Tear Proteins Research Articles

Differentially Expressed Tear Proteins in Sjögren's Syndrome Keratoconjunctivitis Sicca.

The purpose of this study was to determine if there are significant differences in the concentrations of tear proteins in Sjögren's syndrome keratoconjunctivitis sicca (SS KCS) compared to healthy controls. Tear samples were collected with unmarked Schirmer strips from 15 patients with SS KCS and 21 healthy controls. Tear protein was eluted and the concentration measured. Inflammatory mediators were assayed with a Raybiotech L-507 glass slide array and normalized by strip wetting length. All patients underwent an ocular surface exam to evaluate tear break-up time (TBUT), corneal fluorescein (CF) staining, and conjunctival (CJ) staining. The symptom assessment questionnaire in dry eye (SANDE) scores were collected for all patients. Two hundred fifty-three of the 507 tear proteins analyzed were significantly different in patients with SS compared to controls. Two hundred forty-one if the proteins were upregulated and 12 were downregulated. One hundred eighty-one differentially expressed proteins were significantly correlated with all four clinical parameters: TBUT, CF staining, CJ staining, and SANDE score. These findings indicate that hundreds of factors can be assayed in tear proteins collected from a Schirmer strip. The results suggest tear protein concentrations are altered in patients with SS KCS compared to controls. The upregulated tear proteins correlated with clinical measures of dry eye symptoms and disease severity. Tear proteins could serve as important biomarkers for studying pathogenesis and in clinical diagnosis and management of SS KCS.

Tear Fluid Biomarkers and Quality of Life in People with Type 2 Diabetes and Dry Eye Disease.

Dry eye disease (DED) can be extremely distressing and is common in type 2 diabetes (T2D). To investigate potential biomarkers of DED in T2D, panels of proteins in tears, alongside clinical signs and symptoms of DED, were assessed. Patients were classified into four groups: T2D + DED (n = 47), T2D-only (n = 41), DED-only (n = 17) and healthy controls (n = 17). All patients underwent the Ocular Surface Disease Index (OSDI) and Dry Eye-Related Quality of Life (DEQS) questionnaires, tear evaporation rate (TER), fluorescein tear break-up time (fTBUT), corneal fluorescein staining (CFS) and Schirmer 1 test assessments. Six metabolic proteins and 14 inflammatory cytokines were analyzed with multiplex bead analysis. Interleukin (IL)-6 and IL-8 concentrations in tears were significantly higher in the T2D + DED group, and these biomarkers were positively correlated with CFS. In addition, tear IL-6 was negatively correlated with fTBUT in the T2D + DED group. Clinical signs of DED in the T2D + DED group were similar to the DED-only group. The T2D + DED group had more patients with moderate and severe DED (versus the DED-only group), suggesting a different pathogenesis for DED in T2D versus DED-only. Therefore, IL-6 and IL-8 could potentially be diagnostic biomarkers of DED in T2D.

Experimental Analysis of Tear Fluid and Its Processing for the Diagnosis of Multiple Sclerosis.

A pilot analysis of the tear fluid of patients with multiple sclerosis (MS) collected by glass microcapillary was performed using various experimental methods: liquid chromatography-mass spectrometry, Raman spectroscopy, infrared spectroscopy, and atomic-force microscopy. Infrared spectroscopy found no significant difference between the tear fluid of MS patients and the control spectra; all three significant peaks were located at around the same positions. Raman analysis showed differences between the spectra of the tear fluid of MS patients and the spectra of healthy subjects, which indicated a decrease in tryptophan and phenylalanine content and changes in the relative contributions of the secondary structures of the polypeptide chains of tear proteins. Atomic-force microscopy exhibited a surface fern-shaped dendrite morphology of the tear fluid of patients with MS, with less roughness on both oriented silicon (100) and glass substrates compared to the tear fluid of control subjects. The results of liquid chromatography-mass spectrometry showed downregulation of glycosphingolipid metabolism, sphingolipid metabolism, and lipid metabolism. Proteomic analysis identified upregulated proteins in the tear fluid of patients with MS such as cystatine, phospholipid transfer protein, transcobalamin-1, immunoglobulin lambda variable 1-47, lactoperoxidase, and ferroptosis suppressor protein 1; and downregulated proteins such as haptoglobin, prosaposin, cytoskeletal keratin type I pre-mRNA-processing factor 17, neutrophil gelatinase-associated lipocalin, and phospholipase A2. This study showed that the tear proteome in patients with MS is modified and can reflect inflammation. Tear fluid is not a commonly used biological material in clinico-biochemical laboratories. Experimental proteomics has the potential to become a promising contemporary tool for personalized medicine, and it might be applied in clinical practice by providing a detailed analysis of the tear-fluid proteomic profile of patients with MS.

