[Improvement of dry eye symptoms with polyunsaturated fatty acids]. / Amélioration de la symptomatologie chez des patients atteints de sécheresse oculaire et traités oralement par des acides gras polyinsaturés.

INTRODUCTION: Polyunsaturated fatty acids (PUFAs) are involved in inflammatory pathways via prostaglandins. Conjunctival inflammation is a hallmark of all dry eye syndromes. We investigated the role of dietary n-6 and n-3 fatty acids in patients suffering from ocular dryness. PATIENTS AND METHODS: Seventy-one patients presenting with mild to moderate dry eye syndromes were randomly assigned to Nutrilarm or placebo capsules, twice a day for 6 months. The Schirmer test, BUT, fluorescein staining, and lissamin green stainings were performed at inclusion and after 1, 3, and 6 months. Furthermore, a questionnaire related to the dry eye symptoms and global discomfort was provided at every visit. RESULTS: The Schirmer test, BUT, fluorescein staining, and lissamin green stainings were improved with treatment when compared to placebo but the difference was not statistically significant. The efficacy evaluated by the patients and the investigator were nearly significant (p=0.052 and p=0.054, respectively). For some signs, such as reflex tearing and conjunctival hyperemia, the improvement reached the threshold of significance (p=0.047 and p=0.045, respectively). The same results were found with skin quality and emotional condition, which were improved (61% with treatment versus 36% with placebo). CONCLUSION: This double-masked pilot study shows that PUFAs seem to be an interesting tool to alleviate the symptoms related to dry eye syndrome. These results should be confirmed using a larger study population.

[Treating severe dry eye syndromes with autologous serum]. / Traitement des syndromes secs graves par sérum autologue.

BACKGROUND: Dry eye syndrome with tear deficiency can be improved with artificial tears, which can be associated with topical anti-inflammatory agents. Autologous serum can provide the ocular surface with beneficial growth factors and vitamins. PATIENTS AND METHODS: Twenty-one patients suffering from severe dry eye due to Sjögren's syndrome were treated with 20% autologous serum for 2 Months. The Schirmer I test, break-up time, and fluorescein and lissamine green stainings were performed before and after treatment. Subjective complaints such as burning, foreign body sensation, dryness and photophobia were assessed by a questionnaire as well as a face score reflecting the current condition of patients' eyes. RESULTS: Lissamine green and fluorescein scores improved significantly as well as subjective symptoms of burning, foreign body sensation and dryness (p<0.05). The face score was significantly improved. Bacterial culture of serum delivered to the patients all remained negative. DISCUSSION: Autologous serum provides growth factors and vitamins that are useful for an altered ocular surface due to Sjögren's disease. However, some problems still remain: risk of contamination, arbitrary dilution of autologous serum, and a current lack of regulations for use of autologous serum. A close collaboration between ophthalmologists and the Etablissement Français du Sang (French Blood Bank) is mandatory because autologous serum should be considered as a useful tool to treat severe ocular surface disorders. CONCLUSION: The use of autologous serum improved symptoms and objective signs caused by severe Sjögren's syndrome. Currently, a lack of clear regulations prevents its widespread use in severe ocular surface disorders.

[Current treatments of xerophthalmia in Sjögren's syndrome]. / Traitements actuels de la xérophtalmie dans le syndrome de Gougerot-Sjögren.

PURPOSE: To describe the large variety of treatments currently used in Sjögren's syndrome for one of its major manifestations, keratoconjunctivitis sicca or xerophthalmia. CURRENT KNOWLEDGE AND KEY POINTS: Sjögren's syndrome causes a diffuse immunoinflammatory disturbance of main lacrimal glands and the whole ocular surface. Dry eye syndrome is responsible for chronic and deep impairment of quality of life. Many different tear substitutes have been widely developed that are poorly efficient for relieving patients from their complaints. Tear substitutes of various viscosity from standard artificial tears to synthetic gels may be used. Hyaluronic acid is currently the most promising tear substitute, but all eye drops and gels are only efficient in mild to moderate dry eyes and keratoconjunctivitis sicca mostly resists to lubricants. Moreover, the latter may increase patients' complaints when they are associated to preservatives, antiseptic drugs that have widely demonstrated their toxic or irritating potential. Preservatives are, therefore, to be avoided whenever possible in keratoconjunctivitis sicca, by using monodose disposable packaging or specific bottle filtering or eliminating the preservative. Stimulation of lacrimal and salivary secretions with systemic pilocarpine, or obturation of lacrimal puncta in order to limit the drainage of tears in lachrymal ducts may be useful in most severe forms of Sjögren's syndrome. However, the development of topical cyclosporine and other immunomodulating agents is the most relevant progress in the treatment of keratoconjunctivitis sicca in Sjögren's syndrome. PERSPECTIVES: The future for treating Sjögren's syndrome is most likely to pass through the use of new drugs capable of treating the disease or at least its mechanisms, and not only to try to relieve symptoms with poorly efficient tear substitutes.

