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C Baudouin

Publications and source records attributed to C Baudouin.

At least 73 records · Page 4Linked to original sources
PubMed2002

[Contribution of intravitreal infracyanine green to macular hole and epimacular membrane surgery: preliminary study].

PURPOSE: To evaluate the contribution of intravitreal infracyanine green to macular hole and epimacular membrane surgery. PATIENTS AND METHODS: Twenty-five consecutive patients were operated on for macular hole (seven cases) or epiretinal membrane (18 cases) using intravitreal infracyanine. In most cases, 25mg of infracyanine powder was diluted in 5ml of solvent, then in 5ml of BSS and used within 1 hour. The solution was injected at the end of the vitrectomy and aspirated after 1-2 minutes. The internal limiting membrane was removed in both diseases. All specimens were studied using confocal microscopy or an immunocytochemical preparation. RESULTS: In cases of macular hole, the internal limiting membrane was bright green colored. Opening and peeling were greatly facilitated. In cases of epiretinal membrane, staining was variable: the most frequent aspect was an uncolored central area similar to a geographic map surrounded by a green stained zone. Sometimes the coloration appeared as sparse spots on the posterior pole. Occasionally the staining was homogeneous as in the macular hole cases. After the removal of the membrane, a second injection made it possible to check for the persistence of the internal limiting membrane and proceed to its ablation when necessary. A good correlation was found between surgical and histopathological aspects. CONCLUSION: Intravitreal infracyanine green is very helpful for the removal of internal limiting membrane in macular holes and epiretinal membrane surgery.

Epiretinal Membrane↗
PubMed2001

The pathology of dry eye.

Homeostasis of the tear film involves delicate hormonal and neuronal regulatory mechanisms. The eye appears to be a target organ for sex hormones, particularly the androgens, as they modulate the immune system and trophic functions of the lacrimal glands and the functioning of the meibomian glands. The cornea, lacrimal glands, mucous cells, and meibomian glands are all richly innervated, indicating the importance of nervous regulation in their function. Parasympathetic, sympathetic, and sensory innervation play complex stimulatory or inhibitory roles, and neuronal pathways interact via complex surface results cascades. Abnormalities at any point in these pathways can cause overall dysregulation of lacrimal function. Whatever the initial causes of dry eye, chronic dryness of the ocular surface results in inflammatory reactions and gradual destruction of the lacrimal glands and conjunctival epithelium. Once dry eye disease has developed, inflammation is the key mechanism of ocular surface injury, as both the cause and consequence of cell damage. In practice, dry eye can be associated with Sjögren's syndrome, allergies, infection, blepharitis, and preservative-containing eye drops.

Dry Eye Syndromes↗
PubMed2001

Evaluation of corneal stromal changes in vivo after laser in situ keratomileusis with confocal microscopy.