Effect of vincristine on intraocular pressure and tear fluid oxidative stress biomarkers in canine transmissible venereal tumor.

The ocular side effects of cancer chemotherapeutic drugs are relatively uncommon. Nonetheless, the ocular system has a potentially high sensitivity to toxic substances. This study proposed a framework to assess the effect of vincristine chemotherapy on intraocular pressure, tear protein, and oxidative stress in canines with transmissible venereal tumor (TVT). The study group comprised 10 dogs with TVT, whose diagnosis was based on cytology, and all dogs were treated with vincristine for 4 weeks. Each animal was given a complete ophthalmic examination, followed by a standard Schirmer tear test. Before and 20 min after administering vincristine, intraocular pressure (IOP) was measured in the eyes with a noncontact tonometer. At any of the times mentioned, tear samples were collected using the Schirmer test procedure and were subjected to protein analysis-oxidative stress index (OSI), total antioxidant capacity (TAC), total oxidant status (TOS), nitric oxide (NO), and malondialdehyde (MDA) were determined, and standard statistical analysis was applied. No significant differences were found in protein in tears, but mean Pre and Postinjection IOP revealed a significant decrease in the eyes each week. Also, results indicated significant differences in oxidative stress markers: increased OSI, NO, and MDA, and reduced TAC. The importance of an increase in oxidative stress levels in the tears of vincristine-treated patients should be taken seriously, as it appears to play a role in the pathogenesis of eye disease. Therefore, during the treatment weeks prior to prescribing vincristine, eye diseases should be evaluated and considered.

Effect of Contact Lens Solutions in Stabilizing the Activity of Tear Lysozyme.

Interactions between tear proteins and the interfaces of contact lenses can be complex and can influence contact lens wear success. Tear proteins, including lysozyme, function to maintain the balance of ocular surface homeostasis, as evidenced by the effects of its conformation relative to stabilizing the tear film and its potential impact on corneal epithelial cells. Contact lens manufacturers include components in lens care and blister package solutions to help stabilize the tear film and preserve homeostasis. This in vitro study was performed to evaluate the ability of daily disposable contact lens package solutions to stabilize lysozyme and preserve its native conformation under denaturing conditions. Lysozyme was added to contact lens solutions sampled from kalifilcon A, etafilcon A, senofilcon A, narafilcon A, nelfilcon A, verofilcon A, delefilcon A, somofilcon A, and stenfilcon A blister packages, then mixed with the protein denaturant sodium lauryl sulfate. Lysozyme activity was evaluated by adding test solutions to a suspension of Micrococcus luteus. Native lysozyme lyses the Micrococcus luteus cell wall, which decreases suspension turbidity. Stabilization of lysozyme activity was determined by comparing suspension turbidity before and after exposure to test solutions. Lysozyme stabilization was 90.7% for kalifilcon A solution, a statistically significant improvement (p < 0.05) compared to phosphate buffered saline (PBS, negative control). No significant improvement was observed with any other contact lens solution (all lysozyme stabilization < 5.00%). The representative tear protein lysozyme was significantly more stable in the novel kalifilcon A contact lens solution containing multiple moisturizers and osmoprotectants than in PBS or other daily disposable contact lens solutions. The lysozyme activity assay provides mechanistic evidence that the kalifilcon A contact lens solution can stabilize proteins under conditions that typically denature proteins, which may contribute to maintaining ocular surface homeostasis.

