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Bacterial keratitis (BK) is an infection that causes inflammation of the cornea and, if severe, can result in blindness. Topical fluoroquinolones combined with corticosteroids have been shown to be useful in the treatment of BK. A rapid, selective, and sensitive bioanalytical method for simultaneous quantification of Gatifloxacin (GAT) and Dexamethasone (DEX) has been developed and validated using tandem mass spectrometry (LC–MS/MS). Optimal separation was accomplished in under 5 min using an Agilent Zorbax C18 column (100 mm × 4.6 mm, 3.5 μm). The mobile phase was composed of a blend of 0.2% formic acid in triple distilled water and methanol with a flow rate of 0.65 mL/min in isocratic mode. GAT and DEX were detected in positive electrospray ionization multiple reaction monitoring mode (MRM), and the retention time was found to be at 1.64 and 2.93 min, respectively. The linearity of GAT and DEX was found to be in the range of 1.56–400 ng mL−1 with good precision and accuracy. The method was validated according to USFDA regulatory guidelines. The validated method was effectively utilized for preclinical pharmacokinetic analysis of GAT and DEX in rabbit tear fluid following the topical application of a commercial formulation. [ABSTRACT FROM AUTHOR]

Purpose: To determine whether there is a significant association between inflammatory cytokines in the tear fluid and the severity of Retinopathy of Prematurity (ROP). Study Design: Retrospective cohort study. Methods: The cytokine levels in tear fluids were determined in 34 eyes with ROP and 18 eyes without ROP. There were 15 eyes with severe ROP requiring treatment and 19 eyes with mild ROP not requiring treatment. For severe ROP eyes, tear fluids were collected before treatment. Results: Significantly higher levels of CCL2 and vascular endothelial growth factor (VEGF) were detected in eyes with severe ROP compared to eyes with mild ROP and no ROP. When assessed for cytokine levels that discriminate each disease group, CCL2 showed a significant odds ratio of 1.76 for severity change (/quintile, P = 0.032, after adjusting for birth weight). Correlation analysis showed that birth weight correlated with IL-1α levels, and decreased weight gain increased IFN-γ levels. We next determined tear fluid cytokines which discriminate severe ROP using receiver operating characteristics' analysis. We found that combination of higher CCL2 levels, higher VEGF levels, and lower IFN-γ levels in the tear fluid had a stronger predictive value for severe ROP (area under curve, 0.85). Conclusion: The levels of CCL2, VEGF, and IFN-γ in tear fluid may serve as useful biomarkers for assessing the severity of ROP. [ABSTRACT FROM AUTHOR]

Purpose: To determine the association between systemic vitamin D (VD) and immunoglobulin E (IgE) levels with severity and ocular surface inflammatory profile in patients with epidemic keratoconjunctivitis (EKC). Methods: 210 eyes of 105 patients who were clinically diagnosed with EKC were included in the study. The levels of serum VD and serum IgE were measured. Schirmer’s strip-based tear fluid (TF) was used to determine levels of IL-1β, IL-6, IL-10, IL-17A, TNFα, MMP9, sICAM1, and VEGF-A in a subset of patients. Results: Levels of VD were significantly (P < 0.05) lower and levels of IgE were significantly higher in patients with severe forms of conjunctivitis compared to those with nonsevere forms. Majority of the patients with severe forms of the disease exhibited VD deficiency and/or abnormally high IgE. A negative correlation (r = –0.682; P < 0.0001) was observed between VD and IgE levels. TF levels of IL-1β, IL-6, TNFα, and sICAM1 were significantly higher in eyes with severe forms of conjunctivitis compared to those with nonsevere forms and controls. These factors showed a positive correlation (P < 0.05) with IgE levels and a negative correlation (P < 0.05) with VD levels. Conclusion: Patients with severe forms of EKC exhibited VD deficiency and higher levels of IgE. Increased TF inflammatory factors demonstrated a disease causal relationship with VD and IgE. Hence, restoring the altered levels of VD and IgE to normal range would be pivotal in the prevention and management of severe conjunctivitis.

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. [ABSTRACT FROM AUTHOR]

Purpose: To determine the association between systemic vitamin D (VD) and immunoglobulin E (IgE) levels with severity and ocular surface inflammatory profile in patients with epidemic keratoconjunctivitis (EKC). Methods: 210 eyes of 105 patients who were clinically diagnosed with EKC were included in the study. The levels of serum VD and serum IgE were measured. Schirmer's strip-based tear fluid (TF) was used to determine levels of IL-1β, IL-6, IL-10, IL-17A, TNFa, MMP9, sICAM1, and VEGF-A in a subset of patients. Results: Levels of VD were significantly (P < 0.05) lower and levels of IgE were significantly higher in patients with severe forms of conjunctivitis compared to those with nonsevere forms. Majority of the patients with severe forms of the disease exhibited VD deficiency and/or abnormally high IgE. A negative correlation (r = -0.682; P < 0.0001) was observed between VD and IgE levels. TF levels of IL-1β, IL-6, TNFα, and sICAM1 were significantly higher in eyes with severe forms of conjunctivitis compared to those with nonsevere forms and controls. These factors showed a positive correlation (P < 0.05) with IgE levels and a negative correlation (P < 0.05) with VD levels. Conclusion: Patients with severe forms of EKC exhibited VD deficiency and higher levels of IgE. Increased TF inflammatory factors demonstrated a disease causal relationship with VD and IgE. Hence, restoring the altered levels of VD and IgE to normal range would be pivotal in the prevention and management of severe conjunctivitis. [ABSTRACT FROM AUTHOR]

Managing ocular microbial infections typically requires pharmacotherapy using antibiotic eye drops, such as moxifloxacin hydrochloride (MFX), combined with an antifungal agent like amphotericin B (AB). We carried out and validated an LC–MS/MS assay to quantify these compounds in rabbit tear fluid in order to look into the pharmacokinetics of these two drugs. We employed a protein precipitation technique for the extraction of drugs under examination. A Waters Symmetry C18 column was used to separate the analytes and internal standard. The composition of the mobile phase was like (A) 0.1% v/v formic acid in water and (B) methanol. The detection of MFX and AB was accomplished through the utilization of positive ion electrospray ionization under multiple reaction monitoring mode. The linearity curves for both analytes exhibited an acceptable trendline across a concentration range of 2.34–300 ng/mL for MFX and 7.81–1000 ng/mL for AB in surrogate rabbit tear fluid. The lower limit of quantitation for MFX was 2.34 ng/mL, while for AB, it was 7.81 ng/mL. The approach was strictly validated, encompassing tests of selectivity, linearity (with r2 > 0.99), precision, accuracy, matrix effects, and stability. Consequently, we employed this method to evaluate the pharmacokinetics profiles of MFX and AB in rabbit tear fluid following single topical doses. [ABSTRACT FROM AUTHOR]

