Tear Cytokines Research Articles

Tear cytokine levels in Sjogren's syndrome-related dry eye disease compared with non-Sjogren's syndrome-related dry eye disease patients: A meta-analysis.

As a common complication of Sjogren syndrome (SS), SS-related dry eye disease (SS-DED) significantly affects the patients' quality of life. Pro-inflammatory cytokines in tears are widely believed to play a crucial role in the pathogenesis of SS-DED. A systematic literature review with meta-analyses was conducted to provide a quantitative summary of tear cytokine levels in SS-DED compared with non-SS-DED and healthy controls. The PubMed, Embase, Web of Science, Ovid, and Scopus databases were searched until June 2022. Original case-control studies investigating tear cytokines in SS-DED patients compared with non-SS-DED or healthy individuals were included. Differences of cytokines levels were compared with random-effects standardized mean differences ± 95% confidence intervals calculated as the effect size. A total of 15 articles, 809 subjects (302 for SS-DED, 220 for non-SS-DED, and 287 for healthy controls) were included in the study. SS-DED patients had higher tear levels of interferon-gamma, interleukin (IL)-17, IL-1β, IL-2, IL-4, IL-6, and IL-8 as compared to healthy controls. As for comparison between SS-DED and non-SS-DED group, the levels of IL-12p70, IL-17, IL-4, IL-6, IL-8, and tumor necrosis factor-alpha were significantly higher in SS-DED patients compared with the non-SS-DED group. The level of epidermal growth factor was significantly lower in SS-DED patients compared with both non-SS-DED patients and healthy controls. The findings from this study provide evidence for levels of tear cytokines in SS-DED to become potential diagnostic biomarkers or therapeutic targets. Further studies with a higher number of subjects and improved quality are necessary.

Efficacy of 0.05% Cyclosporine-A eye drops (II) and 3% Diquafosol ophthalmic solution in the treatment of dry eye after cataract surgery.

To compare the efficacy of 0.05% cyclosporine A (CsA) eye drops (II) and 3% Diquafosol ophthalmic solution (DQS) in the treatment of dry eye (DE) after cataract surgery (CS). Clinical data of 123 patients with DE after CS treated at the Fourth Hospital of Hebei Medical University from June 2022 to August 2023 were retrospectively analyzed. Patients were divided into three groups based on the treatment: Conventional group (tobramycin & dexamethasone eye drops combined with pranoprofen eye drops, n=41), DQS group (3% DQS based on the conventional treatment, n=42), and CsA group (0.05% CsA eye drops(II) based on the conventional treatment, n=40). The therapeutic effects; Schirmer I test (SIt), tear film breakup time (TBUT), levels of serum inflammatory factors, and tear cytokine levels before and after treatment were compared between the groups. Chief complaint score, conjunctival congestion score, corneal fluorescein staining score, and ocular surface disease index score of the DQS group and the CsA groups were significantly lower than those of the conventional group (P<0.05). After treatment, the improvement in SIt, TBUT, serum inflammatory factors, and tear cytokine levels in the DQS group and the CsA group was significantly better than that in the conventional group (P<0.05). However, these indexes were comparable in the DQS and the CsA group (P>0.05). Compared to the conventional treatment alone, the addition of 3% DQS or 0.05% CsA eye drops (II) to the conventional treatment both are effective and might more effectively alleviate DE in patients undergoing CS.

CD4+ T cells drive corneal nerve damage but not epitheliopathy in an acute aqueous-deficient dry eye model