Proteomic analysis of tears in dry eye disease: A prospective, double-blind multicenter study

PurposeTo identify specific dry eye disease (DED) tear biomarker(s) using tear proteomic analysis, clinical parameters, and their correlations before and after DED treatment. MethodsA prospective, double-blinded, national multicenter clinical study was performed using data from 80 DED patients. The patients were treated with 0.1% cyclosporine (CsA, n = 28), 0.05% CsA (n = 26), or 3% diquafosol (DQS, n = 26) eye drops, and tear proteome changes and clinical outcomes (tear break-up time [TBUT], corneal erosion [Cor-Er], conjunctival erosion [Conj-Er], and symptom assessment in dry eye [SANDE] scores) were observed at 4, 8, and 12 weeks. For all clinical parameters, correlation analysis was performed between the three drug conditions and the differentially expressed proteins (DEPs) from the proteomic analysis. ResultsAFM, ALCAM, CFB, H1-4, PON1, RAP1B, and RBP4 were identified in all treatment groups and were downregulated after treatment. All clinical parameters significantly improved at 12 weeks than at baseline (p-value <0.0001); however, their values were not significantly different among groups, except for Cor-Er (p-value = 0.007). Compared with the DQS group, Cor-Er score significantly improved after treatment with 0.1% and 0.05% CsA. The seven DEPs identified in all groups were not consistently correlated with the clinical parameters (p-value >0.05). ConclusionsDespite differences in drug concentration and action mechanisms, the improvement levels of TBUT, Cor-Er, and SANDE scores were clinically adequate. However, useful tear protein biomarkers, clinically acceptable biomarker combinations correlating with clinical parameters, and clinically acceptable levels of specificity and sensitivity were not identified.

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.

Optimization and evaluation of tear protein elution from Schirmer's strips in dry eye disease.

Extraction of tear protein from Schirmer's strip is a prerequisite for the proper identification and screening of biomarkers in dry eye disease. The study compares different methods of extraction of tear proteins from the Schirmer's strip. Reflex tear was collected from healthy controls (HC; n = 12), Stevens-Johnson syndrome (SJS; n = 3) and dry eye disease (DED; n = 3) patients using capillary tube. This tear was used to measure the volume absorbed by Schirmer's strip per microliter. Different buffers (6) were used to compare the protein yield from the Schirmer's strip in four different conditions. The tear proteins extracted using the highest protein yield buffer were analyzed by mass spectrometry. A linear relationship between the tear volume and wetting length was observed (r = 0.0.997, n = 6). The highest yield was observed after incubation of the Schirmer's strip in 100 mM ammonium bicarbonate (ABC) with 0.25% Nonidet P-40(NP-40) at 4°C for an hour (P < 0.00005). The in-solution digestion of tear eluted in the above condition 100 Mm ABC + 0.25% NP-40 with one-hour incubation yielded a total of 2119 proteins in HC, SJS, and DED. The unique protein observed in SJS and DED was 0.6% and 17.9%, respectively. The significantly expressed proteins are associated with innate immune response, proteolysis, wound healing, and defense response. A method for extraction of protein from Schirmer's strip was optimized for increase in protein yield from the tear sample. SJS and DED tear samples have unique protein signature. The study will aid in better design of tear protein-based experimental study.

Morphological alterations in corneal nerves of patients with dry eye and associated biomarkers