Comparison of morphological and functional characteristics of primary-cultured human conjunctival epithelium and of Wong-Kilbourne derivative of Chang conjunctival cell line.

PURPOSE: To analyze the relevance of a human conjunctival cell line in a study of conjunctival epithelium. We investigated and compared the effects of IFNgamma and TNFalpha in a primary culture of human conjunctiva and in a human conjunctival cell line. METHODS: A primary-cultured human conjunctival epithelium and a human conjunctival cell line (Chang cells) were treated for 72 hr with 20, 200, 400 and 600 U ml(-1) IFNgamma or with 1100 and 11,000 U ml(-1) TNFalpha. Then, the expression of HLA DR, CD40, CD44, CD63, CD80, CD86, Fas receptor, E-cadherin, ICAM-1, MUC1, cytokeratins and vimentin were investigated by flow cytometry. Cell morphology was studied with phalloidin staining. Apoptosis was detected by flow cytometry with Annexin V and via cell cycle analysis. RESULTS: The primary culture of human conjunctival epithelium expressed cytokeratin K4, non-keratinized squamous epithelial marker. Chang cells presented a more dedifferentiated phenotype and were cytokeratin K4 negative. In primary-cultured cells, IFNgamma (600 U ml(-1)) induced only a low level of apoptosis and a significant upregulation of most tested proteins such as HLA DR, Fas, ICAM-1, CD40 and CD63. In the Chang cell line, IFNgamma induced a significant level of apoptosis at concentrations of 200, 400 and 600 U ml(-1). HLA DR and CD63 were induced at lower levels than in primary-cultured cells. Other proteins were modified in a similar manner after IFNgamma treatment in both systems. In the primary-cultured cells, TNFalpha induced an important upregulation of ICAM-1, Fas and CD40 whereas CD44 and CD63 were significantly decreased. Conversely, only a very weak alteration of CD63 and ICAM-1 was observed in the Chang cell line after TNFalpha treatment. CONCLUSIONS: A primary culture of a human conjunctival epithelium demonstrated well-defined epithelial features. TNFalpha and IFNgamma, two inflammatory cytokines, induced different effects in both cellular systems, in a primary-cultured conjunctival epithelium and a human conjunctival cell line. Inflammation-related molecules were highly upregulated in the primary culture and, to a lesser extent, in the Chang cell line. Thus, the Chang cell line differs in certain features from a primary culture of human conjunctival epithelium, a fact which emphasizes the complexity of interpretation of in vitro data and this should be taken into consideration in in vitro studies of human conjunctival epithelium.

[Effect of preservatives on the conjunctiva: a comparative study of beta-blocker eye drops with and without preservatives in glaucoma patients]. / Retentissement conjonctival des conservateurs: étude comparative de collyres bêta-bloquants conservés et non conservés chez des patients glaucomateux.

PURPOSE: To compare the effect of timolol with or without preservatives on the conjunctival epithelium of glaucoma patients. METHODS: A retrospective study using impression cytology (IC) was conducted on patients treated with 0.5% timolol with or without 0.01% benzalkonium chloride (BAC). Fifteen eyes from 15 patients treated with timolol, BAC+ and 17 eyes from 17 patients treated with timolol, BAC- were included in two groups comparable for age and duration of treatment lasting at least 1 year. Specimens were analyzed by flow cytometry for inflammatory profile (using antibodies directed against HLA DR and ICAM-1) and mucin detection (anti-M1/MUC5AC antibody). RESULTS: IC analyses showed a significant increase in the expression of the two inflammatory markers, HLA DR and ICAM-1 in the timolol, BAC+ group and also a significant decrease in goblet cell density in the same group as compared to the timolol, BAC- group. CONCLUSION: The use of long-term preserved beta-blocker in glaucoma patients is associated with a direct subclinical epithelial toxicity in the conjunctiva, as already demonstrated by previous experimental studies.

[Flow cytometry in impression cytology during keratoconjunctivitis sicca: effects of topical cyclosporin A on HLA DR expression]. / Cytofluorimétrie sur empreintes conjonctivales au cours de la kératoconjonctivite sèche: effets de la ciclosporine topique sur l'expression d'antigène HLA DR.