PURPOSE: To assess by in vivo confocal microscopy the modifications of the corneal stroma after laser in situ keratomileusis (LASIK) for myopia. DESIGN: Nonrandomized comparative (self-controlled) trial. PARTICIPANTS: Sixteen eyes of 13 patients were examined before surgery and at days 8, 30, and 90, and 9 eyes were examined at 6 months postoperatively using an in vivo confocal microscope. TESTING/INTERVENTION: Stromal morphologic changes, keratocyte density, flap thickness, and subclinical haze were evaluated and compared at different time points. LASIK was performed with a Flapmaker microkeratome (Solan Ophthalmic products, Jacksonville, FL) and a Lasersight LSX excimer laser (LaserSight Technologies Inc., Winter Park, FL). MAIN OUTCOME MEASURE: Confocal microscopy results. RESULTS: Microfolds at the Bowman's layer were found in most eyes, as well as variable reflectivity particles (pa) located at the interface level in all eyes examined postoperatively. The density of these particles significantly decreased with time with, respectively, 504 +/- 101 pa/mm2 at day 8 and 380 +/- 111 pa/mm2 at day 30 (P = 0.003), 332 +/- 100 pa/mm2 at month 3 and 312 +/- 40 pa/mm2 at month 6. The mean flap and the activated-cells area thicknesses were, respectively, 102 +/- 26 microm and 61 +/- 19 microm and showed significant negative correlation (P < 0.0001). The intensity of the added peak (47.3 microm 8.6%), corresponding to the subclinical haze, realized by Z-scan measure, was also negatively correlated with flap thickness (P = 0.01). Keratocyte (k) density quantified in the posterior stroma significantly increased from day 0 (480 +/- 67 k/mm2) to day 8 (701 +/- 41 k/mm2, P < 0.0001 compared with day 0) and day 30 (917 +/- 143 k/mm2, P = 0.0006, compared with day 0) but significantly decreased at 3 months postoperatively (597 +/- 56 k/mm2, P < 0.0001 compared with day 30) to reach the initial level at month 6 (502 +/- 41 k/mm2, nonsignificant compared with day 0). There was no correlation between preoperative or postoperative spherical equivalent and the density of particles, keratocytes, and the haze intensity. CONCLUSIONS: This study confirms the presence of microfolds and particles at the interface level, as well as subclinical impairment. Evaluation of keratocyte density constitutes a major contribution of confocal microscopy toward an understanding of the keratocyte response to corneal wound healing after corneal refractive surgery. Moreover, flap thickness seems to be involved in the postoperative cellular activation with a higher response when thin.

Adult↗
PubMed2001

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.

Adult↗
PubMed2001

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.

Administration, Topical↗
PubMed2001

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.

Apoptosis↗
PubMed2001

[Evaluation of confocal microscopy in the analysis of the external trabecular membrane during deep nonpenetrating sclerectomy].

PURPOSE: Deep non penetrating sclerectomy (DNPS) is a new filtering surgical procedure whose aim is a selective ablation of the external portion of the trabecular meshwork (TM) which is involved in the aqueous outflow resistance, i.e. the inner wall of Schlemm's canal (SC) and the external trabecular layers, especially the cribriform TM. We evaluated, with a confocal microscope, the structural characteristics of this part of the TM in glaucomatous patients. METHODS: Thirty-six external trabecular membranes (ETM) were obtained from 33 consecutive glaucomatous patients (mean age: 56.5+/-14.5 years) and from four post-mortem normal donors (60.5 +/-7.7 years), which underwent DNPS according to the same surgical procedure. Under conjunctival and scleral flaps, the roof of the SC was opened and removed. A deeper dissection led to the removal of the inner wall of the SC and the adjacent ETM, i.e the TEM, which allowed a satisfactory aqueous flow through the remaining internal TM layers. After fixation with acetone or triton X100 and immunostaining with anti-fibronectin or vimentin antibodies, the samples were analyzed with a confocal microscope (Nikon EZ 2000). RESULTS: The mean thickness of the ETM was 34.4+/-7.3 microm in glaucomatous eyes, not significantly different from the controls (39.0+/-10.7 microm). The main characteristic of the glaucomatous ETM membrane is a paucicellularity as compared to the controls (respectively 21.6+/-12.1 cells/area and 156.1+/-28.8 cells/area). The confocal microscope analysis shows that the ETM involves two different portions of the trabecular meshwork. The architectural characteristics of the outermost portion of the ETM with its star-shaped cells arranged in a homogenous extra-cellular matrix, suggest that it is the trabecular cribriform meshwork. The inner portion of the ETM shows cells arranged regularly in a fibrillar extra-cellular matrix as described in the corneoscleral trabecular meshwork. CONCLUSION: These results confirm the previous reported histopathological changes of the glaucomatous TM. The thickness of the removed ETM and its structural characteristics show that the ablation of the TM layers necessary to obtain a satisfactory aqueous filtration, not only involves the cribriform layers but also one of the inner parts of the TM, i.e. the corneoscleral TM. This result suggests that aqueous humor resistance not only involves the cribriform trabecular meshwork but also a part of the corneoscleral trabecular meshwork.

Aged↗
PubMed2001

[Antioxidant effects of mast cell inhibitors in a human conjunctival cell line].