The purposes of the present study were to (1) identify the relationship between dry eye symptoms and morphological changes in corneal subbasal nerves/ocular surfaces, and (2) discover tear film biomarkers indicating morphological changes in the subbasal nerves. This was a prospective cross-sectional study conducted between October and November 2017. Adults with dry eye disease (DED, n = 43) and healthy eyes (n = 16) were evaluated based on their subjective symptoms and ophthalmological findings. Corneal subbasal nerves were observed using confocal laser scanning microscopy. Nerve lengths, densities, branch numbers, and nerve fiber tortuosity were analyzed using ACCMetrics and CCMetrics image analysis systems; tear proteins were quantified by mass spectroscopy. Compared with the control group, the DED group had significantly lower tear breakup times (TBUT) and pain tolerance capacity, and significantly higher corneal nerve branch density (CNBD) and corneal nerve total branch density (CTBD). CNBD and CTBD showed significant negative correlations with TBUT. Six biomarkers (cystatin-S, immunoglobulin kappa constant, neutrophil gelatinase-associated lipocalin, profilin-1, protein S100-A8, and protein S100-A9) showed significant positive correlations with CNBD and CTBD. The significantly higher CNBD and CTBD in the DED group suggests that DED is associated with morphological alterations in corneal nerves. The correlation of TBUT with CNBD and CTBD further supports this inference. Six candidate biomarkers that correlate with morphological changes were identified. Thus, morphological changes in corneal nerves are a hallmark of DED, and confocal microscopy may help in the diagnosis and treatment of dry eyes.

Abstract P6-03-07: Development of an at-home breast health assessment test, to increase compliance with screening mammography using proteins from tears

Abstract Anna E. Daily1,*, Prashanth Ravishankar1, Wanyi Wang3, Ryan Krone3, Steve Harms1,2, and V Suzanne Klimberg1,4,5 1Namida Lab Inc, Fayetteville, Arkansas; 2The Breast Center-Medical Associates of Northwest Arkansas, Fayetteville, Arkansas; 3Elite Research LLC, Irving, Texas; 4Department of Surgery, University of Texas Medical Branch, Galveston, Texas; 5Breast Surgical Oncology, University of Texas MD Anderson Cancer Center, Houston, Texas. *anna@namidalab.com Background: There is a growing body of evidence to support tears as a non-traditional biological fluid in clinical laboratory testing. In addition to the simplicity of tear fluid processing, the ability to access key cancer biomarkers in high concentrations quickly and inexpensively make them an attractive biofluid source. Here we report our biomarker discovery study on tears to identify and validate candidate biomarkers for breast cancer and develop a model that is significantly associated with a positive breast cancer diagnosis. Methods: Participants were recruited from individuals having a yearly screening mammogram, biopsy, and/or recently diagnosed with breast cancer. Imaging results were obtained from clinical sites and samples were then classified as: control (normal imaging no biopsy) or diagnosed breast cancer pre-treatment (diagnosed by biopsy). Biomarker discovery was conducted using 102 individual tear samples collected using the Schirmer strip collection method. Liquid chromatography/tandem mass spectrometry (LC-MS/MS) was performed to identify protein biomarker candidates with altered expression levels in breast cancer patients. ELISA assay to confirm LC-MS/MS trends for biomarkers of interest was conducted using 171 tear samples. An additional round of validation utilizing 848 samples was performed which included protein concentrations determined by ELISA and collection of demographic and clinical covariates. The resulting concentration data, combined with the demographic and clinical covariates, was analyzed using logistic regression analysis to build a model for classification of samples as positive or negative. Results: A total of 301 proteins were identified by LC-MS/MS and narrowed to a list of 14 proteins (p-value &amp;lt; 0.05) with potential significance in breast cancer patients. Three biomarkers, S100A8 (p-value = 0.0069), S100A9 (p-value = 0.0048), and Galectin-3 binding protein (p-value = 0.01) with an increased expression in breast cancer patients were selected for validation using ELISA. Logistic regression analysis produced three models, which were then evaluated on breast cancer cases and controls at two diagnostic thresholds and resulted in sensitivity ranging from 52% - 90% and specificity from 31% - 79%. Conclusions: Our results demonstrate clinical feasibility for tear proteins to detect breast cancer and includes the most extensive published data set of individually analyzed tear samples. This analysis suggests that models developed using tear fluid have clinical validity and could be used in further development of a biological assay. We envision positioning this assay as a tool for activation around breast health screening for low to average risk patients who may be screening avoidant or adverse to encourage participation in screening mammography. Citation Format: Anna Daily, Prashanth Ravishankar, Victoria S. Klimberg, Steve Harms. Development of an at-home breast health assessment test, to increase compliance with screening mammography using proteins from tears [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-03-07.

