Tear Proteins Research Articles

Dry Eye and Clinical Disease of Tear Film – Diagnosis and Management

Dry eye disease (DED) is one of the most frequent conditions in ophthalmology. The early diagnosis and the monitoring of therapy efficacy may help to prevent the severe complications and the impairment of visual function. This article presents the advances in the aetiology and diagnostic procedures of dry eye and clinical disease of tear film with a particular focus on SDS-agarose gel electrophoresis of tear proteins (Hyrys-Hydrasys SEBIA system). The test was used for the first time in the author’s laboratory and demonstrated an important clinical value in ocular and systemic diseases, as well as in high-risk groups for DED. The measurement of many target tear proteins (lactoferrin, lysozyme, albumin, immunoglobulin, proteins 20–60 kDa) in a single analysis using only 5 μl of tears with minimal cost and rapid results are strong arguments for including of SDS-agarose gel electrophoresis of tear proteins in routine test panel for DED.

Lacritin-mediated regeneration of the corneal epithelia by protein polymer nanoparticles.

The avascular corneal epithelium plays an important role in maintaining normal vision and protecting the corneal interior from environmental infections. Delayed recovery of ocular wounds caused by trauma or refractive surgery strengthens the need to accelerate corneal wound healing and better restore the ocular surface. To address this need, we fused elastin-like polypeptide (ELP) based nanoparticles SI with a model mitogenic protein called lacritin. Lacritin fused at the N-terminus of the SI diblock copolymer is called LSI. This LSI fusion protein undergoes thermo-responsive assembly of nanoparticles at physiologically relevant temperatures. In comparison to ELP nanoparticles without lacritin, LSI showed potent signs of lacritin specific effects on a human corneal epithelial cell line (HCE-T), which included enhancement of cellular uptake, calcium-mediated signaling, and closure of a scratch. In vivo, the corneas of non-obese diabetic mice (NOD) were found to be highly responsive to LSI. Fluorescein imaging and corneal histology suggested that topical administration of LSI onto the ocular surface significantly promoted corneal wound healing and epithelial integrity compared to mice treated with or without plain ELP. Most interestingly, it appears that ELP-mediated assembly of LSI is essential to produce this potent activity. This was confirmed by comparison to a control lacritin ELP fusion called LS96, which does not undergo thermally-mediated assembly at relevant temperatures. In summary, fusion of a mitogenic protein to ELP nanoparticles appears to be a promising new strategy to bioengineer more potent biopharmaceuticals with potential applications in corneal wound healing.

Analysis of Graves’ ophthalmopathy patients’ tear protein spectrum

Background Graves’ ophthalmopathy/orbitopathy (GO) patients often suffer ocular surface damages and tear fluid proteins play a significant role in maintaining healthy ocular surfaces, while changes in tear protein components reflect the changes ocular surface abnormalities. In this study proteomics techniques were used to investigate tear protein compositions in GO patients. Methods We carried out a case-control study by comparing tear fluid contents of GO patients with that of healthy subjects. In the first step the tears were subjected to SDS-PAGE electrophoresis and then single protein bands were analyzed by to in-gel trypsin digestion and nano-flow liquid mass spectrometry (LC-MS/MS) using a MS software. Results In tear samples of GO subjects, the protein fractions of inflammation-related protein immunoglobulin kappa chain C region (IgKC) and serum albumin were essentially reduced, whereas a novel isoform of complement component 3 (C3), which we detected in control subjects, was completely absent in the GO patients’ tears. Conclusions Reduced protein concentrations of particularly IgKC and complement C3 as well as albumin in the tears of GO patients may contribute to changes in their ocular surfaces via diminished reactive oxygen species (ROS) depletion and adaptive immune responses. The completely absent of C3 in the GO patients’ tears, may imply that an important inflammatory signaling pathway is affected, which needs further investigation.

