Short-Term Efficacy of Ophthalmic Cyclosporine: A 0.1% Cationic Emulsion in Dry Eye Patients Assessed Under Controlled Environment.

INTRODUCTION: To evaluate the short-term efficacy of cyclosporine A (CsA)-0.1% cationic emulsion (CE) in patients with dry eye disease (DED) and mitigation of the inflammatory flares triggered by desiccating stress environments. METHODS: A single-center non-randomized clinical trial was performed at a tertiary care setting. Twenty patients with DED treated with CsA 0.1% CE were exposed to a normal controlled environment (NCE) (23 °C, 50% relative humidity) and an adverse controlled environment (ACE) (23 °C, 10% relative humidity, 0.43 m/s localized airflow) during baseline and the 1- and 3-month visits. Patients underwent the following evaluations: conjunctival hyperemia and staining, corneal fluorescein staining (CFS) using the Oxford and Cornea and Contact Lens Research Unit (CCLRU) scale, meibomian gland (MG) secretion quality, Dry Eye Questionnaire-5, Symptom Assessment in Dry Eye (SANDE II), and Change in Dry Eye Symptoms Questionnaire. Multivariate models were adjusted for statistical analysis. RESULTS: Nineteen women and one man (mean age, 58.9 ± 12.3 years) completed the study. All symptom questionnaires, CFS, conjunctival hyperemia and staining, and MG secretion quality improved (p ≤ 0.003) with 1 month of treatment; improvements were maintained after 3 months (p ≤ 0.02), except for SANDE II (p ≥ 0.07). The CFS worsening (total CCLRU) after baseline ACE exposure (from 8.6 to 10.1) was higher, although not significant (p = 0.64), compared with 1 month (from 5.4 to 5.8) and 3 months (from 5.0 to 5.9) after treatment. CONCLUSION: Topical CsA-0.1% CE improved DED signs and symptoms after 1 month of treatment under controlled environmental conditions. Future studies should confirm the benefit of CsA-0.1% CE in desiccating stress environments. TRIAL REGISTRATION: ClinicalTrials.gov identifier, NCT04492878.

Analysis of the mucosal chemokines CCL28, CXCL14, and CXCL17 in dry eye disease: An in vitro and clinical investigation.

Mucosal chemokines have antimicrobial properties and play an important role in mucosal immunity. However, little is known about their expression on the ocular surface. This study aimed to analyze the expression of the mucosal chemokines CCL28, CXCL14 and CXCL17 in corneal and conjunctival epithelial cells under in vitro dry eye (DE) conditions, and in conjunctival samples from healthy subjects and DE patients. Human corneal epithelial cells (HCE) and immortalized human conjunctival epithelial cells (IM-HConEpiC) were incubated under hyperosmolar (400-500 mOsM) or inflammatory (TNF-α 25 ng/mL) conditions for 6 h and 24 h to measure CCL28, CXCL14, and CXCL17 gene expression by RT-PCR and their secretion by immunobead-based analysis (CCL28, CXCL14) and ELISA (CXCL17). Additionally, twenty-seven DE patients and 13 healthy subjects were included in this study. DE-related questionnaires (OSDI, mSIDEQ and NRS) evaluated symptomatology. Ocular surface integrity was assessed using vital staining. Tactile sensitivity was measured with Cochet-Bonnet esthesiometer, and mechanic and thermal (heat and cold) sensitivity using Belmonte's non-contact esthesiometer. Subbasal nerve plexus and dendritic cell density were analyzed by in vivo confocal microscopy. Conjunctival cells from participants were collected by impression cytology to measure mucosal chemokines gene expression by RT-PCR. Our results showed that HCE and IM-HConEpiC cells increased CCL28, CXCL14, and CXCL17 secretion under hyperosmolar conditions. The gene expression of CCL28 was significantly upregulated in conjunctival samples from DE patients. CCL28 expression correlated positively with symptomatology, corneal staining, heat sensitivity threshold, and dendritic cell density. CXCL14 expression correlated positively with age, ocular pain, conjunctival staining, tactile sensitivity, and image reflectivity. CXCL17 expression correlated positively with corneal staining. These results suggest that corneal and conjunctival epithelial cells could be a source of CCL28, CXCL14, and CXCL17 on the ocular surface and that CCL28 might be involved in DE pathogenesis.