Experimental design considerations for studies of human tear proteins

PurposeHuman tears contain abundant, diverse sets of proteins that may serve as biomarkers of ocular surface health. There is a need for reproducible methods that consider multiple factors influencing the tear proteome, in addition to the variable of interest. Here we examined a workflow for proteomic analysis of tear proteins without the need to pool tear samples from multiple individuals, thus allowing for analyses based on individual factors, and increasing opportunities for protein biomarker discovery. MethodsTears were collected by Schirmer strip following topical ocular anesthetic application then individually stored at −80 °C prior to processing for proteomics. Tear proteins were extracted from Schirmer strips, digested using suspension trapping spin columns (S-Trap), and labeled with high multiplicity tandem mass tags (TMT). Peptide digests were then extensively fractionated by two-dimensional chromatography and analyzed by mass spectrometry to identify and measure changes in protein abundance in each sample. Analysis of select samples was performed to test protocols and to compare the impact of clinically relevant parameters. To facilitate comparison of separate TMT experiments, common pool samples were included in each TMT instrument run and internal reference scaling (IRS) was performed. ResultsDifferences in subsets of tear proteins were noted for: geographic site of tear collection, contact lens use, and differences in tear fluid volume among individuals. ConclusionThese findings demonstrate that proteomic analysis of human tear proteins can be performed without the need to pool samples, and that development of analytic workflows must consider factors that may affect outcomes in studies focused on diverse clinical samples.

Cellular mechanisms and effects of IL-4 receptor blockade in experimental conjunctivitis evoked by skin inflammation.

Ocular surface diseases, including conjunctivitis, are recognized as common comorbidities in atopic dermatitis (AD) and occur at an increased frequency in patients with AD treated with biologics targeting IL-4 receptor α (IL-4Rα) or IL-13. However, the inflammatory mechanisms underlying this pathology are unknown. Here, we developed a potentially novel mouse model of skin inflammation-evoked conjunctivitis and showed that it is dependent on CD4+ T cells and basophils. Blockade of IL-4Rα partially attenuated conjunctivitis development, downregulated basophil activation, and led to a reduction in expression of genes related to type 2 cytokine responses. Together, these data suggest that an IL-4Rα/basophil axis plays a role in the development of murine allergic conjunctivitis. Interestingly, we found a significant augmentation of a number of genes that encode tear proteins and enzymes in anti-IL-4Rα-treated mice, and it may underlie the partial efficacy in this model and may represent candidate mediators of the increased frequency of conjunctivitis following dupilumab in patients with AD.

Pre-analytical sample handling effects on tear fluid protein levels

Tear fluid is emerging as a source of non-invasive biomarkers, both for ocular and systemic conditions. Accurate quantification of tear proteins can be improved by standardizing methods to collect and process tear fluid. The aim of this study was to determine sample handling factors that may influence the tear protein biomarker profile. Tear fluid was collected using Schirmer’s strips. Tear proteins were extracted by elution through centrifugation. Total protein content was determined using the bicinchoninic acid assay. Key concepts that apply to the entire sample processing cycle are tear sampling, tear storage, protein extraction and data normalization. Differences in wetting or migration length were observed between Schirmer’s strips from different manufacturers, and between protein-free and protein-rich solutions. One unit of migration length (mm) did not correspond to one unit of volume (µL). A positive correlation (r = 0.6671, p < 0.0001) was observed between migration length and total tear protein content. The most beneficial storage conditions were strips that were not stored (+ 21.8%), or underwent ‘wet’ storage (+ 11.1%). Protein recovery was the highest in 400 µL extraction buffer and independent of protein molecular weight. This study helps to explain inter- and intra-variability that is often seen with tear biomarker research. This information is critical to ensure accuracy of test results, as tear biomarkers will be used for patient management and in clinical trials in the near future. This study also highlights the need for standardization of Schirmer’s strip manufacturing, tear fluid processing and analyte concentration normalization.