A novel research objective is to identify new molecules in more readily accessible biological fluids that could be used in the diagnosis of multiple sclerosis (MS) and other demyelinating disorders. To compare the level of selected cytokines in tears between patients with MS or other demyelinating disorder and healthy controls. 84 patients with diagnosed MS during remission or with other demyelinating disease of the CNS and 70 healthy controls were enrolled in the study. Tears were collected without any stimulation and stored till the day of assessment. The concentration of selected cytokines was measured by the Bio-Plex Pro Human cytokine screening panel 27 cytokines assay according to the manufacturer's instructions. Statistical analysis was performed with Statistica 13. IL-1b level was significantly lower in the study group compared to the control group [3,6 vs 8.71, p < 0.001]. The same pattern was observed for IL-6 [3,1 vs 5.26, p = 0.027] and IL-10 [1,7 vs 10.92, p < 0.001] (Table 1). In the study group, IL-1RA (p = 0.015), IL-5 (p = 0.04), IL-9 (p = 0.014), and IL-15 (p = 0.037) showed significant correlations with age. In the total sample, IL-1Ra (p = 0.016) and IFN-g (p = 0.041) were significantly correlated with age, while in the control group, IL-8 (p = 0.09), MIP-1a (p = 0.009), and RANTES (p = 0.031) showed significant correlations. Our results show that MS and other demyelination diseases lead to decrease in the overall level of cytokines in tears. Further research is needed to determine the role of tear fluid in the assessment of demyelinating disorders like MS. [ABSTRACT FROM AUTHOR]

Abstract The tear fluid is a readily accessible, potential source for biomarkers of disease and could be used to monitor the ocular response to contact lens (CL) wear or ophthalmic pathologies treated by therapeutic CLs. However, the tear fluid remains largely unexplored as a biomarker source for RNA-based molecular analyses. Using a rabbit model, this study sought to determine whether RNA could be collected from commercial CLs and whether the duration of CL wear would impact RNA recovery. The results were referenced to standardized strips of filtered paper (e.g., Shirmer Strips) placed in the inferior fornix. By performing total RNA isolation, precipitation, and amplification with commercial kits and RT-PCR methods, CLs were found to have no significant differences in RNA concentration and purity compared to Schirmer Strips. The study also identified genes that could be used to normalize RNA levels between tear samples. Of the potential control genes or housekeeping genes, GAPDH was the most stable. This study, which to our knowledge has never been done before, provides a methodology for the detection of RNA and gene expression changes from tear fluid that could be used to monitor or study eye diseases.

The aim of this study was to assess the impact of physiological factors, namely tear fluid and lysozyme enzyme, as well as surfactant polysorbate, on the release profile from solid lipid microparticles (SLM), in the form of dispersion intended for ocular application. Indomethacin (Ind) was used as a model drug substance and a release study was performed by applying the dialysis bag method. Conducting release studies taking into account physiological factors is expected to improve development and screening studies, as well as support the regulatory assessment of this multi-compartment lipid dosage form. The effect of the lysozyme was directly related to its effect on lipid microparticles, as it occurred only in their presence (no effect on the solubility of Ind). Polysorbate also turned out to be an important factor interacting with the SLM surface, which determined the release of Ind from SLM. However, in study models without tear fluid or lysozyme, the release of Ind did not exceed 60% within 96 h. Ultimately, only the simultaneous application of artificial tear fluid, lysozyme, and polysorbate allowed for the release of 100% of Ind through the SLM dispersion. The examination of the residues after the release studies indicated the possibility of releasing 100% of Ind from SLM without complete degradation of the microparticles' matrix. The incubation of SLM with tear fluid confirmed a similar influence of physiological factors contained in tear fluid on the surface structure of SLM as that observed during the in vitro studies. [ABSTRACT FROM AUTHOR]

Background: Tear fluid (TF) production is an important component of normal ocular function. It is regulated by parasympathetic and sympathetic innervation. Because parasympathetic nerve fibers originate in the brainstem, pathology in this brain region may affect TF production. For example, a reduction in TF production has been described in patients with Parkinson's disease (PD). Methods: TF was collected at one center from 772 individuals, 708 of which were patients with different neurological diseases, and 64 healthy controls. Wetting lengths (WL) were recorded using Schirmer test strips with a collection time of 10 min. Results: WL correlated negatively with age and was significantly reduced in subgroups of patients with neurodegenerative diseases (NDDs) (PD, Amyotrophic lateral sclerosis (ALS), other motor neuron diseases (MNDs)), as well as inflammatory/autoimmune/infectious central nervous system (CNS) diseases and vascular CNS diseases (VCDs), even if corrected for age or sex. While temperature had a significant negative effect on TF production, other environmental factors, such as hours of sunlight and humidity, did not. Conclusion: WL was altered in many neurological diseases compared to healthy controls. Most importantly, we observed a reduction of WL in NDDs, independent of age or sex. This study highlights the potential of WL as an easily obtainable parameter and suggests functional alterations in the autonomic innervation in various neurological disorders. [ABSTRACT FROM AUTHOR]

Ciliary neurotrophic factor (CNTF) is a pluripotent neurotrophic factor with a high neuroprotective potential, a neurocytokine that has shown potential in therapy of neurodegenerative, mental and metabolic diseases. Pre-clinical data support the general concept of its promising survival and trophic effects, while recent clinical data support the theoretical role of CNTF for treating neurodegeneration and obesity. The data of occasional studies on CNTF levels in invasive (blood) and non-invasive (tears) human biomaterial suggest its potential use as a biomarker of selected brain diseases, though additional studies should be performed to validate it. [ABSTRACT FROM AUTHOR]

Purpose: In this study, we determined the positive rates of allergen-specific immunoglobulin E (IgE) in the tear fluid of Chinese patients with common allergic conjunctivitis (AC) in autumn and winter, compared systemic and ocular allergen tests, and explored the correlation between the numbers and categories of allergens and clinical AC features. Methods: This cross-sectional study recruited 44 patients with AC (86 eyes). Specific IgEs for allergens common in China (house dust mite, cat/dog dander, mugwort/ragweed pollen, cottonwood/willow/elm pollen, milk, egg whites, soybeans) were measured in collected tears using kits for allergen-specific IgE antibodies. AC signs and symptoms were graded according to severity. Results: Specific IgE in tears was positive in 87.2% of eyes. House dust mite was the most common allergen (86.0%), followed by cat (24.4%) and dog (7.0%) dander; tree and grass pollen accounted for only 4.7% and 2.3%, respectively. Food allergens were not detected. The positive rates of the systemic allergen tests were lower than in tear fluid tests in both eyes, especially for house dust mites (P = 0.000). In patients with more allergens, itching was more severe (P = 0.035), while conjunctival hyperemia was milder (P = 0.002). Conclusion: In autumn and winter, the most common AC allergen in Chinese patients was house dust mites. Compared with systemic allergen tests, measuring specific IgE in tears may be a non-invasive method to diagnose and evaluate AC severity, which may be more suitable to reflect the local conditions of ocular surface inflammation due to its high positive rate and convenience. [ABSTRACT FROM AUTHOR]

