Tear Fluid Samples Research Articles

The effect of different potency glucocorticosteroids treatments on tear inflammatory factors and corneal optical density after femtosecond-assisted laser in situ keratomileusis.

To assess the impact of glucocorticosteroids with varying potencies on inflammatory mediators in tears and corneal optical density after femtosecond-assisted laser in situ keratomileusis (FS-LASIK). In a prospective study, 110 patients (220 eyes) who underwent FS-LASIK were divided into 2 groups: 55 patients (110 eyes) received dexamethasone, and another 55 patients (110 eyes) received fluorometholone. Visual acuity, intraocular pressure, and corneal optical density were measured before, 1 week, and 1 month after surgery. Tear fluid samples were also collected to assess expression levels of TNF-α, IL-1α, IL-6, and TGF-β1. One week after the procedure, the dexamethasone group exhibited elevated intraocular pressure (IOP) levels (P > .05) and a decreased expression of TNF-α in tears (P < .001) compared to the fluorometholone group. Within the 0 to 2 mm range from the corneal apex, the anterior corneal layer's optical density in the fluorometholone group surpassed that of the dexamethasone group (P < .05). At 1 month post-surgery, the IOP in the fluorometholone group was higher than that in the dexamethasone group (P < .05). In both the 0 to 2 mm and 2 to 6 mm intervals from the corneal apex, the optical density of the anterior corneal layer was significantly higher in the fluorometholone group compared to the dexamethasone group (P < .05). There was no statistically significant difference in visual acuity between the 2 groups at any postoperative time point. Short-term use of potent corticosteroids after FS-LASIK can swiftly address ocular surface inflammation, enhance corneal wound healing, reduce corneal edema, and accelerate the restoration of corneal transparency, in contrast to prolonged use of milder corticosteroids post-surgery.

Differential Ophthalmological Profile in Patients with Coronary Artery Disease Coexisting with Type 2 Diabetes Mellitus: Elevated Tear Cytokine Concentrations.

Background/Objectives: Coronary artery disease (CAD) and type-2 diabetes mellitus (T2DM) are characterized by chronic low-grade inflammation. However, measuring cytokines typically involves invasive blood sampling, which can be problematic for CAD patients. This study aimed to assess ophthalmological parameters and tear cytokines in patients with CAD, comparing those with comorbid T2DM to those without to understand their inflammatory profiles. Methods: One hundred subjects with suspected chronic or acute CAD were initially included in this single-center cross-sectional study after clinical stabilization. Seventy-two patients with confirmed CAD were divided into two groups: 32 patients with T2DM and 40 patients without T2DM. A total of 144 eyes were examined, and tear fluid samples were collected to determine cytokine concentrations. Ophthalmological parameters and tear concentrations of cytokines were analyzed, controlling for age, sex, and other cardiovascular risk factors. Results: Patients with CAD and T2DM exhibited decreased ophthalmological parameters and increased cytokine concentrations in comparison to those without T2DM. Significant inverse correlations between ophthalmological parameters and cytokine concentrations were observed. Following adjustment, a full logistic regression model for distinguishing patients with CAD and comorbid T2DM included macular cube volume, mean macular thickness, interleukin (IL)-4, IL-5, IL-6, IL-8, IL-9, IL-13, granulocyte colony-stimulating factor (G-CSF), CCL3, CCL4, and CCL11/eotaxin-1, demonstrating excellent discriminatory power (Area Under the Curve = 0.95, 95% Confidence Interval = 0.91-0.99; p < 0.001). Subsequently, IL-5 (Odds Ratio = 1.68, 95% CI = 1.26-2.24; p < 0.001), G-CSF (OR = 1.06, 95% CI = 1.02-1.11; p < 0.01), and CCL11/eotaxin-1 (OR = 1.56, 95% CI = 1.19-2.05; p = 0.001) emerged as the most distinguishing variables in a reduced model (AUC = 0.89, 95% CI = 0.84-0.95; p < 0.001). Conclusions: Differences in ophthalmological variables, mainly in cytokine concentrations, suggest distinct pathophysiological mechanisms in patients with CAD based on the presence of T2DM. These findings demonstrate that the inflammatory profile can be readily detected through tear sample cytokines, proving valuable for establishing more accurate prognoses and monitoring in cardiometabolic disorders.

