addition, complement‐mediated islet lysis was examined using factor D‐deficient and C1‐deficient sera. An adenovirus encoding DAF under the control of the cytomegalovirus promoter, Ad.pCMV‐DAF, was then constructed, and used for transducing NPCC. The amelioration of complement‐dependent cytotoxicity of the NPCC by the transduced DAF was assessed as an in vitro hyperacute rejection model of a pig to human xenograft.
Results: The NPCC clearly expressed the α‐Gal epitope, and the human natural antibodies, IgG and IgM, and the anti‐H–D antibody also reacted with the NPCC. Treatment of NPCC with tunicamycin led to a drastic reduction in the extent of deposition of IgG, indicating the importance of N‐linked sugars on the islets, presumably related to α‐Gal expression on N‐linked sugars. Neuraminidase treatment indicated the presence of, not only the H–D antigen, but also other sialic acid antigens which reacted with the human natural antibody, especially IgG. The complement deposition of factor B on NPCC was clear, and the alternative pathway‐mediated NPCC killing accounted for approximately 30% of that by the total complement pathway. On the other hand, approximately 90% of the NPCC could be transduced to express DAF by the adenovector, Ad.pCMV‐DAF. The expressed DAF showed an approximately 50–62% suppression in complement‐dependent NPCC lysis.
Conclusion: The origin of the antigenicity of NPCC is mainly N‐linked sugars including α‐Gal and sialic acid antigens, and NPCC expressed the transduced molecule in high efficiency by the adenovector.
The pig cDNA encoding C1 esterase inhibitor (C1-INH) was isolated and the homology of the sequence was compared with that from other animals. The structure of pig C1-INH contains a two disulfide ...bridge pattern identical to the human C1-INH. In the amino acid sequence of the first Cys-91 to the C-terminal end, the pigC1-INH has a 76.2% homology with the human protein, and the sequence of the reactive site is close to the human. A surface-bound form of pig and human C1-INH, pC1-INH-PI and hC1-INH, respectively, were next constructed. Stable Chinese hamster ovarian tumor (CHO) cell lines and pig endothelial cell (PEC) lines expressing these C1-INH-PI were prepared by transfection. The basic function and the species specificity of pCI-INH were then investigated using these transfectants. pC1-INH and hC1-INH have almost the same suppressive effect on pig, human, dog and rabbit sera in complement-dependent cell lysis, indicating little species specificity.
: Background: It is difficult to produce a transgenic animal with high expression of decay‐accelerating factor (CD55: DAF) or other molecules. The purpose of this study was to assess the effect of ...tandem forms of DAF on a xenogeneic cell membrane against human complement.
Methods: cDNAs of the delta‐Short Consensus Repeat (SCR) 1‐DAF, the double‐DAF, the triple‐DAF, and the tetra‐DAF with a FLAG‐tag were established. Chinese hamster ovary (CHO) cell lines and a pig endothelial cell (PEC) line expressing these molecules were established. The amelioration of complement‐mediated lysis by the transfectant molecules on these cells was examined. The CHO cell transfectants were also incubated with normal human serum, and the amount of C3 deposited was determined by FACS analysis.
Results: Stable CHO cells and PEC transfectants, in which each molecule was clearly expressed, and Western blots showed that each band corresponded to the expected molecular weight. The extent of amelioration of complement‐mediated lysis by these four molecules was then examined. A clear tendency was found, as follows: The higher the tandem number of DAF, the greater was the effect on cytotoxicity. Additional experiments focusing on triple‐DAF and tetra‐DAF did not indicate any significant difference in complement‐mediated lysis. Consistent with the complement‐regulatory ability, the inhibitory effect of the deposition of C3 fragments by these molecules was closely related to the degree of amelioration.
Conclusion: These data indicate that tandem DAF, especially a triple‐DAF, is a very effective form for protecting against complement activation.
To suppress C3 fragment deposition in the classical pathway complement activation on xenogeneic membranes, decay accelerating factor (DAF) was the most effective molecule among the complement ...regulatory proteins (CRPs) used in the present study. C3 fragment deposition was closely related to subsequent xenogeneic cell lysis. However, other molecules were also very effective in different ways and include phosphatidylinositol (PI)-anchored short consensus repeat (SCR) 2–4 of membrane cofactor protein (MCP-PI), PI-anchored C1 esterase inhibitor (C1-INH-PI), and PI-anchored SCR8–11 of complement receptor type 1 (CR1-PI). On the other hand, regarding a strategy for downregulating C4 fragment deposition, the use of only C1-INH-PI and PI-anchored SCR1–3 of the C4b-binding protein (C4bp-PI) was found to be effective.
PDF
