Tear Fluid Proteins Research Articles

A review: In-situ gel drug delivery system

The eye is the body's most delicate organ. Pharmaceutical scientists find that designing ocular drug delivery systems is the most difficult because only 5% of administered drugs reach the eye due to the complex anatomical structure of the eye, the cornea's small absorptive surface and low transparency, the cornea's lipophilicity, pre-keratitis (from nasolacrimal drainage), the drug's bonding with proteins in tear fluid, blinking, and the conjunctival sac's low capacity, all of which limit the entry of drug molecules at the site of action and ultimately result in suboptimal ocular therapy. Considerable research is being done on developing more advanced drug delivery methods for ocular administration in an attempt to increase the bioavailability of ophthalmic medications. These innovative drug delivery methods have many benefits over traditional ones, including better release profiles that boost drug delivery efficiency and lower drug toxicity. Numerous studies in this field support the idea that in situ gelling devices can be helpful for the transport of drugs into the eyes. Drug delivery systems known as "in situ gel-forming systems" are in solution before being supplied to the body, but thereafter go through a process called "in situ gelation" to form a gel in response to an external stimulus like pH or temperature. A brief overview of in situ gels, several methods for in situ gelling systems, types of polymers utilized in in-situ gels, their mechanisms of gel formation, and an assessment of polymeric in situ gel are all included in this review.

Ocular tear fluid biomarkers collected by contact lenses

PurposeTo collect tear fluid biomarkers from contact lenses (CLs) and determine the impact of CL wear duration. MethodsRabbits were fitted with commercial etafilcon A CLs, which were collected after 1 min, 4 and 8 h (n = 4/time point). Tear fluid proteins and cytokines were extracted from the CLs and quantified. An exploratory comparison was performed between CLs and Schirmer Strips (SS) for a 1 min duration. ResultsThe concentration of MUC5AC was significantly higher after 4 h of CL wear. The expression of all investigated cytokines (IL-1α, IL-1β, IL-8, IL-17A, IL-21, Leptin, MIP-1β, MMP-9, NCAM-1, and TNF-α) was detectable after 1 min of CL wear, and over time, all showed significant variations throughout the 8-h CL wear period. Notably, IL-1α significantly increased by 8 h of CL wear, while MMP-9 decreased. Albumin and lysozyme did not show significant variations with CL wear. Differences between CLs and SS after 1 min were statistically significant for albumin, Leptin, TNF-α, IL-1α, IL-1β, and IL-8. ConclusionsThe duration of CL wear significantly affects the collection of some tear fluid biomarkers. Albumin, MUC5AC, and cytokines may have individual and synergistic diagnostic or prognostic potential. CLs and SS were similar for lysozyme and MUC5AC but differed in the collection of albumin and some cytokines. CLs are a viable tear fluid collection method for biomarker analyses and can be immediately added as a routine clinical test by being FDA-approved medical devices.

Neutrophil-driven and interleukin-36γ-associated ocular surface inflammation in chronic Stevens-Johnson syndrome.

This study aims to elucidate the tear proteome and understand the underlying molecular mechanisms involved in the ocular complications following Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN). Mass spectrometry (MS) was performed to quantify the tear fluid proteins from chronic SJS/TEN patients (n = 22 eyes) and age- and gender-matched controls (n = 22 eyes). The candidate proteins were validated using ELISA (n = 80 eyes) in tear samples and immunohistochemistry (IHC; n = 12) in eyelid margin specimens. These proteins were compared for significant differences based on age, gender, disease duration, and ocular severity. A total of 1692 tear fluid proteins were identified, of which 470 were significantly differentially regulated in chronic SJS/TEN. The top 10 significantly upregulated proteins were neutrophil secretions including neutrophil elastase (p < .0001), defensin (p < .0001), and matrix metalloproteinase 8 (p < .0001). The presence of neutrophils was confirmed by the upregulation of IL-8 (p < .001) in tears, a key cytokine known for recruiting neutrophils. Additionally, positive expression of myeloperoxidase was observed in the keratinized eyelid margins of SJS/TEN to validate the presence of neutrophils. Among 41 unique proteins identified by MS, IL-36γ (p < .01) was expressed in three SJS/TEN patients and was confirmed in SJS/TEN tears and eyelid margins by ELISA and IHC, respectively. IL-36γ was specifically expressed in the superficial layers of eyelid margin keratinized conjunctiva. The majority of the significantly downregulated proteins were lacrimal gland secretions such as lacritin (p < .0001) and opiorphin (p < .002). Neutrophil elastase (p < .02) was significantly elevated in patients with severe eyelid margin keratinization. Our observations indicate a clear correlation between eyelid margin keratinization and the expression of IL-36γ, potentially mediated by neutrophils recruited via IL-8. Future experimental studies are needed to test the role of therapies targeting IL-8 and/or IL-36γ in reducing eyelid margin keratinization and its associated ocular complications in SJS/TEN.