Development of lacrimal gland organoids from iPSC derived multizonal ocular cells.

Lacrimal gland plays a vital role in maintaining the health and function of the ocular surface. Dysfunction of the gland leads to disruption of ocular surface homeostasis and can lead to severe outcomes. Approaches evolving through regenerative medicine have recently gained importance to restore the function of the gland. Using human induced pluripotent stem cells (iPSCs), we generated functional in vitro lacrimal gland organoids by adopting the multi zonal ocular differentiation approach. We differentiated human iPSCs and confirmed commitment to neuro ectodermal lineage. Then we identified emergence of mesenchymal and epithelial lacrimal gland progenitor cells by the third week of differentiation. Differentiated progenitors underwent branching morphogenesis in the following weeks, typical of lacrimal gland development. We were able to confirm the presence of lacrimal gland specific acinar, ductal, and myoepithelial cells and structures during weeks 4-7. Further on, we demonstrated the role of miR-205 in regulation of the lacrimal gland organoid development by monitoring miR-205 and FGF10 mRNA levels throughout the differentiation process. In addition, we assessed the functionality of the organoids using the β-Hexosaminidase assay, confirming the secretory function of lacrimal organoids. Finally, metabolomics analysis revealed a shift from amino acid metabolism to lipid metabolism in differentiated organoids. These functional, tear proteins secreting human lacrimal gland organoids harbor a great potential for the improvement of existing treatment options of lacrimal gland dysfunction and can serve as a platform to study human lacrimal gland development and morphogenesis.

Analysis and comparison of tear protein profiles in dogs using different tear collection methods

BackgroundTear proteomic analysis has become an important tool in medical and veterinary research. The tear collection method could influence the tear protein profile. This study aims to evaluate the protein profiles of dog tears collected using microcapillary tubes (MT), Schirmer tear strips (ST), and ophthalmic sponges (OS).MethodsThe tear samples were collected using MT, ST, and OS. Tear protein profiles were analyzed using two-dimensional electrophoresis (2-DE) and the different protein spots’ expression was compared. Fourteen protein spots were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS).ResultsTear protein concentrations ranged from 2.80 to 4.03 μg/μL, with no statistically significant differences among collection methods. Protein expression in each collection method differed in terms of both the number and intensity of the spots. There were 249, 327, and 330 protein spots found from tears collected with MT, ST, and OS, respectively. The proteins albumin, haptoglobin, and lactoferrin identified from OS were found to have higher spot intensities than other methods of collection. The use of MT demonstrated the downregulation of nine proteins.ConclusionsThe recent study supported that tear protein analysis is affected by different tear collection methods. Although ST is commonly used for tear collection, it provides insufficient information to study particular tear proteins.

Relationship between dry eye and expressions of CXCR3 and CCR5 after ocular acid burn