PURPOSE: The aim of this study was to evaluate in vitro antioxidant effects of two mast cells inhibitors. METHODS: Cytotoxicity tests were done on a continuous human conjunctival cell line using microplate cold light cytofluorimetry. Membrane integrity (neutral red test), DNA condensation (Hoechst 33342 test), and reactive oxygen species (ROS) production (dichlorofluoresceine diacetate and hydroethidine tests) were evaluated on living cells treated with sodium cromoglycate and N-acetyl-aspartyl glutamic acid (NAAGA) preserved (benzalkonium chloride: BAC at 0.01%) and unpreserved after 60 minutes of treatment or 60 minutes and 24 hours of cell recovery. They were tested pure and at a 1/10 dilution. ROS production was also evaluated after a 60 minute pretreatment with antiallergic drugs and a 15-minute treatment with BAC, according to previous experiments performed on BAC showing its ROS production properties. RESULTS: No cytotoxicity was observed with the unpreserved formulations of antiallergic drugs. An apoptotic phenomenom was suggested with preserved drugs after a 1-hour treatment, whereas a necrotic mechanism appeared after a 24-hour cell recovery period. A ROS production decrease was observed with the two preserved and unpreserved drugs tested (p<0.001 compared to BAC) even if it was significantly higher with cromoglycate formulations. A ROS production decrease also was detected after a pretreatment with antiallergic drugs and treatment with BAC (p<0.001 compared to BAC alone). CONCLUSION: In vitro, no cytotoxicity was found with the two unpreserved mast cell inhibitors tested. An antioxidant effect also was observed with these two molecules; sodium cromoglycate appeared to be the best free radical scavenger.

Anti-Allergic Agents↗
PubMed2001

[The lipid layer of the lacrimal tear film: physiology and pathology].

The preocular tear film (POTF) is composed of a deep aqueous-mucin phase that supports a thin superficial lipid phase. The tear lipid layer (TLL), although thin, stabilizes the POTF providing a 25% surface-tension decrease and a 90-95% aqueous evaporation reduction. TLL is formed from lipids secreted by tarsal meibomian glands and spread onto the ocular surface by blinking. The TLL itself is composed of two phases. A thin and deep polar phase, adjacent to the aqueous-mucin layer, has a surfactant role. A thicker and superficial nonpolar phase has antievaporative properties. At the same time, tear lipocalins help the TLL spread and stabilize the lipid-aqueous interface. For clinical examination, TLL is directly observed with the Tearscope. POTF stability and the evaporation rate depend on the lipid layer pattern. When chronic, POTF qualitative trouble (evaporative syndrome) due to a TLL anomaly, leads to secondary ocular surface impairment with increased tear instability and self-propagation of ocular dryness. Meibomian gland dysfunction (MGD) results from local pathology, dermatologic disease (ocular rosacea) or iatrogenic etiology. Cosmetic use is the other principal cause of TLL destabilization. Lid hygiene is the mainstay of MGD treatment. Systemic antibiotics (cyclins) can be associated in cases of severe symptoms. Topical treatment is useful if there is marginal lid inflammation or infection.

Humans↗
PubMed2001

Mitochondrial activity and glutathione injury in apoptosis induced by unpreserved and preserved beta-blockers on Chang conjunctival cells.