Dry eye disease (DED) is characterized by a dysfunctional tear film in which the corneal epithelium and its abundant nerves are affected by ocular desiccation and inflammation. Although adaptive immunity and specifically CD4+ T cells play a role in DED pathogenesis, the exact contribution of these cells to corneal epithelial and neural damage remains undetermined. To address this, we explored the progression of a surgical DED model in wild-type (WT) and T cell-deficient mice. We observed that adaptive immune-deficient mice developed all aspects of DED comparably to WT mice except for the absence of functional and morphological corneal nerve changes, nerve damage-associated transcriptomic signature in the trigeminal ganglia, and sustained tear cytokine levels. Adoptive transfer of CD4+ T cells from WT DED mice to T cell-deficient mice reproduced corneal nerve damage but not epitheliopathy. Conversely, T cell-deficient mice reconstituted solely with naïve CD4+ T cells developed corneal nerve impairment and epitheliopathy upon DED induction, thus replicating the WT DED phenotype. Collectively, our data show that while corneal neuropathy is driven by CD4+ T cells in DED, corneal epithelial damage develops independently of the adaptive immune response. These findings have implications for T cell-targeting therapies currently in use for DED.

Evaluation of ocular surface inflammation and systemic conditions in patients with systemic lupus erythematosus: a cross-sectional study

ObjectiveThe cross-sectional study was designed to evaluate the association of ocular surface inflammation with systemic conditions in patients with systemic lupus erythematosus (SLE).MethodsThe study enrolled 30 SLE patients and 30 controls. Ocular symptoms were evaluated using the Ocular Surface Disease Index (OSDI) questionnaire. Tear samples from all participants were collected for tear multi-cytokine and chemokine concentration analysis. All participants were assessed for dry eye disease (DED), including Schirmer I test, tear break-up time (TBUT), corneal fluorescein staining (CFS), meibomian gland secretion (MGS), lid-parallel conjunctival folds (LIPCOF), corneal clarity, and symblepharon. Besides, all participants were also examined for conjunctival impression cytology to measure the density of conjunctival goblet cells (CGCs). The peripheral blood indicators from SLE patients were also collected to measure the SLE-associated autoantibody specificities and systemic inflammatory indicators. Pearson and Spearman’s analysis were uesd to examine the correlation between tear cytokines, CGCs, DED-related indicators, and systemic conditions.ResultsThe two groups were matched for age and gender in this study. 36.67% of eyes (11 in 30) of SLE patients and 13.33% of eyes (4 in 30) of controls were diagnosed with DED. OSDI scores, abnormal TBUT percentages, CFS percentages, and DED grading were all higher in SLE patients than in control group, while density of CGCs was lower. There were no significant differences in Schirmer I test, MGS, LIPCOF, corneal clarity, and symblepharon between SLE patients and controls. The levels of tear chemokine (C-X-C motif) ligand 11 (CXCL11) and cytokine interleukin-7 (IL-7) in patients with SLE were significantly higher than those in control group. Moreover, among SLE patients, the severity of DED and the level of tear chemokine CXCL11 were significantly positively correlated with SLE-associated autoantibody specificities.ConclusionDry eye and tear cytokines and chemokines-mediated ocular surface inflammation persist in SLE patients and are associated with systemic conditions. Therefore, it is necessary for patients with SLE to combine systemic and ocular assessments.

Ocular tear fluid biomarkers collected by contact lenses

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

Longitudinal Analysis of Tear Cytokine Ratios in Association with Dry Eye Signs and Symptoms in the Dry Eye Assessment and Management (DREAM) Study

Purpose To extend cross-sectional data on cytokine ratios (CRs) in dry eye disease (DED) signs by investigating longitudinal change in pro- to anti-inflammatory CRs and associations with change in DED signs and symptoms. Methods Secondary analysis of fifty-four subjects [mean age 57.3 (SD 13.2) years, 85.2% female; 68.5% white] with ≥ 4 uL pooled tear volumes at months 0, 6, and 12. Pro-inflammatory (IL-1b, IL-6, IL-8, IL-17A, IFN-g, and TNF-a) to anti-inflammatory (IL-6, IL-10) cytokine ratios (CR) were calculated. DED signs (corneal and conjunctival staining scores, tear break-up time, Schirmer test, Meibomian gland plugging, tear osmolarity, composite sign severity score) and symptoms [Ocular Surface Disease Index (OSDI)] were measured. Changes over time in DED signs, symptoms, and CRs were evaluated using longitudinal models. Correlations between changes in CR and changes in DED signs and symptoms were evaluated using Spearman correlation coefficients (rho). Results DED signs which improved over time (p < 0.001) included corneal and conjunctival staining score, tear break-up time, tear osmolarity, and composite sign severity score. Using IL-10 as anti-inflammatory, changes in corneal and conjunctival staining and composite severity score significantly correlated with changes in pro- to anti-inflammatory CRs from month 0 to 6 (|rho|: 0.29-0.45, p: 0.003-0.04) but not between month 0 to 12 (|rho|: 0.01 to 0.24, all p > 0.08). DED symptoms decreased across one year (p < = 0.001) for all OSDI measures; these changes did not correlate with changes in CRs (|rho|: 0.00 to 0.29, all p > 0.04). Conclusions Improvement in some DED signs across one year correlated weakly with decreases in pro- to anti-inflammatory CRs, in alignment with the understanding of DED as inflammatory. CRs may provide greater insight than absolute tear cytokine concentrations as possible DED biomarkers. Additional studies that provide standardized clinical information and tear collection and analysis are needed to validate CRs in DED.

