Tear Proteins Research Articles

Effect of Daily Disposable Contact Lens Solutions in Stabilizing the Activity of a Tear Film Protein

Purpose: Interactions between tear proteins and the interface of contact lenses (CL) can be complex and can influence CL wear success. Tear proteins, including lysozyme, function to maintain the balance of ocular surface homeostasis, evidenced by research of its conformation relative to stabilizing the tear film, its lubricating properties, and the potential impact of denatured lysozyme to corneal cells. A novel silicone hydrogel daily disposable CL, kalifilcon A, has integrated multiple moisturizers infused into the lens material to help stabilize the tear film. This in vitro study investigates the ability of daily disposable CL solutions to stabilize lysozyme in its native state.

Dysregulation of Receptor for Advanced Glycation End Products (RAGE) Expression as a Biomarker of Keratoconus.

Background Because of the implications of Receptor for Advanced Glycation End Products (RAGE) in keratoconus (KC), we describe a differential expression of RAGE transcripts and proteins in corneal tissues and tears of KC and healthy patients. Methods Using a case-controlled study, corneal epitheliums and tears of KC and healthy subjects were obtained during corneal collagen cross-linking and photorefractive keratectomy (PKR) and during usual consultations. Quantitative reverse transcription (RT-qPCR) and Western-Blot were performed to analyze RAGE transcripts and proteins' expression in corneal tissues and tears. Results One hundred and six patients were included in this study. The characteristics of the patients were as follows: 56 KC (25 corneal epithelium and 31 tears) and 50 control subjects (25 corneal epithelium and 25 tears). Transcripts of RAGE, HMGB1, and S100 family ligands were quantified by RT-qPCR, identifying a significantly higher expression of RAGE and HMGB1 in the healthy group than in the KC group (p = 0.03 and 0.04, respectively). Western Blot showed a significantly higher fl-RAGE expression in KC corneal epithelium than control (p < 0.001) and lower s-RAGE expression in KC tears than control (p = 0.04). Conclusions Linked with the inflammatory process occurring in KC pathophysiology, we propose for the first time that the RAGE expression (total and truncated forms of receptor and ligands) in KC corneal tissues and tear samples provides viable biomarkers.

Potential Tear Biomarkers for the Diagnosis of Parkinson's Disease-A Pilot Study.

Parkinson’s disease (PD) is the second most common neurodegenerative disease after Alzheimer’s disease. In this study, the tear proteome profile of patients with idiopathic PD (iPD, n = 24), carriers of the E46K-SNCA mutation (n = 3) and healthy control (CT, n = 27) subjects was analyzed to identify candidate biomarkers for the diagnosis of PD. An observational, prospective and case-control pilot study was carried out, analyzing the participants tear samples by nano-liquid chromatography–mass spectrometry (nLC–MS/MS) and assessing their neurological impairment. The proteomic data obtained are available at ProteomeXchange with identifier 10.6019/PXD028811. These analyses led to the identification of 560 tear proteins, some of which were deregulated in PD patients and that have been implicated in immune responses, inflammation, apoptosis, collagen degradation, protein synthesis, defense, lipid transport and altered lysosomal function. Of these proteins, six were related to neurodegenerative processes and showed a good capacity to classify patients and controls. These findings revealed that certain proteins were upregulated in the tears of PD patients, mainly proteins involved in lysosomal function. Thus, in this study, tear proteins were identified that are implicated in neurodegeneration and that may be related to an aggressive disease phenotype in PD patients.