Tear Proteomics in Young Malays with Dry Eyes

Introduction: Changes in tear protein concentrations may reflect ocular surface health. This study analyzes changes in tear protein concentrations of young Malays with dry eye (DE) and determines its association with the clinical findings. Methods: Subjects were screened using McMonnies questionnaire (MDEQ) and flourescein tear break up time (TBUT). Total tear protein concentration (TTPC) was determined using Bradford's technique and specific tear protein (sIgA, lysozyme, lactoferrin and human serum albumin (HSA)) concentrations were determined using SDS-PAGE. Parametric and nonparametric tests were used to compare means between groups. Spearman correlation was used to determine the association between variables measured. Results: A total of 42 subjects (21 DE and 21 NDE) were included. Mean MDEQ score for DE was 16.00±1.48 and NDE was 8.47±3.47. Mean TBUT for DE was 3.47±0.47s and NDE was 4.98±0.43s. Mean TTPC for DE and NDE was 9.84±2.40mg/ml and 8.96±1.84mg/ml respectively. Mean sIgA, lysozyme, lactoferrin and HSA for DE was 0.54±0.10mg/ml, 1.68±0.17mg/ml, 1.47±0.25mg/ml, 0.06±0.03mg/ml and for NDE was 0.57±0.09mg/ ml, 2.04±0.19mg/ml, 1.75±0.23mg/ml, 0.06±0.03mg/ml accordingly. Significant differences were noted in MDEQ score (p=0.01), TBUT (p=0.01), lactoferrin (p=0.01) and lysozyme (p=0.01) but not in TTPC (p=0.19), HSA (p=0.74) and sIgA (p=0.24) between groups. Significant correlations were noted between TBUT with lactoferrin (r=0.02, p=0.02) and lysozyme (r=0.63, p=0.01) and between MDEQ score with lactoferrin (r=-0.34, p=0.02) and lysozyme (r=-0.64, p=0.01). Conclusions: There are changes in specific tear protein in dry eye patients, which correlate well with clinical results. Tear protein analysis may play an important role in the diagnosis of the dry eye.

The effects of wearing rigid gas permeable contact lens on tear protein concentrations

The effects of wearing rigid gas permeable contact lens on tear protein concentrations

Zinc Finger Protein in Severe Dry Eye Syndrome

Purpose: Zinc finger protein known to induce squamous metaplasia and regulate vitamin A expression has been few investigated as tear protein. We investigated tear protein variations in patients with dry eye syndrome (DES).Materials and methods: Tears from healthy subjects as control and patients with DES were collected. Tear proteins were separated by one-dimensional electrophoresis. The protein bands were analyzed by nano liquid chromatography–mass spectrometry.Results: Compared to the controls, significant down-regulation of lactoferrin and lysozyme was detected, while significant up-regulation was observed for serum albumin in patients with DES. DES grade 4 patients showed different protein patterns. Zinc-finger motif-enhancer binding-protein-1 gamma and bromodomain adjacent to zinc finger domain 2B were detected in DES grade 4 patients.Conclusions: Tear protein changes are valuable to diagnosis DES. Zinc finger proteins may be associated with pathophysiology of severe DES. Further studies are needed to better understand the relationship of zinc finger proteins in tear of patients with DES.

Bilateral acute pyogenic conjunctivitis with iritis induced by unilateral topical application of bacterial peptidoglycan muramyl dipeptide in adult rabbits

