María J. González-García

Dry Eye Disease as an Inflammatory Disorder

Purpose: Dry eye disease (DED) is a prevalent inflammatory disorder of the lacrimal functional unit of multifactorial origin leading to chronic ocular surface disease, impaired quality of vision, and a wide range of complications, eventually causing a reduction in quality of life. It still is a frustrating disease because of the present scarcity of therapies that can reverse, or at least stop, its progression. Methods: A comprehensive literature survey of English-written scientific publications on the role of inflammation in DED. Results: New investigations have demonstrated that a chronic inflammatory response plays a key role in the pathogenesis of human DED. Additionally, correlations between inflammatory molecules and clinical data suggest that inflammation can be responsible for some of the clinical symptoms and signs. Conclusions: Research efforts to clarify its pathophysiology are leading to a better understanding of DED, demonstrating that inflammation, in addition to many other factors, plays a relevant role.

Dry eye exacerbation in patients exposed to desiccating stress under controlled environmental conditions.

PURPOSE To determine if controlled environmental conditions can induce acute exacerbations of signs and symptoms in dry eye and asymptomatic subjects. DESIGN Prospective cross-sectional study. METHODS Nineteen patients with dry eye and 20 asymptomatic controls were exposed to controlled low humidity (5% relative humidity, desiccating environment) for 2 hours in our Controlled Environmental Research Laboratory at the University of Valladolid. The patients completed the Single-Item Score Dry Eye Questionnaire and the following diagnostic tests were performed before and after exposure: tear osmolarity, phenol red thread test, conjunctival hyperemia, fluorescein tear film break-up time, Schirmer test, and ocular surface vital staining. Sixteen molecules in the tears samples were analyzed by multiplex bead analysis. RESULTS After exposure, the patients and controls had a significant (P ≤ .003) increase in corneal staining (from 0.68 ± 0.15 to 1.16 ± 0.14 and from 0.50 ± 0.15 to 1.30 ± 0.19, respectively), significantly decreased (P ≤ .01) fluorescein tear film break-up time values (from 2.78 ± 0.56 seconds to 1.94 ± 0.24 seconds and from 2.81 ± 0.24 seconds to 2.13 ± 0.19 seconds, respectively), and significantly increased (P ≤ .03) matrix metalproteinase 9 tear levels (from 10 054.4 ± 7326.6 pg/mL to 25 744.5 ± 13 212.4 pg/mL and from 10 620.5 ± 4494.3 pg/mL to 16 398.7 ± 5538.3 pg/mL, respectively). In the control group, the epidermal growth factor tear levels decreased significantly (P = .007; from 1872.1 ± 340.7 pg/mL to 1107.1 ± 173.6 pg/mL), and interleukin 6 levels increased significantly (P < .001; from 29.6 ± 5.8 pg/mL to 54.3 ± 8.3 pg/mL) after exposure. CONCLUSIONS Adult patients with mild-to-moderate dry eye and asymptomatic subjects of similar ages can experience acute exacerbation in an environmental chamber that resembles the sudden worsening that patients with dry eye experience daily.

Influence of a Controlled Environment Simulating an In-Flight Airplane Cabin on Dry Eye Disease

PURPOSE To evaluate symptoms, signs, and the levels of 16 tears inflammatory mediators of dry eye (DE) patients exposed to an environment simulating an in-flight air cabin in an environmental chamber. METHODS Twenty DE patients were exposed to controlled environment simulating an in-flight airplane cabin (simulated in-flight condition [SIC]) of 23°C, 5% relative humidity, localized air flow, and 750 millibars (mb) of barometric pressure. As controls, 15 DE patients were subjected to a simulated standard condition (SSC) of 23°C, 45% relative humidity, and 930 mb. A DE symptoms questionnaire, diagnostic tests, and determination of 16 tear molecules by multiplex bead array were performed before and 2 hours after exposure. RESULTS After SIC exposure, DE patients became more symptomatic, suffered a significant (P ≤ 0.05) decrease in tear stability (tear break up time) (from 2.18 ± 0.28 to 1.53 ± 0.20), and tear volume (phenol red thread test), and a significant (P ≤ 0.05) increase in corneal staining, both globally (0.50 ± 0.14 before and 1.25 ± 0.19 after) and in each area (Baylor scale). After SSC, DE patients only showed a mild, but significant (P ≤ 0.05), increase in central and inferior corneal staining. Consistently, tear levels of IL-6 and matrix metalloproteinase (MMP)-9 significantly increased and tear epidermal growth factor (EGF) significantly decreased (P ≤ 0.05) only after SIC. CONCLUSIONS The controlled adverse environment conditions in this environmental chamber can simulate the conditions in which DE patients might be exposed during flight. As this clearly impaired their lacrimal functional unit, it would be advisable that DE patients use therapeutic strategies capable of ameliorating these adverse episodes.