Distribution of proteins in the different parts of the schirmer strips

AstractPurposeTo analyze and compare protein composition in different parts of the Schirmer strips (called the bulb and the rest of the strip) by using a comprehensive proteomics approach based on highly sensitive trapped ion‐mobility spectrometry coupled quadrupole time‐of‐flight (timsTOF Pro).MethodsSchirmer strips were collected from healthy subjects on 4 different visits over two consecutive days to rationally prepare three groups: 4 whole strips, 4 bulbs and 4 rests of the strips. Each group was extracted in ammonium bicarbonate before analysis with nanoElute UHPLC (ultra‐high‐pressure liquid chromatography) coupled timsTOF Pro. Protein Gene Ontology classification was performed by using Panther. Mass spectrometry data were processed using MaxQuant for protein identification.ResultsThe number of identified proteins increased by 49.6% when the bulbs are separated from the rests of the strips, each group being processed independently. Most of the identified proteins (%50.5) were common in the bulbs and the rests, 26.3% identified only in the bulbs and 23.2% only in the rests. Among all, 1650 proteins were identified from three groups, binding (44%) and catalytic activities (38.3%) constituted the main groups of molecular functions. The cellular (31.3%), metabolic (20.9%), biological regulation processes (13%) formed the main classes in the biological processes of identified proteins. A high number of enzymes (480), formed of hydrolases (47.5%), oxidoreductases (22.1%), transferases (16.7%), ligases (10%), isomerases, (2.1%) and lyases (1.7%) were identified in tear proteome.ConclusionsThe separate study of the bulbs and the rests identified several proteins that are not found in the entire strip alone. Probably, treating bulbs and rests apart allowed to elute more proteins from each part. A decrease in the concentration of abundant tear proteins might have enabled the identification of less abundant ones too. This methodology could improve the pre‐analytical steps before MS analysis. The dataset created can help to model and compare multiple signaling pathways associated with ocular surface pathologies. Moreover, the TimsTOF Pro could also add a technical improvement for the investigation of biomarkers in ocular diseases.

Analyzing changes in tear proteins of Sjogren’s syndrome patients with timsTOF pro

AbstractPurposeTo investigate changes in the tear proteome of patients with Sjögren's syndrome (SSp) using a high‐resolution trapped ion mobility spectrometry (TIMS) powered with parallel accumulation–serial fragmentation (PASEF).MethodsTear samples were collected using Schirmer strips from healthy controls (HC) and SSp, then extracted in ammonium bicarbonate. The protein content of each sample was normalized to 250 ng. Proteomics analysis was performed by using TIMS coupled quadrupole time‐of‐flight (timsTOF Pro) followed by data processing using MaxQuant software for protein identification. Differentially expressed proteins were detected by a Limma statistical test with a false discovery rate (FDR)˂1%. Protein Gene Ontology classification was performed by using Panther.ResultsThe proteins identified decreased by 23.9% in SSp compared to HC. Off the proteins identified in SSp, 90% were common with HC while only 68% of the proteins identified in HC were detected in SSp. A total of 175 proteins were significantly modulated in SSp with a fold‐change ≥ 1.5 (p‐value ≤0.05). None of the proteins detected only in SSp was among significantly modulated proteins but 25 proteins identified only in HC were significantly modulated. Catalytic activity (46.9%) and binding proteins (38.5%) formed the major molecular function groups among significantly modulated proteins. Proteins that exhibit binding activity were mainly down‐regulated in SSp while proteins involved in apoptosis such as caspase‐3 and proteasome subunits were up‐regulated. Several oxidoreductase enzymes (18) such as sulfhydryl oxidase‐1, Glutathione peroxidase‐3 and lactoperoxidase have been down‐regulated. Actins, actin‐binding proteins, tubulins, microtubule‐binding proteins were decreased in SSp. These results highlight an alteration in cellular process and antioxidase activity.ConclusionsA large amount of proteins expressed differently in SSp that involve important biological processes and signalling pathways have been revealed. The improved spatial resolution, sensitivity, speed, and specificity of timsTOF Pro can dramatically increase the ability to fully investigate the molecular factors behind the pathophysiology of SS.

The Effect of Different Cleaning Methods on Protein Deposition and Optical Characteristics of Orthokeratology Lenses.

Orthokeratology lenses are commonly used for myopia control, especially in children. Tear lipids and proteins are immediately adsorbed when the lens is put on the cornea, and protein deposition may cause discomfort or infection. Therefore, we established an in vitro protein deposition analysis by mimicking the current cleaning methods for orthokeratology lens wearers for both short-term and long-term period. The results showed that the amounts of tear proteins accumulated daily and achieved a balance after 14 days when the lens was rubbed to clean or not. Protein deposition also affected the optical characteristics of the lens regardless of cleaning methods. Our results provided an in vitro analysis for protein deposition on the lens, and they may provide a potential effective method for developing care solutions or methods that can more effectively remove tear components from orthokeratology lenses.