Relationship of Inflammatory Mediators (Interleukin and Cortisol Concentrations) with Corneal Epithelial Quantifiable Metrics

PurposeTo investigate the relationship of inflammatory biomarkers with corneal epithelial quantifiable metrics in patients with keratoconus and in healthy eyes. DesignProspective observational comparative study. ParticipantsThis study included 100 eyes of 100 patients, 48 eyes of 48 patients with KC, and 52 healthy eyes of 52 healthy controls. MethodsThe concentrations of tear cytokines were investigated in both groups: interleukin (IL) 1B, IL6, IL8, IL10, IL12p70 and TNFα were obtained by capillary flow and measured using flow cytometer. Cortisol concentrations were determined in both groups from the most proximal hair segment as an index of cumulative secretion and measured by liquid chromatography mass spectrometry. Epithelial variables were obtained with optical coherence tomography (OCT). Pearson correlation (r) was used to measure linear dependence between two different variables. Main Outcome MeasuresInvestigating the existence, strength, and significance of any correlation between inflammatory biomarkers (IL1B, IL6, IL8, IL10, IL12p70, TNFα and hair cortisol concentration) and OCT corneal epithelial quantifiable variables such as minimum and maximum epithelial thickness of the map, difference between the minimum and maximum (Epithelial Min-Max) and standard deviation of the epithelial thickness of the map (Epithelial Std Dev) and average epithelial thickness of the superior and inferior regions of the map. ResultsEyes with keratoconus presented statistically significantly higher levels of IL1b (p=0.02), IL6 (p <0.0001), IL8 (p <0.0001), and TNFα (p <0.0001) and hair cortisol concentration (p=0.01) compared to healthy controls.There was a significant correlation between IL6 and measurement Epithelium Min-Max [Pearson= -0.59 (-0.69, -0.47); p<0.0001] and Epithelial standard deviation (Std Dev) [Pearson= +0.56 (0.44, 0.67); p<0.0001]. There was a significant correlation between hair cortisol concentration and Epithelium Min-Max [Pearson=-0.27 (-0.42, -0.1); p<0.0001] and Epithelium Std Dev groups [Pearson= +0.2 (0.03, 0.36); p=0.021]. There was also a significant correlation between TNFα and Epithelial Max. [Pearson= -0.37 (-0.55, 0.17); p<0.0001]. We found no significant correlation between the concentration of IL1b, IL8, IL10 and IL2p70 with any epithelium parameters. ConclusionsThe higher concentration of inflammatory markers (IL6 and hair cortisol) in eyes with keratoconus present a significant correlation with OCT metrics identifying epithelial variability, such as Epithelial Min-Max and Std Dev. These findings demonstrate the role of chronic inflammation in eyes with keratoconus, and that these epithelial changes detectable with OCT are sensitive to this inflammatory process.

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.

Tear cytokines as potential markers of inflammation in feline conjunctivitis.