Influence of controlled environmental conditions in potential salivary ocular pain biomarkers for enhancing the assessment of ocular pain.

PURPOSE: We endeavored to identify objective salivary biomarkers for pain, a subjective sensation with a biological basis, using molecules already described related to pain. The study aimed to analyze inter-individual differences and intersession variability in salivary potential ocular pain biomarkers on healthy subjects, in samples obtained under the influence of controlled environmental conditions. METHODS: Thirty-four healthy subjects, 20 male, 14 female, median age 35.44 years (range 30-40) were exposed for 30 minutes under standard environmental conditions (T: 22°C, 50% relative humidity) in the Controlled Environmental Research Laboratory (CE-Lab, Vision R&D, Valladolid Spain) in two separate visits (V1, V2) at least 24 hours apart. Saliva was collected after the exposure in each of the visits, and cortisol, α-amylase (sAA), secretory IgA (sIgA), testosterone, and soluble fraction of TNFα receptor II (sTNFαRII) were analyzed by ELISA. Repeatability of inter-subject inter-session measurements was assayed by intraclass correlation coefficient (ICC). RESULTS: There were no significant inter-session differences in testosterone (p = 0.2497), sTNFαRII (p = 0.6451) and sIgA (p = 0.9689) salivary levels. The reproducibility for salivary cortisol, sAA, testosterone, sTNFαRII and sIgA were 0.98 ng/ml, 20.58 U/ml, 21.07 µg/ml, 24.68 pg/ml and 0.19 pg/ml, respectively. Salivary cortisol, sAA, testosterone, sTNFαRII and sIgA yielded the following ICCs: 0.506, 0.569, 0.824, 0.870 and 0.4295, respectively; all these ICCs (except that for cortisol and sIgA) were found to be improved compared to those found previously by our group in a previous study in salivary samples obtained from healthy subjects under non-controlled environmental conditions; Cortisol´s ICC didn´t improve and was in both cases at the limit of acceptability. CONCLUSION: Environmental factors such as temperature and relative humidity affect the reproducibility of measurement of some salivary molecules which have been proposed as potential pain biomarkers. The exposure of subjects to standard controlled environmental conditions before salivary sample obtention would improve the reproducibility of these molecule measures' as potential biomarkers of chronic ocular pain.

Gene expression changes in conjunctival cells associated with contact lens wear and discomfort.

PURPOSE: This study aimed to analyze the differences in the expression of pain-related genes in conjunctival epithelial cells among symptomatic contact lens (CL) wearers (SCLWs), asymptomatic CL wearers (ACLWs), and non-CL wearers (non-CLWs). METHODS: For this study, 60 participants (20 non-CLWs, 40 CLWs) were enrolled. The CLW group comprised 20 ACLWs and 20 SCLWs according to the Contact Lens Dry Eye Questionnaire short form©. Conjunctival cells were collected using impression cytology, and RNA was isolated and used to determine the expression levels of 85 human genes involved in neuropathic and inflammatory pain. The effects of CL wear and discomfort were evaluated using mixed-effects ANOVA with partially nested fixed-effects model. Gene set enrichment analysis was performed to assign biological meaning to sets of differentially expressed genes. RESULTS: Six genes (CD200, EDN1, GRIN1, PTGS1, P2RX7, and TNF) were significantly upregulated in CLWs compared to non-CLWs. Eleven genes (ADORA1, BDKRB1, CACNA1B, DBH, GRIN1, GRM1, HTR1A, PDYN, PTGS1, P2RX3, and TNF) were downregulated in SCLWs compared to ACLWs. These genes were mainly related to pain, synaptic transmission and signaling, ion transport, calcium transport and concentration, and cell-cell signaling. CONCLUSIONS: CL wear modified the expression of pain- and inflammation-related genes in conjunctival epithelial cells. These changes may be in part, along with other mechanisms, responsible for CL discomfort in SCLWs.

Tear and Plasma Levels of Cytokines in Patients with Uveitis: Search for Active Disease Biomarkers.