PURPOSE: Immune-based inflammation has been observed as a common mechanism of keratoconjunctivitis sicca (KCS). In KCS-affected eyes, up-regulated expression of HLA DR by conjunctival epithelial cells has been demonstrated in impression cytology (IC) specimens using a technique of flow cytometry. The purpose of this study was to monitor the effects of topical cyclosporin A on the expression of this marker over a 12-month period of treatment. METHODS: Patients with moderate-to-severe KCS included in a large European multicenter clinical trial (Cyclosporin Dry Eye Study, Allergan, Irvine, CA) underwent collection of IC specimens at baseline, month 3, month 6, and month 12. They randomly received 0.05% or 0.1% cyclosporin A or vehicle. Patients randomized to receive vehicle received 0.1% cyclosporin A from month 6 onwards. Specimens were processed and analyzed in a masked manner by flow cytometry, using monoclonal antibodies directed to HLA DR. RESULTS: We included 169 patients in this study. HLA DR expression, both in percentage of positive cells and level of expression, was highly significantly reduced after 0.05% and 0.1% cyclosporin A treatment at months 3, 6, and 12 compared with baseline values, whereas vehicle did not induce any change in HLA DR expression over time. The 0.05% and 0.1% cyclosporin emulsions were significantly more effective than the vehicle in reducing HLA DR at months 3 and 6 (0.05%) and at month 6 (0.1%). CONCLUSIONS: Topical cyclosporin A strikingly reduced HLA DR, whereas the vehicle, used as a control tear substitute, had almost no effect. This study confirms that cyclosporin A may be effective in reducing conjunctival inflammation in moderate-to-severe KCS and is consistent with clinical results in this indication.

[Apoptosis and the ocular surface]. / Apoptose et surface oculaire.

Apoptosis, or programmed cell death, is an active phenomenon that plays a major role in most mechanisms of regulation, differentiation and wound healing. Mostly studied in the retina, apoptosis is also extensively involved in the anterior segment, especially the ocular surface. Apoptosis of keratocytes is a rapid phenomenon following excimer refractive surgery. Any epithelial aggression stimulates a series of mechanisms leading to death of deep keratocytes. The role of epithelial cell mediators may explain the superiority of LASIK compared to PRK in terms of functional rehabilitation. Conjunctiva is also a major site in which inflammation and apoptosis are combined. Proinflammatory cytokines may both amplify immune reactions and stimulate epithelial apoptosis, which is most likely to result in elimination of injured tissues. Toxic drugs also play a major role and iatrogenic apoptosis should be avoided as much as possible, especially by eliminating preservatives from eyedrops, most of which use both proinflammatory and proapoptotic agents.

[Human trabecular cells and apoptosis: in vitro evaluation of the effect of betaxolol with or without preservative]. / Apoptose et cellules trabéculaires humaines: évaluation in vitro de l'effet du bétaxolol avec ou sans conservateur.

PURPOSE: Trabecular meshwork, which is involved in aqueous outflow resistance, is deeply modified in glaucoma patients, with a decrease in the trabecular cell number. Trabecular toxicity of antiglaucoma medications cannot be excluded. On a human cultured trabecular cell line, we investigated the potential proapoptotic effect of a beta-blocker with or without preservative, benzalkonium chloride (0.01% BAC), by flow cytometry and confocal microscopy. MATERIAL: and METHODS: A human immortalized trabecular cell line (HTM-5) obtained from a normal donor was cultured under normal conditions. Preserved 0.25% betaxolol suspension (betaxolol BAC +), unpreserved 0.25% betaxolol suspension, and 0.01% BAC were respectively added to the culture medium in a 1/10 or 1/100 dilution for 15 minutes. After a 24-hour recovery period in normal culture conditions, cell size and the expression of an apoptotic marker, Apo 2.7, were evaluated by flow cytometry and confocal microscopy. Untreated trabecular cells were used as control cells. RESULTS: Preserved and unpreserved betaxolol in a 1/10 dilution induced a significant decrease in trabecular cell size compared to controls. However, this cell size decrease was less pronounced than that induced by BAC at the same dilution. Similar results were obtained with betaxolol and BAC in a 1/100 dilution. Trabecular cell Apo 2.7 expression was significantly increased after treatment with betaxolol BAC + and BAC- in a 1/10 dilution compared to controls (36.8%, 28.1%, and 15.4%, respectively p<0.005). However, this proapoptotic activity was much less pronounced than that induced by BAC- at the same dilution (96.9%, p<10(-4)). Unpreserved betaxolol in a 1/100 dilution had no apoptotic activity on trabecular cells. Trabecular cell Apo 2.7 expression slightly increased with betaxolol BAC + at a 1/100 dilution (24.9%, p=0.04), while it was greatly increased with BAC at the same dilution (39.9%; p<10(-4)). CONCLUSION: In our model, unpreserved betaxolol at a low concentration displayed no proapoptotic activity on trabecular cells. On the other hand, preserved betaxolol displayed a moderate proapoptotic activity by triggering cell death of around 25% of cells. Trabecular cell toxicity appeared to be mainly due to the preservative benzalkonium chloride (BAC). Taken together, our results demonstrated that the strong apoptotic activity of BAC was greatly reduced within the preserved eye drops, probably through the interaction of BAC with the active compound.