Effects of model tear proteins and topical ophthalmic formulations on evaporation inhibition and biophysical property of model tear lipid nanofilm in vitro

Biophysical property and water evaporation retardation through a lipid nanofilm can be altered by model tear protein and topical ophthalmic formulation. Evaporation rate and dynamic surface pressure were measured using a sessile drop technique. Water evaporations from 5 individual protein solutions, their mixture, and 6 ophthalmic formulations were quantified. Biophysical property and evaporation through model lipid nanofilms spread on model electrolyte solutions, tear protein solutions, and ophthalmic formulations were assessed. Model lipid nanofilms spread on electrolyte solution reduced evaporative fluxes by 43–60%. Evaporative fluxes from individual protein solutions without lipids were 3–19% lower than from electrolytes solution. Evaporative fluxes through lipid nanofilms were decreased by the presence of albumin or lactoferrin in solutions but increased by lysozyme and mucin. Evaporative fluxes from ophthalmic formulations were 10–43% lower than from electrolyte solution. Evaporations through lipid nanofilms spread on formulations were higher than through lipids on electrolyte solution. Model lipid nanofilms spread on Diquas appeared more rigid than on electrolyte solution but showed softening when spread on Refresh Mega-3. Some proteins and ophthalmic formulations altered model lipid nanofilms evaporative barriers. Ophthalmic formulation induced changes in biophysical property of model lipid nanofilms in vitro may suggest possible tear film destabilization in vivo.

Cytokine profile in tear and aqueous humor of primary open-angle patients as a prognostic factor for trabeculectomy outcome.

To evaluate the cytokine profile in tear and aqueous humor in primary open-angle glaucoma before trabeculectomy and correlate preoperative cytokine levels with the surgical outcome. Prospective study. Twenty-nine patients with primary open-angle glaucoma undergoing primary trabeculectomy were included. Levels of 27 cytokines were measured in tear an aqueous humor using the Bio-Plex Pro Human Cytokine 27-Plex Immunoassay kit (Bio-Rad Laboratories, Hercules, CA, USA). 29 patients who underwent trabeculectomy were included and their first-year follow-up visits were recorded. Mean age was 76.0 ± 7.0 years (range 56-84), mean intraocular pressure was 18.2 ± 3.6 mmHg and mean number of topical medications was 2.3 ± 0.9. At the one-year visit, 5 patients were classified as surgical failure. In aqueous humor, preoperative cytokine levels of regulated on activation normal T cell expressed and secreted (RANTES) were significantly higher in those patients with surgical failure at one year. IL-8 in tear and interferon gamma-induced protein (IP-10) in aqueous humor correlated positively with one-year IOP reduction. No statistically significant correlations were found with changes in visual field mean defect or global peripapillary retinal nerve fiber layer thickness (all, p >0.05). Preoperative RANTES levels in aqueous humor as well as other cytokines could serve as useful biomarkers for trabeculectomy outcome.

Looking deeper into ocular surface health: an introduction to clinical tear proteomics analysis.

Ocular surface diseases are becoming more prevalent worldwide. Reasons for this include the ongoing population ageing and increasing use of digital displays, although ophthalmologists have a wide selection of tools, which can be implemented in the evaluation of the ocular surface health, methods, which enable the in-depth study of biological functions are gaining more interest. These new approaches are needed, since the individual responses to ocular surface diseases and treatments can vary from person to person, and the correlations between clinical signs and symptoms are often low. Modern mass spectrometry (MS) methods can produce information on hundreds of tear proteins, which in turn can provide valuable information on the biological effects occurring on the ocular surface. In this review article, we will provide an overview of the different aspects, which are part of a successful tear proteomics study design and equip readers with a better understanding of the methods most suited for their MS-based tear proteomics study in the field of ophthalmology and ocular surface.

Electrostatic and Covalent Assemblies of Anionic Hydrogel-Coated Gold Nanoshells for Detection of Dry Eye Biomarkers in Human Tears.

Although dry eye is highly prevalent, many challenges exist in diagnosing the symptom and related diseases. For this reason, anionic hydrogel-coated gold nanoshells (AuNSs) were used in the development of a label-free biosensor for detection of high isoelectric point tear biomarkers associated with dry eye. A custom, aldehyde-functionalized oligo(ethylene glycol)acrylate (Al-OEGA) was included in the hydrogel coating to enhance protein recognition through the formation of dynamic covalent (DC) imine bonds with solvent-accessible lysine residues present on the surface of select tear proteins. Our results demonstrated that hydrogel-coated AuNSs, composed of monomers that form ionic and DC bonds with select tear proteins, greatly enhance protein recognition due to changes in the maximum localized surface plasmon resonance wavelength exhibited by AuNSs in noncompetitive and competitive environments. Validation of the developed biosensor in commercially available pooled human tears revealed the potential for clinical translation to establish a method for dry eye diagnosis.

