Ben J. Glasgow

The International Workshop on Meibomian Gland Dysfunction: Executive Summary

DOI:10.1167/iovs.10-6997a Investigative Ophthalmology & Visual Science, Special Issue 2011, Vol. 52, No. 4 Copyright 2011 The Association for Research in Vision and Ophthalmology, Inc. 1922 ドライアイ疾患の原因としては、マイボーム腺機能不全 (MGD)がおそらく最も多い。この疾患によって数百万人 もの健康と幸福が損なわれているにもかかわらず、MGD の定 義、分類、診断、治療について世界的なコンセンサスはない。 そうしたコンセンサスに達する目的で、非営利団体である Tear Film and Ocular Surface Society( TFOS; http://www. tearfilm.org)が International Workshop on Meibomian Gland Dysfunction(国際マイボーム腺機能不全ワークショップ、 www.tearfilm.org/mgdworkshop/index.html)を起ち上げた。こ のワークショップの目的は以下の通りである:

Ocular toxoplasmosis in patients with the acquired immunodeficiency syndrome

In seven of eight cases of presumed ocular toxoplasmosis in patients with AIDS, the diagnosis was supported by a reduction or resolution of intraocular inflammation and healing of necrotic retinal lesions after initiation of antiparasitic drug therapy including one or more of the following medications: pyrimethamine, sulfadiazine, clindamycin, tetracycline, or spiramycin. In two cases the diagnosis was confirmed histologically. The cases differed clinically and histopathologically from those in immunocompetent patients. There was no evidence that disease originated in preexisting retinochoroidal scars. Lesions frequently were bilateral and multifocal. Vitreous inflammatory reaction was a common clinical finding, but histopathologic examination demonstrated scant retinal inflammation in areas of necrosis. Ocular toxoplasmosis in these patients with AIDS probably resulted from newly acquired infection or dissemination of organisms from nonocular sites of disease. Infections became clinically inactive with drug therapy in all treated patients, but reactivation and progression of disease occurred when therapy was stopped in two of three patients. Severe retinal necrosis led to retinal tears or detachment in three cases. Ocular lesions were the first manifestation of Toxoplasma gondii infection in four of five patients with evidence of multisystem infection.

Tear lipocalins bind a broad array of lipid ligands

To identify the native ligands of tear lipocalins, tear proteins were separated by size exclusion chromatography and the lipid content in the major protein fractions identified. Lipids extracted from native tears and purified tear lipocalins comigrated with fatty acids, fatty alcohols, phospholipids, glycolipids, and cholesterol on thin layer chromatograms. Abundant stearic and palmitic acids as well as cholesterol, and lesser amounts of lauric acid were specifically identified in extracts of purified lipocalins by gas chromatography-mass spectroscopy. A preliminary study of the ligand-protein interaction was carried out using nitroxide spin-labeled lipids.

The International Workshop on Meibomian Gland Dysfunction: Report of the Subcommittee on Tear Film Lipids and Lipid-Protein Interactions in Health and Disease

Understanding the molecular composition (e.g., proteins and lipids) of the tear film (TF) and the contribution of the meibomian gland to the TF is critical in gaining knowledge about TF instabilities, dry eye syndromes, contact lens (CL) incompatibilities, and other eye diseases. Among its functions, the lipid layer of the TF slows evaporation of the aqueous component, preserves a clear optical surface, and forms a barrier to protect the eye from microbial agents and organic matter, such as dust and pollen.1 The TF contains a complex mixture of proteins, enzymes, lipids, mucins, and salts that allows the TF to perform its functions (Fig. 1). Researchers believe the outer lipid layer is 5 to 10 molecules thick and is composed primarily of wax and sterol esters, possibly intercalated with each other and with proteins rather than forming distinct repeating layers of molecules.2,3 Evidence from interferometric studies indicate that the TF lipid layer thickness ranges from 20 to 160 nm.4 If the size of a lipid molecule is approximately 2.2 nm (22 Å), then the calculated thickness for one layer would be 11 to 44 nm. The addition of polar and nonpolar layers would add to the lipid thickness, which indicates that the lipid component of the TF may be multiple layers thick or have other contributing sources to correspond with reported thickness measurements.5 Figure 1. A proposed model of the precorneal tear film showing the relationship and interaction of lipid-binding proteins and the outer lipid layer. While the signs and symptoms of TF instability are reasonably well characterized, we are only beginning to understand the specific molecular components of the TF and their relationship with disease and TF stability. The purpose of this review is to examine the meibomian glands contribution to TF lipids and lipid–protein interactions in health and disease.