Analyzing Tear Fluid Composition by Synchronous Fluorescence for Diagnosing Dry Eye Disease and the Role of Phytotherapy Intervention

Purpose Tear fluid gained attention as a representative biological fluid. Its simple and non-invasive collection methods as well as richness of candidate biomarkers made it a potential diagnostic tool for different diseases such as dry eye. Synchronous fluorescence spectroscopy is a highly sensitive analytical tool that results in narrowing and enhanced peak resolution, and has a potential role in disease diagnosis, biomarker identification, and therapeutic monitoring. We applied synchronous fluorescence spectroscopy to monitor variations of tear fluid composition during the development of dry eye disease and to evaluate the potential effects of phytotherapy. Methods Dry eye model was induced in Chinchilla rabbits by instillation of 1% atropine sulfate ophthalmic solution. Then, the tear fluid was collected at 3, 7, and 14 days and subjected to synchronous fluorescence spectroscopy. Phytotherapy was achieved by topical instillation of 20 µl of water extracts of pomegranate peel or green tea powders. Results The fluorescence results revealed changes in the structure of tear fluid over time and the eye is subjected to toxification due to oxidative stress. In addition, dry eye disease was found to affect the metabolic/energetic state of the eye. On the other hand, phytotherapy led to enhancement of the metabolic/biosynthesis state due to activation of flavin adenine dinucleotide-associated proteins. Conclusion There was change in the electrical conductivity of tear fluid proteins. In the case of dry eyes, they became electrical insulators, while in the case of treatment with extracts, their electrical conductivity properties improved. The effects of phytotherapy can be related to the high content of ellagic acid and anthocyanin of pomegranate extract, while in green tea, they are related to catechins and phenolic compounds.

Unraveling the Intraday Variations in the Tear Fluid Proteome.

Tear fluid is a complex and dynamic biological fluid that plays essential roles in maintaining ocular homeostasis and protecting against the external environment. Owing to the small sample volume, studying the tear proteome is challenging. However, advances in high-resolution mass spectrometry have expanded tear proteome profiling, revealing >500 unique proteins. Tears are emerging as a noninvasive source of biomarkers for both ocular and systemic diseases; nevertheless, intraday variability of proteins in tear fluid remains questionable. This study investigates intraday variations in the tear fluid proteome to identify stable proteins that could act as candidate biomarkers. Tear samples from 15 individuals at four time points (10 am, 12 pm, 2 pm, and 4 pm) were analyzed using mass spectrometry to evaluate protein variation during these intervals. Technical variation was assessed by analyzing pooled samples and was subtracted from the total variation to isolate biological variability. Owing to high technical variation, low-abundant proteins were filtered, and only 115 proteins met the criteria for further analysis. These criteria include being detected at all four time points in at least eight subjects, having a mean peptide-spectrum match count greater than 5, and having a technical variation less than 0.10. Lactotransferrin, lipocalin-1, and several immunoglobulins were among the 51 stable proteins (mean biological coefficient of variation < 0.10). Additionally, 43 proteins displayed significant slopes across the 4 time points, with 17 increasing and 26 decreasing over time. These findings contribute to the understanding of tear fluid dynamics and further expand our knowledge of the tear proteome.

High Levels of Mutant Huntingtin Protein in Tear Fluid From Huntington's Disease Gene Expansion Carriers.

