Biomedical subjects
C Baudouin
Publications and source records attributed to C Baudouin.
[Evaluation of free radical production by conjunctival impression cytology of patients treated with long-term antiglaucoma drugs or of contact lens wearers].
PURPOSE: To investigate free radical production in impression cytology specimens using microplate cold light cytofluorimetry. METHODS: 60 impression cytology specimens (IC) were harvested in 30 patients aged from 20 to 90 years. IC were taken in 10 healthy subjects, 10 glaucoma patients receiving longterm treatment for glaucoma and 10 contact lens wearers for more than five years. A complete ophthalmologic examination (BUT, corneal staining) was performed. Evaluation of free radical production was done using a dichlorofluoresceine diacetate (DCFHDA) test for H(2)O(2) evaluation and a hydroethidine test for O(2)(.)- measurement. RESULTS: The BUT values in the two pathological groups were significantly lower than those of controls (p<0.001). A free radical production was found in both contact lens wearers (p<0.001 compared to control values) and glaucoma patients (p<0.001 compared to control values). Both hydrogen peroxyde and superoxide anion productions were significantly correlated to the decrease of BUT values and the increase of superficial punctuate keratitis. CONCLUSION: The results of this preliminary study confirm that conjunctival epithelial cells may abnormally produce oxygen free radicals in chronic ocular surface disorders. This may participate to the cellular alterations observed in inflammatory diseases of ocular surface.
[Consensus on neovascular glaucoma].
Neovascular glaucoma is a dreadful pathology with a rapid spontaneous evolution responsible for painful and blind eye. The main cause is an anterior neovascular proliferation following a broad retinal ischemia. Early diagnosis and treatment are required in order to maintain a good visual status and a satisfactory IOP control with medical, surgical or cylodestructive procedures. In any case, the treatment of the retinal ischemia has to be performed. One must keep in mind that the most efficient way to avoid the incidence of neovascular glaucoma is a strict control of clinical situations potentially responsible for retinal ischemia, namely VRO in elderly patients and diabetic retinopathy in younger patients.
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.
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.
[Cytotoxicity evaluation of three tear substitutes used in the treatment of dry eye syndromes].
PURPOSE: To investigate cell tolerance of three tear substitutes used in the treatment of dry eye syndromes. METHODS: Cytotoxicity tests were done on a continuous human conjunctival cell line using microplate cold light cytofluorimetry. Membrane integrity, DNA condensation and reactive oxygen species (ROS) production (hydrogen peroxyde and superoxide anion) were evaluated after 15 minutes of treatment or 15 minutes and 24 hours of cell recovery. Hyaluronic acid, hydroxypropyl methylcellulose associated with sodium perborate (HPMC) and carbomer 934P were tested at their commercial concentrations (respectively 0.18%, 0.3% and 0.3%) and after a 1/10 dilution. RESULTS: Cell viability and chromatin condensation were not altered by hyaluronic acid for all concentrations and times tested. A decrease in membrane integrity was observed with 0.3% carbomer 934P after 24 hours of cell recovery and with 0.3% HPMC after 15 minutes. This decrease was amplified after 24 hours and associated with an apoptotic phenomenon. A H(2)O(2) production was observed with HPMC associated with sodium perborate and an O(2) production was found with hyaluronic acid diluted at 1/10. CONCLUSION: Hyaluronic acid, carbomer and HPMC are in vitro well-tolerated even if HPMC induces a more important decrease of cell viability compared to the other drugs. Hyaluronic acid, with its rheologic properties, with no in vitro toxicity, seems to be efficient for dry eye patients.
[Relevance of quality of life and treatment compliance measurement in patients with chronic open-angle glaucoma].
Chronic glaucoma is a severe disease that can induce blindness.Early diagnosis and symptomatic treatment reduce the risk of blindness. Treatment that will be started before the onset of clinical signs and will remain lifelong thereafter is troublesome, and therapeutic compliance is usually poor. Thus, quality of life (QOL) measurement in patients with chronic glaucoma has a particular purpose: to measure patients' perception of the disease and treatment in order to maintain good treatment compliance to ensure therapeutic management efficacy and to preserve visual function. No glaucoma-specific instrument is available in the medical and QOL literature. Various generic(SF-36, SF-20 and SIP) and specific(VAQ, VF-14, NEI-VFQ) QOL questionnaires,one glaucoma-specific symptomatic scale (GSS),and one treatment preference scale (COMTol) have been used to measure QOL in glaucoma patients. These instruments do not sufficiently measure the psychosocial dimension of the disease and the QOL impact of treatment. An instrument able to measure all dimensions needs to be developed in order to help ophthalmologists in the therapeutic management of their patients and to measure QOLin patients in the successive stages of the disease.