The factors responsible for the conjunctivitis and iritis associated with acute ocular infection and post enteric inflammatory disease are not fully known. The pro-inflammatory activity of unilateral topical application of muramyl dipeptide (MDP; the smallest bio-active Gram-positive and Gram-negative bacterial cell wall component) was investigated in adult rabbits. The resultant bilateral conjunctivitis/iritis and pyogenic responses were characterized. Bilateral symptoms were graded by slit lamp examinations; tear fluid, Schirmer tests (tear production), blood and aqueous humor (AH) samples were obtained from MDP-treated and untreated rabbits. MDP concentration, gamma-glutamyltranspeptidase activity (GGT; key enzyme in glutathione recapture, xenobiotic detoxification, eicosanoid synthesis and neutrophil function), protein concentration, and tear cell density, cytology, and immunofluorescent antibody reactivity to GGT and calreticulin (CRT; MDP-binding protein) were determined. MDP was cleared from ipsilateral tears and serum by 6 h, but was undetected in mock-treated contralateral tears. Bilateral signs of acute transient pyogenic conjunctivitis, characterized by tearing, lid edema, conjunctival hyperemia, chemosis and leukocytic infiltrate with iritis (erythema and aqueous flare) were detected. Milder symptoms occurred in the mock-treated contralateral eyes. Bilateral symptoms, tear production, tear protein, GGT activity, and mucopurulent discharge (containing up to 2.5–5.0 × 106 cells/mL) were elevated 4–8 h post MDP and resolved to near pre-treatment levels by 24 h. Tear GGT activity and protein levels were higher in MDP-treated and mock-treated contralateral eyes than in eyes of untreated adult rabbits (p's < 0.001). Elevated tear GGT activity was associated with histopathology and increased vascular and epithelial permeability to serum protein, GGT-positive epithelia cells, macrophages and heterophils. Repeat MDP applications induced recurrent induction and resolution patterns of bilateral conjunctivitis/iritis and tear GGT activity, but ipsilateral GGT responses were lower. The results suggest unilateral topical MDP application to adult rabbit eyes induces a bilateral acute pyogenic conjunctivitis/iritis (PCI) characterized by increased vascular and epithelial permeability similar to acute bacterial conjunctivitis in man. The detection of CRT/GGT positive heterophils in tears suggests efferocytosis (phagocytosis of dead/dying cells). Tear GGT activity may be a useful means to quantify MDP-induced toxicity and extraocular inflammation.

Analysis of protein composition and protein expression in the tear fluid of patients with congenital aniridia

Aniridia is a rare congenital genetic disorder caused by haploinsuffiency of the PAX6 gene, the master gene for development of the eye. The expression of tear proteins in aniridia is unknown. To screen for proteins involved in the aniridia pathophysiology, the tear fluid of patients with diagnosed congenital aniridia was examined using two-dimensional electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Two-dimensional map of tear proteins in aniridia has been established and 7 proteins were differentially expressed with P<0.01 between aniridia patients and control subjects. Five of them were more abundant in healthy subjects, particularly α-enolase, peroxiredoxin 6, cystatin S, gelsolin, apolipoprotein A-1 and two other proteins, zinc-α2-glycoprotein and lactoferrin were more expressed in the tears of aniridia patients. Moreover, immunoblot analysis revealed elevated levels of vascular endothelial growth factor (VEGF) in aniridia tears which is in concordance with clinical finding of pathological blood and lymph vessels in the central and peripheral cornea of aniridia patients. The proteins with different expression in patients' tears may be new candidate molecules involved in the pathophysiology of aniridia and thus may be helpful for development of novel treatment strategies for the symptomatic therapy of this vision threatening condition. This study is first to demonstrate protein composition and protein expression in aniridic tears and identifies proteins with different abundance in tear fluid from patients with congenital aniridia vs. healthy tears.

Neutrophil Collagenase, Gelatinase, and Myeloperoxidase in Tears of Patients with Stevens-Johnson Syndrome and Ocular Cicatricial Pemphigoid

To investigate the levels of matrix metalloproteinases (MMPs), myeloperoxidase (MPO), and tissue inhibitor of metalloproteinase-1 (TIMP-1) in tears of patients with Stevens-Johnson syndrome (SJS) and ocular cicatricial pemphigoid (OCP). Prospective, noninterventional cohort study. Four SJS patients (7 eyes), 19 OCP patients (37 eyes), and 20 healthy controls who underwent phacoemulsification (40 eyes). Tear washes were collected from all patients and were analyzed for levels of MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-12, MPO, and TIMP-1 using multianalyte bead-based enzyme-linked immunosorbent assays. Total MMP activity was determined using a fluorometric assay. Correlation studies were performed between the various analytes within study groups. Levels of MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-12, MPO, and TIMP-1 (in nanograms per microgram of protein) and total MMP activity (in relative fluorescent units per minute per microgram of protein) in tears; MMP-8-to-TIMP-1 ratio; MMP-9-to-TIMP-1 ratio; and the correlations between MMP-8 and MMP-9 and both MMP and MPO. MMP-8, MMP-9, and MPO levels were elevated significantly in SJS and OCP tears (SJS>OCP) when compared with controls. The MMP activity was highest in SJS patients, whereas OCP patients and controls showed lower and similar activities. The TIMP-1 levels were decreased in SJS and OCP patients when compared with those in controls, with levels in OCP patients reaching significance. The MMP-8-to-TIMP-1 and MMP-9-to-TIMP-1 ratios were markedly elevated in SJS and OCP tears (SJS>OCP) when compared with those of controls. Across all study groups, MMP-9 levels correlated strongly with MMP-8 and MPO levels, and MMP-8 correlated with MPO, but it did not reach significance in SJS patients. There was no relationship between MMP-7 and MPO. Because MMP-8 and MPO are produced by inflammatory cells, particularly neutrophils, the correlation data indicate that they may be the common source of elevated enzymes, including MMP-9, in SJS and OCP tears. Elevated MMP-to-TIMP ratios and MMP activity suggest an imbalance in tear MMP regulation that may explain the predisposition of these patients to demonstrate corneal melting and chronic complications associated with persistent inflammation. Myeloperoxidase in tears may be a sensitive and specific marker for the quantification of ocular inflammation.

