title: Local gene transfer to modulate rat corneal allograft rejection.
abstract: OBJECTIVE
  Allograft rejection is the leading cause of corneal graft failure. CD4(+) T cells control the allograft response and represent targets for antirejection therapy. The purpose of this study was to transfer cDNA encoding a monomeric anti-CD4 antibody fragment to donor corneal endothelium, to attempt to modulate orthotopic corneal allograft rejection in the rat.
  
  
  METHODS
  A replication-deficient adenoviral vector (AdV) encoding anti-CD4 single-chain, variable-domain antibody fragment (scFv) and enhanced green fluorescent protein (eGFP) was constructed (AdCD4GFP). AdV encoding eGFP alone (AdGFP) was used as a control. Transgenic product was detected by reverse transcription-polymerase chain reaction (RT-PCR), Western blot, flow cytometry, and fluorescence microscopy. The alloinhibitory capacity of anti-rat CD4 scFv was measured in the one-way mixed lymphocyte reaction (MLR). The survival of Wistar-Furth corneas transduced with AdV either immediately or 3 days before orthotopic transplantation in Fischer 344 recipients was examined.
  
  
  RESULTS
  ScFv and eGFP mRNAs were detected in rat corneas transduced in vitro, and active scFv secreted in corneal supernatants peaked at days 4 to 5 after transduction at 23 +/- 4 ng of protein per cornea per day. Antibody and scFv against rat CD4 blocked alloproliferation in MLR. However, transduction of corneas with AdCD4GFP ex vivo, immediately before transplantation, or in vivo, 3 days before transplantation, did not significantly prolong corneal allograft survival (P > 0.05).
  
  
  CONCLUSIONS
  Anti-CD4 scFvs were capable of blocking allostimulation, but their local expression within the eye did not prolong corneal allograft survival, suggesting that sensitization may still occur.
authors.affiliation: Department of Ophthalmology, Flinders University of South Australia, Flinders Medical Centre, Bedford Park, SA 5042, Australia.
authors.forename: Claire F
authors.initials: CF
authors.lastname: Jessup
authors.forename: Helen M
authors.initials: HM
authors.lastname: Brereton
authors.forename: Pamela J
authors.initials: PJ
authors.lastname: Sykes
authors.forename: Michael A
authors.initials: MA
authors.lastname: Thiel
authors.forename: Douglas J
authors.initials: DJ
authors.lastname: Coster
authors.forename: Keryn A
authors.initials: KA
authors.lastname: Williams
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D015153:Blotting, Western; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D049109:Cell Proliferation; D003315:Cornea / Q000276:immunology; D016039:Corneal Transplantation; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006084:Graft Rejection / Q000378:metabolism / Q000517:prevention & control*; D006085:Graft Survival; D049452:Green Fluorescent Proteins; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D007959:Lymphocyte Culture Test, Mixed; D008856:Microscopy, Fluorescence; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011916:Rats, Inbred F344; D011920:Rats, Inbred WF; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D057127:Single-Chain Antibodies; D014184:Transplantation, Homologous
chemical_list: D007135:Immunoglobulin Variable Region; D008164:Luminescent Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; C410726:scFV green fluorescent protein, recombinant; D049452:Green Fluorescent Proteins