Huntington's disease (HD) is an autosomal dominant, fully penetrant, neurodegenerative disease that most commonly affects middle-aged adults. HD is caused by a CAG repeat expansion in the HTT gene, resulting in the expression of mutant huntingtin (mHTT). Our aim was to detect and quantify mHTT in tear fluid, which, to our knowledge, has never been measured before. We recruited 20 manifest and 13 premanifest HD gene expansion carriers, and 20 age-matched controls. All patients underwent detailed assessments, including the Unified Huntington's Disease Rating Scale (UHDRS) total motor score (TMS) and total functional capacity (TFC) score. Tear fluid was collected using paper Schirmer's strips. The level of tear mHTT was determined using single-molecule counting SMCxPRO technology. The average tear mHTT levels in manifest (67,223 ± 80,360 fM) and premanifest patients (55,561 ± 45,931 fM) were significantly higher than those in controls (1,622 ± 2,179 fM). We noted significant correlations between tear mHTT levels and CAG repeat length, "estimated years to diagnosis," disease burden score and UHDRS TMS and TFC. The receiver operating curve demonstrated an almost perfect score (area under the curve [AUC] = 0.9975) when comparing controls to manifest patients. Similarly, the AUC between controls and premanifest patients was 0.9846. The optimal cutoff value for distinguishing between controls and manifest patients was 4,544 fM, whereas it was 6,596 fM for distinguishing between controls and premanifest patients. Tear mHTT has potential for early and noninvasive detection of alterations in HD patients and could be integrated into both clinical trials and clinical diagnostics.

Tear nanoDSF Denaturation Profile Is Predictive of Glaucoma.

Primary open-angle glaucoma (POAG) is a frequent blindness-causing neurodegenerative disorder characterized by optic nerve and retinal ganglion cell damage most commonly due to a chronic increase in intraocular pressure. The preservation of visual function in patients critically depends on the timeliness of detection and treatment of the disease, which is challenging due to its asymptomatic course at early stages and lack of objective diagnostic approaches. Recent studies revealed that the pathophysiology of glaucoma includes complex metabolomic and proteomic alterations in the eye liquids, including tear fluid (TF). Although TF can be collected by a non-invasive procedure and may serve as a source of the appropriate biomarkers, its multi-omics analysis is technically sophisticated and unsuitable for clinical practice. In this study, we tested a novel concept of glaucoma diagnostics based on the rapid high-performance analysis of the TF proteome by differential scanning fluorimetry (nanoDSF). An examination of the thermal denaturation of TF proteins in a cohort of 311 ophthalmic patients revealed typical profiles, with two peaks exhibiting characteristic shifts in POAG. Clustering of the profiles according to peaks maxima allowed us to identify glaucoma in 70% of cases, while the employment of artificial intelligence (machine learning) algorithms reduced the amount of false-positive diagnoses to 13.5%. The POAG-associated alterations in the core TF proteins included an increase in the concentration of serum albumin, accompanied by a decrease in lysozyme C, lipocalin-1, and lactotransferrin contents. Unexpectedly, these changes were not the only factor affecting the observed denaturation profile shifts, which considerably depended on the presence of low-molecular-weight ligands of tear proteins, such as fatty acids and iron. Overall, we recognized the TF denaturation profile as a novel biomarker of glaucoma, which integrates proteomic, lipidomic, and metallomic alterations in tears, and monitoring of which could be adapted for rapid non-invasive screening of the disease in a clinical setting.

Biomedical Applications of Lactoferrin on the Ocular Surface

Lactoferrin (LF) is a first-line defense protein with a pleiotropic functional pattern that includes anti-inflammatory, immunomodulatory, antiviral, antibacterial, and antitumoral properties. Remarkably, this iron-binding glycoprotein promotes iron retention, restricting free radical production and avoiding oxidative damage and inflammation. On the ocular surface, LF is released from corneal epithelial cells and lacrimal glands, representing a significant percentage of the total tear fluid proteins. Due to its multifunctionality, the availability of LF may be limited in several ocular disorders. Consequently, to reinforce the action of this highly beneficial glycoprotein on the ocular surface, LF has been proposed for the treatment of different conditions such as dry eye, keratoconus, conjunctivitis, and viral or bacterial ocular infections, among others. In this review, we outline the structure and the biological functions of LF, its relevant role at the ocular surface, its implication in LF-related ocular surface disorders, and its potential for biomedical applications.

Putative Biomarkers in Tears for Diabetic Retinopathy Diagnosis.