Proteomic analysis revealed the altered tear protein profile in a rabbit model of Sjögren's syndrome-associated dry eye

Sjögren's syndrome (SS) is an autoimmune disease that results in pathological dryness of mouth and eye. The diagnosis of SS depends on both clinical evaluation and specific antibodies. The goal of this study was to use quantitative proteomics to investigate changes in tear proteins in a rabbit model of SS-associated dry eye, induced autoimmune dacryoadenitis (IAD). Proteomic analysis was performed by iTRAQ and nano LC-MS/MS on tears collected from the ocular surface, and specific proteins were verified by high resolution MRM. It was found that in the tears of IAD rabbits at 2 and 4 weeks after induction, S100 A6, S100 A9, and serum albumin were upregulated, whereas serotransferrin (TF), prolactin-inducible protein (PIP), polymeric immunoglobulin receptor (pIgR), and Ig gamma chain C region were downregulated. High resolution MRM with mTRAQ labeling verified the changes in S100 A6, TF, PIP, and pIgR. Our results indicated significant changes of tear proteins in IAD rabbits, suggesting these proteins could potentially be used as biomarkers for the diagnosis and prognosis of dry eye. Several of these proteins were also found in the tears of non-SS dry eye patients indicating a common basis of ocular surface pathology, however, pIgR appears to be unique to SS.

Lipid peroxidation is increased in tears from the elderly

We describe a procedure in which tears, obtained from Schirmer strips, are used to measure a marker of lipid peroxidation, malondialdehyde (MDA). We also compared the levels of proteins and MDA in tears from two groups of people: young adults (18–30 years old) and elderly adults (65–85 years old), because the data related to total protein concentration of human tears vary widely and because the majority of people over the age of 65 experience some symptoms of dry eyes and this condition has been recognized as an oxidative stress-induced disease. Our results show a significant difference in the protein concentration of the tears taken from the two age categories, younger adults (18–30 years old) and older adults (65–85 years old). Herein, we report for the first time an increase in MDA concentrations determined by HPLC in human tears based on age. It is possible that alterations in the tear lipid layer may lead to an increase in lipid peroxidation. Further studies are needed to understand the nature and function of tear film and stability in order to obtain new methods to analyze tears in patients with different diseases. In this sense, it would be interesting to compare MDA concentration in tears from control subjects and from people with meibomian gland dysfunction.

A proposed model of the response of the anophthalmic socket to prosthetic eye wear and its application to the management of mucoid discharge

Mucoid discharge associated with prosthetic eye wear can be a distressing condition that affects the quality of life of people who have lost an eye. Discharge is the second highest concern of experienced prosthetic eye wearers after health of the companion eye and is prevalent in anophthalmic populations. Specific causes of mucoid discharge such as infections and environmental allergens are well understood, but non-specific causes are unknown and an evidence based protocol for managing non-specific discharge is lacking. Current management is based on prosthesis removal and cleaning, and professional re-polishing of the prosthesis. Tear protein deposits accumulate on prosthetic eyes. These deposits mediate the response of the socket to prosthetic eye wear and their influence (good and bad) is determined by differing cleaning regimes and standards of surface finish.This paper proposes a three-phase model that describes the response of the socket to prosthetic eye wear. The phases are: An initial period of wear of a new (or newly-polished) prosthesis when homeostasis is being established (or re-established) within the socket; a second period (equilibrium phase) where beneficial surface deposits have built up on the prosthesis and wear is safe and comfortable, and a third period (breakdown phase) where there is an increasing likelihood of harm from continued wear. The proposed model provides a rationale for a personal cleaning regime to manage non-specific mucoid discharge. Professional care of prosthetic eyes is also important for the management of discharge and evidence for effective surface finishing is reported in this study. Taken together, the proposed regimes for personal and professional care comprise a protocol for managing discharge associated with prosthetic eye wear. The protocol describes prosthetic eye cleaning methods and frequency, and suggests minimum standards for professional polishing. If confirmed, the protocol has the potential to resolve the current varied and contradictory opinions about the management of discharge, and to clarify advice given to patients about how to personally care for their prosthetic eyes.