Uveitis accounts for up to 20% of blindness in Europe, making the development of new non-invasive biomarkers which could help in its management a field of interest. It has been hypothesised that tear levels of cytokines and chemokines could be used as a potential biomarker in patients with anterior uveitis, and this could be correlated with their concentration in plasma. Therefore, we measured twelve cytokines/chemokines in tear and plasma samples of 22 patients diagnosed with active anterior uveitis. Levels of these molecules in tears and plasma were compared and associated with the degree of activity of the uveitis. It is notable that the percentage of tear interleukin (IL)-6 detection was significantly reduced in the inactive phase (p < 0.05). However, the tear concentration in epidermal growth factor (EGF), fractalkine, IL-8, IL-1RA, interferon-inducible protein (IP)-10/CXCL10, vascular endothelial growth factor (VEGF) and IL-6, comparing the active and inactive period, was not statistically different. Apart from the tear VEGF levels, the cytokine/chemokine concentration in tears in the active/inactive phase was statistically different (p < 0.05) from the counterpart levels in plasma. In conclusion, no isolated cytokine/chemokine in the tears has been found in a concentration which could be used as a potential biomarker of disease activity and treatment response.

Effect of a single vectored thermal pulsation treatment of Meibomian gland dysfunction patients under controlled environmental conditions.

To assess the prophylactic effect of LipiFlow treatment in Meibomian gland dysfunction (MGD) patients exposed to an adverse environmental humidity. MGD patients were exposed to normal (23 °C; 50% relative humidity; 30 min) and adverse (23 °C; 10% relative humidity; 2 h) controlled environments consecutively during baseline and follow-up visits (3, 6, and 12 months) after a single LipiFlow treatment. Ocular Surface Disease Index (OSDI), lipid layer thickness (LLT), fluorescein tear break-up time (TBUT), corneal and conjunctival staining, change in dry eye symptoms questionnaire (CDES-Q), and Meibomian gland yielding liquid secretion (MGYLS), were assessed. Linear mixed-effects and cumulative logit mixed models were fitted to assess the effect of the LipiFlow treatment over time and within the controlled environments. Seventeen females and 4 males (59.6 ± 9.4 years) completed the study. LLT and TBUT did not vary significantly (p > 0.05) after LipiFlow treatment. OSDI, corneal and conjunctival staining, and MGYLS scores were improved (p ≤ 0.01) 12 months after treatment. After the adverse exposure, corneal staining increased at all visits (p = 0.01), and there was no significant improvement in CDES-Q scores after LipiFlow treatment (p ≥ 0.07). One LipiFlow treatment improved objective and subjective outcomes in MGD disease for at least one year. Further studies are needed to support that LipiFlow might also help as an adjuvant to avoid acute flares against an adverse environmental humidity.

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-ß2 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.

Phenotypic characterization of patients developing chronic dry eye and pain after refractive surgery: A cross-sectional study.

PURPOSE: To describe the clinical characteristics of patients suffering from chronic dry eye (DE) and pain after refractive surgery (RS). METHODS: Cross-sectional, observational, single-visit study. DE-, pain- and psychological-related symptoms were evaluated with specific questionnaires. DE-related tests evaluated tear osmolarity, conjunctival hyperemia, Meibomian gland dysfunction, tear stability and production, and ocular surface staining. Corneal mechanical sensitivity (Cochet-Bonnet) was measured pre/post topical anesthesia, and symptomatic variation post-anesthesia (anesthetic challenge test) was recorded. When pain was present, it was further categorized as neuropathic or nociceptive based on published criteria. RESULTS: We recruited 104 patients (39.5 ± 9.5 years). Most, 85.6%, had corneal RS as opposed to intraocular RS. Migraines, anxiety, depression (p < 0.0001), and central sensitization syndromes (p = 0.0214) were more frequent post-RS than pre-RS. Persistent DE-symptoms, severe in 86.5% patients, developed in a range of 0-204 months post-RS. Dryness and pain were the two most frequent symptoms. The only DE-related tests showing abnormal values were tear osmolarity (315.2 ± 17.1 mOsm/L; normal ≤308) and tear break-up time (4.1 ± 2.5 s; normal >7). Corneal sensitivity was 55.4 ± 7.0 mm, and decreased (p < 0.0001) after topical anesthesia, 6.0 ± 10.4 mm. However, it remained pathologically elevated, ≥10 mm in 61 (58.7%) patients. The normal symptomatic post-anesthesia improvement was absent in 58 (55.7%) patients. Ocular pain was present in 82 (78.8%) patients, and it was categorized as neuropathic in 66 (80.5%) of them, 63.5% of the entire cohort. CONCLUSIONS: Chronic ocular pain and its neuropathic subtype were diagnosed in 78.8% and 63.5% respectively of patients seeking consultation for persistent symptomatic DE post-RS.