Correlation between surface friction and the hydrophobicity of structure-related side-chain exposure of albumin on contact lens

The management of contact lens discomfort remains a challenge leading to the increased contact lens dropout rates. Tear protein accumulates on the lens surfaces with different configurations observed are correlated to the lens friction, with the improved comfort experienced by reduced surface friction in the eye. However, protein adsorption is a complex process with the combined protein-protein interactions (PPI) and protein-surface interactions (PSI) involved, which is difficult to explain the complicated tribological behavior in terms of protein structural shifts alone on lens surfaces. On the other hand, the type of solvent-exposed side chains from specific protein conformations on lens surfaces should be more important to the lens friction involved. We aim to investigate the correlation between the structure-related side-chain exposure and corresponding lens friction of adsorbed tear proteins on lens surfaces under varied PPI and PSI. Albumin was the model protein adsorbed onto the conventional lens material. Such as polymethylmethacrylate (PMMA) or the poly-2-hydroxyethyl methacrylate (PHEMA) surfaces applied here. Adsorption was conducted under varying protein solution concentrations to saturate the model surface to change the PPI effects over a wide range. Our results indicate that PPI effects help stabilize protein structures on both surfaces. When PPI is minimized, a distinct correlation was observed between the surface friction and the hydrophobicity of structure-related side-chain exposure of albumin on lens surfaces depending on the different PSI involved. At a fundamental understanding, our results would provide insights for developing new lens materials or the lens care solution designs to reduce the lens discomfort.

Longitudinal Tear Protein Changes Correlate with Ocular Chronic GVHD Development in Allogeneic Hematopoietic Stem Cell Transplant Patients

Ocular graft-versus-host disease (oGVHD) is a manifestation of chronic GVHD, frequently occurring in patients after allogeneic hematopoietic stem cell transplant (HSCT). We analyzed tear protein changes before and after allogeneic HSCT, and correlated their levels with the oGVHD development. This retrospective study included 102 patients, and data were recorded before the conditioning treatment, and after 3 to 6 months postoperatively. Tear protein analysis was performed with the Agilent-2100 Bioanalyzer on individual tears sampled by aspiration. Total protein (TP), Lysozyme-C (LYS-C), Lactoferrin (LACTO), Lipocalin-1 (LIPOC-1), Transferrin (TRANSF), Albumin (ALB), and Zinc-alpha-2-glycoprotein (ZAG-2) levels were retrieved and statistically analyzed. Following HSCT forty-three patients developed oGVHD. TP, LACTO, LYS-C, and ZAG-2 levels significantly decreased post-HSCT as compared to pre HSCT levels. In univariate analysis, TP, LACTO, and ZAG-2 decrease was associated with an increased development of oGVHD (OR = 4.49; 95% CI, 1.9 to 10.5; p &lt; 0.001; OR = 3.08; 95% CI 1.3 to 7.6; p = 0.01; OR = 11.1; 95% CI 2.7 to 46.6; p &lt; 0.001, respectively). TRANSF post-HSCT levels significantly increased (OR 15.7; 95% CI, 4.1 to 52.2; p = 0.0001). No pre-post-HSCT changes were shown in ALB and LIPOC-1 levels. Data suggest that TP content, LACTO, TRANSF, and ZAG-2 pre-post changes might be significant predictors of oGVHD development.

