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JE Moore, CB T McMullen, IL Campbell, R Rohan, NA Afshari, Y Kaji, T Usui, DB Archer, AP Adamis; The Inflammatory Milieu Associated with Conjunctivalised Cornea and its Alteration Using IL-1 RA Gene Therapy . Invest. Ophthalmol. Vis. Sci. 2002;43(13):28.
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Purpose: This study was designed to gain insight into the inflammatory milieu into which a donor limbal graft is routinely introduced. The objective was to modulate this environment via gene therapy with the anti-inflammatory cytokine IL-1 RA. Methods: In a mouse model, the ocular surface cytokine environment of a conjunctivalised cornea four weeks post injury was assessed. Total corneal epithelial and limbal debridement was carried out using a combination of alkali and scrape injury. Cytokines and adhesion molecules measured included: IL-1 alpha, IL-1 beta, IL-6, VEGF, ICAM-1 and VCAM-1 using real time PCR or ELISA. Injured corneas were transfected with IL-1 RA by injection of naked plasmid pIRES-EGFP-IL-1 RA immediately after injury while corneas transfected with pIRES-EGFP served as controls. Expression of corneal IL-1 RA was assessed over a 2 week period using real time PCR and Western blotting. In addition, limbal stem cell grafts transfected with IL-1 RA were assessed for leukocyte influx. Results: Conjunctivalised corneas showed increased expression of IL-1 alpha (p=0.05, n=5), IL-1 beta (p=0.03, n=5), IL-1 RA (p<0.001), IL-6 (p=0.0003, n=9), VEGF (p=0.007, n=6), ICAM-1 ( p=0.0008, n=6) and VCAM-1 (p=<0.001) compared to normal cornea. Transfection efficiency experiments indicated corneal expression of IL-1 RA peaked at 12-24 h and lasted 2 weeks. IL-1 RA corneal gene therapy resulted in a down regulation of IL-1 beta (p<0.001, n=6)and VCAM-1 (p=0.007, n=12) expression at 4 weeks post injury, while down regulation of IL-6 expression was evident only at 1 week post injury. Corneal neovascularization was also reduced by IL-1 RA gene therapy (p=0.007, n=24). In addition, corneal limbal stem cell grafts transfected with IL-1 RA showed a decreased leukocyte influx (p<0.001, n=6) compared with that of control grafts. Conclusion: Corneal transfection with IL-1 RA immediately after epithelial injury altered the cytokine profile of the resulting conjunctivalised cornea and suppressed corneal neovascularization. Transfection of corneal limbal donor tissue with IL-1 RA prior to engraftment reduced graft leukocyte influx. These findings demonstrate the feasibility of using transient cytokine gene expression, either in donor or recipient corneal tissue, to beneficially alter the ocular surface environment.
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