Glypican-4 serum levels are associated with cognitive dysfunction and vascular risk factors in Parkinson’s disease

Glypicans are biomarkers for various pathologies, including cardiovascular disease, cancer and diabetes. Increasing evidence suggests that glypicans also play a role in the context of neurodegenerative disorders. Initially described as supporting functionality of synapses via glutamate receptors during CNS development, Glypican 4 (GPC-4) also plays a role in the context of dementia via tau hyperphosphorylation in Alzheimer’s disease, which is also a co-pathology in Parkinson’s disease dementia. However, clinical evidence of circulating GPC-4 in Parkinson’s disease (PD) is missing so far. We therefore investigated GPC-4 in biofluids of PD patients. We analyzed GPC-4 levels in cerebrospinal fluid (CSF, n = 140), serum (n = 80), and tear fluid samples (n = 70) of PD patients and control subjects in a similar age range by ELISA (serum, CSF) and western blot (tear fluid). Expression of circulating GPC-4 was confirmed in all three biofluids, with highest levels in serum. Interestingly, GPC-4 levels were age-dependent, and multiple regression analysis revealed a significant association between GPC-4 serum levels and MoCA score, suggesting an involvement of GPC-4 in PD-associated cognitive decline. Furthermore, stratification of PD patients for vascular risk factors revealed a significant increase of GPC-4 serum levels in PD patients with vascular risk factors. Our results suggest GPC-4 as a clinical biomarker for vascular risk stratification in order to identify PD patients with increased risk of developing dementia.

Comparison of immunological and molecular methods for laboratory diagnosis of ocular toxoplasmosis in blood, serum and tears in Brazil.

Ocular toxoplasmosis (OT) is caused by protozoan T. gondii. Ophthalmological examination is considered the gold standard for OT diagnosis, and laboratory tests are used for diagnostic confirmation. However, these tests can present different results, which change depending on their basis, on sample type and on patients' clinical alteration. Thus, the aim of the present study is to assess immunodiagnostic and molecular techniques applied in blood, serum and tear fluid to diagnose T. gondii infection in patients seen at an Ophthalmology Clinic. In total, 160 patients were included in the study, 40 of them had OT with active lesions (G1); 40 had OT with healed lesions (G2), 40 had non-toxoplasmic uveitis (G3) and 40 had no ocular alterations (G4). Serum samples were subjected to Immunoenzymatic Assay (ELISA) and to Indirect Immunofluorescence Reaction (IFAT) to search for anti-T. gondii IgM and IgG. Tear fluid samples were analyzed through ELISA for IgA research. All blood and tear fluid samples were subjected to conventional polymerase chain reaction (cPCR) and in a Nested PCR model for T. gondii DNA amplification with targets B1, GRA7 and REP 529. IgG and IgM anti-T. gondii was detected in serum samples from 106 and 15 patients, respectively, when combining ELISA and IFAT results. Anti-T.gondii IgA antibodies were detected in 9.2% of the tear material. Nested PCR with GRA7 target showed higher positivity in blood samples (24.4%); Nested PCR with B1 target showed a higher frequency of positivity in tears (15%). Biological samples of patients with active lesions showed the highest positivity frequencies in all immunodiagnostic assays, as well as in most PCR models. The present results highlighted the need of associating techniques with different fundamentals to confirm OT diagnosis. Furthermore, further tear fluid analyses should be performed to validate this biological material as lesser invasive alternative for the more accurate OT diagnosis.