Ocular surface changes induced by contact lens wear.

PURPOSE: To evaluate subclinical inflammation and mucus production of the conjunctiva in asymptomatic contact lens (CL) wearers, and to obtain an estimation of the chronologic variations in each group. METHODS: Eighteen eyes fitted with rigid CL (RCL) and 28 eyes with soft CL (SCL) worn daily were compared with 10 eyes from five healthy non-CL wearers. Impression cytology (IC) specimens were collected after clinical examination and were analyzed by flow cytometry using antibodies directed to HLA DR and intercellular adhesion molecule type 1 (ICAM-1) (CD 54), as inflammatory markers, and to the peptidic core of the conjunctival mucin (M1/MUC5AC) for mucus and goblet cell detection. The percentage of positive cells was calculated, and levels of fluorescence expression were quantified and compared between each group. RESULTS: A significant increase of HLA DR and ICAM-1 was observed in the SCL group in comparison with the control group. The two inflammatory markers were highly positively correlated with each other. Mucin detection with M1/MUC5AC did not find a significant difference between each group in terms of percentage of positive cells, but analyses of mean levels of fluorescence showed a significant decrease in the two CL groups. Evolution in time was different for each group, with a regular low level of inflammation in the RCL group in the first 10 years in comparison with the SCL group. In the SCL group, inflammation seemed to be higher before 2 years and after 10 years of wear. Mucin expression was variable in time, but without significant difference at any time. CONCLUSION: This study confirms difference in expression of subclinical conjunctival inflammation in asymptomatic CL wearers, with lower levels for RCL than SCL wearers with daily or extended wear. The mucin system is also modified by this low but chronic aggression of the ocular surface, with a tendency to decrease with time in the RCL and SCL groups.

Quaternary ammoniums and other preservatives' contribution in oxidative stress and apoptosis on Chang conjunctival cells.

PURPOSE: To investigate some of the toxicity mechanisms of 10 preservatives currently used in ophthalmic solutions in vitro. METHODS: A continuous human conjunctival cell line was treated with different concentrations of various preservatives for 15 minutes and for 15 minutes followed by 24 hours of cell recovery: three benzalkonium chlorides (BACs) with different hydrocarbon chain length, benzododecinium bromide (BOB), cetrimide (Cet), phenylmercuric nitrate (PM), thimerosal (thi), methyl parahydroxybenzoate (MPHB), chlorobutanol (clb), and EDTA. An inhibition study was then conducted using a 1-hour vitamin E pretreatment followed by a 15-minute BAC treatment. Membrane integrity was assessed using a neutral red test and chromatin condensation with a Hoechst 33342 test. Reactive oxygen species were measured using dichlorofluorescein diacetate test for H2O2 production and hydroethidine test for O2.- production. These tests were performed using microplate cold light cytofluorometry. Cell size and DNA content were also analyzed using flow cytometry. Confocal microscopy was used to explore morphologic changes. RESULTS: A significant decrease of membrane integrity with chromatin condensation was observed with all the quaternary ammoniums tested at concentrations of 0.005% and higher. The effect was amplified after 24 hours of cell recovery. The other preservatives tested did not decrease membrane integrity. H2O2 production was observed with all the preservatives, whereas O2.- production was significantly higher with the quaternary ammoniums at 0.005% and 0.01%, compared with the other preservatives. Flow cytometry results confirmed the cytotoxicity observed with cold light cytofluorometry. CONCLUSIONS: The quaternary ammoniums tested (BAC, BOB, and Cet) were the most cytotoxic preservatives in the current model. An apoptotic mechanism appeared to be present at low concentrations of quaternary ammoniums, whereas a necrotic process appeared at higher concentrations. Superoxide anions may play an important role in tissue damage induced by preservatives in ocular surface disorders.

