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Normal Human Tear Film Lipid Vesicles
TEAR FILM & OCULAR SURFACE NORMAL HUMAN TEAR FILM LIPID VESICLES. Pamela Steele and Douglas Borchman. Department of Ophthalmology and Visual Sciences, University of Louisville, Louisville KY, USA. Purpose. A significant amount of lipid is present in the aqueous-mucin layer of human tears. Studies show that the surface tension of tears is due to lipocalin-lipid complexes. The purpose of this study was to determine if liposomes are present in tears and whether they are associated with proteins. Methods. Reflux tears were collected by sterile pipette. Liposomes were isolated from acidified tears by centrifugation at 1,100 X g. Liposomal pellets were identified by scanning electron microscopy and lipid was confirmed by infrared spectroscopy. Liposome pellets were run on SDSPAGE to determine the presence of proteins. Isolated tear lipid was combined with normal human tear fluid and tear fluid absent of proteins and mucins. Tears were examined by infrared, fluorescence and matrix –assisted laser desorption ionization-mass spectroscopy. Results. Transmission electron micrographs revealed liposomes ranging in size from 500 to 2000 nm. Infrared analyses of prepared liposomes confirmed the presence of lipids. Tear lipid hydrocarbon order, lipid-lipid interactions and sphingomyelin were relatively higher in younger individuals (~18 y) as compared to mature individuals (~50 y), while triacylglycerol levels were lower in younger individuals. Three major proteins with molecular masses of approximately 15 kDa, 20 kDa and greater than 250 kDa and two minor bands 9 kDa and 6 kDa were associated with purified liposomes. Fluorescence data indicated a component of tears greater than 5 kDa interacted with the lipid head group region but not the hydrocarbon chain region, and upon binding with the lipid, excluded water as at the tear fluid – tear lipid interface. Conclusions. A significant amount of lipid is present in aqueous human tears and its composition and physical properties appears to be age dependent. Tear lipids form liposomes and an unidentified component in tears binds to the lipid, expelling water. Proteins appear to be associated, perhaps bound to liposomes. The present spectroscopic and biochemical data provide insight into the molecular properties and modifications that may affect human tear film stability. Support: EY13860, EYO7975 (PS), the Jewish hospital Foundation and an unrestricted grant from Research to Prevent Blindness Inc., NY
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EVALUATION OF OCULAR SURFACE INFLAMMATION IN THE PRESENCE OF DRY EYE AND ALLERGIC CONJUNCTIVAL DISEASE. Michael E. Stern,1 Karyn F. Siemasko,1 Jianping Gao,1 Margarita Calonge,2 Virginia L. Calder,3 Stephen C. Pflugfelder,4 Jerry Y. Niederkorn.5 Allergan, Inc. Irvine, CA, USA;1 IOBA, Univ. of Valladolid, Spain;2 Univ. College London, London, United Kingdom;3 Baylor College of Medicine, Houston, TX;4 Department of Ophthalmology, Univ. of Texas Southwestern Medical Center, Dallas, TX, USA.5 Purpose. The effects of IFN-J, a classic TH1 type inflammatory mediator, on ocular inflammatory events in a mouse model of allergic conjunctivitis (classic TH2 animal model) alone or combined with a desiccating environment (classic TH1 animal model) were evaluated in BABL/c WT mice and IFN-J knockout mice. Methods. BALB/c WT mice or IFN-J knockout (KO) mice were sensitized on day 1 with 20 Pg SRW in alum followed by multiple SRW topical challenges on days 10-16 in the absence or presence of animals additionally being treated with scopolamine 3X/day in the presence of a low humidity chamber and constant air flow. Results. Exposing SRW sensitized and challenged mice to a desiccating environment (TH1-type stimulus) significantly increased the number of inflammatory cells infiltrating the conjunctiva as compared to SRW sensitization and multi-hit challenge alone. SRW sensitized and challenged IFN-J KO mice had a significantly reduced conjunctival inflammatory cell infiltrate as compared to the SRW challenged WT group (77% decrease in eosinophils; 35 % decrease in neutrophils). Conclusions. Inflammatory cell infiltrations into the conjunctiva of SRW sensitized and challenged animals in the presence of a desiccating environment were significantly increased suggesting that the presence of TH1 cytokine mediated disease can further exacerbate a TH2-like disease pathology. IFN-J is unexpectedly essential for the immunopathology of allergic conjunctivitis disease. IFN-J regulation of adhesion molecules required for extravasation of inflammatory cells into sites of disease are currently under investigation. Commercial Relationship(s): M. E. Stern, Allergan, Inc. E.; K. F. Siemasko, Allergan, Inc. E; J. Gao, Allergan, Inc. E; M. Calonge, Allergan, Inc. C; V. L. Calder, Allergan, Inc. C; S. C. Pflugfelder, Allergan, Inc. C; J. Y. Niederkorn, Allergan, Inc. C.