Olive Pomace Phenolic Compounds: From an Agro-Industrial By-Product to a Promising Ocular Surface Protection for Dry Eye Disease.

Dry eye (DED) is a prevalent disease with immune-mediated inflammation as the principal pathophysiological etiology. Olive pomace, the major by-product of the olive oil industry, is rich in high-value polyphenols. Their anti-inflammatory and immunomodulatory activities were determined on human CD4+ T cells (hTCD4+) and in a DED animal model. The viability of hTCD4+ cells isolated from peripheral blood and activated with phytohemagglutinin-M was evaluated after treatment for 48 h with an olive pomace extract (OPT3, 0.10-0.40 mg/mL) and its major compound, hydroxytyrosol (25-100 µM). Regarding the DED animal model, 100 µM hydroxytyrosol, 0.20 mg/mL OPT3, or vehicle (borate buffer) were topically administered to 14 days-desiccating stress-exposed (constant airflow/scopolamine administration) C57BL/6 mice. Tear volume, corneal fluorescein staining (CFS), CD4+, and CD8+ T cell count in lymph nodes (flow cytometry), and IP-10 and TNF-α gene expression (qRT-PCR) in the cornea, conjunctiva, and lacrimal glands were evaluated. OPT3 (0.2-0.4 mg/mL) and hydroxytyrosol (100 µM) significantly reduced hTCD4+ proliferation. In mice, both treatments reduced lacrimal gland IP-10 gene expression. OPT3 also decreased CFS, and conjunctival IP-10 and corneal TNF-α gene expression. In lymph nodes, hydroxytyrosol reduced CD3+, OPT3, and CD8+ count. Thus, a high-value application as a promising DED protection was proposed for olive pomace.

Diurnal variation on tear stability and correlation with tear cytokine concentration.

PURPOSE: To investigate the effect of time of day on tear evaporation rate (TER) and tear break-up time, and its possible relationship with the concentration of inflammatory tear molecules (cytokines) in healthy subjects. METHODS: Participants with healthy ocular surfaces attended 3 visits, including the screening visit (V0), the 2nd visit (V1) and the 3rd visit (V2). There were 7-day intervals between visits. Participants with Dry Eye Disease (DED) were excluded by using appropriate clinical tests during V0. Clinical evaluation (TER and Non-Invasive Tear Break-Up Time (NITBUT)) and tear collection were performed during V1 and V2, between 9 and 10AM and 3-4PM. The relative humidity and temperature of the examination room were also measured. The tear fluid concentrations of 15 cytokines were measured by multiplex bead analysis. RESULTS: Seven men and 10 women (mean age ± S.D; 25.1 ± 6.63 years old) participated in the study. There were no differences in neither the TER and NITBUT outcomes, nor humidity and temperature among times or visits. Eleven out of the 15 cytokines measured were detectable in tear fluids in > 50% of the participants. In the tear levels, no significant (p > 0.05) inter- and/or intra-day differences were detected for EGF, fractalkine, IL-1RA, IL-1ß and IP-10. However, significant inter-day differences were found in the tear levels of IL-10 (p = 0.027), IFN-γ (p = 0.035) and TNF-α(p = 0.04) and intra-day differences in the tear levels of IL-8/CXCL8 (p = 0.034) and MCP-1 (p = 0.002). A significant correlation between TER and IL1-ß, IL-2, and Fractalkine (p = 0.03, p = 0.03 and p = 0.046, respectively) was found at V1. CONCLUSIONS: NITBUT and TER values had no significant variability over the course of a day (AM versus PM), or on different days in healthy participants when humidity and temperature were constant. However, some tear molecule levels did show inter- and intra-day variability, having an inconsistent and moderate correlation with TER diurnal variation.

Inflammation-related molecules in tears of patients with chronic ocular pain and dry eye disease.