Flow cytometric analysis of inflammatory markers in KCS: 6-month treatment with topical cyclosporin A.

PURPOSE: Immune-based inflammation has been observed as a common mechanism of keratoconjunctivitis sicca (KCS). In KCS-affected eyes, upregulated expression of HLA DR and various immune- or apoptosis-related markers by conjunctival epithelial cells has been demonstrated in an earlier study, by a technique of flow cytometry in impression cytology (IC) specimens. The purpose of this study was to monitor the effects of topical cyclosporin A on the expression of these markers throughout a 6-month period of treatment. METHODS: Patients with moderate to severe KCS included in a large European multicenter clinical trial (Cyclosporin Dry Eye Study, Allergan, Irvine, CA) underwent collection of IC specimens at baseline, month 3, and month 6. For 6 months, they randomly received 0.05% or 0.1% cyclosporin A or vehicle. Specimens were processed and analyzed in a masked manner by flow cytometry, using monoclonal antibodies directed to HLA DR, CD40, CD40 ligand, Fas, and the apoptotic marker APO2.7. Percentages of positive cells were calculated and levels of expression quantified after conversion into standardized units of fluorescence. RESULTS: One hundred fifty-eight patients had at least two IC specimens available for flow cytometry analysis. HLA DR expression, both in percentage of positive cells and level of expression, was highly significantly reduced after 0.05% and 0.1% cyclosporin A treatment at months 3 and 6 compared with baseline values, whereas vehicle did not induce any change in HLA DR expression over time. The 0.05% and 0.1% cyclosporin emulsions were significantly more effective than the vehicle in reducing HLA DR at months 3 and 6 (0.05%), and at month 6 (0.1%). CD40 expression was significantly reduced at month 3 and partially at month 6, compared with baseline, with no reduction in patients who received the vehicle. CD40 ligand expression also decreased at months 3 and 6 in patients taking both concentrations of cyclosporin A. APO2.7 expression was significantly increased in all three groups, whereas percentage of Fas-positive cells decreased only in patients treated with 0.05% cyclosporin A at months 3 and 6. CONCLUSIONS: Flow cytometry provided an objective technique to monitor the effects of topical cyclosporin A on immune- and apoptosis-related markers in the conjunctival epithelium of patients with KCS enrolled in a large multicenter trial. Topical cyclosporin A strikingly reduced HLA DR and to a lesser extent, other inflammatory and apoptotic markers, whereas the vehicle, used as a control tear substitute, had almost no effect. This study confirms that cyclosporin A may be efficient in reducing conjunctival inflammation in moderate to severe KCS and is consistent with clinical results in this indication.

Flow cytometric analysis of conjunctival epithelium in ocular rosacea and keratoconjunctivitis sicca.

PURPOSE: To investigate by flow cytometry and impression cytology (IC) specimens the inflammatory status of the conjunctival epithelium and goblet cell density in two series of patients with rosacea and dry eye syndrome compared with a population of healthy subjects. DESIGN: Nonrandomized, prospective, comparative case series. PARTICIPANTS: Twenty-six eyes of 13 patients with rosacea, 26 eyes of 13 patients with dry eye syndrome, and 24 eyes of 12 control subjects were included in this study. METHODS: IC specimens were collected after clinical examination of the ocular surface and analyzed by flow cytometry, using antibodies directed to human lymphocyte antigen-DR (HLA-DR), intercellular adhesion molecule-1 (ICAM-1) (CD 54), and the peptidic core of the conjunctival mucin (M1/MUC5AC). The percentage of positive cells was calculated and levels of fluorescence expression quantified and compared with those obtained in a series of 12 healthy subjects. MAIN OUTCOME MEASURES: Tear break-up time (TBUT), Schirmer test, fluorescein and lissamin green stainings, and IC were realized in this study. RESULTS: A significant increase of HLA-DR and ICAM-1 expressions by epithelial cells was consistently found in the two pathologic groups compared with levels calculated in normal eyes. The two markers were well correlated with each other and inversely with TBUT and Schirmer test. The percentage of goblet cells was significantly decreased in rosacea patients and in dry eye patients compared with the normal group with a significant negative correlation with both HLA DR and ICAM-1 markers. CONCLUSIONS: Ocular rosacea and keratoconjunctivitis sicca were associated with severe ocular surface changes, such as an overexpression of inflammatory markers and a significant decrease in the number of goblet cells.