Conjunctivitis is a prevalent feline ocular surface disorder, often accompanied by inflammation. Inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), play a crucial role in the pathogenesis of conjunctival inflammation. This study aimed to evaluate the levels of TNF-α and IL-6 in the tears of cats with conjunctivitis and compare them with healthy controls, thereby enhancing our understanding of the inflammatory processes in feline conjunctivitis. Tear samples were collected from cats of various breeds diagnosed with conjunctivitis (n = 15) and healthy control cats (n = 5) using Schirmer strips. The levels of TNF-α and IL-6 were measured using the enzyme-linked immunosorbent assay (ELISA) method. Protein concentration were measured using Bradford assay and data were expressed as pg/mg protein of tear sample. Our results revealed a statistically significant increase in the levels of both TNF-α and IL-6 in cats with conjunctivitis compared to the control group (p < .0001). Positive correlation were observed between tear IL-6 (p < .001, r = 0.902) and TNF-α (p < .001, r = 0.919) with clinical grades of conjunctivitis. The results demonstrated a significant elevation in the levels of TNF-α and IL-6 in the tears of cats with conjunctivitis, suggesting that these cytokines are involved in the inflammatory response of feline conjunctivitis. These findings could pave the way for new diagnostic and therapeutic approaches, focusing on cytokine modulation, to manage feline conjunctivitis more effectively.

Ocular involvement in Steven-Johnson syndrome/toxic epidermal necrolysis: recent insights into pathophysiology, biomarkers, and therapeutic strategies.

To review the pathophysiology, recent biomarkers related to the ocular aspects of Steven-Johnson syndrome (SJS)/toxic epidermal necrolysis (TEN), and to highlight notable evidence published in recent years. Several studies reveal the relationship between tear cytokines and the pathological components in eyes of SJS/TEN patients. Specific clinical features and associated risk factors in the acute stage have shown significant correlations with chronic ocular sequelae. Recent treatment protocols, including early pulse systemic and topical steroids, as well as tumor necrosis factor-α inhibitors, have demonstrated positive effects on ocular outcomes. In addition to conventional surgical treatment, a new surgical technique, simple oral mucosal epithelial transplantation (SOMET), has been introduced as a simple ocular surface reconstruction for patient with SJS. Advancements in knowledge and management strategies have notably enhanced ocular outcomes for SJS/TEN eyes. A deeper understanding of the biomarker changes in these eyes could facilitate the development of future targeted treatment options.

Multimodal mucosal and systemic immune characterization of a non-human primate trachoma model highlights the critical role of local immunity during acute phase disease.

Trachoma is a leading cause of infection-related blindness worldwide. This disease is caused by recurrent Chlamydia trachomatis (Ct) infections of the conjunctiva and develops in two phases: i) active (acute trachoma, characterized by follicular conjunctivitis), then long-term: ii) scarring (chronic trachoma, characterized by conjunctival fibrosis, corneal opacification and eyelid malposition). Scarring trachoma is driven by the number and severity of reinfections. The immune system plays a pivotal role in trachoma including exacerbation of the disease. Hence the immune system may also be key to developing a trachoma vaccine. Therefore, we characterized clinical and local immune response kinetics in a non-human primate model of acute conjunctival Ct infection and disease. The conjunctiva of non-human primate (NHP, Cynomolgus monkeys-Macaca fascicularis-) were inoculated with Ct (B/Tunis-864 strain, B serovar). Clinical ocular monitoring was performed using a standardized photographic grading system, and local immune responses were assessed using multi-parameter flow cytometry of conjunctival cells, tear fluid cytokines, immunoglobulins, and Ct quantification. Clinical findings were similar to those observed during acute trachoma in humans, with the development of typical follicular conjunctivitis from the 4th week post-exposure to the 11th week. Immunologic analysis indicated an early phase influx of T cells in the conjunctiva and elevated interleukins 4, 8, and 5, followed by a late phase monocytic influx accompanied with a decrease in other immune cells, and tear fluid cytokines returning to initial levels. Our NHP model accurately reproduces the clinical signs of acute trachoma, allowing for an accurate assessment of the local immune responses in infected eyes. A progressive immune response occurred for weeks after exposure to Ct, which subsided into a persistent innate immune response. An understanding of these local responses is the first step towards using the model to assess new vaccine and therapeutic strategies for disease prevention.

