title: Trial using pig cells with the H-D antigen knocked down.
abstract: OBJECTIVE
  This report describes an attempt to reduce the expression level of Hanganutziu-Deicher (H-D) antigens by small interfering RNA (siRNA) for pig cytidine monophospho-N-acetylneuraminic acid hydroxylase (pCMAH).
  
  
  METHODS
  A pig endothelial cell (PEC) line, and PEC and fibroblasts from an α1,3galactosyltransferase knockout (GalT-KO) piglet were used. Real-time PCR was used to evaluate the degradation of mRNA by siRNA. The H-D antigen was stained, and then the cells were incubated with human serum for the FACS analysis. The extent of lysis in human serum was next calculated using an LDH assay.
  
  
  RESULTS
  Suppression of the mRNA of pCMAH by each siRNA was first determined. The mixture of siRNAs for pCMAH reduced the expressions of the H-D antigen on the PEC and fibroblasts to a considerable extent. The further reduction in the xenoantigenicity for human serum of the GalT-KO cells was then confirmed. In addition, the PEC line showed a significant downregulation in complement-dependent cytotoxicity by the siRNAs, thus indicating that the anti-H-D antigen in human serum is capable of causing lysis of the pig cells.
  
  
  CONCLUSIONS
  pCMAH silencing by siRNA reduced the expression of the H-D antigen and its antigenicity, thus confirming that the H-D antigen is one of the major non-Gal antigens in this situation.
authors.affiliation: Division of Organ Transplantation (E9), Department of Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka, 565-0871, Japan.
authors.forename: Aki
authors.initials: A
authors.lastname: Yamamoto
authors.forename: Kosuke
authors.initials: K
authors.lastname: Ikeda
authors.forename: Dandan
authors.initials: D
authors.lastname: Wang
authors.forename: Shino
authors.initials: S
authors.lastname: Nakatsu
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Takama
authors.forename: Takehisa
authors.initials: T
authors.lastname: Ueno
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nagashima
authors.forename: Akihiro
authors.initials: A
authors.lastname: Kondo
authors.forename: Masahiro
authors.initials: M
authors.lastname: Fukuzawa
authors.forename: Shuji
authors.initials: S
authors.lastname: Miyagawa
mesh_terms: D000818:Animals; D015478:Antigens, Heterophile / Q000378:metabolism*; D002460:Cell Line; D042783:Endothelial Cells / Q000276:immunology*; D005347:Fibroblasts / Q000276:immunology; D005700:Galactosyltransferases / Q000235:genetics; D055785:Gene Knockdown Techniques / Q000379:methods*; D020868:Gene Silencing; D006801:Humans; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000502:physiology; D013552:Swine; D014183:Transplantation, Heterologous
chemical_list: D015478:Antigens, Heterophile; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D005700:Galactosyltransferases