Fas- and interferon gamma-induced apoptosis in Chang conjunctival cells: further investigations.

PURPOSE: Previously interferon (IFN)gamma-induced apoptosis and expression of inflammation-related proteins in a human conjunctival cell line were demonstrated. The aim of this study was to further investigate the mechanisms of IFNgamma-, Fas-, and cycloheximide (CHX)-induced programmed cell death, with special attention to the role of transcriptional factors NF-kappaB and STAT1. METHODS: In a human conjunctival cell line (Chang conjunctival cells) apoptosis was induced with 500 ng/ml anti-Fas antibody (anti-Fas ab) alone (24 or 48 hours) or, as previously reported, with 300 U/ml of human recombinant IFNgamma alone (48 hours). To study the role of IFNgamma on Fas-induced apoptosis, cells were treated first with IFNgamma at 30 U/ml during 24 hours (nontoxic dose), and then anti-Fas ab was applied for 24 hours. Moreover, to study the influence of CHX on Fas- and IFNgamma-induced apoptosis, cells were treated for 24 hours with 300 U/ml IFNgamma together with a nontoxic concentration (1 microg/ml) of CHX, or with 500 ng/ml anti-Fas ab together with 1 microg/ml CHX (24 hours). After treatment, cell viability (neutral red assay), mitochondrial membrane potential (rhodamine 123 assay), chromatin condensation (Hoechst 33342 assay), and the index Hoechst/neutral red were studied by cold light microplate cytometry. The apoptotic process was sought for by contrast phase microscopy and DAPI staining and was confirmed by immunoblotting of PARP. Activation of caspase-3 (CPP32) and caspase-8 were investigated by Western blot analysis. NF-kappaB and STAT DNA-binding activities were studied by electrophoretic mobility shift assays (EMSA). RESULTS: After 24 and 48 hours of treatment with anti-Fas ab alone, 15% to 20% and 30%, respectively, of apoptotic cells were observed. When anti-Fas sera were applied after IFNgamma pretreatment or together with CHX, 50% to 80% of cells demonstrated morphologic characteristics of programmed cell death. Apoptosis was confirmed by a cleavage of PARP and CPP32, by caspase-8 activation, and by an index Hoechst/neutral red greater than one. All these modifications were preceded by a decrease in mitochondrial membrane potential. EMSA revealed that NF-kappaB was activated after IFNgamma and anti-Fas ab treatments and inhibited after CHX treatment. STAT1 was strongly activated after IFNgamma treatment and only in a minor degree after anti-Fas ab treatment. STAT1-binding activity persisted after CHX treatment. CONCLUSIONS: The relative resistance of Chang cells toward Fas-induced apoptosis could be related to the activation of NF-kappaB. IFNgamma-induced programmed cell death preferentially involves the activation of STAT1 that counterbalances NF-kappaB antiapoptotic effects. In fact, Fas-induced apoptosis was potentiated by IFNgamma or CHX treatments. These results suggest that NF-kappaB activation could maintain cell viability as well as participate in IFNgamma-induced inflammatory modifications, whereas STAT1 activation could provide, in this model, a proapoptotic signal.

Flow cytometric analysis of inflammatory markers in conjunctival epithelial cells of patients with dry eyes.

PURPOSE: To investigate in impression cytology (IC) specimens the expression of inflammatory and apoptosis-related markers by conjunctival epithelial cells from patients with dry eye as a rationale for treatment with topical cyclosporine. METHODS: Immunologic anomalies were identified at baseline, before treatment with the masked medication, in a homogeneous series of patients with dry eye syndrome, who were enrolled in a large European multicenter clinical trial (Cyclosporin A Dry Eye Study; Allergan, Irvine, CA). IC specimens were collected in 243 patients with moderate to severe keratoconjunctivitis sicca (KCS), with or without Sjogren's syndrome (SS). Fifty normal subjects were separately examined to provide normal control values. Specimens were analyzed in a masked manner by flow cytometry, using antibodies directed to markers of the immune system and/or apoptotic pathway: HLA DR, CD40, CD40 ligand, Fas, and APO2.7. Levels of expression were quantified, and results were compared with those obtained in the 50 normal patients. RESULTS: One hundred sixty-nine specimens were successfully interpreted at baseline, including 41% from patients with SS. A highly significant increase of HLA DR expression by conjunctival cells was found in KCS-affected eyes compared with normal eyes, which did not express this marker or did so very weakly. HLA DR expression in eyes with SS was significantly higher than in KCS-affected eyes without SS. Fas and APO2.7 were found at low levels in all normal and KCS-affected eyes. CD40 and CD40 ligand expressions were significantly increased in eyes with KCS compared with normal eyes. HLA DR, CD40 and Fas were found at significantly higher levels in the SS group than in the non-SS group. CONCLUSIONS. Conjunctival cells from patients with dry eye with moderate to severe KCS, with or without SS, overexpress inflammatory and apoptosis-related markers. Whether inflammation is a primary phenomenon in KCS or is the consequence of repetitive abrasion of the ocular surface after tear film deficiency remains to be determined. These data, nevertheless, support the use of immunomodulatory and/or anti-inflammatory drugs in the treatment of patients with KCS.