Clinical Characteristic and Tear Film Biomarkers After Myopic FS-LASIK: 1-Year Prospective Follow-up.

To assess the long-term (1-year) effect of myopic femtosecond laser-assisted in situ keratomileusis (FSLASIK) on clinical characteristics and tear film biomarkers. Eighty eyes from 80 patients who underwent FSLASIK were evaluated. Ocular surface symptoms and signs were evaluated using specific questionnaires and tests. The corneal nerves and dendritic cells were examined using in vivo confocal microscopy. Corneal sensitivity was evaluated using a Cochet-Bonnet esthesiometer. Tear inflammatory cytokines and neuropeptides were evaluated using Luminex immunoassay. These examinations were performed preoperatively and at 1, 3, 6, and 12 months postoperatively. Seventy-three participants completed all follow-up visits. Following FS-LASIK, ocular symptoms and signs (except Schirmer I test) worsened at 1 month but corneal and conjunctival stainings improved by 3 months. The numbers of dendritic cells and activated dendritic cells increased at the 3-month postoperative visit and recovered to preoperative levels by the 6-month visit. Ocular symptoms and corneal sensitivity recovered to preoperative levels at the 12-month visit. Tear break-up time and corneal nerve morphology were not recovered to preoperative status at the 12-month visit. Interleukin (IL)-1β, IL-17A, tumor necrosis factor-α, and substance P tear levels significantly increased at all postoperative visits compared to preoperative levels. Corneal staining scores positively correlated with tear IL-1β and IL-17A levels, whereas corneal nerve morphology positively correlated with corneal sensitivity and negatively correlated with substance P levels. Although most clinical variables improved at 12 months postoperatively, some tear inflammatory cytokines and substance P remain altered beyond 12 months, indicating that ocular homeostasis is not completely recovered. [J Refract Surg. 2024;40(8):e508-e519.].

Tear fluid cytokine analysis: a non-invasive approach for assessing retinopathy of prematurity severity.

To determine whether there is a significant association between inflammatory cytokines in the tear fluid and the severity of Retinopathy of Prematurity (ROP). Retrospective cohort study. 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. 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). The levels of CCL2, VEGF, and IFN-γ in tear fluid may serve as useful biomarkers for assessing the severity of ROP.

Tear Cytokines as Predictive Biomarkers of Success in Contact Lens Discomfort Management

ABSTRACT Purpose To analyze changes in tear levels of inflammatory mediators in symptomatic contact lens (CL) wearers after refitting with daily disposable CLs and to identify potential biomarkers of success in CL discomfort (CLD) management. Methods Symptomatic CL wearers (CLDEQ-8 ≥ 12) were refitted (V1) with daily disposable CLs (Delefilcon A). After one month (V2), participants were classified into the post-fitting non-symptomatic (CLDEQ <12) and symptomatic (CLDEQ ≥12) groups. At each visit, the participants were clinically evaluated, tears were collected, and 20 inflammatory mediators and substance P (SP) were measured using multiplex immunobead analysis and ELISA, respectively. The detection rates and concentrations were compared between visits and groups, and logistic regression models were performed. Results Forty-three subjects (32 women/11 men; mean age: 23.2 ± 4.9 years) were enrolled. The IL-1β and IL-9 detection rates were higher at V2 (p ≤ 0.044). The detection rates of IL-1β, IL-9, MIP-1α/CCL3, and MMP-9 at V1 (p ≤ 0.045) and IL-17A at V2 (p ≤ 0.014) were higher in the post-fitting symptomatic group. The tear IL-9 concentration was increased at V2 (p = 0.018). The tear concentrations of fractalkine/CX3CL1, IL-2, IL-6, IL-10, MCP-3/CCL7, MIP-1β, NGF, RANTES/CCL5, and TNF-α were higher in the post-fitting symptomatic group (p ≤ 0.044). Additionally, levels of fractalkine/CX3CL1, IL-2, IL-6, IL-10, RANTES/CCL5, and TNF-α at V1 were significantly associated with the post-fitting grouping (p ≤ 0.044). Conclusions Low tear concentrations of specific inflammatory mediators may be used as a predictive biomarker of success for refitting symptomatic CL wearers with daily disposable CLs. However, complementary treatments might be required for symptomatic CL wearers with higher levels of these inflammatory molecules.