Clinical and Molecular Inflammatory Response in Sjögren Syndrome-Associated Dry Eye Patients Under Desiccating Stress.

PURPOSE To evaluate the response of the lacrimal function unit in Sjögren syndrome (SS)-associated dry eye patients exposed to 2 simulated daily life environmental conditions. DESIGN Prospective crossover pilot study. METHODS Fourteen female SS dry eye patients were exposed for 2 hours to a controlled normal condition (23 C, 45% relative humidity, and air flow 0.10 m/s) and a controlled adverse condition that simulates desiccating stress (23 C, 5% relative humidity, and air flow 0.10 m/s). The following dry eye tests were performed before and after the exposure: tear osmolarity, phenol red thread test, conjunctival hyperemia, fluorescein tear break-up time, corneal fluorescein staining, conjunctival lissamine green staining, and Schirmer test. Levels of 16 molecules were analyzed in tears by multiplex immunobead analysis. RESULTS Clinical evaluation showed lacrimal functional unit impairment after the desiccating stress: significantly increased tear osmolarity (315.7 ± 3.0 vs 327.7 ± 5.1 mOsm/L, P = .03), conjunctival hyperemia (1.3 ± 0.1 vs 1.6 ± 0.1, P = .05), and corneal staining in temporal (3.5 ± 0.5 vs 4.7 ± 0.4, P = .01) and nasal (3.6 ± 0.5 vs 4.5 ± 0.5, P = .04) areas. Tear concentrations increased for interleukin-1 receptor antagonist (16 557.1 ± 4047.8 vs 31 895.3 ± 5916.5 pg/mL, P = .01), interleukin-6 (63.8 ± 20.2 vs 111.5 ± 29.6 pg/mL, P = .02), interleukin-8 (2196.1 ± 737.9 vs 3753.2 ± 1106.0 pg/mL, P = .03), and matrix metalloproteinase-9 (101 515.6 ± 37 088.4 vs 145 867.1 ± 41 651.5 pg/mL, P = .03). After the simulated normal condition, only a significant increase in nasal corneal staining (2.9 ± 0.5 vs 3.6 ± 0.5, P = .03) was observed. CONCLUSIONS Even a short exposure to a desiccating environment can produce a significant deterioration of the lacrimal function unit in female SS dry eye patients. The often unnoticed exposure to these conditions during daily life may increase inflammatory activity rapidly, triggering an ocular surface deterioration.

Intra- and inter-day variation of cytokines and chemokines in tears of healthy subjects

Tear levels of certain cytokines/chemokines can potentially serve as biomarkers for dry eye and other ocular surface diseases if they remain stable from day-to-day in healthy eyes. The aim of this study was to determine the normal intra- and inter-day variation of selected tear cytokines/chemokines. Tear samples from 24 young, healthy adults were collected 11:00 AM-1:00 PM (mid-day) and 5:00-7:00 PM (evening) on three non-consecutive days. Concentrations of 18 cytokines/chemokines (EGF, eotaxin, CX3CL1/fractalkine, GM-CSF, IFN-γ, IL-10, IL-1β, IL-13, IL-17A, IL-1RA, IL-5, IL-6, CXCL8/IL-8, IL-9, CXCL10/IP-10, CCL5/RANTES, TNF-α, and VEGF) were measured by multiplex bead analysis. Ocular surface disease was ruled out by clinical tests. A random-effects ANOVA model was used to evaluate intra- and inter-day effects on cytokine/chemokine levels. Repeatability of intra-subject inter-day measurements was assayed by coefficient of variation. Ten out of the 18 molecules had detectable tear levels in >50% of the subjects. Of those, only IL-10 and IL-1β levels had significant inter-day variations. EGF, CX3CL1/fractalkine, CXCL10/IP-10, and VEGF were consistently higher in the evening compared to the mid-day measurements. EGF, CXCL10/IP-10, VEGF and CXCL8/IL-8had good intra-subject reproducibility. In conclusion, tear cytokines/chemokines can be measured reproducibly over time, with most not having significant inter-day variability. Some varied significantly depending upon the time of tear collection, and these variations should be taken into account when comparisons are made. The good intra-subject reproducibility for EGF, CXCL10/IP-10, CXCL8/IL-8, and VEGF indicates that these molecules could potentially serve as biomarkers of ocular surface disease.