The purpose of this study was to analyze inflammation- and pain-related molecules in tears of patients suffering from chronic ocular pain associated with dry eye (DE) and/or a previous corneal refractive surgery (RS). Based on history, symptomatology, and clinical signs, the subjects (n = 180, 51.0 ± 14.7 years, 118 females, 62 males) in this cross-sectional study were assigned to one of five groups: DE and chronic ocular pain after RS (P/DE-RS, n = 52); asymptomatic subjects, i.e., without DE and chronic ocular pain, after RS (A-RS, n = 30); DE and chronic ocular pain without previous RS (P/DE-nonRS, n = 31); DE, no pain, and no previous RS (DE-nonRS, n = 35); and asymptomatic subjects with no previous RS (controls, n = 32). The tear concentrations of 20 cytokines and substance P (SP) were analyzed by immunobead-based assay and enzyme-linked immunosorbent assay, respectively. We found that tear levels of interleukin (IL)-10 and SP were increased in the RS groups. There were significant differences in IL-8/CXCL8 among the five groups. Nerve growth factor (NGF) tear levels were significantly higher in P/DE-RS than in DE-nonRS and controls. IL-9 had the highest percentage of detection in the P/DE-RS and P/DE-nonRS groups, while macrophage inflammatory protein (MIP)-1α, IL-2, and interferon (IFN)-γ were higher in the P/DE-RS, A-RS, and P/DE-nonRS groups. IL-17A was detected only in the A-RS group. Moderate correlations were observed in the A-RS, P/DE-nonRS, DE-nonRS and controls groups. A positive correlation was obtained between growth related oncogene concentration and tear break-up time (rho = 0.550; p = 0.012), while negative correlation was found between monocyte chemoattractant protein-3/CCL7 and conjunctival staining (rho = -0.560; p = 0.001), both in the A-RS group. IL-10 correlated positively with ocular pain intensity (rho = 0.513; p = 0.003) in the P/DE-nonRS group. Regulated on Activation Normal T Cell Expressed and Secreted/CCL5 correlated negatively with conjunctival staining (rho = -0.545; p = 0.001) in the DE-nonRS group. SP correlated negatively with corneal staining (rho = -0.559; p = 0.001) in the controls. In conclusion, chronic ocular pain was associated with higher IL-9 tear levels. IL-10, SP, MIP-1α/CCL3, IL-2, and IFN-γ were associated with previous RS. Higher levels of IL-8/CXCL8, MIP-1α/CCL3, IL-2, and IFN-γ were associated with DE-related inflammation, while NGF levels were related to chronic ocular pain and DE in RS patients. These findings suggest that improved knowledge of tear cytokines and neuromodulators will lead to a more nuanced understanding of how these molecules can serve as biomarkers of chronic ocular pain, leading to better therapeutic and disease management decisions.

Potential Ophthalmological Application of Extracts Obtained from Tuna Vitreous Humor Using Lactic Acid-Based Deep Eutectic Systems.

A green technique was developed to extract hyaluronic acid (HA) from tuna vitreous humor (TVH) for its potential application in managing dry eye disease. Deep eutectic solvents (DES) were used to extract HA and were synthesized using natural compounds (lactic acid, fructose, and urea). The DES, the soluble fraction of TVH in DES (SF), and the precipitated extracts (PE) were evaluated for their potential use in dry eye disease treatment. In vitro experiments on human corneal epithelial cell lines and the effect on dry eye-associated microorganisms were performed. The influence of the samples on the HCE viability, their intracellular reactive oxygen species (ROS) scavenging capacity, inflammatory response, and antimicrobial properties were studied. According to the results, all samples displayed an antioxidant effect, which was significantly higher for PE in comparison to SF. Most of the tested samples did not induce an inflammatory response in cells, which confirmed the safety in ophthalmic formulations. In addition, the DES and SF proved to be efficient against the studied bacterial strains, while PE did not show an antimicrobial effect. Hence, both DES and SF at defined concentrations could be used as potential compounds in dry eye disease management.

Olive Pomace Phenolic Compounds Stability and Safety Evaluation: From Raw Material to Future Ophthalmic Applications.