The simplicity of collecting and evaluating tear fluid (TF) can potentially provide a convenient non-invasive diagnostic tool that easily fits into a personalized approach to medicine based on risk assessment. Though, to date, most tear biomarkers are not yet ready for routine use due to problems with their clinical validation, given the huge clinical advantage of TF and the emerging advanced technical approaches developed for proteomic, lipidomic and metabolomic analysis of tears, TF studies will doubtless become a routine test for health monitoring in the near future. A number of associations between the levels of different substances in TF and the brain makes TF an invaluable source of brain disease biomarkers helpful in early diagnostics and personalized treatment. TF is a promising biological material, an invaluable source for predictive, diagnostic, prognostic, and mechanistic biomarkers. [ABSTRACT FROM AUTHOR]

Abstract Background Alzheimer’s disease (AD) is the most common cause of dementia, and due to increasing life expectancy the number of patients is expected to grow. The diagnosis of AD involves the use of biomarkers determined by an amyloid PET scan or cerebrospinal fluid analyses that are either invasive or expensive, and not available in each hospital, thus limiting their usage as a front-line screener. The TearAD study aims to use tear fluid as a potential source for AD biomarkers. In previous reports, we demonstrated that AD biomarkers amyloid-beta and tau, are measurable in tear fluid and are associated with disease severity and neurodegeration. This study aims to validate previous results in a larger cohort and evaluate the diagnostic accuracy of tear biomarkers to discriminate between individuals with and without neurodegeneration as determined by hippocampal atrophy. Methods The TearAD study is an observational longitudinal multi-center study that will enroll 50 cognitively healthy controls, 50 patients with subjective cognitive decline, 50 patients with mild cognitive impairment and 50 patients with AD dementia from the memory clinic. Participants will be examined at baseline, after one year, and after two years follow-up. Study assessments include neuropsychological tests and ophthalmic examination. All participants will receive a MRI scan, and a subset of the study population will undergo cerebral spinal fluid collection and an amyloid PET scan. Tear fluid will be collected with Schirmer strips and levels of Aβ38, Aβ40, Aβ42, t-tau and p-tau in tear fluid will be determined using multiplex immunoassays. Blood samples will be collected from all participants. Images of the retina will be obtained with a standard, hyperspectral and ultra-wide field fundus camera. Additionally, macular pigment optical density will be measured with the macular pigment reflectometer, and cross-sectional images of the retina will be obtained through optical coherence tomography imaging. Discussion The TearAD study will provide insight into the potential diagnostic use of tear biomarkers as a minimally invasive and low cost tool for the screening and diagnosis of AD. Trial registration Retrospectively registered at clinicaltrials.gov (NCT05655793).

Purpose: Scleritis is an extremely painful and potentially blinding inflammation of the sclera with unknown pathogenesis and unpredictable course. To gain insight in its disease process and identify biomarker candidates, we performed extensive proteomics in serum and tear fluid. Design: Prospective multicenter cohort study. Participants: A total of 121 patients with noninfectious scleritis (of which 39 active cases), 30 healthy controls, and 23 disease controls (uveitis and rheumatoid arthritis) were enrolled in the Netherlands from 2020 to 2022. Methods: Serum, tear fluid of both eyes, and clinical data were gathered. The level of 368 inflammatory proteins was measured using proximity extension assays. Results were validated in an independent cohort of 15 patients with scleritis, and using addressable laser bead immunoassay, or enzyme-linked immunoassays. In addition, we studied an extended panel of matrix metalloproteinases in tear fluid of necrotizing scleritis with addressable laser bead immunoassay. Main Outcome Measures: Statistically significant differences in the level of inflammatory proteins between patients with scleritis and control groups. Results: Proteomics revealed 18 significantly upregulated or downregulated serum proteins in active scleritis cases compared with all control groups in both the discovery cohort and the validation cohort. The most upregulated protein was nuclear migration protein nudC (NudC; P = 0.0032), a protein involved in neurogenesis. The other significant hits included proteins involved in T-cell activation, apoptosis, epithelial barrier maintenance, and angiogenesis. Our tear fluid analysis showed matrix metalloproteinase 9 (MMP9) to be upregulated in the tear fluid of patients with scleral necrosis. Conclusions: The results of our proteomics analysis suggest a role for neurogenesis, T-cell activation, disruption of epithelial barrier, and angiogenesis in the pathogenesis of scleritis, and highlight MMP9 and NudC as biomarkers with potential clinical relevance. Funding Disclosure(s): The authors have no proprietary or commercial interest in any materials discussed in this article.

Purpose: To measure the levels of inflammatory factors in tear fluid of pre-term infants with and without retinopathy of prematurity (ROP). Methods: The cross-sectional pilot study included 29 pre-term infants undergoing routine ROP screening. Pre-term infants were grouped as those without ROP (no ROP; n = 14) and with ROP (ROP; n = 15). Sterile Schirmer's strips were used to collect the tear fluid from pre-term infants. Inflammatory factors such as interleukin (IL)-6, IL-8, MCP1 (Monocyte Chemoattractant Protein 1; CCL2), RANTES (Regulated on Activation, Normal T Cell Expressed and Secreted; CCL5), and soluble L-selectin (sL-selectin) were measured by cytometric bead array using a flow cytometer. Results: Birth weight (BW) and gestation age (GA) were significantly (P < 0.05) lower in pre-term infants with ROP compared with those without ROP. Higher levels of RANTES (P < 0.05) and IL-8 (P = 0.09) were observed in the tear fluid of pre-term infants with ROP compared with those without ROP. Lower levels of tear fluid IL-6 (P = 0.14) and sL-selectin (P = 0.18) were measured in pre-term infants with ROP compared with those without ROP. IL-8 and RANTES were significantly (P < 0.05) higher in the tear fluid of pre-term infants with stage 3 ROP compared with those without ROP. Tear fluid RANTES level was observed to be inversely associated with GA and BW of pre-term infants with ROP and not in those without ROP. Furthermore, the area under the curve and odds ratio analysis demonstrated the relevance of RANTES/BW (AUC = 0.798; OR-7.2) and RANTES/MCP1 (AUC = 0.824; OR-6.8) ratios in ROP. Conclusions: Distinct changes were observed in the levels of tear inflammatory factors in ROP infants. The status of RANTES in ROP suggests its possible role in pathobiology and warrants further mechanistic studies to harness it in ROP screening and management. [ABSTRACT FROM AUTHOR]