HPLC-LED-Induced Fluorescence Analysis of Tear Fluids: An Objective Method for Primary Open Angle Glaucoma Diagnosis

PurposeThe study showcased the application of the lab-assembled HPLC-LED-IF system to analyze proteins in tear fluid samples collected from individuals diagnosed with primary open-angle glaucoma (POAG).MethodsClinical application of the said technique was evaluated by recording chromatograms of tear fluid samples from control and POAG subjects and by analyzing the protein profile using multivariate analysis. The data analysis methods involved are principal component analysis (PCA), Match/No-Match, and artificial neural network (ANN) based binary classification for disease diagnosis.ResultsMahalanobis distance and spectral residual values calculated using a standard calibration set of clinically confirmed POAG samples for the Match/No-Match test gave 86.9% sensitivity and 81.8% specificity. ANN with leaving one out procedure has given 87.1% sensitivity and 81.8% specificity.ConclusionThe results of the study revealed that the utilization of a 278 nm LED excitation in the HPLC system offers good sensitivity for detecting proteins at low concentrations allowing to obtain reliable protein profiles for the diagnosis of POAG.

The detection of phosphorylated tau in tear fluid

AbstractBackgroundThe recent development of ultrasensitive immunological methods has allowed for the detection of biomarkers related to neurodegenerative disorders in blood, e.g., phosphorylated tau (p‐tau). This allows for a cost‐effective and scalable assessment of the causes behind cognitive decline. Nevertheless, other biofluids, e.g., tear fluid, have the potential to surpass these metrics. Previously, tear fluid from patients with neurodegenerative conditions has been explored both with mass‐spectrometry and immunoassays. Hereby, we established a method for the detection of p‐tau181, p‐tau217 and p‐tau231 in tear fluid from patients.MethodA total of 40 participants with neurodegenerative diseases (amyotrophic lateral sclerosis [n = 11], dementia [n = 9], Parkinson’s disease [n = 20]) and control subjects without clinical signs of neurodegeneration [n = 20], were recruited at the Department of Neurology at the Klinikum rechts der Isar, München, Germany. Tear fluid from both eyes was collected with Schirmer strips. The strips from both eyes were combined and eluted by centrifugation (16,000g) in sample buffer. The eluate was then analysed for p‐tau181, p‐tau217 and p‐tau231 by Single molecule array (Simoa). Results were normalised to tear fluid running length.ResultAll tear fluid samples were above the lower limit of detection (LLOD) for all p‐tau assays ([mean] p‐tau181, 92.1pg/ml; p‐tau217, 0.398pg/ml; p‐tau231, 52.9pg/ml) improving on previous attempts found in literature. There was a highly significant correlation between all p‐tau biomarkers in tear fluid (r = 0.90–0.98; P&lt;0.001). The normalised concentration of tear fluid p‐tau had a significant positive correlation with age (R p‐tau181 = 0.28, R p‐tau217 = 0.27, R p‐tau231 = 0.39; P&lt;0.05) but there was no association with the corresponding p‐tau in serum. No significant group differences in tear fluid p‐tau were observed but a trend towards increased serum p‐tau181 was found in the neurodegenerative group (6.37pg/ml) compared to controls (4.37pg/ml).ConclusionIn this pilot study, we describe quantifiable levels of p‐tau181, p‐tau217 and p‐tau231 in tear fluid samples. However, the detectable levels of p‐tau in tear fluid do not seem to correlate to p‐tau levels in serum. Further studies will show p‐tau in tear fluid of patients with suspected AD, stratified by CSF biomarkers, and thus establish the relevance of tear fluid as a diagnostic biofluid in AD.