Effects of Keratin and Lung Surfactant Proteins on the Surface Activity of Meibomian Lipids

In vitro studies indicate that surface tension and surface viscosity of the tear film lipid layer (TFLL) are governed by interactions between meibomian lipids and proteins from the aqueous layer. The role of minor tear proteins with strong lipophilic properties or those correlated with pathological states is still unknown. The discovery of lung surfactant proteins (SPs) in tears and keratin in normal and abnormal meibomian gland excretions warrants investigation into their effects on the surface activity of meibomian lipid films. Commercial keratin and bovine lung SPs were used in vitro to assess the surface pressure of meibomian lipid films using a Langmuir trough. The pressure-area profiles of meibomian lipid films seeded with SPs (2.5 μL; 0.1 μg) demonstrated hybrid characteristics between meibomian lipid films alone and SPs alone but reached much higher maximum surface pressures (approximately 30 vs. 24 mN/m). Microscopically, the appearance of meibomian lipid films was not altered by SPs. Maximum surface pressure of meibomian films premixed with keratin was much higher than meibum alone. The pressure-area isocycles appeared more like those of meibomian lipids with a low concentration of protein and more like pure keratin films at a high concentration. The data strongly indicate that SPs and keratin likely interact with the TFLL. SPs are likely to act as strong surfactants and to reduce the surface tension of the lipid layer. Excess concentrations of keratin as identified in patients with meibomian gland dysfunction could disrupt the normal structure of the meibomian lipid film.

Lacritin-Induced Secretion of Tear Proteins From Cultured Monkey Lacrimal Acinar Cells

During inflammation of the ocular surface, increased proinflammatory cytokines depress tear protein secretion, suggesting that a decline in lacrimal cell function contributes to dry eye. Lacritin, a glycoprotein secreted from lacrimal acinar cells, may function as an autocrine factor to stimulate tear protein secretion. The purpose of the present experiment was to investigate lacritin-induced protein secretion in normal and cytokine-pretreated (inflammation model) monkey acinar cells. Acinar cells from monkey lacrimal glands were cultured with or without tumor necrosis factor alpha (TNF-α) plus interferon gamma (IFN-γ). Protein secretion was induced by lacritin or carbachol (Cch, positive control). Proteins were detected and identified by immunoblotting and immunocytochemistry. Intracellular Ca(2+) was measured with the fluorophore Calcium-4, and cell damage was determined by LDH leakage into the culture medium. In cultured monkey acinar cells, lacritin stimulated tear protein secretion in normal cells without elevating intracellular Ca(2+). In contrast, Cch elevated intracellular Ca(2+) and release of tear proteins. This contrast suggested an alternate, calcium-independent mechanism for lacritin-induced protein secretion. TNF-α plus IFN-γ caused LDH leakage from sensitive human corneal epithelial cells, but even higher doses of TNF-α plus IFN-γ did not cause LDH leakage from monkey acinar cells, suggesting a higher tolerance against these cytokines. In cytokine-treated acinar cells, lacritin stimulated protein secretion as much as that in normal cells. In contrast, Cch-induced elevation of Ca(2+) and release of proteins were depressed by cytokines. Lacritin might be a useful biotechnology-based treatment agent against ocular surface diseases where endogenous lacritin is inadequate.