PurposeTear fluid biomarkers may offer a non-invasive strategy for detecting diabetic patients with increased risk of developing diabetic retinopathy (DR) or increased disease progression, thus helping both improving diagnostic accuracy and understanding the pathophysiology of the disease. Here, we assessed the tear fluid of nondiabetic individuals, diabetic patients with no DR, and diabetic patients with nonproliferative DR (NPDR) or with proliferative DR (PDR) to find putative biomarkers for the diagnosis and staging of DR.MethodsTear fluid samples were collected using Schirmer test strips from a cohort with 12 controls and 54 Type 2 Diabetes (T2D) patients, and then analyzed using mass spectrometry (MS)-based shotgun proteomics and bead-based multiplex assay. Tear fluid-derived small extracellular vesicles (EVs) were analyzed by transmission electron microscopy, Western Blotting, and nano tracking.ResultsProteomics analysis revealed that among the 682 reliably quantified proteins in tear fluid, 42 and 26 were differentially expressed in NPDR and PDR, respectively, comparing to the control group. Data are available via ProteomeXchange with identifier PXD033101. By multicomparison analyses, we also found significant changes in 32 proteins. Gene ontology (GO) annotations showed that most of these proteins are associated with oxidative stress and small EVs. Indeed, we also found that tear fluid is particularly enriched in small EVs. T2D patients with NPDR have higher IL-2/-5/-18, TNF, MMP-2/-3/-9 concentrations than the controls. In the PDR group, IL-5/-18 and MMP-3/-9 concentrations were significantly higher, whereas IL-13 was lower, compared to the controls.ConclusionsOverall, the results show alterations in tear fluid proteins profile in diabetic patients with retinopathy. Promising candidate biomarkers identified need to be validated in a large sample cohort.

Association of tear fluid amyloid and tau levels with disease severity and neurodegeneration

There has been increasing interest in finding non-invasive biomarkers for neurodegenerative diseases such as Alzheimer’s disease (AD). This observational study investigated AD-specific biomarkers in tear fluid. Tear fluid was collected from a total of 65 subjects, including 23 patients with subjective cognitive decline (SCD), 22 patients with mild cognitive impairment (MCI), 11 dementia patients and 9 healthy controls (HC). Levels of amyloid-beta peptides (AB38, AB40, AB42), total-tau (t-tau) and phosphorylated-tau (p-tau) were determined using multiplex immunoassays. Levels of AB40 and t-tau were detectable in the vast majority (> 94%) of tear fluid samples. Cerebrospinal fluid (CSF) was available from a subset of patients. In this group, tear t-tau levels were significantly higher in people with dementia compared to SCD patients. Tear t-tau levels were elevated in patients with neurodegeneration (classified according to the A/T/N system) compared to patients without neurodegeneration. Negative correlations were found between CSF AB42 and CSF t-tau, and between CSF AB42 and tear t-tau. In summary, this study shows the potential of tau proteins in tear fluid to be associated with disease severity and neurodegeneration.

Establishment of functional epithelial organoids from human lacrimal glands

BackgroundTear deficiency due to lacrimal gland (LG) dysfunction is one of the major causes of dry eye disease (DED). Therefore, LG stem cell-based therapies have been extensively reported to regenerate injured lacrimal tissue; however, the number of stem cells in the LG tissue is low, and 2D long-term cultivation reduces the differentiation capacity of stem cells. Nevertheless, 3D LG organoids could be an alternative for a DED therapy because it is capable of prolonged growth while maintaining the characteristics of the LG tissue. Here, we report the development of LG organoids and their application as cell therapeutics.MethodsDigested cells from human LG tissue were mixed with Matrigel and cultured in five different media modified from human prostate/salivary organoid culture media. After organoid formation, the growth, specific marker expression, and histological characteristics were analyzed to authenticate the formation of LG organoids. The secretory function of LG organoids was confirmed through calcium influx or proteomics analysis after pilocarpine treatment. To explore the curability of the developed organoids, mouse-derived LG organoids were fabricated and transplanted into the lacrimal tissue of a mouse model of DED.ResultsThe histological features and specific marker expression of LG organoids were similar to those of normal LG tissue. In the pilocarpine-treated LG organoid, levels of internal Ca2+ ions and β-hexosaminidase, a lysosomal protein in tear fluid, were increased. In addition, the secreted proteins from pilocarpine-treated lacrimal organoids were identified through proteomics. More than 70% of the identified proteins were proven to exosome through gene ontology analysis. These results indicate that our developed organoid was pilocarpine reactive, demonstrating the function of LG. Additionally, we developed LG organoids from patients with Sjogren’s syndrome patients (SS) and confirmed that their histological features were similar to those of SS-derived LG tissue. Finally, we confirmed that the mouse LG organoids were well engrafted in the lacrimal tissue two weeks after transplantation.ConclusionThis study demonstrates that the established LG organoids resemble the characteristics of normal LG tissue and may be used as a therapy for patients with DED.

