Response profiles to a controlled adverse desiccating environment based on clinical and tear molecule changes.

PURPOSE: To investigate response profiles in the lacrimal functional unit of dry eye disease (DED) and healthy volunteers after exposure to a controlled adverse desiccating environment (CADE) by identifying groups of individuals with similar clinical and molecular changes. METHODS: Clinical parameters and tear molecule levels of 20 mild-moderate DED patients and 20 healthy volunteers were evaluated pre- (baseline) and post-CADE exposure. Clustering based on relative change from baseline values was used to identify response profiles. One-vs-all logistic regression was used to identify baseline predictors for response clusters. RESULTS: Four response profiles were identified. Cluster 1: tear break-up time (TBUT) decrease and matrix metalloproteinase 9 (MMP-9) increase. Cluster 2: marked increase in corneal staining, up-regulation of both MMP-9 and interleukin (IL)-6 levels, and down-regulation of epithelial growth factor (EGF). Cluster 3: increase in fractalkine, vascular endothelial growth factor (VEGF), MMP-9, IL-6, IL-8, IL-1 receptor antagonist (IL-1Ra) and RANTES (regulated on activation, normal T expressed and secreted) tear levels; and increased corneal staining and decreased TBUT and phenol red thread scores. Cluster 4: decreased single-item score dry eye questionnaire (SIDEQ) scores and increased corneal staining. Predictive models using baseline variables found that cluster membership depended on: corneal and conjunctival staining, SIDEQ score, interferon gamma-induced protein (IP)-10, VEGF, and IL-1Ra concentrations. CONCLUSIONS: The response of both mild-moderate DED and healthy asymptomatic individuals to environmental stress (CADE) can be predicted based on baseline (pre-exposure) clinical and tear molecular parameters. Thus, identifying individuals with a predictable response could improve patient enrollment in DED clinical trials.

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.

Influence of Climate on Clinical Diagnostic Dry Eye Tests: Pilot Study.

PURPOSE: To analyze dry eye disease (DED) tests and their consistency in similar nonsymptomatic population samples living in two geographic locations with different climates (Continental vs. Atlantic). METHODS: This is a pilot study including 14 nonsymptomatic residents from Valladolid (Continental climate, Spain) and 14 sex-matched and similarly aged residents from Braga (Atlantic climate, Portugal); they were assessed during the same season (spring) of two consecutive years. Phenol red thread test, conjunctival hyperemia, fluorescein tear breakup time, corneal and conjunctival staining, and Schirmer test were evaluated on three different consecutive visits. Reliability was assessed using the intraclass correlation coefficient and weighted kappa (κ) coefficient for quantitative and ordinal variables, respectively. RESULTS: Fourteen subjects were recruited in each city with a mean (± SD) age of 63.0 (± 1.7) and 59.1 (± 0.9) years (p = 0.08) in Valladolid and Braga, respectively. Intraclass correlation coefficient and κ values of the tests performed were below 0.69 and 0.61, respectively, for both samples, thus showing moderate to poor reliability. Subsequently, comparisons were made between the results corresponding to the middle and higher outdoor relative humidity (RH) visit in each location as there were no differences in mean temperature (p ≥ 0.75) despite RH values significantly differing (p ≤ 0.005). Significant (p ≤ 0.05) differences were observed between Valladolid and Braga samples on tear breakup time (middle RH visit, 2.76 ± 0.60 vs. 5.26 ± 0.64 seconds; higher RH visit, 2.61 ± 0.32 vs. 5.78 ± 0.88 seconds) and corneal (middle RH, 0.64 ± 0.17 vs. 0.14 ± 0.10; higher RH, 0.60 ± 0.22 vs. 0.0 ± 0.0) and conjunctival staining (middle RH, 0.61 ± 0.17 vs. 0.14 ± 0.08; higher RH, 0.57 ± 0.15 vs. 0.18 ± 0.09). CONCLUSIONS: This pilot study provides initial evidence to support that DED test outcomes assessing the ocular surface integrity and tear stability are climate dependent. Future large-sample studies should support these outcomes also in DED patients. This knowledge is fundamental for multicenter clinical trials. Lack of consistency in diagnostic clinical tests for DED was also corroborated.

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.