Topical Betamethasone Treatment of Stevens-Johnson Syndrome and Toxic Epidermal Necrolysis with Ocular Involvement in the Acute Phase

To clarify the importance of administering topical steroids for the treatment of Stevens-Johnson syndrome (SJS) / toxic epidermal necrolysis (TEN) with ocular involvement in the acute phase. Retrospective case series. Using the medical records of acute SJS/TEN patients treated at the Kyoto Prefectural University of Medicine Hospital, Kyoto, Japan, between July 2006 and July 2017, the ocular findings, topical steroid dosage, systemic steroid dosage, and ocular sequelae were retrospectively examined. The level of cytokines in tear fluid and serum samples was also analyzed. This study involved 13 cases. In 10 cases in whom the clinical courses were recorded before the start of steroid therapy, the mean acute ocular severity score (AOSS: 3=very severe; 2=severe; 1=mild; 0=none) was 2.8 ± 0.4 points in the severest phase. The mean systemic steroid dose after steroid pulse therapy was 694 ± 386 mg and the mean topical steroid (0.1% betamethasone eye drop and ointment) dose was 13.4 ± 3.3 times daily in the severest phase. Analysis of cytokine levels of 4 cases showed that a cytokine storm occurred in the tear fluid after the steroid pulse therapy. At final follow-up, 16 eyes of 8 patients had a logMAR visual acuity of ≤0, and no serious ocular sequelae were observed. In patients with SJS/TEN, ocular surface inflammation remains strong even after systemic inflammation has improved post steroid pulse therapy, thus suggesting that both systemic and topical steroid therapy should be administered appropriately.

Comparative analysis of local cytokine profile and corneal regenerator process after LASIK and Femto-LASIK surgery

Introduction. The existing data in the scientific literature on the role of cytokines as a special biological system, a function of which is the local regulation of regeneration, justifies the relevance of research task in this direction.Purpose: to study changes of the cytokines concentration in the lacrimal fluid in patients after excimer laser vision correction with LASIK and Femto-LASIK surgery and its correlation with postoperative patients’ parameters.Methods. The study included 20 patients (40 eyes) with mild myopia and compound myopic astigmatism. The prospective study was carried out in January-August 2022. The patients were divided into 2 groups. In the comparison group (n = 10, 20 eyes) patients underwent LASIK surgery, in the main one (n = 10, 20 eyes) – Femto-LASIK. During the study, the tear fluid was taken and its further biochemical study was carried out to determine the level of cytokines: IL-1β, IL-8, TNF-α.Results. In the main group, frequency detection of the cytokine IL-1β that is the main pro-inflammatory agent was 80%. In the comparison group it was detected in 90% of the tear fluid samples. Mean IL-1β values were the highest in the comparison LASIK group. Mean TNF-α scores were the highest in the comparison LASIK group. In the same time, differences of the average values between the main and comparison groups were statistically significant (p &lt; 0.05). Mean IL-8 values were the highest in the main group who underwent Femto-LASIK surgery.Conclusion. The course of regenerative process in patients after excimer laser vision correction depends on concentration of the pro-inflammatory cytokines IL-1β and TNF-α and the anti-inflammatory cytokine IL-8. Based on this, a higher level of the pro-inflammatory cytokines in the lacrimal fluid determines the prolongation of pain relief and epithelialization after surgery.

Sex Hormones and Calcitonin Gene-Related Peptide in Women With Migraine: A Cross-sectional, Matched Cohort Study.

Sex hormones may modulate calcitonin gene-related peptide (CGRP) release in the trigeminovascular system. We studied CGRP concentrations in plasma and tear fluid in female participants with episodic migraine (EM) and a regular menstrual cycle (RMC), female participants with EM and combined oral contraception (COC), and female participants with EM in the postmenopause. For control, we analyzed 3 corresponding groups of age-matched female participants without EM. Participants with an RMC had 2 visits: during menstruation on menstrual cycle day 2 ± 2 and in the periovulatory period on day 13 ± 2. Participants with COC were examined at day 4 ± 2 of the hormone-free interval (HFI) and between days 7 and 14 of hormone intake (HI). Postmenopausal participants were assessed once at a random time point. Plasma and tear fluid samples were collected at each visit for determination of CGRP levels with an ELISA. A total of 180 female participants (n = 30 per group) completed the study. Participants with migraine and an RMC showed statistically significantly higher CGRP concentrations in plasma and tear fluid during menstruation compared with female participants without migraine (plasma: 5.95 pg/mL [IQR 4.37-10.44] vs 4.61 pg/mL [IQR 2.83-6.92], p = 0.020 [Mann-Whitney U test]; tear fluid: 1.20 ng/mL [IQR 0.36-2.52] vs 0.4 ng/mL [IQR 0.14-1.22], p = 0.005 [Mann-Whitney U test]). In contrast, female participants with COC and in the postmenopause had similar CGRP levels in the migraine and the control groups. In migraine participants with an RMC, tear fluid but not plasma CGRP concentrations during menstruation were statistically significantly higher compared with migraine participants under COC (p = 0.015 vs HFI and p = 0.029 vs HI, Mann-Whitney U test). Different sex hormone profiles may influence CGRP concentrations in people, with current or past capacity to menstruate, with migraine. Measurement of CGRP in tear fluid was feasible and warrants further investigation.