Purpose: This study aims to investigate the effects of maqui-berry extract (MBE) in improving signs and symptoms of dry eye disease (DED) along with ocular surface inflammation in patients with DED. Methods: Twenty patients were randomly assigned to a MBE or a placebo group (PLC). DED parameters including Schirmer's test 1 (ST1), tear film break-up time (TBUT), ocular surface disease index (OSDI), and corneal staining were assessed before treatment and 2 months post-treatment. Tear fluid samples before and after treatment from a subset of these patients were collected from the study subjects using sterile Schirmer's strips, and the levels of interleukin (IL)-1β, IL-10, IL-6, IL-17A, tumor necrosis factor-α (TNFα), matrix metalloproteinase-9 (MMP9), soluble intercellular adhesion molecule-1 (sICAM1), and vascular endothelial growth factor-A (VEGF-A) were measured using a microfluidic cartridge-based multiplex ELISA. Results: The MBE group demonstrated a significant (p < 0.05) decrease in OSDI scores along with a significant increase in Schirmer's test 1 compared to the PLC group. No significant change in TBUT and corneal staining was observed between the study groups. Levels of proinflammatory factors such as IL-1β, IL-6, IL-17A, TNFα, and MMP9 were observed to be significantly reduced, along with a significant increase in IL-10 levels following treatment in the MBE group compared with the PLC group. Conclusion: Consumption of MBE resulted in the resolution of DED signs and symptoms, along with a reduction in ocular surface inflammation.

Purpose: To study ocular surface signs, symptoms, and tear film composition following prophylactic thermal pulsation therapy (TPT) prior to refractive surgery, and to compare these outcomes with those who underwent TPT after refractive surgery. Methods: Patients with mild-to-moderate evaporative dry eye disease (DED) and/or meibomian gland dysfunction (MGD) undergoing refractive surgery were included. Group 1 patients received TPT (LipiFlow) prior to laser-assisted in situ keratomileusis (LASIK; n = 32, 64 eyes), and Group 2 patients received TPT three months after LASIK (n = 27, 52 eyes). Ocular Surface Disease Index (OSDI) score, Schirmer's test (ST1, ST2), Tear Breakup Time (TBUT), meibography, and tear fluid were obtained preoperatively and at three months postoperatively in Groups 1 and 2. Additional postoperative evaluation was performed three months after TPT in Group 2. Tear soluble factor profile was measured by multiplex enzyme-linked immunosorbent assay (ELISA) using flow cytometry. Results: Postoperative OSDI score was significantly lower and TBUT was significantly higher when compared with matched preoperative values of Group 1 participants. On the other hand, the postoperative OSDI score was significantly higher and TBUT significantly lower when compared with matched preoperative values of Group 2 participants. TPT significantly reduced the postoperative elevation in OSDI and significantly reduced the postoperative reduction in TBUT in Group 2 participants. Tear Matrix metalloproteinase-9/ Tissue inhibitor matrix metalloproteinase 1 (MMP-9/TIMP1) ratio was significantly higher, postoperatively, when compared with matched preoperative levels in Group 2. However, MMP9/TIMP1 ratio remained unaltered in Group 1 participants. Conclusion: TPT prior to refractive surgery improved postsurgical ocular surface signs and symptoms and reduced tear inflammatory factors, thereby suggesting the plausibility of reduced post-refractive surgery DED in patients.

The endocrine system influences all tissues and cells in the human body. The ocular surface is constantly exposed to circulating hormones and expresses their specific receptors. Dry eye disease (DED) is a disorder with multifactorial etiology, and endocrine anomalies are one of the inciting factors. The endocrine anomalies that cause DED include physiological conditions such as menopause, menstrual cycle variations, pathologies such as polycystic ovarian syndrome, androgen resistance, iatrogenic conditions such as contraceptive use, and antiandrogen treatment. This review highlights the status of these hormones in DED along with the mechanism of action of different hormones on the ocular surface structures and the clinical implications of these effects. The influence of androgens, estrogens, and progesterone on the ocular surface tissues, and the implications of androgen-deficient states in DED are also discussed. The physiological and pathological effects of menopause and sex hormone replacement therapy are discussed. The effects of insulin and insulin resistance on the ocular surface and DED, and the growing potential of topical insulin therapeutics for DED are mentioned. Thyroid-associated ophthalmopathy, its impact on the ocular surface, and the tissue effects of thyroid hormone in the context of DED are reviewed. Finally, the potential role of hormonal therapeutics in the management of DED has also been discussed. The compelling evidence suggests that it would be clinically beneficial to consider the possibility of hormonal imbalances and their impact while treating patients with DED.

This article was focused on the evaluation of protein biomarkers related to thrombosis, hypoxia, angiogenesis, and tissue remodeling in tear fluid of patients with non-penetrating corneal trauma. 32 patients with non-penetrating corneal injures were enrolled in the study, the control group consisted of 15 healthy patients. Samples of tear fluid were collected from the patients and control volunteers with the use of a disposable end micropipette. Protein levels of D-dimer, hypoxia-inducible factor 1α (HIF-1α), angiostatins, and matrix metalloproteinase 9 (MMP-9) in tear fluids were determined by western blot analysis. Proteolytic activity values of MMP-9 were measured by gelatin zymography. Results of western blot and zymography assay were calculated by densitometry analysis and expressed as arbitrary units. Significant increase of D-dimer and HIF-1α levels in tear fluid of patients with injured cornea by 7.3 (p

Background: Alzheimer's disease (AD) is the most common cause of dementia, and due to increasing life expectancy the number of patients is expected to grow. The diagnosis of AD involves the use of biomarkers determined by an amyloid PET scan or cerebrospinal fluid analyses that are either invasive or expensive, and not available in each hospital, thus limiting their usage as a front-line screener. The TearAD study aims to use tear fluid as a potential source for AD biomarkers. In previous reports, we demonstrated that AD biomarkers amyloid-beta and tau, are measurable in tear fluid and are associated with disease severity and neurodegeration. This study aims to validate previous results in a larger cohort and evaluate the diagnostic accuracy of tear biomarkers to discriminate between individuals with and without neurodegeneration as determined by hippocampal atrophy. Methods: The TearAD study is an observational longitudinal multi-center study that will enroll 50 cognitively healthy controls, 50 patients with subjective cognitive decline, 50 patients with mild cognitive impairment and 50 patients with AD dementia from the memory clinic. Participants will be examined at baseline, after one year, and after two years follow-up. Study assessments include neuropsychological tests and ophthalmic examination. All participants will receive a MRI scan, and a subset of the study population will undergo cerebral spinal fluid collection and an amyloid PET scan. Tear fluid will be collected with Schirmer strips and levels of Aβ38, Aβ40, Aβ42, t-tau and p-tau in tear fluid will be determined using multiplex immunoassays. Blood samples will be collected from all participants. Images of the retina will be obtained with a standard, hyperspectral and ultra-wide field fundus camera. Additionally, macular pigment optical density will be measured with the macular pigment reflectometer, and cross-sectional images of the retina will be obtained through optical coherence tomography imaging. Discussion: The TearAD study will provide insight into the potential diagnostic use of tear biomarkers as a minimally invasive and low cost tool for the screening and diagnosis of AD. Trial registration: Retrospectively registered at clinicaltrials.gov (NCT05655793). [ABSTRACT FROM AUTHOR]