Intravital Imaging of the Human Cornea Reveals the Differential Effects of Season on Innate and Adaptive Immune Cell Morphodynamics

Defining how the invivo immune status of peripheral tissues is shaped by the external environment has remained a technical challenge. We recently developed Functional invivo confocal microscopy (Fun-IVCM) for dynamic, longitudinal imaging of corneal immune cells in living humans. This study investigated the effect of seasonal-driven environmental factors on the morphodynamic features of human corneal immune cell subsets. Longitudinal, observational clinical study. Sixteen healthy participants (aged 18-40 years) attended 2 visits in distinct seasons in Melbourne, Australia (Visit 1, November-December 2021 [spring-summer]; Visit 2, April-June 2022 [autumn-winter]). Environmental data were collected over each period. Participants underwent ocular surface examinations and corneal Fun-IVCM (Heidelberg Engineering). Corneal scans were acquired at 5.5 ± 1.5-minute intervals for up to 5 time points. Time-lapse Fun-IVCM videos were created to analyze corneal immune cells, comprising epithelial T cells and dendritic cells (DCs), and stromal macrophages. Tear cytokines were analyzed using a multiplex bead-based immunoassay. Difference in the density, morphology, and dynamic parameters of corneal immune cell subsets over the study periods. Visit 1 was characterized by higher temperature, lower humidity, and higher air particulate and pollen levels compared with Visit 2. Clinical ocular surface parameters and the density of immune cell subsets were similar across visits. At Visit 1 , corneal epithelial DCs were larger, with a lower dendrite probing speed (0.38 ± 0.21 vs. 0.68 ± 0.33 μm/min; P < 0.001) relative to Visit 2; stromal macrophages were more circular and had less dynamic activity (Visit 1, 7.2 ± 1.9 vs. Visit 2, 10.3 ± 3.7 dancing index; P < 0.001). Corneal T cell morphodynamics were unchanged across periods. Basal tear levels of interleukin 2 and CXCL10 were relatively lower during spring-summer. This study identifies that the invivo morphodynamics of innate corneal immune cells (DCs, macrophages) are modified by environmental factors, but such effects are not evident for adaptive immune cells (T cells). The cornea is a potential invivo window to investigate season-dependent environmental influences on the human immune system. Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Framework Nucleic Acids Combined with 3D Hybridization Chain Reaction Amplifiers for Monitoring Multiple Human Tear Cytokines.

Existing tear sensors are difficult to perform multiplexed assays due to the minute amounts of biomolecules in tears and the tiny volume of tears. Herein, the authors leverage DNA tetrahedral frameworks (DTFs) modified on the wireless portable electrodes to effectively capture 3D hybridization chain reaction (HCR) amplifiers for automatic and sensitive monitoring of multiple cytokines in human tears. The developed sensors allow the sensitive determination of various dry eye syndrome (DES)-associated cytokines in human tears with the limit of detection down to 0.1 pgmL-1, consuming as little as 3 mL of tear fluid. Double-blind testing of clinical DES samples using the developed sensor and commercial ELISA shows no significant difference between them. Compared with single-biomarker diagnosis, the diagnostic accuracy of this sensor based on multiple biomarkers has improved by ≈16%. The developed system offers the potential for tear sensors to enable personalized and accurate diagnosis of various ocular diseases.

Vitamin D and tear fluid cytokines in predicting outcomes in viral conjunctivitis - A new outlook.

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). 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. 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. 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.

The level of cytokines in tears as a novel indicator of demyelinating diseases

ABSTRACT Introduction 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. Aim To compare the level of selected cytokines in tears between patients with MS or other demyelinating disorder and healthy controls. Material and methods 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. Results 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. Conclusions 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.