The levels of hypoxia- and angiogenesis-related regulators and matrix metalloproteinase 9 activity in tear fluid of patients with non-penetrating ocular traumas

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&lt;0.05) and 56 (p&lt;0.001) folds, respectively was shown, compared with control, indicating thrombotic events and hypoxia condition to be involved in pathogenesis of ocular trauma. Dramatically elevated levels/activity of MMP-9 enzyme (by 105 folds vs. control, p&lt;0.001) suggests intense tissue remodeling and degradation of extracellular matrix in the damaged cornea. Up-regulation of angiostatin level, products of proteolytical cleavage of plasminogen, in tear fluid collected from patients with traumatic eye in comparison with healthy volunteers (by 7.3 folds, p&lt;0.05), could represent an adaptive mechanism, which counteracts excessive hypoxia-induced neovascularization of injured cornea. It is summarized that there was a strong association between elevation of D-dimer, HIF-1α, angiostatins, and MMP-9 levels suggesting thrombosis- and hypoxia-mediated mechanisms triggering wound healing of injured cornea. The findings of this study are novel and provide a basis for further investigations of the reparation mechanisms during non-penetrating ocular trauma. Studied proteins of tear fluid can serve as relevant biomarkers of corneal wound healing and are appropriate for diagnostic and prognostic purposes.

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.

Biological processes and clinical variables affecting trabeculectomy surgery outcome

AbstractPurpose: To establish what preoperative clinical factors and tear protein levels influence the trabeculectomy outcome.Methods: In this prospective study, we followed 80 glaucomatous eyes for 5 years after trabeculectomy. Preoperatively, tear fluid samples and clinical data were collected from all patients. Postoperative statuses, i.e., qualified success (QS) in case of 5‐fluorouracil (5FU), needling or restart of glaucoma medication and failure (F) in case of reoperation, were recorded. The patients' postoperative statuses were then compared against the preoperative tear proteomics and clinical variables with Wilcoxon rank sum test. Proteomic pathway analyses were performed using Ingenuity Pathway Analysis (IPA) software.Results: Within 1 year of the surgery, 15 (19%) QS + F patients were detected. Patients with early failure were younger than those with a successful early outcome (64.7 ± 9.5 vs 70.1 ± 7.6 years, respectively, p = 0.04) and according to pathway analysis results, had increased activation of immune responses prior to surgery (all p‐values &lt;0.05). Overall, 24 (31%) QS + F cases occurred during the 5‐year follow‐up. Pathway analysis revealed that these patients had increased levels of apoptosis prior to surgery (activation z‐score = 2.2, p‐value = 0.02).Conclusions: These results further emphasize the importance of observing the preoperative state of the ocular surface. The tear proteomic profiles reveal biological functions that are associated with the trabeculectomy outcome.

Sex Steroid Hormone Analysis in Human Tear Fluid Using a Liquid Chromatography—Mass Spectrometry Method

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.

