F. Brignole

Immunopathological findings in conjunctival cells using immunofluorescence staining of impression cytology specimens.

The conventional technique of impression cytology provides a non-invasive method for the evaluation of conjunctival epithelium alterations. Using indirect immunofluorescence procedures two inflammatory markers, class II MHC antigens HLA DR and the receptor to IgE (CD23), were sought in impression cytology specimens obtained from 80 patients. In normal subjects conjunctival epithelial cells did not show any reactivity. Only scattered dendritic cells were found to express class II antigens but not the receptor to IgE. In contrast patients with chronic conjunctivitis of various aetiologies, mainly infectious or allergic, had 40-100% of brightly positive conjunctival cells for one or both antigens. In these cases epithelial cells and goblet cells reacted similarly. Twenty four eyes in 12 patients with idiopathic dry eye syndrome disclosed results similar to those from normal conjunctival specimens. However 18 other specimens from patients suffering from idiopathic tear deficiency but undergoing multiple substitutive treatments for dry eye had moderate to strong positivity for HLA DR and/or the receptor to IgE (20-100% of cells). As these results were independent of the degree of squamous metaplasia the expression of these membrane markers may reflect local inflammation in addition to tear deficiency, possibly due to sensitisation to the eye drops used. These immunocytological techniques thus provide useful methods of investigating conjunctival inflammation and allergy. They may constitute valuable aid in the diagnosis and appropriate treatment of ocular surface disorders.

Relationship between in Vitro Toxicity of Benzalkonium Chloride (BAC) and Preservative-Induced Dry Eye

The term “dry eye syndrome” encompasses various ocular surface defects with a common element: deterioration in quantity and/or quality of lacrymal film. A constant step in the evolution of dry eye is an increase in tear osmolarity, leading in turn to other anomalies of the ocular surface. These include decreased conjunctival goblet cell density, increased corneal epithelial desquamation and destabilization of the cornea-tear interface. Iatrogenically induced dry eye is commonly encountered in clinical practice, especially while long-term treatments are employed for chronic ophthalmic affections such as glaucoma or allergy.1–4 On the cellular level, long-term use of topical, preserved drugs is associated with conjunctival metaplasia, stromal infiltrates, and epithelial expression of inflammation-dependent molecules (HLA DR), as well as apoptotic markers.5,6 The toxic and pro-inflammatory actions of preservatives on surface epithelia, as well as their detergent effect on the lipid phase of lacrymal film, seem to be responsible for these alterations.7,8 The role of active compounds of topical drugs in these pathological processes is not well established, although some in vivo and in vitro studies have demonstrated the lack of any noxious, tissue or cellular side effects.9,10 The aim of the present study was to investigate the relationship between the possible in vitro toxic effects of benzalkonium chloride (BAC), the most commonly used ophthalmic preservative, and the pathology of dry eye.

MHC class II antigen expression by ocular cells in proliferative diabetic retinopathy

Summary— An immunohistological study was performed on ciliary biopsies of the pars plana obtained surgically in 10 patients suffering from diabetic retinopathy and on 15 surgical specimens of pre‐retinal neovascularized membranes. Using immunofluorescence and immunoperoxidase procedures, linear deposits of IgG, IgA and complement components were found in the 8 pars plana from patients with proliferative diabetic retinopathy, at the basal pole of the pigment epithelial cells and within the stroma. In contrast, these deposits were absent from normal pars plana and from the cases of background retinopathy. Moreover, pigment and non‐pigment epithelial cells were found to express HLA DR and DQ determinants, in six of the eight patients with proliferative retinopathy. Immunohistological examination of pre‐retinal membranes showed deposition of immunoglobulins and complement components within the connective stroma and along the new blood vessels. Endothelial cells of the newly formed vascular walls strongly expressed class II antigens on their membrane, as well as scattered stromal cells. As neither pigment epithelial cells nor retinal vascular endothelial cells normally express class II determinants, our results suggest the involvement of immunological phenomena in intraocular proliferative diseases and eventual interactions between the immune system and peptide growth factors. However, whether or not this immune reaction plays a role in the initiation or extension of intra‐ocular proliferation remains to be determined.