Alterations in the Tear Proteome of Dry Eye Patients—A Matter of the Clinical Phenotype

Previous studies demonstrated alterations in the tear proteome of dry eye patients. The aim of the present study was to analyze tear protein patterns of dry eye patients considering different clinical phenotypes in order to examine their influence on tear film protein composition. We applied a surface-enhanced laser desorption/ ionization-time-of-flight (SELDI-TOF)/matrix-assisted laser desorption/ ionization-time-of-flight (MALDI-TOF)/TOF mass spectrometry (MS)-based strategy to detect/identify candidate biomarkers. Tear samples of 169 patients, enrolled in two independent studies, were analyzed. Patients were subdivided into healthy controls(CTRL: N = 39), aqueous-deficient dry eye (DRYaq: N = 40), lipid-deficient dry eye (DRYlip: N = 40), and a combination of the two (DRYaqlip: N = 40). We uncovered six peptide/protein markers matching the stringent criteria applied for selection of reliable markers (P < 5.0E-03 in both studies). For example, proline-rich protein 4 was found to be diminished in DRYaq and DRYaqlip patients when compared to healthy subjects. Mammaglobin B and lipophilin A were found to be increased in these patients, as well as calgranulin S100A8. Remarkably, DRYlip patients revealed only slight alterations; these patients strongly deviated from the DRYaq or DRYaqlip group. With regard to classification of patients, we achieved discrimination from healthy subjects with a sensitivity and specificity ≈100% for DRYaq and DRYaqlip patients (receiver operating characteristic curve [ROC curve]: area under the curve [AUC] = 1) through use of the six-biomarker set. This study demonstrates that different clinical phenotypes of dry eye are reflected by specific alterations of the tear film proteome. Especially a deficiency of the aqueous phase of the tear film seems to strongly influence the expression patterns of several proteins.

Evaluation of the Frequency of Ophthalmic Solution Application: Washout Effects of Topical Saline Application on Tear Components

Purpose: To determine and compare the effects of single and frequent topical applications of saline solution on tear protein concentration in clinically normal subjects.Materials and methods: Tears were collected from both eyes of 11 normal volunteers using Schirmer’s strips. Saline solution (0.9% sodium chloride) was applied once in the right eye and five times with an interval of 1 min each in the left eye. Tears were collected before and 5, 15, 30 and 60 min after application of the solution. Total tear protein concentration in the samples was measured by the Bradford method and major tear protein concentration (secretory immunoglobulin A, lactoferrin, lipocalin-1, lysozyme and sialic acid) was measured using a high-performance liquid chromatography assay.Results: A significant decrease was observed in the concentration of total tear protein, major tear proteins and sialic acid after topical application of saline solution. This decrease was attributed to the washout and dilution effect. A low protein concentration persisted longer with more frequent application of the solution. The concentration of secretory immunoglobulin A and sialic acid concentration recovered slowly compared with that of other proteins.Conclusions: Even a single application of saline solution resulted in significant changes in major tear protein and sialic acid concentration in the tears of normal subjects. Differences in the recovery of tear protein concentration may be related to the process of protein production and secretion. A balance between normal tear function and the therapeutic effects of ophthalmic solutions should be considered when deciding the frequency of application, particularly in patients with dry eye.

Conserved Regional 3′ Grouping of Rare Codons in the Coding Sequence of Ocular Prosecretory Mitogen Lacritin

Lacritin is a prosecretory mitogen in tears and, although a tear protein, it promotes basal tearing and lacrimal gland secretion. Since scale up is relevant to its potential use in the treatment of dry eye, we explored various mutagenic strategies to alter the stability, solubility, and translational efficiency of nascent lacritin, and discovered 3' clustering of rare human codons. Site-directed mutagenesis of lacritin coding cDNA "pLAC" generated 24 different nonsynonymous and 13 synonymous mutations. Nonsynonymous mutations altered amino acids with nonpolar, basic or acidic side chains to serine. Synonymous mutation progressively optimized human codons that are rare or uncommon in Escherichia coli without changing the amino acid specified. These changes were validated by sequencing and protein production, and analyzed via the "rare codon calculator" (RCC). Nonhuman primate and nonprimate lacritin coding sequences were extracted from Ensembl, and analyzed via RCC using codon usage appropriate for each species. Superior yields were obtained by modification of individual hydrophobic residues or a predicted salt bridge, suggesting that production was limited by lacritin stability. Accordingly, elimination of rare codons increased yields less effectively. Importantly, RCC analysis of human, nonhuman primate (mouse lemur) and nonprimate (cat, tree shrew) lacritin coding sequences revealed remarkable 3' clustering of rare codons, unlike human lipocalin-1 and 21 other widely expressed human tear genes. Lacritin protein yields were improved primarily by hydrophobic or salt bridge mutagenesis and less so by elimination of rare codons. The 3' clustering of rare codons is conserved in all lacritin orthologs examined.