PRODUCTION OF ANTI-LACTOFERRIN ANTIBODIES AND THEIR APPLICATION IN ANALYSIS OF THE TEAR FLUID IN HEALTH AND CORNEAL INJURIES

Lactoferrin is a ubiquitous and multifunctional protein, which has antimicrobial and immunomodulatory activities. Lactoferrin plays an important role in the maintenance of ocular health. The aim of the study was to produce polyclonal antibodies against human lactoferrin in order to apply them in evaluation of lactoferrin levels in tear fluid collected from healthy eye and after corneal injury. Materials and methods. Affine chromatography on Protein A-sepharose was applied in order to isolate immunoglobulin G (IgG) fraction from the blood serum of lactoferrin-immunized rabbits. Each step of protein purification was monitored by denaturing gel electrophoresis (SDS-PAGE). Target antigen recognition by produced antibodies was established by western blot analysis with the use of diluted IgG fraction. Lactoferrin levels in the tear fluids collected from healthy individuals (n = 4) and patients with non-penetrating corneal injures (n = 6) were determined immunochemically with the use of purified antibodies. The results of western blot of lactoferrin levels in the tear fluids of healthy individuals and patients with corneal wounds were analysed using Mann-Whitney U-test. The difference between group mean values was considered significant at P&lt;0.05. Results. Using affine chromatography on Protein A-sepharose, antibodies against human lactoferrin were purified as IgG fraction from blood serum of lactoferrin-immunized rabbits. Western blot analysis showed that obtained antibodies recognize the antigen as a 75-kDa band, which corresponds to the intact human lactoferrin polypeptide. The same major polypeptide band was visualized by western blot with enhanced chemiluminescence detection in the tear fluid samples. Densitometry analysis of 75-kDa lactoferrin band showed 3.2-fold decrease in lactoferrin level in the tear fluid samples obtained from patients with non-penetrating corneal traumas as compared with samples collected from healthy persons (P&lt;0.05). Besides, tear fluid of patients with injured corneas contained large amounts of truncated lactoferrin immunoreactive polypeptides as well as high molecular weight bands, which could correspond to lactoferrin complexes with other proteins occurring during inflammation. Conclusions. According to our data, obtained anti-lactoferrin antibodies can be used as a valuable tool for development of advanced tests and procedures for diagnostics of eye diseases associated with the corneal lesions. Reduced lactoferrin concentration might represent a potential prognostic biomarker for diagnosis of ocular diseases including non-penetrating corneal injuries in a simple and non-invasive way.

Conjunctival Intraepithelial Lymphocytes, Lacrimal Cytokines and Ocular Commensal Microbiota: Analysis of the Three Main Players in Allergic Conjunctivitis.

Conjunctival intraepithelial lymphocytes, tear soluble molecules and commensal microbiota have important roles in the ocular mucosal immune response in healthy and diseased subjects. For the purpose of this study, the cellular and microbial populations of the conjunctiva and the lacrimal soluble molecules were analyzed to find the main biomarkers in allergic conjunctivitis. A total of 35 healthy subjects, 28 subjects with seasonal allergic conjunctivitis and 32 subjects with perennial allergic conjunctivitis were recruited to obtain peripheral blood, conjunctival brush cytology, tear fluid and microbiota samples. Flow cytometry for lymphocytes, multiplex bead assays for cytokines and high-throughput DNA sequencing for microbiome analysis were used. For perennial allergic conjunctivitis, an increased proportion of Th2 and NKT lymphocytes was found, while CD3+TCRγδ+ lymphocytes and double negative MAIT cells were decreased. In contrast, seasonal allergic conjunctivitis was distinguished by an increase in Th17 and Th22 cell proportions, while the Th1 cell proportion decreased. Among tear fluid, the vast majority of pro-inflammatory cytokines (especially Th2 and Th17 cytokines) in perennial allergies and MMP-9 together with IgA in seasonal allergies were increased. In contrast, TGF-β2 was decreased in both forms of conjunctivitis. Finally, fungal (Malassezia species) and bacterial (Kocuria and Propionobacterium acnes species) colonization were observed in the perennial allergic conjunctivitis group. These results provide the basis for the development of a disease profile for perennial allergic conjunctivitis and open the door to new therapeutic and diagnostic strategies.