Toxicity of Preserved and Unpreserved Antiglaucoma Topical Drugs in an In Vitro Model of Conjunctival Cells

PURPOSE To compare the toxicity of a short-time application of timolol with benzalkonium chloride (timolol-BAC+) and unpreserved timolol (timolol-BAC-) in a human conjunctival cell line. METHODS Changs conjunctival cell line (ATCC CCL 20.2) was treated for 15min. with 0.1%, 0.25% or 0.4% timolol-BAC(+) or BAC(-) and then examined immediately or 24h later. Cell viability, chromatin condensation and free radicals production were studied by microplate cold light cytometry. Moreover, relative cell number was evaluated by crystal violet colorimetric test. The comparison was done with an oxidative stress model of cells treated with 0.001-0.000001% hydrogen peroxide (H(2)O(2)). In addition, cell size and the expression of an apoptotic marker Apo2.7 were evaluated by flow cytometry. RESULTS Timolol-BAC(+) induced a rapid decrease in cell viability ranging from 40% immediately after treatment to 85% 24h later. A small initial decrease in cell viability was also observed with all tested concentrations of timolol-BAC(-) but, 24h later, cell viability either tended to remain constant or cells completely recovered. Cell viability fell down after 24h exposure to 0.001% H(2)O( 2) whereas it was not modified at lower concentrations. 24h after treatment with 0.25% timolol-BAC(+), the relative cell number was reduced by 55% whereas it did not vary after 0.25% timolol-BAC(-) treatment. Only timolol-BAC(+) induced chromatin condensation and cell size reduction. Moreover, cells treated with timolol-BAC(+) overexpressed the apoptotic marker Apo2.7. Both timolol-BAC(+) and BAC(-) induced reactive oxygen species (ROS) production which was significantly more important when 0.25% or 0.4% timolol-BAC(+) were applied. Only 0.001% and 0.0001% H(2)O(2) generated a significant free radicals production. CONCLUSION In our model of conjunctival cells in vitro timolol-BAC(+) induced irreversible cytotoxic damage with some characteristics of apoptosis. The active compound of timolol-BAC(-) could be responsible for reactive oxygen species production and for cell viability variations. The role of oxidative stress in timolol-BAC(+)-induced toxicity seems not to be predominant. in vitro toxic effects of antiglaucoma drugs could, in part, explain some ocular surface disorders in long-term treated patients.

Class II Histocompatibility Antigen Expression by Cellular Components of Vitreous and Subretinal Fluid in Proliferative Vitreoretinopathy

Proliferative vitreoretinopathy (PVR) is the major cause of failure in retinal detachment surgery. It is characterized by the formation of membranes extending along both surfaces of the detached retina and within the vitreous, but the nature of the growing cells has not yet been determined. Using cytologic and immunocytologic procedures with 13 different monoclonal antibodies directed against Class II histocompatibility antigens and various markers of epithelial and immunocompetent cells, 30 specimens were studied of vitreous or subretinal fluid removed from patients with PVR. Five main types of cells could be identified: heavily pigmented cells, poorly pigmented ones, large totally unpigmented macrophage-resembling ones, smaller unpigmented cells, and lymphocytes. Analysis of intravitreal pigment granules, using autofluorescence by epiillumination and cytologic procedures, showed two different populations of pigmented cells: one with autofluorescent lipofuscin granules and the other with exclusively melanin pigment. Immunostaining procedures confirmed the epithelial nonmacrophage lineage of the intravitreal and subretinal cells because most of these cells were positive for cytokeratin but negative for macrophage markers. In addition, 40-100% of these epithelial-derived cells strongly expressed Class II histocompatibility antigens HLA-DR and -DQ. Lymphocytes were found in 13 specimens; B-cells were seen, but no T-lymphocytes could be identified. These results confirm the involvement of retinal pigment epithelial cells and the strong morphologic changes they undergo during the course of PVR. Moreover, the expression of Class II histocompatibility antigens by the growing cells may be related to inflammatory phenomena, but their eventual role in the development and the extension of periretinal proliferation has not been determined.