Nowadays, increasing interest in olive pomace (OP) valorization aims to improve olive's industry sustainability. Interestingly, several studies propose a high-value application for OP extracts containing its main phenolic compounds, hydroxytyrosol and oleuropein, as therapy for ocular surface diseases. In this work, the stability and accessibility of OP total phenolic and flavonoid content, main representative compounds, and antioxidant activity were assessed under different pretreatment conditions. Among them, lyophilization and supercritical CO2 extraction were found to increase significantly most responses measured in the produced extracts. Two selected extracts (CONV and OPT3) were obtained by different techniques (conventional and pressurized liquid extraction); Their aqueous solutions were characterized by HPLC-DAD-MS/MS. Additionally, their safety and stability were evaluated according to EMA requirements towards their approval as ophthalmic products: their genotoxic effect on ocular surface cells and their 6-months storage stability at 4 different temperature/moisture conditions (CPMP/ICH/2736/99), together with pure hydroxytyrosol and oleuropein solutions. The concentration of hydroxytyrosol and oleuropein in pure or extract solutions was tracked, and possible degradation products were putatively identified by HPLC-DAD-MS/MS. Hydroxytyrosol and oleuropein had different stability as standard or extract solutions, with oleuropein also showing different degradation profile. All compounds/extracts were safe for ophthalmic use at the concentrations tested.

Ocular Surface Pathology in Patients Suffering from Mercury Intoxication.

PURPOSE: To report the ocular surface pathology of patients suffering from acute/subacute mercury vapor intoxication. DESIGN: Cross-sectional study. PARTICIPANTS: Male workers intoxicated with inorganic mercury referred for ophthalmic involvement and healthy control subjects. METHODS: The following tests were performed: dry eye (DE)-related symptoms indicated by the ocular surface disease (OSDI) index questionnaire; tear osmolarity; analysis of 23 tear cytokine concentrations and principal component and hierarchical agglomerative cluster analyses; tear break-up time (T-BUT); corneal fluorescein and conjunctival lissamine green staining; tear production by Schirmer and tear lysozyme tests; mechanical and thermal corneal sensitivity (non-contact esthesiometry); and corneal nerve analysis and dendritic cell density by in vivo confocal microscopy (IVCM). RESULTS: Twenty-two out of 29 evaluated patients entered the study. Most had DE-related symptoms (OSDI values > 12), that were severe in 63.6% of them. Tear osmolarity was elevated (>308 mOsms/L) in 83.4% of patients (mean 336.23 (28.71) mOsm/L). Corneal and conjunctival staining were unremarkable. T-BUT was low (<7 s) in 22.7% of patients. Schirmer test and tear lysozyme concentration were low in 13.6% and 27.3% of cases, respectively. Corneal esthesiometry showed patient mechanical (mean 147.81 (53.36) mL/min) and thermal thresholds to heat (+2.35 (+1.10) °C) and cold (-2.57 (-1.24) °C) to be significantly higher than controls. Corneal IVCM revealed lower values for nerve density (6.4 (2.94) n/mm2), nerve branching density (2 (2.50) n/mm2), and dendritic cell density (9.1 (8.84) n/mm2) in patients. Tear levels of IL-12p70, IL-6, RANTES, and VEGF were increased, whereas EGF and IP-10/CXCL10 were decreased compared to controls. Based on cytokine levels, two clusters of patients were identified. Compared to Cluster 1, Cluster 2 patients had significantly increased tear levels of 18 cytokines, decreased tear lysozyme, lower nerve branching density, fewer dendritic cells, and higher urine mercury levels. CONCLUSIONS: Patients suffering from systemic mercury intoxication showed symptoms and signs of ocular surface pathology, mainly by targeting the trigeminal nerve, as shown by alterations in corneal sensitivity and sub-basal nerve morphology.

Olive Pomace Phenolic Compounds and Extracts Can Inhibit Inflammatory- and Oxidative-Related Diseases of Human Ocular Surface Epithelium.

Oxidative- and inflammatory-related ocular surface diseases have high prevalence and are an emerging issue in ophthalmology. Olive pomace (OP) is the olive oil's industry main by-product, and is potentially environmentally hazardous. Nevertheless, it contains phenolic compounds with important bioactivities, like oleuropein (OL) and hydroxytyrosol (HT). The antioxidant and anti-inflammatory effects of four OP extracts (CONV, OPT(1-3)), pure OL and HT, and mixtures thereof were screened on human corneal (HCE) and conjunctival epithelial (IM-ConjEpi) cells. CONV was conventionally extracted, while OPT(1-3) were produced by pressurized liquid extraction. Thanks to their improved activity, CONV and OPT3 (HT-enriched) were selected for dose-dependent studies. Cells were stimulated with tumor necrosis factor-α or ultraviolet-B radiation, measuring interleukin (IL)-1ß, IL-6, IL-8, and IL-17A as well as interferon γ-induced protein [IP]-10 secretion or intracellular ROS production, respectively. On HCE, both extracts and HT inhibited the secretion of most measured ILs, demonstrating a strong anti-inflammatory effect; while in IM-ConjEpi, all samples decreased IP-10 secretion. Moreover, HT, OL, and both extracts showed strong dose-dependent antioxidant activity in both cell lines. Compared with CONV, OPT3 was active at lower concentrations, demonstrating that intensified extraction techniques are selective towards targeted biomarkers. Hence, a high-value application as potential ocular surface therapy was proposed for the OP valorization.