Phenylephrine increases tear cathepsin S secretion in healthy murine lacrimal gland acinar cells through an alternative secretory pathway

Little is known about the relationship between stimulation of lacrimal gland (LG) tear protein secretion by parasympathetic versus sympathetic nerves, particularly whether the spectrum of tear proteins evoked through each innervation pathway varies. We have previously shown that activity and abundance of cathepsin S (CTSS), a cysteine protease, is greatly increased in tears of Sjögren's syndrome (SS) patients and in tears from the male NOD mouse of autoimmune dacryoadenitis that recapitulates SS-associated dry eye disease. Beyond the increased synthesis of CTSS detected in the diseased NOD mouse LG, increased tear CTSS secretion in NOD mouse tears was recently linked to increased exocytosis from a novel endolysosomal secretory pathway. Here, we have compared secretion and trafficking of CTSS in healthy mouse LG acinar cells stimulated with either the parasympathetic acetylcholine receptor agonist, carbachol (CCh), or the sympathetic α1-adrenergic agonist, phenylephrine (PE). In situ secretion studies show that PE significantly increases CTSS activity and protein in tears relative to CCh stimulation by 1.2-fold (***, p = 0.0009) and ∼5-fold (*, p-0.0319), respectively. A similar significant increase in CTSS activity with PE relative to CCh is observed when cultured LGAC are stimulated in vitro. CCh stimulation significantly elevates intracellular [Ca2+], an effect associated with increases in the size of Rab3D-enriched vesicles consistent with compound fusion, and subsequently decreases in their intensity of labeling consistent with their exocytosis. PE stimulation induces a lower [Ca2+] response and has minimal effects on Rab3D-enriched SV diameter or the intensity of Rab3D-enriched SV labeling. LG deficient in Rab3D exhibit a higher sensitivity to PE stimulation, and secrete more CTSS activity. Significant increases in the colocalization of endolysosomal vesicle markers (Lamp1, Lamp2, Rab7) with the subapical actin suggestive of fusion of endolysosomal vesicles at the apical membrane occur both with CCh and PE stimulation, but PE demonstrates increased colocalization. In conclusion, the α1-adrenergic agonist, PE, increases CTSS secretion into tears through a pathway independent of the exocytosis of Rab3D-enriched mature SV, possibly representing an alternative endolysosomal secretory pathway.

Tear dynamics testing and quantitative proteomics analysis in patients with chronic renal failure

Ocular surface changes may develop in patients with chronic renal failure (CRF) undergoing hemodialysis. In recent years, an association of CRF with dry eye syndrome has been emphasized. However, tear proteomics of CRF patients has not been analyzed. Here, we performed systematic profiling of the tear film proteins in CRF patients through use of isobaric tags for relative and absolute quantitative (iTRAQ) MS/MS, aiming to identify associations between dry eye symptoms and expression of tear proteomic changes in patients with CRF undergoing hemodialysis. Twenty CRF patients and ten healthy subjects underwent a series of ophthalmic examinations. Tear samples from the participants were analyzed by iTRAQ approach. A total of 1139 tear proteins were screened, and 212 differentially expressed proteins were identified. The pattern changes included 77 whose expression levels were upregulated (fold increase >1.2) whereas 135 others that were downregulated (fold decrease <1/1.2). Bioinformatics analysis showed that these proteins were significantly enriched in lipid metabolism, inflammatory, and immune response pathways. Furthermore, APOA1, APOA4, APOB, APOE, S100A8, S100A9, S100A4, HSP90B and other molecules were significantly changed. Our study elucidated the characteristics of tear dynamics and protein markers in CRF patients undergoing hemodialysis.Significance: Despite the association of chronic renal failure (CRF) with dry eye disease, there are no reports describing potentially important differentially expressed tear proteins in CRF patients undergoing hemodialysis. It is still a challenge to obtain a comprehensive description of the pathogenesis of dry eye in CRF patients which hinders establishing a patient specific therapeutic scheme. Our study is the first iTRAQ proteomics analysis of the tears of patients with CRF, which reveals the changes in the protein expression profile in CRF patients afflicted with dry eye disease. The identity was verified of some relevant differentially expressed proteins, and they may be candidate diagnostic markers of dry eye disease in patients with CRF. These tear film protein constituents found in hemodialysis patients can be of important clinical significance in treating this condition. SignificanceDespite the association of chronic renal failure (CRF) with dry eye disease, there are no reports describing potentially important differentially expressed tear proteins in CRF patients undergoing hemodialysis. It is still a challenge to obtain a comprehensive description of the pathogenesis of dry eye in CRF patients which hinders establishing a patient specific therapeutic scheme. Our study is the first iTRAQ proteomics analysis of the tears of patients with CRF, which reveals the changes in the protein expression profile in CRF patients afflicted with dry eye disease. The identity was verified of some relevant differentially expressed proteins, and they may be candidate diagnostic markers of dry eye disease in patients with CRF. These tear film protein constituents found in hemodialysis patients can be of important clinical significance in treating this condition.