SARS-CoV-2 RNA and antibodies in tear fluid

Background/aimsSARS-CoV-2 is highly contagious. More evidence concerning extrapulmonary transmission routes such as the eyes is urgently needed. Although the humoral immune response is important in the viral containment, the local...

Electrospun GelMA fibers and p(HEMA) matrix composite for corneal tissue engineering

The development of biocompatible and transparent three-dimensional materials is desirable for corneal tissue engineering. Inspired from the cornea structure, gelatin methacryloyl-poly(2-hydroxymethyl methacrylate) (GelMA-p(HEMA)) composite hydrogel was fabricated. GelMA fibers were produced via electrospinning and covered with a thin layer of p(HEMA) in the presence of N,N′-methylenebisacrylamide (MBA) as cross-linker by drop-casting. The structure of resulting GelMA-p(HEMA) composite was characterized by spectrophotometry, microscopy, and swelling studies. Biocompatibility and biological properties of the both p(HEMA) and GelMA-p(HEMA) composite have been investigated by 3D cell culture, red blood cell hemolysis, and protein adsorption studies (i.e., human serum albumin, human immunoglobulin and egg white lysozyme). The optical transmittance of the GelMA-p(HEMA) composite was found to be approximately 70% at 550 nm. The GelMA-p(HEMA) composite was biocompatible with tear fluid proteins and convenient for cell adhesion and growth. Thus, as prepared hydrogel composite may find extensive applications in future for the development of corneal tissue engineering as well as preparation of stroma of the corneal material.

Tear Levels of IGFBP-3: A Potential Biomarker for Diabetic Nerve Changes in the Cornea.

Type 2 diabetes mellitus has reached epidemic levels in the United States and worldwide. Ocular complications from this disease include diabetic retinopathy and keratopathy, both of which can lead to significant vision loss. While frequently underappreciated, diabetic keratopathy is associated with painful ocular surface disorders, including corneal erosions and delayed wound healing. Recent work in our laboratory has focused on the role of the insulin-like growth factor (IGF) system in diabetic corneal disease. Here, we review recent findings on the presence of IGF-1, insulin, and the insulin-like binding protein (IGFBP-3) in human tear fluid and evaluate their potential use as biomarkers in diabetes. We further examine clinical evidence using in vivo confocal microscopy as an important imaging biomarker in diabetes and discuss associations between tear film changes in diabetes and corneal nerve loss. IGFBP-3 was the only tear film marker significantly associated with nerve loss in type 2 diabetes, whereas tear levels of IGF-1 were associated with aging. Interestingly, tear levels of IGFBP-3 were not directly related to serum levels of HbA1c, suggesting that hyperglycemia alone is not driving increased secretion of this protein. Overwhelming evidence supports the use of in vivo confocal microscopy as a tool to evaluate corneal nerve and epithelial changes induced by diabetes in research settings. The newly identified relationship between morphological changes in the corneal subbasal nerve plexus in diabetes and the increase in tear levels of IGFBP-3 suggest that this protein may represent an innovative new biomarker to assess risk of ocular and nonocular complications in type 2 diabetes mellitus.

Contact lens-based lysozyme detection in tear using a mobile sensor.