Choroidal thickness and granulocyte colony-stimulating factor in tears improve the prediction model for coronary artery disease

BackgroundCoronary artery disease (CAD) detection in asymptomatic patients still remains controversial. The aim of our study was to evaluate the usefulness of ophthalmologic findings as predictors of the presence of CAD when added to cardiovascular classic risk factors (CRF) in patients with acute coronary cardiopathy suspicion.MethodsAfter clinical stabilization, 96 patients with acute coronary cardiopathy suspicion were selected and divided in two groups: 69 patients with coronary lesions and 27 patients without coronary lesions. Their 192 eyes were subjected to a complete routine ophthalmologic examination. Samples of tear fluid were also collected to be used in the detection of cytokines and inflammatory mediators. Logistic regression models, receiver operating characteristic curves and their area under the curve (AUC) were analysed.ResultsSuggestive predictors were choroidal thickness (CT) (OR: 1.02, 95% CI 1.01–1.03) and tear granulocyte colony-stimulating factor (G-CSF) (OR: 0.97, 95% CI 0.95–0.99). We obtained an AUC of 0.9646 (95% CI 0.928–0.999) when CT and tear G-CSF were added as independent variables to the logistic regression model with cardiovascular CRF: sex, age, diabetes, high blood pressure, hypercholesterolemia, smoking habit and obesity. This AUC was significantly higher (p = 0.003) than the prediction derived from the same logistic regression model without CT and tear G-CSF (AUC = 0.828, 95% CI 0.729–0.927).ConclusionsCT and tear G-CSF improved the predictive model for CAD when added to cardiovascular CRF in our sample of symptomatic patients. Subsequent studies are needed for validation of these findings in asymptomatic patients.

MiR-223 inhibits hyperosmolarity-induced inflammation through downregulating NLRP3 activation in human corneal epithelial cells and dry eye patients

We previously showed that increases in reactive oxygen species (ROS) generation upregulate NLRP3 inflammasome and inflammation through increases in both caspase-1 activity and rises in IL-1β expression levels in animal models of dry eye (DE). As changes in microRNA (miRNAs) expression levels can modulate inflammasome function, we determine here if there is a relationship in DE between changes in miR-223 expression levels and NLRP3 activation induced in an intelligent controlled environmental system (ICES) in mice. In parallel, ROS, miR-223 and NLRP3 expression levels were assessed in conjunctival impression cytology and tear fluid samples obtained from DE patients and normal subjects. MiR-223 expression levels were modulated by transfection of either a mimic or its negative control (NC) in a human corneal epithelial cell line (HCECs) exposed to a 500 mOsm hyperosmotic medium for 4 h. The dual-luciferase reporter assay confirmed that miR-223 controls NLRP3 gene expression readout through directly interacting with the 3’ UTR of its mRNA. Hyperosmolarity-induced NLRP3 activation was confirmed based on recruitment and colocalization of NLRP3 with ASC as well as increases in IL-1β expression. The miR-223 expression level decreased by 55% in the conjunctiva and cornea of the murine DE model from the level in the control group (P ≤ 0.047), while NLRP3 protein expression rose by 30% (P ≤ 0.017). In DE patients, miR-223 expression decreased in conjunctival impression cytology samples (P = 0.002), whereas IL-1β tear content rose significantly (P < 0.001).The relevance of this decline was confirmed by showing that exposure to a 500 mOsm stress decreased the miR-223 expression level whereas ROS generation as well as the NLRP3, and IL-1β expression levels rose in HCECs (P ≤ 0.037). In contrast, miR-223 mimic transfection reduced the NLRP3 protein expression level by 30% (P = 0.037), whereas both ROS generation and IL-1β secretion rose compared to their corresponding levels in the control group (P ≤ 0.043). Thus, miR-223 negatively regulates NLRP3 inflammasome activity via suppressing NLRP3 translation in DE. This inverse regulation between miR-223 and NLRP3 expression levels suggests that selective upregulation of miR-223 expression may be a novel option to suppress chronic inflammation in DE.