A technique of collection of the tear fluid with Schirmer strips for evaluation of the activity of the main antioxidant defense enzymes (superoxide dismutase, catalase, and glutathione peroxidase) in the tear fluid was proposed. The degree of extraction of the studied enzymes from the Schirmer strip is >85%. Cytometry showed that conjunctival and corneal cells can be transferred to the Schirmer strips during tear collection, which leads to sample contamination with intracellular fractions of the antioxidant enzymes. The approach proposed by us allows avoiding this contamination during tear fluid sampling. This technique makes it possible to increase the accuracy of determining the activity of antioxidant protection enzymes in the tear fluid and can be used for diagnostics of ocular surface pathologies in clinical practice. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The endocrine system influences all tissues and cells in the human body. The ocular surface is constantly exposed to circulating hormones and expresses their specific receptors. Dry eye disease (DED) is a disorder with multifactorial etiology, and endocrine anomalies are one of the inciting factors. The endocrine anomalies that cause DED include physiological conditions such as menopause, menstrual cycle variations, pathologies such as polycystic ovarian syndrome, androgen resistance, iatrogenic conditions such as contraceptive use, and antiandrogen treatment. This review highlights the status of these hormones in DED along with the mechanism of action of different hormones on the ocular surface structures and the clinical implications of these effects. The influence of androgens, estrogens, and progesterone on the ocular surface tissues, and the implications of androgen‑deficient states in DED are also discussed. The physiological and pathological effects of menopause and sex hormone replacement therapy are discussed. The effects of insulin and insulin resistance on the ocular surface and DED, and the growing potential of topical insulin therapeutics for DED are mentioned. Thyroid‑associated ophthalmopathy, its impact on the ocular surface, and the tissue effects of thyroid hormone in the context of DED are reviewed. Finally, the potential role of hormonal therapeutics in the management of DED has also been discussed. The compelling evidence suggests that it would be clinically beneficial to consider the possibility of hormonal imbalances and their impact while treating patients with DED. [ABSTRACT FROM AUTHOR]

Purpose: This study aims to investigate the effects of maqui‑berry extract (MBE) in improving signs and symptoms of dry eye disease (DED) along with ocular surface inflammation in patients with DED. Methods: Twenty patients were randomly assigned to a MBE or a placebo group (PLC). DED parameters including Schirmer’s test 1 (ST1), tear film break‑up time (TBUT), ocular surface disease index (OSDI), and corneal staining were assessed before treatment and 2 months post‑treatment. Tear fluid samples before and after treatment from a subset of these patients were collected from the study subjects using sterile Schirmer’s strips, and the levels of interleukin (IL)‑1β, IL‑10, IL‑6, IL‑17A, tumor necrosis factor‑α (TNFα), matrix metalloproteinase‑9 (MMP9), soluble intercellular adhesion molecule‑1 (sICAM1), and vascular endothelial growth factor‑A (VEGF‑A) were measured using a microfluidic cartridge‑based multiplex ELISA. Results: The MBE group demonstrated a significant (p < 0.05) decrease in OSDI scores along with a significant increase in Schirmer’s test 1 compared to the PLC group. No significant change in TBUT and corneal staining was observed between the study groups. Levels of proinflammatory factors such as IL‑1β, IL‑6, IL‑17A, TNFα, and MMP9 were observed to be significantly reduced, along with a significant increase in IL‑10 levels following treatment in the MBE group compared with the PLC group. Conclusion: Consumption of MBE resulted in the resolution of DED signs and symptoms, along with a reduction in ocular surface inflammation. [ABSTRACT FROM AUTHOR]

Purpose: To study ocular surface signs, symptoms, and tear film composition following prophylactic thermal pulsation therapy (TPT) prior to refractive surgery, and to compare these outcomes with those who underwent TPT after refractive surgery. Methods: Patients with mild‑to‑moderate evaporative dry eye disease (DED) and/or meibomian gland dysfunction (MGD) undergoing refractive surgery were included. Group 1 patients received TPT (LipiFlow) prior to laser‑assisted in situ keratomileusis (LASIK; n = 32, 64 eyes), and Group 2 patients received TPT three months after LASIK (n = 27, 52 eyes). Ocular Surface Disease Index (OSDI) score, Schirmer’s test (ST1, ST2), Tear Breakup Time (TBUT), meibography, and tear fluid were obtained preoperatively and at three months postoperatively in Groups 1 and 2. Additional postoperative evaluation was performed three months after TPT in Group 2. Tear soluble factor profile was measured by multiplex enzyme‑linked immunosorbent assay (ELISA) using flow cytometry. Results: Postoperative OSDI score was significantly lower and TBUT was significantly higher when compared with matched preoperative values of Group 1 participants. On the other hand, the postoperative OSDI score was significantly higher and TBUT significantly lower when compared with matched preoperative values of Group 2 participants. TPT significantly reduced the postoperative elevation in OSDI and significantly reduced the postoperative reduction in TBUT in Group 2 participants. Tear Matrix metalloproteinase-9/ Tissue inhibitor matrix metalloproteinase 1 (MMP-9/TIMP1) ratio was significantly higher, postoperatively, when compared with matched preoperative levels in Group 2. However, MMP9/TIMP1 ratio remained unaltered in Group 1 participants. Conclusion: TPT prior to refractive surgery improved postsurgical ocular surface signs and symptoms and reduced tear inflammatory factors, thereby suggesting the plausibility of reduced post‑refractive surgery DED in patients. [ABSTRACT FROM AUTHOR]