We report a method for sensing analytes in tear-fluid using commercial contact lenses (CLs) as sample collectors for subsequent analysis with a cost-effective and field-portable reader. In this study we quantify lysozyme, the most prevalent protein in tear fluid, non-specifically bound to CLs worn by human participants. Our mobile reader uses time-lapse imaging to capture an increasing fluorescent signal in a standard well-plate, the rate-of-change of which is used to indirectly infer lysozyme concentration through the use of a standard curve. We empirically determined the best-suited CL material for our sampling procedure and assay, and subsequently monitored the lysozyme levels of nine healthy human participants over a two-week period. Of these participants who were regular CL wearers (6 out of 9), we observed an increase in lysozyme levels from 6.89 ± 2.02 μg mL-1 to 10.72 ± 3.22 μg mL-1 (mean ± SD) when inducing an instance of digital eye-strain by asking them to play a game on their mobile-phones during the CL wear-duration. We also observed a lower mean lysozyme concentration (2.43 ± 1.66 μg mL-1) in a patient cohort with dry eye disease (DED) as compared to the average monitoring level of healthy (no DED) human participants (6.89 ± 2.02 μg mL-1). Taken together, this study demonstrates tear-fluid analysis with simple and non-invasive sampling steps along with a rapid, easy-to-use, and cost-effective measurement system, ultimately indicating physiological differences in human participants. We believe this method could be used in future tear-fluid studies, even supporting multiplexed detection of a panel of tear biomarkers toward improved diagnostics and prognostics as well as personalized mobile-health applications.

Kinetics of Tear Fluid Proteins after Endothelial Keratoplasty and Predictive Factors for Recovery from Corneal Haze.

Endothelial keratoplasty (EK) is less invasive with faster recovery as compared to conventional penetrating keratoplasty, however, it relies on the clarity of the host corneal stroma. Corneal transplantation involves the induction of immune tolerance for allogeneic tissues as well as the corneal wound healing process, in which coordinated interactions between cytokines and growth factors are critical. In this study, we profiled the expression of 51 soluble factors in the tear fluid over the course of EK and have provided evidence of dynamic changes in cytokine expression in the ipsilateral and contralateral eyes. Cluster analyses classified the cytokine expression kinetics into five groups. Group 1 proteins included TGF-b1, IL-1b, and innate proinflammatory cytokines, which bilaterally increased after surgery, despite the use of topical corticosteroid in the transplanted eyes. Local corticosteroids suppressed cytokines involved in adaptive immunity in the transplanted eyes but not in the contralateral eyes. We found tear protein expression at baseline and one week post-surgery to be a potential predictive biomarker of delayed recovery after EK in terms of the corneal haze and visual acuity. Furthermore, Group 1 tear proteins were most associated with persistent corneal haze pre-surgery as well as visual acuity at one month-post transplant.

Tear proteome in uveitis

AbstractPurposeUveitis represents the fourth commonest cause of legal blindness in adult patients. Analysis of tear fluid proteins in acute anterior uveitis (AAU) patients could represent a novel diagnostic modality. This study aimed at detecting potential tear fluid biomarkers in patients with AAU and bacterial keratitis (BK).MethodsFourteen patients with unilateral AAU, seven patients with bacterial keratitis (BK) and 14 healthy age‐ and sex‐matched control subjects were included. Tear fluid samples were obtained by inserting two Schirmer’s test strips behind the lower eyelid of each eye for five minutes without anesthesia. Proteins were then identified by liquid chromatography tandem mass spectrometry. Pooled tear fluid samples from healthy controls were compared with samples from AAU and BK patients, respectively. Ingenuity Pathway Analysis (IPA) was used to explore biological function.ResultsA relative protein level was obtained for 235 proteins in AAU patients and 198 proteins in BK patients, while using healthy control subjects as reference. In AAU patients, nine proteins were at least two‐fold up‐regulated, whereas in BK patients, 12 proteins were up‐regulated more than two‐fold. None of the top ten upregulated proteins in AAU and BK overlapped. The top canonical pathway identified by IPA was ‘Acute Phase Response Signaling’ in AAU and ‘LXR/RXR activation’ in BK. Top upstream activated regulators included peroxisome proliferator activated receptor delta in AAU and interleukin 6 in BK.ConclusionsTear fluid protein analysis represents a promising diagnostic modality for ocular inflammatory diseases and demonstrate differentially expressed proteins in acute anterior uveitis and bacterial keratitis.

Combined Targeted Analysis of Metabolites and Proteins in Tear Fluid With Regard to Clinical Applications.

