[Show abstract][Hide abstract] ABSTRACT: To examine the role of the CD28-CD80-CD86 pathway of T-lymphocyte costimulation in corneal allograft rejection and the effect of blockade of that pathway on graft survival.
Kinetics of CD80 and CD86 expression in the cornea and draining lymph nodes were examined by RT-PCR and immunohistochemistry in untreated allograft recipients in a high-responder rat model. The effect of blockade of CD28-mediated costimulation was first examined by ex vivo incubation of excised Brown Norway rat donor cornea with the inhibitory protein CTLA4-Ig or an adenovirus vector (AdCTLA) expressing CTLA4-Ig, before grafting into Lewis rat recipients. A second group of graft recipients received systemic posttransplantation treatment with either CTLA4-Ig or AdCTLA.
Expression of CD80 mRNA was increased in both donor and recipient cornea 16 hours after transplantation, whereas CD86 was detected constitutively, with no significant early increase. Immunohistochemistry on day 5 after transplantation demonstrated major histocompatibility complex (MHC) class II expression, no CD80, and only a trace of CD86 in corneal allografts. In lymph nodes strong MHC class II, weak CD80, and moderate CD86 expression was noted. Both donor cornea and recipient treatment with CTLA4-Ig resulted in prolonged allograft survival. AdCTLA was found to induce sustained secretion of bioactive CTLA4-Ig from corneas infected ex vivo. Survival of corneal allografts incubated with AdCTLA was marginally prolonged, and systemic treatment with AdCTLA significantly prolonged survival.
Protein- or gene-based administration of CTLA4-Ig prolongs allograft survival by treatment of either the recipient or the donor tissue ex vivo before grafting.
[Show abstract][Hide abstract] ABSTRACT: Background. The application of gene therapy to prevent allograft rejection requires the development of noninflammatory vectors. We have therefore investigated the use of a nonviral system, transferrin-mediated lipofection, to transfer genes into the cornea with the aim of preventing corneal graft rejection.
Methods. Rabbit and human corneas were cultured ex vivo and transfected with either lipofection alone or in conjunction with transferrin. The efficiency of transfection, localization, and kinetics of marker gene expression were determined. Strategies to increase gene expression, using chloroquine and EDTA, were investigated. In addition to a marker gene, a gene construct encoding viral interleukin 10 (vIL-10) was transfected and its functional effects were examined in vitro.
Results. Transferrin, liposome, and DNA were demonstrated to interact with each other, forming a complex. This complex was found to deliver genes selectively to the endothelium of corneas resulting in gene expression. Treatment of corneas with chloroquine and EDTA increased the transfection efficiency eightfold and threefold, respectively. We also demonstrated that constructs encoding vIL-10 could be delivered to the endothelium. Secreted vIL-10 was shown to be functionally active by inhibition of a mixed lymphocyte reaction.
Conclusions. Our data indicate that transferrin-mediated lipofection is a comparatively efficient nonviral method for delivering genes to the corneal endothelium. Its potential for use in preventing graft rejection is shown by the ability of this system to induce vIL-10 expression at secreted levels high enough to be functional.
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to examine the kinetic profile of bioactive TNF levels in aqueous humour of rabbit eyes undergoing corneal allograft rejection and to investigate the effect of locally blocking TNF activity after corneal transplantation. In a rabbit corneal transplantation, endothelial allograft rejection was identified and correlated with increase in central graft thickness. Samples of aqueous humour obtained on alternate days following transplantation were tested for TNF mRNA and bioactive TNF protein. To investigate the effect of locally blocking TNF activity in allograft recipients, the fusion protein TNFR-Ig was administered by injections into the anterior chamber after transplantation. Pulsatile increases in levels of this cytokine were found in 14 of 15 allograft recipients. Peaks of TNF bioactivity preceded by varying intervals the observed onset of rejection in allograft recipients. TNF levels were not elevated in aqueous humour from corneal autograft recipient controls or in serum of allografted animals. mRNA levels were elevated before onset of and during clinically observed allograft rejection. In three of seven animals receiving TNFR-Ig injections on alternate days from day 8 to day 16 post-transplant, clear prolongation of corneal allograft survival was demonstrated. Bioactive TNF is present in aqueous humour following rabbit corneal allotransplantation. Rather than correlating directly with endothelial rejection onset, pulsatile peak levels of TNF precede and follow the observed onset of endothelial rejection. Blockade of TNF activity prolongs corneal allograft survival in some animals, indicating that this cytokine may be a suitable target in local therapy of corneal allograft rejection.
[Show abstract][Hide abstract] ABSTRACT: Gene transfer to the corneal endothelium has potential for modulating rejection of corneal grafts. It can also serve as a convenient and useful model for gene therapy of other organs. In this article we review the work carried out in our laboratory using both viral and nonviral vectors to obtain gene expression in the cornea.
American Journal of Respiratory and Critical Care Medicine 11/2000; 162(4 Pt 2):S194-200. · 11.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background. Allogeneic rejection is the most common cause of corneal graft failure. The aim of this work was to establish the kinetics of cytokine and chemokine mRNA expression before and after onset of corneal graft rejection.
Methods. Intracorneal cytokine and chemokine mRNA levels were investigated in the Brown Norway→Lewis inbred rat model in which rejection onset is observed at 8/9 days after grafting in all animals. Nongrafted corneas and syngeneic (Lewis→Lewis) corneal transplants were used as controls. Donor and recipient cornea was examined by quantitative competitive reverse transcription-polymerase chain reaction (RT-PCR) for hypoxyanthine phosphoribosyltransferase (HPRT), CD3, CD25, interleukin (IL)-1β, IL-1RA, IL-2, IL-6, IL-10, interferon-γ (IFN-γ), tumor necrosis factor (TNF), transforming growth factor (TGF)-β1, and macrophage inflammatory protein (MIP)-II and by nonquantitative RT-PCR for IL-4, IL-5, IL-12 p40, IL-13, TGF-β2, monocyte chemotactic protein-1 (MCP-1), MIP-1α, MIP-1β, and RANTES (for regulated upon activation normal T cell expressed and secreted).
Results. A biphasic expression of cytokine and chemokine mRNA was found after transplantation. During the early phase (days 3-9), there was an elevation of the majority of the cytokines examined, including IL-1β, IL-6, IL-10, IL-12 p40, and MIP-II. There was no difference in cytokine expression patterns between allogeneic or syngeneic recipients at this time. In syngeneic recipients, cytokine levels reduced to pretransplant levels by day 13, whereas levels of all cytokines rose after observed rejection onset in the allografts, including TGF-β1, TGF-β2, and IL-1RA. The T cell-derived cytokines IL-4, IL-13, and IFN-γ were detected only during the rejection phase in allogeneic recipients.
Conclusions. There is an early cytokine and chemokine response to the transplantation process, evident in syngeneic and allogeneic grafts, that probably drives angiogenesis, leukocyte recruitment, and affects leukocyte functions. After an immune response has been generated, allogeneic rejection results in the expression of Th1 cytokines (IL-2, IL-12 p40, IFN-γ), Th2 cytokines (IL-4, IL-6, IL-10, and IL-13), and anti-inflammatory/Th3 cytokines (TGF-β1/2 and IL-1RA).