ObjectiveTo investigate the manifestation of dry eye and its relationship with CXCR3 and CCR5 expression in patients with ocular acid burns.MethodsThis is a case–control study. A total of 27 eyes of 22 cases ocular with acid burns of I-V degrees from Jan.2020 to Feb.2021 in Jinshan Hospital of Fudan University were selected as observation group, and 8 eyes of 8 cases of normal people were selected as control group. The follow-up period was 3 months. The visual acuity, intraocular pressure (IOP), corneal fluorescein staining scores (CFS), breakup time of tear film (BUT), Schirmer I test, corneal thickness and tear meniscus height (TMH) were observed at 1 day, 1 and 3 months after injury. The protein expressions of CXCR3 and CCR5 were examined by ELISA and compared among groups at each time point.ResultsBUT and Schirmer I tests value in the observation group were lower than those in the control group 3 months after injury (BUT: Group I ~ IV p = 0.0266, p = 0.0222, p = 0.0003, p = 0.0059, respectively; Schirmer I test: Group I ~ IV p = 0.0027, p = 0.0033, p = 0.0016, p = 0.0032, respectively). CFS scores were higher than those in the control group at 1 day after injury (all p < 0.0001), but decreased gradually at 1 and 3 months after injury (Group I ~ IV p = 0.0042, p = 0.0096, p < 0.0001, p < 0.0001, respectively). The corneal thickness and TMH 1 day after injury were higher than those in the control group (corneal thickness: Group II ~ IV p = 0.0010, p < 0.0001, p < 0.0001, respectively; TMH: Group II ~ IV p = 0.0002, p < 0.0001, p < 0.0001, respectively), and also higher than those at 1 month and 3 months after injury (corneal thickness: Group II ~ IV p = 0.0010, p < 0.0001, p < 0.0001, respectively; TMH: Group II ~ IV p = 0.0345 and p = 0.0045, p = 0.0005 and p < 0.0001, p = 0.0114 and p = 0.0019, respectively). The expression levels of CXCR3 and CCR5 protein were significantly negatively correlated with BUT (all p < 0.0001), and CXCR3 and CCR5 were also significantly negatively correlated with Schirmer I test value (p < 0.0001, p = 0.0004, respectively).ConclusionOcular acid burns can cause dry eye, and the expression of CXCR3 and CCR5 protein in tears may be related to the occurrence of dry eye after ocular acid burn.

Smartphone-based fluorescent sensing platforms for point-of-care ocular lactoferrin detection

Lactoferrin is a critical glycoprotein that accounts for the major component in tear protein composition. Tear lactoferrin has been indicated as a potential biomarker in ocular health screening, such as dry eye disease diagnosis. Fluorescent biosensors are a desirable alternative to current diagnostic methods, due to their high selectivity and sensitivity, and rapid and cost-effective detection technologies at point-of-care (POC) platforms. Herein, fluorescent lactoferrin sensing is examined and applied on nitrocellulose membrane, capillary tube, and contact lens platforms in cooperation with a developed smartphone software for data collection and analysis. Fluorescent sensors based on trivalent terbium (TbCl3) are integrated into different platforms, including nitrocellulose membranes, capillary tubes, and contact lenses, and tested in artificial tear fluid. A bespoke 3D printed readout device integrated with a smartphone camera was employed for image acquisition and readout. The detection range of the lactoferrin sensors could be varied from 0 to 5 mg mL−1 with a strong linear relation and yielded a limit of detection (LOD) of 0.57 mg mL−1 for lateral flow sensing, 0.12 mg mL−1 for capillary tube sensing, and 0.44 mg mL−1 for contact lens sensing. The contact lens sensor obtained the highest linear regression, while the capillary tube sensor achieved a lowest LOD. This work could pave the way towards accessible lactoferrin monitoring at POC and could induce hospitalization at the UK national health service (NHS) for future clinical trials and examinations.

Phosphorylcholine-Based Contact Lenses for Sustained Release of Resveratrol: Design, Antioxidant and Antimicrobial Performances, and In Vivo Behavior.

Design of advanced contact lenses (CLs) demands materials that are safe and comfortable for the wearers and that preserve the normal eye microbiota, avoiding chronic inflammation and biofilm development. This work aimed to combine the natural antibiofouling phosphorylcholine and the antioxidant and prebiotic resveratrol as integral components of CLs that may have the additional performance of preventing oxidative-stress related eye diseases. Different from previous uses of 2-methacryloyloxyethyl phosphorylcholine (MPC) as coating, we explored the feasibility of adding MPC at high proportions as a comonomer of 2-hydroxyethyl methacrylate (HEMA)-based hydrogels while still allowing for the loading of the hydrophobic resveratrol. Homogeneous distribution of MPC along the hydrogel depth (confirmed by Raman spectroscopy) notably increased solvent uptake and the proportion of free water while it decreased Young's modulus. Relevantly, MPC did not hinder the uptake of resveratrol by CLs (>10 mg/g), which indeed showed network/water partition coefficients of >100. Protocols for CLs sterilization and loading of resveratrol under aseptic conditions were implemented, and the effects of tear proteins on resveratrol release rate were investigated. CLs sustained resveratrol release for more than 24 h in vitro, and sorption of albumin onto the hydrogel, although attenuated by MPC, slowed down the release. The combination of MPC and resveratrol reduced P. aeruginosa and S. aureus growth as tested in a novel hydrogel disk-agar interface biofilm growth setup. The developed CLs showed excellent anti-inflammatory properties and biocompatibility in in ovo and rabbit tests and provided higher and more prolonged levels of resveratrol in tear fluid, which favored resveratrol biodistribution in anterior and posterior eye segments compared to eye drops. Correlations between the release profiles of resveratrol in vitro and in vivo were assessed. Relevantly, the CLs preserved the antioxidant properties of resveratrol during the entire 8 h of wearing. In sum, CLs prepared with high proportion in MPC may help address safety and comfort requirements while having drug releasing capabilities.