PurposeTo establish a robust workflow for combined mass spectrometry–based analysis of metabolites and proteins in tear fluid with regard to clinical applicability.MethodsTear fluid was taken from 12 healthy volunteers at different time points using specially designed Schirmer strips. Following the liquid extraction of metabolites from standardized punches, the remaining material was processed for bottom-up proteomics. Targeted metabolite profiling was performed adapting a metabolomics kit, which targets 188 metabolites from four different analyte classes. Proteomics was performed of the identical samples targeting 15 tear proteins relevant to ocular health.ResultsSixty metabolites could be consistently determined in all tear samples (98 metabolites were detectable in average) covering acylcarnitines, amino acids, biogenic amines, and glycerophospholipids. Following normalization, the majority of metabolites exhibited intraindividual variances of less than 20%, both regarding different times of sampling, and the individual eye. The targeted analysis of tear proteins revealed a mean intraindividual variation of 23% for the three most abundant proteins. Even extreme differences in tear secretion rates resulted in interindividual variability below 30% for 65 metabolites and two proteins.ConclusionsThe newly established workflow can be used for combined targeted detection of metabolites and proteins in one punch of a Schirmer strip in a clinical setting.Translational RelevanceOur data about intra- and interindividual as well as intereye variation provide a valuable basis for the design of clinical studies, and for the applicability of multiplexed “omics” to well accessible tear fluid with regard to future routine use.

Severity of clinical dry eye manifestations influences protein expression in tear fluid of patients with primary Sjögren's syndrome.

Ocular dryness is a characteristic feature of primary Sjögren’s syndrome (pSS). This may result in dry eye disease (DED), leading to damage of the ocular surface. Additional, non-invasive diagnostic techniques are needed when evaluating pSS patients. Hence, screening for disease-specific biomarkers in biological fluid could be promising. We have previously examined the proteome of tear fluid from pSS patients through Liquid chromatography-mass spectrometry (LC-MS), and conducted a thorough ocular evaluation of patients with pSS. In this study we further explored the association between dry eye manifestations and protein expression in tear fluid of pSS patients. Medical history of 27 patients and 32 healthy controls was gathered. Subjective complaints were registered through questionnaires. Objective findings including tear osmolarity, tear film break up time (TFBUT), Schirmer’s test, and ocular and corneal surface staining were also recorded. LC-MS was conducted formerly on tear fluid from all subjects in order to generate proteomic biomarker profiles. Scaffold was employed to analyse the LC-MS data for quantitative differences between patient and control groups, and the mean spectral counts were calculated for the five most upregulated proteins in relation to DED manifestations. Dysregulated cellular processes were identified in pSS patients using FunRichv3 enrichment analysis. The five most upregulated proteins previously identified in pSS patients were DNA (apurinic or apyrimidinic site) lyase (APEX1), thioredoxin-dependent peroxidase reductase (PRDX3), copine (CPNE1), aconitate hydratase (ACO2), and LIM domain only protein 7 (LMO7), in descending order. A significant increase in mean spectral counts for these proteins were observed in pSS patients with pathological DED manifestations compared to healthy controls (p<0.0001). Consequently, dysregulated cellular pathways involving innate and adaptive immunity were also detected. In conclusion, our observations suggest a relationship between presence of dry eye signs and upregulated proteins in tear fluid from patients with pSS. Further studies are needed in order to replicate the concepts explored and analyses performed in a greater cohort of pSS patients, where sensitivity and specificity of the methods conducted can also be verified further.

Proteomic analysis of human lacrimal and tear fluid in dry eye disease

To understand the pathophysiology of dry eye disease (DED), it is necessary to characterize proteins in the ocular surface fluids, including tear fluid (TF) and lacrimal fluid (LF). There have been several reports of TF proteomes, but few proteomic studies have examined LF secreted from the lacrimal gland (LG). Therefore, we characterized the proteins constituting TF and LF by liquid chromatography mass spectrometry. TF and LF were collected from patients with non-Sjögren syndrome DED and from healthy subjects. Through protein profiling and label-free quantification, 1165 proteins from TF and 1448 from LF were identified. In total, 849 proteins were present in both TF and LF. Next, candidate biomarkers were verified using the multiple reaction monitoring assay in both TF and LF of 17 DED patients and 17 healthy controls. As a result, 16 marker proteins were identified (fold-change > 1.5, p-value < 0.05), of which 3 were upregulated in TF and 8 up- and 5 down-regulated in LF. In conclusion, this study revealed novel DED markers originating from the LG and tears by in-depth proteomic analysis and comparison of TF and LF proteins.