Comparison of two methods of tear sampling for protein quantification by Bradford method

The aim of this study was to compare two methods of tear sampling for protein quantification. Tear samples were collected from 29 healthy dogs (58 eyes) using Schirmer tear test (STT) strip and microcapillary tubes. The samples were frozen at -80ºC and analyzed by the Bradford method. Results were analyzed by Student's t test. The average protein concentration and standard deviation from tears collected with microcapillary tube were 4.45mg/mL ±0.35 and 4,52mg/mL ±0.29 for right and left eyes respectively. The average protein concentration and standard deviation from tears collected with Schirmer Tear Test (STT) strip were and 54.5mg/mL ±0.63 and 54.15mg/mL ±0.65 to right and left eyes respectively. Statistically significant differences (p&lt;0.001) were found between the methods. In the conditions in which this study was conducted, the average protein concentration obtained with the Bradford test from tear samples obtained by Schirmer Tear Test (STT) strip showed values higher than those obtained with microcapillary tube. It is important that concentration of tear protein pattern values should be analyzed according the method used to collect tear samples.

Pathogen Induced Changes in the Protein Profile of Human Tears from Fusarium Keratitis Patients

Fusarium is the major causative agent of fungal infections leading to corneal ulcer (keratitis) in Southern India and other tropical countries. Keratitis caused by Fusarium is a difficult disease to treat unless antifungal therapy is initiated during the early stages of infection. In this study tear proteins were prepared from keratitis patients classified based on the duration of infection. Among the patients recruited, early infection (n = 35), intermediate (n = 20), late (n = 11), samples from five patients in each group were pooled for analysis. Control samples were a pool of samples from 20 patients. Proteins were separated on difference gel electrophoresis (DIGE) and the differentially expressed proteins were quantified using DeCyder software analysis. The following differentially expressed proteins namely alpha-1-antitrypsin, haptoglobin α2 chain, zinc-alpha-2-glycoprotein, apolipoprotein, albumin, haptoglobin precursor - β chain, lactoferrin, lacrimal lipocalin precursor, cystatin SA III precursor, lacritin precursor were identified using mass spectrometry. Variation in the expression level of some of the proteins was confirmed using western blot analysis. This is the first report to show stage specific tear protein profile in fungal keratitis patients. Validation of this data using a much larger sample set could lead to clinical application of these findings.

Comparison of IgA, TNF-α and surface tension of the tear film in two different times of the day

The main goal was to study the biochemical composition of the tear film in two different times of the day. Tear samples were collected from 10 individuals, non contact lenses wearers, from the university population without pathologies. To assess daily variations in the tear film, samples were collected twice in the day, one early in the morning and another in the evening using capillary tubes. Tear protein profile was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), tumor necrosis factor α (TNF-α) and immunoglobulin A (IgA) were quantified by Enzyme-Linked ImmunoSorbent Assay (ELISA). Tear film stability was obtained through measurements of Non-Invasive Break Up Time and tear surface tension was measured by obtaining the Langmuir isotherms. The stability of the tear film was higher in the morning than in the afternoon; corresponding to a higher value of surface tension in the afternoon. Protein electrophoresis tear profile is variable during the day as IgA concentration decreased from morning to afternoon (p<0.05). TNF-α concentration also decreased, but there were not significant statistical differences (p=0.089). We concluded that there are daily variations in the composition and properties of the tear film, indicating that changes occur without being caused by contact lenses wear or by the presence of ocular and systemic pathologies. Presence of TNF-α is not a direct indication of inflammatory pathology, since this cytokine was identified in relatively high amounts in subjects without inflammatory pathology.