Effects of EGb761 and superoxide dismutase in an experimental model of retinopathy generated by intravitreal production of superoxide anion radical.
BACKGROUND: A study was carried out to investigate the effect of two antioxidants--Ginkgo biloba extract (EGb761) and superoxide dismutase (SOD)--in an experimental model of vitreoretinopathy obtained by direct production of oxygen free radicals in the vitreous cavity. METHODS: Twenty-eight pigmented rabbits were used. Vitreoretinopathy was induced by intravitreal injection of 50 microliters of a mixture composed of 40 nmol of xanthine and 0.001 IU of xanthine oxidase. Rabbits were randomly distributed into four groups: Group 1 (n = 8) did not receive any treatment and served as a positive control. Groups 2 (n = 8) and 3 (n = 8) received for 1 month EGb761 given orally at a dose of 100 mg/kg/day, respectively 1 day after and 1 week before induction of retinopathy. Group 4 (n = 4) was treated by three intramuscular injections of 15,000 IU/kg of SOD, 24 h before induction and 24 and 48 h thereafter. Clinical evaluations and electroretinograms (ERG) were repeatedly performed until the animals were killed at day 28. Histological examinations and immunohistological procedures were performed to ascertain the origin and characteristics of the cellular proliferation and to compare vitreoretinal structures in the four groups. RESULTS: Intravitreal injection of xanthine-xanthine oxidase produced a strong inflammatory response with vitreous infiltrates and epiretinal membrane formation, inconstantly associated with retinal detachment. ERG showed a decrease of the a-, b- and c-waves beginning within a few hours after injection. Histologic evaluation found an intravitreal and epiretinal infiltration by leukocytes and epithelial-derived cells, dense vitreoretinal membranes and retinal detachments with occasional neovascularization. In the treated groups (groups 2-4), all clinical, electric and histologic data were significantly improved compared to the control group. However, no difference could be found among the three treated groups. CONCLUSION: This study demonstrates the strong pathologic effects of free radical production on the retina and the close relationships between free radicals, inflammatory pathways and vitreoretinal proliferative disorders. It also confirms the pharmacological interest of prevention by antioxidants and free radical scavengers.
Ocular surface inflammatory changes induced by topical antiglaucoma drugs: human and animal studies.
OBJECTIVES: To investigate conjunctival and trabecular specimens from patients with glaucoma according to the duration and number of drugs received before filtration surgery, and to confirm, in a complementary experimental model, the role of preservative by comparing the effects of preserved and nonpreserved timolol. STUDY DESIGN: Experimental animal and human tissue study. PARTICIPANTS: Paired specimens of conjunctiva and trabeculum were taken from 61 patients undergoing trabeculectomy. Twenty-six patients were treated with 2 or more drugs for at least 1 year; 30 had received a beta-blocker for more than 1 year and 5 underwent primary surgery. A second study was performed in 25 rats receiving topical solutions in both eyes for 1 month. INTERVENTION: Immunohistochemistry was performed in all biopsy specimens using 12 different monoclonal antibodies. Ocular structures from rats treated for 1 month with preserved 0.5% timolol, nonpreserved 0.5% timolol, or 0.01% benzalkonium chloride were similarly investigated in an experimental study. MAIN OUTCOME MEASURES: Inflammatory cell infiltrates and fibroblasts were evaluated in biopsies, as well as in animal specimens, together with histologic changes induced by the drugs applied. RESULTS: Twenty-four of 26 conjunctivae and 21 of 24 trabecular pieces from multitreated patients were found to be abnormally infiltrated by cells expressing inflammatory or fibroblastic markers or both. Nineteen of 30 conjunctivae and 9 of 22 trabeculums in the monotherapy group and only 1 of 5 specimens from the primary surgery group were abnormal. In rats, preserved timolol and benzalkonium similarly showed infiltrates together with toxic histopathologic changes as compared to the nonpreserved timolol and control groups. CONCLUSIONS: These two combined studies confirmed histopathologic effects of antiglaucomatous drugs on the conjunctiva and showed similar effects in the trabecular meshwork. The experimental study showed that benzalkonium chloride is at least, to a large part, responsible for these toxic or immunoinflammatory effects or both on the ocular structures.