PURPOSE: Quaternary ammonium ions have been demonstrated to induce apoptosis correlated with superoxide anion production in vitro. The purpose of this study was to further investigate the mechanisms of benzalkonium chloride (BAC), unpreserved and preserved beta-blocker eye-drops-induced programmed cell death, with special attention to the roles of mitochondrial transmembrane potential and intracellular reduced glutathione. METHODS: Chang conjunctival cells were incubated with different concentrations of unpreserved or preserved timolol (0.1%, 0.25%, and 0.4%), or carteolol (1% and 2%), or BAC (0.0001% to 0.01%) for 15 minutes, or for 15 minutes with a 24-hour recovery period in normal medium. Cellular viability (neutral red test), mitochondrial activity (rhodamine 123 test), intracellular reduced glutathione (monochlorobimane test), DNA condensation (Hoechst 33342 test), and reactive oxygen species (ROS) production (dichlorofluorescein diacetate and hydroethidine tests) were evaluated using microplate cold-light cytofluorometry. RESULTS: A significant, concentration-dependent decrease in cellular viability was found with preserved beta-blockers and with BAC alone, whereas unpreserved preparations did not show any toxicity. Only preserved beta-blockers induced chromatin condensation associated with an alteration of mitochondrial activity and a decrease of glutathione, suggesting an apoptotic phenomenon. BAC increased glutathione after 15 minutes, whereas a decrease was observed after a recovery period. ROS production was found with preserved formulations at significantly higher levels than those observed with unpreserved drugs. CONCLUSIONS: This in vitro study demonstrates that oxidative stress, evidenced by enhanced ROS production and mitochondrial injury rather than by cellular glutathione depletion, is a mechanism involved in apoptosis induced by preservative-containing eye-drops.

Adrenergic beta-Antagonists↗
PubMed2001

[When should glaucoma be surgically treated?].

The medical treatment of primary open angle glaucoma has progressively become more and more efficient and safe, and surgery is therefore mostly restricted to failure of and intolerance to antiglaucoma eyedrops. Glaucoma surgery may thus cause severe complications and a high risk of failure has tempered its prognostic. Nevertheless, when efficacious, glaucoma surgery definitively resolves two major pitfalls of medical treatment: patient compliance and eyedrops tolerance. Moreover, new surgical developments of surgery have come from the new technique of non-penetrating deep sclerectomy, which is actually an external trabeculectomy involving removal of the area of maximal resistance to aqueous outflow. This procedure has a very low risk of complications, much lower than that of standard trabeculectomy, but its efficacy is still controversial. Whatever the technique chosen for filtering surgery, antimetabolites may be used in order to limit the risk of postoperative fibrosis, but they also expose to specific, sometimes sight-threatening, complications. Therefore, the most important--and also the most difficult--choice for treating glaucoma patients still remains the best timing for surgery, either excessive, useless, and aggressive medical treatment, or systematic primary surgery.

Glaucoma↗
PubMed2000

Distinct Chk2 activation pathways are triggered by genistein and DNA-damaging agents in human melanoma cells.

Genistein, a natural isoflavone found in soybeans, exerts a number of biological actions suggesting that it may have a role in cancer prevention. We have previously shown that it potently inhibits OCM-1 melanoma cell proliferation by inducing a G(2) cell cycle arrest. Here we show that genistein exerts this effect by impairing the Cdc25C-dependent Tyr-15 dephosphorylation of Cdk1, as the overexpression of this phosphatase allows the cells to escape G(2) arrest and enter an abnormal chromatin condensation stage. Caffeine totally overrides the genistein-induced G(2) arrest, whereas the block caused by etoposide is not bypassed and that caused by adriamycin is only partially abolished. We also report that genistein activates the checkpoint kinase Chk2 as efficiently as the two genotoxic agents and that caffeine may counteract the activation of Chk2 by genistein but not by etoposide. In contrast, caffeine abolishes the accumulation of p53 caused by all the compounds. Wortmannin does not suppress the Chk2 activation in any situation, suggesting that the ataxia telangiectasia-mutated kinase is not involved in this regulation. Finally, unlike etoposide and adriamycin, genistein induces only a weak response in terms of DNA damage in OCM-1 cells. Taken together, these results suggest that the G(2) checkpoints activated by genistein and the two genotoxic agents involve different pathways.

Caffeine↗
PubMed2000

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.

Adolescent↗
PubMed2000

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.

Adult↗
PubMed2000

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.

Adrenergic beta-Antagonists↗
PubMed2000

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.

Adult↗
PubMed2000

[Toxicity of preserved and unpreserved beta-blocker eyedrops in an in vitro model of human 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 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.

Adrenergic beta-Antagonists↗