Type 2 diabetes (T2DM) defined as the adult-onset type that is primarily not insulin-dependent, comprises over 95% of all diabetes mellitus (DM) cases. According to global records, 537 million adults aged 20-79 years are affected by DM that means at least 1 out of 15 persons. This number is projected to grow by 51% by the year 2045. One of the most common complications of T2DM is diabetic retinopathy (DR) with an overall prevalence over 30%. The total number of the DR-related visual impairments is on the rise, due to the growing T2DM population. Proliferative diabetic retinopathy (PDR) is the progressing DR and leading cause of preventable blindness in working-age adults. Moreover, PDR with characteristic systemic attributes including mitochondrial impairment, increased cell death and chronic inflammation, is an independent predictor of the cascading DM-complications such as ischemic stroke. Therefore, early DR is a reliable predictor appearing upstream of this "domino effect". Global screening, leading to timely identification of DM-related complications, is insufficiently implemented by currently applied reactive medicine. A personalised predictive approach and cost-effective targeted prevention shortly - predictive, preventive and personalised medicine (PPPM / 3PM) could make a good use of the accumulated knowledge, preventing blindness and other severe DM complications. In order to reach this goal, reliable stage- and disease-specific biomarker panels are needed characterised by an easy way of the sample collection, high sensitivity and specificity of analyses. In the current study, we tested the hypothesis that non-invasively collected tear fluid is a robust source for the analysis of ocular and systemic (DM-related complications) biomarker patterns suitable for differential diagnosis of stable DR versus PDR. Here, we report the first results of the comprehensive ongoing study, in which we correlate individualised patient profiles (healthy controls versus patients with stable D as well as patients with PDR with and without co-morbidities) with their metabolic profiles in the tear fluid. Comparative mass spectrometric analysis performed has identified following metabolic clusters which are differentially expressed in the groups of comparison: acylcarnitines, amino acid & related compounds, bile acids, ceramides, lysophosphatidyl-choline, nucleobases & related compounds, phosphatidyl-cholines, triglycerides, cholesterol esters, and fatty acids. Our preliminary data strongly support potential clinical utility of metabolic patterns in the tear fluid indicating a unique metabolic signature characteristic for the DR stages and PDR progression. This pilot study creates a platform for validating the tear fluid biomarker patterns to stratify T2DM-patients predisposed to the PDR. Moreover, since PDR is an independent predictor of severe T2DM-related complications such as ischemic stroke, our international project aims to create an analytical prototype for the "diagnostic tree" (yes/no) applicable to healthrisk assessment in diabetes care. [ABSTRACT FROM AUTHOR]

Proliferative diabetic retinopathy (PDR) the sequel of diabetic retinopathy (DR), a frequent complication of diabetes mellitus (DM), is the leading cause of blindness in the working-age population. The current screening process for the DR risk is not sufficiently effective such that often the disease is undetected until irreversible damage occurs. Diabetes-associated small vessel disease and neuroretinal changes create a vicious cycle resulting in the conversion of DR into PDR with characteristic ocular attributes including excessive mitochondrial and retinal cell damage, chronic inflammation, neovascularisation, and reduced visual field. PDR is considered an independent predictor of other severe diabetic complications such as ischemic stroke. A "domino effect" is highly characteristic for the cascading DM complications in which DR is an early indicator of impaired molecular and visual signaling. Mitochondrial health control is clinically relevant in DR management, and multi-omic tear fluid analysis can be instrumental for DR prognosis and PDR prediction. Altered metabolic pathways and bioenergetics, microvascular deficits and small vessel disease, chronic inflammation, and excessive tissue remodelling are in focus of this article as evidence-based targets for a predictive approach to develop diagnosis and treatment algorithms tailored to the individual for a cost-effective early prevention by implementing the paradigm shift from reactive medicine to predictive, preventive, and personalized medicine (PPPM) in primary and secondary DR care management. [ABSTRACT FROM AUTHOR]

Background: The patho‐mechanism of ocular surface disease (OSD) in dupilumab‐treated atopic dermatitis (AD) patients remains unclear. The aim of this study is to measure dupilumab levels in tear fluid and serum, and relate these findings to the severity of OSD during dupilumab treatment in AD patients. Methods: This prospective study included dupilumab‐treated moderate‐to‐severe AD patients who were seen by a dermatologist and an ophthalmologist before the start of dupilumab (baseline), and after 4 and 28 weeks of dupilumab treatment. Dupilumab levels in tear fluid and serum were measured by liquid chromatography coupled with tandem mass spectrometry (LC‐MS/MS). Additionally, a pilot study was conducted to measure dupilumab on conjunctival epithelial cells using flow cytometry and LC‐MS/MS. Results: At baseline, 89.6% (n = 43/48) of the patients had OSD, with 50.0% having moderate‐to‐severe OSD. After 28 weeks of dupilumab treatment, the median dupilumab tear fluid levels were 0.55 mg/L (IQR 0.35–1.31) and 0.29 mg/L (IQR 0.16–0.60) in patients with moderate‐to‐severe OSD and patients with no or mild OSD, respectively (p = 0.02). Dupilumab levels could be detected on conjunctival epithelial cells of 5 AD patients treated with dupilumab for 4 weeks. Conclusion: Patients with moderate‐to‐severe OSD had higher dupilumab tear fluid levels compared to patients with no or mild OSD, indicating that dupilumab reaches the ocular surface. Dupilumab was also detected in conjunctival cell suspensions and was found to directly bind CD45‐conjunctival epithelial cells. This suggests that AD‐induced changes of the conjunctival epithelium may play a role in the development of OSD as well as increased local drug availability. [ABSTRACT FROM AUTHOR]

Dysregulation of intraocular pressure (IOP) is one of the main risk factors for glaucoma. γ-synuclein is a member of the synuclein family of widely expressed synaptic proteins within the central nervous system that are implicated in certain types of neurodegeneration. γ-synuclein expression and localization changes in the retina and optic nerve of patients with glaucoma. However, the mechanisms by which γ-synuclein could contribute to glaucoma are poorly understood. We assessed the presence of autoantibodies to γ-synuclein in the blood serum of patients with primary open-angle glaucoma (POAG) by immunoblotting. A positive reaction was detected for five out of 25 patients (20%) with POAG. Autoantibodies to γ-synuclein were not detected in a group of patients without glaucoma. We studied the dynamics of IOP in response to IOP regulators in knockout mice (γ-KO) to understand a possible link between γ-synuclein dysfunction and glaucoma-related pathophysiological changes. The most prominent decrease of IOP in γ-KO mice was observed after the instillation of 1% phenylephrine and 10% dopamine. The total protein concentration in tear fluid of γ-KO mice was approximately two times higher than that of wild-type mice, and the activity of neurodegeneration-linked protein α2-macroglobulin was reduced. Therefore, γ-synuclein dysfunction contributes to pathological processes in glaucoma, including dysregulation of IOP. [ABSTRACT FROM AUTHOR]

The marked sexual dimorphism prevalent in inflammatory/autoimmune diseases is mostly due to sex hormone actions. One common eye disease that disproportionately affects women is dry eye. Thus, our aim was to optimise our highly sensitive liquid chromatography–tandem mass spectrometry method for steroid hormone quantification in tear fluid (TF). We used tears and matched serum samples from 10 heathy individuals. Estrone, estradiol testosterone, progesterone, androstenedione, and dehydroepiandrosterone, were quantified with an HPLC coupled with a Triple Quad 5500 MS. Estrone was measured in 80% of female and 20% of male TF samples (mean ± SD, 68.9 ± 62.2 pmol/L), whereas estradiol was undetectable in tears. Progesterone was identified in half of the female tear samples (2.91 ± 3.47 nmol/L) but in none of the male samples, whereas testosterone was quantifiable only in male tears (0.24 ± 0.1 nmol/L). TF hormone levels were, on average, from 1.4% to 55% of systemic values. Estrone, progesterone, and testosterone levels in tears correlated with the matching serum samples (r = 0.82, 0.79, and 0.85, respectively), but androstenedione and dehydroepiandrosterone showed no correlations. Our LC–MS/MS method could detect five out of the six steroid hormones studied in individual human TF samples and could therefore be used to analyse the role of sex steroids in eye diseases. [ABSTRACT FROM AUTHOR]

