title: Adenovirus-mediated gene transfer of triple human complement regulating proteins (DAF, MCP and CD59) in the xenogeneic porcine-to-human transplantation model. Part I: in vitro assays using porcine aortic endothelial cells.
abstract: We assessed whether the adenovirus-mediated gene transfer of triple human complement regulating proteins (hCRPs) to the porcine aortic endothelium (PAE), could possibly exert a synergistic effect to inhibit human complement activation. Adenovirus vectors, encoding E.Coli beta-galactosidase (AxCALacZ), human membrane cofactor protein (MCP) (AxCAMCP), decay-accelerating factor (DAF) (AxCADAF), and CD59 (AxCACD59) were produced by the COS-TPC method. AxCALacZ was transfected to porcine aortic endothelium cells (PAECs) under various multiplicities of infection (MOI) to determine the efficiency of adenovirus-mediated gene transfer by 5-bromo-4-chloro-3-indolyl beta- D-galactopyranoside (X-gal) staining. The mRNA expressions of transfected CRPs were examined by reverse transcriptase-polymerase chain reaction (RT-PCR). Cellular damage to the PAEC was assessed by an MTT assay. PAEC was most efficiently transfected with the LacZ gene at 10(3) MOI/60-min incubation time (89.1%). In all samples transfected with the CRP gene, the corresponding mRNAs were detected in the RT-PCR. In the MTT assay, PAECs co-cultured with 20% human serum, showed the highest cellular viability after gene transfer of triple CRPs (117.7%), when compared with those of marker LacZ, single or double CRPs. The adenovirus-mediated multiple gene transfer of CRPs may thus be an efficient method for suppressing complement activation in the porcine-to-human model of hyperacute rejection.
authors.affiliation: First Department of Surgery, University of Ryukyus School of Medicine, Uehara 207, Nishihara-cho, Okinawa 903-0215, Japan.
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Nagahama
authors.forename: Masayuki
authors.initials: M
authors.lastname: Shiraishi
authors.forename: Takashi
authors.initials: T
authors.lastname: Oshiro
authors.forename: Kaoru
authors.initials: K
authors.lastname: Taira
authors.forename: Hideki
authors.initials: H
authors.lastname: Sugawa
authors.forename: Eiji
authors.initials: E
authors.lastname: Nozato
authors.forename: Hironori
authors.initials: H
authors.lastname: Nomura
authors.forename: Masaru
authors.initials: M
authors.lastname: Nagamine
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Muto
mesh_terms: D000256:Adenoviridae; D000818:Animals; D000910:Antibodies, Heterophile / Q000032:analysis; D015703:Antigens, CD / Q000235:genetics* / Q000276:immunology; D001011:Aorta; D001483:Base Sequence; D018958:CD55 Antigens / Q000235:genetics* / Q000276:immunology; D018957:CD59 Antigens / Q000235:genetics* / Q000276:immunology; D017690:Cell Transplantation; D003165:Complement System Proteins / Q000502:physiology*; D017931:DNA Primers; D004730:Endothelium, Vascular / Q000637:transplantation*; D004797:Enzyme-Linked Immunosorbent Assay; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis; D007075:Immunoglobulin M / Q000032:analysis; D051916:Membrane Cofactor Protein; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000276:immunology; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D013552:Swine; D014183:Transplantation, Heterologous / Q000276:immunology / Q000379:methods*; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: D000910:Antibodies, Heterophile; D015703:Antigens, CD; C497857:CD46 protein, human; D018958:CD55 Antigens; D018957:CD59 Antigens; D017931:DNA Primers; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D051916:Membrane Cofactor Protein; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D003165:Complement System Proteins; D001616:beta-Galactosidase