Development of a Questionnaire for Detecting Changes in Dry Eye Disease-Related Symptoms.

OBJECTIVES: Determining the changes in symptomatology suffered by dry eye disease (DED) patients after an intervention is difficult because there is only one validated questionnaire specifically designed to measure these changes and it is somewhat complex. This work uses a simplified questionnaire to evaluate the changes in DED-related symptoms. METHODS: A new questionnaire based on a global rating of change scale was designed. The Change in Dry Eye Symptoms Questionnaire (CDES-Q) consists of 2 questions: CDES-Q1 asks for the change in symptoms ("better," "same," or "worse") relative to a determined previous time and CDES-Q2 quantifies this change (range: 0 to +100). To evaluate the CDES-Q, a prospective observational study was performed. At baseline (V1; day-0), DED-related symptoms were evaluated using the ocular surface disease index (OSDI). In the post-treatment visit (V2; day-90), OSDI, Symptoms Assessment Questionnaire in Dry Eye (SANDE) II, and CDES-Q were used. Also, clinical evaluations were performed in each visit. RESULTS: Thirty-six patients were included. At V2, OSDI, SANDE II, and CDES-Q showed a significant reduction in symptoms (-7.17±12.73, P=0.0021; -11.29±20.95, P=0.0035; -25.28±42.28, P=0.0011, respectively). Patients who answered "better" in CDES-Q1 showed a significantly lower SANDE II than those who answered "same" or "worse," while SANDE II did not discriminate between these groups. CONCLUSIONS: CDES-Q can be a useful tool for the evaluation of changes in DED-related symptoms. It is simple and better discriminates patients without changes from those who suffered a worsening than SANDE II.

Conjunctival Neuropathic and Inflammatory Pain-Related Gene Expression with Contact Lens Wear and Discomfort.

Purpose: To identify alterations in neuropathic and inflammatory pain gene expression associated with contact lens (CL) wear and CL discomfort (CLD).Methods: Eight non-wearers, eight asymptomatic CL wearers (CLWs) and eight symptomatic CLWs were included. Conjunctival cells were collected by impression cytology and the mRNA expression levels of 85 genes were analyzed. Differentially expressed genes between non-wearers and CLWs and between asymptomatic and symptomatic CLWs were analyzed. An enrichment analysis was also performed.Results: Twelve genes were upregulated (including IL10, PDYN and PENK) and 28 downregulated (CCL2, IL1A, IL1B, IL2 and NGF) in CLWs (p ≤ 0.050). Eleven genes were upregulated (CCL2, IL1A, IL1B, IL2 and NGF) and nine downregulated (PDYN and PENK) in symptomatic CLWs (p ≤ 0.035). Enriched overrepresented terms were related to pain, neuronal transmission and inflammation.Conclusion: Contact lens wear might produce a desensitization-like mechanism responsible for comfortable CL wear. A malfunction of this mechanism might contribute to CLD.

Substance P Level in Tears as a Potential Biomarker for Contact Lens Discomfort.

Purpose: To analyze the effects of contact lens (CL) wear, time of the day, and CL discomfort (CLD) on clinical signs, tear inflammatory mediators and substance P.Methods: Thirty symptomatic and 30 asymptomatic CL wearers attended two visits (morning and afternoon) on two days (non-CL and CL wearing days). Comfort, meniscus area, noninvasive breakup time (NIBUT), tear collection, hyperemia, lid parallel conjunctival folds, fluorescein staining, and sensitivity were performed. The tear levels of 23 inflammatory mediators and substance P were measured.Results: Comfort, meniscus area, NIBUT, and MMP-9 were lower while conjunctival staining and EGF higher (p ≤ 0.015) on the CL wearing day. Comfort, IL-8/CXCL8, and VEGF were lower while EGF, IP-10/CXCL10, and MCP-1/CCL2 higher (p ≤ 0.047) in the afternoon. Comfort was lower and substance P higher (p ≤ 0.006) in symptomatic wearers.Conclusion: Substance P may be implicated in CLD etiology; its role and potential application as a biomarker should be further studied.