[Toxicity of preserved and unpreserved beta-blocker eyedrops in an in vitro model of human conjunctival cells]. / Toxicité des collyres bêta-bloquants avec ou sans conservateur dans un modèle in vitro de cellules conjonctivales humaines.

PURPOSE: To compare the toxicity of a short-time application of timolol with benzalkonium chloride (timolol-BAC+) and unpreserved timolol (timolol-BAC-) in an in vitro model of human conjunctival cells. METHODS: Chang's 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, mitochondrial mass and activity, free radicals production were studied by microplate cold light cytometry. Moreover, relative cell number was evaluated by crystal violet colorimetric test. In addition, cell size and the expression of an apoptotic marker Apo2.7 were studied 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, significantly less important decrease in cell viability was also observed with all tested concentrations of timolol-BAC(-). 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, decrease in mitochondrial membrane potential and cell size reduction. Moreover, cells treated with timolol-BAC(+) overexpressed the apoptotic marker Apo2.7. Also reactive oxygen species (ROS) production was significantly more important after cell exposure to timolol-BAC(+). 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 ROS production and for cell viability variations. Oxidative stress could also play a role in timolol-BAC(+)-induced toxicity. In vitro toxic effects of antiglaucoma drugs could, in part, explain some ocular surface disorders in long-term treated patients.

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: Chang's 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.

Expression of CD40 and CD40 ligand in the human conjunctival epithelium.

PURPOSE: CD40 antigen is a membrane receptor that plays a role in the regulation of immune reactions. The expressions of CD40 and CD40 ligand (CD40L) were investigated ex vivo and in vitro in conjunctival epithelial cells, in correlation with HLA DR class H antigen, previously shown to be upregulated in conjunctival inflammatory conditions. METHODS: Impression cytology specimens were collected in 186 patients: 52 normal ones, 65 with keratoconjunctivitis sicca, and 69 with chronic conjunctivitis. Cells were processed for flow cytometry, by using monoclonal antibodies to CD40, CD40L, and HLA DR antigens. Chang conjunctival cells were also used and treated with human recombinant interferon (IFN)-gamma or tumor necrosis factor (TNF)-alpha. CD40, CD40L, and HLA DR expressions were studied by flow cytometry after 24 and 48 hours of treatment. RESULTS: CD40 was found in both normal and pathologic eyes. Quantitation of levels of fluorescence showed a significantly higher expression in pathologic eyes than in normal ones (P < 0.0001). CD40L was variably and inconstantly expressed by conjunctival cells. A strong expression of HLA DR was observed in pathologic eyes, whereas normal eyes showed very low levels (P < 0.0001). Significantly positive correlations were found among CD40, CD40L, and HLA DR levels. Chang conjunctival cells expressed CD40 in basal conditions, whereas CD40L and HLA DR were negative. CD40 expression significantly increased after 24 hours of IFNgamma treatment and after 48 hours' exposure to TNFalpha. These cytokines had no effect on CD40L expression. HLA DR was upregulated after 24 hours of treatment with IFNgamma but remained negative after exposure to TNFalpha. CONCLUSIONS: Human conjunctival epithelial cells normally express CD40 antigen, and, more inconsistently, CD40L. Flow cytometry showed higher expression of these molecules in inflammatory eyes than in normal ones in correlation with class II antigen expression, as well as CD40 and HLA DR upregulation after treatment with proinflammatory cytokines in vitro.

Correlation between tear IgE levels and HLA-DR expression by conjunctival cells in allergic and nonallergic chronic conjunctivitis.