Tear Proteomics Approach to Distinguishing Primary from Secondary Sjögren’s Syndrome for Dry Eye Patients with Long-Term Instillation of Eyedrops

The diagnosis and monitoring of Sjögren syndrome (SS) is often difficult, requiring a multidisciplinary approach with invasive procedures. Our aim is to elucidate the tear protein alterations of dry eye disease (DED) with primary SS (pSS) and secondary SS (sSS) with the long-term instillation of eyedrops. We collected clinical demographics and tear fluid (TF) samples from DED patients with no autoimmune diseases (non-SS-DED), pSS-DED, and sSS-DED patients, followed by TF screening with tandem mass tagging-labeling gel-free proteomics assay. Bioinformatic analysis via Ingenuity Pathway Analysis was used to identify functional pathways and interacting networks. Validation of candidate proteins with enzyme-linked immunosorbent assay on the tear samples was done. The top functional pathways of the two comparisons (sSS-DED vs. pSS-DED and sSS-DED vs. non-SS-DED) were both associated with inflammation and stress-related signaling. After constructing an interaction network model with the selected candidate proteins, five proteins were identified. A Disintegrin and Metalloproteinase domain-containing protein 10 (ADAM10) was found to be an important candidate biomarker in all groups, followed by epidermal growth factor (EGF) in TF. This study revealed novel DED markers, ADAM10 and EGF, in differentiating between primary and secondary SS patients from tears by in-depth proteomic analysis.

Tear proteome profile in eyes with keratoconus after intracorneal ring segment implantation or corneal crosslinking

AbstractPurpose: Intracorneal Ring Segment (ICRS) and Corneal collagen Crosslinking (CXL) are widely used treatments in keratoconus (KC) disease, but the alterations they cause in biomechanical mediators in the cornea, are still poorly understood. The aim of this study was to analyse the tear proteome profile before and after treatments to identify biomarkers altered by surgery.Methods: An observational, prospective, case–control pilot study was conducted, analysing tear samples from KC patients by nano‐liquid chromatography‐mass spectrometry (nLC‐MS/MS). Patients with KC who underwent ICRS surgery (n = 4), CXL (n = 4), and healthy subjects (CT, n = 4) were included in this study. Clinical parameters were measured and tear samples were collected before and 18 months after surgery. Proteins with ≥2 expression change and p‐value &lt;0.05 between groups and times were selected to study their role in postoperative corneal changes.Results: These analyses led to the identification of 447 tear proteins. In comparisons between the two surgical groups and CTs, the biological processes that were altered in KC patients at baseline were those that were dysregulated as a consequence of the disease. Among the biological processes seen to be altered were: immune responses, cytoskeleton components, protein synthesis, and metabolic reactions. When comparing the two treatment groups, A‐kinase anchor protein 13 (AKAP‐13) was the most overexpressed protein before and after surgery in CXL group.Conclusions: The changes observed in tears after 18 months postoperatively could be due to the treatments performed and the pathology. Among the deregulated proteins detected, AKAP‐13 deserves special attention for its involvement in corneal thinning, and for its strong overexpression in the tears of patients with more active KC and faster disease progression. However, it should be kept in mind that this is a pilot study conducted on a small number of patients.