Inflammatory status predicts contact lens discomfort under adverse environmental conditions.

PURPOSE: To characterize and predict the clinical and tear molecular response of contact lens (CL) wearers exposed to a controlled adverse desiccating environment (CADE). METHODS: Objective and subjective variables and tear cytokine levels were evaluated of monthly silicone hydrogel CL wearers pre- and post-90 min of CADE exposure. Unsupervised hierarchical agglomerative clustering based on relative change from baseline values was used to identify response profiles (clusters). A multiple logistic regression model was used to identify cluster membership predictors. RESULTS: Forty-seven CL wearers were divided into 3 clusters having similar age (mean: 27.7 ± 7.7 years) and sex distribution. All of them showed a significant (p ≤ 0.05) increase in limbal hyperemia and staining after CADE exposure. Additionally, Cluster-1 (n = 22, 46.8%) membership was characterized by a significant (p ≤ 0.05) higher worsening of corneal and limbal staining, increased CL wear symptoms, and reduced epidermal-growth-factor and increased interleukin (IL)-4 and IL-6 tear levels. Cluster-2 (n = 22, 46.8%) showed no changes (p > 0.05) in symptoms after CADE; however, their IL-12p70, monocyte-chemoattractant-protein-1 and regulated-on-activation, normal-T-cell-expressed-and-secreted (RANTES) post-exposure tear levels significantly (p ≤ 0.05) increased. Finally, Cluster-3 (n = 3, 6.4%) mainly showed significant higher blink rate (78.1 ± 21.7) during CADE. Corneal staining and tear IL-12p70 levels were identified as Cluster-1 membership predictors. CONCLUSIONS: Most of silicone hydrogel CL wearers exposed to CADE showed a worsening of the ocular surface integrity and an upregulated tear inflammatory status. However, only half of them reported worsening of CL wear symptoms. These CL wearers were detected based on corneal integrity and tear inflammatory status. These findings can help reduce CL wear discontinuation and drop out.

Clinical and tear cytokine profiles after advanced surface ablation refractive surgery: A six-month follow-up.

Neuropathic dry eye is one of the most frequently seen complications after corneal refractive surgery, however, its incidence decreases in a significant manner along the first six months postoperative, reaching between 10 and 45% incidence. However, little is known on the inflammatory status of the ocular surface during this recovery process. We aim to analyze the clinical and tear molecule concentration changes along six months after advanced surface ablation for myopia correction, in a prospective study including 18 eyes of 18 subjects who bilaterally underwent advanced surface ablation corneal refractive surgery. Clinical variables (uncorrected distance visual acuity, symptoms, conjunctival hyperemia, tear osmolarity, tear stability, corneal fluorescein staining, conjunctival lissamine staining, Schirmer test, and corneal esthesiometry) and a panel of 23 pro and anti-inflammatory cytokines/chemokines concentration in tears preoperatively and at 1, 3 and 6 months postoperatively were evaluated. We found that uncorrected distance visual acuity improved significantly from baseline at 1-month visit, symptoms improved and tear osmolarity decreased significantly from baseline at 3-month visit and there was a decrease in mechanical corneal threshold between 1-month and 3- and 6-month visits. Regarding tear molecules, IL-4, IL-5, IL-6, IL-13, IL-17A, and IFN-γ tear levels were significantly increased at all the three visits, compared to preoperative levels at V0; IL-2 and VEGF were also significantly increased at 1-month and 6-month visits, but not at 3-month visit, whereas IL-9 IL-10 and IL-12 were only significantly increased at 6-month visit. Although we found that there is a recovery in clinical variables at 6 months postoperatively (i.e. neuropathic dry eye was not developed in the sample), ocular surface homeostasis is not completely restored, as it can be seen by the changes in concentration of some pro and anti-inflammatory molecules measured in tears.