BACKGROUND: Chronic conjunctival inflammatory diseases may depend upon various mechanisms. Discriminating allergy from nonspecific inflammation has become of striking importance for diagnosis and treatment. We investigated conjunctival inflammatory response by comparing two objective biological tools, tear IgE and HLA-DR expression by conjunctival epithelium, as indirect indicators of activation of the Th2 and Th1 subsets, respectively. METHODS: Eighty-two patients with chronic conjunctivitis underwent tear IgE measurement by an ELISA technique and quantitation of HLA-DR expression in impression cytology specimens. Forty-two had direct or indirect clinical indications of allergic mechanisms, 26 had chronic conjunctivitis without any sign of allergy, and 14 suffered from isolated nonallergic dry eyes. RESULTS: Patients clinically considered as allergic only showed positive IgE in 47 of 84 eyes (56%), whereas 21% and 25% of eyes with nonspecific conjunctivitis and dry eyes respectively were also positive. IgE levels were significantly higher in the allergic group than in the other two groups. HLA-DR positivity in epithelial cells was found in 28.5%, 48% and 50% of eyes, respectively. HLA-DR expression by epithelial cells was negatively correlated with tear IgE, as most specimens positive to one criterion were negative to the other one (49 eyes DR+, IgE-; 47 eyes DR-, IgE+; only 9 eyes positive to both criteria; chi-square: P = 0.0001). CONCLUSION: As IgE synthesis and HLA-DR induction may represent indirect indicators of the activation of the Th2 and Th1 subsets, association of these two simple tests could be interesting for the routine assessment of the mechanisms of inflammatory ocular surface diseases.

Interferon-gamma induces apoptosis and expression of inflammation-related proteins in Chang conjunctival cells.

PURPOSE: The purpose of this study was to investigate the effect of interferon (IFN)gamma on cell viability, cell growth, and apoptosis and on expression of apoptotic and inflammation-related proteins in epithelial conjunctival cells in vitro. Some aspects of transduction pathways of IFNgamma-induced alterations were also investigated, especially the role of protein kinase C (PKC) and IFNgamma transcriptional factor STAT1. METHODS: A human conjunctival cell line was treated with different concentrations (30 and 300 U/ml) of human recombinant IFNgamma. After 24, 48, and 72 hours of treatment, cell viability and relative cell number were studied with 3-(4,5-dimethylthiazol-2yl)2,5-diphenyl tetrazolium bromide (MTT) and crystal violet colorimetric assays. The apoptotic process was sought by phase-contrast microscopy, 4',6'-diamidino-2-phenylindole dihydrochloride (DAPI) staining, and transmission electron microscopy and was confirmed by DNA electrophoresis and immunoblotting of poly(ADP-ribose) polymerase (PARP). The cell cycle and expression of apoptotic proteins Fas, bax, and p53; of inflammation-related proteins HLA-DR and intercellular adhesion molecule (ICAM)-1; and of IFNgamma signal-transducing factor STAT1 were evaluated by flow cytometry and/or western blot analysis. To investigate PKC-related transduction pathways, two PKC modulators, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and staurosporine, were applied for 3 hours, followed by IFNgamma treatment for 72 hours. Moreover, the effects of PKC depletion were studied after a 24-hour application of TPA, also followed by IFNgamma treatment for 72 hours. Then, Fas, ICAM-1, and HLA-DR expressions were studied by flow cytometry. RESULTS: IFNgamma at 30 U/ml induced no change in cell cycle and in cell viability. Cell viability significantly decreased after 48 hours of treatment with 300 U/ml IFNgamma, associated with cell cycle alterations (decrease in number of cells in the S-M phase), apoptotic chromatin condensation and fragmentation, ladder pattern on DNA electrophoresis assay, and cleavage of PARP. Moreover, IFNgamma-treated cells overexpressed plasma membrane Fas, HLA-DR, and ICAM-1 in a dose- and time-dependent manner, and STAT1 in both nuclear and cytosolic cell fractions. Only 300 U/ml IFNgamma-treated cells overexpressed bax, whereas Bcl-2 and p53 proteins were not modified. HLA-DR and Fas were upregulated after addition of staurosporine or after PKC-depleting treatment and repressed with TPA. Staurosporine, PKC depletion, and TPA all enhanced ICAM-1 expression. CONCLUSIONS: In our model, IFNgamma induced expression of inflammatory molecules and apoptotic mediators, cell growth arrest, and apoptosis of Chang conjunctival cells. Moreover, our results suggest that activation of PKC is not involved in some IFNgamma cellular effects that possibly imply the upregulation and nuclear translocation of STAT1. IFNgamma-induced apoptosis could explain in part the recently reported coexistence of inflammation and programmed cell death in ocular surface inflammatory disorders such as Sjögren's syndrome.