Reduced Level of Tear Antimicrobial and Immunomodulatory Proteins as a Possible Reason for Higher Ocular Infections in Diabetic Patients.

(1) Background: Diabetes mellitus is one of the most common metabolic disorders and a risk factor for bacterial ocular infections. Our aim was to examine the antibacterial activity of tears from patients with diabetes mellitus with and without diabetic retinopathy and to link this activity to the level of tear proteins. (2) Methods: Non-stimulated basal tears were collected from 39 eyes of 35 subjects. The antibacterial activity of tear pools was tested against pathogenic Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 26922 and Pseudomonas aeruginosa ATCC 27853 strains. The levels of 10 antimicrobial and immunomodulatory proteins were analyzed in the individual tear samples of the studied groups by SRM-based targeted mass spectrometry analysis. (3) Results: Disease stage-specific antimicrobial effect was observed in case of Staphylococcus aureus ATCC 29213 strain, and a non-disease specific inhibitory effect was observed in case of Pseudomonas aeruginosa ATCC 27853 strain. Changes in the levels of the studied antimicrobial and immunomodulatory proteins in the tears of the studied groups were also observed. (4) Conclusions: The higher ocular infection rate observed in diabetic patients may be the consequence of the decreased antimicrobial activity of tears possibly caused by the changes in the levels of antimicrobial and immunomodulatory proteins.

Nuclear lamin A in rotator cuff tear margin tenocytes: an antiapoptotic and cell mechanostat factor

BackgroundThe network of intermediate filament proteins underlying the inner nuclear membrane forms the nuclear lamin. A- and B-type lamins are the major components of the nuclear lamina. Lamins function in many nuclear activities. The role of lamin A and transcription factors (NF-kB) as anti-apoptotic is well documented. Recently, lamin A has also been considered as a mechanosensor protein that is able to maintain nuclear integrity from mechanical insults.We aimed to verify how lamin A expression varies in healthy cuff cells and in those with different-sized tears where various mechanical stresses are present.MethodsForty-three patients with rotator cuff tear (RCT) [23M–20F, mean age (SD): 63.5 (6.1)] were enrolled. Tissue samples excised from the most medial point of tear margins were analyzed for lamin A expression by immunohistochemistry. Controls were represented by samples obtained by normal supraspinatus tendons excised from patients submitted to reverse shoulder prosthesis implant [8M–7F, mean age (SD): 67.9 (7.1)]. The intensity of staining was graded, and an H-score was assigned. Statistical analysis was performed.ResultsOur study revealed a moderate intensity of lamin A in the healthy cuff tendons, a higher expression of this protein in the small tears, and a significant decrease of lamin A with increasing tear size (p < 0.0001).ConclusionsOur study emphasizes the importance of early repair of small RCTs since nuclear stability is maintained, and the cellular function is protected by lamin A overexpression. High re-tear of massive cuff repair could be due to cellular apoptosis and nuclear modifications induced by lamin A lack.Level of evidenceIII

A Pilot Proteomic Study of Normal Human Tears: Leptin as a Potential Biomarker of Metabolic Disorders

The concentrations of insulin, leptin, active ghrelin, C-peptide and gastric inhibitory polypeptide (GIP) and their inter-day variations were examined in normal human tears. In addition, correlations between the concentrations of these metabolic proteins and ocular surface parameters were determined. Subjects with healthy ocular surfaces attended three visits, with 7-day intervals. Tear evaporation rate (TER) and non-invasive tear break-up time (NITBUT) were assessed, and a total of 2 µL tears were collected from all subjects. Tear fluid concentrations of insulin, leptin, active ghrelin, C-peptide and GIP were measured by multiplex bead analysis. Insulin was the most highly expressed metabolic protein, followed by leptin, C-peptide, active ghrelin and GIP. Of these, only active ghrelin had a significant inter-day variation (p &lt; 0.05). There was no inter-day variation in the mean concentrations of the other metabolic proteins. Leptin had a strong intra-class reproducibility. No correlation was detected between tear metabolic protein concentrations and ocular surface parameters. This pilot study shows, for the first time, that active ghrelin and GIP are detectable in healthy tears. The strong intra-class reproducibility for leptin shows that it could be used as a potential tear fluid biomarker and, possibly, in determining the effects of metabolic disorders on the ocular surface.

