Epithelial component and intraepithelial lymphocytes of conjunctiva-associated lymphoid tissue in healthy children.

Conjunctiva-associated lymphoid tissue (CALT) plays a key role in protecting the eye surface by initiating and regulating immune responses. The aim of this study was to investigate in healthy children the proportion of intraepithelial lymphocytes (IELs), the degree of viability and/or apoptosis and cell proliferation in three different topographic areas of the conjunctiva. Superior tarsal, superior bulbar, and inferior tarsal-bulbarfornix conjunctival cells were collected by brush cytology (BC) from 24 healthy paediatric subjects (13 boys and 11 girls, mean age 6±2 years) who were to undergo strabismus correction surgery under general anaesthesia. Subsequently, these cells were analysed phenotypically and functionally by flow cytometry (FC). Flow cytometry analysis showed that not all the cells obtained by BC were of the epithelial lineage, but that there was a population of CD45+ cells (IELs) regularly present in the conjunctiva of healthy children. These IELs were mostly T-lymphocytes (CD3+) and B-lymphocytes (CD19+), with higher levels of T-lymphocytes (CD3+) in the upper areas than in the inferior tarsal-bulbar-fornix, where the highest levels of B-lymphocytes (CD19+) were found. In the apoptosis assay, two groups of cell populations were differentiated by cell size and complexity (cytoplasmic granularity), with more complex cells predominating in the upper areas of the conjunctiva and less complex cells being more abundant in the inferior tarsal-bulbar-fornix. Finally, the proliferative capacity of the conjunctival epithelium was significantly higher in the upper tarsal zone than in the rest of the zones analysed. These results suggest that the epithelial component and the IELs of CALT are also regularly present in the conjunctiva of the healthy child, varying in phenotype, viability and cell proliferation according to the different conjunctival regions analysed, which could lead us to believe that each conjunctival zone plays a different, specific role in the regulation of the immune response at the ocular level.

Efficacy and safety of microspheres of cyclosporin A, a new systemic formulation, to prevent corneal graft rejection in rats.

PURPOSE: To evaluate the efficacy and safety of a new systemic formulation of cyclosporin A (CsA)-loaded microspheres in a rat model of penetrating keratoplasty rejection. METHODS: Female Lewis rats received orthotopic corneal allografts from inbred female Fisher donors. The rats were divided into three groups: 1, untreated controls; 2, daily subcutaneous injection of 10 mg/kg of the commercially intravenous CsA formulation starting after surgery (time 0) and for 15 days; and 3, one subcutaneous injection of 150 mg/kg of CsA microspheres. The grafts were evaluated clinically for 30 days and the rejection index, mean survival time, and rejection rate were calculated. Serum levels of CsA were measured at 5, 10, 15, 20, and 30 days in groups 2 and 3. Eyes, liver, and kidneys were histologically evaluated at the end of the experiment. RESULTS: Graft rejection was significantly reduced in group 3 at day 30 (P < 0.05) and serum levels of CsA were constant (range, 73.28 +/- 43.93 to 183.33 +/- 83.69 ng/ml). High levels (>3000 ng/ml) were obtained in group 2 as long as CsA was injected. Both formulations delayed rejection onset, but only the microspheres decreased the rate of corneal graft rejection (100% in group 2 and 70% in group 3 at day 30). Histologic examination showed no hepatic lesions with either formulation, but both resulted in deposition of a hemoglobin-like material in the kidneys. CONCLUSIONS: Although subcutaneous CsA-loaded microspheres delay rejection onset and decrease the rate of corneal graft rejection in an orthotopic keratoplasty rejection model in rats, administration of the microspheres did not prevent acute renal toxicity.