Tear fluid is an emerging source of disease biomarkers, drawing attention due to its quick, inexpensive, and non-invasive collection. The advancements in detection techniques enable the measurement of ultra-low biomarker levels from small sample volumes typical of tear fluid. The lack of standardized protocols for collection, processing, and analysis of tear fluid remains a significant challenge. To address this, we convened the Tear Research Network Review Taskforce in 2022 to review protocols from the past three decades, providing a comprehensive overview of the methodologies used in tear fluid biomarker research. A total of 1,484 articles published from January 1974 to May 2024 from two electronic databases, Embase and Ovid MEDLINE, were reviewed. An exponential increase in the number of articles on tear fluid biomarkers was observed from 2015 onwards. The two most commonly reported collection methods were; glass capillaries (45.2%), and Schirmer's strips (25%), with glass capillary tube collection remaining the most frequent method until 2019, when Schirmer's strips became the leading method. Most articles analyzed tear fluid proteins (65%) and focused on a single analyte (32.3%). In recent years, an increase was observed in the type and number of examined analytes. The differences in the reported methodologies and protocols underscore the need for standardization and harmonization within the field of tear fluid biomarkers to minimize methodological differences and reduce variability in clinical outcomes. Consistent and detailed reporting is essential for improving the reproducibility and validity of tear fluid studies, in order to advance their potential clinical applications., Competing Interests: Declaration of Competing Interest No conflicts of interest, (Copyright © 2025. Published by Elsevier Ltd.)

Purpose: Thyroid eye disease (TED) is a complex autoimmune disease. Early detection with routine disease monitoring using biomarker assessment would help in mitigating TED-associated vision loss. Hence, we performed a non-invasive tear fluid (TF) based screening in patients with TED as part of the South Indian Thyroid Eye Disease Study (SITED)., Materials and Methods: We used TF from healthy controls (HC;13 eyes;13 subjects), patients with thyroid dysfunction but without TED (No TED;11 eyes;11 subjects) and patients with TED (18 eyes;18 subjects). TED subjects were further sub-divided into those with and without an active form of the disease. Patients with dysthyroid optic neuropathy (DON) were analyzed separately. The diagnosis of TED was based on Gorman and Bartley's criteria. Activity was defined as scoring more than 4 in the Vision, Inflammation, Strabismus and Appearance (VISA). Schirmer's strip was used to collect TF and the levels of IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-13, IL-17A, IL-17F, IL-22, IFNγ, TNFα, PDGF-AA and PDGF-BB were determined by multiplex ELISA using flow cytometry., Results: Significantly ( p < 0.05) higher levels of IL-6 and IL-10 were observed in TED patients compared to HC and No TED subjects. TF levels of IL-6 and IL-10 were significantly higher in active TED patients compared to No TED subjects. Interestingly, TF levels of PDGF-AA were observed to be negatively associated with IL-4 and IL-13., Conclusion: Elevated TF levels of IL-6 and IL-10 can be explored for their role as a non-invasive risk stratification biomarker or as targets to modulate management of TED.

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.

Thyroid eye disease (TED) is a poorly understood autoimmune manifestation of thyroid diseases most commonly associated with Graves' disease. Due to a lack of specific biomarkers and uncertain signs and symptoms, Graves' orbitopathy (GO) is challenging to diagnose early and treat effectively. Nowadays, there is great interest in searching for precise molecular biomarkers for early detection, disease monitoring, and clinical management. Researchers are keen to identify novel methods to predict and diagnose diseases and to monitor patient therapeutic response. Tears, due to their direct contact with the eye and the fact that lacrimal glands can also be affected by the disease, could give new insights into the mechanisms taking place in thyroid-associated orbitopathy (TAO) and reveal potential promising biomarkers. Tear fluid offers the possibility of the non-invasive acquisition of a sample with a high protein content, thereby attracting continuously growing interest in the discovery of novel biomarkers. This article provides an up-to-date overview of the various putative tear-fluid biomarkers that have been identified. In this review, we present the potential use of tears as a diagnostic fluid and tool to investigate the mechanism of ocular diseases and discuss the future research directions in this area. [ABSTRACT FROM AUTHOR]

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Human tear film, with a flow rate of 1–3 µL/min, is a rich bodily fluid that transmits a variety of metabolites and hormones containing proteins, lipids and electrolytes that provide clues about ocular and systemic diseases. Analysis of disease biomarkers such as proteins, mRNA, enzymes and cytokines in the tear film, collected by noninvasive methods, can provide significant results for sustaining a predictive, preventive and personalized medicine regarding various diseases such as glaucoma, diabetic retinopathy, keratoconus, dry eye, cancer, Alzheimer's disease, Parkinson's disease and COVID-19. Electrochemical impedance spectroscopy (EIS) offers a powerful technique for analyzing these biomarkers. EIS detects electrical equivalent circuit parameters related to biorecognition of receptor–analyte interactions on the electrode surface. This method is advantageous as it performs a label-free detection and allows the detection of non-electroactive compounds that cannot be detected by direct electron transfer, such as hormones and some proteins. Here, we review the opportunities regarding the integration of EIS into tear fluid sampling approaches. [ABSTRACT FROM AUTHOR]

Abstract Background To identify candidate tear fluid biomarkers in patients with unilateral acute anterior uveitis (AAU) that can aid in the differentiation between these patients and patients with bacterial keratitis or healthy controls. Methods Thirteen patients (40.1 ± 16.2 years of age) with unilateral AAU, seven patients with unilateral bacterial keratitis (40.2 ± 15.3 years of age), and 14 healthy subjects (41.1 ± 11.6 years of age) were included. The tear proteome of affected eyes was compared with that of the unaffected eye or healthy controls. Proteins were identified by liquid chromatography tandem mass spectrometry and enzyme-linked immunosorbent assay. Results Relative protein ratios were detected and calculated for 272 unique proteins. Compared with healthy controls and the unaffected eye, the top upregulated proteins in AAU eyes were submaxillary gland androgen regulated protein 3B (SMR3B) and SMR3A. Similarly, the top upregulated proteins in bacterial keratitis were S100 calcium-binding protein A9 and orosomucoid 2. The acute phase response protein Serpin Family A Member 3 (SERPINA3) was increased in the healthy eye of AAU patients (P = 0.019) compared with healthy controls. Laser flare measurements in affected eyes of AAU patients showed positive logarithmic correlation with SERPINA3 in tear samples of the unaffected eye (P = 0.022). The use of SERPINA3 as a tear biomarker yielded a sensitivity of 85% and a specificity of 71% in detecting patients with AAU in the study population. Conclusions The acute phase response protein SERPINA3 was increased in tear samples of unaffected eyes of patients with unilateral AAU compared with healthy controls. This study highlights SERPINA3 as a potential biomarker for AAU. Future research should explore the dynamic properties of SERPINA3 in the tear fluid of active and quiescent uveitis eyes.