PROTEIN MARKERS OF HYPOXIA AND ANGIOGENESIS IN TEAR FLUID OF PATIENTS WITH TRAUMATIC CORNEAL INJURY

The aim of our study was to evaluate tear levels of some protein endpoints that can reflect intensities of hypoxia, angiogenesis and tissue remodeling in wounded cornea. Methods. We examined 21 patients (21 eyes) with nonpenetrating corneal injuries. The patients underwent standard ophthalmological examination including previous history and ocular symptoms, visual acuity test, complete anterior and posterior eye segments examination using slit lamp biomicroscopy, evaluation of corneal staining with fluorescein, ophthalmoscopy. Healthy volunteers (n = 10) served as a control. Tear fluid was collected from patients and control volunteers with the use of a disposable tip micropipette. From the lower arch of the conjunctiva without instillation of anesthetic, tears were collected in a sterile plastic Eppendorf tube and frozen at -20 oC before laboratory examination. Proteins of tear fluids were separated by SDS-PAGE (loading 50 µg total protein per track). Then, levels of hypoxia inducible factor 1α (HIF-1α), vascular endothelial growth factor (VEGF), and angiostatins were measured by western blot. Active MMP-9 levels were evaluated by gelatin zymography. The results of blot and zymography assays were processed by densitometric software and then analyzed statistically with the use of Mann-Whitney U-test. Results. Elevated HIF-1α (P&lt;0.001) and angiostatins (P&lt;0.05) levels were revealed by western blot in tear fluid samples collected from patients with injured cornea in comparison with the control group. It is noteworthy that extremely low amounts of VEGF were detected in tear fluid from injured eyes, in spite of abundance of its transcription inducer HIF-1α. Dramatically increased levels of active MMP-9 were found in the tear fluids of patients with corneal wounds, while no significant collagenolytic activity was observed in tears from healthy eyes. There is a strong correlation between extent of corneal lesions and changes in markers expression. Conclusions. Tear levels of HIF-1α and angiostatin as well as MMP-9 activity could represent valuable biomarkers of corneal injury severity in traumatic eye.

Clinical and Molecular Outcomes After Combined Intense Pulsed Light Therapy With Low-Level Light Therapy in Recalcitrant Evaporative Dry Eye Disease With Meibomian Gland Dysfunction.

Dry eye disease (DED) is a leading cause of ocular morbidity worldwide. This study evaluates the effects of combined light therapy [intense pulsed light (IPL) and low-level light therapy (LLLT)] on clinical and molecular outcomes in evaporative DED with meibomian gland dysfunction (MGD). This prospective study evaluated 94 eyes (47 subjects) with chronic MGD treated with combined light therapy. Patients underwent a detailed evaluation of MGD and DED using the Ocular Surface Disease Index, dry eye tests-tear breakup time and Schirmer test, ocular surface staining, meibomian gland expressibility scoring, and meibography. Patients underwent a single session of combined light therapy (IPL + LLLT treatment) using the Eye-light device. All these tests were repeated at 3 and 6 months after treatment. Tear fluid and ocular surface wash samples were collected from a subset of patients before and after treatment for cellular and secreted immune factor profiling by flow cytometry. Combined light therapy (IPL + LLLT) demonstrated a marked improvement in the clinical metrics studied. Three months after treatment, Ocular Surface Disease Index showed a significant reduction in 95.6% ( P < 0.0001), tear breakup time increased in 72.3% ( P < 0.0001), and meibomian gland expressibility scoring increased in 80.8% ( P < 0.0001) of the eyes. These effects were observed to be sustained during the 6-month follow-up visit. Significant ( P < 0.05) reduction in tear fluid levels of interleukin-1β, interleukin-17F, and MMP9; MMP9/TIMP1 ratio; and ocular surface B-cell proportions was observed. Combined light therapy shows promising results in patients with chronic MGD and DED, even in recalcitrant cases. Clinical and molecular factor alterations support the improved symptomatology and reduced inflammation.

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

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