Effects of benzalkonium chloride on growth and survival of Chang conjunctival cells.
PURPOSE: The aim of this study was to investigate the action of benzalkonium chloride (BAC), used as a preservative in most ophthalmic topical solutions, on epithelial conjunctival cells in vitro. METHODS: A continuous human conjunctival cell line (Wong-Kilbourne derivative of Chang conjunctiva) was exposed to BAC solutions at various concentrations (0.1%-0.0001%) during a period of 10 minutes. Cells were examined before treatment and 3, 24, 48, and 72 hours later, after reexposure to normal cell culture conditions. Cell number and viability were assessed with crystal violet and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide colorimetric assays. The expression of the apoptotic marker Apo 2.7, nuclear antigen p53, membrane proteins Fas and Fas ligand, and DNA content was studied by flow cytometry. Morphologic aspects of cell nuclei were analyzed on slides with a nucleic acid-specific dye, 4',6'-diamidino-2-phenylindole dihydrochloride. Cytoskeleton was labeled with a monoclonal anti-pancytokeratin antibody. In addition, apoptosis was measured by DNA electrophoresis assays in agarose gel. RESULTS: Cell exposure to 0.1% and 0.05% BAC induced cell lysis immediately after treatment. All cells (100%) treated with 0.01% BAC died in a delayed manner within 24 hours, with most of the characteristics of apoptosis (chromatin condensation and DNA fragmentation, reduction in cell volume, expression of the apoptotic marker Apo 2.7, and apoptotic changes in DNA content). Aliquots of 0.005%, 0.001%, 0.0005%, and 0.0001% BAC induced growth arrest and apoptotic cell death in a dose-dependent manner between 24 and 72 hours after treatment. The expressions of Fas and p53 did not vary after BAC treatment. Fas ligand was always negative. CONCLUSIONS: These results suggest that BAC induces cell growth arrest and death at a concentration as low as 0.0001%. The mode of BAC-induced cell death is dose-dependent. Cells die by necrosis after BAC treatment at high concentrations and by apoptosis if low concentrations of BAC are applied. This new aspect of in vitro toxicity of BAC could in part explain some ocular surface disorders observed in patients undergoing long-term topical treatments with preservative-containing drugs.
[The patient with glaucoma and his treatment: is there real perception?].
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[Clinical tolerance of antiglaucoma eyedrops with and without a preservative. Results of an unpublished survey in Europe].
PURPOSE: To evaluate the clinical tolerance of antiglaucoma eye drops with and without a preservative in routine practice. METHODS: A total of 125 ophthalmologists in private practice located throughout France examined altogether 919 glaucomatous patients treated with eye drops containing a preservative or not. For some patients the examination was repeated during a second visit. Functional signs as well as conjunctival and corneal examination results were recorded. RESULTS: The proportion of patients experiencing discomfort or pain during instillation was 58% for eye drops containing a preservative and 30% for eye drops with no preservative (p < 0.001). Moreover, the proportion of patients presenting at least one symptom of eye irritation (sensation of itching or burning, sensation of a foreign body in the eye, and flow of tears) was greater with preservative-containing eye drops (53% vs 34%; p < 0.001). The experience of discomfort during instillation was more often associated with problems later on. The patient's complaints were correlated with objective signs of conjunctival (conjunctival redness, conjunctival follicles), or corneal (superficial punctuate keratitis) damages. A higher proportion of patients treated with eye drops containing a preservative showed at least one conjunctival sign (52% vs 35%; p = 0.001) or superficial punctuate keratitis (12% vs 4%; p = 0.01). In 164 patients, whose treatment was changed from eye drops containing a preservative to eye drops with no preservative and who were examined a second time (mean interval between visits: 3.3 months) the frequency of all symptoms and objective signs fell by a factor of 3 to 4 (p < 0.001). CONCLUSION: Patient complaints and objective damage to the ocular surface are more frequent among patients treated with eye drops containing a preservative. Ocular surface damage due to preservatives is partially reversible. Patient complaints should consequently be taken into account and preservative-free eye drops should be prescribed when ever possible.
[Anterior chamber and retinal detachment].
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[Clinical exploration of the lipid phase of the laryngeal film using Tearscdope plus].
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[Conjunctivitis and ocular parasitic diseases].
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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.