To collect tear fluid biomarkers from contact lenses (CLs) and determine the impact of CL wear duration. Rabbits 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. The 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. The 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. • CLs are a viable tear fluid sampling method for ocular biomarkers. • CL wear duration significantly affects the collection of some tear proteins. • A protocol to isolate tear proteins from CLs can be applied in disease models. • Similarities and differences exist between CLs and SS in collecting tear fluid. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Sensitive and rapid diagnosis of the early stages of glaucoma from tear fluid is a great challenge for researchers. Methods: Tear fluid was analyzed using three-dimensional synchronous fluorescence spectroscopy (3D-SFS). Our previously published results briefly describe the main methods which applied the second derivative to a selected synchronous spectrum Δλ = 110 nm in distinguishing between healthy subjects (CTRL) and patients with glaucoma (POAG). Results: In this paper, a novel strategy was used to evaluate three-dimensional spectra from the tear fluid database of our patients. A series of synchronous excitation spectra were processed as a front view and presented as a single curve showcasing the overall fluorescence profile of the tear fluid. The second derivative spectrum provides two parameters that can enhance the distinction between CTRL and POAG tear fluid. Conclusions: Combining different types of 3D-SFS data can offer interesting and useful diagnostic tools and it can be used as input for machine learning and process automation. [ABSTRACT FROM AUTHOR]

The detection of biomarkers in tears has aroused great interest owing to the advantages of non‐invasive and rapid collection. The combination of ultrasensitivity and label‐free detection of surface‐enhanced Raman spectroscopy (SERS) sensors is expected to achieve real‐time diagnosis in home medical care. However, the surface of SERS sensors is susceptible to biofouling and inactivation by biological impurities in tears, resulting in rapid degradation of sensitivity, limiting the commercialization of point‐of‐care devices. Herein, a binary nanosphere array with dual properties is constructed as a separation‐sensing platform for the diagnosis of target molecules in tears. The upper part of the structure is composed of Au nanoparticles (AuNPs) and a sputtering Au layer, which can bind the target molecules that interact with Au and provide high‐strength and high‐density SERS hotspots. The lower half is an inactive SiO2 nanosphere array with periodic large pores that allows biological impurities to penetrate the lower part and be separated from the target analyte. Furthermore, this substrate was integrated into homemade tear kits, enabling simultaneous tear collection, pre‐separation, and detection. Combined with the Raman spectra of tears and LDA analysis, we successfully identified patients with jaundice in clinics. This platform is expected to provide an opportunity for early disease screening based on biological fluids. [ABSTRACT FROM AUTHOR]

Dry eye disease (DED) and allergic conjunctivitis affect a large number of patients, and many patients usually have both symptoms. We investigated the interactions between DED and allergic conjunctivitis in mice. Four experimental groups were compared: control, DED, allergy, and allergy with DED. DED was induced by removing the extraorbital lacrimal glands of the mice. Allergic conjunctivitis was induced by intraperitoneal administration of ovalbumin and antigen eye drops. The early phase reaction of the allergy was evaluated using the clinical score, scratching behavior, and vascular permeability in the conjunctiva. Epithelial barrier function was assessed by an LC-biotin assay. Tear fluid volume and corneal fluorescein staining decreased in the DED and allergy with DED groups. LC-biotin penetrated the entire epithelium of both the cornea and conjunctiva in DED mice. The clinical score of the early phase reaction was higher in allergy-induced mice than in non-allergy mice. Edema of the eyelid and conjunctiva were aggravated in mice with DED. The number of scratching episodes and leakage of Evans blue into the conjunctiva were higher in allergy-induced DED mice than in control mice. The presence of aqueous-deficient dry eye caused ocular surface epithelial damage and exacerbated allergic signs and symptoms. [ABSTRACT FROM AUTHOR]

Prolonged daily face mask wearing over several months might affect health of the ocular surface and is reported to be associated with complaints of discomfort and dry-eye-like symptoms. We studied the ocular surface clinical parameters, tear soluble factors and immune cell proportions in ophthalmologists practicing within similar environmental conditions (n = 17) at two time points: pre-face-mask period (Pre-FM; end of 2019) and post-face-mask-wearing period (Post-FM; during 2020 COVID-19 pandemic), with continuous (~8 h/day) mask wear. A significant increase in ocular surface disease index (OSDI) scores without changes in tear breakup time (TBUT), Schirmer's test 1 (ST1) and objective scatter index (OSI) was observed Post-FM. Tear soluble factors (increased—IL-1β, IL-33, IFNβ, NGF, BDNF, LIF and TSLP; decreased—IL-12, IL-13, HGF and VEGF-A) and mucins (MUC5AC) were significantly altered Post-FM. Ex vivo, human donor and corneoscleral explant cultures under elevated CO2 stress revealed that the molecular profile, particularly mucin expression, was similar to the Post-FM tear molecular profile, suggesting hypercapnia is a potential contributor to ocular surface discomfort. Among the immune cell subsets determined from ocular surface wash samples, significantly higher proportions of leukocytes and natural killer T cells were observed in Post-FM compared to Pre-FM. Therefore, it is important to note that the clinical parameters, tear film quality, tear molecular factors and immune cells profile observed in prolonged mask-wear-associated ocular surface discomfort were distinct from dry eye disease or other common ocular surface conditions. These observations are important for differential diagnosis as well as selection of appropriate ocular surface treatment in such subjects. [ABSTRACT FROM AUTHOR]

Patients with head and neck cancer (HNC) and patients with primary Sjögren's syndrome (pSS) may exhibit similar symptoms of dry mouth and dry eyes, as a result of radiotherapy (RT) or a consequence of disease progression. To identify the proteins that may serve as promising disease biomarkers, we analysed saliva and tears from 29 radiated HNC patients and 21 healthy controls, and saliva from 14 pSS patients by mass spectrometry-based proteomics. The study revealed several upregulated, and in some instances overlapping, proteins in the two patient groups. Histone H1.4 and neutrophil collagenase were upregulated in whole saliva of both patient groups, while caspase-14, histone H4, and protein S100-A9 were upregulated in HNC saliva only. In HCN tear fluid, the most highly upregulated protein was mucin-like protein 1. These overexpressed proteins in saliva and tears play central roles in inflammation, host cell injury, activation of reactive oxygen species, and tissue repair. In conclusion, the similarities and differences in overexpressed proteins detected in saliva from HNC and pSS patients may contribute to the overall understanding of the different pathophysiological mechanisms inducing dry mouth. Thus, the recurring proteins identified could possibly serve as future promising biomarkers [ABSTRACT FROM AUTHOR]