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Tear Proteomics in Evaporative Dry Eye Disease

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Tear Proteomics in Evaporative Dry Eye Disease Eye (2010) 24, 1396–1402 & 2010 Macmillan Publishers Limited All rights reserved 0950-222X/10 $32.00 www.nature.com/eye 1 2 3 4 LABORATORY STUDY Tear proteomics in P Versura , P Nanni , A Bavelloni , WL Blalock , M Piazzi4, A Roda2 and EC Campos1 evaporative dry eye disease Abstract Introduction Purpose: To analyze tear protein variations in ‘Dry eye’ is now recognized as a disruption of patients suffering from dry eye symptoms in the lachrymal functional unit,1 a system the presence of tear film instability but comprising the ocular surface (cornea, without epithelial defects. conjunctiva, and limbus), the lachrymal and Methods: Five microlitres of non-stimulated meibomian glands and the lids, interconnected tears from 60 patients, suffering from by an integrated neural arc. The updated evaporative dry eye (EDE) with a break-up time definition states that ‘Dry eye is a multifactorial (BUT) o10 s, and from 30 healthy subjects as disease of the tears and ocular surface that control (no symptoms, BUT 410 s) were results in symptoms of discomfort, visual collected. Tear proteins were separated by mono disturbance, and tear instability with potential and bi-dimensional SDS-PAGE electrophoresis damage to the ocular surface. It is accompanied and characterized by immunoblotting and by increased osmolarity of the tear and enzymatic digestion. Digested peptides were inflammation of the ocular surface’.2 As 1Ophthalmology Unit, analyzed by liquid chromatography coupled to postulated already in the previous report by University of Bologna, electrospray ionization quadrupole-time of Lemp et al in 1995,3 the major classes of dry eye Bologna, Italy flight mass spectrometry followed by are mainly related to tear deficiency or comparative data analysis into Swiss-Prot increased tear evaporation. 2Department of human protein database using Mascot. Pharmaceutical Science, This last form, in particular, is increasingly University of Bologna, Statistical analysis were performed by applying observed as a consequence of environmental Bologna, Italy a t-test for independent data and a Mann– factors such as forced air dry heat, wind, air Whitney test for unpaired data (Po0.05). pollution, or reduced blinking because of 3Laboratory of Cell Biology Results: In EDE patients vs controls, a driving, TV watching, and computer work. Tear and Electron Microscopy, significant decrease in levels of lactoferrin (data evaporation is considered the main contribution Istituto Ortopedico Rizzoli, in %±SD): 20.15±2.64 vs 24.56±3.46 (P ¼ 0.001), 4 Bologna, Italy to tear thinning and break-up, although neither lipocalin-1: 14.98±2.70 vs 17.73±2.96 the complete mechanism of stability nor the role ± 4Department of Human (P ¼ 0.0001), and lipophilin A–C: 2.89 1.06 vs of tear composition have been elucidated to date. Anatomical Sciences, Cell 3.63±1.37 (P ¼ 0.006) was revealed, while a Proteomic methods have identified nearly 500 Signalling Laboratory, significant increase was observed for serum proteins in human tears,5–8 but for only a University of Bologna, albumin: 9.45±1.87 vs 3.46±1.87 (P ¼ 0.0001). minority of these proteins has the biological role Bologna, Italy No changes for lysozyme and zinc a-2 in dry eye disease or, more generally, in ocular glycoprotein (P ¼ 0.07 and 0.7, respectively) were Correspondence: P Versura, surface physiology been clarified. The most Ophthalmology Unit, shown. Proteomic analysis showed a representative tear proteins have been analyzed University of Bologna, downregulation of lipophilin A and C and either in Sjogren’s and non-Sjogren’s dry eye.9,10 Azienda Ospedaliera lipocalin-1 in patients, which is suggested to be Abnormal changes in these protein profiles S.Orsola-Malpighi, Pad. 1 associated with post-translational modifications. were suggestive of impaired lachrymal gland Palagi, Via Palagi, 9 40138 Conclusions: Data show that tear protein Bologna, Italy function, related to autoimmune disease or Tel/Fax: þ 39 051 6362846. changes anticipate the onset of more extensive apoptotic events. E-mail: piera.versura@ clinical signs in early stage dry eye disease. The purpose of this work was to analyze the unibo.it Eye (2010) 24, 1396–1402; doi:10.1038/eye.2010.7; behaviour of the main tear proteins in patients published online 12 February 2010 suffering from mild evaporative dry eye (EDE), Received: 2 August 2009 the most frequent form of the disease, which Accepted in revised form: Keywords: proteomics; human tears; dry eye 5 January 2010 consists in an excessive water loss from the Published online: 12 disease; mass spectrometry; western-blot; ocular surface even in the presence of adequate February 2010 electrophoresis tear production. Tear protein analysis in dry eye disease P Versura et al 1397 Materials and methods Monodimensional electrophoresis Subjects