THE OCULAR SURFACE / JANUARY, 2005, VOL. 3, NO. 1 / SUPPLEMENT
S116
EVALUATION OF OCULAR SURFACE INFLAMMATION IN THE PRESENCE OF DRY EYE AND ALLERGIC CONJUNCTIVAL DISEASE. Michael E. Stern,1 Karyn F. Siemasko,1 Jianping Gao,1 Margarita Calonge,2 Virginia L. Calder,3 Stephen C. Pflugfelder,4 Jerry Y. Niederkorn.5 Allergan, Inc. Irvine, CA, USA;1 IOBA, Univ. of Valladolid, Spain;2 Univ. College London, London, United Kingdom;3 Baylor College of Medicine, Houston, TX;4 Department of Ophthalmology, Univ. of Texas Southwestern Medical Center, Dallas, TX, USA.5 Purpose. The effects of IFN-J, a classic TH1 type inflammatory mediator, on ocular inflammatory events in a mouse model of allergic conjunctivitis (classic TH2 animal model) alone or combined with a desiccating environment (classic TH1 animal model) were evaluated in BABL/c WT mice and IFN-J knockout mice. Methods. BALB/c WT mice or IFN-J knockout (KO) mice were sensitized on day 1 with 20 Pg SRW in alum followed by multiple SRW topical challenges on days 10-16 in the absence or presence of animals additionally being treated with scopolamine 3X/day in the presence of a low humidity chamber and constant air flow. Results. Exposing SRW sensitized and challenged mice to a desiccating environment (TH1-type stimulus) significantly increased the number of inflammatory cells infiltrating the conjunctiva as compared to SRW sensitization and multi-hit challenge alone. SRW sensitized and challenged IFN-J KO mice had a significantly reduced conjunctival inflammatory cell infiltrate as compared to the SRW challenged WT group (77% decrease in eosinophils; 35 % decrease in neutrophils). Conclusions. Inflammatory cell infiltrations into the conjunctiva of SRW sensitized and challenged animals in the presence of a desiccating environment were significantly increased suggesting that the presence of TH1 cytokine mediated disease can further exacerbate a TH2-like disease pathology. IFN-J is unexpectedly essential for the immunopathology of allergic conjunctivitis disease. IFN-J regulation of adhesion molecules required for extravasation of inflammatory cells into sites of disease are currently under investigation. Commercial Relationship(s): M. E. Stern, Allergan, Inc. E.; K. F. Siemasko, Allergan, Inc. E; J. Gao, Allergan, Inc. E; M. Calonge, Allergan, Inc. C; V. L. Calder, Allergan, Inc. C; S. C. Pflugfelder, Allergan, Inc. C; J. Y. Niederkorn, Allergan, Inc. C.
THE OCULAR SURFACE / JANUARY, 2005, VOL. 3, NO. 1 / SUPPLEMENT