Toll-like receptors (TLRs) expressed by the corneal epithelium represent a first line of host defense to microbial keratitis. The current study examined the role of TLR2, TLR4, and TLR9 and the common adaptor molecule myeloid differentiation factor 88 (MyD88) in a Staphylococcus aureus model of corneal inflammation. The corneal epithelia of C57BL/6, TLR2 ؊/؊ , TLR4 ؊/؊ , TLR9 ؊/؊ , and MyD88 ؊/؊ mice were abraded using a trephine and epithelial brush and were exposed to heat-or UV-inactivated S. aureus clinical strain 8325-4 and other clinical isolates. Corneal thickness and haze were measured by in vivo confocal microscopy, neutrophil recruitment to the corneal stroma was quantified by immunohistochemistry, and cytokine production was measured by enzyme-linked immunosorbent assay. The exposure of corneal epithelium to S. aureus induced neutrophil recruitment to the corneal stroma and increased corneal thickness and haze in control C57BL/6 mice but not in TLR2 ؊/؊ or MyD88 ؊/؊ mice. The responses of TLR4 ؊/؊ and TLR9 ؊/؊ mice were similar to those of C57BL/6 mice. S. aureus-induced cytokine production by corneal epithelial cells and neutrophils was also significantly reduced in TLR2 ؊/؊ mice compared with that in C57BL/6 mice. These findings indicate that S. aureus-induced corneal inflammation is mediated by TLR2 and MyD88 in resident epithelial cells and infiltrating neutrophils.

Previous attempts to prepare monoclonal antibodies (MAbs) against S-antigen, a photoreceptor cell protein involved in the visual process and a potent autoantigen for the induction of experimental autoimmune uveitis (EAU), have yielded MAbs which define only carboxyl terminal epitopes. In this study we devised alternate strategies to prepare five MAbs directed to other regions of the molecule. MAbC10C10 and MAbH11-A2 were prepared against synthetic peptides known to be uveitopathogenic and they were selected for more detailed studies. MAbC10C10 was generated against synthetic peptide BSA281-302 which contains a predictive consensus sequence for defined T cell epitopes (GIALD) as well as a consensus sequence for GTP-binding proteins. One human adenosine deaminase synthetic peptide containing an extensive amino acid sequence homology to BSA281-302 was a potent inhibitor of MAbC10C10 binding in a competitive inhibition radioimmunoassay. MAbH11-A2 was generated against peptide BSA303-332 which also contains a uveitopathogenic site. The binding site of MAbH11-A2 was determined to be within amino acid positions 305 to 314 (NLASSTIIKE) in S-antigen. This binding site corresponded closely to the binding site of an affinity-purified rat polyclonal antibody raised to human S-antigen. MAb5C6.47 was isolated from a mouse hyperimmunized with bovine S-antigen and was specific for a highly conserved sequence near the amino terminus, amino acid residues 42 to 48 (DGVVLVD). Both MAbC10C10 and MAb5C.47 were useful in screening gt11 cDNA libraries expressing S-antigen polypeptides as fusion proteins. Our results demonstrate the feasibility of producing site-specific MAbs potentially useful in the study of T cell-mediated immune mechanisms in EAU as well as in the phototransduction of vision.

These results suggest that retinal pathology may be a primary immunological event in equine uveitis, provide evidence that leptospira-associated uveitis may be a distinct subset of equine uveitides, underscore the relevance of the study of equine uveitis to human uveitis, and support the plausibility of a post-infectious immunopathogenesis of some naturally occurring uveitides in both humans and horses.

Since immunization of guinea pigs with homologous retina can elicit experimental allergic uveitis concomitant with development of tissue specific antibodies, an attempt was made to localize a uveitogenic soluble retinal antigen in the normal guinea pig eye by an indirect fluorescent antibody method. Specific green fluorescence was seen in the outer portions of the retina, surrounding the nuclei of the outer nuclear layer and extending in both directions outward to the outer limbs of the photoreceptor cells and inward to the outer plexiform layer. This specific fluorescence was not detectable in other ocular structures, notably the uvea. Use of a homologous system in an indirect fluorescent antibody technique also resulted in a demonstration, by FA anti-GP, of distribution of γ-globulins in the normal guinea pig eye. Green fluorescence due to γ-globulins was seen brightly in the corneal stroma and in the connective tissue external to the sclera, less brightly and diffusely throughout the uvea, very faintly in the vitreous, and not at all in the retina. The demonstration of normal γ-globulins by this procedure did not interfere with the detection of tissue specific antigen. The relationship of antigen in the retina to possible mechanisms of autoimmune uveal disease is discussed.

PurposeAn increased risk of corneal infiltrative events has been noted with the use of certain contact lenses and multipurpose solutions (MPS). This study was designed to evaluate tear cytokine assay as a sensitive, objective, and quantitative measure of the ocular surface response to contact lens/MPS and to consider the assay’s clinical relevance in the context of other measures of ocular surface response.MethodsTwo MPS, ReNu® Fresh™ (RNF) and Opti-Free® RepleniSH (OFR), were used with daily wear silicone hydrogel contact lenses in a randomized, prospective crossover study involving 26 subjects. Clinical data collection (conjunctival hyperemia, ocular surface sensitivity, solution induced corneal staining (SICS) test score, and subjective responses) and tear cytokine assays were conducted masked. Responses were tracked as change from baseline throughout the experimental schedule.ResultsSimilar response patterns for several inflammatory cytokines were seen throughout both phases: subjects who received OFR in Phase I had mean tear concentrations that were generally higher than those of the RNF Phase I group. OFR Phase I subjects had significant (P < 0.01) increases over baseline at day 1 and/or following washout for 13 cytokines (cc chemokine ligands [CCL] 3, CCL5, CCL11, granulocyte-macrophage colony-stimulating factor [GM-CSF], interferon [INF]-γ, interleukin [IL]-2, IL-4, IL-5, IL-6, IL-13, IL-15, IL-17, tumor necrosis factor [TNF]-α). These changes were not observed in RNF Phase I subjects, even though SICS test scores increased. Phase I OFR subjects also had increased dryness, while RNF Phase I subjects had decreased bulbar hyperemia. No changes were detected with respect to limbal hyperemia or surface sensitivity thresholds.ConclusionThe tear cytokine assay can detect and differentiate contact lens/MPS induced increases in inflammatory cytokines. Changes in cytokine levels were consistent with measurement of hyperemia and dryness but not with SICS scores, thereby suggesting a proinflammatory response to OFR compared to RNF that is not related to SICS test score. Tear cytokine profiles may be useful for reconciling clinical relevance of test results and in revealing signaling involved in the development of corneal infiltrative events.