Effect of tear fluid sampling and processing on total protein quantity and electrophoretic pattern.

Human tears contain more than 1500 proteins that could be diagnostically relevant. To date, numerous candidates on a biomarker of protein origin were identified for ocular and systemic diseases. However, the suitable sampling method is still the subject of discussion. To address the need for a description of sampling methods properties for possible clinical analyses, we studied a total protein concentration and electrophoretic pattern of tear fluid collected by capillary tubes, Schirmer strips, cellulose microsponges, and flushing. The total protein concentration was 4.339 μg/μL ± 1.905 μg/μL, 0.967 μg/μL ± 0.117 μg/μL, 0.022 μg/μL ± 0.016 μg/μL, and 0.008 μg/μL ± 0.006 μg/μ for the capillary tubes, Schirmer strips, flushing, and cellulose microsponges, respectively. Sodium dodecyl sulfate polyacrylamide electrophoresis showed the different patterns of tear proteins obtained by the above-mentioned sampling methods. These differences could originate from the use of a bigger amount of extraction reagent that was not used in the case of capillary tubes, and retention of the proteins by strips and sponges. Taken together, capillary tubes, Schirmer strips, cellulose microsponges, and flushing represent sensitive and convenient sampling methods for tear fluid collection. For the isolation of proteins from strips and sponges, and for the flushing, less than 100 μL of a reagent should be used to ensure the sufficient concentration of the biomarkers in a trace amount.

Changes in tear protein profile in dogs with keratoconjunctivitis sicca following topical treatment using cyclosporine A.

Background and Aim:Keratoconjunctivitis sicca (KCS) is a chronic inflammatory ocular disease that occurs in many dog breeds worldwide. This study aimed to investigate the tear protein pattern of healthy dogs, KCS dogs, and KCS dogs after treatment with cyclosporine A (CsA).Materials and Methods:Twenty-eight dogs of any breed were enrolled in the study. The subjects were divided into three groups: Healthy, KCS, and CsA-treated dogs. Tear samples were collected using Schirmer strips. Tear proteins extracted from the strips were analyzed using two-dimensional electrophoresis. For the first dimension, total protein from tears was separated by isoelectric focusing. The second dimension was performed using 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gel images were analyzed and the protein spots of differential expression were manually cut for protein annotation using mass spectrometry.Results:In total, 12 protein spots were excised and subjected to protein identification. Associated with KCS, six protein spots were a downregulated protein, namely, lysozyme. The other six protein spots were upregulated in KCS dogs, consisting of heat shock protein beta-1, protein S100-A12, and keratin type II cytoskeletal 1 and 5. After treatment with CsA for 45 days, the lysozyme protein was still decreasing and the inflammation protein (S100-A12) was not identified.Conclusion:Inflammatory tear proteins and proteins involved in cellular stress were present in KCS dogs and appeared to be reduced in medicated eyes. Treatment with topical CsA in the short term may not improve the activity of antibacterial proteins. Changes in the expression patterns of these four proteins might be useful for disease severity and progression assessment, as well as for exploring a novel method for dry eye management in dogs.

Diagnostic biomarkers in tear fluid: from sampling to preanalytical processing

Tear fluid is receiving growing attention as a source for novel diagnostic biomarkers. Multiple techniques are available for its collection and impact the composition of acquired samples. We sought to provide a direct comparison of two collection methods with regard to implementation, acceptance, and impact on sample composition. Tear fluid was collected from fifteen healthy volunteers with capillary tubes and Schirmer strips and analyzed for total protein and IgG concentrations. Sampling parameters and perception by test persons were compared. The use of capillary tubes was more convenient for the participants while causing more effort for the collector. Tear flow rates as well as the relative and absolute amount of IgG were higher when Schirmer strips were used. Consecutive collections with Schirmer strips significantly influenced tear flow rates, IgG, and protein concentrations. A moderate correlation was observed between tear flow rates and IgG concentrations for both methods. Samples collected with both methods can be analyzed by isoelectric focusing, a potential diagnostic application in the field of neurology. The specific advantages and limitations of tear fluid sampling with either capillary tubes or Schirmer strips demonstrate the need for a thorough investigation of collection methods with regard to the application of interest.