Characterization by Belmonte's gas esthesiometer of mechanical, chemical, and thermal corneal sensitivity thresholds in a normal population.

PURPOSE We used a prototype gas esthesiometer to measure corneal threshold sensitivity values for mechanical, chemical, and thermal stimuli. We also evaluated the reproducibility of the esthesiometer measurements, the influence of previous corneal symptoms, and the safety of this technique. METHODS Forty healthy subjects participated in the study. Mechanical, chemical, and thermal (hot and cold) thresholds were determined at the center of the cornea using a prototype Belmontes gas esthesiometer. To determine reproducibility of the results, the sensitivity thresholds were measured for each eye on 2 days. Corneal fluorescein staining and bulbar hyperemia after completion of the tests were analyzed. RESULTS There were no differences for any sensitivity threshold between eyes or between the first and second esthesiometries. The reproducibilities of mechanical and hot thresholds were higher than for chemical and cold thresholds. Men had significantly higher chemical intensity thresholds than did women (men: 23.50 ± 5.10; women: 10.20 ± 2.16, P = 0.021). There were no alterations of the ocular surface after completion of the measurements. CONCLUSIONS Mechanical, chemical, and thermal corneal sensitivity thresholds in the central cornea have been established in healthy men and women of different age groups. The use of the Belmonte gas esthesiometer is safe and reproducible, with the highest reproducibility in determining mechanical and hot thresholds.

Precision of higher-order aberration measurements with a new Placido-disk topographer and Hartmann-Shack wavefront sensor

Purpose To assess the intrasession and intersession precision of ocular, corneal, and internal higher‐order aberrations (HOAs) measured using an integrated topographer and Hartmann‐Shack wavefront sensor (Topcon KR‐1W) in refractive surgery candidates. Setting IOBA‐Eye Institute, Valladolid, Spain. Design Evaluation of diagnostic technology. Methods To analyze intrasession repeatability, 1 experienced examiner measured eyes 9 times successively. To study intersession reproducibility, the same clinician obtained measurements from another set of eyes in 2 consecutive sessions 1 week apart. Ocular, corneal, and internal HOAs were obtained. Coma and spherical aberrations, 3rd‐ and 4th‐order aberrations, and total HOAs were calculated for a 6.0 mm pupil diameter. Results For intrasession repeatability (75 eyes), excellent intraclass correlation coefficients (ICCs) were obtained (ICC >0.87), except for internal primary coma (ICC = 0.75) and 3rd‐order (ICC = 0.72) HOAs. Repeatability precision (1.96 × Sw) values ranged from 0.03 μm (corneal primary spherical) to 0.08 μm (ocular primary coma). For intersession reproducibility (50 eyes), ICCs were good (>0.8) for ocular primary spherical, 3rd‐order, and total higher‐order aberrations; reproducibility precision values ranged from 0.06 μm (corneal primary spherical) to 0.21 μm (internal 3rd order), with internal HOAs having the lowest precision (≥0.12 μm). No systematic bias was found between examinations on different days. Conclusions The intrasession repeatability was high; therefore, the devices ability to measure HOAs in a reliable way was excellent. Under intersession reproducibility conditions, dependable corneal primary spherical aberrations were provided. Financial Disclosure No author has a financial or proprietary interest in any material or method mentioned.

Biomarkers in Ocular Chronic Graft Versus Host Disease: Tear Cytokine- and Chemokine-Based Predictive Model

