Purified S-antigen was used to induce experimental autoimmune uveitis in 42 guinea pigs. 16 animals were used as controls. Footpad immunization with fresh bovine S-antigen in physiologic saline mixed with complete Freunds's adjuvant induced a clinical disease in 95% of the eyes in test animals. In aqueous humour the increase in phospholipase-A2 and proteins as well as myeloperoxidase measured from iris-ciliary blocks correlated well with the severity of uveitis evaluated by clinical grading. Leukotriene C4 and prostaglandin E2 were only elevated in aqueous humour drawn from eyes showing a mild form of uveitis. Neither leukotriene C4 nor prostaglandin E2 were detected in eyes graded as clinically moderate or severe. In serum samples phospholipase A2, leukotriene C4, prostaglandin E2 and gamma glutamyl transpeptidase were measured. Of these biochemical parameters, only gamma glutamyl transpeptidase was significantly elevated in test animals with experimental autoimmune uveitis. Histological analysis revealed focal mononuclear cell infiltrations in the choroid. Mononuclear as well as polymorphonuclear cell infiltration was seen predominantly in the pars plana region of the ciliary body of test animals with uveitis. Simultaneous destruction of the outer segments of the photoreceptor layer was seen.
Topical cyclosporin A is increasingly being used in the treatment of ocular surface immune-mediated disorders. The availability of the drug in oil-based vehicles or collagen shields has restricted its use because of ocular irritation or blurring of vision. Although topical cyclosporin is being used more frequently, its effect on the immunocompetent cells of the conjunctiva is not known. Our aim was to study the effect of cyclosporin instillation on the immunocomponent cells of conjunctiva-associated lymphoid tissue (CALT) of Lewis rat, using a novel method of topical drug delivery. A suspension of collagen bits impregnated with cyclosporin A was instilled into eyes of Lewis rats for 4 days (group 1) or 8 days (group 2). Control rats (group 3) received the suspension without cyclosporin. Frozen sections of eyelids and conjunctiva were immunostained with the following monoclonal antibody markers: W3/13 (CD3), W3/25 (CD4, macrophages), OX-8 (CD8), MARD-3 (B cells), ED1, ED2 (macro/monocytes), OX-6 (class II MHC, Ia) and OX-39 (CD25, IL-2 receptor). Intraepithelial (IE) and substantia propria cells for each subset were counted and expressed as numbers per section. By day 8, intraepithelial and substantia propria cells for all the above markers, except B cells, showed a significant reduction in numbers. The p values were < 0.02 for W3/13 (CD3), W3/25 (CD4), OX-8 (CD8), OX-39 (CD25) (IE only), ED1, ED2 and OX-6 positive cells. Goblet cells of control animals showed strong positive reaction with OX-39 (CD25) antibody. This was completely abolished following 8 days of topical cyclosporin. This study demonstrated that topical cyclosporin A induces a marked reduction in numbers of all subtypes of immunocompetent cells in the conjunctival epithelium and substantia propria.
SUMMARY Bovine S-antigen was purified by gel filtration and ion exchange chromatography according to previously described techniques. An enzyme linked immunosorbent assay (ELISA) using antiserum to bovine S-antigen raised in guinea-pigs was employed to detect S-antigen in the chromatographic fractions. The purity of the S-antigen was determined by SDS-PAGE electrophoresis, where a single band was found. The purified S-antigen in microgram quantities together with Freund's complete adjuvant induced uveitis in rats two weeks after injection into the foot pad. Serum samples from children suffering from chronic uveitis and healthy children were tested for antibodies to S-antigen by the ELISA. A statistically significant difference in the level of specific antibodies between patients and controls was found. There was no clear-cut correlation between the severity of uveitis and antibody titre, but cases with retinal involvement and aggressive uveitis all showed definite elevations of antibodies to S-antigen.
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