Human Tear Lipocalin Exhibits Antimicrobial Activity by Scavenging Microbial Siderophores

ABSTRACT Human tear lipocalin (TL; also known as Lcn1) is a secretory protein present in large amounts in fluids that cover epithelial surfaces such as tears and respiratory secretions. It is supposed to act as a physiological scavenger of hydrophobic, potentially harmful molecules, but there is evidence that it also inhibits bacterial growth. In the present study, we reconsidered the possibility that TL might interfere with microbial growth by scavenging of siderophores, as described for human neutrophil gelatinase-associated lipocalin (NGAL). Indeed, our experiments revealed that TL binds to microbial siderophores with high affinities. In contrast to NGAL, which was shown to have some specificity for bacterial catecholate-type siderophores, TL binds to a broad array of siderophores, including bacterial catecholate-type enterobactin and hydroxamate-type desferrioxamine B, and all major classes of fungal siderophores. By adding exogenous TL, bacterial and fungal growth could be inhibited under iron-limiting conditions. Thus, TL might be a novel member of the innate immune system especially involved in mucosal defense against fungal infections.

Keratectasia after laser in situ keratomileusis (LASIK): evaluation of the calculated residual stromal bed thickness

PURPOSE To report corneal histopathology associated with keratectasia after laser in situ keratomileusis (LASIK) and to evaluate the thickness of the calculated residual stromal bed in two cases and those in the literature. DESIGN Interventional case reports. METHODS Three eyes of two patients developed keratectasia after LASIK. Corneal specimens after penetrating keratoplasty in one eye of each patient were studied histopathologically, and the residual stromal bed was directly measured. For comparison, residual stromal bed thicknesses were calculated from published cases of keratectasia. RESULTS Two eyes of a 26-year-old woman and one eye of a 22-year-old woman developed keratectasia after LASIK. Calculated residual stromal bed thicknesses were 210, 213, and 261 microm. Histologic sections revealed focal scarring in the flap plane. The cornea specimens measured 75 and 118 microm thinner than calculated values immediately after LASIK. Transmission electron microscopy of one case revealed an average lamellar thickness of 0.94 microm. In 28 (49%) of 57 previous cases of keratectasia, the calculated residual stromal bed thicknesses were greater than 250 microm. CONCLUSIONS Both the flap and the stromal bed of the cornea may thin after LASIK. A residual stromal bed thickness of 250 microm does not preclude the development of keratectasia after LASIK.

Quantitation of tumor seeding from fine needle aspiration of ocular melanomas.

Twenty-two fine needle (30 gauge) aspirations were performed in eyes enucleated for the clinical diagnosis of melanoma. Cytologic preparations were evaluated for adequacy of material, and needle tracts were evaluated for tumor implantation. A scleral marking method was used to identify all needle tracts. The number of tumor cells in tracts of direct transscleral aspirates was compared to those in tracts of indirect aspirates that traversed the anterior chamber or vitreous. Cellular material obtained with 30-gauge needles was sufficient for the diagnosis of malignant melanoma in all but one case. While 14 of 21 (67%) of all fine needle aspiration tracts and eight of 15 (53%) of indirect tracts contained tumor cells, the number of tumor cells was less than that associated with tumor growth in experimental models. Indirect aspirate tracts contained significantly fewer cells than tracts of direct aspirates (P less than .001).