Tear samples were diluted 1 : 2 with 0.125 M Tris-Cl pH 6.8, 4% sodium dodecyl sulfate (SDS), 20% glycerol, 10% A total of 90 subjects, including 60 patients affected by 2-mercaptoethanol buffer, and boiled for 5 min. The EDE (18 men and 42 women; mean age 64.2±22.3 years) proteins were separated using a 18% acryl amide -TRIS- and 30 healthy control subjects (8 men and 22 women; HCl Ready-gel (Bio-Rad, Laboratories, Milano, Italia) mean age 61.1±17.8 years), were enrolled for this study. applying a voltage of 200 V for 1 h at room temperature. Patients were classified as grade 1 dry eye severity A pre-stained standard of low molecular weight proteins according to DEWS2 scheme (Table 1). For patients, (Bio-Rad) was used to monitor the electrophoretic the inclusion criteria were a Schirmer test I value higher separation. Gel staining was performed using Brilliant than 10 mm/5 min, a tear film break-up time (TFBUT) Blu G (Sigma-Aldrich, Milano, Italy) for 12 h at room value p10 s, mild subjective symptoms of ocular temperature. The gel image was acquired with the discomfort as evaluated by an OSDI questionnaire11 densitometer scanner Umax (Amersham Biosciences-GE (score 12–24), and use of tear substitutes only, for Healthcare, Milano, Italia) and analyzed for the percent treatment, which were suspended for at least 3 days abundance of each protein of interest in the samples before tear collection. using Gel-Pro Analyzer software (MediaCybernetics Inc., Inclusion criteria for healthy control subjects were a Bethesda, MD, USA). Schirmer test I value higher than 10 mm/5 min, a TFBUT value X10 s, and absence of subjective symptoms of Immunoblotting ocular discomfort (OSDI score o12). In both groups, excluding criteria were the presence of Total protein from 1 ml of tears was subjected to SDS- punctuate keratopathy and/or autoimmune diseases, the polyacrylamide gel electrophoresis at 200 V constant use of contact lens, and having undergone ocular surgery using a Mini-Protean III (Bio-Rad) and transferred onto a in the last 6 months. nitrocellulose membrane (Hybond-C extra, Amersham) The study was conducted according to the declaration applying a voltage of 100 V (1 h, 4 1C) in buffer containing of Helsinki involving human subjects. 0.3% Tris, 1.4% glycine, and 20% methanol using a Bio- All the tear samples were provided by the Ophthalmic Rad wet-blotting apparatus. The correct transfer of the Unit at S Orsola-Malpighi University Hospital of Bologna proteins was evaluated using the reversible coloration (Italy) after obtaining informed consent from the subjects with Red Ponceau. The nitrocellulose membrane studied and according to DEWS guidelines.2 From each containing the transferred proteins was saturated with subject, a minimum amount of 10 ml of unstimulated 3% bovine serum albumin (Sigma) in phosphate-buffered tears was collected using a micropipette with sterile tips, saline (PBS) þ 0.1% Tween 20 for 1 h at room avoiding any reflex tearing. Patients were requested to temperature. For immunoblotting, anti-lactoferrin position the head slightly reclined in such a way that (sc-25622), anti-lipocalin (sc-34680), anti-lysozyme tears are driven to the most outer side of the lower fornix. (sc-27956), anti-lipophillin A (sc-48324), and anti- After 30 s, the tip of the micropipette was carefully lipophillin B (sc-48327) antibodies were purchased from positioned and tears aspired. Samples were centrifuged Santa Cruz Biotechnology, Santa Cruz, CA, USA; the at 13 200 g for 15 min and stored frozen in plastic vials at anti- zinc-alpha-2-glycoprotein (ZAG) antibody was À80 1C until use. purchased from BioVendor GmbH, Heidelberg, Germany. Table 1 DEWS dry eye severity grading system. Patients included in this study were classified as grade 1 Dry eye severity grade 1 2 3 4 Severity and frequency of Mild and/or Moderate, episodic or Severe frequent or Severe and/or constant discomfort episodic chronic constant Conjunctival injection None to mild None to mild þ /Àþ/ þþ Conjunctival staining None to mild Variable Moderate to marked Marked Corneal staining None to mild Variable Marked central Severe punctuate erosions Lid/meibomian glands MGD variably MGD variably present Frequent Trichiasis, present symblepharon TFBUT (s) Variable p10 s p5 s Immediate Schirmer score (mm/5 min) Variable p10 mm p5mm p2mm Eye Tear protein analysis in dry eye disease P Versura et al 1398 Primary antibodies were diluted in PBS containing with a electrospray interface to a quadrupole-time of 0.1% Tween 20 and incubated overnight at 4 1C. The blots flight (QTOF) micro (Micromass, Manchester, UK). The were washed three times with PBS þ 0.1% Tween 20 and peptide separation was performed on an Atlantis dC18 incubated with horseradish peroxidase-conjugated NanoEase column (150 Â 0.3 mm, 3 mm) (Waters) with an secondary antibodies diluted (1 : 10000) in PBS þ 0.1% Atlantis dC18 NanoEase precolumn (5 Â 0.3 mm, 5 mm Tween 20 for 1 h at room temperature.
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