Is Meibomian Gland Dysfunction One Disease? Heterogeneity Among Phenotypes.

The purpose of this study was to examine ocular surface symptoms, tear metrics, and tear cytokines by Meibomian gland dysfunction (MGD) features. Symptom questionnaires and an ocular surface evaluation were performed on 40 individuals with varied MGD signs (Meibomian gland [MG] plugging, eyelid vascularity, meibum quality, and MG dropout). Tear proteins were extracted off Schirmer strips and analyzed for 23 human inflammation-related proteins. Statistical analysis was performed to examine associations between dry eye metrics inflammatory proteins and MGD features. The study involved 40 South Florida veterans with a mean age of 61 ± 13 years; most individuals were male (95%), White (31%), and non-Hispanic (85%). MGD features differentially related to dry eye signs. Eyelid vascularity, meibum quality, and MG dropout, but not MG plugging, correlated with higher corneal staining and lower tear production. MGD features also differentially related to tear cytokines. Eyelid vascularity most closely related to inflammation with significant correlations for interferon-gamma-γ (r = 0.36, P = 0.02), interleukin-4 (IL-4) (r = 0.43, P = 0.006), IL-17A (r = 0.42, P = 0.007), matrix metalloproteinase-2 (r = 0.39, P = 0.01), C-X-C motif chemokine ligand 5 (Regulated upon Activation, Normal T-Cell Expressed and presumably Secreted [RANTES]) (r = 0.32, P = 0.04), and tumor necrosis factor α (r = 0.36, P = 0.02). The other 3 MGD signs were less related to inflammation. Multivariable models revealed IL-4 to be most closely related to eyelid vascularity (standardized β = 0.39, P < 0.0001). Eyelid vascularity was the MGD sign most closely related to inflammatory cytokines, suggesting that different MGD features may be driven by different pathophysiological mechanisms.

5 Janus Kinase 1/2 inhibition Effect on Cytokine Levels in Tears of Patients with Ocular Graft Versus Host Disease

OBJECTIVES/GOALS: The goal of this study is to identify if ocular graft versus host disease (oGVHD) patients treated with a systemic JAK inhibitor have a change in their tear cytokine profile (a possible bio-marker) and oGVHD score. oGHVD is a severe inflammatory dry eye disease and major cause of morbidity after a hematopoietic stem cell transplant. METHODS/STUDY POPULATION: Janus Kinase (JAK) is an upstream regulator of cytokine production. A JAK 1/2 inhibitor, Ruxolitinib, was recently FDA approved for the treatment of chronic GVHD. We propose that JAK inhibition results incytokine changes in tears and improvement of oGVHD. To study this, we will quantify tear cytokines in patients with oGVHD, with and without systemic JAK inhibition treatment. Patients with ‘definite’ oGVHD based on the international chronic oGVHD diagnostic criteria (ICOGVHD) whom we have collected tears will be grouped based on JAK inhibition treatment. Tear cytokines are analyzed using Iso spark Meteor bulk quantitative proteomic analysis. RESULTS/ANTICIPATED RESULTS: Seven patients were identified from our patient cohort who met inclusion criteria (oGVHD; tears collected while on Ruxolitinib), five patients were identified whom we have collected tears with oGVHD who have not taken ruxolitinib. The following 10 cytokines will be analyzed in the tears by the Iso spark Meteor bulk quantitative proteomic analysis: GM-CSF, IFN-g, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-17A, TNF-a. The change in cytokine levels will be compared with the ICOGVHD score, corneal fluorescein staining, schirmers test (measurement of tear production), conjunctival injection score, ocular surface disease index score (validated symptomatic score of dry eye disease). DISCUSSION/SIGNIFICANCE: OGVHD is a major cause of morbidity for patients who undergo a hematopoietic stem cell transplant and is the result of a highly complex immune process including dysregulation of pro-inflammatory cytokines. It is critical to understand the effect of cytokine changes on the eyes to potentially identify a biomarker and possible treatment targets.