Binding studies of tear lipocalin: the role of the conserved tryptophan in maintaining structure, stability and ligand affinity

The principal lipid binding protein in tears, tear lipocalin (TL), binds acid and the fluorescent fatty acid analogs, DAUDA and 16-AP at one site TL compete for this binding site. A fluorescent competitive binding assay revealed that apo-TL has a high affinity for phospholipids and stearic acid (Ki) of 1.2 microM and 1.3 microM, respectively, and much less affinity for cholesterol (Ki) of 15.9 of the hydrocarbon chain. TL binds most strongly the least soluble lipids permitting these lipids to exceed their maximum solubility in aqueous solution. These data implicate TL in solubilizing and transporting lipids in the tear film. Phenylalanine, tyrosine and cysteine+ were substituted for TRP 17, the only invariant residue throughout the lipocalin superfamily. Cysteine substitution resulted in some loss os secondary structure, relaxation of aromatic side chain rigidity, decreased binding affinity for DAUDA and destabilization of structure. Mutants of TL, W17Y, and W17F showed a higher binding affinity for DAUDA than wild-type TL. Comparison of the results of the tryptophan 17 substitution in lipocalin with those of tryptophan 19 substitution in beta-lactoglobulin revealed important differences in binding characteristics that reflect the functional heterogeneity within the lipocalin family.

Tissue expression of lipocalins in human lacrimal and von Ebner's glands: colocalization with lysozyme

Abstract• Background: Tear-specific prealbumin is a group of proteins recently renamed as the tear lipocalins. These proteins were initally described as unique to lacrimal fluid. The tissue distribution and localization have never been thoroughly studied.• Methods: The distribution of purified tear lipocalins was studied in many human secretions and tissues by western blots, immunohistochemistry and immunoelectron microscopy. • Results: Tear lipocalin species of the same molecular weights were observed in western blot lanes loaded with tears, saliva, and protein extracts from the lacrimal and lingual von Ebners glands. Lacrimal and von Ebners glands contained tear lipocalins; other human tissues and secretions, including other salivary glands and taste buds, did not. Tear lipocalins colocalized with lysozyme in serous acinar cells of lacrimal and von Ebners glands. Ultrastructurally, tear lipocalins were present on polyribosomes, endoplasmic reticulum, and Golgi areas. Lipocalins were concentrated in lacrimal secretory granules in amounts commensurate with a regulated pathway.• Conclusion: Tear lipocalins are expressed and truncated similarly in lingual von Ebners and lacrimal glands, but not at all in other human tissues. Lipocalins are expressed and secreted with lysozyme. Lipocalins are concentrated in secretory granules in an amount consistent with a regulated secretory pathway.

Structural changes in human tear lipocalins associated with lipid binding

Structural and conformational changes in tear lipocalins were detected in association with ligand binding and release. Circular dichroism measurements demonstrated that ligand binding induces beta structure formation, aromatic side chain asymmetry, and a more rigid state in tear lipocalins (TL). The exposure of the tyrosyl component is less in apo-TL than in holo-TL. The sole tryptophan residue, Trp17, is buried in both holo- and apo-TL. The steady state exposure of Trp17 is the same in holo- and apo-TL, but the dynamic exposure is two-fold greater in apo-TL. Maneuvers to unfold the protein with urea or incubation in an acidic environment resulted in increased exposure of aromatic amino acids. Electron paramagnetic resonance studies verified that lipids are liberated from TL in an acidic environment. Acidic pH promotes conformational changes in TL involving aromatic residues, particularly the conserved residue Trp17. These changes are associated with lipid release. The liberation of lipid from the cavity of TL under acidic conditions involves a molten globule state of the protein. We postulate that TL, exposed to the steep surface pH gradient that exists at lipid-aqueous interfaces, would release lipid in association with a molten globule transition. The data suggest a plausible regulatory mechanism for lipid delivery from lipocalins at the tear film surface.