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-1ß, 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-1ß 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.

Basal values, intra-day and inter-day variations in tear film osmolarity and tear fluorescein clearance.

PURPOSE: The aim of this study was to determine the normal inter-day and intra-day variations in tear film osmolarity and the tear fluorescein clearance test (T-FCT) in healthy subjects. METHODS: Tear samples from 24 young, healthy adults were collected from 11:00 AM to 1:00 PM (midday) and 5:00 PM to 7:00 PM (evening) on three non-consecutive days. Tear osmolarity measurement and the T-FCT were performed to assess the basal values and inter-day and intra-day variations of the test results. A freezing point depression osmometer was used to analyze the tear osmolarity, and the T-FCT was performed using a fluorophotometer. RESULTS: The mean osmolarity value was 270 ± 4.4 mOsm/l and the mean T-FCT result was 2.97 ± 0.17 fluorescence arbitrary units. The inter-day or intra-day tear osmolarity values did not differ significantly. The T-FCT results varied significantly during the day, with significantly (p = 0.0004) higher results in the evening; no significant differences were found in the inter-day analysis. CONCLUSIONS: Tear osmolarity was unaffected by intra-day variations; however, the T-FCT showed an inter-day variation, which indicated that the time of day when the test is performed must be considered when it is used to evaluate the diagnosis of dry eye disease, disease progression or therapeutic effectiveness.

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.

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 Belmonte's 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.

Gómez-López-Hernández syndrome: another consideration in corneal neurotrophic ulcers.

PURPOSE: To report a case of a Gómez-López-Hernández syndrome (GLHS) variant with corneal neurotrophic ulcer. METHODS: Case report and review of the literature. RESULTS: A 6-year-old child presented with watering in the right eye for 3 days without ocular inflammation or pain. He had a peculiar facial phenotype and scalp alopecia in the right side. Slit-lamp examination showed an epithelial defect in the right eye and a corneal scar around the defect. Belmonte noncontact esthesiometry showed reduced corneal mechanosensory and thermal sensitivity. In vivo confocal microscopy revealed the absence of innervation in the right cornea. There was also an evident insensitivity in the alopecic region. Despite normal magnetic resonance imaging, the phenotypic manifestations along with ocular features suggested the diagnosis of a GLHS variant. CONCLUSIONS: Patients with GLHS remain asymptomatic even when they develop a corneal ulcer. Parents should be advised regarding the susceptibility of an affected child to the development of corneal lesions and the importance of regular follow-up and prompt treatment to prevent vision-threatening abnormalities.

Cytokine and chemokine levels in tears from healthy subjects.

PURPOSE: There is growing evidence for the existence of an 'immune tone' in normal tears. The aim of this study was to determine the levels of a large panel of cytokines and chemokines in tears obtained from healthy subjects. These levels can then serve as baseline values for comparison with patients suffering from ocular surface diseases. SUBJECTS AND METHODS: Nine healthy subjects participated in this study, and normal ocular surface health was documented by the results of a dry eye questionnaire, Schirmer strip wetting, and vital staining of the cornea. Four microliters of tears were collected from each eye and analysed separately with multiplex bead-based assays for the concentration of 30 cytokines and chemokines. RESULTS: Twenty-five cytokines/chemokines were detected. CCL11/Eotaxin1, GM-CSF, G-CSF, IFN-γ, IL-2, IL-3, IL-4, IL-5, IL-10, IL-13, IL-12p70, IL-15, CX3CL1/Fractalkine, TNF-α, epidermal growth factor, and CCL4/MIP-1ß were present at 5-100 pg/ml. IL-1ß, IL-6, IL-7A, CXCL8/IL-8, and CCL2/MCP-1 were present at 100-400 pg/ml. IL-1Ra, CXCL10/IP-10 and vascular endothelial growth factor were present at more than 1000 pg/ml. CONCLUSION: Multiplex bead-based assays are convenient for cytokine/chemokine detection in tears. Fracktalkine has been detected in human healthy tears for the first time. The knowledge of cytokine/chemokine concentrations in tears from normal subjects is an important reference for further comparison with patients suffering from ocular surface diseases. Variability in their levels can reflect a phenomenon of potential importance for the understanding of the ocular surface cytokine pattern.