PURPOSE To develop a tear molecule level-based predictive model based on a panel of tear cytokines and their correlation with clinical features in ocular chronic graft versus host disease (cGVHD). METHODS Twenty-two ocular cGVHD patients and 21 healthy subjects were evaluated in a controlled environmental research laboratory (CERLab). Clinical parameters were recorded, and tears were collected. Levels of 15 molecules (epidermal growth factor [EGF], IL receptor antagonist [IL-1Ra], IL-1β, IL-2, IL-6, IL-8/CXCL8, IL-10, IL-12p70, IL-17A, interferon inducible protein [IP]-10/CXCL10, IFN-γ, VEGF, TNF-α, eotaxin 1, and regulated on activation normal T cell expressed and secreted [RANTES]) were measured by multiplex-bead assay and correlated with clinical parameters. Logistic regression was used to develop a predictive model. Leave-one-out cross-validation was applied. Classification capacity was evaluated in a cohort of individuals with dry eye (DE) of other etiologies different from GVHD. RESULTS Epidermal growth factor and IP-10/CXCL10 levels were significantly decreased in ocular cGVHD, positively correlating with tear production and stability and negatively correlating with symptoms, hyperemia, and vital staining. Interleukin-1Ra, IL-8/CXCL8, and IL-10 were significantly increased in ocular cGVHD, and the first two correlated positively with symptoms, hyperemia, and ocular surface integrity while negatively correlating with tear production and stability. Predictive models were generated, and the best panel was based on IL-8/CXCL8 and IP-10/CXCL10 tear levels along with age and sex, with an area under the receiving operating curve of 0.9004, sensitivity of 86.36%, and specificity of 95.24%. CONCLUSIONS A predictive model based on tear levels of IL-8/CXCL8 and IP-10/CXCL10 resulted in optimal sensitivity and specificity. These results add further knowledge to the search for potential biomarkers in this devastating ocular inflammatory disease.

Gene Expression-Based Predictive Models of Graft Versus Host Disease-Associated Dry Eye.

PURPOSE To develop a predictive model based on inflammatory gene mRNA expression in conjunctival cells of graft versus host disease (GvHD)-associated dry eye (DE) patients, as well as to find meaningful correlations between gene signals and clinical signs. METHODS Twenty GvHD-DE patients and 14 healthy controls were recruited. Patients discontinued medications for 1 week before examination. Dry eye-related symptoms and signs were recorded, and conjunctival epithelial cells were collected by impression cytology after spending 20 minutes under standard conditions within a Controlled Environmental Research Laboratory. Gene expression of inflammatory molecules was determined by polymerase chain reaction, and the results were correlated with clinical signs. Shrinkage discriminant analysis, support vector machine, and k-nearest neighbor classifier methods were used to develop predictive models that were validated considering accuracy, calibration, and discriminant capability. RESULTS Out of the 84 genes analyzed, 34 showed significant differences in expression. IL-6, IL-9, CCL24, CCL18, IL-10, IFN-γ, and CCL2 were highly increased (>6-fold); 26 genes were moderately upregulated (2- to 6-fold), whereas EGFR was downregulated (2.63 fold) in GvHD-DE samples. A panel based on EGFR, IL-6, IL-9, and NAMPT had an area under the receiver operating characteristic curve of 0.994, a sensitivity of 100%, and a specificity of 92.9%. EGFR expression correlated negatively with ocular surface damage markers, while IL-6, IL-9, and NAMPT correlated positively with these tests. CONCLUSIONS EGFR, IL-6, IL-9, and NAMPT have the greatest potential as diagnostic biomarkers, with excellent sensitivity, specificity, and clinical relevance to the ocular surface status of GvHD.

Influence of environmental factors in the in vitro dehydration of hydrogel and silicone hydrogel contact lenses

PURPOSE To analyze in vitro the influence of different environmental conditions on the dehydration pattern of seven currently marketed hydrogel (Hy) and silicone hydrogel (Si-Hy) contact lenses (CL). METHODS Three Hy and four Si-Hy CLs were evaluated. CLs were exposed to four different relative humidity (RH) conditions (5%, 30%, 50%, and 70%) and two air flow (AF) rates (0 and 2.75 m/seg) within an environmental chamber. Dehydration was assessed using the gravimetric method. Data were taken at baseline, 5, 10, 15, 20, 30, 45, 60, 90, and 120 minutes of exposure. Dehydration rate (DR), valid dehydration (VD) and stabilization time were calculated. RESULTS The interaction between RH, AF and the type of the CL material had a significant effect (p ≤ 0.03) on DR up to 60 minutes. The maximum differences in VD values among CL occurred around 15 minutes exposure varying from 25.16% to 42.75%. Stabilization time was quicker under the 5%RH with AF condition than under 70% RH without AF one for most CLs. CONCLUSIONS Lower RH seems to increase CL dehydration being further accelerated with the AF presence. The dehydration pattern is material dependent, thus current marketed CLs behave differently under several controlled environmental conditions. Future in vivo studies should confirm these outcomes.